precedent to that of the nitric, but the two processes go on so nearly
together as to prevent the accumulation of any large quantities of the
lower salt in the soil.
Whether or not the formation of ammonia precedes that of nitrous acid is
still a subject for experimental demonstration. Chemically, both nitrous
acid and ammonia may be produced by the reduction of nitric acid. In
nature, the reverse of this process may be the customary method.
=417. Production of Ammonia in the Soil by the Action of Microbes.=—It
is highly probable that organic nitrogen in the soil in passing into the
form of nitric acid exists at some period of the process in the form of
ammonia.
Marchal has isolated and studied some of these ammonia-making
bacteria.[276] Bacillus mycoides is the most active of these organisms.
It occurs constantly in surface soils and is present in the air and in
natural waters. In decomposing albumen it produces a strongly alkaline
solution due to ammonium carbonate. Organic carbon, during this process,
is converted chiefly into carbon dioxid, but small quantities of formic,
propionic, and butyric acids are also produced. Any organic sulfur which
is present is converted into acid. No hydrogen or nitrogen is eliminated
in a free state. While slight alkalinity is favorable to the development
of this bacterium, yet it may be propagated in a feeble sulfuric acid
solution when the acid is less than one per cent.
The greatest activity of this organism is manifested at 30°. Below 5°
and above 42° no ammonia is produced. The bacillus will not develop in
an atmosphere of hydrogen or carbon dioxid, except in solutions of
organic matter and nitrate. In addition to its action on egg albumen it
decomposes other proteid bodies as well as leucin, tyrosin, creatin, and
asparagin. It, however, does not oxidize urea, nor does it develop in
solutions of ammonium salts and nitrates, except as mentioned above.
When soluble carbohydrates are present, acids are formed. It is
concluded from these experiments that the final oxidation of organic
nitrogenous matter is preceded by its conversion into ammonium
carbonate.
=418. Summary of Statements.=—All nitrogenous matters which would be
naturally present in the soil may become subject to nitrification when
the proper conditions are supplied. Munro has also succeeded in
nitrifying ethylamin, thiocyanates, and gelatin, urea, asparagin, and
the albuminoids of milk and rapeseed.
The products of nitrification are ammonia, nitrous or nitric acid,
carbon dioxid, and water. The ammonia and nitrous acid may not appear in
soils as the final products of nitrification, as the nitric organism
attacks the latter at once and converts it into nitric acid. Nitrous
acid and ammonia may also be produced in soils as one of the retrograde
steps in denitrification.
To summarize the conditions necessary for nitrification it may be said
that first, the proper material must be supplied; _viz._, an organic or
inorganic nitrogenous compound capable of oxidation. In the second
place, the medium must be faintly alkaline, the temperature must not be
too high, the nitrifying organisms must have abundant food, and the
process must take place in the dark.
=419. Order of Oxidation.=—It is quite definitely determined that
activity of the ammoniacal and nitrous organisms is the first step in
the process, since the nitric organism appears to have no power whatever
to oxidize proteid compounds; while, on the other hand, the nitrous
organism can not, in any case, complete the conversion of nitrous into
nitric acid.
The conditions which permit certain organisms to oxidize free nitrogen
have not been definitely determined. The presence of such bodies in the
tubercles attached to the rootlets of certain leguminous plants has been
established. Lately, Winogradsky has isolated from the soil a nitrifying
organism which is capable of converting free nitrogen into forms suited
to nourish plant growth. This organism is cultivated in dextrose with
careful exclusion of all nitrogen, save that which exists in the air
carefully freed of every trace of ammonia or oxidized nitrogen.
Under the influence of the growth of this organism the sugar undergoes a
butyric fermentation, and nitrogen in an oxidized form is assimilated in
an amount apparently equal to about one five-hundredth of the sugar
consumed.
This result leads Warington[277] to remark that it is a fact of
extraordinary interest, both to the physiologist and chemist, that a
vegetable organism should be able to acquire from the air all the
nitrogen it needs.
=420. The Nitrification of Ammonia.=—The same organism which converts
organic nitrogen into nitrous acid acts also on ammonia and its
compounds with a similar result. In fact, the formation of ammonia may
be regarded as one of the stages on the road from albuminoid to nitric
nitrogen.
Data have been collected by Schloesing on the nitrification of ammonia
taking place in arable soil, tending to show that this phenomenon is
accomplished without appreciable loss of nitrogen in the gaseous
state.[278] This, however, does not hold good when the quantity of
ammonium carbonate introduced into the earth is largely increased. In
two experiments, conducted by Schloesing, with a larger quantity of
ammonium carbonate, the loss of nitrogen was very notable. In certain
conditions the production of nitrous acid may take place, and it is
interesting to know whether the appearance of nitrites has any influence
on the disengagement of free nitrogen. In order to determine this
question a solution of calcium nitrite was prepared by decomposing
silver nitrite with calcium chlorid. From the results of the experiments
made it was seen that the nitrites were only the results of a retarded
and partially incomplete nitrification. They are, moreover, thus an
obstacle to the normal work of the nitrifying organisms. It is also
established that when they are present a disengagement of gaseous
nitrogen takes place, whether the nitrites are formed during the
progress of the experiment, or whether they were originally present.
However, it is not best to say that the nitrites themselves have been
the cause of the disengagement of the nitrogen. It may happen that the
disengagement of the nitrogen and the presence of nitrites are simply
simultaneous and due to one and the same cause. The destruction of
nitrates in the midst of reducing agents furnishes, according to the
nature of these bodies and the circumstances, nitrous acid, nitrogen
dioxid, nitrogen protoxid, free nitrogen, and even ammonia.
This destruction of nitrates and the appearance of oxids of nitrogen and
of free nitrogen are more likely to be due to the presence of a separate
denitrifying ferment as pointed out by Springer than to have arisen in
the manner mentioned above by Schloesing. In the present state of our
knowledge, moreover, we can hardly regard the presence of nitrites as an
obstacle to complete nitrification. On the other hand, it seems to be
well established that the production of nitrites or ammonia is a
necessary step between organic nitrogen and nitric acid.
=421. Occurrence of Nitrifying Organisms.=—According to the observations
of Schloesing and Müntz the nitrifying organisms are widely
distributed.[279] Arable soil containing considerable humus seems to be
the medium in which they grow most freely and in which they accomplish
their most important functions. Sewage waters are also rich in
nitrifying ferments, and, in fact, all waters containing organic matter.
They are also found in running waters but not in great numbers. They
affect chiefly the surface of bodies, and especially are found on the
bottom of culture-flasks.
These authors have not found the nitrifying organisms in normal air.
They could not seed sterilized flasks by admitting air freely. The
absence of these ferments from the air is explained by reason of their
sensitiveness to desiccation.
The method used by Schloesing and Müntz for the separation of the
organism consisted in the preparation of original and subcultures in
sterilized solutions containing nitrifiable matters. The proof of
isolation was assumed when a given subculture contained only one kind of
organism as seen with the microscope. The appearance of this organism,
as described by the authors, was that of the later isolations by
Warington and Winogradsky, but the method used could hardly now be
regarded as decisive.
=422. Determination of Nitrifying Power of Soils.=—In studying the
distribution of the nitrifying organisms in a soil the general method of
procedure is based on the production of nitrification in a convenient
solution by the organisms present in a given sample of soil. If the
solution seeded with the given portion of soil remain unaffected, it
will show that there were no nitrifying organisms present in the seed
used. On the other hand, the vigor of the nitrifying process when once
it is started, may be taken as an evidence of the number and activity of
the organisms in the soil, a sample of which was used for seed.
=423. Composition of the Culture Medium.=—The solution recommended by
Warington for the culture and isolation of the nitrifying ferments has
the following composition:
Ammonium chlorid 80 milligrams.
Sodium potassium tartrate 80 „
Potassium phosphate 40 „
Magnesium sulfate 20 „
calcium carbonate about 200 „
Pure bacteria-free water to make one liter.
=424. Apparatus and Manipulation.=—The experiments are conducted in
short, wide-mouthed bottles. The initial volume of the solution in each
bottle is 100 cubic centimeters, and the bottle should be of such size
as to give a depth of liquid of from three to five centimeters.
The neck of the bottle is closed with a plug of cotton and this is
protected from dust by tying over it a cap of filter paper. Arranged in
this way, filtered air has free access to the solution. The bottle with
the solution thus protected is placed in a water-oven and kept near the
temperature of boiling water for six to eight hours to destroy any
organisms present. When cool, the solution is ready for use.
The calcium carbonate used should be prepared by precipitation and added
in a moist state. The calcium carbonate solution should be added after
the sterilization of the liquid, the precipitated carbonate being boiled
just before it is added.
_Preparation of Seed._—The seed employed to start the nitrification
should be a small quantity of fresh soil, usually about one-tenth of a
gram. If a previously nitrified solution be used for seed it should be
thoroughly shaken and about one cubic centimeter of the solution removed
for seeding the new bottle.
In introducing the nitrifying liquor into the bottle the plug should be
lifted slightly and a small pipette inserted by means of which the
liquor is added. The operation should be carried on in a room perfectly
free from dust and to which no one but the operator has access. The
greatest care should be exercised to prevent any particles of matter
entering the solution except that which is purposely added. In
withdrawing the liquor from the nitrifying solution cotton wool should
be pressed around the top of the pipette so that the entering air may be
filtered before admission to the interior of the bottle. The pipette
which is used should be kept in boiling water until it is required for
use. After use it should be washed and replaced in boiling water until
again required.
After seeding, the bottles should be placed in a dark cupboard and
exposed to the ordinary temperature of the laboratory. If a higher or
stated temperature be desired, the bottle should be placed in a metal
box the temperature of which can be regulated to any degree.
_Test of the Commencement of Nitrification._—The beginning of the
nitrification can be determined in a solution by testing it with
diphenylamin. One cubic centimeter of the solution withdrawn as above
indicated, is placed in a small beaker, a drop of solution of
diphenylamin sulfate in sulfuric acid added, and then two cubic
centimeters of concentrated sulfuric acid and the contents of the beaker
well shaken. The development of a violet-blue color shows the presence
of nitric or nitrous acid. This test will detect one part of nitric
nitrogen in twenty million of water.
_Determining the Progress of Nitrification._—The progress of
nitrification is determined by repeated examinations for ammonia by
nesslerizing, and for nitrous acid with metaphenylenediamin. Each
experiment is made with five cubic centimeters of the solution withdrawn
as above indicated and placed in test-tubes, always of the same size.
The reaction with the nessler solution is then made by adding it in the
usual way. The colorations are recorded as, trace, small, moderate,
considerable, large, and abundant.
If the change produced by the organism consisted in the formation of
nitrites only, the ammonia in the original solution would fall from
_large_ to _trace_, while the nitrous acid would increase from _trace_
to _large_. If the nitrification consisted in the production of nitrates
only, the ammonia would diminish without any corresponding production of
nitrous acid. In mother solutions which contain ammonium carbonate
instead of sulfate, it should not be forgotten that the ammonia might
gradually disappear owing to the volatilization of the carbonate without
any corresponding production of free nitrites or nitrates. The complete
disappearance of the ammonia in the above experiments shows the
completion of the process.
=425. To Determine the Distribution of the Nitrifying Organism in the
Soil.=—The principle on which the determination of the distribution of
the nitrifying organism in the soil depends, rests upon seeding the
growth solutions with samples of soil taken at different depths and
carefully protected from the time of sampling until the time of seeding
from any admixture of accidental organisms.
The method of Warington is the simplest and best to follow.[280] The
samples of soil are taken by digging a pit of convenient depth usually
from eight to ten feet. A fresh surface is then cut on one of the sides
of the pit at the spot selected for sampling. This surface is scraped
with a freshly ignited platinum spatula. The spatula should then be
washed, re-ignited, and cooled, and a small portion of the soil, at the
depth required, detached with the spatula and transferred at once into
one of the growth bottles already described.
The growth solution best suited for the purpose contains four cubic
centimeters of urine per liter. Each bottle should also contain some
freshly precipitated calcium carbonate. In sterilizing urine solutions
the calcium carbonate should be added before the heating instead of
afterwards. The quantity of soil taken for each seeding should be about
one-tenth of a gram.
Inasmuch as the cotton stopper has to be lifted to introduce the soil,
opportunity is given for the entrance of any organisms floating in the
air. Experience, however, has shown that air free from soil dust very
seldom contains nitrifying organisms. The seeded bottles are placed in a
dark cupboard of moderate temperature as already described.
=426. Sterilized Urine Solution.=—The sterilized urine solution used for
the determination of the distribution of the nitrifying organisms in the
soil, is made by taking four cubic centimeters of healthy urine,
diluting to one liter, adding some freshly precipitated calcium
carbonate, stoppering with cotton wool and heating for several hours at
the boiling temperature of water.
As a result of Warington’s experiments it was shown that the nitrifying
organism in the soil did not exist, at least in portions of one-tenth of
a gram, to a greater depth than eighteen inches. In only one case was
nitrification produced from a sample of soil taken at a greater depth
and this may have been due to the accidental introduction of organisms
from other sources. It may be assumed that any long delay in the
commencement of nitrification under favorable conditions, implies the
presence of a very limited quantity of organisms in the solution. Thus a
comparative study of the period of incubation and the progress of
nitrification in solutions seeded with soils taken at different depths
or at different places, becomes a fair index of the number and vitality
of the nitrifying organisms contained therein.
=427. Depth to Which Micro-Organisms are Found.=—Koch states that at the
depth of about one meter, the soil is nearly free from every kind of
bacteria.[281] These observations have been corroborated by Pumpelly and
Smyth who find that no infection of a bacterial nature is produced in a
sterilized solution from samples of clay taken at the depth of nine feet
below the surface.[282]
It is evident from the nature of the experiments above described that
the nitrifying processes go on almost exclusively in those portions of
the soil which are subject to cultivation, while in the subsoil and
below the processes of nitrification are either retarded or arrested.
Any stores of nitrogenous matter, therefore, in an insoluble state,
resting in the subsoil, are preserved from oxidation and consequent
waste until such time as they may be removed to near the surface.
=428. Isolation of the Nitrous and Nitric Organisms in the Soil.=—The
action of the organisms which produce nitrification either in form of
nitrites or nitrates, having been thoroughly established, and the method
of testing the soil therefor given, it remains to describe a method by
means of which these organisms in the soil may be isolated and obtained
in a state of purity. The difficulties attending this process are
extremely great on account of the similarity of the two organisms. All
earlier attempts to make pure cultures of the two separate organisms
were attended with but little success.
According to Winogradsky the method of culture on gelatin so long
practiced is not to be relied upon.[283] It is very difficult to
eliminate by this process the organisms which grow rapidly in gelatin
and which mature their colonies in two or three days, but where they
require eight or ten days to produce a colony the method is successful.
In fact, by the gelatin process as it was at first practiced, a good
deal was owing to chance, but sometimes by a happy accident a pure
nitro-bacterium might be isolated.
Formerly it was considered that a liquid could be regarded as sterile if
it gave no growth upon gelatin. It has, however, now been demonstrated
that a liquid may contain large numbers of nitro-bacteria and still
produce no growth upon gelatin. However, for the organisms which
accompany the nitro-bacteria in soils, it is regarded as certain that if
no growth on gelatin is produced by them they are absent. Therefore in
the case of a solution which has been seeded with a soil, if it can be
brought to such a state as to produce no growth on gelatin, it may be
safely assumed that it contains no bacterial organisms save those which
are capable of producing nitrites or nitrates. Therefore if such a
solution produce nitrification and at the same time no growth upon
gelatin, it may be considered as a proof of the isolation of the
nitro-organisms from all others.
This method was also worked out independently by Mr. and Mrs.
Frankland.[284]
Winogradsky says further he confesses that he has advanced these views
only provisionally and without being convinced of their infallibility.
Strictly speaking, the proof of seeding gelatin is not sufficient alone
because the absence of growth can not be regarded as the exclusive
privilege of the nitro-bacteria. Such might be the case sometimes for an
accidental mixture of microbes, introduced with any given sample of soil
into the cultures, but the criterion is not absolute. Microbes, for
example, of a sulfurous or ferruginous nature may be cited, for which
the gelatin layer is not only unfavorable but even fatal. It may thus
happen that there may be eliminated from the solution all that will grow
upon gelatin without freeing it from some special kinds of cultures,
refractory like the nitro-bacteria, but which might reappear if they
should be resown in some favorable nutritive solution. On account of
this fault in the process, Winogradsky has been impressed with the
necessity of bringing out a better method.
In using the gelatin media it is necessary to find the one that is
suited to nourish these organisms, which would evidently be the way
promising the greatest success. This having been found, and those
organisms which produce colonies being easily recognizable, a great step
towards the solution of the problem will have been made and the more so
as the medium would be at the same time absolutely unfavorable to other
forms of microbes. On account of the slow degree of development of the
nitro-organisms, all others would probably have opportunity to grow and
strengthen to their exclusion, unless these interfering organisms could
be completely removed.
=429. The Culture Solution.=—The culture-solution, first proposed by
Winogradsky, had the following composition:
To ten grams of gelatin or one part of agar-agar in 100 cubic
centimeters of water add potassium phosphate, one-tenth of a gram;
magnesium sulfate, five-hundredths of a gram; calcium chlorid, trace;
and sodium carbonate, half a gram. The solution being sterilized in the
usual way by heating, there are added to it a few cubic centimeters of a
sterilized solution containing two-tenths per cent of ammonium sulfate.
Such a solution has been proved to be very favorable to nitro-organisms.
Nevertheless the experiments with such solutions gave no definite
results and they were abandoned.
The non-success of this method led Winogradsky to adopt a nitrifying
solution which absolutely excluded all organic substances. Instead of
using an animal or vegetable gelatinous substance he used one of a
mineral nature, first proposed by Graham and Kühne.[285] Two of these
gelatinous mineral substances were considered; _viz._, the aluminum
hydroxid and the hydrate of silica. The latter was chosen.
=430. Preparation of the Mineral Gelatinous Solution.=—The soluble glass
which is found in commerce is generally of a thick, sirupy consistence.
It is first diluted with three times its volume of water. One hundred
cubic centimeters of this liquid are poured with constant stirring into
fifty cubic centimeters of dilute hydrochloric acid and the mixture
placed in a dialyzer. It is useless to employ a standard solution of
silica. All that is necessary is to submit to dialysis a liquid with an
excess of acid and sufficiently dilute not to be exposed to the danger
of being spontaneously gelatinized in the dialyzer. The dialyzer is left
for one day in running water and two days in distilled water, often
renewed. The solution is then ready for use. This is the case when it is
no longer rendered turbid on the addition of silver nitrate, showing
that the hydrochloric acid has been entirely extracted. The solution is
then to be sterilized by boiling, and preserved in a glass flask closed
with a plug of cotton.