Further investigations on the chemistry of fungal “humic acids”

FURTHER

INVESTIGATIONS ON THE CHEMISTRY OF FUNGAL “HUMIC ACIDS’ M. SCHNIKZR and J. A. NEYROUD* Soil Research Institute, Agriculture Canada, Ottawa, Ontario KIA OC6. Canada

Summary--in view of the considerable interest in laboratory-prepared fungal “humic acids” as possible precursors or incorporated structural components of soil humic substances, we degraded four fungal “humic acids” by the relatively mild alkaline cupric oxide oxidation. The oxidation products were extracted into organic solvents, methylated, separated by thin-layer chromatography and identified on a gas chromatographic-mass spectrometric-computer system. Average yields of major degradation products were: (a) aliphatic compounds, 38 per cent; (b) benzenecarboxylic compounds, 25 per cent; and (c) phenolic compounds, 21 per cent. The remaining 16 per cent consisted of a number of dialkyl phthalates. Our data agree with those that we reported earlier when we degraded a number of fungal “humic acids” by the more drastic alkaline permanganate oxidation and show that fungal “humic acids” are enormously complex organic materials containing aliphatic and aromatic structures, (some of which contain N), but only a relatively small proportion of which is phenolic. Most of the aliphatics isolated consisted of alkanes and fatty acids, which are known to persist in soils over long periods of time and are frequently firmly retained by soil humic substances.

INTRODUCTION

There is currently considerable interest in laboratoryprepared fungal “humic acids” (“HA’s”) as possible precursors or incorporated structural components of soil humic substances. Based mainly on their work involving reductive degradation with Na-amalgam, Haider and Martin (1970), Martin and Haider (1969) and Martin, Haider and Wolf (1972) have concluded that fungal “HA’s” were phenolic polymers. The Naamalgam reduction method has been criticized by several workers (Mendez and Stevenson, 1966; Stevenson and Mendez, 1967; Schnitzer, 1974b). The method appears to exaggerate the relative importance of phenolic structures at the expense of aliphatic, benzenecarboxylic and complex N-containing chemical structures, all of which are sign~cant components of fungal “HA’s” (Schnitzer, 1974b). To obtain further information on the chemistry of fungal “HA’s”, Schnitzer et al. (1973) first methylated three fungal “HA’s” and then degraded the methylated preparations by alkaline permanganate oxidation. The oxidation products were identified by gas chromatography-mass spectrometry. The results showed that, compared with soil-humic and fulvic acids, fungal “HA’s” were chemically more complex, containing aliphatic and aromatic components, only a relatively small proportion of which was phenolic. Claims by Martin, Haider and Wolf (1972) that simple phenols and phenolic acids were the most significant structural components of these “HA’S’ were not confirmed. All fungal “HA’s” were found to contain considerably greater proportions of aliphatic than of phenolic structures.

* Visiting scientist from the Federal Research Station at Lausanne. Switzerland.

In view of the potential importance of fungal “HA’s” and the great interest that work along these lines has generated among soil biochemists, we decided to continue investigations on the chemistry of these materials. using a different approach. Since alkaline permanganate oxidation is a relatively harsh method, we set out to examine the possible use for this purpose of the relatively mild alkaline cupric oxide oxidation (CuO-NaOH), a degradative method that has been widely used for the oxidation of lignins and similar materials and which does not destroy aromatic nuclei (Chang and Allan, 1971). The method has also been employed on humic acids (HA’s) and fulvic acids (FA’s) by Greene and Steelink (1962), Schnitzer and Ortiz de Serra (1973) and Schnitzer (1974a). Since the latter found that the CuO-NaOH oxidation of humic substances was relatively selective for the isolation of phenolic constituents, we felt that the method would enable us to reassess the relative importance of phenolic structures in fungal “HA’s”. Following oxidation, the degradation products were extracted into ethyl acetate, methylated, separated by thin-layer chromatography (TLC) and identified on a gas chromatography-mass spectrometry-computer system. The results were then compared with those obtained by the use of the same methods on soilhumic substances.

Agricultural 365

MATERIALS

AND METHODS

The Stuchybotrys chartarum-“HA” was produced as described previously (Schnitzer et al., 1973). One portion was characterized as prepared, another one was hydrolyzed with 6 N I-ICI for 20 h (Schnitzer et ul., 1973) and the acid-insoluble residue used for further investigations. “HA’s” formed by S. atra and by Epicoccum pwpurescens were kindly provided by K.

hl. SCHNITZER

366 Table

c

and .I A.

NEYROUD

1. AnaIytical characteristics of fungal “HA’s” (dry. ash-free)

U.)

51.3

66.5

53.1

H U.1

7.0

7.2

5.7

7.1

N (2)

6.8

3.8

5.6

3.4

s (%I 0 U.) Ash*

(by :W

difference)

1.2

0.9

1.5

27.7

21.6

34.8

0.6

0

Haider. The former material was unhydrolyzed, the fatter was the residue remaining after 6 N HCI hydrolysis. A number of analytical characteristics of the four fungal “HA’s” are presented in Table 1. cupric oxide oxiduGbn The oxidation procedure was the same as that described earlier (Schnitzer, 1374a), except that the “‘HA’s” were oxidized in the unmethylated form. Following oxidation, suspended CuO was removed by centrifu~tion and washed with distilled H,O. The supernatant solution plus washings were acidified with 4 N HzS04 solution to pH 2. With the unhydrolyzed S. chartarum and S. atra ‘“HA’s”, portions of the H,O soluble oxidation products began to coagulate in the acidified solutions. Weights of the coagulates were, per l.Og of initial material, 47Y mg for the S. charturttm “HA+’and 62Omg for the E. purpuresccn~ “HA”. With the remaining two “WA’s”, which had beefi hydrolyzed before oxidation, no coagulation occurred when the aqueous oxidation products were acidified. The clear, acidified, aqueous solutions containing soluble oxidation products were transferred to a liquid-liquid extractor and extracted with ethyl acetate for 24h. The ethyt acetate soluble materials were dried first on a rotary evaporator and then in a vacuum desiccator over P,O, at room temperature. Weights of ethyl acetate-soluble materials resulting from the CuU -NaOH oxjdatjon of 1-Og fungal “HA” were 120, 105, 208 and 230mg for the unhydrolyzed S. chartarum “HA”, the hydrolysed S. churk~~~n “HA”, the unhydrolyzed S. arra “HA” and the hydrolyzed E. purpurescens “‘HA”, rcspcctively. Addine

Separation of oxidation products In order to make the extracts sufficiently volatile for gas chromatographic (GC) separations and mass

1.5

64.0

1.0 2lr. 5 I.7

spectrometric analyses, they were methylated with diazomethane generated from Diazald (Schnitzer, 1974a). Preliminary GC separations of the methylated extracts showed a profusion of peaks, so that further chromatographic separations were attempted. This was done by TLC on SKI, with toluene-ethyl acelate (1:1j as devebping reagent. Following inspection of each developed TLC plate under a U.V.lamp, five fractions were marked out, scraped off the plate and extracted with ethyl acetate. Fraction I. was near the solvent front, fraction 5 near the line of application. Weights of the different TLC fractions are shown in Table 2. Identijicatlon qf oxidation products Each TLC fraction was dissolved in benzene and examined as a 25 per cent (w/v) solution by analytical GC (Hewlett-Packard, mode1 5700 A, flame ionization detector, 1500 x 6mm coiled glass column packed with 3 per cent OV 17 on Chromosorb W HMDS, 80 to 100 mesh, programmed from 90 to 310°C at a rate of 8°C min-’ and a Nz Row rate of 30 ml min- ‘). Compounds represented by welldefined peaks were isolated by preparatke GC under the same conditions as those outlined above, except that aliquots from 10 per cent (w/v) solutions in benzene were injected into the gas chromatograph. Materials eluting from the gas chromatograph were collected in capillary tubes and analysed by mass spectrometry and micro-i.r. spectrophotometry (Ogner atid Schnitzer, 1971; Khan and Schnitzer, 1971). Each fraction was also analyzed on a gas chromatographic-mass spectrometric (GC-MSjcomputcr system. The instrument used was a Finigan 3100 D GC-MS, interfaced with a model 6ooO data system. The GC MS conditions were as follows: GC column: I SO0x 6 mm, 3 per cent OV 17 on Chromosorb W,

Table 2. Weights of TLC fractions

1

2

10.0

8.0

15.0

IS.0

3.3

8.0

42.0

67.0

3

26.0

22.0

42.0

39.0

4

7.0

2.0

75.0

61.5

5

56.0

54.0

47.0

40.0

107.0

94.0

221.0

221.0

148.0

126.0

250.0

272.0

Chemistry

of fungal

HMDS, glass column; injector temperature: 250°C; oven programmed from 90 to 270°C at 10°C min- 1 and a He-flow of 30 ml min- ‘. The separator temperature was 25O”C, MS pressure: 1 x 1O-5 mm Hg, MS resolution: 1:500, electron energy: 70 eV, and mass range 40 to 500. Preliminary identification of GC peaks was first made by recording “mass chromatograms” for fragments characteristic of specific compounds or groups of compounds expected to occur in the mixture by searching through the stored spectral data for specific m/e ratios. The identity of the compound in each peak was confirmed by: (1) running its mass spectrum; (2) eluting it from the GC and recording its micro-i.r. spectrum; (3) matching the mass and i.r. spectrum with the known compound to which it corresponded; and (4) co-chromatography (on the gas chromatograph) of known and unknown. Quantitative estimates of each compound were made by triangulation of peak areas on the gas chromatograms. All solvents were purified by distillation through high-efficiency columns.

“humic

acids”

-?

.-6 E

5 +0,

,\”

Analytical rnethods Total C and H were determined by dry-combustion, total N by the automated Dumas method, total S by oxygen-flask combustion and 0 was calculated by difference. Infra-red spectra were run as smears between NaCl plates or KBr pellets (0.5 mg/400 mg KBr) on a Beckman i.r.- 12 spectrophotometer.

4(200 34002800

I 2200 1800 1500 1200 900 700

Wavenumber,

RESULTS

Fig. 2. Infra-red spectra of: (a) unhydrolyzed S. chartarum “HA”; (b) acid precipitate of (a) after CuO-NaOH oxidation; (c)-(h) TLC fractions 1-4 and 5a and 5b.

AND DISCUSSION

Analytical characteristics

of fungal “HA’s”

Compared to unhydrolyzed fungal “HA’s”, residues resistant to 6 N HCl hydrolysis contained more C but less N and 0 (Table 1). This is due to removal by the strong acid of carbohydrates, low-molecular weight N compounds and other adsorbed materials that were low in C but high in N and 0. Considerable proportions of the total N appeared to resist hydrolysis by 6 N HCl. The ash content of all preparations was relatively low.

k

0

4

8

I2

Time,

b

I6

20 24

28

32

cm“

36

min

Fig. I. Gas chromatograms of: (a) E. purpurescens, TLC fraction I (near solvent front), and (b) S. atra. TLC fraction 2 (in the centre of plate).

Table 2 shows that between 72 and 88 per cent of methylated, ethyl-acetate soluble oxidation products were recovered in the five TLC fractions, so that irreversible adsorption on SiO, (on the plates) was not a serious problem. Figure la is the gas chromatogram of TLC fraction 1, separated from methylated, ethyl acetate-soluble products resulting from the oxidation of the E. purpurescens “HA”, while Fig. lb is the gas chromatogram of TLC fraction 2 separated in the same way from the S. atra “HA”. The two gas chromatograms show many overlapping peaks and high base lines which indicate that the two fractions are very complex even after repeated fractionations. Especially noteworthy are the well-d&led, high-temperature (>3WC) peaks in Fig. lb, with retention times of 30min and longer, which we have so far never observed among soil-HA and soil-FA oxidation products degraded by the same procedure as the fungal “HA’s”. These relatively high-molecular weight compounds with low volatilities were present in substantial concentrations in all TLC fractions higher than 1. It is in this respect that the fungal “HA’s” differ significantly from soil-HA’s and soilFA’s. Infia-red

spectra

To obtain information on the general chemical characteristics of the fungal “HA’s” and fractionated oxidation products derived from them, we ran i.r. spectra of the different materials. Since i.r. spectra of

36X

M.

SCHNITZERand

the initial “HA’s” and of the corresponding fractions were quite similar to each other, we have selected those produced by S. ckartarurn for illustration purposes. Infra-red spectra of the unhydrolyzed S. chartarum “HA”, of acid-insoluble oxidation products and of TLC fractions of ethyl acetate-soluble oxidation products are shown in Fig. 2. Curves g and h correspond to TLC fraction 5, which was split into two fractions, 5a and 5b. In curve a (the initial “HA”), main absorbances are near 34OOcm- ’ (hydrogenbonded, OH, NH,), between 2960 and 2930cm-’ (C-H stretch of aliphatic CH, and CH, groups), near 1660cm- ’ (amide) and 1470cm- ’ (aliphatic CH2). Smaller bands occur near 1630cm-’ (COO-. aromatic C==C, hydrogen-bonded c--O, C===C conjugated with C==O), at 1390cm-’ (C-CH3), 1350 and 1330cm- ‘. 1260cm-’ (C-&C stretch), 1090 and 1040cm- ’ (possibly primary and secondary C-N stretch), 990,940.900 and 840 and 770 cm- I (primary N-H deformation). The i.r. spectrum of the CuG NaOH oxidation products precipitating from acidified aqueous solution (curve b) generally resembles curve (a) with the following exceptions: (a) bands near 1660 cm- ’ in curve (a) are resolved into two sharp bands at 1700cm-’ (likely C==O of CO,H) and 1610cm-’ (COO-); (b) most of the smaller bands in the 150~12OOcm-’ region are also better defined; (c) a series of sharp bands appear at 1090, 1070, 1055 and 1015cm-’ (probably due to alcoholic OH groups) and between 1000 and 900cm-’ ; and (d) a small well-defined band can be observed at 7 15 cm- ’ [(CH&]. The i.r. spectrum for TLC fraction 1 (near the solvent front) (curve c) is relatively simple and consists essentially of four major bands at 2930 and 2860cm-r (aliphatic C-H), 1725 (CO,CH,) and 1465 cm- ’ (aliphatic CH2). The spectrum suggests that the main component of TLC fraction 1 is an aliphatic ester. The i.r. spectrum for TLC fraction 2 (curve d) is more complex than the preceding curve,

J. A.

NEYROLD

but also conveys the impression that this fraction is highly aliphatic. This is indicated by strong bands at 2930, 2860 and 1470 to 1440cm-‘. The presence of CO,CH, groups is shown by strong absorbance at 1725 cm-‘. Other well-defined bands occur at 1610cm~‘. 1335cm-‘, 1250cm-’ (C-&C of aromatic ether). near I1 IO and 1lOOcm~ ’ (aliphatic ether),1010cm~‘(tertiaryC-N).940cm~’,915cm~’, 865cm-’ (tertiary N-H), 815 cm-’ and 775 cm-* (tertiary N-H). The spectra for TLC fractions 3 and 4 (curves e and f) are very similar to curve d. All three curves show the presence of predominantly aliphatic esters and ethers, some of which could contain C-N and N-H bonds. The spectrum of TLC fraction Sa (curve g) exhibits broad, overlapping bands which suggest the presence of a complex mixture. The relatively strong bands near 2930 and 1460 cm- ’ indicate considerable aliphaticity, the strong band near 1740cm- ’ shows the presence of C0,CH3 groups. The spectrum of TLC fraction 5b (curve h) shows better definition than that of TLC fraction 5a (curve g), but the main absorbances are at the same wave numbers except for the strong band (at 1115 cm- r) due to Si-(X-C, which most probably originated from organic matter than had combined with SiO, (used for TLC) and which had become soluble in the polar methanol used as eluant.

Nature

of o.xid.atiorz products

In view of the low OCH,-content of the initial “HA’s” (< 1%) and also because the oxidation products were methylated before the GC separations. it is likely that in the original “HA’s” most of the C02H and OH groups occurred as carboxyls and phenolic hydroxyls rather than as esters and ethers. It may, therefore. be appropriate to refer here to the compound isolated and identified as phenolic and benzenecarboxylic acids rather than as esters and ethers.

Table 3. Compounds (mg) produced by the CuOPNaOH oxidation of I,Og of fungal “HA”

Total ldenrified Weight Of methylated, % identified

ethyl

acetate-soluble

OxIdario”

products

20.8 148.0 14

14.6 126.0 12

87. 7 250.0 35

76.1 272.0 78

Chemistry

of fungal

The most abundant oxidation product formed by three of the four “HA’s” was dioctyl adipate plus small amounts of other dialkyl adipates (Table 3 Fig. 3). The unhydrolyzed S. charturum “HA” also afforded an appreciable amount of dialkyl phthalates, the most conspicuous representative of which was dioctyl phthalate. The two S. chartarum “HA’s” yielded small amounts of a large variety of compounds. Of these 35.6 and 38.0 per cent were aliphatic (in case of the unhydrolyzed and hydrolyzed S. chartarum HA’s, respectively), 15.9 and 29.5 per cent phenolic and 11.5 and 21.2 per cent benzenecarboxylit. Dialkyl phthalates constituted 31.3 per cent of the unhydrolyzed S. chartarum “HA” oxidation products. Following hydrolysis, this group of compounds accounted for only 0.1 per cent of the oxidation mixture, while dialkyl adipates were reduced to only 0.2 per cent of the oxidation products under similar conditions. In agreement with previous degradation studies on the same “HA” in which alkaline per1.

CH3(CH2)“CH3

~P3 (CH ) I 2n C02CH3

“humic

369

acids”

manganate oxrdatron was employed (Schnitzer et al., 1973), only small amounts of fatty acids were found among the products resulting from the CuO-NaOH oxidation of the two S. charturum “HA’s”. Of special interest was the isolation of small amounts of n-C, to n-c,, alkanes from the hydrolyzed S. chartarum “HA” (Table 3). Apparently, acid hydrolysis was required to liberate these compounds as we did not detect them among oxidation products from the unhydrolyzed S. chartarum “HA”. The S. atra and E. purpurescens “HA’s” yielded considerably larger amounts of oxidation products per unit weight of initial material than did the two S. charturum preparations. Especially prominent among aromatic degradation products in the case of S. atra were phenolic acid 22 and benzenecarboxylic acids 5 and 20. The n-C,, fatty acid (13) was the most abundant aliphatic compound that we isolated. Aliphatics constituted 44.5 per cent of the oxidation mixture, with n-fatty acids accounting for almost one half *=5toIl=9

3.

n=*

4.

n=4

10.

9. 23.

Fig. 3. Chemical

”

= 8

”

= 12;

12.

D = 13;

13.

”

= 14;

17.

”

= 15;

n = 16

structures

of compounds

identified.

370

M. SCHNITZERand J. A. NEYROUD

Table 4. Major types of products (mg) resulting from the degradation of l,Og of fungal “HA” and of l.Og of a soil HA and soil FA

*

““hydrolyzed

xx hydrolyzed

of the aliphatics, phenolics were 24.2 per cent and benzenecarboxylic acids 28.6 per cent. Major compounds produced by the CuO-NaOH oxidation of the E. purpurescens “HA” were benzenecarboxylic acids 20, 5, 26 and 27, phenolic acids 25 and 22 and n-fatty acids 23 and 13. Aliphatics made up 41.1 per cent of the total oxidation products, with n-fatty acids accounting for two-thirds of this percentage, phenolic acids constituted 16.3 per cent and benzenecarboxylic acids 41.9 per cent. As shown in Table 3. only between 12 and 35 per cent of the methylated, ethyl acetate-soluble oxidation products were identified. By contrast we were able to identify between 81 and 92 per cent of the oxidation products when we degraded a soil-HA and a soil-FA by the same procedure (Neyroud and Schnitzner, 1974a). Our lack of success in identifying greater proportions of the oxidation products is most likely due to the presence among the fungal “HA” degradation products of considerable amounts of high-molecular weight, non-volatile organic materials that were resistant to degradation by the relatively mild CuO-NaOH. Although i.r. spectra (see Fig. 1) indicate the presence of N in several TLC fractions, we did not isolate any N compounds, which suggests that the N is associated with the high-molecular weight, resistant materials. The Cu&NaOH substances

oxidation

of fungal and soil humic

Table 4 compares major types of products formed by the CuO-NaOH oxidation of the four fungal “HA’s” with those obtained under the same experimental conditions from a soil-HA and a soil-FA. Probably the most interesting data in this Table are the different weight ratios. For example weight ratios of aliphatic/phenolic compounds for the fungal “HA’s” were significantly higher than those for the soil humic substances. Compared to one average unit weight of soil&HA or soil-FA, one average unit weight of fungal “HA” contains about three times as many aliphatic as phenolic compounds. Also, weight ratios of benzenecarboxylic/phenolic compounds for the fungal “HA’s” oxidation products were considerably higher than those for soil humic substances. This again points to the presence of only relatively small amounts of phenolics in the fungal “HA’s”. It may be relevant to refer here to recent

work by Schnitzer (1974a) who found that the alkaline CuO-NaOH oxidation of humic substances was especially effective in splitting C-O bonds and in so producing relatively large amounts of phenolic compounds but that the method was relatively inefficient for cleaving C-C bonds, Thus, the low yields of phenolics resulting from the CuGNaOH oxidation of the fungal “HA’s” lend additional support to our findings that phenolic compounds constitute on the average not more than between 20 and 25 per cent of the total weights of the degradation products. This is in contrast to soil humic substances (see Table 4) where phenolic compounds may account for up to 5.5 per cent of the Cue-NaOH oxidation products. The chemistry of jiqal

“HA’s”

The i.r. data and the qualitative and quantitative analyses of the Cu&NaOH oxidation products show that aliphatic structures constitute (on the average) 38 per cent of the fungal “HA’s”, phenolics account for 21 per cent and benzenecarboxylic acids for 25 per cent. The remaining 16 per cent is made up mainly of dialkyl phthalates. The repeated isolation of the latter and also of appreciable amounts of dialkyl adipates suggests that the fungi are able to bring about the esterification of phthalic and adipic acids with long-chain alcohols. We have during the past several years isolated on several occasions (Khan and Schnitzer, 1971; 1972; Schnitzer and dc Serra, 1973; Schnitzer, 1974a; Schnitzer and Skinner, 1974; Neyroud and Schnitzer. 1974b) dialkyl adipates and dialkyl phthalates from HA and FA degradation products, so that the isolation of these compounds in this investigation can no longer be ascribed to contamination from organic solvents, which we have always very carefully purified. In case of the S. atru and E. purpurescens “HA’s” about 50 per cent of the aliphatic oxidation products consisted of n-fatty acids. Close to 90 per cent of the aliphatic oxidation products from the hydrolyzed S. chartarum “HA” was made up of n-C, to /z-C,, alkanes. The results of this investigation are in agreement with those reported previously (Schnitzer rt al.. 1973) and show that fungal “HA’s” are complex organic materials containing aliphatic and aromatic structures, only some of which are phenolic. We found fungal “HA’S to be considerably more complex than any naturally-

M. SCHNITZERand

occurring HA and FA that we have so far investigated. The data presented in this and in our earlier paper (Schnitzer et ai., 1973) clearly show that fungal “HA’s” are extremely complex mixtures of a wide variety of organic materials and that there is little justiflcation in referring to them as ‘phenolic polymers”. Our data indicate that fungi make important contributions to soil humic substances by synthesizing alkanes and fatty acids (Schnitzer et al., 1973 and this paper). Recent work in our laboratory (Schnitzer and Neyroud, 1975) indicates that alkanes may constitute up to 2 per cent of the dry, ash-free weights of humic substances and that fatty acids may account for up to 10 per cent. Most of the alkanes were in the n-C,s to n-C,, range and had an odd to even C ratio of 1.0. The fatty acids ranged from n-C,, to r&as, with the majority being in the n-Cl4 to n-CJz range and with the n-Cl6 and n-C,, fatty acids constituting between 60 and 80 per cent of fatty acids extracted. The C-even to C-odd ratios of the fatty acids extracted from the humic materials ranged from 2.6 to 9.6 (Schnitzer and Neyroud, 1975). The C-odd to C-even ratios of the alkanes and fatty acids indicate a microbiological origin and the data presented herein and previously (Schnitzer et al., 1973) show that soil fungi can synthesize alkanes and fatty acids which are capable of persisting in soils over long periods of time. While most of the alkanes seem to be physically adsorbed on HA and FA structural networks, most of the fatty acids appear to form esters with phenolic humic “building blocks”. By this mechanism fatty acids are stabilized and preserved (Schnitzer and Neyroud, 1975). The role of the other major components of fungal “HA’s” in the synthesis and general chemistry of soilHA’s is much more difficult to assess than that of alkanes and fatty acids. It is likely that the phenolic, benzenecarboxylic and complex N-containing compounds would have to undergo many chemical transformations before becoming integral components of soil-HA and soil-FA structures. It will require much ingenuity and hard work to unravel the chemical reactions involved in these transformations.

J. A.

NEYROUD

371 REFERENCES

CHANG H.

M. and ALLANCi. G. (1971) Oxidation. In Lignins, Occurrence, For~tio~, Structure und Reactions (K. V. Sarkanen and C. H. Ludwig, Eds.) p. 448. Wiley-Interscience, New York. GREENEG. and STEELINKC. (1962) Structure of soil humic acid-II: Some copper oxidation products. J. org. Chem. 27. 17&174. HAIDER K. and MARTIN J. P. (1970) Humic acid-type phenolic polymers from Asperyil/us sydowi culture medium. Soil Bid. Biochem. 2, 145-156. KHAN S. U. and SCHNIXZERM. (1971) Further investigations on the chemistry of fulvic acid, a soil humic fraction Can. .!. Gem. 49, 2302-2309. KHAN S. U. and SCHNI~ZERM. (1972) The retention of hvdrouhobic organic compounds by humic acid. Geockm. Losmochimy Acta 36, =/45-754.

.

MARTIN J. P. and HAIDER K. (1969) Phenolic nolvmers of Stachy~o~ys atra, Stach~bo~_ys chartarum and E&coccum ~~r~f~ in relation to humic acid formation. Soil Sci. 107, 260-270. MARTINJ. P., HAIDERK. and WOLF D. (1972) Synthesis of phenols and phenolic polymers by Hendersonula in relation to humic acid formation. hoc. Soil Sci. Sot. Am. 36, 311-315. MENDEZ J. and STEVENWNF. J. (1966) Reductive cleavage of humic acid with sodium amalgam. Soil Sci. 102, 8593. NEYROUDJ. A. and SCHNITZER, M. (1974a) The exhaustive alkaline cupric oxide oxidation of humic acid and fulvic acid. Proc. Soil Sci. Sac. Am. 38. 907-913. NEVROUDJ. A. and SCHNITZERM. (1974b) The chemistry of high-molecular weight fulvic acid fractions. Gun. J. Chem. 52, 4123-4132. OGNER G. and SCHNITZERM. (1971) Chemistry of futvic acid, a soil humic fraction, and its relation to fignin. Can. J. Chem. 49, 1053-1063. SCHNITZERM. (1974a) Alkaline cupric oxide oxidation of a methylated fulvic acid. Soil Biol. Biochem. 6, 1-6. SCHNITZERM. (1974b) Letter to the Editor. Geoderma 11, 323-326. SGINITZERM.

and NEYROL’DJ. A. (1975) Alkanes and fatty acids in humic substances. Fuel 54, 17-19. S~HI-IIIZER M. and ORTIZ DE SERRA M. I. (1973) The chemical degradation of humic acid, Can. d. Chem. 51, 15541566. SCHNITZERM., ORTIZ DE SERRAM. I. and IVAR~~NK. (1973) The chemistry of fungal humic acid-like polymers and of soil humic acids. Proc. Soil Sci. Sot. Am. 37, 229-236.

Acknowledgei~nrs-We thank K. Haider of the Institut fur Biochemie des Bodens for sending us two funnal “HA” preparations and S. I. M. Skinner for CC-MS-computer analyses. This is Contribution No. 529. from the Soil Research Institute.

SCHN~T~ER M. and SKINNERS. I. M. (1974) The peracetic acid oxidation of humic substances. Soil Sci. 118. 322331. SEVENYJN F. J. and MENDEZJ. (1967) Reductive cleavage products of soil humic acids. Soil Sci. 103, 383-388.