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     

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     

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     

Biodegradation of atrazine under denitrifying conditions

Anaerobic biodegradation of atrazine by the bacterial isolate M91-3 was characterized with respect to mineralization, metabolite formation, and denitrification. The ability of the isolate to enhance atrazine biodegradation in anaerobic sediment slurries was also investigated. The organism utilized atrazine as its sole source of carbon and nitrogen under anoxic conditions in fixed-film (glass beads) batch column systems. Results of HPLC and TLC radiochromatography suggested that anaerobic biotransformation of atrazine by microbial isolate M91-3 involved hydroxyatrazine formation. Ring cleavage was demonstrated by ¹⁴CO₂ evolution. Denitrification was confirmed by detection of ¹⁵N₂ in headspace samples of K¹⁵NO₃-amended anaerobic liquid cultures. In aquatic sediments, mineralization of uniformly ring-labeled [¹⁴C]atrazine occurred in both M91-3-inoculated and uninoculated sediment. Inoculation of sediments with M91-3 did not significantly enhance anaerobic mineralization of atrazine as compared to uninoculated sediment, which suggests the presence of indigenous organisms capable of anaerobic atrazine biodegradation. Results of this study suggest that the use of M91-3 in a fixed-film bioreactor may have applications in the anaerobic removal of atrazine and nitrate from aqueous media. Received: 3 September 1997 / Received revision: 4 December 1997 / Accepted: 2 January 1998     

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     

Involvement of manganese peroxidase in the transformation of macromolecules from low-rank coal by Phanerochaete chrysosporium

Manganese peroxidase (Mn peroxidase) catalyses the oxidation of Mn(II) to Mn(III), a diffusible non-specific oxidant likely to be involved in the transformation of polyphenolic macromolecules from brown coal by the white-rot fungus Phanerochaete chrysosporium. We report here that solubilised macromolecules from Morwell brown coal were depolymerised by Mn(III) ions when incubated under hyperbaric O₂. However, under N₂ or air they were polymerised, suggesting that net depolymerisation by Mn(III) requires molecular oxygen to inhibit coupling of coal radicals. Coal macromolecules were also polymerised when separated by a semipermeable membrane from a culture of P. chrysosporium or from a solution of Mn peroxidase, Mn(II) and H₂O₂, probably by Mn(III) crossing the membrane. In oxygenated cultures in which Mn peroxidase␣was up-regulated by Mn(II), the extent of depolymerisation correlated with cumulative Mn peroxidase activity suggesting that Mn-peroxidase-generated Mn(III) has a central role in initial depolymerisation of coal molecules in vivo. However, mutant ME446-B17-1, which produces Mn peroxidase but not lignin peroxidase, polymerised coal macromolecules in oxygenated cultures. In sum, it appears Mn peroxidase can both polymerise and depolymerise brown coal macromolecules and that, in vivo, both hyperbaric O₂ and lignin peroxidase are also required to force net depolymerisation to products assimilable by cells. Received: 4 September 1997 / Received revision: 29 January 1998 / Accepted: 30 January 1998     

Formation and degradation of a synthetic humic acid derived from 3-fluorocatechol

A synthetic fluorinated humic acid (FHA) was prepared by the spontaneous oxidative polymerization of 3-fluorocatechol. The ¹³C-solid-state NMR spectrum showed signals in the region for aromatic carbons with different substituents (aryl-H, aryl-C, aryl-O carbons) and for carboxyl-carbon. The latter indicated the formation of carboxylic groups, probably caused by ring cleavages during the polymerization process. An indication of the formation of carboxylic groups was also found in the infrared spectrum (band at 1715 cm⁻¹). The dissolved FHA was degraded with active mycelium of the agaric white-rot fungus Nematoloma frowardii as well as with its isolated manganese peroxidase. In both cases, decolorization of the brownish FHA solution and partial defluorination (45–60%) took place. Degradation proceeded via formation of lower-molecular-mass fulvic acid-like substances. The results demonstrate that halogenated humic substances, e.g., resulting from the humification of xenobiotic compounds (bound residues), can in principle be eliminated by ligninolytic fungi (e.g., soil colonizing litter decomposers) and their manganese peroxidase system. Received: 28 June 1999 / Received revision: 14 October 1999 / Accepted: 16 October 1999     

Glutathione-mediated mineralization of 14C-labeled 2-amino-4,6-dinitrotoluene by manganese-dependent peroxidase H5 from the white-rot fungus Phanerochaete chrysosporium

Manganese-dependent peroxidase (MnP) H5 from the white-rot fungus Phanerochaete chrysosporium, in the presence of either Mn(II) (10 mM) or GSH (10 mM), was able to mineralize ¹⁴C-U-ring-labeled 2-amino-4,6-dinitrotoluene (2-A-4,6-DNT) up to 29% in 12 days. When both Mn(II) and GSH were present, the mineralization extent reached 82%. On the other hand, no significant mineralization was observed in the absence of both Mn(II) and GSH, suggesting the requirement of a mediator [either Mn(II) or GSH] for the degradation of 2-A-4,6-DNT by MnP. Using electron spin resonance (ESR) techniques, it was found that the glutathionyl free radical (GS^•) was produced through the oxidation of GSH by MnP in the presence as well as in the absence of Mn(II). GS^• was also generated through the direct oxidation of GSH by Mn(III). Our results strongly suggest the involvement of GS^• in the GSH-mediated mineralization of 2-A-4,6-DNT by MnP. Received: 18 February 2000 / Received revision: 24 May 2000 / Accepted: 26 May 2000     

Rapid atrazine mineralisation in soil slurry and moist soil by inoculation of an atrazine-degrading Pseudomonas sp. strain

The evaluation of pesticide-mineralising microorganisms to clean-up contaminated soils was studied with the widely applied and easily detectable compound atrazine, which is rapidly mineralised by several microorganisms including the Pseudomonas sp. strain Yaya 6. The rate of atrazine removal was proportional to the water content of the soil and the amount of bacteria added to the soil. In soil slurry, 6 mg atrazine kg soil⁻¹ was eliminated within 1 day after application of 0.3 g dry weight inoculant biomass kg soil⁻¹ and within 5 days when 0.003 g kg soil⁻¹ was used. In partially saturated soil (60% of the maximal water-holding capacity) 15 mg atrazine kg soil⁻¹ was eliminated within 2 days by 1 g biomass kg soil⁻¹ and within 25 days when 0.01 g biomass kg soil⁻¹ was used. In unsaturated soil, about 60% [U-ring-¹⁴C]atrazine was converted to ¹⁴CO₂ within 14 days. Atrazine was very efficiently removed by the inoculant biomass, not only in soil that was freshly contaminated but also in soil aged with atrazine for up to 260 days. The bacteria exposed to atrazine in unsaturated sterile soil were still active after a starvation period of 240 days: 15 mg newly added atrazine kg soil⁻¹ was eliminated within 5 days. Received: 31 October 1997 / Received revision: 16 January 1998 / Accepted: 18 January 1998     

Enhanced mineralization of pentachlorophenol by κ-carrageenan-encapsulated Pseudomonas sp. UG30

A pentachlorophenol(PCP)-degrading Pseudomonas sp. strain UG30 was encapsulated in κ-carrageenan for use in PCP degradation. Free and encapsulated cells were compared for their ability to dechlorinate and mineralize 100–800 μg/ml sodium pentachlorophenate in broth. Dechlorination was measured with a chloride ion electrode, and mineralization was measured by ¹⁴CO₂ evolution from radiolabelled [U-¹⁴C]PCP. Free and encapsulated Pseudomonas sp. UG30 cells mineralized up to 200 μg/ml and 600 μg/ml PCP, respectively, after 21 days. Encapsulation of UG30 cells provided a protective effect, allowing dechlorination and mineralization of high levels of PCP to occur. Received: 3 May 1996 / Received revision: 4 September 1996 / Accepted: 13 September 1996     

Ultrahigh-cell-density culture of a marine green alga Chlorococcum littorale in a flat-plate photobioreactor

To test the feasibility of CO₂ remediation by microalgal photosynthesis, a modified type of flat-plate photobioreactor [Hu et al. (1996) Biotechnol Bioeng 51:51–60] has been designed for cultivation of a high-CO₂-tolerant unicellular green alga Chlorococcum littorale. The modified reactor has a narrow light path in which intensive turbulent flow is provided by streaming compressed air through perforated tubing into the culture suspension. The length of the reactor light path was optimized for the productivity of biomass. The interrelationship between cell density and productivity, as affected by incident light intensity, was quantitatively assessed. Cellular ultrastructural and biochemical changes in response to ultrahigh cell density were investigated. The potential of biomass production under extremely high CO₂ concentrations was also evaluated. By growing C. littorale cells in this reactor, a CO₂ fixation rate of 16.7 g CO₂ l⁻¹ 24 h⁻¹ (or 200.4 g CO₂ m⁻² 24 h⁻¹) could readily be sustained at a light intensity of 2000 μmol m⁻² s⁻¹ at 25 °C, and an ultrahigh cell density of well over 80 g l⁻¹ could be maintained by daily replacing the culture medium. Received: 20 October 1997 / Received revision: 19 December 1997 / Accepted: 24 January 1998     

Thermophilic biodegradation of BTEX by two consortia of anaerobic bacteria

Two thermophilic anaerobic bacterial consortia (ALK-1 and LLNL-1), capable of degrading the aromatic fuel hydrocarbons, benzene, toluene, ethylbenzene, and the xylenes (BTEX compounds), were developed at 60 °C from the produced water of ARCO'S Kuparuk oil field at Alaska and the subsurface water at the Lawrence Livermore National Laboratory gasoline-spill site, respectively. Both consortia were found to grow at 45–75 °C on BTEX compounds as their sole carbon and energy sources with 50 °C being the optimal temperature. With 3.5 mg total BTEX added to sealed 50-ml serum bottles, which contained 30 ml mineral salts medium and the consortium, benzene, toluene, ethylbenze, m-xylene, and an unresolved mixture of o- and p-xylenes were biodegraded by 22%, 38%, 42%, 40%, and 38%, respectively, by ALK-1 after 14 days of incubation at 50 °C. Somewhat lower, but significant, percentages of the BTEX compounds also were biodegraded at 60 °C and 70 °C. The extent of biodegradation of these BTEX compounds by LLNL-1 at each of these three temperatures was slightly less than that achieved by ALK-1. Use of [ring-¹⁴C]toluene in the BTEX mixture incubated at 50 °C verified that 41% and 31% of the biodegraded toluene was metabolized within 14 days to water-soluble products by ALK-1 and LLNL-1, respectively. A small fraction of it was mineralized to ¹⁴CO₂. The use of [U-¹⁴C]benzene revealed that 2.6%–4.3% of the biodegraded benzene was metabolized at 50 °C to water-soluble products by the two consortia; however, no mineralization of the degraded [U-¹⁴C]benzene to ¹⁴CO₂ was observed. The biodegradation of BTEX at all three temperatures by both consortia was tightly coupled to sulfate reduction as well as H₂S generation. None was observed when sulfate was omitted from the serum bottles. This suggests that sulfate-reducing bacteria are most likely responsible for the observed thermophilic biodegradation of BTEX in both consortial cultures. Received: 12 July 1996 / Received revision: 31 December 1996 / Accepted: 31 January 1997     

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     

Metabolism of 2,4,6-trinitrotoluene by the white-rot fungus Bjerkandera adusta DSM 3375 depends on cytochrome P-450

Degradation of 2,4,6-trinitrotoluene (TNT) by the white-rot fungus Bjerkandera adusta DSM 3375 was studied in relation to extracellular ligninolytic activities. The Mn(II)-dependent peroxidase, the only ligninolytic enzyme detectable, reached a maximum activity of 600 ± 159 U/l after incubation in mineral medium with a sufficient nitrogen source. In contrast, the highest extent of [¹⁴C]TNT mineralization was detected in malt extract broth, so that the ability of B. adusta to mineralize TNT did not parallel ligninolytic activity. The microsomal fraction of cells grown in the presence of TNT was found to contain 11 pmol cytochrome P-450/mg protein. In cells grown without TNT, no microsomal cytochrome P-450 could be found. Instead, 14 pmol P-450/mg protein was present in the cytosolic fraction of these cells. Cytochrome P-450 apparently affected the TNT metabolism, as shown by inhibitory studies. Addition of the cytochrome P-450 inhibitor piperonyl butoxide diminished the ¹⁴CO₂ release from 21% to 0.9%, as determined after 23 days of incubation, while 1-aminobenzotriazole and metyrapone decreased the mineralization to 8.6% and 6.3% respectively. Mass-balance analysis of TNT degradation in liquid cultures revealed that, by inhibition of cytochrome P-450, the TNT-derived radioactivity associated with biomass and with polar, water-soluble metabolites decreased from 93.9% to 15.0% and the fraction of radiolabelled metabolites extractable with organic solvents fell to 92.6%. The TNT metabolites of this fraction were identified as aminodinitrotoluenes, indicating that this initial transformation product of TNT may function as a substrate for cytochrome-P-450-dependent reactions in B. adusta. Received: 27 May 1999 / Received revision: 19 August 1999 / Accepted: 19 August 1999     

Mineralisation of 14C-labelled synthetic lignin and ligninolytic enzyme activities of litter-decomposing basidiomycetous fungi

Within a screening program, 27 soil litter-decomposing basidiomycetes were tested for ligninolytic enzyme activities using agar-media containing 2,2′-azinobis(3-ethylbenzthiazoline-6-sulphonate), a humic acid or Mn²⁺ ions as indicator substrates. Most active species were found within the family Strophariaceae (Agrocybe praecox, Stropharia coronilla, S. rugosoannulata) and used for mineralisation experiments with a ¹⁴C-ring-labelled synthetic lignin (¹⁴C-DHP). The fungi mineralised around 25% of the lignin to ¹⁴CO₂ within 12 weeks of incubation in a straw environment; about 20% of the lignin was converted to water-soluble fragments. Mn-peroxidase was found to be the predominant ligninolytic enzyme of all three fungi in liquid culture and its production was strongly enhanced in the presence of Mn²⁺ ions. The results of this study demonstrate that certain ubiquitous litter-decomposing basidiomycetes possess ligninolytic activities similar to the wood-decaying white-rot fungi, the most efficient lignin degraders in nature. Received: 20 April 2000 / Received revision: 12 July 2000 / Accepted: 16 July 2000     

Screening for fungi intensively mineralizing 2,4,6-trinitrotoluene

Within a screening program, 91 fungal strains belonging to 32 genera of different ecological and taxonomic groups (wood- and litter-decaying basidiomycetes, saprophytic micromycetes) were tested for their ability to metabolize and mineralize 2,4,6-trinitrotoluene (TNT). All these strains metabolized TNT rapidly by forming monoaminodinitrotoluenes (AmDNT). Micromycetes produced higher amounts of AmDNT than did wood- and litter-decaying basidiomycetes. A significant mineralization of [¹⁴C]TNT was only observed for certain wood- and litter-decaying basidiomycetes. The most active strains, Clitocybula dusenii TMb12 and Stropharia rugosa-annulata DSM11372 mineralized 42 % and 36 % respectively of the initial added [¹⁴C]TNT (100 μM corresponding to 4.75 μCi/l) to ¹⁴CO₂ within 64 days. Micromycetes (deuteromycetes, ascomycetes, zygomycetes) proved to be unable to mineralize [¹⁴C]TNT significantly. Received: 8 August 1996 / Received revision: 16 December 1996 / Accepted: 20 December 1996     

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     

Effects of nitrogen limitation on biofilm formation in a hydrocarbon-degrading trickle-bed filter

The effect of nitrogen limitation on young and mature steady-state biofilm in a trickle-bed filter was studied. Toluene and n-heptane were the sole carbon source. Biomass concentration, respiration, substrate-induced respiration, metabolic quotient, and total hydrocarbon degradation efficiency were measured. The aim of the experiment was to control excess biomass production in the trickle-bed filter by limiting the mineral nutrients and to achieve increased mineralization of the carbon source. Biofilm growth responded strongly to the amount of available nitrogen, whereas hydrocarbon degradation efficiency reached a maximum of 60% and could not be increased even by further addition of nitrogen. The experiments showed that 95% of the adsorbed carbon was mineralized completely and only 5% was used for biofilm formation. This complete mineralization can also be concluded from the metabolic quotient. The value of the latter was about 6–10 mg CO₂-C g⁻¹C_mic h⁻¹, indicating an expanded energy demand due to stress effects in the presence of nutrient deficiency. It was postulated that determination of the metabolic quotient could be an simple instrument to measure the rate of mineralization of carbon sources and also the rate of biomass formation in trickle-bed filters or biofilters. Received: 11 November 1998 / Accepted: 5 December 1998     

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     

Mineralization and solubilization of synthetic lignin by manganese peroxidases from Nematoloma frowardii and Phlebia radiata

Crude and purified manganese peroxidase from the white-rot fungi Nematoloma frowardii and Phlebia radiata catalyzed the partial depolymerization of a [¹⁴C-ring]labelled synthetic lignin into water-soluble fragments (30–50%). The in vitro depolymerization of the ¹⁴C-labelled lignin was accompanied by a release of ¹⁴CO₂ ranging from 4 to 6%. Small quantities of the thiol mediator glutathione stimulated the depolymerization of lignin resulting in a mineralization and solubilization of up to 10 and 64%, respectively. Most of the water-soluble substances formed had molecular masses around 0.7 kDa, although a higher-molecular mass fraction was also detectable (>2 kDa). Photometric assays using 2,2′-azinobis(3-ethylbenzothiazolinesulphonate) as an indicator demonstrated that high levels of Mn(III), which were very probably responsible for the depolymerization and mineralization of the ¹⁴C-labelled lignin, were adjusted within the first 24 h of incubation. The manganese peroxidase catalyzed depolymerization process was not necessarily dependent on H₂O₂; also in the absence of the H₂O₂-generating system glucose/glucose oxidase, effective solubilization and mineralization of lignin dehydrogenation polymerizate occurred, due to the in part superoxide dismutase sensitive, ‘oxidase-like’ activity of MnP which probably produces radical species and peroxides from malonate.