ANNALS OF THE NEW YORK ACADEMY OF SCIENCES
Volume 108, Art. 2 Pages 339-616
LIFE-LIKE FORMS IN METEORITES AND THE PROBLEMS OF
ENVIRONMENTAL CONTROL ON THE MORPHOLOGY
OF FOSSIL AND RECENT PROTOBIONTA
Consulting Editor
Bartholomew Nagy ^
J. Joseph Lynch, SJ. (Conference Chairman) ^ ^
AUTHORS
E. Anders, E. S. Barghoorn, R. Berger, J. L, Blum, P. Bourrelly, R. E.
Cameron, B. J. Cholnoky, G, Claus, L. Dienes, H, Dombrowski, D. L.
EuROPA, F, W. Fitch, S. W. Fox, D. J. Hennessy, J. H, Johnson,
W. G. Meinschein, B. Nagy, J. Oro, C. M. Palmer, A. Papp, R. Patrick,
R. Ross, A. T. Soldo, P. Tasch, S. A. Tyler, J. R. Vallentyne, S. Yuyama
Editor Managing Editor
Harold E. Whipple Stanley Silverzweig
Q
11
.N4
V. 108
NEW YORK
PUBLISHED BY THE ACADEMY
June 29, 1963
THE NEW YORK ACADEMY OF SCIENCES
(Founded in 1817)
BOARD OF TRUSTEES
BORIS PREGEL, Chairman of the Board
Class of 1960-1963
HENRY C. BRECK
GORDON Y. BILLARD
HILARY KOPROWSKY
LOWELL C. WADMOND
BORIS PREGEL
G. W. MERCK
Class of 1962-1964
FREDERICK A. STAHL
Class of 1962-1965
HARDEN F. TAYLOR
Class of 1963-1966
W. STUART THOMPSON
CHARLES W. MUSHETT, President of the Academy
FREDERICK Y. WISELOGLE, Past President JAMES B. ALLISON, Past President
EUNICE THOMAS MINER, Executive Director
SCIENTIFIC COUNCIL, 1963
CHARLES W. MUSHETT, President
J. JOSEPH LYNCH, S.J., President-Elect
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CHARLES R. NOBACK
Recording Secretary
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EMERSON DAY
Elected Councilors
1961-1963
1962-1964
1963-1965
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Corresponding Secretary
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SECTION OF BIOLOGICAL AND MEDICAL SCIENCES
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DIVISION OF ANTHROPOLOGY
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DIVISION OF INSTRUMENTATION
WALTER E. TOLLES, Chairman CARL BERKLEY, Vice-Chairman
DIVISION OF MICROBIOLOGY
H. CHRISTINE REILLY, Chairman EUGENE L. DULANEY, Vice-Chairman
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SECTION OF CHEMICAL SCIENCES
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DIVISION OF BIOCHEMISTRY
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SECTION OF GEOLOGICAL SCIENCES
BARTHOLOMEW S. NAGY, Chairman BRUCE C. HEEZEN, Vice-Chairman
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Conferences are also held at irregular intervals at times announced by special programs.
ff J
^ ANNALS OF THE NEW YORK ACADEMY OF SCIENCES K
Volume 108, Art. 2 Pages 339-616
June 29, 1963
Editor Managing Editor
Harold E. Whipple Stanley Silverzweig
LIFE-LIKE FORMS IN METEORITES AND THE PROBLEMS
OF ENVIRONMENTAL CONTROL ON THE MORPHOLOGY
OF FOSSIL AND RECENT PROTOBK
Consulting Editor
Bartholomew Nagy
CONTENTS
itroductory Remarks. By J. Joseph Lynch, S. J 341
nvironmental Biophysics and Microbial Ubiquity. By J. R. Vallentyne 342
"he Influence of Water Currents on the Life Functions of Algae. By John L. Blum. . 353
'he Structure of Diatom Communities under Varying Ecological Conditions. By Ruth
Patrick 359
^'eli Structure and Environment. By B. J. Cholnoky 366
The Morphology of PPLO and Bacterial L Forms. By Louis Dienes 375
Axenic Culture of Paramecium — Some Observations on the Growth Behavior and Nu-
tritional Requirements of a Particle-bearing Strain of Paramecium aurelia 299X.
By Anthony T. Soldo 380
The Effect of Pollution on River Algae. By C. Mervin Palmer 389
Ultrastructure Research as an i\id in the Classification of Diatoms. By R. Ross 396
Morphology of Representative Blue-Green Algae. By Roy E. Cameron 412
Loricae and Cysts in the Chrysophyceae. By Pierre Bourrelly 421
Morphological Trends among Fossil Algae. By J. Harlan Johnson 430
Paleoecological Considerations of Growth and Form of Fossil Protists. 5y Paul Tasch. 437
Fossil Organisms from Precambrian Sediments. By Elso S. Barghoorn and Stanley
A. Tyler 451
Bacteria from Paleozoic Salt Deposits. By Heinz Dombrowski 453
Fossil Protobionta and Their Occurrence. By A. Papp 461
Studies in Experimental Organic Cosmochemistry. By J. Or6 464
Evaluation of Radiation Effects in Space. By Rainer Berger 482
Abiotic Production of Primitive Protein and Formed Microparticles. By Sidney W.
Fox AND Shuhei Yuyama 487
Observations on the Nature of the "Organized Elements" in Carbonaceous Chondrites.
By Frank W. Fitch and Edward Anders 495
On the Origin of Carbonaceous Chondrites. By Edward Anders 514
* This series of papers is the result of a conference on The Problems of Environmental Control
^-^ on the Morphology of Fossil and Recent Protobionta held by The New York .\cademv of Sciences
^ on April 30 and May 1, 1962.
Aqueous, Low Temperature Environment of the Orgueil Meteorite Parent Body. By
Bartholomew Nagy, Warren G. Meinschein, Douglas J. Hennessy 534
Evidence in Meteorites of Former Life: The Organic Compounds in Carbonaceous Chon-
drites Are Similar to Those Found in Marine Sediments. By W. G. Meinschein,
Bartholomew N.\gy, Douglas J. Hennessy 553
Further Observations on the Properties of the "Organized Elements" in Carbonaceous
Chondrites. 5,v George Claus, Bartholomew Nagy, Dominic L. Europa.... 580
Discussion of the Identity of the "Organized Elements." HARf)LD C. Urey, Moderator . 606
^V^NOf».«^^
MBLAJ7HOI
Library
Copyright, 1Q63, by The Neic York Academy of Sciences
INTRODUCTORY REMARKS
J. Joseph Lynch, S. J.
Seismology can contribute nothing to the problem of extraterrestrial life.
One naturally wonders then why a seismologist should be called upon to open
this symposium. Dr. Nagy must be blamed for that. He and I occupy offices
in adjacent buildings and when either of us has a problem in Earth science we
mull it over together. When Dr. Nagy first found evidence of organic fossils
in the Orgueil meteorite he came to me and discussed the evidence with me.
He thought that somehow I had helped him by my encouragement and as an
acknowledgment insisted that I give these opening remarks.
The possibility of life outside of our planet has been a question in man's mind
almost as far back as man himself. The divergence of views on the matter is
about as broad as it could be. Only a century and a half ago the great English
astronomer, Sir William Herschel, first President of the Royal Astronomical
Society and discoverer of the planet Uranus said in one of his Presidential ad-
dresses that he was convinced that life existed within the Sun. Unfortunately
he did not elaborate upon what kind of life he had in mind. The present Secre-
tary of the same Royal Astronomical Society, Michael Ovenden, in his recent
book, Life in ihe Universe, as his view states that life is probably possible any-
where in the universe except within a Sun! It would be hard to imagine two
more divergent views on the same subject by members of the same society. It
has even been suggested that life is older than Earth itself and came to us from
another galaxy. However, confining ourselves to our own solar system, most
thinkers on the subject would restrict the possibility of life — for reasons of tem-
perature— to that part of our solar system between Venus and Mars. Beyond
Venus the temperature would be too hot — beyond Mars and some of the as-
teroids, the temperature would be too cold. Where within this region did the
fossils on the Orgueil meteorite originate?
Dr. Nagy and his co-workers in presenting their evidence for organic fossils
on the Orgueil meteorite have adequately ruled out the possibility of their
origin by contamination since the meteorite fell to Earth. How and where the
organisms — if they were organisms — originated, are questions that this sym-
posium should throw much light on. Did they originate on Earth and later
return to Earth via the moon? Or did they originate on an asteroid or a planet
outside of the Earth? The organizing committee deserves great credit for hav-
ing brought together such a distinguished group of experts. They cover not
only every phase of the subject, but represent the views of almost every coun-
try. Because you are gathered to hear their evidence and not any rambling
conjectures of mine, I shall cut my remarks short and let the session chairman
get the program started.
The which if you with patient ears attend,
Whence came these forms, you'll find out at the end.
{With apologies lo William Shakespeare)
341
ENVIRONMENTAL BIOPHYSICS AND MICROBIAL UBIQUITY
J. R. Vallentyne
Department of Zoology, Cornell University, Ithaca, N.Y.
Since the downfall of the near-collision theory of the origin of the solar sys-
tem and the revival of the dust cloud hypothesis it has generally been assumed
that planetary systems must be common in the universe. There has also been
a strong tendency to regard the formation of life within a planetary system as
the probable outcome of a series of nonbiological events operating within a re-
stricted range of physicochemical conditions. These points of view contrast
markedly with those held even as little as 30 years ago. Few persons today
would attempt to maintain that Earth is the sole place in the universe where
life resides.
In spite of this drastic change in attitude and the recent reports of organized
matter in carbonaceous chondrites (Nagy et al., 1961; Claus and Nagy, 1961),
there are still many who hesitate to beUeve that life within the solar system can
exist beyond the confines of Earth. In relation to the cjuestion of life on Mars,
for example, it is customary to tmd opinions clouded in a mass of delicately
phrased intellectual jargon that is designed to be all inclusive and noncommit-
tal. Much of the criticism levelled against the notion of life on Mars is made
from what the self styled Soviet astrobotanist, G. A. Tikhov (1955), would
term a geocentric point of view. Thus, it is often questioned whether organ-
isms could survive the rigors of a Martian climate: an average temperature
50° C. below that of the earth; daily temperature fluctuations of about 60° C.
at the equator; an atmosphere richer in CO2 , and decidedly lower in O2 and
total pressure than that characteristic of Earth; an environment in which water
is scarce and in which the level of ultraviolet radiation may reach "lethal"
proportions.
This, however, is absolutely the wrong approach to the question. The whole ap-
proach assumes a curious lack of adaptation on the part of the presumed Mar-
tian organisms, almost forcing them to adapt to terrestrial conditions in a
Martian locality. At least two assumptions seem to be involved in the reason-
ing: (1) that a complete body of information exists defining the environmental
limits beyond which life, as known on Earth, is impossible; and (2) that these
geoenvironmental limits of life are not exceeded on a cosmic scale. The first
of these assumptions is clearly erroneous as the present paper will show, and
the second seems rather questionable.
My main purpose here is to summarize current knowledge and ignorance re-
garding the environmental boundaries that delimit the "stability field" of liv-
ing matter. The problem is approached purely on an empirical basis. Most
of the discussion is limited to conditions that permit growth and reproduction
because this is the central cjuestion that has to be faced; however, some remarks
are made concerning survival because of its pertinence to life in fluctuating
environments. The review is not intended to be exhaustive, nor comprehen-
sive in anything other than a qualitative sense; only to serve as a reminder of
forgotten or little known facts concerning some of the extreme types of environ-
ment inhabited by living organisms. Attention is focussed on microorganisms
342
y.
Vallentyne: Environmental Biophysics & Microbial Ubiquity 343
because of their great environmental and physiological diversLty as compared
to the so-called "higher" forms of Hfe. ^~ "^
Temperature
The temperatm-e range for growth and reproduction of different microor-
ganisms extends from —18° to 104° C. These Hmits exceed those defining the
stability field of pure water under one atmosphere of pressure, but they do not
exceed the stabihty field of water in the liquid state when it is impure and under
variable pressure.
Let us first consider some cases of microbial activity at temperatures below
0° C. It is important in this connection to realize that ice does not form in sea
water with a salinity of iS per thousand until the temperature drops below
— 1.9° C, and also that 90 per cent of all sea water has a temperature less than
5° C. It is thus not surprising to find that many marine bacteria will grow at
subzero temperatures. Bedford (1933) was able to culture 65 of 71 marine
bacteria from the north Pacific at subzero temperatures, and ZoBell (1934)
independently showed the same for 76 out of 88 marine bacteria in his collec-
tion. Ten of the taxa cultured by Bedford (1933) were capable of growth and
reproduction in nutrient-enriched salt solutions at — 7.5°C. Twelve others
grew at — 5°C. Horowitz-Wlassowa and Grinberg (1933) found 5 bacteria
that would grow at —5° C, and 14 others that grew at —3° C. Bacteria are
known to multiply in ice cream stored at —10° C. (Weinzirl and Gerdeman,
1929) and on fish stored at -11° C. (Redfort, 1932).
Fungi, and probably algae as well, also multiply at these low temperatures.
Thus, the mold Sporotrichum carnis grows at —7.5° C. and very slowly even
at —10° C. (Haines, 1931). ChoelosLylum fresenii and Horniodendrou cladospo-
roides also grow at — 10°C. (Bidault, 1921). Tchistiakov and Botcharova
(1938) similarly found several different fungi that were capable of growth at
— 8° C., although none of these would grow at —12° C. The flagellate Pyra-
mimonas (Pyramidomonas?) has been observed swimming in saline water at
— 7.7°C. under the cover of ice in Lake Balpash, Kazakh S.S.R. (Zernow,
1944). Populations of 12 other photosynthetic forms were found in the same
water, presumably also alive and metabolizing. Zernow (1944) even observed
swimming Pyramidomonas and Dunaliella in drops of Lake Balpash water de-
rived from soft ice that had formed at —15° C.
The most extreme cases of growth at low temperatures are those referred to
by Borgstrom (1961) who states that some molds and pseudomonads will grow
in concentrated fruit juices and sugar solutions at temperatures of — 18° to
— 20°C. He has also observed the growth of Aspergillus glaucus kept in
glycerol at —18° C. A report of pink yeasts growing on oysters at tempera-
tures of —18° to —30° C. (McCormack, 1950) needs independent verification.
No experiments seem to have been undertaken on the possibility of algal
photosynthesis in saline media at subzero temperatures, but such a result would
not be unexpected. Although slightly out of context, it is worth noting that
some terrestrial plants are able to carry out a limited photosynthesis at —2°
to — 3°C., and respire down to — 7°C. (Zeller, 1951). In the last century,
Jumelle (1891) reported that certain lichens and conifers could photosynthesize
at temperatures between —20° and —40° C., but modern studies have failed
344 Annals New York Academy of Sciences
to corroborate these findings (Rabinowitch, 1945; Zeller, 1951). Before leav-
ing the subject of growth at low temperatures it must be stressed that in all
cases the growth is slow, usually requiring weeks and sometimes months before
definitive results are obtained.
At the upper end of the temperature scale it has long been known that some
bacteria and blue-green algae exist in hot springs with temperatures in the
range of 80° to 88° C. For summaries of existing information the works of
Copeland (1938), Precht et al. (1955), and Allen (1960) should be consulted.
Baker et al. (1955), have cultured a strain of Bacillus stearothermophilus at
80° C. No attempt was made to determine whether growth would still occur
at higher temperatures. According to ZoBell (1958) thermophilic sulfate re-
ducing bacteria isolated from subterranean deposits have been cultured in the
laboratory at temperatures to 65° to 85° C. These forms were originally ob-
tained from depths of 6000 to 12,000 feet, at which temperatures in situ ranged
from 60° to 105° C. and hydrostatic pressures from 200 to 400 atmos. ZoBell
(1958) also states: "The maximum temperature at which the thermophilic cul-
tures are active is increased by compression. At 1000 atmospheres one culture
reproduced and produced HoS at 104° C. No attempt has been made to as-
certain whether bacteria will grow at temperatures higher than 104° C. when
compressed, but indications are highly suggestive of the possibilities in view of
the protective effect of high pressure on the thermal tolerance of bacteria."
The case referred to represents the highest temperature so far recorded for the
growth and reproduction of any organism.
Eh and pH
The best general treatment of the environmental limits of Eh and pH
for growth and reproduction is that given by Baas Becking et al. (1960).
These workers have summarized paired Eh-pH data for the growth of diverse
microorganisms in natural environments and laboratory cultures. Although
the Eh values may in some cases not represent truly reversible potentials they
at least give a reproducible and reasonably accurate picture. Their results
are shown graphically in figure 1. When the data for all microorganisms are
combined and compared to Eh-pH measurements in natural surface waters of
the earth, a complete overlap is observed. This suggests that there is probably
no major aqueous environment that cannot be colonized by some microor-
ganism. The range for growth and reproduction of microorganisms was found
to lie between 850 mv. and —450 mv. on the Eh scale (when expressed as Eh at
the prevailing pH) ; and between values of 1 .0 and 10.2 on the pH scale. These,
however, do not represent the true extremes because the authors considered
only data for which paired measurements of Eh and pH were available.
Some environmental extremes of pH that can be tolerated by reproducing
populations may now be cited. Thiobacilli are well known for their abihty to
grow in acid solutions. In fact, they tend to show optimal growth in the pH
range of 1 to 3, many growing poorly above pH 7. Carbon dioxide is the sole
carbon source, and energy is obtained from the oxidation of reduced forms of
sulfur to sulfate under aerobic conditions. Growth and reproduction can
occur at pH values in the neighborhood of 0, and cultures receiving no initial
Vallentyne: Environmental Biophysics & Microbial Ubiquity 345
supply of H2S()4 can contain concentrations up to 2.08 n H2SO4 at the end of
growth (Starkey, 1925).
Several molds are capable of growth at a pH of 1.7 (Johnson, 1923). The
most acid tolerant fungi known are Acontiuni velatum and fungus D (an un-
identified member of the Dermatiaceae), originally isolated from strong acid
B
H
J K L
Figure 1. Eh-pH characteristics of diverse microorganisms. A, green algae and diatoms;
B, DunalieUa; C, Enteromorpha; D, blue-green algae; E, photosynthetic ])urple bacteria;
F, photosynthetic green bacteria; G, sulfate reducing bacteria; //, thiobacteria; /, iron bac-
teria; J, denitrifying bacteria; A', three species of heterotrophic bacteria; L, methane producing
bacteria. Redrawn from Baas Becking el al. (1960). Eh is expressed in millivolts.
346 Annals New York Academy of Sciences
solutions containing 4 per cent CUSO4 in an industrial plant (Starkey and
Waksman, 1943). These forms grow well when submerged in nutrient-enriched
sulfuric acid solutions at pH values between 0.4 and 7.0. Some growth occurs
at pH 0 (2.5 normal H2SO4) even when solutions are saturated with CUSO4 .
No study was made of the permeability of the cells to copper and hydrogen
ions, but presumably there was little to no penetration.
One alga is notable for its growth in acid solutions, a strain of Cyanidhim
caldarium originally isolated from a hot spring containing 0.1 N H2SO4 . Allen
(1959) has cultured this form in 1 n H2SO4 . No attempt was made to deter-
mine whether growth would still occur in more concentrated solutions or acid
solutions at elevated temperatures.
At the upper end of the pH scale many microorganisms are known to grow
actively at a pH of 10, some at a pH of 11, and a few others possibly at still
higher pH values. Johnson (1923) reported that limiting growth of Penicillium
var labile occurred in the pH range of 10.1 to 11.1. Two other fungi, Fusarium
hullatum and F. oxysporum, were limited by pH values in the range of 9.2 to
11.2. Many alkaline lakes are known with pH values in the range of 9 to 11,
and these are by no means sterile. Jenkin (1936) found populations of 13
algae, 4 rotifers, and 2 copepods living in the alkaline lakes of Kenya. In lakes
Elementeita and Nakuru, in which the pH was commonly in the range of 10 to
11, large concentrations (10^ individuals per ml.) of the blue-green a\a.ga.Arthrop-
sira platensis were found (Jenkin, 1936). Still more extreme cases of growth
at high pH have been reported by Meek and Lipman (1922) for Nitrohacter and
Nitrosomonas. They state that these forms multiplied in solutions with initial
pH values of 13.0, although not when the initial pH was as high as 13.4. These
results, however, seem rather surpising because of the apparent lack of a toxicity
effect due to ammonium hydroxide which would be expected for these forms
under the culture conditions used. Other workers have failed to corroborate
the findings of Meek and Lipman for Nitrobader and Nitrosomonas. Kingsbury
(1954) has reported that the blue-green alga Plectonema nostocorum will grow
in solutions of Ludox (a DuPont 30 per cent SiOo solution) adjusted to an initial
pH of 13, however the growth in this case was apparently limited to the surface.
Salinity
The range of salt concentrations tolerated by microorganisms during growth
and reproduction is enormous. Kalinenko (1957) has shown that some hetero-
trophic bacteria will multiply in double distilled water. (The water in this
case contained only 70 /xg. of organic matter per liter.) On the upper side it is
known that the fungi Aspergillus oryzae and A. terricola will grow in 4.1 m
MgS04 , a concentration equivalent to about 500 g. of salt per liter of solution
(Johnson, 1923). HalophiUc bacteria in nature grow abundantly in salt
Hmans, saturated brines, and on animal hides dried with concentrated salt solu-
tions. Even the Dead Sea with its salinity of 280 to 320 per thousand and high
bromide concentration is not sterile. A small gram negative rod, a yeast-like
form, and a green filamentous form were all found to grow and reproduce in
Dead Sea water enriched with 1 per cent peptone (Wilkansky, 1936). Other
bacteria and algae were also present. Some of the bacteria failed to grow in
Vallentyne: Environmental Biophysics & Microbial Ubiquity 347
media containing less than 15 per cent salt. See Clifton (1958, p. 262) for a
summary of Volcani's study of the Dead Sea biota.
Solar evaporation ponds are often discolored by the growth of halophilic
bacteria and algae. According to Carpelan (cited by Gibor, 1956) photosyn-
thetic production rates in such environments are comparable to those in the
most productive parts of the oceans. Gibor (1956) has shown that the osmo-
tolerant brine flagellate, DunaUella salina, grows well in 10 X concentrated
artificial sea water. Some halophilic bacteria isolated from salt brines fail to
grow in salt solutions containing less than 16 per cent NaCl, and will survive
on dry crystals of salt obtained by the evaporation of brines (Browne, 1922).
According to Gibbons and Payne (1961) the most rapid growth rates of several
halophilic bacteria (Halobacterium spp. and Sarcina littoralis) occur in solutions
containing 20 to 25 per cent NaCl at temperatures in the range of 40° to 45° C.
ZoBell (1958) states that sulfate reducing bacteria grow naturally and can be
cultured in waters with salinities up to 300 per thousand.
Pressure
The effect of varying atmospheric pressure on the growth and reproduction
of microorganisms seems not to have been investigated in much detail. Strug-
hold (1961), however, passingly refers to the cultivation of soil bacteria under
an atmosphere with the composition and total pressure (0.1 Earth atmos.) of
that presumed to exist on Mars. The existence of barophilic bacteria in sub-
terranean deposits and deep sea sediments has been demonstrated by ZoBell
et al. Most organisms living in the surface regions of Earth fail to grow and
are killed by hydrostatic pressures of a few hundred atmospheres. In contrast
to these, barophilic bacteria isolated from the deep sea bottom can be cultured
only under hydrostatic pressures comparable to those in their natural environ-
ment, i.e., pressures of 1000 atmos. or more (ZoBell and Morita, 1956). The
viability of some barophiles is unaffected by alternate compression and decom-
pression between 1 and 1000 atmos. of hydrostatic pressure when applied 10
times within 10 minutes (ZoBell, 1958). ZoBell (personal communication) has
cultured deep sea bacteria under 1400 atmos. of hydrostatic pressure.
Water
Water is the most concentrated single molecule in protoplasm. Its depletion
can therefore be expected to restrict growth and reproduction. Most organ-
isms, microbes included, survive periods of extreme drought in dormant states,
often as spores. On the other hand, in the case of Pleurococcus vulgaris slightly
modified vegetative cells suffice to withstand prolonged drought (Fritch, 1922;
Fritch and Haines, 1923). According to Zeuch (1934) cell division of Pleurococ-
cus vulgaris can still occur at relative humidities of 68 per cent at 1° C, 55 per
cent at 10° C, and 48 per cent at 20° C. Aspergillus glaiicus is well known for
its growth on substrates where the activity of water (a„,) is as low as 0.65 to 0.70
(Scott, 1961). Kordyum and Bobchenko (1959) hold the opinion that many
microorganisms can actually use air as a habitat for growth and reproduction.
The growth of lichens on bare rock surfaces, bacteria and fungi in flour, and
many microorganisms in strongly saline media represent ecological instances of
348 Annals New York Academy of Sciences
growth in environments in which the chemical potential of water is low. Noth-
ing more than speculative attention has been given to the possibility of micro-
bial growth in nonaciueous media. It should not be forgotten, however, that
the water dependent metabolism of all living organisms that are known must
be at least to some extent the end result of selection on a water rich earth. It
is not known whether life could form on a planet on which the predominant
lifjuid was some other compound than water. One should also remember that
under aerobic conditions of metabolism water is one of the main excretory com-
pounds formed by living organisms. Mechanisms for the selective retention of
metabolically formed water might enable some organisms to persist and grow
in liquid media with low water contents.
Other Factors
In relation to natural radiations, direct sunlight is known to be lethal for
many microorganisms, but the effects probably result from dehydration and
high temperatures in most cases. ZoBell and McEwen (1935) were unable to
detect any lethal effect when marine bacteria were exposed in layers of water
greater than 5 mm. in thickness to full noon sunlight on a roof top in La JoUa,
Cahfornia. Two halophilic bacteria isolated by Browne (1922) withstood in-
definite exposure to "the brightest sunlight."
The effect of ultraviolet light on microorganisms has been studied by many
workers; however, most of the data refer to high dosages for short times. It
would be of much interest to know the maximal levels of continuous ultraviolet
radiation that can be tolerated by actively growing cultures. Although ultra-
violet light in high doses is harmful to all organisms, it must be remembered
that deleterious effects are much less pronounced above M)() m/x- than below for
equal energies of incident light (Meier, 1936). There is also a great variation
in the sensitivity of different microorganisms to ultraviolet light. Siliceous
tests of diatoms apparently afford no protection (Ursprung and Bloom, 1917).
Because the possibility of shielding and the well known photoreactivation phe-
nomenon, whereby the lethality of ultraviolet light is partly reversed by later
application of visible light, it is probably incorrect to assume, as many have
done, that an ozone free earth would necessarily be sterile.
The biological effects of gamma- and other types of ionizing radiations have
also been studied by many investigators. Single large doses have usually been
used. Populations of many microorganisms will survive single doses in the
range of 10'' r. (Shields et al., 1961). Saccharomyces cerevisiae has been cultured
under continuous exposure to 50 mr. per day of radium emanations (Maisin et
al., 1960), however, this is doubtlessly far below the maximal level that can be
tolerated. According to Prince (1960) a good place to look for radiation re-
sistant microorganisms would be in nuclear reactors. He states that it is
"common knowledge that some bacteria can adapt even to the water in a swim-
ming-pool-type nuclear reactor."
A few other case histories will serve to round out the picture that has been
presented. Some of these refer to survival rather than to growth and repro-
duction. The cases are as follows.
(1) The growth of several bacteria and fungi in concentrated CuS04 solu-
tions. The subject has been reviewed by Starkey and Waksman (1943).
Vallentyne: Environmental Biophysics & Microbial Ubiquity 349
(2) Bacteria that grow actively in solutions containing 1 g. of phenol per
liter (Putilina, 1959).
(3) Growth of the fungus Aspergillus in a 40 per cent solution of citric acid
(Johnson, 1923).
(4) An aerobic bacterium (Hydrogenomonas?) , originally isolated from sewage
sludge that shows poor growth in air, but develops well in an atmosphere con-
taining 20 per cent by volume O2 and 80 per cent by volume CO (Kistner, 1953).
(5) Heterotrophic growth of algae in lakes during the sunless arctic winter
(Rhodhe, 1955) and reproduction of algae in subterranean caves (Claus, 1955).
(6) The survival of some bacterial spores after 5 hours' immersion in non-
aqueous media at temperatures approaching 140° C. (Rodenbeck, 1932).
(7) The survival of bacterial and fungal spores, and even vegetative cells of
Mycobacterium smegmatis, after 5 days' exposure to ultrahigh vacuum at pres-
sures below 10~^ mm. of Hg. (Portner et al., 1961).
Table 1
Environmental Limits of Temperature, Eh (at the Prevailing pH), pH, Hydrostatic
Pressure, and Salinity for Growth and Reproduction
OF Microorganisms
Factor
Lower limit
Upper limit
Temperature
-18° C. (fungi, bacteria)
104° C. (sulfate reducing bacteria
under 1000 atmos. hydrostatic
pressure)
Eh
-450 mv. at pH 9.5 (sulfate re-
ducing bacteria)
4-850 mv. at pH 3 (iron bacteria)
pH
0 {Acontium velatum, fungus D,
Tbiobacillus iliiooxidans)
13 (?) (Plectonema nostocorum)
Hydrostatic pres-
Essentially 0
1400 Atmos. (deep sea and bac-
sure
teria)
Salinity
Double distilled water (heterotro-
Saturated brines {Dunaliella, halo-
phic bacteria)
philic bacteria, etc.)
(8) Survival of many microorganisms after prolonged exposure to tempera-
tures approaching absolute zero (Belehradek, 1935; Becquerel, 1950). Life
may, in some cases, be capable of almost infinite preservation under such condi-
tions.
One could multiply the examples at greater length, but those already pre-
sented suffice to make the point.
General Remarks
In TABLE 1 are summarized the ranges of temperature. Eh, pH, hydrostatic
pressure, and salinity that still permit growth and reproduction of one or more
microorganisms. It is not maintained that growth is anywhere near maximal
under the extreme conditions referred to, merely that it does occur. Selection
and mutation over long periods of time could doubtlessly result in a further
widening of the observed limits. It should also be stressed in this connection
that scientists are inchned to study single factors taken one at a time. When
two or more environmental factors show antagonistic effects, as is the case
with temperature and pressure, one can expect to find an increased tolerance
to each factor using combined action.
350 Annals New York Academy of Sciences
The microorganisms referred to in this paper are pecuhar in that they grow
in environments that are lethal to most other forms of Ufe. One can instruc-
tively reverse the point of view that has been taken here and ask why it is
that most organisms live under "common" conditions. The answer is, of
course, because life as a whole is selectively adapted to growth in common
environments. If the waters of the earth were predominantly acid, growth
at neutral pH values would be regarded as an oddity. Thus, the fact that
most living species conform physiologically and ecologically to average Earth
conditions should not be taken to indicate any inherent environmentally based
physicochemical conservatism of living matter. Adaptation has taken place.
Environments of the Earth that are sterile or nearly so mostly fall into one
of two categories: nonaqueous environments, and noncirculatory aqueous en-
vironments. The first category is so obviously restrictive in a biological sense
that it requires no further comment. The second refers to rock-enclosed waters
that do not readily enter into the hydrological cycle. Oil brines, for example,
that are perfectly sealed in place, seem to be sterile (Shturm, personal communi-
cation), and deeply buried wet sediments usually have low to negligible bac-
terial populations. In small enclosed systems extinction becomes increasingly
probable with time because of the small numbers of organisms involved, the
accumulation of metabolic waste products, and the general decrease in free
energy of the system as a function of time. Continuous circulation negates
these factors and in addition permits occasional injections of diverse micro-
organisms into new environments, to which they may become adapted over
many generations. Given the presence of circulating water, it seems rather
unlikely that any aqueous environment could remain indefinitely sterile over
geologically long periods of time. The powers of microbial reproduction and
variation are so immense in an evolutionary sense as to make this a virtual
impossibility. This assumes, of course, that some energy source is available
for metabolism in the environment concerned; but this is not a restrictive
limitation either biologically or geochemically.
Returning to the cjuestion of extraterrestrial life, the problem involved seems
not so much to be whether organisms could live elsewhere under conditions
that we would regard as unusual on Earth, as it is to account for the origin of
life itself. In relation to the possibility of life on Mars, for example, the ques-
tions should be of two types: (1) whether conditions there were ever favorable
for the origin or introduction of life; and (2) whether subsequent conditions
have been favorable for the persistence of such life as might have been formed.
The second question is far less critical at the present time than is the first.
To appreciate the potentiahties of adaptation one need only contemplate how
an Ordovician observer might have viewed the likelihood of birds flying in the
air, the possibility of an animal maintaining a temperature of 37° ± 1° C. for
virtually all of its lifespan over a period of 100 years, or the existence of plants
that trap and feed on animals. What can the leper know of the scorpion's
sting? And what does the blind man know of the firefly's light?
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THE INFLUENCE OF WATER CURRENTS ON
THE LIFE FUNCTIONS OF ALGAE*
John L. Blum
Canisius College, Bn_ffalo, N. Y.
Selective effects by the current. Of the many habitats on Earth which are
colonized and exploited by sessile organisms, those which are in contact with
a mass of air or water in relatively rapid movement are likely to constitute a
convenience or a necessity to the uptake and excretory systems of the organ-
ism but to represent, at the same time, a major threat to the organism's se-
curity. Metabolizing organisms as we know them are inhabitants of fluids.
These fluids when laden with small cjuantities of nutrients and motionless may
or may not be suitable for successful growth and reproduction. When the
fluid is in unidirectional or turbulent motion and the organism remains in place,
the possibilities for successful growth of many sessile organisms are greatly
enhanced, but security is likely to be threatened by factors like evaporation
or physical buffeting by the current, and by the molar agents which are flung
at the organism. Areas where surface or subsurface currents run in close
proximity to the bottom or other stable objects are successfully exploited by
numerous sessile marine plants and invertebrates; in fresh water currents ses-
sile invertebrates are relatively few and inconspicuous, but the algae have
successfully colonized what to most animals is a peculiarly dangerous spot, the
rapids of streams both large and small. So unicjue is this habitat that some
of the algae which are found in the rapid water habitat are seldom if ever found
anywhere else.
The present paper concerns algae which inhabit and are essentially limited
to fresh water currents, that is, algae which have moving water all around them
or in very close proximity; but inasmuch as the current has varied influences
as well on organisms which are in it only temporarily, I shall make occasional
mention of other river algae. The true current-inhabiting species are not
adequately described by the term "river algae" because the latter category
includes many forms which cannot attach and which are often unable to remain
in place in a strong current. Essentially all surface streams are inhabited by
some such forms, many of which are found as commonly or more commonly in
standing water.
From source to mouth a freshwater stream consists of alternating shallow
(rifHe) areas and pools. These respective habitats differ in many ways and
it is usual to find that each is inhabited by a distinctive assemblage of animals
and plants. Current rate is influenced by a number of well known variables;
in small streams these variables act so as to subject different but adjacent
points to quite different pressures. Such pressures are likely to fluctuate
greatly from moment to moment but minute differences in depth and pre-
sumably in average current rate between points distant by only a few milli-
meters on the stream floor make of each shallow area a mosaic of differing
* This study was aided in part by funds from the National Science Foundation (G-10898).
353
354 Annals New York Academy of Sciences
microhabitats whose existence and individuality is attested by striking differ-
ences in the algal populations which colonize them at certain seasons.
A primary influence of current on algae, therefore, is the exclusion of certain
species from pool areas or other places where current is minimal, or the en-
hancement of growth of such species in the most favorable, frequently the
fastest current. The fact that algae colonize so dangerous a habitat as flowing
water suggests that they can be provided some unique service by this habitat.
The relationship of algal photosynthesis and respiration to water movement
has been discussed by various investigators including Gessner (1937) and
Steeman-Nielsen (1944). Oxygen consumption in the dark and the photo-
synthetic rate are increased in moving water above the respective values for
standing water. More recently, respiratory rate and P uptake by Oedogonium
kurzii Zeller have been studied by Whitford (1961). Radioactive P uptake
in water moving at 18 cm. per second was found to be over 10 times that in
still water. He concludes that the cause for "inherent current demand" by
lotic organisms is the need for rapid exchange of materials with the water and
that the steep diffusion gradient in a current satisfies this demand.
This inherent current demand and the gradients involved may be of sig-
nificance to algae in two ways: for materials which are brought to the algae
by the current and for removal downstream of substances which might be
harmful. At least some algae are known to excrete substances which eventu-
ally retard their own growth rate. That such materials would be flushed
away from an alga growing in a current is evident, and may explain the limita-
tion of at least certain species to rapid water. It may also explain the high
cell density achieved by many current algae.
EJfecls of current on algal size or shape. Precisely how current influences the
structure of an individual algal cell or thallus has received relatively little
attention. Many benthic stream algae are so flexible that the current con-
tinually bends and twists them without visible damage or effect. Unlike a
tree which bends permanently under the influence of prevailing winds, there
is nothing about their structure which would even betray the usual direction
of the current if by some means the current were suddenly averted or brought
to a stop. The same is true of certain less flexible bottom-inhabiting forms.
The Phormidium-Audouinella-Schizothrix community which is known from
streams of the North Temperate Zone (Blum, 1956) does not, in the surface
topography of its crust, show any very evident polarity with respect to the
current. Others — and relatively few cases are known — show by the form or
orientation of their thallus the effects of unidirectional current as in the Phor-
midium community described by Wehrle (1942), a composite community of
Vaucheria and Plectonema described by WaUner (1934), or in the colonies of
Cocconeis growing on a vertical cylindric stake as described by Gessner (1955).
How the current controls the size of certain benthic algae is shown by work
done by Picken on the alga Rivularia. In regions of relatively rapid flow
thallus size was found to be proportional to the size of the stones to which the
thallus was attached. In slower water, however, thallus size was independent
of stone size. The bulk of this alga increases more rapidly than the area of
its attachment, and the current limits the maximal size of the thallus, either
Blum: Water Currents & Algae 355
tearing the thallus away from the stone, or transporting both stone and thallus
to a slower part of the stream (Picken, 1936).
Influence of the current on algal reproduction. In their reproduction current
algae take full advantage of the medium of dispersal which is at their doorstep.
It is commonly observed that many algae which colonize stream bottoms
achieve in certain seasons almost saturation coverage of available and favor-
able sites. Thanks to the mixing done by the current these algae are able to
introduce their reproductive units into what must be a very high percentage
of rock fissures, cracks, scratches, and roughened areas, into enough, at least,
of such depressions to permit subsequent growth from the colonizing cells to
cover close to 100 per cent of the available surface. In southern Michigan
streams which I investigated colonization of rock surfaces is very rapid, and
successful in very high percentages of the space available. The winter dom-
inant diatoms Gomphonema olivaceum and Diatoma vulgare, for example, achieve
good growth in winter on newly submerged rock surfaces in as little as 10 days.
Both of these forms were at the same time colonists and seasonal dominants,
no evidence being found of succession before the establishment of the com-
munities they represent. The period within which G. olivaceum colonized
bare rock surfaces extended from late November to early April, and coloniza-
tion seemed to be possible at any time within this period (Blum, 1954).
Evidence that planktonic forms reproduce as they are carried downstream
has been presented by various workers but there remains the suspicion that
much of the actual cell division occurs on the bottom and that the apparent
increase in phytoplankton downstream is largely the result of more extensive
nutrient beds there and of more dense populations of benthic individuals,
many of which rise every day into the plankton. I observed the vegetative
dissemination of Spirogyra and Oscillatoria communities on warm summer
days in the Saline River in southern Michigan. These communities were
especially characteristic of cjuiet shoals or bays. Here the algae remained
on the bottom in contact with nutrient-rich silt deposits, as masses of filaments
easily visible from a distance. The surface waters of such shoals or bays is
usually in slow circular movement set up by the main current of the stream,
which by-passes the shoal or the bay in a tangent to the circular current which
it produces there. At times of rapid photosynthesis, individual masses of the
algal filaments are detached and buoyed upward by trapped oxygen bubbles.
Once the algal mass has quit the floor of such a shoal, it is carried slowly along
in the eddying surface water. After moving for some time in this circular
manner it may eventually be picked up by the tangential current of the main
stream which removes it definitively from the shoal. As the algal mass travels
downstream, it disseminates live filaments along the way. The progress of
these filaments is arrested on obstructions or on new shoal areas or other sedi-
ments downstream, which in this way are themselves colonized. The elevation
of algal masses by entrapped bubbles can be observed from about noon until
about 2 to 3 p.m. on sunny days in summer and the movement downstream
of these floating masses can be observed throughout an entire afternoon.
The evolution of current-inhabiting algae. I believe the first attempt to clas-
sify the body types of current algae was made by Cedergren (1938). His
356
Annals New York Academy of Sciences
classification included 4 groups, namely (1) richly branched thalli; (2) long,
flexible cylinders, (3) spherical cushions, and (4) simplified platelike forms.
The second of these groups should probably be modified to include forms with
laciniate, reticular or lacunate bodies which float downstream from a point of
attachment; it should also be pointed out that certain algae with short un-
branched filaments, although they indeed qualify as cylinders, nevertheless
have a somewhat unique superficial form since, as in Vaucheria, they frequently
constitute a virtual turf but do not become interwoven to form massive skeins
as in the first group. If body form is a major criterion for these groups, at
Cladophora glomerata
£22^i-tli'.- v,.ii.?jV-t^:„'.-e
Figure 1. Cladophora glomerata. The illustration at bottom represents several algal
thalli X'2 attached to a portion of rock (stippled). The upjier drawings represent increasing
magnifications of small portions of the thallus.
least one other category should probably be added for forms with a rigid,
cylindrical, but pseudoparenchymatous body like Lemanea.
The first 2 groups as outlined by Cedergren can be summarized by the
qualification that they live in the current and permit water to run among their
filaments or at least on more than one side of the thallus. Hence they e.xpose
a large surface area directly to the surrounding water. A common example
of this type is Cladophora glomerata (L.) Kiitz. (figure 1). These groups can
be further subdivided into gelatinous and nongelatinous types. The gelat-
inous types in general have relatively small filament or trichome diameter.
The last 2 groups of Cedergren can be qualified by virtue of their position
mostly below the current — in other words they become a part of the stream
bottom. The current docs not flow among their filaments but only in their
^
^^-., .. "^
^^ ;, '?^
^
Gomphonema ottvaceum
Figure 2. GompJionema olivaceum. The illustration at the bottom represents several
soft thalli attached to and completely covering a rock. The mottled dark area represents
bare rock at a point where an algal thallus has been cut away. The upper drawings represent
successive magnifications of the area cut away. Insect larvae which feed on these diatoms are
shown within the algal mass.
^i-M^^ji'.^^ -^-x
l^ivularici sp
Figure 3. Rivularia sp. Stippled portion represents a rock to which several subspherical
colonies of Rivularia are attached. The upper drawings show increasing magnification of a
single colony or thallus. Calcium carbonate cr)stals are shown as they appear in the gelatin
between adjacent algal filaments.
357
358 Annals New York Academy of Sciences
vicinity. These include massive sheets which cover rocks in the current and
may extend partly into the current as in Phormidium spp.; parenchymatous
or pseudoparenchymatous collections of cells; soft, gelatinous masses that
move slightly in the current (some diatoms such as Gomphonema olivaceum
(Lyngb.), Kiitz. (figure 2); and firmer, spherical, or hemispheric masses which
are frequently gelatinous as in Rivularia spp. (figure 3). The gelatin in these
types serves to lubricate the alga-current interface and to reduce friction and
injury to the plant but it also serves to separate adjacent trichomes or filaments
and to keep, in many algae, a rather precise spatial relationship between fila-
ments as they lie in their intercellular material (figure 3).
When fresh water algae, generally, are compared and contrasted with marine
algae, the essential absence from the former of massive plant bodies, leathery
and foliose types which are so common in the marine Rhodophyta and Phaeo-
phyta is noteworthy. Although the Phaeophyta have proven generally un-
successful in fresh water and would not really be expected to produce such
plant forms in any event in fresh water, the same is not true of the Rhodophyta
or of the Chlorophyta. Nevertheless, the latter groups are not represented
in fresh water by forms more massive than Tuomeya, Lemanea, Chaetophora,
or Monostroma.
The evolution of fresh water algae has thus been successful largely for the
smaller, more delicate forms which are characteristic of standing water rather
than of currents. If we suppose that the rather specialized current algae have
evolved at least in part from their fresh water relatives that are tolerant of
standing water, it must be granted that their form has not been greatly modified
by the change in habitat.
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Cedergren, G. R. 1938. Reofila eller det rinnande vattnets algsamhallen. Svensk. Bot.
Tidskr. 32: 362-373.
Gessner, F. 1955. Hydrobotanik. Vol. 1. VEB Deutscher Verlag der Wissenschaften.
Berlin.
Lastochkin, D. 1945. Achievements in Soviet hydrobiology of continental waters. Ed.
G. E. Hutchinson. Ecology. 26: 320-331.
PiCKEN, L. E. R. 1936. Mechanical factors in the distribution of a blue-green alga, Rivularia
haematites. New Phytol. 35: 221-228.
Steeman-Nielsen, E. 1947. Photosynthesis of aquatic plants with special reference to
the carbon sources. Dansk Botan. Arkiv. 12: 1-71.
Wallner, J. 1934. Beitrag zur Kenntnis der Vaucheria-Tuffe. Zentr. Bakteriol. Parasi-
tenk. 2(90): 150.
Wehrle, E. 1942. Algen in Gebirgsbachen am Sudostrande des Schwarzwaldes. Beitr.
Naturk. Forsch. Oberrheingebiet. 7: 128-286. PI. 1-3.
Whitford, L. a. 1960. The current effect and growth of fresh-water algae. Trans. Am.
Microscop. Soc. 79: 302-309.
THE STRUCTURE OF DIATOM COMMUNITIES UNDER
VARYING ECOLOGICAL CONDITIONS
Ruth Patrick
Department of Limnology, Academy of Natural Sciences of Philadelphia,
Philadelphia, Pa.
During the preceding 15 years we at the Academy of Natural Sciences of
Philadelphia have spent a great deal of time studying the composition of diatom
communities in the eastern and southern United States. The findings from sec-
tions of streams characteristic of this area which have not been adversely
affected by pollution are discussed in this paper. To understand these com-
munities of diatoms not only the species which compose them but also the
sizes of the populations of these species must be known. This necessitates
collecting species from all types of habitats in the community. It also neces-
sitates counting sufiEicient specimens to determine most of the species compos-
ing the community. Obviously, when studying a community which has one
or two species with large numbers of individuals, many more specimens must
be counted to discover the species composed of small populations.
From our studies it is evident that 7000 to 8000 or more specimens usually
must be counted before a reliable picture of a diatom community can be ob-
tained if one wishes to compare the quantitative characteristics of commu-
nities. From TABLE 1 it is evident that only a small percentage of the species
composing the community are seen when only 200 or 500 specimens are counted
and approximately 50 to 75 per cent of the number of species are seen when
1000 specimens are counted when compared with the number seen when several
thousand specimens are counted. Similarly, the percentage of the population
composed of specimens of dominant species in some cases varies greatly when
based upon counts of a few hundred specimens as compared with counts of a
few thousand specimens. As seen in table 2 the numbers of species compos-
ing the diatom community remain fairly similar when similar segments of the
communities are analyzed if no serious change in the environment occurs. As
seen in table 3 the percentage of the population composed of dominant species
does not vary greatly for similar environments when similar segments of the
community are studied.
When the structure of these populations was plotted by representing the
number of species as the ordinate and the number of individuals composing
each species as the abscissa, the data approached the shape of a truncated
normal curve, figure 1. To determine what mathematical formula might
best express the results of these studies several formulae were tried (Patrick
et al., 1959) and the truncated normal curve provided a little better fit than
the other methods investigated. The use of a truncated normal curve to ex-
press the structure of communities of organisms has been supported by the
work of MacArthur el al.
By using this method we objectively compared similar segments of diatom
populations. For example, if enough specimens are counted and enough species
are identified to always place the mode in approximately the same interval,
359
360
Annals New York Academy of Sciences
a similar segment of the community will have been studied regardless of the
dominance of any species that may be present.
We have found in natural rivers which are relatively free from pollution the
communities are composed of many species with most of them having relatively
small populations. These findings support the theory set forth by Thiene-
mann (1939) that optimal environments support many species composed of
relatively small populations. Furthermore, the numbers of species do not
change greatly from season to season in the same area nor do they change very
much from area to area collected at the same time. For example, in table 2
Table 1
The Number of Species and the Percentage of the Specimens in Populations of
Dominant Species Observed when Varying Numbers
OF Specimens are Counted
River
Specimens counted
Number of species
Percentage of
dominance*
Wateree River, South Carolina,
September 22, 1961
200
558
1009
5970t
33
52
81
117
62.5
62.2
51.2
27.2
Assunpink Creek, New Jersey,
September 19, 1959
200
569
1219
12,5841
35
65
97
178
51.5
31.6
61.5
39.5
Potomac River, Maryland, Octo-
ber 18, 1960
206
558
1637
I7,911t
24
37
76
148
89.3
87.3
7t.S
62.2
Sabine River, Texas, October 18,
1960
211
511
1348
7369t
24
39
68
105
75.8
72.2
54.7
60.0
* The percentage of specimens counted composing the dominant species. A dominant
species is one that is represented by 1000 or more specimens when 5000 or more specimens are
counted.
t These are the number of specimens which had to be counted to place the mode in the
second interval when a truncated normal curve is constructed from the data.
are shown the data for these statements derived from studies of the Savannah
River.
When the numbers of species found in different natural soft water rivers, for
example the Savannah River, the Red Clay Creek, and the Wateree River,
are compared, they do not vary greatly. The total number of species for the
Savannah River (South Carolina) was 188; Red Clay Creek (Delaware), 145;
Wateree River (South Carolina), 181. Considering only those species repre-
sented by more than 6 specimens when 7000 or more specimens are counted,
we find Savannah River, 85; Wateree River, 89; Red Clay Creek, 76. The
reason that 6 or more .specimens have been used for estimating that a species
is established in a given area is that if a truncated normal curve is constructed
those species represented by 4 to 8 specimens will have better than a 50 per
Patrick: Structure of Diatom Communities
361
cent chancL- of not shifting their position in the curve (Preston, 1948) and,
therefore, will remain a part of the community.
However, if the kinds of species in similar sections of various rivers are ex-
amined, a great variation as to the kinds of species is seen as described by
Patrick (1961). Also, in studies of the same area of the Savannah River at
about the same season (late August, early September) of the year in different
years, only 34 per cent of the species were common to both studies. A sim-
ilar, but not as great, variation is seen when two different areas in the same
Table 2
Savannah River
Summary of Catherwood Diatometer Readings at Station 1
October 1953 to January 1958
Date
Specimen number
in modal interval
Oct. 1953
Jan. 1954
Apr. 1954
July 1954
Oct. 1954
Jan. 1955
.\l)r. 1955
July 1955
Oct. 1955
Jan. 1956
.Vpr. 1956
July 1956
Oct. 1956
Jan. 1957
.\pr. 1957
July 1957
"Oct. 1957
Jan. 1958
(Apr. 1954-1958 averages)
4-8
2-4
2-4
4-8
4-8
2-4
2-4
2-4
2-4
4-8
2-4
2-4
2-4
2 4
4-8
2-4
2-4
Species in mode
22
19
24
23
21
19
25
20
27
30
35
24
23
29
21
29
25
27
24
Species observed
150
151
169
153
142
132
165
132
171
185
215
147
149
177
132
181
157
152
151
Species in theo-
retical universe
178
181
200
193
168
166
221
180
253
229
252
185
206
233
185
203
232
212
194
Table 3
Dominant Species in Two Areas, Guadalupe River
Station 1
Station 2
9 Sept. 59
9 Sept. 59
Gom plionema affine var. insigne
1272
2850
G. parvulum
4346
2700
Naviciila sp.
1900
N . tripunclatus var. schizonemoides
30,634
23,750
Nitzschia palea
2400
Percentage of total count composed of domi-
95
90
nant species
362
Annals New York Academy of Sciences
river which have the same types of ecological habitats are studied at the same
time. The kinds of species in common are more variable than the numbers.
For example, Stations 1 and 6 on the Savannah River which are about 30 miles
apart were studied in June of 1960 and 187 species were identified at Station 1
and 54 per cent of these were found at Station 6. At Station 6146 species were
identitied and 75 per cent of these were found at Station 1. In October of 1960
when these two areas were studied the number of species at Station 1 was 184
and the number at Station 6 was 185. However, 75 per cent of the species at
Station 1 were at Station 6 and 75 per cent of the species at Station 6 were at
Station 1.
This same principle as to similarity of numbers of species but differences in
kinds of species also holds for the hard water rivers we have studied. Often
the numbers of species are slightly less in natural hard water rivers than in
40 r
INDIVIDUALS = 1-2 2-4 4-8 8-|6 16-32 32-64 64" 128" 256" 512" 1024- 2048-4096" 8192-16384-32768-
128 256 512 1024 2048 4096 8192 16384 32768 65536
INTERVALS =0 1 2 3 4 5 6 7 8 9 1 0 1 1 12 13 14 15 16
Figure 1. Ridley Creek, Pennsylvania
soft water rivers. For example, in the Potomac River, a hard water river, from
April of 1959 to October of 1960 in one area studied the observed species varied
from 130 to 148 (average 144) as contrasted with a variation from 118 to 185
(average 161) in the Savannah River which is a soft water river, over a similar
period of time.
In brackish waters such as the estuary of the York River the numbers of
species composing a diatom community sometimes are a little less than in a
soft water river. From November of 1956 to May of 1959 the number of
observed species varied from 108 to 147 (average, 130). However, in all of
these three types of rivers — soft, hard, and brackish water — the communities
are made up of many species most of which have relatively small populations
if the rivers are natural and not polluted.
A different picture is found when the structure of diatom communities in
dystrophic streams is examined. In these there is a restricted diatom flora
which can live in these naturally acid streams high in humates. They are
species largely confined to the genera Eunotia and Frustidia and certain species
Patrick: Structure of Diatom Communities
363
of genera such as Pinnularia, Adinella, Anomoeoneis and Surirella. Thus, we
have a community composed of fewer species with populations that are much
more variable in size (figure 2). The truncated normal curve representing
the structure of the community has a much lower mode, fewer observed spe-
40
35
<" 30
UJ
in
^ 20
o
a 15
m
2 10
o
INDIVIDUALS
INTERVALS
1-2 2-4 4-8 8-16 16-32 32-64 64" 128- 256" 512" 1024- 2048-4096" 8192-16384-32768-
128 256 512 1024 2048 4096 B 1 92 16384 32768 65536
= 0 1
10 II
12
13 14 15 16
Figure 2. Egg Harbor River, N.J.
40
35
<^ 30
UJ
o
Q. 25
"- 20
o
Q: I 5
UJ
CQ
2 10
INDIVIDUALS = 1-2 2-4 4-8 8-16 16-32 32-64 64" 128" 256- 512" 1024- 2048-4096" 8192-16384-32768-
iinuiviuuml;^ l^g 255 512 1024 2048 4096 8192 16384 32768 65536
INTERVALS =0
10 II 12 13 14
15
Figure 3. Back River, Maryland
cies, a much greater a^, which means more variability in the sizes of the diatom
populations, and covers more intervals, because a few species have very large
populations.
This is the type of curve often found for the structure of diatom populations
which are subjected to pollution (figure 3). In both cases one or more eco-
364 Annals New York Academy of Sciences
logical factors have operated to greatly limit the numbers of species which
can survive in these particular kinds of ecological conditions.
A few studies which we have done indicate that in springs in which the en-
vironment is fairly constant the numbers of species composing a diatom com-
munity may be much less than in the very variable environment of an eutrophic
or mesotrophic, natural river. It seems that it is the highly variable, yet con-
tinuously favorable, environment of natural rivers of these types that is largely
responsible for the great diversity of species that make up these communities.
The fact that the numbers of species remain fairly similar, although the kinds
of species vary considerably, suggests that there are a similar number of niches
for diatom species in ecologically similar natural areas and more species are
available than there are niches for them. Thus, each niche is occupied by a
different species. The lack of similarity in kinds of species present is probably
in part due to the highly variable environment in a natural river and the avail-
ability of species which have their best development in different variations of
the environment. Because diatoms have very rapid reproduction under favor-
able conditions the populations of certain species can quickly increase, whereas
populations of other species decrease beyond the limits of collectability or dis-
appear.
Another important consideration in the study of diatom communities is the
kinds of species composing the communities. By careful consideration of the
kinds of species associated together, a qualitative evaluation of many of the
characteristics of the environment can be made. However, because of a lack
of data as to the complete physiological requirements of any species in nature,
it is very dangerous to say that the lack of any species indicates that the spe-
cific characteristic of the environment under consideration is not there, be-
cause the lack of any factor essential for the life of an organism may eliminate
it, although all other factors of the environment may be favorable to it. Also,
it is hazardous to use changes in the population sizes of specific species as a
basis for saying that the quantitative nature of a given environmental factor
has changed. For example, we studied two areas in the Guadalupe River
which were not over 500 yards apart. The structural environmental charac-
teristics of the two areas were very similar. Because no tributaries or pollu-
tion entered the river between these two areas during the time of this study,
the characteristics of the water were very similar. This was substantiated by
chemical analyses. When similar segments of the communities of diatoms
were studied the percentages of the community composed of specimens of dom-
inant species were very similar, 95 and 90 per cent, respectively (table 3).
However, the sizes of the populations of the dominant .species and the kinds
of species varied considerably. At Station 2, the population size of Gompho-
nema affinis var. insigue was twice that found at Station 1. The population
of Gomphonema parvulum was 38 per cent larger at Station 1 than at Station 2.
At Station 2 Navicula sp. had a population of 1900 specimens and Nitzschia
palea had a population of 2400 specimens yet neither of these species were
present at Station 1. Only one of the dominant species, .\avicula Iripunciala
var. schizonemoides had populations of similar size at the two stations.
It is only as a result of thorough and continuous study of an environment
and the species living in it that one can venture to describe the quantitative
Patrick: Structure of Diatom Communities 365
changes in the natural environment of a river by changes in the quantitative
abundance of specific kinds of species.
In conclusion, our studies have shown that diatom communities can be best
characterized by consideration of the kinds of species, the numbers of species,
and the relative sizes of the populations of the species that comprise the com-
munity. An excellent way to consider the relative sizes of the populations of
all the species studied is by the construction of a truncated normal curve. The
presence of certain kinds of species may tell us much as to the quaUtative char-
acteristics of an environment. The best means for determining quantitative
shifts in the environment is by considering the shift in numbers of species and
the ratio of the number of species with small populations to those with large
populations. Perhaps the reasons that the numbers of species do not vary
greatly is that there are similar numbers of niches for species occupancy in
ecologically similar types of streams. Also, at any one time there are prob-
ably more species available to inhabit natural eutrophic or mesotrophic areas of
streams than there are niches available for species occupancy, thus, each niche
is filled with a different species. The reasons that the kinds of species vary con-
siderably in streams of these types are the continually varying yet favorable en-
vironment; the availability of species which have their best development in
different conditions of the environment; and the ability of diatom populations
to quickly expand or contract with changes in the environment.
References
Patrick, R. 1961. A study of the numbers and kinds of species found in rivers in eastern
United States. Acad. Nat. Sci. Phila. 113(10): 215-258.
Patrick, R., M. H. Hohn & J. H. Wallace. 1954. A new method for determining the
pattern of the diatom flora. Acad. Nat. Sci. Phila. No. 259.
Preston, F. W. 1948. The commonness, and rarity, of species. Ecology. 39: 254-283.
Thienemann, a. 1939. Grundzuge einer allgemeinen Okologie. Arch. Hydrobiol. 35;
267-285.
CELL STRUCTURE AND ENVIRONMENT
B. J. Cholnoky
Council for Scientific and Industrial Research, National Institute for Water
Research, Pretoria, South Africa
During the so-called classical period of the study of cells, algal cells were
frequently used in cytological investigations. The great discoveries of Weis-
mann, Biitschli, Ramon y Cajal, and Flemming were made possible with fixed
and stained objects, but as the equipment and the microscopical methods then
available were unsuitable for living and especially for unstained objects, living
algal cells were only rarely used for cytological purposes. Listead, methods
were developed which were supposed to leave the fixed protoplasm unaltered,
and differential staining procedures were used which rendered visible to the
human eye structures which, it was believed, occurred in the living cell. Vio-
lent, but barely scientific controversy which often led to personal insults and
verbal battles ensued, during which the living cell was more and more for-
gotten. This was also due to the exemplary, or not so exemplary preparations
which were made to support sophisticated hypotheses which arose from staining
techniques. These techniques often resulted in works of art rather than
impressions of the living cell.
No matter how perfectly fixation for specific purposes has been accomplished,
the living constituents of the cell must necessarily undergo alteration when
fixed (otherwise they would continue to live), and minute changes in the
protoplasm due to environmental factors cannot, therefore, be detected. The
difficulties were increased because colloidal physics had not yet been developed,
and because the changes were generally of a submicroscopical nature. Investi-
gations of these changes in the living protoplasm, therefore, were only later
tackled.
Seen against this background, the accidental discovery by Benecke (1901)
of the reduction in size and the ultimate disappearance of the chromatophores
in Niizschia putrida (Synedra hyalina Provazek), by means of which he sought
to show a clear connexion between the size of the chromatophores and the
pollution (as he called it) of coastal waters, was surprising and also important.
The approach adopted by Benecke was, however, soon abandoned, and the
observation of living algal cells continued only for the purpose of systematics
and morphology. The observed structures, cell components, etc., were re-
garded as something rigid and unchangeable, or, as we would express it today,
genotypically determined. Consequently, the results of these observations
were used only as characteristics: they were used, and frequently misused, for
describing species, and as a result, plant physiologists and the early ecologists
did not want to do anything with them.
On the other hand, investigations into the causes of adaptation of algae have
begun. These investigations at first pursued a course which was of importance
to Man but not to the algae. Apart from the vague conjectures of the 19th
century, which were mainly concerned with descriptions of the habitat or with
plant geography, the first ecological study of algae was the so-called Saprobic
366
Cholnoky: Cell Structure and Environment 367
System of Kolkwitz. This was, however, not based upon precise observations
or experiments but on an untenable hypothesis (Kolkwitz and Marsson, 1902,
1908, 1909; Kolkwitz, 1950; Liebmann, 1951, etc.). As a basis for the hy-
pothesis it was assumed that the substances (of which no one then bothered to
ascertain the chemical nature) which were responsible for the pollution of
waters could be removed first by reduction and subsec^uently by oxidation.
The so-called reduction phase was called polysaprobic and the oxidation phase
mesosaprobic. Naumann (1932), however, had shown that this hypothesis
was untenable: he demonstrated the nonexistence of a reduction phase and
consequently it was found impossible to judge the quality of waters, let alone
to purify them, on Kolkwitz principles. Under these circumstances it was not
surprising that cytologists found no reason to study protoplasmatic changes
attributable to "pollution" according to Saprobic System concepts.
It was only much later that greater stimulus was given by the ecological
work of Kolbe (1927, 1932), who showed that certain diatom species are better
adapted to a high salt concentration than others. In his opinion it was the
chloride ion of sodium chloride which was responsible for the phenomenon of
adaptation. He also attempted to prove that in the absence of the afore-
mentioned ions (oligohalobic conditions), a moderate concentration (meso-
halobic conditions), or a high concentration (polyhalobic conditions) simulating
salt water was responsible for the distribution and adaptation of certain
species.
Almost at the same time it had been shown (Cholnoky, 1929) that the diatom
associations of the soda lakes of Hungary (which contain carbonates and not
chlorides) were identical with those of Kolbe's mesohalobic waters of Speren-
berg. From these observations it was possible to deduce the fact that pri-
marily it was not the chemical composition of the salt molecules but rather
their concentration which was responsible for the halobic phenomena. In
other words, it was not the chloride ion at all, but the prevailing osmotic
pressure, i.e., molarity. It also became clear very soon after that the prevail-
ing osmotic pressure in the Hungarian soda lakes can be as high as, or even
higher than, that of the sea (a concentration of 2 mol. sodium carbonate is not
exceptional in the lakes), and that these high values do not necessarily give
rise to the growth of typical marine algae. It was recognized that it was not
the absolute salt content or molarity, but the variation of osmotic pressure
which produces the necessary conditions for the so-called brackish water
species; or put more precisely, the ability to withstand the molarity variations
gives advantages to these brackish water species.
Because the variation of osmotic pressure mainly affects the protoplasm of
the brackish water organisms, it was clear that protoplasmic differences must
exist, and that these differences could only be discovered by studying the
living cells.
After the classical studies of de Vries (1871, 1885), one could assume, as a
matter of course, that an increase in osmotic pressure would cause plasmolysis,
and also that plasmolysis could be neutralized by permeance to, or active uptake
of, the plasmolyzing substances. Hofler showed (1918, and more accurate
concept 1931) that de Vries's concept of semipermeability was untenable.
368 Annals New York Academy of Sciences
Thus, the causes of adaptation to the conditions of brackish water, i.e., the
considerable variation of osmotic pressure, were to be found in plasmolysis
which must necessarily and at least temporarily occur.
The experiments which were undertaken (Cholnoky, 1928a, 19306, 1932)
showed that apart from certain fundamental morphological features which
seem to be genotypically determined, and which are characteristic for the
various algal groups during plasmolysis, there are large morphological differ-
ences between the plasmolyses of freshwater and brackish water species.
Among other things, the distribution of viscosity of the protoplasmic colloids
is characteristic for the species. It was ecjually evident that the brackish water
species poses a high degree of permeabihty in regard to the salts in solution
in their habitat. In Hungary the high degree of permeability is confined to
the carbonates, and only to a lesser degree to the chlorides, although the cells
show only slight or nonpermeability to such plasmolytica as nitrates, sugar,
urea, etc. The same species when found along the South African coast are
mostly permeable to the chlorides, whereas when they occur in the South
African sodium carbonate rich waters of the Jakkals River for instance, the
same permeability to carbonates as in Hungary was observed.
These observations forced me to the conclusion that owing to the high degree
of permeability of the protoplasts, the brackish water species are ecologically
favored. It follows that if such an assumption were true, there would be far-
reaching colloid-physical effects. The permeating salt molecules would, under
normal circumstances, alter the electrical charge of the mono- or polymolecular
micelles and thus be the cause of coacervation and ensuing coagulation and
death. The protoplasm of brackish water species appears to be extremely well
protected against such alterations of electrical charge, and further study will
probably provide important information on the submicroscopical structure of
the protoplasm.
Typical freshwater algae which were treated with a plasmolyticum consisting
of some partly evaporated brackish water from another habitat speedily died
as a result of permeation (Cholnoky, 19306, 1931<z, 19316). Others, however,
remained plasmolysed for an extraordinarily long time without showing any
sign of protoplasmatic damage and without the least trace of permeation.
Other chemical compounds for which the protoplasts of the investigated species
were more or less permeable, acted immediately on permeation as poisons,
during which it was seen that the gradual destruction of the protoplasm indi-
cated an unecjual resistance of the protoplasmic components of the cell (Chol-
noky, 1953).
Hofler (1951) obtained similar results and found that Na2C03 acted in a
specific manner on the diverse species of bog algae (Desmidiales). The cells
of some species were slightly permeable, others were barely permeable. The
nonpermeable ones were able to survive plasmolysis lasting several days with-
out sustaining any visible protoplasmatic damage. (It was possible com-
pletely to deplasmoly.se Euastrum after pla.smolysis lasting 72 hours.) I was
able to confirm that certain brackish water algae were even more resistent to
plasmolysis than Euastrum. These species built a superficial inner cell wall
on the site of positive plasmolysis; i.e., at those places where the protoplasm
body had withdrawn from the original cell wall. As a result of the possible
Cholnoky: Cell Structure and Environment 369
repetition of ihis operation, the formation of the so-called inner cell wall is
explained: it arises from an increase in the osmotic pressure of the environment
during the gradual drying up of the waters in the summer (Cholnoky, 19286,
1954; Kamija, 1938; Kuster-Winkelmann, 1949). These phenomena are clear
proof that the otherwise generally fatal plasmolysis does not alter, or alters
only to a Hmited extent, the structure of the protoplasmic colloids in the brack-
ish water species which are adapted to variations in osmotic pressure. These
species not only survive the ordeal but actually build a cell wall during the
process — a procedure which would be hardly thinkable if the metabolism had
been upset.
The repeated reductions in pressure due to variations of osmotic pressure do
not occur without having any side effects. With brackish water diatoms the
reductions in pressure are the triggers for sexual reproduction (Cholnoky,
19296), because the dilution caused by the culture medium always gives rise,
in the diatoms, to sexual propagation, i.e., auxospore formation.
A sudden dilution causes plasmorhexis in brackish water species. This is
evidence for their having comparatively many free salt molecules in the water
mantles of the micelles of their colloids, which can cause an osmotic pressure
(Cholnoky 1928a). Such phenomena do not occur in brackish water species
if the dilution is carefully made. But with marine species, a dilution, no
matter how carefully made, causes plasmorhexis and death. This can be
accepted as proof that the salts causing isotonia, are indispensable to the proto-
plasmic colloids, and are structurally part of, and inseparable from, the micelles.
But further experiments will be necessary to be able to evaluate the position
fully.
Without further experiments it will be equally impossible to explain the
mechanism of the phenomena which Lenk (1953) called Seasonal Variation of
Permeability. Variation of permeability without change of protoplasmic
structure is unthinkable. Consequently it can be assumed that submicro-
scopical protoplasmic structure is also subject to seasonal variation which can
only be due to adaptation to altered conditions of the habitat.
As I have already suggested, the behavior of freshwater algae which have
been killed by the permeation of salt molecules, indicates that they undergo
coacervation and lethal coagulation due to the penetration of the molecules.
These protoplasmic changes are a kind of a poison effect and leads from a study
of adaptation to the important study of resistance (Biebl, 1937, 1952). It
would, however, be inappropriate here to discuss fully all of the hitherto known
cytological resistance phenomena.
From the point of view of cytophysiology, a study of the poisonous effects of
salts and the resulting cytomorphological changes (which are often submicro-
scopical) is all the more important, as far reaching deductions regarding
adaptation phenomena will be possible. The studies on Melosira areiiaria
(Cholnoky, 1934) may be regarded as a beginning; and subsequent work on
cellular changes in other species and other algal groups (Ulothrix, Oedogonium,
Zygnemales, Desmidiales, and Siphonocladiales) led to important ecological
and cytological regularities being discovered. The notes, manuscripts, and
data, however, remained unpublished as they were lost at the end of the war.
The poisonous effects of some salts {e.g., sodium carbonate) could only be
370 Annals New York Academy of Sciences
characterized if the cytomorphological changes which they caused could be
compared with the poisonous effects of other substances. For this purpose
cuhures were utiUzed of which the culture fluids were displaced by cocaine and
colchicine, both of which are known to be cell toxic to a high degree. The
effect of cocaine on Cladophora (Cholnoky, 1930a) showed that this alga was
able to tolerate appreciable concentrations and that it can react in a very
characteristic manner. Without any microscopically visible protoplasmatic
changes occurring, resting stages developed, which were independent of the
seasons, and appeared to be completely resistant to cocaine so that when re-
moved to a normal habitat {i.e., cocaine free) they were able to germinate.
That these observations remained comparatively unknown, may be due to the
title of the paper having been arbitrarily changed by the editor of the journal
to which the paper was sent. The observations made may explain how algae
are able to survive temporary poisoning, as a result, for example, of industrial
effluents.
As is well known, colchicine affects the development of the spindle during
nuclear division and is, therefore, often used for obtaining polyploids. This
substance was also used for culture experiments. Surprisingly, only a high
concentration of colchicine (10 ppm) resulted in damage to the nuclear division,
but no polyploids were obtained. With Cladophora the number of nuclei in
the polyenergid cells was reduced. With Spirogyra, etc. pseudosexual condi-
tions quickly developed which often became lethal after only a lapse of several
weeks. The observed phenomena may explain why certain industrial wastes
produce no poisoning of the cocaine type (certain cells become impermeable to
poisons), but many abnormalities instead. The results of this series of experi-
ments were also entirely lost owing to the war, and as no further opportunity
to repeat them has been given me, it is up to some other researcher to undertake
this work. Nevertheless, they do seem to elucidate the effects of the waste
products produced by human activities as far as the terms "pollution" and
"poisoning" of natural waters are concerned.
I am familiar with only a few of the cytological effects of other poisons:
among these are the studies on aluminium salts and "cramp" plasmolysis
(Weber, 1924, 1933; Hofler, 1958) which clearly show that the salts have ren-
dered impossible the functions of the investigated cells through colloidal
changes etc., and very probably also through interference with the electrical
charge of the micelles. Although the quoted papers do not mention the colloid-
physical significance of the phenomena, it seems to me that they must be due
to coacervation and coagulation.
Colchicine as well as cocaine cause radical changes in the structure of the
protoplasm which are to a certain extent discernible by experiment, but the
mechanism of the effects of poisoning can be better seen microscopically if the
poisons can be seen or can be made to be seen. When the first algal investiga-
tions were started, the results of the experiments which had been made with
the cells of the higher plants led one to suspect that the process known to the
workers during the classical period of cytology as vital staining was actually
a microscopical manifestation of poisoning and destruction of the cells. It was,
therefore, possible without further ado to use stains which were formerly re-
garded as harmless, i.e., which did not kill the protoplasm suddenly.
Cholnoky: Cell Structure and Environment 371
As this exposition is mainly concerned with the efifects of the environment
on the structure of the protoplasm, I shall have to omit a detailed description
of what is known about the general principles of stain uptake and storage or
changes in the stain molecules, e.g., ionization in the cell or its environment.
It would also go beyond the scope of this paper to draw attention to the present
state of our knowledge derived from investigations with the fluorescence micro-
scope. As far as I am aware, those studies have hitherto only been made with
material divorced from its natural habitat, and have, in many cases, degen-
erated merely into a study of stains, without reference to colloidal structure
or the changes it undergoes. Such work often led Biitschli el al., into fruitless
hypothetical discussions.
This scarcely scientific approach is regrettable because even the first experi-
ments on stain uptake in algal cells (Cholnoky, 1934, 1935a, 1935&, 1935c)
showed that uptake and storage of the stain molecules, or the ion gradients in
the protoplasm was far reachingly dependent on the conditions under which
the algae lived before the experiments. When stained with methylene blue or
neutral red, the disassociation of the stain molecules remained dependent upon
the conditions of the culture before the staining experiments were done. Also
in those cases in which the stain fluid (unlike the culture fluid) possessed con-
stant physicochemical characteristics {e.g., stains dissolved in distilled water,
buffer solutions or plasmolytica) the effects on the protoplasm of increased
osmotic pressure, pH, and light conditions could be clearly proved.
The environmental conditions before the staining experiments generally hav-
ing remained neglected; this explains why so many apparently contradictory
results were obtained. The use of fluorochromes increased still further the
complexity of an already complicated position, as conclusions were drawn re-
lating to the storage of stain molecules and ions which incorporated many hypo-
thetical assumptions, such as "full" and "empty" ceU-sap (Hofler and Schindler,
1955), which did not attempt to reconcile observed facts with the environmental
factors under which the algal cells were living before the experiments.
This change of concept became apparent as preliminary work (Cholnoky and
Hofler, 1950) had already been done which went so far as to enable one to dis-
tinguish between the cytological behavior of species (Loub, 1951).
Regarding Loub (1951), it should be remembered that his material came from
ecologically dissimilar environments. After arrival in the laboratory they were
rough cultured and only examined after a more or less lengthy period. Apart
from the fact that the culture conditions were uncontrolled in the rough culture,
Loub did not investigate the natural conditions of the habitat. By his method
he was able to examine only adapted associations. He thus lost the oppor-
tunity to investigate the protoplasmic changes caused by ecological factors.
It will be clear from what has been said that most protoplasmic experiments
(such as plasmolysis and staining) were done without reference to the condi-
tions in which the algae lived in nature or in cultures. Although the cytological
results obtained are of very great value, it is indispensable that the methods
so far used should be thoroughly changed. Ecological studies have shown on
the one hand that not only the conditions of life prevaihng at the time of the
experiments but also their fluctuations must affect protoplasmic structure. On
the other hand, it now seems certain that Naumann's trophic conditions of the
372 Annals New York Academy of Sciences
waters (1932) play a much greater part in cell protoplasm than was formerly
believed. All future experiments must, therefore, take place under rigid control
of the culture conditions. Only in this way shall we discover protoplasmic
adaptation phenomena.
We shall first have to consider the possible effects of changes of pH and the
nitrogen content of waters, the latter having a direct bearing on trophic condi-
tions enabling one to distinguish between autotrophic and heterotrophic algae
(Algeus, 1946; Fogg, 1953; Saubert, 1957).
It seems obvious, finally, that the permeability and uptake of dissolved com-
pounds depends principally upon the structure of the protoplasm, so that one
can no longer think in terms of a specific filter system. Such hypothetical sys-
tems are, however, still accepted by some, although Seifriz (1936) has indicated
that the permeation of the whole protoplasm was responsible.
The correctness of this concept was confirmed by later experiments (Chol-
noky, 1952a, 19526; Hofler, 1959). On this basis, it seems to me highly proba-
ble that the structure of the protoplasm (after obligatory or optional nutrition
of the algal cells) is subject to changes which are also necessarily manifest in
the uptake of stain molecules. As the protoplasm of the purely autotrophic
algae must be adapted to small molecules and even ions, its microstructure
must be very different from that of the nitrogen heterotrophic species, the
protoplasm of which can take up amino acids or even bigger molecules (protein
particles, amino acid groups). These differences in protoplasmic structure,
which are due to the nutritional requirements of the cell and must also be mani-
fest in the uptake and storage of such substances as stains, seem to me so prob-
able that I am presently engaged in appropriate culture experiments. These
experiments will include the uptake of stains and fluorochromes in algae of the
same species which have been given different nutrients and also with algae
which are genotypically different for a study of their metabolism.
Owing to circumstances beyond my control, these experiments have just be-
gun. It has, however, been supposed that the uptake of dissolved substances
represents an active function on the part of the protoplasm, i.e., that it must
be a dynamic process, and not one influenced by static structures such as lamel-
lae or filters. That is why it is hardly likely that the results of these experi-
ments will ever be reconcilable with the static concepts of such researchers as
Frey-Wyssling (1955). Electron microscopical observations cannot be re-
garded as a basis of research on the living substances concerned with the uptake
of dissolved molecules that Frey-Wyssling called "Grundplasma".
I would like to recall what I said when I referred to the classical period of
cytology. The fixing and staining procedures then used could not lead to a
knowledge of protoplasmic structure, let alone changes due to physiological
causes. Electron microscopy must of necessity use similar, if more refined,
methods, as it is technically impossible to study living protoplasm with this
kind of microscope. The images obtained with the electron microscope are
only of static structural elements, and not of dynamic functions and changes in
the protoplasm. More succinctly, fixed protoplasm under the electron micro-
scope is at least partially an artificial product, as otherwise it would continue to
live unchanged.
Cholnoky: Cell Structure and Environment 373
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etc. Botan. Notiser. 1946: 129.
Benecke, W. 1901. tJber farblose Diatomeen der Kieler Fohrde. Pringsheim's Jahrb.
wiss. Botan. 35: 535.
BiEBL, R. 1937. Okologische und zellphvsiologische Studien an Rotalgen der englischen
Sudkiiste. Beih. Bot. Centr, Abt. A. 57: 381.
BiEBL, R. 1952. Ecological and non-environmental constitutional resistance of protoplasts
of marine algae. J. Marine Biol. Assoc. V. K. 31: 307.
Cholnoky, B. J. 1928a. Uber die Wirkung von hyper- und hypotonischen Losungen auf
einige Diatomeen. Intern. Rev. ges. Hydrobioi. Hydrog. 19: 452.
Cholnoky, B. J. 1928i. Uber mehrfache Schalenbil'dungen bei .\nomoenoneis sculpta.
Hedwigia. 68: 297.
Cholnoky, B. J. 1929a. Adnotationes criticae ad floram Bacillariearum Hungariae.
TV. Floristisch-okologische Untersuchungen in den siidlichen Teilen der ungarischen
Tiefebene (Alfold). Magyar Botan. Lapok. 1929: 100.
Cholnoky, B. J. 1930a. Die Dauerorgane von Ciadophora glomerata. Z. wiss. Botan.
22: 545.
Cholnoky, B. J. 19306. Untersuchungen iiber den Plasmolyse-Ort der Algenzellen 1 u.
2. Protoplasma. 11: 278.
Cholnoky, B. J. 1931a. Untersuchungen iiber den Plasmolyse-Ort der Algenzellen. III.
Die Plasmoiyse der ruhenden Zeilen der fadenbildenden Conjugaten. Protoplasma. 12:
321.
Cholnoky, B. J. 19316. Untersuchungen uber den Plasmolyse-Ort der Algenzellen. IV.
Die Plasmoiyse der Gattung Oedogonium. Protoplasma. 12: 510.
Cholnoky, B. J. 1932. Neue Beitrage zur Kenntnis der Plasmoiyse der Diatomeen.
Intern. Rev. ges. Hvdrobiol. Hydrog. 27: 306.
Cholnoky, J. B. 1934.' Plasmoiyse und Lebendfarbung bei Melosira. Protoplasma. 22:
161.
Cholnoky, B. J. 1935a. Protoplasmatische Untersuchungen durch Lebendfarbung und
Plasmoiyse. Mathematikai es Termeszettudomanyi Ertesito. Akad. Wiss. Budapest.
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Cholnoky, B. J. 19356. Farbstoffaufnahme und Farbstoffspeicherung lebender Zeilen
pennater Diatomeen. Osterr. Botan. Z. 84: 91.
Cholnoky, B. J. 1935c. Zur Kenntnis der Cyanophytenzelle. Protoplasma. 28: 524.
Cholnoky, B. J. 1952a. Beobachtungen uber die Wirkung der Kalilauge auf das Proto-
plasma. Protoplasma. 41: 57.
Cholnoky, B. J. 19526. Fin Beitrag zur Kenntnis des Plasmalemmas. Ber. Deut. Botan.
Ges. 65: 369.
Cholnoky, B. J. 1953. Fin Beitrag zur Kenntnis der Oedogonium-Zelle. Osterr. Botan.
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Cholnoky, B. J. & K. Hofler. 1950. Vergleichende Vitalfiirbungsversuche an Hoch-
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DE Vries, H. 1871. Sur le permeabilite du protoplasme de betteraves rouges. Arch.
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Botan. 16: 465.
Fogg, G. E. 1953. The Metabolism of Algae. Methuen's Monographs on Biological
Subjects, I, 1. Catalogue No. 4122/U. London.
Frey-Wyssling, a. 1955. Die submikroskopische Struktur des Cytoplasmas. Proto-
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Hofler, K. 1918. Permeabihtiitsbestimmung nach der plasmometrischen Methode. Ber.
Deut. Botan. Ges. 36: 414.
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Deut. Botan. Ges. 49: 79.
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49: 248.
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KoLKWiTz, R. & M. Marsson. 1902. GrundsJitze fiir die biologische Beurteilung des
Wassers nach seiner Fauna und Flora. Kleine Mittcilungen Kgl. Priifungsanstalt f.
VVasserversorgung u. Abwasserbeseitigung. 1.
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Math.-Natwiss. Kl., Abt. I. 162: 235.
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R. Oldenbourg. Miinchen.
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Seifriz, W. 1936. Protoplasm. McGraw-Hill Book Co., Inc. New York and London.
Weber, F. 1924. Krampfplasmolyse bei Spirog>ra. Arch. ges. Physiol., Pfliigers. 206: 629.
Weber, F. 1933. Aluminiumsalz-Wirkung und Plasmolyse-Permeabilitat. Protoplasma.
17:471.
THE MORPHOLOGY OF PPLO AND BACTERIAL L FORMS*
Louis Dienes
Departments of Medicine and Bacteriology, Massachusetts General Hospital, and the
Robert W . Lovett Memorial Foundation for the Study of Crippling Diseases,
Harvard Medical School, Boston, Mass.
The smallest organisms growing without the help of other cells are found
in the cultures of pleuropneumonia-like organisms (PPLO). Some are as
small as 0.15 to 0.25 /x- The majority of the organisms in the culture is
considerably larger. Size is only one of the distinctive characteristics of
these organisms. Their structure, the appearance of their colonies, their
chemical makeup and their reproductive processes also differ at first sight from
those of other microorganisms. However, many similarities to bacteria are
present. Their organization is as simple as is that of the bacteria. They do
not have distinct nuclei. Their growth requirements, metabolism, and sensi-
tivity to antibiotics are quite similar to those of the bacteria. An important
exception is that the PPLO are not sensitive to penicillin. The basic difference
between PPLO and bacteria is the absence in PPLO of a rigid cell wall, and
most of the distinctive properties of PPLO are the consequence of the lack of
this structural property characteristic of bacteria. The organisms are soft,
fragile, and easily distorted. Their size varies within wide limits from 0.15 /x
to 10 M, or larger. On agar media the structure and appearance of the colonies
of PPLO are characteristic and differ markedly from those of bacteria. Finally,
the method of reproduction seems to be more complex than that of bacteria,
although basically it is probably similar. In the fight of these similarities and
differences some authors propose to create a special class for PPLO,^ while
others regard them as a subdivision of the class of bacteria.-'^
The PPLO were discovered as parasites causing disease in animals or living
on their mucous membranes. They were isolated also from sewage, well water
and soil. The saprophytic strains differ in some respects from the parasitic,
but we have no information to suggest that they are part of the microflora
other than those related to animal organisms.
The suggestion that the PPLO might be an independent subdivision of mi-
croorganisms is made unlikely by the observation that bacteria under certain
conditions assume a growth form which presents aU the distinctive properties
of PPLO.-* These bacterial forms, usually designated as L forms, like the
PPLO are soft and fragile, lack a rigid cell waU, and are considerably smaller
than the usual bacteria. The appearance of the colonies, the morphology of
the organisms, their reproductive processes and their sensitivity to antibiotics
are similar to that of PPLO, and include resistance to penicillin. The best
illustration of the similarity between the two groups is the fact that 15 years
passed before it was generally recognized that the L forms were growth forms
of bacteria and not PPLO mixed with the cultures and thus foreign to the bac-
* The work reported in this paper was supported by a grant from the National Institute
of Arthritis and Metabolic Diseases, Public Health Service, Bethesda, Md. This paper is
pubHcation No. 322 of the Robert W. Lovett Memorial Foundation for the Study of Crip-
pling Diseases.
375
376 Annals New York Academy of Sciences
teria with which they were associated. At present the impression of the ma-
jority of bacteriologists interested in these organisms is that, although they
are fixed in their form of growth, PPLO derived from the bacteria at some time
in the past. This status would correspond to that of the fungi imperfedi. The
impression of some investigators is that PPLO may represent a primitive stage
in the phylogenetic development of bacteria to which under certain conditions
bacteria may return. It should also be mentioned that some authors^ '^'^ re-
gard the similarity between PPLO and bacteria as superficial and without
significance.
Information on morphology and reproductive processes of PPLO has been
confused for a long time, and to some extent it still is today. This confusion
exists not so much because of their small size but because of their fragility and
the ease with which they may be distorted. For these reasons, use of the
electron microscope thus far has yielded hardly more information than a
better definition of the smallest elements in the cultures of PPLO.
In 1935, Turner- gave an excellent description of the morphology of the
organism of bovine pleuropneumonia in broth cultures with dark field illumina-
tion. His basic observations are as follows: "An old broth culture contains
only small granules less than 0.5 /jl in diameter. Transferred to fresh media
these granules increase in size to about 1 n. One or more areas appear on their
peripheries from which short filaments may grow out. The structures thus
formed suggested the first name of the organism "Asterococcus." The ends
of the short filaments grow to a larger size and repeat a similar reproductive
process. The filaments may grow longer and either differentiate into small
granules or develop swellings from which filaments again grow out. In addi-
tion, rather large spherical or irregularly-shaped bodies, several m in diameter
appear in the culture. Under appropriate conditions these reproduce the
granules and filaments. Very long straight filaments, sometimes visible in the
cultures, are apparently artefacts."
The development of colonies of PPLO in agar cultures was carefully studied
by Liebermeister.'^ With the phase microscope he examined several strains.
Like Turner, he observed the extrusion of short filaments from the granules
and the development of new organisms at the end of the filament. It is char-
acteristic that the smallest organisms seem to divide but that the daughter
organisms usually are not closely associated but seem to be at the ends of a
short rod. Liebermeister did not observe the development of multiple fila-
ments from a granule nor the development of long filaments in the strains
which he studied.
The size of the organisms, especially on the surface of agar colonies, varies in
the cultures, and the smallest forms visible with the light microscope usually
make up a very small fraction of the culture. Autolysis and deformation of
the larger forms often produce a bewildering pleomorphism in aging cultures.
Klieneberger^ has published beautiful photographs indicating that the larger
organisms are aggregates of small ones enclosed in a common envelope. This
structure of the large forms is clearly visible in electron micrographs. Under
appropriate conditions granules grow out from the large bodies.
From this short discussion it seems that the morphology and reproductive
Dienes: PPLO & Bacterial L Forms 377
processes of the organisms are very simple. The basic elements are small gran-
ules between 0.15 and 0.3 ^ in diameter that multiply by fission after elonga-
tion. Somewhat larger forms may divide by extruding short filaments. In
addition the granules may form more or less large aggregates enclosed in a
common envelope out of which they again grow. The structure of such large
bodies is essentially similar in cultures of bacteria, L forms, and PPLO. In
the organisms of bovine pleuropneumonia, and possibly in a few other strains,
the granules also can grow into thin filaments. This form of growth was not
observed in most strains.
L forms, like PPLO, do not have rigid cell walls. This lack of a rigid cell
wall is demonstrated in thin sections of L forms examined with the electron
microscope. Chemical studies indicate that the L forms do not have the
chemical complexes that are responsible for the rigidity of bacterial cell walls.
A large part of the similarity of L forms to PPLO is the consequence of this
lack. However, some of the similarities to PPLO do not seem to be the im-
mediate consequence of the absence of a rigid cell wall. One of these is the
small size of both PPLO and the L forms. According to filtration measure-
ments by Klieneberger, the size of viable granules in the L forms of Streptoba-
cillus moniliformis is similar to, or only slightly larger than, the size of PPLO.
The electron microscope shows granules of similar size in both groups. An-
other property not directly connected with the cell wall, common to both
groups, is the tendency of the growing organisms to embed themselves in agar.
Multiplication in agar cultures occurs mainly inside the agar. The charac-
teristic appearance of the colonies in both groups is the consequence of this
tendency. Both groups of organisms invade agar only and not other solid
media. Finally, a remarkable property of both groups is the tendency to grow
into large bodies. This tendency is greater in the L forms than in PPLO.
The L forms in broth or in gelatin multiply only by the growth of granules to
large bodies and by the liberation of granules from the large bodies.
As noted above, bacteria also tend to grow into large bodies. Transforma-
tion of bacteria to L forms is always preceded by the appearance of large
bodies, and the L forms grow out of the large bodies. In a few instances
large bodies were observed during forniation from bacteria,** and like bac-
terial filaments, these bodies developed by multiplication without separation
of the bacteria. In the early stages large bodies disintegrated into a group
of bacteria by the development of cell walls between the constituent organisms.
After this period, the large bodies reproduced bacteria for a certain length of
time. Later, an increasing number lost the ability to develop or they pro-
duced L forms. Some of the L forms so produced, like the large bodies de-
veloping from bacteria, return immediately to bacterial form when the influ-
ence resulting in these transformations, e.g., penicillin, is eliminated. Most L
forms revert to a bacterial form of growth only occasionally and after long
cultivation may lose this ability completely.
The large bodies are formed in these instances, under conditions which in-
hibit the multiplication of the single organisms, by multiplication of organisms
possessing the full potentialities of bacteria. After some time the abihty to
return to bacteria is lost, but the organisms are able on agar media to multiply
378 Annals New York Academy of Sciences
outside the large body. The agar seems to offer a suitable physical environ-
ment similar perhaps to that present in the large bodies and necessary for
multiplication of L forms.
Bacterial large bodies have been known since the beginning of bacteriology
and are usually referred to as involution or dying forms. They are produced
by a great variety of influences on the bacteria that prevent normal multipU-
cation. Large bodies occur in the natural environment of bacteria. In some
cases, in contrast to older opinion, it is apparent that they remain viable and
able to reproduce for a longer period than single bacteria. Hence, the forma-
tion of large bodies is probably a useful process for bacteria in their natural en-
vironment and can be thought of as a phenomenon of adaptation and not
merely the result of degeneration.
At present, L forms can not be regarded in the same light. In most in-
stances they develop and can be propagated only under artificial conditions.
It seems likely that they represent the growth of forms in the laboratory that
naturally occur only in the large bodies derived from bacteria. Small size,
growth into agar, and a tendency to produce large bodies (characteristics of L
forms) may be the result of this natural site of growth.
It is remarkable that bacteria cultivated directly from pathological processes
relatively often have the tendency to grow into large forms and to produce L
colonies. This may be the result of injury to the organism by the defensive
forces of the host. On the other hand, it may be an adaptation of the bac-
teria to parasitism. In one case of peritonitis, for example,^ it seemed that a
bacteroides strain continued to multiply in the L form inside the phagocytic
cells of the host. Such an observation suggests that although L forms may be
produced under artificial conditions, this process might occur naturally and
thus might have been the origin by stabilization of strains of PPLO that have
continued life in this form. The PPLO not only appear to be bacteria without
the usual cell wall but also bacteria that have passed through the processes in-
volved in the growth of the large bodies. The most marked difference between
L forms and PPLO is that PPLO are better adapted to grow in artificial media
and especially to grow in the small granular form. The L forms grow usually
only from large inocula and have a pronounced tendency to grow into large
bodies as well as to undergo autolysis.
At present PPLO do not seem to be of the main stream of phylogenetic
development or to be a link in it. These organisms probably represent the
result of the simplification of the structure of bacteria as a consequence of
parasitism. They are not complex and occasionally are very small but, like
the viruses, they offer no direct clues for the origin of life.
For illustration of the morphology of PPLO and L forms we refer to articles
previously published in the Annals of this Academy.^ ■!"
References
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Bacteriol. Rev. 5: 331.
2. Turner, A. W. 1935. A study on the morphology and life cycles of the organism
of pleuropneumonia contagiosa bovum (Borrelomyces peripneumoniae nov. gen.) by
observation in the living state under dark ground illumination. J. Pathol. Bacteriol.
45: 1.
Dienes: PPLO & Bacterial L Forms 379
3. Freundt, E. a. 1958. The Mycoplasmataceae. Munksgaard. Copenhagen.
4. Dienes, L. & H. J. Weinberger. 1951. The L forms of bacteria. Bacteriol. Rev.
15: 245.
5. Klieneberger-Nobel, E. 1962. Pleuropneumonia-like Organisms (PPLO) Myco-
plasmataceae. Academic Press, Inc. London & New York.
6. 0RSKOV, J. 1942. On the morphology of peripneumonia-virus, agalactia-virus and
Seiffert's microbes. Acta Pathol. Microbiol. Scand. 19: 586.
7. Liebermeister, K. 1953. Untersuchungen zur Morphologic der Pleuropneumonia-
(PPLO-)Gruppe. Z. Naturforsch. 12: 757.
8. Dienes, L. & VV. E. Smith. 1944. The significance of pleomorphism in Bacteroides
strains. J. Bacteriol. 48: 125.
9. Madoff, S. 1960. Isolation and identification of PPLO. Ann. N. Y. Acad. Sci. 79:
383.
10. Dienes, L. 1960. Controversial aspects of the morphology of PPLO. Ann. N.Y.
Acad. Sci. 79: 356.
AXENIC CULTURE OF PARAMECILMSOME OBSERVATIONS ON
THE GROWTH BEHAVIOR AND NUTRITIONAL REQUIRE-
MENTS OF A PARTICLE-BEARING STRAIN OF
PARAMECIUM AURELIA 299X
Anthony T. Soldo*
Department of Cancer Chemotherapy, Schering Corporation, Bloomfield, New Jersey
The genus Paramecium comprises a group of free Uving ciUates noted for
their morphological and genetical complexity. For these reasons and because
the organisms represent an end point in a divergent course of evolution, this
genus has been an object of interest. Certain members of this group exist in
association with self reproducing, intracytoplasmic particles.'- Recent ad-
vances in the knowledge of the nutritional requirements of Paramecium has
made it possible to cultivate these particle-bearing paramecia in sterile medium.
The purpose of this paper is to summarize the present state of knowledge of
the nutrition of Paramecium and to present the results of some detailed studies
on a particle bearing strain, Paramecium aurelia 299X.
Nutrition of Paramecium. In the past, Paramecium was cultivated in a
medium consisting of plant extracts, notably cerophyl and lettuce infusion,
supplemented with living bacteria, usually Aerobacter aerogenes? The first
successful report of axenic cultivation was made by Johnson and Baker in
1942.4 These workers grew Paramecium multimicronucleata in a medium con-
sisting of pressed yeast juice and proteose peptone. Two components of the
pressed yeast juice were recjuired for growth. One proved to be heat labile
which they assumed to be a protein, but was later replaced by a mixture of
ribosidic derivatives of a purine and a pyrimidine; the other was a heat stable
component. In 1949, van Wagtendonk and Hackett successfully established
P. aurelia in a medium composed of equal parts of 0.5 per cent yeast autolysate
and a 24-hour culture of A. aerogenes in lettuce extract.^ This medium could
be heat sterilized and provided the basis for later work which led to the de-
velopment of a more complex bacteria free medium.^ Folic acid, riboflavin,
thiamine, and a steroid proved to be absolute requirements for the growth of
stock 5L7 of P. aurelia; the steroid requirement could be satisfied by ^- and
7-sitosterol, fucosterol, brassicassterol, stigmasterol, and A'*'"-stigmastadie-
none.'^'* Miller and van Wagtendonk found that P. aurelia required 11 amino
acids, nicotinic acid, panothenic acid, and possibly, pyridoxal.'^ Also, one or
more essential growth factors remained in the yeast. Miller and Johnson
studied further the nutrition of P. multimicronucleata, and demonstrated, in
addition to the purine and pyrimidine requirement for that organism, a need
for an exogenous source of a fatty acid. '"■'•' Recently Lilly et al. cultivated
Paramecium caudatuni in a medium chemically defined, except for a single
component.''* Their medium was similar to the one used for the cultivation of
P. aurelia and P. multimicronucleata, except that it was necessary to add mi-
crogram quantities of a protein concentrate obtained from dried green peas.
* Present address: Department of Contractile Proteins, Institute for Muscle Diseases, Inc.,
New York, New York.
380
Soldo : Axenic Culture of Paramecium
381
Purification of the protein factor and subsequent analysis led to the qualitative
identification of 16 amino acids. The nutritional role of this protein has not
been satisfactorily explained. In table 1 is given the composition of a typical
medium which supports the growth of most strains of Paramecium.
Axenic cultivation of X-bearing Paramecium. Lambda particles were dis-
covered in the cytoplasm of stock 299X of P. aurelia by Schneller, in 1958.^^
She noted that animals containing these particles possessed the ability to kill
sensitive or particle free animals when members of the appropriate types were
placed in the same container. In this respect, this particle-protozoan system
is similar to the well known k system.^^
Table 1
Axenic Medium for Paramecium
Amino acids
Mg./ml.
Vitamins
Mg./ml.
L-Alanine
25
Biotin
0.125
*L-Arginine-HCl
100
*Ca-pantothenate
5
L-Aspartic acid
50
*Folic acid
2.5
L-Glutamic acid
75
a-Lipoic acid
0.05
Glycine
25
*Nicotinamide
5
*DL-Histidine
50
*PyridoxaIHCl
5
*DL-Isoleucine
150
Pyridoxamine • HCl
2.5
*DL-Leucine
150
*Riboflavin
5
*L-LysineHCl
125
♦Thiamine -HCl
15
*DL-Methionine
150
Inorganic salts
*L-Phenylalanine
75
Fe(NH4)2(S04)2-6H20
15
L- Proline
50
ZnCla
2
*DL-Serine
200
EDTA
5
*DL-Threonine
150
MnS04-4H20
2
*L-Tryptophan
50
CuS04-5H20
0.3
*L Tyrosine
50
CoS04-7H20
0.5
DL-Valine
75
MgS04-7H20
50
Purines and pyiimidines
Other factors
*Guanylic acid
500
*Stigmasterol
1
*Uridylic acid
500
*Na oleate
40
Na acetate
500
*Yeast factorf
50-500
* Components known to be absolute requirements for the growth of one or more species
of Paramecium.
t For preparation see (9). May be replaced by Pea factor for P. caudatum (14).
Efforts to cultivate X-bearing animals in media used for the growth of parti-
cle free strains were unsuccessful. It was necessary to supplement a crude
medium consisting of proteose peptone, a dialyzable component of hot water
extract of Baker's yeast and salts, with Edamine S, an enzymatic digest of
lactalbumin.'^ This medium supported the growth of the protozoans and main-
tenance of the particles through serial subcultures for a period of 2 years.
Particles of axenically cultivated animals number several hundred per cell,
contain RNA, little or no DNA, and are similar in size to the bacterium,
Escherichia coli.^^ They are gram-negative and may be stained with most
bacteriological dyes. Examination under phase microscope reveals a rod or
diplorod type structure. A furrow which divides the particle into almost
382
Annals New York Academy of Sciences
equal halves suggests that the particles may reproduce by longitudinal divi-
sion; occasionally they appear to be vacuolated.
Particle reproduction is synchronized with the division of the protozoan.'^
Further evidence in support of this view is given in figure 1. Animals re-
SYNCHRONOUS DIVISION
7--
& 5
CL
o
CL
(3 4
o
3--
LAMBDA PARTICLES
Growth Medium
Resting Medium
PARAMECIUM
'Growth Medium
Resting Medium
0
3 4 5 6
DAYS
Figure 1. Growth medium: see table 1.
jjhosphate buffer, pH 7.0.
7 8 9
Resting medium: isotonic saline, 0.01 M
moved during the log phase of growth, washed to remove all traces of the
original medium and resuspended in a buffered salt solution, "resting medium,"
failed to multiply. Estimates of the particle population revealed that they,
too, did not increase in number. Synchronous division may account for the
ability of the particles to keep pace with the host, although it seems likely that
this may be only a partial answer to the phenomenon. It has been observed
that occasionally one or more of the animals loses all its particles. Clones
Soldo : Axenic Culture of Paramecium
383
derived from these animals are also particle free. It has not yet been possible
to rule out mutation as an explanation for this phenomenon.
The existing synchronism between the particles and the host cell makes it
possible to quantitatively evaluate agents that may selectively inhibit the
particles themselves. Of interest here is the number of antibiotics that possess
this capability (table 2). ID50 values, derived in a manner previously de-
scribed/* reflect the relative effectiveness of these substances to inhibit particle
populations. This selective action correlates with the toxicity produced by
these agents in man. Antibiotics such as penicillin and tetracycline which
exhibit the least toxicity in man prove to be excellent particle inhibitors; those,
Table 2
A- Comparison of the Activity of Antibiotics in the X System
WITH Chronic Toxicity in Man*
Antibiotic tested
Actinomycin D
Actidione
Bacitracin
Neomycin
Polymyxin
Candicidin
Streptomycin
Cephalosporin C
Novobiocin
Oleandomycin
Chloramphenicol
Aureomj'cin
Terramycin
Penicillin
Tetracychne
ID50 ratio
IDso
protozoan
protozoan
particlef
only
X
0.9
1
0.9
> 1,000
0.9
370
1.1
32
1.2
32
0.9
> 1,000
1.8
350
10
> 1,000
10
320
14
450
22
220
39
40
116
370
312
> 1,000
930
220
Toxicity in man
Very high toxicity.
Toxic — fatal to rats— 1 mg./kg. orally.
Nephrotoxicity, proteinuria.
Nephro- and ototoxicity.
Causes renal damage.
Toxic — used topically.
Low toxicit} — damage to eighth cranial nerve on
prolonged therapy.
Low toxicity — mice tolerate 5,000 mg./kg. intra-
venously.
Low loxicit> — 7 mg./kg. intravenously tolerated
in man.
Low toxicity — 40 mg./kg. orally tolerated in
children.
Low toxicity — 30 mg./kg. tolerated in man.
Low toxicity — 15-30 mg./kg. tolerated in man.
Low toxicity — 15-30 mg./kg. tolerated in man.
Verj' low toxicity — very well tolerated.
Very low toxicity — very well tolerated.
* Toxicity data obtained from Spector, W. S. 1957. "Handbook of Toxicology," vol. II.
t IDso ratios of greater than 1.2 indicate selective inhibition.
such as actinomycin and neomycin produce varying degrees of toxicity in man
and are not selectively inhibitory for the particle. Thus, the particle-Pam-
mecium system might be useful in predicting chronic human toxicity of poten-
tially useful antibiotic substances. In figure 2 the effectiveness of penicillin
in reducing the X population is shown. Under the conditions of the experi-
ment complete destruction of the particles is achieved in 1 day at a concen-
tration of 100 units per milliliter of the antibiotic.
Xutrilional requirements of X-beariiig Paramecium. A nutritional study was
made with particle-bearing and particle-free strains. The latter were obtained
by treating axenically cultivated, X-containing animals with penicillin to re-
move the particles. Both require a factor (or factors) present in a nondialyza-
ble aqueous extract of Baker's yeast. Chemical fractionation resulted in a
384
Annals New York Academy of Sciences
partially purified material which is not absorbed on anion or cation exchange
resins; the material may be precipitated with 67 per cent ethanol in the cold,
contains carbohydrate, protein, and no nucleic acid or lipids. Attempts to
replace this fraction with known substances, thus far, have been unsuccessful.
However, it has been possible to demonstrate a purine and pyrimidine require-
ment for the organisms, as well as their need for a number of vitamins, in a
medium (table 1) supplemented with this factor.
Purine requirements for particle containing and particle free animals are
summarized in table 3. The need for exogenous source of a purine derivative
THE EFFECT OF PENICILLIN ON LAMBDA
POPULATION
None
lUvml
7
Figure 2.
is apparent and may be met by guanosine and guanylic acid. The free base,
and adenosine and its derivatives, do not replace the purine. Apparently,
Paramecium converts guanosine to adenosine and its derivatives, whereas
the reverse reactions do not occur. Inosine, its derivatives, and xanthosine
and its derivatives failed to replace guanosine as a growth requirement.
The pyrimidine requirements may be satisfied by uridine, cytidine, uridyhc
and cytidylic acids (table 4). The free bases uracil, cytosine, and thymine,
as well as thymidine and thymidylic acid were not effective in replacing uridine
or cytidine. These data confirm earlier work with P. miillimicronucleala,
P. caudahim, and other strains of P. aurelia.
By means of C^'^-labeled purines, it has been shown that adenosine is in-
corporated into nucleic acid adenine only, whereas exogenously supplied guano-
Soldo: Axenic Culture of Paramecium
385
sine is incorporated into both nucleic acid-guanine and -adenine. ^^ These data
confirm the nutritional findings. Similar data obtained with isotopically la-
beled pyrimidines are in agreement with the nutritional evidence that cytidine
and uridine are interconvertible and serve as precursors for thymidine and thy-
midylic acid. The data further illustrate that similar pathways exist for
Table 3
Purine Requirements of Paramecium
Population den
sity* (No./ml.)
Purines tested (2/iM/ml.)
299X
299 S
Adenine
0
0
Guanine
0
0
Hvpoxanthine
0
0
Xanthine
0
0
Adenosine
0
0
Guanosine
9200
10,200
Inosine
0
0
Xanthosine
0
0
Adenylic acid (5')
0
0
Guanviic acid (5')
8700
9200
Inosinic acid (5')
0
0
* Values obtained after iirst transfer.
Table 4
Pyrisodine Reqltirements of Paramecium
Population density* (No./ml.)
Pyrimidines tested (2/iM/ml.)
299X
299 S
Cvtosine
0
0
Uracil
0
0
Thymine
0
0
Cytidine
6600
5800
Uridine
8200
7600
Thymidine
0
0
Cytidylic acid (5')
5200
7200
Uridylic acid (5')
4800
6500
Thymidylic acid (5')
0
0
* Values obtained after first transfer.
purine and pyrimidine utilization in both particle free and particle bearing
animals.
Generally, the requirements for vitamins for particle bearing and particle
free animals are similar (table 5). The need for nicotinamide, riboflavin, and
thiamine becomes apparent in the second transfer, whereas the requirement for
pyrido.xal is evident only after three or four serial subcultures. An absolute
requirement for calcium panthentate has not been shown. Some degree of
growth, appro.ximating 10 per cent of the control, remains even after several
transfers. Biotin and lipoic acid are not required. Particle bearing animals,
386
Annals New York Academy of Sciences
in the absence of folic acid, may be subcultured indefinitely. Particle free
animals, on the other hand, show an absolute requirement for this substance,
as judged from their inability to grow beyond the second transfer.
Particles may produce sufficient quantities of folic acid to provide for the
nutritional needs of the protozoan. To test this possibihty, particle bearing
animals were treated with penicillin in the presence and the absence of folic
acid (table 6). As expected, particle free animals did not grow in the con-
Table 5
Vitamin Requirements of Paramecium
Population (% control)
299X
299 S
Serial subculture
Serial subculture
1
2
3
4
5
1
2
3
4
s
Biotin
105
123
94
99
105
110
69
77
112
105
Ca pantothenate
95
38
38
7
12
105
73
74
36
8
Folic acid
100
71
71
69
75
40
0
a-Lipoic acid
101
103
99
85
95
87
57
96
121
112
Nicotinamide
97
0
75
0
Pyridoxal
110
98
82
0
75
48
37
26
0
Riboflavin
35
0
0
Thiamine
25
0
12
0
Table 6
The Effect of Penicillin upon the Folic Acid
Requirement of Paramecium
/
Population density (No. /ml.)
Addition
Medium plus folic acid
Medium minus folic acid
299X
299 S
299X
299 S
None
Penicillin, 1000 U/ml.
5200
9000*
7400
10,200
3800
0
0
0
* Animals particle free.
trols, or in penicillin-treated tubes in the absence of folic acid. Addition of
folic acid to the medium restored the ability of these animals to grow in the
presence or absence of peniciUin. Particle containing animals, on the other
hand, failed to grow in folic acid free medium containing penicillin, whereas
animals under similar conditions retained their particles and grew well in the
absence of penicillin. (Irowth of particle bearing animals in which folic acid
was present in both the control and penicillin-treated tubes was good. Peni-
cillin-treated animals contained no particles. Subsequent deletion of folic
acid from the medium containing these penicillin-treated animals resulted in
\ the death of the protozoan. These data support the view that folic acid pro-
Soldo: Axenic Culture of Paramecium 387
duction is dependent upon the presence of the particle in the cytoplasm; im-
plicit here is that the vitamin is produced by the particles themselves.
Discussion
The symbiotic association between X particles and the host Paramecium is
an example of what is doubtless a widespread phenomenon in nature. Para-
mecium bursaria harbors an alga of the genus Chlorella in its cytoplasm in what
has been described as a symbiotic system.-'' Colicins in bacteria,-^ extranu-
clear particles responsible for cytoplasmic inheritance in yeast," and particle-
Uke inclusions found in many insect tissues"-^ -^ may be further examples. A
well documented case of an endosymbiote has been found for the flagellated
protozoan, Strigomonas}^ This organism, apparently, exists in association
with cytoplasmic bipolar-like bodies. The presence of these particles, as with
Paramecium, alters the nutritional requirements of the host.
A distinctive feature of the A system is the ability of particle bearing animals
to release a toxin which causes the death of certain particle free detector strains,
but is without effect upon the X bearers themselves. In this respect the X
system bears a striking resemblance to colicin producing systems."^ Colicins
are antibiotic substances produced by certain bacteria, notably members of the
family Enterobacteriaceae. The ability of these bacteria to produce these
substances is believed to be due to the presence of a transmissible pathogenic
agent which is regarded as a bacterial virus. The analogy serves to illustrate
the degree to which the X particles may have incorporated themselves into the
genetic structure of the protozoan.
Yet X particles, unlike viruses, are highly complex structures which resemble
bacteria in size, morphology, staining characteristics, chemical composition,
and, possibly, manner of reproduction. Studies concerning the chemistry of
the particles reveal the presence of protein, carbohydrate, phospholipid, and
nucleic acid (W. J. van Wagtendonk and R. Tanguay — personal communica-
tion). Moreover, antibiotics are particularly effective in reducing or eliminat-
ing the particles from the cytoplasm of the protozoan. (These data, obtained
for the first time with axenically cultivated animals, provide the strongest
evidence to date on the action of antibiotics on particles in Paramecium.)
Finally, the finding that X particles produce amounts of folic acid sufficient to
support the growth and reproduction of the protozoan carries with it the im-
plication that the complex enzymatic machinery necessary for the synthesis of
this compound is present in the particles themselves.
A cknowledgments
I wish to express my sincere thanks to Andrea M. Pascale and William E.
Ronca for their excellent technical assistance.
The work described in this article was supported in part by a Government
Contract, SA-43ph-1929, to The Cancer Chemotherapy National Service Cen-
ter, National Cancer Institute, National Institutes of Health, Bethesda, Md.
References
1. SoNNEBORN, T. M. 1959. Kappa and related particles in Paramecium. Adv. Virus
Res. 6: 229.
388 Annals New York Academy of Sciences
2. WiCHTERMAN, R. 1953. The biology of Paramecium. The Blackston Company, Inc.
New York.
3. SoNNEBORN, T. M. 1950. Methods in the general biology and gene genetics of Para-
mecium aurelia. J. Exp. Zool. 113: 87.
4. Johnson, VV. H. & E. G. S. B.aker. 1942. The sterile culture of Paramecium mulli-
nucronucleata. Science. 9: Hi.
5. v.AN Wagtendonk, W. J. & P. L. Hackett. 1949. The culture of Paramecium aurelia
in the absence of other living organisms. Proc. Natl. Acad. Sci., U.S. 35: 155.
6. van Wagtendonk, W.J. , R. L. Conner, C. A. Miller &M. R. R. Rao. 1953. Growth
requirements of Paramecium aurelia var. 4, stock 51.7 sensitives and killers in axenic
medium. Ann. N.Y. Acad. Sci. 56(5): 929.
7. Conner, R. L., W. J. van Wagtendonk & C. A. Miller. 1953. The isolation from
lemon juice of a growth factor of steroid nature required for the growth of a strain of
Paramecium aurelia. J. Gen. Microbiol. 9(3): 434.
8. Conner, R. L. & W. J. VAN W.agtendonk. 1955. Steroid requirements of Paraweciww
aurelia. J. Gen. Microbiol. 12(1): 31.
9. Miller, C. A. & W. J. Wagtendonk. 1956. The essential metabolites of a strain of
Paramecium aurelia (stock 47.8) and a comparison of the growth rate of different
strains of Paramecium aurelia in axenic culture. J. Gen. Microl)iol. 15(2): 280.
10. Johnson, W. H. & C. A. Miller. 1956. A further analysis of the nutrition of Para-
mecium. J. Protozool. 3: 221.
11. Johnson, W. H. & C. A. Miller. 1957. The nitrogen requirements of Paramecium
muliimicronucleatum. Physiol. Zool. 30: 106.
12. Miller, C. A. & W. H. Johnson. 1957. A purine and pyrimidine rec|uirement for
Paramecium muliimicronucleatum. J. Protozool. 4: 200.
13. Miller, C. A. & W. H. Johnson. 1960. Nutrition of Paramecium. A fatty acid re-
quirement. J. Protozool. 7(3): 297.
14. Lilly, D. M. & R. C. Klosek. 1961. A protein factor in the nutrition of Paramecium^
caudatum. J. Gen. Microbiol. 24: 327.
15. Schneller, M. V. 1958. A new type of kiUing action in a stock of ParaweciwiM awre/ja
from Panama. Proc. Indiana Acad. Sci. 67: 302.
16 Sonneborn, T. M. 1938. Mating tvpes in Paramecium aurelia. Proc. Am. Phil.
Soc. 79:411.
17. Soldo, A. T. 1960. Cultivation of two strains of killer Paramecium aurelia in axenic
medium. Proc. Soc. Exp. Biol. Med. 105: 612.
18. Soldo, A. T. 1961. The use of particle-l)earing Paramecium in screening for potential
anti-tumor agents. Trans. N.Y. Acad. Sci. 23(8): 653.
19. Soldo, A. T. & W. J. van Wagtendonk. 1961. Nitrogen metabolism in Paramecium
aurelia. J. Protozool. 8(1): 41.
20. Siegel, R. W. 1960. Hereditary endosymbiosis in Paramecium busaria. Exp. Cell
Research. 19: 239.
21. Eredericq, p. 1950. Rapports entre colicines et bacteriophages. Bull. Acad. Roy.
Med. Belgique. 15:491.
22. Ephrussi, B. 1953. Nucleo-cytoplasmic relations in micro-organisms. Oxford Univ.
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23. Steinhaus, E. A. 1946. Insect Microbiology. Cornell Univ. Press (Comstock).
Ithaca.
24. Buchner, p. 1953. Endosymbiose der tiere mit pflanzlichen mikroorganismen.
Birkhauser. Basel.
25. Newton, B. A. & R. W. Horne. 1957. Intracellular structures in Strigomonas onco-
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26. Eredericq, P. 1957. Colicins. Ann. Rev. Microbiol. 11: 7.
THE EFFECT OF POLLUTION ON RIVER ALGAE
C. Mervin Palmer
U. S. Department of Health, Education, and Welfare, Public Health. Service,
Robert A. Taft Sanitary Engineering Center, Cincinnati, Ohio
A large majority of algae are affected adversely by the gross pollution of
streams with organic wastes such as domestic sewage. After partial self-purifi-
cation of the stream has occurred, however, the populations and kinds of algae
become much more numerous than are present in the clean portion of the
stream above the area of pollution. This increase is due to the nutrients that
are made available from the decomposing organic wastes.
The undecomposed organic wastes affect the algae by causing chemical and
physical changes in the stream. Increased turbidity reduces the light availa-
ble for photosynthesis. Increased organic content in the water stimulates
saprophytic and saprozoic organisms which then compete for space with the
algae. Certain constituents of the waste are toxic to many algae. Thus,
many factors of the environment that are changed by the organic wastes have
an effect on the algae.
Information on the physiological and morphological effects of organic pollu-
tion on algae is very limited at present. There have been, however, many
studies of the change in the algal tlora as a result of pollution. Gross pollu-
tion causes a great reduction in the number of kinds of algae in the stream.
Those able to remain have frequently been called "indicators" of pollution,
but no specific kinds individually are reliable indicators of grossly polluted
water. Polluted water varies too much to ensure an environment satisfactory
for the growth or persistence of any one particular algal species. Any indi-
vidual species tolerant of pollution may also be found in unpolluted areas of a
stream or may be absent in some areas of pollution.
When a number of the tolerant genera and species are considered, it becomes
likely that a high percentage of these will be present in all areas of streams
grossly polluted with organic wastes. The presence of such a community of
algae in a stream, therefore, is a reliable indicator of the condition of the water.
Many workers have listed the genera and species of algae found in polluted
waters, particularly in the United States and in Europe. The number of
kinds which they have considered to be pollution tolerant is generally quite
limited for any one area or survey, but becomes very large when all of the
results of many investigators are combined.
The lists of pollution-tolerant algae reported by 110 workers have been ex-
amined by the writer to date. The genera and species of algae tolerant to
sewage or to related conditions have been recorded, and a total of more than
600 species and varieties has been compiled.
To tabulate the information, the writer has allotted arbitrary numerical
values to each author's record of an alga. A value of 2 was given to each
alga reported as very highly tolerant, and a value of 1 to each alga highly tolerant
to the presence of organic matter. Lightly tolerant and nontolerant algae
were not recorded in the compilation. The total points from all of the 110
389
390
Annals New York Academy of Sciences
authors were then determined for each genus and species. The algae were
arranged in the order of decreasing emphasis by the authors as a whole as in-
dicated by the comparative total scores for each alga. Theoretically an alga
considered as very highly tolerant by all 110 authors would have had a perfect
score of 110 multiplied by 2, or 220 total points.
For studies in sanitary science the algae are frequently placed into four
groups. All flagellates containing photosynthetic pigments constitute one of
the four groups. The other three groups are the blue-green algae, the diatoms,
and the green algae, the last group including all of the nonflagellated green,
yellow-green, and other related forms.
Table 1
Pollution Algae
Most tolerant genera, by groups
Highest
4
10
50
Blue-greens
Greens
Diatoms
Flagellates
1
1
0
2
2
3
3
2
8
15
15
12
Table 2
Pollution Algae
Most tolerant species, by groups
Highest
4
10
so
Blue-greens
Greens
Diatoms
Flagellates
1
1
1
1
3
4
1
2
15
10
11
14
All four groups are well represented among the genera and species with high
scores as pollution-tolerant algae. For example, of the 10 genera with the
highest scores, 2 are blue-green algae, 2 are flagellates, 3 are diatoms, and 3 are
green algae (table 1). Of the four species with the four highest scores, each
belongs to a different group. Among the 50 most tolerant species, the range in
number per group is from 10 to 15 (table 2).
The 52 most tolerant genera are listed in table 3. Leading the list, in order
of decreasing total scores, are Euglena, Oscillaloria, Clilamydomonas, Scenedes-
mus, Chlorella, and Xilzschia. The first two were considered as tolerant genera
by 62 and 61 authors and rated 110 and 105 total points, respectively. These
are in contrast with the fiftieth genus, Cocconeis, which was referred to by only
8 authors for a total score of eight.
Palmer : Effect of Pollution on River Algae
391
Table 3
Pollution Tolerant Genera of Algae List of 52 Most Tolerant Genera in
Order of Decreasing Emphasis by 110 Authorities
Genera
Group
No. of authors
Total points*
1
Euglena
F
62
110
2
Oscillatoria
B
61
105
3
Chlamydomonas
F
42
70
4
Scenedesmus
G
40
65
5
C/dorella
G
36
63
6
Nitzschia
D
38
63
7
Navicula
D
35
55
8
Stigeodonium
G
34
50
9
Phormidiiim
B
30
45
10
Synedra
D
25
a
11
F ha ens
F
23
32
12
Ankistrodesmus
G
19
31
13
Gomplionema
D
20
30
14
Spirogyra
G
19
29
15
Cydotella
D
22
29
16
Pandorina
F
18
25
17
Closterium
G
19
25
18
Lepocindis
F
14
24
19
Melosira
D
18
24
20
Chlorogoniiim
F
14
23
21
A nabaena
B
17
23
22
Ulothrix
G
17
23
23
Micractinium
G
13
21
24
FragUaria
D
15
20
25
A nacystis
B
16
20
26
Frachelonwnas
F
16
20
27
Art/irospira
B
11
19
28
Carleria
F
12
19
29
Surirella
D
14
19
30
Cryptomonas
F
15
19
31
AgmeneUiim
B
11
18
32
Lyngbya
B
11
18
33
Eudorina
F
12
18
34
Fediastrum
G
14
18
35
Oocystis
G
12
16
36
Fyrobotiys
F
10
15
37
Cymbella
D
10
14
38
Steplianodisciis
D
10
14
39
Coelaslrum
G
12
14
40
Cladophora
G
13
14
41
Golenkinia
G
9
13
42
Spondylomonim
F
9
13
43
Achnantlies
D
11
13
44
Actinastntm
G
11
13
45
Hanlzschia
D
9
12
46
Spindina
B
9
12
47
Finnitlaria
D
8
11
48
Ski Krone! s
D
9
11
49
Tribonema
G
6
10
50
Coeconeis
D
8
10
51
Selenasfrnm
G
8
10
52
Cosmarium
G
9
10
* Tolerance by author, "Very High," 2 points. Tolerance by author, "High," 1 point.
Table 4
Pollution Tolerant Species of Algae: A List of the 60 Most Tolerant
Species in Order of Decreasing Emphasis by 110 Authorities
Species
Group
No. of
authors
Total points*
1
Euglena viridis
F
34
63
2
Nitzscfiia palea
D
30
46
3
Stigeoclonium lenne
G
17
26
4
Oscillaloria tenuis
B
17
25
5
Oscillaloria limosa
B
14
21
6
Scenedesnms quadricauda
G
12
18
7
Chlorella vulgaris
G
11
17
8
Pandoritia morion
F
12
17
9
Arthrospira jenneri
B
9
16
10
A nkistrodesmiis Jalcatus
G
11
16
11
Cydotella meneghiniana
D
12
16
12
Chlorella pyrenoidosa
G
8
15
13
Gomphonema parvuluni
D
8
15
14
Euglena gracilis
F
9
15
15
Oscillatoria clialybea
B
10
15
16
Synedra ulna
D
12
15
17
Oscillatoria cidorina
B
9
14
18
Nilzschia acicularis
D
10
14
19
Oscillatoria formosa
B
10
14
20
Oscillatoria princeps
B
10
14
21
Oscillatoria putrida
B
8
13
22
Euglena oxyuris
F
9
13
23
Navicula cryptocepliala
D
9
13
24
Flwrmidium uncinatum
B
11
13
25
Agmenellum quadriduplicatum
B
7
12
26
Chlorogonium euchlorum
F
7
12
27
Hantzscliia ampliioxys
D
9
12
28
Phormidium autumnale
B
9
12
29
Surirella ovata
D
9
12
30
Euglena acus
F
10
12
31
LepocincUs ovum
F
7
11
32
Micractinium pusillum
G
7
11
33
Eunorina elegans
F
8
11
34
Euglena deses
F
8
11
35
Oscillaloria splendida
B
9
11
36
Oscillatoria lauterbornii
B
6
10
37
Euglena polymorplta
F
7
10
38
LepocincUs texta
F
7
10
39
Spondylomorum quaternarium
F
7
10
40
A ctinaslru m hantzschi
G
8
10
41
Closterium acerosum
G
8
10
42
A nabaena constricta
B
6
9
43
Anacystis montana
B
6
9
44
Pliacus pyrum
F
6
9
45
Scenedesmus obliquus
G
6
9
46
Cocconeis placentida
D
7
9
47
Achnanthes minutissima
D
8
9
48
Coelastrum micro poruni
G
8
9
49
Melosira varians
D
8
9
50
CItlamydomonas rein liardi
F
5
8
51
Pediastrum horyanum
G
5
8
52
Scenedesmus dimorphus
G
5
8
53
Chlorogonium elongatum
G
6
8
54
Euglena intermedia
F
6
8
55
Euglena pisciformis
F
6
8
56
Phacus pleuronectes
F
6
8
57
Tetraedron mulicum
G
6
8
58
Anacystis cyanea
B
7
8
59
Melosira granulata
D
7
8
60
Phormidium faveolarum
B
8
8
* Tolerance by author, "Very High," 2 points.
392
Tolerance by author, "High," 1 point.
Palmer: Effect of Pollution on River Algae
393
The 60 most tolerant species are given in table 4. Euglena viridis, followed
by Nitzschia palea, are at the top of the list with total scores of 6v^ and 46,
respectively.
The names and total points for the 10 most tolerant species of a genus are
shown for the two leading genera, Euglena and Oscillator ia (tables 5 and 6).
In the former genus, the first species, E. viridis, is far ahead of the other nine
species. In the latter genus there is a more gradual change in total points from
Table 5
Species of Euglena: Ten Most Tolerant of Pollution
Authors
Points
E. viridis
34
63
E. gracilis
9
15
E. oxvuris
9
13
E. aciis
10
12
E. deses
8
11
E. polvmorpha
7
10
E. intermedia
6
8
E. pisciformis
6
8
E. proxima
5
7
E. spirogyra
6
7
Table 6
Species of Oscillatorla: Ten Most Tolerant of Pollution
Authors
Points
0. tenuis
17
25
0. liniosa
14
21
0. clialvhea
10
IS
0. clilorina
9
14
0. fonnosa
10
14
0. prince ps
10
14
0. putrida
8
13
0. splendid a
9
11
0. lauterbornii
6
10
0. brevis
6
7
one species to the next. Eight of the 10 species of Euglena and 9 of Oscillatoria
are among the 60 most tolerant forms as noted in table 4.
It would be interesting to know what species of Chlamydomonas was con-
sidered most tolerant of organic pollution, but unfortunately very few of the
110 investigators have determined and recorded the species for this genus. For
the genus Xavicula, numerous species have been recorded by the investigators,
but there is little indication that there may be one or two species which are
much more tolerant than others that they have named.
Additional records by other workers would undoubtedly change the com-
parative total points and the relative positions of the algae in both the genus and
species lists. This is particularly so for the algae near the low ends of the lists
where a relatively few reports are responsible for their present positions.
394 Annals New York Academy of Sciences
The lists of algae in the tables are meant to be aids for persons engaged in
stream pollution surveys or related projects. They give a general consensus
of opinion as to the relative significance of the many algae tolerant of organic
wastes which have been reported. Particular care can thus be taken in bio-
logical surveys to check for the presence of these genera and species of algae
during the microscopic examination of samples.
The references given represent many of the more exhaustive studies that were
included in the preparation of this report.
References
Blum, J. L. 1956. The ecology of river algae. Botan. Rev. 22: 291-341.
Butcher, R. W. 1949. Pollution and repurification as indicated by the algae. Fourth
International Congress for Microbiology (held) 1947. Report of Proceedings.
Cholnoky, B. J. 1958. Hydrobiologische Untersuchungen in Transvaal. II. Selbstrei-
nigung im Jukskei-Crocodile Flusssystem. Hydrobiologia. 11(3-4): 205-266.
FjERDiNGSTAD, E. 1950. The microflora of the River M^lleaa with special reference to
the relation of the benthal algae to pollution. Folia Limnol. Scand. No. 5.
FoEBES, S. A. & R. E. Richardson. 1913. Studies on the biologj' of the upper Illinois
River. Bull. Illinois State Lab. Nat. Hist. 9(Art. 10): 481-574.
HORNUNG, H. 1959. Floristischokologische Untersuchungen an der Echaz unter be-
sonderer Beriicksichtigung der Verunreinigung durch Abwasser. Arch. Hydrobiol.
55: 52-126.
Hynes, H. B. 1960. The Biology of Polluted Waters. Liverpool Univ. Press. Liverpool.
KoLKWiTZ, R. 1950. Oekologie der Saprobien. Uber die Beziehungen der Wasserorga-
nismen zur Umwelt. Schriftenreihe des Vereins fiir Wasser-, Boden- und Lufthygiene
Berlin-Dahlem. Piscator-Verlag. Stuttgart.
Lackey, J. B. 1941. The significance of plankton in relation to the sanitary condition of
streams. In Symposium on Hydrobiology. : 311-328. LTniv. of Wisconsin, Madison.
Lackey, J. B. 1956. Stream enrichment and microbiota. Public Health Repts. 71: 708-
718.
Liebmann, H. 1951. Handbuch der Frischwasser- und Abwasserbiologie. R. Olden-
bourg. Miinchen.
Mackenthun, K. M., L. a. Lueschow & C. D. McNabb. 1960. A study of the effects
of diverting the effluent from sewage treatment upon the receiving stream. Trans.
Wisconsin Acad. Sci. 49: 51-72.
McGauhey, p. H. & H. F. Eich. 1922. A study of the stream pollution problem in the
Roanoke, Virginia, MetropoHtan District. Part 3. Third portion: The plankton of
the waters and muds. Bull. Va. Polytech. Inst. (Eng. Expt. Stat. Ser. No. 51). 35:
64-88.
Oliff, W. D. 1960. Hydrobiological studies on the Tugela River system. Part II. Or-
ganic pollution in the Bushmans River. Hydrobiologia. 16(2): 137-196.
Palmer, C. M. 1957. Algae as biological indicators of pollution. Biology of Water Pol-
lution: Trans. Seminar on biological j^roblems in water pollution held in 1956. : 60-
69. Robert A. Taft Sanitary Engineering Center. Cincinnati, Ohio.
Palmer, C. M. 1959. Algae in water supplies. U.S. Public Health Service Publ. No. 657.
U.S. Government Print. Off. Washington, D.C.
Palmer, C. M. 1932. Plankton algae of White River in Marion County and Morgan
County, Indiana. Butler Univ. Botan. Studies. 2: 125-131.
Patrick, R. 1948. Factors effecting the distribution of diatoms. Botan. Rev. 14(8):
473-524.
Purdy, W. C. 1930. A study of the pollution and natural purification of the Illinois River.
II. The plankton and related organisms. U.S. Pubhc Health Bull. No. 198. : 1-212.
Silva, p. C. & G. F. PAPENFU.SS. 1953. A systematic study of the algae of sewage oxida-
tion ponds. Calif. State Water PoUution Control Board. Publ. No. 7.
Sramek-Husek, R. 1956. Zur biologischen Charakteristik der hoheren Saprobitatsstufen.
Arch. Hydrobiol. 51: 376-390.
Uherkovich, G. 1961. Limnologia, a tiszai algak a szaprobionta rendszerben. Hidrol.
Kozlony. 1: 85-88.
Weston, R. S. & C. E. Turner. 1917. Studies on the digestion of a sewage-filter effluent
Palmer: Effect of Pollution on River Algae 395
by a small and otherwise unpolluted stream. Contrib. from Sanitary Res. Lab. and
Sewage Expt. Station. Mass. Inst. Technol. Vol. 10.
Whipple, G. C, G. M. Fair & M. C. Whipple. 1948. The Microscopy of Drinking Water.
Ed. (4). J. Wiley & Sons. N. Y.
WiEBE, A. H. 1927. Biological survey of the upper Mississippi River with special refer-
ence to pollution. Document No. 1028. Bull. Bur. Fisheries. 43(2): 137-167.
WiSNiEWSKi, T. F. 1961. The Badtish River before and after diversion of sewage plant
effluent. Algae and MetropoUtan Wastes. Trans. 1960 Seminar. Robert A. Taft
Sanitary Engineering Center, Cincinnati, Ohio, Tech. Rept. W61-3: 118-124.
Wysocka, H. 1961. Periphyton des lamelles en verre comme I'indicateur de la pollution
d'eau. Verhandel. Intern. Verein. Limnol. 14: 1063-1070.
ULTRASTRUCTURE RESEARCH AS AN AID IN
THE CLASSIFICATION OF DIATOMS
R. Ross
British Museum {Natural History), London S.W . 7, England
Present Knowledge of the Ultrastrudure of Diatoms
The frustules of diatoms were among the first biological objects to be exam-
ined with the electron microscope (Mahl, 193^), and in the preceding 20 years
a large number of works dealing with the subject have appeared. These have
been listed comparatively recently by Hendey (1959), and at the time at which
he wrote information about some 300 species was available. Although it is not
important to review the results of these studies in detail, there are two points
about them which need to be emphasized here. The tirst is that none of this
work has been done with any particular taxonomic problem in mind. For the
most part it would seem that investigators took the material which came readily
to hand, mounted drops of it on electron microscope grids, and took pictures of
the forms they found there. This has on occasion led to doubt as to the true
identity of the species studied, as in the case of the illustrations published by
Kolbe (1951, plate 2, tig. 4, plate 3, figs. 5 and 6) as Navicula subtUissima Cleve,
but said by Hustedt (1952, 1955) to be of Anomoeoneis exilis (Kiitz.) Cleve or
A. serians var. brachysira (Breb.) Cleve (Kolbe, 1954, 1956, 1959). A more im-
portant consequence, however, is that there is not any group of supposedly re-
lated species of which more than a small proportion have been studied with the
electron microscope. In no case do we know the patterns of similarity and
difference and the range of ultraslructure to be found within a single genus,
with the possible exception of Pinnularia Ehrenb., of which electron micro-
graphs of some 15 species in a genus totaling at least 250 suggest that the
ultrastructure is as uniform as that revealed by the light microscope.
The other important point is that the interpretation of electron micrographs
is by no means easy, and also that, in some cases, those published do not give
an adequate picture of the structure of the species illustrated, either because the
specimen was damaged in preparation or because the resolution is insutlicient.
Interpretation is ditlftcult because of the great depth of focus of the electron
microscope and the considerable opacity of silica to electrons. Even in pic-
tures of complete frustules, the whole is equally in focus. In the light micro-
scope it is possible to build up a picture in depth from a series of optical sections
obtained by alterations of focus, but this technique is not available to the elec-
tron microscopist. When more than one layer is visible it is often not possible
to tell from single pictures which lies above which. Much of the valve, also, is
completely opaque to electrons, and where this is so there is no information
about differences in thickness from differences in transmission of electrons.
Stereomicrographs accordingly provide much more information than single
prints, as may be seen from the large number published by Helmcke and Krieger
(1953, 1954, Helmcke et al., 1961). These authors have applied stereogram-
metric techniques to the study of their stereoscopic pairs and have produced
models of the structure of a number of species, thus obtaining the maximal
amount of information from the data recorded on the micrographs.
396
Ross: Classification of Diatoms 397
The possibility of being misled by photographs with inadequate resolution
or of damaged specimens is best illustrated by particular examples. Hendey's
(1959) list of the species examined with the electron microscope includes an
indication of the ultrastructure of the valve. Both Stanroneis anceps Ehrenb.
and S. phoenicenteron (Nitzsch) Ehrenb. are said to have laminar valves per-
forated by fully open holes. His information about S. anceps is derived from a
picture published by Helmcke and Krieger (1953, plate 67) and that about
S. phoenicenteron from three pictures pubUshed by Okuno (1949, plate 3, fig. 8,
1952, plate 19, fig. 4, 1955, plate 9, fig. 1). In both species, however, the
striae consist of a series of elongated chambers with a membrane pierced by a
sUt on the outside and a membrane with fine pores in triangular tesselation on
the inside. The outer membrane is visible with a lens on negatives taken at
XlOOO, but not easily so, whereas the inner membrane, in which the repeat
distance of the pores is only about 170 A, can only be seen on negatives taken at
X5000. Helmcke's and Krieger's and Okuno's pictures seem to have been
taken at a much lower magnification than this and enlarged in reproduction.
Recently Helmcke et al. (1961, plates 289-290) have published pictures of S.
phoenicenteron showing the two membranes, but not all the detail described
below (p. 401). The ultrastructure can also be damaged either by chemical
cleaning or in fossilization. Figure 2 (p. 402) of a postpleistocene fossil speci-
men of S. phoenicenteron^ which may be compared with the pictures of the
species published by Helmcke et al., shows an example of this.
When features are misinterpreted or imperfectly understood, and especially
when, in consequence, like things are considered unlike or unlike things are
grouped together, they will not provide satisfactory taxonomic characters. It
is, therefore, necessary to base any taxonomic use of the ultrastructure of
diatoms upon a proper understanding of that structure. Hendey (1959) has
presented a classification of the types of ultrastructure in which the primary
division is into laminar valves, consisting of one layer of siliceous substance,
usually perforate, and locular valves, which are formed of a double layer of
siliceous substance separated by vertical walls. In my opinion, however, such
a distinction cannot be drawn. In most cases, at least, the diatom valves are
pierced by chambers; these may occasionally be completely open on both sides,
when they may properly be described as pores, but more usually they have a
membrane, itself perforate, on one or both sides. In a number of cases, what
were originally thought to be pores have been found, when more critically
examined, to be closed by membranes on one or both sides. This makes it seem
possible that such membranes will be found to be normal throughout the
diatoms, and that only the mucilage pores that occur singly or in small numbers
in some species will prove to be true pores. What Hendey classes as partially
occluded perforations through a single-layered wall are exactly similar in
structure to what he classes as loculi open on one side; the only difference lies
in the closeness of their packing. His failure to realize this may be due in part
to the difficulty of establishing relations in depth from single electron micro-
graphs and his not recognizing in consequence that the membranes occluding
the perforations were at the level of one or other surface of the valve. Ac-
cordingly, the classification of Helmcke et al. (1961), based entirely upon the
structure of the individual chambers, is much more satisfactory. This sepa-
398 Annals New York Academy of Sciences
rates pores, open at both ends, from chambers, with a septum at one or both
ends, and classifies these according to the position and type of perforation of
the septum or septa.
The Use of Diatom Ultrastructure in Taxonomy
In spite of the large amount of information available about the ultrastructure
of diatoms, it has until now been of little use in their taxonomy. Hustedt
(1952, 1955), in the course of an interchange of opinion on the subject with
Kolbe (1954, 1956), maintained that ultrastructure is more uniform than the
features that can be seen with the light microscope, and that its variations
show no correlation with the characters used to distinguish genera; ultrastruc-
ture, accordingly, cannot be regarded as having any taxonomic significance
above the specific level (Hustedt, 1959, pp. V-VI). Hendey (1959) came to a
similar conclusion, but added that when a large number of species have been
examined it may be possible to subdivide the genus NavicuJa Bory. Views
similar to Hustedt 's are presented by Lund (1962) in his recent review of the
criteria adopted in classifying algae.
It is probably not an unfair generalization to suggest that taxonomists are
conservative in their outlook, especially in their views about which characters
are important in classifying a particular group. They do not seize every
opportunity of using a newly discovered set of characters to produce a new
system supplanting the current one. They tend rather to keep alterations to
a minimum, apart from the addition of numerous new species and taxa of lower
rank. One of the most gratifying results of the study of diatom frustules with
the electron microscope has been that it has brought to light nothing really
surprising. Structure too fine to be resolved with the light microscope has been
demonstrated, but this was only to be expected. Nothing which could be seen
with the light microscope has been found to have a structure markedly different
from that which it was thought to have. This represents a great tribute to the
skill and acumen of those who used the light microscope at the limit of its
potentialities to elucidate the structure of the diatom valve, especially O. Miiller
(1889, 1895, 1896a, b, 1898, 1899, 1900, 19()la, b, 1909) and Hustedt (1926o,
h, 1928o, b, 1929a, b, 1935a, b). On the other hand it has meant that no re-
visions of the system have been forced upon diatom taxonomists, and in the
absence of any such pressure they have not actively pursued the question of
how far knowledge gained with the electron microscope could influence classifi-
cation above the specific level.
Although conservatism has played its part in persuading diatomists that
ultrastructure can only play a minor role in the taxonomy of the group, they
have been helped to reach that conclusion by two other factors. Both of
these have already been discussed; they are the inadequate number of species
investigated with the electron microscope and the inadequate information
about the ultrastructure of many of those examined. Thus, Hendey's (1959)
list of the diatoms investigated with the electron microscope includes only 28
identified species of Navicula, out of at least 1000 at present known, and it is
probable that the information about the structure of many of these is as inade-
quate as that which he gives about Stauroneis anceps and S. phoenicenteron
(cf., p. 397). For all other genera fewer species have been investigated, and
Ross: Classification of Diatoms 399
only in Chaetoceros Ehrenb. and Pinnularia of the larger genera is the proportion
studied greater than in Navicula.
There are two parts of the system of classification of the diatoms in which the
currently accepted taxonomy above the specific level is patently unsatisfactory:
the famiUes Navicuiaceae and Biddulphiaceae. In both, species are grouped
with others to which they seem only distantly related and separated from those
which seem close to them. In a taxonomic investigation of a small group
of species in the Navicuiaceae on which I was recently engaged, I decided
that electron stereomicrographs would be useful in elucidating a particular
point about the structures connected with the central nodule. Through the
kindness of K. Little of the Nuffield Orthopaedic Centre, Oxford, England, who
is responsible for all the micrographs illustrating this paper, these were obtained.
They showed not only the details of the central structure but also the ultra-
structure of the perforations through the valve, and the correlations between
these two suggested that ultrastructure might well form a guide to a revision
of the limits of Stauroneis Ehrenb. and possibly certain other genera, and that
the attitude of Hustedt (1959), Hendey (1959), and Lund (1962) to its use for
this purpose was unduly defeatist. These observations are being extended, and
much more needs to be done before any firm conclusions can be reached. This
paper cannot, in consequence, be anything more than a report on progress to
date, but its object will be fulfilled if it dissipates doubts about the value of
ultrastructure as a source of taxonomic characters and stimulates others to
work on similar lines.
Technique
This approach necessitates the accumulation of electron micrographs of a
large proportion of the species in the group under investigation. Many species
of diatom occur only as comparatively rare members of the assemblage con-
tained in a particular gathering. To obtain the electron micrographs needed
in a taxonomic investigation accordingly demands the use of a techniciue similar
to that used in the making of selected slides of individual specimens for the
light microscope. Reliance on serendipity, which has hitherto been the normal
practice when choosing specimens for investigation with the electron micro-
scope, will not suffice.
Each worker who makes selected slides of individual diatoms develops a
technique which suits the resources of his own laboratory and his personal
characteristics, in particular the steadiness of his hand. This account of the
method I have used for selecting individual diatoms for study with the electron
microscope, which is based upon that which I use when making selected slides
for examination with the light microscope, should be taken only as a general
guide and not as a model to be rigidly followed in all of its details.
One starts with a suspension in distilled water of chemically cleaned diatom
frustules (for methods see Hustedt, 1927, 1958, Swatman, 1937, Hendey, 1938,
1951, Leboime, 1952, van der Werff, 1955, Barber, 1962) which is known to
contain the diatom which it is desired to study. A few drops of this are allowed
to evaporate, preferably on a mica surface, to which diatoms adhere less than
they do to glass. Heat should not be used as convection currents cause the
diatoms to clump together. Diatom frustules apparently adsorb some of the
400 Annals New York Academy of Sciences
chemicals used in cleaning and liberate these slowly into the water in which
they are washed or stored. If these chemicals are present, they cause the
diatoms to stick to the mica. It is, therefore, desirable to leave the diatoms in
at least the last two washing waters for a period of 2 days or more, and to pour
off the water in which they have been stored and replace with fresh distilled
water immediately before the preparation of the strews from which specimens
are to be selected.
The actual selecting is most conveniently done under a binocular dissecting
microscope at a magnification of about XlOO. Except with the larger forms,
it is not possible at this magnification to recognize the species to be selected with
certainty. It is, therefore, necessary to locate them under an ordinary micro-
scope and to note their position relative to prominent specimens that can act
as markers. This process is facilitated if a grid is ruled on the back of the slip
of mica with the point of a needle and the scratches filled with India ink. The
micashp can then be mounted with balsam on a microscope slide. It is usually
more convenient to assemble specimens of each species to be investigated in
separate groups near the edge of the mica shp before transferring them to the
grids. When small diatoms are being dealt with, each group can then be
examined under the ordinary microscope to see that all the specimens are of the
correct species.
The necessary number of formvar-coated grids are attached to an ordinary
microscope sUde by tiny drops of gum arable at their edge. They are held
steady by this during mounting but can be readily detached for insertion in the
microscope. A label can be placed at one end of the slide giving a numbered
key to the grids. The diatoms can then be taken up individually on a bristle
from the mica shp and placed on the formvar film over the spaces in the grid.
This can usually be done without tearing the film. When the work is done in a
dry atmosphere, the diatoms at times accjuire an electrostatic charge, which
causes them to fly off the grid when it is lifted off the slide. This trouble can
be obviated by breathing gently on the grids after the diatoms have been trans-
ferred to them. After the thin film of water thus condensed on them has
evaporated, they adhere sufficiently not to fall off when the grid is placed in
the electron microscope, and will normally remain in position through a number
of insertions into and removals from the instrument.
I find it possible to transfer the diatoms freehand, even specimens the major
axis of which is between 10 and 15 n in length. For this I use a bristle mounted
on a cylindrical rod of wood about as thick as a pencil and sharpened like one
to a point at one end. The bristle is stuck to this point with about 2 mm.
protruding. Pelletan (1888) and Hustedt (1927) recommend a pig's eyelash
as the most suitable bristle and I find one very satisfactory.
A number of types of mechanical fingers for the selection of diatoms have
been developed, the most widely used probably being that designed by Meakin
(1939). These could no doubt also be used for transferring diatoms to electron
microscope grids. Stiffer bristles than those used for freehand mounting are,
however, normally used in mechanical fingers and these would be more likely
to tear the formvar films on the grids. When a mechanical finger is used to
mount diatoms for the electron microscope it will probably be advantageous to
replace its normal bristle by a more flexible one.
Ross: Classification of Diatoms 401
It has already been pointed out that stereomicrographs are much more
informative than single ones. The techniques for obtaining these and mount-
ing them for examination have been described by Little (1958, 1962). It is
also important to ensure that the micrographs are taken at a magnification and
with a resolution sufficient to show the true structure of the valve. Low
power micrographs of the specimen, which will enable its identity to be checked,
should also be taken.
New Observations on Diatom Ultrastrudure
The species originally described as Schizostauron crucicula Clrun. ex Cleve and
S. karstenii Zanon are currently placed in the genus Stanroneis, the structure
associated with their central nodule being interpreted as a bifid stauros. Speci-
mens of these two species were recently encountered in some gatherings from
Lake Tanganyika, and in the same material two undescribed species which
seemed related were also found. One of these was very similar to the two
known species, but the other had the asymmetry characteristic of the genus
Amphora Ehrenb., i.e., both its apical and its pervalvar axes were curved. Al-
though it was possible to be reasonably certain under the light microscope that
the structures associated with the central nodule were not very similar to an
ordinary stauros, details of their form could not be made out with certainty.
There was also need to confirm that the asymmetric species differed from the
others only in shape and not in any point of structure. Specimens of Schizo-
stauron crucicula, S. karstenii, and the asymmetric form were therefore examined
in the electron microscope and stereomicrographs of them were obtained.
Specimens of the type species of Stauroneis, S. phoenicenteron, and of 5". anceps
and S. smithii Grun. were also examined for comparison. These observations,
which are reported in detail by Ross (1963), confirmed that the species with a
so-called "bifid stauros" were so different from S. phoenicenteron that they
should be placed in a separate genus, for which the correct name is Caparto-
gramma Kuff. Also, S. phoenicenteron and S. anceps were found to be very
similar, but to differ greatly from 5. smithii. The results may be summarized
as follows.
(1) Stauroneis phoenicenteron (figures 1 and 2) and S. anceps (figures 3 and
4) have a stauros which is a wide but not very deep thickening of the valve.
The chambers that form their striae are elongated along the direction of the
stria, especially near the inner surface, where they are separated by a very
narrow wall. These chambers are closed on their inner side by a membrane
with fine pores in triangular tesselation and on the outer side by a membrane
with a broad slit along the direction of the stria. The length of this slit is
shorter than the length of the main part of the chamber.
(2) Stauroneis smithii (figures 5 and 6) has a deep and narrow thickening
across the valve. Its chambers are not close; they are approximately circular
and are closed on the inner side by a membrane with fine pores in triangular
tesselation and on the outer side by a membrane with a narrowly elliptical
opening of which the major axis is across the direction of the stria and is longer
than the diameter of the main part of the chamber.
(3) All three species of Capartogramma (for illustrations see Ross, 1963) have
on either side of the central nodule two, or occasionally three, deep and very nar-
Figures 1-2. Stauroneis phoenicenleron (Nitzsch) Ehrenb. In figure 2 are shown
artifact structure caused by too rigourous cleaning (r/., Helmcke el al., 1961, plate 289 to 290)
for true structure of this species. Figure 1, X2500. Figure 2, X40,000.
Figures 3-4. Stauroneis anceps Ehrenb. Figure 3, X2000. Figure 4, X40,000.
Figures 5-6. Stauroneis smitliii Grun. These specimens are somewhat eroded but in
figure 6 it is shown that the slits in the outer membrane run across the striae, figure 5,
X2500. Figure 6, X 40,000.
402
Ross : Classification of Diatoms 403
row flanges running from the central nodule to the valve margin, projecting at
right angles to the valve surface but turned towards the apices at their free
edges. Their chambers are not close; they are approximately circular and are
closed on the inner side by a membrane with fine pores in triangular tesselation
and on the outer side by a membrane with a broad slit that runs across the
direction of the stria and is longer than the diameter of the main part of the
chamber.
These observations not only confirmed that it is correct to separate the
species with a "bifid stauros" from Stauroneis and to associate the species with
amphoroid asymmetry and the symmetrical ones; they also suggested that
other species now grouped in Stauroneis might belong to separate genera. To
see whether examination of more species would provide evidence to confirm this,
S. acuta W. Sm., 5*. amphioxys Greg., and S. salina W. Sm. were examined under
the electron microscope, and more species will be as opportunity offers. The
stauros of the first two species appears under the light microscope to be broad
and narrow. The electron micrographs showed their structure to be as follows:
(4) Stauroneis acuta (figures 7 and 8) has a broad and rather shallow stauros,
as in S. phoenicenteron and S. anceps, and the ultrastructure of its striae is
similar to that in those two species.
(5) Stauroneis amphioxys (figures 9 and 10) has a broad and shallow stauros,
which extends for less than two-thirds of the width of the valve. Its striae
consist of distant circular chambers closed on the inner surface by a fine
membrane with pores in triangular tesselation. The chambers taper outwards,
i.e., they have the shape of truncated cones, but they have no membrane on
their outer side.
(6) Stauroneis salina (figures 11 and 12) has a stauros that is rather deep
at the center of the valve and becomes narrower and shallower toward the
margin. The striae consist of distant circular chambers closed on the outer
side by an oblique parallel- sided slit that is slightly longer than the diameter of
the main part of the chamber and on the inner side by a fine membrane with
pores in triangular tesselation. The valve surface is depressed between one-
third and two-thirds of the distance from the raphe to the margin and through-
out this area the chambers in the striae are more distant than elsewhere.
Taxonomic Implications
Attention is here drawn to some similarities and differences in ultrastructure
that may have a taxonomic significance; not only the original observations
recorded above but also published micrographs of various species of Navic-
ulaceae are considered. The present state of our knowledge provides only a
very tenuous basis for taxonomic speculations; the justification for indulging
in these and putting them on record is that others may be stimulated to collect
further data that will tend to confirm or refute them.
Stauroneis. Until recently the presence or absence of pseudosepta has been
treated as a character distinguishing sections within this genus (Cleve-Euler,
195vS). The close similarity which S. acuta, in which these are present, bears
in all other respects to S. phoenicenteron, in which they are absent, confirms the
view put forward by Hustedt (1959) that they are of httle taxonomic signifi-
cance. Also, Hustedt's (1959) contention that S. amphioxys (which he in-
404
Annals New York Academy of Sciences
Figures 7-8. Stauroneis acuta W.Sm. In figure 7 is shown the extension of the cham-
bers along the line of the striae, figure 8 the inner membrane with tine pores in triangular
tesselation and the iiroad slit along the line of the striae. Figure 7, X5000. Figure 8,
X 40,000.
Figures 9-10. Stauroneis amphioxys Greg. Figure 9, X2500. Figure 10, X40,000.
Figures 11-12. Stauroneis salina W.Sm. Figure 11, X2000. Figure 12, X40,000.
Ross : Classification of Diatoms 405
correctly calls S. gregorii Ralfs) and .S". salina are quite distinct species is con-
firmed,
Ultrastructure confirms the view that S. phoenicenteron , S. anceps, and
S. acuta should be placed in the same genus. S. amphioxys, S. saliua, and
S. smithii differ considerably from these and from one another. Meresch-
kowsky (1903a), on the basis of endochrome structure, removed .S\ amphioxys
and .5. salina from Slauroneis and created a new genus, Slaurophora, for the
two species. Although their ultrastructure indicates that they should perhaps
be removed from Slauroneis, it provides no contirmation for grouping them
together. Information about many more species is needed before any firm
conclusions can be drawn about the correct position of these species. S.
smithii, however, seems to be close to Capartogranima both in the structures
associated with the central nodule and in the ultrastructure of the chambers,
and S. salina bears some resemblance. It is noteworthy that Frustulia rhom-
boides var. saxonica (Rabenh.) De Toni (Helmcke et al., 1961, plates 279 to
280) has an ultrastructure almost identical with that of Caparlogramma and
S. smithii, and so also has Scoliopleura tumida (Breb.) Rabenh. (Helmcke and
Krieger, 1954, plate 177), a species grouped by Cleve (1894) not with the
other members of that genus but in his Naviculae Microstigmaticae, in which he
also included Slauroneis. This ultrastructure has certain similarities to that
found in most of the species of Pleurosigma and Gyrosigma examined. Whether
the species that possess this type of ultrastructure in common form a group of
genera more closely related to one another than to the rest of the Naviculaceae
is a question that can only be determined as more knowledge is accumulated,
but it seems that it is a possibiUty.
Amphora. As mentioned, there is a species which differs from the others
placed in the genus Caparlogramma only in shape of frustule; it has that char-
acteristic of the genus Amphora although the other species of the genus are,
like most Naviculaceae, symmetrical about the apical, transapical and pervalvar
planes. Cleve, in 1896, (p. 99) made the suggestion that the species placed in
the asymmetric genera Amphora and Cymbella Ag. were more closely related to
symmetrical species of similar valve structure than they were to one another.
The discovery of this new species of Caparlogramma adds further evidence for
the view that symmetry by itself is not a proper basis for delimiting genera.
The only species of the large and variable genus Amphora the ultrastructure of
which is known are A. cofeiformis (Ag.) Kiitz. (Helmcke and Krieger, 1953,
plate 76), A. deUcalissima Krasske (Helmcke el al., 1961, plate 294) and A.
ovalis (Kiitz.) Kiitz. (Helmcke and Krieger, 1953, plate 77, 1954, plate 181).
In A. cofeiformis and A. ovalis the ultrastructure resembles that found in
Anomoeoneis exilis (Helmcke and Krieger, 1954, plate 169) and A. serians
(Breb.) Cleve (Helmcke and Krieger, 1953, plate 68), which may indicate
relationship. Amphora deUcalissima has a cjuite different structure.
Cymbella. This is another genus which, like Amphora, is distinguished from
Navicula solely on the basis of asymmetry. Cleve (1894, p. 157) considered
that its species were most closely related to those of Navicula subgen. Navicula
(his Naviculae Lineolatae) . As far as ultrastructure is concerned, this is true
of C rabenhorslii Ross (Kolbe and Golz, 1943, plate 1, fig. 3, Helmcke and Krie-
ger, 1953, plate 75, as C. gracilis (Rabenh.) Cleve), C. turgida Greg. (Desika-
406 Annals New York Academy of Sciences
chary, 1952, figs. 17 and 18), and C. venlricosa Ag. (Desikachary, 1952, figs. 19
and 20, Helmcke and Krieger, 1953, plate 75) (c/., Xavicula cryptocephala
Klitz., Helmcke and Krieger, 1953, plate 69, N. digitoradiata (Greg.) A.
Schmidt, Helmcke et al., 1961, plate 292 and 293, N. radiosa Klitz., Helmcke
and Krieger, 1954, plate 172, and A^. viridula (Kiitz.) Kiitz., Helmcke and
Krieger, 1953, plate 73). Cymbella delicatnla Klitz. (Helmcke and Krieger,
1954, plate 180) and C. mexicana (Ehrcnb.) Cleve (Okuno, 1956, plate 21,
fig. 2), however, each have an ultrastructure which is different from that of
these species and from each other's. Electron micrographs of other species of
the genus have been published but none give adequate pictures of the ultra-
structure.
Maslogloia. The ultrastructure of M. braunii Grun. (Helmcke and Krieger,
1953, plates 57 and 58, 1954, plate 159) and M. smitliii Thwaites ex. W. Sm.
(Helmcke and Krieger, 1954, plate 160) is similar and resembles that of the
only two species of Navicula subgen. Lyraneis Freng. of which adequate electron
micrographs are available, viz.: N. forcipata Grev. (Helmcke et al., 1961, plate
291) and N. pygmaea Klitz. (Helmcke and Krieger, 1953, plate 71). Maslogloia
angulata Lewis (Okuno, 1957, plate 7, fig. 2) and M. fimbriata Cleve (Okuno,
1953, plate 1, fig. 3) resemble each other in their ultrastructure, but this is
quite different from that of M. braunii and M. smithii.
Discussion
The principles of taxonomy have recently been much discussed, and from this
discussion it has emerged that the amount of overall similarity is the only basis
for a satisfactory taxonomic classification (Cain, 1962, Sneath, 1962). To
accord overriding importance to a particular character, or to characters derived
from a particular structure, even if there are a priori grounds for considering
these of particular importance, results in an artificial and unsatisfactory system.
Almost without exception, however, diatoms have been classified solely on
the basis of the symmetry and structure of their siliceous frustule as seen under
the light microscope: although this provides comparatively few characters,
some of these, in particular symmetry, have been treated as having an im-
portance overriding that of the others. This concentration of attention on the
frustule has not been based upon any a priori reasoning but purely on con-
venience; in both fossil and recent material the valves are always present and
recognizable, and provide sufficient information for identification at the specific
level.
The current classification of the Naviculaceae rests on such a basis. The
species are separated into genera on the common possession of a single charac-
ter, or a combination of only two or three, all drawn from the structure of the
frustule. Some of the genera so characterized are probably natural groups,
e.g., Diploneis Ehrenb., Neidium Pfitz., and Pinnularia; others contain very
diverse elements, e.g., Amphora and probably Maslogloia Thwaites ex. W. Sm.
and Slauroneis. The species that do not possess any characteristic that has
been seized on as a mark of generic distinction are left in the very large genus,
Navicula, a hotchpotch of species of diverse affinity. The little that we already
know of the ultrastructure of the Naviculaceae shows that it provides a series
Ross: Classification of Diatoms 407
of characters to some extent culling across the present classification. Ultra-
structure, however, provides few characters and a system based solely upon it
would be as open to criticism as one based solely upon the structure of the
valve as seen under the light microscope. All of the information about the
frustule, whether obtainable with the light microscope or the electron micro-
scope, must be taken into consideration with any that can be obtained about
other characters.
A few authors have attempted to use characters from the cell contents, in
particular the form of the chromatophores, for delimiting genera within the
Naviculaceae (Pfitzer, 1871, Mereschkowsky, 1901a,6, 1902, 1903(7,6) or sub-
genera within Navicula (Karsten, 1899). However, except where these groups
could also be readily distinguished by characters of the valve, e.g., Anomoeoneis
Pfitz. and Neidium, they have not been adopted by subsecjuent authors. The
principal reason that there has been no further work along these lines is a matter
of technique. The greatest possible amount of detail in the structure of the
valves of diatoms can be seen most easily under the light microscope if all of
the organic matter is removed and the frustules mounted in a medium of high
refractive index. Diatomists have rarely used any other method of making
preparations and all collections of diatoms consist almost entirely of specimens
treated in this way. They provide information perfectly adecjuate for identifi-
cation, and hence workers on floristics and ecology have had no incentive to
change their technique. These have been the chief fields of work of virtually
all diatomists throughout this century and even when they have turned their
attention to true taxonomy they have not altered their methods. It may be
that it would not have been possible before the phase-contrast microscope was
available to devise a technicjue which made both the fine detail of the valve
structure and the cell contents visible in the same specimen. It would seem,
however, that it was not attempted. The justification for ignoring the cell
contents in taxonomic work has been the contention, also used in connection
with ultrastructure, that a classification by chromatophore number, shape, and
disposition within the cell runs counter to the currently accepted one (Peragallo,
1907). This criticism is valid insofar as it is directed against a classification in
which characters of the chromatophore are accorded overriding importance, but
it is not a reason for ignoring the cell contents completely.
It has been pointed out that the classification of the Naviculaceae is on a very
unsatisfactory basis, at least above the specific level, and there is no reason for
supposing that it is much better in other families of diatoms. Cell contents
and ultrastructure provide characters of which the distribution does not, in
places, accord with the current classification. There is no justification for
arguing from this that variations in these features occur at random and have no
taxonomic significance. To do so is to attach overriding importance to the
particular characters of the frustule on which emphasis is placed in the current
classification; not even a priori grounds have been advanced for this. Instead
of arguing in this way from the lack of correspondence between the current
classification and the distribution of types of cell contents and ultrastructure,
this discrepancy should be regarded as an indication that there is a need for a
new classification based upon the extent of overall resemblance with these
features taken into account.
408 Annals New York Academy of Sciences
Future Developments
At present the data required to construct a classiiication by this method is
not available. Progress in diatom taxonomy depends upon its being obtained.
So far as ultrastructure is concerned, there are techniques for collecting the
data (cf., p. 399). The more difficult problem is to make it available. As can
be seen when Helmcke and Krieger's (1953, 1954, Helmcke et al., 1961) work is
compared with other published electron micrographs of diatoms, the only
method of reproduction that is really adequate is the making of photographic
prints. The cost of pubUcation of sufficient of these to cover most species of
diatoms would be prohibitive. The most feasible method of building up files
of micrographs will be by the exchange of duplicate prints between workers, or
their institutions, in much the same way as herbarium specimens are now ex-
changed. It is to be hoped that diatomists who have the facilities for electron
microscopy will enter into such a scheme. The desirability of stereomicro-
graphs has already been stressed, and also the necessity for adequate resolution.
A low magnification micrograph permitting verification of identity should
accompany those showing the detail of the ultrastructure, and adequate docu-
mentation of the origin of the specimen is essential.
Collection of information about cell contents, on the other hand, depends
upon the development of a technique of preparation that will enable details of
both this and the valve structure to be seen in the same specimen. Now that
the phase-contrast microscope is available, this should be possible. I plan to
attempt it in the immediate future, but, in the words of the old proverb, two
heads are better than one, and there is more likelihood of success if others also
try to find a method. When such a technique is available, the same problem
as with ultrastructure will arise: the examination of large numbers of species
and the dissemination of the resulting information so that, as far as recent
diatoms are concerned, a volume of knowledge about cell contents comparable
to that about valve structure is available. Here again, the quantity involved
is Ukely to make publication impossible and the most satisfactory alternative
will probably be exchange of preparations.
Not until we know the ultrastructure and the cell contents of most of the
species in a group will it be possible to consider whether, and if so in what way,
the taxonomy of the group can be remodeled on sounder lines. At present all
that is pertinent is to suggest that the methods of numerical taxonomy (Sneath
and Sokal, 1962) are likely to be of great use at that stage. As Sneath (1962)
has pointed out, at least 40 or 50 independent characters of each operational
taxonomic unit {e.g., individuals being classified into species or species being
classified into higher groups) need to be taken into consideration when using
the method of numerical taxonomy to construct a natural classification. If, as
has been normal practice, we rely on intuition rather than calculation to eval-
uate overall resemblance, our judgments are likely to be sound only if we take
note of a comparable number of characters. It is this which makes it essential
that diatom taxonomists should no longer confine themselves to studying
cleaned frustules under the light microscope, but should observe the cell con-
tents and the ultrastructure and make use of the information these provide in
their classifications.
Ross: Classification of Diatoms 409
Summary
Our present knowledge of the ultrastructure of diatoms covers only a very
small proportion of the total number of species, and some of the published infor-
mation is inadecjuate or misleading. The variations in types of ultrastructure
found do not, in a number of cases, correspond with the current classification,
which is based almost entirely upon characters of the valve as seen under the
light microscope. On the other hand, the observations made with the light
microscope have not been contradicted by work with the electron microscope.
For these reasons it has been contended that ultrastructure does not provide
information that can be used in diatom taxonomy. This view is criticized.
If the characters of the ultrastructure are to be used in diatom taxonomy,
information about most species in a group is needed. As many species are
often sparsely represented in gatherings, individual specimens need to be
selected and mounted for examination in the electron microscope. A technique
is described.
In a study just completed, electron microscopy has confirmed that a small
group should be removed from Stauroneis and placed in a separate genus. A
continuation of this work now in progress points to the need for further division
of Stauroneis, and there are indications that ultrastructure may provide infor-
mation that will assist in a revision of the present unsatisfactory generic classi-
fication of the Naviculaceae. In such a revision the characters of the frustule
structure as seen under the light microscope, of the ultrastructure, and of the
cell contents should all be given equal weight. It is, therefore, necessary to
obtain information about the ultrastructure and cell contents of a large propor-
tion of the species in the family: a prerequisite for this is the development of
a technique for preparing specimens in such a way that both their cell contents
and the structure of their frustules can be studied.
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MORPHOLOGY OF REPRESENTATIVE BLUE-GREEN ALGAE
Roy E. Cameron
Jel Propulsion Laboratory, California Inslilule of Technology, Pasadena, Calif.
The blue-green algae, of the phylum Gyanophyta or Schizophyta, belong to
the class designated variously as the Cyanophyceae, Schizophyceae, or Myxo-
phyceae, and are plants of a gelatinous, stony, mealy, or leathery nature.
They are tirm or soft, extremely tough, and impregnated with salts or mucus
and easily disrupted when the gelatinous material surrounding them is of slight
viscosity. Their forms vary in size from micro- to macroscopic and in shape
they occur as spheres, cushions, strata, or cyhnders. The growth habit is
frequently centripetal, and depending on the inherent characteristics of the
plant and its environment, the adult plant can be a single cell and of less than
1 /x in diameter or a spreading plant of up to 1 m. in extent. They are cosmo-
politan in nature and are found chiefly on soil and in water but also in a variety
of other habitats wherever moisture, temperature, energy supply (sunlight),
/>H, biogenic salts, respiratory gases (CO2 and O2), and other conditions for
growth and reproduction are favorable. They share with the bacteria a
unique ability to survive, as well as reproduce, at the extreme limits of the
natural environment.
The blue-green algae are considered to be an ancient group of plants ex-
tending back to the Archeozoic (Tilden, 1935) although the geological record
is difficult to determine because they have few hard parts. The evidence of
their presence is attributed frequently to calcareous and sihcious strata and
cushions and very ancient deposits of tufa, marl, travertine, and sinter de-
veloped by activities of mainly filamentous forms (Drouet, in press). The
fossil remains that have been attributed to blue-green algae have not provided
evidence as to their evolutionary sequence (Fritsch, 1942).
Characteristics of blue-green algae show that they resemble nonalgae as well
as other algae. Because they resemble bacteria in some respects, i.e., in
having no organized nuclei or true cell walls and a similar mode of cell division,
they have been classified as coordinate with the bacteria in the Schizophyceae
(Breed el al., 1958). It is also known that both groups contain members that
produce spores, and some have demonstrated the ability to fix atmospheric
nitrogen. Most contain a ,a'-diaminopimehc acid, and their concurrence in
similar ecological habitats and in cultures attests to similarities in certain
physiological characteristics. Sufficient differences, however, are found for
separation of bacteria from blue-green algae. Blue-green algae are rarely
parasitic, pigmentation is not comparable with that of the bacteria, oxygen is
evolved as a result of photosynthesis, movement is of a gUding or oscillating
nature — there are no flagella — and the size range of cells and plants is much
greater. Heterotrophic, colorless forms of blue-green algae usually can be
attributed to bacteria or fungi which have been misinterpreted (Drouet, in
press), unless one accepts an organism such as Beggialoa as a colorless form
(Pringsheim, 1949). Morphologically, the Myxophyceae show a greater
structural complexity and diversity than bacteria, but less so than other algal
412
Cameron: Morphology of Blue-Green Algae 413
groups. It is recognized that the major taxa of algae may show Httle afi&nity
with each other (Papenfuss, 1955), but they are still grouped on the basis of
an "algal-type" of organization, the parallelism cited in the evolution of plant-
body types, the morphology and physiology of the individual cells (Smith,
1950), photosynthates, and especially in regard to the principal protoplasmic
pigments (Dougherty and Allen, 1960). Blue-green algae contain the phyco-
bilins C-phycoerythrin and C-phycocyanin not found in other algae or bacteria
although phycobilins are characteristic for red algae and have also been found
in other groups, e.g., green algae and cryptomonads (Eocha, 1960). Chlo-
rophyll a, and /3-carotene are shared in common with other algal groups, but
certain carotenes and xanthophylls are unique to blue-green algae (Goodwin,
1960). By means of a fluorescence microscope, the pigments are found to show
an orangish red, red, or reddish brown fluorescence in a darkened background.
Photosynthates include polysaccharides and glycoproteins, and cell contents
may become brown when treated with I-KI solution.
In some species cells form reproductive spores which are denoted from
other cells by their larger size, thick walls, and more resistant nature. Color-
less cells, or heterocysts, are also formed in some species. Their function and
necessity are doubtful although they have been observed to germinate (Geit-
ler, 1921), and they have been noted to anchor the trichome to the firm sheath
(Bornet and Flahault, 1886). Endospores, undifferentiated reproductive cells,
are formed by 1 family, the Chamaesiphonaceae, but for filamentous taxa, the
random death of individual cells permits segments of trichomes, or hormogonia,
to propagate the species when moisture is available. Cell division is by fission,
i.e., constriction into two parts, or by centripetal progression of a dividing
membrane through the protoplast. Reproduction is frequently by fragmenta-
tion. Sexual reproduction, although recently reported for a strain of Nostoc
muscorum (Lazaroff and Vishniac, 1961), is not considered characteristic for
the group.
Cytologically, the cells are found to have the aforementioned pigments, pro-
tein granules, pseudovacuoles of a gaseous nature, and occasionally vacuoles,
within a containing membrane. Pseudovacuoles are characteristic of plank-
tonic "water-blooms"; they appear black in transmitted light, red in reflected
fight, and are dissolved when treated with detergent. Vacuoles occur in old
or degenerated cells, particularly as the environment becomes anaerobic. The
protoplast is said to be clearly divisible into two parts (Desikachary, 1959) : the
pigmented, peripheral chromoplasm and the central colorless centroplasm.
It also may be recognized, however, that such a strict differentiation is super-
ficial. Feulgen positive granules are found particularly in the centroplasm
(Cassel and Hutchinson, 1954). Pigments are reported to be in grana-like
lamellae of the chromoplasm according to electron microscope studies (Niklo-
witz and Drews, 1956). Few studies on nucleoproteins of blue-green algae
have been undertaken although it has been reported that these are similar to
those recorded for tissues of other organisms (Biswas, 1961).
Classification
The Myxophyceae have been classified in one or more orders. The classi-
fication followed here considers the blue-green algae to be in a single order.
414 Annals New York Academy of Sciences
the Chroococcales, and 8 families which diverge in morphological characteristics
in a single evolutionary sequence (Drouet, in press). The coccoid families
include the Chroococcaceae, Chamaesiphonaceae and Clastidiaceae. Fila-
mentous famihes consist of the Stigonemataceae, Nostocaceae, Rivulariaceae,
Scytonemataceae, and Oscillatoriaceae. Consideration of the first three
families is given according to a recent comprehensive revision (Drouet and
Daily, 1956), that of the other famihes follows the starting points according
to the International Rules of Nomenclature (Gomont, 1892; Bornet and
Flahault, 1886-1 888a and b).
A representative member of each family is given (figures 1 to 8). These
members are not to be construed as "typical" because there can be wide varia-
tion inter- and intraspecifically in nature as well as in culture. However,
Anacystis montana (figure 1) is the most frequently collected of the coccoid
species (Drouet, 1954). Nostoc musconim (figure 5) is of common occurrence
on soil, and Calothrix parielina (figure 6) is of wide distribution in moist
habitats (Fan, 1956). Scytonema Iwfmannii (figure 7) is also a frequently
encountered species, and Microcoleus vaginatus (figure 8) is an oscillatorioid
member often found on soil as well as in aquatic habitats. These species have
been recently described with others found in the United States north of the
Rio Grande River (Drouet, 1959).
The Chroococcaceae consist of uni- or multicellular, micro- or macroscopic
plants which are subaerial or aerial, free, as cushions or strata. The cells are
spherical, discoid, ovoid, ellipsoid, cylindrical, or pyriform, in regular or ir-
regular order, each cell dividing into 2 ecjual daughter cells which become sepa-
rated from each other by the gelatinous matrix. Reproduction is by fragmen-
tation as for most of the blue-green algae, but in some cases by cell division.
Under most conditions, except for Coccochloris, cells are found in the process
of division. Species of Anacystis, represented here by A. montana (figure 1)
have cells at first hemispherical, later becoming spherical. The cells then di-
vide in 3 planes perpendicular to each other. Coccochloris resembles Anacystis,
but has subspherical to long cylindrical cells and division at right angles to the
long axis. Other genera include Johannesbaptistia which has a linear series of
discoid cells within an elongate gelatinous matrix, and AgmeneUum, Micro-
crocis, and Gomphosphaeria which have cells that divide successively in 2
planes perpendicular to each other. Plants of the first two genera are plate-
like, whereas those of the latter genus are unique in that the cells are frequently
cordiform in division and the remains of individual sheaths form branched
stalks radiating from the center of the plant.
The Chamaesiphonaceae contain one genus, represented here by Entophy-
salis lemaniae (figure 2). Plants of this family are uni- or multicellular,
aquatic, micro-, or macroscopic. The cells are at first solitary and affixed to
the substratum, each dividing serially into first unequal then equal daughter
cells which are not separated by gelatinous material. Subsequently, a stratum
or cushion is developed above the substratum, and branched filaments grow
downward from this into the substratum. Any cell is then capable of enlarg-
ing and dividing internally into a few or many endospores. Reproduction is
by fragmentation as well as by endospores.
Plants of the Clastidiaceae are infrequently collected. The plants consist
1
rdd^ ^
4
A
W
rVii'V
Figure 1. Anacystis montaiia (Lighlfoot) Drouet & Daily.
Figure 2. Entophysalis lemaniae (Agardh) Drouet & Daily.
Figure 3. Slichosiplwn sansibaricus (Hieronymusj Drouet & Dail\-.
Figure 4. Sligonema pani/onne (Agardh) Bornet & Flahault.
Figure 5. Nostoc niuscontm Agardh.
Figure 6. Calothrix parietina (Nageli) Thuret.
Figure 7. Scylonema kofmannii Agardh.
Figure 8. Microcoletis vaginatus (Vaucher) Gomont.
415
416 Annals New York Academy of Sciences
of elongate, epiphytic unicells contained in thin gelatinous sheaths and at-
tached to the substratum by basal developments of the sheath. The entire
protoplast is found to divide into a uniseriate chain of rounded or compressed
spherical cells which usually remain united by their membranes. As the cells
enlarge, the trichome then bursts through the sheath of the mother cell, and
the cells upon dissociation from each other then elongate into a new unicell
and secrete new sheaths. The family is represented by two small genera,
Clastidium and Stichosiphon, each containing one species. 5. sansibarkus
(figure 3), has a smooth apex, whereas plants of C. setigerum terminate in a
spinelike projection of the sheath at the apex.
Plants of the Stigonemataceae are floccose, feltlike, cushion-shaped, or
spherical. The filaments are free or imbedded in a gelatinous matrix, the
trichomes are branched, and the cells are uni- or multiseriate with division
occurring in planes perpendicular to or parallel with the axis of the filament.
Heterocyst formation is random, intercalary or terminal on short branches.
Cell division in planes perpendicular to the axis of the trichome is followed by
a growth in length of cells at filament apices which forms the resulting elongate
and branched filaments. Cell division also occurs in planes parallel to the
axis of older filaments with consequent increase in diameter and in the forma-
tion of subsequent branches. Reproduction is by fragmentation. The family
is represented here by Stigonema panniforme (figure 4). Members of this
genus have filaments which soon develop multiseriate cells connected by proto-
plasmic strands. Other prominent genera include Capsosira which has up-
right and parallel filaments that form compact cushion-shaped plants, Nosto-
chopsis which has radial filaments within a gelatinous matrix of coalesced
sheaths and develops intercalary, pedicellate, or sessile heterocysts, and Hapa-
losiphon and Fischerella which contain trichomes of uniseriate cells except in
the older basal portions of the plant. The latter two genera also exhibit
scytonematoid branching.
The Nostocaceae contain aquatic or terrestrial plants which are free or at-
tached to a substratum. The sheaths are mucous, gelatinous, membranaceous,
or well hydrolyzed and absent. Trichomes are unbranched, frequently twisted
and entangled; all of the cells divide at relatively the same time, and inter-
calary or terminal heterocysts are present. Reproduction is by fragmentation
or by spores that are formed in most species. The trichomes of Anabaena are
free or form a fragile layer; the matrix is composed of hyaline, hydrolyzed
sheaths. Spores are variously situated in relation to the heterocysts. Tri-
chomes of planktonic Raphidiopsis and Aphanizomenon resemble those of
Anabaena except that the end cells are pointed in Raphidiopsis and colorless in
Aphanizomenon. Trichomes of Nostoc, and Wollea develop within a gelatinous
matrix of definite shape; all cells may apparently become spores or hetero-
cysts. In species of Nostoc, e.g., N. muscorum (figure 5), the trichomes
become much contorted, whereas i^ Wollea they are relatively straight. Cylin-
drospermum has comparatively short trichomes with terminal, solitary hetero-
cysts and adjacent spores. Cells and spores of Nodularia are compressed or
disciform in rather straight trichomes. Hydrocoryne, a rarely collected species,
apparently forms no spores and has discrete although readily hydrolyzed cylin-
drical sheaths.
Cameron: Morphology of Blue-Green Algae 417
In the Rivulariaceae plants are aquatic or in moist habitats, spherical, cush-
ion-shaped, crustaceous, velvety, feltlike, or brushlike. The filaments are
branched or unbranched, radiate from the center of the plant outward, or are
parallel and tufthke. Trichomes are unbranched, thick at the base, tapering
above, each ending in a colorless hair. Heterocysts are basal or intercalary,
although absent in some species. Cell division is transverse and primarily in
the middle of the trichome above the heterocyst. Reproduction is by frag-
mentation and spores. Amphithrix is a thin crustaceous plant, which lacks
heterocysts and has terminal ephemeral hairs. Filaments of Calotlirix, as
represented by the most frec^uently collected species, C. parietina (figure 6)
(Fan, 1956) is usually unbranched, whereas the filaments of Dichothrix are
more or less dichotomously branched, the bases of the branches included for a
short distance within the parent sheath. Rkularia and Gloeotrichia have
filaments of coalesced sheaths that develop radially to form spherical or cush-
ion-shaped plants. No spores are formed in Rivularia but in Gloeotrichia they
are thick walled and next to the basal heterocysts.
The Scytonemataceae contain irregularly cushion-shaped or matlike plants
with branched filaments that are single or geminate. The sheaths are firm,
tubular, at first colorless, but later yellow, or brown. Trichomes each consist
of a single row of cells, one or more included in a sheath. Heterocysts and
spores are variously disposed. Cell division primarily occurs behind the tip
of the trichome, resulting in lateral perforation of the sheath by dividing and
elongating cells which then give rise to single or geminate branches. Repro-
duction is usually by fragmentation of the trichome or filament, although one
genus, Aulosira, is unique in that all vegetative cells are capable of forming
thick walled cylindrical spores or heterocysts. Branching varies with the
genera, depending upon its relation to the heterocyst. In species of Scylonema,
e.g., S. hofmannii (figure 7), branches may be single and near a heterocyst,
but commonly arise at a point somewhat remote from the heterocyst and are
geminate. Branches in Tolypothrix are single and arise at the heterocysts.
Branches of Desmonema are included within a common sheath. Filaments of
Fremyella are short, uncommonly branched, and have basal heterocysts.
The Oscillatoriaceae is the largest family of the group. It is comprised of
plants developing as layers or cushions and is differentiated from other families
in that the trichomes do not form spores, heterocysts, or hairs. The cylindri-
cal trichomes consist of 1 row of cells in branched or unbranched filaments;
the broken ends or hormogonia regenerate in a mode characteristic for the
various taxa. In many species, a terminal cell develops a thickened outer
membrane. Cell division occurs throughout the entire trichome and at rela-
tively the same time. Reproduction is by fragmentation. The current divi-
sion of the genera is based largely upon the structure of the sheath (Gomont,
1892) and is in need of further study for clarification. The sheaths of Oscilla-
tor ia, Arthrospira, and Spirulina are seldom discernible even by application of
various staining technicjues. The sheaths of Microcoleiis, e.g., M. vaginatus
(figure 8), and Sckizothrix contain one to many trichomes within diffluent
or firm sheaths. Usually only one trichome is found in firm sheaths of Plec-
lonema, Lyngbya, and Porphyrosiphon. Sheaths of the latter become red or
purple; sheaths of Lyngbya may be hyaline or become yellowish-brown.
418 Annals New York Academy of Sciences
Plectonema may show scytonematoid branching. In Symploca, the sheaths
are discrete and contain one trichome; adhering filaments form fascicles at
the surface of the plant. The sheaths of Phormidium are thin, hyaline, and
become diffluent.
General Ecology
Ecological studies on the Myxophyceae are quite limited. Most attention
has been given to the collection of organisms from a variety of habitats and
some information is available on their geographical distribution. In general,
the blue-green algae occur in all parts of the world where light and water are
available. Individual species may be distributed in the various climatic
zones, but others are found at extreme limits of the environment, from cold
regions such as the Antarctic or in the cryoconite of Greenland (Gerdel and
Drouet, 1960), and from the low elevation of the Dead Sea to mountains over
14,000 feet in altitude. They are a part of the salt marsh flora (Chapman,
1960), occur in extremely saUne Great Salt Lake (Flowers), hard and soft
waters (Palmer, 1959) and hot, dry desert soils (Cameron, 1961; KiUian and
Feher, 1939). Planktonic forms, frequently a single species, may grow pro-
lifically in favorable seasons when nitrates and phosphates are high and in some
cases release obnoxious toxins (Prescott, 1959). Aquatic species have also
been found in the lower subUttoral zone where Ught intensity is low (Ruttner,
1953), and in hot springs where the temperature may reach 86° C. (Kaplan,
1956). Other aquatic habitats can include industrial wastes with a high con-
tent of metals and acids (Palmer, 1959). More exotic habitats include associa-
tions with animals such as sponges, corals, and snails. In barren, eroded soil,
on wood, in sewage, on and under light transmitting rocks, and even in areas
of comparatively recent volcanic activity (Treub, 1888), it has been found that
blue-green algae are able to grow and survive. Furthermore, it has been de-
termined that the Eh range of blue-green algae is from —0.200 to +0.700 volts
and the ^H from 1.5 to 11 (Baas Becking et al., 1960). That they can resist
desiccation for decades has been shown in the revival of species from old, stored
soils (Bristol, 1919). Reproduction can be quite rapid, and oscillatorioid
forms can develop macroscopic growth in a few hours on desert soil which has
remained dry for a number of years. Prolonged resistance to desiccation has
been found in a dried herbarium specimen of nonsporeforming Nostoc commune
previously revived after 88 years of storage (Lipman, 1944), and later revived
after an additional time period of 19 years (Cameron, in press). Resistance is
also found to low temperatures. At —80° C, algae, in combination with
fungi as Uchens have been found to survive, and at —30° C. to even photo-
synthesize slowly (James, 1955). Parasitism of certain species of blue-green
algae by fungi is not uncommon (Drouet, 1954), and where optimal conditions
prevail for one of the organisms, the other is overwhelmed. The association
between the alga and the fungus in forming and maintaining the hchen is ex-
ceedingly complex and although the alga excretes antibiotic substances, the
fungus can have a lethal effect on the alga (Henriksson, 1961).
Environmental conditions which are most favorable for the entire group of
blue-green algae are difficult to determine and correlate. Many species have
been named as distinct on the basis of the kind of environment in which they
Cameron : Morphology of Blue-Green Algae 419
occur. Distinctions have also been made between plants which differ morpho-
logically in some details but are actually only growth forms of the same species
found in a slightly different environment. Microcoleus, for example, has been
considered as a multitrichomatous organism occurring only on soils, and blue-
green algae are said to be more abundant in cultivated than in noncultivated
areas (Tiffany, 1951). Such restrictions have not been found valid upon fur-
ther study. An exhaustive review of specimens and their subsequent enumera-
tion on the basis of pertinent characteristics, as for the coccoid Myxophyceae
(Drouet and Daily, 1956) is needed for the other blue-green algae. Culture
studies, although valuable, are often confusing in that the cultured plant can
lose its identity with more famiUar forms occurring in the natural environ-
ment. Changes in any one of the environmental conditions can result in
plants differing from the original organism in form and structure, as well as
regeneration rate, cell division, size, shape, and contents. Pleomorphism
among the blue-green algae will remain as a confusing factor until an extensive
review has been made of all available material in herbaria and in other collec-
tions, and investigations performed on the growth of organisms in both natu-
ral and induced environments.
A cknowledgment
Appreciation is expressed to Francis Drouet, who has made suggestions,
loaned herbarium specimens and reference materials, and given generously of
his time and assistance.
Rejerences
Baas Becking, L. G. M., I. R. Kaplan & D. Moore. 1960. Limits of the natural en-
vironment in terms of pH and oxidation-reduction potentials. J. Geol. 68: 243.
Biswas, B. B. 1961. Studies on the nucleoproteins of Nostoc muscorum. Trans. Bose
Research Inst. Calcutta. 24: 25.
BoRNET, E. & C. Flahault. 1886. Revision des Nostocacees heterocystees contennes dans
les principaux herbiers de France. Ann. sci. nat. Botan. et Biol, vegetale. 3: 323.
BoRNET, E. & C. Flahault. 1887. Revision des Nostocacees heterocystees contennes
dans les principaux herbiers de France. Ann. sci. nat. Botan. et Biol, vegetale. 4: 343.
BoRNET, E. & C. Flahault. 1888ti. Revision des Nostocacees heterocystees contennes
dans les principaux herbiers de France. Ann. sci. nat. Botan. et Biol, vegetale. 5: 51.
BoRNET, E. & C. Flahault. 1888&. Revision des Nostocacees heterocyste'es contennes
dans les principaux herbiers de France. Ann. sci. nat. Botan. et Biol, vegetale. 7: 177.
Breed, R. S., E. G. D. Murray & N. R. Smith, Eds. 1957. Bergey's Manual of Deter-
minative Bacteriology. Ed. 7. The Williams & VVilkins Co. Baltimore.
Bristol, B. M. 1919. On the retention of vitality by algae from old stored soils. . New
Phytol. 18: 92.
Cameron, R. E. 1961. Algae of the Sonoran Desert in .\rizona. Ph.D. Thesis. Library,
Univ. of Arizona. Tucson.
Cameron, R. E. Species of Nostoc Vaucher occurring in the Sonoran Desert in Arizona.
Trans. Am. Microscop. Soc. In press.
Cassel, W. a. & W. G. Hutchinson. 1954. Nuclear studies on the smaller Myxophyceae.
Exptl. Cell Research. 6: 134.
Chapman, V. J. 1960. Salt Marshes and Salt Deserts of the World. Interscience Pub-
lishers. New York.
Desikachary, T. V. 1958. Cyanophyta. Indian Council of .Agricultural Research. New
Delhi, India.
Dougherty, E. C & M. B. .\llen. 1960. Is pigmentation a clue to protistan phylogeny?
In Comparative Biochemistry of Photoreactive Systems. : 129. M. B. Allen, Ed.
Academic Press. New York.
Drouet, F. 1954. Parasitization by fungi in the coccoid Myxophyceae. Vlllth Int. Bot.
Cong. Paris, Rapp. et Comm. 17: 48.
420 Annals New York Academy of Sciences
Drouet, F. 1959. Myxophyceae. In Fresh-water Biology, Ed. 2. : 95. W. T. Ed-
mondson, Ed. John Wiley & Sons. New York.
Drouet, F. Cyanophyta. Encyclopedia of Science & Technology. McGraw-Hill Book
Co. New York. In press.
Drouet, F. & W. A. D.ailv. 1956. Revision of the coccoid Myxophyceae. Butler Univ.
Botan. Studies. 12: 1.
EocHA, C. 1960. Chemical studies of phycoerythrins and phycocyanins. In Compar-
ative Biochemistry of Photoreactive Systems. : 181. M. B. Allen, Ed. Academic
Press. New York.
Fan, K. C. 1956. Revision of Calotlinx Ag. Rev. Alg. N.S. 2: 154.
Flowers, S. Undated. The blue-green algae of Utah. Mimeograph. Univ. of Utah
Press. Salt Lake City.
Fritsch, F. E. 1942. The interrelations and classification of the Myxophyceae (Cyano-
phyceae). New Phytol. 41: 134.
Gerdel, R. W. & F. Drouet. 1960. The cryoconite of the Thule area, Greenland. Trans.
Am. Microscop. Soc. 79: 256.
Geitler, L. 1921. Versuch einer Losung des Heterocysten-problems. Sitzber. Akad.
VViss. Wien, Mat.-Naturw. Kl. Abt. 1. 130: 223.
Goodwin, T. W. 1960. Algal carotenoids. In Comparative Biochemistry of Photoreac-
tive Systems. : 1. M. B. Allen, Ed. Academic Press. New York.
GoMONT, M. 1892(7. Recherches des Oscillariees (Nostocacees Homocystees). Ann. sci.
nat. Botan. et vegetale. 15: 263.
GoMONT, M. 1892/). Recherches des Oscillariees (Nostocacees Homocystees). Ann. sci.
nat. Botan. et vegetale. 16: 91.
Henriksson, E. 1961. Studies in the phvsiology of the lichen Collema. IV. Physiol.
Plant. 14: 813.
James, P. E. 1955. The limits of life. J. Brit. Inler])lanet. Soc. 14: 265.
Kaplan, I. R. 1956. Evidence of microbiological activity in some of the geothermal
regions of New Zealand. New Zealand J. Tech. 37: 639.
KiLLiAN, C. & D. Feher. 1939. Recherches sur la microbiologic des sols desertiques.
Encvclopt'die periodifjue sci. mc'd-biol. 21: 1.
Lazaroff, N. & VV. ViSHNiAC. 1961. The ])articipation of filament fusion in the develop-
mental cycle of Nostoc musconini. Bacteriol. Proc. 61: i^.
LiPMAN, C. B. 1944. Longevity in microorganisms. /;; Science in the University. : 211.
Univ. of California Press. Berkeley, Calif.
NiKLOwiTz, VV. & G. Drews. 1956. Beitriige zur Cytologic der Blaualgen. Arch. Mikro-
biol. 24: 134.
Palmer, C. M. 1959. Algae in water supplies. Public Health Service Publication No. 657.
U. S. Govt. Print. Off.
Papenfuss, G. F. 1955. Classification of the algae. In A Century of Progress of the
Natural Sciences, 1853-1953. : 115. Cahf. Acad. Sci. San Francisco, Calif.
Prescott, G. W. 1959. Biological disturbances resulting from algal populations in stand-
ing waters. In The Ecology of Algae. : 22-37. Special Publication No. 2. Pyma-
tuning Laboratory of Field Biology. Univ. of Pittsburgh Press. Pittsburgh, Pa.
Pringsheim, E. G. 1949. The relationship between bacteria and Myxophyceae. Bac-
teriol. Rev. 13: 47.
Ruttner, F. (Frey, D. G. & F. E. J. Fry, Trans.). 1953. Fundamentals of Lmnnology.
Ed. 2. Univ. of Toronto Press. Toronto.
Smith, G. M. 1950. The Fresh-water Algae of the United States. Ed. 2. McGraw-Hill
Book Co. New York.
Tiffany, L. H. 1951. Ecology of fresh-water algae. In Manual of Phycology. : 293.
G. M. Smith, Ed. Chronica Botanica Co. Waltham, Mass.
Tilden, J. E. 1935. The Algae and Their Life Relations. Univ. of Minnesota Press.
Minneapolis, Minn.
Treub, M. 1888. Notice sur la nouvelle flora de Krakatau. Ann. Jard. Botan. Buiten-
zorg. 7:221.
LORICAE AND CYSTS IN THE CHRYSOPHYCEAE
Pierre Bourrelly
Museum National d'Hisioire Natiirelle, Paris, France
The unicellular algae, solitary or colonial, often have their cytoplasm enclosed
within shells of various shapes and kinds called loricae or thecae. These
thecae are found in numerous phyla of algae: Euglenales (Trachelomonas,
Strombomonas), Volvocales (Phacotus, Coccomonas), Dinophyceae (Peri-
dinium, Dinophysis, Exuviella), and in numerous Chrysophyceae and Craspedo-
monadinae.*
The Chrysophyceae may have, in addition, a phase of dormancy or of resist-
ance in the form of siliceous cysts or statospores. These cysts always have an
endogenous origin, and may arise from a simple encystment of a vegetative
cell, or, on the contrary, of a zygote resulting from a autogamy or from an
isogamic fusion.
The cysts of the Chrysophyceae are exclusively siliceous, and are of highly
varied forms, but they exhibit a pore closed by a silicopectic plug. The forma-
tion of a siliceous cyst with a pore and plug is the basic characteristic which
enables us to identify the whole Chrysophyceae group without any possibihty
of error.
Certain loricae of the Chrysophyceae {Chrysococcus, for example), are si-
liceous, and have very small pore openings. In the absence of a flagellum and
the plug which closes the pore, one might easily confuse the cyst and the lorica.
In fact, in the Chrysophyceae, the thecae are pierced with a pore opening from
which the flagellum (or the flagella) or the pseudopodia emerge.
Loricae
If we take as an example of loricated Chrysophyceae, the genus Dinobryou
(figure 8) and the kindred genus Hyalobryon, we note that the morphology
and the structure of the loricae vary with the species. In the two genera cited,
the shell is in the form of a conical or cylindroconical horn, more or less flared out
at the apex opening; the cellular body is bound to the lorica by a retractile cysto-
plasmic filament, the epipode. The shell is hyaline, of a cellulose-pectic nature,
with a marked dominance of the cellulose. The outline of this lorica is either
straight or undulating, according to the species The action of coloring agents
(Congo Red) causes the appearance of a very fine heHcoidal striation of the
wall, accompanied at times by a spiral torsion indicated already by the undulat-
ing edge of the theca {Dinobryon divergens (figure 1)). In all of the colonial
Dinobryon which were studied, the basic helicoidal striation has the same direc-
tion of rotation (counter-clockwise), whereas the marginal undulations display
a coiling in the opposite direction.
Dinobryon suecicum (figure 1), a solitary species, free, with a smooth cellu-
lose-pectic lorica, hyaline, with an helicoidal, projecting execresence, brown in
color and of an unknown nature (calcareous substance impregnated with iron
salt?) running throughout the greater part of its length.
An analogous feature is found in some Pseudokephyrion. The solitary fixed
* We will leave out the Silicoflagellates and the family of the Coccolithophoraceae, as
these might constitute the subject of a special stud>-.
421
422
Annals New York Academy of Sciences
Dinobryons: Dinohryon utricidiis (figure 1), have a lorica which is very rich
in pectin, and made up of small elliptical scales, imbricated in helicoidal series.
This structure presages the one which appears in the Synura and the Mallo-
monas.
Figure 1. Loricae after Bourrelly, 1957. 1: Dinohryon cylindncum var. pahistre; 2:
Dinohryon sp.; 3: Lagynion Janei; 4: Dinohryon siiecicuin; 5: Ilyalohryon ramosiim; 6: H.
Borgei; 7: Dinohryon utriculiis; 8: D. sertularia; 9: D. divergens (1, 8, 9: after staining).
O CD
Figure 2. Loricae of Clirysococcus (after Bourrelly). 1: Chrysococcus rufescens; 2:
C. tesselatus; 3: C. ovoides; 4: C. elegans; 5: C. umhonatus; 6: C. porifer; 7: C. minutus; 8: C.
rufescens var. compressa; 9: C. cordiformis; 10: C. rufescens fo. tripora; 11: C. dokidophonts;
12: C. radians; 13: C. 6we/!w; 14: C. spinosus; 15: C. klehsianus; 16: C. heverlensis; 17: C.
ornatus; 18: ('. areolatus; 19: C scidptus.
Finally, a genus very close to Diuobryon: Hyalobryon (figure 1) is charac-
terized by its very long lorica, cellulose-pectic, formed by pieces of encased
cylindrical tubes, of unequal length, the widest one being the one at the base,
and the narrowest one being at the top, presenting a flagellate opening.
Bourrelly: Loricae & Cysts in Chrysophyceae
423
With the genera Chrysococcus and Pseudokephyrion, we have loricae which are
often very much embelhshed and are of a yellow-brown color. These loricae
have a very fine pectic membrane entirely impregnated with calcareous sub-
FiGURE 3. lyoricae of Pseudokephyrion (after Bourrelly). 1-2: conictim; 3; Eulzii; 4, 5,
6: Entzii fo. granulata; 7: lieveiiensis; 8: pocuhim; 9: miniitissimiDu; 10: Rutlneri; 11-12:
cylindricum; 13: depressum; 14: cinctum; 15: obtusum; 16-19*: latum; 20: Skujae; 21: pilidum;
22: Scliilleri; 23: urnula;2i: ehgans; 25: ampullaceum; 26: undulatum; 27: acuiuin; 28: />«/-
cherrimum; 29: lintirniahidum; 30: circumcisum; 31: uiidiilatissimum; 32: spirale; 33: pseudo-
spirale; 34: gallicum; 35: Klarnelii; 36: form os is si mum; 37: ellipsoideum; 3^: ovum; 39: or-
natum; 40-41: circumvallalum.
stance. Acetic acid dissolves the brown and brittle lorica quite well, and there
remains a thin membrane which takes Ruthenium red color admirably.
Along with the numerous Chrysococci (figure 2) with calcareous theca, two
species embellished with spines or needles, have a sihceous wall. We note
that the metabolism of the calcareous type and that of the sihceous type may
co-exist in the same species. Also, some Pseudokephyrion with a calcareous
shell produce siliceous cysts hke the other Chrysophyceae.
424
Annals New York Academy of Sciences
With these calcareous or siHceous impregnations, the lorica becomes thick,
and then presents a stable ornamentation in the same species, but quite variable
from one species to another. Spines, bristles, warts, webs, dots, rings, and
checks decorate the surface of the lorica.
The ma.ximal diversity in ornamentation is obtained in the following two
genera: Pseudokep/iyrion (figure 3) and Kephyrion. Here we find forms with
Figure 4. l.oricae of Kephyrion (after Bourrelly). 1: silla; 2: doUolum; 3-4: nuislign-
phoruni; 5: ciipidijonne; 6: littorale; 7: liltorale var. conslricla; 8: nibri-daustri; 9: ruhri-daus-
tri var. amphora; 10: impletum; 11: cylindricum; 12: hetnispliaericum; 13: petasatum; 14
campantdiforme; 15: amphonda; 16: ovale; 17: cinctum; 18: Valkanovii; 19: globpsum; 20
Starmachii; 21: wo.sqiiensis; 22: spirale; 23: hacillijorme; 24: densalum; 25-29'': as per; 30
Schmidii; 31: imonstans; 32: lalicollis; ii: parvidiim; 34: moniliferum; 35: circnmvallalinn; 36
prismaikum; 37: velatum.
marked calcareous impregnation, and forms with little or no calcification. The
small, more or less calcified cells, such as in Pseudokephyrion undulatum or
Psendokephyrlon latum (figure 3) recall the loricae of the Dinobryons in the
undulating appearance of the edges, but they do not show the hehcoidal torsion.
But the forms with heavily colored, thick lime incrusted walls, have by con-
trast, a more varied ornamentation. One may recognize with them: (1) granu-
lations or striations arranged in regular transversal circles: {Pseudokephyrion
Entzii fo. granulala, Ps. Skujae (figure 3)) or irregular ones (Ps. circum-
Bourrelly: Loricae & Cysts in Chrysophyceae
425
vallalum), (2) helicoidal protruding excrescence (Ps. Klarnelii, Ps. pseudo-
spirale (figure 3)), (3) regular cross checks {Ps. ovum, Ps. ornatum (figure
3)), (4) longitudinally projecting sides {Ps. formosissimuni (figure 3)).
The same remarks may be applied to the genus Kephyriou (figure 4) in
which we note the same diversity in the form of the small cells, but a smaller
variety in the ornamentation of the walls.
In the genus Lagynion (figure 5), the cells do not have flagella, but have
more or less ramified pseudopoda issuing from the oral pole. This genus with
Figure 5. Loricae of Lagynion (after Bourrelly). \: fiihiim; 2: oblongum; 3: arachne;
4: rliizopodicHm; 5: notostomum; 6-7 : rednctiim; %-\\: Seller ffelii; \2:ampullaceiim; 13-15: sub-
ovaium; 16 and 18: maerotraehehim; 17: triangularis; 19 and 20: triangularis var. pyramida-
tiim; 21-23: reflexiini; 24: sphagnieolum; 25: vasieola; 26: Janei; 27-28: cystodinii; 29: globosum
var. undidatum.
calcified pectic lorica does not show any characteristic ornamentation, the
lorica is always brown or yellow, thickened, finely granulated. The forms are
highly varied and the evolutive process comes to bear on the neck terminating
the lorica. In some species {Lagynion Janei (figure 5), for example), the
wall of the theca is double, the inside is thin and hyaline, the outside brown,
thick and calcified. This structure is found in the Diploeca series among the
Craspedomonadines, a large group of collared flagellates related to the Chryso-
phyceae.
In the family of the Stylococcaceae, we note a large variation in the form of
the loricae: along with sessile loricae, there are genera with pediculate shells
{R/iizasler, Slylococcus). We also find genera in which the thecae show numer-
426
Annals New York Academy of Sciences
ous pores, from which issue the pseudopods (Chrysocrinus, Slepkanoporos) for
the sessile forms; Porostylon for the pedicular small cells.
The loricae held by a pedicel are found with other Chrysophyceae belonging
to families very remote from the family of the Stylococcaeae: we cite only the
Figure 6. Loricae of Dcre pyxis (after Bourrelly). 1: Derepyxis amphora; 2-3: ollula;
4: bidbosa; 5: anomala; 6: maxima; 7: tubulosa; 8: dilalata; 9: amphoroides; 10: dispar; 11:
crater; 12: hacchanalis.
Figure 7. Scales of Mallomonas and Synitra (after Bourrelly). 1-2: Mallomonas fasli-
gata var. Kriegeri; 3-6: Mallomonas Leboimei; 7-11: Mallomonas reginae; 12: Mallomonas
tonsitrata; 13-15: Synura Bioretii.
Bourrelly: Loricae & Cysts in Chrysophyceae
427
Derepyxis (figure 6), monads with two flagella and the Lepochromiilina (figure
8), with single flagellum. We mention also, the extraordinary Chrysopyxis of
which the cellulose lorica, in the form of a saddle, attaches itself to the fila-
mentary algae by a thin cellulose cord which completely entwines the support-
ing algae. ^
Alongside of the Chrysophyceae with loricae of homogeneous structure, we
may place the species of the family of the Synuraceae in which the lorica is
replaced by a covering of siliceous scales (figure 7).
Figure 8. Loricae and cysts (after Bourrelly). 1: Dinobryon divergens, lorica and cyto-
plasm; 2: Lepochromulina calyx, lorica and cytoplasm; 3-5: IleterocltromuUna vhipara var.
minor, building of cyst; 6: Dinobryon niriculus, lorica and division; 7: c\st of Chrysostomacea
Outesia; 8: cyst of Ouiesia; 10: cyst of Clericia; 11: cyst of Deflandreia (?). bb: mouth-band;
Cm: muciferous bodies; cv-vc: contractile vacuole; cy: cytoplasm; ep: epipode (contractile
thread); /-/2: flagella; gg: oil-drop; k: membrane of cyst; /: leucosin; Ic: cellulosic lorica; n:
nucleus; p: parabasal body; pi: chromatophore; s-s s2: stigma; sy: symbionts.
The scales arranged in helicoidal series, such as these of the Dinobryon
utriculus (figure 1*), have been the subject of fine studies in electronic mi-
croscopy.
The systematization of the genus Mallomonas (about 100 species) of the genus
Synura (12 species) is almost solely based upon the form of the scales and of the
bristles which adorn them. The observation of a single siliceous scale is some-
times enough to permit the identification of the species. This is not the case
with the true lorica, in which we have a convergence of form to such an extent
that it is impossible in certain instances to decide from the study of an empty
* The scales of Dinobryon utriculus are not siliceous, but pecto-cellulosic.
428
Annals New York Academy of Sciences
lorica whether it is a Chrysophycean, Craspedomonadina, or even one of the
colorless flagellates of the Bicoeca group.
Cysts
The same problem will arise for the Chrysophyceae cysts. We will have, at
all times with the present forms, siliceous cysts with their pore and plug. But
Figure 9. Cysts of Uroglena (after Bourrelly). 1: Uroglena americana; 2: U. Conradi
var. gallica; 3: U. botrys; 4: V. Nygaardii; 5: U. volvox var. uplandica; 6: U. volvox; 7: U.
soniaca; 8: U. Lindii;9: IL marina; 10: IL europaea; 11: U. notabilis.
Figure 10. Cysts of Chrysaslrella furcala (Chr\sostomataceae): polymorphism (after
Bourrelly).
within the same genus, the cysts have a highly varied ornamentation, and the
identity of forms in the cysts does not seem at all related to the organism. The
endogenous cyst is built within the cell, around the nucleus (figure 8). The
cytoplasmic parts left out of the cyst contribute to the external ornamentation
of the cyst wall. The cyst is siliceous, but as was the case with the Diatomae,
a pectic substance remains bound to the silica. The plug which closes the
pore of the cyst is itself siliceous, but with a substantial pectic tendency.
Bourrelly : Loricae & Cysts in Chrysophyceae 429
The genus Uroglena (figure 9) which shows a great structural and cytologic
homogeneity, is an excellent example of the diversity form of the cysts, in fact,
here knowledge of the cyst is indispensable for the determination of the species.
The cysts of many unicellular Chrysophyceae are still unknown. On the
other hand, many cysts are known in which the free vegetative phase is un-
known. This has led the protistologists and the micropaleontologists to give
genus and species names to the cysts of which the vegetative phase is unknown.
It is a convenient method, but these are not true species, only provisional
names without classification value.
The fresh water cysts, both fossils and recent have been placed in the
pseudofamily of the Chrysostoniataceae, whereas the fossil marine cysts make up
the Archaemonadaceae.
The Chrysostomalaceae (figures 8 and 10) are abundant in the present
and fossil peat bogs, and in the Diatomae lacustrine deposits. More than
200 forms have been observed from the Tertiary period to the present time.
The fossil marine forms of the Archaemonadaceae are found in association
with Diatomae from the Cretaceous and Tertiary periods (less than 100 fos-
sil forms are known).
The fossihzation of the cysts is often perfect (the pore plug usually being
missing, however) whereas that of the loricae of Chrysophyceae seems much
more diiBcult, and observations of fossil loricae have been very rare (2 or 3
observations only).
In closing, it must be noted that although the present Chrysophyceae are
well known in fresh waters, the forms of marine nanoplankton are very scant
because their study has been much neglected. There is a vast domain in which
investigation has only begun, and the rare current projects in this field have
already yielded a harvest of interesting and novel facts.
Reference
Bourrelly, P. 1957. Bull. Micr. Appl. n. s. 7(5): 118-124.
MORPHOLOGICAL TRENDS AMONG FOSSIL ALGAE
J. Harlan Johnson
Colorado School of Mines, Golden, Colorado
The algae may be considered as a vast subkingdom of primitive plants that
exhibit an enormous range in structure, reproduction, and life history. Struc-
turally at the base are unicellular forms, often motile, that are indistinguishable
from similar unicellular animals except for the presence in the cell of color spots
or chromatophores, which contain photosynthetic pigments. At the other ex-
treme are tree-sized multicellular plants in which there is some differentiation
of tissue for dilTerent functions.
For convenience in study and classification the algae are divided into a num-
ber of major groups. These groups have been considered as classes by the older
authors but the tendency today is to think of them as phyla. They are named
on the basis of the pigmentation, for example, the Chlorophyta or green algae;
the Rhodophyta or red algae. Nine such groups are recognized in most classi-
fications, 11 in others.
Before considering the morphological trends among fossil algae it will be de-
sirable to review two things regarding recent algae. (1) The structural trends,
and (2) structural parallelism among the major groups of algae.
Algal Morphology
General. The algae show a great range in form, size, and structural develop-
ment. At the bottom are the microscopical unicellular forms. These occur
in all but two of the major groups and in a number of them no higher structural
types have ever developed. A majority of the unicellular forms are motile
flagellate types or at least in their life cycle pass through a flagellate stage.
Structural evolution seems to have followed the steps shown in table 1, with
the first three, either 4 or 6, and 5, forming an evolutionary series.
Parallelism. One of the most striking facts facing a student of algal morphol-
ogy is the evidence of parallel evolution and development among the members
of the various groups (tables 2 and 3).
Marked structural complexity of the plant occurs only in two groups, the
Rhodophyceae and the Phaeophyceae, with some of the green algae (Chloro-
phyceae) reaching a high medium of complexity. It should be emphasized
however, that even in these three groups a majority of the known species have
simple types of structure. The highest structural features have developed
among the brown algae, with some of the reds not far behind. The green algae
probably show the greatest diversity of structural types with, however, the
highest types missing.*
Fossil Algae
General. A review of the structural types and evolutionary trends among
Recent algae, as briefly summarized in the previous section, and a study of
* This has been explained by numerous writers on the basis that the highest types moved
ashore and gave rise to the land plants.
430
Johnson : Morphological Trends among Fossil Algae 431
fossil algae, bring out 2 basic facts. (1) The beginnings of the algae are to be
found in very remote ages of geological time, at or very close to, the origins of
life upon Earth. They were among the earliest forms of life to appear and the
evidence available suggests that each of the major groups started independently,
Table 1
Structural Types
Simple types
Unicell
Palmelloid and dendritic
Coccoid habit
Filamentous habit
Heterotrichous habit (^ creeping basal portion
(an upright portion above
Siphoneous habit
Advanced types
Heterotrichous filaments
Discoid
Crusts or cushions
Elaborately erect type
Compact (uniaxial)
Compact (multiaxial)
Foliose
Tubular
Table 2
Parallelism in Development of Simpler Types of Growth Forms
Algal group
Type of algal structure
1)
<u
u
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Motile holophvtic unicell
X
X
X
X
Motile colorless unicell
X
X
X
X
X
X
X
Encapsuled unicell
X
X
X
Motile colony
X
X
X
X
Dendroid colony
X
X
X
X
X
Palmelloid colonv
X
X
X
X
X
X
Coccoid (zoosporic)
X
X
X
X
X
Coccoid (azoosporic)
X
X
X
X
Simple filament
X
X
X
X
Heterotrichous filament
X
X
X
X
X
X
Siphoneous type
X
X
Holoph\tic amoeboid type
X
X
X
Holozoic amoeboid type
X
X
X
X
Plasmoidial type
X
X
X
X
probably at appro.ximately the same time, and have developed along more or
less parallel courses since. (2) By the beginning of the Paleozoic Era (earliest
Cambrian time) roughly 500 million years ago, the algae had developed to the
point where the algal population was probably equal to that found today, with
432
Annals New York Academy of Sciences
all the major groups present, and even some of the existing orders and families
already present and showing their characteristic features.
Our knowledge of fossil algae is limited and very spotty with many vacant
spaces both in time, and in algal groups. The study is still in its early child-
hood. There are a number of reasons for this. (1) Geologists and paleon-
tologists have only recently become interested in fossil algae, and to begin to
search for and to study them. (2) The nature of the fossils, (table 4); and
(3) the difficulties in accurately identifying and classifying the fossils will be
discussed later.
Thanks to the fact that the oil companies have discovered that algal lime-
stones make good reservoir rocks, petroleum geologists and paleontologists are
becoming interested in fossil algae. However, as yet, very few have the knowl-
edge and experience to use them. I seriously doubt if there are 10 people in
the world with a good working knowledge of the subject. In the Western
Hemisphere there are only 3 people working full time in the field, and 2 of these
Table 3
Parallelism in Development of Advanced Forms
Group of algae
Type of growth form
Chlorophyceae
Phaeophyceae
Rhodophyceae
Heterotrichous filament
X
X
X
Discoid type
X
X
X
Crusts or cushions
X
X
X
Elaborate erect type
X
X
X
Compact (uniaxial)
X
X
Compact multiaxial
X
X
X
Foliose
X
X
X
Tubular
X
X
are interested only in certain groups. However, there are a number who are
learning, and are studying either certain groups or the fossil algae present dur-
ing certain geological periods.
From the very nature of the majority of the algae their chances of being pre-
served as fossils are very slight.
A tiny drop of jelly surrounded by a thin wall of organic material will only be
preserved under very exceptional conditions, and even then the chances of it
iDeing found are very slight. Normally only those microscopical forms which are
encased in a covering of siUca or other mineral material are hkely to be pre-
served, as in the case of diatoms and silicoflagellata. Among the larger forms
it is also true that the chances of the bodies of such soft organisms being pre-
served are almost nil. The only common exceptions are those higher types
which have developed the habit of secreting or depositing calcium carbonate
within or around the plant tissues, and the microscopical forms which are en-
veloped in a siliceous or calcareous covering, or have a hardened encysted stage.
Groups wilh kmmn fossil representatives (tablf, 5). This program deals with
the Protobiota, so emphasis is placed on the microscopical forms. However,
other speakers are giving detailed papers on the diatoms, dinoflagellates, and
Johnson : Morphological Trends among Fossil Algae 433
other types of the chrysophyceae and the siUcoflagellates, and because my work
has been largely with the megascopic limestone building forms, I cannot refrain
from discussing them briefly. (From the point of view of evolutionary trends
Table 4
Methods of Preservation of Fossil Algae
Type
Information given by fossil
Value for accurate
classification
Impressions
Give a general idea of size and
shape, some surface markings.
No internal structure
Very little
Molds and casts
Same as above
Verv little
Preservation in chert
Variable. Some remarkable pres-
Often very good.
ervation of microorganisms and
Probably the best
small megafossils
Carbon films
Size, shape, and surface features
beautifully preserved, some-
times. Rarely traces of internal
structure
Fair
In coal or peat
At times remarkable preservation
of microfossils, and internal
structure of larger ones
Good to very good
Calcareous algae. Original
Good internal structure. Even
Good to very good
material or calcified or silic-
the size, shape, and arrangement
ified
of cells in the tissue in case of
coralline algae
Table 5
Groups with Fossil Representatives
Group
Representatives
Size
Age range
Chlorophyceae
Codiaceae (some
genera)
Dasycladaceae
Mega.
Mega.
Cambrian-recent
Cambrian-recent
Chrysophyceae
Silicoflagellata
Micro.
Miocene-recent
Bacillariophyceae
Diatoms
Micro.
Jurassic ?-recent (pos-
sibly older)
Dinophyceae
Dinoflagellata
Micro.
Ordovician-recent
Rhodophyceae
Solenoporaceae
Corallinaceae
Gymnocodiaceae
Mega.
Mega.
Mega.
Cambrian-cretaceous
Jurassic-recent
Permian-cretaceous
Myxophyceae (Cyano-
phyceae)
Stromatolites
Mega.
Precambrian-recent
these are of interest as they have made much of their development since Cam-
brian time with a fair fossil record to document the development.)
Megascopic fossil algae. The remains of multicellular algae, as well as micro-
scopical ones, may be preserved in a number of ways. The nature of the fossils,
resulting from the way in which they were formed and preserved, is of great
434 Annals New York Academy of Sciences
importance as it controls the amount of information, especially with regard to
structure (table 4).
The most common and the most useful algal fossils are those of calcareous
algae. If not recrystallized these commonly show not only the external form
and surface features but at least some details of the microstructure. In the
case of the coralline algae they actually show the size, shape, and arrangement
of the cells in the tissue and details of the reproductive organs permitting def-
inite, accurate classification.
Identificalion and classification of the fossils. This is the most important and
frequently the most dithcult part of the study of fossil algae. The remarkable
parallelism in structural development and growth form in several of the major
groups and numerous orders and families gives a perplexing choice of possible
assignments for the fossils, which can only definitely be decided on the basis of
internal structure and reproductive organs. As just pointed out, very few of
the fossils can give this information except the calcareous algae.
This means that the calcareous algae are the only groups of megascopic fossil
algae for which we have enough solidly based information to be able to discuss
the evolutionary morphological trends.
Morphological trends. Among the green algae two families, the Dasyclada-
ceae and the Codiaceae have a long fossil record. Both appear in the record
during the Cambrian and continue down to the present.
Dasycladaceae. The general form for most members of this family suggests
a test tube brush, consisting of a central stem from which develop more or less
regularly spaced whorls of primary branches. From the tips of the latter may
arise tufts of secondary branches, which in some genera may produce tertiary
branches. In the earlier, primitive forms the primary branches are not col-
lected in regular whorls, but may be irregularly spaced, or develop in more or
less regular rows which spiral upward around the central stem. However,
genera with regular whorls of primary branches are definitely present during the
Silurian period and characterize most of the genera thereafter. From Silurian
times on the general trend is toward greater structural complexity, involving
greater numbers of whorls, the development of secondary, and tertiary, rarely
even quaternary branches, and the differentiation of the branches into whorls
of purely vegetative branches, and whorls of fertile sporangia bearing branches,
with, in some cases, the modification of certain branches into elaborate holders
of sporangia or spores. This trend toward greater elaboration of structure
reaches its climax during the Jurassic period, after which a tendency toward
simplification begins. This has continued to the present.
Codiaceae. The early Paleozoic record of this family is meager but sufficient
to show that by Ordovician times some members had reached a high structural
level quite close to that of present day types, like Halimeda which thev closely
resemble. Since then "increased structural complexity suggesting evolutionary
changes, such as are seen in the Dasycladaceae, can scarcely be recognized
among the Codiaceae. This fact suggests that, as a consequence of vegetative
differentiation and evolution from primitive plants sometime during the Pre-
cambrian, the family was already well established," (Konishi, 1961, p. 233).
Actually, from the Mississippian up into the Lower Cretaceous various mem-
Johnson : Morphological Trends among Fossil Algae 435
bers of this family are probably numerically the most common fossil algae.
They changed in detail, but the general morphology and structure changed but
Httle.
Red algae. Calcareous red algae were relatively rare throughout the Paleo-
zoic. From the Cambrian to the Pennsylvanian, all found to date appear to
belong to two genera of the family Solenoporaceae. During that time they
show little morphological change. Then, during the Pennsylvanian, several
quite different types of red algae appear. One of these, the genus Archaeolitho-
phyllum, has much higher structural features with the tissue differentiated into
a well developed hypothallus and perithallus, and definite conceptacles. Also
the Pennsylvanian genera show a much greater variety in growth form.
Whether this rapid rise of new types represents an evolutionary surge, or for
some reason long established groups of plants acquired the calcareous habit
and begin to be preserved as fossils, we do not know.
During the Permian another family of calcified red algae, the Gymnocodia-
ceae, appear and in the Late Permian become abundant and widespread, adding
additional morphological types.
The record of Triassic red algae is scanty, but during the upper half of the
Jurassic the group undergoes a strong evolutionary push. Within the family
Solenoporaceae many developments and new growth forms appear, and repre-
sentatives of the family Corallinaceae begin to emerge. The first recorded
articulated corallines appear during the Middle Jurassic, and the earliest known
crustose corallines during the Late Jurassic. By the end of the Cretaceous,
almost all of the common genera of the coralline algae had appeared. They
were well established by the middle Eocene and had developed essentially all
the morphological features known today.
Myxophyceae {Cyanophyceae). The only other important type of calcareous
algae are the stromatolites. These are calcareous masses of distinctive form
and surface markings, commonly showing thin arched laminae, built largely or
entirely by the activity of certain types of glue-green algae. They have been
reported from rocks as old as the late Archaeozoic, and are faily abundant in
the Huronian of few areas. They were the Hmestone building organisms of the
Proterozoic and Early Cambrian. With the appearance of limestone building
animals in the Cambrian and Ordovician their importance decreases greatly,
but they have continued in considerable numbers down to the present day.
However, in morphology and structure, they show practically no change after
Late Cambrian times, consisting of mats or felts of tiny algal filaments which
often trapped some silt or organic debris and was encased in a mold of fine cal-
careous dust precipitated by the algae. Commonly they developed colonies of
a consistent shape, show growth laminae, but little or no microstructure.
Bibliography
Doty, M. S. 1957. Fxology of marine Algae (annotated bibliography). Treatise on marine
ecology and paleoecology. Geol. Soc. America Memoir 67. 1: 1041-1050.
FosLiE, M. & H. Printz. 1929. Contributions to a Monograph of the LUhothamnia.
Royal Norwegian Museum of Natural History. Trondheim.
436 Annals New York Academy of Sciences
Fritsch, F. E. 1956. The Structure and Reproduction of the Algae. Cambridge Univ.
Press. Cambridge, England.
Johnson, J. H. 1960. Paleozoic Solenoporaceae and related red algae. Colorado School
of Mines Quart. 55(3): 77.
Johnson, J. H. 1961. Limestone building algae and algal limestones. Colorado School of
Mines, special ])ubl.
KoNiSHi, K. 1961. Studies of Paleozoic Codiaceae and allied algae. Part I. Codiaceae
(excluding systematic descriptions). Kanazawa Univ. Science Repts. 7(2): 159-261.
PALEOECOLOGICAL CONSIDERATIONS OF GROWTH
AND FORM OF FOSSIL PROTISTS
Paul Tasch
Department of Geology, University of Wichita, Wichita, Kansas
Protists known as fossils range from bacteria (0.5 n in size) lo foraminifers
and tintinnids (from 10 to 1000 n or greater in size). Some protists, for exam-
ple, bacteria and lime secreting algae, are first recorded as fossils in rocks of
Pre-Cambrian age; others, including radiolarians, hystrichosphaerids,* and
foraminifers, apparently make their first appearance in Paleozoic time. Finally,
Mesozoic rocks bear the first record of a dominant element of the living phyto-
plankton, namely, the diatoms, as well as other protists, such as, the coccolitho-
phorids, silicoflagellates, tintinnids, and the Chrysomonadina.
There are numerous studies by protozoologists on variations in size and form,
as well as structure and physiological characteristics of protozoans. They have
found it necessary to distinguish races, varieties, and strains within a given
species to italicize the observed variation.- By contrast, studies on the skeleton
of various protists in which the differential morphology, as well as causative
factors, is considered, are relatively few.
Thompson^ approached protist skeletal morphology and factors influencing
it from a different point of view. With mathematical-physical considerations,
he reached some insightful conclusions. One need but regard the minute mass
of protoplasm that is involved in secreting a protist skeleton as a fluid drop
and subject to all the physical forces known to affect such a drop to explain its
form by the laws of surface tension. It is apparent that many protists tend to
have skeletons of spherical configuration. The sphere, of course, offers the
least surface area for a given volume. Because a chain of such drops is possible,
and any individual drop can be acted on by gravity, the observed variation in
protist skeletal morphology can be simply explained.
Ecology and Paleoecology of Protists
There is a very extensive literature on ecological factors that influence
growth of living phytoplankton.^"^ Nitrogen and phosphorus are primary
nutrient factors.^ Other elements of importance include: silicon, ^'^^ iron,i-'i*
and possibly manganese.^ A sudden increase in vitamin B12 may be the stimu-
lus for certain phytoplankton blooms.^* Among physical factors, tempera-
ture and salinity are effective "selective agents" on the species level. The
species specific salinity response has recently been attributed to "special re-
quirements for the concentration of sodium ions in the medium. "^^ Radiation
is obviousl}^ of primary importance affecting as it does, latitudinal and seasonal
variations in phytoplankton production. In addition, the photic zone must
be replenished by nutrients from deeper waters. This redistribution is attained
in coastal waters by vertical circulation.^'''
* Affinities to dinoflagellates are indicated for several, but not all, hystrichosphaerids.'
Tasch (in press) has found undoubted dinoflagellates in the Permian of Kansas. These were
associated with hystrichosphaerids.
437
438 Annals New York Academy of Sciences
It seems valuable to reiterate, with fossil protists in mind, that "we may
assume that during the period for which we have good fossil evidence, the sea
has remained very much the same in overall chemical composition."^*^ Cer-
tainly, this is applicable to the Tertiary and Mesozoic. By extrapolation, for
protists such as the radiolarians, it may be referred back to the older Paleozoic.
Ecological studies of living marine biotas suggest "dim outlines of food chains
that must have had links similar to those of the present day"' in the geological
past.
In thinking about assemblages of fossil protists, their growth and form in
ancient seas, coastal and inland waters, one can refer to the same or equivalent
physical-chemical factors known to influence living phytoplankton.
The Diatom Frustule and Dinoflagellale Armor
Certain physical realities of the environment have to be satisfied to ensure
survival for various protists including pelagic diatoms and dinoflagellates.
We may speak of these as "fence" or limiting conditions. These restrictions
influence not only distribution but growth and form as well. The first "fence"
is the specific weight of living protoplasm, which is 1.02 to 1.06, and hence
heavier than pure water."* There will be a tendency to sink if the added incre-
ment of a skeleton (test or armor) is superimposed on this naked weight.
Whether the protist is a passive floater like the pelagic diatoms, or capable of
feeble flagellar locomotion like the dinoflagellates, the fence condition will
apply. The second "fence" is established by the requirements of photosynthe-
sis. Pelagic protists need to be physically positioned, or located in a specific
zone of the sea, the photic zone, or both.
Given these fence conditions, a selective advantage will favor individual
pelagic diatoms and dinoflagellates with slight variations in skeletal morphology
that tend to retard the rate of sinking. Natural selection would then become
effective within the available band of skeletal variation characterizing a given
population.
Projecting spines, chains of cells, disc-shaped tests or needle and hair types,
curvature of cells, bevelled ends of tests, are all structural adaptations to resist
the gravitational force. Spines, for example, aid flotation, as do spiral or
flattened chains of cells. This last feature produces more surface area and
hence greater frictional resistance.'^ It should be emphasized that test shape
and modification of the ends of tests do not prevent sinking. Rather, these
features either facilitate a return to the horizontal from a vertical position, or
expand the path of passive descent from a straight line to a zigzag path or a
widely circular one. In this way, removal from the photic zone is slowed down
or delayed.** 'i^''**^
Weight and spination of diatom frustules have been observed to vary ac-
cording to species, season, and habitat. Generally, pelagic species tend to be
thin shelled, whereas bottom and littoral forms are not. Viscosity, which
varies inversely as the temperature, is a factor in flotation of pelagic protists.
Heavier frustules tend to sink under reduced viscosity. It follows then, that
cold water or winter forms will have heavier shells.'' '^'^ In all such instances,
silicon metabolism and the supply of silicon are also involved.'""
Tasch: Growth and P'orm of Fossil Protists 439
The shapes of some nonmarine diatoms can be influenced by other ecological
factors. Individuals of the genus Desmogoniurn were long and had barely
capitate ends in fast flowing water but were short and had broadly capitate
ends in standing water. ^"
Similar considerations also apply to the morphology of armored dinoflagel-
lates.^'^^-^"'''^ Kofoid studied skeletal development {i.e., an armor of loosely
cemented cellulose plates) in the genus Gouyaulax.-^ He found that all modifi-
cations in this genus were variants of the spherical configuration (cf., Lejeune-
Carpentier-' for fossil Gonyaula.x). In turn, this ensures least surface area,
and hence confers an advantage on protists leading a pelagic existence.
Braarud^'^ stresses that form variation is observed in every species. Study
of Schiller's work on dinoflagellates brings this out clearly. Dinophysis
hastata^^ for example, shows a whole spectrum of variation from ovate to sub-
ovate and subelliptical conligurations, and corresponding size and shape varia-
tions in epithecal and hornlike structures. Braarud''' noted that in some
instances, form variation appeared to be "phenotypic" and "tentatively re-
lated" to a whole series of ecological factors such as salinity, temperature,
nutrient salt concentration, and day length. An excellent example of such
infraspecific form variation is found in the fossil record of the dinoflagellate
Nannoceralopsis recovered from beds of Jurassic age.-^
Twenty specimens of N . deflandrei show variations in form from ovate to
subovate hanging drop configurations. These may bear weakly defined an-
tapical horns or lack them. Other forms are broadly and acutely subtriangular
with the base faintly or markedly concave between short horns. This strik-
ingly contrasts with the long horn type, X . pelliicida.^^
It is apparent from our previous discussion that these variants are adaptive
modifications for flotation. Something similar to pelagic diatom adaptation
in thickness of test is found in the armored dinoflagellates. Thus, in colder
waters of the South Ecjuatorial Currents in which viscosity was greater, the
horns of Cerialia were found to be longer than those of equivalents taken in the
warm water of the Guinea stream."*
The short horn, X. deflandrei, may be related to warmer waters, whereas
.V. peUucida, the long horn species, would then indicate colder waters. A third
type tentatively assigned to Xannoceraiopsis has been found in the Permian.
This form is flask-shaped and bears stubs in place of horns (Tasch, in press).
Other structural modifications that have adaptive value in armored dino-
flagellates, include a variety of specializations to ensure suspension or flotation
when the flagella are at rest or swimming is feeble.^ •-^■"•^■-'^ In the genus
Triposolenia, the ends of the antapical horns are deflected. The significance of
this asymmetry has engendered speculation. Kofoid"* thought that the asym-
metry must bear a "profound relationship" to the survival of the forms in
which it appears. It occurs in more than one species and in the genus Amphiso-
lenia also.-'' Still other genera have analogous structures. After a descent of
about 10 times the body length, the asymmetrical horns serve to orient the
long axis horizontally, i.e., the position of greatest resistance to downward
pun.4 19
A few of the morphological variants in armored dinoflagellates include:
round and egg-shaped skeletons (Glenodinium) sometimes bearing spines on the
440 Annals New York Academy of Sciences
hypotheca; hanging-drop configurations to subspherical forms with a tapered
hypotheca and bearing or lacking antapical horns (Peridinium), an eccentric
expression of the same configuration with partly deflected apex and horns
(Heterodinium); bizarre, multihorned Ceratium in which horns may deflect at
all angles and in all directions.^^ -^
Nine varieties of Ceratium hirundella were found in various European
waters.- '^^ Size variants have been reported from different Swiss lakes: 92 ju
in Lake Como, to 707 n in Lake Schwendi.-'' SampHng several ponds in the
vicinity of Darmstadt, Germany at 2-week intervals over a 5-year period,
Ljg|-29a found that population density fluctuated with rise and fall of tempera-
ture. He noted seasonal variation in both horn length and horn number in
C. hirundella. In the summer four-horned type, for example, horns were
shorter during very hot summers than they were during cooler ones. Ap-
parently, in fresh, as well as marine waters, horn development is an adaptive
modification to resist sinking below optimal food and sunUt levels.
Flaring, sail-like, structures from the girdle, inverted umbrella and parachute
type membranes as in Ornithoceras, Diuophysis, and other forms'* ■-'' all tend to
increase the surface area of the anterior over the posterior. In turn, this helps
to orient properly the given protist.
A third group of protists of polyphyletic origin are the hystrichosphaerids.
Many forms classified under this group are apparently dinoflagellate cysts.i -^^
Fossils often show dinoflagellate plates although many forms lack a distinguish-
able plate system. Configuration of the central body is often globular but all
variations are known from subround and ovate to subelUptical. Arising from
the central body are spines or tubular processes, or both, with flattened or
bifurcate terminations. It has become clear that these tubular processes were
originally connected to a circumscribing membrane. A recently found globular
hystrichosphaerid from the Kansas Cretaceous bears a short, tubular process
that terminates in two fine flagella-Hke extensions within the body of the en-
closing membrane. ^^
For those hystrichosphaerids which are definitely dinoflagellate cysts,
morphology was determined by encystment. HystrichospJiaera furcata and
H. speciosa bear an equatorial girdle and dinoflagellate plates, and are good
examples. Generally, hystrichosphaerid form is a variation of the spherical
configuration. Why this is so can best be understood if one observes a cyst
inside a subtriangular-to-buUet-shaped dinoflagellate like Deflandrea phos-
phoritica?- The spherical shape is the most efficient configuration that can be
enclosed in the volume available.
Radiolarian Scleratoma and Tiniinnid Lorica
Radiolarians and tintinnids occur together in the Mesozoic fossil record in
the Mediterranean area^^ and hence it seems desirable to discuss them together.
Both groups have living representatives which occur in great abundance.
Radiolarians found in the fossil record are almost invariably "upper-zone
pelagic types"^^ although abyssal forms are known.
Although radiolarians are incapable of horizontal locomotion, tintinnids can
swim rapidly by the aid of bristles and cilia.^* Both forms had to solve the
problem of resisting passive sinking below optimal levels of the sea.
Tasch: Growth and Form of Fossil Protists 441
The form of radiolarian species seems to be adaptive to environmental
conditions'*'^ although experimental study of factors influencing shell mor-
phology are wanting.'^^ Thompson/'* as noted previously, provided some useful
insights into radiolarian morphology.
Free floating radiolarians, among both fossil and living assemblages, tend to
be spherical and elliptical, with a foamy or spongy appearance. Such forms
occur in the Spumellina, Nasselina, and Acantharina. The shells are delicate,
small, and bear various structures such as, numerous slender apophyses, large
pores, thin bars between pores, and varied spinose development. Inhabitants
of deeper layers (Phaeodarina and some Nassellina) are heavier, more massive,
and tend to bilateral symmetry. They are infrequently burrlike. Structures
found in such forms include: short apophyses and small pores with thick tra-
beculae.^'*'^^ .
In some forms (Semantidae, etc.)'*^ one can observe configurations not too
different from those of the silicoflagellates.^® The shell in the Challengeridae
bears a fine hexagonal mesh resembhng similar structures of the diatom frus-
tule.^^ Some configurations of radiolarians are analogous to those of armored
dinoflagellates, for example, Coelodecas?^ Hexaspyris papilio^^ is reminiscent
of the bizarre spinose development in the dinoflagellate Ceratium.
It is generally agreed that variations in scleratoma configuration and in
skeletal structures found in radiolarians reflect adaptations to retard sinking
below certain depth levels of the sea. Within a given species of course, varia-
tions of shape and structure are merely those of a normal population spread.
The gelatinous or pseudochitinous cuplike or elongate lorica of tintinnids is
frequently agglutinated. Foreign particles encrusted or included in the deli-
cate membranous wall may consist of fine mineral grains, coccoliths, diatoms,
and organic debris.^^'^'^'^^ Shape of skeleton in both fossil and living tintinnids
is extremely diverse. ^^ Surface markings of the lorica include: ribs, ridges,
plications, flutings, shelves, reticulations, fenestrae, and lacunae. Among
aboral structures are apophyses, pedicel, knob, lance, and skirt. ^'"^
Because tintinnids move like squids with oral end directed backwards,
streamline configuration of the aboral tip would offer less frictional resistance
to forward movement. It is also likely that the lorica may aid flotation.^^
The total effect of such configurations is to check descent below optimal levels.
Modification of Shape and Form in Foraminifera
Work on living foraminiferal distribution and ecology has clearly established
characteristic faunal suites in distinct brackish and marine environments. '"'"■*''
Although the majority of foraminifers are vagrant benthos, planktonic forms
that float at or near the surface such as the Hantkennidae, Orbulinidae, and
Globorotalidae have been more closely studied in the past decade.'*" '^^
Bandy^* found a striking correlation in form, structure, and environment in
benthonic foraminiferal assemblages in modern seas. Among the variations
he observed are overall size, shape, and size of chambers, chamberlets, coiled
and uncoiled forms, spinosity, surface sculpture of the test (costa, striae).
These were found to vary with bathymetry (bay, shelf, and bathyal zones).
Phleger'*" believes that the influence of temperature may have been overstated
442 Annals New York Academy of Sciences
in the literature and suggests a whole spectrum of ecological factors that may
have been involved.
In both modern tropical and subtropical waters, spindle-shaped tests seem
to characterize definite depth zones (20 to 80 meters). By extrapolation,
Bandy^^ ascribed equivalent depth zones to fusulinids — an extinct Paleozoic
family — and to the Cretaceous spindle-shaped Loftusia. Similarly, he noted
that deeper water assemblages seem to show a size increase and coarser surface
features.
The planktonic foraminifers show a variety of morphological and structural
adaptations for their floating existence.^^ The variations are ascribed to
temperature and saUnity. Thin walled shells, for example, characterize surface
Orbulina miiversa and Globigerina in contrast to thick walled shells for indi-
viduals living at greater depths. Reduction in the specific gravity of the
planktonic test is also affected by increase in pore size, aperture enlargement,
or the development of supplementary apertures.
Resistance to sinking which is the critical problem facing all pelagic protist
inhabitants, is attained in planktonic foraminifers as follows: flattening of the
body accompanied by a radial test, and elongate or clavate chambers. In the
Orbulinidae and Hantkennidae spinose projections develop. Other adaptations
include: globose chambers that increase in size as added, large primary aper-
tures, and in such forms as Globigerinoides, development of many secondary
openings.
Although all of the above named variations may be related to genetical
events and the operation of natural selection, there are other nongenetical
factors known to influence foraminiferal morphology. On the Argentine shelf,
a depauperate foraminiferal fauna was found to be characterized by its small
size, partial or complete loss of ornamentation, a tendency toward asym-
metry, and growth retardation. Spectrographical study of trace elements in
the shells revealed the presence of lead in depauperate, as compared to, normal
faunas in which it was absent.^^ ■'*^ Study of Allogromia laticoUaris in culture
revealed occasional populations with a large number of flattened discoidal
individuals. In this instance, the flattening was directly attributed to "down-
ward pressure exerted by rapidly multiplying algal filaments."'*^ Dwarfed
foraminifers are reported from poorly ventilated basins.^-
An unusual example of a testate protozoan, Difflugia oblonga, can be cited
here although it belongs to a different order than the foraminifers. A small
pond (10 X 6 meters) in the environs of Prague, Czechoslovakia, contained
numerous individuals of this species. They exhibited an astonishing morpho-
logical variation. Every variant was observed from a globose bowl with a
smooth base, to elongate figures with tapered basal projections variously
curved. Some specimens took on the configuration of an Erlenmeyer flask
with knoblike projections from each basal edge. The heavy discharge of
industrial waste gas (CO2) in the environs was thought to be the causative
factor .^^
Classes Chrysophyceae, Coccolithophorida, and Silicoflagellata
The several flagellates cited in the subtitle of this section, with the diatoms
discussed earlier, constitute the phylum Chrysophyta. Members of the order
Tasch: Growth and Form of Fossil Protists 443
Chrysomonadina are either solitary or colonial. They are widespread in both
fresh- ■'*^ and marine waters'^ and have fossil representatives in the family
Archaemonadaceae Deflandre.^"
Formation of siliceous resting spores or cysts is a "most characteristic feature
of the order. "2 '18 '51 Such cysts have a funnel-shaped opening or neck and
resemble a stoppered or plugged spherical jar. The plug is formed of cyto-
plasm retracted from outside the cyst wall.
In the cyst of Microglena,^^ Conrad has distinguished "numerous minute
lens-shaped masses of silica" embedded in an outer layer of pectic substances.
A delicate, inner smooth layer of cellulose underlies this outer layer. This
genus with other Chromulineae is closely related to the coccolithophorids in
cell structure although it differs from the latter in flagellation and composition
of its cyst.
Cysts are usually spherical but variants from this configuration occur.
Archeomonadopsis, which is flask-shaped, is such a variant. Surficial orna-
mentation finds diverse expression: ridges that may form a reticular network;
encircling equatorial flanges; spine and knob structures on ridges, and peripheral
spines. The size range of cysts is 10 to 25 /x.
Although little is known about the marine Chrysomonadina, it is apparent
that the morphology and small size of the cyst, together with the cytoplasmic
plug, would favor both resistance to sinking below the pelagic zone and wide-
spread passive distribution. Fossil cysts also indicate a broad geographical
spread.^" The same types of adaptive modification found in living representa-
tives occur in fossils.
A third large group of planktonic algae are the Coccolithophoridae.'^'^^"'
52a ,53 ,54 fhey arc typically open sea biflagellates although in places like the
Oslo fjord, they may occasionally occur in such densities, that the water looks
like milk."* Fresh water forms like Hymenomonas are also known.^^
One may study a form like Coccochrysis,^^ Discosphaera,^ or Syracosphaera
and CoccoUlhus^^ and observe a subovate configuration in the first and third
and a more spherical form in the second and fourth. Lohman^- figures several
different species of Pontospliaera, Calyptrosp/iaera, and Coccolilhop/iora, as well
as species of the second and third genera named above. All of these species
show the same trend in configuration. Generally, therefore, the shape of
coccolithophorids are modifications of a sphere.
The formation of the coccolithophorid skeleton is gradually achieved. At
fairly equal intervals, numerous, minute, variously shaped, calcareous discs
(coccoliths) are "imbedded in an investing membrane. "'^^ This envelope of
variable thickness is gelatinous initially. The coccoliths become "rigidly
united when the mucilage calcifies in older individuals."'** Coccoliths have a
central perforation or are imperforate."^ Although living biflagellates commonly
range from 5 to 20 n,'^ sizes can attain 50 )u.^^ Coccoliths found in sediments
range between 2 and 30 fj.}^
Several coccoliths bear anteriorly and medially spinelike processes. Of
interest, is the successive formation of new coccoliths within the old as the old
are gradually dislodged'^ and contribute to oceanic bottom deposits. Although
today coccoliths are but a "minor part" of oceanic carbonate muds, in Miocene
and Oligocene time, for example, they formed "coccolith ooze."^^
444 Annals New York Academy of Sciences
Braarud'^ and others have experimented on variation in salinity and its
influence on the growth of the coccolithophorids, Hymenocaras carterae and
Coccolillnis liuxleyi. For the tirst species, salinity was excluded as an important
environmental influence on growth. This corresponded to the littoral habitat
in which it is most abundant and in which salinities are quite variable. The
second species, C. huxleyi, is distributed worldwide in oceanic waters (35 per
thousand)" and in northern European coastal waters (15 to 20 per thousand).
Experiments have shown that between these ranges of salinity there was good
growth.
Salinity apparently does act as an ecologic fence in excluding C. huxleyi
from brackish waters. A vertical size distribution of coccohthophorids at
equatorial stations has been reported." Small forms were abundant in the
upper 50 meters. Near surface temperatures are also a probable factor in
distribution. It is thought that variety, large size, and abundance of Eocene
coccolithophorids indicate "warmer seas. "•''•''
The life cycle of coccolithophorids has recently been shown to be more
complex than previously thought.'** A motile stage and a cyst stage have been
experimentally demonstrated for Coccolithus pelagiciis}^
From these data, shape, size, and encystment seem to be adaptations similar
to those in the closely related siliceous Chrysomonadina. Coccolilh formation,
shape, their even spacing in the membrane, and spinelike processes arising from
some coccoliths, are all adaptive devices to aid flotation. Abnormal amounts
of calcite in some Tertiary coccoliths are thought to reflect calcium carbonate
rich waters and not a diagenetic effect.''^ Conceivably, this abnormal deposi-
tion may have served to aid buoyancy or to adjust specific gravity.
One can confidently transfer the general interpretation given to Tertiary
coccolithophorids.
The silicoflagellates have a siliceous skeleton which is covered by a delicate
layer of cytoplasm containing chromatophores. This occurs in early develop
ment when the skeleton is internal, whereas in the adult individual it is ex-
ternal.-''^ The skeleton ranging in size from 10 to 150 fx is essentially a "lattice-
work case of hollow siliceous bars."^" Dislephanus ( = DictyocIia) speculum
with 6 radial spines may be taken as an example of the group. In most silico-
flageUates, the spines give the skeleton a stellate appearance. There may also
be accessory and basal spines. The basal body ring may be from 3 to 10-sided
with as many radial spines. Radial spines issue from the point of intersection
of any 2 sides. The basal body ring of .some fossil forms like Mesocena and
Corbisema^^ is 3-sided with a small spine at each angle. Others, like Dictyocha
crux are 4-sided and have longer spines. D. speculum is 6-sided, and D.
flcfonaria is 8-sided .'^^
The siliceous skeleton is most often a complex of 2 rings or polygons joined
by a series of rods."* Dictyocha speculum is a good example of this construc-
tion. The basal body ring of Mesocena forms an ellipse, and in Corbisema, it
forms a triangle.
Silicoflagellates are exclusively marine plankton'^'' and are found in colder
seas. Frequently they occur associated with diatoms and radiolarians in
ancient and modern sediments.^^ Although they are not uncommon in food
I
Tasch: Growth and Form of Fossil Protists 445
vacuoles of tintinnids, quantitatively they are a minor contributor to the food
economy of the sea.^
Thompson's explanation^ of the basket-shaped skeletal units of Dictyocha
envisioned 4 or more vesicles side by side in one plane and separated by a
"polar furrow." The radial spines normal to the main basket or lattice work
were interpreted to be uncompleted portions of a larger basket. This last
interpretation seems unacceptable in light of the work of K. Gemeinhardt.^^
He demonstrated that adult individuals had a smaller skeleton fitted into the
larger one. In this instance, the inner set of radial spines were not the be-
ginnings of a still larger skeleton, but rather parts of the skeleton of a daughter
cell, and its appearance preceded division. Hovasse confirmed this finding, in
1932,^^ and noted that the new skeleton was a mirror-image of the old one.
Thus, the opaline silica lattice work may be envisioned as derived by secre-
tion on a tiny sphere of protoplasm that had a vesicular surface. Open space,
ovate, elliptical, and polygonal skeletal configurations can then be readily
explained. The radial spines which confer a stellate appearance are most
Ukely adaptive modifications to sustain flotation when the single flagellum is
at rest. All other accessory spines and ornamentation, such as beads and pits
on the discs,*^ may constitute minor adjustments of specific gravity of the
skeleton that had selective value.
In the evolution of the silicoflagellates there is a tendency to increase the
number of radial spines from 3 or 4, to 6, 8, and 10. That trend clearly de-
notes the adaptive value of particular skeletal modification.
Miscellaneous Protists
In this section, bacteria and lime secreting algae will be considered from the
special point of view of our discussion. Despite the frequency of pleomorphism,
there are three common or fundamental forms of true bacteria; spherical or
ovoid (coccus), rod-shaped {bacillus), and spiral (spirallum). Spherical forms
may grow in pairs, in fours, or in chains. Rods vary in configuration from
cylindrical to ellipsoidal with rounded-to -flattened ends. In young cultures
and favorable media, bacteria tend to "exhibit characteristic morphology,''
whereas in senescence, there are a decrease in size and considerable form varia-
tion. Other factors influencing shape are: temperature and age of culture,
concentration of substrate, and composition of medium.^^ A barophilic
property (pressure-dependence) has also been reported. Near their threshold
of pressure-tolerance, cells of many bacteria grow into long filaments and
mutations are promoted.""''^
Bacteria are commonly about 0.5 fj, in size but range to 10 fx. Fossil bacteria
are generally identifiable by size, shape, and arrangement alone.*- However,
viable bacteria of Permian age have since been reported from the United States*^
and from Germany (Dombrowski, 1960). In such instances, physiological
activities which distinguish modern bacterial species can also be studied in
ancient populations.
The descriptive literature on lime secreting algae known as fossils is very
extensive. "^"^^ A good review of recent stromatolites and their ancient ana-
logues is given by Ginsburg.*^ Types of stromatoUte configurations include:
446 Annals New York Academy of Sciences
kiminaled algal-mats such as can be observed forming today at Turner and
Price Falls, Oklahoma, or equivalent forms described by Black from the
mudtlats of Andros Island, Bahama; domes, heads, and more extensive digitate
masses. Onkolites (unattached forms) can have the shape of the nucleus or
be variously shaped biscuits or flattened discs. '^''
Factors such as a slight increase in iron above tolerance amounts have long
been known to retard growth, affect size, and ultimately, the shape of several
nonmarine, nonlime secreting, filamentous algae.'"^ Cyclicity in occurrence of
the Cambrian form Cryptozoon nndulatum was attributed to inhibition of
growth due to increasing turbidity caused by transported sediments.'^- Pre-
Cambrian bioherms of Northwestern Montana show different forms — columns,
domes, sheets — which were "apparently" determined by physical conditions
such as water movements.''^ The ultimate external form of Recent algal bis-
cuits is credited to two determinants: stability of the surface on which the
biscuits grow and the strength of attachment to it.*^^
Work in progress (Tasch, unpublished) on newly discovered algal reefs and
onkolites in the Kansas Permian provide some evidence on controls of ultimate
form. An influx of mud over the growing algal mat (stromatolite) inhibited
growth in certain directions only. Turbidity, of course, can exclude or diminish
hght penetration and hence interfere with photosynthesis. If, however, sedi-
ment influx is negligible (4 to 5 mm.), filamentous algae can "move up through
the sediment and reestablish themselves on the surface. '"^^ The topography
of the substrate on which the original filamentous algal mat spread, also can
be a partial determinant of shape of a stromatolite.
Sporadic circular to elliptical perforations of algal blades in the fossil genus
Eugenophyllum appear to represent adaptive modifications. Although these
forms lived below normal wave agitation, the perforations would help to dissi-
pate even gentle current action against the upright blades which are several
inches in height.''"
Among factors influencing growth of stromatolites and onkolites are: sub-
strate, turbidity, amount of light penetration, depth of water, wave and current
action. Influence of metallic cations can also be inferred.
. Terrestrial M icroproblemal ica
Microproblematica are apparent fossils observed in sections of rock suffi-
ciently thin to transmit light. They are primarily of Mesozoic age, but are
also known from the Paleozoic and Pre-Cambrian. Distinctive structure and
form characterize them. However, they cannot confidently be assigned to
any known taxa. Occasionally, additional study and collection permits ulti-
mate resolution of assignment.^' •'"-
The microfossil Xamioconus kanipliicr, 1931, is a good example. The object
ranges from 5 to 50 /u in length, with an average of 15 to 20 fx; width varies
from 5 to 15 /x. It is definitely an "organized object." There is a distinctive
wall composition (spirally arranged calcite wedges, 1 m in thickness). In
longitudinal section, it is either conical, spherical, barrel-, or pear-shaped, or
cylindrical U-shaped. There is an axial canal or a basal cavity, or both, and
2 apertures opposite each other. Through time, it shows apparent speciation.''*
Nannoconus is widespread in distribution, having been reported from the
Tasch: Growth and Form of Fossil Protists 447
Mesozoic (U. Jurassic-Lower Cretaceous) pelagic deposits in the Mediterranean
area, Rumania, Cuba, and Mexico.^^ It is always associated with the pelagic
facies containing radiolarians and tintinnids, and occasionally, with smooth
ammonites. ^
The following affinities have been suggested: (1) the object represents an
embryonic stage of the tiask-shaped foraminifer Lagena; (2) it is a unicellular
chlorophyllous alga; (3) it is of inorganic origin having formed from calcite
crystals in a highly saturated medium; (4) it belongs to the oogonia of certain
Charophyta; and (5) it represents a little known coccolithophorid.''^'^^
There are then a whole set of constants and some variables to explain. Con-
stant factors include: distinctive wall composition and structure, 50 ju or less
in length; persistence of faunal associations; and occurrence in pelagic facies
intermittently deposited over a span of tens of millions of years in different
parts of the globe. Variable factors include: nine species of X annoconus based
upon variations in axial canal and basal cavity, overall shape and size; three
distinct Xaiiiiocoiius faunas in as many zones of the Lower Cretaceous.'^^
In light of our previous discussion on form in many protists, the likelihood
is that configurations in Xannoconus are variants of a sphere.^^ Thus, selective
modification of the sphere gives an elongate type or a cylindrical type. The
basal cavity of circular types have no axial canal when seen in thin section.
Circular types were spherical in life. Elongate, conical, and subovate types
do have an axial canal. The size and configuration of axial canal and basal
cavity could be a function of compression of an original sphere. Although this
can account for the variation in morphology and inner spatial relationships of
the object, it is unclear whether mechanical compaction or genetics was the
active agent.
Although we assume the first of these possibilities, the list of constant fea-
tures still remains to be explained. Colum'^" notes that Xannoconus at times
appears in great numbers in pure limestone lithotopes. Population density is
thus another variable.
What is the likelihood that inorganic precipitation of calcium carbonate and
mechanical distortion alone can account for Xannoconus? The nearest ap-
proach to a regular type of inorganic carbonate deposit is the example of oolites.
These may be radiate in internal structure or bear concentric bands around a
nucleus. In size range, oolites are also restricted wherever they are found.
Mineralogy of the bands tends to be relatively uniform although alterations
are known. There is a definite spherical-to-elliptical configuration. When
compressed, flat, pelletiferous shapes result. Why cannot Xajinoconus be an
object of this type?
The best argument against an inorganic origin is the persistent crystallization
of minute calcite wedges, all of which are perpendicular to, and form a band
about a hollow basal cavity or axial canal. Inorganic origin cannot account
for the discrete thickness of the wall in this case as it can for the successive
bands of oolites. If the calcite wedges were invested in an organic membrane
that surrounded a cavity or canal, both wall thickness and mineral orientation
could be readily explained.
Once the conclusion is reached that Xannoconus is of organic origin, the other
448 Annals New York Academy of Sciences
array of factors readily supports the interpretation that it represents a pelagic
protist of uncertain affinities.
Among other organized micro-objects of uncertain position are Favreina,
Globochaeta, Eothrix, Lomhardia,''^ Pilhonella,^^ and objects described by
Elliott.^i"
Discoaster, an object 3 to 15 /x in diameter, is represented by calcareous,
stellate, or rosette-shaped plates. In many species the central area bears a
stem. These objects are abundant in pelagic sediments of Tertiary age. The
sediments containing discoasters also bear coccohths, Globigerina, and other
pelagic foraminifers.^*'^^ These objects are now thought to be the skeletal
remains of nannoplanktonic organisms of uncertain affinities.
One ecological observation has been made about discoasterids. Across the
Eocene-Oligocene boundary, Riedel found not merely a change of radiolarian
fauna, but "surprisingly," a change in discoasterid assemblage. This is thought
to reflect "some change in surface waters. "^^ The active factor here might be
surface temperature.
A whole series of related forms of uncertain position among the calcareous
nannoplankton include: Claihrolithus, Discoasteroides, Fasciculilhus, Heliolithus,
Tsthmolithus, Polycladolithus, Sphenolithus, and Rhomboasier.^^ Even though
Rhomboasler "is suggestive of some unusual habit of inorganic calcite growth,"
three considerations refer it to the nannoplankton: specimens occur in abun-
dance; they are found only with coccolithophorids; their occurrence in time
is restricted. ^^
Numerous reports of minute sporelike and other types of bodies and "mesh-
work filaments" from Pre-Cambrian algal stromatolites are now at hand from
Russia, Scandinavia, France, West Africa, and the United States (Gunflint
formation of Northern Michigan). The biological organization of Barghoorn's
material "is supported by geochemical evidence" {i.e., the quantity of C'^ per
mil).^^ There is no equivalent verification of pyrite spherules thought to have
replaced microorganisms.^*
Stimmary
Pelagic protists tend to contigurations of least surface area. The sphere and
its modifications is a recurrent shape. The many shapes and structures
(spinosity, for example) of the scleratoma of radiolarians, the lorica in tin-
tinnids, the frustule of pelagic diatoms, the armored skeleton of dinoflagellates,
the test of planktonic foraminifers, the siliceous and calcareous skeleton of
chrysophytes, appear to be adaptations to resist sinking below optimal food
and photic levels of the sea.
Examples of nongenetic factors affecting differential morphology of protists
include: variable oceanic temperature, salinity, depth, and turbidity; presence
of lead, excess iron and copper as well as carbon dioxide; condition of encyst-
ment; nature of substrate, barophilic property, nutrient salt concentration,
and amount of light penetration. Some of these factors apply only to specific
protists.
Fossil microproblematica of Mesozoic age seem to be nannoplankton of un-
certain affinities among the protists (for example, Nannoconus). Micro-
objects of Pre-Cambrian age may represent spores and algae.
Tasch: Growth and Form of Fossil Protists 449
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55. Bramlette, M. N. & W. R. Riedel. 1954. J. Paleontol. 28: 385-403. Pis. 38 39.
Figs. 1-3.
56. Bramlette, M. N. 1961. In Oceanographv. A.A.A.S. Publ. 67: 345-366.
57. Hasle, G. R. 1959. In Intern. Oceanog. Cong. A.A.A.S. : 156-157. Preprints.
58. Hanna, G. D. 1928. J. Paleontol. 1: 259-263. PI. 41.
59. Salle, A. J. 1954. Fundamental Principles of Bacteriology. : 59-61. McGraw-Hill
Book Co. New York.
60. ZoBell, C. E. 1959. In Intern. Oceanog. Cong. A.A.A.S. : 395-396. Preprints.
61. ZoBell, C. E. & C. H. Oppenheimer. 1950. J. Bacteriol. 60: 771-781.
62. ZoBell, C. E. 1957. Bacteria. In Treatise Marine Ecol. and Paleoecol. G.S.A. Mem.
67: 693-698.
63. Reiser, R. & P. Tasch. 1960. Trans. Kansas Acad. Sci. 63: 31-34.
64. PiA, J. 1926. Pflanzen als Gesteinsbildner. Berlin.
65. PuGH, W. E. 1950. Bibliography of Organic Reefs, Bioherms, and Biostromes. Seis-
mograph Serv. Corp. Tulsa, Okla.
66. Maslov, V. P. 1956. P'ossil Calcareous Algae. Moscow.
67. GiNSBURG, R. N. 1960. /« Proc. 21st Internat. Geol. Cong. Part 22. : 26 35. Copen-
hagen.
68. Tasch, P. 1951. Am. Midland Naturalist. 46: 751-753. Table 1.
69. Fenton, C. L. & M. a. Fenton. 1957. In Treatise Marine Ecol. and Paleoecol. G.S.A.
Mem. 67: 105-106.
70. KoNisHi, K. & J. L. Wray. 1961. J. Paleontol. 35: 659-665. PL 1.
71. Elliott, G. F. 1948. Micropaleontology. 4: 419-428. Pis. 1-3.
72. Elliott, G. F. 1962. Micropaleontology. 8: 29-44. Pis. 1-6.
73. Bronnimann, P. 1955. Micropaleontology. 1: 28-51.
74. Cloud, P. E. & P. H. Abelson. 1961. Proc. Nat. Acad. Sci., U.S. 47: 1705-1712.
FOSSIL ORGANISMS FROM PRECAMBRIAN SEDIMENTS
Elso S. Barghoorn
Department of Biology, Harvard University, Cambridge, Mass.
Stanley A. Tyler
Department of Geology, University of Wisconsin, Madison, Wise.
In widely scattered outcrops of the Gunflint Iron Formation, Lake Superior
region, Ontario, Canada, dense, black, nonferruginous, fossiliferous cherts oc-
cur as thin units in the sedimentary sequence of black shales, argillites, and
dolomites. In its geological setting, the Guntiint Iron Formation is generally
regarded as comprising the middle unit of the Animikie Series (Middle Hu-
ronian equivolent) of the Lake Superior region. Absolute age of the Gunflint
Formation has been determined by P. W. Hurley by measurement of the po-
tassium-argon ratios in authigenic minerals which occur in direct association
with the cherts and interbedded in the Gunflint sedimentary sequence. Repli-
cate determinations have yielded consistent values of 1900 M years (1.9 X 10^
years) .
The cherts have been studied with the use of thin sections, acid maceration,
and a variety of chemical techniques. Thin sections of the chert, when viewed
in transmitted light, reveal that its black color, as seen en masse, is caused by
the abundance of finely disseminated organic matter that appears light amber
to dark brown in color in sections 50 /x or less in thickness. In this respect the
chert behaves petrographically much as a typical bituminous coal, which in
thin section exhibits a range in color of the petrographical components from
light yellow through amber to dark orange red to opaque. In the Gunflint
chert a large fraction of the organic constituents reveal a distinct morphological
organization consisting of filaments, septate and nonseptate, spheroidal or
spherical bodies, and more complex asymmetrical structures. The discrete en-
tities are all microscopical in size and present an appearance analogous to
masses of anastomosing algal filaments in which are enmeshed other microor-
ganisms. The chert matrix in which the organisms are embedded varies from
clear and hyaline to granular and crystalline. In polarized light the chert is
microcrystalline. Crystals of pyrite, calcite, and apatite vary in abundance,
but in no case are more than minor petrographical constituents.
The biological affinities of the organisms preserved in the Gunflint chert
present a curious paleontological problem inasmuch as a number of the distinct
entities or "types" possess a morphology that is quite unlike that in existing
microscopical crganisms, either plant or animal. In this connection it should
be emphasized that the organic structures are 3-dimensionally preserved and
not flattened or unilaterally distorted. They are hence amenable to morpho-
logical and histological study.
The most abundant organisms in the assemblage are filaments ranging in
diameter from 0.6 to 6.0 n. In the most favorably preserved state these are
found to be both septate and nonseptate. The septate types exhibit a form
indistinguishable from that of filamentous blue green algal {vis., Oscillator ia,
Lyngbya, etc.). The nonseptate types are more difficult to interpret in terms
of biological affinities. With exceedingly few exceptions they are unbranched
451
452 Annals New York Academy of Sciences
and visibly devoid of internal structures or inclusions. Whether these repre-
sent coenocytic algae or fungi is not possible to determine, although the general
form and undulating outline of the filaments is more characteristic of algae than
of aquatic fungi. Among the larger nonseptate filaments very occasionally
forms have been observed in which the lumen of the filament contains numerous
spherical sporelike bodies. In living organisms a somewhat comparable
morphology may be found among certain of the iron bacteria {Crenothrix poly-
spora).
The sporelike bodies which are ubiquitous and irregularly distributed
throughout thin sections of the chert vary in size between 1.0 to 16.0 /x in
diameter (measured along the long axis if ellipsoidal). They are predom-
inantly spheroidal and are not appendaged. The range in size, thickness
of wall, and variation in the sculpture pattern of the wall residues indicates that
they comprise an assemblage of forms the morphology of which gives little clue
to phylogenetic aflinity.
A very common and distinct organism in certain facies of the Guntiint chert
is an entity whose closest morphological comparison among living organisms
can be found in certain groups of the phylum Coelenterata. Rather than to
accept the existence of coelenterate animals in an assemblage of such geological
age as the Gunflint sediments exhaustive efforts have been made to compare
these structures with algae, various of the larger colonial bacteria, and protozoa.
It has not been possible, however, to find morphologically comparable struc-
tures in these diverse groups and the authors have been forced to conclude, on
the grounds of morphology, that the organisms most probably represent meta-
zoons, the closest structural affinity of which is among the Coelenterata. A de-
tailed description of these organisms and other microstructures occurring; in the
chert will be made in a forthcoming paper dealing with the detailed geology and
paleontology of the (iunflint chert.
The organic fraction of the darker and more organic samples of the Gunflint
chert varies between 0.2 to 0.6 per cent by dry weight. As previously noted
Si02 comprises the major mineral component and constitutes more than 99 per
cent of the dry weight of much of those chert samples that exhibit the best
preservation of organic structures. The organic residues yield small amounts
of benzol-acetone-methanol soluble substances, probably hydrocarbons of
molecular weights C20 or above. These extracts fluoresce strongly in ultra-
violet light. Upon destructive distillation at 400° C. the insoluble organic resi-
dues yield small amounts of aliphatic hydrocarbons, chiefly methane (87 ppm),
ethane (4 ppm), and propane (0.7 ppm) and traces of aromatic hydrocarbons
(benzene, 0.34 ppm; toluene, 0.15 ppm; xylenes, 0.45 ppm). Degassification
of the chert at room temperature yields methane (6.0 ppm) and butane (0.2
ppm). The chemical data, although limited, are entirely consistent with the
paleontological interpretation that the black chert represents the silicified re-
mains of a biocoenose of microscopical organisms the organic matter of which is
partially retained, although highly modified through time by very low thermal
and metamori)hical alteration. For these reasons the (iunflint chert is uni(|ue
among earlier Precambrian sediments in exhibiting the morphological organi-
zation of an assemblage of very ancient and primitive organisms, some of which
have counterparts among existing primitive group.^
JS.
BACTERIA FROM PALEOZOIC SALT DEPOSITS
Heinz Dombrowski
Justus-Liebig University, Giessen, Germany
Stimulated by the bacteriological tindings in the mineral springs of Bad
Nauheim, which carry salts from Permian deposits, I investigated from a bac-
teriological point of view the Zechstein salts, obtained by means of mining and
drilling. Mliller and Schwartz (1953), Rippel (1945), and Strong (1956) only
achieved the isolation of dead bacteria from Zechstein salts. Reiser and Tasch
(1960) reported the living isolation of a diplococcus from Permian salts. We
now succeeded in isolating living bacteria. Yet, this achievement seemed rather
improbable; for if we had actually extracted living bacteria from Zechstein
salts, then we have to assume that we found creatures of the highest individual
age ever registered.
The following is a description of the isolating technique we used.
In bacteriological work it is obviously very easy to get unwanted secondary
infection. To be sure that this secondary effect would not spoil our results,
we used extraordinary precautions. (1) We chose a small research laboratory
in which an ultraviolet sterilization lamp was kept burning for four days before
the experiment. No one entered this room during these four days. (2) The
two researchers entered the laboratory in sterile clothes and sterile rubber gloves
after thorough disinfection of their hands and arms. (3) The table and neces-
sary tripods were covered with sterile towels. (4) All necessary instruments,
glassware, and apparatus were thoroughly sterilized. (5) The research ma-
terial, i.e., the piece of salt under consideration, was suspended on thin, sterilized
wire from the tripod. (6) This suspended piece of salt was then flamed for one
minute with a hot bunsen flame. (7) Immediately afterwards a glass with a
culture solution was brought under the piece of salt, so that it was suspended
in the solution. (8) The supporting wire was then cut and the glass was closed
after sterilizing the rim and the stopper also with the bunsen flame. (9) The
cultivation was carried out at a temperature of 40° C. (10) As soon as the cul-
ture began to grow, the elaboration to the pure culture proceeded in the usual
bacteriological manner.
To working procedure 6, I must add that the necessary time for the surface
treatment of the salt with the bunsen flame was ascertained in preliminary
experiments. Salt-pieces, which were brought into a fresh suspension of living
Pyocyaneus — about 80,000 per cm.^ — could be sterilized in 45 seconds.
Because salt is a poor heat conductor, the temperature fell rapidly toward the
center of the crystal. We heated the surface for 45 seconds. Then 3 cm. from
the surface, the temperature rose only by 6.2° C. Thus, we achieved a sterility
of the surface and regions close to the surface without producing sterilizing
temperatures in deeper layers. Of course, the crystals must be large enough;
they must have a diameter of at least 6 cm. Such specimens have a weight of
about 250 to 300 gm. A crystal this large saturates about 1 liter of culture
solution; a saturated solution is necessary for the cultivation of halophil and
halotolerant organisms.
453
454 Annals New York Academy of Sciences
For the duration of this work we set up cuhure plates on which germs in the
air coukl germinate, which in most cases did not happen. If the germs of
the air did germinate, however, they were brought into saHne solutions to
prove their tolerance to salt. This test always showed an intolerance to salt,
so that there was no identity to the bacteria that came from the salt specimens.
In counter-checks we sterilized salt crystals for 4 hours at 200° C., before
investigating them bacteriologically in the prementioned manner. These
crystals proved to be sterile. We also examined crystals coming up from a
depth of more than 4300 m.; in the Mesozoic era these salts lay about 1000
meters deeper than today. At this depth the temperature is at least 160° C,
and as expected these salt specimens also showed no sign of life.
Now, how can we find an explanation for the conservation of life over such
an extended period of time, that is for over 180 million years? There are two
possibilities. First, one is reminded of the method for conserving bacteria that
is practiced today, i.e., dehydration at low temperatures. If one extracts al-
most all the water from the protein of micro-organisms, it is possible to preserve
them for years without changing any of their particular characteristics, although
there is no metabolic activity whatsoever. We know of certain germs, which
lived for more than 30 years, although their metabolism was totally inhibited.
Starke and Harrington (1931) consider the vitality of dried bacteria as un-
limited. If this is correct, then the hypothesis of finding living organisms in
Paleozoic layers could not have received better support, and we would then
have found a way of understanding the survival of these organisms over such
long periods of time. Second, there is the possibility of reversibly denaturing
protein by salification. This method can also be used on higher organisms with
good results. For instance, the protein from the eggs of sea urchins can be de-
naturized in a saturated solution of ammonium sulfate. After months, this
process is reversible by simply removing the salts. The eggs retain the ability
to be fertilized. Perhaps in our specific case both methods, that of dehydration
and that of salification, were in effect.
If this interpretation was true, then the method should be reproducible in a
laboratory experiment. For this experimental reproduction we used Pseu-
domonas halocrenaea, which were isolated from Zechstein salts. This bacterium
does not bear spores.
If the nutrient solution in which it started growing is slowly dehydrated,
the bacterium will die. This will not happen if one slowly saturates the
solution by adding 1 gm. of salt per week. This substratum is now slowly de-
hydrated, until all salts are completely dry and crystalline. In this dry state
it can be kept for long periods of time. When bringing these salts into a fresh
nutrient solution again, the original vitality of the bacterium can be re-estab-
lished.
I would like to point out a further peculiarity: the optimal temperature for
many of the germs that we found lies between +45 and +vS5° C, which is
astonishingly high. But, elucidating enough, this temperature corresponds
exactly to that temperature which, geologists say, was present when the Zech-
stein sea was slowly drying up.
I believe that this correspondence of temperatures is certainly not accidental.
Because the bacteria were embedded in the crystals, they were assured against
Dombrowski: Bacteria from Paleozoic Salt Deposits 455
destruction by mechanical pressure. After considering the depth of our find-
ings, we can estimate a maximum of 1400 m. With the normal geothermic
gradient, which gives the temperature at a certain level, we get a maximal
value of +42° C, which the germs were exposed to during their long latent
life. This temperature in no way prevents the preservation of life.
The cjuestion of which geological specimen is to be examined is of foremost
importance. At first I used all sorts of Zechstein salts, while trying out the
bacteriological working procedure. But later, I carefully selected the speci-
mens to be investigated. All specimens, which came from questionable
regions, such as near faults or the upper salt level, were discarded. Specimens
showing signs of recrystallization were also discarded. We used only pieces
which definitely showed signs of being primary Zechstein salts, and of these only
those which came from perfectly undisturbed points in the middle of larger suc-
cessions of rock salt, the layers of which were formed normal-hypidiomorphic
to allot riomorphic. Their grain size lies in the order of millimeters. But even
with this careful selection of specimens, only about every second culture showed
results.
Because it is very probable that the organisms are of primary genesis, we can
undertake an estimation of the age of these isolated living bacteria. Because
pollen grains were isolated, which served as characteristic fossils, it was rela-
tively easy to establish the age of the bacteria.
We also centered our attention on another aspect of the problem: in undis-
turbed geological layers the rock salt has practically no pores, if we disregard
the lye enclosures. If the salt is taken out from its natural environment, it
will not be subject to the pressure of the overlaying strata anymore. It relaxes
and thus increases in volume by a few per cent. Due to this loosening, pores
begin to form and air can automatically enter the salt. This would make
possible the entering of bacterial contamination from the outside. To prove
that this was not happening, we prepared petrographic thin sections of the salt.
In examining these, we found the bacteria to be embedded in the crystalline
structure of the salt and not in the capillary crevices (figure 1).
Contrary to the previously shown Paleozoic microorganisms, this form (fig-
ure 2) is a direct decendent of the Paleozoic germ, which was obtained by cul-
tivation, and identified as Bacillus circulaus. I found this form in three differ-
ent Zechstein formations. It is a very rare specimen, which has been described
only eight times since 1890. A comparison of the Paleozoic and the Recent
representatives of this group is of special interest. When the Recent germs are
compared from an evolutionary point of view they are neither older nor younger
than the Paleozoic ones, but the Recent type has gone through completely
different stages of development. They were not preserved in a latent stage of
life, but have probably gone through an immensely great number of cell divi-
sions. If it were not for the phenomenon of circular migration, which is pecu-
liar to both the Paleozoic and the Recent type, it would be very difficult to find
a relationship between the two.
Comparing them biochemically, we find very distinct differences. Our 3
Paleozoic strains show almost identical biochemical properties. The strain
found by Kienholz lost all its saccharolytic characteristics, which its Paleozoic
relatives had. The only new characteristic is their ability to liquefy gelatine.
456
Annals New York Academy of Sciences
Beyond this fact, a comparison over such long periods of tune gives the
following results: (1) The paleozoic strains of the Bacillus circulans have quite
a lot more biochemical characteristics than those described in the preceding 70
^
^
^
\
i
%
■%
4. m
-"W
ms
Figure 1 (Top). Bacterium in the center of a thin section of a thickness of 15 ^u, enlarge-
ment 3600:1.
Figure 2 (Bottom). Bacillus circulans from the Zechstein salt, enlargement 950:1.
years. (2) It seems that the long, latent life of about 180 million years has
brought about no loss of characteristics for the Paleozoic species. (3) A loss of
characteristics was proved, however, for the Recent representatives of Bacillus
circulans, which have gone through a vast number of cell divisions. (4) Al-
though the differences in biochemical behavior are very distinct, there is an
Dombrowski: Bacteria from Paleozoic Salt Deposits 457
absolute accord in the morphological characteristics between the Paleozoic and
the Recent representatives of the Bacillus circulans. (5) This leads us to be-
lieve that the genes responsible for the morphological differentiation are much
more stable than those leading to the biochemical characteristics of a species.
There is no doubt that this goes for other species as well, but at the moment
we are only considering Bacillus circulans.
We could not have made these statements, if this species did not have the
characteristic of migration. Relying only on the peripherally whipped bac-
terium and its micromorphology, as with Bacillus circulans, any definite deter-
mination would have been impossible. Even biochemical investigations and
comparisons would lead nowhere, because there are great doubts concerning
the cjuestion of whether or not characteristics of the Paleozoic germs came to a
4
Figure 3. Bacterial strain VIII/D from the Middle-Devonian, enlargement 1200:1.
further development in Recent types. Therefore, it should be very difficult to
show the identity of other types of bacteria, isolated in mineral salts, with
Recent species beyond the probable affinity to a species.
If all of these considerations were true, then it should be possible to cultivate
bacteria from salts of even older origin than those of the Permian age, provided
that these salts come from regions where no tectonic movement had occurred
since their original formation. These experiments had positive results. In
FIGURE 3 are shown bacteria from Middle-Devonian salts from Saskatchewan.
All in all we achieved the isolation of six different species from Middle-Devonian
salts. We were also fortunate to be able to isolate three different species from
Silurian salts, coming from Meyers, New York (figure 4).
Because it was possible to cultivate 2 bacterial species out of Precambrian salt
specimens from Irkutsk, we have reached a sort of absolute level of research.
It is highly improbable that scientists will find even older individual life than
Precambrian, alread}^ approximately 650 million years old.
In FIGURE 5 is shown a bacterium from the Precambrian salt after silver
458
Annals New York Academy of Sciences
impregnation by the method of Zettnow. Both bacteria found in the Pre-
cambrian seem to be closely related to each other.
A list of biochemical data of the isolated germs from paleozoic salts is given
in TABLE 1.
#
8^ •wi^^^^-
«♦• •
'4^'
^%'~^ >•
W%Bi
I V
Figure 4 (Top). Bacterium from the Silurian, strain XV/1, enlargement 1200:1.
Figure 5 (Bottom). Bacterium from the Precambrian salt, strain XXX/1, enlargement
1200:1. (The pictured bacteria are probably the oldest known living organisms with their
approximate age of 650 million years.)
I have not yet examined salts from the Carboniferous. The bacteria from the
Precambrian, Silurian, and some from the Devonian show only few biochemical
properties. The "younger" these germs are, the more they are able to perform
biochemically, only to lose this ability in later life, as shown in the comparison
'S
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Morphology
Spore forming
Motile with flagella
Gram
Physiology
Starch hydrolysis
Nitrate reduction
Indol production
Pigment production
Gelatin liquefaction
H2S production
Salt tolerance
Methyl red test
Voges-Proskauer test
Hemolysis:
Acid from:
Glucose
Laevulose
Sucrose
Maltose
Lactose
Raffinose-hydrate
Rhamnose-hydrate
1-arabinose
Salicin
Inulin
Xylose
Trehalose
Dulcitol
Inositol
Mannitol
O
459
460 Annals New York Academy of Sciences
with Bacillus circulans. A final proof of my findings is now in preparation.
Because it is now possible to find the bacteria in thin sections of the salts, I
want to isolate each bacterium individually with a micromanipulator and let
it grow in a microculture. During this process it will be kept under constant
observation until it shows the germination of spores, or until it starts the first
cell division after being dormant for more than 650 million years. I hope soon
to be able to show this exciting moment in a motion picture film.
Other institutes are now doing research on the coenzymes and proteins of
these Paleozoic bacteria.
Summary
For the first time it became possible to isolate and cultivate bacteria from
Permian deposits. The methods of isolation are described in detail and the
arguments, which lead to the assumption that the discovered microbes are
living representatives of the oldest known individual ages, are sunmiarized.
(1) Only such salt deposits were investigated, which showed indications of being
of primary genesis. (2) From these salt specimens pollen grains were isolated,
which served as characteristic fossils for establishing the age of the deposit.
(3) None of the geological prerequisites, such as tectonics, orogenesis, and geo-
thermic gradients, proved to be contrary to the findings. (4) The method of
isolation, as well as the precautionary measures and the controlling experiments,
are discussed in detail. (5) The results of dehydration at low temperatures
and the reversible method of denaturation by salification are pointed out. (6)
The embedded bacteria are shown optically in thin sections of the examined
salts.
Studies on other salt deposits were made, and living bacteria were isolated
from salt deposits from the Middle-Devonian, the Silurian, and the Precam-
brian. A comparison of the biological characteristics of the Paleozoic germs
with Recent bacteria was carried out.
References
DoMBROWSKi, H. 1960(;. Fundamental balneobiokim. 1: H3.
DOMBROWSKI, H. 1960/). Zentr. Bakteriol. Parasitenk. 178: 83.
DoMBROwsKi, H. 1960c. Munch. Med. Wochschr. 102: 526.
DOMBROWSKI, H. 1960(/. Arztl. Mitt. 4: 143.
DOMBROWSKI, H. 1961(7. Arch. Phys. Therapie. 13(H2): 191.
DoMBROWSKi, H. 19616. Monatsh. iirztl. Forlbild. 11: 78.
DOMBROWSKI, H. 1961c. Zentr. Bakteriol. Parasitenk. 183: 173.
DOMBROWSKI, H. 1961(/. Therap. Gegenw. 100(H9): 442.
DOMBROWSKI, H. Wiss. Arheits. Burgenld. In press.
DoMBROwsKi, H. 1962(1. Kosmos. 58: H3.
DOMBROWSKI, H. 19626. Heilbad u. Kurort. 14: S50.
MtiLLER, A. & W. Schwartz. 1953. Z. Geol. Ges. 105:
Reiser, R. & P. Tasch. 1960. Trans. Kansas Acad. Sci. 63: 31.
RIPPEL, A. 1945. Arch. Mikrobiol. 6: 350.
Starke, C. N. & B. L. Harrington. 1931. J. Bacteriol. 21: 13.
Strong, M. W. 1956. Adv. Sci. 12(49): 583.
FOSSIL PROTOBIONTA AND THEIR OCCURRENCE
A. Papp
Department of Paleontology, University of Vienna, Vienna, Austria
The body of paleontological research consists essentially of knowledge of
organisms having a preservable skeleton. Therefore, one cannot expect that
the oldest organisms will have been preserved. Amino acids, but not the
organisms themselves, have been found in lower Precambrian limestone. The
earliest phase — origin of the basic building blocks, their development into
primitive organisms, as well as the primary evolutionary forms — is beyond the
scope of present paleontological basic research.
By the Cambrian (600 million years ago) many highly diversified skeleton-
forming organisms had developed. Therefore these organisms are within the
focus of paleontological research. At that time life had already attained a
considerable level of evolution, with almost all the invertebrate families present.
In spite of this fact, our knowledge of fossil protobionta is still incomplete; it is
quite possible that a whole array of different organisms is still unknown. How-
ever, the following groups may be classified: (1) bacteria; (2) protobionta with
a preservable outer skeleton of organic material; (3) protobionta with an outer
skeleton of inorganic material; and (4) sporomorpha and spores.
(1) The presence of fossil bacteria has been revealed by different techniques.
Since 1960 H. Dombrowsky's observations regarding bacteria from paleozoic
salt deposits have shown, however, that paleobacteriology is still in its infancy.
The role of anaerobic or sulfate-reducing bacteria in the development of Ufe
lies outside the field of paleontology.
(2) In the group of protobionta with preservable outer skeleton those com-
posed of organic material are believed to be the older species. In this group
only the already relatively complicated structures are known, among them such
Dinoflagellate forms as Chitinozoa and Hystrichosphaeridae. Fossil Hystri-
chosphaeridae with shells of material resembling cutin, which developed in the
Cambrian, cannot be readily distinguished from later forms. In fact, their
close resemblance to spores of fungi (Zygospores) is noteworthy. Skeletons of
fossil protobionta show remarkable resistance under suitable conditions of
fossilization.
(3) Microorganisms of simple structure, such as Archaemonadidae, Sili-
coflagellatae, Diatoms, and Radiolaria, deposit silica in their shells. Fora-
minifers built shells by cementing stone particles with calcite, or occasionally
of calcite alone. In rare instances they employed chitin-like substances. In
contrast, Dinoflagellata seusu lata, undoubtedly represent a later phase in
evolution and offer a vast amount of material for research. Coccolitho-
phoridean skeletons show small calcite particles which may form rock strata
under favorable conditions of fossilization.
(4) The terms sporomorpha and spores imply a state of reproduction con-
siderably different from the fully developed organism. Their resistant outer
layer (exine) composed of sporopoUinin ensures preservation under favorable
conditions. Sporomorpha and spores result from reduction division, which
461
462 Annals New York Academy of Sciences
may take place in a limited number of ways of which the following are known:
(a) tetrahedral — resulting in trilete spores; (b) abortive — same as (a) except
that only one spore develops fully; and (c) rhomboidal — resulting in monolet
spores.
The most common form, tetrahedral meiotic division, necessarily results in
trilate spores with three-sided symmetry. Some of the organized elements
from carbonaceous chondrites described by G. Claus and B. Nagy appear to
resemble such trilete spores. It should be emphasized that in CirciiUna sp.
of the upper Trias — although similar to the above-mentioned organized ele-
ments— the tetradic (trilete) mark is not evident.
The existence of truly multicellular organisms is allied to the function of
reduction division; otherwise, polyploidy would result. We know, however,
that in terrestrial conditions tetraploidy may cause sterility.
A so-called Dauerstadimn is usually linked with the formation of spores or
sporomorpha. Protecting the plasma is a strong hull which consists of the
exine or the sporoderm and the intine. The sporoderm is made up of sporopol-
linin, a terpene derivative, which can become soluble in the presence of oxygen
but is very resistant and capable of fossilization in the absence of oxygen when
minerals are present. It can bind iodine, bromine, and chlorine. During coal
and peat formation, where bacterial activity is reduced because of the acidic
environment, spore preservation is enhanced. Under suitable conditions rich
deposits of sporoderms may occur (fimminit).
The Dauerstadmm allows the organisms to live through highly unfavorable
periods — an especially important consideration if they are subjected to wide
variations in environment, such as extremes of cold or drought.
The majority of skeleton-forming fossil protobionta lived in the oceans of
primeval tmies, although sporomorpha and spores form in marine, limnetic,
and terrestrial biotopes. Adequate preservation of all residues of organisms
depends upon the particular fossilization process. Skeleton-forming proto-
bionta have been described mainly in marine sediments. Sporomorpha and
spores occur in both marine and limnetic deposits and very exceptionally in
terrestrial deposits. Quick embedding in all instances is favorable to the
preservation of fossils. Concentration of residues depends upon the following:
(1) mass of the organisms; (2) mass of the inorganic material involved in the
sedimentation process; {?>) resistance of the organic substance; and (4) destruc-
tive factors before and during fossilization (diagenesis). Ciranting factors 1 and
v3 even relatively small organisms may affect the mineral composition of rocks,
e.g., enrichment of Coccolithophoridae will affect the lime content of marl.
The rule for concentration of fossil spores or pollen is: 20,000 to 40,000 exines
per gram represents the accumulation of normal flora in a given sediment. A
larger number per gram is positive proof of autochthonous flora. However,
the occurrence of fewer and scattered exines points to contamination.
Minute fossilia, except for spores and pollen, are found principally in the
marine biotope. An aquatic medium is usually necessary for preservation and
fossilization of such organisms.
Theories of extraterrestrial life are based on existing conditions on earth.
Each organism, wherever it occurs, must fultill certain regular functions in line
with a given physical law in order to remain alive. The most simple organism
Papp: Fossil Protobionta 463
is a single cell whose plasma is protected by a resistant cell wall. The stronger
the wall, the more likely it is to have perforations (pores or marks) which allow
the plasma to come in contact with the surrounding environment. Only the
most primitive organisms reproduce by simple cell division. All higher forms
of life depend upon sporomorpha to survive hostile periods and to reproduce.
According to G. Erdtmann, sporomorpha in the broadest sense are spores whose
position in the system in unknown. Although they do not always exhibit
trilete markings, their three-sided symmetry may indicate that reduction
division has taken place. One of the criteria of survival is that during the
Dauerstadium substances needed to maintain life be reduced to a minimum.
The basic importance of reduction division (meiosis) to genetic propagation
has already been mentioned. It should also be noted that tetrahedral meiotic
division results in spores with three-sided symmetry. However, three-sided
symmetry is the rule with the widespread trilete spores and the exception with
protobionta and, in fact, with the total animal and plant kingdom. From the
above definition of sporomorpha, it is reasonable to apply this term to the
organized elements of extraterrestrial life having three-sided symmetry, i.e.,
the triporate or trilete forms. This does not specify their position in the sys-
tem, nor does it suggest that an organism similar to an organized element is
equivalent to it. The possibility that organized elements with three-sided
symmetry result from reduction division may not be excluded in the case of
extraterrestrial life. The function of such division is also a possibility in an
extraterrestrial environment .
Residues of extraterrestrial organisms could not be preserved at all except
for a process which may be called fossilization. The following rule holds in all
circumstances: the more residues, the more favorable were the conditions of
fossilization. This requirement is undoubtedly best fulfilled on earth in the
aquatic medium which offers conditions for suitable embedding.
The organized elements w'ith resistant exines or organic material must have
depended on the functions of protein molecules. In this event the extrater-
restrial temperature range of the organized elements' environment would have
to be similar to that on the earth.
A cknowledgments
I wish to thank Dr. W. Klaus, Dr. A. Freisinger, and Dr. K. Turnovsky for
many stimulating discussions on this subject.
STUDIES IN EXPERIMENTAL ORGANIC COSMOCHEMISTRY
J. Oro
Chemistry Department, University of Houston, Houston, Tex.
The four most abundant elements in the universe, with the exception of
the noble gases, are hydrogen, oxygen, carbon and nitrogen,'"'^ which are also
precisely the four major constituent elements of organic compounds and of
living matter. Indeed, as has recently been said, "the composition of living
matter turns out to be a better sample of the universe than the dead earth."''
These four elements exist mainly as atoms and diatomical combinations,
such as CN, CH, C2 , CO, NH, and OH, in the atmospheres of relatively cool
stars,^'^ including the sun,^ and in interstellar or circumstellar space.^'^-^ They
also exist as di- and polyatomic combinations in planets,^"" comets,^-'i^ and
meteorites. ^^'^^ Thus, simple and complex compounds of carbon are found
widely distributed in the universe. In principle, these compounds will exist
wherever the prevailing temperatures are compatible with the stability of the
bonds between carbon and the other elements. If the carbon containing
diatomic combinations, CN, CH, C2 , and CO, are considered, it is observed
that their thermal stability ranges from the low temperatures of interstellar
space to the relatively high temperatures prevailing above the surface of stars.
In fact, such diatomical combinations have been detected in the atmospheres of
supergiant carbon stars at temperatures of the order of 6000° K. at which some
of the most thermally stable oxides, namely titanium and zirconium oxides,
are dissociated into their metallic ions.'
Observations bearing on the distribution of simple and complex compounds
of carbon in cosmic bodies and on the natural formation of these compounds,
form part of a space science which may be called "organic cosmochemistry."
Because of the limited observational data so far obtained and the importance
of the fundamental problems involved ,-'''^^ attempts have been made to follow
an experimental approach in this study. As a result of the initial experiments
of Garrison et al.p Miller,^" '^^ and the more recent ones carried out in this^- and
other laboratories,^^ it has become apparent that processes of organic synthesis
which may have occurred in the primitive Earth's atmosphere, or may be
occurring in certain cosmic bodies such as comets, can be partially reproduced
in the laboratory. These experiments have opened a field of investigation
for which the name "experimental organic cosmochemistry" is proposed.
Models for Organic Synthesis
Any experimental approach to duplicate an incompletely known natural proc-
ess requires the formulation of assumptions about the experimental model to
be used. It is recognized that it would be difficult to determine with certainty
all the conditions applying to a cosmic model for organic synthesis. However,
if it is understood that organic reactions pathways are determined by rather
general laws, then it becomes possible to obtain significant knowledge about
natural organic synthetic processes even with only partially complete models.
We have focused our attention on a cometary modeP" primarily because
464
Oro: Experimental Organic Cosmochemistry 465
comets are supposed to contain large amounts of reactive carbon compounds
and because it is considered that their composition reflects approximately the
composition of the primordial solar nebula and protoplanets.'*'* Indeed, a
recent model for the protoplanets of the solar system,-''^ as suggested by Fowler,
is almost identical to a cometary model proposed some time ago by Whipple^^
and recently revised by the same author.'*^ Cn the basis of this physical and
chemical similarity it is reasonable to assume that the chemical processes which
occur in comets by the action of solar radiation, when these bodies are at dis-
tances of less than 3 A.U. from the sun may have also occurred, but in a much
larger scale in the Earth protoplanet.
Furthermore, it is possible that the conditions for organic synthesis were
quite favorable during the transformation of the gravitationally undiffer-
entiated protoplanet into the primitive planet. This would result from the
mixing of the reactive precursors of organic compounds with inorganic particles,
such as silicate and metallic grains, which could have acted as surface catalysts.
Due to the low density of the synthesized organic compounds, these compounds
would migrate toward the exterior of the planet during the process of gravita-
tional differentiation. The nonvolatile combinations, ionic or high molecular
weight compounds, would accumulate on the surface of Earth, whereas the
gases and the compounds volatile at the prevailing temperatures, would be
evaporated into the outer region of the solar system where comets originate
presently .^^ The difiicult problem of the escape of gases and volatile com-
pounds from primitive planetary atmospheres has been discussed mainly by
Suess'^ and Urey.-'-^"
With regard to the composition of the model, it is known that the spectra of
comets show fluorescence emission bands corresponding to the molecules or
radicals CN, CH, CH. , C2 , C3 , NH, NHo , and OH, to the radical ions CH+,
0H+, C0+, N2+, and CO2+ and to the atoms of Fe, Ni, Cr, and other ele-
ments.'-'^^ ■■*"•*' These emission bands are observed in the heads or in the tails
of comets when these bodies are at less than 3 A.U. from the sun. The band
corresponding to the CN radical is generally the first emission band to appear
on the tails of comets during the travel of these bodies toward the sun, and it is
also the band with the largest degree of extension into the comets' heads fol-
lowed in intensity by the C2 (Swan) and C-i bands.
The above compounds exist in the nuclei of comets either as frozen free
radicals,^--^^ or as "ices"'*'^-'*^ (or crystalline clathrate type hydrates^^) of mole-
cules, which are vaporized and dissociated into radicals by the solar radiation.
In general, it is considered that the parent molecules of CN, NH2 , and OH are
hydrogen, cyanide or cyanogen, ammonia, and water, respectively. The
parent molecules of the carbon radicals are supposed to be methane, acetylene,
and other hydrocarbons. Therefore, a simplified experimental model could be
made of hydrogen cyanide, ammonia, and water. A slightly more complex
model could contain, in addition, cyanogen, acetylene, carbon monoxide, carbon
suboxide, and other compounds. There are certain relations between this
model and the 2 atmospheric models which have been studied previously,
namely, the "primitive planetary atmosphere" model,-^ and the "volcanic
atmosphere" model. ■*'^'^' These models should not be considered as providing
466 Annals New York Academy of Sciences
alternative, but rather complementary, approaches to the study of the forma-
tion of organic compounds on the abiotic earth.^^'** In fact, they represent
progressive stages in the development of the earth. An important condition
which is common to all of these models is thai they are essentially reducing or
at least nonoxidizing in character, of which we have cosmochemical^"-^' and
geochemical^- evidence. Additional evidence for the reducing conditions of
the atmosphere of magmatic origin is provided by the fact that the terrestrial
rate of oxygen production by photolysis of water is less than the rate of vol-
canic carbon monoxide production.*^
Energy Sources
Several sources of energy were available for the synthesis of organic com-
pounds during the transformation of the Earth from protoplanet into planet.
The main source was, of course, the sun providing ultraviolet light and ion-
izing radiation at a rate 10^ times as high as that observed at the present time.^*
A second source was the earth itself with its natural radioactivity^^ ■*■* and the
heat derived from gravitational compression and radioactivity.^^
However, I wish to emphasize that if, as indicated above, some of the primor-
dial constituents of the earth protoplanet were radicals or reactive chemical
compounds, then organic synthesis could have occurred spontaneously at
relatively low temperatures during the melting of the protoplanetary ices in
the absence of highly activating forms of energy. It is surmised that these
spontaneous syntheses were responsible for the formation of substantial
amounts of organic and biochemical compounds. Furthermore, due to the rela-
tively low prevailing temperatures and the reducing conditions of the proto-
planetary environment, the compounds thus formed would have been pre-
served for very long times.
During the further stages of geological development additional sources of
energy were available on the surface and atmosphere of the earth. It is
likely that in addition to ultraviolet light and ionizing radiation, electric
discharges and the heat from plutonic processes contributed also to the forma-
tion of organic compounds.
Synlliesis of Amino Acids and Hydroxy Acids
The synthesis of amino acids and hydroxy acids under possible primitive
Earth conditions has been accomplished by several investigators who used
electrical discharges, ultraviolet light, and ionizing radiation. Moreover,
when some of the reactive carbon compounds detected in comets were used, the
formation of amino acids and hydroxy acids was observed to occur spon-
taneously at moderate temperatures.
(/) By electric discharges. In particular, Loeb,** Miller, *^"*^ Hough and
Rogers,*^ Abelson,^'' Heyns e/ a/.,''^ Pavlovskaya and Pasynskii,*^- Franck,^^ and
Oro and Engberg,^'' applied silent and spark discharges to aqueous mixtures of
totally reduced (CH4 , NH3) or partially oxidized carbon and nitrogen com-
pounds. The products obtained include the amino acids glycine, alanine,
(3-alanine, sarcosine, a-amino-n-butyric acid, a-aminoisobutyric acid, glutamic
acid, aspartic acid, valine, and leucines, and the hydroxy acids glycolic, lactic,
succinic and hydroxybutyric.
Oro: Experimental Organic Cosmochemistry 467
The yield of total amino acids in these experiments was usually less than 5
per cent of the theoretical and the relative yield of each individual amino acid
was approximately inversely proportional to the number of carbon atoms in the
molecule. When methane was used the amino acids formed contained almost
exclusively from 2 to 4 carbon atoms. When methane was replaced partially
by ethane or higher hydrocarbons, valine and leucines were formed in addition
to the other amino acids. ^^ Aside from these and other small variations, the
overall qualitative composition of amino acids obtained in different experiments
by several investigators is very similar, if not identical.
Although the mechanisms of synthesis have not been studied in detail, it
seems that the first phase of one of the possible mechanisms involves the forma-
tion of radicals which recombine to form many compounds including hydrogen
cyanide, aldehydes, amines, nitriles, and aliphatic hydrocarbons. The primary
formation of methyl radicals has been suggested by the experiments of Franck,^^
with either isooctane or methanol in the presence of ammonia and water.
When methanol was used, the observed amino acid yield was increased more
than 50 per cent as compared to that obtained from methane. This is in
line with the fact that 20 per cent less energy is required to form a methyl
radical from methanol than from methane. ^^ That methyl radicals are formed
can also be deduced from a study of the products formed by the action of
electrical discharges upon methane, ^^ and upon mixtures of methane and
ammonia. ^^ Because of the high thermal stability of the triple bonded radical
C2H derived from acetylene®* one would expect that this radical should act as a
trap for other radicals giving rise to the formation of methyl, ethyl, vinyl, and
ethynyl derivatives of acetylene. In fact, these compounds were precisely
the products identified in the aforementioned experiments.*® In a similar
manner the nitrile analogues of the above compounds, namely, acetonitrile,
propionitrile, acrylonitrile, and cyanogen should also be expected to be formed
from the thermally stable triple bonded CN radical derived from hydrogen
cyanide. And in fact some of these compounds were detected by Sagan and
Miller" in model experiments with Jovian atmospheres.
The second phase of this mechanism of amino acid synthesis does not seem to
occur in the gas phase, but rather in aqueous solution. It involves a Strecker
condensation of aldehydes with hydrogen cyanide in the presence of
ammonia.^® '^^ The resulting a-amino acid nitriles which can be detected during
the first hours®'* are progressively hydrolyzed into the corresponding amides
and acids.
In addition to a-amino nitriles, (8-aminonitriles have also been detected in the
reaction product. In particular, |S-aminopropionitrile which is a precursor of
/3-alanine and of pyrimidines has been detected by paper chromatography,®^
This nitrile gives a characteristic green derivative when it reacts with ninhydrin.
An alternative mechanism for the formation of amino acids in the experiments
with electrical discharges is suggested by the presence in the reaction product of
polymers of hydrogen cyanide which are known to be converted into amino
acids (section (4)).
(2) By ultraviolet light. Studies on the photochemical synthesis of amino
acids in aqueous systems were reported some time ago by several investigators.
Baudisch®^ claimed the formation of amino acids from potassium nitrite, carbon
468 Annals New York Academy of Sciences
monoxide, and ferric chloride. Dhar and Mukherjee observed the formation of
glycine from glycol, and of arginine from glucose. Nitrates were used as a
source of nitrogen and titanium dioxide or ferrous sulfate as catalyst. More
recently, Bahadur ef a/.,^^"'^ also with the use of nitrates and ferric chloride
have observed the formation of serine, aspartic acid, and asparagine from
paraformaldehyde. Other amino acids formed in these experiments as detected
by paper chromatography (without previous separation from other ninhydrin
positive compounds by ion exchange) were glycine, alanine, and threonine and
in particular C5 and Ce amino acids which are formed with difficulty in the
experiments with electric discharges. These include valine, ornithine, arginine,
proline, glutamic acid, histidine, leucine, isoleucine, and lysine. The above
amino acids comprise essentially all the building blocks of proteins with the
exception of the aromatic and sulfur containing amino acids.
It would be difficult to visualize the presence of nitrates in a primitive Earth
environment or in a cosmic body. However, the nitrate ion per se should not
be considered as the immediate precursor of the amino group of amino acids.
It is clear that the nitrates must be reduced at the expense of the oxidation of
part of the carbon compounds, such as formaldehyde, which are always present
in a large excess in these experiments. In fact it is known that in the presence
of metallic ions and partiahy reduced carbon compounds, nitrates,'^^ and ni-
trites^^ are rapidly reduced by the action of light to some nitrogen compound of
a lower oxidation level.
Hydroxylamine was suggested by Oro et al.^^ as one of the nitrogen com-
pounds which may be involved more directly in the formation of amino acids.
In fact, this could also be deduced from the synthesis of amino acids from
formhydroxamic acid and formaldehyde by Baly et alP The preferred partici-
pation of hydroxylamine in the comparative photochemical synthesis of amino
acids from formaldehyde and either nitrates, nitrites, hydroxylamine hydro-
chloride, or ammonium chloride has been coniirmed in our laboratory .^^ The
same conclusion has been arrived at by Ferrari^^ •^'' from similar comparative
photochemical experiments but with more complex carbon compounds instead
of formaldehyde.
From a conceptual point of view, ammonia and ammonium chloride are
perhaps the most logical precursors of the amino group of amino acids in a
primitive Earth environment. Experiments carried out by Miller,^** and by
Groth and von Weyssenhoff ,*i '^^ have given evidence that the amino acids
glycine and alanine can be synthesized by irradiating wjth short wave ultra-
violet light (Krypton 1165, 1235 A, Xenon 1295, 1470 A, and mercury vapor
1850 A), aqueous mixtures containing ammonia as the nitrogen source and
either methane or ethane as the carbon source. A higher amino acid yield was
obtained when ethane was used instead of methane. On exposing a mixture
of methane, ammonia, carbon monoxide, and water to the radiation of a hy-
drogen lamp through a thin LiF window, Terenin**^ observed the formation of
the alanines and of several other amino acids.
On the basis of the experimental quantum yields obtained by Groth and
recent theories of solar evolution, Sagan^'' has calculated that the synthesized
organic compounds in the contemporary atmospheres of the Jovian planets.
Oro: Experimental Organic Cosmochemistry 469
and in the primitive reducing atmospheres of the terrestrial planets is of the
order of 1000 g. per cm.^ of planetary surface.
Experiments carried out by Pavlovskaya and Pasynskii"- and also in this
laboratory/^ have shown that several amino acids can be synthesized by irradia-
tion with ultraviolet light of aqueous mixtures containing formaldehyde and
ammonium salts. The synthesized amino acids, which were separated by ion
exchange resins and detected by paper chromatography, include glycine, serine,
alanine, and glutamic acid. The Russian investigators found also vaUne,
isoleucine, phenylalanine, and basic amino acids.
With regard to the mechanism of photochemical synthesis of amino acids it
has been pointed out previously, that the amino group may be derived from
either ammonia or hydroxylamine. However, very little is known about the
mechanism of formation of the hydrocarbon chain. Perhaps monosaccharides
of 2 to 6 carbons are lirst formed photochemically and then transformed by
redox processes into a-keto acids which upon transamination are converted into
amino acids.
That hexoses and hydroxy acids or their lactides are formed by the irradiation
of formaldehyde solutions with ultraviolet light was shown by Baly^^ and Irvine
and Francis.'^'' Moreover, when the syrupy product, thus obtained, was
heated with a trace of acid at 100° C. it was found to resinify into a polymeric
material. This suggested the additional presence in the reaction product of
furfuryl alcohols or polyhydroxyphenols. If phenolic compounds were formed
from formaldehyde these compounds may be the precursors of the aromatic
amino acids.
That hydroxy acids and also keto acids and dicarboxylic acids react photo-
chemically with ammonia, ammonium salts, or other nitrogen compounds to
produce amino acids has been shown by Deschreider*^ and by Cultrera and
Ferrari.^^'*^ Nonphotochemical transamination reactions are also well known.
The synthesis of amino acids containing straight chains with 5 or 6 carbon
atoms could be explained by the intermediate formation of Cs or Ce mono-
saccharides, respectively. These compounds become stabilized by the forma-
tion of furanose and pyranose cyclic structures, stopping the growth of the
monosaccharide chain by preventing the condensation of additional formalde-
hyde molecules. Therefore, essentially no monosaccharides and amino acids
with a linear chain of more than 6 carbon atoms are formed. Branched chain
amino acids could be derived from branched chain monosaccharides such as
dendroketose.
It is of interest that the same maximal amino acid chain length is observed in
these photochemical experiments as in the experiments with electric discharges.
Whereas in the present case the maximal chain length may be determined by
the stability of cyclic structures, in the experiments with electrical discharges it
may be the result of the decreased probability of formation of long chains by
processes of methyl radical recombination.
(3) By ionizing radiations. The synthesis of organic compounds by ionizing
radiation was reviewed by Swallow.'^* After the pioneering investigations in this
area by Garrison et al.,-^ the formation of amino acids by the action of ionizing
radiations has been studied by several investigators. Hasselstrom et al.,^^
470 Annals New York Academy of Sciences
obtained glycine, aspartic acid and possibly diaminosuccinic by irradiating
with j8-rays an aqueous solution of ammonium acetate. Paschke et al.,^^
irradiated solid ammonium carbonate with the 7-rays from a cobalt-60 source
and obtained glycine, 2 other ninhydrin-positive compounds, 1 of which was
tentatively identified as alanine, and ammonium formate.
It is known that formic acid and simple aldehydes are formed by the action
of ionizing radiation over aqueous solutions of carbonic acid.-^-^^ It is also
known the glycolic acid is produced by the irradiation of formic acid.''^ There-
fore, it is conceivable that glycine and other amino acids could also be obtained
by the irradiation of aqueous solutions of ammonium carbonate.
Although from the above experiments it is evident that amino acids can be
synthesized from partially oxidized compounds such as ammonium carbonate,
it would seem more logical, on the basis of theoretical considerations,^^ to study
the irradiation of aqueous mixtures of reduced carbon and nitrogen compounds,
such as methane and ammonia. This has been done by Dose et o/.,^'*'^^ and a
larger number of amino acids and bases have thus been obtained. More
recently, Calvin^® and Palm and Calvin^'' have irradiated mixtures containing
C^*-methane, ammonia and water, among other compounds, with 5 MeV elec-
trons and have obtained a number of amino acids including glycine, alanine,
and aspartic acid. Radiochemical and nonradiochemical mechanisms of syn-
thesis may be involved in this case because hydrogen cyanide, which is known
to condense into products which yield amino acids, was also formed in sub-
stantial amounts in these experiments.
Apart from these amino acid syntheses, it may be added that the 7-irradia-
tion of mixtures of carbon dioxide and ethylene at room temperature yields
significant amounts of long chain carboxylic acids containing as many as 40
carbon atoms. ^^ Also, high energy proton or electron irradiation of methane,
ammonia, and water at 77° K., in a simulated cometary model, yields a number
of organic compounds. ^^
(4) From reactive precursors. As pointed out earlier it is known from astro-
nomical observations that in the atmospheres of carbon stars, very reactive
diatomic combinations of carbon, nitrogen, oxygen and hydrogen are formed.
These combinations are presumed to diffuse out and eventually become part of
interstellar matter, cosmic bodies and protoplanets, being converted in the
process into simple but reactive compounds. These may include hydrogen
cyanide, acetylene, carbon monoxide, formaldehyde, acetaldehyde, ammonia,
hydrazine, and hydroxylamine among others. Some of these compounds have
also been produced in the laboratory from aqueous ammonia-methane mixtures.
Thus, it was considered of interest to discover whether some of these com-
pounds are sufficiently reactive to yield amino acids, and other biochemical
compounds in the absence of electrical discharges, ultraviolet light, or ionizing
radiation.
It was first shown in our laboratory^® that aqueous mixtures of formaldehyde
and hydroxylamine hydrochloride at moderate temperatures and under slightly
acidic conditions yield large amounts of glycine and smaller amounts of alanine,
(3-alanine, serine, threonine, and aspartic acid, the last 3 having been only
identified by paper chromatography. Amino acid amides, glycinamide in
Oro: Experimental Organic Cosmochemistry 471
particular, were found as intermediates, and formic, lactic, and glycolic acids
as side products.
It was found^'' that the mechanism of synthesis involves the initial formation
of formaldoxime and its dehydration into hydrogen cyanide. Strecker and
cyanohydrin condensations yield nitriles which are hydrolyzed first into amides
and then into acids. Condensation of formaldehyde with glycinamide is
presumed to yield serinamide which can be converted into serine and alanine.'""
A similar formation of serine and threonine involving aldol type condensations
of formaldehyde and acetaldehyde with methylene-activated glycine deriva-
tives, such as glycine chelates or polyglycine, was also shown by Akabori
et a/.i"'"i"^ It may be added here that when the formaldehyde-hydroxylamine
hydrochloride mixtures were made slightly basic, pyridines were also formed
in addition to amino acids.
A subsequent study in our laboratory of the products formed by refluxing
aqueous mixtures of formaldehyde and hydrazine revealed the formation of
glycine, vaHne, and lysine as detected by paper chromatography.'"* The
mechanism of lysine formation is thought to involve the intermediate formation
of hexoses and their reduction-oxidation by hydrazine. It is well known that
hexoses are formed from formaldehyde by base catalysis, that hydrazine is
formed by the action of electric discharges on ammonia,'"^ and that hydrazines
can be both reducing and oxidizing reactants.
As mentioned earlier, 3 of the major compounds which are supposed to
exist in comets are hydrogen cyanide, ammonia, and water. For this reason, a
study of the products formed with mixtures of these 3 compounds was subse-
quently undertaken in our laboratory. It was observed that the amino acids
glycine, alanine, and aspartic acid, and other biochemical compounds were
formed spontaneously at moderate temperatures in these mixtures.'*"^ Oli-
gomers of hydrogen cyanide are presumed to be the intermediates of the amino
acids. In fact, tetrameric hydrogen cyanide was observed to be one of the
first products formed in the above mixtures,'"^ and it is known that tetrameric
hydrogen cyanide can be hydrolytically degraded into glycine.'"*''"^ Two
possible degradation mechanisms of tetrameric hydrogen cyanide into glycine
have been suggested by Loquin"" and Ruske."' Other mechanisms involving
processes of reductive deamination can be postulated for the formation of
alanine and aspartic acid.
The formation of amino acids in the hydrogen cyanide-ammonia-water
mixtures has been confirmed and extended by Lowe et al}^'^ In addition to the
above 3 amino acids, Lowe and co-workers have also detected the presence of
(8-alanine, a,(8-diaminopropionic, a-aminoisobutyric, glutamic acid, arginine,
leucine, and isoleucine in the reaction product. The formation of hydroxy
amino acids could conceivably take place in these mixtures if aldehydes were
present, because it is known that formaldehyde and acetaldehyde condense
with methyleneaminoacetonitrile to form serine and threonine, respectively."^
It can thus be seen that, with the exception of the aromatic and sulfur con-
taining amino acids, most of the building blocks of proteins can be synthesized
nonenzymatically in aqueous sytems from very simple precursors in the absence
of highly activating forms of energy.
472 Annals New York Academy of Sciences
With regard to the formation of sulfur containing amino acids, simple
nonenzymatic pathways can also be visualized. Cysteine could be formed in a
similar manner as serine by condensation of thioformaldehyde"'* with a methyl-
ene-activated glycine derivative, such as glycine nitrile, glycinamide, poly-
glycine or a metal chelate of glycine. Methionine could be formed by the addi-
tion of methyl mercaptan to acrolein, followed by the condensation of the
resulting methional"'^ with hydrogen cyanide and subsequent hydrolysis of the
nitrile. One of the possible pathways for the synthesis of aromatic amino acids
could be through monosaccharides or similar compounds obtained from form-
aldehyde.**^
Synthesis of Monosaccharides
Since the early studies of Butlerow,"'"' Loew,"^ and Fischer"* it has been
known that formaldehyde in aqueous solutions condenses into sugars by the
action of basic catalysts. As a result of the work of Fischer"* and others,"^-'-"
fructose, sorbose, xylulose, and glycolaldehyde were identilied among other
compounds in the formaldehyde reaction product.
Relatively recently, Mariani and Torraca'^^ analyzed by two-dimensional
paper chromatography the product of the base catalyzed condensation of
formaldehyde and confirmed and extended the previous results. They detected
the presence of the hexoses galactose, glucose, mannose, fructose and sorbose,
and the pentoses arabinose, ribose, ribulose, xylose, xylulose, and lyxose in
addition to 10 more unidentified monosaccharides. More recent studies by
Mayer and Jaschke^- and by Pfeil and Ruckert^-^ have shown the formation of
glycolaldehyde, glyceraldehyde, dihydroxyacetone and tetroses in addition to
pentoses and hexoses. Dendroketose was also obtained as the product of the
condensation of two moles of dihydrox3^acetone.
The reaction is supposed to be initiated by the condensation of two moles of
formaldehyde into glycolaldehyde which occurs at a very slow rate (induction
phase) .1-* This is followed by aldol condensations which lead to the formation
of trioses, tetroses, pentoses, and hexoses and use up all the formaldehyde in a
very short time (autocatalytic phase) .^-^ The overall reaction is catalyzed by
calcium carbonate, calcium oxide, and other bases.
Because no attempts had been reported on the synthesis of 2-deoxypentoses.
in particular 2-deoxyribose, we undertook the synthesis of this compound,^"
which is known to be one of the essential building blocks of deoxyribonucleic
acid. This deoxypentose and its isomer, 2-deoxyxylose, were obtained in
yields of about 5 per cent by the condensation of acetaldehyde with glyceral-
dehyde in aqueous systems. The reaction occurs very rapidly at room tem-
perature when catalyzed by calcium, magnesium and other divalent metallic
oxides. Results from our laboratory have shown that the reaction is also
catalyzed by ammonia and other simple nitrogen bases which may have been
the predominant bases in the primitive Earth's environment. In contrast to
the fast reaction which divalent metallic oxides catalyze, the reaction occurs
in a slow and controllable manner when ammonium hydroxide is used as cata-
lyst. In fact, the continuous synthesis of this compound was observed for an
uninterrupted period of more than 2 months. 2-Deoxyribose was also obtained
Oro: Experimental Organic Cosmochemistry 473
in smaller yields from aqueous solutions of formaldehyde and acetaldehyde in
the presence of calcium oxide.^-^
Synthesis of Purines and Purine Intermediates
The formation of purines on the primitive Earth or in cosmic bodies pose^
a priori a difficult conceptual problem because it requires the formation of two
fused heterocyclic structures, an imidazole and a pyrimidine.
In principle, there are, however, two relatively simple mechanisms or path-
ways which can be visualized for the formation of the purine ring. One involves
condensation of a 3-carbon compound with a 1-carbon reactant to form a 4,5-
disubstituted imidazole and the other involves condensation of a C3 compound
with a Ci reactant to form a 4,5-disubstituted pyrimidine. The reaction
terminates by cyclization of either the disubstituted imidazole or the disubsti-
tuted pyrimidine with another mole of the Ci reactant.
It is known that the formation of purines in living organisms occurs by a
pathway involving 4,5-disubstituted imidazole derivatives,^-'' and it has also
been observed that the acid degradation of adenine yields 4-aminoimidazole-5-
carboxamidine as an intermediate.'-^ On one hand we have the very mild
conditions of enzymatic synthesis and on the other hand the very drastic
conditions of acid hydrolysis, yet in both cases a 4,5-disubstituted imidazole
shows as an intermediate. Shortly after these observations were made it
became apparent to the author that if a nonenzymatic synthesis of purines
under possible primitive Earth conditions was discovered, it may likely proceed
through the imidazole pathway. The first demonstration of the spontaneous
synthesis of adenine from hydrogen cyanide under conditions presumed to
have existed on the primitive Earth was made relatively recently in our labora-
tory,^^* and in line with the above reasoning 4,5-disubstituted imidazoles were
found in the reaction product as intermediates.
Adenine was synthesized in substantial amounts by heating a solution of
hydrogen cyanide (1 to 15 m) in aqueous ammonia for 1 or several days at
moderate temperatures (27 to 100°). The insoluble black polymer of hydrogen
cyanide was removed by centrifugation and adenine was isolated from the
red-brown supernatant solution by chromatographic methods. The main
ultraviolet absorbing compound of the reaction product was identified as
adenine by a number of different procedures including ultraviolet spectro-
photometry and the melting point of its picrate derivative. The synthesis was
found linear with time at room temperature, and in a typical experiment at the
end of 4 days more than 100 mg. of adenine per liter of reaction mixture were
obtained.^'^^
Since adenine is an essential building block of nucleic acids and of the most
important coenzymes, and since hydrogen cyanide, ammonia, and water are
presumed to be common natural constituents of the solar system, these findings
were considered to be of special significance in relation to the problem of the
origin of life.
In addition to adenine several purine precursors, namely 4-aminoimidazole-
5-carboxamide (AICA), 4-aminoimidazole-5-carboxamidine (AICAI), form-
amide, and formamidine were also found in the reaction product.'^^'^^' The
474 Annals New York Academy of Sciences
mechanism of adenine synthesis is supposed to be initiated by the base catalyzed
polymerization of hydrogen cyanide into nitriles.^^- The role played by am-
monia in this synthesis is 2-fold. It acts as a basic catalyst and it causes the
ammonolysisof hydrogen cyanide into formamidine and of nitriles into amidines.
One of the resuhing nitriles, possibly aminomalonodinitrile, condenses either
directly or after transformation to its mono- or diamidine with formamidine to
form AICAI. In the last step, AICAI condenses with another mole of formami-
dine to yield adenine. This last step has been confirmed in a separate experi-
ment in our laboratory. ^^^
The other purines were postulated to be formed from 4-aminoimidazole-5-
carboxamide.^^^ Recent experiments in our laboratory have confirmed this
assumption. 1^* It has been observed that AICA and guanidine condense in
aqueous ammonia systems to yield guanine. Moreover, when AICA is allowed
to react with urea under similar conditions, guanine and xanthine are formed. '^^
The formation of the 1-carbon reactants, guanidine and urea, in the absence of
free oxygen, poses no special problem because compounds of this oxidation
level, such as urea, were detected by Miller, ^^ Berger,^* and Palm and Cal-
vin,^^ in their respective experiments with electric discharges, high energy
protons, and high energy electrons, which were carried under reducing condi-
tions. Other workers have also observed the formation of guanidine^^- and
urea^^^'^^^'^^^ from cyanides, cyanogen, or cyanates.
The above experiments on the synthesis of adenine from mixtures of hydrogen
cyanide, ammonia, and water have been confirmed by Lowe et al}^^ who have
found an additional purine, hypoxanthine, among the reaction products. A
significant extension of these experiments has been carried out recently by
Calvin, ^^ and Palm and Calvin," who have observed the formation of adenine
by irradiating with 5 MeV electrons a mixture containing methane, ammonia,
and water among other reduced compounds. In summary, it seems to be well
established that the 4 major biological purines can be synthesized, from very
simple precursors, in aqueous systems under possible primitive Earth conditions.
From a historical point of view, it should be said that at the turn of the last
century, cyanogen^^^ and hydrocyanic acid^^^'^^^ were thought to be involved in
the synthesis of proteins and purines in living organisms. These have since
been found to be erroneous concepts. Nevertheless, it is of interest that such
early ideas may apply to the abiogenic formation of these compounds. Studies
on the polymerization of hydrocyanic acid were initially carried out more than
150 years ago,'^- and, therefore, it is highly probable that purines, purine inter-
mediates, and other compounds of biological significance were synthesized in
the laboratory many times since then, yet have remained unidentified until the
present time. Interesting observations bearing on the synthesis of purines from
hydrogen cyanide were made by Gautier,''"* Fischer,'*'' Salomone,^'" and Johnson
and Nicolet,'*- and they are discussed in some detail in a recent paper from our
laboratory.'^' Aside from these early unsuccessful attempts on the synthesis
of purines from hydrogen cyanide, it should be added that uric acid was syn-
thesized from glycine and urea by Horbaczewski,''*^ and purine from formamide
and other simple compounds by Bredereck et «/.'■" '^^ However, none of the
biochemical purines found in nucleic acids was isolated or identified in these
experiments.
Oro: Experimental Organic Cosmochemistry 475
Synthesis of Pyrimidines
With regard to the formation of pyrimidines it was proposed recently^- that
derivatives from the C3 molecular species found in comets could be the source
of these heterocyclic compounds. One of these C3 derivatives is malonamide
semialdimine or its isomer /3-aminoacrylamide which by condensation with urea
could be expected to yield uracil.
Because (S-aminoacrylamide was not available to us, we tested some of the
C3 compounds which are formed in the experiments with electric discharges
and which are considered to be intermediates in the formation of i3-alanine.
These intermediates are acrylonitrile, /3-aminopropionitrile, and /3-aminopro-
pionanide. When each of these compounds was allowed to react with urea in
aqueous ammonia systems at 130° C, the formation of small amounts of uracil
was observed in each case.''*'^ Uracil was characterized by paper and ion
exchange column chromatography and by ultraviolet spectrophotometry. The
yields obtained from /3-aminopropionanide were approximately 2 and 5 times
higher than those obtained from /(i-aminopropionitrile and acrylonitrile, respec-
tively. This is what would be expected if acrylonitrile has to undergo first
amination into /3-aminopropionitrile and this, in turn, has to undergo hydrolysis
into /3-aminopropionanide. Because this amide is, in fact, the dihydroderiva-
tive of /3-aminoacrylamide it is obvious that the mechanism of the reaction
must involve a dehydrogenation step either before or after the cyclization.
The mechanism of uracil formation involving |S-aminoacrylamide or its
isomer, malonamide semialdimire, is in line with the well known chemical
synthesis of uracil from malic acid and urea in the presence of a strong mineral
acid.^''^''^'^ A strong mineral acid transforms malic acid into malonic semialde-
hyde which then condenses with urea to form uracil. '^^ Also, in line with the
above mechanism, it is known from the work of Bredereck et al.,^'^^ that the
pyrimidine ring can be formed in good yield from either aminoacrolein or
malonodialdehyde. In theory the 3 pyrimidines found in nucleic acids could
conceivably be formed in aqueous systems under possible primitive earth
conditions by the mechanism described above. In addition to /3-aminoacryl-
amide yielding uracil, /3-aminoacrylamidine could be expected to condense with
urea into cytosine, and a-methyl-;3-aminoacrylamide into thymine.
A possible pathway for the conversion of the symmetrical C3 species of comets
into ;8-aminoacrylamide or malonamide semialdimine is through the formation
of carbon suboxide (C3O2), which has been suggested to exist in several cosmic
bodies.''^*' By the addition of hydrogen and ammonia to carbon suboxide,
malonamide semialdehyde or malonamide semialdimine might be obtained. In
fact, malonic acid derivatives have been obtained recently in the laboratory
from carbon suboxide.^^^ In addition to purines and pyrimidines, preliminary
data have been obtained on the synthesis of other heterocyclic compounds and
fluorescent pigments. ^•^-
Synlhesis of Polypeptides
The early literature on the direct polymerization of unsubstituted amino
acids has been previously reviewed in some detail. '•^^"^'•^ Current studies on
the synthesis of peptides and of polymers containing amino acids, under condi-
tions presumed to have existed on the primitive Earth were initiated by Fox and
476 Annals New York Academy of Sciences
Middlebrook/^^ and by Akabori.^^^ This work has been reviewed recently'^*"^^^
and has been extended by other workers. As a result of these investigations a
number of different pathways for the formation of polypeptides in a cosmic
body or on the primitive Earth seems possible.
Polymers containing many of the amino acids found in proteins can be pre-
pared by heating a mixture of these amino acids in the presence of an excess of
dicarboxylic"'- ■^'^^ or diamine amino acids.^^^ This synthesis requires anhydrous
conditions and heating at high temperatures for relatively short periods of
time.
The formation of homo- and heteropolypeptides can occur also under aqueous
conditions and at moderate temperatures, as shown by other workers. Thus,
unsubstituted amino acids'^' '^^^ and their corresponding amides^ *''^"'^^ and
nitriles'®^'^^'^^^ have been observed to polymerize directly, or by the action of
basic (ammonia) or surface (silicates) catalysts.
A pathway which seems to be particularly good for the formation of poly-
peptides containing hydroxy acids is that of Akabori et al.,^^^ which is based
upon the condensation of aldehydes (also olefins) with polyglycine. The
natural occurrence of this process would be quite probable because, as has been
shown in our laboratory, polyglycines are readily formed from glycine in
aqueous ammonia systems. Furthermore, in practically all of the abiogenic
synthesis of amino acids studied, glycine has been found to be the predominant
amino acid formed.
Another interesting pathway has been described recently by Schramm
et al™ Polyarginine (mol. wt. 4000 to 5000) was prepared from arginine
with the help of polyphosphate esters. Using the same method, polyleucine,
polyvaline, and polyserine were prepared in our laboratory. '^^
In addition to the above pathways of polypeptide formation other obser-
vations have been made which indicate that peptides or polymers containing
amino acids can also be obtained by the action of ultraviolet light^^- and
electric discharges."^ It should be added that some of the products obtained
by thermal polymerization have the ability to form microspheres with internal
structure,"* and of displaying some catalytic activity.^^^
Finally, a very significant recent development is the isolation of polymers
containing several amino acids from the reaction product of mixtures of hy-
drogen cyanide, ammonia, and water. ^'^ This is the same reaction mixture
that has been shown to give rise to the formation of amino acids, purines, purine
intermediates, and fluorescent pigments among other compounds. Because
nitriles are formed in this system it is possible that the above polymers result
from nitrile condensation reactions. Hydrogen cyanide has been suggested as
an amino acid condensing agent by Calvin."^ Hydrogen cyanide and also
cyanamide (formed by combination of CN and NHo radicals), were probably
abundant in the primordial cosmic bodies of the solar system. It is quite
possible that these reactants were responsible for the formation of a number of
polymeric compounds including polypeptides. In fact, it is known that un-
substituted cyanamide can be used for the synthesis of peptides."^
Synthesis of Polymicleotides
A possible abiogenic mechanism for the formation of a high energy phos-
phate compound, carbamyl phosphate, was proposed some time ago."^ F'orm-
Oro: Experimental Organic Cosmochemistry 477
iminyl phosphate, obtained by condensation of hydrogen cyanide with mono-
hydrogen phosphate, is suggested here as another possibiUty of a primitive
high energy phosphate compound. More recently, Schramm et al.,^'^ have
shown that mononucleosides, mononucleotides, and polynucleotides can be
synthesized at moderate temperatures, from their building monomeric blocks,
with the help of polyphosphate esters. The polymers obtained seem to have
the v3',5'-phosphate diester linkages which are common to RNA and DNA.
Strand complementarity, which is the principle of molecular self duplication,
and autocatalytic activity, have also been observed in the above polynucleo-
tides. The role that nucleic acids and other macromolecules may have played
in directing prebiochemical evolution has been discussed in some detail by
several authors.'''^ •^^*"^*''
Conclusion
There is no doubt that carbon compounds exist widely distributed in the
universe. Whether the more complex biochemical compounds described in
this paper are present in cosmic bodies other than the earth will only be
answered with certainty by space probes. Probes to the moon. Mars, and
Venus are feasible and should provide valuable information about the organic
and inorganic chemistry in these bodies. However, more information about
the chemistry prevailing during the beginning of the solar system would be
obtained by sending probes to Jupiter and to comets passing sufficiently close
to the earth's orbit.
From the experimental studies presented here it is reasonable to say that if
the Earth protoplanet had some of the simple organic constituents of comets,
a large number of biochemical compounds (including carbohydrates, amino
acids, purines, pyrimidines, and polymers containing amino acids) would have
been spontaneously synthesized during the development of this cosmic body.
The formation of complex biochemical compounds from simple organic mole-
cules is not in disagreement with thermodynamic principles. In fact, these
syntheses can occur because the initial precursors (nitriles, aldehydes, olefins,
etc.) are compounds of high energy content which, in their tendency to acquire
lower energy states and to become stabilized, react and are ipso facto trans-
formed into biochemical compounds.
The possibility that organic chemical synthesis may have occurred in inter-
stellar dust and planetesimal bodies before the Earth was formed has also been
suggested by Lederberg and Cowie^^' and Fowler, Greenstein and Hoyle.^^^
Acknowledgment
Some of the work from our laboratory reported in this paper was supported
in part by research grants from the National Science Foundation (G-13117)
and the National Aeronautics and Space Administration (NsG-257-62).
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EVALUATION OF RADIATION EFFECTS IN SPACE
Rainer Berger
Lockheed-California Company, Biirbank, California
In addition to such radiation effects in space as nuclear transformations, the
breakage of chemical bonds and other physical phenomena, the formation of
chemical compounds by radiation synthesis is of great importance.
The aspects of the synthesis of more complicated organic compounds from
simple predecessors are primarily to be discussed in this paper, because they
offer clues to the evolution of organic compounds and to some degree to ques-
tions connected with studies on the origin of life.
Figure 1 illustrates the overlapping successive evolutions which occurred
ever since the body of the Earth accreted. It is evident how the span of chem-
ical evolution interacts with that of organic evolution, the period in which
somewhere life began.
Radiation reactions of the kind discussed here are believed to have occurred
on the primitive Earth. They proceeded in the past and still do on planets,
their satellites, comets, meteors, and even particles of the smallness of inter-
stellar grains. However, each type of reaction may not be applicable every-
where in space.
One of the first experiments carried out in this area of research is the dis-
charge experiment of Miller.' Theories of Oparin- and also Urey^ held for
some time that the atmosphere of the primitive Earth was essentially com-
posed of methane, water, ammonia, and hydrogen. When these compounds
were subjected to an electrical discharge in the laboratory to simulate condi-
tions in nature during a thunderstorm or in the proximity of corona discharges,
a host of different biologically important compounds was detected in the reac-
tion mixture. A number of the resulting compounds are listed in table 1.
The most interesting species are the synthesized amino acids, which as is
generally known are the building blocks of all proteins. It is significant that
none of the complicated amino acids such as tryptophane or serine are produced
in this way (figure 2).
A number of similar confirming experiments were performed by Abelson'*
who used various mixtures of H2 , CHi , CO2 , NH3 , N2 , O2 and H2O. Heyns,
Walter, and Meyer'^ in addition to confirming Miller's work used also HoS in
their investigations and obtained ammonium thiocyanate, thiourea, and thio-
acetamide. Pavlovskaya and Passynsky® equally checked the discharge experi-
ments.
Generally speaking, amino acids were obtained from reducing mixtures only
containing an excess of either H2 , CH4 , CO, or NH;j . No amino acids could
be obtained from an oxidative environment. The mechanism of amino acid
production follows essentially the path of a Strecker synthesis. First HCN
and aldehydes are obtained in the gas phase by the action of the electrical
discharge, then these compounds give amino nitrilcs in the aqueous phase.
Finally, hydrolysis leads to the amino acids.
The experiments on the reaction mechanism show that special conditions of
482
Berger: Radiation Effects in Space
483
the electron bombardment are not necessary, which make it the more plausible
that these radiation syntheses were responsible for the occurrence of amino
acids in the oceans of the primitive earth." It was there that they could be
used to further evolution.
EVOLUTIONS
ORGANIC EVOLUTION CHEMICAL EVOLUTION FORMATION OF THE EARTH
TODAY
a'llO^ 3'«I03 4«I0^ YEARS AGO
Figure 1.
Table 1
Yields! from Sparking a Mixture of CH4 , NH3 , H3O , and H2 ; 710 mg. of Carbon
Was .\dded as CH4
Compound
Yield (moles (XIO^))
Glycine
Glycolic acid
Sarcosine
63
56
5
Alanine
Lactic acid
N-Methylaianine
34
31
1
a-Amino-«-l)Ut\ric acid
a-Aminoisobutjric acid
a-Hydroxybutyric acid
5
0.1
5
)3-Alanine
Succinic acid
15
4
Aspartic acid
Glutamic acid
Iminodiacetic acid
Iminoacetic-propionic acid
0.4
0.6
5.5
1.5
Formic acid
Acetic acid
Propionic acid
233
15
13
Urea
N-Methyl urea
2.0
1.5
Because the composition of gas mixtures may be varied at will in the labora-
tory, conditions in the gas envelope of other planets may be approximated.
Sagan and Miller^ used hydrogen, methane and ammonia resembling the at-
mosphere of Jupiter. The reaction mixture contained afterward a number of
different lower hydrocarbons and acetonitrile. Recent investigations on mag-
netic fields around Jupiter indicate that very strong ones are indeed present.^
Therefore, Jupiter may contain currents of molten material which cause the
fields. It may be deduced that the possibility of warmer zones on Jupiter has
484
Annals New York Academy of Sciences
to be reckoned with. In such zones further chemical reactions are entirely
possible to yield more compUcated systems.
The effects of ultraviolet light on a mixture of methane, water, and ammonia
have been studied also by Miller. ^° Only a very small yield of amino acids
could be obtained. Groth, and separately, Terenin, examined similar reac-
tions.'^ Ellenbogen irradiated a reaction mixture containing FeS, NH4CI,
H2O, and CH4 with ultraviolet light and observed the formation of a substance
the infrared absorption spectrum of which indicated peptide bonds. ''^ Ap-
parently other similar experiments to synthesize amino acid sequences held
together by peptide bonds have not yet been successful.
The effects of visible light on the formation of many different organic com-
pounds have been examined extensively in the literature on photochemistry
and need not be discussed in this paper.
AMINOACID AND PROTEIN STRUCTURE
R R R
— NH — CH— CO— NH — CH — CO— NH — CH-
CO —
V.
H
I
/
Y
H
I
^N —
-C C-
II
0
H
II
H
/
c4_
-N-
C
II
0
Figure 2.
In a number of experiments high energy particle radiation as well as x- and
7-rays were utilized. Dose and Rajewsky obtained amino acids and amines
from gaseous mixtures of NH3 , N2 , H2O, CH4 , and CO2 with X-rays.'^ The
action of 2 Mev electrons on CH4 , H2O, and NH3 yielded also amino acids.''*
Calvin irradiated CH4 , NH3 , H2O, and PH3 in the gas phase with electrons.'^
Radiochemical analysis showed the presence of small quantities of nucleic acid
bases, substances which are of vast importance in genetic material (figure 3).
Garrison et al.,^^ used 40 Mev helium ions to obtain formic acid and formal-
dehyde from carbon dioxide. Aqueous formic acid yielded formaldehyde and
oxalic acid.""' Hasselstrom and Henry also obtained oxalic acid from Ca-
(HC03)2 and NH4HCO3.'' Succinic, tricarbolic, malic, citric, and malonic
acid were isolated from the reaction of aqueous acetic acid with helium ions.'*
Also, glycine and aspartic acid were the products in the 2 Mev electron bom-
bardment of acjueous ammonium acetate.'^
All of the previously mentioned radiation reactions occur in gaseous or liquid
systems, but even reactions in the solid state may be carried out. For example,
high doses of 7-rays on solid (NH4)2C03 yielded formic acid and glycine.^^
Berger: Radiation Effects in Space
485
In another experiment, methane, water, and ammonia were condensed to a
solid icy mixture and irradiated with 12 Mev protons.''^ These conditions
approximate the environment thought to exist on comet heads. Analysis of
the reaction mixture indicated the presence of urea, acetamide, and acetone.
The mechanism of this reaction proceeds presumably through a free radical
stage. Either the radicals react with each other in the cold when radical
concentrations become too high, or reaction takes place when the reaction site
warms up to a higher temperature. Similar reactions may not only occur on
comets and icy meteors, but also on the colder outer planets of the solar system
and their satellites.
Based upon astronomical, chemical, and physical observations, it is clear
that not all reactions apply to the same body in space; rather certain reac-
tions will not occur in some instances but play a major role in others. There-
fore, it is essential to consider carefully the environment of the object in
NUCLEIC ACID STRUCTURE
OH 0 0 OH °V/°"
-0 P^— 0 — CH-CH-CH— 0 — ^^P — 0 — CH-CH — 0 > 0 —
2
ADENINE N
/ \
CM2 0
H "CH^
/ \
^ I
THYMINE
H.
H
/•^-c-^°
/ \
CH» O
N C
GUANINE
/C^^/'^N^"
"^ ^c^ ^c-
CYTOSINE
H >• ' O'' Nh2
CH,
\ /
-0— ^P.— 0-CH,-CH — CH_o — P 0 — CHi-CH-CH— 0-^--0 —
0 OH
0' OH
Figure 3.
OH ^0
space before assigning which reactions may predominate. Glasel bombarded
solid D2O with electrons and observed the liberation of considerable amounts
of D2 .^^ Because the bond energy of the D — O bond is higher than that of the
C — H bond in organic compounds, it is to be expected that over periods of
time unshielded organic compounds will be destroyed in space. Therefore,
organic material initially produced on cosmic grains will not remain intact.
Similar destructive radiation effects will occur elsewhere; it is only there, where
shielding from damaging radiation comes into play, that organic compounds
will be available for further reactions.
If I may speculate a little, it may very well be that initially radiation may
have been the agent which at least in part built up molecules to such systems,
which finally were able to handle in a controlled manner radiation or rather
light quanta. The first such successful system to use radiation energy for the
synthesis of organic compounds was the beginning of photosynthesis.
In conclusion, let me say that with the aid of radiation as a form of energy,
one can synthesize chemical species which are the building blocks of proteins,
nucleic acids and other important biological compounds.
486 Annals New York Academy of Sciences
It is reasonable to assume that vast quantities of organic material are or
were formed in space from which a fraction under special circumstances was
the substrate for the evolution of life. To what extent radiation was involved
is hard to assess quantitatively at the moment but the experimental evidence
points to a major role in the processes leading to the creation and functioning
of life.
References
1. Miller, S. L. 1953. Science. 117: 528. 1955. J. Am. Chem. Soc. 77: 2351. 1957-
Biochim. et Biophys. Acta. 23: 480.
2. Oparin, a. I. 1957. The Origin of Life. Academic Press. New York.
3. Urey, H. C. 1952. The Planets. Yale Univ. Press. New Haven, Conn.
4. Abelson, P. H. 1956. Science. 124: 935.
5. Heyns, K., W. Walter & E. Meyer. 1957. Naturwissenschaften. 44: 385.
6. Pavlovskay'a, T. E. & A. G. Passynsky. 1957. Reports of the Moscow Symposium
on the Origin of Life.
7. Miller, S. L. & H. C. Urey. 1959. Science. 130: 245.
8. Sagan, C. & S. L. Miller. 1960. \m. .Astronom. Soc. Meeting, August 1960. : 106.
1960. Astronomical J. 65: 499.
9. Morris, D. & G. L. Berge. Astrophvs. J. In press.
10. Miller, S. L. 1957. Ann. N.Y. Acad. Sci. 69: 260.
11. Groth, W. 1957. Angew. Chem. 69: 68T.
12. Ellenbogen, E. 1958. Abstract of Am. Chem. Soc. Meeting, Chicago.
13. Dose, K. & B. Rajewsky. 1957. Biochim. et Biophys. Acta. 25: 225.
14. Miller, S. L. Unpublished experiments.
15. Calvin, M. In press.
16. Garrison, W. M. c/ (3/. 1951. Science. 114: 416. 1952. J. Am. Chem. Soc. 74: 4216.
17. Hasselstrom, T. & M. C. Henry. 1956. Science. 123: 1038.
18. Garrison, W. M. et al. 1953. J. Am. Chem. Soc. 75: 2459.
19. Hasselstrom, T., M. C. Henry & B. Murr. 1957. Science. 125: 350.
20. Paschke, R., R. Ch.ang & D. Young. 1957. Science. 125: 881.
21. Berger, R. 1961. Proc. Natl. Acad. Sci., U.S. 47 (9): 1434.
22. Gl.\sel, J. A. In press.
ABIOTIC PRODUCTION OF PRIMITIVE PROTEIN AND FORMED
MICROPARTICLES*
Sidney W. Fox and Shuhei Yuyama
Institute for Space Biosciences, The Florida State I'niversity, Tallahassee, Florida
This presentation of results with a thermal model of origins will focus par-
ticularly on some of the properties of the microparticles which emerge there-
from. The more purely chemical aspects of the model have been treated else-
where.'"^ Although the significance of the particles found is much ditTerent
in this context than in others, salient features of the experimentally derived
scheme of origins will, however, again be reviewed here.
One of the popular assumptions which had to be abandoned before the re-
search could proceed was the widely held belief that heating amino acids above
the boiling point of water will yield only dark, unworkable products. This
evaluation has been a common one in the experience of many organic and
biological chemists and has been documented many times.** If, however, one
follows the suggestions from analyses of evolution at the molecular leveF it
becomes possible simultaneously to condense thermally all of the amino acids
common to protein. The products contain each of these amino acids and
have many of the properties of protein. The necessary conditions are the use
of a sufficient proportion of aspartic acid or lysine and an initially dry state.
Heating can be at 170° for 3 hours. '^•' The product is a light amber in color
when sufficient aspartic acid is used, and, like protein, it may then be further
purified by dialysis and reprecipitation by salting out the polymer from aque-
ous solution.
A second heresy concerns the belief that heat has generally been thought to
be a reliable agent for denaturation of protein. Not so generally known is the
fact that this process is "extraordinarily sensitive'"^ to the amount of water
present.^ Also, enzymes are more stable when dry}'* Accordingly, the pro-
duction of biologically significant polymers by heating amino acids is not
precluded.
After extensive study of thermal copolymerization of simple combinations of
amino acids, initial evidence that these processes could be effected simultane-
ously was obtained by chromatography.'' End group assay'' '^ showed that
molecular weights were above that of insulin' (6000 for insulin, or approxi-
mately 3000 per end group). With lysine, thermal polymers of mean molecu-
lar weight over 300,000 have been demonstrated in the ultracentrifuge.^^ The
two criteria of qualitative composition and molecular weight are common to
the only two textbook definitions of protein that we have found." ■'-
Of particular interest is the fact that polymerization is aided by phosphoric
acid,'5 7.i3 polyphosphoric acid, or ATP,!^!^ and especially, that the minimal
*The work reported in this paper was supported in part by Grant no. C-3971(04) of the
National Institutes of Health, U.S. Puljlic Health Service, and Grant no. NsG- 173-62 of the
National Aeronautics and Space Administration. Presented in part at the Symposium on
Extraterrestrial Biochemistry and Biology, American Association for the Advancement of
Science meeting, December 27, 1961, Denver, Colorado. Contribution no. 5 of the Institute
for Space Biosciences.
487
488 Annals New York Academy of Sciences
temperature for polymerization is lowered by addition of polyphosphoric acid
to about 70°/ '^^ as well as its contribution to the formation of uracil.^*^ The
recent report of Schramm/^ in which is claimed the polymerization of nucleo-
tides as earlier proposed in a thermal mode/ occurs under similar conditions.
A principal difference is Schramm's use of the ethyl ester of polyphosphoric
acid.
The polyamino acids obtained are referred to as proteinoids because of
molecular weif^ht and (jualitative composition, but they have in addition many
properties in common with protein.^ '-"
Two properties of most interest are those of catalytic activity and morpho-
genicity. Catalytic activity has been found and studied for the hydrolysis of
/»-nitrophenyl acetate. This is an unnatural substrate popularly used in studies
by enzyme model chemists.-' This substrate is unstable and hydrolyzes spon-
taneously over a large range of pH. Histidine, which has been implicated as
part of the active site of many enzymes/" catalyzes this hydrolysis. Simple
derivatives of histidine also have this effect and some which are several times
as active as histidine have been reported, e.g., carbobenzoxyhistidine.-- Pro-
teinoids have been found to be many times as active as that, and in fact 2 of
them are more than 15 times as active.
Of more interest is the fact that the catalytically active proteinoids are in-
activated by heat at 100° for 20 minutes in aqueous buffer solution at pH 6.8.
This effect has been observed in numerous repetitions and the percentage of
inactivation has been found to be greatest for those proteinoids possessing the
highest relative activity.
In an overall view, one interesting relationship involves the fact that cat-
alytically powerful macromolecules are formed under almost dry conditions
by heating and that this activity is later lost also by heating, but the loss
occurs in acjueous solution. The signiiicance of understanding the intimate
and subtle effects of water is emphasized by this relationship. Also demon-
strated is the fact that very elaborate molecules, approximately as complex as
protein molecules, can be produced by a process which, although mechanis-
tically complicated, is remarkably simple in operation.
The kind of morphogenicity observed also depends upon the intrusion of
water into the system, under conditions different from those for inducing loss
of catalytic activity. Acid proteinoid is typically heated in boiling water or
salt solution (1 part of solid to 2000 parts of aqueous phase) for 10 seconds, the
hot supernatant decanted and allowed to cool. There result, for each milli-
gram of solid, approximately 10^ to lO'^ microspheres of the kind shown in
FIGURE 1. The fact that intrusion of water is required for formation of spher-
ules demands a relative absence of water from the system before the macro-
molecules are organized into supramolecular entities.
These formed units are of interest as precell models alternative to Oparin's
coacervate droplets, also studied as precell models.-^ They and derivatives
are of interest also for their morphological similarity to some microfossils-^
and to formed elements found in meteorites.''-^ Interesting differences between
microspheres and coacervate droplets are known; for example, both the micro-
spheres and bacteria retain their integrity on centrifugation, whereas the
coacervate droi)lets coalesce easily.-^ The microspheres also emerge from a
/ Fox & Yuyama : Abiotic Production of Primitive Protein 489
continuum of conditions which can explain the origins of enzymes and of
"^-inetaBblism, whereas the coacervates are fabricated from such materials as
gelatin and gum arabic, which arose late in evolution.
The units in figure 1 are slightly less than 2.0 ^u in diameter. They have
the size and shape of the cocci, which have been thought of as the most primi-
tive of the bacteria.-^
In FIGURE 2 are microspheres which have been transferred to a solution
saturated with proteinoid and containing 38 per cent calcium chloride. Two
boundaries can be seen. The effects are not optical, as indicated by acentricity
in some of the units. It was later learned that double boundaries could be
'1
f\
D
O
Figure 1. Microspheres. Photomicrograph courtesy of Dr. K. Harada. Lhiits are
approximately 2 fi in diameter.
more easily produced by raising the pH, as from 3.0 to 5.5. Time lapse photo-
micrographic studies demonstrate that the interior can be completely dissolved,
yet the outer membrane remains. This behavior poses the provocative ques-
tion of the difference between the nature of the outer membrane and the inner
material.
In FIGURE 3 is seen a field in which appears a form resembling a cell in divi-
sion. In fact, this one is very similar to an object carefully referred to by
Claus and Nagy in figure 5 of their paper as an organized element resembling
cell division. Preliminary time lapse studies suggest neither division nor
fusion is occurring in the majority or all of these units. The appearance of
such phenomena, however, is provocative in the sense of the properties and
behavior found in the units. An additional field of twinned microspheres is
490
Annals New York Academy of Sciences
seen in figure 4. This figure also shows filamentous structures which arise
from proteinoid.
In FrcuRE 5 are seen the effects of pressure on the microspheres. This seg-
mentation resulted from digital pressure on the coverglass.
In FIGURE 6 are seen algal-like associations of microspheres. These were
produced by making them under a coverglass on the microscope slide. The
resemblance is to Auaboeiia or Xostocr^ We are indebted to Dr. Chester S.
Nielsen for aid in verifying the superficial, albeit incomplete, resemblance.
The resemblance of alleged fossils of this type is also imperfect.
#
U'
Figure 2. Microspheres with double boundaries following increase in pH. Larger figures
are approximately 10 yu in length.
The microspheres are also found to be birefringent, indicating internal order.
When we review the results of almost a decade of experimental studies of
models of biochemical origins we can perceive: (1) amino acids have been pro-
duced by many workers under many laboratory conditions and from many
reactants that plausibly existed on or in the prebiological Earth; (2) in a
majority of such experimental reports, the key aspartic acid appears as a prod-
uct; (3) the polymerization of amino acids has now been accomplished in
hundreds of variations over a range of conditions; and (4) similarly, the forma-
tion of spherular forms has been accomplished in thousands of variations in the
laboratory. We now regard processes 3 and 4 as so rugged and so inexorable
as to believe that they could and should have occurred on many occasions
in many places in the universe. Also, the origin of the necessary amino acids
seems to be inexorable, by one process or another.-^
Fox & Yuyama: Abiotic Production of Primitive Protein 491
-*«
Figure 3. Twinned microspheres produced by rise in pH. Size as in figure 2.
^
J :mm
W
I
Figure 4. An additional field of twinned microspheres. Size as in figure 2.
492
Annals New York Academy of Sciences
C
Figure 5. I'^ffect of digital pressure on microspheres. Size as in figure 2.
■k^ "'•-%;
3»g'
^^c^"^^
-^-r
-2X
Figure 6. Associations of microspheres with resemblance to algae. Size as in figure 2.
<\
Fox & Yuyama : Abiotic Production of Primitive Protein 493
In the context of the orighi of livhig units, one inference is that nature had
almost endless opportunities to experiment with precellular forms until the
necessary apparatus for repHcation was included by chance.
In the context of the present conference, the presumed protobionta observed
in fossils and meteorites may actually be prebionta. If they are, they would be V
in one sense more significant than if they are protobionta. A third possibility ^
is that they are meaningless artifacts, easy for nature to come by because of the
simpUcity of the processes leading to their formation. This point of view has^x
a semantic flavor, because of the position that no natural experiment is truly
without meaning. Also of interest is the fact that Dr. Philip Morrison inde- J)
pendent ly reached the same conclusion from data presented at the Denver
meeting of the American Association for the Advancement of Science.^" In
essence, however, and either with or without regard to the difficult questions
of terrestrial contamination of meteorites, the conclusion at present is that
there cannot yet be a conclusion on the cjuestion of whether the inclusions in
meteorites are protobionta.
References
1. Fox, S. W. 1960. How did life begin? Science. 132: 200-208.
2. Fox, S. W. & K. Harada. In press. Experiments related to the chemical origins of
protein. G. Bourne, Ed. Space Flight. : 261-270. Academic Press. New York.
3. Harada, K. 1961. On the formation of primordial protein and the thermal theory
(Title transl.). Proteins, Nucleic Acids, Enzj-mes (Tokyo). 6: 65-75.
4. Fox, S. W., K. Harada & A. Vegotsky. 1959. Thermal polymerization of amino
acids and a theory of biochemical origins. Exjjerientia. 15: 81-84.
5. Fox, S. \V. 1956. Evolution of protein molecules and thermal synthesis of biochemical
substances. Am. Scientist. 44: 347-359.
6. Fox, S. W. & K. Har.ada. 1958. Thermal copolymerization of amino acids to a product
resembling protein. Science. 128: 1214.
7. Fox, S. \V. & K. Harada. 1960. The thermal copolymerization of amino acids com-
mon to protein. J. .\m. Chem. Soc. 82: 3745-3751.
8. Altman, R. L. & S. W. Benson. 1960. The etYect of water upon the rate of heat de-
naturation of egg albumin. J. Am. Chem. Soc. 82: 3852-3857.
9. Barker, H. A. 1933. The effect of water content upon the rate of heat denaturation
of crj'Stallizable egg albumin. J. Gen. Physiol. 17: 21-34.
10. Dixon, M. & E. C. Webb. 1958. Enzymes. : 153. Academic Press. New York.
11. Fruton, J. S. & S. SiMMONDS. 1958. General biochemistry. : 16. John Wiley and
Sons. New York.
12. Mitchell, P. H. 1948. A textbook of general physiology. : 245. McGraw-Hill Book
Co. New York.
13. Fox, S. W. & K. H.AR.A^DA. 1960. Thermal copolymerization of amino acids in the
presence of phosphoric acid. .\rch. Biochem. Biophys. 86: 281-285.
14. Vegotsky, A. & S. W. Fox. 1959. Pyropolymerization of amino acids to proteinoids
with phosphoric acid or polyphosphoric acid. Federation Proc. 18: 343.
15. Vegotsky, .4. 1961. Thermal copolymers of amino acids. Ph.D. dissertation. Florida
State University.
16. Harada, K. & S. W. Fox. 1960. Thermal copolymerization of amino acids at tem-
peratures below 100°. : 28C-29C. .\merican Chemical Society meeting, Cleveland.
Ohio. Abstracts.
17. Genaux, C. & S. W. Fox. Unpubhshed experiments.
18. Fox, S. W. & K. H.ARADA. 1961. Synthesis of uracil under conditions of a thermal
model of prebiological chemistry. Science. 133: 1923-1924.
19. Schramm, G. 1962. Nicht-enzymatische s}nthese von polysacchariden, nucleosiden
und nucleinsauren. .\ngew. Chem. 74: 53-59.
20. Fox, S. W., K. Harada & D. L. Rohlfing. 1962. The thermal copolymerization of
a-amino acids. : 47-54. M. Stahmann, Ed. Polyamino Acids, Polypeptides and
Proteins. Univ. of Wisconsin Press. Madison.
21. Bender, M. L. 1960. Mechanisms of catalysis of nucleophilic reactions of carboxylic
acid derivatives. Chem. Revs. 60: 53 113.
494 Annals New York Academy of Sciences
22. NoGUCHi, j. & T. Saito. 1962. Studies on the catalytic activity of synthetic poly-
amino acids having an imidazole group in the active site. : 313-327. M. Stahmann,
Ed. Polyamino Acids, Polypeptides and Proteins. Univ. of Wisconsin Press. Madi-
son.
23. Oparin, a. I. 1961. Life: Its Nature, Origin and Development. Oliver and Boyd.
Edinburgh.
24. Barghoorn, E. In P. E. Cloud, Jr. & P. H. Abelson. 1961. Woodring conference on
major l)iological innovations and the geologic record. Proc. Natl. Acad. Sci. U.S. 47:
1705-1712.
25. Claus, G. & B. Nagv. 1961. A microlnological examination of some carbonaceous
chondrites. Nature. 192: 594-596.
26. Bungenbf.rg DeJong, H. G. 1949. Morphology of coacervates. 433-482. In
Colloid Science. II. H. R. Kruyt, Ed. Elsevier Publishing Co. New York.
27. Lamanna, C. & M. F. Mallette. 1959. Basic Bacteriology. : 44-47. The Williams
and Wilkins Co. Baltimore.
28. Smith, G. M. 1950. The Fresh-water Algae of the United States. McGraw-Hill
Book Co. New York.
29. Fox, S. W. 1957. The chemical problem of spontaneous generation. J. Chem. Educ.
34: 472-479.
30. Morrison, P. 1962. Carbonaceous snowflakes and the origin of life. Science. 135: 663-
664.
OBSERVATIONS ON THE NATURE OF THE "ORGANIZED
ELEMENTS" IN CARBONACEOUS CHONDRITES
Frank W. Fitch
Department of Pathology, University of Chicago, Chicago, III.
Edward Anders
Enrico Fermi Institute for Nuclear Studies, Departments of Chemistry and Geophysical
Sciences, University of Chicago, Chicago, III.
Our interest in the morphological study of carbonaceous chondrites was
stimulated by reports of Claus and Nagy^ and of Nagy et al.,- describing a
variety of "organized elements" found in Class I carbonaceous chondrites.
The organized elements had been classified by Claus and Nagy into 5 types on
the basis, primarily, of morphology. The various properties of the organized
elements are tabulated in table 1. Types I and II which were circular or
spherical were most numerous; the other types were much less abundant. A
total of about 1700 organized elements per milligram were reported.' ■"'
In an attempt to confirm these observations and to characterize further the
composition of the organized elements, we examined samples of the carbona-
ceous chondrites Orgueil and Ivuna. One sample of Orgueil was obtained
several years ago from the Musee d'Histoire Naturelle, Paris. Another sample
was obtained through the courtesy of Henderson of the U.S. National Museum,
and was from the same fragment given to Nagy. A sample of Ivuna was ob-
tained through the courtesy of Roy of the Chicago Natural History Museum.
Conventional brightfield, phase contrast and fluorescence microscopy were
used. Other methods included staining with biological stains, and the use of
x-ray diffraction and electron microprobe analysis.
Microscopical Observations
Both samples of Orgueil and the single sample of Ivuna had crumbled apart
and consisted of fragments ranging in size from a fine dust to several millimeters
in diameter. Fragments were inspected visually to be certain that they were
free from fusion crust, paint markings, and other visible contaminants. To
minimize sampling errors, observations were made on the fine dust as well as
fragments broken from larger pieces. This dust that had accumulated at the
bottom of the sealed glass containers came from the surface of many individual
fragments and should, therefore, be fairly representative of the meteorite as a
whole. Because of the friable nature and the porosity of the carbonaceous
chondrites, it is not feasible to clean the meteorite surface. For microscopy,
samples of the meteorite weighing about 1 mg. were placed in a drop of glycerin
on a microscope slide which had been cleaned with 95 per cent ethanol. The
sample was gently crushed with a glass rod cleaned with ethanol. Samples
subjected to density separation were lightly crushed in an alcohol-cleaned agate
mortar.
Initially, particles were sought which had the general morphological charac-
teristics of the organized elements. Because Types I and II elements were
circular or spherical, particles with this morphology were sought. As reported
495
496
Annals New York Academy of Sciences
in an earlier paper,^ the most conspicuous particles with this shape and oc-
curring in the abundance of several thousand per milligram were opacjue and
highly magnetic. They could be concentrated by density separation in the
fraction with a density greater than 3.33. Although opaque, many had trans-
parent, yellow-brown mineral fragments attached to the surface. When viewed
with phase-contrast microscopy, the diffraction pattern around the particles
frequently gave a false impression of a double outer wall, especially when the
particles were slightly out of focus. X-ray diffraction and electron microprobe
studies of isolated particles of this type indicated that they were composed of
troilite or magnetite.^ Although possessing several characteristics of the or-
ganized elements, these troilite and magnetite particles were opacjue. Subse-
Table 1
Reported Properties of Organized Elements*
T3 J-.
V a
O
Shape
Surface
Color
size
Abundance
I
Circular
Double wall, thickening
Yellow-
4-10
Abundant
II
III
Circular
Shield-shaped
and sculpturing
Spines, appendages, furrows
Thickening and sculpturing
green
8-30
15
Abundant
Less com-
mon
IV
Cylindrical
Thick wall, sculpturing
10-12 X 20
Less com-
mon
V
Hexagonal
Appendages
20
Rare
Other reported general properties:
Fluorescence in ultraviolet light
Staining with biological stains
Appearance suggesting cell division occasionally
Resistance to HF treatment
* From Claus and Nagy' and Nagy et al.^
quently, Nagy et al.^^ emphasized several differences between these particles
and the "organized elements."
Other spherical particles were found in some samples of the meteorite which
had been subjected to a density separation with organic liquids. These ranged
in size from about 1 to 20 fx and were transparent and colorless or yellow. Some
appeared to have a double wall. These had a bluish fluorescence of the outer
portion when viewed with ultraviolet light. The smaller particles had uniform
bluish fluorescence. A number of tests indicated that these were hydrocarbon
droplets and droplets of supercooled liquid sulfur coated with hydrocarbon.^
They could be removed by repeated washing of the sample with chloroform
or acetone and therefore did not seem to be organized elements.
A variety of hexagonal particles varying in size from about 2 to 20 yu were
also found. Some hexagonal particles were transparent and yellow-brown
with an opaque, irregular central area; these particles were highly magnetic.
They may be goethite pseudomorphs after troilite, probably formed by pre-
terrestrial oxidation of troihte. Other hexagonal particles were quite
Fitch & Anders: "Organized Elements" in Chondrites 497
small, colorless, and transparent. These were probably silicate or carbonate
minerals. Other hexagonal particles were opac|ue and nonmagnetic. These
were probably one form of troilite which is non-magnetic. None of these
hexagonal particles had appendages cjuite like those found in the type V
hexagonal particle illustrated by Claus and Nagy.^ It should be noted, how-
ever, that the type V organized element is quite rare; only two and a fragment
of a third were found by them in Orgueil.
No other particles of distinctly spheroidal shape could be found. The bulk
of the meteorite consists of a brownish-yellow hydrated silicate (Orgueil LM).*
Most of the silicate particles had a very irregular shape, but a few were roughly
spherical (figure 1). However, even these ovoid to spherical fragments had
at least a partially irregular surface, and none had any definite internal struc-
ture or double walls. They were not magnetic. Although some variation in
color and refractility was noted, the spheroidal particles had numerous irregular
counterparts which matched them in every way except shape. It seems likely
that all of these particles were mineral fragments.
Although each of these types of particles had some of the characteristics of
the organized elements, none seemed to possess all of the primary morphological
properties. However, other properties of the organized elements have been
described.''^ These include fluorescence in ultraviolet light, staining with
biological stains, and insolubility in hydrofluoric acid. Particles with these
characteristics were then sought.
Fluorescence in Ultraviolet Light
Crushed, but otherwise untreated, Orgueil from the U. S. National Museum
was examined with the fluorescence microscope and all of the tkiorescent
particles seen were photographed in visible and ultraviolet light. Based upon
the information of Claus and Nagy,' 39 organized elements should have been
found in the area of the slide examined. Actually, 15 fluorescent particles
were found, but they did not seem to resemble the published illustrations or
descriptions of the organized elements (table 2). They were quite irregular
and when viewed with ordinary illumination were colorless or slightly yellow.
Two typical particles selected from the 14 photographed are illustrated in
FIGURE 2.
Biological Stains
Since the organized elements have been reported to stain with various
biological staining reactions including Feulgen and PAS, these as well as other
staining procedures were used on samples of the meteorite. Many of the
irregular yellow-brown grains stained slightly with the PAS and Feulgen reac-
tions. Although many particles stained slightly, none stained the brilliant
magenta usually achieved in biological materials, and many of the rounded
grains did not appear to stain at all (figure 1). Similar results were obtained
with the Feulgen reaction.
To interpret these staining results it is necessary to examine the nature of the
PAS and Feulgen reactions. The color in both reactions is produced by using
Schiff's reagent, prepared by decolorizing basic fuchsin with sulfurous acid.^
498
Annals New York Academy of Sciences
4
■■■"" «*
"■10 ■
f
%
imtHmtm
Fitch & Anders: "Organized Elements" in Chondrites 499
If aldehydes are reacted with Schiff's reagent, a red- violet color develops that
is different from the original fuchsln. In addition to aldehydes, certain ketones,
certain unsaturated compounds, and various oxidants can colorize Schiff's
reagent.^'* The solution must be fairly freshly prepared; oxidation, aging,
exposure to air, and sunlight can recolorize Schiff's reagent stored in the
laboratory/
In addition to any aldehyde groups present initially, Schiff's reagent will
react with any artificially produced aldehyde groups. For example, periodic
acid oxidizes 1,2 glycol linkages to aldehyde groups. If one of the hydroxyl
groups is substituted with amino alcohol, alkylamino alcohol or carbonyl, it is
also oxidized to give a positive reaction. In biological materials, the reaction
is relatively specific for carbohydrates, mucoproteins and glycolipids. Un-
saturated lipids which can also react are usually removed from biological
samples during preparation for microscopical examination.'''^
Table 2
UV Fluorescence in Orgueil
Color
Size range
Number of particles
Regular
Irregular
Yellow
Bluish
Bluish
Bluish
2-10
2-10
10-50
>50
0
0
0
0
5
3
5
2
In biological tissues the Feulgen reaction is usually considered to be specific
for desoxyribonucleic acid (DNA).^'^ As the first step in the procedure, DNA
is partially hydrolyzed by 1 n HCl to produce the aldehyde form of desoxyribose
phosphate. The aldehyde groups then react with Schiff's reagent to produce
the same magenta color found in the PAS reaction. In biological samples,
substances which will react directly with Schiff's reagent are usually not
present. With meteorite samples it is essential to determine whether or not
materials are present that will react directly with Schiff's reagent. Such
substances would give a false positive Feulgen reaction and simulate the pres-
ence of DNA. To correctly interpret the results of the staining reactions on
the meteorite samples, proper controls are necessary.
To control the staining reactions, sections of rat spleen tissue fixed in Carnoy's
solution and embedded in paraffin, as well as samples of kimberlite and Orgueil
were studied. Kimberlite, the diamond-bearing rock usually believed to have
come from deep within the earth, was chosen becaues it is perhaps more similar
to Orgueil in mineral composition than other terrestrial rocks. Both Orgueil
and kimberlite consist primarily of serpentine-like hydrated silicates produced
from olivine by alteration under aqueous, reducing conditions. For the stain-
ing reactions, samples of Orgueil and kimberhte were suspended in 6 per cent
gelatin and the mixture was spread on microscope slides and allowed to dry.
500
Annals New York Academy of Sciences
4J Ov
I"
r
J-l '-'
-4—' Ct
c -0
nj O
U "*-
O en
O
U
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c
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oj ra JJ
JJ O r-
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-to ^_rt
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Fitch & Anders: "Organized Elements" in Chondrites 501
Gelatin was used to adhere the samples to the slides because preliminary experi-
ments showed that there was little staining of the gelatin.
The Feulgen reaction was carried out in the routine manner on these samples
with the usual hydrolysis with 1 n HCl at 60° C. for 8 minutes followed by
Figure 3. Orgueil, rat spleen, and kimberlite stained with the Feulgen reaction. (.4)
Orgueil, Feulgen reaction. Most particles stain irregularly. (B) Rat spleen, Feulgen reac-
tion. Nuclei have brilliant magenta staining of DNA. Other tissue comi)onents do not
stain. (C) Kimberlite, Feulgen reaction. Most particles stain irregularly. Some of the
sample was dissolved by the HCl treatment. (Z?) Orgueil, Schiff's reagent only. Staining is
as intense as with the Feulgen reaction. (E) Rat spleen, Schiff's reagent only. There is no
staining of nuclear DNA. (F) Kimberlite, Schiff's reagent only. Staining is as intense as
with the Feulgen reaction.
treatment with Schiff's reagent for 1 hour.^ As a control procedure, samples
were reacted with Schiff's reagent for 1 hour without previous treatment with
acid. In the rat spleen sections, nuclear DNA stained brilliantly after acid
hydrolysis (figure S, B). In sections treated with Schiff's reagent alone, no
staining occurred (figure 3, E). However, samples of Orgueil and kimberlite
stained equally well whether treated with acid or not (figure 3; A, D, C, F).
Something is present in the meteorite and in kimberlite which reacts directly
502 Annals New York Academy of Sciences
with the Schiff's reagent. Therefore, the development of a magenta color
with the Feulgen reaction is, in this instance, not specific for DNA.
Similar results were obtained with the PAS reaction. There seemed to be
no additional staining produced when samples were treated with periodic acid
before reaction with Schiff's reagent, as compared with reaction with Schiff's
reagent alone. Attempts to inhibit the staining produced by Schiff's reagent
by previous treatment of samples with aniline chloride and hydroxylamine, to
block the aldehyde groups,'^ were only partly successful in the samples and in
periodic acid treated starch controls. Hence, the nature of the reactive groups
is not known at present.
The presence of DNA in the organized elements would be powerful evidence
of their biologic origin. Because the results of the Feulgen reaction had been
interpreted in published reports as indicating the presence of nucleic acid in
the meteorite,^-* it was desirable to confirm this interpretation with another
histochemical test for DNA. Methyl green is freciuently used for this purpose.^'
The characteristic reaction of DNA with this stain is thought to be the result
of binding of the dye by phosphoric acid radicals in the intact, polymerized
DNA.* Thus, the mechanism of this reaction is altogether different from that
of the Feulgen reaction.
Samples of rat spleen, Orgueil and kimberlite were stained with methyl
green.* As a control procedure, samples were treated with 10 per cent per-
chloric acid for 4 hours and 30 minutes, a procedure which depolymerizes and
extracts DNA from biological samples.'-' In rat spleen sections stained directly
with methyl green, there was brilliant green staining of the nuclei (figure 4, B).
In spleen sections treated with perchloric acid to remove DNA before reaction
with methyl green, there was no nuclear staining (figure 4, E). However,
the samples of Orgueil and kimberlite stained brilliantly with methyl green
whether treated previously with perchloric acid or not (figure 4; A, D, C, F).
It is evident that when biological staining reactions are applied to nonbiolog-
ical materials, great care is necessary in the interpretation of results. Because
of the presence of other reactive groups the usual tests for DNA are no longer
specific. Positive or negative reactions of any DNA present would be masked
by the intense, nonspecific staining due to other groups. Under these condi-
tions, the staining tests cannot be regarded as evidence for the presence of
DNA in the meteorite.
Treatment with Hydrofluoric Acid
The "organized elements" were reported by Nagy el at., not to be seriously
affected morphologically by treatment with boiling hydrofluoric acid (HF) for
15 minutes, whereas silicate minerals should be dissolved.- We treated a
sample of Orgueil with boiling HF for 15 minutes; 49 per cent of the sample
remained (tabi.e 3). Because the carbon content of the meteorite is only 3.1
per cent, the bulk of this residue must have been inorganic. Consideration of
the pertinent solubility products indicates that calcium, magnesium, and
possibly other major constituents of the meteorite should remain as insoluble
fluorides or fluosilicates. Thus, persistence after HF treatment is not a suffi-
cient criterion for the organic nature of a particle.
To dissolve the mineral residue, the sample was first treated with HF for 17
Fitch & Anders: "Organized Elements" in Chondrites 503
hours at 60° C. and whh 6 N HCl for 18 hours at 25° C. Treatment with
HF-HCl is a standard palynological technique which leaves organic materials
of biological origin, including various pollen grains, morphologically unaf-
fected.'" After this treatment only 3 per cent of the sample remained. X-ray
Figure 4. Orgueil, rat spleen, and kimberlite stained with methyl green. (.4) Orgueil
methyl green stain. Many particles stain irregularly. (B) Rat spleen, methyl green stain
Nuclei are stained a dark green. Other tissue components do not stain. (C) Kimberlite
methyl green stain. Many particles stain irregularly. (D) Orgueil, methyl green stain after
HCIO4 treatment. Staining is as intense as before extraction. (E) Rat spleen, methyl
green stain after HCIO4 treatment. There is no staining of nuclei; DNA has been depoly-
merized and extracted. iF) Kimberlite, methyl green stain after HCIO4 treatment. Stain-
ing is as intense as before extraction.
diffraction and infrared spectrophotometry indicate that this residue is mainly
amorphous carbon with traces of MgFo and organic matter. Microscopical
examination of the residue showed finely granular, black to brown material
virtually devoid of any structure (figure 5, C, D). Often, it was present in
large irregular aggregates (figure 5, B). Very rarely, spherical transparent
particles were seen (figure 5, A), but only 2 were found in an area where
several thousand organized elements should have been present. Granular
504
Annals New York Academy of Sciences
material was adherent to their surface, and httle structural detail could be
resolved with either phase-contrast or brightfield microscopy. The possible
nature of these particles will be discussed in the following section.
Table 3
Orgueil Meteorite: Treatment with HF
Reagent
Temperature
Time
Residue
Composition of residue
HF 24 M
HF24M
HF24M
HC16M
75°
100°
60°
25°
hoius
24
18/
%
>50
49
3
MgF2, CaFo, Fe304, FeS, or-
ganic matter
MgF2 , CaF2 , organic matter
Carbon + organic matter
1
*
_ .^y
-^r
V
.Bjr-%
r,. '
'^
M
m. J'
#
D
Figure 5. Orgueil after HF-HCl treatment. (.4) One of 2 transparent spherical par-
ticles seen. Irregular black-brown material is adherent to the surface. (B, C, D) Amorphous
residue remaining after HI-'-HCl treatment. Most of this material is amorphous carbon with
traces of MgF2 and organic matter. The line is 20 n in length.
A ttempls at Identification of Some Organized Elements
It is evident that there are discrepancies between our findings and those of
Nagy et al. In an attempt to resolve these differences, we visited the labora-
tories of Claus and Nagy at their invitation. They examined our material
and we examined their material. It became evident that there were several
reasons for the differences.
4
I
Fitch & Anders: "Organized Elements" in Chondrites 505
First, their material contained a few particles of striking morphology which
we had not found and which they did not find in our material. Examples of
such particles found in their material are shown in figure 6, A and B, and
FIGURE 7, A and B. These were classified by Claus and Nagy as type II
organized elements with double wall and spiny surface. Particles of strik-
ingly similar morphology are illustrated in figure 6, C and D, and figure 7,
C and D. These are common ragweed pollen grains. The particles in figures
6, A and B, and 7, A and B, were suggested by them to be extraterrestrial life
Figure 6. (A and B) Organized element from preparation of Claus and Nagy. The
different levels of focus indicate double wall structure and spin\- surface. (C and D) Ragweed
pollen grain. Double wall and surface spines are shown at different levels of focus. The
line is 20 n in length.
forms resembling hystrichospherids, spiny fossil algae. The appearance of
these algae and some pollen grains may be similar. It seems that in this in-
stance, morphological criteria alone may not be a sufficient basis for identifica-
tion.
Two other particles from their material identified by them as type II or-
ganized elements are illustrated in figure 8, A and B. A third organized
element of similar appearance was also seen in their material. All 3 particles
were found on a slide reportedly stained with the Feulgen reaction. They
show a resemblance to starch grains (figure 8, C and D), stained with the
PAS reaction. The difference between the Feulgen and PAS reactions may
not be of significance in this instance, since we have noted that Schiff's reagent
Figure 7. (A and B) Another organized element from preparation of Claus and Nagy
(C and D) Ragweed pollen grain. The line is 20 m in length.
^ir
B
D
Figure 8. (.1 and B) 'F'wo different organized elements from prej)aration of Claus and
Nagy stained with i'eulgen reaction. (C and D) Starch grains stained with PAS reaction.
See text for discussion of significance of staining. The line is 20 /u in length.
506
1
Fitch & Anders: "Organized Elements" in Chondrites 507
alone will stain some starch grains. This staining was more pronounced when
an aged batch of Schiff's reagent was used, and was somewhat stronger for
"Biosorb" (modified starch prepared by Ethicon Laboratories) than for potato
starch. We cannot exclude the possibility that the particle in figure 8, A is
actually a juniper pollen grain. Again, morphological criteria seem to be in-
adequate to establish the identity of a given particle.
Another organized element, classified by Claus as a type II element resem-
bling a Thecamoeba, is shown in figure 9, A. Illustrated in figure 9, B is an
object with similar morphology found in the airborne pollen sample collected
on July 20, 1961 by Siegel at the Jewish Hospital in Brooklyn, N.Y. These
microscope slides, prepared for the New York City annual pollen survey, were
t'iGURE 9. (.4) Organized element from preparation of Claus and Nagy. (B) Particles
with similar appearance found in pollen survey slide. See text for discussion.
kindly loaned to us by Siegel. We are not certain as to the identity of this
object, but the resemblance between the organized element from the meteorite
and the airborne particle is evident.* More recently, we have found several
similar particles in dust samples from the American Museum of Natural His-
tory.
Pollen, mold, and fungus spores, and a variety of other objects are present in
large numbers in the atmosphere at certain seasons, with daily totals of up to
100 ragweed pollen grains per cm.- " and up to 363 mold spores per cm.- ^~ being
* Gregory (i)rivate communication) has suggested that these particles might be furnace
ash spheres.
508
Annals New York Academy of Sciences
reported for New York City. Several of these objects are illustrated in figure
10. It is extremely difficult to prevent contamination by this type of ma-
terial. These types of particles are often present in great abundance in the
air and are deposited as dust that later forms a secondary source for con-
tamination.
Siegel has pointed out in personal communication that he had found it
extremely difficult during the summer and fall to prepare Vaseline-coated
slides free of pollen contamination, although working in a dust free, "sterile"
Figure 10. Objects found in pollen survey slides. (A) Unidentified object. (B)
Ragweed pollen grains. (C'j Oak pollen grain. (D) Unidentified object. The line is 20 n
in length.
room. Also, ragweed pollen grains were occasionally seen by Siegel in pollen
study slides exposed long after the period of bloom, and probably represent
contamination from the laboratory or other sources.
Thin Sedions
Organized elements embedded in mineral veins in thin sections of the Orgueil
meteorite have been described and illustrated.- It is extremely important to
characterize these particles because they are undoubtedly indigenous to the
meteorite. However, the nature of the thin sections makes adecjuate morpho-
logical study difficult. The sections are relatively thick, 10 to 25 ^i, and al-
though the veins are composed of transparent minerals, there are irregularities
and impurities which cause optical distortion. It is difficult to be certain of
tine surface detail because the practical limit of resolution for the microscope
Fitch & Anders: "Organized Elements" in Chondrites 509
under ideal conditions is only 0.2 to about 0.3 fx for the objectives that must be
used with this sort of preparation. Akhough the organized element illustrated
by Nagy et al.~ had to be viewed through a layer of optically imperfect mag-
nesium sulfate, the presumed spines illustrated in the drawing were spaced
only 0.3 yu apart.
Judging from both visual inspection and the published illustration [tigure 4d
in reference 2] this organized element appears to be opaque. Previously, it
was emphasized that all organized elements in crushed preparations were
transparent.^ - Also, none of the particles in the thin sections seems to have
the highly structured morphology, although about 8000 organized elements
should have been present in a thin section I4 inch in diameter and 20 yu in
thickness.
Some organized elements in the thin sections were described as having pink
fluorescence [tigure 5 in reference 2]. We encountered occasional particles in
crushed preparations which appeared red against the dark background when
illuminated with ultraviolet light. However, this did not prove to be true
fluorescence. These particles when viewed with polarized visible light were
doubly refractile. The fluorescence microscopes commonly used in biological
investigations use darkfield illumination. The usual light source is a high
pressure mercury arc with various filters placed in the light path to absorb the
visible light. All of the 5 filters commonly used transmit ultraviolet and some
blue light but they have an appreciable transmittance in the red portion of the
spectrum as well.'^ Hence, doubly refractile particles should be expected to
appear red when viewed with ultraviolet light in the fluorescence microscope.
Perhaps additional study of thin sections will reveal particles with a more
conclusive combination of properties.* In our opinion the present evidence is
inadequate to suggest a biological origin for the indigenous particles.
Discussion
Several features make it difficult to accept the highly structured particles as
extraterrestrial in origin. They are absent from our preparations of Orgueil,
although material from the same stone was used. They have not been ob-
served in thin sections, and they often show a morphological resemblance to
common airborne contaminants. Although a strong case can be made for the
biological origin of some of these structures, the probability of a terrestrial
contamination has not been ruled out in their case.
The situation is altogether different in the case of the small, brownish-yellow,
somewhat irregular, roughly spherical grains which apparently make up most
of the 1700 particles per milligram reported previously by Claus and Nagy^
and Nagy ei air Although our own experience suggests that this number
represents an appreciable overestimation, there is no doubt that such particles
do exist.
They are undoubtedly indigenous to the meteorite, but their morphology is
so featureless that an inorganic origin cannot be ruled out. None of the other
* Additional observations on thin sections are reported in another paper (Anders and
Fitch, Science, in press).
510 Annals New York Academy of Sciences
criteria for a biological origin seems to hold for these particles. They do not
fluoresce and they do not take biological stains in a manner that will distinguish
them from irregular silicate fragments in Orgueil and in kimberlite. Because
they disappear after treatment with acids, we believe that they are most Ukey
grains of minerals, although they are classilied as organized elements by Nagy
et al. The 2 particles remaining in our sample after HF-HCl treatment re-
semble terrestrial contaminants. Moreover, it must be emphasized that only
2 were seen where several thousand should have been found.
Even if organic particles should be found, a biological origin need not be
inferred. Both the polypeptide particles of Fox'* and the hydrocarbon poly-
mer particles of Wilson'^ have an appearance at least as organized as the less
structured organized elements. These materials are produced in vitro, by dry
polymerization of amino acids,^* and the Miller-Urey type synthesis,'*'''^ re-
spectively. In FIGURE 11 is illustrated a preparation obtained through the
courtesy of Wilson in which most of the polymer occurred in the form of sheets
containing thickened, round spots about 10 /x in diameter. Much of the ma-
terial was fluorescent, but some of the larger spots were not.
It may well be that life did exist in meteorites, but we feel that the present
evidence is not adequate to suggest an extraterrestrial biological origin for the
particles found in the carbonaceous chondrites.
Criteria for Identification of Life Forms
If the present data are inadequate, what kind of information is needed to
decide whether or not a particle is, in fact, a life form? This requires an initial
definition of life. Life has three essential qualities. Life requires reproduc-
tion of itself with the possibility of mutations developing along the way. Regu-
lated and integrated anabolical and catabolical chemical processes are a second
feature of life. Structural organization at the molecular and supramolecular
levels is a third feature. Probably for simple, small organisms, it is necessary
to demonstrate all of these features — reproduction, metabolism, and organiza-
tion— to establish the presence of life.
What is needed to establish that life had been present at some time in the
past? Ideally, remnants of all these features should be found. In reproduc-
tion of all terrestrial forms, nucleic acids carry information from one genera-
tion to the next. Nucleic acids or breakdown products from them may remain
after life has ceased. Evidence of metabolic processes frequently remains.
Many carbohydrates and lipids are rather resistant and persist for long periods.
Persistence of the organization of any organism forms the basis for terrestrial
paleontology. This morphology may be the result of partial or complete
replacement of biological materials with nonbiogenic compounds. If replace-
ment has been complete, probably one can never be entirely certain that a
given structure was originally of biological origin. In terrestrial materials,
this is occasionally an important question but it is never a critical one. For
nonterrestrial materials it is a critical question.
If "fossilization" or replacement has been incomplete, then metabolical
products of various sorts will remain. In pre-Cambrian rocks containing ap-
parent fossil forms, there are, in fact, substances that resist the acid treatments
used to remove the mineral materials.'" With cytochemical as well as other
Fitch & Anders: "Organized Elements" in Chondrites 511
microscopical techniciues, il should be possible to gain considerable information
about the composition of these substances. Once characterized at the micro-
scopical level, the substances can be isolated in larger quantities and other
parameters including optical activity and isotopic composition can be measured.
•
G
t'
!»».
O
1B3p
^ i
1%"^
y •
% %
A* A \
,^
J*
^^
♦
IT •
Figure 11. Hytlrocailmn polymer prepared by Wilson (1960). Thickened sjjots are
present in the sheet. Viewed in ultraviolet light, the spots and the sheet had a yellowish
fluorescence. The line is 20 /x in length.
512 Annals New York Academy of Sciences
The observed properties of the resistant material can be compared with proper-
ties of biological compounds as well as with those of various synthetic materials
including polypeptide particles prepared by Fox^'' and hydrocarbon polymer
particles prepared by Wilson. ^^ It is evident from their work as well as that
of Miller,"^ '^^ Palm and Calvin/- '^^ Or6,-° Berger'-^ and others that complex
organic materials can be prepared through nonbiological processes.
This approach assumes to some extent at least that extraterrestrial life re-
sembles terrestrial life chemically. This may be a provincial idea, but com-
parison of unknown materials with terrestrial forms would seem to be a good
starting place. It may be that even after this information is gathered and
analyzed, no detinite conclusions can be drawn. However, this information
should provide a broader basis for critical evaluation than morphology alone.
Summary
"Organized elements" described by Claus and Nagy^ and by Nagy et air
are a heterogeneous group of particles which, in our opinion, are best classified
into two types: those that have a highly structured morphology and those
that have a much simpler appearance. The particles with highly structured
morphology are less numerous than the simpler type. They have not been
seen in thin sections and many appear to have a strong resemblance to com-
mon terrestrial contaminants. The particles of simpler morphology which
do not fluoresce, which either do not stain or stain atypically with biological
stains, and which are soluble in acids seem to be of an inorganic composition
and origin. It is possible that life did exist in meteorites, but we think that
the present evidence is not adequate to suggest an extraterrestrial biological
origin for the particles found in the carbonaceous chondrites.
A cknowledgments
The authors express their gratitude to the Argonne Cancer Research Hospital
for allowing the use of its facilities for some of the experiments, and to the
staff of the Allergy Laboratory of the Jewish Hospital of Brooklyn for the loan
of pollen slides. We are also indebted to Dr. George Claus and Prof. Bartholo-
mew Nagy for permission to study and photograph their samples, in exchange
for our preparations which they described in reference 2.
This work was supported in part by the U.S. Atomic Energy Commission.
References
1. Claus, G. & B. Nagy. 1961. A microbiological examination of some carbonaceous
chondrites. Nature. 192: 594.
2. Nagy, B., G. Claus & D. J. Hennessy. 1962. Organic particles embedded in minerals
in the Orgueil and Ivuna carbonaceous chondrites. Nature. 193: 1129.
3. Fitch, F., H. P. Schwarcz & E. Anders. 1962. "Organized elements" in carbona-
ceous chondrites. Nature. 193: 1123.
4. DuFresne, E. R. & E. Anders. 1962. On the chemical evolution of the carbonaceous
chondrites. Geochim. et Cosmochim. Acta. 26: 1085.
5. Pearse, a. G. E. 1960. Histochemistry, Theoretical and Applied. Little, Brown &
Co. Boston.
6. LisoN, L. 1960. Histochemie et Cytochemie Animates, Principes et Methodes. Vol.
I. Gauthier-Villar. Paris.
7. McManus, J. F. A. 1961. Periodate oxidation techniques. In General Cytochemical
Methods. 2. : 171. J. F. DanieUi, Ed. Academic Press. New York.
Fitch & Anders: "Organized Elements" in Chondrites 513
8. Bernal, J. D. 1962. Comments. Nature. 193: 1127.
9. Seshachar, B. R. & E. VV. Flick. 1949. Application of perchloric acid technique to
protozoa. Science. 110: 639.
10. FuNKHOUSE, J. VV. & W. R. EviTT. 1959. Preparation techniques tor acid-insoluble
microfossils. Micropaleontology. 5: 369.
11. DuRH.AM, O. C. 1950. Report of the Pollen Survey Committee of the .\merican Acad-
emy of Allergy for the season of 1949. J. Allergy. 21: 442.
12. DuRH.AM, 0. C. 1938. Incidence of air-borne fungus spores. II. Hormodendnim,
Aliertiaria and rust spores. J. Allergy. 10: 40.
13. Richards, O. W. 1955. Fluorescence microscopy. In Analytical Cytology. Ed. 1 :
5/1. R. C. Mellors, Ed. Blakiston Diy., McGraw-Hill Book Co! New York.
14. Fox, S. VV. & S. YuYAM.A. 1963. Abiotic production of primitiye protein and formed
microparticles. Ann. N.V. Acad. Sci. 108(2): 487-494.
15. Wilson, A. T. 1960. S\nthesis of macromolecules vmder j)ossible primeval Earth
conditions. Nature. 188: 1007.
16. Miller, S. L. 1953. A production cf amino acids under possible primitive Earth
conditions. Science. 117: 528.
17. Miller, S. L. 1955. Production of some organic compounds under possible primitive
Earth conditions. J. Am. Chem. Soc. 77: 2351.
18. Palm, C. & M. Calvin. 1961. Primordial Organic Chemistry. I. Compounds re-
sulting from electron irradiation of C'^H4 . J. .\m. Chem. Soc.
19. Palm, C. & M. Calvin. 1961. Electron irradiation of aqueous solutions of HCN. : 65.
Bio-Organic Chemistry Quarterly Report UCRL-9900.
20. Oro, J. 1963. Studies in experimental organic cosmochemistry. Ann. N.Y. Acad. Sci.
108(2): 464-481.
21. Berger, R. 1963. Evaluation of radiation effects in space. .\nn. N.Y. Acad. Sci.
108(2): 482-486.
ON THE ORIGIN OF CARBONACEOUS CHONDRITES*
Edward Anders
Enrico Fermi Institute for Nuclear Studies, and Departments of Chemistry and Geophysical
Sciences, University of Chicago, Chicago, III.
Carbonaceous chondrites are related to other classes of meteorites in many
ways, and much of what has been said about the origin of meteorites, in gen-
eral, appUes to carbonaceous chondrites as well. Like all other meteorites,
they are fragments of larger bodies. To reconstruct their history, we must
try to learn more about the nature of these bodies, that is, their size, number,
and location, and the chemical and physical processes that produced the de-
tailed structural and compositional features of the meteorites.
Some of the principal hypotheses on the origin of meteorites are outlined
in TABLE 1. (A more complete review of the subject has been given by Anders
and Goles, 1961.) Each of these hypotheses can account for some 90 to 95
per cent of the properties of the meteorites, and it is only the last 5 to 10 per
cent that causes difficulties. There is just as much disagreement on the origin
of the carbonaceous chondrites (table 2). Mason (1960, 1961) and Ring-
wood (1961) assume that they represent some of the primitive material from
which the solar system formed; Urey (1961) believes that they are alteration
products of the high iron group chondrites, which are themselves several steps
removed from primitive material. Finally, Wood (1958, 1962) and others
believe that they are alteration products of a hypothetical, primitive chon-
drite, similar to Renazzo or Ornans (Fish et al., 1960; DuFresne and Anders,
1962a).
Clues to the Origin of Carbonaceous Chondrites
Mineralogy. Some clues to the origin of the carbonaceous chondrites can
be obtained from a study of their mineralogy. Results for 9 of these meteor-
ites are shown in table 3 (DuFresne and Anders, 1962a). The estimated
relative abundances are expressed as negative logarithms of 2; the entry 3,
for example, stands for 2"^ or 1/8. The minerals found can be divided into
three classes: conventional, "high-temperature" minerals; "characteristic"
minerals pecuhar to this class of meteorites; and trace minerals. In addition,
these meteorites also contain appreciable amounts of sulfur, hydrated MgS04,t
elemental carbon, and organic compounds. On the basis of their mineral
composition, the carbonaceous chondrites can be divided into 5 subclasses.
These show a fair degree of correspondence with Wiik's (1956) three classes,
established on the basis of chemical composition only.
One can prove rather convincingly that the characteristic minerals are al-
teration products of the high-temperature minerals, rather than vice versa.
* This work was supported in part by the U.S. Atomic Energy Commission.
t The state of hydration varies with the temperature and the relative humidity at the
time of measurement. Very probably, the MgS04 was present as the anhydrous salt or as
the monohydrate at the time of fall, and became hydrated after exposure to atmospheric
moisture. Boato's (1954) measurements show that the water released below 180° C. has a
normal D/H ratio, and is probably of terrestrial origin.
514
Anders: Origin of Carbonaceous Chondrites
515
X-ray diffraction and optical studies of composite grains of olivine and Murray
F (a hydrated silicate), show that the olivine sometimes occurs in thin parallel
plates of the same crystallographic orientation, although the individual plates
are separated by a thin layer of exceedingly finely grained, randomly oriented,
Murray F mineral. The common orientation of the olivine plates can be
understood only if single crystal olivine served as the starting material (Du-
Fresne and Anders, 1962a). Still, one cannot exclude the possibility that some
fraction of the characteristic minerals is primordial, rather than being derived
from the olivine.
Many of the other characteristic minerals, too, seem to be hydrated silicates.
This fact, and particularly the occurrence of MgS04 in distinct veins (figure 1)
Table 1
Properties of Meteorite Parent Bodies
Size
Location
Number
Heat source
Lovering (1957)
Planetary
2-5 a.u.
One
Long-lived
radioactivity
Urey (1959)
Lunar
1 a.u.
One
Chemical reac-
tions; adiabatic
compression
of gases
Fish et al. (1960);
Wood (1958, 1962)
Asteroidal
2-5 a.u.
Several
Extinct radio-
activity
Ringwood (1961)
Lunar
2-5 a.u.
Several
Radioactivity
Table 2
Origin of Carbonaceous Chondrites
\. High-iron group chondrites altered by infiltration of water, carbonaceous matter, and
hydrogen sulfide from some other source (Urey, 1961).
2. Primitive material accreted at low temperatures from solar nebula (Mason, 1960, 1961;
Ringwood, 1961). Other chondrites were derived from this material by heating and
reduction.
3. Primitive material expelled from the sun at high temperatures (Wood, 1958), accreted at
low temperatures into asteroidal-sized bodies (Wood, 1958, 1962; Fish et al., 1960),
altered by liquid water and sulfur compounds (DuFresne & Anders, 1962a).
suggests that licjuid water must once have acted on these meteorites. This
raises three interesting questions. First, what were the chemical and phys-
ical conditions (pH, reduction potential, and temperature) during this aqueous
stage, and how long did it last? Second, what was the source material of the
carbonaceous chondrites, i.e., where did the high temperature minerals come
from? And third, in what setting did this aqueous stage occur?
Former environment. To answer the first question, one can turn to the sta-
bility diagrams of Garrels (1960), which give the stabihty regions for various
minerals and ions as a function of pH and reduction potential (Eh) . In figure
2 is shown a composite diagram based upon Garrels' data. Looking up the
stability regions of the principal constituents of carbonaceous chondrites on
this diagram, one finds that nearly all of them [Fe304 , (Mg,Fe)C03 , MgS04 ,
S, organic matter] can coexist under equilibrium conditions at pH 8 to 10 and
516
Annals New York Academy of Sciences
Eh > —0.2 V. This conclusion was reached independently by Nagy el al.
(19626). The only exception is FeS, in place of which one would expect FeSa .
It is not too difficult to find an ad hoc assumption that accounts for this dis-
crepancy. For example, one can argue that the FeS was first made under
conditions in which it was stable, possibly even at high temperatures, and
that it was then brought in contact with solid sulfur at such low temperatures
that the rate of reaction was very slow.
It is quite remarkable that the carbonaceous chondrites are so close to chem-
ical equilibrium, because intuitively one would think of an assemblage of highly
Table 3
Mineralogy of Carbonaceoits Chondrites*!
Orgueil
Ivuna
Hari-
pura
Cold
Bok.
Mighei
Murray
Ornans
Lance
Mokoia
Wiik's class
I
I
II
II
11
II
III
III
III
Subclass
A
A
B
C
C
C
D
D
E
Clinopyroxene
Olivine
a-Iron
7-Iron
Magnetic troilile
Orgueil LM
Magnetite
Murray F
Haripura M
Mokoia HT and SW
Epsomite
Sulfur
Dolomite
Breunnerite
Pentlandite
Higli Temperature Minerals
3
?
3
3
1
1
0-1
0-1
9
10
10
7
5
10
5
5
5
"C
liaracle
nslic" j1
lineraL
f
1
1
1
1
3
1
3
1
lit
1
I
?
?
3
3
6
6
6
6
>16
10
6
6
6
9
9
9
>20
13
5
6
6
Trace Minerals
9
10
8
11
* After DuFresne and Anders (1962a).
t Estimated abundances are given as negative logarithms of 2. Thus Mighei is about 50
per cent olivine and 50 per cent "Murray F" mineral, with mere traces of iron, pent-
landite, magnetite, epsomite, and sulfur. Italicized values are of lower accuracy.
I Trace associated with metallic iron.
oxidized (S04=, Fe.s04 , CO,r) and reduced (S, FeS, C, organic matter) species
as being far from chemical equilibrium. The source for the basic pH might
be ammonia, and for the negative Eh, hydrogen (< 10""^ atmos.). Both would
conveniently disappear as the water evaporated.
The temperature at which the aqueous stage occurred is a little harder to
determine. A lower limit near 0° C. is implied by the condition that the water
was liquid; an upper limit of 200° to 400° C. is provided by various other ob-
servations, e.g., the strained glass found in the Mighei carbonaceous chondrite
(DuFresne and Anders, 1961). As shown in figure 3, the strain disappears
after annealing for 48 hours at 206° C, so that after the incorporation of this
Anders: Origin of Carbonaceous Chondrites
517
glass into the meteorite the temperature of Mighei could never have exceeded
206° C. for as long as 48 hours. Other time-temperature combinations can be
read off the graph, although it is doubtful whether any extrapolation beyond
the measured points is valid. One can infer that temperatures were much lower
from the fact that the characteristic minerals are quite finely grained, judging
from the diffuseness of their x-ray diffraction patterns. It seems likely that
the aqueous stage occurred at approximately room temperature. There is
hope of obtaining a more accurate value by measuring the O'YO^'' fractiona-
tion between carbonate and magnetite (Clayton, 1962). Presumably the
Figure 1. A fragment of Orgueil, showing white vein of magnesium sulfate running hori-
zontally across specimen. This vein must have deposited from water solution, thus offering
evidence of the onetime presence of liquid water in the meteorite parent body. (Reproduced
from DuFresne and Anders, 1962a, with permission of the editor.)
carbonate was made during the aqueous stage, by the action of CO2 on basic
oxides. The CO2 was, in turn, probably evolved from the interior of the body
during reduction of iron oxides to metallic iron. If the carbonate and mag-
netite reached isotopic ecjuiUbrium during the aqueous stage, the temperature
of this stage may be determined by means of Urey's paleotemperature method.
A clue to the duration of the aqueous stage is given by the relatively high
degree of ordering of the Ca++ and Mg++ ions in the dolomite from Orgueil
and Ivuna. From a comparison with terrestrial dolomites. Goldsmith has
estimated a formation time of > 10^ years.
Ancestral material of carbonaceous chondrites. It is a little harder to get an
answer to the second question, concerning the origin of the high temperature
minerals. Edwards and Urey (1955) and Urey (1961) have pointed out that
518
Annals New York Academy of Sciences
the carbonaceous chondrites have a variable, and frequently lower, content of
Na and K than the ordinary chondrites. In the most extreme case, Nogoya,
this depletion amounts to a factor of ^^4. Urey, therefore, suggested that the
carbonaceous chondrites were derived from the ordinary chondrites [specif-
0.8-
FiGURE 2. Stability relations among some of the important constituents of carbonaceous
chondrites, as a function of reduction potential and hydrogen ion concentration. Solid lines
show boundaries between solids and aqueous species at an activity of the latter of 10"" m;
dashed boundaries, those between aqueous species at 1:1 ratios. Temperature = 298° K.;
total pressure = 1 atmos. Total activity of dissolved sulfur species = 0.1; of carbonate
species, 0.01. Most of the constituents of carl)onaceous chondrites could coexist under equi-
librium conditions at Eh -^ —0.2 and pH 6 to 10. The exceptions are FeS (in place of which
FeSo would be expected) and (Mg,Fe)CO:! . The absence of FeS.> was discussed in the text.
The presence of (Mg,Fe)CO:j is not surprising: although i)ure FeCOn is unstable under the
particular conditions indicated, magnesium-rich breunnerite is likely to be stable. Also, an
increase in the total carbonate, and a decrease in the total sulfur activity will make FeCOs
stable in the triangular field bounded bv the dotted line. This figure has been adapted from
Garrets (1960), figures 6.11, 6.18, 6.19,' 6.20, and 6.21. (Rejjroduced from DuFresne and
Anders, 1962a, with permission of the editor.)
ically, the high iron group, Fe/Si ^ 0.85, Urey and Craig (1953)], by an altera-
tion process that depleted the alkalis while introducing S, C, and a few other
elements in free or combined form.
This picture has become less satisfactory now that the abundances of various
trace elements in meteorites have been determined. Most elements occur in
meteorites in approximately their "cosmic" abundances, as given by the semi-
Anders: Origin of Carbonaceous Chondrites
519
empirical abundance curves of Suess and Urey (1956) and Cameron (1959).
Other trace elements, including most chalcophile ones, do not conform to this
pattern. They occur in approximately their predicted abundances in car-
bonaceous chondrites, but are depleted by factors of up to 1000 in ordinary
chondrites (figure 4). If the carbonaceous chondrites were derived from
ordinary chondrites, as suggested by Urey, one would have to assume that
the depleted elements were somehow added to the carbonaceous chondrites
during the alteration process. In that case, it would be a remarkable coinci-
dence if 6 of the 7 elements happened to be restored to just their cosmic abun-
dances. (The seventh, mercury, may be exceptional because of its high
Annealing of Mighei Glass
• Almost Complete Anneal
— I Completion Observed
X Discontinued
300
o
260 I
0.1
10 100
Annealing Time (hrs)
1,000
Figure 3. .\nnealing of strained glass from Mighei carbonaceous chondrite. .\fter the
incorporation of the glass, the meteorite cannot have been heated to temperatures as high as
206° for as long as 48 hours, or the strain would have disappeared. (After DuFresne and
Anders, 1961.)
volatility, but it should be noted that the point in figure 4 is based upon a
single measurement.)
The olivine in carbonaceous chondrites has a highly variable iron content
(Ringwood, 1961), whereas it is of nearly constant composition in ordinary
chondrites (Mason, 1962). This factor, too, makes it difficult to derive car-
bonaceous chondrites from ordinary chondrites by any simple process.
Another clue comes from the primordial noble gases which seem to be present
in all carbonaceous chondrites (figure 5). All meteorites contain noble gases
produced by cosmic rays or the decay of long lived radioactivities, but the car-
bonaceous chondrites also contain primordial noble gases that can be distin-
guished from cosmogenic or radiogenic noble gases by their isotopic and ele-
mental composition (Stauffer, 1961 ; Anders, 19626). With the exception of He^
and Ar^", most of which is radiogenic, the noble gases in an ordinary chondrite
520
Annals New York Academy of Sciences
are produced chielly by the action of cosmic rays on iron, silicon, and other sta-
ble elements in the meteorite. For example, the 3 neon isotopes are made in
nearly equal amounts in this process (Eberhardt and Eberhardt, 1961) whereas
in primordial neon (represented by neon in the earth's atmosphere) the ratio
Ne^o/Ne^VNe-- is 90.8/0.26/8.9. The elemental ratios differ too, as can be
seen in figure 5. The bulk of the primordial noble gases once associated
with the matter of the terrestrial planets and the asteroids seems to have been
lost at a very early stage in the history of the solar system. It is not very
plausible to assume that these gases were first lost from the ordinary chon-
100
.10
c
o
-o
c
■D
XI
<
"(J
e
(/>
o
o
■ Carbonaceous Chondrites
n Ordinary Chondrites
Pb
Pb
Tl
Bi
10
10
In
Ls_
-4
10'^ 10' "^
Observed Abundance (atoms/ 10 atoms Si )
Figure 4. Trace element abundances in carbonaceous chontlrites and ordinary chondrites.
x\lthough strongly depleted in ordinary chondrites, most of these trace elements occur in
carbonaceous chondrites in nearly their "cosmic" abundances. This suggests that carbona-
ceous chondrites are more closely related to primordial matter than the ordinary chondrites.
[Data were taken from the following sources: Bi, Hg, Pb, and Tl, Reed et al. (1960), and
Ehmann and Huizenga (1959); Cd, Schmitt (1961); I and Te, Goles and Anders (1962); In,
Schindevvolf and Wahlgren (1960); Sb, Anders (1960).]
drites, then stored somewhere, and finally incorporated somehow in the car-
bonaceous chondrites.
The spheroidal troilite and magnetite particles found in Orgueil also suggest
a high-temperature stage (Fitch et al., 1962). Their chemical identilacation was
confirmed by electron microprobe analysis (Smith, 1962). Spheroidal par-
ticles might be expected from the condensation of vapors in the liquid field,
but in the presence of cosmic proportions of hydrogen, metalUc iron rather
than FeS or Fe;j04 would result (Urey, 1952). Such "primary" metal spherules
might be transformed to FeS or Fe304 by the action of HoS or HoO at lower
temperatures. It is interesting that Sztrokay et al. (1961) have observed
spherical, opaque particles in olivine chondrules from the Kaba carbonaceous
chondrite. Similar particles are found in chondrules of many ordinary chon-
Anders: Origin of Carbonaceous Chondrites
521
drites as well (Fredriksson, 1%2). Alteration of the olivine by water would
release these spherules, possibly in altered form, from their chondrule matrix.
But it is also possible that the spherules formed at a later stage. The particles
in Orgueil are quite similar to the troilite globules in meteorite veins (Anders
and Goles, 1961) and may well be of similar origin. The association of many
#-
• Carbonaceous Chondrite (Murray)
o Ordinary Chondrite (Holbroolt)
»20
.21
.22
.36
.38
He He Ne'^"' Ne"' Ne'^'" Ar'^ Ar""
FiGURE 5. Noble gases in a cariionaceous and an ordinary chondrite. In Holhrook, these
gases (except for radiogenic He-") are produced by cosmic-ray induced spallation reactions on
iron and other stable nuclides. The 3 neon isotopes are made in nearly ef|ual aliundance.
In Murray, the isotopic abundances resemble those in Earth's atmosphere, suggesting that
these gases, too, are of primordial origin. A small amount of cosmogenic gas is present in
Murray as indicated l)y the increased abundances of He^ and Ne-^ relative to their atmospheric
abundances.
of the Orgueil spherules with firmly attached silicate fragments is consistent
with either hypothesis.
The trace element abundances, the variations in the olivine composition, and
the primordial gas content are most easily e.xplained by assuming that both
the carbonaceous chondrites and the ordinary chondrites were derived from
still more primitive ancestral matter. Perhaps the most embarrassing require-
ment for this material is that some of it at least must have passed through an
earlier, high-temperature stage without losing its primordial gases completely.
It is possible to accomplish this in the meteorite parent body, but some
special assumptions are required (DuFresne and Anders, 19626). A more
522 Annals New York Academy of Sciences
attractive possibility is offered by Wood's (1958, 1962) hypothesis, according to
which planetary matter, expelled from the sun at high initial temperatures,
cooled by adiabatic expansion, so that progressive expansion could take place.
The least volatile constituents would condense to high-temperature minerals
(olivine, pyroxene, nickel-iron, and later, magnetite), which would trap some of
the surrounding primordial gas. Other substances, e.g., H2O, NH3 , and carbon
compounds, would condense on temperature drop. The further accretion of
the (now cold) dust into solid bodies, and the separation of the solids from the
noncondensable gas would proceed along the path outlined by Urey (1952,
1954, 1956, 1957, 1958) or Fish et al. (1960). Incidentally, if such a high-tem-
perature stage ever took place, then cometary matter, too, must have passed
through it. This raises some new possibilities in regard to the mineral com-
position of comets. In particular, the presence in comet tails of metal (or mag-
netite?) spherules, inferred from scattered light and polarization measurements
(Liller, 1960), is somewhat easier to understand if part of the cometary material
had a high temperature history, even though its final accretion occurred at low
temperatures. This view gains further support from the discovery in cosmic
dust of metal flakes with amorphous organic attachments. The fall dates of
these particles seem to be correlated with several meteor showers of cometary
origin (Parkin, Hunter, and Brownlow, 1962). Perhaps Herbig's (1961) sug-
gestion that the carbonaceous chondrites were derived from comets should be
re-examined in the hght of this possibility.
Aqueous stage and the prerequisites for life. What about the third question,
the setting in which the aqueous stage took place? This is one point in which
the large planet hypothesis has an advantage over all others. A planet of
terrestrial size can hold water vapor gravitationally, and can maintain bodies of
liquid water, from ponds to oceans. Surely, the surface temperature must be
high enough to allow liquid water to exist, but the temperature is controlled
not only by the distance from the sun, but also by the composition of the
atmosphere. If Venus, with its CO^-rich atmosphere, were located in the
asteroidal belt, it would have a comfortable surface temperature near 300° K.,
instead of the 600° K. prevailing at its present location. If it were not for the
fact that the planetary hypothesis runs into so many other ditficulties (Anders
and Goles, 1961), one could stop here.
Of all the parent bodies discussed, the asteroids are least likely to retain
liquid water at their surfaces, owing to their small size and consequent low
escape velocities. But there is a way in which they could retain liquid water in
their interiors. If the asteroids were ever heated by an internal heat source
{e.g., extinct radioactivity), some temperature distribution resembling the
curves in figure 6 would result. The surface temperature of the body would
be controlled by the amount of solar radiation reaching it, and might be around
100 to 200° K. Farther inward, the temperature would rise until the melting
point of ice was reached. Liquid water could exist in this zone, down to a
depth at which the boiling point at the prevailing pressure was reached. In
FIGURE 7 is shown the location of this zone of liquid water for a body with a
central temperature of 1900° K. In this case, some 5 per cent of the volume of
the body will contain liquid water.
The water will not last forever, of course. Above the zone of liquid water,
Anders: Origin of Carbonaceous Chondrites
523
there will be a permafrost zone,* and the ice from this zone will evaporate at a
rate determined by its vapor pressure (Watson et al., 1961). The vapor pres-
sure depends upon the temperature, which in turn depends on the distance from
the sun. For a body with 100-km. radius, with an initial water content of 10%,
these times are indicated in table 4.
Unfortunately, this water zone is located in a dark, underground region,
where photosynthetic organisms could not grow or reproduce. To support
4000
0.00
Relative Fractional Volume
Figure 6. Temperature distribution of asteroids heated by radioactivit}- or some other
uniformly distributed internal heat source. The 2 solid curves are calculated for different
heating rates, assuming heat transport by conduction only; the daslied curve includes an allow-
ance for convective heat transport as well. In all 3 cases, some 5 per cent of the body will
find itself in the temiierature range 273° to ^400° K., in which liquid water can exist. Melt-
ing points of important meteorite minerals are indicated by horizontal lines. (Reproduced
from fish el al., 1960, with permission of the editor. Copyright, 1960 by the University of
Chicago.)
life, some source of free energy must be available. Sunlight could provide this
free energy indirectly, if some mechanism existed for bringing photosynthetic
products from the surface to the interior. It is hard to see how this might be
accomplished without a liquid vehicle. Hence, the principal remaining possi-
bility is to derive the free energy from a local source, as first suggested by Sagan
(1961). A nonequilibrium assemblage of minerals might provide such a source.
* This permafrost zone can serve to retain an "internal atmosphere" within the meteorite
parent bodN', and mav have played a role in the retention of noble gases (DuFresne and Anders,
1962a,b).
524
Annals New York Academy of Sciences
The free energy change in the conversion of high-temperature minerals to char-
acteristic minerals cannot be calculated with any accuracy, because no thermo-
dynamic data exist for the latter or their terrestrial counterparts, the serpentine
and chlorite minerals. As a crude approximation, the following reaction may
be considered:
MgoSiOj + H,0 (1) -> MgSiOs + Mg(OH)o
for which AFoys is —20 kcal. per mole. This corresponds to about 0.1 kcal.
per gram of olivine, and because the products in this reaction are capable of
rZ^ Wholly Molten (>I620°K)
^ Zone of Fe-FeS Eutectic ( 1260° < T<I620°)
H Zone of Liquid Water
Figure 7. Temperature distribution in an internally heated asteroid, for a central tem-
perature of 1900° K. The location of the zone of liquid water is indicated.
Table 4
Times for Water Loss from Asteroids
reacting further to give hydrated siUcates, this value is probably conservative.
A chondrite of the type suggested as a possible precursor of the carbonaceous
chondrites, e.g., Ornans or Warrenton, contains more than 75 per cent olivine
on a normative basis. Thus, although possible contributions by the other
minerals are neglected, the average amount of free energy released in the forma-
tion of the characteristic minerals is likely to be close to 0.1 kcal. per gram.
The extractablc organic matter in Orgueil comprises about 10 per cent of the
total carbon content (3.1 percent, Wiik, 1956). Thus, approximately 3 X 10"*
calories would be available for each gram of organic matter, assuming that none
Anders: Origin of Carbonaceous Chondrites 525
of this energy is wasted by direct reactions between the minerals. At most,
only a few thousand calories per gram would be required to produce biochemical
compounds from simpler starting materials. If some form of life arose at this
point, the remaining chemical energy could sustain it for many generations.
Any such life form would be doomed from the outset, because its energy
supply, once exhausted, would no longer be replenished. But the total amount
of energy available from this source is appreciable, f'or a liquid water zone
comprising 5 per cent of the volume of a 100-km. body, as much as 8 X 10'^ cal.
could be stored in this manner. At a typical asteroidal distance of 2.8 a.u.,
this corresponds to the total solar energy received by the body in 2 X 10^ years.
Of course, the futility of a doomed subterranean life form based upon a finite
supply of energy makes it less appealing to the human mind than a photosyn-
thetic form with a life expectancy approaching that of the planet or its central
star. But if life arose by a spontaneous event, without guidance from above,
then the probability of this event would have depended upon the chemical and
physical conditions in the environment only, and not upon the perpetuity of
the energy supply.
The suitability of asteroidal bodies as abodes of life would thus seem to
hinge mainly on three questions. First, were the times for water retention
(table 4) long enough for life to arise spontaneously? All we known about this
"induction period" for the origin of life is that it lasted less than 0.5 AE on
Earth (Kulp, 1961). Hence the asteroids cannot be disqualified on this count
alone. Second, were the necessary organic compounds present? From the
work of Calvin and Vaughn (1960), and Briggs (1961), it seems that this ques-
tion can be answered in the atfirmative, although Degens and Bajor's (1962)
observations on the bacterial production of some of these compounds may
require a reevaluation of the evidence. Third, could the initial hfe forms learn
to utilize the particular inorganic energy sources present (e.g., reactions of HoO
with olivine, Fe°, etc.)? No definite answer to this question is possible, al-
though it is perhaps relevant to point out the known, high adaptability of
modern terrestrial microorganisms.*
Thus, one cannot conclude a priori that the asteroids were never capable of
supporting life. The question of whether life ever existed in meteorites may,
therefore, be examined on its own merits, because the size of the parent body
does not impose any major limitations.
Isotope measurements. Further clues to the history of these meteorites come
from isotope measurements, although the interpretation of the data is not
always free from ambiguities. If we assume a simple, monotonic cooling
history for the meteorites, the K'^'VAr^'^ ages in table 5 give the time at which
the temperature of the meteorite fell to a low enough value to permit the
retention of radiogenic Ar'*" from the decay of K'"'. Judged from the heating
experiments of Stauffer (1961), interpreted according to the model of Goles et al.
(1960), this temperature probably lies near 200° K. Of course, short K-Ar
ages would also result if the meteorite were reheated at some later stage in its
* If such subterranean life forms ever arose on the meteorite parent bodies, they are likeh'
to have arisen on Earth and on the moon as well. This would somewhat reduce the chances
of finding prebiotic organic matter on the moon (Sagan, 1961). Moreover, much of the
Earth's initial endowment of organic matter would have been transformed by biological
activity at a very early stage in its history.
526
Annals New York Academy of Sciences
history (e.g., during close approaches to the Sun), or if its parent body happened
to remain at a temperature somewhat above, say, 200° K., where sHght, but
continuous argon losses by diffusion would occur.
That the short exposure ages are not due to diffusion losses at perihelion has
been shown conclusively at least for Cold Bokkeveld (Anders, 1962c). Here,
the content of a nonvolatile cosmogenic nuclide, Al^*^, is consistent with the Ne'-^
Table 5
Ages of Carbonaceous Chondrites
Meteorite
Group
K-Ar age
Cosmic ray exposure
age
AE
m.y.
Cold Bokkeveld*
c
1.2
0.2
Felixt
D
4.5
56
Felix*
D
4.1
48
Ivunaf
A
1.4
1.6
Lancet
D
<3.9
5
Migheit
C
4.3
Mighei*
C
2.4
2.4
Mokoiaf
E
3.4
13
Murrayt
C
2.5
4
Murray*
C
1.6
4
Orgueil*
A
1.3
3
*Zahringer (1962).
tStauffer (1961).
J Gerling and Rik (1955).
Table 6
Carbon Isotopic Composition in Carbonaceous Chondrites (Boato, 1954)
Meteorite
Class
C
«C"
%
%c
Ivuna
A
3.3
-6.6
Orgueil
A
2.8
-11.4
Cold Bokkeveld (London)
C
1.55
-9.4
Cold Bokkeveld (Paris)
C
1.6
-5.2
Mighei
C
2.6
-9.9
Murray
C
1.9
-3.9
Lance
D
0.34
-15.7
Mokoia
E
0.84
-17.4
Forest City
Ordinary Ch.
0.08
-24.3
Richardton
Ordinary Ch.
0.02
-24.6
content, so that diffusion losses of the latter seem to be ruled out. The short
exposure age (0.1 to 0.2 m.y.) would seem to suggest a lunar origin, as proposed
by Urey (1962), but this hypothesis has its difficulties (Anders, 1962f).
Other isotope measurements exist that have a bearing on the origin of car-
bonaceous chondrites. Boato (1954) has measured the carbon isotopic compo-
sition in these meteorites (table 6). The C^'/O^ ratio is variable from meteor-
ite to meteorite, and even within the same meteorite (Cold Bokkeveld). It
is known that living organisms have a preference for C^', so that biogenic ma-
Anders: Origin of Carbonaceous Chondrites
527
terials are generally depleted in C^^ relative to the source material: atmospheric
CO2 or oceanic bicarbonate (Craig, 1953). This effect is quite pronounced if the
biogenic carbon comprises only a small fraction of the total available carbon
(figure 8).
o
-36
-34
-32
-30
-28
-26
-24-
-22-
-20
-18
16
fO
O -14
<:50
-12
-10
-8
-6
-4
-2
0
+ 2
+ 4
+ 6
+ 8
+ 10
UJ
<
Z
o
CD
a:
<
- o
o
■ o
' o
••0
CO
UJ
<
cr
CD
UJ
h-
cc
UJ
>
z
UJ
z
CC
<
2
A
if)
I-
z
<
UJ
Z
<
2
UJ
_J LJ
Q.
Quj
Z UJ
< q:
_i I-
•
5:
S
Q
o
o
Z
UJ
u
UJ
cc
Q
o
o
CO
O
dip
_i
<
o
u
UJ CO
ZUJ
h- *<
COO
0
o
I
U
OJ
O
U
H
c/)
I-
Z
UJ
Q
UJ
UJ
_l
O
CC
f-
UJ
Q.
UJ
H
X
Q.
t
0
o
q:
C/)
D
O
UJ
Z
o
o
o
o
cr
UJ
X
Q.
CO
o
if)
Q
<
Figure 8. Isotopic composition of carbon from various sources. Processes involving a
partial loss of carbon in the form of volatile compounds {e.g., the formation of petroleum from
the remains of organisms) result in the depletion of C. Such a depletion is also observed in
the case of the meteorites (table 6) in which the C" content declines with decreasing total
carbon content. [Reproduced from Craig (1953), with permission of the editor.]
528
Annals New York Academy of Sciences
Unfortunately, Boato measured only the total combustible carbon, and not
the fractionation among the several forms of carbon in the meteorite. There
seems to be a correlation between decreasing C'^ content and decreasing total
carbon in the meteorite. Boato suggested that this imphed preferential loss
of C" during partial volatilization, and pointed out that a similar depletion
had been observed in terrestrial processes that were accompanied by a loss of
volatiles, e.g., the conversion of dead organisms to petroleum. For the purpose
of the present discussion, it is immaterial whether this last process is abiotic or
biotic; any low-temperature process will lead to qualitatively similar fractiona-
tions.
Urey (1962) suggested that sulfur metabolizing organisms might be responsi-
ble for the oxidized sulfur compounds (S and MgS04) in the carbonaceous
chondrites. However, as seen in table 7, the elemental sulfur in Orgueil is
enriched in S^^ relative to the sulfate (Thode and DuFresne, 1961), whereas
sulfur bacteria as well as inorganic processes occurring under equilibrium condi-
Table 7
Sulfur Isotopic Composition in Carbonaceous Chondrites
SSHfoc)
Object
S04=
S°
Orgueil*
Gulf Coast salt domes (11 samples) f
Sulfur Lake, Cj-renaica, N. Africaf
-1.30
+41.4
+ 15.8
+3.04
+2.5
-15.3
* DuFresne & Thode (1961).
t Thode, Wanless & VVallouch (1954).
tions tend to produce just the opposite fractionation, depleting elemental S in
S''^ (Thode et al., 1954). The equilibrium constant for the reaction
s^-'Or + H Ss^^ ^ S3404= + H Ss'^
is 1.071 at 25° C. (Tudge and Thode, 1950), so that the sulfur and sulfate in
Orgueil are clearly out of equilibrium. Perhaps the origin of the higher oxida-
tion states of sulfur will be clarified by further isotope measurements on the
troilite in Orgueil (Thode and Anders, 1962).
Boato (1954) also measured the hydrogen isotopic composition of the hy-
drated silicates in carbonaceous chondrites. His results (table 8) show that
the D/H ratio in Ivuna, Orgueil, and Mokoia was considerably higher than that
in terrestrial waters. This fractionation may have been caused by kinetic
isotope effects during formation of the hydrated silicates (Clayton, 1961) or
by extensive evaporation of the water in the meteorite parent body.
Hydrocarhous. Finally, a few words should be said about the hydrocarbons
(Nagy el al., 1961). This matter has been discussed in greater detail elsewhere
(Meinschein, 1961; Anders, 1961, 1962a; Nagy el al., 1962a; Meinschein et al,
1962). For the present discussion, only three of the most salient points will be
restated.
Meinschein and his associates certainly deserve great credit for determining
Anders: Origin of Carbonaceous Chondrites
529
the mass spectrum of the hydrocarbons in the meteorite, and for drawing atten-
tion to its possible resemblance to the mass spectra of biogenic hydrocarbons.
One point on which we disagree, however, is the extent of such resemblance.
FIGURES 9 and 10, plotted from their data, show the worst and the best cases,
respectively. If the comparison is extended to the entire mass spectrum, and
to a larger variety of biogenic reference materials, certain additional resem-
blances, but also certain differences appear. It seems very difficult to decide,
on purely objective grounds, whether these resemblances are strong enough to
prove a biological origin.
There is also a question to what extent the peak height at a given mass num-
ber may be taken as a measure of the amount of parent hydrocarbon of this
mass. This is a good assumption for the [CnHiH+o]"^ ions derived from the
CnH.2n+2 paraffius. But as one goes to compounds progressively poorer in
hydrogen, the ambiguity increases. The [CnH-in-e]"*" ions are derived not only
from the C„H-2„_6 ( = tetracycloalkane) series, but also from the CuH2u+2 ,
Table 8
Hydrogen Isotopic Composition in Carbonaceous Chondrites (Boato, 1954)
Meteorite
Class
H2O
6D
%
%
Ivuna
A
7.0
-1-35.8
Orgueil
A
7.3
-1-29.0
Cold Bokkeveld (London)
C
7.8
-13.0
Cold Bokkeveld (Paris)
C
8.0
-5.8
Mighei
C
8.6
-6.4
Murray
C
6.8
+9.6
Lance
D
0.9
-7.7
Mokoia
E
0.8
+25.9
Terrestrial waters
-15 to +5
CnHon , CnH2n-2 , aud CnH2n-4 families, with possible additional contributions
from nitrogen and oxygen compounds. Thus, it seems fair to attribute most
ob the observed peak height in the C„H2n+2 series to paraffins. However, just
in the case of this series, the resemblance is rather poor (figure 9), and the
great difference between the spectra of the original Orgueil distillate (Nagy
et al., 1961) and the chromatographically separated hydrocarbon fraction
(Meinschein, 1961) shows that even in this favorable case, some 70 to 90 per
cent of the originally observed peak height came from compounds other than
saturated hydrocarbons.* In figure 10, the resemblance is very good, and the
changes have been moderate, but as pointed out, the peaks in this series contain
substantial contributions from so many different sources, that it seems unsafe
to infer a similarity in parent hydrocarbon distribution from a similarity in
peak heights.
Finally, one must not overlook the possibility that the observed hydrocarbon
* This sample is not strictly comparable to the original distillate, collected in the range
250° to 400° C, because it also contains the 400° to 500° C. fraction. But differences of the
same order are found between the original distillate and a chromatographically separated
hydrocarbon fraction of a solvent extract of the whole meteorite (Meinschein et al., 1962).
530
Annals New York Academy of Sciences
distribution was made abiotically by Miller-Urey type reactions in the solar
nebula. Such reactions are known to produce carbon chains of varying length,
presumably by free radical reactions. The hydrocarbons in comets and the
organic material in cosmic dust (Parkin et al., 1%2) may have been produced
in this way. Meinschein (1961) has argued that such reactions would be
highly nonselective, showing little preference among the billions of possible
I.OOOr
100-
X
o
<v
a.
10-
- 1 1 1 1 1 1 1 1 1
-
: C,H2,,2 Series
-
A
/ 1 ,. _^
-
^^,,^1 _,.^
■-——-'' — 7 j*^^" ' /
^•'~"-— — J-"' \ \. 1
y'' ' * ^V /
/ * ^X ^ /
~ / \ \/' '\^ J/\
~
I / ', / \^ *^ // ^^
-
/ ' ' ^"^"^..J ''^
-
"">.
-
-y N^-A
-
V^
~
\ / —
~
\ /
\ /
—
'-- \ /
-
» /
-
\'
-
_ OrgueiKdistillate ) V
-
Orguell (saturated HC fraction )
~ Butter
Recent sediments
1 1 1 1 1 1 1 1 1
15
20
Carbon Number
25
Figure 9. Mass spectrum of meteoritic hydrocarbons and 2 Ijiogenic reference materials
(Nagy el al., 1961). The observed peak heights in the C„Ho„+.. series are probably due,
mainly, to parent ions of saturated hydrocarbons, although fragment ions of other substances
also contribute. The difference between the original Orgueil distillate and the chemically
separated, saturated hydrocarbon fraction indicates that large amounts of other substances
were present in the distillate.
isomers. But it is essential not to equate the concepts "abiotic" and "non-
selective." Industrial chemical syntheses, from polyethylene to medicinals,
are highly selective, favoring one or a few products over the multitude of others.
Even Miller-Urey type reactions can be quite selective, as shown by Wilson
(1960). He obtained products mainly in the mass ranges Ci to C5 and C20
and up. Although the product distribution in that particular experiment
(and in the industrial Fischer-Tropsch synthesis of hydrocarbons) may not be
an accurate match of the Orgueil hydrocarbon distribution, one must remember
that only an intinitesimal fraction of the possible combinations of conditions
Anders: Origin of Carbonaceous Chondrites
531
(composition, temperature, pressure, time, energy input, catalysts, availability
of surfaces, etc.) has been explored.
Some chemical evidence has become available on the Orgueil hydrocarbons
(Yang and Tsong, 1962). A cyclohexane extract of the meteorite shows nothing
but C — H groups in its infrared spectrum, indicating that it consists mainly of
hydrocarbons. The ultraviolet absorption spectrum shows a broad band near
270 m/i, but virtually no absorption above 300 m/x. Hence, aromatic ring
systems larger than naphthalene or biphenyl seem to be ruled out. Pre-
sumably, 1- and 2-ring aromatic systems with aliphatic side chains are present.
Chromatographic separation on silica gel resolved in the material into 5 spots, 2
of which fluoresced weakly under ultraviolet light. The material possessed a
strong, terpene-hke odor. More complex materials, including polynuclear
1,000
5 100
o
Q.
20
z — 1 r- "T 1 1 1 1 1 1 1 1 1 1
-
^ ^^^^^^^ ^>— -.'1 /^*.i*ll oio. \
^
Orgueil (distillate)
- /\ Orgueil (saturated HC fraction)
—
"/'^^ Butter
jr _^ ^^^ Recent sediments
^v \ '^5v /" - — — ■-' ^
y
^^^C^^^^''*^--.. "/^ ^
v~
- C^Hg^.g Series ~~— -'
1 1 1 1 1 1 1 1 1 1 1 1 1
\
15
20
Carbon Number
25
Figure 10. Mass spectrum of meteorilic hydrocarbons and 2 biogenic reference materials
(Nagy et al., 1961). In the CnHsn.e series, the meteorilic and terrestrial mass spectra show a
strong resemblance to each other, but because the contribution of fragment ions to the peaks
is quite large in this series, the similarity in peak heights does not necessarily imply a simi-
larity in hydrocarbon distribution.
hydrocarbons of higher molecular weight, bearing polar substituents, were
extracted from the meteorite with more polar solvents, but it seems that none
of these higher polynuclear hydrocarbons were present in the free state. This
relative simplicity of the aromatic hydrocarbon fraction was already noted by
Meinschein el al. (1962), on the basis of mass spectrometric analysis.
Perhaps the hydrocarbons in Orgueil are of biogenic origin. But in our
opinion, the present evidence is not suthcient to justify this conclusion.
Summary
The carbonaceous chondrites seem to have been produced by the action of
liquid water on a more primitive source material. Their mineralogy implies
that this exposure to water occurred at temperatures near 300° K., a pH of 6-10,
a reduction potential of <— 0.2 volts, and that it lasted for at least 10^ years
(DuFresne and Anders, 1962). Their high content of chalcophile trace ele-
532 Annals New York Academy of Sciences
imiits and primordial nobk- gases suggests a source material more primitive
than ordinary chondrites; yet the presence of high-temperature minerals
implies that this source material j)assed through at least one high-temperature
stage. These conditions would be satisfied by material expelled from the sun
in a gaseous state (Wood, 1958), and accreted to soUd bodies after condensation
and cooling (Urey, 1952).
The exposure to liquid water could have occurred in subsurface regions of an
asteroid heated by extinct radioactivityor anotherinternal energy source. Sun-
light for photosynthesis would not reach these regions, but an appreciable
amount of free energy would be available from the conversion of olivine to
hydrated silicates. Although this source of energy is finite, it may have
served as the basis for the evolution of a nonphotosynthetic life form.
None of the isotopic data suggest the presence of life, however. The fractiona-
tion between sulfur and sulfate in Orgueil is in the opposite direction from that
observed for terrestrial sulfur bacteria. The carbon data are inconclusive, hav-
ing been determined on the total combustible carbon only, rather than on
individual compounds or fractions. The hydrocarbon data are also not con-
clusive, since the degree of resemblance to biogenic hydrocarbons and the ability
of nature to produce such a hydrocarbon distribution by purely abiotic (Miller-
Urey) reactions are still open to dispute.
A cknowledgments
I am greatly indebted to E. R. DuFresne, whose work provided many of the
basic data cited in this paper. I also want to express my gratitude to N. C.
Yang and Maria Tsong, who made available their unpublished data on the
organic matter in Orgueil, and to Frank W. Fitch, who contributed many valu-
able criticisms.
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AQUEOUS, LOW TEMPERATURE ENVIRONMENT OF THE
ORGUEIL METEORITE PARENT BODY
Bartholomew Nagy
Department of Chemistry, Fordliam University, New York 58, N. Y.
Warren G. Meinschein
Esso Research and Engineering Co., Linden, N.J.
Douglas J. Hennessy
Department of Chemistry, Fordliam University, New York 58, N.Y.
Sources of knowledge of the composition of the universe have been limited to
(1) information which can be deduced from radiated energy and nuclear parti-
cles, and to (2) the results of the studies of meteorites. The presence of hydrous
silicates, iron oxide, water soluble salts, and organic matter in the carbonaceous
chondrites makes the study of this rare group of meteorites especially intriguing.
There are 19 known carbonaceous chondrites (table 1). All 19 meteorites
were observed to fall. They all show a few millimeters thick fusion crust.
Various investigators have found, however, that below the crust the stones are
unaltered. Carbonaceous chondrites usually have loose textures and many of
them have a mineralogical composition indicating that they were never sub-
jected to temperatures higher than 300 to 400° C.
The Orgueil meteorite, the principal object of the present investigation, fell
at 8 P.M. on May 14, 1864. Fragments were collected in and about the villages
of Orgueil, Nohic, and Campas in southern France. The appearance of the
luminous meteor, the subsequent detonations and the fall were observed by the
local residents. It is reported that the sound of the detonations was heard
within an area of approximately 75-miles radius. The combined weight of the
fragments which are now in various museum collections is approximately 11.5
kg. Based upon present knowledge of the attrition a stony meteorite under-
goes when it enters the atmosphere and the loss of fragments scattered by the
explosions that accompany most of these falls, it is probably safe to assume
that the Orgueil stone weighed several tons before it fell to earth.
Carbonaceous chondrites are characterized by the few per cent of carbona-
ceous matter that they contain, by their water content, and as Urey and Craig^
pointed out, by the highly oxidized state of their iron content. Mere traces of
carbon and water, however, have been found in a large number of stony and in a
few metallic meteorites, all of which had high temperature histories. Only 4
of the 19 carbonaceous chondrites have been subjected to organic analysis.
As recently as 1956, Wiik- observed that". . . the organic compounds are the
least well known substances in the carbonaceous chondrites." This lack of
information is probably caused by the fact that only small quantities of organic
matter can be extracted from these chondrites and that this organic substance
is difficult to analyze. There are a number of early investigations of varying
reliability described in the literature; most of these vaguely refer to "bitumi-
nous" substances, specifying odor, color, etc.
534
Nagy et al. : Environment of Orgueil Meteorite Parent Body 535
Organic Analyses
Meteorite organic analyses may be divided into two types: (1) the classical
type analyses (which may involve the combustion of the organic matter and
the subsequent gravimetric determination of CO2 , the reacting of the extracts
with acids or alkalies); and (2) the analyses which were based upon spectro-
scopical (infrared, ultraviolet, mass spectrometry) and chromatographic tech-
niciues. Many of the former type analyses are either incompletely recorded
or seem to be unreliable for other reasons. Consequently, only 4 of these
analyses will be discussed briefly. These are: Berzelius' analysis^ of Alais in
1834; Wohler's analysis-* of Kaba, in 1858; Berthelot's analysis^" of Orgueil,
in 1868; and Mueller's analysis^ of Cold Bokkeveld, in 1953.
Berzelius was the first to ascertain the presence of organic matter in a stony
meteorite.* He suggested that the Alais organic matter resembled humic acids
Table 1
List of Known Carbonaceous Chondrites
Locality of fall
Date of fall
Locality of fall
Date of fall
Alais, France
1806
Mighei, U.S.S.R.
1889
Cold Bokkeveld, South Africa
1838
Mokoia, New Zealand
1908
Crescent, United States
1936
Murray, United States
1950
Felix, United States
1900
Nawapali, India
1890
Haripura, India
1921
Nogoya, Argentina
1879
Indarch, U.S.S.R.
1891
Orgueil, France
1864
Ivuna, Tanganyika
1938
Santa Cruz, Mexico
1939
Kaba, Hungary
1857
Simonod, France
1835
Lance, France
1872
Staroye Boriskino, U.S.S.R.
1930
Tonk, India
1911
or similar organic materials and observed that the meteorite disintegrated in
water.
Within approximately 1 year after its fall, Wohler obtained what was appar-
ently an uncontaminated sample of the Kaba meteorite. He suggested that the
meteorite may contain remnants of humic matter. One year later, in a shorter
note,* Wohler reported that he had identified bituminous material resembling
ozocerite in Kaba, and stated that this matter "has undoubtedly organic
origin." This rather important statement came 31 years after this same in-
vestigator had first discovered that a biochemical (urea) could be synthesized
from inorganic matter. One must keep in mind, of course, that facilities for a
comprehensive evaluation of organic compositions were somewhat limited in
1858.
Berthelot was the first investigator who obtained hydrocarbons from Orgueil.
* Berzelius' comments are of interest: ... "Es leidet folglich keinen Zweifel, dass der
untersuchte Stein, ungeachtet aller seiner Verschiedenheiten im Aeussern, ein Meteorstein
ist, welcher, aller Wahrscheinlichkeit nach, aus der gewohnlichen Heimath der Meteorsteine
herstammt." and "Giebt diess moglicherweise einen Wink iiber die Gegenwart organischer
Gebilde auf anderen VVeltkorpern?"
536 Annals New York Academy of Sciences
Gaseous, liquid, and solid hydrocarbons were found to be present after treat-
ment with hydriodic acid.*
In 1953, Mueller reanalyzed the Cold Bokkeveld stone. (This meteorite
had already been studied by Wohler.) Mueller extracted a soft resinous sub-
stance. Reactions of the organic matter with alkalies suggested that the
extract consisted basically of complex organic acids, containing some nitrogen,
sulfur, and halogen. It must be pointed out, however, that the high organic
halogen content has not yet been confirmed by other investigators. Mueller
searched for but could not detect graphite in the organic substance; on the other
hand, he observed 10 to 12 per cent crystalline sulfur. The Cold Bokkeveld
meteorite was found to contain water; rehydration experiments demonstrated
that this water was not a terrestrial contamination. This author also suggested
that the extract resembled humic acid. The author was able to reject the
carbide theory of hydrocarbon synthesis in meteorites on various experimental
grounds. An alternate theory was proposed by Mueller, namely, that carbona-
ceous chondrites are fragmental aggregates, and that the organic matter is the
result of low temperature condensation from the atmosphere of the meteorite
parent body. He concluded that the temperature of the meteorite never
exceeded 200 to 350° C.
There are few analyses of the second type. In 1959, Sisler^ ran infrared
spectra on an extract of the Murray meteorite and recorded carbon-hydrogen
and the carbonyl absorptions. Calvin^" obtained water extracts from Murray
and Orgueil. The extracts probably contained some hydrocarbons and hetero-
cyclic bases. It was reported that the ultraviolet absorption curves of the
extracts, taken at different pH values, showed that there was a pH sensitive
absorption at the wave length corresponding to the cytosine absorption.
Amino acid analysis led to negative results; on the other hand, mass spectro-
scopical data showed what may have been hydrocarbons containing up to 12
carbon atoms.
Boato's study" of the distribution of the hydrogen and carbon isotopes in
carbonaceous chondrites is of considerable interest. This author found that
vvater, which was distilled from Orgueil, Murray, Ivuna, and Mokoia in vacuo
and above 180° C. temperature, showed hydrogen isotope ratios that were
definitely outside the terrestrial range. On the other hand the water which
was distilled below 180° C. seemed to be a terrestrial contamination. Carbon
isotope ratios were found to be similar to those on earth. Although Boato
thought that the carbon compounds could not be derived from living things,
the C^^ depletion in the Orgueil meteorite which he observed is typical of the
depletions found in some marine organisms. The author pointed out that
carbonaceous chondrites are heterogeneous bodies, and suggested that the
meteorite organic matter is indigenous.
In a recent pubUcation^^ tj^g results of an analysis of organic matter in the
Orgueil meteorite were reported. Saturated hydrocarbon groups were identi-
fied, some of which contained up to 29 carbon atoms per molecule. The
* Berthelot's original statement reads: J'ai applique la meme methode a la matiere char-
bonneuse de la meteorite d'Orgueil. J'ai reproduit, en effet, quoique plus peniblemenl qu'ayec
la houille, une proportion notable de carbures formeniques, C2"H-"+2, comparables aux huiles
de petrole."
Nagy et al. : Environment of Orgueil Meteorite Parent Body 537
preliminary observation was made that the type of molecular species present in
the meteorite hydrocarbon mixture and the molecular weight range of the
mixture resembled in many important aspects the hydrocarbons in the products
of organisms and in sediments on earth. Studies in progress are to extend the
preliminary investigation.^- The purpose of the present study is to determine
whether the physical-chemical conditions on the meteorite parent body may
have been suitable to sustain a form of life.
Inorganic Analyses
Carbonaceous chondrites contain only a small percentage of organic matter,
the remainder consists of inorganic minerals. Their history can be determined
most clearly only if one has a satisfactory understanding of both their organic
and inorganic composition. Most stony meteorites, have been subjected to
inorganic analyses. As early as 1878 Nordenskiold'^ noted the pronounced
uniformity in the chemical compositions of chondrites. In 1953, Urey and
Craig^ reviewed some 350 chemical analyses, selected the reliable ones, and
came to the conclusion that chondrites fell into two distinct groups, a high and
a low group as far as their total iron content and the oxidation state of their
iron was concerned. They suggested that the cause of this phenomenon was
related to the genesis of meteorites. The parent asteroids went through a low
temperature accumulation process, a high temperature melting and evaporation
process, a stage of collision with smaller objects and finally a collision of 2
asteroidal sized bodies. These authors, and later Wiik,- observed that the
carbonaceous chondrites belonged to the high iron group. Urey and Craig
suggested that the material forming the carbonaceous chondrites had been
infiltrated on the parent body by water, carbon compounds, and hydrogen
sulfide. It is generally agreed that more information is a necessary prereq-
uisite to a satisfactory understanding of the genesis of these meteorites.
Wiik- has shown that there are three types of carbonaceous chondrites. The
first type (Orgueil, Ivuna, Tonk) contains approximately 20 per cent water,
approximately 22 per cent Si02 and 15 to 18 per cent "FeS." All forms of
sulfur, including elementary sulfur, were hypothetically combined with the
iron in the "FeS" reported, but x-ray diffraction data on Orgueil does not show
any FeS. The second type (Cold Bokkeveld, Murray, Mighei, Staroye
Boriskino) contains approximately 13 per cent water, 27.5 per cent Si02 , and
9 per cent "FeS." Neither the first nor the second group contains any metalUc
iron, nickel, or cobalt. The third type (Lance, Mokoia) contains approximately
33 to 34 per cent Si02 , less than 1 per cent water and between 5 and 6 per cent
"FeS." Metallic nickel and iron are present in the third group. Edwards,^*
using an analytical method developed by Edwards and Urey,^^ found that the
sodium and potassium distributions in carbonaceous chondrites agreed with
Wiik's classifications. They noted that there was one exception, the Murray
meteorite, which gave abnormally low alkali metal values.
Inorganic analyses of meteorites point out certain important relationships,
which can serve to supplement mineralogical data. Structural and synthetic
mineralogy, an active field of study during the preceding 15 years, has been
repeatedly applied with success to investigations concerned with determining
the physical-chemical environment during rock and mineral genesis. The
538 Annals New York Academy of Sciences
identification of the mineral content of noncarbonaceous chondrites is usually a
rather straightforward process. Mineral analysis in carbonaceous chondrites
is more complicated.
In 1864, Pisani/*'' who was one of the first analysts of Orgueil, noted the
presence of magnetite and a "serpentine-like" mineral. More recently, Kvasha^^
reported finding chlorites in Staroye Boriskino. Stulov^*^ concluded that
Orgueil, Cold Bokkeveld, and Staroye Boriskino contained chlorite-serpentine
type minerals. Mason^^ suggested that all carbonaceous chondrites may con-
tain chlorites. Calvin'" found that the water soluble salts in Orgueil and
Murray were magnesium sulfate and calcium sulfate, respectively. Sztrokay,
Tolnay and Foldvary-Vogl''" performed ore microscopical studies and chemical
analyses on the Kaba meteorite. They suggested that carbonaceous meteor-
ites may represent an arrested phase of meteorite development.
Layer lattice silicates (such as chlorite and serpentine) lose structural water
at elevated temperatures; Mueller'' and Boato^' came to the conclusion that
water lost at high temperature was not a terrestrial contamination. Conse-
quently, it is probably safe to conclude that the layer lattice silicates are prod-
ucts of the meteorite parent body.
Experimental Studies
The experiments were designed to examine the mineral composition and,
through this, the parent environment. Six stony meteorites were studied:
Orgueil, Murray, Ivuna, Holbrook, St. Marks, and Bruderheim. The last 3
are not carbonaceous chondrites; they were used as controls. There were 3
different samples of the Orgueil meteorite. One sample (A) was obtained from
the collection ot The American A^Iuseum of Natural History, New York. Sam-
ple (A) has only recently been acquired by this museum; previously it formed
part of an academic collection in the United States. The hydrocarbon analysis
reported in the preliminary publication'- was performed on sample (A).
The second sample (B) was broken off from meteorite specimen No. 519 of
The American Museum of Natural History. Sample (B) has been in the
museum collection for several years. The third sample (C) was obtained from
the U.S. National Museum, Washington, D.C. It was Usted as part of meteor-
ite specimen No. 234 and it was noted that the museum originally obtained it
from S. Meunier. The samples of the Ivuna, Holbrook, and St. Marks meteor-
ites were obtained from The American Museum of Natural History. The
Murray sample was received from the Institute of Meteoritics, The University
of New Mexico, Albuquerque, New Mexico, where it had been labeled as I.
O. M. No. CRi-102. The Bruderheim meteorite was obtained from the Depart-
ment of Geology, University of Alberta, Edmonton, Alberta, Canada; it had
been part of specimen B-79. The chemical analyses of the 3 carbonaceous
chondrites are Usted in table 2. The analysis of 1 of the noncarbonaceous
chondrites (Holbrook) is included in the table for comparison. The samples
were examined for visible impurities with a microscope or by visual examination,
or both.
Trace Element Analysis
The origin of carbonaceous chondrites has been discussed repeatedly since
Berzelius' research in 1834. Recently, Bernal" proposed that the Orgueil
Nagy et at. : Environment of Orgueil Meteorite Parent Body 539
meteorite may be part of the primitive earth ''shot off some hundreds of milUons
of years ago and again united to its parent body." It was, therefore, deemed
necessary to determine whether Orgueil is really a meteorite of extraterrestrial
origin.
Chondrites, as well as sedimentary and igneous rocks on earth, have char-
acteristic trace element distribution patterns. Fifteen trace elements were
Table 2
Chemical Analyses of 4 Meteorites
Carbonaceous chondrites
Noncarbonaceous
chondrite
Orgueil*
Ivuna*
Murray*
Holbrookf
Fe
7.18
Ni
—
—
—
1.09
Co
• — -
—
—
0.052
FeSt
15.07
18.38
7.67
7.94
SiO.
22.56
22.71
28.69
40.11
TiO.
0.07
0.07
0.09
0.14
AI2O3
1.65
1.62
2.19
1.90
MnO
0.19
0.23
0.21
0.37
FeO
11.39
9.45
21.08
12.01
MgO
15.81
16.10
19.77
25.18
CaO
1.22
1.89
1.92
1.74
Na.>0
0.74
0.75
0.22
0.93
K2O
0.07
0.07
0.04
0.10
P2O5
0.28
0.41
0.32
0.40
H,0+
HoO-
19.89
18.68
9.98
2.44
0.27
CroOs
0.36
0.33
0.44
0.45
NiO
1.23
1.34
1.50
CoO
0.06
0.06
0.08
C
3.10
4.83
2.78
Loss on ignition (or-
6.96
4.10
0.62
ganic matter)
Sum
100.65
101.02
100.64
99.98
* After Wiik.6
t After Mason and Wiik.^'
t Includes ail forms of sulfur, including elementarj^ sulfur. There is no X-ray diffraction
evidence that FeS, as such, occurs in Orgueil.
Note.
H2O, C, and S have been reduced from the value of the loss on ignition. The oxidation
of FeO, Fe, Ni, and Co have been taken into consideration. The ignition loss as given, is
an approximate estimate of the amount of organic matter.
H20~ refers to water removed below 110° C. temperature, H2O+ to water obtained above
that temperature.
determined in Orgueil sample (A) by emission spectroscopy. Another ele-
ment, phosphorus, was determined spectrophotometrically by the molybdenum
blue method. A Jarrell-Ash, 3.4 m. spectrograph (15,000 lines per inch grating)
was used for the trace element analysis. All determinations were made in
duplicate. Germanium was used as internal standard for cobalt, chromium,
copper, manganese, nickel, and vanadium. No internal standard was used for
barium, gallium, lithium, strontium, zirconium, scandium, cesium, and rubid-
ium because these elements were below the limits of detection. No internal
540
Annals New York Academy of Sciences
standard was used for titanium. Kodak SA No. 1 (2200-4650A) and Kodak
I-N (6700-9500A) plates were used to record the spectrum. The following
wave lengths ranges were covered (1; 2200-3500A (for Co, Cr, Cu, Ga, Mn,
Ni, Ti, V, Zr, and Sc) ; (2) 3500-4650A (for Ba and Sr) ; (3) 6700-9500A (for
Li, Cs, and Rb).
The trace element data in Orgueil are consistent with the average abundances
of trace elements in chondrites. Note particularly the Ni, Cr, Co, Ti, Ba, Sr,
and Rb values. Table 3 also shows that the Orgueil analysis does not agree
with the average abundances of trace elements in shales and in igneous rocks
(the latter is commonly referred to as the crustal abundance).* The trace
Table 3
Trace Element Abundances in the Orgueil Meteorite, in Chondrites, and in
Igneous Rocks and Shales
Meteorites
Terrestrial rocks
Element
Orgueil carbon-
aceous chondrite
Chondrites*
Shalest
Igneous rockst
Ba
<10
8
570
1,000
Co
400
800
18
20
Cr
2,600
2,200
no
100
Cs
10
0.13
5
5
Cu
200
90
18
55
Ga
<10
5.3
13
19
Li
<3
2.7
55
32
Mn
1,900
1,900
620
1,000
Ni
11,000
13,400
64
35
Rb
<10
3.7
140
115
Sc
<10
9.4
14
20
Sr
<10
10
300
450
Ti
200
790
4,920
4,400
V
30
39
120
110
Zr
<20
33
160
156
P
790
Concentrations in parts per million.
* After Goldschmidt.2'
t After Shaw;--* supplemented by Taylor and Sachs" with recent data from the literature.
{ After Ahrens and Taylor.-^
element abundances support the view that the Orgueil sample had an extra-
terrestrial or precrustal origin.
Electron Microscopy
Most of the mineral particles in Orgueil were too small to be visible under the
polarizing microscope. Therefore, 2 of the samples, (A) and (B), were examined
with North American Phillips EM-IOOB electron microscopes. Detailed
measurements were made on sample (A) after a survey has shown that both (A)
and (B) contained particles which had identical crystal habits. Specimens
were prepared by dusting with a Q-tip on I'\)rmvar film. Specimens were given
* It should l)e noted, that it is difficult to establish average values for trace elements in
shales; this was pointed out by Shaw.-''
J
i
Nagy et al. : Environment of Orgueil Meteorite Parent Body 541
a light coating of carbon evaporated under vacuum for stabilization and for
improved heat conductance under the electron beam. Some specimens were
shadowed with platinum.
Figure 1. Electron micrograph of the Orgueil meteorite. A, micaceous particle. Ao,
micaceous particle with one edge rolled up; B, aggregate of micaceous particles; C", opaque,
equidimensional particle (probably magnetite).
In FIGURE 1 is shown 1 of the electron micrographs. It was found that the
Orgueil meteorite consisted mainly of thin, sheethke particles, and of the
aggregates of such particles. The flaky crystals had irregular shapes. Their
average particle size was appro.ximately 0.1 to 0.2 fx. The thickness of the
flakes was not estimated; it appeared that they were quite thin. Some of the
flakes showed a tendency of rolling up along one or more edges under the elec-
tron beam. The flaky particles resembled layer lattice silicates, and particu-
larly the thin, irregular, and fluffy flakes of montmorillonite clay. In addition
542 Annals New York Academy of Sciences
to the flaky mineral, a few opaque and equidimensional particles were also
visible. These were probably octahedral or dodecahedral crystals of magnetite.
Their average diameters were 0.2 to 0.5 fi.
X-ray and Electrott Diffraction Studies
X-ray and electron diffraction techniques were used to identify the mineral
matrix of the Orgueil meteorite. The x-ray data were obtained from diffrac-
tometer patterns, from manual, step-scanning counts, as well as from flat film
and Debye-Scherrer photographs.
The 6 meteorites, (including 2 Orgueil samples, A and C), were x-rayed. In
addition, x-ray patterns were obtained from a sample of salt, extracted with
water from Orgueil, from 5 samples of Orgueil heated with water in sealed glass
tubes for a period of several days at 105°, 240°, 350°, and 400° C, respectively,
and, from samples of Orgueil, Murray, and Holbrook, after being subjected to
rapid heating in air to 980° C. temperature. The results were compared with
published data and with the diffraction patterns of the following standards:
chlorite (clinochlore) from Brinton Quarry, West Chester, Pa.; magnetite from
Mineville, Adirondack Mts., N.Y. (both were obtained from the Mineral
Collection of the Department of Geology, Columbia University); serpentine
(mainly antigorite) from Havana, Cuba (from the Genth Collection, The
Pennsylvania State University), and iron (metal) powder, C.P. grade.
The carbonaceous chondrites gave poor diffraction patterns. Apparently,
this was caused by small particle size and by a strong fluorescence of the sample,
when subjected to CuKa radiation. Magnetite lines appeared on all Orgueil
patterns; many of the silicate lines were made visible on photographic film by
reducing the exposure of the diffuse background with another strip film, put in
front of the one that was to be used for the diffraction record. Manual, step-
scanning in the low angle region established a diffuse band related to the char-
acteristic basal reflections of layer lattice silicates. The counting pattern,
however, did not show the same resolution as the photographs, where at least
one of the 001 reflections stood out as a very weak but as a still shghtly notice-
able line. The hydrothermal treatment of Orgueil failed to improve the
quahty of the diffraction effects.
Diffraction data from the Orgueil sihcates are shown in table 4, with some
layer lattice silicates containing magnesium. Low angle counts obtained from
oriented slides are shown in figure 2. The oriented samples were prepared by
subjecting the powdered meteorite to shearing stress with a pestle on abraded
glass slides. This produced a thin, glossy film in which the mineral flakes
appear to have been aligned parallel to the glass, as prescribed by earlier experi-
ments and theory.-^ Each 0.2 degree 26 increment was counted in the 2.0°-
16.0° 2d range for a period of 134 seconds, with CuKa radiation and a scale
factor of 256 on a Norelco X-ray diffractometer unit. The statistical probable
error in the counts, under such experimental conditions, is 0.4 per cent.
Because of the uncertainty in the position of the 001 reflections positive
identification was not possible. Chlorite and/or montmorillonite may be
present; the former is a likelier constituent, considering that chlorites rich in
iron give weak 1st and 3rd order basal reflections. Reflections extending above
7 A preclude serpentine.
i
Nagy et al. : Environment of Orgueil Meteorite Parent Body 543
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544
Annals New York Academy of Sciences
7.6A
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ORGUEIL, (C), ORIENTED SAMPLE
(U.S. NATIONAL MUSEUM,
WASHINGTON, D.O" ■
ORGUEIL, (A), ORIENTED
SAMPLE, (THE AMERICAN
MUSEUM OF NATURAL
HISTORY, NEWYORK, N.Y.
26
Figure 2. Record of manual, step-scanning counts in the low angle region. Norelco
X-ray diffractomeler, CuKa radiation, fixed time basis. Each point on the graph was counted
for 134 seconds, and the scale factor was 256.
Nagy et al. : Environment of Orgueil Meteorite Parent Body 545
Magnetite (Fe304) is a component in Orgueil;* the magnetite lines were
sharp enough to ensure that they were not caused by chromite (FeCr204),
which gives a similar pattern. The 2.97 A, 2.53 A, 1.71 A, and 1.62 A magnetite
hnes were recorded. Chromite peaks at 4.83 A and 1.91 A were not observed;
the strongest diffraction effect was sharp and always appeared at 2.53 A. The
x-ray diffraction patterns showed no evidence for hematite (a-Fe203), pyrite
(FeS2), troilite (FeS), pyrrhotite (FeySs), metallic iron and nickel, fayalite
(Fe2Si04), forsterite (Mg2Si04), enstatite (MgSiOs), or gibbsite (Alo03-3H20)
in Orgueil. The X-ray data indicated that the mineral composition of the
sample was heterogeneous to some extent.
The water soluble salt was obtained by heating the sample in water in sealed
glass tubes at 104° C. for a period of 2 days, after which the supernatant liquid
was poured off, filtered, and evaporated. The crystalline product was MgS04-
6H2O; there were a few minor peaks which have not been identified. The
diffraction patterns showed that subjecting the Orgueil sample to rapid heating
(6.5° C. per minute) to 980° C. temperature in air, led to the formation of a
limited cjuantity of olivine (forsterite) and hematite. When the chlorite and
the serpentine standards were subjected to the identical heat treatment they
seem to have fully recrystallized into the high temperature minerals.
The diffraction pattern of Ivuna was almost identical to Orgueil, but Murray
showed signs of containing olivine. The diffraction patterns of the noncarbona-
ceous chondrites were sharp and distinct; the results were in agreement with
published data.
Electron diffraction studies were conducted in an attempt to confirm the
x-ray data. Specimens were prepared by dusting with a Q-tip because it was
thought that this method would lead to a random orientation of the flakes.
Patterns were taken in selected areas and in manipulator positions. A "beam-
stop" was used for some patterns; the centers of the patterns were reduced with
Farmer's reducer. Measurements were made both on plates and on enlarge-
ments.
The electron diffraction diagrams showed a series of concentric rings, with a
hexagonal (or pseudohexagonal) array of spots overimposed on most rings.
There were also 2 diffuse bands present. 001 reflections were not recorded.
The electron and X-ray diffraction data were in good agreement (d-values in
TABLE 4 are based on both). There were only two differences. Electron
diffraction diagrams did not show magnetite lines (probably because of the
scarcity of magnetite in the fields that were examined). Furthermore, electron
diffraction diagrams were always sharp and distinct. The hexagonal pattern
of spots was related apparently to diffractions from the basic hexagonal building
units of layer silicate structures.
Thermogravimetric A nalysis
In addition to the X-ray and electron diffraction methods, there are 2 thermal
methods of layer lattice silicate analysis: differential thermal analysis and
thermogravimetric analysis. Faust^- obtained differential thermal curves on
Orgueil and Mighei but was unable to interpret the data because of the inter-
* Part of the magnetite may contain Ni, as NiFe204 .
546
Annals New York Academy of Sciences
ference of a wide range of exothermic effects (caused probably by the combus-
tion of organic matter). Thermogravimetric analysis was selected because it
was thought that the temperature-weight curves of untreated meteorite samples
could be meaningfully interpreted even if there was organic matter present.
Experiments were performed with the 6 meteorites and with mineral standards,
as well as with mineral-organic mixtures. A "Stanton Thermo-recording"
instrument was used; the samples were heated in platinum crucibles. The
instrument was calibrated for both temperature and weight effects. In addi-
100 200 300 400 500 600 700 800 900 1000
TEMPERATURE, "C.
100 200 300 400 500 600 700 800 900 1000
TEMPERATURE, °C.
Figure 3. Thermalbalance curves of meteorites (left) and of mineral standards, and
of a mineral-organic mixture (right). Heating rate 6.5° C. per minute; each sample weighed
0.302 gm.
tion, standard kaolinite (API-No. 17) and montmorillonite (API-No. 25)
samples were run for the purpose of calibration. Each sample was run at a
6.5° C. per minute heating rate; sample weights were held identical: 0.302 g.
The thermobalance curves are shown in figure 3 (left and right).
The Orgueil curve shows a gradual decrease in weight to the inflection point
(Ai) at approximately 600° C. temperature. At approximately 900° C. there
is a second inflection point (Bi). The distillation residue of Orgueil sample
(A), heated in an initial vacuum of lO"'^ mm. Hg at 510° C. for a period of 2
hours, showed only the effect at Bi . The weight gain in the 400 to 500° C.
temperature range may have been caused by the oxidation of magnetite made
apparent by the removal of volatile organic matter and part of the water.
Nagy et al. : Environment of Orgueil Meteorite Parent Body 547
The initial weight loss of the distillation residue may have been caused by the
loss of rehydrated water. The Ivuna thermobalance curve was very similar
to that of Orgueil, although the position of point Ai was less well defined.
Murray lost less weight than either Orgueil or Ivuna. The 3 noncarbonaceous
chondrites gained weight during heating, caused probably by the oxidation of
metal. Note that the Holbrook and Bruderheim curves were actually identi-
cal. The cause of the small inflection at approximately 870° C. (Ci) is not
known.
One may compare the meteorite curves with published data and with those
in Figure 3 {right) in an attempt to evaluate the meteorite compositions. The
thermogravimetric patterns and the differential thermal curves of chlorites are
characterized by two high temperature dehydration effects; see Mielenz,
Schieltz and King,-''^ and Nutting.-^* Serpentine (antigorite), seems to show
only one principal, dehydration reaction at high temperatures.^^ Certain
chlorites, containing 2 polymorphic (14A and 7 A) units, had been observed to
exhibit 3 high temperature, dehydration effects.^^ Montmorillonite may give
complicated patterns; in most montmorillonites, however, the first dehydration
reaction occurs at temperatures lower than Ai . Some montmorillonite-
organic complexes show endothermic reactions in the 850 to 950° C. temperature
range.^" Talc^^ seems to have only 1 principal, dehydration effect, which occurs
close to 1000° C. Gibbsites seem to lose the majority of their water below
425° C. temperature.^^
The Orgueil curve (untreated) has 2 inflection points, Ai and Bi , similar to
chlorite, A2 and B2 . The thermobalance curve of a synthetic mixture con-
sisting of 68 weight per cent chlorite, 14 per cent magnetite, 8 per cent Mg2S04-
6H2O, 4 per cent elementary sulfur, and 6 per cent humic acid,* was in part
similar to the Orgueil curve. Other mixtures, containing either bituminous
petroleum, asphaltene, graphite, and/or serpentine, were less similar. The
thermogravimetric pattern of a chlorite sample, ground in and saturated with
piperidine, has shown that point Bi shifted to higher temperatures. Thermo-
gravimetric analysis of mineral-organic mixtures suggests that the gradual
decrease in weight below 600° C. temperature is caused by the volatilization of
complex organic matter.
The following samples yielded curves which were dissimilar to Orgueil:
Recent marine sediment (from the Eastern Atlantic Ocean, 35°57'N, 07°30'W,
from a depth of 1350 feet, and 575 cm. below the sea bottom); top soil (from an
oak forest in Hartsdale, N.Y.) ; and a low temperature silicate reaction product.
The latter sample was prepared by mixing, in stoichiometric proportions,
sodium silicate and magnesium chloride solutions and allowing them to stand
for several days at room temperature. It has been claimed in the literature^^'^^
that such a low temperature process might yield a product resembling serpen-
tine. X-ray diffraction patterns of the product did not show serpentine lines
and most of the weight loss occurred below 300° C. temperature on the thermo-
balance curve. The soil sample also lost most of its weight at low temperatures.
The Recent sediment was indicative of clay minerals other than chlorites, and
also probably other than montmorillonite.
* The humic acid was prepared by I. A. Breger of the U.S. Geological Survey from Minne-
sota peat by low temperature alkali extraction, followed by acid precipitation and dialysis.
548 Annals New York Academy of Sciences
A Discussion of Conditions on the Parent Body
Experimental data establish that the Orgueil meteorite consists chiefly of the
following substances (Hsted in an approximate order of decreasing abundance).
(1) Hydrous layer lattice sihcate mineral(s), (probably chlorite or, less likely,
montmorillonite); (2) magnetite; (3) magnesium sulfate; (4) organic matter;
and (5) elementary sulfur.
The terrestrial occurrence of the minerals (1, 2, 3, 5) must be briefly con-
sidered before one attempts to evaluate the environment of the Orgueil parent
body. As an initial consideration, one may note that hydrous, layer lattice
silicate minerals can form only in the presence of water (licjuid or vapor).
Clearly, the parent body must have contained water.
The chlorite minerals occur in crystalline, metamorphic schists (which had
high temperature histories), in altered, basic igneous rocks as well as in soils
and sediments (low temperature history). Serpentine and talc have either
hydrothermal origins or they are alteration products of igneous rocks. The
montmorillonite minerals are known to occur both in soils and sediments and
in rocks altered by hot hydrothermal solutions. Layer sihcate minerals occur
under a rather wide range of temperatures.
Magnetite is present in many igneous rocks (which crystallized from molten
silicates), and in sediments. Epsomite is known to crystallize from (low
temperature) mineral water; it is often found in limestone caves. Sulphur
may be the result of either volcanic activity, of the decomposition of H2S in
thermal springs or of bacterial action in rocks and Recent sediments.
On the other hand, phase equilibria studies,^^"'*' have demonstrated that
certain, characteristic high temperature minerals, absent in Orgueil, beg'n to
form above 450 to 500° C. temperature. This then may be safely assigned as
the upper limit of the Orgueil temperature history. As to the lower limit of
the parent body, one must resort to speculation. It is difficult to visualize
how a great mass of crystalline siUcates could have formed through solid state
reactions, at temperatures below the freezing point of water.
Other considerations may narrow down the temperature range. The fact
that some sulfur and hydrocarbons can be liberated from the stone at tempera-
tures as low as 150 to 200° C, at slightly reduced pressure, suggests that the
upper limit of the temperature range could not have been much higher than
200° C* Furthermore, the composition of the organic matter seems to have
been altered when the meteorite was heated with water in sealed glass tubes at
temperatures substantially higher than 200° C. DuFresne and Anders"*- noted
recently that some strained glass fragments found in the Mighei carbonaceous
chondrite indicated that the meteorite could not have been subjected to a
temperature of 180° C. for a period longer than a few weeks. The authors
claimed that Mighei temperatures could not have exceeded 300° C. It was
also suggested that the magnesium sulfate veins in Orgueil were produced by
liquid water.
There are 2 other useful indications of environment : the oxidation-reduction
potential (Eh) and the pH. It is known from Pourbaix's*^ fundamental work
* Gas chromatographic and mass spectrometric analyses indicate that hydrocarbons as
small as C9 are present in Orgueil. The boiling point of H-nonane is 150° C. at atmospheric
pressure.
Nagy et al. : Environment of Orgueil Meteorite Parent Body 549
on the thermodynamics of dilute aqueous solutions that /ih-pH relationships
may be used to detine mineral stability. £h-pH diagrams had been used to
deduce environmental conditions from low temperature mineral paragenesis
and from sedimentation data; Carrels'^ presented a comprehensive treatise on
the geological aspects of £h-pH relationships.
Because of a lack of information, an evaluation of the parent environment in
(UPPER LIMITED OF WATER
.STABILITY)
^^0^
Fe203
(S04--)
0.0
-0.2
-0.4
-0.6
-0.8-
-1.0
0
(LOWER LIMIT OF
WATER STABILITY)
8
10
12 14
pH
Figure 4. Stability relationships of iron sulfides and oxides in water at 25° C. and 1 atmos
total pressure and total dissolved sulfur activity of 10~^ After Garrels.^^
terms of £h-pH relations must be basically speculative in nature. Speculation
is possible, however, if it can be assumed, that: (1) the temperature range was
approximately 0° to 200° C; (2) organic matter and other minor components
do not substantially affect known £h-pH relationships; and (3) minerals of the
Orgueil suite are gentically related. It is known^* from the stability of iron
oxides in water that pressure has only a limited affect on /?h-pH relationships.
A change of the temperature from 25° to 100° C. causes a shift in the stability
fields of solids relative to the £h-pH axes but it does not affect the shape and
size of the fields.
550
Annals New York Academy of Sciences
In FIGURE 4 is illustrated a common geological phenomenon, i.e., the inter-
relations between iron oxides and iron sulfides in water at different Eh and
pH values. The stability relationships on the diagram were calculated by
Garrels^ for 25° C. temperature and for 1 atmos. pressure at a total dissolved
sulfur activity of 10~^ Under such circumstances, magnetite is stable under
mildly reducing conditions and at a pH higher than 7. The SO^T ion is stable
UJ
+1.0
+0.8
+0.6
+0.4
+0.2
0.0
-0.2
-0.4
-0.6
-0.8
-1.0
OXIDIZING
VANADIUM
^^DE POSITS
%
V.
^a
%
%,
MINE '3"
WATERS,
'A/ /^ OXIDIZING \ '
'/3 DEPOSITS
RAIN, "^C
V.
>
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WATER, ^ /■/
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pH
Figure 5. Approximate position of some natural environments as characterized by Eh.
and pH. After Garrels.^^
in the magnetite field, but the stability field of elementary sulfur extends
toward a more oxidizing environment and an acidic pH. The missing mineral
phases in a given suite are also indicative of environment. Note that py-
rite forms at the same pH as magnetite but under a more reducing environment
than magnetite; whereas, hematite forms under a more oxidizing environment.
Neither pyrite nor hematite has crystallized out in Orgueil, although their
elementary components are present. Layer lattice silicates are known to occur
under conditions similar to the magnetite environment in figure 4.
Nagy et al. : Environment of Orgueil Meteorite Parent Body 55 1
The relationship shown in figure 4 may be appUcable, in general, to the
Orgueil meteorite parent body. From this relationship one may then speculate
that the Orgueil parent body had an aqueous, low temperature, slightly alkaline
and slightly reducing environment. It seems that sulfur was formed by some
unrelated process.
The approximate positions^ of some terrestrial environments as characterized
by Eh and pK are shown in figure 5. It is interesting that the proposed
Orgueil environment resembles those terrestrial environments which are isolated
from the earth's atmosphere (organic rich saline waters).*
Summary
The Orgueil meteorite has long been known to contain bound-water, organic
matter, and sulfur, in addition to silicate, iron oxide, and magnesium sulfate.
Trace element data in Orgueil, obtained during the present study, were found
to be consistent with the average abundances of trace elements in chondritic
meteorites but they appeared dissimilar to average abundances in terrestrial
shales and igneous rocks. Electron microscopy showed that the meteorite
consists mainly of micaceous minerals. X-ray, electron diffraction studies and
thermogravimetric analysis confirmed the occurrence of hydrous layer lattice
silicates and of magnetite, in addition to some magnesium sulfate. The
mineral suite prescribes an aqueous environment of the parent body. The
parent body temperature seems to have been low to moderate and one may
speculate that the environment was slightly reducing and that the pH was
slightly alkaline.
Acknowledgments
A part of these studies was supported by the National Science Foundation;
this support is gratefully acknowledged. The authors wish to thank Vincent
Modzeleski of Fordham University for his able assistance in several experi-
ments. The authors would like to thank Brian Mason, R. F. Folinsbee,
Lincoln LaPaz, and E. P. Henderson for providing the meteorite samples and
Ralph J. Holmes for providing the mineral standards. The recent marine
sediment sample was received from the Lamont Geological Observatory of
Columbia University. The trace element analysis was performed by Ledoux
and Company, Teaneck, N.J.; the electron micrographs and electron diffraction
patterns were taken by the Ladd Research Industries, Burlington, Vt. The
authors would like to thank I. Fankuchen of the Polytechnic Institute of Brook-
lyn for his helpful suggestions regarding the x-ray diffraction analysis and for
permitting the use of his laboratory for making certain measurements. The
authors would like to thank Harold C. Urey of the University of California,
Brian Mason of The American Museum of Natural History, Robert M. Garrels
of Harvard University, I. Fankuchen of the Polytechnic Institute of Brooklyn,
* Sagan^*" suggested that indigenous organic matter may exist buried under the surface
of the moon. He observed that "organisms shielded from solar illumination, perhaps in
congealed dust matrix interstices, might survive cosmic radiation for 10" years or more; lunar
subsurface temperatures are too low to impede survival." The Orgueil meteorite may repre-
sent the remnant of such an undergrounfl habitat, but the experimental data gathered in this
study do not preclude the possibilitj- that the parent body was of sufficient size to hold an
atmosphere and thus, bodies of water.
552 Annals New York Academy of Sciences
Lincoln LaPaz of The University of New Mexico and J. D. Bernal of Birkbeck
College, University of London, for reading the manuscript.
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28. MacEwan, D. M. C. 1951. X-ray Identification and Structure of the Clay Minerals.
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29. Gruner, J. W. 1934. Z. Krist. 88: 412.
30. Selfridge, G. C. 1937. Am. Mineralogist. 22: 97.
31. Brindley, G. W. & K. Robinson. 1951. X-ray Identification and Structure of the
Clay Minerals. Mineral Soc. Gr. Brit. Monograjjh. : 173.
32. Faust, G. T. Unpublished work.
33. Mielenz, R. C, N. C. Schieltz & M. E. King. 1954. Proc. 2nd Nat. Conf. on Clays.
: 285.
34. Nutting, P. G. 1943. U.S. Geol. Survey. Bull. 197.E: 197.
35. Nelson, B. C. & R. Roy. 1954. Proc. 2nd Nat. Conf. on Clays. 335.
36. Byrne, P. J. S. 1954. Proc. 2nd Nat. Conf. on Clays. : 241.
37. Strese, H. & U. Hofmann. 1941. Z. anorg. u. allgem. Chem. 247: 65.
38. Epprecht, W. 1941. Schweiz. mineral. Petrog. Mitt. 27: 1.
39. BowEN, N. L. & 0. F. Tuttle. 1949. Bull. Geol. Soc. Am. 60: 439.
40. Yoder, H. S. 1952. Am. J. Sci., Bowen. 569.
41. TuRNOCK, A. C. 1960. Ann. Rept. Director Geophys. Lab. 98.
42. DuFresne, E. R. & E. Anders. 1961. Geochim. et Cosmochim. Acta. 23: 200.
43. PouRBAix, M. J. N. 1949. Thermodynamics of Dilute Aqueous Solutions. Edward
Arnold & Co. London.
44. Garrels, R. M. 1960. Mineral Equilibria. Harper and Bros. N.Y.
45. Sagan, C. 1960. Proc. Natl. Acad. Sci., U.S. 46: 393.
46. Sagan, C. 1960. Proc. Natl. Acad. Sci., U.S. 46: 396.
EVIDENCE IN METEORITES OF FORMER LIFE: THE ORGANIC
COMPOUNDS IN CARBONACEOUS CHONDRITES ARE
SIMILAR TO THOSE FOUND IN MARINE SEDIMENTS
W. G. Meinschein
Esso Research and Engineeriufi Co., Linden, X.J.
Bartholomew Nagy, Douglas J. Hennessy
Fordham I'nhersity, New York, N.Y.
Recently, the composition of the hydrocarbons in the Orgueil carbonaceous
chondrite has been proposed as evidence for biological activity in the parent
body.' This apparently novel use of hydrocarbons has created great interest
and is the subject of appreciable controversy.
The proposal that certain meteorites were once a part of an extraterrestrial
biosphere is not new. Analyses of carbonaceous substances in chondrites
were published first more than 120 years ago. Berzelius,"' in 1834, speculated
about and decided against the possibility that the humic acid type substances
in the Alais meteorite were biological products. Wohler,'*-^ however, thought
that the ozocerite type constituents in the Kaba chondrite, which he investi-
gated, in 1858, were "undoubtedly of organic origin;" but Berthelot did not
share Wohler's belief that a resemblance to terrestrial organic matter was
proof of a biological origin. Berthelot'''' hypothesized a reaction of metallic
carbides and water to explain the presence of "petroleum-like" hydrocarbons
in the Orgueil stone. Although Mueller'^ noted experimental evidence against
the carbide theory of Berthelot, the "chlorobitumens" which were reportedly
isolated from the Cold Bokkevelt by Mueller^ were not suggested as organic
products. All of these observational and elemental analyses were far less
definitive than the analyses that have been made possible by modern technicjues
and instruments and recent accjuisition of paleobiological reference data.
Investigations of terrestrial biotic matter and modern analytical methods,
now, provide a basis for speculations about extraterrestrial life.
Spectrometric, chromatographic, and new microscopic methods were not
utilized in the study of the carbonaceous substances in meteorites before 1954.
Boato"* nieasured the abundances of stable carbon and hydrogen isotopes in
14 meteorites. He noted that "the carbonaceous material is, of course, not
derived from living sources, but it is noteworthy that the range of variation
in C'-^ in the meteorites is of the order of the depletion observed in a terrestrial
process involving loss of volatile compounds."^ Recent investigations, how-
ever, do not support the view that a loss of volatile compounds leads to a
depletion of C''* in sedimental organic matter. Silverman and Epstein'^ and
Park and Epstein" have found that ecology exerts the principal control on
terrestrial, organic C'-^ contents. Lipids, which are the major volatile con-
stituents of plants and animals, have lower C'^ contents than other organic
compounds."
Boato's deuterium determinations provided evidence of the extraterrestrial
origin of meteorites. He found much greater concentrations of deuterium in
the combined water which were removed from the Orgueil stone at temperatures
553
554 Annals New York Academy of Sciences
exceeding 180° C. than have been found in natural waters on earth. Neither
deuterium** nor trace nietaP- abundances in the Orgueil meteorite support
Bernal's''^ contention that this carbonaceous chondrite "may be a part of the
primitive earth shot ofT hundreds of milHons of years ago and again united to
its parent body."
Calvin'^ obtained the ultraviolet absorption spectra of water extracts of the
Murray and Orgueil meteorites in acidic and basic solutions, and he concluded
that the absorption wavelengths and the variations in these lengths induced
by the addition of acids were suggestive of cytosine, a building block of nucleic
acids which are essential components of all living cells. Briggs'''' reported
evidence of purines and imidazoles in the Orgueil, Murray, and Mokoia meteor-
ites.
Detailed analyses of the amino acids, amino sugars, and sugars in the Bruder-
heim chondrite and the Murray carbonaceous chondrite have been run by
Degens and Bajor."'' They determined the quantities of 20 amino acids, 3
sugars, and 2 amino sugars as well as the presence of cytosine, uracil, and/or
hypoxanthine in the 2 samples.^'' "After complete extraction of all hydrolyz-
able matter in the meteorite," the investigators^^ found that "amino acids and
sugars could be generated within 3 weeks' exposure to open air in c|uantities
about 20 per cent of the original values." Because of the regeneration, com-
position, and stabilities of the amino, sugar, and nucleic acid constituents in
the Bruderheim and Murray, Degens and Bajor"' proposed that the "organic
constituents analyzed are with great probability terrestrial in origin rather
than fossil remnants of extraterrestrial life."
Claus and Nagy have reported "organized elements" in the Orgueil and
Ivuna'^ and have observed similar elements in the Alais and Tonk carbonaceous
chondrites.'^ These "organized elements" are dissimilar to any known mineral
forms but resemble, yet are not identical to certain species of algae. "'^ Recog-
nizable, well-preserved terrestrial type organisms, also, were seen in the 4
carbonaceous chondrites, but the latter species were present in much smaller
numbers than the microfossil-like elements. ''' The terrestrial type organisms
were assumed to be contaminants acquired on earth, and they represented the
only specimens resembling microorganisms that were found in the Bruderheim
and Holbrook chondrite.'^
Palynological treatments have provided additional information about some
of the "organized elements" in meteorites. Staplin'^ added: (1) hydrochloric
acid to remove the carbonates; (2) hydrofluoric acid to remove the silicates;
and (3) Schulz solution (nitric acid and potassium chlorate) to bleach the
residue that he had obtained by these acid treatments of an Orgueil fragment.
The residue contained "recent (organic) contaminants, a very few well-pre-
served Cretaceous microfossils, and relatively numerous less well-preserved
microfossils of unknown age or affinities. The unidentified microfossils, mostly
in the 10 to 100 n size range, superficially resemble certain of the unicellular
algae if size, texture, and the presence of an acid resistant pellicle are con-
sidered."
Fox^" has made abiotically double walled carbonaceous particles which he
believes may be formed in shapes and sizes that resemble the "organized ele-
ments" in meteorites. Fitch et al.,^^ noted that the "organized elements" in
I
Meinschein et al. : Evidence in Meteorites of Former Life 555
carbonaceous chondrites may be minerals or sulfur droplets; whereas Briggs
and Kitto" conclude that the "complex organic microstructures" in the Mokoia
meteorite may be either of biogenic or abiogenic origin. It remains to be
demonstrated, however, that these abiotic products can duplicate the fluores-
cence, size, and numerical distributions, structural details, biological stain
acceptance, and behavior during palynological treatment which have been
reported'^ '^ for the microfossil-like "organized elements" in meteorites. In
the opinion of Nagy el al.,~^ no organic particles have yet been prepared that
possess all the properties of "organized elements" or cell remnants. Bernal-^
states, "the question of whether the objects admittedly composed of sulfur or
mineral fragments are or are not identical with the 'organized elements' . . .
clearly requires for its resolution careful comparisons by a panel of impartial
experts." Urey^^ feels that "although the present evidence is not conclusive,
there are good reasons for exploring possible origins of lifelike forms in the
carbonaceous chondrites other than contamination after their arrival on earth."
Biological Indicators
Living things may be grossly regarded as unique assemblages of parts or
molecules that possess efficient means of synthesizing, using highly select
arrays of complex molecules, and of reproducing their specie. Plants can con-
vert several per cents of the solar energy that they receive into molecular
energy or food. Compounds which form a major portion of the constituent
parts of organisms comprise an exceedingly small fraction of the compounds
which theoretically can be made by abiotic reactions. Sagan'''^ reports "the
most optimistic extrapolation from existing laboratory ultraviolet experimental
data" for the quantum yield of organic molecules by Miller-Urey-*^ type syn-
theses is 1 part in 1()(),()()0 parts, and the products of these syntheses are neither
solely nor entirely the compounds made by living things. Organisms are
apparently in excess of a thousand times more efficient than abiotic reactions
which may have occurred in a primordial environment.-^
Although some nonbiological process under some presently undelinable
conditions may duplicate the productive capacities of living cells, available
data support the view that detectable concentrations of complex molecular
mixtures composed of compounds resembling those in living cells are products
of life.'-^
Because organisms are efficient and apparently unique producers of certain
arrays of molecules, plant and animal matter has probably exerted a major
control on the compositions of many carbonaceous substances in terrestrial
sediments for the last 2 or more billion years.-^ Either the preserved or the
altered biosynthetic products in Earth's sediments may provide a valuable,
legible record of prehistorical life and its evolution. Analyses of extracts of
terrestrial sediments indicate that ancient plants and animals have left evidence
of their existence and that some extractable substances of natural samples
may be used as biological indicators.
In this investigation, the compositions of the benzene extracts of soils and
marine sediments froni various regions on earth have been used as references.
It is postulated that the terrestrial extracts retain evidence of biological activity,
and it is assumed that similarities between terrestrial and meteoric extracts
556 Annals New York Academy of Sciences
constitute evidence that the meteorites were either contaminated while on
Earth or a part of a parent body which supported hfe. Careful consideration
will be given to the compositions of the extracts of the sediments and meteorites
so as to determine, as well as these compositions permit, whether the extractable
fractions of carbonaceous chondrites are indigenous or contaminants.
Experimental Procedure
Solvents and glassware. Reagent Cirade solvents were used exclusively.
Before use, solvents were distilled through 6 plate glass helices columns, and
100 gm. aliquots of each solvent batch were blown to constant weight in the
sample recovery system. Solvents accepted for use contained less than 0.1 mg.
residue per 100 gm. of solvent and these residues did not absorb detectably in
either the 2 to 15 yu or 220 to 400 m/i regions. All glassware and porcelain
used in preparing and analyzing meteorite samples were cleaned with acid
and carefully rinsed with the accepted solvents.
Blanks. A blank, which omitted only the meteorite sample, was run on
each step of sample preparation and analysis.
Extractions. Consolidated fragments of the l-Orgueil(B) (1.7 gm.), 2-
Orgueil(C) (14.5 gm.), 1-Murray (1.9 gm.), 2-Murray (10.2 gm.), and Hol-
brook (1.8 gm.) meteorites were placed individually on glass wool plugs in 5
glass funnels.-'' Each fragment was rinsed separately with several portions of
a 1 volume methanol to 9 volumes benzene (9:1 benzene-methanol) solvent.
Meteorite samples smaller than 2 gm. were rinsed with 25 ml. of solvent, and
the 2-Orgueil and 2-Murray fragments were rinsed with 80 and 50 ml. of solvent
respectively. The rinses from each meteorite were analyzed separately.
The rinsed fragments were crushed to 20 to 40 mesh size, placed separately
on a glass wool plug above a sintered glass partition between a boiling tiask
and a water-cooled condenser in an all glass, single piece, Soxhlet-type extractor.
Small (<2 gm.) and large (>10 gm.) fragments were extracted by slightly
different procedures. A 25-ml. aliquot of 9:1 benzene-niethanol was added
to each of the extractors containing the crushed small fragments. After 6
hours at reflux, the extracts were withdrawn and a second 25 ml. of the solvent
was added to each unit. The extractions were continued an additional 14
hours. In this manner, a rinse, a 6-hour extract, and a 6- to 20-hour extract
of each small fragment was obtained. The large 2-Murray and 2-Orgueil frag-
ments were extracted for 20 hours with 50 ml. of solvent, so that only a rinse
and a 20-hour extract of each of these samples was recovered.
Sample recovery. Solvents were evaporated from rinses, extracts, and from
the eluates of colloidal copper and silica gel columns. Sample bottles con-
taining organic solutions of meteorite rinses, extracts, or eluates were placed in
receptacles or aluminum cups in a constant temperature bath maintained at
40 ± 1° C. Nitrogen filtered through silica gel was blown over the organic
solutions for 4 to 6 hours. This recovery procedure removes the solvents and
most organic compounds from the meteorites that have vapor pressures greater
than Ci:i w-paraffins. Thus, the hydrocarbons recovered in the meteorite
samples consisted primarily of Ch and larger molecules.
In TABLE 1 are presented the weights of the extracts recovered from the
sulfur removal step.
I
Meinschein et al. : Evidence in Meteorites of Former Life 557
Although a semimicro balance was used in all weighings, 1 or more removals
of solvent and 2 or more weighings were required to obtain a residue weight.
Therefore, the weights listed are probably accurate to only ±0.2 mg.
Sulfur removal. Elemental sulfur was removed from all meteorite rinses and
extracts by means of colloidal copper columns.^"
Chromatograpluc separations. The 6-hour extracts of 1-Orgueil and 1-
Murray, and 20-hour extracts of 2-Murray and 2-Orgueil fragments were
fractionated on 9-gm. silica gel columns. ^^ The Holbrook extract was too
small to fractionate. This method of chromatographic separation on silica
gel has been previously investigated. Thousands of crude oils and organic
extracts of sediments and organisms have been fractionated by this chromato-
graphic procedure. Infrared, ultraviolet, and mass spectrometric analyses,
elemental analyses, and aluminum chromatographic analyses^^''^** of numerous
fractions of these silica gel eluates have established: (1) the M-heptane eluates
Table 1
Organic Residues
(Weights in milHgrams)
* A portion of sample lost when solvent "bumped."
t Not determined.
X Nonvolatile residue — inorganic salts.
§ Contained visible traces of colloidal copjjer from sulfur removal step.
\ Estimated from mass spectra peak heights.
Sample
Rinses
6-Hour e.xtracts
6 to 20-Hour
extracts
1-Orgueil (1.7 gm.)*
2-Orgueil (14.5 gm.)
1 -Murray (1.9 gm.)
2-Murray (10.2 gm.)
Holbrook (1.8 gm.)
Blank
0.1
0.3
0.1
t
0.2t
o.i§
6.0
75.0 (20-hour)
1.1
6.7 (20-hour)
0.4§ (O.OH)
o.m
0.6
t
0.1
t
0.1
0.0
are composed primarily of saturated hydrocarbons; (2) carbon tetrachloride
fractions contain saturated hydrocarbons, olefins, traces of some nonpolar
organic nitrogen and sulfur compounds, and alkyl- and cycloalkylbenzenes;
(3) benzene eluates contain most of the aromatic hydrocarbons, some organic
esters, alcohols, and other organic nonhydrocarbons;^i and (4) methanol eluates
are composed predominately of organic nonhydrocarbons.
Weights of the chromatographic fractions of 1-Orgueil, 1-Murray, 2-Murray,
and blanks, which were in most ca.ses too small to be determined accurately,
are presented in table 2. The 2-Orgueil extract was of sufficient size for a
triplicate analysis, and the chromatographic data on the 3 alic|uots of this
sample are given in table 3.
Infrared spectroscopy. All blanks, rinses, extracts, and the methanol eluates
of 1-Orgueil and 2-Murray were scanned in the 2 to 15 m region. Infrared
spectra were obtained of the //-heptane, carbon tetrachloride, benzene, and
methanol fractions of 2-Orgueil. Scans were run on a Baird Associates Model
4-55 spectrometer with a sodium chloride prism. Sample and blank cells
had 0.1-mm. cell lengths and were equipped with sodium chloride windows.
558
Annals New York Academy of Sciences
Blanks, rinses, Holbrook extracts, and 1-Murray 6- to 20-hour extract did not
absorb significantly in the 2 to 15 ^ infrared region. The infrared spectra of
the total blanks, and total Holbrook, l-()rgueil, and 2-Murray organic extracts
are presented in figure 1. In figure 2 are shown the spectra of the individual
chromatographic fractions of the 2-Orgueil extract.
Ultraviolet and visual spectroscopy. All blanks, extracts, rinses, and chro-
matographic fractions were scanned in 220 to 400 ni/u region. Scans were run
on a Gary model 14 spectrometer with matched cells of 1 cm. in length and
methanol as solvent. Visual spectra were run on the benzene and methanol
chromatographic fractions of the 1-Orgueil, and 1-Murray. Absorption
decreased continuously from 400 to 800 m^ in all of these fractions. The
Table 2
Silica Gel Chromatographic Fractions
(Weights in milligrams)
1-Orgueil (6.0)
1 -Murray (1.1)
2-Murra'v (6.7)
Blank
w-Heptane
0.1 (0.3*)
0.0 (0.1*)
0.4
0.1 (0.0*)
Carbon
tetrachloride
0.2
0.1
0.1
0.0
Benzene
0.6
0.0 (0.1*-
0.4
0.0
t)
Methanol
4.4
0.7
4.3
0.1 (O.Ot)
* Estimated from mass spectra peak heights.
t Estimated from ultraviolet absorption.
Table 3
Silica Gel Chromatographic Fractions of 2-Orgueil
(Weights in Milligrams)
I
2-Orgueil aliquots
Awt. 24.50
B wt. 25.09
C wt. 24.19
/(Heptane
2
2
2
32 (9.5%,)
45 (9.8%,)
56 (10.6%,)
Carbon
tetrachloride
1.46 (6.0%)
1.32 (5.3%,)
1.17 (4.8%,)
Benzene
0.76 (3.\%)
0.82 (3.3'^)
0.76 (3.1%)
Methanol
15
15
15
00
92
62
(61.2%,)
(63.4%)
(64.6%)
Left on column
4.96 (20.2%)
4.58 (18.3%)
4.08 (16.9%)
Orgueil extracts ab.sorbed the strongest in the visual range, but the visual
spectra of all extracts lacked any suggestion of a specific absorption at a particu-
lar wavelength.
The blanks did not absorb and the chromatographic fractions of the Holbrook
absorbed only slightly in the 220 to 400 niyu range. The ultraviolet spectra of
the 1-Orgueil and 2-Murray chromatographic fractions are presented in figures
3 and 4, respectively. In figure 5 are given the ultraviolet spectra of the
total Holbrook extract and the total procedure blank. The Murray and
Orgueil extracts fluoresced in ultraviolet light.
Mass spectroscopy. Blanks and the rinse; 6-hour and 6- to 20-hour extracts
of Holbrook; and carbon tetrachloride, benzene, and methanol eluates of the
Orgueil distillate did not produce measurable peaks at masses greater than 150
in the mass spectrometer. Measurable mass spectra were obtained of the 4
individual chromatographic fractions of the 6-hour extract of 1-Orgueil and
1
Meinschein et al. : Evidence in Meteorites of Former Life 559
of the 2-Murray extract; of the composited //-heptane, carbon tetrachloride,
and benzene eluates and the methanol eluate of the 6-hour extract of l-j\Iurray;
the rinses and 6- to 20-hour extracts of 1-Orgueil and 1 -Murray and of the
w-heptane eluate of the Orgueil distillate. Additional fractionations and
analyses are being run on the 2-Orgueil fractions. All spectra except those
of the 2-Murray eluates were obtained on a Consolidated 21-103C mass spec-
3000 2500 2000
1500
WAVENUMBERS IN CM"I
1300 1200 1100 1000
600
700
625
6 9 10 II
WAVELENGTH IN MICRONS
Figure 1. Infrared spectra of total Ijenzene-methanol extracts (free sulfur removed) and
blanks. Only minor absori)tions appear in the spectra of the blank and Holbrook extract.
Infrared absorption bands in the 2-Murray and 1 -Orgueil extracts are similar to the bands
otjserved in the benzene extracts of some terrestrial sediments.
5000
3000 2500 2000
WAVENUMBERS IN CM~'
1300 1200 MOO 1000
625
8 9 10
WAVELENGTH IN MICRONS
Figure 2. Infrared spectra of silica gel chromatographic fractions of the 2-Orgueil ben-
zene-methanol extracts. That «-he]itane and carbon tetrachloride eluates absorb only at
wavelengths that may be attributed to C — C and C — H bonds. The benzene eluate has a
small carbonyl absorption near .S.8 fi and absorption bands at 9.7, 12.3, and 13.4 n. These
latter bands appear in the infrared spectra of the benzene eluates of ancient sediment extracts,
crude oils, and many recent marine sediment extracts. Absorption Imnds in the methanol
eluate are typical of the bands found in the spectra of the methanol eluates of many terrestrial
sediment extracts.
trometer which is modified for the analysis of high molecular weight organic
compounds. Operating conditions for the Consolidated instrument were:
ionization potential, 70 volts (benzene and methanol eluates of 1 -Orgueil were
run also at 12 volts); ionization current, 45 yuamp.; magnet current, 1.30 amp.;
scan rate, accelerating potential 3300 to 4000 volts in 20 minutes; temperature,
ionization chamber, 250° C, and inlet system, 300° C.
The mass spectra of the 2-Murray eluates were obtained by the Analytical
Research Division of Esso Research and Engineering Company with a General
560
Annals New York Academy of Sciences
Electric 12-in. mass spectrometer that is modified for high mass analyses.
Operating conditions of the General Electric instrument were: ionization
potential, 31 volts (benzene and methanol eluates of 2-Murray were run also
at 12 volts); ionization current, 50 yuamp.; accelerating potential, 2500 volts;
scan rate, magnet current 50 to 500 mamp. in 20 minutes; temperatures, sample
evaporator 255° to 262° C, volume 233° to 240° C, inlet lines 213° to 217° C,
ionization chamber 174° to 177° C.
o
<
OD
tr
o
to
CD
<
, \.\ I -•■n-HEPTANE
\ 1/ ll \ • • -CARBON-TET.
I l|i V- -BENZENE
' \) ' I— METHANOL
*O.OImg/ml lo.025 mg/ml \
\ I Vl
I 10.2 mg/ml
, U-V SOLVENT \
\ -METHANOL \
\ \
"200 240 280 320
MILLIMICRONS
360
400
Figure 3. Ultraviolet spectra of the silica gel chromatographic fractions of 1-Orgueil
6-hour extract. Saturated hydrocarbons do not absori) in the ultraviolet range. Aromatic
hydrocarbons do absorb. These spectra indicate that w-heptane eluate is composed primarily
of saturated hydrocarbons and that the aromatics in the 1-Orgueil extract are concentrated
in the benzene eluate.
<
GD
q:
O
en
CD
<
200
240
280 320
MILLIMICRONS
360
400
Figure 4. Ultraviolet spectra of the silica gel chromatographic fractions of the 2-Murray
20-hour extract. Chromatographic fractions of the 2-Murray extract are similar in composi-
tion to those of 1-Orgueil extract, but the relative concentrations of the aromatic hydrocarbons
in these extracts vary in much the same manner as do the aromatic contents of different
terrestrial sediments.
Meinschein et al. : Evidence in Meteorites of Former Life 561
The Consolidated and G.E. instruments can be used for accurate analyses
of mixtures of known hydrocarbons. Concentrations of individual compounds
can normally be determined within ±2 per cent of the true concentrations.
Preparation of mass spectral data sheets. The mass spectra were obtained as
photographic records of the galvanometer deflections produced when the
ions of each particular mass are brought sequentially into focus. The deflec-
tions were measured and the heights of the individual peaks were recorded.
These measured peak heights were corrected by a computer for ions containing
C'^ and H- (corrections made on the basis of terrestrial abundances of C^'^ and
H-). The computer totaled and normalized the corrected peak heights to a
value of 30(),(K)() and printed these heights as a 14-column array.
UJ
o
<
CD
cr
o
03
<
1.0
0.9
Q8
07
06
05
0.4
03
02
Ol
O.Q
— TOTAL EXTRACT
--BLANK (REAGENTS.
ANALYTICAL PRO-
CEDURES)
^<.00lmg
PHENANTHRENE/ml
il/2 TOTAL EXTRACT IN
3 ml METHANOL
200 240 280 320
MILLIMICRONS
360
400
Figure 5. Ultraviolet spectra of total Holbrook 6-hour extract and total procedures
blank. The blank does not absorb significantly in the ultraviolet. This indicates that no
aromatic contaminants were added to the meteoritic extracts during their analyses. Absorp-
tion of the total Holbrook extract is very small. The amount of aromatics acquired by the
Holbrook fragment in almost 50 years of storage could not have exceeded a few micrograms.
Descriptions of the 14-column array have been published.'*' ••^■''••^'' The com-
puter arbitrarily labels the 14 columns from left to right with values of .f
which range as integers from —11 to +2. Use of 14 columns results in the
placement of homologous ions {i.e., ions of the same structural type which
differ by CH2 or 14 mass unit groups) of a particular type in a single column.
The integers heading the columns may represent .v values in the general hydro-
carbon formula C„H2„+j . Each horizontal row of peak heights is marked
with a value of n or C f^ which may indicate the number of carbon atoms in
the ions forming the various peaks.
The C i^ and x values assigned to the rows and columns of the spectra in
TABLES 4, 5, and 7 are correct in most cases because saturated hydrocarbons
in nature yield ions with .v numbers that are predominantly in the x = —11 to
+ 2 range. However, the x values and thus the C ^ shown in tables 6 and 8
are subject to a different interpretation. Aromatic molecules in the meteoritic
extracts have .v values ranging chiefly from .v = —25 to —12, and the columns
in T.'VBLE 6 and 8 would be more accurately labeled by .v values that are 14 less
562
Annals New York Academy of Sciences
and C # values that are one greater than the values shown. These apparent
discrepancies in x values and C )^ values in the mass spectral data sheets of
aromatic fractions are commonly accepted because they simplify the problem of
Table 4
Mass Spectrum of the w-Heptane Eluate from Silica Gel of the 1-Orgueil 6-Houe
Extract. Saturated Hydrocarbon Fraction
Total ionization = 58022.28
Normalized isotope corrected peak heights (300,000)
-11
-10
-9
-8
-7
-6
-5
-4
-3
-2
-1
0
+ 1
+2
0
0
-0
-0
-0
-0
-0
3
4
-0
0
0
0
-0
-0
-0
-0
-0
-0
-0
-0
-0
-0
4
5
-0
0
-0
-0
-0
-0
-0
-0
9389
3350
19430
5997
16219
74
5
6
175
265
223
221
2025
573
3593
1234
10154
4045
13601
2908
10411
67
6
7
263
86
285
229
5338
2511
3087
1175
9135
3219
11029
2118
2317
70
/
8
110
112
267
290
1885
436
2621
1208
5861
1812
6230
1651
1639
47
8
9
479
183
603
258
1418
405
1880
854
3944
1173
3360
1079
1338
376
9
10
440
175
617
245
872
361
1706
707
2465
734
1418
813
1192
176
10
11
354
161
566
171
687
312
1682
572
1768
642
921
713
985
131
11
12
270
137
433
141
581
266
1354
462
1409
479
698
650
867
114
12
13
208
101
286
142
544
280
1178
482
1112
433
555
580
751
103
13
14
234
74
263
127
585
263
1166
373
982
410
461
511
668
59
14
15
172
139
252
265
1099
335
962
252
626
324
344
422
585
29
15
16
120
64
233
116
574
293
594
296
515
284
296
385
519
44
16
17
96
62
248
105
429
215
472
220
429
254
248
350
532
10
17
18
106
59
222
87
344
176
427
178
394
215
237
318
478
67
18
19
192
49
252
88
382
158
386
156
360
208
194
319
459
44
19
20
108
60
168
73
262
114
318
152
303
196
195
265
436
39
20
21
180
53
162
72
292
83
312
124
312
185
215
294
477
60
21
22
173
41
174
63
262
103
286
121
265
203
209
292
385
200
22
23
118
44
163
79
273
133
255
176
219
262
193
312
294
274
23
24
78
56
155
84
229
218
201
221
164
287
101
260
177
189
24
25
85
81
142
126
170
210
171
211
108
265
59
214
132
129
25
26
65
82
121
140
180
188
107
192
72
171
69
152
85
96
26
27
49
150
85
167
114
261
80
169
36
114
44
88
57
59
27
28
29
69
73
106
59
129
32
80
33
77
81
67
45
42
28
29
24
66
32
98
37
105
8
57
0
73
0
477
25
157
29
30
0
31
13
38
5
0
1
0
-0
-0
-0
-0
-0
-0
30
Humble paraffin-naphthene type analyses
in percentage
Paraffins
17.57
Noncondensed naphthenes
28.86
2-Ring naphthenes
17.16
3-Ring naphthenes
11.83
4-Ring naphthenes
11.25
5-Ring naphthenes
6.41
6-Ring naphthenes
6.91
Total
100.00
programming the computer. No problem other than a simple arithmetic
calculation enters the interpretation of these data sheets. All peaks in tables 6
and 8 are recorded at their proper mass positions, and the masses and sizes of
these peaks supply in conjunction with the ultraviolet spectra of the meteoritic
aromatics all of the information subsequently discussed.
Meinschein et al. : Evidence in Meteorites of Former Life 563
Mass spectral data. Mass spectra of complex saturated hydrocarbon mix-
tures suggest the structures and molecular weight distributions of certain of
the compounds and compound types that comprise the mixtures. Spectra
Table 5
Mass Spectrum of the Carbon Tetrachloride Eluate from Silica Gel of the
1-Orgueil 6-Hour Extract. Saturated, Olefinic, plus Minor Concentrations
OF Aromatic Hydrocarbons
Total ionization = 18452.57
Normalized isotope corrected peak heights (300,000)
-11
-10
-9
-8
-7
-6
-5
-4
-3
-2
-1
0
+1
+2
-0
0
1
-0
-0
-0
-0
3
4
-0
0
0
0
-0
-0
-0
-0
-0
-0
-0
-0
-0
-0
4
5
-0
0
-0
-0
-0
-0
-0
-0
5456
2229
15614
5938
13054
169
5
6
325
395
244
253
2155
795
3026
849
7492
3040
11285
2830
9047
66
6
7
297
0
271
70
4296
981
2525
999
6527
2071
7929
2087
2119
66
7
8
226
169
466
586
3859
661
2426
899
4490
1235
4827
1999
1766
187
8
9
1434
535
1203
653
4590
626
2041
649
2911
871
2267
933
1483
1157
9
10
1366
505
1670
604
1966
538
1923
591
1836
600
1250
729
2035
754
10
11
1150
419
1658
294
1280
356
1860
350
1206
836
973
812
1708
592
11
12
917
307
1132
231
1043
309
1082
286
1361
477
862
671
1344
377
12
13
615
211
705
167
792
236
1448
628
995
315
881
578
1056
290
13
14
916
142
595
176
789
308
1156
412
892
370
706
513
1060
310
14
15
486
137
534
505
2732
380
3586
237
655
286
457
368
690
150
15
16
366
122
519
160
478
427
626
900
475
204
421
354
558
165
16
17
313
160
413
157
672
222
444
247
443
214
433
347
701
66
17
18
522
51
331
56
285
130
320
90
311
206
223
384
611
140
18
19
279
208
334
130
349
177
306
201
367
172
258
325
439
154
19
20
244
163
260
143
297
249
61
136
287
235
276
235
422
154
20
21
307
64
230
52
219
136
232
98
306
179
252
238
500
130
21
22
242
44
187
141
165
177
222
80
276
200
213
163
402
85
22
23
205
36
140
54
154
82
276
99
252
120
182
186
362
73
23
24
239
57
133
167
188
35
205
84
240
202
207
142
241
54
24
25
100
93
61
70
69
167
58
231
138
158
155
302
98
84
25
26
79
32
0
79
53
57
56
56
154
44
153
126
178
50
26
27
85
79
80
0
114
74
161
39
72
35
126
68
163
55
27
28
67
35
109
23
79
98
106
21
128
82
92
74
96
25
28
29
95
26
94
124
111
227
109
515
97
358
114
279
75
150
29
30
21
92
0
121
51
100
87
105
100
117
46
101
0
6
30
Humble parafRn-naphthene type analysis in percentage
Paraffins 16.78
Noncondensed naphthenes 22.52
2-Ring naphthenes 9.86
3-Ring naphthenes 15.87
4-Ring naphthenes 17.00
5-Ring naphthenes 7. 10
6-Ring naphthenes 10.87
Total
100.00
shown in tables 4, 5, and 7 indicate the relative numbers of ions of various
masses that were formed when gaseous, predominantly saturated hydrocarbons
were bombarded by 70-volt electrons. The ions measured consisted chiefly
of fragment and "parent" ions. Fragment ions are made by the rupture of
carbon-to-carbon and/or carbon-to-hydrogen bonds of hydrocarbon molecules.
564
Annals New York Academy of Sciences
"Parent" ion.s are formed when a single electron is removed from a molecule.
Only the positively charged ions with masses greater than 66 are recorded in
TABLES 4 through 8.
A saturated hydrocarbon may yield a variety of ions. Any compound can
form a greater number of small than of large fragments, and large molecules
can produce more fragments than can small molecules. It is for these reasons
that the values (peak heights) in tables 4, 5, and 7 generally decrease from the
Table 6
Mass Spectrum of the Benzene Eluate from Silica Gel of the 1-Orgueil
6-HouR Extract. Aromatic Hydrocarbon Fraction
Total ionization = 3748.80
Ionizing potential = 12 volts
Normalized
isotope
corrected peak heights (300,000)
-11
-10
-9
-8
-7
-6
-5
-4
-3
-2
-1
0
+1
-0
+2
0
3
-0
-0
-0
-0
3
4
-0
0
0
0
-0
-0
-0
-0
-0
-0
-0
-0
-0
-0
4
5
-0
0
-0
-0
-0
-0
-0
-0
56
1493
1254
0
0
0
5
6
0
0
0
3i
0
0
628
1375
1565
11. SO
1017
0
0
0
6
7
0
0
0
86
0
329
672
1204
2602
2412
503
0
0
0
7
8
0
0
0
0
0
11
136
364
504
2171
982
121
0
0
8
9
0
0
0
0
391
0
0
538
563
558
1598
0
452
0
9
10
0
0
0
1105
815
200
589
150
196
0
620
0
463
0
10
11
0
6
0
379
90
123
120
0
300
3141
551
447
477
315
11
12
0
0
0
137
358
8
52
0
1180
12303
890
1918
1150
526
12
13
0
215
185
0
62
0
0
38880
706
9265
379
871
83
515
13
14
0
0
0
0
0
815
2653
36
2021
461
442
221
336
14
15
0
51
134
1074
917
130
1062
438
0
295
/
211
341
0
15
16
0
0
182
428
0
308
243
969
121
0
0
0
0
0
16
17
0
1128
0
416
0
308
560
1546
0
141
271
83
0
0
17
18
0
244
0
129
0
446
614
4647
0
0
0
2460
284
593
18
19
89
506
0
970
16
673
0
494
0
0
0
599
96
664
19
20
0
256
0
378
0
0
0
1074
0
133
0
313
157
895
20
21
0
167
0
226
0
702
0
320
0
686
0
555
18
1799
21
22
0
496
0
954
0
65
0
639
953
1487
0
954
707
4257
22
23
0
585
0
403
0
1376
0
947
1498
8527
0
1308
341
5767
23
24
0
239
0
1275
0
2294
29
3413
101
14557
167
2635
187
6071
24
25
0
533
0
4600
0
2176
0
695 V
44
6348
0
3832
0
5896
25
26
0
758
0
2588
0
1820
98
5077
22
4726
0
3537
0
6088
26
27
0
3057
0
1957
0
1185
0
2664
0
1676
0
3262
0
1772
27
28
0
1077
0
525
0
710
0
722
0
722
0
1628
0
1788
28
29
0
847
0
1036
0
1069
0
49
0
-0
-0
-0
-0
-0
29
Humhie aromatics type analysis in i)ercentage
Alk}l l)cnzenes
Najjhthalenes
5.11
16.56
Acena])iithenes
17.75
Acenaphthyienes
Phenanthrenes
11.10
20.95
Diacenaphthylenes
Pyrenes
Chrysenes
4.01
1.43
0.55
Acejjyrenes
Benzpyrene and other organic
compounds
1.09
21.45
Total
100.00
Meinschein et al. : Evidence in Meteorites of Former Life 565
top toward the bottom of each x column. However, there are exceptions.
Certain ions have peak heights which exceed or approach in size peaks imme-
diately above them on the data sheets. These anomalously large peaks provide
structural and distributional information about some of the compounds in
mixtures of saturated hydrocarbons.
Table 7
Mass Spectrum of w Heptane Eluate from Silica Gel of the 2-Murray 20-Hour
Extract. Saturated Hydrocarbon Fraction
Total ionization = 127759.59
Normalized isotope corrected peak heights (300,000'
-n
-10
-9
-8
-7
-6
-5
-4
-3
-2
-1
0
+1
+2
0
0
-0
-0
-0
-0
-0
3
4
-0
0
0
0
-0
-0
-0
-0
-0
-0
-0
-0
-0
-0
4
5
-0
0
-0
-0
-0
-0
-0
-0
2678
1227
8838
3342
12892
186
5
6
110
105
52
46
189
96
719
763
5669
3153
9558
2430
9621
151
6
7
101
48
38
20
475
177
1155
984
6975
3298
10467
1765
3703
87
7
8
73
38
53
70
581
233
1361
950
5738
2329
7673
1671
2742
81
8
9
98
51
164
99
624
295
1645
911
4587
1738
4935
1409
2134
85
9
10
127
58
256
108
616
326
1941
963
3490
1243
2556
1104
1702
79
10
11
139
67
296
114
595
326
2120
826
2654
939
1700
913
1385
77
11
12
147
71
281
108
615
345
1803
710
2252
773
1302
835
1175
68
12
13
131
60
274
122
670
342
1622
659
1849
664
1049
793
1108
64
13
14
135
49
282
118
761
348
1657
613
1578
629
815
697
932
48
14
15
134
66
283
159
773
325
1138
483
1135
511
633
613
793
45
15
16
146
65
329
147
845
400
990
396
899
468
523
550
707
49
16
17
142
64
324
154
698
340
845
360
787
439
432
549
706
45
17
18
155
64
346
146
624
295
765
337
731
394
367
510
661
38
18
19
179
88
360
162
651
261
725
304
646
366
343
499
648
59
19
20
187
76
347
128
565
233
611
283
582
345
363
450
600
53
20
21
189
78
360
142
589
227
563
280
511
359
318
449
581
169
21
22
196
111
345
159
543
269
522
312
455
409
285
476
518
260
22
23
207
131
335
197
486
339
478
378
414
438
250
471
444
317
23
24
192
147
311
224
425
381
426
389
332
437
209
424
365
247
24
25
183
132
275
226
364
327
358
250
288
380
164
343
294
205
25
26
156
141
260
212
346
274
306
299
222
303
137
292
230
160
26
27
147
129
236
226
290
318
243
257
174
250
109
235
177
140
27
28
135
116
204
175
250
251
191
191
132
184
86
175
138
106
28
29
110
98
176
213
181
250
156
155
105
152
57
138
112
109
29
30
90
91
122
175
137
182
114
123
76
117
43
122
83
73
30
31
74
66
95
116
88
117
81
85
52
84
24
71
60
64
31
32
54
57
77
81
79
89
62
78
34
63
17
55
44
37
32
33
47
51
56
65
54
60
37
48
22
52
19
36
32
33
ii
34
26
42
36
47
36
46
24
41
13
30
11
34
17
26
34
35
17
34
21
32
21
32
16
22
7
21
0
24
4
15
35
36
8
19
17
13
17
16
9
19
0
2
0
-0
-0
-0
36
Humble paraffin-naphthene type analysis in percentage
Paraffins 16.49
Noncondensed naphthenes 17.28
2-Ring naphthenes 18.06
3-Ring naphthenes 13.87
4-Ring naphthenes 13.18
5-Ring naphthenes 10. 12
6-Ring naphthenes 10.99
Total
100.00
566
Annals New York Academy of Sciences
Table 8
Mass Spectrum of Benzene Eluate from Silica Gel of the 2-Murray 20-Hour Extract.
Aromatic Hydrocarbon Fraction
Total ionization = 38025.84
Ionizing potential = 12 volts
Normalized isotope correc
ted peak heights (300,000)
-11
-10
-9
-8
-7
-6
-5
-4
-3
-2
-1
0
+1
-0
+2
0
0
-0
-0
-0
-0
3
4
-0
0
0
0
-0
-0
-0
-0
-0
-0
-0
-0
-0
-0
4
5
-0
0
-0
-0
-0
-0
-0
-0
121
114
272
334
433
57
5
6
149
64
0
0
0
36
47
9
249
286
417
290
416
53
6
7
85
4
0
0
93
137
78
122
356
274
540
259
304
57
7
8
21
0
0
130
315
172
112
221
424
288
512
264
237
59
8
9
75
129
344
252
489
292
176
244
419
236
435
179
219
82
9
10
175
235
402
241
447
267
270
200
373
228
298
141
245
235
10
11
148
307
453
214
368
224
343
188
332
276
511
340
289
280
11
12
274
280
452
201
296
214
216
161
326
257
1180
277
350
276
12
13
246
257
389
228
289
203
239
439
363
364
814
244
314
222
13
14
306
213
384
162
312
283
444
325
428
342
749
237
286
191
14
15
308
273
379
20919
891
675
545
1124
447
1317
629
322
326
335
15
16
296
316
881
7558
966
1345
604
1996
522
637
622
426
374
2359
16
17
444
5966
1060
5834
715
1750
524
3305
561
843
612
748
410
1584
17
18
476
2587
741
4114
556
1731
518
1972
489
956
518
10436
665
2274
18
19
437
1837
552
4092
510
1718
398
1475
438
1180
484
2912
409
1534
19
20
375
1401
384
2298
375
1365
374
4454
487
1659
460
2195
337
1808
20
21
362
1319
363
1761
237
1219
270
1619
413
1449
416
1610
315
1243
21
22
284
1107
291
2101
270
1233
281
1357
360
1594
312
1434
286
1074
22
23
265
1095
211
1310
204
1013
271
1147
322
1169
267
1119
233
940
23
24
193
915
203
1004
199
830
246
941
230
934
201
894
157
734
24
25
193
791
153
877
154
708
200
780
195
794
174
752
147
635
25
26
173
638
141
724
117
649
124
663
155
695
140
627
144
258
26
27
148
838
82
588
104
497
131
509
142
544
91
487
142
419
27
28
121
522
102
577
108
422
132
477
83
443
24
407
54
375
28
29
46
379
34
406
58
365
69
379
80
343
67
357
32
344
29
30
53
322
57
329
54
329
46
284
61
290
35
251
41
219
30
31
28
241
43
226
47
217
65
243
48
224
23
178
41
183
31
32
23
220
16
196
57
143
49
165
19
167
28
148
42
152
32
33
31
155
45
157
0
159
0
121
12
147
0
106
4
97
33
34
0
134
0
123
27
110
5
95
13
85
0
99
0
102
34
35
3
104
8
110
0
108
0
90
0
102
0
27
0
89
35
36
0
92
0
94
0
94
0
56
0
45
0
36
0
31
36
37
0
69
0
54
0
37
0
39
0
71
0
61
0
44
37
38
0
3
0
-0
-0
-0
-0
-0
-0
-0
-0
-0
-0
-0
38
Humble aromatic type analysis in percentage
Alky] benzenes 4.73
Naphthalenes 3 . 65
Acenaphthenes 7.72
Acenaphthylenes 2.81
Phenanthrenes 6.36
Diacenaphthylenes 4.75
Pyrenes 13.14
Chrysenes 7.57
Acepyrenes 2 . 93
Benzpyrenes & other organic compounds 46.34
Total
100.00
Meinschein ct al. : Evidence in Meteorites of Former Life 567
Large peaks may appear at masses equal to those of molecules, fragments
containing stable ring systems, or fragments of different structure and common
mass. w-Parafiftns and polycycloalkanes form more molecular or "parent" ions
than do branched chain parat^ins or monocycloalkanes. FVagments containing
the stable, partially hydrogenated phenanthrene ring system frequently give
large peaks. Isoprenoids usually have a number of methyl branches or substit-
uents, and several different fragments of the same mass can be formed when
an isoprenoid loses a methyl group. Common mass ions of isoprenoids, also,
yield large peaks in the mass spectra of naturally occurring saturated hydro-
carbons.
"Parent," stable ring, or common mass ions can be identitied in most cases.
For convenience, the whole number atomic masses of C = 12 and H = 1 are
used in calculations of ion mass numbers, and fragment ions generally have odd
mass numbers. Therefore, stable ring and common mass ions are found
predominantly in odd numbered .v columns. All "parent" ions of hydrocarbons
have even mass numbers, and these ions appear in even numbered .v columns.
Common mass ions normally appear a row above and one column to the left of
"parent" ions; whereas most stable ring fragment ions have masses 50 or more
units less than the masses of the molecular ions. Thus, stable ring ions appear
chietly in odd .v columns several rows above the "parent" ion region.
Some stable ring ions, however, are formed by breaking 2 bonds. These
ions have even mass numbers. An example of an even mass stable ring ion is
the -v = —6 and C^ = 16, mass 218, ion in the cholestane'*^ mass spectrum.
The even mass numbers of some fragment ions lead to ambiguity in the identi-
fication of "parent" ions. This ambiguity can be removed in many cases by
additional fractionations or by information gathered from the mass spectra
of reference compounds. Studies of thousands of mass spectra of saturated
hydrocarbon mixtures from plant and animal lipids, sedimental extracts, and
crude oils have shown that certain fragment and "parent" ions commonly
yield large peaks at particular masses. Some of these large peaks may be
absent in the mass spectra of highly paraffinic fractions, but further separations
usually yield cycloalkyl concentrates in which these large peaks appear as they
do in most biological and sedimental hydrocarbons.
The peaks that are normally large in the spectra of naturally occurring
saturated hydrocarbon mixtures have been mentioned in previous publica-
tions,^^"^^ and many of these peaks will be indicated, again, in the subsequent
interpretation of the meteoritic hydrocarbon spectra. But, it is noteworthy
in the spectra of cholestane^^ that the .v = — 7, C ^ = 26 (common mass ion)
and 17 (stable ring ion); x = —6, C ;i^ = 27 ("parent" ion) and 16 (stable
ring ion) ; and .r = — 5, C ^ = 11 (stable ring ion) peaks are large. These same
mass peaks have been observed to be large in either total or refined saturated
hydrocarbon fractions which have been isolated from extracts of terrestrial
sediments. Peaks at x = —6, C# = 28, 29, and/or 30, which may be
''parent" ions of compounds structurally related to parent sterol hydrocarbons
other than cholestane, are also prominent in most naturally occurring alkane
fractions.
Mass spectra of the meteoritic hydrocarbons presented in tables 6 and 8
568 Annals New York Academy of Sciences
were obtained at an ionization potential of 12 volts. At this low potential
aromatic hydrocarbons yield chiefly "parent" ions. This technique cannot
be used in the analyses of saturated hydrocarbons because the energies required
to break bonds and remove electrons are approximately equal in alkanes.
Nearly all of the large peaks in table 6 are produced by molecular ions, but
apparently the wide range of electron energies in the ion source of the General
Electric instrument caused appreciable fragmentation. In table 8 the data on
many large fragments as well as "parent" ions are presented. Ultraviolet
spectra provide a valuable assistance in the interpretation of the mass spectra
of aromatic hydrocarbons. Many of the aromatic hydrocarbons in naturally
occurring aromatic mixtures can be identified by the combined use of mass and
ultraviolet spectroscopy.
Blanks and the Holbrook meteorite. Because limited amounts of carbonaceous
chondrites are available, minimal sample sizes were used in these investigations.
To ensure that laboratory contaminants did not significantly affect the results of
the analyses obtained on the minimal sized samples, an elaborate system of
blanks was used. In addition, a high temperature meteorite, the Holbrook,
which should not have contained significant amounts of indigenous organic
matter served as an indicator of the type of contaminants a meteorite might
acquire during its fall to earth, contact with earth, and storage in a museum.
None of the blanks contained organic matter that could be detected by in-
frared, ultraviolet, or mass spectrometric analyses; and the Holbrook extracts
showed very small infrared and ultraviolet absorptions as shown in figures 1
and 5. These controls indicate: (1) that laboratory contaminants did not
measurably alter the meteorite analyses; (2) that the amount of organic matter
acquired by meteorites (Holbrook fell in 1912) may be negligible.
Rinses and extracts. The process of first rinsing and of then extracting the
meteorites was used to detect contaminants. It was assumed that surface and
near surface contaminants could be rinsed from the surfaces of the stones.
Analysis of the rinse fractions (all of which were relatively small) and the
extracts did not show any marked changes in concentrations of hydrocarbons
between the exterior and interior portions of the meteorites. These analyses
suggest that the surfaces of the meteorites had not been contacted or con-
taminated by significant quantities of extractable organic matter during
storage.
Authenticity of meteorite fragments. A complementary publication^- lists
the reliable sources of the fragments studied in these investigations. This
complements^ also reviews and presents data that indicate that meteorites
are of extraterrestrial origin and that the samples used in this study are authen-
tic meteorites.
Records of Life on Earth
Terrestrial organisms or their remnants represent the only established
references for detection of biological materials. Variations in the appearances,
behaviors, and compositions of organisms make it apparent that qualitative,
rather than precise, quantitative measurements or observations provide the
best means of recognizing previously unseen or unanalyzed forms or remnants
of life. Presently the extensive data on the fossil remains and organic matter
Meinschein et al. : Evidence in Meteorites of Former Life 569
in the sediments on earth are the most acceptable standards for the identifica-
tion of former life in meteorites.
Numerous soils and marine sediments have been analyzed by methods
analogous to those used in this investigation of the Murray and Orgueil carbona-
ceous chondrites. Additional analyses have been run on saturated hydro-
carbons from sediments, plants, and animals. A review of these investigations
of terrestrial samples will serve as a basis for evaluating the analyses of the
meteoritic hydrocarbons.
Smith^" found that geologically young hydrocarbons (C* ages 9000 to 14000
years) isolated from recent sediments have optical activities, infrared spectra,
elemental and type compositions, and chromatographic properties equivalent
to hydrocarbons in ancient crude oils. Oakwood^' plotted optical activities
versus distillation temperatures for hydrocarbons from kelp (a seaweed) and
crude oil, and he observed that these similar plots peaked in the same tempera-
ture region.*-' Saturated hydrocarbons in mixtures of plant and animal lipids,
recent and ancient sediments, and crude oils seem to have equivalent chromato-
graphic properties and infrared spectra, and similar mass spectrometric cracking
patterns.^'* C^^ ages^"'*'* and //-paraffin distributions^^ •^**-''^'^^-*^-"^ provide the
only reported means of distinguishing between the Cu and larger saturated
hydrocarbons in recent sediments and those in crude oils.
Wax-esters, closely re.sembling beeswax, have been found in a variety of
types of soils from arid and humid areas of tropical and temperate regions of
the world.^^ Blumer^*^ has identified in soils the same aromatic hydrocarbons
that have been identified in marine sediments.^^ Hunf*^"*^ and Brenneman^"
have reported similarities between hydrocarbons dispersed in ancient sediments
and concentrated in crude oils. Bray^' has observed that the aromatic frac-
tions of all crude oils absorb near 12.35 and lvS.45 m in the infrared.*^ He has
referred to these absorptions as "oil bands."
2,6,10,14-Tetramethylpentadecane, pristane, is a norisoprenoid hydro-
carbon constituent of fish oils.^-'^'' Pristane forms 0.2 and 0.5 per cent, respec-
tively, of the two crude oils in which it was measured. ^^ 2,6,10,14-Tetra-
methylhexadecane, phytane, is a diterpenoid or isoprenoid which is, also, a
common component of fish and crude oils.^^ The concentrations of phytane
in 10 Iranian oils is to be reported. ^^ It is of interest that the first Cu, or
larger, branched paraffins, pristane and phytane, isolated from crude oils are
of an isoprenoid type.'^^
When the possible complexity of petroleum is considered, the relatively
high concentrations of pristane and the measurable quantities of phytane in
crude oils is noteworthy. There are in excess of 100,000 possible isomers of
Ci9 and 366,319 possible isomers of Con paraffins.^'' Because crude oils contain
hydrocarbons composed of more than 50 carbon atoms, the number of paraffins,
cycloalkanes, and aromatic compounds that might form petroleum is astronomi-
cally large and of the order of 10'** different hydrocarbons. Because of the
diversity of compound types and the large range of carbon atom contents of
the hydrocarbons in petroleum, the abundances of pristane and phytane in
crude oils suggest a highly selective synthesis of these compounds. The com-
mon presence of the precisely structured pristane and phytane in fish and
crude oils may be more than fortuitous. Possibly certain of the stable saturated
570 Annals New York Academy of Sciences
hydrocarbons from prehistoric Hfe have been preserved for geological periods
of time in nature, and these compounds may be used to define and study the
existence of ancient organisms.
It has been suggested that saturated isoprenoid type hydrocarbons synthe-
sized either by living things*-'^* or from sterol and isoprenol remnants of
organisms^'* •^^•^^•"■^'^ are major sources of naturally occurring alkane hydro-
carbons. Disagreement on the origin of terrestrial alkanes centers about the
issue of whether living things*""^^'^^ or chemical reactions acting on plant and
animal remains'*^ ■^''•^''•^'^''^"'®^ make most of the saturated hydrocarbons in
nature. Either of these sources would yield biosynthetically controlled prod-
ucts which could serve as reliable biological indicators.
Similarities and differences in the benzene and analogous extracts of terrestrial
sediments may be generally summarized and partially explained. An average
sediment contains between 20 and 80 parts per million of Cm and larger hydro-
carbons.^'■■*" •'*^ ■** These hydrocarbons usually comprise between 10 and 30
per cent of most extracts, and the hydrocarbons can be separated chromato-
graphically from the organic oxygen, nitrogen, and sulfur containing molecules
which make up 70 to 90 per cent of the extract.^' ■^"•^^ Hydrocarbons are
eluted primarily in the w-heptane, carbon tetrachloride, and benzene eluates;
whereas the organic nonhydrocarbons appear chiefly in the methanol eluates
from silica gel chromatographic cokmns.
The extractable organic nonhydrocarbon fractions from sediments normally
show hydroxyl or amino (2.9 to 3.0 fx), carbon-hydrogen (3.3 to 3.5 m), carbonyl
(5.7 to 5.9 fx) and broad absorptions in the 7 to 15 /jl regions of the infrared.
Saturated and aromatic hydrocarbons from sedimental extracts show the
usual carbon-hydrogen absorptions in the 3.3 to 3.5 n and 7.2 to 7.8 m regions.
In addition, the saturated hydrocarbons in most cases absorb at the carbon
chain frequency near 13.9 n which is indicative of //-paraffins, and aromatics
generally absorb at the "oil band" frequencies of 12.4 and 13.4 fx.
Nonlinear polyring aromatics are dominant in sedimental hydrocarbon
mixtures. Phenanthrenes, chrysenes, pyrenes, and perylenes appear with and
without alkyl and cycloalkyl substituents in many soils and marine sediments.
Anthracenes, naphthacenes, and larger linear polyring aromatics have not been
identified in extracts of soils or marine sediments. Carruthers^'' has isolated
some alkylanthracenes from crude oils; but in petroleum also, phenanthrenes
are much more abundant than anthracenes.^^
Sedimental extracts can be divided into two broad classes: (1) soil or aerobic,
and (2) marine sediment or anaerobic. Overlaps do exist. Anaerobes and
aerobes both live in soils and marine sediments, but aerobes appear dominant
in most soils. Soil extracts normally contain more wax-esters and less free
sulfur and aromatic hydrocarbons than do extracts of marine sediments.^' •''^
Although the same molecular structures appear to be present^^'^^'*^ in sedi-
mental hydrocarbons, the ratio of saturated to aromatic hydrocarbons is
usually greater in soils than in marine sediments.'*' •^'*'^"''*^
Apparently, the compositions of sedimental extracts can be grossly explained
on the basis of the stabilities of plant and animal constituents in different
natural environments. Chemically and/or biochemically active carbohy-
drates, proteins, fats, oils, and porphyrins (hemin, etc.) comprise all but a
Meinschein et al. : Evidence in Meteorites of Former Life 571
small fraction of the substances in most living cells. Yet, only traces of these
substances are found in some sediments. i^'®*"*^* Stable saturated hydrocarbons,
extremely minor constituents of the lipid fractions of most organisms*^'^^'^^'^^-
^^''^* are present in measurable concentrations in essentially all sediments.
Likewise, wax-esters, which form thin protective coatings of plants and in-
sects,^^'''* are apparently stable in aerobic environments and are found com-
monly in soils.^^
Differences in the compositions of the extractable fractions of soils and
marine sediments may be traceable to the anaerobic activity in sea bottoms.
Organic acids and alcohols combine to form wax-esters. Anaerobes utilize
these acids and alcohols and sulfate ions.^^ Conversions of olefinic steroid and
isoprenoid acids and alcohols into stable aromatic molecules result in large
energy releases. Anaerobes need energy. Most common foods are almost
completely consumed by aerobes in surface sediments. To survive, anaerobes
must frequently use unusual energy sources and reduce sulfates to hydrogen
sulfide which is oxidized to free sulfur.''* Marine sediments may contain more
free sulfur and aromatic hydrocarbons and less wax-esters than soils,^^ merely,
because marine environments are normally more anaerobic^* than nonmarine
environments. Thus, analyses of sedimental extracts may serve as environ-
mental as well as biological indicators.
Meteoritic Extracts
Because minimal sample sizes were used in this investigation, most meteorite
extracts were too small to provide reliable data by all of the analytical methods
used. Only the 2-Orgueil extract was of sufficient size to permit accurate
chromatographic analysis and to supply ;/-heptane, carbon tetrachloride, and
benzene fractions which absorbed significantly in the 2 to 15 micron regions
(table 3, FIGURE 2). However, because the ultraviolet and mass spectro-
metric data (figures 3 and 4 and tables 4, 5, 6, 7, and 8) indicate that the
Murray and Orgueil extracts are related much as are terrestrial sediment
extracts, analyses of the 2-Orgueil sample may be approximately representative
of other extracts of carbonaceous chondrites.
The 2-Orgueil chromatographic data in table 3 fall in the terrestrial sedi-
ment range.^i ■'*'''** Infrared spectra of the total meteorite extracts in figure 1
and of the chromatographic fractions of the 2-Orgueil in figure 2 indicate
that all of the major absorption bands may be traceable to hydroxyl or amino
(2.9 to 3.1 m), carbon to hydrogen {i.i to 3.6 and 7.1 to 7.9 /x), carbonyl (5.6 to
6.0 and 8.0 to 9.0 pi), aromatic or olefinic (10 to 14.5 m), and carbon-to-carbon
chain (13.8 to 13.9 /x) groups. The carbonyl absorptions in the Murray and
Orgueil extracts suggest a complex mixture of carbonyl compounds and the
mass spectra (tables 6 and 8) show that these samples do not contain apprecia-
ble concentrations of either fatty acids or beeswax-like esters (absence of
large peaks at odd carbon numbers in x = — 10 column). Ultraviolet (figures
3 and 4) and mass (tables 6 and 8) spectra show that the Murray and Orgueil
extracts contain significant quantities of aromatic hydrocarbons. The Murray
extracts contained small and the Orgueil extracts contained copious amounts of
free sulfur. The preceding cursory examination of chromatographic and
spectrometric data indicate that the Murray and Orgueil extracts resemble
572 Annals New York Academy of Sciences
terrestrial marine sediment extracts. Like most marine sediments and unlike
most soils, the Murray and Orgueil meteorites have benzene extractable frac-
tions which contain complex mixtures of carbonyl compounds (which are
not wax-esters) and significant concentrations of aromatic hydrocarbons and
free sulfur.
Further consideration of the spectrometric data provides evidence of addi-
tional similarities and some dissimilarities between meteoritic and terrestrial
sedimental extracts. In figure 2, the 12.3 and 13.4 m absorption bands of the
benzene eluate of 2-()rgueil are similar to the "oil bands''^' found in the in-
frared spectra of the aromatic fractions of all crude oils,'*^'^' but the ultraviolet
(figure 5) and mass spectra (tables 6 and 8) show that the meteorite aro-
matic fractions are unusually simple. Aromatic mixtures in crude oils and
ancient sediments^^'"'-^^'^^ greatly exceed in complexity these meteoritic frac-
tions. Apparently, certain recent sediments are the only terrestrial samples^^'
39,45,46 containing naturally formed aromatic mixtures which even approach
in simplicity the aromatic hydrocarbons from these meteorites.
Structural types of the major aromatic species in the Orgueil and Murray
fractions can be deduced from the ultraviolet (figures 3 and 4) and mass
(tables 6 and 8) data. The ultraviolet spectra indicate the possible aromatic
nuclei, and the mass data permits the elimination of some possible nuclei
which do not yield large parent ions in the mass spectra. Based upon the
ultraviolet and mass data the principal aromatic nuclei are in order of decreas-
ing abundance: (1) Phenanthrenes, pyrenes, and chrysenes in the Orgueil extract.
Most abundant aromatic hydrocarbon is phenanthrene. See 178 mass peak
in X = —4 column at C^ =13 in table 6. (2) Pyrenes, chrysenes, benz(j)-
fluoranthenes (indicated but not completely identified) and phenanthrenes in
the Murray extract. Most abundant aromatic hydrocarbon is pyrene. See
202 mass peak in x = —8 column at C j^ = 15 in table 8.
Large "parent" ions appear in the 22 to 29 carbon number range of the even
.r columns in table 6. These ions are made from complex aromatic molecules
many of which differ in carbon and hydrogen content from aromatics reported
in the literature. It has been proposed^'* '^^ that naturally occurring aromatics
may be products of transformations of isoprenoids and steroids, and this
proposal has recently been supported by the identification of 21 aromatic
compounds in petroleum. Mair and Martinez-Pico'^'^ note that "most of the
('21 aromatic') compounds are related to steroids. The results . . . give strong
support to the theory that steroids and other natural products related to
phenanthrene are petroleum precursors." Conversions of olefinic steroids
and terpenes to aromatics necessitate the loss of some alkyl, usually methyl
substituents, from the highly substituted isoprenoid ring systems. Conse-
(juently, aromatics formed from terpenes and steroids would contain a lower
number of carbon atoms than their precursors. The high concentrations of
largely unreported C2.S through C28 phenanthrenes, chrysenes, and pyrenes
indicated by the large peaks in table 6 suggest that C27 through C^o steroids
and triterpenoids may have been a source of the aromatics in the Orgueil
meteorite. This suggestion is amplified by the common prevalence of the
same types of nonlinear polyring aromatics in meteoritic and sedimental
Meinschein et al. : Evidence in Meteorites of Former Life 573
hydrocarbon mixtures, as is further indicated by the "oil band" absorptions of
the benzene eluate in figure 2.
C23 through C28 aromatics are less abundant in the Murray (table 8)
than in the Orgueil. The lower abundance may indicate that the Murray
was subjected to a high temperature, more severe environment than the
Orgueil and the polyalkyl substituted aromatics may have been partly de-
graded in the Murray. Olivine, a high temperature mineral, is a constituent
of the Murray meteorite. The simplicity of the aromatic fractions in these
meteorites may be explained by assuming that more restricted varieties of
organisms existed in the meteorites than are normally found in recent terrestrial
sediments.
/i-Paraffin^'*'^'''^^'*^'*^ and polycycloalkane distributions have been cited as
evidence of the biologic origin of some of these compounds.-^ •^^•^^■^^ Most
sediments and organisms contain greater abundances of: (1) odd- than even-
carbon number //-paraffins in the C21 to C35 range. (2) C24, C27, C28, C29
and/or C30 than of other C17 and larger tetra-, penta-, and higher polycyclo-
alkanes. Although alterations, which change some organic molecular struc-
tures and distributions, decrease the features characterizing biologically
derived hydrocarbons, these features apparently persist even in the hydro-
carbons from ancient sediments.^'* ■^^•'*^'^'*
Many of the features noted previously for terrestrial hydrocarbons appear
in the mass spectra of the meteoritic hydrocarbons. In the x = -\-2 columns,
the 23 and 29 carbon number peaks in tables 4 and 7 and the C25, C27, and
C29 peaks in table 5 are larger than the peaks immediately above and below
them in the x = -\-2 column. Peaks in the .v = -1-2 are produced by ions
that have masses equal to paraffins or heptacycloalkanes. Branched paraffins
do not produce "parent ions" to a measurable degree in the mass spec-
trometer,^^'^* and most heptacycloalkanes would contain more than 25 carbon
atoms in their ring nuclei. Therefore, the above designated peaks probably
contain w-paraffin "parent peaks," and the "peakings" at odd carbon numbers
in the C23 to C29 range in these x = -\-2 columns of tables 4, 5, and 7 are similar
to "peakings" observed in the mass spectra of saturated hydrocarbons from
various ancient terrestrial sediments.^^
Other "peakings" and "inflections" (anomalously small differences in sizes
between successive peaks in an x column) in tables 4, 5, and 7 are note-
worthy. These are: C20 through C30 peaks in the odd numbered x columns of
tables 4, 5, and 7 are approximately as large as the even numbered x peaks
in this carbon number range. These large odd x peaks and certain of the
"peakings" in the odd x columns are indicative of poly alkyl-substituted or
branched chain alkanes such as isoprenoids. Saturated hydrocarbons, made
by hydrogenating products of the abiotic Fischer-Tropsch synthesis, do not
yield as large odd .v peaks as do meteoritic and terrestrial alkanes. A com-
parison of abiotic and meteoritic hydrocarbons will be presented in a subsequent
publication.
Because of the chromatographic properties of the fractions and the odd
mass numbers of the ions the large peaks at x = — 7, C^ = 15 in tables 4
and 5 and at .v = ~5, C^ = 13 and 15 in table 5 are produced apparently
574 Annals New York Academy of Sciences
by nonbasic cyclic nitrogen compounds. These compounds are slightly more
polar than alkanes and are more concentrated in the carbon tetrachloride
eluate (table 5) than in the »-heptane eluate (table 4). In table 4, the
peaks at .v = —6 and — 7, C^ = 16 are larger than those of homologous ions
at C^ 13, this suggests that the nitrogen ions may have obscured the "peak-
ings" at X = —6 and — 7, Ci^ = 16 which are generally observed in the mass
spectra of the terrestrial alkanes. "Peakings" at x = +1, C^ = 21 in
tables 4 and 5 have an odd mass number and also are suggestive of nitrogen
compounds. "Peakings" in the 19 to 25 carbon number ranges of the x = 0,
— 2, and —4 columns which appear in tables 4, 5, or 7 are uncommon in
sedimental hydrocarbon spectra but are present in the mass spectrum of the
saturated hydrocarbons from oysters.'^^ Neither crude oil nor sedimental
organic contaminants are probable sources of the mono-, di-, and tricyclo-
alkanes which form the ions producing the latter peakings. Nevertheless,
differences in the carbon numbers at which the "peakings" maxima occur in
the various columns of tables 4, 5, and 7 as well as the alternate high or low
values of odd and even C ^ "parent ions" in table 5 may be more suggestive
of a biological product than of an abiotically formed mixture that is thermo-
dynamically at equilibrium.
Contamination
Carbonaceous chondrites are friable, seemingly porous stones. Olivine, a
mineral that forms at temperatures above 400° C. is present in the Murray
but apparently not in the Orgueil stone. Associated minerals in the Orgueil
suggest that it may have formed in an environment resembling an organic rich
saline environment on Earth,'- and the compositions of the extractable car-
bonaceous fractions of the Murray and Orgueil, also, are indicative of marine
type sedimentary deposits. Although the compositions and the intimate
associations of the mineral and carbonaceous materials in carbonaceous
chondrites are not incongruous with a marine ecology, meteorites are likely to
be contaminated by terrestrial substances. It is important to consider the
most probable contaminants and the effect that they may have upon the
composition of the carbon constituents of meteorites.
All meteorites accepted as carbonaceous chondrites were observed during
their fall to Earth. Many of the carbonaceous chondrite fragments that have
been collected are partially coated with a heat altered layer. Charred crusts
were apparently formed on the lead surfaces of the meteorites when these
areas were heated to incandescence on entry into Earth's atmosphere.
In transit to Earth carbonaceous chondrites break-up. Boato's- measure-
ments show that meteoritic waters released at temperatures above 180° C.
apparently have not been exchanged with terrestrial waters. Carbonaceous
chondrites give off sizeable quantities of water at temperatures in excess of
180° C.,* and additional evidence has been presented'- that these meteorites
did retain substances in space which normally boil below 180° C. The volatile
constituents of carbonaceous chondrites suggest that they have restricted the
egress of gases to the vacuum of space. Perhaps, the interiors of these frag-
ments are less accessible than their porous structures may indicate, but re-
gardless of the permeabilities of carbonaceous chondrites, their fall was over
Meinschein et al. : Evidence in Meteorites of Former Life 575
quickl}^ and, in the thin molten surface layers of the falling meteorites, tem-
peratures far in excess of 180° C. were reached. Even from these thin melts,
sizeable volumes of gases may have been expelled. During their plummet to
Earth, carbonaceous chondrites probably lost more volatile carbonaceous
substances then they received from the atmosphere.
When the meteorites struck Earth, their hot surfaces may have distilled or
decomposed organic matter, and cool portions of the chondrites may have
condensed and collected the vapors. But, the burned crusts of recovered
fragments of carbonaceous chondrites cover only a fraction of the stones.
These crusts are very thin, and the fragments are friable. It is doubtful that
the heat or impact energies of these stones could have vaporized more than a
trace of terrestrial organic matter. If carbonaceous chondrites are con-
taminated appreciably, they probably acquired most of the contaminants
after the fragments were collected.
Analyses of the Holbrook chondrite show that this stone was not greatly
contaminated either during its fall or almost 50 years of storage on Earth.
Nevertheless, the carbonaceous matter in the Murray and Orgueil meteorites
may have been defiled. Carbonaceous chondrites are more porous than
ordinary chondrites, and the Murray and Orgueil contain higher concentrations
of carbon than the Holbrook. Organic materials may be strongly adsorbed
on carbonaceous substances. Thus, the Murray and Orgueil fragments were
probably more susceptible to contamination than the Holbrook. Notwith-
standing, the high concentrations of benzene extractable materials in the
Murray and Orgueil cannot be easily explained by natural contaminants.
Why in 10 years should the Murray, or in 100 years should the Orgueil stones
accumulate much more extractable carbonaceous substances than an average
soil collects in thousands of years? It seems likely that most of the carbon
compounds in the Murray and Orgueil fragments were either indigenously
formed in the parent body or carelessly added by man.
Meteorites are handled, and some are marked for identification. Oily
hands, paints, wax pencils, polishes on display cases, plasticizers in plastic
storage cases, microorganisms, pyrolysis products of fossil fuels in urban
atmospheres, lacquer coatings, and other carbonaceous substances may have
contacted and contaminated the Murray and Orgueil fragments. Contamina-
tion has been considered a major problem throughout this investigation, and
appreciable attention has been paid to this problem.
Fragments of the Orgueil meteorite were obtained from two museums and
the Murray fragments came from another collection. Contaminants from
these various locations should have been quite different, but none of the
variations in the compositions of the meteoritic extracts suggested significant
organic contamination. All of the fragments were carefully inspected and no
evidence of markings or coatings were observed. Microscopical examina-
tions'^"^ show that recent terrestrial type organisms are present in the Murray^''
and Orgueil in numbers that are two or more orders of magnitude less than in
the average terrestrial sediment. Microorganisms usually contain from 1 to 3
parts per thousand by weight of hydrocarbons. These concentrations are only
slightly greater than those of the hydrocarbons in the Orgueil (table 2).
Terrestrial organisms which have existed in the meteorites, seem to have been
576 Annals New York Academy of Sciences
too small in number to have supplied more than a trace of the meteoritic
hydrocarbons.
Analytical data provide additional evidence against significant contamina-
tion of the Orgueil and Murray fragments. Benzpyrenes are common pyrolysis
products. The air in urban areas contains from 1.5 to 25.5 parts per trillion
by weight of benzpyrenes.^^ Pyrolysis products of wood include methyl
alcohol, ketones, organic acids, Cg and smaller alkanes, as well as olefinic
hydrocarbons. Colored markings usually contain pigments that absorb
sharply in the visual range. Wax pencils are frequently composed of wax-
esters or petroleum waxes. Drying oils in paints and lacquers are mixtures of
olefinic compounds which have carbonyl functional groups. Crude oil dis-
tillates and polishing oils have limited boiling point ranges and contain chiefly
C20 and smaller compounds. The aromatic fractions in petroleum are more
complex than the aromatics in recent sediments or the meteorites.^* ■^^•''^•^^
Analyses of the Murray and Orgueil extracts show that they contain:
(1) negligible concentrations of olefins (and in the Orgueil extract of benz-
pyrenes); (2) no substances which absorb sharply in the visual region; (3)
alkane and aromatic hydrocarbons which are distributed as they are in ter-
restrial marine sediments; (4) hydrocarbons and benzene extractable nonhy-
drocarbons in the same relative abundances that they are found in sedimental
extracts. Because the Murray contains about 5 times and the Orgueil more
than 50 times the amount of benzene extractable materials that is found in an
average sediment, it is unlikely that these extracts could have been obtained
from terrestrial sediments which are the only previously reported sources of
extracts of these compositions. The low recent organism counts'^ '^^ make it
improbable that terrestrial organisms were a source of the extracts. Qualita-
tive and quantitative considerations support the view that the Murray and
Orgueil carbonaceous extracts were predominantly indigenous.
Nonbiological Sources of Hydrocarbons
Anders^' suggests that hydrocarbons resembling those in terrestrial organ-
isms may have been made abiotically in the solar nebula and incorporated
later in the bodies of the solar system. When life evolved on Earth, he believes
that these hydrocarbons favored the evolution and survival of organisms which
could utilize and synthesize hydrocarbons of the types which are now present
in most solar bodies.^^ Innumerable other speculative sources of hydro-
carbons may be proposed. Many of these proposals are neither clearly
supported nor directly denied by experimental data.
Although the extensive literature on organic reactions define what many
reactants will form under various conditions, our imaginations may specify
reactants and conditions that are either untried or unobtainable on Earth.
Nevertheless, organic chemistry is, in part, a record of the means that have
been devised by intensive study and extensive research to synthesize biotic
type products. This record clearly attests that it is extremely difficult by the
use of abiotic reactions to duplicate most of the individual biological com-
pounds that have carbon numbers in the range covered by the compounds in
the benzene extracts of carbonaceous chondrites. After failing to synthesize
a pristane reference, Bendoraitis et al.,^* isolated this hydrocarbon, which is a
Meinschein et al. : Evidence in Meteorites of Former Life 577
minor constituent of fish oils. To avoid the problems of total synthesis, Dean
and Whitehead-^^ used phytol, a biological product, and exchanged a single
hydroxyl group for a hydrogen atom to make phytane. Thermodynamically,
it is, also, difficult to deiine feasible conditions under which hydrocarbons in
terrestrial sediments may have formed abiotically. Amossow and Wasso-
jewitsch-"'' have observed that the ecjuilibrium temperatures calculated from
the abundances of various hydrocarbons in crude oils range between 0° and
225° C. for a Nebit-Dagh oil, 90° and 1075° C. for a Kara-Tschuchura oil, and
— 70° and 225° C. for a Kostschage oil. These temperature ranges are in
great disagreement with the temperatures that are believed to have existed in
sedimentary basins during petroleum formation.
Research efforts carried out over a 100-year period have failed to provide
any evidence that abiotic, radiological, or chemical reactions were a significant
source of hydrocarbons in terrestrial sediments. A summary of the API
research on the origin of hydrocarbons notes the inability of radioactive
induced reactions to make products similar to those found in nature.^*
C()iicl!isio)is
Aromatic hydrocarbons have been identified as common constituents of
meteoritic and terrestrial sedimental extracts. Saturated hydrocarbons
isolated from the Murray and Orgueil carbonaceous chondrites have infrared
spectra, molecular weight ranges, and cracking patterns in the mass spec-
trometer that resemble those of sedimental saturated hydrocarbons. The
relative amounts of hydrocarbons and nonhydrocarbons, the infrared spectra
of the nonhydrocarbons, and the free sulfur contents of the benzene extracts
of the Orgueil and terrestrial marine sediments are similar. Except for the
relative simplicity of the aromatic fraction from the Orgueil fragment, analyses
of both the Orgueil and Murray extracts fall within the range of compositional
variations observed in terrestrial sediment extracts of plant and animal
hydrocarbons.
Although further research may provide an alternative explanation for the
amounts and overall compositions of the benzene extracts of the Murray and
Orgueil carbonaceous chondrites, many similarities of these extracts to the
extracts of terrestrial marine sediments have been demonstrated. Lacking
another experimentally established explanation, we propose that the amounts
and compositions of the benzene extracts of the Murray and Orgueil are evi-
dence for biological activity in the parent body of these meteorites. Because
of the apparent stabilities of certain hydrocarbons in natural environments,
these compounds may provide a means of tracing the evolution of life in
primordial times.
Acknowledgments
The authors thank Brian Mason, R. F. Folinsbee, Lincoln LaPlaz, and E. P.
Henderson for providing the meteorite samples. T. C. Menzel, G. G. Wanless,
and J. J. Waters of Esso Research and Engineering Company ably assisted in
the analyses of some meteorite extracts. The authors thank the various
individuals who critically read the manuscript.
578 Annals New York Academy of Sciences
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FURTHER OBSERVATIONS ON THE PROPERTIES OF THE
"ORGANIZED ELEMENTS" IN CARBONACEOUS
CHONDRITES
George Claus
New York University, Medical Center, Neii< York 16, N.Y.
Bartholomew Nagy
Department oj Chemistry, Fordham University, New York 58, N.Y.
Dominic L. Europa
Department of Pathology, BeUeviie Hospital, New York 16, N.Y.
Independent studies, conducted at various laboratories, indicate that the
"organized elements" do not seem to be terrestrial contaminants. This
evaluation is based mainly upon a consideration of fine morphological criteria.
New experiments with biological stains revealed that the organic microstruc-
tures in carbonaceous meteorites may selectively take stains in the presence
of mineral matter.
Claus and Nagy (1961, 1962) and Nagy el al. (1962), described organic
microstructures that were found embedded in some of the indigenous minerals
of certain carbonaceous meteorites. These findings have been confirmed, or
partially confirmed by independent investigators. Reimer (1961), Staplin
(1962), Palik (1962, 1963), Cholnoky (1962)," and Skuja (1962) examined the
same meteorite sample as did Claus and Nagy (1961, 1962). Briggs and Kitto
(1962) described what they thought to be indigenous, stainable microstructures
in the Mokoia meteorite. However, the last two authors were unable to reach
a conclusion regarding the origin of these particles. Ross (1962) examined
another sample of the Orgueil meteorite, from the collection of the British
Museum, and found microstructures which he believed were of biological origin
and which were most likely indigenous to the meteorite. Recently, Engels
(1962) isolated HF-resistant pellicles from another sample of the Orgueil me-
teorite. Timofeev (1962) found fossilized and indigenous microflora in the
Mighei carbonaceous chondrite. The microscopical preparations of Claus
and Nagy have been examined by approximately 100 microbiologists including
Erdtman, Bourrelly, Papp, Deflandre, Palmer, Durham, Dienes, and Gregory.
Tentative but divergent identifications were offered by some of these inves-
tigators.
Fitch and Anders (1963) argued that the organized elements were silicate
mineral grains, opaque mineral particles, hydrocarbon globules, coacervates,
Fox (1961) spheroids, sulfur droplets, pollens, and starch grains or spores, or
other unknown terrestrial contaminants. Deflandre (1962) stated that the
organized elements are unspecified terrestrial contaminants or artifacts (except
those which are embedded in minerals). Urey (1962a) reviewed the available
information and suggested that the organized elements may indicate, but they
cannot yet be regarded as conclusive proof for the existence of extraterrestrial
life.
Other experimental information which may suggest the presence of extra-
580
J
Claus et al.: "Organized Elements" in Chondrites 581
terrestrial biological processes includes the finding of what seems to be biochemi-
cal compounds in carbonaceous meteorites. Nagy et al. (1961a), and Mein-
schein et al. (1963), reported isolation of complex, saturated, and aromatic
hydrocarbons, respectively. Anders (1962) and Krejci-Graf (1962) criticized
these findings and the interpretations. However, a rebuttal has been offered
(Nagy et al., 1962). Calvin (1961) and Briggs (1961) reported the finding of
compounds which might be cytosine or purines, respectively. It should be
noted that bituminous organic matter was isolated from the Orgueil meteorite
by Cloez (1864), only a few weeks after the meteorite fell. This fact suggests
that a sizeable portion of the meteoritic organic matter is likely to be of extra-
terrestrial origin.
Mineralogical and petrographical studies have shown that the parent body
(ies) of carbonaceous chondrites was capable of supporting a form of life. It
seems that liquid water was present and that this low-to-moderate temperature,
aqueous environment was slightly alkaline and somewhat reducing. Extrap-
olation of the parent body environment from the known mineral assemblage
in terms of phase equilibria data in />H-redox systems has been published by
Nagy et al. (19616), and in more detail (1963). Similar conclusions, arrived
at by independent studies, have been advanced by DuFresne and Anders (1962).
Petrographical observations by Nagy and Claus (1963) showed that the meteor-
ite parent body had been subjected at one time to mechanical stresses that
produced fractures which were later filled with magnesium sulfate. The
textural patterns of the Orgueil and Ivuna carbonaceous chondrites resemble
certain terrestrial rocks, such as pyroclastic rocks, deposited in water from
fragmental volcanic debris. They also resemble silicate rocks altered by
hydrothermal solutions. These petrographical studies have also shown that
the interior of the meteorites does not contain evidence for high temperature
effects acquired during the fall through the Earth's atmosphere. Conse-
quently, organic microstructures and unorganized biochemical type compounds
could have arrived in the meteorites without destruction by heat. The study
of petrographical thin sections also suggests that the average pore size of the
Orgueil and Ivuna meteorites is too small (<1 n) to permit the entrance of
most airborne terrestrial contaminants.
The micropaleontological examinations, the biochemical analyses, and the
mineralogical and petrographical measurements strongly suggest that biologi-
cal activity was active at one time on the meteorite parent body. A full
evaluation of the origin of the organized elements must involve a consideration
of fine morphological structures, the applicability of biological stainings and
other microchemical methods.
Two suggestions had been advanced to explain the origin of the organized
elements, provided that they will prove to be indigenous microfossils in the
carbonaceous chondrites. Bernal (1962) suggested that life may have evolved
along similar lines at various places in the Universe. Urey (19626) suggested
that the organized elements are terrestrial forms that contaminated the moon
from Earth during early geological times. According to Urey's concept, bio-
logical matter and water may have been transferred to the moon, which was
at that time closer to earth, by the impact of meteorites into terrestrial bodies
of water. The carbonaceous meteorites are thought to come from the moon.
582 Annals New York Academy of Sciences
The Usefulness of ike Microscopical Evaluation of Morphological Criteria
Organisms consist of highly organized organic mailer. Consequently,
they have a characteristic and speciiic chemical composition which reveals
itself in specific morphology. Most morphological features serve the specific
life functions of the organisms.
Morphological features develop through 2 basic processes: hereditary proc-
esses transmitted through genes from parent to offspring, and environmental
influences affecting the individual. The first process results in genotypical
morphological features; the second leads to phenotypical morphologies. Geno-
typical features are constant within a narrow limit (Dobshansky, 1951),
whereas the phenotypical features are apt to show wide variations. Identifi-
cations based upon morphology must be restricted to genotypical features
(Cholnoky, 1960). This means, of course, that a particular species cannot be
identified through the examination of a single individual. A series of specimens
must be examined to define the limits of phenotypical variation. The geno-
typical and the phenotypical morphological features are functional. However,
genotypical morphology reflects hereditary needs (phylogenetic adaptation),
whereas phenotypical features represent individual needs bearing on environ-
ment (ontogenetic adaptation; Goldschmidt, 1940).
The relationship between function and morphology is apparent among
plants of higher and lower orders. For example, two species of the flowering
plant genus Ambrosia, A. elatior and A. artemisiaefoUa, live in habitats which
are exposed to different degrees of sunshine and contain different amounts of
moisture. The latter species, A . artemisiaefoUa, lives in a semidesert environ-
ment. Consequently, the size of the foliage is smaller than that of the former
species, A. elatior, and scleral elements are abundant in the leaves to provide
mechanical support during periods of severe loss of turgor.
The Ambrosia pollen, i.e., ragweed pollen, shows a characteristic genotypical
morphology, a solid, spinose exo-exine. Clearly, the spines represent a geno-
typical feature because they must develop from the tapetal layer of the pollen
sack. The pollen grains, during their ontogenesis, are not directly exposed to
environmental influences. The spines are formed by apposition. For the
same reason the spines of the ragweed pollen are solid rather than hollow.
The solid intine can be penetrated by 3 pores only. The spines may facilitate
the transportation of the pollen grains. The characteristic tricolpate structure
is always observable upon proper focusing of the microscope (Erdtman,
1952, 1957; Faegri and Iversen, 1950; Jonas, 1952; Hyde and Adams, 1958).
See FIGURE 1, pollen grain of Ambrosia trijida; figure 2 the same in optical
section; figure 3, Hystrichosphaeridium sp. from the Upper Cambrian; fig-
ure 4, pollen grain of Dahlia pinnata; figure 5, Hystrichosphaeridium from
the Upper Cambrian. (Figures 1, 2, and 4 were taken from Wodehouse,
1942, and correspond to his numbers; 118, 119, and 115, respectively; figures
3 and 5 were taken from Timofeev, 1956, and correspond to his numbers 20
and 19, respectively.)
There are similar looking species of unicellular, aquatic plants. For ex-
ample, Hystrichosphaeridium Deflandre is covered with spines. These spines,
however, serve a different function, develop through different embryological
Claus et al.: "Organized Elements" in Chondrites 583
processes and are constructed differently (Deflandre, 1936; Timofeev, 1956;
Evitt, 1961a, 1962). The spines of real Hystrichospherids grow out from the
outer layer of the cell wall through intussusception. These hollow spines help
the organisms to float in water (Schiller, 1933-37). Both pollen and Hystri-
chosphere spines help to protect the species. On casual observation ragweed
pollen grains and certain Hyslricliosp/ieres may look alike (cf. figures 1 and 3,
10 /u,
Figures 1 to 5. 1, 2, and 4, pollen grains; 3 and 5, Hyslrichospheres.
and 4 and 5). Very careful microscopical examination is required by experi-
enced microscopists to distinguish the hollow Hystrichospheridmm spines from
the solid ragweed pollen spines. This example may emphasize that detailed
and careful observations are necessary for the identification of all microscopical
plants and parts of plants.
Fitch and Anders (1963) questioned the validity of using fine morphological
criteria in the identification of microorganisms. They claimed that structural
features less than 1 /x in size are difficult to observe and they suggested that
the resultant identification must be subjective. Yet the science of systematic
584 Annals New York Academy of Sciences
microbiology and micropaleontology provides numerous examples of the
successful use of fine morphological characteristics (involving features less
than 1 /x in size) in the identification of protobionta.
The blue-green algal genus Oscillatoria has 160 morphologically distinct
taxa, 28 of which are less than 1 ^t in diameter but they are still amenable to
morphological characterization (Hollerbach et al., 1953). As early as 1871,
optical microscopy was sufficiently advanced to allow Pfitzer to establish two
new genera Neidiuni and Anomoeoneis, that were formerly included in Navicnla,
by observing morphological features less than 0.3 ju in size. The recent work
of Hay flick (1962) established that primary, atypical pneumonia in humans
is caused by a pleura pnetimouia-Uke organism (PPLO), less than 0.3 n in size.
The same detailed morphological observation is also used in characterization of
microfossils. Recently, Evitt (19616 and 1962) has shown that the group
of Hyslrichospheres (Precambrain to Recent) consists of polyphyletic members.
This finding was again based upon the observation of fine morphology, which
involved the examination of the number of processes, spines, and the plate
structures. Most biologists agree that the microscopical examination of fine
morphological features (1 /z or less in size) is not only possible but it is a common
practice in systematic zoology and botany.
Modern biological microscopes, if properly used by experienced investi-
gators, can resolve objects as small as 0.2 /x in diameter. The theoretical
limit of resolution is 0.10 ^t. Clearly, it is quite possible to observe morpho-
logical features in the 0.3 to 0.5 n range. Fitch and Anders argued that one
of the organized elements embedded in mineral matter in one of the thin sec-
tions of Nagy et al. (1962) cannot be characterized because the spines are
approximately 0.3 ji long and the resolution of a microscope which they be-
lieved to be equipped with a regular, high dry objective is 0.3 ju- This argu-
ment is in error because the spines on this organized element were observed
with a X/O oil immersion objective, with a numerical aperture of 1.15, which
gave a lower limit of resolution of 0.22 ^i. The transparency of the embedding
mineral (magnesium sulfate), its lack of color in the thin section and the lack of
significant differences in refractive indices in this portion of the microscopical
preparation prevented any serious interference from attaining the necessary
resolution.
Fitch and Anders (1963) proposed a set of criteria to establish that certain
objects are microfossils. Their criteria are essentially the customary defini-
tion of life. They suggested that to be able to prove that the organized ele-
ments are indigenous and extraterrestrial microfossils one must show (1) that
they have characteristic morphologies, (2) show some evidence of propagation,
and (3) show signs of metaboHc processes.
Their first point needs no further discussion. One may reply to their second
requirement by noting that adjoining organized elements have been observed
embedded in minerals. This raises the possibility of either copulation or
division (figurp: 6c). Similar objects (but solitarily) were often found in the
mineral matrix (figure db). A less direct indication may be derived from the
possible presence of deoxyribonucleic acid- (DNA)-type material, which will
be discussed in another chapter. Finally, the presence of what may be bio-
J
Claus et al.: "Organized Elements" in Chondrites 585
genie compounds, such as certain hydrocarbons, cytosine and purines, should
have already provided an answer to their last postulated requirement.
The Applicahilily of Biological Stains to the Investigation
of Meteorite Samples
The use of biological stains for demonstrating cellular structures is indispen-
sable in microscopical biology. During the preceding 2 decades a better under-
standing of the chemical nature of dyes and the chemical reactions involved,
has made it possible to use the color developed as a specific indicator for the
presence of a certain compound. With the development of color indices and
standardization of the marketed dyes (Conn, 1953) many of the unpredictable
results or uncertainties originating from the varied staining procedures have
been eliminated. At present there are still some major gaps in the knowledge
of the reaction mechanisms of several dyes and their exact specificity in several
cases is not known; however, one is able to use them with a certain degree of
confidence if one fulfills the following three criteria. (1) Use a whole array
of structurally different dyes on the same substrate. (2) Use adequate amounts
of controls. (3) Rely more on stains which chemically react with the sub-
strate (or before application are in colorless form) than on those which are
merely adsorptive in nature.
SUdes were prepared essentially in 2 ways. (1) The sample was either
crushed or dispersed on the slide in double distilled water, was covered, and
the aqueous staining solution was, during the period of observation, constantly
sucked through the preparation. (2) The meteorite and soil samples were
crushed between 2 slides the surfaces of which were coated with fresh egg
albumin. The other materials like pollen or starch grains, etc. were dusted
over the albumin covered slides with a fine brush. The slides prepared in this
way were then subjected to the staining procedures, washed with tridistilled
water, dehydrated, mounted in balsam and coverslipped. In cases of the
eosin and hemato.xylin-eosin staining instead of eggwhite, collodium was used
for adhesion. The staining with ninhydrin was performed in small test tubes,
and the material was transferred to glycerin and examined in it. During the
staining procedures special care was taken not to contaminate or crosscontami-
nate the preparations, therefore the stains were freshly made up with tri-
distilled water and the different specimens were stained in separate sets of
copUn jars.
As can be seen from table 1, 19 widely differing biological stains were used,
of which only 1, i.e., safranin, is considered to be a true adsorptive stain (the
nature of dyeing with the eosin stains, like Azure II, and Dienes PPLO blue
stain is still debated). Of the 19 stains only 1, Sudan IV, gave negative re-
sults with the organized elements. All of the other stains were found to be
positive. One has to emphasize, however, that only a portion of the organized
elements stained with the different stains and the proportion of the stained to
the unstained particles varied from stain to stain. One has also to admit that
several of the dyes used stained not only the organized elements but also a
portion of the mineral material. However, one could easily distinguish by
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Claus el al.: "Organized Elements" in Chondrites 589
the intensity or shade of the color the organized elements from the minerals.
The staining of the soil samples gave similar results. One may make the fol-
lowing comments regarding the stains.
The use of chlor-zinc-iodide in the taxonomy of the OsciUaloriaceae (Cyano-
phyta) is mandatory. It is one of the best cellulose reagents and the system-
atics of the sheathed genera of the above mentioned family is based upon the
positivity or negativity of this reaction, i.e., whether the sheath turns blue or
does not stain at all. The 2 other color reactions ascribed to this stain are,
however, somewhat less specitic. It is accepted that chitinous substances
turn brown, whereas pectic compounds show a yellowish brown coloration.
The presence of proteinaceous moiety disturbs this reaction, as proteins will
also assume a yellowish brown color. There is, however, some difference be-
tween the color given by pectins and that produced by proteins. To differ-
entiate between these 2 colors requires either color charts or materials for com-
parison. In the case of the organized elements, chlor-zinc-iodide invariably
gave a yellowish brown color in the walls, characteristic for pectic substances.
Although it would be premature to conclude on the basis of this color reaction
that the organized elements possess walls made up by pectins, one is able to
rule out the possibility that they are recent pollen or spore contaminants
because then they should either become blue (cellulose) or dark brown
(chitin) in their walls. Types 1, 2, 3, and 4 have been seen reacting with this
stain.
The blue stain of Dienes was developed for the dyeing of pleuropneumonia-
Uke organisms (PPLO) as a substitute for the more complicated Giemsa staining
(Dienes, 1939). It is an alcoholic solution of methylene blue and Azure II.
Viable PPLO or bacteria will stain deep blue with the stain but will later be-
come faint due to decolorization of the methylene blue, whereas dying or dead
bacteria stain pink or do not stain at all. Fungus hyphae or spores usually
stain very dark blue; cellulose elements, however, stain lilac. This stain was
selected not so much to study its effect on the organized elements as to enable
us to recognize terrestrial contaminants. However, in the samples under
study, no viable bacteria were seen; fungus hyphae were absent and only a
single gonotokont was observed. The organized elements turned either bluish
or lilac by the stain but the majority did not stain at all. Types 1, 2, and 3
have been seen taking up the stain. The mineral matrix in the Orgueil or
Ivuna meteorites and in the soil samples turned light bluish.
One of the most surprising results was obtained with the Feulgen stain.
This staining technique was developed for the selective staining of nuclei and
chromosomes. It involves the use of the Schiff reagent (leuko-basic-fuchsin)
and its reaction with the aldehydes obtained by the acid hydrolysis (HCl) of
deoxyribonucleic acids. The staining is considered to be extremely sensitive
and very specitic. Since its first description, in 1924, by Feulgen, there has
been published voluminous literature dealing with the questions of sensitivity
and of specificity of the technique (Pearse, 1960). Several modifications were
proposed and, at present, the Feulgen staining has become one of the most
reliable and one of the most widely used techniques for the demonstration of
DNA in cells, and for the study of nuclear movements during cell division.
There are other substances besides DNA that are, however, known to give a
590 Annals New York Academy of Sciences
positive Feulgen reaction. These are the plasmalogens (acetal phosphatides),
(Schubert, personal communication, N.Y.U.). These latter compounds, how-
ever, are not very likely to occur in either the meteorites or in the soils as they
are quite unstable and are known to be present only in the central nervous
system and in the muscles of animals. Another, as yet unidentified substance
yielding a positive Feulgen reaction is the binding material among the cells of
some of the species of the green algal genus Oedogonium (Woes-Tschermak,
personal communication, Vienna, Austria). This material according to our
observations seems to possess a faint pinkish color even in the unstained,
living algae, if viewed in the microscope in dimmed oblique light and it may be
a compound similar to that described by Palik (1928) in Hydrodictyon (Chloro-
phyta) and named as erythropectin because of its pink color. In the case of the
Oedogonia the acid hydrolysis seems only to strengthen the pink color already
present and in reality we may not be dealing with a positive Feulgen reaction.
This question, however, deserves further investigation.
By using the Feulgen technique on our samples it was found that a con-
siderable number of the organized elements of type 1 and type 2, stained
homogeneously pink with this stain, whereas the mineral material remained
unstained or took a faint greenish color. It was most instructive to see the
results of the staining on the different controls; organisms present in the soil
or dust samples did not stain, except for their nuclei, which turned red. The
mineral and possible organic material in these samples, similarly to the material
in the Orgueil and Ivuna meteorites, either remained unstained or took a pale
greenish color. The same color developed also in the walls of the pollen grains,
whereas their nuclei stained pink (see figure 7, a io e). Pollen grains as a
whole never turned pink after the use of this staining method. In 1937, Shuita
investigated the nucleic divisions of pollen grains and found the Feulgen stain-
ing (by virtue of its complete noninterference with any other cellular element
of the grains except their chromatins), was the most suitable stain for such
type of studies.
Another interesting observation was made regarding the starches. Because
we could not find any literature data dealing with the effect of Feulgen stain-
ing on starch grains it seems to be worthwhile to deal with this problem at
some length. Starch (amylum) occurs in the phylogeny of plants compara-
tively early. It is present in the green algae and it remains characteristic for
the main line of plant evolution up to the Monocotyledoneae. Starch is always
an intracellular product and in most of the green algae and in the leaves of
higher plants it is formed in the chloroplasts. In the green algae usually a
separate organell, the pyrenoid, is in the center of the grains, other grains may,
however, directly be deposited in the stroma of the plastids or even in the cyto-
plasm (Czurda, 1928). Fritsch writes (1949, p. 67): "The grains . . . appear
to grow by apposition of layers on all sides, and their polyhedral form (giving
the entire group the shape of a shell) is a result of the fact that free deposition
can take place on the external surface." In cases of grains directly develop-
ing in the stroma or in the few cases in which they arise in the cytoplasm their
shape becomes more or less spherical and the layering takes a concentric shape.
Similarly, in any other starch producing plant the grains consist of an inner.
Figure 6a Petrographic thin section of the Figure 6b Petrographic thin section of the
Orgucil meteorite showing two, adjoining Orgueil meteorite showing a singular object,
organized elements. Magnification 590 x . similar to those in Fig. 6 a. Magnification 590 x .
Figure 7a Type 1 organized element after Figure 7b A type 2 organized element after
Feulgen staining. Magnification 1000 x. Feulgen staining. Magnification llQx.
Figure 7c Cassava starch grain after Feulgen
staining. Magnification 590 x ,
Figure 7 d Cassava starch grain after periodic
acid Schiff / PAS / staining. Note that Feulgen
staining, when administered according to stan-
dardized techniques, does not stain starch
whereas PAS does. Magnification 590 x .
Figure 7e Elm pollen grain after Feulgen
staining. Note that only the nuclei are stained.
Magnification 1000 x.
Figure 8 a A type 2 organized clement in
optical cross section from a powdered pre-
paration of the Orgueil meteorite. Magnifi-
cation 1000 X.
{■im-
^<^V ^'
Figure Sb The same organized element pho-
tographed by focusing on its top. Note that
the protrusions have dark centers indicating
hollow tubes. Magnification 770 x .
Figure Sc Cross section of an organized ele-
ment similar to that shown in Figs. 8 a and
8 b. This object occurs in a petrographic thin
section of the Ivuna meteorite. Magnification
770 X .
Figure 8d Ambrosia pollen grain after Grid-
ley staining in optical cross section showing
solid protrusions. Magnification 590 x .
Figure 8e The same preparation showing the'
characteristic tricolpate, triporate structure of
the Ambrosia pollen grain. Magnification
590 X.
w
•"•iC
• •*
fclKgy
Figure 9a Organized element resembling a
Dinoflagellate cyst focused to show the ridges
and canals of its surface. Magnification 770 x .
Figure 9b Same object focused on the sur-
face appendages. Magnification 770 x .
Figure 10a A type 2 organized element from
the Orgueil meteorite. Magnification 590 x .
Figure 10b A similar object from the Ivuna
meteorite shown in UV light. Ribs appear in
the interior of the organized element. Mag-
nification 400 X .
Figure 10c Petrographic thin section of the
Ivuna meteorite seen in UV light, showing an
organized element in a magnesium sulfate
vein. Magnification 400 x .
Figure 10d The same organized element in
UV light after the blueish colored fluores-
cense of the mineral material had been elimi-
nated by an appropriate set of filters. Mag-
nification 400 X .
Figure 11a Two type 1 organized elements
and mineral grains in a powdered sample of
the Orgueil meteorite. Magnification 590 x .
Figure 1 1 b An opaque, magnetic particle from
the Orgueil meteorite. Magnification 590 x.
$■••*
• 4.'
Figure 12a An organized element resembl-
ing the features of a Thecamoeba, focused on
the sculptured top of the object. Magnifi-
cation 770 X .
Figure 12b Same object focused on the spines
of its lower surface. Note that in the hollow
interior a bubble of / possibly / air occurs.
Magnification 770 x .
Figure 13a A type 5 organized element after
Gridley staining. Focused to show the three
tubular protrusions of the body. Magnifi-
cation 590 x .
Figure 13b Same object focused on two of
the protrusions only. Magnification 590 x .
Claus et al.\ "Organized Elements" in Chondrites 591
variedly shaped "nucleus" or hilum and the layered starch is around it. The
mcoposition of the "hilum" is unknown. It seems that it is proteinaceous in
nature. The sole exception from this is represented by Rhodophyta, in which
the so-called Floridean starch develops always in the cytoplasms, is unlayered
and is lacking a "nucleus." This compound, however, is closer to glycogen in
its composition as it stains brown instead of blue with iodine. The shape of
starch grains, their mode of layering and the form of their "nuclei" is constant
and species specific, thus, it is a true genotypical feature which gives a good
basis to establish relationships between different plants. The pictures of the
starch grains in the endospermia of varied Gramineae, the so-called amylo-
grams, were successfuUy used in the ehicidation of several important micro-
systematic problems (Soo, 1953).
None of the 6 starches showed a positive reaction after Feulgen staining.
Actually, the grains became so translucent that it took considerable time to
recognize them in regular transmitted light. There were several dark bhie
dots on the slides, however, which in phase contrast were found to be the
"nuclei" of the grains; they were invariably surrounded with a translucent
envelope, showing concentric rings and corresponding in size and shape to the
grains before treatment. In the case of the Cassava starch (grains of Manihot
utilissima) the envelope occasionally turned faint blue on exposure to the
Feulgen stain. It seems that the starch grains are inhomogeneous in their
chemical composition and the circular striations are caused by a very thin
layer of a matter different from amylum. The envelope covering the grains
must be of the same material as those that cause the circles. That we were
dealing with true structural elements that upon the action of P'eulgen staining
occasionally turned faint blue could be determined by investigating ruptured
or broken grains. In these cases fringes of the envelope and of the circles
could be observed on the edges of the ruptures. No other staining and not
even the unstained materials gave similar results. The possibility exists that
the starch was at least partially hydrolyzed by the HCl and that this procedure
made conditions favorable for the detection of the envelope. The staining
pattern of the starch "hila" and that of the envelope after the Feulgen stain
do not allow us, as yet, to conclude anything about their nature. We may,
however, conjecture, per analogiam, to the green algae that the starch "nuclei"
may be proteinaceous in nature.
It is clear from these experimental results that the claim of Fitch and Anders'
(1963) that one of the type 2 organized elements (stained with Feulgen staining
and showing a diffuse pink coloration) is a starch grain is unfounded. These
authors demonstrate that the Schiff reagent is a nonspecific carbohydrate stain,
which is even capable of reacting with several inorganic materials, including
clay minerals. They show pictures of deep magenta colored starch grains,
dyed with the Schiff reagent and conclude that as Feulgen staining uses the
Schiff reagent, it is nonspecific, hence the organized element in question is most
probably a starch grain. This conclusion is in error as shown by figures
la-Id. Figure la shows a type 1 ; figure lb a type 2 organized element after
Feulgen staining. In figure 7c is shown the Cassava starch with Feulgen and
in figure Id with PAS staining.
592 Annals New York Academy of Sciences
Similarly, the assumption of Gregory (1962) that the organized element in
question may either be a "Hulle" cell of Aspergillus nidiilans or the pollen
of a Juiiiperus sp. or Taxus baccata can be rejected on the basis of the stain-
ing alone, even without taking into account the basic morphological differences.
It has already been mentioned that with Feulgen staining only the nuclei of the
pollen grains stain and not the body as a whole.
Interpreting the results of the diffuse Feulgen staining of the type 1 and
type 2 organized elements presents a different cjuestion. Bacteria and blue-
green algae are known to contain chromidial apparatus instead of a compact
nucleus. It is customary to refer to the central body or centroplasm of the
Cyanophyta as nuclear equivalents, containing diffused nucleic material. In-
deed, Feulgen staining in such organisms will result in a diffuse pink coloration
of the protoplasm. One may thus think that some of the organized elements
indeed contain diffused nucleic material in their interior. The present evidence,
however, is not sufficient to substantiate such a supposition. We can only
state that, according to one of the most specific biological staining techniques
developed, there is an indication that nucleic acids are present in the interiors
of some of the type 1 and type 2 organized elements. It is, however, equally
possible that an as yet unknown material is present in these organized elements,
which will react with the Feulgen reagent to produce the pink color.
The Gridley staining was primarily developed (195vS) as "A Stain for Fungi
in Tissue Sections." It is the combination of Gomori's aldehyde-fuchsin stain
and the Hotchkiss-McManus technique. As Gridley states (p. 303): "The
problem of positive tissue elements with the periodic acid-Schiff reaction was
eliminated by hydrolyzing in chromic acid instead of periodic acid." For
counter staining Metanil yellow is used. Fungal conidia and hyphae are
stained deep blue to purple by this method while other tissue elements, mostly
the proteins, stain yellow with the counter stain. The results of this technique
on our samples were interesting for 3 reasons. (1) Some of the organized ele-
ments of type 1, type 2, and type 3, stained lilac with the stain, whereas the
mineral matrix of the Orgueil and Ivuna meteorite in many instances took a
dirty purple color. A similar staining pattern of the minerals was observed
in the soil samples, in which, however, some algae stained rose and fungi be-
came blue. It was, therefore, surprising to see that the minerals of the Murray
meteorite did not stain with this stain. The starch grains took a vivid magenta
color. (2) Type 5 organized elements stained orange with the Gridley stain-
ing and the surrounding halo turned yellow from the counter stain. The
meaning of this reaction is obscure. (3) The forms described by Staplin (1962)
as Coelesliles sexaugulalus from the Orgueil meteorite stained yellow, probably
from the Metanil yellow. These forms have originally a yellowish-orange
shade, however, after the exposure to the Gridley staining the change in their
color was striking. On the slides prepared from the meteorites not a single
spore or fungus hypha could be detected. In the preparation from Holbrook
1 specimen of Nitzschia acicularis (a Diatom) was seen.
A surprising result was obtained by the application of a watery solution of
Janus green B. Many of the type 1 and a few of type 2 organized elements
developed a blue-stained reticulate structure in their inside. This stain is a
Claus et al.: "Organized Elements" in Chondrites 593
vital or supravital stain generally used for the demonstration of mitochondria.
It is considered to be more or less specific for ribonucleic acids. The reticulum
that developed in the organized elements was similar to that obtained after
their treatment with 6 N HCl. The meaning of this staining pattern is ob-
scure.
Metanil yellow alone, in a watery solution was applied to rule out the possi-
biUty that the observed yellow coloration of Staplin's form after Gridley stain-
ing was due either to the hydrolysis with chromic acid or to the Schiff reagent.
As was the case in the Gridley stained preparations, after treatment with the
simple watery preparations, Staplin's Coelestites turned into a striking yellow.
A few of type 1 organized elements also became yellow. With our present
knowledge, we cannot satisfactorily interpret these results.
Neutral red, usually used in biology for its nontoxic character as a vital or
supravital stain, stained some of type 1, 2, and 3 organized elements a homo-
geneous red.
Ninhydrin, this most sensitive amino-acid or protein reagent, stained lilac
several type 1 and 2 organized elements; however, it gave a purple coating
to the mineral debris in the carbonaceous meteorite and soil samples. The
ordinary stony meteorites gave negative results.
Periodic acid-Schifif (PAS) reagent seemed to be the least adequate for
differential staining. Being a general carbohydrate stain, it dyed magenta
color the pollen and starch grains and algal cells. Several of the organized ele-
ments of types 1, 2, and 3 also took this stain and developed a color similar to
those of the algal cells or starch or pollen grains. The minerals of the Orgueil
and Ivuna meteorites and those of the soil samples also took up the stain.
There was a significant difference between the shades of the organized elements
and the starch or pollen grains on one hand and that of the minerals on the
other; the latter having a more "dirty" magenta color. Furthermore, some
type 1 organized elements remained totally unstained among well stained
mineral aggregates. It was interesting to see that the minerals of the Murray
meteorite did not, or only very occasionally, stain with PAS and that the
staining of both the Holbrook and Bruderheim meteorites was negative. The
quality and intensity of the color of the stained organized elements resembled
closely the color of the controls. One may speculate that some kind of chemical
similarity may exist between the organized elements and the controls that
contain carbohydrates. Because of the nonspecificity of this reaction, how-
ever, it seems advisable not to attempt to reach any premature conclusion in
this matter.
It was previously mentioned that Sudan IV was the only stain which left
unaffected the organized elements, although it stained vivid red the oil drop-
lets of the terricole Diatoms.
Toluidine blue gave a blue or pink color with some types 1, 2, and 3 organized
elements. In several cases the minerals of the carbonaceous meteorites and
of the soil samples also turned blue. Metachromasia, however, was not ob-
served with the mineral particles. Samples of Orgueil, after being treated
with boiling HF for 10 minutes, left acid resistant peUicles of the types 1, and
2 organized elements, which after staining with toluidine blue showed signs of
594 Annals New York Academy of Sciences
metachromasia. It would, however, be somewhat premature to conclude from
this staining pattern that there are acidic or basic polysaccharides in the acid
resistant pellicles of the organized elements.
One may draw the following conclusions from the staining experiments.
(1) The specificity of some of these stains is not known. However, it seems
unhkely, that 18 of the 19 stains used, gave positive results by chance. To
evaluate the meaning of a single staining reaction often seems to be impossible.
One cannot argue that a sample is of biogenic origin on the basis of a single
staining. However, if a whole array of different stains are applied, which are
widely differing in their chemical composition and in their specificity, one can
point out biogenic material.
(2) The use of a great variety of stains (some of them specifically developed
for the scanning of certain microorganisms, like Dienes blue-stain for PPLO
or the Gridley staining for fungi) facilitated the recognition and, thus, the
elimination of earthly contaminants in the meteorite samples. As only small
meteorite fragments or powdered material could be used for these studies, the
question of contamination could be settled only on the basis of elimination.
But by the use of the numerous stains and the relatively great number of con-
trols (including soil from the impact area, and dust from the museum) one
could recognize and exclude the common contaminants.
(3) An examination of the soil and dust samples has shown that micro-
organisms stained differently from the mineral constituents, i.e., the latter
did not stain at all or took a different color. These and the starch and pollen
controls have confirmed the specificity of the Feulgen reaction.
(4) It has been pointed out that not all of the organized elements stained.
A gradation in the staining was observed with almost every stain (with the
possible exception of safranine). One reasonable explanation for this phenom-
enon may be that different degrees of mineralization are present in the or-
ganized elements. In terrestrial bitumens microfossils are often differen-
tially mineralized (Andreanszky, 1954).
Physical and Chemical Observations on the Organized Elements
Fluorescence in ultraviolet light. When preparations of the 4 carbonaceous
meteorites were examined with a fluorescent microscope a number of particles
became readily visible. Most of these particles fluoresced with a greenish-
yellow light when excited by ultraviolet radiation and when Corming 7-59 +
Wratten 2B filters were used. A Zeiss fluorescent microscope was applied
in these studies. Less frequently, particles fluoresced with a green, pink or
red color upon excitation by ultraviolet light. An examination of the thin
sections and of the crushed samples has shown that some of the mineral con-
stituents (possibly those that were coated with bituminous matter) seem to
have fluoresced with a bluish- white color. The fluorescence of the organized
elements, in our opinion, can be readily distinguished from the fluorescence of
the mineral matter. It is, of course, possible to select the right combination
of filters to exclude the bluish-white fluorescence from the yellowish-green or
pink fluorescent light. Several of the type 1 organized elements were found to
fluoresce with greenish-yellow light, contrary to the argument of Fitch and
Anders (1963). Fitch and Anders also claimed that pink colored fluorescence
Claus et al.: "Organized Elements" in Chondrites 595
of organized elements is not a true fluorescence. They suggested that bire-
fringent particles may appear red when viewed with ultraviolet light in the
fluorescent microscope. The reason for this could be that the usual filters
transmit a portion of the red part of the spectrum. However, the particles
which were described previously were not birefringent when examined in a
polarizing microscope and they were photographed with an additional set of
filters (Corming 7-59 + Wratten 2B + Zeiss 064 + 061) that permitted mainly
blue light to enter the microscope. Consequently, the argument of Fitch and
Anders is not valid in this case.
The microscopical assembly used for the fluorescence studies enabled one to
view objects in UV darkfield illumination, as weU as in regular transmitted
light. In this way it was possible to select organized elements of distinct
morphologies for the fluorescence studies. It is true that a few irregularly
shaped particles also fluoresced in greenish-yellow light. These particles
could be easily fragments of organized elements, broken during the crushing
of the meteorite samples. On the other hand, the majority of the irregularly
shaped particles fluoresced with a different color than the organized elements;
they emitted bluish-white light and they were probably mineral particles (or
particles coated with bituminous matter).
Fluorescent microscopy is also useful to demonstrate certain morphological
features which are not readily visible in transmitted light. Mitochondriatic
granules become visible in the UV microscope when they emit fluorescent light
with or without fluorochromation (Drawert and Metzner, 1956). Similarly,
fluorescent microscopy was found to be useful to visualize certain morphological
features that were not visible when the organized elements were examined in
regular, transmitted fight or with phase contrast microscopy. Figure 106
shows an organized element that fluoresced with greenish-yeUow light.
Centripetal ribs were found to be present around the walls. This morphological
feature is rather unusual, because only a few Diatom species are known to show
centripetal ribs (Hustedt, 1930). This feature is not identical with the internal
septae of Coccolilhoplwrideae, Silicoflagellatae, Foramiiiiferae, and certain Di-
atoms such as Naviculaceae etc., the septae of which extend much farther into
the cell. The general habit, as seen in transmitted fight, figure 10a, resembles
a Trachelomonas, a genus of aquatic protophyta, except of the location of the
pore. However, a Trachelomonas does not have centripetal ribs. The presence
of the centripetal ribs are of particular interest regarding the argument of
Fitch and Anders about pollen grains. The exo-exine of pollen grains show
centrifugal thickenings but they never show centripetal ribs. Fames and
MacDaniels (1947) state on p. 49 in their "Introduction to Plant Anatomy"
that: "The external wall layers and surface projections of spores and pollen
grains are formed in part by tapetal fluid or mother cell cytoplasm." (There-
fore, only centrifugal thickenings can occur on walls of pollen grains.) It
seems unlikely, that the particles shown in figure 10, a and 6, are pollen grains
or spores. It is known that spores of fungi do not fluoresce. Hofler and
Pecksieder write (1942, p. 117): "Angesichts der weiten Verbreitung primar
Fluoreszenz im Gewebe der Pilzkorper beriihrte uns die Beobachtung urn so
auffalliger, dass die Sporen der Hulpilze im UV-Lichl nicht fluoreszierten, viel-
mehr meisl vdllig unsichtbar waren."
596 Annals New York Academy of Sciences
The abundance of the organized elements. The number of the organized
elements (per milligram) in the Orgueil, Alais, Ivuna, and Tonk meteorites has
been reported previously (Nagy et al., 1962). The numbers were arrived at
by counting all types of organized elements or such fragments thereof, which
appeared to be larger than 50 per cent of 1 particle. The type 1 organized
elements of Claus and Nagy (1961) are the most abundant; they comprise
approximately 80 to 90 per cent of all microstructures. This type has also the
simplest morphology. Consequently, certain investigators were unable to
distinguish this type of organized elements from mineral particles or were of
the opinion that the morphological criteria are not sufficient to distinguish
them from mineral particles. If one chooses to exclude the type 1 organized
element one will arrive at a count that is substantially lower than that given
by Claus and Nagy (1961). It should be noted, however, that some biologists,
on critical examination, were inclined to include this type 1 particle among
the organized elements (Papp, 1963; Cholnoky, 1963; Skuja, 1962; Palik,
1963). Also, as Urey (1962o) pointed out, one only needs to have some bio-
genic and indigenous microstructures in a meteorite to ascertain the existence
of extraterrestrial life. The total count of organized elements, including type
1 (1300 to 1700 per mg.) shows good agreement with counts of microplanktons
in fossil marine populations (1200 per mg.), as reported by Kolbe (1952) and
with the counts of stainable organic microstructures in the Mokoia meteorite
(1000 to 1700 per mg.) described by Briggs and Kitto (1962).
Solubility in acids. The effect of acids and organic solvents on the organized
elements has been reported previously (Nagy et al., 1962). It is necessary,
however, to comment again on this subject because Fitch and Anders (1963)
claimed to have dissolved 97 per cent of an Orgueil meteorite sample by heating
it for 17 hours at 60° C. in HF and for 18 hours in 6 N HCl at 25° C. The
remaining residue was reported to be an aggregation of "finely granular, black
to brown material virtually devoid of any structure." Milder treatments in
concentrated HF (Urey, 1962o; Staplin, 1962; and Nagy et al., 1962) and
6 N HCl resulted in a residue which contained several transparent and acid
resistant pellicles. Organized elements, including type 1, retained their
characteristic morphologies upon exposure to 6 A^ HCl at room temperature
for varying periods of time. The type 1 organized elements were not de-
stroyed when boiled in concentrated HF for 15 minutes. The statement
made by Fitch and Anders ". . . since they disappear after treatment with HF,
we believe they are most likely grains of silicate minerals although they are
classified as organized elements by Nagy and coworkers" seems to be in error.
In spite of the rather severe treatment they used, Fitch and Anders were still
able to find some transparent and highly organized structures of undoubtedly
biogenic nature.
Problems of contamination. Several claims were made in the literature to
the effect that the organized elements are terrestrial contaminants (Fitch and
Anders, 1963; Deflandre, 1962; Gregory, 1962; and Pearson, 1962). Contami-
nation is, of course, a serious problem and it cannot be fully excluded at the
present time. However, it should be borne in mind that no trained micro-
biologist or micropaleontologist who has actually worked with an Orgueil sam-
Claus et al.: "Organized Elements" in Chondrites 597
pie, for any length of time, has yet (at the time of this writing) positively
identified the organized elements as known terrestrial species. Certain com-
ments, that to us seem to be somewhat vague, such as Deflandre's (1962)
statement: "Positive identifications in this case are unnecessary and super-
fluous" cannot be taken too seriously. Deflandre, to our knowledge, has
never examined a carbonaceous meteorite. Similarly, the criticisms of Gregory
and Pearson cannot be accepted as strong evidence against the extraterrestrial
nature of the organized elements because these authors made their identi-
fications from a set of photographs and sketches that were reproduced rather
poorly in a scientific journal. (Ciregory saw some of the microscopical prep-
arations after he submitted his paper to the press.) Gregory and Pearson
identified the same organized element as 2 different terrestrial contaminants.
On one occasion a single organized element, when it was briefly shown in a
microscope to 18 microbiologists, was "identified" as 18 different species of
protobionta or organic artifacts.
Fitch and Anders (1963) claim that only a few of their particles, which ac-
cording to them are mere terrestrial contaminants, survive the combined HF,
HCl treatment. We found that these particles, when we examined their
preparations under the microscope, showed morphological features that were
dissimilar to common airborne contaminants (Wodehouse, 1942, 1945; Gregory,
1961). They appeared to us identical to some of the forms that we found in
our preparations. As a matter of fact, we found one of these forms ourselves
in Fitch and Anders' preparations, in their presence, during their visit to our
laboratories.
Fitch and Anders claim that some of these particles are ragweed pollen.
However, accorchng to their own measurements, given in their report, some of
those particles seem to be too small to be Ambrosia pollen. It is clear that one
must both critically evaluate the fine morphology and make accurate measure-
ments of size to establish a particle as a known terrestrial contaminant. Fur-
thermore, Fitch's and Anders' contention that some of the organized elements
in our preparation are ragweed pollen is also untenable because it is based upon
the comparison of photographs of rather low resolution which do not permit
the evaluation of fine morphological criteria. In figure 8g are shown some
ragweed pollen grains, to demonstrate the solid spines of theexo-exine. Figure
M is an optical cross-section of the same. In figure 86 is shown the type 2
organized element (note the hollow protrusions). In figure 8c is .shown a
similar object embedded in minerals. The identification of another organized
element (figure lb) as either a starch grain or a recent Juniperus pollen is also
in error because the structure of starch grains shows concentrical layering.
Juniper pollens are much larger than the object in question, they do not have
papillae and have rugate exo-exines (Erdtman, 1957).
Additional sources of possible terrestrial contaminations have been exam-
ined recently. Soil samples and outcrop samples have been collected in the
vicinity of the villages of Orgueil and Nohic in Southern France near the lo-
cation where the meteorite fell. It has been suggested (Bourrelly, 1962) that
soil and rock samples from the impact area be examined to evaluate the degree
of contamination from the local environment. Bourrelly noted that the culti-
598
Annals New York Academy of Sciences
Montouban 12 Km
iJ
. . /' I.Perllfrot
^ 2 Lescure
^_Compsas
T
^
"""^-^^^C^^ River
=^^
^^^^r^^ Orgueil
J
\
OI<ind2 Locotions of falls of
the two Orgueil meteorite
samples, received from
the Montouban Museum
I Locations of the
soil somples
1=50,000
Figure 14. Map of the Orgueil and Campsas area.
CROSS SECTION ALONG THE ORGUEIL METEORITE TRAJECTORY BETWEEN
THE VILLAGES OF LAPEYRIERE AND ORGUEIL.
Lopeyriere
Campsas
Ofgueil
mmmmfi.
GEOLOGICAL COLUMN AT ORGUEIL
1 : recent alluvium of the Tarn.
2;ancient alluvium of the middle
terrace of the Tarn.
3:ancient alluvium of the lower
terrace of the Tarn,
/♦ifluvlal terraces of the Garonne^
5:molasse (Stamplen-Ol Igocene)
Tert lary
soils about 30 cm thick
alluvium (clay), about 60-90 cm thick
o; o
O.'o sand and pebbles of the terrace,
about 5 ni- thick
molasse (Stampien), clay and sand,
at least 200 m. thick
Figure 15. Geological cross-section in the vicinity of Orgueil, in France.
vation of land in this part of France is basically the same today as it was at
the time of the fall of the Orgueil meteorite. One may expect, therefore, that
a microbial population similar to that of 1864 is present in the soil. In figure
14 are shown the locations of the falls (on May 14, 1864) of the stones and the
locations where soil samples were collected on March 29, 1^62. In figure 15
a geological cross-section of the area is shown. The sedimentary strata that
underlies the alluvium in the Orgueil-Nohic area consists of Tertiary formations
extending to a depth of at least 600 feet. Staplin (1962) suggested that a few
Cretaceous microfossil contaminants might have been included in the Orgueil
meteorite from the soil in the impact area. Our studies, based upon the recent
field work of Henri Coustau, revealed no Cretaceous outcrops near Orgueil and
Claus et al.: "Organized Elements" in Chondrites 599
Nohic. Staplin (1962) was undecided about the Cretaceous identity of the
forms in cjuestion. It seems that these forms are not Cretaceous contaminants,
after all.
Microbiological and micropaleontological examination of the soil and rock
outcrop samples revealed no forms that were morphologically identical to the
organized element in the Orgueil meteorite. The species of microorganisms
that had been identitied from the Orgueil soil samples are listed in table 2.
These samples still contained a considerable amount of their original water
content when they arrived at our laboratories, thus several forms could be
studied while still alive. The soil and rock samples were treated identically
to the meteorite samples, including the biological staining techniques. We
concluded that the organized elements in the Orgueil meteorite are not identical
with the organisms and microfossils that were collected on March 29, 1962,
by Henri Coustau, in the soils and rocks of the impact area.
Another source of contamination may be the microorganisms in the air.
When a meteorite enters the earth's atmosphere it "breathes in" air because of
the reduced pressure in its interior. It is conceivable that some organisms
may be sucked in at such time (although the average pore size of the Orgueil
meteorite is estimated to be less than 1 /x). In order to gather some informa-
tion about this possibility, particles collected in the atmosphere have been ex-
amined. The airborne particle samples were received through the courtesy of
C. W. Phillips, U.S. Army Chemical Corps, Fort Detrick, Md. They were col-
lected on precleaned microscope slides at the elevation of the collection. It is
known (Proctor and Parker, 1942) that at the height of between 10 and 30,000
feet mainly bacteria exist. An examination of the slides revealed no organisms
that were morphologically identical to the organized elements in the carbon-
aceous meteorites. There are a number of reports in the literature (Hyde and
Adams, 1958) on airborne pollen grains and spores; and a few reports on algae in
the air (Schlichting, 1961). It seems that the organized elements of the meteor-
ites do not correspond to known airborne contaminants.
Other possible sources of contaminations, such as chemicals used, including
the water, have been evaluated previously (Nagy et al., 1962).
Finally, 2 samples of the Orgueil meteorite were recently obtained from the
Montauban Museum (through the courtesy of A. Cavaille). These samples
have been in Montauban, France, which is near Orgueil, continuously since
approximately 2 weeks after the fall of the meteorite. The samples were kept
under glass jars; however, they were not stored in a sterile environment.
A microscopical examination of the Montauban samples revealed identical
organized elements (except type 5) to those from other museums. It is very
difhcult to believe that 6 samples of the Orgueil meteorite (from the American
Museum of Natural History in New York, the U.S. National Museum in
Washington, D.C., from the British Museum, from the Musee d'Histoire
Naturelle, Paris and the 2 from Montauban) would have been contaminated
by identical microorganisms in storage. The organized element that has been
claimed to be a ragweed pollen by Fitch and Anders, was also found to be pres-
ent in the Montauban sample. Ragweed (Ambrosia) is a native American
plant. It was introduced to Europe only in the early part of the twentieth
century and it is still not a common plant there (Soo, 1953).
Table 2
Biological Material Found in Soil and Sedimentary Rock Samples Near the
Village of Orgueil
Name of species
Soil sample A,
surface
Soil sample B,
from 40 cm. depth
Rock sample from
quarry
Achnanllws cf. micrccephala
+
+
Actinomyceles 2 different spp.
+
+
Adelomycetes spore 5 dilYerent spp.
+
+
Amoeba 3 different spp.
+
AmpJiora oval is
+
A nkistrodesm us Jalcatiis
+
Arcella sp.
+
Aspergillus sp.
+
BotryococcHS sp.
+
+
Chlorella vulgaris
+
Chloi-ococcuin h umicolum
+
+
+
Chroococcus tiirgidus
+
Chrysophyta cysta, 2 types
+
+
Ciliata 4 different spp.
+
+
Closlerium ehrenbergii
+
Cocconeis spp. 2 t>'pes
+
+
Cosmarium sp.
+
Cosmarium granulatunt
+
Cymhella ventricosa
+
Cymhella prostrala
+
Difflugia cf. pyriformis
+
Dinohryon serkdaria
+
Euglena deses
+
Euglena sp. cysta
+
Fragilaria pinnala
+
+
Gomphonema olivaceuni
+
Kephyrion sp.
+
Melosira varians
+
Navictda rhincliocepl/ala
+
Navicula hungarica var. capitata
+
Navicula pupula
+
Nematoda 2 different s|)p.
+
+
Nitzschia acicularis
+
+
Nilzschia fruslulum
+
Nitzschia sigmoidea
+
Nostoc fusiforme
+
Oocystis pusilla
+
Oscillaloria animalis
+
+
Oscillatoria teyebrijormis
+
Oscillatoria tenuis
+
Pediastrum boryanum var. gramdatum
+
Peridinium sp. (fragment)
+
Phormidium autumnale
+
+
Phortnidium foveolarum
+
Pinnularia viridis
+
Pleurococcus naegelii
+
Pollen grains of
Abies sp.
+
Achillea cf. millefoHum
+
Agrostis cf. vulgaris s. alba
+
+
Alnus sp.
+
Bettda sp.
+
+
Cirsium sp.
+
Hordeum cf. vulgare
+
+
Knautia arvensis
+
Pinus sp.
+
+
Polygonum cf. convolvulus
+
Prunus s. Crategus sp.
+
Quercus cf. puhescens
+
Sa gill aria sagiltifolia
+
Salix cf. cinerea s. caprea
+
Senecio cf. vulgaris s. viscosus
+
+
Trifolium cf. arvense
+
+
Triticnm aestivum
+
+
Tussilago farfara
+
i
(
tinn
Claus et al.: "Organized Elements" in Chondrites
Table 2 — Continued
601
Soil sample A,
Soil sample B,
Rock sample from
Name of species
surface
from 40 cm. depth
quarry
Scenedesmiis aciiminalus
+
Scenedesmus bihtgatiis
+
Scenedesmus obliquns
+
Scenedesmus quadricauda var. longispina
+
+ .
Steril ni\celia 3 types
+
+
+
Stephanodiscus lianlzsckii
+
SlicltococcHS minor
+
Strombomonas sp.
+
Surirellii ovata
+
SynechococcHS elongatus
+
Synedra nimpens
+
+
Synedra ulna var. oxyrliynchus
+
Tetraedron muHcum
+
Ulollirix syi.
+
Unidentitiahle arthrospores (Noslocalesf)
+
+
3 types
Unidentifiable conidiospores 4 types
+
+
+
Unidentifiable green algal zygotes or
+
+
+
zygospores (Oedogoniaceaef Conjii-
galesff) 5 types
Unidentifiable moss-protonema
+
+
Vampyiella sp.
+
Windier id cf. sessilis
+
Xanthidium sp.
+
On occasion fungi are known to grow on mineral specimens in museums.
In the growth process the hyphae get attached to or penetrate into the samples.
Microscopical examination of the thin section of such samples reveals the spores
and hyphae. No hyphae or remnants of hyphae were yet seen in carbonaceous
meteorites. This renders unlikely the possibiUty that the organized elements
are spore contaminants from fungi that grew on the samples in the museums.
Such a fungal growth would be rather unusual and could occur only in the
presence of adequate moisture. Some of the mineral components of the Orgueil
meteorite point out that the samples were kept in dry museum storage.
Terrestrial contaminations should have been able to enter the pores if the
interiors of the meteorite are contaminated. Organized elements are embedded
in minerals and in the mineral aggregates in meteorites, as was reported pre-
viously. A petrographical study of the thin sections (Nagy and Claus, 1963)
led to the estimation of the average pore sizes as less than 1 n in diameter.
The size of the organized elements varies between 3 to 60 m- Although there
may be a few wider fractures going through the samples it is thought to be
impossible for organisms to penetrate the dense and unfractured areas of the
mineral matrix.
Microscopical preparations of the carbonaceous meteorites have now been
prepared from time to time, over a period of 1 year (before, during, and after
the pollination time of ragweed and other flowering plants). No correlation
has yet been found between the types and numbers of the organized elements
and the time of preparation of the slides. This suggests that the organized
elements were not introduced into the sample when the microscopical prepara-
tions were made. Identical organized elements, such as the particle that is
602 Annals New York Academy of Sciences
claimed to be ragweed pollen by Fitch and Anders, have also been found by
different investigators at different laboratories at different times.
In our opinion the probability of terrestrial contamination is a most serious
problem. However, the control experiments described in this report and
previously (Claus and Nagy, 1961; Nagy el al., 1962) strongly indicate that
the organized elements (or most of them) are not terrestrial contaminants. Yet
it must always be borne in mind that even unusual contaminants may become
included easily in a sample. Microscopical preparations of an Orgueil meteor-
ite sample provided through the courtesy of Fitch and Anders for us to study,
contained fragments of a Compsopogou filament (a not too common species
of Rhodophyta), individuals of Chlorella, a rare species of Ndgeliella, antennae
of Cladocerae, sqamae of Tilia leaves, and emergentia of unknown origin.
Although the organized elements were clearly visible, the presence of the
aquatic contaminants suggested more of a sample of a Recent sediment than
that of a carbonaceous meteorite.
The Diverse Morphology of Organized El erne ills
An examination of approximately 400 microscopical preparations of car-
bonaceous meteorites, and related material, has as of now revealed 30 distinct
morphological types of organized elements. Other investigators (Staplin,
1962; Palik, 1962, 1963; Ross, 1963) found several other types. None of these
organized elements seems to be identical to known terrestrial species, although
they resemble them.
Organisms can be classified into four symmetry groups. The simplest
symmetry group is the sphere, and the most advanced one is the bilateral type.
(Asymmetrical categories can be derived from each of the four groups.) Trig-
onal symmetry is the least common among terrestrial organisms. Organized
elements, however, often fall into this class. Organized elements contain
examples of each of the symmetry categories (see figures 6, a and b; 1, a and
b; 8, a, b, and c; 9, a and b; 10, a, b, c, and d; 11a; 12, a and b; and 13, a and b).
Conclusions
Consideration of the fine morphology, physical and chemical tests, staining
with biological stains, and further evaluation of contaminations suggest that
the Orgueil, Ivuna, Tonk, and Alais carbonaceous meteorites contain indige-
nous, organic microstructures which seem to be of biogenic origin. Full proof of
the indigenous and biological nature of the organized elements is still not
available but the indications seem to be strong.
It has been shown that fine morphological criteria are of diagnostic value.
As a matter of fact, microbiologists and morphologists are using such criteria
every day in a variety of problems. It has been shown, as it is known to many
investigators, that morphological features of 0.3 ^ size can be observed and
identified by optical microscopy. The value of morphological criteria was
noted by Fournier (1962) when he stated at the First International Conference
on Palynology, that a worker in biology . . . "classifies his pollen based on
morphological features alone, a fact that has proven no detriment to his work."
The criticisms of Fitch and Anders have been considered and found to be
unacceptable. A critical, systematic and objective evaluation of the organized
Claus et al.: "Organized Elements" in Chondrites 603
elements is essential if an accurate identification* of these particles is to be
achieved.
A ck nowledgments
We wish to thank the microbiologists who examined the microscopic prepara-
tions. Special acknowledgments are made to A. Cavaille of the Montauban
Museum for providing our new, Orgueil meteorite samples; to Henri Coustau
of SNPA in Pau for collecting the soil and rock samples and C. W. Phillips of
the U.S. Army Chemical Corps, Fort Detrick, Aid. for providing the airborne
dust samples. The pollen samples were obtained from B. Siegel of the Brook-
lyn Jewish Hospital in New York. We wish to thank Professor Harold C.
Urey of the University of California for his encouragement and continued in-
terest.
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PANEL DISCUSSION
The Identity of the "Organized Elements"
H. C. Urey (Moderator; University of California, LaJolla, Calif.): In re-
viewing the events that led to this symposium, the moderator recalled that
approximately one year ago at The New York Academy of Sciences a presenta-
tion was made by Nagy el al. regarding the finding of what might be biogenic
hydrocarbons in the Orgueil meteorite. The moderator stated that although
he viewed their finding with skepticism at that time, he later looked over the
mass spectrometric data collected by the investigators and was sufficiently im-
pressed to suggest that additional analyses be run, such as infrared and ultra-
violet spectra. Once these suggestions had been carried out, the moderator
viewed microscopic preparations obtained from the Orgueil and Ivuna meteor-
ites that reminded him of biological matter. The moderator said he then posed
the following question to himself: Suppose these were living things, how did
they become imbedded in dolomite? In a subsequent published article, the
moderator suggested that these "organized elements" might be earthly forms
that had somehow been transferred from the earth to the moon in early geo-
logical times and later had returned to earth in carbonaceous meteorites.
Reactions among scientists to this theory varied; doubts were expressed, and
the moderator himself was (and still is) unsure of it. The moderator believes,
however, that the study of carbonaeceous meteroties for Ufe-like forms is not
an unreasonable pursuit, particularly when one considers that the United
States plans to spend some 25 billion dollars to put a man on the moon.
The moderator acknowledged, on the other hand, that he was also impressed
by the arguments of Fitch et al. that the "organized elements" might be merely
terrestrial contaminations, such as ragweed pollen. He noted, however, that
investagators Nagy, Claus, Meinschein, and Hennessy have been willing to
show their sample preparations freely and to solicit the opinions of others. He
noted, also, that they are enthusiastic; and while it is true that enthusiasm
may lead to errors, it is also true that lack of enthusiasm is not an especially
strong motivation for further work.
J. D. Bernal {Department of Physics, Birkbeck College, University of London,
London, England) : This discussant suggested that the problem be defined in
terms of the question, "What is it we are looking at?"; also that the problem be
approached in terms of all related subjects and that carbonaceous meteorites
be considered only as related to other meteorites. He raised the question of
whether carbonaceous meteorites represent the beginning or the end of the
development of meteorite bodies; the origin of these objects, he pointed out, is
an extremely important question. In his opinion, the "organized elements"
could be contaminations, "jokes of nature," or remnants of organisms. One
approach to the contamination problem is to determine whether anything could
have gotten into the samples since the meteorite fell on earth; another is to
ascertain whether the "organized elements" resemble any known biological
forms. If they are not contaminations, the burden of proof lies with Nagy
et al., and it is the biologists who must evaluate such proof. As for being "jokes
of nature," the "organized elements" might, for example, be mineral concretions
606
Panel Discussion: Identity of "Organized Elements" 607
that give the appearance of microfossils. Some of the Precambrian micro-
fossils shown earUer in the sessions might also be such particles. Bernal stated
that if one could prove that the Precambrian forms were indeed fossils, it
would perhaps aid in the identification of the "organized elements." Finally,
if the "organized elements" are neither contaminations nor "jokes of nature,"
one might then wonder whether anyone had "faked" them. Again the burden
of proof would lie with those who say that the "organized elements" are truly
indigenous fossils in the meteorites. If they are indigenous microfossils, what
would this mean? One must consider where these objects might have origi-
nated, whether life was brought to earth on meteorites, and where hfe might
first have arisen, on earth or on another body.
Among other considerations mentioned by Bernal was that the carbonaceous
meteorites might contain between six and eight per cent of organic matter, most
of which is definitely not terrestrial contamination. According to Meinschein
and Nagy, the meteorite hydrocarbons are products of life; but can we really
say this, or might they be abiotic matter from which life originated? Some
scientists even question whether petroleum hydrocarbons are of biological
origin. It was recalled that in an earlier paper in this symposium the syntheses
of biochemicals from abiotic sources was described; however, no mention was
made of lipids. Meinschein's and Nagy's evidence depends on Hpids, but
one does not known whether lipids can be produced abiotically.
Next, according to Bernal, there is the question of the mineral composition
of the meteorites. Water must have been present when these minerals were
synthesized. Were the serpentine minerals the decomposition products of
other silicates? The discussant pointed out that one fact is known: some of the
serpentine particles were euhedral. Mason postulated that the primitive
meteorite aggregated from dust particles, but the carbonaceous meteorites do
not fit into this picture. Sztrokay found veins filled with bituminous material
in the Kaba carbonaceous meteorite, which would suggest an elaborate chemical
history. All of this was very puzzling according to Bernal. One might ask
why it is necessary to bother with all this when in ten years time someone will
land on an asteroid and settle the question of extraterrestrial life; however one
must remember that the solution to this problem depends on many people,
specialists in their various fields, who must get together and coordinate their
efforts since no one man can settle this problem alone.
H. C. Urey: The moderator stated that the subject of extraterrestrial life
was of such great importance that it might affect scientific thinking about the
moon and, in fact, about the entire solar system. He did not, however, agree
with Bernal regarding the origin of meteorites. Moreover, to understand
better the "organized elements," one must also question what primitive life
forms would be like. Would they survive as long as present life forms do?
What would be the biochemical composition of primitive life forms? Would it
be the same as it is today? Would the porportions of amino acids be the same
as they are today? These, he felt, were all questions which would yet have to
be answered.
B. ]. Cholnoky (National Institute jar Water Research, Pretoria, Republic of
South Africa) : Cholnoky stated that the only important consideration is whether
there are or are not fife forms in carbonaceous meteorites. It matters not which
608 Annals New York Academy of Sciences
scientist is right or wrong. The problem is basically biological. To the biolo-
gist it should be of no importance, as far as identitication goes, whether the
"organized elements" came from meteorites, from outer space, or from some-
where else; all that should matter is whether the "organized elements" can be
identified as remnants of once living matter. Cholnoky emphasized that he is
a microbiologist, who has spent 51 years studying microorganisms. He is not
particularly interested, he stated, in fossils in meteorites, as such, but only in
life forms in general.
There were then two main points which he wanted to make: First, he ex-
pressed his surprise that physicists and chemists seemed willing to offer critical
evaluations regarding the biogenicity of organic microstructures. As a biolo-
gist, he would never think himself competent to comment on purely chemical
and physical problems. He suggested that physicists and chemists adopt a
similar attitude regarding biological problems. Consecjuently, he believed
that the suggestion put forth in an earlier paper by investigators from Florida
i.e., that protenoid coacervates may resemble living cells in appearance, must be
rejected. The methods of identification of these must be judged as insufficient;
any identification of strains of coacervates must be submitted to experts.
Although experiments with coacervates go back to the work of dejong, and were
designed to investigate vacuole formations, dejong never said anything about
cell walls.
Secondly, Cholnoky commented on claims that the "organized elements" in
carbonaceous meteorites were only grains of starch or pollen contaminations.
He said he has seen starch grains under microscopes on innumerable occasions
and could not identify the "organized elements" as starch grains. To argue a
point at meetings with photographic evidence was not satisfactory, since micro-
organisms are three dimensional and their morphology cannot be adequately
represented in two dimensional photographs.
Sidney W. Fox {Institute for Space Biosciences, Florida State University,
Tallahassee, Florida) : The discussant stated that he had heard the word Florida
mentioned, so he assumed Cholnoky's first point was in reference to his work.
He wondered if Cholnoky had made correct distinctions. The Florida group
works with microspheres, which can be separated by centrifugation; these
microspheres are more stable than the Oparin coacervate droplets. He wanted
to make another point, which he had forgotten to mention at the earlier session
that day. Should the micropaleontologists and meteorite investigators con-
clude that the "organized elements" were not fossils of micro-organisms, but
preprotobionata, i.e., a type of abiotic microspheres, then this would be an even
more significant finding, because it would indicate the discovery of precursor
organic particles from which life forms could have later evolved.
Robert Ross {Department of Botany, British Museum of Natural History,
London, England) : This discussant reported on his own studies of the Orgueil
meteorite:
The Orgueil meteorite fall consisted of about 20 stones. Two of the speci-
mens at the British Museum (Natural History) arrived there as complete
stones. He had studied one of these, which had not yet been examined for
"organized elements" by other workers. In straight crushed preparations, he
Panel Discussion: Identity of "Organized Elements" 609
did not find the large number of particles that he had expected to find after
reading the report by Claus and Nagy. Nevertheless, he did find a small
number of particles which, if found in terrestrial samples, he would have said
to be of biological origin. Two colleagues at the British Museum agreed that
these particular objects looked as if they might be of biological origin.
Plans have been made to conduct more refined experiments on these organic
particles. Electron microprobe analysis. X-ray microanalysis, and electron
microscopy are being contemplated. Certain additional experiments have
already been performed, however. In addition to the examination of straight
crushed preparations, density separations were carried out with aqueous cad-
mium borotungstate solutions; this liquid was used instead of the organic
liquids used by Nagy et al. It was thought that the use of inorganic liquids
would eliminate some of the criticisms raised against these investigators, i.e.^
that the "organized elements" were mere droplets of bituminous matter, dis-
solved and then precipitated from organic liquids.
A fragment of the Orgueil meteorite was used, and its surface was scraped off
with sterilized instruments. The sample in water was then subjected to re-
peated and prolonged freezing and thawing in an attempt to break up the
mineral matrix and to disintegrate the stone. This process was partially suc-
cessful. The disintegrated material was then suspended in cadmium boro-
tungstate solution and centrifuged. Four fractions were obtained, one of
which sank in liquids of 2.4 density. In the three light fractions, representing
densities of below 1.6, equal to 1.6, and between 1.6 and 2.4, a number of the
Type I "organized elements" of Claus and Nagy were found. In these density
range fractions, furthermore, there were also other objects, which resembled
collapsed spore membranes. Finally, two unusual forms were found in the
lightest fraction. Each of these objects consisted of a hollow tube, approxi-
mately 25/i long and X^in wide. The tubes contained an infilling •'^4m wide,
that had a refractive index different from the tube walls and quite different
from the Canada balsam in which these objects were mounted. The fillings
were probably air bubbles. One end of each of these tubes blended smoothly
into what looked like a torn piece of membrane, approximately lO^i wide. The
overall appearance of these forms, the tubes and the torn membranes together,
approximated a mushroom shape. These forms had been associated with the
meteorite matrix; the sterile procedures used suggested that they were not con-
taminations acquired during the study, but were part of the Orgueil meteorite.
The forms reminded Ross somewhat of the fossil hystrichospheres that Papp
had described earlier during the sessions. They might be parts torn from such
an organism. He concluded they were of biogenical origin. Claus and Nagy
had shown that the objects they found take up biological stains and resist
acids. These crude tests suggested that they consisted of carbon compounds.
All this evidence, he believed, adds up to a strong indication, but not proof,
that there are indigenous remains of living organisms in the Orgueil meteorite.
H. C. Urey: He expressed the opinion that Ross's findings were quite im-
pressive. He thought, however, that one might still wonder about what hap-
pens to living matter when it is fossilized for four and a half billion years; also,
what would the very earliest forms of life look like? He stated that it would be
610 Annals New York Academy of Sciences
significant if one could find many objects with only narrow variations in their
morphology, since the same types of organisms should not vary widely in
morphology.
George Claus {Department of Microbiology, New York University Medical
Center, New York, N.Y.): Claus stated that Type I of the "organized elements"
is by far the most common. Morphological variations, as well as size distribu-
tion of organisms, follow a Gausian distribution curve. "Organized elements"
follow the same pattern.
F. W. Fitch (Department of Pathology, University of Chicago, Chicago, III.) :
According to Fitch, many different kinds of particles described in Orgueil me-
teorite preparations have been called "organized elements." In his opinion,
one deals with a heterogeneous population of objects which can be divided into
two general classes — particles having a simple appearance and particles having
highly structured morphology. The rather featureless objects are numerous
but seem to have no specific properties indicating biological origin. Particles
having complex morphology are quite rare and some may have a biological
origin. However, there is no proof that they are not terrestrial contaminants.
Fitch wondered what would be adequate criteria for identifying the "organized
elements" as extraterrestrial forms having biological origin. He did not be-
lieve that morphology alone was adequate evidence. There are at least 250,000
plant species on the earth. It is impossible for any individual to be familiar
with more than a fraction of these and to identify isolated plant fragments.
There were no experts specializing in the study of pollen and of fungi at this
meeting, according to Fitch; therefore evaluation of the objects at this meeting
must necessarily be incomplete.
Bartholomew Nagy (Department of Chemistry, Ford ham University, New
York, N. Y.) : Nagy stated that because there were no experts on pollen at the
meeting, he and Claus took their microscopic preparations to a meeting they
attended during the previous week of the First International Congress of
Palynology at Tucson, Arizona. At this meeting there were approximately
300 experts on pollen from 22 different countries. The slides were exhibited in
public, and anyone who wished to examine them under the microscope could
do so. Approximately 80 specialists did so and to his knowledge, no one
definitely identified the "organized elements" as recent pollen contaminations.
Since, however, Anders et al. have argued that the "organized elements"
were ragweed pollen grains and starch grains, he thought it might be interesting
to recall Erdtman's comments on the "organized elements." Erdtman is a
Swedish pollen expert. His first impression was that the "organized elements"
were indeed pollens; however, after more careful examination, he concluded that
this was incorrect because they were similar to hystrichospheres, a pelagic form
of protobionta.
H. C. Urey: Urey noted that enthusiastic people can make mistakes, but a
mistake should not stop anyone. If one cannot identify these objects, one
should consult others. Photographs of objects projected on the screen do not
settle the question.
Rainer Berger (Lockheed California Company, Bjtrbank, Calif.) : Berger
agreed that undoubtedly more experimentation is needed. As Cholnoky
pointed out, biochemical tests could be inconclusive when applied to fossils.
Panel Discussion: Identity of "'Organized Elements" 611
For example, people buried in Pompeii in ash from the eruption of Vesuvius
apparently have no carbon left, because the tissues have been fully replaced by
mineral matter. Nobody doubts that they are remnants of people, yet bio-
chemical tests on them would give negative results.
Berger went on to say that one does not know what happens to meteorites
during their passage through the atmosphere. There is a question as to
whether air or air-borne pollen is sucked in. He wondered if it might be pos-
sible that the pollen could become imbedded in the meteorite and become
fossilized during museum storage. He also wondered how long it takes to
fossilize organisms.
Warren Meinschein (Esso Research and Engineering Company, Linden,
N. J.) : Meinschein's opinion was that it requires a long time to fossilize organ-
isms and it certainly requires water.
R. Berger: Berger wondered if there was enough water for this to occur in
the museum.
D. J. Hennessy {Department of Chemistry, Fordham University, Xew York,
N. Y.) : According to Hennessy, the issue at the present time was whether these
"organized elements" were terrestrial or extraterrestrial. Since Orcel in the
Paris Museum has large, single pieces of the Orgueil meteorite, perhaps he could
be persuaded to permit drilling into one with a sterile drill to obtain a sample
from the interior.
Edward Anders {The Enrico Fermi Institute for Nuclear Studies, University
of Chicago, Chicago, III.): Anders suggested that it was utterly misleading to
speak of "organized elements" as if they were a single, well-defined family of
particles with certain generic properties. Instead, it appeared that the or-
ganized elements fell into two sharply distinct classes. Particles of the first
class have a striking morphology, and most of them are probably biogenic.
However, they are quite rare, even in Nagy's samples, and they have not been
seen in the Orgueil samples studied at Chicago. Most of them show a strong
resemblance to common airborne contaminants, such as pollen grains, fly ash,
etc., and it seems hkely that most of them are in fact terrestrial contaminants.
Particles of the second class are probably indigenous to the meteorite. But
they seem to lack all other properties suggestive of a biological origin: their
morphology is nondescript, and resembles that of mineral grains; they do not
take biological stains, or take them atypically; they do not fluoresce in ultra-
violet light; they dissolve in acids; and they have nearly the same density as
the mineral grains in the meteorite. In view of these findings, Fitch and
Anders believed that two questions needed to be settled before all others. For
the particles of the first class, what is the evidence that they are not terrestrial
contaminants? And for the particles of the second class, what is the evidence
that they are not in fact mineral grains?
[Note added by the discussant in proof. Most of the evidence obtained since
the meeting has favored the view that the majority of the organized elements
are either contaminants or mineral grains. The spiny Type II elements
("hystrichospherids"), alleged by us to be ragweed pollen grains, have in fact
been identified as ragweed pollen by several pollen experts. The Type V ele-
ment ("dinoflagellate"), discovered by Claus and Nagy on a Gridley-stained
slide of Orgueil was shown to resemble Gridley-stained ragweed pollen (Fitch
612 Annals New York Academy of Sciences
and Anders, Science, 1%.^, in press). The particles of simple morphology,
which we said resembled mineral grains, do indeed have the chemical composi-
tion of limonite (hydrated ferric oxide), according to electron microprobe
analyses by Nagy, Fredriksson, Claus, Anderson, Urey, and Percy (Nature,
1963, in press), and they dissolve in acids without leaving a structured organic
residue (Anders and Fitch, Science, 1962, 138: 1392). The case for their bio-
logical origin now rests entirely on their featureless morphology.]
H. C. Urey: Ross's findings impressed the moderator. Ross had worked
with a complete stone which had probably been heated during passage through
the atmosphere. The moderator wondered whether there had been any signs
of contamination on Ross's sample, and whether it had been marked with
paint or mounted on wax.
R. Ross: The samples of the Orgueil meteorite in the British Museum were
kept in a box with a glass cover on it. There were no precautions taken during
the years to keep them sterile in storage. He had used, however, sterilized
instruments to work with the samples. He had also scraped away exposed
surfaces of the meteorite with sterile instruments before taking a sample, and
before placing the samples into water and subjecting them to freezing and
thawing.
P. Morrison {Department of Physics, Cornell University, Ithaca, N. Y.):
Morrison stated that he would like to see a count of the relative distribution
of the "organized elements."
G. Claus: According to Claus, "organized elements" were present in Orgueil
on the average of 1700 per milligram. Type I was by far the most common and
on one side Type II represented approximately five per cent of the total count.
Claus noted, regarding the staining, that there are many "organized elements"
which do not take stains.
H. C. Urey: This was a puzzle to Urey, and he wondered if the simpler ones
could be artifacts and the more complicated ones, contaminations.
Paul Tasch {Department of Geology, University of Wichita, Wichita, Kansas) :
Tasch observed that, exclusive of magnetic particles, the "organized objects"
found in some carbonaceous chondrites fall into three classes: (1) terrestrial
contaminants in addition to those already cited by Claus and Nagy; (2)
proteinoid microspheres of Fox or organic-chemical analogues of Morrison; and
(3) indigenous microfossils. He believed that allowance for (1) and (2) had
been made, but (3) still remained to be explained. The discussant had observed
three distinct objects in Claus and Nagy's thin sections. Two of these were on
display at the International Palynological Conference at Tucson, Arizona, and
one was presented by Claus in his talk. The discussant's first impression of
one of these objects embedded in salt was that it resembled a chrysophyte;
another object, named Daidaphore Berzelii, had a hystrichospherid-like organi-
zation. A third object appeared to have antapical horns and a girdle, thus
suggesting a dinoflagellate.
Ross, according to Tasch, had reported how he isolated a distinct object from
a carbonaceous meteorite, and had indicated his conviction that it was not a
contaminant, but resembled a process of hystrichosphere.
Tasch also pointed out that F. L. Staplin of Imperial Oil, Ltd. processed a
sample of the Orgueil meteorite and wrote a report, soon to be published, which
Panel Discussion: Identity of "Organized Elements" 613
the discussant had ah-eady seen in a preprint. Staplin found, after palynologi-
cal processing of the sample, a group of micro-objects in the fmal residue. In
his judgment, these were the closest in affinities to a hystrichosphere-leiosphere
assemblage. Among the objects, he also found some chrysophytes.
Now, continued Tasch, these three observations, in addition to those of
Claus, all seemed to be consistent.
Tasch suggested that to advance this discussion the problem of contamina-
tions be bypassed and the objects found in the meteorites be accepted as in-
digenous. What would then follow? Pelagic protists closely resembling
terrestrial types must have lived on the parent body; once this is admitted,
then it follows that there were water bodies in which they lived. In addition,
there must also have been a supply of phosphorous, nitrogen, and other nutrient
substances. That, according to Tasch, would be as far as paleobiology can
take it. It would then be necessary for Urey, Bernal, and others to explain
where it is possible for such water bodies to exist.
A. Papp {Department of Paleontology, University of Vienna, Vienna, Austria) :
Papp emphasized that the basic rule of the natural sciences is that an experi-
ment must be repeatable before one can accept the tindings as valid. Research
on the "organized elements" started only a short while ago, yet the experiments
had already been successfully repeated, and independently, by Ross in England,
Staplin in Canada, and Skuja in Sweden. Now the Anders group came up with
negative results. A most important cjuestion would be whether any one else
has found the "organized elements" besides Nagy et al; the answer to this is
yes. They were found in England, in Canada, in Sweden and, according to
Papp, one would venture to say they were also found in Chicago.
There is then the question as to the differences in yield; the answer to this
would be that the greatest number of objects were found by those who did
most of the work. Papp illustrated this by pointing out that in two kilograms
of sedimentary rock one would find more microfossils than in only one gram of
rock. It would seem that the problem of differences regarding the number of
"organized elements" would be related to the relative amounts of time spent on
the problem by the different investigators.
With respect to attempts to identify and to classify these forms, Papp con-
cluded that it was unimportant whether they look somewhat like dinoflagel-
lates or something else. They are something different, and it would be impos-
sible to include them in terrestrial systems. One could only compare them with
terrestrial forms and state that they resemble certain terrestrial species, and
even with this, one would be saying very much.
In summary, Papp beUeved that the "organized elements" had been proven
to be organic and that their organized nature had been confirmed independently
in four countries. He was impressed that "organized elements" were not
identical to, but only resembled terrestrial organisms and, therefore, he con-
sidered that the question of their origin was closed.
H. C. Urey: He and his associates in Lajolla could not find anything in their
sample of Orgueil. They then sent their samples to Nagy and the "organized
elements" were encircled on the slides and returned to them. They still could
not find one. Finally, however, a technician did find an "organized element."
A. Papp: Six months previously one of Papp's colleagues, a mineralogist in
614 Annals New York Academy of wSciences
Vienna, had said that the "organized elements" were mineral spherolites.
Papp stated that this only showed that one needs extensive trainmg in micro-
biology to recognize these forms.
H. C. Urey: Urey mentioned that Volcani, the microbiologist at Lajolla,
was not discouraged.
C. M. Palmer {Division of Water Supply and Pollution Control, Robert A.
Taft Sanitary Engineering Center, Cincinnati, Ohio) : He stated that it should be
mentioned that Claus was capable of observing and finding structural details
that other people often overlooked; i.e., details on filamentous algae which had
never been seen, although the forms had been known for more than 100 years.
Pierre Bourrelly (Department of Cryptogamic Botany, Miisee d'Histoire
Naturelle, Paris, France) : Bourrelly saw the microscopic preparations. He
believed that the "organized elements" were definitely organisms. They did
not look much like hystrichospheres, and they did not seem to be contaminants.
He was puzzled because they resembled terrestrial forms; he thought they should
exhibit greater differences.
R. Berger: He recalled that Papp had implied that the terrestrial evolu-
tionary sequence might have occurred elsewhere. There might be an equiva-
lent biochemistry which would lead to similar organisms.
J. D. Bernal: He expressed the opinion that the carbonaceous meteorites
could not be of terrestrial origin because of their unusual mineral content.
If they were not terrestrial, he questioned what the origin might be. Only
Earth and Mars are capable of holding water; therefore, if the "organized
elements" arose elsewhere than on earth, one would be forced to choose between
two possibilities: that the same biochemistry is prescribed for every origin of
life and, therefore, life always follows the same trend; or that all life forms
originated from the same ultimate life source. In other words, one is faced
with these questions: did life originate in several places as a result of the same
biochemical mechanism, or did Ufe evolve only once and then spread to different
places? He added that four billion years might not be a sufficient amount of
time for biochemical evolution on earth.
H. C. Urey: According to Dr. Urey, life might have transferred to the moon
from earth.
P. Tasch: He was one who considered the moon transfer theory a serious
possibility, though a difficult one to accept.
H. DoMBROWSKi {Department of Balneology, Justus-lJebig University
Giessen, Germany): Dombrowski stated that there is an analogy between the
problem of "organized elements" and his work obtaining living bacteria from
salt deposits. Chemists have known for a long time that salts contained less
than 0.01 per cent of nitrogen. The origin of this nitrogen could not be ex-
plained until biologists started to work on salt samples. Now it is known that
this nitrogen content is associated with the bacteria embedded in the salt.
Brian Mason {Department of Mineralogy, The American Musuem of Natural
History, New York, N. Y.) : Mason thought that it would be very difficult not
to contaminate a meteorite in a museum. The American Museum of Natural
History acquired its Orgueil sample in 1901, but it is not known what happened
to it before this date. The sample was kept in an open box. It should be
Panel Discussion: Identity of "Organized Elements" 615
mentioned that magnesium sulfate in the meteorite in New York is MgS04-
4H2O; in Chicago it is MgS04- 7H2O. This would only show how the environ-
ment can effect the "organized elements."
J. D. Bernal: He mentioned that the Orgueil sample in the Bombay Mu-
seum in India fell to a fine dust because of the humidity.
W. G. Meinschein: The hydrocarbon distribution in Orgueil resembled
biogenic hydrocarbons in Recent marine sediments. Mass spectra showed
that these hydrocarbons could not have been contaminated by accident, much
less by intent. The hydrocarbon concentrations were above Recent sediment
concentration levels.
B. Mason: This discussant was of the opinion that the organic matter in
Orgueil was clearly indigenous, but that one must prove that it was not formed
by inorganic processes.
W. G. Meinschein: It was emphasized by Meinschein that 23 saturated
hydrocarbon compounds had been identified. The parent sterol hydrocarbons
were present and the aromatic hydrocarbons were those which are found in
Recent sediments. Clearly, he believed, they were of biogenic origin.
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