125 633 THE FEMUON OF AMMONIA AND NITRAYE BY ORGANIC MATERIALS Thests for Hm Degree of M. 5. MICHIGAN STATE UNIVERSITY Sadao Shoji 1958 THE FIXATION OF AI-‘fl-IONIA. AND NITRATE BY ORGAI‘II’ NATL RIALS By SADAO SHOJI AN ABSTRACT Submitted to the College of Agriculture Michigan State University of Agriculture and Applied Science in partial fulfillment of the requirements for the degree of MASTER OF SOL-DICE Department of Soil Science 1958 I 1 _...¢/ ', Approved _[ fl/(Zflr 7/??? ///r r I325; [(1% 2/ fl /%«€4afi Sadao Shoji ABSTRACT Non—biological fixation of nitrogen by soils and lignin was studied in the laboratory in relation to oxida- tion and associated changes in cation exchange capacity, methoxyl content and nethylatinj capacity. a No nitrate was f xod at any pH oy any of the materials. Extensive oxidative fitatior of rnnonia by muck soils and 1 ov p1, concentration of ammonia or U lignin was influenced ammonium ion and by the nature of associated cations. ‘ Nethylation of orvanic materials witn Cimethyl sulfate blocked ammonia fixation and decreased cation exchange capa— city about one-third. Oxidation with Each resulted in a seven-fold increase in cation exchange capacity of lignin and a 10 to 15$ increase in exchange capacity of ruck soils. Oxidation with KHMOH resulted in a two-fold increase in exchange capacity of 115- nin but a 10 to 15¢ decrease in that of muck. It appeared that oxidativc ammonia fixation involved two opposing pro- cesses with reference to its effect on cation exchange Capacity: Cxidative processes increas d exchange capacity, as in the case of NaCH, whereas -ssociated reactions with ammonia resulted in disappearance of active exchaige sites joined during oxidation. Acid leaching and M30 dist'llation removed similar quantities of fixed nitrogen. A smaller proportion of fixed nltPOCen was reaoved fnam lignin than in the case of the muck Sadao Shoji materials. Ultraviolet absorbance spectra of lisnin showed a well-defined minimum at 262 mpand a sharp maximum at 283 m». Oxidation with NaOH shifted these two points i the direction {3 of longer wavelengths, bu: did not materially alter the shape of the curve. Oxidative ammonia fixation altered the shape of the absorbance curves for lignin by large y obscuring the minimum and maximum peaks in thksrange, producing curves with gentle lepes, similar to the spectra for muck materials. THE FIXATIOIJ OF ADIIICTIIA AITD NITRATE BY ORGANIC hATJdIALS By SADAO STIOJ I A TITLES IS Submitted to the Gollege of Agriculture Michigan State University of Agriculture and Applied Science in partial fulfillment of the requirements for the degree of F3 MAS ER OF SCIENCE Department of Soil Science 1958 Approved ACKNOWLEDGEMENTS The writer wishes to thank Dr. A. R. welcott and Dr. M. Mortland for their helpful suggestions and guidance throughout the progress of this work. He also wishes to thank Dr. J. F. Davis, Dr. H. L. Sadoff and Dr. A. Timnick for their advice and assistance. . , a"! .0. Id ‘_0 I 1 4:1 TMBLE OW CONTEST-ITS 222.22:- I. mmonrjcnow II. REVIIMOFLITHIATLBE........... . III. OBJBETIVES.......'................ IV. MATHiIAISAICDMETHODS ................. quaerhentalliaterials. . .. .. .. . .. .. .. . Ebcperimental‘lreatments . . .... . .. .. . .'. . Fixation of nitrate nitrogen . . . . . . . . . . . . Fixation of ammonia nitrogen . . . . . . . . . . . Oxidation and methylation studies .... . . . . . . . Chenical and Physical Determinations . . . . ... . . . V. RESULTS ........................ Preperties of Materials . . . . . . . . . . . . . . . Fixation oi‘ Nitrate Nitrogen . . . . . . . . . . . . . Fixation of Armenia Nitrogen and Factors Affecting It Influence of pH on oxidative ammonia fixation . . . Influence of bases and concentration of NH’ Cl on ammonia fixation at pH 7.5 e e o e 0*. o o e 0 Influence of methylation on ammonia fiXation . . . . Cation Exchange Capacity of Materials and Factors AffmtingIto0.0000000000000000 Influence of oxidation with NaOH on cation exchange capaCiw Of'the materials 3 o e o e o e o o o 0 Influence of oxidative ammonia fixation on cation exchange capacity of materials . . . . . . . . . Influence of methylation on cation exchange capacity ofmaterials..........o.o.o... 21 26 26 26 Chapter Methoxyl Content of Materials and Factors Affecting It . Stability of Fixed Nitrogen When Subjected to Treatment withACidsorAlkali............o.. [El-Wielet Absorption . o o e o o o o o o e o o o e 0 VI. DBCIBS ION e o o o e o o o o o o o e o o o o o e o o o 0 VII. SUMY . O O O O O O O O O O “ .- 0 ‘ O C - I O O O 0 O O O O O VIIIOBBLIommoooooooeoOoQOOOoooooooo Sea}; 315;? h? 1. 2. 3. h. 5. L13 T OF TABLES RUPERTIESOFTHEORIGINALMATI‘BIAIS . . . . . ... . . .. FIXATION OF NITROGEN FROM 1N NaNO3 SOLUTIONS ADJUSTED TO VARIOIE pH'S . C . O O O O O O O O O O I O O C O C C O O NFLUENCE OF pH ON OXIDATIVE FIXATION FROM IN N'HhCl SOLUTIONS INFLUENCE OF BASES AND CONCETTRATION OF NHhCl 0N AI’J’IONIA FmeN AT pH 7.5 0 O O O O O O O O C O O O O O O O O C INFLUENCE OF IETHYLATION ON E'IONIA FDCATION FROM 12% NHhOH SOLUTION . O O C O C O O C C C O O O O O O C O O C O . . INFIUENCE OF OXIDATION WITH N33}! ON CATION EXCHANGE CAPACITY INFLUENCE OF OXIDATIVE FIXATION OF AMMONIA FRG‘I 12% NHhOH SOLUTION ON CATION EXCHANGE CAPACITY . . . . . . . . . . INFLUENCE OF IJZETH'EATION ON CATION EXCHANGE CAPACITY OF mmms O C O O I O O O O C O O O O O O O O O O O O O O METHOXYL CONTENT OF ORIGINAL AND NETHYLATED I~IA‘I'ERIAIS BEFORE ALVD ”Tm OXIDATION O O O O O O O O O O O O O O O O O O O INFLUENCE OF OXIDATION AND AI'EIONIA FIXATION ON METHYLATION OF LIGNIN MID MTEK O O O C O . C O O O O O C C O O O O . REMOVAL BY MgO DIS TILLATION AND ACID LEACHING OF NITROGEN OXIDATIVELY Fun) FROM 12% NHhOH SOLUTION . . . . . . . . 22 22 27 31 31 32 32 33 2. 3. LIS T OF FIGURES Page Alkoxyl appa Iatlls .7 O I I O O O O I O O O O O O O O O O O 16 Influence of pH on the oxidative fixation of ammonia nitrOgen .. o o o o e o e e o e e o o o o o o e e o o 23 Influence of bases and concentration of NHhCl on ammonia fixation'by virgin muck at pH 7.5 . . . . . . . . . . . 2h Influence of bases and concentration of NH Cl on ammonia fixation by fertilized muck at pH 7.5 . . . . . . . . 2h Influence of bases and concentration of NHhCl on ammonia fixa‘tionbyligninatpH’IS ............. 25 Influence of methylation on ammonia fixation and cation GXChange capaCity o e e e e o o o e e e e o o o o e o o 28 Effects Of NaOH oxidation and oxidative ammonia fixation on cation exchange capacity . . . . . . . . . . . . . . 29 Ultraviolet absorbence Spectrum of lignin . . ._ . . . . . . 36 Ultraviolet absorbence spectrum of fertilized.muck . . . . 36 Soaplw. 215 INTRODUCTION Classical studies with organic matter in plant residues, litter and.humus have shown that ammonia is extensively fixed in chemically and biologically resistant forms by the lignin or lignin—like constituents of these materials. Such fix- ation may have a significant influence on the availability of fertilizer nitrogen when high concentrations of nitrogen are introduced into localized zones of the soil by.band place- ment techniques and by applicators used to apply ammonia in gaseous form or in water solutions to soils. This may be particularly true in soils which are high in organic matter. Experience in fertility studies on organic soils in Michigan has shown that crops which respond to spring applications or top dressings of ammonia fertilizers frequently fail to respond to fall applications. The present studies were carried out to determine the extent of fixation of nitrogen by lignin and muck soil, and to study some of the conditions which might influence it. Also, it was of interest to determine something of the nature of the mechanisms whereby fixation occurs and the changes in cation exchange prOperties which result. REVIEW OF LITERATURE Soil organic matter has been the subject of'many investigations. A great difficulty in these studies has been that humic materials can not be precisely separated from.the Cunhumified organic matter. In other words, the soil organic matter consists of a whole series of products ranging from undecomposed plant and animal tissues to so-called "humified", stable, amorphous, black material. Some of these organic materials are intimately associated with the clay minerals in complexes from which they can be released with difficulty by extracting procedures which probably alter their essential chemistry. Bremner (3) has summarized the present status of knowledge about soil organic matter by saying that although no great progress has been made since Waksman's comprehensive review (2h) of the subject in 1938, much useful information has been obtained and several points have been clarified. .Marshall (11) reviewed the literature on physico- chemical studies of soil organic matter and concluded that knowledge of the physical chemistry of soil humic matter has advanced very little since the classical work of Oden published in 1912. Physico-chemical work on humus since 1920 has largely centered around four topics: Characterization by pH titration curves, chemical and physical properties under saturation by various cations, and utilization of the scanty X-ray evidence available to throw light upon molecular structure. Many investigators have worked on the structure and molecular weight of humic acid. waksman (2h) found that the chemistny of various "humic acids" depended upon the method used in their separation, especially upon the nature and concentration of the alkali and acid, and upon the length of treatment. It is not surprising, therefore, that no exact comparison could be made between the results obtained by different investigators. Odén's (2h) physico-chemical work on humus suggested the formula Cg“ H52 032 (mol. wt.= 1332) for "humus acids" containing three to four -COOH groups. Fuchs (2h) showed that the molecular weight of humdc acid obtained fr:m.peat was 1300 to lhOO and it contained three to four -COOH groups, three to four -OH groups, one methoxyl group and one carbonyl group. The -OH groups were found to be active in meflnflatrni and base-binding. Broadbent and Bradford (6) showed that the probable cation-exchange groupings in soil organic matter were carboxyl groups and phenolic hydrbxyl or enolic hydroxyl groups. I The study of the processes of humus formation is one of the most interesting problems. The restricted knowledge of humic matter constitution makes the study difficult. The humic matter in the soil has been considered to be derived from two chief sources: (a) lignins in plant residues and (b) produzts of microbial metabolism. Haksman and Iyer (25) proposed the ligno-protein complex theory. Though there is b no direct proof of this theory, it is supported indirectly by. the fact that protein is resistant to microbial decomposition in the presence of lignin (S). From Bremner's (3) fractionations of organic nitrogen, it appears that a third to two thirds of soil organic nitrogen is present as protein derivatives. Some of the remainder is thought to exist as heterocyclic nitrogen compounds. Mattson and Kouttler-Anderson (12, 13, 11;) presented the lignO-ammonimn complex theory, suggesting that same of the stable nitrogen compounds in the soil might be produced by the reaction of oxidized lignin and ammonia (or possibly aromatic amines) at the sites of phenolic hydroxyl groups to form.amidophenols. Upon oxidation and condensation, these would form polymers containing ring nitrogen. The change of lignin to humic matter seems to involve two processes: .oxidation and enrichment with nitrogen. The oxidation results in an increase of carboxyl groups (16) and probably phenolic groups. This oxidation takes place spon- taneously in the presence of air in an alkaline medium.(h, 9). If ammonia is present during oxidation, some of the ammonia is incorporated chemically with the lignin and appears in the oxidized peoducts (9, 12). The mechanisms of these reactions are not known. Some of the early studies on ammonia fixation seem.to have been made in connection with coal chemistry. Schrader (18) conducted experiments on the auto-oxidation of lignin ‘ S and natural humins. He found that lignin was fairly rapidly converted into humic acid in an NaQH medium containing NH3, oxygen being absorbed from the air concurrently with ammonia fixation. Very little NH3 was absorbed in the same medium when exposed to an atmosphere of N2 gas. Kappen et a1 (10, 23) worked on problems relating to the potential value of brown coal as a fertilizer. They observed that treatment of coal with gaseous or liquid ammonia caused same combination of nitrogen in unavailable forms. Springer (21) performed experiments on the influence of ammoniation on the organic material in high moor peats. He concluded that by ammoniation the humus forming complexes and organic pre-humzs materials could be converted more or less completely into humic materials. The extent of ammoniation was greater at higher temperature and pressures, and was dependent upon other reaction conditions.- Sohn and Peech (20) reported that heating an ammoniated organic matter reduced the ammonia-retention but increased the ammonia-fixing capacity. Problems d? oxidation and nitrogen enrichment in soil organic matter were approached by Junker (9). He checked the absorption of'ammonia by dioxane Spruce lignin and a Scholler lignin, using Bancroft's phase rule apparatus and obtained curves showing three flat portions in the sorption isotherm. These plateaus occurred at 0.72, l.hh and 2.76 milliequivalents NH3 per gram of sample. The curve indicated a superposition of absorbed and chemically combined ammonia on the lignin. He suggested that the combination of ammonia with lignin took place in three different stages, two of which might correspond to the combination of ammonia with phenolic hydroxyl groups, the third might indicate the combination of ammonia with aldehyde groups or alcoholic hydroxyl groups. He also carried out experiments involving the absorp- .tion of oxygen increased with increasing NaOH concentration 'of the lignin sol. Equivalent concentrations of NaOH, KOH and Ba(OH)2 gave equal oxygen absorption and the absorption in alkaline earth solutions decreased in the order-~Ba(OH)2:> Ca(OH)2>Mg(OH)2. Lignin in distilled water also absorbed oxygen. The absorption of oxygen in alkaline solutions was decreased in the presence of Mn(0H)3 or Fe(OH)3. Oxidized spruce lignin was more easily dispersed and had better base- binding prOperties than electrodialized lignin. Mattson and Kouttler-Andersson (12) investigated ammonia fixation by different forms of litter and humus, by water-soluble organic matter and lignin fractions, and by a series of organic compounds of known constitution. The ammonia fixation was greatest under conditions of simultaneous - auto-oxidation. Very small amounts of ammonia were fixed under anaerobic conditions. The water-soluble and lignin fractions fixed more ' ammonia per'unit weight of organic matter than the whole sam- ple. The lower the.base status of the litter and humus, the greater was the absorption of oxygen and the fixation of ammonia. The ammoniated complex was very stable to acids, the nitrogen being largely carried over in the lignin fraction obtained by 72% sulfuric acid treatment. Strong alkali re- moved nearly half of the fixed ammonia. They suggested that the stable nitrogen compounds in soil might be produced by the reaction of ammonia, or possibly aromatic amines, with oxidized lignin at the sites of phenolic hydroxyl groups to form amidOphenols. These would form polymers containing ring nitrogen when oxidized and condensed. More recently, studying peat formation, the same authors (13, 1h) found that while auto-oxidation increased the base- binding capacity of peat, ammonia fixation depressed this. capacity. Finally they proposed that the resistant residue of organic decomposition and transformation which forms the permanent humus is a ligno-nitrogen complex. Bennet's (1) methylation studies with lignin seem to support Mattson's theory. Compared with the amount of - 'ammonia fixed during oxidation, the increase of lrlethoxyl content in methylated and oxidized lignin was disproportion- ately low. In the process of change of lignin to humic matter, Amethxmyl groups decrease and cation exchange capacity increases, possibly because of an increase in carboxyl groups (16, 23). Using conifer seedlings, Themlitz (22) studied the availability of nitrogen fixed by raw humus. He showed that- the nitrogen fraction was firmly held in ammoniated humus, possibly in a heterocyclic bond. It was not distilled off in the presence of MgO and was poorly available to the seedlings. Spectrographic absorption studies over the ultra-violet and visible ranges show differences between humic materials and lignin. Absorption curves for lignin have much steeper 310pes than those for humic matter. Maximum absorbance for lignin occurs at about 280 mu and the minimum.at about 260 mu (2, 8, 17, 19). Spectra for humic materials show a generally declining absorbance with increasing wavelengths, with no distinct maxima or minima. OBJECTIVES ‘ The objectives of the present study were to study the non-biological fixation of nitrogen by organic soils and by lignin. It was desired to study fixation as affected by ‘ nitrogen source and conditions of reaction, including pH, concentration and associated bases. The relation of fixation to cation exchange capacity was of interest. By the use of methylating procedures it was hOped that some information might be derived relative to the mechanisms whereby nitrogen is fixed. 10 MATERIALS AND METHODS Experimental Materials Experimental materials used in this experiment in- cluded the following: commercial lignin*, Sims silt loam -(humic glei soil), and samples of Houghton muck soil, taken from a virgin area, a reclaimed area unfertilized for fifteen years and a reclaimed area which had been heavily fertilized for fifteen years. These materials were air-dried and passed Ithrough a 2 mm. sieve before being subjected to the various laboratory treatments. Experimental Treatments Fixation of nitrate nitrogen Fifty ml. of 1N NaNO3 and a drop of toluene were placed in a 500 ml. Erlenmeyer flask containing 2 g. material (10 g. for Sims silt loam). The pH in different flasks was adjusted with NaOH and/or HNO3 to 2, u, 7, and 9. The flasks were closed with two-hole rubber stoppers to allow for aeration. They were then shaken continuously for three days. The contents d? the flasks were then neutralized with dilute HCl or NaOH. The precipitate in each flask was filtered off and was waShed with neutral 0.5N KCl to remove free and exchangeable NEE and N05. The total nitrogen of the precipi- tate was determined by the Kjeldahl method using'Devarda's *"Indulin A", a product of the west Virginia Pulp and Paper Co. ‘ “.1.- ‘- \x.,uw , 4. a ,3, .4 5.49 u'l alloy to reduce any N03 that may have been fixed. A similar quantity of the original material was taken, neutralized with dilute HCl or NaOH, and washed with neutral 0.5 N KCl as the above. Total nitrogen was determined by the same procedure. The differedce between these two total nitrogen determinations was expressed as me. per 100 g. of material. Fixation of ammonia nitrogen A 2-g. sample of the original material (10 g. for Sims silt loam) was placed in a 500 ml. Erlenmeyer flask. Fifty ml. of NHuCl or 12% NHhOH solution and one drop of toluene were added to the flask. In the case of the HHuCl solution, the pH of the contents was adjusted to a given vahu: inung NHhOH, HCl, NaOH or Ca(OH)2, according to the purpose of the experiment. The flask was fitted with a two-hole rubber stopper through which air could freely enter. The flask was shaken continuously for three days. Then the con- tents were neutralized with dilute HCl or NaOH, the precipi- tate was filtered off and was washed with neutral 0.5N KCl until ammonia was no longer detected in the fiItrate. The total nitrogen of'the residue was determined by the Kjeldahl Procedure. This nitregen determination was considered to contain both the nitrogen in the original material (No) and the ammonia fixed during treatment (Nf), A separate 2-g. aliquot of the original material was neutralized, washed as the above and the total nitrogen of 12 the residue (No) was determined. "Fixed nitrogen" was deter- tuned as the difference between total nitrogen in the treated sample (No+'Nf) and total nitrogen in the original material (No). This was converted to me. per 100 g. Oxidation and methylation studies Eight treatments were designed to study the relation- ships between ammonia fixation, methylating capacity and cation exchange of lignin, fertilized reclaimed muck,and virgin muck. 'Ihese treatments were as follows: (a) Original material. (b) Original material subjected to oxidative ammonia fixation as previously described, using 12% NHhOH and a three-day shaking period. (c) Original material subjected to methylation, as follows: Two grams of the original material were allowed to react for 2h hours with 1 ml. of dimethyl sulfate in the presence of 50 m1. of 0.2N KOH in an atmoSphere of nitrogen. After the treatment, the suspension was acidified with di- lute acid. The precipitate was centrifuged and’ washed with distilled water. This treatment was repeated five times on each sample to assure com- . plete methylation. (d) Ammonia-treated material (b) subjected to (e) (f) (a) (h) The methylation as in (c). Methylated material (c) subjected to ammonia treatment as in (b). Original material subjected to oxidation with NaOH as follows: Fifty m1. of 0.5N NaOH were placed in a 500 ml. Erlenmeyer flask containing 2 g. of the material. The flask was fitted with a two-hole rubber stOpper and shaken continuously for three. days. Then, the suspension was neutralized with dilute HCl and the precipitate was washed with distilled water. . Sodium hydroxide-treated material (f) subjeCted to methylation as in (c). hethylated material (c) subjected to NaOH- oxidation as in (f). ' Chemical and Physical Determinations following chemical and physical determinations were made on the original materials and/or the materials after various treatments: 1. 3. The pH of original materials was determined using a glass electrode and a 1:1 soil to water ratio. Ash content of original materials was determind by ignition at 70003. Cation exchange capacity of original and treated materials was determined using the barium acetate lb method described by Wilson and Staker (27). Total nitrogen of original and treated materials was determined by the Kjeldahl method, using a mixture of CuSOu and K2th (1:9) as the catalyst. Devarda‘s alloy was used when it was desired to include NO3'. . The stability of fixed nitrogen was determined by .its resistance to removal by leaching with 0.1N H01 or by distillation of the KCl-extracted mate- rial in the presence of MgO. The procedure used was as follows: . Using 12% NHhOH’ a 2-g. sample of the original material was subjected to oxidative ammoniatnni as in the (b) treatment described above. This treated material was leached with successive por- tions of 0.1N HCl to a final volume ofa.bout 1000 ml. The total nitrogen of the residual material was determined. Another treated sample was leached with neutral 0.5N KCl until ammonia disappeared in the filtrate. ‘The total nitrogen of the residual material was determined. The difference between these two total nitrogen determinations was ex- pressed as "nitrogen removed by acid treatment?. A 2-g. sample of material subjected to oxi- dative ammonia fixation as above and another untreated sample were leached with neutal 0.5N K01 .15 until exchangeable and free ammonia were removed. Then the nitrogen released during distillation in the presence of excess MgO for MS minutes was determined. The difference between the nitrogen removed by MgO from treated and untreated materi- als was calculated as me. per 100 g. of material. The methoxyl content of original and treated materi- als was determined, using a semi-micro alkoxyl apparatus, by a modification of the official AOAC procedure (15) suggested by the chemistry section of the West Virginia Pulp and Paper Co. (26). The apparatus used is shown in Figure 1. The trap of the apparatus was filled with distilled water. A 10% solution of potassium acetate in glacial acetic acid, to which had been added 6 drOps of bromine, was introduced into the absorption tubes. Six ml. of this reagent was poured in the first tube and h ml. in the second. About 20 to 30 mg. of sample material and one drop of Hg were placed in the digestion flask. Melted phenol (2.5 m1.) and 5 ml. of HI were added. The digestion flask was connected to the absorption 'tubes. Nitrogen gas was passed through the appar- atus from the side arm of the flask at a uniform rate. The liquid in the digestion flask was boiled at such a rate that vapors of boiling cm 4% f 7 5 H i L a u ;! ME E U l‘ é it ‘3' g i‘ i? s W . E ; ~ ' 35 1: C re- «J I v w“ in cm . . : . a if . 31 E E' l V L 4 h g f l D F 3 E w ei ; } A; N2 gas inlet (capillary tip) .59; q} fir B; Reaction flask (gas/20) M L, C; Condenser (diam. 0.7 cm.) G D; Trap l | f i E, E', E"; Joint ($7/25) . F, G; Absorption tubes (pyrex, 10 ml.) I H; DrOp catcher (diam. 2 cm.) Fig. l. Alkoxyl apparatus 17 liquid rose about half way up the condenser. Boiling was continued for 60 minutes. The flask was disconnected and the absorption tube was re- moved. The contents of the tubes were washed into 125 ml. Erlenmeyer flasks containing 5 ml. of 25% aqueous sodium acetate solution. The volume was adjusted to about 50 ml., and formic acid was added drOpwise until excess Bra was destroyed. Any Bra vapors were removed by blowing air over the liquid. .Then 0.5 g. of KI and 5 ml. of 10% HZSQH were added. The solution was swirled to dissolve KI and to mix the contents. Liber- ated 12 was titrated with Kg Na28203 solution. A blank determination was run, using all reagents, without the sample. The percent methoxyl content was calculated as follows: (ml. of Na28203 in determination- ml. in blank)xn%15.l7*100 sample weight (mg) Ultralviolet Absorption Ultraviolet absorption Spectra were developed for the fOllOIJ ing: (a) (b) (o) (d) Original lignin and fertilized muck. Lignin and fertilized muck after oxidative ammonia fixation. Lignin and muck after methylation. Lignin and muck after NaOH oxidation. '18 A Beckman DK—2 recording spectrophotometer was used. The materials were suspended in 0.1N NaOH in suitable dilu— tions for effective observation over the range of wavelengths from 2&0 to 350 mu. 19 RESULTS Preperties of Materials , Table 1 shows the properties of materials used for this research. Sims silt loam and virgin muck both had a pH of 6.2. Lignin was the lowest at pH 3.3. The Sims silt loam contained the highest percentage ash, 9h.h% and lignin had the lowest, 9.2%. All the mucks contained less than 20% ash. The nitrogen in the Sims soil and in lignin was much less than that in muck. Among mucks, nitrogen content decreased in the order: Fertilized>virgin>unfertilized. The greatest cation ex- change capacity was found in the muck: A maximum of 16h.u me. per 100 g. was found in the nonfertilized reclaimed muck, 161.0 me. in the fertilized, and lh2.l me. in the virgin muck. On the other hand, the cation exchange capacities of the Sims soil and of lignin were 22.3 and 32.9 me. per 100 g., respec- tively. Fixation of Nitrate Nitrogen The results of experiments conducted to determine the effect of pH on the fixation of nitrate nitrogen by lignin and soil materials are presented in Table 2. No nitrate nitrogen was fixed by any of the materials. Negative fixation was obtained for virgin muck, and the net loss of nitrogen was slightly greater in both acid and alkaline solutions than at neutrality. In any case, the amounts of nitrogen lost were less than u me. per 100 g. TABLE 1. 20 PROPERTIES OF THE ORIGINAL EXPERIMENTAL MATERIALS Materials pH Ash content Total N CEC % % me./100g. Sims silt loam 6.2 9uoh 0.20 22.3 Virgin muck 6.2 17.9 3.27 ' lh2.1 Nonfertilized muck 5.u 19,5 3,07 16h.h Fertilized muck 5.7 18.7 3.32 161.2 Lignin' 3.3 9.2 0.17 32.9 TABLE 2. FIXATION OE NITROGEN FROM IN NaNOg SOLUTIONS ADJUSTED TO VARIOUS pH' 1011* Sims (silt loam Virgin muck Lignin me./100g. me./lOOg. —2.2 -2.0t -2.0 -3.8 me./100§T %) pH was adjusted with NaOH 0 .l.aun'v at. '3’- bl’KtE . l ... NI». .1. I 21 Fixation of Ammonia Nitrogen and Factors Affecting It Influence of pH on oxidative ammonia fixation For this experiment, IN NHuCl was used and pH was adjusted to h, 7, 9, and 10.5 with NHAOH, and/or H01. The results are shown in Table 3 and Figure 2. Sims silt loam fixed a small amount of ammonia from acid solution but none on the alkaline side of neutrality. All the organic materials fixed much more nitrogen on the alkaline side than at neutral- ity or below. In the case of lignin, the amount of fixed nitrogen was 100.h me. per 100g. at pH 10.5. This was the highest for any material. Though fertilized muck fixed slightly more ammonia than virgin muck, both fixed less than 30 mo. per 100 g. When the materials were shaken in 12% NHAOH without any adjustment of pH, amJonia fixation was in- creased to 32.h, 35.3 and 168.0 me. per 100 g. for virgin and fertilized mucks and lignin, respectively. Influence of bases and concentration of NHuCI on ammonia fixation at pH 7.5. In Table A and Figures 3, h and 5 are presented data showing the influence of bases used for pH adjustment and concentration of NHuCl solution on the oxidative fixation of ammonia. It was found that the amount of ammonia fixed in— creased with increasing concentration of NHuCl with all .materials, whether NaOH or Ca(OH)2 were used to adjust the solution.to pH 7.5. However, at any given pH, more ammonia 22 TABLE 3. INFLUENCE OF pH ON OKIDATIVE AI-’13.~’10NIA FIXATION FROM IN NHuCl SOLUTIONS pH* Sims silt 10am Virgin muck Fertilized muck Lignin A me./IOOg. ram/100$. me./100g. rum/105$. u ' ' 3.5 2.2 8.6 u.1 7 1.0 3.3 9.h u.1 9 - V12.1 lt-h 29.7 10.5 - 25.h 27.6 100.h 12% NHhOH — 32.u 35.3 168.0 at) pH was adjusted with NH 0H and/or HCl after sus... pending 2 g. of material'in 50 ml. of IN NHuCI solution. TABLE u. INFLUENCE OF BASES Are CONCENTRATION OF NHu01 0N AMMONIA_FIXATION AT pH 7. . Virgin muck Fertilized muck Lignin NaQH Ca ( 0H)~2 NagH Ca.‘(.:0H)2 Na'QH Ca_.(_.9‘H)2 HEEL-(13g. ngfiOCg. flog. mjmge m8.r1038. Concentration of 118,401 0.05 N 1.0 1.0 2.3 1.0 ‘ 3.5 3.0 0.1 N 1.5 1.0 3.0 1.8 5.0 3.h 1.0 N u.5 1.5 11.5 2.3 ' 9.5 5.5 *) After addition of neutral NHuCl, PH was adjusted to 7,5 with NaOH. am) After addition of neutral I‘HMCl, pH was adjusted to 7,5 with Ca(OH)2. 23 5 Sims silt loam e o OVir gin musk ‘___x xF'ertilized muck A—A ALignin 100 b ‘i’ I" 90 . I." 'I 80 . .5 8 70' H ‘l a 1'! a 60 L II o‘ E 50 e .’ j I I 'd I a no- I H I s-I . z I 30 ~ e I l . 113 I, g 20 I ,I O I E " “ .. 4: 10.5 pH of suspension Fig. 2. Influence (1‘ pH on the oxidative fixation of ammonia nitrogen if!!! .9... ill 1' I . I I .‘ o I 6“ I m . I ~ _ 4 4 III. . ‘11..- 2b .10 n o—-—-o O NaOH series 0) a II o----O---°-Ca(OH)2 series 0 F O H I ‘4 I 0 a. 5.. e I - >4 ..4 G—I I z . M. ._-___-—0 g —--—~---.o—----------’ o 1‘ J— L-._-._....._..- _J. 0.05 0.1 0.6 1.0 N Normality of NHuCl Fig. 3. Influence of bases and concentration of NHLLCI on ammonia fixation by virgin muck at pH 7.5 r O lOr 0 0 “ NaOH series . o—-—o---—o Ca(0H)2 serie I Me. N fixed per 100 gms. \n T A j J - 0.05 0.1 0.5 . 1.0 N Normality cf NHuCl Fig. )4. Influence of bases and concentration of NHLLCI on ammonia fixation by fertilized muck at pH 7.5 25 0 NaOH series 0....--o..-—-o Ca(OH)2 series ...: O t U'l Ivie . N fixed per 100 gms. l 0.05 0.1 0.5 1.0 Normality of NHuCl Fig. 5. Influence of bases and concentration of NlihCl on ammonia fixation by lignin at pH 7.5 26 was fixed in the presence of sodium than in the presence of calcium. This difference became greater with increasing (concentration of NTIhCL In any case, the maximum amount of ammonia fixed was not more than 12 me. per 100g. Influence of methylation on ammonia fixation In another experiment, the original materials were first methylated and then subjected to oxidation in 12% NHLLOH. The results are presented in Table 5 and Figure 6. Methyla- tion completely destroyed the capacity for oxidative fixation of ammonia by virgin and fertilized muck and resulted in a net loss of nitrogen from these materials. Although the methylated lignin fixed a small amount of ammonia, the capacity for doing so was greatly reduced, fmm 168.0 me. per 100g. in the original material to 9.5 me. after methylation. Cation Exchange Capacity and Factors Affecting It Influence cf oxidation with NaOH on cation exchange capacity 6f the materials Table 6 and Figure 7 show the effect of oxidation with (NaOH on the cation exhange capacity of organic materials. The cation exhange capacity of all materials was increased by oxidation. In oxidized lignin, cation exchange capacity was increased from 32.9 me. in the original material to 261.1 me. per 100g. after oxidation, or about 7-fold. The increases for the two muck samples were much less. The increase for virgin muck was somewhat greater than for the fertilized reclaimed muck. 27 TABLE 5. Hit LUST-T CE 0? 1-113 'L‘HleA '1' I CI? 01: AI-EMGNIA F IKAT ION FROM 12% NHuOH SOLUTION me. of NH3 - N fixed per 100 g. Material Original material Methylated material Virgin muck 32.h -8.3 Fertilized muck 35.3 “-7.8 Lignin 168.0 +9.5 -TABLE_6. INFLUENCE OF OAIDATION NITH NaOH ON CATION EXCHANGE CAPACITY Cation exchange capacity — me. per 100g. Material Original material fiaCH giigztion Net change Virgin muck lb2.1 _ 16h.1 22.0 Fertilized muCk 161.2 177.6 16.h Lignin 32.9 , 261.1 228.2 28 I; Amount of ammonia fixed by original sample II; " " " " by methylated sample III; Cation exchange capacity of original sample of) IV; " " " of methylated sample 0 o H 18 6.: °f 9 III ‘53:; 160* I III , ' 11m? F- ! e 2 1’3 ‘ g g 120} g 3' ‘ ‘ ° = a, 100, X E. w I s C i 3: 3 8 L i g o 0 s - :5 ’4 3 55 in ES 0 ; E l g. g 60* _ f g i E 33' ii '3 EE ‘ o ’40? I E; I III p 5% y 7 IV 201- i; / é a z in n /. II {2 _ _ O»—— -m -..; . ._ “m" L. ...... --- - g.-- _ _208___—-. _- - ._t_l,_.__.-___,_ . Virgin Ferflilized Lignin muck muck Fig. 6. Influence of methylation on ammonia fixation and cation exchange capacity I; Material after NaOH oxidation 29 280 P II; Original material (a) III; Material after oxidative I 260 r- ammonia fixation Z / 21m t g “3 ¢ 8 220 r g? H ¢ " ¢ 3 200 I % ‘ é i a I 180 ." ¢ ,5 . +9 L V .3 160 1 % g I - / . / a m; g LP 3 ¢ III 2 g) 120 . / 33f? / 3 ¢ 5&5? g I % :55 o 100 r g :5: 8 2 g L»? H ‘ / :‘r'; 4:5 80 r ¢ 2'" O ' % III 6° / F232 3 3 ”°' ? :2; II ¢ 2°” 3 é v i Lignin Fig. 7. Effects of NaOH oxidation and oxidative ammonia muck fixation on cation exchange capacity. 30 Influence of oxidative ammonia fixation on The cation exchange capacity of materials The effect of oxidative fixation of ammonia from 12% NHhOH solution on the cation exchange capacity of materials is shown by data presented in Table 7 and Figure 7. Ammonia fixation decreased cation exchange capacity of virgin muck and fertilized muck, 16.1 and 22.6 me. per 100;?” reSpectively. 0n the other hand, an increase of 32 me. per 100 g. was observed in ammonia-fixed lignin. This is an approximate 2 fold increase, which is in sharp contrast with 7-fold increase in cation exchange capacity due to oxidation with NaOH. Influence of methylation on cation exchanre capacity of materials The data in Table 8 and Figure 6 indicate that methylation decreased the cation exchange Cnpnc ity of all materials by 30 to 140%. I-Iethoxyl Content of I~‘Iaterial and Factors Affecting It The methoxyl content was determined for materials after they had been subjected to various treatments, as follows: (a) original materials; (d) after oxidative ammonia fixation followed by methylation; (f) after oxidation with NaOH; (e) after methylation; (g) after NaOH oxidation followed by methylation; and (h) after methylation followed by oxidation with NaOH. The results are presented in Tables .9 and 1.0. Methoxyl content of virgin muck and fertilized muck was 1.8% and 1.14%, reapectively, and was much less than that I 4‘91 33’..- F . f‘ .1... 9.1.9-. g A... .. 31 TABLE 7. INFLUENCE OF OXIDATIVE FIKATION 0F AMMONIA FROM 12% NH,OH SOLUTION on CATICN ' uxcahrca CAPACITY Cation exchange capacity — me. per 100 g. F t i V- 7 1a er 31 Original material After oxidative Let ammonia fixation change Virgin muck 1h2.1 126.0 -16.1 Fertilizcd muck 161.2 138.6 -°2.6 Lignin 32.9 6h.9 +32.0 TABLE 8. INFLUENCE CF Lifll‘T—EYLFLLION 01"? ClTIC‘I‘I EXCHANGE CAPACITY OF MATERIALS Cation exchange capacity - me. per 100g. Material N Original material Methylatcd material at change Virgin.ruuflc lh2.1 91.6 -50.S Fertilized muck 161.2 109.6 -Sl.6 Lignin 32. 21.0 -11.9 . 1. '-_""’~‘1“— 4 _ em... a... . ‘z 32 TABLE 9. METHOXYL CONTENT OF ORIGIINAL AND PEMATED MATERIALS BEFORE AND AFTER OXIDATION Methoxyl content - percent Material am i"!- c-x- h-x- Virgin muck 1.8 1.6 7.0 6.9 Fertilixed MUCk 1.11 1.3 507 506 Lignin 12.8 11.6 2303 23.1 Lignin-H - 12.1 - - *) a original material f " " subjected to oxidation with HIGH c " " subjected to methylation h methylated material subjected to oxidation with NaOH H) Lignin after three days oxidative ammonia fixation TABLE 10. INFLUENCE OF OXIDATION AND AMMONIA F DCATION ON METHYLATION OF LIGNIN AND MUCK Methoxyl Methoxyl content after methylation of content Material before Original NaOH oxidized NHhOH oxidized methylation material material material ~— II II a . ? Virsln muck 1.8 7.0 6.8 6.h Fertilized muck 1.1: 5.7 5.6 5.3 Lignin 12.8 23.3 18.5 16.2 -—¥ 33 TABLE 11. REJIOVAL BY NgO DISTILLATION AND ACID LEACHING OF NITROGEN OXBJATIVELY FIXED FROM 12% NHhOH SOLUTION Nitrorzen removed by Nitrogen ranoved by Material M30 distillation “ leaching withfir no: me./100g. % of fixed N me./100g. 5% of fixed N Virgin muck 1.1.1 314.2 ' 11.8 36.11 Fertilized muck 12.5 35 .7 12.7 35.9 Lignin . 29.5 16.9 32.7 19.5 3b of lignin, 12.8%. Oxidation with NaOH did not materially affect the methoxyl content of the mwflc soils. The methoxyl content of lignin was reduced 1.2% by NaOH oxidation. After methylation, all materials were much higher in methoxyl con- tent than were the original. The methoxyl content of methy- lated mucks was about h times greater and of methylated lignin about 2 times greater than in the materials before methylation. Oxidation with NaOH changed the methoxyl con- tent of methylated materials very slightly, if at all. Data in Table 10 show the effects of prior oxidation with NaOH or NHNOH on the methylation of lignin and muck. When virgin and fertilized mucks were first oxidized with NaOH and then subjected to methylation, the increase in methoxyl groups was essentially equivalent to the increase when the original materials were methylated without prior oxida- tion. When lignin was oxidized with NaOH, its capacity for reacting with methyl groups was reduced. The reduction was even greater when lignin was oxidized in 12% NHhOH9 and a similar trend was noted for the two mucks. Stability of Fixed Nitrogen When Subjected to Treatment with Acids or Alkali Data in Table 11 indicate that both alkali and acid treatment of virgin muck and fertilized muck after oxidative amonia fixation in 1275 T‘U'IuOH removed nitrogen equivalent to one third of the fixed nitrogen. However, the amount of nitrogen removed from the lignin was not more than 20%: 16.9% 35 by distillation and 19.5% by leaching with Ig'HCI' Ultraviolet Absorption In Figures 7 and 8 are shown ultraviolet absorption curves for variously treated lignin and fertilized muck. On Athe whole, untreated lignin produced a complicated absorbance Spectrum. The original material (a) had a minimum absorbance at 262 m». and a maximum at 283 m”. Methylated lignin (0) had a minimum.at 263 nun and a maximum.at 280 mm” So. the spectrum.for methylated lignin (c) was fairly similar to that for the original numerial (a) except that the maximum and' minimum.peaks were no longer so clearly defined. There was a slight shift towards longer wavelengths below the minimum absorbance point and a shift towards shorter wavelengths above that point as a result of methylation of the lignin. Oxidation with NaOH and oxidative ammonia fixation both.shifted the absorption curves towards greater wave- lengths in the range below 310 mm. The curve for lignin subjected to oxidation with NaOH still had a distinct minie mum.at 278 mpand a maximum.at 289 mm. However, the oxida- tive fixation of ammonia resulted in a spectrum.in which the slope reversal in the curve between these two points was completely eliminated. This difference between the curve for lignin oxidized in the presence of .NHLLOH and that for lignin oxidized in the presence of NaOH is similar to the effect of Emethylation on the curve for lignin. The reactions between lignin and ammonia and those involved in methylation both - iii-(3h Absorbance Absorbance ch- .3 Fig. 36 (a)*——*——*—— original material (b)4- - - + - —x~ -- after oxidative ammonia fixation \ .\\.‘ ff 1 ”K \V (c H——~4———«—after methylation ' \ ~ \- \.\(f)—.. - —.-—---4after oxidation with NaOH \ \'\ ?" \ i 4 “mug-a—uuL -- fin .5 ‘ 250 260 270 280 290 3OO 310 320 330 3N0 350 Wavelength -- mpL 8. Ultraviolet absorbance spectrum of lignin (a) a a a original sample (b) —+---fu--—u-- after oxidative ammonia fixation (r) :7 g a after oxidation with NaOH 2B0 260 270‘ 286 290 300 310 320 330 380 350 Wavelength -- up 9. Ultraviolet absorbance Spectrum of fertilized muck 3? resulted in damping of absorbance in the range from 270 to. 300.m;s 0n the other hand, fertilized.muck,--well-humidified ratter,--produced curves with simple and gentle slopes. The effects of NaOH oxidation and Oxidative ammonia fixation did not produce such.striking changes in the shape of the absorbance curves as were observed in the case of lignin. Oxidation with NaOH did result in a shift toward higher wavelengths, whereas oxidation in the presence of NHhOH resulted in a shift towards lower wavelengths. These results are similar to the relative effects of NaOH oxidation and NHuOH oxidation on absorbance by lignin in Figure 7. Since absorbance in the ultraviolet is principally due to resonance associated with double bonds (C-O, C=C, etc.), it would appear that oxidation 222 fig gives rise to an in- crease in such double-bonded groups. On the other hand, methylation and ammonia fixation would appear to result in the disappearance of double bonds. To some extent, at least, both processes would appear to involve similar reaction sites on the lignin molecule or on.humic complexes. 38 DISCUSSION Direct nitration of organic materials is known to take place under drastically anhydrous conditions in the laboratory. The failure to show fixation of nitrate by lignin and soil materials under the mild conditions of the present study might have been ex ected. There appears to be no reason t) believe that direct chemical fixation of nitrate occurs in soils under field conditions. - Numerous workers have shown that direct chemical fixation of ammonia by lignin and soil humic materials does occur and that the process is intimately associated with oxidation of the organic materials. Oxygen absorption by lignin and humic materials is promoted by alkaline conditions. Data by Junker, Bremner and others indicate that basic ions decrease in their effectiveness in promoting oxygen absorp- tion in the order Na> K> NH“) Ba> Ca) Mg) Fe. This is also the order of their decreasing efficiency in peptization of colloidal suspensions of these organic materials, or con- versely the order of their increasing effectiveness in flocculating such suspensions. Thus oxygen absorption, or the rate of autoxidation of lignin and humic materials appears to be in part a function of particle size and the .active surface exposed. In the study, much more ammonia was fixed in the pre- seruns of NaOH than in the presence of Ca(OH)2. This appears to Tnxve been due to the greater peptizing action of the sodium 39 ion and the nmre rapid and complete oxidation which resulted. Ammonia fixation by the organic materials studied increased with increasing pH. The rate of increase was accelerated above pH 7.0. Ox‘gen absorption has also been shown to increase rapidly in the alkaline range. This may be due in part to increaseipeptization, but also to the activation at high pH of potential sites for oxygen absorption. At a given pH, the fixation of ammonia was a direct function of the concentration of ammonia. The increase in fixation with increasing ammonia concentration was logarithmic rather than linear, indicating that fixation involved a series of interdependent reactions rather than a single reaction. Methylation with dimethyl sulfate before treatment with 1““ NHuOH almost completely blocked the oxidative fix- C- If) ation of ammonia by lignin. A net loss of nitrogen occurred (he to hydrolysis by the HON used in the methylation procedure. Thus it appeared that ammonia fixation was specifically associated with methyl reactive groupings in these organic Imiterials. Methylation blocked approximately one-third of the cation exchange sites in the muck materials and in lignin. .Dinuithyl sulfate reacts with alcoholic-OH and phenolic-OH The empirical formula submitted by the manufacturers groupS. of’iflse lignin used, which corresponds to the formula preposed by'Iirauns, shows five hydroxyl groups, one of them.phenolic. The actual increase in methoxyl content due to methylation -»T""-"‘_'“"-“-‘ f - .. .. ho would have blocked approximately two-thirds of these hydroxyls. No carboxyl or other groups with recognized cation exchange activity are present. Thus there appears to be no stoichio- metric relationship between total alcoholic and phenolic-OH groups of lignin and its cation exchange capacity. Oxidation with NaOH resulted in a 10 to 15% increase in cation exchange capacity of the muck materials and a 7-fold increase in exhange capacity of lignin, or an increase of 228 me. per 100 g. Oxidation in 12% NHuOH resulted in a 10 to 15% decrease in cation exchange capacity of the muck materials. A moderate 21fold increase for lignin was in sharp contrast to the 7-fold. increase which resulted fnim NaOH oxidation. Ammonium hydroxide oxidation resulted in a decrease in methylating capacity equivalent to 7.1 grams of -OCH3 per 100 grams, or 229 me. If it is assumed that oxidation 222.29 in th£:presence of NHuOH resulted in loss of the same methyl reactive groupings as those affected by NQOH oxidation, then additional loss in methylating capacity with NHhOH oxidation nunit have been due to the formation of non-dissociable com- binations of ammonia with methyl reactive sites which were Inuiffected by NaOH oxidation. Significatly, the total loss of1methylating capacity of lignin due to NHMOH oxidation (229 mm“) was equivalent to the total gain in cation exchange capacity'with NaOH oxidation (228 me). By this line of reasoning, the increase in cation hi exchange capacity due to NaOH 0x1 accounted for as the sum of methyl reactive sites destroyed by oxidation plus ammonia reactive groups formed by oxidation at sites not active in methylation. Such ammonia reactive groups could include carboxyls resulting from oxidative degradation of saturated side chains which would not have had methylating activity. Subsequent or concurrent processes of ammonia fixation and ceidensation would have resulted in the tying up of an equivalent number of methyl reactive groups. The loss of methylating capacity in lignin with NHuOH oxidation was greater than the loss with NaOH oxidation by an amount equivalent to 2.3 grams of -OCH3 per 100 grams. If the_products of the reaction of ammonia with these in- form of imino- ( activated methylation sites were all in the phenols, this would be equivalent to a lOou of 7a me. of cation exchange capacity in the oxidized lignin. If linear condensation reactions occurred of the type inferred in the previous paragraph, involving side-chain carboxyls and imino- :flacnols to form nonecyclic imides, the loss in exchange capa- city would have been twice as great, or lh8.:me. per 100 grams. If condensation reactions involving ring closure had occurred, a.ifliird anionic grouping with cation exchange activity could turve been affected for each molecule of NH3 fixed. In this Chase, a total cancellation of 222meq. of exchange capacity vflfllld have esulted. The actual depression of exchange cajnicity due to oxidation in the presence of ammonia as .n...‘ _ .. ..V l u2 compared with oxidation in the presence of NaOH was 196 me. per 100 grams. The logarithmic increase in ammonia fixation with increasing ammonia concentration previously discussed is consistent with this concept of multiple reactions involved in ammonia fixation. The results with lignin provide a basis for interpret- ing the results obtained with the muck materials. Differences in cation exchange capacity and aunonia fixing capacity of virgin muck and fertilized reclaimed muck appeared to be due to differences in degree of oxidation and degree of saturation of potential a'monia fixing sites. The fertilized reclaimed muck appeared to be more highly oxidized than the virgin This was indicated by higher ash content, lower meth- The higher muck. oxyl content and lower methylating capacity. cation exchange of the fertilized much was apparently due to its higher oxidation status and the increased number of oxidized groupings {-COOH and CrC-OH) which could be inferred therefrom. Chemical oxidation in NaOH did not increase exchange capacity of the fertilized muck to as great an extent as it did in the virgin muck. The ammonia fixing capacity was greater, particularly at lxna pH and with higher eorcentration of ammonia, in fertil- ized tflmu11n.virgin.muck. The depression in cation exchange capacity due to ammonia fixation was also greater. Thus the degree of samration of potential a;.-m1onia fixing sites in fertilized muck was less than in the virgin muck. Apparently processes of ammonia fixation during fifteen years of b3 cultivation had not kept pace with processes of oxidation. In the muck materials, all of the ammonia fixing activity appeared to be associated with oxidation of methyl reactive groups, since prior methylation completely suppressed oxidative ammonia fixation. Increases in cation exchange capacity with NaOH oxidation approached the quantitative equivalence to losses in methylating capacity due to oxidation and ammonia fixation which was found in lignin. In the virgin muck, NaOH oxidation increased exchange capacity 22 me. per 100 g. While oxidative ammonia fixation reduced methylating capacity 20 me. per 100 g. In the fertilized reclaimed muck, there-was an increase of 16 me. in exchange capacity with NaOH treatment and a decrease of 13 me. in methylating capa- city with NHuOH. Ammonia fixation, NaOH oxidation and methylation studies were not conducted on the non-fertilized muck. It had a higher ash content and a somewhat higher cation exchange capacity than the fertilized muck, which suggests that it was nmme highly oxidized. It had a lower nitrogen content tkmui either the virgin or fertilized mucks. From this fact 11:1night be expected to be lower in degree of saturation of potmnitial ammonia fixing sites and hence higher in ammonia- fixing capacity than the other two muck materials. The data on losses of fixed ammonia with MgO distilla- tion and acid leaching indicate that added ammonia is fixed less tenaciously by humified soil materials than by fresh hh lignin. This is doubtless correlated with the higher degree of saturation of potential ammonia fixing sites in humified materials. The differences in exhange capacity and ammonia fixa- tion noted between muck and lignin appear to be essentially due to differences in degree of oxidation and degree of saturation of potential ammonia-fixing sites. Ultraviolet absorbance studies showed a shift towards higher wavelengths and an accentuation of characteristic mimimum and maximum peaks in the curves for lignin after NaOH oxidation. Oxida- tive ammonia fixation of lignin tended to eliminate charac- teristic maxima and minima. Absorbance spectra for mucks showed gentle curves without maximum.or minimum peaks. The shape of the curves for muck materials were unaffected by NaOH oxidation or ammonia fixatiOn. Methylation had no effect on the shape of curves for lignin or for mucks. b5 SUMI-IARY The non-biological fixation of nitrate and ammonia by mineral and organic soils and by lignin was studied in the laboratory. The results of these studies say be summarized as follows: 1. No nitrate was fixed at any pH by any of the materials. A small amount of aitluonia was fixed by mineral soil at pH n.0, very little at pH 7.0, and none above neutral- ity. Fixation of ammonia by organic soils and lignin increased with pH at a fixed concentration of NHlLCl and with concentra- tion of NHlLCl at a fixed pH. Fixation was much greater in the-presence of NaOH than when Ca(OH)2 was used to adjust pH. 2. Leaching with N/lO HCl an d distillation with MgO removed 314. to 36% of fixed ammonia from mucks and 16 to 19% from lignin. There was little difference in the proportion removed by acid leaching or by 34530 distillation. 3. Fixation of ammonia by lignin and muck soils was related to the oxidation of methyl reactive groupings to groupings active in cationic exchange, in the following manner: When parallel samples were oxidized in NaOH and in NHZLOH, an increase in cation exchange capacity was observed in NaOH oxidized samples which was equivalent to the loss in the methylating capacity of samples oxidized in NHLLOH. Each molecule of ammonia fixed, however, resulted in the inactiva- tion of-2 to 3 of the cation exchange sites which were formed by oxidation. In the muck soils this resulted in a net loss 116 in cation exchange capacity as the result of oxidative fixa- tion of ammonia. h. Differences in cation exchange capacity and ammonia fixing capacity of virgin and cultivated mucks appeared to be related to differences in degree of oxidation and in degree of saturation of potential ammonia fixing sites. The cultivated muck was higher in ash, lower in methoxyl con- tent, lower in methylating capacity, and higher in cation exchange and ammonia fixing capacity than the virgin soil. 5. Ultraviolet absorbance spectra for lignin exhibi- ted complicated curves with steep slepes and a distinct minimum.at 262 mu and a distinct maximum.peak at 283 mu. Oxidation with NaOH resulted in a shift towards higher wave— lengths of these characteristic minimum and maximum.peaks; ammonia fixation tended to eliminate them. hethylation had no effect on the shape of the lignin curve. Curves for muck materials displayed gentle lepes without maximum.or minimum . peaks and their shape ms unaffected by NaOH oxidation, ammonia fix- ation or methylation. 1. 10. 11. 12; 13. 1:7 BIBLIOGRAPHY Bennett, E., Fixation of ammonia by lignin. Soil Sci. 68: 399-3’40. 19m Brauns, F. E., The Chemistry 93 Lignin. Academic Press Inc., New York, 1952. Bremner, J. M., A review of recent work on soil organic matter. 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Schrader, H., The auto-oxidation of lignin and the natural humins and coals, and the effect of alkalies upon it, BrennstofT-(hcm, 3: 161-167, 1922. Schubert, w. J. and Nerd, F. F., Investigations on lignin and lignification: I. Studies on soft wood lignin. J. Amer. Chem. Soc. 72: 997-981, 1950. .— Sohn, J. B., and Peech, H. Retention and fixation of ammonia by soils. Soil Sci. 85: 1-9, 1958. Springer, U. The influence of ammonification on the organic material in high moor peats. Z.Pflanz. Bodenk, 28: 160-186, 19h2. (C. A. referred). Thomlitz, R. Zur Frags der Ausnutzung dos von Rohhumus aus Armoniak in foster Iiindurg aufgenonunenen Stickstoff und der Stikstoffs und Gogenwart von Rohhumus durch Nadelhalzsamlingo. Z. Pflanz. Dung. Bodenk., 70: 207-220, 1955. , Die Stickstoffostlegung aus schwefelsaurom Ammoniak durchlfichtenrohhumus boi verschicdener Reaktion. Z. Pflanz. Dung. Bodenk., 73: 202-209, 1956. ‘Waksman, S. A. Humus. The Williams and fiilkins Company, Baltimaee, 1938. , and Iyer, K. R. N. Contribution to our knowledge of the chemical nature and origin of humus: I. On the synthesis of the ”humus nucleus". Soil Sci, west Virginia Pulp and Paper Co. Indulin. Bulletin L-6 of the Develorment Department, I951. 27. Wilson, B. soils. D., and Staker, 3. Cornell Univ. Agr. h9 V. 'Ionic exchange of peat pr. Sta. Hem. 172, 1935. .- -vxr.__....‘._n.-n_.-—n_ . ‘ ‘\ ®‘-~ ”£535 circulation dept. $3.)" AUG ”2' 9 195% ,3"! 7 May w, ...... i ‘34 FT“ Ag»; "‘ “ .‘i ‘- L—J