UNIVERSITY OF CALIFORNIA PUBLICATIONS
IN
AGRICULTURAL SCIENCES
Vol. 1, No. 7, pp. 141-172 April 25, 1914
STUDIES ON AMMONIFICATION IN SOILS
BY PURE CULTURES
BY
C. B. LIPMAN and P. S. BURGESS
The study of the physiological efficiency of soil bacteria
rather than their number is admittedly the dominant method in
soil bacteriological investigations. In view of this fact it is
singularly striking to note how little work has been accomplished
in the study of some phases of the physiological efficiency of pure
cultures of certain groups of soil bacteria. This is especially so,
since the introduction of radical changes in our soil bacterio-
logical methods have made it necessary to repeat some, if not all,
of the work which had been carried out by the old methods.
However this may be, it remains a fact that, since the publication
of MarchalV splendid work on ammonification in solution
cultures, but scant information has been adduced from studies
of soil bacteria which relate to the physiology as well as the
physiological efficiency of even the more common ammonifying
bacteria. Certainly, the work carried out along this line in
direct soil cultures, which recent work has shown to be so far
superior to solution cultures, has been very meager indeed.
In order, therefore, to glean some useful information relative
to the physiological efficiency of pure cultures of a number of
ammonia-producing bacteria, the writers deemed it wise to select
a number of organisms and to compare their power to produce
ammonia not only from one form of organic matter, but from
several forms, most of which have found use in farm prac-
tice as fertilizers. Accordingly, the following organisms in pure
142 University of California Publications in Agricultural Sciences [Vol. 1
culture were selected for the experiments : B. mesentericus vul-
gatus, Ps. putida, B. vulgatus, B. megatherium, B. mycoides, B.
sub t His, B. tumescens, Sarcina lutea, B. proteus vulgaris, B.
icteroides, B. ramosus, Streptothrix, sp., Ps. fluorescens, B. vul-
garis (Novy strain), Mic. tetragenus. The organic materials
experimented with were dried blood (13.16% N), tankage
(9.62% N), cottonseed meal (5.5% N), sheep and goat manure
(2.13% N), peptone (14.14% N), fish guano (8.63% N), and bat
guano (3.96% N).
Method of Experiments
Fifty-gram portions of soil were placed in tumblers and
thoroughly mixed with the organic material to be tested. The
tumblers were covered with Petri dish covers and sterilized in
the antoclave at a pressure of thirty pounds for three hours.
After cooling, the soils were each inoculated with a 1 cc. suspen-
sion of the organisms to be tested, made up by shaking with some
sterile water a young slope culture grown on bouillon agar.
The soil was then stirred with a sterile spatula after enough
sterile water had been added to make a moisture content in the
soil about equal to the optimum. The soil cultures thus pre-
pared were incubated at 28° to 30° C for twelve days. After
the incubation period the soils were transferred to copper dis-
tilling flasks, 400 cc. of distilled water and an excess of Mg O
added, and distilled into standard H/10 HC1. The ammonia
was then determined in the usual way.
No attempt was made to run all the series with the different
forms or organic matter at the same time, because only the
relative powers of the different organisms to produce ammonia
were sought. For the same reason amounts of organic matter
were chosen in the different series which would least affect the
physical conditions obtaining in the cultures rather than amounts
employed which would make the total amount of nitrogen added
the same in all scries.
For the reasons above given, therefore, the effects of the
various organisms on any given form of organic material will
be treated below as a separate series in the case of each soil and
1914] Lipman-Burgess : Ammoniftcation in Sails by Pure Cultures
143
comparisons between the different series made only where per-
missible. The data, moreover, are presented so that the dupli-
cate determinations which were carried out in all cases may be
compared. The averages of duplicate determinations, however,
are also given as well as the percentages of nitrogen in the
organic matter which was transformed to ammonia.
Three different California soils were tested with each one of
the ammonifiable materials. The soils were a sandy soil from
Anaheim, a clay loam from Davis, and a black clay-adobe soil
from Berkeley. The mechanical and chemical analyses of these
soils are given in Tables I and II which follow.
TABLE
I
Mechanical Analyses op Soils
Hyd. value
Sandy
Clay-Loam
Clay-Adobe
Clay
5.78
19.12
31.93
0.25 mm.
14.59
40.93
25.77
0.50
1.04
3.35
3.40
1
2.30
6.60
4.77
2
5.28
7.75
7.49
4
9.62
8.78
6.20
8
11.58
8.10
.87
16
4.87
3.30
2.78
32
15.23
4.15
7.66
64
29.40
TABLE
II
3.07
5.44
Chemical Analysis
of Soils
Sandy
Clay-Loam
Clay-Adobe
Insoluble matter
73.59
53.55
I 77.84
Soluble
silica
11.17
19.77
K20
.64
.75
.45
Na20
.15
.11
.07
CaO
1.39
.82
1.05
MgO
.93
1.39
1.21
Mn304
.04
.04
.08
Fe203
5.10
7.56
1.68
A1203
3.92
10.04
7.79
PA
.12
.13
.23
S03
.02
.03
.08
Water and
organic
matter
2.88
5.62
5.72
144 University of California Publications in Agricultural Sciences [Vol. 1
The pure cultures of the organisms employed in these investi-
gations were obtained by one of us from the bacteriological
laboratories of the University of Illinois. Fresh strains of
B. subtilis and B. mycoides were, however, isolated by us from
California soils in order to check the stock cultures of the same
organisms. The same relative powers of producing ammonia
were, however, found to obtain with the fresh as with the old,
and different strains thus strengthening the validity of the
results below discussed.
Series I. Experiments with Dried Blood
Sandy Soil
Two per cent of finely sifted dried blood was added to the
soils in this series, or one gram per fifty grams of soil. In the
case of the sandy soil three series in duplicate were tried, and the
same relation under different conditions having been found to
obtain between the ammonifying powers of the different organ-
isms, we give only one duplicate set of the determinations. The
incubation period was twelve days throughout at a temperature
varying between 27° C and 30° C. The results of the ammoni-
fication determinations with all the soils using dried blood
throughout are given in Table III. The percentage of nitrogen
of the total amount added which is made available is also given
in every case.
The data in Table III most strikingly indicate the superiority
of Ps. putida, B. vulgaris and Sarcina lutea to all other organisms
in their efficiency at the production of ammonia from the nitrogen
of dried blood. The next fact of singular interest is that B.
mycoides, which has, in the hands of several investigators, shown
such marked superiority over other ammonifying organisms in
the production of ammonia from organic nitrogen in solutions,
manifests a relatively low power to transform the nitrogen of
dried blood in soil cultures into ammonia. This, moreover,
cannot be considered accidental, since different strains of B.
mycoides, as above explained, exhibited that same lack of vigor in
three duplicate sets of cultures run at different times and under
slightly varying conditions of temperature and period of incuba-
1914] Lipman-Burgess : Ammonification in Soils by Pure Cultures
145
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146 University of California Publications in Agricultural Sciences [Vol. 1
tion. Of the three organisms showing the highest efficiency, as
above indicated, in transforming the nitrogen of dried blood to
ammonia. B. vulgaris appears in its turn to be the most vigorous,
though the other two approach it closely and are about equal
among themselves.
Clay-Loam Soil
"When the clay-loam soil is used as the medium with dried
blood, marked differences are apparent in the efficiency of all
the organisms. The poorer air supply of the clay-loam soil, due
to its fineness and much greater tenacity, are evidently inimical
to ammonia production, even though the same source of nitrogen
— dried blood — is supplied for the ammonification process. The
largest amount of ammonia produced in this series was that by
B. proteus vulgaris, and even that was little more than one
quarter of the amount produced by B. vulgaris as above noted
in the sandy soil.
Moreover, the most efficient transformers of the nitrogen in
dried blood in the sandy soil medium are not necessarily the
same as those in the clay-loam soil. For example, in the case of
the clay-loam soil B. proteus vulgaris is the most efficient
ammonia producer with dried blood as ammonifiable material,
while in the sandy soil the same organism manifests less than half
the ammonifying efficiency of B. vulgaris. This latter organism,
however, stands second to B. proteus vulgaris in efficiency in
the clay-loam soil and yields 7.49 mgs. of ammonia nitrogen as
against 9.10 mgs. produced by the last-named organism. There
appear to be four other organisms which approach the efficiency
of the two just discussed in the clay-loam soil in the following
order: Mic. tetragenus, B. ramosus, Streptothrix, sp., and Ps.
putida. While the latter does not compare in efficiency in the
clay-loam soil with that shown by it in the sandy soil, it still
manifests a notable efficiency. Sarcina lutea, however, appears
to have lost in the clay-loam soil the marked ammonifying power
possessed by it in the sandy soil.
Of course il must be again emphasized that the amounts of
ammonia produced by all the organisms in the clay-loam soil
1914] Lipman-Burgess : Ammonification in Soils by Pure Cultures 147
series are relatively so small that the margin allowable for safe
comparison must of necessity be much decreased and therefore
comparisons are more difficult.
Clay- Adobe Soil
Passing on to a study of the data obtained with the clay-adobe
soil as a medium, we find again that he physical condition of the
soil is a powerful factor in determining the amount of ammonia
produced in soils by pure cultures of organisms possessed of
ammonifying powers, if the fifteen different organisms used are
a suitable criterion. This confirms the findings of J. G. Lipman
in his long series of ammonification experiments with mixed cul-
tures. For practical purposes, we may add that most of the
bacteria ammonify dried-blood nitrogen equally well in the clay-
adobe soil and in clay-loam soil, though there does appear to be
a slight though consistently greater amount of ammonia produced
in the first-named soil. Again, we find in the clay-adobe soil
an organism which stands out as far superior to all others in
ammonifying efficiency and again also it is not the same organism
as manifested that superiority in the preceding soil. While the
duplicate determinations here do not agree as well as might be
desired, they indicate amounts so much greater than the quan-
tities of ammonia produced by the other organisms of the series
that there can be no doubt of the marked and superior efficiency
of B. tumescens as an ammonia producer from the nitrogen of
dried blood in the clay adobe soil as a medium. Two other
organisms appear to be in the second class in this series and they
are Mic. tetragenus and B. mycoides. The first, it can be seen
from Table III, occupied third place in efficiency in the clay
loam soil, but the second has thus far been relatively inefficient.
All the other organisms of the series do not manifest differences
in efficiency of sufficient magnitude to warrant further comment,
except that it is curious and interesting to note that the most
efficient organism in the sandy soil is the least efficient organism
in the adobe soil.
It should be remarked here that the generally excellent agree-
ment between duplicate determinations, as shown in the table.
148 University of California Publications in Agricultural Sciences [Vol. 1
eliminates the fear that physical conditions in the individual
cultures might operate to produce the rather marked effects noted.
In a comparison of the three soils it appears that the follow-
ing organisms are among those which show the highest efficiency
in transforming the nitrogen of dried blood into ammonia: Ps.
putida, Sarcina lutea, B. vulgaris, B. proteus vulgaris, Mic.
tetragenus, B. tumescens. Of these organisms only Mic. t et ra-
ge nus shows a high efficiency in all three soils. Of the others
B. vulgaris shows a high efficiency in both the sandy and adobe
soils and Ps. putida in the sandy and clay-loam soil. The rest
namely Sarcina lutea, B. proteus vulgaris and B. tumescens, are
markedly efficient only in one soil each, namely, the sandy soil for
the first, the clay-loam soil for the second, and the clay-adobe
soil for the third. It appears to us particularly worthy of note,
also, that of the last three organisms named B. proteus vulgaris
and B. tumescens each holds a pre-eminent position of efficiency
in its own soil which no other organism of the fifteen has
approached; and even in the case of Sarcina lutea we find that
it occupies a position in efficiency in its favorite soil which is
second only to that of B. vulgaris and not very far behind the
latter. This rather remarkable condition would hardly seem to
be accidental and appears to us to indicate for certain organisms
marked preferences for certain physical characteristics in media
in which they are grown.
It must be added here, too, that only six of the fifteen
organisms show marked ammonifying efficiency as regards the
nitrogen of dried blood even when tested in three widely different
soil types. The others vary but little from one another in all
soils. Oddly enough, the organism which has shown the highest
ammonifying efficiency because it maintained it through all soils,
namely, Mic. tetragenus, has never before been looked upon, so
far as we are aware, as an important ammonifier. That it should
be the only one of fifteen organisms tested which should be about
equally efficient in all soils used is not unworthy of note.
Tt is further significant to note here that B. mycoides, which
has always been regarded as one of the most efficient soil organ-
isms at ammonification, does not in our experiments show any
unusual activity in that direction, at any rate when dried blood
1914] Lipman-Burgess : Ammonification in Soils by Pure Cultures 149
is used as the ammonifiable material, no matter what the soil
medium. A possible exception to this statement may be found
in the case of the adobe soil, in which B. mycoides seems to be
superior to all but three or four of the fifteen organisms tested.
Such superiority, under the circumstances noted, is probably of
little significance.
Series II. Experiments with Tankage
This series was arranged in a manner similar to the preced-
ing, the sandy, clay loam, and clay-adobe soils again being used
as media, but the ammonifiable material in this case was a high-
grade tankage, the nitrogen content of which was 9.62 per cent.
The results of the ammonia determinations were as shown in
Table IV on page 150.
Discussion of Series II
The Sandy Soil
When we study in the foregoing table, the ammonification of
tankage by pure cultures of bacteria, and compare the results
with those of Table III, we see at once some very striking differ-
ences between the ability of the same micro-organisms to produce
ammonia from tankage and from dried blood respectively. Not
only do more of the organisms show a high efficiency in trans-
forming the tankage nitrogen to ammonia, but the point of
highest efficiency is not reached by the same organisms as before,
others having taken their places in this series with sandy soil as
the culture medium. For example, we find that B. mesenteric us,
which in the preceding series showed throughout an extremely
low ammonifying efficiency even in the sandy soil, now manifests
in the same culture medium, which, however, has tankage in
place of blood added to it, the highest efficiency of all of the
organisms tested. Indeed, it occupies a position of its own in
that direction, much as does B. vulgaris in the sandy soil of the
last series. The organism which approaches it most closely in
the same medium is B. proteus vulgaris which, however, falls 25
per cent short of producing the amount of ammonia yielded by
150 University of California Publications in Agricultural Sciences [Vol. 1
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1914] Lipman-Burgess : Ammonification in Soils by Pure Cultures 151
B. mesentericus. Then follow, not far behind B. proteus vulgaris,
B. tumescens, B. ramosus, and B. vulgaris in the order named,
the latter being more than 33% per cent short of the efficiency
exhibited by B. mesentericus. Nearly all of the other organisms
fall more than 50 per cent short of the efficiency of the last-
named organism under this set of circumstances, namely, sandy
soil with tankage. There are, therefore, but five organisms out of
the fifteen tested which can be adjudged distinctly efficient
ammonifying organisms under these conditions. B. mycoides
again exhibits a low efficiency, and not far different from, though
slightly below, that manifested by it in the same soil in the dried-
blood series.
It must be added here that, with the exception of B. mesen-
tericus, the efficient organisms in this part of Series II have also
shown more or less marked efficiency in the preceding series.
The Clay-Loam Soil
Most of the organisms tested in the clay-loam soil seem to
have found the latter a more congenial medium for ammonifica-
tion with tankage than they did when dried blood was present.
In this part of Series II, three of the organisms, namely, B.
mesentericus, B. vulgatus, and B. tumescens, were not only the
most efficient ammonifiers but also about equal in their ammoni-
fying power. It appears, therefore, that so far as tankage is
concerned B. mesentericus is an equally efficient ammonifier in
the clay-loam and sandy soils. B. tumescens has previously
established its pre-eminent position among the fifteen organisms
as an ammonifier of dried-blood nitrogen in the adobe soil, but
B. vulgatus enters here for the first time as a markedly efficient
ammonifier.
Only slightly behind the three organisms just discussed in
their ammonifying efficiency as regards tankage nitrogen in the
clay-loam soil are, in the order named, B. subtilis, B. mycoides,
and B. vulgaris. While the latter has manifested its high
efficiency in other series above described, the first two organisms
named for the first time in the work thus far described show
marked ammonifying ability. Only three organisms in this part
152 University of California Publications in Agricultural Sciences [Vol. 1
of Series II have shown themselves to be really weak ammonifiers,
and they are B. proteus vulgaris, B. icteroides, and Mic. tetra-
genus. The first and the third of these, it will be remembered,
have given evidence of marked efficiency under other circum-
stances, but the second has thus far been throughout an organism
of low efficiency. It is striking to note the much greater uni-
formity which exists in this portion of Series II in the ammoni-
fying powers of four-fifths of the organisms tested than that
which obtains in other parts of this series and of other series.
The Clay-Adobe Soil
Here again we find the great uniformity in ammonifying
power between the larger number of bacteria tested which is
characteristic of the foregoing section of Series II. On the other
hand, the clay-loam soil seems to have been a more congenial
medium than the adobe soil for the ammonification of tankage
nitrogen, for larger amounts of ammonia are produced in it by
the same organisms in the same period of incubation.
Sarcina lutea shows the highest efficiency as an ammonifier
in this part of the series, but is only slightly more efficient than
B. tumescens. The organism taking third place is B. mycoides
which, indeed, is not far behind the other two. Next in order of
importance and still very efficient ammonifiers are B. subtilis and
Streptothrix, sp. The other organisms are considerably weaker
ammonifiers than those just mentioned and B. icteroides again
proves to be distinctly the weakest. Both Sarcina lutea and
B. tumescens have, as mentioned above under other circum-
stances, plainly evidenced their high efficiency as ammonifiers
and B. mycoides and B. subtilis have taken similar positions with
respect to tankage in the clay-loam soil. Streptothrix, sp., it will
be remembered, has also shown a high ammonifying power before
in the case of the clay-loam soil when dried blood was used.
Comparing the three soils in this series with the same ones
in the preceding series, it appears quite clear that taking them
by and Large, ammonifying bacteria manifest a much higher
efficiency with high-grade tankage than with dried blood under
similar conditions. Likewise also, in Series II a small number of
1914] Lipman-Burgess: Ammonification in Soils by Pure Cultures , 153
organisms mostly identical with those in Series I seem to mani-
fest a distinctly high efficiency which the much larger number
of the balance do not in most cases even approach. It is worthy
of remark, moreover, that B. mycoides attains or rather ap-
proaches in portions of Series II its position of prime importance
which has thus far so generally been accorded it among ammoni-
fying organisms.
Series III. Cottonseed Meal
The cottonseed meal used in this series showed on analysis a
nitrogen content equal to 5.5 per cent. The experiment in this
series was otherwise conducted like those of Series I and II,
2 per cent of cottonseed meal being used, or 1 gram per 50 grams
of soil. The results are shown in Table III.
Again the efficiency of the organisms tested is much greater
in the sandy soil than in either the clay-loam or adobe soils.
Sandy Soil
In this part of Series III the organism of greatest ammonify-
ing efficiency is B. tumescens. Those approaching it closely in
efficiency are B. vulgatus, Sarcina luiea, and B. mycoides in the
order named. B. ramosus takes fifth place and the next three
organisms fall more than 33 % per cent short of producing the
amount of ammonia yielded by B. tumescens. They are B.
mesentericus, B. megatherium, and B. proteus vulgaris. The
balance of the organisms show only about half the efficiency of
B. tumescens and in one case, B. icteroides, which has in all pre-
vious series shown a very low efficiency, only about 25 per cent
of the maximum efficiency is manifested. Taken as a whole, the
data given in Table III for the Anaheim sandy soil reflects
favorably on cottonseed meal as a source of available nitrogen
under the conditions named. Again, the greatest efficiency is
manifested in this section of Series III by organisms which in all
cases have shown high efficiency in other series above reported.
As in the case of the heavy soil in the preceding series with
tankage, B. mycoides also shows marked efficiency in the light soil
when cottonseed meal is used.
154 University of California Publications in Agricultural Sciences [Vol. i
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1914] Lipman-Burgess : Ammonification in Soils by Pure Cultures 155
The Clay-Loam Soil
The most notable thing in this part of Series III is as above
intimated, the very low efficiency of all of the organisms tested.
Indeed only two organisms manifest any notable activity as
ammonifiers of nitrogen in cottonseed meal in the clay-loam soil
as a medium. These two in the order of their importance are
B. ramosus and Streptothrix, sp. To these, in view of the dis-
agreement of the duplicates as above shown, B. vulgaris may
probably be added, and perhaps also B. vulgatus. The other
organisms are all distinctly below the first two mentioned, and
the lowest efficiency thus far noted is that exhibited by Mic.
tetragenus in this soil. Considering the high efficiency of the
latter organism in Series I, the results just discussed are puzzling.
The Clay-Adobe Soil
Even a casual glance at the data obtained in the clay-adobe
soil as a medium indicates the distinct superiority of that medium
to the clay loam soil for ammonification of the nitrogen of cotton-
seed meal. Not only relatively but absolutely the data obtained
show the production of much larger amounts of ammonia in this
portion of the work.
As is the case in the sandy soil with cottonseed meal as the
ammonifiable material, B. tumescens shows its distinct superiority
to the other organisms as an ammonifier in the adobe soil. The
next most efficient organism is B. subtilis, which, however, is con-
siderably less efficient ; and the next two organisms, about as far
below B. subtilis in efficiency as the latter is below B. tumescens,
are B. ramosus and B. mycoides. The other organisms are all
low in efficiency, though in nearly all cases absolutely better than
the same organisms in the clay-loam soil. Thus far we find that
B. subtilis shows itself markedly efficient, for the first time, in
the clay-adobe soil with cottonseed meal. All the other organisms
above named have manifested marked efficiency in some parts
of the foregoing series.
Looking at Series III as a whole it is interesting to note that
the nitrogen of cottonseed meal seems to be made available
through the activity of pure cultures of ammonifying bacteria,
156 University of California Publications in Agricultural Sciences [Vol. 1
with much greater rapidity than has heretofore been believed.
The next striking fact brought out in these results is the clearly
indicated superiority of the clay-adobe soil to the clay-loam soil
as a medium for the ammonification of the nitrogen in cottonseed
meal. The third point worthy of mention in Series III is the fact
that we find again in it, as in the preceding series, only a few
of the fifteen organisms tested which show marked ammonifying
efficiency.
The culture of Ps. fluorescens died in the midst of these
investigations and was not replaced.
Series IV. Fish Guano
Fish guano, in accordance with the teachings of Voorhees and
other agricultural chemists, has always been esteemed a good
source of available nitrogen; in the words of Voorhees,2 "rank-
ing in availability well up to blood and tankage." It seemed to
us therefore of importance to compare in these pure culture
studies fish guano with the other organic materials discussed
above. Accordingly a series was started similar to those above
described, except that 1% grams of finely sifted fish guano was
the ammonifiable material used per 50 grams of soil. The fish
guano used contained 8.63 per cent nitrogen. The results
obtained are shown in Table VI.
The data in Table VI not only seem to confirm the opinion of
Voorhees as above stated, if ammonia production by pure cul-
tures may be taken as a criterion for determining the availability
of fish guano, but they indicate in most striking fashion what
was not shown in any of the foregoing series for the fertilizers,
and particularly in the sandy soil, namely, the obliteration of
the marked physiological differences obtaining in other series
between the different organisms. Of the four nitrogenous fer-
tilizers thus far discussed, fish guano seems to contain the form
of nitrogen most generally ammonified by a large group of
bacteria.
1914] Lipman-Burgess : Ammoniftcation in Soils by Pure Cultures 157
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] 58 University of California Publications in Agricultural Sciences [Vol. 1
The Sandy Soil
The good ammonifying power manifested by most of the
organisms tested in this soil with fish guano finds its maximum
in the case of B. vulgaris, which, it will be remembered, has
already given evidence of notable ammonifying efficiency with
other nitrogenous fertilizers. B. tumescens, likewise an organism
with a record as an ammonifier well established, is a very close
second to B. vulgaris in its efficiency at the ammonification of the
nitrogen in fish guano. Four other organisms distinctly in the
second class, and about alike in ammonifying efficiency in this
part of Series IV, are, in the order of their efficiency, Ps. putida,
B. vulgatus, B. ramosus, and Sarcina lutea. Peculiarly enough,
B. icteroides shows an ammonifying efficiency here which places
it in the second class, a position Which it has thus far never even
remotely approached in the series of experiments above discussed,
nor, for that matter, in other series which are described below.
All other organisms in this part of Series IV are distinctly in
the third class, but nevertheles manifest notable ammonifying
efficiency.
In no other series of results have we obtained such sharp
lines of demarcation between the classes of organisms here tested,
and arbitrarily grouped, in accordance with their respective
powers of transforming organic nitrogen into ammonia.
The Clay -Loam Soil
The change in the physical condition of the soil from the sand
to the clay loam shows a marked effect on the ammonifying
power of the same organisms. Nevertheless we find, on the whole,
the best set of results thus far obtained with the clay loam when
the series with fish guano is studied. In a class by itself under
these conditions is B. megatherium, which is markedly superior
to all other organisms in this part of Series IV, except B. mesen-
tericus, which is a close second. The organism, however, which
takes third place, B. vulgaris, falls about 20 per cent short of
m11 fining the efficiency of B. megatherium under these conditions.
Distinctly lower in efficiency in this part of Series IV are, in
the order of their importance, but only slightly different from
1914] Lipman-Burgess : Ammonification in Soils by Pure Cultures 159
one another, B. vulgatus, Ps. putida, B. ramosus, and B. tumes-
cens. All of these, however, fall more than 30 per cent short of
attaining the ammonifying efficiency of B. megatherium under
the conditions here considered. Two other organisms, B. subtilis
and Sarcina lutea, fall 40 per cent short of the efficiency of B.
megatherium, and the others all fall far below even that figure.
It is singular, here again, .that the physical nature of the soil
medium employed should so strikingly and so variously influence
the efficiency of the ammonifying bacteria. The two organisms
which are distinctly superior in ammonifying ability as regards
the nitrogen of fish guano in the clay loam soil were only of
moderate efficiency with the same form of nitrogen in the sandy
soil. And again, the organism which in the latter soil was
paramount in its position retreats in the clay-loam soil to third
place.
The Clay-Adobe Soil
This is the first series of those we have thus far considered,
as the data in Table VI shows, in which the clay-adobe soil proves,
on the whole, to be inferior to the clay-loam soil. Seven organ-
isms out of fourteen here show an extremely low ammonifying
efficiency, and two others are by no means efficient organisms.
That leaves five organisms in this group which may be considered
of importance. Of these, B. vulgaris is the most efficient, but
B. tumescens is not far behind it. B. subtilis belongs to the
second class in this group and B. proteus vulgaris and B.
mycoides to the third class. It is rather unfortunate that it was
not possible in this part of Series IV to obtain better agreement
between duplicate determinations. It would not appear to us,
however, that the discrepancies in question militate against the
justice of the conclusions above drawn.
B. megatherium holds a very good place in the sandy and
clay loam soils of Series IV and a fair place in the adobe soil.
The same is even more strikingly true of B. vulgaris, and in a
minor degree this is also true of B. tumescens. The other organ-
isms do not manifest such consistent efficiency under the three
widely varying soil conditions.
160 University of California Publications in Agricultural Sciences [Vol. 1
Miscellaneous Series
For the purpose of comparing sheep and goat manure as well
as phosphatie guano with the other organic materials above
described and with peptone, it was deemed of interest to obtain
data exemplifying the ammonification in the same soil of all the
different materials above used with the three additional ones just
mentioned. The sandy soil was chosen for this series and when
peptone (Witte) was used, 0.5 gram of it was added to 50 grams
of soil. The peptone contained 14.14 per cent N. The sheep
and goat manure (2.13 per cent N), owing to its low nitrogen
content, was added to the extent of 3 grams per 50 grams of soil,
and the phosphatie guano (3.96 per cent N) was also added at
the rate of 3 grams per 50 grams of soil. The results obtained
are recorded in Table VII.
As was to be expected, the very available form of nitrogen
in the peptone allows of the production of much larger quantities
of ammonia than do the less available forms of the other materials.
The fineness of division and easy solubility of the peptone, as well
as the form of nitrogen which it contains, doubtless have con-
tributed to the results. The more or less uniform decomposition
of it, however, by most of the organisms tested shows peptone to be
unsuited, as has been claimed by other investigators, for ammoni-
fication studies with pure or mixed cultures when the application
of the data obtained, to field conditions, is contemplated. How-
ever that may be, Sarcina lutea shows the highest efficiency at
ammonifying peptone nitrogen and at least six other organisms
approach it rather closely. Relatively speaking, all but two of
the organisms tested are efficient ammonifiers of peptone nitrogen.
But their position with respect to peptone, as can be seen from
Table VII, is no criterion as to their efficiency with respect to
the other materials.
One of the interesting facts about the ammonification of the
sheep and goat manure by pure cultures is that only one organism
showed ammonifying efficiency worthy of the name, and that was
B. megatherium. The other organisms showed a very slight
power only of ammonifying the nitrogen in it. Just why this
large discrepancy should exist with respect to this manure
between B. megatherium and the other organisms still remains
1914] Lipman-Burgess : Ammonification in Soils by Pure Cultures
161
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162 University of California Publications in Agricultural Sciences [Vol. 1
to be explained. If, therefore, the readiness of the transformation
of its nitrogen into ammonia by pure cultures of ammonifiers is
to be taken as a criterion, sheep and- goat manure must be
adjudged to contain a relatively unavailable form of nitrogen.
The most amazing evidence portrayed in Table VII is the
very high availability of the nitrogen of bat guano, which is a
phosphatic guano. "While without question the fact of the large
amount of the guano used, as well as its low content of nitrogen,
preclude an accurate and wholly justifiable comparison of it with
the other nitrogenous materials, one cannot help being struck
by the large transformation of its nitrogen into ammonia which
nearly all of the organisms tested can accomplish. In many
ways, the transformation of the nitrogen of bat guano into
ammonia resembles that of the transformation of peptone
nitrogen. The differences between the ammonifying powers of
the different organisms are, however, unquestionably more
marked in the case of the bat guano. In its efficiency as an
ammonifier of bat guano nitrogen, B. mycoides appears for the
first time in all the series studied to be distinctly superior to all
the other organisms tested. B. vulgaris easily takes second place.
Not far behind, however, and about equal in efficiency, are B.
megatherium, B. vulgatus, B. tumescens, and Mic. tetragenus.
In the third class are B. ramosus, B. subtilis, and Ps. putida, B.
proteus vulgaris, and B. mesentericus. In the fourth class are
Streptothrix, sp., Sarcina lutea, B. icteroides, and Ps. fluorescens.
A Comparison of the Relative Availabilities of the Organic
Materials above Employed based on the Percentage
of Nitrogen Contained in Them that was
Transformed to Ammonia
Thus far we have been considering only the relative degrees of
efficiency as ammonifiers of the different organisms among them-
selves as respecting a given organic form of nitrogen in a given
soil. There is possible, however, a further very interesting study
of the data above given as a basis. We refer to the percentage
of nitrogen which is transformed in the different materials into
ammonia so as to give them a relative rating as to availability as
1914] Lipman-Burgess : Ammonification in Soils by Pure Cultures
163
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164 University of California Publications in Agricultural Sciences [Vol. 1
regards the work of pure cultures. The columns on availability
of the various tables show a comparison in different soils of all
the materials as attacked by the same organisms, and give the
percentages of nitrogen in those materials which were trans-
formed to ammonia. In Table VIII all of the columns indicating
percentages of nitrogen of different fertilizers made available
are brought together from the other tables and one is enabled to
compare with much greater ease the different materials on the
basis of availability in the same soil and with the same organism
and in different soils, with different organisms.
The Sandy Soil
So far as this soil is concerned, the data in Table VIII
indicate clearly the superiority, from the point of view of the
availability of its nitrogen, of cottonseed meal to the other organic
nitrogenous fertilizers with which it is compared. Not only are
the absolute amounts of ammonia produced in most cases larger
from cottonseed meal nitrogen than from other forms, but there
are more organisms of the fifteen tested which can vigorously
ammonify this form of nitrogen. So that to illustrate, there are
but five organisms which have shown the power to transform
15 per cent or more of the nitrogen of dried blood into ammonia
in the sandy soil. Under similar conditions there are ten
organisms which hold such a record for cottonseed meal. Tank-
age shows itself to be the next important nitrogenous fertilizer
to cottonseed meal from the point of view of the availability of
its nitrogen. Thus, comparing it with dried blood as above, we
find that there are nine organisms which transform 15 per cent
or more of the nitrogen of tankage into ammonia. No such high
availability is obtained at all in the case of the fish guano.
When we consider these nitrogenous fertilizers from the point
of view of the transformation of 10 per cent or more of their
nitrogen into ammonia, we find that there are thirteen of the
fifteen organisms tested which possess that power as regards
cottonseed meal nitrogen and another comes very close to that
point. In the case of tankage nitrogen there are thirteen organ-
isms with a similar power. In the case of dried-blood nitrogen
] 914] Lipman-Burgess : Ammonification in Soils by Pure Cultures 165
there are but ten organisms which can accomplish that task, and
but seven such in the case of fish-guano nitrogen.
From the point of view of availability by pure cultures
therefore in the sandy soil the four nitrogenous fertilizers are
to be rated as follows : cottonseed meal, tankage, dried blood, and
fish guano. The first two are nearly alike and are far superior
to the last two, which are nearly alike, but much more different
from each other than the first two.
The greatest efficiency at ammonification manifested by any
organism in the sand}^ soil is that of B. mesentericus with tankage
nitrogen, which transforms 32.52 per cent of the nitrogen present
into ammonia in twelve days. It should be noted in this con-
nection also that absolutely higher amounts of ammonia are
produced from tankage nitrogen than from any other form in
the sandy soil, even if there are fewer organisms which attack
it readily than there are in the case of the cottonseed meal.
The Clay -Loam Soil
Conditions are evidently entirely different for ammonification
in this soil. Not only is the ammonia production very low so
far as all the fertilizers are concerned, but they no longer bear
to one another the relation which obtained between them in the
sandy soil. There are thus but few organisms which possess the
power of transforming 10 per cent or more of the total nitrogen
in any of the four fertilizers into ammonia in twelve days. In
fact, there are none such in the case of the dried blood, only two
such each in the cases of cottonseed meal and fish guano, and
seven such in the case of the tankage. No organism attains to
the production of ammonia equivalent to 13 per cent of the total
amount present in the clay-loam soil regardless of the kind of
fertilizer at its disposal.
The tankage, however, is superior to the cottonseed meal in
the clay loam and distinctly so as above indicated. The cotton-
seed meal takes second place, the fish guano third place, and the
dried blood is by far the poorest. Indeed, no organism was
capable of producing an amount of ammonia in excess of 6.91
166 University of California Publications in Agricultural Sciences [Vol. 1
per cent of the total amount of dried-blood nitrogen furnished,
and that occurred in only one case, all the other organisms pro-
ducing much less.
The Clay-Adobe Soil
Some very striking facts become apparent when the avail-
ability of the four fertilizers in clay-adobe soil are considered.
While on the whole dried-blood nitrogen is only slightly more
efficiently transformed into ammonia than in the clay-loam soil,
cottonseed meal and tankage, particularly the former, are more
vigorously acted on in the clay-adobe soil by most of the organ-
isms. Fish guano, while not markedly so, is none the less superior
here again to dried blood.
Again comparing the different fertilizers on the basis of the
amounts of their nitrogen transformed into ammonia by the
organisms tested, we find that four organisms transform 10 per
cent or more of the nitrogen in cottonseed meal into ammonia,
a like number accomplish similar results in the case of tankage,
and none succeeds in that direction in either fish guano or dried
blood. While thus cottonseed meal and tankage appear alike,
a study of table VIII reveals the superiority of the former in the
larger absolute amounts of nitrogen which are transformed there
than in the case of the latter.
The first striking fact shown in Table VIII is the marked
superiority of the sandy soil as a medium for ammonification by
pure cultures. Likewise the added fact of its superiority as a
medium for most of the organisms tested must be noted in this
connection. The second point of great interest is the surprising
fact of the superiority of the clay-adobe soil to the clay-loam
soil as a medium for ammonification. From its tenacious nature
one would suppose the former type to be a much poorer medium
for ammonification than the clay loam and yet it is distinctly
superior to the latter as regards tankage and cottonseed meal.
As regards fish guano, it is slightly inferior to the clay loam and
again as regards the dried blood about equal to the clay loam
or possibly slightly superior.
Considering all the data given in Table VIII from all points
of view, tankage must be given first place as regards the avail-
1914] Lipman- Burgess : Ammonification in Soils by Pure Cultures 167
ability of its nitrogen, cottonseed meal easily takes second place
and, owing to its superiority in the sandy soil, dried blood takes
third place. Considering the superiority of the fish guano to
the dried blood in the other soils, however, it is probably fairer
to adjudge dried blood and fish guano of equal availability from
the point of view of the transformation of their nitrogen into
ammonia by pure cultures of ammonifying bacteria.
General Discussion
Several of the facts which have come to light in the investiga-
tions above described demand a word of comment with respect
to their general significance. First as regards the relative
efficiencies at ammonification of the different organisms tested,
we find that there is marked variation. Indeed it is difficult to
find an organism among the fifteen tested which consistently
stands as the best ammonifier regardless of the soil and the
ammonifiable material employed. There are, however, one or two
organisms which nearly approach such a description. In other
words, it appears that, viewing ammonification of organic
nitrogen from the standpoint of pure cultures, every organism
will do best with a definite combination of soil and organic
matter. To be sure there are some organisms of those tested, even
though they be in the minority, which are consistently weak
ammonifiers. B. icteroides and Ps. fluorescens serve to exemplify
such.
That B. mycoides is by no means always the most efficient of
ammonifying bacteria as has heretofore been believed is clearly
indicated above. On the other hand, it does possess and manifest
marked superiority in certain cases. Thus, for example, while
showing poor or mediocre ammonifying power in different soils
with dried blood, tankage and fish guano, it manifests great vigor
in the case of cottonseed meal and succeeds in making the record
for the percentage of nitrogen transformed in the case of bat
guano, in which it transforms to ammonia 36.06 per cent of the
nitrogen present.
168 University of California Publications in Agricultural Sciences [Vol. 1
Comparing the organisms in any one given soil as a medium
we find some interesting facts. In the sandy soil, for example,
with dried blood B. vulgaris is the most efficient ammonifier,
making available or transforming into ammonia 24.36 per cent
of the nitrogen present. With tankage, B. mesentericus shows
the highest efficiency, transforming 32.52 per cent of the nitrogen
present into ammonia. With cottonseed meal, B. tumescens is
paramount, yielding an amount of ammonia equivalent to 24.30
per cent of the amount of nitrogen present. With fish guano,
B. vulgaris again manifests its superiority over the other organ-
isms by changing 13.09 per cent of the nitrogen present into
ammonia. When bat guano is used, B. mycoides stands distinctly
superior to all others, as above shown, by transforming 36.06 per
cent of the nitrogen present into ammonia. In the case of sheep
and goat manure there is but one efficient organism and that is
B. megatherium, which transforms 24.97 per cent of the nitrogen
present into ammonia. Lastly when peptone is used Sarcina
lutea stands pre-eminent, and when all materials are compared,
regardless of whether they were used in all soils or not, the last-
named organism makes the record for availability by transform-
ing 41.98 per cent of the nitrogen present into ammonia. There
are thus six organisms out of the fifteen which make records in
one and the same soil but with different forms of organic matter.
One of the six stands superior in the cases of two nitrogenous
materials, namely dried blood and fish guano, and that is
B. vulgaris.
Comparing the same organisms with the same nitrogenous
materials, above used, except the last three named, in the clay-
loam soil, we find that with dried blood B. proteus vulgaris is
most efficient, transforming 6.9 per cent of the nitrogen present
into ammonia. With tankage, B. tumescens is pre-eminent and
transforms 12.74 per cent of the nitrogen present into ammonia.
With cottonseed meal, Streptothrix, sp., is superior, transforming
12.84 per cent of the nitrogen present into ammonia; and lastly
with fish guano, B. megatherium is again pre-eminent, trans-
forming 11.66 per cent of the nitrogen present into ammonia.
We see again, therefore, that in one and the same soil, with four
different materials, four different organisms make records for
1914] Lipman-Burgess : Ammonification in Sails by Pure Cultures 169
availability. To be sure, two of these organisms have shown
themselves superior to all others in the sandy soil but with
different materials.
In the case of the clay-adobe soil we find that with dried
blood B. tumescens stands at the top of the list and transforms
9.41 per cent of the nitrogen present into ammonia. With tank-
age in the same soil, Sarcina lutea is the most efficient ammonifier
and transforms 12.44 per cent of the nitrogen present into
ammonia. With cottonseed meal, B. tumescens is again superior
to all others and transforms 18.20 per cent of the nitrogen present
into ammonia ; and finally with fish guano B. vulgaris again
assumes the ascendency and transforms 8.64 per cent of the
nitrogen present into ammonia. B. tumescens is very nearly as
efficient in this latter case as B. vulgaris. We have thus seen
that organisms which in all cases have shown their superiority
in other soils are also very efficient in the clay-adobe soil.
It is clear, therefore, that only about half of the fifteen
organisms tested show greatest efficiency in some soil or with
some form of organic matter. Scrutinizing more closely the
efficiencies of each of these, we must concede to B. tumescens the
paramount place among them, for it has stood pre-eminent, in
five combinations of soil and fertilizer, out of fifteen, and has in
addition been close to first place in several other instances.
Comparing our findings with those of Marchal, the following
critical statements must be made. First, that results of solution
cultures are no criterion as to results to be obtained in soils.
Secondly, that no two forms of organic nitrogen are attacked and
ammonified with the same vigor by any one organism. Thirdly,
that different soils will modify an organism 's power to ammonify
any one given form of nitrogen very markedly, so that it may be
efficient in one case and feeble in another. Fourthly, that the
ammonifying efficiency of organisms is greater in sandy soil, and
possibly in others, than in solutions, for we have obtained a
transformation of 41.98 per cent of peptone nitrogen and 36.06
per cent of bat guano nitrogen into ammonia by Sarcina lutea
and B. mycoides respectively in twelve days at temperatures
between 27° C and 30° C, while Marchal only obtained similar
transformations in thirty days at 30° C in albumin solutions.
170 University of California Publications in Agricultural Sciences [Vol. 1
Owing to the general distribution of the efficient ammonifying
organisms above described in most soils, it is extremely improb-
able, to judge from our results, that we may look forward
to a profitable form of inoculation of soils with ammonifying
bacteria. The choice, however, of the form of nitrogenous fer-
tilizer for a soil, which will be most readily made available, when
available nitrogen is needed, may indeed be something of much
greater practical significance, as our data would appear to
indicate. Particularly emphatic are our results in illustrating
that, at least so far as pure cultures are concerned, and as also
partly shown by J. G. Lipman3 and others, with mixed cultures,
prevalent ideas with respect to the relative availability of common
nitrogenous fertilizers are incorrect. Both tankage and cotton-
seed meal, and, in some cases, fish guano, show higher avail-
abilities than dried blood, which we have always been in the
habit of regarding as the most available of organic nitrogenous
fertilizers (based only on vegetation tests).
Nor do we argue too far afield from our subject when we
make the remarks just preceding. We recognize fully that
availability as measured by ammonification does not necessarily
denote availability as measured by nitrification nor by assimila-
tion of nitrogen by plants. We cannot, however, help noting,
also, that a persistent preference exists among practical orchard-
ists in this state for tankage as against dried bood and we
therefore feel that, judged by other critera, similar conclusions,
must be drawn to those adduced from our experiments with pure
cultures of ammonifying bacteria.
We have decided, however, to go much further in these experi-
ments and are now prosecuting more elaborate investigations on
nitrogen transformation not only to ammonia but also to nitrates.
In these studies we shall deal with the soil flora as existing in a
large variety of soil types freshly collected from the field for our
purposes. Moreover, we shall employ the raw, unsifted fertilizer
material just as it is used by the farmer. From these investiga-
tions we hope to glean much more evidence which will be of
great practical significance, and, like the results above described,
also of marked scientific interest.
1914] Lipman-Burgess : Ammonifieation in Soils by Pure Cultures 171
Summary
Results are above given which deal with :
1. The marked differences in ammonifying efficiency of fifteen
organisms in pure culture.
2. The soil was used as a medium and three types employed,
sandy, clay loam and clay adobe.
3. Four common fertilizers as sources of nitrogen were used,
in all soils, and peptone, bat guano, and sheep and goat manure
employed only in the sandy soil, besides. The four fertilizers
were dried blood, tankage, cottonseed meal and fish guano.
4. The nature of the soil, as well as the nature of the nitro-
genous material, markedly modify an organism's ammonifying
power.
5. There is no regularity in these variations and they cannot
be foretold.
6. While it is difficult to make an exact decision, B. tumescens
appears, on the whole, to have been the most efficient organism
above tested.
7. The highest efficiency in a single culture with a fertilizer
was shown by B. mycoides, which transformed in twelve days at
27° to 30° C, 36.06 per cent of the nitrogen in bat guano into
ammonia.
8. The highest efficiency in a single culture with peptone was
shown by Sarcina lutea, which transformed 41.98 per cent of the
nitrogen present into ammonia under similar conditions.
9. A comparison of availability of nitrogenous fertilizers with
ammonifiability as a criterion, according to our experiments,
markedly changes the positions of tankage, fish guano, and
cottonseed meal with respect to dried blood, showing them in
most or in many cases to be superior to the latter.
10. One half of the number of organisms tested is far superior
to the other half in ammonifying ability.
11. Interesting comparisons with Marchal's work are made.
12. B. icteroides has shown itself throughout to possess but
little ammonifying efficiency.
172 University of California Publications in Agricultural Sciences [Vol. 1
13. Experiments are following those above described to make
the latter more complete. Elaborate tests are being made with
many soil types using the original soil 's mixed flora from freshly
collected samples in the field. Not only ammonifying powers of
these mixed flora in the different soil types will be studied, but
also the corresponding nitrifying powers. A large variety of
nitrogenous fertilizers as employed in practice will be tested here.
REFERENCES OF MORE OR LESS DIRECT PERTINENCE
i Bull. Acad. Roy. Soc. Belg., 3 ser., vol. 25, p. 727.
- Voorhees, Fertilizers, Macmillan Co., 1898.
3 N. J. Sta. Bulletin, no. 246.
4 Ann. Sci. Agron., vol. 19, p. 209.
s Rpt. Del. Exp. Sta., 1899, p. 76.
e Cent, fur Bakt., 2le Abt., vol. 20, p. 322.
7 Ibid., vol. 15, p. 433.
s Ibid., vol. 29, p. 238.
o Ibid., vol. 31, p. 49.
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