The distribution and activities of bacteria in soils of the arid regions

UNIVERSITY OF CALIFORNIA PUBLICATIONS

IN

AGRICULTURAL SCIENCES

Vol. 1, No. 1, pp. 1-20 Issued October 15, 1912

THE DISTRIBUTION AND ACTIVITIES OP

BACTERIA IN SOILS OF THE

ARID REGIONS^

BY

CHARLES B. LIPMAN

INTRODUCTION

The student of soils in the humid region, when for the first
time exploring soils in the arid region, is invariably struck with
the extraordinary depth of the latter as against the very shallow
nature of the former. Taken by and large, and excepting the
faulty soils, including those underlaid at no great depth by stiff
clay, coarse gravel, hardpan, or original rock, respective^, the
soils of the arid region very commonly show a depth of at least
eight to ten feet, and, when viewed in section, exhibit such a
striking uniform it.y in texture and color as to attach to this
unusual condition, in the mind of the observer, a certain marked
practical and scientific interest. The full significance to crops of
the arid region of this extraordinary condition in our soils was
first realized and pointed out by Hilgard and was made the
subject, by him and Loughridge, of a comprehensive investiga-
tion on the ''soil-columns" of California, a large part of which
is completed, but some of which is still in progress. The study
of the soil-columns of California comprised what might be
looked upon as a very thorough partial soil survey of Cali-
fornia. It was the intention of the investigators above named,
at the inception of the work, to obtain columns of soil repre-
senting depths of twelve feet, including a sample for every foot

* Eead before the Society of American Bacteriologists, Washington,
D. C, December 27, 1911.

2 University of California Publications in Agrindtural Sciences \ Vol. 1

ill depth, and to obtain a kno\vled<iT of the chemical constitu-
tion and the texture of the soils by makino^ systematic chemical
and mechanical analyses of all the samples thus collected. The
information thus obtained in the several years in which the soil-
columns Avere studied by Ililj^ard and Louo'hrid^e and the large
number of types of soils considered, alons: Avith the most striking'
circumstance of the depths to which plant root-systems of the
arid regions penetrated, led Hilgard to believe that the striking-
chemical and mechanical differences between the soils of the arid
and humid regions, as well as the differences in the development
of the root-systems in these regions, respectively, might find
a parallel also in a difference between the bacterial flora at
various depths in the soil. It was this belief on Hilgard's part
and his valuation thereof as being of exceeding scientific interest
as well as practical value, that led to the association with him
something over three years ago of the writer, and it was then
that I undertook, among other biological problems in soils, a
study of the nitrogen-transforming and nitrogen-fixing bacteria
in the different layers of soils in the arid region. This study.
while it has progressed considerably, is still in the first stage
of its development and the complete results thereof are intended
ultimately to be combined with the mechanical and chemical
analyses of these soils in a comprehensive report on the whole
work. For the purposes of this paper, it is sufficient to give a
resume of some of the important results obtained in these inves-
tigations with an account of the methods employed in the work,
so that it may serve as a preliminary communication on the sub-
ject and bring out certain striking facts with reference to the
distribution of bacteria in California soils.

:\rp7rTT0T)s employp]d in these investigations

One of the most difficult problems in connection with these
investigations was to find a method for the collection of soil
sam[)les at the several depths which would fairly represent the
actual conditions which obtain there, so far as the bacterial
Hora are concerned. Our first attempts in this direction were
made with an auger of the typc^ manufactured by Iwan Brothers
at South Bend, Indiana, by means of which we tried, through

1912] Lipman: Bacteria in Soils of Arid Eegions 3

successive sterilization of the auger (before taking each sample),
to obtain a sample which represented, uncontaminated, each of
the soils as they are found in their natural state in the field.
With a sterile spatula there were taken from the samples thus
obtained with the auger representative samples which were im-
mediately placed in sterile cotton-stoppered bottles. It was
soon found, however, that this method could not be relied upon
for accurate results, since no matter how carefully the samples
were thus taken, there were many chances for contaminating
samples from the lower layers with soil from the upper layers
and thus obtaining results which were erroneous. After much
experimenting we finally decided on the following plan for
taking the soil samples, which, so far as I know, is as free from
chances of error as any method that can be adopted in a series
of investigations which must of necessity be so extensive. In-
deed, I believe the chances for error here are so small that they
cannot affect the validity, to any appreciable extent, of the
results obtained. Our method consists in having dug, a day
or two prior to sampling, a hole twelve feet in depth with at least
one vertical wall and large enough for a man to stand in. The
samples are taken as follows: With a sterilized spade, a layer
of soil of about five or six inches in depth is sliced down along
the whole length of the wall which is to be sampled. After this
is done, to remove the soil that in the one or two days' exposure
may have become contaminated, the fresh surface thus obtained
on the vertical wall is sterilized by means of a plumber's torch
on the surface surrounding every spot, previously marked off,
at which a sample from each foot in depth is to be taken. When
this is done a sterile cylindrical tin tube, a little over one inch
in diameter and about ten inches long, is driven at right angles
to the wall into the spot selected for sampling, immediately
drawn out when sufficient soil has thus been obtained, and the
cotton plug replaced. In our first experiments, glass tubes of
the size described were employed, with paraffined corks at one
end and cotton stoppers at the other. We found this to be a
poor method, however, and have replaced the glass by tin tubes,
closed at one end and plugged with cotton at the other. These
are sterilized at 150 deg-rees centierrade for one hour and a half

4 University of California Publications in Agricultural Sciences [Vol. 1

before using. In this way by the use of a plumber's torch at
every depth as we descend from the surface of the soil down
to the twelve-foot depth, we obtain, by starting at sterile sur-
faces, a sample of soil representing as nearly as possible the true
condition which obtains at very depth. The samples are marked
properly, taken to the laboratory, and examined for their am-
monifying, nitrifying and nitrogen-fixing powers by means of
a modified Remy method, the solutions employed for the work
being prepared in accordance with the formulae used by J. G.
Lipman.^ Every 50 c.c. portion of the medium in a 250 c.c.
Erlenmeyer flask is inoculated with 5 grams of soil.

DESCRIPTION OF SOILS EMPLOYED IN THESE
EXPERIMENTS

The descriptions given below represent the soils which were
employed for bacteriological examinations and sampled for the
purpose as above described. The numbers employed below are
used throughout all the following tables so as to make unneces-
sary any further descriptions.

Soil No. 1. Eed clay loam mesa soil, from Riverside, California, on
which good orange trees were growing at time of sampling. The soil is
well supplied with potash, but rather poor in phosphoric acid and very
poor in humus and nitrogen. It is underlaid by hardpan at six feet from
the surface, which continues on down to the twelve-foot depth, "With the
careful cultivation which is given it, along with proper fertilization and
tillage, the soil produces profitable crops of oranges and lemons.

Soil No. 2. Silty alluvial loam, from Davis, California. The samples
used were obtained from between some fig trees at the University Farm.
This soil is practically uniform in color from the first foot to the twelfth
and only becomes slightly different in texture below the fifth foot, becoming
gradually coarser and sandier as we descend to the lower layers. It is
well supplied with potash, phosphoric acid, and lime and has, for a soil of
the arid region, a normal content of humus.

Soil No. 3. Sandy alluvial loam, from Davis, California. Samples were
taken from a wheat field at the University Farm, only to a depth of ten
feet. This soil is well supplied with j)hosphoric acid, potash and lime,
but rather poor in humus and nitrogen. The sand is of a coarse nature
and becomes rapidly coarser, descending from the first foot down to the
twelfth, where it is found as very coarse sand.

1 Bulletin 180. N. J. Agr. Expt. Station.

1912] Lipmau: Bacteria in Soils of Arid Eegions 5

Soil No. 4. Sandy alluvial loam, from Davis, California. Samples ob-
tained in almond orchard at the University Farm. This soil is not nearly
so coarse as soil No. 3 and shows a more uniform texture throughout a
seven-foot depth, but after that becomes coarser in texture. It is better
supplied with humus and nitrogen than soil No. 3 and is well supplied
with potash, phosphoric acid, and lime.

Soil No. 5. Alluvial loam, from Davis, California. Samples obtained
in a pear orchard at the University Farm. The soil is uniformly of a
fine sandy loam texture for a depth of nearly five feet and then rapidly
becomes much coarser than the soil at similar depths in No. 3. The upper
soil is well supplied with potash, phosphoric acid, and lime, and fairly well
supplied with humus and nitrogen. The lower layers are rather poor in
phosphoric acid, humus and nitrogen.

Soil No. 6. Fine silty soil, from Hanford, California. Samples taken
from a vineyard at Hanford, from the first to the ninth foot only. No
sampling was done below the ninth foot because of the fact that the water-
table was reached at about that point and it was almost impossible to get
samples uneontaminated. This soil is almost devoid of humus and contains
but little nitrogen, but is fairly well supplied with phosphoric acid, potash,
and lime. No alkali is present in the soil.

Soil No, 7. Silty alluvial loam, from Davis, California. Samples taken
at the University Farm, close to a young eucalyptus tree, about twenty
months old. The soil is fairly uniform in texture throughout the entire
depth studied and is fairly well supplied Avith humus and nitrogen, and
well supplied with phosphoric acid, potash, and lime. No alkali is present.

Soil No. 8. Alkali soil from Tulare, California. Taken only to a depth
of ten feet, owing to water conditions such as those described in soil No. 6.
This soil contains very little humus and is strongly impregnated with salts,
especially ' ' black alkali. " It is otherwise well supplied with phosphoric
acid, potash, lime, and the other minerals. Hardly any vegetation can exist
on this soil after the salts have risen to the upper layers.

Soil No. 9. A very stiff and tenacious silty clay adobe, from Imperial,
California. Uniform in texture from the surface down to the eighth foot,
at which there is found a layer of fine sand for a foot and a half in
depth and then a silty sand below to the twelve-foot depth. The soil
throughout is almost devoid of humus and contains but very little nitrogen;
It is very rich, however, in phosphoric acid, potash, and lime. The upper
layers of the soil consist of particles of silt and clay which are so fine
as to become cemented together into an extremely hard, refractory material,
which is almost of the consistency of a dry, but not heated, brick. A
considerable quantity of common salt is present in this soil. This soil has
never been cultivated or cropped.

Soil No. 10. Fine, sandy soil from the desert of Coachella Valley. In
this, as in the Imperial Valley soils, there are to be found narrow layers
of an inch or two, and sometimes more, of very fine shells of former life
which existed in the water at one time covering this land. The soil has very

6 University of California Publications in Agricultural Sciences [Vol. 1

little or no humus and nitrogen. It is, however, rich in phosphoric acid and
lime and -well supplied with potash. The soil is uniform in texture through-
out the twelve-foot depth and becomes only a little coarser at twelve feet.
The only changes visible in color and texture in the vertical wall are
merely those of the shell layers above noted. The soil from which these
samples w^ere taken has never been cropped, but similar soil, with a good
water supply, produces very fine alfalfa. Very little alkali is present in
this soil.

Soil No. 11. Fertile, alluvial loam from Hayw^ard, California. Uniform
in texture for seven feet and then rapidly becoming quite coarse and re-
maining so down to a depth of twelve feet. This soil is very fertile,
producing good crops of cherries, walnuts, potatoes, and other agricultural
plants. It is well supplied with humus and nitrogen, judged by the
standard for soils of the arid region, throughout the twelve-foot depth.
The phosphoric acid, potash, and lime are also plentiful in all the soil layers.
The samples used in these investigations were obtained in a cherry orchard.

AMMONIFICATION IN SOIL COLUMNS

Second only to the importance of soil bacteria in maintaining
the total nitrogen supply in soils is their power to supply con-
stantly available nitrogen to plants. The essential nature of
this important phase of the activities of soil organisms is in no
wise detracted from by the recent research which has made it
clear that some plants at least can take their nitrogen from the
soil in forms other than the nitrate. While many of them may
not absorb their nitrogen in the form of nitrates, it seems quite
certain that practically all of them must take their nitrogen in
forms much simpler than the proteid. This being undeniably
the case, some agency in the soil is necessary to accomplish the
transformation of the organic nitrogen (no matter what the
source of the latter to the soil may be) into a simpler, more
available, or more assimilable form. These agencies we have
found to be the various types of soil organisms which constitute
what we now designate by the term "ammonifying flora" of the
soil.

With these statements admitted, it seems reasonable to sup-
pose that any increase in the activities of the organisms, included
under this head, is a distinct advantag^e to the plant. Under our
climatic conditions, where, as above stated, the plant roots very
deeply, besides making a large lateral root-development, it is
necessary to have the activities of the ammonifying organisms

1912] Lipman: Bacteria in Soils of Arid Regions 7

not only in the upper layers of the soil, but in the lower layers
where an actual examination of the root-systems of plants shows
a large development of fibrous or feeding roots. A study, there-
fore, of the ammonifying powers of the different layers of soil,
or, rather, of the microorganic flora which they contain, is of
practical moment, since it is bound to throw light on the soluble
nitrogen supply for roots in the greater depths of soil and
indicate what practical measures may be taken toward sustain-
ing and encouraging the growth and activities of the organisms
responsible for that soluble nitrogen supply. Since, therefore,
we assume ammonia production to be the first great step recog-
nized by our analytical methods in the transformation of soil
nitrogen, I have first determined the ammonifying powers at
various depths of soils, which may be considered typical of well-
defined areas and conditions in the arid region.

For this purpose there were inoculated into sterile 50 c.c. por-
tions of 1 per cent peptone solution, 5 grams of soil from every
foot from the surface down to the last depth taken, as above
described. After four days incubation at about 28 degrees
centigrade, the cultures were washed into copper distilling flasks,
sufficient distilled water added, as well as a slight excess of
magnesia, and distilled. The distillate was caught in standard
tenth normal hydrochloric acid, the excess of which was titrated
with standard tenth normal ammonia. Table I gives the results
of determinations of the ammonifying power of the soils chosen,
as above described. The ammonifying power of only one soil,
namely No. 6, is not given, for the reason that the soil column
had inadvertently become contaminated before we were ready
to use it.

The numbers of the soils refer to corresponding numbers
under the descriptions given above, and the amounts of ammonia
produced, as given in the table, represent milligrams of nitrogen
as ammonia.

The data given in table I prove very clearly two facts. First,
that in the typical deep and normal soils of the arid region, the
activities and the distribution of the ammonifying flora seem to
run parallel with the texture, the chemical composition, and the
root-development in these soils. Second, that in the absence of

University of California Publications in Agricultural Sciences [Vol. 1

TABLE

I

Ammonification in

Soil Columns

Soil No, 1

2

3

4

5

6 7

8

9

10

11

mg.

mg.

mg.

mg.

mg.

mg.

mg.

mg.

mg.

mg.

1st ft.

59.80

67.76

68.25

61.60

54.67

72.05

7.28

25.46

12.15

42.84

2nd ft.

55.46

77.00

72.66

54.46

49.14

68.90

4.90

8.27

6.46

36.84

3rd ft.

52.30

69.02

68.40

38.50

32.76

70.43

5.46

8.20

3.21

14.14

4th ft.

55.83

70.63

43.05

44.80

lost

65.72

2.80

8.69

1,99

7.28

5th ft.

49.72

67.48

34.23

39.90

8.40

63.69

4.76

4.87

1,25

7.84

6th ft.

49.00

84.63

31.64

47.60

15.68

60.50

4.41

8.27

1,61

21.19

7th ft.

31.70

72.17

42.00

44.80

17.85

56.90

2.45

6.03

1,57

7.14

8th ft.

30.54

52.57

35.00

22.54

9.80

50.43

10.22

5.40

1.34

7.48

9th ft.

28.65

52.43

35.70

32.90

15.40

45.69

10.64

2.18

1,24

5,60

10th ft.

25.40

36.89

29.40

10.22

43.25

2.23

2,11

16,38

11th ft.

15.65

21.56

25.48

11.06

40.76

1.34

4.87

16,64

12th ft.

14.30

35.84

38.22

10.50

38.45

2.62

1.44

10,85

humus and moisture, or in the presence of alkali salts, the activi-
ties of ammonifying organisms are seriously handicapped.

To discuss these more in detail we find, for example, in soil
No, 1, derived from the mesa soil at Arlington Heights, River-
side, a strong ammonification, varying but little from the first
foot down to the seventh, below which depth we find a sudden
marked decrease in ammonia production, for the reason, doubt-
less, that from the sixth foot down to the twelfth we find a
layer of hardpan which, owing to its poor aeration and poor
water conditions, is unfit for the development of a vigorous bac-
terial flora. In other words, we find in this soil-column, through
the ammonifying power of the various depths of soil, an expres-
sion of the vigor and numbers of bacteria present in these soil
layers and also of the amounts of soluble nitrogen which can
there be expected to be made available through the agency of
soil organisms.

In soil No, 2, however, which represents a good, deep alluvial
soil, we find a very vigorous ammonification from the first seven
feet, and only slightly reduced ammonia production in the eighth
and ninth feet, after which we find a large reduction of about
50 per cent in ammonia production for the other three feet.
We have here, therefore, good vigorous ammonia production down

1912] Lipman: Bacteria in Soils of Arid Regions 9

to the tenth foot and therefore an indication that in these soils
there is constantly being made available nitrogen, if organic
nitrogen be present from humus and other sources, for the needs
of plants with deep root systems.

In soil No. 3, which is more sandy than the other alluvial
soil described and which rapidly becomes coarser in texture as
we descend into the lower layers, we find vigorous ammonifica-
tion to obtain down to the fourth foot, below which we find a
considerable decrease in ammonifying power, owing to the fact
that in that coarse soil neither water nor humus, nor soluble
minerals, are present in sufficient quantity to encourage bacterial
development. Here, however, we find the general tendency for
ammonifying organisms to penetrate to the greater depth quite
plainly visible. The remarks made for soils 2 and 3 are just
as truly applicable to the other alluvial soils from the same
district represented by Nos. 4, 5, and 7. The marked produc-
tion of ammonia, even in the twelfth foot of No. 7, is in accord
with the fine physical and chemical condition of that soil to
that depth and therefore deserves additional mention here.

As to other types of soils, the data in the table show plainly
enough what a profound effect strong alkali salts (both black
and white alkali salts among them) may exert on the ammoni-
fying flora and their vigor. Here ammonification is indeed very
feeble in the surface soil, becoming feebler as we go down until
the eighth foot is reached, at which depth, as well as in the ninth
foot, we find quite a marked increase in ammonia production.
This is doubtless due to the fact that the total salt-content is
at that depth much lower and therefore not so seriously affecting
the activities of the organisms there contained. As for the
desert soils, which never have contained much humus and very
frequently contain too much alkali, it is natural to expect a
rather feeble ammonifying power on the part of the soils. Table
I shows that in this case the expected happens. In soil No. 9.
for example, not only the lack of humus and moisture, but the
very unfavorable physical condition, above referred to under
the description of that soil, along with its salt-content, have so
far affected the ammonifying power of that soil as to reduce it
to a little over one-third of what the normal vallev soils de-

10 University of California Publications in Agricultural Sciences [Vol. 1

scribed have exhibited. Moreover, it would seem that the salt-
content in the lower layers of this soil, which increases as we
go down, has very seriously checked the development of these
organisms there and was probably assisted by the unfavorable
physical condition mentioned. In soil No. 10, while the salt-
content is only meager, we have a rather coarse, sandy soil with
hardly any humus, which is therefore for that reason an unfavor-
able medium for the development of bacteria, to say nothing of
the lack of moisture there and the great heat which these desert
soils must absorb from the sun. We therefore have a very much
smaller ammonifying power in the upper layers of the soil than
exists even in soil No. 9, from Imperial, and then a very rapid
decrease to almost no ammonifying power in the lower layers.

By a general survey of all of these data, it would certainly
seem that we are justified in drawing the conclusion that am-
monification and the ammonifying flora of soils are vigorous for
several feet down in the arid region and are limited in their
activities only by the presence of large amounts of salt or a
lack of humus and moisture. Since, however, California soils,
taken by and large, are deep, we have reason, from the facts
above given, to suppose that the ammonifying power in most
of these soils, which are not in any way "abnormal," is vigorous
at great depths.

NITRIFYING POWERS OF SOIL COLUMNS

By very many and perhaps by most plants, nitrate is the
form of nitrogen taken up. It is therefore of importance not
only to study ammonia formation in soils, but nitrate formation
as well. In these investigations we have studied qualitatively
and quantitativel}^ the production of nitrites and nitrates in
ammonium sulfate solution by soils from the different depths
in every case. Here also, as in the ammonification work, 5 grams
«of soil were used to inoculate 50 c.c. of culture solution. The
results obtained in this work are set forth in a qualitative man-
ner, as to nitrate formation merely, in table II, since it is
sufficient for the purpose of this preliminary paper to know to
what depths in the soil nitrates are produced. Later publica-
tions, giving the more complete data of these investigations, will

1912] Lipman : Bacteria in Soils of Arid Begions 11

give the quantitative results as they are given for ammonification
in table I. The plus sign represents nitrate formation and the
minus sign the absence thereof. The numbers of the soils are
referred again to the descriptions above given.

. TABLE II

Nitrification in Soil Columns

Soil No. 1 2 3 4 5 6 7 8 9 10 11

1st ft. + + + + + + + ___ +

2nd ft. + + + + + — + — — — +

3rd ft. + + + + + _ + ___ +

4th ft. + + + + + _ + ___ +

5th ft. + + + + + _____ +

6th ft. trace + — — + — ___ — +

7th ft. — — — — — — — — — — J^

8th ft. _ — _______ — +

10th ft. — — — — — ___
11th ft. — — — — — ___
12th ft. — — _— _ ___

From the data in table II we see again a striking resemblance
between nitrification in the soil depths and ammonification in
the same. All the alluvial soils in particular show very uniform
nitrate formation. The latter seems to be as much inhibited
in soil No. 1 by the hardpan layer as is ammonification. In
soils 2, 3, 4, and 5, as well as 7, we find a general tendency for
nitrates to be formed in the first five feet and then an enfeebled
powTr of nitrate formation, in some cases for one foot and in
other cases a total loss of that power. In nearly all cases these
run parallel with a similar decrease in ammonia formation, but
it seems that nitrate formation is more seriously hampered by
the conditions which curtail ammonia formation and particularly,
it appears, by the lack of oxygen in the lower layers of the soil.
This is an account, therefore, of the first case which has come to
my notice of nitrification at any depths below two or three feet
in the soil, and shows a marked difference in itself between soils
formed and existing under humid and those formed and exist-
ino: under arid conditions. Nitrogen therefore is available in

12 University of California Publications in Agricultural Sciences [Vol. 1

these soils, not only for those plants which are able to absorb
ammonia nitrogen, but also for that larger class of normal
plants which absorb their nitrogen in the nitrate form. It must
be said here, however, that nitrate formation proceeded always
more rapidly in soils from the first foot than in cultures pre-
pared from the other depths. This may indicate a smaller num-
ber of nitrifying organisms in the lower layers of the soil or
perhaps a less vigorous flora, but their activities are uniform
from the second foot down to the last depth in which they
show no activity as indicated in table II. In soil No. 6, which
w^e find on analysis contained merely a trace of humus, that cir-
cumstance seems to have made the soil unfit for the develop-
ment of the vigorous bacterial flora and is supported by the
data in tables II and III. As to the nitrate formation in
culture solutions by the inoculation with this Hanford soil,
nitrates were produced only after a month's incubation and only
in small quantities in the culture prepared from the upper foot
of soil, whereas all other surface soils, Avhen inoculated into solu-
tions with the exception of those which show no nitrification at
all, showed nitrate formation before the end of two weeks. In
culture solutions from soil No. 6, kept about three months and
prepared from the lower layers of soil, no nitrates were ever to
be found. The depressing effect of alkali on the bacterial flora,
as well as the inhibiting effect of a lack of humus, moisture,
and the proper physical condition, are again exemplified in
table II in soils 8, 9, and 10, as they w^ere for the same soils in
table I referring to ammonification. Even after one month's
incubation, not one of these soil-samples showed any nitrate
formation, whether the culture was prepared with the soil from
the upper layers or from the lower. There seems to be a total
absence of nitrifying bacteria of one kind or another.

The best example of the penetration of nitrifying bacteria
to great depths was obtained in soil No. 11, a fine alluvial loam
from Hayward, where nitrate formation was obtained down to
the ninth foot in the soil. In this case also there was, besides
a mere formation of nitrates, as shown by a qualitative test, an
actually vigorous nitrate formation in the lower layers as well
as in the upper layers of the soil. It would seem again here

1912] Lipman : Bacteria in Soils of Arid Regions 13

therefore, in general, that where soils in the arid region are
supplied with a moderate amount of humus, with the proper tex-
ture and chemical constitution, as well as freedom from alkali,
all of which is true of the large majority of our soils, nitrification
as well as ammonification is found to obtain vigorously in the
lower layers of the soil for four feet at least, and in some cases
to six and to nine foot depths.

NITROGEN FIXATION IN SOIL COLUMNS

The next point of interest to determine in these soil-column
investigations from the bacteriological standpoint was to show
whether or not the supply of nitrogen, at the disposal of the
ammonia-forming and nitrate-forming organisms, which we have
found developed to such great depths, and enabling roots to
have a soluble nitrogen supply there, was provided merely by
the humus content of the soil at those depths and produced from
decaying roots, or carried dow^n from the upper layers; or, was
that nitrogen supply in part a new one obtained directly from
the atmosphere by nitrogen-fixing bacteria. If such were the
case, we should, of course, have enormous quantities of nitrogen
fixed per acre, since the fixation would not be limited to the
upper foot of soil. Accordingly, experiments were inaugurated
to obtain the facts which exist with reference to this matter.

Here the necessary mannite solution w^as inoculated with five
grams of soil in each case, and a culture prepared from every
foot in depth in the case of every soil. Table III shows in
tabular form the results obtained, which are set forth qualita-
tively. The numbers at the heads of the columns refer again
to the numbers used in the description of soils, and one plus
sign is intended to show the presence of Azotobacter, two of
a fairly vigorous development of these organisms, and three of
a very vigorous development. In this qualitative way, there-
fore, nitrogen fixation has been judged by the development of
Azotobacter as a criterion. It may justly be argued against this
that other organisms are capable of fixing nitrogen and that the
quantitative figures would be preferable to the qualitative one
showing merely the presence of Azotobacter. While this argu-
ment may in part be true, it appears from my results, which

14 University of California Fuhlications in Agricultural Sciences [Vol. 1

show quantitative as well as qualitative figures in these as well
as other experiments, that in the absence of Azotobacter only
very slight fixations of nitrogen or none are obtained.

From the results set forth in table III, it appears that only
one soil of the eleven tested shows the presence of Azotobacter
as deep down as the fourth foot and six others show the pres-
ence of these organisms in the third foot. Most of them, how-
ever, show the presence of Azotobacter in vigorous form only
in the first two feet. It is therefore not sufficient, evidently, for

TABLE III

Nitrogen Fixation in Soil Columns

Soil No. 1 2 3 4 5 6 7 8 9 10 11

1st ft. +H-+ + + + + + + + + + +++ - + + + — — — + +

2ndft. ++ + + + + + + + + + + + + — + + + — — — + +

3rd ft. + — ++ + + + + + + — ++ — — — + +

4th ft. — — — — + + + — — — — — +

5th ft. — — — — — — — _ _ _ _

6th ft. — — — — — — — -. _ _ _

7th ft. — — — — — — — _ _ _ _

8th ft. — — — — — — — _ _ _ _

9th ft. — — — — — — — _ _ _ _

10th ft. — — — — — — — — — — —

11th ft. — — — — — — — — — — —

12th ft. — — — — — — — ____

soils to be chemically and physically as favorably constituted as
these soils are for ammonification and nitrification to encourage
the deeper penetration of Azotobacter. As is well known, these
organisms are extremely sensitive to a lack of oxygen and it
would appear that this circumstance regulates and controls the
penetration of Azotobacter organisms as above portrayed. I think
in addition, however, it may fairly be argued that the presence
of Azotobacter in more than half of these soils in the third foot
is, in itself, a favorable indication of the nature of the soils in
question. It is of interest also that, in soil No. 5, Azotobacter
organisms, with the nitrogen-fixing power as vigorous as those
above, were found in the fourth foot. This, so far as the writer is

1912] Lipman: Bacteria in Soils of Arid Eegions 15

aware, constitutes the only published case of even this extent of
penetration of Azofobacter organisms. It has been reported to
me, however, that Azobacter organisms have been found in the
twelfth foot of soil in some of the very favorably constituted loess
soils of Nebraska. The question put in the introduction to this
subject of nitrogen fixation is therefore answered in the negative.
For the greater depths, at any rate, in which ammonification
manifestly is vigorous in our soils, Azotobacter organisms do
not penetrate and are not the source of the supply of nitrogen
which can be transformed at those depths by the ammonifying
organisms or by the nitrifying organisms. The nitrogen supply
of these, therefore, in the lower layers of the soil must be the
humus produced from the decaying roots at those depths, or
the humus brought down in solution from the upper layers of
the soil.

As regards soil No. 6 we have here again a total absence
of Azotobacter organisms, possibly due in part, at least, if not
wholly, to the absence of any but very small amounts of humus,
by which I have already tried to explain the feeble nitrification
only in the first foot of this same soil. The same remarks
also which were made above, with reference to soil Nos. 8, 9, and
10, as regards their ammonifying and particularly their nitrify-
ing power, apply again in the case of their nitrogen-fixing power.
No Azotobacter organisms and no fixation of nitrogen were ever
observed in any of these soils, no matter from what depth of soil
the cultures were prepared.

In justice to this subject it must further be stated here that
the comparatively slight penetration of Azotobacter organisms
in our soils may be due to factors other than merely a lack of
a plentiful supply of oxygen. There is evidently some other
circumstance which controls the presence or absence in many
of our soils of Azotobacter organisms and that may also limit
the depth to which these organisms may penetrate. Just what
this factor may be is not at present clear to the writer, but the
fact remains that frequently soils with a good chemical and
physical constitution and producing good crops, will yet show
no Azotobacter organisms.

16 University of California Puhlications in Agricultural Sciences [Vol. 1

GENERAL DISCUSSION

As I have already pointed out in an earlier publication,^ the
slow formation of clay substances in soils of the arid region,
owing to the peculiar climatic conditions there obtaining, is
doubtless responsible for a much greater degree of aeration in
soils because of the larger volume of pore spaces made possible
through a lack of large quantities of cementing substances. Thus
when soils first begin to form from disintegrating rock we have
much more complete aeration with an encouragement for bac-
teria, probably the earliest inhabitants of the soil, to penetrate
to greater depths. Such penetration on the part of bacteria is
invariably accompanied by the production of more favorable
physical and chemical conditions in the soil for the roots of
plants. These in their turn, through physical and chemical
changes which they bring about in the soil in their search for
water and food, make better conditions for a deeper penetration
of bacteria and so through mutual aid the latter and the higher
plants are able, under our arid climatic conditions, to make the
deeper layers of soil a more congenial medium for each other.
The changes thus brought about result in a more uniform tex-
ture of soils at great depths, uniformity of chemical composi-
tion, including humus content, in all the soil layers, and a
much closer approximation of the bacterial flora in the lower soil
layers to those of the upper layers than can be found in the
average soils of the humid region, where climatic conditions are
unfavorable to good aeration, because tendencies opposite to those
above described for our soils are in operation. An estimate of
the biological condition of our deep soils was thus similarly made
by Hilgard on a priori considerations and the investigations
above recorded serve, in general, to confirm his surmise.

Viewing the subject in its entirety, we find that the organ-
isms forming ammonia in soils penetrate to greater depths than
the nitrifying or nitrogen-fixing bacteria studied. While am-
monification is usually most vigorous in the surface, four to six
feet, it is none the less very pronounced in the lower layers from
six to ten feet in depth in all of our normal deep soils. Hardpan,

- Ijii)rnan, C. B., New Facts about Bacteria of California Soils, Science
N. S., June 11, 1909.

1912] Lipman : Bacteria in Soils of Arid Regions 17

alkali, and a lack of humus and moisture decrease the ammoni-
fying powers of our soils or are not favorable to the develop-
ment of vigorous ammonifying flora, but their effects are just as
pronounced in the upper layers of these abnormal soils as in
the lower layers which, therefore, cannot be fairly compared
with our deep average soils as to bacterial content. To what a
serious extent alkali salts may affect ammonification has been
shown by me in a recent paper.^ That the humus content alone
may profoundly affect the number and vigor of bacteria is well
exemplified in both soils No. 6 and 10, where all other condi-
tions but the humus content are favorable and where both the
number and physiological efficiency of the organisms is small.

It would therefore seem, in brief, that ammonification is
vigorously active in the lower soil layers in soils of the arid region
where humus is present and hardpan and alkali are absent.
Since these conditions are complied with in the average of our
cropped soils, the opinion is justified that the deep penetration
of bacteria is a distinctive characteristic of soils in arid regions
which results from much better aeration, as a starting point,
than can be attained in soils of the humid region. The experi-
mental data above given amply confirm this opinion and help
to explain why deep plowing is not only harmless in our soils
but directly beneficial, and why three or four feet of upper soil
may be removed in grading, and alfalfa and fruit trees may be
grown on the newly uncovered subsoil without difficulty, a feat
which cannot be accomplished on soils of the humid regions.

As for nitrification my data present again features of striking
interest. They go to prove that nitrate formation, like ammoni-
fication, goes on at much greater depths in soils of arid than in
soils of the humid region, and thus render distinctly sectional the
observations of Dyer* on this subject, and makes them applicable
only to soils of the humid region. While the nitrifying organ-
isms are doubtless more susceptible to a lack of oxygen than the
ammonifying bacteria, the differences obtained above between
the two groups of organisms, so far as soil fertility is concerned,
are rather those of degree than of kind. The same relationships

4 Bulletin 106, p. 55, O. E. S., U. S. D. A.
3 Centrallblatt fiir Bakt., 2 Abt., vol. 32, p. 58.

18 University of California Publications in Agricultural Sciences [Vol. 1

displayed by the ammonifying bacteria toward hardpan, alkali,
and a lack of humus and moisture, hold in a more exaggerated
way as regards the nitrifying organisms. More specifically, the
writer has also shown^ the distinct effects of each of the alkali
salts on nitrifying bacteria in work quite recently completed. It
would appear in general, however, that in our deep soils, a supply
of nitrate as well as of ammonia is at the disposal of plants for
a depth of five or six feet. As regards the nitrogen-fixing powers
of soils of the arid region, my results show plainly that they do
not differ strikingly from those of soils in the humid regions,
if the presence and vigor of Azotobacter organisms be taken as
a criterion. While it is true that in one or two cases Azotobacter
organisms were found in our soil-columns below the depth at
which they occur elsewhere, and perhaps at a slightly greater
depth in all soils in which they were found, I feel loath to believe
that these are expressions of a rule for soils of the arid region.
Other observations indeed lead me to believe that Azotobacter
development has not gone so far in our soils as it has in soils
of other regions. For example, I have studied many soils in Cali-
fornia with a favorable physical and chemical constitution which
were absolutely devoid of Azotobacter organisms. If therefore
the results set forth above, with reference to nitrogen fixation,
are to be considered representative, the nitrogen supply in the
lower layers of the soil must be replenished in this region as
well as. in the humid region, not from direct fixation by Azoto-
bacter, but from the nitrogen of the upper soil layers.

With reference to these investigations in general, one or two
additional points need more than passing consideration. First,
as to the method of collecting the soil-samples for examination,
it appears to the writer that every possible precaution was used
to prevent contamination and it would be difficult to devise a
method which takes into consideration and avoids more of the
avenues of contamination by which any results might be vitiated.
Moreover, I find strong confirmation of this belief in the facts
brought out in the data above given, viz., that any abnormality
in the soil was sure to be reflected in the results obtained with
cultures prepared from that abnormal soil. Thus hardpan layers

5 Cent, f iir Bakt., 2 Abt., vol. 33, p. 305.

1912] TApman : Bacteria in Soils of Arid lief/ionn 19

never ^^ave evidence of vigorous bacteria, nor did alkali soils
or soils devoid of humus.

Secondly, the writer desires to anticipate criticism on the
method used in culturin^i' the organisms of the various soil
samples, viz., a modified Remy sohition method. No one is more
ready than I am to admit the just criticism made of the solu-
tion-culture methods in soil bacteriology. Indeed I believe that
I was one of the first to put into practice on a lar^ic scale the
direct soil-culture method in the laboratory. But when problems
of the nature involved in these investigations must be attacked,
regard must be had for the chances of contamination in the
method employed, and for the feasibility of obtaining, uncon-
taminated, large volumes of soil for use in these experiments.
When these were considered from all points of view, only one
feasible and reliable method of culturing the soils seemed avail-
able and that was the solution method. The difficulties, prac-
tically insurmountable, which must arise with any other method,
when such work is carried out on a large scale as it must of
necessity be, can be fully appreciated by those who have ever
attempted it. The gratifying results obtained in this work, how-
ever, seem to me a further justification of the methods em-
ployed.

It seems of particular moment now, to call the attention of
soil bacteriologists in particular, and soil scientists in general,
to the important field explored in these investigations and the
striking results obtained therefrom, not only because it repre-
sents a new field of research, but because it emphasizes more
strongly than ever the radical differences which obtain between
soils of the humid and arid regions. It also helps to explain
the extraordinary appearance of our subsoils (if subsoils they be)
and the marvellous root developments of which plants under our
climatic conditions are capable. While these studies have not yet
departed from the realms of the preliminary, they are replete
with facts which are already of considerable practical and scien-
tific significance and which are doubtless destined to become
more so as time progresses. As a part especially of a comprehen-
sive soil study they are invested with unusual importance and
may help to solve problems now perplexing and difficult to study.

20 University of California Publications in Agricultural Sciences [Vol. 1

CONCLUSIONS

Investigations of the distribution and activities of bacteria in
soils of the arid region show:

1. That samples of soil for studying the flora of each layer
of soil can best be obtained from a hole twelve feet in depth
with at least one vertical wall, the latter when sterilized being
sampled.

2. That tin tubes ten inches long and about one inch in
diameter closed at one end and cotton-stoppered are best for
collecting the samples.

3. That the solution method for stud3ang the soils, despite
its many drawbacks, is the most feasible one to emplo3^

4. That soils of the arid region at all depths studied show
ammonifying powers which, however, are generally most
vigorous in the first six or eight feet. In one case ammonifica-
tion was noted in soil from a depth of fifteen feet, or adjoining
the water-table.

5. That nitrification is found commonly down to a depth of
five to six feet in soils of the arid region. In one case soil from
the eight-foot depth showed a vigorous nitrifying power.

6. That nitrogen fixation through Azotobatcer does not go
on below two feet in the soil usually, but has been found in
some soils at three feet and in one soil down to four feet. Many
soils in the arid region, otherwise favorably constituted, do not
contain Azotobacter organisms.

7. That from the point of view of ammonification and nitri-
fication soils in the arid region differ markedly from those in
the humid region when the lower layers of soil are considered.
The difference is not marked as regards nitrogen fixation.

8. The results above recorded help to explain the favorable
physical and chemical constitution of our soil and also the deep
rooting of plants so characteristic of the arid regions.

Transmitted April S, WIS.