The Chemistry of Fungal Humic Acid-like Polymers and of Soil Humic Acids1

M. SCHNITZER, M. I. ORTIZ DE SERRA, AND K. IVARSONS

ABSTRACTA. niger, E. nigrum, and S. chartarum were grown in glucose-

asparagine media according to Martin et al. The fungal "humicacids" synthesized were isolated, purified, and characterizedby elementary and functional group analyses and by permanga-nate oxidation of methylated preparations. The oxidationproducts were separated by extraction into organic solvents,followed by preparative gas chromatography. The major com-ponents were identified by mass spectrometry and microinfra-red spectrophotometry. The experimental data for the fungal"humic acids" were then compared with those obtained byidentical methods on soil and peat humic acids.

There were a number of similarities in surface functionalgroups between the fungal and soil preparations. The majoroxidation products from the fungal materials were: (i) aliphaticmono- and dicarboxylic acids; ( i i ) benzenecarboxylic acids;(iii) phenolic acids; and (iv) aromatic compounds containingS and N. Compared with peat and soil humic acids, the fungalmaterials produced, per gram of initial material, relatively smallamounts of aromatic but more aliphatic compounds. The E.nigrum "humic acid" yielded substantial amounts of n-Ci4 ton-C18 fatty acids. The results show that fungal "humic acids"are complex organic materials containing aliphatic and aro-matic structures, only some of which are phenolic. Claims byMartin et al. that simple phenols and phenolic acids weresignificant constituents of fungal "humic acids" were not con-firmed.

Additional Index Words: Aspergillus niger, Epicoccum nig-rum, Stachybotrys chartarum, functional groups, permanganateoxidation of methylated preparations.

1 Contribution no. 429 from the Soil Research Institute, Can-ada Agr. Ottawa, Ont. Received Aug. 16, 1972. Approved Oct.12. 1972.2 Senior Research Scientist; Visiting Scientist from the Uni-versidad National del Sur, Bahia Blanca, Argentina; and Re-search Scientist, respectively. The authors thank G.F. Morrisfor elementary analyses.

WHILE it is often assumed that the main structural unitsin soil humic substances originate from lignins and

other phenolic plant constituents, the possible occurrencein humic materials of complex chemical structures of mi-crobial origin has been stressed in a series of papers byMartin, Haider, and associates (1, 7, 8, 13, 14, 15, 16).These workers have reported that cultures of Epicoccumnigrum, Stachybotrys atra, Stachybotrys chartarum, Asper-gillus sydowi, and Hendersonula toruloidea, when providedwith relatively simple organic C and N sources, were ableto produce dark-colored polymeric substances which re-sembled humic materials in resistance to decomposition inthe soil, elemental analysis, exchange capacity, total acid-ity, molecular weight distribution, release of amino acidson hydrolysis with 6N HC1 or proteolytic enzymes, andphenols, toluenes, and phenolic acids released after Na-amalgam reduction of 6N HCl-hydrolyzed materials. Bothenzymatic and nonenzymatic oxidative and coupling me-chanisms were assumed to be active in the formation of thefungal polymers, which were thought to arise from inter-actions of phenols synthesized by the fungi with peptidesor amino acids. Ether-soluble reduction products includedthe following phenols, toluenes, and phenolic acids (8,14): phloroglucinol, resorcinol, pyrogallol, orcinol, 2,4-dihydroxytoluene, 2,6-dihydroxytoluene, methyl phloro-glucinol, methyl pyrogallol, 2,3,5-trihydroxytoluene, 2,4,5-trihydroxytoluene, orsellinic acid, m-hydroxybenzoic acid,p-hydroxybenzoic acid, salicylic acid, 2,4-dihydroxyben-zoic acid, 3,5-dihydroxybenzoic acid, 6-methyl-salicyclicacid, gallic acid and 2,3,4-trihydroxybenzoic acid.

Ether-soluble degradation products accounted for 12-32% of the fungal "humic acids" (13). Crude estimatesof the quantities identified, based on sizes and intensitiesof spots separated on thin-layer plates, ranged from 2-6%of the starting materials for E. nigrum up to 15-20% of

230 SOIL SCI. SOC. AMER. PROC., VOL. 37, 1973

the starting materials for "humic acids" produced by S. atraand S. chartarum (13). Addition of montmorillonite, ver-miculite, and kaolinite to aerobic cultures of Hendersonulatoruloidea, Stachybotrys spp., and Aspergillus sydowigreatly accelerated the formation of humic acid-like poly-mers (1).

Martin, Haider, and associates (1, 7, 8, 13, 14, 15, 16)used two approaches to demonstrate similarities betweenfungal "humic acids" and natural humic acids: (i) theymeasured oxygen-containing functional groups, which pro-vided information on the chemical properties of the poly-meric surfaces, and (ii) they degraded the two types ofmaterials with Na-amalgam in order to find out what thechemical make-up of the "cores" or "inside" of the differ-ent polymers was.

It may be appropriate to discuss at this point some ofthe problems associated with Na-amalgam reduction whichwas the sole degradative chemical method used by Martin,Haider, and associates, and on which they based many oftheir conclusions regarding structural similarities betweenfungal "humic acids" and natural humic acids.

An examination of the literature shows that oxidativeand thermal degradations of humic substances have so farprovided more useful information on the chemical struc-ture of humic materials than have reductive degradations.As has already been pointed out by Zetsche and Reinhart(26) in 1939, it is difficult to reduce humic acids; theyhave a natural tendency to be oxidized. Zetsche and Rein-hart (26) reduced a number of synthetic and natural humicacids with Na-amalgam in aqueous solutions *at about100C. The solutions underwent a series of color changeswhich ranged from dark brown, through green, reddish,orange to light yellow. The reduction products were foundto be extremely unstable and were readily reoxidized byair-oxygen. It was, therefore, necessary to completely ex-clude oxygen. The reduced humic materials could be sta-bilized by methylation, followed immediately by reductionwith Zn-dust in glacial acetic acid and additional methyla-tion with diazomethane. Zetsche and Reinhart (26) con-cluded that the reductive action of Na-amalgam appearedto be limited mainly to the removal of oxygen. They didnot isolate any simple compounds from the reductionproducts.

Burges et al. (4, 5) reported recoveries up to 30% ofinitial soil humic acids of ether-soluble substances afterreductive cleavage with Na-amalgam. They described theether extracts as rich mixtures of phenolic acids and alde-hydes, including Ce-C1 and C6-C3 units. However, Men-dez and Stevenson (18), Stevenson and Mendez (25),Felbeck [see footnote in Martin et al. (16)], and Schnitzer(unpublished data) were unable to confirm the findings ofBurges et al. (4, 5) that most of the ether-soluble materi-als consisted of phenols and phenolic acids. They noted theoccurrence of substantial amounts of aliphatic substances inthe reduction products from natural humic acids. Stevensonand Mendez (25) failed to detect any of the phenols iden-tified by Burges et al. (4, 5) such as phloroglucinol, resor-cinol, methylphloroglucinol, 2,4-dihydroxytoluene, andpyrogallol. Only vanillic and syringic acid were positively

identified and strong evidence was obtained for the occur-rence of vanillin and syringaldehyde in the humic acid re-duction product (22). These compounds were estimatedto account for no more than 1 % of the original humic acid.Mendez and Stevenson (18) have also shown that knownphenols and phenolic acids were degraded by Na-amalgamreduction and that substances enriched in aliphatic struc-tures were formed. This suggests that phenolic constituentsreleased from humic acids during reductive cleavage maybe chemically modified, so that the chemical structures ofthe compounds isolated may have little or no relation tothose occurring in the initial humic acids.

From what has been stated above, we can conclude thatNa-amalgam reduction is an experimentally difficult pro-cedure and that we know relatively little about the reactionsinvolved in it. The procedure appears to work well on somehumic acids and in some laboratories but does not do so onother humic acids and in other laboratories. Some of thedifficulties referred to above can be overcome by check-ing the results obtained by a different but independentmethod. Thus, we set out to produce a number of fungal"humic acids" according to the conditions described byMartin et al. (16) and characterized the fungal "humicacids" by a number of analytical methods and by per-manganate oxidation of methylated materials, one of themost useful degradative procedures presently available forthe characterization of humic materials. Methylation priorto oxidation protects phenolic OH groups against attackby electrophilic KMnO4, and so permits the isolation ofphenolic in addition to aliphatic and benzenecarboxylicacids. During the past 2 years this procedure has beenwidely used in this laboratory (9, 10, 11, 19, 24) for thecharacterization of a large number of humic acids, fulvicacids, and humins extracted from soils of widely differentgeographical origins and pedological histories. Our proce-dure entailed oxidation of the methylated materials with4% aqueous KMnO4 solution, extraction of the oxidationproducts into ethyl acetate, remethylation, separation bypreparative gas chromatography into relatively pure com-pounds which were identified by mass spectrometry andmicroinfrared spectrophotometry. The analytical data forfungal "humic acids" were then compared with those ob-tained by identical methods on soil and peat humic acids.It was hoped that the results so obtained would shed addi-tional light on the contribution of microbes to the synthesisof naturally occurring humic acids, a subject that has beenof concern to soil scientists for many years.

METHODS AND MATERIALSThe A. niger was isolated from a soil and supplied to us by

E.A. Peterson, Chemistry and Biology Research Institute, Can-ada Department of Agriculture, Ottawa, Ontario. The E. nigrumused was culture D-64 isolated from decaying Pinus strobus andwas supplied by J. K. Shields of Canadian Forestry Service,Department of the Environment, Ottawa, Ontario. The S. char-tarum culture employed was University of Alberta MicologyHerbarium No. 1690 which was isolated from a soil and ob-tained from J. W. Carmichael, University of Alberta. For"humic acid" production the procedure described by Martinet al. (16) was used with the following modifications: Dextrosewas sterilized and added to 1.2-liter portions of sterile medium

SCHNITZER ET AL.: CHEMISTRY OF ACID-LIKE POLYMERS AND SOIL HUMIC ACIDS 231

Table 1—Elementary analysis of untreated and 61V HC1-hydrolyzed fungal "humic acids" and of soil and peat

humic acids ( % dry, ash-free)

Table 2—Elementary analysis of methylated untreated and6N HCl-hydroIyzed "humic acids" ( % dry, ash-free)

Elementor

group

CHNS0

OCH,Asht

Weight loss on6NHC1hydrolysis, %

A. nlgerHumic acidb*

52.35.89.41.5

31.01.11.0

at63.54.21.10.7

30.60.50.0

47.0

E. nigrumHumic acid

b*.49.17.2

10.61.7

31.51.31.4

at60.76.13.40.7

29.20.00.6

82.0

S. chartarumHumic acidb'

57.37.06.81.2

27.70.70.6

Range Cor soil andpeat humtc acids

at66.7.3.0.

21.0.0.

49.

5289710

0

53.3.0.0.

32.

b*6-58.72-5.58-4.76-1.52-36.5

0.3-1.90. 9-11.2

55.3.0.0.

31.0.

31.

at9-59.42-4.53-1.55-0.65-37. 53-1.60-0.6

8-50.0' Before 6N HC1 hydrolysis,t After 6JV HC1 hydrolysis,t On air-dry basis.

in ten 5-liter Roux bottles. Ten milliliters of spore suspensionsof 5-day cultures of the fungi were added to each bottle. Thebottles were incubated on the flat side for 7 weeks at 22C. Toobtain "humic acids," the cultures were filtered and the filtrateswere transferred to dialysis tubing and dialyzed against distilledwater for 48 hours. "Humic acids" were coagulated by adjust-ing the pH of the solutions to 2.0 with IN HC1 solution. Afterstanding overnight, the coagulates were separated by centrifu-gation, dialyzed until free of Cl~ and then freeze-dried. Yieldsof fungal "humic acids" from 12 liters of culture solutions wereas follows: A. niger: 4.1 g; E. nigrum: 3.5 g; and S. chartarum:4.0 g. Attempts to produce "humic acid" with the aid of A.Sydowi were unsuccessful.

Carbon and H were determined by dry-combustion, N by theautomated Dumas method, S by oxygen-flask combustion, andO was calculated by difference. The OCH3 content was mea-sured by the Zeisel method. Moisture was determined by heat-ing separate samples at 105C for 24 hours and ash by heating at700C for 4 hours. Total acidity and carboxyl groups were mea-sured by methods described by Schnitzer and Gupta (21), car-boxyl groups by oximation (6), and total hydroxyls by acetyla-tion (3). Phenolic and alcoholic hydroxyls were considered tobe equal to the difference between total acidity and carboxyls,and between total hydroxyls and phenolic hydroxyls, respec-tively. Quinone groups were measured amperometrically (23).E4/E6 ratios were determined by dissolving samples in 0.057VNaHCO3 solution and measuring optical densities at 465 and665 nm on a Beckman model B spectrophotometer. The ratioof the optical densities at the two wavelengths was the E4/E6ratio.

To lower the N-content, separate 1-g samples were refluxedwith 100 ml of 6N HC1 solution for 20 hours. The residue wasseparated from the supernatant solution by filtration through asintered glass funnel and washed thoroughly with distilled wateruntil free of chloride. The residue was then dried over P2O5 ina vacuum desiccator at room temperature.

Two methylation procedures were used: One-gram sampleswere suspended in 10 ml of methanol and methylated with anether solution of diazomethane generated from Diazald. Themethylation procedure was repeated with fresh diazomethaneuntil the OCH3-content remained constant. The other proce-dure involved methylation with dimethyl sulfate over anhydrousK2CO3 in acetone (2).

The procedures used for oxidation, separation, and identifica-tion of oxidation products were identical to that described byKhan and Schnitzer (10).

RESULTS

Elementary Analysis—As shown in Table 1, the C con-tent of untreated fungal "humic acids" synthesized by A.niger and E. nigrum was somewhat below the range of thatfor soil and peat humic acids, but the C content of theuntreated "humic acid" produced by S. chartarum fellwithin the range. The data for soil and peat humic acids

Elementor

groupCHNSO

OCHj

A. nigerHumic acid

b*

It53.26.57.91.4

31. .113.8

It64.35.53.61.0

25.620.7

atn§

66.95.31.61.0

25.111.8

E. nigrumHumic acidb*

It49.27.3

10.41.5

31.79.3

atIt

64.96.73.40.8

24.315.1

S. chartarumHumic acid

b'

It57.17.48. 11.1

26.311.9

It68.27.44.80.6

19.213.5

itIII

60.96.81.93.7

26.810.8

• Before 6JVHC1 hydrolysis,t After 6N HC1 hydrolysis.t Methylated with diazomethane.§ Methylated with dlmethylsulfate.

were obtained by analyzing 14 different humic acids bythe same methods as the fungal "humic acids." The soil andpeat humic acids were extracted from samples taken fromthe following soils: the O and Bh horizons of a Podzol;the Ap horizon of a Gray Wooded soil; the Al horizonof a Volcanic Ash soil; the Al horizon of a Diluvial soil;the Ah horizon of a Chernozem; the Al horizon of a Chest-nut; the Ah and Al horizons of a Solonetz; the Ah andAl horizons of a Solod, the Al and B2 horizons of a Bru-nizem and the surface horizon of a peat. The H content ofthe untreated fungal "humic acids" (Table 1) was higherthan that for soil and peat humic acids. Especially note-worthy was the high N-content of the fungal "humic acids"when compared with the soil and peat humic acids. The Scontent of the fungal "humic acids" was slightly higherwhile the O content was somewhat lower than the corre-sponding ranges for the soil and peat humic acids. TheOCH3 content of the fungal "humic acids" fell within therange of that for soil and peat humic acids.

In order to lower the N-content and to remove carbo-hydrates, phenols, phenolic acids, metals, and other ad-sorbed materials, each humic preparation was hydrolyzedwith 6N HC1 solution. As has been shown previously byRiffaldi and Schnitzer (19), 6/V HC1 hydrolysis appearsto purify humic acids without causing significant changesin chemical structure and analytical characteristics. Thus,acid hydrolysis produces more homogenous starting mate-rials for subsequent analytical and structural investigations.

Following 6N HC1 hydrolysis (Table 1), the C contentof fungal "humic acids" increased significantly and was inall cases higher than the range for soil and peat humicacids. Except for the A. niger "humic acid," the H con-tent of the fungal "humic acids" after acid hydrolysis alsotended to be higher than that for soil and peat humic acidstreated similarly. Hydrolysis with 6/V HC1 solution reducedthe N content of the fungal "humic acids" by between 44%(for the 5. chartarum "humic acid") and 88% (for the A.niger "humic acid"). Losses of N on 6N HC1 hydrolysiswere approximately 70% for the soil and peat humic acids.Both the fungal "humic acids" and the soil and peat humicacids lost considerable proportions of their S content on acidhydrolysis. Methoxyl groups in fungal "humic acids" wereless resistant to acid hydrolysis than were those occurringin soil and peat humic acids. Of special interest was theunusually high weight loss on acid hydrolysis of the E.nigrum "humic acid."

Elementary analyses of methylated (with diazomethane)

232 SOIL SCI. SOC. AMER. PROC., VOL. 37, 1973

Table 3—Major oxygen-containing functional groups in untreated and 6N HCl-hydrolyzed"humic acids" and in soil and peat humic acids (meq/g, dry, ash-free)

Type of humicacid

A. nlger humic acidE. nigrum humic acidS. chartarum

Total tb*

4.65.5

icldlty

bt7.97.0

CO]Hb a

1.5 2.01.3 1.7

Total OH Phenolic OHb

9.76.8

a b9.6 3.17.5 4.2

a5.95.3

Alcoholic OH Total 0=0 Qulnoneb

6.62.6

a b3.7 2.52.2 1.6

a b5.23.8

a2.72.1

Ketonlc COb a

2.51.7

humic acid 3.5 4.5 1.8 2.1 7.5 5.9 1.6 2.5 5.9 3.5 2.8 1.3Range for soil and

peat humic acids 6.2-8.9 4.5-8.1 3.9-5.7 3.5-5.5 2.4-6.2 4.4-5.8 2.1-4.3 1.0-3.6 0-3.2 0.6-4.4 2.7-5.6 4.8-6.0 1.5-2.1

1.0 0.3

1.2-3.5' Before 6N HC1 hydrolysis. t After 6N HC1 hydrolysis.

untreated fungal "humic acids" are shown in Table 2. TheN content ranged from 7.9 to 10.4% and the OCH3-con-tent from 9.3 to 13.8%. Following 6N HC1 hydrolysis,separate samples of the fungal "humic acids" were methy-lated with diazomethane and dimethylsulfate. Methylationwith the dimethylsulfate was done to check whether diazo-methane (CH2N2) would add N across double bonds toform pyrazolines. As the data in Table 2 show, acid hy-drolyzed "humic acids" methylated with diazomethanecontained more N than did those methylated with dimethyl-sulfate but the latter added S to the 5. chartarum "humicacid." Diazomethane was more efficient in increasing theOCH3 content of acid-hydrolyzed "humic acids" than wasdimethylsulfate. Not sufficient E. nigrum acid-hydrolyzed"humic acid" methylated with dimethylsulfate was avail-able for elementary analysis so that data for this prepara-tion methylated with diazomethane only are shown inTable 2.

Functional Groups—The total acidity and CO2H groupsof untreated fungal "humic acids" (Table 3) were lowerbut total OH groups, especially alcoholic OH groups, werehigher than those in soil and peat humic acids. Phenolic

OH and total C =: O groups fell approximately within therange for naturally occurring humic acids. Following acidhydrolysis, total acidity and, to a lesser extent, CO2Hgroups increased (Table 3). Total acidities were wellwithin the range for soil and peat humic acids, whereasCO2H groups were still below the range. Acid hydrolysisappeared to have little effect on total OH groups, but in-creased phenolic OH groups while simultaneously loweringalcoholic OH groups. In case of two out of three fungal"humic acids" acid hydrolysis increased total C = O groups,which were made up of slightly more quinone than ketonicC = O groups. In addition, for two out of three fungal"humic acids" total C = O groups after hydrolysis werewell within the range for the same groups in soil humicand fulvic acids analyzed under the same conditions.

Distribution of Oxygen and E4/E6 ratios—The distri-bution of oxygen in CO2H groups tended in the case of theA. nlger and E. nigrum "humic acids" to be lower thanin natural humic acids (Table 4). The opposite was truefor proportions of total oxygen in phenolic and alcoholicOH groups, which appeared to be higher in fungal "humicacids" than in natural humic acids. The proportion of oxy-

2025

27

25

10 15 20 25 30

23

24 25

10 15 20 25 30 35

TIME

35 0 5

(MIN)

Fig. 1—Gas chromatographic separations of: S. chartarum "humic acid", methylated with diazomethane (upper curve on left),with dimethylsulfate (lower curve on left); E. nigrum "humic acid", methylated with diazomethane (upper curve on right), withdimethylsulfate (lower curve on right).

SCHNITZER ET AL.: CHEMISTRY OF ACID-LIKE POLYMERS AND SOIL HUMIC ACIDS 233

Table 4—Distribution of oxygen in functional groups andE4/Ee ratios of 6N HCl-hydrolyzed fungal "humic

acids" and soil and peat humic acids

Type of humicacid

A. nlgerHumic acid

E. nlgrumHumic acid

S. chartarumHumic acid

Range for soil andpeat humic acids

OasCO2H

Total O

0.21

0.19

0.31

0. 23-0. 53

O asphen. OH

Total O

0.31

0.29

0.17

0.09-0.25

O asalcoh. OH

Total O

0.19

0.12

0.26

0-0.16

OasCO

Total O

0.27

0.21

0.09

0. 04-0. 27

Oaccounted

forTotal O

0.98

0.81

0.83

0.61-0.94

VE«

3.12

3.14

3.61

3. 80-5. 12

gen in total C = O groups was within the range of that forsoil and peat humic acids. E4/E6 ratios of the fungal"humic acids" were of the same order of magnitude butwere somewhat lower than those for soil and peat humicacids, indicating a relatively high degree of condensationor molecular complexity.

Separation and Identification of Oxidation Products—Gas chromatographic separations of oxidation products of"humic acids" synthesized by E. nigrum and S. chartarumand methylated with diazomethane and with dimethylsul-fate are shown in Fig. 1. Chemical structures of the com-pounds unambiguously identified are listed in Fig. 2.

Identification of Compounds 13, 14, and 17—Sincecompounds 13, 14, and 17 had not been found in previousinvestigations (9, 10, 11, 19, 24), attempts were made toidentify them by additional mass spectrometric and IRmethods. Following analysis on a GEC model 21-490 massspectrometer, each of the five samples was reanalyzed inconsiderable detail on a Hitachi-Perkin-Elmer RMU-6Dmass spectrometer.

Compound 13 appeared to have a molecular weight ofeither 201 or 199. The odd mass implied that either anodd number of N atoms was present in the molecule orthat m/e = 199 and 201 were fragment ions from an un-seen parent ion. The base peak was at m/e, = 199. Majorfragment ions, in the order of relative intensities, appearedat m/e = 129, 69, 70, 59, 97, 141, 55, 130, 139, 83,and 53.

The IR spectrum showed the following bands: 2930(m)*,1785(m), 1740(str)**, 1430(m), 1375(w)***, 1255(m),1190(w), 1110(w), 1080(m), 1020(w), 860(w), 790(w)cm"1 (* medium; ** strong, *** weak).

There was no evidence for an aromatic character. Themolecule appeared to contain a CO2CH3 group and themajor fragmentation 199+ —————* 129+ involved mostlikely the loss of a C3H4NO group. The IR bands at 1785and 1740 cm"1 suggested the presence of a /3-lactam, thatis, a four-membered ring, containing a C-NH group, at-

Otached to a straight-chain acid ester. A possible structurefor compound no. 13, suggested by mass and IR spectra isthe following:

ONXC — NH

I ICH3-CH -CH -CH2-CH2—CO2CH,

1. CO2CH3-(CH2)3-CO2CH3

2. C02CH3-(CH2)4-CO2CH3

3. CH3-(CH2)I3-CH3

4. C02CH3-(CH2)5-CO2CH3

5. CH3-(CH2)14-CH3

6. CO2CH3-(CH2)6-CO2CH3

7. CH3-(CH2),0-C02CH3

8. R, = R2 = CO2CH3, R3 = R, = R5 = R,,

9. CH3-(CH2)-,2-C02CH3

10. R, = R3 = R, = R5 = R, = H, R2 = CH.-SOjOQH,,

11. R, = C02CH3, R3 = R, = OCH3, R2 = R, = R, = H

12. R, = R3 = CO2CH3, R2 = OCH3, R, = R5 = R, = H

15. R, = CO2Ch3, R3 = R, = R5 = OCH3, R2 = R, = H

16. CH3-(CH2)14-CO2CH3

17. R, = R3 = R, = R5 = R* = H, R2 = CH2SO2NHCH3

H

18. R, = R2 = R, = C02CH3, R3 = R5 = R, H

19. CH3-(CH2)I6-C02CH3

20. R, = R2 = R3 = R, = CO2CH3> R; = R<, = H

21. R, = R2 = R3 = Rs = C02CH3, R, ='R, = H

23. R, = R2 = R3 = R, = RS = C02CH3, R, = H

24. R, = R2 = R3 = R, = R5 = CO2CH3, R« = H

25. R, = R2 = R3 = R, = RS = R,, = CO2CH3

Fig. 2—Chemical structures of compounds isolated and identi-fied.

Compound 14 had the same mass spectrometric frag-mentation pattern and IR spectrum as compound 13. Smallpeaks at m/e 213 and 227 suggested that compound 14contained one or two more CH2-groups than 13.

Compound 17 had an intense ion at m/e = 185. Theodd mass suggested the presence of an odd number ofnitrogen atoms. The intense M + 2 peak indicated thepossible presence of sulfur.

185 +185 +

185H

155-1

-» 167+ +H2O-+ 155+ + 30 (NH-CH3 or CH2NH2)

O

-» 92+ + 93+ (S-NH-CH2, H transfer)IIO

^91+ +S02

The IR spectrum showed bands at 3250(str), 2950(m),2900(m), 1600(m), 1450(m), 1430(m), 1300(str), 1290(m),1190(w), 1160(str), 1130(w), 1100(str), 1070(m), 1025(w),840(w), 822(str), 710(w).

234 SOIL SCI. SOC. AMER. PROC., VOL. 37, 1973

Table 5—Compounds (mg); produced by the oxidation of 1 g of methylated fungal"humic acids" and range for soil and peat humic acids

A. nlger

a)(2)(3)(4)(5)(6)(7)(8)

(9)(10)(H)(12)(13)(14)(15)(16)(17)

(18)

(19)(20)(21)

(22)(23)(24)(25)(26)(27)

* 1 =t 11 =

ComponentDimethyl glutarateDimethyl adlpaten-C15 alkane •Dimethyl pimelaten-C16 alkaneDimethyl suberaten-C13 fatty acid methyl ester1, 2-benzenedIcarboxylic acid dimethyl ester1,3-benzenedlcarboxyllc acid dimethyl ester1,4-benzenedicarboxyllc acid dimethyl estern-CH fatty acid methyl esterbenzylethylsulfonate3,4-dlmethyoxybenzolc acid methyl ester2-methoxy-l,3-benzene-dlcarboxyIic acid dimethyl esteraliphatic methyl ester with p-lactam groupaliphatic methyl ester with p-lactam group3,4, 5-trlmethoxybenzolc acid methyl estern-C16 fatty acid methyl esterBenzylsulfonyl methylamlne1, 2, 3-benzenetrlcarboxyllc acid trimethyl ester1, 2,4-benzenetrlcarboxyllc acid trimethyl ester1,3,5-benzenetrlcarboxylic acid trimethyl estern-C18 fatty acid methyl ester1, 2, 3,4-benzenetetracarboxylic acid tetramethyl ester1, 2, 3, 5-benzenetetracarboxyllc acid tetramethyl ester1, 2,4, 5-benzenetetracarboxyllc acid tetramethyl ester5-methoxy-l, 2, 3,4-benzenetetracarboxyllc acid tetramethyl esterDtmethoxy-benzenetetracarboxyllc acid tetramethyl esterBenzenepentacarboxyllc acid pentamethyl esterMethoxy-benzenepentacarboxyllc acid pentamethyl esterBenzenehexacarboxylic acid hexamethyl esterComplex aromatic anhydrideComplex aromatic anhydrideDehydrodlveratrlc acid dimethyl esterIdentifiedTotal weight of oxidation products% of products Identified

Methylated with dlazomethane.= Methylated with dlmethylsulfate.

5

0

I«__

———.96—.93———

2.28

1

4

2

51

——.93—.14———.28——.17.63——

2

nt——————--.23———

E. nigrumI*

_--___-—-_7.75———

74.49

nt__——__—5.74————9.23

1. 17

3

21

2

672

—_ _——.50—.17—.82—.80.67.82——

—_———

178.40——_—7.04__-__-—

2.824.521.58

13.954.21

8262

.94

.91

.51

.91

3.13———

—__———

29.1745.64

—_—

40.4020.20

_ _-_—

10.9724.1913.726.73—

5.17

567476

—.57.17

—69.45

10267

.94

--270. 81316. 57

85

—205. 99343.64

59

S. chartarumI*_—

1.540.633.122.33

_ _2.37

———

8.05—„

9.826.232.21

—2.56--

3.51—

2.057.89

———

4.422.521.856.271.580.35--

69.3098.5170

nt1.151.652.141.813.951.32

—2.63

———

9.550.661.154.449.387.57

—12.67

--4.03

—6.905.76

———

3.793.130.991.65

—_—

86.41115.2175

in_--__—__

2. 89- 7.—

6.00- 21.1.07- 3.1.67- 8.

--__—_—--—__—

6.67- 55.3.22- 23.2.50- 10.

—

17

804000

671590

13.61- 75.604.57- 15.5.67- 35.4.28- 26.

—11.39- 68.13.12- 20.13.44- 70.7.15- 13.9.393.89- 26.

131.67-396.228. 00-588.58- 87

327537

45550045

651800

1 Range for soli and peat humic acids.

A possible structure for 17 is:

OII

-CH,— S — NH-CH,2 II 3

0

benzylsulfonyl methylamine.Compounds 26 and 27 were complex aromatic anhy-

drides with molecular weights of 452 and 466. The massspectrometric fragmentation patterns, IR spectra and pos-sible chemical structures of these compounds have beendiscussed in a previous paper (24).

Major Oxidation Products—The nature of the methylat-ing reagent appeared to affect the qualitative and quantita-tive distribution of the major products. This was especiallymarked in the case of the E. nigrum "humic acid" (Table5). The oxidation product from this "humic acid" initiallymethylated with diazomethane consisted almost entirely ofn-C12, n-C14, n-Cj6 and n-Ci8 fatty acid methyl esters.By contrast, the products from the same "humic acid"methylated with dimethylsulfate prior to oxidation con-

tained appreciable amounts of fully methylated phenolicand benzenecarboxylic acids in addition to fatty acidmethyl esters. A number of fully methylated benzenecar-boxylic and phenolic acids that were identified in oxida-tion mixtures resulting from soil and peat humic acids werenot found among the oxidation products of the fungal"humic acids."

Except for the E, niger "humic acids," the total weightsof oxidation products from the fungal "humic acids" wereconsiderably lower than those from natural humic acids.Between 59 and 85% of the products from the fungal"humic acids" were identified, which was within the rangefor proportions of products identified in oxidation mixturesfrom soil and peat humic acids. The experimental data aresummarized in Table 6.

DISCUSSION

Elementary and functional group analyses show thatunhydrolyzed fungal "humic acids" contained more H andN and, per unit weight, more alcoholic OH but fewerCO2H and phenolic OH groups than did unhydrolyzed

Table 6—Major oxidation products (mg) resulting from the oxidation of 1 g of methylated materialType of humic acid

A. nlgerType of product

Aliphatic acid methyl estersBenzenecarboxylic acid methyl estersPhenolic acid methyl esters and ethersOthersTotal IdentifiedBenzenecarboxylic: AliphaticPhenolic: AliphaticBenzenecarboxylic: Phenolic

I'7.89(13.9)1

28.48(50.3)17.92(31.7)2.28(4.0)

56.573.632.271.59

nt6.80( 9.8)

30.99(44.6)9.32(13.4)

22.34(32.2)69.454.561.373.33

267300

2700

E. nigrum

I". 68(98. 8). 13( 1.2)

.81

.01

nt84.54(41.0)51. 12(24. 8)24.69(12.0)45.64(22.2)

205. 990.600.292.07

S. chartarum

I*25.72(37.1)22.56(32.6)10.41(15.0)10.61(15.3)69.300.880.402.17

nt32.74(37.9)17.29(20.0)14.16(16.4)22.22(25.7)86.410.530.431.22

Volcanic ashnt

7.17( 2.4)225.51(75.3)66.74(22.3)

0299.4231.45

9.313.38

Chernozem

5.45( 1.349.71(89.33.87( 8.0

389.0364.176.21

10.32

4)9)7)

Peat

2.89( 2.6)77.67(71.1)28.72(26.3)0

109. 2826.879.942.70

• I =• Methylated with dlazomethane.t n = Methylated with dlmethylsulfate.t Numbers In parentheses refer to % of total Identified.

SCHNITZER ET AL.: CHEMISTRY OF ACID-LIKE POLYMERS AND SOIL HUMIC ACIDS 235

soil and peat humic acids. The C and N content of 6NHC1 hydrolyzed humic acids were higher but the O contentlower than those of corresponding soil and peat humicacids. Acid hydrolysis also increased the content of phe-nolic OH groups in fungal "humic acids"; this was accom-panied by a decrease in alcoholic OH groups. E4/E6 ratiosof unhydrolyzed fungal "humic acids" were lower thanthose for soil and peat humic acids, indicating a high degreeof molecular complexity for the fungal materials. Diazo-methane was more effective in increasing the OCH3 con-tent of the methylated fungal "humic acids" than was di-methylsulfate (see Table 2). The data in Tables 1 to 4show a number of similarities in analytical characteristics,especially surface functional groups, between the soil andfungal materials but provide little information on the"nuclei" or "inside" of these substances.

As shown in Table 5, the major oxidation productswere: (i) aliphatic mono- and dicarboxylic acid esters andalkanes; (ii) benzenecarboxylic acid methyl esters; (iii)fully methylated phenolic acids, and (iv) aromatic com-pounds containing N and S.

In view of the low methoxyl content of the unmethylated"humic acids," it is likely that most of the aliphatic, benzenecarboxylic, and phenolic acids were methylated in thelaboratory and that they occurred as acids and phenolsin the untreated "humic acids." It may therefore be morerealistic to refer to the compounds isolated as acids ratherthan as esters and ethers.

As can be seen from the data in Table 6, amounts ofbenzenecarboxylic and phenolic acids produced per gramof initial methylated material ranged from 3.13 to 75.81mg for the fungal "humic acids" as compared with 106.39,292.25, and 383.58 mg for the Peat, Volcanic Ash soil,and Chernozem humic acids, respectively. On the otherhand, the oxidation of methylated fungal "humic acids"tended to produce, per unit weight, more aliphatic com-pounds than did that of soil and peat humic acids. Thiswas especially true for the E. nigrum "humic acid" whichdiffered from the other fungal "humic acids" in that itsoxidation yielded substantial amounts of n-C14 to n-C18fatty acids, especially when methylated with diazomethaneprior to oxidation. The effect of the methylating reagent(diazomethane vs. dimethylsulfate) on the types of com-pounds produced on subsequent oxidation was quite strik-ing with this material and is difficult to explain at this time.It is likely that with a chemically complex material suchas this "humic acid" certain parts of the structure are morereadily methylated with one reagent than with another. Theoccurrence of substantial amounts of n-C14 to n-C18 fattyacids among the E. nigrum "humic acid" oxidation prod-ucts points to a possible fungal origin for the correspond-ing fatty acids isolated by Schnitzer and Ogner (22) andKhan and Schnitzer (12) from fulvic and humic acids. Thelow weight ratios of benzenecarboxylic to aliphatic andphenolic to aliphatic compounds (Table 6) for fungal"humic acids" as compared with natural humic acids fur-ther demonstrate the significantly greater aliphatic char-acter of the fungal materials. Of the relatively large num-ber of "primary phenolic polymer constituents" isolatedby Martin, Haider, and associates (1, 7, 8, 13, 14, 15,

16) only the following three were found in this investiga-tion, with yields shown in brackets: gallic acid, isolatedas 3,4,5-trimethoxybenzoic acid methyl ester, from A. niger"humic acid" (0.41 and 0.35%), from E. nigrum (0%),and from S. chartarum (0.22 and 0.76%). Another phe-nolic compound that could have originated from vanillicor protocatechuic acid was 3,4-dimethoxybenzoic acidmethyl ester, which was obtained from the S. chartarum"humic acid" only in a yield of 0.66%. In addition, a smallamount (0.12%) of 2-methoxy-l,3-benzenedicarboxylicacid dimethyl ester was isolated from the S. chartarum"humic acid." It may be appropriate to mention here thatOgner and Schnitzer (20) isolated small amounts of3,4-dimethoxybenzoic acid and 3,4,5-trimethoxybenzoicacid from a Podzol Bh fulvic acid but not from humicacids. More complex phenolic acids such as dihydroxy-benzenetetracarboxylic acid (isolated as dimethoxyben-zenetetracarboxylic acid tetramethyl ester) and hydroxy-benzenepentacarboxylic acid (obtained as methoxybenzene-pentacarboxylic acid pentamethyl ester) were isolatedin varying amounts from the different fungal preparations.It is not possible to assign simple precursors for these com-plex phenolic compounds. Considering the relatively highphenolic OH content of the hydrolyzed fungal "humicacids" (Table 3), the yields of phenolics isolated were low.This suggests that either most phenolic OH groups in thefungal preparations occur in complex structures that donot yield simple phenolics on permanganate oxidation orthat the phenols and phenolic acids formed are destroyedduring oxidation.

Thus, yields of relatively simple phenolic acids resultingfrom the permanganate oxidation of the methylated fungal"humic acids" were of the order of 1 % or less, and onewonders whether Martin and Haider et al. were justifiedto refer to these materials as phenolic polymers. Asidefrom aliphatic compounds, the major oxidation productsfrom fungal "humic acids" were benzenecarboxylic acids.The latter could have arisen from aromatic compounds,including polycyclics, not substituted by OH groups orfrom saturated or hydroaromatic structures that becamearomatic during oxidation (24). By comparison with soilhumic acids, the total amounts of aromatic compoundsproduced per gram of initial material were small. It is pos-sible that eventually aliphatic mono- and dicarboxylic acidsthat occur in these "humic" substances are transformedby microorganisms in the soil to aromatic compounds(14). Whether this does indeed occur requires further in-vestigation. One could argue with equal justification thatphenolic and other aromatic and nonaromatic compoundsreleased from lignin are as likely to contribute to humicacid synthesis as the fungal "humic acids" studied in thisinvestigation. Further work is needed to shed light on thesepoints.

The results of this investigation show that fungal "humicacids" are complex organic materials containing aliphaticand aromatic structures, only some of which are phenolic.Claims by Martin et al. (15), based on information obtainedwith the aid of Na-amalgam reduction, that simple phenolsand phenolic acids are significant constituents of the fungalpolymers were not confirmed.

236 SOIL SCI. SOC. AMER. PROC., VOL. 37, 1973