Mineralization of synthetic humic substances by manganese peroxidase from the white-rot fungus Nematoloma frowardii

A manganese peroxidase preparation from the white-rot fungus Nematoloma frowardii was found to be capable of releasing up to 17% ¹⁴CO₂ from ¹⁴C-labelled synthetic humic substances. The latter were prepared from [U-¹⁴C]catechol by spontaneous oxidative polymerization or laccase-catalysed polymerization. The ex-tent of humic substance mineralization was considerably enhanced in the presence of the thiol mediator glutathione (up to 50%). Besides the evolution of ¹⁴CO₂, the treatment of humic substances with Mn peroxidase resulted in the formation of lower-molecular-mass products. Analysis of residual radioactivity by gel-permeation chromatography demonstrated that the predominant molecular masses of the initial humic substances ranged between 2 kDa and 6 kDa; after treatment with Mn peroxidase, they were reduced to 0.5–2 kDa. The extracellular depolymerization and mineralization of humic substances by the Mn peroxidase system may play an important role in humus turnover of habitats that are rich in basidiomycetous fungi. Received: 25 September 1997 / Received revision: 12 January 1998 / Accepted: 13 January 1998     

Degradation of lignite (low-rank coal) by ligninolytic basidiomycetes and their manganese peroxidase system

Ligninolytic basidiomycetes (wood and leaf-litter-decaying fungi) have the ability to degrade low-rank coal (lignite). Extracellular manganese peroxidase is the crucial enzyme in the depolymerization process of both coal-derived humic substances and native coal. The depolymerization of coal by Mn peroxidase is catalysed via chelated Mn(III) acting as a diffusible mediator with a high redox potential and can be enhanced in the presence of additional mediating agents (e.g. glutathione). The depolymerization process results in the formation of a complex mixture of lower-molecular-mass fulvic-acid-like compounds. Experiments using a synthetic ¹⁴C-labeled humic acid demonstrated that the Mn peroxidase-catalyzed depolymerization of humic substances was accompanied by a substantial release of carbon dioxide (17%–50% of the initially added radioactivity was released as ¹⁴CO₂). Mn peroxidase was found to be a highly stable enzyme that remained active for several weeks under reaction conditions in a liquid reaction mixture and even persisted in sterile and native soil from an opencast mining area for some days. Received: 31 July 1998 / Received revision: 29 September 1998 / Accepted: 2 October 1998     

Depolymerization of low-rank coal by extracellular fungal enzyme systems. II. The ligninolytic enzymes of the coal-humic-acid-depolymerizing fungus Nematoloma frowardii b19

The production of ligninolytic enzymes was studied in surface cultures of the South American white-rot fungus Nematoloma frowardii b19 and four other strains of this ecophysiological group (Clitocybula dusenii b11, Auricularia sp. m37a, wood isolates u39 and u45), which are able to depolymerize low-rank-coal-derived humic acids with the formation of fulvic-acid-like compounds. The fungi produced the three crucial enzymes of lignin degradation – lignin peroxidase, manganese peroxidase and laccase. In the case of N. frowardii b19, laccase and the two peroxidases could be stimulated by veratryl alcohol. Manganese (II) ions (Mn²⁺) caused a rapid increase of Mn peroxidase activity accompanied by the complete repression of lignin peroxidase. Under nitrogen-limited conditions the growth as well as the production of ligninolytic enzymes was partly repressed. During the depolymerization process of coal humic acids using solid agar media, gradients of ligninolytic enzyme activities toward 2,2′-azinobis(3-ethylbenzthiazoline-6-sulphonate) and syringaldazine were detectable inside the agar medium. Received: 5 August 1996 / Received revision: 13 November 1996 / Accepted: 15 November 1996     

A simple and rapid method to gain high amounts of manganese peroxidase with immobilized mycelium of the agaric white-rot fungus Clitocybula dusenii

The agaric basidiomycete Clitocybula dusenii was used for the production of the extracellular ligninolytic enzyme, manganese (Mn) peroxidase. An immobilization technique is described using cellulose and polypropylene as carrier for the fungal mycelium. High amounts of Mn peroxidase were obtained with agitated cultures of immobilized fungus (up to 3,000 U l⁻¹) while the biomass was recovered and used for further production cycles. Purification of Mn peroxidase revealed the existence of two forms: MnP1 (molecular mass 43 kDa, pI 4.5) and MnP2 (42 kDa, pI 3.8). Received: 30 July 1999 / Received revision: 1 December 1999 / Accepted: 3 December 1999     

Depolymerization of low-rank coal by extracellular fungal enzyme systems. III.In vitro depolymerization of coal humic acids by a crude preparation of manganese peroxidase from the white-rot fungus Nematoloma frowardii b19

The in vitro depolymerization of humic acids derived from German lignite (low-rank coal, brown coal) was studied using a manganese peroxidase preparation from the white-rot fungus Nematoloma frowardii b19. The H₂O₂ required was continuously generated by glucose oxidase. Mn peroxidase depolymerized high-molecular-mass humic acids by forming fulvic-acid-like compounds. The depolymerization process was accompanied by the decolorization of the dark-brown humic acid fraction soluble in alkaline solutions (decrease in absorbance at 450 nm) and by the yellowish coloring of the fraction of acid-soluble fulvic-acid-like compounds (increase in absorbance at 360 nm). The Mn peroxidase of N. frowardii b19 has been proved to be highly stable; even after an in vitro reaction time of 7 days in the presence of humic acids, less than 10% loss in total oxidizing activity was detectable. Received: 16 September 1996 / Received revision: 16 December 1996 / Accepted: 20 December 1996     

Production of ligninolytic enzymes and synthetic lignin mineralization by the bird's nest fungus Cyathus stercoreus

Production of ligninolytic enzymes and degradation of ¹⁴C-ring labeled synthetic lignin by the white-rot fungus Cyathus stercoreus ATCC 36910 were determined under a variety of conditions. The highest mineralization rate for ¹⁴C dehydrogenative polymerizates (DHP; 38% ¹⁴CO₂ after 30 days) occurred with 1 mM ammonium tartrate as nitrogen source and 1% glucose as additional carbon source, but levels of extracellular laccase and manganese peroxidase (MnP) were low. In contrast, 10 mM ammonium tartrate with 1% glucose gave low mineralization rates (10% ¹⁴CO₂ after 30 days) but higher levels of laccase and manganese peroxidase. Lignin peroxidase was not produced by C. stercoreus under any of the studied conditions. Mn(II) at 11 ppm gave a higher rate of ¹⁴C DHP mineralization than 0.3 or 40 ppm, but the highest manganese peroxidase level was obtained with Mn(II) at 40 ppm. Cultivation in aerated static flasks gave rise to higher levels of both laccase and manganese peroxidase compared to the levels in shake cultures. 3,4-Dimethoxycinnamic acid at 500 μM concentration was the most effective inducer of laccase of those tested. The purified laccase was a monomeric glycoprotein having an apparent molecular mass of 70 kDa, as determined by calibrated gel filtration chromatography. The pH optimum and isoelectric point of the purified laccase were 4.8 and 3.5, respectively. The N-terminal amino acid sequence of C. stercoreus laccase showed close homology to the N-terminal sequences determined from other basidiomycete laccases. Information on C. stercoreus, whose habitat and physiological requirements for lignin degradation differ from many other white-rot fungi, expands the possibilities for industrial application of biological systems for lignin degradation and removal in biopulping and biobleaching processes. Received: 29 January 1999 / Received revision: 5 July 1999 / Accepted: 9 July 1999     

Polymerization of guaiacol by lignin-degrading manganese peroxidase from Bjerkandera adusta in aqueous organic solvents

 Lignin-degrading manganese (II) peroxidase (MnP) purified from the culture of a wood-rotting basidiomycete, Bjerkandera adusta, was used in the polymerization of guaiacol. MnP was found to catalyze polymerization of guaiacol in 50% aqueous acetone, dimethyl formamide, methanol, ethanol, dioxane, acetonitrile, ethylene glycol and methylcellosolve. Maximum yield of polyguaiacol was achieved in 50% aqueous acetone. The weight average molecular weight (M _w) of the polymer was estimated to be 30 300 by gel permeation chromatography. However, matrix-assisted laser desorption ionization time of flight mass spectroscopy (MALDI-TOF-MS) analysis gave a more reliable M _w of 1690. IR, ¹³C-NMR, MALDI-TOF-MS and pyrolysis GC-MS analyses showed the presence of C–C and C–O linkages and quinone structure in polyguaiacol. It was also indicated that polyguaiacol has a methoxy-phenyl group as the terminal moiety. This suggests that polyguaiacol is a branched polymer in which guaiacol units are cross-linked at the phenolic group. Thermal gravimetric and differential scanning calorimetric analyses were also carried out. MnP also catalyzed the polymerization of o-cresol, 2,6-dimethoxyphenol and other phenolic compounds and aromatic amines. M _w of these polymers ranged from around 1000 to 1500. Received: 2 August 1999 / Received revision: 10 December 1999 / Accepted: 4 January 2000     

Biological bleaching of water-soluble coal macromolecules by a basidiomycete strain

There is need for new effective technologies to convert coal into environmentally acceptable liquid fuels. Thermochemical coal-conversion processes occur under extreme conditions. Thus there is a potential to use the biotransformation of coal as a cheap alternative method. A basidiomycete strain, which decomposes coal macromolecules, was isolated from humic-acid-rich soil of a lignite surface-mining region. The isolate showed the ability to decolorize liquid dark-brown media containing water-soluble coal-derived substances (humic acids). The presence of an easily available substrate is necessary for the biodegradation. The influence of different culture conditions on the bleaching effect was studied. Evidence for decomposition of water-soluble coal substances was provided by measuring the decrease of absorbance and the modification in the distribution of molecular masses. The degradation process resulted in a complete decolorization of the coal-derived humic acids and was also combined with massive alterations in their molecular structure. Solid-state #13C-NMR spectroscopy showed an increase of carboxylic groups as well as hydroxylated and methoxylated aliphatic groups, which indicates an oxidative attack. Enzymatic analysis showed the presence of a Mn peroxidase in the culture supernatant. Extracellular lignin peroxidase and laccase activities were not detectable. The production of the peroxidase was induced by addition of humic acids. But, in vitro, this enzyme did not cause a decolorization or reduction in molecular mass of the coal-derived humic acids. Received: 30 May 1996 / Received revision: 11 September 1996 / Accepted: 13 September 1996     

Combined effects of an oxidative enzyme and dissolved humic substances on 13C-labelled 2,4-D herbicide as revealed by high-resolution 13C NMR spectroscopy

Phenoxyalkanoic acids are a widely used class of herbicides. This work employed high-resolution ¹³C NMR to study the structural changes induced by humic substances and horseradish perodixase on 2,4-dichorophenoxyacetic acid (2,4-D) ¹³C-labelled in the side chain. NMR spectra showed that humic substances chemically catalyze abiotic splitting of [¹³C]2,4-D into 2,4-dichlorophenol and [¹³C]acetic acid at pH 7 but not at pH 4.7. Peroxidase did not catalyze the oxidative degradation of [¹³C]2,4-D at any pH tested and inhibited the effect of humic substances. Catalytic degradation by humic substances was attributed to free-radical reactions enhanced by the stereochemical contribution of large conformational structures formed by heterogeneous humic molecules at neutral pHs. Inhibition of 2,4-D degradation when humic substances were combined with peroxidase was explained by modification of both chemical and conformational humic structure due to peroxidase-promoted oxidative cross-coupling among humic molecules. Our findings show for the first time that the abiotic degradation of 2,4-D is catalyzed by dissolved humic substances at neutral pH. Journal of Industrial Microbiology & Biotechnology (2001) 26, 70–76. Received 09 February 2000/ Accepted in revised form 22 May 2000     

Fungal biosolubilization of Rhenish brown coal monitored by Curie-point pyrolysis/gas chromatography/mass spectrometry using tetraethylammonium hydroxide

Residues and coal fractions that remained after the biosolubilization of Rhenish brown coal by strains of Lentinula edodes and Trametes versicolor have been studied by Curie-point pyrolysis/gas chromatography/mass spectrometry using tetraethylammonium hydroxide (NEt₄OH) at 610 °C. To differentiate methyl derivatives of esters and ethers from free or bound hydroxyl and carboxyl groups NEt₄OH was used in the thermochemolysis experiments instead the commonly used tetramethylammonium hydroxide. A comparison of humic acid fractions before and after fungal attack shows considerable alteration of the soluble macromolecules of coal. Depending on the coal fraction studied and the fungi used, the assortment of fatty acid esters released during the pyrolysis varies significantly. Furthermore, dicarbonic acid ethyl diesters as well as ethyl derivatives of aromatic ethers and acids yield information about humic acid structure and the biosolubilization of brown coal. Variations in the mixture produced are possibly caused by differences in the pattern of extracellular enzymes secreted that attack the macromolecular structural elements of brown coal. Therefore pyrolysis of native and microbiologically altered geomacromolecules using NEt₄OH allows one to differentiate between free hydroxyl groups as well as substances that are attached to humic substances via ester or ether bridges, and their methylated counterparts. Received: 13 July 1998 / Received revision: 12 October 1998 / Accepted: 16 October 1998     

Manganese and malonate are individual regulators for the production of lignin and manganese peroxidase isozymes and in the degradation of lignin by Phlebia radiata

 The effects of high manganese [180 μM Mn(II)] concentration and addition of malonate (10 mM) were studied in nitrogen-limited cultures of the white-rot fungus, Phlebia radiata. High levels of manganese alone showed no systematic influence on the production of lignin peroxidase (LiP), manganese peroxidase (MnP) or laccase. In contrast, high-manganese containing cultures of P. radiata showed lower efficiency in the mineralization of ¹⁴C-ring-labelled synthetic lignin ([¹⁴C]DHP). The highest rates of mineralization, up to 30% in 18 days, were reached in low- manganese(2 μM)-containing cultures when malonate was omitted. Degradation of [¹⁴C]DHP was substantially restricted by the addition of malonate. The combination of high manganese and malonate resulted in increased levels of MnP and laccase production, whereas LiP production was repressed. Also, the profiles of expression of the MnP and LiP isozymes were affected. A new P. radiata MnP isozyme of pI 3.6 (MnP3) was found in the high-manganese cultures. Addition of malonate alone caused some repression but also stimulating effects on distinctive MnP and LiP isozymes. The results indicate that manganese and malonate are individual regulators of MnP and LiP expression and have different roles in the degradation of lignin by P. radiata. Received: 30 August 1995/Received revision: 10 January 1996/Accepted: 12 February 1996     

Cloning and characterization of an epoxide hydrolase-encoding gene from Rhodotorula glutinis

We cloned and characterized the epoxide hydrolase gene, EPH1, from Rhodotorula glutinis. The EPH1 open reading frame of 1230 bp was interrupted by nine introns and encoded a polypeptide of 409 amino acids with a calculated molecular mass of 46.3 kDa. The amino acid sequence was similar to that of microsomal epoxide hydrolase, which suggests that the epoxide hydrolase of R. glutinis also belongs to the α/β hydrolase fold family. EPH1 cDNA was expressed in Escherichia coli and resting cells showed a specific activity of 200 nmol min⁻¹ (mg protein)⁻¹ towards 1,2-epoxyhexane. Received: 2 August 1999 / Received revision: 4 October 1999 / Accepted: 10 October 1999     

Differential expression of manganese peroxidase and laccase in white-rot fungi in the presence of manganese or aromatic compounds

White-rot fungi (basidiomycetes) play an important role in the degradation of lignin which is, beside cellulose, the major compound of wood. This process is catalyzed by ligninolytic enzymes, which are able to cleave oxidatively aromatic rings in lignin structure. Manganese peroxidase and laccase of white-rot-fungi are the most important of these among the ligninolytic enzymes. In addition, they are able to degrade xenobiotic aromatic polymers, persisting as environmental pollutants. Manganese and aromatic compounds have often been discussed as being inducers, enhancers or mediators of these ligninolytic enzymes. It is known that supplementing the growth medium with either Mn²⁺, veratryl alcohol or coal-derived humic acids leads to significantly enhanced extracellular ligninolytic activities. Measuring the amount of expressed mRNA of the two enzymes by quantitative RT-PCR provided evidence that the expression of manganese peroxidase was induced in the three tested white-rot fungi, Clitocybula dusenii b11, Nematoloma frowardii b19, and a straw-degrading strain designated i63–2. Laccase, on the other hand, was expressed in all three fungi with a significant basic activity even without inducer added. However, since the level of laccase mRNA was higher in cultures supplemented with any one of the tested inducers, we conclude that both manganese and the aromatic substances also increase the expression of laccase. Received: 4 February 2000 / Received revision: 11 May 2000 / Accepted: 12 May 2000     

Effects of aeration intensity on formation of phenol-fed aerobic granules and extracellular polymeric substances

Effect of air aeration intensities on granule formation and extracellular polymeric substances content in three identical sequential batch reactors were investigated. The excitation–emission–matrix spectra and multiple staining and confocal laser scanning microscope revealed proteins, polysaccharides, lipids, and humic substances in the sludge and granule samples. Seed sludge flocs were compacted at low aeration rate, with produced extracellular polymeric substances of 50.2–76.7 mg g⁻¹ of proteins, 50.2–77.3 mg g⁻¹ carbohydrates and 74 mg g⁻¹ humic substances. High aeration rate accelerated formation of 1.0–1.5 mm granules with smooth outer surface. The corresponding quantities of extracellular polymeric substances were 309–537 mg g⁻¹ of proteins, 61–109 mg g⁻¹ carbohydrates, 49–92 mg g⁻¹ humic substances, and 49–68 mg g⁻¹ lipids. Intermediate aeration rate produced 3.0–3.5 mm granules with surface filaments. Reactor failure occurred with overgrowth of filaments, probably owing to the deficiency of nutrient in liquid phase. No correlation was noted between extracellular polymeric substances composition and the proliferation of filamentous microorganisms on granule surface.     

Chemical changes in humic substances from compost due to incubation with ligno-cellulolytic microorganisms and effects on lettuce growth

 Differences in the chemical properties of the organic matter from a highly lignocellulosic compost after incubation with two ligno- and cellulolytic microorganisms were studied in this work. Inoculation with either of the two microorganisms assayed, Trichoderma viride or Bacillus sp., of soil-compost mixtures enhanced degradation processes and the degree of organic matter humification. According to the humification index, inoculation with T. viride produced the highest humification rate in all the compost-soil proportions studied (10, 20 and 30%). To evaluate the quality of the extracted humic substances according to their electrofocusing behaviours a new index was established. This index showed an increased yield of humic substances of the lowest electrophoretic mobility (highest molecular weight) in treatments inoculated with Bacillus sp., whereas inoculation with T. viride enhanced the formation of molecules of the fastest electrophoretic mobility. These results, together with the fibre analysis performed, showed that the nature of the humic substances produced after incubation appeared to depend greatly on the degradation pathway carried out by the inoculated microorganism, T. viride or Bacillus sp.. Both degradation-humification pathways beneficially affected lettuce growth, demonstrating that inoculation with any of these two microorganisms may be a useful tool to modify agronomic properties of unripe composts. Received: 2 August 1995/Received revision: 14 November 1995/Accepted: 11 December 1995     

Effects of Mn2+ and NH+ 4 concentrations on laccase and manganese peroxidase production and Amaranth decoloration by Trametes versicolor

Extracellular lignin peroxidase (LiP) was not detected during decoloration of the azo dye, Amaranth, by Trametes versicolor. Approximately twice as much laccase and manganese peroxidase (MnP) was produced by decolorizing cultures compared to when no dye was added. At a low Mn²⁺ concentration (3 M), N-limited (1.2 mM NH₄ ⁺) cultures decolorized eight successive additions of Amaranth with no visible sorption to the mycelial biomass. At higher Mn²⁺ concentrations (200 M), production of MnP increased and that of laccase decreased, but the rate or number of successive Amaranth decolorations was unaffected. There was always a 6-h to 8-h lag prior to decoloration of the first aliquot of Amaranth, regardless of MnP and laccase concentrations. Although nitrogen-rich (12 mM NH₄ ⁺) cultures at an initial concentration of 200 M Mn²⁺ produced high laccase and MnP levels, only three additions of Amaranth were decolorized, and substantial mycelial sorption of the dye occurred. While the results did not preclude roles for MnP and laccase, extracellular MnP and laccase alone were insufficient for decoloration. The cell-free supernatant did not decolorize Amaranth, but the mycelial biomass separated from the whole broth and resuspended in fresh medium did. This indicates the involvement of a mycelial-bound, lignolytic enzyme or a H₂O₂-generating mechanism in the cell wall. Nitrogen limitation was required for the expression of this activity. Received: 19 May 1998 / Received revision: 22 October 1998 / Accepted: 7 November 1998     

New ways of determining structural groups in brown coals and their bioconversion products by FT IR spectroscopy

New methods of determining the structural groups —COOH and —CH₂— have been developed. The investigation of carboxyl groups is possible both after derivatization with p-fluorophenacylbromide and by quantitative interpretation of the Fourier transform infrared (FT IR) spectra. There exists a linear relationship between the results of these two methods that is generally valid for the analysis of all brown coal components. The maximum extinction coefficient of the symmetric stretching vibration band of the CH₂ groups has been determined using model substances. This allows quantification of this structural group directly from the FT IR spectrum. The results agree with the contents of methylene groups as determined by ¹³C-cross polarization–magic angle spinning–nuclear magnetic resonance (¹³C CPMAS NMR) spectroscopy. Using these methods, the COOH and CH₂ groups contained in brown coals of the North Rhine region and in their bioconversion products have been quantified. Received: 21 December 1999 / Received revision: 25 April 2000 / Accepted: 1 May 2000     

Role of microorganisms in corrosion inhibition of metals in aquatic habitats

In acetate-limited chemostat cultures of Acinetobacter johnsonii 210A at a dilution rate of 0.1 h⁻¹ the polyphosphate content of the cells increased from 13% to 24% of the biomass dry weight by glucose (100 mM), which was only oxidized to gluconic acid. At this dilution rate, only about 17% of the energy from glucose oxidation was calculated to be used for polyphosphate synthesis, the remaining 83% being used for biomass formation. Suspensions of non-growing, phosphate-deficient cells had a six- to tenfold increased uptake rate of phosphate and accumulated polyphosphate aerobically up to 53% of the biomass dry weight when supplied with only orthophosphate and Mg²⁺. The initial polyphosphate synthesis rate was 98 ± 17 nmol phosphate min⁻¹ mg protein⁻¹. Intracellular poly-β-hydroxybutyrate and lipids served as energy sources for the active uptake of phosphate and its subsequent sequestration to polyphosphate. The H⁺-ATPase inhibitor N,N′-dicyclohexylcarbodiimide caused low ATP levels and a severe inhibition of polyphosphate formation, suggesting the involvement of polyphosphate kinase in polyphosphate synthesis. It is concluded that, in A. johnsonii 210A, (i) polyphosphate is accumulated as the energy supply is in excess of that required for biosynthesis, (ii) not only intracellular poly-β-hydroxybutyrate but also neutral lipids can serve as an energy source for polyphosphate-kinase-mediated polyphosphate formation, (iii) phosphate-deficient cells may accumulate as much polyphosphate as activated sludges and recombinants of Escherichia coli designed for polyphosphate accumulation. Received: 23 October 1998 / Received revision: 18 January 1999 / Accepted: 22 January 1999     

Fermented whey – an inexpensive feed source for a laboratory-scale selenium-bioremediation reactor system inoculated with Thauera selenatis

It is critical that an inexpensive electron- donor/carbon-source be found for selenium bioremedia-tion using the selenate-respiring bacterium, Thauera selenatis. Since acetate is a preferred substrate for growth of this organism, a method was developed for fermenting the lactose in whey to large amounts of acetate. Indigenous whey microorganisms fermented the whey lactose in this manner when grown in continuous culture at a very slow dilution rate (D = 0.05 h⁻¹). The successful use of the fermented whey lactose as the carbon-source/electron-donor feed for a laboratory-scale selenium-bioremediation reactor system, inoculated with T. selenatis, treating selenium-contaminated drainage water was also demonstrated. Selenium oxyanions and nitrate were reduced by 98%. Received: 30 October 1998 / Received revision: 26 January 1999 / Accepted: 5 February 1999     

Degradation of Soil Humic Extract by Wood- and Soil-Associated Fungi, Bacteria, and Commercial Enzymes

Abstract An alkaline humic extract (HE) of a black calcareous forest mull was exposed to 36 fungal and 9 eubacterial isolates in liquid standing culture. At 21 d in fungi, and 4 d in bacteria, the groups of wood-degrading basidiomycetes, terricolous basidiomycetes, ectomycorrhizal fungi, soil-borne microfungi, and eubacteria had reduced the absorbance (A ₃₄₀) of HE media by 57, 28, 19, 26 and 5%, respectively. Gel permeation chromatography revealed that the large humic acid molecules were more readily degraded than the smaller fulvic acid molecules and served as a sole source of carbon and energy. The more active HE degraders reduced the overall molecular weight of humic and fulvic acids by 0.25 to 0.47 kDa. They also reduced the chemical reactivity of HE to tetrazotized o-dianisidine, indicating the degradation of hydroxylated aromatic molecules (which are responsible for this reaction). Decreases in absorbance, molecular weight, and reactivity were caused by fungal manganese peroxidase, horseradish peroxidase, β-glucosidase, and abiotic oxidants such as H₂O₂ and Mn(III) acetate. It is concluded that fungi, some of which are propagated in contaminated soils to control xenobiotics, metabolize HE compounds enzymatically. They use enzymes which are also involved in the degradation of soil xenobiotics. Because of reductions in the molecular weight of HE, which is a potential carrier of heavy metal ions and xenobiotics, solubility and motility of humic substances in soil and surface waters are increased. Received: 4 March 1998; Accepted: 1 June 1998