14

Microbial Degradation andModification of Coal

Dr. Martin Hofrichter1

Dr. RenØ M. Fakoussa2

1 Department of Applied Chemistry and Microbiology, University of Helsinki, ViikkiBiocenter, P.O. Box 56, 00014 University of Helsinki, Finland, Tel: �358-919159321,Fax: �358-919159322, E-mail : Martic@gmx.net

2 Institute of Microbiology and Biotechnology, Rheinische Friedrich Wilhelms-University of Bonn, Meckenheimer Allee 168, 53115 Bonn, Germany,Tel: �49-228737219, Fax: �49-228737576

1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 394

2 Historical Outline . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 397

3 Modification of Hard Coal . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3983.1 Bacteria . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3983.2 Fungi . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 399

4 Bioconversion of Brown Coal . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4034.1 Mechanisms: Solubilization, Depolymerization, Utilization . . . . . . . . . . 4034.2 Organisms . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4064.3 Solubilization of Brown Coal . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4064.3.1 Alkaline Solubilization . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4064.3.2 Solubilizing Effects of Chelators . . . . . . . . . . . . . . . . . . . . . . . . . . 4084.3.3 Involvement of Hydrolases in Brown Coal Solubilization . . . . . . . . . . . . 4094.4 Depolymerization of Brown Coal by Oxidative Enzymes . . . . . . . . . . . . . 4104.4.1 Lignin Peroxidase (LiP) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4104.4.2 Manganese Peroxidase (MnP) . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4124.4.3 Other Peroxidases . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4144.4.4 Laccases . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4154.4.5 Use of Decarboxylases for the Hydrophobation of Lignite . . . . . . . . . . . . 417

5 Anaerobic and other Approaches to Convert Coal . . . . . . . . . . . . . . . . . 417

6 Outlook and Perspectives . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 418

393

7 Patents . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 419

8 References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 421

ABCDE-system microbial effector system consisting of alkaline substances, oxidative)biocatalysts, chelators, detergents and esterases

ABTS 2,2'-azinobis(3-ethylbenzthiazoline-6-sulphonate)DMF N,N-dimethylformamideESR electron spin resonanceGC-MS gas chromatography-mass spectrometryGPC gel permeation chromatography3-HAA 3-hydroxyanthranilic acidHRP horseradish peroxidaseLiP lignin peroxidaseMnP manganese peroxidaseMW molecular weightNADPH reduced nicotinamide adenine dinucleotideNMR nuclear magnetic resonancePAH polycyclic aromatic hydrocarbonpI isoelectric pointSBP soy bean peroxidaseSDS sodium dodecyl sulfateTHF tetrahydrofuran

1

Introduction

Relatively few microbiologists, and perhapseven fewer geochemists and fuel scientists,have seriously considered that microorgan-isms might be able to modify the physico-chemical structure of coal. There are twomain reasons for this. Microbiologists usu-ally prefer simple sugars, organic acids andthe like as substrates for microbial activity,and they try to avoid the use of too complexsubstrates such as coal. On the other hand,the resistance of geochemists and fuel scien-tists stems from an equally specializedknowledge of the physico-chemical processesand extreme conditions (temperature, pres-sure) which were involved in the genesis ofcoal (coalification) and are also required forthe industrial coal conversion.

Nevertheless, there are several reasons toinvestigate microbial activities towards coal.Besides crude oil, coal is the most importantfossil fuel and thus, a basic energy as well as araw material source. The worldwide coaldeposits are considerably larger than those ofoil and therefore, coal could become again themain resource of raw materials (feedstock)for the chemical industry (see Chapter 19). Inthis context, new conversion technologies forcoal are urgently needed to reduce environ-mental damages caused by the classic carbo-chemistry processes. One approach, which isstill at the fundamental research stage, mightbe the utilization of biotechnological proc-esses to convert coal into value-added prod-ucts which can be used for further biotechno-logical or chemical synthesis. Another reasonto study the microbial conversion of coal isattributed to environmental problems of

14 Microbial Degradation and Modification of Coal394

former coal mining areas (e.g. the huge open-cast mines in East Germany). When coalmining finishes in a region, the landscapethat remains is usually devastated, withinfertile soil that has to be recultivated. Thedegradative activities of microbes towards theresidual coal may be of significance if soilfertility is to be improved and intact soils re-created by mobilizing the humic substancesin coal (see Chapter 9). In addition to theseeconomic and ecological considerations,there is also a general interest on the part ofcoal chemists with regard to the degradativeactivities of microorganisms, which might behelpful in elucidation of the coal structure bygradually decomposing the coal components.

Although coals have been of major eco-nomic importance for more than 100 years,the structure of coals ± low rank coals andeven hard coal ± is still under discussion (VanKrevelen,1993; see alsoChapters14and15 inthis volume). Research into this field hasbeen inadequate, especially in the case ofbrown coal (� lignite, low-rank coal; Stefa-nova et al. , 1993), and only a few structuralmodels of coals are to be found in theliterature. The so-called `coal structure'changes with the rank of the coal. Figure 1provides a survey of different coal structuremodels (modified from Schumacher, 1997).Schulten and Schnitzer (1993) have publish-ed the first detailed structural model of ahumic acid from brown coal; in addition,several different structures of the so-calledfulvic acids, which are also present in soils,are discussed by Stevenson (1994). Sinceresearch into coal microbiology focuses onbrowncoal (lignite),wemust face thefact thatlignite has an even more complex structurethan hard coal because it consists of severaldistinct classesof constituents. These includethe mainly hydrophobic bitumen, the alkali-soluble humic and fulvic acids, and theinsoluble residue designated as matrix orhumine. In spite of the great economic

importance of brown coal, there have beenfew investigations of the humic substances inlignite, compared for example with researchintohumicacids fromwaterorsoil (Stefanovaet al. , 1993; Stevenson, 1994). Even thoughsome Australian, American and East Euro-pean lignites have been characterized (Wild-enhain, 1969; Hatcher et al. , 1981, 1988;Verheyen and Johns, 1981; Verheyen et al.,1982, 1985; Chaffee et al. , 1983; Künstneret al. , 1986; Hatcher, 1990), these resultscannot be transferred to lignites of otherorigins, because the coal diagenesis differsstrongly with each coal deposit depending onplant input, coal generating conditions, etc.(Stach et al. , 1982; van Krevelen, 1993). Untilrecently, therewasneitherareliable measure-ment of the molecular weight (MW) distri-bution of humic acids from lignite (Henninget al. , 1997) nor any publication about thisimportant basic characteristic. Only for mi-crobiologists has the MW of lignite com-pounds become a research topic. Linehanet al. (1991) described how the apparent MWswere influenced by the method used. For thesame humic acid solution, they determinedaverage MWs between 340,340 and800,000 Daltons (Da), depending on themethod. Only Hofrichter and Fritsche(1996) have established a rapid method tocompare the MWs of humic acids by gelpermeation chromatography (GPC) usingsimple HPLC equipment.

In the first instance, coal appears to beresistant to microbial degradation; undernormal circumstances, no extensive micro-bial growth can be seen on coal piecescollected in open-cast or underground mines.However, when the conditions for microbesare improved (humidity, minerals, additionalcarbon source), growth of both fungi andbacteria on coal can be achieved. This chapteraims to demonstrate that, even though coal isindeed comparatively resistant to microbialattack, there are microorganisms which are

1 Introduction 395

14 Microbial Degradation and Modification of Coal396

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capable of modifying the coal structure bydifferent mechanisms. With respect to theorigin of coal from fossil lignocelluloses, theactivities of ligninolytic fungi and theirextracellular enzymes are considered in par-ticular. The chapter focuses on the microbialconversion of brown coal and the two maintransformation principles: solubilizationand depolymerization, since most studieswere carried out with this type of coal. Inaddition, a summary of the microbial mod-ification of hard coal is also provided.

2

Historical Outline

The idea that coals might be acted on bymicrobesorutilizedasgrowthsubstrate isnotnew. For example, as early as 1908 Potterreported that bacteria acted as biocatalyticagents in the oxidation of amorphous browncoal (Potter, 1908). Two years later, Galle(1910) first isolated pure cultures of bacteriagrown on brown coal samples, whilst someyears later Fischer and Fuchs (1927a,b)published two articles about the growth offungi on various types of coal. They hadobserved by chance that white and greenishmycelia had arisen readily on untreated,moist coal samples stored in the laboratory.This observation prompted them to study thisphenomenon in greater detail, and resultedin the discoveryof various filamentous micro-fungi (molds) that were able to colonizebrown coal; later, even coal-briquettes, cokeand hard coal were found to be growthsubstrates for these fungi. Microscopic stud-ies indicated that the microfungi responsiblebelonged to the deuteromycetes (Penicilliumspp., Aspergillus spp.) as well as to the yeast-like fungi (Torula spp.). In addition, Fischerand Fuchs referred to their correspondencewith a Dutch colleague (D.W. Kreulen,Amsterdam) who observed the development

of the zygomycete Mucor mucedo on bitumen-rich brown coal (`Fettschlammkohle') andspeculated that the fungus somehow oxi-dized the coal to assimilateand solubilize partof the humic substances during this process(Fischer and Fuchs, 1927b). First detailedinvestigations into the microflora of naturalcoal deposits were carried out by Lieske andHofmann (1928), and resulted in the descrip-tion of a wide range of microorganisms inmining areas. The same author first consid-ered the idea of (bio)technological applica-tionsofcoalandcoal-colonizingmicrobes, forexample, the use of coal as fertilizer inagriculture (Lieske, 1929, 1931). All theseinvestigations came to a preliminary end in1932, marked by the publication of thesummarizing article `Biologie und Kohlefor-schung' (Biology and coal research; Fischer,1932).

Unfortunately, no further interest wasshown in this field until 1981, when RenØFakoussa (working at the institute in Mül-heim where Fischer and Fuchs had workedsome 50 years earlier) demonstrated thatcertain bacteria were able to utilize organicextracts of hard coal as sole carbon source andto solubilize part of the native coal, resultingin the formation of colored liquids (Fakoussa,1981). The same author also recognized thebiotechnological potential of coal-modifyingmicroorganisms, and published the firstdetailed considerations into this topic in1983 (Fakoussa and Trüper, 1983). At almostthe same time, in the USA Cohen andGabriele (1982) found that wood-decayingbasidiomycetes (white-rot and brown-rotfungi) could form black droplets from leo-nardite particles (a special kind of highlyoxidized brown coal). Both findings were thestarting point for a number of intensiveresearchprogramsthatwereconducted intheUS, and later also in Germany, Spain andAustralia, to find suitable microorganismsfor the biological conversion of coal (prefer-

2 Historical Outline 397

entially browncoal) intouseful products suchas chemicals and fuels. The progress in thisfield during the past 20 years is summarizedin Table 1 (Fakoussa and Hofrichter, 1999).Another aspect of coal microbiology that isalso of biotechnological significance con-cerns the bacterial desulfurization of coal,and this topic is detailed in Chapter 15.

3

Modification of Hard Coal

3.1

Bacteria

Certain bacteria can grow by utilizing hardcoal as sole carbon source. In a screening ofabout 3100 cultivation experiments, micro-

14 Microbial Degradation and Modification of Coal398

Tab. 1 Advances in coal microbiology and biotechnology during the past two decades

Year Achievement Reference(s)

1981 Effects on hard coals by bacteria (Pseudomonasspp.), simultaneous biotenside secretion

Fakoussa (1981)

1982 Solubilization of lignite to droplets on agar platesby action of wood-decaying basidiomycetousfungi

Cohen and Gabriele (1982)

1986 Acceleration of solubilization by pretreatment ofcoal (fungi � bacteria)

Scott et al. (1986), Grethlein (1990) and others

1987 First solubilization mechanism elucidated: pro-duction of alkaline substances (fungi � bacteria)

Quigley et al. (1987, 1988a, 1989a)

1988 Second solubilization mechanism elucidated:production of chelating agents (fungi)

Cohen et al. (1990), Quigley et al. (1988b, 1989b)

1989 First product on market: solubilized lignite asfertilizer

Arctech, Virginia (USA)

1991 Evidence that chelators alone are not responsiblefor all effects

Fakoussa and Willmann (1991), Fakoussa (1994)

1994 Decolorization and reduction of MW of solublelignite derived humic acids proves catalytic, i.e.,enzymatic attack (basidiomycetous fungi)

Willmann (1994), Ralph and Catcheside (1994),Hofrichter and Fritsche (1996, 1997a)

1991 Improved analysis by 13C-solid state NMR, MWdetermination, e.g., ultrafiltration, gel permea-tion chromatography

Willmann and Fakoussa (1991), Polman andQuigley (1991), Ralph and Catcheside (1996),Hofrichter and Fritsche (1996, 1997a), Henninget al. (1997) and others

1996 In-vitro systems shown preferentially to poly-merize humic acids without regulation of thefungus (laccase)

Willmann (1994), Frost (1996) and others

1997 In-vitro systems based on fungal Mn peroxidasewas shown to depolymerize humic acids and toattack coal particles, including the matrix

Hofrichter and Fritsche (1997b), Hofrichter et al.(1999)

1997 Involvement of unspecific esterases in the solu-bilization of coal (deuteromycetous fungi)

Hölker et al (1997, 1999)

1997 First fine chemical produced successfully fromheterogeneous humic acid mixtures by bacterialpure cultures: polyhydroxyalkanoates (PHA,`Bioplastic')

Steinbüchel and Füchtenbusch (1997), Füchten-busch and Steinbüchel (1999)

organisms from suitable locations were en-riched using five types of hard coal, each withdifferent volatile matter. The coals were notchemically pretreated, but ground into piecesof an average size of 2.5 mm in order toincrease the surface area. Forest fire regions(0.5 to 20 years old) were used as screeninglocations, since the charcoal structure issomehow related to that of hard coal. Growthwas observed in 0.2% of all cases, which is anexceptionally low rate.

Initially, pleomorphic bacteria were isolat-ed, probably belonging to the group ofmycobacteria or nocardia. These pleomor-phic bacteria had extremely hydrophobic cellwalls. Thus, the cell aggregations could onlyfloat on the surface of the medium liquid andcould only be dispersed by using detergentsor oily substances. The poor growth rates, thedistance to the (sedimented) coal particlesand investigation of the fate of substances inthe culture supernatant indicated that thesebacteria only grow on water-soluble coalsubstances, which are released through sur-face pores (Fakoussa, 1981, 1990). Suchstrains can also be observed on old solutionsof humic acids obtained from lignite.

During this screening, a more interestingbacterium was enriched and subsequentlyidentified as Pseudomonas fluorescens. Thisstrain showed some remarkable properties:

. The bacterium released a very effectivesurfactant into the medium, which low-ered the surface tension to about25.5 mN m±2. This value is even lower thanthat of a saturated solution of sodiumdodecyl sulfate (SDS) (for comparison, thesurface tension of water is 72.6 mN m±2,while that of acetone is 23.5 mN m±2).

. The coal particles were altered during thecultivation in several characteristics, suchas color, wettability, and extractability.

. After some time the culture supernatantturned brown in color, indicating that coal

substances were being released. Whenisolated, these substances had molecularweights of 50,000 ± 100,000 Da. Infraredspectra and esterification experimentsshowed a high content of carboxylic andhydroxyl groups, which is consistent withan oxidative attack on the coal particles(Fakoussa, 1988).It canbeassumedthatanysecretedenzyme

converted the coal substances to a morehydrophilic status, i.e. , they become morewater-soluble, and were then taken up by thebacterium. Control experiments with differ-ent surfactants indicated that these mole-cules adhere to the hydrophobic surfaceof thehard coal particles, thus clogging the pores.Consequently, the use of pure water willresult in a more extensive extraction of hardcoal than would a solution of surfactant.

To summarize, the results of these studiessuggested that hard coals with a high contentof volatile organic matter are more easilyattacked by bacteria, though the bacterialattack on the coal particles is not efficientenough for application to commercial coalconversion processes.

3.2

Fungi

A number of comprehensive screening pro-gramshavebeencarriedout to identify fungalstrains capable of modifying the physico-chemical structure of hard coal or derivedproducts (e.g., asphaltenes, organic hard coalextracts). In addition to testing a largenumber of bacteria (Section 3.1), Fakoussa(1981, 1988, 1990) also investigated whetheryeasts and filamentous fungi from suitablelocations (e.g., former forest fire sites, hardcoal samples) could utilize hard coal as solesource of carbon and energy and releasebrown-colored substances from powderedhard coal. As a result of these studies, threefilamentous fungi and two yeasts were en-

3 Modification of Hard Coal 399

riched that would grow (albeit at an excep-tionally slow rate) with powdered hard coal assole carbon source. Attachment of coalparticles (average size 2.5 mm) to the fungalcell wall was observed both for filamentousfungi and yeasts, and attributed to a hydro-phobic interaction (Figure 2A,B). Interest-ingly, when the fungal hyphae had justgerminated, there was no affinity to the coal;however, hyphae in a more mature stageshowed many coal particles adhering to thecell wall, and older hyphae even becamecoated with a layer of coal. There was noembedding of hard coal particles into muci-laginous substances.

Similar results have been reported byStewart et al. (1990), who investigated thecolonization of differently pretreated bitumi-nous coals placed on agar plates by a numberof filamentous fungi. A Penicillium sp. strainandaCunninghamellasp.strainwerefoundtobe the most active fungi. By using scanning

electron microscopy, an extensive surfacecolonization (including conidia formationand a tightattachmentof fungal hyphae to thecoal particles) was observed. Interestingly,only air-oxidized coal samples, which hadpreviously been exposed to 150 8C for 7 days,were overgrown by the molds, whereas un-treatedparticlesshowedonly littleevidenceofcolonization. The gravimetric quantitativeanalysis indicated that up to 10% of the pre-oxidized coal was converted to water-solubleproducts.

An extensive screening involving morethan 750 strains of filamentous fungi wascarried out to select strains which modify anuntreated German hard coal (Bublitz et al.,1994; Hofrichter et al. , 1997a). Among thestrains tested were representatives of differ-ent taxonomic groups of filamentous fungi:zygomycetes, ascomycetes and deuteromy-cetes, aswell asbasidiomycetes.Thehardcoalparticles were exposed for 6 weeks to agar

14 Microbial Degradation and Modification of Coal400

Fig. 2 (A) Germinating conidia-spore of afilamentous microfungus (mold). Hard coalparticles start to attach at the tip of the younghypha. (B) Hypha of a filamentous microfungusthat grows in a liquid medium in the presence ofhard coal particles. Attachment of the coalparticles to the fungal hypha is clearly visible(Fakoussa 1981, 1988)

plates which had been overgrown by thefungalmycelia.Onlysixofthe750strainstestedacted noticeably on the hard coal. Tight con-nections were developed between the fungalhyphae or rhizomorphs and the coal particles(Figures 3 and 4), which in turn were splitinto smaller pieces. Moreover, the wettabilityof the particles increased, this becomingvisible by the attachment of fungal guttationdroplets on the hydrophobic hard coal sur-face, suggesting either the secretion of fungalsurfactants or oxidation of the surface.

Interestingly, all hard coal `eroding fungi'belongedtothe litter-decomposingandwood-decaying basidiomycetes. The most activefungus, Coprinus sclerotigenis, was studied inmore detail with respect to the formation oflow-molecularmassproducts frompowderedhard coal. 2-Hydroxybiphenyl, alkylated ben-zenes, polycyclic aromatic hydrocarbons

(PAHs) and branched alkanes were extractedwith tetrahydrofuran (THF) from coal sam-ples treated with this fungus (Figure 5). Itwas assumed that the nonoxidized com-pounds (alkylated benzenes and alkanes,PAHs) might be part of the mobile phase ofhard coal, and were liberated from the micro-pores through mechanic effects of the fungalhyphae (biodeteriorationofcoal). Incontrast,the formation of 2-hydroxybiphenyl wasprobably brought about by an enzymatic,bond-cleaving process, because the samecompound was also detected after the fungaltreatment of hard coal-derived asphaltenepowder lacking micropores and a mobilephase. However, it remained unclear whichenzyme was responsible for this, since themost promising candidates ± ligninolyticperoxidases and phenol oxidases ± seemedto be lacking in Coprinus sclerotigenis.

3 Modification of Hard Coal 401

Fig. 3 Formation of rhizomorphs by Coprinussclerotigenis C142-1 which are tightly attached tothe surface of a hard coal piece (1 3 mm)(Hofrichter et al., 1997a)

Fig. 4 Mycelium of Coprinus sclerotigenis C142-1on the surface of a hard coal particle (scanningelectron micrograph; original magnification,�1000) (Hofrichter et al. , 1997a)

Powdered Polish hard coal and its chloro-form extracts were exposed to the basidiomy-cetous fungus Piptoporus betulinus belongingto the wood-decaying white-rot fungi (Osipo-wicz et al., 1994). The agitated cultures usedcontained maltose and peptone as actualgrowth substrates, while the coal served as co-substrate. Under these conditions, the fun-gus acidified the medium drastically duringits growth (from pH 6.0 to 1.0!) , which wasassociated with the production of organicacids. Piptoporus betulinus acted on both thenative coal and its organic extract, this beingdemonstrated on the basis of spectroscopicdata. According to the latter, the biotransfor-mation caused dearomatization and depoly-merization of the coal substrates. Thus, adecrease in aromatic absorption in the UVspectra and a reduction in intensity of thebroad signal of aromatic protons, togetherwith an increase in alkene protons in the 1H

NMR spectra were observed. On the basis ofthe IR spectra, it was concluded that thebiotransformation process was also accom-panied by decarboxylation reactions. Further-more, high-performance size-exclusion chro-matography (HPSEC) using chloroform asthe solvent revealed a substantial shift inmolecular mass distribution towards lowervalues. Attempts to explain the alterations ofhard coal caused by the fungus were notmade. Another white-rot fungus, Panus tigri-nus, was found to convert an asphaltene ±obtained through the hydrogenation of Ger-man hard coal ± in a similar manner. Theasphaltene, representing a complex mixtureof aromatic compounds with molecularmasses between 0.25 and 0.6 kDa was ex-posed to solid-state cultures of the fungusgrowing on wood shavings (Hofrichter et al.,1997a). As the result of a 6-week incubation,the predominant molecular masses of the

14 Microbial Degradation and Modification of Coal402

Fig. 5 Gas chromatogram of a THF extract of powdered German hard coal (<200 mm) treated with thebasidiomycetous fungus Coprinus sclerotigenis (modified after Hofrichter et al. , 1997a). The liberatedcompounds were putatively identified by mass spectroscopy (GC-MS analysis). Except for 2-hydroxybiphenyl(12), the substances most likely originate from the coal micropores (according to Hofrichter et al., 1997a). (1),1-methyl-2-ethylbenzene; (2), 1,3-dimethylbenzene; (3), 1,2-dimethylbenzene; (4), 1,2-diethylbenzene; (5), 1,4-diethylbenzene; (6), 1,3,4,5-tetramethylbenzene; (7), 1,3,4, 5-tetramethylbenzene; (8), naphthalene; (9), 3,4,5-trimethylbenzyl alcohol; (10), biphenyl; (11), 1,2-dimethylnaphthalene; (12), 2-hydroxybiphenyl; (13), 2,6,10-trimethylundecane; (14), 1,7-di(tert-butyl)-4-methylheptane; (15), tetramethyl-biphenyl; (16), fluorene; (17),pyrene

asphaltenedecreasedfrom0.4to0.3 kDa,anda new low-molecular mass fraction (0.1 kDa)consisting probably of monoaromatic com-pounds was formed. Itwas concluded that theligninolytic enzyme system of Panus tigrinusattacked the asphaltene unspecifically whileit degraded the lignin in the wood shavings.Because manganese (Mn) peroxidase is thepredominant ligninolytic enzyme of thisfungus (Maltseva et al. , 1991) and one ofthe most powerful peroxidases (see Section4.4.2), it was proposed that this enzyme isinvolved in the asphaltene depolymerizationby this fungus.

The most effective modification of hardcoal observed so far has been reported forSpanish bituminous and sub-bituminouscoals which were converted under co-meta-bolic conditions (Sabouraud maltose brothwas used as growth medium) to a tar-likemass by the action of a deuteromycetousfungus isolated from native hard coal sam-ples (Monistrol and Laborda, 1994). Unfortu-nately, thestrain lost itshardcoal-solubilizingability over the following years, probably dueto spontaneous degeneration processes. Theauthors, however, succeeded in isolating newmolds (Trichoderma sp. M2, Penicillium sp.M4) with hard coal-modifying and -solubiliz-ing activities, although the formation of tar-like products was not observed again (Labor-da et al. , 1997, 1999). The authors detectedoxidative and hydrolytic enzyme activities infungal supernatants which had been growninthepresenceofhardcoal,but theactual roleof these enzymes (phenoloxidases, peroxi-dases, esterases) in hard coal transformationis still not clear.

In summary, hard coal is much moreresistant towards fungal attack than lowerrank coals, this being a result of its higherhydrophobicity, the higher proportion ofcondensed aromatic rings, and the loweroxygen content (Fakoussa and Trüper, 1983).Nevertheless, there are some fungal organ-

isms ± both deuteromycetes and basidiomy-cetes ± which are capable of modifying thephysico-chemical structure of hard coal andeven liberating low-molecular mass com-pounds. The biochemical processes, howev-er, underlying these phenomena are onlypoorly understood. It is most likely that astring of factors, e.g., mechanical effects offungal hyphae, surface-active substances(surfactants), and extracellular enzymes (hy-drolytic and/or oxidative) are responsible forthe structural changes.

4

Bioconversion of Brown Coal

4.1

Mechanisms: Solubilization,Depolymerization, Utilization

Distinction must be made between twodifferent principles of the structural modifi-cation of brown coal (� low-rank coal andlignite), namely solubilization and depoly-merization (Catcheside and Ralph, 1997;Hofrichter et al. , 1997b; Fritsche et al.,1999; Klein et al. , 1999) (Figure 6). Thesolubilization of brown coal, which leads tothe formation of black liquids (Figure 7), is amainly nonenzymatic desolving process thatoccurs preferentially at higher pH values (pH7 ± 10) and is due to the microbial formationof alkaline substances and/or chelatingagents and surfactants. In addition, recentstudies have provided a novel indication thatcertain hydrolytic enzymes may enforce thesolubilization process (fordetails, see below).Coal solubilization does not result in asubstantial decrease in the molecular massof coal humic substances; on the contrary, itmay even be accompanied with polymerizingreactions and an increase in the predominantmolecular mass (Hofrichter et al. , 1997b).

4 Bioconversion of Brown Coal 403

The term `liquefaction', although often usedin earlier publications, should be avoided inconnection with microbial activities towardscoal, because it is already occupied by the`pure' chemical processes of coal conversion(see Chapter 17).

The depolymerization of brown coal orderived macromolecules (coal humic acids)is an enzymatic process that occurs at lowerpH values (pH 3 ± 6) and results in thecleavage of bonds inside the coal molecule,leading to the formation of yellowish, fulvicacid-like substances (`bleaching') with lowermolecular masses. As an example, Figure 8shows the decolorization of an agar platecontaining a high-molecular mass humicacid from coal as a test substrate.

In general, coal solubilization ± and prob-ably also depolymerization of coal ± arefacilitated when oxidatively pretreated ornaturally highly oxidized coals (e.g., weath-ered lignite, leonardite) are exposed to mi-crobes (Scott et al. , 1986; Ward et al., 1988;Hofrichter et al. , 1997b). Artificial oxidativepretreatment can be performed in the labo-ratory using nitric acid (HNO3), hydrogenperoxide (H2O2), ozone (O3), or radiation(Strandberg and Lewis, 1987; Kitamura et al.,1993; Achi, 1993; Gazsó, 1996; Henninget al. , 1997; Hofrichter et al., 1997b).

In addition to the structural modificationsmentioned above, a number of microorgan-isms (bacteria, molds, yeasts) is able to growon brown coal by utilizing parts of the mobile

14 Microbial Degradation and Modification of Coal404

Fig. 6 The two main structural modifications of brown coal by microorganisms (modified after Fritsche et al. ,1998)

Fig. 7 Solubilization of brown coal by a fila-mentousmicrofungus(Alternariasp.).Theblackdroplet was formed from a brown coal piece (13 mm) placed on a pre-grown agar plate

phase (Hodek, 1994), which comprises acomplex mixture of low-molecular massaromatics and wax-like aliphatics, as solecarbon source (Kucher and Turovskii, 1977;Ward, 1985; Ralph and Catcheside, 1993;Willmann, 1994; see also Section 2). Atpresent, no information is available aboutthe exact nature of these organic compounds,but studies using low-molecular mass mod-els have indicated that they include substan-ces such as phenols, benzoic acids, biphenylsand biphenylethers, as well as various cyclo-alkanes, n-alkanes and n-alkanols (Engesseret al. , 1994; Ralph and Catcheside, 1994a;Schumacher and Fakoussa, 1999; Fritscheand Hofrichter, 2000). Residual cellulose andhemicelluloses that can be found in certainbrown coals (e.g., xylite� coal that preservedthe wood structure) might be an additionalcarbon source for microorganisms (Cohenand Gabriele, 1982). Usually, the microor-ganisms grow slowly on coal particles, but thegrowth is noticeably stimulated when natu-

rally weathered or chemically pre-oxidizedcoals are used (Ward, 1985) (Figure 9); theoxidation most likely enhances the bioavail-ability of available compounds. Furthermore,the addition of mineral solutions (N, P, S,metal ions or mineral salts) stimulates themicrobial growth, indicating limitations ofessential elements in native coal. In somecases, the utilization of brown coal might beaccompanied by its solubilization. Thus,Hölker et al. (1999b) have reported that14CO2 was evolved from radioactively labeledcoal (14C-methoxylated German lignite) by acoal-solubilizing fungus.

Fakoussa (1991) has proposed the so-calledABC-system, that was later extended to theABCDE-system (Fakoussa and Hofrichter,1999), to describe all possible mechanismswhich are involved in brown coal bioconver-sion:

. Alkaline substances

. Biocatalysts (oxidative enzymes)

4 Bioconversion of Brown Coal 405

Fig. 8 Depolymerization of high-molecularweight coal substances by a ligninolytic fungus(Nematoloma frowardii b19). The agar platescontained coal humic acids extracted with NaOHfrom Rhenish brown coal (modified after Hof-richter and Fritsche, 1996). Right: control with-out fungus; center: 10-day-old culture; left: 20-day-old culture

Fig. 9 Growth of a filamentous microfungus(Penicillium sp.) on a piece of brown coal.Almost the entire surface of the coal particle iscovered with conidia-forming mycelium. Thefungus utilizes part of the mobile phase of coalas carbon source

. Chelators

. Detergents (surfactants, biotensides)

. EsterasesThese mechanisms are illustrated in Fig-

ure 10, using the example of a brown coalmodel structure.

4.2

Organisms

While the process of brown coal solubiliza-tion is typical for microfungi (molds, yeasts)and bacteria (actinomycetes, pseudomo-nads), the depolymerization of coal is evi-dently limited to the ligninolytic basidiomy-cetes (wood-decaying and litter-decomposingfungi,�white-rot fungi; see Figure 4). Nev-ertheless, there is some overlap of theseabilities. Thus, some white-rot fungi (e.g.,Trametes (Coriolus) versicolor, Phanerochaetechrysosporium), which are active decolorizersof coal humic substances (Ralph and Catche-side, 1994b; Frost, 1996), can also solubilizecoal, but under other conditions and/or byusing other mechanisms (Cohen and Gabri-ele, 1982; Torzilli and Isbister, 1994).

Microorganisms utilizing part of coal asgrowth substrate can be found among manygroups of aerobic microorganisms, and itshould also be mentioned that brown coal-derived products are used as fertilizers andsoil conditioners in agriculture (Yang et al.,1985; Fortun et al., 1986; Gyori, 1986; Alek-sandrov et al. , 1988). Table 2 provides aconcise overview of microorganisms whicheither solubilize or depolymerize brown coal,or utilize it as a growth substrate.

4.3

Solubilization of Brown Coal

4.3.1

Alkaline SolubilizationThe alkaline solubilization of brown coal wasthe first discovered mechanism of microbialcoal conversion (Quigley et al. , 1987, 1988a),and was later confirmed by several authors(Maka et al. , 1989; Runnion and Combie,1990; Torzilli and Isbister, 1994). This phe-nomenon is attributed to the high content ofcarboxylic groups in the coal humic acids,which can be deprotonated at higher pH (>8),

14 Microbial Degradation and Modification of Coal406

Fig. 10 The so-called ABCDE-mechanism of biological conversion of brown coal (modified after Fakoussa,1991).Thearrows indicatestructures thatcanbeattackedbydifferentmicrobialagents.A:alkalinesubstances;B: biocatalysts (oxidative enzymes); C: chelators; D: detergents; E: esterases

4 Bioconversion of Brown Coal 407

Tab. 2 Selected microorganisms that have solubilizing or depolymerizing activities towards brown coal, orutilize it as a growth substrate

Organisms Effects on brown coal and derivedproducts

References

BacteriaActinomycetesStreptomyces spp.Streptomyces setoniiEubacteriaBacillus sp.Bacillus lichiniformisPseudomonas cepacia

SolubilizationSolubilization

SolubilizationSolubilizationSolubilization (depolymerization)

Gupta et al. (1988)Strandberg and Lewis (1987)

Quigley et al. (1989a)Polman et al. (1994a)Crawford and Gupta (1991)

Basidiomycetous fungiWood-decaying white-rot fungiClitocybula dusenniNematoloma frowardii

Phanerochaete chrysosporium

Trametes (Coriolus) versicolorLitter-decomposing fungiAgrocybe praecoxStropharia rugosoannulataIsolates RBS 1k1, RBS 1b1

Wood-decaying brown-rot fungiPoria monticola

DepolymerizationDepolymerizationSolubilization, depolymerizationSolubilization, utilization, depoly-merization

DepolymerizationDepolymerizationDepolymerization

Solubilization, utilization

Ziegenhagen and Hofrichter (1998)Hofrichter and Fritsche (1996)Torzilli and Isbister (1994), Ralphand Catcheside (1994)Cohen and Gabriele (1982), Frost(1996), Fakoussa and Frost (1999)Steffen et al. (1999)Hofrichter and Fritsche (1996)Willmann (1994), Willmann andFakoussa (1997a,b)

Cohen and Gabriele (1982)

Deuteromycetous and ascomycetous fungiAlternaria sp.Aspergillus terreusFusarium oxysporumNeurospora crassaPaecilomyces spp.Penicillium citrinumTrichoderma atroviride

SolubilizationUtilizationSolubilizationSolubilizationSolubilization, utilizationSolubilization, utilizationSolubilization

Hofrichter et al. (1997b)Ward (1985)Hölker et al. (1995)Patel et al. (1996)Ward (1985), Scott et al. (1986)Polman et al. (1994b)Hölker et al. (1997b, 1999a)

Yeast-like fungiCandida bombicolaCandida sp.Candida tropicalis

SolubilizationUtilizationUtilization

Breckenridge and Polman (1994)Ward (1985)Kucher and Turovskii (1977)

Zygomycetous fungiCunninghamella sp.

Mucor lausannesis

Solubilization

Utilization

Ward and Sanders (1989), Ward(1993)Ward (1985)

1 Enriched and isolated from brown coal from an open-cast mining region.