Chapter XX: Introduction (19)

Fifteen grams of fine earth, dried and mixed with a little sugar, are mixed with thirty grams of soda-lime in powder. The bottom of the combustion tube contains a little moist soda-lime, which is heated at the end of the operation at the same time that a current of pure hydrogen is made to pass through it, and the temperature of the tube is raised, little by little, to a distinct redness. The contents of the receiving bulbs are distilled, after the addition of water and soda, in the same apparatus which served in the estimation of nitrogen, by the kjeldahl method; the determinations and titrations are made also under the same conditions.

Blank determinations are also made under the same conditions to determine the amount of correction to be made by the two methods. Soda-lime, heated with pure sugar, gave 0.0002 gram of nitrogen for a total weight of fifty-five grams of the soda-lime contained in the tube. The fuming sulfuric acid gave 0.0011 cubic centimeters of ammoniacal nitrogen for the volume of thirty cubic centimeters.

The numbers obtained by the kjeldahl method in general, are lower than those obtained by the soda-lime method when no stearic or benzoic acid is used. The numbers obtained when stearic acid alone was used were sometimes inferior to those obtained by the soda-lime method. The numbers obtained when benzoic acid is used are, in general, about the same as those obtained by the soda-lime method.

It would seem that the double distillation, outlined above, for the kjeldahl method, would not be necessary if due care were exercised in the first distillation. This variation, therefore, seems to be unnecessary.

In the soda-lime method, time would be saved by the reception of the ammonia in standard acid, and its titration in the usual way, unless a further purification of the nitrogenous products of the combustion by the final distillation be desired.

=405. Volumetric Determination of the Nitrogen.=—Instead of separating the nitrates, the total nitrogen in the soil can be determined directly by the classic method of Dumas, which consists in bringing the whole of the nitrogen into a gaseous state and afterwards measuring its volume.

The following method illustrates the general principles of the determination:

A glass combustion tube closed at one end, about one meter in length, is selected. In the bottom of this tube is placed some potassium bicarbonate in a crystalline form, in small pieces, filling the tube to a distance of about twenty centimeters. Afterwards copper oxid is placed to the depth of ten centimeters and finally a mixture of from twenty to thirty grams of the earth with thirty to forty grams of copper oxid in a fine state of subdivision, and about ten grams of metallic copper obtained by reducing the copper oxid by hydrogen. Next the tube is filled with copper oxid to a depth of from twenty to twenty-five centimeters, and afterwards with reduced copper to the depth of at least twenty-five centimeters, and after this another layer of copper oxid of about five centimeters, and finally a plug of asbestos. The combustion tube is closed with a stopper carrying a glass tube of about ninety centimeters in length, of which the extremity, bent into the form of a =ᥩ=, extends to a mercury trough. The glass combustion tube is surrounded with brass gauze, except that part which contains the potassium bicarbonate. The beginning of the operation consists in heating the tube to decompose a part of the potassium bicarbonate, until the whole of the apparatus is filled with carbon dioxid. In order to determine that the whole of the air has been expelled and that the apparatus is entirely filled with carbon dioxid, a part of the gas which is disengaged, is received into a jar filled with mercury, in which a little potash-lye has been placed. If the gas is entirely absorbed by the potash, so that there remain only unappreciable particles, the tube can be regarded as completely free of air. When assurance is given that the air is all out of the apparatus, a jar of about 300 cubic centimeters capacity, filled with mercury and containing from thirty to forty cubic centimeters of a solution of potash of a density of 42° baumé, is placed over the outlet tube. The combustion is commenced by heating the anterior part of the tube, avoiding the heating of the part containing the earth. When the first part of the tube has reached the red stage the part containing the earth is gradually heated in order to obtain a gentle evolution of gas. The temperature of the tube is carried to redness and the heating gradually carried back toward the closed extremity, but avoiding raising the temperature of the part containing the potassium bicarbonate. The red heat is continued as long as bubbles of gas are discharged into the reservoir. When the evolution of gas has ceased the apparatus is again filled with carbon dioxid for the purpose of driving out the last traces of nitrogen, by heating again the part of the tube containing the potassium bicarbonate. The evolution of the carbon dioxid should be maintained for about fifteen minutes. At the end of this time all the nitrogen will be found in the receiving jar. Sometimes a small quantity of nitrogen dioxid is formed incidentally in the operation. After waiting for a quarter of an hour, in order to permit all the carbon dioxid which may have escaped into the reservoir to be completely absorbed, the receiving jar is carried to a water-basin and the mercury allowed gradually to escape; its place being taken by the water. The gas is then transferred into an azotometer where its volume and temperature are read in the usual way.

In order to absorb any nitrogen dioxid which may be admixed with the nitrogen itself, a little crystal of ferrous sulfate is introduced. The reservoir containing the nitrogen is carried to the mercury trough, and the water which it contains is nearly all run out in such a way as to be replaced with mercury, great care being exercised to avoid any escape of gas. Afterwards there is introduced over the mercury a crystal of ferrous sulfate and the azotometer is shaken until this crystal is dissolved by the water which it still contains. It is then allowed to remain for twenty hours. At the end of this time the nitrogen dioxid is absorbed and the volume of the gas is again read as before. One-half only of the total loss should be subtracted, since the volume of the nitrogen dioxid is twice the volume of the nitrogen itself. For the practice of this method, in connection with the use of a mercury pump, the directions which will be given under fertilizers may be consulted.

=406. Estimation of Ammonia.=—Ammonia exists ordinarily only in very small quantities in the soil, since it is incessantly transformed into nitrate or diffused in the air. Nevertheless, it is sometimes interesting to determine its quantity.

The method of determining the ammonia in soils is one of extreme delicacy on account of the small proportion therein, and the difficulty of expelling it without at the same time converting some of the organic nitrogen into ammoniacal compounds. The various methods employed for this purpose may be classified as follows:

1. Treatment of the soil with soda-lye in the cold, and the absorption of the ammonia given off by standard sulfuric acid.

2. The method of Boussingault, which consists in replacing the soda-lye with magnesia and distilling the ammonia at a boiling temperature, absorbing the distillate in a standard acid.

3. A modification of the above method, due to Schloesing, which consists first in extracting the ammonia by hydrochloric acid and subjecting the extract to distillation with magnesia.

4. The method of Knop consists in treating the soil in a closed cylinder with soda-lye containing bromin. The ammonia set free by the lye is decomposed in the presence of bromin into free nitrogen and hydrochloric acid. The nitrogen is collected and measured in an azotometer. The brom-soda-lye is prepared by dissolving 100 grams of sodium hydroxid in 1,200 cubic centimeters of water and adding twenty-five cubic centimeters of bromin.

5. The process described under 4, as shown by Baumann,[270] does not give accurate results and it has been modified by him as follows: Two hundred grams of soil are treated with 100 cubic centimeters of dilute hydrochloric acid (one part acid and four of water) free of ammonia; 300 cubic centimeters of ammonia-free distilled water are added and the whole digested for two hours with frequent stirring. If a soil contain much calcium carbonate larger quantities of acid must be used. Two hundred cubic centimeters of the filtrate are placed in an evolution flask, connected with an azotometer, with five grams of freshly burned magnesia. The mixture is then oxidized as follows: Ozone is generated by adding three parts by weight of sulfuric acid to one part of dry and powdered potassium permanganate. A stream of air is drawn through the ozone generator by an aspirator, and the ozone is conducted into a flask containing the hydrochloric acid extract of the soil and magnesia. The oxidation is completed in about ten minutes. The mixture is then brought into the evolution flask of the azotometer and the nitrogen set free and measured in the usual way.

It has been shown that if asparagin or glutamin be present in the soil they are decomposed by the soda-lye and the results obtained are too high. It has been further proved that soils which contain a notable quantity of humus give, with soda-lye in the cold, a practically continuous evolution of ammonia. Moreover, soils which are rich in humus and which have been treated by distillation with magnesia give, on subsequent treatment with soda-lye, considerable additional quantities of ammonia.

_Comparison of Methods of Estimating Ammonia._—Baumann has determined the ammonia-nitrogen in various soils by the soda-lime method; distillation of the hydrochloric acid extract with magnesia, and the azotometric method modified as indicated above. These methods will be designated as 1, 2, 3, respectively in the following table.

METHOD.
1. 2. 3.
Ammonia-nitrogen in one
kilogram of soil.
—————— —————— ——————
No. of sample. Gram. Gram. Gram.
1 0.0448 0.02227 0.02781
2 0.0168 0.01105 0.01326
3 0.0336 0.01771 0.02214
4 0.0056 0.00443 0.00443
5 0.0280 0.02337 0.02894
6 0.0196 0.01243 0.01672

From the above figures it is seen that the method usually attributed to Schloesing gives uniformly higher numbers than either of the other processes, while the third gives slightly higher values than the second.

=407. The Magnesia Distillation Process.=—If a sample of soil be distilled directly with magnesia and water, there is danger on the one side of not extracting all the ammonia, by reason of the absorbing power of these bodies, and on the other, of transforming into ammonia the nitrogen of the organic matters. It is therefore preferable to separate the ammonia from the soil in the form of chlorid, and to subject this extract to distillation.

In fifty grams of the soil the humidity is determined by drying at 100° until there is no further loss of weight. The quantity of moisture being known, 200 grams of soil are taken and moistened with water, and then there is added, in small portions, some dilute hydrochloric acid, shaking frequently until the whole of the calcium carbonate present is decomposed. The liquor should remain acid at the end of the operation, but without containing a notable excess of acidity. Knowing beforehand the quantity of moisture contained in the 200 grams, water is added until the total quantity shall be equal to 500 cubic centimeters. The whole is then shaken and allowed to repose, and filtered rapidly, covering the funnel with a glass vessel and receiving the liquid which runs through in a flask with a narrow opening. Two hundred and fifty cubic centimeters of this liquor, or mixture, represent 100 grams of earth of known humidity. This quantity is introduced into a flask for determining the ammonia and five grams of calcined magnesia added. Before commencing the distillation, assurance should be had that the magnesia has completely saturated the acid in excess, and that the liquor is alkaline.

If, by chance, the liquor should be still acid it would be necessary to add sufficient magnesia in order that the reaction should be manifestly alkaline. Afterwards the distillation is begun and the ammonia is received in an appropriate vessel containing one-tenth normal sulfuric acid and titrated in the usual way, or nesslerized.

Inasmuch as the quantities of ammonia contained in the earth are generally very small it is necessary to be very particular in order to avoid errors. The distilled water which is employed should be deprived of all traces of ammonia by prolonged ebullition, and the hydrochloric acid should be distilled in the presence of a little sulfuric acid. The treatment with hydrochloric acid is for the purpose of destroying the absorbing properties of the soil for ammonia, and to permit this last to enter into solution as chlorid. When there is need of very great precision it is convenient to make a blank operation with the hydrochloric acid and water which are employed, in order to make a correction for the traces of ammonia which these reagents may contain.

=408. Estimation of Ammoniacal and Amid Nitrogen by the Method of Berthelot and André.=[271]—Heat one hundred grams of earth for thirty hours on a steam-bath with about .500 cubic centimeters of dilute hydrochloric acid (fifteen grams of hydrochloric acid to 500 cubic centimeters of water). At the end of this time throw the contents of the flask on a filter and wash with hot water until acid reaction has ceased. Determine both the ammoniacal and amid nitrogen in the soluble, and the total nitrogen in the insoluble portion, the ammoniacal by distillation with magnesia, and the amid and total with soda-lime.

_Example._ A soil contained 0.1669 per cent total nitrogen. Of this there were obtained:

As ammoniacal nitrogen 13.7 per cent
In the soluble part as amid nitrogen 56.2 „ „
In the insoluble part, total nitrogen 29.7 „ „
————
Sum 99.6 „ „

_Treatment of the Insoluble Portion._—Treat the part insoluble in hydrochloric acid with a three per cent solution of potash on a steam-bath for thirty hours. Estimate the nitrogen remaining insoluble, from which the part dissolved can be determined by difference. The potash will dissolve usually about two-thirds of the remaining nitrogen.

About ninety per cent of the total nitrogen present in an arable soil will be rendered soluble by successive treatment with acid and alkali. The reverse treatment will give practically the same result. It is therefore immaterial, from an analytical standpoint, whether the acid or alkali be used first.

=409. Estimation of Volatile Nitrogenous Compounds Emitted by Arable Soil.=—The following method, due to Berthelot and André,[272] may be practiced:

Porcelain pots, containing one kilogram of soil, are placed under bell-jars of fifty liters capacity adjusted to glass dishes designed to receive the waters of condensation.

During the first period the pots are to be sprinkled from time to time, during the duration of the experiment, through the upper tubulature, so as to prevent the soil from becoming dry. The water is partly condensed on the sides of the bell-jar. It is removed each week through the inferior tubulature, treated with a little dilute sulfuric acid, and preserved for further study. A small vessel containing dilute sulfuric acid is placed near the porcelain pot for the purpose of collecting, as far as possible, the evolved ammonia.

During the second period the pots are not sprinkled, the soil becomes dry and there is no longer any condensation of water on the walls of the bell-jar. The two periods should include about five months, from May to October.

At the end of the second period the following determinations are to be made:

1. The ammonia absorbed by the dilute sulfuric acid.

2. The ammonia set free by distillation with magnesia, such as may have accumulated in the condensed water.

3. The organic nitrogen contained in the latter after elimination of the ammonia. This is determined by adding a slight excess of acid, evaporation to dryness, and combustion with soda-lime, or by moist combustion with sulfuric acid.

Example:

_Earth Employed._—One kilogram of sandy clay containing total nitrogen, 0.09 gram. Nitrogen in sprinkling water, 0.000048 gram.

_Nitrogen in Exhaled Products._—

FIRST PERIOD. SPRINKLING.
Ammoniacal nitrogen collected in the dilute sulfuric 0.00012 gram.
acid
Ammoniacal nitrogen collected in the condensation waters 0.00012 „
Organic nitrogen in condensation waters 0.00220 „
————————
Sum 0.00244 „

SECOND PERIOD. NO SPRINKLING.
Ammoniacal nitrogen in dilute sulfuric acid 0.000007 gram.
„ „ „ condensed water 0.000007 „
Organic „ „ „ „ 0.000040 „
————————
Sum 0.000054 „

_Conclusions._—The exhalation of nitrogenous compounds takes place with a certain relative activity, about two milligrams in two months and a half, as long as the soil is kept moist by sprinkling.

In the second period, without sprinkling, the exhalation is reduced to a mere trace.

The vessel containing the dilute sulfuric acid placed near the porcelain pot absorbs only about one-half of the ammoniacal nitrogen set free. The nitrogen emitted under other forms than ammonia is, in every instance, greatly superior in quantity, and this is the most important of the observed phenomena. This is true at least with the kind of soil with which the experiment was made. With arable soil containing twenty times as much nitrogen as the soil described above this order is reversed,[273] the ammoniacal prevailing over the non-ammoniacal nitrogen volatilized.

These phenomena are doubtless greatly influenced in soil under culture by microbes, and the lowest orders of vegetation to which are doubtless due the traces of non-ammoniacal volatile nitrogenous compounds, a sort of vegetable ptomaines.

=410. General Conclusions.=—In the light of our present knowledge concerning the methods of nitrogen determination in the soil in the form of organic compounds and ammonia, moist combustion with sulfuric acid is to be preferred to the older soda-lime process. For the nitrogen combined as ammonia, the extraction of the sample with hydrochloric acid and subsequent distillation with an excess of freshly calcined magnesia, are recommended. For the study of the progressive decomposition of the nitrogenous compounds, the various processes devised by Berthelot and André are the best.

The origin of the nitric acid in the soil, the methods of studying the various nitrifying organisms, and of estimating the nitric acid produced, will form the subject of the next part.

NOTE.—At the Eleventh Annual Convention of the Association of Official Agricultural Chemists, held in Washington, August 23, 24 and 25, 1894, the following process of soil extraction was adopted as the official method:

_Preparation of the Sample._—500 grams or more, of the air-dried soil, which may be either the original soil or that which has been passed through a sieve of coarser mesh, are sifted upon a sieve with circular openings one-half millimeter in diameter, rubbing, if necessary, with a rubber pestle in a mortar, until the fine earth has been separated as completely as possible from the particles that are too coarse to pass through the sieve. The fine earths thoroughly mixed and preserved in a tightly stoppered bottle from which the portions for analysis are weighed out.

The coarse part is weighed and may be subjected to further examination, (as in _Bulletin 38, Div. of Chem._, pp. 65, 75 and 200.) It may sometimes be necessary to wash the soil through the one-half millimeter sieve with water, in which case proceed as directed on pp. 65 and 75 of the above _Bulletin_. The use of water is to be avoided whenever possible.

_Determination of Moisture._—Heat two to five grams of the air-dried soil in a flat-bottomed, tared platinum dish; heat for five hours in a water-oven kept briskly boiling; cover the dish, cool in a desiccator, and weigh.

Repeat the heating, cooling, and weighing at intervals of two hours till constant weight is found, and estimate the moisture by the loss of weight. Weigh rapidly to avoid absorption of moisture from the air. An air-bath must not be used in this determination.

_Determination of Volatile Matter._—The platinum dish and soil used to determine moisture are used also to determine volatile matter. Heat the dish and dried soil to full redness until all organic matter is burned away. If the soil contain appreciable quantities of carbonates, the contents of the dish, after cooling, are to be moistened with a few drops of a saturated solution of ammonium carbonate, dried and heated to dull redness to expel ammonium salts, cooled in the desiccator and weighed.

The loss in weight represents the organic matter, water of combination, ammonium salts, etc.

_Extraction of Acid-Soluble Materials._—In the following scheme for soil analysis it is intended to use the air-dried soil from the sample bottle for each separate investigation. The determination of moisture, made once for all on a separate portion of air-dried soil, will afford the datum for calculating the results of analysis upon the soil dried at the temperature of boiling water. It is not desirable to ignite the soil before analysis or to heat it so as to change its chemical properties.

The acid digestion is to be performed in a flask so arranged that the evaporation of acid shall be reduced to a minimum, but to take place under atmospheric pressure and at the temperature of boiling water. Any flask resistant to acids is suitable, but it is not necessary to use a condenser, as a simple bohemian glass tube eighteen inches in length will answer the purpose of preventing loss of acid. Where it is not desired to determine sulfur trioxid, an erlenmeyer fitted with a rubber stopper and hard glass tube will answer. The flask must be immersed in the water-bath up to the neck or at least to the level of the acid and the water must be kept boiling continuously during the digestion.

In the following scheme, ten grams of soil are taken, this being a convenient quantity in most soils, in which the insoluble matter is about eighty per cent. If desired, a larger quantity of such soil may be taken, using a proportionately larger quantity of acid and making up the soil solution to a proportionately larger volume. In very sandy soils, where the proportion of insoluble matter is ninety per cent or more, twenty grams of soil are to be digested with 100 cubic centimeters of acid and the solution made up to 500 cubic centimeters or a larger quantity may be used, preserving the same proportions. It is very important that the analyst assure himself of the purity of all the reagents to be used in the analysis of soils before beginning the work.

_Acid Digestion of the Soil._—Place ten grams of the air-dried soil in a 150 to 200 cubic centimeter bohemian flask, add 100 cubic centimeters of pure hydrochloric acid of specific gravity 1.115, insert the stopper with condensing tube, place in a water or steam-bath and digest for ten hours continuously at the temperature of boiling water, shaking once each hour. Pour the clear liquid from the flask into a small beaker, wash the residue out of the flask with distilled water on a filter adding the washings to the contents of the beaker. The residue after washing until free of acid, is to be dried and ignited as directed below. Add one or two cubic centimeters of nitric acid to the filtrate, and evaporate to dryness on the water-bath, finishing on a sand or air-bath to complete dryness; take up with hot water and a few cubic centimeters of hydrochloric acid, and again evaporate to complete dryness. Take up as before, filter and wash thoroughly with cold water or with hot water slightly acidified at first with hydrochloric acid. Cool and make up to 500 cubic centimeters. This is solution “A.” The residue is to be added to the main residue and the whole ignited and weighed, giving the insoluble matter.

The determination of the various components of the solution remains essentially as described in the provisional methods of the Association which have already been given.

It is directed that all results of soil analysis be calculated on the basis of the sample dried to constant weight at the temperature of boiling water.

AUTHORITIES CITED IN PART SIXTH.

Footnote 189:

Annales de Chimie et de Physique, sixiéme serie, Tome 25, pp. 292, et
seq.

Footnote 190:

L’Analyse du Sol, p. 14.

3. Die Landwirtschaftlichen Versuchs-Stationen, Band 38, S. 311.

Footnote 191:

Bulletin 38, Chemical Division United States Department of
Agriculture, p. 201.

Footnote 192:

Untersuchung Landwirtschaftlich und Gewerblich Wichtiger Stoffe, S.
14.

Footnote 193:

Vid. op. cit. supra.

Footnote 194:

Vid. op. cit. 1.

Footnote 195:

Vid. op. cit. 3, Band 37, S. 279.

Footnote 196:

Journal of the Chemical Society, September, 1880, p. 617.

Footnote 197:

Wanklyn, Philosophical Magazine, Series 5, Vol. 5, p. 466.

Footnote 198:

Vid. op. cit. 1.

Footnote 199:

Traité d’Analyse des Matiéres Agricoles, p. 148.

Footnote 200:

Bulletin 38, Division of Chemistry, United States Department of
Agriculture, pp. 84, et seq.

Footnote 201:

Encyclopedie Chimique, Tome 4, p. 182.

Footnote 202:

Die Landwirtschaftlichen Versuchs-Stationen, Band 37, S. 280.

Footnote 203:

Vid. op. cit. supra, Band 28, S. 229.

Footnote 204:

Comptes rendus, 1890, pp. 290, et seq.

Footnote 205:

Annales Agronomiques, 1890, p. 558.

Footnote 206:

Bulletin 13, Division of Chemistry, p. 590.

Footnote 207:

Bulletin de la Société Chimique, Serie 3, Tome 2, pp. 483, et seq.

Footnote 208:

Zeitschrift für analytische Chemie, Band 3, S. 165.

Footnote 209:

Petermann, L’Analyse du Sol, p. 20.

Footnote 210:

Zeitschrift für analytische Chemie, Band 3, S. 92.

Footnote 211:

Journal of the Chemical Society, March, 1894, p. 141.

Footnote 212:

Agricultural Science, January, 1894, p. 2.

Footnote 213:

American Journal of Science, Vol. 7, 1874, p. 20.

Footnote 214:

Geological and Agricultural Report of Kentucky, Vol. 3.

Footnote 215:

Bulletin 38, Division of Chemistry, p. 77.

Footnote 216:

Op. cit. supra, p. 83.

Footnote 217:

Die Landwirtschaftlichen Versuchs-Stationen, Band 37, S. 311.

Footnote 218:

Zeitschrift für analytische Chemie, Band 3, S. 92.

Footnote 219:

Traité d’Analyse des Matiéres Agricoles, p. 144.

Footnote 220:

Op. cit. 28, pp. 202, et seq.

Footnote 221:

Op. cit. 28, pp. 77, et seq.

Footnote 222:

Op. cit. 22, p. 21.

Footnote 223:

Manuscript communication to author.

Footnote 224:

Annales de la Science Agronomique, Huitiéme Année, Tome 1, p. 278.

Footnote 225:

Die Landwirtschaftlichen Versuchs-Stationen, Band 37, S. 311.

Footnote 226:

Comptes rendus, 1890, p. 289.

Footnote 227:

Thoms, Zur Werthschätzung der Ackererde, S. 120.

Footnote 228:

Le Stazioni Sperimentali Agrarie Italiane, Vol. 16, p. 679.

Footnote 229:

Crookes’ Select Methods in Chemical Analysis.

Footnote 230:

Chemiker Zeitung, Band 13, S. 1391.

Footnote 231:

Annales de Chimie et de Physique, serie sixiéme, Tome 15, p. 309.

Footnote 232:

Vid. op. cit. 37, pp. 270, et seq.

Footnote 233:

Chemiker Zeitung, Band 13, S. 726.

Footnote 234:

Die Agrikultur Chemische Versuchs-Station, Halle, a/S., S. 80.

Footnote 235:

Comptes rendus, Tome 107, pp. 999 and 1150.

Footnote 236:

Die Landwirtschaftlichen Versuchs-Stationen, Band 41, S. 453.

Footnote 237:

Bulletin de la Société Chimique de Paris, 1893, p. 343.

Footnote 238:

Die Agrikultur Versuchs-Station, Halle, a/S., S. 70.

Footnote 239:

Op. cit. supra., S. 68.

Footnote 240:

Op. cit. 37, p. 267.

Footnote 241:

L’Analyse du Sol, p. 20.

Footnote 242:

Op. cit. 1, pp. 303, et seq.

Footnote 243:

Op. cit. 40, S. 114.

Footnote 244:

Bulletin 38, Division of Chemistry, p. 80.

Footnote 245:

Zeitschrift für analytische Chemie, Band 3, S. 92.

Footnote 246:

Chemisches Centralblatt, 1861, p. 3.

Footnote 247:

Berichte der deutschen chemischen Gesellschaft, Band 26, S. 386.

Footnote 248:

Manuscript communication to author.

Footnote 249:

Op. cit. 37, p. 285.

Footnote 250:

Op. cit. 44, pp. 305, et seq.

Footnote 251:

Die Landwirtschaftlichen Versuchs-Stationen, Band 37, S. 284.

Footnote 252:

Op. et loc. cit. 58.

Footnote 253:

Op. et loc. cit. 41.

Footnote 254:

Op. et loc. cit. 37.

Footnote 255:

Op. et loc. cit. 58.

Footnote 256:

L’Analyse du Sol, p. 17.

Footnote 257:

Op. cit. 44, p. 308.

Footnote 258:

Op. cit. 37.

Footnote 259:

Op. cit. 64, Band 40, S. 251.

Footnote 260:

Bulletin de la Société Chimique, May, 1891, pp. 643, et seq.

Footnote 261:

Bulletin 38, Division of Chemistry, p. 204.

Footnote 262:

Bulletin 38, Division of Chemistry, p. 81.

Footnote 263:

Op. cit. 44, March, 1891, pp. 393, et seq.

Footnote 264:

Op. cit. 58, Band 31, S. 525.

Footnote 265:

Relatorio Annual do Instituo Agronomico do Estado de San Paulo
(Brazil), 1892, p. 107.

Footnote 266:

Op. cit. 37, pp. 253, et. seq.

Footnote 267:

Op. cit. 44, Tome, 25, pp. 299, et seq.

Footnote 268:

Petermann, L’Analyse du Sol, p. 17.

Footnote 269:

Op. et. loc. cit. 76.

Footnote 270:

Die Landwirtschaftlichen Versuchs-Stationen, Band 33, Ss. 247, et seq.

Footnote 271:

Op. cit. 44, Tome 25, pp. 327, et seq.

Footnote 272:

Op. cit. 44, Tome 25, pp. 330, et seq.

Footnote 273:

Op. et loc. cit. supra.

PART SEVENTH.

THE ORIGIN AND ESTIMATION OF OXIDIZED NITROGEN IN SOILS, RAIN AND
DRAINAGE WATERS.

=411. Introductory Considerations.=—The estimation of oxidized nitrogen in the soil would properly find a place in the preceding part; but on account of the late progress in our knowledge of the source of this indispensable and costly plant food it has become necessary to give it especial attention. The present part will, therefore, be devoted to a brief statement of our present knowledge in respect of the origin of oxidized nitrogen, a description of the nitrifying ferments and methods for their isolation and determination and finally the most approved methods of estimating the ammonia, nitrous, and nitric acids formed thereby both in the soil and the waters pertaining thereto or proceeding therefrom. It is scarcely necessary to caution the reader not to consider this part in any sense a treatise on the bacteria active in soil chemistry. Its object is rather to place in the hands of the soil analyst data which will enable him to intelligently study the soil phenomena depending on these organisms and to determine the extent and character of their biological and chemical functions. These are matters which, up to the present time, have found no place in manuals dedicated to agricultural analysis.

=412. Organic Nitrogen in the Soil.=—With the exception of the small quantities of nitric acid added to the soil directly by rain water, the whole of the supply of this substance is derived from the products of the oxidation of nitrogenous bodies. These products are either stored as the results of past nitrification or are formed synchronously with their consumption by the growing plant. Nitrogenous compounds are present as organic vegetable or animal remains and as humus. All vegetable and animal material deposited in or on the soil contains more or less of these proteid or nitrogenous matters while the amount of nitric acid supplied in this way is probably represented entirely by the quantity in the organism of the plant or animal and unabsorbed at the time of its death. In other words it is not demonstrated that nitrates or nitrites are in any sense a special product of plant growth save in the case of nitrifying organisms themselves which are supposed to be of a vegetable nature. Animal organisms do not in any sense assimilate nitric nitrogen.

With most plants, the quantity of proteid nitrogen which they can deliver to the soil is in no case greater than the sum of organic and nitric nitrogen supplied in their food and they can therefore be regarded only as the carriers and conservers of this substance. On the other hand there are some plants notably those belonging to the leguminous family which permit of the development on their rootlets of colonies of bacteria which have the faculty of rendering atmospheric nitrogen available for plant growth. Whether or not there exist plants other than the micro-organisms mentioned which are capable of directly oxidizing and fixing atmospheric nitrogen is still an unanswered question. It is not probable, however, that the difficult task of oxidizing atmospheric or free nitrogen would be accomplished in nature in only one way. In fact it has already been established that organisms do exist which are capable of oxidizing free nitrogen in a manner wholly independent of other plant life and to produce weighable quantities of nitric acid when developed in media of mineral matters and pure carbohydrates to which free nitrogen has access. It is, therefore, fair to assume that the fixation of free nitrogen is a function of chemical activity quite independent of ordinary plant life and that the leguminous plants take no further part in this process than that of providing in their radical development a favorable nidus for the growth of the nitrifying organism.

By the action of denitrifying organisms a portion of the nitrogen of nitric acid is constantly restored to a free state, a far larger portion, perhaps, than is fixed in the atmosphere itself by the action of electricity. Were it not, therefore, for the activity of the nitrifying ferments the stores of nitrogen available for growing plants would constantly become less. Instead of this being the case, however, it is probable that the contrary is true and that, by a wise system of agriculture, the total nitrogen at the disposal of plants may become greater and greater in quantity.

=413. Development of Nitric and Nitrous Acids in Soils.=—Owing to the solubility of nitrates there can be but little accumulation of them in soils in those countries where there is any considerable amount of rain-fall. On the other hand in arid regions there may be found extensive deposits of nitrates. The occurrence of a certain quantity of nitrates in the soil, however, is essential to the growth of plants. Until within a few years little was known of the origin of nitric acid in the soil. The presence of nitrates in drainage waters was well established, likewise the consumption of nitric acid by the growing plant, but the method of its supply was unknown. In a general way it was said that the nitric acid came from electrical action and the oxidation of the albuminous bodies in the soil, but without specifying the manner in which this change takes place. The researches of Schloesing and Müntz, of Springer, Winogradsky, Frankland, Warington and others have demonstrated the fact that this oxidation is caused by means of bacteria and that the nitrates formed can be consumed and destroyed by other species of this organism. In the one case the process has been called nitrification and in the other denitrification.[274]

The influence of these low organisms both in producing fertility in a soil and maintaining it in a state of fertility is of the highest importance.

=414. Conditions Necessary for Nitrification.=—In order to properly understand the reasons for many of the steps in investigating a soil for nitrifying organisms, it will be useful to state the general conditions on which nitrification depends.

The nitrifying organism, like every other one, first of all feels the necessity for food. In general, food which is given to microbes of all kinds consists of some organic matter together with the addition of mineral substances necessary to growth. These substances in general are phosphoric acid, potash, and lime. Of these articles of bacterial food phosphoric acid seems to be the most important. With the nitrifying organisms, however, it has been found that the organic matter can be omitted. In fact, as will be seen further on, the omission of organic matter supplies the best condition for the proper isolation of the organisms. In other words some forms of the nitrifying organisms have the property of subsisting wholly on mineral substances, _i. e._, are true vegetables.

The presence of oxygen is also necessary to the growth of the common nitro-organisms. In an atmosphere deprived of oxygen or in which the oxygen is reduced to a very low percentage, the process of nitrification is retarded or stopped as the oxygen diminishes or disappears.

The presence of a base with which the nitrous or nitric acid formed may unite is also essential to the proper conduct of the process. For this reason the nitrification should take place in a solution which is feebly alkaline or in the presence of a base which can be easily decomposed so that no acidity can take place. Calcium carbonate is a base well suited to favor the nitrifying process and its presence in a soil favors the rapid oxidation of proteid matter. The mistake must not be made, however, of supposing that an excess of alkali would favor nitrification. The contrary is true. A slight excess of alkali may prevent nitrification altogether when it is due to the common organisms present in an arable soil. It may be that in soils charged with alkali a different organism exists which is capable of exercising its functions when the alkali is in excess.

The temperature to which the nitrifying body is subjected is also a matter of importance. The nitrifying organisms have the property of remaining active at lower temperatures than most bodies of their class. On the contrary their action is retarded and destroyed by high temperatures. The most favorable temperature for nitrification is about that of blood heat; _viz._, 37°. At 50° the organism shows very little activity and at 55° its activity ceases altogether. Nitrification, however, according to Warington, cannot be started in a solution if the initial temperature is 40°.

Desiccation has the same retarding influence on nitrification that a high temperature has. Even thoroughly air-drying a soil may destroy its nitrifying qualities.

Darkness is also necessary to the proper progress of nitrification. In a strong light, the activity of the organism is very much diminished or destroyed altogether. A bright light like sunshine may even stop nitrification which has set in.

=415. Effect of Potassium Salts on Rate of Nitrification.=—Dumont and Crochetelle have described some experiments to determine the effect of potassium salts alone and in combination with lime on nitrification.[275]

Soil rich in vegetable mold (18.5 per cent of humus and 0.29 per cent of lime) was treated with varying amounts of potassium sulfate and carbonate and kept for twenty days at 25°. In the untreated soil the amount of nitric acid produced was twenty-five parts per million. When potassium carbonate was applied in quantities of from one-tenth to six per cent the amount of nitric acid increased from forty-seven parts per million to 438 parts when four and one-half per cent of the potassium salt were used. Larger quantities caused a decrease in the amount of nitric acid produced. Very little effect, on the contrary, was produced by the action of potassium sulfate. When one-half per cent was employed the quantity of nitric acid formed rose to fifty parts per million, while with quantities as high as five per cent it fell below the normal; _viz._, twenty-five parts per million.

When calcium carbonate was added to the soil in conjunction with potassium sulfate there was a marked increase in the amount of nitrogen oxidized. The activity of potassium sulfate in promoting nitrification is therefore increased by the presence of the calcium salt, potassium carbonate and calcium sulfate being formed.

=416. Production of Nitrous and Nitric Acids.=—In the following pages the study of the methods of isolating the nitrous and nitric ferments will be considered as one process, the final isolation of the two classes of bodies being the result of their synchronous cultivation in appropriate media. The special process of the production of ammonia by oxidation is not so well-known, and will therefore be described in brief.

It is now generally conceded that the action of the nitrous organism is 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.