Short Communication Degradative Oxidation Products of the Melanin of Ulocladium Atrum

It is known that many soil fungí synthesize brown, large aromatic polymers (fungal melanins), chemically related to soil humic acids (Martin and Haider, 1971; Schnitzer and Neyroud, 1975). Since the chemical constituents (or at least, their degradation products) and the physicochemical properties of fungal melanins are comparable to those of humic acids, it is possible that fungal metabolism may represent an important source of constituents for humus formation. Production of humic acid-like compounds by Uloc/adium Preuss was reported by Almendros et al. (1985). The alkali-soluble melanin of Uloc/adium atrum Preuss was extracted together with a green pigment having similar properties to the Pg fraction found by Kumada and Sato (1962) in soil humic acids, and of a fungal origin confirmed by Kumada and Hurst (1967). This species also produced a significant proportion of a soluble polysaccharide (Martínez et al., 1984), and is an interesting organism for studies of synthesis and transformation of soil organic matter. The fungus was grown (30°C; 200 rey min -1) in a medium containing malt extract, 30 g; NaNO}. 2 g; K2HP04, 1 g; KCI, 0.5 g; MgS04 0.5 g; FeS04 10 mg; water, 11 (PH-5). After 2 weeks, the freeze-dried fungal biomass was washed with water and ether, the melanin extracted with 0.1 N NaOH under N2, precipitated with HCl and purified by centrifugation in alkaline pH (20,000 rey min -1), reprecipitation and electrodialysis. Melanin was degraded by two different methods: persulfate in acidic medium, and alkaline permanganate oxidations, the former reagent being milder than the latter. Persulfate oxidation was carried out according to Martín et al. (1981): 200 mg of freeze-dried melanin were oxidized with a saturated K2S20 g solution in a digestion bomb at 140°C. The same weight of melanin was diazomethanemethylated, in order to protect phenolic groups on aromatic rings (Matsuda and Schnitzer, 1972), and oxidized at 90°C with KMn04: 70 mI of 4% KMn04 solution were progressively added to the sample in 4-ml aliquots every 15 mino After cooling, the mixture was acidified and the excess of permanganate was destroyed with Na2S20S' The digests obtained by both oxidation procedures were extracted with ethyl acetate in a liquid-liquid extractor for 12 h. The organic solvent was dehydrated with anhydrous CaS04' evaporated at 40°C, the oxidation products were suspended in methanol and methylated three times with ethereal diazomethane (Schnitzer, 1974). The degradation compounds were separated and identified using a Hewlett-Packard 5992 B GC-MS computer system equipped with a 25-m cross-linked fused silica capillary column coated with OV-IO!. Oven temperature was programmed from 100 to 270°C at a heating rate of 6°C min -l. The flow rate of the carrier gas (He) was adjusted to 1 mI min 1, and the ionizing voltage was 70 e V. Chromatographic peaks were identified by searching computer Iibraries and bibliographic data, and when possible, by comparison with the standard compounds. Potassium persulfate degraded 40.5% ofthe initial weight, while no residue was obtained with KMn04' Table 1 shows the yields of the oxidation products (Fig. 2) as percentages of the total volatile methylated extract. Quantitative yields respective to the initial weight of the sample were not calculated, following the criteria of Maximov et al. (1977). Permanganate oxidation of U. atrum methylated melanin yielded similar compounds to those described for soil humic acids (Matsuda and Schnitzer, 1972), but their quantitative proportions differed greatly. Methyl esters of normal and branched fatty acids were the major peaks (Fig. 1). On the other hand, persulfate oxidation released the most interesting compounds, the major oxidation products being methyl palmitate, fluorene, nonacosane and biphenyl. It is noticeable that there were significant proportions of flourene (9), naphthalene (1) and a compound having similar mass spectra to a trimethylstyril naphthalene (31), because it seems that the production of such compounds would be more characteristic of a more drastic degradative method. Similar compounds, when obtained by Zn-dust distillation, are frequently considered as artifacts, but in the present case, the mild character of persulfate oxidation suggest that they were indeed present in the humic-like molecule, though probably loosely linked to the polymer. Biphenyl (6) is not a typical degradation product of soil humic acids, but the presence of dehydrodiveratric acid, a polysubstituted biphenyl, has been frequently reported in these soil components (Schnitzer and Khan, 1972). The occurrence of sorne of the above-mentioned compounds could be explained in relation to the biosynthetic pathways of compounds derived from binaphtyl or perylene, whose structure and reactions were studied in Daldinia concentrica (Bolt. ex Sr.) Ces. & de Not., a fungus producing pigments similar to the green fraction of Uloc/adium melanin (Allport and Bu'Lock, 1(60). A dihydroxyacetophenone comparable to compound 5 was described in the residue from steam-distillation of chromamone in D. concentrica, and has also been found in "fungal humic acids" (Schnitzer and Neyroud, 1975).