STUDIES ON THE CHEMICAL. PHYSICAL AND BIOLOGICAL PROPER'FIES OF SOIL ORGANIC MATTER Thesis {or III“: Degree 0‘ pk. D. MICHIGAN STATE UNIVERSITY Harry H. Johnston 1958 I—II f ES THESIS , II I III IIIIIII This is to certify that the thesis entitled Studies on the Chemical, Physical and Biological Properties of Soil Organic Matter presented by Harry H. Johnston has been accepted towards fulfillment of the requirements for 43% degree in Mae Rtem/ Major professor Date May 1 5, 1 9 58 0-169 LIBRARY Michigan Stan University MSU RETURNING MATERIALS: Place in book drop to remove this checkout from UBRARms 1—”. your record. FINES will be charged if book is returned after the date stamped below. 1-“.‘0'0' I -- 1 m 3&9: Jury. = « (. llalv . STUDIES ON THE CHEMICAL, PHYSICAL AND BIOLOGICAL PROPERTIES OF SOIL CRGflNlC EATTER Submitted to the Schov] it? Advanced Graduate Stuiirs of Wichi‘en State Universitv of Agriculture and Applied Science in partial fulfillment of the requirements for the degree 2" f r‘."vm ’ l_‘- ('3 "3-: '1' ;' __’- ' '31:, bew -L-» (k- l f$;$.-‘v--\( '1 :ad- Department of Soil Science 1958 Approved: Date: H. H. Johnston ABSTRACT: Studies on the chemical, physical and biological preperties of soil organic matter. Ph. D. thesis submitted to the School for Graduate Studies, Michigan State University, by Harry H. Johnston. 1958 In a greenhouse experiment, 12% and 25 tons per acre of sawdust, acid—extracted ("lignlfied") sawdust, corn stalks, wheat straw and alfalfa hay were added to Csntemo sand. Three levels of supplemental nitrogen added as urea were used with non- leguminous materials. One series of pots was not cropped during a hOdweek decomposition period. Separate series were cropped to wheat or alfalfa. Periodic sail samplings were made for 1 esthnation of microbial manners and pi. All pots were planted uniformly to wheat after LO weeks. Nitrogen mined on the harvested portions ;P all wheat Qfld alfalfa crops. Soil samples were taken at the and oi" the l;0d3''AAFU _ v. a .a...-,;_(.. O Figure 7. 8. 9. 10. 11. 12. —x- LIST OF FIGURES (Con't) Page Relation of non-acid-hydrolyzable nitrogen in the soil to the mineralization of carbon and nitrogen in Oshtemo sand ....................... 71 Carbon and nitrogen mineralized during incubation as related to soil C:N ratio and plant residues applied No weeks previously to Oshtemo sand .... 72 Effects of various organic amendments and nitrogen on numbers of bacteria and fungi over a ho week period with and without cropping ..... 75 Correlation between yield and nitrogen uptake of three crops of wheat grown on Oshtemo sand in the greenhouse experiment ................... 81 Total nitrogen taken up by three crops of wheat following treatment of an Oshtemo sand with organic amendments and nitrogen according to the schedule presented in Table 13 ................. 8h Nitrogen uptake by wheat as related to soil C:N ratio and various organic amendments, supplemental nitrogen treatment and previous cropping. (OShthO sand) ooooooonoooooOooooooooo 88 , .a(.‘..,.t. Figure 13. 15. 16. 17. 18. 19. 20. LIST or FIGURES (Con't) Nitrogen uptake of wheat as related to water—soluble nitrate in the soil at planting time. (Oshtemo sand) ................. Nitrogen uptake by wheat as related to the sum of water-soluble nitrate in the soil at planting time plus nitrifiable nitrogen re- leased as nitrates during 1h days incubation at 350C. 0.0.0..........OOOOOOOOOOOOOO0.0...... Infrared absorption spectrum of acid lignin ... Infrared absorption spectrum of alkali ligrljn Oboooooooooooooooooooooooooooooooooooooo Infrared absorption spectrun of casein.......... Infrared absorption spectrum of acid lignin- Casein 00mp1ex (6 to l ratiO) 0.0000000090000000 Infrared absorption spectrum of alkali lignin— casein complex (6 to 1 ratio) .................. Infrared absorption spectrum of alpha humus extracted from mUCk 00.000000.0.0000000000000000 Page 89 90 119 120a 121a 122a 123a 12ha 4.." ...- .7 .... ,9..—..‘. ...i g t . .r( a.« . .- . . A - . c .4 a. _. .. r a. f ... . _ , a . e .-.; .... .. 4 r . e . s .uu-c. :. ‘ n « ,4 . .. - . » . .nr ,, V r Figure 21. 22. 23. 2b. 25. 26¢ 27. — xii — LIST OF FIGURES (Con't) Page Infrared absorption spectrum of alpha humus extracted with NaOH from an Oshtemo sand. The soil had been incubated for 30 weeks following an application of acid lignin ..................... 128 Infrared absorption spectrum of alpha humus extracted from sims clay loam with sodium phrophosphate. Check treatment .................. 129a Infrared absorption spectrum of hydrogen saturated'Wyoming bentonite ...................... 130a Infrared absorption spectrum of alpha humus prepared from four parts muck complexed with one part bentonite .............................. 131a Ultraviolet spectrum of alpha humus from 8 81.1118 Clay 103111 00000000000.000000000000000000000 133 Paper electrophoresis separation of alpha humus fraction from a Sims clay loam ................. 135 Ultraviolet spectrum of fluorescent component separated by paper electrophoresis ............. 136 Figure 28. 29. 30. 31. 32. - xiii — LIST OF FIGURES (Con't) High frequency (A) and Conductometric (B) titration curves for barium-saturated acid lignin in 100 m1. of water and 50 m1. of 8.1001101 00000000000000000...0.0000000000000000. High frequency (A) and conductometric (B) titration curves for barium-saturated alkaline lignin in 100 m1. of water and 50 ml. of 8.1001101 0.0...0.......0.........OOOOOOOOOOOOOOC High frequency (A) and conductometric (B) titration curves for barium-saturated casein in 100 ml.of water and 50 m1. of alcohol ...... High frequency titration curves for barium— saturated acid lignin-casein complex in 100 m1. of water and 50 ml. of alcohol ................ High frequency titration curves for barium- saturated alkali lignin-casein complex in 100 ml. of water and.SO ml. of alcohol ............ Page lhOa lhla lh2a lh30 lhha o r h ‘ s. r a {A ~ u n - r r A ' 7 1 a a A < ,, '- u v r a s . 1, u A Q : I n ' a h V - o 3 I I .1 ‘ l l A GENERAL INTRODUCTION Characterization of individual soil components is one of the major goals of the soil scientist. ‘Nith the help of X-ray, dL Wf cntial thermal analysis, and the electron microsc0pe, clay components can now be fairly well identified and their structure written with a reasonable degree of accuracy. Organic matter and clays together comprise the Jeater per- centage of the "active fraction" of the soil; therefore it is logical that an expanded program of research on these fractions be initiated. Knowled e of the vrgan‘1 :raction c: soils is ar~ C") tremely'limited. Its structure is mere postulati n and as yet no easy or simple way has oeen devised to separate it fron the soil. The degree to which various organit extraction is not known r.=5ts any certainty. Organic residues addec to the soil are c: ogre because thev are a source of food and energy for microorganisms. Latcriil" resistant+ o decompositicn and. by- croducts of decomposition remain in the soil and impart to the soil some of its; nost important physical and chemical properties. The fact that no sing 1c method of IvoLawior has been fo nd for the separation and characterization of soil organic matter 1 - dicates its complexity. “vdrolv of soil organic matter with acids or bases has given the best in cumetion rsgariing its composition thus far. Extraction of soil organic matter with alkali or me itral :3 $011 Q r (I ~ ‘J *4 r b reagents pennits the study of ”some of its ph ,sical pr 0pm as its chemical proper Jti s. Even if unaltered organic matter could be isolated from the inorganic fraction of thee oil, its structure would be so complex that no one tool or instrument c.uld identify its structure and characteristics. Studies on soil organ c matter must then be deal.t wi th in a stepwise manner until encug? injzrmstion is account for its structu ure, characteristics finite approaches can be mace: (a) Synthesi cqnpounds and comparison with. :zc-‘ural so and (b) isolation of the crvan ic soil ccnst methods. lpI‘O‘Ce- “" w J1? S. Tr: o flee—- s of postulated m;;;1 organic matter fractions, itm 3nts by new an dwimp ~"cd Objectives of this study on soil argcnic mat,er include the characterization of some of its properties amendments, soil type and nitrogen S‘pply, a I o c 3 W 3. Lil ' :- . .. -. ‘ 1., . . ° 3 inilucnch Oy organic special emphasis on nitrogen transformations; and a oreta il 3d1 studv of individual componm ants of certain organic matter irici;;ons. LITERATURE REVIEW The literature on soil organic matter is so extensive that a separation of the various investigations into related sections should give a clearer and more understandable presentation. Soil Organic Phosphorus Soil organic phosphorus has proven difficult to determine quantitatively because of it complexity, and the large amount of in- organic phosphorus present in some soils. Present biochemical methods, especially the use of exchange columns, have permitted more detailed study of specific phosphorus containing organic compounds, including inositol phosphates (91), Several methods have been devised for the gross determination of organic phosphorus in soils. As yet no direct method has been intro- duced. One of the first methods used was that of Pearson (78). The organic phosphorus is extracted with alkali and determined by the difference between total and inorganic phosphorus content of the ex- tract. Another method based on extraction is that of Mehta and Legg (67) where NaOH is used for extraction instead of NHuOH. A more recent method uses ignition (56) to determine the amount of phosphorus before and after heating the soil to 2hOOC. Only the more labile organic phosphorus compounds would appear to be broken down at this temperature, however. There still exists considerable controversy about each of the mentioned methods. Legg found 20 per cent more phosphorus extracted with the Wrenshall and Dyer (118) method than by the method of Pearson. A Black 23 a; (13) have prepared a comprehensive review of soil organic phosphorus. Further work is xeeded on procedures for the determination of organic phosphorus. Chang (28), working with pure cultures of.microorganisms, found that between 0.3 and 0.h per cent phosphorus was assimilated in organic form per unit of cellulose decomposed. Kaila (53) reported similar results with the use of pure cultures and the use of other organic materials as sources of carbon. If the phosphorus content of the organic material was below 0.3 per cent phosphorus, then mineral phos~ phorus was taken from the soil and incorporated into organic form. Kaila found that the ratio of organic carbon to organic phosphorus in mineral soil was about 100:1 to 150:1. The ratio of organic nitrogen to organic phosphorus was about 8:1 to 10:1. Predominant evidence indicates that most of the organic phosphorus exists in the form of nucleic acids and phytin (15) with as much as ho to 50 per cent in the latter form. The availability to plants of these two forms has been tested (16, 12) but no definite conclusions have been reached as to their effectiveness when compared to inorganic forms. The presence of large amounts of o‘ganic phos,horus in s.me soils would certainly warrant further investigation of these phosphorus compounds and their relation to plant nutrition. The Uronide Gr Polyuronide Fraction Of The Soil Based on the method of Lefevre and Tollens (55), which depends on the liberation of carbon ioxide when uronic acids are boiled in 12 per cent HCL, many investigations have been conducted on the uronide or polyuronide fraction of soil organic matter. Shorey and Martain (89), Norman and Bartholomew (7h),‘Waksman and Reuszer (111), and Fuller (bl) " Tr_— .1 — n have shown that from 10 to to per cent of the total carbon may be present in this form. Brenner has criticized the large portion of the total carbon attributed to this fraction, since no proof has been given that uronites are stabilized in the soil to such a large extent. Lynch (59) used a chromatographic technique to separate some of the sugars present in the fulvic acid fraction of soils. The same carbOhydrates were found following various treatments of the fraction, but mild acid hydrolysis gave higher yields of these materials. Fuller (h2), doing work on the uronic fraction, concluded that this fraction is of microbial origin because of the high preportion of CO2 produced when compared to plant uronides. He also reasoned that if uronides were not intimately combined with other fractions, then their isolation would be easily made. Fractionation of the soil, however, was found to bring into solution various other materials in the presence of which positive identification of uranideS'was impossible. Mattson and Kouttler-Andersson (63) found that in beech lignin the content of uronic anhydride increased 2 to 10 per cent as a result of auto-oxidation in alkali. This would show that sources other than uronic acids are capable of releasing 002 under the conditions of the Lefevre-Tollens determination for uronic carbon. From the existing evidence it would seem that some of the uronides are of microbial origin, but the method that has been used for the determination of this particular fraction is too empirical. The method in its present application gives no assurance that only uronides release 002 under these conditions, even though uronic acids are decarboxylated 1umntitatively by this method. Composition of Soil Organic Matter Literature references to the gross organic fraction are rather difficult to evaluate, since no uniform method has been accepted for the isolation of the humus or humic acid fractions. Previous to 1900, much work had been done on the characterizan tion of the dark colored material present in the soil but little work was done on the chemical compounds present in the organic fraction. Schreiner and Shorey (90) brought to the attention of investigators the fact that specific organic constituents could be isola‘ (1“ ed from the soil. Robinson (86) found that acid hydrolysis of soils liberated amino nitrogen. The amount of amino nitrogen released increased up to a point and then decreased with further hydrolysis. Gortner and Morrow (70) fractionated the nitrogen present in mineral and organic soil according to a protein hydrolysis. Their pioneering work (Table 1) showed that a large part of the organic nitrogen present was in the form of protein or proteinaceous compounds. Other work of this kind led several workers to conclude that a large part of the protein present in soil was in the form of yeast and bacteria. Gortner found that ammonia and sodium hydroxide do not extract the same subs stances from the soil, and that humus does not consist of a black colored compound alone, but that a portion of almost colorless pro- ducts is masked by it. Little interest was shown in this work until recently when similar studies on soil organic matter have received considerable attention. Hobson and Page (50), and Page (7?) performed numerous studies on soil organic matte‘ and concluded that the humic materials contain mo.m pm.m oN.m mm.m oa.m mm.m mom aom mp mwnm pmm z Ofimwm :©.HOH mm.wm oo.ooa >>.moa ©:.mm mm.moa humboomm R pm.» mzé mm; :46 masm omfi $me 90 z oqfle< mm.©m mm.:m mm.mm >N.©N do.wm mm.om HHow qfi sass: mannaomcH mm.ma wo.wa HH.NH wm.m mm.ma mw.m z Oflmwm ;~.mm mm.mm oa.o: Hm.om mm.N: H~.m4 z.oafle< awe: ma.m om.ma Hm.m Nfimovmo hp opam ofiasm mo.mm mm.om om.ow oo.gm m:.ma mo.mm z wficoea¢ mmooa mmmoa awed awed Uonoboo pmonom vmnoboo mwnfimhm pawn ownwm hwao ompmm x052 eanwwnam Amav amcpnoo scam cwnflmpno «pan .nmwogpfln HHom Hw¢oa mo pave hmm aw acwpsnanpmwv ammonpfin mmmym>d_ I .H magma 8 a complex of nondnitrogenous humic acids and protein. A smaller pro— portion of the total nitrogen extracted from soils with cold soda was found to be in the amino form than in proteins of animal or vegetable origin. LFrom this they concluded that the protein was of a different source than plant of animal protein. They also found that humic acids prepared from sucrose and farfural did not behave as acids but did give a dark color. At a cut the same tine‘daksman and Iyer (107, 108, 109, 110) postulated that protein exieted in soils in the “can of a resistant ligno-protein complex, and that this accounted for its apparent stability in soil. From synthetic preparations it was found that such a higher exchange capacity than the csielnal protein or ligei“. how- ever, other workers (77, 61) have failed to observe an increase in exChange capacity during formation of such complexes. :ecause cf the great influence of‘uakenan't work cn other workers in this area of investigation (105, 113, 11%), it was not until recently that the 1 ligno—prctein coupler theory was subjected to serious criticise. Mchcrge {Ch} :tuni tear the exchange capacity 6? highly organic soils is approximately a linear :hEPtl?J vi the per cent of carhcr in the soil. he attributoi the ILChafiu: aetivit" Ht licnin or lignin— hemicellulose. The mechanism of clay~tr¢arir ecmbinati': r\ 2 p 01 studied with pure clay systems by Gieseklng (h?) and Ensmqnger and flieseking {33), The organic molecules and proteins used interacted with the clays J... and resisted hydrolysis to a much greater deérce than she :whrtarees themselves. There was some ones the enzyme and caused its inactivation. that inorganic colloids exerte .d some organic materials. tion E‘Il Montmorillonite was til 55"} \IA as to whether -¢'i4 e clay assort) Allison, gt gl, (L) found Iiiluence on the dc?0mho ition cf mcst effective in stabiliz~ ing carbon, and kaolinite the 3est. L:Jn h.2:‘:l, {60) studied some carbohydrate-clay complexes :ni £1113 that complex sr gar w”79L110° were absorbed hy clays. McLean (65) rel~ orted that the act!” ties of claywhaelc systems were not decreased too much firs; L:cs: cf the clay itself. He stated that th accizlpics cf ions «3 tie clay could still be p1011c,co at the vz1lole 301 1. He attritutfi ttc 15c '=;se in exclan5e capacity of the complex to reacticns bv-Vpuh the “lay ani the organic materiel and not to the mechanical coveric5 of the exch an5c sits: the clay. Gillman (hh) used acetylaticn and methylaticn to strdy the exchange reactions of humic acids. humic acids “ere methylated fin} acetylaseo and showed reduction of exchange capacity by these treatments. The I -. reduction was less than ec._xrien to the incrtzcc in acetyl and lethyl content. More recent work alcn5 t1 ese lines vet conducted Broad- bent (26} , with the use of ’czcmethane and dimethyl sulfate. he found that, ir Oil freed Ere LV» i.cr5~-i: Trccticn by hydrofluoric acid, diazomethane reduced tie oatizn archange capacity to e greater extent than methyl sulfcie. free this he concl1oeo that the exchange activity in humic matcrj.;ls resides in carboxylic, phenolic, or enolic groups. Gottlieb and hendricks (hf) “at —-'\— nitrobenzene reduction 3 method '5‘ ""1 ..‘t ‘1 VA. .~.. .ru ‘1 - .. .g .1 .Vorc513 11 silo. 1 iscl ti(n of con; (unds fron the m 1. {11" lO soil organic matter withovt too much surx'ess. They concluded that the lignin molecule undergoes condensation to form a fused ring structure more resistant than native lignin. More recent work on acid hydrolysis of soil organic matter by Kojima (Sh), Stevenson (96, 97, 98) and Bremner (1?, 13 22) showed that: (a) About 29 to 35 per cert of the t1tal soil nitrnm u o o‘ \J F." ....- :J C F 5...) ...J ’S U C 5 H (L ”J t". x“ (. gen is present as alpha amino nitrogen ammonia in acid hydrol v:a , es could com: fro4 hydrclviis c? pr17‘ .ein (r ‘- amides so that the abcve .“' burn is a micirhm for prctcinacaous nitrogen; (0) compounds like amino sugars are present to some extent and may account for 7 to 10 per cent of the total nitrogen; and (d) that 65 to 80 per cent of the total nitrogen is hvdrolvsed in acid. Hock (51), along with othfir norhcrs triad to use th: color of alkali extracted material as an index of the amount of humus present. Bremner (21) showed that cflor is a poor indr‘ :rro aplit; “no . ' . A . , I-“ .. .1 .: f._ ‘ . —.. ~ \1 fl 1"" run- -\ \' .' -.. supplemental nitrogen ta :. i_~- An vce~.wl_ ’i _t :ltt. ana A — — A. ' I\/' v .- -.. " '1": ‘o r. ' . J" c '- {- n -- . P) -5 . " hundred-twenty pounds ~,: 3 - A12- :-ur.g ; '59 .?I11 o Jitn cor“, v.- - - q n '1 . ‘§/1." ’4." any ,—~v rlr VP. "-. 4‘ - —— Hy: -\- hO pounds wi th boa): an. 2 psuhw- en: 3 12* -t i _oil_-:e; ' r; . J’ ' ”5' ~ K. I": applied on the ~WP ywar: e , slow. . - _ ’r" ,'.'L,‘ :1 fl “,'1 fl ._,, p‘_\ a ‘ In the spring of 17:6 composii sol; ”Pmkweg wa.e taken :32? tea 0 ‘ _ .L "'1' ‘0 , ‘ ‘v ,. \I_.'- I 3. ,— _ . fix? ,, ‘T' .' o}. nitrogenwtreated half of pl oos nllCL 37$ secehvcd 35 toes - satires F———" _fir 211 fl. 1 ' l I" . ~r, h 7- v:4‘u ‘1 -; ' . xx , , A. _ a,‘ v .. ‘ one, three ano “lfc jegre pre‘luaalf. A eke: samplu W9? tompssltei from corre3ponding plots wPicn haa received Sertili er and sup loaontal nitrogen but no additional plant residues. "rcrwlbrrvest late Data in Table "\3 (’1' I .J \ .5 .,‘ MI 3 + "5 I ‘ J if) 4 ‘ 4.. .\ J- . v ‘ . .1 ., n 1 . -. 1"- - -, ‘ . , . r. [‘J. .. ,2 . - 1. the sawdust ureaanent. “lelue v- earn an» “43bL Joar ear sanuxst '. I ' . “ " ' ' '1 3I ' - 'r ’ " " ‘ v—n “' """ " ”‘ Fx“ (" '3" - "‘ " "' " " {‘J' appllcatlsn wee; faauoel .a we; C;“ho Ltrle; is4-s anr¢~ -carc alas? 1 o 1" . W.A ' . ._ .‘ -.d_v r».", .». "_.“.. ..._ ... ‘4- sawaust appllca.;cn w»). r so :oluoed. l“ a Janna on “xuuell-uoulo; C?” r I was an importan J ‘l ‘ ‘ , ’J_. .t D 1 _ ' v n ‘ ‘ ’_ ‘ _. ‘ . o 4L w ‘ ' .-~. '- ‘ - . ‘ face tnat tne adultlon CL lQO peanue .I? 4,4; F; n;ufio up _;e::aseu ’.| 1. ‘ 1' _- 5 ‘. '0 ,- 1 ’\ ~ ‘r - .‘~ - , ‘ “. . - u 1 ' . ' ‘, '» \ " .L - .' o' ‘- ‘ '- ‘ I :'~ L COI‘n yl'gxlb v.1 1-)! L. LPN“; ’ r" , ....»..“w ; ... A; ‘ .- - .v , - i; - -- f": -'.-'. ..‘Tx :~.3'."."..1.1.;. v o , ’_\, .' ‘\‘| "F‘ ”I - '_ t _ k '_ \ '. : ‘l v ( -.3‘ _',, ‘ ' 1" ‘ plots ralsed barlef jlzkag be away, V__ Jame Lava}: my Jp p10»; uh hi 1 A ~ “‘3'; 7'06 7" .. 06": ‘ “t . #17,. ~71 F 1 ._ .. ”p .-‘... .- A”... A.» 2‘._- \ ... war). .5.“ la». lept, ..\ v.1 .J. -J-‘J ‘. ..-.I '9 v r._* ...k;. .2 . -_:.‘.‘, »,., .. I "of ._ - ,' . ._ k 2'...’ ' '"J ‘. ‘1 " ‘v “‘2‘ fl "‘ ‘f‘ ‘ - " ’. -‘-"« ' , n‘ ‘- ‘la'l‘ I x I . v ~ - ‘ however’ were L2 will}- 5:31;]... 7‘-" .;. -...‘ 3U .1.“ . '. '3‘ L. 3V7 Kw T’ué g, ;'l"-\"“'LC“’ .' a J ' . 1-1- a H . .. J ,— .. . .. . .. ‘ — «L'- , ... .3 .- ' . - Bean flt‘1‘g9 in my- ah; .. ‘ - «4 ,, t; ULLC _r-.l-;.cr_.:i_.1.cn r.» '11.le at d '33“ OTC“: «mm M "H "w“ at“ "1x '. ~. .‘ - . fl' .7 ’v 7 7‘.'\.. -‘-‘v‘ 1!"! C‘ "11-3. 3 "3 St 19 an U-sua .-4 “a- A \_ , w _, L -_ _--.a __.__ . _ .. V -. “mi, ‘ — ‘ ' -\ L. a- ‘L -- » ‘ ‘ 0 . ~- ‘ ~. 1 r - . l- . 0'," . :- ~-,— 'I\ ~ 9 -‘ It mav have Leer t mu page autnla; .a ._ a a, L;.roben was reunceu ° ‘ J . .. - .--“: fi-.'.‘!—'.L—w :31 W‘- .-...,.+?.. . to 11.1711.th 1 :‘4 L31.” ”if 3-,. ram-I 13L)_‘.L, ' L} . 3" .~ .~_ n 3, ,._ ;-. :c. ._- fag", ~13" ‘ - 1 ,. .“o- -:. t -. :l -. .a—La‘l -.. “n 1- -\ “a-“tafi 1 +». 1183/ ’3. OGLC c"; '.\ '. ‘ ".1. ' "a '3' T' l_‘ '. *5 ‘ ‘.' t9 tr. a A 7 _'_" .;.A_; ,: gave)? 0 C qr 09° :-—1 {3011.5 \H ‘ ;‘ p. r ' 1 »«",~. (5(3- ‘I. ,l_ ‘ .1. .LC L up -- - - .lk 1.1. M' . .L. .< ..3— -~ _ r‘ - _ s‘ Q '1 D K L J. 1‘ our ye? 1“) QAL t'o.“ .LL’ v: " 4‘," _'_L-.l_"?J~—-, ‘v ‘pL‘.->(' h. .. .w - .. - Lr— , J v \4' ‘ I... V—nlC yields of alfalfaétroau. Total nitrogen, Wfliorflfleafarle ¢?*erlal ab; 4; ‘ t» J_‘ ._ , . . .. , .4. ._ a . - . _. + o" N I x':‘ n-v MK . A.‘ , I'D/\fi ‘ 71":- V 2.3.3» 1.) "‘ -. Do Fr! I I; \‘\_, _ Talflxa 3 SLJn«s scan! CL m.- Lp\\_s ; , -azee.rass Anu.e \ we“- .--Jo 25 .hmHHmn go %ON paw .mqmop no %04 «choc co whom you z.*o~a ** .coflpwpm pnmsflhomam HdeQHSOHpm< :mwflnowa map Ho hmopnsoo esp nmsonsp dopaommhm mpmv nonmaapsmq: * ...... 23 so; Amfifiso ES unsannn wasp mm.a Ameappzo pmav mgpmam: a (38 mm suntan: weep no.a chappso eqmv IIIIIII m:o¢ NN.H Amcflppzo pmH/ meoanma¢ a 950% 0:02 63 mém 53 4.3 $28 m 083 mm .msn m.mm .msn m.mm hoanwm N moan» mnoz .mnn m.wm .mdn m.mm mummm m 039 mm .25 :Am .25 fimm 23mm m 25 8% .mnn m.mm .mdn ©.H: snow a mac mm .mdp 2.5m .mfin o.mm £900 a one onoz **:mmonpan :mwounaq omwhobm ca mnwmh noapwowammw whom you mnop spas.vaoaw ascend: vHoHH mono mono no 903552 hopmm awow pzosvwonp pmscsmm $.pamauzmew pmdvzwm w pdosvfls cam spas amoa zwao mawm no coflpmpoa hawk o>flg a ma mvamfih mono I .w manna soil samples taken one, th"c* and five Years a”trr sawl ct abfili- and from the checks to whicr We aifiitifinal Crganic materials had applied. The initial depression (Til 12-; it: tr.“-.:’:"i“.*?‘. 21‘7“; 3:51”: ‘ .‘L. . a, . - ‘ r e 4. ' -- . - ’ —. - a ' .. fluently reporteo she? iai-—- meter. 3 “re dc;.mnus nu ratio]? ‘ - . ‘ m ,. ’ A . . . ' , . - . .... . $011 (3). ihere was an incre.u: -1”.zn‘eg iv L? its: yer a'ro - “1 __ v vn _ _~ _ "r.“ ff): _ ‘ I1 _ ' . _ materials floatanle in nat~r one ;»ri a_te: the Q? t3 I 4 g g ‘9 9 g ?0 D- .g 0‘ 2 a 98—5 3 " o s :22 i Z Taoswaoaus - PPM 55 0% :3 a § 5: <3 I 1 ‘ l l I I ) ' id s ‘ ‘ é \\\\\\\\ mu 3 c: s “ ‘§ 2222;; “my TE!""‘“& z///////////4: d a W3. 2 so:;»\\\\\\\§ nL'i' E, ZWHE o. A . ‘ WIT” ¥ 2 ’,”,’:«\\\\\\\\\\\\\ , m: ‘6 2|’///////////// 3 8'. a a 32 é e e. é a 9'. ' "HOS :)O SNVB‘D 017 was N $9M 37 increased.most probably by incorporation of nitrogen into residual lignaceous materials which accumulated as the sawdust decomposed. This latter process is presumably not micrdbial but is associated with the chemical oxidation of lignin or similar compounds involving benzene ring structures (61, 62, 63). Alkali-extractable nitrogen The results of alkali extraction and fractionation of nitrogen in Sims clay loam samples are presented in Table 6. Figures for non- acidéhydrolyzable nitrogen are also shown for purposes of comparison. Nitrogen extractable in alkali reflected the sharp drOp in total nitrogen the first year after sawdust application. However, the upward trend in total nitrogen in the third and fifth years' samples was not paralleled by similar increases in alkali extracted nitrogen. A somewhat closer correlation was Observed between the percent- age of total N appearing in the humic acid fraction and the percentage not hydrolyzed by acid, both showing a distinctly downward trend. This might be expected, since insolubility in acid was the basis for separation of both fractions. Humic acid nitrogen in the alkali extract continued to decline in percentage over the whole fiveiyear period, whereas the percentage nitrogen not hydrolyzed by acid de- finitely leveled off between the third and fifth years. Thus it would appear that certain soil constituents may be common to both fractions, while others are more specifically associated with one or the other of these two fractions. The fulvic acid fraction showed a sharp increase percentagewise 38 Table 6. -»Effects of sawdust (35 tons per acre) and time after application on the humic and fulvic acid fractions of Sims clay'loam.* Years after sawdust application Nitrogen fraction Check lst year 3rd year 5th year Total soil nitrogen mgs per'100 gms 233 193 2h2 268 Total soil nitrogen extracted in alkali mgs per 100 gms 79.5 68.0 69.0 62.2 Percent of total soil nitrogen extracted in alkali 36.3 38.8 28.7 25.8 Humic acid nitrogen mgs per 100 gms 57.2 hh.5 51.7 hh.h Percent of total 2h.5 23.0 21.3 16.5 Fulvic acid nitrogen mgs per 100 gms 22.3 23.5 17.3 17.8 Percent of total 9.5 12.1 7.1 6.6 Non-acidéhydrolyzable nitrogen-mgs per 100 gms ww-69. 51 59 66 Percent of total soil 29.5 26.3 2u.6 25.5 nitrogen *Fifty grams of soil were twice extracted with 2% NaOH for two four- hour periods at room temperature with shaking. The centrifuged supernatant was acidified with HCl until the dark colored material was precipitated. This acid precipitated material is the humic acid fraction and the supernatant the fulvic acid fraction. *tNon-acid-hydrolyzable nitrogen from Table 5. 39 one year after sawdust was applied. This may have reflected a tempor- ary increase in free, -or loosely complexed, -amino acids or protein moieties resulting from micrObial activity stimulated by the large addition of fresh energy materials. The declining level of fulvic acid nitrogen in the third and fifth years parallels that for hwmic acid nitrogen. The significance of these changes is not clear. They do indicate that soil organic constituents were being actively transformed as a result of sawdust treatment and that the influence of the sawdust on these transformations was still in evidence five years after treatment. It appears likely that many of the changes noted were related to increased complexing activity of lignin as it was pro- gressively exposed and oxidized during decomposition of the sawdust. Organic phosphorus The work of Chang (28) has shown that phosphorus to the extent of 0.3% of added cellulose was assimilated during decomposition. This corresponds to numerous reports that a phosphorus content of 0.2% to 0.3% in organic materials represents the critical level between release and.immobilization of mineral phosphorus during decomposition (13). Phosphorus in the sawdust was not determined, but analyses of similiar materials reported in the literature (3) indicate that it would.have been of the order of 0.01%. An increase in organic phosphorus would be expected where sawdust is allowed to decompose in soils. Changes in total, inorganic, and organic phosphorus with ho sawdust treatment and time are tabulated in Table 7. Total phos- phorus was maintained at rather constant levels in all soils. Two- fold increases in per cent organic phosphorus were found in the third year and fifth year samples. These increases in organic phosphorus 'were:made principally at the expense of the inorganic phosphorus. There was a sharp decrease in acid—soluble phosphorus the first year after sawdust applications, at which time 29 ppm (58 pounds per acre) was found. In the Michigan system of fertilizer recommendations based on soil tests, 50 pounds per acre of phosphorus soluble in .135N HCl represents the dividing line between "high" and "low" levels of available phosphorus for soils below pH 6.5. ‘When the combined requirements of a growing crOp and the microfloral population supported by the residues is considered, the 58 pounds of acid-soluble phosphorus found in this sample may well have been deficient. This may be one of the reasons that corn and barley did not give larger responses to nitrogen fertilizer. The continued increase in organic phosphorus Observed through the third and fifth years suggests that microbial competition for phosphorus as well as nitrogen may have been a signi- ficant factor in crop yields over the entire period. Organic phosphorus increased with amino nitrogen, as is shown graphically in Figure 1 (P. 36). It may be inferred that carbon added to each of these organic fractions came largely from added saw- dust by'microbial compounding with nitrogen and phosphorus from soil and fertilizer sources. This does not imply that the increased quanti- ties of organic phosphorus found in the third and fifth years' samples ‘were present entirely in the form.of microbial cell substance. hl Actually, only a small amount of phosphorus could be considered to have been in this form. Rather, a selective accumulation of phoSphorus canpownds released by the death and decay of successive generations of1microbia1 cells would appear to have occured. The possibility that decomposition products of lignin might form.complexes with phosphorus compounds has never been investigated. Table 7. - The effect of sawdust 0n the total, inorganic and organic phosphorus in Sims clay 10am.* Years after Repli— Total P Inorganic P 0r anic P .135N HCl - ppm % of sawdust , cation ppm ppm %7of soluble P application total total ppm % of total Check 1 832 6h0 192 57.5 2 800 6&2 138 58.0 average 816 651 79.77 165 20.23 57.8 7.1 lst year 1 870 66h 206 27.7 2 900 700 ' 200 30.0 average 885 682 77.06 203 22.9h 28.8 3.3 3rd year 1 8h0 th h06 52.00 2 700 376 32h 55.0 average 770 hOS 52.60 365 h7.h0 53. 6.9 5th year 1 870 too u70 50.00 2 800 h7O 330 h0.0 average 835 h35 52.10 uoo u7.90 h5.0 5.u *-Method of Pearson (78) GREENHOUSE EXPERIMENT Design of Greenhouse Experiment An experiment was initiated in the greenhouse to study the effects of decomposition of various organic amendments on the dis- tribution of soil nitrogen. So that relative changes might be more easily observed, a sandy soil low in carbon and nitrogen was selected. It was also considered that the effects of clay minerals on the de- composition would be minimized in a sandy soil. Oshtemo sand was used. Cation exchange capacity and exchange— able cations were determined by the ammonium acetate method (32). Available phosphorus was estimated as that extracted in 0.1N H01 containing 0.03 N NHhF, according to the method of Bray (32). The initial pH of the soil was determined with the glass elegtrode. The soil.was screened and thoroughly mixed. Four thousand grams of soil was placed in each of the one gallon pots used in the study. Calcium hydroxide was added in an amount calculated to bring base saturation to 80 per cent with respect to calcium. Primary and secondary nutrients other than nitrogen were added at double the re- quired rates calculated from soil tests, in an attempt to make nitrogen the only limiting nutrient. Two mls of a minor element mixture suggested by Hoagland (68) was added in solution. The dry mineral amendments which were added to each pot were as follows: Ca(0H)2............ CaHPOh ............ 2 l Ego oooccooooooo 00, KC]- 000000000000 1 ............ u3 The following plant materials were used as organic amendments: Sawdustfi, lignified sawdust**, wheat straw, corn stalks¥§ and alfalfa hay. These materials were dried at 70°C before the prescribed aliquots for each treatment were weighed.and added to the soil. The materials were added at two rates of application (50 and 100 gms per poW) and mixed thoroughly with all of the soil in each pot. Three levels of nitrogen treatment were employed with all materials except alfalfa hay. At the 50-gm rate of organic amendment, the N6, N1 and N2 levels of nitrogen treatment corresponded to addi- tions to each pot of nitrogen from urea as follows: Sawdust (0, .392 and 1.177 gms); lignified sawdust (O, .6hl and 1.923 gms); straw (0, .073 and .219 gms); corn stalks (0, .126 and .379 gms). At the lOO—gm rate of organic amendment, urea-nitrogen additions per pot to achieve No’ N and N2 levels of nitrogen treatment were as follows: Sawdust l (0, .785 and 2.355 gms); lignified sawdust (0, 1.281 and 3.8h6 gms); straw (0, .lh6 and .h38 gms); corn stalks (0, .252 and .757 gms). *- Hardwood sawdust. *t'Acid-extracted sawdust prepared as follows: Hardwood sawdust w 3 treated with 72 per cent sulfuric acid w/y for two hours at 10 C and then hydrolyzed according to the method described by Norman (T3). The acid-treated, acid-hydrolyzed material was then washed with water until free of sulfates. This treatment is designed to remove cellulose and other carbohydrates, leaving a residue which, in the case of non—proteinaceous materials such as wood, is con- sidered to be principally lignin. ,1 The corn stalks included cobs and hulls in addition to the stalks themselves. /¥‘Equivalent to 12.5 and 25 tons per acre, respectively. Assuming that the materials used all contained 50 per cent carbon, the N0’ N1 and N2 levels of nitrogen treatment represented additions of carbon and.nitrogen in the following ratios: Sawdust (353:1, 60:1 and 20:1); lignified sawdust (577:1, 36:1 and 13:1); straw (66:1, 55:1 and h2:l); corn stalks (llh:l, 72:1 and bl:l). The ratio of carbon to nitrogen in the alfalfa hay was 18:1. Two check treatments were included. In one case neither organic amendments nor nitrogen were added. In the other, 1.923 gms of urea nitrogen was added per pot without organic amendment. Water was added to bring the soils to 10 per cent moisture (approximately field capacity). The soils were incubated for 110 weeks in the greenhouse. One series of duplicated pots of all treatments was planted to wheat, two crops of wiich were grown and harvested during the first 25 weeks of the incubation period. Soils were re— ,moved from the pots and remixed after harvest of the first cr0p and before planting of the second cr0p of wheat. The other series of dup- licated pots were not cropped during the hO weeks of the incubation period but were maintained at the same moisture content as the cropped series by periodically adding water to constant weight. The sawdust and lignified sawdust treatments were also applied in duplicate to a third series of pots which were then planted to alfalfa. Three cuttings of alfalfa were harvested from these pots during the hOdweek incubation period. Daily maximum and minimum temperatures were recorded from January through September, or through all but the first month of the period. Daily'maximum temperatures fluctuated moderately in the 70's and 80's 1:5 through February, when the first cr0p of wheat was harvested. From March through May while the second cr0p of wheat was growing, in- creasingly extreme fluctuations in temperature were recorded, with increasingly frequent.maxima in the 90's, or above. From June through September,:mean monthly maximum temperatures were consistently above 100 degrees F, and daily minima ranged between 60 and 70 degrees. Because of these high temperatures, no attempt was made to grow wheat through the summer, although all pots were maintained at the moisture level which had been established in the beginning. Alfalfa was grown continously through September in the pots which were planted to alfalfa at the beginning of the experiment. During this hOdweek period soil samples were taken periodically for determination of pH and estimation of microbial numbers. At the end of the to weeks, soil samples from representative treatments were taken for use in the laboratory determinations outlined in Chapter IE. At the end of the to weeks, wheat was planted in all pots as a biological indicator of residual nitrogen availability. Top growth harvested from these plants, as well as from the two crops of wheat and the three cuttings of alfalfa from the two previously cropped series of pots, were dried at 65 degrees C, weighed and ground for analysis for total nitrogen. A detailed description of all treatments included in the experi- ment is presented in Table 17 of the Appendix. Results of Greenhouse Experiment Because of the time-consuming nature of a number of the laboratory he determinations, it was not feasible to make use of soil samples from all treatments in the laboratory. Representative treatments were selected for this purpose. For the most part, samples from the high rate of residue addition (100 gms per pot) were subjected to detailed analysis, since it was felt that maximum.variations relatable to treatment would be found at the higher rate. Soil samples from the duplicate pots of each of these selected treatments were bulked and most analysis were performed in duplicate on aliquots of the bulked sample. The experimental treatments have been described in the preceeding section and in Table 17 of the Appendix. The following code will be used to relate laboratory results with the experimental treatments im- posed prior to and during the course of incubation in the greenhouse: LS: Lignified sawdust SD: Sawdust CS: Corn stalks ST: Wheat straw ALF: Alfalfa hay CK: Check 'W: The pots were cropped twice to wheat during the first 25 weeks of the decomposition period. 'W': No crop was grown during the hO week decomposition period. N : Highest C:N ratio of organic amendment (no supplemental nitrogen). N1: Intermediate C:N ratio of organic amendment (lower level of supplemental nitrogen). 2: Lowest C:N ratio of organic amendment (higher level of supplemental nitrogen). CK +~N: 1.923 gms of urea.nitr0gen added per check pot without organic amendment. Soil pH and nitrates Data in Table 8 show that the pH of the check soil to which urea 1:7 was added had increased to 7.7 two weeks after the start of the exper- iment. This was due to ammonia released by hydrolysis of the urea. Similar increases over the no-nitrogen check were observed for alfalfa hay and for the uncropped sawdust and lignified sawdust treat- ments at the N2 level of nitrogen. Soil pH was depressed at this time by lignified sawdust at the NO and N1 levels of nitrogen. This re- flects the high baseébinding capacity reported for lignaceous acidoids (61. 62, 63). Soil acidity after to weeks appeared to be closely related to the level of nitrate nitrogen in the soil. Relatively high nitrate levels following addition of alfalfa were less effective in depressing the pH than was the case with the other materials. 0f the materials used, alfalfa would have contributed the greatest quantities of mineral cations to neutralize organic and mineral acids produced during decom- position. The oxidation of lignin is known to result in an increase in acid groups and an increase in cation exchange capacity. This would explain the fact that the increase in acidity after to weeks was dis- proportionately greater for a given level of nitrate with lignified sawdust than with the other:materials. The original organic components of all cr0pped soils had been augmented by the root residues from two crops of wheat grown during the decomposition period. Evidence will be presented later to show that these root residues contributed greatly to the supply of energy materials and.narkedly influenced the biological and chemical proper- ties of these soils at the end of the decomposition period. From 1:8 Table 8 it is apparent that the net effect of cr0pping in the case of the lignified sawdust, sawdust and corn stalk treatments was to reduce the level of nitrate and retard the development of acidity following high rates of nitrogen treatment. In the alfalfa-treated soih,cr0p- ping had no apparent effect on pH or nitrate level after no weeks. 'Water-floatable materials and total carbon and nitrogen The transformation of organic materials added to these soils was probably more rapid and extensive than would have been the case in the field because of the abnormally high temperatures which attained in the greenhouse. A rough indication of the extent to which the various materials were altered in to weeks is given by the recovery of materials which floated off when the soils were suspended in water (Table 8). The percentage recovery of materials originally added without supple- mental nitrogen decreased in the order lignified sawdust >’sawdust;> corn stalks;> alfalfa. This is the order which would be expected in the light of the original carbonenitrogen ratios of these materials and what is known about the relative decomposabilities of similar plant materials. These water—floatable materials accounted for roughly % to 2/3 of the increases in total carbon which were observed in all soils to which organic materials were added. This was true in all cases ex- cept where the high level of supplemental nitrogen was used with lignified sawdust. Here the greatest increase in total carbon for any treatment (LSAbeNZ) was associated with a very low recovery of water- floatable material. The amount of relatively unaltered lignin 1:9 Table 8. - Effects of various soil amendments on soil pH after 2 and to weeks and their relation to final levels of nitrate, 'water-floatable materials and total carbon and nitrogen in Oshtemo sand.* Treatment Soil_pH Levels after to weeks After After N0 -N ‘Water-floatable Total Total 2 wks. ho wks. l 3. materials C N per acre % % lbs. of original of soil per acre amendment """""""""""""" we"--'-aw*-‘aw-w*-- ls-w-No 6.7 6.3 1 78.0 1.38 111:0 LSAWANl 6.5 6.2 32 62.8 1.h6 1320 LS-W-NZ 6.9 5.2 79 16.6 1.38 1th LSJWbéNZ 7.2 h.8 blO 22.h 1.53 1860 SID-‘Wl-No 7.0 6.7 0 5h.8 1.22 1180 sn-w-N1 7.0 6.3 77 53.2 1.11 1500 SD-X‘FN2 7.1 5.6 150 51.2 1.11 15210 CS‘J‘V-No 60 8 6 o 9 2 ’48 ch 1002 1260 CSJN-‘N2 7.0 6.6 28 30.8 1.00 lh60 CSJWb-NQ 6.8 6.2 98 23.2 1.03 1380 mar-No 7.5 6.5 136 13.2 1.00 1580 ALFJWoéNO 7.5 6.5 116 15.2 1.01 1580 CKJWéNO 7.0 6.6 h2 -- .8h 1060 CK-w-N 7.7 5.3 28h -- .78 1220 '* Organic amendments were added at the rate of 25 tons per acre. *w-Percent recovery of water-floatable materials was calculated after substracting the amount found in the check from the amounts recover- ed from the other treatments. *wt-Total carbon based on ignition of 50 gm samples of soil. 'water-floatable materials. *wtfi-Nitrates were not removed prior to determination of total Kjeldahl nitrogen. Includes 50 recovered in the four lignified sawdust treatments decreased as the level of nitrate nitrogen increased. Thus, increasing the level of mineral nitrogen in the decomposition medium hastened the transforma- tion of lignin, even though the transfonnation did not always result in proportionate losses of carbon. The failure of the transformed lignin to float may have been due in part to physical changes which increased its wettability. These changes would appear to have been related to processes of oxida- tion and complex formation which were enhanced in the presence of nitrogen. Increasing exchange capacity which results from oxidation of lignin would have increased the susceptibility of lignin trans- formation products to precipitation by the Ba012 which was added to the water in which the soils were suspended. Relative values for total carbon and nitrogen in these samples are presented in Table 9. Increases in total nitrogen over the check ranged from 7 to 75 per cent, or from 80 to 800 pounds per acre. The smallest increases were with the lignified sawdust and sawdust treat- ments which were orOpped without supplemental nitrogen. The largest increases occurred with the uncropped, N2 levels of the same materials. Increases with alfalfa, cr0pped, were intermediate and of about the same order as the increases with lignified sawdust, sawdust and corn stalks when these were cropped at the N2 levels of nitrogen. Cropping had little effect on the nitrogen level where alfalfa hay and corn stalks were used. Except for the lignified sawdust treatments, there was no marked tendency for total carbon to increase with total nitrogen as the 51 Table 9. - Relative carbon and nitrogen contents of variously treated oshtemo sand after forty weeks in the greenhouse. Treatment Relative carbon and nitrogen contents Percent of check c N IS-W-No 161; 107 ls-w-N1 17h 12h IS-W-Nz 16h 135 IS-Wo-N2 182 175 SD-W—NO 11:5 111 SD-W-Nl 132 1141 3134,7412 .. 132 1’45 SM: 0412 1110 173 cs-w-No 121 118 CS-WO-‘NZ 122 130 ALF-W-No 119 1119 ALF-Wo-N 119 1h? 0 CK-W-No 100 100 CK-w + N 92 115 52 result of nitrogen treatment. A close relationship was observed between nitrate nitrogen and total nitrogen after hO weeks. This relationship is shown graphically in Figure 2. The points for all residue-treated soils cluster about the curve established by the values for lignified sawdust and sawdust. The values for the check soils fall away from this curve but show the same upward trend. Nitrates were not removed prior to the Kjeldahl digestion for total nitrogen. Thus it is possible that some nitrates were included in the determination of total nitrogen. The quantities so included may be inferred to be small. The magnitude of the increases in total nitrogen in residue-treated soils is such as to warrant the assumption that increasing nitrate level was associated with greatly increased quantities of nitrogen in forms other than nitrate. Hydrolytic nitrogen fractions The results of acid hydrolysis and fractionation of nitrogen in these samples is presented in Table 10. The addition of sawdust or lignified sawdust alone without supplemental nitrogen resulted in very little change in the levels of total or non—acid-hydrolyzable nitrogen as compared with the check. The addition of supplemental nitrogen with residues low in combined nitrogen and the addition of alfalfa containing a high percentage of combined nitrogen resulted in marked increased in total and non-acid—hydrolyzable nitrogen. The proportion of hydrolyzable nitrogen to the total decreased as the level of non—acid-hydrolyzable nitrogen increased. Relative changes FIGURE 2 Relationship between total and nitrate nitrogen in Oshtemo sand ’40 weeks after incorporation of various organic residues. 53 CwTs.’PER ACRE-TOTAL NrrRerN 53a IQ- /" //Y/ l8L ,/’ //// l7- /, [6» ,// o LSAwDUST AA” x SAwOUST / X .51 LKS '5” x // o c A n / A ALFALFA ‘4 /' 0 CHECK *' / , a a- / / DI [2L/ 0 I H 0 [O L I s n 1 n 1 1 4 A 0 4o 80 ‘20 160 200 240 280 320 360 4—00 DOUNDS PER ACRE Nos-N 5h Table 10. - Acid-hydrolyzable nitrogen fractions in an Oshtemo sand to weeks after incorporation of various residues.* Total Non—acid Acid hydrolyzable nitrogen N hydrolyzable fractions in per cent of total Treatment % of N soil nitrogen soil % of Total acid hydroly— NH -N soil zable N were 3 *w *r* LS-W-No .058 .015 7b.98 15 .146 LS-WFNZ .065 .018 71.77 lh.6h LS-JNOAN2 .078 .032 58.89 lb.6o SD-W-No .057 .013 76.51; 16.53 SD-W-N2 .067 .018 72 .59 111.07 SD4NO-N2 .07h .026 65.50 12.15 ALFJN-No .083 .025 69.68 1h.17 CK-w-No .057 .0117 75.39 16.15 Virgin Houghton muck 3.090 .O7h 76.10 12.23 * Determinations on samples sieved through hO-mesh screen. hO grams of soil hydrolysed with 6N H01 for l2-lh hours under a reflux column. *w'Total Kjeldahl nitrogen as per cent of sieved samples.(Nitrates were previously removed by leaching with water.) *** Non-acid hydrolyzable nitrogen as per cent of sieved sample, by difference. **** Nitrogen in acid hydrolyzate as per cent of total Kjeldahl N. (Nitrates were removed prior to hydrolysis by leaching with water.) Table 10. - (Continued) 55 Acid hydrolyzable nitrogen fractions in percent of total soil nitrogen Ratio of humin N to non-acid hydrolyzable N Treatment Amino Basic Acid hydr. humin N N N 35.29 12.03 9.88 .098 LS-W-No 30.08 7.92 8.2h .083 LS-W-N2 30.60 7.77 8.b9 .085 LSJWOéNZ 36.31 8.72 9.58 .096 SD-W-No 33070 8.70 10.70 .107 513.1”qu 28.1.1 7.91 8.88 .089 SD-wO—N2 31.50 8.51; 9.26 .093 AIFJN-No 33087 10071 12 all 0121 CK'JN’NO 1.16003 7077 6031 .063 Virgin Houg‘hton muck 56 among the various hydrolyzable nitrogen fractions were due principally to changes in the absolute levels of amino nitrogen and non-hydrolyzable nitrogen, as may be seen in Figure 3. The absolute level of amino nitrogen in Figure 3 was much higher following alfalfa and cornstalks treatments than in the check. It was higher for these two treatments than for any of the other residue treatments for which data was obtained. Microbial numbers (see page 75) were three to ten times larger throughout the decomposition period with alfalfa and corn stalks than with any of the other treatments represented in Figure 3. This supports the interpretation that the acidéhydrolyzable amino nitrogen fraction represents principally materials which have been rather recently synthesized by soil micro- organisms and are present either in the form of microbial cells or as products of microbial metabolism. The amino nitrogen fraction varied erratically with the treat- ments which involved sawdust or lignified sawdust. With these two materials, however, the non-acid—hydrolyzable nitrogen fraction in- creased consistently when supplemental nitrogen was used. This increase was much greater in the uncropped soils. As has been pointed out, cropping reduced the level of mineral nitrogen in the decomposition medium. That this may have been a factor in the accumulation of nitrogen in the acid~resistant fraction would appear to follow from the data plotted in Figure h. The increase in non-acid—hydrolyzable nitrogen with increasing nitrate level in Figure h was essentially linear for sawdust and lignified sawdust over the range of values encountered. It is known FIGURE 3 Fractional distribution of nitrogen in Oshtemo sand )40 weeks after treatment with various residues applied at the rate of 2.5 grams per 100 grams of soil. 57 ‘fl «1 m 773 £88 23;” , 7///////7///7/////7/77/7////7777777ng ('1’: N a”! \\\\\\\l\\\)\\ \‘l 2 E ,1, . 777/7/7/‘7777/777.o 3% gm a;;:\\\:\\\\\1\\\\ .. g E “1,7122, \\\\\\\ 7 :33 t 3 - ‘ 5 2 , ,,,,,,,,,,, 777/777777/77/777/77gg i g #:17\\\X\\\\\\\\‘ ‘1 77777777/7/77g5.§ g g 72’7,’7\\\\\\\\\\\\\‘ J is u < 7 9 z' IW/ v g; #7 3 7:722:\\\\\\\\\\\i 1 1 1 ._ 1m 95 >~ 1,. 777777777771 . 2}; 197177,’x;.\\1\\\\\\\\\‘ W“ 1 .- . 77777777777735}; 7222”2:77;.\\\\\\\\\\\\\\\\\) 5 :2 77777777 . gig} ‘ \\\\\\\\\\\\\\ . , f 3 s 5? 3 Q 8 S f; 3:“ mos swvse om use N Sew 83$? 58 that complexing reactions between lignaceous constituents of plant residues or soil humic materials and ammonia or proteinaceous nitrogen compounds give rise to chemically resistant substances. In this connection, it is pointed out that maximum levels of non-hydrolyzable nitrogen were found here in materials known to contain relatively large amounts of lignin (alfalfa, sawdust and lignified sawdust). The curve for total Kjeldahl nitrogen in the upper half of Figure b shows a distinct tendency to level off at high levels of soil nitrate. This would suggest that incorporation of nitrogen into re- sistant fractions may become increasingly important as rates of nitrogen fertilization are increased or as the ratio of carbon to nitrogen in the soil system decreases. Such a relationship was found in the soils studied here and is depicted in Figure 5. If the points for lignified sawdust treated samples in Figure 5 are considered by themselves, non-acid—hydrolyzable nitrogen was found to decrease with increasing soil C:N ratio in a curvilinear pattern, approaching the level in the check soil at the widest C:N ratio. The points for the other materials and the check sample clustered along a similar curve which was displaced in the direction of lower C:N ratio, or a higher nitrogen content. Thus it would seem that an increasing level of nitrogen in the soil promoted an increasingly intense fixation or immobilization of nitrogen in chemically resistant combinations. The quantity fixed was a function, not only of nitrogen level, but also of the quantity of carbonaceous compounds present that were capable of fixing nitrogen in this form. For example, the displacement to the left of the curve FIGURE b Total soil nitrogen and non-acid-hydrolyzable nitrogen in Oshtemo sand as related to nitrate nitrogen after I10 weeks. 59 a 6‘ 0 M65 0? wA‘rt-Q \NSOLUBLE N PER 100 6M5 0F Son. M 8’- 3 o N 0 IO A o I, ’A /’ x —. u ///’ , 3” TOTAL N ’3’ x O,” / / / I / 1.. / it” X 0 O Ll6N\FIED SAwouST X SAWDUST .. CJ CORN STALKS A ALFALFA 0 CHECK O L. _, /’?< A I’l’.’ c: O’,,*” X ’I-” Non-Amo-HYORO LYzABLE M -’ 0 FOR $753005? AND LlGNnHED sAwous-r Y: we + .1721x T: . 4'7 1 1 | l l J L l _L l o 2 4 6 8 7012147678 20 nos OF WATER SOLUBLE Nos-N ‘Peaxoo 6M5 on son. FIGURE 5 Relation of non-acid-hydrolyzable nitrogen to C:N ratio of the soil in Oshtemo sand 110 weeks after treatment. 60 60a J 8 32— w 0 LSAwoos‘r 7- X ANGUST \D 30" 5 o D c.5TALKs 2 A ALFALFA 328% o caecx e m at f: “’24” cf 22* N 3 cc 20" o )s I (8’ [1 X 9 (J _ < 16 6 z (4’ e _.V (2... lo I l L l :2 I4 16 (8 7.0 22 C:N RATIO OF SOIL 61 for sawdust, alfalfa and corn stalks would appear to have been due to a greater respiration loss of carbon resulting from preferential micro- bial attack on the readily available carbohydrates and proteins with which the lignin (or lignin-like) constituents of these materials were diluted. The fixation of ammonia by lignin in the laboratory is influenced by pH as well as ammonia concentration. Appropriately high pH's and ammonia concentrations did exist during the early stages of decompo- sition in the soils to which large amounts of nitrogen were added either as urea or in the form of alfalfa hay. ‘Whether active complex formation occurred primarily during this early period or more or less continuously throughout the to weeks is not clear. The performance of successive wheat crops grown with lignified sawdust suggests that initial fixation of ammonia may have been by relatively weak exchange mechanisms, and that ammonia so fixed became progressively more re- sistant to release as decomposition progressed. These results will be discussed later (page 85). In these Oshtemo samples there was no evidence of an initial attack on this resistant nitrogenous fraction in the presence of fresh plant materials such as was found in the Sims clay loam samples one year after a field application of sawdust. No simple explanation appears at this point to account for this result. Differences in mineral nitrogen supply, differences in quantity and degree of oxidation of humic materials originally in the soil, and differences in aeration related to soil structure and conditions of moisture and temperature in the field and in the greenhouse may have been involved. 62 On the other hand, two distinct and essentially independent mechanisms of nitrogen accumulation were seen to be operative in both soils. One was the accumulation of amino nitrogen associated with a large and active microbial popilation in the presence of a ready supply of energy materials. The second mechanism was the chem- ical fixation of nitrogen by lignaceous constituents of plant residues and soil humic materials. The latter became increasingly important as the ratio of carbon to nitrogen in the soil decreased. The ratio of humin N to non-acid-hydrolyzable N in these samples (Table 10) was much lower and more variable than in the case of the Sims samples (Table 5). This tends to undermine the hypothesis that acidéhydrolyzable humin constituents represent equilibrium products of hydrolyzable materials represent the resistant core (cf. page 33). However, it would be expected that such an equilibrium would be in- fluenced by the state of oxidation of the parent substances, as well as by the extent to which clay minerals might be integrated with the parent structures. The relationships which exist between these two fractions would appear to deserve further study. Alkali-extractable nitrogen Table 11 shows the amount of nitrogen extracted in alkali and its distribution between the humic acid and fulvic acid fractions for four selected treatments of the Oshtemo sand. Nitrogen not hydrolyzed by acid (from Table 10) is also presented for comparison. Approximately 55 to 60 percent more nitrogen was extracted by alkali from the three residue-treated soils than from the check. 63 .OH edema seam somOHpH: oHemNhHoapheleflowIsoz.*** .Haom mo new OOH mod wemhm m.m «penance mo deem .sowoppfls oa.eo>Hoooa soars_xooeo one pdooxo «mamboa «2 as poor; on someone mpooEpmoap HH<.** hmlwamwewl .smoH mmao msflm pom eobwaomoe mm eons oaaeooond 08mm * ”.mm w.nm n.6N a.am sameness Hence no encoded o.®H 0.:H o.©a o.wH new 00H hog mweloowoapwo rte oanmmaaoaean ease eoz m.wH m.ma m.©a :.m m.m m.wa m.mH Hmpop we assumed new OOH pom owe m.ma m.a m.m m.a o.a N.ma r.ma momenta: sacs oasesa o.mw N.NH ~.>H 0.0m o.mm N.mm ©.mm Aesop mo essence new OOH pod mws o.oa a.m d.m m.mm m.mm o.ra m.oa assesses sacs oases Hamxam ma eopomupxo sowouvfls 4.0: m.mm p.4m N.mm m.H: m.o: Q.H: Hmom Hmpop mo pnoouom new 00H nod mwe Hamxam ea sweetness m.mm :.mH :.ma o.Hm m.om m.mm m.mm :owonpae HHom Hosea m.ma m.am m.mm o.da m.ma o.ma a.me new ooa sod ems oowohpfis Hflom Hence H .eom HH .aom H .aom HH .aom H .eom HH .aom H .eom madden esoo moose sarcasm peasant eoaeaswaq **.peoevmoup cowpownm sowoapfiz *.coapanomsoooe .mxoos o: nevus esmn osopnmo or» mo scenery“ cabaam new cash: new no emanates msofihme mo upcommm I .HH canoe 61.: Most of this increase occurred in the humic acid fraction. The increases in alkali-soluble nitrogen corresponded to a 28 percent increase in non-acid-hydrolyzable nitrogen in residue-treated soils relative to the check. In the Sims clay loam, both alkali-soluble nitrogen and non-acid hydrolyzable nitrogen were reduced initially by sawdust treatment (Table 6). This difference between the two soils was most likely due to the higher levels of supplemental nitrogen used with the organic amendments applied to the Oshtemo soil. A high level of nitrogen in the decomposition medium promoted the incorporation of nitrogen into the non-acid-hydrolyzable fraction, as has been shown (Figures h and 5). The fact that non-acidehydrolyzable and alkali-extractable nitrogen fractions showed a tendency in both soils to vary together in their relative and absolute proportions supports the contention that certain organic constituents were common to both fractions. In the Sims samples, the decline in alkali-extractable nitrogen continued over a longer period of time than did the decline in non- acidehydrolyzable nitrogen. In the Oshtemo samples, the increase in alkali-extractable nitrogen was twice as great as the increase in the acid-resistant fraction. both of these results support the view that the alkali-extractable materials include a significant portion of compounds which represent intermediate stages of transformation. As such, they would be expected to accumulate during the early stages of decomposition of fresh plant materials. Where mineral nitrogen in the soil was low, increases in alkali- soluble nitrogen might be noted principally in the fulvic acid fraction, 65 as was Observed the first year after addition of sawdust to the Sims clay loam (Table 6). In the presence of nigh levels of nitrogen, processes of complex formation involving lignin-like materials would be enhanced and their products would be expected to appear in the humic acid fraction, as was the case in the Oshtemo samples (Table 11). At later stages of decomposition, this humic acid nitrogen might appear in either the more highly oxidized materials resistant to acid hydrolysis or in the hydrolyzable amino nitrogen fraction, de- pending on the degree of oxidation or the supply of ready energy materials to promote microbial assimilation. In the Sims soil increases in both the hydrolyzable amino fract- ion and the acid-resistant fraction of nitrogen concurred with a de- cline in alkali-soluble nitrogen in the third and fifth years (compare Fig. l with Table 6). The Oshtemo data represents only the early stages of decomposition and provides no basis for postulating later trends. Mineralization of carbon and nitrogen Nitrogen and carbon bmnobilized in organic compounds is released, or mineralized, primarily by microbial decomposition of these organic compounds. In a given soil, the net mineralization of organic nitro- gen will depend on the total quantity present, its availability to microbial attack, and the proportion of carbon to nitrogen in the medium to meet the assimilative requirements of the microorganisms. It was of interest to know to what extent the observed differences in quantity and distribution of carbon and nitrogen in tin; Sims and 66 Oshtemo samples.might be correlated with differences in the rates of mineralization of carbon and nitrogen during incubation under controlled conditions. In Table 12 are assembled pertinent data for the Oshtemo samples. Carbon mineralized was taken as a measure of the availability or decomposability of organic materials left in the soil after the ho- week decomposition period. As seen in Table 12, there was no correla- tion between carbon mineralized and the total carbon in the soil. 'With all materials, however, except alfalfa, carbon released as 002 decreased sharply as the level of nitrate in the soil increased. It was pointed out earlier (Table 8) that the quantity of unaltered or water-floatable materials which were recovered at the end of no- weeks decreased generally with increasing nitrate level. This suggests that carbon mineralized during incubation might have been related to the amount of water-floatable materials, since these might have re- presented the most readily available energy materials in the incubating samples. The upper half of Figure 6 shows that a generally direct relationship did exist in the case of all treatments except sawdust. A.much closer inverse relationship was found between 002 evolution and total nitrogen as is seen in the lower half of Figure 6. The same inverse relationship between nitrogen content and de- composability of soil organic materials was observed in the sawdust- treated Sims clay loam samples (Table h), where a one-third increase in nitrogen content from the first to the third year was accompanied by a two-thirds reduction in C02 production. As shown in Figure h, increases in total nitrogen in Oshtemo 67 Table 12. - Total carbon and nitrogen and nitrate nitrogen in Oshtemo sand ho weeks after incorporation of residues and their relation to the mineralization of carbon and nitrogen during incubation. Total C and N Pounds_per acre Nitrate Nitrifiable Carbon C:N of Treatment 0 N C:N N N mineral-l mineral- % % ized ization Lsewewo 1. 38 .057 2h. 2 (l7 (87 figi (h; Lsewuwl 1.h6 .066 22.2 32 35 h09 1h.3 LSJWéNZ 1.38 .072 19.0 79 37 2h2 6.5 Ls-wo-w2 1.53 .093 16.5 510 19 131 6.9 spewswo 1.22 .059 20.7 0 0 790 0° snawle 1.11 .075 1h.8 77 38 311 8.2 spew-4N2 1.11 .077 lb.h 150 32 258 8.1 SD-wo-N2 1.18 .092 12.8 372 to 159 11.0 cs-w-No 1.02 .063 16.2 2 1.3 615 117.3 cs-w-N2 1.00 .073 13.7 28 us 368 8.1 cs-wo-N2 1.03 .069 117.9 98 A9 302 6.2 ALFJWANO 1.01 .079 12.8 136 58 310 5.3 ALF-Wo-No 1.00 .078 12.7 110 55 30h 5.5 gK-W-No .81. .053 15.8 A2 27 11.2 5.3 cxaweN .78 .061 12.8 28h 17 128 7.5 (l) Nitrate nitrogen in initial water extract of air dried soil. (2) Nitrifiable nitrogen by incubation at 35°C for 1h days. (3) Total carbon evolved as 00 in 10 days at 35°C. (h) Ratio of carbon mineralize8 to nitrifiable nitrogen. FIGURE 6 Comparison of carbon dioxide evolution for various soil residue treatments with water-floatable material and total nitrogen after 1:0 weeks' decomposition. 68 8 TOTAL M65 OF CO; EVOLVE D 68a 0 CH ECK 0 CHECK+ N A ALFALFA Ocean STALKS 0 L. SAWDUST /’ X SANDUST / t I l l I [50- e 9 TOTAL. mes 06 C02 EVOLVED a T l L 1 1 _‘ _ .2, .4 .b .8 [.0 (.2. (.4 (.4, (.3 2.0 one PERIOOGMS H10 FLOATABLE MATeRlAL l 4 L I l 4 1 5 5‘ 6 o 6 5‘ 7O 75 80 85 90 05 loo PERCENT TOTAL sou. M (:00 3) 69 samples were accompanied by increases in non—acid-hydrolyzable nitrogen, although the rates of increase were not parallel. In the upper half of Figure 7, carbon mineralized during incubation is plotted against non-acid—hydrolyzable nitrogen. The values for lignified sawdust and sawdust fall together on a smooth curve. Those for corn stalks and alfalfa fall above this curve, an observation which appears to be related to the higher content of hydrolyzable amino nitrogen which was found in these samples (Fig. 3) and the greater availability of associated energy materials which may be inferred from the nature of the orininal materials themselves. The significance to nitrogen mineralization of the close relation- ship Observed for sawdust and lignified sawdust treatments between non-acidrhydrolyzable nitrogen and decomposability is suggested by the data plotted in the lower half of Figure 7. The mineralization of nitrogen during incubation was suppressed at low levels of non-acid- hydrolyzable hydrogen. This suppression was due to microbial immobil— ization in the presence of excess energy materials, as may be inferred from the fact that no supplemental nitrogen was applied.with the cor- responding sawdust and lignified sawdust treatments. Intense microbial immObilization of nitrogen may also be inferred from the high rate of CO2 evolution at corresponding nitrogen levels in the upper graph. Maximum.mineralization of nitrogen in Figure 7 coincided.with moderate levels of microbial activity and intermediate levels of acid- resistant nitrogen. Reduced mineralization of nitrogen at high levels of non-acidrhydrolyzable nitrogen was associated with a high degree of resistance to microbial attack, as reflected by the low rate of 70 002 production in the upper graph. The curves in Figure 7 have been drawn to emphasize the ideal relationships which appear to be reflected by the data for sawdust and lignified sawdust treatments. The chemically resistant materials in soil do not appear to be inert. It is generally recognized that they represent products of complex formation, and that they may them- selves represent active complexing agents. The data for the two saw- dust materials in Figure 7 suggest that the complexing potential of materials resistant to acid hydrolysis may exert a strong controlling influence on the transformations of carbon and nitrogen in the soil. The degree of control expressed by this fraction will vary with the quantity and nature of associated organic materials. This is shown by the fact that values in Figure 7 for the check soil and for soils treated with corn stalks and alfalfa do not fall on the same curve as those for the sawdust and lignified sawdust. The effects of associated.materials, however, appear to resolve themselves primarily into differences in their availability as energy substrates for the soil micrObial population. This point is brought out more clearly in Figure 8, where the carbon and nitrogen mineralization data are plotted against C:N ratio. In the upper graph in Figure 8 it is seen that the mineralization of carbon was greater at any given C:N ratio for the sawdust treatment than for lignified sawdust. This would be expected since the more readily available energy materials had been removed from the latter by acid extraction. The residual materials from corn stalks and alfalfa were decomposed.more rapidly than those from sawdust, reflecting the FIGURE 7 Relation of non—acid—hydrolyzable nitrogen in the soil to the mineralization of carbon and nitrogen in Oshtemo sand. 71 71a 80w- K \ 700L \\ o L. 5Awou 5T \ g} X X SAwousr g 6 \ Q C. STALKS O 04 \\ A ALFALFA ma \ 0 CHECK 0:3 500‘ \‘ U \\. 't a \ Q- 5 «hi- \\ a4 x a < \ m {g \ 3 2 h" \\\ A «2 v to § 2m. \\~- (g ~~~~~~~~~~~~~ < O --------- u too- I I l 1 1 IO (4 £8 22. 26 30 a a a a LBS PERACRE M n‘RerN MINERAHZED N I4 0&5 6 Mes NON ACID HVDKOLYzABLEN-PER 100 6M5 sou. A (1 /”’ ‘\\\\ z X \ O/x/ / ’X I / \ O ,1/ \\ l/ \\ / / I I I / I I X L' 4: \ l I L :0 l4- :8 22 26 30 M65 NON ACID HYDROLYZABLE N PEK. I00 ONE 50! L. FIGURE 8 Carbon and nitrogen mineralized during incubation as related to soil C:N ratio and plant residues applied 11,0 weeks previously to Oshtemo sand. 72 72a AA 3 ' (6 Son. (1: N RAT|O 22 20 ’8 f4 _ . t p F M «... 3 w m 5 mica 0‘ 3 ommiekali 29320 30% 9.5 03 Fun we: (2.. O LSAvasT K 5Aw0u5‘r a (LSTALKS fl mum LCC \ AEE NH.“ ACC Ado r p b _ — p #44. .v N... MW W2~E WMQQNJE muons \ 3 Q Stu. i J.Om.m.zw 09ka wt?! 20 ‘6 son. C:N RATco l4 I7. 73 normally greater decomposability of these plant materials, as well as the much higher hydrolyzable amino nitrogen content found in these soil samples. The mineralization of nitrogen from sawdust treated soils (lower graph, Fig. 8) was inversely related to C:N ratio." This was true also for the alfalfa-and cornstalk-treated soils considered as a group, although the release of nitrogen was more rapid at a given C:N ratio for these than for the sawdust series. The release of nitrogen in these samples was inversely related to 002 evolution, or micrdbial activity. In other words, increasing rate of mineralization of nitrogen was due in part to decreasing intensity of microbial immobilization which was associated with decreasing C:N ratio. The three lignified sawdust treatments and the checks showed a different behavior. Maximum.mineralization occured in the lignified sawdust sample which had an intermediate C:N ratio and fell off sharp- 1y at lower and higher ratios. A heavy application (962 lbs per acre) of nitrogen to the check soil, which resulted in a narrower C:N ratio, also resulted in a reduced rate of nitrogen mineralization. From the carbon mineralization data in the upper graph in Figure 8, it is seen that both the check and lignified sawdust treated soils contained.smaller amounts of readily available energy materials at any given C:N ratio than the soils to which other organic amendments had been added. The decline in nitrogen mineralization rate following high levels of nitrogen amendment in these soils of low energy content is presumed to have been due to the formation of resistant chemical complexes of nitrogen with lignin or soil humic materials. As was 7h seen in Figure 5, the formation of such complexes was greatly increased in soils of low C:N ratio. Numbers of bacteria and fungi Periodically during the ho week incubation period, plate counts for bacteria and fungi were taken to determine the effect of the various treatments on the soil micrObial population. The results of these counts are tabulated in Table 22 of the Appendix. Data for re- presentative treatments are presented graphically in Figure 9. The counts shown in Figure 9 for the second week of decomposition were made on samples taken after the first crop of wheat was up in the pots in which wheat had been planted. Those for 15 and 25 weeks were taken after the harvest of the first and second wheat crops respective— ly. Consequently, the first three counts were influenced by the presence or absence of living roots and root residues from an associated wheat crop. All pots were uncropped during the period from 25 to ho weeks, so the counts at hO weeks reflect the residual effects of the initial treatments plus modifying residual effects of previous cropping as carried over a ldeeek fallow period. In Figure 9-A, it is seen that, where no nitrogen was applied, bacterial pOpulations were consistently higher with lignified sawdust and sawdust than in the checks. Comparison with Figure 9-B shows that bacteria were three to ten times more numerous where alfalfa hay was incorporated than with the sawdust materials. These results are in keeping with recognized differences in decomposability, or energy- availability, of similar plant materials. These results have already Figure 90 A. B. C. D. E. 75 Effects of various organic amendments and nitrogen on numbers of bacteria and fungi over a ho week period with and without crOpping.* Effects of lignified sawdust and sawdust without sup— plemental nitrogen on bacterial numbers compared with the unamended check with and.without nitrogen. Effects of alfalfa hay on bacterial numbers. Effect of nitrogen level and crop on bacterial numbers in soil treated with lignified sawdust. Effect of nitrogen level and crOp on bacterial numbers in soil treated with sawdust. Effects of nitrogen level and crop on fungal numbers in soil treated with lignified sawdust. Effects of nitrogen level and crop on fungal numbers in soil treated with sawdust. All amendments were added at the rate of 2.5 grams per pot, or 25 tons per acre. k 9 BACTERIA x/ob BACTERIA x/o" Fun/<71 x10; 25‘ $’ 2? a 75a J r L l 1 627‘l ‘W’\ «s \ 0 '\ 0’ \ - ‘7/ 40 \x \ ’ \ \ /’~S / \ y, \0. \\ / \ \ ‘(v 30" \/ \ "\\ . \ \ \ \ zo« \ \ \\ wa ‘ / \ x‘, \ ,2" \ ..W ‘~v . ’/ IO \30/ 1 t 1 25- Q33) ’} \ 20« \\ F \ \ 15 ‘ ”‘ - \ // \d‘ \ .I’ t \O\‘ ’A.\ \ik [Us I, \ \ \ I, \ \" / \\ N d - Z; 5“ I I \O'Spvw- ' - ' ‘ x \ so—YBS—da’” " A A p _l 2, [5‘ 25‘ 4-0 76 been alluded to in support of the conclusion that the acid-hydrolyzable amino nitrogen found in these soils after ho weeks represented materials recently synthesized by soil microorganisms (page 56). 'When a large amount (equivalent to 962 pounds per acre) of urea nitrogen was added to the check soil in Figure 9-A, bacterial numbers were depressed relative to the no—nitrogen check through the first three samplings. ‘When nitrogen was added with lignified sawdust and sawdust (Figs. 9-0 and 9-D), bacterial numbers in the first two samplings were generally larger than with the same materials at the No level of nitrogen treatment (Fig. 92A). However, in the last two samplings, bacterial numbers were sharply depressed by the N2 level of nitrogen treatment. The same depressive effect of high nitrogen levels was expressed on the fungal population, as is shown in Figures 9-E and 9-F. The low numbers of microorganisms which were found after no weeks in the lignified sawdust and sawdust treated soils following extremely high levels of nitrogen treatment correspond to the low rates of 002 evolution which were obtained during subsequent incubation of the same soils (Fig. 6). Thus it would appear that both microbial numbers and CO? evolution reflected the decreasing availability of energy mater- ials which was associated with increasing levels of non-acidéhydroly- zable nitrogen (Fig. 7). It may be argued that microbial numbers and activites in these samples were influenced by pH rather than by intrinsic differences in decomposability of residual energy substrates. It is true that pH levels in the soils which had received N2 levels of nitrogen treatment 77 were low (Table 8). However, no smoothly regular relationship was found between 002 evolution and pH such as was found between CO2 evolution and non-acid—hydrolyzable nitrogen with sawdust and lignified sawdust. Levels of nitrate in the soil after ho weeks were obviously im— portant in determining the final pH of the variously treated soils. However, the pH at any given nitrate level was also influenced by plant origin of the organic amendments (see discussion on pagelf7). It must be assumed that nitrates were in equilibrium with a dynamic soil system in every case. Since nitrates in soil are the end products of a system of oxidative processes, their absolute level in the absence of cropping or leaching losses must reflect the degree of oxidation attained in the total system. A linear relationship was found between nitrates and non-acid-hydrolyzable nitrogen in these Oshtemo samples (Fig. h). This resistant nitrogen fraction appears to have been the product of processes of complex formation involving lignin and ammonia, which are known to be oxidative in nature. Thus, increasing levels of nitrate and non-acid hydrolyzable nitrogen both appear to have been the result of the increasing degree of oxidation of organic residues in these soils. Increasing levels of initially added nitrogen hastened the dissipation of masking high- energy materials and the subsequent oxidation of resistant lignaceous constituents. The associated decrease in availability of energ: appears from this data to have been at least as significant a factor as pH in the decline in microbial numbers with progressive decomposition, as well as in the Observed relationship between 002 evolution and 78 acid-resistant nitrogen. Striking effects of cropping on micrObial numbers at N2 levels of nitrogen were observed (Fig. 9). Bacterial numbers were higher in the first sampling in the pots where wheat was grown in the pre- sence of lignified sawdust at the N2 level of nitrogen treatment than where no wheat was planted (Fig. 9-C). The same thing was true in the first two samplings from the corresponding sawdust treatments (Fig. 9-D). 'The behavior of fungi was essentially the same as for bacteria with both materials (Fig. 9+E and 9-F), except that numbers were increased by cropping to an even greater extent in the first sampling than with bacteria. Three distinctly different effects of cropping may be inferred from the data at hand. As has been pointed out (P.h8 ), a principle effect of the two wheat crops, in the case of sawdust and lignified sawdust treatments, was to reduce the level of mineral nitrogen in the soil. A part of this reduction would have resulted from uptake and removal of nitrogen in the harvested portions of the wheat. A part would have resulted from microbial immobilization in the presence of energy materials contributed to the soil in the form of root residues and, in the case of the growing crop, in the form of root secretions or abraded fragments. The removal of mineral nitrogen by these two processes associat- ed with the crop would have moderated the effects described earlier of nitrogen level on oxidative processes. A third effect of the crop is indicated by the magnitude of the stimulus to microbial numbers two weeks after planting of the first wheat crop. At this stage of F‘v'w 79 development the wheat could not have contributed greatly to the supply of energy'materials in the soil. The effect here appears to have been of catalytic prOportions. Numerous heterotrophic organisms have been shown to require small amounts of certain amino acids, organic acids or vitamins as essential growth factors in their external environment (58). The supply of such growth factors would be extremely low in such materials as sawdust, and even lower after acid extraction as in lignified saw- dust. On the other hand, it has been shown that a number of such compounds are secreted by living plant roots (29). The marked stimulus to:microbial numbers of the first wheat crOp two weeks after planting would appear to have been due to rhizosphere effects of such secre- tions. Following sawdust treatments, this apparent rhizosphere effect was still in evidence prior to planting of the second wheat crop 13 weeks later (Figs. 9-D and 9-F). With lignified sawdust, however, it had completely disappeared by the time of this second sampling (Figs. 9-C and 94B). Since most of the more readily available energy materials had been removed from the latter material by acid extraction, only a very transient benefit from catalytic root secretions would have been expected. A final point of interest in Figure 9 is the fact that microbial numbers were better maintained to the end of the decomposition per- iod where no nitrogen was used with sawdust and lignified sawdust than where high levels were employed. The high levels of energy materials indicated by bacterial numbers in these no-nitrogen samples was re- 80 flected again by their high rate of CO evolution during the subse- 2 quent respiration experiment. The distribution of nitrogen among hydrolytic fractions was essentially unaltered from that in the check by lignified sawdust or sawdust when no supplemental nitrogen was used (Fig. 3). This would imply that the larger numbers of bacteria found were the result of more rapid recycling of nitrogen contained in microbial cells, rather than by net increases in microbial proteins, since the latter would have resulted in increased amounts of amino nitrogen. Large changes in nitrogen distribution occured only when supplemental nitrogen was used. The data for microbial numbers and CO2 evolution both point to rapid depletion of readily available energy materials and extensive oxidation of resistant lignin constituents as factors responsible for the increase in acid-resistant nitrogen when supplemental nitrogen was used with sawdust and lignified sawdust. Nitrogen uptake and crop yields Yields and nitrogen uptake by three successive wheat crops are tabulated by greenhouse pot numbers in Table 18 of the Appendix. The treatments which correspond to each pot are described in Table 17. The overall relationship between yields of wheat and nitrogen uptake is shown in Figure 10. The high correlations obtained for each crop indicate that nitrogen availability was a controlling factor in determining wheat yield in most pots. A number of divergent points plotted for each crop, however, suggest that other factors may have been operating also. This is further substantiated by the extremely 81 FIGURE 10 Correlation between yield and nitrogen.gptake of three crops of wheat grown on Oshtemo sand in the greenhouse. YIELD 6M5 g F\QST CROP 7. 6— 5.. 4- 3‘ 0 Z- \og 9 :1 ... t4e+.447 \ogx3 5,,‘2170 v = .80?“ L._ \ I \ 1 i I l l I L J O 97 ° 3+ 7., SECOND CROP 61 5+ 4... 3‘ 2+ ‘0892.—8\§+j43logx‘) SxytJZD r=.84o“‘ n 0 \ J l I l ‘ I t \ \ I 10 30 40 50 too 200 300 NFC ROGEN uPTmomqw who: :mmoppac mo movom * .mxooz m ca do :oxmp amMthfl: pom om05o0hm hmppwa hum *¥* .mmawmam how quflvpsO m pom pawns you macho N seam smppme and Hmpoe ** .HHom mo msmhm coo“: mom manna * Jaum NOH HH.m me wa.OH = % H40. OOH = om.m 4m HH.~ wmw 0:.w mmamma4 o 00H = Hm.m 5m om. HAM om.:H = me.H OOH pmsvsmm SA a Na. 2 4m; pawns o 03 smashing mm.m om wm.m men mm.HH = % Hmm.a om = mm.m mm mm.m wwm om.m «mammH4 o om : 97m ma E. new 5.1” ._ So. om inseam $4 ow we; 3 3. pass» 0 om earning am.m mm mm.m NNH mm.m = map. OOH = mm.m mm pm. HON m~.w mmamma< o 00H 2 moom MN 0m.m Hp :H.m = mm». OOH z Hw.H : ma. m Mm. among 0 OOH pmzpsmm Hm.m mm om.m paw om.m = mam. om = N:.N m: mc.m cam Hm.m mmamma< o om = NHom mm wmoH MN. mmom = Nam. Om 2 2. N 3 as. 0 me. pass; 0 om $5st om.: wHH H~.m owm mm.w = mmm.H = mn.m mm Nm.H NOH om.w among 0 Hohscoo EEWS diam $5 54% *a® *Em ofimmafi mummvmfi ®prm§ EOHm Umfifim Paofivfimfiw cw z a oomohpfiz UHon cowohawz macaw mono aomonpwz ovum aflaowso mxoms o: nmpmo popqwdw pawns mo oncommon Hwoofimom axon: o: pmnflm namepomne .mono poms: mnwomoooSm a no oxmpmo ammonpan use uaoflh no mpoommo HmoUHmon esp one «phoneme Umamacmwa one pn§UIMn nvfis damn campamo Ho semapmonp mnHBOHHom exact o: Pnuflh 02¢ wnwhfio nichm wHHwMHn Ho mMQprfio owns» vow pawns mo macho 03D Mo oxMPma sameness tau oHoHH I oafi bands Fxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxw 9h 'Where no nitrogen was used, total yield and nitrogen uptake of the two crops of wheat grown during the first to weeks were severely curtailed by sawdust and lignified sawdust treatments. By contrast, the dry'metter produced and the nitrogen taken up by three cuttings of alfalfa during the same period in the presence of these materials without added.nitrogen compared favorably with dry matter yield and nitrogen taken up by wheat on the control soil to which supplemental nitrogen had been applied. The addition of nitrogen with lignified sawdust increased yields and nitrogen uptake of alfalfa. 'With the higher level of raw sawdust the addition of nitrogen depressed alfalfa yield and nitrogen uptake. Presumably this reflects the competitive microbial tie-up in the presence of sawdust of some other nutrient, probably phosphorus, since the results with and without nitrogen at the low level of sawdust addition indicate that the ability of alfalfa to.fix atmospheric nitro- gen was not greatly impared by the presence in the soil of rapidily decomposing sawdust. It may be assumed that nitrogen taken up by wheat during the first hO weeks represented the actual availability of nitrogen from soil sources during this period, particularly where no nitrogen was used in conjunction with the organic amendments. Nitrogen taken up by alfalfa in excess of this may be ascribed to symbiotic fixation from the atmosphere. On this basis, alfalfa fixed 20h mgs of nitrogen as an average for the two levels of sawdust treatment without supple- mental nitrogen and 2h? mgs as an average for the two levels of ligni- fied sawdust. On an acre basis, these would have been equivalent to E_.___—_ 95 102 and 123 pounds per acre of nitrogen fixed during this hOdweek period. This compares favorably with reported annual rates of nitrogen fixation by alfalfa in field and greenhouse studies (88). The higher figure for the lignified sawdust reflects the less intense microbial competition for nitrogen and other nutrients in the presence of this relatively refractory lignaceous material, as well as the greater availability of ammonia nitrogen which was presumably held on the lignaceous complexes by base exchange forces during the early stages of decomposition. These results were paralled by those for wheat, since the highest yields and nitrogen uptake for any treatment during this period were achieved where wheat was grown with supplemental nitrogen in pots treated with lignified sawdust. The residual availability after hO weeks of applied nitrogen in the control soils was high. This is shown in Table lb by the fact that 118 mgs of nitrogen was taken up from the nitrogen treated con- trol by the third wheat crop to produce tissue containing b.36 per— cent nitrogen. These values were the highest for any treatment. Where this third crOp of wheat followed wheat grown in pots treated with sawdust or lignified sawdust without nitrogen, the avail- ability of nitrogen was about as low as it had been in the case of the first two crops. Where supplemental nitrogen had been added with these materials before the first two wheat crops, the residual avail- ability of nitrogen to the third crOp was greatly increased in the case of sawdust but not in the case of lignified sawdust. It is evident that rapid dissipation of energy materials in the sawdust in the presence of supplemental nitrogen had shortened the period of intense micrObial immobilization and that nitrogen from dead cells 96 left by'a declininglnicrObial population was being mineralized and was contributing to the available supply after to weeks. The low residual availability of nitrogen with lignified sawdust appears to be related to the fact that maximum yields of dry matter had been produced in the two proceeding crOps of wheat with this treatment. Maximum quantities of root residues would have been added to the soil and microbial immobilization at the time of the third crop would have been intensified relative to the comparable sawdust treatments. ‘Where no nitrogen was used with the two sawdust materials and alfalfa was grown during the first ten months, a large residual benefit to the following wheat crop from alfalfa was observed in all cases except the high rate of sawdust application. That nitrogen was still limiting with this treatment is indicated by the fact that only a moderate level of nitrogen was found in the harvested wheat tissue (2.38 percent). That the growing alfalfa had contributed to some ex- tent to the more rapid decomposition of the sawdust is shown by the fact that nitrogen uptake by the third wheat crop was greater than where wheat had been grown during the first hO weeks without nitrogen and in sawdust treated pots. however, at the higher rate of applica- tion, nitrogen from alfalfa was inadequate to promote maximum decomposi- tion of energy materials. As a result, microbial immobilization was still a dominant factor in nitrogen availability after to weeks. Where nitrogen had been applied in the beginning, the maximum residual benefit from this applied nitrogen in terms of wheat yields was found.where alfalfa was grown during the first ten months in pots treated with lignified sawdust. The residual benefit was less hlIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIII-IIIIIIIIIIIIIIIIIIII ---J 97 where sawdust had been used, but it was still greater after alfalfa than after'wheat. Although wheat yields were greater after alfalfa with nitrogen treated organic amendments than after wheat in the nitrogen treated control, nitrogen uptake and nitrogen content of wheat tissue was less. The extremely high nitrogen content of wheat from the nitrogen treated control suggests that the nitrogen level in the soil was ex— cessively high and that a toxic effect may have limited yields with this treatment. Certainly an excessive amount of nitrogen was used (962 pounds per acre), and a distinctly toxic effect was observed on the first crop of wheat (Fig. 11). The highly beneficial residual effects of lignified sawdust Observed in Table 1h where nitrogen was applied in the beginning at rates of .6hl and 1.281 gms per pot (320 and 6h0 pounds per acre) may be interpreted in part as a protection against excessive nitrogen concentrations afforded by the buffering action of the lignin. The results with sawdust show that the injurious effects of massive additions of such low nitrogen materials can be materially re- duced if sufficient nitrogen is used. They show, further, that such corrective fertilizer treatment may be much more effective if combined with the planting of a stronglegtme such as alfalfa. The broadcast- ing of carbonaceous materials such as sawdust on established stands of alfalfa suggests itself as a practical means for handling such materials. This would minimize the economic losses which occur due to micrdbial.immobilization of nitrogen when non—legumes are planted after incorporation of residues low in nitrogen. If organic materials high 98 in lignaceous constituents are used, the favorable effects of lignin which were Observed.here in the case of the acid-extracted sawdust may enhance the residual benefits from their use. SUMMARX AND CONCLUSIONS: PART ONE Mechanisms of Nitrogen Immobilization Two distinct mechanisms were Observed whereby nitrogen was immdbilized in organic combinations in soils to which plant materials were added: A. During the early stages of decomposition, immobilization by microbial assimilation was a dominant process. In field and green- house experiments the intensity of microbial immobilization was reflected in crop yields, microbial numbers, mineralization of carbon and nitrogen in incubated soils, or in the level of amino nitrogen in acid hydrolysates. B. At advanced stages of decomposition, moderate to large in- creases in the soil nitrogen fraction which was not hydrolyzed by acid were Observed in both soils. At the end of a hoaweek decomposition period in the greenhouse experiment, the level of non-acid-hydrolyza— ble nitrogen increased as a continuous geometric function of decreasing soil C:N ratio. The curves for soils with different organic amendments were different. For any given soil C:N ratio, much larger quantities of acid-resistant nitrogen were found following the addition of sawdust or acid-extracted ("lignified") sawdust than with the more readily decomposed.materials. The curves for sawdust and lignified sawdust were congruent, which indicated that the acid-resistant nitrogen found.was associated with a constituent common to both materials and presumed to be lignin. The lignin content of hardwood sawdust is known to be higher than in La— _ lOO corn stalks. These and other considerations lead to the conclusion that increases in non-acid-hydrolyzable nitrogen observed in the greenhouse samples were due to chemical fixation of ammonia and pro- teinaceous nitrogen compounds by lignin or lignaceous constituents of the organic amendments. Similar increases in non-acid-hydrolyzable nitrogen were observed in soil samples taken three and five years after addition of sawdust in the field experiment and were similarly ascribed to the formation of chemical complexes with lignin. Factors Affecting Micrdbial Immobilization The intensity of micrdbial immobilization was directly related to the quantity and availability of energy'materials contained in the various organic amendments or in their residues at various stages of decomposition. The intensity of microbial immobilization was also increased by the addition of nitrogen in_high nitrogen materials or as supplemental nitrogen with materials low in contained nitrogen. This latter effect was reflected in increased micrcbial numbers or increased quantities of amino nitrogen recovered in acid hydrolysates, even though it was rarely reflected in reduced crOp yields at the ab- normally high rates of supplemental nitrogen which were used. The length of time over which intense microbial immobilization occurred following the addition of various organic amendments was in- versely related to the availability of energy substrates contained in the original materials. Effects of nitrogen on the duration of micro- bial immObilization were difficult to separate from its effect on lOl chemical fixation. However, an earlier decline in microbial numbers where nitrogen was added with residues low in contained nitrogen in- dicated an inverse relation between level of nitrogen treatment and the duration of intense microbial immobilization. The presence of a young, growing wheat crOp stimulated micrdbial assimilation of nitrogen, as reflected in greatly increased microbial numbers two weeks after the start of the greenhouse experiment. This rhizosphere effect was of catalytic preportions. Microbial numbers were maintained at higher levels in crepped than in uncropped soils all through the decomposition period by reason of energy materials contributed by root residues from two successive wheat crops. The enhanced microbial immobilization of nitrogen attributable to cropping was reflected at the end of the hOdweek decomposition period by in- creased CO evolution and decreased mineralization of nitrogen during 2 incubation, as well as by reduced uptake of nitrogen by the third crop of wheat. Factors Affecting Chemical Immobilization The chemical fixation of ammonia by lignin is known to occur at pH's around neutrality or above. It is also known that the amount fixed in chemically resistant form increases with ammonia concentra- tion and the degree of oxidation of the lignin. In the greenhouse soils to which supplemental nitrogen was added as urea, appropriately high pH's and ammonia concentrations for ammonia fixation were attained during the first two weeks. CrOp re— sponse and pH data showed that ammonia was quickly absorbed by the 102 lignified sawdust and somewhat less rapidly by sawdust. The sorbed ammonia nitrogen was readily available to the first wheat crop but declined rapidly in availability to the next two crops. The decline in availability of nitrogen to the wheat was accompanied by sharply reduced numbers of bacteria and fungi, from which it was inferred that masking energy materials were rapidly dissipated, exposing the more resistant lignin to oxidation. The level of acid-resistant nitrogen found after hO weeks was a function of the degree of oxidation of energy materials in these soils. This was shown by the fact that CO2 evolved during a lO—day incubation declined geometrically with increasing non-acid—hydrolyzable nitrogen. Carbon dioxide evolved from soils treated with a given organic amend- ment decreased linearly with soil C:N ratio. At any given C:N ratio, the 002 evolution for different materials was of the order expected from the known decomposability of the original materials. Thus, the chemical immobilization of nitrogen in the greenhouse experiment appears to have been related directly to level of nitrogen treatment, the quantity of lignaceous materials added in the various organic amendments, and the degree of decomposition or dissipation of associated energy materials. The effect of cropping was to reduce the level of acid-resistant nitrogen, presumably by reducing nitrogen level through crop removal and by adding to the energy supply in the form of root residues from the wheat. l03 Factors Affecting Release of MicrObially Immobilized Nitrogen Comparison of nitrogen taken up by successive cropsof wheat with micrObial numbers showed that nitrogen initially immobilized by microbial assimilation was released again as the more readily available energy materials were dissipated and the numbers and activity of soil microorganisms declined. This was also shown after to weeks by the in- verse relationships that were found between nitrogen released and micro- bial activity when the carbon and nitrogen mineralized during incuba- 3 tion were both plotted against soil C:N ratio. Factors Affecting Release of Chemically Fixed Nitrogen Bremner and Shaw (23) have shown that ammonia or protein nitrogen in chemical complexes with lignin is very resistant to mineralization by soil microorganisms. In the present study, the decomposability of organic residues in soils hO weeks after treatment declined accord- ing to a geometric function of increasing content of non-acid-hydrolyza- ble nitrogen, as shown by 002 evolution from incubated soil samples. Mineralization of nitrogen during incubation also appeared to bear a continuous relationship to acid—resistant nitrogen, but the shape of the curve for sawdust and lignified sawdust reflected the action of two independent variables, namely decreasing microbial immobilization and increasing resistance to mineralization of chemically fixed nitrogen. The data were fragmentary, but the values for sawdust and 10h lignified sawdust showed a distinct tendency for nitrogen mineralized to increase with non-acid—hydrolyzable nitrogen over the lower range where decreasing intensity of microbial immobilization was indicated by rapidly decreasing 002 evolution. In the higher range of non—acid- hydrolyzable nitrogen where microbial activity had been reduced to very low levels, nitrogen mineralized during incubation was sharply reduced. In these more highly oxidized soils, the resistance toxnicro- bial mineralization of chemically fixed nitrogen was clearly seen. One year after application of sawdust to the Sims clay loam in the field experiment, the proportion of non-acid-hydrolyzable nitrogen was less than in the check soil and the proportion of amino nitrogen was greater. This result suggested that chemically fixed nitrogen may have been attacked and utilized by microorganisms in the presence of fresh energy.materials. In the greenhouse, non-acid-hydrolyzable nitrogen after ten months was not reduced below the level of the check by any treatment. However, from the shapes of the curves relating acid-resistant nitro- gen to soil C:N ratio, it was apparent that decreases in this fraction following addition of carbonaceous energy materials would be much easier to detect in soils of narrow C:N ratio. The C:N ratio of the Oshtemo sand used in the greenhouse experiment was 16.031, that of the Sims clay loam in the field experiment was ll.h:l. These differences in soil C:N ratio help to explain the fact that decreases in non-acid hydrolyzable nitrogen were detected in the field after sawdust applica- tion and not in the greenhouse. The addition of root residues from two crops of wheat in the 105 greenhouse resulted in wider C:N ratios and lower levels of non-acid- hydrokyzable nitrogen than in the corresponding uncropped soils. Thus, non-acidshydrolyzable nitrogen appeared to be an equilibrium product of oxidative processes in the soil. As such, its level in the soil would be expected to decrease when fresh organic materials containing reduced carbon compounds of high energy content are added to the soil. This process would be analogous to the "priming" action of fresh re- sidues on decomposition of soil humic materials which has been report- ed in tracer studies. The conflicting evidence that has been reported regarding this "priming" phenomenon may well have arisen by reason of differences in oxidative status and associated levels of chemically fixed nitrogen in the soils used by different workers. Significance to Crop Response It is generally assumed that nitrate is the principal form in which nitrogen is taken up by most plants. Nitrogen taken up by the third crOp of wheat planted after hO weeks in the greenhouse experi- ment was found to be very closely related to water-soluble nitrate in the soil at the time the wheat was planted. No discernible re- lationship existed between nitrogen uptake and incubation tests for nitrifiable nitrogen. However, when nitrate released during incubation was added to nitrate already in the soil at planting time, the apparent functional relationship between nitrogen uptake and nitrate nitrogen was improved for a majority of soils. A direct linear relationship was found between nitrate nitrogen and non-acidéhydrolyzable nitrogen in these soils at the time this 106 third wheat crop was planted. This indicated that both forms of nitro- gen were equilibrium products of the oxidation of organic materials in the soil. In confirmation of this interpretation, it was found that nitrogen availability to the third wheat crop was an inverse function of soil C:N ratio for soils with any given organic amendment. The residual availability of nitrogen in previously cropped soils appeared to have been determined largely by the nature of the original organic amendment and the soil C:N ratio after to weeks, with only a general and erratic relation to the total quantity of nitrogen originally added. The failure of the incubation tests for nitrifiable nitrogen by themselves to reflect levels of nitrogen availability was due partly to the fact that large amounts of nitrate had accumulated in a number of these soils prior to planting of the wheat. However, with uncropped soils and soils treated with alfalfa hay, unusually large amounts of nitrogen were taken up by wheat at given levels of nitrate or nitrate- plus-nitrifiable nitrogen. Several of these soils were unusually high in non-acid-hydrolyzable nitrogen. Apparently, rhizosphere activities associated with the growing wheat crop greatly enhanced the availa- bility of chemically fixed nitrogen to the crop. Of these irregular soils, those treated with sawdust or lignified sawdust at high levels of nitrogen without previous cropping were unusually high in total nitrogen, so that a capacity factor, as well as a rate factor, was involved in the net release of nitrogen to the wheat. ‘With all uncropped soils, microbial immobilization was less intense (shown by reduced CO evolution) than in the corresponding 2 107 cropped soils because of the absence of energy materials contributed from root residues from preceding wheat crOps. The abnormally high uptake of nitrogen with the previously cropped alfalfa treatment was consistent with experience in other areas where it has been found that the incubation test for nitrifiable nitrogen fails to reflect the high nitrogen supplying potential of a legume preceding corn. The complexity of factors that were found to influence the tie-up and release of nitrogen during incubation in the greenhouse and in the laboratory and the additional interactions of these factors with the succeeding crop indicate that no single chemical or incubation test can be relied on to predict the availability of nitrogen to that crop. Practical Implications From the results that have been reported here, a number of pract- ical implications may be drawn: 1. The quantity of humified soil organic matter can be materially increased by massive additions of plant materials. Such increases are probably only temporary. Decomposition is relatively rapid. Materials high in lignin, such as sawdust or extracted wood products will have residual effects detectable for at least five years after application. Effects of rapidly decomposable materials such as corn stalks or cereal straws will be dissipated much more quickly. Alfalfa hay is decomposed more rapidly in the initial stages than cereal straws or corn stalks, but leaves a larger residue of resistant organic matter because of its higher lignin content. 108 2. The rate of transformation of fresh plant materials to humic substances is hastened by the addition of nitrogen. Nitrogen retained in organic combinations in the soil is increased by nitrogen treatment. Except with very highly lignaceous materials, the quantity of carbon retained in resistant humic materials at advanced stages of decomposition appears to be relatively unaffected over a very wide range of nitrogen treatment. 3. The initial injurious effects on non-leguminous cropsof large additions of plant materials low in nitrogen can be corrected by the addition of large quantities of nitrogen (of the order of 30 to hO pounds per ton of sawdust). 'With plant materials low in phos- phorus, extra fertilizer phosphorus will also be needed (5 pounds per ton for sawdust). h. A leguminous crop such as alfalfa is not seriously re— duced in yield by the presence of large quantities of sawdust. The addition of extra phosphate may be all that is needed to eliminate any interference with alfalfa production. The broadcasting of sawdust on established stands of alfalfa would appear to be an economical method for using sawdust for soil building purposes. 5. Lignin materials extracted from sawdust have a large capacity for absorbing ammonia. The sorbed ammonia was found to be highly available to the first crop of wheat in concentrations which were highly toxic to wheat grown without lignin treatment. The lignin- ammonia complex quickly became resistant to microbial attack and yet retained a surprisingly high level of availability to subsequent wheat crops. These properties suggest the need for investigating the use of 109 lignin-ammonia complexes in fertilizer formulations, particularly foerocalized placement of heavy rates of application and for con- trolled release effects. PART m9 PHYSICO-CHEMICAL PROPEiTIES OF SOME SOIL HUMIC MATERIALS AND SYNTHETHIC MODELS INTRODUCTION TO PART THC A large number of organic constituents Lave been isolated and identified in hydrolysates of soil humic materials. These include hexose and pentose sugars and their uronic acids and amino derivatives, numerous amino acids characteristic 02 plant and microbial proteins, nucleic acids and inositol phosphates, resins and waxes, and a complex of phenolic compounds combined with nitrogen. In their natural state, the humic materials are largely insoluble in water, WliCh indicates a I-’ C fairly high degree of polymerizat 0. Cl the simple organic constitu- ,3 soil organic matter extract- l‘o} ents. The behavior or gross fractions 0 ed from soils by various extracting procedures does not correspond to that of polysaccharides, proteins or other natural polymer isolated from living plant, animal or microbial tissues. Thus, many of the larger organic groupings in soil organic matter appear to arise by processes of chemical and physical complex formation rather than by well defined patterns of biological condensation and polymerization. It must be assumed that the actual processes whereby simple organic building blocks are com 0 complex structures in the soil will be controlled or modified by the interplay of chemical, physical and biotic forces. Qualitative and quantitative diirerences in the composition and structure of humic materials will occur from soil to soil, reflecting differences in parent materials, topography, age, climate, vegetation and cultural practices. 0n the other hand, certain patterns of recombination will recur in all soils. The qualitative composition of plant materials which 112 are the principal source of organic matter in soils varies but little with the species of plant. ‘When various plant residues are attacked by decay organisms, an essentially similar assortment or spectrum of active organic groupings are exposed. These include carbonyl groups, ketones, carboxyls and hydroxyls, amino, imino, amido and.imido nitro- gen groups, sulfhydryl groups and various phosphate esters, to mention only a few. The chemical properties of these active groupings will determine the nature of the reactions in which they can participate in the chemico-physical environment represented by the soil solution and the interfaces between the gaseous, liquid and solid phases of the soil. For these reasons, a certain skeletal similarity in mole- cular structure of soil humic materials would be expected regardless of soil type or climatic situation. A primary goal of fundamental soil organic matter studies has long been to define this "humus skeleton". A major stumbling block to realizing this goal has been the difficulty of separating organic materials in unaltered form from the mineral constituents of soils. The results of chemical studies on extracted fractions cannot be directly projected to soil organic matter ig.§itg. One approach to this difficulty has been to synthesise hypotheti- cal model substances and draw inferences from observed similarities in chemical or physical behavior between these models and organic matter in natural soil systems. This was the approach taken by Waksman and others in developing the concept of a ligno—protein complex (107-110). It is again being vigorously pursued by Flaig and his c0dworkers in 113 Germany in their studies of synthetic polymers of quinones and hydroquinones (35-37). This remains an essentially undeveloped area of experimentation. Further limitations in fundamental organic matter studies are the tedious, time-consuming nature of chemical procedures available for characterizing the organic constituents of soils and the low specificity of many of them. Chromatographic and electrophoretic procedures have come into the picture to greatly facilitate suCh studies (9h, 96, 99). However, these are not entirely satisfactory for extensive surveys of large groups of soils. Furthermore, they pro- vide no direct evidence as to the way in which the simpler components are combined into larger groupings in the soil. The absorption of infrared and ultraviolet light by appropriately prepared solutions or films of matter has in recent years been de- veloped into tools for the elucidation of molecular structure. The criteria presently available for the interpretation of absorption spectra in these extradvisible wavelengths are still rather limited, but rapid advances are being made. The techniques involved are rapid and should provide a means for quickly surveying the chemical structure of the organic constituents of large groups of soils, and for determin- ing the nature of their association with soil minerals. The theoreti- cal basis for interpretation of the spectra derived with natural soil preparations can be developed by corollary studies with synthetic models prepared to test hypothetical modes of chemical combination or complex formation. Preliminary studies were conducted employing this approach. 11h Infrared spectra were developed using organic fractions extracted frcm three soils with alkali or with neutral salt solutions. Syn- thetic models used for comparison included wyoming bentonite and a mixture of'Wyoming bentonite and alpha humus extracted from muck, and complexes of casein with varying proportions of lignin prepared from sawdust by acid extraction and by alkali extraction. Ultraviolet absorption studies were conducted on two'mdbile components separated from the alpha humus of Sims clay loam by paper electrophoresis. MATERIALS AND METHODS Preparation of lignins Acid lignin was prepared by removing non-lignaceous constituents from hardwood sawdust by hydrolysis with cold 72 percent sulfuric acid followed by hydrolysis with hot dilute sulfuric acid according to the method described by Norman (73). Alkali lignin was extracted from hardwood sawdust, using h per— cent sodium hydroxide under pressure, as described by Waksman (108). Both materials were freed from chlorides and sulfates by dialysis. Preparation of lignin—casein complexes Casein and lignin were mixed in the dry, powdered form. Separate mixtures were made with each type of lignin in two proportions, one- half gram of casein being added to 1% and 3 grams of each lignin mat- erial. The mixtures were dissolved in 2 percent NaOH and shaken inter- mittently for eight hours. The complexed materials were then precipi— tated by addition of HCl, washed free of chlorides, dried at hS-SO degrees C and ground to a fine powder. Table 15 shows the carbon and nitrogen contents of the lignins and the changes in nitrogen content where casein was complexed with six parts of lignin. Preparation of alpha humus A sample of Oshtemo sand which had been treated with lignin in the greenhouse experiment reported in Part One and samples of muck and Sims clay loam were extracted with 2 percent NaOH overnight at 116 room temperature and were then centrifuged. The black supernatant liquid was recovered by decantation and acidified until a precipitate was formed. This precipitate was centrifuged out, washed free of chlorides by dialysis, dried at hS-SO degrees C and then ground to a fine powder. Table 15. - Carbon and nitrogen found in lignin-casein complexes used for infrared spectrum analysis. .._‘ _r—~1—¢_- A Material 0 N Theoretical N Percent Percent Percent Acid lignin 57.3 0.3 Alkali lignin 53.5 0.2 Casein --—— 13.6 Alkali lignin- casein complexw -~~- 1.9 2.1 Acid lignin— casein complex*- ~—-- 2.2 2.2 % Both analysis from complex of one part casein to six parts of as Carbon by dry combustion. Soil organic matter extracted with neutral salt solution A sample of Sims clay loam was extracted overnight at room tem- perature with .OSM sodium pyrophosphate. The dissolved organic matter was separated from the soil by centrifugation and then precipita— ted, freed of chlorides, dried and ground in the same manner as the alpha humus. 117 Infrared spectra Materials for infrared spectrum analysis were mounted in a Nujol mull in a Perkin-Elmer recording infrared spectrophotmometer. Absorb- ancy curves were developed over a range of wavelengths from 2 to lh.5 microns. Ultraviolet spectra Ultraviolet spectra were developed on a Beckman DK-2 recording spectrophotometer. Materials were dissolved in dilute sodium hydroxide, and introduced into the light path in quartz absorption cells. Ab— sorbancy curves were developed over a range of wavelengths from 220 to 3&0 microns. Paper electrophoresis Electrophoretic separations from the alpha humus fraction of Sims clay loam were made using a Spinco Model R paper electrophoresis cell. The transporting medium was a barbiturate buffer at pH9.0 with an ionic strength of .l gamma per ml. Runs were made for fouréhour periods with a current of lO millamperes. Migrant spots were locat- ed visually under white or ultraviolet light, then cut from the paper and redissolved in .001 M NaOH in ethyl alcohol for ultraviolet analysis. EXPERIMENTAL RESULTS Infrared Spectra Infrared spectra for acid lignin, alkali lignin and casein are shown in Figures 15, 16, and 17. The three major absorption peaks which appear at wavelengths of 3.h5, 6.85 and 7.25 microns are char- acteristic of the Nujol dispersing medium and are to be observed in all infrared spectra reported here. The sharp break at 9.2 microns is a mechanical irregularity which results when the instrument is switched over from a lower to a higher range of wavelengths. In the spectra for alkali lignin and casein an absorption band appears at 3.00 microns which is not to be seen in the spectrwm for acid lignin. This is a region where characteristic absorbence by N-H or O-H bonds has been shown. At 8.h to 8.5 microns, an absorbence band for casein and acid lignin coincides with an abrupt damping of absorbence in the spectrum of alkali lignin. Again at 8.9 and 9.8 microns, there are absorbance bands in the alkali lignin spectrum which are not found in the spectra of acid lignin or casein. All three spectra show a peak of absorbence at 1h microns. The significance in terms of molecular structure of these simil— arities and dissimilarities in infrared absorption by these three materials is not known, but they do reflect the fact that these mat- erials differ in their chemical constitution. More exact interpreta- tion of such spectra is possible through comparison with absorption spectra that have been reported for numerous reference compounds. Such data were not readily available at the time this report was Figure Figure Figure Figure Figure Figure 15. 16. 17. 18. 19. 20. Infrared absorption spectrum of Infrared absorption spectrum of Infrared absorption spectrmn of Infrared absorption spectrum of complex (6 to 1 ratio) Infrared absorption spectrum of casein complex (6 to 1 ratio) Infrared absorption spectrum of from muck 119 acid lignin alkali lignin casein acid lignin-casein alkali lignin- alpha humus extracted FIGURE IS WM“— A BSORBANCE l i l l J l l l 2 3 4 5 6 7 8 9 IO II I2 l3 I4 MICRONS .- ... ABSORBANCE ,- 7 8 MICRONS FIGURE (e, ABSORBANCE ... ... l 7 l l 8 9 MICRONS FlGURE l7 ABSORBANCE p— .— MICRONS 5:90:16 (‘3 F ((9025 19 LIJ 0 2 <1 (I) (I O (D (I) <1 I I I I l I I I I l I L__ 2 3 4 5 6 7 8 9 IO || I2 I3 I4 MICRONS ABSORBANCE .— I 7 l 8 MICRONS FlGURE ZO ... ‘1'! r‘ '3 125 prepared. In Figures 18 and 19 are shown the spectra for the synthetic complexes of casein with acid lignin and.with alkali lignin. The Shape of the two absorption curves was essentially similar, even to the exact correspondence of numerous absorption maxima.and.minima at the same wavelengths for both materials. Similar curves were Obtained whether three or six parts of lignin were mixed with one of casein. 'When Figures 18 and 19 are compared with Figures 15, 16 and 17, it will be seen that numerous characteristic absorption peaks in the parent materials have been sharply reduced in the complexes. This is particularly evident in the region from 7.5 to 12 microns. Here the damping effect of the mixture might reflect merely the interference of opposing regions of maximal and minimal absorbance contributed.by elements of the two parent materials in a mechanical mixture. However, in the case of the alkali lignin—casein comples (Fig. 19), if a purely'mechanical mixture were involved, it would be expected that there should be reinforcement of the absorption peak exhibited at 3.00 microns by both alkali lignin (Fig. 16) and casein (Fig. 17). Instead, this peak was less pronounced than in either parent material. Apparently a chemical combination had taken place which resulted in the disappearance of N-H group or 04H group or both. In the case of the acid lignin-casein complex (Fig. 18) there was a similar damping of absorption at 2.3 microns where reinforce- ment would have been expected, since peaks occurred here in acid lignin (Fig. 15) and casein (Fig. 17). Again a chemical transformation is indicated. No postulation is offered as to active groups or bonds which may have been involved. 126 In Figure 20 is shown the infrared absorption diagram for alpha humus extracted from muck. There is a striking similarity between this spectrum and that for acid lignin (Fig. 15). Both have a sharply defined absorption peak at 2.3 microns. Neither shows the peak at 3.0:microns that was shown by alkali lignin and casein and weakly by the two complexes. In the region above 7.5 microns numerous maxima and.ninima coincide exactly, although they are less sharply defined in the alpha humus from muck. This close similarity in absorbance behavior between humic material from muck and lignin isolated from sawdust by virtue of its resistance to hydrolysis by strong acid is consistent with the generally accepted concept that resistant ligna- ceous residues from plant decomposition are a principle constituent of soil humus, particularly in poorly drained situations where aeration is poor. An alpha humus was also extracted from one of the Oshtemo sand samples which had been treated with acid lignin and incubated in the greenhouse for 30 weeks in the experiment reported in Part One. The infrared spectrum of this preparation is shown in Figure 21. There are sharp absorption peaks at 3 and 6 microns which correSpond to those shown by casein (Fig. 17). The intensity of these peaks compared with those obtained where casein was complexed with lignin at ratios of l to 3 (Figs. 18 and 19) provides indirect evidence that proteins were extensively stabilized by the lignin treatment in this soil. This had been inferred from the large increases in acidehydrolyzable amino nitrogen and non-hydrolyzable nitrogen reported in Part One. The fact that the peaks were more pronounced for the alkali -127 extract from the incubated soil (Fig. 21) than for the complex formed in alkaline solution (Figs. 18 and 19) may reflect quantitative dif- ferences in the proportion of proteinaceous materials present. 0n the other hand, the mechanisms of complex formation or protein stab- ilization in the incubated soil may have been different than under the conditions existing in the laboratory synthesis. In Figure 22 the spectrum for alpha humus extracted from Sims clay loam.with neutral sodium pyrophosphate is presented. Rather weak but distinct absorption peaks at 3 and 6 microns appear. No attempt at comparison with previous spectra can be made since a dif— ferent extractant was used as well as a different soil. However, the point is made that characteristic peaks are recognizable regardless of extractant. This suggests that successive fractional extraction using different extractants may provide one means for identifying chemical groups responsible for specific peaks in infrared spectra of soils. The pattern of absorbance in Figures 21 and 22 for alpha.humus extracted from mineral soils showed no similarity above 7.5 microns to any of the previous spectra. A broad absorption band around 9.5 mic- rons has been reported to be characteristic of clay and other silicate minerals. Accordingly a spectrum was developed for hydrogen—saturated wyoming bentonite and for a mixture of Nyoming bentonite and alpha humus extracted from muck. These are presented in Figures 23 and 2h. Major peaks occur in the bentonite spectrum at 2.3, 6.1, 9.h to 9.8, and at 1h microns. These are also the major peaks in the spectrum of the mixture. 128 Figure 21. - Infrared absorption spectrum of alpha humus extracted with NaOH from an Oshtemo sand. The soil had been incubated for 30 weeks following an application of acid lignin. Figure 22. - Infrared absorption spectrum of alpha humus extracted from Sims clay loam with sodium pyrophosphate. Check treatment. Figure 23. - Infrared absorption Spectrum of hydrogen saturated WVOming bentonite. Figure 2h. - Infrared absorption spectrum of alpha humus prepared from four parts muck complexed with one part bentonite. FIGURE 2| ABSORBANCE I I I I l I 2 3 4 5 6 7 .— .— — 8 MICRONS ABSORBANCE ,— _. 7 I 8 MICRONS 9 M V FIGURE 2.3 moz OO POT. DIAL READING .bU'IU'I O O I I I __I 20 4O 6O 80 MILLIEQUIVALENTS Mg $04 PER IOO GRAMS 115 of 29 m.e. in total exchange capacity over that of the acid lignin alone was largely accounted for by the appearance of 25 m.e. of ex— change capacity in the titration preceding the first end point. Table 16. - Cation exchange capacities of lignins, casein and lignin- , casein complexes as detenmined by high frequency titration of Ba—saturated materials with N/lO MgSOh. Cation exchange capacity, m.e. per 100 gms Material Material Complex with casein alone First Ratio endpoint Total * Acid lignin 19 6:1 11 30 3:1 25 AB Alkali lignin hl 6:1 11 AS 3:1 11 A6 Casein 35 _.— __ __ * Ratio of lignin to casein in the complex. The behavior of the alkali lignin complex was quite different from that of the acid lignin complex. There were no differences in either lower energy or total exchange capacities between the 6:1 and the 3:1 ratios of lignin to casein. With both ratios, the total ex- change capacity of the complex was only A or 5 m.e. greater than that of the alkali lignin alone. It is interesting to note that the total capacity of the complex was 10 or 11 m.e. greater than that of casein alone, which is equivalent to the capacity of new exchange sites represented by the first endpoint. %; 1146 The significance of the observed cation exchange phenomena is not known. The different results with acid lignin as compared with alkali lignin help to explain why some investigators have found that the exchange capacity of lignin—protein complexes was greater than that of lignin alone,c whereas other investigators have been unable to show such changes. The manner in which lignin is extracted fron natural sources greatly influences the chemical properties of the product. This was indicated by the infrared spectra as well as by the differences in cation exchange properties of the materials them- selves and of their complexes with casein. The appearance of new exchange sites in lignin-casein conplexes substantiates the inferences made from infrared spectra that actual chemical combinations had occurred between active groups in the two materials when they were mixed in alkaline solution. SUMMARY AND CONCLUSIONS The physico—chemical studies reported here represent an attempt to apply several new techniques to the study of the fundamental nature and properties of soil organic matter. It is recognized that the results Obtained are fragmentary and that their theoretical significance is not clear. However, the experiences recorded.may be a guide to future studies along these lines. Several specific results appear to justify further investigation. Infrared absorbance spectra were develOped for a number of ex- tracted soil organic matter fractions and synthetic models involving casein, acid- and alkali-extracted lignin and clay minerals. The spectra for organic materials were highly characteristic; for example distinctly different absorbance patterns were exhibited.by lignins extracted from hardwood sawdust with acid and.with alkali. Struct- ural differences revealed by infrared spectra were reflected in differences in cation exchange properties of acid and alkali lignins alone and in synthetic complexes with casein. The infrared spectrum for alpha humus extracted with alkali from.muck was very similar to that for acid extracted lignin, but was distinctly different from that of alkali lignin extracted from sawdust. This result is consistent with the accepted concept that chemically resistant lignaceous constituents of plants accumulate as the principle component of humus in poorly drained situations where biological oxidation is limited. Alpha humus extracted from mineral soils gave spectra in which the patterns characteristic for organic fractions were Obscured 11:8 above 7.5:microns by mineral impurities. Comparison of these spectra with those Obtained with bentonite alone and in mixture with alpha humus from muck indicated that the mineral impurities were probably silicates. Future infrared studies with the organic fractions of mineral soils will need to consider means for eliminating these mineral impurities. 0n the other hand, studies with synthetic clay-organic matter systems in the regions of the infrared spectrum where silicates interfere may reveal clues as to the active groups involved in inter- actions between clays and organic compounds. The usefulness of the infrared absorbance diagrams was limited by the lack of an extensive reference list of absorbance data for pure compounds. The absorption of electromagnetic energy by matter is known to be a function of the bonding energy between units of molecular structure. Specific absorption wavelengths have been de— termined for numerous interatomic combination. A necessary phase of any investigation involving radiation absorbance phenanena should be the tabulation from the literature of available data of this sort. Infrared analysis appears to have considerable promise as a means for surveying large numbers of soils with a.view to characteri- zing gross organic fractions which can be readily freed from mineral impurities. Ultraviolet absorption, on the other hand, has more limited application as a.means of tentative identification of certain fundamental humic components after they have been isolated in rather pure form. Presumably these will include compounds with aromatic ring structure or heterocyclic compounds derived from lignaceous 1A9 materials. Ultraviolet absorption Spectra may serve as criteria of the purity of isolates to be used for further chemical analysis. Preliminary experiments with paper electrophoresis suggest that this may provide a.method for isolating components of alpha humus with sufficient purity to give characteristic ultraviolet absorbance spectra. 'Whereas others have reported a single mobile component in alpha humus preparations employing free electrophoresis in a Tiselius cell, in the present work with paper electrOphoresis two distinct components were observed. One of these was a colorless material which fluoresced under ultraviolet light. The separation of the two mObile components was not complete, but their ultraviolet absorbance spectra showed much better resolution than those for the original alpha humus. The extreme complexity of soil organic matter and its intimate association with soil minerials has made it a rather unrewarding ob- ject of study in the past. A number of new instruments and techni- ques, including those used here have opened new possibilities for the fruitful examination of soil organic matter. No one method can be expected to accomplish much by itself. There is considerable promise, however, in an integrated approach involving several methods on both natural soil systems and synthetic models. LITERATURE C ITED 2. 3. 7. 8. 9. 10. LITERATURE CITED Allen, M. B., and Van Neil, C. B. 1952. Experiments on bacterial denitrification. Jour. Bact. 6h:397. Allison, F. E. 1927. Nitrate assimilation by soil microorganisms. Soil Sci. 2h:79. Allison, F. E.,and Anderson, M. S. 1951. The use of sawdust for mulches and soil improvement. U. S. D. A. Ciro. 891. Allison, F. E., Sherman, M. s., and Pinck, L. A. 19h9. Maintenance of soil organic matter. Inorganic soil colloid as a factor in retention of carbon during the formation of humus. Soil Sci. 68:h63. Arnold, P.'W. 195A. Losses of nitrous oxide from the $011. Jouro SOil SCio 5:116. Audus, L. J. l9h6. A new soil perfusion apparatus. Nature l58:hl9. Barnes, R. B., Gore, R. C., Liddel, U., and Williams, V. Z. 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Canadian Jour. Res. 17B:199. 'Wrensnall, C. A., and Dyer,'E. J. 19h1. Organic phosphorus in soils: II. The nature of the organic phosphorus compounds. A. Nucleic acid. B. Phytin. Soil Sci. 51:235. Van Slyke, D. D. 1911. A method for quantitative determination of aliphatic amino groups. Jour. Biol. Chem. 10:185. APPEND IX Table 17. - Treatments used with Oshtemo sand in greenhouse experiment 162 Pot Material added Rate C:N ratio C:N Nitrogen Crop number gms. of original ratio added (d) grown (a) material actual gms. 1,2 Lignified 100 577:1 12.7:1 3.8h6 wheat(b) sawdust 3,h " " " 36.5:1 1.281 " 5,6 " " " 12.7:1 3.8h6 -—~(o) 7,8 " " " 36.5:1 1.281 -— 9,10 " SO " 12.7:1 1.923 Wheat 11,12 " n " 36.5:1 .6u1 H 13,1h " " " 12.7:1 1.923 -—- 15,16 " " " 36.5:1 .6hl -—— b9,50 Sawdust lOO 353:1 20:1 2.355 Wheat 51,52 " " ., 60.5:1 .735 " 53,5h " " " 20:1 2.35; --l 55,56 " " " 60.5:1 .785 -—- (a) All pots contained hOOO gms of a uniformly sieved Oshtemo sandy loan. (b) Five plants were grown in each pot, 2 crops were grown during first 25 weeks of decomposition period. (c) No plants were grown. (d) Nitrogen added as urea. Table 1? (Continued) Pot Material added Rate C:N ratio C:N Nitrogen CrOp number gms. of original ratio added (d) grown material actual gms. 57,581 Sawdust 50 353:1 20:1 1.177 wheat 59,60 " " " 60.5:1 .392 " 61,62 " " " 20:1 1.177 -~ 63,6h " " " 60.5:1 .382 -—- 17,18 Straw 100 65.7:1 h1.6:l .h38 wheat 19,20 " n " 55.1:1 .1h6 " 21,22 " " " h1.6:1 .h38 ——— 23,2b " " " 55.1:1 .1h6 -—— 25,26 " So " u1.e:1 .219 wheat 27,28 " SO " 55.1:1 .073 " 29,30 " " " h1.6:1 .219 ——_ 31,32 " " " 55.1.1 .073 -—- 33,3h Corn stalks 100 113.6:1 no.9:1 .757 wheat 35,36 " " " 72.2.1 .252 " 37,38 " " " b0.9:l .757 -~ 39,uo " " " 72.2:1 .252 —-— hl,h2 " SO " no.9:1 .379 wheat 13,1u " " " 72.2:1 .126 " 115,116 " n " 11-0.9.1 .379 -- Table 1? (Continued) 16h Pot Material added Rate C:N ratio C:N Nitrogen Crop number gms. of original ratio added (d) grown material actual gms. h7,u8 Corn stalks SO 113.6:1 72.2:1 .126 -- 65,66 none ——~ ~---— ~-- 1.92, wheat 67,68 Lignified 50 577:1 —_— _____ n sawdust 69,70 Sawdust " 353:1 ——— __1._ n 71,72 Straw " 65.7:1 —-— __.__ n 73,70 Corn stalks " 113.6:1 —-- __-__ H 5,76 Lignified 100 577:1 ..- _____ n sawdust 77,78 Sawdust " 353:1 -—— ___-_ u 79,80 Straw " 65.731 ..- _____ n 81,82 Corn stalks " 113.6:1 --- _____ n 83,8h none -—— ———»-— -_~ ___-_ n *85,86 Alfalfa 100 18.0:1 --— ----- wheat 87,88 n u n ___ _____ ___ 89:90 " 50 " --— ----- wheat 91,92 u n u ___ _____ ..- 101,102 Sawdust 100 353:1 -—— --—-- alfalfa 103,10h " " " 60.5:1 .785 " 105,106 " SO " -.. ___-- n % 2.708 gms. of nitrogen added per pot as alfalfa hay. Table 17 (Continued) 165 Pot Material added Rate C:N ratio C:N Nitrogen Crop number gms. of original ratio added (d) grown material actual gms. 107,108 Sawdust 50 353:1 60.5:1 .392 alfalfa 109,110 Lignified " 577:1 ——- _____ u sawdust 111,112 " " " 18.2:1 1.281 " 113,11h " 100 " --- ______ n 115,116 " " " 68.7:1 .6h1 " 117 , 118 Sawdust " 353 :1 ...—— ----.. n 119,120 " 5O " ...—— ..---- u . _ an . . . n I a .- . C I I 166 Table 18a. - Yield and nitrogen uptake of wheat crops in greenhouse experiment lst Cro 2nd Cro Pot Yield 7N Nitrogen Yield 5N Nitrogen number gms. uptake mgs. gms. uptake mgs. 1 6.805 u.o 272.2 7.535 2.88 217.0 2 7.195 2.72 267.6 6.870 2.8u 195.1 3 7.885 2.h3 191.6 3.250 1.3h h3.5 u 1 11.680 2.uu 280.0 6.830 1.51 103.1 5 6 7 8 9 10.130 3.11 38&.h 6.100 2.70 16h.7 10 2.990 3.70 110.6 7.770 2.67 207.8 11 8.670 2.05 177.7 1.950 .99 19.3 12 9.590 1.92 18u.1 2.260 1.13 25.5 13 1h 15 16 h9 2.850 h.h0 125.b 6.8bo 2.92 199.7 50 1.925 8.60 88.5 2.110 1.53 109.1 51 5.210 1.hl 73.u .810 1.62 7.1 52 n.015 1.20 h8.2 .620 2.22 13.7 53 5h Table 18a (Continued) 167 3rd Crop Totals Average Yie1d pN Nitrogen N Yield N Yield Pot gzns . uptake m gs . mgs . gms . mgs . gms . number 3.777 1.95 186.7 675.9 18.113 1 612.5 17.930 3.683 3.98 116.5 609.2 17.718 2 .223 2.60 5.8 210.9 11.358 3 338.0 15.655 1.193 3.22 18.0 135.1 19.953 1 2.193 1.78 119.1 5 150.3 3.198 3.903 1.65 181.1 6 30u73 hol3 lh3oh 7 131109 30823 1.173 3.03 126.1 8 1.953 1.30 83.9 597.0 18.183 9 501.7 15.728 2.513 1.21 106.5 121.5 13.273 10 .173 2.10 9.9 206.9 11.093 11 221.1 12.003 1.063 2.12 25.7 235.3 12.913 12 2.883 3.80 109.5 13 111.2 3.583 1.283 1.01 173.0 11 3.213 1.10 131.7 15 117.9 3.633 1.053 1.05 161.1 16 2.133 3.86 93.9 119.0 12.123 19 359.8 9.565 2.573 1.01 103.1 300.7 7.008 50 2.103 2.16 51.7 132.2 7.753 51 113.1 7.500 2.613 3.59 93.8 155.7 7.218 52 3.736 1.31 162.1 53 132.3 3.171 2.613 3.93 102.6 51 .I. 168 Table 18a. — Yield and nitrogen uptake of wheat crops in greenhouse experiment lst Cro 2nd Cro Pot Yield %N Nitrogen Yield 5N Nitrogen number gms. uptake mgs. gms. uptake mgs. 55 56 57 10.255 2.19 255.3 2.690 2.39 61.2 58 11.160 2.63 293.5 1.555 1.63 71.2 59 1.175 1.16 52.0 1.110 1.88 18.9 60 5.630 1.11 61.1 .110 2.75 9.9 61 62 63 61 17 2.870 1.31 37.5 3.795 1.15 55.0 18 2.370 1.02 27.7 2.100 1.73 36.3 19 .210 .7 1.1 .700 2.01 11.0 20 .260 .9 2.31 1.310 1.97 26.3 21 22 23 21 25 2.585 1.03 26.6 1.270 1.78 21.6 26 3.060 1.02 31.2 1.160 1.56 22.1 27 .385 1.71 6.5 1.210 1.72 21.3 28 .100 1.63 6.5 1.110 1.78 21.1 Table 18a (Continued) 169 3rd Crop Totals Average Yield %N Nitrogen N Yield N Yield Pot gms. uptake mgs. mgs. gms. mgs. gms. number 2.813 2.70 75.9 55 91.1 3.133 1.013 2.78 112.3 56 2.581 1.16 107.1 113.1 15.378 57 112.1 16.833 2.183 3.31 72.7 170.8 18.288 58 1.673 2.98 19.8 122.7 7.588 59 115.2 8.135 2.083 3.26 67.9 167.8 8.683 60 2.783 2.66 71.0 61 96014 30058 3.333 3.57 118.9 62 2.363 3.96 93.5 63 92.0 2.718 3.073 2.95 90.6 61 3.213 3.23 101.7 197.2 9.908 17 180.3 8.150 2.523 3.91 99.1 163.1 6.993 18 3.083 3.58 110.3 121.5 3.993 19 101.2 3.703 1.813 3.22 58.3 86.9 3.113 20 3.613 3.81 138.7 21 130.1 3.658 3.673 3.31 121.5 22 3.233 3.98 128.6 23 133.5 3.388 3.513 3.91 138.5 21 2.193 1.20 92.1 110.3 6.018 25 152.8 6.825 3.063 3.61 111.5 165.1 7.583 26 2.283 302h 7b00 101.8 3.908 27 113.7 3.960 2.123 1.05 98.1 125.7 1.013 28 170 Table 18a. - Yield and nitrogen uptake of wheat crops in greenhouse experiment lst Cro 2nd Cro Pot Yield 7N Nitrogen Yield 5N Nitrogen number gms. uptake mgs. gms. uptake mgs. 33 7.000 1.17 81.9 .91 2.19 20.5 31 7.880 1.16 103.0 1.000 1.56 15.6 35 .210 1.16 3.5 .280 1.61 1.6 36 .160 .81 1.3 1.370 1.63 22.3 37 38 39 10 11 5.525 1.19 65.7 1.215 1.71 21.6 12 6.380 1.11 72.7 1.260 1.82 23.0 13 .380 1.71 6.5 .510 1.92 9.9 11 .765 1.72 13.1 .510 2.31 11.9 15 16 17 18 65 .210 3.10 8.1 8.550 2.91 251.3 66 .920 1.03 37.0 8.190 1.96 212.1 67 .370 .95 3.5 .395 1.85 7.3 68 .120 1.03 1.3 .100 1.92 7.6 Table 18a (Continued) 171 3rd Crop 393% Average Yield %N Nitrogen N Yield ‘3 Yield Pot gms. uptake mgs. mgs. gms. mgs. gms. number 1.713 2.59 15.2 117.5 9.683 33 ‘ 201.2 10.163 2.353 5.80 136.1 251.8 11.211 31 3.193 2.81 90.6 98.9 3.713 35 97.5 3.793 2.313 3.10 72.6 96.2 3.873 36 3.663 3.80 139.2 37 116.1 3.793 3.813 1.00 153.7 38 3.323 2.73 90.7 39 79.2 3.018 2.713 2.50 67.8 10 2.223 3.35 71.1 161.7 8.993 11 168.6 9.388 2olh3 3073 7909 175.6 90783 h2 1.863 2.68 19.9 66.3 2.753 13 71.5 3.105 2.183 2.65 57.7 82.7 3.158 11 2.353 2.91 68.1 15 2.573 3.78 97.2 16 1.703 3.65 152.2 17 108.8 3.658 2.613 2.51 65.5 18 2.213 1.13 99.3 358.7 11.033 65 387.1 11.658 3.173 1.29 136.1 115.5 12.283 66 1.103 2.09 23.0 33.8 1.868 67 31.6 1.875 1.063 1.65 17.5 29.1 1.883 68 172 Table 18a. — Yield and nitrogen uptake of wheat crops in greenhouse experinent lst Cro Yield %N 2nd Cro Yield 5N Pot Nitrogen Nitrogen number gms. uptake mgs. gms. uptake mgs. 69 .510 .80 1.0 .170 1.29 2.1 70 .395 .87 3.1 .220 1.13 2.5 71 .300 .90 2.7 .130 2.11 10.1 72 .260 .70 1.8 .780 1.57 12.2 73 .215 .70 1.5 .220 1.90 1.1 71 .210 .70 1.5 .280 1.98 5.5 75 .890 .95 8.1 .115 .80 1.1 76 1.290 .91 11.7 .160 .63 1.0 77 .135 .93 1.2 .115 .87 1.0 78 .255 .61 1.6 .160 .80 1.2 79 .210 .55 1.3 .215 .60 1.1 80 .215 .15 .9 .185 .75 1.3 81 .170 .53 .9 .160 1.20 5.5 82 .165 .51 .8 .155 .78 1.2 83 8.015 1.11 92.3 1.530 .95 11.5 81 6.610 1.16 77.0 1.590 1.28 20.3 85 9.000 3.11 306.9 9.625 2.33 221.0 86 7.700 3.13 261.1 10.505 1.79 188.0 87 88 89 9.700 2.67 258.9 5.315 1.19 63.2 90 9.530 2.57 211.9 5.070 1.33 67.1 Table 18a (Continued) 3rd Cro Totals Average Yield 5N Nitrogen N Yield N Yield Pot gms. uptake mgs. mgs. gms. mgs. gms. number 20.6 1.310 .763 2.11 16.0 21.9 1.378 70 2.201 2.95 61.7 77.8 2.901 71 78.3 2.978 2.203 2.95 61.9 78.9 3.051 72 1.923 2.52 18.1 51.0 2.113 73 13.5 1.878 1.123 2.32 26.0 33.0 1.613 71 .173 1.15 2.0 11.5 1.208 75 15.0 1.165 .273 1.83 5.0 17.5 1.723 76 .213 2.05 5.0 7.5 .193 77 6.2 .525 .113 1.39 2.0 1.9 .558 78 1.613 2.82 15.1 18.1 2.098 79 55.1 2.531 2.563 2.37 60.7 62.8 2.963 80 .923 2.31 21.5 27.9 1.553 81 19.5 1.213 .533 1.73 9.2 11.2 .873 82 1.763 2.90 51.1 157.9 11.337 83 110.1 10.120 1.273 2.00 25.1 122.1 9.503 81 1.073 1.11 167.1 698.5 22.698 85 635.1 22.038 3.173 3.76 119.3 571.1 21.378 86 3.513 1.11 111.3 87 151.1 3.208 2.903 5.11157.9 88 1.313 3.91 52.5 371.6 16.358 89 385.7 16.787 2.613 3.21 81.6 396.9 17.217 90 171 Table 18a. — Yield and nitrogen uptake of wheat crops in greenhouse experiment 1st Cro 0nd Cro Pot YieIH %fi Nitrogen Yiela 5N number gms. uptake mgs. gms. Nitrogen uptake mgs. 91 92 175 Table 18a (Continued) 3rd Crop Totals Average Yield /oN Nitrogen N Yield N Yield Pot gms . uptake mgs . mgs . gms . mgs . gms . number 3.973 3.76 1149-3 91 151.7 1.008 1.013 3.96 160.1 92 176 Table 18b. — Yield and nitrogen uptake of alfalfa and succeeding wheat crop * Al£21£§ Pot Cuttings, Total N Total number lst Crop 2nd Crop 3rd Crop uptake yield mgs. gms. 101 2.250 1.895 3.390 217.2 7.212 102 2.300 1.670 2.920 165.9 6.890 103 1.860 1.185 1.885 113.1 5.230 101 2.070 1.805 2.870 201.1 6.715 105 2.610 2.095 3.210 218.5 7.915 106 2.950 2.085 ‘ 3.350 238.8 8.385 107 2.180 2.185 1.250 260.6 9.215 108 2.310 2.300 2.710 228.8 7.380 109 2.350 2.135 1.690 301.0 9.117 110 1.870 2.070 1.505 252.2 8.115 111 2.600 2.580 1.777 327.3 9.957 112 3.730 3.600 6.350 129.5 13.680 113 2.880 2.110 1.090 250.1 9.110 111 2.280 1.890 3.510 223.6 7.680 115 3.070 2.865 5.510 310.1 11.175 116 2.950 2.695 1.830 301.2 10.175 117 1.720 1.730 3.180 183.7 6.630 118 2.710 2.130 1.715 236.5 9.885 119 2.190 1.805 3.605 203.7 7.600 120 2.110 2.155 3.115 205.0 8.010 * Three cuttings of alfalfa followed by one crop of wheat Table 18b (Continued) 177 Alfalfa - Ave. ‘Wheat Average N. uptake Yield Yield I. uptake Yield N. uptake Pot mgs. gms. gms. mgs. gms. mgs. number 1.723 39.9 101 191.5 7.001 .968 22.5 .213 5.0 102 2-513 73.1 103 172.2 5.987 2.988 87.5 3.133 101.6 101 1.833 15.1 105 228.0 8.165 2.028 18.1 2.223 51.5 106 2.763 76.8 107 215.7 8.297 2.693 95.3 3.163 113.8 108 2.333 59.2 109 278.1 8.960 2.290 58.1 2.253 57.0 110 3.363 101.2 111 378.1 11.818 3.368 96.2 3.373 88.0 112 1.513 33.7 113 237.0 8.395 2.108 51.0 2.673 71.3 111 3.693 122.2 115 320.6 10.975 3.113 107.2 2.533 92.2 116 .263 7.1 117 210.1 8.258 .318 8.1 .373 9.6 118 1.253 30.8 119 201.1 7.855 1.813 19.0 2.373 67.3 120 a . n u a . . 1 , u 1 ... A . . - 178 Table 19. — Average yield and nitrogen uptake by three crops of wheat grown upon various soil amendments lst Crop Treatment Yield gms. Nitrogen uptake, mgs N0 N1 N2 N0 N1 N2 1.09 9.76 6.99 10.1 237.8 269.9 Lignified sawdust .39 9.13 6.56 3.9 180.9 217.5 .19 1.61 2.38 1.1 60.8 106.9 Sawdust .15 5.05 10.70 3.7 58.1 271.1 .28 .23 2.62 2.1 11.8 32.6 Straw .20 .39 2.82 2.2 6.5 28.9 .16 .20 7.91 .9 2.1 92.1 Corn stalks .21 057 5095 105 908 6902 8.35 285.5 Alfalfa 9.61 251.9 Check 7 03h *059 8,-1.6 W307 * Check plus nitrogen H=1OO grams per pot of residue L= 50 grams per pot of residue 25 tons/acre 12.5 tons/acre NO: no nitrogen, N1 = low level N, "N2: High level of N, (see Table 17). 179 Table 19 (Continued) 2nd Crop Treatment Yield gms. Nitrogen uptake, mgs. N0 N1 N2 N0 N1 N2 H .15 5.01 7.20 2.0 73.3 206.1 Lignified sawdust L .39 2.11 6.91 7.5 22.1 186.2 H .11 .53 1.62 1.1 10.1 151.1 Sawdust L .19 1.00 3.62 2.3 11.1 69.2 H .21 1.02 2.91 1.1 20.2 15.6 Straw L .60 1.22 1.36 11.3 21.2 22.0 H .31 .82 .97 3.1 13.2 18.0 Corn Stalk L .25 .51 1.025 L108 1009 22.3 H 10.06 206.0 Alfalfa L 5.19 65.3 Check 1.56 8.37 17.1 216.8 180 Table 19. - Average yield and nitrogen uptake by three crops of wheat grown upon various soil amendments 3rd Crop Treatment Yield gms. Nitrogen uptake mgs. N0 N1 N2 N0 N1 N2 H .23 .86 3.73 3.7 26.9 166.6 Lignified sawdust L 1.13 .76 2.23 20.2 17.8 95.2 H .19 2.35 2.50 3.7 72.7 98.5 Sawdust L .69 1.88 2.38 11.6 18.9 88.8 H 2.08 2.15 2.88 53.1 81.3 102.1 Straw L 2.20 2.31 2.62 61.9 86.1 101.8 H .71 2.77 2.01 15.3 81.6 90.0 Corn stalks L 1.07 2.02 2.18 37.2 53.8 77.2 H 3.62 113.1 Alfalfa L 2.98 68.5 Check 1.07 2.71 38.2 117.7 Table 19 (Continued) 181 Uncropped Pots* Total three crops Yield gms N-uptake Yield gms Nitrogen uptake mgs N1 N2 N1 N2 N0 N1 N2 N0 N1 T2 H 2.82 3.19 131.9 150.2 1.1 16.6 17.8 15.0 338.0 611.5 L 3.63 3.58 117.9 111.3 1.8 12.0 15.7 31.6 221.1 501.7 H 3.12 3.17 99.1 132.3 .5 7.5 9.5 6.2 113.1 359.8 1 2.72 3.06 92.1 96.5 1.3 8.1 16.8 20.6 115.2 112.1 H 3.39 3.65 133.5 130.1 2.5 3.7 8.5 55.1 101.2 180.3 L 3.19 2.66 108.1 78.1 3.0 3.9 6.8 78.9 133.7 152.8 H 3.02 3.75 79.2 116.1 1.2 3.7 10.1 19.5 97.5 201.2 L 3.16 2.16 108.1 78.1 1.8 3.1 9.3 13.5 71.5 168.6 3.20 151.1 .2230 635.1 2.01 101.7 16.7 385.7 1001.1 1196 111-0011 387.]. JL Treatments cropped only once, after 10 weeks of decomposition. 182 Table 20. - Daily rate of CO evolution by soils during 10 day incubation perioa at 35 oC. Production of CO by days of incubation Treatment or Mgs 00 2f8ay/100 gm soil3 pot number Ferden farm samples Check 26.1 11.0 10.5 7.5 7.2 5.1 1.2 lst year 51.8 56.5 60.8 67.3 66.3 52.5 50.8 3rd year 15.0 26.2 17.6 11.7 13.3 10.8 9.5 5th year 53.2 25.1 19.6 11.9 12.5 10.1 8.6 Greenhouse samples 1 7.7 6.2 5.3 3.6 3.8 2.7 2,5 2 10.3 7.3 5.1 3.8 6.1 3.1 3.2 3 16.7 12.6 11.2 9.3 6.7 10.5 10.1 1 8.0 6.5 5.7 1.1 1.6 2.8 2.1 5 1.6 3.8 1.5 1.8 1.6 1.8 1.3 6 5.6 1.0 3.5 1.7 2.0 2.1 2.0 33 13.6 8.6 5.0 5.7 5.9 5.5 5.6 37 10.7 6.9 6.1 6.0 5.5 1.1 3.8 19 12.2 7.3 5.2 5.0 3.8 1.0 3.1 50 9.8 6.9 1.3 3.8 1.3 2.9 2.1 51 11.6 8.1 5.3 5.0 1.5 1.5 1.1 52 12.6 7.7 6.7 6.9 5.3 1.2 3.7 53 9.6 5.2 2.2 3.2 2.8 2.2 1.6 51 7.6 1.9 2.2 1.8 2.1 1.7 1.1 * All figures represent average of duplicate determinations 183 Table 20 (cont.). - Daily and cumulative CO2 production Average of replicates Mgs COO/day/loo _g_n_s of soil Treatment or L pot number Days of incubation Cumulative totals 1 2 3 1 5 8 10 Check 89.1 lst year 579.0 3rd year 167.5 5th year 173.7 1 9.0 6.8 5.2 3.7 5.1 3.0 2.9 11.5 2 3 12.1 9.5 8.1 6.8 5.6 6.6 6.2 75.0 1 5 5.1 3.9 2.5 1.8 1.8 2.0 1.6 211.0 6 33 6.7-5 37 55:5 19 11.0 7.1 1.7 1.1 1.1 3.1 2.8 17.2 50 51 12.1 7.9 6.0 5.9 1.9 1.3 3.9 57.6 52 53 8.6 5.1 2.2 2.5 2.1 1.9 1.3 29.2 . . '- - o . . a. . . .. . . I . ...-.. . . i .. . I _ . I I u! l I l I n l o l . O I I I I I . . I 1 I I Q o o l I O I I I I I 181 Table 20 (Cont.) — Daily rate of 00 evolution by soils during 10 day incubation period at 35 C. Production of CO by days of incubation Treatment or Mgs Gog/gay/loo gm soi1* pot number 1 2 3 1 5’ 8 10 65 5.0 2.7 1.6 2.1 3.2 2.0 1.1 66 5.5 3.8 1.5 2.1 2.9 1.5 1.3 75 12.5 8.1 8.5 8.7 9.5 8.2 7.5 76 9.6 8.5 8.2 8.6 8.8 8.2 10.7 77 20.1 15.1 11.8 12.1 12.2 12.2 12.2 78 21.0 18.2 16.2 11.9 15.8 11.5 10.8 81 . 18.8 15.2 13.6 11.7 11.7 9.2 7.1 83 ' 6.1 3.8 2.7 2.8 2.5 1.7 1.1 81 6.7 3.8 2.6 2.9 2.5 1.6 1.3 85 11.3 7.1 1.9 1.8 1.2 1.1 3.6 86 10.9 8.3 5.2 1.6 1.0 1.1 6.8 87 12.1 5.9 5.2 1.9 1.6 1.2 3.7 88 11.6 6.8 3.8 1.1 1.1 5.6 1.1 103 18.1 15.8 13.6 12.1 11.3 10.9 10.9 113 13.7 12.2 9.7 8.7 8.5 7.1 8.1 115 10.3 7.8 6.2 5.3 1.9 1.1 1.1 * All figures represent average of duplicate detenminations 185 Table 20 (Cont.) - Daily and cumulative 002 production Average of replicates Mgs 002 [day/100 gms of soil Treatment or pot number Days of incubation Cumulative totals l 2 3 1 5 8 10 65 . ‘ ' 5.2 3.2 1.6 2.1 3.1 1.9 1.2 23.5 66 75 10.9 8.5 8.3 8.6 9.1 8.2 9.1 88.2 76 77 22.0 1608 15.5 1305 11100 13.3 110).} 1111109 78 81 112.7 83 ' ‘ 6.6 3.8 2.7 2.8 2.5 1.7 1.1 26.1 81 85 12.6 7.7 5.1 1.7 1.1 1.1 5.2 56.9 86 67 13.3 6.3 1.5 1.6 1.3 1.9 1.1 55.9 88 113 91.2 115 56 .1 186 Table 21. - Nitrates and nitrifiable nitrogen in experimental soils by the Iowa test Treatment or Nitrate-nitrogen’x- Nitrifiable nitrogen‘n" pot number (Initial extraction) (Final extra_ction) ‘ Rep. I Rep. II E Rep. 1 Rep. II E' Ferden Fann Samples Check 1 2 3 78 56 67 lst year 1 7 1 1 O 0 0 3rd year 6 2 1 126 126 126 5th year 1 l l 99 122 112 Greenhouse Experiment 1 58 56 57 28 32 30 2 89 111 100 30 57 11 3 1 3 2 21 28 26 1 57 66 62 11 12 13 5 800 672 736 30 32 31 6 278 288 283 8 3 6 33 32 21 28 18 12 15 37 102 93 98 13 51 19 19 118,118 116,126 129 12,13 33,30 31 50 169 ~ 171 172 31 27 29 51 81 81 83 38 13 11 52 71 66 70 35 33 31 53 310,266 360,380 336 12,38 35,31 39 51 108,100 366,160 108 12,30 26,23 10 * Values give in lbs/acre N03—N 187 Table 21 (Continued) Treatment or Nitrate-nitrogen* Nitrifiable nitrogen* pot number (Initial extraction) Final extraction) Rep. I Rep. II E Rep. I Rep. II x 65 312,305 288,373 329 32,23 21,21 25 66 201,251 256,236 238 16,6 8,5 9 75 3 0 2 0 0 0 76 0 0 0 0 0 0 77 0 0 o 0 0 0 78 0 0 0 0 0 0 81 3 1 2 12 11 13 83 62,31 ’ 50,11 17 36,21 26,25 28 81 38 31 36 25 21 25 85 201,192 136,159 172 63,51 57,60 59 86 83,111 87,111 99 52,51 61,53 56 87 156,111 136,102 129 51,57 18,17 52 88 100 81 92 58 56 57 101 8 6 7 38 11 10 113 53 50 52 38 37 38 115 12 11 13 11 12 13 % Values given in lbs/acre NOB-N O - I I I I . . y l . . w . . . . _ . -\ fix -\ 1 . o .x _ . m... . u. . c 1 z .1 . a.\ -\ ..x I n\. I .\ - 188 Table 22: Numbers of bacteria and fungi in Oshtemo sand at various tine intervals after treatment * 6 * 5 Treatment Bacteria x 10 Fun 1 x 10 2 wks. lkas. 25wks, hOwks. 2wks. 1 wks. 25wks. howks. Lsaw-NO 56 9 11 21 20 3 1 7 LS4W—Nl 51 31 28 39 17 11 11 11 Ls-JN-N2 '11 17 11 12 7 2 3 3 13410-12 36 23 63 12 2 9 11 1 SDJN-Ne 36 51 11 18 33 12 5 6 SD-w—Nl 11 29 38 28 5 13 17 8 SDJW-N2 26 6 20 12 1 2 3 1 SDJNO—NZ 38 31 35 32 1 3 25 1 STJH—Nl 216 99 37 16 17 29 ST-JM-N2 168 88 10 35 1 18 STJNO-NZ 31 5 22 CSJN-Nl 170 85 36 25 9 3 CSJN-Nz 153 129 56 27 3 12 05410-12 179 65 63 10 25 27 ALFJH-NO 300 201 300 119 21 23 36 6 ALFJNO-NO 291 218 202 112 38 28 25 9 CKAN—NO 15 15 33 17 1 2 2 1 CKJN plus N 6 2 19 18 .3 .1 1 6 % Counts represent averages of duplicate plates from each of duplicate pots of each treatment (1 plates per count . 189 Table 230 - A comparison of ignition and dry combustion determina- tions of carbon is soil containing plant residues * Treatment or pot number Ignition Method %** Corrected %C Check lst year 3rd year 5th year N OUT-IT'U) 37 19 50 51 52 53 51 Dry Combustion 1% Actual %C %C Ferden Farm 1.10 3.32 1.01 1.31 3.21 1.08 3.06 3.99 Greenhouse Samples 1.28 1.73 1.51 1.73 2.85 1.69 2.11 1.96 2.19 2.10 2.09 1.72 1.00 1.25 1.08 1.29 1.33 1.36 1.30 1.12 1.31 1.63 1.31 1.13 1.31 1.13 1.32 1.57 2.61 3.16 3.25 3.18 1.38 1.38 1.35 1.57 1.68 1.38 1.00 1.03 1.08 1.11 1.30 1.11 1.11 1.25 190 Table 23 (Cont.). — A comparison of ignition and dry combustion detenninations of carbon is soil containing plant residues % Treatnent Ignition Method %%* or Dry Combustion Actual Corrected pot number %C 710 730 65 .73 .95 .76 66 .81 1.00 .80 75 1.71 1.36 76 1.75 1.10 77 1.19 1.19 78 1.56 1.21 81 1.08 1.28 1.02 83 .78 1.01 .83 81 .76 1.01 .81 85 1.07 1.25 1.00 86 1.01 1.27 1.01 87 1.26 1.01 88 1.26 1.01 * MWsmCManmmmflwm By selection of a rather uniform sample (in this case number 33) the following method was used for the correction of the carbon content by the ignition method. Sample number 33 was run six times by the dry combustion method so as to obtain as accurately as possible a sample that could be a standard to samples run by the ignition method. This sample was then included in all determinations of carbon run by the ignition method and the actual value for the test samples was corrected by the use of this standard fonnula: Wt loss of Standard Wt loss of unknown % carbon in standard : ,5. carbon of unknown 191 Table 23 (Cont.). — A comparison of ignition and dry combustion determinations of carbon is soil containing plant residues * An example is given: 1.2539 1.3198 x ., 3.1 7!, Carbon 1.00 3 X The soil was first dried at 105°C before ignition to eliminate the variation due to water held by the soil and crop residues. This method was chosen for carbon analysis because of the wide variation encountered with samples taken from the sawdust treatments. With dry combustion only 1-2 gram samples can be used for a determin- ation, but in the ignition method 25—50 grams of soil could be analyzed. The use of such large sample was thought to minimize the great variations of non-uniform residues encountered in the samples. A reference sample has limitations in that it is not desirable when changing from one soil type to another. There is little doubt that the dry combustion method is one of the most accurate for detennin- ing carbon content when a sample can be uniformly sampled. For most of the samples taken in this experiment one gram was too small for accurate sampling. Both methods have limitations but the use of a larger sample was thought to elmninate more error in relative values for different treatments than with the use of a smaller sample and the dry caubustion method. ** Dry cambustion of 1-2 gm sample at 950°C in carbon train *%% Ignition of 50 gm sample at 500°C in muffle furnace sllll. . IlILlrr .:.. r , a .- 1... 192 Table 21. - Description of media used for bacterial and fungal plate counts Modified Formula of Soil Extract - Tryptone Agar (See Ref. 19) (for bacteria and eatinomycetes) Magnesium Sulfate (MgSOh.7E2O . . . . . . . . . . . . . 0.2 grams Dibasic Potassium Phosphate (K2HP01) . . . . . . . . . I 0.5 grams Ferric Chloride (FeClB) . . . . . . . . . . . . . . . . 10 mgs. Tryptone . . . . . . . A . . . . . . . . ... . . . . . 0.5 grams Glucose (Dextrose) . . . . . . . . . . . . . . . . . . 1.0 grams Agar O O O O O C O O O 0 O O O O O I O O O O O 0 O O O 3.5 O O gI‘a-Ins SOilEXtraCtSOlIl-tionoooooccoo coco... 500113.180 TapWa‘ber...................... 50011115. Reaction of media: pH 7.2 Martin's Medium for Fungi (See Ref. 92) Dextrose O I I O D O O C O O I O O I C O 0 O O O O O O 1000 m. Peptone o o o o o o o o a o o o o o o o o a o o o o o 5.0 gnlo Potassium dihydrogen phosphate . . . . . . . . . . . . 1.0 gm. MagleSim sulfate. 0 o o o o o o o o o o o c o o o o o 0.5 an. Rosebengal 00000000 000 ooooolpartjll30,000part80f medium Agar . . . . . . . . . ° . . . . . . C . . C C O . O . 20.0 $11. Streptomycin solution 0 o o o o o o o o o o a o o o o 30Yper ml. Distilledwatero o o o oo o no. 0 o 000 o o. 0 01000001111. 1 A ,1 _ ROOM USE ONLY ROOM USE emy "I7'11111111’171T