M. Hofrichter · W. Fritsche. Depolymerization of low-rank coal by extracellular fungal enzyme systems. I. Screening for low-rank-coal-depolymerizing activities.
( Springer-Verlag 1996
Appl Microbiol Biotechnol (1996) 46:220—225
OR I G I N A L P AP E R
M. Hofrichter · W. Fritsche
Depolymerization of low-rank coal by extracellular fungal enzyme systems. I. Screening for low-rank-coal-depolymerizing activities
Received: 5 February 1996/Received revision: 15 April 1996/Accepted: 22 April 1996
Abstract A miniscale screening system was developed to detect depolymerizing activities of fungi toward lowrank coals. This system was suitable for the determination of changes in molecular masses as well as for the measurement of the enzymes responsible. A total of 486 fungal strains of different ecophysiological and taxonomic groups were tested for their ability to decolorize agar media containing coal-derived humic acids; 38 wood- and litter-decaying basidiomycetes caused a strong bleaching effect and 49 a weak effect. In contrast, micromycetes were proved to be unable to decolorize the coal substances. The wood-decaying fungus Nematoloma frowardii b19 most effectively bleached the medium. It could be demonstrated by gel-permeation chromatography that the strain really depolymerizes the high molecular-mass fractions of coal humic acids by forming fulvic-acid-like compounds. Extracellular enzyme activities of oxidases and peroxidases towards 2,2@-azinobis(3-ethylbenzthiazolinesulphonate) were extractable from the agar media.
vert various coals to soluble substances (Cohen and Gabriele 1982; Cohen et al. 1987; Faison and Lewis 1988; Monistrol and Laborda 1994). In addition, several bacteria, mostly prominent strains of the genus Streptomyces, have been reported to degrade low-rank coals (Strandberg and Lewis 1988; Crawford and Gupta 1993). However, in most cases the coal solubilization is not accompanied by its depolymerization (Crawford and Gupta 1993). Pyne et al. (1988) reported that cell-free extracts of Coriolus versicolor could solubilize leonardite and form a product with lower average molecular masses. Wondrack et al. (1989) demonstrated a partial depolymerization of highly oxidized lignites (pretreated with HNO ) under in vitro condi3 tions by lignin peroxidase from Phanerochaete chrysosporium, the accuracy of their gel permeation procedure, however, has been questioned (Crawford and Gupta 1993). In the present study we developed a special screening system to select fungi with a high potential for the depolymerization of low-rank coals. The method makes it possible to detect the decrease of molecular masses of coal substances as well as the activities of responsible extracellular enzymes reproducibly.
Introduction Some microorganisms have been reported to attack low-rank coals (lignites) and form water-soluble poducts. Fakoussa (1981) first demonstrated that microbes, above all filamentous fungi, could solubilize solid particles of low-rank hard coal. Since then, reports have been published describing fungi of different ecophysiological groups that have the ability to con-
M. Hofrichter ( ) · W. Fritsche Friedrich-Schiller-University Jena Institute for Microbiology, Department Technical Microbiology, Philosophenweg 12, D-07743 Jena, Germany
Materials and methods Organisms Most of the fungal strains used in the present study were our own isolates from a wide variety of habitats; they were predominantly wood- and litter-decaying fungi as well as micromycetes from soil. They were collected in different geographical and climatic regions of Europe, South America and Asia. Some other fungi were provided by the Stock Culture Collection Weimar (Germany), from DSM Braunschweig (Germany) and from CBS Baarn (The Netherlands). Stock cultures were maintained on 2% malt extract/agar at 4°C. Some sensitive wood-decaying fungi were also maintained on shavings (wood mill) to preserve high degradation activities for a prolonged time.
221 Coal Coal humic acids (water-soluble low-rank coal polymers) were purchased as sodium salts from Aldrich Chemie AG (Neu Ulm, Germany). In addition, humic acids were prepared from German lignite (Rheinbraun AG, Lithotype A, Cologne) by extraction with 0.1 M NaOH (100 g coal/l) and following evaporation to dryness. The ash-free coal material obtained was black-coloured and shone metallically.
c
b
Medium The inoculation material was precultivated on 2% malt extract/agar. The medium used for coal depolymerization consisted of modified Kirk- and Czapek-Dox agar media. The modified Kirk medium contained (per litre) 1 g coal humic acids (Aldrich or German lignite), 0.2 g glucose, 0.05 g yeast extract, 2.2. g 2,2-dimethylsuccinate, 0.5 g ammonium tartrate, 2 g KH PO , 0.5 g 2 4 MgSO ·7H O, 0.1 g CaCl , 28 g agar agar; the Czapek-Dox me4 2 2 dium contained 1 g coal humic acids, 0.2 g glucose, 0.05 g yeast extract, 0.5 g NaNO , 1 g K HPO , 0.5 g MgSO ·7H 0, 0.5 g KCl, 3 2 4 4 2 0.01 g FeSO ·7H O, 18 g agar agar. Both agar types were dark 4 2 brown. The pH values were adjusted to 4.5 and 5.0 for Kirk and Czapek-Dox medium respectively.
Culture conditions The screening was performed by using plastic petri dishes (90 mm inner diameter) containing about 40 ml solid coal acids/agar mentioned above. The plates were inoculated with agar plugs taken from active mycelia precultivated on malt extract/agar (0.05%—2%). Wood- and litter-decaying fungi (basidiomycetous fungi) were cultivated on Kirk agar, micromycetous fungi (deuteromycetes, ascomycetes, zygomycetes) on Czapek-Dox agar. The agar plates were incubated at 24°C in the dark for 3 weeks and were periodically observed (once a day) for decolorization (bleaching) of the darkbrown agar around the fungal growth areas.
a
Fig. 1 Agar plate containing coal humic acid (Aldrich) inoculated with a fungus. The frames show the sample areas. a Sample decolorized by the fungus (for gel-permeation chromatography, GPC, analyses), b sample not yet decolorized (for GPC analyses), c sample for extraction of extracellular enzymes
centrifugation the supernatant served as the source of enzymes (Fig. 1). Oxidases and peroxidases were measured by the oxidation of 2,2@-azinobis(3-ethylbenzthiazoline-6-sulphonate (ABTS) at 420 nm (e : 36 000 M~1 cm~1; Wolfenden and Willson 1982). The 420 assay mixture for the oxidases contained 50 mM malonate buffer pH 4.5 and 0.5 mM ABTS. The reaction was started by addition of 100 ll enzyme extract. Peroxidase activity was determined by the same assay, but after addition of MnSO (0.2 mM) and hydrogen 4 peroxide (0.4 mM). Peroxidase activity was corrected by oxidase activity. All activities are expressed in units defined as follows: 1 U"1000 mU"1 l mol ABTS oxidized/min.
Results Gel-permeation chromatography (GPC) This method for determining the average molecular masses was performed on a Merck-Hitachi HPLC-system (L-6200) equipped with a diode-array detector (L-4500/D-6500 DAD system manager) and a 10-lm HEMA-Bio 40 column (300]8 mm inner diameter) from Polymer Standard Service (Mainz, Germany). The solvent consisted of 80% bidistilled water (containing 5 g/l NaNO and 2 g/l 3 K HPO ) and 20% acetonitrile. It was adjusted to pH 10.0 by 1 M 2 4 NaOH; the flow rate was 1 ml/min. The sodium salts of polystyrene sulphonic acids (3.8—46 kDa; Polymer Standard Service, Mainz) as well as NADPH (0.839 kDa) and biphenyldicarboxylic acid (0.246 kDa) were used for calibration. Samples of the coal agar corresponding to 4.5 cm3 were extracted after disruption with 5 ml 0.1 M NaOH (1 min vortex shaking and 5 min sonication; Fig. 1). After centrifugation, 1 ml supernatant was acidified to pH 1.5 with HCl and centrifuged (7000 rpm). The precipitated coal pellet was resuspended in 1 ml 0.1 M NaOH. The acid supernatant as well as the resuspended precipitate were injected (25 ll) in the HPLC/GPC system.
Enzyme assay Extracellar enzymes in the fungal growth areas were extracted from agar samples (1 cm3) with 0.05 M McIlvaine buffer pH 4.5. After
Growth and coal decolorization A total of 486 fungal strains (253 soil-derived micromycetes, 233 wood- and litter-decaying basidiomycetes) were tested during the first screening step using coal humic acids (Aldrich) as substrate. The mycelial growth on humic acid agar of all fungi tested was less in comparison to those on a comparable malt extract/agar (0.05%), only a few basidiomycetes were able to form mycelial mats on the agar surface (Auricularia sp. i12, Stropharia rugosa-annulata E1 and E2). Most of the micromycetes produced large amounts of spores instead of hyphae (e.g. strains of the genera ¹richoderma sp., Aspergillus sp, Penicillium sp, Chaetomium sp.); conspicuous mycelia were only formed by a few strains, among them Xylaria sp. i70 and Botrytis cinerea H1. The ability to decolorize the dark-brown agar and form yellowish products was limited to the basidiomycetes; 38 strains of this group strongly bleached the agar within 3 weeks, 49 strains weakly (Fig. 2). In contrast, micromycetes were proved to be
222 Table 1 Strains selected during screening step 2 that strongly decolorized the humic acid agar (humic acids derived from German lignite). # Growth rating: minor growth (formation of poorly visible hyphae), ## formation of conspicuous hyphae, ### formation of mycelial mats. Decolorization rating: # diffuse decolorization of the agar medium, ## decolorization and formation of distinct bleaching areas
Fig. 2 Result of the two screening steps. Step 1 Screening test of 486 fungal strains for their ability to decolorize (bleach) agar containing coal humic acid (Aldrich). Step 2 test of the 38 most active strains from step 1 using coal humic acids derived from German lignite. A No decolorization, B weak decolorization, C strong decolorization
Fungal strains
Growth
Decolorization
Nematoloma frowardii b19 Auricularia sp. i37a Clitocybula dusenii b11 Pholiota sp. X9 ¸entinus edodes 301 Kuehneromyces mutabilis X1 Pleurotus cornucopiae P21 ¹rametes versicolor u1 Stropharia rugosa-annulata E1 Isolate u45 Isolate i63-2
# ### # ## ## # ## # ### # #
## ## # # # # # # # ## ##
unable to decolorize coal humic acids (Fig. 2). Comparative tests could demonstrate that the decolorizing activities did not depend on the medium, thus basidiomycetes also decolorized the coal in CzapeckDox agar. On the other hand, the use of Kirk agar did not lead to decolorization in the case of micromycetes. The bleaching effect was stable for more than 6 weeks before a gradual repolymerization began. The second screening step was performed with humic acids derived from German lignite. Only the 38 strongly decolorizing strains selected in the first screening step were used for this purpose. The change of coal substance did not influence the growth of fungi; the bleaching activities, however, were less obvious. Thus, only 11 strains could strongly decolorize the agar, most of them diffusely without distinct bleaching zones (Table 1). The wood-decaying fungus Nematoloma frowardii b19 was the most active strain, bleaching the agar around its growth area rapidly and forming a distinct decolorizing zone. The whole agar was completely decolorized. This organism was selected for further analyses by gel-permeation chromatography.
Gel-permeation chromatography Gel-permation chromatography using a HEMA-Bio 40 column combined with HPLC proved to be a suitable method for the rapid determination of average molecular masses of low-rank coal macromolecules in aqueous solutions. To obtain reproducible chromatograms, interactions of the column matrix and the coal macromolecules had to be prevented by a special solvent system containing high salt concentrations and acetonitrile. The column calibration with sodium salts of sulphonated polystyrenes, NADPH and biphenyldicarboxylic acid gave a high reproducibility of the retention times (over 1 year of using this method the retention times varied only between 0 and 2 s). A linear
Fig. 3 Calibration curve of the HEMA-Bio 40 GPC column using sodium salts of sulphonated polystyrenes (3.8—46.5 kDa), NADPH (0.839 kDa) and biphenyldicarboxylic acid (0.242 kDa) as calibration substances. Column: HEMA-Bio 40; wavelength: 280 nm; solvent: water/acetonitrile (80/20) containing 5 g/l NaNO and 2 g/l 3 K HPO (pH 10.0); flow-rate: 1 ml/min; injection volume: 25 ll 2 4
relationship between retention times and molecular masses (logarithmic values) was detectable between 0.25 kDa and 20 kDa (Fig. 3). Gel-permeation chromatograms of coal extracts from agar plates inoculated with N. frowardii b19 are given in Fig. 4. The coal humic acids were extracted from differently coloured areas of the same agar plate
223 Table 2 Extracellular enzyme activities towards 2,2@-azinobis(3-ethylbenthiazoline-6-sulphonate) of selected fungal strains that decolorize coal humic acids (basidiomycetes) or grow well on humic acid/agar (micromycetes). The enzymes were extracted with buffer after 14 days of growing
Fig. 4A, B GPC chromatograms of extracted agar samples which show the average molecular masses of the coal humic acids and demonstrate the real depolymerization of the high-molecular-mass fraction. A Acid supernatant after precipitation of the extracted coal humic acid with HCl. B Humic acids precipitated and resuspended in 0.1 M NaOH. The conditions were the same as in Fig. 3. -L-LDark-brown agar from the edge of the plate, ——— bleached agar from the fungal growth zone (compare Fig. 1)
(the bleached growth zone, the edge of the plate not yet decolorized; Fig. 1), precipitated with HCl and resuspended in NaOH. In comparison to the controls, the bleached growth areas showed obvious differences in the distribution of molecular masses. In the alkaline extracts of resuspended coal the high-molecular-weight fraction ('20 kDa) was nearly completely depolymerized and the low-molecular-weight part was conspicuously reduced (Fig. 4A). In agreement with this, the amount of the low-molecular-mass fraction was enhanced in the acid supernatant (Fig. 4B). This increase of acid-soluble bleached coal substances had to be caused by cleaving of chemical bonds inside the coal macromolecule.
Enzyme activities Table 2 shows unspecific extracellular activities of oxidases and peroxidases in the decolorized growth zones of different fungi. As a function of the culture time the amounts of these enzymes ranged from 0 to 100 mU/cm3. The most active enzyme producers were the white-rot fungi N. frowardii b19, Auricularia sp. i37a and Kuehneromyces mutabilis (oxidase and peroxidase activities). The highest enzyme activities for N. frowardii b19 could be measured at the edge of the lighter zone where the depolymerizing reactions were still pro-
Fungal strain
Oxidase activity (mU/cm3)
Peroxidase activity (mU/cm3)
Nematoloma frowardii b19 Auricularia sp. i37a Kuehneromyces mutabilis X1 Stropharia rugosa-annulata E1 Pleurotus cornucopiae P21 Xylaria sp. i70 Alternaria sp. G5 Botrytis cinerea H1
103 78 82 — 52 — — 32
10 9 16 18 34 23 22 —
ceeding. In addition to white-rot fungi a few strains of other taxonomic groups also produced such enzymes; bleaching effects, however, could not be observed. Thus, a few micromycetes growing well on coal humic acid-agar and secreted peroxidases (Xylaria sp. i70, Alternaria sp. G5) and oxidases (Botrytis cinerea H1; Table 2).
Discussion It has been demonstrated that the decolorization (bleaching) of agar media containing coal humic acids is a suitable and easy method for the rapid selection of low-rank-coal(lignite)-depolymerizing fungi. With the exception of a few strains, most of the fungi tested grew slowly and produced a small amount of mycelium. An inhibitory effect of humic acids on microbial growth was also reported for various deuteromycetous fungi as well as bacteria (Flaig and Schmidt 1957; Ladd and Butler 1969; Macor 1979). In spite of the slight growth of most fungi on agar containing coal humic acids, some of them rapidly decolorize the dark-brown polymers. This bleaching effect around the growth area of fungi is due to a real breakdown of the coal structure. High-molecular-mass coal fractions are depolymerized through the formation of acid-soluble substances with lower molecular masses. Similar findings were made by Fakoussa et al. during bleaching of water-soluble German lignite molecules by two basidiomycetous fungi while submerged conditions (personal communication, 1993; Willmann 1994). In soil chemistry non-precipitable, acid-soluble substances with molecular masses between 300 Da and 1000 Da are known as fulvic acids distinguishing them from the acid-precipitable high-molecular-mass humic acids (Stevenson 1994). Therefore, a conversion of coal humic substances to fulvic-acid-like compounds by certain fungal strains is assumed. Above all, wood-decaying basidiomycetes (white-rot fungi) are able to degrade coal
224
macromolecules. In many publications these fungi were reported to be the crucial microorganisms in lignin depolymerization (Ka¨a¨rik 1974; Ander and Eriksson 1977; Blanchette 1991). Hurst et al. (1962) as well as Ziechmann (1980) could show that some white-rot fungi decolorized soil humic acids as well as a highly oxidized brown coal (‘‘Kasseler Braun’’). In particular Gloeoporus adustus (Bjerkandera adusta) was suitable for degradation of humic acids connected with the formation of non-acid-precipitable fulvic acids. Ziechmann (1980) further concluded that the repolymerization of lignin fission products to humic acids is prevented by similar reactions. Because of their size, lignin as well as coal humic acids cannot be taken up into the hyphae, thus an extracellular enzymatic attack has to be assumed. Many authors have described unspecific oxidative enzymes (lignin peroxidase, manganese peroxidase, laccase) that attack lignin polymers and a wide range of low-molecular-mass aromatic compounds outside the cell (Leisola and Fiechter 1985; Hammel 1989; Schoemaker and Leisola 1990; Yadav and Reddy 1993; Hatakka 1994). These enzymes are able to form reactive radicals (aryl cation radicals) within the lignin polymer, which can lead to a complete breakdown of the high-molecular-mass structure (Lundell et al. 1992, 1993). The detection of oxidase and peroxidase activities toward ABTS inside the bleached growth areas of various fungi used in our studies indicates that those unspecific radical-forming enzymes also play an important role during coal depolymerization. In addition to typical white-rot fungi, some litter-decaying basidiomycetes (e.g. Stropharia rugosa-annulata E1) show decolorizing activities towards coal humic acids. Surprisingly, the ascomycete Xylaria sp. i70 as well as the deuteromycetous moulds Alternaria sp. G5 and B. cinerea H1 also produce extracellular oxidative enzymes, although they are not able to effect bleaching. As far as we know, this is the first report on the production of extracellular peroxidases by the genera Xylaria spp. and Alternaria spp. An extracellular laccase of Botrytis cinerea was first described by Marbach et al. (1984) and further by Slomczynski et al. (1995). Studies with cell-free culture supernatants of Polyporus versicolor indicated that oxidases (e.g. laccases) are probably involved in low-rank coal solubilization (Pyne et al. 1987; Cohen et al. 1987). Wondrack et al. (1989) first demonstrated the oxidizing effect of lignin peroxidase from Phanerochaete chrysosporium on coal polymers in aqueous solution. Lignin peroxidase caused a disappearance of high-molecularmass coal fractions and the formation of smaller ones. The depolymerizing activity could be stimulated by addition of the mediator veratryl alcohol. Further investigations have to clarify which enzymes exactly are involved in coal depolymerization, what are their functions and by what mechanisms fungi control this process in order to effect a real breakdown of the high-molecular-mass structure and to prevent re-
polymerization reactions. In addition, the method of selecting coal-depolymerizing fungi could be used in degradation studies of humic-acid-bound residues of environmental pollutants in order to clarify whether a liberation of hazardous compounds is possible. Acknowledgements The present work was supported by the Bundesministerium fu¨r Bildung und Forschung (BMBF), grant 0329309E3: Depolymerisation von Braunkohle sowie kohlesta¨ mmigen Produkten durch Pilze as well as by the Fonds der Chemischen Industrie. We thank I. Schwabe for excellent technical assistance.
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