CHEMICAL NEWS, May 18, 1917 THE Organic Nitrogen Compounds of Soils and Fertilisers A STATISTICAL STUDY OF ORGANIC SERIES. By W. R. FORBES, B.Sc. (Lond.). In the subjoined an attempt is made to compare organic series by finding their coefficient of variation. The statistical method employed is outlined in Watts's "Heredity." The boiling-points of the series of organic compounds are expressed in absolute temperature. The average is then obtained. Each datum is subtracted from the average (1) and the result squared (2). The sum of these squares is then divided by the sum of the data giving us the square of the standard deviation. The deviation expressed as a percentage of the average gives us the coefficient of variation. The series are selected so as to show in each case equal additions of (CH2). Series. 229 In the paraffin group which contains no oxygen or hydroxyl group, it appears that the coefficient of variation lies right apart those of the other series considered. The coefficient for the benzine series comes remarkably near that for the alcohol and carboxylic acid series, which are almost identical. In considering the alcohol and carboxylic acid series we must remember that each contains an OH group. It therefore appears that the (H2) of the alcohol series corresponds to the (O) of the carboxylic acid series in its influence on the boiling point for equal additions of (CH2). The benzine series contain no oxygen or hydroxyl groups, but the difference of the coefficient of variation from that of the paraffin series can be accounted for by the structure of the benzine nucleus. The approximation to the coefficient of variation in the alcohol and carboxylic series points to an equivalence of the doubtful benzine bond to (O or H2), and so would tend to support the double bond formula for the benzine nucleus rather than the centric formula. Therefore this statistical study appears to furnish additional evidence as to the nature of the benzine ring and to the theory of the double bond. Marlborough, New Zealand. The Decay of Proteins in the Soil. THAT Constant decomposition of proteins is going on in agricultural soils is clearly shown by the two preceding investigations. This is evidenced, first, by the fact that a number of primary protein decomposition products are regularly occurring soil constituents, and by the additional fact that side by side with these simpler nitrogenous compounds there are present in soils, complexes, themselves proteins or nucleoproteins, or very nearly akin to these. Such evidence shows the importance of a thorough knowledge of the biochemistry of the decay of protein materials in soils in finally elucidating the changes which take place normally in the organic nitrogenous matter of soils, and the nature of the compounds which result from these changes, This subject is not only of theoretical importance, but is of practical value as well. Not only do proteins from the débris of plants growing in the soil accumulate there, but there are added to most agricultural soils stable manure, organic fertilisers, and green manuring crops. All of these manures contain considerable quantities of proteins or complexes closely related to the proteins. The question of the availability or agricultural value of these fertilisers is only to be answered either by directly determining the effects of the products of decay of these materials upon the plants growing in the soil, or by determining by chemical means certain facts concerning their nature. It is obvious that a knowledge of the normal decay of protein material in soils will materially aid in solving such a problem. This investigation was undertaken for the purpose of studying the changes taking place in the protein material of a high-grade organic nitrogenous fertiliser when added to an agricultural soil. Since ammonia formation is but one step in this process it becomes of interest to know from what portion of the protein molecule this ammonia is formed; for how long the protein itself or the primary decomposition products of the protein can persist in the soil, and finally to gain some insight into the nature of the compounds formed by the action of the micro-organisms in their life processes. The Proteins Studied. Dried blood, which was chosen for this investigation, is composed almost entirely of various animal proteins and the commercial product is of fairly constant composition. Abderhalden (1898) reports figures on the composition of the blood of cattle, sheep, pigs, horses, and goats, which show that a mixture of the blood of these animals should contain about 200 parts of solid matter per 1000 parts of blood. Of these solids about 54 per cent is hemoglobin, and about 32 per cent albumin, or approximately 86 per cent is composed of proteins, exclusive of any nucleoproteins or nucleic acids, which undoubtedly are also present. The dried blood used in this investigation was purchased in the open market, and contained 13'92 per cent of total nitrogen. Two samples of dried blood were hydrolysed by boiling with hydrochloric acid, and the various forms of nitrogen in the hydrochloric acid extract were then estimated according to the nitrogen partition method proposed by Van Slyke. The results so obtained are presented in Table III. Cystine nitrogen was not determined, and the small amounts of cystine nitrogen in the blood would be included with nitrogen in the form of arginine, histidine, and lysine. TABLE III.-The Forms of Nitrogen in Dried Blood and Amide .. Melanin Dried blood. Soil. Arginine 7'517 Histidine The soil chosen for use was a Norfolk fine sandy loam taken from a cantaloupe field near Raleigh, N.C. The soil was in a bigh state of cultivation, and had received both mineral fertilisers and stable manure. It was found to contain o'0301 per cent of total nitrogen. The soil was passed through a 40-mesh sieve and dried in vacuum. Forty pounds of soil were mixed with about 3 pounds of dried blood by sieving the two together until samples taken from different parts of the mixture gave duplicate analyses for total nitrogen. The total nitrogen in the soil thus prepared was found to be 0.8945 per cent. The ammonia in the prepared soil was found to be o'0005 per cent. The soil was made up to a 10 per cent moisture content, and was kept in a stoneware jar covered with perforated wrapping paper to exclude dust. The decomposition was allowed to proceed at the temperature of the laboratory. During the first period of eighteen days the soil was maintained at a moisture content of 10 per cent, and was mixed several times by hand during that period to promote aëration. Later on, however, the soil was restored to the 10 per cent moisture content every five to eight days, and on two occasions it was allowed to dry out completely; At each addition of water to the soil it was turned out of the jar, and thoroughly mixed. The total length of the experiment was 240 days, during which time samples of the soil were taken at the following intervals: Nitrogen Partition. By the use of the methods outlined the nitrogen was separated into the following: -(1) Total nitrogen in the soil; (2) total nitrogen in bydrochloric acid solution; (3) ammonia nitrogen in the soil; (4) ammonia nitrogen in hydrochloric acid solution; (5) melanin nitrogen; (6) nitrogen precipitated by phosphotungstic acid, reported as arginine, histidine, and lysine nitrogen; (7) nitrogen in the filtrate from the phosphotungstic acid precipitate, reported as monoamino acid nitrogen and non-amino nitrogen. By subtracting the amount of ammonia nitrogen found in the soil (3) from the amount of ammonia nitrogen found in the hydrochloric acid (4) the amount of nitrogen in the soil in the form of the amide group in proteins or as acid amides may be obtained. This is reported as amide nitrogen. The amount of nitrogen in the soil in the form of proteins or protein decomposition products, with the exception of ammonia nitrogen, may be obtained by subtracting the amount of ammonia nitrogen in the soil (3) from the amount of total nitrogen in hydrochloric acid solution (2). This is reported as" hydrolysable" nitrogen. The amount of nitrogen in all of the various fractions was determined by the Kjeldahl method, which does not include nitrate nitrogen unless large amounts of reducing substances are present; such may be the case, however, with some of the Kjeldahl analyses, and any nitrate nitrogen therefore included in a Kjeldahl determination would be reported as non-amino nitrogen. In regard to the nitrogen reported in this investigation as amide nitrogen it might be stated that it is difficult to conceive in the present state of our knowledge of any other soil compounds than the various proteins which contain the amide group, or the acid amides themselves which would resist heating in vacuo with calcium hydroxide, and subsequently split off ammonia on heating with hydrochloric acid. The melanins are at present undefined and no significance can be attached to the figures obtained. The nitrogen reported as monoamino acid nitrogen includes all nitrogenous compounds not precipitated by calcium hydroxide or not volatile in its presence in vacuo, not precipitated by phosphotungstic acid in the concentrations used, and containing a free amino group which will react with nitrous acid to produce free nitrogen. The greatest inaccuracies occur in the diamino acid fraction, and these are distributed between arginine, histidine, and lysine nitrogen. This group includes all nitrogenous compounds which are precipitated by phosphotungstic acid, excepting ammonia and melanin nitrogen. all nitrogenous compounds not accounted for in the above, The nitrogen reported as non-amino nitrogen includes and may include small amounts of nitrogen present in the soil in the form of nitrates. The results obtained by the methods outlined are presented in Tables V. and VI. The amount of dried blood added to the Norfolk fine sandy loam is far in excess of the amount ever used in good agricultural practice. However, this amount was found to be necessary in order to obtain accurate analytical results, and to assure d fferences of a large enough order between the various samples of soil analysed; furthermore, it seemed desirable to add enough dried blood protein to the soil to render the small amount of soil proteins negligible, so that only the fertiliser nitrogen would be under observation. By reference to Table III., in which the results of the analysis by the Van Slyke method as applied to the mixture of blood and soil are reported, it will be observed that the figures obtained for the various forms of nitrogen correspond very closely to those obtained from the dried blood alone, except the figures for melanin and non-amino nitrogen, but the reason for this is not altogether clear. Ammonia Formation. Assuming that ammonification of protein materia! in soil must precede nitrification and denitrification and that CHEMICAL NEW Organic Nitrogen Compounds of Soils and Fertilisers. 231 TABLE VI.-Forms of Nitrogen in the Soil at the End of each Period. Hydrolysable Nitrogen at the End of each Sampling Period = 100. Time in days from the beginning of the experiment. all loss of nitrogen in this investigation is due to ammonia | TABLE V.-The Forms of Nitrogen in the Soil at the End evaporation, nitrification, or denitrification, and that free of each Period. Hydrolysable Nitrogen in the Original nitrogen is not split off from compounds other than nitrates Soil = 100. or nitrites, then it is possible to arrive at the amount of ammonia formation in the soil during each period of time. It should be stated that this is ammonia formation exclusive of ammonia assimilation, there being no way in which ammonia assimilation could be accurately determined in these experiments. This ammonia formation may be calculated by the following equations : Total N-NH3 nitrogen in the original soil = A. Total N-NH3 nitrogen at the end of each period = B. Then A-B = X, or ammonia formation during the period. X/A per cent of nitrogen changed to ammonia during the period. In Table IV. are presented the results obtained by the use of these equations. TABLE IV. Per Cent of Total Nitrogen in the Soil Ammonified at the End of each Period of Sampling. Time in days from the beginning of the experiment. 18 44 86 148 240 Per cent of total nitrogen. 18.72 54'03 72.66 78.13 78.92 The Results obtained by the Van Slyke Method. By comparing the results obtained by the Van Slyke analysis of each soil sample during the experiment with the results obtained on the original soil the amounts of gain or loss in the eight different forms of nitrogen can be arrived at. It is thus possible to determine how rapidly any par ticular form of nitrogen compound disappeared from the soil during the course of the decomposition, and, further, to determine the relative amounts of nitrogen in these fractions in respect to the total amount of nitrogen present in the soil at the end of any period. When an increase in any particular form of nitrogen over the amount present in the soil during the previous period is observed, it is not possible in all cases to state the compound in which this nitrogen existed. But when a certain form of nitrogen shows a loss during a period it is an absolute indication that that particular kind of nitrogen was disappearing or had disappeared from the soil, although the rate could not be determined. The results obtained by this analysis are presented in Table V., in which the amounts of nitrogen in the various fractions are reported in percentages of the hydrolysable nitrogen of the original soil. The figures presented in Table VI. represent the relative amounts of the various forms of nitrogen in percentages of the hydrolysable nitrogen of the soil present at the end of each period. From this table the fluctuating composition of the hydrolysable nitrogen of the soil may be followed, and the final composition of the hydrolysable nitrogenous matter of the soil may be established. Form of nitrogen. Amide Original soil. 86. 148. 240. 7:008 7'515 6 025 5'429 3'454 3°222 4'767 5080 4'374 2.276 1.391 1.698 7.601 5162 3.041 1857 1342 1*395 12.366 12.975 5'547 2912 2.382 2.010 10.093 7.610 1'110 0'429 0'528 0.972 58.220 40 493 18.612 8.970 7.938 7.187 0.312 1120 1675 2:191 0.738 0.297 100'000 79'660 40'598 24'070 17.740 16'741 of loss of nitrogen in each form from the soil at the end of The figures presented in Table VII. show the amounts each sampling period. The amount of loss is stated in percentages of the largest amount of any form of nitrogen in the soil at any time; for example, in the case of the amide nitrogen the amount is largest at the end of eighteen days, and this figure is taken as 100. In this table the word " indicates an increase in the amount of gain nitrogen over that present at the end of the preceding period. loss from each would be about the same. The fact that there is a difference of about 11 per cent between the losses from these fractions leads to the supposition that nitrogen split off of the monoamino acids has been assimilated by the micro-organisms in the formation of their protoplasm. From Table VI. it will be observed that the proportion of the monoamino acids present in the soil at the various times of sampling fluctuates. The lowest figure is 37 per cent at the end of eighty-six days. Lysine Nitrogen. The analytical results show that lysine disappears from the soil quite rapidly. At the end of forty-four days 89 per cent of the lysine originally present in the proteins has been decomposed, and at the end of eighty-six days 96 per cent. During the remaining and longer part of the decomposition period there is a continual gain in lysine nitrogen, indicating that synthetic processes are at work. The gain in lysine nitrogen, after the original had practically vanished from the soil, is to be attributed to the action of the micro-organisms in synthesising some compound or compounds which give the analytical reactions for lysine. That this increase is due entirely to lysine cannot be stated, but lysine no doubt makes up a part of the gain observed. It will be noted from Table VII. that the two fractions which show the greatest amount of loss during the experiment are lysine nitrogen and monoamino acid nitrogen. It is not surprising that these two show the greatest loss when their chemical composition is considered. The monoamino acids are straight chain acids with the amino group in the a-position to the carboxyl group. Lysine, a diamino acid, is also a straight chain acid containing two amino groups, one in the a position to the carboxyl group, and one at the extreme end of the chain from the carboxyl group, or in the w-position. The relationship between lysine and the amino acids may be clearly shown by presenting the structural formulæ for lysine and for leucine; for example: NH2 NH2-CH2-CH2-CH2-CH2.CH COOH However, it is observed that the lysine vanishes more quickly from the soil than the monoamino acids. This may be due to the fact that the w-amino group of the lysine exists free in the molecule of the native proteins which occur in the dried blood. Under such conditions this group is subject to deaminisation by the action of the micro-organisms before hydrolysis takes place, while in the case of the monoamino acids hydrolysis must precede deaminisation, since these acids are linked in the protein molecule in anhydride structure. Furthermore, if the w group be split off from the lysine while it is still a constituent part of the protein molecule it is changed into an amino acid with but one amino group, and would be determined analytically as monoamino acid nitrogen. From Table VI. it will be observed that there are very marked fluctuations in the proportions of lysine nitrogen in the soil at the end of each period. The lowest amount occurs in the soil at the end of eighty-six days, which was the low point for monoamino acids. The final amount is about half that originally present in the dried blood. Histidine Nitrogen. At the end of eighteen days the histidine nitrogen showed a gain. Although the compounds which cause this increase cannot be arrived at it is possible that they are, in part at least, the purine and pyrimidine bases, which by the analytical methods would be classed as histidine nitrogen. It is well known that the protoplasm of micro-organisms is made up of considerable amounts of nucleoproteins and nucleic acid, which on hydrolysis would yield the purines and pyrimidines. At the end of forty-four days 60 per cent of the histidine nitrogen had disappeared, at the end of eighty-six days 80 per cent, and after 240 days 83 per cent. The proportion of the histidine nitrogen in the soil at the various times of sampling is about constant, with the exception of the eighteen-day sample. Arginine Nitrogen. After eighteen days 31 per cent of the arginine had vanished from the soil, while at the end of 148 days 83 per cent had gone. From the 148th to the 240th day, a period of ninety-two days, a gain in arginine nitrogen is observed. This may be due to nitrogen in the form of arginine, or nitrogen in the form of compounds which give the analytical reactions for arginine. It is nitrogen formed by the action of micro-organisms, probably a synthesis of protoplasm, and possibly in the form of proteins. The relative amount of arginine nitrogen shows little fluctuation throughout the experiment, and is a little greater at the end of the experiment. Amide Nitrogen. The analysis of the figures for amide nitrogen brings out some interesting points. After eighteen days there is an increase in amide nitrogen. It may be safely assumed that the compounds which this increase represents are acid amides formed by the action of the micro-organisms, existing in the soil either free or combined in the molecule of some new proteins contained in the protoplasm of the organisms. That there was actually an increase in this form of nitrogen after eighteen days was, however, unexpected, since it is well known that micro-organisms when grown in solutions of acid amides can use them for the building up of their protoplasm, and, furthermore, Jodidi (1912) has shown that acid amides are very easily and quickly ammonified when placed in an agricultural soil. It was therefore expected before the results were obtained that the amide nitrogen would be among the forms which would most quickly disappear from the soil. From Tables V. and VII. it will be observed that this fraction disappears least completely and most slowly." The question arose as to whether the soil used was capable of ammonifying acid amides. Consequently, I grm. of pure asparagine, one of the two acid amides considered to be present in the protein molecule, was added to 100 grms. of air-dried Norfolk fine sandy loam to which no dried blood had been added. The soil was made to about a 10 per cent moisture content, and allowed to stand for four days. On analysis for ammonia it was found that the soil had converted 734 mgrms. of asparagine nitrogen into ammonia nitrogen in this time, or, in other words, the soil in four days had ammonified 39.3 per cent of the total asparagine nitrogen. This shows that if acid amides were free in the soil they would have been to a very large extent converted into ammonia during the eighteen days of the experiment, and points unquestionably to the fact that the increase in this form of nitrogen is due to the synthetic action of the micro-organisms in the building up of their own protoplasm. Society of Chemical Industry.-The Committee and Members of the Chemical Industry Club invite all those members of the London Section who have not already joined the Club to attend its first meeting, which will be held on Monday, May 21, at 8 p.m., at Lyons's Birkbeck Café, Higa Holborn. An interesting programme has been arranged, and, in addition to other items, Mr. M. C. Lamb will read a paper on "The Commercial Utilisation of Discarded Army Boots and other Waste Leather." Mr. Lamb has undertaken the investigation of this matter at the request of the War Office. He will include in the paper the suggestions of other investigators. The object of this publication, which is being made at the express wish of the War Office, is to incite, if possible, discussion and the formulation of further suggestions for the utilisation of this waste product on a commercially profitable scale. CHEMICAL NEWS, May 18, 1917 International Movement of Fertilisers, &c. THE INTERNATIONAL MOVEMENT OF FERTILISERS AND CHEMICAL PRODUCTS USEFUL TO AGRICULTURE.* THE unsatisfactory results afforded by the last cereal crops throughout the greater part of both hemispheres have induced Governments and other persons interested to seek for the means of obtaining better yields in the coming season, in order to minimise the risk of another deficient harvest. The judicious use of fertilisers is among the principal of these expedients; investigations as to their production and disposal therefore take rank as questions of the greatest usefulness. The review now under consideration, which appeared in the March, 1917, number of the Bulletin of Agricultural and Commercial Statistics of the International Institute of Agriculture of Rome, deals fully with this subject. It consists of more than seventy pages, and includes a considerable number of statistics, some official, some from other trustworthy sources. Phosphatic, potash, and nitrogenous fertilisers are dealt with, as well as the principal chemical products useful to agriculture. We proceed to the résumé or reproduction of the most important items of information. I. THE WORLD'S PRODUCTION. It is noteworthy that the American production for 1915 indicates a decided decline. While in 1913 the United States extraction of phosphates was 3,161,146 tons, the quantity made available in 1914 was 2,777,917 tons, both of these being abont normal, but in 1915 the total fell away to 1,865,123 tons. This decline indicates very clearly the difficulties encountered in this industry ever since the outbreak of war. A considerable proportion of the hands previously employed in the phosphate industry took up other descrip: tions of work which have been increasingly in demand for the past two years, with remunerative pay. On the other hand, the European demand has fallen off very decidedly as compared with the requirements in 1913 by more than a million tons. Shipment for central Europe, where the consumption was very large, have almost entirely ceased, and for other parts of this continent the traffic has not asserted itself very vigorously. Business has been interfered with by scarcity of steamers and advanced rates of freight. Lastly, in regard to markets within the country itself, the large demand for sulphuric acid for war purposes has also been a factor tending to diminish the output of phosphates intended for the United States. The price of the acid has advanced so seriously that the manufacture of superphosphates in that country has fallen off to a marked extent. Turning to the data of American shipments, we find that those of Florida hard rock were reduced in 1916 to such small quantities that they scarcely merit attention. It seemed probable in 1915 that the shipments then going forward would be the minimum for this trade, but the continued decline during 1916 indicates that the trade in this description of phosphate has become quite unimportant. The chief reasons for this decrease in the traffic arise from the war. In 1916, less than 33,000 tons were placed on board ship, and of this 4600 tons were for destinations in the United States. The decrease in the trade of land pebble phosphate was not quite so marked, though equally noticeable. In 1916 and in 1915, shipments to countries other than the United States were practically identical in quantity, but they both show a deficiency of nearly 700,000 tons as compared with the shipments of this description of phosphates effected prior to the war. • International Institute of Agriculture, Rome, April 30, 1917. 233 In North Africa, although excepting for Tunis we have no data as to production of phosphates in 1916, we can assume that a considerable increase is shown. While in 1915 exports were no more than 226,000 tons, they reached in 1916 a total of 380,000 tons, somewhat larger than those of 1914 with 355,000. In Egypt, on the other hand, the exports of 1916 show a deficiency of over one-third as compared with those of 1915 (21,000 tons against 33,000), and these latter did not reach one half of the exports in 1914 (87,000 tons). For Tunis, also, though in a less degree, there has been some decline in exports in 1916, almost entirely due to the returns of the second half of the year. Production, on the other hand, was greater in 1916 than 1915. The Government of Tunis informs us that there are considerable reserve stocks. It would appear that, among the deposits of the islands in the Pacific there has been considerable renewed activity. We hear from private sources in Japan that, since the beginning of 1916, imports of phosphates to the extent of 80,000 tons have taken place from Rasa, 70,000 from Ocean Island, and 30,000 from Argaur, making a total of 180,000 tons. We embody in Table I. the principal figures recently to hand and published in the Review of the International Institute of Agriculture of Rome. TABLE I.-Natural Phosphates. During the second half year of 1916, the production showed a slight reduction as compared with the first half of the year, in opposition to the usual course prevailing in peace times. This reduction did not, however, prevent the output of the second half of the year from exceeding those of previous corresponding periods. From July to December, 1916, the production was 1,425,751 tons, while in 1913, when the second half of the year provided the largest figures on record, the output was 1,393,610 tons. For the corresponding periods in war time the figures were respectively 1,175,763 tons in 1915, and 984,550 tons in 1914. Stocks on the Chilean coast did not reach more than 718,315 tons at the close of 1916, although the production during that year had distanced all previous figures, while at the close of 1915 these stocks were 789,700 tons, and at the end of 1914 amounted to 1,087,910 tons. It should also be borne in mind that on June 30, 1916, the stocks were 888,621 tons, and that this total was the largest on record for that period of the year. The quantity delivered for consumption during the last half of 1916 was also a maximum, and, in fact, in spite of scarcity of tonnage and continually advancing rates of freight, shipments amounted |