Dec.,
1915
T H E J O U R N A L O F I N D U S T R I A L A N D E N G I N E E R I ATG C H E M I S T R Y
I049
necessary precautions, b u t this leads t o a great deal of inconvenience. S T A N D A R D H Y D R O C H L O R I C ACID s o ~ u r ~ o x - - - A 0.4 N 111-A new form of tower, described above, makes solution is employed. possible t h e use of Ba(0H)S as a n absorbing medium for B A R I U M HYDROXIDE SOLUTION-A 0.4 N solution is COSunder nearly all conditions. I t also eliminates pracprepared as follows: Dissolve 80 g. of B a ( O H ) S . 8 H 2 0 tically all contamination of COSfrom t h e air, a n d greatly in 1000 cc. of water, heat t o boiling a n d t h e n filter with facilitates t h e washing. The titration has t h e ada Buchner funnel into a stock bottle which has been vantage of a very good end point. The standard freed of C O S . A double filter paper is carefully laid solution of Ba(OH)2 is easily prepared free of carbonon t h e funnel a n d drawn down a n d then t h e funnel is ates. never allowed t o suck dry till t h e solution has all gone DEPARTMENT OF SOILS, WISCOXSIN EXPERIXENT STATION through. I n this way a solution free of carbonates is r K I V E R S I T Y OF WISCOWSIN.MADISON much more quickly a n d easily prepared t h a n b y allowing t h e precipitate t o settle a n d siphoning over. T h e THE AMINO ACID NITROGEN OF SOIL burette a n d appropriate guards are t h e n attached a n d B y R. S. POTTERA N D R. S. SNYDER Received June 24, 1915 t h e proper dilution is made after titrating against t h e standard acid. INTRODUCTION PHENOLPHTHALEIN s o L u T I o N - O n e gram of t h e inT h e importance of t h e amino acids as the chief dicator is dissolved in I O O cc. of ethyl alcohol. products of t h e chemical, a n d under some conditions, of t h e biological decomposition of protein material, ACCURACY O F METHOD makes desirable a quantitative knowledge regarding I n order t o test t h e efficiency of t h e apparatus and their occurrence in t h e soil. While there is, no doubt, technique in preventing t h e contamination of t h e present in t h e soil representatives of all t h e classes B a ( O H ) 2 b y t h e COS of t h e air, measured quantities of compounds between undecomposed protein a n d of t h e B a ( O H ) 2were placed in t h e tower in t h e regular ammonia a n d nitrates, yet there are no methods way, and after aspirating C02-free air through t h e which have been applied t o t h e quantitative determinaapparatus for some time, t h e solution was removed in tion of any of these classes of compounds except amt h e regular way a n d titrated. T h e titrations disclosed monia, nitrates, a n d nitrites, which together make no measurable quantities of COS, a n d hence the con- up but a small proportion of t h e total nitrogen comtamination is practically entirely prevented. I n order pounds. It would seem, therefore, t h a t a more comt o test t h e efficiency of t h e absorption with this tower, plete knowledge of the nitrogen of t h e soil is very measured quantities of t h e Ba(OH)z solution were important from a n academic a n d a practical standtreated with COS a n d after boiling t o expel t h e excess point. I n this investigation, our attention has been of C 0 2 , t h e precipitate of B a C 0 3 was transferred t o confined t o t h e amino acid nitrogen. Many investia n evolution flask a n d t h e evolved COS drawn through gators have found amino acids in soil after subjecting t h e absorption tower a n d determined b y titration. i t t o hydrolysis, b u t there has been no quantitative The results never varied from t h e calculated by more determination of t h e amino acids as such. I n fact, t h a n a drop of 0.4 N acid, which represents less t h a n t h e only investigations showing t h e presence of amino 0.5 mg. of CO2. The Ba(OH)* in t h e tower will acids in t h e soil were made by Shreiner and Shorey, absorb efficiently with rapid aspiration t o nearly one- who found histidine, arginine,' a n d lysine,2 in a weak half saturation, which represents about 0.1j g. of COS. alkali extract of soil.a With slow aspiration still more may be safely absorbed. Except for t h e work of Chardet, which has not been The tower is conveniently attached t o almost any form confirmed, t h e only proof there is of t h e presence of of apparatus in which COz is evolved for measurement. amino acids in t h e soil is the work of Schreiner a n d For more accurate determinations of small amounts Shorey. Until quite recently, there has been no of COS, more dilute solutions of B a ( O H ) 2 a n d HC1 method sufficiently sensitive t o permit of t h e accurate should be used. determination of the amino acids in t h e soil, but t h e 5 U M MARY copper method of K ~ b e r being ,~ capable of detecting, I-The gravimetric determination of COS by weigh- with considerable accuracy, one part of amino acid ing of absorption apparatus gives accurate results in nitrogen in soo,ooo parts of solution, has been found t h e case of small amounts of COS, slow aspiration, and very suitable for soil work. By this method j t o due precautions in weighing a n d drying. I n t h e case 30 parts of amino acid nitrogen per million parts of of rapid aspirations carrying considerable amounts of soil have been found, so i t can easily be seen how t h e earlier methods would be entirely unsuited. The COS t h e gravimetric method is troublesome,. formol titration method of Soerensenj has been shown 11-The determination of COS by absorption in J . B i d . Chem., 8 (1910), 382. alkali hydroxide a n d double titration has many dis2 U. S. Dept. Agric., Bur. of Soils, Bull. 88 (1913). advantages. T h e amount of indicator used needs t o 3 Since t h e experimental p a r t of this paper was completed, Chardet (Rev. g i n . chem., 17, 137) reports t h e analysis of 4 soils for amino acid be and standard end points nitrogen by t h e Soerensen method. He found from 49 t o 68 per cent of be used. Under t h e best conditions, t h e end points t h e total nitrogen was amino acid nitrogen. Following his directions, are not sharp, making it absolutely unusable by Some as given, we have been unable t o find a n appreciable amount of amino acid people. Good results can be obtained in t h e case of people having a good eye for color by observing t h e 5 Z. physiol. Chem., 64 (1910). 120. REAGEISTS
1
~~,y,o,lns.e,v,~~~~i~~~~~~~~~~
I O jo
T H E J O U R N A L OF I N D U S T R I A L A N D ENGINEERING C H E M I S T R Y
b y Loebl t o be accurate t o only one p a r t in 1600, while t h e Van Slyke2 method has been shown b y its originator t o be accurate t o one p a r t in 10,000,a n d besides is specific for t h e amino group, not for amino acids. T h e reader is referred t o t h e papers b y Kober3 a n d collaborators for t h e details of t h e copper method for amino acid nitrogen. I n this report t h e t e r m “amino acid nitrogen” is t h e nitrogen in t h e amino group in t h e a a n d p position .with reference t o a n unsubstituted carboxyl group; “free amino acid nitrogen” is t h e a a n d /3 amino nitrogen in “free” amino acids; “peptide nitrogen” is t h e CY a n d /3 amino nitrogen of t h e amino acids from t h e hydrolyzed peptide. E X P E RI hl E S T A L
,
T h e technique of t h e Kober method as recommended b y its originator was followed, with some modifications, in all its essentials in this work. T h e cupric hydroxide was prepared in a n ice mixture a n d centrifuged instead of filtered as Kober recommends. T h e buffer solution was titrated from time t o time, a n d it was found t o hold its original strength. T h e thiosulfate solutions were standardized from time t o time against s t a n d a r d iodine solutions. S. a n d S. No. 589, Blue Ribbon filter paper was used throughout, instead of S. a n d S. N o . j90, as recommended b y Kober. T h e No. 590 paper, although more rapid, often caused trouble. Any other deviations from t h e original method are cited in t h e proper place. It is well known t h a t dilute acids in t h e cold dissolve only a small p a r t of t h e soil nitrogen, yet i t is known t h a t I per cent HC1 dissolves some organic n i t r ~ g e n , hence ~ i t was thought possible t h a t a n y amino acids present would be dissolved. One hundred a n d fifty g. of a soil which contained 0.181per cent nitrogen were shaken with 300 cc. of 0 . 2 N hydrochloric acid for one hour. T h e mixture was poured u p o n a paper filter, a n d 80 cc. of t h e clear filtrate were transferred t o a I O O cc. flask: 7 cc. of a saturated solution of lead acetate a n d 7 cc. of ammonia (sp. gr. 0 . 9 0 ) were added a n d water t o complete t h e volume t o I O O cc. T h e mixture was thoroughly shaken a n d t h e n filtered. T o 7 5 cc. of t h e filtrate z j cc. of a s a t u r a t e d solution of barium hydroxide were added, a n d t h e ammonia removed by,boiling a t a pressure of a b o u t 2 5 mm.5 T h e liquid was t h e n made u p t o 7 5 cc.; t h e barium carbonate filtered off and jc cc. of t h e filtrate were transferred t o a I O O cc. flask. T h e solution was t h e n made barely alkaline t o phenolphthalein, 40 cc. of “buffer” solution added, a n d after cooling for a b o u t t w o hours or more in t h e ice box, one cc. of a cold suspension of cupric hydroxide in water was pipetted in, t h e whoIe vigorously shaken, water added t o t h e mark a n d t h e mixture filtered 1 2
3
Ber. d . chem. G e s , 46 (1913). 696. J . B i d . Chem., 7 (1910). LOC.cii., also J . Biol. Chem.. 13 (1912). 1; A m . Chem. J . , 48 (1912),
383. Kelley and Thompson, J . A m . Chem. SOL.,36 (1914). 438. After this procedure, Kober found i t necessary to remove lead either with potassium sulfide or sulfate. In our experience, the lead is entirely precipitated by the ammonia. This is in harmony with the statement found in text books that lead hydroxide is insoluble in excess of ammonia. 6
.
Vol. 7, NO. 1 2
through S. a n d S. No. j 8 9 Blue Ribbon filter paper: jo cc. of the filtrate showed no trace of copper. Other soils treated in t h e same way gave t h e same result, Upon adding a. small amount of alanine t o some soils a n d t h e n extracting with 0 . 2 ;V hydrochloric acid in t h e same way, no amino acid nitrogen was found. This result is somewhat in harmony with t h e fact t h a t only from 6 0 t o 70 per cent of small amounts of ammonia added t o t h e soil are extracted with 0.2 i V acid.’ Dilute alkali is known t o dissolve more organic matter from t h e soil t h a n a n y other of t h e milder reagents, therefore 2 per cent sodium hydroxide was next tried as t h e extraction agent a n d was found t o be quite satisfactory. Much t h e same procedure was used here as for t h e acid extraction, except t h a t 600 CC. of t h e alkali were used with I jo g. soil. It is almost impossible t o filter this extract b y a n y of t h e ordinary methods, so i t was cleared b y centrifuging in t h e machine described in a bulletin from this Station.* After whirling in this machine a few minutes, t h e clear b u t dark-colored solution was drawn off through t h e silver t u b e : 80 cc. of this solution were approximately neutralized with I : I HC1 a n d t h e n t h e same procedure carried o u t t h a t was used in t h e acid ext r a c t u p through t h e filtering off of t h e excess copper hydroxide. Although with all t h e soils tested copper was always found in this filtrate, yet there was present some reducing substance which took u p t h e iodine liberated b y adding potassium iodide. Hence, instead of titrating directly, j o cc. were placed in a beaker, acidified slightly with nitric acid, heated t o boiling a n d bromine water added until a permanent color was given. The solution was t h e n boiled down t o about I O O cc. a n d t o make sure t h e last traces of free bromine were given off 3-40 cc. water added a n d again boiled down t o I O cc. T h e solution was cooled, neutralized with sodium carbonate, a very slight excess of I per cent hydrochloric acid added a n d after t h e addition of t h e potassium iodide a n d starch solution, t h e free iodine was titrated with 0.001N sodium thiosulfate solution. E a c h cc. of this solution equals 0.0280 mg. of amino acid nitrogen, or 0.0140 mg. peptide amino nitrogen. For t h e determination of t h e free amino acid nitrogen, 40 cc. of t h e solution were used a n d Kober’s directions were exactly followed. As a check upon our work, analyses were r u n on some pure glycine a n d alanine solutions, a n d also known amounts of these substances were added t o soil a n d t h e increase in amino acid nitrogen found. For t h e analysis of t h e pure amino acid solutions, t h e indicated amounts, obtained by aliquoting strong solutions, were placed in I O O cc. flasks, made u p t o a b o u t 80 cc. with sufficient sodium hydroxide t o give a 2 per cent solution of t h e alkali a n d from t h e n on r u n exactly as was t h e soil. The amino acids were added t o t h e soil b y aliquoting t h e same solutions, a n d t h e n sufficient sodium hydroxide solution Potter and Snyder, THIS JOURNAL, 7 (1915), 2 2 1 . Stevenson, Wells and Coover, Iowu BulZ., 134 (1911). A glass tube was originally recommended for drawing off the solution. We are indebted to Prof. R . E. Smith for the suggestion of the use of a silver tube which has been found much more satisfactory. 1 2
Dec., 191j
T H E JO17RAFTALO F I N D U S T R I A L A LYD E N G I N E E RI ,VG C H E M I S T R Y
1051
incubated at room temperature for from six t o t e n days with soils containing 18 per cent moisture: in a m o u n t s varying from 0.1t o 0 . j g. amino acid per 5 0 g. soil increased t h e a m o u n t of ammonia nitrogen in t h e soil b y from 44 t o 74 per cent of t h e a m o u n t of amino acid nitrogen added. T h e amino acids tyrostine a n d phenylalanine, containing carbocyclic TABLEI--AMIGO h71TROGES A D D E D , F O U N D A S D R B C O V E R E D ADDED Amino PUT found Amino N recovered groups, under similar conditions give rise t o approxiSUBSTAKCE hlg. XIg. dv. R hlg. Av. 70 mately 2; per cent less ammonia. . As pointed out b y Glycine . . . . . . . . . . 0 . 2 2 4 0.203 . . . . . . . . . 0.224 0.210 92.4 . . . . . . . . . . . . . . Jodidi, t h e actual production of ammonia might have Soil A , , . . . . . . . . . . . . . . . 0.168 . . . . . . . . . . . . . . . . . . . . . . . been really greater, because p a r t of t h e ammonia . . . . 0 . l i 4 0.171 . . . . . . . . . . . . . . . . . . Soil X and glycine 0.224 0.370 . . . . . . . . . 0.199 . . . . . produced might have been nitrified. N o doubt, , 0.224 0 . 3 6 4 0.367 .... 0.193 0 . 1 9 6 8f:S Alanine.. . . . . . . . . 0.224 0.208 . . . . . . . . . . . . . . . . . . also, p a r t of the nitrogen was assimilated b y bacteria. 0.224 0 . 2 1 6 0 . 2 1 2 9416 . . . . . . . . . . . . . . T h e a m o u n t of amino acid added t o t h e soil b y Jodidi Soil A a n d alanine 0.224 0.3i5 . . . . . . . . . 0,204 . . . . . 0.224 0.380 0 . 3 i X , , , , 0 . 2 0 9 0 . 2 0 7 Si,'4 mas far in excess of t h a t actually present in a n y soil F r o m t h e results set forth in Table I i t is seen which we have examined, yet since he found t h e pert h a t b y t h e analysis of t h e pure glycine 92.4 per centage transformations were about t h e same whether cent of t h e theoretical q u a n t i t y was found, while he added 0 . 1 g. or 0.j g. of amino acid, no doubt t h e in t h e case of t h e alanine 94.6 per cent was found. r a t e of transformation of still smaller amounts would Since t h e samples used were Kahlbaum's C . P. material, be similar. I t is a well-known fact t h a t t h e action of bacteria a n d since practically t h e theoretical amount of nitrogen was found in each case b y t h e Kjeldahl analysis, a n d boiling acids upon proteins gives rise t o about t h e material is, no d o u b t , very close t o 100 per cent t h e s a m e products, namely, proteoses, peptones, peppure. K h e n it is considered what small a m o u n t s tides a n d amino acids, b u t as pointed out above, t h e were dealt with in each case, t h e low percentages action of bacteria does not stop with t h e amino acids. obtained are not surprising. In solutions of glycine Another important factor t o be t a k e n into account containing approximately t h e same amounts, Kober when considering t h e transformation of soil nitrogen found in one case 94.8 per cent a n d in another 9j.7 is t h e assimilation of t h e nitrogen b y bacteria. While per cent of t h e theoretical q u a n t i t y . T h e recovery d a t a are lacking on t h e chemical constitution of bacof t h e 87. j per cent of t h e added glycine a n d 92.4per teria, usually present in soil, yet their composition is cent of t h e added alanine, when t h e many operations probably not far different from t h a t of some of t h e through which t h e soil solutions must pass are con- pathogenic organisms. I t has been shown b y Cramer,' for instance. t h a t t h e spirillum cholerae has a protein sidered, seems, on t h e whole, not unsatisfactory. In order t o determine if t h e a m o u n t of amino acid content varying f r o m 4 5 t o 6j per cent according nitrogen found was influenced b y t h e duration of t h e t o whether i t was grown on a media poor or rich in t r e a t m e n t with t h e alkali, samples of t w o soils were protein material. Therefore, in soil there are t w o opshaken Eor I , 2 , 4 a n d 6 hours with 2 per cent sodium posing tendencies, namely, t h e degradation of t h e comhydroxide a n d t h e amino acid nitrogen was determined plex nitrogenous material into its constituent p a r t s in t h e extract from each t r e a t m e n t . Within t h e ex- a n d subsequent ammonification a n d nitrification of perimental error t h e results for the four periods for these p a r t s , a n d t h e n t h e assimilation of t h e degradaeach soil were t h e same. This was expected from t h e tion products b y t h e microflora of t h e soil t o give known stability with alkali of t h e amino acids a n d again material more or less complex t h a n originally. protein material as far as amino acid production is Of course, there are other changes, such as assimilation of t h e nitrogen from t h e atmosphere a n d denitrificaconcerned. I n Table I 1 are given t h e results for t h e various tion, b u t these a n d other changes need not be entered kinds of nitrogen for t w o soils. T h e differences are into here. It is a well-known fact ' t h a t t h e introduction of notable a n d t h e relations for other soils will be studied large a m o u n t s of fresh organic m a t t e r in soils causes in t h e f u t u r e in this laboratory. a great depression or t h e complete disappearance of TABLE11-NITROGENI S P. P. 11. A I R - D R Y SOIL (DUPLICATES) nitrates therefrom, a n d also prevents a n y appreciable SOIL Amino acid Free amino acid Total peptide N A . . . . . . . . 8.1 7.8 5.6. 5.3 32.6 30.4 formation a n d accumulation of nitrate nitrogen. 15.0 14.4 30.0 31.8 Various explanations have been offered t o account T h e consistency with which t h e a m o u n t of amino for this, such as denitrification, assimilation, or t h e acid nitrogen is so small in t h e soils reported above depression of nitrification because of accumulated points t o t h e fact t h a t , as with ammonia, there is not intermediate products of degradation. Whether there much tendency for i t t o accumulate in soils. This is a continuous production of nitrates which are is, perhaps, t o be expected from t h e fact t h a t amino subsequently assimilated, or whether t h e organisms acids are known t o be very good nutritive media for get their nitrogen from t h e ammonia or amino acids2 bacteria.' I t has also been shown b y Jodidi2 t h a t or other compounds cannot be definitely stated, b u t those amino acids made u p of a1iphati.c nuclei, when 1 Archia. f. Hygiene, 12, 1 5 i ; 13, 76; 16, 171. t o give 600 cc. of z per cent alkali, a n d after shaking t w o hours a n d centrifuging, 80 cc. of t h e extract were t a k e n a n d t h e regular procedure carried o u t . T h e results are given in Table I a n d are calculated i n every case t o t h e a m o u n t of amino acid nitrogen in t h e 80 cc. of solution or extract.
Czapek, Hofmeisleu's Beit., 1 (1902). 538; 2 (1902). 5 5 7 ; 3 (1902). 4 7 ; Emmerling, B e y . d . chem. Ges., 35 (1902), 2289. * I o w a Reseavch Bull., 9 (1912).
It h a s been shown b y Bierima (Cenlr. Bakt. Parasitenk., I I Abl., 23 (1909). 672) t h a t amino acids, as well as ammonia are assimilated b y soil bacteria.
1052
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T H E J O U R N A L OF I N D U S T R I A L A N D ENGINEERING C H E M I S T R Y
in well aerated soils not too wet, one would certainly expect nitrification t o be taking place, a n d for t h e same reasons, none or only a negligible amount of denitrification. T h e use of t h e t e r m “acid soluble” b y Jodidi’ in t h e following statement has led t o some confusion on this question:. “ T h e principal portion of t h e acid-soluble organic nitrogen contained in t h e soil herein investigated is made u p of acid amides, monamino acids a n d diamino acids.’’ T h e Kober method for amino acids, as we have applied it t o soils, will give definite information as t o t h e amounts of amino acids actually present in ,313 az
gII
4 9 .
k ¶
4
g5 n
G3 SAMPLING
soil, a n d will, therefore, show whether under various conditions there is a n y tendency for these products t o accumulate in soil. Interest in t h e question is also raised b y t h e accumulating evidence t h a t green plants may utilize amino acids2 ,for their nitrogen nutrition. While it is not thought t h a t green plants would utilize a n appreciable amount of amino acid in t h e presence of nitrates a n d ammonia, yet it is possible t h a t in soils temporarily deprived of nitrates, t h e amino acids, as well a s t h e ammonia, might be used b y t h e plant. It was t o s t u d y t h e .problems suggested b y t h e above discussion t h a t t h e following experiment was performed: Several hundred pounds of Soil A, which was acid t o litmus a n d showed a lime requirement of 3800 lbs. of calcium carbonate per 2 , 0 0 0 , 0 0 0 lbs. of soil b y t h e Veitch3 method a n d which contained 0.151 per cent of nitrogen, were procured. The sample was air-dried, shaken through a four mm. sieve and very thoroughly mixed. Twenty pot experiments, each pot containing 30 Ibs. of air-dry soil, were r u n with this soil with t h e treatments indicated below. TABLE111-TREATMENTIN TONSPER 2,000,000 LBS. SOIL Experiment N o . . . . . . . . . . . 1 2 3 4 5 6 7 8 9 1 0
{i
TREATMENT: CaC08 ...................... 0 Stable manure.. . . . . . . . . . . . . . 0
3 4
5
.4
0 10
0
6
7
8
9 10
11 12
0 0 20 30 50 0
13 15 1416
17 19 1820
4
4 4 30 50
4 10
20
The manure used was collected from t h e stalls of horses a n d was made u p of about equal parts of t h e droppings a n d of t h e wet straw. I t was cut u p , mixed while still moist a n d added t o t h e soil in t h a t condition. T h e object of adding t h e moist manure was t o avoid too great a modification of t h e bacterial flora. Because of t h e condition of t h e manure, it was impossjble t o obtain a perfectly uniform mixture, or t o mix it with t h e soil a s well as was desirable. Manure gathered as above was analyzed for total amino acid nitrogen, ammonia nitrogen a n d total Iowa Research Bull., 3 (1911). 121. Molliard. Bull. SOC.botan. Fvance, 57 (1910), 541-547; Hutchinson and Miller, J . A g y . Sci., 4 , 282. J . A m . Chem. SOL.,24 (1902),1120. 1
2
12
nitrogen. For t h e amino acid nitrogen IOO grams were stirred with 400 cc. of I per cent sodium hydroxide for a few minutes, t h e coarser particles were strained out a n d t h e liquid was t h e n filtered through a folded, d r y filter. Aliquots were t a k e n for duplicate determinations a n d t h e solution was t h e n carried through precisely t h e same procedure as t h e soil extract. For t h e ammonia determination, jo g. of t h e manure were placed in 500 cc. Kjeldahl flasks, 2 0 0 cc. of water a n d about 5 grams of sodium carbonate added a n d t h e ammonia determined b y aeration. Aeration was continued until practically no more ammonia was being given off. T h e t o t a l nitrogen waS determined in a-gram samples obtained by drying a n d grinding a larger sample. T h e results are given in Table IV. TABLEIV-NITROGEN IN P. P. M. WET MANURE AMINOA C I D . ......... . 31.0 24.7 32.6 Average 29.4 AMMONIA .............. 140 161 155 152 TOTAL . . . . . . . . . . . . . . . . 5100 4795 5460 5118
$ 7
Pat N o ......................
Vol. 7 , No.
It should be pointed out t h a t t h e determinations were made on separate samples; t h a t is, ammonia a n d amino acids were not determined on t h e same sample. T h e discrepancies of t h e results illustrate t h e nonuniformity of mixing. T h e results are calculated t o t h e wet basis, t h a t is, just as i t was added t o t h e soil. All of t h e pots were maintained a s closely as possible a t 2 0 per cent moisture b y replenishment of t h e evaporated water every two or three days with distilled water. T h e experiment was s t a r t e d on M a y 15, 1915; t h e first sampling made M a y 18th a n d t h e n every two weeks‘ until in all fine samplings had been made. On each of t h e samples t a k e n , amino acid, ammonia, a n d nitrate nitrogen were determined b y t h e following methods: AMINO ACID NITROGEK-At t h e first sampling, t h e moisture was immediately accurately determined, enough of t h e fresh soil t a k e n t o give 150 grams of d r y soil was p u t in a bottle a n d sufficient water a n d 4 per cent sodium hydroxide t o give 600 cc. of 2 per cent alkali, a n d after shaking t w o hours, t h e t o t a l amino acid nitrogen was determined. Then t h e fresh
g
-1
80
2 70
2 2 60 4
50 n
W 40 K a
5 30 S A M PLl NG
soil, a s soon as t h e soil had been withdrawn for t h e determinations of t h e amino acid nitrogen i n t h e wet soil, was quickly dried, with t h e aid of a n electric fan. T h e t o t a l amino acid nitrogen was then determined in t h e air-dry soil in t h e usual way. T h e results using t h e fresh soil a n d t h e air-dried soil were practically t h e same, so for t h e remaining samplings, air-dried soil was used. T h e dissolved copper was t i t r a t e d with 0.001 N sodium thiosulfate. Duplicate determinations were always r u n . A M M O N I A NITROGEX-7 j grams air-dried soil, zoo cc. distilled water, I O grams magnesia a n d a small
Dec., 1 9 1 j
T H E J O L ' R S - i L O F I N D C S T R I A L A iV D E LVGI-VE E RI N G C H E M I S T R Y
I053
piece of paraffin were placed in a copper flask a n d distilled, 1 5 0 cc. of distillate being collected in 0.1 N acid.' T h e results b y this method are, no d o u b t , high. b u t conditions were k e p t as nearly constant as possible, so t h e results are probably a t least approximately comparable. NITRATES-The phenoldisulfonic acid method as modified b y Cliamot? a n d collaborators \\.as used throughout. A s is well knon-n, this method gives somewhat lorn results with higher a m o u n t s of nitrates: IOO grams of soil a n d z grams calcium carbonate were shaken for about a half hour with 2 0 0 cc. of water a n d immediately filtered a n d a n aliquot of t h e solution so obtained immediately evaporated t o dryness; treated with t h e phenoldisulfonic acid reagent a n d after dilution potassium or sodium hydroxide3 added until t h e maximum color was produced.
striking thing brought out is t h a t t h e curves for t h e unmanured soils are below t h e curves for t h e manured soils, in t h e case of t h e unlimed soils for t h e first three samplings a n d for t h e limed soils for t h e first four samplings. This points t o denitrification or assimilation, or a combination of t h e tn-0. If denitrification takes place t o a n y great extent, i t might be followed b y total nitrogen determinations. I n this laborat o r y a t present some very carefully controlled experiments along this line are being carried o u t . coscLusIoss I-The work of Chardet, who used t h e Soerensen method a n d found from 49 t o 68 per cent of t h e soil nitrogen t o be amino acid nitrogen, was n o t confirmed. Practically n o amino acid nitrogen could be found ' in soil b y this method. T h e Kober method as applied t o soils gave t h e folRESULTS lowing results: I n Table T', because of lack of space, only t h e s-KO amino acid nitrogen could be found in t h e averages of t h e results from t h e samplings of t h e dilute acid extract of soils. duplicate pots are given. These results have been 3-Upon adding small quantities of amino acid t o
TABLEV-THREE FORMSOF NITROGEN (RESULTS I N PARTSPER MILLIONAIR-DRYSOIL) ACID h-ITROGEL----AMMONIA NITROGEN-ITRA IT RATE NITROGEN3 4 5 1 2 3 4 5 1 2 3 4 10.1 5 . i 4 7.42 5,46 5.18 48.3 25.6 46.0 49.0 45.2 30.8 6.4 23.3 22.4 1 0 . 0 6 . 16 6.16 5.04 4.76 53.9 33.6 41.8 45.5 44.5 31.5 6.4 20.3 28.2 10.68..31 6.44 5.32 6.44 55.3 40.0 49.0 45.9 30.8 3.1 8.8 15.2 26.4 . . . . . 10.8 7.70 6.86 7.84 6.44 60.9 44.1 53.2 47.3 34.7 2.2 4.8 10.4 25.2 ..... 12.98.40 7.98 8.54 6.72 64.4 43.9 55.3 47.6 34.7 0.8 2.2 8.2 31.2 6. . . . . . . . . . 12.9 9.10 9.24 8.68 8.12 70.4 1.45 11.3 40.8 42.0 62.3 54.3 33.6 0.6 7. . . . . . . . . . 10.3 6.72 9.38 6.30 3.64 60.2 43.1 56.0 55.8 33.6 3.2 9.8 9.8 31.2 8.......... 11 . O 6 . 8 6 9.52 Lost 3.71 58.8 45.9 32.0 4.5 14.0 53.2 49.0 35.4 1.9 9. . . . . . . . . . 11.2i.00 10.1 7.28 3.85 47.6 48.8 19.2 52.2 49.9 2.0 6.7 35.4 0.82 10 12.5 7 . 0 0 11.2 7.28 5.32 53.2 49.3 49.3 49.7 37.5 1.0 4.4 17.6 1.5 Di s' between duplicates: MAXIMUM ... 2.2 2.24 0.6 1.40 0.84 14.0 8.1 9.8 15.4 9.1 2.6 8.2 3.4 6.4 AVERAGE.. , 0 . 7 1.27 0.23 0.6 0.37 4.2 5.4 2.7 4.1 3.3 0.4 1.7 2.0 3.7
Expt. No. Sampling 1. . . . . . . . . . 2. . . . . . . . . . 3. . . . . . . . . .
___- 2 No. I
AMIXO
plotted (Figs. I , 2 a n d 3) a n d t h e points so found have been joined b y straight lines. I n Fig.1 i t is seen t h a t in general there is a decrease of amino acid nitrogen compounds. T h e values for t h e manured soils a r e b u t slightly higher t h a n for t h e u n m a nured. This is, perhaps, t h e most striking fact brought o u t b y t h e experiment. There i s n o tendency for a n accumulation of these products under t h e conditions of t h e experiment. Another noteworthy fact emphasized b y Fig. I is t h e greater consistency of t h e results for
c.
t h e limed pots. Perhaps a reason for this is t h a t t h e microbiological activities proceed more normally in a medium of t h e reaction induced b y t h e lime. T h e ammonia results, plotted in Fig. 2 , in a general way follow t h e amino acid results. When t h e experim e n t stopped, a s with t h e amino acid, t h e ammonia content was decreasing. In Fig. 3, which shows t h e nitrate results, t h e most 1 Since t h e experimental p a r t of this paper was completed, t h e authors have worked out a method f o r t h e determination of ammonia in soils (Loc. cit.) which is much more satisfactory t h a n t h e magnesia method. 2 J . A m . Chem. SOL.,33 (1911), 381. s The original directions call for potassium hydroxide.
21.2 5.9
a soil a n d extracting with dilute acids, no amino acid could be found. 4-Vpon adding small quantities of amino acids t o a soil a n d extracting with dilute alkali, practically t h e entire a m o u n t added was recovered. 5-There was found t o be no difference in t h e quant i t y of amino acid nitrogen extracted b y dilute alkali in one, two, four a n d six hours. 6--A few soils were analyzed for free amino acid nitrogen, total amino acid nitrogen a n d total peptide nitrogen, with t h e following results: ~'ITROGEN-P. P. hI. AIR-DRIED SOIL Total nitrogen Total Free Soil Per cent of soil amino acid amino acid 7.95 5.45 A . . . . . . . . 0,151 B . . . . . . . . 0,180 23.1 14.7 . . . . . . . 0.21 23.1 ....
SAMPLING
5 30.4 30.4 30.5 20.8 39.2 39.2 32.0 37.6 27.2 25.8
Total peptide 31.5 30.9
....
T h e conclusions t o be drawn from t h e pot experiments are as follows: T-Thefe is no tendency for t h e amino acid t o accumulate under t h e conditions of t h e experiment, namely in a limed a n d unlimed acid soil, in a heavily manured a n d limed, a n d a heavily manured unlimed acid soil. 8-The amino acid nitrogen was present in t h e soil in less amounts t h a n t h e ammonia nitrogen, b u t in a general way it fluctuates with t h e ammonia nitrogen. 9-The soils with t h e higher amounts of manure show a decided decrease in t h e a m o u n t of nitrate nitrogen at first, b u t after from four t o six weeks, there is a decided increase. LABORATORY OF SOIL CHEMISTRY IOWASTATE COLLEGE EXPERIMEXT ST.4TlON AMES. IOWA
