MBLAVHOI

Library

Soil Microbiology

c

Soil Microbiology

SELMAN A. WAKSMAN

Professor of Microbiology
Rutgers University

JOHN WILEY & SONS, INC., NEW YORK
CHAPMAN & HALL, LIMITED, LONDON

Copyright, 1952

BY

John Wiley & Sons, Inc.

All Rights Reserved

This hook or any part thereof must not
he reproduced in any form -without
the ivritten permission of the publisher.

Library of Congress Catalog Card Number: 52-9965

PRINTED IN THE UNITED STATES OF AMERICA

Preface

This is a book about the Hfe in the soil, the soil under our feet, the
soil in which our culti\'ated and unculti\'ated plants grow and from
which they derive most of their sustenance, and which in their turn
support animal life, including that of man himself. This book deals
with the numerous biological, physical, and chemical processes which
continuously go on in the soil and in which microbes are involved.
Without soil microbes, life on this planet would soon come to a stand-
still.

The soil is not a mass of dead debris, merely resulting from the
physical and chemical weathering of rocks; it is a more or less ho-
mogeneous system which has resulted from the decomposition of
plant and animal remains. It is teeming with life. The numerous
living forms which spend all or part of their life in the soil range
from the submicroscopic viruses and phages, through the micro-
scopic bacteria, actinomycetes, fungi, algae, and protozoa, to the
lower animal forms, the worms, insects, and rotifers, many of which
can be seen and recognized with the naked eye. These organisms,
comprising both the living forms and their dead bodies, as well as
the products of theii- decomposition, such as carbon dioxide and or-
ganic acids, interact with the rock constituents to give rise to soil.
The soil thus gains the characteristic properties that make it a suit-
able medium for plant and animal life.

The aims of this book are to survey the nature and abundance of
microorganisms in the soil, to review the important role that they
play in soil processes, and, so far as possible, to show the relation be-
tween them and soil fertility ( including plant nutrition and crop pro-
duction ) . I have attempted to point out some of the more promising
lines of advance in the field of soil microbiology and to suggest some
likely paths for future study.

This book presents broadly a discussion of the soil microbiological
population, the general flora and fauna of the soil, and the mutual
interrelationships among microorganisms; the decomposition of plant
and animal residues and the formation of humus; the transformation
of various elements essential for plant growth; and the general appli-

VI

Preface

cations of soil microbiology to other fields of knowledge, especially
soil formation and plant nutrition. It should not be considered a
reference book, but rather a textbook. No attempt has been made
to give complete bibliographies. Appended to each chapter, how-
ever, are references to some of the more important papers and books
available in the English language, where the student and general
reader can find up-to-date, concise statements concerning the prob-
lems discussed in the respective chapters and dealing with the various
phases of soil microbiology.

This book follows along the general lines of two previous volumes,
Principles of Soil Microbiology (Williams & Wilkins Co., 1st Ed.,
1927; 2nd Ed., 1932) and The Soil and the Microbe (John Wiley &
Sons, 1931). I have drawn heavily upon the texts as well as upon
the tables and illustrations of these earlier publications. This book
may, therefore, be looked upon as a logical outgrowth of the older
volumes, standing midway between the one that was more detailed
and the other that was more concise and even more elementary.

In the preparation of this book, no attempt was made to cover in
a comprehensive manner every aspect of the subject of the micro-
organisms inhabiting the soil, or of the various processes for which
they may be responsible, or of the importance of these in soil fer-
tility. It may even appear to some of those who will read this book
that some aspects of the subject are treated rather sketchily, whereas
others are discussed in great detail. This was bound to be so in a
book covering such a vast subject as the complex microbiological
population of the soil and its role in soil fertility and plant growth.
My intention was to present here a broad outline of the subject, one
might even say a philosophy of soil microbiology. The supplemen-
tary books and papers recommended at the end of each chapter will
help to fill the gaps and suggest additional sources of information
on the various aspects of the subject to those who are eager to enlarge
upon the information supplied in this \'olinTie.

Selman a. Waksman

Neiv Brunswick, New Jersey
June, 1952

Contents

CHAPTER

1 • Historical 1

2 • The microbiological population of the soil as a whole . 29

3 • Occurrence of specific microorganisms in the soil ... 59

4 • Decomposition of plant and animal residues in soils and in

composts 95

5 • Humus: nature and formation 124

6 • Decomposition of soil organic matter and evolution of car-

bon dioxide 149

7 • Transformation of nitrogen in soil; nitrate formation and

nitrate reduction 166

8 • Nitrogen fi.xation— nonsymbiotic 191

9 • Nitrogen fixation— symbiotic 20S

10 ■ Transformation of mineral substances in soil by micro-

organisms 230

11 • Higher plants and soil microorganisms 246

12 • Associatixe and antagonistic effects of soil microorganisms 261

13 ■ Disease-producing microorganisms in the soil and their

control 282

14 • Stable manures, composts, and green manures 303

15 • Microorganisms and soil fertility 323

16 • Recent developments in soil microbiology .... 337

Index 347

vu

/

Historical

It has been recognized since the dawn of microbiology that the
soil is inhabited by a living microscopic population which is respon-
sible for the numerous reactions that take place in the soil and that
affect the life and economy of man in many ways. Some of these
reactions brought about by the microorganisms in the soil are highly
beneficial, such as the destruction of various dead plant and animal
residues that find their way into the soil; other reactions are injurious
to plant and animal life, such as development of organisms which
serve as potential sources of many plant and animal diseases. Within
recent years, our knowledge of microbes in general and of soil mi-
crobes in particular, as they affect the cycle of life in nature, has
been greatly advanced to a point where the role of these organisms
in the transformation of matter and their importance in soil processes
and plant growth have been appreciated.

Less than a century ago a battle was in progress among chemists,
plant physiologists, agronomists, and microbiologists. The contro-
versy centered about the role of microbes in the decomposition or
"fermentation" processes and in a number of other important reac-
tions that occur in nature, such as the effect of legumes on the
subsequent growth of cereals.

The chemists, as typified by Justus von Liebig, as well as by
Berzelius and Wohler, maintained that the evolution of carbon
dioxide that takes place during the disintegration of sugars and
other organic materials was a purely chemical reaction, which was
described as "eremacausis." Liebig wrote: "All plants and vegetable
structures undergo two processes of decomposition after death. One
of these is named fermentation, the other decay, putrefaction, or
eremacausis. Decay is a slow process of combustion, a process,
therefore, in which the combustible parts of a plant unite with the
oxygen of the atmosphere." Berzelius believed that yeast was not a

1

2 Historical

living organism but a noncrystalline chemical substance similar to
a precipitate of alumina. The chemists looked with contempt upon
the experiments of the biologists as frivolous and nonscientific. Fer-
mentation was considered a mysterious catalytic force.

The plant physiologists, as typified by Dumas and Boussingault,
also failed to recognize the role of microbes in the cycle of life in
nature. They considered the animals and the plants the only living
forms that participate in this cycle. They stated, "Tout ce que I'air
donne aux plantes, les plantes le cedent aux animaux, les animaux
le rendent a I'air; cercle eternel dans lequel la vie s'agite et se mani-
feste, mais ou la matiere ne fait que changer de place" (Chemical
Statics of Organized Bodies). Matter was thus believed to be in
a state of change between plant and animal bodies.

It was Pasteur, the microbiologist, who emphasized the micro-
biological nature of the processes of transformation of organic mat-
ter in nature. He established beyond question that, in addition to
plants and animals, a third group of living forms, the microbes, par-
ticipate in the cycle of life; they bring about the mineralization of
residues of both plant and animal life and retransform the elements
into forms available for fresh plant growth.

Since the epoch-making contributions to the new science of micro-
biology by Pasteur and others, there have gradually emerged two
distinct phases of this science, the medical and the biochemical.
These have frequently overlapped and just as frequently diverged
from one another. Their attitudes toward the role of microorganisms
in natural processes can be summarized from their respective view-
points.

1. Microorganisms as Disease-Producing Agents. The role of
microorganisms as causative agents of disease gave rise to a branch
of microbiology which is usually described by the terms "medical
bacteriology," "medical mycology," and "plant pathology." It deals
with the causation of disease, with infections and epidemics, among
human beings and other animals, as well as among plants. This
newly acquired knowledge has revolutionized our whole concept of
public health and disease.

2. Microorganisms as Chemical Reagents. A study of the chem-
ical activities of microorganisms led to the development of microbial
physiology and biochemistry. It has resulted in numerous applica-
tions of microbiology to various fields of human endeavor, as indi-
cated by the terms "dairy bacteriology," "soil microbiology," "sewage
bacteriology," "microbiology of foodstuffs," and "industrial micro-

Historical 3

biology." These sciences are based on the appHcation and practical
utilization of the activities of microorganisms. They also deal with
the cycle of life in nature, notably the numerous decomposition
processes whereby microorganisms liberate products that are useful

Fig. 1. Sir John Lawcs established the Rothamsted Experimental Station, in
which the most continuous experimental studies on soil fertility have been car-
ried out for more tlian a century. Some of the fundamental studies of the
microorganisms of the soil have been carried out at that institution.

or even essential for the growth of plants and animals. The knowl-
edge thus gained has revolutionized many agricultural practices and
has contributed to the discovery of many new processes, ranging from
industrial fermentations to the manufacture of chemotherapeutic or
disease-combating agents.

Pasteur established the fact that the mechanism of decomposition
of organic matter by microorganisms depends not only upon the
natiu'e of the organisms but also upon the environmental conditions
under which the process is taking place. In the presence of oxygen,

4 Historical

or under aerobic conditions, a carbohydrate will be completely de-
stroyed with the production of carbon dioxide. In the absence of
oxygen, or under anaerobic conditions, the carbohydrate will be
only incompletely attacked, with the formation of alcohols, organic
acids, and certain gases, such as hydrogen, methane, and carbon
dioxide. The latter process, resulting from "life without air," came to
be known as "fermentation," although this term has often been
applied, quite incorrectly, to microbial life as a whole.

The Concept of Soil Microbiology

With the growing recognition of the numerous processes carried
out by the bacteria, fungi, and other microbes in the soil, there
gradually emerged a branch of microbiology which came to be
known as "soil microbiology." This deals with the microscopic
population of the soil, its role in the various transformations taking
place in the soil, and its importance in plant nutrition and crop pro-
duction. It concerns itself not only with the enumeration and classi-
fication of soil-inhabiting microorganisms, but also with the measure-
ment of their activities in the soil, notably the decomposition of or-
ganic substances that are present in the soil or that find their way
into the soil, with the production of ammonia and nitrates, with
the fixation of nitrogen, and with numerous other transformations.
The soil microbiologist is thus concerned with the isolation, identi-
fication, and description of the important groups of microbes occur-
ring in the soil, as well as with the part they play in the physical and
chemical changes that are brought about in that complex natural
substrate.

Four distinct phases of soil microbiology have gradually emerged:

1. The ecological phase, which comprises the study of the quanti-
tative and qualitative composition of the microscopic and ultra-
microscopic soil population.

2. The experimental or physiological phase, which includes the
study of the physiology and the biochemistry of the organisms, their
role in the cycle of life in nature, and their utilization for the forma-
tion of valuable metabolic products.

3. The agronomical phase, or the application of microbiological
activities to soil fertility and crop production.

4. The pedological phase, or the importance of microorganisms in
soil formation and soil structure.

The Concept of Soil Microbiology 5

From the point of \'icw of their economic value, the nmnerous soil-
inhabiting microorganisms include forms which are useful and highly
important for plant and animal life; these take part in the decomposi-
tion of organic residues and in the liberation of the nutrient elements

Fig. 2. Sir Henry Gilbert, the first chemist of the Rothamsted Experimental

Station.

in available forms, as well as in numerous other transformations of
various elements and compounds which are essential for the con-
tinuation of life on this planet. Other groups of soil-inhabiting
microorganisms include other forms of economic importance which
are injurious to plant and animal life; this effect may either be direct,
by attacking and destroying the higher forms of life, or indirect, by
transforming certain chemical compounds into substances injurious
to living processes. Here belong the numerous plant and animal
pathogens that find in the soil a permanent or temporary habitat.

6 Historical

Our knowledge of the microbiological population of the soil and
its importance in the continuation of life on this planet has been
gradually accumulating during the last two and a half centuries,
but it is only during the last seventy-five years that rapid progress
has been made in dealing with this important branch of biology.
Various groups of investigators have contributed to this progress.

Fig. 3. J. B. J. D. Boussingault initiated during the first half of the nineteenth
century some of the most comprehensive investigations in agricultural chemistry

and physiology.

The medical bacteriologists were interested in the soil as a medium
for the growth and survival of disease-producing organisms. The
agricultural chemists were interested in the soil processes that are
important for the growth of cultivated plants and that result from
the activities of microorganisms. The general bacteriologists, bot-
anists, and zoologists were interested in certain special groups of
organisms found in the soil, because they presented special problems
in microbiology, either from theoretical consideration or from the
point of view of practical utilization, as illustrated in the recent
search for antibiotic-producing organisms. Finally, the soil micro-
biologists proper studied the soil population, either independently

Beginnings of Soil Microbiology 7

of liny practical applications or in an attempt to solve certain prac-
tical problems. Thus the science of s(")il microbiology came into
being.

Beginnings of Soil Microbiology (P-1860)

Although the beginnings of soil microbiology are attributable to
farm practices, on the one hand, and to the growing knowledge of
plant nutrition and of soil transformations, on the other, ample ref-
erence is found among the writings of the ancients and of the natural-
ists of the Middle Ages to the presence in the soil of certain minute
living organisms that affect, in various ways, our own life, as well
as that of our cultivated plants and animals.

First historical mention of the possible presence in the soil of
microscopic organisms which may directly influence the life of man
is usually credited to a Roman writer who said that swamps give
rise to minute animals which infect the an* and cause human diseases.
Columella wrote about 60 b.c. of the marshes throwing up "noxious
and poisonous steams" and breeding "animals armed with poisonous
stings," whereby "hidden diseases are often contracted, the causes
of which even physicians cannot properly understand."

Actual obser\ations of the presence of microorganisms in the soil
were reported in 1671 by Athanasius Kircheus, who wrote, "That the
air, water, and soil are inhabited by numerous insects is so certain
that it can be recognized with the naked eye; it was also known that
worms are formed on putrefying bodies; but only since the wonder-
ful discovery of the microscope has it become recognized that all rot-
ting bodies swarm with a numerous mass of worms not recognized
with the naked eye. I would have never believed that myself, had
I not been convinced by experiments frequently repeated during
many years." He cautioned about marshy lands near a home as
follows: "If they become dry, certain animalcules which the eye
cannot discern get into the body by the mouth and nose and propa-
gate obstinate diseases."

This report was soon followed by the classical studies of van
Leeuwenhoek, the "father of bacteriology," who used his now-famous
microscope to examine various materials for the presence of "ani-
malcules." These were found in rain water, in scum on teeth, and
in other materials.

The next one hundred years brought forth various investigations
of bacteria and other microorganisms found in natiu-e, but these
studies dealt primarily with the development of the microscope.

8 Historical

with determination of the size and shape of bacteria, and with at-
tempts at rudimentary classification. The activities of the organisms
and their role in natural processes either were not mentioned at all
or were merely a subject for speculation, although the important rela-
tion of the lower organisms to the higher forms of life, namely,
plants and animals, was gradually established.

It is sufficient to refer in this connection to the detailed knowledge
that existed concerning the production of nitrates in soils and in

Fig. 4. G. J. Mulder investigated in detail the chemistry of soil organic matter,
or humus, and its role in soil fertility.

composts. When, after the Revolution, the French Republic found
itself blockaded by the English Navy and deprived of the possibility
of importing nitrate from India, instructions were given to French
farmers to accumulate nitrate by the proper composting of their
stable manure. These instructions were so clear cut, and the bio-
logical processes involved so well understood, that it is obvious that
nearly a century before the nitrate-forming bacteria were isolated
their activities and importance were well appreciated.

By the middle of the last century, it was definitely established
that stable manure becomes a nutrient to plants only after it has
undergone a period of decomposition, or "fermentation," although
the exact nature of this process was hardly understood. The pro-
duction of ammonia and the liberation of heat, which are now recog-
nized to be the important phases of protein decomposition and of

Beginnings of Soil Microbiology 9

energy transformation by microorganisms, were, however, looked
npon as two distinct characteristics of the first stage of manure
decomposition.

Tlic be^innins; of our systematic knowledge of soil microorganisms

Fig. 5. Justus von Liebig contributed fundamental information to our knowledge
of soil processes and plant nutrition.

had to wait until the birth of general systematic bacteriology. In
1836-1839, several distinct contributions appeared which had an
indirect bearing upon knowledge of the activities of the microbial
population of the soil. These were the first careful studies on the
role of microorganisms in the decomposition of organic matter and
in the formation of alcohols and acids. The work of Schwann and
of Cagniard-Latour on the fermentation of wine and on the various

10 Historical

"putrefactions" due to living organisms was soon followed by that
of Kiitzing, in 1837, on the production of acetic acid. In 1841,
Boutron and Fremy studied the formation of lactic acid from sugar;
in 1844, Pelouze and Gelis studied the formation of butyric acid
under anaerobic conditions; and, in 1850, Mitscherlich made a care-
ful investigation of the decomposition of the cell wall of plants by
microorganisms.

Although Mitscherlich emphasized in 1843 that the two well-
known decomposition processes, designated as "fermentation" and
"putrefaction," were brought about by two different groups of micro-
organisms, the first by yeasts and the second by vibrios, it remained
for Pasteur to elucidate clearly in 1857 the principle of anaerobiosis
or life without atmospheric oxygen. These two men thus clearly
indicated the role of microorganisms, especially of bacteria, in a
number of processes which are found to be of the greatest importance
in the soil. Mitscherlich emphasized the role of bacteria in the de-
composition of carbohydrates, and Pasteur the role of bacteria and
other microorganisms in the decomposition of urea and other ni-
trogenous materials, processes of great importance in the degradation
of plant and animal residues in soils and in composts.

Foundation of Soil Microbiology (1861-1890)

About the middle of the last century three distinct biological
processes had been clearly outlined and were partly understood.
These were decomposition of organic matter, nitrification, and nitro-
gen fixation.

Organic matter decomposition was known to give rise to humus,
which was believed to be one of the fundamental factors in soil
fertility. Some investigators considered that humus was only an
intermediary product and not a plant food. Organic matter was
believed by chemists to decompose slowly by chemical oxidation.
The work of Wollny and others finally led to a better understanding
of the microbiological nature of the process.

The accumulation of nitrates in the soil as a result of decomposi-
tion of organic matter was known in the seventeenth and eighteenth
centuries, but only Boussingault and Schloesing connected this process
with soil fertility. Thus began a series of studies on nitrification
which was to have an important eft'ect upon our knowledge of soil
microbiological processes.

Foundation of Soil Microbiology

11

The use of legumes for enriehment of the soil was knowu to the
ancient Romans. Berthelot was the first to suggest that nitrogen
fixation may be accoinphslicd also l^y uonsymbiotie l)acteria. The

Fig. 6. Louis Pasteur established the role of microorganisms as causative agents
of diseases and fermentations.

organisms concerned both in nitrification and in nitrogen fixation
were not isolated until toward the close of the last century.

Thus, the work of the bacteriologists and chemists, of plant physi-
ologists and agricultural chemists, contributed to the understanding
of the cycle of life in the soil and laid the foundation for soil micro-
biology. Microbes, especially bacteria, were found to occur abun-

12 Historical

dantly in the soil and to be responsible for many soil transformations.
Attention may be directed, for example, to the work of Kette,
who emphasized in 1865 that the importance of addition of stable
manure to the soil was due to the fact that the manure could not
be replaced by inorganic nitrogen compounds and minerals or by
purely vegetable matter, because these lack "a true vibrion fermen-
tation."

Fig. 7. Ferdinand Cohn laid the basis for die classification of bacteria, including
many that occur in the soil.

Robert Koch introduced in 1881 the gelatin plate for the study of
bacteria, thus laying the basis for a systematic study of soil micro-
organisms. The gelatin was soon replaced by agar-agar as a solidify-
ing agent for bacteriological media. Those who followed Koch were
medical men who were more interested in public health and hygiene
than in soil processes. They limited themselves to a study of various
soil layers for the presence of bacteria and fungi that would develop
on the gelatin plate. Any organism that did not develop on the
plate was considered to be of no importance. The occurrence of
specific bacteria was studied primarily for the purpose of establishing
whether the soil contained pathogenic organisms. The pure-culture
techniques that were thus developed made, however, an important
contribution to bacteriology and had a significant effect upon the
study of soil bacteria.

In a study of the purification of sewage water, Pasteur made the
passing suggestion in 1862 that nitrification was due to bacterial
action. Schloesing and Mijntz found that, when a stream of sewage

Foundation of Soil Microbiology

13

was allowed to pass \ xuy slow!)' through a column of sand and lime-
stone, the ammonia in the sewage was at first unaffected, but, after
twenty days, it became conxertcd into nitrate, so that later the am-
monia disappeared and nitrate was found in its place. Addition of
a little chloroform vapor stopped the process completely; when the

Fig. 8. Robert Koch introduced the gelatin plate method for the enumeration of
bacterial population of the soil and made other important contributions to micro-
biology.

chloroform was removed and a little soil suspension added, the
process was started again. The role of "organized ferments" in the
process of nitrification was thus established.

The enrichment culture method was introduced by Beijerinck. It
consisted in using a selecti\e medium containing special nutrients,
for the purpose of stimulating the development of specific bacteria,
which were then isolated in pure culture. This method was utilized
by many bacteriologists, especially Winogradsky, and resulted in
the isolation of a number of organisms concerned in important soil

14

Historical

processes. This method contributed greatly to the solution of sev-
eral important problems in soil microbiology.

Among the numerous soil microbiological processes, none is of
greater importance than the fixation of nitrogen by leguminous
plants. This has an important historical background, which can be
separated into the following stages : ( 1 ) Our knowledge that legumes
enrich the soil, which dates back to the time of the Romans. (2)

Fig. 9. J. J. T. Schloesing demonstrated the biological nature of the process of

nitrification.

Boussingault's emphasis in 1838 that the favorable action of legumes
upon the soil is due to their power to fix atmospheric nitrogen.
(3) Lachmann's demonstration in 1858 and Woronin's in 1866, to
be followed by those of various other botanists, that nodules are
formed on the roots of leguminous plants. (4) Frank's demonstra-
tion in 1879 that the nodules on the roots of the plants are formed
as a result of inoculation with microorganisms. Hellriegel and Wil-
farth found in 1885 that, whereas the growth of nonleguminous
plants is proportional to the amount of nitrate added to the soil,
there is no such relationship in the case of leguminous plants. There
was no gain in nitrogen when the nitrogen content of the plant was
added to that of the sand in which nonleguminous plants were
growing, but there was a considerable gain in combined nitrogen

Foundation of Soil Microbiology 15

1)\ the leguminous plants. Hellriegel and Wilfarth concluded, there-
tore, that the legumes took the nitrogen 'from the air through the
agency of bacteria existing in the nodules of their roots. (5) Schloes-
ing and Laurent's discovery that the weight of the nitrogen absorbed
from the air by the leguminous plant is al:)out ecpial to the gain in
nitrogen by the plant and the soil. (6) The final isolation, by Beije-
rinck, of the organism responsible for the formation of nodules.

Numerous other important contributions to our knowledge of the
microbiological population of the soil were made during this period.
The work of Adametz on the fungus flora of the soil, of Miquel and
others on urea bacteria, of Gayon and Dupetit on denitrifying bac-
teria, of Hoppe-Seyler and others on anaerobic cellulose decomposi-
tion, and of Warington and others on the bacteria concerned in
the process of nitrification may serve as examples.

Determination of the role of microorganisms in decomposition
processes, unfortunately, did not make such rapid progress. The
clear and logical ideas of Pasteur were not always successfully fol-
lowed by the bacteriologists and especially by the chemists, who
attempted to apply them to various natural processes. They were
very frequently confused, especially by the introduction of numerous
terms to explain processes that were but vaguely understood. This
is best illustrated by the ideas of Wollny, who summarized the
status of the knowledge of decomposition of organic matter at the
end of the nineteenth century. According to Wollny, there were
three different classes of decomposition, as follows:

a. Decay or Aerobic Decomposition. This process was consid-
ered to be virtually a chemical "slow-burning" or "direct oxidation"
of the organic matter. It was believed to be similar to the "fire-
fanging" of manure, carried out "with or without the aid of minute
organisms" and "leaving essentially ash behind." The organic sub-
stances were said to be volatilized, the nitrogen changed to am-
monia, the carbon to carbon dioxide, the hydrogen to water, and
the sulfur to sulfuric acid.

b. Putrefaction. When the oxygen tension was low, the processes
were supposed to be different. Instead of carbon dioxide, water,
ammonia, and minerals, various gaseous products consisting largely
of methane, hydrogen, carbon dioxide, hydrogen sulfide, nitrous ox-
ide, and nitrogen gas were believed to be formed, and the organic
material left behind was described as rather dark colored and
highly resistant to further decomposition. This residual material
was reported to contain, in addition to nitrogen-free compounds

16 Historical

(various fatty acids such as formic, butyric, acetic, propionic, valeri-
anic), various nitrogen compounds (leucin, tyrosin, indol, skatol,
amines, amides, as well as ammonia and sometimes nitrite); the
minerals were said to be found largely in unassimilable forms.

c. Other Decomposition Processes. These were frequently
placed between the two previously mentioned categories, as in the
case of "Vermoderung," which was said to occur in nitrogen-poor
substances with a moderate water content and in the presence of air.
"Decay" and "putrefaction" were believed to take place at times
side by side, according to the oxygen content of the various layers,
as in manures. The various alcoholic and acid "fermentations" were
also included in these intermediary reactions.

These confusing ideas took no consideration of the microbes as
living organisms which possess a distinct metabolism and which use
organic matter as a source of energy and of nutrients. This con-
fusion carried over to more recent times. It was particularly evident
in the ideas current even as late as the end of the second decade of
this century, concerning the "humification" of organic matter in
nature, especially in soil, in peat bogs, and in coal formation.

The Golden Age of Soil Microbiology (1891-1910)

Once the foundation for soil microbiology was laid, the field was
open for contributions dealing with the population of the soil, espe-
cially the bacteria, and its role in soil processes. Numerous organ-
isms were described and many important soil processes were eluci-
dated. Among the outstanding investigators during this period,
especially during its early phases, were M. W. Beijerinck and S. N.
Winogradsky, who laid the foundation for much of the subsequent
work. A number of chemical processes involved in the various trans-
formations of nitrogen in soils and in composts were studied in detail.
This element was recognized as frequently limiting plant growth. It
was established that the transformation of nitrogen in nature is largely
dependent upon the activities of various groups of soil microorgan-
isms, especially the processes of ammonification, nitrification, and
nitrogen fixation. These comprise the liberation of nitrogen, in an
available form as ammonia, from complex organic nitrogenous sub-
stances; the oxidation of ammonia to nitrate; and the fixation of
atmospheric nitrogen by microorganisms when an available source
of energy is supplied. The final elucidations of these processes were
made, respectively, by Marshal and by Miintz and Coudon in 1893,

The Golden Age of Soil Microbiology

17

by Winogradsky in 1891, and by Winogradsky and Beijerinck in 1893
and 1901.

The fixation of nitrogen by the association of bacteria and legu-
minous plants formed an important chapter of its own which was
begun in the previous period and which has continued to the present.

Fig. 10. S. N. Winogradsk>' first isolated in pure culture the bacteria concerned
w itli the processes of nitrification and nitrogen fixation, and later made a com-
prehensive study of the microbiological population of the soil.

The brilliant contributions of the earlier investigators were followed
by numerous studies that led to the solution of this important prob-
lem, which has a significant bearing upon microbiology, plant physi-
ology, and agronomy.

Among the most interesting investigations carried out during this
period should be listed the attempts to coordinate the activities of
bacteria with soil fertility. It was believed that a full appreciation
of this relationship not only would modify our outlook upon soil
economy, but also might revolutionize the whole agricultural prac-
tice. The agriculturist, as typified by Caron in 1895, believed that
knowledge of the soil bacteria would do for soil management what
knowledge of disease-producing bacteria had done for medicine.

18 Historical

In 1902, Remy attempted to utilize some of the soil microbiological
processes as a measure of the sum total of activities of the microbial
population of the soil and to develop methods for measuring soils
of different fertility. The assumption was made that the fertility of
the soil is directly correlated with the activities of its microbiological

Fig. 11. M. W. Beijerinck first isolated some of the most important soil bacteria,

including the root-nodule organisms of leguminous plants, Azotobacter chroococ-

cum, Thiobacillus thioporiis, and numerous others.

population and that, by inoculating proper organisms into soil or by
controlling the population by proper treatment of soil, the fertility
of the latter could be regulated. Culture solutions containing pro-
teins or their derivatives as a source of energy were used to favor
the development of protein-decomposing organisms and the accumu-
lation of ammonia. Such cultures were inoculated with soils of dif-
ferent fertility and the amounts of ammonia produced after a given
incubation period were measured. The amounts of ammonia were
thought to correspond to differences in the fertility of the soils. These

The Golden Age of Soil Microbiology

19

methods were further de\eloped and improved by Lohnis in 1904-
1905.

Unfortunately, detailed studies of these methods have shown that
a liquid culture medium in the laboratory does not present conditions
comparable to those that exist in normal soils. The soil itself was
then substituted for measuring the rate of chemical change that
could be produced in a given substance. Such substance was added
to the soil and was allowed to remain there for a definite period of

Fig. 12. V. L. Omeliansky first isolated tlie anaerobic cellulose-decoinposiug

bacteria.

time under favorable temperature and moisture conditions. This led
to the development of the "tumbler" or "beaker" method by J. G.
Lipman and P. E. Brown. To measure the rapidity of ammonia for-
mation from an organic nitrogenous material, a protein or a protein-
rich substance, such as dried blood or peptone, was added to the
soil. To measure the rapidity of nitrate formation, ammonia in the
form of one of its salts or an organic nitrogenous substance was
added to the soil. To measure the nitrogen-fixing capacity of a soil,
a soluble carbohydrate was used; the soil was brought to optimum
moisture condition and allowed to incubate for 7-30 days; the amount
of nitrogen fixed was then measured.

20

Historical

These methods were variously modified, according to the nature
of the substances added to the soil and the processes studied. Ni-
trates were utilized for stimulating the growth of nitrate-reducing
organisms, which produced nitrite, ammonia, or gaseous nitrogen.
Cellulose was used to bring about the development of cellulose-

FiG. 13. F. Lohnis wrote the first comprehensi\e book on soil bacteriology; he
also studied the methods for the evaluation of the role of bacteria in soil fertility.

decomposing bacteria. The evolution of ammonia and the forma-
tion of nitrates also found application in determining the liberation
of nitrogen from fertilizer materials, especially of organic origin.
Hopes were aroused that the results thus obtained would find appli-
cation in the measurement of soil fertility. It was believed that
such methods could be utilized for routine examination of soils to
foretell their potential productive capacity.

Unfortimately, the results obtained by those methods did not
justify the expectations. After thousands of tests had been made

Soil Microbiology as an Independent Science 21

and correlated \\ ith erop yields, the arbitrary conditions of the tests
were found to limit their value, and the methods have now been
largely abandoned.

The appearance in 1910 ot Lohnis" monumental work, Ilandbiich
der landwirtschaftlichen Bakteriologie, tended to summarize the
developments in this subject and to point a way to futiue investiga-

FiG. 14. Jacob G. Lipnian first initiated in tlie United States a comprehensive
study of the microbiological population of the soil and its role in soil processes.

tions. This book was followed in 1911 by Lipman's somewhat dif-
ferent volume, Bacteria in Relation to Country Life.

Soil Microbiology as an Independent Science (1911-1940)

The thirty-year period from 1911 to 1940 brought forth extensive
developments in the field of soil microbiology. Particular emphasis
was laid not only on the bacterial population of the soil, but also on
the other groups of microorganisms inhabiting the soil, notably the
fungi, actinomycetes, algae, protozoa, nematodes, and insect larvae.
Attention was also focused upon the complex interrelationships among
the various groups of microorganisms and their significance in soil
fertility.

22 Historical

It was at first believed that the bacteria are the all-important agents
concerned in soil fertility. Information began to accumulate, how-
ever, indicating that other groups may also have important func-
tions, Ehrenburg had shown in 1839 that the soil contains numerous
protozoa. Darwin directed attention in 1881 to the role that earth-
worms play in certain soil processes. Adametz found in 1886 that
fungi occur abundantly in the soil, and the abundance of actino-
mycetes was studied by Hiltner and Stormer. Algae and other
organisms had also received their share of attention as important
groups of soil-inhabiting microorganisms. It thus gradually be-
came established that the soil harbors an extensive population repre-
senting all the foregoing groups in varying degrees of abundance.
The activities of these organisms were found to result in products
which are essential for plant growth. Some are important agents of
decomposition, whereas others may exert injurious effects, such as
causing plant and animal diseases.

This complex soil population can be considerably modified by
soil treatment, such as liming, cultivation, addition of organic mat-
ter, and partial sterilization. Some of the soil processes, for example
nitrification, nitrogen fixation, and sulfur oxidation, are carried out
by specific groups of organisms, whereas other processes, such as
decomposition of proteins, cellulose, and complex plant residues,
are carried out by a variety of organisms or by large groups of
organisms.

The complexity of the relationships between various microorgan-
isms inhabiting the soil can best be illustrated by the theory of
Russell and Hutchinson. According to this theory, protozoa are
able to consume bacteria and thus exert a controlling effect on the
bacterial processes in the soil and thereby on soil fertility. Any
treatment that leads to elimination of the protozoa would, theo-
retically, result in an improvement of soil fertility. Although sub-
sequent studies did not confirm this theory, the abundance of proto-
zoa in the soil was definitely established.

Other important soil microbiological processes received much con-
sideration. Among these was the decomposition of cellulose by
microorganisms. In spite of the fact that cellulose makes up 20-50
per cent of most plant residues, little was previously known about
the nature of the organisms concerned in decomposition of this
important material. As late as 1902, when Omeliansky published his
studies on the anaerobic cellulose bacteria, no consideration was
given to the role of fungi or other microorganisms in this process.

Soil Miciobiolog)' as an Independent Science

23

Most of the discussion following Omeliansky's work was concerned
with the question of purity of the cultures and with the uncertainty
as to whether aerobic or anaerobic bacteria were more important
and which group was the primary agent in cellulose decomposition.
Numerous studies demonstrated that a large number of fungi are
capable of decomposing cellulose. In fact, under certain conditions,
as in acid soils or in composts, fungi may be more important than

Fig. 15. Charles Thorn made a detailed study of the tungus population of the

soil; he is responsible for the identification of numerous soil organisms, notably

members of tlie genera Penicillhim and Aspergillus.

bacteria. Actinomycetes may also play an important part in cellu-
lose decomposition, especially in high-temperature composts. Even
protozoa may be able to transform cellulose. Considerable knowl-
edge of the aerobic bacteria capable of bringing about cellulose de-
composition was also obtained. The nature of the material, the
natiu-e of the soil or compost, and the conditions of decomposition,
especially aeration and temperature, determine which organisms will
attack the cellulose in soils, in peat bogs, in sewage, or in composts
of manures and \'arious plant materials.

The work of Jensen, Goddard, Waksman, and others on the fungi
of the soil, which was followed later by the studies of Melin and
Rayner on mycorrhiza fungi; the studies of Krainsky, Conn, Waks-
man, and Curtis on the actinomycetes of the soil; and the work of

24

Historical

Cutler on the soil protozoa and of Pringsheim, Robbins, Esmarch,
Chodat, and Bristol on the soil algae, opened new fields for the
study of the microbial population of the soil. These investigations
and numerous others soon following broadened the subject of soil
microbiology and reaffirmed the great abundance in the soil of

Fig. 16.

E. B. Fred made a comprehensi\e stud>' of the root-nodule bacteria
of leguminous plants.

various groups of organisms besides the bacteria. In spite of the
broadened scope of investigations, however, the bacteria were not
neglected. It is sufficient to mention the work of Topping, Lochhead,
Conn, and Winogradsky on the general composition of the bacterial
population; of Ford and N. R. Smith on the spore-forming bacteria;
of Hutchinson and Clayton, Krzemieniewska, Issatchenko, and others
on the aerobic cellulose-decomposing bacteria; of Khouvine, Pochon,
and Fred on the anaerobic cellulose-decomposing bacteria; of Lip-
man, Waksman, and Starkey on the sulfur-oxidizing bacteria; of
Lohnis, Hansen, Fred, Baldwin, McCoy, Stapp, and others on the

Recent DcNclopmciits and Perspectives 25

specificity of legume bueteria; of Viitanen, P. S. Wilson, F. E. Allison,
and others on the chemistry of the nitrogen-fixation process; of
Lohnis on the life cycle of Azotobactcr; and of Christenseii, Gainey,
and others on the influence of environmental conditions, such as
reaction, upon the growth and activities of these bacteria in the soil.

This period was also marked by new methods for the study of
soil microorganisms, notably the soil-staining, contact slide, and
direct soil examination, introduced, respectively, by H. J. Conn and
Winogradsky, by Rossi and Cholodny, and by Kubiena. These meth-
ods yielded additional information on the soil microscopic population.

The appearance in 1927 and in 1932 of Principles of Soil Micro-
biology by \\^iksman closes this chapter in the history of the subject,
just as Lohnis' and Lipman's books closed the previous chapter.

Recent Developments and Perspectives (1940-1950)

World War II disorganized the normal course of development of
many sciences, including that of soil microbiology. When the plow-
share was set aside for the sword, peaceful pursuits had to give way
to those which would help in winning the war and in alleviating
human suffering. The soil microbiologist did not set aside his micro-
scopes and his test tubes, his Petri dishes and his retorts; he used
them for other purposes. Chief among these were studies resulting
directly or indirectly from the war eftbrt. The search centered on
finding (a) methods for combating fungi causing spoilage of sup-
plies and essential materials, (b) means of meeting possible attacks
from the enemy, who might be tempted to utilize poisonous gases
and bacterial warfare or to use microorganisms for killing cultivated
plants, and finally (c) microorganisms capable of producing chem-
ical substances which could be used as chemotherapeutic agents for
combating infections and epidemics.

In the development of antibiotics, the soil microbiological popula-
tion has contributed more than its share. It is to the soil that the
microbiologists came in search of new antibacterial agents. In the
isolation of the numerous antibiotics, organisms were utilized that
came either directly from the soil or indirectly through the dust of
the atmosphere. Thus came gramicidin and tyrocidine, as well as
the many other bacterial products; penicillin, gliotoxin, claxacin, and
other fungus products; actinomycin, streptothricin, streptomycin,
chloramphenicol, aureomycin, terramycin, and neomycin, produced
by actinomycetes. This field is still far from exhausted; and, although

26

Historical

the significance of these studies in terms of soil fertihty processes
and plant growth still remains to be determined, the great importance
of these findings to human health is beyond question.

Where is soil microbiology headed now? What are its problems
and what is its future? One of the great masters of soil microbiology,

Fig. 17. Sir John Russell, Director of the Rothamsted Experimental Station,

stimulated the study of the protozoa in the soil and their role in soil fertility

and of numerous other microbiological processes in the soil.

Winogradsky, considers that what is called at the present time "soil
microbiology" is nothing but a chapter of general microbiology, the
treatment of microorganisms isolated from the soil and hypothetically
admitted as taking part in some of the processes which are charac-
teristic of the soil. He considers the information available at the
present time merely an introduction to soil microbiology, not soil
microbiology itself. Insufficient attention is believed to be paid to
the study of the biological agents responsible for various soil proc-
esses, as they take place in nature, in the original soil and under
specific soil conditions. General microbiology is based upon the

Recent Developments and Perspectives 27

obligatory pure culture method and upon the reactions carried out
by these cultures under various conditions. In view of the fact that
a specific organism has to compete in a certain process in the soil
with numerous other organisms, some of which are much more active
and more specialized, the ability of a given organism to carry out a
certain function under laboratory conditions and in pure culture is
no proof that the organism will carry out the same function in the
soil. Stress should be laid on the crude cultures of an elective char-
acter, arranged in a manner to allow the observation of the biological
acti\"ities in the soil itself.

It thus becomes evident that the scope of soil microbiology can-
not be narrowed down to one or two specific methods for determin-
ing the nature and abundance of the microbiological population, or
to one or two processes for measuring the activities of one or more
members of this population. The scope of the science is much
broader. It avails itself of the methods of the botanist, the zoologist,
the mycologist, and the bacteriologist, for determining the nature
of the organisms present in the soil and their abundance. It avails
itself of the methods of the chemist and of the physicist, for measur-
ing the nature of the processes carried out by these organisms. It
attempts to correlate the information thus obtained with that of the
soil chemist, the soU physicist, and the agronomist, thus contributing
its share to the building up of our knowledge of the science of the
soil.

Like every other science, soil microbiology calls upon some of
the older and some of the closely related sciences for specific methods
and for the elucidation of its results. Soil microbiology is not and
cannot be merely a theoretical or a strictly applied science, as be-
lieved by some. It is a science in itself with many theoretical phases
and practical applications. From this point of view, one is hardly
justified in saying that up to now we have had only contributions to
general microbiology and that the applied science of soil micro-
biology is still to come. One might be more justified in saying that
up to now the general ecological and biochemical phases of soil
microbiology have been dominant and that the application of this
science to our knowledge of the soil is still of limited significance.
Even this, however, would hardly be fully justified, since consider-
able information has been accumulated concerning the interrelations
between the soil processes and the microscopic population of the
soil.

28 Historical

Selected Bibliography

1. Beijerinck, M. W., Verzamelde Geschriften, 6 vols., Delft, Holland, 1921-
1946.

2. Bulloch, W., The History of Bacteriology, Oxford Unixersity Press, London
and New York, 1938.

3. Lohnis, F., Handbuch der landwirtschaftlichen Bakteriologie , Borntraeger,
Berlin, 1st Ed., 1910, 2nd Ed., 1938.

4. Medical Research Council, A System of Bacteriology in Relation to Medi-
cine, Vol. 1, London, 1930.

5. Waksman, S. A., Principles of Soil Microbiology, Williams & Wilkins Co.,
Baltimore, 1st Ed., 1927, 2nd Ed., 1932.

6. Waksman, S. A., Humus; Origin, Chemical Composition and Importance in
Nature, Williams & Wilkins Co., Baltimore, 1st Ed., 1936, 2nd Ed., 1938.

7. Waksman, S. A., Three decades with soil fungi, Soil Sci., 58:89-115, 1944.

8. Waksman, S. A., Soil microbiology as a field of science, Science, 102:339,
1945.

9. Waksman, S. A., Sergei Nikolaevitch Winogradsky; the story of a great
bacteriologist. Soil Sci., 62:197-226, 1946.

10. Winogradsky, S. N., Microbiologic dii sol; prohlemes ct mcthodes, Masson
et Cie, Paris, 1949.

2- .

The Microbiological Population
of the Soil as a Whole

Composition of the Soil

The soil represents a medium or substrate in which numerous
microorganisms Hve and bring about a great variety of processes
which are responsible for continuation of the cycle of life in nature.
A normal soil is made up of solid, liquid, and gaseous constituents.
These can be broadly divided into five groups:

1. Mineral Particles. These vary greatly in size and in the de-
gree of their mechanical and chemical disintegration. They include
particles ranging from large pebbles to fine sand, clay, and silt.

2. Plant and Animal Residues. These comprise the freshly fallen
leaves and other plant stubble and dead remnants of a variety of
insects and other animal forms. Some of the materials are largely
undecomposed; still others are partly or thoroughly decomposed, so
that the original structure can no longer be recognized. In the last
state they are spoken of as humus or humified materials.

3. Living Systems. These include the living roots of higher plants;
the great number of living animal forms, which range from protozoa,
insects, and earthworms to rodents, as well as the numerous algae,
fungi, actinomycetes, and bacteria.

4. Water. The liquid phase of the soil, comprising both free and
hygroscopic water, contains in solution varying concentrations of in-
organic salts and certain organic compounds.

5. Gases. The soil atmosphere consists of carbon dioxide, oxygen,
nitrogen, and a number of other gases in more limited concentrations.

The composition of a typical podzol soil is shown in Table 1.
The microbiological population which inhabits the soil, together
with the roots of higher plants and with animal forms, makes the

29

30

Microbiological Population of the Soil

Table 1. Composition' of a Gray Forest or Podzol Soil (from Glinka)

Horizon Ai:

Horizon A2:

Horizon B:

Organic-

Bleaclied

Brownish

Horizon C

Matter-Rich

Whitish

Yellow

Granitic

Soil Constituent

Material

Horizon

Layer

Base

-per cent

per cent

per cent

per cent

Loss on

ignition

12.78

5.02

6.00

1.21

Organic

matter

10.94

1.25

2.29

SiOa

66.86

74.01

63.60

74.87

AI2O3

13.38

13.78

17.10

13.82

Fe203

1.71

1.95

4.50

1.92

Mn304

0.04

0.04

0.08

0.04

CaO

1.38

0.92

0.69

0.63

MgO

0.14

0.13

0.45

0.40

K2O

2.36

2.28

4.12

3.96

NaaO

1.56

1.75

3.46

2.62

soil a living system and not a mere dead mass of mineral matter and
organic residues. This can be clearly seen in Fig. 18.

The quantitative composition of the population and its qualita-
tive nature depend largely upon the origin and nature of the soil
and the relative composition of its inorganic and organic constitu-
ents. The prevailing climate and the growing vegetation also influ-
ence greatly the nature and abundance of the microorganisms that
inhabit the particular soil. Among the other factors that have a
marked effect upon the relative composition of the microbiological
population, it is sufficient to mention the reaction of the soil, its
moisture content, and the conditions of aeration.

Microbiological Population of the Soil

Study of the soil population has progressed along several distinct
lines. Some investigators have devoted their major attention to the
quantitative composition of the microbiological population; others
were interested in the nature of the organisms making up this popu-
lation; still others were concerned with the chemical processes
brought about in the soil by the various organisms and their im-
portance in soil fertility and in plant growth.

The methods for the enumeration of bacteria and other micro-
organisms in the soil have undergone various changes during the
last fifty years. The same is true of the concepts concerning the
relative importance of the various constituent groups of micro-
organisms inhabiting the soil. These facts, as well as the discovery

Microbiological Population of tlio Soil

31

that certain groups of organisms occur in some soils and not in others,
as affected by \arious cultural and environmental conditions, have
all influenced the prexailing ideas concerning the nature and abun-
dance of the microbiological population of the soil and its importance
in the transformation of organic and inorganic materials. They have

55

45

35

25

15

5 -

~ ■

i

w
6

Ml

ml
Iff.

m

m
It

i-ii-

-

Organic
matter

Mineral

Air

Water

Fig. 18. The relati\e distribution of materials that compose a soil under con-
ditions fa\orable to plant growth (from Waksman and Starkey).

modified considerably our understanding of the various processes
which go on in the soil and determine its fertility.

At first, a general belief prevailed among agronomists and soil
chemists that it was sufficient to count the numbers of bacterial
colonies developing from a suspension of a given soil upon an agar
or gelatin plate to obtain an accurate idea not only of the relative
abundance of the particular organisms, but even of the composition
of the soil microbiological population as a whole. The introduction
of elective culture methods for the study of soil microbiological proc-
esses permitted a broader insight into the nature and activities of
this population. These methods had also certain serious limitations.
When artificial culture media are inoculated with small amounts

32 Microbiological Population of the Soil

of soil, they allow the development of only a small part of the micro-
organisms which are present in a particular soil, depending on the
composition of the medium and conditions of incubation. The re-
sults thus obtained tended to obscure the importance of the micro-
biological population in the various soil processes. The information
obtained by these simple cultural methods gave, therefore, only a
limited insight into the composition of the complex population, into
the numerous processes for which this population was responsible,
and into the complex associative and antagonistic interrelationships
among the various soil-inhabiting microorganisms.

At first, bacteria were considered to comprise the all-important
group of microorganisms; the various processes which influence soil
fertility and for which microbes were known or were believed to be
responsible were associated with the occurrence and abundance of
bacteria. When it was recognized that various other groups of micro-
organisms must receive attention, and their importance in numerous
soil processes could no longer be ignored, there came a change in
the general concept of the population and its importance.

These changes in our understanding of the nature of the soil popu-
lation were also accompanied by a growing realization of the rela-
tion of this population to soil processes. In order to measure the
activities of the population in the soil, a small amount of soil was at
first added to aqueous solutions containing varying concentrations
of different substances, such as peptone, cellulose, ammonium salts,
or nitrates. These solutions were incubated for a few days in the
laboratory, and the chemical changes that took place in the composi-
tion of the specific substance were determined by various analytical
procedures. The results thus obtained were believed to serve as a
measure of the activities of the microbiological population in the soil.
It was later found that the chemical changes brought about in the
solution cultures might not have been due at all to the important
groups of organisms, but only to those forms that could adapt them-
selves more readily to the artificial conditions created by the par-
ticular selective media. This tended to give to such organisms an
exaggerated importance, out of all proportion to the part that they
actually play in soil processes. The more predominant and perhaps
the more important groups of microorganisms frequently had great
difficulty in developing in the artificial media and under the arti-
ficial conditions of culture.

The "solution" methods were gradually replaced by the "soil"
methods, whereby a small amount of a known substance was added

Microbiological Population of the Soil 33

to a gi\cMi ([iiantity of soil, the moisture adjusted to optimum, and
the soil kept in the laboratory for a given time. The chemical
changes that took place in the soil as a result of such treatment were
analyzed b\" simple teehniciues. These procedures were often called
"beaker" or "timibler" methods after the container in which the soil
was kept. The cliemieal ehanges brought about in the added sub-
stance by the soil microbiological population also gave an incom-
plete idea of the importance of certain groups of organisms; others
may not ]ia\e been recognized at all.

The introduction of microscopic methods for the examination of
soil microorganisms proved to be a valuable tool in supplying infor-
mation concerning the distribution of microorganisms in the soil
and the nature and abundance of special groups of organisms. These
methods, usually based upon the staining and microscopic observa-
tion of a small amount of soil, were likewise subject to certain limi-
tations and did not necessarily convey a true picture of the popula-
tion, especially from the point of view of its importance in soil
processes. The greatest difficulty thereby encountered was that some
of these methods could not be developed as routine laboratory pro-
cedures. They could be used only by specialists, and the results thus
reported varied so considerably, especially when different modifica-
tions of the methods were employed, that the information obtained
proved to be of but limited value. This is brought out by the fact
that, after long experimentation, the conclusion was reached that
the direct microscopic methods can, at best, supplement but not
replace the plate and culture methods for evaluating the abundance
and activities of the soil population. Among the microscopic meth-
ods, the contact slide proved to be most valuable, since it could
present a picture of the microbiological state of the soil under given
conditions of treatment.

Various discrepancies have been encountered in an attempt to
correlate the presence of certain groups of microorganisms with the
part that they were believed to play in certain soil processes. When
such organisms were isolated from the soil and grown in pure cul-
ture, it was found that the chemical reactions brought about by
them under those conditions did not necessarily correspond to simi-
lar reactions taking place in the soil itself. The importance of the
actual or potential actixities of such organisms in the soil under
various conditions of cultivation was, therefore, questioned.

Studies made of the effect of various soil treatments and changes
in environment demonstrated that the microbiological population in

34 Microbiological Population of the Soil

the soil is far from constant, that it undergoes a number of variations
and changes which may be seasonal and even more frequent, and
that these changes are subject to a variety of influences. Thus the
many discrepancies in the interpretation of results obtained by dif-
ferent investigators, using different methods, were due more fre-
quently to the procedure employed than to underlying fundamental
differences in the soil population. The depth of the soil was found
(Tables 2 and 3) to influence not only the total number of organ-
isms, but especially the distribution of the various constituent groups.

Table 2. Distribution of Microorganisms in the Different Horizons of a
PoDZOL Soil Profile (from Gray and Taylor)

Microorganisms in thousands per gram dry soil.

jrizon

Moisture

Organic C

Bacteria

Act

Inoraycetes

Fung

per cent

per cent

Ai

73.3

22.7

9,792

1,104

191

A2

16.8

0.9

369

53

10

Bi

14.1

0.4

400

<1

<1

B2

19.0

0.9

1,006

<1

<1

Table 3. Bacteria and Actinomycetes at Various Depths of Soil
(from Waksman)

Numbers in thousands per gram, determined by plate method.

Deptli

Bacteria

Actinomy(

:;etes

inches

numbers

per cent

numbers

T

:r cent

1

7,340

91

743

9

4

5,300

85

933

15

8

2,710

82

612

18

12

950

80

239

20

20

259

51

246

49

30

124

35

240

66

Composition of the Soil Microbiological Population

As has been pointed out, most of the investigators who devoted
themselves, as late as the beginning of this century, to the study
of occurrence and activities of soil microorganisms looked upon the
bacteria as responsible for the most important, if not all, soil processes.
A mere reading of the papers dealing with soil microorganisms,
published during the final decade of the last century and the first
decade and a half of this century, will tend to prove this generaliza-

Composition of Soil Microbiological Population 35

tion. This is illustrated by the work of Russell and Hutchinson in
England; of J. G. Lipman, H. J. Conn, and E. B. Fred in this coun-
try; of Lohnis and others in Cermany. Little attention was paid to
the other groups of soil microorganisms. The protozoa were looked
upon merely as "injurious forms" or as "enemies of bacteria"; the
fungi were considered cither nuisances or "dust contaminants."

Admittedly, it has been known since the time of Ehrenburg, in
1839, that the soil harbors numerous protozoa; since Darwin, in 1881,
that earthworms may play an important role in certain soil processes;
since Adametz, in 1886, that fungi are found in great abundance in
the soil; and since Hiltner and Stormer, in 1904, that actinomycetes
form an important constituent group of the soil population, as could
be measured by simple plating procedures. None of these organ-
isms, however, were given sufficient consideration in any systematic
study of the soil population; if they were considered at all, only
feeble attempts were made to coordinate their occurrence and activi-
ties with important soil processes.

It has become definitely established only during the last three
and a half decades that the soil is characterized by a distinct micro-
biological population, which is made up of specific groups. These
exert a great variety of associative and antagonistic effects upon one
another. These actixities markedly influence the fertility of the soil
and the growth of cultivated and uncultivated plants.

The following major groups make up the soil microbiological
population, or its flora and fauna:

1. Bacteria. These include spore-forming and non-spore-forming
rods, cocci, vibrios, and spirilla. They vary considerably in size,
shape, oxygen requirements (aerobic and anaerobic), energy utili-
zation (autotrophic and heterotrophic), slime formation, and rela-
tion to plants and animals (saprophytic and parasitic).

2. Actinomycetes. Three of the genera of actinomycetes are well
represented in the soil. Species of Nocardia are closely related to
some of the bacteria, especially the mycobacteria and corynebac-
teria. Species belonging to the genera Strepfomyces and Micro-
monospora are more closely related to the true fungi. Actinomy-
cetes vary greatly in their biochemical properties, in their relation to
higher plants and animals (saprophytic vs. parasitic), and in their
effect upon soil bacteria ( associative and antagonistic interrelations ) .

3. Fungi. These include large groups of organisms, known as
Phycomycetes, Ascomycetes, Hyphomycetes or Fungi Imperfecti,
and Basidiomycetes. They produce extensive mycelium and spores

36

Microbiological Population of the Soil

in soils and in composts. Their growth throughout the soil may be
so extensive as to hold the mass of particles together by means of
a very fine microscopic network of mycelium and its excretion prod-

«^

2^

^ C

^

^'

^'->.

V

Fk;. 19. Micr()bii)l()gical population of soil, as shown by contact slide method:
a, h, d, different types of liacteria; c, fungus mycelium (from Cholodny).

nets. Fungi vary greatly in their relation to higher forms of life,
notably plants (saprophytic vs. parasitic), to soil bacteria (forma-
tion of antibiotic substances), and to other members of the soil
population.

4. Algae. These organisms comprise the grass-green Chloro-
phyceae, the blue-green Cyanophyccae, and the Diatomaceae. Their

Composition of Soil Microbiological Population 37

al)ilit\ to produce chloropln 11 makes their life in the soil, especially
oil its surface, independent of the presence of organic matter.

5. Protozoa. These comprise amoebae, flagellates, and ciliates.
The \egetati\e \s. cyst condition of the protozoa in the soil has at-
tracted considerable attention. This is true also of their relation to
the bacteria, since it was suggested at one time that protozoa func-
tion in the soil as the natural enemies of the bacteria. By feeding
upon bacteria, protozoa exert, it was believed, a controlling effect
upon the abundance of bacteria, thus affecting adversely a variety
of soil processes.

6. Higher Animal Forms. These include nematodes, rotifers,
earthworms, and lar\ae of insects. These organisms have a variety
of functions in the soil. The ability of some of the soil-inhabiting
insects to attack certain higher plants frequently makes them of
great economic importance. The action of earthworms as "soil cul-
tivators" places them in an important category. The fact that some
of the injurious insects spend part of their life cycle in the soil
suggests certain methods of control.

7. Filterable Organisms. These include phages and other
viruses. Although our knowledge of the importance of these forms
in soil processes is still very limited, their ability to attack both lower
and higher forms of life, ranging from the bacteria and actinomycetes
to cultivated and wild plants and animals, makes them of great
potential importance.

8. Higher Plant Forms. In addition to the microscopic and ultra-
microscopic organisms, the soil also harbors the root systems of
higher plants. The activities of these are frequently dovetailed with
those of the microorganisms.

Although the soil-inhabiting organisms form only a very small part
of the total soil mass, they are responsible for the major changes that
take place in the soil. These organisms are disti-ibuted throughout
the soil, primarily in the upper layers, where the plants send down
their roots and where they obtain their necessary nutrients. When
the roots die, they are rapidly decomposed by the fungi, bacteria,
and other groups of organisms. The constituent chemical elements,
notably the carbon, nitrogen, and phosphorus, are thereby returned
to circulation and again made available for the growth of new roots
and new plants. In these processes of decomposition, microorgan-
isms build up extensive cell substance, which contributes to the
organic matter of the soil or the soil humus. The microbial cells

38 Microbiological Population of the Soil

not only serve as a reservoir for the further activities of microorgan-
isms, but also act as binders for the soil particles.

Many of the groups of microorganisms found in the soil are cos-
mopolitan in nature, whereas others are of only limited occurrence.
Some are found in a number of soil types, and others only in certain
soils and under specific environmental or cultural conditions. Among
the bacteria, the Bacillus subtilis and the B. mijcoides groups are
cosmopolitan in nature, whereas the Rhizobium legwninosarum is
limited largely to soils in which specific legumes are growing. Azoto-
bacter chroococcwn is found only in soils that have a pH above 6.0,
whereas A. indicum can withstand much more acid reactions. Fungi
are more abundant in acid soils, and actinomycetes in alkaline. Many
organisms are controlled by the nature and abundance of the organic
matter, by climatic conditions, aeration, and reaction, and by the
specific vegetation. The mycorrhiza fungi and the various plant-
pathogenic fungi and bacteria are particularly influenced by vege-
tation.

The associative and antagonistic effects among microorganisms
are often believed to exert a controlling influence upon the specific
nature of the soil microbiological population. The inhibition of
many bacteria, notably of the spore-forming rods and cocci, by anti-
biotic substances produced by fungi; the feeding of certain fungi
upon nematodes and protozoa; the feeding of many protozoa upon
bacteria; the attack of many bacteria and fungi upon insect larvae;
the ability of various phages to attack bacteria— all contribute to the
modification of the soil population.

The addition of large amounts of organic matter, especially fresh
plant and animal residues, to the soil completely modifies the nature
of its microbiological population. The same is true of changes in
soil reaction which are brought about by liming or by the use of acid
fertilizers, by the growth of specific crops, notably legumes, and by
aeration of soil resulting from cultivation. The results of fertiliza-
tion and liming upon the microbiological population, as determined
by the agar plate method, are brought out in Table 4. Liming of
soil favors the bacteria and actinomycetes, but not the fungi. Acid
mineral fertilizers, like ammonium sulfate, favor the fungi, but not
the other two groups. Manure favors all groups.

Often a sequence of forms occurs after a certain treatment, one
group of organisms following another. The addition of cellulose-
rich materials, for example, first favors the development of fungi,
notably species of Chaetomiiim, Fusarium, Aspergillus, Penicillium,

KiiisMiids |)iM- K'nuii-

Soil

Reaction

Actiiio-

pU

Bacteria

inycetes

Fungi

4.(i

3,000

1,150

60

(1.4

.5,210

2,410

22

5 . ')

5,160

1 , 520

38

5A

8,800

2 , 020

73

4.1

'2,690

370

111

,).S

7,000

2 , 520

30

5.5

7,600

2,530

4(!

Methods of StiidNing the Soil Population 39

Tari.k 4. Influence of Soil Tkeatment on Numheh of Mkuooiu; anisms
IN THE Soil (from Waksman)

Nniiilicrs

Treat nu-nt of Soil *
Unfertilized
Lime alone
Minerals

Manure ami minerals
Minerals and ammonium sulfate
Minerals, annnonium sulfate, an<l Hi
Minerals and sodium nitrate

* Minerals = 320 pounds KCI and (i4() pounds acid ]>liosphate per acre every year.

and Trichodcrma, and of bacteria, especially myxobacteria and spe-
cies of Cytopliaga; these organisms may be followed by the growth
of various spore-forming bacteria and finally by actinomycetes. The
sequence of various groups of microorganisms can best be studied
in composts, since the changes in temperature and the degree of
decomposition of the materials in the compost greatly influence the
nature of the organisms present: fiist bacteria begin to multiply
rapidly, accompanied by the nematodes, the protozoa, and certain
fungi, notably the mucorales; these are followed by other filamentous
fungi, such as penicillia and aspergilli; finally the actinomycetes ap-
pear, and certain bacteria, notably the thermophilic types.

Methods of Studying the Soil Population

To understand the theoretical and practical significance of the
results, a critical e\'aluation of the methods used in determining
the nature and abundance of this population is of the greatest im-
portance.

Some of the methods, like the agar plate method and \arious liquid
culture methods, supply information concerning the abundance of
viable or reproducible members of the population; they also indicate
the nature of the biochemical processes for which these organisms
are responsible in the soil. These methods are based upon the
development of the living cells in the form of colonies on the agar
plate or upon their growth in specific cultme media. The numbers
of organisms obtained by either of these methods are commonly
reported to range from 1 to 50 millions per gram of soil. These num-

40 Microbiological Population of the Soil

bers may represent only a very small fraction of the total soil popula-
tion, such as 1 per cent or even less. This is due to the fact that
many organisms actually present in the soil in a living state are
unable to develop in the artificial culture media employed for their
evaluation. In some cases, very special procedures have to be de-
vised before one is able to determine even the presence of a certain
organism in the soil.

On the other hand, the microscopic methods may give a highly
exaggerated picture of the abundance of the microbiological popula-
tion, since the counts may include not only living but also dead cells
of various organisms. Hence counts of 1-10 billions of bacteria per
gram of soil (Table 5), as frequently reported by use of these

Table 5 Microorganisms in 1 gm of Soil as Determined by the Direct
Microscopic Method (from Richter)

Numbers in thousands per gram.

Bacteria

1

1

Pieces of

Azotobacter

Fungus

Type of Soil

Depth

cm

Cocci

Bacilli

Cells

Mycelium

Forest

0

1,379,000

1,212,000

1 , 000

47,000

10

991,000

466,000

31,000

34,000

20

281,000

169,000

7,000

Brown loam

0

870,000

376,000

84,000

5,000

10

569,000

106,000

1,000

3,000

Sandy soil

0

.")19,000

192,000

79,000

3,000

10

■107,000

153,000

23,000

19,000

20

269,000

139,000

8,000

3,000

methods, may not be fully correct. These and similar methods may,
therefore, also be open to criticism, since they do not give an ac-
curate picture of the soil microbiological population.

Whatever the methods used, however, for e\'aluating the num-
bers of soil microorganisms, it may well be recognized that the actual
living mass of microorganisms in the soil is considerable. Most of
the organisms found in the soil are indigenous members of the
population. Some are found there because they seek shelter and
protection beneath the soil surface. Others are carried into the soil
by dead and dying plants and animals, by wind, or by rain, to live
or to die there. Numerous groups of organisms pass their whole

Methods of Stuching the Soil Popuhition 41

lite in the soil, find their food there, and eventually leave their bodies
to become a part of the soil mass, just as the roots of the higher
plants penetiate and ramify throughout the whole of its surface area,
and leave their remains to decompose in the soil. In the many
changes that take place in the life cycle of the various microorgan-
isms in the soil, a definite equilibrium has become established be-
tween the various groups. This equilibrium is not stable, however,
but undergoes many changes as a result of the treatment that the
soil undergoes.

The methods most commonly used for the enumeration of micro-
organisms found in the soil are commonly divided into several
groups:

I. Microscopic methods.

1. Staining of soil and diuxt niicroscoi)ic examination.

2. Contact slide method.

3. Direct examination of unstained .soil.
II. Culture methods.

1. Plate culture methods.

2. Electi\e cultiu-e methods.

3. Soil enrichment methods.

The numbers and types of organisms vary considerably, depending
upon the method. Each one of these methods has its advantages
and limitations. Some of the methods may have to be further modi-
fied in the study of a specific problem, or for the purpose of estab-
lishing the presence or abundance of a particular type of organism
under a particular set of conditions.

Microscopic Examination of Stained Soil Preparations. This
method consists in the preparation of a suspension of soil in a dilute
fixative solution; one or two drops of the suspension is spread upon
a clean slide, which is then dried and stained with an acid dye, and
finally examined with a high-magnification microscope. The fixa-
tive solution is prepared by dissolving 0.15 gm of gelatin in 1 liter
of distilled water. The staining solution consists of 1 gm erythrosin
or rose-bengal dissolved in 100 ml of a 5 per cent aqueous solution of
phenol, containing sufficient CaClo (0.001-0.1 per cent) to give a
very faint precipitate of the calcium salt of the dye.

The process of staining is carried out by placing a loopful of
the soil suspended in the fixative solution upon a glass slide and
spreading out with a needle until it covers a known area. The smear
is allowed to dry over a boiling water bath. A drop of the staining
solution is added to the smear and allowed to remain for 1 minute,

42 Microbiological Population of the Soil

while the slide rests on the bath. The stain is then washed off with
water and the preparation allowed to dry.

Various modifications of the above method have been proposed.
In one such modification, a measured amount of soil is suspended in
a molten agar gel. Small drops of the agar are removed, placed
in a hemacytometer slide of known depth, and allowed to solidify.
The films are dried and stained in a solution of acetic-aniline blue,
followed by dehydration in alcohol and mounting in euparal. Differ-
ential counts of a measured area of the film will give a quantitative

4 * 9 9

'^^ ^ \:

Fig. 20. Soil examination by direct microscopic method (from Winogradsky ) .

estimation of the microorganisms present in a given amount of soil.
By adding a known suspension of bacteria to sterile soil, Jones and
Mollison were able to recover 95.7-98.4 per cent. According to this
method, the numbers of bacteria per gram of soil varied from 2,275
to 5,420 millions. The method will also detect large amounts of
mycelium and spores of fungi and actinomycetes.

Thornton mixed a soil with a known amount of indigotin; by
establishing the relation between the numbers of erythrosin-stained
bacteria and indigotin particles, he was able to measure the abun-
dance of microorganisms in the soil. He found that 1 gm of soil
contained 1-4 billions of bacteria, whereas only 2 per cent of these
bacteria are measured by the plate method. Detailed studies of
stained soil preparations have established the fact that the numbers
of bacteria in the soil are far greater than those that have been ob-
tained by the plate culture methods.

Cholodny believed, however, that the direct staining methods
cannot give a complete picture of the soil population in its natural
habitat, because the shaking of soil with water allows the distribution

Methods of Stiidving the Soil Population 43

of the organisms in a manner not comparable with their existence
in the natural soil itself. An especially distorted picture is thus ob-
tained of the actinomycetes in the soil.

Contact Slide Method. Rossi and Cholodny proposed a method,
designated as "contact slide," "soil plate," or "surface growth plate."
This method consists in making a slit in the soil with a sharp knife
and inserting into the slit a clean cover slide. The soil is then pressed
gently to bring it in contact with the slide, which is left in position
for 1-3 weeks. The slide gradually becomes covered with some of
the soil solution and with soil particles. When the slide is removed
from the soil, it is cleaned on one side with a cloth to remove the
soil particles and allowed to dry in the air. The preparation is fixed
by passing the slide over a flame; it is then washed gently in tap
water to remove the coarse soil particles, followed by distilled water,
and stained with phenol erythrosin for 30 minutes, at room tempera-
ture. The slide is finally washed, dried, and examined under the
microscope.

The microscopic population observed on the contact slide may not
be exactly the one that may be found in the soil at a given moment,
since it results from the development of specific organisms on the
slide in contact with the soil. In experiments on decomposition of
organic matter in soil, Jensen found that the results obtained by the
direct method agreed with those secured by the plate method; hence
the two methods were believed to be able to compensate each other.

In their physical relation to the soil, the microorganisms are found
chiefly upon the solid soil particles; only a small number of organ-
isms are found in the liquid phase, chiefly because of the adsorption
of the organisms by the soil particles. Thus the mechanical compo-
sition of the soil, its chemical nature, especially its reaction, and the
nature of the base in the adsorbing complex are all significant in
determining the degree of adsorption of soil organisms.

Conn found that an increase in the moisture content of the soil
results in a rapid change from a natural flora of fungi and actinomy-
cetes to one in which bacteria predominate. In a natural soil and
under normal conditions of wetting and drying, filamentous organ-
isms are active, especially in acid soils high in colloidal material.
With an increase in the moisture content of such a soil, due to ex-
cessive rainfall, bacteria became more active; such increased activity
is even greater than that following addition of lime.

The contact slide method was found to offer great possibilities
not only for the quantitative evaluation of the soil microscopic popu-

44 Microbiological Population of the Soil

lation, but also for establishing certain important qualitative changes
in the population, such as those resulting from fertilization of soil
or use of antiseptics. It was also suggested that this method is
effective for determining the influence of important plant nutrients,
notably nitrogen, phosphorus, and potassium salts, upon the numbers
of microorganisms in soil. The method also lends itself readily to
the study of the influence of soil treatment upon the rhizosphere or
the relationship between plant roots and microorganisms.

In spite of these favorable results, various investigators reported
that the microscopic examination of soil, either by direct staining or
by the contact slide method, is inadequate for determining the
functions of microorganisms in the soil. These methods were be-
lieved, therefore, to be of importance only as supplements to the
plate and other culture methods.

Among the limitations of the contact slide method is the fact that
bacteria produce zooglea upon the glass. To overcome this, Cho-
lodny proposed the "soil chamber" method, which consists in placing
fine particles of soil upon moist slides, keeping the slides in moist
chambers, and examining microscopically the organisms growing
out of the soil.

Direct Examination of Soil by Microscope. Kubiena developed
a special microscope for the direct examination of soil in an undis-
turbed condition. The microbiological population may thus be
observed in a natural state. Special surface illumination is attached
to the microscope. By means of micromanipulators, some of the
larger organisms, notably the fungi, can be removed from the soil
for staining purposes and for closer study. This method has not
been used very extensively, and only limited information, dealing
primarily with the fungi, is so far available.

The nature of the soil population, as determined by the direct
microscopic method, has been summarized by Jones and Mollison.
The soil bacteria are largely coccoid and adherent to the humic mat-
ter, few or none being attached to mineral particles. They may be
in the form of large zoogleal colonies or may consist of smaller
clumps or single individuals. Frequently groups of large cocci
resembling Azotobacter cells are seen. Long rods are but rarely
observed in fresh soil. Fungal mycelium shows variable staining.
There is, in fact, strong evidence of correlation of intensity of stain-
ing with viability. Progressive loss of the protoplasm from the
hyphac, due either to decomposition or to its migration to the hyphal
tip, can be frequently observed, most of the hyphal fragments lack-

Methods of Stud)ing the Soil Popiihitioii 45

ing organized contents. Such hyphae are stained purple in contrast
with the deep blue coloration of those filled with protoplasm. This
was confirmed by inoculating sterilized soil with fungal mycelium,
allowing it to incubate for several days, and making films from a
sample of this soil; on these films only deeply stained fungal frag-
ments were seen. In normal soils, mycelium is scanty, and because
of its filamentous nature and very variable length is not amenable to
accurate statistics, though useful comparative results may be ob-
tained. There were significantly fewer pieces of mycelium present
in the soils receiving mineral fertilizer than in those receiving
farmyard manure.

Lengths of well-stained mycelium frequently have organic matter
adherent to their walls, probably through secreted mucilage. This
may have an important bearing on the formation of soil crumbs.
Few fungal spores are seen. Fibers may be distinguished from hy-
phae by their lack of staining and their polarization colors under
crossed nicols. Other plant tissue absorbs but little dye and, at most,
has a greenish hue. Stained nematodes are sometimes seen, and
what are thought to be earthworm setae can be distinguished from
fragments of mycelium by their tapering apices.

Plate Culture Methods. The gelatin plate followed by the agar
plate was the first method used for the enumeration of soil organisms.
It still remains the one most commonly employed. The method con-
sists in suspending a given portion of soil in a given volume of sterile
tap water, and making a series of dilutions, such as 1 : 100, 1 : 1,000,
to 1:10,000,000. The final dilutions of soil are prepared in such a
manner as to allow 40-200 colonies to develop on each plate. One-
milliliter portions of the final dilutions ai'e placed in plates to which
suitable agar media are then added; the contents of the plates are
carefully mixed; the plates are then incubated at 28-30° C, and the
colonies counted after varying periods of time.

The plate method is very convenient, but its chief limitation lies
in the fact that it allows the development of only the heterotrophic
aerobic bacteria, certain yeasts, molds, and actinomycetes. Fre-
quently, special media are used for the enumeration of fungi,
whereby bacterial development is suppressed either by acidifying
the medium or by the addition of antibacterial agents. This makes
possible the use of much lower dilutions of soil than those required
for the enumeration of the larger numbers of bacteria.

The plate method, supplemented with other methods, has made
it possible to establish that different soil types possess characteristic

46

Microbiological Population of the Soil

microbiological populations. Correlations between microbial activi-
ties and crop yields have been derived only for certain soils. Definite
relations have been observed between optimum conditions for the

Fig. 21. Colonies of bacteria, actinomycetes {lower plnfe), and fungi (upper

plate), developing on jilates used in counting these organisms in soils (from

Waksman and Starkey).

growth of higher plants and for microorganisms. The possibility of
correlating the numbers and activities of certain groups of micro-
organisms and soil conditions, notably moisture and temperature,
has also been indicated.

Methods of Stuchiiig the Soil Population

47

By use of the plate method, it was possible to demonstrate that
an extensive microbiologieal population is found in field and garden
soils, in forest soils, in peat bogs, and even in the ash of volcanoes
and in desert sands.

Fig. 22. Plate preparation, slun\ing dc\ clopiiiLiit of spreading colonies of bacteria

(from Lipman).

The results obtained by the plate method, fully substantiated by
other methods, emphasize the fact that the various groups of micro-
organisms are largely concentrated in the surface layer of the soil.
This is true particularly of podzol soils, in which the surface layer
corresponds to the Ai horizon. In cultivated soils, the changes
in the numbers of microorganisms with depth of soil are more
gradual. Although bacteria and actinomycetes diminish with an
increase in depth, the proportional reduction is far greater for the
bacteria than for the actinomycetes; whereas at a depth of 1 inch the
actinomycetes make up only 9.2 per cent of the organisms develop-
ing on the plate, their proportion may be increased to 65.6 per cent
at a depth of 30 inches.

48 Microbiological Population of the Soil

Among the \aiious factors which influence the abundance of
microorganisms in the soil, the most important are organic matter,
reaction, moisture, temperature, aeration, and nature of crop grown.
The distribution of microorganisms in the soil is, therefore, controlled
by numerous ecological factors, comprising climatic or atmospheric,
edaphic or soil, and biotic or living. Although no important relation
has been found between the numbers and kinds of microorganisms
and the climatic conditions, the edaphic and biotic factors are of
great significance. The amount of organic matter in the soil influ-

FiG. 23. Colonies of fungi and bacteria, as determined by plate method (from

Lohnis and Fred)

ences markedly the numbers of all groups of microorganisms, whereas
the reaction governs largely the ratio of the fungi to the bacteria
and actinomycetes. According to Feher, the total number of bac-
teria in the soil diminishes as one proceeds northward; however, the
proportion of fungi to bacteria increases. This change was found
to be correlated with a reduction in pH value and a change in tem-
peratiue. There is also a decrease in numbers of bacteria with in-
creasing altitude.

The effect of moisture is well illustrated in Table 6. With an
increase in moisture from a fairly dry state to 80 per cent saturation,
there is an increase in the number of bacteria; at the saturation point,
there is again a decrease.

Among the other soil treatments that modify greatly the micro-
biological population of the soil, additions of organic matter are of
greatest importance. Addition of manure favors bacteria and actino-
mycetes. Addition of acid fertilizer, such as ammonium sulfate,
favors fungi and is detrimental to bacteria and actinomycetes; lime
has the opposite effect.

Methods ot Studying the Soil Popuhitioii 49

Tablk 6. Infiaknch ok Moisti-hk Content ok Sfyi, on thk Ximukks or |{\(Tkhi\

(from Eiigl)or<liiig)

Niiinbors of hactcria in IIkiiisiiikIs ])c'f Kram dry soil.

IVrfonlage of

Moisture Moisture-Holding Total Relative

Content Capacity Bacteria Numbers

per cent per cent

6.5 30 !),!)80 33

10. !> 50 11,890 40

U.l 65 16,410 5.5

17.4 80 29,960 100

'21.7 100 25,280 84

The numbers of microorganisms in the soil vary with the season
of year, being highest in spring and fall and lowest in summer and
winter. The abundance of the individual constituent groups of
bacteria may also \ary with the season of year. Hiltner and Stormer
reported that actinomycetes make up 20 per cent of the microbial
population de\eloping on the plate in spring, 30 per cent in autumn,
and 13 per cent in winter. Conn found larger numbers of bacteria
in winter, e\en in frozen soil, than in summer; he explained this
by the existence of two types of bacteria, "winter" and "summer."
Further studies suggested a simpler explanation for this observation:
the freezing of the soil and subsequent thawing result in the break-
ing up of the clumps of bacteria usually present; this gives an appar-
ent increase in numbers, as determined by the plate method.

In addition to seasonal variations, there are also short-term varia-
tions among the microorganisms in the soil. These are believed to
arise from different competitive factors among the microorganisms.
The long-term fluctuations reflect the seasonal changes in climatic
conditions, as affecting the supply of energy for microorganisms pro-
vided by plant materials. These variations offer a more logical ex-
planation than the "inherent urge" concept suggested by some soil
investigators. The abundance and distribution of microorganisms
in soil, as well as the composition of the population of different soil
types, are influenced primarily by additions of organic matter.

The bacteria of the sofl are capable of adapting themselves readily
to changes in temperature. A definite correlation was found be-
tween the a\erage yearly temperature of the air and soil and the
optimum temperature for bacterial development; this optimum tem-
perature is considerably higher than the soil temperature even dur-
ing the warm periods of the year.

50

Microbiological Population of the Soil

Elective Culture Methods. These methods consist of diluting
the soil with sterile water and adding definite volumes, such as 1-ml
portions of the final dilutions, to special nutrient media adapted for
the growth of specific groups of microorganisms. The highest dilu-
tion which allows positive growth permits the calculation of the

55

1 , 1 1 1 1
_ A Crop yield for 1908-1921 y^

A -

40

/ \ X^^^^

\

25

\^^

^-^^

10

i i 1 1 1 1

1 1

4A 5A 7A 7B 9B llA IIB 18A 19A 19B

Fig. 24. Crop yields and bacterial numbers (from Waksman).

approximate number of specific organisms present in the particular
soil. Thus, if a dilution of soil of 1:100,000 placed in a pectin
medium allows the decomposition of the pectin, whereas a dilution
of 1 : 200,000 does not, one may conclude that the soil contains 100,000
pectin-decomposing organisms per gram.

These methods are rather cumbersome, since they involve the
preparation of a large number of media for the development of vari-
ous physiological groups of organisms, and the need for a number
of containers for making the \arious dilutions. Furthermore, the

Methods of Studying the Soil Population

51

f^^^^^oW II with phosphorus

0 1000 2000 3000 4000 5000 7500 10,000

Pounds calcium carbonate per 2,000,000 pounds soil

Fig. 25. Effect of CaCOs upon bacterial numbers in soil (from Bear).

results show great ^'ariability. These methods, however, may find
certain applications in the solution of special problems.

Table 7. Abuxdanxe of Physiological Groups of Bacteria in Soil
Numbers per gram soil.

After Hiltner

Bacterial Group

and Stormer

After Lohnis

Peptone-decomposi

ng

3,750,000

4,375,000

Urea-decomposing

50,000

50,000

Nitrifying

7,000

5,000

Denitrifying

50,000

50,000

Nitrogen-Bxing

25

388

Table 7 shows the results obtained by two investigators. They
are rather consistent and tend to throw some light upon the rela-
tive abundance of different physiological groups of bacteria in the
soil.

Soil Enrichment Methods. These methods consist in adding
various organic and inorganic nutrients to portions of soil placed in
beakers or tumblers. The soil is adjusted to moisture and kept in
an incubator for varying periods. The chemical changes that have

52 Microbiological Population of the Soil

been brought about in the particular substance by the microorgan-
isms in the soil are then determined. The extent of change is used
as a measure of the activity of the microbiological population of the
soil.

These methods, whatever their value for measming the rate of
certain soil processes, do not lend themselves readily to the quantita-
tive enumeration of soil microorganisms.

Further details of the abundance of specific groups of bacteria
will be found in the respective chapters.

MiCEOORGANISMS IN MANURE AND IN CoMPOSTS

The microbiological population of manures and composts can
be studied by methods similar to those used for the study of the
soil microorganisms.

By the direct microscopic method, manure was found to contain
37,600 millions of bacteria per gram, the greatest number occur-
ring in stable manure kept in heaps, and the smallest number in the
manure that was undergoing a "hot fermentation" process. A definite
correlation was found in the microbiological population of com-
posted manure, whether determined by the plate or by the contact
slide method.

The numbers of bacteria and other microorganisms in stable manure
vary greatly, depending on the composition of the manure, especially
on the nature and amount of solid excreta, and on the degree of its
decomposition. During the decomposition of manure, a marked
change takes place in the nature and abundance of its microbial
population. Fresh manure is very rich in cells of E. coli and other
enteric bacteria. During the process of composting, these soon dis-
appear. At first, multiplication of various bacteria takes place, to
be followed later by a reduction in numbers. The temperature at
which the decomposition of the manure is taking place exerts a
marked influence upon the nature and abundance of the microbio-
logical population, at different stages of decomposition, as shown in
Table 8.

The addition of manures and other organic materials greatly in-
creases the nimibers of microorganisms in the soil. There may also
be a shift in the relative abimdance of different groups. An increase
in numbers of saprophytic organisms may be accompanied by a
reduction in numbers of plant-disease-producing fungi and bacteria.

Microorganisms in Manure and in Composts 53

Table 8. Infliexce of TEMrEnvTiRE ipov the ^IicROBiouKiUAL Population of
Manure Composts (from Waksmaii, Cordon, and Ilulpoi)

Nnmhcrs per fjrani of moist compost.

'lVm])iTaliiro

of Period of Bacteria and

Decomposition Decomposition Actinomycetes

"C days

28 0

8
21
39

50 0

2

8

39

65 0

39

75 0

8

21

It was at first believed that the increase in bacterial numbers fol-
lowing the addition of manure was due to the introduction of organ-
isms present in the manure. The \'alue of addition of manure to
the soil was even ascribed, at least partly, to its bacterial content.
It was later demonstrated, howexer, that when manure is sterilized
before its addition to the soil the effect upon the bacterial popula-
tion is similar. It is the organic materials in the manure which serve,
therefore, as nutrients for the bacteria and other microorganisms and
which are responsible for their multiplication. Certain bacteria,
however, such as the thermophilic forms, may be introduced into
the soil by the manure.

The influence of organic materials in controlling the development
of disease-producing bacteria and fungi in the soil is due to the fact
that the antagonistic organisms favored by the manure play an im-
portant role in suppressing the growth of plant parasites, as will be
shown later.

inomycetes

Fungi

m ill torts

thotisands

i,Goy

200

14,000

0

175

85

11,000

r>o

600

1 , GOO

200

100

2,000

6

1 , 000

1,600

200

100

0

106

0

8

0

1,600

200

4

0

2

0

54 Microbiological Population of the Soil

Distribution of Fungi and Other Nonbacterial
Microorganisms in the Soil

Fungi are important constituent groups of the soil population.
They are widely distributed, certain forms being characteristic of
one type of soil as a natural medium for their development, and
others of other soils. Fungi exist in the soil in the form of vegetative
mycelium and of spores. A colony of a fungus developing on the
agar plate may thus represent either a spore or a piece of mycelium.
The latter produces a fine network around the soil particles. The
mycelium is sensitive to drying of soil, as a result of which dry soil
contains fewer fungi.

The mycelium of some fungi does not break up readily into fine
particles, so that each of these would develop into a colony on the
plate. Because of this, the plate method does not give a fair idea
of the abundance or distribution of various fungi in the soil. A high
plate count of certain fungi may merely indicate a high sporulating
capacity of these organisms. This is true particularly of species of
Aspergillus and Pcnicillium, the mycelium of which also breaks up
readily. On the other hand, the mycelium of the mucorales does
not break up readily, which accounts for their relatively low num-
bers as determined by the plate method. Although it has been def-
initely demonstrated that the normal fungus population in the soil
is present extensively in the mycelial state, the question is still raised:
to what extent does the plate count represent the actual abundance
of fungi in the soil?

Brierley found that the plate count of fungi is open to certain
criticisms: (1) The slow-growing Basidiomycetes are almost all
eliminated in plating the soil and are not found among the colonies
developing on the plate. (2) The same is true of some of the slow-
growing Ascomycetes and Fungi Imperfecti. (3) Some of the Phy-
comycetes require special techniques for their isolation and do not
develop on the plate at all. Most of the published lists of fungi
found in the soil, especially when determined by the plate method,
thus represent only a fraction of the total fungus population.

There is no basis for comparing the relative abundance of the
bacterial and fungus flora of the soil with their potential activity in
the soil, especially when it is not known whether the fungi represent
mycelium or spores. The presence in a given amount of soil, as
measured by the plate method, of a thousand fungi may indicate a

Distribution of Nonbacterial Microorganisms

55

far greater degree of potential acti\ity than the presence of a mil-
Hon bacteria. This is particularly true when a certain process, such
as cellulose or protein decomposition, is stndied. If the number of
fungus colonies is a result of de\'elopment of inactive spores, the
significance of such a comparison may be further questioned.

Among the factors which control the abundance of fungi in the
soil, the reaction occupies a prominent place. An acid medium, ad-

40,000

15 22 29 5 12 19 26 3
October November

10 17 24 31
December

Fig.

7 14 21 28
January

26. Changes in numbers of bacteria and flagellates in soil (from Cutler
and Crump).

justed to ;jH 4.0, is frequently used for determining the numbers
of fungi in soil, since at that reaction most of the bacteria are elim-
inated. This reaction is not the optimum for the growth of fungi,
which lies rather at pH 4.5-5.5. As the acidity of the soil decreases,
the number of fungi decreases and the actinomycetes and bacteria
increase.

Singh found a direct correlation between soil fertility and the
number of fungi and actinomycetes in the permanent mangel and
wheat fields at Rothamsted. The evidence concerning the periodic-
ity of these organisms was inconclusi\e, the numbers being usually
lower in winter. The nature of the crop did not exert a dominant
eflfect, the actinomycetes being relatively higher in the wheat fields
and the fungi in the mangel fields.

Protozoa are also abundant in the soil, their numbers and distri-
bution being influenced greatly by the soil and environmental fac-

56 Microbiological Population of the Soil

tors that influence the bacteria. Among the various counts reported,
the following may serve as an example: the flagellates ranged from
100,000 to 1,000,000 per gram, the amoebae from 50,000 to 500,000,
and the cfliates from 50 to 1,000 per gram. Plots of soil treated with
manure and with organic fertilizers had much larger numbers of
amoebae than untreated plots, as shown in Table 9,

Table 9. Influenxe of Soil Fertilizatiox upox the Xumbers of Amoebae ix

Soils (from Singh)

Numbers

per gram soil.

Amoebae

Barnfield

Broadbalk

Soil Treatment

Soil

Soil

Manure

.'34,000

72,000

Arti6cial fertilizer

^26,000

48,000

T'ntreated

8,000

17,000

The distribution of algae in the soil is controlled largely by hu-
midity and by depth of soil. Although the subterranean numbers
of algae appear to bear no relation to the abundance of carbon and
nitrogen compounds in the soil, manuring was found to have a de-
cided influence upon the development of specific types. The effect
of manure upon the distribution of algae in different depths of soil
is illustrated in Table 10.

Table 10. Ixfluence of Maxure axd Soil Depth upox the Distributiox of
Algae ix Soil (from Bristol-Roach)

Numbers per gram

soil.

Depth

Unmanured

Manured

inches

0-1

16,000

62,000

1-2

10,000

28,000

3-4

28,000

56,000

5-6

4,000

15,000

Earthworms and nematodes are also widely distributed in the soil.
A typical enumeration of nematodes in different soils is given in
Table 11.

Selected Bibliography 57

TAm.K 11. .\m\nAXCK of Xkmatodes i\ Differe.vt Soiijs (from (;<>l)l) aiul Sk-iiicr)

Numliers per acre of soil.

Tliousaiids of
Nematodes in
Location and Xatnir of Soil Surface 6 Inches

Missouri corn fioKl 648,000

New Jersey corn field 129,600

New Hampshire corn field 99,600

Vermont corn fiehl 580,000

Acid forest soil in Virginia 320,000 *

rtah sugar-heet field U , 044 , 000 f

* Surface 3 cm.
t Surface 2 feet.

Selected Bibliography

1. Brierley, W. B., The Microorganisms of the Soil, E. J. Russell, Longmans,
Green and Co., London, 1923.

2. Cholodny, N., t)ber eine neue Methode zur Untersuchung der Bodenmikro-
flora, Arch. Microb., 1:620-652, 1930.

3. Conn., H. J., The microscopic study of bacteria and fungi in soil, N. Y. Agr.
Expt. Sta. Tech. Bull. 64:1918; also 129:1927; 204:1932; Soil Sci., 26:257-
260, 1928; J. Bad., 17:399-405, 1929.

4. Feher, D., Untersuchungen iiher die Mikrobiologie des Waldhodcns, J.
Springer, Berlin, 1933.

5. James, N., and Sutherland, M. L., The accuracy of the plating method for
estimating the numbers of soil bacteria, actinomyces, and fungi in the dilu-
tion plates, Can. J. Research, C, 17:72-86, 1939.

6. Jones, P. C. T., and MoUison, J. E., A technique for the quantitative esti-
mation of soil microorganisms, /. Gen. Microb., 2:54-69, 1948.

7. Kubiena, W., Ein Bodenmikroskop fiir Freiland- und Laboratoriumgebrauch,
Internad. Soc. Soil Sci., Soil Research, 3:91-102, 1932; Arch. Mikroh., 3:507-
542, 1932.

8. Rossi, C, Preliminary note on the microbiology of the soil and the possible
existence therein of invisible germs. Soil Sci., 12:409-412, 1921.

9. Russell, E. J., and Hutchinson, H. B., The effect of partial sterilization of
soil on the production of plant food, J. Agr. Sci., 3:111-144, 1909; 5:152-
221, 1913.

10. Russell, E. J., et al.. The Microorganisms of the Soil, Longmans, Creen and
Co., London, 1923.

11. Singh, B. N., Selection of bacterial food by soil flagellates and amoebae,
Ann. Appl. Biol, 29:18-22, 1942; /. Gen. Microb., 3:204-210, 1949.

58 Microbiological Population of the Soil

12. Starkey, R. L., Some influences of tlie development of higher f)lants upon
the microorganisms in the soil. VI. Microscopic examination of the rhizo-
sphere, Soil Sci., 45:207, 1938.

13. Taylor, C. B., Short-period fluctuations in the numbers of bacterial cells in
soil, Proc. Roy. Soc, B, 119:269-295, 1936.

14. Thornton, H. G., and Gray, P. H. H., The numbers of bacterial cells in field
soils as estimated by the ratio method, Proc. Roy. Soc, B, 115:522-543,
1934.

15. Waksman, S. A., Microbiological analysis of soil as an index of soil fertiUty.
III. Influence of fertilization upon numbers of microorganisms in the soil.
Soil Sci., 14:321-346, 1922.

16. Waksman, S. A., Principles of Soil Microbiology, WiUiams & Wilkins Co.,
Baltimore, 1st Ed., 1927, 2nd Ed., 1932.

17. Waksman, S. A., The Actinomycetes, Chronica Botanica Co., Waltham,
Mass., 1950.

18. Waksman, S. A., Cordon, T. C, and Hulpoi, N., Influence of temperature
upon the microbiological population and decomposition processes in com-
posts of stable manure, Soil Sci., 47:83-113, 1939.

19. Winogradsky, S., Etudes sur la microbiologic du sol. I. Sur la methode,
Ann. Inst. Pasteur, 39:299-354, 1925.

3

OccuiTence of Specific Microorgaiiisiiis
ill the Soil

The soil microbiological population has been divided by Wino-
gradsky into two broad groups: (a) the autochthonous or native mi-
crobes, which are characteristic of the particular soil and which may
be expected always to be found there; {h) the zymogenic microbes,
or those which develop under the influence of specific soil treat-
ments, as addition of organic matter, fertilization, or aeration. To
these two groups, another may be added, (c) the transient microbes,
comprising organisms that are introduced into the soil intentionally,
as by legume inoculation, or unintentionally, as in the case of agents
producing animal and plant diseases; these may die out rapidly or
may sur\i\e in the soil for \'arying periods, especially in the presence
of plant or animal hosts.

Very few organisms can be identified whfle they are still living in
the soil or in the compost. It is necessary to isolate them in culture,
and preferably in a purified state. For physiological studies, pure
cultures of organisms are absolutely essential. Certain fungi, actino-
mycetes, and heterotrophic bacteria can easily be isolated and culti-
vated in pure culture by means of ordinary bacteriological pro-
cedures and simple media. In the case of other organisms, however,
isolation of pure cultures involves considerable skfll, use of special
techniques, and expenditure of much time. This is true, for ex-
ample, of the autotrophic bacteria, most of the protozoa, and certain
fungi. The methods to be used in the isolation and study of differ-
ent microorganisms must, therefore, be adapted to the nature and
nutrition of the organisms.

For identification of different microorganisms, known treatises,
such as Bergey's manual, or special monographs, such as Waksman's
Actinomycetes and Oilman's Soil Fungi, are used.

59

60 Occurrence of Microorganisms in Soil

Soil Bacteria

For the classification of soil bacteria, the Bergey system is now
almost universally used. It will also be adopted here, with certain
slight modifications. The following five orders are now recognized:

I. Simple and undifferentiated forms, not producing any tlireads and not

branching under normal conditions of culture Eubacteriales.

II. Rod-shaped, clubbed, or filamentous cells, with decided tendency to true
branching Actinomycetales.

III. Filamentous, largely aquatic forms, some showing false branching.

Chlamydobacteriales.

IV. Cells enclosed in a slimy mass, forming a pseudoplasmodium-hke aggrega-
tion before passing into a cyst-producing resting stage .... Myxobacteriales.

V. Cells slender, spiral, flexuous Spirochaetales.

Another system of classification of bacteria based upon their physi-
ological activities has frequently been employed in soil studies.

A. Autotrophic and facultative autotrophic bacteria, deri\ ing their carbon prima-
rily from the COj of the atmosphere and their energy from the oxidation of
inorganic substances or simple compounds of carbon.

I. Bacteria using simple nitrogen comiDounds, ammonia and nitrite, as

sources of energy.
II. Bacteria using sulfur and simple inorganic sulfur compounds as sources
of energy.

III. Bacteria using iron (and manganese) compounds as sources of energy.

IV. Bacteria using hydrogen as a source of energy.

V. Bacteria using simple carbon compounds (CO, CH4) as sources of
energy.

B. Heterotrophic bacteria deriving their carbon and energy from organic com-
pounds.

I. Nitrogen-fixing bacteria, deriving their nitrogen from the atmosi^here as
gaseous atmospheric nitrogen.

1. Nonsymbiotic nitrogen-fixing bacteria.

a. Anaerobic, but>'ric acid organism.

b. Aerobic Azotohacter, Radiohacter, Aerobacter, etc.

2. Symbiotic nitrogen-fixing, or root-nodule, bacteria.
II. Bacteria requiring combined nitrogen.

1. Aerobic bacteria.

a. Spore-forming bacteria.

h. Non-spore-forming bacteria:

( 1 ) Gram-positi\'e bacteria.

(2) Gram-negative bacteria.

2. Anaerobic bacteria, requiring combined nitrogen.

Autotrophic Bacteria 61

Autotrophic Bacteria

Autotrophic hactcria arc characterizect by certain physiological
properties that differentiate them sharply from all the other bacteria.
The principles originally laid down by Winogradsky for the growth
of these bacteria still hold today with only slight modifications. The
characteristic properties of these organisms can be summarized as
follows :

1. Their development in nature takes place in strongly elective
mineral media, which contain specific oxidizable inorganic sub-
stances.

2. Their existence is connected with the presence of such inor-
ganic elements or simple compounds, which undergo oxidation as
a result of the life activities of the organisms.

Fig. 27. Nitrite-forming bacterium, Nitrosomonas europea (from Winogradsky).

3. The oxidation of the inorganic substances supplies the only
source of energy for the growth of these organisms.

4. They do not need any organic nutrients either for cell synthesis
or as a source of energy.

5. They are almost incapable of decomposing organic substances
and may even be checked in their development by certain com-
pounds.

6. They use carbon dioxide as an exclusive source of carbon,
which is assimilated chemosynthetically.

The isolation of autotrophic bacteria forms one of the most fas-
cinating chapters not only in the history of soil microbiology but
also in the history of microbiology as a whole.

Nitrifying Bacteria. Among the autotrophic bacteria, the nitri-
fying organisms have received the greatest consideration, because of
the importance of the process of nitrification in the soil, in composts,
in sewage-disposal systems, and in fresh and salt waters. During the
last three decades of the nineteenth century, the elucidation of the

62 Occurrence of Microorganisms in Soil

process of nitrification engaged the attention of some of the most
brilHant minds in the fields of agronomy, soil science, and micro-
biology. These studies cuhninated in the isolation by Winogradsky
in 1891 of the organisms concerned.

Various purely chemical theories were at first suggested to explain
the formation of nitrates in nature. Pasteur was the first to suggest
that the oxidation of ammonia to nitrate is accomplished by the
agency of microorganisms. This view was confirmed in 1877 by
Schloesing and Miintz. When a soil capable of transforming am-
monia to nitrates was heated to 100 °C or treated with antiseptics,
such as chloroform, the process of nitrification was prevented. When
a fresh portion of soil was added to the soil that had been heated
or chloroformed, its power to transform ammonia to nitrate was
restored.

Aeration was found to be essential to nitrification. Proper aera-
tion could be obtained either by bubbling air through the medium
or by placing the medium as a thin layer over the bottom of the
container. It was soon established that the quantity of oxygen con-
sumed during nitrification bore a definite ratio to the amount of
nitrogen nitrified. The addition of calcium carbonate or alkaline
carbonates in low concentrations (0.2-0.5 per cent) had a favorable
effect.

The conditions commonly utilized in saltpeter heaps were thus
found to correspond to the essential factors favorable for the activi-
ties of the nitrifying organisms. These are: (a) presence of ni-
trogenous compounds; (b) thorough aeration; (c) proper moisture
content; (d) presence of bases, like calcium or magnesium car-
bonate. Nitrate formation was noticeable at 5°, became prominent
at 12°, and reached a maximum at 37°C. Higher temperatures, such
as 45 °C, exerted an injurious eflFect, and at 55 °C the process came to
a standstill.

These observations of the French investigators concerning the
biological nature of the nitrification process were confirmed by
Warington. A dilute aqueous solution of ammonia, containing
chalk and sodium-potassium tartrate, proved to be a favorable
medium; addition of sugar to replace the tartrate exerted an injuri-
ous effect upon the process of nitrification. Upon inoculation with
soil, the ammonia was first oxidized to nitrite, and the latter to
nitrate. When organic nitrogenous compounds or nitrogen-rich
materials, such as urine, milk, and asparagine, were added to the
medium, they could be nitrified only after they were first converted

Autotiopliic Bacteria 63

to aniinonia. The process of ammonia formation was, however,
sharply distinguished from that of ammonia oxidation, the latter
being an essential part of the nitrification process, although the
existence of two different organisms was not suspected at first.

The various reactions involved in nitrification of nitrogenous com-
pounds were clearly elucidated toward the middle of the ninth
decade of the last century. All efforts to isolate the specific organ-
isms concerned in the process failed, however. This was chiefly
due to a lack of recognition of the specific mode of nutrition of the
organisms concerned in nitrification. As long as the characteristic
manner of energy utilization by these organisms was not understood,
no suitable methods could be developed for their isolation. Although
numerous inxestigators asserted that certain organisms, some even
pathogenic in nature, were able to produce nitrates, the observations
were not fully confirmed. It is even doubtful whether the results
obtained were properly interpreted; the traces of nitrate observed
in these experiments may haxe come from the atmosphere, and the
nitrite reported may have been a result of the reduction of nitrate
present in the medium. Many of the investigators who approached
this problem were primarily chemists or agronomists, whereas the
bacteriologists were at that time so much under the influence of the
gelatin plate method of Robert Koch that the fact that an organism
produced no growth on this medium was sufficient to justify the
conclusion that such an organism did not exist.

Winogradsky was fully prepared to undertake this study by his
previous investigations of sulfur and iron bacteria, carried out in
1885-1888, which brought out the fact that these organisms were
able to deri\ e their energy from inorganic compounds. He reasoned
by analogy that the nitrifying bacteria could probably use the am-
monia as a soiu^ce of energy. These organisms might, therefore, pos-
sess properties similar to those which have the capacity to oxidize
other elements or simple inorganic substances. The principle of
elective culture was used, with ammonium salts as the only available
source of energy. Conditions were thereby made unfavorable for
the development of all those microorganisms that are unable to
oxidize ammonium salts and utilize the energy thereby liberated.

Flasks containing a salt solution free from organic carbon com-
pounds and with an ammonium salt were inoculated with soil. Bac-
terial growth took place after 4-5 days' incubation at 25-30°C; some-
times a longer period was requued. Manured and culti\ated soils
contained nitrifying organisms in greatest abundance, especially in

64 Occurrence of Microorganisms in Soil

the upper layers. Warington showed that the process of nitrification
consists of two stages: (a) the oxidation of the ammonium salt to
nitrite; (b) the oxidation of the nitrite to nitrate.

In crude cultures prepared from soil, the nitrite-forming bacteria
are present together with the nitrate-formers, and, even when the
development of the former reaches its maximum, the latter may still
be dormant. As soon as all the ammonia has been transformed into
nitrite, the nitrate-formers become active. When transfers are made
from the crude cultures thus obtained into fresh media, the stage of
oxidation of the ammonium salt will influence the type of organism
that will be prevalent in the subsequent culture. If transfers are
made at an early stage of oxidation, when the ammonium ion is still
present, the nitrate organism may be entirely eliminated from the
culture even after only a few such consecutive transfers. If, however,
nitrite is substituted for the ammonium salt in the medium, which is
then inoculated with soil or with a previous culture at an active stage
of nitrate formation, the nitrite-forming organism may be entirely
eliminated. The two bacteria can thus be separated from one an-
other when their characteristic metabolism is recognized.

The culture media in which these organisms develop show at
first no turbidity or pellicle formation, because of the scarcity of
growth of the corresponding organisms. After repeated additions
to the media of ammonium salt or of nitrite, a bluish slime is pro-
duced on the bottom and on the wall of the flask. When this slime
is examined microscopically, it is found to consist of a layer of minute
rods staining with difficulty. After several transfers into fresh media,
the culture becomes sufficiently enriched so that plates can be pre-
pared for the isolation of pure cultures.

All soils that are not very acid in reaction contain bacteria capable
of oxidizing ammonium salts to nitrites and the latter to nitrates.
The limiting acidity for the dexelopment of these bacteria is pU. 4.0-
3.7, whereas their optimum reaction is at pH 6.8-7.3. When a sofl
more acid in reaction than the minimum and lacking the nitrifying
organisms is treated with lime, the organisms will gradually appear,
although inoculation with a good fertile soil is often practicable, so
as to introduce the organisms immediately. This is true of acid peat
soils and certain acid forest sofls.

The nitrifying bacteria are not very sensitive to drying, but steam
or volatile antiseptics are highly injurious, resulting in their rapid
destruction in the soil.

Autotrophic Bacteria 65

Sexeral typos of nitrite-forming organisms are found in various
soils. These bacteria were classified by Winogradsky into four
groups:

1. Nitrosonionas. Free, motile forms, present in the soil as cocci
or as rods with rounded ends. Optimimi reaction is at pH 8.6-8.8;
some strains may ha\e their optimum at /jH 9.1-9.2, and others at
pH 7.5-7.8; growth ceases at pH 6.0.

2. Nitrosocystis. Masses of cocci surrounded by a membrane.
Optimum pH 7.4-7.8.

3. Nitrosospira. Spiral-shaped forms.

4. Nitrosogloca. Zoogloea-producing organisms.

Fig. 28. Nitrate-forming bacterium, Nitrobacter sp. (from Fred and Davenport).

Not all the various types of nitrite-forming bacteria occur in all
soils; the last group is found, for example, only in uncultivated soils.
They differ greatly in their activity, the Nitrosomonas being most
active and the Nitrosospira least. The Nitrosocystis is found in for-
est soils, including both mull and raw-humus soils.

The numbers of the nitrifying bacteria per gram of soil vary
greatly, from a few cells to as many as 24,000. The dilution method
is commonly used for this determination. In view of the fact, how-
ever, that many cells are usually added to a liquid medium before
growth can take place, since the artificial conditions of culture are
not so favorable for their development as in normal soil, the actual
number of living cells in the soil is far greater than indicated by this
method. In humid soils, the nitrifying bacteria are present in the

66

Occurrence of Microorganisms in Soil

upper few inches and rapidly disappear in the subsoil. In arid soils,
they occur to a depth of many feet.

Sulfur Bacteria. The sulfur bacteria, or those bacteria which
are capable of obtaining the energy necessary for their growth from
the oxidation of sulfur or its compounds, should be distinguished
from other bacteria taking part in the sulfur cycle, such as those
liberating hydrogen sulfide in the hydrolysis of proteins or in the
reduction of sulfates.

The sulfur bacteria do not form any uniform morphological or

physiological group of organisms,
as do the nitrifying bacteria.
Morphologically they are found
among the Desmobacteriaceae
and among the Bacteriaceae.
Physiologically they oxidize hy-
drogen sulfide and other sulfides,
elementary sulfur, or thiosulfate;
they act either in an acid or in an
alkaline reaction. Some are obli-
gate autotrophic and some are
facultative. They are widely dis-
tributed in nature, occurring in
water basins, soils, and other nat-
ural substrates. Those sulfur bac-
teria that are found in normal,
fertile soils, or that become active
in such soils when introduced, are limited chiefly to the genus Tliio-
hacillus.

At least eleven species of TJiiobacilliis are found in the literature,
and twelve others have been described but not named. There is
considerable overlapping among the various forms, many of them
having been only incompletely described. Some, like TJi. tliiooxi-
dans, also oxidize sulfiu and are obligate autotrophic. The thio-
sulfate-oxidizing bacteria have been separated into the strictly auto-
trophic (Th. thioparus), facultative autotrophic (TJi. noveUus), and
heterotrophic (Pseudomonas fluorescens) forms: the first two groups
increase the acidity of the medium, and the third group decreases
its acidity.

When sulfur is mixed with soil, it is oxidized slowly at first and
then, as the soil becomes acid, more rapidly. If powdered rock phos-
phate is added to the mixture of soil and sulfur, the insoluble phos-

FiG. 29. Sultur-uxidizing bacterium,

Thiobacillus thiooxidans (from Waks-

man and Joffe).

Aiitolropliic Ikicteria 67

pluitc is tianslonncd into soluble loims by the acid protluccd from
the sulfur. A direct correlation has been found between the acid
formed and the amount of phosphate going into solution. When a
fresh mixture is inoculated with some material from an old compost,
the reaction goes on more rapidly, indicating the biological nature
of the process.

15y inoculating, with some of the above compost, a medium con-
taining sulfur as the only source of energy, certain mineral salts, and
tricalcium phosphate as a neutralizing agent, the culture of a bac-
terium capable of oxidizing sulfur to sulfuric acid is obtained. The
acid produced interacts with the tricalcium phosphate and trans-
forms it into calcium sulfate and monocalcium phosphate and finally
into phosphoric acid.

By use of very acid media, with an initial reaction of pH 2.0 and
a high dilution of the crude culture (1:100,000), a pure culture of
an organism was obtained from such composts. This culture gave
no growth when inoculated into broth or other mecha favorable for
the growth of bacteria and fungi. Microscopic examinations further
established the purity of the organism described as Tli. thiooxidans.
It is a small, nonmotile rod, 0.75-1.0 by 0.5-0.75 /i; it produces cloudi-
ness tliroughout the medium but does not form any pellicle.

The organism is strictly aerobic, the particles of sulfur in the cul-
ture being surrounded by the bacterial cells. The medium becomes
very acid. In the presence of calcium phosphate or carbonate, the
sulfuric acid produced in the medium interacts with the calcium
to gi\ e crystals of CaS04 • 2H2O, which hang down from the particles
of sulfur floating on the surface of the medium; gradually they fill
the flask with gypsum crystals.

The organism forms no spores and is destroyed at 55-60 "C in sev-
eral minutes. The limiting reactions are pH 6.0 and 1.0. It is pos-
sible, howe\"er, to accustom the organism to a neutral and even an
alkaline reaction, when transferred from one soil to another before
the reaction becomes too acid.

Most of the other sulfur bacteria, especially the filamentous forms
{Beggiatoa, TJiiotlirix), occur largely in water basins.

Van Niel has shown that the metabolism of purple and green
sulfur bacteria (Thiorhodaceae) is a truly photosynthetic process
of the general reaction:

CO2 + 2H2A = CH2O + H2O + ^2A
The green bacteria dehydrogenate the H2S only to S, whereas the

68

Occurrence of Microorganisms in Soil

purple bacteria oxidize H2S, S, sulfite, and thiosulfate to sulfate. In
the absence of oxidizable sulfur compounds, the purple bacteria can
develop in the presence of organic compounds under anaerobic
conditions, but only in the presence of radiant energy.

Other Autotrophic Bacteria. Among the other autotrophic bac-
teria should be mentioned those that oxidize hydrogen, carbon mon-
oxide, and ferrous iron; not all of these bacteria are strictly soil
inhabitants, although certain conditions make some of them abundant
in the soil.

Heterotrophic Bacteria

Heterotrophic bacteria comprise the great majority of soil organ-
isms. They depend on organic materials for then energy sources,
and are primarily concerned with the decomposition of cellulose and

hemicelluloses, starches and sug-
ars, proteins and other nitroge-
nous materials, fats and waxes.
These bacteria vary greatly in
structure and physiology, in abun-
dance, and in importance. Some
are aerobic; others are anaerobic.
Some are spore-forming; others
are non-spore-forming. Some are
Gram-positive; others are Gram-
negative. Some are able to fix
atmospheric nitrogen; others de-
pend upon fixed forms of organic
or inorganic nitrogen.

Spore-Forming Bacteria. The
soil harbors a large number of
spore-forming bacteria. The three
most common forms can be read-
ily recognized by the gelatin plate
method. Bacillus mycoides is a rapidly liquefying form; it produces
large filamentous to rhizoid colonies. Bacillus cereus liquefies gela-
tin almost as rapidly and usually forms round colonies with entire
edges; the surface membrane contains granules that tend to be ar-
ranged concentrically. Bacillus megatherium liquefies gelatin more
slowly; its colonies are seldom more than 1 cm in diameter and are
characterized by a flocculent center composed of white opaque gran-
ules, surrounded by a zone of clear hquefied gelatin; the smaller

OOooOOo

Fig. 30. Heterotrophic, spore-form-
ing bacterium, Bacillus megatherium:
a, young cells showing flagella; h,
young cells showing connections into
chains of rods; c, older cells; d, vari-
ations in size and shape of spores
( from Conn ) .

Heterotrophic Bacteria

69

colonies ha\e no surrounding zone and ajc recognized only by their
granular structure. The occurrence of dilFerent spore-forming bac-
teria in the soil is illustrated in Table 12.

Table h2. Occiurenck of Spoue-Forming Bacteria in Different Soils
(from Ford rt al.)

Presence in Number

of Soil Samples

Baltimore

Nazareth

Organism

Soil

Soil

B. pctasitcs

73

IIG

B. cereus

134

41

B. megatherium

29

13

B. subtilis

24

9

B. mesentericus

9

11

B. vulgatus

12

6

B. mycoidcs

15

2

B. mesentericus var

.flavus

9

B. cereus \av.fluorcsccns

3

B.fusiformis

3

2

B. brevis

3

B. simplex

1

B. cohaerens

1

2

B. agri

2

Total isolations

306

214

Non-Spore-Forming Bacteria. The aerobic, heterotrophic, non-
spore-forming bacteria usually produce punctiform colonies on agar
and gelatin; they are chromogenic or nonchromogenic, motile or non-
motile; some liquefy gelatin rapidly, whereas others do it only slowly
or not at all. The most important representative of the group of
rapid-liquefying organisms is Pseudomonas fliiorescens. The whole
group is often spoken of as the fliiorescens group, although many of
the organisms never produce any fluorescence.

Conn divided these organisms into five groups on the basis of
their growth upon synthetic media:

1. Organisms forming small short rods, usually less than 0.5 /x in
diameter, nonmotile or having one or possibly two polar flagella; no
tendency to change in morphology but very variable in physiology,
such as liquefaction of gelatin and gas formation from nitrate. Bac-
terium parviilitm belongs to this group.

2. Organisms that appear, for a day or two after inoculation on
a new medium, as small short rods, less than 0.5 /x in diameter, then

70 Occurrence of Microorganisms in Soil

shorten and begin to look like micrococci. All liquefy gelatin slowly.
Bacterium globiforme can be considered a representative of this
group; it is most abundant in the soil; its presence was believed to
be a good index of the availabiHty of the soil nitrogen.

3. Small short rods, with a tendency to produce long filaments,
usually unbranched, but frequently branched.

4. Organisms consisting mostly of branching forms, especially in
young cultures; they are apparently produced by the germination
of small spherical artlnospores. The branching types disappear in
a few days, leaving the coccoid forms.

5. Organisms occurring normally as cocci, but with a tendency to
produce rods and filaments after a few days of growth on ordinary
media. This group is more abundant in manure than in soil.

Topping suggested classification of the non-spore-forming, non-
acid-fast, rod-shaped soil bacteria into four groups:

1. Gram-positive, motile bacteria, capable of producing branching
variants.

2a. Gram-positive, nonmotile, rod-shaped bacteria.

2b. Gram-positive, nonmotile, mycelium-shaped bacteria.

3. Gram-negative bacteria (motile and nonmotile).

Some of the forms included under 2b are identical with Nocardia
corallinus, which Jensen placed between Corynebacterium and No-
cardia. The forms included in 2a are related to the Nocardia but
are classified with C. liquefaciens.

Thermophilic Bacteria. Miquel was the first to isolate, in 1879,
bacteria capable of developing at 72°C. These organisms were
found in river mud, sewage, animal excreta, dust, and soil. It was
soon established that various bacteria capable of growing at 50-70°C,
but not at room temperature, are found in the soil. Organisms ca-
pable of growing at temperatures up to 79.5 °C were also found in
stable manure. The self-heating and burning of hay, cotton, peat,
and manure are caused by bacteria which Schloesing designated in
1892 as "thermogenic bacteria." Some of these organisms may be
rather thermotolerant than strictly thermophilic. Their distribution
depends on the nature of the soil: the sands of the Sahara Desert
contain such organisms, but forest soils do not. The nature and
amount of manure and fertilizer applied to the soil have a marked
effect: heavily manured garden soils may contain 1-10 per cent
thermophilic forms, as measured by the plate method; field soils
contain only 0.25 per cent or less of these bacteria; uncultivated soils
may be entirely free from them.

llftcrotiophic liacteria 71

In addition to the bacteria, other groups of microorganisms also
contain thermophihc forms. This is indicated by such names as
Thcrmonujces, Thcrmoactinoimjces, and Actinomyces thermophilits.

Myxobacteria. Myxobacteria occur abundantly in manure and
in soil. The total number of these organisms depends upon the
nature of the soil. Some are found only in alkaline, neutral, or
faintly acid (pH 8.0-6.0) soils, others in very acid soils (pH 3.7-5.9),
and still others are independent of the reaction. Moist soils are more
favorable for their development than dry soils; cultivation of soil has
also a fa\orable effect. Peat bogs and moist forest soils contain a
specific flora of myxobacteria.

To demonstrate their presence, balls of rabbit manure, previously
moistened with water and sterilized, are placed on the surface of
the soil. As many as 7-10 species may thus be obtained from a
single soil sample. Certain myxobacteria can be isolated by the use
of living fungus mycelium, such as VerticiUium, growing in a dish;
the bacteria cause the destruction of the mycelium.

Some of the myxobacteria play an important role in the decompo-
sition of vegetable residues in soil, notably the cellulose. It has even
been suggested that the very active cellulose-decomposing group
Cytophaga represents a group of myxobacteria (Myxococcus).

Denitrifying Bacteria. A large number of microorganisms are
able to reduce nitrates to nitrites or to ammonia. Only specific bac-
teria, however, can reduce, under certain conditions, the nitrate to
elementary nitrogen and to oxides of nitrogen, which can thus escape
into the atmosphere. Under anaerobic conditions, the nitrate may
serve as a source of oxygen for these bacteria, with organic carbon
compounds as sources of energy. This process is usually referred to
as complete denitiification, and the bacteria are spoken of as denitri-
fying bacteria.

Various denitrifying bacteria have been isolated from horse
manure, cattle excreta, and soil. Van Iterson demonstrated the
presence in soil of organisms designated as B. stutzeri, B. denitro-
fluorescens, and B. viilpinus. The same soil which favors nitrifica-
tion under aerobic conditions will favor denitrification in absence of
free oxygen.

Several organisms reducing nitrates are capable of obtaining their
energy from inorganic compounds. ThiohaciUus denitrificans, an
organism widely distributed in the soil, oxidizes sulfur and reduces
nitrate to nitrogen gas. Thiosulfate can be oxidized under anaerobic
conditions only in the presence of nitrate as a source of oxygen. The

72

Occurrence of Microorganisms in Soil

energy obtained by oxidation of hydrogen gas has been utihzed for
reduction of nitrates by Hydro genomonas agilis.

Decomposition of cellulose in the soil may be due to the symbiotic
action of two bacteria, one reducing nitrate to atmospheric nitrogen
and the other decomposing cellulose; the products of the cellulose-
decomposing bacteria are used by the other organism as sources of
energy, thus enabling it to reduce the nitrate, whereas the oxygen
thus liberated makes it possible for the cellulose-decomposing or-
ganism to live under anaerobic conditions.
Sulfate- Reducing Bacteria. Several
organisms capable of reducing sulfate to
hydrogen sulfide have been described.
Vibrio desulfuricans was isolated from
soil and other substrates. It is a strictly
anaerobic, Gram-negative form, growing
at 30-55 °C, and able to use salts of or-
ganic acids as sources of energy. The
cultures grown at the lower temperature
(30°) consist of small vibrios or spirals,
motile by means of one or two polar
flagella, and asporogenous; at higher tem-
perature (55°C), the cells are largely
vibrios, containing granules. Starkey
found that they form spores and sug-
gested that the name be changed to
Sporovibrio desulfuricans.
Urea-Decomposing Bacteria. Pasteur was the first to recognize,
in 1860, that ammonia formation from urea is brought about by a
living organism, which he designated as Torula ammoniacale. It was
later established that organisms capable of decomposing urea are
found in most families of bacteria, actinomycetes, and fungi, but that
only certain specific bacteria, whose metabolism is closely connected
with the transformation of this substance, are termed "urea bacteria."
These are divided into cocci and bacilli; the former are usually de-
stroyed at 60-70°, whereas the latter, because of their ability to form
endospores, can withstand heating at 90-95° for several hours. The
optimum temperature is about 30°C. These organisms usually thrive
best in media containing urea (2 per cent), particularly when made
alkaline with ammonium carbonate. The accumulation of ammonia
from the hydrolysis of the urea in the culture is so great as to kill, in
many instances, the organisms themselves. Rapid urea decomposi-

FiG. 31. Sulfate-reducing
bacterium, Spirillum desul-
furicans ( from Beijerinck
and Omeliansky ) .

llctcrotropliic l^acteiia 73

tion docs not necessarily accompany rapid growth. The urea bac-
teria differ in their oxygen tension: most of them are aerobic, although
the amount of oxygen required may be rather small.

The urea bacteria have a pH limit of 6.6 for Urobacillus duclauxii,
7.0 for Ur. maddoxii, and 8.1 for Ur. pasteurii. The favorable effect
of acid peat in prc\enting losses of ammonia from manure is be-
lie\'ed to be due to the checking of the growth of the urea bacteria.

Anaerobic Bacteria. By selective culture methods, Diiggeli
found the following numbers of anaerobic bacteria per gram of soil:
1,000-1,000.000 but>Tic acid, 0-1,000 cellulose-decomposing, 100-
1,000,000 nitrogen-fixing, 100-1,000,000 protein-decomposing, and
100-1,000,000 pectin-decomposing forms. By the deep tube method,
only between 19,000 and 900,000 anaerobic bacteria were found per
gram of soil. No single solid medium can be devised which would
be fa\orable for the development of all anaerobic bacteria.

These bacteria take an active part in the composting of manure
in the heap, whenever there is an insufficiency of aeration. The
phenomenon of "putrefaction" is chiefly a result of the decomposi-
tion of proteins under anaerobic conditions, due to incomplete oxida-
tion resulting from insufficient aeration. The absence of air in the
deeper layers of the manure pile, the slightly alkaline reaction, and
the presence of large amounts of undecomposed organic substances
make conditions favorable for the development of anaerobic bacteria.
Various anaerobic urea bacteria and thermophflic organisms also
find conditions in the composting manure heap favorable for their
development.

Well-rotted horse manure contains spore-forming, anaerobic,
thermophilic bacteria, the limiting temperature for their growth be-
ing 60-65°C and the thermal death point 110-120°C. Some of these
organisms are actively proteolytic. A number of anaerobic spore-
bearing bacteria are no doubt brought into the soil in great abun-
dance with the feces; certain types have been found in intestinal
excreta.

The role of anaerobic sulfate-reducing bacteria in bringing about
iron corrosion appears to be very important. Localized anaerobiosis
is believed to exist in the vicinity of buried jDipes; in the presence
of sulfate and a certain amount of organic matter, the bacteria are
able to bring about the formation of hydrogen sulfide, which will
precipitate the iron dissolved from the pipe to give iron sulfide.

Various anaerobic pathogenic bacteria are able to survive in the
soil. Clostridium welchii was demonstrated in 100 per cent of all

74

Occurrence of Microorganisms in Soil

soils examined, CI. piitrificus verucausus in 71 per cent, CI. putrificus
tenuis in 21 per cent, CI. amylobacter in 65 per cent, CI. tetanomor-
plium in 14 per cent, and CI. tetani in 11 per cent of the soils.

Cellulose-Decomposing Bacteria. Cellulose decomposition in
nature is carried out by numerous groups of microorganisms. Among

..""-'•n

Fig. 32.

Anaerobic, cellulose-decomposing bacterium, Bacillus ccUulosac dis-
solcens (from Khouvine).

these, bacteria occupy a prominent place. The anaerobic bacteria
were at first believed to be the most important agents in the decom-
position of cellulose. It was later found, however, that aerobic
bacteria and various fungi are far more important than the anaerobic
bacteria. In peat bogs, however, and in the digestive tracts of ani-
mals, the anaerobic bacteria are most active. In addition to these,
certain special groups of bacteria are often recognized, such as the
thermophilic forms, actively concerned with the decomposition of

Hetorotropliic Bacteria 75

cellulose in niuuuie, and the denitrifying forms which decompose
cellulose only in the presence of nitrates.

Omeliansk)' found that the gases liberated in the decomposition
of cellulose by anaerobic bacteria contained either hydrogen or
methane. On further study, he observed that these two gases seemed
to be produced by two different organisms: when the inoculum was
added without preliminary heating, methane was formed; when the
inoculum was heated for 15 minutes at 75 °C, conditions favored the
de\'elopment of bacteria which formed hydrogen. The spore of the
methane organism germinated earlier than that of the hydrogen
form. When the cultin-e was transferred, the former organism pre-
dominated and the latter could finally be eliminated. By heating the
inoculum of a young culture, the vegetative cells produced from the
spores of the methane form, which had already germinated, were
killed, whereas the ungerminated spores of the hydrogen form sur-
vived and proceeded to develop. By heating the culture several
times, at an early stage of development, the hydrogen-producing
type could be obtained free from the methane-producing form.

Kellerman and associates could not confirm the results of Omelian-
sky. They isolated from Omeliansky's cultures an aerobic cellulose-
decomposing organism and suggested that the cellulose was decom-
posed by aerobic bacteria; the anaerobic organisms accompanying
the aerobic bacteria were able to form gas from the products of cellu-
lose decomposition. This interpretation has not been universally
accepted, howev er.

Khouvine isolated from the intestine of man an obligate anaerobic
organism, B. cclhilosae dissolvens, capable of decomposing cellulose
vigorously, especially in mixed culture. The vegetative cells were
2.5-12.5 iJL long and produced no Hagella; the spores were 2.5 by
2 IX. The organism was cultivated upon a medium containing fecal
matter as a source of nitrogen. The spores were killed only on boil-
ing for 45-50 minutes. Cellulose was decomposed at a range of 38-
51°C.

Werner isolated from the intestinal tract of the larvae of Protosea
cuprea an anaerobic organism, B. ceUulosam fermentans, capable of
attacking cellulose but no other carbohydrates. The anaerobic nature
of the cellulose-decomposing bacteria in the digestive tract of horses,
cattle, termites, and insect larvae was fully confirmed. Under an-
aerobic conditions, cellulose decomposition is canied out entirely
by bacteria. The mechanism of cellulose decomposition, especially
in relation to the nutrition of herbivorous animals, has been studied

76

Occurrence of Microorganisms in Soil

in detail by Woodman. Methane was produced by unheated cul-
tures and was probably due to accompanying forms. The thermo-
philic organisms occupy an important place among the anaerobic
bacteria.

Probably no other group of bacteria has been so much confused
as the aerobic cellulose-decomposing organisms. Many names have
been proposed for different members of this group, one organism

receiving a number of names from dif-
ferent investigators. In 1918, Hutch-
inson and Clayton reported the isola-
tion from the soil of an organism
which develops first as a sinuous fila-
mentous cell ( 3-10 IX by 0.3-0.4 fi ) and
which goes through several phases in
its life cycle, terminating in the pro-
duction of a spherical body or sporoid.
This body differed in a number of re-
spects from the true spores of bac-
teria. Germination of the sporoid
gave rise to a filamentous form which
possessed perfect flexibility and was
feebly motile, although no flagella
were observed. This organism was
named Spirochaeta cytophaga.

A detailed systematic study of vari-
ous aerobic cellulose-decomposing bacteria found in the soil has been
made by Winogradsky, who divided these organisms into three
genera:

1. Cytophaga: slender, flexible filaments, 3-8 fi long, and pointed
at each end; only cellulose can be used as a source of energy; the
cellulose is changed into a colloidal gel, colored yellow, orange, rose,
red. Four species of this organism were described, including Cyt.
hutchinsoni, the organism previously described by Hutchinson and
Clayton.

2. Cellvibrio: slender, bent rods with rounded ends, 2-5 jx long;
actively motile, with one flagellum; cellulose decomposition is not
invariably specific; cream- to ocher-colored pigment, readily diffus-
ing; very abundant, although only two species were described.

3. Cellfalcicula: spindle- or sickle-shaped cells, not exceeding 2 fi in
length, with pointed ends; motile, with one flagellum; paper stained

Fig. 33. Aerobic, cellulose-de-
composing bacterium, Cytophaga
lutea ( from Winogradsky ) .

Actinomvcctcs 77

green and creani-coloied, ne\er distinctl)' yellow, red, or orange, as
the first two genera are; three species were described.

According to Krzemieniewska, Cyf. hiitcJiinsoni is a totally differ-
ent species from Sp. cytojjJiaga, since the former docs not form
microcysts and the latter does. Spirochaeta cytophaga is believed
to be quite distinct from other species of Cytophaga in its life cycle,
which resembles more closely that of the Myxococcus of the myxo-
bacteria. The name Cyt. myxococcoides was suggested for this or-
ganism. Germination of the microcysts and their transformation into
rods are influenced by the reaction of the medium, temperature, and
oxygen tension. Similar results concerning the life cycle of this
organism were obtained by Issatchenko, who suggested, however,
that the name given by Winogradsky be reserved for the organism.
Another organism belonging to this group was described by Rippel
under the name Itersonia fcrriiginea. Under certain conditions, the
cellulose bacteria are adapted to a specific mode of nutrition, as
shown for the organisms found in rice fields or iron-rich soils; these
bacteria require a certain amount of iron to make their optimum
growth. Their optimum pH is 8.0; growth ceases at pH 4.5 even
in presence of sufficient iron.

Other Bacteria. Many other groups of bacteria are found abun-
dantly in the soil. Among these are mycobacteria, corynebacteria,
\arious anaerobic bacteria in addition to those listed above, and a
host of other bacteria characterized by specific physiological or mor-
phological properties. Some of them are adapted to a special mode
of nutrition and may possess various biochemical properties which
render them of great economic importance. These include the nitro-
gen-fixing bacteria, which are treated in detail elsewhere (p. 191);
antibiotic-producing bacteria, like B. subtilis, B. brevis, and B.
polymyxa; bacteria capable of decomposing the capsular material of
the pneumococcus and of oxidizing p-aminobenzoic acid and an-
thranilic acid; and various coliform bacteria.

ACTINOMYCETES

Actinomycetes form, taxonomically, a link between the bacteria,
tlirough the genera Mycobacterium and Conjnebacterhim, and the
true fungi. They are characterized by the formation of a unicellu-
lar mycelium, composed of hyphae, which show true branching,
similar to that of fungi. The hyphae are rather long and are usually

78

Occurrence of Microorganisms in Soil

0.5-0.8 fx in diameter. The mycelium develops either in the sub-
strate or on the surface of the substrate as aerial growth. The
mycelium breaks up into short fragments, which may look like bac-
terial rods and resemble true bacteria in their protoplasmic proper-
ties. When examined directly under the microscope, the aerial my-
celium is found to consist of ^'ery fine, characteristic, long or short

branching hyphae, with distinct

4^k Hf^> 1/

spore-bearing hyphae.

The reproductive conidia, which
are characteristic of the genus
Streptomijces, are produced by a
simultaneous division of the proto-
plasm in the sporogenoiis hyphae,
progressing from the tip toward
the base. The spores possess a
somewhat greater power of re-
sistance to environmental factors
than the vegetative hyphae. They
resemble bacteria in size, shape,
and staining properties, are 0.5-
1.5 fx in diameter, 1-2 jx long, oval
to rod-shaped.

All actinomycetes, particularly
in young preparations, are Gram-
positive. In stationary liquid
media, they never cause trn-bidity,
but grow either on the surface of
the medium or in the form of
flakes or small colonies through-
out the medium; they may sink to
the bottom of the container or adhere to the glass. The surface
colonies may grow together to form a smooth or wrinkled surface
membrane. The colonies on solid media are usually tough, leathery,
smooth or wrinkled, often growing high above the surface of the
medium, and are broken up only when appreciable effort is applied.
When transferred to suitable media, the spores germinate readily.
The older the mycelium, the more reduced is the germinating power
of the individual fragments. In shaken cultures, they grow in the
form of "clumps" or "colonics" throughout the medium. This mass of
growth can easily be removed by filtration, leaving a clear fluid.

Fig. 34. Streptomijces griseus, with
a short mycelium and abmidant
branching: a, b, c, portions of aerial
mycelimn; d, f, spores germinating
with one and two germ tubes, re-
spectively (from Drechsler).

Actinomycetes

79

The aerial nneeliuin may be white, gray, laxeiider, red, yellow,
brown, green, or of some other type of pigmentation. The aerial
hyphae may be short, giN'ing the growth a chalky appearance, or long,
forming a thick mat o\er the snrface of the vegetative growth; or

Fig. 35. Soil actinomycetes showing difPerent types of sporulation.

they may form a fine network. The colonies are often brilliantly
colored. Some cultures produce soluble pigments which vary in
color and intensity in accordance with the effect of the composition
of the medium. Most species are characterized by the production of
a peculiar sharp odor, characteristic of the soil (earthy odor). All
species of Streptomyccs liquefy gelatin; the rapidity of liquefaction
depends upon the nature of the organism and previous cultivation.
Most of the actinomycetes produce acti\e diastatic enzymes; fewer
produce invertase; still fewer produce tyrosinase, which enables

80

Occurrence of Microorganisms in Soil

■■■■P

t

♦ ' if^^Bi

^^^^^^

V*

. 4

iik^^.U.lL..

mmr^

Actinomycetes

81

thcin to com ort the t)iosin of the protein molecule into dark-colored
melanins.

The numerous species differ primarily in the length of their vegeta-
ti\e mycelium, nature of their aerial mycelium, absence or presence
of spores, method of spore formation, shape and color of colony,
pigmentation of colony and formation of soluble pigment, oxygen
requirement, production of diastatic and proteolytic enzymes, and

Fig. 37. Growth of two typical strains of Micromonospora.

a number of other morphological and physiological characters. These
vary in quantity as well as in quality, not only under the influence
of environmental conditions but even on continued cultivation under
the same conditions. The characteristic pigments produced by many
species may be lost or changed in kind; the color of the aerial my-
celium may be modified, and even the very property of forming such
mycelium may be lost.

The ability of actinomycetes to produce antibiotics has recently
attracted considerable attention. Nearly seventy-five compounds or
preparations have now been obtained. They vary greatly in chemical
composition, toxicity to animals and to plants, in vivo activity, and
chemotherapeutic potentialities. Some, like streptomycin, chloram-
phenicol, aureomycin, terramycin, and neomycin, have found ex-

82 Occurrence of Microorganisms in Soil

tensive application in human and in animal therapy. Their function
in soil processes is still unknown.

According to the system of classifying actinomycetes proposed by
Waksman and Henrici and adopted in Bergey's manual, four genera
are now recognized: Actinomyces, Nocardia, Streptomyces, and

Fig. 38. Effect of actinophage upon Streptomyces griseus ( from Reilly, Harris,

and Waksman).

Micromonospora. The first genus includes the animal pathogens;
the second comprises both parasites and saprophytes; the third em-
braces most of the soil forms, including the plant-pathogenic scab
producer; and the fourth is the most abundant in lake bottoms and
in high-temperature composts.

Soil Fungi

Although fungi are not represented in the soil by so many physio-
logical groups as are the bacteria, many thousands of species find in
the soil a temporary or permanent habitat. Of the various genera of
fungi found in the soil, the most common, both in the number of

Soil Fungi 83

species and in the Ireciuenc) ot occurrence, are 7Aj<:,oihijiichus, Mu-
cor, Rhizopus, PcnicilUum, Aspergillus, frichodenna, Fusarium, and
Clodosporiuni. The wide distribution of different genera of fungi
in the soil is demonstrated in Table 13. Brierley tabulated sys-

T.xni.K l.'{. Isolation- ok Common Gknera of Soil Fungi ky Dikkkkent
Investigators (from Waksiuan)

Mclycan
and

Genus Koiiing Dale Jensen Goddard Wilson Waksman

Acrosialagimts * * * . . *

Alternaria * * * * *

Aspergillus * * * * * *

Cephalosporium * . . . . . . . . *

Cladosporium . . * * * * *

Fusarium * . . * . . *

Mucor * * * * * *

Pejiicillium * * * * * *

Rhizopus .. * * * * *

Trichodcrma * * * * * *

Verticillium . . * . . * . . *

Zygorhynchus . . * * . . * *

* Found to be present.

tematically all the fungi which have been found in the soil. Of these,
56 species belonged to 11 genera of Phycomycetes; 12 species be-
longed to 8 genera of Ascomycetes; 197 species belonged to 62
genera of Fungi Imperfecti. Many more groups have since been
added from all parts of the world. Niethammer and Gilman pub-
lished comprehensive summaries of the fungi isolated from various
soils.

When fresh plant materials are added to the soil, the fungus popu-
lation is greatly stimulated. There is usually a sequence of forms,
depending on the chemical composition of the materials and the
extent of their decomposition. On the basis of their relation to or-
ganic matter, the fungi were divided into seven groups: (1) humi-
coloiis forms, which grow on practically pure humus; (2) terrestrial
(geophilic, terricolous) forms, which grow in soil containing more
or less organic matter; (3) coprophilic (fimicolous) forms, growing
on manure; (4) hijpogeous forms, which grow below the surface of
the soil; (5) lignicoleous forms, growing on the lignins of plant ma-
terials; (6) pseudoparasitic forms, which are wound parasites,
mycorrhiza-formers, facultative parasites; and (7) true parasites.

84

Occurrence of Microorganisms in Soil

Garrett divided the root-infecting fungi into soil inhabitants and soil
invaders, the former being primitive or unspecialized parasites with
a wide host range and widely distributed in the soil, and the latter
including the specialized parasites which depend upon the host
plant.

From an ecological point of view, one may recognize certain spe-

Oo 0^0^

fT II

Fig. 39. Microscopic structure of a soil Penicilliiim (from Thorn).

cific groups of fungi, depending on the nature of the substrate or the
particular nutrients in the substrate which favor their development.
Thus, one may speak of (1) "sugar fungi" (comprising largely Phy-
comycetes); (2) "cellulose-decomposing fungi" (comprising various
Ascomycetes and Fungi Imperfect!); (3) "lignin-decomposing fungi"
(comprising some Basidiomycetes ) ; (4) "humus fungi"; (5) "root-
inhabiting fungi"; (6) "soil-inhabiting parasitic fungi"; (7) "cop-
rophilous fungi"; (8) "predaceous fungi"; etc. When a fresh supply
of nutrients is made available in the soil, as by the penetration and
subsequent death of plant roots, there is a rapid sequence in the
flare up of the various groups of fungi, the "sugar" forms coming
first and the "lignin-decomposing" types last (Garrett).

The effect of lime and manure upon the numbers of fungi in the
soil is shown in Table 14.

Higher Fungi

85

Tmu.i: I I. T\i-i.ri-,\cE of Cki,i,uix)se upon tji^o Xvmhkrs ok Fungi in Soil
(from Waksman and Starkoy)

XiiiulxTs |)(-r firaiii of soil.

NaNOg r
React ion Added

Numbers of Fungi

Soil Soil with

of Soil to without 1 Per Cent

\atiu"e of Soil pW Cultures Cellulose Cellulose

Unlimed, unmanuml . k 1 - 115,700 160,000

Unhmed, unmamiml .">. 1 + 115,700 4,800,000

Limed, umuamu-ed 6.5 - ^20,000 47,000

Limed, unmanured 6.5 + 20,000 i>!)0,000

Unlimed, manured 5.5 - 87,300 320,000

Unlimed, manured ^.') + 87,300 3,100,000

Higher Fungi

The occurrence of Basidiomycetes in soil has been studied largely
by obser\'ations with the naked eye, and findings are not based upon
isolations from soil and cultivation in the laboratory. Hence, only
those fungi which produce fruiting stages visible to the naked eye
have been reported. Gilbert found that the nature and the concen-

FiG. 40. Microscopic structure of a soil Aspergillus (from Thorn).

tration of organic matter in the soil are the most important factors
influencing the development of these fungi. Reaction, moisture con-
tent, light, temperature, season of year, topography, and nature of
higher plants are among the other factors of importance in this con-
nection. Some of the organisms are highly specialized, growing
only under specific conditions and upon very few organic materials,
whereas others are less specific, growing under a great variety of
conditions.

86 Occurrence of Microorganisms in Soil

The higher fungi have been divided into two general groups:

(1) Calcofilic fungi, including Amanita ovoidea, Lepiota granulosa,
Clitocybe geotropa, Tricholoma album, Russula macnlata, Cortinar-
ius fulgens, Boletus satanas, Clavaria flava, and Lycoperdon caelatum.

(2) Calcofugic fungi, including Ainanita virosa, Lepiota procera,
Clitocybe clavipes, Lactarius turpis, Russula amoena, Cortinarius
mucosus, and Boletus bovinus.

Cellulose-Decomposing Fungi

The addition of cellulose to the soil brings about an extensive de-
velopment of fungi, most of which possess very strong cellulose-
decomposing power. These include various species of Penicillium,
Aspergillus, Trichoderma, SporotricJuwi, Fusarium, Chaetomium, and
other forms. McBeth suggested that the fungi play a much more
important part in cellulose decomposition in moist soils, particularly
in humus soils, than in dry soils. Daszewska found Verticillium cel-
lulosae, V. glaucum, Sporotrichum olivaccum, and various other
sporotricha, fusaria, monosporia, alternariae, and moniliae among the
strongest cellulose-decomposing fungi in the soil. She also con-
cluded that the Hyphomycetes play a much more important part
than the bacteria in the decomposition of cellulose in the soil, the
color of the humus being due to the color of the mycelium and the
spores of fungi. Sugars and alcohols were formed as intermediary
products.

More recent studies have fully confirmed these observations. It
may now be concluded that the fungi play a highly important part
in the decomposition of cellulose in soils and in composts. Their
part in the decomposition of cellulosic materials under tropical con-
ditions became particularly important during World War II.

Mycorrhiza Fungi

The mycorrhiza fungi form a special group of organisms. They
are capable of attacking the subterranean organs of plants, feeding
upon their organic constituents. The plant cells may recover, how-
ever, and in their turn digest the fungus mycelium. In this instance,
the subterranean part of the plant and the fungus mycelium form an
association which is frequently of benefit to both, this union being
known as mycorrhiza or fungus-root.

Frank divided the mycorrhiza into two groups: (1) Ectotrophic
mycorrhiza, in which the fungus produces an external investment of
the root, in the form of a crown of hyphae, without penetrating into

Mycorrhiza Fungi

87

cells other than tliose of the epidermis; there is an extensive intcr-
celluhu' de\ elopmcnt between the cortical cells of the roots which is
especially characteristic of forest trees. (2) Endotrophic mycorrhiza,
in which the hyphae of the fungus penetrate to the inner parts of

Fic. 41. Ectotropliic mycorrhiza growing in flask culture (from Melin).

the roots, into definite root layers, and into the cells, and have little
connection with the mycelium in the soil. This is true of plants
belonging to the Orchidaceae, Ericaceae, and Eparidaceae, and
is now known also for many other plants. Root hairs are frequently
absent in ectotrophic mycorrhiza and are replaced by hyphae of
fungi.

Melin described three types of mycorrhiza formations on pine
trees: (1) Forked mycorrhiza, best developed in the presence of an
abundant layer of raw humus; it is golden-brown to black in color.
(2) Tuber mycorrhiza, which is pale at first and later becomes gray
to brownish gray. (3) Simple mycorrhiza, or the unbranched form
characteristically found on the pine; this may be a young stage of

88 Occurrence of Microorganisms in Soil

the forked or tuber type, or it may be a result of conditions unfavor-
able for optimum growth of the fungus. Melin also recognized
pseudomijcorrhiza, which are endotrophic in nature but are not
comparable to the true endotrophic forms in orchids; the hyphae
are not digested and the fungus is largely parasitic.

The stimulating effect of fungi on the growth of Ericaceae is
believed to be due to inactivation, destruction, or absorption of toxic
substances in the rooting medium, rather than to the secretion of
substances stimulating to the higher plants. Rayner claimed that
Phoma radicis is capable of bringing about systemic infection and
results in an obligate mycorrhizal relationship with ericaceous plants.
This concept has not been confirmed.

Numerous species of fungi, nearly all Basidiomycetes, largely
Agaricineae, are capable of forming mycorrhiza. Many of the my-
corrhiza fungi are especially adapted to certain trees, some are less
specific, and still others grow without association with the living
tree. When a forest is removed, the obligate mycorrhizal fungi dis-
appear from the soil and reappear only when a new crop begins to
develop. The spores of these fungi do not germinate on artificial
media, and the mycelium and the fruiting bodies do not develop
when not connected with living tree roots.

Algae

Algae are widely distributed in the soil. Although they are largely
confined to the surface layer and are controlled by the moisture con-
tent, they may also be found below the surface and even in fairly dry
soils. Since they depend on sunlight for their growth, the subter-
ranean forms must either lead heterotrophic existence or remain there
largely in an inactive state.

The soil algae comprise the Myxophyceae, or the blue-greens; the
Chlorophyceae, or the grass-greens; and the Bacillariaceae, which in-
clude the diatoms. Some of the blue-greens are able to fix atmos-
pheric nitrogen. The grass-greens are very abundant in acid soils.

Protozoa

Protozoa are unicellular organisms, varying in size from a few
microns to 4-5 mm. Some protozoa are also able to form colonies
which consist of numerous individuals. The majority of species, par-
ticularly the soil forms, are microscopic and can be studied in detail

Protozoa

89

only with the highest inugnifieations. Their protoplasm is in a col-
loidal state and contains chromatic or nuclear substance, generally
forming nuclei readily distinguishable from the protoplasmic body,

rf?5

o

Fig. 42. Different h'pes of soil algae (from Bristol).

which is either naked at the surface or enclosed by a cell membrane.
Usually one or two nuclei are present; in some cases, several. Con-
tractile \acuoles, when present, are for the elimination of waste
fluids or possibly for the adjustment of the osmotic pressure of the
protoplasm. Some Mastigophora contain in their endoplasm green,
yellow, or brown chromatophores. The most important constituents
of the cell are the complex proteins, particularly the nucleins and

90

Occurrence of Microorganisms in Soil

Fig. 43. Flagel-
late, Bodo cauda-
ttis ( from Martin
and Lewin).

nucleoproteins. In addition to these, carbohydrates, Hpoids, and
enzymes are always present in the hving cell. Also found in the
protozoa are undigested food particles, waste materials, or foreign
elements, which take no part in the physiology of
the organism; algae and bacteria may often be
present in the endoplasm, either as ingested food
or as a result of a certain symbiotic relationship.
Many species of protozoa are subject to attacks by
parasitic organisms.

The protozoa are classified on the basis of loco-
motion, as follows:

1. Sarcodina or Rhizopoda. Motility by means
of pseudopodia, which are extensions of the proto-
plasm of the cell body. The pseudopodia are
broad, blunt, finger-like or filiform, simple, or
branched. In some, the ray-like pseudopodia are
usually supported by axial filaments. Some of the
rhizopods are naked. Others form shells, which
are composed of secreted materials, as chitin,
silica, and calcium carbonate; they may also be constructed from
foreign materials, as diatoms, sand grains, and clay particles. Some
shells are delicate, transparent, whereas others are composed of dis-
tinct plates, arranged more or less regularly.

2. Mastigophora or Flagellata. Motility by means of flagella.
These flexible whip-like processes are usually

attached at one end of the body. Either one
or more flagella may be present. When single,
the flagellum is usually directed forward and
draws the body forward by its movement.
When more than one flagellum is present, one
or more may be directed backward. Some
low flagellates can form pseudopodia.

3. CiUata or Infusoria. Motility by means
of numerous cilia or short hair-like processes
present during the entire existence of the
protozoa or during their embryonic stage only.
The cilia are either evenly distributed over
the surface of the organisms or restricted to
certain regions. Large spine-like cirri or setae, or vibrating mem-
branelles, may be formed from fusion of cilia. Most ciliatcs are free
swimming; some are attached by rigid or flexible stalks or pedicels.

«»

V

Fig. 44. Ciliate, Col-

poda steinii ( from

Goodey ) .

Protozoa 91

4. Sporozoa. These are parasitic forms, the motility of which is
greatly reduced.

Ciliates are present in the soil largely in an encysted condition
and cannot, therefore, function as a factor limiting bacterial activity
in the soil, a property often ascribed to protozoa. Smaller amoebae
and flagellates were at one time beliexed to play the most important
part in the phenomenon of "sick" soils. The limiting factor as
regards their activity in the soil is the quantity of water. An ex-
tensive protozoan fauna nonnally occurs in the soil in a trophic
state; this fauna is most readily demonstrated in moist soil well sup-
plied with organic matter, like heavily manured soils, sewage soils,
and especialh' greenhouse "sick"
soils. The forms predominating
in the soil are not necessarily the
same as those that develop on
artificial media, such as ha>' in-
fusions inoculated with soil. ^ ,^ „ ., , t. 777 r

biG. 45. Sou amoeba, \ ahlkampfia

Protozoa that are present in the „,,,• (f,„„, j^j^^.tin and Lewin).

soil in the form of cysts, especially

after a continuous dry period, will be rapidly transformed into a
trophic state by the first rain that brings the moisture content of the
soil to optimum. Some protozoa are found in an actixe state even
in soils containing a low percentage of water. The flagellate Cer-
comoims crassicauda is capable of excysting and reproducing in
air-dried soils brought to one-sixth of their water-holding capacity.
Various other common protozoa behave normally in soil previously
dried and restored to one-half to one-third of its water-holding
capacity.

Some investigators have reported that ciliates and flagellates are
more abundant in the soil than are amoebae; others have found
amoebae and thecamoebae to be most prevalent. The discrepancy
may be due to the difference in methods used, especially in view
of the sensitiveness of the amoebae and thecamoebae to the compo-
sition of the medium. The largest numbers of protozoa are present
in the soil in spring, after the thawing of snow, or in summer, after
heavy rainfall; only cysts are found in dry soils.

The protozoan fauna is largely confined to the top 6 inches of soil.
In arid regions, especially in poor sandy soils, protozoa are found in
greatest abundance somewhat below the surface. Irrigation of arid
sofls stimulates considerably the development of numerous proto-

92 Occurrence of Microorganisms in Soil

zoa. The richer the soil is in organic matter, the richer it is in
protozoa, especially in amoebae and thecamoebae.

The majority of soil protozoa are cosmopolitan, since they are
found throughout the world, although not all the species are found
in every soil.

Cutler and associates found six species of protozoa occurring con-
stantly in the soil in sufficient numbers to admit the application of
statistical methods to the results. These are: (1) Dimastigamoeba
gruberi, (2) a small limax amoeba, (3) Heteromita sp. resembling
Bodo repens, (4) a small soil flagellate, 3-6 by 2-3 ju; (5) Cercom-
onas sp., and (6) Oicomonas termo.

Sandon found the following average number of species of protozoa
in 107 soils examined: 7.2 flagellates, 3.4 ciliates, 2.45 amoebae, and
2.0 testaceous rhizopods. Some species grew in all media employed;
others developed only in special media. In all, Sandon recorded 250
species of protozoa, some of which were observed in every soil, often
in very large numbers. The flagellates Heteromita globostis,
Oicomonas termo, and Cercomonas sp.; the ciliates Colpoda ciicul-
lus and C. steinii; and the limax amoebae Ndegleri gruberi and Hart-
manella hijalina were most common and most abundant. Most pro-
tozoa found in the soil are also present in various other habitats,
such as standing and flowing fresh waters, sea water, and plankton;
a few are found only in the soil. The extreme climate of arctic land
is not in itself an obstacle to the abundant development of protozoa,
provided the soil is well manured and in good condition.

In general, the soil contains an extensive population of protozoa,
consisting largely of amoebae and flagellates, and to a lesser extent of
ciliates. These organisms are specifically adapted to a terrestrial
form of life. The protozoa, in comparison with other groups of
microorganisms, form only a small part of the microbial population
of the soil. Theii- ability to reduce the numbers and control the ac-
tivities of other groups of microorganisms in soil is very limited.
Some protozoa feed only upon certain types of bacteria, others con-
sume protozoa, and still others take an active part in the decomposi-
tion of plant and animal residues; even by consuming certain specific
bacteria, they may favor the process for which these bacteria are
responsible. Partial sterilization of soil does not destroy all the
protozoa.

Viruses and Phages

93

Other Animal Forms

Animal forms larger than protozoa also occur abundantly in the
soil. They range from microscopic nematodes to large earthworms
and insect larvae. Some nematodes
(Hetcrodera schachtii) and certain in-
sects are parasitic on plants; some
(hookworm larvae) are parasitic on
animals; others, such as nematodes that
attack Japanese beetle larvae, parasi-
tize plant parasites and are thus bene-
ficial. Many are saprophytic; these

comprise the earthworms, which macerate the soil, mix the organic
with the inorganic contents, and thus greatly improve soil fertility.

Fig.

46. Parasitic nematode
(from Cobb).

Viruses and Phages

Certain viruses and various phages exist independently in the soil.
The mosaic \'irus of wheat can be transmitted from the soil. Heating
the soil for 10 minutes inactivates this virus. The survival in the soil

Fig. 47. Saprophytic nematode attacking parasitic form (from Cobb'

of phages active upon legume bacteria may become an important
economic problem in successful legume inoculation; the selection of
phage-resistant strains of bacteria may be the answer. Various ac-
tinophages have also been demonstrated in the soil.

94 Occurrence of Microorganisms in Soil

Selected Bibliography

1. Bergey's Manual of Determinative Bacteriology, Williams & Wilkins Co.,
Baltimore, 6th Ed., 1948.

2. Cutler, D. W., and Crump, L. M., Problems in Soil Microbiology, Longmans,
Green and Co., London, 1935.

3. Darwin, C, The Formation of Vegetable Mould through the Action of
Worms, with Observations on Their Habits, John Murray, London, 1881.

4. Garrett, S. D., Root Disease Ftingi, Chronica Botanica Co., Waltham, Mass.,
1944.

5. Garrett, S. D., Ecological groups of soil fungi: A survey of substrate rela-
tionships, The New Phytologist, 50:149-166, 1951.

6. Gilman, J. C, A Manual of Soil Fungi, The Collegiate Press, Ames, Iowa,
1945.

7. John, R. P., An ecological and taxonomic study of the algae of British soils.
L The distribution of the surface-growing algae, Ann. Botany, N. S., 6:323-
349, 371-395, 1942.

8. Melin, E., Untersuchungen iiber die Bedeutung der Baummykorrhiza,
G. Fischer, Jena, 1925.

9. Niethammer, A., Die Mikroskopischen Bodenpilze, N. V. Van de Garde &
Co., Drukkerij, Zaltbommel, 1937.

10. Rayner, M. C, Mycorrhiza, Wheldon and Wesley, London, 1927.

11. Russell, E. J., et al.. The Microorganisms of the Soil, Longmans, Green
and Co., London, 1923.

12. Sandon, H., The Composition and Distribution of the Protozoan Fauna of
the Soil, OHver and Boyd, London, 1927.

13. Smith, N. R., Gordon, R. E., and Clark, F. E., Aerobic mesophilic spore-
forming bacteria, 17. S. Dept. Agr. Misc. Pub. 559, 1946.

14. Starkey, R. L., Products of the oxidation of thiosulfate by bacteria in min-
eral media, /. Gen. Physiol., 18:32.5-349, 1935; Isolation of some bacteria
which oxidize thiosulfate, Soil Sci., 39:197-220, 1935.

15. Van Niel, C. B., Advances in Enzymology, pp. 263-328, Interscience Pub-
lishers, New York, 1941; The culture, general physiology, morphology, and
classification of the non-sulfur purple and brown bacteria, Bact. Revs., 8:1-
118, 1944.

16. Waksman, S. A., Principles of Soil Microbiology, Williams & Wilkins Co.,
Baltimore, 1st Ed., 1927, 2nd Ed., 1932.

17. Waksman, S. A., The Actinomycetes, Chronica Botanica Co., Waltham,
Mass., 1950.

18. Winogradsky, S. N., Microbiologic du sol; problemes et methodes, Masson
et Cie, Paris, 1949.

19. Wolf, F. A., and Wolf, F. T., The Fungi, 2 vols., John Wiley & Sons, New
York, 1947.

4

Decomposition of Plant

and Animal Residues
in Soils and in Composts

For dust thou art, and unto dust shalt thou return. Genesis 111:19

Nature of Plant and Animal Residues

With the exception of autotrophic bacteria, the green or chloro-
phyll-bearing plants are the only living forms on this planet capable
of synthesizing organic matter out of inorganic elements and simple
compounds. These essential nutrients are obtained partly from the
atmosphere and partly from the soil. By utilizing the photosynthetic
energy of sunlight, plants are able to produce, from carbon dioxide
and water, sugar and stai'ch, which serve as the starting point for
the synthesis of numerous other carbohydrates, fats, proteins, and
various other compounds. The soluble forms of nitrogen and the
minerals required by the plant for synthetic purposes are obtained
from the soil; certain few plants, the legumes, are able, in association
with root-inhabiting bacteria, to obtain their nitrogen from the ele-
mentary form in the atmosphere.

Plant materials are partly used for animal feeding and are partly
returned to the soil in the stubble and other plant residues. The
animals and their excretion products also find their way, sooner or
later, into the soil. These materials are subject to decomposition by
numerous groups of microorganisms and thereby contribute to the
soil organic matter. The various organic residues which undergo
decomposition in soils and in composts can be classified as follows:

1. Plant and animal remains decomposing on the surface of the
soil; here belong the leaves, needles, branches, and twigs of all plant
life.

95

96 Decomposition of Plant and Animal Residues

2. Plant residues plowed into the soil; these include plant stubble
and special crops which are grown specifically for this purpose, as
cover crops or green manures.

3. Stable manures; these consist of the solid and liquid animal
excreta and bedding.

4. Artificial manures and composts.

5. Organic commercial fertilizers; these include a \ariety of ani-
mal and plant products, such as bone meal, dried blood, tankage,
cottonseed meal, linseed meal, peat.

6. Microorganisms and their dead bodies.

The plant residues are made up of tliree groups of constituents:
water, organic materials, and inorganic compounds. The water con-
tent of plant residues varies from 50 to 95 per cent, depending on
the nature and degree of maturity of the plant, usually about 80 per
cent for young and 60 per cent for mature plants. The water-free
plant material consists of 88-99 per cent organic matter, and 1-12
per cent mineral or inorganic matter. The organic constituents com-
prise a large number of chemical compounds containing the ele-
ments carbon, hydrogen, oxygen, and nitrogen and, in lesser amounts,
sulfur, phosphorus, potassium, and a variety of others, some of which
are usually present in mere traces.

When the organic matter synthesized by the plants undergoes
digestion by herbivorous animals, many of the constituents are de-
stroyed and the elements changed back into simple gases or inorganic
compounds such as COo, H^O, NH3, phosphates, sulfates, potassium
salts. Out of the plant materials, directly or after they have been
transformed into simpler compounds, the animals synthesize their
own tissues. These animal bodies may now be used, in their turn,
as food by other animals. The bodies of these omnivorous or carniv-
orous animals also undergo a series of transformations; part of the
elements and compounds which they consumed as nutrients are lib-
erated as waste products, in the form of gases ( CO;., NH3 ) , as simple
organic compounds (urea, organic acids), or as complex organic
materials comprising the residual and partly digested plant and
animal residues found in the feces.

Animals depend upon plants and some of them upon other ani-
mals for their necessary energy, for the organic nitrogenous com-
pounds, for some of the fatty substances, and for the vitamins. On
the other hand, animals are able to synthesize, out of the complex
materials supplied to them by the plants and other forms of life,
new organic compounds, largely of a protein and fatty nature. Some

Nature of Plant and Animal Residues 97

ot the plant eonstituents, sueh as the sugars, starches, fats, and pro-
teins, are utilized by animals for their own metabolism and for
suppUing their energy needs. Other plant eonstituents, like the
hemieelluloses, cellulose, lignins, and waxes, are used not at all
in the animal system or only to a very limited extent. The cow and
other ruminant animals are able to digest a large part of the cellu-
lose, with the help of bacteria lixing in their digestive tracts. The
undigested residues are excreted by the animals and sooner or later
find their way into the soil.

The bacteria and certain protozoa may play an important part in
the digesti\e mechanism in the animal body: (a) by digesting the
cellulose and certain other carbohydrates to organic acids, they make
these constituents axailable to the animal for its nutrition; (b) by
synthesizing certain vitamins and other complex substances in the
animal, they supply nutrients which the animal is unable to synthe-
size; (c) they may also form certain products that are undesirable
or even toxic to the animal body.

The plant and animal residues find their way into the soil either
directly or after preliminary decomposition in composts or on the
surface of the ground. These residues comprise either the whole
plant, stems, lea\es, and seed, or only certain parts of the plant,
needles and lea\es, surface stubble, and subsurface roots. The sur-
face portion of certain crops, such as grasses used for pasture, cereals,
and corn, may be largely removed for cattle food or for other pur-
poses. In some cases the straw may be returned to the soil, either
as such or as a constituent part of the stable manures. In crops like
peas and beans, only the seed may be removed from the land, whereas
the rest of the plant may find its way into the soil. In still other
crops, used for soil cover or as green manures, the whole plant may
be returned to the soil. In pastures and in forests, where the soil
is not plowed at all, the plant residues are attacked by microorgan-
isms either in the soil itself or on the surface of the ground; the
products of decomposition gradually find their way into the soil
through leaching or by land cultivation.

Thus the cycle of life is completed, from the soil back to the soil.
In this broad cycle, numerous secondiuy cycles occur, in which one
or more elements are concerned. In the transformation of each one
of these, microorganisms play a highly important part. Without
them, life would soon come to a standstill; upon their activities, the
continuation of life on the planet depends.

98 Decomposition of Plant and Animal Residues

Abundance and Chemical Composition of Plant Residues

Sachs calculated that the leaves of an ordinary sunflower plant,
having a surface area of 1.5 square meters, absorb two-thirds of a
liter or 1.3 gm of COo per hour. If the growing day is taken to be
10 hours, the plant will absorb 400 gm of COo a month. On the
basis of a milHon plants per square kilometer of land and a 3-month
growing period, the sunflower will consume annually 1,200,000 kg
of CO2. In view of the fact that the COo content of the atmosphere
is very small, only 0.03 per cent, the available supply of this essential
plant nutrient would soon become exhausted if it were not for the
continuous liberation of the COo from the soil by the action of micro-
organisms upon the plant and animal residues and upon the soil
organic matter.

According to Lundegardh, the amount of CO2 produced in the
soil by microorganisms approaches that which is required by the
plants for the photosynthesis of organic matter. If it is assumed that
the average content of the organic matter in the upper 15 cm of
soil is 2-4 per cent, an acre of soil will contain 20,000-40,000 kg of
organic matter. Since the carbon content of the latter is 58 per cent,
it is possible to conclude that the average amount of organic carbon
in an acre of soil is 10,000-22,000 kg. In some soils, like prairie and
peat soils, the organic matter content may be considerably higher
(10 per cent or more), whereas, in poor sandy sofls, it may be 1 per
cent or less.

A study by Waksman and Starkey of the evolution of COo from
soil revealed that, under favorable moisture and temperature condi-
tions, 1 kg of soil may give off, in 24 hours, 5-30 mg of carbon as COo.
Taking an average of only 10 mg of carbon and a period of active
annual decomposition of 4 months, we find that an acre of soil con-
taining 10,000 kg of carbon will give off during the wann months
1,000 kg of carbon in the form of COo. Under these conditions of
decomposition, the soil organic matter would become exhausted
within 10 years. If it were not for the constant addition of plant,
animal, and microbial residues to the soil, the amount of available
COo from the above source would soon also become a limiting factor
in plant growth.

Ebermeyer calculated that the vegetation on 1 hectare of field soil
consumed annually 2,000 kg of carbon and on 1 hectare of forest
soil 3,000 kg, corresponding to 7,300-11,000 kg COo. The plant

Chemical Nature of Constituents 99

vegetation of the whole eartli (allowing for 25 per cent of the earth
surface as being nnproducti\c), coxering 10,160 million hectares,
will require an annual consumption of 90 billion kg of COo. The
whole atmosphere contains 2,100 billion kg of CO^, thus allowing
only for about 25 annual crops, a xery small figure indeed. Others
have calculated, howe\er, that the green plants consume annually
only one-se\ enteenth of the CO:., of the air in 1 year, which amounts
to 30 billion kg annually. Ebermeyer further reported that, out of
the 3,000 kg of carbon synthesized by 1 hectare of forest, 1,491-1,792
kg was con\erted to wood and 1,196-1,467 kg to litter. The latter
is returned immediately to the soil and becomes subject at once to
decomposition by microorganisms.

Chemical Nature of Plant and Animal Constituents

The plant and animal bodies are made up of numerous organic
compounds. Attention will be directed to only the more important
and more abundant substances, the decomposition of which by
microorganisms has been studied in greater detail and contributes to
oiu- knowledge of the cycle of life in the soil:

1. Fats, oils, waxes, sterols, and terpenes.

2. Carbohydrates, including the simple sugars or the mono-, di-,
and tri-saccharides, the starches, the hemicelluloses (comprising the
pentosans and hexosans ) , the polyuronides ( pectins, gums, and muci-
lages), and true cellulose.

3. Organic acids, including saturated fatty acids, oxy-fatty acids,
and unsaturated acids.

4. Aldehydes, ketones, and alcohols, including aliphatic, poly-
valent, and unsaturated alcohols.

5. Lignins, compounds which are frequently spoken of as "in-
crustants." They are believed to form definite chemical or physical
compounds with the celluloses. Some believe, however, that cellu-
lose and other carbohydrates do not form any chemical compounds
with lignins and may not e\en form any homogeneous mixtiu'cs.
This concept is substantiated by the fact that the lignin content of
plants varies considerably, depending on the plant and on the stage
of growth, and may even vary in the different tissues of the same
plant.

6. Cyclic compounds, including hydrocarbons, phenols, quinones,
tannins.

100

Decomposition of Plant and Animal Residues

7. Alkaloids and organic bases, including purine bases, pyridine,
and piperidine compounds.

8. Proteins, polypeptides, amino acids, amines, and other ni-
trogenous compounds.

9. Enzymes, hormones, vitamins, pigments, antibiotics, and other
important products of living systems, the exact chemical nature of
some of which still remains unknown.

Fig. 48.

Cellulose

Influence of age upon the chemical composition of rye ijlants (from
Waksman and Tenney).

10. Mineral constituents: phosphates, silicates, sulfates, carbonates,
chlorides, nitrates, and potassium, sodium, calcium, magnesium, and
other salts.

It is almost impossible to make a complete quantitative analysis
of plant and animal materials, whereby all the chemical constituents
are accounted for. For most purposes, it is sufficient to account for
some of the more important groups of compounds, to obtain a
fairly good idea of the chemical composition of the materials which
undergo decomposition. Such an analysis need be only proximate
in nature. It may be supplemented by special determinations of
certain compoimds wliich are either characteristic of a given ma-
terial or essential for the understanding of a certain process. In a
proximate analysis, only those compounds which occur most abun-
dantly in the plant and the decomposition of which is best under-

Decomposition Processes 101

stood arc taken into consideration. The common loodstult analyses
are not of sufficient value for this purpose, since they give a rather
limited concept of the chemical composition of the plant materials
that find their way into the soil.

A series of analyses of various plant materials, as obtained by
the proximate method, is gi\ en in Table 15. The chemical composi-
tion of the plant \ aries not only with its nature, but also with its age
and with conditions of growth and nutrition. Tables 16 and 17
indicate the effect of age upon the chemical composition of rye and
corn plants. At an early stage of growth, plants are rich in water-
soluble substances, including sugars and amino acids, in proteins,
and in mineral constituents; the older the plants become, the less is
the proportion of these constituents and the greater is the concen-
tration of cellulose and lignin and, to some extent, of the hemicellu-
loses and polyuronides. This change in composition has an im-
portant bearing upon the rapidity of decomposition of the plant
materials. In leguminous plants, such as alfalfa, the protein content
also decreases with the maturity of the plant, and the cellulose and
lignin contents increase. This affects both the digestibility of the
plant materials by animals and their decomposition in soil.

Decomposition Processes

When plant and animal residues are added to the soil or are placed
in composts under favorable conditions of moisture and aeration,
they are attacked by a great variety of microorganisms, including
bacteria, fungi, actinomycetes, protozoa, worms, and insect larvae.
As a result of the activities of these organisms, considerable portions
of some of the constituent chemical elements in the residues, notably
the carbon, nitrogen, phosphorus, and potassium, are rapidly liber-
ated in forms available for plant growth. The process is at first
rapid but gradually slows down; the rate of decomposition depends
upon the nature of the residues and upon the conditions under
which decomposition is taking place. If the residues are low in
nitrogen, as in straw, this element wffl not be liberated for some
time and is, therefore, not made available for plant growth.

This is illustrated in Table 18, where the formation of nitrate in
the soff is used as a measure of liberation of the nitrogen in an avail-
able form. A definite quantity of root material obtained from several

102 Decomposition of Plant and Animal Residues

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Decomposition Processes

103

Taui.i; Hi. l\KLLi;.NCE OK Atiio UK Hyp; I'i-ants ipdn Tiikiu Chkmicu- ('i)\ii'i>.srrr(i\
(from AVaksiiiaii ami 'I'cimry)

IVr fciil of (Ir\- iii;il('ri;il.

Constituent
Fats and waxes
Cold- water-soluble
Pentosans
Cellulose
Lignin
Ash
Total nitroKen

Age

of Plant

Just l)efore

10-14

.Just before

Hloom, Stems

Mature Plants,

Inches

Head For-

and Leaves

Stems and

(I)

mation (II)

(III)

Leaves (IV)

2.60

2.60

1.70

1.26

34.24

22.74

18.16

9.90

16.60

21.18

22.71

22 . 90

18.00

26.9a

;50.59

36.29

9.90

11.80

18.00

19.80

7.66

5.90

4.90

3.90

2.50

1.76

1.01

0.24

Table 17. Composition of Indian Corn at Different Stages of Growth
(from Morrison and Henry)

Pounds

per acre.

Dry

Crude

Stage of Growth

Matter

Ash

Protein

Four feet high, .luly 24

731

90

149

First tassels, Aug. 6

2,245

195

360

Silks drying, Aug. 28

4,567

272

436

Milk stage, Sept. 10

0,174

328

544

Glazing stage, Sept. 24

8,104

389

566

Silage stage, Oct. 1

8,929

369

660

Ready to shock, Oct. 8:

corn and cobs

5,186

76

492

stalks and blades

4,226

307

199

Total

9,412

383

691

plants and containing 0.6 gm of nitrogen was added to 13 kg of soil;
the nitrate produced during 3 months was measured. With an in-
crease in the nitrogen content of the plant material, there is an
increase in the formation of nitrate. Only when the nitrogen con-
tent is 1.7 per cent is the rate of decomposition sufficient to supply
the requirements of the microorganisms for cell synthesis. The
lower the nitrogen content of the plant residues, the more nitrogen
is required by the microorganisms from an outside source, the soil,
and, therefore, the lower is the nitrate content of the soil itself or
the amount of nitrate a\ailable for plant growth.

104 Decomposition of Plant and Animal Residues

Table 18.

IXFLUEXCE OF NiTROGEN CoNTENT OF PlANT RESIDUES ON THE LIBERATION

OF Nitrogen as Nitrate (from Lyon, Bizzell, and Wilson)

Nature

Nitrogen

Weight

Nitrogen

of Root

Content

of Roots

Found as

Material

of Roots

Used

Nitrate

per cent

gm

mg

Control soil

0

946.6

Oats

0.45

133.3

207.3

Timothy

O.fi^Z

96.8

398.4

Corn

0.79

75.9

510. G

Clover

1.71

35.1

9-24.4

To hasten the decomposition of straw and of similar plant ma-
terials, some available nitrogen and phosphorus may have to be
added in the form of inorganic salts. The microorganisms bringing
about decomposition of the plant and animal residues are living sys-
tems. They grow and multiply; they require considerable amounts
of energy and nutrients. In the straw and in the stubble of cereals
and other plants, they find sufficient energy but not enough essential
nutrient elements, especially nitrogen and phosphorus. Hence, these
must be added to favor the activities of the organisms which thus
bring about the rapid destruction of the plant materials. When
green plants, however, such as young rye and clover, or plant and
animal residues high in nitrogen and in phosphorus are added to the
soil, the microorganisms are able to decompose them rapidly, without
the addition of inorganic salts; the plant nutrients are liberated.

The results presented in Table 19 fully confirm those given in
Table 18, even though totally difi^erent types of material are used.
Plant substances high in nitrogen decompose rapidly; a large part
of the nitrogen is liberated as ammonia, and comparatively little

Table 19. Products of Decomposition of Rye Plants Harvested at Different
Stages of Growth (from W'aksman and Tenney)

Stage of
Growth

of Plant

I

II

III

TV

CO2
Given Off
mg C
286.8
280.4
199.5
187.9

Nitrogen

Liberated

as Ammonia

7ng N

22.2

3.0

0

0

Nitrogen Consumed

from Ammonium

Salt Added to Soil

vig X

0

0
7.5
8.9

*See Taljle Hi

Decomposition Processes

105

humus is loft. Materials low iu nitrogcu decompose slowly, liberat-
ing at first no available nitrogen and leaving a large amount of
humus. This is illustrated in Fig. 49. There are many e.xceptions
to this rule, depending on the nature of the residues and their treat-
ment and on the nature of the soil.

25

20 -

15 -

10 -

5 -

< /

/

-S' /

if

A 7

40°-

/ /"^X /

„ ''"' 35°

r'
1

1

P A\

f / * N
/ \ \

/ * \

/'''Hem>ce\\uLoses_

1
1

1 I
1 / ,

/ ^ \

-Y'--^" 30° -

n/

>

\

1 /J

i/i

/ \

' \
/
/

\ Temperature 25°

//

/
/

/

--,^C^>'-

/ r

/

•

/ //

•

///

Xylan in cellulose 20° -

\ / /^

^^■-^ '

X / yv ^

V/Xf /

A^ ' ^-^

^^

fU"-: 1^'

1 1 1 [ 1

, 1,1,1,

60

-40

- 20

0 2 4 6 8 10 12 14 16 18

Time, days

Fig. 49. Decomposition of oat straw (from Norman).

Straw, stubble, and forest litter, unless properly supplemented
with essential nutrients, are useful primarily as sources of humus
and less as fertilizing materials for plant growth; they may leave,
after 3-10 months of decomposition, as much as 50-60 per cent
hiunus.

In the process of humus formation from plant residues, three dis-
tinct phases are recognized: (a) rapid decomposition of some of the
chemical constituents by microorganisms, (/?) synthesis of new sub-

106 Decomposition of Plant and Animal Residues

stances by these organisms, (c) formation of resistant complexes by
various processes of condensation and polymerization. During the
process of decomposition, a considerable amount of microbial cell
substance is synthesized. This substance is later attacked again by
other microorganisms. The processes of decomposition are contin-
ued until most of the organic complexes in the original plant ma-
terials are gradually changed into simple elements or inorganic
compounds. The final processes of decomposition of organic matter
by microorganisms and the final liberation of the elements in min-
eralized form complete the cycle of transformation of the essential
chemical elements which are used for the building up of organic life
in nature. The microbes thus tend to complete the cycle begun by
plants.

The plant and animal residues do not decompose as a whole. The
various chemical constituents are attacked at different rates. The
sugars and starches, some of the hemicelluloses, and some of the
proteins undergo a most rapid decomposition by a great variety of
microorganisms. The cellulose, certain hemicelluloses, and some
of the fats, oils, and other plant constituents are decomposed more
slowly and, commonly, by specific orgauisms. The lignins and some
of the waxes and tannins are most resistant to decomposition; some
of the lignins may even afl^ect the decomposition of the proteins by
rendering the latter more resistant to attack. This is illustrated in
Table 20.

Table 20. Chemical Changes in Corn Stalks as a Resi'lt of Decomposition by
Microorganisms (from Tenney and Waksman)

On basis of dry material.

Original After Days of Decomposition
Chemical Corn < ■ <

Constituent Stalks 27 68 20.5 405

per per per per per

cent cent cent cent cent

Ether-soluble 1.80 2.22 0.80 0.64 0.2")

Cold-water-soluble 10.58 3.43 5.27 JJ.OG 4.59

Hot-water-soluble 3.56 2.45 3.20 5.36 8.71

Hemicelluloses 17.63 15.56 16.41 10.68 10. .39

Cellulose 29.67 23.80 21.93 6.28 5.05

Lignin 11.28 17.70 19.12 23.83 21.30

Crude protein 2.50 4.81 6.84 10.93 12.13

Ash 7.53 26.12 29.43

Products of Microbial Decomposition

107

The rate of decomposition of plant and animal materials can be
measm-ed by a number of different methods. These are based upon
the products of decomposition; the disappearance of specific plant
and animal constituents, such as the sugars, the cellulose, the pento-
sans, or some of the nitrogenous bodies; the formation of resistant
products of decomposition, such as accumulation of lignins and their
transformation into humus compounds. As shown in Table 20, the
accumulation of the ash may be used as a measure of total decom-
position.

Products of Microbial Decomposition

When plant and animal residues undergo decomposition in the
soil and in composts, the various constituent elements, especially the
carbon, nitrogen, sulfur, and phosphorus, are liberated in mineralized
forms.

15

45 50

65

Fig. 50.

25 30 35 40
Days of composting

Course of decomposition of stable manure as measured by temperature
chans;es (from Waksman and Nissen).

The carbon is liberated, under aerobic conditions, as COj, and
under anaerobic conditions, as methane, organic acids and alcohols,
and COo. Even imder the most favorable conditions of decomposi-
tion, however, the carbon of the organic matter is not all transformed
to COo at once. This is due to the assimilation of a large part of
the carbon by the microorganisms concerned in the decomposition

108 Decomposition of Plant and Animal Residues

process, for the synthesis of their cell material. The decomposition
of 100 gm cellulose (containing 40 per cent carbon) in the form of
straw or other plant material, for example, gives rise to 20 or 30 gm
carbon as COo; the rest of the carbon may be tied up in the syn-
thesized bodies of the bacteria and fungi. Under anaerobic condi-
tions, not more than 10 gm of the carbon may be liberated as CO2,
whereas the larger part of it may be left in the form of organic acids
or methane.

When proteins undergo decomposition, they are first hydrolyzed,
by proteolytic enzymes produced by microorganisms, to polypep-
tides, amino acids, and other nitrogen derivatives. These are further
acted upon by a variety of organisms. The nitrogen is finally con-
verted to ammonia; the amount thus liberated depends upon the
abundance of the proteins and also upon the other constituents of
the plant material, especially the carbohydrates. In a comparison
of the decomposition of rye plants harvested at different stages of
growth, young plants were found to decompose very rapidly, as
shown by the evolution of CO2; some of the nitrogen was liberated
as ammonia, as shown in Table 19. As the plants grew older, they
decomposed more slowly, and less nitrogen was liberated as am-
monia, until a point was reached at which additional nitrogen was
required to hasten decomposition of the plant materials.

When plant residues contain more than 1.5 or 1.7 per cent of
nitrogen, some of it will be liberated as ammonia, the actual amount
depending upon the original concentration of the nitrogen in the
plant residues. When the nitrogen content is less than 1.5 per cent,
however, very little ammonia will be liberated, even after several
months of decomposition. The decomposition of cereal straw, which
contains only 0.2-0.5 per cent nitrogen, requires the addition of
available nitrogen to enable the microorganisms to decompose the
carbohydrates in the straw. This process is utilized in the prepara-
tion of artificial manures, as shown later.

The ammonia which is produced in the soil in the decomposition
of plant and animal residues does not accumulate there except under
very special conditions, but is rapidly oxidized by the nitrifying
bacteria to nitrate. Some of the ammonia may also be consumed
by various microorganisms responsible for the decomposition of
the carbohydrates.

Decomposition of Carbohydrates 109

Decomposition of Carbohydrates

The mechanism of decomposition of carbohydrates by microorgan-
isms depends entirely upon the nature of the carbohydrate, the
natine of the organisms, and the conditions of decomposition, espe-
cially the oxygen supply. Thus, if glucose is attacked by fungi, the
following reactions are involved:

Citric
acid

CeHioOo + 4^02 = 3C2H2O4 + '3H2O

Oxalic
aciii

C6H12O0 + 6O2 = OCO2 + 6H2O

If the glucose is attacked by anaerobic bacteria and by yeasts, the
following reactions are invohed:

C6H12O6 = 2C3H6O3

Lactic
acid

CfiHiaOe = 2C2H5OH + 2CO2

Alcohol

C6Hi20fi = C4H8O2 + 2CO2 + 2H2

Butyric
acid

If the glucose is attacked by anaerobically growing fungi, another
reaction may take place:

CgHisOo + £H = C4H6O4 + C2H5OH + H2O

Glucose Fumaric Alcohol

acid

Under aerobic conditions, the alcohol is further oxidized, through
the acetic acid stage, to fumaric acid:

C2H5OH + 20 = CH3COOH + H2O

2CH3COOH + 0 = C4H0O4 + H2O

Starch is first hydrolyzed by diastatic enzymes, to give rise to
dextrins, and finally to maltose and glucose:

(CoHh,05)2„ + [n - ljH2() - /K:i2H220n

Starch Maltose

Ci2H220n + H2O = 2C6Hi20fi

Maltose Glucose

110 Decomposition of Plant and Animal Residues

Starch is readily decomposed by a large numlDer of microorgan-
isms. Among the fungi, certain species of Aspergillus, such as

A. orijzae, and, among the bacteria, various spore-formers, such as

B. amylovorus, B. incsenfcrictis, and B. macerans, are particularly
capable of attacking starch. The products of starch hydrolysis are
further broken down by microorganisms, through some of the reac-
tions shown above for the sugars. In addition to the highly special-
ized starch-decomposing organisms capable of producing powerful
diastatic or amylolytic enzymes, numerous other fungi, bacteria, and
actinomycetes are also capable of utilizing starch.

Decomposition of Cellulose

Cellulose, like starch, is a polymer of glucose. Because of its spe-
cific physical structure, however, and its resistance to most enzymes
and chemical reagents, it presents distinct problems as regards
decomposition in soils and in composts. The formation of specific
ceilulolytic enzymes can be demonstrated only with great difficulty.
Cellulose represents chemically a single type of compound. Be-
cause of difterences in the nature of the accompanying impurities,
celluloses of different origin may show distinctly different physical
properties.

Cellulose predominates in fibrous and woody materials, such as
straw, stubble, weeds, grasses, leaves, branches, and twigs. In young
and succulent plants, the cell-wall material is proportionally low,
whereas the sugars, proteins, and soluble minerals are high. In
mature plants, the straw, stems, leaves, and twigs are high in cellu-
lose. Cellulose is resistant to various oxidizing agents and is hy-
drolyzed only by concentrated acids. It is also resistant to attack
by the great majority of soil-inhabiting microorganisms. It can be
decomposed readily, however, by certain specific organisms found
among the bacteria, fungi, actinomycetes, and lower animals.

Various systems have been proposed for classifying the cellulose-
decomposing organisms. They can be divided into a number of
distinct groups, on the basis of either morphological or physiological
differences. One such system comprises (1) aerobic bacteria, (2)
myxobacteria, (3) anaerobic bacteria, including thermophilic forms,
(4) actinomycetes, (5) filamentous fungi, (6) higher or mushroom
fungi, (7) protozoa, (S) insects and other animal forms.

The mechanism of the breakdown of cellulose by microorganisms
depends entirely upon the nature of the organism and the condi-

Decomposition of Cellulose

11

tions of decomposition. Tlie aerobic bacteria and fungi break down
the cellulose completely, producing only 'COo, some slimy material,
certain pigments, and a considerable amount of microbial cell sub-
stance. As much as 30-40 per cent of the cellulose decomposed may
be converted into the cell material of the organisms decomposing
the cellulose.

175

150

125

5 100 -

g 75 -

50 -

25

a
I

a

c :!;
d

a

bA
~ de

a

b i

:■■ c

a

b

b

c

c

b

a

c
d
e

d -
- e

Total Cold-water- Hemi- Cellulose Lignin

material soluble celluloses

Crude
protein

Fig. 51. Course of decomposition of \arious chemical constituents of corn
sto\er under aerobic conditions (from Tenney and Waksman).

Anaerobic bacteria, howexer, decompose the cellulose with the
formation of \arious organic acids and alcohols in accordance with
the following reactions:

(CgHioOs),, + (n - IjHsO = nL^U.^Oe

Cellulose Glucose

CeHisOe - C3H0O3 + C2H5OH + CO2

Lactic
acid

Ethyl
alcohol

2C2H5OH = C2H4O2 + 2CH4

Ethyl Acetic

alcohol acid

Methane

The animal forms capal)le of utilizing cellulose as a foodstuff range
from termites and other wood-destroying insects to herbi\orous ani-

112 Decomposition of Plant and Animal Residues

mals, especially the ruminants. The latter carry out the digestion
largely by means of an extensive population of bacteria and proto-
zoa that inhabit their digestive tracts. The breakdown products of
cellulose, the sugars, alcohols, and organic acids, are utilized by the
animals for their nutrition. Association or symbiosis is thus estab-
lished, the animal providing a habitat or shelter for the microbes,
and the latter digesting the food for the host. Whether this associa-
tion holds true also for shipworms and other mollusks capable of
digesting cellulose in wood still remains to be determined.

Among the environmental factors that influence the nature of the
microorganisms concerned in the destruction of cellulose under a
particular set of conditions, the most important are moisture, reaction,
aeration, temperature, and a sufficient supply of nitrogen and other
nutrient elements.

A high moisture content (80-95 per cent) favors the development
of anaerobic bacteria and is injurious to the growth of fungi and of
most actinomycetes. A medium moisture (50-75 per cent) is fa-
vorable to filamentous fungi and to aerobic cellulose-decomposing
bacteria; some of the fleshy fungi, like the wood-destroying forms,
develop at a lower moisture than the filamentous forms. A very low
moisture ( 10 per cent or less ) completely stops the activities of most
cellulose-decomposing organisms, although some, such as insects,
may still be able to make a certain amount of growth; destruction of
paper in books and in paper files takes place at a rather low moisture.

The reaction of the medium also has a marked influence upon
the nature of the microbiological population responsible for the
process of cellulose decomposition. The aerobic bacteria belonging
to the Cytophaga group are able to grow at pH 6.1-9.1. Soils more
acid than pH 6.0 may be lacking in this group of organisms entirely,
although other cellulose-decomposing bacteria are able to develop
at pH 5.0-6.0. Actinomycetes grow at pH 5.5-9.5, whereas fungi
develop within much wider reaction ranges, at pH 3.0-9.5. Some
cellulose-decomposing fungi, like Trichoder)na, are able to grow even
at as high an acidity as pH 2.1-2.5. A slightly alkaline reaction (pH
7.5) favors, therefore, the growth of bacteria, whereas an acid reac-
tion is injurious to bacteria and is favorable to fungi. Addition to
the soil of acid-reacting fertilizers, which results in a low pH, favors
the development of fungi concerned in cellulose decomposition; addi-
tion of alkaline fertilizers, especially lime, reduces considerably the
numbers of fungi and leads to the development of bacteria which
are responsible for decomposition of the cellulose.

Decomposition of (lclliilo.se

113

The aerobic cellulose-decomposing bacteria ha\c their tempera-
ture optimum at 20-28°C; the anaerobic organisms grow best at
37''C, the thermophilic fungi at 45-55°C, and the thermophilic bac-
teria and actinomjcetes at 50-65'^C. Diiferent temperatmes may,
therefore, fa\or the development of different groups of organisms

100
90
80

^ 40

Carbohydrates-aerobic

,,,g>;^S^:t!-S-

^^ Carbohydrates-anaerobic ________ —

405

498

Fig. 52.
the total

205

Days of decomposition

Influence of aeration upon the decomposition of the whole alfalfa plant,
water-insoluble carbohydrates, and the lignins under aerobic and an-
aerobic conditions ( from Tenney and Waksman ) .

and thus modify the nature and extent of the process of cellulose
decomposition.

The oxygen supply also influences the nature of the cellulose-
decomposing microorganisms developing in a given substrate, as
well as the speed of their activities. If horse manure is to be stored
for some time before it is needed for the preparation of composts
for mushroom production, it is kept in a well-compacted state;
this creates anaerobic conditions and results in comparatively little
cellulose decomposition. When the manure is needed for the mush-

114 Decomposition of Plant and Animal Residues

room house, it is thoroughly aerated; this leads to active cellulose
decomposition, which is accompanied by a rise in temperature.
Jensen made a detailed study of cellulose decomposition in lab-

30

Cellulose decomposed

CO;, evolved

N assimilated

1100

- 1000

900

700
600
500
400
300
200
100

Weeks

;^ Cellulose decomposed as carbon

|C assimilated (27 4 x 9)
I CO . evolved as C

Fig. 53. Relation between cellulose decomposition by microorganisms and nitro-
gen assimilation, or its transformation into microbial cell substance (from Waks-
man and Iloukolekian).

oratory experiments, using well-manured soil. He reached certain
general conclusions concerning the course of decomposition and
accompanying microl)iological processes:

1. When farmyard manure was added to neutral (pH 6.5-7.0)
soils, cellulose-decomposing organisms of the genus Vibrio devel-

Decomposition of Ilcmicclliiloses 115

oped. When the leaetioii of the soil was aeicl (/;il 5.7-6.2), the
N'ibrios were reduced and Spirocluicta cijtophaga grew abundantly.
At a still greater aeidit\ , fungi were the only eellulose-d(^stroyiug
forms; among these Trichodcrma and VeniciUium were most active.
The fungi found in neutral soils included Mijcogonc nig,ra, Stachij-
botnjs, Coccospora agricola, and Botr{/<).s))()ritnn.

2. The cultures of bacteria isolated from the \arious soils behaved
in pure culture in a manner similar to that in the natural soil. Four
species of Vibrio were not active upon cellulose below ;;H 6.4 but
gave optimum growth at pH 7.1-7.6. Spirochacta cijtophaga, how-
ever, was able to grow at pH 5.6-6.0.

3. All cultures thus isolated were able to decompose not only pure
cellulose but also lignified cellulose of straw.

4. The nitrogen required for cellulose decomposition varied from
1 part nitrogen to 25-54 parts cellulose.

5. The cellulose-decomposing bacteria did not produce any humus-
like substances. The fungi Mijcogone nigra and Stachyhotrys gave
rise to humus.

Dubos used a simple mineral salt solution for studying cellulose-
destroying aerobes. This medium consisted of 0.5 gm NaNOs, 1.0
gm K.HPO^, 0.5 gm MgS04-7HoO, 0.5 gm KCl, 0.01 gm FeS04-
7H2O, and 1,000 ml water. Strips of filter paper were placed in the
tubes containing this medium, and the tubes were inoculated with
various dilutions of soil. This method proved to be very favorable
for the isolation of Cytophaga and other cellulose-destroying bac-
teria. The medium could also be used for determination of the
quantitative distribution of cellulose-decomposing bacteria in soil.

Decomposition of Hemicelluloses

Hemicelluloses represent a great variety of chemical compounds,
usually divided into polysaccharides, or those compounds which give
on hydrolysis simple sugars (CcHioOo, C5H10O5), and polyuronides,
or those that gi\e on hydrolysis sugar acids (CuHioOt) or mixtures
of sugars and sugar acids. The designation of individual hemicellu-
loses is based on the sugar produced on their hydrolysis by acids or
enzymes. On hydrolysis, pentosan gives pentose sugar; araban yields
arabinose; xylan gives xylose; hexosans yield hexose sugars, galactan
giving galactose and mannan giving mannose.

The polyuronides are more complex. Pectin is an abundant con-
stituent of fruits and vegetables and is made up of galactose, arabi-

116 Decomposition of Plant and Animal Residues

nose, galacturonic acid, acetic acid, and methyl alcohol. Some poly-
uronides are simple polymers of uronic acid, (CeHioOo)^, whereas
others are even more complex than pectins.

Hemicelluloses are attacked by a great variety of bacteria and
fungi; they can also be digested by most animal forms. There is
greater variation in the digestibility and in the rate of decomposi-
tion of the hemicelluloses than of cellulose, because of greater differ-
ences in chemical nature between various hemicelluloses. Some,
like the mannans, are attacked readily, similarly to the starches,
whereas others, like the galactans, are more resistant to decomposi-
tion and can be attacked only by highly specific organisms. In the
rotting of fruits and vegetables, either in a growing state or in stor-
age, the breakdown of the pectins is particularly important. This
is carried out first by a group of enzymes, designated as pectase,
pectinase, and pectolase, as follows:

C41H60O36 + 9H2O = CeHiaOe + CsHioOs +

Pectin Galactose Arabinose

2CH3COOH + 2CH3OH + 4C6H10O7

Acetic acid Methyl Galacturonic

alcohol acid

Similar reactions are involved in the retting of flax and other
fibers by aerobic bacteria and fungi; anaerobic bacteria change the
sugars and sugar acids of the pectin to alcohols and lower acids.

Since arabans and xylans make up 20-30 per cent of cereal straw,
of corn cobs, and of other plant residues, their breakdown in com-
posts and in soil is of great importance. They are usually attacked
by a variety of organisms somewhat more readily than is cellulose.
Hemicelluloses also form an important group of constituents of
microbial cell substance ( capsular material ) and may thus contribute
materially to the humus produced.

The decomposition of cellulose and hemicelluloses in oat straw
harvested at different stages of growth is brought out in Table 21.
When the plant is young, and its cellulose and lignin contents are
low, it decomposes very rapidly: as much as 56.3 per cent of the
total material has been destroyed by the microorganisms in 59 days.
As the plant grows older and as its cellulose and lignin contents in-
crease, its rate of decomposition decreases; that this is due largely
to an unbalanced nitrogen condition is brought out by the fact that,
when a soluble nitrogen compoimd is added, the mature plant ma-
terial decomposes as rapidly and as extensively as the young plant.

Decomposition of Nitrogenous Substances 117

Taiii.k '21. Influence of Age of Plant (Oats) upon the DEroMrosmov oi- Its
Constituent Carbohydrates (from Gerretsen and Waksiiiiini

IVr rent of docoinposilioii aftt-r 1'2.) days.

Age

of Plant,

days

Constituent

59

8()

112

Ilcinicellulose

15.3

17.4

19.. T

Ct'UuIose

24.6

34.5

39 . 1

Lignin

6.7

11.7

15.7

Total decomposed, no nitrogen added

56.. '5

37.4

27.1

Total decomposed + (NH4)2HP04

62 . 8

60.2

llemieellulose decomposed

14.4

12.6

10.4

Hemicellulose decomposed + (NH4)2HP04

16.0

17.3

Cellulose decomposed, no nitrogen added

20.8

24 . 3

20.1

Cellulose decomposed + (NH4)2HP04

31.8

3(».0

The effect of the added nitrogen is largely concerned with the
greater decomposition of the cellulose and hemicelluloses.

Numerous other transformations take place in the process of
decomposition of complex plant materials.

Decomposition of Proteins and Other
Nitrogenous Substances

Proteins make up 1-20 per cent of all plant residues. They are
complexes of amino acids. They contain, on an average, 50-55 per
cent carbon, 15-19 per cent nitrogen, 6-7 per cent hydrogen, 21-23
per cent oxygen, and small amounts of sulfur; some proteins also
contain phosphorus.

Proteins vary considerably in nature and in functions, depending
upon their amino acid make-up. On hydrolysis by specific enzymes
or by chemical reagents, the proteins are split first into various
polypeptides and finally into simple amino acids. The latter are
further attacked by a great variety of bacteria and fungi, gi\'ing rise
to ammonia, carbon dioxide, and various organic acids and alcohols.
Under anaerobic conditions, various amines and mercaptans are also
formed; these are responsible for the "putrefactive" odors produced
in the decomposition of protein-rich materials.

The great majority of soil organisms are capable of attacking pro-
teins. The amount of nitrogen finally changed to ammonia depends
upon the nature of the organism, nature of the protein, presence of
available carbohydrates, and soil conditions. Since in the decom-

118 Decomposition of Plant and Animal Residues

position of proteins and amino acids energy is also liberated, a cer-
tain amount of cell material will be synthesized by the organisms.
Some of the nitrogen will thus be consumed and transformed into

100

90

70

60

S 50

^ 40

E 30 -

20

10

A\

1 1

t\ "^

--________^^

-Total

nitrogen

"\A

'^■^

^-.

^,,^ -^

—

—

~ \\\

„^ Total dry matter

\\\

^-

—

- \\

\.

y^

^■^'

- \

^^ -^Celluloses

■v.

/
/
/

/
/

,--

\

I ^Pentosans

/
/

-

-

^■^-'

-■"

^

-

-

1 1

\ 1

-

30

50 75

Per cent moisture

75'

85

Fig. 54. Influence of moisture and aeration upon decomposition of horse manure

( from Egorov).

microbial cell substance. The amount of aiumonia liberated in the
decomposition of protein will thus be a resultant of the breakdown
of the protein and the destruction of the amino acids, on the one
hand, and of cell synthesis, on the other. The amount of ammonia
finally liberated may thus range from 50 to 80 per cent of the total
nitrogen of the protein decomposed. In the presence of carbohy-
drates, more of the ammonia will be consumed by the organisms;

l^c'coinposition of Ligniiis 119

iherclore, the greater the relati\e eoneentratioii ot the carbohy-
drates to the proteins, the less ammonia will be liberated.

The decomposition of different proteins by pnre cnltnres of dif-
ferent microorganisms is bronght ont in Table 22. In comparison

T.MM.K '2'2. Im)I(.\I VTIO.N OK AmMOMA (M(i) HY MlCKUOlUiAMSMS FHOM O..') (]\l OK

PwoTKiNs IX 40 Days (from AVaksman and Starkeyj

Organism

Proteolytic

Bacillus

Streptomyces

Rhizopus

Protein

Harterium

siibfilis

coelicolor

sp.

Gelatin

•25 A-)

4'i . 82

39.99

18.98

Casein

37.57

23.43

21.81

18.58

Gliadin

^>9.91

14.55

21.41

18.59

Fil)rin

19.76

18.55

16.12

18.55

Albumin

15.75

14.54

15.35

11.31

Zein

^25.86

7.68

8.89

2.43

with the fungus and actinomyces cultures, the bacterium synthesized
less cell material and liberated the greatest amount of ammonia.

Plant and animal residues contain, in addition to proteins, various
other nitrogenous substances, such as urea, purine bases, hippuric
acid, lecithin, choline, cyanamide, cyanide, alkaloids, and chitins.
These compounds are also decomposed by a great variety of micro-
organisms in soils and in composts; the mechanism of their decompo-
sition depends upon the nature of the organism and conditions of
decomposition (Table 23).

Cyanamide is first changed in the soil to urea, which is decom-
posed further; it may also polymerize to give dicyanodiamide, which
is toxic to some bacteria, such as the nitrifying forms. Choline is
transformed to trimethylamine by a variety of bacteria. Urea is
decomposed to ammonia:

/NH2
C0< + H2O = 2NH3 + CO2

\NH2

Decomposition of Lignins

Lignins are complex plant materials characterized by a benzol
ring structure with certain side chains. This is shown by the fol-
lowing formula:

C4oH3o06(OCH3)4 • (0H)5 CHO

120 Decomposition of Plant and Animal Residues

Table 23.

Protein

Edestin

Gli

Zeiii

Casein

Decomposition of Vegetable and Animal Proteins by Different
Microorganisms (from Waksman and Starkey)

Period of decomposition, 9-15 days.

Nitrogen

Dry Weight

Content

Organism

of Residue

of Residue

NH2-N

NH3-N

mg

mg

mg

mg

Control

978

164.2

0

Trace

T. koningi

604

85.1

28.3

32.6

S. viridochromogenus

862

140.8

4.5

11.7

B. cereus

408

65.0

22.8

40.1

Control

954

135.0

0

0

T. koningi

271

28.0

36.8

32.8

S. viridochromogenus

792

109.8

3.5

15.2

B. cereus

51

7.3

36.2

42.6

Control

966

144.8

0

0

T. koningi

718

97.3

14.0

26.8

B. cereus

128

17.9

27.8

46.5

Control

• • •

140.2

7.5

1.2

T. koningi

232

17.2

12.6

44.7

S. viridochromogenus

95

10.2

13.3

19.2

B. cereus

105

12.3

38.9

40.9

This formula is not generally accepted. Certain investigators
proposed the formula of substituted phenyl-propane groups linked
together. The phenyl group has a hydroxyl in the para position
and a methoxyl in the meta. The propane may have a double bond,
thus becoming a propene, and a hydroxyl. It is also claimed that
nitrogen is present as a tertiary amine in a linkage similar to that
in pyridine. Bondi and Meyer claimed that lignins of various plants
are built out of three of these units, having a molecular weight of
about 650. They formed two methoxyls in grass lignins and one
methoxyl in leguminous lignins, each lignin containing two phenolic
hydroxyls and one aliphatic.

Lignins are found in virtually all plants in varying concentrations,
depending upon the nature of the plant and the degree of its ma-
turity, usually to the extent of 5-30 per cent. The more mature the
plant, the higher its lignin content; young plants have comparatively
little lignin, whereas mature plants have a high lignin content.

In the decomposition of plant materials under natural conditions,
lignin tends to accumulate, since it is more resistant to decomposi-
tion than are the carbohydrates and proteins (Table 24). Fir wood,

Decomposition ot Ligniiis 121

Table 'ii. CiiANtiES in" the Chemral C().MrusiTio,\ of Wood as a Result oe Its
Decomposition (from Rose and Lissc)

On basis of dry wood.

(VI Ill-

rcMlo-

Mctho.\yl

Alkali-

Melhyl-

Clioiuical Coiistitiicut

lose

saii

Groups

Soluble

pentosan

per cent

per cent

per cent

per cent

per cent

Fresli wootl

58. 9G

7.16

3.94

10.61

2.64

Partly decomposed svood

41.60

6.79

5.16

38.10

3.56

Fully decomposed wood

8.47

'2.96

7.80

65.31

6.06

for example, lost all or nearly all of its cellulose constituents, but
still contained 85.55 per cent lignin, after considerable decomposition.

W^hen plants are attacked by soil microorganisms, the lignins are
aflPected only to a very limited extent, especially when present in
mature plants and under anaerobic conditions of decomposition. As
a result, lignin contributes considerably to the formation of humus
in soils, in composts, and in certain types of peat bogs.

Under aerobic conditions, lignin is not absolutely resistant to de-
composition but can be gradually oxidized. The exact nature of the
organisms concerned in the oxidation of the lignins in soils and the
nature of the products formed are not yet clearly understood. It is
known, however, that certain organisms, like some of the higher or
fleshy fungi, including some of the wood-destroying forms, are
capable of attacking lignins very rapidly. Falck distinguished two
processes in the decomposition of wood by fungi, namely, "destruc-
tion" and "corrosion." In "destruction," cellulose is decomposed,
whereas the lignin accumulates; organisms like Merulius lacnjmans
and species of Coniophora, Poria, and Lenzites are concerned in this
process. In "corrosion," the lignin as well as the cellulose is at-
tacked. Polyponts annosus is responsible for this process in spruce
wood. Tramc'tes pint attacks the lignin in pine wood. In the "de-
struction" of wood, the cellulose diminished from 56 per cent in the
original material to 7.8 per cent, whereas the lignin increased from
23.5 to 56.5 per cent; in "corrosion," the lignin diminished from 23.5
to 15.1 per cent and the cellulose from 56.0 to 48.2 per cent. Several
other fungi, like Stereum rugosum, are also capable of attacking lig-
nins. Agaricus nebitlaris destroys lignin, cellulose, and pentosan,
whereas Coniophora ccrehella is able to decompose cellulose but not
lignin.

The common edible mushroom, Fsalliota campestris, is capable of
utilizing lignin for its nutrition. In 51 days, 18 per cent of the lignin

122 Decomposition of Plant and Animal Residues

in a compost and only 14.5 per cent of the cellulose were decom-
posed. Other fleshy fungi, like Coprimis, are also capable of attack-
ing lignin; from fresh horse manure, C. radians removed, in 51 days,
22 per cent of the total lignin and 70 per cent of the cellulose.

Decomposition of Other Plant Constituents

Plant and animal materials contain organic compounds that
undergo rapid or slow decomposition by microorganisms. Of par-
ticular importance are the oils, fats, and waxes, the sterols and alco-
hols, the organic acids and tannins, the paraffins, cutins, and gums,
and a variety of compounds that occur in varying concentrations
from a fraction of 1 per cent to more than 2 per cent. These are all
decomposed, sooner or later, giving rise to numerous products. Oils
and fats, for example, are hydrolyzed to glycerol and fatty acids; the
glycerol is readily oxidized to COo and water, and the fatty acids may
give rise to certain resistant and, sometimes, toxic products.

The transformation of fatty substances under anaerobic conditions
is a process that is beheved to have contributed materially to the
origin of petroleum. Clostridium perfringens, for example, has been
shown to form, from alkaline oleates prepared from olive oil, a black
combustible liquid immiscible with water and resembling a petroleum
fraction.

Decomposition of Plant Materials as a Whole

Rapid decomposition of plant materials is favored by the following
conditions :

1. A low lignin and wax content of the plant material.

2. The presence of an adequate supply of available nitrogen.

3. A fine state of mechanical disintegration.

4. A favorable pH.

5. Favorable aeration and an adequate supply of moisture. An-
aerobic conditions result in a restricted bacterial population, with
lower nitrogen requirements.

6. A high temperature, usually within the range of 30-45° C.
Mixed materials frequently decompose more quickly than single

types of materials. This is true, for example, of a mixture of straw
and alfalfa, bedding and excreta in animal manures, and mixed
litter from several species of trees.

Selected Bibliography 123

Selected Bibliography

1. Dubos, R. J., The decomposition of cellulose by aerobic bacteria, J. Bad.,
15:223-234, 1928.

2. Jensen, H. L., Microbiology of farm>ard manure decomposition in soil
II. Decomposition of cellulose, J. Agr. Sci., 21:81-100, 1931.

3. Lundegardh, II., Environtncnt and riant Development, Edward Arnold &
Co., London, 1931.

4. Norman, A. G., The Biochemistry of Celhilose, Pohjuronides and Ligmn,
Oxford University' Press, New York, 1937.

5. Pringsheim, H., The Chemistry of the Monosaccharides and of the Pohj-
saccharides, McGraw-Hill Book Co., New York, 1932.

6. Smith, F. B., and Brown, P. E., Decomposition of lignin and other organic
constituents by certain soil fungi, J. Am. Soc. Agron., 27:109-119, 1935.

7. Waksman, S. A., Principles of Soil Microbiology, Williams & Wilkins Co.,
Baltimore, 2nd Ed., 1932.

8. Waksman, S. A., and Diehm, R. A., On die decomposition of hemicelluloses
by microorganisms. I. Nature, occurrence, preparation, and decomposition
of hemicelluloses. Soil Sci., 32:73-95, 1931.

9. Waksman, S. A., Humus, Williams & Wilkins Co., Baltimore, 2nd Ed., 1938.
10. Wise, L. E., Wood Chemistry, Reinhold Publishing Corp., New York, 1944.

5

Humus: Nature and Formation

What Is Humus?

In the past, various meanings attached to the term "humus." Some
used this term to designate a certain fraction of the organic matter
in soils and in composts; others used it to indicate all the organic
matter of the soil; still others recognized as humus the organic ma-
terials of natural origin in advanced stages of decomposition, whether
in soils, in composts, or in peat bogs, and whether plant or animal
in nature. Fresh plant roots and stubble, fresh stable manures and
green manures, fresh kitchen wastes and garbage, undecomposed
bodies of worms and insects, fresh tankage and fish, and numerous
other products of plant and animal life, when their origins are still
recognizable, are not in a humified or in a humus state. All these
serve as sources of humus; upon their decomposition by microorgan-
isms, humus is produced.

When various plant and animal residues are plowed into the soil
or are made up into composts, they are immediately attacked by
numerous microorganisms, including bacteria, actinomycetes, fungi,
protozoa, and worms. As a result of their decomposition, some of
the constituents of the fresh materials are volatilized, others are used
by the microorganisms for the building of microbial cell substance,
and still others are gradually transformed into a uniform, dark-
colored, amorphous mass, which is designated as "humus." The
rate of formation of humus and the amounts produced depend upon
the physical and chemical nature of the residues, the nature of the
soil or of the compost in which decomposition is taking place, the
nature of the microorganisms concerned, and the environmental
conditions, notably temperature, moisture supply, aeration, and
reaction. In humus, the products of decomposition can no longer
be distinguished from the original plant and animal materials from
which they have been formed.

124

What Is Humus? 125

This bioatl concept ot huinus must l)c tUllcrcntiatcd iroin its nar-
row definition, whereby only certain co'nstitutents ot the organic
matter of soils, composts, and peats, possessing characteristic prop-
erties, such as dark pigment, solubility in alkalies, and insolubility in
certain oxidizing agents, are designated as humus. Frequently, the
narrow definition does not difi^erentiate between "humus" and "humic
acid," another ill-defined term, occasionally used to designate the
alkali-soluble or the alcohol-soluble humus constituents. A highly
complex terminology has been introduced for distinguishing a num-
ber of "humic acids" on the basis of their solubility in certain re-
agents. In view of the great confusion that has resulted from this
definition, this concept of the "humic acids" can no longer be ac-
cepted in classifying humus types and humus constituents.

By the use of selective adsorption techniques, Forsyth separated
four fractions from humus: (1) a fraction that is usually present in
small quantities and contains water-soluble organic compounds, such
as sugars and amino acids; (2) a fraction containing phenolic glyco-
sides or tannins; (3) a polyuronide of the glucuronic acid type con-
taining d-glucose, rf-xylose, /-rhamnose, and another sugar; this frac-
tion seems to have a composition independent of the soil from which
it has been extracted, and may be of bacterial origin; (4) a fraction
rich in nitrogen and containing pentose sugars and organic phos-
phates.

Bremner used neutral pyrophosphate for dispersing the humic
fractions of the soU humus. The proportions of the fractions ap-
peared to depend on the treatment. These results indicate that
artifacts are produced by hydrolysis of the humus material, espe-
cially when sodium hydroxide solution is used for dispersion. Other
side reactions may also be brought about.

The so-called humins in the soil are not dispersed in caustic soda.
They appear to be polymerization products of some of the humus
constituents and also contain some of the undecomposed or partially
decomposed plant and microbial residues.

Certain constituents of humus or even certain types of humus are
frequently designated as "true humus" or "pure humus," especially
in speaking of organic materials in advanced stages of decomposi-
tion. This is true of various peats, forest fitter, well-composted plant
materials, and the humus of mineral soils. These must not be con-
sidered superior forms of humus, but merely types of humus, or
designations of certain humus forms. Under different conditions, a
variety of different humus types is produced. One can thus speak of

126 Humus: Nature and Formation

field, orchard, and garden soil humus, of pasture soil humus; of forest
humus, comprising not only the litter, but also the underlying humus
layers; of highmoor, lowmoor, and forest peat humus; of composts
produced from stable manures and from other farm residues; of
sewage sludge and garbage humus; and of water humus or marine
humus, that form of organic matter which is found in flowing and
in standing fresh- or salt-water basins.

Some plant residues decompose very rapidly in the soil but leave
comparatively little humus, whereas others decompose much more
slowly and leave large amounts of humus. Not only the nature of
the plant material, but also its age or degree of maturity influences
the rapidity of its decomposition. Among the soil factors that influ-
ence formation of humus from the plant residues, the mechanical
composition of the soil; its physical conditions, notably its texture;
and its chemical properties, especially reaction and presence of avafl-
able nutritive elements, are most important. The formation and
nature of humus are also influenced by the system of crop rotation,
by fertilizer treatment, by utilization of green manures, by abun-
dance of animals on the farm, by climatic conditions, and by other
factors.

Humus may be considered the more or less final and stable product
into which some of the plant and animal residues are transformed in
the process of decomposition. Humus is not an absolutely resistant
product, since it is also decomposed, but only very slowly, in the soU.
The rate of its destruction, however, is far less than that of the plant
and animal materials from which it originated.

Humus thus represents a natural organic system, in a state of a
more or less dynamic equilibrium. Since humus originates from
plant, animal, and microbial residues, its composition depends upon
the chemical nature of these residues. Since humus is formed as a
result of various decomposition processes, its composition will also
depend upon the microorganisms concerned in the decomposition of
the residues, and upon the conditions under which this process takes
place. Because of these factors, a number of humus types are pres-
ent in nature. One is thus able to difterentiate between the humus
of lowmoor and of highmoor peats; between the humus in conifer-
ous and deciduous forests; between the humus found in mineral soils,
notably of the podzol, chernozem, and gray-desert types; and be-
tween the humus present in lake and in marine bottoms. Although
all these forms of humus vary markedly in chemical composition.

Nature and Functions of Humus

127

the>' repicstMit a t\pc of organic matter which has a number of com-
mon physical, chemical, and biological pi'operties. An appreciation
of the chemistry of humus is based largely upon a knowledge of the
processes of decomposition of plant and animal residues under
natural conditions.

Fig. 55. Humus podzol (from Thom).

Nature and Functions of Humus

Humus possesses certain properties that distinguish it from the
plant and animal materials from which it has been formed. These
properties of humus can be briefly characterized as follows: it is
dark brown to black in color; it is virtually insoluble in water, al-
though a part of it may go into colloidal solution in pure water; it
dissolves to a large extent in dilute alkali solutions, especially on
boiling, giving a dark-colored extract; a large part of this extract
precipitates when the alkali solution is neutralized by mineral acids.

128 Humus: Nature and Formation

Certain constituents of humus may also dissolve in acid solutions
and may be precipitated at the isoelectric point, which is at pH 4.8.

Chemically, humus contains a somewhat larger amount of carbon
than do plant, animal, and microbial bodies; the carbon content of
humus is about 55-60 per cent, usually averaging 58 per cent.
Humus contains considerable nitrogen, from 3 to 6 per cent; these
figures may frequently be lower, as in certain highmoor peats, which
contain only 0.8-1.0 per cent nitrogen; they may also be higher,
especially in certain subsoils, where they may reach 10-12 per cent.

Humus contains the elements carbon and nitrogen in a ratio which
is close to 10:1; this is true of many soils and of the humus in sea
bottoms. This ratio varies somewhat with the nature of the humus,
the stage of its decomposition, the nature and depth of soil from
which it has been obtained, and the climatic and other environ-
mental conditions under which it has been formed. Humus is not
chemically static or nonvariable, but is rather in a dynamic condition,
since it is constantly formed from plant and animal residues and is
continuously decomposed further by microorganisms. Humus serves
as a source of energy for the development of various groups of
microorganisms, and as a result of its decomposition a continuous
stream of carbon dioxide and ammonia is given off.

Humus possesses a high capacity for base exchange, the ability
to combine with various other inorganic soil constituents, to absorb
water, and to swell. It is also characterized by other physical and
physicochemical properties that make it a highly valuable constitu-
ent of natural substrates, such as soils, which support plant and
animal life.

The importance of humus in the soil is manifold: it serves as a
source of nutrients for plant growth; it modifies, in various ways, the
physical and chemical nature of the soil; it regulates and determines
the nature of the microbial population and its activities, by supply-
ing various organic and inorganic nutrients essential for growth of
these organisms and by making the soil a more favorable substrate
for their development. An abundance of humus in the soil is
practically equivalent to a high rate of fertility of the soil. Humus
characterizes the soil type, since differences in its origin, abundance,
and chemical nature result in the development of a particular type
of soil.

Humus may be looked upon as a storehouse of important chemical
elements essential for plant life, especially of carbon and nitrogen,
and to a less extent of phosphorus, calcium, magnesium, iron, man-

The Nature of the Clay-Humus Complex 129

ganese, and many others. The utihzation of some of these elements
held in the inorganic fraction of the soil is also inflnenced by the
soil humus, through its chemical interaction with the inorganic com-
plexes. One should consider further the colloidal effects of humus
on the soil; its buffering properties, which modify the soil reaction;
its combining power with bases; its influence upon the oxidation-
reduction potential of the soil; its adsorption of certain toxic ma-
terials injurious to plant growth; its ability to supply certain catalytic
agents and small quantities of various trace elements essential for
plant growth; its influence upon soil structure, upon the moisture-
holding capacity of the soil, and upon soil temperatiue. Humus also
brings about in the soil numerous other reactions which are of direct
or indirect importance to plant growth.

The functions of humus in the soil may thus be considered three-
fold: (1) physical, thereby modifying the color, texture, and struc-
ture of the soil, as well as its moisture-holding capacity and aera-
tion; (2) chemical, influencing the solubility of certain soil minerals,
forming compounds with some of the elements, such as iron, which
thus become more readily available for plant growth, and increasing
the buffering properties of the sofl; (3) biological, by serving as a
source of energy for the development of microorganisms, by making
the soil a better medium for the growth of higher plants, and by
supplying certain essential nutrient elements and compounds re-
quired by higher plants.

The Nature of the Clay-Humus Complex

Among the important aspects of humus in the soil, the most sig-
nificant is its interaction with the clay constituents, which gives rise
to clay-humus. In this respect, two types of soil are frequently recog-
nized: in one the clay and humus particles are held together by
calcium ions; in the other, iron, and possibly also manganese and
aluminum, may replace calcium, although aluminum has not yet been
demonstrated to play this part. Tiulin classified the sofl colloids into
two groups: in one (group H colloids) the iron acts as a cement,
holding the clay and humic particles together; iron, and possibly
also aluminum, appear to be responsible for the binding of the humus
to the sand particles in the B horizon of a podzol. The organic mat-
ter dispersed from the B horizon is rich in iron and possibly also in
aluminum; it is comparable to the ^-humus of Waksman. When dis-
persed in acid solutions, these substances may become negatively

130 Humus: Nature and Fomiation

charged, act as cations, and move toward the cathode during the
electrodialysis of soils. They are usually not considered to confer a
favorable structure on the soil.

The group I colloids of Tiulin comprise the clay-humus complex
which is held together by calcium ions. Agriculturally, it is believed
to be the most important type. This complex is largely responsible
for the favorable physical conditions found in various soils and com-
posts. The clay appears to have an appreciable base-exchange ca-
pacity. The presence of calcium is essential if only for ensuring the
formation of the correct type of humus.

Increasing the amount of soil organic matter in this combination is
of great importance. The addition of clay to sandy soil may be as
important as the addition of organic matter itself. A certain quantity
of stable manure or compost or mass of plant residues can be con-
verted into this type of complex only when it is composted with a
certain type of clay before it is applied to the soil.

A third organic-inorganic colloid has been prepared, although it
has not been isolated from field soil. Ensminger and Gieseking
found that proteins can be strongly adsorbed by bentonites through
their basic groups acting as an exchangeable base; this process of
adsorption renders the protein more resistant to proteolytic enzymes.
In view of the fact that only little is known about the nitrogenous
constituents of the soil organic matter, it is difficult to postulate the
formation of protein complexes and the reasons for their stability.

How Humus Is Formed

The various plant and animal residues on the farm and in the
home, the crop wastes, and those crops which are specially grown
as a source of humus, all vary considerably in chemical composition
and in the rapidity of decomposition. The rate of liberation of the
chemical elements, notably the carbon and the nitrogen, in forms
available for crop growth, and the nature and amount of humus pro-
duced from these residues will, therefore, also vary. Among the
sources of humus, plant stubble, stable manures, green manures, and
artificial composts occupy a leading place.

Plant stubble includes the root systems of the plants, as well as
the stems, leaves, and other residues left after the crop has been
harvested. There is considerable variation in the amount and chem-
ical composition of the stubble, depending on the nature and abun-
dance of the crop, method of soil cultivation, and fertilizer treat-

How Huniiis Is Foriiiod

131

20

16

Fig. 56.

Aerobic decomposition

Anaerobic decomposition

2 3 4 5 6 7 8

Period of decomposition, weeks

Decomposition of sheep manure under aerobic and anaerobic conditions
(from Joshi).

nient. Because of the difficulty in removing the mass of roots left
by the crop, only the surface portions are usually determined. The
root mass may equal if not exceed the surface stubble, as shown in
Table 25, where the results obtained by Woods in 1888 are reported.

Table 25. AMOtrxT of Stubble and Roots Left by Various Crops (from Woods)

Pounds (air-dry) per acre.

Weight of

Weight of

Weight of

Plant

Tops

Stubble

Roots

Clover, in bloom

1,657

898

Clover, ripe

1,272

1,460

Wheat, heading

2,554

226

591

Wheat, ripe

7,092

595

591

Oats

5,037

216

293

Barley, heading

2,449

187

408

Barley, ripe

7,154

355

336

Timothy, ripe

5,254

2,056

5,215

The plant residues vary considerably in chemical composition, the
stubble and roots of cereals containing about 0.5 per cent nitrogen,
0.1 per cent phosphorus, and 0.5 per cent potassium, whereas the
corresponding concentrations of these elements in legume residues
are 2-3, 0.5, and 2-2.5 per cent. The chemical composition of stable
manures, green manures, and other sources of humus is given in
Chap. 14.

132

Humus: Nature and Foniiation

In the decomposition of plant and animal residues by microorgan-
isms in soils and composts, the materials are not attacked as a whole.
Some of the organic constituents are decomposed very readily, others
less quickly, and still others are fairly resistant and tend to disappear
only very slowly. Some of the compounds are decomposed com-
pletely; others are transformed into various products which are more
resistant. The sugars and starches are rapidly destroyed, followed
by some of the hemicelluloses, the proteins, and the celluloses. The

1 1 \ \ \ r

A = Humus production

Bi = Humus destruction with plenty of air

62= Humus destruction under water

50

Humus accumulation
in aerated soil

Temperature, °C

Fig. 57. Humus accumulation and humus decomposition in tropical soils (from

Mohr).

lignins and some of their derivatives, certain proteins and hemicellu-
loses, as well as the waxes, tannins, and other materials, are more
resistant to decomposition and therefore gradually accumulate. The
processes of decomposition are accompanied by the synthesis of mi-
crobial cell substance comprising fungus mycelium, bacterial bodies,
worms, and insects.

When plant and animal residues are added to the soil or placed
in composts, rapid decomposition sets in at first. This is followed by
consumption of oxygen, evolution of heat, liberation of considerable
carbon dioxide and ammonia if the material is rich in nitrogen, and
darkening of the residual material. As the decomposition progresses
and as the more readily decomposable constituents disappear, the
process becomes slower until a certain level is reached, when the
residual mass has become brown to black.

This mass of slowly decomposing and decomposed material, to-

Methods of Anahsis of Humus 133

gctlicr with the nc\\l>- s\ntheiiized microbial cell substance, com-
prises the humus. Humus is formed frorn the plant and animal resi-
dues which ha\e lost the readily decomposable constituents, have
gained the synthesized microbial substances, and have accumulated
the more resistant constituents. Humus is, therefore, chemically not
always the same.

Humus accumulates under conditions not favorable to its further
decomposition. This is true particularly when humus is formed in
a water-saturated en\ ironment, as in peat bogs, or in an acid environ-
ment, as in raw humus layers in forest soils, or at very low tempera-
tures, as in high altitudes, when it is frequently designated as "alpine
humus."

Methods of Analysis of Humus

The total humus content can be determined by the loss on igni-
tion, especially in peats, composts, and other humus-rich materials.
In mineral soils, the best method of determinating humus is to calcu-
late it from the organic carbon content, by using the factor 1.724.
The fact that humus is not simple in chemical composition and that
it comprises a number of complex substances, both organic and
inorganic, can be demonstrated by the proximate method of analysis,
when it is possible to show that different types of humus have dif-
ferent chemical compositions (Tables 26 and 27). Not only does

Table 26. Chemical Composition of the Organic Matter in Different

Mineral Soils

On basis of total dry soil, surface samples.

Total

SoU

Loss on

Carbon

Total

C/N

No.

Description of Soil

pH

Ignition
per cent

X 1.72
per cent

Nitrogen
per cent

Ratio

4

Summit

6.8

7.9

4.5

0.24

11

6

Chernozem, Hays, Kansas

7.6

6.0

2.7

0.15

10

16

Chernozem, Edmonton, Alberta

6.4

17.1

11.2

0.67

10

18

Brown soil at Indian Head, Saskatchewan

8. .3

10.3

6.2

0.33

11

21

Chernozem at Brandon, Manitoba

8.3

10.0

7.4

0.40

11

29

Carrington loam, dark colored prairie

7.8

10.2

6. .5

0.32

12

forest humus vary from peat humus and from humus in composts,
but all these \ary considerably from the humus in mineral soils. The
humus in the different layers of forest soils varies greatly in chemical
composition.

134

Humus: Nature and Formation

Table 27. Chemical Nature of the Organic Matter of Soils Described in

Table 26

On basis of total organic matter (C X 1.72).

Lignin-

Soil

Ether-

Alcohol-

Hemi-

Humus

No.

Soluble

Soluble

cellulose

Cellulose

Complex

Protein

•per cent

per cent

per cent

per cent

per cent

per cent

4

3.6

0.6

5.4

3.6

43.4

33.8

6

4.7

1.5

8.6

5.2

40.8

34.7

16

0.8

0.8

5.5

4.1

41.9

37.4

18

1.0

0.9

7.0

3.5

42.0

33.3

21

0.5

0.8

8.5

2.8

42.8

33.4

29

0.6

0.6

8.2

3.6

42.3

30.4

Peat as Humus

Peat represents a type of humus which has originated as a result
of decomposition of plant materials in areas submerged in water.
It comprises various organic formations spoken of also as "muck,"

Woody peat
Hypnum peat
'.I Glacial till

llllllllllll Sphagnum peat
I I Sedge peat
I I Reed peat

Fig. 58. Different layers in peat profile (from Stokes).

"turf," "peat moss," and "black humus." The physical and chemical
differences found among the different types of peat are due largely
to the nature of the plants from which they have been formed,

Lowmoor peat, frequently spoken of as "muck," is produced prin-
cipally from reeds and sedges; it is slightly acid in reaction (pH 5.0-
6.0 ) and contains 5-10 per cent mineral matter and 2-4 per cent nitro-
gen. Peat moss, or highmoor peat, is formed from sphagnum and

Hiiimis in Forest Soils 135

other nioss)" plants; it is more fibrous in nature, very acid in reaction
( pH 3.5-4.5 ) , low in ash and nitrogen ( less than 1 per cent ) . After
proper drainage, the lowmoor peat, as well as a third type of jocat,
known as sedimentary, forms good agricultural soil. Lowmoor peat
is also harN'ested and marketed for making lawns. Highmoor or
sphagnum peat is used as litter in stables and for horticultural pur-
poses. A number of other peats, intermediary in nature, are formed
from different types of vegetation and under different environmental
conditions.

Most peats are \aluable as sources of humus, but they are poor as
plant nutrients, and even the nitrogen in the peats is not so readily
available for plant growth as is that in animal manures and green
manures. Peat cannot, therefore, take the place of fertilizers for
plant nutrition; it serves primarily to improve the physical condition
of the soil. Peat is frequently mixed or composted with soil, before
its application to the soil, in order to prevent the formation of layers
and to improve the uniform structure of the soil.

The chemical composition of a group of peats is given in Table 28.

Table 28. Proximate Chemical Composition of Some Typical Peats
On basis of dry material (from Waksman).

Lowmoor,

Saw-

Lake

Forest

Sphagnum

Sphagnun

New

Grass,

Peat,

Peat.

Peat,

Peat,

Peat Constituent

Jersey

Florida

Florida

New York

Germany

Maine

per cent

per cent

per cent

per cent

per cent

per cent

Ether-soluble

0.7

3.0

0.4

3.2

3.1

2.5

Water-soluble

3.1

1.7

0.7

Hemicellulose

10.3

6.4

4.2

5.4

16.9

20.9

Cellulose

0

0.3

0

2.7

19.4

1G.2

Lignin-like complex

38.4

46.1

35.2

60.7

34.0

25.4

Protein

22.5

23.1

13.1

14.3

5.2

5.7

Ash

13.2

10.0

39.6

3.9

1.7

1.8

pH

5.9

6.2

7.3

4.7

4.1

4.0

The marked variation in reaction, total organic matter content, and
chemical nature of the organic matter is due to differences in the
vegetation from which peat has been formed, the nature of the
waters finding their way into the peat bogs, the topography of the
region, and the climate.

Humus in Forest Soils

Forest humus varies with the nature of the vegetation and the
soil. The forest floor usually consists of several distinct layers of

136

Humus: Nature and Formation

Fig. 59.

Slight Medium Good

Degree of decomposition of peat

Decomposition of plant constituents in the jjrocess of peat formation
(from Maliutin).

vegetable remains. The surface layer, or litter, is made up of partly
decomposed leaves, needles, roots, twigs, cones, and other tree
residues. This layer is superimposed on another layer of partly
decomposed plant residues and is underlain by a third layer of
thoroughly decomposed material which is said to be completely
humified. The total organic layer of the surface of forest soil is 0.5
to more than 6 inches deep. In evergreen forests, the largely organic
surface layers are usually not mixed with the inorganic soil layers;
the former are referred to as the "raw hiuuus" or "duff." In decidu-
ous forests, the organic residues and their decomposition products
are well mixed with the inorganic part of the soil, giving rise to a
type of soil known as "mull." This soil is less acid and is more active
biologically.

Decomposition of Hiinuis

137

1 1

1 1 , ,

V 1 i

1 1 ' /, '

* 1 1

\

O)

~

/

\

J3

(0

-

/
/

\
\

> c

^ DO

o o

TO O

/y^

^^^'\ , — Ammonia

-n\ -

\
\
\

0) 1/1

//

\ ^Nitrate-N

\

>

1 1

x>

]to

//

cc

V ,

1 1 1 1

1 1

8.0

7.5

5.0

4.5

7.0 6.5 6.0 5.5

Reaction of soil, pH

Fk;. fiO. InHuc'iKc of reaction upon the nitrate and aiuiuoiiia nitrogen lil)erati()n
in forest soil (from Aaltonen).

Decomposition of Humus

When compared with fresh plant residues, humus is rather re-
sistant to decomposition; otherwise it would not accumulate at all
in the soil, and it would certainly not persist there for long periods.
Under favorable conditions, however, humus can decompose further.
Were this not the case, the soil long ago would have become covered
by a surface layer of organic debris of varying degrees of thickness,
similar to that formed in peat bogs, in certain forests, and especially
in coal. If humus in soil were as resistant to decomposition as humus
in coal and in peats, the surface of the earth would have become
organic and would not have remained predominantly inorganic in
nature; the limited supply of available carbon would soon have be-
come exhausted, making all further life impossible. In fact, under
certain conditions, the decomposition of humus in the soil may be so
rapid that the farmer experiences considerable difficulty in keeping
up the supply, especially when he cultivates his soil year after year
and does not return to it sufficient plant residues and organic manures,
as in the growing of intertilled crops.

When the temperature, moisture, reaction, and aeration of the soil
are faxorable, the soil humus undergoes constant decomposition, as
evidenced by the continuous stream of carbon dioxide given off into
the air and by the nitrate that accumulates in the soil; the nitrogen
is first liberated as ammonia, which is changed rapidly to nitrate by
certain bacteria. The gradual disappearance of the humus becomes

138 Humus: Nature and Formation

especially evident when the soil is kept fallow or free from all plant
growth and no plant residues in any form are added to it.

In the cultivation of soil, the top layer, including both living and
dead materials to a depth of 4-8 inches, is turned over. The rapidly
decomposing organic residues, which previously were on the surface,
are now placed under the surface. Root systems filling the upper
layer of soil are thus killed and mixed with the residues already dead
and decomposing. The lower layer of soil, which was hitherto pro-
tected by a surface layer, is in its turn brought to the surface.
Harrowing, hoeing, or other cultivating brings this fresh soil into
more or less intimate contact with air, sunlight, and the daily varia-
tion of heat, cold, and drought.

The whole mass of soil from the surface to the lower layer of the
furrow-slice becomes an aerobic environment in which microorgan-
isms find favorable conditions for development. This results in great
changes in the soil flora and fauna and in the soil organic matter.
Smith and Humfeld, in their studies of decomposition of green ma-
nure, showed that great activities were localized wherever plant
remains were left to decompose in such a mixture of materials.

Humfeld demonstrated that the whole mass above the green
manure is quickly flooded with carbon dioxide, due to the respiration
of the dying vegetation and to the microbiological reactions. When
optimum moisture was present, the process was accompanied by the
disappearance of the various fungi, and even by the partial suppres-
sion of the whole fungus flora. This was true especially of the brown-
and black-walled fungi. With a low moisture content, certain fungi
developed; the mycelium was colorless, however, and soft-walled;
this was associated with a low content of lignin or related substances,
which are characteristic of the flora of the surface decomposition
process.

Under conditions of regular cultivation, as demonstrated by King
and Doryland in Kansas, the organic matter content of the soil was
gradually decreased by cultivation. Conditions were made favorable
for decomposition of the soil organic matter. This was accompanied
by a release of nitrogen, phosphorus, and potash. As a result, crop
production increased. Continuance of this system of cropping, how-
ever, without compensating return of organic manures, might be
expected to deplete the humus supply of the soil.

Difficulties encountered in continuous cropping of soils in prairie
areas led Alway and others to make extensive comparisons of the
organic matter in various soils.

Decomposition of Humus

139

When a virgin soil is brought under cultivation, the humus con-
tent is reduced, at first rapidly, and theVi more slowly, depending
upon the soil, its climatic conditions, and the manner of cultivation.
Although a crop, whether cultivated or unculti\ated, leaves certain

32

28

24

E
Q0 20

16

>/> 12

\i<

\oe

/

/

//

//

/

! /

V^^^'

,<^®^

^^l

/''

/

SV.eepJ^l^l!^'-

Sheep manure + straw

3 4 5

Period of decomposition, weeks

Fig. 61
and

. Influence of straw upon the liberation of nitrogen as nitrate from urine
cow and sheep manure, in the process of decomposition ( from Joshi ) .

residues in the form of roots and stubble, which are gradualh'
changed to humus, the amount of humus decomposed as a result
of culti\ation is not fully compensated by the humus produced from
these residues. To keep up the fertility of the soil in certain types
of farming, it is essential to increase the humus content above that
made possible by the residues of a cultivated crop grown on the soil.
This can be done by growing a special crop to be plowed under for
green manuring purposes, by adding stable manures, or by intro-

140

Humus: Nature and Formation

ducing special forms of humus, such as composts, forest htter, or
peat. Addition of sufficient inorganic fertiHzer will also result in
an increase in the amount of plant residues, which will yield greater
amounts of humus to replace, partly at least, the loss resulting from
cultivation.

Shutt found that, on cultivating a virgin prairie soil, there was a
loss of over 100 pounds of nitrogen per acre annually for 22 years;

0.9

-z

1 1

§ 0.7

(V
Q.

— 1

1
1

—

"c

\

^0.5
o

u

c
<u

00

2 0.3

- \

X

-

0.1

-

1 _

7.0

5.0

3.0 o

1.0

16

20

0 4 8 12

Temperature, °C

Fig. 62. Schematic representation of the widening C:N ratio of soil humus with
decreasing temperature ( from Jenny ) .

only one-third of the loss was accounted for by the crop grown.
About 25 per cent of the total organic matter of the soil was lost as
a result of cultivation during this time. Lipman and Blair also
found an annual loss, for 20 years, of 70-100 pounds of nitrogen
from a heavily manured soil. This loss could not be accounted for
in the crop, but could be accounted for largely by the nitrogen in
the drainage water. Some losses may also occur in the gaseous forms
of nitrogen.

In dry-land farming, the most important soil problem, aside from
that of water, is maintenance of organic matter. Russell calculated
the loss of organic matter under dry-land farming to be 6.5 per cent
during the first 3-7 years under cultivation, 12.4 per cent in 8-15
years, 26.8 per cent in 17-30 years, and 28.0 per cent in 45-60 years.
The depletion of soil organic matter leads to erosion by wind and

Decomposition of Hunuis 141

water. Creator losses ot organie matter, up to 60 per cent in 40 years,
have been reported under conditions of i:?oor soil management.

The losses of organic matter as a result of cultivation are at first
rapid, later becoming slower, until an equilibrium is attained after
about 30 years; a base level of organic matter has then been reached,
which is 25-30 per cent lower than that of the virgin soil. Jenny cal-
culated that the loss of nitrogen during cultivation of a virgin soil
is 25 per cent the first 20 years, 10 per cent during the second 20
years, and 7 per cent during the third 20 years. Alway (Table 29)

Table '29. Influence of Cultivation upon Loss of Ohganic Matter from
Prairie Soils (from Alway)

30-40 year.s in cultivation.

Organic

Matter

Content

Cultivated

Virgin

Loss on

Treatment

Soil

Soil

Cultivation

per cent

per cent

per cent

15 years in wheat, 15 in corn

2.74

3.89

29.5

Corn, oats, and wheat

3.21

4.43

27.5

Alfalfa last 12 years

3.86

4.43

12.9

Alfalfa last 7 years

3.38

5.03

32.8

Alfalfa last 7 years, badly blown 1 year

2.47

5.03

50.9

Alfalfa last 12 years

4.29

5.41

20.7

Orchard, clean cultivation, last 7 years

2.76

5.41

49.0

Grass last 5 years

3.57

5.41

34.0

Heavily manured for W years

4.68

5.05

7.3

has shown that the growth of a legume or addition of stable manure
reduces considerably the loss of organic matter. The greatest dan-
ger to the humus content of the soil was found in clean cultivation
and in wind or water erosion.

Detrimental changes in soil structure and tilth accompany losses
of organic matter, which imparts stability to the soil granules. In
analyzing the results of 50 years of field experiments at the Woburn
Experimental Station, Russell and Voelcker came to the conclusion
that soil deterioration under continuous cropping can be prevented
only by a combination of farmyard manure with crop rotation. The
plots receiving about 8 tons of manure a year showed no deteriora-
tion in crop yield and no loss of nitrogen from soil. The fact that
artificial fertilizers did not completely stop deterioration suggested
that the latter was due to the exhaustion of some soil constituent

142 Humus: Nature and Formation

necessary for plant growth under field conditions. By eliminating
the possibility that minor elements were concerned, the conclusion
was reached that exhaustion is associated with loss of organic matter.
The possibility was suggested that productiveness of a given soil
rises and falls with the percentage of humus present.

The influence of treatment upon the rate of humus decomposition
can be best illustrated when a peat bog is drained. When the bog
is saturated with water, humus accumulates, the rate of accumula-
tion depending upon the nature of the vegetation, climate, topog-
raphy, and chemical composition of the waters entering the bog.
When the bog is drained, humus ceases to accumulate and begins to
disintegrate. The rate of disintegration of the humus is controlled
by the nature of the peat, climatic conditions, and the depth of the
drainage system. The rapid rate at which certain peats decompose
in warm climates points to the great danger of their rapid disappear-
ance by improper handling.

In recent studies Broadbent and Norman used isotopic nitrogen
and carbon compounds. They found that, when decomposable or-
ganic matter was added to the soil, a considerable increase took place
in the rate of decomposition of the soil organic matter present. In
one experiment they added 1 and 2 gm of sudan grass to 100 gm of
soil, incubated the soil for 11 days, and measured the COo produced.
Since the sudan grass contained 2.67 per cent of the isotopic carbon,
they could calculate the proportion of the COo which resulted from
the oxidation of the soil organic matter. When the soil alone was
incubated, 49 mg CO2 was produced from the soil organic matter;
when 1 and 2 gm of sudan grass were added, 216 and 329 mg were
produced, respectively, from the soil, in addition to the 418 and 651
mg of CO2 from the decomposition of the sudan grass. For every
100 mg of CO2 produced from the sudan grass, an additional 40 mg
was formed from the soil organic matter. No explanation could be
given for the fact that soil organic matter became a better source
of nutrient for the soil population when readily decomposable or-
ganic matter was added.

Abundance and Nature of Humus in Different Soils

The hiunus content of soils varies considerably, from extremely
small amounts, as 0.1 per cent in some poor sandy soils, to as much
as 20 per cent, in prairie soils; it may be even higher (50-80 per
cent) in certain soils which have originated from peat bogs that have

Humus in Different Soils 143

ht'CMi brought iimlcr tultixatioii. Light-textured soils eontuin less
organie matter than soils of heavier texture. Typieal analyses oi
sandy loams ga\e, on an a\erage, 0.06 per cent nitrogen, which is
equi\alent to 1.03 per cent organic matter; clay loams in the same
series of soils were found to contain 0.1 per cent nitrogen or 1.72
per cent organic matter. The stage of development of a soil influ-
ences considerably its organic matter content.

Humus is not distributed evenly through the soil depth. It is
largely concentrated in the upper 10 or 12 inches, or the surface soil
layer, and decreases rapidly in the subsoil. In brown forest soil and
in the red and yellow soils, there is a rapid drop in organic matter
content below the upper 6 inches; it becomes \'ery low at compara-
tively shallow depths. In prairie soils, chernozems, and chestnut-
brown soils, there is a gradual decrease of organic matter with depth.

It has already been emphasized that the accumulation of humus or
its further decomposition depends entirely upon the \'egetation, the
soil, its topography, and the environmental conditions, especially the
climatic, under which humus has been produced. These are the
major factors which influence the abundance and nature of the
humus. In heavy soils, especially those rich in lime, humus will be
fixed and will tend to decompose only \ery slowly. In light sandy
soils, humus will not accumulate to so great an extent. Under favor-
able conditions, humus will decompose more rapidly; under certain
conditions, it may also be readily leached out, as illustrated by the
process of podzolization.

The nature of the vegetation has a marked effect upon the accumu-
lation of humus. In pasture and prairie soils, where the land is cov-
ered by a growing crop and is not cultivated, the gradual disintegra-
tion of the roots in a soil environment lacking suflBcient oxygen will
give rise to a considerable amount of humus. If this soil is high in
lime, the humus produced will be fixed and will gradually accumu-
late. This results in the course of time in a type of soil which is
designated as "chernozem." In forest vegetation, the annual drop of
lea\es, needles, and other tree residues will bring about an extensive
accumulation of organic matter on the surface of the soil. If this
organic matter is gradually worked in with the underlying mineral
part of the soil, a phenomenon brought about largely through the
activities of the earthworms, insects, and other animals inhabiting
some of these soils, a type of soil will result which is designated as
"mull"; this soil is characterized by one type of humus. If the tree
residues are not mixed with the underlying mineral layers, however.

144 Humus: Nature and Foiination

a soil will result which is designated as "raw humus"; this may be
due to the nature of the vegetation and type of soil. The surface
layer of the raw humus soil may undergo considerable leaching,
whereby certain of its organic and inorganic constituents are re-
moved and are reprecipitated in the subsurface; a type of soil is thus
formed which is designated as a "podzol."

A comparison of the chemical nature of the humus in different
soil types shows that, in warmer regions, the carbon-nitrogen ratio
of the humus tends to become narrower. This is due to a greater
decomposition of the carbohydrates and other nonnitrogenous con-
stituents of the humus and the greater accumulation of the proteins,
the latter originating largely through microbial synthesis.

The two major factors which control the abundance and nature
of the humus in different soil types are, first, the formation and
accumulation of the humus, as influenced by nature of vegetation,
type of soil, aeration of soil, reaction, and environmental factors,
especially temperature and rainfall; and, second, the decomposition
of the humus, as controlled by the treatment of the soil, especially
drainage and cultivation, farming practices, and climate.

Humus and Soil Fertility

Among the various factors which contribute to the fertility of
the soil, none occupies a more prominent place than soil organic
matter. It has a fourfold effect upon the soil:

1. It serves as a storehouse of plant nutrients: the slow but gradual
decomposition of the organic matter by microorganisms results in
the liberation of a continuous stream of carbon dioxide; of available
nitrogen as ammonia, which is soon changed to nitrate; of phos-
phorus; and of other elements essential for plant growth.

2. It has important physical effects upon the soil: it improves the
soil structure; it provides better aeration; it has a binding effect
upon the soil particles; it increases the water-holding capacity of the
soil; it helps the soil to absorb more heat; it increases the buffering
properties of the soil, preventing rapid increases in acidity or
alkalinity.

3. It has certain chemical effects upon the soil constituents, such
as rendering phosphorus and other elements more soluble, and
neutralizing substances which tend to be toxic to plants; it has also
a very high base-holding power.

Humus and Soil Fertility

145

4. It has an important cUcct upon the biological state of the soil,
making it a more favorable medium for tlie development of the root
svstoms of plants and for the growtli of microorganisms essential for
soil processes.

Color of Soil

\

4,0

35

3.0

'^ 2.5

2.0

1.5

C J2

Is

5 -Q

■\

\

X) ^

^_

. '^,

^^^

^

if

St?;

\

^

5 .-

o o

■^ c"

^^X

:v

Color of Soil

c
5
o

-Q

c
S
o

c
o

c
o

o

X)

I/)

5

g

5

o

o

"33

o

<u

5-

c

><

a

>^

o

5

o

5

o

o

o

■^^

a>

JZI

5

XI

c
5

5

jr

.c

o

c

S

J3

S

X3

c

J2

§

o

DO

o
"oj

TO

i

DjO

CO

>

_l

>■

Q

CD

_l

\

/

\ \

//

\

\

//

\
\

\

//

;/

\

\

/

\\

^<\

/

\ \

0?^"

\

\

/

\

^^v°^

f>\

]

\

<^

\

'^

0.40

0.35

0.30

0.25 .^

0.20

0.15

Surface Soil Subsoil

Fig. 63. Carbon and nitrogen content of different soils and subsoils (from

Brown and O'Neill).

For countless generations, man depended upon the humus of the
soil to supply the necessary plant nutrients, through the activities of
the numerous microbes which inhabit the soil in thousands of mil-
lions per single gram. These nutrients were built up by the plants
into plant tissues, which were partly consumed by animals, including
man, and partly returned to the soil in the form of leaves, needles,
stems, and roots. The animals and their excreta found their way,
sooner or later, into the soil, to serve again as sources of humus, to
be later again decomposed, with the liberation of the constituent
nutrient elements for renewed plant growth. Humus can thus be

146 Humus: Nature and Formation

considered the granary of plant nutrients in the soil. The benefits
to plant growth resulting from an increase in the concentration of
carbon dioxide about the leaves cannot be overemphasized. In addi-
tion to being an important source of plant nutrients, notably nitrogen
and phosphorus, humus also has solvent effects upon relatively in-
soluble elements.

The major effects of humus upon the soil and upon the growing
plant are not associated with its direct fertilizing value, however
important this may be. The physical and biological functions of
humus are of great significance. The improvement in the physical
condition of the soil as a result of addition of humus is associated
with improved texture, structure, and tilth, better water and air
relations, influence upon soil temperature and reaction, retention of
plant nutrients, and neutralization of toxic effects of certain com-
pounds formed in the soil. Humus tends to make soils granular,
causing the individual particles to form aggregates, thus preventing
baking when the soil is dry and stickiness when it is wet. The effects
of humus are particularly evident on sandy soils and on heavy silt or
clay soils. In the latter, a more open structure develops, which
favors increased circulation of the air and more rapid movement of
the water. In the sandy soils, humus exerts a binding effect, thus
retarding rapid percolation of water and giving to the soil the prop-
erties of heavier texture with higher moisture-holding capacity.

Soils receiving the proper amount of organic matter hold water to
better advantage, because sufficiently large pore spaces are created
to permit drainage of the excess water, while at the same time the
moisture-holding capacity of the organic matter is sufficiently high
to keep the soil from drying out too rapidly. This enables the plants
to resist drought not only because of the increased moistiue content
at the surface but also because of the deeper root penetration favored
by the improved soil structure. Air circulation in the soil is essential
for good root growth and plant development. Root penetration may
be favored by making the soil more porous; by improving the gas
relationships in the soil, whereby the water table is lowered, root
persistence is made possible. Soils receiving organic manures are
also found to be less subject to seasonal variations than those receiv-
ing artificial fertilizers only, as brought out by the investigations of
the Rothamsted Station,

In the presence of sufficient humus, plant nutrients are washed
out from the soil less readily by the percolating waters. This is par-
ticularly true of the basic elements comprising a number of important

Selected Bibliography 147

coinpouucls, such as aninionia and the salts of potassium, calcium,
and magnesium. Rapid changes in reaction to either higher or lower
acidity are also prc\ontod by the "bufrering" properties of soil or-
ganic matter. Plant poisons become less toxic in a soil high in
humus; high salt concentrations are less injurious; and aluminum
solubility, and thus its specific injurious action, are markedly de-
creased (Hester and Shelton). Plant deficiency diseases are usually
less severe in soils well supplied with organic matter, not only be-
cause of the increased vigor of the plants but also because of an-
tagonistic effects of the various soil microorganisms which become
more active in the presence of an abundance of organic matter.
Although some of these diseases may sometimes be controlled by
treatment of the soil with organic matter, the effectiveness of this
procedure cannot be fully relied upon in all cases.

Selected Bibliography

1. Albrecht, \V. A., Methods of incorporating organic matter with the soil in
relation to nitrogen accumulations, Missouri Agr. Expt. Sta. Res. Bull. 249,
1936.

2. Alway, F. J., Changes in the composition of the loess soils of Nebraska
caused by cultivation, Nebraska Agr. Expt. Sta. Bull. Ill, 1909.

3. Hester, J. B., and Shelton, F. A., Soil organic matter investigations ui^on
coastal plain soils, Virginia Truck Expt. Sta. Bull. 94, 1937.

4. Jenny, H., Soil fertiht>' losses under Missouri conditions, Missouri Agr. Expt.
Sta. Bull. 324, 1933.

3. Lipman, J. G., and Blair, A. W., Nitrogen losses under intensive cropping.
Soil Sci., 12:1-16, 1921.

6. Lyon, T. L., Bizzell, J. A., and Wilson, B. D., Depressive influence of cer-
tain higher jjlants on the accmnulation of nitrates in soil, J. Am. Soc. Agron.,
15:457-467, 1923.

7. Russell, E. J., Plant Nutrition and Crop Production, University of California
Press, 1926.

8. Russell, E. J., and Voelcker, J. A., Fifty Years of Field Experiments at the
Woburn Experimental Station, Longmans, Green & Co., London, 1936.

9. Russell, J. C, Organic matter problems under dry-farming conditions, /. Am.
Soc. Agron., 21:960-969, 1929.

10. Shutt, F. T., Influence of grain growing on the nitrogen and organic matter
content of the western prairie soils of Canada, Can. Dept. Agr. Bull. 44,
N.S., 1925.

11. Smith, N. R., and Humfeld, II., Effect of rye and vetch green manures on
the microflora, nitrates, and hydrogen-ion concentrations of two acid and
neutralized soils, J. Agr. Research, 41:97-123, 1930.

148 Humus: Nature and Formation

12. Sprague, H. B., The value of winter green manure crops, N. J. Agr. Expt.
Sta. Bull. 609, 1936.

13. Waksman, S. A., Principles of Soil Microbiology, Williams & Wilkins Co.,
Baltimore, 2nd Ed., 1932.

14. Waksman, S. A., Humus; Origin, Chemical Composition and Importance
in Nature, Williams & Wilkins Co., Baltimore, 1st Ed., 1936, 2nd Ed., 1938.

15. Waksman, S. A., and Tenney, F. C, The composition of natural organic
materials and their decomposition in the soil. II. Influence of age of plant
upon the rapidity and nature of its decomposition— rye plants, Soil Sci.,
24:317-333, 1927.

16. Woods, C. D., Roots of plants as manure. Conn. (Storrs) Agr. Expt. Sta.,
1st Ann. Kept., 1888, pp. 28^3; Stubble and roots of plants as manure,
2nd Ann. Kept., 1889, pp. 67-83.

• 6-.

Decomposition of Soil Organic Matter
and Evolution of Carbon Dioxide

Evolution of COo in Decomposition of Plant and Animal

Residues

Carbon makes up an average of about 50 per cent of all the
elements in plant and animal tissues. In certain carbohydrates and
organic acids, it may be somewhat less than 40 per cent, and in fats
and waxes it is above 60 per cent. In the decomposition of these
residues by microorganisms, most of the carbon is liberated as CO2;
the e\olution of this gas can, therefore, be taken as a measure of the
rate and extent of the decomposition process. Some of the carbon
is assimilated by microorganisms for cell synthesis, whereas an-
other part is left in the form of intermediary products, in both aerobic
and anaerobic decomposition. The total amount of CO2 liberated
depends on the nature of the material, the microorganisms con-
cerned, and the conditions of decomposition.

When cellulose, hemicelluloses, sugars, and starches are decom-
posed by fungi and by aerobic bacteria, as much as 50-80 per cent
of the carbon is liberated as COo. In a comparative study of the de-
composition of rye straw by pure and mixed cultures of microorgan-
isms, the mixed soil population decomposed only about one-third as
much of the total material in absence of added nitrogen as when
ammonium salt was added. The corresponding amount of carbon
liberated as CO^ was about one-fourth in absence of added nitrogen.
Of the total carbon in the material, 72 and 83 per cent were liberated
as COo. Less COo was liberated by pure cultures of fungi because
of greater consumption of the carbon and greater quantities of inter-
mediary products left.

Plant materials in a young green state decompose much more
rapidly than those in a mature state, and much more COo is produced

149

150

Decomposition of Soil Organic Matter

in a given time. The ratio of COo liberated to the organic matter
decomposed is similar, however, and depends on the organisms and
conditions of decomposition. In nitrogen-rich materials, liberation
of COo is accompanied by production and accumulation of ammonia,
which is soon changed in field and garden soils to nitrate; the ratio
between the carbon and nitrogen liberated depends on the nitrogen

H^U

_l

1 1 1 1 1 1 1 1 1 1 M_

-

r-. ._x - . .i^r

360

— -

Second cutting —

^

- -

Third cutting >/-

tab

-300

—

Fourth cutting y^ -

—

sf —

oT

y ^~

■g

y -^

X

—

/ ^cr-' —

o

s< ^

-5 240

—

/ ^

c

/ ^ ^

o

/ y --o'l-o

(13

—

J/ ^-'°'^^' ~

o 180

—

P o' / —

o

/ -'^^'

c

/ >>' /

o

/ /y

"3 120

o

~

/ /y

>

jj

~

// /y

—

H^

60
n

i

M 1 1 1 1 1 1 1 1 1 1

0 4 8 12 16 20 24 28 0 4 8 12

Time, (days) Time, (weeks)

Fig. 64. Influence of age of plant (stems and leaves) on its decomposition in
sand medium as indicated by the evolution of carbon dioxide (from Waksman

and Tenney).

content of the material undergoing decomposition and on the rate
of decomposition.

Decomposition of plant residues low in nitrogen is controlled by
the amount of available nitrogen present. Ammonia is not liberated
and may actually be consumed. The wider the carbon-nitrogen ratio
in the plant and animal residues, the greater is the proportion of COo
to ammonia liberated, until the latter becomes a negative figure;
then nitrogen must be added for active decomposition.

Figures 65-67 illustrate the course of COo evolution in the de-
composition of different plant materials under different conditions,
both in soils and in composts.

Although some COo may be formed by purely chemical processes
in the soil and although considerable COo is evolved by the roots of

Evolution of CO..

151

green plants during their respiration, in whieh the plants obtain their
oxygen as a part of the soil air and return CO2 to the gas mixture,
most of the COo is a result of decomposition processes carried out by
microorganisms.

30

20

- JS 10

E 2
00 u

e s

10

^20

30

40

Aog

vrm

V V

V V

vbV

V

1

Dg

Corn Potatoes

Table beets Sweet clover

I I I I I I II I I I I I II I I

^ m ^ 00 n
^ vo 00 m 1^

•<* ro lO 00 ■ . -. -• -

fH rt n n M-

in ^ r^ cTi ^ tx> o

Period of growth (days)
Fig. 65. Influence of plant tle\elopment upon the e\olution from the soil of
carbon dioxide of microbial origin: V, height of vegetati\e de\elopment; B,
blooming; Dg, degeneration; D, death (from Starkey).

The total CO2 given off is a result of the decomposition of plant
remains by microorganisms, chemically produced CO2, and CO2
given off during respiration by the roots of green plants. Lunde-
gardh calculated the percentage of total COo due to respiration, and
reached the conclusion that about 30 per cent of the total COo was
due to the presence of the roots. He believed that here, too, micro-
organisms associated with the roots had much to do with the COo
liberation. He concluded that, when oats were grown in sterilized

152

Decomposition of Soil Organic Matter

soil, 45 per cent of the increase in CO2 evolution due to the roots
was microbial in origin, which would reduce the contribution of the
oat roots to 16.5 per cent. One may thus conclude that about 85 per
cent of the total CO2 liberated from the soil is due to the activity of
microorganisms.

Evolution of COo from Soil Humus

The study of COo evolution from soil has followed several lines,
depending on the methods of determination. These are:

1. Methods based upon the extraction of gas from soil samples
removed from the field and taken to the laboratory. Leather estab-
lished that soil gases contain varying percentages of COo ranging
from 3.84 per cent to 15.29 per cent.

39

290

96 157

Decomposition, days

Fig. 66. Changes in composition of horse manure compost during different stages
of decomposition ( from Waksman and Diehm ) .

2. Methods in which an apparatus is placed on or thrust into the
soil, and the gas present is extracted by suction, as measured by
Appleman, by Russell and Appleyard, by Potter and Snyder, and by
Lundegardh.

3. Methods in which representatixe soil samples are taken and
tested in the laboratory for capacity to evolve COo under arbitrarily
prescribed conditions, as done by Waksman and Starkey and by
Marsh.

Evolution of CO^. from Soil Humus 153

4. Methods in which the e\okitioii of CO2 from a measured area
in a given time is determined in the field, as by Lundegardh and by
Humfeld.

Some of the results may be summarized as follows:

When soil samples are taken in the field for extraction in the
laboratory, the greatest precautions are necessary to devise apparatus
for taking the sample without driving out the gases already present
or allowing diffusion between the sample and the atmosphere.

z\ppleman devised a sampling tube for collecting the soil atmos-
phere. This consisted of two brass tubes, the smaller fitting tightly
inside the larger and extending about 1 inch at each end. One end
was groo\"ed for a rubber tube; the other was fitted with a point
the size of the larger tube, thus leaving a space between the point
and the large tube, in which twelve holes were drilled to drain the
soil gases from the free space. The outer tube was slipped down
o%er the holes, and the instrument was thrust into the soU to the
desired depth; then the smaller tube was pushed in far enough to
uncover the holes while the outer tube was stationary. Rubber
tubing was then attached and suction applied. The first 100 ml of
gas collected over mercury was discarded; then 250-ml samples were
taken for analysis.

In the cooler part of the season, between May 1 and June 1, the
COo from the soil varied from 0.13 to 0.38 per cent. Later in the
season, July 16, Appleman found 6.91 per cent in the soil under al-
falfa; 2.97 per cent under cabbage leaves; 5.05 per cent in a check;
and only 1.4 per cent in the soil between the rows. The air 1 foot
above the soil contained 0.03 per cent COo. When the plots high in
CO2 were cultivated, the total COo dropped off 90 per cent in the first
day, showing that cultivation brings aeration and dissipates the
accumulated gas.

The difficulty of thrusting an instrument into the soil without
opening channels, cutting across wormholes, insect burrows, or other
cracks which bring atmospheric air to the instrument in but slightly
changed condition, is readily seen.

In the study of the capacity of a soil to evolve COo, the sample is
collected as representative of the area. It may be partly dried, at
least to a known water percentage. It is crumbled or ground, sifted,
and weighed, in lots of 50 or 100 gm, into flasks with or without the
admixture of specific fertilizing or organic manuring substance.
Water content is brought to a definite point, and the flask is con-
nected into the collecting system to be held for a definite time at con-

154 Decomposition of Soil Organic Matter

trolled temperature. Marsh forced a stream of C02-free air through
the mass of soil, whereas Waksman and Starkey placed the soil in a
layer 2-3 cm deep in the bottom of an Erlenmeyer flask, through the
stopper of which a tube brought C02-free air into the flask, while a
second tube, reaching nearly to the surface of the soil, removed the
C02-laden stratum of air as fast as it was formed. The CO2 was
absorbed by alkali and determined by titration.

The main weakness of these procedures is that conditions are arbi-
trarily chosen; they may not reproduce in any definite way the
demands made upon a soil in the field. They do not measure any-
thing which is definitely found in nature.

Lundegardh devised a zinc bell— a pyramidal cover with straight
margins to be forced down into the soil, giving a collecting space of
a known volume. After the collecting bell remains in position for a
specified time, gas samples are pumped into containers and taken to
the laboratory for analysis. By standardizing the operation of his
collector, Lundegardh found it possible to move from plot to plot
across the field and take a sample every 20 minutes, accumulating
the receiving tubes and taking them all back to the laboratory for
microanalysis.

Lundegardh's apparatus has the advantage of placing a known
container over undisturbed soil for a brief period and collecting for
analysis a part of the air and CO2 mixture resulting from interruption
of the difl:usion of the COo evolved during the period. The sample
taken can be calculated to milligrams of COo evolved per square
meter of surface per hour or to any other desired unit. Whether
confinement of the CO2 evolved during that period results in delay-
ing evolution of COo may be questioned. If so, the results may be
low. The work of Lundegardh does, however, give a comparison,
of actual COo evolved, between different plots of ground in the field.

In the studies reported by Humfeld, the collecting apparatus was
modified in appearance from Lundegardh's bell, into a box 3 by 8
inches and 3 inches high, with the open side pressed into the soil
about 1 inch. At one end a collecting tube about ^ inch in diameter
was attached, and at the other a vent to which usually a tube was
attached with its open end carried 2-3 feet above the soil to give a
supply of atmospheric air to the collector. In this apparatus, the air
passing over the soil carried CO2; hence the extra tension of COo-
free air was eliminated, but the percentage of the COo present in
the atmosphere was determined regularly and deducted from the
totals found in the collection apparatus.

Evolution of CO.. from Soil Humus

155

With this apparatus, one can list the rate of CO;, evolution day
hy day o\or a season, or hour by hoiu- through some special period.
It recjuires no abstruse calculation but makes a demand for a con-
tinuous suction with a steady source of power; it also calls for regular
attention.

100
90

Carbohydrates-aerobi^
erotiic

Carbohydrates-anaerobic ^____^— — -

Lignin-anaerobic

405

498

Fig. 67.

205

Days of decomposition

Influence of aeration upon decomposition of alfalfa plant (from Tenney
and Waksman).

This procedure has weaknesses such as the small size of the col-
lecting box; the continuous position that interrupts processes that
go on near or on the surface; the irregularity of suction apparatus;
and inability to adjust absorbent, rate of flow, and frequency of
changes to the great changes in rate of COo evolution brought about
by some types of fertilization. It is probably most valuable in fol-
lowing the effects of some soil treatment from the time of applica-
tion for a period limited to a few days.

Smith and Brown emphasized that COo diffuses downward into
the soil from the area of biological activity as well as upward into

156

Decomposition of Soil Organic Matter

the air; that absorption by soil solution with subsequent loss in drain-
age water carries away part of the CO2 produced. Total production
of COo is, therefore, much greater than the amount which escapes
into the atmosphere. Hence the amount of CO2 collectible over a
given area cannot be regarded as a measure of the COo produced in
the soil.

175

150-

125

5P100

75

50

25

6 ^

Total
material

Cold-water-
soluble

Hemi-
celluloses

Celluloses Lignins

Fig. 68.

Crude
protein

Decomposition of ^'arious chemical constituents of rye straw with addi-
tional nutrient salts ( from Tenney and Waksman ) .

As shown previously, the humus in the soil is not absolutely re-
sistant to decomposition, but undergoes slow but continuous decom-
position. The rate of evolution of CO2, especially under aerobic con-
ditions, is the most accurate and simplest method for measuring
humus decomposition. This can be measured either (a) as total
COo arising from a given volume of soil during a certain period of
time, or (Z?) as the amount of COo found in the soil atmosphere.
The mineralized nitrogen liberated as a result of humus decomposi-
tion can be measured as ammonia or as nitrate, or both; in most soils
ammonia does not accumulate as such but is rapidly oxidized to ni-
trate. In addition to determining the products of humus decompo-

Evolution of CO^ from Soil Humus 157

sition, measurements may also be made of the actual disapiDcarancc
of humus as a whole or of some of its sp'ecific chemical constituents.
This can be done by measuring the total caibon or nitrogen content
of the soil or by making a proximate analysis of the humus. The
latter is of particular advantage in the study of peats, forest soils,
and composts.

Other methods can be utilized to measure humus decomposition,
as, for example, the change in calorific value of the humus in the
soil or the e\olution of heat accompanying processes of humus de-
composition. These methods amply demonstrate the fact that humus
is not stable, that it can disintegrate and disappear from the soil,
but that the rate of its disappearance is rather slow. Different farm
practices may result in the preservation and even accumulation of
humus or in its destruction. Which of these is more desirable de-
pends entirely upon the nature of the soil, the nature of the crop
grown, and general problems of soil utilization and soil conservation.

As a result of extensive decomposition, humus may reach a
definite chemical equilibrium, as shown by its more or less constant
carbon-nitrogen ratio. This equilibrium is particularly characteristic
of humus in field and garden soils. In this condition, further decom-
position of humus results in a parallel liberation of carbon as COo
and of nitrogen as ammonia, rapidly oxidized to nitiate. In com-
posts, forest soils, and peats, the ratio of COo liberated to nitrate pro-
duced varies considerably, depending upon the nature of the ma-
terial and the state of its decomposition.

A distinct parallelism was thus found to exist between the abun-
dance of microorganisms in the soil and the decomposition of the
soil humus; the more fertile a soil is, the greater will be the amount
of CO2 liberated in a given time. Wollny reported in 1880 that the
COo content of the soil rises and falls with the amount of organic
matter present.

One of the early accurate studies on humus decomposition was
carried out by Deherain and Demoussy. They placed the soil under
examination in closed glass containers and kept them at different
constant temperatures. At the end of a definite period of incubation,
the gas was extracted from the soil and container and the COo pres-
ent determined. These workers were among the first to demonstrate
that the formation of COo was due almost entirely to the action of
microorganisms; it increased with an increase in temperature to
about 65°C, then decreased; at 90°C, another increase in COo for-
mation took place, which was due to the chemical oxidation of the

158

Decomposition of Soil Organic Matter

humus at this high temperature. A certain amount of moisture was
required for the maximum production of COo by microorganisms.
The state of division of the soil and its aeration were found to affect
greatly the rate of decomposition of the humus. Sterile soils pro-
duced only small amounts of COo; when a soil infusion was added,
the process was increased twenty-five times. One of the most im-
portant points brought out in these investigations was the fact that
sterilized and inoculated soil gave two to five times as much COo

125

100 - A

^ 75

50

25

~

a

a

a

a

a

J)

a

b

b^

U

C '

t

M

C

1

r*

c

d_

b

c

d

Cj — 1

c

0

-^

€

de

.c

-

1"

^

Total

Cold-water-

Hemi-

Celluloses

Lignins

Crude

material

soluble

celluloses

protein

Fig. 69. Decomposition of various chemical constituents of alfalfa witliout addi-
tional nutrient salts (from Tenney and Waksman).

as unsterilized and uninoculated soil. This indicates definitely that
the process of sterilization rendered the soil humus more susceptible
to decomposition. There is also an optimum moisture content for
the formation of COo; this is influenced by the state of division of
the soil and its aeration.

A distinct parallelism was found to exist between the amount of
oxygen absorbed and the amount of COo produced from different
soils. A similar parallelism was also found between COo evolution
and nitrogen accumulation in the form of ammonia and nitrate.
Russell measured the amount of oxygen absorbed by the soil as an
index of soil oxidation instead of determining the COo produced.
The rate of absorption of oxygen was found to increase with tempera-
ture, the amount of water (up to a certain point), and the amount
of calcium carbonate present in the soil. Since these factors also
paralleled soil fertility, Russell suggested the use of soil oxidation as

Evolution of CX)^ from Soil Humus 159

a measure of the fertility of the soil. The amount of oxygen absorbed
was thus belie\ ed to measure the total aotion of soil mieroorganisms,
whieh are responsible for the decomposition processes in the soil.

Stoklasa and Ernest placed 1-kg portions of sieved soil in glass
exlinders through which a current of air was passed at the rate of
10 liters in 24 hours. They observed that the evolution of COo by a
soil, under certain conditions of moisture and temperature, in a given
time, can furnish a reliable and accurate method for the determina-
tion of bacterial activities in the soil; the presence of organic matter
and the temperatures were found to be of greatest importance. The
e\olution of COo was shown to be greatest in neutral or slightly alka-
line soils abundantly supplied with readily assimilable plant nutrients
and well aerated. The production of CO^ from the soil was found to
be in direct proportion to the available organic matter in the soil
rather than to the total organic matter. The evolution of COo was
thus found to be an index of the availability of the soil humus, or of
the ease with whieh it decomposes (Table 30).

Table 30. Ixflcence of Soil Depth and Soil Treatment upon the Decomposition
OF Humus, as Measured by the Rate of CO2 Evolution (from Stoklasa)

Soil Treatment

Cultivated,

Manured,

Soil

T'ncultivated.

Fertilized,

Fertilized,

Deptli

I'n fertilized

under Clover

under Beets

cm

mg

mg

mg

10-!20

16.5

38.6

47.5

20-30

19.4

38.8

49.7

30-50

9.8

20.2

28.5

.50-80

3.3

6.3

6.6

80-100 2.1 2.7 2.3

Van Suchtelen passed a current of air, usually 16 liters in 24 hours,
through 6 kg of soil placed on pure sand in a jar. The intensity of
CO2 production was much greater at the beginning of the experi-
ment and decreased rapidly after a short time. The amount of COo
produced was measured until it reached a uniformly low level; the
average amounts of CO2 produced per unit time from the different
soils served as a basis for comparison. The conclusion was reached
that the determination of COo formation from different soils fur-
nishes a better means for estimating the bacterial activities in the
soil than the numbers of bacteria. Cultivation, aeration, and nutri-

160 Decomposition of Soil Organic Matter

tive salts exerted a stimulating effect upon COo production; moisture
and organic matter content of the soil are among the most impor-
tant factors.

On comparing the curves for bacterial numbers, nitrate accumula-
tion, and COo content of the soil air, Russell and Appleyard found
them to be sufficiently similar to justify the view that all these phe-
nomena are related. A rise in bacterial numbers was accompanied
by a rise in the CO2 content of the soil air and somewhat later by a
rise of nitrate in the soil. The rate of decomposition of organic mat-
ter in the soil, as measured by CO2 evolution, was, therefore, looked
upon as a function of bacterial activities. The rate of these activities
in the soil attained maxima in late spring and autumn, and minima
in summer and winter. In autumn the bacteria increased first, then
the CO2 content rose, and finally the nitrate increased.

Neller measined the C02-producing capacity of the soil by adding
plant material to 200-gm portions of soil placed in tumblers under
bell jars, and passing C02-free air for 16 days. On comparing two
limed and two unlimed soils, he obtained distinct correlation between
crop yield, nitrate accumulation, and numbers of bacteria, but these
did not correlate with ammonia accumulation.

The evolution of COo from soil resulting from the decomposition
of humus was thus found to be the best index of the rate of decom-
position of this humus. This is further brought out in Table 31,

Table .SI. Effect of Liming upon Microbiological Activities in Soil (from Xeller)

Carbon

Soil

Dioxide

Nitrifi-

Nitrogen

Bacterial

rreatinent

Evolution *

cation

Eixation *

Numbers f

7ug

VI g

v}g

millions

Unliuied

2-Zl

10.4

-0.02

2.5

Limed

320

21.3

+ 1.35

6.2

L^nlimed

199

16.1

-0.90

5.1

Limed

333

39.9

+7.00

6.5

* Per 200 gra soil in 16 days.

t Per gram soil, as determined by plate method.

where the respiratory power of soils has been measured for a group
of plots variously treated and of varying degrees of fertility. The
curves for bacterial numbers, accumulation of nitrate, and evolution
of CO2 were found to be sufficiently similar to justify the view that
they are closely related. A rise in bacterial numbers was found to

Evolution of CO^ from Soil Humus 161

be accompanied by an increase in the (X)j content of tlie soil air
and somewhat later by a rise in the amonnt of nitrate in the soil.
The rate of decomposition of hnmns in the soil may, therefore, be
looked upon as a fnnetion of microbiological activities. Cultivation,
aeration, and presence of nutrient salts exert a stimulating effect
upon these processes. Among the most important factors, however,
are moisture content and abundance and nature of humus. A direct
relation exists between temperature and humus decomposition; the
process of humus decomposition goes on at temperatures below 0°C,
but it is greatly hastened by a rise in temperature. Variations in the
production of COo with season of year are due largely to variations
in temperature and to the available organic matter. The amount of
CO2 evohed in 24 hours from 1 square meter of soil was shown to
range from 2 to 20 gm calculated as carbon; the actual amount of
COo liberated depends on the nature of the soil, treatment, season
of year, and various other factors.

Since evolution of COo from soil is a measiu*e of the rate of
decomposition of the humus, one may conclude that the rate of
decomposition depends upon the abundance of humus in the soil,
the physical and chemical nature of the soil, its treatment, and the
crops grown (Table 32). In fertile soils, humus decomposes more
readily, thus resulting in the liberation of a greater amount of COo
and ammonia. Those soils in which humus decomposes very slowly
are called infertile. Frequently this condition can be corrected by
addition of lime, by culti\ation, by drainage, or by other special
treatments. An increase in the rate of decomposition of humus will
be accompanied by an increase in the fertility of the soil.

The decomposition of humus is brought about by the activities of
a large number of bacteria, fungi, and other microorganisms inhab-
iting the soil. These activities are rather slow as compared with the
decomposition of fresh plant and animal residues. The amount of
humus decomposed in a season is usually between 2 and 5 per cent
of the humus content of the soil. Under special conditions, the de-
composition may be greater; under other conditions, it may be less.

Another early student of humus decomposition in soil, Bous-
singault, recorded, in 1873, observations on a humus-rich soil. He
found that one-half of the total organic carbon in the soil became
changed to COo in 11 years; one-third of the nitrogen appeared as
nitrate in that period. The more extensive experiments at Rotham-
sted, however, showed a loss of only one-third of the nitrogen in 50
years, from soils free from crops but cultivated. In prairie soils, a

162

Decomposition of Soil Organic Matter

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Evolution of CO.> from Soil Humus

163

loss {)t oiie-tliircl of the nitrogtMi was sliown to takt> place in 22 years,
as a result ot (.iiltiNatioii; one-tliird of the' nitrogen liberated durinj^

80 100 120

Days of incubation

200

Fic. 70. Influence of moisture upon decomposition of peat (from Waksman and

Purvis ) .

the decomposition was recovered in the crops. The nature and the
treatment of the soil are of considerable importance in determining
the extent of these changes (Table 32).

TArtLE 3:3. Influence of Carbon-Xitrogen Ratio in Soil upon the Decomposition
OF Soil Organic Matter (from Sievers and Holtz)

Carbon Nitrogen C/N Ratio of
Liberated Liberated Mineralized

Carbon

Nitrogen

C/N Ratio

asC02

as Nitrate

Elements

■per cent

■per cent

mg

ppm

0.91

0.09

10.0

120

15.4

7.8

1.68

0.14

11.8

188

18.3

10.3

1.86

0.16

1-2.0

168

17.6

9. .5

2.89

0.23

1-2.4

•2.'J1

26.3

8.8

164 Decomposition of Soil Organic Matter

Influence of COo on Soil Minerals

An increase in the evolution of COo, as a result of the decomposi-
tion of plant and animal residues added to the soil or of the soil
humus, leads to an increase in the COo content of the soil atmosphere.
This results in an increase in the hydrogen-ion concentration of the
soil.

CO2 + HoO = H2CO3

H2CO3 = H+ + HCO3-

The hydrogen ion will interact with the various soil minerals, espe-
cially the phosphate and silicates, and bring about their greater solu-
bilization and availability for plant growth. This is shown in the
following reactions :

Ca3(P04)2 + 2H2CO3 = 2CaHP04 + Ca(HC03)2

NagSiOs + 2H2CO3 = 2NaHC03 + HaSiOa

H2Si03 = H2O + SiOa

The COo content of the soil atmosphere thus hastens the whole
process of weathering of rock constituents of the soil. It exerts an
important solvent effect upon the soil minerals, bringing them into
solution and making them more readily available for plant growth.
An increase in CO2 concentration in the soil will thus influence the
availability of various minerals essential for plant growth. This
affects first of all the solubility of the phosphates and silicates and
also that of other anions (borates). It also results in bringing into
solution greater concentrations of various cations, such as potassium,
calcium, and magnesium.

Influence of COo on Plant Growth

Carbon dioxide affects plant growth both directly and indirectly.
It has been definitely established by Lundegardh and others that
plants depend a great deal upon the COo liberated from the soil
humus for their nutrition, to supplement the COo present in the
atmosphere above the plants. It has even been said that the major
function of the addition of manure to soil is to increase the amount
of COo liberated, thus rendering larger concentrations of this im-
portant element available for plants.

Selected liihliography 165

The inicrooigauisins are thus fotiiul to act as regulators of the CO2
tension in the atmosphere and of the an'iount of CO2 available to
plants. Were these or<j;anisins less aeti\e in the liberation of the
COo from the dead plants and animals or from the waste products of
their metabolism, the earth would soon become covered with unde-
composed plant and animal debris, while the limited supply of CO2,
ha\ing become exhausted, would gradually bring to an end all forms
of life. The actixities of microorganisms which bring about the
decomposition of this organic debris prevent its abundant accumula-
tion and make axailable to plants a constant stream of COo, as well
as of nitrogen and other nutrient mineral elements. On the other
hand, were these organisms more active in bringing about these
decomposition processes, the surface of the earth would soon become
covered with a layer of sand or clay free from all traces of organic
matter; this would soon prevent the normal growth of the majorit)^
of economic plants by pro\ iding an unfavorable physical and chem-
ical medium for plant growth, and even more by preventing any
possible accumulation of nitrogen, which is also important for plant
growth.

Selected Bibliography

1. Appleman, C. O., Percentage of carbon dioxide in soil air. Soil Sci., 24:241—
245, 1927.

2. Humfeld, H., A method for measuring carbon dioxide e\olution from soil.
Soil Sci., 30:1-9, 1930.

3. Lundegardh, H., Carbon dioxide evolution of soil and crop growtli. Soil
Sci., 23:417-450, 1927.

4. Marsh, F. W., A laboratory apparatus for the measurement of carbon dioxide
evolved from soils, Soil Sci., 25:253-260, 1928.

5. Smith, F. B., and Brown, P. E., Soil respiration, /. Am. Soc. Agron., 23:
909-916, 1931; 24:577-583, 1932.

6. Starkey, R. L., Some observations on the decomposition of organic matter
in soils. Soil Sci., 17:293-314, 1924.

7. Waksman, S. A., and Starkey, R. L., Microbiological analysis of soil as an
index of soil fertility. VII. Carbon dioxide evolution, Soil Sci., 17:141-161,
1924.

7

Transformation of Nitrogen in Soil;
Nitrate Formation and Nitrate Reduction

Nitrogen is added to the soil in organic and inorganic forms. The
organic forms are found largely in plant residues, such as stubble,
weeds, leaves, and pine needles; green manure crops; stable manures
and composts; and special organic fertilizers, such as dried blood
and cottonseed meal. The inorganic forms of nitrogen are added
largely in the mineral fertilizers; these include mostly ammonium
salts, cyanamide, and nitrates. Both organic and inorganic fertiliz-
ers are subject in the soil to various changes brought about by
microorganisms.

Decomposition of Proteins and Their Derivatives

The amount of protein in organic material is usually measured
by multiplying the total nitrogen, as determined by the Kjeldahl
method, by the factor 6.25. The protein content of plant and ani-
mal residues added to the soil varies from 1.5 per cent or even less,
as in cereal straw and wood shavings, to 15 or even 20 per cent, as
in legume plants, young rye plants, and certain animal manures, such
as chicken manure. The protein content of certain defatted meals,
such as cottonseed meal, or of certain animal residues, such as dried
blood, may run to 30 or even 60 per cent.

The primary stage of the breakdown of proteins by microorgan-
isms consists in the formation of smaller units known as peptides,
and these in turn break down into the individual component amino
acids. The enzymes produced by the organisms and responsible for
these reactions are known as proteases or proteolytic enzymes. The
amino acids are then reduced to certain derivatives, depending on
the organisms and the conditions of decomposition, and finally to

166

Organism

Per C(

B. mycoidcs

46

Bad. vulgare

36

B. mesenterieii.f nih/iiliis

;5(!

S. luiea

il

B. subtilis

23

B. janlJdnus

'■v^

Ps. fluorcscfH{i iiutidiis

-li

Decomposition of Proteins 167

iiininoiiiu (Table 34). The enzymes coneeined in these reactions
arc known as deaminases and amidases.

Table 3-t. TuANSFciiniATiox ov Photioin Xithouen' into Ammonia hy IVIicuoouganisms

Orgaiiisni Per Cent

B. arborcscens 19

Ps.fluorescens liquvfaciens 16

Cephalothecium roseum 37

A. terricola 32

Botryotrichum pilulifcnini 24

Stemp/iylium sp. 5

Actinomyces sp. 21

Many proteins are present in \arious residues, especially in micro-
bial products, in the form of compounds with nucleic acids, known
as nucleoproteins. The composition of a typical protein is NHo-
CHRCO(NHCHRCO)„NHCHRCOOH. A typical nucleic
acid has the composition C3CH4SO30N4P4 and is made up of a purine
or pyrimidine base, a carbohydrate (hexose or pentose), and phos-
phoric acid.

Proteins can be broken down by microorganisms through one or
more of the following processes:

1. Hydrolytic deaminization. The hydrolysis of an amino acid
may result in the formation of a lower fatty acid and ammonia; or
of an alcohol, COo, and ammonia; or of an aldehyde, lower acid,
and ammonia, as shown by the general formulas:

R CH XH2 COOH + H2O = R CHOH COOH -f NH3 (1)

R CH • NH2 • COOH + H2O = R CH2OH -f CO2 + NH3 (2)

R CH XH2 COOH + H2O = R CHO + H COOH -f NH3 (3)

2. Decarboxylation with amine formation:

R CH NHo COOH - R CH2 NH2 + CO2 (4)

R CH2 XH2 + H2O = R CH2 OH + NH3 (5)

3. Reductive deaminization:

RCH NH2COOH + H2 = R CH2 COOH -f NH3 (6)

or

RCH NH2COOH + H2 = R CH3 + NH3 + CO2 (7)

168 Transformation of Nitrogen

4. Ammonia formation without reduction. Anaerobic bacteria may
produce ammonia from amino acids, without reduction.

RCH2 CH NH2 COOH = R CH:CH COOH + NH3 (8)

5. Oxidative deaminization:

R • CH • NH2 • COOH + O2 = R COOH + NH3 + CO2 (9)

This process is carried out by aerobic organisms, especially by fungi.
As examples of this reaction, the transformation of leucine into iso-
valeric acid, as shown above, and of glutamic acid into succinic
acid may be cited:

COOH CH2 • CH2 • CH • NH2 COOH + O2

Glutamic acid

= COOH •CH2CH2- COOH + NH3 -f CO2

The decomposition of one amino acid may involve the reactions of
oxidative deaminization, decarboxylation, and reduction. The same
organism may bring about a series of these reactions, while different
results may be obtained by the same organism under different con-
ditions.

The action of microorganisms on proteins and amino acids results
in the formation of various indols, some of which, such as indol-
acetic acid, act as growth-promoting substances, hormones or auxins,
upon plants. The favorable effect upon plant growth of organic
manures and animal excreta may thus be due to the action of these
plant hormones, or, as they have been called, "phytohormones."
Certain fungi and probably bacteria are able to synthesize these
hormones, as in the case of rhizopin.

In addition to proteins, other organic nitrogen compounds, like
lecithin, methylated amines, purine bases, and other substances pres-
ent in plant or animal tissues and found in the soil, are acted upon
by microorganisms to form simpler compounds. Ammonia is one
of these.

Lecithin is first split to choline, glycerophosphoric acid, and fatty
acids.

The choline is decomposed into ammonia, trimethylamine, carbon
dioxide, and methane. Betaine, creatinine, guanidine, and purine
bases, like uric acid, also undergo decomposition by microorganisms.

Uric acid can also be decomposed by various bacteria according
to the following reaction:

C5H4N4O3 -f 8H2O + 1^(02) = 4NH4HCO3 -I- CO2

Decomposition of Proteins

169

}uaD J3d 'u9§oj)!u paoijo^suejj^

•^ 00 ' ":

}u9D J9d 'uaSojjiu leuiSuQ

oijej ua3oj}iu-uoqje3

Ijos /^jp-jie io uj§ 001 -lad §uj 'm'HN + M'^ON

170

Transformation of Nitr

ogen

63

57

— NHj-N, Bac. cereus
and Pseud, fluorescens

' / NH^-N, Bac. cereus

t / / and Pseud, fluorescens

1 2

15

4 6 9

Days

Fig. 72. Course of accumulation of amino- and ammonia-nitrogen from casein
6y Bacilhis cereus and Pseudomonas fltiorescens (from Waksman and Lomanitz).

A large number of microorganisms are able to use hippuric acid
as a source of carbon and nitrogen. The acid is usually first hydro-
lyzed by an enzyme produced by these organisms:

CeHs CONHCH2 COOH + II2O = Cells COOII

Hippuric acid Benzoic acid

+ NH2CH2COOH

Giycocoll

Urea is hydrolyzed, with the formation of ammonia, by a large
number of soil microorganisms as well as by specific groups of bac-
teria, which utilize the energy liberated in the process:

C0< + 2H.0 = (NIl4).C0a = COs + 2NH3 + HoO

\NH2

Decomposition of Proteins

17]

The tnu- stnictiire of iiroa luis been postulated, however, to be as
follows:

AH

HN = C< I

Cyanamide readily breaks down in the soil to ammonia, whieh
is th(Mi nitrified \irtually quantitatively. Cyanamide may first be

40

^y^2^^ + dicyanodia

mide

Days

Fig. 73. Accumulation of ammonia from cyanamide and dried blood, as influ-
enced by the presence of dicyantjdiarnide (from Cowie).

decomposed in the soil into urea by a purely chemical process, under
the influence of catalyzers, or it may polymerize into dicyanodiamide
(especially in the presence of catalyzers such as ZnClo).

Soils differ markedly in the rate with which they decompose cal-
cium cyanamide, but very few are deficient in the required catalyst,
which changes the cyanamide to urea. The urea is transformed in
the soil to ammonia within a few days, especially in soils active micro-
biologically. Nitrate formation proceeds more slowly in soils treated
with cyanamide, the retardation depending on the soil and the
environmental conditions. A great many soil organisms are capable
of decomposing dicyanodiamide.

172 Transformation of Nitrogen

Chitin is found among the synthesized constituents of the cells
of microorganisms, especially fungi, and is constantly added to the
store of soil organic matter. It consists of one molecule of glucos-
amine and three molecules of acetyl-glucosamine, from which four
molecules of water have been removed (C30H50O19N4).

Ammonia Formation by Microorganisms

The earlier investigators of bacterial metabolism, like Hoppe-
Seyler, Bienstock, and Hauser, found that mixtures and pure cultures
of bacteria, like Proteus vulgaris. Bacillus subtilis, Serratia mar-
cescens, Clostridium putrificus, and Fseudomonas fluorescens lique-
faciens, are capable of breaking down proteins with the formation
of various end products, one of which is ammonia. Proteins of both
plant and animal origin are decomposed by a number of bacteria,
giving a great variety of products.

Marchal used a solution containing 1.5 per cent nitrogen, in the
form of egg albumin made insoluble by means of 0.01 per cent ferric
sulfate, which was inoculated with various bacteria; ammonia was
determined, after 20 days' incubation at 30°C, by distilling with
MgO.

Strains of B. rnycoides derived from different som-ces varied in
their power to produce ammonia from proteins. With one strain,
Marchal obtained a transformation of 58 per cent of egg-albumin
nitrogen into ammonia, accompanied by a marked change of the
reaction of the medium to alkaline. The more dilute the solution
of the protein, the greater was the transformation.

The great majority of soil organisms developing on the plate were
found to produce ammonia from proteins. The gelatin-liquefying
bacteria are capable of inducing greater protein decomposition with
more abundant ammonia formation. Since the members of this
group form at times more than 15 per cent of the total number of
soil bacteria developing on the plate, they were believed to do the
initial work in rendering soluble the protein nitrogen in the soil, so
that it might be further decomposed by the same or other soil organ-
isms.

According to Conn, however, the non-spore-forming bacteria are
much more active in manured soil than the spore-forming organisms.
Bacillus cereus was found to decompose proteins to amino acids,
whereas Fs. fluorescens acts largely upon amino acids. In the pres-
ence of a mixture of the two organisms, the protein is rapidly changed

Ammonia Formation by Microorganisms

173

to ammonia. Thus tluTc is a possibility that different organisms take
an acti\ e part in different stages of the pJrocess of protein decompo-
sition; bacteria hke B. ccrciis may be active in the first stages of
hydroKsis, and bacteria hke Ps. jiiiorescens, in the hitter stages lead-
ing to the formation of ammonia.

0 5 10

Days incubated

Fig. 74. Rate of decomposition of cottonseed meal in soil, as shown by the evo-
lution of carbon dioxide and accumulation of ammonia (from Gainey).

The rapidity of ammonia formation from proteins by bacteria de-
pends not only upon the nature of the organism but also upon the
kind of protein. The process of ammonia formation from casein
was completed in a few days, whereas it continued for more than
a month from gliadin. The amino-nitrogen contents of the gliadin
and casein media were 0.57 and 0.68 mg before hydrolysis; 42.56
and 99.31 mg after acid hydrolysis; and 17.03 and 46.00 after hy-
drolysis with B. subtilis. All the nitrogen forms of the protein mole-
cule are changed more or less by the action of bacteria, the end

174

Transformation of Nitrogen

product being ammonia; in no case, however, is one form of nitro-
gen completely destroyed. A similarity was found in the chemical
changes produced by acid hydrolysis and by bacteria. Carbohy-
drates exert a depressing effect upon the liberation of ammonia
from proteins (Table 35).

Table 35. Influence of Various Concentrations of Glucose on the Formation
OF Ammonia from Casein (from Doryland)

Casein + 0.05

Casein + 0.1

Casein + 0.2

Casein

Per Cent

Per Cent

Per Cent

Organism

Incu-
bation

Glucose

Glucose

Glucose

NHs

Bact.*

NHg

Bact.

NH3

Bact.

NH3

Bact.

days

mg

mg

mg

mg

B. subtilis

2

15.7

2.9

13.0

4.6

12.0

4.9

13.2

4.0

4

25.1

11.4

20.0

22.8

15.9

30.9

13.0

33.0

6

49.5

49.0

55.0

54.7

48.7

54.3

17.0

59.0

Pr. vulgaris

2

13.8

5.1

10.9

6.0

11.8

6.8

10.9

6.9

4

20.2

21.1

22.0

39.2

13.7

48.3

15.0

50.1

6

30.3

86.6

32.0

80.1

30.1

89.1

15.7

92.2

B. mycoides

2

22.6

4.7

14.8

4.4

13.9

3.8

14.0

4.8

4

64.0

25.9

20.1

32.1

14.4

33.0

15.0

37.0

6

69.6

63.2

60.0

65.9

52.0

66.7

11.9

66.1

S. lutea

2

12.9

1.9

11.2

2.0

11.9

2.8

10.8

3.1

4

22.4

5.9

10.2

7.6

11.9

6.1

12.0

7.4

6

26.1

7.9

20.0

19.0

11.3

10.7

11.0

8.9

* Bact. = bacteria in millions.

Fungi are able to decompose proteins very vigorously. Different
species vary greatly in this respect; the nature of the protein, re-
action of medium, and presence of available carbohydrates affect the
process. A large part of the nitrogen may be left in the form of
intermediary products. Organisms like Aspergillus iiiger, which pro-
duce large amounts of acid (oxalic and citric) from carbohydrates
and even from proteins and which are thus enabled to neutralize the
ammonia, accumulate only very small amounts of amino acids in
artificial cultures; at the same time appreciable quantities of am-
monia are formed in the medium. But when the oxalic acid is neu-
tralized with CaCO-?, or when the formation of both oxalic acid
and ammonia is prevented by means of insufficient aeration, amino
acids accumulate.

Protein Decomposition 175

When the protein is the oiih' souree of earbon avaihible for fungi,
a definite parallehsm is found between the grovvtli of the mycehum
and the produetion of ammonia. Different protein derivatives are
not utiHzed aHke, and their nitrogen is not hberated ahke in the
form of ammonia. As))crgiIUis ni'^cr grows best with leucine, fol-
lowed by peptone, asparagine, and glycocoll. The difference in the
nature of the carbon compounds accompanying the proteins or of
the protein carbon itself accounts for the difference in the amount of
fungus growth and ammonia formation, because, in the absence of
a\ ailable carboln drates, the fungus uses the protein both as a source
of energy and as a source of nitrogen. The amount of nitrogen lib-
erated as ammonia depends not only upon the nitrogen content of
the protein, but also largely upon the availability of other sources of
earbon. The nitrogen is then either liberated as a waste product,
ammonia, or reassimilated and changed into microbial protein (Table
36).

Table 36. Influence of Composition of Amino Acid upon Ammonia Production
BY Microorganisms (from Waksman and Lomanitz)

Dry

Growth

Growth

Amino Acid

C/N

Organism

of CelLs
mg

NH3-N

mg

NH3-N

Glycocoll

1.7

Trichoderma

50

24.28

2.0

Glycocoll

1.7

Actinomyces

59

30.46

2.0

Alanine

ii.57

Trichoderma

80

21.98

3.6

Alanine

2.57

Actinomyces

126

39.17

3.2

Glutamic acid

4.28

Trichoderma

218

29.12

7.5

Glutamic acid

4.28

Actinomyces

169

28.36

5.9

Glutamic acid

4.28

P.S-. fluorescens

128

28.50

4.5

Nitrogen Transformation in Decomposition of Organic Matter

When nitrogenous organic substances are added to the soil, a group
of complex reactions result, so far as the nitrogen is concerned:

(1) Hydrolysis of the proteins into polypeptides and amino acids,
with liberation of some ammonia, takes place. (2) This is followed
by the decomposition of the amino acids and other products of pro-
tein hydrolysis, with a further liberation of ammonia. (3) Synthesis
of microbial protoplasm leads to a storing away of a part or the
whole of the ammonia nitrogen; the greater the quantity of available
non-nitrogenous organic matter accompanying the nitrogenous sub-

176

Transformation of Nitrogen

stances, the greater will be the synthesis of microbial protoplasm,
leading to a greater assimilation of the nitrogen and to a smaller
accumulation of ammonia. (4) Various soil conditions, as well as
differences in the composition of the nitrogenous and the accom-
panying non-nitrogenous organic substances, lead to the develop-
ment of different microorganisms capable of decomposing the ni-
trogenous materials; the carbon-nitrogen metabolism of these micro-
organisms is different; this leads, therefore, to differences in the
amounts of ammonia liberated in a free state.

These reactions result in a transformation of a larger or smaller
part of the nitrogen of the organic complexes into ammonia, which,
either as such or after it has been oxidized to nitrate, is available as
a source of nitrogen for the growth of cultivated plants. In view of
the great economic importance of the liberation of ammonia, numer-
ous contributions have been made to the subject, known as "ammoni-
fication." These studies have been chiefly limited to adding about
1 gm of the nitrogenous organic material to 100 gm of soil, mixing,
placing in timnblers, then bringing the moisture content of the soil
to optimum (60 per cent saturation), incubating for 4-14 (usually
7) days, then measuring the amount of ammonia present in the soil
by distilling with MgO.

Thus, in the presence of available carbohydrates (Table 37), two

Table 37. Influence of Carbohydrate upon Ammonia Formation in Soil

(from Kelley)

1 gni of

].'?'-2.9 mg Organic

Each Organic

Nitrogen plus

Material

Enough Starch to

.\dded to

Make Equivalent

Nitrogen

Nitrogen

100 gm Soil

Amounts of Carbon

Source

Content

NH3-N

NH3-N

per cent

mg

mg

Casein

12.40

50.2

31.4

Dried blood

13.29

42.4

18.9

Soybean cake

8.28

40.9

34.1

Cottonseed meal

5.10

27.1

34.0

Linseed meal

5.00

26.0

34.1

factors are at work: first, less of the protein is decomposed, since
the bacteria and fungi prefer the carbohydrate to the protein as a
source of energy; second, the ammonia that has been formed from
the decomposition of the proteins may be reassimilated by the micro-

Ammonia Formation

177

organisms which ntih/.i> the carbohydrate as a source of energy.
These microbes are, tlierefore, competing -with higher plants for the
avaihible nitrogen compounds in the soil. As a result of these
studies, Doryland defined ammonification as "an expression of an
unbalanced ratio for microorganisms, in which the nitrogen is in
excess of the energy-nitrogen ratio." If the available energy ma-

o t)U —

uu
90

1

1 1

MM

1 1 1 II 1 II

80

-

/o

70

—

/

60

-

/

50

-

/

40

-

/

30

-

-

20

-

-

10
n

^^

rr

Mil

M

o 4U —

0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18
Incubation, days

Fig. 75. Rate of aniinonia formation from peptone by Aspergillus niger (from

Waksman ) .

terial is equal to or in excess of the energy-nitrogen ratio required
by the flora, the coefficient of ammonia formation tends to approach
zero; it tends to approach a maximum if the available energy material
is less than the energy-nitiogen ratio. Depending on the proportion
of energy material to nitrogenous substances, "beneficial" bacteria
may become "harmful."

The nature and the composition of the organic matter greatly influ-
ence its decomposition. The ratio between the carbon and nitrogen
of the material used is of special importance in this connection. The
same is true of the natnre of the non-nitrogenous organic materials
introduced into the soil in addition to the nitrogenous substances.
Table 38 has been compiled from the results of Lipman and associ-
ates, who added different organic nitrogenous materials to 100-gm

178

Transformation of Nitrogen

Table 38.

Influence of Carbohydrates upon the Accumulation of Ammonia
FROM Nitrogenous Organic Materials (from Lipman)

Total

Ammonia Formed

Nitrogenous

in

No Carbo-

Glucose

Sucrose

Starch

Substance *

Material

hydrate

2 gm

2 gm

2 gm

mg

mg

mg

mg

mg

Rice flour

46. -i

1.26

1.30

1.48

0.87

Corn meal

51.2

1.18

1.30

1.04

0.69

Wheat flour

94.8

5.14

3.66

5.84

1.56

Cowpea meal

156.8

50.88

31.71

28.57

23.70

Linseed meal

247.0

110.69

96.01

60.73

63.34

Soybean meal

245.6

129.64

108.03

94.88

54.36

Cottonseed meal

246.1

123.63

99.67

97.23

54.54

* Four grams of organic material added to 100 gm of soil.

portions of soil; the moisture content was brought to an optimum,
and after the soils were incubated for 7 days, the ammonia was de-
termined by distilling with MgO. Rice flour and corn meal, with a
wide carbon-nitrogen ratio, allowed no accumulation of ammonia,
either with or without additional carbohydrate. The substances rich
in nitrogen allowed an accumulation of almost 50 per cent of the
nitrogen as ammonia, but this was considerably reduced when more
available energy in the form of carbohydrates was added ( Table 39 ) .

Table 39. Influence of Added Nitrogen Salt upon the Decomposition of Rye
Straw * and Its Constituent Carbohydrates by Microorganisms

Units in milligrams.

Organisms

Ammonium
Salt Added

CO2
Liberated

Total Plant

Material
Decomposed

Hemi-

celluloses

Decomposed

Cellulose
Decomposed

NH3-N

Assimi-
lated

Control
Control
Trichoderma
Trichoderma
llumicola
llumicola
Soil inoculum
Soil inoculum

+
+
+

+

37

37

333

648

588

1,106

799

2,964

251
504
602
908
601
1.940

190
280
199
339
240
609

47
327
125
461
116
1,079

25
31
53

* Five-gram portions.

Nitrification in Soil

179

Nitrification in Soil

The production of nitrates from ammonia was known and utilized
long before the microbiological nature of the process was under-
stood. This is illustrated by the fact that during the Napoleonic
Wars careful instructions were given to the French farmers for
preparing composts of stable manure, favoring nitrate formation and

Fig. 76. Influence of reaction on nitrification in soil, after 11 days (I), 22 days
(II), and 29 days (III) (from C. Olsen).

accumulation. During our own Civil War, especially in the South,
"niter plantations" or "nitriaries" were common (Taber),

The decomposition of proteins and of other nitrogenous organic
substances leads to the formation and often the accumulation of
ammonia in the soil. Under favorable conditions, this is rapidly
oxidized to nitrites and then to nitrates. Under certain conditions,
when the nitrifying bacteria are killed, as in the partial sterilization
of soil, or when conditions do not favor nitrification, as with exces-
si\e soil acidity, ammonia may accumulate in the soil.

The oxidation of ammonia to nitrate can be accomplished by
three types of processes, namely, chemical, physicochemical, and,
most important, biological.

At high temperatures and in the presence of catalysts, ammonia
may be oxidized chemically to nitrate, as in the electrolytic oxida-
tion of ammonia in the presence of copper oxyhydrate. Oxidation
may also take place, to a limited extent, in an atmosphere saturated

180

Transformation of Nitrogen

with ammonia and in the presence of ferric hydrate. Ammonia is
also oxidized to nitrite by ultraviolet radiation. According to Weith
and Weber, hydrogen peroxide and ammonia react with each other,

120

8.0 1 9.0
NaHCOj
Reaction -pH

10.0 I
Na.,CO

11.0

100

Free H. CO3
Fig. 77. Influence of reaction upon nitrate formation (from Meyerhof).

giving rise to nitrous acid. The interaction between ozone and am-
monia to give ammonium nitrate has been known for several decades:

(NH3)2 + 4O3

NH4N02 + H202

NH4N02 + H202 + 402

NH4NO3 + HoO

It has been reported recently that oxidation of ammonia and
its salts in solution and in soil may be effected by sunlight, in the
presence of certain photosensitizing substances, TiO and ZnO being
most active; basicity favored oxidation and acidity impeded it ( Table
40). It was believed that a part of the nitrification in soil is due at
least to photochemical oxidation at the surface of xarious photo-
catalysts present in the soil. The process was believed to be of
special importance in tropical countries, such as India (Dhar). Rossi
also suggested that the process of nitrification is of a purely physico-

Nitrification in Soil 181

Taulk 40. 1{ki,aii()\siiii' hktwkkn Suil Rkaction and Am .\1)a.n( !•; oi' Ammoma-
()xiDizix(i Organisms (from' Wilson)

Al)iiii(laiic('
j)\l ut' Organisms

iK-i 1,000

(i.l .'{,.500

().(i (i.-iSO

(i.S '2.5, ()()(»

7.0 .T),!^^)

chemical nature at tlie \ery surface sod layer. The evidence sub-
mitted to substantiate both these theories is still insufficient. On
further study, at least in tropical countries, negative results were
obtained; the results obtained by Rossi were explained by the spe-
cific effect of drying upon the removal of nitrate already present in
the soil. Nath concluded that sunlight and ultraviolet hght have no
effect on the oxidation of ammonia in soil; actually nitrates are de-
composed under these conditions.

At best the quantities of nitrite and nitrate formed by chemical
agencies are insignificant and of httle importance in the soil. The
biological process of nitrification, as established by Schloesing and
Miintz, Warington, and Winogradsky, is by far the most important.

Mechanism of Biological Oxidation of Ammonia. Various re-
actions have been suggested to explain the mechanism of oxidation
of ammonia to nitrite by the nitrite-forming bacteria. The follow-
ing two reactions are most probable:

2XH3 + 3O2 -> ^2HX02 + ^2H20

2NH3 + 3O2 -> X2O3 + 3H2O

The free energy efficiency of Nitrosomonas is usually taken to be
about 6 per cent. Lees and Hofmann have recently calculated such
energy for different stages of growth of the organism. At the early
stages of growth, the \'alue was found to be about 50 per cent, falling
rapidly as nitrite accumulated; with a nitrite concentration of 1.5 mg
nitrogen per ml, it reached about 6 per cent. This rapid fall in effi-
ciency was beliex ed to be due to an increased respiration loss follow-
ing the maintenance of a low intracellular nitrite concentration with
an increasing nitrite content. Using paper chromatography, Hof-
mann found that the amino acid content of the protein of nitrifying
bacteria is similar to that of the proteins of other organisms.

182 Transformation of Nitrogen

Mechanism of Nitrite Oxidation. The oxidation of nitrite to
nitrate takes place according to the following reaction:

NaNOa + ^Oa = NaNOg

This was demonstrated by measuring the nitrite and oxygen con-
sumption. With optimum concentration of the nutrients and proper
aeration of the culture, the nitrate-forming organisms, in liquid
culture, may oxidize 4-5 gm NaNOo per liter in 24 hours.

Winogradsky observed the interesting phenomenon that am-
monium salts injuriously affect the growth of nitrate bacteria. This
seemed rather strange in view of the fact that the nitrate bacteria
are active side by side with the nitrite bacteria which use the am-
monium salt as a source of energy. It was then suggested that
the two processes follow in two successive periods in the soil, nitrate
formation beginning only after all the ammonium salt is converted
into nitrite. On decreasing the amount of NaoCOs, which would
lead to a lower alkalinity, Boulanger and Massol found that the
injurious effect of ammonium salt is less, and concluded, therefore,
that the growth of nitiate bacteria is not injured by the salt but by
free ammonia. This was confirmed by Meyerhof, who established
that the injurious influence of ammonia and its derivatives ( aliphatic
amines) consists in the penetration of the base into the cell (which
does not take place in the case of ammonium salt) and in a specific
action of the NH3 and NHo groups. Lipoid-insoluble amines, like
the diamines, are not injurious. The injurious effect of amines and
cations depends upon their ability to penetrate into the cell and
upon the reaction of the media; respiration is usually less affected
than growth. The intermediary products of the oxidation of sulfur
( hyposulfite ) are a decided deterrent to the process of nitrate forma-
tion in soil; the nitrifying bacteria as such are not injured, since the
process is resumed as soon as these intermediary products have dis-
appeared.

Schloesing compared the formation of nitrates from various am-
monium salts added to the soil and fouud that the following relative
amounts of nitrogen (in milligrams) are nitrified per day: NH4CI,
3.4; (NH4),S04, 9.0; (NH4)2CO:i, 4.0. Ammonium salts of organic
acids are also nitrified rapidly.

It was thought at first that organic matter can be nitrified directly.
Miintz has shown, however, that organic matter has to be decom-
posed first, and ammonia liberated, before nitrates can be formed.
Omeliansky later obtained negative results also for urea, asparagine,

Denitrificatiou in Soil 183

methylamino, diiiu'th\ laminr, and egg albumin, lie concluded that
all forms of organic nitrogen have to be transformed first into am-
monia before the\ can be nitrified. This was found to hold true also
for calcium cyanamide. When the processes of nitrate formation
from ammonium salts and from amino acids are compared, the latter
is found to take place more slowly. This is probably due to the fact
that the amino acids ha\ e to be changed first to ammonia and also
to the fact that some of the nitrogen is stored away in the microbial
cells which use the carbon of the amino compounds as a source of
energy.

When ammonium sulfate is used as a source of nitrogen for nitrate
formation and the reaction of the soil is acid to begin with, there will
be an increase in acidity in absence of sufficient buffer or base, as a
result of formation of nitric acid from the oxidation of the ammonia
and the accumulation of the residual sulfuric acid. Nitrate accumu-
lation will proceed until the reaction of the soil has reached a pH of
about 4.0. The amount of nitrate formed under these conditions
depends upon the initial reaction of the soil and its buffer and base
content; the higher the buffer and base content of the soil, the larger
will be the amount of nitrate formed for a certain change of reaction.
The continuous use of ammonium sulfate as a fertilizer without the
addition of lime will, therefore, lead to a gradual increase in soil acid-
ity. However, nitrates may be found even in very acid soils. This
was explained by Hall and associates as due to the fact that, under
acid conditions, nitrate formation takes place in films surrounding
the small isolated particles of CaCOs. The addition of CaCOs has,
therefore, a decidedly stimulating effect on nitrate formation, par-
ticularly in acid soils. In alkaline soils which are deficient in organic
matter, CaCOs may have the opposite effect, since it tends to liberate
from ammonium salts free ammonia, which retards nitrification.

Conditions that tend to promote nitrate formation in the soil are
temperature of 27.5 ^C, an abundant supply of air (oxygen), proper
moisture supply, a favorable reaction (pH greater than 4.6), pres-
ence of carbonates or other buffering agents, and absence of large
quantities of soluble organic matter. The nature of the crop grown
also influences the nitrate content of the soil.

Denitrification in Soil

Just as aerobic conditions in soil favor oxidation processes, so do
anaerobic conditions (exclusion of free oxygen) fa\'or processes of

184

Transformation of Nitrogen

reduction. Either organic or inorganic compounds may be formed
as a result of these processes, depending upon the composition of
the medium. It is not necessary for the soil to be saturated with
water for the conditions to be anaerobic. Winogradsky demon-
strated, by the development of anaerobic nitrogen-fixing bacteria.

10

20 30 40 50
Years of cultivation

60

70

Fig. 78. Nitrogen le\'el of soils culti\atcd for a number of years (from Jenny).

that, when a soil contains water equivalent to only about 40 per cent
of its moisture-holding capacity, anaerobic bacteria find conditions
favorable for their development even at the very surface of the soil.
The disappearance of nitrates in soil as a result of activities of
microorganisms may be due to three groups of phenomena: first,
direct utilization of nitrates by microorganisms as sources of nitro-
gen, in the presence of sufficient energy material; second, reduction
of nitrates to nitrites and ammonia in the process of nitrate assimila-
tion; third, utilization of nitrates as sources of oxygen (nitrates as

DcMiitrification in Soil 185

hydrogou acceptors). In i1k> last process oxygen is ntilized by the
organism lor the oxidation ol earhon eoniponnds or inorganic sub-
stances, such as sulfur. The energy thus derived is used for the
reduction of the nitrate to nitrite, to free nitrogen gas, to oxides of
nitrogen, or to ammonia. The formation of nitrogen gas from nitrate
may be so rapid under fa\ orable conditions that the gas can actually
serve as a measure of the amount of nitrate reduced.

The disappearance of nitrates in the soil due to the various proc-
esses of nitrate reduction and nitrate assimilation has often been
referred to as "denitrification." However, the reduction of nitrates to
nitrites and annnonia, as well as their assimilation by microorganisms,
involves no losses of nitrogen, but merely indicates that the nitrates
are for the moment taken out of circulation and changed into forms
from which nitrate can be again produced. The nitrates may com-
pletely disappear without in\olving any loss of nitrogen, as in their
assimilation by fungi and various bacteria in the presence of avail-
able energy. The term "denitrification" ( or complete denitrification )
should designate the complete reduction of nitiates to atmospheric
nitrogen and oxides of nitrogen, whereas the other processes involv-
ing disappearance of nitrates may be referred to as "nitrate reduc-
tion" and "nitrate assimilation."

Certain bacteria are capable of reducing nitrates to nitrites, am-
monia, and atmosplieric nitrogen or oxides of nitrogen. Goppels-
roder was the first to observe that nitrates are reduced in the soil
to nitrites. He ascribed this property to the organic matter of the
soil. Schoenbein in 1868 and Meusel in 1875 recognized the bac-
terial nature of the process. This idea was developed further by
Gayon and Dupetit and others, as shown previously.

In absence of free oxygen but in presence of nitrate, various aerobic
bacteria are capable of existing anaerobically. Some organisms bring
about complete denitrification; others reduce the nitrate to the nitrite
stage only, a smaller amount of oxygen thereby becoming available:

2HNO3 - 2HNO2 + O2
When the nitrite is reduced to atmospheric nitrogen,

2HNO2 = N2 + 1^02 + H2O

In the reduction of nitrate to ammonia, the following reaction takes
place:

HNO3 + H2O = NH3 -f 2O2

186

Transformation of Nitrogen

The more nearly complete the reduction of the nitrate, the more
oxygen becomes available, and, therefore, the greater is the amount
of carbohydrate that can be oxidized and the greater is the gain in
energy. In the case of many aerobic microorganisms, nitrate can
act as the hydrogen acceptor, whereby it is first reduced to the NO2
ion, and this, through the hypothetical dioxyammonia (HON -HON),

7.0 6.5 6.0 5.5 5.0 4.5

Reaction of soil, pH

Fig. 79. Influence of reaction upon nitrogen liberation in forest soil (from

Aaltonen ) .

to NHo-OH ( hydroxylamine ) and then to NH3.
the reduction can be presented as follows:

The first stage of

HCOOH + HNO3 -^ CO2 + HNO2 + H2O

Nitrate reduction can be brought about readily by a number of
soil bacteria, under anaerobic conditions, when carbon complexes
are available as sources of energy. Nitrates enable many facultative
anaerobes to develop under anaerobic conditions, using sources of
carbon which would otherwise not be available.

The reduction of nitrates to atmospheric nitrogen always goes
through the nitrite stage. The following reaction was at first sug-
gested to explain the complete reduction of the nitrate molecule:

SCeHiaOe + 24HNO3 = -24H2CO3 + GCOo + ISHoO + I2N2

The carbohydrates or organic acids of the media are decomposed
with the formation of carbon dioxide and nascent hydrogen; the
nitrate is then used by the organism as the hydrogen acceptor, which
results in the reduction of the nitrate. When tartaric acid is oxidized
by atmospheric oxygen or by reduction of nitrates, nearly equal

DcMiitiificatioii in Soil

187

amoiiuts ol c'iu'iij;y aw libtMatcd, siiico tlic rctliictioii of nitratos to
atmospheric nitrogen does not consume 'a large amount of energy.
Most of tlie denitriUing bacteria reduce nitrate to nitrogen gas
and NjO in varying proportions, B. nifroxits being particularly active
in the process. A 5-12 per cent solution of nitrate inoculated with
soil gives, at 20-37'"C, a current of gas which is 80 per cent NoO.

16

•Nitrate

30 40 50

Time, days

Fig. 80. Reductiun of nitrate by niicrobos (from Korsakowa).

Various other denitrifying bacteria, like Ps. aeruginosa and B. stutzeri,
give in solutions of nitrate ( particularly NH4NO3 ) a gas rich in NoO.
Of 100 cultures of bacteria tested by Maassen, 31 were found capable
of reducing nitrate to nitrite; the latter is then reduced to atmospheric
nitrogen and various oxides of nitrogen. This process was rather
slow and independent of the oxygen supply. Tacke found that 38
per cent of the gas mixture formed during the process of nitrate re-
duction by bacteria may consist of NoO. The formation of nitric
oxide in the reduction of nitrates has also been demonstrated by-
other investigators.

In general, the liberation of atmospheric nitrogen by reduction of
nitrate depends upon changes in oxidation-reduction potential of
medium, pH value, presence of nitrite, and nature of available car-
bon sources. The presence of certain growth-inhibiting substances
is also of importance. The addition of KCN to a culture of Micro-
coccus dcnitrificans inhibited the formation of elementary nitrogen;
the last stage in the reduction process, that of hyponitrite to gaseous

188 Transformation of Nitrogen

nitrogen, was believed to be affected, the hyponitrite breaking up
to the oxide of nitrogen and free base.

The presence in the soil of bacteria capable of reducing nitrates
to atmospheric nitrogen and oxides of nitrogen was definitely estab-
lished in 1882 by Gayon and Dupetit and by Deherain and Ma-
quenne. The same year, Lawes, Gilbert, and Warington pointed
out that considerable quantities of nitrogen may be given off when
a soil receives heavy applications of manure and is saturated with
water or is improperly aerated. Breal announced in 1892 that many
substances of organic origin, especially straw, can serve as sources
of energy which would enable the bacteria to liberate atmospheric
nitrogen from nitrates. In 1895, Wagner reported that the addition
of manure to liquid cultures containing nitrates greatly increased de-
nitrification; this observation led him to the conclusion that the same
process takes place in the soil. He found confirmation of this in
field experiments where organic nitrogen and nitrates were added
simultaneously before the crop was planted. Wagner declared, on
the basis of these experiments, that denitrification may take place
extensively in cultivated soils; the application of manure (cow or
horse) to the soil may often be not only unprofitable but even harm-
ful. This was believed to be due to the fact that manure carries
microorganisms which destroy the nitrates in the soil, not only ni-
trates added as such, but even those formed by the nitrifying
bacteria.

These and similar other investigations created the impression that,
when nitrates are added to the soil, denitrification sets in and may
produce an injurious action by causing the transformation of the
nitrate into gaseous nitrogen. It was soon found that these results
were greatly exaggerated. Losses of nitrogen were found possible
only when considerable amounts of organic matter were added
together with the nitrate, but this is not commonly done. Pfeiffer
and Lemmermann demonstrated that very little actual denitrification
takes place in the soil as a result of addition of manure. The lack
of nitrogen often observed is due to otlu>r causes rather than to
the loss of nitrogen. Nitrate reduction sets in when the soil is
saturated with water. Only in the presence of a great abundance of
organic manures is there any fear of loss of nitrate nitrogen in a
gaseous form from the soil. When soils are submerged in water,
the nitrates are rapidly reduced. Nitrites may be formed not at all
or only in too small amounts to cause plant injury. Ammonia is
formed in some cases. As a result of this reduction, the reaction of

Selectocl Bil)liogiap]iy 1S9

the soil 1h'c(miu\s more alkaline. A similar inercase in alkalinity is
obserxed when green manures are applied to flooded soils. In high-
moor peat soils, the addition of lime leads to acti\'e mtrifieation;
when the nitrates are redueed by denitrifying baeteria, the nitrogen
in the soils is rapidly depleted.

Great losses of nitrogen may take plaee in a humid, hot climate;
the rate of loss is increased by liming; bare fallows in rainy season
were found to be especially wasteful because of the leaching of
nitrates in drainage waters. There is little danger from denitrifica-
tion in normal soils. The partial reduction of nitrates to nitrites and
ammonia, which is more extensive and carried out by larger num-
bers of microorganisms, does not involve any actual losses of nitro-
gen. The nitrates may completely disappear from the medium with-
out loss of nitrogen. The products formed from the nitrates (nitrites
and ammonia) can be further acted upon by nitrifying bacteria; the
part of the nitrate assimilated by microorganisms is merely stored
away in the soil in an organic form.

It is often observed that addition of large quantities of undecom-
posed organic matter to a soil particularly rich in carbohydrates and
poor in nitrogen injures crop growth. This is not due to denitrifica-
tion, to which it has often been ascribed, but to the fact that, in the
presence of an excess of available organic matter, the fungi, actino-
mycetes, and \'arious heterotrophic bacteria synthesize an extensive
protoplasm. For this purpose, they assimilate the nitrates and am-
monium compounds present in the soil and thus compete with higher
plants.

The conclusion ma)' be reached that the phenomenon of denitri-
fication is of no economic significance in well-aerated, not too moist
soils, in the presence of moderate amounts of organic matter or
nitrate. In soils kept under water for some time, as rice soils, how-
ever, addition of nitrates may prove injurious because of the forma-
tion of toxic nitrite. It may be added here that there is also no dis-
tinct parallelism between plant communities, the geological substrate,
and the presence and activities of denitrifying bacteria.

Selected Bibliography

1. Barritt, X. W., The liberation of elementary nitrogen by bacteria, Biochem.
J., 23:196.5-1972, 1931.

190 Transformation of Nitrogen

2. Bonazzi, A., On nitrification. V. The nicclianism of ammonia oxidation,
/. Bact., 8:343-363, 1923.

3. Bright, J. W., and Conn, II. J., Ammonification of manure in soil, N. Y. Agr.
Expt. Sta. Tech. Bull. 67, 1919.

4. Corbet, A. S., The formation of hyponitrous acid as an intermediate com-
pound in the biological or pliotochcmical oxidation of ammonia to nitrous
acid, Biochem. J., 28:1575-1582, 1934.

5. Dhar, N. R., Bhattacharya, A. K., and Biswas, N. N., Influence of liglit on
nitrification in soil, /. Indian Chem. Soc, 10:699-712, 1933; Nature, 133:
213-214, 1934.

6. Dhar, N. R., and Rao, G. C, Nitrification in soil and in atmo.sphcre. A
photochemical process, J. Indian Chem. Soc, 9:81-91, 1933.

7. Doryland, C. J. T., Tlie influence of energy material upcm the relation of
soil microorganisms lo soluble plant food, N. Dakota Agr. Expt. Sta. Bull.
116, 19J6.

8. Gainey, P. L., Parallel formation ol carbon dioxide, ammonia, and nitrate
in soil, Soil Sci., 7:293-311, 1919.

9. Kluyver, A. J., llw Chemical Activities of Microorganisms, University of
London Press, 1931.

10. McLean, II. C, and Wilson, G. W., Ammonification studies with soil
fungi, N. J. Agr. Expt. Sta. Bull. 270, 1914.

11. Ncller, J. R., Studies on the correlation between the production of carbon
dioxide and the accumulation of ammonia by soil organisms, Soil Sci., 5:225-
242, 1918.

12. Osborne, T. B., The Vegetable Proteins, Longmans, Green & Co., London,
1924.

13. Rao, G. G., and Dhar, N. R., Photosensitized oxidation of ammonia and
ammonium salts and the problem of nitrification in soils. Soil Sci., 31:379-
384, 1931; 38:143-159, 183-189, 1934.

14. Taber, S., The production of saltpeter in the south during tlic (>i\il War,
Science, 96:535-536, 1942.

15. Temple, J. C., Nitrification in acid or non-basic soils, Cleorgia Agr. Expt.
Sta. Bull. 103. 1914.

16. Thorne, C. E., Farm Manures, Orange Judd Publishing Co., New York, 1914.

17. Voorhees, E. B., and Lipman, J. G., A review of investigations in soil bac-
teriology, U. S. Dept. Agr. Office Expt. Sta. Bull. 194, 1907.

18. Waksman, S. A., and Lomanitz, S., Contribution to the chemistry of decom-
position of proteins and amino acids by various groups of microorganisms,
J. Agr. Research, 30:263-281, 1924.

19. Waksman, S. A., and Starkey, R. L., The decomposition of proteins by
microorganisms with particular reference to purified \egetable proteins,
J. Bad., 23:405-428, 1932.

20. Workman, C. H., and Wood, II. G., On the metabolism of bacteria, Botan.
Rev., 8:1-68, 1942.

♦o* .

Nitrogen Fixation — Nonsynibiotic

Nitrogen Fixation in Nature

The supply of fixed nitrogen in the soil is very limited, ranging
normally from less than 0.1 to 0.2 per eent, and higlier in e.xeeptional
cases. Rainfall brings down small quantities of nitrogen that have
been fixed by electric discharges in the atmosphere. These are
chiefly in the form of nitrogenous oxides. The chemical and physico-
chemical fixation of nitrogen, through the agency of sunlight, for
example, may also be considered of very limited importance. The
major part of the elementary nitrogen that finds its way into the
soil and that is used for synthesis of plant and animal life is due
entirely to its fixation by certain groups of microorganisms.

Two major groups of bacteria, usually designated as nonsymbiotic
and symbiotic forms, are primarily concerned in this process. Aside
from these, there is also a limited fixation of nitrogen by a variety of
different bacteria and fungi, and especially by blue-green algae. The
capacity of nonsymbiotic bacteria to fix atmospheric nitrogen and
the amount of nitrogen fixed depend largely upon the nature and
concentration of the available energy. Since soil humus cannot be
used as a source of energy and since nitrogen fixation is inhibited by
the presence of available forms of nitrogen in the soil, the significance
of the nonsymbiotic organisms in normal soil is still a matter of
speculation. On the other hand, the fixation of nitrogen through
the symbiotic action of leguminous plants and bacteria that grow
in the plant roots and produce nodules is of great economic im-
portance in agriculture.

The root-nodule associations were the first to be recognized for
their ability to fix atmospheric nitrogen. The ability of leguminous
plants to improve the soil by increasing its supply of available nitro-
gen has been known for more than two thousand years. The role of
the bacteria in the process was established more than six decades

191

192 Nitrogen Fixation— Nonsymbiotic

ago, but it is only within the last decade or two that the true bio-
logical nature of the process has been established. Numerous other
claims concerning the ability of various organisms to fix atmospheric
nitrogen have been questioned. An organism is considered unable
to fix nitrogen if no increase in combined nitrogen can be demon-
strated by chemical analysis. Even then, if such an increase can be
demonstrated, the importance of the reaction in the soil itself may
still be open to question.

Berthelot claimed in 1885 to have demonstrated that, when a soil
is exposed to the air, its nitrogen content gradually increases and the
fixation of nitrogen is biological in nature. This claim was not sub-
stantiated. It was not until six years later that the capacity for
nitrogen fixation by nonsymbiotic organisms was established by Wino-
gradsky. Clostridium pasteiiriamim, an anaerobic organism belong-
ing to the group of butyric acid bacteria, was the first organism to
be found capable of bringing about an increase in the amount of
combined nitrogen in the medium. An available source of energy
was required for this purpose. A definite ratio was found to exist
between the carbohydrate consumed and the amount of the nitrogen
fixed.

Following Winogradsky's work, Beijerinck demonstrated in 1901
that nonsymbiotic nitrogen fixation can be carried out by aerobic
bacteria belonging to the genus Azotohacter. Other organisms, desig-
nated as Graniilobacter, were also found capable of fixing atmos-
pheric nitrogen. In addition to these, numerous other bacteria
in the soil were found capable of fixing small amounts of nitrogen
on artificial culture media, especially when freshly isolated from
the soil.

It has been claimed that various other organisms, in addition to
the bacteria, have been found capable of fixing varying amounts of
atmospheric nitrogen. These organisms ranged from different groups
of algae and fungi to a variety of animal forms. Most of these claims
have remained unsubstantiated. However, some of the blue algae
and some of the purple ( nonsulf ur ) bacteria have been found capable
of fixing molecular nitrogen.

Classification of Nitrogen-Fixing Organisms

The nitrogen-fixing bacteria recjuire sources of energy that they
are able to obtain from certain organic compounds of carbon, which
are also used for cell svnthesis. These organisms can be classified

Anaerobic Bacteria 193

on the basis of their abilit\ to iitili/.c the available sources of energy
in a nonsymbiotic manner. Other organisms are able to obtain the
carbon for their energy and for cell synthesis from the growing plant
with which they live symbiotically. These organisms are not obli-
gate, so far as the nitrogen is concerned, since they are also able to
obtain their nitrogen from organic or inorganic compounds.

I. Nonsymbiotic nitrogen-fixing bacteria.

1. Anaerobic organisms.

a. Clostridium pastcttriantim, comprising the group of non-starch-fer-
menting type of Clostridia.

b. Bacillus saccharobuitjricits, comprising the starch-fermenting Clostridia
and occasional plectridia.

c. Plcctridium group, including the starch-fermenting Tlcctridium,
which differ from the plectridia of the previous group by forming
long, slender, often curved rods, with thick oval spores as their ex-
treme ends, and by being more proteolytic and less fermentati\e in
nature.

d. But>l-alcohol-forming group, morphologically related to the second
group of starch-fermenting Clostridia.

2. Aerobic organisms.

a. Azotohacier, comprising fi\e distinct species: Az. cJiroococcum, Az.
beijerinckii, Az. vinelandii , Az. agilis, and Az. indicum.

b. Diplococcus pneumoniae, Aerobacter aerogenes, and other non-spore-
forming bacteria.

c. Bacillus astcrosporus and other spore-forming bacteria.
II. Symbiotic nitrogen-fixing bacteria.

1. Bacteria living in the roots of leguminous plants.

2. Bacteria living on and in the roots of nonleguminous plants.

3. Bacteria li\ing in the leaves of certain plants.
III. Nitrogen fixation by blue-green algae.

Anaerobic Bacteria

The nitrogen-fixing capacity is well distributed among the an-
aerobic butyric acid bacteria but to a varying degree.

The number of nitrogen-fixing Clostridia in the soil has been found
to be greater than 100,000 per gram. They are much more abundant
than the members of the Azotohacier group. This led various inves-
tigators to conclude that the genus Clostridium rather than Azoto-
hacter is the most important group of nonsymbiotic nitrogen-fixing
bacteria. Diiggeli reported 100-1,000,000 anaerobic bacteria and
0-100,000 aerobic nitrogen-fixing bacteria per gram of soil. Plots
receiving sodium nitrate as a source of nitrogen contained 10,600-
12,000 cells of Clostridium and 4,900-6,300 Azotohacier. Plots re-
ceiving no nitrogen, but potassium and phosphorus fertilizers, con-

194 Nitrogen Fixation— Nonsymbiotic

tained 1,120,000 Clostridiwn and 98,700 Azotobacter cells per gram
of soil.

It is essential to keep in mind that, out of a hundred living cells of
CI. pasteurianwn found in a culture, only very few are able to de-
velop into colonies or give positive growth on artificial culture media.
For this reason, the abundance in the soil of anaerobic bacteria
capable of fixing atmospheric nitrogen must be considered very ex-
tensive. Further, CI pasteurianwn is found in soils that are much
too acid for the favorable development of Azotobacter, in the growth
of which an acid reaction becomes a limiting factor. This tends to
add further weight to the claim of the potentially greater importance
of the anaerobic than the aerobic bacteria as nonsymbiotic nitrogen-
fixing organisms in the soil.

When freshly isolated from the soil, the Clostridia fix more nitro-
gen than after they have been cultivated for a long time in artificial
media. Cultures kept in collections for a long time can be invig-
orated by growing them in liquid media to which enough ammonium
sulfate is added to offer the organism less nitrogen than is needed
for the complete decomposition of the sugar. By transferring such
cultures, when gas formation ceases, to fresh media, normal growth
and nitrogen fixation are obtained.

Nitrogen fixation was shown by Bredemann to be a common prop-
erty of the butyric acid bacilli. Some of these organisms are strict
anaerobes, whereas others are less sensitive to oxygen. Although
more tolerant of acidity than Azotobacter, they have a definite opti-
mum at approximately neutral reaction. In pure cultures, 2-3 mg
nitrogen is fixed per gram of sugar decomposed, although some
strains may fix as much as 5 to more than 6 mg. The mechanism of
fixation is explained as a direct reduction of elementary nitrogen to
ammonia by nascent hydrogen. Bacillus asterosporus is a faculta-
tive anaerobe. It was found capable of fixing small amounts of
nitrogen, 1-3 mg per gram of sugar.

Aerobic Bacteria

When a simple medium containing tap water, 0.02 per cent
K2HPO4, and 2 per cent glucose is inoculated with soil and incu-
bated, anaerobic and certain other bacteria are obtained. When
the glucose is replaced by mannitol or by propionate of potassium
or sodium, aerobic bacteria predominate. Beijerinck first isolated
one of these organisms, which he described as Azotobacter chroococ-
cum. It is found in soils and manures. On repeated transfer to fresh

Aerobic Bacteria

195

lots of: sterile media, the organism was gradually purified from most
of the contaminating forms and was finalK isolated in pure culture
on mannitol agar.

Although five species of Azotobacter are now known, Lohnis and
Smith recognized only two species, Az. chroococcum and Az. a^lis.
They considered Az. bcijcrinckii Lipman and Az. vitreum Lohnis to
be varieties of Az. aii,i]is. Khuver has shown, however, that Az. aailis

r /

r

r /"

'- ■ ^

•'

,'-

^ f"^ ,''

r - f

( -— --

' ^

* ' ' .

r- ■■ ''

^ - ^ --

r-

r ■• ■ r-

'^ , •'

__ '-•, ''■

'

J' , -

•' '^ "^ r— f

r-

' ^ (

t"

*

/- ^ '•■-.'''',-

^

■" , _ /-

O'

i

•' ■

Fig. 81. Azotobacter chroococcum, young culture (from Beijerinck).

is quite distinct from Az. vinelandii and is found only in canal waters,
whereas the latter is characteristic of soil. Winogradsky proposed
the name Azomonas for the noncystogenic organisms found in waters.
Azotobacter indicum is certainly a distinct form.

Azotobacter represents a highly interesting group of strictly aerobic
organisms. Its temperature range lies between 10° C and 40° C,
with an optimum at 30-35 °C. It is highly sensitive to acidity, with
an optimum at pH 7-8. A large number of organic compounds can
be used as sources of energy and carbon. They include fatty acids
and oxyacids, higher and lower alcohols, and mono-, di-, and poly-
saccharides.

The fixation of nitrogen by Azotobacter is brought about by a sys-
tem of enzymes designated as "azotase." Nitrogenase, one of the
components of this system, is capable of combining directly with
elementary nitrogen. This enzyme complex has not been isolated,

196

Nitrogen Fixation— Nonsymbiotic

although there is found in the hterature a statement by Bach that
such enzyme complexes can be obtained. Apparently the production
and activity of this enzyme are inseparably linked with the synthesis
of cell substance. Burk designated the enzyme as "growthbound,"
for which the name "phyo-enzyme" was suggested. The enzyme
nitrogenase requires a certain concentration of calcium (or stron-
tium) for its activity. It is incapable of exerting any effect at reac-

FiG. 82. Azotohactcr agilis, showing Hagella stain (from Beijerincki

tions below pH 6. It is strongly activated by minute concentrations
of molybdenum and to a lesser degree by vanadium.

The primary product of nitrogen fixation is not fully known, as
shown later. Winogradsky reported it to be ammonia. Virtanen
and others have said that an oxime compound seems involved. Most
of the fixed nitrogen is present in the cultures as cell material. Small
quantities of combined nitrogen may be secreted into the medium as
long as growth takes place. When the medium is exhausted of
nutrients and growth has ceased, a rapid production of ammonia
from the cell material sets in, because of the lytic processes that
take place.

Azotohactcr can also assimilate various combined forms of nitro-
gen, such as nitrate, ammonia, and simple amino compounds. The
presence of these compounds in the medium represses the fixation
of free nitrogen. This appears to be due not so much to preferential
assimilation, as to the inactivation of the nitrogenase. Burk and

Aerobic Bacteria

197

Linc\vca\cr obscr\ccl tliis cfFcct in conci'utrations ot 0.5 iiig NO3-N
or NH4-N per 100 ml of nietliuin.

Azotohactcr transforms the carbon componnds into carl)on dioxide,
water, and cell substance. The amount of mtro<ji;en fixed is about 10

Fig. 83. Clostridium pasteiiiiamim (from Winogradsky ) .

mg per gram of sugar consumed. Other carbon compounds are util-
ized in proportion to their availability, as shown in Table 41. The

Table 41. Nitrogen Fixed by Azotohader chroococcian with Different Sources

OF Energy

Milligrams of nitrogen fixed per 100 gm of carbon available.

Pine needles

57.3

Plant roots

and stubble

596.8

Oak leaves

126.9

Lupines

711.5

Maple leaves

89.5

Alfalfa

319.5

Wheat straw-

325.4

Clover

1237.9

Corn stover

280.3

filuco.se

14.56.5

efficiency of nitrogen fixation also depends upon the type of organ-
ism, experimental conditions, and presence of contaminants, as
shown in Tables 42 and 43.

Azotobacter is widely distributed in the soil, although only a rela-
tively limited number of cells are found in any one soil. The reac-
tion of the soil, the abundance of organic matter, the concentration
of certain mineral elements, notably phosphate, and the absence of

198 Nitrogen Fixation— Nonsymbiotic

Table 42. Nitrogen Fixation by Strains of Azofobacter with and without a
Contaminant (from Lind and Wilson)

Nitrogen Fixed f

Strain Pure Culture Plus

Hours No.* of Azotobadcr Contaminant

Azotobactcr and BacUhis from Mixed Culture
21 1 6.9 11.1

2 9.5 16.2

3 8.2 16.2

12 1 7.0 19.8

2 (5.7 27.2

3 8.3 26.4

12 1 5.0 20.1

2 8.9 22.3

3 7.3 21.7

41 1 16.5 31.4

2 20.0 32.5

3 18.9 32.5

12 2 10.8 18.8

3 10.4 26.7

Azotobactcr and Clo.stridlum pa.sfrurianuin
12 2 (5.7 19.5

12 3 8.9 21.0

* Strain 1 was Azotobactcr stock culture; strains 2 and 3 were pure Isolates of .(;.')/()-
bacter from mixed culture.

t Values in milligrams N per 100 ml; this includes nitrogen in inoculum, which was
1 ml in 15 ml.

antagonistic or competing agents are among the factors that control
the presence and abundance of this organism in the soil.

Out of 105 soil samples examined, Burri found Azotohacter miss-
ing in 34, chiefly heavy clay soils. Jones and Murdoch isolated Azo-
tohacter from 9 out of 17 soil types examined, and from 22 out of 29
soil samples representing 9 types. The maximum number of Azoto-
hacter cells found per gram of soil was 18. Rossi reported an average
of 1,815 cells per gram of Italian soil, the number ranging from 0 to
21,400. Swaby found Azotohacter in 26 per cent of all soil samples
examined, usually 30 cells per gram of soil. Clostridium hutijlicum
was found, however, in most of the soils, usually 100 spores per
gram.

Aerobic Bacteria 199

Tahi.k 4;J. Efkixt ok Soiuc k ok Iuon on Xituockn Fixaiion uy Pikk and MrxKi)
CuLTUHES OF Azolohaclrr (from Lliid and Wilson)

Nitrogen J

Fixed *

Pure

I'lns

Source of Iron

Hours

Culture Coulaminant

New humate

8

1.9

1.6

17

10.3

9.8

23

18.4

23.8

Old humate

16

5.9

II. 0

New humate

16

13. 1

l(i.4

Old humate

18

G.5

13.4

Old humate

23

10.5

25.8

9.8

25.3

Old Immate + Fe

26.(5

27.8

27.2

26.7

* Values in milligrams N per 100 ml; this includes nitrogen in inoculum, which was
1 ml in 15 ml.

Azotobacter usually cannot develop in a soil having a pH of less
than 6.0. As soon as the reaction of the soil is adjusted by means of
lime, so that the pH becomes higher than this minimum, a txpical
Azotobacter flora will develop. The carbonate-phosphate ratio in
soil was found to influence markedly the development of this organ-
ism in soils of different reaction. In Malayan soils, however, certain
strains of Azotobacter have been found capable of growing at a
wide pH range, 3.6 being the limit on the acid side. Starkey and
De isolated an organism {Az. indictim) capable of growing at reac-
tions lower than pH 6.0.

Abundance of Azotobacter in the soil was found to be influenced
by cropping and by fertilizer treatment, the numbers being higher
in unfertilized than in fertilized soils.

Azotobacter may live symbiotically with algae, especially with
Nostoc and Anabaena, as well as with other bacteria. The quanti-
ties of nitrogen fixed by this association are frequently considerable,
as pointed out in Tables 42 and 43. The symbiotic action between
CI. pasteurianum and Azotobacter, whereby the latter uses up the
oxygen, making conditions favorable for the former, has also been
demonstrated. The various acids produced by Clostridium are
neutralized by the soil bases and can be utilized by Azotobacter as

200

Nitrogen Fixation— Nonsymbiotic

sources of energy; this symbiotic action leads to a niaxiinuni economy
in the utihzation of energy. In the presence of RJiizo])ium legumino-
sartim, Azotohactcr was found to fix luore nitrogen tlian alone. Sym-

FiG. 84. Clostridium pasteurianiim, showing spore formation (from Wino-

gradsky ) .

biosis between pure cultures of cellulose-decomposing and nitrogen-
fixing bacteria has also been reported.

Blue-Green Algae

The ability of blue-green algae to fix atmospheric nitrogen is now
definitely established. Drewes first observed this phenomenon in
1928 for species of Anahaena and Nostoc. His discovery was con-
firmed by Allison and Morris in 1930. Certain strains of Myxophy-
ceae, isolated from nitrogen-deficient warm springs, were also found
to be capable of utilizing atmospheric nitrogen. A species of Nostoc
punctifonne was isolated from different host plants and found ca-
pable of fixing up to 1.95 mg of nitrogen per 50 ml of culture solution,
in presence of a suitable sugar source. A culture of Nostoc mus-
corum isolated from the soil was found capable of obtaining both
its carbon and its nitrogen from the air; it fixed as much as 10 mg
of nitrogen in 45 days and 18 mg in 85 days per 100 ml of medium
from carbohydrate; in the dark, it fixed 10-12 mg of nitrogen per
1 gm of glucose consumed.

Mecluuiisni of Nitrogen Fixation

201

Mechanism of Nitrogen • Fixation

The amount of nitrogen fixed by various bacteria depends upon
the nature of the energy source, the presence of available nitrogen
and minerals, the soil reaction and other environmental conditions,
and the presence of various specific bacteria. Some species utilize
one source of energy more readily than another. Lipman recorded

1

1 1 1

1

'

1 1 1

/

/

/ —
/
/

~ Heat of combustion -V f

C — ^

\i ^-«^^^ /

^-^""""^ /

^^^ y

y

^^r

^

X

/'^^.^itrogen fixed,
^^ Gainey's data

y

~ /

"~

-/ ^'^

•^ N^Nitrogen fixed,

Mockeridge

—

^ ^^y^

— ~.y^ /

—

■^ y'

y

y

y 1

1 1 1

'' 1

1 1 1

-6

4 a>

-3

- 1

3
CD

Fig. 85. Comparison between nitrogen fixation and heat of combustion per gram
of organic acid, used as a source of energy (from Gainey).

an increase in the amount of nitrogen fixed with an increase in
molecular weight of fatty acids, in the form of sodium salts, includ-
ing acetic, propionic, and butyric; the next number of the homologous
series (valerianic acid) presented a poor source of carbon; the sodium
salts of succinic and citric acids were not utilized at all.

With glucose as a source of energy, Az. chroococcum was reported
to liberate 70 per cent of the carbon as CO2; 12 per cent was assimi-
lated in the bacterial cells, and 18 per cent was left among the vari-
ous decomposition products other than CO2. These were made up
of ethyl alcohol, aldehyde, and formic, acetic, lactic, tartaric, and
other acids. The bacterial cells contained 30 per cent protein, a
considerable amount of fat, and phosphatides.

202 Nitrogen Fixation— Nonsymbiotic

Kostytschev and Winogradsky demonstrated that ammonia is pro-
duced in cultures of Azotobocter. They conckided that this am-
monia is the first stage in the fixation of nitrogen b)- bacteria. Burk
found that ammonia was present in the older cultures and concluded,
therefore, that it is rather a secondary decomposition product. The
inability to find ammonia in \oung cultures of the organism speaks
against its being the first step in the fixation process.

Blom suggested the following reactions as explanations of the
mechanism of the fixation of nitrogen, through the h\drox\lamine
stage. This was produced by cultures of Az. ogilis, iron serving as a
catalyst:

1. N^N (atmospheric) :;=i N^X (solution).

2. 2(Cat. Fe++) + N^N ^ (Cat. Fe++)2-N=^N.

3. (Cat. Fe++)2-N=N + iR.O ^

(Cat. Fe++)2 • HOXH— HXOH.

4. (Cat. Fe++)2- HOXH— HXOH + ^2H+ ^

2(Cat. Fe+++) + '^HOXH..

5. (Cat. Fe+++) + H ^ (Cat. Fe++) + H+.

The hydrox) lamine, once produced, will iuteract with oxalacetic
acid to give rise to oximes. which are changed to aspartic acid, as
shown later.

According to Winogradsky, both anaerobic and aerobic bacteria
produce ammonia out of the nitrogen gas and nascent hydrogen with
which they come in contact. In the case of the anaerobes, the
hydrogen is formed during the butxric acid fermentation. In the
case of Azofobacter, an enzyme, azoh)'drase, concerned in the am-
monia synthesis is believed to be produced. A part of the am-
monia is immobilized by the growing cells, and a part is excreted
into the medium. The molecular nitrogen acts as a h\drogen ac-
ceptor, the action of the enzyme continuing even after the death of
the cells.

Wieland also considered that the action of the hydrogen acceptors
formed in the cells of nitrogen-fixing bacteria does not depend upon
oxygen for hydration, but rather upon the molecular nitrogen with
which the hydrogen forms ammonia, perhaps through the hydrazine
stage in a manner similar to the Haber synthesis.

Virtanen found aspartic acid in young cultures of Azotobacter
before ammonia could be detected; this led him to conclude that

NU'fhaiiisin of Nitrogen Fixation 203

amino acid is tlu' first product of nitrogen fixation. This is brought
out in tlu- iollowiuu; series of reactions: '

X2 -* •■' -^ XH.OII

Hydr<)\.\l:uninc

C0H12O,, -> HOOCCOCHo COOH

Oxalacetic acid

HOOC C(X0H)CH2 COOH -^ HOOC CIKMLjCH. COOll

Oxiiue Asijartic acid

HOOC CH(XH2)CH2 COOH + HaC ( O COOH ->

Aspartic acid Pyruvic acid

HOOC CO CH2 COOH + CH3 CHIXH.) COOH

Oxalacetic acid Alanine

The first step in the reaction has also been presented by Virtanen as
follows:

Xo ^ HX— XH ^ XH. or X.> -> HX--XH -^> XH,OH

11"'

OH OH OH

According to Wilson and his associates, the biochemical nitrogen
fixation b\- Azotobacter has much in common with that of the
legumes. The following points were recognized:

1. Hydrogen and carbon monoxide are specific inhibitors for both
types of fixation.

2. Aspartic acid, possibK with an oxime as a precursor, occupies a
key position.

0. MoKbdenum acts as a specific catalyst in Azotobacter and
appears to have a similar effect in legumes.

4. Azotobacter produces a hydrogenase which seems to be con-
nected with the nitrogen fixation; this enzyme is not found in nodules
or in cultures of the nodule bacteria in vitro.

5. Nitrogen fixation by Azotobacter has, except in one species, an
optimum at /;H 7.0-7.5, and ceases at pW 6.0, which seems to rep-
resent the normal reaction of the nodule tissue and therefore pre-
sumably also the optimum for the s\ nibiotic process of fixation.

6. Azotobacter fixes nitrogen only during active cell multiplication,
and uses virtually all the fixed nitrogen for cell synthesis. This does
not seem to appK' to the legmnes, where the fixation appears more
like a kind of respiration process which results in a steady transfer
of some 80-90 per cent of the fixed nitrogen from the nodules to the

204

Nitrogen Fixation— Nonsymbiotic

rest of the host plant. Therefore, symbiotic nitrogen fixation requires
a much smaller expenditure of energy. Azotohacter consumes at its
optimal rate of growth at least 40-50 units of carbohydrate, and
usually twice as much, per unit of nitrogen fixed, whereas Bond cal-
culated that the corresponding figure for the root-nodule bacteria in

a bed

Fig. 86. Growth of Azotohacter in soils treated with starch to test for deficiencies
in available nutrient elements. Upper row, soil deficient in phosphate; lower
row, soil not deficient in either potash or phosphate. Left to right, a, check,
nothing added; h, potasli added; c, phosphate added; d, phosphate and potash

added (from Sackett).

soybeans is only about 15 units, and that the total respiration of the
nodules consumes some 16 per cent of all the carbohydrate produced
in photosynthesis (Jensen).

Effect of Humus on Nitrogen Fixation

The beneficial action of humus on nitrogen fixation is frequently
ascribed to its inorganic constituents, particularly to its content of
aluminum and silicic acid. This assumption is confirmed by the
following two facts: («) artificial humus has no such effect; (h) the
source of the natural humus influences markedly the degree of its
beneficial action. The claim that the action of the humus is due to
its inorganic constituents has been further substantiated by the fact
that purified humates do not possess any stimulating effect. The
role of the colloid is probably due chiefly to its catalytic action and
its protective action against poisons; the protective action of the

Effect of Hiiniiis on Nitrogen Fixation 205

colloid has also bci-n astrihcd to the (listribution ol the phosphorus
and to the buffering effect upon the rcact'ioii ol the nieihum.

Hurk conchided that the favoral)le effect of hunuis upon the nitro-
gen-fixation process is chie entireU to its iron content. Accorch'ng to
Hirch-Hirschfeld, soil extract contains se\'eral components that have
a fa\'orable effect upon growth and nitrogen fixation by Azotohacter.
These components are of botli an organic and an inorganic nature,
the growth stimulation being due to the organic complex. Molybde-
num cannot take the place of soil extract, although both favor about
alike the amount of nitrogen fixed per unit of sugar consumed.

Burema and Wieringa demonstrated that the role of molybdenum
in the fixation of nitrogen is that of a reducing agent. Less molybde-
num is required for the reduction of nitrate than for the reduction
of free nitrogen. Jensen reported that Az. indicum requires molyb-
denum for nitrogen fixation; molybdenum could not be replaced by
vanadium.

The nitrogen fixed through nonsymbiotic processes can at best
restore only a part of the losses of nitrogen from soil by crop re-
moval and by leaching. The common estimates are 20-50 pounds
fixed per acre annually, but the actual amounts may be much smaller.

The frequently expressed opinion that soils from arid climates
have an extraordinary nitrogen-fixing power and may be employed,
by the use of crop residues by nonsymbiotic nitrogen-fixing organ-
isms, for cereal cultivation without depletion of nitrogen, has been
denied by Jensen. In Australian wheat soils no gain at all is usually
expected, and only under exceptionally favorable circumstances was
a fixation obtained corresponding to one-third of the nitrogen re-
quirements of the crops on wheat land worked on the usual wheat-
fallow rotation. The activity of nonsymbiotic nitrogen fixation in
nature appears to be largely confined to uncultivated soils where no
crops are carried away and the vegetable debris is allowed to de-
compose.

Jensen further concluded that the practice of growing wheat
alternating with fallow and without use of nitrogenous fertilizers
is to be regarded as a gradual consumption of the nitrogen reserves
of the soil, from which some nitrate is produced during fallowing.
The nonsymbiotic nitrogen fixation and the effect of the rain will
compensate for this loss only incompletely. If continued, it must
in time lead to permanent loss of fertility. The growth of leguminous
crops in the rotations and the judicious application of nitrogenous
fertilizers are the logical correctives.

206 Nitrogen Fixation— Nonsvni])iotic

Further information on the inability of nonsymbiotic nitrogen-fix-
ing bacteria to supply available nitrogen when natural plant residues
high in cellulose are added to the soil is found in Tables 44 and 45.

TaBLK 44. I.NKH'KNCK OF Si (iAIi ll'ON CuDI' ^'IK1,I) AND NiTllOfiKN CoNTKXT OF ( 'ifOI'

(from .\. Kocli)

(irams ol' dry suhstaiicc per \m>\, lor IS-ycar ix'rioil.

Control (ihicosc * Sucroso Sucrose

Crop yield 4^20 . .J 4S() . 7 49^2 . i oii . 6

Excess over control (iO.'-l li.l ]'ii.l

IVlilliiiranis of Nitrofjcn in Crop
Nitrogen '•2,.'5(i:5 ^2,!)1(> .3, 000 .'5,040

Excess over control .m.S 0.'57 1,'28.'5

* .'500 gni of sugars added, over the IS-year |)eriod, to llic treated j)ols.

Tabi.e 4.5. IxFLTfEN'CK OF Celu'lose I'pox (]rop Yikld (froiH A. Kr)cll)
(iranis of dry suhstance jx-r i)ot, for ;5-year periods.

Paper,

V2() gm

Manure

Paper,

+ Manure

Infusion

Years

Confroi

1^20 gni

Infusion

-Mone

1911 1, '5

(IS.. '5

1^2. S

17.9

()7.0

1!)14 10

00.0

81.7

87.3

(H . 8

1917 19

Oti.O

77.4

8-2.8

09.9

19'20 "21

0.>.'2

71.1

7-2.8

07.0

Total cro]) -2.">9.;) '24f5.0 •200.8 '2fifi.7

With glucose and sucrose as sources of energy, considerable nitrogen
was fixed. With cellulose, on the other hand, there was no increase
in the nitrogen supply even after 11 years' treatment. Unfortunately,
plant residues that usually find their way into the soil are poor in
sugar and rich in cellulose.

Selected Bibliography

1. Allison, F. K., llooNcr, S. 1^, antl Morris, II. J., Pli\siological studies with
tlie nitrogen-fixing alga Nostur mitsconim, Botan. (laz., 98:433-463, 1937.

2. Bond, G., Quantitati\e observations on the fixation and transfer of nitrogen

Selected Bil)liogiaphy 207

in tlic sonIk-uh with spivial irit-reiKf to tlu" iiuxlianisiii ot translcr of fixed
nitrogen Ironi hacillus to liost, Aim. Botamj^ 50:559-578, 1936; Syinhiosis
of Icguniinons plants and nodule bacteria. I. Ol)si'r\ations on resjiiration
and on the extent of utilization of host carholndratcs In- tlie nodule bac-
teria. Ann. Botany, N.S., 5:313-337, 1941.

3. Burk, D., and Horner, C. K., The origin and significance of annnonia
formed by Azotobacter, Soil Set., 41:81-132, 1936.

4. Burk, D., and Burris, R. If., Biochemical nitrogen-fixation, Ann. Rev. Bio-
ehem., 10:587-618, 1941.

5. Jensen, H. L., Contributions to the nitrogen econonu' of Australian wlieat
soils, with particular reference to \e\v South Wales, Proe. Linnean Soe.
N. S. Wales, 45:1-122, 1940.

6. Jensen, H. L., Symbiotic nitrogen-fixation. Austral. J. Sci., 6:162-165, 1944.

7. Jones, D. H., and Murdoch, F. G., Quantitati\e and (jualitatixc bacterial
analysis of soil samples taken in fall of 1918, Soil Sei., 8:259-267, 1919.

8. Kluy\er, A. J., and \an Reenen, W. J., Uber Azotohaeter af^ilis Beijerinck,
Arch. Mikrob., 4:280-300, 1933.

9. Lind, C. J., and Wilson, P. W., Nitrogen-fixation by Azotobacter in asso-
ciation with other bacteria, Soil Sci., 54:105-111, 1942.

10. Starkey, R. L., and De, P. K., A new species of Azotobacter, Soil Sci., 47:
329-343, 1939.

11. Stumbo, C. R., and Gainey, P. L., An apparent induced loss of nitrogen-
fixing ability in Azotobacter, J. Agr. Re.search, 57:217-227, 1938.

12. Van Xiel, C. B., A note on the apparent absence of Azotobacter in soils,
Arch. Mikrob., 6:215-218, 1935.

13. Mrtanen, A. I., Cattle Fodder and Human Nutrition, with Special Reference
to Biological Nitrogen Fixation, Cambridge Uni\ersity Press, 1938.

14. \'irtanen, A. I., Biological nitrogen-fixati(m, Ann. Rev. Microb., 2:485-506,
1948.

15. Wilson, P. W., The Biochemistry of Symbiotic Nitrogen Fi.xation, Uni\ersity
of Wisconsin Press, Madison, Wis., 1940.

16. Wilson, P. W., and Lind, C. J., Carbon monoxide inhibition of Azotobacter
in microrespiration experiments, /. Bact., 45:219-232, 1943.

9

Nitrogen Fixation — Symbiotic

Early Observations

Many centuries before the discovery was made that bacteria exist
in the root nodules of leguminous plants and that these bacteria live
in symbiosis with the plants, thus enriching the soil with combined
nitrogen, the practical agriculturist came to consider the growth of
legumes on his land as equivalent to manuring or fertilizing the soil
for the succeeding crop. The use of leguminous plants for green
manuring was described in great detail by Greek and Roman writers,
notably Virgil, Varro, and Columella. Directions were given for
preparing the soil and for sowing, cultivating, and harvesting the
crop. Lupines, vetches, and alfalfa were frequently mentioned in
these books as specific crops to be turned over when the plants were
young.

With the beginning of the nineteenth century, when the basis was
laid for modern agricultural science, more accurate information
gradually began to accumulate. Sir Humphry Davy, in his book
Agricultural Chemistry, published in 1813, observed: "Peas and
beans in all instances seem well adapted to prepare the ground
for wheat . . . they contain a small quantity of a matter analogous
to albumen; but it seems that the azote which forms a constituent
part of this matter is derived from the atmosphere."

These observations were fully borne out in the classical studies of
Boussingault, published in 1837-1838. This French agronomist and
chemist was the first to develop systematically the idea of nitrogen
nutrition of leguminous and cereal plants. A typical field experiment
on crop rotation is shown in Table 46. Boussingault established the
fact that, when clover is grown in unmanured soils, there is a consid-
erable gain of nitrogen; wheat, on the other hand, showed no gain
or loss of nitrogen. He suggested that leguminous plants assimilate
nitrogen from the atmosphere, whereas cereal plants cannot do so.

208

Earlv Observations

209

'r\iii.K K>. ( 'uoi'-Korv ri<)\ Kxphuimknt hk Uoissinc; aii/i

Rotation

Nitrogen

Gain

1

-

3

4

5

0

In
Crop

pounds
per
acre

In
Manures

Used

pounds

per

acre

Total

pounds
per

acre

Per
Year

pounds
per
acre

Potatoes

Wheat

Clover

Wheat

Turnips

229

185

44

9

Mangel beets

Wheat

Clover

Wheat

Oats

231

185

4()

9

Potatoes

Wheat

Clover

Wheat

Peas

Rye

323

223

100

17

Fallow

Wheat

Wheat

80

76

4

1

Alfalfa

Alfalfa

Alfalfa

Alfalfa

Alfalfa

Alfalfa

980

205

775

130

Boussingault made an effort to repeat these experiments under more
carefully controlled conditions. He ignited the sand, thereby killing
the bacteria, and found that neither cereals nor legumes were capable
of assimilating nitrogen from the atmosphere.

The German chemist Liebig (1843) could not accept the idea that
atmospheric nitrogen can be assimilated by plants. The beneficial
effects of leguminous plants were explained by the fact that the
plants form a large leaf surface and thus expose a greater area for
absorption of ammonia from the atmosphere. The results of Bous-
singault's rotation experiments, which occupied sixteen years, were
considered to be due to errors in the analysis of the manure. Since
the farm manure was dried in a vacuum at 110°C before being
analyzed for nitrogen, at least half the ammonia nitrogen could have
been volatilized. Liebig suggested that, had such errors been taken
into account, the results would lose much of their significance.

To prove or disprove Liebig's ideas, Lawes, Gilbert, and Pugh, of
the Rothamsted Experimental Station, began in 1857 a series of
crucial experiments. They were so careful in handling the soil that
they destroyed the organism fixing the nitrogen symbiotically with
leguminous plants. They thus failed to become the discoverers of
the symbiotic fixation process. In absence of bacteria, the legume
behaved like cereals. This phenomenon was later confirmed by a
number of other investigators who showed that legumes do not
fix nitrogen when the soil has been ignited but do fix nitrogen in
unignited soil. Schulz-Lupitz grew lupines for fifteen consecutive
times, without application of nitrogen fertilizer and without diminish-

210

Nitrogen Fixation— Symbiotic

ing yields. Much higher yields were obtained when cereals followed
lupines than when the cereals were grown on the same soil not
preceded by a leguminous crop; the nitrogen content of the soil
was thereby found to increase.

Role of Bacteria in Fixation Process

The presence of nodules on the roots of leguminous plants was
known long before their significance was recognized. At first they

S\

.v

J ^'.....^

FiG. 87. 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 Hellriegel and Wilfarth,

after Russell).

were looked iii)()n as root galls. Althougli Lachmanu observed in
1858 that motile ])acteria cause the formation of tlie nodules and that

Kolo ol Bacteria in I'^'xation Fiocrss

211

these are resp()n.sil)le for nitrogen fixation, W'oronin, who also lonnd
in 1866 that the nodnles eonsist of bacteVia, eonsidered the iKxhilcs
pathologieal ontgrowths. In 1879, Frank demonstrated that th(>
formation of nochiles ean he prevented 1)\- sterih/ation ol soil; he
snggested that the nodules are eansed in ontside infeetion.

Atwater and ^^'oods, working in C'onneetieut, reeogni/cni in 1884
the possihilitx that both plants and baeteria are faetors in the process
of fixation of atmospheric nitrogen. Soon afterward, Hellriegel and
^^'i]farth in German) demonstrated tliat the nodules on the roots of
k'guminons plants are due to bacterial infection, and that this is a

Fig. 88. Life histon' of root-nodule bacteria in the tissue of the alfalfa nodule

( from Thornton ).

beneficial process, since within these nodules the bacteria fix the
atmospheric nitrogen. Plants could be grown on artificial soils
containing only traces of combined nitrogen, provided the mineral
elements necessary for the nutrition of the plant were present and
nodules were formed. In absence of nodules, the plants were unable
to utilize the atmospheric nitrogen for their growth. When sterilized
soil was treated with a suspension of fresh soil, nodules were formed
and the plants grew normally. The growth of the cereals depended,
however, on the nitrate content of the soil. These results were soon
confirmed by Lawes and Gilbert and others.

The causative organism responsible for nitrogen fixation in pure
culture was isolated in 1888 by Beijerinck, who named it Bacillus
radicicola. He described three stages in the development of the
organism :

1. The bacterium is present in the soil in the form of small rods
which can penetrate the root hairs of the leguminous plants and
from there are transferred to the "infectious tissue."

2. The organism later changes into a motile form.

3. Within the plant tissues, the cells are transformed into bac-
teroids, which function as the symbiotic mechanism.

212 Nitrogen Fixation— Symbiotic

The mechanism of root infection by the legume organism was
studied in detail by Prazmowski in 1889. The amounts of nitrogen
obtained by the association of the plant and the bacterium were
worked out by Schloesing and Laurent. Leguminous plants were
grown in sterile glass cylinders containing sterile sand and watered
with sterile water. The composition of the gas in the cylinder was
determined. It was found that the uninoculated plants showed a
gain of only 0.6 mg of nitrogen and no nodule formation; the inocu-
lated plants, however, showed a gain of 34.1 and 40.6 mg of nitro-
gen and abundant nodule formation. These results were confirmed
by numerous investigators. It is sufficient to cite those obtained by
Virtanen (Table 47).

Table 47. Gkowth of Red Clover lv Quartz Sand (from Virtanen)
pYL 6.5; 10 plants in eadi pot; age of plant.s, 106 days.

Dry Weight

N Nutrition

Inoculation

of Plants

gm

KXO3

Not inoculated

23.78

KNO3

Not inoculated

24.07

(NH4)2S04

Not inoculated

22.32

(NH4)2S04

Not inoculated

18.00

N-free medi

ium

Inoculated

31., 38

X-free niedi

iuin

Inoc-ulated

.30.27

Different plants vary greatly in the amount of nitrogen fixed, as
.shown by Wilson (Table 48).

T.\BLE 48. Daily Uptake of Nitrogen by Various Leguminous Plants

(from Wilson)

Milligrams of N

Milligra

ms of N per

Plant

per Gram of
Dry Weight of

*"

Plant

Minimum

Maximum

Average

Nodule

Horsehean

14.0

27.7

17.1

38

Pea

5.6

10.3

8.7

98

Bean

6.9

9.8

7.8

67

Lupine

3.4

8.4

5.8

65

Vetch

3.0

3.4

3.2

80

Alfalfa

1.0

1.5

1.2

67

Red clover

0.9

1.0

1.0

55

Strain of Organisms and Nodule Formation 213

Strain ok Organisms and Nooule Formation

Numerous studies were made of strain variations of l:)acteria as
iiiHuencing the fixation of nitrogen. It is sufficient to cite the results
of Fred, Baldwin, and McCoy.

1. Different isolations of organisms from a nodule or from the soil
may varx- widely in their ability to benefit the host plant through
the fixation of nitrogen, even though nodules are readily formed. A
strain that fixes little nitrogen in association with the host is called
"poor," and one that is beneficial is considered a "good" strain.

2. The nodules formed by poor strains are usually small, round,
and white and are scattered over the entire root system. Nodules
from a good strain are fewer in number but much larger; they are
pink in color and elongated, and are located near the main roots.
This is true of certain legumes but not necessarily of others.

3. The plant species determines largely whether a given strain is
poor or good. Cultivation on certain media often causes a good
strain to lose its effectiveness. Successive passage through a host
plant modifies the effectiveness of a strain. A poor strain may im-
prove, and a good one may deteriorate.

4. Many of the strains found in the natural habitat, either in the
soil or in the nodules of wild legumes, are of the poor type.

5. Although a given host may possess nodules of both effective and
ineffective t\^pes, plants already infected with one strain of the or-
ganism resist infection of a contrasting strain to a greater degree
than do nodule-free plants.

The plants exert a chemotactic effect upon the bacteria, which
congregate around the plant roots; the bacteria, in their turn, secrete
a substance of the nature of an auxin, which causes the curling of
the root hairs of the plant. During the early stages of growth of
the plant the bacteria act as parasites and enter the host through the
root hairs or through ordinary epidermal cells. When nitrates are
present, formation of nodules is repressed. The plant may also form
a substance which inhibits the growth of the bacteria and causes
their destruction, which may explain the inefficiency of certain bac-
terial strains. On entering the root, the bacteria multiply, forming
a thread of infection, which branches out into the parenchymatous
cells of the root. The bacteria elaborate certain stimulating sub-
stances which cause the cells to enlarge. On reaching the inner cells

214 Nitrogen Fixation— Symbiotic

of the roots, the bacteria favor the multiphcation of the surrounding
cells. This leads to the formation of a young nodule, which pro-
duces a swelling on the side of the root by pushing out the overlying
cortical parenchyma and epidermis.

In some plants, bacterial infection results in a rapid division of the
infested cells, which give rise to bacteroidal tissue. In these plants
the nodules usually arise in the cambium layers.

A third type of infection is known, in which the intercellular zo-
ogloea plays the important part. The bacteria which enter the root
change into rods and multiply rapidly in the slime filament or in the
zoogloeal mass. Many of these rods change into bacteriods.

Wilson reported three types of plant response with respect to
nitrogen fixation:

1. The association of certain strains of bacteria with all the species
of host plant tested.

2. The association of certain strains of bacteria with only one
species of host, not with another.

3. The association of certain strains of bacteria with certain species
of plants, producing erratic responses due to the carbohydrate-nitro-
gen relationship in the plant.

Morphology and Life Cycle of Nodule Bacteria

The bacteria responsible for the formation of root nodules vary
greatly in size and shape. Beijerinck described the small, oval forms
as "swarmers." In the young nodules, there are present normal rods
together with large club-shaped or branching forms (bacteroids).
In old decomposing nodules, the branching forms are vacuolated,
showing small, oval, deep-staining bodies within, which are the
motile swarmers or the branching form dividing into bacilli.

The organism produces short Gram-negative rods, motile by means
of flagella when young. The bacteroids may also be formed on
artificial media, when such substances as acid phosphate, sodium
succinate and glycerol, caffeine and cumarine are present. Caffeine
and other vegetable alkaloids, like guanidine, pyridine, and chinoline,
will stimulate the formation of involution forms in pure culture. It
was suggested, therefore, that the formation of the bacteroids in root
nodules is due to the presence of alkaloids in the plant. The bac-
teroids are produced in the medium or in the nodule as a result of
specific nutrition or of unfavorable conditions. According to Zipfel,

Moi plu)l()g\ ol Nodule Ikiclcria

215

the hraiichiug lonns arc not degeneration lornis, but may be looked
upon as normal and necessary stages in the life cycle of the organism
with specific biological functions. Some in\'estigators cjuestion, how-
ever, the reproducibility of the bacteroids.

Unbanded
nonmotile rods

Motile rods

Swarmers

Banded rods
(bacteroids)

Coccoid forms

Preswarmers
Fig. 89. The life cycle of Bacilhis radicicola (from Tliornton and Gangulee).

Five stages in the life cycle of the root-nodule organism are now
recognized :

1. Nonmotile, preswarmer stage, observed in cultures kept in a
neutral soil solution.

2. Larger, nonmotile coccus, obtained in the presence of certain
carbohydrates and phosphates.

3. Motile, swarmer stage, the cell becoming ellipsoidal and devel-
oping high motility.

4. Rod form, resulting from the finther elongation of the swarmsr,
with decreasing motility.

5. Vacuolated stage produced in absence of carbohydrates, the
chromatin dividing into a number of bands. These bands become
rounded and escape from the rod as the coccoid preswarmer.

216

Nitrogen Fixation— Symbiotic

Fig. 90. Different t>iie.s of nodules of leguminous plants.

Specific Differentiation

Three groups of methods are usually employed for the specific
differentiation of the root-nodule bacteria: (1) plant inoculation,
(2) morphological and cultural studies, (3) serological and immuno-
logical reactions.

Zipfel made agglutination tests and concluded that the various
root-nodule bacteria do not represent merely varieties of the same

Specific DifFercntiation 217

species but arc distinct species. He rec(),<2;iii/.ctl six groups of organ-
isms capable of iufccting (1) lAipinus, (2') Trifolium, (3) Medicago,

(4) Pistim, (5) Falxi, and (6) PIkiscoIiis. Otlier investigators rec-
ognized nine groups of Icgiune liactcria on the basis of serological
investigations: (1) Ijipintis and Ornithopiis, (2) McIHotiis, Medi-
cago, and Trigoiu'lla, (3) Vicia (V. sativa), (4) PisiiDi, (5) Vicia
faha, (6) TrifoUum pratense, (7) Pliascohis, (8) Glycine (Soja),
and (9) Onobrycltis saliva.

Bergey placed the root-nodule organism in a separate genus,
Rliizol)ium, and divided the different forms into two species: (1)
RJi. leguminosaniin Frank, inoculating Pistim, Vicia, Lathyrus, etc.,
and (2) Rh. radicicola Beij., producing nodules on TrifoUum, Pliasc-
ohis, and others.

Fred, Baldwin, and McCoy classified these bacteria into seven
groups: (1) alfalfa group, RJi. meliJoti; (2) clover group, Rh. trifolii;
(3) pea group, Rh. legiiminosarum; (4) bean group, Rh. phaseoli;

(5) lupine group, Rh. hipini; (6) soybean group, Rh. japoniciim;
(7) cowpea group, Rliizobium sp.

Within each species, there are various strains, which differ pri-
marily in their effectiveness, or ability to fix free nitrogen in associa-
tion with the proper host plant. Various explanations have been
suggested for the specificity of the root-nodule organisms, based on
soil reaction and climate. It was at first believed that this is a case
of specific enzymes produced by the bacteria or of differences in the
root sap. The various members of each cross-inoculation group are
closely related with respect to protein characteristics of their seeds.

It was at first believed that some plants will interact with several
strains of Rhizobium, whereas other plants are limited to particular
strains. Cross-pollinating plants were said to be inoculated by more
bacterial strains than are self-pollinating plants. The application of
serological reactions brought out the fact that various strains of bac-
teria may form nodules on the same host plant, but only one type
is found in the same nodule. That not all strains of the organism
are capable of inoculating one type of plant suggests the existence
of various biotypes even for the same plant. Two types of the
organism can form nodules on the soybean plant. Both are iden-
tical morphologically, but they are different physiologically and
serologically.

218

Nitrogen Fixation— Symbiotic

Physiology of Nodule Bacteria

The various strains of Rhizohium are strictly aerobic. They are
unable to fix atmospheric nitrogen when grown in artificial media.
Different carbohydrates can be used as sources of energy, maltose,
sucrose, glucose, and mannitol being best; cellulose, pectin, or starch
cannot be utilized. Laurent found that Rhizobium can be cultivated
on nitrogen-free media containing 0.1 per cent KH2PO4, 0.01 per
cent MgS04, and 5-10 per cent of an available energy source. Beije-
rinck insisted, however, that a source of nitrogen is also required.
Some of these strains produce considerable acidity, whereas others
do not, the acid producers giving rise to peritrichous flagellation.
Some of the strains grow very fast, others very slowly, requiring 3-4
weeks. The slow growers produce gum (Table 49).

Table 49. Rate of Fixation of Nitrogen by Various Leguminous Plants
Fixation of Atmospheric Nitrogen

'

Per Gram

Per Cent of

Dry Weight

Nitrogen

Data

Period of

Per

Per

of Nodules

Transferred

Reported

Growth

Plant

Day

per Day

to Plant

by

days

mg

mg

mg

Soybeans

35-43

3.98

0.50

27.6

80

Bond

49-63

12.30

0.88

15.4

83

70-84

14.34

1.39

10.3

87

99-108

19.50

2.16

8.1

89

108- 12o

23.09

1.36

4.5

92

125-141

0.80

0.05

0.2

350

Soybeans

25-31

12.04

2.01

33.5

84

Wilson and

38-48

27.55

2.76

21.2

88

Umbreit

48-60

54.08

4.51

19.6

90

25-29

2.98

0.75

10.7

69

35-44

34.08

3.84

29.5

87

Cowpeas

14-22

4.96

0.62

71

Whiting

30-41

26.84

2.44

82

41-58

63.32

3.73

93

The optimum reaction for the growth of Rhizobium is /;H 5.5-7.0,
with limiting reactions of pll 3.2-5.0, on the acid side, and pH 9.0-
10.0, on the alkaline. Maze was the first to draw attention to the
fact that the nodule bacteria comprise both acid-resistant and acid-

Physiolog)' of Nodule Bacteria 219

sensitive types. The alt alia organism is most sensitive to acidity, and
the kipine organism most resistant.

The Hmiting temporatm-cs for the growth of nodule bacteria are
0^ and 50^C. The thermal death point is at 60-62^ and the opti-
mum varies between 18° and 28° C. The bacteria are not injured by
diffused sunlight and can readily withstand direct sunlight. Drying
is injiuious but not fully destructive. As a result of direct and rapid
drying of soil, the numbers of RJiizobiiim diminish rapidly, as de-
termined by the plate method. The organism can persist in the soil
for several \ears, even in absence of the host plant; however, it is
seldom found in soils where specific plants have not grown. Rhi-
zohium is not found in manures. The bacteria move through the
soil very slowly, and are largely distributed by seed, soil, and ground
waters.

The rate of photosynthesis of the plant and the available supply
of combined nitrogen have an important effect upon nitrogen fixa-
tion. When photosynthesis is suppressed by permanent darkness,
nitrogen fixation ceases and the bacteria become parasitic upon the
host plant. With moderately rapid photosynthesis, nitrogen fixation
reaches its maximum and may even exceed the rate of protein syn-
thesis, so that excretion of combined nitrogen takes place. At an
excessive rate of photosynthesis, nitrogen fixation is again depressed.
Optimum fixation depends on a balance between the supplies of
carboh)xlrate and nitrogen; under these conditions the fixation process
is stimulated by nitrate, which otherwise is inhibitive. In the fixa-
tion of nitrogen, nitrate has two functions: (a) it counteracts the
deformation of the root hairs, which is necessary for entrance of the
bacteria, thus reducing the number of nodules; (b) it affects the
activities of the nodules already formed by reducing the volume of
bacterial tissue and by influencing the carbohydrate-nitrogen bal-
ance in the host plant.

The fixation of nitrogen depends on certain relations between the
bacteria and the host plant. The bacteria may produce on one host
plant nodules of abnormal nature and yield little or no fixed nitro-
gen; in a host of a different species, normal nodule formation and
nitrogen fixation may occur. Clovers may show this phenomenon
of "host-plant specificity," a concept which has recently come into
prominence and which takes the place of the separation of bacterial
strains into the "effective" and the "ineffective." Chen and Thornton
(1940) showed that nodules produced by "ineffective" bacteria con-

220 Nitrogen Fixation— Symbiotic

tain a small volume of rapidly degenerating bacterial tissue; the
quantity of nitrogen fixed per unit volume of bacterial tissue, how-
ever, is the same in "effective" and "ineffective" nodules. An "in-
effective" or "poor" strain is not one, therefore, which lacks the power
of causing nitrogen fixation, but one which in a given host plant
evokes the formation of specific substances inhibitory to the devel-
opment of the bacterial tissue, as pointed out by Jensen. Certain
strains may be effective, however, on some species and not on other,
closely related species.

Nodule Formation by Nonleguminous Plants

In addition to leguminous plants, certain nonlegumes, such as
Ceanothtis (redroot), Elaeagniis (silver berry), Alniis (alder), and
Myrica (sweet gale), are also capable of forming nodules on their
roots. These nodules are perennial and branch in all directions,
finally developing round aggregates of considerable size.

These nodules were at first believed to be of fungus origin. It
was shown later that they are caused by bacteria closely resembling
the Rhizobitim group, and that they are capable of causing fixation
of nitrogen. Burrill and Hansen emphasized, however, that some
of the nodules are not caused by Rhizobiiim. The concept of their
ability to fix atmospheric nitrogen was not considered conclusive.
In some plants {Myrica) the organism seems to be of the nature
of an actinomyces. Coriaria japonica produces nodules similar to
those of Ahms, due also to an actinomyces (A. myricae of Peklo).
This plant, when it forms nodules, is able to grow vigorously and
accumulate nitrogen in a medium free from combined nitrogen. The
plants free from such nodules show signs of nitrogen starvation.

The presence of Rhizohiiim, Azotobocter, and certain algae {Ana-
baena, Nostoc) was noted in the roots of Cycas. The ability of most
of these plants to fix nitrogen is still questionable, although it is
reported that some, like Casuarina, are able to grow readily in poor
sandy soil. There is no doubt that some of these bacteria found in
the roots of the plants are responsible for symbiotic nitrogen fixa-
tion. The production of a growth-promoting substance by the bac-
teria has also been suggested.

There are also certain leguminous plants that do not form nodules.
These include various members of the Caesalpinaceae, such as
Gymnocladus, Cercis, and Gleditsia.

Mccliaiiisin of Nitrogen Fixation

221

Fig. 91. Scnbeaiis grown in sand culture. Left, no humus; right, abundant
suppK of luimus. Note how luinius has stimulated root and plant growth (from

Blair).

Mechanism of Nitrogen Fixation by Leguminous Plants

The mechanism of nitrogen fixation by the leguminous plants has
long been, and still is, a subject of controversy. These plants fix
atmospheric nitrogen through their roots and not through their
leaves, as was first assumed for some plants. In the early stages of
growth, the roots contain the larger part of the nitrogen in the
plant; at the time of harvest, however, 74 per cent of the nitrogen is
found in the tops. The fixation of the nitrogen takes place in the

222

Nitrogen Fixation— Symbiotic

early stages of growth of the seedling. The mechanism of the fixa-
tion process has been elucidated by Virtanen as follows:

^2 (atm. nitrogen)
i

NH

COOH

II (di-imide)?

NH

COOH

COOH

CO

i

CNOH

CHNH2

^6Hi206 -

CH2

+ NH2OH

CH2

1

CH2

COOH

COOH

COOH

Carbo-
hydrates
in plants

Oxal-
acetic
acid

Hydroxylamine

Oximino-

succinic
acid

/-Aspartic
acid

The presence of various amino acids and amides in leguminous
plants has been used as substantiation of the above concept. These
acids are believed to be excreted from the plant into the soil and
are made available for the growth of nonleguminous plants. The
data presented in Table 50 tend to substantiate these conclusions,
with which Wilson, however, could not fully agree.

Table 50. Growth and Nitrogen Content of Red Clover, Peas, Barley,
AND Wheat Plants in Quartz Sand (from Virtanen)

Nitrogen

N Nutrition

Dry Weight

in Plant

gm

mg

Red clover

KNO3

2.329

50.0

Aspartic acid

4.428

90.9

Without N nutrition

0.028

0.14

Peas

KNO3

1 . 402

40.1

Aspartic acid

1.474

40.0

Without N nutrition

0.325

6.2

Barley *

KNO3

0.433

13.3

Aspartic acid

0.049

1.9

Without N nutrition

0.063

0.7

Wheat

KNO3

2.143

35.3

Aspartic acid

0.113

3.7

Without N nutrition

0.117

0.8

* Culture of l)arley liarvesled at mucli cariicT stage tiiaii oilier cultures.

Bacteriophage 223

Inoculation of loguminous plants increases the protein content of
the plant, olten without increasing the crop yield. Plants that de-
pend largel)' upon the bacteria for their nitrogen show a high alka-
loid content. When the plants obtain their nitrogen from inorganic
compounds, they are poor in alkaloids. This is especially true of
lupines.

Molybdenum is said to have an important effect on nitrogen fixa-
tion by leguminous plants. This subject was investigated recently in
detail by Jensen, who concluded that nitrogen fixation by alfalfa
and white clover in agar culture is not stimulated by additions of
molybdenum in quantities exceeding 0.03-0.05 y per plant. As much
as 37,000 parts of nitrogen could be fixed per part of molybdenum
present. A relatively small but significant response was found to
1 part of moKbdenum per 80,000 parts of nitrogen fixed. At a ratio
of molybdenum to nitrogen of 1:20,000, further addition of mo-
Kbdenum had no effect. The root nodules of leguminous plants
grown at low molybdenum concentration contained 5-15 times more
molybdenum than did the roots, and the latter were richer in mo-
Kbdenum than the tops. Alfalfa plants took up more molybdenum
when fixing free nitrogen than when utilizing combined nitrogen.
These results suggested that molybdenum stimulates the process of
s)mbiotic nitrogen fixation and is undoubtedly required for general
metabolism. Vanadium cannot replace molybdenum.

Bacteriophage

Root-nodule bacteria are subject to the action of phage, which is
quite specific for the different organisms. This phenomenon repre-
sents a complicating factor in the host-bacteria relationship. The
phage is widely distributed. It has been isolated from nodules,
roots, and stems of leguminous plants, as well as from soils in which
legumes have grown. Demolon and Dunez found the bacteriophage
in the neighborhood of the roots of leguminous plants, but not a few
inches away from the roots. As in the case of other phages, resistant
strains can easily be obtained. This is complicated by the fact that
different phages vary in their ability to cause the lysis of a given
sensitive strain. Vandecavaye and Katznelson isolated a phage which
caused lysis in a dilution of 10~". According to Demolon and
Dunez, phages from clovers, lupine, and pea are able to lyse prefer-
entially the particular species of bacteria, but some phages seem to
be more general in their effectiveness upon different strains of rhi-

224 Nitrogen Fixation— Symbiotic

zobia. When a large number of legumes were planted on an alfalfa
field suffering from "fatigue," believed to be due to the presence of
the bacteriophage, the nodules of all species contained abnormal,
vacuolated forms of the organisms as well as the specific phage. The
importance of the phage in the phenomenon of fatigue may still be
considered questionable, however. According to Katznelson, a phage
for alfalfa was absorbed from suspensions only by members of the
alfalfa-sweet-clover cross-inoculation group, thus suggesting a tech-
nique which may be useful in distinguishing members of the differ-
ent species of Rhizobium.

Excretion of Nitrogen by Legumes

The first demonstration that nonleguminous plants are able to
benefit from association with a legume was presented by Lipman.
He suggested that the beneficial effect was due to nitrogenous com-
pounds excreted by the leguminous plants and consumed by the non-
legumes. Lipman used a half-and-half mixture of peas and oats
grown in a medium consisting of soil plus sand. The total nitrogen
and dry weight of the oats associated with the peas exceeded those
of the crop of oats grown alone. The yields of oats and peas in the
mixture were greater than those of the crops grown alone.

Lipman further demonstrated in a series of experiments that the
benefit to the associated nonlegume arises from excretion of nitro-
gen by the leguminous plant. He used as the medium a pure quartz
sand treated with all the necessary plant nutrients except nitrogen
and placed a small pot within a larger one so that the two plants
grew in separate containers. Whenever the inner pot was porous,
or would allow the passage of substances in solution, the nonlegume
developed normally and, on analysis, showed considerable quantities
of nitrogen. If the inner pot was glazed, the nonlegume grew poorly
and contained little nitrogen. On the basis of these results, Lipman
suggested that nonlegumes benefit from association with legumes as
a result of the excretion of nitrogen by the latter. This work was
repeated by many others, some quite independently of the above
experiments.

Virtanen observed in 1927 that oats grown in association with peas
on a nitrogen-free quartz sand developed as though combined nitro-
gen had been supplied. Similar results were later obtained with
other combinations of legumes and nonlegumes. The fact that

Excretion of Nitrogen by Legumes

225

legumes can excrete nitrogen from the nodule is substantiated by
the following evidence:

1. The quantitN' of nitrogen excreted is too large to be explained
by the sloughed-off nodules and portions of the roots. Frequently
50 per cent or more of the total nitrogen fixed is excreted, more
nitrogen than is usually present in tlu^ entire root system.

Fig. 92. Influence ot a legume (peas) on growth of a nonlegume (oats). Oats
in inner pot, peas in outer. Porous inner pot on left, glazed inner pot on right

( from Lipman ) .

2. Virtanen grew the plants under bacteriologically controlled
conditions. Excretion took place, thus proving that the origin of
the nitrogen is the legume bacteria and not other soil microorganisms.

3. Nitrogen compounds were excreted even in the absence of non-
legumes. They were identified as comprising largely aspartic acid I ^
and beta-alanine. The formation of the beta-alanine was ascribed l

to decarboxylation of the excreted aspartic acid by the root-nodule
organisms. If the nitrogen originated from sloughed-off portions of
the plant, the presence of more than one amino acid would be
expected.

226 Nitrogen Fixation— Symbiotic

4. Excretion may occur fairly early in the development of the
plant, before sloughing of nodules or roots would be expected.

Wilson further developed this concept by considering the effect of
length of day upon the excretion of nitrogen by the leguminous plant,
as measured by absorption by the nonlegume (Table 51).

Table 51. Influence of Length of Day upon Fixation of Nitrogen

(from Wilson)

Nitrogen

I Fixed

Short Day

1
Long Day

mg

mg

Canada field pea

36.2

52.3

Associated barley

43.8

50.1

Canada field pea

30.1

.37.0

Associated barley

47.8

51.1

Associated barley control 36.7 19.8

Importance of Symbiotic Nitrogen Fixation in the Soil

Though the amounts of nitrogen fixed nonsymbiotically under field
conditions are still subject to doubt, the symbiotic fixation of nitro-
gen is of great economic importance. The amount of nitrogen
added to the soil by leguminous plants depends entirely upon the
abundance of available nitrogen in the soil. The poorer the soil is
in nitrogen and the richer it is in lime, phosphorus, and potash, the
greater will be the gain in nitrogen from the growth of legumes.
The nature of the legume, soil conditions, and season will affect the
amount of nitrogen fixed (Table 52).

Tahle 52. Influence of CaCOs upon Nitrogen Fixation by Alfalfa
Grown in Sand (from Jensen)

Gain of N

per Gram

Total N in Plants

Dry Nodule

Days

CaC03* +CaC03

t

-CaCOa

+ CaC03

mg mg

mg

mg

GO

16 36

712

85

127 144

1,298

1,506

JOO

175 258

1,209

2,321

of sand 5.1.

t;;II

of sand 7.2.

Warington demonstrated in 1891 that an increase of about 350
pounds of nitrogen per acre may be obtained as a result of the growth

Importance of Nitrogen Fixation 227

of inoculated clover. All the subsequent reports point to large in-
creases in soil nitrogen due to the gro\Vth of leguminous plants in
the presence of specific bacteria. Poor soils usually show larger

0.50

0.45

0.40

0.35

0.30

0.25

0.20

0.15

0.10

0.05

^H N obtained from soil

E22 N added in NaNOj

I I N obtained from atmosphere

^

^

'/..

0

1200

200 400 600 900
NaNOa pounds per acre
Fig. 93. Relation between soil and atmospheric nitrogen obtained by a crop
of inoculated alfalfa growing on soil variously treated with sodium nitrate ( from

Giobel ) .

gains than rich soils. Soils to which lime and phosphorus compounds
have been added show greater increases in combined nitrogen than
do soils in which these are lacking. Inoculated soils give higher
increases than uninoculated, especially if the particular legume has

228 Nitrogen Fixation— Symbiotic

not been grown previously on the same soil. Hiltner obtained by
inoculation an increase of 1.7-.31 times the yield for lupines and 15-
80 times for serradella. On the average, there may be a gain of
50-100 pounds of nitrogen per acre of soil due to the growth of
legumes. Lipman and Blair obtained a gain of 54 pounds annually
over a period of 7 years from the growth of legumes in rotation with
corn, potatoes, oats, and rye in cylinders.

Hopkins reported that a 3-ton crop of cowpea hay adds 86 pounds
of nitrogen per acre, a 25-bushel crop of soybeans with 2.25 tons
of straw adds 106 pounds, a 4-ton clover crop adds 106 pounds, and
a 4-ton alfalfa crop adds 132 pounds. On the average, about two-
thirds of the nitrogen in the legumes grown in the soil is obtained
from the air. Under optimum conditions and on a relatively poor
soil, as much as 400 pounds of nitrogen may be added per acre
yearly. In a light sandy soil, clover was reported to produce an
annual gain of 50 pounds of nitrogen. If the crop is removed, the
nitrogen content of the soil may not be greatly increased, since the
amount fixed may be just sufficient to fulfill the need of the tops.
Perennial legumes, like alfalfa, may not show an increase in soil
nitrogen, although the nitrogen is higher than in the same soils
upon which grains are grown.

Because of the associated bacteria, the economic importance of
legumes in agriculture is so great that it has been said many times
that, had the whole subject of soil microbiology contributed nothing
more of practical value than a knowledge of the legume bacteria, it
would have more than fully justified itself.

Selected Bibliography

1. Allen, E. K., and Allen, O. N., Biochemical and symbiotic properties of the
rhizobia, Bad. Revs., 14:273-330, 1950.

2. Allison, F. E., and Ludvvig, C. A., The cause of decreased nodule formation
on legumes supplied with abundant combined nitrogen, Soj7 Sci., 37:431-
443, 1934.

3. Bond, G., Quantitati\c obser\ations on the fixation and transfer of nitrogen
in the soy bean, Ann. Botany, 50:559-578, 1936.

4. Chen, H. K., and Thornton, II. G., The structure of "ineffecti\e" nodules
and its influence on nitrogen fixation, Proc. Roy. Soc. London, B, 129:208-
229, 1940.

Selected Bibliography 229

5. Fred, E. Ik, Baldwin, I. L., and McCoy, E., Root Nodule Hdctciia and
Leguminous Plants, Uni\t'isity of WisconsiiT Press, Madison, Wis., 1932.

6. Giobel, G., The relation of the soil nitrogen to nodule de\ elopment and
fi.xation of nitrogen by certain legumes, N. J. Agr. Expt. Sla. Bull. 436:
125, 1926.

7. Jensen, H. L., Nitrogen fixation in leguminous plants, I-VI, Proc. Linncun
Soc. N. S. W(dcs. 67:98-108, 1942; 68:207-220, 1943; 69:229-237, 1944;
70:20a-210, 1946.

8. Jones, F. R., and Tisdalc, W. B., Effect of soil temperature upon the de\el-
opment of nodules on the roots of certain legumes, J. Agr. Research, 22:17-
32, 1921.

9. Lipman, J. G., and Con\l)eare, A. B., Preliminary note on the in\entory
and balance sheet of plant nutrients in the United States, N. J. Agr. Expt.
Sta. Bull. 607, 1936.

10. Thornton, H. G., The influence of the host plant in inducing parasitism in
lucerne and clover nodules, Proc. Roy. Soc. London, B, 106:110-122, 1930.

11. Thornton, H. G., and Nicol, II., Reduction of nodule numbers and growth,
produced by the addition of sodium nitrate to lucerne in sand culture,
J. Agr. Sci., 26:173-188, 1936.

12. Virtanen, A. I., Cattle Fodder and Human Nutrition, ivith Special Reference
to Biological Nitrogen Fi.xation, Cambridge University Press, 1938.

13. Virtanen, A. I., and Laine, T., Investigations on the root nodule bacteria of
leguminous plants. XXII. The excretion products of root nodules. The
mechanism of N-fi.\ation, Biochem. J., 33:412-427, 1939.

14. Wilson, P. W., The Biochemistry of Symbiotic Nitrogen Fixation, University
of Wisconsin Press, Madison, Wis., 1940.

15. Wilson, P. W., and Burris, R. H., The mechanism of biological nitrogen
fixation, Bad. Revs., 11:41-73, 1947.

16. Wilson, P. W., Hull, J. F., and Burris, R. H., Competition betv^'een free
and combined nitrogen in nutrition of Azotobacter, Proc. Natl. Acad. Sci.,
29:289-294, 1943.

17. Wyss, O., Lind, C. J., Wilson, J. B., and Wilson, P. W., Mechanism of
biological nitrogen fixation. 7. Molecular Ho and pN-2 function of Azoto-
bacter, Biochem. J., 35:845-854, 1941.

10

Transformation of Mineral* Substances
in Soil by Microorganisms

Through their roots, plants take up from the soil a number of dif-
ferent elements in the form of salts. These are frequently classified
as essential and minor or trace elements. The first group includes
sulfur, phosphorus, potassium, calcium, magnesium, iron, manganese,
and sodium. The second group includes zinc, molybdenum, cad-
mium, chlorine, aluminum, boron, copper, silicon, and a variety of
others. In the transformation of these elements in the soil, micro-
organisms frequently play a direct part, though often their effect
upon the various elements is merely indirect. Even such inert ele-
ments as silicon may be essential in the growth of certain bacteria
and of various algae, notably the diatoms. Molybdenum has been
found to play a role in the fixation of atmospheric nitrogen. Other
elements are found to have a neutralizing effect upon plant toxins.
Still others, like cobalt, may influence the growth of the cell to retard
multiplication. Some of the elements, notably phosphorus, are essen-
tial constituents of the cell nucleus and its cytoplasm. Still others,
like sulfur, may serve as sources of energy to various bacteria and
also form essential constituents of certain amino acids.

Transformation of Sulfur

Sulfur is one of the elements essential for plant growth. This ele-
ment makes up about 0.11 per cent of the earth's crust. Phosphorus
occurs in about the same concentration. It appears from analyses of
river water that sulfur is removed from the soil faster than any other
element, however, since its ions, SO4, tend to dissolve in the water,
whereas the PO4 ions tend to precipitate in the soil.

*■ The term "mineral" is used to designate all elements and compounds not
containing carbon or nitrogen.

230

Transformation of Sulfur

231

The removal of sulfiu- b\- farm crops may be far greater than has
usualK' been assumed; it may be entirely out of proportion to the
reserve of this element in the soil. It has been suggested that the
depletion of sulfur ma\ , in time, have an important effect on soil
fertility. The annual loss of sulfur from uulimed soil througli crop-

10,800

9600

8400

7200

>- '-> 6000

4800 -

3600

2400 -

1200

0

1 1 1 1

1

so., inmg

/

J, ^^ ^

/

^^

, —

-

/

-

/

-

/

/

-

: /

~

/

-

/

^^^

: /

y"^^ z

/ y

^

- 1 /

—

- / /

_

- / /

-

/ /

1 1

0

35

5 10 15 20 25 30

Period of incubation in weeks

Fig. 94. Course of accumulation of citrate-soluble P2O5 and SO4 in composts of
soil, rock phosphate, and sulfur (from Lipman, McLean, and Lint).

ping and drainage has been reported to amount to 44 pounds per
acre. This loss was increased by applications of lime. One-half to
two-thirds of the sulfur applied to the soil in the form of potassium
sulfate was found to be removed in the drainage water.

Sulfur is added or finds its way into the soil in a number of dif-
ferent forms: (fl) in certain organic compounds, which form con-
stituent parts of the plant and animal residues added to or left on
the soil; (Z?) as elementary sulfur, which is usually added to the soil
as a fertilizer or which is continuously brought down in the rain

232 Transformation of Mineral Substances

water; (c) as sulfates, which are added to the soil in certain mineral
fertilizers, such as superphosphates and gypsum.

The sulfur content of plant materials varies from a small fraction
of 1 per cent, as in rye straw, to almost 1 per cent, as in turnip tops.

Sulfur and sulfur compounds in the soil are subject to numerous
transformations resulting directly or indirectly from the activities of
different groups of microorganisms. When plant and animal resi-
dues undergo decomposition, whether in the soil or in the compost,
the sulfur-bearing constituents, notably the proteins and certain glu-
cosides, are hydrolyzed. The proteins give rise to the sulfur-bearing
amino acid cystine; the glucosides yield mustard oil and other sul-
fur compounds.

The chemical structure of two typical sulfur-bearing compounds
may be presented as follows:

dl2 S b 0x12

I I

NH, HC CH NH2

I I

HOOC COOH

Cystine
CH2SO0OH

I
CH2NH2

Taurine

In recent studies on antibiotic products of microbial metabolism,
sulfur has also been found as an essential constituent of such im-
portant compounds as penicillin.

These compounds can be attacked by various bacteria and other
microorganisms. Only a small part of the sulfur is consumed by
these organisms. Most of it is liberated as hydrogen sulfide. Under
anaerobic conditions, other sulfur compounds, such as mercaptans,
may also be produced. These are largely responsible for the pungent
odor which is always present when animal residues and certain
protein-rich materials undergo decomposition in absence of avail-
able oxygen. Under aerobic conditions, both hydrogen sulfide and
mercaptans are rapidly oxidized further to sulfates.

Among the bacteria responsible for the transformation of sulfur
in nature, those that are able to oxidize the elemental sulfur and
simple compounds of sulfur are of particular interest. These bacteria
are known as "sulfur bacteria." They belong to different morpho-
logical and physiological groups, some being small, a micron or so

1 laiislorniatioii oi Sullur

2.33

in size. Others are large and filamentous. Some are obligate auto-
trophic, that is, they depend on the energy liberated in the oxidation
of sulfur, and others are facultative autotrophic, or are also able to
derive their energy from organic compounds.

The most numerous of these bacteria in the soil population are tlu>
autotrophic organisms. They function in a manner somewhat similar
to green plants, though the latter use the energy of sunlight (photo-
SNuthesis), whereas the autotrophic bacteria are able to utilize the
chemical energy liberated in the oxidation process ( chemosynthesis ) .
Both the bacteria and the green plants use the carbon dioxide of
the atmosphere as sources of carbon for cell synthesis.

The following chemical reactions are involved in the oxidation of
sulfur and its compounds by bacteria:

-2H2S + (),
So + 3O2 + ^^H.O

-iHoO + S2

^2H2S04

Xa2S04 + H2SO4

2Na2S203 + O2 = 2Na2S04 + S2

The rate of oxidation of the sulfur and its simple compounds to
sulfuric acid and to sulfates is influenced not only by the nature of
the organism but also by the environmental conditions (Table 53).

Table 5.3. Oxiuatidn of Klementaky Sulfur to Sulfuric Acid by
Thiobacilhis fhloo.ridans (from Waksman and Starkev)

Amount of

Control Flask

Inocula

ted Flask

Elementary

Increase

Iiiouhation

Culture in
Flask

Sulfur
Disappeared

in

Sulfate

Sulfur

Sulfate

Sulfur

Sulfate

days

ml

ma S

mg S

mg S

mg S

mg S

mg S

l.T

100

1,001

86.4

788

302.1

213

215.7

30

100

992

90.5

735

354.0

257

263.5

1.5

300

3,002

112.2

2.496

633.0

506

520.8

30

300

2,997

126.5

1.974

1.168.0

1.023

1,041.5

Of particular importance is the reaction of the soil or substrate, salt
concentration, and presence of organic materials.

Each of the above reactions can be brought about by different
types of bacteria. The first reaction, which results in the precipita-
tion of sulfur, takes place largely in water basins and in peat bogs

234 Transformation of Mineral Substances

and can be carried out by a great many bacteria. This type of reac-
tion has recently attracted much attention as an outgrowth of an
effort to increase the supply of available sulfur by utilizing the waste
products. In well-aerated soils, however, sulfur does not accumu-
late as a result of oxidation of hydrogen sulfide, but is rapidly oxi-
dized further to sulfate, as shown by the second reaction.

When elementary sulfur is the starting point, only very few bac-
teria are able to bring about its oxidation to sulfuric acid. The acid
interacts with the bases and other buffering substances present in
the soil to give rise to various sulfates. When it is desirable to reduce
the alkalinity of certain soils, such as black alkali soils, or when it is
advisable to make the soil more acid for control of certain disease-
producing organisms, such as potato scab, addition of sulfur, and its
resultant oxidation to sulfuric acid, may become of great economic
importance. This reaction can be summarized as follows:

Ca3(P04)2 + ^2H2S04 = 3CaS04 + Ca(H2P04)2

Among the other transformations of sulfur compounds in the soil
and in other natural substrates, the reduction processes are of great
significance. The reduction of sulfate to hydrogen sulfide is brought
about by certain specific bacteria, usually designated as sulfate-
reducing organisms. One of these organisms has been designated by
Beijerinck as Microspira dcsulfiiiicans, and more recently by Starkey
as Sporovibrio desttlftiricans. The reduction process takes place in
the presence of a suitable source of energy as follows:

CaS04 + C Hg C OOH = H.S + CaC Og + ( O2 + HgO

Anaerobic conditions, or absence of free atmospheric oxygen, is
essential for this reaction, and a form of organic matter must be
available. The sulfate is used by the bacteria as a source of oxygen
for the oxidation of the organic substances; the energy liberated in
the oxidation process is partly consumed in the reduction of the
sulfate. The hydrogen sulfide produced in this reaction is character-
istic of certain water basins, of peat bogs, and of other water-
saturated environments, where there is a lack of free oxygen. In the
presence of iron, black iron sulfide is produced, which is character-
istic of the organic muds laid down under anaerobic conditions.
This reaction may lead to corrosion of iron in steel pipes, which may
become of considerable economic importance.

Tiaiisforniation of Phosphorus 235

Thi" h\ tlioiicn siilficU' loniiccl in the rt'ductioii ol sullalcs iiia\' attain
he oxidized to sulfate upon coming in contact with an oxygen source
and in the presence of the specific sulfur-o\i(h/.ing hacteria.

Transformation of Phosphorus

Phosplu)rus is coiitinuonsK' added to the soil in organic residues
and in fertilizers. It is also found in untreated soil in a niunher of

'J'aui-k .)4. PiKisi'iioiii s AND TorAfssnM (Ontknt of Somk Tvi'ical Soil Hacteuia

Asli Total IM)5 Total K'.jO

per cent per cciil per cent

Az.cliroororcuin 8. '2-8. (5 4.<J3,5.'-2 '2.41 -'2. Co

li. mycoidcs 7.'> 4.07 2.27

Ps.fI)iorr.\Tciisli(jiirf(iriciis (i.48 5. Si 0.8,S

different forms. Briefly these forms of phosphorus may be classified
as follows:

1. Organic compounds present in plant and animal residues added
to the soil. They are also abundant in the microbial cell substance
^^'hich is synthesized in the soil. Organic compounds of phosphorus
also form a constituent part of the humus complexes of the soil.

2. Rock phosphate and other insoluble phosphates are usually
present in the native rocks from which the soil is derived. They
are also frequently added to the soil in the form of various fertilizers
as well as in the bones of dead animals (Table 55).

Table 55. Effect ok Xithificatiox ox the Souijility ok Thicalcilm
Phosphate in Soil (from Kelley)

After 28 Days

After 57 Days

Materials Added

NO3-N

Ca

P2O5

NO3-N

Ca

P2O5

ppm

ppm

ppm

ppm

ppm

ppm

Control

20 . (J

4 0.0

13.1

25.5

50 . 6

11.0

CaC0:i

22 . 0

:>(\.::>

11.9

29.0

70.8

13.2

Ca3(P04)2

21.0

.3.3..^

24.2

28.0

58.8

25.0

CaCOs + Ca3(PO.i)2

22.0

59 . 1

17.3

28.0

70.1

22.4

(NH4)2S04

!J8.0

219.4

18.5

99.0

225.4

19.4

(NH4)2S04 + CaCO:i

97.0

254.4

18.5

98.0

270.5

7.4

(NH4)2S04 + Ca:,(P04)2

'J'J . 0

217.7

.52.1

99.0

229. G

38.0

(NH4)2S04 + CaCO:i + Ca:

i(P04)2

100.0

25.3.4

26. G

11)1.0

230 . 4

13.9

Dried blood

91 .0

107.7

9.7

90.0

113.9

10.0

Dried blood + CaCOj

89.0

107.2

9.8

90.0

140.2

11.5

Dried blood + Ca3(P04^2

82.0

111.7

24.3

88.0

117.7

22.2

Dried blood + CaCOj + Ca

i-i(VOi)->

81.0

118.2

19.5

87.5

138.1

18.3

236 Transformation of Mineral Substances

3. Soluble inorganic phosphates, like those of sodium, potassium,
calcium, and magnesium, are added to the soil in fertilizer materials
and in plant and animal residues.

Among the organic phosphorus compounds which find their way
into the soil, lecithin, nucleic acids, and phytin occupy a prominent
place.

Lecithin contains 9.39 per cent Pi-O.-,, 1.6 per cent N, and 65.36
per cent C. It contains two fatty acid radicals, such as pahnitic
and stearic or oleic acids, as shown by the following structural
formula:

CHo OR

CH OR^

I /OH

CHoOPO< /(CHgjg + ,SHo()

\0-(CH9).,\<

\0H

Lecithin

CHoOH

= CHOH

I /OH /(CH3)3

CHoO P0< + ROH + RK)H + (( H,).,OH X<

^OH \0H

( Uyccro- I'atty acids Choline

phosphoric acid

Nucleic acids are found abundantK' in the microbial cell substance.
In their decomposition, some of the groups are broken down more
readily than others. In the presence of readily available sources of
carbon and nitrogen, various bacteria and fungi are capable of break-
ing down both lecithin and nucleic acids and liberating the phos-
phorus as phosphate. As much as 66 per cent of the lecithin phos-
phorus was transformed into soluble phosphate in 60 days. The rest
of the phosphorus was assimilated by the bacteria for the synthesis
of cell material.

Phytin is a hexaphosphate, which occurs abundantly in plant ma-
terials, notably in the seeds. It contains about 26 per cent phosphorus
in the form of phytic acid. C(;Ho4027P,;. This compound is acted
upon by fungi and bacteria by means of an enzyme, designated as
"phytase," with the result that the organic phosphorus is transformed
into phosphate:

C6H24O27P6 + «02 = 6H3PO4 + 6CO2 + 3H2O

Transfoniuitioii ot Pliospliorus 237

Niiclooprotcins contain 7-9 per cent i^hosphorus and 13-14 per
cent nitrogen. When attacked by microorganisms, they give rise to
phosplioric acid, sngar, pnrine, and pyrimi(Hne bases. These com-
pounds are decomposed further by a variety of bacteria and fungi.
Certain bacteria, designated as the Niicleohacter group, have been
found to l)e specifically concerned in the decomposition of nucleins,
through the nucleic acid stage, into phosphoric acid.

A number of other organic phosphorus compounds, notably inosite
monophosphate (C(;H,;;Oi,P), which is found in wheat bran, are
commonly added to the soil. Their decomposition is similar to the
transformation of phytin and nucleic acids.

The insoluble phosphates added to the soil are subject to the
activities of microorganisms largely in an indirect manner. The
\arious organic and inorganic acids produced by microorganisms
interact with the insoluble phosphates, giving rise to soluble com-
pounds. This is illustrated bv the following reactions:

Ca3(P04)2 + ^2HX()3 = ^>raHP04 -f ( alXOs),

Ca3(P04)2 + 4HX()3 = Ca(H2P()4)2 + '2Ca(N03)2

ra3(P()4)2 + H2SO4 = ^2CaHP04 + ("aS04

The relation between sulfur oxidation and phosphate solubilization
is brought out in Fig. 95.

Gerretsen presented evidence that the intake of phosphorus by
plants from basic slag is markedly improved by the activity of soil
microbes. The solvent action of the soil microorganisms on insoluble
phosphates was obtained with the aid of glass plates, covered with
an agar film, in which calcium phosphate was precipitated, and
buried aslant in the culture vessels. Clear solubilization zones were
observed in distinct spots, especially underneath root tips and young
branches. In sterile cultures these solubilization zones were absent,
proving that the solvent action of the roots was negligible. When
the roots excrete organic substances with a low C : N quotient, there
is a possibility that phosphates may be precipitated by microbio-
logical activity.

hi the decomposition of organic matter by microorganisms, usually
not all the phosphorus is liberated as phosphate, a certain amount
being assimilated by the organisms for the synthesis of fresh cell
material. When the organic complexes contain little or no phos-
phorus, the bacteria and fungi are able to remove from the soil some

238

Transformation of Mineral Substances

of the soluble phosphate that they require for their eell synthesis.
This is similar to the needs of the microorganisms for nitrogen,
although the amounts of phosphorus required for this purpose are
less.

2.4

E 120 -

E
= 3.4 2

4.4

5.4

1 i 1 1 1 1 1 1 1 1 1 1 1 1 1

'

150

: A

-

/

—

/ \ L^

-

11 f \ J

-

120

h y
p A

-

100

/ '' \

-

-

-

^ / y

-

70

" -v -'

-

yV

fin

^~J^ 1 — \ — \ — \ \ \ \ \ \ \ \ \ L

1

0

15

240

200

E
160 1"

- 120 .£

80

40

5 10

Incubation period, days

Fig. 95. Course of sulfur o.xidation and transformation of insoluble phosphate
into soluble forms l>y Tliiolxicilhis tliiooxiclans in lifjnid media (from Waksman

and Joffe ) .

The estimation of available phosphate in the soil, or the potential
response of a crop to a dressing of phosphate, is complicated by sev-
eral factors, notably the root system of the plant, the depth of pene-
tration of the root system, aud rainfall. The favorable effect of addi-
tion of available phosphate to the soil will be an increase in the root
system, which leads to a greater uptake of the soil phosphate. The
ability of the roots of plants to absorb the less easily available phos-
phates depends on the phosphate nutrition of the plant and its root
systeui. Some plants excrete organic acids from their roots. All

Traiisiorniation of Pliosplioiiis 239

plants ha\X' certain coiifcntratioiis ol carbon dioxide aronnd their
roots, because ol respiration of the niicroliiological popnhition of the
soil, especially in the rhizosphere. This resnlts in greater availability
of insoluble phosphates and may result in a transfer of some of the
phosphate thus made available to the roots of the crop. Soil condi-
tions, especialh' aeration, are of great importance in this connection.

The methods no\\' in use for estimating the available phosphate in
the soil are based upon treatment of the soil with a suitable solvent,
such as citric or acetic acid or ammonium fluoride, and determina-
tion of the amount of phosphate in the extract. A number of bio-
logical methods are also in use. These include measuring the amount
of phosphate taken up by rye seedlings, or the amount of growth
made by phosphate-requiring bacteria, such as Azotobacter, or fungi,
such as Aspergillus, with soil as the only source of phosphorus. These
methods allow a proximate evaluation of the availability or shortage
of phosphate in the soil. No single method, however, is sufficient for
evaluating the potential responsiveness of soils to a given crop treated
with a certain amount of phosphate, since different crops vary
greath' in their ability to extract phosphate from the soil.

The microbiological methods for evaluating the available phos-
phorus in the soil are based upon the fact that a certain parallelism
has been observed between microbial cell synthesis and phosphate
consumption. This observation has been used to advantage in de-
termining the available phosphate present in the soil. Various bac-
teria, notably Azotobacter cJiroococctim, and fungi, such as Asper-
gillus niger, Cimninghcniiella, Trichoderina, and A. oryzae, are util-
ized as test organisms.

The method of analysis is usually carried out as follows: A certain
quantity of the soil in question is added to a sugar-salt solution free
from phosphorus and nitrogen and is inoculated with a suitable strain
of Azotobacter. After incubation for 14-30 days at 28"C, the amount
of nitrogen fixed is determined. The fixed nitrogen is a measure of
Azotobacter growth, which is controlled by the available phosphorus.
Since the ratio of cell nitrogen to cell phosphorus is 2:1, the amount
of phosphorus consumed can be calculated from the amount of
nitrogen fixed, thus giving the available phosphorus in the soil sample
added to the solution.

This method has been \ariousl\' modified. In one of these modifi-
cations a carbohydrate ( sugar, starch, mannitol ) is added to the soil
in (iuestion. Some CaCO.-j is also added if the soil is acid. The soil
is then inoculated with Azotobacter and packed into small dishes.

240

Transformation of Mineral Substances

the surfaces of which are smoothed out. The amount of available
phosphorus in the soil is measured by the growth of Azotobacter on
the surface of the soil, as determined by the appearance of slimy
colonies. The available phosphorus in a given soil is measured from
the actual amount of growth of the Azotobacter colony.

Some of the methods are based upon the use of fungi. These
methods are similar to the above, except that available nitrogen is
also added to the solution or to the soil. The fungus growth is used
as a measure of the available phosphorus.

Transformation of Potassium, Calcium, Magnesium, and Iron

In addition to the elements already discussed, others that are
required for plant and animal nutrition and that are present in or
introduced into the soil are also subject, directly or indirectly, to
activities of microorganisms. Some of these elements, including po-

1 1
Soil No

1

8

\ 1 1

210

180

~ fpH

y^

150

- \

/SO3 ^

S"l20
90

: 1/

/

/Acidity

60

7/

J _--^

30

\/^

^

^T^o -

n

>r 1 1 1 1 II

0 1 3

6 9 12
Weeks

15 18

13

9 o
5 ^
1

Fig. 96. Relation lictwueii sultur oxidation and water-soluble potassium in
composts containing sulfur and greensand marl (from Rudolfs).

tassium, calcium, magnesium, and iron, are utilized for the metabolism
of the soil organisms. They are used either as nutrients or as cata-
lysts and are, therefore, required in very small amounts. Their trans-
formation by microorganisms and their role in microbial metabolism
depend on the nature of the element, on the nature of the organism,
and on soil conditions.

Potu'isiuni is found in soil both in organic forms and in the form
of zeolitic and nonzeolitic silicates. It is added to the soil in soluble

Transformation of K, Ca, Mg, Fe 241

inorganic ionns, notabU' salts of sulfates, chlorides, and phosphates;
in insoluble inorganic form, known as marl; and in the form of stal)le
maniues and plant residues. The K_.0 content of plant residues
ranges from 0.5 to 2.0 per cent. Fresh manure contains 0.288-0.504
per cent K^.O. Its concentration in the ash content of bacterial cells
is 4.0-25.6 per cent, and in that of fungus mycelium, 8.7—39.5 per
cent. The presence and activities of microorganisms influence greatly
the a\'ailabilit\- of potassium in soil to plant growth. Microbial ac-
tivities may lead to an increase in the available potassium, as when
organic matter is decomposed by microorganisms and when acids
interact with the zeolites, liberating the potassium. Orthoclase inter-
acts with certain microbial products to give soluble potassium salts:

AUOs-KoOOSiO, + 4H2S()4 - Al2(S04)3 + K2SO4 + ()Si02 + 4H2O

AlaOa-KoOCiSiOo + ra(H(X)3)2 = AL^Oa-CaO GSiOs + ^2KHC()3

Potassium compounds present in the soil or in culture are assimi-
lated by bacteria and fungi and stored away in the cell material.
When the latter is decomposed, the potassium again becomes avail-
able. The potassium replaces in the soil the zeolitic bases Ca, Mg,
and Na. The concentration of available potassium in the soil is thus
controlled not only by the total concentration of the element, but
also by the form in which it is present in the soil, by the degree of
saturation of the soil zeolites, by the soil reaction, by the available
organic matter, and finally by the activities of various groups of
microorganisms.

The Azotobacter method has often been used to determine the
concentration of available potassium in the soil, which was found to
vary between 2 and 30 per cent of the total, depending upon the
fertility of the soil.

Calcium and viagnesium are also essential in the nutrition of soil
microorganisms. In addition, they play important parts as buffering
substances for neutralizing the organic and inorganic acids formed in
the soil.

Iron may undergo in the soil a variety of transformations through
the activities of microorganisms. It is essential for cell synthesis.
Certain bacteria are capable of oxidizing ferrous salts to ferric com-
pounds and of utilizing the energy liberated for the assimilation of
carbon dioxide, in a manner similar to the action of nitrifying and
sulfur-oxidizing bacteria, according to the following reaction:

2FeS04 + 8H2O + 2Ca( O3 + O = ^2Fe(OH)3 + !2CaS04 + 2CO2

242

Transformation of Mineral Substances

Iron interacts with the humus compounds of the soil to give rise
to iron humates. It is more readily available in this form to plants
growing in alkali soils when it is not precipitated out as inorganic
phosphate.

logC,
4.0

5.0

6.0

-

1 1 1

1

1 1

-

Solution of /

Solution of

1

potassium y

magnesium^

/

/

//

1

-

/
/

X

'L

Solution of

-

\ 1 1

•

/•

•
•

calcium

>

/

1

1 1

4.0

3.0
Solution of minerals increase-

2.0iOgCK,Ca

Fig. 97. Influence of acidity created by growth of Azotobacter on solution of
calcium, magnesium, and potassium from the mineral biotite (from Wright).

The Role of Microorganisms in the Transformation of
Rare or Trace Elements

Molybdenum, copper, zinc, cobalt, boron, and certain other ele-
ments act primarily as catalyzers for the activities of different soil
organisms. Molybdenum is essential for the fixation of nitrogen by
Azotobacter, a fact utilized for determining the concentration of
available molybdenum in the soil. Boron is highly essential to the
growth of legume bacteria, though high concentrations are injuri-
ous. Copper is essential for a variety of microbial processes, but
high concentrations of this element, too, are injurious. Its concen-
tration in the soil can be determined by the use of certain fungi and
bacteria. Zinc and cobalt form essential constituents of certain en-
zyme and vitamin systems. Zinc also favors growths of fungi and
represses spore formation.

Tiaiisloiniatiou oi ArstMiic and Soleuiiini 243

Transfohmahox of Ahsenic'and Selenium

Arsenic is wideK' distributt'tl in natnre. It is iar('l\ lonnd, how-
ever, in toxic anionnts in tlie soil. Althongh arsenic has come into
Ueneral nse in \arions insecticides, only small amonnts find their way
into thc> soil. In some cases, however, as when arsenical dnst is
applied to combat certain insects, the amonnts left in the soil may
resnlt in the stnnting or dwarfing of the sncceeding crop. This is
trne also of soil in coniferons nnrseries where lead arsenate is nsed
to combat insect larvae. Microbial activities in the soil may be af-
fected nnless this arsenic is rendered inactive or insolnble.

Certain soil fnngi have the capacity to volatilize arsenical snb-
stanees b\- redncing them to arsine. Cnltnres of snch fnngi readily
give off the odor of arsine from arsenic-containing media. Members
of the genns Scopiikiriopsis or PeniciUiiim brevicaule, certain asper-
gilli, notabl\- A. sydowi, A. fuinigatiis, and A. ochraceiis, species
of Fusariioii, and various dematiaceae are responsible for these
activities.

The transformation of selenium by microorganisms in the soil is
of importance in connection with the selective absorption of this
element b)- crop plants. Certain geological formations contain se-
lenium. Plants grown in these areas accumulate the element in
their cell material. Various bacteria and fungi have the capacity of
volatilizing selenium, producing markedly offensive odors. Tliese are
readily detected in culture tubes and in pot experiments in the
greenliouse. When selenium-containing plants undergo decomposi-
tion, the activities of the various microorganisms result in the pro-
duction of strong odors. Microbiological activities in the soil render
the selenium-containing substances available to green plants under
conditions in which the plants are not otherwise able to obtain
selenium. The organisms concerned in the volatilization of selenium
include most of the arsenic fungi, especially the Scopiikiriopsis brevi-
caule group, and certain soil bacteria, notably Psetidomonos fiio-
rescens.

Selenium compounds are subject to a variety of other bacterial
activities, in the reduction of selenates to selenites and in the oxi-
dation of elementarv selenium.

244 Transformation of Mineral Substances

Transformation of Other Elements

In addition to the above elements, a variety of others either are
subject to transformation by microorganisms or play a part in their
metabolism. It is sufficient to mention hydrogen, oxygen, and silicon.

Hydrogen enters into the composition of microbial cells in the
form of water. It forms an important part of the various organic
and inorganic constituents of the microbial cell. It is subject also
to characteristic oxidation reactions by specific bacteria, considerable
energy being liberated in this reaction:

H2 + O = HoO

Oxygen is highly important in all biological reactions, including
both anaerobic (fermentation) and aerobic (respiration) processes.
It is important in cell synthesis and in organic matter decomposition.
Without it, no life would exist.

Silicon is present abundantly in the mineral framework of the
soil. It is found extensively in the cell substance of many soil organ-
isms, notably the diatoms, certain protozoa, fungi, and bacteria.
Silicon undergoes various transformations as a result of the direct
and indirect activities of microorganisms. When plant residues are
decomposed, silicon is liberated as silica and is allowed to accumu-
late. Silica may be rendered soluble through the action of carbon
dioxide and through the organic and inorganic acids produced by
microorganisms. This plays an essential role in rock weathering and
soil formation.

The action of microorganisms on silica has been little studied, even
though a high silica content is found in the stems of various plants.
In the cereals the rigidity of the straw is largely due to silica. An
abundance of sodium nitrate added to the soil was found to depress
the silica content of straw.

Selected Bibliograi)hy

1. Fred, E. B., and Haas, A. R. C, The- ctcliing of inarhlr In roots in tlie pres-
ence'of bacteria, J. Gen. Physiol., 1:631-638, 1919.

2. Gerretsen, F. C, Manganese deficiency of oats and its relation to soil bac-
^ teria, Ann. Botamj, N.S., 1:207-230, 1937.

Selected Bi]:)li()graphv 245

3. Iloiikins, C C, ami Wliitiiiji, A. L., Soil bacteria and phospliatrs, ///. Afir.
Expt. Sta. Bull. 190, 1916.

4. JiMison, C. A., Efh'tt of ck'foinposint^ orsfanif matlrr on llu' soluhility ol
certain inorganic constituents ot tlie soil, J. Agr. Research, 9:253-268, 1917.

5. Jofle, J. S., The role of snUnr in ajiricnltnrc, N. J. Aur. Expt. Sta. Bull. 374,
1922.

6. Joflc, J. S., and McLean, II. C, Alkali soil in\ estit^ations, Soil Sci., 17:395-
409, 1924; 18:13-30, 133-149, 237-251, 1924.

7. Kelley, W. P., Effect of nitrifying bacteria on the solnbilit\- of tricalcinni
phosphate, /. Afii: Research, 12:671-683, 1918.

8. Lipnian, J. C, McLean, H. C, and Lint, H. C, Snlfnr o.xidation in soils
and its effect on the a\ailabilit\' of mineral phosphates, So/7 Sn'., 2:499-538,
1916.

9. Rudolfs, W'., and Hellbroniier, A., Oxidation of zinc snifide liy microorgan-
isms. Soil Sci., 14:459-464, 1922; Compt. rend., 174:1378-1380, 1922.

10. Teakle, L. J. H., Phosphate in the soil solution as affected by reaction and
cation concentrations. Soil Sci., 25:143-162, 1928.

11. Waksman, S. A., and Joffe, J. S., The chemistry of the oxidation of sulfur
by microorganisms to sulfuric acid and the transformation of insoluble phos-
phates into soluble forms, 7. Biol. Chem., 50:35-45, 1922.

12. ^^'aksman, S. A., and Starkex', R. L., On the growth and respiration of sulfur-
oxidizing bacteria, J. Gen. Physiol, 5:28.5-310, 1923.

13. Whiting, A. L., Inorganic substances, especially aluminum, in relation to
the acti\ities of soil organisms, J. Am. Soc. Agron., 15:277-289, 1923.

14. Wright, D., Equilibrium studies with certain acids and minerals and their
probable relation to the decomi^osition of minerals by bacteria, Univ. Calif.
Publ. Agr. Sci., 4:247-337, 1922.

w/

Higher Plants and Soil Microorganisms

The microorganisms of the soil exert a variety of effects on the
growth of higher plants. Most of these effects are beneficial, but a
few can be injurious. Higher plants, on the other hand, influence
the growth of microorganisms in different ways, both stimulating
and injurious. The mutual interrelations between the higher plants
and the microorganisms may be summarized under the following
groups of reactions:

1. Microorganisms favor the growth of higher plants by affecting
the availability of various nutrient elements essential for plant growth,
notably carbon as carbon dioxide, nitrogen, and phosphorus.

2. Microorganisms favor plant growth through the production of
specific growth-stimulating or growth-regulating substances, such
as auxins and phytohormones.

3. Certain groups of microorganisms form a variety of symbiotic
relationships with higher plants.

4. Different microorganisms may compete with higher plants for
some of the nutrients present in the soil.

5. Some microorganisms have injurious effects upon higher plants,
either by directly attacking them as plant parasites, or by producing
certain toxic substances.

6. Certain viruses, notably bacteriophages, have the capacity to
attack useful bacteria and may thus prove to be indirectly injurious
to plant growth.

The plants, in turn, supply microorganisms with various nutrients
in the form of plant residues and excretion products. They also offer
a favorable medium for the growth of various groups of microorgan-
isms, either in the immediate vicinity of the roots or directly upon
the roots. By the excretion of toxic products, plants may also exert
various injurious effects upon the growth of microorganisms.

246

The Cvoncopl of Hliizosplu'io 247

The Concept of RhiJiiosphere

In 1904 Hiltncr introduccxl tlic concept oF the "ihi/osplu'it'" to
express tlie zone of increased niierohiological activity ininiechately
around the roots (jf higher phuits. This term came, in time, to desig-
nate the intimate relations between soil microorganisms and the root
s\ stems of higher plants. More recently, two zones of influence of
plant roots upon the microbiological population came to be recog-
nized, the root surface and the rhizosphere, both being often grouped
under the term "root region." Although considerable information has
now accumulated concerning the mutual effects of these two bio-
logical systems, we are still lacking a clear idea of the importance of
this phenomenon in the growth of higher plants.

These relationships may be considered midway between those of
true S)mbiosis, on the one hand, as in the case of the root-nodule
bacteria and the leguminous plants and of some of the mycorrhiza
formations, and, on the other, of the phenomena of parasitism. Some
of the relationships, like the utilization by the plants of the metabolic
products of microorganisms, are no doubt highly beneficial; others,
such as the possible curling and even more toxic effects of certain
antibiotics on the leaves of some plants, may be injurious.

The rhizosphere can be studied conveniently by means of the
contact slide method of Rossi and Cholodny. This consists in bury-
ing glass slides or cover glasses in contact with the plant roots; these
slides or cover slips are removed at various intervals, stained, and
examined microscopically. This method allows us to study the effect
of root growth upon the development of specific microorganisms.
Unfortunately, the larger forms like the nematodes and protozoa do
not adhere to the slides. This method established the fact, however,
that various bacteria, actinomycetes, and fungi find the root zone
a highly favorable medium for their development.

The nature of the plant and its age and the nature of the soil and
its treatment will influence considerably the nature and abundance
of the organisms. Starkey showed that alfalfa roots had only a slight
stimulating effect upon filamentous fungi, whereas eggplants had an
appreciable effect; sugar beet and corn were least effective. Legumes
exerted a particularly marked effect upon bacteria. Starkey was able
to distinguish two sources of nutrients provided by roots: (a) soluble
excretions, (h) sloughed-off dead root cells. Although root excre-
tions and detritus are the principal cause of the rhizosphere effect.

248

Higher Plants and Soil Microorganisms

Fig. 98. Microorganisms pliotographed on roots processed with iormalin-acetic-
alcohol and lactophenol with acid fuchsin: a, optical section of a massi\e bac-
terial colony completely encircling the base of a lettuce root Iiair; /;, bacterial
cf)lonies distributed over young maize root; c, relati\ely large spherical to o\oid
cells of undetermined nature clustered on pineapple root hairs ( from Linford ) .

The Concept of Khizospliere

249

other possible causes were eousidered, iiaineK, lowerinu; ol the con-
centration of certain mineral nutrients in* the soil due to absorption,
partial desiccation of the soil by absorption of water, increase in soil
carbonates following root excretion of carbon dioxide, contribution
of microbial foods by sloughed-off root portions or excretions.
Thom and Humfeld found that alfalfa, rye, and vetch stimulated

0

Beans

Cucumbers

Tomatoes,

vegetative

growith

Tomatoes,
fruit

0.1

0.02 0.04 0.06 0.08

Per cent carbon dioxide in the atmosphere

Fic. 99. Influence of concentration of carbon dioxide in the atmospliere on
growth of plants (from Lnndegardh).

the bacteria more than the fungi, and the latter more than the actino-
mycetes. In other studies, the rhizosphere population of manured
soil was reported to be much greater than that of unmanured soil,
although the effect of manuring was much greater upon the non-
rhizosphere population.

Garrett emphasized that it is necessary to distinguish between
rhizosphere effects characteristic of living roots and the increase in
the microflora of the root region in diseased roots and in healthy
but senescent roots; the latter phenomena are associated with the
initial stages of the microfloral succession occurring in moribund
and dead roots, whether a pathogen is the primary colonizer or not.
Thom and Humfeld observed a marked increase in numbers of root

250 Higher Plants and Soil Microorganisms

surface microorganisms in a root-rot-susceptible variety of tobacco
grown in infective soil, as compared with the same variety of tobacco
grown in healthy soil; similar effects for wheat were also demon-
strated. Starkey illustrated the intense development of microorgan-
isms in and about senescent and dying roots or portions of roots.

According to West and Lochhead, there exists in every soil inves-
tigated a balance between two general nutritional classes of bacteria.
On the one hand are bacteria capable of rapid growth on a single
substrate, and in opposition to them are others dependent for their
development on an ample supply of certain specific compounds. If
conditions favor an increase in one group, the incidence of the other
must correspondingly fall. These two workers demonstrated strik-
ing differences in distribution of these nutritional groups between
rhizosphere and control soils: in the rhizosphere soil, the percentage
incidence of organisms capable of growth on the basal medium alone
declined, whereas that of organisms with more complex growth re-
quirements increased. The increase in percentage incidence of the
group requiring growth factors was especially marked. It was found
later that a requirement for amino acids, and not for growth factors,
was the most important characteristic of the rhizosphere population.
Fertile soil is usually well supplied with growth factors, and the
growth factor heterotrophy does not, therefore, necessarily limit
growth of a microorganism in soil.

Influence of Soil Microorganisms upon Plant Growth

Among the numerous soil processes that are carried out by micro-
organisms and that directly affect the growth of higher plants, the
following may be analyzed in further detail:

Microorganisms decompose the plant and animal residues added
to the soil and the organic matter or humus in the soil itself. They
thus liberate the nitrogen and minerals necessary for the growth of
higher plants and produce considerable quantities of COo, which
is essential for plant growth.

Microorganisms oxidize and otherwise transform into forms readily
available to plants various minerals either introduced into the soil,
such as ammonium salts and sulfur, or formed there in the decompo-
sition of the organic residues.

Microorganisms synthesize a variety of organic substances from
the inorganic compounds in the soil and thus compete with higher
plants for available nutrients, notably the nitrogen and the minerals.

Microorganisms upon Plant Growth

251

Since tlu' soluhlc niatcrials toud to Ic^acli from the soil, this process
fax'ors conscr\ation of the nutrients in tlie soil in the absence of a
growing crop.

Under certain conditions, microorganisms reduce various oxidized

450

400

S 350

w 300

250

I 200

150

100

I I Average effects of all plants

■■■ Average effects of individual plants

2258,1 _^

527.4 ^IL. I

530.6— '
609 7 —

m

Q- u~) ca u<ri— OS
Colonies of
radiobacter

Fig. 100.

Carbon dioxide
evolved

Influence of plants upon the growth and acthities of bacteria in soil
(from Starkey).

substances, hke sulfates and nitrates, to substances which may be
to.xic to higher plants or which result in losses of available elements.
Microorganisms enter into various associations with plants that
are highly important in the growth of the plants. The growth of
leguminous plants is directly affected by the symbiotic nodule-form-
ing bacteria, making these plants independent of the supply of soil
nitrogen. The growth of many trees and other plants depends largely
upon the fungi that form mycorrhiza on their roots. This represents

252 Higher Plants and Soil Microorganisms

a phenomenon of symbiosis or of commensalism, whereby the fungi
favor in some way the growth of the plants. It has even been sug-
gested that fungi play a role in tuberization in plants as well as in
protein formation in certain seeds, as in legume association or my-
corrhiza formation.

Various groups of bacteria, fungi, and actinomycetes are capable
of penetrating the roots of plants and developing there, or of living
in close proximity, giving rise to a rhizosphere effect. It has been
suggested that certain soil organisms are capable of bringing about
a decided stimulation of the development of plant roots, either
through the production of hormones or through some other mech-
anism.

The formation by microorganisms of CO:., and various organic
acids results in a greater solubility of the soil minerals, particularly
the carbonates and phosphates, and some of the zeolites. The vari-
ous inorganic acids produced as a result of bacterial action, includ-
ing nitrous, nitric, and sulfuric acids, are also known to exert marked
effects in the solubilization of minerals in the soil. Since microorgan-
isms influence the concentration of gases in the soil atmosphere,
notably oxygen and CO^, root development of higher plants may
thus be appreciably affected.

Gerretsen found that, in sterile sand cultures with Cas(P04)2 as
the source of phosphorus, more of this element was absorbed when
the sand was inoculated with 1 per cent of garden soil. Various
experiments indicated that microorganisms play a highly important
role in making phosphorus available to the plants. When plant roots
were placed on an agar medium containing tricalcium phosphate,
the microorganisms developing about the roots caused the solution
of the phosphate. The factors affecting this process are the nature
and quantity of root excretions, presence and number of phosphate-
dissolving microorganisms in the soil, chemical composition of the
phosphate, and pH and temperature of the soil.

Microorganisms have often been reported to exert a highly favor-
able effect upon the germination of plant seeds and their subsequent
growth. This relation was even said to be symbiotic in nature.
More careful studies, however, established the fact that the normal
microflora of the seed is essentially that derived from the soil, and
that none of the organisms are specific for the seeds themselves.
Seeds and soil microorganisms are associated in an indirect way,
but they are physiologically unrelated. The growth and activities
of the two do not depend on one another, although some studies seem

Microorganisms upon Plant (.rowth

253

to indicate that ct-rtain seeds, at least, may eariy a typical microhio-
logical population. More recent studies tend to indicate that micro-
organisms ma\- produce substances which have a direct beneficial
t>flect upon plant growth, due to production of hormones, auxins, or
similar substances.

240

c 200
o

■-= ™ 160
; t Q.

i a> 120

Q. y

80

40

0

April May

June

July

Aug

Sept

Oct Nov

If) r±

II 1 1 IM 1 III
Bare soil-

i III ' III Lx-l-LJ^ 1

1 1

—

\'

~^-^

—

_ Maize soil —

*"'r'i i II 1 1 II 1

*• _j^^

1 1 1

1 1 III 1 III 1

1 1

oj VI

40

iS n

.-!()

c;

o --J

?n

Q. E

10

240

c

200

TT

I I

— (/)
rl:5 160

' S; o 120

CL nj

in "5 80

40 h
0

II 1

1 III 1 III 1 III

1 1 1 1 J III 1 II

-

Bare soil-^ .x--^

/

-

Potato soil--- / /-"^^

-

II 1

__^_^_^x:>^

\ III 1 1 M 1 II

1 III 1 III 1 II

40

QD fO

i2 CL

:-i()

O 03

20

0) O

Q. Q.

1 n

Fig. 101. Influence of crop upon nitrate content of the soil (from Ly(

Bizzell).

When a green manure crop is turned under and seeds are planted
immediately, the seedlings may be injured, partly by the action of
microorganisms, and partly by the presence in decomposing plant
residues of certain substances toxic to seedlings or germinated seeds.
As a result of the decomposition of the young plants, numerous
fungi develop, some of which are destructive to seedlings. The rapid
evolution of CO- and consumption of oxygen also produce conditions
imfavorable to oxidation, a phenomenon essential for the germina-
tion of the seeds. When seeds are planted two weeks after the plow-

254 Higher Plants and Soil Microorganisms

ing of the green manure, there is no serious injury to germination.

Various attempts have been made to explain the unproductiveness
of certain soils by the presence of substances that are injurious to
plant growth. These substances, designated as soil "toxins," were
believed to be formed in the soil as a result of activities of certain
groups of microorganisms. The "toxin" theory of soil fertility was
based upon the injurious action of such substances on the growth
of higher plants. The treatment of soil by heat, volatile antiseptics,
or simple liming was believed to overcome this injurious effect.

Under certain conditions, the presence of decomposed organic
residues and of microbial cell substance has a favorable effect upon
the growth of plants and microorganisms. Whether this is due to
the production by microorganisms of plant-stimulating substances, in
the nature of auxins, "auximones," or "phytohormones," or to the
"buflfering" or "poising" effect upon the oxidation-reducing potential
of the medium, or to the production of stimulating substances, what-
ever their nature may be, remains to be determined.

Krassilnikov and his associates have shown that various bacteria
and fungi may exert a highly stimulating or inhibiting effect upon
the growth of isolated roots of plants, depending on the nature of
the organism and of the plant. The inhibitory action may be due to
competition for nutrients, to injurious action of high concentrations
of growth-stimulating substances, or to the physical effect of bacterial
slimes in preventing the roots from obtaining sufficient nutrients.

Influence of Plants upon Microorganisms

The growing plant exerts a variety of influences upon the activi-
ties of the microorganisms in the soil. These can be briefly summar-
ized as follows:

1. Plants secrete soluble organic and inorganic compounds that
offer a favorable medium for the growth of microorganisms. Among
the chemically defined products, it is sufficient to mention formic,
oxalic, and malic acids, certain reducing and nonreducing sugars,
phosphatides, and various nitrogenous compounds. All these favor
the growth of many soil fungi and bacteria.

2. Plants supply large amounts of energy for the growth of micro-
organisms, in the form of dead roots and root hairs, root cap cells,
epidermal cells, and other waste products of plants.

3. Through their roots, growing plants continuously remove from
the soil various soluble minerals, including nitrates, phosphates, and

Influence of Plants upon Microorganisms

255

potassium salts. This results in a change in the composition of the
soil solution and in a modification of the activities of microorganisms.
4. Plants excrete considerable COj into the soil. This tends to
change the reaction of the soil. It also increases the solubility of
certain inorganic soil constituents and changes the composition of
the soil atmosphere.

Fig. 102. Influence ot 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, 12.5°C, 150°C for 2 hours (from Pickering).

5. Plants remove from the soil considerable moisture, thus exerting
an injurious influence upon the growth of microorganisms.

6. Plants modif)- the structure of the soil and thereby produce a
medium that is more favorable for the development of micro-
organisms.

7. Plants remove the nitrates from the soil, leaving the bases be-
hind and thereby affecting the reaction of the soil.

To measure the influence of plants upon microbiological activities
in the soil, certain well-defined procedures may be utilized. These
comprise some of the common measures of the microbiological state
of the soil, such as (a) the numbers of microorganisms, (b) nitrate
accumulation or nitrifying capacity of the soil, (c) oxidizing power
of the soil as expressed in terms of oxygen absorption or COo pro-
duction, (d) a. variety of other biological activities. It is often diffi-
cult to differentiate between the direct influence of the growing
plant and the influence of the plant products.

Among the bacteria which are particular!)' influenced by growing
plants, the Radiobacter group occupies a prominent place. A crystal

256 Higher Plants and Soil Microorganisms

violet medium is used for plating out the soil. The dye inhibits the
growth of the actinomycetes and the Gram-positive bacteria, but not
of the Gram-negative bacteria. Radiobacter colonies are raised,
smooth, glistening, with opaque centers and transparent edges.
When legumes are grown in a given soil, there is an increase in the
number of Radiobacter in that soil. The greatest numbers of this
group of organisms are found close to the plant, but frequently none
at all one foot away from the plant. Cowpeas, field peas, vetch, and
soybeans stimulate these bacteria, the increase being accompanied
by an increase in the consumption of nitrates and in the evolution
of CO..

When a single type of plant is continuously grown in a soil, it
leaves residues that may result in a change in the chemical compo-
sition of the soil, which in turn influences the bacterial population.
Some kinds of plant species favor certain types of bacteria and
inhibit others, and thus bring about a change in the bacterial equi-
librium in the soil. The new bacterial population produces a change
in the composition of the soil which may subsequently affect plant
growth, whereby some plant species are favored and others are
retarded. The continuous growth of a single type of plant, such as
wheat, flax, or clover, will bring about the development of fungi
pathogenic to this plant, making the soil "sick" for the particular
plant.

Starkey has shown that higher plants may affect some groups of
microorganisms differently from other groups; the extent of the
influence of various plants upon different organisms may vary. The
greatest increase appears to be brought about in the Radiobacter
group, although some striking effects may be observed upon the
general bacterial population. Potato increased the numbers of bac-
teria only to a slight extent, whereas rape produced some striking
changes. The influence of various plants upon the soil population
as measured by the number of Radiobacter per gram of soil depends
upon the stages of growth of the plant ( Table 56 ) . Slight effects are
observed in the early stages. The maximum occurs when plants
reach considerable size. Because of their longer growing period,
bieimials show a much more prolonged effect upon the soil organisms
than do annuals. The effect of higher plants upon the microbio-
logical population of the soil may be an important factor in bring-
ing about the seasonal fluctuations of microorganisms.

Neller measured the total CO- liberated from oxidation processes
taking place in the soil during plant growth. He found much more

InflucMK'c of Plants upon Microorganisms 257

Tmim-; j(>. Im'li kncic ok Dkvelopmknt ok IIkmiku Plants ui'ov Am noanck
OF Radiobactvr (froiii Sfarkey)

Number of Radiobacter, thousands per gram of soil

Avcrafie

U 6.'5 8G MIH 17;5 of All

ri:iiit D.iys l);iys Days Days Days I'nidils

Fallow 540 [h>{) !)()() ;{-2() 4'->0 (!2()

Oals 780 7,800 (J.S'iO 8G0 ()70 ;5,'->90

(nrn ()80 2,0^>0 .'{,180 .'5,340 IHIO ^2,040

Hcaiis 1,980 2,540 4,400 360 l,(i40 2,180

rotatoes 780 980 5,340 1,200 500 1,760

Table beets 840 1,540 3,560 2,180 1,380 1,900

Mangel beets 1,400 6,400 3,160 4,000 1,400 3,270

Rape 46.600 8,600 6,360 5,120 3,640 14,060

Sweet clover * 1 . 1 40 2 , 000 1 , 900 620 2 , 820 1 , 700

* For the sweet elover, the sampling periods were 25, 44, 67, 119, and 154 days.

rapid oxidation in a soil in which plants were growing than in the
corresponding nncropped soil kept under the same conditions of
moisture, aeration, and temperature. The growing roots exerted a
direct influence upon the decomposition of the organic matter in the
soil. This also brought about a greater liberation of available plant
nutrients and thus stimulated further plant growth. Neller suggested
that a symbiotic relationship exists between the growing plant and
the oxidizing organisms in the soil. The influence of nature of crop
upon the numbers of bacteria and evolution of COi> from soil is
illustrated in Table 57.

Table 57. Lnfluexce of Plant upon the Numbers of Bacterta and
Evolution of COo (from Neller)

COo Produced

by 1 Kg Soil

Ik.

[•teria

l)er

Soil

in

24 Hours

Plant

Gi

:am Soil

Reaction

i

It 20°C

millions

pH

nig

Triticum rulgare

49

6.75

69.4

Secede cereale

42

6.44

68 . 2

Avena satira

45

6 . 42

79.0

Beta vulgaris

78

6.89

74.3

Medicago sativa

120

() . 89

86.8

Tri folium pratrnsr

6.66

82.4

According to Starkey, the evohition of CO^ is greater from soils
in which plants are growing than in unplanted soil. The course of

258 Higher Plants and Soil Microorganisms

formation of the CO^ during the growing season is different for each
of the plants. There is a parallel influence of the plants on the
formation of CO2 and changes in the bacterial population in the
soil. This is expressed by a slight effect in the early stages of growth
of the plants, and by a greater effect with advance in vegetative
development and fruiting; the effect becomes less when the plants
begin to degenerate and die. The oxidation of the soil nitrogen to
nitrate was affected in a somewhat similar manner.

The fact that the roots of plants are surrounded by a film of bac-
teria actively respiring was taken as explaining the formation of COj
about the roots.

Grass growing in the vicinity of trees is usually observed to have
a harmful effect upon the trees. This was explained by the fact that
the surface roots of the trees are deprived of combined nitrogen by
the grass roots. Furthermore, by producing a soil atmosphere rich
in CO2, the grass causes the surface roots to grow down and thus
suffer from lack of oxygen. It has frequently been suggested that
the injurious effect is due to the formation of a toxin by the grass;
however, no evidence of this has been presented.

The root residues, in the form of sloughed-off portions and finer
rootlets, may influence greatly the nature of the population develop-
ing in their neighborhood. This is likewise true of the excretion
products of the plants. Plants also produce a variety of gases which
greatly influence the nature of the organisms developing in the par-
ticular area. Further evidence of the marked influence of growing
plants on the microbiological population of the soil is found in the
fact that a given soil decomposes cellulose with varying rapidity
according to the nature of the plants that have been growing in it.
The nature of the organisms taking part in the decomposition of
cellulose in a given soil varies with the plants grown in the soil.

It thus seems to be definitely established now that larger numbers
of microorganisms find a more favorable condition for their develop-
ment in close proximity to plant roots than at a distance. Nitrogen-
fixing and cellulose-decomposing bacteria are particularly prominent.
This may be because the plants excrete or leave in the form of
residues a certain amount of available energy; this would favor the
development of the nitrogen-fixing organisms. There is no evidence,
however, that fixation of nitrogen is increased around the roots. The
cellulose-rich residues would naturally favor the development of
cellulose-decomposing bacteria and fungi in the soil. The nature of

Selected Bihliogiaphv 259

those oruaiiisins c1c[)i'ik1,s u[-)()n tlic nature ot the residues, tlie nature
ot the soil, and the cmu ironnuMital eonditions.

Bacterization

Tlie term "baeterization" lias been applied to soil and seed inocula-
tion with bacteria and otlier microorganisms either to stimulate plant
growth or to combat the attack on plants by various pathogenic fungi
and bacteria. There is no doubt of the favorable effect upon the
growth of leguminous plants of seed or soil inoculation when prop-
erly adapted cultures are used. This is true also of certain mycor-
rhiza fungi upon nursery plants, especially on trees not previously
grown in a given area. The favorable effect, however, upon wheat
or other cereal plants or upon sugar beets of inoculation with Azoto-
bacter or other bacteria has not been established. In summarizing
these results, Jensen questioned the premise of nitrogen fixation by
Azotobacter, although he was inclined to accept the favorable effect
of growth factors elaborated by bacteria. Allison, as well, submitted
to severe criticism the value of the experiments on the use of Azoto-
bacter as a seed inoculant. He examined in detail the claims made
for the beneficial effects of such inoculation, that (a) nitrogen is
fixed by the bacteria living in the rhizosphere, largely on the root
excretions; (b) the added bacteria protect the plants against patho-
genic microorganisms either by discouraging their growth or by
destroying them; (c) the bacteria stimulate plant growth through
the production of hormones, auxins, vitamins, and other growth ac-
celerators or regulators. The last claim was considered the only
plausible one that may be of any significance as regards seed inocu-
lation.

Selected Bibliography

1. Allisun, F. E., Azotobacter inoculation of crops. I. Historical, Soil Sci., 64:
413-429, 1947.

2. Clark, F. E., Soil microorganisms and plant roots, Advances in Af^ron., 1:
241-288, 1949.

3. Crafts, A. S., Mo\ement ot \iruses, auxins, and chemical indicators in plants,
Botan. Rev., 5:471-504, 1939.

4. Garrett, S. D., Ecology of the root-inhabiting fungi, Biol. Revs., 25:220-254,
1950.

5. Gerretsen, F. C, The influence of microorganisms on the phosphate intake
by the plant, Plant and Soil, 1:51-81, 1948.

260 Higher Plants and Soil Microorganisms

6. Gustafson, F. G., Inducement of fruit de\elopment by growth-promoting
chemicals, Pwc. Natl. Acad. Sci., 22:628-636, 1936.

7. Harley, J. L., Mycorrhiza and soil ecology, Biol. Revs., 23:127-158, 1948.

8. Jensen, H. L., Bacterial treatment of non-leguminous seeds on agricultural
practice, Austral. J. Sci., 4:117-120, 1942.

9. Katznelson, H., Lochhead, A. G., and Timonin, M. I., Soil microorganisms
and the rhizosphere, Botan. Rev., 14:543-587, 1948.

10. Linford, M. B., Methods of obser\ing soil flora and fauna associated with
roots, Soil Sci., 53:93-103, 1942.

11. Lipman, J. G., A method for the study of soil fertilit>' problems, /. Agr. Sci.,
3:297-300, 1909.

12. Lochhead, A. G., and Thexton, R. H., Qualitati\e studies of soil micro-
organisms. VII. The "rhizosphere effect" in relation to the amino acid
nutrition of bacteria. Can. J. Research, C, 25:20-26, 1947.

13. Neller, J. R., The influence of growing plants upon oxidation processes in
the soil. Soil Sci., 13:139-159, 1922.

14. Nicol, H., Plant Growth Substances, Chemical Publishing Co., New York,
2nd Ed., 1941.

15. Parker-Rhodes, A. F., Preliminary experiments on the estimation of traces of
heteroauxin in soils, /. Agr. Sci., 30:6.54-671, 1940.

16. Schreiner, O., and Skinner, J. J., Nitrogenous soil constituents and their
bearing on soil fertility, U. S. Dept. Agr. Bur. Soils Bull. 87, 1912.

17. Starkey, R. L., Some influences of the dexelopment of higher plants upon
the microorganisms in the soil. VI. Microscopic examination of tlie rhizo-
sphere. Soil Sci., 45:207-249, 1938.

18. West, P. M., and Lochhead, A. G., Qualitati\e studies of soil microorganisms.
IV. The rhizosphere in relation to the nutritive requirements of soil bac-
teria. Can. J. Research, C, 18:129-135, 1940; Soil Sci., 50:409-420, 1940.

19. West, P. M., and Wilson, P. W., Biotin = co-enzyme R as a growth stimu-
lant for the root-nodule bacteria, Enzymologia, 8:152-162, 1940.

20. Wilson, J. K., and Lyon, T. L., The growth of certain microorganisms in
planted and in unplanted soil, .V. Y. (Cornell) Univ. Agr. E.xpt. Sta. Mem.
103, 1926.

♦/2-.

Associative and Aiita<>:()iiistic Effects
of Soil Microors>:aiiisins

Microorganisms live in the soil, not in the form of pure cultures,
hut as complex populations. Each particle of soil, no matter how
small, contains more than one type of organism. Many of these
organisms depend upon one another for direct and indirect nutrients;
some compete with one another for energy sources and for the ele-
ments and compounds used as nutrients. This results in the forma-
tion of numerous associations among the soil microorganisms in
which \'arious relationships exist, some favorable to one another
and others injurious. The abundance, in the complex soil population,
of each type of organism, the rate of its multiplication, and its physio-
logical activities are greatly influenced by the presence and abun-
dance of other organisms.

What Is a Soil Microbiological Population?

The quantitative and qualitative composition of a complex popu-
lation is controlled by the nature and availability of the nutrients;
the physical, chemical, and biological nature of the habitat; and the
environmental conditions, especially aeration, temperature, and mois-
ture supply. This is also true of the soil microbiological populations
(Table 58). The examination by suitable methods of a sandy or
clay soil, which is as free from organic materials as possible, will
reveal a microbiological population that is very limited in numbers
and types. Snow, for example, made a study of the abundance of
microorganisms in wind-blown soils. She found as few as 17,000
organisms per gram of soil containing about 0.3 per cent organic
matter; these organisms were largely bacteria, together with some
(10-15 per cent) actinomycetes and a few (0.56-2.0 per cent) fungi.
Another soil with 0.45 per cent organic matter gave, on the average,

261

262 Associative and Antagonistic Effects

59,666 organisms per gram, with only 0.61 per cent actinomycetes
and 0.27 per cent fungi. These organisms, even in such low num-
bers, were found to be made up of various distinct types, as brought
out by differences in pigmentation, staining reactions, and spore for-
mation. The limited quantity of nutrients brought in by subter-
ranean drainage or rainfall, by localized growth of a plant or an
animal, will result in formation and liberation of small amounts of
nutrients for keeping the microbial population alive. The organisms
capable of living in this environment find comparatively little com-
petition.

Table 58. Influence of Growing Plants on Number of
Microorganisms in Soil (from Starkey)

Microorganisms Found *

Sample of

'

Plant

Soil Taken

Bacteria

Actinomycetes

Fungi

Rye

Near roots

28,600

4,400

216

Away from roots

13, 200

3,200

162

Corn

Near roots

41,000

13,400

178

Away from roots

24,300

8,800

134

Sugar heet

Near roots

57,800

15,000

222

Away from roots

32,100

12,200

176

Alfalfa

Near roots

93 , 800

9,000

268

Away from roots

17,800

3,300

254

In thousands per gram of soil.

Among these organisms, the autotrophic bacteria are of prime im-
portance. These are highly specialized forms, capable of using as
sources of energy the traces of ammonia brought down by the rain
or the traces of hydrogen and methane found in the atmosphere.
Although very little fixed nitrogen is available to the autotrophic
bacteria, the traces produced by atmospheric discharges and brought
down by the rainfall will suffice, since energy is the all-important
limiting factor, and nitrogen-fixing bacteria are hardly able to exist
under these conditions until specialized higher plants become estab-
lished or complex forms of energy are made available. Microbial
life is thus at a minimum under these conditions and competition is
limited, since the carbon source required for cell synthesis, COu>, is
all-abundant. Only upon death of the microorganisms, when they
become themselves nutrients for other organisms, does competition
set in. A certain amount of association is possible, as when nitrate-

What Is a Soil Microbiological Population? 263

torining bacteria utilize the nitrite produced by ammonia-oxidizing
forms.

The next step in the development of a microbial population comes
when organic materials are made a\'ailablc. To simplify the reactions
in\olved, it is sufficient to consider the effect of the various chemical
constituents of plant life upon the growth of a complex population.
Since 80-99 per cent of the organic matter in plant materials is made
up of carbohydrates and lignins, the effect is a group of important
reactions. One ma\' take, for illustration, three groups of the non-
nitrogenous constituents, the glucose, the cellulose, and the lignin.

The simple carbohydrate can be attacked by many types of organ-
isms. When the nitrogen supply is low, only organisms capable of
fixing nitrogen of the atmosphere will be able to grow and utilize
glucose. Under these conditions there is very little competition,
since the two groups of bacteria capable of bringing this process
about, in the absence of the green plant, are aerobic forms {Azoto-
hactcr) and anaerobic types {Clostridium). These can only supple-
ment one another, the first assisting the second by consuming the
oxygen, and the second helping the first by breaking down its waste
products. Thus it has been shown that collaboration of these two
groups of organisms leads to greater fixation of nitrogen.

The cellulose leads to the development of totally different groups
of organisms, since it cannot be utilized directly by nitrogen-fixing
bacteria. Its decomposition is, therefore, controlled entirely by the
amount of available nitrogen. The abundance and nature of the
nitrogen and the nature of the environment influence greatly the
nature of the organisms developing at the expense of the cellulose.
A variety of associative and competitive phenomena may result. The
first is manifested when the cellulose is broken down by some organ-
isms to dextrin-like compounds or to simple carbohydrates. These
are transformed by other organisms to organic acids, which are finally
broken down by still other organisms to COo and water or to COo
and methane. Competitive processes result when the cellulose is
attacked by bacteria, lower or higher fungi, actinomycetes, or even
invertebrate animals. Whether one group or another becomes domi-
nant depends on the reaction of the soil, nature and amount of avail-
able nitrogen, oxygen supply, and temperature (Table 59).

Finally, lignin presents a different problem in regard to microbial
development. This substance, the chemical nature of which is still
in dispute, is more resistant to decomposition than most other organic
compounds synthesized by plant or by animal life. Although it is

264 Associative and Antagonistic Effects

known that various fungi, such as certain basidiomycetes, certain
actinomycetes, and certain bacteria, are capable of decomposing lig-
nin, very Httle is known concerning the mechanism of its breakdown.
This is due primarily to the inability of most organisms to attack
isolated lignin, which has apparently been changed chemically in
the process of its isolation, and to the changing nature of lignin with
the growth of the plant and in different types of plants.

Table .59. Decomposition of Alfalfa by Pure and Mixed ("rLTt'REs
OF Microorganisms

Total Hemi-

Alfalfa celluloses Cellulose NHs-N

Organism Decomposed Decomposed Decomposed Produced

per cent

per cent

per cent

my

Trirlioderma

9.3

\.l

0

61

Rhizopiis

6.(i

hi. 8

•2.9

53

Trichodcrma + Rhizopus

13.7

ii . 6

10.6

63

Trichoderma -\- Vitniiinghamdht

1.5.0

15.4

5. 7

47

Trichodcrma -\- Pa. fluorcsccns

10.5

14.5

6.4

3i

Streptomyccs 3065

10.6

43.0

'•23.'-2

5-1

Trichodcrma + Streptomyccs .30(5,5

Vl.o

14.6

4.8

56

Soil infusion

'28.4

40.9

50.8

21

These few illustrations will suffice to emphasize the fact that the
composition of a microbiological population is influenced by the
composition of the plant residues and by the environmental factors.
It leaves considerable room for numerous types of interrelationships
among the organisms making up this population.

The general interrelationships among living systems, as influenced
by environment and available foods, are frequently expressed by the
terms "biotic populations," "ecologic relationships," and "struggle
for existence." In most instances, only scanty consideration has thus
been given to microbial populations. This is due, on the one hand,
to the dominant interest that has centered upon higher forms of life,
and, on the other, to the fact that most knowledge gained from
the study aud utilization of microorganisms, especially their physio-
logical activities, has been derived largely from pure cultures rather
than mixed populations. Although the microbiological population
of soils and of water basins offers unusual opportunities for the study
of such relationships, the student of these tried to steer clear of the
complicated problems thus involved, and concerned himself as much
as possible with single organisms and with specific processes brought

Associative Effects 265

about hy tlu-in. Tlu' probltMu was considered nearly solved, once a
pure culture was obtained. On the other' hand, the investigator who
worked with the soil population as a whole usually polluted it to
such an extent b\- the addition of an excess of a single type of
material that any natural relationship among microorganisms was
thereb)' erased. The information gained from studies of "pure" and
"mixed" cultures was patched together to fit the complex natural
processes occurring in soils or in water basins, with the result that
often a "crazy quilt" arrangement resulted rather than a clear pic-
ture of the natural processes. This was due entirely to failure to
recognize that many of the processes carried out by microorganisms,
and often the very existence of these organisms, are greatly modi-
fied in the natural environment as compared with their growth in
pine cultures and in the test tube.

The study of the associative and antagonistic interrelationships
among microorganisms, especially the marked interest that has re-
cently been centered upon the production by these organisms of
antibiotic substances, has resulted in the accumulation of many
facts that permit more systematic generalization dealing with the
subject under consideration.

Associative Effects

The associative influences among microorganisms living in the soil
are numerous. They may be classified briefly as follows:

1. Effect of aerobic organisms upon the growth of anaerobes.
The aerobes consume the free oxygen in the soil atmosphere, thus
creating conditions that are favorable for the growth of organisms
not requiring oxygen, the anaerobes.

2. Preparation of an essential nutrient or substrate by one organ-
ism for the growth of another. This type of relationship is very
common in the soil. Nitrite-forming bacteria oxidize ammonia to
nitrite, thus producing a substrate which is required for the activi-
ties of the nitrate-forming bacteria, since the latter are not able to
use any other source of energy. Proteolytic bacteria hydrolyze pro-
teins to amino acids, thereby producing substances which are essen-
tial for the activities of peptolytic bacteria or of organisms that
cannot attack native proteins. Cellulose-decomposing bacteria give
rise to organic acids and other intermediary products essential for
the activities of various organisms which themselves cannot attack
cellulose.

266 Associative and Antagonistic Effects

3. Production by certain organisms of specific substances which
are essential for the growth of other organisms. These are fre-
quently designated as growth-promoting substances or as vitamins.

4. Utilization and destruction by various microorganisms of the
metabolic waste products of other organisms. In this process, the
former organisms create conditions which are favorable for the
continued growth of the latter.

5. Dependence of certain organisms upon others for carrying out
life activities; this association becomes one of symbiosis. Miscel-
laneous associative relationships exist among microorganisms; for
example, the living together of algae and Azotobacter, the former
synthesizing carbon compounds and the latter fixing nitrogen. This
is true of associations of leguminous plants with root-nodule bacteria,
of coniferous trees and other higher plants with mycorrhiza fungi,
of insects with fungi and bacteria; in the association between an
insect and an actinomyces, the latter provides some growth substance
for the former.

Antagonistic Effects

Antagonistic interrelationships are also very common among mem-
bers of the soil population, whereby one organism, directly or indi-
rectly, affects injuriously the activities of another organism. These
interrelationships may also be briefly summarized:

1. Competition among microorganisms for available nutrients.
This may occur between organisms belonging to the same group,
as between two types of bacteria, or between organisms belonging to
different groups, as between bacteria and fungi.

2. Creation by one organism of conditions which are unfavorable
for the growth of another, as by changing the reaction of the medium
to acid, by the production of inorganic (nitric, sulfuric) or organic
(citric, oxalic, fumaric, butyric, lactic) acids.

3. Production by one organism of specific substances which are
injurious to growth of other organisms (Table 60). Here belong
such well-defined compounds as alcohols and quinones, as well as
the numerous antibiotics. These substances are frequently classified
as soil toxins, the exact nature of most of which still remains un-
defined (Table 61).

4. Direct parasitism of one organism upon another. Here belong
the various effects of fungi upon bacteria, of bacteria upon fungi, of
fungi and nematodes upon insect larvae. One of the significant as-

Antagonistic Effects 267

pects of parasitism among soil microorganisms is the attack of various
bacteria and fungi upon plant-parasitic Insects and nematodes.
5. Prcdaceous effects, or the feeding of one organism upon an-

FiG. 103. Dexelopnient of antagonistic fungi on bacterial-agar plate (from
Waksman and Horning).

Other, as in the consumption of bacteria by protozoa, of fungi by
insects, of nematodes by one another.

Many organisms are capable of producing substances that are in-
jurious to their own development {isoantagonistic) or to the growth
of other organisms in close proximity (heteroantagonistic) . This is
largely the reason why certain fungi and bacteria are capable of
growing in virtually pure cultures even in a nonsterile environment.
It is sufficient to cite the production of lactic and butyric acids by

268

Associative and Antagonistic Effects

Table 60. Antagonistic Action of Pseudomonas fluorescens upon Various
Microorganisms (from Lewis)

Percentage of Aged Medium in the Agar

Organism

0.5

1.0

^2.5

5.0

10

15

^20

30

40

50

B. cereus

+

B. mycoides

-

-

+

B. anthracis

-

+

B. vulgatus

-

-

+

B. subtilis

-

-

+

B. megatherium

-

+

R. cinncharcus

—

+

R. roseus

-

-

+

M. fiavus

-

-

-

+

N. catarrhalis

-

-

-

+

Ps. aeruginosa

-

-

-

-

-

-

-

-

-

-

Ps. fluorescens

—

-

-

—

-

-

-

-

-

-

S. lutea

-

-

-

+

S. marcescens

-

-

-

-

-

+

S. albus

-

-

+

S. aureus

—

-

-

+

S. citrens

-

-

+

K. pneumoniae

-

-

-

+

V. comma

-

+

Ch. violaceum

-

+

E. typhi

-

-

+

Sh. paradysenteriae

-

-

+

S. enteritidis

-

-

-

+

S. suipestifer

-

-

-

+

S. pullorum

-

-

-

+

E. coli

-

-

-

-

-

-

+

A. aerogenes

-

-

-

-

-

-

+

Ph. bovlesii

-

-

+

Sac. marianus

—

—

—

—

—

—

—

—

—

—

Sac. ellipsoideus

-

-

-

-

-

-

-

+

Sac. pastorianus

-

-

-

-

-

-

-

-

+

Zygosac. priorianus

-

-

-

-

-

-

-

+

Torula sphaerica

-

-

-

-

-

-

-

-

-

-

A. nigcr

—

—

—

—

"

"

"

"

+ denotes complete iiiliihition.

Antagonistic Effects 269

'l\iii,i-: (II. SiuvivAL OK I'Jucluri cilia coli. in Soil and l)i:\Kr.oi'MK\T
OF Antagon'Ists (from Waksmaii and W Inili)

N'umluTs o( orf^aiiisms in I lioiisands |)cr gram (lr\\- soil.

\uinl)or of Total

liuuhatioM ICiiricliiiuMit.s K. coli Xumber of Nuiiihcr of

of Soil witli K. roll Cells Haclcria .\nlaK'(>iiisls *

days

0 .. Few !»,]()(» t

5 I fi , 800

33 ."> i;JO

127 11 0 40,000 Tj.TOO

* An antagonistic colony is ()m<> surrounded hy a lialo of dissolwd K. coli ct'lls on the
plate.

t Control soil, not receiving any enrichments.

the corresponding bacteria; of citric, oxalic, and gluconic acids by
Aspergillus niger; of fumaric and lactic acid by Rhizopus nigricans;
of a number of alcohols by various yeasts, bacteria, and fungi; and
of certain phenols and quinones by various fungi. These substances,
as well as a great number of other compounds which, for lack of

Fig. 104. Bacterial plates made from soil, showing clear zones surrounding colo-
nies of antagonistic organisms ( from Stokes and Woodward ) .

more exact information, are usually designated as "lethal," "toxic,"
or "growth-inhibiting" compounds, and more recently as "antibiotics,"
have frequently been looked upon as protective metabolic products
formed by microorganisms in "their struggle for existence." Some
may play a highly significant part in the life of many organisms; the
role of others is still a matter of speculation.

270 Associative and Antagonistic Effects

Microbial Equilibrium

The numerous microorganisms inhabiting the soil are Hving largely
in a state of mutual equilibrium. Any modification of this equi-
librium results in a number of changes in the nature and abundance
of the microbiological population. The numerous interrelationships
among these organisms permit not only an understanding of their
specific ecological nature under a certain set of conditions, but also
a better understanding of the metabolic products resulting from
the activities of this population. Since the complex nature of this
population does not permit its treatment as a whole, certain relation-
ships among different organisms may be isolated and examined sep-
arately. Attention may be directed, for example, to the relations
between the non-spore-forming bacteria in the soil and the spore-
formers, of the actinomycetes and the bacteria, of some fungi and
other fungi, of bacteria and fungi, of nonpathogenic organisms and
pathogens, and of protozoa and bacteria.

Conn and Bright found that, when Bacillus cereus and Pseudo-
monas fliiorescens were inoculated simultaneously into sterile manured
soil, the former failed to develop, whereas the latter grew abun-
dantly. Lewis reported that Ps. fliiorescens repressed the growth of
B. mycoides and of other spore-forming bacteria and micrococci;
however, Aerobacter aerogenes and Serratia marcescens were highly
resistant; fungi were not inhibited; yeasts were inhibited only to a
limited extent, and actinomycetes were more sensitive. Lewis also
confirmed results of other investigators that the production of bac-
tericidal and inhibitory substances by bacteria depends on the
amount of available oxygen; these substances were found to be
thermostable and were adsorbed by charcoal and by soil.

Greig-Smith demonstrated that various actinomycetes are capable
of producing substances toxic to bacteria. The fact that actinomy-
cetes grow rather slowly suggested to him the possibility that they
comprise the factor limiting bacterial development in the soil. Cer-
tain actinomycetes were later found to be antagonistic to S. pyogenes
and to spore-forming bacteria, but not to Ps. aeruginosa. The latter,
because of its capacity to produce pyocyanase, was believed to be
capable of vaccinating the substrate against the growth of other
microorganisms.

The antagonistic activities of microorganisms have received par-
ticular attention as potential agents for suppressing the growth and
even for destroying bacteria and other microorganisms capable of

Microbial Equilibiiuin

271

producing luiinan and animal diseases, and possibly als(j fungi and
bacteria causing plant diseases. A nunlber of theories have been
proposed at xarious times in an effort to explain the mechanism of

Fig. 105. Antagonistic effect of one fungus, Pseiideurotium zonatum (center),
upon another, Trichoderma lignorum (from Goidanich et al.).

antagonism of one organism to another. These theories may be sum-
marized as follows:

1. Exhaustion of available nutrients in the medium or substrate.

2. Physicochemical changes, produced by growing one organism
in a certain medium, which affect the activities of another. These
include changes in osmotic pressure, surface tension, oxidation-reduc-
tion potential, and reaction.

3. Certain types of reactions, such as radiation effects, which may
be designated as action at a distance.

4. Space antagonism or competition for available space in a given
medium.

5. Production of specific enzymes, either by the antagonist itself
or as a result of autolysis of the antagonized cells, which have the
capacity of lysing or dissolving the cells of other organisms.

272 Associative and Antagonistic Effects

6. Destruction of certain organisms by others, as that of bacteria
by protozoa, or the parasitizing of some organisms upon others, as
of certain nematodes upon Japanese beetle larvae.

7 Production by certain microorganisms and liberation of specific
substances that have a selective bacteriostatic and bactericidal effect,
or fungistatic and fungicidal action, or both, namely, antibiotics.

Effect of Protozoa upon Soil Bacteria 273

Of these theories, oiiK tlie last two deserve careful consideration
iroin the point of view of soil microbiological processes and their
effect upon plant growth.

Effeci^ of Protozoa upon Soil Bacteria

"The protozoan theory of soil fertility" was suggested by Russell
and Hutchinson. It was based upon the belief that the capacity of
protozoa to consume some of the bacteria is responsible for the
infertility of certain soils.

The results of later and more detailed investigations on the rela-
tion of protozoa to bacteria, however, fail to support this theory.
When protozoa are added to cultures of specific bacteria concerned
in known important soil processes, such as ammonia-forming and
nitrogen-fixing bacteria, they are able to feed upon these bacteria
and to bring about considerable reduction in their numbers. This
capacity is not necessarily accompanied by a detrimental effect upon
the specific processes for which these bacteria are responsible; the
effect of the protozoa may actually be beneficial. It has, therefore,
been suggested that the presence of protozoa in the soil, even if
accompanied by the consumption of bacteria, may result in keeping
the latter at a level of maximum efficiency.

The theory that protozoa play a controlling part in soil fertility
was based upon the changes in bacterial numbers and activities as a
result of partial sterilization. When the protozoa were destroyed by
heat or chemicals, the bacteria were found to multiply rapidly. This
was believed to lead to more active decomposition of the organic
matter, to greater liberation of nitrogen, and to improvement in soil
fertility. This explanation was based upon several assumptions
which were not fully justified. It was assumed, for example, that
bacteria are the only important soil organisms responsible for the
decomposition of organic matter; actually it has been repeatedly
shown that fungi, actinomycetes, and other organisms are also ca-
pable of bringing about this process. It was further assumed that
protozoa, by consuming some of the bacteria, especially those de-
composing organic matter and forming ammonia, restrict bacterial
development and, ipso facto, organic matter decomposition. The
fact was overlooked that fungi and actinomycetes of the soil could
bring about, just as well as the bacteria, the decomposition of soil
humus and liberate the nitrogen as ammonia, a process which could
thus take place even with the elimination of all the bacteria.

274 Associative and Antagonistic Effects

When protozoa were found to exert a favorable effect upon various
processes brought about by bacteria in controlled laboratory experi-
ments, it was assumed that similar action is exerted in the soil. The
protozoa were thus found to be not injurious but actually favorable
to soil processes. This assumption, however, may also be open to
question; no consideration is given to the fact that the presence of
numerous other organisms in the soil modifies considerably the
activities of the protozoa. The use of artificial media may give a
one-sided concept of the significance of protozoa in soil processes.

Direct microscopic methods of soil examination have revealed the
fact that protozoa make up only a small portion of the soil popula-
tion, both in numbers and in the total amount of active cell sub-
stance. Their ability to reduce bacterial numbers in normal soil is
very slight. The indirect method of studying protozoa in solution
media, where the types developing and the activities resulting are
quite different from those occurring in the soil, has been largely re-
sponsible for the exaggerated importance attached to these organ-
isms. Certain observations have also been made on the toxic action
of different bacteria upon protozoa. In some cases protozoa were
able to develop a certain resistance to the action of bacterial prod-
ucts.

It is now generally agreed that partial sterilization of soil brings
about the destruction of most parasitic insects and fungi. In this
process, a large amount of organic matter is made available for
the surviving bacteria (Table 62). These soon begin to develop at
the expense of the available organic matter and bring about the
liberation of large amounts of nitrogen as ammonia. This ammonia
accumulates in the soil, since it cannot be nitrified, because of the
destruction of the nitrifying bacteria by the treatment. It favors
increased plant growth.

The effect of organisms destructive to pathogens and their use
in controlling various plant diseases offer great practical potentialities.

Production of Antibiotic Substances by Microorganisms

Tremendous interest has been aroused in recent years in the sub-
ject of antibiotic substances, especially from the point of view of
their possible utilization as chemotherapeutic agents. These sub-
stances are produced largely by soil-inhabiting microorganisms.
They are classified on the basis of the organisms producing them,
such as penicillin, actinomycin, or streptomycin, or on the basis of

Production of Antibiotic Substances

275

T.vHi.E G^i. Srn\ ivAL OF Bactkria Added to Sqil and THf:iii Kkfect upon the
Soil Micuouiologicai. Pf)PULATiON

Organisms Ili-covcrcd *

Inoculuiu

Control soil
E. coli added f
E. coli added J
E. coli added
Control soil
E. coli added
E. coli added J
E. coli added

* In thousands per gram of soil.

t Washed suspension of E. coli cells added at start and after 5 days.

I CaCOs added to soil.

Inc

uhation

Tem-

Time

perature

days

°C

5

28

5

28

5

28

5

37

33

28

33

28

33

28

33

37

Col if or m

Total

Bacteria

21,400

200

25 , 600

6,800

39,700

3,500

22,800

4 , 700

5,900

10

22,100

130

17,600

140

23,000

10

Fig. 107. Cup method for quantitative measurements ot concentration of anti-
biotics.

276 Associative and Antagonistic EflFects

the organisms affected by them, such as mycocidin, or on the basis of
their chemical composition, such as chloramphenicol. They differ
greatly in their chemical properties, toxicity to animals, and in vitro
vs. in vivo activities.

Cup method against fungi.

Many microorganisms isolated from the soil have been found ca-
pable of producing antibiotics. Among the actinomycetes, for
example, 10-50 per cent of all organisms tested were found to have
such properties. Both spore-forming bacteria and non-spore-formers
are able to produce antibiotics. Those produced by the spore-formers
include tyrothricin, bacitracin, subtilin, and polymyxin; those pro-
duced by the non-spore-formers include pyocyanase, pyocyanin,

Production of Antibiotic Substances 277

prodigiosin, nisin, and colicincs. The soil fungi have yielded a large
number of antibiotics, most important of which is penicillin; others
include mycophenolic acid, gliotoxin, clavacin, gladiolic acid, che-

FiG. 109. Streptomyces griseus, streptomycin-producing strain. Vegetati\e and

aerial mycelium.

tomin, penicillic acid, fumigatin, and fumigacin (Table 63). The
actinomycetes have already yielded more than seventy antibiotics,
some of which, notably streptomycin, chloramphenicol, aureomycin,
terramycin, and neomycin, have found extensive practical applica-
tion. Others include actinomycin, streptothricin, actidione, strep-
tocin, xantomycin, viomycin, antimycin, fungicidin, and fradicin.

Table 63. Antagonistic Interrelationships among Different Fungi

Antagonist
Acrostalagmus sp.
Alternaria tenuis
A. clavatus
A.flavus
A. niger
Botrytis allii
Botrytis cinerea
Cephalotheciiim roseum
Curmiiighamclla elegans
Fusarium lateritiu m
Fusariitm sp.
Gliocladiiim sp.
HelmintJioaporium sp.
H. teres
H. sativum
Mucor sp.
Penicillium sp.
Peziza sclerotiorum
Peziza trifolioriiin
Sclerotiuin rolf.s-ii
Sterigmatocy.s-fis sp.
Thu m n idiu rn elegans
Torula suganii
Torulopsis sp.
T. lignorum
Verticillium sp.

Organisms Affected
Rhizodonia
Ophiobolus
Various fungi
Peziza

Peziza, Rhizodonia
Monilia, Botrytis, etc.
Rkizoctonia
Helminthospori um
Monilia
Rhizodonia
Deuterophoma

Helminthosporium, Mucor, etc.
Colletotrichum, Fusarium, Botrytis, etc.
Fusarium, Ustilago, Helminthosporium, etc.
Ophiobolus
Ophiobolus, Mucor

Peziza, Rhizodonia, Ophiobolus, Fusarium
Mucor, Trichothecium, Dcmatium, etc.
Peziza

Helminthosporiu m
Alternaria
Mucor

Aspergillus, Monascus, etc.
Blue-staining fungi

Rhizodonia, Armillaria, Phytophthora, Pythiitni, etc.
Rhizodonia

Tryptone consumption

pH changes

Streptothricin production

2 4 6

Incubation period, days

10

2 4 6 8

Incubation period, days

10

Fig. 110. Metabolism of S. lavenduhe and production of streptothricin (from

Woodruff and Foster).
278

Production of Antibiotic Substances

279

In view of the fact that the organisms producing antibiotics are
of soil origin, the question has naturally been raised of what im-
portance those substances are in soil processes. It has been estab-
lished, for example, that various bacteria, both beneficial and harm-

FiG. 111. Electron micrograph of actinophage, type I, of streptomycin-producing
Streptomtjces griseus X31,000 (courtesy of Squibb Institute for Medical Re-
search ) .

ful, are affected by certain antibiotics; the former include the root-
nodule bacteria and Azotohacter, and the latter include bacteria
causing various blights and other plant infections.

The effect of antibiotics upon the soil microbiological population
has given rise to various speculations.

On the one hand, certain claims have been put forth that this
phenomenon is of only minor importance in soil processes. The
following reasons have been presented to substantiate this view:

280 Associative and Antagonistic Effects

(a) the production of antibiotics is dependent upon the presence of
specific nutrients which usually are not found in normal soil; (b)
some antibiotics are readily destroyed by different soil-inhabiting
microorganisms; (c) the soil organisms exposed to the action of
antibiotics tend rapidly to develop resistance to them; (d) the sur-
vival or predominance of various microorganisms in the soil does not
appear to be correlated with the capacity of such organisms to
produce antibiotics.

On the other hand, claims have been made which tend to suggest
that, under certain conditions at least, antibiotics may play a part
in soil processes. These are based on the following observations:
(a) the presence of small amounts of antibiotic substances in the
soil; (b) the formation of antibiotics by various pure cultures of
microorganisms in sterile soil; (c) the persistence of certain anti-
biotics added to the soil; (cZ) the capacity of various soil-inhabiting
organisms to inhibit the growth of plant pathogens; (e) the favor-
able eflFect upon the control of certain plant pathogens exerted by
stable manures, green manures, and other materials which favor the
development of antibiotic-producing organisms. Claims, not fully
confirmed, have been made that inoculation of soil with antagonistic
organisms will result in a depression in the development of the
pathogens.

The presence in soil of substances toxic to plant growth has also
been definitely established. It is not known whether these are re-
lated to the antibiotics. It is known that some antibiotics, like actino-
mycin and glutinosin, have the capacity of causing certain plant
diseases, such as curly tips. There are not enough established facts,
however, to permit generalizations concerning the importance of
antibiotic substances, or of the organisms producing them, in the
control of soil fertility.

Selected Bibliography

1. Baron, A. L., Hamlhook of Antibiotics, Rdnliold Puljlishing Corp., New
York, 1950.

2. Florey, H. W., Chain, E., Heatley, N. C, Jennings, M. A., Sanders, A. C,
Abraham, E. P., and Florey, M. E., Antibiotics, a Survey of Penicillin,
Streptomycin, and Other Antimicrobial Substances from Fungi, Actinomy-
cetes. Bacteria, and Plants, 2 vols., Oxford Uni\ersity Press, New York, 1949.

Selected Bibliography 281

3. Karcl, L., ami Koath, 1'",. S., A Diclionani of .\ntil)ii>sis, (loluiuhia Um'\crsity
Press, New Yi)rk, 1951.

4. Pratt, R., and Dulrciun, J., Antil)ititics, J. li. Lippiucoll (Jo., rliiladclpliia,
1949.

5. Waksman, S. A., Microbial Antagonisms and Antibiotic Substances, The
Commonwealth Fund, New York, 1947.

6. Waksman, S. A., ct iil., Strcptontt/cin, Williams & Wilkins Co., Baltimore,
1950.

13

Disease-Producing Microorganisms
in the Soil and Their Control

Survival of Human and Animal Pathogens in the Soil

Microbes capable of causing various human and animal diseases
find their w^ay into the soil and into water basins in very large num-
bers, either in the excreta of the infected host or in the dead and
infected residues of the latter. If one considers the millions of years
that animals and plants have existed on this planet, one can only
surmise the great numbers of microbes causing the numerous dis-
eases of all forms of life that must have thus been introduced into
soils and surface waters. What has become of all the bacteria caus-
ing typhoid fever, dysentery, cholera, diphtheria, pneumonia, bu-
bonic plague, tuberculosis, and leprosy in man, mastitis and abortion
in cattle, and numerous diseases of other animals? This question
was first raised by medical bacteriologists in the eighties of the last
century. The soil was searched for the presence of bacterial agents
causing infectious diseases and responsible for epidemics. The re-
sults obtained established beyond doubt that, with very few excep-
tions, organisms pathogenic to man and to animals do not remain
alive in the soil for very long.

A few disease-producing microorganisms, however, are able to
survive in the soil for considerable periods. One need only mention
the organisms causing tetanus, gas gangrene, skin infections, actino-
mycosis and blackleg in cattle, coccidiosis of poultry, hookworm in-
fections, trichinosis, enteric disorders in man. To these must be
added diseases caused by various other bacteria, actinomycetes, and
fungi. This is also true of numerous plant diseases, such as potato
scab, root rots, take-all of cereal crops, and the damping-off diseases
of vegetables. The great majority of disease-producing microorgan-
isms, notably the human and animal pathogens listed above, are able
to remain in an active and reproducible state in the soil for only

282

Survival of Pathogens in the Soil 283

very short periods. It is also important to cite the fact that typhoid
and dysentery bacteria, which are known' to contaminate watersheds
and water suppHes, sooner or later disappear. No one now raises
the question concerning the role of the soil as the carrier of these
disease-producing agents or as the cause of severe or of even minor
epidemics. This rapid disappearance of disease-producing bacteria
mav be due to several factors, such as unfavorable environment,
lack of sufficient or proper food supply, destruction by predaceous

Fig. 112. Eflect ot soil organisms against parasitism b\' P. vohitiuu on Agrostis

(from van Luijk).

agents, such as protozoa and other animals, and destruction by vari-
ous saprophytic bacteria and fungi considered antagonists.

Jordan and his associates found that Eberthella typhosa survived
in sterilized tap water for 15-25 days, as against 4-7 days in fresh
water; it died off even more rapidly (in 1-4 days) in raw river or
canal water. The degree of survival of this organism in water was
found to be in inverse ratio to the degree of contamination of the
water, the saprophytic bacteria being directly responsible for the
destruction of the pathogen. Freshly isolated organisms survived
a shorter time than laboratory cultures, and higher temperatures were
more destructive than lower ones.

The presence of certain bacteria in water is often found to hinder
the survival of E. typhosa. When Tseudomonas aeruginosa, on the
other hand, is present in drinking water, it may not be accompanied
by any other bacteria. Media inoculated with this organism and
with Escherichia coli gave, after 13 days' cultivation, cultures of

284 Disease-Producing Microorganisms

only Ps. aeruginosa; however, the two organisms can coexist in
sterihzed water. Vibrio cholerae does not survive very long in fresh
water, although long enough to cause occasional epidemics.

The addition of typhoid bacteria to a well-moistened and culti-
vated soil brings about rapid destruction of the organisms. The
same phenomenon occurs when a culture of these organisms is added
to that of a soil microbe. An antagonistic relation is often found to
exist in some soils but not in others; this is traced to the presence
of specific bacteria. Frost reported a marked reduction in numbers
of typhoid bacteria added to the soil, 98 per cent of the cells being
killed in 6 days. It was suggested that in the course of a few more
days all these cells would have disappeared from the soil. On the
other hand, under conditions less favorable to the antagonists, the
typhoid organism survived not only for many days, but even for
months.

Escherichia coli is rapidly crowded out by other organisms in
manure piles and in soil. The dysentery and typhoid organisms dis-
appear rapidly, in 12 and 16 hours, in sea water; the paratyphoid
organisms have been found to survive for 21 and 23 days. Sea water
appears to contain an agent, other than its salts, which exerts a
bactericidal effect.

Under conditions prevailing in southern England, Mycohacterium
tuberculosis was found to remain alive and virulent in cow's feces,
exposed on pasture land, for at least 5 months during winter, 2
months during spring, and 4 months during autumn; in summer, no
living organisms were demonstrated even after 2 months; under pro-
tection from direct sunlight, the survival period was longer. Bovine
tubercle bacteria have been detected in soil and manure, and on
grass up to 178 days after infection, but not later. When M. tubercu-
losis was added to nonsterile soil, it was slowly destroyed; the plate
coimt was reduced to about one-sixth of the original in 1 month.
Brucella melitensis survived in sterilized tap water for 42 days, as
compared to 7 days in unsterilized water; it survived in sterilized
soil 72 days, as compared to 20 days in unsterilized soil.

In addition to the above pathogenic organisms, others which have
the capacity of causing infections or of producing potent toxins in
human foodstuffs under proper conditions are found abundantly in
the soil. These include the tetanus and gas gangrene organisms,
on the one hand, and the botulinus, on the other. These organisms
may be present even in virgin soils.

Sui\i\al ot Piithoiicns in the Soil 285

'ti

Clostiicliuni tcUiiti appears to be also uiiivt'isalK tlistrihulcd in
soils fertilized with animal manures and' subject to the dust of the
streets. Nicolaier demonstrated the presence of this organism in
more than 50 per cent of the soils examined, an observation later
confirmed by others. Of 100 Scottish soils examined, 4 gave cultures
producing botulinus toxin and 5 tetanus toxin. It was even sug-
gested that the tetanus organism develops in rotting straw or manure,
taking a part in processes of decomposition. The presence of this
organism in the soil has also been ascribed to fecal excretions, be-
cause of its de\'elopment in the intestine.

The subject of the gas-gangrene-producing bacteria has received
special consideration in connection with the study of war wounds
and trench fever. Spores of CI. sporogencs, Cl. welchii, CI. teiiius,
CI. ocdemoticns, Cl. bifermentans, Cl. cochleariits, Cl. tetani, and of
other bacteria haxe been found in all soils of Central Europe.

The nature of the soil, or its physical, chemical, and biological
conditions, have a marked influence upon the survival of these or-
ganisms in the soil. The bacterimn causing fowl typhoid {Shigella
gallinorinn) will not remain in the soil for more than a week at a
reaction of pH 6.2-6.4 or lower. At a pH of 6.7-7.0, however, the
organism does not seem to be affected and will survive in the soil
for 40-70 days. The organism causing white diarrhoea in chickens
(Sh. pullorum) shows somewhat greater susceptibility to acid soils
than Sh. gallinarum; it survived for more than 64 days in soils of pH
7.0. In moist soils, the organism was more viable and less sus-
ceptible to lower pH than in dry soils; it survived for 8 days in soils
of pH 6.2-7.0. Mycobacterium tuberculosis will survive in the soil
for many years, without losing its virulence.

The causative agents of human and animal actinomycotic diseases
are often claimed to be brought about by soil organisms or forms
harbored upon plants. Klinger drew attention to the fact that none
of the aerobic actinomycetes commonly found on grasses and in
straw infusion (also in soil) were isolated by him in any actinomy-
cotic case. Only anaerobic forms were obtained from the latter;
these developed on most media at temperatures above 30° C, and
only seldom were cultures obtained which made a scant growth
under aerobic conditions. Mixed infections consisting of anaerobes
growing at body temperature together with aerobes are often ob-
tained. We have to do here with species which have adapted them-
selves to a svmbiosis with warm-blooded animals, and which have
almost nothing in common with aerobic saprophytes. There is no

286

Disease-Producing Microorganisms

doubt, however, that some aerobic actinomycetes are capable of
causing infections of men and animals.

The hookworm disease, caused by Ancijlosfoma duodenale and
Necator americamis, is primarily due to soil pollution. The larvae
were found to develop for as long as 6 months in soil protected by

— 2

12

10

7.0

Fig. 113. Relation of soil reaction to the growth of scab organism and occur-
rence of potato scab (from Dippenaar).

vegetation. The physical, chemical, and biological soil conditions
have a very important influence upon the development of hookworm
larvae from infected feces and upon the continued life of these larvae
in the soil. The larvae are found largely in the capillary film of mois-
ture surrounding the soil particles.

In spite of the gradual and even rapid destruction of some patho-
genic microorganisms in the soil, the survival of others presents im-
portant problems to farmers raising hogs, cattle, poultry, and other
domestic animals. To overcome this condition, rotation of crops is
usually practiced; several years are generally required to render

Siii\ i\al of Plant Pathogens in the Soil

287

infected pastures safe for use. A better understanding of the an-
tagonists that are responsible for the rapid destruction of pathogenic
organisms in the soil may throw hght upon this problem and im-
prove the methods of control.

Fig. 114. Influence of the hydrogen-ion concentration on the incidence of potato
scab (from Dippenaar).

Survival of Plant Pathogens in the Soil

The organisms causing plant diseases can be divided into five
distinct groups: fungi, actinomycetes, bacteria, animal forms, and
viruses. They are all found in the soil, and most are able to survive
there for long periods, especially in the presence of the host plant.
The fungi are by far the largest and most important group.

These fungi belong to the Myxomycetes {Plasmodiophora brassi-
cae, causing club root of cabbage), Phycomycetes (Phytophthora
infestans, Aphanomyces laevis, Syncliytriiim endohioticinn, Pytliium
debaryamim) , Ascomycetes {Botrytis cinerea, Sclerotinia trifoliorum,
Corticium vagiim). Fungi Imperfecti {Phoma betae, Verticilliiim
alboatriim, Hehninthosporium gramineum, Fusarium lini, Fiisarium
vasinfectiim) , and finally certain Basidiomycetes, including smuts.

Various fungi have been isolated from both cultivated and virgin

288 Disease-Producing Microorganisms

soils on which the particular host plant has never been grown be-
fore. Fusarium radicicola and Rhizoctonia solani, known to be para-
sitic on the Irish potato, were isolated from soils never cropped
with potatoes and from virgin desert lands. Disease-free seed
planted on new lands frequently yielded a diseased product. Soils
in which clover or grain was previously grown are better adapted
to the production of disease-free potatoes than is virgin land. Some
plant-pathogenic fungi are able to persist in soil for many years;

'»'yx;' A."^,

\-.x

"^^

Fig. 115. An antagonistic fungus, Tridwdcrma, attacking a plant pathogenic
fungus, Sclewthim roJfsii, showing one break of a septum (from WeindUng).

flax, for example, must be grown only on new soils. Various species
of Phytophthora can withstand low winter temperatures without
much injury; they can also resist some desiccation; Ph. infestans can
live saprophytically in soil, growing on old, partly decomposed
plants. The pathogenicity of these fungi is not diminished by living
in the soil. Many plants are infected by fungi, the spores of which
may not live in the soil but may adhere to the seeds and produce a
mycelium, which, on the germination of the seed, is able to attack
the seedlings.

Many of these fungi are facultative parasites, since they are able
to grow in soil in absence of the host plant. The spores of Sclerotinia
trifoUoriini can give rise to a mycelium which is at first saprophytic
and then becomes facultative parasitic. These spores germinate on
the vegetable residues in the soil; the mycelium spreads over the
soil at a rate which depends on environmental conditions.

Siini\al of Plant Pathogens in the Soil 289

The soil fusaria have been divided into true soil inhabitants and
soil invaders. The latter are dependent on the host for their con-
tinued existence in the soil; once the host plant is removed, the
fungus gradually dies out.

Cabbage- and tomato-sick soils may show as many as 40,000 colo-
nies (on plate) of the parasitic organisms per gram of soil. Tricho-
dcrma koniniii and T. U<i,nonn)i, two common saprophytic soil fungi,
are known to cause rots of stored sweet potato, as well as "ring

Fig. 116. Sweet-orange seedlings in nonsterilized soil: A, control; B, RJiizoctonia

inoculated into soil layer in bottom of jar; C, RJiizoctonia as in B, plus Tricho-

dcrma in top layer of peat (from Weindling and Fawcett).

rots." The common soil and dust fungus Rhizoptis nigricans is known
to be the cause of soft rot of sweet potatoes.

The plant-pathogenic actinomycetes are represented in the soil
by several species, the most important of which is Streptomyces
scabies, the causative agent of potato scab. The limiting reactions
for growth of these organisms are pH 5.5-6.0 and 9.0, with an opti-
mum at pH 7.0-7.5. A greater acidity, pH 5.0 or less, either con-
trolled or reduced the disease but did not eliminate the organism.
At pH 4.8 or lower, the potato plant as well was injured. An exam-
ination of some 100 fields for the presence of scab organisms in
Nebraska gave no field free from scab, 24-30 per cent causing de-
cided infection of the tubers. Previous cultivation of the soil, fre-
quent growth of potatoes, heavier soil, and larger numbers and
percentages of actinomycetes in soil corresponded with a higher
percentage of scab.

290 Disease-Producing Microorganisms

Comparison of a large number of strains of actinomycetes isolated
from scabby potatoes shows that one is dealing here not with a
single species, but with a large group of organisms. As many as
thirty species of actinomyces have been described; these are be-
lieved to be causative agents of potato scab, the type of lesion being
influenced by the species. The existence of more than one type of
actinomyces capable of causing mangel-beet scab has also been
suggested.

The formation of "pox" on sweet potatoes was shown to be due
to an actinomyces found in the soil. This organism has been desig-
nated as Actinomyces poolensis.

Bacteria capable of causing plant diseases include Bacillus tiime-
faciens, the crown-gall organism; B. campestris, causing black rot of
cruciferae; and a number of other bacteria causing bacterial rot of
potatoes and other plants.

Among the plant and animal pests present in the soil, we find also
protozoa, nematodes, wireworms, crustaceans, myriapods, and in-
sects. The plant-parasitic nematodes include Heterodera schachtii,
causing the disease of mangels; Tylenchiis tritici on wheat; Het.
radicicola, causing swellings or knots on roots of tomatoes and
cucumbers; Tylenchiis dipsae, causing the root knot on oats, tulip
root, and clover; Aphelenchus olesistus, causing leaf blight; and
Tylenchiis dipsaci, capable of causing galls on stems, leaves, and
tubers of potatoes.

The plant-parasitic insects include a number of Coleoptera, Lepi-
doptera, and Diptera. Wireworms may cause considerable damage
to crops, as when old meadows are plowed under and planted to
corn or potatoes. These worms are capable of traveling considerable
distances below the surface of the soil. The nematodes and other
worms, as well as the various insect pests, are favored by the addi-
tion of organic matter.

There is an association between the intensity of plant disease
caused by Het. schachtii and the cyst content of the soil, in those
cases where the disease was observed recently. The nematode dis-
ease may be due to an association between the fungus Rhizoctonia
solani and Het. schachtii. Infestation of soil with Phylloxera has
been found to be most frequent in heavy-textured soils and less
frequent in sandy soils.

There is also a possibility that the soil harbors organisms which
are parasitic upon plant and animal parasites, as was shown to be

Survi\'al of Plant Pathogens in the Soil 291

true of a nematode parasitie on the Japanese beetle, Popillia japonica,
which spends a large part of its life cycle in the soil.

The mosaic disease of tobacco, caused by a filterable virus, may
persist in the soil. Tomato mosaic was also found to be able to live
for 4-6 weeks in field soils, but there was no evidence of overwinter-
ing of the virus.

Different soil-borne parasites are affected very differently by en-
vironmental factors. High soil temperatiu'es stimulate the develop-
ment of the Fiisariiim disease of cabbage, and low temperatures
inhibit it. TJiielavia root rot of tobacco is checked in warm soils
and is seriously injurious only in cool soils. Jones emphasized this
seasonal contrast. Of two successive summers one was very cool,
with a midsummer soil temperature averaging about 5°C lower than
that of the succeeding summer; the root rot of tobacco was unusually
severe, whereas cabbage remained relatively free from disease. The
succeeding year with its warm midsummer period caused destruc-
tion of the cabbage crop because of the yellows disease, whereas the
tobacco was free from root rot. High soil temperatures favor vascu-
lar diseases, including flax wilt, tomato wilt, and cabbage yellows.
High temperature (25-27°C) also favors Sclerotium rolfsii and
certain other plant-pathogenic fungi.

Soil moisture also exerts an important influence on many plant
parasites. Sanford found dry soils favorable and wet soils inhibitory
to potato scab. Soil fungi causing "damping off" are favored by
high moisture; wet soils, even to the saturation point, favor the club-
root parasite of cabbage. Powdery scab develops best in periods
of damp, rainy, and cloudy weather and is favored by poor drainage.

The amount of organic matter present in the soil influences plant
infection, since it offers a source of energy for the saprophytic ex-
istence of the organisms. Certain pathogens cannot infect the host
plant in pure sand, but will do so in the presence of organic matter,
which allows the myceliimi to exist for some time. Clay soils are
more favorable to infestation than are sandy soils. The root rot of
peas is not disseminated by the seed, but spreads through the soil
and is especially favored by a high content of organic matter. The
cotton and alfalfa root rot spreads through the soil radially with a
growth similar to fairy rings; it is favored by heavy soils, humid
weather, and dense cover crops. Addition of organic matter to soil
also favors development of nematodes and rainworms. In some
instances, the application of organic matter has helped to check
the spread of a disease, for example, cotton rot.

292 Disease-Producing Microorganisms

In addition to organic matter, the use of artificial fertilizers, espe-
cially nitrogen and phosphorus, and the nature of the soil have a
considerable influence on the development of different organisms
causing plant diseases. The other most important treatments which
have a significant effect are those leading to changes in soil reaction.

Organisms Antagonistic to Plant-Disease-Producing Microbes

The antagonistic action of soil organisms to fungi causing plant
diseases results in a modification of the virulence of the pathogens
that find a temporary or permanent habitat in the soil. Various bac-
teria capable of inhibiting the development of the corn smut have
been isolated. These bacteria are able to destroy the smut fungi.
The widespread distribution of such bacteria in the soil was believed
to check the multiplication of the pathogen. Some of the antago-
nistic bacteria produce enzymes which have the capacity to dissolve
the chemical constituents of the cell walls of the fungi. Chudiakov
isolated two bacteria which lysed different species of Fusariiim and
other pathogenic fungi. These bacteria were widely distributed in
the soil, but were absent in certain flax-sick soils. When the pathogen
was introduced into soils containing active antagonistic bacteria, it
did not develop, and no disease was produced.

The virulence of the root rot of cereals caused by Ophiobolus
graminis received special attention. It was believed that this or-
ganism could be completely controlled by the activities of various
soil-inhabiting microorganisms; filtrates of these organisms were
nearly as effective in repressing the pathogen as were the living
organisms. The fact that potato scab can be reduced by plowing
under a green rye crop has also been explained by the development
of other organisms, such as saprophytic actinomycetes, which sup-
press the growth of the pathogenic S. scabies.

The "biological control" of various soil-borne diseases was thus
suggested. This method consists in modifying the soil so as to
encourage the maximum development of the antagonizing sapro-
phytic soil population. Inoculation of sterilized soil with the sapro-
phytic fungus Trichoderma, for example, was found to prevent in-
fection of citrus seedlings by the pathogenic Rhizoctonia (Table 64).

The spoilage of fruits can also be suppressed by inoculation with
mixtures of known organisms. According to Potter, the organism
causing rot of turnips produces a potent, heat-resistant toxin, which
is also destructive to the pathogen itself. Spraying turnips with this

Plant-Disease Antagonists

293

Tarlk (U. Efkkct of Tricliodcrma Ugnorum on Germination' and Gkowth of

Uaulky Infected with Uclminthosporium sativum in Sterilized Soil

(from Christensen)

Percentage of Plants

Sfraiii of

uncrgtM

Stunted

('..nt

)rl(Ml L(

■a ves

//. .idtinnn

H

H

H

H

H

H

H*

+ T

+ SI

II

+ T

+ SI

II

+ T

+ SI

'21

84

94

94

46

1-2

6

5^2

3^2

15

•2 '2

88

94

98

33

8

6

57

^27

14

'23

86

88

90

'io

17

8

78

31

21

'24

88

98

94

10

4

3

17

15

10

* H = II(lniintlinsj>oriiuii, T = Triclindcrma, SI = soil infusion.

bacterial product checked the disease. The same principle was
found to hold true for oranges infected with PeniciIIiiim itaUcum.
The injurious action of certain common soil bacteria upon Pseudo-
monas c'ltri, the cause of citrus canker, has also been reported.
Wheat seedlings were protected from infection by Helminthosporium
and flax seedlings from Fusarium by use of antagonistic bacteria. A
watermelon disease caused by Phymatotrichiim omnivorum was re-
duced when certain fungi (Trichoderma Ugnorum) and bacteria
were present in the soil together with the pathogen. The severity
of the seedling blight of flax, caused by F. lini, was diminished when
the pathogen was accompanied in the soil by certain other fungi.
The pathogenicity of H. sativum on wheat seedlings was suppressed
by the antagonistic action of TricJiothecium roseum, which is be-
lieved to produce a toxic substance.

The role of microbiological antagonism in the control of soil-borne
plant diseases has been outlined as follows: The soil population is in
a dynamic biological equilibrium. When a certain crop is grown
continuously, various parasites capable of attacking the roots of that
crop multiply. Organic manures stimulate the development of vari-
ous saprophytes in the soil. These multiply at the expense of the
pathogens and are able to check their activity, either by preventing

294 Disease-Producing Microorganisms

their growth or by attacking and destroying the mycehum of the
parasites. The biological control of plant diseases is particularly
effective against those organisms which have become highly special-
ized to a parasitic form of life.

Van Luijk obtained biological control of plant parasites by inocu-
lation of soil with microorganisms selected for their antagonistic
capacity, or by addition of their growth products. Broadfoot em-
phasized that antagonism of a saprophyte to a plant pathogen, as
measured by growth on artificial media, is not necessarily a measure
of the actual control that may be exerted upon the parasite in the
soil. Inoculation of soil with an antagonistic organism, such as
T. lignorum, may have only a temporary effect in changing the
microbiological balance of the soil population. Weindling and Faw-
cett attempted to control R. solani by use of T. lignorum, and Cordon
and Haenseler by use of B. simplex, with similar effects. Daines
reported that T. lignorum produces a substance toxic to S. scabies.
This substance is rapidly destroyed in the soil on aeration. It was,
therefore, believed doubtful that the fungus could be of much
assistance in combating potato scab.

Fellows obtained field control of the take-all disease of wheat
(O. graminis) in Kansas by application of chicken and horse manure,
alfalfa stems and leaves, and other organic materials. Garrett at-
tempted to prove that the factor chiefly controlling the spread of the
pathogenic fungus along the roots of the wheat plant was associated
with the accumulation of carbon dioxide and a corresponding lower-
ing of the oxygen tension. This could best be maintained by addi-
tions of organic manures. Organic matter low in nitrogen was more
effective than high-nitrogenous materials; it was, therefore, suggested
that the soil microflora uses the mycelium of the pathogen as a
source of nitrogen. Addition of nitrogenous materials, either in an
organic or in an inorganic form, protected the parasite by offering
a more readily available source of nitrogen. Differences in the
microflora associated with the decomposition of various composts are
believed to be largely responsible for differences in persistence and
virulence of pathogens causing root rots of cereals.

Green manures, when added to soil before planting, cause consid-
erable reduction in slime disease of tomato plants. Organic ma-
terials high in nitrogen, and supplementary additions of nitrogen
sufficient for complete decomposition of the organic material, are
most effective. Thom and Morrow found that organic matter, dur-

Methods of Control of Plant Diseases 295

ing the period of its active decomposition, is most effective in de-
pressing pathogenic fungi. Inactivation of pathogenic fungi in the
soil b\' use of the antagonistic action of soil microorganisms has
been variously attempted. Organic manures were added to the soil
to control PJiymatotrichum omnivoruin, the root rot of irrigated cot-
ton under continuous cultivation in Arizona. With the Rossi-Cho-
lodn\- slide technique, it was possible to demonstrate that micro-
biological antagonism represents the true mechanism of the control
process. Development of saprophytic organisms was most profuse
in the slides buried in the manured plots, whereas the mycelium of
P. omnivorum was most abundant on the slides in the unmanured
plots. King suggested that parasitism of the fungal strands by bac-
teria is one of the reasons for the decline of the pathogen in manured
soils. Henry believed that the biological control by the soil micro-
flora could even be directed against internal seed infection, since
appreciable infection of surface-sterilized flaxseed was found to occur
in sterilized but not in unsterilized soil.

Attention has already been called to the fact that numerous
observations have been made concerning the favorable effect of
bacteria in depressing various plant diseases. This is true, for
example, of the addition of bacteria to unsterilized soil exhausted
by growing flax; the percentage of plants diseased by F. lini was
thereby lowered. The term "bacterization" was applied to the
process of treatment of seed with active bacteria to support the
plant against pathogenic fungi. It has been suggested that the
effect of bacteria on germinating seeds is due to the liberated bac-
terial products capable of depressing the development of parasitic
fungi. Although not in all cases conclusive, the results fully justify
the hope that a better knowledge of the soil antagonists may lead,
if not to complete control, at least to a certain amount of control
over the numerous plant diseases caused by pathogenic fungi, espe-
cially those that persist for a time in the soil.

Methods of Control of Plant Diseases

The methods of treatment of soil for the control of injurious micro-
organisms are divided into five distinct groups:

1. Proper rotation, or withholding of the host plant, since various
parasites accumulate as a result of continuous growth of the same
or closely related plants; use of resistant plant varieties.

296 Disease-Producing Microorganisms

2. Special physical methods of soil treatment, such as soil cultiva-
tion, change of soil reaction, use of organic matter, use of specific
fertilizers.

3. Partial sterilization of soil.

4. Use of chemicals for the destruction of specific disease-produc-
ing organisms.

5. Biological control or introduction of organisms destructive to
the parasites.

Crop Rotation

In practicing crop rotation, one should remember that many of
the disease-producing organisms can persist in the soil for a number
of years and some are capable of leading there a normal saprophytic
existence. A rotation of at least 5-6 years should be practiced
against the club root of cruciferous plants and the sugar-beet
nematode.

Physical and Chemical Methods of Soil Treatment

Among the most efficient methods of control of soil-borne infec-
tions is the adjustment of the soil reaction, by the use of either
alkali-forming (lime) or acid-forming (sulfur, ammonium sulfate)
materials. Addition of sulfur or inorganic acids to soils having a
reaction of pH 5.9 or above is recommended; the amount of sulfur
or acid to be applied depends, of course, on the initial reaction and
buffer content of the soil. Hov^ever, the action of sulfur in control-
ling the wart disease of potatoes depends not alone upon the acidity
produced, but also upon some other mode of action of the sulfur,
probably thiosulfuric acid produced at an early stage of oxidation
of the sulfur.

For the control of potato scab, sulfur is effective. Sweet-potato
scurf and pox can also be checked by the application of sulfur.
Lime, which reduces the acidity of the soil, and stable manure favor
the development of scab. The addition of fertilizers (acid phos-
phate) to make the soil reaction acid tends to decrease the develop-
ment of scab. According to Millard, sufficiently liberal dressings of
green manure added to the soil will inhibit the disease. This is
probably due to the temporary increase in soil acidity, as a result of
the decomposition of the organic matter by the soil fungi, and to an
increase in soil moisture. Scab is much more prevalent in dry
seasons, since actinomycetes are much less active in very moist soils.
Sanford suggested that the soil reaction may not be the important

Soil Sterilization and Partial Sterilization

297

factor in controlling the clc\elopnient of potato scab in the soil.
Moisture was found to be directly or indirectly the main factor, a
high moisture content controlling the disease, whereas abundant

20 40 60 0
Time, days

40 60

Fig. ir

Influence of toluol and heat upon the numbers and acti\ ities of micro-
organisms in soil (from Waksman and Starkey).

scab was formed in dry soils. The development of scab is influenced
also by the temperature of the soil, the optimum for scab being
22°C.

Soil Sterilization and Partial Sterilization

The soil may be sterilized completely or only partially, whereby
not all the organisms are destroyed, but only certain groups. Com-
plete sterilization is difficult to accomplish in the field or in the green-
house, since the soil readily becomes reinfected again; it is not even
desirable. In the laboratory, it often becomes necessary to sterilize
a soil for growing pure cultures of organisms, for testing the purity
of certain strains, and for invigorating laboratory-kept cultures. To
sterilize a soil, it is placed in glass or clay containers and heated
under pressure, at 15-20 pounds, for 2-3 hours. By using flowing
steam for 1-2 hours, on 6 consecutive days, complete sterilization

298 Disease-Producing Microorganisms

can also be obtained. The sterility of the soil thus treated must be
carefully checked.

Though we know little about transmission through the soil of
virus diseases of animals, we do know that various virus diseases
of plants may be thus transmitted; for example, mosaic virus of
wheat. To inactivate the virus, the soil, according to Johnson, must
be heated for 10 minutes at 50-60° C.

Origina
100

CSo added 1

5 6 7

Month

Fig. 118. InHuence of CS2 upon the numbers of bacteria and actinomycetes in
soil (from Hiltner and Stormer).

It often appears necessary to partly sterilize the soil to destroy
certain injurious insects or pathogenic fungi, but not to kill the whole
soil population. Such partial sterilization can be brought about by
use of heat, as by steam and dry heat, or by means of various vola-
tile and nonvolatile antiseptics, the first comprising carbon bisulfide,
toluol, formaldehyde, and hydrocyanic acid; the second including
phenol, cresol, and chloropicrin. These disinfectants do not accumu-
late in the soil, but are either lost by volatilization or destroyed by
soil organisms. The treatments have a selective eflPect upon the soil
microbiological population, affecting particularly the fungi, many of
the protozoa, and certain bacteria, such as the nitrifying organisms.

special Chemicals for Treatment of Soil 299

Many bacteria are able to resist these treatments and soon begin to
multiply rapitlK after the disinfectant has been removed. Active
multiphcation of these bacteria results in extensive decomposition
of the soil organic matter, which is accompanied by abundant lib-
eration of ammonia. The latter is used by plants, resulting in in-
creased plant growth. Partial sterilization of soil may, therefore, be
compared to fertilization with nitrogen. The effect of partial sterili-
zation of soil with toluol and heat upon the bacterial and fungus
popidations, as well as upon the liberation of the nitrogen in an
available form, is shown in Fig. 117.

Several theories have been proposed to explain the effect of par-
tial sterilization in increasing the fertility of the soil. It has been
asserted by some that disinfectants, when used in small amounts,
have a direct stimulating effect upon microorganisms and plant
roots; others have assumed that the toxins of the soil are destroyed
by such treatments; still others have emphasized the destruction of
plant-pathogenic fungi and bacteria in the soil. The theory that has
received the greatest consideration, in an attempt to explain the
phenomenon of partial sterilization, is the "protozoan theory of soil
fertility," discussed previously.

Use of Special Chemicals for Treatment of Soil

A number of chemical compounds have been recommended for
control of various fungi and nematodes in the soil. The saturation
of soil with formaldehyde to prevent spreading of disease-producing
organisms has often been practiced. Formaldehyde in concentra-
tions of 0.045-0.05 per cent was found to give very good results in
combating the sugar-beet nematodes. It is difficult to reach the
nematodes at a depth lower than 60 cm, and it is difficult to cause
the poison to penetrate the whole mass of soil. The nematodes
present in the lower depths of soil and in the form of cysts can be
made to develop and come nearer the surface by the use of catch
crops and chemical stimulants. In absence of the host plant, the
nematode larvae die off. Formalin, sometimes following crude ben-
zol treatment for control of potato wart, mercury bichloride, and
other disinfectants are recommended for control of various plant
diseases. The treatment of soil with a 1:1,000 or 1:1,200 solution
of mercuric chloride was found to be effective in controlling root
maggot, black rot, club root, and damping-off diseases.

Arsenic, mixed with ashes for soil dressing, is rather widely em-
ployed in China for destruction of worms; a similar practice is used

300

Disease-Producing Microorganisms

for golf greens. Acetic acid (1.2 per cent), applied 10 days before
planting, has also been recommended for destruction of soil-infesting
damping-off fungi.

Various other soil fungicides and volatile antiseptics, like carbon
bisulfide and toluol, have been frequently employed for destruction
of pathogenic fungi. Carbon bisulfide can be used with success
against a number of disease-producing fungi. This disinfectant
should be applied to the soil free from plants; otherwise the chemical

7.4 7.2 7.0 6.8 6.6 6.4 6.2 6.0
Hydrogen-ion concentration

5.8 5.6

Fig. 119. Effect of soil acidity on the percentage of club root (from Chupp).

will result in plant injury. Good results have also been obtained
with toluol in controlling various diseases.

It is impossible to sterilize the soil completely, especially by the
use of high doses of disinfectants, without injuring the plants. The
amount of disinfectant necessary to destroy the pathogenic organ-
isms in the soil is considerably less than that necessary to sterilize
the soil as a whole. Various chemicals vary greatly in this respect.
Mercuric chloride is far more active than organic mercuriates; the
microbicidal dose for mercury compounds is about the same as the
antiseptic dose, whereas for copper salts the antiseptic dose is much
lower than the microbicidal.

When soil is sterilized, the fungi and other plant and animal
parasites are readily destroyed. Once certain parasitic organisms
are introduced, however, they may develop readily in the treated
soil and even cause a larger amount of infection. Treatment of soil
with a disinfecting agent followed by inoculation with saprophytic

Use of Soil Inoculants

301

fungi may prove to be most effieient in increasing the value of the
treatment. Whether the saprophytic fungus uses up the available
nutrients rendered sokible on steaming of soil or whether the favor-

100

80

"5 60

S 40

20

5.0

9.0

Fig. 120. Effect of pH upon occurrence of root rot and wilt (from Taubenhaus

et al. ) .

able effect is due to the production of a substance directly injurious
to the plant pathogen, remains to be determined.

Use of Soil Inoculants

The methods of biological control of disease-producing organisms
are still insufficiently studied. Here belong the introduction of
birds and other higher animals, as well as of certain insects feeding
upon specific injurious insects and worms, or the use of predaceous
nematodes against plant-pathogenic nematodes, or of entomogenous
fungi and bacteria parasitic upon insects. The phenomena of an-
tagonism may also be listed here.

Numerous attempts have been made to inoculate the soil with
antagonistic organisms for the purpose of controlling plant diseases.
These efforts have proved to be, in most instances, complete failures,
as pointed out previously. This is largely because, unless the condi-

302 Disease-Producing Microorganisms

tions of the soil are modified by supplying more nutrients to the
antagonists or by creating a favorable reaction, the antagonists will
not develop. The introduction of organic materials, such as green
manures and stable manures, may correct such a condition, thus
favoring development of the antagonists, which bring about, directly
or indirectly, suppression of the disease-producing agent.

Selected Bibliography

1. Daines, R. H., Control of plant diseases by use of inorganic soil amend-
ments, Soil Sci., 61:55-66, 1946.

2. Johnson, F., Heat inactivation of wheat mosaic virus in soils. Science, 95:
610, 1942.

3. Garrett, S. D., Root Disease Fungi, Chronica Botanica, Waltham, Mass.,
1944.

4. Newhall, A. C, Volatile soil fumigants for plant disease control, Soil Sci.,
61:67-82, 1946.

5. Sanford, S. B., Some soil microbiological aspects of plant pathology, Sci.
Agr., 13:638, 1933; Soil Sci., 61:9, 1946.

6. Waksman, S. A., The Actinomycetes, Chronica Botanica, Waltham, Mass.,
1950.

7. Weindling, R., Microbial antagonism and disease control, Soil Sci., 61:23-
30, 1946.

W4v

Stable Manures, Composts,
and Green Manures

Nature of Stable Manures

A large part of the plant residues removed from the land in the
form of harvested crops is returned to the soil as various waste
materials, ranging from factory to kitchen and farm wastes, or after
the plant residues have been used for bedding purposes and been
partly consumed by animals, or after they have passed through the
digestive system of these animals.

Stable manures consist of three groups of components: (a) bed-
ding or litter, (b) solid excreta of animals, (c) liquid excreta or
urine. The nature and relative concentration of these components
vary greatly in different manures, depending on the animals and the
methods of feeding and handling the animals. Since the various
components of the manures also differ considerably in chemical
composition, it is natural to expect that the composition of different
manures should vary.

Plant residues used for bedding purposes are usually high in car-
bohydrates, especially in cellulose, and low in nitrogen and minerals.
Urine is high in nitrogen and minerals and has very little, if any,
carbohydrate material. Solid excreta contain considerable amounts
of proteins, and thus tend to give a more balanced medium for the
growth of microorganisms. The chemical composition of three
diflFerent types of stable manures is shown in Table 65, Sheep
manure is high in protein, in cold-water-soluble organic materials,
and in ash; it is low in cellulose. Horse manure is low in protein
and high in cellulose and hemicelluloses. Cow manure falls be-
tween these two. A comparison of the nitrogen and mineral compo-
sition of a number of manures is given in Table 66. Chicken and
pigeon manures are highest in nitrogen, phosphorus, and potassium,

303

304

Manures, Composts, Green Manures

Table 65. Chemical Composition of Various Fresh Manures
(from Waksman and Diehm)

On basis of drv, litter-free material.

Sheep

Horse

Cow-

Chemical Constituents

Manure *

Manure f

Manure *

per cent

per cent

per cent

Ether-soluble substances

2.8

1.9

2.8

Cold-water-soluble organic matter

19. €

3.2

5.0

Hot-water-soluble organic ma

tter

.5.7

2.4

5.3

Hemicelluloses

18. .5

23.5

18.6

Cellulose

18.7

27.5

25 . 2

Lignin

20.7

14.2

20.2

Total protein

25.5

6.8

14.9

Ash

17.2

9.1

13.0

* Solid and liquid excreta.
f Solid excreta only.

or the most important nutrients required for plant growth. Cattle
and horse manures contain the lowest quantities of these essential
ingredients.

On the basis of numerous analyses, stable manure is found to
contain, in a fresh state, about 70-80 per cent water, 0.3-0.6 per
cent nitrogen, 0.1-0.4 per cent phosphorus as P2O5, 0.3-1.0 per cent
potassium as KoO. A ton of fresh manure thus carries about 400-600
pounds of dry matter; about 10 pounds is nitrogen, 6 pounds P2O5,
and 10 pounds potash. About half the nitrogen and a large part of
the other two elements are in water-soluble forms and are thus
immediately available for plant growth.

Cow manure and horse manure are about equal in nutritive value
to plants and as sources of humus. To eliminate certain undesirable
characteristics of fresh stable manure, to destrov the weed seeds

Table 66. Chemical Nature of Different Manures (from Jenkins)

Composition of Dry Matter

Manure

Moisture

Nitrogen

P2O5

KoO

per cent

per cent

])('r cent

per cent

Cattle

80

1.67

1.11

0.56

Horse

75

2.2!)

1.25

1 . ;58

Sheep

68

3.75

1.87

1 . 25

Pig

82

3.75

3.13

2.50

}hn

5(!

6.27

5.92

3.27

Pigeon

52

5.68

5.74

3.23

Decomposition of Stable Manures 305

that nun be present, and to t)btain a piodnct whieh ean be readily
pulverized, such manures are sometimes composted before their
introduction into the soil. Although stable manures contain appre-
ciable amounts of plant nutrients, their value as sources of humus
increases their importance in soil. A large part of the organic mat-
ter in some of the manures decomposes rapidly and, therefore, has
a relatively short period of effectiveness. Poultry manure and sheep
manure particularly are frequently used as organic fertilizers and
not as sources of soil organic matter. As fertilizers they are gen-
erally expensive; they are applied to the soil directly or mixed with
certain proportions of peat or soil.

Decomposition of Stable Manures

When placed in a compost that is kept under conditions of favor-
able moisture and aeration, the various organic constituents of stable
manures are immediately attacked by a great variety of microorgan-
isms, including not only fungi, actinomycetes, and aerobic and an-
aerobic bacteria, but also protozoa and other forms of life. These
organisms do not attack the manures as a whole or all the chemical
constituents of the manures at the same rate. The decomposition of
the compost and the various changes brought about in its specific
organic constituents depend to a large extent upon the nature and
composition of the manure and upon the conditions under which
the decomposition of the manure is taking place.

The microorganisms bringing about the decomposition of stable
manures either inhabit the manures or are derived from the soil.
These microorganisms bring about a rapid destruction of the carbo-
hydrates and some of the proteins; this is accompanied by synthesis
of considerable microbial cell substance. Although the various
processes involved in the decomposition of stable manures are
closely interrelated, they may be considered from three distinct
angles: (a) the decomposition of the organic matter as a whole in
the manures and the formation of humus; (b) the liberation, oxida-
tion, reduction, and synthetic processes involving the nitrogen com-
plexes; (c) the influence of the microorganisms found in the manures
upon the microbiological population of the soil and upon soil
processes.

The transformation of the nitrogen in the manures, leading to its
final liberation in a form available for the growth of higher plants.

306

Manures, Composts, Green Manures

or as ammonia, is closely connected with the general processes of
decomposition of the organic constituents in the manures. The
nature and activities of the microorganisms in the manures are also

20

10

0

A. Total material

A. Hemicellulose

A. Cellulose

A Lignin

AN. Total material

AN. Hemicellulose

AN Cellulose

AN. Lignin

0

100

400

500

200 300

Days

Fig. 121. Course of decomposition of alfalfa plant: A = aerobic, AN = an-
aerobic (from Tenney and Waksman).

dependent on and are closely connected with the transformation of
the organic and inorganic complexes. The rate of decomposition
of stable manures, the liberation of the nitrogen into available forms,
and the formation of humus, all depend upon the nature and abun-
dance of the three components of the manures; namely, the bedding
or litter, solid excreta, and urine.

Decomposition of Stable Manures

307

According to Deherain, the function of the Httcr in manure consists
in absorbing the hquids excreted by the animals and in supplying
celluloses, substances which characterize the nature, decomposition
processes, and products resulting from the manure, and which give
the manure its special value for soil improvement. A knowledge of

Original straw

5^5

f'n?-

Wc^

v^'i;

^•^■o

1/1

c'.-v:

'!"'■

•J^^

ro

5

8

ro

-■•-..V

^^

i

Straw compost

Fig. 122. Comparati\ e chemical composition of oat straw and of a compost pre-
pared from it ( from Waksman and Gerretsen ) .

the chemical composition of the litter was, therefore, considered to
be of great importance. Konig emphasized that decomposition of
stable manures involves, first of all, the transformation of carbon
compounds present in the manures, and that 75 per cent of the car-
bon disappears within a year after application of the manure to the
soil. The decomposition is more rapid during the warmer periods
of the year than during the colder periods.

Among the organic constituents of the manure, the pentosans and
cellulose are decomposed more rapidly than the total organic mat-
ter, whereas the lignins are decomposed more slowly. Decomposi-
tion of manure is thus accompanied by a rapid reduction in the car-
bohydrates and a gradual enrichment in the lignins. Both the total

308 Manures, Composts, Green Manures

and easily soluble nitrogenous compounds in the manure diminish
rapidly after its application to the soil. This was originally believed
to be due either to denitrification or to a removal of the nitrogen to
the subsoil. It was later found that large parts of these soluble
forms of nitrogen are transformed into insoluble forms by the micro-
organisms responsible for the decomposition of the carbohydrates.
Only about a third of the nitrogen and a third of the phosphoric
acid in the manure were found to be made available to the growing
crops during the first 2 years after application of the manure; 70
per cent of the potash was made available in that time.

Barthel and Bengtsson freed stable manure from ammonia by
distillation. When the manure thus treated was added to the soil,
no nitrate was formed; this tended to prove that ammoniacal nitro-
gen and urea nitrogen in the manure, but not the nitrogen present in
the form of organic compounds, undergo active nitrification. The
organic nitrogen was believed to be tied up in the manure in a form
unavailable for growth of higher plants. According to Egorov, one-
half to two-thirds of the nitrogen in the solid excreta of the manure
is in the form of microbial cell substance. By fixing the nitrogen in
their cells, during the process of decomposition of manure, fungi and
other microorganisms are thus able to reduce the losses of nitrogen.
Moisture and aeration exert an important effect upon the rapidity
and nature of the decomposition. This is illustrated in the compost-
ing of fresh horse manure (Fig. 54).

Although fresh stable manure contains an extensive population of
characteristic microorganisms, especially bacteria and protozoa ( cop-
rophilic forms), many of these organisms die out in the process of
composting and are replaced by others which are characteristic of
the compost or of the soil. Among these, the thermophilic micro-
organisms, comprising various fungi, actinomycetes, and bacteria,
occupy a unique place. They develop in the composts after the tem-
perature has reached 50-60° C. Some of them are able to grow even
at 65-75° C, bringing about active decomposition of the organic con-
stituents of the manure. These organisms are quite distinct from
the typical soil population. This has bearing upon the various re-
ports that manure should be considered a source of bacteria for soil
inoculation.

Conservation of Stable Manures

A number of processes are utilized in the treatment of manures,
to conserve the nutrients and render the manures highly beneficial

Conservation of Stable Manures

309

tor soil fertilization and soil improvement. Each of the processes
has certain distinct advantages and disadvantages. The major ob-
jective of snch conservation is prevention of the nitrogen losses which
nsnally take place in the decomposition of manures. These losses
can be threefold: (a) volatilization of the nitrogen as ammonia, (b)

20

0 5 10 15

Moisture content, per cent

Fig. 123. Influence of moisture upon nitrate formation (from Traaen).

losses of nitrogen by denitrification of the nitrate to atmospheric
nitrogen, (c) losses of nitrogen by leaching of the nitrate and of
soluble organic forms. Some of the phosphate and potassium in
the manure may also be lost by leaching, as a result of careless
handling. Another purpose in devising special methods of treating
manures is conservation of as much of the organic matter as possible
(Table 67).

Among the different processes of conserving manures, those desig-
nated as "hot manure" and "cold manure" are the most common.
The "hot fermentation" process has recently received particular at-
tention. When the manure is first permitted to undergo aerobic de-
composition for a few days, to allow a rapid rise in temperature, and

310 Manures, Composts, Green Manures

Table 67. Losses from Exposure of Manure in the Open Yard
(from Salter and Schollenberger)

Canada,!

New York, §

New Jersey,*

3 Months,

Ohio,J

6 Months,

2\i Months,

Apr.-July,

3 Months,

Apr.-Sept.

Early Summer,

One-Half Horse,

Jan. -Apr.,

'

Constituent

Cow Manure

One-Half Cow

Steer

Horse Cow

per cent

per cent

per cent

per cent per cent

Organic matter

60

39

Nitrogen

31

29

30

60 41

Phosphoric acid

19

8

24

47 19

Potash

43

22

59

76 8

* Thorne, C. E., Farm Mannres, p. 146, Orange Judd Publishing Co., New York,
1914.

fShutt, M. A., Barnyard manure. Can. Dept. Agr. Cent. Expt. Farm Bull. 31, 1898.

X Thorne, C. E., ei al.. The maintenance of soil fertility, Ohio Agr. Expt. Sta. Bull.
183, 1907.

§ Roberts, I. P., and H. H. Wing, On the deterioration of farmyard manure by leach-
ing and fermentation, Cornell Unir. Agr. Expt. Sta. Bull. 13, 1889.

is then compressed to produce anaerobic conditions, a compost is
formed which behaves differently from the commonly composted
manure. Hot fermented manure has also the advantage that many
of the disease-producing organisms carried in the manure are de-
stroyed.

In the decomposition of stable manures, both in composts and in
soils, considerable quantities of humus are formed. The processes
involved are similar to the general processes of decomposition of
plant and animal residues discussed previously. Because of the spe-
cific nature of the manure and because of the particular conditions
under which the decomposition may take place in composts, a num-
ber of special problems are involved. Most important among these
are the conservation of the nitrogen in the manure, as pointed out
above, hygienic treatment of the solid human excreta to prevent
epidemics and infectious diseases, and maximum production of
humus. It is believed by many that, when equal amounts of nutri-
ent elements are added to the soil in the form of mineral fertilizers,
on the one hand, and of stable manures, on the other, the resulting
equal increases in plant growth tend to prove that the manure is not
superior to the fertilizer. The fact is frequently overlooked that
manure may not give immediate superior effects because it does not
contain sufficient concentrations of available nitrogen. Further,
insufficient consideration is usually given to the importance of the

Stable Manures and vSoil FtMtilitv

311

300

manured

1880 1890 1900 1910 1920
Fig. 124. Influence of manures upon the conservation of soil fertility (from

Russell ) .

Table 68. Manure Spread ox Field Compared wnn Manure Left in Piles
(from Salter and Schollenberger)

Relative Value
in Increasing
Crop Yields
100

71

80

Manure spread and plowed under immediately

Manure spread 2 days before plowing

Manure in piles 2 days before spreading and plowing

Manure spread 14 days before plowing

Manure in piles 14 days before spreading and plowing

49
55

residual effect of manure in building up the humus content of the
soil (Table 68).

Effect of Stable Manures upon Plant Growth and Soil Fertility

The important role of stable manures in plant growth and in soil
fertility has been variously ascribed to six distinct factors: (1) Ma-

312 Manures, Composts, Green Manures

nures offer a readily available supply of nitrogen, phosphoric acid,
and potash for growth of higher plants. (2) In the decomposition of
the carbon complexes in the manures by microorganisms, consider-
able carbon dioxide is liberated, which is essential for plant growth.
(3) The organic matter of the manures replenishes the supply of soil
humus. (4) Manures exert an important influence upon the micro-
biological activities in soil. (5) Because of their bacterial content,
stable manures are important for their inoculating properties. (6)
Manures influence the colloidal properties of soil and the state of
its aggregation.

The phosphorus and potassium in stable manures are present
largely in forms readily available to higher plants, as are inorganic
salts. The nitrogen in the manure is only about one-third to one-
half as available as that of inorganic fertilizer; this has been brought
out in numerous field tests as well as by nitrification experiments.
There is little justification, therefore, for comparing the availability
of the nitrogen in organic manures and in inorganic fertilizers, with-
out considering the cumulative effects of the manure on the fertility
of the soil and especially on its physical condition; this error has been
frequently made in fertilizer trials, where only given crop yields for
one year have been measured. Stable manures were found to give
particularly significant results, as compared with inorganic fertiliz-
ers, in dry years and on light soils (Table 69).

Table 69. Comparative Cumulative Effects of Manure and
Chemical Fertilizers (from Salter and Scliolleiiborger)

Ohio Agricultural Experiment Station, Wooster, Ohio; .5-year rotation
fertility experiment

Average Increase in

1 Total Produce per

Increase as Per Cent

Rot at

ion *

of That for Chemicals

First 10

Entire 4.3

First 10

Entire 43

Plot

Treatment

Years

Years

Years

Years

pounds

pounds

per cent

per cent

18

Manure

(),U^2

11,120

79

106

12

Chemicals

7,7;u

10,, 528

100

100

20

Manure

4,0^24

6,231

68

86

14

Chemicals

5.891

7,263

100

100

Averages for limed and unlimed soils.

Artificial Maniiies 313

Artificial Manvres

With the introduction of machine power to replace horse and other
animal power, the amount of stable manure available to the farmer
was appreciabl)' reduced. Furthermore, the growth of large urban
areas recjiiired intensive truck-garden systems which consumed large
quantities of the available manure. This resulted in a decrease in
the amount left for use in fields and gardens. Recourse was had,
therefore, to composting plant residues with mineral fertilizer, result-
ing in a product similar in every respect to that obtained from stable
manures. To distinguish such composts of plant residues from com-
posts of stable manures, the former are usually designated as "arti-
ficial manures."

To prepare artificial manures similar in chemical composition and
in their effects upon the soil to composts commonly obtained from
stable manures, cereal straw or other plant residues are utilized.
These are supplemented with an inorganic source of nitrogen and to
some extent also with available phosphorus and lime. When they
are properly moistened, decomposition of the composts sets in im-
mediately and is accompanied by a rise in temperature. The micro-
biological population and the chemical processes of decomposition
involved are similar to those which commonly are found in like com-
posts of stable manures. The value of inoculating such composts
with active microorganisms is still debatable. Some fresh garden
soil or actively decomposing stable manure is occasionally used for
inoculation, with very satisfactory results.

The rapidity of decomposition of the plant materials in the com-
post and their transformation into humus depend upon the nature
of the materials; their chemical composition; the amount and nature
of the inorganic nutrients added, especially the nitrogen; the mois-
ture content of the compost; its proper aeration; and temperature.
These factors also influence the chemical nature of the humus pro-
duced in the compost and its effect upon soil processes. The nature
of the plant residues and the conditions and extent of decomposition
are particularly important. The humus of a compost produced from
cereal straw will, therefore, vary from that formed from corn stalks,
or from oak leaves, or from pine needles, or from soybean stover
(Table 70).

The compost is usually turned several times, especially after a
temperature of 65-80° C has been attained. The turning must be

314

Manures, Composts, Green Manures

Table 70. Chemical Composition ov Plant Residues, Manures,
AND Soil Humus (from Pic-hard)

Per cent of total organic matter.

Cereal
Straw

Artificial Manures

Horse
Manure

for
Mush-
rooms

Humus

in

Peat

Humus

Constituents

1

2

in
Mineral

Soil

F'atty substances

1.90

0.73

0.9]

0.14

1.12

12.00

Resins

4.48

1.95

4 . 83

3.93

2.24

0.44

Pentosans

28.40

23.57

11.26

11.47

4.72

3.44

Hexosans

5.05

10.81

4.80

3.48

Cellulose

37.35

29.66

19.59

13.17

2.77

2.98

Lignin

14.35

20 . 76

15.54

29.60

16.42

13.65

Soluble "huniic acids"

3.56

16.62

19.25

26.98

27.46

"Humins"

4 . 25

5.50

3.50

16.95

6.21

Nitrogen

0.41

0.56

1.36

2 . 42

3.37

2.15

frequent enough for proper aeration, which favors development of
aerobic fungi, actinomycetes, and bacteria. The moisture content
of the compost must be adjusted to 75-80 per cent. If excess water
is added, anaerobic conditions, which will retard decomposition, are
created. With insufficient water, especially in loose, open heaps,
nitrogen losses may result, and an inferior product is obtained.

Figure 122 gives the relative composition of artificial manure and
of the original plant material from which it was prepared. Oat straw
was allowed to decompose for 273 days at 37°C in the presence of
added inorganic nutrient salts. In that time, the cellulose disap-
peared almost completely, the hemicelluloses and fats were markedly
reduced, the lignin increased appreciably in relation to the other
constituents, and the relative amounts of ash and protein increased
to an even larger extent. The increase in the ash content is due to
its gradual accumulation, resulting from destruction of the organic
constituents. The increase in protein content is relatively greater
than the increase in ash; this is due both to the relative accumulation
of the protein parallel to the destruction of the carbohydrates and
to the synthesis of microbial proteins from the inorganic nitrogen
added to the compost. The lower the original nitrogen content of
the plant material used in the preparation of the compost, the greater

Artificial Manures

315

the amount of nitrouen re([uirocl, and the jfreater tlie amount of
protein subsequentK- s^ntliesized. The hgnin in the compost also
increases during the process of decomposition, but to a relatively
sniallc>r degree than tlie increase in ash and protein; this increase in

•r-

^

> *'

J

■k

^^

'i

^^,
.^^,?

«

%f:

\<

V %.

... ^?^

Fig. 12.5. Effect of artificial manure on growtli of alsike clo\er. Left to right,

lime and acid phosf)hate; lime, acid phosphate, and straw; lime, acid phosiihate,

and artificial manure (from Albrecht).

lignin content is due to the greater resistance of lignin than of the
other plant constituents to microbial decomposition. The tempera-
ture and moisture content of the compost have an important influ-
ence upon the rapidity of decomposition of the straw and upon the
formation of the humus.

A number of formulas have been suggested for supplementing
straw and other plant residues to obtain a good artificial compost.
Two such formulas follow;

316 Manures, Composts, Green Manures

Formula 1

AiuLuonium sulfate 67.5 pounds

Acid phosphate 22 . 5

Ground limestone 60

Formula 2

Ammonium sulfate

60 pounds

Acid phosphate

30

Ground limestone

50

Potassiixm nitrate

25

Use 165 pounds of this n

lixt

ure per ton

of straw.

Use 150 pounds of this mixture per ton
of straw.

Other sources of nitrogen may be employed, such as urea, calcium
cyanamide, or ammonium phosphate. The amounts of phosphate

1 *1S:~^J»*^ Jf*;^

Fig. 126. Effect of temperature upon composting: 1, 7°C; 2, 7°C; 3, 18°C; 4,

27°C; 5, 37°C. All except 1 received supplementary additions of nitrogen and

mineral salts (from Waksman and Gerretsen).

and lime added are adjusted according to the nature of the nitrogen
source used.

When the moisture content of a heap of plant residues is not high
enough to allow normal decomposition and is not low enough to
prevent it, a phenomenon known as self-heating may occur. Animal
manures as well as plant residues, especially hay, kept in heaps, may
undergo limited decomposition; this may lead to the formation of
certain volatile substances, which, on coming in contact with the
air, ignite spontaneously. This is also true of heaps of peat. The
heap acts as an insulator, preventing radiation of heat from the inside
and penetration of oxygen from the outside. The lack of sufficient

Green Manures

317

moisture prevents absorption of the heat. A low moisture, a high
teuiperaturc, and a lack of oxjgen penetration may thus create con-
ditions favorable for spontaneous heating. Losses of organic matter
during spontaneous heating of alfalfa were found to be largely at
the expense of the fats, sugars, and hemicelluloses, and to a lesser
degree of the cellulose and protein; lignin suffered no loss at all.
Absorption of oxygen by the lignin, accompanied by a rise in tem-

1

1 1 I

V

Continuously manured 1852-1911

~ \

—

- \

-

\

^Manured 1852-1872, none thereafter

:

-

^""^■^^---^^^0^

-

*■ -

« , , • ,

o

-

.\ •

^Continuously untreated 1851-1911

0-

^-1871

1 1 1

100

90

80
2 70
>• 60
'^ 50
"^ 40

30

20

10

0
1870 1880 1890 1900 1910

Fig. 127. Residual effects of liea\y applications of manure (from Hall).

perature to the ignition point, was believed to lead to the actual
ignition. It has been suggested that addition of salt ta moist alfalfa
hay will inhibit bacterial development and thus delay the process of
decomposition long enough to permit curing.

Green Manures

Green manures comprise plant crops grown in a given soil to a
certain stage of development and plowed under while still green.
Both leguminous and nonleguminous plants are utilized for this
purpose. The nature of the plant to be selected for green manuring
depends upon the soil and the climate and upon farming practice.

Green manures serve several distinct purposes for plant growth
and soil improvement: (1) To increase the supply of total and avail-
able nitrogen in the soil. Various leguminous plants are utilized for
this purpose. The nature of the legume thus selected depends
largely on the geography of the region, the season of the year when

318 Manures, Composts, Green Manures

the land is free from a crop, the nature of the soil, and the rotation
system. (2) To prevent the nutrient elements of the soil, especially
the nitrates, from leaching out during the part of the year when no
cultivated crops are being grown on the soil. (3) To increase the
supply of organic matter in soil. (4) To protect the soil against
erosion.

The plants used for green manuring are high in water-soluble
constituents, in nitrogen, and in minerals; they are comparatively
low in cellulose and in lignin. As a result, decomposition of a green
manure crop plowed into the soil sets in very rapidly. This is accom-
panied by rapid liberation of the nitrogen and the minerals in avail-
able forms; comparatively little humus is produced. Figure 48
illustrates the difference between the chemical composition of young
plants and of mature plants and the influence that this has upon the
decomposition of the various organic constituents of these materials.
As the plants grow older, their ash and nitrogen contents decrease,
and their cellulose and lignin contents increase. Decomposition of
younger plants results in liberation of some of the nitrogen as am-
monia; the younger the plant and the higher the nitrogen content,
the more rapidly is ammonia liberated and the greater is the amount
liberated. Beyond a certain stage, no ammonia will be liberated;
actually, nitrogen may have to be added to hasten decomposition,
as already shown.

The plants grown for green manuring and plowed into the soil
can be divided into three categories : ( 1 ) those that contain a cer-
tain balanced proportion of available carbohydrates to nitrogen; (2)
those that contain an excess of nitrogen, or more than is required for
decomposition of the carbohydrates; and (3) those that contain an
excess of carbohydrates and lignin over nitrogen. The third group,
comprising both legumes and nonlegumes, decompose more slowly
than the plants of the other two categories.

Table 71 shows the relative amounts of total plant material and
nitrogen in a number of plants used for green manuring, and the
effect of such manures on crop growth and loss of humus from soil,
as compared to the plowing under of weeds only. Vetch and crim-
son clover produced the largest amounts of growth, contained the
highest amounts of nitrogen, and gave the highest crop yields of
corn after plowing under of the green manure. Wheat and rye used
as green manures gave the highest percentages of plant material in
the roots, the lowest nitrogen contents in the plant material, and the
poorest effects upon the corn crop; they also resulted in the smallest

Green Manures

319

loss of humus. Whereas the liunuis loss for the weed plot was 0.08
per eent of the carbon, losses lor tlu- wheat and r\e plots were only
0.01 pi-r eent. It is important to note that, even with heavy apph'-
eations of green manures for 5 years, the humus content of the soil
decreased.

TaHI-K 71. \'li:i.I)S AM) CoMI'O.SITIOX OK (iKKlON MaM HE ( 'UOPS SkKDED IX

Staxdixg C'ohn" axi) I'lowei) Indeu the F()LL<)\vix(i Si>Rix(; (from Sprague)

Weight of Crop,
5- Year Average

Nitrogen

Content of

Tops and

Roots,

2- Year

Average

per cent

Total
Nitrogen
per Acre
in Green
Manure

pounds

Yields of

Shelled Corn

Following

Green

Manure

Crops

per cent

Lo.ss in

Nature of Plant

Used as Green

Manure

Total

Dry

Weight

per Acre

pounds

Per Cent
of Dry
Weight

in Roots

Soil Humus
as Carbon

per cent

Winter vetch
Crimson clover
Red clover
Sweet clover
Alsike clover
Winter wheat
Winter rye
Weeds only

3.812
3.049
1,786
1.436
1.983
2.089
2,463
1.263

20.8
21.3
17.8
19.7
20.6
39.3
35.5
7.6

3.49
3.03
2.82
2.75
2.68
1.63
1.29
1.50

1.33.0
92.4
.50.4
39.5
53.1
34.1
31.8
18.9

127.8
115.6
114.7
113.7
104.4
99.7
95.3
100.0

0.02
0.10
0.05
0.07
0.11
0.01
0.01
0.08

When comparatively young plant materials are used as green
manures, there is danger of a loss of nitrogen through volatilization
as ammonia, the loss depending upon the amount of total nitrogen,
as well as of readily decomposing nitrogen compounds in the green
plant material. Young plants, low in lignin and in cellulose, but
high in water-soluble substances and in nitrogen, decompose much
more rapidly than do mature plants; they leave a much smaller resi-
due in the form of humus; and only a small part of the original nitro-
gen is stored away in this humus. In the case of more mature plants,
a considerably larger amount of humus is left in the soil, because of
slower decomposition of the plant material and because of the higher
lignin content; smaller quantities of the plant nutrients are liberated
in decomposition of these materials. In many instances, consider-
able time may elapse before the nutrient elements, especially the
nitrogen, are liberated in forms available for plant growth. The age
of the plant used for green manuring exerts an important influence
upon the amount and rapidity of liberation of the nutrient elements

320 Manures, Composts, Green Manures

in available forms and upon the chemical nature and abundance of
the humus produced. The extent of the liberation of the nutrients
and the amount of humus produced can thus be controlled by proper
selection of plants for green manuring and of the time when these
plants are plowed under.

The humus left from the decomposition of green manures does not
completely replace the humus lost from the soil as a result of culti-
vation. Mooers has shown that, when cowpeas were grown on a
soil and the whole crop was turned under annually, there was a loss
of 0.11 per cent of humus, or a total of 2,200 pounds per acre during
a 20-year period. When the cowpea crop was removed and only
the stubble turned under, the loss of humus from the soil was con-
siderably greater: at the end of the 20 years, the total loss was 0.24
per cent, or 4,800 pounds per acre. As a result of the turning under
of 20 annual crops of cowpea hay containing about 20 tons of dry
matter, there was left in the soil 2,600 pounds of humus, that is, only
6.5 per cent of the total plant material. Stable manure, on the other
hand, not only could fully replace the losses of humus from the soil
but actually brought about an increase in humus content. When the
soil received stable manure, at the rate of 4 tons per acre annually
for 20 years, a gain of 0.11 per cent of humus took place.

Use of green manures is recommended where an available supply
of nitrogen and carbon dioxide is required but where the amount of
humus left is not of great importance. When it is essential to in-
crease the supply of humus in the soil, stable manures or mature
plant residues are to be preferred, either after they have been com-
posted or when supplemented with available nitrogen and phos-
phorus upon addition to the soil.

Selected Bibliography 321

Selected Bibli()graj)liy

1. Alhrcclil, \\'. A., Artificial manure production on the farm, Univ. Missouri
Agr. Expt. Sto. Bull. 258, 1927.

2. Albrccht, W. A., Methods of incorporating organic matter with the soil in
relation to nitrogen accumulations, Univ. Missouri Agr. Expt. Sta. Bull. 249,
1936.

3. Barnettc, R. M., Jones, H. W., and Hester, J. B., Lysimetcr studies with the
decomposition of summer c()\er crops, Univ. Florida Agr. Expt. Sta. Bull.
327, 1938.

4. Daji, J. A., The decomposition of green manures in soil, /. Agr. Sci., 24:15-
27, 1934.

5. Dunn, L. E., and Wheeting, L. C, Utilization of barnyard manure for Wash-
ington soils. Wash. State Coll. Agr. Expt. Sta. Bull. 395, 1941.

6. Humfeld, H., and Smith, N. R., The decomposition of vetch green manure
in relation to the surrounding soil, J. Agr. Research, 44:113, 1932.

7. Jenkins, S. H., Organic Manures, Imperial Bureau of Soil Science, Harpen-
den, England, 1935.

8. Jensen, H. L., The microbiology of farmyard manure decomposition in the
soil. I. Changes in the microflora and their relation to nitrification, J. Agr.
Sci., 21:38-80, 1931.

9. Leukel, W. A., Barnette, R. M., and Hester, J. B., Composition and nitrifi-
cation studies on Crotalaria striata. Soil Sci., 28:347-371, 1929.

10. Mooers, C. A., Effects of hming and green manuring on crop yields and on
soil supplies of nitrogen and humus, Univ. Tenn. Agr. Expt. Sta. Bull. 135,
1926.

11. Piper, C. v.. Green manuring, U. S. Dept. Agr. Farmers' Bull. 1250, 1922.

12. Salter, R. M., and SchoUenberger, C. J., Farm manure, Ohio Agr. Expt. Sta.
Bull. 605, 1939.

13. Smith, F. B., Stevenson, W. H., and Brown, P. E., The production of arti-
ficial farm manures, Iowa State Coll. Agr. Bull. 126, 1930.

14. Smith, F. B., and Thornton, G. D., Production of artificial manure, Univ.
Florida Agr. Expt. Sta. Bull. 415, 1945.

15. Sprague, H. B., The \alue of winter green manure crops, A''. J. Agr. Expt.
Sta. Bull. 609, 1936.

16. Thome, C. E., Farm Manures, Orange Judd Publishing Co., New York, p.
146, 1914.

17. Turk, L. M., The composition of soybean plants at \arious growth stages
as related to their rate of decomposition and use as green manure, Univ.
Missouri Agr. Expt. Sta. Bull. 173, 1932.

18. Waksman, S. A., Chemical and microbiological principles underlying the
decomposition of green manures in the soil, J. Am. Soc. Agron., 21:1-18,
1929.

322 Manures, Composts, Green Manures

19. Waksman, S. A., Humus; Origin, Chemical Composition and Importance in
Nature, Williams & Wilkins Co., Baltimore, 2nd Ed., 1938.

20. Waksman, S. A., Tenncy, F. G., and Diehm, R. A., Chemical and micro-
biological principles underlying the transformation of organic matter in the
preparation of artificial manures, /. Am. Soc. Agron., 21:533-546, 1929.

/5-.

Microorgaiiisins and Soil Fertility

DOMESTlCATIOx\ OF MICROORGANISMS

The control of a given reaction that occurs in nature and the ap-
plication of such reaction or system for the benefit of man and his
economy, especially when this involves complex biological processes,
gradually lead to the domestication of this system and these proc-
esses. Men learned to domesticate animals and plants in prehistoric
times. Only very few animals and very few plants have been intro-
duced into human economy since history began. In the case of
microorganisms, the picture is quite different. The ability of man
to domesticate microorganisms, including those living below ground
and those living above it, those that are able to control diseases and
those that bring about useful processes, may be looked upon as one
of the greatest triumphs of modern civilization. This has been ac-
complished in the brief span of less than about three-quarters of a
century. Some of these domestications have been brought about
only within the last decade, as in the manufacture of antibiotics.
Soil microorganisms have contributed their share to the growing
family of domesticated forms of life which man has placed under
his control.

Within this category, by far the most important group of organ-
isms, from the point of view of soil processes and crop production,
are the root-nodule or legume bacteria, or those organisms that form
nodules on the roots of leguminous plants. The solution of the prob-
lem of soil inoculation and development of an understanding of the
strain specificity of the organisms concerned in the inoculation of
specific plants have in many instances revolutionized agricultural
practice. Such organisms have come to occupy a highly important
place in rural economy and also in soil conservation and soil im-
provement.

In addition to root-nodule bacteria, various other microorganisms
have been utilized for soil inoculation. Although but seldom is a

324 Microorganisms and Soil Fertility

soil found to be so lacking in specific organisms that their introduc-
tion is required to bring about a particular soil process, occasionally
the growth of certain crops and the need for specific reactions in the
soil make such inoculation desirable. This is true, for example, of
the growth of various forest trees, for which certain mycorrhiza
fungi are required. It is also true for certain types of orchids and
other plants. In addition, the use of sulfur bacteria in very specific
cases and of nitrifying organisms in others nearly exhausts the occa-
sional needs for artificial introduction of organisms into the soil. All
other claims for the favorable effects obtained by inoculation of soil
with various bacteria or fungi, ranging from Azotobacter and spore-
forming bacteria, namely, the "all-crop inoculants," to certain fungi
or earthworms, are exaggerated claims, based more on hope than on
fact, and always with an eye on the immediate benefit to the seller
of those cultures.

Among the other important processes in which considerable im-
provement has resulted from knowledge of the microbiological popu-
lation, the preparation of composts, discussed in the preceding chap-
ter, the preservation of manures, and the conservation of soil deserve
particular attention.

Preservation of Manures

When stable manures or plant residues supplemented with in-
organic fertilizer are placed in composts and conditions are made
favorable to the activities of microorganisms, through proper aera-
tion and sufficient moisture, numerous microbiological reactions im-
mediately set in. These are accompanied by a rapid rise in tem-
perature. Among the major chemical changes that take place during
the process of composting, the reduction of the cellulose and hemi-
celluloses and the relative increase in ash, lignin, and protein are
most significant. The latter occurs at the expense of the water-
soluble forms of nitrogen, which are utilized by the microorganisms
for their synthetic needs and are thereby converted into complex
organic forms. There is hardly any need for specific inoculation.
Plant residues and soils carry enough organisms which will imme-
diately become active when favorable conditions are established.
Addition of a few more organisms will scarcely modify the many
changes set in motion by the microorganisms already present.

One of the major economic problems involved in the preservation
of stable manures is the loss of nitrogen, which may amount to as

Exaluation of Soil Fertility 325

much as 20-50 per cont of tlic total nitrogen present in the manure.
\'arious procedures have been utih/ech for preventing such losses.
One of the simplest principles is to hasten the activities of the micro-
organisms which bring about the destruction of the cellulose and the
hemicclluloses in the manure. If conditions are favorable, an active
microbiological population will bring this about during the early
stages of decomposition, thereby t.'ansforming the soluble forms of
nitrogen in the manure into complex organic forms. The supple-
mentar\- addition of superphosphate will often tend to neutralize the
ammonia liberated, thus preventing its volatilization.

If the decomposition of the manure has been allowed to proceed
too far and if oxidation of the nitrogen to nitrate has begun, there
is great danger of this nitrate's being reduced to atmospheric nitro-
gen. A compost offers ideal conditions for such a reaction. To pre-
vent such losses, the compost must be made anaerobic, so as to
hinder the activities of the nitrifying bacteria, thus avoiding the
conversion of the ammonia into nitrate and the subsequent reduc-
tion of the latter to gaseous forms of nitrogen.

Evaluation of Soil Fertility by Measuring
Microbiological Activities

Numerous attempts were made during the first decade of this cen-
tury, beginning \\'ith Remy and Lohnis and followed by Lipman and
Brown and many others, to interpret the fertility potential of a soil
on the basis of its microbiological activity. Several methods of ap-
proach were usually followed, of which these may serve as illus-
trations :

1. A small amount of a given soil was added to a nutrient solution
of known composition and, after a few days' incubation, a single
biological change was measured. The reactions most commonly
studied were the formation of ammonia from peptone, or ammonifi-
cation; the formation of nitrate from ammonium salt, or nitrification;
the destruction of nitrate, or denitrification; and the fixation of ni-
trogen.

2. A chemical substance, simple or complex in nature, was added
to a given quantity of soil, the moisture of which was adjusted to
60 or 70 per cent of water-holding capacity. The soil was incubated
at 20-30 ^C for 7-30 days, and changes, similar to those listed above,
were measured.

326

Microorganisms and Soil Fertility

3. An examination was made, by the plate or other suitable
method, of the abundance of certain organisms in the soil. An at-
tempt was then made to evaluate the fertility of the soil on the
basis of the numbers and biochemical potentialities of these or-
ganisms.

4. Microorganisms were used for determining the concentration
of certain important plant nutrients in the soil.

''^^j^

m

Acttnomfcete

-HP'"

Actinomycete f'ungus

Fig. 128. Effect of microorganisms on soil aggregation ( from S\\ab>-

Without analyzing the voluminous literatiue that dealt with this
subject, it is sufficient to say that none of these methods yielded
results that could meet the test of severe criticism. They are now
largely abandoned, in spite of the fact that certain very definite cor-
relations have often been reported between the results obtained by
these methods and the fertility of soils. The major difficulty in-
volved in the use of such methods was that reactions brought about
by microorganisms in the soil under natural conditions are subject
to too many variables. These comprise not only inherent differences
in soil conditions, but also the effects of climate and soil manage-
ment. Not all these could possibly be taken into consideration in
the various laboratory studies. As long as a knowledge of the effects
of these variables was lacking, the information obtained by micro-

MicTooriianisnis and Soil Conscrxation

327

biological pioccdurrs was liiiiitccl in scope and had little ai^plication
to practical agricnltnrc. •

Microorganisms and Soil (x:)nservation

The role of microorganisms in improxing the physical condition
of the soil. notal)l\ soil aggregation, has recently received consider-

100

90 -

-5 80

o

Co
"D

§ 60
o

^ 50

c

^ 40

a;

Z 30

a
o

S 20

10

0

Plamfield sand
Spencer silt loam
Superior red clay

r

Aspergillus Penicillium Alternaria Fusartum StemphyUum Uporolnchum

Mold species
Fig. 129. Influence of .specific fungi on aggregation of three soils treated with
alfalfa (from Gilmour, Allen, Truog).

able attention. The strnctiire of the soil is greatly affected by the
m\'celium of fnngi and the slimy cells of bacteria, as well as by their
metabolic products. The effect of these consists in binding the loose
soil particles into water-stable aggregates. Various microorganisms
vary greatly in this respect. Whereas some bacteria have very little
effect, certain fungi, by means of their long hyphae, entangle the soil
particles into stable aggregates. The slimy substances, of a hemi-
cellulose or polyuronide nature, produced by various bacteria are
also highly effective in this respect.

Addition of organic materials to the soil, notably glucose, starch,
straw, clover, and stable manures, favors greatly the state of aggre-
gation by favoring development of various groups of microorganisms.

328

Microorganisms and Soil Fertility

According to Swaby, pure cultures of Absidia glauca and Aspergillus
nidulans growing in sterilized soil enriched with glucose produced
242 and 374 meters of mycelium per gram, as measured by methods
of Jones and Mollison. They entangled, respectively, 96.5 and 80.3
per cent of soil into stable aggregates. Fresh soil contained 38.8
meters of hyphae per gram. The presence of 38 per cent of aggre-
gates in such soil could thus be ascribed to the function of fungus
mycelium (Fig. 129). This is also brought out in Table 72. The

Table 7'2. The Ixfluexce of Mold Species and Alfalfa on the Dispersion
Ratios of Three Soils (from Gilmour et al.)

Dispersion Ratios

Mold Spscies Added

Plainfield Sand

Spencer

silt Loam

Superior

Red Clay

No

No

No

Organic

Alfalfa

Organic

Alfalfa

Organic

Alfalfa

Matter

Added

Matter

Added

Matter

Added

Added

Added

Added

None

41.5

37.2

43.5

30.4

38.0

20.0

.4. niyer

42.6

29.7

26.4

27.5

18.1

10.5

Penicillium sp.

41.4

28.8

24.7

21.4

16.8

9.2

Aliernaria sp.

40.1

29.9

27.3

11.6

21.3

8.2

Fusarium sp.

42.6

25.0

28.2

18.2

35.3

2.9

Helminthonporium sp.

42.6

24.8

25.2

27.5

36.7

9.9

CuTvularia sp.

40.1

22.8

28.4

12.6

37.6

4.1

Stemphyliiim sp.

44.7

15.7

29.6

14.2

35.0

8.2

Sporotrichum sp.

41.5

15.7

28.4

21.4

34.8

11.8

aggregating effect of bacteria was calculated to be only about 2 per
cent of the total.

That microorganisms differ greatly in their soil-aggregating prop-
erties and that the products of some of these organisms are excellent
soil-binders have also been brought out by Martin, McCalla, and
others. Fungi and certain polysaccharide-forming bacteria were
found to be more effective than actinomycetes, which in turn were
better than yeasts; certain bacteria were least effective.

Many of the soil-aggregating substances and even the mycelium
and cells produced by microorganisms are later destroyed by other
microorganisms, thus producing an effect of disaggregation. Micro-
bial associations alone could not account for the formation of per-
manent crumbs, especially when no fresh organic materials were

Microorganisms and Soil Conservation

329

added; this suggests the probabihty that other cementing substances,
such as chiy and hunuis, play essential rolffs in this process, as shown
recently by S^^'ab\'.

The inoculation of soil with fungi, such as Trichoderma Ugnorum,
to improve soil structure has been recommended. Such inoculation
is effecti\e, howcNcr, only when accompanied by addition of freshly
decomposable organic matter to the soil.

Table 73. AtiCiUi-ujATiNc; Ei^'fect of MicitooiuiANisMs upon Various Silt and Clay

Fractions of Collington Sandy Loam with Complex Organic Materials as

Energy Sources (from Martin and Waksman)

Incubation period, days

20

50

90

Fraction, m

<50

<20

<5

<50

<20

<5

<50

<20

<5

Inoculation

Energy
Source *

Agt

Ag

Ag

Ag

Ag

Ag

Ag

Ag

Ag

.4. niger

Control

0

0

0

0

0

0

0

0

0

Alfalfa

49

45

31

57

52

36

52

46

30

Manure

24

22

10

29

41

31

32

36

40

Peat

4

9

3

19

31

30

15

23

34

Soil suspension

Control

0

0

0

0

0

0

0

0

0

Alfalfa

57

61

61

71

77

74

68

68

64

Manure

32

41

27

44

54

56

40

47

50

Peat

8

17

18

15

29

34

13

20

25

* All organic materials used in 2 per cent concentration.
t Ag = Percentage aggregation.

Gilmour, Allen, and Truog concluded that inoculated soils to
which no organic materials had been added underwent only a slight
to moderate degree of aggregation. On the other hand, the addition
of oat straw and alfalfa decreased considerably the percentages of
unbound silt and clay in the soils studied. When no fungi were
present, there were lesser decreases in the unbound fractions. In
the presence of alfalfa and fungi, there was a marked reduction in
the susceptibilities of the soils to erosion. The effectiveness of fungi
in the aggregation process was related to the ejffectiveness of the in-
dividual organisms, the type of organic matter, and the physical
composition of the soil.

330 Microorganisms and Soil Fertility

Table 74. Ixfluence of Fungi on Soil Aggregation (from Swaby)

Mean Weight

Growth on Agar,

of Aggregates,

Total

Number of Strains

> 1 Mm/50 Gm

Number of

' '

Aggregation

Soil
gm

Strains

Woolly Prostrate

Excellent

37-45

21

18 3

Very good

29-37

15

10 5

Good

21-29

11

3 8

Fair

13-21

3

0 3

Totals 50 31 19

Soil Inoculation

It often becomes necessary to introduce into the soil bacteria and
certain other microorganisms that may be lacking there. Among
these, root-nodule bacteria occupy a pre-eminent place, as brought
out previously. At first, soil in which the legume was grown suc-
cessfully was used for inoculation. Soon after, however, artificial
cultures in liquid and solid media were substituted for the soil. In
recent years, peat material has been utilized as a carrier for legume
bacteria.

Although soils in which legumes have once grown contain for
some time the organisms responsible for formation of nodules on
the corresponding plants, it was found that these bacteria may de-
teriorate in the soil, either by loss of vitality or through the effect
of antagonistic microorganisms. It may, therefore, become advisable
to inoculate a soil frequently for a certain legume. The existence of
various strains of bacteria, which vary greatly in activity, the forma-
tion of bacteriophages active against the specific bacteria, and the
potential effect of antibiotics produced by other microorganisms
lead more and more to recognition of the importance of repeated
inoculation of soils with vigorous cultures of organisms.

On a much smaller scale, but of potential importance, are the
mycorrhiza fungi. These are capable of producing associations with
various higher plants, notably certain evergreens, resulting in in-
creased plant growth. It has been found advisable to inoculate
nursery beds with a small amount of soil from an old bed in which
the corresponding trees have been grown successfully. So far, no
pure cultures of fungi have been utilized for this purpose. In cer-

Modification of Soil Reaction

331

tain soils, however, it seems to have been estabhshed beyond donbt
that the presence of fungi is essential for normal tree development.
In addition to these two groups of microorganisms— the legume
bacteria and mycorrhiza fungi— it has also been found that occa-
sionally inoculation of soils with other organisms may result in
increased plant growth. Among these organisms, it is sufficient to
mention the nitrifjing bacteria, sulfur-oxidizing bacteria, bacteria
pathogenic to Japanese beetles or other insects, and nematodes para-

FiG. 130. Ectotroi^hic mycorrhiza developing on roots of Piniis sylvcstris (from

Melin).

sitic upon insects or destructive to other injurious nematodes. In
some cases the advisability of microbial inoculation of soil is ques-
tionable, unless accompanied by certain soil treatments. This is true
of the use of certain saprophytic fungi which are believed to act as
a check upon the development of pathogenic fungi, of fungi for im-
proving soil structure, and of "all-soil inoculants" ( Azotogen ) .

Enrichment of the soil with organisms not present there originally
may lead to development of antagonists, which bring about the de-
struction of the introduced bacteria.

Modification of Soil Reaction and Microbiological Activities

There is no one particular reaction which is favorable alike to all
groups of soil microorganisms. When the soil is acid, especially at
a reaction less than /;H 6.0, it may become injurious to the growth
of many bacteria, notably the nitrifying and the nitrogen-fixing types,
and favorable to the development of fungi. This may be because

332 Microorganisms and Soil Fertility

the competition of the bacteria for the available nutrients in the soil
is repressed by increased acidity. On the other hand, a less acid or
shghtly alkahne reaction of the soil may become unfavorable to the
development of fungi and have a favorable effect upon many of the
soil bacteria. Thus, when conditions are made unfavorable to the
development of one group of organisms in the soil, another group
may be favored.

Addition of calcium carbonate to an acid soil was found to stimu-
late greatly the multiplication of bacteria, accompanied by an in-
crease in the decomposition of the soil organic matter. This is
shown in Table 75. Addition of excess calcium carbonate and espe-

Table 75. Influence of CaCOs on Evolution of CO2 from Soil (from Konig)

COo Evolved

CaCOs Added

per Day

fer cent

mg

0

181.3

O.OJ-

223.6

0.10

308.4

0.20

416.4

0.40

455.4

cially of magnesium carbonate, however, may become injurious to
many of the soil bacteria.

Effects of Cultivation and Fertilization

Cultivation of soil, which results in conservation of the soil mois-
ture, is favorable to the development of various groups of micro-
organisms. It brings about an increased production of nitrate be-
cause of improved soil aeration. By favoring the development of
aerobic organisms, cultivation stimulates greater decomposition of

Table 76. Influence of Tillage upon Abundance of Bacteria in Soil

(from Chester)

Numbers in thousands

; per gram,

Period of Time

Bacteria

At start

2,040

After 7 days

5,495

After 9 days

6,171

After 14 days

11,326

After 24 days

12,600

Microorganisms and Plant Growth 333

the organic matter, leading to increased carbon dioxide evolution
and greater liberation of the nitrogen a's ammonia. This explains the
favorable effect of fallowing upon the activities of the soil micro-
biological population. The "ripening" of soil in spring is a result of
treatments that are favorable to the activities of soil microorgan-
isms.

Fertilization and crop rotation also have an important effect upon
the microbiological population. The nature of the fertilizer, the
residual effect upon the reaction of the soil, the nature of the crop
grown, and the treatment of the crop will influence in one way or
another the nature and abundance of microorganisms of the soil.
This is true especially of the addition of available energy in the
plant residues, the excretion by plants of substances favorable to
microbial development, the increase in soil nutrients, and the im-
provement in the buffering capacity and physical condition of the
soil.

Microorganisms and Plant Growth

Plants and microorganisms exert numerous effects upon one an-
other. Plants supply to the microorganisms most of the energy and
nutrients in the form of the numerous residues in the roots and
stubble. They also secrete soluble substances which affect in various
ways the growth of microorganisms. Plants control the chemical
composition of the soil solution, thus modifying the nature of the
medium in which most of the activities of microorganisms take place.
By removing some of the nutrients from the soil, plants may exert an
injurious effect upon the growth of microorganisms, or may actually
compete with them for some of these nutrients. Plant roots influ-
ence the structure of the soil and bring about an improvement in soil
aeration, thus affecting greatly the growth of microorganisms.

Microorganisms, in their turn, exert numerous influences upon the
growth of higher plants. By decomposing the plant and animal
residues in the soil, thus bringing about their mineralization, micro-
organisms liberate the nutrients required for plant growth, espe-
cially the carbon dioxide, nitrate, phosphate, and sulfate. The sym-
biotic nitrogen-fixing bacteria, through their association with the
roots of leguminous plants, effect the fixation of large quantities of
nitrogen. Plants and microorganisms form a variety of other sym-
biotic associations, designated as mycorrhiza (roots and fungi) and
bacteriorrhiza (roots and bacteria). Although the importance of

334 Microorganisms and Soil Fertility

mycorrhiza in plant development has been definitely established,
the effect of the bacteriorrhiza formations is still under discussion.
Various microorganisms are believed to produce plant-growth-
stimulating substances, including vitamins and hormones. Although
this is still open to debate, the fact remains that plants may benefit
considerably from addition of certain hormones and vitamins to the
soil. To what extent bacteria and other organisms are responsible
for the production of such substances has not been estabhshed as
yet. There is no doubt, however, that addition of organic matter,
especially stable manures, to the soil results in favorable effects upon
plant growth, which cannot be ascribed to the mere inorganic fer-

Table 77. 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

Bacteria

.\ctinomycetes

Fungi

CO2 Fori

millions

millions

thousands

mg

Bean

15 inches from main roots

18.6

7.6

24.6

9.7

Bean

9 inches from main roots

32.8

10.0

21.6

12.0

Bean

3 inches from main roots

36.2

8.0

20.0

12.0

Bean

Close to main roots

55.4

6.2

19.2

15.1

Bean

Superficial layer of the roots

199.4

12.6

55.2

Beet

15 inches from main root

18.6

10.0

25.8

11.2

Beet

9 inches from main root

27.0

11.4

25.0

13.6

Beet

3 inches from main root

33.4

10.4

25.8

14.9

Beet

Close to main root

57.4

6.8

30.0

18.2

Beet

Superficial layer of the roots

427.4

10.6

156.0

Corn

15 inches from main root

22.8

8.4

29.6

10.3

Corn

9 inches from main root

26.2

11.8

23.2

15.2

Corn

3 inches from main root

44.8

8.8

29.6

15.2

Corn

Close to main root

93.2

10.2

49.6

25.0

Corn

Superficial layer of the roots

653.4

8.6

278.0

* Age of plants, 113 days.

tilizer constituents of the manure. The favorable action, resulting
from decomposition of the organic residues, upon plant growth must
be definitely ascribed to the activities of microorganisms.

The effect of antibiotics produced by microorganisms upon plants
is another debatable question. Certain compounds, like actinomycin
and clavacin, are formed in artificial media by soil-inhabiting micro-
organisms. These compounds have a toxic effect upon plant growth,
resulting in a type of wilting. The question remains, however, to
what extent these substances are produced in the soil itself and
how their activities are modified by the inorganic and organic soil
colloids.

Soil Population 335

Soil Population as a Whole

The numerous interrelationships existing in the soil between plants
and microorganisms, on the one hand, and between soils and micro-
organisms, on the other, demonstrate the manifold activities of the
extensive microbiological population inhabiting the soil. These
microorganisms are responsible for numerous chemical reactions
taking place in the soil. The organisms do not exist and multiply
in the soil in a fixed manner. Their growth and activities are con-
stantly modified, depending upon the nature of the soil, its treat-
ment, the crop grown, and various changes in environmental condi-
tions. The microbiological population of any soil, at a given mo-
ment, ma\- be in a state of equilibrium. Any modification of this
equilibrium will bring about a marked change, both in qualitative
composition and in quantitative interrelations, among the constituent
members of this population.

Under natural conditions, modifications of this equilibrium take
place constanth'. The freezing of soil in winter, the melting of
snow and the thawing of ice in spring, the frequent wetting and dry-
ing of soil in summer and in fall, the addition of leaves, roots, and
other plant stubble from the growing vegetation, will continuously
modify the soil population by changing the conditions of the soil.
The nature of the crop and the treatment of the soil, especially culti-
\'ation and fertilization, further influence in many ways the nature
and composition of the soil microbiological population.

Aside from those modifications, man has learned to influence the
soil population through various specific treatments, such as addition
of lime or of acid-reacting fertilizers, air-drying or steam-steriliza-
tion, or treatment with various antiseptics. This is true particularly
of greenhouse soils and of nursery beds. All these treatments bring
about marked changes in the composition of the microbiological
population.

Selected Bibliography

1. Ensminger, L. E., and Gieseking, J. E., Resistance of clay-adsorbed pro-
teins to proteolytic hydrolysis, Soil Sci., 53:205-209, 1942.

2. Geltser, F. Y., Influence of the type of organic matter on soil structure,
Trans. Sov. Sect. Intern. Soc. Soil Sci., 5:115-120, 1936.

336 Microorganisms and Soil Fertility

3. Gilmour, C. M., Allen, O. N., and Truog, E., Soil aggregation as influenced
by the growth of mold species, kind of soil, and organic matter, Proc. Soil
Sci. Soc. Am., 13:292-296, 1949.

4. McCalla, T. M., Influence of microorganisms and of some organic substances
on soil structure. Soil Set., 59:287-297, 1945.

5. McCalla, T. M., Influence of some microbial groups on stabilizing soil struc-
ture against falhng water drops, Proc. Soil Sci. Soc. Am., 11:260-263, 1946.

6. Martin, J. P., Microorganisms and soil aggregation. I. Origin and nature of
some aggregating substances. Soil Sci., 59:163-174, 1945; II. Influence of
bacterial polysaccharides on soil structure. Soil Sci., 61:157-166, 1946.

7. Myers, H. E., and McCalla, T. M., Changes in soil aggregation in relation
to bacterial number, hydrogen-ion concentration, and length of time soil
was kept moist. Soil Sci., 51:189-200, 1941.

8. Swaby, R. J., The relationship between microorganisms and soil aggregation,
J. Gen. Microb., 3:236-254, 1949.

♦ /6-

Recent Developments in Soil Microbiology

General Trends

Soil microbiology is a borderline science. It deals with micro-
organisms and their importance in soil processes. It involves prob-
lems in ecology, physiology, and biochemistry. Since it is concerned
with soils as the natural substrate for the growth of microorgan-
isms, it embraces physical, chemical, and biological phenomena. An
understanding of the relationships of microorganisms to higher plants
and of the effect of microorganisms upon the activities of other
microorganisms is essential.

Soil microbiology has certain theoretical and practical considera-
tions. It involves (a) knowledge of the microscopic, ultramicro-
scopic, and near-microscopic populations of the soil, as influenced by
the nature and composition of the soil, by climatic and environmental
conditions, and by plant growth; (b) knowledge of the activities of
these microorganisms, which result in a variety of processes and in the
formation of numerous metabolic products, influencing directly or
indirectly the nature and composition of the soil and the growth of
cultivated and uncultivated plants; ( c ) methods of control of micro-
biological activities, and their domestication, thus harnessing them
for the service of man as well as of those plants and animals upon
whom man has come to depend for his existence.

Although the numerous groups of microorganisms inhabiting the
soil form only a very small part of the soil mass, they are responsible
for many of the chemical transformations, and even for some of the
physical changes, that take place in the soil. They result in making
the soil a living system rather than a mass of dead debris. The
microbiological population is largely distributed through the upper
layers of the soil mass, where the living plants send down their roots
and where they obtain the necessary nutrients. When the roots
die, they are rapidly attacked by the soil organisms, with the result
that some of the nutrient elements are returned to circulation and

337

338 Recent Developments in Soil Microbiology

made available again for the growth of new roots and new plants.
In this process, the microorganisms build up extensive cell material,
comprising bacterial cells and slimy substances produced by bac-
teria, mycelium of fungi and of actinomycetes and their products, as
well as numerous other living and dead bodies of microscopic forms
of life. All these contribute to the formation of soil humus. They
not only serve as reservoirs for further activities of microorganisms,
but also exert various physical and chemical effects upon the soil, as
by binding the soil particles and interacting with the various cations
and anions of the soil organic and inorganic constituents.

As a result of these microbiological activities in the soil, a continu-
ous stream of carbon dioxide, ammonia, nitrate, phosphate, and
other nutrient elements is made available for plant growth. The
humus supply of the soil may either increase or be gradually de-
stroyed, depending on the rate of formation of new plant material
and its decomposition. This dark-colored, amorphous, highly char-
acteristic soil constituent possesses certain important physical and
chemical properties which give to the soil its specific characteristics.
The formation and disintegration of humus are closely bound with
the activities of the microbiological population of the soil, on the
one hand, and with soil conditions and plant growth, on the other.

Many attempts have been made to develop inocula for various
nonleguminous plants; these comprise the so-called all-crop soil
inocula, and the inocula of nonsymbiotic nitrogen-fixing bacteria.
All these have failed to accomplish useful results. The suggestion
that the favorable effect of small amounts of stable manures upon
plant growth is due to the introduction of large numbers of bacteria
into the soil has likewise remained unsubstantiated. When soil con-
ditions are not favorable to the development of particular organisms,
mere introduction of these organisms will not result in their estab-
lishment in the soil. When conditions are made favorable for the
development of new organisms, as by drainage of salt lands and peat
bogs, by liming of acid soils, and by planting specific host crops,
certain organisms may be introduced to advantage. This is particu-
larly true of the legume bacteria, and occasionally of nitrifying bac-
teria, sulfur-oxidizing bacteria, and mycorrhiza fungi.

The preparation of composts represents another important process
in which considerable improvement has resulted from knowledge of
the microbiological population. When stable manures or plant resi-
dues supplemented with inorganic fertilizer are placed in a compost
and conditions made favorable to the activities of aerobic micro-

General Trends 339

(Mganisins, as b\ proper aeration and provision of sufficient moisture,
nimierous reactions immediately take place. These are accompanied
1)\- a rapid rise in temperature, which may serve as a measure of the
rapidity of the decomposition process. The microbiological popula-
tion of the compost changes with a change in temperature and with
the nature of the materials undergoing decomposition. Among the
major chemical reactions that take place during the process of com-
posting, the destruction of the cellulose and hemicelluloses, and the
resulting increases in ash, lignin, and protein contents are most sig-
nificant. Protein synthesis is brought about by the activities of the
microorganisms.

One of the major economic problems involved in the preservation
of stable manures is the potential loss of nitrogen, as pointed out
previously. Various methods have been utilized for the conservation
of the manure, the major purpose being the prevention of these losses.
One of these methods consists in hastening the activities of micro-
organisms which bring about the destruction of the cellulose and
hemicelluloses in the manure; if the microbiological population is
sufficiently active to bring this about during the early stages of com-
posting, the soluble forms of nitrogen in the manure will be rapidly
transformed into complex insoluble organic forms.

The survival in the soil of organisms causing plant and animal
diseases has also received considerable attention. Among the plant
diseases, the root rots, take-all diseases of cereals, soft rots, scabs,
club roots, and numerous others brought about by fungi, actinomy-
cetes, and bacteria are particularly important. To these should be
added the many insect pests which pass a part of their life cycle in
the soil, and the various diseases caused by worms and other animal
forms. Numerous methods of control have been developed, ranging
from partial sterilization by heat and chemicals to the introduction
of bacteria, fungi, and nematodes destructive to the parasite.

The fate of bacteria causing epidemics of animal diseases, and that
of fungi and actinomycetes causing less widespread outbreaks of
skin diseases and deep-seated diseases, have received considerable
attention. The study of antagonistic organisms found in the soil
and their formation of substances destructive to the pathogens is now
making rapid progress.

Recent trends in soil microbiology have thus centered upon a
better understanding of the nature and complexity of the soil popula-
tion, the conditions which influence its quantitative and qualitative
composition, the activities of these organisms in the soil, and the

340 Recent Developments in Soil Microbiology

utilization of these activities for soil improvement, soil conservation,
plant productivity, and combating of plant and animal pathogens.

The Soil as a Living System

Because of the extensive microbiological population inhabiting it,
the soil must be considered not merely a dynamic or even a biological
system, but a living system. This assumption can be substantiated
as follows: (a) living organisms, belonging both to plant and to
animal systems, have taken an active part in the processes of rock
weathering and soil formation; ( b ) these organisms have contributed
to the formation and accumulation of one of the most important
and characteristic soil constituents, humus, which is largely re-
sponsible for differentiating a soil from a mere mass of inorganic
debris; (c) the soil processes are continuous both in summer and
in winter, and are affected by temperature, aeration, moisture, and
supply of fresh plant and animal residues; (d) the extensive flora and
fauna representing numerous forms of life that inhabit the soil
range from the smallest bacteria to the large burrowing animals and
the roots of higher plants.

The Soil Microbiological Population

One could discover in the soil most forms of life, within proper
dimensions of size and space, if one would only search for them long
enough and develop the proper methods for their demonstration.
Exclusive of higher plants, which find in the soil a support and a
medium for their growth and from which they derive most of their
nutrients, and exclusive of the numerous animals that spend the
whole or a part of their life cycle in the soil, there exists in the soil
an extensive population of microorganisms. This comprises forms
which are characteristic of the soil and which seldom live in a natural
state under other conditions, as well as forms which find in the soil
only a temporary habitat.

The soil population also varies considerably, both in kind and in
abundance, depending upon the nature of the soil, its treatment,
and various environmental conditions. This can easily be demon-
strated by comparing the population of an undistiu-bed virgin soil
with that of the same soil after it has been cultivated and has re-
ceived various added organic and inorganic substances. In the
virgin soil, the microorganisms are in a state of equilibrium, where

The Soil Microbiological Population 341

till' ri>lati\o ahundance of the various bacteria, fungi, actinomycetes,
and protozoa depends upon the nature of the soil and its condition.
In the treated soil, however, this equilibrium is often disturbed,
and certain organisms develop in great abundance, out of all propor-
tion to the others. The specific nature of these organisms depends
either upon the chemical nature of the material added or upon the
nature of the changes produced in the soil by the treatment. The
disturbance thus brought about in the microbiological equilibrium
may be of a lasting nature, whereby one group of organisms may
become predominant, to be followed later by the rapid development
of other groups; or it may be only temporary, that is, after a short
time the interrupted equilibrium may become re-established on the
same quantitative basis or in a modified form.

The changing activities brought about by the microbiological popu-
lation of the soil can be best illustrated by following the course of
decomposition in the soil of fresh plant and animal residues. Pro-
tein-rich materials lead to an extensive development of bacteria and
actinomycetes; cellulose-rich materials bring about extensive devel-
opment of fungi and certain bacteria. Among the fungi, the Phyco-
mycetes may come first when fresh plant residues are added; they
are followed by Ascomycetes and Fungi Imperfecti, and finally by
Basidiomycetes. A large part of the synthesized fungus mycelium
will be gradually destroyed by bacteria. The bacteria may be fol-
lowed by protozoa. This sequence of forms does not follow under
all conditions. Many of the microorganisms are specific and are
adapted to one process; others are omnivorous and are capable of
performing a number of functions. The nature of the material under-
going decomposition, environmental conditions, and incidental oc-
currence of specific microbial types will influence the predominance
of certain forms over others.

The changing numbers and types of organisms in the complex soil
population, particularly when influenced by a number of soil and
environmental factors, do not lend themselves readily to ordinary
statistical treatment. Under these conditions, one is likely to over-
look the forest because attention is focused upon single trees. Sta-
tistics alone, when not properly interpreted, may tend to overempha-
size certain members of the population, frequently of very little sig-
nificance in soil processes, and to overlook others of much greater
importance.

342 Recent Developments in Soil Microbiology

Interrelationships of Members of the Soil Population

The interrelations of members of the soil population, on the one
hand, and of higher plants and other soil microorganisms, on the
other, have received considerable attention. Of particular interest
are the antagonistic and associative effects among microorganisms.
The antagonistic effects have received recognition by those interested
in combating soil-borne plant diseases. The specific effects of fungi,
bacteria, and actinomycetes in depressing various disease-producing
fungi, such as cotton root rot, various root diseases of cereals, and
damping-off diseases of other plants, have been ascribed to the pro-
duction of toxic substances by saprophytes or to the competition
with the parasites for the available food. In some cases, the depres-
sion of the parasite has been brought about by controlling the
activities of specific soil saprophytes. Potato scab may be controlled
by addition of sulfur, which is oxidized by specific bacteria to sul-
furic acid; the resulting acidity becomes unfavorable to the actino-
myces producing the scab.

An attempt has been made to interpret the ability of certain or-
ganisms to produce antibiotic substances in terms of survival of
certain microorganisms in the struggle for existence in nature, and
especially in the soil. One cannot, of course, deny the fact that
certain substances produced by some organisms are toxic to others,
and may thus tend to control the development or even the survival
of the latter in the soil. If one considers, however, the artificial
conditions under which antibiotics are produced by various selected
strains of organisms, the fact that these antibiotics are selective in
their action upon other organisms, and that these can readily develop
strains which are resistant to the action of antibiotics, one wonders
how effective these substances are in controlhng the soil population
under natural conditions. Penicillin, produced by various species of
Penicillium and Aspergillus, offers a good illustration. It is produced
only in highly specific media, of which the soil is hardly a type.
It is readily destroyed by various organisms inhabiting the soil. It
has but little activity upon the fungi and most of the bacteria living
in the soil.

Effect of Changing Conditions

The numerous bacteria and fungi living in the soil will not always
react in a similar manner to a change in conditions of nutrition and
environment. Many of the important soil bacteria, such as the

Effect of Changing Conditions 343

nitrifying organisms, the nonsymbiotic nitrogen-fixing forms, and
some of the celkilose-decomposing organisms, are highly sensitive
to acidity and will nsnally fail to grow at a pH less than 6.0; other
bacteria, however, snch as some of the snlfur-oxidizing forms and
the facultative anaerobic bacteria, seem to be able to withstand
considerable acid concentration. The same is trne of the response
of different bacteria to the addition of specific organic substances,
to a change in soil aeration, and to other soil changes. The fungi
also show considerable variation in response to changing soil or
nutrient conditions: some are more sensitive than others to increasing
acidity or to diminished aeration; some attack the water-soluble sub-
stances more readily, others attack by preference the cellulose, and
still others prefer the lignins and the proteins. There is also consid-
erable \ariation in response to changes in environment and in food
supply among the various actinomycetes and protozoa.

The stimulation of specific groups of organisms, whereby the nor-
mal microbiological equilibrium in the soil is interrupted and one
particular type or group, previously present only in limited numbers
or even in a latent state, becomes predominant, is due to the speciali-
zation of various microorganisms. Usually the energy source intro-
duced into the soil can be utilized only by the particular organism
under specific soil conditions, or the soil is modified to such a degree
as to favor the development of one organism in preference to others.
Winogradsky distinguished between the "autochthonous" bacteria,
or those organisms which attack primarily the organic substances of
the soil, and the "zymogenic" forms, or those which develop rapidly
as a result of addition of fresh organic substances.

When complex plant and animal materials are added to the soil,
the stimulating effect upon the development of various bacteria or
fungi is difficult to analyze, because of the changing nature of the
organisms with the progress of the decomposition process. The
chemical composition of the material added, which varies with the
nature of the material and the degree of its maturity in the case of
a plant substance, the chemical and physical soil conditions, and the
environmental factors, all modify the microbiological response to
such treatments. As a plant matures, it contains smaller quantities
of water-soluble substances, such as sugars and amino acids, and it
becomes poorer in nitrogen and minerals and richer in cellulose and
lignin. The addition to the soil of residues of a young plant will
favor an abundant development of many bacteria, including the
lactic acid forms, which attack the sugars and other water-soluble

344 Recent Developments in Soil Microbiology

substances; mature plant residues favor extensive development of
fungi, especially when available nitrogen is present in the soil or is
added to it.

To illustiate further the effect of changing conditions upon the
development of specific microorganisms, the population of a compost
may be examined in further detail. If a compost is kept at 28° C,
the population will consist largely of bacteria, fungi, protozoa, and
nematodes; actinomycetes develop only to a limited degree; aerobic
cellulose-decomposing bacteria, especially members of the Cyto-
phaga group, are most active. At 50 °C, where the rate of decom-
position is highest, certain thermophihc fungi and actinomycetes pre-
dominate; bacteria are also present but they are not the most abun-
dant forms; and the animal population is almost completely lacking.
At 65 °C, the fungi are eliminated entirely; certain actinomycetes,
belonging to the Micromonospora type, and the thermophihc bac-
teria are most abundant; cellulose decomposition is brought about
by anaerobic, spore-forming thermophilic bacteria. At 75°C, decom-
position is limited and takes place largely at the expense of the
proteins and hemicelluloses; cellulose is not attacked at all; certain
bacteria of the Plectriditim type and certain species of Micromono-
spora make up the population. The most rapid decomposition of
the manure takes place first at 65 °C. At this temperature, the nitro-
gen is completely consumed. The inoculation of hot composts with
an active thermophihc population has been found to hasten the
process of decomposition. Animal pathogens present in the manure
are also destroyed at the high temperature.

Role of Microorganisms in Soil Processes

The role of microorganisms in the minerahzation of waste ma-
terials in soils, water basins, and composts no longer requires em-
phasis. One need not dwell upon the function of microorganisms in
bringing about the liberation of nitrogen in an available form, as
ammonia, and in the oxidation of the ammonia to nitrate. R is now
universally recognized that the growth of legumes and their associ-
ated bacteria are of tremendous economic significance to agriculture.
Numerous other microbiological reactions have been elucidated and
are at present well understood. R is sufficient merely to mention
the oxidation of sulfur by bacteria, a process which frequently be-
comes of considerable importance; the reduction of sulfates, nitrates,
and arsenates, processes which involve the activities of various groups

Role of Microorganisms in Soil Processes 345

of microorganisms and max lunc, under certain conditions, great eco-
nomic significance; the composting of stable manures and plant resi-
dues for the production of artificial composts, which involves the
activities of large microbial populations; and the growth of plant and
animal parasites, involving fungi, actinomycetes, bacteria, nema-
todes and N'arious other worms, and insect larvae, resulting in condi-
tions which require radical modification of soil management.

The importance of microorganisms in a number of other soil
processes is still a matter of dispute, if not of mere speculation.
Here belong the activities of nonsymbiotic nitrogen-fixing bacteria,
in spite of the fact that the occurrence and physiology of these organ-
isms have been studied extensively. The specific effect upon plant
growth of various substances produced by microorganisms, including
vitamins, hormones, and other growth-promoting substances, is also
still a matter for speculation. The effect of the saprophytic popu-
lation of the soil upon plant and animal parasites which live in or
find their way into the soil is still insufficiently understood. The
influence of microorganisms and of their metabolic products upon
the physical condition of the soil, especially in aggregating the finer
soil particles, a problem of great importance in soil conservation, is
becoming more and more clearly recognized. The mycorrhizal rela-
tionships, in spite of the progress made during the last few years,
are still to be unraveled, and the processes involved are yet to be
understood and utilized for practical purposes.

These and numerous other processes resulting from the activities
of the soil-inhabiting microorganisms are frequently complicated and
involved. So far, only very few of them have been recognized and
still fewer utilized. Further progress will undoubtedly result with
the development of new methods and with the growing appreciation
of the interlocking activities of the complex microbiological popula-
tion of the soil.

Soil microbiology has made only a beginning. It is still facing
open vistas for further investigation. A highly complex population
active in a most complex medium, the soil, and bringing about a
number of most complicated processes, fully deserves the interest
not only of the soil microbiologist and of the soil chemist, but also
of the agronomist, the botanist, the zoologist, the pedologist, and the
biochemist, in finding the answers to some of the riddles which
Mother Earth still propounds for us.

Iiide

X

Absidia glauca, 328

Acetic acid, 300

AciditN', effect of, 242, 289, 299, 300,
301

Acrostalagmus, 83, 278

Actinomyces, 82, 167, 175

A. imjricue, 220

A. poolensis, 290

A. thennopliilus, 71

Actinoinycetales, 60

Actinomycetes, 23, 34, 35, 39, 47, 53,
55, 77-82, 252, 270
production of antibiotics by, 75

Actinophage, 279

Aeration, effect of, 62, 113

Aerobacter, 60

A. aerogenes, 193, 268, 270

Aerobic nitrogen-fixing bacteria, 194-
200

Agarictis nebularis, 121

Agar-plate method, 39

Age of plant, 103

Aggregation of soil, 326, 329

Agronomical phase of soil microbiol-
ogy, 4

Alfalfa, and soil aggregation, 327
decomposition of, 155, 158, 306

Algae, 36, 56, 88

nitrogen fixation b\ , 193, 200

All-crop inoculaiits, 324, 338

All-soil inoculants, 331

Altemaria, 83, 328

A. tenuis, 278

Amanita ovoidea, 86

A. virosa, 86

Amine formation, 167

Amines, effect on nitrification, 182-
183

Amino acid decomposition, 167-168
effect of, 175

Ammonia, formation ot, 18, 19, 63,
108, 118-119, 137, 167, 168, 170,
172-178, 188

oxidation of, 13, 62, 181
Ammonia-oxidizing organisms, 181
Annnonification, 176
Amoebae, 91, 92

in soil, 55, 56
Anabena, 199, 200, 220
Anaerobic bacteria, 73

cellulose decomposition, 74—75

nitrogen-fixing bacteria, 184, 193-
194
Animal forms, 37

pathogens in soil, 282

residues, 95, 149
Antagonistic effects, 38, 261, 266-269
Antagonistic fungi, 267
Antibiotics, 25, 269, 272, 274-280

effect on plants, 334

in soil, 280
ApJuniomt/ccs lacvis, 287
Aphclcnchus olesistns, 290
Armillaria, 278
Arsenic, 243, 299
Artificial manures, 313-317
Ascomycetes, 54, 83, 287, 341
AspergiUus, 23, 38, 54, 65, 83, 85, 86,

110, 239, 278, 342
A. clavatus, 278
A. fiavus, 278
A. fumigatus, 243
A. nidulans, 328
A. niger, 88, 174, 175, 177, 239, 268,

269, 278, 328, 329
A. ochraceus, 243
A. oryzae, 110, 239
A. sijdowi, 243
A. tcrricola, 167

347

348

Index

Association of plants and microor-
ganisms, 251

Associative effects, 17, 38, 261, 265-
266

Autochthonous bacteria, 59, 343

Autotrophic bacteria, 60, 61-68, 262

Auximones, 254

Azohydrase, 202

Azomonas, 195

Azotase, 195

Azotobacter, 25, 40, 44, 60, 192, 193,

194, 195, 196, 197, 198, 199, 200,
202, 203, 204, 220, 239, 241, 242,
259, 263, 279

inoculation of soil, 324
Az. agilis, 193, 195, 196, 202
Az. beijerinckii, 193, 195
As. chroococcum, 18, 38, 193, 194,

195, 197, 201, 235, 239

Az. indictim, 38, 193, 195, 199, 205
Az. vinelandii, 193, 195
Az. vitreum, 195

Bacilli in soil, 40

Bacillus agri, 69

B. ainylovorus, 110

B. anthracis, 268

B. arhorescens, 167

B. asterosporus, 193, 194

B. brevis, 69, 77

B. campestris, 290

B. cellulosae dissolvem-, 74, 75

B. cellulosam fermenians, 75

B. ceretis, 68, 69, 120, 170, 172, 173,

268, 270
B. cohuerens, 69
B. fusiformis, 69
B. janthinus, 167
B. macerans, 110
B. megatherium, 68, 69, 268
B. mesentericus, 69, 110, 167
B. mtjcoides, 38, 69, 167, 172, 235,

268, 270
B. nitroxus, 187
B. pctasites, 69
B. pohjmyxa, 77
B. radicicola, 211, 215
B. .saccharobutijriciis, 193
B. s-implcx, 69, 294

B. stutzeri, 187

B. subtilis, 38, 69, 77, 119, 167, 172,

173, 268
B. vidgatus, 69, 268
Bacteria, classification of, 60

in manure, 52

in soil, 32, 34, 35, 39, 47, 55, 60-
76, 160
Bacterial-agar plate, 267
Bacteriophage, 223-224
Bacteriorrhiza, 333, 334
Bacterium denitroftuorescen.<i, 71
Bact. globiforme, 70
Bact. parvulum, 69
Bact. radicicola, 211, 215
Bact. stutzeri, 71
Bact. tumefaciens, 290
Bact. vulgar e, 167
Bact. vulpinus, 71
Bacterization, 259, 295
Bacteroids, 214
Basidiomycetes, 54, 84, 85, 88, 287,

341
Beaker method, 19, 33
Beggiatoa, 67
Beijerinck, M. W., 13
Bergey's system, 60
Biological control, 81, 292, 294
Biodc population, 264
Blue-green algae, 200
Blue-staining fungi, 75
Bodo cantatus, 90
B. re pens, 92
Boletus bovinus, 86
Bol. satanas, 86
Boron, 243
Botnjosporium, 115
Botnjotrichum piluliferum, 167
Botrytis, 278
Bot. alia, 278
Bot. cinerea, 278, 287
Boussingault, J. B. J. D., 2
Brucella melitensis, 284

Calcium carbonate, effect of, 51, 226,

332
Calcium transformation, 241
Calcofilic fungi, 86
Calcofugic fungi, 86

Iiulfx

319

(,'arl)()li>clrali's, tU-t()iiii)ositi(Hi ol,

109-1 17
effect on aninionia fomiation, 174,

178
Carbon bisnlfidc, effect of, 298
Carbon dioxide, exolution of, 41-44,

107-108, 142, 149-163
formation of, 252
liberation of, 256, 258
in soil, 98, 249
Carbon monoxide oxidation, 68
Carbon-nitrogen ratio, 140, 145, 157,

163
Cellfalcicula, 76
Cellulose, effect on nitrogen fixation,

206
Cellulose-decomposing bacteria, 74-

76
Cellulose-decomposing fungi, 84
Cellulose decomposition, 15, 20, 23,

72, 110-114, 263
CeUvibrio, 76
Cephalosporium, 83
CepJudothecium roseum, 75, 167, 278
Cercomonas, 92
Cer. crasscauda, 91
Chaetomium, 38, 86
Chemical treatment of soil, 296, 299
Chernozem, 143
Chitin, 172

Chlamydobacteriales, 60
Choline, decomposition of, 119, 168,

236
Chromobacteriutn violaceum, 268
Ciliata, 90
Cladosporium, 83
Clavaria flava, 86
Clay-humus complex, 129-130
Clitocybe clavipes, 86
Cl. geotropa, 86
Clostridium, 193, 194, 199, 263
Cl. amylobacter, 74
Cl. bifermentans, 285
Cl. butylicum, 198
Cl. cochlearius, 285
Cl. oedematiens, 285
Cl. pa.'fteiirianum, 192, 193, 194, 197,

198, 200
Cl. perfringens, 122

(7. puhificus, 172
Ct. ptitrificus tenuis, 74
Cl. putrijicus vcrucausus, 74
Cl. sporogenes, 285
Cl. tertius, 285
Cl. tetani, 74, 285
Cl. tcfaunmorphus, 74
Cl. welchii, 73, 285
Club root, 300
Cobalt, 242
Cocci in soil, 40
Coccospora agricola, 115
Cold manure, 309
Colletotrichum, 278
Colpoda cucidlus, 92
C. steitiii, 90, 92
Combustion, 1

Composition of soil, 29, 30, 31
Composts, 52, 53, 99, 307, 313, 338-
339

of sulfur, 231
Concept of soil microbiology, 4
Coniophom, 121
C. cerebella, 121

Contact slide method, 36, 43, 295
Control of plant diseases, 295
Copper, 242
Coprinus, 122
C. radians, 122
Coprophilic fungi, 83, 84
Com, composition of, 103, 106

decomposition of. 111
"Corrosion," 121
Corticium vagtim, 287
Cortinarius fulgens, 86
C. mucosas, 86
Corynebacterium, 70, 77
C. liquefaciens, 70
Crop, effect of, 253
Crop rotation, 209, 296
Crop yields, 50, 162
Cross inoculation, 224
Culti\ation, effect of, 332
Culture methods, 41
Culture solutions, 18
Cunninghamella, 239, 264
Cun. elegans, 278
Curvularia, 328

350

Inde>

Cyanamidc ck-coiiipositioii, 119, 171

effect of, 183
Cystine, 232

Cytophaga, 39, 76, 112, 115, 344
Cyt. huicJiinsoni, 77
Cyt. hitea, 76
Cyt. myxococcoides, 77

Damping off, 291
Deaiiiinization, 167
Decarboxylation, 167
Decay, 15, 16

Decomposition, of organic matter, 3,
15, 16, 37, 53, 101

of plant and animal residues, 95-
123

of stable manures, 52
Dematium, 278
Denitrification, 183-189
Denitrifying bacteria, 71, 187-188
Depth of soil, effect of, 34
"Destruction," 121
Deuterophoma, 278
Dicyanodiamide, decomposition of, 171
Dimastigamoeha, 92
Diplococcus pneumoniae, 193
Direct examination of soil, 44
Disease-producing microorganisms, 2,

282-302
Distribution of microorganisms, 34
Domestication of microorganisms,

323
Dry-land farming, 140
Duff, 136

Earthworms, 37, 45, 56

in soil, 15
Eberthella typhi, 268
E. typhosa, 283
Ecologic factors, 48
Ecologic relationships, 264
Ecological phase, 24
Ectotrophic mycorrhiza, 86—87
Electi\'e culture methods, 31, 32, 50
Endotrophic mycorrhiza, 87
Enzymes, production of, 274
Equilibrium of microorganisms, 41,

270-272, 335
Eremycausis, 1

Escherichia culi, 52, 268, 275, 283,

284
Eubacteriales, 60
Excretion of nitrogen, 224—226
Exhaustion of nutrients, 271

Fatigue of soil, 224

Fatty substances, decomposition of,

122
Fermentation, 4, 8, 10, 16
Fertility of soil and humus, 139

and microorganisms, 323
Fertilization, effect of, 38-39, 332
Fertilizers for soil treatment, 292
Filterable organisms, 37
Fimicolous fungi, 83
Fixation of nitrogen, sec Nitrogen

fixation
Flagellata, 90
Flagellates, 92

in soil, 55, 56
Food stuffs, microbiology of, 2
Forest humus, 135-137
Forked mycorrhiza, 87
Formaldehyde, 299
Foundation of soil microbiology, 10
Fungi, and soil aggregation, 327, 328-
330

distribution of, 54

in soil, 35, 39, 53, 55, 82-88
Fungi Imperfecti, 54, 287, 341
Fungicides, 300
Fusarium, 38, 83, 86, 243, 278, 291,

292, 293, 328
F. lateritium, 278
F. lini, 287, 293, 295
F. radicicola, 288
F. vasinfectum, 287
Fusarium wilt, 301

Gases, 29

Gelatin plate method, 12
General microbiologx-, 26
Geophilic fungi, 83
Gilbert, Sir Henry, 5
Gliocladium, 278
Glucose, effect of, 174
GranuJohacter, 192
Grass, effect on trees, 258

Index

351

Crcrn inaiuucs, 25'3, 317— 320

tor control of scab, 296
Croon sulfur haotoria, 67
Crowth-proinoting suhstancos, 67, 33 1

llnrtmanclla liyaliiui. 92

Hclniinthosporium, 278, 293, 328

H. grainincum, 287

H. sativum, 278, 293

H. teres, 278

Heniicelluloses, doooinposition of,
11.5-117

Heteroantagonistio, 267

Hctcrodcra radicicoUi, 290

Het. schaclUii, 93, 290

Heteromita, 92

H. glohosus, 92

Heterotrophic bacteria, 68-76

Higher fungi, 85-86

Hippuric acid decomposition, 170

Historical, 1-28

Hookworm disease, 286

Hookworm lar\ae, 93, 286

Hormones, 168, 334

Horse manure, 118, 314

Horse manure compost, 152

Host plant specificity, 219

Hot fermentation of manure, 53, 309

Human pathogens in soil, 282

Humic acids, 125

Huniicola, 178

Humicolous fungi, 83

Humification, 16

Humus, analysis of, 133
and soil fertility, 144-147
decomposition of, 131, 132, 137-

142, 143, 157, 159
effect on leguminous plants, 221
effect on nitrogen fixation, 204-205
formation of, 105-106, 130-133,

143
importance of, 128
in green manure, 319
in soil, 133, 134, 142-144
nature of, 124-130, 142

Humus fungi, 84

Humus podzol, 127-130

Hydrogen oxidation, 244
as source of energy, 60, 68

lli/diDfii'notnotui.s (inilis, 72
Ihuroxylamine, 186
Hxphao in soil, 45
H\ pliomycctes, 86
HvpogcoTis fungi, 83

"Incrustants," 99

Indigotin, 11

Industrial microbiology, 2-3

Infusoria, 90

Inoculants, 301

Inoculation, 223

of soil, 227, 330

of sulfur, 63
Interrelationships in soil, 342
Iron transformation, 241
Isoantagonistic, 267
Itcrsonia ferruginea, 77

Japanese beetle lar\ac, 93

Klebsiella pneumoniae, 268

Lactarius turpis, 86
Lawes, Sir John, 3
Lecithin, 236

decomposition of, 168
Legumes, use of, 226-228
Leguminous plants, 14, 208
Lenzites, 121
Lepiota granulosa, 86
L. procera, 86
Liebig, J. von, 9
Life cycle of nodule bacteria, 214-

215
Lignicoleous fungi, 83
Lignin, decomposition of, 119-122,

263
Lignin-decomposing fungi, 84
Liming of soil, 160
Lipman, J. G., 19
Li\ing matter in soil, 29
Lohnis, F., 19
Lycoperdon caelatum, 86

Magnesium, transformation of, 241
Manganese as source of energy, 60
Manure, and soil fertility, 311-312
composition of, 304

352

Index

Manure, decomposition of, 118, 139,
152, 30.5-307, 310

effect of, 38, 48, 113-115, 294

microorganisms in, 52

preservation of, 308, 310, 324, 339
Mastigophora, 89
Medical bacteriology, 2
Medical mycology, 2
Mercury compounds, use of, 300
Merulius lacrymans, 121
Methods, 19, 39

for evaluation of soil fertility, 325
Microbial equilibrium, 270-272
Microbiological antagonism, 293
Microbiological population, 33, 192,

193
Micrococcus denitrificans, 187
M. fiavus, 268

Micromonospora, 35, 81, 82, 344
Microorganisms, effect on plants,

250-254
Microscopic methods, 33, 40, 41, 42
Microspira desulfuricans, 234
Mineral substances, transformation

of, 230-245
Minerals in soil, 29, 164
Moisture, effect of, 48, 49, 112, 163,

291, 309
Molybdenum, 230, 242

effect on nitrogen fixation, 223
Monascus, 278
Monilia, 86, 278
Monosporia, 86
Morphology of bacteria, 214
Muck, 134
Mucor, 83, 278
Mulder, J. G., 8
Mull soil, 136, 143
Mycelium in soil, 36, 40, 44, 45, 54
Mycobacterium tuberculosis, 284, 285
Mycogone nigra, 115
Mycorrhiza, 86-88, 259
Mycorrhiza fungi, 38, 86-88, 330
Myxobacteria, 71, 77
Myxobacteriales, 60
Myxococcus, 71, 77
Myxomycetes, 287
Myxophyceae, 200

Ndegleri gruberi, 92

Nematodes, 45, 57, 93, 290, 299

"Niter plantations," 179

Nitrate accumulation, 160

Nitrate assimilation, 185

Nitrate formation, 104, 137, 139, 171

Nitrate reduction, 184, 185

Nitrification, 12-13, 15, 160, 179-
183, 235

Nitrifying bacteria, 61-66

Nitrite oxidation, 182

Nitrobacter, 65

Nitrogen, assimilation by plants, 209

Nitrogen fixation, 14, 160

mechanism of, 201-204, 222-223
nonsymbiotic, 191-207
symbiotic, 14, 208-229

Nitrogen-fixing bacteria, 60, 184

Nitrogen-fixing capacity, 19

Nitrogen liberation, 104

Nitrogen losses, 140, 141, 188, 189

Nitrogen transformation in soil, 166-
190

Nitrogenase, 195

Nitrogenous substances, decomposi-
tion of, 117-120

Nitrosocystis, 65

Nitrosogloea, 65

Nitrosomonas, 61, 65, 181

ZV. europea, 61

Nitrosospira, 65

Nocardia, 35, 70, 82

N. catarrhalis, 268

N. coraUinus, 70

Nodule formation, 210-212, 213-214
in nonlegumes, 220

Non-spore-forming bacteria, 69-70

Nonsymbiotic nitrogen fixation, 191-
207

Nostoc, 199, 200, 220

N. muscorum, 200

Nucleic acid, 167

Nucleobacter, 237

Nucleoproteins, 237

Numbers of bacteria, 39, 53

Oats, decomposition of, 105, 117, 307

Oicomonas termo, 92

Oils, decomposition of, 122

Index

353

(li.K-liansky, W L., lU
0))hi()ho}us\ 278
(). fii-aminis. 292, 294
Or<ianic-iiu)rganic colloid, 130
Organic matter decomposition, 149-
163, 175

effect of, 291, 294

in soil, 130, 133
Organized ferments, 13
0\idati\e deaminization, 168
Ox>gen, importance of, 244

Partial sterilization of soil, 74, 255,

273-274, 297-299
Pasteur, L., 2
Pathogens in soil, 282
Peat, 134-135

decomposition of, 163

formation of, 136
Peat humus, 314
Peat moss, 134
Pectin, 115-117
Pedological phase, 4
Penicilliwn, 23, 38, 54, 83, 84, 86,

93, 115, 278, 328, 342
P. brevicaule, 66, 243
P. italicum, 293
Peziza, 278
P. sclerotiorum, 278
P. trifoliorum, 298
Phages, 37, 93
Phoma betae, 287
Ph. radicis, 88
Phosphorus, availability of, 252

transformation of, 235-240
Photochemical nitrification, 180
Photosynthesis and nitrogen fixation,

219
Phycomycetes, 54, 83, 84, 287, 341
Phylloxera, 290

Phymatotridum omnivorum, 293, 295
Phyo-enzymes, 196
Physical methods of soil treatment,

296
Physiological groups of bacteria, 51
Ph\'siological phase, 4
Physiology of nodule bacteria, 218
Phytin, 236
Phytohormones, 168, 254

Phyluphthorii, 278, 288

Ph. bowlesii, 268

Ph. infest cms, 288

Plant diseases, control of, 295, 339

Plant forms, 37

Plant growth and microorganisms,

164, 246-260, 333
Plant materials, composition of, 103
Plant residues, 29

composition of, 98, 150
decomposition of, 35-36, 40, 95-
123, 126, 149
Plant roots, 334

Plants, and microorganisms, 262
effect on microbial population, 250-
258
Plasmodiophora brassicae, 287
Plate culture methods, 45, 46, 47
Plectridium, 193, 344
Podzol, 29, 30, 47, 127, 144
Poly poms annosus, 121
Polyuronides, 1 16
Popillia japonica, 291
Population, microbiological, 6, 17, 18,
22, 27, 29-58, 261, 275, 293,
335, 340
Poria, 121
Potassium, transformation of, 240-

241
Potato scab, 286, 287, 292, 296
Powdery scab, 291
"Pox" of sweet potatoes, 290, 296
Predaceous fungi, 84
Protein decomposition, 45, 48, 108,

117-120, 166-172
Proteus vulgaris, 172
Protosea cuprea, 75
Protozoa, 88-92

effect on bacteria, 273
in soil, 37, 55
Protozoan theory, 22, 273, 299
Psalliota campestris, 121
Pseudomonas aeruginosa, 187, 268,

270, 283, 284
Ps. citri, 293
Ps. fluorescens, 66, 69, 170, 172, 173,

175, 243, 264, 268, 270
Ps. fluorescens liquefaciens, 172, 235

354

Index

Ps. fiiioresccns putidus, 167
Ps. zonatum, 271
Pseudomycorrhiza, 88
Pseudoparasitic fungi, 83
Pure humus, 125
Purple sulfur bacteria, 67
Putrefaction, 10, 15, 16, 73
Pijthium, 278, 287

Radiobactcr, 60, 251, 255, 256, 257
Raw humus, 136, 144
Reaction, effect of, 179, 180, 181,
186, 301
of soil, 331
Reduction of nitrates, 186
Reducti\e deaminization, 167
Remy-Lohnis solution method, 325
Rhizobium, 217, 218, 219, 220, 224
Rh. japonicum, 217
Rh. leguminosurum, 38, 200, 217
Rh. lupini, 217
Rh. meliloti, 217
Rh. phaseoh, 217
Rh. radicicola, 217
Rh. trifolii, 111
Rhizoctonia, 79, 278, 292
Rh. solani, 288, 290, 294
Rhizopoda, 90
Rhizopus, 83, 119, 264
R. ingricans, 269, 289
Rhizosphere, 70, 247-250
Rhodococcus cinnebareus, 268
R. roseus, 268

Roman writers, 7, 11, 14, 208
Root infection, 212
Root-inhabiting fungi, 84
Root-nodule bacteria, 191
Root region, 247
Root rots, 301

Roots, effect on microorganisms, 248
Rossi-Cholodny techniciue, 25, 295
Rotation of crops, 77, 90
Russell, Sir John, 22
Russula amoena, 86
R. maculata, 86

Rye plants, composition ot, 100, 104
Rye straw, decomposition of, 156,
178

Succharomyces eUipsuideus, 268
Sac. murianus, 268
Sac. pastorianus, 268
Salmonella enteriditis, 268
Sal. pullorum, 268
Sal. suipestifer, 268
Sarcina lutea, 167, 268
Sarcodina, 90
Schloesing, J. J. T., 14
Sclerotinia trifoltorum, 287, 288
Sclewtium rolfsii, 78, 278, 291
Scopulariopsis brevicaule, 243
Season of year, effect of, 49
Selenium transformation, 243
Serratia marcescem; 172, 268, 270
Shigella galliuanim, 285
S/i. parady.senferiac, 268
S/i. pullorum, 285
"Sick" soils, 69, 91, 256
Silicon transformation, 244
Simple mycorrhiza, 87
Smuts in soil, 287
Soil, as li\ing system, 337, 340
composition of, 7
conserxation of, 327
fertility of, 17, 18, 87-91, 323-
335

Soil aggregation, 326

Soil atmosphere, 164, 165

Soil chamber, 44

Soil-enrichment methods, 51

Soil-inhabiting fungi, 84

Soil inoculants, 301, 330

Soil methods, 32

Soil plate, 43

Soil processes, 344-345

Soil reaction, effect of, 286

Soil sterilization, 296

Solution methods, 32

Space antagonism, 274

Spirillum desulfuricans, 72

Spirochaeta cytophaga, 76, 77, 115

Spirochaetales, 60

Spore-forming bacteria, 68-69

Sporotricha, 86

Sporotrichum, 86, 328

Sp. olivaceum, 86

Sporovibrio desulfuricans, 72, 234

Sjiorozoa, 91

Iiulcx

.355

St.il)!.' iiiaimus, 107, .■M).?-;M2; .scf

(iImi Manure
Stachyhiytnj.s, 115
Stainod soil preparations, 41
Staphylococcus alhtts. 2fi8
S. aureus, 268
S. cUrcus, 26<S
S. enter it idis, 268
Starch decomposition, 110
Steam sterilization, 255
Stemphylium, 167, 328
Stercuni ru^osuin, 121
Stcri^ntatocystis, 278
Sterilization of soil, 297-299
Stinuilation of organisms, 93
Strains ot nodule bacteria, 213-214
Straw, composition ot, 314

efFect of, 139
Streptococcus pyogenes, 270
Streptomyces, 35, 78, 79, 82, 264
S. coelicolor, 119
S. griseus, 78, 82, 277, 279
S. lavendulae, 278
S. sca/;/c*, 289, 292, 294
S. viridochromogenus, 120
Struggle for existence, 264
Stubble, 131

Sugar and nitrogen fixation, 206
Sugar fungi, 84
Sulfate-reducing bacteria, 72
Sulfur, effect of, 230-235, 296

transformation of, 62-63
Sulfur bacteria, 66-68, 232
Sulfur composts, 231, 240
Sweet-potato scurf, 296
Symbiotic nitrogen fixation, 52, 61,

208-229
Symbiotic nitrogen-fixing bacteria,

193
Synchytrium endobioticum, 287
Systematic bacteriology, 9

Taurine, 232

Temperature, effect of, 49, 52, 53,

219, 316
Terrestrial fungi, 83
Terricolous fungi, 83
Thamnidiuni elegans, 278
Thecamoebae, 91

Tlicrniodclinoniyccs, 7 I

Thermogenic bacteria, 70

Tliernioniyces, 71

Thermopiiilic bacteria, 53, 70-71, 73

Thermophilic microorganisms, 73

Thiclavia, 291

Thiohacillus, 66

Th. denitrificans, 71

Th. novellus, 66

Til. tliiooxidans, 66, 67, 233, 238

Th. thioparus, 18, 66

Thiotlirix, 67

Thorn, C, 23

Tillage, effect of, 332

Torula ammoniacale, 72

T. .sphaerica, 268

T. .suganii, 278

ToruJopsis, 278

Toxic substances, 280

Toxins in soil, 254

Trametes pini, 120

Transient microbes, 59

Trichoderma, 39, 83, 86, 112, 175,

178, 239, 264, 292
T. koningi, 120, 289
r. lignorum, 271, 278, 289, 293, 294,

329
Trichok)nia aUnitu, 86
Trichothecium, 278
T. roseum, 293
True humus, 125
Tuber mycorrhiza, 87
Tumbler method, 19, 33
Turf, 134

Tylenchus dipsaci, 290
T. dipsae, 290
r. tritici, 290

Urea bacteria, 72-73

Urea, decomposition of, 119, 170-

171
Uric acid, decomposition of, 168
Urine, decomposition of, 139
UrohaciUus duclauxii, 73
Ur. maddoxii, 73
Ur. pesteurii, 73
Ustilago, 278

356

liid

ex

Vahlkampfia, 91

Verticillium, 71, 83, 86, 278

V. alboatrum, 287

V. cellulosae, 86

V. glaucum, 86

Vibrio, 114

V. cholerae, 284

V. comma, 268

V. desulftiricans, 19

Viruses, 37, 93, 298

Vitamins in soil, 334

Water, 29

Wind-blown soils, 71

Winogradsky, S. N., 13

Wireworms, 290

Wood, decomposition of, 121

Zinc, 242
Zijgorhijnchus, 83
Z. priorianus, 268
Zymogenic bacteria, 343
Zymogenic flora, 59

All