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The Soil and the Microbe. By Professors Sel-
mnn A. Waksman and Robert L. Starke v pp xi
+ 260. $3 50. 1931. John Wiley and Sons. Inc.

When one recalls that our knowledge of the
role of microoro-anisms in soil processes and plant
growth has fleveloped chiefly in the past hnlf
century, it is remarkable how' large a bodv of in-
formation has already Ijeen accumulated. And
mncli of the most significant of this Dr. Waksman
and Dr. Starkey marshall briefly in lan inter-
esting and instructive manner in "The Soil and
the Microscope", so that the reader obtains a
vivid picture not only of soil organisms and their
multitudinous physiological reactions, but also of
the relation of these processes to the origin and
development of soils, to the cycle of the elements
in nature, and to plant nutrit'on. Tlie present
volume affords an excellent introduction to the
somewhat encyclcpnedic "Principles of Soil
Microbiography" by the senior author, a world
n.uthoritv in the field. — L. L. Woodruff.

4

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THE WILEY AGRICULTURAL SERIES

EDITED BY

J. G. LIPMAN

THE SOIL AND THE MICROBE

^ u

THE SOIL AND THE MICROBE

An Introduction to the Study of the Microscopic

Population of the Soil and Its Role in

Soil Processes and Plant Growth

BY

SELMAN A. WAKSMAN

Professor of Soil Microbiology
Rutgers University

AND

ROBERT L. STARKEY

Assistant Professor of Soil Microbiology
Rutgers University

NEW YORK

JOHN WILEY & SONS, Inc.

London: CHAPMAN & HALL, Limited

1931

Copyright, 1931, by
Selman a. Waksman

AND

Robert L. Starkey

All Rights Reserved
This book or any part thereof must not
be reproduced in any form without
the written permission of the publisher.

PRIMTED IN U. S. A.

PRESS or

BRAUNWORTH Bt CO.. INC.

BOOK MANUFACTURERS

BROOKLYN. NEW YORK

THIS BOOK IS DEDICATED TO

SIR JOHN RUSSELL

INVESTIGATOR AND WRITER, WHOSE BOOKS

ON SOIL FERTILITY AND PLANT GROWTH

HAVE DISSEMINATED WIDELY THE

KNOWLEDGE OF THE SOIL

AND ITS PRACTICAL

APPLICATION

FOREWORD

The soil is not a mass of dead debris, resulting simply from the
physical and chemical weathering of rocks and of plant and
animal remains through atmospheric agencies, but it is teeming
with life. Every small particle of soil contains numerous types
of living organisms belonging both to the plant and animal king-
doms, yet so small that they cannot be recognized with the naked
eye. These organisms are, therefore, called microbes, micro-
organisms or microscopic organisms. These microbes comprise
numerous types of bacteria, fungi, algae, protozoa, nematodes and
other invertebrates which vary considerably in their structure,
size, mode of living and relationship to soil processes.

In the cycles of transformation of elements in nature, the
microbes play an important, if not a leading, role. Were it not
for them, the soil would soon become covered with a considerable
mass of undecomposed plant and animal residues; life would soon
cease, since the very Hmited supply of carbon and available
nitrogen, the most essential elements in the growth of hving
organisms, would become exhausted. It should be recalled that
carbon dioxide, the source of carbon for the growth of plants,
which in their turn supply the food for animals, is present in the
atmosphere only in a concentration of 0.03 per cent. This is
equivalent to 5.84 tons of carbon over each acre of land. A good
yield of sugar-cane will consume about 20 tons of carbon in a
single growing season; of course most of the surface of the earth
supports less vegetation than this, and diffusion tends to create
a uniform distribution of gases. It has actually been calculated
that the plant world consumes 64.8 millions of tons of carbon
annually, which amounts to 1,'35 of the total carbon content of
the atmosphere. The atmospheric supply of carbon dioxide is,
however, constantly replenished from the decomposition products
of the organic substances in the soil; only as a result of this does
plant growth not cease entirely through a deficiency of an available
supply of carbon. In the absence of microbes the available
nitrogen would also become very rapidly exhausted, as can be

viii FOREWORD

appreciated from the fact that this nitrogen is never present in
the soil in forms available to plant growth, as ammonia or nitrate,
in amounts of more than a few pounds per acre. It is made avail-
able to plants only through the constant activity of the microbes.

The microorganisms, through their various activities, thus
enable organic life to continue uninterruptedly on our planet.
They keep in constant circulation the elements which are most
essential for plant and animal life. They break down the complex
organic molecules, built up by plants and animals, into the simple
mineralized constituents, making the elements again available
for the growth of cultivated and uncultivated plants which in
their turn supply further food for animals.

Just as man and other animals, as well as higher plants, find
their habitat on the surface of the soil or immediately below it,
so do the microbes live largely within the upper few inches of the
earth's crust, where they carry out their important activities,
supplying a continuous stream of nutrients in an available form
for the growth of higher plants. This surface pellicle of the earth
is thus found to be the seat of numerous processes of incalculable
importance in the life of man, animals, and plants, enabling them
to carry out their normal existence on our planet. Just as man and
animals are determined in their development by the supply of
plant food, so is the growth of plants determined by the activities
of microorganisms in the soil. The microbes were probably among
the first living organisms which appeared on our planet millions of
years ago. Although their presence in ancient rocks is largely
speculative, it is reasonable to assume, from an appreciation of
their specific physiological processes, that they may have lived
normally on the earth long before it was a fit habitat for higher
plants and animals.

Our knowledge of the soil microbes and their role in soil
processes and plant growth has developed in the last fifty years.
However, a large body of information has since accumulated
which enables us to construct a clear picture not only of the
microscopic population of the soil, of its numerous physiological
reactions, but also of the relation of these processes to the
origin and formation of soil, to the cycle of elements in nature, and
to plant nutrition. Selman A. Waksman

New Brunswick, N. J. Robert L. Starkey

October 18, 1930

CONTENTS

CHAPTER PAGE

I. The Soil and the Plant 1

The nature of the soil. Weathering of rocks. Effect of
cUmate upon chemical composition of soil. Soil formation.
Movement of water in soil. Organic matter of soil. Soil
phases. Role of microbes in plant growth. The nature of
plant nutrients. Absorption of nutrients by plants. Sum-
mar}'.

II. The Microbe .\nd Its Activities 21

The microbe and its importance in the soil. Nature of soil
microbes. Nutrition of soil microbes. Bacteria of the soil.
Activities of soil bacteria. The fungi of the soil. The
actinomyces of the soil. The algae of the soil. The protozoa
of the soil. Worms and insects in the soil. Soil organisms
causing plant and animal diseases. Symbiotic relationships.
Summary-: The complex soil population.

III. The Soil Population and Its Distribution 44

The occurrence of microbes in soil. Relation of microbes to
plants and animals. QuaUtative and quantitative distribution
of microbes in soil. Methods for determining numbers of
microorganisms in the soil. Methods for studying activities
of soil microbes. Summary of methods. Direct microscopic
methods. Plate method for counting microbes. Elective
culture method. Abundance of bacteria in soil. Influence of
soil conditions and treatment upon the distribution of bacteria.
Influence of organic matter. Influence of moisture. Influence
of reaction. Influence of season of year. Development of
actinomyces in soil. Development of fungi in soil. Develop-
ment of protozoa in soil. Development of algae in soil.
Lower invertebrates in soil. The activity of the soil popu-
lation as a whole. Summary.

IV. Role of Microbes in the Decomposition of Organic Sub-

st.\nces in the Soil 75

Principles of decomposition of organic matter by microbes.
Composition of plant and animal substances. Factors affect-
ing decomposition. Decomposition of sugars and their deriva-
tives. Decomposition of hemicelluloses. Decomposition of
ix

31 ^SZ

K CONTENTS

:hapter page

cellulose. Fungi decomposing cellulose. Bacteria decom-
posing cellulose. Influence of soil conditions upon cellulose
decomposition. Lignin and its decomposition. Decomposi-
tion of fats. Decomposition of other plant constituents.
Decomposition of the plant as a whole. Metabolism of
microbes and decomposition of organic matter. Summary —
Decomposition of organic matter in soil and formation of
soil humus or soil organic matter.

V. Transformation of Nitrogen by Soil Microbes 102

Sources of nitrogen in soil. Transformation of nitrogen in
soil. Fixation of atmospheric nitrogen. Non-symbiotic nitro-
gen-fixing bacteria. Importance of non-symbiotic nitrogen
fixation. Symbiotic nitrogen fixation. Nature of the bacteria
and classification of leguminous plants. Influence of nitrate
upon nitrogen fixation. Amounts of nitrogen fixed. Influence
of soil conditions upon nitrogen fixation. Decomposition of
proteins by microorganisms. Decomposition of nitrogenous
substances of a non-protein nature. Formation of ammonia
by soil microbes.

VI. Transformation of Nitrogen by Soil Microbes (Continued) . . 136
Nitrate formation. Influence of ammonia upon nitrate forma-
tion. Influence of soil reaction upon nitrate formation.
Influence of soil aeration upon nitrate formation. Organic
matter and nitrate formation. Moisture and nitrification.
Nitrate reduction. Importance of denitrification in soil.
Summary.

VII. Transformation of Mineral Substances in Soil through

the Direct or Indirect Action of Microorganisms 154

Relationships of microorganisms to the elements occurring in
nature. Mineral elements and their inorganic compounds as
sources of energy. I'se of inorganic salts as sources of
oxygen. Interaction of insoluble inorganic salts with in-
organic and organic acids produced by microbes. Carbon
dioxide in soil. Nitric acid in soil. Organic acids in soil.
Change in soil reaction. Mineral assimilation by micro-
organisms. Transformation of sulfur in soil by microbes.
Transformation of phosphorus by soil microbes. Transforma-
tion of iron by soil microbes. Transformation of potassium by
soil microbes. Importance of mineral transformation by
microbes in soil formation.

VIII. Interrelationships between Higher Plants and Soil Micro-

ORG.ANISMS 181

Interdependence of higher plants and microbes. Evolution
of carbon dioxide. Influence of root excretions. Conditions

CONTENTS xi

HAPTER PAGE

favoring nitrogen fixation. Absorption of organic compounds
by plants. Associative growth of green plants and microbes.
Mj'corrhiza. Nodule formation by leguminous plants. Bac-
teriorrhiza. Summary.

IX. Modification of the Soil PoprL.A.Tiox 203

The soil population subject to alteration. Influence of soil
treatment upon the nature and abundance of microbes. Influ-
ence of organic matter upon the soil microbes. Influence of
green manures. Influence of stable manure. Artificial
manures. Influence of soil cultivation. Effect of Hming.
Influence of reaction upon soil microbes. Influence of artificial
fertiHzers upon soil microbes. Influence of plant gro'Ri:h.
Influence of partial steriUzation of soil upon the soil popula-
tion and its activities. Inoculation of soil with microorgan-
isms. The estimation of soil fertiUty by microbiological
methods. The activity of physiological groups of soil microbes.
Abundance of microbial inhabitants of soils. Biological
activity of the soil population as a whole. Determination of
the availability of specific nutrients in soils. Summary.

X. Importance of Microbes in Soil Fertility 242

Relationships of microbes to soil processes. Role of micro-
organisms in the cycle of elements in nature. Synthetic activi-
ties of microorganisms. Disappearance of nitrogen from the
soil. Nitrogen fixation in soil. Role of microbial metabohc
products in soil transformations. Reduction and oxidation
processes. Summary.

Index 251

THE SOIL AND THE MICROBE

CHAPTER I
THE SOIL AND THE PLANT

The Nature of the Soil. — The upper layer of the earth's
surface, varying in thickness from 6 to 18 inches in the case of
some humid soils and up to 10 or 20 feet in the case of arid soils,
possesses certain characteristic properties which distinguish it
from the underlying rocks and rock ingredients. This very thin
surface layer of the earth's pellicle is spoken of as the soil. It is
distinguished from the lower layers by its mechanical, physical,
and chemical properties, but especially by the presence of hving
organisms including a variety of microbes, lower animals, and
roots of plants. Dead bodies of these organisms also occur in the
soil in all stages of decomposition. The science of the soil is
frequently spoken of as Pedology.

The type of soil that has developed upon the underlying rock
is a result of climate and the organic life upon it or within it,
including the action of higher plants, animals, and microorgan-
isms. The soil is arranged in a series of characteristic layers or
horizons, which make up the soil profile, which is a direct result
of the conditions under which it has been developed. A soil
profile is obtained by making a vertical cut through the soil,
showing its various horizons (Fig. 1). The upper horizon is more
or less dark colored on account of the presence of organic matter
in different stages of decomposition. The color of the soil may
become darker or hghter with depth, depending on the accessi-
bility of air and movement of water through the profile, the
penetration of roots, and the activity of microorganisms.

The soil is characterized morphologically by the texture,
structure, color, and chemical composition of the various horizons.
These horizons are designated by letters: A, usually at the sur-

THE SOIL AND THE PLANT

face, is that horizon from which certain material has been removed
by mechanical or chemical means. B, is that horizon into which

material has been car-
ried. C, designates the
parent material. These
horizons are frequently
subdivided into Ai, A2,
etc. The microbiologi-
cal processes in the soil
are carried out largely in
the A horizons, and it is
here that most of the
plant remains become in-
corporated. A consid-
eration of the composi-
tion of the various hori-
z o n s reveals marked
differences, especially in
the content of organic
matter, as shown in
Table 1.

Weathering of
Rocks. — The surface of
the earth is modified in
] physical appearance so
slowly or in such ways
that one is inclined to
create a mental picture
of the soil as a static or
fixed formation. Violent
natural changes are so
few or so seldom noticed that the transformations which we may
read in the rocks and soils as occurring through geologic ages
appear to be widely separated from the times we live in. Only
as our attention may become attracted by floods, earthquakes,
volcanoes, or glaciers do we begin to appreciate the fact that
physical forces are active in modifying the surface of the earth.

Such phenomena and others of a less apparent nature have
been active in producing the earth's surface as we know it, and
are active continually in creating new changes. The layer of fine

Fig. 1. — Profile of podsol soil with raw humus.
The light-colored leached layer (A2) so typical
of a podsol, is clearly apparent (from Tamm).

WEATHERING OF ROCKS

TABLE 1
Composition of a Gray Forest Soil* (from Glinka)

Horizon

Ax

A,

B

C

Description

Organic matter

or peat-like

material

Whitish
horizon

Brownish-
yellow

Granite

Soil Constituent
H2O at 100° C . . . .
Organic matter. . . .
Loss on ignition . . .

Si02

AI0O3

Per Cent

3.06

10.94

12.78

66.86

13.38

1.71

0.04

1.38

0.14

2.36

1 . 56

Per Cent
1.69
1.25
5.02
74.01
13.78
1.95
0.04
0.92
0.13
2.28
1.75

Per Cent
4.10
2.29
6.00
63.60
17.10
4.50
0.08
0.69
0.45
4.12
3 . 46

Per Cent
0.9S

1.21

74.87
13 82

Fe.03

1 92

Mn304

CaO

MgO

KoO

0.04
0.63
0.40
3 96

NaaO

2.62

* Podsol.

material has all originated from the compact rocks by slow proc-
esses of disintegration due to weathering, encouraged by the action
of waters, heat and cold, other atmospheric agencies, and biologi-
cal factors. Some of this material remains superimposed upon
the rocks from which it was formed; some becomes translocated
by waters and winds and finds its resting place at regions far
distant from the place where it originated. The disintegration
of rocks finally leads to an accumulation of granulated material
of the fineness of sands and clay. Soon after or even during rock
disintegration, and also greatly assisting this process, vegetation
springs up. The inorganic materials which are soluble in water
or dilute acids, such as carbonic, are then removed from the soil
by plants and percolating waters. Some of these materials again
enter the soil and become incorporated with it on the death of
the vegetation. Here also the plants undergo partial or complete
decomposition by microorganisms.

The chemical processes involved in the weathering of rocks are
those of hydrolysis, oxidation, hydration, solution, and carbona-
tion, or carbonate formation. The following reactions illustrate

4 THE SOIL AND THE PLANT

the chemical changes involved in the weathering of orthoclase
and olivine, two rock-forming minerals:

2KAlSi308 + 2H2O + CO2 = H4Al2Si209 + K2CO3 + 4Si02

Orthoclase Water Carbon Kaolinite Potas- Silica

dioxide sium

carbonate

12MgFeSiOi + 26H2O + 3O2

Olivine

= 4H4Mg3Si209 + 4Si02 + 6Fe203-3H20

Serpentine Silica Limonite

The soils thus owe their composition largely to the rocks from
which they are formed. Rocks are not homogeneous substances,
but aggregates of minerals which themselves are chemical entities
and which vary in complexity, from the elements, as graphite (C)
and free iron (Fe), to complex molecules like muscovite mica
(Al3KH2Si30i2). The relative abundance of any one or groups
of these minerals in the particular rock and the degree of their
consolidation determine not only the nature of the rocks but also
the soils which are formed from them.

TABLE 2

«
The Relative Abundance of Chemical Elements Existing in Greatest
Quantities in the Earth's Crust (from Clarke)

Element Per Cent

Oxygen 47 . 33

Silicon 27.74

Aluminum 7 . 85

Iron 4 . 50

Calcium 3 . 47

Magnesium 2 . 24

Sodium 2.46

Potassium 2 . 46

All others 1 . 95

Total 100.00

The common soil minerals contain only 21 of the known
chemical elements. Eight of these elements compose 98 per
cent of the total mineral matter of the earth's crust, as shown in
Table 2. The five elements, hydrogen, sulfur, carbon, titanium,
and phosphorus occur in many important minerals, and each
comprises from 0.1 to 0.5 per cent of the inorganic part of the soil.
The remaining eight (fluorine, chlorine, zirconium, boron, nitro-

WEATHERING OF ROCKS

gen, barium, manganese, and chromium) make up together only
0.35 per cent of all soil mineral matter.

It is worthy of note that carbon, one of the most important
elements in the life of plants and animals and which plays such
an important role as a source of fuel and in the synthesis of hun-
dreds of compounds used by man, makes up only a very small
fraction of the surface of the earth, as well as of the whole litho-
sphere, hydrosphere and atmosphere. The constant circulation
of this element in nature is necessary to keep life from becoming
rapidly extinguished, and it is the soil microbes that bring about
certain important phases of the transformation of this element.

Seventy-five per cent of all the sohd surface of the earth's
crust is composed of the two elements oxygen and siUcon, while
silica (Si02) as the compound and as combined in silicates makes
up 60 per cent of the crust. This silica thus comprises the major
part of the inorganic portion of the soil, which results from the
disintegration of the rocks, due not only to its being the most
abundant material in rocks but also as a result of its resistance to
solution by water or dilute acids excreted by plants or formed by
microorganisms in the soil.

Weathering agencies remove certain rock constituents quite
rapidly, and others only in very small amounts even after long
periods of time. Table 3 brings out further the changes which

TABLE 3

Chemical Composition of a Rock and of a Residual Soil

Formed from It (from Clarke)

Fresh rock

Residual soil

SiOa

Per Cent
60.69
16.89
9.06
1.06
4.44
2.82
4.25
0.62
0.25

Per Cent
45 31

A1003

26 55

FesOs and FeO

12 18

MgO

0 40

CaO

Trace

NaaO

0 22

KoO

1 10

Ignition

P.,Oi

13.75
0 47

Totals

100.08

99 98

6

THE SOIL AND THE PLANT

have taken place in the transformation of a micaceous gneiss to a
soil, by the decomposition processes which occurred in situ under
weathering agencies of a humid climate.

The general changes in the chemical composition during the
process of rock weathering and the formation of the earth's crust
consist in the separation of the silica and of the bases, the oxida-
tion of the compounds of iron, the removal of bases by processes of
leaching and replacement, the general hydration of the remaining
silicates, aluminum and iron, and a very appreciable addition of
organic matter coincident with the invasion of plants and of
microorganisms.

In order to characterize soils as to texture they may be ana-
lyzed mechanically, to separate the particles into groups of cer-
tain sizes including various sands, silt and clay. Typical mechan-
ical analyses of two soils are given in Table 4.

TABLE 4

Classification of Soil Particles and Mechanical Analysis of two

Soils*

Fraction

Fine gravel . . .
Coarse sand . .
Medium sand.
Fine sand . . . .
Very fine sand

Silt

Clay

Size

Millimeters
2.00-1.00
1.0 -0.50
0.50-0.25
0.25 0.10
0.10-0.05
0.05-0.005
0.005 and below

Fine sandy
loam

Per Cent

1

2

3
22
35
24
13

Clay

Per Cent

1

2

2

6
11
41
37

* On the basis of methods used at the U. S. Dept. of Agriculture.

Effect of Climate upon Chemical Composition of Soil. —
Climate is the most important factor determining the type of
changes brought about in the transformation of the rock con-
stituents and therefore in the development of the soil. Among
the various climatic factors, temperature and precipitation are
of major importance. Differences in climate affect the rate of
change, the course of mechanical and chemical transformation,

CLIMATE AND CHEMICAL COMPOSITION OF SOIL 7

the types and amount of vegetation, and the kinds of residues
resulting and accumulating in the soil. In general, the cooler the
temperature and the lower the precipitation, the slower the trans-
formation of the soil materials. Table 5 presents certain differ-
ences obtained by the determination of the average composition
of 466 soils in humid regions of the southern portion of the United
States as compared with the composition of 313 soils from arid
areas within the states of California, Washington and Montana.

TABLE 5

Comparative Chemical Composition of Soils of Humid and
Arid Regions (from Clarke)

Humid Soil

Arid Soil

Insoluble in HCl

Soluble SiOa

AI2O3

FejOg

Mn304

MgO

CaO

NazO

K2O

P2O5

SO3

Water and organic matter

Totals

Per Cent
84.03
4.21
4.30
3.13
0.13
0.23
0.11
0.09
0.22
0.11
0.05
3.64

100.25

Per Cent
70.57
7.27
7.89
5.75
0.06
1.41
1.36
0.26
0.73
0.12
0.04
4.95

100.41

It is quite apparent that arid conditions are conducive to the
accumulation of much more soluble substances than the humid
environment. These arid soils are frequently very fertile and owe
their scant plant growth entirely to the small amounts of water
which reach them. It requires only the introduction of moisture
to transform them from dry, brown deserts to flowering gardens.

The mineral compounds in the rocks undergo modifications
which result in the solution and fragmentation of the large aggre-
gates. Many of the more soluble substances initially present in
the parent materials, such as chlorides and sulfates of the alkalies,
are removed from the soil quite rapidly in humid regions and

8

THE SOIL AND THE PLANT

• Prairie
oTimber

finally reach the ocean, which becomes a vast reservoir of these
removed materials. In general the basic minerals enter solution
more rapidly than the acidic rock components. This weathered
soil material consists largely of silicates, aluminum and iron — a
mere skeleton of the parent material, but enriched by a clothing
of the organic residues of the decomposing vegetative cover.

The climate exerts even a more important influence upon the

organic than upon the
inorganic soil fraction.
The soils in cool and
moist regions are, as a
rule, richer in organic
matter than those in
warm and dry regions.
As shown in Fig. 2 the
nitrogen contents of soils
of cool regions are higher
than the nitrogen con-
tents of soils of warmer
regions. Since there is
generally a close rela-
tionship between the
nitrogen content of a soil
and the total amount of
organic matter, these
results may be inter-
preted as indicating the
presence of greater
amounts of organic mat-
ter in the cooler soils.
Not only is the total organic matter in the soil influenced by
climate, but also its chemical nature, such as the relation between
the elements carbon and nitrogen.

We may consider then that the soil is continually exposed to a
variety of influences which modify its physical structure, chemical
composition, and even its location. Strictly non-biological
factors may exert pronounced effects, but the development of
higher plants and microorganisms is to a large degree responsible
for the creation of fertile agricultural soils from the inorganic
substances.

N%

0,3

0.2

0.1

Wisconsin Illinois Ky. Tenn. Mississippi

40°

50°

60° Annual Temp. F.

Fig. 2. — Influence of temperature upon the ni-
trogen content of prairie and timber soils (after
Jenny).

SOIL FORMATION

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Soil Formation. — In the initial processes of formation of
soils, lichens, mosses and other small organisms attack and weaken
the rock constituents by re-
moving certain of their more
soluble elements or com-
pounds. Changes in tempera-
ture cause expansion and
contraction which open seams
and gradually form small frag-
ments which, when mixed
with the decomposing organic
remains of the first invaders,
supply footholds for the de-
velopment of larger plants. Fig. 3.— Physical structure of soil in rela-
In turn they further disinte- ^ion to root development. Schematic

, , V 1 J. • 1 1 representation of the sohd soil particles,

grate the rock materials by r\.u ■ i r ^ i i I

° ''or the au- spaces and of root development

physical and chemical forces. (from France).

Gradually the organic residues

of these plants become mixed with the coarse and fine rock
materials, thus giving rise to the beginnings of agricultural soils
(Fig. 3).

The abrasion of soil particles is carried out further by the
action of air, water and ice, thus adding to the disintegrated
material. IVIuch of this fine substance reaches the lowlands by
water and ice removal and tends to fill in the valleys. Here,
temperature conditions being more conducive to plant growth, a
vegetation develops, the abundance of which is determined
principally by the available moisture. The repeated develop-
ment of plants and the incorporation of their remains with the
soil, where they undergo partial disintegration, finally produces
a material having very few characteristics in common with the
rocks from which it originated. The greatest modification takes
place near the surface, where pronounced disintegration and
chemical changes have been produced. This region is exposed to
the most marked processes of leaching and receives also the largest
amount of plant residues. The deeper layers of soil, or the
so-called subsoils or B horizons, contain much less organic matter
and consequently consist almost entirely of mineral substances
some of which may have originated from the surface and accumu-
lated at deeper zones as the result of leaching. Below this

10 THE SOIL AND THE PLANT

material, large partially disintegrated aggregates of the parent
rock are found superimposed upon the solid bedrock.

The organic substances are important in determining the
physical properties of soils. They are largely colloidal in nature
and thus have pronounced absorptive properties. This charac-
teristic makes their presence particularly desirable in the coarse,
open, sandy soils which are so readily leached by percolating
water. To soils containing large amounts of fine particles, as
clay, the organic residues give a more open granular structure,
so necessary for proper cultural treatments, for the penetration
of gases and for the movement of water.

The soil which has undergone changes through numerous
generations is thus found to consist of an inorganic framework,
of coarse and small particles, some existing separately but most
occurring as aggregates, surrounded with a colloidal jelly-like
layer, made up of very fine inorganic materials and substances of
organic origin. This colloidal material is extremely fine, being
smaller than 0.00004 of an inch in diameter. The average size
of the colloidal soil particles appears to be close to 0.000004 of
an inch. Such particles are only visible with the most powerful
microscopes. It is these particles which determine to a large
degree the physical properties of the soil. The tendency of
soU particles to adhere in a plastic mass is largely determined
by them. By reason of the large surface exposed per unit
weight they have great absorptive capacity for gases, liquids
and dissolved substances, and to them we owe the characteristic
of soils to retain water and basic substances. The spaces between
the solid particles are filled with water and gases. Diffusion
tends to bring these gases to the same composition as the normal
atmosphere, but there are always marked differences between the
two. The speeds of decomposition of organic materials and per-
meability of the soils largely determine their differences. Since
carbon dioxide is formed in large amounts in soils by the decom-
position of organic materials, and since oxygen is consumed in
the process, there are larger amounts of carbon dioxide and smaller
amounts of oxygen in the soil air than in the normal atmosphere.
Where penetration of air is greatly retarded, other gases such as
methane and hydrogen, may appear in considerable quantities.

Movement of Water in Soil. — The soil air may be partly
replaced by water, which forms films around the solid par-

ORGANIC MATTER OF SOIL 11

tides. At times, the water may entirely fill the pore spaces,
pressing out all of the air. This film of water, or the free water,
carries in solution the minerals which are dissolved from the
inorganic soil constituents, the carbon dioxide and other sub-
stances produced from the decomposition of the organic matter.
This film of water forms the soil solution. The growing plants
obtain the nutrients necessary for their growth by absorbing
them largely from the solution by means of their roots and root
hairs, which penetrate between the inorganic soil particles.

Some of the plant nutrients are only slightly soluble in water
or in the weakly acidified aqueous solution, and may be present,
therefore, in the soil solution only in small amounts at any one
time. However, as these minute quantities are removed by the
growing plant or by drainage waters they are replaced by further
solution from the crystalline or colloidal soil materials.

Water moves in soils in many ways, depending upon its
abundance, the physical condition of the soil and the vegetation.
Where large amounts reach the soil in a short period of time,
much of it may disappear by running off from the surface into
drainage channels. Some of the water penetrates the soil and,
where there is more than the soil particles can hold about them-
selves, it sinks to the level of the water table by precolation.
When moisture is not descending upon the soil there is loss by
evaporation from the surface and transpiration from the leaves of
the vegetative cover. This water may be drawn up from the
lower levels of the soil by capillary forces as more and more water
is lost from the surface. Hea\y rainfall alone may not solve
the problem of supplying water to plants. The solution fies
rather in the proper distribution of the water in the soil over the
period of time in which the plants develop. In humid regions
the problem may become one of drainage; in regions of very low
rainfall, irrigation may be resorted to. A combination of ade-
quate drainage and irrigation systems makes possible almost
complete control of the soil water, but unfortunately such equip-
ment is too expensive for general practical application.

Organic Matter of Soil. — In addition to the soHd inorganic
particles or the mineral framework, the soil also contains solid
organic particles, namely, roots and sloughed-off portions of
roots, residues of stems, leaves and branches, as well as numerous
organic complexes which have originated by the partial disin-

12

THE SOIL AND THE PLANT

>^

tegration of the plant materials. To this is added a large quan-
tity of organic matter in the form of bodies of microorganisms
and their various decomposition products.

In addition to the mineral soils, which are predominantly
inorganic (95 per cent), there are soils which are largely organic
in nature. Here belong the peat soils, which originate from bogs.
These soils contain 30 to 98 per cent organic matter and only 2
to 70 per cent inorganic material. The surface layers of certain
types of forest soils are also predominantly organic in nature.
These soils are characteristically organic, since the accumulation
of plant materials takes place more rapidly than the decomposition
of these residues by microorganisms, due either to saturation
with water (as in peat bogs) or high acidity of soil combined with
other factors unfavorable to the activities of the organisms.

Soil Phases. — The soil thus consists of three definite phases

(see Fig. 4): (1) The
solid phase, which, in
the case of mineral
soils, is composed
largely (up to 99.5
per cent) of inorganic
rock constituents and
only to a limited ex-
tent (0.5 to 10 per
cent) of organic ma-
terials; in the case of
peat soils, the organic
matter may make up
30 to 99 per cent of
the solid phase of the
soil constituents on
a water-free basis.
(2) The liquid phase,
consisting of water
which contains in
solution various or-
ganic and inorganic

<S45

35

25

15

Air

Water

Organic Mineral
Matter
Fig. 4. — The relative distribution of materials
which compose a soil under conditions favorable
to plant growth.

materials. This water may be combined with the soil colloids
as hygroscopic water which is held very tenaciously or it may be
present as capillary film water or as free gravitational water.

MICROBES IN PLANT GROWTH

13

It may make up 5 per cent (in the case of sands) to 93 per cent
(in the case of peats) of the total volume of soil. (3) The gaseous
phase, or the soil air, which may differ greatly in composition
from ordinary atmosphere, because of the higher content of
carbon dioxide, lower content of oxygen, and frequently the
presence of some methane and hydrogen.

The existence of these three phases alone would hardly be
sufficient to make the soil a medium fit for the growth of higher
plants. The presence in the soil of another factor, namely, the
microbes, makes the soil a living medium and renders it dynamic.

Fig. 5. — Schematic representation of the relation of microorganisms to the
phj-sical structure of soil (from France).

Role of Microbes in Plant Growth. — The soil may be
looked upon not only as a medium supporting growth of higher
plants but as a complex natural environment inhabited by a popu-
lation more diverse and active in a greater variety of affairs than
the visible inhabitants of the earth. Economically the major
interest in soils is concerned with the growth of higher plants,
but, since there is such a close correlation between the develop-
ment of plants and microbial activities, a comprehensive appre-
ciation of the microbial life is of much more than academic interest.

These microbes are largely responsible for the numerous

14 THE SOIL AND THE PLANT

chemical changes constantly going on in the soil, principally as
agents of destruction of the complex organic molecules synthe-
sized by plants and transformed by animals. Coincident with
this destruction is the construction or formation of new com-
pounds. Some of the end products of such transformations are
inorganic or mineral materials. There is no breaking down that
is not accompanied by a proportionally equivalent building up.
The activities of microorganisms upon the mineral constituents
of soils are numerous and varied. These may be of the nature
of oxidation, reduction, hydrolysis, and carbonation.

Were these microbes eliminated from the soil, the organic
matter constantly added would accumulate until all the combined
nitrogen, phosphorus, and some of the potassium would be in
that form. These elements which are so important for the
growth of cultivated and uncultivated plants would not be avail-
able to a new crop, simply because plants cannot utilize in any
amounts materials in complex organic forms, but must have them
supplied as simple mineralized substances. To understand these
relationships we must know something of the nutrition of the
plants and of their composition.

The Nature of Plant Nutrients. — All plants require for
their nutrition the elements, carbon, hydrogen, oxygen, nitrogen,
phosphorus, potassium, calcium, magnesium, iron, sulfur, and
possibly some other elements in small amounts (see Fig. 6).
Very few of these elements, however, become factors hmiting
plant growth as a result of their presence in insufficient amounts
in soils. This is due to the fact that either many of the elements
are required by plants only in very small amounts or because
some of these elements exist in soils in considerable abun-
dance. Under continuous culture of soils in humid regions it is
frequently necessary to replenish the store of three elements in
particular — nitrogen, phosphorus, and potassium, and occasionally
sulfur. These are required by plants in greater amounts than
any of the other mineral elements obtained from the soil, and,
therefore, become depleted most rapidly. To overcome this
deficiency a great variety of substances are utilized. Nitrogen
is added in such inorganic forms as compounds of ammonia,
nitrate, cyanamid, as well as in organic substances such as urea,
packing-house refuse, and guano. This last is the natural com-
post of excrements and carcasses of such animals as bats and

THE NATURE OF PLANT NUTRIENTS

15

sea-fowls. Besides nitrogen, it generally also contains considera-
ble quantities of both phosphorus and potassium. The recently-
developed industries fixing nitrogen from the air have supplied
products which largely replace the natural substances. These
may be obtained as substances containing ammonia, cyanamid,
nitrate, and urea. Certain plants, as the legumes, may draw
upon the store of nitrogen in the air through the agency of bac-

Indispensable in appre-
ciable amounts

ELEMENT

Carbon

(C)

Oxygen

(0)

Hydrogen

(H)

Nitrogen

(N)

Phosphorus (P)

Potassium

(K)

Calcium

(Ca)

Magnesium (Mg)

Iron

(Fe)

Sulfur

(S)

Manganese

(Mn)

Silicon

(Si)

Boron

(B)

Zinc

(Zn)

Fluorine

(F)

Iodine

(I)

Chlorine

(CI)

Aluminum

(Al)

Copper

(Cu)

PRINCIPAL SOURCE

From the carbon dioxide of
the atmosphere.

From oxj'gen and carbon di-
oxide of the atmosphere
and from water.

From Mater.

From nitrate or ammonia
in soil. Can be obtained
from gaseous nitrogen by
leguminous plants.

From phosphates in soil.

From salts in the soil.

From salts in the soil.

From salts in the soil.

From ferrous or ferric salts
in the soil.

From sulfates in the soil.

• From salts in the soil.

Indispensable in small
amounts, exerting
stimulating effects or
correcting soil condi-
tions

Fig. 6. — Sources of plant nutrients.

terial action. These plants are used quite generally as cover
crops which, when plowed into the soil, greatly increase its nitrogen
content. The return of barnyard manures to soils not only adds
nitrogen but also both phosphorus and potassium.

Phosphorus is most frequently introduced into the soil as the
relatively insoluble tri-calcium phosphate, as the soluble super-
phosphate, or as basic slag, which is a by-product of the steel

16

THE SOIL AND THE PLANT

industry. Ground bone may also be utilized as a source of this
element. Potassium is used as the sulfate and chloride which are
obtained from natural salt deposits.

Continuous cultivation of soils may lead to a depletion of the
basic substances and to the development of an acid reaction,
particularly where artificial fertilizers are injudiciously used.
Both of these conditions are unfavorable to the development of
many of the important agricultural plants. To offset these
effects, calcium is applied in various forms such as the oxide,
hydroxide, or carbonate. Its addition to the soil is not so much
for the purpose of overcoming a deficiency of an element
required for plant growth as to correct certain other unfavor-
able soil conditions.

An idea of the abundance of some of the most important
constituents in soils may be derived from Table 6. Although

TABLE 6

Concentration of Important Constituents in Mineral Soils
(from Lyon and Buckman)

Constituent

Percentage
range

Average,
per cent

Pounds per acre in
upper 6 inches (on
basis of 2,000,000

pounds
of soil per acre)

Organic matter

1.00-10.00
0.05- 0.50
0.01- 0.40
0.50- 4.50
0.05- 1.50
0.05- 0.50

4.00
0.20
0.15
2.00
0.40
0.15

80,000

Nitrogen (N2)

Phosphoric acid (P2O0) ....
Potash (K^O)

4,000

3,000

40,000

Lime (CaO)

8,000

Sulfur trioxide (SO 3)

3,000

these data suggest that a soil may have sufficient nutrients to
supply the requirements of large crops for many decades, this
conclusion is not justified. Very small amounts of these sub-
stances are available to plants at any one time. Most of the
nitrogen is locked up in organic compounds which are slowly
decomposed by microbes. The phosphorus may exist in organic
combination, in mineral materials such as apatite, and as other

ABSORPTION OF NUTRIENTS BY PLANTS 17

compounds of calcium, iron, or aluminum. Most of these com-
pounds are only slightly soluble. Crops which are grown on
soils which contain large total amounts of phosphorus frequently
develop much better following the application of phosphatic
fertiUzers. Similarly, the potassium, calcium, and sulfur exist
in combinations from which they are not liberated rapidly into
the soil solution.

Some soils contain such an abundance of basic salts that plani
growth is markedly depressed. The influence of this condition
on plant development may be due to at least two effects: (1)
In the so-called alkali soils of arid regions the concentration of
soluble salts may be so great that the plants fail to grow. (2)
In other soils the reaction may be so basic that iron will not go
into solution in amounts sufficient to satisfy the requirements of
the plants ; this brings about a yellowing or mottHng of the green
surfaces of the plants, stunts the growth and may lead to
death.

Absorption of Nutrients by Plants. — Carbon is taken from
the carbon dioxide which is present in limited concentrations
(0.03 per cent) in the atmosphere. Only green plants, which
contain chlorophyll and which can utihze, by photosynthesis,
energy of the sun, and a few bacteria, are able to use this source
of carbon. The carbon dioxide is reduced in the leaves and com-
bined with water to give formaldehyde, which is then built up to
form carbohydrates. Soil activities are particularly important
in this process, even if indirectly, by replenishing the carbon
dioxide of the atmosphere by a constant stream of the gas origi-
nating from the activities of the microbes in the soil. Oxygen
comes from the carbon dioxide obtained from the air, from the
water drawn from the soil, and from numerous other substances.
Hydrogen is derived principally from water.

Nitrogen in various combinations is absorbed by the plant
roots from the soil. Although from 75 to 80 per cent of the
gases of the atmosphere consists of elemental nitrogen, none of it
can be used by the plants, since they can assimilate only com-
bined nitrogen in the form of ammonia (NH4+) or nitrate
(NOs"). Plants store in their tissues considerable quantities of
nitrogen, which becomes available to subsequent plant growth
only after these tissues undergo decomposition through the
agency of the soil microbes. Very few plants (legumes) are

18 THE SOIL AND THE PLANT

able to make use of the vast store of gaseous elementary nitrogen,
but even here they require the cooperation of soil organisms.

The elements phosphorus and sulfur are present in the soil
both in organic and in inorganic forms. They undergo various
transformations in the soil through the activities of microbes,
either directly or indirectly, before they can be assimilated by
plants. Here again, plants cannot assimilate appreciable amounts
of phosphorus either in the form of organic compounds or in the
form of insoluble rock phosphate. In the first case, it has to be
decomposed by microbes; in the second case, it is made soluble
by the various inorganic and organic acids formed by the soil
microbes.

Potassium, iron, magnesium, and calcium are basic constituents
of soils which are required by plants in certain amounts. Potas-
sium frequently occurs in available forms in amounts in-
sufficient to satisfy the requirements of plants. Calcium and
magnesium are recjuired by plants in smaller quantities, but they
play important roles in the neutralization of soil acids and in the
coagulation of the dispersed soil particles. All of these elements
are obtained by the plants from the soil, and all of them are
changed from one form to another, directly or indirectly, by the
soil organisms.

Higher plants may be grown in the absence of other forms of
life and in the absence of organic materials under artificial con-
ditions when they are supplied with the necessary salts as they are
required. Such conditions never develop in the natural habitats.
Here the soil organic matter acts as a reservoir from which many
of the elements required for development of the plant are supplied
throughout the growing season. The mineral fraction of the soil
slowly furnishes other elements to the soil solution, partly as
a direct result of microbial activities. This may be brought about
either through the solvent action of carbonic or other acids or by
the oxidation or reduction of these minerals.

Summary. — Four distinct relationships between soils, plants,
and microbes should be considered: namely (1) the role of microbes
in soil formation and soil transformation; (2) the role of microbes
in the liberation of nutrients for plant growth; (3) the soil as a
medium for the growth of microbes; (4) the role of higher plants
in supplying nutrients for microbes.

Through their activities, microbes liberate large quantities of

SUMMARY 19

carbon dioxide, various organic acids as well as certain inorganic
acids (notably nitric and sulfuric) which have a marked solvent
action upon rocks and rock constituents and thus assist materially
in the weathering processes which lead to soil formation. By
assimilating certain chemical elements or compounds, by chang-
ing the nature of other chemical compounds, by the processes of
decomposition of organic matter, as well as by the synthesis of
new organic complexes, microbes play a very prominent part in
the constant transformation of the soil.

The elements which plants require for their nutrition come
directly or indirectly from the soil. The microbes play prominent
roles in transforming these elements into forms available for
plants. Out of these elements the plants synthesize their tissues:
the roots, the stems, the branches, the leaves, the grain, and the
fruits. Only a part of these plant products are utilized by men
and animals for their food. Another, frequently the larger part,
is returned to the soil. Even the very bodies of men and animals
that live by consuming plant substances will sooner or later also
return to the soil, and with them a large part of the elements
removed from the soil through the agency of the plants.
These are acted upon by the soil microbes, liberating the important
elements, carbon, nitrogen, phosphorus, sulfur, and potassium,
into circulation, where they are again available for plant growth.

The soil is an excellent medium for the development of microbes
since it contains all the elements essential for their activities.
The plant and animal residues offer suitable sources of energy to
keep the various microbes in a state of constant activity and repro-
duction. The relationships between soils and plants, on the one
hand, and the microbes, on the other, are mutual. The soil and
plant supply the medium for the growth of the microbes, and the
energy and other nutrients for their activities and reproduction;
the microbes carry out the processes which keep the elements in
constant circulation, thus enabling the plants to develop with a
limited supply of nutrients.

20 THE SOIL AND THE MICROBE

LITERATURE

1. Clarke, F. W. The data of geochemistry. Bulletin 695, U. S. Geological

Survey. Washington, 1920.

2. Glinka, K. D. The great soil groups of the world and their development.

Transl. C. F. Marbut. Edward Bros. East Lansing, Michigan,
1928.

3. HiLGARD, E. W. Soils. The Macmillan Co. New York, 1906.

4. Lyon, T. L., and Buckm.^n, H. O. The nature and properties of soils.

The Macmillan Co. New York, 1929.

5. Ramann, E. The evolution and classification of soils. Transl. C. L.

Whittles. Hetfer. Cambridge, 1928.

6. Russell, E. J. Soil conditions and plant growth. 5th ed. Longmans,

Green & Co. New York, 1927.

7. WoLFANGER, L. A. The major soil divisions of the United States. J.

Wiley & Sons. New York, 1930.

CHAPTER II
THE MICROBE AND ITS ACTIVITIES

The Microbe and its Importance in the Soil. — The
various forms of life which develop directly at the expense of the
chemical constituents of the soil and the decomposing plant and
animal residues are extremely numerous and diverse. Included
among these forms are all plants, ranging from the large trees to
the tiniest of microbes, with an almost limitless number of tran-
sition forms bridging this wide gap, and besides these are various
representatives of the animal kingdom. Indirectly, all animal
forms depend for their nutrition upon the elements which are
liberated in the soil; the microbes of the soil keep the elements in
circulation. Were it not for them, all plant and animal life would
soon cease because of the exhaustion of the supply of available
elements. A knowledge of these microbes and their acti\'ities is,
therefore, of prime importance for the understanding of soil
processes and plant growth.

The study of the nature and activities of soil microorganisms
would be of considerable interest even if the existence of these
organisms had no great influence upon the growth of other plants
and animals. Their identity, habits of growth, and influences
become of much wider interest and extreme importance with the
realization that to these tiny living things w^e owe the continued
development of higher plants. Studies of the reactions with which
they are associated in their strife for existence, and the variety
of influences which they exert directly and indirectly upon other
living organisms, open a fascinating chapter in biology.

The microorganisms are the least differentiated of all forms of
life — they are organisms either unicellular in structure or, at the
most, simple aggregates of a few cells with comparatively minor
differentiation. They are too small to be seen as individuals
with the naked eye, and in some cases too small to be seen even
with the aid of the most powerful microscope, and are known

21

22

THE MICROBE AND ITS ACTIVITIES

only through their activities, as in the case of the bacteriophage
and filterable viruses. However, they vary considerably in the
chemical processes that they bring about in the soil and in arti-
ficial cultures.

The importance of a study of the nature and development of
higher plants is accepted without question because these plants
are so evident in appearance and in general usefulness to man and

his domesticated animals. In
a similar manner, one has come
to recognize the importance of
those microorganisms which
have been discovered to be
the agents responsible for the
various diseases of man, domes-
tic animals, and plants, and
the need for combating them.
However, the microscopic
forms of life in the soil are so
obscured by their environment
that they are not given such
general consideration. They
are frequently given no atten-
tion or considered to play an
insignificant part in natural
processes. The transformations
with which they are concerned have been at times completely
overlooked or called non-biological. Provided microbes were not
generally distributed in soils, their presence in certain localities
would be very apparent from the enhanced plant growth. Under
the prevailing conditions, however, it might be considered that
their very ubiquity tends to conceal their activity.

These soil organisms are largely saprophytic, acting upon the
organic plant and animal residues and the inorganic soil con-
stituents. Occasionally certain of the representatives may
attack living tissues of the higher plants ; the soil may also harbor
a number of animal parasites. As compared, however, with the
saprophytes, either in numbers, kinds, or importance, these occa-
sional parasites are quite insignificant, except in certain special
instances.

A qualitative survey of the microscopic organisms found in the

d {)OOoOOO

Fig. 7.- — Heterotrophic, spore-forming
bacterium, Bacillus megalhermm; (a)
young cells showing flagella, (b) young
cells showing connections into chains of
rods, (c) older cells, (d) variations in
size and shape of spores (after Conn).

NATURE OF SOIL MICROBES

^3

soil discloses a heterogeneous assortment of forms belonging to no
single classified group, either physiologically or morphologically.
They vary considerably both in their general appearance and in
the nature of the processes which they bring about. The indi-
vidual organisms are so small that they cannot be seen with the
naked eye and their structures can be studied only by means of
powerful microscopes. However, a mass of growth of micro-
organisms, frequently referred to as a colony, can be obtained on

Fig. 8. — Aerobic cellulose-decom-
posing bacterium, Cytophaga lutea
(from Winogradsky).

Fig. 9. — Anaerobic, cellulose-decom-
posing bacterium, Bacillus cellulosae
dissolvent (from Khouvine).

natural or artificial substrates, large enough to be studied with
the naked eye.

Nature of Soil Microbes. — Both in respect to numbers and
abundance of cell substance in the soil, most of the microbes
belong to the plant kingdom. These include various bacteria,
actinomyces or ray fungi, a number of true fungi including the
molds and the mushrooms, as well as various green and blue-green
chlorophyll-bearing algae. The animal kingdom is numerically
less abundant in the soil. Representatives include the protozoa,
the nematodes or round-worms, the rotarians or wheel animal-
cules, a number of earthworms, and larvae of insects. In view of
the fact that, in the case of some microbes, it is difficult to decide
whether they should be classified with plants or with animals,

24

THE MICROBE AND ITS ACTIVITIES

Fig. 10. — Aerobic nitrogen-fixing bac-
terium, Azotobacter chroococcum (from
Krzemieniewski) .

they have frequently been referred to as Protista (first hfe). All
these microbes have one thing in common, namely, they are all

very small, and, with the ex-
ception of the higher animal
forms, such as earthworms and
larvae, and the fruiting bodies
of mushrooms and certain
other fungi, they cannot be
seen with the naked eye.

Various methods have been
developed for the study of soil
microbes. These include (1)
microscopic methods for the
study of the organisms as
found in the soil; (2) cultural
methods for the study of the
activities of the microbes in
the soil itself; (3) methods for
the enumeration of different
groups of soil microbes; (4) methods for the isolation and culti-
vation in pure culture of
various microbes.

In order to understand
the role of a certain microbe
in a known soil process, it
is desirable to isolate this
microbe from the soil,
grow it in pure culture on
artificial media, then re-
inoculate it into sterile soil
and bring about the proc-
ess in question under con-
trolled conditions. A pure
culture of a microbe is an
isolated cell which repro-
duces a number of times
giving identical cells. Such
a culture allows an ex-
aggeration of the activities of the particular microbe, so as to
make accessible to observation the elementary principles of cell

Fig. 11. — Anaerobic nitrogen-fixing bac-
terium, Clostridium pastorianum (from Omeli-
ansky and Solounskoff ) .

NUTRITION OF SOIL MICROBES

25

^cr

^

nutrition. It is not always possible or even necessary to isolate
a soil organism in pure culture; valuable information concerning
soil processes may be obtained even with so-called crude cultures.
For physiological investigations, however, pure cultures of organ-
isms are very desirable and frequently necessary.

Nutrition of Soil Microbes. — The microbes bring about in
the soil various transformations which result in the liberation of
plant food in forms available to cultivated and uncultivated
plants. They find the soil a favorable medium for their activi-
ties, and also obtain from the soil a sufficient supply of energy and
nutrients required for
growth and reproduction.
Most of the numerous
microbes which inhabit
the soil are useful, while
some are or may become
harmful from the stand-
point of the practical agri-
culturalist. Since the
activities of soil microbes
are so closely associated
with plant growth, an
economic management of
the soil requires a thorough
understanding of these ac-
tivities and their direction
into the proper channels, f''V 12^- Sy^J^iotic nitrogen-fixing bac-
. terium; different strains of Bacterium radi-
SO as to reduce the m- ^jg^fa obtained from: (1) garden bean, (2)
jurious effects to a mini- goats rue, (3) vetch, (4) alfalfa (after Shunk).
mum and increase the

useful activities to a maximum. Frequently the development of
toxic or unfavorable conditions for plant growth may be com-
pletely avoided or quickly overcome if, in the treatment of the
soil, the activities of the soil microorganisms and not alone the
higher plants are taken into consideration.

In nutrition, microorganisms have much the same require-
ments as higher forms of life, but their existence as single cells
is much simpler than that of their multicellular associates. All
microbes require for their growth and synthesis of cell substance,
supplies of energy and several nutritive elements, essential for

\

r^

26 THE MICROBE AND ITS ACTIVITIES

the building up of their cells, including carbon, hydrogen,
oxygen, nitrogen, phosphorus, potassium, sulfur, and a few
others.

With the exception of the algae, all the microbes of the soil
are devoid of chlorophyll. They are thus compelled to derive
their energy either from the oxidation of simple inorganic sub-
stances, as in the case of the limited groups of autotrophic bacteria,
or from complex organic substances, as in the case of the majority
of bacteria or the heterotrophic organisms, and of all fungi and
protozoa. The great majority of soil organisms are thus dependent
upon the complex organic substances of the soil for their carbon and
energy supply. The algae and autotrophic bacteria can obtain their
carbon from the carbon dioxide of the atmosphere, the former using
by means of their chlorophyll the energy of the sun, and the latter
using energy which is liberated in the oxidation of simple inor-
ganic substances, such as ammonia, sulfur, or hydrogen. The
elements hydrogen and oxygen are present in sufficient amounts
in water and in the gases of the atmosphere to supply all the needs
of the microbes. Frequently, however, conditions arise (such as
deficiency of elementary oxygen) under which microbes obtain
their oxygen from inorganic compounds of nitrate or sulfate.

One of the most important elements in the nutrition of microbes

is nitrogen. This is utilized by

i\ the microbe either in the form
w C of complex organic substances,

^ \ ^ W such as proteins and amino
I ^ J acids, or as simple inorganic

X ^ compounds, such as nitrates or

ammonium salts. Certain
limited groups of bacteria are
capable of utilizing the gaseous

atmospheric nitrogen. The
Fig. 13. — Nitrite-forming bacterium, , , , . i . • i i_

^r.. , f. „r. other elements are obtamed by

JSIitrosomonas europea (alter Wmo- "^

gradsky). tlie microbes from the minerals

of the soil, phosphorus being
used in greater quantities than any of the others.

In the utilization of the organic materials incorporated in the
soil, the microorganisms make use of an elaborate store of enzymes,
or organic catalysts. These may be active in attacking the
insoluble protein or carbohydrate molecules, or in oxidizing.

f-v

BACTERIA OF THE SOIL

27

reducing, or hydrolyzing the numerous substances which serve as
sources of energy and as materials for cell construction.

Bacteria of the Soil. — The most abundant group of organ-
isms in the soil are the bacteria, exceeding both in numbers and
in the variety of their activities all the other soil organisms, but
not, however, in the bulk of the organic cell substance found in the
soil in the form of living and dead microbes. The fact that
bacteria are so abundant and that they participate in numerous
soil processes is largely responsible for the frequent reference to
the total microscopic population of the soil by* the term bacteria
of the soil, and reference to the whole subject of soil microbiology
as soil bacteriology. These bacteria are so small that if 500 to 1000
of them were placed in one hne, end to end, they would only
extend for a length of one millimeter. They are in general about
the size of colloidal particles, the maximum size of which is about
5 to 8 microns — one micron being 1/10,000 of a centimeter. It
takes about 1,000,000 to 1,000,000,000 of them to weigh one
milligram, or one-thousandth part of one gram; even then this
mass of cell material
will be found to consist
of 80 to 85 per cent of
water.

It is not exceptional
to find hundreds of
millions of bacterial
cells per gram of soil,
particularly where or-
ganic substances have
been added. Let us
assume that there are
one hundred million
bacteria in one cubic
centimeter of soil and
that each cell occupies
only one cubic micron
of space. Since there
are 1,000,000,000,000 cubic microns in one cubic centimeter,
and since the one hundred million bacteria would occupy only
100,000,000 cubic microns, the cells would occupy only one-ten
thousandth of the total volume of the soil. It is not surprising

Fig. 14. — Nitrate-forming bacterium, Nitro-
bader sp. (from Fred and Davenport).

28

THE MICROBE AND ITS ACTIVITIES

that the activities of such large numbers of hving things may
continue unnoticed in a small particle of soil and become re-
vealed only by a careful search using a highly specialized
technique.

These minute organisms are capable of bringing about very
rapid and extensive transformations of the substrates upon which
they are growing. A few hours are sufficient for the complete
decomposition of a quantity of sugar to carbon dioxide and water,
or of protein to ammonia, carbon dioxide, water, and other com-
pounds. The extent of the development of these microbes is
limited only by the supply of available energy, by the environ-
mental conditions favorable or unfavorable to their development,
and by the formation of certain products injurious to their own
activities. Although modifications in the supply of oxygen,
moisture, and of inorganic compounds, or a change in tempera-
ture, may affect the develop-
ment of the soil population,
the greatest response in activ-
ity is brought about by the
incorporation of organic sub-
stances with the soil.

In general, bacteria are not
easily differentiated morpho-
logically. The shapes which
they may assume are very
limited in number, and one
must rely largely upon physio-
logical distinctions for the
identification of their numer-
ous species. The soil bacteria
are divided into three main
groups: (1) The cocci or the
spherical forms. (2) The hacilli or the rod-shaped forms, with
rounded or square ends, differing both in length and in thickness.
Some of them are able to form spores which are more resistant
to unfavorable environmental conditions, such as dryness, changes
in temperature, and action of antiseptics, than the vegetative cells;
those rod-shaped bacteria which are unable to form spores
are less resistant to adverse changes in environment. Some of
these bacteria depend upon the movements of the fluids in which

Fig. 15. — Sulfur-oxidizing bacterium,

Thiohacillus tidooxidans (from Waks-

man).

BACTERIA OF THE SOIL

29

they live to transfer them, while others are able to migrate by-
means of their organs of locomotion, namely, the flagella. (3)
The spirilla are thin spiral-shaped forms, which have different
numbers of turns characteristic for the different species; those
forms which produce only one-half turn are intermediate between
rods and spirilla, and are spoken of as vibrios. Numerically, the
rod-shaped forms are much the largest group of the soil bacteria.

Figs. 7 to 16 show several typical soil bacteria. Fig. 7 repre-
sents the general group of spore-forming, rod-shaped, hetero-
trophic bacteria, living by the processes of decomposition of
various organic compounds in the soil. Figs. 8 and 9 represent
two typical bacteria capable of decomposing cellulose, one being
aerobic and one anaerobic in nature, living in the presence and
in the absence of atmospheric oxygen, respectively. The aerobic
and anaerobic non-symbiotic nitrogen-
fixing bacteria are shown in Figs. 10
and 11. Species of symbiotic legume
bacteria are shown in Fig. 12. The
important groups of nitrifying bacteria,
namely, the nitrite- and nitrate-forming
organisms, are shown in Figs. 13 and 14.
Bacteria capable of obtaining their en-
ergy from the oxidation of elementary
sulfur to sulfuric acid are shown in
Fig. 15. A bacterium which can re-
duce sulfates to sulfides is represented
in Fig. 16. The activities of some of
these bacteria and their roles in soil
processes will be described in subse-
quent chapters.

Nearly all the bacteria are so small
that they are able to pass through ordi-
nary filters. To remove them from liquids, filtration is accom-
plished by the use of Chamberland candles made of unglazed
porcelain, or Berkefeld filters made of diatomaceous earth.
The pore spaces of these filters are so small that they will pre-
vent the passage of bacteria which are \isible with the micro-
scope. It has been estabUshed, however, that there are certain
ultra-visible bacteria which can even pass through these filters.
These are spoken of as ultramicroscopic orga7iis7ns. Such tiny

Fig. 16. — Sulfate-reducing

bacterium, Spirillu7n desul-

furicans (from Beijerinck

and Omeliansky).

30

THE MICROBE AND ITS ACTIVITIES

bits of life appear to be incapable of existing upon inert material
and, so far as is known, can develop only in conjunction with living
cells. The presence of such organisms in soil is speculative for
the most part. There is, however, a certain kind of ultramicro-
scopic substance parasitic upon bacteria — the bacteriophage —
which is of common occurrence in soil. It has been found that
these organisms are about the size of a protein molecule, or only
20 to 30 millimicrons (mm) in diameter. Some appreciation of
this infinitesimal size may be gathered from the fact that 1 mm is
equal to 1/1,000 n, or 1/10,000,000 of a centimeter. It has also
been shown that the ultramicroscopic organism causing mosaic
diseases in plants may survive in soil for a short period of time.

Fig. 17. — Penicillium sp.: (c, d) ter-
minal sporulating filaments highly mag-
nified; 0', k, m) lower magnifications
of sporulating organs (from Thom).

Fig. 18. — Aspergilhis sp. showing

various sporulating characteristics

and differences in sizes of spores

(from Henrici).

Activities of Soil Bacteria. — The activities of the bacteria,
especially those concerned with the decomposition of organic
matter, are frequently referred to by the layman as processes of
"putrefaction," "fermentation," and "decay." These terms
(with the possible exception of the use of the term "fermentation"
as apphed to the decomposition of carbohydrates under anaerobic
conditions) are significantly important only historically. In times
past they were used in reference to a variety of processes previous
to an understanding of the nature of the chemical changes and the
biological agents which were involved. These terms will, there-
fore, be carefully avoided in the following pages.

ACTIVITIES OF SOIL BACTERIA

31

Some of the soil bacteria are strictly aerobic in nature, devel-
oping only under conditions where there is free access of air;
some exist in the complete absence of oxygen, under so-called
anaerobic conditions. Most of the soil bacteria develop best at
reactions close to neutraUty, where there is no appreciable excess
acid or base. Some, however, can exist at a much higher acidity
than others. Thermophihc bacteria, or those which are favored
by high temperature, such as 50 to 60° C, also occur in soils, but
the great majority of the soil bacteria grow best at reactions
between 20 and 30° C. All the bacteria require a considerable
supply of water to permit their active
development. Under dry conditions, they
either change to resistant spores, become
dormant vegetative cells in the moist films
which cover the soil particles, or die from
the effects of desiccation. In the presence
of excessive moisture where rapid circula-
tion of oxygen is prevented, the aerobic
microorganisms are depressed and the
anaerobic cells find conditions more con-
ducive to their growth.

As a result of microbial attack upon
proteins, there are formed carbon dioxide,
ammonia, and various incompletely de-
composed organic substances. From the
decomposition of carbohydrates, carbon
dioxide, organic acids, and alcohols are
produced. The oxidation of ammonia
leads to the formation of nitrites which are
oxidized to nitrates. These various sub-
stances may be further acted upon by soil organisms. The same
organism may also produce different end products from different
initial compounds, or from the same compound under different
conditions. The numerous processes brought about by soil
organisms are usually interdependent and follow one another.
Irrespective of their source, these substances are waste products
of the metabolism of the microbial cells, and many of the
nutrients obtained by higher plants as a result of the activities of
bacteria in the soil are actually waste products of the nutrition of
these bacteria.

Fig. 19. — Rhizopus sp.
(from Jensen).

32

THE MICROBE AND ITS ACTIVITIES

Among the soil processes in which bacteria are important and
frequently play a predominant role, the following need only be
enumerated here in order to suggest the
diversity of the changes: the fixation of
atmospheric nitrogen symbiotically by bac-
teria in association with leguminous plants,
and non-symbiotically by the bacteria in a
free-living condition in the soil ; the decom-
position of various organic constituents of
plant and animal residues, including cellu-
lose, hemicelluloses, sugars, proteins, amino
acids, fats, and waxes, both under anerobic
and aerobic conditions; the liberation of
ammonia as a result of the decomposition of
various proteins, protein derivatives, and
other organic nitrogenous substances; the
formation of nitrites and nitrates from am-
monia; the reduction of nitrates to nitrites,
to ammonia and to atmospheric nitrogen;
the oxidation of sulfur and of hydrogen, as
well as of incompletely oxidized compounds
of sulfur and of iron, and the oxidation of various other simple
inorganic and complex
organic substances.

Truly, bacteria are
active in all phases of
transformation of inor-
ganic complexes and of
organic matter in the
soil, this fact partly jus-
tifying the early concep-
tion that bacteria are
the only significant soil
organisms. However, as
the important role of
other groups of soil mi-
crobes is understood, it
becomes evident why
the general conception of the soil population was changed to
include a consideration of many other soil microbes. The science

Fig. 20. — Trichoderma
sp. : (a) arrangement of
spores on sporulating
hyphae; (6) higher
magnification of one of
the branches (from
Henrici).

Fig. 21. — Acrosfalagmus sp. : (a) arrangement of
spores on sporulating branches, (6) higher mag-
nification (after Goddard).

THE FUNGI OF THE SOIL

33

of soil microbiology may be interpreted as embracing a study of
the microscopic soil population which is responsible for the numer-
ous transformations occurring in the soil environment. How-
ever, many of the soil microorganisms are not limited to this
environment alone but may exist also in a variety of other
habitats.

The Fungi of the Soil. — Fungi are characterized by their
filamentous structure. This is termed mycelium, and consists
of numerous hyphae, either unicellular or multicellular. This
mycelium frequently attains considerable dimensions and forms
a complicated, profusely branched, vegetative growth with
specialized, spore-forming, fruiting bodies. Although numeri-
cally, as determined by the number of single cells, they are fewer
in the soil than the
bacteria, their actual
abundance, as meas-
ured by the amount
of cell substance pro-
duced, may be con-
siderably greater than
that of bacterial
growth. They vary
considerably in struc-
ture and size from the
simplest yeasts and
molds to the more
complex forms, as the

mushrooms and ^ ^ , . , ,

, , f. . 1 Fig. 22. — Mucor s\>: (a) young sporulating head,

bracket tungl Wiiose ^^^ mature sporulating organ, (c) spores becoming
sporulating bodies can liberated from sporangium, (d) columella after the
be seen with the naked scattering of the spores,

eye. Fungi are devoid

of chlorophyll, and consequently cannot obtain their carbon from
the carbon dioxide of the air as green plants do.

Fungi can be classified into three principal groups on the basis
of their morphology: (1) Filamentous fungi, which are fre-
quently spoken of as molds. These comprise various Phycomy-
cetes, Ascomycetes, and Fungi Imperfecti or Hyphomycetes. The
important soil genera, Mucor, Rhizopus, and Zygorhynchus are
found in the first class; Aspergillus and PenicilHum in the second,

34

THE MICROBE AND ITS ACTIVITIES

while a number of other forms, including Fusarium, Hormoden-
drum and Trichoderma, producing both colorless and black
mycelium, belong to the third class. (2) The yeasts, which belong
to the Ascomycetes, are found in the soil only to a limited extent.
(3) The mushroom fungi, belonging to the Basidiomycetes, are
found abundantly in forest and other soils in the form of a very
extensive mycelium, sometimes producing fruiting bodies in the
form of various mushrooms, toadstools, and puffballs. Many
fungi belonging to this group also produce an associative growth
with the roots of higher plants, especially forest trees, referred to
as mycorrhiza (or fungus root). In such associations, the fungi
assist the plants in obtaining their nutrients from the soil. As a
rule, the fungi show
such differences in
structures that their
classification into
species is almost en-
tirely based on mor-
phological features ;
unlike the bacteria,
httle resort to physi-
ological tests is used
in their differentia-
tion. Figs. 17 to 23
show a series of typi-
cal soil fungi.

The fungi vary
considerably in the
nature of the proc-
esses which they
bring about in the soil, but they are almost all associated with the
decomposition of complex organic substances, non-nitrogenous as
well as nitrogenous. They have the capacity for producing more
cell material per unit of organic substance decomposed than the
bacteria. They are usually able to grow only under aerobic
conditions, although most yeasts and some Mucors can also live
in the absence of free gaseous oxygen, without, however, making
much increase in cell substance under such anaerobic conditions.
Certain Basidiomycetes may grow extensively in anaerobic
environments, providing portions of the growth are exposed to an

Fig. 23. — Fusarium sp: {n) hyphal segments,
(6) spores (after Sherbakoff).

THE ACTINOMYCES OF THE SOIL

35

abundant supply of oxygen. Their deep penetration into woody
tissues and thick masses of decomposing organic materials illus-
trate this characteristic development.

The filamentous fungi are probably more tolerant to wide
changes in reaction than any other large group of soil micro-
organisms. A great number develop equally well under acid
and under alkaline conditions, although spore formation may be
somewhat delayed at high alkalinity. They are particularly
favored by a relatively humid aerobic environment.

The soil fungi are very active in the decomposition of proteins
and of various complex carbohydrates, such as cellulose and hemi-
celluloses. Some fungi are far
more active than bacteria in such
transformations. As a group,
they are more versatile than any
others in their ability to decom-
pose a great variety of organic
compounds. Some of the most
resistant substances succumb to
the attacks of the fungi: these
include a wide variety of nitrog-
enous compounds, cellulose,
starch, pentosans, vegetable gums,
paraffins, and hgnin. Nearly all
of the simple and complex or-
ganic compounds are attacked by
one group or another of the soil
fungi, and it is through these
activities that they play a par-
ticularly prominent part in the
transformations in the soil.

The Actinomyces of the Soil. — The actinomyces are similar
to the bacteria in that they are of about the same size in cross-
section. They are unlike the true bacteria and resemble the fila-
mentous fungi in that they produce a very extensive unicellular
filamentous network, very profusely branched and in many
cases reproducing by sporulation on specialized structures.
Microscopically these spores appear like bacterial cells. Some-
times the entire mycelial growth breaks up into fragments which,
as individuals, are indistinguishable from bacterial cells. As a

Fig. 24. — Young mycelium of Ac-
tinomyces (from Lieske).

36 THE MICROBE AND ITS ACTIVITIES

group, the actinomyces may be considered distinct, having char-
acteristics which place them in a position intermediate between
the bacteria and the filamentous fungi, although the majority of
the soil forms somewhat more closely resemble the fungi than the
bacteria. The mycelium is, however, much smaller in cross sec-

FiG. 25. — Branching mycelium of Actinomyces (from Drechsler).

tion than that of the filamentous fungi. The actinomyces are
relatively abundant in the soil, making up from 10 to 60 per cent
of the colonies developing on the plates which have been prepared
from soil, by the use of artificial media. Actinomyces are active
in the decomposition of a variety of organic substances in the
soil.

Most of the actinomyces are aerobic, although possibly less

THE ALGAE OF THE SOIL

37

strictly so than most aerobic bacteria. They are as a rule more
sensitive to changes in reaction and hve over a narrower range of
acidity and alkaUnity. They appear to make more extensive
development than the bacteria in soils of low moisture content,
but they develop quite well in fairly moist soil under aerobic
conditions. Fig. 24 shows a typical actinomyces myceHum as it
appears under the microscope, using low magnification. A much
more complicated structure is shown in Fig. 25; this schematically
represents highly magnified portions of growth.

The Algae of the Soil. — The chlorophyll-bearing micro-
scopic plants, namely the algae, also form an extensive group of
soil organisms. They vary greatly in size and in shape; but the
soil forms are largely
microscopic species,
unicellular or filament-
ous in structure. Since
they contain chloro-
phyll and are capable
of utilizing the energy
of the sun, they are
independent of the
energy sources in the
soil so long as they
have free access to
light. They are capa-
ble of making an ex-
tensive growth, deriv-
ing from the soil the
required nitrogen and minerals. They live both on the surface
of the soil and at various depths beneath the surface. Below the
soil surface they act in a manner similar to fungi, that is, living
at the expense of the energy derived from the utilization of organic
materials.

Soil algae are divided into three groups: (1) Cyanophyceae or
blue-greens; (2) Chlorophyceae or grass-greens; and (3) Diatomaceae
or diatoms. Representatives of these three groups are found in
the soil in considerable numbers, generally less abundantly, how-
ever, than the bacteria and the fungi. Their development in the
soil results in increasing the supply of organic matter and in
temporarily transforming soluble forms of nitrogen and minera

Fig. 26. — Soil alga, Bumillaria exilis
(from Bristol).

38

THE MICROBE AND ITS ACTIVITIES

into organic or insoluble forms. Figs. 26 and 27 show two of
the most common types of soil algae. Fig. 28 shows a soil
diatom.

The Protozoa of the Soil. — The protozoa are the most
elementary forms of life belonging to the animal kingdom. They
are all microscopic, unicellular, larger than bacteria and more
complex in their activities. They move either by means of cilia,
flagella, or pseudopodia. The nature of their locomotion is used
as a basis for their classification. They may be conveniently
grouped as follows: (1) The Ciliata, which include the ciliates
or infusoria; (2) the Mastigophora, which include the flagel-

Fifi. 27. — Soil alga, Chlamydomonns
communis (from Bristol).

Fir:. 28. — Soil diatom, Navicula
terricola (from Bristol).

lates; and (3) the Rhizopoda, which include the amoebae. They
vary from naked forms to those which produce chitinous or
siliceous envelopes. All three groups are abundantly represented
in the soil, the smaller flagellates and amoebae being especially
abundant. Representatives of the various groups of protozoa
are shown in Figs. 29-33.

Physiologically the protozoa vary considerably. Due to the
fact that it is very difficult to free protozoa from bacteria, the
exact nature of the physiology of these infinitesimal animals is
still a matter of dispute. Some of them at least are capable of
utilizing dead organic and inorganic substances from solution
and from solid particles. Many forms are capable of ingesting

WORMS AND INSECTS IN THE SOIL

39

living bacteria and using them as food. A nutrient solution
inoculated with some fresh soil first shows an extensive develop-
ment of bacteria; this is soon followed by the appearance of
numerous protozoa, which use the bacteria as food.

This succession of microscopic forms
need not indicate that all the protozoa
depend for their nutrition upon the
bacterial cells and that they appreciably
limit bacterial development. It simply
points further to the complex inter-
relationships of the soil organisms. It
gave rise to a theory which tended to
explain soil fertility on the basis of the
interrelationships of the protozoa and
the bacteria. The protozoa feeding
upon bacteria were supposed to limit
soil fertility, since the bacteria were
looked upon as the sole organisms bring-
ing about the important processes of
decomposition of soil organic matter.
However, this theory has not found
confirmation on further study and investigation.

Worms and Insects in the Soil. — The higher animal popu-
lation is represented in the soil by numerous worms and insect
larvae which inhabit the soil permanently and take an active part
in soil processes. Some live in the soil only for certain periods of
their life. Some of these groups are very abundant in most
soils; others occur only in certain soils under a given set of con-
ditions. Among the various worms, it is sufficient to mention
nematodes and earthworms. The nematodes (Figs. 34 and 35)
alone are found in billions per acre of soil. The insects may find
in the soil a temporary habitat, by passing there a certain
part of their life cycle; some of them spend in the soil the
major part of their life, only coming to the surface occasionally.
All these members of the animal population are very active in
bringing about an intimate mixture of the soil and the organic
matter, in macerating the organic matter and the fungus mycelium
attacking it, and thus assisting in the disintegration of the dead
materials. Some of them cause damage to living plants by
attacking their roots or certain parts growing above ground.

Fig. 29. — Soil flagellates
(from Fellers and Allison).

40

THE MICROBE AND ITS ACTIVITIES

Some nematodes are predacious in nature, feeding upon plant
parasites.

Soil Organisms Causing Plant and Animal Diseases. —
The soil also harbors a number of organisms which are causative
agents of disease, either in plants or in animals. Some of them
find in the soil only a temporary habitat, while others persist in
the soil for long intervals. The animal pathogens are repre-
sented in the soil by such important organisms as Clostridium
tetani, causing tetanus, Clostridium hotulinus, causing food poison-
ing, and various organisms causing sporotrichosis and actinomy-
cosis in man and animals. The number of plant pathogens which

, ^/^

Fig. 30.— Flagellate. Fig. 31.— Ciliate, Boiari- Fig. 32.— Ciliate, CoZpoda
Bodo caudatus (from tiophorus elongatiis (from steinil (from Goodey).
Martin and Lewin). Goodey).

find a temporary or permanent habitat in the soil is quite large.
This group includes organisms causing such bacterial diseases as
various rots, wilts, leaf spots, and galls; such fungus diseases as
numerous root rots, dam ping-off fungi and blights; such actin-
omycotic diseases as scab in potatoes and sugar beets and pox
in sweet potatoes; various nematodes causing diseases which
appear as swellings on the roots of a number of plants.

The soil microorganisms are frequently classified into bene-
ficial and injurious forms, on the basis of their relation to man and
his cultivated plants and domestic animals. The beneficial

SYMBIOTIC RELATIONSHIPS

41

Fig. 33. — Soil amoeba, Vahlkampfia soli
(from Martin and Lewin).

microbes are considered as taking part in the various transforma-
tions in the soil which result in the liberation of nutrients neces-
sary for the growth of plants, which in their turn supply the food
for domestic animals and man. The injurious organisms either
attack the plants and animals directly, by causing various diseases,
or injure them indirectly by bringing about transformations in
the soil which are unfavorable to plant growth.

Such a strict classification of microorganisms as either bene-
ficial or injurious forms is too arbitrary. Some organisms may
be beneficial at one time and injurious at another, depending
upon the soil conditions and
plant growth. Even certain
organisms, as various Fusaria,
Pythia, and Rhizoctonia, which
cause plant diseases, grow ex-
tensively in soil, leading a
saprophytic existence and tak-
ing a part in the decomposi-
tion of organic matter. Other common soil saprophytes may
cause nitrogen starvation of plants at one time and produce an
abundance of nitrogen available for plant consumption at another
time, depending upon the cultural treatment of the soil.

Symbiotic Relationships. — Certain soil microorganisms live
in definite symbiosis with plants. Included in this group are
various fungi that form mycorrhiza with forest trees and other
plants, such as orchids. The activities of these fungi are of

decided importance in the nutrition
of the specific plants; some of the
higher plants may actually be
parasitic upon the fungi which
supply them with nutrients from
the soil, or play another important
part in their metabohsm. In the
case of legume bacteria, which form
nodules on the roots of leguminous plants, the symbiosis appears
to benefit both the plants and the bacteria.

Summary: The Complex Soil Poplxation. — All the micro-
organisms living in the soil and included in the numerous groups
of bacteria, actinomyces, fungi, protozoa, algae, and small inverte-
brate animals make up the soil population. They do not live in

Fig. 34. — Parasitic nematode
(from Cobb).

42

THE MICROBE AND ITS ACTIVITIES

the soil in separate communities, but are variously intermixed, so
that minute particles of soil contain many representatives of the
various groups of soil microbes. The reactions produced by
a single organism are far from simple, and the complexity of the
mixed population makes the reactions taking place in the soil
even more complicated. One set of conditions favors the devel-
opment of certain groups in preference to others. Many microbes
compete with one another for the available nutrients; many
complete one another, one using as food
the waste products of other organisms,
some depending for their nutrients upon
the activities of others.

The sum total of the activities of the
various organisms is the liberation of
nutrients from the dead plant and animal
debris into forms available for the nutri-
tion of higher plants, as well as the
storage of some of the nutrients in the
microbial cells which contribute directly
or indirectly to the soil organic matter.
The environmental conditions modify
the nature of the soil population; the
nature of the organisms influences the
form in which the nutrients again be-
come available; the nature of these
nutrients influences the nature of the
plant vegetation which a given soil will
support. So intimately interwoven are these numerous reac-
tions that their products appear to be formed by a single
reaction from a single body.

Fig. 35. — Beneficial soil

nematode, Mononchus pa-

pillatus (from Cobb).

LITERATURE

1. Greaves, J. E., and Greaves, E. O. Bacteria in relation to soil fertility.

D. Van Nostrand Co. New York, 1925.

2. LoHNis, F., and Fred, E. B. Textbook of agricultural bacteriology.

McGraw-Hill Co. New York, 1923.

3. LtJTMAN, B. F. Microbiology. McGraw-Hill Book Co., Inc. New York,

1929.

4. Marshall, C. E. Microbiology. P. Blakiston's Son and Co. Phila-

delphia, 1921.

LITERATURE 43

5. Russell, E. J., and others. The Micro-organisms of the soil. Longmans,

Green & Co. London, 1923.

6. Stephenson, M. Bacterial metaboHsm. Longmans, Green & Co. 1930.

7. Tanner, F. W. Bacteriology — A Textbook of Microorganisms. John

Wiley & Sons, Inc. New York, 1929.

8. Waksman, S. a. Principles of soil microbiology. The Wilhams & Wilkins

Co. Baltimore, 1927.

CHAPTER III
THE SOIL POPULATION AND ITS DISTRIBUTION

The Occurrence of Microbes in Soil. — Because of the
fact that microbes are so small in size and that many of them
are capable of growing on a variety of foods in a wide range of
environments, they are found universally distributed in nature.
They are present in great abundance in water, in various food-
stuffs, upon all growing and dead plants, in the digestive tracts
of animals, upon dust particles, and in soil. Because of their
great abundance in nature and the ease with which they are dis-
tributed by means of air, water, moving animals, living and dead
plants, as well as through man's activities, it is reasonable to
assume that in soils of the same composition in identical environ-
ments the numbers and abundance of types of microorganisms
should be nearly identical.

However, microbes do not develop actively upon all sub-
strates where they may be located. The mere fact that many
bacterial cells are found upon clothing and dust does not indicate
that they grow and develop upon these substrates; it merely
indicates that clothing and dust are carriers of these microbes
and that the organisms can be distributed in nature through
these vehicles. Likewise certain microbes may be found in the
soil merely because they have been introduced there by dust,
waters, or through man's activities, as in the application of
manures of domesticated animals. The fact that a certain microbe
is found in the soil is no proof that it is a normal inhabitant of the
soil. The presence of large numbers of Bacterium coli in a soil
may merely indicate that this organism has recently reached the
soil in animal excreta and not that it finds the soil a favorable
medium for its development.

One should thus differentiate between microbes found in the
soil as a result of their fortuitous introduction and those which
lead a normal existence in the soil. If conditions are not favor-

44

DISTRIBUTION OF MICROBES IN SOIL 45

able for the development of specific organisms, repeated intro-
duction of even great numbers of these microbes will fail to estab-
lish them as permanent members of the soil population. The
development of a specific microbe in a soil depends upon the
chemical composition of the soil, especially the presence of nutri-
ents essential for its growth, and the environmental conditions,
such as temperature, moisture, and reaction. The soil and
environmental conditions thus determine to a large extent the
abundance and kinds of organisms that will inhabit the soil and
the reactions which they perform there.

Relation of Microbes to Plants and Animals. — In their
relation to plants and to animals, microorganisms can be con-
sidered as being either parasites or saprophytes. The parasitic
forms are capable of growing upon the living plant or animal,
causing a diseased condition of the tissues, leading to an abnormal
condition of the host and possibly to death, unless the host is
able to withstand the attack of the parasite.

The saprophytic microorganisms grow only upon dead plant
and animal tissues or their decomposition products. They are
thus of extreme importance in the transformation of complex
organic substances into simple forms, making the nutrient ele-
ments locked up in those substances again available for plant
nutrition. The typical soil population consists of microorganisms
which are largely saprophytic in nature. However, certain
plant and animal parasites may persist in the soil for some time,
and a few are even capable of leading a normal existence there.
Certain microbes are capable of acting parasitically upon other
microbes which are either beneficial or injurious to plants. The
saprophytic microorganisms are widely distributed in the soil
and occur in greater or less abundance practically everywhere.
There is no soil known, ranging from the Sahara Desert to the
polar regions, that should be free from saprophytic microorgan-
isms. Certain specific groups of organisms may of course be
absent, where conditions are unfavorable for their development.

Qualitative and Quantitative Distribution of Microbes
in Soil. — In most soils we find much the same qualitative dis-
tribution of organisms, but there are very marked differences in
the quantitative relationships. A change of soil conditions, such
as an increase in acidity or alkalinity, a change in food supply,
moisture content, aeration, or other physical or chemical soil

46

THE SOIL POPULATION AND ITS DISTRIBUTION

conditions, is followed by marked changes in the qualitative and
quantitative distribution of the soil microflora and microfauna.

The microbes occur in greatest abundance in the surface layer
of the soil (Fig. 36). This layer varies in thickness from a few-
centimeters, as in the case of humid soils, to two or three meters,
as in the case of arid soils. The greatest abundance of individuals
and variety of microorganisms are found either at the very
surface of the soil, as in forests, meadows, and other shaded soils,
or just below the surface, as in the case of the open cultivated

I Numbers in A Horizons
I Numbers in B Horizons
I Numbers in C Horizons

25 1,000 5

20 800

15 o

600 -2 3

■5510 ^AOO ^2

200 1

NUMBERS OF
BACTERIA

NUMBERS OF
ACTINOMYCES

NUMBERS OF
FUNGI

A.AjAjBC.C^

AjAjAjB C

Aj A2A3 B CjCj

A,AjA3BC

A,AjA3BC,C3

AjAjAjBC

Cultivated
Soil

Virgin
Sod

Cultivated
Soil

Virgin
Sod

Cultivated
Soil

Virgin
Sod

Fig. 36. — -Vertical distribution of microbes in soil
(after Brown and Benton).

soils. The numbers of microorganisms diminish with depth, the
rapidity of the diminution varying with the soil conditions, espe-
cially the distribution of organic matter and degree of aeration.
The decrease in numbers and activities of microorganisms is very
rapid in the shallow, humid soils ; in the case of the deep arid and
semi-arid soils, the decrease is comparatively slow.

Although there are certain differences in the abundance of
organisms from place to place in the soil, the general distribution
of microorganisms in any limited soil mass is more or less uniform.
However, the relative abundance of the different types of organ-

DISTRIBUTION OF MICROBES IN SOIL

47

isms found in different soils varies considerably, depending upon
the composition of the soil and the environmental conditions.
The physical characteristics of the soil (whether it is sand, loam
or clay, porous or compact, well drained or poorly drained), the
reaction, abundance and nature of organic matter, the amount
of rainfall, average temperature, period of the year, nature of the
vegetation, type of cultivation, and numerous other factors — all
influence the nature, abundance, and activities of microorganisms
in the soil. Largely as a result of the influences of these factors,

2.1 35

1.8 30

= 1.5 25 -

§ 1.2 = 20 -

h 0.9 § 15

■G 0.6 10 -

0.3 5 -

1 1 1 1 1 1 1 lll

1 II II II

Production of CO2

Ml II 1 1 1 I

A

-

\ A^^l

- \l

1 \ / V' 1 1 /
' \ ' \ a'

I

/
/

- /

K ^ r \ ' '

/

1 1 1 1 1 1 1 1 1 1

1 I 1 1 M II 1 1

/ —
' /

I /

II II II ll

Jan. Feb. Mar. Apr. May June July Aug. Sept. Oct. Nov. Dec.

Fig. 37. — Abundance of bacteria and carbon dioxide in soils at different seasons
of the year (after Russell).

marked differences are found in the numbers of different groups of
microorganisms during different seasons of the year (Fig. 37).
Differences may be found even within a few days or a few hours.
It is important to keep in mind the fact that, since the physical
and chemical soil conditions even at the same depth of soil are not
homogeneous, the distribution of microorganisms throughout the
soil as a whole is not uniform. There are pronounced differences
in the abundance of microorganisms in different samples of soil
from an apparently uniform field. Twice as many organisms may
be found in one gram of soil taken at one spot of the field than in

48 THE SOIL POPULATION AND ITS DISTRIBUTION

another gram taken a few feet or even a few inches distant from the
first. Results obtained from studies of the influences of environ-
mental factors on the abundance of microorganisms in field soils
should therefore be interpreted with an appreciation of the natural
variation in the normal distribution of these organisms in the soil.

Methods for Determining Numbers of Microorganisms
IN THE Soil. — There is no one method available at the present
time which can measure the total abundance and activities of soil
microorganisms. The various methods commonly employed are
adapted to the enumeration of only a certain few groups of organ-
isms commonly found in the soil or to the measurement of one
product of the development of some microbes. The organisms
which are counted make up only a part, frequently a very small
one, of the total soil population, and the product measured is
but one of many. The methods used for counting soil organisms
are based either upon the observation of microorganisms in stained
preparations of soil, or upon their development in solid or liquid
culture media which arc specially prepared for the cultivation of
these organisms. In either case only a part of the total soil popu-
lation is measured. In the case of stained preparations, some of
the organisms are either destroyed in the process of preparing the
specimen, as in the case of the protozoa, or are not visible at all, or
are found in clumps, so that the individual cells cannot be counted.
In the case of those methods which are based upon the develop-
ment of microbes in culture media, the greatest difficulty is experi-
enced in finding any single medium which will permit the growth
of a large fraction of the soil inhabitants.

Each medium is more or less selective as far as the growth of
certain organisms is concerned, but some media permit the devel-
opment of fewer forms than others. Some media are purposely
made favorable for the development of only those microbes that
are capable of bringing about a specific transformation. Such
media can be used for the determination of the abundance of
specific physiological groups of organisms which play known roles
in certain important soil processes.

Methods for Studying Activities of Soil Microbes. —
For the determination of the activities of soil organisms, the
methods commonly employed are based upon a knowledge of those
soil processes which are considered to be essential to soil fertility.
These may deal with the transformation of a single element of

METHODS FOR STUDYING ACTIVITIES OF SOIL MICROBES 49

known importance to plant growth, such as the elements, nitrogen,
carbon, or sulfur, or with a single change in a group of transfor-
mations in which a certain element or compound is involved In
the decomposition of cellulose in the soil, for example, either
the disappearance of cellulose, the formation of intermediary
substances such as organic acids, or the formation of an end prod-
uct of the reaction, such as carbon dioxide, can be used as an index
of the microbial action. In studying the degradation of a protein
in the soil, the disappearance of the protein, the formation of
amino acids, the accumulation of ammonia, and frequently even
the transformation of ammonia into nitrate have served as meas-
ures of transformation. Many different groups of organisms are
concerned in such reactions, and the value of any single measure-
ment depends upon the object of the study.

Cellulose and proteins are only two groups of chemical com-
plexes contained in the organic matter which is added to the soil
by the growing plants, either in the form of residues of the aerial
portions of the plant, such as leaves, needles, stems, and fruiting
parts, or in the form of the subterranean portions, namely, the
roots. The quantity of organic substances reaching the soil is
also increased through the introduction of stable manures,
green manures, various animal and plant residues, and such arti-
ficial organic preparations as urea and cyanamid. In the decom-
position of the organic complexes, various organisms are con-
cerned which in their development bring about numerous processes
of oxidation and reduction, decomposition and synthesis, which are
of extreme importance to soil fertility.

To measure the activities of all these organisms, just as
to determine their total abundance, is beyond the scope of
any single method or group of methods. The nearest approach
to the ideal method is that which allows the determination
of an end product of the sum-total of the activities of a large
fraction of the soil microbes. One usually studies only those
transformations which appear to be of greatest economic impor-
tance to man, who utilizes these activities for the growth of his
crops and the nutrition of his animals. However, microorganisms
participate in a great many processes which take place in the soil
of which little or nothing is known. With the advance of our
knowledge of the soil microbes and their activities, the relation-
ships of soil microorganisms to the transformation of soil constitu-

50 THE SOIL POPULATION AND ITS DISTRIBUTION

ents will be more accurately appraised and their activities will be
more profitably utilized.

Summary of Methods. — The present methods for studying
soil microorganisms can be listed as follows:

(1) Those methods which are used for the determination of
the number of microorganisms in the soil as well as the specific
nature of some of these organisms. These methods can be
conveniently subdivided :

(a) Direct microscopic methods.

(6) Cultural methods, based upon the development of
microorganisms upon solid media in plates or in tubes. By
adjusting the concentration and nature of specific nutrients,
such as the energy source, nitrogen supply, and other inor-
ganic salts, by modifying the hydrogen-ion concentration and
air supply, these media can be prepared so as to allow the
development of either large numbers of a variety of microbes,
or only representatives of specific groups.

(c) Cultural methods, based upon microbial growth or
upon a specific chemical transformation brought about by
microbial development in a medium containing a specific
chemical substance, after inoculation with high dilutions of
soil. Either liquid or solid media are employed for such use.

(2) Methods which are devised primarily for obtaining
information about the activities of the total soil flora and fauna,
or of only one specific transformation, irrespective of the nature
of the organisms concerned.

(3) Methods for determining the availability in soil of
elements essential for plant growth. These will be discussed
in Chapter IX.

Direct Microscopic Methods. — The direct microscopic
methods have been devised with the idea of determining the total
abundance of organisms in the soil in order to supplement cultural
methods which are selective. A small particle of soil or a dilute
soil suspension is first mixed with a dilute solution of agar or gela-
tin, dried and fixed upon a slide, and then stained with acid dyes
such as rose bengal or erythrosine. The bacteria can be distin-
guished and counted with the aid of a microscope, since they are

DIRECT MICROSCOPIC METHODS 51

stained deep red, while the organic and inorganic soil particles are
either not stained at all or stained yellowish or brown. By the use
of a graduated slide, a calibrated microscope, and a definite volume
of the known soil suspension, for making the stained preparation,
the approximate number of bacteria in any mass of soil can be
determined. This method has the advantage that it stains all
the soil bacteria, irrespective of their nutritive peculiarities, and
thus permits the determination of their abundance in the soil.
It also permits the recognition of the relative abundance of various

Fig. 38. — Presence of bacteria in soil, as indicated by direct staining of soil

(from Cholodny).

morphological groups of soil organisms, as well as the physical
relationship between the soil and its microbiological population.
On the other hand, there are certain factors which limit the use-
fulness of the method. Some of the bacteria occur in aggre-
gates, which makes their counting difficult; there is also great
variation in the distribution of the cells in the stained preparation,
thus decreasing the accuracy of the enumeration; actinomyces
spores can seldom be distinguished from bacteria by this procedure ;
many of the small bacteria are difficult to distinguish from soil
particles, and only a minute portion of the soil (one-millionth or
one-five-millionth of a gram) can be examined by the microscope

52

THE SOIL POPULATION AND ITS DISTRIBUTION

at any one time. Figs. 38 and 39 are preparations of soil showing
the occurrence of some of the microbial cells in stained specimens.
The results obtained by this method indicate that bacteria
exist in the soil not only in millions but in tens and hundreds of
millions per gram. Most of the bacteria found by the direct
microscopic method are non-spore-forming, rod-shaped or spherical
cells. Many of the non-spore-forming organisms are found within
the colloidal films which surround the soil particles, while the
spore-forming organisms occur in the spaces between the individual
particles. The cells may occur in large aggregates or colonies
enclosed in an abundance of capsular material. The protozoa,
fungus mycelium, and portions of actinomyces growth are fre-
quently disintegrated by the process of staining. Table 7 shows
the large numbers of bacteria and fungi which have been observed
in soil by the direct microscopic method.

TABLE 7

Numbers of Microorganisms in One Gram of Soil as Determined
BY THE Direct Microscopic Method (from Richter)

Depth

Numbers of Bacteria

Azoto-

bacter

cells

Pieces of

Type of soil

Cocci

Bacilli

mycelium

Surface
10 cm.
20 cm.

Surface
10 cm.

Surface
10 cm.
20 cm.

1,379,000,000
991,000,000
281,000,000

870,000,000
569,000,000

519,000,000
407,000,000
269,000,000

1,212,000,000
460,000,000
169,000,000

376,000,000
106,000,000

192,000,000
153,000,000
1.39,000,000

1,000,000
31,000,000

47,000,000

34,000,000
7,000,000

Brown loam

Sandy soil

84,000,000
1,000,000

79,000,000

23,000,000

8,000,000

5,000,000
3,000,000

3,000,000

19,000,000

3,000,000

Plate Method for Counting Microbes. — The plate method
for counting bacteria as well as certain other soil microbes makes
use of the fact that media containing agar and gelatin can be kept
liquid at temperatures which are not destructive to microbes and
form gels at temperatures favorable to growth of the organisms.
A highly diluted suspension of soil is prepared in sterile water in
such a manner that the liquid contains only a limited number of
microbes; these are as completely dispersed in the water as pos-

PLATE METHOD FOR COUNTING MICROBES

53

sible. When a small portion of this suspension is added to the
medium which has been brought to the Uquid condition (by warm-
ing), mixed well and the medium then allowed on cooling to solidify-
in Petri dishes, the individual cells of the various organisms
become fixed in separate positions on the dish. If the medium
contains all of the nutrients required for the growth of these
microbes and if other conditions, such as reaction, temperature,
and circulation of gases are favorable, the organisms multiply

Fig. 39. — Azotobacter cells in soil, as demonstrated by direct staining
(from Winogradsky).

rapidly, with the result that colonies are formed from each of the
original cells present in the soil suspension. After incubation for a
few hours or days, the colonies become visible to the naked eye,
and their total number can be determined by simple enumeration
(Fig. 40). If the dilution has been so conducted that a known
amount of soil was added to the medium, one can calculate, from
the number of visible colonies which appear on the plate, the
number of organisms per unit of soil which are able to develop
under this particular set of conditions.

The plate method possesses several distinct advantages.
Determinations can be made of the abundance in the soil of such

54 THE SOIL POPULATION AND ITS DISTRIBUTION

organisms as are capable of developing upon the media which are
used. Since the various microbes form typical colonies, the
method allows the determination of the abundance of specific

Fig. 40. — Colonies of bacteria and actinomyces (below), and fungi (above),
developing on plates as used in counting these organisms in soils.

types of organisms in the soil. Each medium is selective for cer-
tain groups of microbes, and each set of conditions under which the
organisms are allowed to develop upon the medium is selective for
certain organisms. A medium containing proteins will favor the

ELECTIVE CULTURE METHOD 55

development of proteolytic bacteria; a medium containing starch
or cellulose will favor the growth of organisms capable of utilizing
starch or cellulose. The incubation of the plates under aerobic
conditions will favor the development of aerobic organisms;
under anaerobic conditions the development of anaerobic bacteria
is favored. At high temperatures (50 to 65° C) thermophilic
organisms develop. An acid medium favors the development of
acid-tolerant microbes. The value of the method as an agency for
measuring the total numbers of microbes in the soil is further lim-
ited by the fact that not all of the cells of even those organisms for
which the medium is suited develop and form colonies. Fre-
quently only 3 to 10 cells out of 100 will grow into colonies. Many
fungi and protozoa fail to grow even upon the most carefully
prepared media. Many bacteria and fungi require special media
and conditions of incubation which are adapted to a highly specific
type of development.

Elective Culture Method. — To meet the peculiar nutrition
of the numerous soil organisms, the third method, namely, the
solution or elective culture method, has been introduced. It con-
sists in adding a definite quantity of soil to definite volumes of
sterile water, thus diluting the soil to a desired degree, then adding
measured quantities of the various dilutions to media favorable
for the development of the particular organisms. The cultures
are then incubated under favorable conditions, and observations
are made to determine at what dilutions growth of the specific
microbes has taken place. One can thus measure the approxi-
mate number of the specific microbes in a given quantity of soil.

To determine, for example, the number of aerobic cellulose-
decomposing bacteria in a certain soil, the following method has
been used. A series of flasks or tubes containing a liquid or solid
medium favorable for the development of cellulose-decomposing
bacteria is prepared and sterilized. A medium containing cellulose
in the form of filter paper or cotton and the other nutrients as
inorganic salts in dilute solution was found favorable for this
purpose. The soil is diluted with sterile water so that 1 cc.
quantities of the dilution contain the following portions of a gram
of soil in suspension: 1 100, 1/400, 1/1,000, 1/5,000, 1/10,000,
1/25,000. One cubic centimeter portions of the suspensions are
added to the flasks or tubes of the medium, and the flasks are
incubated at 25 or 28° C. After a few days, the cultures are

56 THE SOIL POPULATION AND ITS DISTRIBUTION

examined to determine whether or not growth of microbes has
taken place. Development will be readily detected by the disin-
tegration of the paper in the culture, especially in contact with the
surface of the liquid. If the flask that has been inoculated with
1 5,000 of a gram of soil shows evidences of growth, and those
flasks or tubes to which smaller amounts of soil were added show
no growth, one is justified in concluding that one gram of the
particular soil contains at least 5,000 cells of bacteria which are
capable of decomposing cellulose under aerobic conditions. Certain
formulae have been devised which can be used for accurate deter-
minations of the number of specific organisms in a given quantity
of soil from the number of cultures giving positive and negative
growth.

It may be necessary to modify the method greatly so as to
encourage the growth of the various specific microbes found in the
soil. For the development of protozoa, sterile portions of nutrient
meat extract agar media are placed in sterile Petri dishes; these
are inoculated with 1 cc. portions of various dilutions of soil and
incubated for at least 28 days. The surface of the agar is kept
moist by frequent additions of sterile water. At various inter-
vals, a loopful of material is removed from the surface of the agar
and examined under the microscope to determine the presence of
protozoa in general and of specific forms in particular.

For the development of nitrogen-fixing bacteria, media are
employed which are free from combined organic or inorganic nitro-
gen, but which contain the mineral elements required for growth
and a source of energy, such as simple sugars or higher alcohols.
Thus, by modifying the nature and composition of the culture
media and conditions of growth, the development of various soil
microbes can be favored.

In spite of the fact that all of the methods mentioned previ-
ously are limited in their usefulness for determining the abundance
of the microscopic organisms in soils, they become quite useful
for comparative studies of different soils when interpreted in the
realization of their deficiencies.

Abundance of Bacteria in Soil. — The numbers of bacteria
found in different soils depend entirely upon the method employed
for their enumeration. When the plate method is used, the total
number of bacteria is found to vary from 10,000 per gram, as in the
case of desert sands, sand dunes, and very poor sandy soils, to

ABUNDANCE OF BACTERIA IN SOIL

57

50,000,000 frequently found in well-manured and cultivated
soils. The nature of the medium employed for preparing the
plates is of considerable importance in this connection, peptone-
meat-extract media giving a smaller number of organisms than the
so-called synthetic media, since the former media favor the rapid

Fig. 41. — Plate preparation showing development of spreading colonies of
bacteria (from Lipman).

development of certain spreading colonies which rapidly over-
grow the plate (Fig. 41).

When the direct microscopic examination of stained soil prep-
arations is used, the bacteria per gram of soil are numbered in the
hundreds of millions or even billions (Table 7). By the use of
elective culture methods, lower numbers are found and the organ-
isms are limited to those microbes favored by the specific media.
Table 8 gives a comparison between the abundance of bacteria
determined by the use of various elective culture methods and the

58

THE SOIL POPULATION AND ITS DISTRIBUTION

plate method. All these methods give, however, only a fraction
of the total bacterial population of the soil.

TABLE 8

Numbers of Bacteria in One Gram of Soil as Determined by Plate
AND Dilution Methods (from Duggeli)

Soil type

Numbers of aerobic bacteria
developing on gelatin
plate

Numbers of anaerobic bac-
teria

Aerobic nitrogen-fixing bac-
teria

Anaerobic nitrogen-fixing
bacteria

Nitrifying bacteria

Anaerobic cellulose-decom-
posing bacteria

Garden

,116,000

622,000

2,620

8,200
2,620

401

Field

10,640,000

820,000

6,040

4,420
2,201

420

Deciduous

forest

1,422,000

44,020

0

460
2,000

Lowland

1,010,000

80,200

0

440
2,020

Influence of Soil Conditions and Treatment upon the
Distribution of Bacteria. — The physical and chemical composi-
tion of the soil, as well as the environmental conditions, determine
the relative abundance of the microbes present at any given
time in the soil. In general, light sandy soils, poor in organic
matter, contain fewer microbes than fertile clays and loams rich
in organic matter. The number of microbes need not necessarily
indicate their potential activity, since such processes of decom-
position and oxidation as are of the greatest importance for plant
growth may be as active, if not more so, in the lighter than in the
heavier soils. As soon as available nutrients are added, the
response in microbial development becomes even greater in sandy
soils than in heavy clay soils; there is a more rapid increase in the
numbers of microbes and quicker transformation of the microbial
foods.

Undrained peat and water-logged soils contain considerably
smaller numbers of fungi, actinomyces and aerobic bacteria than

INFLUENCE OF SOIL CONDITIONS

59

the corresponding drained soils, although the number of anaerobic
bacteria may be greater in the former. Acid soils, especially acid
forest or raw-humus soils, are very rich in fungi and poor in bac-
teria. The fungus development is so extensive that the soil is
profusely permeated with fungus mycelium (Fig. 42). As soon
as an acid soil is limed, there is a rapid increase in the abundance
of bacteria.

In general, the numbers of bacteria, as determined by the
common plate method, range from 1,000,000 to 20,000,000 per
gram of soil. These figures refer only to the aerobic bacteria that
are capable of developing upon certain solid media. They are

Fig. 42. — Fungus development on surface litter of forest soil (from Rayner).

only relative figures and should not be interpreted as representing a
constant number of organisms in a particular soil; they refer only
to the relative abundance of certain types of cells at the time that
the determination was made. It should be kept in mind that
these numbers change greatly from time to time, even without any
apparent alteration in the conditions of the soil. Much greater
changes occur when the environment is considerably modified.

Injudicious cropping without fertilization will in time greatly
deplete the soil organic matter and the potential sources of nutri-
ent substances for plants, and will lead to depressions in the abun-
dance of microorganisms. Certain fertilizer practices, as con-

60 THE SOIL POPULATION AND ITS DISTRIBUTION

tinued application of ammonium sulfate without calcium carbon-
ate, increase the acidity of the soil to such a degree as to greatly
lower the numbers of bacteria. On the other hand, the applica-
tion of inorganic fertilizers considerably increases the microbial
population through both direct and indirect factors. The effect
may be direct, by satisfying the deficiencies of certain elements
required for microbial growth. It may be indirect, by affecting
the physical condition of the soil, by modifying the soil solution,
creating a greater or less solvent action, or by changing the soil
reaction.

The environmental factors affecting the abundance of bacteria
in soil as well as their distribution at different depths are numerous,
most important being the factors of organic matter, moisture,
reaction, temperature, and air penetration.

Influence of Organic Matter. — Additions of organic
materials to the soil probably exert more pronounced effects upon
the microbial population than any other treatment, especially
under humid conditions, but the microbial response is different with
different substances, depending upon the relative ease with which
they are decomposed and the types of organisms which are able
to attack them. The effects of introduction of plant or animal
residues are pronounced upon the physical condition of the soil,
by binding loose sandy soil more closely together, and by bringing
heavy clay soil to a more porous state. These effects are in general
desirable for the development of both plants and microorganisms.
Soil conditions resulting from the introduction of plant substances
will generally be associated with extensive development of the
microbial inhabitants. Soils not receiving any fresh additions of
organic matter become progressively depopulated, irrespective of
other soil conditions.

What course the microbial changes follow will be largely deter-
mined by the nature of the organic materials added. Tree prod-
ucts are quite resistant to decomposition, and produce marked
effects on the organisms only over a considerable period of time.
The lower carbohydrates and protein substances produce effects
which are very quickly apparent but which persist for compara-
tively short periods. Owing principally to the chemical composi-
tion and abundance or residues which they may leave in the soil,
different plants affect the development of soil organisms differ-
ently, as will be shown in Chapter IV.

INFLUENCE OF MOISTURE

61

TABLE 9

Influence of Moisture Content of Soil on Numbers of Bacteria

(from Engberding)

Per cent saturation
of the moisture-
holding capacity

Moisture
content,
per cent

Numbers of bac-
teria per gram
of dry soil

Relative
numbers

30
50
65

80
100

6.51
10.85
14.10
17.35
21.69

9,980,000
11,890,000
16,410,000
29,960,000
25,280,000

33.3

39.7

54.8

100.0

84.4

Influence of Moisture. — Bacterial development is gener-
ally at a maximum when the moisture content is relatively high
(Table 9). The maximum development of the aerobic bacteria,
which are concerned with some of the most important soil proc-
esses, takes place when the moisture content is between 50 and 70
per cent of the moisture-holding capacity. This corresponds to
the moisture content best adapted to development of most culti-
vated plants. Although bacterial activity in air-dry soil, with 2 to
5 per cent moisture, is almost negligible, drying of soil has a striking
effect on the growth of both microbes and higher plants, after the
dried soil has been remoistened. Drying renders the soil sub-
stances much more available for decomposition, due to physical
and chemical changes in the soil organic matter, to the killing of
many microbial cells by the desiccation process, and to modifica-
tions in the nature of the soil population. Whatever the respon-
sible factors may be, the intermittent drying and moistening of the
surface soil is quite beneficial in rendering nutrient elements
available for plant growth. Fig. 43, taken from laboratory studies,
indicates the course of microbial development following the
moistening of a dried soil. Over the same period of time, the
soil which had not been desiccated would have shown no pro-
nounced increase or decrease in abundance of bacteria or fungi
or in the formation of carbon dioxide.

In humid regions the root systems of plants are confined much
nearer to the surface than in semi-arid zones, consequently the
plant residues are more generally concentrated near the surface

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fa

INFLUENCE OF REACTION

63

in regions of heavy rainfall and may appear more abundantly in the
deep soil layers in regions of scant precipitation. The factor of
major importance in determining the conditions in the dry regions
is the lack of sufficient moisture at the surface to support a vigor-
ous growth of either the plant or the microbial population. In
compact soils the physical condition is such as to resist the deep
penetration of roots and, even though there were an abundance
of organic matter at considerable depth, the material would be
incompletely disintegrated on account of the lack of oxygen pen-
etration.

The influence of climate is quickly impressed upon the bac-
terial activities in the soil. Even when soils are brought from one
region to another there is a rapid modification, chemically, bio-
logically, and physically (Table 10). The numbers of bacteria of
arid soils usually increase when the soils are placed under humid
conditions, and bacterial numbers in humid soils decrease when soils
are placed under arid conditions.

TABLE 10

Influence of Climate on Bacteria in Soils (from C. B. Lipman)
Numbers of bacteria per gram of soil in the surface foot

In California
813,000

Califoryiia Soil
In Kansas
5,067,000

In Maryland
5,467,000

In California
1,507,000

Kansas Soil

In Kansas

5,167,000

In Maryland
1,370,000

In California
6,140,000

Maryland Soil

In Kansas

880,000

In Maryland
546,000

Influence of Reaction. — Among the environmental factors
influencing the development of bacteria in soil, the reaction,
that is, the degree of acidity or alkalinity, is of particular impor-
tance. When a humid soil is treated with acid-forming fer-
tilizers, such as ammonium sulfate which on oxidation by nitri-
f3dng bacteria gives nitric and sulfuric acids, or with sulfur
which on oxidation by sulfur-oxidizing bacteria gives sulfuric acid,

64 THE SOIL POPULATION AND ITS DISTRIBUTION

the total number of bacteria, as well as the development of certain
specific types, will decrease. When an acid soil of humid regions
is treated with lime, the numbers of bacteria rapidly increase.

As a result of this, the idea prevails that bacteria are more
favored by an alkaline reaction while fungi are more abundant
in acid soils. The results given in Table 11 show the effects
upon bacterial development of differences in reaction resulting
from differences in treatment of soil. These results bring out
further the fact that the reaction is not the sole limiting factor
in the development of microorganisms. Plot 5A is slightly more
acid in reaction than 9A, but still contains a much larger number
of bacteria because it contains more organic matter, as shown by
the higher nitrogen content. Plot 9A is more acid than plot 7B,
but still it contains a greater number of bacteria, because of the
higher organic matter content.

When a black alkali soil having a pH value of 9.6 to 10.0 is
treated with sulfur, sulfuric acid, aluminum sulfate, or other acid-
reacting substances, thus making the soil less alkaline, an increase
in the number of bacteria takes place, because of the fact that
conditions become more favorable for their development.

Influence of Season of Year. — The greatest numbers of
bacteria are usually found in the soil in the spring time and in the
fall (Fig. 37). During the winter months the soil becomes so
altered chemically and physically by the low temperatures that
there is a pronounced acceleration of microbial development when
the soil habitat becomes warm again. After this period of
unusual activity, the microorganisms decrease in numbers some-
what and fluctuate from this lower level, in response to changes in
their environment brought about by rainfall, plant development,
and cultural treatments. In the fall of the year, the organisms
reach a second peak in development which may be explained in
part by the incorporation of considerable amounts of organic
material accompanying maturity of plants, in part by the higher
moisture content resulting from decreased transpiration and evap-
oration. During the winter months, when the soil is frozen, the
organisms are quite inactive, although a large portion of the
microbial population persists in spite of the adverse conditions
(Fig. 44).

Appreciable changes in temperature, in the moisture films
about the soil particles, or in the amount of available food materials,

DEVELOPMENT OF ACTINOMYCES IN SOIL

65

are followed by changes in the abundance and activities of the
microscopic population of the soil.

Development of Actinomyces in Soil. — QuaHtatively, the
actinomyces react to changes in the soil environment in much the
same way as most of the soil bacteria do. They respond markedly
to the addition of organic materials, such as root residues or other
plant and animal remains, and may be concerned with the slow
decomposition of some of the most resistant organic substances

20 -.

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50 100

40

30

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60

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Fig. 44. — Microorganisms in soils during the winter and spring months
(after Lochhead).

Nov.

Dec.

Dec.

Jan.

Jan.

Feb.

Feb.

Mar.

Apr.

Apr.

14

1

19

7

28

11

29

21

8

22

in the soil. Thej^ are favored by much the same moisture content
as the bacteria, but may develop better than the bacteria when
the soil is relatively low in moisture. They are more sensitive
than bacteria to soil acidity, and develop best at neutral or slightly
alkaline reactions. These organisms appear to be more constant
in abundance than either the bacteria or fungi and show less
marked fluctuations due to environmental changes. Although
they decrease in abundance from the surface to the deeper layers of

66 THE SOIL POPULATION AND ITS DISTRIBUTION

soil, the decrease is more moderate than that of the numbers of
bacteria. Consequently, at the deep layers of many soils the plate
count reveals a proportionally large abundance of actinomyces.
This may be due to their greater tolerance to low concentrations
of oxygen as well as to their abihty to thrive upon the organic sub-
stances of a complex nature which may be washed into these low
levels. The earthy odor of plowed sod land is due to certain
aromatic substances elaborated by the actinomyces.

Since actinomyces are essentially aerobic organisms they are
absent in peat bogs, except near the surface, since the bog condi-
tions favor the development of anaerobic organisms. Most
actinomyces are sensitive to acidity, and are, therefore, practically
absent from raw-humus forest soils and unlimed highmoor peats.
Certain few species of actinomyces, however, are known to thrive
at reactions even more acid than pH 4.0. Since they are quite
resistant to dry conditions they are found abundantly in arid and
semi-arid soUs as well as in sandy soUs.

In general, the numbers of actinomyces range from a few
thousands to many millions per gram of soil. However, in view
of the fact that they occur in the soil both in the form of spores and
vegetative mycehum, a large part of which may not develop readily
upon artificial media, the numbers determined by the plate method
are only relative, and serve merely to indicate the approximate
abundance of these organisms in a given soil under a given set of
conditions.

From observations of the actinomyces in differently treated
plots of soil which were originally the same (Table 11), it is apparent
that, when the soil had become acid following repeated additions of
ammonium sulfate, the numbers of actinomyces diminished even
to such a low level as 370,000 per gram. In the same soil kept at
pH 6.7 by liming (19B), the organisms were as numerous as
2,820,000 per gram. However, with the actinomyces as with
the bacteria, the organic matter content of the soil is an important
factor in determining their abundance. In Plot 5A, with a pH
of even 5.4, as many as 2,920,000 actinomyces were found per gram
of soil.

Development of Fungi in Soil. — Fungi occur in soil in the
form of both vegetative mycelium and spores; this is true both of
soils acid in reaction and rich in organic matter, or neutral and
even alkaline in reaction and containing little organic residues. As

DEVELOPMENT OF FUNGI IN SOIL

67

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68

THE SOIL POPULATION AND ITS DISTRIBUTION

seen from Table 12, it is likely that fungi exist in soils in the vege-
tative state to a much greater extent than in the form of spores.
It has been observed that, when vegetative growth is rapidly-
desiccated in soils, it undergoes destruction; under the same con-
ditions fungus spores are unaffected. When soils containing the
soil organisms in their normal condition are rapidly desiccated,
the numbers of fungi are very largely reduced. Therefore, it
seems likely that when one counts colonies of fungi on agar plates
prepared from soil dilutions, one is counting colonies which have
developed from pieces of mycelium of the organisms. What the
sizes of the pieces of mycelium may have been at the time of the
determination is unknown. Undoubtedly the individual pieces
varied greatly in size.

TABLE 12

Influence op Desiccation of Soils on Development of Colonies of
Fungi on Agar Pl.\tes (from McLennan)

Average Number of Colonies per Plate

Preparation

Plated before
desiccation

Plated after
desiccation

Only spores present in the soil

Only mycelium i)resent in the soil ....
Field soil No. 1

31.0

20.0

10.6

11.6

8.8

25.2

199.0

326.3

30.0
0.8
0.6

Field soil No. 2

1.0

Field soil No. 3

1.1

Field soil No. 4

3.0

Field soil No. 5

13.7

Field soil No. 6

5.6

The value of the plate method for determining the abundance
of fungi in the soil is further limited by the fact that many fungi
fail to grow on the media commonly used for their determination.
In this respect there is a similarity to the plate method for deter-
mining the numbers of bacteria. The results of these fungus
counts should, therefore, be interpreted only as rough quantitative
measurements of certain representatives of the fungus population.

The types of fungi which are found in different cultivated soils
are much the same, but the relative abundance of any of the species
is determined by the environmental conditions. One of the most

DEVELOPMENT OF PROTOZOA IN SOIL 69

important factors affecting their growth and development in the
soil is the abundance of organic food. However, even in soils low
in organic substances there may be relatively extensive fungus
development; such a condition develops most frequently in
quite acid soils. This does not necessarily signify that fungi
develop best in soils under extremely acid conditions, for it has
been observed that they develop quite well over a wide range of
reaction. Since the bacteria and actinomyces may be at a low
order of activity in the acid environment, the fungi, which can
develop under these conditions as well as in neutral soils, have less
competition for the available food supply and consequently appear
in greater abundance.

The results of determinations of the numbers of fungi in variously
treated plots, as shown previously in Table 11, are illustrative of
the above conclusions. Limed soils contain fewer fungi than un-
limed soil, while soils treated with acid fertilizers contain the largest
numbers of fungi. In soils more nearly neutral in reaction, there
is such a large number of microorganisms which are capable of
developing under these conditions, that the factor of competition
for food by the cells may keep the fungi from making an extensive
development.

The fungi are favored by a relatively high oxygen content in
the air since they are distinctly aerobic organisms. Consequently,
we would expect to find fungi developing best under the aerobic
conditions close to the soil surface. The fact that there is greater
abundance of organic matter in the surface soil is also responsible
for the most extensive fungus development being restricted to the
superficial layers.

From the response of the actinomyces and fungi to the soil
reaction it becomes apparent that frequently the total abundance
of these organisms, and also the portion of the total soil population
composed of either fungi or actinomyces, are functions of the soil
reaction. However, the responses of these two groups to reaction
are opposite, one appearing in greatest abundance in acid soils,
the other in more nearly neutral or basic soils.

Development of Protozoa in Soil. — To a large degree, the
biological transformations in soil are brought about by the bac-
teria, actinomyces, and fungi. The lower animal life may be of
particular importance under certain soil conditions and in connec-
tion with some processes, but it would appear that it exerts minor

70

THE SOIL POPULATION AND ITS DISTRIBUTION

influences upon the activities brought about by microorganisms in
a normal soil environment.

The conditions which favor the development of bacteria also
appear to favor the growth of protozoa. The explanation becomes
apparent when the fact is considered that bacteria are an impor-
tant part of the protozoan diet. In fertile soils rich in organic
matter and relatively high in moisture, the protozoa are found in
greatest abundance. The small flagellates and amoebae are con-
siderably more numerous than the ciliates. They develop best
at slightly alkaline reactions, but appear to be as tolerant to acid
conditions as most of the soil bacteria. Under unfavorable con-
ditions, the soil protozoa go into a dormant state in the form of
cysts, which again become active when the environment is favor-
able. The active trophic stage of protozoa is probably more com-
mon in moist soils. Although they may occur in the soil in much
smaller numbers than bacteria, as shown in Table 13, their cells
occupy considerably more space, as brought out in Table 14.
As with most of the other members of the soil population, the
protozoa occur in greatest numbers near the surface.

TABLE 13
Numbers of Protozoa in American Soils (from Sandon)

pR

Flagel-
lates

Amoebae

Ciliates

Irrigated land, no manure

Irrigated land, 30 tons of manure . .
Humid Sassafrass soil, unma-

nured, unlimed

Humid Sassafrass soil, manured . . .
Humid soil (Penn loam), under

alfalfa

7.5-8.0
7.5-8.0

5.3
5.2

450
14,380

121
6,780

10,785

4,795

3,596

28,770

165
95,067

10,785

81,495

225
114

0
145

98

Humid soil (Penn loam), fallow
after corn

263

Development of Algae in Soil. — Algae occur in greatest
numbers in cultivated land and to depths of even 40 to 50 cm.
below the surface. As chlorophyll-bearing organisms, they are
capable of utilizing the rays of the sun photosynthetically when
growing on the surface, but, at lower depths of soil, they function as
heterotrophic organisms. Since their distribution below the sur-

DEVELOPMENT OF ALGAE IN SOIL

71

TABLE 14

Comparative Numbers and Weights of Cell Subst.^nce of Protozoa

AND Bacteria in the Soil (from Cutler)

Period of Low Numbers

Period of High Numbers

Number

per gram

of soil

Pounds
per acre

Number

per gram

of soil

Pounds
per acre

Flagellates

Amoebae

Bacteria

350,000

150,000

22,500,000

78

156

24

770,000

280,000

40,000,000

174

294

40

face is controlled by factors similar to those which regulate the
distribution of other organisms, they are more numerous near the
surface, and have fewer species represented and less total number
of individuals in the deeper layers. Rarely do they appear in
abundance in soils of low water content. Grassland appears to
be a more favorable environment for their development than the
more highly cultivated soils. It has been estimated that, per
volume, they occupy about three times as much space per unit of
soil as the bacteria, and about one-third the space occupied by the
protozoa. Naturally they can hardly be present in such abun-
dance without exerting profound changes in certain soil processes.
According to Bristol-Roach, the actual numbers of algae in the
cultivated layers of agricultural soils range from 700 per gram to
many thousands, the numbers changing with depth of soil, season
of year, and soil treatment (Table 15).

TABLE 15

Influence of M.anure and Soil Depth upon the Distribution of Algae

IN English Soils (from Bristol-Roach)

Depth

Xum])ers per gram of soil

Unmanured

Manured

Surface inch . . .
Second inch. . . .

16,000

10,000

28,000

4,000

62,000
28,000

Fourth inch

56,000

Sixth inch

15,000

72 THE SOIL POPULATION AND ITS DISTRIBUTION

Lower Invertebrates in Soil. — The lower invertebrates
occur in greatest abundance in light soils, other conditions being
alike. As with the other soil organisms, environmental conditions
greatly affect their prevalence; probably these effects are even
more marked than with those forms which do not have the ability
to migrate appreciable distances. The abundance of organic
matter is a particularly important factor. In unmanured field
soil cultivated to wheat, Morris found 4,885,400 of the larger
invertebrate animals per acre. In the manured soil there were
14,795,000 individuals. However, these figures represent only a
very small fraction of the total numbers of invertebrate animals
present in the soil. The nematodes alone have been found in hun-
dreds of millions of cells per acre of land (Table 16).

TABLE 16
Abundance of Nematodes in Soils (from Cobb and Steiner)

Soil

Number per acre in the surface
6 inches

Missouri corn field

648,000,000

New Jersey corn field

129,600,000

99,600,000

580,000,000

Acid forest soil in Virginia

320,000,000 (in surface 3 cm.)

Utah sugar-beet field

12,044,111,000 (in surface 2 feet)

The effects which these small animals exert are probably more
physical than chemical. They aid in the decomposition of organic
substances to some extent, but it is doubtful that they play roles
which could be compared with the bacteria and other related
microorganisms.

The Activity of the Soil Population as a Whole. — Many
methods have been used to determine the response of the total soil
population to changes in its environment. Of these methods the
determination of the amounts of carbon dioxide formed in a certain
unit of time has proved to be most valuable. The carbon dioxide
of soils originates almost entirely through biological agencies.
Practically all of the soil microbes produce carbon dioxide as one
of the principal waste products of their development, and the
amounts are closely proportional to the degree of their activity.

SUMMARY 73

Consequently, results of determinations of the amounts of carbon
dioxide produced give information which indicates, more accurately
than determinations of the numbers of any single group of organ-
isms, the level of biological activity, irrespective of the organisms
that are concerned.

The method does not discriminate between those organisms
which can be counted by plate methods and those failing to grow
on artificial media. Although one cannot distinguish what organ-
isms are responsible for producing the carbon dioxide, one has in
this method a device for determining to what extent microbial
activity as a whole becomes depressed or favored by particular
soil conditions.

In general, those factors, considered in the preceding pages,
which favor development of aerobic bacteria, actinomyces, and
fungi, also favor rapid formation of carbon dioxide. High carbon
dioxide evolution is associated with productive soils, and factors
which increase crop production also increase formation of carbon
dioxide. Such factors as relatively high temperature and moisture
content, thorough aeration, presence of an abundance of organic
matter, inorganic plant nutrients, and lime, all exert favorable
effects on biological activity as measured by the rate of formation
of carbon dioxide.

The resultants of some of these effects are indicated in the sea-
sonal variation of the gas formation which is shown in Fig. 37.

The rate of formation of nitrates, oxygen absorption by soil,
and heat evolution, as a result of the activities of microorganisms,
can also be used as measures of the activity of the soil population,
but none of these is as simple as the determination of evolution of
carbon dioxide.

Summary. — The soil is inherently a very complex system,
where chemical, physical, and biological systems are active. All
of these agencies tend to become adjusted to the environmental
conditions existing in the soil at any one time. If a soil is kept
under uniform conditions, the speeds of the numerous processes
taking place in the soil also tend to become uniform. Any influ-
ence which tends to disturb this adjustment in the soil conditions
alters these processes, which will now tend to become adjusted to
the new conditions. Thus, a change in soil reaction causes a series
of changes in the biological condition of the soil; after a lapse of
time, the new population becomes adjusted to the new reaction.

74 THE SOIL POPULATION AND ITS DISTRIBUTION

The magnitude of the effects of different factors in bringing about
changes among the microorganisms of the soil, including numbers,
types, and activities, varies greatly. Cultivating a soil shows only
slight effects on the population. Addition of inorganic fertilizers
increases the activities slightly more. Of the various soil treat-
ments in humid regions, the introduction of organic substances
affects the numbers and activities of microorganisms more than
any other.

The soil in its natural environment is constantly exposed to
fluctuating conditions of temperature, moisture, pressure, and air
movements, numerous factors being active at any one time.
Furthermore, addition and removal of organic and inorganic
materials, through the agency of man, animals, and plants, are
always occurring. These factors are undoubtedly responsible
for the perpetuation of the active biological conditions in the soil.
They exert their action either directly upon the microbes, or
only indirectly, by first influencing the plant and the soil con-
stituents, and these in their turn modifying the development of the
microorganisms.

The composition of the soil microbial population, both qualita-
tively and quantitatively, is thus a resultant of numerous factors,
which can be traced to the soil and atmospheric agencies, as well
as to the nature of the plant and animal populations.

LITERATURE

1. Buchanan, R. E., and Fulmer, E. I. Physiology and biochemistry of

bacteria. Volume 2. The Williams & Wilkins Co. Baltimore, 1930.

2. Conn, H. J. A bacteriological study of a soil type by new methods.

Soil Science, 25, 1928: 263-272.

3. Henrici, a. T. Molds, yeasts, and actinomycetes. John Wiley & Sons,

Inc. New York, 1930.

4. Jordan, E. 0., and Falk, I. S. The newer knowledge of bacteriology and

immunology. Chapters 23, 24, 26. The University of Chicago
Press, 1928.

5. Russell, E. J. Soil conditions and plant growth. 5th ed. Chapter 6.

Longmans, Green & Co. London, 1927.

6. Russell, E. J. The microorganisms of the soil. Longmans, Green &

Co. London, 1923.

7. Waksman, S. a. Principles of soil microbiology. Chapter 1. Williams

& Wilkins Co. Baltimore, 1927.

chapter iv

rOle of microbes in the decomposition of
organic substances in the soil

Principles of Decomposition of Organic jMatter by
Microbes. — Undoubtedly the most important function of micro-
organisms, from the point of view of man's economy, is their ability
to bring about the decomposition of organic plant and animal re-
mains not only in soils but in all natural environments. The sim-
plification of the chemical structure of the organic materials, or
their transformation from complex to simpler forms, leading finally
to the liberation of completely oxidized inorganic substances in
forms available to higher plants, is brought about by most of the
microorganisms in the soil. The end products of the complete
decomposition of non-nitrogenous materials are carbon dioxide,
water, and synthesized microbial cells. The nitrogenous sub-
stances give also, in addition to these, ammonia, and other simple
compounds of nitrogen, as well as compounds of sulfur and phos-
phorus.

The attack of any complex compound by microorganisms sel-
dom results directly in complete decomposition even under aerobic
conditions; various intermediate products are usually formed.
The incompletely decomposed residues left from the action of cer-
tain organisms are further attacked by other members of the soil
population which have the ability to decompose them. Sooner
or later the initial material completely loses its identity and is
transformed principally to the completely oxidized end products,
and partly to a variety of synthesized substances in the form of
microbial cells. The various soil organisms differ from one an-
other greatly in their physiological characteristics; what may be a
readily available food for one species would not be available to
others. The great diversity of nutrition exhibited by the many
soil microbes makes possible the rapid digestion of whatever mate-
rial may be added or formed in the soil.

75

76 DECOMPOSITION OF ORGANIC SUBSTANCES IN SOIL

Scarcely any organic compounds are unlikely to be introduced
into the soil. The numerous substances synthesized by the great
variety of higher plants are continuously added in the form of plant
roots, stubble, leaves, twigs and branches. Immature plants are
also abundantly incorporated with the soil as green manures.
Plant residues are added to the soil in the form of stable manures.
Various waste products reach the soil subsequent to threshing,
milling, and utilization of various cereal crops, as well as cotton-
seed and linseed. Partly digested plant products, which have
been used in animal feeding, are contained in the manures. Various
animal products are also introduced into the soil, other than the
partially digested excreta. Those most commonly added are
by-products of industry, such as dried blood, meat scraps, bone
meal, hair, hoofs, and feathers. There are also large numbers of
small invertebrate and vertebrate animals which find in the soil
a temporary or permanent habitat. Finally there is a continuous
addition of considerable quantities of microbial cell substance to the
soil. Consequently, we may feel secure in assuming that there
may be added to the soil any compound contained in higher plants,
animals, or microorganisms, as well as any substance which might
be formed from these compounds as they are attacked by the
microorganisms in the soil.

Composition of Plant and Animal Substances. — The plant
and animal residues comprise a large number of chemical com-
pounds which can be classified conveniently into the following
groups :

I. Carbohydrates.

1. Monosaccharides.

a. Hexoses (CeHi-iOe), such as glucose, fructose, mannose.

b. Pentoses (CaHioOj), such as arabinose and xylose.

2. Disaccharides (Ci2H2:Oii), the most important of which are
sucrose and maltose.

3. Trisaccharides (CisHsaOie), rafSnose.

4. Polysaccharides.

a. Starch, glycogen, inulin, and dextrins.

b. Cellulose.

c. Hemicelluloses and polyuronides.

(1) Hexosans, as mannans and galactans.

(2) Pentosans, of the structure (C6H804)j.

(3) Pectins and other uronic acid compounds.

II. Lignins.

COMPOSITION OF PLANT AND ANIMAL SUBSTANCES 77

III. Tannins.

IV. Glucosides.

V. Organic acids, salts, and esters.
VI. Fats, oils, waxes, and related compounds.
VII. Resins.

VIII. Nitrogen compounds.

1. Proteins.

2. Amino acids.

3. Amines.

4. Alkaloids.

5. Purines.

6. Nucleic acids.

IX. Pigments.

1. Chlorophyll — green coloring matter.

2. Carotinoids — pigments of leaves, stems, flowers, and fruit.

3. Anthocyanins — pigments of leaves, fruit, and flowers.

X. Mineral constituents.

L Bases, especially Ca, Mg, K, Fe.

2. Phosphates.

3. Chlorides.

4. Sulfates.

5. Silicates.

The various plant constituents can be divided into the following
groups, which are the most extensive and important, since they
make up the larger part of the plant and influence the nature and
rapidity of its decomposition:

(1) The water-soluble constituents, including the simple
carbohydrates, starches, amino acids, and various organic
acids.

(2) The hemicelluloses, which are condensation products of
hexoses, pentoses, or both of these with uronic acids.

(3) Cellulose, which is a condensation product of glucose.

(4) Lignin, the exact chemical nature of which is still
unknown. It is known to comprise a benzene ring group, sev-
eral methoxyl and hydroxyl groups, and an aldehyde group.
The following formula is typical for this complex:

C4oH3o06- (0CH3)-t • (0H)5- CHO

78 DECOMPOSITION OF ORGANIC SUBSTANCES IN SOIL

In the plant it forms absorption or chemical compounds with
cellulose. The young plant contains largely cellulose in its
structural tissues, but with the maturing of the plant, the cel-
lulose fibers absorb (or combine with) lignin, thus forming
the ligno-cellulose of the plant tissues.

(5) Proteins, composed of various amino acids. These
are the most important nitrogen constituents of the plant.

(6) Fats, oils, and waxes, which are esters of alcohols and
one or more higher fatty acids.

(7) The ash or the mineral constituents of the plant.

A typical series of analyses of various plant products is given in
Table 17.

TABLE 17

Proximate Composition of Natural Organic Matter (from
Pringsheim)

Substance

Cellu-
lose

Pento-
san

Lignin

Pro-
tein

Fats

and

waxes

Ash

Hay

per cent
28.50
35.43
34.27
37.66
30.56

per cent
13.52
21.33
21.67
31.50
23.54

per cent
28.25
20.40
21.21
14.70
15.13

per cent
9.31
4.70
3.00
2.11
3.50

per cent
2.00
2.02
0.67
1.37
0.77

per cent
6.05

Oat straw

4.81

Wheat straw

4.33

Corn-cobs

1.80

Corn -stover

6.15

The remaining substances not given in the above table include
the water-soluble constituents (sugars, organic acids), various
hemicelluloses, in addition to the pentosans, the pigments, and
other substances present in smaller fractions.

Not only do various plants vary in their composition, but the
composition of the same plant varies at different stages of growth,
as seen in Table 18.

Factors Affecting Decomposition. — The various chemical
complexes are decomposed in the soil with different degrees of
rapidity and frequently by different organisms. Since different
plants vary so greatly in the relative abundance of the various
constituents, the speeds of their changes and the types of the resi-
dues are very dissimilar even under the same environmental con-

DECOMPOSITION OF SUGARS AND THEIR DERIVATIVES 79

TABLE 18

Composition of the Rye Plant (Stems and Leaves) at Different
Stages of Growth* (from W.\ksman and Tenney)

stage of plant growth

Fats

and

waxes

Water-
soluble

sub-
stances

Pen-
tosans

Cellu-
lose

Lignin

Total
nitrogen

Ash

10-14 inches high ....
Just before heads

begin to form

Just before bloom. . .
Mature plant

per cent
2.60

2.60
1.70
1.26

per cent
34.24

22.74

18.16

9.90

per cent
16.60

21.18
22.71
22.90

per cent
18.06

26.95
30.59
36.29

per cent
9.90

11.80
18.00
17.10

per cent
2.50

1.76
1.01
0.24

per cent
7.66

5.90
4.90
3.90

* Calculated on the dry basis.

ditions. To understand why certain plant residues may be decom-
posed when they are introduced into a soil and become an avail-
able source of nutrients to higher plants, and why other plant
materials, or the same material under different conditions, may
accumulate and fail to undergo destruction, is a problem involving
a consideration of many factors. The chemical nature of the com-
plex, the other substances with which it is associated in the plant
structure, the nature of the soil (such as physical structure,
chemical composition, aeration, moisture content, reaction, and
temperature) with which the materials are incorporated, are all
important factors determining the types of organisms active in the
transformation and the nature of the resulting material. Under
some conditions there are such accumulations of organic matter as
peat; in other instances, grassland formations or forests develop.
Further, the environmental conditions determine the kind of natural
vegetation and consequently the nature of the organic substances
which become added to the soil.

Decomposition of Sugars and their Derivatives. — After
organic matter reaches the soil, sugars and other water-soluble sub-
stances are the first to be decomposed. These substances largely
disappear within the first few days subsequent to their attack by
various bacteria and fungi. A part of these is completely oxidized
to carbon dioxide and water, while some are incompletely decom-
posed with the formation of a number of various organic acids and
alcohols. Some fungi decompose the sugars with the formation of

80 DECOMPOSITION OF ORGANIC SUBSTANCES IN SOIL

glucuronic, citric, oxalic, fumaric, and succinic acids, as shown in
the following reactions:

CsHisOe + O2 = CHO • (CH0H)4 • COOH + H2O

Glucose Glucuronic

acid

CeHioOe + UO2

Glucose

= CH2 • COOH • COH • COOH -CHs- COOH + 2H2O

Citric acid

C6H12OC + 4IO2 = 3(COOH)2 + 3H2O

Glucose Oxalic acid

C6H12OC + 6O2 = 6CO2 + 6H2O

Glucose

Bacteria decompose sugars in a manner quite different from
that of the fungi, and the resulting end products are determined
principally by the presence or absence of free oxygen. Lactic,
butyric, acetic, propionic, formic, and valerianic acids, methane
and hydrogen are some of the products formed under anaerobic
conditions; frequently butyl alcohol, ethyl alcohol, and acetone
are also produced under these conditions.

CgHioOo = 2CH3-CHOHCOOH

Glucose Lactic acid

CoHioOg = CH3-CH2CH2COOH + 2CO2 + 2H2

Glucose Butyric acid

CgHioOg = 2CH3CH2OH + 2CO2

Glucose Ethyl alcohol

In the presence of oxygen, the sugar molecule is broken down,
through the lactic acid, acetaldehyde, and pyruvic acid stages, to
carbon dioxide and water, as shown by the following reactions:

CeHiaOo = 2CH3-CO-COOH + 2H2

Glucose Pyruvic acid

CHs-CO-COOH + Ho = CHs-CHOH-COOH

Pyruvic acid Lactic acid

CHs-COCOOH + Ho = CH3CHO + Ho + CO2

Pyruvic acid Acetaldehyde

CH3CHO + Ho = CH3CH2OH

Acetaldehyde Ethyl alcohol

DECOMPOSITION OF HEMICELLULOSES 81

CH3COCOOH + H2O = CHs-COOH + H-COOH

Pyruvic acid Acetic acid Formic acid

CH3-CH20H + 3O2 = 2CO2 + 3H2O

CHa-COOH + 2O2 = 2CO2 + 2H2O

H-COOH + ^02 = CO2 + H2O

The starch content of plants, with the exception of a number of
various seeds and other reserve food bodies, is very small. The
decomposition of the starches is very rapid, the process being
similar to the decomposition of the sugars. Some of the microbes,
especially the anaerobic bacteria, attack the starch molecule with
the formation of various organic acids, alcohols, aldehydes, acetone,
hydrogen, and carbon dioxide. Numerous soil microbes are able
to produce an enzyme, diastase, which hydrolyzes the starch to
maltose, and another enzynie, maltase which hydrolyzes the di-
saccharide maltose to two molecules of glucose.

Decomposition of Hemicelltjloses. — Hemicelluloses are
present in considerable amounts in higher green plants and to some
extent in microscopic green and chlorophyll-free plants, where
they may occur as reserve food material. On hydrolysis, they are
changed to simple sugars, hexoses or pentoses :

(C5H804)/i + nH20 = nCCsHioOo)
(CsHioOs)?! + nHsO = ^KCeHioOe)

The most abundant group of hemicelluloses are the pentosans,
present in quantities ranging from 7 per cent in pine needles to 32
per cent in corn-cobs.

Hemicelluloses, especially pentosans, are readily decomposed
by a large number of fungi, actinomyces, and bacteria. In the
degradation of fresh plant materials in the manure heap or in the
soil, the decomposition of the pentosans proceeds somewhat more
rapidly than that of cellulose. However, the organic matter in
certain peats and in mineral soils is apt to be free from cellulose,
while still containing considerable quantities of hemicelluloses.
This is due to the greater resistance of some of the hemicelluloses
to decomposition and to the formation of new hemicelluloses
through the synthesizing activities of the microorganisms. Such
substances formed by the organisms are commonly referred to as

82 DECOMPOSITION OF ORGANIC SUBSTANCES IN SOIL

gums and slimes, which are produced by a variety of bacteria
and fungi, either as excretion products of the cells, in the form of
capsules, or as cell constituents.

Decomposition of Cellulose. — With certain few exceptions,
cellulose forms the largest single group of constituents of plant
materials added to the soil. Such fibrous materials as flax, hemp,
cotton, and jute are largely made up of pure cellulose. Cellulose
is not present in a free state in any great abundance in the
vegetative structure of the plant, but is combined with lignin,
cutin, and pectin, the compounds thus formed giving rise to
ligno-celluloses, cuto-celluloses, and pecto-celluloses.

Cellulose is an amorphous polysaccharide, giving on hydrolysis
with strong mineral acids or with appropriate enzymes the di-
saccharide cellobiose, which in its turn is readily hydrolyzed to
glucose. When acted upon by soil microorganisms, sugars can
be demonstrated as intermediate products only in exceptional
cases. Cellulose can be readily decomposed by a large number of
organisms which are present in normal soils, including various
fungi, bacteria, and actinomyces, and possibly also some protozoa.
The nature of the microbes responsible for the decomposition of
cellulose varies with the conditions; it is different in soil than in
the manure pile or in the peat bog; the chemistry of the process of
cellulose decomposition depends upon the environmental condi-
tions and the organisms concerned in the process.

Fungi Decomposing Cellulose.— The fungi capable of
decomposing cellulose include a large number of organisms found
among the Ascomycetes, Basidiomycetes, and Fungi Imperfect i.
The following genera have been shown to contain one or more
species capable of decomposing cellulose: Trichoderma, Asper-
gillus, Penicillium, Fusarium, Cephalosporium, Verticillium,
Sporotrichum, Monosporium, Alternaria, Hormodendrum, Humi-
cola, Chaetomium, various Polyporynae, and Agaricinae. These
fungi are especially active in the so-called raw-humus forest
soils, which are acid in reaction and are only seldom favorable for
the development of cellulose-decomposing bacteria. The forest
humus, comprising the upper few centimeters of forest soil,
consists largely of a mass of partly or completely disintegrated
leaves, needles and their residues; this is so much interwoven with
colorless and brown fungus mycelium that, on examining the mass
of partly disintegrated organic residues under the microscope,

FUNGI DECOMPOSING CELLULOSE

83

it is difficult to say whether the residue from the plants or the
mycelium of the fungi is more predominant. Even in field,
garden, and greenhouse soils, where the reaction is quite favorable
for the development of the other cellulose-decomposing organisms,
the fungi also play an active part in the disintegration of this
abundant plant constituent, especially at the early stages of
decomposition. This is brought out in Table 19, which shows the
influence of the addition of cellulose to the soil upon the develop-
ment of fungi, the latter being determined by the plate method.

TABLE 19

Influence of Cellulose upon the Numbers of Fungi in Soil
(from Waksman and Starkey)

Reaction

of soil,

pR

NaNOg
added to
cultures

Numbers of Fungi in
1 gm. of Soil

Nature of soil

Soil
without
cellulose

Soil with

1 per cent

cellulose

Unlimed, unmanured

Unlimed, unmanured

Limed, unmanured

Limed, unmanured

Unlimed, manured

Unlimed, manured

5.1
5.1
6.5
6.5
5.5
5.5

+
+

+

115,700
115,700
20,000
20,000
87,300
87,300

160,000

4,800,000

47,000

290,000

320,000

3,100,000

Accompanying the decomposition of the cellulose the fungi
assimilate considerable quantities of nitrogen, which are required
by the organisms to enable them to synthesize their cell sub-
stance. There is a fairly definite ratio between the amount of
cellulose decomposed and the nitrogen required, which is between
30 : 1 and 50 : 1; in other words, for every 30 to 50 parts of cellu-
lose decomposed, the organisms require 1 part of available nitrogen.
A large part of the carbon of the cellulose decomposed is also used
by the fungi for the synthesis of their mycelium. Since the
mycelium or microbial cell substance has a definite chemical com-
position and the ratio between its carbon and nitrogen content is
more or less constant, and since the cellulose decomposed is used

84 DECOMPOSITION OF ORGANIC SUBSTANCES IN SOIL

both as a source of energy and as a source of carbon for the building
up of the microbial cell substance, the ratio between the carbon
used for energy and the carbon used for cell synthesis is constant
for a given organism under a given set of conditions. There is also a
ratio between the amounts of cellulose decomposed and the nitro-
gen assimilated. For every 100 units of cellulose decomposed, 20
to 30 units of cell substance are synthesized, which contain about
1.5 to 2.5 units of nitrogen. Since cellulose is free from nitrogen,
this element has to be obtained from some other compounds to
enable the microbes to bring about the decomposition of the cellu-
lose. The amount of available nitrogen in the soil very frequently
becomes the limiting factor in the decomposition of cellulose.
The effect of the presence of available nitrogen on development of
fungi is apparent from Table 19. The influence which soil reaction
and available nitrogen may have is shown in Table 20.

TABLE 20

Influence of Reaction and Available Nitrogen upon Development
OF Cellulose-decomposing Bacteria and Fungi (from Dubos)

Nature
of soil

Treatment

Soil
reac-
tion,

pH

Mgm. of C
given off as
CO2 from
500 mgm.
cellulose in
36 days

Cellu-
lose
decom-
posed,
per cent

Number of
cellulose-
decom-
posing
bacteria

Number of
fungi

Control

Cellulose added
Cellulose and

(NH4)2S04 added
Control

Cellulose added
Cellulose and

(NH4)2S04 added

5.4
5.4

5.4

0.8
(5.8

6.8

0
50,000

10,000

1,000

5,000,000

10,000,000

87,000

Unlimed

Unlimed

C3.C
122.8

55
9G

353,000

2,700,000
43,000

Limed

Limed

00.4
130.2

53
99

110,000
490,000

The beneficial effects which additions of animal manures and
many different fertilizer materials exert on the decomposition of
cellulose may frequently be ascribed to the greater amounts of
available inorganic nitrogen which these substances put to the
disposal of the microbes in the soil. Since phosphorus, and to a
less extent potassium, sulfur, and other nutritive elements, are
also required for the synthesis of microbial cell substances, these
elements must be present in the soil in appropriate amounts in

BACTERIA DECOMPOSING CELLULOSE

85

available forms to permit the microbes to use the cellulose as food.
Phosphorus is required in greater amounts than any of the other
mineral substances, and may comprise two-fifths of the total ash
of microbial cells. However, phosphorus (calculated as P) may
be required for the synthesis of cells in only about one-tenth the
amounts that nitrogen is needed. Other minerals are required in
much smaller quantities.

Bacteria Decomposing Cellulose. — The bacteria decompos-
ing cellulose can be divided conveniently into two groups, repre-
sentatives of which differ considerably in their morphological
characters and physiological activities: (1) aerobic bacteria and
(2) anaerobic bacteria. Among the aerobic forms there are spore-
forming and non-spore-forming rod-shaped bacteria, spherical
organisms and spirochaete-like or flexuous organisms (see Fig. 8).
It is characteristic of all that free atmospheric oxygen is re-
quired for development, just as in the case of the cellulose-
decomposing fungi, but, while the fungi will grow readily in very
acid soils of a pH 4.0 and even less, the aerobic bacteria will not
grow at an acidity greater than pH 5.6-6.0, as shown in Table 21.

TABLE 21

Effect of Reaction of the Soil on the Number of Cellulose-
Decomposing Bacteria (from Dubos)

Soil reaction.

Number of cellulose-
decomposing
bacteria

Soil reaction,
pH

Number of cellulose-
decomposing
bacteria

8.7
8.5
8.2
7.5
7.0

0

250,000

25,000,000

25,000,000

25,000,000

6.5
6.0
5.2
4.5
4.0

25,000,000

250,000

0

0

0

Neither fungi nor aerobic bacteria will grow in marshes or
undrained peat soils which are anaerobic in nature. In forest,
field, and garden soils, both groups of organisms will develop, the
reaction of the soil determining the group that will predominate.
Soils more acid than pH 5.5 will show largely a flora of fungi which
will be the active agents in cellulose decomposition; soils less acid

86 DECOMPOSITION OF ORGANIC SUBSTANCES IN SOIL

than pH 5.6 will favor the development of aerobic bacteria, with-
out, however, being unfavorable to fungi (see Table 20).

Many of the aerobic bacteria use cellulose as the exclusive
source of energy and are unable to develop upon the simple sugars,
indicating that this process of decomposition may not proceed
through the sugar stage or at least does not lead to the accumula-
tion of the lower carbohydrates. Bacteria decompose cellulose,
leaving a mucilaginous mass consisting of bacterial cells imbedded
in certain gum-like compounds (hemicelluloses) synthesized by the
organisms. Small amounts of various acids and certain yellow
and orange pigments are also formed. The aerobic bacteria also
require considerable quantities of nitrogen and phosphorus for
the decomposition of the cellulose, for the same reasons that they
are required by fungi.

Anaerobic bacteria (see Fig. 9) capable of decomposing cellu-
lose occur in arable soils only in limited numbers. They are
found abundantly in peat and marsh soils, in rivers, in manure
piles, and in other habitats where fresh organic matter is abundant
and free access of air is prevented. Some of the anaerobic bacteria
are thermophilic in nature, being capable of active growth and
decomposition of cellulose at 60° C. Both the organisms that
grow at normal temperatures and those that grow best at high
temperatures (55 to 65° C) can bring about very rapid decomposi-
tion of cellulose with the formation of various organic acids, alco-
hols, and gases, such as methane, hydrogen, and carbon dioxide.
Omeliansky found that out of 3.3 gm. of cellulose decomposed,
there were produced 2.2 gm. fatty acids, 1.0 gm. CO2, and 0.014 gm.
hydrogen. A thermophilic bacterium produced from the decom-
position of 42 gm. cellulose, 21.6 gm. acetic acid, 10.3 gm. ethyl
alcohol, and 11.9 gm. CO2, in addition to considerable quantities
of hydrogen. In addition to acetic acid, formic, lactic, and butyric
acids have been found as products of decomposition of cellulose by
anaerobic bacteria. The formation of several of these products
was explained by the following reactions:

(C6Hio05)n + ^iHoO = 2n(CH3-CO-COOH) + 2nH2

Cellulose Pyruvic acid

CH3COCOOH + Ho = CH3CHOHCOOH

Pyruvic acid Lactic acid

CH3COCOOH = CH3CHO + CO2

Pyruvic acid Acetaldehyde

INFLUENCE OF SOIL CONDITIONS 87

2CH3-CHO + H2O = CH3CH2OH + CH3COOH

Acetaldehyde Ethyl alcohol Acetic acid

2CH3CH2OH = CH3COOH + 2CH4

Ethyl alcohol Acetic acid Methane

2CH3-CHOHCOOH = CH3 • CH2 • CH2 • COOH -^ 2CO2 + 2H2

Lactic acid Butyric acid

Various bacteria are capable of decomposing cellulose in the
absence of atmospheric oxygen if nitrate is present; under such
conditions the nitrate is used as a source of oxygen. The nitrate
is reduced to gaseous nitrogen, and the oxygen thus liberated is
used for the decomposition of the cellulose.

A number of actinomyces decompose cellulose but at a com-
paratively slow rate. The nature of the reaction is still unknown.

Influence of Soil Conditions upon Cellulose Decompo-
sition.— The above considerations tend to explain why cellulose is
decomposed in different soils with different degrees of rapidity.
The soil conditions, such as moisture, reaction, aeration, organic
matter content, and supply of available nutrients, influence the
types of organisms present in the soil and the types that take an
active part in the decomposition of the cellulose; these organisms
in their turn influence the nature of the chemical reactions brought
about and the nature of the products formed. However, the
microbes which have the capacity of decomposing cellulose are so
numerous in the soil, they are of such various forms, and they
differ so greatly in physiological requirements, that, wherever
sufficient inorganic nutrients are available and wherever there is
adequate moisture and the temperature conditions are favorable,
cellulose is rather quickly decomposed. Either under moderate or
excessive moisture, at acid, neutral, or basic reactions, at moderate
or high temperatures, some microbes will find conditions conducive
to their activity. However, the mechanism of the decomposition
and the chemistry of the products formed will be different. The
formation of organic substances through the synthesizing agencies
of microorganisms contributes one of the sources of the soil organic
matter frequently referred to as soil humus.

Fig. 45 shows the comparative growth of the three major groups
of microorganisms, namely the bacteria, fungi, and actinomyces,
during the decomposition of cellulose at different moisture
contents.

DECOMPOSITION OF ORGANIC SUBSTANCES IN SOIL

LiGNiN AND ITS DECOMPOSITION. — The lignin in plants increases
in abundance as the plants become older; this complex makes up a
considerable portion of the mature plants. Since Ugnin is very-
resistant to decomposition by the common soil fungi and bacteria,
it tends to accumulate with the advance in decomposition of the
plant materials, while the sugars, proteins, hemicelluloses, and

Mgm.
180

160-

cul40
1 120

-iioo

O

80 -

1 1 I 1

Bacteria

' /'

/

/ /

/ _

Fungi

y / -

^

/ /

/

/
f

II 1- ^^ -

Mcunornyces

1 1 1 1

/ /

1

/ /

1

/ /

1

/ /

1

/ /

/

/ / •■■' ' ~

/

/ '

/ ..-:

1 i

/ ry^

I 1 ■■■■■'

i /

I 1 ••■■■

' /^

/ * —

// /

I A'

- J.y

>/ .*' ~

/ ' /

f *' '

/■•■ V

A*

J^ , 1 1

1 i/-' 1 1 1 1

o

1 60
o

^ 40
20

0

0 10 20 30 40 50 0 10 20 30 40 50
Time (Days)

Fig. 45. — Comparative growth of cellulose-decomposing bacteria, fungi, and
actinomyces in a neutral soil. Left curves at 30 per cent of the moisture-
holding capacity of the soil; curves at right at 50 per cent of the moisture-
holding capacity (after Dubos).

cellulose are quite readily decomposed. Only certain groups of
Basidiomycetes (species of Polyporus, Merulius) and certain
Actinomyces are capable of attacking the lignin; however, even
the action of these organisms is much slower than in the decom-
position of other plant constituents. Under anaerobic conditions,
as in peat soils, the lignin remains undecomposed and rapidly
accumulates.

Fig. 46 shows the relative resistance to decomposition of the

DECOMPOSITION OF FATS

89

lignin as compared with cellulose and hemicelluloses, when alfalfa
is allowed to decompose under aerobic and anaerobic conditions.

100

1 1 1 1 1

-

f\ ^' *

1 '^

90

1 \

80

l ^^\

_

iS\ \^

_ 70

•~

ii . V \ *'^.

.^60

- '; * \\ \ **

-

>-

I v^^ \,

o

i\| \>-— -/^^""^--^^

o 50

c
o40

.

k.

\ *'».

Q>

\

O.

\

30

\,

1 1

20

-

A. Hemicellulose

A. Cellulose

— A. Lignin

10

-

-

AN. Total Material

AN. Hemicellulose

AN. Cellulose

n

AN. Lignin

— 1 1 ...

100

400

500

200 300
Days

Fig. 46. — Course of decomposition of total plant material (alfalfa) and of the

hemicelluloses, cellulose, and hgnin under aerobic (A) and anaerobic (AN)

conditions (from Tenney and Waksman).

Decomposition of Fats. — Fats are esters of the higher acids
and a tribasic alcohol, glycerol. The decomposition of a typical
fat proceeds as follows :

90 DECOMPOSITION OF ORGANIC SUBSTANCES IN SOIL

CH2-OOCCl7H33

I
CHOOCC17H33 + 3H20= CHsOH-CHOH-CHoOH

I Glycerol

CH2 • OOC • C17H33 + 3C17H33COOH

Triolein (a fat) Oleic acid

The glycerol and fatty acids are further decomposed in the soil
by a number of various fungi and bacteria, giving lower acids,
carbon dioxide, and water. The decomposition of fats occurs
principally under aerobic conditions.

Decomposition of Other Plant Constituents. — A detailed
discussion of the decomposition of proteins is given in the following
chapter, where other transformations of nitrogen are considered.
The subject is of great importance, since nitrogen forms one of
the most important elements in the protein molecule and since
nitrogen is apt to be the factor most limiting abundant crop
growth. The numerous organic substances present in plants in
quantities ranging from mere traces to one or more per cent, are
sooner or later decomposed by one or more groups of soil organisms,
and either destroyed completely or transformed into other organic
complexes. Even such substances as various aldehydes, vanillin,
toluene, phloroglucinol, phenol, cresol, naphthalene, and their
derivatives, are decomposed more or less rapidly in the soil by
specific or non-specific organisms. As a result, compounds which
might otherwise be toxic to plant growth become innocuous and
may even lead to the formation of substances beneficial to plant
growth. It is necessary, however, that the soil conditions, such
as proper aeration, reaction, and moisture, should be favorable to
the development of the particular organisms.

Methane originates as an intermediate decomposition product
under anaerobic conditions, and may be attacked by a variety of
aerobic bacteria which oxidize it to carbon dioxide and water:

CH4 + 2O2 = CO2 + 2H2O

Some bacteria which are able to oxidize methane are also active in
decomposing benzene and paraffin. Numerous fungi and non-
spore-forming bacteria decompose paraffin, petroleum, vaseline,
benzene and kerosene under aerobic conditions. The disap-
pearance of these substances under anaerobic conditions is much

DECOMPOSITION OF THE PLANT AS A WHOLE 91

slower. The decomposition of such compounds, hke the trans-
formation of other non-nitrogenous substances, necessitates the
presence of considerable nitrogen available from other sources.

The changes which take place in the plant constituents may be
shown in the graphic representation of the decomposition of corn-
stover (Fig. 47) . It is apparent that there is a rather rapid decom-
position of the water-soluble constituents, hemi celluloses and cel-
lulose, and a slow destruction of the lignin. The crude protein, on

Total Cold -Water- Hemi- Cellulose Lignin Crude

Material Soluble Celluloses Protein

A, Original Material; B, Left after 27 Days of Decomposition;

C, Left after 68 Days of Decomposition; D, Left after 205 Days

of Decomposition E, Left after 405 Days of Decomposition

Fig. 47. — -Course of decomposition of various chemical constituents of corn-
stover under aerobic conditions (after Tenney and Waksman).

the other hand, shows a pronounced increase resulting from the
synthesis of an abundance of microbial cells at the expense of the
water-soluble nitrogen. This does not indicate, however, that
the protein constituents are resistant to decomposition. Under the
aerobic conditions, the material as a whole had decomposed to one-
third of the original amount in the period of 68 days.

Decomposition of the Plant as a Whole. — The rate at
which plants or organic fertilizers decompose is regulated to a large
extent by the relative abundance of the various constituents which
compose the organic matter and the state of aggregation of these

92 DECOMPOSITION OF ORGANIC SUBSTANCES IN SOIL

constituents. Some plant residues largely disappear in a few weeks,
while others still retain their original appearance after several
months in the soil. Generally, however, certain portions of prac-
tically all organic materials decompose somewhat rapidly within a
few days subsequent to their addition to the soil. This period is
followed by a uniform but rather abrupt retardation in the speed
of decomposition which is followed by a slow but continuous decom-
position of the remaining material. Even a year or more after the
addition of the organic matter, the soil is still in a more active
biological condition than it was before the treatment.

Although differences exist between the rates with which soils of
different fertility are able to decompose the same organic materials,
these differences are not very pronounced after the first few days.
As a rule, however, conditions favorable to development of culti-
vated plants, such as relatively high temperature and water con-
tent, presence of sufficient available inorganic substances, and
reaction close to neutrality, also exert favorable effects on decom-
position. Consequently, the more fertile the soil, the more rapid
the mineralization of the added organic matter.

Organic materials are applied in farming practice with the object
of producing beneficial effects on plant growth. Whether or not
such effects are produced will depend principally upon the reac-
tions which occur during decomposition of the organic substances.
As has been stated previously, the application of organic materials
results within a very short period in an enhanced development of
the microorganisms in the soil. This is not confined to any
one group, but generally applies to all soil microbes; the extent of
stimulation differs with the various species of bacteria, fungi,
actinomyces, and protozoa, and depends upon the composition and
the amount of the organic material added. The increase in abun-
dance of cells is also accompanied by increases in their activities,
as measured by the formation of carbon dioxide and other end
products.

Coincident with the decomposition of organic matter con-
taining small amounts of nitrogen, there is an immediate and appre-
ciable decrease in the amounts of ammonia and nitrate nitrogen
originally contained in the soil, as is shown in Table 22. In this
experiment, the substances were added in such amounts as
to introduce the same quantity of nitrogen in each case. As a
result of the application of appreciable amounts of organic matter

DECOMPOSITION OF THE PLANT AS A WHOLE

93

containing only small relative concentrations of nitrogen, growth
of higher plants may be greatly depressed.

TABLE 22

Influence of Nature of Organic Matter upon Nitrate Formation
IN Soils (from Lyon, Bizzell and Wilson)

Treatment

Nitrogen
content

Amount of material

added to 28 pounds

of soil

Total NOs-N
in leachings

Soil alone

per cent

0.-15
0.62
0.79
1.71
10.71

gm.

mgtn.
950 0

Soil + oat roots

133.3

96.8

75.9

35.1

5.6

207 3

Soil + timothy roots

Soil + corn roots

398.4
510 6

Soil + clover roots

924 4

Soil -}- dried blood

1751 1

Associated with these changes there is an increase in the abun-
dance of insoluble nitrogen, organic in nature; this increase is
practically quantitatively equal to the decrease in the water-soluble
nitrogen, such as ammonia and nitrate. In the decomposition of
such materials as corn-stover and oat straw, increases of 200 to 300
per cent in the organic nitrogen are not exceptional. Under such
conditions there is seldom any loss of total nitrogen; as a result
of the creation of conditions favorable to nitrogen fixation, there
may even be an absolute increase in the total nitrogen.

Somewhat similar changes occur with sulfur and phosphorus as
with nitrogen. There is a reduction of the amounts of these
elements found in inorganic forms with, however, no loss in the
total amounts found in the soil.

These effects are all directly associated with the activities of the
microbes which are responsible for the decomposition of the organic
materials ; they are also concerned with the normal nutrition of the
microbial cells. The nitrogen, whatever its combination, is built
up from an inorganic into organic cell substance; there is a direct
proportion, in which, for a unit of organic matter decomposed or
per unit of energy liberated, a definite amount of nitrogen is assimi-
lated and a definite amount of cell substance synthesized. A great
abundance of soil microorganisms, including numerous bacteria,
actinomyces, and fungi, are able to assimilate nitrogen provided as

94 DECOMPOSITION OF ORGANIC SUBSTANCES IN SOIL

ammonia, nitrate, or in simple organic combination; the extent of
the assimilation of inorganic nitrogen depends upon the amount
of available energy material and its relative content of organic
nitrogen.

Metabolism of Microbes and Decomposition of Organic
Matter. — Knowing the nature of the organisms effecting the
decomposition of an organic material, the composition of the
microbial cells, the energy balance of these cells, the composition
of the organic matter undergoing decomposition and the environ-
mental conditions, it is possible to interpret in a very definite
manner the end products which will be formed and the speeds with
which these will accumulate. Any one of these five variables
may show differences over a considerable range, but sufficient is
already known to interpret in general terms what may be expected
to take place.

Fungi use the organic substance as a source of energy and as a
nutrient (a source of carbon); they assimilate, in the mycelium
and spores, from 20 to 50 per cent of the carbon contained in the
organic compound decomposed. The rest of the carbon goes off
as carbon dioxide or is left as incompletely decomposed material.
For the sake of simplicity let it be assumed that 35 per cent of the
carbon of the organic matter decomposed is assimilated by the
fungi. Bacteria assimilate much less of the carbon, utilizing
the available energy less economically. They may assimilate
from 1 to 30 per cent of the carbon contained in the organic mate-
rial decomposed. An average of 7 per cent may typify conditions
for many bacteria. Actinomyces are intermediate between the bac-
teria and fungi, assimilating from 15 to 30 per cent of the carbon.

Accompanying the carbon assimilation, there is a consumption
of considerable amounts of nitrogen, which is largely synthesized
in the form of protein complexes. The carbon content of the cells
may be between 45 and 54 per cent. For convenience it may be
assumed that dry microbial cell substance contains 50 per cent of
carbon. The nitrogen content and the ratios of carbon to nitrogen
in the cells may be assumed to be as follows :

Nitrogen content,
per cent

Ratio of carbon
to nitrogen

Fungi

Bacteria

Actinomyces .

3- 8, av. 5
8-12, av. 10
7-10, av. 8.5

10.0 to 1
5.0 to 1
6.0 to 1

METABOLISM OF MICROBES 95

It is apparent that fungi assimilate much less nitrogen per unit of
carbon, but, as has been mentioned above, they use much more of
the total carbon contained in the material during the decomposition
process.

The following calculations show what would happen as the
fungi decompose such substances as cellulose, straw, alfalfa, and
dried blood. For the sake of simplicity, it is assumed that all of
the organic material is decomposed. This is not absolutely the
case, but the errors introduced by this assumption will not greatly
modify the results which are calculated.

Cellulose contains 45 per cent carbon. In 100 pounds of cellu-
lose there are 45 pounds of carbon, 35 per cent of which is assimi-
lated, as assumed previously.

45 X 0.35 = 15.75 pounds of carbon assimilated.

For every ten parts of carbon there is one part of nitrogen assimi-
lated, or 10 per cent as much nitrogen as carbon assimilated.

15.75 X 0.10 = 1.575 pounds of nitrogen assimilated.

Since there is no nitrogen in cellulose, there would be a deficit of
nitrogen, which would have to be added to the medium in which
the decomposition was taking place.

Some straws contain about 37 per cent of carbon and 0.5 per
cent of nitrogen. In 100 pounds of straw there would be 37
pounds of carbon and 0.5 pound of nitrogen. The following figures
indicate the result of its decomposition:

37 X 0.35 = 12.95 pounds of carbon assimilated.
12.95 X 0.10 = 1.295 pounds of nitrogen assimilated.

Since there was 0.5 pound of nitrogen in the straw originally, if this
was all used by the fungi, 1.295 — 0.50 = 0.795 pound of nitrogen
would appear as a deficit which would have to be supplied to the
fungi in order that decomposition of the straw take place.

Alfalfa may be assumed to contain 40 per cent of carbon and 3
per cent of nitrogen. In 100 pounds there would be 40 pounds of
carbon and 3 pounds of nitrogen.

40 X 0.35 = 14.00 pounds of carbon assimilated.
14.00 X 0.10 = 1.40 pounds of nitrogen assimilated.

1.40 — 3.00 = —1.60 pounds of nitrogen, meaning that there

96 DECOMPOSITION OF ORGANIC SUBSTANCES IN SOIL

would be 1.60 pounds of nitrogen in excess of the requirement.
This excess of nitrogen would appear as ammonia as the decompo-
sition progressed, and would represent a waste product of the
nutrition of the fungi.

Dried blood contains about 40 per cent of carbon and 10 per
cent of nitrogen. There would be 40 pounds of carbon and 10
pounds of nitrogen in 100 pounds of the material.

40 X 0.35 = 14.00 pounds of carbon assimilated.
14.00 X 0.10 = 1.40 pounds of nitrogen assimilated.

1.40 — 10.00 = —8.60 pounds of nitrogen, meaning that there
would be 8.60 pounds of nitrogen in excess of the requirements.
Here there would be a great excess of nitrogen over that required
for the nutrition of the fungi.

Similar calculations may be made to indicate the results of the
decomposition of the same organic materials by bacteria. Less of
the carbon is assimilated (only 7 per cent), and there is a greater
amount of nitrogen required per unit of carbon used — ratio of C to
N is 5.0 to 1, or one-fifth as much nitrogen as carbon is required.

It is quite evident that, in the decomposition of organic mate-
rials, such as cellulose and mature straw, there is a marked deficit
in nitrogen which must be supplied from some source, such
as inorganic ammonium salts, nitrates, or available organic nitro-
genous compounds. With organic materials, as in the case of
leguminous plants, which contain from 1.5 to 3 per cent of nitrogen,
there will generally be more nitrogen than is necessary to satisfy
the requirements of the organisms, and consequently there would
soon be some ammonia formed. This may not always occur, since
the plant is not homogenous but a complex aggregate of com-
pounds which decompose with unequal rapidity. It has been
observed that when the plant materials contain less than 1.7 per
cent of nitrogen there will be a more or less extended deficiency of
nitrogen for decomposition to proceed rapidly. With materials
containing more than 1.7 per cent of nitrogen no deficiency appears;
in fact, there will be more nitrogen than is required by the
microorganisms; this excess will appear as ammonia (later as
nitrate), and the amount of the excess will be determined by the
excess of nitrogen over 1.7 per cent which the organic material
contains.

The assimilation of nitrogen by microorganisms should not

METABOLISM OF MICROBES 97

suggest that the nitrogen is permanently stored up in microbial
cells. Cells of the microorganisms lead a transient life in the soil ;
they develop and die within short intervals, depending upon the
environmental conditions. Upon their death they become decom-
posed with the liberation once more of at least a part of the nitrogen
and of the other elements which were assimilated during growth.
It can be appreciated that, in the presence of even small amounts of
nitrogen and large amounts of nitrogen-free organic matter, there
would eventually be no deficiency of nitrogen but even a liberation
of most of the nitrogen as ammonia. The process will proceed
somewhat slowly, since the organisms must die and be decomposed
over a considerable period of time.

Fig. 48 shows schematically the course of the changes under
ideal conditions, or what might be called the perfect case, where
the total plant and the microbial cells which are formed are all
completely decomposed. In the calculations the assumption has
been made that the organisms causing the decomposition assimi-
late into their cell substance one-third of the carbon of the material
decomposed and eliminate the other two-thirds as carbon dioxide.
It is further assumed that the cells of the organisms have a ratio
of carbon to nitrogen of 10 to 1. The nitrogen of the original
organic material (12.5 pounds) permits the organisms to transform
125 pounds of carbon into their cells, since they assimilate ten
times as much carbon as nitrogen. Also, since they assimilate
only one-third of the carbon that they attack, 250 pounds of carbon
will be eliminated as carbon dioxide. This leaves 625 pounds of
carbon in the form of residual undecomposed organic matter.
Providing the microbial cells suffer complete decomposition, 42
pounds of carbon and 4.2 pounds of nitrogen will be synthesized
into the new cells which cause this decomposition (A). Since only
one-third of the carbon is used by the cells, two-thirds of the carbon,
or 83 pounds, will be eliminated as carbon dioxide. Since the cells
which were decomposed contained more nitrogen than the new cells
required for growth, 8.3 pounds of nitrogen will be eliminated as
ammonia. This ammonia permits decomposition of more of the
undecomposed organic matter. As a result of this decomposition,
166 pounds of carbon go to carbon dioxide, 83 pounds of carbon are
assimilated by microbial cells (B), and 376 pounds of carbon still
remain undecomposed (C).

If the total constituents of the microbial cells (A) and (B) are

Original Organic
Matter

1,000 lbs. C
12.5 lbs. N

C : N Ratio 80 : 1

'Microbial Cell

125 lbs. C
12.5 lbs.

ells 1

^

Wast-e Products
-*-CO2-250lbs. C
->-C0, - 83 lbs. C

(A) Microbial Cells

42 lbs. C
4.2 lbs. N

Ammonia

8.3 lbs. N

^Residual Undecomposed
Organic Matter

625 lbs. C

(B) Microbial
Cells

83 lbs. C
8.3 lbs. N

-►CO,- 166 lbs. C

(C) Residual Undecomposed
Organic Matter

376 lbs. C

Combining (A) and (B)

Microbial Cell

125 lbs. C
12.5 lbs

:ells ]

-(D) Microbial Cells

42 lbs. C
4.2 lbs. N

Ammonia

8.3 lbs. N

(C) Residual Undecomposed
Organic Matter

376 lbs. C

^^(£

(F) Residual Undecomposed
Organic Matter

127 lbs. C

-*-C0, - 83 lbs. C

-►CO,- 166 lbs. C

(£■) Microbial
Cells

83 lbs. C
8.3 lbs. N

Combining (D) and (E)

Microbial Cells |

125 lbs. C
12.5 lbs. N

-(C) Microbial Cells

42 lbs. C
4.2 lbs. N

Ammonia

8.3 lbs. N

(F) Residual Undecomposed
Organic Matter

127 lbs. C

Combining (G) and (//)

lial Cells 1
bs. C >
bs. N J

Microb

84 lbs
8.4 lbs.

CN Ratio 10- 1

-► CO, - 83 lbs. C

(//) Microbial

Cells

42 lbs. C

4.2 lbs. N

No Residual
Undecomposed
Organic Matter

-V CO2 - 85 lbs. C
-^ NH,-4.1 lbs. N

-► CO2 - 56 lbs C
-►NH,- 5.6 lbs. N

^Microbial Cells
28 lbs. C
2.8 lbs. N

C:N Ratio 10:1

Fig. 48.— Schematic representation of the changes in the carbon an(i nitrogen
of organic matter during the decomposition of material containing small
amounts of nitrogen. (Braces in each case indicate decomposition taking

place.)
98

SUMMARY 99

combined at this stage we account for 125 pounds of carbon and
12.5 pounds of nitrogen. Continuing with the decomposition as
previously, there is a progressive increase in the amount of carbon
accounted for as carbon dioxide and less present in residual unde-
composed organic matter. After decomposition of the microbial
cell substance represented by (D) and (E) there are eliminated 8.3
pounds of nitrogen as ammonia, which are more than sufficient to
permit the organisms to decompose the 127 pounds of residual
undecomposed organic matter remaining at this time (F). Conse-
quently, the excess nitrogen remains as ammonia, which may now
be considered as a waste product since it will not be further used
by the organisms causing the decomposition. From this point on,
more and more ammonia will be added to the waste products, and
the remaining organic matter will be accounted for entirely as
microbial cells. It can be seen from this outline that eventually
ammonia will be fiberated from the decomposition of organic
materials having as wide an initial ratio of C to N as 80 to 1.
This entails the repeated minerahzation of the nitrogen of the
microbial cells, and eventually leads to a C : N ratio of 10 to 1,
which persists at this figure as long as there is any organic matter
remaining.

In normal soils there is practically always a rather close rela-
tionship between the amounts of organic carbon and nitrogen
present. Even when organic materials of wide ratios of carbon to
nitrogen are added to soils, there is a tendency for these ratios to
become narrower with time, until they become about 10 to 1, at
which level the ratio remains practically indefinitely, although the
total amounts of organic carbon and nitrogen may be continually
decreasing. In the light of the diagrammatic conversion of organic
matter pictured above, it may be apparent why the ratio becomes
10 to 1 and does not become lower. Some variations from this
ratio frequently occur, as determined by differences in the organisms
causing the decomposition and by accumulations of resistant plant
residues of wide C : N ratios.

SuMMAEY. Decomposition of Organic Matter in Soil and
Formation of Soil Humus, or Soil Organic Matter. — A brief
summary of the facts discussed in this chapter allows us to obtain
a clear idea of the general processes involved in the decomposition
of organic materials of plant and animal origin added to the soil
and the formation of the dark-colored substances which comprise

100 DECOMPOSITION OF ORGANIC SUBSTANCES IN SOIL

the soil organic matter, giving to the soil its characteristic proper-
ties, and frequently spoken of as soil humus.

When a ton of fresh organic matter (on a dry basis), in the form
of plant stubble, green manures, stable manures, or organic fer-
tiUzers, is plowed under or worked into the soil, the microorganisms
immediately become active, decomposing first the water-soluble
substances, then the starches, the proteins, the hemicelluloses, and
cellulose. Within 10 to 20 days, under favorable conditions
of moisture, aeration, and temperature, only about 1,000 to 1,200
pounds may be left out of the 2,000 pounds originally added.
These 1,000-1,200 pounds comprise only a part of the cellulose, pen-
tosans and fats, most of the lignin, a large part of the waxes and
cutins, and a large quantity of substance synthesized by the micro-
organisms of the soil. Most of the original protein has disap-
peared, and in its place there has been formed fresh microbial
protein, some ammonia and nitrate, depending upon the relative
nitrogen content of the original plant material. If the nitrogen
was present in amounts less than 2 per cent, only a small amount of
the nitrogen becomes mineralized; if the nitrogen content of the
original plant material was greater than 2 per cent, a large part
may become liberated as ammonia or nitrate within 20 days
under favorable conditions.

Another month has passed, and the residual organic material
represents a new picture. If the original material was in a green
state, or if there was an abundance of nitrogen available, only 400
to 600 pounds may be left out of the original ton of dry material.
This will consist largely of lignin from the original plant or its
transformation products, some fats and waxes, considerable syn-
thesized microbial cell substance in the form of proteins, hemi-
celluloses, chitin, etc. This mass of residual and synthesized
materials is referred to as soil organic matter or humus. If the
original plant material was in the form of cereal straw or corn-
stover, which contain very little nitrogen (about 0.5 per cent), the
decomposition of the plant cellulose and hemicelluloses would have
been slower, since the transformation would have been controlled
by the amount of available nitrogen and by the rapidity with which
the soil microorganisms were able to use the nitrogen in the soil and
in the plant.

The organic matter in the soil is not uniform in composition.
It constantly undergoes numerous changes. The condition of this

LITERATURE 101

organic matter is not static but dynamic, being different from what
it was yesterday, and it will be different to-morrow from what it is
to-day, the changes being both qualitative and quantitative. The
soil organic matter gives off a constant stream of carbon dioxide,
diminishing from day to day, if the soil is left undisturbed. Rain
and dryness, heat and frost, will constantly modify the conditions
of the soil, influencing variously the activities of the microor-
ganisms, the nature and composition of the soil organic matter.
The nature of the growing plants and the management of the soil
still further modify the nature and amount of the soil organic
matter. That the organic matter of the soil which receives httle
or no added material becomes depleted is apparent from the fact
that about 30 mgm. of carbon dioxide may be produced per kilo-
gram of soil of average fertility every day for about 200 days of the
year. This would mean approximately a ton and a half of carbon
per year for each acre of 2,000,000 pounds. Boussingault stated
that in eleven years, one-half of the carbon in a certain soil had
been converted to carbon dioxide.

A knowledge of the chemical composition of the plant and
animal residues undergoing decomposition, of the microorganisms
bringing about the decomposition of the various chemical con-
stituents, and of the environmental conditions under which the
decomposition is taking place, is essential before we can under-
stand the nature and rapidity of liberation of the nutrient elements
in forms available for plant growth and of the formation and
nature of the residual organic matter or soil humus.

LITERATURE

1. Buchanan, R. E., and Fulmer, Ellis I. Physiology and biochemistry of

bacteria. Volume 2, Chapter 12; Volume 3, Chapter 18. The
WilUams & Wilkins Co. Baltimore, 1930.

2. Lyon, T. L., and Buckman, H. O. The nature and properties of soils.

Chapters 5 and 17. The Macmillan Co. New York, 1929.

3. Proceedings and Papers of the First International Congress of Soil Science.

Commission 3, Volume 3. Washington, 1928.

4. Stephenson, M. Bacterial metabolism. Chapter 5. Longmans, Green

and Co. London, 1930.

5. Waksman, S. a. Principles of soil microbiology. Chapters 17, 19, 26.

WiUiams & Wilkins Co. Baltimore, 1927.

6. Waksman, S. A. Chemical nature of soil organic matter, methods of

analysis, and the role of microorganisms in its formation and decom-
position. Transactions of the Second Commission of the Inter-
national Society of Soil Science. Part A, Budapest, 1929.

r-<j

I L I 8 R A R Y

CHAPTER V
TRANSFORMATION OF NITROGEN BY SOIL MICROBES

Sources of Nitrogen in Soil. — The atmosphere is the source
of supply of nitrogen in the soil; a continuous exchange is operating
between the nitrogen that now exists in various combinations in the
soil itself and what remains above the earth in the atmospheric
cloak. The gaseous covering of the atmosphere contains the rela-
tively inert molecular form of nitrogen. This becomes available
to green plants only as it is changed into combined forms by cer-
tain specific microbes or by strictly non-biological processes, such
as union of nitrogen and oxygen to form nitrate through the
agency of electrical discharges, through catalytic combination
of nitrogen and hydrogen to form ammonia, or by a reaction
between nitrogen and calcium carbide to form calcium cyanamid.
Natural electrical discharges add small amounts of inorganic
nitrogenous compounds to the earth; the commercial exploitation
of fixation processes has resulted in the further addition of vast
quantities of combined nitrogen to soils. However, the principal
natural mechanism by which plants obtain their combined nitrogen
and by which the lost quantities of nitrogen are restored to the soil
is that operated by some of the microbes which make the soil their
natural habitat.

The mere presence of nitrogenous compounds in the soil does
not solve the problem of satisfying the requirements of higher
plants for this element. Green plants require certain very specific
nitrogen compounds, and the production of a continuous supply of
these substances is largely the result of the activities of micro-
organisms. The nitrogen compounds in the soil, from which the
supply of nitrogen made available for plant growth originates, are
varied and many are quite complex in composition. They com-
prise largely organic compounds produced as a result of the growth
of green plants and animals, as well as of the numerous microbes.
These compounds include proteins, proteoses, peptones, peptides,

102

SOURCES OF NITROGEN IN SOIL 103

nucleic acids, amino acids, amides, and urea, as well as the alka-
loids and heterocyclic compounds. These various substances
are attacked by soil microbes before they become available to
higher plants. In certain processes, microbes diminish the sup-
ply of combined nitrogen in the soil; they occasionally form molec-
ular nitrogen or nitrous oxide from nitrate and thus decrease the
quantity of nitrogen in the soil.

These numerous interrelated processes resulting from the pres-
ence and activities of microscopic and higher forms of life in and
upon the soil for untold thousands of years have resulted in the
accumulation of considerable quantities of nitrogenous organic
matter in the soil.

The nitrogen content of the superficial 8 to 12-inch layer of soils
of humid regions varies from 0.025 per cent, in the case of the very
poor sandy soils, to practically 4 per cent in the case of certain peat
and forest soils, 90 per cent or more of which may consist of organic
compounds. The nitrogen content of most field soils ranges from
0.05 to 0.5 per cent, with an average of about 0.1 to 0.2 per cent.
Practically all of this nitrogen is found in the soil in complex
organic forms, and only a fraction of 1 per cent of the total nitro-
gen of the soil is generally present in an inorganic form, either
as ammonia, nitrite or nitrate. The nitre spots of certain arid
irrigated soils are exceptions.

The explanation for the lack of any abundance of nitrogen in
the form of inorganic salts is found in the following two facts:

(1) Transformation of ammonia to nitrate occurs more rapidly
under most soil conditions than the formation of ammonia; and

(2) nitrates are so soluble that they do not persist long in the soil
in humid regions, and are either absorbed by the root sj^stems of
plants or become washed out by percolating water. It is, there-
fore, obvious why it is necessary to provide some means for creating
a continuous supply of nitrate during the period of active
absorption by the growing plants. Nature has invested such a
mechanism in the microbial inhabitants of the soil. Their action
is generally supplemented or enhanced by cultural treatments of
intensive agricultural practice.

Practically all of the nitrogen that is brought into the soil by
natural agencies, as in the roots and stubble of cultivated and
uncultivated plants, animal residues, and dead animals, is in the
form of organic compounds, largely proteins and their derivatives.

104 TRANSFORMATION OF NITROGEN BY SOIL MICROBES

The same is true of the forms of nitrogen in the stable manures and
green manures that are added to the soil in varying quantities and,
in the case of some soils, at quite frequent intervals. Various
commercial organic fertilizers, such as guano, dried blood, cotton-
seed meal, and tankage, contain their nitrogen in the form of
organic compounds. Most of the nitrogen added to the soil in
artificial fertilizers is in the form of inorganic compounds, largely
nitrates, ammonium compounds, and amides. However, even
some of the synthetic fertilizers, such as cyanamid and urea, require
the previous interaction of microorganisms before the nitrogen is
made available to growing plants.

Transformation of Nitrogen in Soil. — As long as the nitro-
gen is in an organic form it remains to a large extent unavailable
as far as the growth of higher green plants is concerned. The
organic complexes must be mineralized or decomposed, and, after
a series of transformations, the nitrogen finally becomes liberated
as ammonia. Many plants can absorb their nitrogen in this form.
Others require their nitrogen in the form of nitrite. The trans-
formation of the ammonia first to nitrous acid (nitrite), then to
nitric acid (nitrate), is carried out by specific bacteria, under
favorable conditions, in the soil. The nitrate may be used by the
plants; it may be assimilated by microorganisms in the presence
of available energy ; it may be reduced to nitrite, to gaseous oxides
of nitrogen, to ammonia, and to atmospheric nitrogen; finally, the
nitrate may be leached from the soil.

The various transformations to which nitrogen is exposed in
soil are illustrated in Fig. 49.

Fixation of Atmospheric Nitrogen. — While practically all
living organisms rely for their nitrogen upon organic compounds,
like proteins and their derivatives, or upon inorganic compounds,
such as nitrates or ammonium salts, there are certain microbes
which are able to make use of elementary (molecular) nitrogen to
supply the need of this element for their growth. Such an assimi-
lation of nitrogen by organisms from the molecular state is termed
biological nitrogen fixation.

The ability of utilizing gaseous atmospheric nitrogen and fixing
it in their bodies in the form of complex organic and simple inor-
ganic compounds is limited largely to two groups of bacteria.
These include: (1) the non-symbiotic bacteria which lead a free
existence in the soil and obtain their energy from various organic

FIXATION OF ATMOSPHERIC NITROGEN

105

compounds found
in the soil, prefer-
ably sugars, higher
alcohols and organic
acids; (2) the sym-
biotic nitrogen-fix-
ing bacteria which
live upon the roots
of certain specific
plants, namely, the
Leguminosae, in
specialized bodies
called nodules, and
cause the fixation
of gaseous nitrogen,
which becomes
available to the
plants during their
growth. The nod-
ule-forming bac-
teria are also ca-
pable of leading an
independent exist-
ence in the soil,
their ability to fix
nitrogen under
these conditions
being still a matter
of dispute. It
seems likely, how-
ever, that while
developing inde-
pendently of the
plant these sym-
biotic bacteria fix
no appreciable
quantities of ni-
trogen.

In addition to
these two groups of

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106 TRANSFORMATION OF NITROGEN BY SOIL MICROBES

bacteria, a number of other bacteria may be capable of fixing
small quantities of nitrogen, especially when these microbes have
been recently isolated from the soil. However, neither the
amount of nitrogen fixed nor the specificity of their nature can
place these organisms on an equivalent basis with the other two
groups of bacteria. There are indications that certain highly
specialized forms of fungi, such as species of Phoma, which
cause the formation of endotrophic mycorrhiza, may fix some
nitrogen. Certain of the blue-green algae, as species of Anabena

1.8 2640 12

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Nitrogen Content

Number of Cells

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5 6 7 8 9
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Fig. 50. — Correlation between fixation of nitrogen and growth of bacterial cells
(after Meyerhof and Burk).

and Nostoc, may also be able to fix relatively large amounts of
nitrogen under certain conditions, but the grass-green algae have
no such capacity.

The fixation of nitrogen is thus a physiological inheritance of a
comparatively small number of microorganisms with the principal
representatives included among the bacteria.

Non-symbiotic Nitrogen-fixing Bacteria. — Those bacteria
capable of fixing atmospheric nitrogen which lead a free existence
in the soil are found distributed in two groups: (1) Azotobacter —
large, coccus-like, non-motile, aerobic organisms. This group is

NON-SYMBIOTIC NITROGEN-FIXING BACTERIA

107

represented in the soil by the following species: Az. chroococcum,
Az. agile, Az. heijerinckii, Az. vinelandii, Az. vitreum, and Az.
woodstownii (see Fig. 10). The first species, Az. chroococcum,
occurs much more abundantly in soils than any of the other forms.
(2) Clostridium — spore-forming, rod-shaped organisms, anaerobic
in nature, that is, capable of existing in the complete absence of free
oxygen; the nitrogen-fixing species of this organism are generally
referred to as CI. pastorianum (or Bacillus amylohacter) , but it is
likely that there is a large group of such forms embracing numerous
closely related forms, most of which produce butyric acid when
utilizing carbohydrate material.

For the fixation of nitrogen, these organisms require a source of
energy, some available forms of phosphorus and potassium, suf-
ficient calcium to keep the reaction from becoming acid, and smaller
amounts of various other minerals. These nutrients with nitrogen
from the atmosphere are used for the growth of the cells; as a
result of growth there is an increase in the fixed forms of nitrogen in
the environment where the cells develop. The amount of nitrogen
fixed is closely related to the amount of energy available (as well as
to the available phosphorus when the available energy is not a
limiting factor). This is shown in Table 23. The amount of
nitrogen fixed is very intimately associated with the extent of cell
development, the fixed nitrogen being built up into cell constitu-
ents. Fixation of nitrogen is, therefore, an index of the growth
of the cells and an increase in the cell substance (see Fig. 50).
Cells may remain alive and decompose the available energy source
without fixing nitrogen; such a condition may exist if the cells are
merely respiring without increasing in numbers.

TABLE 23

Influence of Amount of Glucose on Fixation of Nitrogen

BY AZOTOBACTER (fROM HuNTEr)

Glucose concentration,

Nitrogen Fixed (mgm.)

per cent

2 Days

4 Days

6 Days

0.6
1.1
1.5

6.37
7.41
9.1

9.75
17.29
17.68

9.70
18.72
26.0

108 TRANSFORMATION OF NITROGEN BY SOIL MICROBES

As shown by Fig. 51, the largest amount of nitrogen is fixed per
unit of food material used during the early periods of cell develop-
ment of a culture. This decreases rapidly as the culture ages.
In the young culture most of the cells are using the food materials
for growth and multiplication, and nitrogen fixation is propor-
tionately efiicient. In the later stages much of the energy is con-
sumed merely to support respiration of many of the cells, and few
cells are multiplying. Eventually a stage is reached where multi-

-Nitrogen Fixed per Gm. Sugar
-Nitrogen Fixed
"Sugar Decomposed

/

/

/

/..

/•

15 20 25

Time (Days)

30

35

40

Fig.

51. — Relationship between carbohydrate decomposed and nitrogen fixed
(after Omeliansky).

plication ceases entirely and no nitrogen is fixed, even though food
is being consumed to keep alive the cells which already exist.

For similar reasons, the lower the initial concentration of the
energy source in the absence of fixed forms of nitrogen, the greater
are the amounts of nitrogen fixed per unit of energy material
decomposed. This is apparent from Table 24.

Since under soil conditions there is generally a comparatively
small amount of energy materials present at any one time, it is likely
that a relatively large amount of nitrogen is fixed per unit of carbo-
hydrate consumed during the process. In fact, where nitrogen

IMPORTANCE OF NON-SYMBIOTIC NITROGEN FIXATION 109

fixation does occur in soils it appears to be a relatively economical
process.

TABLE 24

Influence of Concentr.\tion of Mannitol on Fixation of Nitrogen
BY Azotobacter (from Lipman)

Concentration of mannitol,
per cent

Nitrogen fixed per gram
of mannitol decomposed,
(mgm.)

0.1
0.2
0.5
1.0
1.5

10.5
8.3
6.4
4.68
3.22

The energy used by Azotobacter and Clostridium may be
obtained from sugars (glucose, galactose, maltose, sucrose), higher
alcohols (glycerol, mannitol), starches, dextrins, a number of
organic acids, and certain hemicelluloses; the Clostridium uses the
latter two sources only to a limited extent. Cellulose, lignin, and
some of the hemicelluloses cannot be utilized by any of these
nitrogen-fixing bacteria as sources of energy.

Importance of Non-symbiotic Nitrogen Fixation. — In
view of the fact that the natural organic materials added to the
soil in the plant residues and in the various organic manures are
largely made up of cellulose, lignin, proteins, and hemicelluloses,
while the content of soluble carbohydrates is very limited, being
not more than 2 to 10 per cent of the total organic matter added
to the soil, the amount of energy available for the nitrogen-fixing
bacteria is very small. The problem is complicated still further
by two facts: (1) The abundant flora of other bacteria and fungi
in the soil will also readily utilize the soluble carbohydrates, if
available forms of nitrogen are present. (2) In the presence of
available forms of fixed or combined nitrogen, such as organic
nitrogenous substances, ammonium salts, or nitrates, even the
nitrogen-fixing bacteria fail to fix nitrogen. This is not due to the
inabihty of the bacteria to develop under such conditions, but
rather to the utilization of the nitrogen which already exists in
fixed forms to supply their requirements. Nitrogen is used from

110 TRANSFORMATION OF NITROGEN BY SOIL MICROBES

the atmospheric sources only in the absence of other available
sources of supply (see Table 25). Growth of most higher plants in
soils is dependent upon a rather extensive and continuous supply
of nitrogen in such fixed forms as ammonium salts or nitrate.
Since the presence of such nitrogen in soils would prevent fixation
processes, it is likely that fixation does not occur very extensively
in soils while plants are growing. This does not eliminate the pos-
sibilities of fixation in some localized regions of the soils or at
certain periods when considerable amounts of plant residues may
become introduced into soils.

TABLE 25
Influence of Nitrates on Transformations of Nitrogen by

AZOTOBACTER (fROM BoNAZZI)

Period of

incubation,

days

Nitrogen Present in
Media (mgm

Culture

)

Nitrate

Organic

Total

Control

5
5
8
8
13
13

8.66
1.12
8.66
0.88
8.66
0.72

0.47
6.53
0.47
7.41
0.47
8.59

9.13

Inoculated

7.65

Control

9.13

Inoculated

8.29

Control

9.13

Inoculated

9.31

Associated with the decomposition of 100 parts of available
organic matter free from nitrogen there may be fixed about one
part of nitrogen. Assuming that an acre of soil received a liberal
application of plant residues, such as 2 tons of water-free material,
the amount of carbohydrates available for the use of non-symbiotic
nitrogen-fixing bacteria would not exceed 800 pounds. Assuming
further that all this energy was used by the specific bacteria, only
about 8 pounds of nitrogen would be fixed per acre per year if the
aerobic organisms were active. Less would be fixed by the anaerobic
organisms. Considering the fact that the 4,000 pounds of dry
organic matter would contain 20 to 60 pounds of nitrogen, some of
which will be liberated through the activities of the numerous
soil fungi and bacteria, the evidence that any appreciable amounts

IMPORTANCE OF NON-SYMBIOTIC NITROGEN FIXATION 111

of energy would be left for the use of nitrogen-fixing bacteria and
that these would have to use the nitrogen from the atmosphere is
not very conclusive. Under field conditions, however, some
nitrogen may be added to soils through the agency of bacteria.
Fixation has been noted even when animal manure was the organic
substance with which the soil was treated. The soil conditions
and the nature of the growing crop probably exert the determining
influences in many cases. It has been stated that in some soils
as much as 40 pounds of nitrogen become added to soils annually
as a result of the activities of non-symbiotic nitrogen-fixing bac-
teria. Certain soils in regions of deficient rainfall show particu-
larly striking increases in nitrogen.

Due to the fact that it is almost impossible to detect even
appreciable increases in the nitrogen content of soils (such as 40
pounds per acre per year), the actual agricultural importance of
the non-symbiotic organisms is practically unknown. With the
numerous facts in mind, it may be assumed that, in most soils,
the increase in the nitrogen content as a result of the activities of
these nitrogen-fixing bacteria is rather limited.

There are two conditions not yet considered under which the
activities of these organisms may tend to increase the store of com-
bined nitrogen in the soil: (1) In the neighborhood of growing
roots of plants there is an excretion of soluble carbohydrates and
addition of other residues to the soil which may serve as food for
the bacteria. Plants rapidly consume most of the available com-
bined nitrogen from this portion of the soil. These two factors,
namely, the presence of available sources of energy and a nitrogen
minimum, would favor the rapid development of Azotobacter and
Clostridium and lead to nitrogen fixation. (2) A similar relation-
ship may exist between certain green algae and the nitrogen-fixing
bacteria. The former ^x the carbon from the carbon dioxide of the
air (by photosynthetic agencies at the surface of the soil) and liber-
ate some of the synthesized carbohydrates, which can be used as
sources of energy by the nitrogen-fixing bacteria; the latter fix
the nitrogen and liberate some of it in a combined form which
can be used by the algae.

The reaction of the soil is one of the factors of major impor-
tance in determining the presence or absence of Azotobacter, as
shown in Table 26. Under the great diversity of soil conditions,
the reaction may limit the distribution of the organisms more fre-

112 TRANSFORMATION OF NITROGEN BY SOIL MICROBES

quently than any other condition. If the reaction is more acid
than pH 6.0, Azotobacter is seldom present; when the soil is limed
and the reaction is changed to pH 6.0 or above, Azotobacter will
develop quite readily, when present or when introduced. The
Clostridium types can resist a somewhat greater acidity than Azo-
tobacter (such as pH 5.2) and occur under a greater variety of
conditions and in considerably greater numbers.

TABLE 26
Soil Reaction and Occurrence of Azotobacter (prom Gainey)

Number of
soils tested

Azotobacter

Reaction (pH)

Present,

Absent,

per cent

per cent

Above 7 . 50

32

100

0

7.00 to 7.49

60

97

3

6.50 to 6.99

45

90

10

6.00 to 6.49

47

80

20

5.50 to 5.99

122

20

80

5.00 to 5.49

43

14

86

4.50 to 4.99

44

7

93

Below 4 . 50

3

0

100

Soil conditions which appear to be aerobic, that is, well-aerated
soils with a good crumb structure and with optimum moisture
content, may be a very favoral^le habitat for the anaerobes. The
conditions within the aggregates of soil particles or close to decom-
posing portions of soil organic matter may be far from aerobic.
Aerobic organisms may consume the oxygen about their cells
during growth and create sufficiently anaerobic conditions to per-
mit neighboring cells of Clostridium to develop. Such associative
development of various organisms is probably more common in
soils than development of single species of organisms entirely
separated from one another. At least, one need not postulate the
fixation of nitrogen by the anaerobic types only under bog condi-
tions or in water-logged soils. In fact, the fixation of small
amounts of nitrogen in soils by the group of non-symbiotic nitrogen-
fixing bacteria may be as much the result of the action of the Clos-
tridium as of the Azotobacter.

SYMBIOTIC NITROGEN FIXATION 113

Large amounts of phosphorus are required by Azotobacter
cells for their growth. Phosphorus is by far the most important
of the mineral substances absorbed during growth of the
cells. The amount of the element required by the bacteria
is in direct proportion to the amount of nitrogen fixed. This
is borne out by a simple analysis of the composition of the
microbial cell which, in a washed condition (freed from adher-
ing capsular material, largely hemicelluloses), contains about
10 per cent nitrogen and 5 per cent P2O5. It has been calculated
that for every gram of glucose used by Azotobacter as a source
of energy, 2.5 mgm. of phosphorus are required. This offers a
good method for determining the amount of available phosphorus
in the soil. For this purpose a definite amount of soil is added to
a definite amount of culture solution, free from phosphorus,
and the solution is then inoculated with Azotobacter; the amount
of nitrogen fixed under these conditions is taken as an index of the
quantity of available phosphorus in the given quantity of soil
added to the solution. A very practical procedure for determining
the available phosphorus in soil, based upon this phenomenon, is
discussed in detail later.

Symbiotic Nitrogen Fixation. — It has been known since
olden times that the growth of leguminous plants, such as clover,
alfalfa, peas, or beans, leaves the soil richer for the succeeding
crops. The reason for this phenomenon was not known, but the
practical farmer recognized that the growth of such a crop was
equivalent to the appfication of manure as far as the succeeding
crop was concerned. It was only toward the middle of the nine-
teenth century that it became an established fact that this bene-
ficial effect of a leguminous crop is due to an increase in the supply
of soil nitrogen; the leguminous plants were found capable of
utilizing the nitrogen gas of the atmosphere, changing it thereby
into combined forms in some mysterious fashion. It was soon
estabhshed that when the soil in which the legume is grown is
previously heated or sterihzed, the leguminous plant behaves like a
cereal plant and is unable to use the gaseous nitrogen. When a
little fresh soil is added to the previously sterihzed soil, the legume
is once more able to draw upon the store of gaseous nitrogen
(Fig. 52). After a series of investigations, to which botanists,
bacteriologists, agronomists, and chemists have contributed, it
became definitely established toward the end of the last century

114 TRANSFORMATION OF NITROGEN BY SOIL MICROBES

that a minute bacterium is responsible for this process. The bac-
terium in question was finally (1888) isolated in pure culture by the
Dutch bacteriologist Beijerinck, and was named Bacillus radicicola.
This organism provokes the development of certain growths,

Fig. 52. — Influence of the legume organisms on growth of peas. All are

growing in sterilized sand, but in the four pots placed alternately, which show

good growth, extract of garden soil has been added (from HeUriegel and

Wilfarth, after Russell).

called nodules, on the roots, and in these newly formed biological
laboratories causes the fixation of nitrogen to take place.

Since the organisms are not contained within the seeds of the
legumes they must enter the plant during its growth in order to
develop their effects. Infection takes place through the root hairs,

SYMBIOTIC NITROGEN FIXATION

115

and the organisms appear to become attracted to these roots by
some substance which is excreted by the young roots. The infect-
ing organisms are probably coccoid cells, either non-motile (pre-
swarmers) or motile (swarmers). Once within the tissues the
bacteria multiply rapidly at the expense of the food material of
the plant. They occur at this stage as short rods enclosed in a
mass of mucoid material. The mass of bacterial growth continues

^ ^-_V

Fig. 53. — Infection of root cells of a legume by the legume bacterium: (1) cells
from the young nodule tissue showing small rod-shaped cells in the infecting
strands; (2) cells from older nodule tissue; many bacteria have separated from
the infecting strand and are still existing as thin rods; (3) cells from quite old
parts of the nodule tissue; the cells have become filled with bacteria in the
swollen "bacteroid" condition (from Brenchley and Thornton).

to pass through cells of the cortex of the root in the form of a fila-
ment with many branches, called the infection thread or strand.
The penetration of the microorganisms results in growth and mul-
tipHcation of the inner cortical cells, which push outward to form
the young nodule. These cells, in turn, become invaded by the
infection threads (see 1, Fig. 53). As the nodule develops, vas-
cular tissue which conducts nutrient materials in the plant grows

116 TRANSFORMATION OF NITROGEN BY SOIL MICROBES

outward into the nodule and favors active division of the plant
cells and growth in the terminal region. The plant cells at the
base of the young nodule swell and become vacuolated and occu-
pied with bacteria freed from the infecting strand (see 2, Fig. 53).
These bacteria appear as thin rod forms. As the nodule ages, the
bacterial cells in these older plant cells become swollen with proto-
plasmic constituents concentrated unevenly; these are known as
the bacteroids (see 3, Fig. 53).

Unhanded
Non ■ Motile Rods

Motile Rods

Swarmers

Coccoid Forms

Pre • Swarmers
Fig. 54. — The life cycle of Bacillus radicicola (after Thornton and Gangulee).

The changes in appearance of the bacterial cells appear to be
closely related to the food supply in the nodule. Where food
materials, presumably carbohydrates, are supplied in abundance,
the bacteria retain the appearance of rod-shaped cells which take
stains uniformly. As the food supply decreases, in the older
regions of the nodule, the bacteria become banded rods and finally
bacteroids. These morphologic changes are characteristic of
bacteria passing from stages of youth to old age and senescence
(see Fig. 54). Thus, if a culture of a legume organism is intro-
duced into a certain culture medium, it soon appears in the " pre-
swarmer " or non-motile coccoid stage. The cells increase in size

SYMBIOTIC NITROGEN FIXATION 117

until the diameter is about doubled, but they still remain non-
motile. These cells become ellipsoidal and develop high motility
in which stage they are known as " swarmers." The swarmers
elongate to form the rod-shaped cells which remain motile for a
time, but gradually become non-motile as the food becomes con-
sumed. When the food material becomes exhausted or other such
unfavorable conditions develop, the rods appear as banded cells
with the protoplasmic contents concentrated in several locations.
Eventually many of these cells become branched, swollen, or other-
wise changed from the rod shapes into the characteristic bac-
teroids. The bands of cell material may give rise to small cocci
once more when placed under suitable conditions of adequate
food supply.

This relationship between the nodule organism and the host
plant has been called symbiosis, since presumably both organisms
benefit from the association. The plant produces carbohydrates
through its photosynthetic activities and supplies such food and
other nutrients to the bacteria as enable them to develop exten-
sively. The bacteria, through some agency, enable the plant to
derive nitrogen for its development through the transformation
of gaseous nitrogen to fixed compounds. Under certain condi-
tions, however, the relationship may not be one of symbiosis but of
actual parasitism. If the plant is unable to make normal growth
on account of a lack of certain essential nutrients, providing that
the bacteria enter the roots, the plant tissue may not react to
develop a normal nodule, but will initiate development of nodular
tissue which soon becomes extensively invaded by the bacteria,
completely destroying the tissue.

The mechanism of the fixation of nitrogen is little understood.
The bacteria related to the process appear to be quite unable to
fix nitrogen while growing independently of the host plants. Like-
wise the legume fails to fix nitrogen while growing free from the
bacterium. It is only in the associative development that appre-
ciable fixation of the nitrogen occurs, and then only when the vas-
cular system of the root develops into the nodule and furnishes
carbohydrates for the growth of bacterial and plant cells; that
system must also carry away the products of cell development,
including presumably nitrogenous compounds resulting from the
fixation. Whether the bacteria fix the nitrogen under the condi-
tions existing in the nodule, or whether the plant fixes the nitrogen

118 TRANSFORMATION OF NITROGEN BY SOIL MICROBES

in the presence of the bacteria, or whether both share equally in
the reaction, is speculative. There are thus many points of

A B C

Fig. 55. — Nodules of cowpeas (A), soybeans (B), and lima beans (C)
(from Lohnis and Leonard).

difference between fixation by symbiotic and non-symbiotic bac-
teria, but the actual chemical reactions involved may not be unhke.

The nodules pro-
duced on roots of
different legumes may
be quite different in
appearance, but the
^^__^^ / general shape is quite

^F* .^S^C^ constant in any one

species of plant. On

some of the beans the

nodules are fairly

large and spherical,

and seldom occur in

clusters (Fig. 55).

Nodules on many of

the clovers and alfalfa

are small elongated

swellings, sometimes

branching or occurring in clusters (Figs. 56 and 57). The nature

of the plant and the soil conditions affect the location of the

nodules. Under conditions particularly favorable for inoculation,

Fig. 56.— Nodules of alfalfa (A) and vetch (B)
(from Lohnis and Leonard).

NATURE OF THE BACTERIA

119

the nodules may appear in abundance close to the main root
near the surface of the soil. Where the moisture content of
the soil is low, as in the semi-arid regions, nodulation may be
prominent at a considerable distance from the surface of the soil.
The abundance and size of the nodules are good indexes of the
extent of fixation of nitrogen. Where the nodules are large and
numerous, nitrogen fixation proceeds rapidly; where the nodules
are smaller and more scattered, fixation is much slower.

Fig. 57. — Nodulation on roots of peas (from Wilson and Leland).

Nature of the Bacteria and Classification of Legumi-
nous Plants. — The bacteria can be readily isolated from the nod-
ules, and grown upon artificial culture media and studied carefully.
It has been found that not all leguminous plants can be equally
infected by the same microbe. In fact, the bacteria infecting
legumes include a number of different species or strains, each of
which can inoculate only one or more leguminous plants. At
least twelve species or strains of the legume bacteria have been
established, each of which will inoculate legumes belonging to only
one of the groups listed below. Consequently the number of

120 TRANSFORMATION OF NITROGEN BY SOIL MICROBES

groups is the same as the number of different cultures of nodule
bacteria required to inoculate these legumes.

(1) Alfalfa, white sweet clover, yellow sweet clover, hubam,
bur clover, yellow trefoil, and fenugreek.

(2) Red, white, crimson, alsike, bersem, and cow clovers.

(3) Garden, canning, and field peas; hairy, spring, and wild
vetches; broad bean; lentil; sweet pea; and perennial pea.

(4) Cowpea, peanut, Japan clover, velvet bean, lima bean,
partridge pea, wild indigo, acacia, dyer's greenwood, and tick
trefoil.

(5) Garden, field, navy, kidney, wax, and scarlet runner
beans.

(6) Lupines and serradella.

(7) Soybean.

(8) Hog peanut.

(9) Lead plant.

(10) Trailing wild bean.

(11) Black or common locust,

(12) Wood's clover.

These varietal differences between the bacteria capable of forming
nodules upon the roots of various plants can be demonstrated by
direct inoculation upon the plants, and also by cultural and sero-
logical tests. Morphologically the organisms belonging to the
different races are quite alike (except for some differences in the
formation of flagella), but physiologically they are quite different.
Influence of Nitrate upon Nitrogen Fixation. — The
amount of nitrogen fixed by inoculated legumes is influenced to a
considerable extent by the amounts of nitrate in the soil on which
the plants are growing. Nitrate-nitrogen is readily absorbed by
legumes, and when present in abundance in the soil may supply
the entire nitrogen requirements of the plants. Since absorption
of nitrate dominates fixation of nitrogen, it is only when the
nitrate available to the plants is less than the nitrogen require-
ments of the plants that appreciable fixation occurs. This is
apparent from Figs. 58 and 59. The amounts of nitrogen in the
plants are not very different irrespective of the amounts of nitrate
supplied to them. However, the amounts of nitrogen fixed are
inversely proportional to the amounts of nitrate available.

INFLUENCE OF NITRATE UPON NITROGEN FIXATION 121

The presence of some nitrate in the soil appears greatly to bene-
fit growth of certain leguminous plants and fixation of nitrogen.
This is particularly the case with legumes, such as alfalfa and the
clovers, which produce tiny seeds. The reserve nitrogenous food
material in the seeds is insufficient to enable the plants to develop

NaNOj-lb. 100
per Acre

200 400 600 800 1,000

Fig. 58. — Relation between soil and atmospheric nitrogen obtained by a crop
of inoculated alfalfa growing on soil variously treated with sodium nitrate

(from Giobel).

substantial root systems without drawing upon some nitrogen
from the soil. It is during this stage that nitrates greatly favor
the plants. After proper root development has taken place, inocu-
lated plants finding no combined available nitrogen in the soil
develop as well as those which are supplied with an abundance of
nitrate. Legumes having larger seeds, as beans and peas, are not
appreciably affected by the presence or absence of available forms

122 TRANSFORMATION OF NITROGEN BY SOIL MICROBES

of soil nitrogen even in the early stages of development, if they are
properly inoculated, since the reserve nitrogenous food materials
in the seeds is generally sufficient to enable the plants to become
established. Subsequent to this initial development, the degree
of inoculation largely regulates the extent to which the legumes
may draw upon the atmospheric nitrogen for their growth.

The explanation for the depressing effects of nitrate on nitrogen
fixation by legumes is not clear, but certain suggestive relationships
exist. Nitrate does not prevent penetration of the root hairs by

Gm
2.0

1.5

10

05

■ N Obtained from Soil

^ N Added in NaNoj

D H Obtained from Atmosphere

II JL fc fc ■

NaN03-lb. 0 150 300 450 600 750 900
per Acre

Fig. 59. — Relation between soil and atmospheric nitrogen obtained by soy-
beans growing on soil variously treated with sodium nitrate (from Giobel).

the bacteria but, if very abundant in soils, may inhibit nodule for-
mation. The bacteria are not themselves injured by the nitrate,
but certain physiological responses of the plants to nitrate depress
nodulation and subsequent fixation of nitrogen. Where nitrates
are abundant in soils the plant juices also contain appreciable
amounts of nitrate or other forms of nitrogen which are readily
assimilated. Such a condition leads to rapid transformation
of the carbohydrates in the plant juices to plant tissues,
decreasing the concentration of carbohydrates to a low level.
Since the carbohydrates are probably the principal food of the
nodule bacteria, the decreased concentration of the food may be

INFLUENCE OF NITRATE UPON NITROGEN FIXATION 123

1^

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Beans

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CO o-i ^

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Soy-
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Vetch

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Crim-
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124 TRANSFORMATION OF NITROGEN BY SOIL MICROBES

responsible for the very limited development of the organisms once
they have entered the plant. The final result is a lack of nodule
development and consequently no fixation of nitrogen within the
plant.

Amounts of Nitrogen Fixed. — Since legumes absorb large
amounts of nitrogen from the soil, if the nitrogen exists in available
forms, it is not possible to state without qualifications how much
nitrogen will be fixed through the use of legumes in agricultural
practice. Under conditions where the legumes make good growth
and the nitrogen supplied by the soil is small, there are usually
between 100 and 200 pounds of nitrogen fixed per acre (Table 27).
Even larger amounts of nitrogen may be fixed under particularly
favorable conditions. However, when the crop is removed for
hay or similar purposes, there may be practically no increase in
the combined nitrogen of the soil, since most of the nitrogen of the
plant (often as much as 75 per cent) is in the stems, leaves, and
seeds, and only a small amount in the roots. The nitrogen con-
tained in the roots may represent no more than such nitrogen as
has been absorbed from the soil during growth. Where a large
portion of the plants is not removed but becomes incorporated with
the soil, there may be great increases in the combined soil nitrogen.

Influence of Soil Conditions upon Nitrogen Fixation. —
Although no other single factor appears to exert such a pronounced
influence on the degree of nitrogen fixation as the nitrate content of
soils, there are numerous other factors which influence the process.
Among these may be mentioned, moisture, soil reaction, tempera-
ture, soil texture, and addition of fertilizer salts. In general, with
the exception of the influence of nitrogen compounds, it maj^ be
stated that there is a close correlation between the effects of these
factors on nodule formation and on the growth of the legumes.
Those conditions most favorable to vigorous development of the
host plants favor nodulation and nitrogen fixation. Conditions
which are unfavorable to the plants lower nodulation and fixation
of nitrogen.

With most legumes, growth is best at reactions close to neu-
trality, and nodulation is greatest under such conditions. Some
legumes are quite acid tolerant, and good nodule development
occurs in acid soils. In general, the degree of acidity which
limits development of the bacteria is also one that is known to be
injurious to the host plant (Table 28). Of the various fertihzing

SOIL CONDITIONS AND NITROGEN FIXATION 125

materials, phosphates and calcium exert the most pronounced
influences on the legumes, although any element essential to plant
growth which is present in insufficient amounts lowers nodulation.

TABLE 28

Critical Reaction (/jH) for the Bacteria of Some Legumes (from Fred)

Alfalfa and sweet clover 4.9

Peas and vetches 4.7

Clovers and beans 4.2

Soybeans 4,0

Lupine 3.2

Since nitrogen fixation is dependent upon the presence and
activity of specific bacteria, it is essential that the organisms be
present in soils in order that the plants may make use of atmos-
pheric nitrogen. Where a legume has never been grown in a cer-
tain soil it is Hkely that the specific bacterium is not present, at
least in sufficient numbers to thoroughly inoculate the plants.
Consequently, the seeds or the soils in which the seeds are planted
are frequently inoculated with the bacteria at the time of planting.
Once the legume has been well nodulated during growth, the bac-
teria persist in the soil for a number of years, provided that the
soil reaction is not too acid. In the absence of the host plant,
the organism will survive longer in a neutral or slightly acid soil
than in a more acid soil.

In addition to the leguminous plants, several non-legumes are
also capable of forming nodules on their roots. These include
such plants as the alder, sweet gale, and red root. The microbe
responsible for the formation of these nodules was found to belong,
in most cases, to the Bac. radicicola group, namely, to the same
group which includes the organisms that cause nodule formation
on the roots of legumes. However, it is still unknown whether
or not nitrogen becomes fixed in these non-leguminous plants.

Some tropical plants, such as Pavetta and Ardisia, form nod-
ules either on the upper or on the lower part of their leaves. Bac-
teria were found to be the responsible agents. These bacteria
are aerobic, rod-shaped cells, developing abundantly in the intra-
cellular spaces of the plants and said to be present even in the
embryo sacs of the seed. With the germination of the seed, they
develop through the plants, so that there is unbroken continuity
of the association throughout the life cycle of the plants. In these

126 TRANSFORMATION OF NITROGEN BY SOIL MICROBES

plants, nitrogen fixation appears to take place, similar to that in the
legumes, and advantage is taken of this fixation in India by using
such plants as green manuring crops.

Decomposition of Proteins by Microorganisms. — Whether
the nitrogen is fixed in the soil through the agencies of nitrogen-
fixing bacteria, whether it is introduced there in the tissues of
plants, animals, and microorganisms in the numerous organic
residues, or whether it is added to the soil in the form of organic
fertilizers — it is not directly available to the growth of higher
plants. This nitrogen occurs to a large extent in the form of pro-
teins and their derivatives, and to a less extent in the form of other
complex organic nitrogenous compounds. Before this nitrogen
can be utilized for the growth of green plants, these complex
organic substances have to be decomposed and the nitrogen
changed to other forms. This process of liberation of nitrogen
is carried on in the soil by numerous fungi, actinomyces, bacteria,
and invertebrate animals, and is one of the most important activi-
ties of the microbes in the soil. It is also one of the processes
common to a large portion of the microorganisms inhabiting the
soil.

Proteins, the most important group of nitrogenous compounds
in the living organism, are made up of chains of certain units or
building stones, namely, the amino acids. So far, between 20 and
30 different amino acids have been isolated from the plant and
animal organism and described. These amino acids can be com-
bined in various ways, giving a number of complex bodies, which
are typical of the proteins and their derivatives found in the living
and dead animals, plants, and microbes. Living processes involve
the building up of new tissue substance or cell material, of which
the proteins are vital constituents; among the processes associated
with the decomposition of the dead bodies of plants and animals
is the decomposition of these proteins into simpler compounds.

Some of the amino acids are comparatively simple in compo-
sition; others are more complex, as shown by some typical
formulae :

Glycocoll or glycine . CH2(NH2) • COOH

Aspartic acid CH2 • COOH -CHCNHg)- COOH

Arginine (NH)C(NH2) NH- (CH2)3CH(NH2) COOH

Tyrosine C6H4(OH) • CH2 • CH(NH2) • COOH

DECOMPOSITION OF PROTEINS BY MICROORGANISMS 127

These various amino acids combine to form simple and complex
peptides. The greater the number of amino acids which are com-
bined together, the more nearly does the complex approach the
characteristics of pure proteins. The protein molecule itself is
built up in a similar manner out of a large number of amino acids,
and has the following general formula, where R may be any of a
large number of radicals:

NHo ■ CHR • CO • (NH • CHR • CO) • NH • CHR • COOH

When a protein is treated with hot alkaline or acid solutions or
with certain proteolytic enzymes, it is hydrolyzed to the individual
amino acids of which it is constituted. This may be illustrated
by the hydrolysis of a dipeptide as follows:

NHo • CH2 • CO • NH • CH2 • COOH

+

HoO

i

NH2 • CHo • COOH + NHo . CH2 • COOH

The amino acids themselves are readily used by most of the soil
microbes as sources of energy, while the nitrogen is liberated as
ammonia. In the process of decomposition of amino acids,
carbon dioxide, ammonia, organic acids, alcohols, and other com-
pounds are formed. In addition to these substances, amino acids
may also give rise, under anaerobic conditions, to amines, hydrogen
sulfide, mercaptans, and a considerable variety of incompletely
oxidized substances. Some amino acids, such as tyrosine, which
contains a benzene group in the molecule, give rise to such com-
pounds as cresol and phenol as intermediary products of decom-
position. Although some of the amino acids are decomposed very
readily, others are resistant to decomposition.

The nitrogen of the amino acid molecule finally appears as
ammonia. A certain part of the nitrogen may be reassimilated
by the organisms, either as the amino acid directly or in the form
of ammonia, and changed into the protein constituents of the
microbial cells. Some of the nitrogen may thus be removed again
from circulation and a part of it may become quite resistant to
decomposition.

128 TRANSFORMATION OF NITROGEN BY SOIL MICROBES

The liberation of ammonia from the amino acid molecule takes
place according to one of the following reactions :

CH3CH(NH2)COOH + H2O = CH3CH2OH + CO2 + NH3

Alanine . Ethyl alcohol

R R R

I I I

H— C— NH2 + H2O -> NHa + H— C— OH -^ CH2OH +CO2

I Alcohol

COOH COOH

Amino acid Hydroxy-acid

HOOC • CH2 • CH2 • CH(NH2) • COOH + H2

Glutamic acid

= HOOCCH2CH2CH3 + CO2 + NH3

Butyric acid

The ammonia thus produced may accumulate in the soil, it may
be used by higher plants or by various microbes (in the presence
of available sources of energy), or it may be changed to nitrate.
In the light of what has been stated previously regarding the
decomposition of such compounds as carbohydrates and the role
of nitrogen in decomposition of plant materials, it is relatively
easy to determine whether or not ammonia will be liberated in the
decomposition of protein materials and about how much ammonia
will appear in the complete decomposition of proteins by any one
type of microorganism. The amount of nitrogen liberated as
ammonia in protein decomposition may be represented as follows:

N of /N assimilated by N left as \

protein de- — I microorganisms + intermediary 1 = N as NH3
composed \ in growth products /

Since pure proteins contain 15 to 18 per cent of nitrogen, ammo-
nia is formed in particularly large amounts in the decomposition
of organic compounds rich in proteins. Ammonia is a waste
product of the metabolism of microorganisms, and is of no further
use to the cells concerned in its formation; it accumulates in the
absence of organisms able to oxidize it. In view of the fact, how-
ever, that organic substances added to the soil do not consist of
pure proteins and frequently the protein part is very small in com-
parison with the carbohydrates and other non-nitrogenous con-
stituents (as in the case of rye or wheat straw which may contain
only 2 per cent protein and 98 per cent of other non-nitrogenous

DECOMPOSITION OF OTHER NITROGENOUS SUBSTANCES 129

organic substances), the liberation of the nitrogen in the form of
ammonia must always be considered in the light of the composition
of the organic matter as a whole.

Decomposition of Nitrogenous Substances of a Non-
protein Nature.' — In addition to proteins and amino acids,
the plant, animal, and microbial residues commonly added to the
soil contain various other nitrogenous complexes, although in some-
what more limited amounts. These substances include nucleic
acids, alkaloids, purine bases, phosphatides, various amines, glu-
cose-amines and amid. Some of these and numerous other com-
plexes are actually produced in the soil by the various microbes
in the processes of synthesis of new cell substances. Some of
these complexes decompose readily; some more slowly. The
decomposition of uric acid takes place as follows:

HN— CO

OC C— NH.

HN— C— NH/

Uric acid

H2N

I

CO2 + OC CO— NH\ + 2H2O

I I /CO >

HN— CH— NH/

Allantoin

CHO /HNo + 2H2O + O

I + 20C< >

COOH \NH2

Glyoxylic Urea

acid

COOH + O

2CO2 + 4NH3 + I > 2CO2 + H2O

COOH

Oxalic acid

Under favorable aerobic soil conditions, calcium cyanamid
breaks down readily to urea and thence to ammonia:

CaCN2 + H2O + CO2 -^ NH2-CN + CaCOs

Calcium Cyanamid

cyanamid

+ 2HoO
NH2CN + H2O -> CO(NH2)2 > 2NH3 + H2CO3

Cyanamid Urea

130 TRANSFORMATION OF NITROGEN BY SOIL MICROBES

Urea is one of the most important nitrogenous substances in
this group. It is excreted by the animal organism in large quanti-
ties; a large part of the nitrogen in stable manure is made up of
urea; it is formed by various microbes; it is produced in the
decomposition of various organic nitrogenous complexes, as in the
case of uric acid; it is produced in the transformation of certain
synthetic nitrogenous fertihzers, such as cyanamid; finally, it
is introduced into the soil as a fertihzer coming from the nitrogen-
fixation industry. Urea is decomposed in the soil by various
organisms according to the following reactions:

C0-(NH2)2 + 2H2O = (NH4)2C03

(NH4)2C03 = CO2 + 2NH3 + HoO

The transformation of urea into ammonia takes place very rapidly,
so much so that in the manure pile there is considerable danger of
actual volatilization of this ammonia. There are certain organ-
isms known as urea bacteria which are able to effect the hydrolysis
of urea by means of an enzyme called urease. The ammonia is
produced so rapidly that the reaction becomes quite alkaline and
the ammonia itself is readily volatilized in the absence of any neu-
trahzing agents. In soil, following the addition of urea, the reac-
tion first becomes distinctly alkaline through the formation of
ammonia; later the reaction becomes more acid than the original
reaction of the soil through the oxidation of the ammonia to nitric
acid.

Chitin is a substance formed in the cells of various micro-
organisms. In the tissues of these microbes it plays a role simi-
lar to that played by cellulose in the tissues of higher plants.
It is a polymer of mono-acetyl-glucoseamine having the formula
(Ci4H26N20io)io. On hydrolysis it gives acetic acid and glucose-
amine. The decomposition of the glucoseamine finally gives
rise to ammonia, carbon dioxide, and water, but its transformation
is quite slow compared to the decomposition of most other organic
nitrogenous substances.

Formation of Ammonia by Soil Microbes. — Decomposi-
tion of various organic substances containing nitrogen is thus
found to give rise to ammonia as an important end product. The
organisms which form ammonia from the organic compounds are

FORMATION OF AMMONIA BY SOIL MICROBES 131

unable to cause further transformation of the ammonia to more
completely oxidized inorganic compounds, such as nitrite or
nitrate. Consequently ammonia may be considered as the
inorganic end product of nitrogen, resulting from the activity of
microbes upon organic substances.

It is apparent that organic nitrogenous compounds which may
find their way into soils are very numerous and vary greatly in
composition. Consequently it is not surprising that a great
number of different organisms are concerned in the formation of
ammonia. In fact, a large percentage of all the microbes found in
soils may be able to produce ammonia from organic materials of
one kind or another. The group includes large numbers of
different bacteria, both aerobic and anaerobic, spore-formers and
non-sporulating species, filamentous fungi and actinomyces.
These organisms vary in their ability to attack different compounds,
also in the speed of ammonia formation and in the environmental
conditions which are favorable for their action. It is apparent
from Table 29 that different compounds decompose with different
rapidity, and microorganisms vary from one another in their ability
to effect the transformation.

TABLE 29

Formation of Ammonia (mgm.) by Microorganisms from 0.5 gm. op
Proteins in 40 Days

Organism

Protein

Proteolytic
bacterium

Bacillus

subtilis

A ctinomyces

violaceous-

r liber

Rhizopus
sp.

Gelatin

Casein

Gliadin

Fibrin

Albumin

25.45
37.57
29.91
19.76
15.75
25.86

42.82
23.43
14.55
18.55
14.54
7.68

39.99
21.81
21.41
16.12
15.35
8.89

18.98
18.58
18.59
18.55
11 31

Zein

2 43

The process of formation of ammonia from organic compounds
of nitrogen is of such importance that, under the name ammonifica-

132 TRANSFORMATION OF NITROGEN BY SOIL MICROBES

tion, it has frequently been considered together with two other
processes, namely, nitrate fonnation and fixation of nitrogen, to
be synonymous with the microbiological activities in the soil.

The amount of ammonia produced in the decomposition of
organic materials varies considerably, as illustrated in Table 30.
A series of different organic substances containing different
amounts of nitrogen were added to 100-gram portions of soil in
tumblers. The low amount of ammonia produced from rice flour,
corn-meal and wheat flour is due to the original low concentration
of nitrogen and comparatively high content of available energy
material.

TABLE 30

Ammonia Formation from Decomposition of Various Plant
Substances* (from Lipman)

Substance used

Nitrogen contained

in the added organic

matter, mgm.

Ammonia formed

in 7 days,

mgm.

Rice flour

Corn -meal

Wheat flour . . . .
Cowpea-meal . . .
Linseed-meal . . .
Soybean-meal . . .
Cottonseed-meal

46.4
51.2
94.8
156.8
247.0
245.6
246.1

1.30

1.22

5.05

50.36

118.31

132.43

123.63

* 4 gm. of organic material added to 100 gm. of soil.

The addition of organic matter free from nitrogen and repre-
senting an available source of energy will depress considerably the
amount of ammonia accumulating from the decomposition of the
organic substance, as seen in Table 31. A small amount of nitro-
gen-free organic matter actually produced an accelerating effect
upon ammonia formation, but larger amounts reduced the ammonia
formed from the decomposition of the nitrogenous substance; the
greater the amount of non-nitrogenous substance added, the less
ammonia was produced. This is due to the fact that a part of
the ammonia is used up by the organisms that utiHze the starch
and sucrose as nutrients; the presence of these also partly depresses
the decomposition of nitrogenous organic matter. The mechanism
of these changes is further discussed in Chapter IV.

FORMATION OF AMMONIA BY SOIL MICROBES 133

TABLE 31

Influence of Starch and Sucrose upon Ammonia Formation from
Dried Blood* (from Lipman)

Substance used

Amount of carbo-
hydrate used, gm.

Ammonia formed
in 7 days, mgm.

Dried blood alone

71.01

Dried blood + starch

0.3
1.0
3.0
5.0
0.3
1.0
3.0
5.0

77.63

Dried blood + starch

56.02

Dried blood + starch

Dried blood + starch

26.92
5.05

Dried blood + sucrose

Dried blood + sucrose

Dried blood + sucrose

Dried blood + sucrose

85.64
83.37
43.99
14.03

* Dried blood containing 155 mgm. of nitrogen added to 100 gm. of soil

The amount of ammonia formed from the decomposition of
organic matter depends upon the following important factors: (1)
the composition of the organic material itself, with particular
regard to its nitrogen content; (2) the organisms concerned in the
decomposition; (3) the prevailing environmental conditions; (4)
period of decomposition.

Organic materials of different origin decompose with different
degrees of rapidity. Even the decomposition of the same plant
will depend upon its age. The nature and abundance of the non-
nitrogenous complexes in the plant, such as sugars, cellulose,
hemicelluloses, waxes, resins, lignin, tannins, and cutins, will
determine to a large extent the rapidity of decomposition. The
rapidity of decomposition of the readily available sources of
energy, such as the sugars, starches, pentosans and other hemi-
celluloses, and cellulose, will influence the rapidity of decomposi-
tion of the proteins and the amount of ammonia reassimilated by
the organisms or left in the soil. A young plant decomposes more
rapidly than an old plant, because the young plant is richer in
readily decomposable substances (sugars, amino acids, phos-
phatides) and poorer in slowly decomposable substances (lignin,
tannins, waxes, resins). For similar reasons the leaves of some
trees decompose more readily than the leaves of others.

The nitrogen content of a substance is equally important in

134 TRANSFORMATION OF NITROGEN BY SOIL MICROBES

determining the amount of ammonia liberated in a given period
of time. The larger the relative nitrogen content the greater is
the relative amount of nitrogen liberated as ammonia in a given
period of time. If one gram of cottonseed-meal containing only
5 per cent of nitrogen and 1 gram of dried blood containing 10 per
cent of nitrogen are added to a given quantity of soil, more ammo-
nia will be produced from the dried blood in a period of 10 to 30
days. Even when 2 grams of the cottonseed-meal are added to a
certain quantity of soil and 1 gram of dried blood is added to
another similar quantity of soil, more nitrogen will still be pro-
duced from the dried blood than from the cottonseed-meal. The
above considerations readily explain the reasons for this difference.
The 2 grams of the cottonseed meal contain three to four times as
much non-nitrogenous energy-yielding substance as the dried
blood. Even assuming that both organic substances decompose
with the same degree of rapidity, still the microorganisms decom-
posing the meal, which offers a greater amount of available energy,
will reassimilate and store a greater amount of the nitrogen which
would have otherwise been liberated as ammonia.

The amount of nitrogen liberated as ammonia will also be
affected by the nature of the flora bringing about the decomposi-
tion. In the case of acid forest soils, for example, fungi are largely
concerned in the decomposition processes. In the arid alkaline
soils, bacteria and actinomyces are more active. Fungi synthesize
larger amounts of cell substance as mycelium, and will, therefore,
liberate less ammonia for the same amount oforganic matter de-
composed. Actually the fungi may be more active than many bac-
teria and, if a short incubation period is used, they may liberate
more ammonia; if a sufficiently long period is employed, the
reverse may be true. The reaction of the soil, nature of the inor-
ganic material, soil aeration, and presence of mineral nutrients all
influence the type of active flora and, therefore, the nature of
decomposition brought about by this flora.

So many different organisms can produce ammonia from
organic materials that comparatively small differences are appa-
rent in ammonia formation from soils varying greatly in chemical
and physical conditions and in general fertility. Since anaerobic
bacteria of many kinds are able to spUt off ammonia quite readily,
there may be large amounts of ammonia formed even where oxygen
is excluded. Further, while ammonia may become oxidized to

LITERATURE 135

nitrate under aerobic conditions, such a transformation is quite
completely inhibited under anaerobic conditions, and accumula-
tion of ammonia over a period of time may be greater in an anaero-
bic environment than where there is free access of oxygen. Under
aerobic conditions a great variety of organisms are active. Some
fungi may be as active under neutral as under acid conditions.
The bacteria and actinomyces are coniined to much narrower
ranges of reaction.

At no time is all the nitrogen of the organic matter liberated
completely as ammonia, but the longer the period of decomposition
the more complete the reaction. The factors considered above are
of particular importance in determining the speed of mineralization
of nitrogen added to the soil in the various plant and animal
residues.

LITERATURE

1. BoNAZzi, A. The mineralization of atmospheric nitrogen by biological

means. Proceedings of the Fourth International Soil Science Con-
ference. 1925. Section 3B, No. 8.

2. Buchanan, R. E., and Fulmer, E. I. Physiology and biochemistry of

bacteria. Volume 3, Chapter 16. The Williams & Wilkins Co.
Baltimore, 1930.

3. Fred, E. B. The root-nodule bacteria of leguminous plants, in The

newer knowledge of bacteriology and immunology, pp. 332-340. The
University of Chicago Press, 1928.

4. GiOBEL, G. The relation of the soil nitrogen to nodule development and

fi.xation of nitrogen by certain legumes. N. J. Agr. Exper. Sta. Bulle-
tin 436. 1926. pp. 1-125.

5. Stephenson, M. Bacterial metaboHsm. Chapter 8. Longmans, Green

& Co. London, 1930.

6. Waksman, S. a. Principles of soil microbiology. Chapters 4 and 22.

The Williams & Wilkins Co. Baltimore, 1927.

CHAPTER VI

TRANSFORMATION OF NITROGEN BY SOIL MICROBES

(Continued)

Nitrate Formation. — Ammonia, the end product of the reac-
tions just considered, becomes the raw material for the process of
nitrate formation. Very few species of soil organisms are con-
cerned in this process. These organisms are conveniently divided
into two groups, in each of which a limited number of bacterial
species is known. One group oxidizes ammonia to nitrous acid
or nitrite, and the other transforms nitrous acid to nitric acid or
nitrate. The process of conversion of ammonia to the more
highly oxidized inorganic compounds of nitrogen as nitrite and
nitrate is frequently referred to as nitrification.

2NH3 + 3O2 = 2HNO2 + 2H2O + energy (calories) liberated

2HNO2 + 02 = 2HNO3 + energy (calories) liberated

None of the bacteria belonging to either group is able to transform
ammonia directly to nitrate, but each is confined to merely one
stage of the reaction. It seems possible that quite a variety of
other microorganisms besides the specific nitrifying bacteria may
be concerned in the production of some nitrite or nitrate from
ammonia, but information concerning the relationship of these
organisms to the oxidation process is indefinite and little more
than suggestive. Nitrification by the specific bacteria is propor-
tionally rapid and considered to be of major importance in the
formation of nitrate in soils.

The conditions suitable for the formation of nitrite and nitrate
by nitrifying bacteria are quite simple and include merely an inor-
ganic medium containing salts of ammonia and several nutrient ele-
ments, a neutral reaction, and aerobic conditions. If soil is intro-
duced into such a medium, active transformation of ammonia

136

NITRATE FORMATION 137

results, first giving rise to nitrite; when a large part of the ammo-
nia has disappeared, nitrate is formed in increasing amounts at
the expense of the nitrite.

The two groups of bacteria concerned in the transformation are
thus quite distinct from one another, but they are alike physio-
logically in that they are both autotrophic, that is, they do not use
organic substances as food but can use the energy liberated in the
specific oxidation processes for their growth and development.
The carbon necessary for the synthesis of their cells is taken from
the carbon dioxide of the atmosphere and used in a manner similar
to its utilization by green plants.

CO2 + H2O = HCHO + O2

Formalde-
hyde

6HCH0 = CgHi20g (sugar) or other organic compounds.

The organisms forming nitrite from ammonia have been
designated as Nitrosomonas or Nitrosococcus, depending upon their
morphology. The organisms producing nitrate from nitrite are
called Nitrobacter. These forms are all very similar in morphology
being spherical or short oval cells, either motile or non-motile
(see Figs. 13 and 14). The difficulties involved in isolating
these organisms in pure culture have delayed the description of
more than a few species. The fact that very few species have been
obtained is of less significance than the fact that nitrifying organ-
isms exist in practically all soils. Since Winogradsky first obtained
the organisms in 1891, they have been found to be active prac-
tically everywhere, with the exception of certain acid forest and
peat soils. After drainage, cultivation, and liming, even these
soils can be made a favorable medium for nitrification.

The optimum reaction for the activities of the organisms is
about pH 7.0 to 8.0, but some strains will still grow at as high an
acidity as pH 3.5 and at an alkalinity of pH 10.0. Under condi-
tions of increasing acidity or alkalinity there develop certain
acid-tolerant or alkali-tolerant strains which have different reaction
requirements from those strains commonly encountered in most
arable soils. However, at an acidity greater than pH 3.5, the
nitrifying bacteria practically cease functioning. Acidity is but
one of the factors inhibiting development of nitrifying organisms in
acid peat bogs. The limited supply of oxygen in the water-

138 TRANSFORMATION OF NITROGEN BY SOIL MICROBES

saturated environment is insufficient to satisfy the requirements of
these strictly aerobic bacteria. The addition of hme to an acid
soil, or the drainage of a water-logged soil, will, therefore, mate-
rially favor the development of nitrifying organisms and the
process of nitrification. The organisms may be found at some
depth in soils, but naturally are much less abundant and little
active in the lower layers, on account of the less aerobic conditions
and the lack of materials upon which to feed. In arid soils these
bacteria are more abundant in deeper layers than in soils of humid
regions, since plant roots penetrate to greater depths and moisture
conditions are more favorable at considerable distances below the
surface.

TABLE 32

Relationship between Soil Reaction and Abundance of Ammonia-
Oxidizing Organisms (from Wilson)

pH Abundance of organisms
6.2 Less than 1,000

6.4 3,500

6.6 6,280

6.8 25,000

7.0 35,000

The numbers of nitrifying organisms in soils vary greatly,
depending upon a number of factors ; in cultivated soils they have
been found to range from a few hundred to more than a milhon
cells per gram. The data in Table 32 show that with increasing
acidity there is a pronounced decrease in the abundance of these
organisms. In many cases the reaction of the soil affects nitrify-
ing organisms only indirectly by modifying plant development,
which, in turn, affects the soil organisms.

Because of the limited number of organisms associated with the
process of nitrification and the similarity of their characteristics
of growth, the soil conditions under which nitrate accumulates
are much more restricted than for ammonia formation. Nitrifica-
tion is affected much more by modification of soil reaction,
aeration, moisture, and salt concentration than the process of
ammonia formation is altered by such changes. The treatment of
soils with steam or volatile antiseptics, known as processes of
partial sterilization of soils, eliminates most of the nitrite- and
nitrate-forming organisms, while leaving many of the ammonia-

NITRATE FORMATION 139

forming organisms uninjured. This results in the accumulation
of ammonia in partially sterilized soils, since once the ammonia
is formed, it persists as such, because of the lack of organisms
capable of further transforming it to nitrate.

In acid soils, such as raw-humus forest soils and certain acid
peats which have been drained and not limed liberally, the decom-
position of the nitrogenous organic substances is associated
with the formation and accumulation of nitrogen as ammonia,
without its being changed to nitrate. In the case of forest
soils this process is of greatest importance in modifying the very
nature of the forest vegetation. In the " raw-humus " types of
soil, only those trees develop which can utilize ammoniacal
nitrogen and are capable of forming mycorrhiza with special fungi,
which decompose the organic matter. However, in the case of
soils favorable for nitrate formation, the " mull " types are pro-
duced. These are considered richer soils; they bring about a more
rapid regeneration of the young forest, and the trees grow rapidly
without having to depend upon the formation of mycorrhiza. In
the case of the " raw-humus " soils, the organic matter is attacked
largely by fungi that allow an abundant accumulation of organic
matter which is characteristically brown; the nitrogen is liberated
as ammonia. In the case of the " mull " soils, decomposition is
more rapid and is brought about by a varied microbial population
including bacteria, actinomyces, and fungi, which give rise to a
black instead of a brown soil; nitrogen is liberated as nitrate.

Undoubtedly some plants utihze nitrogen in forms other than
nitrate, but many plants use nitrate almost exclusively, and some
use other forms than nitrate principally during young stages of
development. The marked effect of the nitrification process on
plant growth is shown in Table 33.

TABLE 33

Influence of Nitrification on Growth of Barley (from Fred)

Weight of
boil conditions , ,

plants, gm.

Control — no nitrogen 1.5

Nitrogen as (NH4)2S04 66.5

Nitrogen as (NH4)2S04 with nitrifying bacteria 116.0

Nitrate formation is controlled or affected by numerous factors,
chief among which are presence of ammonia, soil reaction, aera-

140 TRANSFORMATION OF NITROGEN BY SOIL MICROBES

tion, amount and kind of organic matter, temperature, moisture,
and concentration of inorganic substances.

Influence of Ammonia upon Nitrate Formation. — Since
formation of nitrate is dependent upon the previous formation of
ammonia, all factors affecting the speed of liberation of ammonia
from organic materials will also exert pronounced effects upon
nitrate formation. As pointed out above, the addition of an
abundance of non-nitrogenous organic materials will temporarily
depress the formation of ammonia. The lower the nitrogen con-
tent of the organic matter added to the soil, the greater will be the
depression and the longer will be the period during which the
depression lasts. Nitrate formation follows the production of
ammonia, and takes place readily either when the organic matter
itself contains sufficient nitrogen (over 1.7 per cent) to permit
formation of ammonia or when inorganic nitrogenous compounds
are added. The lower the nitrogen content of the organic matter
the longer will be the period elapsing before its complete decom-
position; the greater the nitrogen content, the shorter will be the
period before nitrate is formed in abundance.

In the presence of readily decomposable nitrogenous materials,
such as proteins or related compounds, ammonia accumulates
rapidly and nitrification follows at a slower rate. This indicates
that the process of nitrification in soils may be a slower process than
ammonia production, when readily decomposable nitrogenous
organic materials are present. However, the organic substances
generally occurring in soils in the humus complex are not rapidly
transformed to ammonia. The nitrifying organisms are able to
oxidize the ammonia more rapidly than it is produced from such
materials in most arable soils, and consequently nitrate and not
ammoniacal nitrogen appears in greater abundance. The speed
of the entire series of reactions is determined by the amount and
availability of the organic nitrogenous compounds which are
undergoing decomposition. The nitrification of the organic con-
stituents of stable manure illustrates the relationship. When
stable manure is added to soil, only a part of the nitrogen (about
one-haK) is readily converted to nitrate. About one-half of the
nitrogen of the manure is in the form of urea, which is rapidly
hydrolyzed to ammonia. This portion of the nitrogen undergoes
further rapid change to nitrate. The other nitrogen of the manure
occurs in compounds which are comparatively resistant to decom-

INFLUENCE OF REACTION UPON NITRATE FORMATION 141

position and is consequently converted to inorganic compounds
very slowly. In certain soils, after the ammonia is changed to
nitrite it may persist in that form for a considerable period of
time and only gradually be changed further to nitrate.

Influence of Soil Reaction upon Nitrate Formation. —
The more acid a soil is, the less nitrate is formed; when the reac-
tion is too acid, nitrate formation may come to a standstill (Fig. 60).

8.0 1

NaHCOj
Reaclion-pH

10.0 /ll.O
Too ^82003

Fig. 60. — Influence of reaction upon nitrate formation
(after Meyerhof).

In slightly alkaline soils, conditions appear to be particularly
favorable for nitrification. The addition of lime to acid soils
generally favors the formation of nitrate.

In certain arid regions of the West, particularly in Colorado and
Utah, there are limited areas of basic soils so rich in nitrate salts as
to prevent plant growth. Although nitrification in these soils is
very active, the large quantity of nitrates probably has not been
formed in these so-called " nitre spots," but has been produced
principally in the soils of the vicinity and concentrated in the spots
by the various movements of the soil water in these areas of
deficient rainfall.

142 TRANSFORMATION OF NITROGEN BY SOIL MICROBES

Soil reaction may be appreciably modified by active nitrifica-
tion, due to the fact that the process results in the oxidation of
ammonia to nitrate which leads to increases in the hydrogen-ion
concentration of soils. When the nitrate is absorbed by plants
or removed by drainage waters it is accompanied by certain cations
(Ca, Mg, Na, etc.) which leave the soil poorer in neutrahzing sub-
stances. The increase in acidity is particularly pronounced
where ammonium salts are added to soils. This change is not
particularly undesirable within certain limits, since it brings more
of relatively insoluble plant nutrients into solution as the acid is
formed, but where large amounts of ammoniacal substances gain

700

J

s

/

\

2 600

/

\

^

(

1 500
o

\

\

2 400

q5

J

r

^

\

1

a.

^ 300

cy^

S

s.

>

^

200

'n

S

>--

^

i

10 20 30 40 50 60 70 80 90 100
Percentage of Oxygen

Fig. 61. — Influence of aeration upon nitrate formation (from Plummer).

entrance to soils, the increase in acidity becomes sufficiently great
to justify the addition of lime to overcome the acid condition.

Influence of Soil Aeration upon Nitrate Formation. —
Since the processes of nitrification are autotrophic, considerable
quantities of carbon dioxide are required. Since the transforma-
tions require large amounts of oxygen they must take place under
aerobic conditions. Certain definite concentrations of these
gases, higher than those that occur in the atmosphere, are most
favorable to nitrification. Under soil conditions, oxygen is the
only one of the two gases which may not be present in sufficient
abundance. The decomposition processes of heterotrophic organ-
isms are responsible for the liberation of large amounts of carbon
dioxide, and the mixture of gases in the soil is always much richer
in carbon dioxide than the atmosphere. The rate of diffusion of

MOISTURE AND NITRIFICATION 143

the gases between the soil and the atmosphere would be the factor
determining the concentration of oxygen. The concentrations of
oxygen most favorable to nitrification lie between 30 and 55 per
cent (Fig. 61), while in soils this gas exists more commonly in
concentrations between 15 and 20 per cent. In the atmosphere,
oxygen comprises close to 20 per cent of the volume. Carbon
dioxide occurs in the atmosphere in concentrations close to 0.03
per cent. However, this gas makes up 0.2 to 2.0 per cent of the
soil air.

It is due to these relationships of the gases to nitrification that
factors favoring the rapid exchange of gases between the soil air
and the atmosphere lead to more rapid nitrate formation. Culti-
vation of soils and other treatments creating a more porous medium
increase the rate of nitrification. Sandy soils nitrify more rapidly
than hea\'y clay soils, other conditions being equal.

Organic Matter and Nitrate Formation. — The early work
of Winogradsky and his colleague Omeliansky indicated that very
small amounts of some organic materials may completely inhibit
nitrification, as determined by studies in solution cultures. Such
substances as glucose, peptone, asparagine, glycerol, and urea were
distinctly injurious in concentrations of 0.2 to 1 per cent. Under
most conditions, the soil solution does not contain such large quan-
tities of these soluble organic substances. Further, the so-called
humic substances which exist in the soil are not nearly as toxic as
many of the organic materials given above. In fact, soil extract
accelerates nitrification rather than depresses it. In the more
fertile soils, which contain relatively greater amounts of organic
materials, nitrification is appreciably more rapid than in the less
fertile soils. Nitrification will also proceed in heaps of animal
manures which contain large amounts of organic matter in solution.
In the preservation of manure from losses of nitrogen one of the
necessities is to keep nitrification down to a minimum, for it is
subsequent to nitrate formation that large amounts of nitrogen are
lost by reduction of nitrate to gaseous nitrogen.

Moisture and Nitrification. — [Moisture conditions most
favorable for nitrification are much the same as are favorable for
many aerobic soil processes, being close to 50 per cent of the amount
of water the soil holds when saturated. The influence of high
moisture content is determined to a considerable degree by the
retarding effect of the water on the circulation of the gases. High

144 TRANSFORMATION OF NITROGEN BY SOIL MICROBES

moisture contents are unfavorable to aerobic processes in general
(Fig. 62).

A number of other factors, such as temperature, concentration
of salts in the soil solution, and previous drying of soil, exert pro-
nounced effects on the rapidity of formation of nitrate from
ammonia, either produced from the decomposition of organic

5 10 15

Moisture Content- Per Cent

Fig. 62. — Influence of moisture upon nitrate formation

(after Traaen).

matter present, or recently introduced into the soil, or added to the
soil in the form of inorganic salts. It was found, for example, that
drying of soil followed by remoistening causes nitrates to form more
rapidly.

In general, the bacteria which bring about the processes of
transformation of ammonia to nitrate are among the most sensitive
of soil organisms. Conditions which can be readily withstood by
other bacteria may prove injurious to these organisms. This is
due to no small extent to their very specific nature.

NITRATE REDUCTION 145

Plant growth may greatly affect nitrate accumulation in soils
but will not necessarily inhibit nitrate formation. In fact, develop-
ment of plants may favor nitrification, as shown later. Through
the absorption of nitrate from the soil by the plants, the amount of
nitrate in the soil will decrease. It is apparent that under such
conditions, the presence of small amounts of nitrate is no indica-
tion of a depression of nitrification. Consequently, because soils
under plant growth contain less nitrate than soils free from vege-
tation, it should not be concluded that plant growth depresses
nitrification. Since nitrate in soils most commonly originates
from the organic nitrogen compounds, the soil containing the
greatest amounts of readily decomposable organic substances will
show the most rapid nitrification, other conditions being favorable
for the processes (Fig. 63) . Where plants are not growing on a soil
no organic substances become incorporated with the soil as resi-
dues of roots and other plant parts. On the other hand, consider-
able amounts of organic matter gain entrance into the soil which is
cropped. In an unplanted soil, the amounts of readily decom-
posable organic matter continuously decrease, while planted soils
receive periodic additions of such substances. These considera-
tions indicate why planted soils may be expected to show a more
active nitrifying population.

Nitrate Reduction. — The nitrogen transformations already
considered do not cause losses of nitrogen from soils. In fact, such
reactions as nitrogen fixation even add nitrogen. With the excep-
tion of nitrogen assimilation by microorganisms, most of the reac-
tions may be interpreted as being quite favorable to plant growth;
even the assimilation processes only temporarily remove the
nitrogen from availability to plants, and may prove to be de-
sirable under some circumstances.

There is at least one set of transformations of nitrogen com-
pounds associated with microbial activities which is generally not
beneficial to plant growth, and may be decidedly undesirable
from the point of view of economics of the soil. These transforma-
tions are concerned with reduction of nitrates, frequently leading
to the formation of gaseous nitrogen and consequent impoverish-
ment of the store of fixed nitrogen in the soil. Associated with the
formation of free nitrogen are a number of other reactions provoked
by microorganisms by which a large number of partly or com-
pletely reduced inorganic compounds of nitrogen are produced.

146 TRANSFORMATION OF NITROGEN BY SOIL MICROBES

paijjj^jN uaSoj^JM aij) p ^usQ J3d

(LuSw) spi'foia uoqjBO io uo!}n|OA3

NITRATE REDUCTION

147

Nitrate may disappear from soils through several channels :

(1) It may be assimilated by plants.

(2) It may pass into deeper regions of the soil in the move-
ments of the water, or it may disappear from the soil dissolved
in drainage waters.

(3) It may be assimilated by microorganisms in the pres-
ence of an available source of energy. In this case the nitrate
is not lost but is temporarily taken from circulation and stored
in the soil in complex organic forms. The reactions involved
in the synthesis of cell constituents from nitrate involve reduc-

HNO3
Nitric Acid

l + H,

H2O +HNO2
Nitrous Acid

H2O + HNO^

Hyponitrous Acid

+H,

-HoO
H2N2O2 > N2O

Nitrous Oxide

-H,

+H;

H,NOH

Hydroxylamine

+H2

H2O+H3N
Ammonia

-2HoO

H4N2O2 — ^N2

i Nitrogen

-H2 +H2

\

HeNsOs

Fig. 64. — Reduction products of nitric acid.

tion processes but need not lead to the accumulation of reduced
inorganic compounds of nitrogen in the medium. The general
transformation may be labeled nitrogen assimilation to distin-
guish it from other changes to be considered below.

(4) It may be reduced to many compounds, such as nitrous
acid, hyponitrous acid, hydroxylamine, ammonia, the gaseous
nitrous oxide, and free nitrogen. The hyponitrous acid and

148 TRANSFORMATION OF NITROGEN BY SOIL MICROBES

hydroxylamine generally do not accumulate but are formed as
intermediary products, but the others may appear in appre-
ciable amounts where active reduction occurs. The relation-
ships of these compounds to each other are schematically
represented in Fig. 64.

These reduction products are the result of either direct reduc-
tion reactions or secondary reactions. The reducing power of the
organism determines the primary products which are formed
(either nitrous acid, hyponitrous acid, hydroxylamine, or am-
monia), these leading to the formation of the gaseous sub-
stances.

It is apparent from Fig. 65 that the reaction takes place in sev-
eral stages, nitrous acid first appearing as the nitrate is reduced.
In turn, the nitrous acid is reduced to other more completely
reduced compounds, and eventually they also disappear coincident
with the formation of elementary nitrogen.

The organisms concerned in the various reductions of nitrate are
quite numerous. Bacteria play by far the most important role in
these transformations. The formation of nitrite from nitrate is
common to actinomyces as well as to numerous bacteria. The
formation of ammonia, nitrous oxide, and nitrogen are reactions
confined to certain species of bacteria. In the absence of uncom-
bined oxygen, the oxygen contained in the nitrate is utilized by
these organisms to perform various oxidation processes. Sulfate
serves a similar purpose under anaerobic conditions, where it is
reduced to sulfide.

Energy is required to effect the reductions, and the reactions do
not occur in the absence of food materials from which micro-
organisms may obtain the necessary energy. Various soluble
carbohydrates, cellulose, organic acids, and alcohols suit the
requirements of the organisms. Some bacteria even use sulfur
as follows:

6KNO3 + 5S + 2CaC03 = 3K2SO4 + 2CaS04 + 2CO2 + 3N2

Table 34 indicates that the amounts of ammonia or gaseous nitro-
gen formed from nitrate differ with the organisms active in the
reaction and the carbohydrates utihzed as foods.

The formation of the gaseous products is the most undesirable
change of all the nitrate reductions, since the volatilization of the

NITRATE REDUCTION

149

"

CM O 00 <i> "*

f-i 1—1

150 TRANSFORMATION OF NITROGEN BY SOIL MICROBES

TABLE 34

Amounts (Percentages) of Ammoni.\ or Gaseous Nitrogen Formed

FROM Nitrate by Bacteria Using Different Carbohydrates as Food

(from Stoklasa and Vitek)

Sugar

Glucose

Levulose

Galactose Arabinose

Xylose

Organism

Formation of Ammonia

B. mycoides

20.69

2.41

14.48

1.9
6.55

1.72
6.22

1.91
12.24
45.55

4.13

3.44

B. svbtilis

8.11

CI. gelatinosum

B prodioiosus

9.68

2.58

Bac. mesentericus vid-
gatus

5.17
2.79

5.52
2.70

Proteus vulgaris

2.79

Formation

of Gaseous Nitrogen

B. Hartlebii

93.97
5.17

87.59
4.31
5.17

57.76
71.55

74.66

5.43

37 93
20.59

66.38
7.08

83.38

B. centropundatum . . . .
B. nitrovorum

B. fluorescens lique-
faciens

84.48
82.76

Bad. pyocyaneum

gases represents a definite disappearance of nitrogen from the soil.
This reduction of nitrate to gaseous nitrogen or nitrous oxide is the
change referred to as denitrification. These various reactions
are considered as processes of nitrate reduction. The forma-
tion of nitrite and ammonia does not necessarily result in losses
of nitrogen from the soil, since, under favorable conditions, these
substances may be oxidized to nitrate again. The reaction (pH) of
the material in which the reductions are taking place will, of course,
affect the loss of ammoniacal nitrogen ; under alkaline conditions,
considerable amounts of ammonia may be lost by volatilization.

Importance of Denitrification in Soil. — Since nitrate plays
such an important part in plant nutrition, the discovery of denit-
rifying organisms in soils suggested that denitrification might be
responsible for the limitation of plant growth by depletion of the

IMPORTANCE OF DENITRIFICATION IN SOIL 151

supply of available nitrogen. However, although nitrate may dis-
appear from the soil in numerous ways, its reduction to gaseous
substances occurs only under very specific conditions, and is not
apt to be of particular economic importance in most arable soils.
The conditions particularly conducive to denitrification are:

(1) Presence of relatively large amounts of nitrate.

(2) Presence of an abundance of decomposable organic
matter.

(3) Lack of free oxygen (anaerobic conditions).

These conditions may appear where soils become water-logged
through faulty drainage or where soils are purposely flooded, as
are rice fields. Conditions may also be favorable where concen-
trations of organic matter as composts of manures or plant residues
undergo transformation in the presence of nitrate.

When nitrates are added to the soil, together with large quanti-
ties of stable manure or green manure, decomposition of the organic
substances may be accompanied by denitrification. The rapid
decomposition of the fresh organic substances leads to an exhaus-
tion of the available oxygen in the soil air and the formation of
equivalent amounts of carbon dioxide. Where carbon dioxide is
produced more rapidly than diffusion permits access of atmos-
pheric oxygen, anaerobic conditions are created and denitrification
may temporarily become a factor of importance. The application
of manures in amounts common to agricultural practice does not
cause such rapid consumption of the oxygen in the soil, and no
denitrification takes place.

Denitrification is responsible for a large part of the loss of
nitrogen from animal manures stored in large quantities under
aerobic conditions. If the manures are kept anaerobic, little loss
of nitrogen in the elementary form occurs. As much as 40 to
50 per cent of the nitrogen may disappear as gaseous nitrogen
under conditions favorable for denitrification. The loss is asso-
ciated with the nitrification reaction. The ammonia produced
from the decomposition of urea becomes changed to nitrate in
aerobic regions of the manure heap. This nitrate in turn may
become washed into lower depths of the heap, where it is rapidly
reduced to gaseous nitrogen. This source of loss may be removed
by preventing the formation of nitrate through compacting and
moistening, thus creating anaerobic conditions.

152 TRANSFORMATION OF NITROGEN BY SOIL MICROBES

In the water-logged condition, rice soils present conditions
favorable for nitrate reduction. If nitrate is added to such soils
as fertilizing material it undergoes reduction to nitrite and gaseous
nitrogen. Either of these transformations is quite undesirable.
The nitrite is toxic to plant growth, and gaseous nitrogen is no
longer available to the plants. Ammonium salts are more suit-
able as nitrogenous fertilizers under such cultural conditions.

Summary. — In soil, nitrogen undergoes a series of transforma-
tions as a result of the activities of microorganisms. The supply of
combined nitrogen in the soil has been furnished to a considerable
extent through the activities of certain groups of microorganisms
capable of utilizing the gaseous elementary nitrogen of the atmos-
phere for the purpose of building up complex organic substances,
namely, the constituents of the microbial cells. These organisms
are either non-symbiotic in nature, leading a free existence in the
soil, or are symbiotic, namely, growing in the cells of higher plants,
as in the roots of legumes. When these bacteria die, the higher
plants obtain the nitrogen fixed by these organisms, either directly
through the action of plant enzymes, or indirectly through the
activities of microbes which bring about the decomposition of the
complexes built up by the nitrogen-fixing organisms.

When the nitrogenous plant residues are added to the soil, in the
form of roots, leaves, twigs, green manures, and stable manures,
they first undergo a series of decomposition processes carried out
by various groups of microbes with the result that the nitrogen is
changed from complex organic substances into a simple inorganic
form (ammonia). The processes of decomposition are accom-
panied by processes of synthesis converting again the simple forms
of nitrogen into complex organic forms of a microbial nature.
The nitrogen content of the plant is apt to be very low, ranging
from 0.2 per cent in certain cereal straws to 3 per cent in certain
leguminous plants. The first result of the decomposition of plant
residues by microbes is a narrowing of the ratio of carbon to nitro-
gen in the decomposed material or increasing the percentage
nitrogen content.

If the amount of nitrogen in the plant residues is sufficiently
large, as 1.7 per cent or more, decomposition proceeds rapidly, and
the liberation of nitrogen in such an available form as ammonia
takes place within 4 to 6 weeks of decomposition under favorable
conditions. If the relative nitrogen content is low, considerable

LITERATURE 153

time may elapse before any of the nitrogen becomes available for
higher plants. Under these conditions, the excess of carbo-
hydrates, especially the cellulose, keeps the available nitrogen at a
minimum until they have nearly all decomposed.

The ammonia liberated as a result of decomposition of the
nitrogenous constituents of plants, animals, and microbes is either
utilized directly by plants or is changed by certain specific groups
of bacteria to nitrites, then to nitrates. This process, known as
nitrification, is of considerable importance in soils because it is
believed that the majority of higher plants obtain their nitrogen
from the soil in the form of nitrate.

The nitrate thus produced, if not assimilated by growing plants,
can be absorbed by microorganisms, in the presence of available
carbohydrates, and changed again to microbial protein. It can
be washed out from the soil by drainage waters. Under certain
conditions, such as hmited aeration, it can be reduced to gases
of nitrogen. The nitrogen thus becomes lost from the soil and
returns to the atmosphere.

LITERATURE

1. Buchanan, R. E., and Fulmer, E. I. Physiology and biochemistry of

bacteria. Volume 3, Chapter 17. The Wilhams & Wilkins Co.
Baltimore, 1930.

2. Greaves, J. E. Agricultural bacteriology. Chapters 21 and 22. Lea

and Febiger. Philadelphia, 1922.

3. Stephenson, M. Bacterial metabolism. Chapter 9. Longmans, Green

& Co. London, 1930.

4. Waksman, S. a. Principles of soil microbiology. Chapters 3, 7, 20, 21.

The Wilhams & Wilkins Co. Baltimore, 1927.

CHAPTER VII

TRANSFORMATION OF MINERAL SUBSTANCES IN

SOIL THROUGH THE DIRECT OR INDIRECT ACTION

OF MICROORGANISMS

Relationships of Microorganisms to the Elements
Occurring in Nature. — Practically all the elements which are
essential, in large or small amounts, for the growth of cultivated
or uncultivated plants, as direct nutrients or as catalysts, are
subject to the action of microorganisms in the soil. The trans-
formations of carbon and nitrogen considered in the preceding
pages are particularly prominent. Some of the other elements
are made available for plant utilization through the direct action
of microorganisms, and still others may be subject to their indirect
action.

Compounds of carbon and nitrogen, available to higher plants,
are apt to become very scarce in nature unless the great variety
of organic materials produced by living things are constantly acted
upon by microorganisms and kept in circulation. The elements
hydrogen and oxygen, which are present in great excess in the
atmosphere and in the lithosphere, in either combined or free
forms, seldom become limiting factors in the growth of plants.
However, these elements are constantly subject to the activities
of microorganisms in one way or another, since they occupy an
important place among the constituents of the microbial nutrients
and of the microbial cells, and take an active part in the numerous
oxidation and reduction processes carried on in the soil by microbes.
It may be sufficient to call attention to only two processes to indi-
cate to what extent these two elements are important in the activi-
ties of microorganisms and in the various soil transformations,
and also what role microbes play in the changes that these two
elements undergo in nature.

In the absence of sufficient uncombined oxygen, as in the case
of peat bogs, where the supply of gaseous atmospheric oxygen is

154

MINERAL ELEMENTS AS SOURCES OF ENERGY 155

excluded as a result of the saturation of the material with water,
complete oxidation of the organic matter is made impossible.
The activities of the aerobic fungi and bacteria are thereby ex-
cluded, and the decomposition of organic matter is dependent
largely upon the activities of anaerobic bacteria. Under these
conditions, some of the constituents of the organic substances,
such as the waxes and the lignin, are scarcely decomposed at all,
and others, like the cellulose and hemicelluloses, are decomposed
very slowly. The formation of peat is thus a result of the con-
tinuous accumulation of certain plant residues in habitats deprived
of free oxygen by saturation with water. The peats formed in
prehistoric times have finally given rise to coal, as a result of
various natural phenomena, including physical and chemical
processes.

When organic materials are decomposing under anaerobic con-
ditions, considerable amounts of hydrogen are produced. When
it reaches an aerobic environment , the hydrogen is utilized as food
by a number of bacteria, and becomes oxidized to water.

Most of the other elements which enter into the composition
of the soil are acted upon by microorganisms in one way or another.
Mineral substances become transformed from one inorganic form
into another, or are brought into solution, or are precipitated as a
result of the direct or indirect action of microorganisms. Some
elements as sulfur and phosphorus are frequently constituents of
organic compounds and consequently pass through a more varied
series of changes. Utilization of these organic compounds as
sources of energy by microorganisms results in the mineralization
of the elements. Autotrophic microorganisms are capable of
using several mineral elements or their simple compounds as sources
of energy. Various microorganisms produce a number of organic
and inorganic acids, which increase the solubility of many mineral
materials. Compounds of potassium, magnesium, calcium, and
iron pky important roles in the decomposition and synthetic
processes which are carried on by the soil organisms. Zinc, man-
ganese, chlorine, sodium, etc., are required only in mere traces
by the soil organisms, and their role in microbial processes is still
imperfectly understood in most instances.

Mineral Elements and Their Inorganic Compounds as
Sources of Energy. — Some of the elements or their simple inor-
ganic compounds, which enter into the composition of the living

156 TRANSFORMATION OF MINERAL SUBSTANCES

cell and which are frequently found in the soil in considerable
amounts, are used extensively as sources of energy by certain
specific types of bacteria. These bacteria are usually designated
as autotrophic bacteria, since they are able to synthesize their cell
substance from mineral materials, using carbon dioxide as their
only source of carbon. This process of carbon assimilation which
is referred to as chemosynthesis, because the energy is obtained
from chemical substances, is similar in many respects to the car-
bon assimilation by green plants which is spoken of as photosyn-
thesis, wherein the energy of the rays of the sun is utilized. The
utilization of hydrogen and of compounds of nitrogen (ammonia
and nitrite) as sources of energy by specific bacteria has been
referred to previously. In this group belong also bacteria which
oxidize elementary sulfur and its incompletely oxidized com-
pounds (sulfides and thiosulfates) to sulfate, organisms oxidizing
ferrous to ferric compounds, and manganous to manganic com-
pounds.

Use of Inorganic Salts as Sources of Oxygen. — Under
conditions which favor the activities of anaerobic bacteria, as where
the supply of free gaseous oxygen is excluded, many bacteria are
capable of using certain inorganic salts rich in oxygen as sources of
oxygen. The reduction of nitrates, sulfates, and phosphates are
cases which can illustrate this group of processes. The reduction
of nitrates gives rise to nitrites, ammonia, nitrous oxide, and even
elementary nitrogen, as discussed previously. The reduction of
sulfates gives rise to sulfites and sulfides, including hydrogen sul-
fide, as shown later. The reduction of phosphates gives rise
to phosphites, hypophosphites, and even phosphine.

Interaction of Insoluble Inorganic Salts with Inor-
ganic AND Organic Acids Produced by Microbes. — The trans-
formation of mineral substances as a result of their interaction in
the soil with organic and inorganic acids formed by microorgan-
isms is of common occurrence. These acids are produced in the
soil by numerous bacteria and fungi. The acids interact with the
insoluble carbonates, phosphates, and silicates, making them
partly or completely soluble. Certain portions of the rock frag-
ments in soil are dissolved, leaving the more resistant silicates.
Minerals of the alkalies, such as sodium and potassium, are more
readily acted upon than minerals of the alkaline earths, such as
calcium. Magnesium is affected to much the same degree as cal-

SOLVENT ACTION OF ACIDS 157

cium. Compounds of iron are generally somewhat more resistant
than any of these.

Among the inorganic acids produced by bacteria, carbonic,
nitric, and sulfuric acids are of principal importance. Nitrous
acid is a precursor to the formation of nitric acid, but it is gen-
erally so rapidly converted to nitric acid that it seldom accumu-
lates in sufficient amounts to exert pronounced effects. Carbonic
acid is produced by all mircoorganisms in appreciable amounts in
their growth processes; nitric acid is the end product of oxidation
of ammonia by nitrifying bacteria; and sulfuric acid is the product
of oxidation of sulfide and sulfur by sulfur bacteria. The group of
autotrophic bacteria as a whole is particularly active in solvent
activities, since strong acids are so frequently produced by their
development. Chief among these are nitric and sulfuric acids.

Algae are frequently recognized as corrosive agents acting upon
stone and rock surfaces. As individuals and as associated with
certain fungi in the form of lichens they represent some of the first
plant forms to develop upon naked rocks. They exert effects
through respiration products, carbon dioxide in particular, and also
indirectly by supplying organic food materials to bacteria which in
turn may bring about solvent effects in different ways.

These inorganic acids vary in strength and in the amounts pro-
duced in soils, and consequently in their solvent action. Sulfuric
and nitric acids are much stronger than carbonic acid, but are
formed in small amounts compared to this weaker acid. Under
most soil conditions nitric acid is produced in much more abun-
dance than sulfuric acid. Exceptions to this may appear in cases
where sulfur is applied to soils in considerable quantities to create
acid conditions.

There may be as much as 7,300 kilograms of carbon dioxide
produced each year per acre of fertile soil. This would represent
about 10,300 kilograms of carbonic acid. Considering that all
of the nitrogen removed by a heavy crop and lost in drainage
waters had been produced as nitric acid in the soil, this would
represent at a maximum close to 250 kilograms of nitric acid per
acre. Similarly, it may be assumed that there would be as much
as 100 kilograms of sulfuric acid produced annually in the same
amount of soil. Fortunately, carbonic acid is a weak acid, exerting
a mild solvent effect and readily disappearing by the volatihzation
of carbon dioxide under soil conditions. The amounts of carbon

158 TRANSFORMATION OF MINERAL SUBSTANCES

dioxide produced by different soils are quite unlike, as is apparent
from the data shown in Table 35.

Carbon Dioxide in the Soil. — Many factors are concerned in
the production of carbon dioxide, and since it is practically all of
biological origin, the effects of various factors on the development
of microorganisms is reflected in the amounts of the products of
microbial metabolism, among which is carbon dioxide. Since

TABLE 35

Carbon Dioxide Produced by Different Soils (from Stoklasa)

Carbon dioxide produced

Soil by 1 kgm. of soil in

24 hrs. at 20° C, mgm.

Soil relatively free from organic substances 8. 14

Meadow soil 10.16

Forest soil poor in organic matter 9. 12

Forest soil rich in organic matter 16. 26

Infertile rye and oat soil 19 . 25

Fertile rye and oat soil 30 . 36

Fertile wheat soil 30 . 48

Fertile clover soil 53 . 60

Fertile sugar-beet soil 56 . 68

Fertile garden mould 62 . 75

most of the organic residues of plants become incorporated in
the superficial portions of the soil, and microorganisms find other
conditions most favorable for their development in this region, the
largest amounts of carbon dioxide are produced near the surface,

TABLE 36

Carbon Dioxide Evolved from Sandy Soil at Different Distances
from the Surface (from Lundegardh)

Depth, CO2 per sq. meter per hour,
cm. gm.

0-10 0.271

10-20 0.084

20-30 0.016

30-40 0.009

Total 0.380

In the deeper layers of soil the gas is produced in smaller amounts
(see Table 36). Although produced most rapidly near the sur-

CARBON DIOXIDE IN THE SOIL

159

face, carbon dioxide occupies a greater portion of the pore spaces
in the deeper regions of the soil (see Table 37) . The air of the soil
is thus considerably higher in carbon dioxide than the normal
atmosphere above the soil. While the atmosphere contains close
to 0.03 per cent of the gas, the soil air shows from 0.05 to 3.8 per
cent, and seldom as low as the smallest figure. Table 38 gives
figures for the composition of the gases in soils under aerobic and
anaerobic conditions.

TABLE 37

Carbon Dioxide in the Soil Air at Different Distances below the
Surface (from Lau)

Depth,

Per cent carbon dioxide

cm.

Sandy soil

Loam soil

Moor soil

15
30
60

0.09-0.19
0.06-0.24
0.11-0.57

0.05-0.27
0.09-0.47
0.20-1.13

0.10-0.75
0.34-1.12
1.01-3.77

Since diffusion of the soil gases to the atmosphere is more rapid
near the surface, the relative carbon dioxide concentration will
be greater at lower depths. In the deeper layers the movement of
the gases is retarded by the longer distance required to be traveled
and the smaller number of channels opening to the surface. Soil
water depresses the speed of movement to an important extent by

TABLE 38

Composition of Soil Gases

Aerobic (from Russell and
Appleyard)

Anerobic or rice paddy

soils (from Harrison

and Aiyer)

Oxygen

Carbon dioxide

Nitrogen

Methane

Hydrogen

18.00 to 21.00 per cent ±

0.06 to 2.0 per cent ±

79 per cent ±

0

0

0 or traces

1-20 per cent

10-95 per cent

15-75 per cent

0-10 per cent

160

TRANSFORMATION OF MINERAL SUBSTANCES

filling the pore spaces. On the other hand, a relatively high tem-
perature and moisture content favor the production of carbon
dioxide, as shown in Table 39. Considering the amount of the
gas produced at the lowest temperature and moisture content cited
in the table as 1, increases in moisture and temperature raised the
production of carbon dioxide to 40. The numerous factors which
favor microbial development increase the rates of carbon dioxide
production, and factors lowering rates of diffusion tend to keep the
gas at a relatively high level in the soil air and consequently also
at a high level in the dissolved state as carbonic acid.

TABLE 39

Influence of Temperature and Moisture on Carbon Dioxide Content
OF SoiL.s (from Wollny)

Water content,

Temperature

per cent

10° C.

20° C.

30° C.

6.8
26.8
46.8

1 vol. of CO2

9.1 vol. of CO2

17.2 vol. of CO2

1.6 vol. of CO2
26.7 vol. of CO2
30.6 vol. of CO2

3.4 vol. of CO2
31.0 vol. of CO2
40.5 vol. of CO2

The action of carbonic acid may be exerted upon a great
variety of soil materials, forming carbonates of the basic sub-
stances, and if present in sufficient amount, producing bicar-
bonates from these carbonates:

CaCOs + H2CO3 = Ca(HC0.3)2.

In spite of the fact that most of the carbonic acid which is produced
in the soil is lost soon after its formation by volatilization of carbon
dioxide, some of the active acid reacts with the soil constituents
and may dissolve considerable amounts of those mineral substances
which are relatively insoluble in water alone. The reactions con-
cerned in these changes may be represented as follows:

Ca2(HP04)2 + 2H2CO3 = Ca(H2P04)2 + Ca(HC03)2

Di-calcium
phosphate

Mono-calcium
phosphate

Ca3(P04)2 + 4H2CO3 = Ca(HoP04)2 + 2Ca(HC03)2

Tri-caleium
phosphate

Mono-calcium
phosphate

NITRIC ACID IN THE SOIL

161

Stoklasa reported a case where distilled water dissolved only 0.07
gm. of phosphate (calculated as P2O5) from 1 gm. of di-calcium
phosphate, while carbonated water dissolved 0.26 gm. The
solvent action of carbonated water on various phosphates is quite
pronounced, as evidenced by the data shown in Table 40. In
general, its action is considerably weaker than that of the organic
acids.

TABLE 40

SoL\'ENT Action of Weak Acids on Pho.sphates (from Stoklasa)

Per Cent Dissolved

Compound

Carbonated
water

Acetic
acid

(0.5%)

Formic
acid

(0.57c)

Di-calcium phosphate

Tri-calcium phosphate

Mono-ferric phosphate

45.75
25.01
27.44
14.68
7.87
33.67

97.13
73.83
19.57
13.51
8.13
99.48
22.09

99.54
90.90
29 . 35

Di-ferric phosphate

Tri-ferric phospliate

Mono-ahiminum phosphate

Tri-aluminum phosphate

27.81
16.00
76.05
93.79

In the vicinity of growing roots of plants, considerable amounts
of carbonic acid appear as a product of respiration of the root
tissues; this is further increased by the microorganisms in their
decomposition of the organic materials originating from the roots.
The solvent action of this carbonic acid in the vicinity of root
absorption is an important factor in the nutrition of plants in an
environment such as soils where the inorganic nutrients occur
in low concentrations.

Nitric Acid in the Soil. — The amount of nitrate which occurs
at any one time in soils supporting plant growth is comparatively
small. This may occasionally be as much as 100 pounds, calcu-
lated as nitrogen, or 450 pounds, calculated as nitric acid, per acre.
Generally the nitrate in soils would be a small fraction of this.
The significance of nitrate formation lies not so much in the fact
that certain amounts exist in soils at any one time combined with
basic substances, but that nitric acid is formed continuously. Its

162

TRANSFORMATION OF MINERAL SUBSTANCES

absorption by plants or removal in drainage waters keeps the
concentration of nitrate low in humid regions. Soon after its
formation it reacts with soil constituents, such as the insoluble car-
bonates, phosphates, and silicates, and, over a period of time,
dissolves large amounts of insoluble substances forming salts of
potassium, sodium, magnesium, calcium, and iron. It has been
stated that, in the conversion of organic nitrogen to nitrate suffi-
cient for a hundred bushel crop of corn, there will be produced
sufficient acid to convert seven times as much insoluble tri-calcium
phosphate into soluble mono-calcium phosphate as would be
required to supply the phosphorus for the same crop. Of course,
the larger part of the nitric acid would take part in other reactions,
and only a small portion of the acid would react with the phos-
phates. Nitrification of ammonium sulfate is responsible for
greater acid formation than nitrate formation from nitrogen origi-
nally contained in organic combination. The solvent action
accompanying nitrification in laboratory cultures containing tri-
calcium phosphate is shown in Table 41.

TABLE 41

Solution of Phosphate and Calcium from Tri-calcium Phosphate by
Nitrification of Ammonium Sulfate in Solution Cultures (from
Hopkins and Whiting)

Nitrogen oxidized,

Phosphorus made

Calcium, made

mgm.

soluble, mgm.

soluble, mgm.

2.54

4.08

3.87

3.81

5.08

5.60

4.88

10.20

18.40

5.52

9.56

14.80

6.40

12.85

22.00

6.40

10.24

23.52

6.88

16.00

31.04

The reaction concerned in this process may be expressed as
follows:

Ca3(P04)2 + (NH4)2S04 + 402 =

Ca(H2P04)2 + Ca(N03)2 + CaS04 + 2H2O

ORGANIC ACIDS IN THE SOIL 163

This reaction can be considered as representative not only of the
solvent effects following nitrification but also of the development
of increased acidity through the absorption or transformation of a
cation of an inorganic salt. By removing or changing the ammo-
nium ion, the sulfate radical takes on hydrogen, which gives to it as
great solvent powers as the nitric acid formed from the oxidation
of the ammonia.

When urea is added to the soil, the reaction of the soil is first
made alkaline, by the transformation of the urea to ammonium
carbonate, then acid following the oxidation of the ammonia to
nitric acid:

CO(NH2)2 + 2H2O = (NH4)2C03

(NH4)2C03 + 4O2 = 2HNO3 + CO2 + 3H2O

These general considerations of the solvent action of nitric
acid on soil minerals are equally applicable to sulfuric acid, with
the exception that sulfuric acid generally appears in soils in smaller
amounts, and consequently its effect is of less importance.

Organic Acids in the Soil. — In addition to the inorganic acids,
various microbes produce in the soil organic acids, which can play
the same function as the inorganic acids but are weaker than nitric
or sulfuric acids although stronger than carbonic acid. Among the
organic acids produced by soil bacteria, the following may be
mentioned: lactic, butyric, acetic, propionic, and valerianic.
Fungi produce the following acids: gluconic, citric, oxalic, fumaric,
and succinic. Various acids may be formed from carbohydrates
under suitable conditions :

C6H12O6 = C4HSO2 + 2CO2 + 2H2 (anaerobic bacteria)

Butyric
acid

CeHioOe + 30 = CsHsOt + 2H2O (fungi)

Citric
acid

CeHiaOe + 90 = 3C2H2O4 + SHoO (fungi)

Oxalic
acid

C6H12OG = 2C3HCO3 (anaerobic bacteria)

Lactic acid

164 TRANSFORMATION OF MINERAL SUBSTANCES

3C6H12O6 = 3C4HSO2 + C2H4O2 + 4CO2 + 2H2O +2H2
^acid" "Idd' (anaerobic bacteria)

3C6H12O6 = 2C4HSO2 + 2C2H4O2 + 4CO2 + 2H2O + 2CH4

(anaerobic bacteria)

The decomposition of a carbohydrate may thus lead to the
formation of butyric and acetic acids as well as certain gases,
largely carbon dioxide and hydrogen or methane, or both together.
This was actually demonstrated to take place in the anaerobic
decomposition of cellulose.

In the decomposition of proteins, the amino acids which are
formed may decompose further with the liberation of fatty acids:

R-CH(NH2)C00H + H2O

Amino acid

= NH3 + RCH(OH)COOH = R-CH20H + CO2

Hydroxy-fatty Alcohol

acid

COOH • CH2 • CH2 • CH • NH2 • COOH + H2

Glutamic acid

= CH3CH2-CH2COOH + NH3 + CO2

Butyric acid

The decomposition of fats and lipoids results in the formation of
various fatty acids:

CH2— OOC— C15H31 CH2— OH

CH — OOC— C15H31 + 3H2O = CH —OH + 3Cir.H3i-COOH

I Fatty acid

CH2— OOC— C15H31 CH2— OH ^'''''""'■•^ '^"''^

fat (glyceryl tri-palmitate) Glycerol

In addition to the organic acids, carbon dioxide is an important
product of most of these reactions. The solvent action of organic
acids compared to carbonic acid is suggested from the data in
Table 40.

Under aerobic conditions, the organic acids do not persist for
long in the soil, but are soon decomposed more completely, even
to the final oxidation products, namely, carbon dioxide and water.
Under anaerobic conditions, the acids may persist for extended
periods of time.

ORGANIC ACIDS IN THE SOIL

165

With all of the acid substances, whether they be weak or strong,
their effects on mineral materials are proportional to the degrees of
acidity produced. Figs. 66 and 67 show this relationship. The
stronger acids naturally cause solvent effects in low concentra-
tions equal to those brought about by the weak acids in propor-
tionately high concentrations.

4.0
Solution of Calcium

2.0 LogCca

Increase

Fig. 66. — The influence of acidity created by lactic acid on solution of calcium
from calcium silicate (after Wright).

Change in Soil Reaction. — Besides being important as
potential supplies of plant nutrients when acted upon by acid
substances, the phosphates, carbonates, and silicates play an
important role as agencies regulating the rapidity of change of
soil reaction. In the absence of an abundance of buffering mate-
rial in soils, the large amounts of acids which are produced would
soon change the reaction to such acidity as to make the soil a
medium unfit for the growth of most higher plants and of various
important soil organisms, especially those that are sensitive to
acid conditions, as Azotobacter, nitrifying bacteria, and Bad.
radicicola. The presence of carbonates, phosphates, and silicates

166 TRANSFORMATION OF MINERAL SUBSTANCES

makes the soil strongly buffered and prevents rapid changes in
reaction.

2CH3CHOHCOOH + CaCOa

= Ca(CH3 ■ CHOH • C00)2 + CO2 + H2O.

Calcium lactate

The acid may become neutrahzed by interacting with calcium car-
bonate. When the lactate is decomposed by other organisms, the
calcium carbonate is regenerated :

Ca(CH3- CHOH -000)2 + 6O2 = CaCOs + 5CO2 + 5H2O

l-ogC^

- Solution of Calcium

1 1

40

-

-

-

-Solution of Magnesium
Solution of Potassium

OJ

_

ra

1

1 1

1

c

-

Y

/ /

/ -

■5,0

"

■ y

• /

■

a.
<

1'

-

/■

/•

/

~

"60

1

1 1

L

1 1-

4 0 3 0 2 0LogCKc. o,Mg

Solution of Minerals — Increase

Fig. 67. — The influence of acidity created by growth of Azotobacter on solu-
tion of potassium, calcium, and magnesium from the mineral biotite (after

Wright).

During the weathering of rocks, which are composed for the
most part of minerals made up of strong bases (as calcium, potas-
sium, magnesium, sodium) bound to a weak acid (silicic), there is a
tendency for the development of an alkaline reaction. This is
what happens in regions of deficient rainfall where the limited
amount of drainage waters fails to remove the soluble salts as
rapidly as they are formed. In the reclamation of alkali soils,
the injurious effects of the basic reaction and high content of basic
salts may be partly overcome by the addition of sulfur, which is
oxidized by bacteria to sulfuric acid, as will be discussed later.
In humid regions the weathering processes are accelerated by the

TRANSFORMATION OF SULFUR IN SOIL BY MICROBES 167

more favorable moisture conditions and the soluble salts fail to
accumulate in appreciable amounts. What amounts are not
fairly rapidly absorbed by growing plants are soon removed in the
percolating waters. The various acids which are so important in
these weathering processes are not removed from the soil in the
form of acids but are combined with various bases which arise
from the compounds in the soil. It is the acceleration of the re-
moval of basic substances in humid regions which so quickly causes
the development of acidity and impoverishment in plant nutrients.

Mineral Assimilation by Microorganisms. — Minerals are
of direct importance to the life of the soil organisms in that they
enter the composition of the cells. Some of the mineral elements
enter into organic compounds of the cell, while others play impor-
tant parts as physiological agents in the cell fluids. Although
required in smaller amounts than nitrogen, these minerals are
nevertheless quite essential in certain amounts to the development
of the living cells. In the decomposition processes carried out by
microorganisms, much of the mineral content of the substances
which undergo decomposition (such as plant residues) becomes
liberated in forms available for subsequent development of higher
plants. The phosphorus, sulfur, potassium, calcium, magnesium,
and other minerals previously absorbed by the plant from the soil
and stored up in its tissues again become liberated and made avail-
able for utiHzation by other plants. On the other hand, a part of
these minerals becomes locked up in the microbial cell substance
which has been synthesized during the decomposition process.
This involves only a fraction of the minerals liberated in the
decomposition of the plant residues, and represents the nutritional
requirements of the microbial cells. The amounts liberated and
not assimilated by the cells represent excess over these require-
ments and may be considered as waste products of the cells.

A detailed analysis is given here of the transformation of four
minerals in the soil, namely, sulfur, phosphorus, iron, and potas-
sium. From what has been said previously, one may conclude
that many of the other minerals are also subject to various changes
in the soil as a result of the activities of microorganisms, and that
many of these minerals play important roles in the growth of
microbes and higher plants even if required only in mere traces.

Transformation of Sulfur in Soil by Microbes. — Sulfur
exists in the soil and is added to it constantly in plant and animal

168 TRANSFORMATION OF MINERAL SUBSTANCES

residues, rain-water, and fertilizing materials, in several distinctly
different forms, namely, as organic sulfur compounds, inorganic
sulfates, sulfides, and elementary sulfur. In the organic matter
of the soil, the sulfur is generally bound up in forms resistant to
decomposition and only after long periods of time liberated in
appreciable amounts as sulfate. The addition of elementary sul-
fur or sulfate in fertilizer materials may be responsible for rela-
tively large amounts of sulfate in the soil solution from time to
time, but, generally, sulfate makes up a small portion of the total
sulfur content of the soil. More commonly from 80 to 90 per cent
of the sulfur is present in organic combination, and only 10 to 20
per cent exists as sulfate.

The plant residues commonly added to the soil contain sulfur
largely in organic combination, varying from 0.05 to 1.0 per cent.
Alfalfa hay, for example, contains 0.29 per cent sulfur; turnip
tops, 0.9 per cent ; and wheat straw, 0.12 per cent. The sulfur in
the organic compounds of such plant residues becomes liberated
as sulfate more rapidly than from the so-called humic matter of
the soil, just as ammonia is formed more rapidly from fresh organic
matter rich in nitrogen than from the residual organic matter in
the humus.

Under natural conditions, many agents, both biological and
non-biological, carry the element through its course of changes.
Microbial effects are largely oxidations and reductions of the inor-
ganic sulfur compounds, and formation and decomposition of
organic compounds containing sulfur. Since sulfur enters into the
composition of all living cells, all forms of life become associated
with its transformation. In the decomposition of the organic
residues by microbes, the sulfur present in organic combination
is changed, after several transformations, into inorganic forms such
as hydrogen sulfide and sulfate.

Some microorganisms utilize sulfate to satisfy their require-
ments for the element. In time, the microbial cells become
destroyed and mineralized. The assimilation of sulfate is more
pronounced when the organic matter which serves as food con-
tains little or no sulfur. Table 42 shows the rapid disappearance
of sulfate when an organism {Aspergillus niger) is feeding on a
sugar.

Sulfur is present in the organic matter largely in the protein
molecule. This molecule contains an amino acid, cystine, which

TRANSFORMATION OF SULFUR IN SOIL BY MICROBES 169

TABLE 42

Disappearance of Sulfate During Growth of Aspergillus Niger on
A Carbohydrate (from Rippel)

Sulfate-sulfur,
Time mgm.

At start 3. 19

After 5 days 1 • 58

After 7 days 0.64

After 12 days 0

is the sulfur carrier. When proteins are hydrolyzed by micro-
organisms, the cystine is first hberated. When the cystine mole-
cule is decomposed by microorganisms in the soil, the sulfur is
usually liberated in the form of hydrogen sulfide:

CHo — S — S — CH2

I I

CH • NHo CH • NHo + 4H2O

I I

COOH COOH

Cystine

= 2CH3COOH + HCOOH + CO2 + 2NH3 + 2H2S

Acetic acid Formic acid

Other organic compounds containing sulfur are frequently intro-
duced into the soil, but generally in very small amounts. These
include taurine, among the animal products, and certain gluco-
sides, among the plant residues.

The nature of the compound as well as the environmental con-
ditions in which it is decomposed, whether aerobic or anaerobic,
will determine to a large extent the resultant form of the sulfur
when it appears in the inorganic state. Under anaerobic condi-
tions sulfides are frequently produced in considerable quantities.
This is the case in water-logged soils, ditches, stagnant pools, and
seas that receive appreciable contributions of organic materials
and sulfates. Coloration of soils may be determined by sulfides
of iron under anaerobic conditions where organic matter is not so
abundant as to conceal this coloration. These sulfides are formed
from the decomposition of organic compounds containing sulfur
and from reduction of sulfates, sulfur, or other inorganic, incom-
pletely oxidized sulfur compounds.

170 TRANSFORMATION OF MINERAL SUBSTANCES

Direct evidence is still lacking to indicate that microorganisms
convert sulfur, associated in organic compounds, directly to sulfate.
Some sulfur may be liberated in this way, but a greater portion is
first changed to sulfide and appears as sulfate only after secondary
attack by organisms able to oxidize inorganic sulfur compounds.
Wherever protein materials undergo decomposition by the agency
of bacteria, sulfide is formed. Under anaerobic conditions it
is not transformed further, but under aerobic conditions it soon
disappears and is oxidized to sulfate, as may be represented by the
following equation:

+ 30
H2S + 0-> H2O + S — > H2SO4

Various specific bacteria (largely of the Thiobacillus group) are
capable of bringing about these processes in the soil (see Fig. 15),
The hydrogen sulfide is first oxidized to elementary sulfur. In the
case of some bacteria, this sulfur actually accumulates within or
without the cells. Other bacteria, however, immediately oxidize
the sulfur to sulfuric acid. This acid interacts with the soil bases:

H2SO4 + CaCOs = CaSOi + H2O + CO2.

Sulfide may be considered as an intermediate decomposition
product. It is unavailable to plants as such, is injurious rather
than beneficial to the activity of most soil microorganisms, and its
presence in soils in any appreciable amounts is indicative of par-
tial anaerobic conditions unsuited for plant growth. Sulfide, like
any other decomposition product such as ammonia, is a waste
product left by the microorganisms as unnecessary in their further
development. Its formation as a product of decomposition of
organic matter indicates that the compounds from which it was
produced contained more sulfur than was required by the organisms
in their growth while using this organic matter as food. As
hydrogen sulfide it represents a source of considerable potential
energy which will become liberated during its transformation to
sulfate.

After elementary sulfur is added to soil, it is oxidized directly
to sulfuric acid if the proper organisms are present. The oxidation
proceeds particularly rapidly where some of the specific sulfur
bacteria are concerned. These organisms are similar in their
nutrition to the nitrifying bacteria in that they are autotrophic,

TRANSFORMATION OF SULFUR IN SOIL BY MICROBES 171

but they use the sulfide or sulfur as their specific foods, oxidizing
them to sulfate. Even in the absence of these organisms, sulfates
are formed, presumably in some way associated with the develop-
ment of various heterotrophic organisms including both bacteria
and fungi. The process of sulfur oxidation can be utilized for
increasing the acidity of the soil, and in view of the fact that certain
organisms producing plant diseases cannot thrive at certain acid
concentrations, it may be desirable to create such acid conditions
as are inhibitive to their development. This is brought about by
adding sulfur to soils. In the soil, it is oxidized to sulfuric acid
and brings about the desired effects as considered in more detail
elsewhere. This process can also be utihzed for reducing the alka-
linity of black alkali soils. The sulfuric acid formed by the oxida-
tion of sulfur interacts with the sodium carbonate, giving sodium
sulfate. The latter salt of sodium is much less injurious to plants
than the former; it has less undesirable effects on the physical
condition of the soil and it can also be more readily removed by
irrigation and drainage waters. The oxidation process is further
utilized in transforming the insoluble phosphate of the rock phos-
phate to more soluble forms. When sulfur is mixed and composted
with rock phosphate and soil, in the proper proportions, the sul-
fur becomes oxidized to sulfuric acid and changes the phosphate
to the di-calcium and mono-calcium forms. The application of
such material to soils gives much the same effects as the applica-
tion of " superphosphate." Similar solution of potassium occurs
when certain insoluble minerals containing potassium are substi-
tuted for the rock phosphate in these composts.

Large amounts of sulfide may be produced in nature by the
reduction of sulfates as well as elementary sulfur itself. These
processes consume appreciable amounts of energy which must be
supplied from other sources, such as the oxidation of organic
compounds. Further, the reaction proceeds only under anaerobic
conditions. In such an environment the reactions take place
somewhat as follows :

2S

+ 2H2O + C6H12O6 = 2CH3-COOH 4- 2H-C00H + 2H2S

Glucose .\cetic acid Formic

acid

3CaS04 + 2(C3H503)Na

Sodium lactate

= SCaCOa -f Na^^COa + 2H2O -f- 2CO2 + 3H2S

172 TRANSFORMATION OF MINERAL SUBSTANCES

In these reductions the sulfur and sulfate act as oxidizing
substances, and serve, under these anaerobic conditions, in
much the same capacity as oxygen itself does in an aerobic
environment.

In case conditions are changed, after the formation of sulfide,
so as to permit the entrance of free oxygen, thus creating aerobic
conditions, the sulfide does not persist but is oxidized again to
sulfate. In certain lakes and seas, as well as in a number of cura-
tive muds, the two processes, namely, the oxidation of hydrogen
sufide to sulfate and the reduction of sulfate to hydrogen sulfide,
may go on side by side. In the lower layers of the lake or sea
where free oxygen is scarce, the reduction process predominates.
The hydrogen sulfide once formed moves upward in the convection
currents or as bubbles of gas, and at or near the surface of the
lake, on coming in contact with the oxygen of the air, it is oxidized
to sulfate by specific oxidizing bacteria. The sulfate may diffuse
downward again, where it is again reduced to sulfide under the
anaerobic conditions.

The reduction of sulfur to sulfide may be brought about by a
great number of different bacteria, but the reduction of sulfate is
limited to very few organisms, although these are widely dis-
tributed. Spirillum desulfuricans is the name generally applied
to the reducing form which is found in fresh water and soils
(see Fig. 16).

As this short discussion indicates, there are striking similarities
between the transformations of various compounds of sulfur and
nitrogen through the agency of microorganisms. Organic com-
pounds of nitrogen decompose to form ammonia, while the sulfur
compounds lead to hydrogen sulfide. Ammonia is changed to
nitrite and nitrate, while sulfide is oxidized to sulfur, sulfate, and
numerous incompletely oxidized inorganic substances. These
oxidations are all the result of the action of specific autotrophic
bacteria. Both nitrates and sulfates may be reduced by anaerobic
bacteria, leading to a variety of products including ammonia
and hydrogen sulfide. Although a process like nitrogen fixation
is not found in sulfur transformations, the reactions associated
with oxidation and reduction of elementary sulfur are similar in
certain respects. Inorganic and organic nitrogen compounds
furnish different microorganisms with their requirements for this
element, and similar sulfur compounds serve a like purpose in

TRANSFORMATION OF PHOSPHORUS BY SOIL MICROBES 173

microbial metabolism. In fact, most of the nitrogen trans-
formations have very close counterparts in changes of sulfur
compounds.

Transformation of Phosphorus by Soil Microbes. — Phos-
phorus is usually added to the soil in the form of various plant and
animal residues (in stable and green manures), in certain organic
fertilizers (tankage, bone meal, guano), and in various inorganic
fertilizers. Phosphorus is present in the latter either as insoluble
rock phosphate, the chief constituent of which is Ca3(P04)2, or
as superphosphate, which consists of rock phosphate previously
treated with sulfuric acid to make it soluble, or as various other
basic phosphates included in slag.

In the plant and animal residues, as well as in stable and green
manures, phosphorus is present in the form of such compounds as
phytin, phospholipids, of which lecithin is the best known repre-
sentative, and nucleoproteins. Before the phosphorus can be
utilized again by higher plants, these organic complexes have to be
decomposed by various soil organisms. These processes of decom-
position can be illustrated somewhat as follows :

C6H24027P(5 + 6O2 = 6H3PO4 + 6CO2 + 3H2O

Phytic
acid

The phytic acid originates in the decomposition of phytin.
Lecithin contains both phosphorus and nitrogen; it is first hydro-
lyzed to cholin (a nitrogen complex), glycerophosphoric acid,
and various fatty acids. Nucleoproteins are compounds of
one or more protein molecules with nucleic acid; the latter con-
tains the phosphorus. Nucleic acids themselves are also very
complex in composition, such as C3GH48O30N14P4. On decompo-
sition, they give, in addition to phosphoric acid, various carbo-
hydrates and organic bases (adenine, guanine, cytosine). The
decomposition processes of some of these substances are frequently
very complicated, involving a number of reactions, but they all
lead to the formation of phosphoric acid.

The phosphorus in organic combination may make up from 20
to 35 per cent of the total phosphorus of many soils. Organic
phosphorus may also compose a large portion of the phosphorus
occurring in solution in soils. In a study of twenty-one soils of
Southern and Central United States, the soluble organic phos-

174 TRANSFORMATION OF MINERAL SUBSTANCES

phorus (calculated as P2O5) was present in 0.176 parts per million
of soil solution, while the inorganic phosphorus was found in only
0.034 parts per million.

The microbial cells which are constantly formed in the soil are
also very rich in phosphoric acid ; the ash of the microbes frequently
contains 50 per cent or more phosphate, calculated as P2O5.
Largely as a result of this consumption of phosphorus by microbes
and its use in cell synthesis, the organic matter of the soil contains
a definite amount of phosphorus. The ratio of the carbon content
of the soil to the organic phosphorus content is more or less con-
stant, just as is the case in the ratio of the carbon to nitrogen.
When the organic matter of the soil or that added to the soil is
decomposed, there is a continuous liberation of phosphorus in an
inorganic form, and the more rapidly the organic matter is decom-
posed the more rapidly the phosphorus is Hberated. In the pres-
ence of an abundance of available organic food material containing
little or no phosphorus, the microbes will reassimilate part of this
phosphorus in the synthesis of their cell substance, and a deficiency
of phosphorus available for plant growth may be created. The
amount of food material used by microorganisms for growth, the
amount of microbial cell substance synthesized from the food, and
the quantities of both nitrogen and phosphorus which are assimi-
lated in these processes are all in definite ratios to one another.
In other words, a definite amount of microbial cell substance is
formed per unit of organic food material consumed, and quite
definite quantities of nitrogen and phosphorus are built into these
cells.

Various fungi and bacteria are capable of liberating phosphorus
from organic complexes in an inorganic form. Some bacteria
are particularly active in this connection since they are
capable of attacking the phosphorus-bearing complexes more
readily.

Processes similar to those causing reduction of nitrates and
sulfates may be concerned in the reduction of phosphates. Nitrates
are quite readily reduced; sulfates, with somewhat more difficulty;
and phosphates, least readily of all. Under anaerobic conditions,
if organic food materials are available, if phosphates are present
in abundance and if the necessary bacteria are active, phosphates
may be reduced to phosphite (H3PO3), hypophosphite (H3PO2),
and phosphine (PH3). The reactions may be illustrated in a gen-

TRANSFORMATION OF PHOSPHORUS BY SOIL MICROBES 175

eral way as follows, where C refers to carbon in organic combina-
tion:

2H3PO4 + C = 2H3PO3 + CO2

Phosphoric
acid

Phosphorous
acid

H3PO4 + C = H3PO2 + CO2

Hypophos-
phorous acid

H3PO4 + 2C = PH3 + 2CO2

Phosphine

The possible significance and importance of these reactions in the
movements of phosphorus in arable soils are unknown. However,
since these processes are favored only by rather extreme reduction
conditions, it seems likely that phosphate reduction is not respon-
sible for very extensive changes under most soil conditions. The
extent of reduction of phosphates to the various substances by
bacteria under conditions favorable to the changes is shown in
Table 43.

TABLE 43

Reduction Products of Phosphate Formed by a Bacterium
(from Rudakov)

Mgm. P2O6 per Liter

Reduced to
phosphine

Reduced to
phosphite
and hypo-
phosphite

Remaining

as
phosphate

Biologically
absorbed or
changed to

other
compounds

Control

2500
1306

Inoculated

102

467

625

The insoluble inorganic calcium phosphate may be present in
the soil in the native rock constituents or occur there subsequent to
the addition of various fertilizer mixtures. This form of phos-
phate is available to plants only in very limited amounts. How-
ever, as a result of interaction with the various organiis and inor-
ganic acids formed by microbes, the phosphate becomes changed

176 TRANSFORMATION OF MINERAL SUBSTANCES

into di- and mono-basic phosphates, which are more soluble and
consequently more readily available to plants. In view of the
fact that the formation of the inorganic and organic acids takes
place in the soil constantly as a result of the decomposition and
oxidation processes, insoluble phosphates tend to become gradu-
ally soluble, especially when accompanied by processes of active
decomposition of organic matter and oxidation of ammonia and
incompletely oxidized sulfur compounds. When the soil contains
free carbonates and the reaction is neutral or alkaline, the acids
will be neutralized by the carbonates in preference to the phos-
phates, and the latter will not go into solution so readily.

The amount of phosphoric acid made available to plants at any
given time is thus found to be a result of various complex biological
and chemical processes which tend either to bring the phosphoric
acid into solution (when the decomposition processes are pre-
dominant) or take it out of solution (when the synthesizing micro-
bial processes are active and the amount of available phosphate is
limited). The amount of available phosphorus in any given soil
can be readily and simply determined by microbiological pro-
cedures as discussed in Chapter IX.

Transformation of Iron by Soil Microbes. — Iron is seldom
added to soils in fertilizing materials and is contained in organic
residues in very limited quantities, but it is one of the elements
present in the soil in very large amounts. In spite of the great
abundance of the element in various compounds in soils and in
spite of the fact that it is required by plants in very small amounts,
it is so slightly soluble in alkaline soils that the supply of plants with
soluble compounds of iron is a serious problem under such condi-
tions. Iron is of considerable importance in the physical compo-
sition of soils, and in extreme cases its solution in the upper layers
and precipitation in localized deeper regions of soils may be so
extensive as to play an important part in the formation of imper-
vious layers called "hardpans."

Microorganisms are associated with the movement of iron in
the soil in various ways. Among these changes may be included
solution and precipitation of iron compounds; these reactions are
frequently accompanied by reduction and oxidation processes.

In the precipitation of iron from soluble inorganic compounds
of the element, the iron bacteria have been considered to play a
preeminent part. These organisms are so named since iron in the

TRANSFORMATION OF IRON BY SOIL MICROBES 177

reduced form serves as their source of energy for growth, and they
live by the oxidation of ferrous to ferric iron and, Hke other auto-
trophic organisms, obtain carbon for their cell development from
the carbon dioxide of the air. Incidentally, subsequent to oxida-
tion of the iron during growth of the bacteria, the ferric com-
pounds precipitate and accumulate about the cells in relatively
large amounts. Organisms of this type are but one group of
many that may be concerned with transformations of iron; how-
ever, they are probably little concerned with the changes of iron
in the soil (Fig. 68).

Fig. 68. — Filament of an iron bacterium, Leptothrix crassa, showing incrusta-
tion of ferric hydrate (after Cholodny).

Certain organic compounds of iron are quite soluble. After
the utilization of the organic portion of these compounds as food
by the heterotrophic microorganisms, the inorganic compounds
of iron which are formed in the process are precipitated, since they
are soluble in only very small amounts. As an example we may
consider ferric ammonium citrate, a double salt of the organic
citric acid. Upon decomposition, the citrate is changed to various
products, including carbon dioxide and water. This liberates the
ammonia as ammonium hydrate and the iron as ferric hydrate.
The iron hydrate precipitates by reason of its slight solubility.
Precipitation by iron bacteria and by the organisms decomposing
organic compounds is more likely to occur under aerobic conditions.
Under anaerobic conditions, iron may be precipitated as sulfide,
which is formed either from organic compounds or from the
reduction of inorganic compounds of sulfur.

Solution of iron may follow the formation of any of the acids
produced by microorganisms in soils, among which carbonic, sul-
furic, nitric, and the various organic acids are especially important.
Since iron is so slightly soluble at reactions most commonly suited
to growth of higher plants, the formation of certain amounts of

178 TRANSFORMATION OF MINERAL SUBSTANCES

these soluble compounds in the vicinity of the developing plant
roots may be of particular importance in their nutrition.

Thus, under aerobic conditions, iron may be brought into solu-
tion provided the reaction becomes more acid. Under anaerobic
conditions, the solution is greatly accelerated by reason of the fact
that iron becomes reduced to the ferrous form and in this condition
is much more soluble than in the ferric form at reactions close to
neutrality. Solution is further favored by the fact that anaerobic
conditions lead to the formation of organic acids which exert con-
siderable dissolving effects.

Most of these changes in the amounts of iron in solution follow
changes in the environmental conditions by microbial activities.
In general, the development of anaerobic conditions or the forma-
tion of acids results in the solution of iron and usually its reduc-
tion. Either a change from anaerobic to aerobic conditions, or a
decrease in acidity, or both, lead to oxidation of iron and may cause
its precipitation. The fact that iron is readily oxidized and
reduced greatly increases the capacity of microorganisms to mod-
ify its condition in the soil.

Transformation of Potassium by Soil Microbes. — Potas-
sium is considerably less susceptible to effects of microorganisms in
its relationships to plant development in soils. It belongs to a
large group of elements in soil which are not affected in a great
variety of ways, on account of the fact that they do not enter into
organic combination to the extent of such elements as nitrogen or
sulfur. Its entrance into organic compounds is more generally
confined to replacing the hydrogen of the acid groups where it
forms the salts of these acids. In plant residues a large portion
of the potassium is present in inorganic form, occurring in the
various fluids of the cells. Upon ignition of the organic substances
the potassium remains in the ash residue. Another factor appre-
ciably limiting the capacities of microorganisms to affect the
element is its stable character. It is not oxidized and reduced
and consequently cannot be carried through the extensive series
of changes common to nitrogen, sulfur, and iron. The influence of
microorganisms on such elements as potassium is confined to caus-
ing its solution from organic and inorganic substances and its
assimilation in growth of the microbial cells.

Considerable potassium becomes added to soils in the form of
various organic and inorganic compounds. Stable manures, green

MINERAL TRANSFORMATION AND SOIL FORMATION 179

manures, plant stubble, and microbial cells all contain potassium.
When these organic substances are decomposed by microorganisms
in the soil, the potassium is liberated in forms available to higher
plants. A small part of this potassium may be reassimilated by
the microbes which bring about the decomposition processes, thus
part of the potassium may be temporarily removed from circula-
tion. The ash of bacteria and fungi usually contains between
5 to 40 per cent of potassium calculated as K2O.

The inorganic forms of potassium added to the soil as fertilizers
are generally soluble, but the minerals in the soil which contain
potassium are quite insoluble. Solution of potassium from these
minerals is accelerated by interaction with various acids produced
by microorganisms, as shown in the case of orthoclase :

Al203-K20-6Si02 + 8HNO3

= 2A1(N03)3 + 2KNO3 + 6Si02 + 4H2O

The products of both nitrification and sulfur oxidation, as well
as the carbonic acid produced by the microbial population in gen-
eral, aid in such solution of potassium.

Importance of Mineral Transformation by "Microbes in
Soil Formation. — The various inorganic constituents of the sur-
face of the earth are subject to a number of changes as a result of
the activities of the numerous soil-inhabiting microbes, especially
in the presence of organic substances. These changes cause con-
tinuous modifications in the nature of the physical and chemical
composition of the surface of the earth, which for convenience is
divided into layers, called the surface soil and subsoil, or horizons
A, B, and C.

There is evidence which tempts speculation regarding the role
played by microorganisms in the development of this soil layer
from the rocks, which were formed subsequent to the cooling of the
earth's surface. The presence of carbon dioxide in the atmos-
phere, the formation of small amounts of ammonia and nitrous
acid by electrical discharges, the presence of sulfur and of various
minerals containing phosphorus, potassium, calcium, magnesium,
iron and other necessary elements in the earth's crust, was sufficient
to allow the growth of autotrophic bacteria. These were capable
of obtaining their required energy for growth from the ammonia,
nitrous acid, and sulfur, and could satisfy their requirements for
carbon from the carbon dioxide of the atmosphere. As a result of

180 TRANSFORMATION OF MINERAL SUBSTANCES

the activities of these primitive organisms, considerable amounts
of organic matter might have been formed. The acids, especially
nitrous, nitric, and sulfuric, would cause dissolution of the rocks
and aid in the weathering processes. Some of the minerals thus
brought into solution might have been appropriated by the organ-
isms for the synthesis of their cell substance. The rest of the min-
erals might be washed away by streams, or accumulate, giving rise
to the beginning of the inorganic part of the disintegrated surface
layer of the earth. The two processes of dissolution of rock con-
stituents and synthesis of organic matter were the beginnings of
the origin of the soil.

To these reactions might be added the activities of the nitrogen-
fixing bacteria, both alone and in symbiosis with algae, which could
have contributed to increases in the supply of fixed nitrogen on the
surface of the earth. Algae themselves are known to have a corrod-
ing effect upon stones, and, in association with various bacteria, the
extraction of mineral substances from rocks is further increased.

The general phenomena considered in these pages should quite
definitely indicate that microbes have played parts of major
importance in rock disintegration and in soil formation. Although
the climatic factors exert regulating influences on the decomposi-
tion processes brought about in the soil, an inquiry into the
activities of microbes reveals a great diversity of action and the
indispensable nature of their roles in soil formation. In some
cases they assist the purely chemical, physical, and mechanical
transformations; in some cases they take an active part in the
transformation processes; and in other cases they play the domi-
nant role, as in the case of formation of forest soils and peat soils.

LITERATURE

1. Bavendamm, W. Die farblosen und roten Schwefelbakterien. Gustav

Fischer. Jena, 1924.

2. Buchanan, R. E., and Fulmer, E. I. Physiology and biochemistry of

bacteria. Vol. 2, Chapter 11; Vol. 3, Chapter 17. The WilUams &
Wilkins Co. Baltimore, 1930.

3. Cholodny, N. Die Eisenbakterien. Gustav Fischer. Jena, 1926.

4. Harder, E. C. Iron-depositing bacteria and their geologic relations.

U. S. Geol. Survey, Professional Paper 113. Washington, 1919.

5. Stoklasa, .T. Biochemischer Kreislauf des Phosphat-ions im Boden.

Gustav Fischer. Jena, 1911.

6. Waksman, S. a. Principles of soil microbiology. Chapters 7, 23, 25.

The Williams & Wilkins Co. Baltimore, 1927.

CHAPTER VIII

INTERRELATIONSHIPS BETWEEN HIGHER PLANTS
AND SOIL MICROORGANISMS

Interdependence of Higher Plants and Microbes. — To
casual observation, the development of higher plants may appear
to be an isolated phenomenon. Each plant seems to be quite
capable of developing independently of other living things, trans-
forming sunlight into available food and drawing from the soil its
requirements of mineral nutrients. However, although one cannot
accurately estimate the absolute extent to which the activities
of other forms of life are concerned with this apparently isolated
development of the higher plants, careful observation of the con-
ditions about plant roots indicates that there is a striking depend-
ence of higher plants upon microorganisms. This is particularly
the case in the absorption by plants of mineral nutrients from the
soil, and in some cases also in the absorption of organic substances
from the soil. The carbon dioxide of the atmosphere, upon which
the plant depends for the synthesis of its body substance, also
results from the activities of microorganisms. These, however,
depend upon the plant and its residues for their energy sources
and other nutrients.

Neither higher plants nor microorganisms can develop long in
nature in the absence of the other without showing certain definite
abnormalities. Consequently, the fact that plant growth con-
tinues in soil and that microorganisms are increasingly active in
this habitat are suggestive of the associative development of roots
and microorganisms in soil.

Some of these effects are only indirect, while others are asso-
ciated with a very intimate development of the plant and the
microbe, even to the actual penetration of the plant by the microbe.
In fact, it is difficult to determine what activities of microorganisms
in soils do not exert certain effects on plant growth; it is equally

181

182 HIGHER PLANTS AND SOIL MICROORGANISMS

difficult to differentiate between those activities of plant growth
which do or do not affect microbial development, directly or indi-
rectly.

The plant withdraws from the soil through its root system a
considerable amount of substances, principally inorganic; it con-
sumes the supply of carbon dioxide which is continually evolving
from the soil; it renders the soil more porous by the penetration
of its roots; it exerts a solvent effect by the excretion of carbon

£2

Beans

Cucumbers

Tomatoes,

Vegetative

Growth

0.02 0.04 0.06 0.08

Per Cent Carbon Dioxide in the Atmosphere

0.1

Fig. 69. — Influence of concentration of carbon dioxide in the atmosphere on
growth of plants (after Lundegardh).

dioxide; eventually the plant itself returns to the soil and under-
goes disintegration through the agency of the microorganisms.
The activities associated with the complex transformations of
organic and inorganic substances in soils are largely regulated by
microbial development. While practically all organic compounds
disappear under favorable conditions in soils, the course and speed
of their transformation, the agents involved in the change, and the
immediate and ultimate results on plant growth vary greatly.
There may be an immediate or delayed benefit to plant growth
determined by the nitrogen content of the substances concerned.

INTERDEPENDENCE OF HIGHER PLANTS AND MICROBES 183

Providing that the nitrogen content of the material under consid-
eration is sufficiently great, there may be a rapid transformation
of the nitrogen to forms available for plant growth.

Non-symbiotic nitrogen fixation may, under certain conditions,
be sufficient to produce appreciable effects upon plant development.
Microbial disintegration of plant residues and organic fertilizers

Interchange with the Free Atmosphere

Movements of Carbon Dioxide about Growing Plants

Soil Surface

Soil Surface

Carbon Dioxide of the Soil of Microbial Origin

Fig. 70. — Diagrammatic representation of the movements of carbon dioxide
between soil and atmosphere about plants (after Lundegardh).

may exert beneficial effects on the development of higher plants,
not only through the roots but also through the aerial parts of the
plant; this effect is associated with the increase in the concentra-
tion of carbon dioxide in the zone of leaf absorption. During the
warm seasons and in periods of bright sunlight the absorption of
carbon dioxide appears to be more rapid than its supply from
the soil. This is clearly apparent from Fig. 69. Where fight

184

HIGHER PLANTS AND SOIL MICROORGANISMS

intensity is low, little or no increase in plant growth follows an
increase in the carbon dioxide content; the assimilation processes
of plants under such conditions are limited by the low light inten-
sity. Any acceleration of decomposition processes by soil organ-
isms tends to offset deficiencies of carbon dioxide in the atmos-
phere. Generally, however, the content of carbon dioxide is
below the level for maximum plant development in brilliant sun-
light. The movements of the gas between soil, atmosphere, and
plant are shown in Fig. 70.

Evolution of Carbon Dioxide. — The carbon dioxide evolved
from soils is almost entirely of biological origin, although some may
arise from soil carbonates. The origin of this gas is divided
between the activities of plant roots and soil microorganisms.
Whether roots or microorganisms produce most of the gas is deter-
mined by the type of plant and soil conditions. Generally the
roots of higher plants produce much less carbon dioxide than the
microbes. In very sandy soils deficient in organic matter, the
roots may give rise to much the greater amount of carbon dioxide,
as shown from certain results obtained by Lundegardh:

Respiration of sandy soil and plant roots . 0 . 45 mgm. of carbon dioxide per hour

Respiration of sandy soil 0.14 mgm. of carbon dioxide per hour

Respiration of plant roots 0.31 mgm. of carbon dioxide per hour

Per cent of the carbon dioxide liberated

from i)lant roots 68 . 9

Under most soil conditions, however, plants produce a smaller
portion of the total carbon dioxide which comes from the soil.

TABLE 44
Carbon Dioxide Produced per Square Meter per Hour in Soil Sup-
porting Plant Growth and in Bare Soil (prom Lundegardh)

Date

Bare soil,
gm.

Soil growing
oats, gm.

Increase due

to plant

development,

gm.

Per cent of
the gas due to
plant devel-
opment

June 29 . . .
July 24...
July 27 . . .
August 10 .
August 14 ,

0.220

0 . 273
0.250

0.216
0.268

0 . 399

0.053
0.034
0.131

19.4
13.6
32.8

EVOLUTION OF CARBON DIOXIDE 185

According to the results given in Table 44, it seems likely that as
much as one-third of the carbon dioxide may originate from roots
at certain stages of plant development. The gas which arises
from the plant roots may come from two sources: (1) from actual
elimination of the gas from the root cells, as a product of cell
respiration; (2) from microbial decomposition of organic exuda-
tion products of the roots. Which of these two sources may sup-
ply the greatest amounts of carbon dioxide is determined by the
age of the plant and its environmental conditions. In general,
the larger portion of the gas comes from root respiration, but at
times as much as 45 per cent of the gas may be the product of
microbial activities, as shown by Lundegardh:

Root respiration in unsterilized sand 5.57 mgm. per hour

Root respiration in sterilized sand 3.05 mgm. per hour

Difference due to microorganisms 2.52 mgm. per hour

This difference due to microorganisms was equal to 45 per cent
of the total amount produced as a result of the plant growth.
Consequently, although gas arising from roots as a product of plant
respiration may be considerable, the amounts proceeding from
microorganisms acting upon root materials and other soil sub-
stances are far greater.

It may be assumed that 30 mgm. of carbon dioxide are evolved
in 24 hours per kilogram of soil to a depth of 16 inches. Assuming
that there are 2,024,000 kgm. of soil per acre to a depth of 16
inches, there would be evolved 60.72 kgm. of carbon dioxide per
day. Since more of the gas is produced during the warm than the
cold months, it may be expected that most of the gas would be
evolved during 200 days of the year in the temperate zone. Thus
there would be produced about 12,144 kgm. of carbon dioxide
from each acre of soil during the year.

It has also been determined that the daily evolution of carbon
dioxide by a cereal plant is about 30 mgm. Assuming a growth
period of 100 days and that there are 810,000 plants per acre, there
would be about 2,430 kgm. of carbon dioxide produced by plant
roots. From these calculations it appears that plants may be an
important source of carbon dioxide which is formed within the
soil. During the active growing period there may be times when
more of the gas comes from the roots than from the microbial
activities in certain regions of the soil, but over the entire year,

186

HIGHER PLANTS AND SOIL MICROORGANISMS

considering the soil both about roots and at a distance from
roots, the soil microorganisms produce a much larger amount of
carbon dioxide.

The development of plants not only increases the production
of carbon dioxide from soils by contributing some gas from root
respiration, but also favors microbial processes to such an extent
that the organic matter already in the soil is more rapidly decom-
posed. This is shown in Table 45.

TABLE 45

The Influence of Plant Development upon Formation of Carbon

Dioxide from the Organic Matter Contained in the Soil Previous

TO Planting (from Neller)

Plant

Soybeans. .
Soybeans. .
Soybeans. .
Soybeans. .
Buckwheat
Field peas .

Wheat

Barley ....

Carbon Dioxide from the Soil (Mgm.)

Planted

2,093.4
2,128.2
4,795.2
2,403.6
953.1
751.6
2,403.6
3,274.9

Unplanted
soil

1,737.0
1,281.8
2,320.7
1,196.8
440.1
440.1
1,196.8
2,532.4

Increase

due to

planting

356.4

846.4
2,474 . 5
1,302.1

513.0

311.5
1,206.8

742.5

Per cent

increase

due to

planting

20.5

66.0
106.6
108.8
116.6

70.8
100.8

29.3

It is further apparent from Fig. 71 that plants increase the
evolution of carbon dioxide in the soil, but the course of forma-
tion of the gas appears to be distinct for each plant and related
to differences in characteristics of growth of the plants. SHght
effects appear in the early stages of growth, and abundant forma-
tion of carbon dioxide occurs when the plants reach stages of
extensive vegetative development. During degeneration the gas
is produced in less abundance, and subsequent to death there is
little excess over that produced where plants are not developing.
Since biennials have a much longer growing period than the
annuals, their influences upon the soil are much more prolonged.

EVOLUTION OF CARBON DIOXIDE

187

The formation of carbon dioxide about plant roots, whatever its
origin, is particularly important in plant development in that its
solvent action is one of the principal agencies responsible for the
solution of relatively insoluble soil minerals containing phos-
phorus, potassium, calcium, and magnesium. The development of

30 r-

—> ^ i-i-H -^rtroro^ —• ^ CO m ^

Period of Growth (Days)

Fig. 71. — Influence of plant development upon the evolution from the soil of

carbon dioxide of microbial origin; V — height of vegetative development,

B — blooming, Dg — degeneration, D — death (after Starkey).

organisms on the roots greatly increases this corrosive action, and
is a very important factor in determining the feeding power of
plants. There appear to be certain factors, not correlated with
either the extent of the root system or the amount of carbon diox-
ide produced, which determine the ability of plants to absorb
certain proportions of the various nutrients, at least where the
plants are dependent upon relatively soluble sources of inorganic

188 HIGHER PLANTS AND SOIL MICROORGANISMS

nutrients. In addition to that, the production of relatively large
amounts of carbon dioxide in soil is very important in determining
the ability of plants to absorb nutrients from relatively insoluble
compounds. Organic acids are eliminated by roots in only
limited amounts under conditions favorable to plant growth
and, consequently, the solvent action is almost entirely exerted
by the carbon dioxide which is evolved.

The extent of the increase in solvent effects due to micro-

FiG. 72. — Etching of marble as a result of root development. No microbes

were present on the roots which etched the marble on the left. The greater

etching of the slab on the right was due to acids produced by microorganisms

developing on the roots (from Fred and Haas).

organisms is suggested by Fig. 72. Plants were grown both under
sterile conditions and in the presence of microorganisms, in such a
manner that the roots passed over marble slabs. Where the roots
were free of microbes there was much less etching of the marble
than where they were present. These effects are largely asso-
ciated with differences in amounts of carbonic acid produced in the
absence and presence of microbes.

The factors responsible for the various changes in microbial

EVOLUTION OF CARBON DIOXIDE

189

activity about plant roots are numerous. There are physical
effects produced by the addition of organic matter from plant
roots, by the liberation of carbon dioxide from the plant roots and
microorganisms, by root penetration opening passages in the soil,
by absorption of inorganic nutrients and large quantities of water.
The dominating influence of the plants on soil organisms is most
commonly exerted by the addition of organic materials in the form
of root excretions and degenerated root tissues. These are sub-
sequently used by the soil microbes as food. Such effects are most
apparent in the zone of root development, frequently referred to
as the rhizosphere, since it is here that the root residues become
introduced. Table 46 indicates that the abundance of bacteria
in soils supporting different plants is different, and that, in general,
the abundance of bacteria about the roots is closely correlated
with the activity of the microbes, as indicated by the formation of
carbon dioxide.

TABLE 46

Influence of Plant Growth upon the Number of Bacteria in Soil and
Production of CO2 (from Stoklasa)

Plant

Millions of

bacteria per

gm. soil

Mgm. CO 2 per

kgm. soil in

24 hours

Alfalfa

120
98
78
63
62
51
49
46
45
42

86 8

Red clover

82 4

Beets

74 3

Buckwheat

78 7

Corn

70 6

Wheat

61 3

Barlev

69 4

Potato

58 5

Oats

59 0

Rve

68 2

Not all elements of the soil population are affected alike by the
development of plants. The influences of any one plant on the soil
population at different stages of plant growth are characteristic,
as shown by Fig. 71. Different plants affect the microbes in dis-
tinct ways. Further, the various microbes in the soil respond

190

HIGHER PLANTS AND SOIL MICROORGANISMS

differently to plant growth. As shown by Fig. 73, the general
bacterial population is affected to a much greater extent that either
fungi or actinomyces; however, some species of bacteria develop
to a greater extent than others in proximity to root systems of
plants. This appears from the fact that the organisms closely
related to the Radiobacter group increase many times more in
response to plant development than do the microbes of the general
bacterial population.

It is on the immediate root surfaces and within the superficial
tissues of the roots that the greatest influences of plants are
exerted, and there are progressively decreasing effects the greater
the distance from the region of soil penetrated by the roots (see
Table 47).

TABLE 47

Influence of Plant Development upon the Abundance of Microbes

AND Their Activity in Soils at Different Distances from the Plant

Roots* (from Starkey)

Plant

Region of sampling

Abundance

of

bacteria,

millions

Abundance

of

actinomyces,

millions

Abundance

of

fungi,

thousands

Formation

of

carbon

dioxide,

mgm.

Bean
Bean
Bean

15 inches from main roots. . .
9 inches from main roots. . .
3 inches from main roots. . .

18.6
32,8
36.2
55.4
199.4

18.6
27.0
33 . 4
57.4
427.4

22.8
26.2
44.8
93.2
653 . 4

7.6

10.0

8.0

6.2

12.6

10.0
11.4
10.4
6.8
10.6

8.4
11.8

8.8
10.2

8.6

24.6
21.6
20.0
19.2
55.2

25.8
25.0
25.8
30.0
156.0

29.6
23.2
29.6
49.6

278.0

9.7
12.0
12.0
15.1

Bean

Beet
Beet
Beet
Beet

Superficial layer of the roots.

15 inches from main root. . . .
9 inches from main root. . . .
3 inches from main root. . . .

11.2
13.6
14.9
18.2

Beet

Corn
Corn
Corn

Superficial layer of the roots.

15 inches from main root". . . .
9 inches from main root. . . .
3 inches from main root ....

10.3
15.2
15.2
25.0

Corn

Superficial layer of the roots .

* Age of plants — 113 days.

Since all plants require some oxygen about their roots and since
microbial activities and root excretions tend to lower the oxygen
concentration and raise the concentration of carbon dioxide, there

EVOLUTION OF CARBON DIOXIDE

191

may be times when microbial activity becomes distinctly injurious
by creating a condition of oxygen deficiency in the root environ-

Comparative Figures (Fallow -100)

OS

o f^
'~^ ^.

ST P

Ig"
„ 3

c^ Co

<L o
o fo

"O o

B -
§ S
"^ 3

O fB
O (jq

2.1
— ■a
o »

B. o

i °

^ 2.

(R B

crt- O
O (ti

0 ►o

1 p*

K> CO

g 3

Potatoes

1

1

1

1

1

1

1

Beans

1

Corn

1

Sweet Clover

■■

ST '3'
0 SL

1

3
3_

0
0
0

S'
0

0

C

■a

9.
5d

Average

1

Oats

Table Beets

^^H^^HI

Mangel Beets
Rape

Potatoes

Beans

Corn

H

■

w 0

" 0

H

J^^

s-s'

^^^*

0 0

H

^^^_

i^H

■

^Hi

Average

Oats

Table Beets

Mangel Beets

Rape

Potatoes

—

^^

1

■

■

Beans

H

1

Corn

1

31

0

I

Sweet Clover
Average

■
Z3

0

1

m

(5"

Oats 1

0

Table Beets

Mangel Beets

Rape

1

0^
m

0

S

CL

<
CL

C

Potatoes

B

3

Beans

^H

O)

(/>

Corn

■

K

9

5r

Sweet Clover |

3
0

0

3

Average

HI

(B

Oats

■1

s;

Table Beets

■

Mangel Beets

■i

Rape

■

1

1

1

1

1

.. 1

1

ment. A moderate reducing condition about roots may not be
injurious to plants. In fact, it is conceivable that such a condi-

102 HIGHER PLANTS AND SOIL MICROORGANISMS

tion may be favorable to certain phases of plant nutrition. Iron
is slightly soluble in a strictly aerobic environment at reactions
close to neutrality, but becomes much more soluble as it is reduced.
Its reduction about plant roots may be important in supplying
available iron to the plants in most soils.

Influence of Root Excretions. — The roots of higher plants
are organs not only of absorption but also of excretion. The sub-
stances that are excreted and the amounts eliminated at any one
period of growth vary with the kind of plant, its stage of develop-
ment, and soil conditions. Although it is apparent that higher
plants must absorb more of the inorganic substances than they
excrete, still at certain stages of growth there is appreciable
elimination of various substances, both organic and inorganic,
nitrogenous and non-nitrogenous. In studies carried out with
plants growing in sterile culture solutions, it was noted that from
1.5 to 3.0 per cent as much organic matter was present in solution
(excreted by the plant) as was contained in the developed plant.
About one-fifth of this organic matter in solution was nitrogenous
in nature. A certain amount of the insoluble organic matter
which was eliminated from the plants consisted of cellular material
sloughed off from the roots.

The amounts of substances entering the soil solution during
growth of certain plants may be sufficiently abundant to modify
greatly the development of other plants which are growing in close
proximity. This is of considerable importance in the growth of
mixed stands of legumes and non-legumes. In such mixtures the
non-legumes may profit from the nitrogen which is fixed through
the agency of nitrogen-fixing bacteria in the roots of legumes.
Certain nitrogenous substances enter the soil solution from the
roots of the legumes, and probably through the action of soil
microbes become modified and are then absorbed by the non-
legumes. Such effects are shown in Fig. 74. The pots were pre-
pared in the following manner. Two large pots were filled with
sand. In the center of one large pot a smaller unglazed pot was
placed. In the other pot a glazed, impervious pot was introduced.
Both small pots contained the same kind of sand as the larger pots.
The unglazed pot permitted diffusion of substances back and forth
between the contents of the small pot and the larger pot, while the
glazed pot prevented such movement. All nutrients required for
plant development with the exception of nitrogen were added

INFLUENCE OF ROOT EXCRETIONS

193

to the sand. A non-legume (oats) was planted in the small pots
and a legume (peas) in the outer pots. As growth progressed, the
oats in the unglazed pot developed far better than the oats in the
glazed pot, showing effects of nitrogen fertiUzation. It is appa-
rent that some nitrogenous substances became available to the oat
plants as a result of development of the peas.

Unfavorable conditions for root development enhance the

Fig. 74. — Influence of a legume (peas) on growth of a non-legume (oats).

Oats in inner pot, peas in the outer. Porous inner pot on left; glazed inner

pot on right (from Lipman).

activity of microorganisms about the roots, on account of death
of the root parts and attack by soil saprophytes. Under condi-
tions of deficient aeration, certain substances may accumulate
which become toxic to plants and microorganisms; they generally
disappear rapidly under conditions favoring thorough aeration
of soils. The conditions brought about by continued cultivation
of cereals and the introduction of appreciable amounts of organic

194 HIGHER PLANTS AND SOIL MICROORGANISMS

matter relatively low in nitrogen appear to be more closely asso-
ciated with nitrogen starvation than toxic excretions by plants.

Among the excretions of roots, phosphatides and certain muci-
laginous substances appear to be of particular importance in certain
cases. It has been noted that such compounds act as stimulants
to the growth of certain microorganisms, increasing growth to a
greater degree than would seem to be likely from the energy which
the substances might furnish in the concentrations in which they
occurred. Such substances may be of importance in stimulating
growth of microorganisms in the rhizosphere, and may even lead to
a penetration of the root cells by these organisms.

That the numerous root excretions enhance development of
microorganisms is quite well defined, but the nature of the organ-
isms affected is unknown except in a few instances such as mycor-
rhiza fungi. With the common cereals and root crops, there
appears to be as varied an assortment about the roots as in the
soil distant from the roots; this assortment includes bacteria,
actinomyces and filamentous fungi of a great variety. Whether
any transformations of the organisms in the rhizophere may be of
particular importance beyond the ways mentioned above is not
known. The limitation of our present knowledge leads to the
conclusion that the general abundance of the population is greater
here and the activity of the cells is greater, as indicated by forma-
tion of carbon dioxide.

Conditions Favoring Nitrogen Fixation. — The following
conditions favor fixation of nitrogen: the presence of available
non-nitrogenous organic matter and necessary inorganic nutrients,
a favorable reaction, and the presence of nitrogen-fixing organisms.
These conditions appear to exist about the roots of many develop-
ing plants, and it is quite likely that the soil becomes enriched in
nitrogen, because of non-symbiotic development of nitrogen-fixing
organisms about plant roots. Although such nitrogen might not
be available to the crop which was responsible for its fixation,
nevertheless it would become available as decomposition of these
fixed nitrogen compounds progressed.

Absorption of Organic Compounds by Plants. — Not only
inorganic substances but also many organic compounds may be
absorbed by plant roots. Some may have pronounced beneficial
effects upon root development. The organic substances which
may be utilized by green plants include carbohydrates, alcohols,

ASSOCIATIVE GROWTH OF GREEN PLANTS AND MICROBES 195

organic acids, peptones, amino acids, and purine bases. In fact,
certain plants, as many of the Orchidaceae, cannot initiate growth
from the seeds without having access to certain assimilable organic
substances furnished either artificially or by means of their sym-
biotic fungus associates.

Long periods of field experiments argue very favorably for the
use of a certain amount of organic fertilizers, or at least of organic
residues in the rotation. While in the first few years crop growth
may be fully as abundant with inorganic fertilizers alone, plants
respond more favorably to organic manures over long periods of
time. It is recognized that organic substances greatly improve
the physical condition of the soil, but it is likely that certain other
properties of the organic materials also contribute to the bene-
ficial effects upon plant growth, and that these effects are not
apparent with the use of inorganic substances alone. It has been
suggested, for example, that animal manures when used as fer-
tilizers result in the production of seeds which are much superior
to those of plants grown on soils either receiving no fertilization
at all or merely mineral fertilizers. Such seeds produced from
soils receiving animal manures were found to grow plants bearing
a much greater abundance of seeds. These results, of course, need
further confirmation and more extensive study.

Under certain conditions, organic substances elaborated by
microorganisms, formerly referred to as auximones, appear to have
a stimulating effect on plant growth; the hypothesis has been
brought forth that certain vitamines, similar to those which are so
prevalent in higher plants, are formed by microorganisms and
absorbed by the plants. Because of the fact that information
on this subject is extremely limited this hypothesis is little more
than suggestive. Organic substances other than those contained
within plant seeds are apparently not essential to plant growth,
but they may prove beneficial as buffering agents and may in some
case even act directly as plant nutrients.

In general, microorganisms in the plant rhizosphere may exert
effects similar to those produced by microbes at a distance from
the roots, but their effects may become more quickly apparent by
reason of the fact that the action occurs nearer to the organ
affected.

Associative Growth of Green Plants and Microbes. —
There are certain much more intimate associations between micro-

196 HIGHER PLANTS AND SOIL MICROORGANISMS

organisms and higher plants than those already mentioned. In
such cases, as with the hchens where there is associative develop-
ment of algae and certain higher fungi, definite dual plant forma-
tions develop which appear Hke single plants. The fungus derives
organic nutrition from the material synthesized by the alga, while
the latter makes use of inorganic nutrients absorbed by the fungus.
Such an association permits the lichens to develop under condi-
tions almost prohibitive to growth of either of the associates
alone.

Under conditions where some of the single-celled algae grow in
association with nitrogen-fixing bacteria, the latter develop at the
expense of some of the organic matter formed by the algae and
fix nitrogen during their growth.

Mycorrhiza. — Various symbiotic and parasitic associations
are known with the higher plants. Penetration by certain micro-
organisms of roots of most plants, including annuals and perennials,
woody plants, and herbaceous plants, is probably the rule rather
than the exception. Roots of all plants growing in soil are liable
to invasion by any fungal hyphae which can effectively penetrate
them. Some of the fungi attack the roots and destroy the vege-
tative tissue. The type of development depends upon the nature
of the parasite, resistance of the plant, and conditions favorable
or unfavorable to the parasite and host.

In some cases, however, the host plant is able to overcome the
attack by the fungus and may even benefit from it. An associa-
tion may thus be established, which is referred to as mycorrhiza,
derived from the words viyces = fungus, and rhiza = root. This
formation is a very widespread and common phenomenon among
plants. It may prove to be of considerable importance in plant
growth, affecting it favorably or unfavorably. The extent of the
penetration of the fungus and degree of its effect upon the growth
of the plant vary with the soil conditions, which determine the
plant vigor and degree of its resistance. Under favorable cultural
conditions, there may be no apparent injury to the plant, but under
adverse conditions of moisture, temperature, or nutrient supply,
the fungus may become a parasite upon the plant and cause
definite injury.

The nature of mycorrhiza produced on various plants, as well
as their role in the nutrition of the plants, differs with different
plants. In some cases, the fungus does not penetrate into the

MYCORRHIZA

197

cells of the roots but forms a mass of mycelial growth around the
root tips, entering the tissues largely between the cells; this type
of mycorrhiza is referred to as ectotrophic (Fig. 75). It is very
common among forest trees, especially evergreens. Other fungi
invade the cells within the roots, producing endotrophic mycorrhiza,
as in the case of the orchids (Fig. 76) and heather. Some fungi
produce slight penetration into the epidermis, and others invade
the cortical cells. Many of the fungi which develop upon roots
of woody trees belong to the Basidiomycetes, or fleshy fungi;
those that grow upon orchids are species of Rhizoctonia; and those

Fig. 75. — Ectotrophic mycorrhiza developing on roots of Pinus sylvestris

(from MeHn).

on Ericaceous plants (heaths) are species of Phoma. In many
cases the specific fungi capable of bringing about mycorrhiza
formations are accompanied by other fungi in their invasion.

The root fungi reach a high state of development in the case of
some of the orchids. These plants have very minute seeds con-
taining small amounts of reserve food material. The develop-
ment of the mycorrhiza appears to be indispensable to the growth
of orchids in nature. The fungi seem to increase the concentra-
tion of soluble organic substances about the embryo and also act
as root hairs for the young plants, furnishing organic and inorganic
nutrients during the early stages of growth. With the advance
in plant development, the fungi do not appear to be as essential.
Orchids have been cultivated from the seeds in the absence of the

198

HIGHER PLANTS AND SOIL MICROORGANISMS

fungus, or endophyte, when high concentrations of sugar were
furnished in the early stages of their development. Subsequent
to growth of the roots, the plants'appear to be able to develop

Fig. 76. — Median longitudinal section of orchid seedling one month after

infection by the mycorrhiza fungus; twisted fungal hyphae appear in many of

the cells (from Bernard).

as other green plants, obtaining their energy from the sunlight and
mineral substances from the soil. With certain non-chlorophyllous
plants, as a few species of orchids and Monotropa, or the Indian
pipe, mycorrhiza fungi play an even more indispensable role.

MYCORRHIZA 199

Here the fungi furnish organic and inorganic nutrients throughout
the growth of the higher plants, which act as parasites upon the
fungi. The interrelationship is so intimate between the roots
of the plant and the fungus that the latter also appears to benefit
from the association; it is not known whether this is similar to the
phenomenon found in chlorophyllous plants, where the fungus
obtains carbohydrates and possibly other food materials from the
plant cells which it penetrates.

In certain of the heaths (Ericaceae) and woody trees (pine,
spruce, fir, larch, beech, maple, oak, hazel, and chestnut), the
mycorrhiza develop into a somewhat less intimate association.
In many cases, the roots are penetrated less deeply, and the fungi
make a more abundant development on the exterior of the roots.
Although mycorrhiza formation may not be indispensable in
any stage of the development of such plants, the fungus associate
undoubtedly favors the growth of its host under certain soil condi-
tions, such as in organic acid soils. Under these conditions, the
fungus breaks down the complex organic substances, supplying
the roots of the plant with the products thus made soluble. The
association is much more pronounced with these forms in the
so-called " raw-humus " and is not as extensive or active in the
" mull " or less acid forest soils. Under these latter conditions,
absorption of plant nutrients may not be so specialized a process
since bacterial decomposition is more active and nitrate is formed
in abundance.

In certain species of the heaths, the fungus invades the host
plant completely through the entire root system, stems, leaves, and
even to the floral parts, remaining absent only from the endo-
sperm and embryo. The seeds carry an abundant fungus growth
which acts as an inoculant; the fungus invades the new plant as
the seed germinates and develops. The fungi grow usually very
poorly in the absence of their vascular hosts; they find food mate-
rials in the cells of these higher plants which greatly favor their
development. In turn, they act as agents for the decomposition
of some of the substances contained in the organic portion of the
leafy detritus of the soil and render these available to the roots of
the host. The fungus may in this way furnish organic and inor-
ganic substances to the higher plant which would not be otherwise
available. In a few cases where the mycorrhiza fungus is a species
of Phoma there is some suggestion that the fungus may fix nitrogen

200 HIGHER PLANTS AND SOIL MICROORGANISMS

and favor plant development much the same as the legume sym-
biont does. With most fungi, however, fixation of nitrogen does
not occur.

The effects exerted by the mycorrhiza differ with different
plants. In some cases there are definite parasitic effects, and the
invading fungus injures the cells which it penetrates without bene-
fiting the host in any way. In other plants the fungus is undoubt-
edly not parasitic, and produces no injury to the living cells
although it may penetrate these cells. The fungus causes a dis-
appearance of starch and other organic substances from the root
cells but does not destroy them ; on the contrary, the plant is much
favored by its development. The fungus mycehum may act as
root hairs absorbing water, inorganic nutrients, and organic sub-
stances, which become available either directly to the plant from
the penetrating parts of the fungus or subsequent to the digestion
of the mycelium, which takes place within the penetrated cells.
These effects, besides possible nitrogen fixation in certain few cases
and the role of decomposing soil organic matter, may explain some
of the more important possible benefits which the higher plants
derive from the fungus association. Undoubtedly in most cases
the mycorrhiza may be considered as symbiotic phenomena in
which both organisms, the fungus and the vascular host, derive
decided benefit from the association.

Nodule Formation on Leguminous Plants. — By far the
best known instance of symbiosis between vascular plants and
microscopic organisms is the association between legumes and a
bacterium called Bacillus radicicola. This type of symbiosis is
apparent in most legumes and some few known non-leguminous
plants. Furthermore, the legume invader is of widespread occur-
rence in soils wherever legumes develop naturally. Domestic
cultivation of legumes frequently requires the inoculation of the
organism in order to insure its presence. By reason of this spe-
cific symbiotic growth, the legumes are particularly favorably
prepared to develop where many other plants could not grow;
they have, therefore, found wide use as cover crops to increase the
fertihty of soils, particularly with reference to nitrogen. This
relationship has been discussed at considerable length in preceding
pages.

Bacteriorrhiza. — Bacterial invasion similar in nature to
fungus invasion by mycorrhiza has been observed in some instances

SUMMARY 201

with cultivated annuals. These developments, called "bac-
teriorrhiza," have been noted in the epidermis, cortex, or even the
bast, both in the interior of the cells and in the intercellular spaces.
Whether they are of general or exceptional occurrence, whether
parasitic to the plants or favorable to their development, is still
undetermined.

Just as different fungi may penetrate plant roots to a greater
or less extent, causing definite injury or exerting favorable effects
on plant development, so bacteria are also not excluded entirely
from entrance into the roots. It has been shown in the preceding
pages that bacteria make very extensive development on the
immediate root surfaces. Some cells of bacteria penetrate the
tissues to greater depths, acting merely as saprophytes upon the
degenerating or weak tissues. Some pathogenic organisms pro-
duce much more extensive penetration and cause pronounced
injurious effects.

Summary. — Microorganisms may thus be considered to affect
root systems of higher plants in numerous ways. At some dis-
tance from the roots, the microorganisms may either (1) act as
general agents of decomposition of organic constituents of the soil
leading to the formation of water, carbon dioxide, ammonia, sul-
fates, and phosphates; (2) act as transformers of such mineral
constituents of the soil as ammonia and sulfur, oxidizing them to
nitrate and sulfate, respectively; (3) act as agents of assimilation
of nutrients which are thus removed at least temporarily from the
zone of absorption of plants; (4) act as agents lowering the oxygen
concentration in the soil system and thus create conditions unfav-
orable to root growth; (5) produce toxic substances or reduce such
substances as nitrates and sulfates to gaseous nitrogen and sul-
fides, thus rendering them unavailable; (6) act as solvent agents
through the organic and inorganic acids which are produced in
various transformations; (7) act as nitrogen-fixing organisms, the
nitrogen sooner or later becoming available to higher plants.

Close to the root systems many similar effects are exerted, but
the changes take place more rapidly because of the accelerated
activity of microbes in the rhizosphere. These influences may
include: (1) decomposition of organic products of excretion from
the roots; (2) solvent action of the carbon dioxide which arises
both from microbial activity and root excretion; (3) fixation of
nitrogen where the organic root excretions contain little or no

202 HIGHER PLANTS AND SOIL MICROORGANISMS

nitrogen; (4) assimilation by plants of such organic compounds as
amino acids or lower carbohydrates which may be produced by
microorganisms as incompletely decomposed products or syn-
thetic products; (5) associative development in such special cases
as algae and bacteria, or fungi and algae.

There may be actual penetration of the roots in certain
instances. Some invasions of the microbes result injuriously;
such effects are produced by the numerous plant pathogens which
inhabit the soil. The invasion of symbiotic agents, as the legume
associates and certain mycorrhiza fungi, produces very evident
beneficial results.

LITERATURE

L LinsTDEGARDH, H. Def Kreislaiif der Kohlensaure in der Natur. 308 pages.
Gustav Fischer. Jena, 1924.

2. Rayner, M. C. Mycorrhiza. An account of non-pathogenic infection by

fungi in vascular plants and Bryophytes. New Phytologist Reprint,
No. 15. 246 pages. Wheldon & Wesley, Ltd. London, 1927.

3. Starkey, R. L. Some influences of the development of higher plants

upon the microorganisms in the soil. Soil Science, 27, 1929: 319-334;
355-378; 433-444.

4. Waksman, S. a. Principles of soil microbiology. Chapters 11 and 29.

The Williams & Wilkins Co. Baltimore, 1927.

CHAPTER IX
MODIFICATION OF THE SOIL POPULATION

The Soil Population Subject to Alteration. — The total
numbers as well as the relative abundance of the numerous kinds
of microorganisms found in the soil can be modified in various
ways by certain soil treatments. These treatments may be
primarily physical, chemical, or biological. The modifications in
the soil population thus brought about may either favor the
development of certain groups of organisms in preference to
others, or they may bring about the entire eUmination of certain
representative types of the soil population, permanently or tem-
porarily.

The introduction into the soil of microbes which have not been
there previously may be another means of modifying the soil popu-
lation. When these organisms find in the soil a favorable habitat,
they will develop readily; when, however, the soil conditions are
not suited to the requirements of these microbes, they will fail to
become estabhshed, irrespective of inoculation by natural or
artificial means. The mere addition of microorganisms to soils
provides no assurance that they will develop there, or that they
will bring about the specific transformations which are expected
of them, even though they are able to grow. The changes in the
nature of the population brought about by any of the soil treat-
ments are accompanied by corresponding changes in the chemical
soil processes, which directly or indirectly influence the fertility of
the soil, as expressed by the response in the growth of higher
plants.

By reason of the fact that microorganisms do not occur in the
same abundance in all soils and that they are generally favored by
conditions that lead to best plant growth, there exists a close
relationship between the biological activity of soils and soil fer-
tility. Since some of the microbial tests are comparatively

203

204 MODIFICATION OF THE SOIL POPULATION

simple and of short duration, they are frequently preferable to
vegetation experiments with higher plants to test the fertility of
soils.

Influence of Soil Treatment upon the Nature ANt)
Abundance of Microbes. — Each modification of the physical
and chemical condition of the soil will be accompanied by a change
in the biological soil condition, as expressed by the nature and
abundance of microbes in the soil. Some treatments result in
profound alterations of the biological activities which persist for
considerable periods of time; other treatments exert only slight
effects of short duration. Among the important treatments of the
soil which might be considered here are the following :

(1) The addition to the soil of organic matter in the form of
stable manures, green manures, artificial manures, plant
stubble, or other plant and animal residues, as well as the
organic fertilizers of commerce.

(2) Physical modification of the soil by plowing, cultivation,
and similar mechanical processes.

(3) The addition of different inorganic fertilizers or lime
in its various forms — carbonate, oxide, and hydroxide.

(4) Cropping the soil according to various systems.

(5) Partial sterilization of soil by heat or antiseptics.

(6) Air-drying of soil.

Influence of Organic Matter upon the Soil Microbes. —
The addition of organic matter to the soil brings about a number of
changes in the soil population, depending upon the nature of the
organic matter and upon the conditions in the soil. Green
manures bring about a rapid development of various bacteria and
fungi which use the water-soluble substances, proteins, cellulose,
and the various hemicelluloses. These organisms are soon fol-
lowed by protozoa that feed upon the bacteria and by various
larvae and worms that feed upon both the residual organic matter
and upon the fungi and bacteria. Finally, when the whole organic
mass has become largely transformed, other bacteria and actino-
myces begin to develop, attacking the more resistant residual
substances. The entire mass of decomposing organic matter
loses its identity as plant material and is transformed into the
finely divided mass which is referred to as soil organic matter or
humus.

INFLUENCE OF ORGANIC MATTER UPON SOIL MICROBES 205

Frequently the decomposition does not proceed very far, but
stops at an intermediary stage, as in the case of the so-called raw-
humus in certain forest soils. The acid nature of the organic matter
(pH 4.0 and even lower), and the fact that this organic matter is
not appreciably mixed with the inorganic portion of the soil but
remains on its surface, are responsible for its accumulation.
These conditions favor an abundant development of fungi, many
of which belong to the Basidiomycetes (mushroom fungi). The
absence of extensive development of bacteria and actinomyces and
the relative absence of an animal population further favors the
persistence of the organic matter which is known as raw humus.
Under these conditions the essential plant nutrients either remain
in the undecomposed plant residues or become removed from
circulation by the fungi which assimilate these elements and build
them up into their own cell substance. The trees can obtain some
of these nutrients only through the agency of certain fungi (mycor-
rhiza) which grow in association with the roots and serve as root
hairs. Many of these mycorrhiza fungi are able to decompose the
resistant organic matter of the forest soil and make the nutrients
contained therein available to the growth of the forest vegetation.
This association is symbiotic in nature and appears to be of benefit
to both the tree and the fungus (see Chapter VIII).

In certain other forest conditions an entirely different series of
changes takes place. When the fresh plant residues which are
first attacked by the fungus population are further acted upon by
the numerous soil bacteria and invertebrate animals, the so-called
mull type of soil develops. This is considered to be a richer soil,
since the nutrients are liberated from it much more readily by
processes of decomposition. This type of organic residue or humus
is formed only when the soil is well supphed with lime, and when
the organic substances are well mixed with the inorganic particles
of the soil. Under these conditions the organic matter has a
reaction of pH 5.0 to 7.0, and its thorough mixture with the soil is
brought about largely through the agency of invertebrate animals
which find these more nearly neutral soils a much more favorable
habitat for their development than the acid raw-humus soils.
In the latter, the nitrifying bacteria are absent and the nitrogen
does not become changed beyond the form of ammonia, but in the
mull type of soil, conditions are favorable to active nitrification
and the ammonia is rapidly changed to nitrate. One need hardly

206

MODIFICATION OF THE SOIL POPULATION

emphasize the advantage of the conditions existing in such soils as
compared with conditions in raw-humus soils. Cultural treat-
ments and appUcation of fertihzers are seldom practical in forests,
but, where the forest land is cleared for other cropping systems,
more elaborate practices are justified. To bring about the decom-
position of the organic matter in the raw-humus soils, the addition
of CaCOs and cultivation are often recommended.

Influence of Green Manures. — The addition of green
manures to soil brings about a considerable modification in the
soil population. Table 48 shows that organic matter exerts a

TABLE 48

Influence of Organic Materials, With and Without NaNOs, on the

Development of Microorganisms in the Soil (from Waksman

AND Starkey)

Treatment of soil

Number of Organisms 14 Days
After Adding the Organic

Matter

Bacteria and
actinomyces

Untreated

0 . 5 per cent straw

0 . 5 per cent straw + 0 . 025 per cent NaNOs .

0 . 5 per cent alfalfa

0 . 5 per cent alfalfa + 0 . 025 per cent NaNOa

87,300
136,000
233,000
297,000
247,000

8,875,000
34,200,000
35,700,000
74,600,000
73,400,000

marked effect not only upon the bacteria and actinomyces but also
upon the fungi. When fresh, undecomposed plant material is
introduced into the soil, the Mucorales generally develop first,
and are later followed by various species of Penicillium, Asper-
gillus, Fusarium, Trichoderma, and others. The increase in bac-
terial growth is closely followed by a rapid development of numer-
ous protozoa. Some of the members of this diverse population
live on the organic substances originally contained in the plant
material which is added to the soil; some exist on intermediary
products of decomposition, while others feed upon the microbial
inhabitants themselves.

INFLUENCE OF GREEN MANURES

207

The degree of the effects thus exerted is related to a great
variety of factors, among which may be included the soil structure
and its reaction, moisture, temperature, and supply of nutrient
salts. Of major importance also are the composition and the
amount of the plant material which is added to the soil, as shown in
Table 49. The development of microbes upon the organic mate-
rials added to the soils follows a changing course (Fig. 77). The
initial growth of the bacteria and fungi upon the readily available
plant constituents is apparent from the rapid formation of carbon

90 40 300

45 60 75

Time (Days)

Fig. 77. — The influence of organic matter on biological activities in soil.

Changes following the addition of 2 grams of ground dry alfalfa meal to

1 kilogram of soil (after Waksman and Starkey).

dioxide and from the increase in numbers of bacteria and fungi
during the period immediately following the addition of the
organic matter. This period of rapid development is followed by a
stage of reduced speed of decomposition of the unattacked por-
tions of the organic substance and of the incompletely decomposed
products. During this stage, there is a decrease in the abundance
and activities of the microorganisms, but the drop is slower than
the initial rise. The available food material continuously decreases
in amount, and the biological conditions slowly approach the more
stabilized state which existed previous to the treatment of the soil.

208

MODIFICATION OF THE SOIL POPULATION

The time elapsing before this stage is reached is determined by a
variety of factors. In most cases it is an extended period and
may consume many months.

TABLE 49

Influence of Different Amounts of Straw on Development of
Bacteria in Soil — Average Numbers over a Period of 71 Weeks
AFTER Treatment (from Murray)

Amount of straw

Number of bacteria

Amount of straw

Number of bacteria

added to the soil,

(counted on nutrient

added to the soil,

(counted on nutrient

per cent

agar)

per cent

agar)

0.0

1,677,000

0.8

11,467,000

0.1

2,163,000

0.9

15,726,000

0.2

3,399,000

1.0

16,157,000

0.3

4,180,000

2.0

28,242,000

0.4

5,650,000

3.0

60,377,000

0.5

8,953,000

4.0

91,040,000

0.6

9,034,000

5.0

94,623,000

0.7

8,497,000

Green manure, whether it consists of leguminous plants, of
young cereal plants, or others, is comparatively rich in water-
soluble substances (sugars, amino acids, etc.), as shown in Table 18.
Such material serves as a readily available source of energy, and
may be quite completely changed within a period of a few weeks in
the soil. After a series of transformations, the nitrogen of the
green manure is liberated as ammonia, then soon oxidized to
nitrite, and further oxidized to nitrate. The amount of nitrogen
liberated from the decomposition of the green manure depends
largely upon the nature and type of plant. The younger the
plant, the more rapidly it decomposes, and the more rapidly its
nitrogen is liberated in an available form. Consequently, less of it
remains to increase the organic matter of the soil over any appre-
ciable period of time.

The addition of green manure thus brings about a series of
changes in the microbial population of the soil, affecting prac-
tically every group of microorganisms, either directly or indirectly.
Organic acids may be formed in the initial stages of decomposition,
but they do not persist for long under aerobic conditions.

INFLUENCE OF STABLE MANURE 209

Influence of Stable Manure. — The addition of stable
manures to the soil results in modifications of the soil population
in three distinctly different ways: (1) The various constituents of
the manure, namely, the straw, faeces and urine, offer favorable
and readily available sources of energy, nitrogen and minerals
(especially phosphates and potassium salts) to many different
microorganisms. (2) Manure contains large numbers of a variety
of microorganisms that have originated in the digestive system of
the animal; the addition of considerable quantities of manure to
the soil may thus considerably modify the soil population through
actual mass inoculation. (3) The addition of manure leads to
modifications of the physical condition of the soil. The general
results of all these effects is the creation of an environment more
suitable for the development of higher plants.

An understanding of the nature and composition of animal
manures makes possible an explanation of the changes taking
place during their decomposition and suggests an interpretation
of the fertilizing values of the manures in comparison with ordinary
plant residues; the latter are quite different in composition from
the stable manures. This is the result of at least three factors:
(1) in many cases the chemical nature of the food of animals is
different from the organic matter entering the soil with the plant
roots and stubble; (2) the organic matter consumed as food by
animals is greatly altered during its passage through the digestive
systems of the animals; (3) a large part of the nitrogen in the
manure is in a readily assimilable form, and another part is in the
form of microbial cells.

During animal digestion, the more readily decomposable sub-
stances of the feeds are removed from the organic matter and,
consequently, the excreted material represents substances more
resistant to decomposition than the original food. The animals
consume practically all of the sugars and a large portion (70 to
80 per cent) of the fats, starch, hemicelluloses, and cellulose. The
lignin is removed to a slighter degree than any other group of the
organic substances in the foods. The manure consequently con-
tains a much higher percentage of lignin than the food which was
consumed. It is apparent that the composition of the food mate-
rial has an effect on the composition of the excreted material;
the foods containing the most lignin will be least digested.

During the passage of the food material through the digestive

210 MODIFICATION OF THE SOIL POPULATION

system of the animals, many bacteria develop and contribute to a
change in the nature of the organic matter. So extensive is this
bacterial development that the cells of the microorganisms com-
pose a considerable part of the total mass of the excreted material.
In the case of man, it has been calculated that as much as 20 per
cent of the faeces consists of bacterial cells. The same is probably
true in the case of other omnivorous and carnivorous animals.
With herbivorous animals, especially those feeding on less con-
centrated foods, the bacterial mass is smaller in amount, due to the
fact that a larger portion of the excreta consists of undigested con-
stituents of the roughage. The numbers of bacteria per gram of
faeces is calculated in billions. Since such large numbers of bac-
teria develop as a result of their feeding on the material that passes
through the animal digestive tract, these organisms must play an
important part in decomposing various organic substances con-
tained in the food.

The nature of the nitrogenous compounds in animal manure is
distinctly different from that of the compounds in plant manures
which have been considered previously. As a result of the
digestive processes, practically half of the nitrogen is excreted
as urea which may be quickly transformed in the soil or in stored
manure into compounds which are available to plants. In fact
the transformation is so rapid that, unless particular care is taken
to preserve this nitrogen, it may soon be lost either by volatiliza-
tion or by leaching.

The decomposition of animal manures is slow in comparison
with the transformation of certain other plant residues. This is
apparent from the curves of Fig. 78. Decomposition, as measured
by the formation of carbon dioxide, indicates that within a period
of about two months considerably less material was decomposed
in ten tons of manure in soil than in one ton of either oats or clover,
these being representative of green manuring crops. During
this period of 53 days, 60.8 per cent of the clover was decomposed,
49.0 per cent of the oats, and only 4.23 per cent of the animal
manure. The slow decomposition of the manure is explained
by the chemical composition of the material. As shown in Table
50, the manure contains relatively large amounts of certain chem-
ical complexes which do not decompose readily. The amount of
water-soluble material in the sheep manure is particularly high,
since both the sohd and liquid excreta were mixed together. It is

INFLUENCE OF STABLE MANURE

211

apparent that, in the transformation of the manures, the hemi-
celluloses and cellulose disappear most rapidly. The crude protein
increases in proportion to the rest of the materials, because of the
growth of microorganisms. The relative amount of Hgnin increases
as a result of its greater resistance to decomposition and as a result

15 30

Time (Days)

Fig. 78. — Amount of carbon dioxide evolved from soils treated with organic
matter (after Potter and Snyder).

of the microbial synthesis of materials included in the lignin com-
plex. Some of the Hgnin actually decomposes, but so slowly as
compared to the rest of the constituents that its relative abundance
is greatly increased.

Decomposition of the sugars, hemicelluloses, and cellulose,
of the urea and proteins, and of the fats and lignin, represents a
definite series of changes brought about by microorganisms. On

212

MODIFICATION OF THE SOIL POPULATION

TABLE 50

Change in Composition of Animal Manures during Decomposition
(from Waksman and Diehm)

Chemical constituents

Ether-soluble substances
Water-soluble substances

Hemicelluloses

Cellulose

Lignin

Crude protein

Ash

Percentage of Dry Material

Sheep Manure ''

Original
manure

2.83
24.92
18.46
18.72
20.68

17.21

Manure

decomposed

for 192

days

2.58
17.89

7.31
12.79
27.31

19.23

Horse Manure f

Original
manure

1.89

5.58

23.52

27.46

14.23

6.81

9.11

Manure

decomposed

for 290

davs

0.95
5.71
12.67
5.97
28.43
16.38
19.32

* Includes the mixed solid and liuqid excreta,
t Includes solid excreta only.

the other hand, the synthesis of microbial cells is always associated
with these changes, whether the decomposition of the manure
takes place in composts or in the soil.

In the decomposition processes, the liquid and solid excreta
should be considered separately, since the transformations asso-
ciated with the two materials are distinctly different. This
applies particularly to the nitrogenous constituents. Urea, which
makes up a large part of the organic material in the liquid manure,
is very rapidly transformed to ammonium carbonate. The soHd
excreta may contain about one per cent of nitrogen on the basis of
the dry material. On the other hand, the liquid manure contains
about 4.50 per cent of nitrogen on the dry basis. Not only do the
solid excreta contain a low percentage of nitrogen, but a consider-
able portion of the organic compounds containing this nitrogen are
only slowly decomposed. There is still another factor which
lowers the rate of liberation of ammonia from the solid manure.
The latter contains appreciable amounts of non-nitrogenous

INFLUENCE OF STABLE MANURE 213

organic matter, which is further increased by strav/ and similar
forms of bedding material. In the decomposition of these organic
substances, considerable nitrogen becomes stored away in micro-
bial cells. These factors preclude the rapid formation of ammonia
from sohd excreta and result in the persistence of the organic
matter in soils for extended periods of time, during which the
nitrogen slowly but continuously becomes liberated in forms
available to higher plants — a very important factor in soil fer-
tihty.

The losses of nitrogen from composting manure are associated
with transformations of the urea which is contained in the liquid
excreta. So much ammonia is rapidly formed that the reaction
becomes sufficiently alkaline to permit the volatilization of some
of the ammonia. The ammonia which remains in the compost
may be further transformed to nitrate, and, as the nitrate may
pass to certain regions of the mass of organic matter where condi-
tions are favorable for reduction, the nitrate may be broken down
to gaseous nitrogen or oxides of nitrogen and become dissipated
into the atmosphere.

The control of such losses of nitrogen is based on several dif-
ferent principles. The losses of ammonia can be prevented by the
addition of acid-reacting substances such as superphosphate, sul-
furic acid, or sulfur. The fact that under these conditions super-
phosphate reverts to the more insoluble phosphates somewhat
decreases the desirability of such treatments. In order to ehm-
inate the losses of nitrogen through denitrification the manure is
so handled as to prevent the formation of nitrate. Since anaerobic
conditions prevent nitrate formation, manures are frequently
packed tightly, kept in tight pits and otherwise handled to prevent
appreciable penetration of air. The addition of disinfectants pre-
vents excessive microbial activity but is not generally practical.

By hastening the processes of decomposition of the carbohy-
drates in the manure, the conversion of the ammoniacal nitrogen
into insoluble forms is accelerated by microorganisms, as they
transform the nitrogen into their cell substance. These processes
are frequently based upon the acceleration of the decomposition
by high temperatures. Preliminary aerobic conditions favor rapid
decomposition. Subsequent tight packing of the manure results in
heating. Under these conditions the temperature rises to about
65° C, and the transformation of the carbohydrates is sufficiently

214 MODIFICATION OF THE SOIL POPULATION

rapid to result in the conservation of most of the original nitrogen
which is present.

Green manure added to the soil is comparatively free from
microorganisms, except those which are carried down by the dust
and may be adhering to the plants, or those organisms which may
actually be pathogenic upon the living plant. Also, some microbes
may have begun an attack of those parts of the plants which have
died off. Leguminous plants are, however, very rich in the spe-
cific nodule-forming bacteria, which become liberated on the dis-
integration of the nodules; the soil thus becomes heavily inocu-
lated.

Stable manure is very rich in bacteria. It has been suggested
that the favorable effect of manure upon soil fertility is due not
so much to the nutrients in the manure (about 10 pounds of nitro-
gen, 2 pounds of phosphorus, and 8 pounds of potassium in one
ton of fresh manure containing 80 per cent of mositure), as to
the presence of large numbers of bacteria. The introduction of
these bacteria into the soil was believed to bring about a modifica-
tion in the soil population of such magnitude as appreciably to
influence the soil processes. If that were the case, the frequent
additions of small quantities of manure might suffice, since even
a hundred pounds of manure would be more than sufficient to
properly inoculate the soil with the various bacteria. However,
the effects of the manure do not appear to be the result of such
modifications of the soil population. In the first place, the kinds
of bacteria in the manure are of a distinctly different nature from
those in the soil. The numerous cells of Bacterium coli and of
other intestinal bacteria present in such abundance in the manure
soon die off, after the manure is plowed into the soil. On the other
hand, the extent of microbial development in the soil is associated
with the amount of available food and with the environmental con-
ditions. If the food supply is increased, or if the environmental
conditions are made more favorable, the numbers of micro-
organisms capable of using this energy will also increase. No
matter how many bacteria are added to the soil (the same is true
of fungi as well), the nature of the population will not be changed,
unless additional food is added or unless the soil is treated in such a
manner as to render the organic matter already present there more
available. There are certain exceptions to this rule, as in the case
when microorganisms are added to soils in which they do not exist,

INFLUENCE OF STABLE MANURE

215

finding in the soil a suitable habitat, and capable of using the soil
organic complexes as sources of energy or bringing about other
reactions more effectively than the organisms already present in the
soil. However, such microbes are only seldom present in animal
manures.

The changes in the abundance of bacteria in soil as a result of
the addition of animal manure is shown in Table 51. There is an
appreciable increase in numbers following the application of fresh
manure; however, there is even a much greater effect as a result
of the treatment with sterile manure. This action of the sterile
manure results from the greater availability of the organic matter,
on account of the killing of the large numbers of bacteria in the
manure, as well as the modification of various organic constituents
during the sterihzation process.

TABLE 51

Influence of Manure (Applied at Rate of Ten Tons per Acre) on
Number of Bacteria in a Loam Soil (from Temple)

Period of
treatment

Untreated soil

Soil plus
manure

Soil plus sterile
manure

At start

5,190,000
6,477,000
5,160,000
5,650,000
4,880,000
4,730,000
4,820,000

5,190,000
8,590,000
6,280,000
6,600,000
6,070,000
7,010,000
6,580,000

5,190,000

7 days

23,600,000

14 days

21 days

28 days

42 days

49 days

13,850,000

11,740,000

10,280,000

8,560,000

7,860,000

The increase in the number of microbes following the addition
of stable manure to the soil may be considered to consist of several
steps. At first there is a decided increase in the abundance of
bacteria in the soil, due to the actual introduction of the bacteria
in the manure. This is soon followed by a drop in the number of
these organisms and by a rapid increase of the bacteria and fungi
of the soil itself, especially those organisms that use the constitu-
ents of the manure as sources of energy. Frequently there is a
sequence of organisms depending upon the nature of the manure
and the soil conditions. This sequence can be illustrated by con-

216 MODIFICATION OF THE SOIL POPULATION

sideration of a single constituent of the manure, namely, the urea.
Half of the nitrogen in the manure is in this form, while the other
half is in the form of proteins and protein derivatives. The urea is
immediately attacked by a number of specific groups of bacteria
and is changed to ammonium carbonate. The latter is soon acted
upon by the nitrite- and nitrate-forming bacteria. The nitrate
may be finally reduced by denitrifying bacteria and changed to
nitrogen gas and oxides of nitrogen, or is assimilated by fungi and
various heterotrophic bacteria (which use the constituents of the
straw in the manure as sources of energy) and changed to microbial
proteins. Thus the addition of one compound to the soil leads
to a number of different reactions which are accompanied by
numerous changes in the microbial population.

Artificial Manures. — Because of the ever-decreasing quan-
tities of stable manure, methods have been developed for the com-
posting of straw and other farm residues with inorganic fertilizer,
yielding a product similar in its chemical composition and action
upon soils and crops, and referred to as artificial manure. The
principles upon which the process is based are the decomposition
of the cellulose and other carbohydrates in the plant residues by
microorganisms; accompanying this process is the synthesis of
large quantities of microbial cell substance for which the inorganic
nitrogen is required. As a result of this action, there is a narrow-
ing in the carbon/nitrogen ratio from 80 : 1, in the straw, to 20 : 1,
in the compost. Although this compost contains an abundance
of various microorganisms, its beneficial effect upon the soil and
the crop is due largely to the nature of the organic and inorganic
complexes introduced.

Influence of Soil Cultivation. — The frequent cultivation
of the soil considerably modifies the numbers and activities of soil
microbes in various ways. The processes of plowing, harrowing,
and cultivating the soil accomphsh several distinct purposes:
(1) They mix the plant residues with the soil itself, thus leading to
better conditions for decomposition of the various residues by
microorganisms. (2) They favor aeration of the soil, thus accel-
erating the exchange of carbon dioxide of the soil air for the oxygen
of the atmosphere; the oxygen is required for the various oxidation
processes in the soil, especially nitrate formation, the decomposi-
tion of organic compounds, and the oxidation of the reduced inor-
ganic substances. (3) Finally, soil cultivation brings about a more

EFFECT OF LIMING 217

uniform distribution of the moisture necessary for the activities
of the soil microbes.

The effect of stirring soil upon the development of bacteria is
shown in Table 52. In this investigation the soil was kept at a
uniform moisture content and thoroughly stirred at frequent inter-
vals. The influence of this treatment was very pronounced, as
shown by an increase in numbers of bacteria from about two mil-
lions per gram present in the soil initially to over twelve millions,
after a period of 24 days. The effect of such mechanical altera-
tion of soil is more pronounced with the deeper layers of soil
than with the surface soil, since the deeper layers are more imper-
fectly aerated in an undisturbed condition.

TABLE 52

Influence of Tillage upon Abundance of Bacteria in Soil

(from Chester)

T, . , , ^ . Numbers of bac-

reriod or time , .

tena per gram

At start 2,040,000

After 7 days 5,495,000

After 9 days 6,171,000

After 14 days 11,326,000

After 24 days 12,600,000

Effect of Liming. — There is no better way of demonstrating
the effects of simple soil treatment upon the nature and activities
of the microbial population than by examining the effects of lime
on an acid soil. Following the addition of lime, the hydrogen-ion
concentration of the soil diminishes (the pH increases). The
extent of the decrease in acidity depends upon the amount of lime
added and upon the nature of the soil. The more lime is added to
the soil, the greater is the change in reaction, up to a certain
point which is distinctly alkaline, namely, at a pH. of 8.2. Above
this point any further addition of lime will exert no effect upon the
reaction, as expressed in terms of hydrogen-ion concentration.

Soils differ in their buffering properties, that is, in their ability
to react with considerable amounts of acids or bases without
appreciably changing in reaction. To effect a definite change in
reaction, a clay soil may require two times as much lime as a loam
soil. The greater the buffering action of the soil (combining power
with base or acid), the greater is the amount of lime required before

218 MODIFICATION OF THE SOIL POPULATION

a definite change in reaction is produced. Since this buffering
capacity resides principally in the colloidal material, the finer-
textured soils require more neutralizing agent to cause a certain
change. Consequently, one would not expect all soils to react
equally to the same applications of lime, even though the reactions
of both were alike previous to the treatment. The effects of lime
are further complicated by the fact that soils differ so greatly in
reaction. Acid soils will generally respond to lime, while soils
which are alkaline in reaction may show no appreciable change in
biological activities or plant growth following the same treatment.
Lime causes its effects principally through its influences on the soil
reaction, but it may produce other changes in the soil micro-
population, both directly and indirectly. Lime is particularly
effective in improving the physical structure of the soil, by causing
flocculation of the colloidal particles with the development of
granular structure. Plant growth may be favored by the creation
of a more suitable soil reaction and physical condition. As a
result of this enhanced growth, the plant residues furnish the soil
organisms with larger amounts of food material.

Thus, the influences of lime on soils cannot be analyzed in
simple terms. The effects are numerous and diverse, and may be
exerted upon plants and microorganisms both directly and indi-
rectly over a period of years. The improved plant growth follow-
ing liming is a suggestion of the extent to which the concealed
microbes are affected. The accelerated activity of the soil popula-
tion contributes to the improvement in plant development, and,
over an extended period of time, one of the factors which causes
the pronounced increases in the abundance of soil organisms is the
modified plant growth itself. Referring again to Table 11, one
can readily observe that liming practices may modify the distribu-
tion of soil organisms to a marked degree. The fertilizer and lime
treatments created marked differences in fertility of these soils,
and the microbial population changed with the growth of plants.
When such soils are compared with respect to the nature and
abundance of their soil population, it is found that, at reactions
below pH. 6.0, the more acid the soil the less favorable it is to the
development of bacteria and actinomyces and the more favorable
it is to the growth of fungi, provided that other conditions are the
same. Table 53 indicates that organic materials added to soils
of different fertility also lead to different responses in the develop-

EFFECT OF LIMING

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220 MODIFICATION OF THE SOIL POPULATION

ment of the microbial population. The greater abundance of
organisms in the limed soils is also reflected in crop yields, as will
be considered later.

Many of the common cultural treatments of soils bring about
changes in the soil population in as varied a manner as lime.
Seldom can the influence of the treatment be ascribed to a single
effect or a single soil change. Even a slight modification of soil
causes a series of changes both in plant growth and microbial devel-
opment, but some treatments have more pronounced and extended
effects than others. Cultivation and the application of fertilizer
salts affect biological activity directly to some extent, but the
indirect effects of such treatments and the direct effects of the
incorporation of organic substances in soils cause a greater degree
of modification.

Influence of Reaction upon Soil Microbes. — Not all
microorganisms are affected alike by acid and alkaline conditions
in soils. Some microbes will develop over a narrow range of soil
reaction with an optimum zone at pH 6.0 to 7.5. Others are tol-
erant to as extreme acid or alkaline conditions as ever appear in
soils; many of the soil fungi, for example, will grow well at a series
of reactions ranging from pH 2.0 to pH 9.0. In general, soil
microbes will tolerate greater concentrations of hydrogen-ions than
hydroxyl-ions, that is, greater degrees of acidity than alkalinity.
In humid regions, soils as acid as pH 5.0 are not uncommon, and
such soils may support good growth of a number of cultivated
crops and a large and varied microbial population. The concen-
tration of hydroxyl-ions in soils as alkaline as pH 9.0 to 10.0 is the
same as the concentration of hydrogen-ions in soils of pH 5.0 to
4.0. Such alkaline soils exist only in regions of deficient rainfall.
They are fairly unproductive and harbor relatively smaller num-
bers of organisms.

Many mycorrhiza fungi grow well at pH 5.0 and make little
growth at neutrality (pH 7.0). Nitrifying bacteria have an acid
minimum at pH 4.0 and an alkaline maximum at pH 9.4. Most
species of Azotobacter have an acid minimum at pH 6.0, so that in
soils which are more acid than pH 6.0 these organisms are either
inactive or absent. However, the anaerobic nitrogen-fixing
organism, Clostridium pastorianum, will grow at a reaction as
acid as pH 5.2, and is, therefore, much more widely distributed in
acid soils than Azotobacter.

ARTIFICIAL FERTILIZERS AND SOIL MICROBES 221

In most soils, practically all actinomyces fail to grow if the
acidity is greater than pH 4.0. Some of these organisms, such as
Actinomyces scabies, the causative agent of potato scab, will not
grow at a pH less than 4.8. Control of potato scab and of sweet
potato pox by treatment of soil with sulfur and other acid-reacting
fertihzers is based upon this sensitivity of actinomyces to high
acidity. Sulfur becomes oxidized by biological agents to sulfuric
acid, which increases the acidity of the soil. If sufficient sulfur is
added to change the reaction of the soil to pH 4.6-4.8 (the amount
to be used depending upon the initial reaction and the buffer
content of the soil), the development of Actinomyces scabies will
be checked, but the potato plants will still make sufficient growth
to produce a good crop. When the soil is to be used the following
year for another crop, it should be properly limed before that crop
is planted, so as to make the reaction less acid. Certain other
microbes which cause plant diseases are inhibited in their develop-
ment by an alkaline reaction of the soil. Such an organism is
Plasmodiophora brassicae, which produces club-root on cabbage.
For the control of this disease, soils are liberally treated with lime.
Thus, a thorough knowledge of the characteristics of the patho-
genic organisms and their response to differences in reaction some-
times enables one to control plant diseases by judicious soil man-
agement.

Different cells of microorganisms of a certain species are not all
alike in their tolerance to different degrees of acidity and alkalinity.
The variabihty among the different individuals results in the
selective development of the forms which are best adapted to the
environment in which they exist. Consequently strains of an
organism obtained from one soil may show certain characteristics
which are different from those of another strain obtained from a
different soil. Some organisms natural to acid soils may show
greater tolerance to acid reactions than strains of the same organ-
isms present in alkaline soils. Even under laboratory conditions,
by growth in certain media, cultures may be obtained which are
more tolerant to acid or alkahne reactions than the mother culture
from which the modified strains were obtained. By gradual suc-
cessive growth of nitrifying bacteria on alkahne media, strains
were obtained which grew at a reaction more alkaline than pH 10.0.

Influence of Artificial Fertilizers upon Soil Microbes. —
Although not as pronounced as many other effects on soil organ-

222 MODIFICATION OF THE SOIL POPULATION

isms, the direct effects of inorganic artificial fertilizing materials
on the abundance and activities of microbes are nevertheless quite
evident. This is apparent from the data presented in Tables 54
and 55. Of the various salts mentioned, phosphates exerted a
somewhat more marked influence than sulfates. Since most of
the inorganic compounds are present in the soil solution, the
greatest stimulating effects might be anticipated when the salts
are added to soils which contain the materials only in small
amounts. Under certain conditions, as in alkali soils, they may
occur in such abundance as to be quite toxic to microbial devel-
opment.

TABLE 54

Influence of Phosphates and Sulfates on the Number of Bacteria
IN Soil (from Fred and Hart)

Bacteria per gram of soil
Treatment (Average during 12 days

of treatment)

Untreated 12,330,000

0.5% CaS04-2H20 14,212,000

0.5% K2HPO4 23,680,000

1 . 0% Ca3(P04)2 12,702,000

1.0% CaH4(P04)2 16,922,000

The effects of inorganic materials are exerted in a variety
of ways: (1) Some constituents of the fertilizing materials may
furnish a supply of elements which are deficient in the soil and may
be consumed by the microorganisms in their nutrition. In the
presence of an abundance of undecomposed plant residues, artificial
fertilizers increase the activities of the various fungi and bacteria
to a much greater extent than would be the case where only the
organic materials were present. The nutritional demands of the
organisms for inorganic substances become multiplied propor-
tionally with the increases in organic food supply. This applies
particularly to available forms of nitrogen, phosphorus, and
potassium. (2) The inorganic materials may affect the physical
properties of the soil, making it a more or less favorable environ-
ment for development of the organisms. (3) The inorganic com-
pounds may affect the composition of the soil solution by either
exerting solvent effects on the insoluble soil minerals, or by pre-
cipitating substances from the soil solution, or by increasing the

ARTIFICIAL FERTILIZERS AND SOIL MICROBES 223

TABLE 55

Influence of Phosphates and Sulfates on Formation of Carbon
Dioxide in Soil (after Fred and Hart)

Carbon dioxide produced
Treatment during 6 days

(comparative figures)

Untreated 100

0.1%K2HPO4 194

0.1% (NH4)2S04 152

0.1% Ca3(P04)2 123

0.1% CaS04-2H20 105

0.1%MgSO4 107

0.1%K2SO4 101

salt concentration as a result of their addition to the solution.
(4) The salts may change the reaction of the soil solution, making
it either more basic or acid as a result of physical-chemical action,
selective adsorption, or transformation of certain portions of the
compounds by soil organisms. (5) Certain catalytic effects may
be exerted, as in the stimulation produced by compounds contain-
ing such elements as manganese or radioactive compounds. (6)
Probably the most pronounced effects upon microbes exerted by the
addition of inorganic fertilizing substances are brought about indi-
rectly, namely, through higher plants, particularly where fertihz-
ing practices have been in operation for extended periods of time.
The response of plants to the fertilizers is transferred to the soil
population, as the plant residues are incorporated in the soil.
The resultant effects of these interwoven reactions is apparent
in the data given in Table 11. Changes in the physical conditions
of the soil accompany the various soil treatments.

Certain fertilizers may affect specific groups of soil organisms to
a far greater extent than the soil population as a whole. This is
particularly the case where certain constituents of the fertilizers
may serve directly as foods for the organisms. The ammonia in
ammonium salts is used by the nitrifying bacteria as a source of
energy. Urea favors the development of specific urea-decompos-
ing bacteria which transform the urea into ammonia. Dicyano-
diamid, frequently an impurity of cyanamid, may even prove
injurious to various important soil bacteria. The numerous
organic compounds of nitrogen which are contained in fertilizers

224 MODIFICATION OF THE SOIL POPULATION

influence a variety of soil organisms, the kinds of organisms affected
and the degree of the effects being determined by the nature of
the fertihzers.

Influence of Plant Growth. — The type of plant grown in a
soil will also considerably influence the microbial population, as
shown in detail in the previous chapter. The influence of the
plant is due to several factors. Considerable material becomes
added to soils from plant roots, either as excretions or as parts of
the roots themselves. The influence of plants on soil moisture,
salt concentration, and physical conditions is largely responsible
for the changes in the population. Subsequent to the death of the
plant, appreciable amounts of organic residues may find their
way into the soil and serve as food for the microbial inhabitants.
Influence of Partial Sterilization of Soil upon the Soil
Population and its Activities. — Soils may be treated in numer-
ous ways for the control or elimination of certain insects and soil-
harbored plant diseases; they may be steamed, treated with vola-
tile antiseptics, as toluene and carbon bisulfide, and also with some
non-volatile substances. These treatments are referred to as
partial sterilization, since they destroy certain members of the
soil population without killing all of the organisms. Some species
or even groups of organisms might be entirely eliminated during
the process, while most of the other microbes become affected to
some extent at least. As a result of studies of the diverse effects
of partial sterilization treatments upon the soil and its population,
it has been shown that plants respond to these treatments in ways
which cannot be explained completely by merely assuming the
ehmination of plant parasites. A brief survey of some of the fac-
tors involved will aid in understanding the phenomenon.

Partial sterilization of soil not only eliminates certain organisms
but greatly modifies the soil as regards its capacity to support plant
growth. The soil acts much as if it had been fertilized with nitro-
genous substances. The extent of the response of plant growth is
apparent from Fig. 79. The effects vary greatly with different
soils as well as with different treatments. Fertile soils rich in a
great variety of organic constituents appear to be most suscep-
tible to changes.

The changes in the soil population are numerous and pro-
nounced; these changes are reflected in the transformations with
which the microbes are associated. There is at first a depression

INFLUENCE OF PARTIAL STERILIZATION OF SOIL 225

in the total number of bacteria. Following this temporary depres-
sion there is a pronounced increase in the number of organisms
over that found in the soil before treatment. After a period of
time, the numbers decrease slowly, and many weeks or months
may elapse before they drop to the original level. The actinomyces
appear to be affected somewhat less than the bacteria. The
increase in these organisms is slower than that of the bacteria, con-
sequently they comprise a proportionately smaller part of the soil
population subsequent to the treatment.

The protozoa and fungi are considerably reduced in numbers,

Fig. 79. — Influence of partial sterilization by heat upon growth of tomatoes.
The soils in the pots from left to right were heated as follows before planting:
30° C, 60° C, 80° C, 100° C, 125° C, 150° C, for two hours (from Pickering).

and are frequently almost all destroyed. These organisms do not
become active in the soil again before an extended interval of time
has elapsed.

A considerable rise in the evolution of carbon dioxide from the
soil accompanies the rise in bacterial numbers, as shown in Fig. 80.
The initial rapid rise in carbon dioxide is followed by a rapid decline
to a certain level; the decrease then becomes comparatively slow,
the rate of carbon dioxide production of the untreated soil not
being reached for a long time. The initial rapid increase generally
precedes the increase of bacterial numbers, and the decrease also
occurs more rapidly than the decHne in the numbers.

This manifestation of increased biological activity is accom-
panied by a rapid formation of ammonia, as shown in Fig. 81.
This nitrogen originates principally from the organic matter of the

226

MODIFICATION OF THE SOIL POPULATION

soil, and it accumulates as ammonia, since the nitrifying organisms
are destroyed by the partial sterilization treatments. When the
nitrifying bacteria again become established in the soil, the ammo-
nia is converted to nitrate and only small quantities of ammonia are
subsequently found in the soil at any one time. The increased
rate of production of both carbon dioxide and ammonia clearly
indicates that the soil organic matter is undergoing more rapid
decomposition as a result of the treatment.

Untreated Soil

Soil Plus Toluene,
Relnoculated

Soil Plus Toluene,

Soil Air -Dried,
Reinoculated

Soil Air -Dried

Soil Plus CSz,
Reinoculated

Soil PlusCSa

Soil Heated,
Reinoculated

Soil Heated

100 150 200

Carbon Dioxide Evolved -Mgm.

250

300

Fig. 80. — The influence of partial sterilization treatments on biological activi-
ties in soil as reflected in the formation of carbon dioxide during the first week
after treatment (mgm. COo per kgm. of soil). In some cases the soils were
reinoculated with infusion of fresh soil after treatments (after Wakeman

and Starkey).

Many compounds in the soil are modified by heat and anti-
septics, as evidenced by the greater solubility of both the organic
and inorganic soil constituents. Besides the modification of the
inanimate material, there is to be taken into consideration also
the destruction of large numbers of living soil organisms. The
soil may thus be altered in many ways by partial sterilization,
these alterations leading to or being responsible for an increase in
productivity.

To explain some of these phenomena, Russell and Hutchinson
suggested the following ingenious hypothesis. It is known that
the soil harbors, in addition to bacteria, various other groups of

INFLUENCE OF PARTIAL STERILIZATION OF SOIL 227

microbes, among which are the protozoa. It is quite generally
agreed that protozoa exist in soils in the trophic state and, since
many of them feed largely upon bacteria, they limit bacterial
development to a certain extent. One may assume that some
of the most important soil processes bearing upon soil fertility are
carried out by bacteria. If it were possible to destroy the protozoa,
the bacteria would begin to develop rapidly, reaching much larger
numbers than in the original soil. If bacteria are the causative

20 40

Time (D a y s>

Fig. 81. — Influence of partial sterilization treatments upon numbers of bac-
teria, numbers of fungi, and accumulation of soluble nitrogen (ammonia plus
nitrate) in an acid soil rich in organic matter (after Waksman and Starkey).

agents of the liberation of plant nutrients, the removal of such
organisms as the protozoa would favor bacterial development,
and also the processes which are of the greatest importance to
soil fertility. The treatment of soil with antiseptics and heat
brings about the destruction of protozoa, and this change has as its
direct result the favorable effect upon soil fertihty.

From such considerations it was concluded that protozoa com-
prise the limiting factor to the development of bacteria in all soils
under various conditions; by their characteristic habits of feeding
they hold the bacteria in restraint and consequently limit soil
fertility. The removal of protozoa, particularly of the amoebae
and ciliates, permits a much enhanced development of the rest

228 MODIFICATION OF THE SOIL POPULATION

of the soil micro-flora. Irrespective of whether soils are dried,
heated, treated with lime, acids, volatile antiseptics, or non-vola-
tile poisons, according to this theory it is the greater susceptibility
of the protozoa, which are injured and partially or completely
eliminated, which results in the accelerated development of bac-
teria.

The value of this explanation is greatly decreased by reason of
the fact that it does not explain all of the numerous changes asso-
ciated with partial sterilization. The increase in the number of
bacteria and in the decomposition of organic materials need not be,
and frequently is not, correlated with the presence or absence of
protozoa; identical changes may occur whether protozoa are or
are not eliminated. In addition to bacteria, other microbes are
capable of decomposing organic matter in the soil and of liberating
the constituents in forms available for plant consumption. Soil
fungi, actinomyces, and algae were left out of consideration in this
hypothesis. A reasonable consideration of the relationship of
fungi to certain of the soil reactions indicates that they are respon-
sible for some of the effects ascribed to protozoa. It has recently
become apparent that the destruction of some of the bacteria in
soils does not necessarily result in an injury to soil processes car-
ried out by these bacteria. One need not deny, however, that
protozoa play a part of some importance in soil processes. Their
great abundance seems adequate evidence to indicate that they are
an important element of the microscopic population of the soil.

The favorable effects of partial sterilization treatments on soil
fertihty are probably due to a number of factors, including (1)
changes in the physical and chemical characteristics of the inor-
ganic and organic soil material; (2) destruction of numerous
organisms, including fungi, protozoa and other invertebrate
animals, bacteria, actinomyces, and algae; (3) direct stimulating
effects of disinfectants, in some instances. Different treatments
differ in their effects because they act upon different soil constit-
uents, both quahtatively and quantitatively. The relative abun-
dance of the different organisms in soil is not determined by one
group of organisms destroying another group, although this may
take place under exceptional circumstances, but it is chiefly a
question of competition for food materials. Under most soil con-
ditions small amounts of available nutrient salts and energy
sources are present at any one time. The partial sterilization

INOCULATION OF SOIL WITH MICROORGANISMS 229

treatments so modify the soil constituents that the supply of avail-
able foods is increased and the soil organisms show a sudden and
pronounced response.

Inoculation of Soil with Microorganisms. — Although
many of the soil organisms appear to be widely distributed and are
quite generally present in most soils, conditions appear where cer-
tain desirable types are absent. This suggests the possibility of
actual modification of the soil population by inoculation of the
organisms which are lacking. An investigation into the causes
for the absence of these organisms may suggest whether inoculation
may be expected to be of any value.

The organisms may be absent because conditions are unfavor-
able. This may be due to the fact that the soil reaction or aeration
is unfavorable. It may also be due to the absence of available
food materials. Certain organisms are limited in their nutrition
to few sources of energy; some sulfur bacteria are able to utilize
only inorganic incompletely oxidized compounds of sulfur, or ele-
mentary sulfur itself. The occurrence of the organisms which
develop in association with leguminous plants within the nodules
on their roots is frequently limited to soils which have recently
grown this particular legume, or representatives of the cross-inocu-
lation group to which this legume belongs. In the presence of the
legume host the organisms find food to satisfy their requirements,
but in the absence of the plant the organisms may disappear in
course of time, especially in acid soils.

On the other hand, certain bacteria may never have become
established in soil even though conditions are favorable to their
development. This may be due to the fact that conditions have
been altered recently by agricultural practices; the new conditions
may be favorable to organisms which could not develop previous
to the alteration.

The possibility also exists that certain strains of organisms may
be developed which are capable of performing important soil
processes more efficiently than those already resident in the soil.
Their addition to soils may improve conditions for plant devel-
opment.

It is known that forest soils, peat soils, and ordinary field and
garden soils possess characteristic floras. In the forest soils, espe-
cially the raw-humus soils, fungi predominate both in the amount
of active cell substance present and in the transformation of

230

MODIFICATION OF THE SOIL POPULATION

the soil constituents. In peat bogs under natural conditions,
both fungi and aerobic bacteria are absent, especially a few inches
below the surface. The conditions are favorable only for the
growth of anaerobic bacteria. When peat bogs are drained, limed
in the case of acid peat bogs, and cultivated, and thus converted
into peat soils, they may profit considerably from inoculation with
fresh garden or field soil. One of the most important groups of
organisms thereby introduced is the group of nitrifying bacteria.

Fig. 82. — Effect of inoculation upon growth of legumes. Left to right —
Soybeans; ten plants from an uninoculated plot, ten plants from an inocu-
lated plot. Pea vines; eight plants from an inoculated plot; eight plants
from an uninoculated plot (from Fred, Whiting and Hastings).

When various leguminous plants are grown upon soil in which
the particular plants have never grown before, it is essential to
inoculate the soil with the specific types of the nodule-forming
bacteria, if a proper crop is to be secured and the best use made of
the particular plant on the given soil (Fig. 82). This is especially
true in the case of plants like alfalfa, soybeans, and cowpeas, when
introduced into regions in which these plants have never grown
before. Although the bacteria in general are quite widespread,

INOCULATION OF SOIL WITH MICROORGANISMS 231

they are by no means ubiquitous. Artificial cultivation of the
legumes over considerable areas widely separated from regions
where legumes have been previously grown necessitates special
treatment to insure the presence of the organisms.

For the purpose of inoculation, some soil in which the particular
crop has been grown before may be used. One disadvantage of
this practice is the likelihood of also introducing plant parasites
and weed seeds at the same time that the bacteria are added.
The practice of inoculating with specially prepared cultures of the
specific legume bacteria has found most satisfactory use. Pure
cultures of the specific organisms grown in liquid or solid media
have proved to be effective. Other useful inoculating material is
prepared by mixing pure cultures with ground peat or similar sub-
stances. Either the seeds themselves or the soils are inoculated
with these special cultures of the specific legume bacteria or with
soil which is known to carry the organisms; this inoculation insures
the presence of the proper bacteria at the time and place where the
seeds develop. Inoculation of the seed is most effective and
requires the least amount of effort. A definite quantity of the
culture is mixed with the seed in a place not exposed to the sun.

Once a soil has been inoculated with the specific organism and
has had a crop of the specific plant grown on it, it need not be rein-
oculated. However, in view of the fact that different strains of
the same organism vary in their vitality, in inoculating power and
in the amounts of nitrogen that they are able to fix, it has been sug-
gested that it is fully worthwhile to reinoculate the soil with fresh
cultures when a new crop is grown. Even a slight increase in crop
yield and in nitrogen content may more than cover the small addi-
tional cost of the culture. This is especially true of acid soils, in
which the nodule-forming bacteria deteriorate more rapidly than
in neutral and alkaline soils, so that within a very few years they
may completely disappear in the acid soils.

Among the other processes which necessitate the artificial
introduction of microbes, it may be sufficient to mention the bac-
terial oxidation of suKur to sulfuric acid. Although there is no
doubt that most soils harbor organisms capable of oxidizing
sulfur to some extent, few soils contain organisms capable of per-
forming the oxidation very rapidly. The introduction of such an
organism as Thiobacillus thiooxidans into the soil with the
sulfur may considerably hasten the formation of acid. The use

232

MODIFICATION OF THE SOIL POPULATION

of suKur for the purpose of making the soil more acid becomes
essential in a system of soil management devised to control such
plant diseases as potato scab, sweet potato pox, and potato wart
(Fig. 83). Other instances where soil inoculation appears to be
quite beneficial have been observed; the inoculation of seed beds

Fig. 83. — Influence of sulfur and inoculation on potato scab. Upper series,

tubers from check plot. Left to right: salable scabby, unsalable scabby.

Lower series, tubers from plot treated with 600 pounds of inoculated sulfur.

Left to right: clean, salable scabby, unsalable scabby (from Martin).

of forest seedlings with mycorrhiza fungi and the inoculation of
soils with certain cannibalistic nematodes which are capable of
destroying the nematodes which cause certain galls on plants
have proved useful. Such treatments have value only in very
specific cases, and their use is still of uncertain practical appli-
cation.

INOCULATION OF SOIL WITH MICROORGANISMS 233

Although the soil is a good medium for practically all of the
desired soil organisms so far known, and although one can isolate
from the soil most of the known saprophytic microbes if one looks
for them long enough, only those microbes are found abundantly
in a particular soil in which the necessary nutrients are available
and where environmental conditions are suitable. Numerous
organisms continually compete with one another for these nutri-
ents. Those microbes become predominant which are best
adapted to the soil and the particular set of conditions and are
able to utilize the nutrients found in that particular soil. A new
microbe can become established only after a change in the condi-
tions of the soil, or in the presence of a specific host plant or a
selective nutrient. When the host plant is removed or the selective
nutrient is exhausted, the specific organisms may die out, unless
the microbe has become adapted to the soil in such a manner as to
find other available nutrients; the microbe may also be resistant
to unfavorable soil conditions and capable of persisting until the
specific conditions again become established. The creation of
conditions favorable for the development of specific organisms
results in the appearance of the desired organisms, without recourse
to inoculation. Conversely, the inoculation of an organism into
the soil which is unsuited to its development cannot be expected
to be of value.

The introduction into the soil of various other organisms than
those mentioned above has been recommended from time to time.
Most of these, on careful investigation, proved to have their value
based upon insufficient evidence. It has been claimed that certain
cultures were composed of numerous organisms capable of bringing
about a more active decomposition of the organic matter of the soil
than the ordinary microbes. Other preparations were claimed to
contain microbes capable of inducing nitrogen fixation in non-
leguminous plants. Still others were claimed to favor plant
growth in general or the growth of some particular plants, in some
unexplained fashion. The interest of the layman is often aroused
by the important activities of the microbes. This interest coupled
with incomplete knowledge of the nature of soil microbes and their
relationships to soil processes unfortunately presents an oppor-
tunity for dishonest exploitation by the charlatan.

In conclusion, it may be restated that, with very few excep-
tions, all soil inoculants, other than those for legume bacteria, have

234 MODIFICATION OF THE SOIL POPULATION

so far proven to be worthless, at best not better than a mere
infusion of some stable manure. Repeated, critical, controlled
tests of the effects of an inoculant under a variety of conditions are
necessary to establish justification for its use in agricultural
practice.

The Estimation of Soil Fertility by Microbiological
Methods. — Even as the numerous and varied soil conditions
determine the intensity of plant development and the kinds of
plants which can be economically grown, so also the environmental
conditions are responsible for the microbiological activity. Growth
of both higher plants and the less conspicuous microbes are reflec-
tions of the soil conditions. The inanimate portion of the soil
itself is the product of diverse activities operating through long
periods of time. Different organisms are contained in some soils
than in others, but the difference in quantitative distribution of
organisms in various soils is more pronounced. It is this differ-
ence in the relative abundance of various species of microorganisms
which gives the soils some of their characteristic properties.

The type and abundance of plant growth are important factors
determining the microbiological soil conditions. Also, the nature
of the activities of the soil microbes is important in determining
the kind and degree of development of the higher plants. Since
the two are so closely related, it is obvious that certain correlations
should exist between the biological activity of a soil and its fer-
tility. Based upon this assumption, numerous studies have been
made to determine what methods could be devised to estimate
conveniently the productive capacity of soils. Some of these
methods have been developed to further determine the desira-
bility of adding fertilizers or Hme to soils.

The Activity of Physiological Groups of Soil Microbes. —
Some of the earliest methods which were used involved attempts to
determine the potential activity of such groups of the soil popula-
tion as could produce ammonia from organic compounds of nitro-
gen. The following procedure is typical of these studies. A
nutrient solution containing peptone, urea, protein, or other nitro-
genous organic substance is inoculated with soil or soil infusion and
then the speed of ammonia formation is determined. On the
assumption that the soils which contained the most organisms or
the most active organisms decompose the nitrogenous compounds
most rapidly, it was suggested that the rapidity of ammonia

ACTIVITY OF PHYSIOLOGICAL GROUPS OF SOIL MICROBES 235

formation be used to determine the microbial condition of the soils.
Such studies are not as valuable as had been anticipated for the
reason that bacteria develop so rapidly in the nutrient solutions
that the microbial conditions of the soils which are used as
inocula are quickly obscured.

Somewhat more suggestive results are obtained when the soils
themselves are used as the media in place of nutrient solutions;
the organic nitrogenous materials are incorporated with the various
soils which are under investigation. Even these studies prove to
be of limited value, since so many and diverse types of organisms
have the capacity of forming ammonia from organic substances
that great differences in soils do not appreciably affect the rate of
transformation of the added nitrogenous compounds.

There are certain other tests which have proved more useful
than the ammonification tests. Principal among these are deter-
minations of the rate of nitrate accumulation, which may be
studied in various ways: in solution media containing ammoniacal
nitrogen and inoculated with soil material; in the soil itself with-
out alteration other than creating proper moisture relationships;
in soil to which is added some source of nitrogen such as an ammo-
nium salt or some organic nitrogenous material. Since nitrifica-
tion is brought about by a limited number of organisms whose
activity is affected in a similar way to many cultivated plants by
similar degrees of reaction, temperature, moisture, and aeration,
it can be readily understood why the nitrifying capacity of fertile
soils may be greater than the nitrifying capacity of infertile soils
(Fig. 84).

A study of the decomposition of cellulose may also be of use in
an interpretation of certain soil characteristics. For such studies
the cellulose is generally added to the soil in a finely divided condi-
tion and the rate of its disappearance is followed. Since large
numbers of various organisms may be associated with the decom-
position, and since different members of the group develop under
different environmental conditions, the rate of decomposition in
soils is not decreased by all of the conditions which limit develop-
ment of higher plants; the transformation is of more value for
determining the readily available nitrogen in the soil. The decom-
position of cellulose is accompanied by the assimilation of consid-
erable quantities of nitrogen, which are used by the organisms that
are effecting the decomposition in order to satisfy their nutritional

236

MODIFICATION OF THE SOIL POPULATION

requirements. If appreciable amounts of cellulose are added to
soils, nitrogen is generally present in insufficient quantities to per-
mit most rapid decomposition. Consequently, the rate of decom-
position is regulated by the rate of liberation of nitrogen from the
soil organic matter.

Minerals

Minerals

Untreated Lime

Minerals

Minerals

Plus

Plus

Plus

Plus

Manure

Manure
Plus
Lime

Soil Treatments

(NH4)2S0^

Plus
Lime

Fig. 84. — Relationships between crop ^yields, abundance of bacteria, and
microbial activities in soils of similar origin but differing in fertility due to
different fertilizer treatments extended over fifteen years (after Waksman and

Stark ey).

Abundance op Microbial Inhabitants of Soils. — One of
the methods first used to obtain information concerning the bio-
logical condition of soils was the plate technique for determining
the abundance of bacteria in the soil. Various other methods have
since been utilized to obtain additional information concerning

AVAILABILITY OF SPECIFIC NUTRIENTS IN SOILS 237

the abundance of certain specific groups of soil microorganisms.
Some methods give information concerning the bacteria as a
whole; others deal with individual physiological groups of organ-
isms such as those that decompose cellulose, fix nitrogen, or oxidize
ammonia to nitrite or nitrite to nitrate. Actinomyces, fungi,
algae, and protozoa are somewhat less frequently the object of
such investigations (see Table 11).

There are many striking correlations between the abundance
of organisms, as determined by these methods, and the pro-
ductive capacity of soils (Fig. 84). The relationships are not
absolute, however, since not all microbial cells even of a single
species work with the same speed in the soil. A small number of
organisms in one soil may be more active than a larger number in
another soil, because of the fact that many individuals of the soil
population are resting forms during a large portion of the time.

Biological Activity of the Soil Population as a Whole. —
A large number of the soil microbes are concerned with the decom-
position of the organic materials in the soil. Consequently,
knowledge of their activity is of considerable value in estimating
the biological condition of the soil. In order to obtain such infor-
mation, measurements have been made of the rate of formation of
carbon dioxide in the soil. Since this gas is one of the principal
products resulting from the decomposition of all organic sub-
stances, and since most of the gas is of biological origin, informa-
tion concerning its formation is of exceptional value as an index
of microbial activity in soils (Fig. 84).

One of the most accurate measurements of biological activity is
the determination of the amount of heat produced during the
development of the organisms. The potential energy contained
in the various substances which are used by microbes as foods
undergoes a change, a part becoming transformed to other forms of
energy as heat and a part remaining in the new compounds which
have been synthesized by the cells of the organisms that grow at
the expense of the food material. Microorganisms are frequently
very inefficient in utilizing the energy contained in food materials,
and a very large portion of the energy is eliminated as heat. Such
heat is formed irrespective of the source of energy that furnishes
the organisms with the power to develop.

Determination of the Availability of Specific Nutrients
IN Soils. — Utilizing the available knowledge of the nutrition of

238 MODIFICATION OF THE SOIL POPULATION

soil organisms, several methods have been developed which may be
used to obtain information concerning the relative amounts of
available phosphorus, potassium, and calcium in soils. Cultures
of Azotobacter have been quite generally used in such studies.
The following procedures are typical of these methods.

For determining deficiencies of phosphate in soil, a solution
medium is prepared to contain all nutrients required by Azotobac-
ter, with the exception of phosphorus. To this medium is added a
certain amount of the soil to be tested. The material is sterilized
and then inoculated with Azotobacter. Since the only source of
phosphorus is the soil which is added, and since considerable phos-
phorus is required by the bacterium to grow, the relative growth
of Azotobacter or the amount of nitrogen fixed is an index of the
amount of available phosphorus contained in the soil. By adding
graded quantities of available phosphate to a series of flasks con-
taining the same medium treated in the manner mentioned above,
still further information can be obtained regrading the response
of the soil to phosphate treatment. By following a similar tech-
nique but with regard to calcium carbonate instead of phosphate,
the studies yield information concerning reaction and lime require-
ment of soil.

Such methods have been modified so that the soil itself is used
in place of solution media. A series of samples of soil are mixed
with starch and with graded quantities of phosphate, calcium car-
bonate, or potassium salt depending upon whether phosphate,
lime, or potash deficiencies are to be studied. The soils are then
made pasty with water and put into dishes for incubation. If
conditions are favorable, Azotobacter grows well and forms raised
colonies on the surface which have the appearance of tiny pearls.
If conditions are unfavorable, no colony development occurs or
there is very hmited growth (Fig. 85).

Growth of certain fungi has also been utilized to give informa-
tion of the same nature. For phosphorus determinations, nutrient
solutions are prepared containing an abundance of all nutrients
with the exception of phosphate, which is added in graded amounts
to a series of flasks containing the medium. A certain amount of
the soil under investigation is added to each flask, and the media
are all inoculated with a fungus such as Aspergillus niger. After
the incubation period, the fungus growth is removed from the
flasks, washed, dried, and weighed. The extent of growth of

AVAILABILITY OF SPECIFIC NUTRIENTS IN SOILS 239

the fungus is limited by the amount of available phosphate in the
soil. For potash determinations the method is the same, with the
exception that the media contain ample phosphate but graded
amounts of some potassium salt (Table 56).

There are thus numerous methods which have been used for
attempting to determine the productive capacity of soils. Some
of these methods are devised to give quite specific information,
while the object of others is to provide more general facts about the
soil. Use of several methods of study is preferred to use of indi-

FiG. 85. — Growth of Azotobacter in soils treated with starch to test for defi-
ciencies in available nutrient elements. Upper row: soil deficient in phosphate.
Reading left to right: check, nothing added; potash added; phosphate added;

phosphate and potash added.

Lower row: soil not deficient in either potash or phosphate. Reading left

to right: check, nothing added; potash added; phosphate added; phosphate

and potash added (from Sackett).

vidual procedures. Even such information is incomplete, and is
most valuable when it is used in conjunction with chemical and
physical determinations in estimating soil fertility. It is because
of the fact that the abundance of the soil population, the types of
organisms composing it and their activity are products of the
environment and are susceptible to change with modifications in
any of numerous conditions, that a certain amount of information
concerning the fertility of a soil can be obtained from a knowledge
of its microbiological condition. One of the principal diflSculties
is to obtain a true conception of the biological status of the soil.

240

MODIFICATION OF THE SOIL POPULATION

TABLE 56

Determination of Potash Requirements of Soils Using Growth of
THE Fungus Aspergillus niger as the Index (from Niklas)

Soil number

Amount of available

potash as tested by

vegetation experiments,

(mgm.)

Weight of growth

of the fungus,

(gm.)

1

7.77

0.82

2

11.98

0.94

3

12.27

1.19

4

10.13

1.21

5

18.00

1.42

6

18.00

1.46

7

21.73

1.61

8

25.99

1.80

9

31.73

2.34

10

35.89

2.52

11

44.89

3.30

12

78.12

5.10

Summary. — The invisible soil inhabitants are the victims of the
circumstances in the regions where they are located. They have
no means of migrating long distances except by occasional for-
tuitous transportation by wind, water, or similar agencies. They
must be able to sustain themselves from the food materials in their
vicinity or else die. They must be able to tolerate sudden changes
in the environmental conditions, since they have little or no means
of regulating the conditions under which they Hve. They must be
able to tolerate high and low temperatures, periods of deficient and
excess moisture, change in reaction, and a variety of other factors
related to their capacity to absorb and utilize the nutrients con-
tained in the soil.

Organisms cannot all tolerate the same conditions, and conse-
quently there is a selective change following any modification in
their habitat. If the conditions are not too severe, numerous
organisms may persist even though they are relatively inactive.
With a change to more favorable conditions they often quickly
respond with renewed growth and activity. These relationships
are responsible for the occurrence of a greater variety of organisms

LITERATURE 241

and more numerous individuals where considerable amounts of
organic substances reach the soil in such forms as plant residues,
animal manures, or commerical fertilizers. Certain inorganic
substances also accelerate microbial development. It is thus
possible to modify the population and consequently its acti\aty
by systematically regulating the cultural treatments. In some
special cases it is desirable to modify the population to an extreme
degree by the addition of certain antiseptics or by heating the soil.
Likewise, soil inoculation is effective where certain leguminous
plants are to be grown.

It is by reason of the fact that the soil population is more active
in fertile soil that microbiological methods are available for esti-
mating soil fertility. Information as to whether or not the organ-
isms are relatively numerous or scant, as well as information con-
cerning what treatments improve their development, is very useful
in judging soils and determining requirements for their improve-
ment.

LITERATURE

1. Fred, E. B., Whiting, A. L., and Hastings, E. G. Root nodule bacteria

of Leguminosae. Wisconsin Agr. Exper. Station Research Bulletin
72, 1926, pp. 1-43.

2. LiPMAN, J. G. Bacteria in relation to country life. Chapters 23, 27-32.

The Macmillan Co. New York, 1921.

3. LoHNis, F., and Fred, E. B. Textbook of agricultural bacteriology.

Chapter 13. McGraw-Hill Book Co., Inc. New York, 1923.

4. Russell, E. J. Soil conditions and plant growth. Chapter 6. Long-

mans, Green & Co. London, 1927.

5. Waksman, S. a. Principles of soil microbiology. Chapters 27, 28, and 31

The Williama & Wilkins Co. Baltimore, 1927.

CHAPTER X
IMPORTANCE OF MICROBES IN SOIL FERTILITY

Relationships of Microbes to Soil Processes. — In conclu-
sion, it may not be amiss to summarize the results presented in the
previous chapters dealing with the occurrence, abundance, and
activities of various microbes in the soil, and point out their impor-
tance for plant growth. The very existence of higher plants and,
therefore, also of animals, including man, depends upon the activi-
ties of the soil microbes. Consequently there is little likelihood
of overestimating the importance of microbes in soil processes;
the growth of soil microbes is intimately concerned with the avail-
abihty of nutrients which determine the fertility of a soil. Any-
thing that hastens the decomposition of the soil organic matter
also favors an increase in soil fertility. A fertile soil is distin-
guished from an infertile soil not by the fact that it contains
more nitrogen, phosphorus, and potassium, but by the fact that
the nutrients present in the soil are liberated with greater rapidity
in the fertile than in the unfertile soil.

Liming and cultivation of soil improve the physical condition;
they provide a more favorable reaction for the activities of the
numerous soil bacteria and admit larger quantities of oxygen which
are necessary for the growth of the aerobic organisms. These
treatments are of great importance in soil fertility, not only because
they produce in the soil a favorable physical and chemical condi-
tion for plant growth. They also create more favorable conditions
for the activities of the microbes which effect more rapid liberation
of the soil nutrients.

Also part of the response in plant growth following the use of
artificial fertilizers is brought about indirectly. The fertilizers
increase microbial activities which lead to more rapid transforma-
tion of the soil constituents.

Even a superficial examination indicates that there are numer-

242

THE CYCLE OF ELEMENTS IN NATURE 243

ous important processes in which microorganisms take an active
part: rock disintegration and soil formation, decomposition of
organic matter in soil, liberation of nitrogen in an available form as
ammonia, liberation of carbon as carbon dioxide, formation of nitrate,
fixation of nitrogen, disappearance of available nitrogen from soil,
synthesis of microbial cell substance, oxidation and reduction of soil
constituents, transformations in composted stable manure, production
of artificial manure, disintegration of green manures, liberation of
mineral elements such as phosphorus from relatively insoluble sub-
stances, transformation of sulfur, the improvement of certain plant
growth following soil inoculation, changes following partial steriliza-
tion treatments, injury to plants and animals by bacterial and fungus
parasites. Some of these processes may be considered at some-
what greater length.

Role of Microorganisms in the Cycle of Elements in
Nature. — The activities of microorganisms in the soil are respon-
sible for completing in nature the cycles of various elements,
especially those that enter into the composition of organic sub-
stances. Here may be included first of all the element carbon
which is the basis of all organic compounds. The element nitrogen
which, in combination with carbon, hydrogen, and oxygen, forms
the most important group of constituents of all hving protoplasm,
is of no less importance. The mineral elements sulfur, phosphorus,
and potassium are likewise absolutely essential for the growth of
green plants and are frequently the controUing factors in develop-
ment of plants in soil. A number of other elements, such as iron,
calcium, and magnesium, are directly or indirectly acted upon by
microorganisms in their cycle in nature.

Chief among the activities of microorganisms is the minerali-
zation of organic matter in nature. The abundant life of green
plants in and on the surface of the earth, from the microscopic
algae to the thousand-year-old evergreen and deciduous trees,
tends to build up organic matter from inorganic elements or from
simple inorganic compounds. From the carbon dioxide in the
atmosphere as the only source of carbon, from the nitrogen present
in the soil in the form of simple compounds (ammonium salts and
nitrates), out of the phosphorus, potassium, sulfur, iron, calcium,
magnesium, and traces of other elements in the form of simple
inorganic compounds, in addition to water and gaseous oxygen, the
green plants are capable of manufacturing every year several tons

244 IMPORTANCE OF MICROBES IN SOIL FERTILITY

of organic matter per acre of soil. A large part of this organic
matter is returned to the soil in the form of plant stubble, leaves,
branches, green manures, stable manures, and various waste
products of plant utiHzation by man, while another part is used as
the source of human and animal food.

Animals, being unable to manufacture their own organic sub-
stances from simple inorganic materials, depend upon the plants
for the organic matter. The synthesized products resulting from
the development of higher plants furnish the sole source of energy
which keeps the other living organisms active in the world. The
animal, using the energy derived from the disintegration of some
of these compounds, changes the composition of the materials
manufactured by the plant, and forms, from these, substances
which are needed for the synthesis of its own tissues and for its
numerous functions. A large part of this animal organic matter
returns to the soil sooner or later in the form of animal excreta
and other waste materials. Finally, as the animals themselves
die, their own bodies, from those of man to the lowest insects or
microscopic worms, are returned to the soil. Consequently,
whatever is taken from the soil and from the atmosphere is sooner
or later returned, but generally in an entirely different form. The
plant has originally taken its nutrients from the soil and from the
air in the form of simple inorganic salts; these are returned in the
form of numerous complex compounds of plant and animal origin.

Since the amount of carbon dioxide in the atmosphere is quite
limited, being not more than three-hundredths of one per cent of
the atmospheric gases, and since nitrogen is present in the soil
in mere traces in the inorganic forms of nitrates and ammonium
salts, it would take but a short time before growth of plants and
animals would stop, were it not for the activities of the micro-
organisms in the soil. The microbes of the soil must constantly
replenish the supply of available nitrogen and carbon for the
plants and keep these two most important elements in constant
circulation. As soon as this stops, which rarely, if ever happens
under natural conditions, the soil is unable to support further life
of plants and animals. The elements hydrogen and oxygen are
present in such great abundance in available forms in the water
and as gases that they rarely become limiting factors in the cycle
of plant and animal life.
, When fresh organic substances, whether of plant or of animal

SYNTHETIC ACTIVITIES OF MICROORGANISMS 245

origin, are introduced into the soil, the numerous bacteria, fungi,
protozoa and other invertebrates immediately become active
and rapidly attack the various constituents, changing them back
to the simple compounds from which the plant started to manu-
facture its tissues. This process or group of processes may be
carried out by one or more than one group of organisms, through
one reaction or through a series of very compUcated reactions,
usually one organism following another or one competing with
another, the nature of the organisms and the mechanism of trans-
formation depending upon the nature of the organic matter and
environmental soil conditions. The sum total of the activities
of the various organisms in bringing about the transformation of
the complex plant and animal organic materials into simple inor-
ganic elements or compounds is known as the nmieralization of
organic matter, frequently spoken of in the older literature as decay
and putrefaction.

In the process of the mineralization of organic matter, many of
the elements are liberated in forms which cannot be assimilated
directly by higher plants. The mineralized inorganic elements or
simple inorganic compounds may have to be transformed first
into compounds which the plants can utilize and assimilate more
readily. For example, in the decomposition of organic matter,
the nitrogen is liberated as ammonia, while most plants prefer and
many even require the nitrogen in the form of nitrate; the sulfur
may be hberated as hydrogen sulfide, while the plant needs its
sulfur as sulfate. A large part of the carbon may be left in the
form of various organic acids, aldehydes, and alcohols, while the
plant uses its carbon as carbon dioxide. The final transformation
of the materials into forms available for higher plants depends
again upon the activities of various groups of microbes, some oxi-
dizing the ammonia to nitrous acid then to nitric acid which com-
bines with the soil bases to give nitrates; others oxidize the sulfides
to sulfates; still others oxidize the various organic acids, alde-
hydes, and alcohols, hberating carbon dioxide.

Synthetic Activities of Microorganisms. — As a result of
their various activities, the microorganisms in the soil build up con-
siderable organic matter of their own. Microorganisms are not
like catalysts, which do not increase or decrease in amount in
bringing about a certain reaction; on the contrary, the reactions
brought about by a microbe result in synthesis of microbial cell

246 IMPORTANCE OF MICROBES IN SOIL FERTILITY

substance, with the result that certain of the simple products
formed in the reaction are once more built up into complex sub-
stances. The microbe may be likened in this respect to any other
living organism which is large enough to be seen with the naked
eye. Food is consumed by an animal in the form of organic
compounds which furnish energy for it« development. Some of
the organic materials are broken down with the liberation of
energy which is utiUzed for synthesizing cell substance and con-
ducting other vital processes. During active growth, an appre-
ciable part of the consumed elements remains in the body as tis-
sues of the animal. The remainder is eliminated as waste products
such as carbon dioxide, water, urea, and a variety of organic sub-
stances. Similarly, in the development of the microbe, the trans-
formations are associated with cell growth and Uberation of waste
products.

Although the results of this synthesis may or may not
be desirable from the point of view of soil fertihty, this is
merely a result of the nutritional development of microorganisms
under the environmental conditions existing in the specific habitat.
The organism develops in competition with its associates to make
the best growth possible under the circumstances. In the absence
of growing plants for a period of time, there is a continuous deple-
tion of sources of energy for cell development, and the waste
products of the cells become the simple inorganic substances from
which further plant growth develops. The renewal of growth of
higher plants again starts the supply of food for the microbe.

Each reaction brought about by microbes is accompanied by a
definite amount of growth as indicated by the amount of cell
substance synthesized, depending upon the nature of the organism,
amount of energy and nutrients available, and environmental con-
ditions. The larger organisms of the soil, namely, those of an
animal nature, feed on the smaller microbes. These numerous
changes thus result in the manufacture of a considerable amount
of organic matter of microbial origin in the soil. Frequently
large quantities of nitrate nitrogen, phosphates, and other nutrient
elements which are so essential to the growth of higher plants are
temporarily stored away in the bodies of the microbes. In carry-
ing out these processes, the microbes compete with the green
plants for the available soil nutrients. Fortunately, even the
microbes are not invulnerable against the attack of their own kind,

NITROGEN FIXATION IN SOIL 247

and the process of mineralization continues even in the case of
microbial cell constituents. A part of the nutrients may remain
for a more or less extended period of time, thus locked up in the
bodies of the microbes, or in the form of disintegration products of
their cells.

Disappearance of Nitrogen from the Soil. — There are
other ways whereby microorganisms may bring about processes
which tend to injure the growth of the green plants, as by the
reduction of nitrates to atmospheric nitrogen carried out under
certain conditions and by certain organisms. Once the nitrogen is
changed into its elementary or gaseous form, it is lost from the soil
as far as the green plants are concerned. Notwithstanding the
fact that nearly 80 per cent of the gases of the atmosphere is made
up of nitrogen, which amounts to many thousands of tons (31,250)
over each acre of soil, nitrogen is usually the limiting element in
the growth of green plants. This is simply due to the fact that
this gaseous nitrogen is inert as far as green plants are concerned.
Any activities of microorganisms which tend to change even a
small fraction of the limited supply of combined nitrogen into the
gaseous form are distinctly injurious to plant growth. As a result
of continued cropping, certain farm practices, and natural environ-
mental conditions, these losses of nitrogen may be far from negli-
gible.

Nitrogen may be removed in crop plants, later fed to animals
and a small portion returned to the soil as excretion products of
these animals. It may be lost as volatilized ammonia from manure
heaps. This may later become precipitated upon the soil and
become available to plants, but, from a practical standpoint, the
nitrogen volatilized is of httle value. Nitrogen may become
stored away in the cells of many microorganisms at least tem-
porarily. Nitrates may be removed from soils in the natural
leaching waters in humid cHmates and in the irrigation waters in
arid regions. Nitrogen may also disappear in some cases in the
gaseous form through the reduction of nitrates.

Nitrogen Fixation in Soil. — To counterbalance such losses
of the combined nitrogen from the soil, nature has provided certain
groups of organisms with the capacity of fixing atmospheric nitro-
gen, i.e., using the nitrogen in its gaseous elemental form and build-
ing from it compounds of nitrogen which sooner or later become
available to green plants. These microbes are known as nitrogen-

248 IMPORTANCE OF MICROBES IN SOIL FERTILITY

fixing bacteria. Some, as the non-symbiotic forms, live in a free
state in the soil and require sources of energy (in the form of various
carbohydrates) to enable them to fix the nitrogen. Others live in
the roots of certain plants (largely legumes), or even in the leaves,
the bacteria fixing the nitrogen and making it available to the
plants, and the latter manufacturing sugars and other carbohy-
drates which they supply to the bacteria as sources of energy.
This mutually beneficial growth of green plants and bacteria is
known as symbiosis, and the bacteria are known as symbiotic
nitrogen-fixing bacteria.

Subsequent to the elimination of nitrogenous compounds or the
death of the bacteria, the complex proteins produced by fixation
processes are sooner or later minerahzed by other bacteria, fungi,
or actinomyces, and the nitrogen made available to free-growing
green plants. These nitrogen-fixing bacteria may be consumed by
protozoa, but no loss of nitrogen is involved in this process. The
protozoa use the organic nitrogen in their nutrition and in turn
die and themselves pass through the mineralization process.
Certain nitrogen-fixing bacteria may five symbiotically not only
with higher plants such as the Leguminosae, but also with certain
microscopic green plants as the algae. This associative develop-
ment appears to favor both the algae, which synthesize carbo-
hydrates from the carbon dioxide of the air, and the bacteria, that
build up complex organic proteins and other nitrogen compounds
from the gaseous nitrogen of the atmosphere; each of the two
groups of organisms is able to use the products synthesized by the
other, thus bringing about processes which lead both to an increase
of soil organic matter and of combined soil nitrogen.

Role of Microbial Metabolic Products in Soil Trans-
formations.— The metabolic processes of the microorganisms
become very involved because of the fact that the medium, the
soil, is so complex, containing an almost limitless variety of
organic and inorganic substances. The environment may be
very different in one locality from that in another, on account of
the inherent soil characteristics and the influence of climate and
vegetation. The conditions favor the development of a micro-
scopic population composed of an abundance of difi"erent forms
which further complicate the mechanism of the transformations
by their varied food requirements and metabolic products. Many
of the transformations of the soil constituents are affected by the

REDUCTION AND OXIDATION PROCESSES 249

microbes only indirectly, as the metabolic products exert their
effects as agents of solution, precipitation, oxidation, and reduc-
tion. A few illustrations may indicate the nature of these effects.

Phosphorus is frequently introduced into the soil in the form of
rock phosphate or as insoluble tri-calcium phosphate. In this
form it can be used by green plants to only a very limited extent.
When the phosphate is acted upon by the various organic acids
which are formed in the soil by fungi and bacteria (gluconic,
citric, oxalic, fumaric, lactic, butyric, formic, acetic, valerianic,
or by the carbonic acid, which is always present in abundance), it
is changed from an insoluble to a soluble form, which is more readily
available to plants.

Of the various acids produced by the microorganisms, the
carbonic and the organic acids exert much weaker effects as solvent
agents than some of the inorganic acids, because of their relatively
low ionization. Much more pronounced effects are exerted by the
inorganic acids formed by microbes, such as nitric, resulting from
the oxidation of ammonia, and sulfuric, formed from elementary
sulfur. Under natural soil conditions, relatively small amounts
of organic and strong mineral acids are produced by microor-
ganisms. On the other hand, carbon dioxide is produced in a con-
tinuous flow of greater or less intensity wherever living organisms
are present. Its effects upon more resistant soil minerals would
not be pronounced except over comparatively long periods. The
strong mineral acids, even if formed in small amounts, can liber-
ate some potassium from its combination with the zeolitic portions
of the soil and even from the more resistant soil minerals.

As a result of the decomposition processes which take place
in the soil, the reaction changes either to more acid or to more
alkaUne, and frequently one way, then another. The decomposi-
tion of urea, for example, leads to the formation of ammonium
carbonate, which tends to make the reaction of the soil more
alkaline; the ammonium carbonate is then oxidized to nitric acid,
which tends to make the reaction more acid than it was initially.

Reduction and Oxidation Processes. — Numerous other
reactions take place in the soil as a result of the activities of micro-
organisms, especially the processes of reduction and oxidation.
The following may be mentioned as suggestions of these reactions:
the reduction of nitrates to nitrites, to ammonia, to oxides of
nitrogen, or elementary nitrogen ; the reduction of sulfates to sul-

250 IMPORTANCE OF MICROBES IN SOIL FERTILITY

fides, of phosphates to phosphites and even phosphene; the reduc-
tion of selenium and tellurium compounds; the reduction of amino
acids and various organic compounds; the numerous oxidation
processes, some of which have been mentioned previously; the
various hydrolytic and other transformations of organic and inor-
ganic compounds in the soil.

Summary. — By comparing, in a very general way, the activities
of the microorganisms in the soil with those of a population of a
large modern country, certain interesting similarities are evident.
One gram of soil possesses as many micro-inhabitants as the total
human population of the United States. These inhabitants also
take part in as great a variety of activities as the human population.
Some are occupied with obtaining raw materials from the earth;
a part of these materials is consumed by those that labor for them,
while a part is turned over to the industries for further exploitation
and processing. The industrial workers consume a part of the
materials either in a raw or in an industrialized form and turn
back a part of the manufactured products to the original cultivators
of the soil. Influences responsible for the concentration of raw
materials in a favorable environment lead to an abnormally abun-
dant population in the region and an accelerated consumption of
these materials. Similarly, limitations of the food supply or lack
of favorable environmental conditions cause a quick depletion of
the population. Even the parasites of human society have their
equivalent in the society of the microscopic population below our
feet — these are the numerous fungi, nematodes, and bacteria that
can cause diseases of plants, or are so cannibalistic as to feed on
their own kind or on other members of the soil population of an
equal or lower social stratum.

Methods used in an approach to the study of the soil organisms
are all quite similar to those used in a study of the human popula-
tion. Certain observations are confined to an enumeration of the
total population, to those of different races or physical types, or
those concerned in specific activities. An entirely different type
of examination might be applied to studies of the activities of the
population in an attempt to estimate the amounts of raw materials
transformed, the rates of transformation, the end products
formed and their fate or further utilization.

INDEX

Absorption of nutrients l)y plants, 17
Abundance of bacteria in soil, 56-58
Acetic acid, 164

Acids, interaction of, with minerals,
156-157, 160-161
organic, formed by bacteria, 80-81,
86-87
formed })y fungi, 80
formed in cellulose decomposi-
tion, 86-87
production of, by microbes, 249
Acrostalagmus, 32
Actinomyces, 35-37, 65, 66, 67
decomposing cellulose, 82, 87
Actinomyces scabies, influence of re-
action on, 221, 231-232
Activities of microbes, 42, 48-50
Alfalfa, nitrogen assimilation during

decomposition of, 95-96
Algae, as corrosive agents, 157

blue-green, fixation of nitrogen

by, 106
green, relationship to nitrogen

fixation, 111
in soil, 26, 37-38, 70-71
symbiosis with bacteria, 248
Amino acids, decomposition of, 127-
128, 164
nature of, 126-127
Ammonia, fate of, in soil, 128, 135
formation of, by microbes, 127-135
from amino acids, 127-128
from animal manure, 130, 140
from nitrate, 147, 150
from proteins, 131
influence of partial sterilization

on, 225-227
influence of carbohydrates on,
132-133

Ammonia, formation of, influence of
environmental conditions on,
134
influence of nitrogen content of
organic matter on, 96, 99, 132-
135, 146
influence of, on nitrification, 140-

141
microbes concerned in formation of,

131, 134
oxidation of, 136-145
Ammonification, 131-134

as an index of soU fertility, 234-235
Amoebae, 38
Anaerobic bacteria, 31
Anaerobic decomposition of organic

matter, 155
Animal products, kinds of, added to

soil, 76
Animal substances, classification of,
76-77
composition of, 76-79
Animals, dependence of, on plants,
244
relation of microbes to, 45
Arid soils, 7
Artificial manure, 216
Ash of plants, 78, 79
Aspergillus, 30, 33
Atmosphere, nitrogen in, 247
Autotrophic bacteria, 26, 137, 156
Auximones, 195

Availability of elements, microbial
methods of determination,
237-240
Azotohacter, 106-113

action of, on minerals, 165
use of, for determining phosphorus
avaUabihty, 238-239
Azotohacter chroococcum, 24

251

252

INDEX

B
Bacilli in soU, 28
Bacillus cellulosae dissolvens, 23
Bacillus megatherium, 22
Bacteria, abundance of, as index of
soil fertility, 236-237
decomposing cellulose, 82, 85-87
effects of partial sterilization on,

224-229
of soil, 27-33

abundance of, 56-58

influence of manure on, 214,
216
activities of, 30-33

influence of tillage on, 216-217
influence of reaction on cellulose

decomposition by, 85-86
nitrogen fixing, 104-126
nodule, life cycle of, 115-117
numbers of, 46-47, 48, 52, 56-65
penetration of roots by, 201
Bacteriorrhiza, 200-201
Bacterium coli, in manure, 214

in soil, 44
Bacterium radicicola, 25, 114, 125, 200
Bacteroids, 116
Balantiophorus elongatus, 40
Bases in soil, 17
Basidiomycetes, 34
Bodo caiidatus, 40
Bumillaria exilis, 37
Butyric acid, 163, 164

C

Carbohydrates, influence of, on am-
monia formation, 132-133
Carbon, assimilation in decomposition
of organic matter, 94-99, 133
changes during decomposition, 97-

99
content of microbial cells, 94-95
effects of microbes on, 243-244
Carbon dioxide, action of, on min-
erals, 160
evolution of, 181
in atmosphere and plant growth,

182-183
in soil, 158-161

Carbon dioxide, in soil, influence of
partial sterilization on, 225-
226
liberation of, by microbes, 18-19
production of, in decomposition
processes, 80-81, 86, 90, 97-
101, 146, 157
in soil, 157

as index of soU fertility, 237
Carbon-nitrogen ratio of soil organic

matter, 99-101
Cellulose, 77, 78

combination of, with lignin, 82
decomposition of, aerobic, 82-86,
89, 91
anaerobic, 85-87, 89
as index of soil fertility, 235
influence of, on formation of soil
humus, 87
on nitrogen assimilation by

bacteria, 95-96
on numbers of fungi in soil, 83
influence of environmental con-
ditions on, 82, 84-86, 89
influence of manures on, 84
influence of moisture on organ-
isms causing, 88
influence of nitrogen on, 83, 84,

86-87
influence of phosphorus on, 84-85
influence of reaction on, 83, 84
influence of soil conditions on, 87
products of, 86-87
occurrence in plants, 82
organisms decomposing, 82
Cellulose bacteria, thermophilic, 86
Cellulose-nitrogen ratio, 83-84
Chemical composition of soil, 5-8, 16
Chitin, decomposition of, 130
Chlamydomonas com?nunis, 38
Chlorophyceae, 37
Ciliates, 38
Citric acid, 163

Climate, effect of, on chemical com-
position of soil, 6-8
influence of, on microbial devel-
opment, 63
Clostridium botulinus, 40

INDEX

253

Clostridium pastorianum, 24, 107

Clostridium tetani, 40

Cocci, 28

Colloidal material in soil, 10

Colpoda steinii, 40

Composition of plant and animal
substances, 76-79
influence of, on decomposition, 78,
79
on products of decomposition,
93, 128, 133, 146
variation of, with growth, 78, 79

Composting sulfur with rock phos-
phate, 171

Cropping, influence of, upon bacteria
of soil, 59

Cultivation of soil, influence of, on
soil microbes, 216, 217

Cultural methods for the study of
microbes, 24, 50, 52-56

Cyanamid, decomposition of, 129

Cyanophyceae, 37

Cycles of elements, 243-245

Cystine, decomposition of, by micro-
organisms, 169

Cytophaga lutea, 23

D
Decay, 30

Decomposition, of cellulose, 82-87,
89, 91
influence of nitrogen on, 84, 86

factors affecting, 78, 79, 92

of amino acids, 127-128

of fats, 89-90

of hemicelluloses, 81-82, 89, 91

of lignin, 88-89

of methane, 90

of nitrogenous compounds, 126-135

of organic matter, 14, 75-101, 204-
216

of pentosans, 81

of plant material, influence of com-
position on, 91-92, 143, 146

of proteins, 126-129, 131

of sugars and their derivatives, 79-
81

products of, 75, 79-81

Denitrification, 150-152

importance of, in soil, 150-152
in manure, 151

in soil, factors affecting, 151-152
Desiccation of soil, influence of, on

fungi, 68
Diatomaceae, 37
Disease-producing organisms in soil,

40-41
Distribution of microbes in soil,

45-48
Dried blood, nitrogen assimilation
during decomposition of, 96,
133

E

Ectotrophic mycorrhiza, 197
Elective culture method, 55-56
Elements, abundance of, in earth's
crust, 4
and growth of microorganisms, 154
as plant nutrients, 15
Endotrophic mycorrhiza, 197
Enumeration of microbes, 24
Environmental conditions, influence
of, on nitrogen fixation, 111-
113
Environmental factors affecting de-
composition, 79, 87
effects of, on soil organisms, 74
Enzymes of microorganisms, 26

Fat, decomposition of, 89-90, 164
Fats and waxes in plants, 78, 79
Fermentation, 30
Fertility, biological estimation of,

234-240
Fertilizer practice, influence of, upon

bacteria of soil, 59-60
Fertilizers, influence of, on soil

microbes, 221-224
nature of effects of, on microbes,

222-224
Filamentous fungi, 33, 35
Fixation of atmospheric nitrogen,

104-126
Flagellates, 38, 39

254

INDEX

Forest humus, nature of, 82-83
Forest soils, inoculation of, 229-230
Formic acid, 171

Fungi, activity of, in raw-humus soils,
82
assimilation of nitrogen by, in
decomposition of cellulose,
83-84, 95-96
decomposing cellulose, 82-85
decomposition of sugars by, 79-81
in soil, 23, 33-35, 59, 66-69

influence of cellulose on number

of, 83
influence of partial sterilization

on, 227-228
influence of reaction on, 220
Fungi Imperfecti, 33
Fusarium, 34, 41

G

Gaseous phase of soil, 13

Gases, formation of, in soil, 10, 13, 184

Glucose, products of decomposition

of, 80-81
Glutamic acid, decomposition of, 164
Growth of microbes, 245-246
Gums and slimes produced by micro-
organisms, 82, 86

H

Hardpans, 176

Heat produced l)y microbes, 237
Hemicelluloses, decomposition of, 81-
82, 89, 91
occurrence of, in plants, 81
synthesis of, by microorganisms,
81-82
Hormodendrum, 34
Humus, forest, nature of, 82-83
formation of, in cellulose decom-
position, 87
soil, formation of, 99-101
Hydrocarbons, decomposition of, 90-

91
Hydrogen from carbohydrate de-
composition, 80, 86
Hyphomycetes, 33

Importance of microbes in soil, 21-23

Infection of legumes by nitrogen-
fixing bacteria, 113-126

Inoculation of soils, 126, 229-234

Inorganic salts as sources of oxygen,
156

Insects in soil, 39-40

Intermediate products, formation of,
during decomposition, 75,
79-81

Invertebrates in soil, 72

Iron bacteria, 176-177

Iron in soil, 18

transformation of, by microbes,
176-178

Isolation of microbes, 24

Lactic acid, 163, 165, 166
Lecithin, decomposition of, 173
Legume bacteria, morphology of,

115-117
Legume nodules, 118-119
Legumes, influence of, on soil ni-
trogen, 124
influence of nitrate on growth of,

120-124
inoculation of soils for growth of,

229-234
invasion of, by nodule bacteria,

114-117
means of inoculation of, 231
nutrition of, 15
Leguminosae, bacteria of, 105, 113-

126
Leptoihrix crassa, 177
Lignin, 77-78

abundance of, in plants, 88
combination of, with cellulose, 82
decomposition of, 88-89, 91
in animal manure, 211-212
Ligno-cellulose of plant tissues, 78, 82
Lime in soil, 16, 18
Liming, influence of, on soil microbes,
217-221
I Liquid phase of soil, 12-13

INDEX

255

M

Magnesium in soil, 18
Manure, animal, composition of,
209-212
decomposition of, 209-216
influence of, on soil microbes, 209,

214-216
losses of nitrogen from, 213-214
microbes in, 209-210, 214
artificial, 216
green, influence of, on soil microbes,

206-208, 214
influence of, on cellulose decompo-
sition, 84
preservation of, 143, 151
Mechanical analysis of soil, 6
Methane, decomposition of, 90
from cellulose decomposition, 86
from sugar decomposition, 80
Microbes, activities of, 21-42
importance of, in soil, 21-23,

242-250
influence of organic matter on,

204-216, 219
modification of, 203-241
nature of, 23-25
role of, in plant growth, 13
in soU, 19

in supplying nitrogen to plants,
103
transformation of nitrogen l)y,
102-153
Microbial activities in soils, 243
correlation of, with soil fertility,
234-240
Microbial cells, carbon content of, 94-
95
decomposition of, 97
nitrogen content of, 94-95
Microbial population, similarities to

human population, 250
Microscopic methods for the study of

microbes, 24, 50-52, 57
Mineral assimilation by microorgan-
isms, 167
Mineral composition of soil, 4-5
Mineral elements as sources of energy,
155-156

Mineral substances, transformation

of, in soil, 154-180
Mineralization of organic matter,

243-245
Moisture, influence of, on carbon
dioxide in soil, 160
on nitrification, 143-144
on numbers of soil microbes, 61-

63
on organisms decomposing cel-
lulose, 88
Molds, 33

Mononchus papillatus, 42
Mucor, 33, 34

Mull, formation of, 205-206
Mull soils, nitrification in, 139

organisms in, 139
Mushroom fungi, 34
Mycorrhiza, 196-200, 205
fungi, nitrogen fixation by, 200
parasitic action of, 200

N

Navicula terricola, 38
Nematodes in soil, 39
Nitrate, avenues of loss from sod,
147-148
formation, 136-146
influence of, on cellulose decom-
position, 87
on nitrogen fixation, 110, 120-124
production of, in decomposition

processes, 93, 146
products of reduction of, 147-148
reduction, 145-152, 156
mechanism of, 147-149
organisms concerned in, 148
Nitre spots, 141
Nitrification, 136-146
bacteria causing, 136-140
factors affecting, 136-145
in manures, 143
in soil, influence of desiccation on,

144
index of soil fertility, 235-236
influence of, on plant growth, 139
on soil reaction, 142

256

INDEX

Nitrification, influence of aeration on,
137-139, 142-143
influence of ammonia on, 140-141
influence of moisture on, 143-144
influence of plants on, 145
influence of organic matter on, 143,

146
influence of reaction on, 137-142
Nitrifying bacteria, abundance of, in
soil, 138
influence of partial sterilization on,

138-139
influence of reaction on, 220-221
occurrence of, 137-138
Nitrobacter, 27

Nitrogen, absorption of, by plants, 17
amounts of, fixed by non-symbiotic
bacteria, 110-111
fixed by symbiotic bacteria, 120-
124
assimilation of, by bacteria, in-
fluence of cellulose on, 95-96
by fungi, influence of cellulose on,

83-84, 95-96
during decomposition of organic
materials, 94-100, 132-135
changes during decomposition, 97-

99
content, of microbial cells, 94-95
of organic matter, influence of, on
products of decomposition,
95-100, 132-135, 146
of soil, 8, 103
importance in nutrition of microbes,

26
in animal manure, 210-216
influence of, on cellulose decompo-
sition, 83, 84
inorganic, removal of, from soil,

103
in plant nutrition, 14, 15
in soil, 16

loss from soil, 145-152, 247
losses from animal manure, 213-214
organic, added to soil, 103-104
role of microbes in transformations

of, 104-105
soil, availability of, 102-103

Nitrogen, soil, influence of decom-
position of organic matter

on, 92-101
sources of, in soil, 102-104
transformation of, by soil microbes,

102-153, 247
Nitrogen-fixation, 104-126, 194, 247-

248
by blue-green algae, 106
by nodule-forming bacteria, 105,

113-126
correlation with growth of bacterial

cells, 106-108
factors associated with, 107
influence of glucose on, 107
influence of nitrates on, 110, 120-

124
influence of phosphorus on, 113
influence of reaction on, 111-113,

124-125
influence of soil conditions on,

124-126
non-symbiotic, importance of, 109-

112
soil factors favoring, 111-113
symbiotic, biological nature of,

113-114
symbiotic, mechanism of, 117-118
Nitrogen-fixing bacteria, influence of,

on plant growth, 114-126
influence of reaction on, 220
non-symbiotic, 104-113
soil inoculation with, 125
sources of energy available to, 109
symbiotic, 105, 113-126
Nitrosomonas europea, 26
Nitric acid, action of, on phosphates,

162-163
in soil, 161-163
production in soil, 157
Nitrite, formation from ammonia,

136-145
occurrence in soil, 141
Nodule bacteria, 105, 113-126

nitrogen fixation by, 105, 113-126
specificity of, 119-120
Nodule formation, 200
Nodules of legumes, 118-119

INDEX

257

Nodules on roots of legumes, 114—117
Non-symbiotic nitrogen-fixing bac-
teria, 104-113
Nucleic acids, decomposition of, 173
Numbers of microbes in soil, 46—47,

48, 52, 56-58
Nutrients, absorption of, by plants, 17
Nutrition, of microbes, 25-27
of plants, 14-19

O

Occurrence of microbes in soil, 23,

44-45, 46-48
Olivine, weathering of, 4
Organic acids in soil, 163

production of, by microbes, 156-
157
Organic compounds, absorption of,
by plants, 194-195
kinds of, added to soil, 76
Organic materials, simplification of,

75
Organic matter, composition of, 78, 79
decomposition of, by microbes, 14,
75-101, 204-216
influence of, on microbial activ-
ities, 204-216
on nitrification, 143, 146
on soil microbes, 60-61, 83-85,

87, 92, 204-216, 219
on soil nitrogen, 92-101
mechanism of, 94—99
effects of microbes on, 244—245
insoU, 8, 9, 11-12, 16
loss of, during decomposition, 98-

101
nitrogenous, decomposition of, 126-

135
soil, carbon-nitrogen ratio of, 99-
101
Organic substances, influence of, on
physical condition of soil,
195
Orthoclase, weathering of, 4
Oxalic acid, 163

Oxygen, influence of, on nitrification,
137-139, 142-143

Parasitic nature of microbes, 22, 45
Parasitic nematodes, 41
Partial sterilization, 224-229
influence of, on nitrif3dng bacteria,
138-139
on soil microbes, 224-229
on soils, 224-228
Peat, formation of, 154-155
Peat soils, 12
Pecto-celluloses, 82
Pedolog}', science of, 1
Penicilliuni, 30, 33
Pentosans, decomposition of, 81
Phoma, fLxation of nitrogen by, 106
Phosphate, reduction of, 172-173
Phosphates, influence of, on soil

microbes, 222-223
Phosphoric acid in soil, 16, 18
Phosphorus, action of microbes on,
249
influence of, on cellulose decompo-
sition, 84-85
on nitrogen fixation, 113
in plant nutrition, 14, 15-16
in soil, 173

soil, availabihty of, determined by
nitrogen-fixing bacteria, 113
transformation of, by microbes,
173-176
Phosphorus availability, Aspergillus
niger method, 238
Azotobacter methods, 238-239
Phycomycetes, 33
Physical properties of soil, 10
Physical structure of soil, 12-13
Physiological methods for studying

microbes, 48
Phytic acid, decomposition of, 173
Plant, decomposition of, as a whole,

91-94, 97-101, 146
Plant constituents, most important,

77-78
Plant growth, importance of microbes
to, 242-243
influence of decomposition of or-
ganic matter on, 93

258

INDEX

Plant materials, kinds of, added to

soil, 76
Plant nutrients, 14-18
Plant substances, classification of,
76-77
composition of, 76-79
Plants, absorption of nutrients by, 17
influence of composition of, on
decomposition, 91-92, 133,
146
on bacterial numbers in soil, 189-

191
on carbon dioxide evolution from

soil, 186-187
on nitrification, 145
on soil microbes, 224
on soil processes, 182-186
proximate composition of, 78, 79
relation of microbes to, 45
variation in composition of, with

growth, 78, 79
water-soluble constituents of, 77,
79
Plants and microbes, 181-202
Plasmodiophora brassicae, control of,

221
Plate method, 52-55

for determination of fungi in soil, 68
Podsol soil, composition of, 3
Population of the soil, 41-42, 44-74
Potassium, availability, Aspergillus
niger method, 238, 240
in plant nutrition, 14, 15
in soil, 16, 18

transformation of, by microbes,
178-180
Potato scab, control, 231-232
Proteins, 78

decomposition of, 49, 90, 91, 126-

129, 131
nature of, 126-127
Protista, 24

Protozoa, effects of partial steriliza-
tion on, 225, 227-228
in soil, 23, 38-39, 69-70, 71
nutrition of, 39
Protozoan theory, 227-228
Pure cultures of microbes, 24

Putrefaction, 30
Pythium, 41

R

Raw humus, formation of, 205-206
soils, activity of fungi and bacteria

in, 82
nitrogen transformations in, 139
Reaction, influence of, on cellulose
decomposition, 83-86
on microbial development, 63-64
on nitrification, 137-142
on nitrogen fixation, 111-113,
124-125
Reaction of soil, 17
influence of, on soil microbes, 220-

221
influence of lime on, 217-218
influence of microbes on, 249
Reduction of nitrate, 145-152
Reduction processes of microbes, 249-

250
Rhizodonia, 41
Rhizopus, 30, 33

Rocks, disintegration of, by microbes,
180
weathering of, 2-6
Root excretions, 192-194
Roots, effects of microbes on, 201-202
influence of, on carbon dioxide

production, 161
solvent action of, as influenced by
microorganisms, 188

S
Saprophytic nature of microbes, 22,

45
Season of year, influence of, on
microbial development, 64-
65
Soil, as a medium for development of
microbes, 19
atmosphere, 159

chemical composition of, 3, 5, 6-8
conditions, influence of, on mi-
crobes, 87, 248
upon distribution of bacteria,
58-60

INDEX

259

Soil, environment, effects on soil mi-
crobes, 240-241
fertility, biological estimation of,

234-240
formation, 9-10, 179
gases, composition of, 159
horizons, 1, 2, 3
humus, formation of, 99-101
inoculation, 125, 203, 229-234
mechanical analysis of, 6
mineral and chemical composition

of, 4-7
moisture and numbers of micro))es,

61-63
morphology, 1-2
nature of, 1-6
nitrogen, influence of legumes on,

124
organic matter, 11-12
phases, 12-13
population, 41-42, 44-74

modification of, 74, 203-241,
242
profile, 1, 2
reaction, 17

change of, by microbes, 166-167
influence of nitrification on, 142
treatment, influence of, upon dis-
tribution of bacteria, 58-60
Soils, nitrogen content of, 103
Solid phase of soil, 12
Solution method, 55
Spirilla, 29

Spirillum desulfuricans, 29, 172
Staining of microbes, 50-52
Starch, decomposition of, 81
Straw, nitrogen assimilation during

decomposition of, 95-96
Sugars, decomposition of, 79-81
products of aerobic decomposition

of, 80-81
products of decomposition of, by
bacteria, 80-81
Sulfate utilization by microorganisms,

168
Sulfates, influence of, on soil microbes,

222-223
Sulfides in soil, 169, 170

Sulfur, elementary, oxidation of, in
soil, 170
in plant nutrition, 14
in plant residues, 168
in soil, 16, 18, 168
oxidation of, 148
reduction of, in soil, 172
transformation of, by microbes,

167-173
use of, to control soil reaction, 221,
231-232
Sulfur bacteria, inoculation of soil

with, 229, 232
Sulfuric acid production in soil, 157
Symbiosis, 41, 117, 125
Symbiotic nitrogen-fLxing bacteria,

105, 113-126
Synthesis, by microbes, influence of
organic matter on, 93-99
by soil microbes, 245-247
influence of cellulose on, 84-85
of hemicelluloses by microorgan-
isms, 81-82
Synthetic processes of microbes, 14

Temperature, influence of, on carbon
dioxide in soil, 160
on nitrogen content of soil, 8
Thermophihc bacteria, 31
Thiobacillus group, 170
Thiobacillus thiooxidans, 28, 231
Toxins, plant, decomposition of, 90
Trichoderma, 32, 34
Trophic state of protozoa in soil, 70

U

Ultramicroscopic organisms, 29
Urea, decomposition of, 129-130, 212,
216
oxidation of, 163
Uric acid, decomposition of, 129

Vahlkampfia soli, 41
Vibrios, 29

260

INDEX

W

Water, movement of, in soil, 10-11
Water-soluble constituents of plants,

77
Water supply to plants, 11
Weathering of rocks, 2-6

Worms in soil, 39-40
Y
Z

Yeasts, 34

Zygorhynchus, 33

V

K\M