SOIL BACTERIA.
_C._ ACTIVITIES CONNECTED WITH THE BUILDING-UP OF BACTERIAL PROTOPLASM.
(1) _Composition of Bacteria._
The activities of the soil bacteria that we have yet to consider are
those connected with the building-up from simpler materials of the
protoplasm of the bacterial cell. It is important to bear in mind that
this process is one requiring an expenditure of energy on the part of
the organism. The sources of energy we have already considered.
The bodies of bacteria contain the same elements common to other
living matter. Analyses of various bacteria have been made by a number
of workers. About 85 per cent. of their weight is made up of water.
This analysis of Pfeiffer’s Bacillus by Cramer[15] shows the typical
percentages of carbon, nitrogen, hydrogen, and ash in the dry matter:--
_Composition of Pfeiffer’s Bacillus (Cramer)._
C 50 per cent.
N 12·3 „
H 6·6 „
Ash 9·1 „
About 65-70 per cent. of the dry matter of bacteria consists of protein.
(2) _Sources of Carbon._
The biggest constituent of the dry matter of bacteria is therefore
carbon. In the soil, bacteria find an abundance of organic matter from
which they may derive their carbon supply. A special case, however,
is furnished by the nitrifying organisms, certain sulphur oxidising
bacteria, and others that derive their carbon from the CO₂ of the soil
atmosphere. The sources from which these special groups obtain the
necessary energy to accomplish this, we have already considered.
(3) _Assimilation of Nitrogen Compounds._
Of chief importance in its consequences are the means adopted by
bacteria to obtain their nitrogen supply.
There is some reason to believe that soil bacteria do not take up
protein and peptones as such, but must first break down these bodies
into simpler compounds. When a sufficient amount of easily decomposable
organic nitrogen is present in the soil, the ammonifying bacteria use
such compounds as sources of energy, and in this case have a nitrogen
supply exceeding their requirements.
But where there is an excess of carbohydrate or other non-nitrogenous
source of energy available in the soil, the case is different. Here
the organisms have a supply of energy which enables them to multiply
rapidly until the organic nitrogen is insufficient for their needs.
Hence they turn to the ammonia and nitrate present in the soil, and
build up their proteins from this source. Doryland[17] has shown that
many common soil ammonifiers assimilate ammonia and nitrate when
supplied with carbohydrate. There may thus be a temporary loss of
nitrate from soil when sugar, starch, straw, or such materials are
added to it.
(4) _Fixation of Free Nitrogen._
The bacteria that we have so far considered take up their nitrogen
directly from compounds containing this element. There remain, however,
a comparatively small but very important group of bacteria possessing
the power of causing elemental nitrogen to combine, and of building it
up into their proteins. This fixation of nitrogen by micro-organisms
is a vital step in the economy of nature. Losses of nitrogen from the
land are continually occurring through the washing-out of nitrates
by rain, and through the evolution of gaseous nitrogen during the
processes of decay. To maintain the supply of combined nitrogen
which is essential to living organisms, there must therefore be a
compensating process by which the supply of nitrogen compounds in the
soil is kept up.
It was discovered in the middle of the nineteenth century that if soil
were kept moist and exposed to the air, there was an increase in the
amount of nitrogen compounds present. Berthelot, in 1893, studied the
nitrogen relationships of soil, and recognised that this fixation of
nitrogen in soil was the work of micro-organisms.
Winogradsky followed up his work and isolated from soil a large
anaerobic spore-forming organism, capable of fixing nitrogen, to which
he gave the name _Clostridium pasteurianum_. In 1901 the investigations
of Beyerinck, in Holland, led to the important discovery of a group of
large aerobic organisms, which he named _Azotobacter_. These were found
to be very active in fixing free nitrogen. More recently, a number of
other nitrogen-fixing bacteria have been described, and the property
has been found to exist to a small extent in several previously
well-known organisms.
It becomes important to determine which are the groups of bacteria
whose nitrogen-fixing powers are of chief importance in the soil.
On account of its energetic fixation of nitrogen in culture media,
_Azotobacter_ has attracted the greatest attention of workers. The
evidence seems to be consistent with the view that _Azotobacter_ is of
importance in the soil. Thus the distribution of _Azotobacter_ would
appear to be world-wide. It is found all over Western Europe and the
United States. Lipman and Burgess[45] found it in soils collected from
Italy and Spain, Smyrna, Cairo, the Fayum, the Deccan in India, Tahiti,
Hawaii, Mexico, Guatemala, and Canada. C. M. Hutchinson[29] found it
to be distributed throughout India. It was found by Omelianski[55]
to be widely distributed in European and Asiatic Russia, and by
Groenewege[28] in Java. Ashby[1] at Rothamsted, isolated it from soils
from the Transvaal, East Africa, and Egypt. Also, an association has
sometimes been found between the ability of a soil to fix nitrogen and
the occurrence and vigour of its _Azotobacter_ flora. Thus Lipman and
Waynick[46] found that if soil from Kansas were removed to California,
its power to produce a growth of _Azotobacter_, when inoculated into a
suitable medium, was lost, and, at the same time, its nitrogen-fixing
power was greatly reduced. Moreover, it is known that conditions
favourable to the fixation of nitrogen by _Azotobacter_ in cultures on
the whole favour nitrogen fixation in soils. The conditions that favour
other aerobic nitrogen-fixing bacteria are, however, not sufficiently
distinct to make such evidence of great value.
It is usually found that nitrogen fixation is most active in
well-aerated soil. Thus Ashby,[1] at Rothamsted, found the
nitrogen-fixing power of a soil to decrease rapidly with depth. Similar
results were obtained in Utah by Greaves. This suggests, at first
sight, that anaerobic nitrogen fixers are unimportant under normal
soil conditions. It is, however, quite possible that they may assume
an importance when acting in conjunction with aerobic organisms.
Thus Omelianski and Salunskov[55] found that beneficial association,
or symbiosis, could occur between _Azotobacter_ and _Clostridium
pasteurianum_, the former absorbing oxygen from the surroundings, and
thus creating a suitable anaerobic environment for the _Clostridium_.
The question of symbiosis of nitrogen-fixing bacteria with each other
and with other organisms offers an inviting field for research.
There is evidence that this factor may have considerable importance.
Beijerinck and Van Delden[3] early recognised that _Azotobacter_
in mixed cultures fixed more nitrogen than in pure cultures.
_Granulobacter_, an organism which they found to be commonly associated
with _Azotobacter_ in crude cultures, appears to increase its
nitrogen-fixing powers (Krzeminiewski).[41] It was also found by
Hanzawa[31] that a greater fixation of nitrogen was obtained when two
strains of _Azotobacter_ were grown together. A symbiosis between
_Azotobacter_ and green algæ has been described, and will be further
discussed by Dr. Bristol. It is likely that this association may be of
importance under suitable conditions on the soil surface where the algæ
are exposed to light.
The combination of elemental nitrogen is an endothermic process which
requires a very considerable amount of energy for its accomplishment.
This fact is well illustrated by the various commercial processes
in use for fixation of atmospheric nitrogen. The nitrogen-fixing
bacteria obtain this energy from the carbon compounds in the soil.
A number of compounds were compared as sources of energy by Löhnis
and Pillai,[47] who tested their effect on the amounts of nitrogen
fixed by _Azotobacter_ in culture. It was found that mannitol and the
simpler sugars give the best results as sources of energy, but that
other organic compounds can also be used. Mockeridge[51] has adduced
evidence that ethylene glycol, methyl-, ethyl-, and propyl-alcohol,
lactic, malic, succinic, and glycocollic acids could also be utilised.
Since so large a part of the organic matter added to soil is in the
form of celluloses, it is of great importance to ascertain how far
these compounds and their decomposition products can be utilised in
nitrogen fixation. Stubble, corn-stalks and roots, oak leaves, lupine
and lucerne tops, maple leaves, and pine needles may all serve as
useful sources of energy to nitrogen-fixing organisms in the soil. Pure
cellulose cannot apparently be used as a source of energy, but when
acted upon by cellulose decomposing organisms, it becomes available as
a source of energy. Hutchinson and Clayton, at Rothamsted, found that
a fixation of nitrogen could be brought about by mixed cultures of
_Azotobacter_, and of the cellulose attacking _Spirochæta cytophaga_,
when grown in cultures containing pure cellulose. It is not known
how far cellulose decomposition must proceed to produce an effective
source of energy, nor what are the substances thus produced that are
utilised. This point will not be decided until something more is known
of the course of changes in the breaking-down of cellulose in the soil.
The amount of nitrogen fixed per unit of energy material decomposed
varies greatly, according to the organism and the conditions.
Winogradsky found that his _Clostridium_ assimilated 2-3 mgs. of
nitrogen per gram of sugar consumed. Lipman found that _Azotobacter_
fixed 15-20 mgs. of nitrogen per gram of mannite consumed.
[Illustration: FIG. 5.
Caption: Azotobacter. Decrease in efficiency in N fixation with age
of culture. (Koch & Seydel.)
X-axis: Days.
Y-axis: Milligrams of Nitrogen fixed per gram of dextrose consumed.]
It is found, however, that in liquid culture, the ratio of nitrogen
fixed to carbohydrates oxidised varies according to the age of the
culture, falling off rapidly as the age increases[42] (Fig. 5). This
decreasing efficiency in cultures may be due to the accumulation of
metabolic products such as would not occur under soil conditions.
Indeed, the efficiency of _Azotobacter_ in a sand culture has been
found by Krainskii[39] to be considerably greater than in solution. It
is thus probable that in soil the nitrogen-fixing organisms are less
wasteful of energy material than under the usual laboratory conditions.
It is to be hoped that future research will indicate what are the
conditions that produce the greatest economy of energy material in
nitrogen fixation.
The fixation of nitrogen in soil is depressed by the presence of
considerable amounts of nitrates. This is, in all probability, due
to the fact that nitrogen-fixing organisms are able to utilise
compounds of nitrogen where these are available. The energy needed
to build up amino-acids and proteins from nitrate or ammonia is, of
course, far less than that required to build up these substances
from elemental nitrogen. It is, therefore, not surprising that where
nitrate is available, _Azotobacter_ will use it in preference to fixing
atmospheric nitrogen.[5]
TABLE III.--ASSIMILATION OF NITRATES.
BY AZOTOBACTER IN PURE CULTURE--(_Bonazzi_).
+---------------------------+--------+-----------+------+
| | Nitrate| Organic |Total |
| | and | Nitrogen |Fixed |
| | Nitrite|and Ammonia| or |
| |Present.| Present. |Lost. |
+---------------------------+--------+-----------+------+
| | mgs. | mgs. | mgs. |
|_Culture with nitrate_-- | | | |
| At beginning | 8·55 | 0·76 | -- |
| After growth | 0·2 | 8·71 | -0·4 |
|_Culture without nitrate_--| | | |
| At beginning | -- | 0·76 | -- |
| After growth | -- | 4·50 | +3.74|
+---------------------------+--------+-----------+------+
(Growth period--24 days at 25° C.)
The chemical process by which nitrogen is fixed is quite unknown,
although a number of speculative suggestions have been made. The
appearance of considerable amounts of amino acids in young cultures of
_Azotobacter_ suggests that these may be a step in the process, but at
present the data are too inconclusive to form a basis for theorising.
_Azotobacter_ is very rich in phosphorus, an analysis of the surface
growth in _Azotobacter_ cultures, made by Stoklasa, giving about 60
per cent. of phosphoric acid in the ash. In cultures it has been found
that a considerable amount of phosphate is needed to produce full
development. As would be expected, therefore, nitrogen fixation in soil
is often greatly stimulated by the addition of phosphates. Christensen
has, indeed, found soils where lack of phosphate was the limiting
factor for _Azotobacter_ growth.
_Azotobacter_ is very intolerant of an acid medium, and is very
dependent on the presence of an available base. In cultures this
is usually provided in the form of calcium or magnesium carbonate.
Gainey[21] found that _Azotobacter_ occurred in soils having an acidity
not greater than P_{H} 6·0, and Christensen,[7],[9] in Denmark, has
found a close association between the occurrence of _Azotobacter_ in
soils and the presence of an adequate supply of calcium carbonate. So
close was this association that he devised a technique based on this
fact for detecting a deficiency of lime in a soil sample.
In addition to the groups already discussed, there is a remarkable and
important group of nitrogen-fixing bacteria that inhabit and can carry
on their functions within the root tissues of higher plants. It has
been known at least from classical times that certain leguminous plants
would, under suitable conditions, render the soil more productive. On
the roots of leguminosæ small tubercles are commonly found. These were
noted and figured by Malpighi in the seventeenth century, and for a
long time were regarded as root-galls. As was described in Chapter I.,
the true nature of these tubercles was finally elucidated by Hellriegel
and Wilfarth in 1886. As the result of a series of pot experiments,
they made the very brilliant deduction that the ability to fix
nitrogen, possessed by the legumes, was due to bacteria associated with
them in the tubercles.
These bacteria were finally isolated and studied in pure culture
by Beijerinck. Since then a very great deal of literature has
accumulated on the subject of the nodule-producing bacteria, which
it is impossible to deal with in a small space. The nodule organism,
_Bacillus radicicola_, when grown on suitable media, passes through a
number of different changes in morphology. The most connected account
of these changes is given in a paper by Bewley and Hutchinson.[4]
In a vigorous culture the commonest type is a rod-shaped bacillus
which may or may not be motile. As these get older they often become
branched, or irregular in shape, the formation of these branched forms
being perhaps due to conditions in the medium. These irregular forms,
known as “bacteroids,” are a characteristic type in the nodules. Their
production in culture media has been found to be stimulated by sugars
and organic acids such as would occur in their environment within the
host plant. In the older rods and bacteroids the staining material
becomes condensed into granules, and finally the rods disintegrate or
break up into coccoid forms. By suitable culture conditions, Bewley and
Hutchinson obtained cultures consisting almost entirely of this stage.
If such a culture be inoculated into a fresh medium rich in sugar,
the swarmer stage appears in great numbers. These swarmers are very
minute coccoid rods, ·9 × ·18 in size, that are actively motile. They
apparently develop later into the rod stage.
[Illustration: FIG. 6.--_Bacillus radicicola._ Stages in the life
cycle. (After HUTCHINSON and BEWLEY.)
Motile Rods
Vacuolated Stage
“Swarmers”
“Bacteroids”
“Pre-swarmers”]
Very little is known as to the life of the organism in the soil.
It is able, however, to fix nitrogen in cultures, and it has been
claimed[35],[48] that it can do so in the soil outside the plant, so
that it is possible that we must take it into consideration in this
connection. More knowledge is needed as to the optimum conditions for
the growth of the organism in the soil. It seems to be more tolerant of
acid soil conditions than _Azotobacter_. The limiting degree of acidity
has been found to vary among different varieties of the organism from
P_{H} 3·15 to P_{H} 4·9.
A long controversy has been held as to whether the nodule organisms
found in different host-plants all belong to one species, or whether
there are a number of separate species, each capable of infecting a
small group of host-plants. As the term “species” has at present no
exact meaning when applied to bacteria, the discussion in this form
is unlikely to reach a conclusion. The evidence seems to show that
the nodule organisms form a group that is in a state of divergent
specialisation to life in different host-plants, and that this
specialisation has reached different degrees with different hosts.
Thus the organisms from the nodule of the pea (_Pisum sativum_) will
also produce nodules on vicia, Lathyrus, and Lens, but seem to have
lost the ability normally to infect other legumes. On the other hand,
the bacteria from the nodules of the Soy Bean (_Glycine hispida_)
have become so specialised that they do not infect any other genus
of host-plant, and soy beans are resistant to infection by other
varieties of the nodule organism. Burrill and Hansen,[6] after an
extensive study, divided the nodule bacteria into eleven groups, within
each of which the host-plants are interchangeable. The existence of
different groups of nodule organisms has been confirmed by the separate
evidence of serological tests (Zipfel, Klimmer, and Kruger).[40] The
results of cross-inoculation tests have sometimes been conflicting. It
seems, indeed, that the host-plant has a variable power of resisting
infection, so that when its resistance is lowered it may be capable of
infection by a strange variety of the nodule organism. The question
that has thus arisen of the ability of the legume to resist infection
is of fundamental importance, and its elucidation should throw light on
the relation of plants to bacterial infection as a whole.
The stage of the organism that infects the plant is not at present
known. It may be supposed that it is the motile “swarmer.” The entry is
normally effected through the root-hairs. The hair is attacked close
to the tip, and an enzyme is apparently produced which causes the tip
to bend over in a characteristic manner. The organisms multiply within
the root hair and pass down it, producing a characteristic gelatinous
thread filled with bacteria, in the rod form. This “infection thread”
passes down into the cells of the root tissue, where it branches
profusely. In young stages of nodule formation the branches can be seen
penetrating cells in the pericycle layer. Rapid cell division of these
root cells is induced. In the course of this cell division abnormal
mitotic figures are sometimes found, such as occur in pathological
growths. The cells push outward the root cortical layer, and so form a
nodule.
Certain of the cells in the centre of the nodule become greatly
enlarged, and in the fully grown nodule are seen to be filled with
bacteria. Differences have been described in the morphology of the
organisms in different parts of the nodule.[62] Whether the different
stages of the organism are equally capable of fixing nitrogen, or what
is the significance of these stages within the nodule, is not certainly
known. It has been held that it is the irregular bacteroid forms that
are chiefly concerned with nitrogen fixation. In older nodules the
organisms become irregular and stain faintly, and the bacteroidal
tissue breaks down, the nodule finally decaying. In the fixation of
nitrogen that occurs in the nodules, the bacteria without doubt derive
the necessary energy from the carbohydrates of the host-plant. There
is evidence that the plant assists the process of fixation by removing
soluble metabolic products from the neighbourhood of the bacteria.
Golding[22] was able to obtain a greatly increased fixation of nitrogen
in artificial cultures by arranging a filtering device so as to remove
the products of metabolism.
The great practical importance of leguminous crops in agriculture has
led to numerous attempts being made to increase their growth, and the
fixation of nitrogen in them, by inoculating the seed or the soil with
suitable nodule-bacteria. This inoculation can be effected either with
soil in which the host-plant has been successfully grown, and which
should consequently contain the organism in fair numbers, or else pure
cultures of the organisms isolated from nodules may be used. Very
varying results have been obtained with inoculation trials.
In farm practice a leguminous crop has often been introduced into a
new area where it has never previously grown. In such soil it is very
probable that varieties of the nodule organism capable of infecting the
roots may not exist. In such cases inoculation with the right organism
or with infected soil often produces good results.
The more difficult case, however, is that in which the legume crop
has been grown for a long time in the locality, and where the soil
is already infected with right organisms. This, the more fundamental
problem, applies especially to this country. Here it would seem that
inoculation with a culture of the organism will benefit the plant only
(1) if the naturally occurring organisms are present in very small
numbers; or (2) if the organisms in the culture added are more virulent
than those already in the soil. The problem of successful inoculation
would therefore seem to be bound up with that of grading up the
infective virulence of the organism to a higher level.
Successful nodule development in a legume crop is also dependent to a
large degree on the soil conditions. The effects of soil conditions on
nodule development have been studied by numerous workers. Moisture has
been found very greatly to affect the nodule development. Certain salts
have a very definite effect on nodule formation.[64] Their effect on
the number of nodules developing has been studied, but the reason for
this effect is unusually difficult to decide. The action is usually a
complex one. Thus phosphates are known to stimulate nodule formation.
They probably act in several ways. In the first place, they may cause
the nodule organisms to multiply in the soil; in the second place, they
produce a greater root development in the plant, thus increasing the
chances of infection; and in the third place, Bewley and Hutchinson[4]
have found that phosphates cause the appearance of the motile stage
of the organism in cultures. A real understanding of the influence of
environment on nodule production will produce great improvements in our
methods of legume cropping.
_D._ THE RELATION OF BACTERIAL ACTIVITIES TO SOIL FERTILITY.
The various activities of the soil bacteria have a vital importance to
the growth of higher plants, which are dependent for their existence
on certain of these processes. In the first place, as we have seen,
bacteria decompose the tissues of higher plants and produce humus
materials, which are essential to the maintenance of good physical
properties in the soil. Then the nitrate supply on which most higher
plants depend is produced by the decomposition of organic nitrogen
compounds by bacteria in their search for energy. The depletion of the
total nitrogen content of the soil through rain and through the removal
of nitrogen in the crops, is to some extent compensated by the fixation
of atmospheric nitrogen by certain bacteria. On the other hand, in the
assimilation of nitrogen compounds to build up protein, the bacteria
are competing with higher plants for one of their essential food
constituents, and their action may, under certain conditions, cause a
temporary nitrogen starvation. One must remember, however, that large
quantities of nitrate are lost from field soils by washing-out through
rain action, especially in winter. The assimilation of nitrate and
ammonia by micro-organisms keeps some of this nitrogen in the soil, and
at certain periods may thus be beneficial.
There is another important respect in which soil bacteria influence
plant growth. Their activities result in the release of inorganic
salts, such as potash and phosphates, in a form available for the use
of plants. The release of phosphorus and potassium compounds takes
place in two ways. In the first place, organic matter containing
phosphorus and potassium, in an insoluble form, is attacked by
bacteria, resulting in these elements being set free as inorganic
salts available to the higher plant. Secondly, much of the phosphorus
supplied to the soil from rock minerals is present as insoluble
phosphates, such as apatite and iron phosphate. Much of the potassium,
too, is derived from insoluble silicate minerals. In both cases the
conversion of the insoluble minerals into soluble phosphates and
potassium compounds is brought about to a large extent by solution in
water containing carbonic and other acids. These acids are largely
produced by micro-organisms, which, in addition to carbonic acid,
produce organic acids, and in specialised cases, sulphuric and nitrous
acids. It has been found, for example, that in a compost of soil
with sulphur and insoluble phosphate, sufficient sulphuric acid may
be produced by the oxidation of the sulphur by bacteria to convert
an appreciable amount of phosphate into a soluble form. When we
consider the functions performed by soil bacteria, therefore, it is
not surprising to find that high bacterial activity in the soil is
associated, as a rule, with fertility.
_E._ CHANGES IN BACTERIAL NUMBERS AND ACTIVITIES, AND THEIR RELATION TO
EXTERNAL FACTORS.
The object of soil bacteriologists is to discover means of favouring
the activity of soil bacteria, especially those activities that are
useful to the higher plant. Knowledge is therefore needed of the
changes in numbers and activities of the soil bacteria, and of the
influence of soil conditions on them. The necessity of studying these
changes has required the development of a quantitative technique by
which the numbers of bacteria in the soil and their activities can be
estimated.
The method commonly used in counting bacteria in soil is a modification
of the plating method of Koch. In counting bacteria two difficulties
have to be overcome--their immense numbers and their small size. The
numbers of bacteria in soil are so large that the bacterial population
of a gram of soil could not, of course, be counted directly. The method
adopted, therefore, is to make a suspension of soil in sterile salt
solution, and to dilute this suspension to a convenient and known
extent, which will depend on the numbers of bacteria expected. In
ordinary field soils it is found convenient, for example, to dilute the
soil suspension so that one cubic centimeter of the diluted suspension
will contain 1/250,000th of a gram of soil. Such a volume will commonly
contain a number of bacteria sufficiently small to count. The second
difficulty is that the organisms are microscopic, and yet cannot be
readily counted under the microscope owing to the presence of soil
particles in the suspension. Hence recourse is had to plating. One
cubic centimeter of diluted suspension is placed in a petri dish and
mixed with a suitable nutrient agar medium, melted, and cooled to about
40° C. The medium sets, and after a few days’ incubation the organisms
multiply and produce colonies visible to the naked eye. By counting
these colonies we obtain an estimate of the number of bacteria in
the one cubic centimeter of suspension, it being assumed that every
organism has developed into one colony, and by multiplying this number
by the degree of dilution we obtain the numbers per gram of soil.
In practice a number of parallel platings are made from one cubic
centimeter portions of the diluted suspension and the mean number of
colonies per plate is taken. By this means the error due to the random
distribution of bacteria in the suspension is reduced, because of the
greater number of organisms counted.
In drawing conclusions from bacterial count data, it is necessary to
distinguish between the indication which the method gives of the
absolute numbers of bacteria in the soil and the accuracy with which
it enables the numbers of two soil samples to be compared. The method
cannot be used for the former purpose at present. We do not know how
far the figures obtained by this counting method fall short of the
actual number of bacteria in the soil. One reason for this is the
difficulty of effecting a complete separation of the clumps of bacteria
into discrete individuals in the suspension. Then again, there is no
known medium upon which all the physiological groups of bacteria will
develop and produce colonies. And even on a suitable medium some of the
individuals may fail to multiply.
In comparing the bacterial numbers in two soil samples, however,
the case is different. Within each bacterial group investigated the
plate method should give counts proportional to the bacterial numbers
in the soil. Thus, by the method one should be able to tell whether
the bacterial numbers are increasing or decreasing over a period of
time, or whether a certain soil treatment produces an increase or
a decrease. With this end in view the technique of the method has
been improved by recent workers. It was found that, when carefully
standardised, the process of dilution of the soil could be carried
out without significant variation in result (Table IV.), and that the
accuracy of the method is limited mainly by the variation in colony
numbers on parallel platings, due in part to random distribution of
bacteria throughout the final suspension, and partly to the uneven
development of colonies on the medium. The question of the medium was
therefore taken up with a view to improving the uniformity of results
obtained with it. Lipman, Conn, and others effected an improvement by
using chemical compounds as nutrient ingredients, thus making their
media more closely reproducible. On most agar media, an important
disturbing factor is the growth of spreading colonies, which prevent
the development of some of the other colonies. A medium has been
devised at Rothamsted on which these spreading organisms are largely
restricted.[61] A statistical examination[19] has shown that on this
medium errors due to the uneven development of colonies, except in
special cases, are prevented, so that in fact the variation in colony
numbers between parallel plates is found to be that produced merely by
random distribution of bacteria in the diluted suspension (see Table
IV.). In this case the accuracy of the counts of the bacteria in the
diluted suspension depend directly on the number of colonies counted,
and can be known with precision.
TABLE IV.--BACTERIAL COUNTS OF A SOIL SAMPLE.
PARALLEL PLATE COUNTS FROM FOUR SETS OF DILUTIONS MADE BY DIFFERENT
WORKERS.
+------------------------------------------+
| Counts of Colonies on each Plate. |
+------+--------+--------+--------+--------+
|Plate.| Set I. | Set II.|Set III.| Set IV.|
+------+--------+--------+--------+--------+
| 1 | 72 | 74 | 78 | 69 |
| 2 | 69 | 72 | 74 | 67 |
| 3 | 63 | 70 | 70 | 66 |
| 4 | 59 | 69 | 58 | 64 |
| 5 | 59 | 66 | 58 | 62 |
| 6 | 53 | 58 | 56 | 58 |
| 7 | 51 | 52 | 56 | 54 |
+------+--------+--------+--------+--------+
| Mean | 60·86 | 65·86 | 64·28 | 62·86 |
+------+--------+--------+--------+--------+
Standard deviation between the four sets = 5·62.
Standard deviation between plates within the sets = 7·76.
The knowledge obtained from counts of soil bacteria is subject to
another serious limitation. We do not know which of the bacteria
counted are the most effective in bringing about the various changes
that take place in the soil. It is not even known which of them are
active in the soil and which are in a resting condition. It is thus
possible to have two soils containing equal numbers of bacteria but
showing widely different biochemical activity, if one soil contains
organisms of a higher efficiency. Moreover, as has been pointed
out, many important groups of soil bacteria do not develop on the
plating media, and so are not counted. These considerations led to
the development of supplementary methods by which it was hoped to
estimate the actual biochemical activity of the soil microflora. The
first of these methods was developed by Remy, who attempted to study
the biochemical activity of a soil by placing weighed amounts into
sterile solutions of suitable and known composition, keeping them
under standard conditions for a definite time and then estimating the
amount of the chemical change that was being studied. Thus, to test the
activity of the organisms that produce ammonia from organic nitrogen
compounds, he inoculated soil into 1 per cent. peptone solution and
measured the amount of ammonia produced in a given time. By similar
methods the power of a soil to oxidise ammonia to nitrate, to reduce
nitrate, or to fix atmospheric nitrogen, is tested. This method has
been extensively used and developed by more recent workers. It suffers,
however, from the same serious disadvantage that it was designed to
avoid, for we cannot be certain that those bacteria that develop in
the nutrient solution are the types that are active in the soil, and,
moreover, even where the same types do function in the two conditions,
we do not know that the degree of their activity is the same in soil
and in solution cultures. For instance, _Nitrosomonas_ appears to show
very different degrees of activity in soil and in culture.
Another method, therefore, of studying the activity of soil
micro-organisms is the obvious one of estimating the chemical changes
that they produce in the soil itself. This method has obvious
advantages over the unnatural methods developed from Remy’s, but it has
a number of limitations that make its actual application difficult.
In the first place, we cannot always tell whether changes found to
occur in soil are due to the activity of micro-organisms, or are
purely chemical reactions unassisted by biological agencies. Then, if
we succeed in showing that the changes are due to micro-organisms, it
is very difficult to determine which organisms are effecting them.
This cannot be definitely tested by isolating suspected organisms and
testing their activity in sterile soil, because in sterilising soil its
nature and composition is altered. In spite of these difficulties,
however, the study of the chemical changes that take place in the soil
has produced valuable knowledge, when it has been combined with a
study of the changes in the number and variety of the micro-organisms
that accompany these reactions. This method of investigation is well
illustrated by the work of Russell and Hutchinson on the effects of
heat and volatile antiseptics on soil, where a study of the chemical
changes such as ammonia production, that occurred in these treated
soils, combined with a study of the changes in bacterial numbers, led
to the realisation that the soil micro-population was a complex one,
containing active protozoa.
A great difficulty in applying quantitative methods to bacteria in the
field is the great variation in the density of the bacterial population
over a plot of field soil, which may be so great that a bacterial count
from a single sample is quite valueless. For example, the distribution
of bacterial numbers over a plot of arable soil near Northampton was
studied by taking sixteen samples distributed over an area about
12 feet square. The result showed that in some cases the bacterial
numbers in samples taken 6 inches apart differed by nearly 100 per
cent. Fortunately, under favourable conditions, a remarkably uniform
distribution of bacterial numbers over a plot of soil can be found.
On such a plot it is possible to investigate the rapidity with which
the numbers of the soil micro-organisms alter in point of time. For
example, on the dunged plot of Barnfield, Rothamsted, which has been
cropped with mangolds for forty-seven successive years, the area
distribution of bacteria has been found to be so uniform that if a
number of samples of soil are taken from the plot at the same time, the
difference in bacterial numbers between the samples cannot be detected
by means of the counting technique (see Table V.). The work of Cutler,
Crump, and Sandon[16] on this plot showed that the bacterial numbers
vary very greatly from one day to the next, and that these fluctuations
took place over the whole plot, since two series of samples, taken in
two rows 6 feet apart, showed similar fluctuations (see Fig. 7). The
discovery of these big daily fluctuations in numbers led to an inquiry
as to how quickly bacterial numbers change, and samples from Barnfield,
taken at two-hourly intervals, showed that significant changes in
numbers took place even at such short intervals.
TABLE V.--BACTERIAL COUNTS OF FOUR SOIL SAMPLES.
FROM BARNFIELD, TAKEN SIMULTANEOUSLY.
+------------------------------------------------------+
| Counts of Colonies on each Plate. |
+------+-----------+-----------+-----------+-----------+
|Plate.| Sample I. | Sample II.|Sample III.| Sample IV.|
+------+-----------+-----------+-----------+-----------+
| 1 | 38 | 45 | 43 | 27 |
| 2 | 32 | 40 | 34 | 41 |
| 3 | 52 | 45 | 52 | 35 |
| 4 | 32 | 31 | 55 | 36 |
| 5 | 40 | 43 | 38 | 45 |
|Mean | 38·8 | 40·8 | 44·4 | 36·8 |
+------+-----------+-----------+-----------+-----------+
Standard deviation between the four samples = 7·25.
Standard deviation between parallel plates within the sets = 7·55.
[Illustration: FIG. 7.
X-axis (top): Days.
Y-axis (left): (Series A) Bacteria--millions per gramme of soil.
Y-axis (right): (Series B) Bacteria--millions per gramme.
Caption: Daily changes in bacterial numbers in field soil.
Counts from two series of soil samples taken 6 feet apart.
(After Cutler.)]
Since the bacteria involved in this fluctuation are of great importance
to the crops, being for the most part ammonia producing types, further
knowledge as to the cause of this fluctuation and of its effect on the
ammonia and nitrate in the soil is of fundamental importance. There is
evidence, which will be discussed later, that the cause is connected
with the changing activities of certain soil protozoa, since the daily
changes in the numbers of active amœbæ in the soil have been found
to be in the reverse direction to those of the bacterial numbers. It
appears, therefore, that we are dealing with an equilibrium between
the various members of the soil population, the point of equilibrium
changing at frequent intervals.
In addition to daily changes, it is possible to detect changes in the
numbers and activity of the soil population related to the season.
There is a well-marked increase in the spring and autumn (see Figs.
15, 16, pp. 89, 90). This is well seen when the fortnightly averages
of the daily bacterial and protozoal counts from Barnfield soil
are plotted. These spring and autumn increases comprise both the
bacterial and the protozoal population, and therefore differ from the
short time fluctuations in being due, not to a disturbance of the
bacteria-protozoa equilibrium, but to a general rise in activity of
both groups of organisms.
When we consider the action of external conditions on the soil
bacteria, the existence of a complex soil population and the
interdependence of its members must be borne in mind. Changes in
external conditions may affect the different components of the
population in different ways or to different degrees, thus upsetting
the equilibrium between the various groups. For example, the addition
of a mild aromatic antiseptic to the soil apparently affects the
protozoa in such a way as to disturb the bacteria-protozoa equilibrium
in favour of the bacteria, while in some cases the aromatic compound
affords a food supply to special bacteria, causing these to increase,
upsetting the equilibrium between the different bacterial groups. When
our knowledge of the effect of external factors on the soil population
becomes sufficient, it will probably be found that in nearly all
cases a change in the soil conditions produces some disturbance in
the equilibrium between the components of the soil population, though
at present there are only certain examples where this disturbance is a
probable explanation of the facts.
Since bacteria are dependent on adequate supplies of energy and food,
it is to be expected that additions of organic matter or of inorganic
food materials will greatly benefit their activities. The effect of
added farmyard manure in increasing bacterial activities has been much
studied.[27] Some of the increased bacterial numbers and activities
in this case may be due to the addition of bacteria with the manure,
but it is thought that this factor is of less importance than the
added energy and food supply which the general soil flora obtain from
it. Nutritive salts such as phosphates and salts of potassium usually
increase the bacterial activities.
The effect of alkali salts on soil bacteria has been especially studied
in the Western United States, where the existence of alkali in the
soil is a serious problem.[23] Soil bacteria are usually stimulated by
small doses of alkali salts that are toxic in higher concentration.
As a rule, chlorides are the most toxic salts, the electronegative
ion playing an important part in the effect of the salt. Salts
affect bacteria both owing to the changes in osmotic pressure which
they produce, and through their specific action on the bacterial
protoplasm.[26] When equal weights of various salts are added to soil,
their toxic action on bacteria shows so little association with their
respective osmotic pressures that we must conclude that this factor is
the less important. There is reason to suppose that the toxic action
of salts on bacteria is often connected with an effect of the specific
ions on the permeability of the bacterial cell-wall. This conclusion
is based on the changes in electrical conductivity of bacterial
suspensions in the presence of various salts.[59]
A definite antagonism between various salts has been found to exist.
It is possible that future work in this line may indicate what are
the proportions of common electrolytes which will produce a properly
“balanced” soil solution so that the harmful excess of one salt may be
antagonised.
Certain salts, such as those of arsenic[24] and manganese, seem to
exercise a stimulating action on bacterial activities; the causes of
this action are not at present understood.
The acidity of the soil has an important effect on the bacterial
processes. The acidity of soils may increase to such a point that the
decomposition of plant tissues by bacteria is hindered, a peat layer
being thus produced. The degree of acidity that is toxic varies very
greatly with different soil bacteria, some of them, like Azotobacter
and Nitrosomonas being very intolerant of acidity.
The conditions of aeration, water content, and temperature are
inter-related in field soil. Ammonifying organisms are not greatly
dependent on aeration, but this factor is sometimes a limiting one in
the case of the very aerobic nitrifying bacteria. Hence efficient soil
cultivation is beneficial to nitrification.
Many attempts have been made to correlate the temperature and moisture
of field soils with the bacterial numbers and activities. These
attempts have given very discordant results. It is generally agreed
that a plentiful moisture supply is beneficial. Thus Greaves, in Utah,
found the optimum water content for ammonia and nitrate production
to be about 60 per cent. of the water-holding capacity. On the other
hand, Prescott[56] found that the summer desiccation of soil in
Egypt was followed by increased bacterial activities. Fabricius and
Feilitzen,[18] using moor soil, found a direct relationship between
soil temperature and bacterial numbers, showing that temperature can
be a limiting factor under certain conditions. With normal arable
soils, however, no such direct effect of temperature or moisture can
be found[16] (see Fig. 8). It has even been found by Conn[11] that
freezing of the soil may cause a marked increase in bacterial numbers.
The erratic effects of temperature and moisture on the soil bacteria
probably afford instances of a disturbance of the equilibrium between
the bacteria and other components of the soil micro-population. Thus
desiccation and freezing, though they harmfully affect the bacteria,
may inhibit other micro-organisms to a greater degree, thus freeing
the bacteria from competition. It is in the investigation of this
equilibrium, and of the factors that can control it to our benefit,
that the great advances in soil biology in the future are to be
expected.
[Illustration: FIG. 8.--Effect of frost on the bacterial numbers in the
soil. (After CONN.)
X-axis: Nov.-May
Y-axis (bottom): Temperature--Degrees C.
Y-axis (top): Bacteria--Millions per Gramme of Soil.]
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