[Footnote 2: A paper by R. Warington, read before the Chemical Section of
the British Association at Montreal.]
By R. WARINGTON.
In the following brief notes I propose to consider in the first place the
present position of the theory of nitrification, and next to give a short
account of the results of some recent experiments conducted in the
Rothamsted Laboratory.
_The Theory of Nitrification._--The production of nitrates in soils, and
in waters contaminated with sewage, are facts thoroughly familiar to
chemists. It is also well known that ammonia, and various nitrogenous
organic matters, are the materials from which the nitric acid is produced.
Till the commencement of 1877 it was generally supposed that this
formation of nitrates from ammonia or nitrogenous organic matter was the
result of simple oxidation by the atmosphere. In the case of soil it was
imagined that the action of the atmosphere was intensified by the
condensation of oxygen in the pores of the soil; in the case of waters no
such assumption was possible. This theory was most unsatisfactory, as
neither solutions of pure ammonia, nor of any of its salts, could be
nitrified in the laboratory by simple exposure to air. The assumed
condensation of oxygen in the pores of the soil also proved to be a
fiction as soon as it was put by Schloesing to the test of experiment.
Early in 1877, two French chemists, Messrs. Schloesing and Müntz,
published preliminary experiments showing that nitrification in sewage and
in soils is the result of the action of an organized ferment, which occurs
abundantly in soils and in most impure waters. This entirely new view of
the process of nitrification has been amply confirmed both by the later
experiments of Schloesing and Müntz, and by the investigations of other
chemists, among which are those by myself conducted in the Rothamsted
Laboratory.
The evidence for the ferment theory of nitrification is now very complete.
Nitrification in soils and waters is found to be strictly limited to the
range of temperature within which the vital activity of living ferments is
confined. Thus nitrification proceeds with extreme slowness near the
freezing-point, and increases in activity with a rise in temperature till
37° is reached; the action then diminishes, and ceases altogether at 55°.
Nitrification is also dependent on the presence of plant-food suitable for
organisms of low character. Recent experiments at Rothamsted show that in
the absence of phosphates no nitrification will occur. Further proof of
the ferment theory is afforded by the fact that antiseptics are fatal to
nitrification. In the presence of a small quantity of chloroform, carbon
bisulphide, salicylic acid, and apparently also phenol, nitrification
entirely ceases. The action of heat is equally confirmatory. Raising
sewage to the boiling-point entirely prevents its undergoing
nitrification. The heating of soil to the same temperature effectually
destroys its nitrifying power. Finally, nitrification can be started in
boiled sewage, or in other sterilized liquid of suitable composition, by
the addition of a few particles of fresh surface soil or a few drops of a
solution which has already nitrified; though without such addition these
liquids may be freely exposed to filtered air without nitrification taking
place.
The nitrifying organism has been submitted as yet to but little
microscopical study; it is apparently a micrococcus.
It is difficult to conceive how the evidence for the ferment theory of
nitrification could be further strengthened; it is apparently complete in
every part. Although, however, nearly the whole of this evidence has been
before the scientific public for more than seven years, the ferment theory
of nitrification can hardly be said to have obtained any general
acceptance; it has not indeed been seriously controverted, but neither has
it been embraced. In hardly a single manual of chemistry is the production
of saltpeter attributed to the action of a living ferment existing in the
soil. Still more striking is the absence of any recognition of the
evidence just mentioned when we turn to the literature and to the public
discussions on the subjects of sewage, the pollution of river water, and
other sanitary questions. The oxidation of the nitrogenous organic matter
of river water is still spoken of by some as determined by mere contact
with atmospheric oxygen, and the agitation of the water with air as a
certain means of effecting oxidation; while by others the oxidation of
nitrogenous organic matter in a river is denied, simply because free
contact with air is not alone sufficient to produce oxidation. How much
light would immediately be thrown on such questions if it were recognized
that the oxidation of organic matter in our rivers is determined solely by
the agency of life, is strictly limited to those conditions within which
life is possible, and is most active in those circumstances in which life
is most vigorous. It is surely most important that scientific men should
make up their minds as to the real nature of those processes of oxidation
of which nitrification is an example. If the ferment theory be doubted,
let further experiments be made to test it, but let chemists no longer go
on ignoring the weighty evidence which has been laid before them. It is
partly with the view of calling the attention of English and American
chemists to the importance of a decision on this question that I have been
induced to bring this subject before them on the present occasion. I need
hardly add that such results as the nitrification of sewage by passing it
through sand, or the nitrification of dilute solutions of blood prepared
without special precaution, are no evidence whatever against the ferment
theory of nitrification. If it is to be shown that nitrification will
occur in the absence of any ferment, it is clear that all ferments must be
rigidly excluded during the experiments; the solutions must be sterilized
by heat, the apparatus purified in a similar manner, and all subsequent
access of organisms carefully guarded against. It is only experiments made
in this way that can have any weight in deciding the question.
Leaving now the theory of nitrification, I will proceed to say a few
words, first, as to the distribution of the nitrifying organism in the
soil; secondly, as to the substances which are susceptible of
nitrification; thirdly, upon certain conditions having great influence on
the process.
_The Distribution of the Nitrifying Organism in the Soil._--Three series
of experiments have been made on the distribution of the nitrifying
organism in the clay soil and subsoil at Rothamsted. Advantage was taken
of the fact that deep pits had been dug in one of the experimental fields
for the purpose of obtaining samples of the soil and subsoil. Small
quantities of soil were taken from freshly-cut surfaces on the sides of
these pits at depths varying from 2 inches to 8 feet. The soil removed was
at once transferred to a sterilized solution of diluted urine, which was
afterward examined from time to time to ascertain if nitrification took
place. These experiments are hardly yet completed; the two earlier series
of solutions have, however, been examined for eight and seven months
respectively. In both these series the soil taken from 2 inches, 9 inches,
and 18 inches from the surface has been proved to contain the nitrifying
organism by the fact that it has produced nitrification in the solutions
to which it was added; while in twelve distinct experiments made with soil
from greater depths no nitrification has yet occurred, and we must
therefore conclude that the nitrifying organism was not present in the
samples of soil taken. The third series of experiments has continued as
yet but three months and a half; at present no nitrification has occurred
with soil taken below 9 inches from the surface. It would appear,
therefore, that in a clay soil the nitrifying organism is confined to
about 18 inches from the surface; it is most abundant in the first 6
inches. It is quite possible, however, that in the channels caused by
worms, or by the roots of plants, the organism may occur at greater
depths. In a sandy soil we should expect to find the organism at a lower
level than in clay, but of this we have as yet no evidence. The facts here
mentioned are in accordance with the microscopical observations made by
Koch, who states that the micro-organisms in the soils he has investigated
diminish rapidly in number with an increasing depth; and that at a depth
of scarcely 1 meter the soil is almost entirely free from bacteria.
Some very practical conclusions may be drawn from the facts now stated. It
appears that the oxidation of nitrogenous matter in soil will be confined
to matter near the surface. The nitrates found in the subsoil and in
subsoil drainage waters have really been produced in the upper layer of
the soil, and have been carried down by diffusion, or by a descending
column of water. Again, in arranging a filter bed for the oxidation of
sewage, it is obvious that, with a heavy soil lying in its natural state
of consolidation, very little will be gained by making the filter bed of
considerable depth; while, if an artificial bed is to be constructed, it
is clearly the top soil, rich in oxidizing organisms, which should be
exclusively employed.
_The Substances Susceptible of Nitrification._--The analyses of soils and
drainage waters have taught us that the nitrogenous humic matter resulting
from the decay of plants is nitrifiable; also that the various nitrogenous
manures applied to land, as farmyard manure, bones, fish, blood, rape
cake, and ammonium salts, undergo nitrification in the soil. Illustrations
of many of these facts from the results obtained in the experimental
fields at Rothamsted have been published by Sir J.B. Lawes, Dr. J.H.
Gilbert, and myself, in a recent volume of the _Journal_ of the Royal
Agricultural Society of England. In the Rothamsted Laboratory, experiments
have also been made on the nitrification of solutions of various
substances. Besides solutions containing ammonium salts and urea, I have
succeeded in nitrifying solutions of asparagine, milk, and rape cake.
Thus, besides ammonia, two amides, and two forms of albuminoids have been
found susceptible of nitrification. In all cases in which amides or
albuminoids were employed, the formation of ammonia preceded the
production of nitric acid. Mr. C.F.A. Tuxen has already published in the
present year two series of experiments on the formation of ammonia and
nitric acids in soils to which bone-meal, fish-guano, or stable manure had
been applied; in all cases he found the formation of ammonia preceded the
formation of nitric acid.
As ammonia is so readily nitrifiable, we may safely assert that every
nitrogenous substance which yields ammonia when acted upon by the
organisms present in soil is also nitriflable.
_Certain Conditions having Great Influence in the Process of
Nitrification._--If we suppose that a solution containing a nitrifiable
substance is supplied with the nitrifying organism, and with the various
food constituents necessary for its growth and activity, the rapidity of
nitrification will depend on a variety of circumstances:
1. The degree of concentration of the solution is important. Nitrification
always commences first in the weakest solution, and there is probably in
the case of every solution a limit of concentration beyond which
nitrification is impossible.
2. The temperature has great influence. Nitrification proceeds far more
rapidly in summer than winter.
3. The presence or absence of light is important. Nitrification is most
rapid in darkness; and in the case of solutions, exposure to strong light
may cause nitrification to cease altogether.
4. The presence of oxygen is of course essential. A thin layer of solution
will nitrify sooner than a deep layer, owing to the larger proportion of
oxygen available. The influence of depth of fluid is most conspicuous in
the case of strong solutions.
5. The quantity of nitrifying organism present has also a marked effect. A
solution seeded with a very small amount of organism will for a long time
exhibit no nitrification, the organism being (unlike some other bacteria)
of very slow growth. A solution receiving an abundant supply of the
ferment will exhibit speedy nitrification, and strong solutions may by
this means be successfully nitrified, which with small seedings would
prove very refractory. The speedy nitrification which occurs in soil (far
more speedy than in experiments in solutions under any conditions yet
tried) is probably owing to the great mass of nitrifying organisms which
soil contains, and to the thinness of the liquid layer which covers the
soil particles.
6. The rapidity of nitrification also depends on the degree of alkalinity
of the solution. Nitrification will not take place in an acid solution; it
is essential that some base should be present with which the nitric acid
may combine; when all available base is used up, nitrification ceases.
It appeared of interest to ascertain to what extent nitrification would
proceed in a dilute solution of urine without the addition of any
substance save the nitrifying ferment. As urea is converted into ammonium
carbonate in the first stage of the action of the ferment, a supply of
salifiable base would at first be present, but would gradually be
consumed. The result of the experiment showed that only one-half the
quantity of nitric acid was formed in the simple urine solution as in
similar solutions containing calcium and sodium carbonate. The
nitrification of the urine had evidently proceeded until the whole of the
ammonium had been changed into ammonium nitrate, and the action had then
ceased. This fact is of practical importance. Sewage will be thoroughly
nitrified only when a sufficient supply of calcium carbonate, or some
other base, is available. If, instead of calcium carbonate, a soluble
alkaline salt is present, the quantity must be small, or nitrification
will be seriously hindered.
Sodium carbonate begins to have a retarding influence on the commencement
of nitrification when its amount exceeds 300 milligrammes per liter, and
up to the present time I have been unable to produce an effective
nitrification in solutions containing 1.000 gramme per liter.
Sodium hydrogen carbonate hinders far less the commencement of
nitrification.
Ammonium carbonate, when above a certain amount, also prevents the
commencement of nitrification. The strongest solution in which
nitrification has at present commenced contained ammonium carbonate
equivalent to 368 milligrammes of nitrogen per liter. This hinderance of
nitrification by the presence of an excess of ammonium carbonate
effectually prevents the nitrification of strong solutions of urine, in
which, as already mentioned, ammonium carbonate is the first product of
fermentation.
Far stronger solutions of ammonium chloride can be nitrified than of
ammonium carbonate, if the solution of the former salt is supplied with
calcium carbonate. Nitrification has in fact commenced in chloride of
ammonium solutions containing more than two grammes of nitrogen per liter.
The details of the recent experiments, some of the results of which we
have now described, will, it is hoped, shortly appear in the _Journal_ of
the Chemical Society of London.
Harpenden, July 21.
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