Potential for Bioremediation of Xenobiotic Compounds by the White

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TOPICAL PAPER Potential for Bioremediation of Xenobiotic Compounds by the White-Rot Fungus Phanerochaete chrysosporium Andrzej Paszczynski” and Ronald L. Crawford Institute for Molecular and Agricultural Genetic Engineering (IMAGE) and Center for Hazardous Waste Remediation Research, University of Idaho, Moscow, Idaho 83844-1052

The white-rot fungi produce a n unusual enzyme system, characterized by a specialized group of peroxidases, that catalyzes the degradation of the complex plant polymer lignin. This ligninolytic system shows a high degree of nonspecificity and oxidizes a very large variety of compounds in addition to lignin. Among these compounds are numerous environmental pollutants. Thus, the white-rot fungi show considerable promise as bioremediation agents for use in the restoration of environments contaminated by xenobiotic molecules. One white-rot fungus, Phanerochaete chrysosporium, has been studied in great detail with regard to ligninolytic enzymes and the degradation of anthropogenic chemicals. It has been widely promoted a s a bioremediation agent. This article examines literature concerning the degradation of xenobiotic compounds by Phanerochaete chrysosporium and attempts to critically assess this organism’s real potential as a bioremediation tool.

Contents Chlorinated Organic Compounds 368 Simple and Polycyclic Aromatic 371 Hydrocarbons Dyes 371 Lignocellulosic Materials 372 Nitro-SubstitutedCompounds 373 Modified Polymers 374 Other Compounds 375 Utility of Phanerochaete chrysosporium 375 in Bioremediation The white-rot fungi are unusual among microorganisms in that they are able to mineralize all components of native lignin to carbon dioxide and water (Eriksson et al., 1990). Lignin is thought to be the second most abundant organic substance in the biosphere, surpassed only by cellulose. This structural polymer of vascular plants consists of an amorphous network of phenylpropanoid subunits linked by a variety of stable carboncarbon and carbon-oxygen bonds that resist attack by most microbes. White-rot fungi, such as the hymenomycete Phanerochaete chrysosporium Burds, however, produce numerous extracellular lignin peroxidases and/ or manganese peroxidases that are able to initiate the depolymerization and degradation of lignin and its oligomers and monomers (Hammel et al., 1993; Warishi et al., 1991). Phenol oxidases such as laccase, which is present in many white-rot fungi but not in P. chrysosporium, are also thought to participate in the oxidative degradation of lignin (Reinhammar, 1984; Morohoshi, 1991; Thurston 1994). Ligninases use HZOZas the terminal acceptor of electrons removed from lignin or other substrates, while laccases use 0 2 . Environmental scientists have become increasingly 8756-7938/95/3011-0368$09.00/0

interested in the most studied of the lignin-degrading white-rot fungi, Phanerochaete chrysosporium, for applications such as the cleanup of toxic organic chemicals in soils and waters. Because the lignin degradation system of P. chrysosporium, which is initiated by lignin peroxidases and manganese peroxidases, is not very substrate specific, the fungus is able to transform and sometimes completely mineralize a variety of persistent environmental pollutants (Bumpus and Aust, 1986). However, environmental pollutants or their degradation products may sometimes inhibit its lignin degradation system (Fenn and Kirk, 1979; Bumpus and Tatarko, 1994). Current lists of the many xenobiotic compounds thought to be degraded by white-rot fungi can be found in recent reviews ( H a m e l , 1992; Field et al., 1993; Barr and Aust, 1994), along with discussions of the mechanisms by which they degrade pollutants (Lamar, 1992; Barr and Aust, 1994). Consequently, we will not discuss these topics in detail here, but instead will examine the organic, man-made chemicals (referred to as xenobiotic compounds) that P. chrysosporium is able to degrade. We will also discuss some of the more interesting degradation pathways and the possible applicability of white-rot fungi as agents promoting the bioremediation of environmental pollution.

Chlorinated Organic Compounds Chlorinated organic compounds have been recognized as environmental hazards for several decades. Examples of chlorinated organic molecules that Phanerochaete chrysosporium is able to degrade are DDT [l,l-bis(4chlorophenyl)-2,2,2-trichloroethane],3,4,3’,4‘-tetrachlorobiphenyl, 2,4,5,2’,4’,5’-hexachlorobiphenyl, 2,3,7,8tetrachlorodibenzo-p-dioxin, lindane (1,2,3,4,5,6-hexachlorocyclohexane), 3,4dichloroaniline,and dieldrin (Bumpus and Aust, 1987a; Bumpus et al., 1985; Morgan et al., 1991). During typical 30-day incubations, mineralization of these compounds to COZhas been reported to

0 1995 American Chemical Society and American Institute of Chemical Engineers

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Ronald L. Crawford is Professor of Microbiology in the Department of Microbiology, Molecular Biology, and Biochemistry at the University of Idaho, where he also holds administrative posts as Co-Director of the Center for Hazardous Waste Remediation Research and Director of IMAGE, the Institute for Molecular and Agricultural Genetic Engineering. His research interests include the biodegradation of hazardous chemicals, the genetics and biochemistry of munitions-degrading microorganisms, microbial immobilization in various matrices, in situ biodegradation of chemical pollutants in groundwater, and subsurface microbiology. He received a B.A. degree in biology from Oklahoma City University and M.S. and Ph.D. degrees in bacteriology from the University of Wisconsin. After 12 years at the Gray Freshwater Biological Institute of the University of Minnesota, he joined the faculty of the University of Idaho in 1987.

Andrzej Paszczynski is a research associate in the Department of Microbiology, Molecular Biology, and Biochemistry at the University of Idaho. His research interests include the biodegradation of recalcitrant dyes, design of biodegradable compounds, properties of ligninases and peroxidases, and biochemistry and molecular biology of higher fungi. Dr. Paszczynski received his M.S. and Ph.D. degrees in biochemistry from the University of Marie Curie-Sklodowska in Lublin, Poland. After several years as a research associate in Poland, he came to the University of Minnesota as a research scientist in 1984 and joined the University of Idaho in 1987.

vary in amount from 1%to 15%. In every case, a majority of the starting compound, if not mineralized, was at least transformed. For example, in an experiment where 4% of an initial DDT substrate was mineralized by P. chrysosporium, more than 75% of the starting compound disappeared from the medium (Bumpus and Aust, 198713). This study also showed that 1,l-dichloro2,2-bis(4-chlorophenyl)ethane(DDE), 2,2,2-trichloro-l,1bis(4-chloropheny1)ethanol(dicafol),and 4,4’-dichlorobenzophenone (DBP) were formed during DDT degradation. The degradation pathway of DDT by P. chrysosporium (Figure 1)differed from that described for bacteria. For example, Alcaligenes eutrophus will hydroxylate and cleave aromatic rings of DDT before chlorine is removed (Nadeau et al., 1994). When the initial concentration of

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DDT was 1.7 ppm, the fungus transformed 50%, but mineralized only 4%, of the DDT during a 30-day incubation period. Additional work showed that lignin peroxidases were involved in DDT catabolism by P. chrysosporium (Bumpus et al., 1985), although this has been questioned by other researchers (Kohler et al., 1988). DDE, one fungal product of DDT, is resistant to microbial degradation and tends to accumulate in animal tissues, inducing cellular cytochrome P-450 activity. Increased activity of P-450 can change steroid hormone levels, thereby interfering with reproduction. In an investigation of the biodegradation of DDE by P. chrysosporium, Bumpus et al. (1993)observed the mineralization of DDE to C02 and proposed a biodegradation pathway for DDT/ DDE (Figure 1). Oxidative dechlorination of aromatic rings catalyzed by lignin peroxidase was shown by Hammel and Tardone (1988), who performed several experiments at pH 3, where chlorinated phenols are insoluble but ligninases are active, by emulsifying the substrates with Tween 80. They proposed a mechanism for 2,4,6-trichlorophenol oxidation by fungal lignin peroxidase, and they observed the oxidative dechlorination of pentachlorophenol (PCP) by crude fungal ligninase preparations. At about the same time, Mileski et al. (1988) used purified ligninase to convert PCP into 2,3,5,6-tetrachloro-2,5-cyclohexadiene-1,4-dione (TCHD). They proposed a metabolic model for PCP degradation by P. chrysosporium whereby PCP can be degraded in two ways: (a)extracellular enzymes first convert PCP to intermediates like TCHD, which are then mineralized to COZ by mycelial enzymes or (b) the fungus directly mineralizes PCP to CO2 without requiring an initial peroxidative reaction (Lin et al., 1990). A silicone membrane biofilm reactor was used to study fungal lignin peroxidase production and PCP degradation. PCP disappeared in the bioreactor at a rate of 10.5 mgwday, 5 times faster than in flasks (Venkatadri et al., 1992). The degradation of PCP and pentachloroanisole (PCA) in soil has also been examined (Lamar et al., 1989,1990). The ability of Phanerochaete species to deplete PCP from soil contaminated with commercial-wood preservatives was confirmed in a field study in which P. chrysosporium and P. sordida removed 88-91% of PCP from soil containing 250-400 ppm of the contaminant. The soil was amended with 2% sterile peat and 3% wood chips overgrown with fungal hyphae (Lamar and Dietrich, 1990). The fbngal treatment of soil contaminated by PCP and polynuclear aromatic hydrocarbons was assessed by the Superfund Innovative Technology Evaluation (SITE) program of the U.S. Environmental Protection Agency (EPA). The field demonstration showed low levels of

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on cation radical structures was thought to be responsible for ring cleavage, as had been postulated earlier by Hammel et al. (1986). I Polychlorinated biphenyls (PCBs), used extensively in industrial applications until the mid-l970s, are extremely unreactive, heat-stable chemicals that are poor conductors of electric current (Hutzinger et al., 1974). Mineralization of polychlorinated biphenyls by P. chrysosporium was first reported by Eaton (1985), who investigated the degradation of Aroclor 1254, a recalcitrant mixture of biphenyls chlorinated up to 54% (w/w) and containing 4-7 chlorines per biphenyl molecule. After a 5-weeks incubation, the fungus mineralized 7% of [U-14ClAroclor 1254 from 300 ppb present in the culture. Mineralization of biphenyl, 2-chlorobiphenyl, and 2,2',4,4'-tetrachlorobiphenyl by submerged cultures of the fungus was also Figure 2. Pathway for the degradation of 2,7-dichlorodibenzoinvestigated (Thomas et al., 1992). Degradation rates p-dioxin, modified from Valli et al. (1992b). were inversely related to the number of chlorine atoms on a biphenyl molecule. Only 1%of tetrachlorobiphenyl leaching of these compounds to the underlying soils and was converted to COZ,but over 40% was found incorpolow rates of volatilization to the atmosphere (Fungal rated into mycelia after 32 days of culture, in experiments Treatment Bulletin, 1993). The data reported from the using 2 ppm of 2,2',4,4'-tetrachlorobiphenyl. This restudy appeared to confirm the feasibility of using Phansearch suggested that a mycelial mat could be used as a erochaete for the bioremediation of soils containing PCP. biological filter to treat low concentrations of PCBs. Lin et al. (1991) coimmobilized Phanerochaete chrysoThe disappearance of PCBs during simultaneous treatsporium mycelium and activated carbon. The mixture ment with UV irradiation and the UV-resistant P. degraded PCP more efficiently than similar mixtures that chrysosporium strain BU-1 was described by Katayama were not immobilized. Only about 10% of the 59 pmol/L and Matsumura (1991). For 2 h per day, cultures were PCP provided was mineralized to COZin 300 h. irradiated with UV light (300 or 254 nm). Up to 4-fold Valli and Gold (1991), observing that P. chrysosporium enhancements of TCDD mineralization were observed degraded 2,4-dichlorophenol by cycles of oxy reduction after 40 days of incubation. Improvements in the degand methylation, recently suggested a pathway involving radation of DDT (254 nm), TCDD (300 nm), Heptachlor the oxidative dechlorination of 2,4-dichlorophenolto yield (254 nm), TCB (3,4,3',4'-tetrachlorobiphenyl) (300 nm), 1,2,3,4-tetrahydroxybenzene,which was then cleaved to and Aroclor 1254 (300 nm) were observed after 28 days produce, after subsequent oxidation, malonic acid. Under of incubation. A rejuvenating effect of glucose on the secondary metabolic conditions (low nitrogen in the combined degradative actions of the fungus and UV medium), P. chrysosporium rapidly mineralized 2,4,5irradiation on TCB was observed. trichlorophenol. The ligninolytic system of this fungus is repressed in medium containing significant amounts P. chrysosporium mineralized 2,4-dichlorophenoxyacetof nitrogen (Keyser et al., 1978). Joshi and Gold (1993) ic acid (2,4-D), one of the most widely used phenoxyalproposed a degradation pathway for this compound that kanoic herbicides, and also 2,4,5-T (2,4,5-trichloropheinvolved multiple oxidative dechlorinations catalyzed by noxyacetic acid). The mass balance analysis of [U-ringfungal peroxidases, followed by quinone reduction, yieldl4C1-2,4-Dmineralization in malt extract cultures showed ing 1,2,4,5-tetrahydroxybenzene.In this pathway, unlike 39% released as I4CO2and 6% assimilated by the mycethose known in bacteria, all three chlorine atoms were lium after 40 days of cultivation (Yadav and Reddy, thought to be removed before ring cleavage occurred. Two 1993b). Degradation of these compounds by the fungus recent reports discuss the involvement of extracellular may have limited application since pure cultures of protein and mycelium in the degradation of 2,4,6-trichlobacteria are known to mineralize 2,4-D much faster. For rophenol by P. chrysosporium (Armenate et al., 1994) and example, bacteria needed only 5 days to bring a 1000 ppm the isolation and characterization of 1,2,3-trihydroxy2,4-D concentration to 0 ppm, while the fungus needed benzene 1,2-dioxygenase from the fungus (Rieble et al., 21 days to degrade 5 ppm of 2,4-D by only 80% CYadav 1994). and Reddy, 1993b; Haugland et al., 1990). Polychlorinated dibenzo-p-dioxins are recognized as The effect of chloromethane on veratryl alcohol and environmental hazards due to their acute toxicity to lignin peroxidase production was examined by Harper animals and humans (Schwetz et al., 1973). In an et al. (1990, 1991). Veratryl alcohol and ligninolytic investigation of the activation of dibenzo-p-dioxin and peroxidases are elements of the extracellular oxidative 2-chlorodibenzo-p-dioxins by ligninase, cation radical system of the fungus. The authors concluded that CH3formation was observed, supporting the conclusion that C1 could be used as a methyl donor for veratryl alcohol this type of mechanism can be generally applied to the synthesis without any significant release of the compound degradation of xenobiotic compounds (Hammel et al., 1986). Mineralization of 2,3,7,8-tetrachlorodibenzo-p- to the external environment. The ability of P. chrysosporium to degrade chlorinated organic compounds could dioxin (TCDD) by P. chrysosporium has been reported be viewed in its broader aspects. Since the production (Bumpus et al., 19851, and degradation mechanisms for of chlorinated organic compounds is very common among 2,7-dichlorodibenzo-p-dioxin(PCDD) have been eluciwood and forest litter-degrading fungi (Jong et al., dated (Valli et al., 1992b). The proposed multistep 1994b), the ability to degrade them by one member of pathway (Figure 2) involved the degradation of PCDD the family is not surprising. Jong et al. (1994a) recently to 1,2,4-trihydroxybenzene via oxidation-reduction and described a possible physiological role of chlorinated aryl methylation reactions. The involvement of lignin peralcohols biosynthesized de novo by the white-rot fungus oxidases, manganese peroxidases, and intracellular enBjerkandera sp. zymes was postulated. The nucleophilic attack of water

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Simple and Polycyclic Aromatic Hydrocarbons Aromatic hydrocarbons are characterized by the presence of a benzene ring. Polyaromatic hydrocarbons (PAHsj are formed during pyrolytic processes or incomplete combustion of organic substrates. Sources of PAHs are exhaust fumes from industrial and private furnaces, car exhaust gases, and tobacco smoke. For about 100 years, chemicals as byproducts in the primary processing of coal to metallurgical coke have been the main industrial source of aromatic compounds used as intermediates in organic synthesis. PAHs are widespread environmental pollutants. Degradation of benzene, toluene, ethylbenzene, and xylenes (BTEX), a group of common organopollutants derived from gasoline and aviation fuels, was observed in work with P. chrysosporium. Degradation of these compounds occurred under nonlignolytic culture conditions in a nitrogen-rich medium. Impressive degradation was found after only 5 days of incubation. Benzene was reduced by 18%, toluene by 41%, ethylbenzene by 99%,o-xylene by 49%, and m-xylene andp-xylene by 67% (Yadav and Reddy, 1993a). These findings suggested to the authors that white-rot fungi could be used for the efficient degradation of complex mixtures of aromatic hydrocarbons at petroleum-contaminated sites. Degradation experiments with the polycyclic aromatic hydrocarbon pyrene showed that pyrene-l,6-dione and pyrene-1,8-dionewere major products of pyrene oxidation by P. chrysosporium. The quinone oxygen introduced into the pyrene molecule came from water (Hammel et al., 1986). At the same time, Haemmerli et al. (1986) showed that ligninase is able to oxidize benzo[alpyrene to 1,6-, 3,6-, and 6,12-quinones. The proposed pathway involved attack by water and oxygen molecules on cation radical derivatives of the substrate. P. chrysosporium rapidly oxidized [14Clbenzo[alpyreneto [14ClCO~, using its ligninolytic enzymes, and the formation of numerous water- and organic solvent-solubleproducts was observed (Sanglard et al., 1986). The oxidation of pyrene was enhanced in the presence of 3,4-dimethoxybenzylalcohol (veratryl alcohol), a fungal secondary metabolite that has a protective role toward lignin peroxidases (Cancel et al., 1993). Metabolism of phenanthrene by the fungus has also been described (Bumpus, 1989; Sutherland et al., 1991). Bumpus (1989) demonstrated the ability of the fungus to degrade PAHs present in anthracene oil (a distillation product of coal tar). At least 22 major components from anthracene oil were degraded by 70100% after 27 days of incubation. Approximately 60% of anthracene was degraded after 21 days in stationary cultures of P. chrysosporium, Coriolopsis polyzona, Trametes versicolor, and Pleurotus ostreatus (Vyas et al., 1994). A mineralization study of [l4C1phenanthrenein nutrient nitrogen-limited cultures of P. chrysosporium showed about 8% conversion into l4CO2,while a large portion of the remaining radioactivity was composed of polar metabolites of [l4C1phenanthrene. An initial degradation pathway for phenanthrene in nitrogen-rich culture medium was proposed (Sutherland et al., 1991). The involvement of monooxygenase and epoxide hydrolase activity was postulated, since lignin peroxidase activity was not detected in the culture during this experiment. Low-nitrogen cultures showed a significant enhancement in the mineralization of phenenthrene and structurally related compounds over high-nitrogen cultures (Hammel et al., 1992). An initial pathway for the degradation of phenenthrene in ligninolytic cultures was proposed. The fungus oxidized phenanthrene and phenanthrene-9,lOquinone at their C-9 and C-10 positions to give the ringfission product 2,2'-diphenic acid (Hammel et al., 1992).

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Results obtained by Dhawale et al. (19921, however, suggest that the degradation of phenanthrene may occur under ligninolytic as well as nonligninolytic conditions of fungal growth. The mechanism of oxidation of PAHs by lignin peroxidase was reviewed by Hammel(1992). To determine whether P. chrysosporium could effectively operate in an actual field sample of oil tarcontaminated soil, the following study was performed by Brodkorb and Legge (1992). Under aerobic conditions, the native soil flora mineralized 20% of [l4C1phenanthrene, but the addition of P. chryrosporium enhanced mineralization to 38% in 21 days of incubation. The authors concluded that with further refinement, this system could prove effective for the bioremediation of soil contaminated with PAH compounds. A pathway for the degradation of phenanthrene under nonligninolytic and ligninolytic conditions of fungal metabolism and the role of ligninase in linking both have been proposed (Tatarko and Bumpus 1993; Figure 3). Moen and Hammel(1994) reported that lipid peroxidation by manganese peroxidase is the basis for phenenthrene oxidation by intact fungi. The detailed mechanism of these reactions awaits explanation.

Dyes The dyestuff, textile, paper, and leather industries, which are the major producers and users of dyes, produce effluents that are usually resistant to biological treatment. Glenn and Gold (1983) first reported on the decolorization of several polymeric dyes by P. chrysosporium more than 10 years ago. Experiments with the dyes Poly B-411, Poly R-481, and Poly Y-606 indicated that the dyes are substrates for lignin-degrading enzymes. The authors suggested that polymeric dyes might be useful as mutant selection tools. Decolorization of Remazol brilliant blue R, a dye whose structure resembles that of Poly B, was reported by Ulmer et al. (1984). The decolorization of the dye Poly B-411 [poly(vinylamine sulfonate)-anthroquinonel and the correlation of color loss with lignin degradation by fungi were investigated by others (Chet al., 1985; Platt et al., 1985). Good correlations between the mineralization of I4C-

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hydroxyl and azo groups in the 1,2-positions were delabeled lignin and decolorization of dye have been graded most rapidly. established. Decolorization of Poly R-478 was used for screening 170 strains of white-, brown-, and soft-rot and Paszczynski and Crawford (1991) found that veratryl alcohol was involved in the oxidation of some azo dyes xylophilous fungi for their peroxidase and phenol oxidase activity. The authors concluded that such a method was by lignin peroxidase. Lignin peroxidase compound I not sensitive enough and that no simple relation existed oxidized azo dyes and was converted to compound 11, which was then reduced by veratryl alcohol t o help between the presence pf manganese peroxidase or lignin complete the catalytic cycle of the enzyme. Ollikka et peroxidase and the decolorization of the dyes (Freitag and al. (1993) found that dyes belonging to four different Morrell, 1992). However, others have shown good corgroups-polymeric, azo, heterocyclic, and triphenylrelations between dye decolorization and peroxidase and methane-were decolorized by three major lignin peroxiHzO2 production. The Poly R decolorization activity of 3 dase isoenzymes (H2, H7, and H8). Although purified of 67 new fungal strain isolates was significantly higher enzymes decolorized all dyes investigated, with isoenthan that of P. chrysosporium (De Jong et al., 1992).P. zymes veratryl alcohol was required for decolorization in chrysosporium degraded (decolorized)triphenylmethane some cases, but not in others (Ollikka et al., 1993). We dyes, including crystal violet (N,N,”,”,N”,N”-hexahave now elucidated some of the major chemical steps methylpararosaniline), pararosaniline, cresol red, broinvolved in the degradation of azo dyes by lignin peroximophenol blue, ethyl violet, malachite green, and brildases and manganese peroxidases (Goszczynski et al., liant green (Bumpus and Brock, 1988). Three identified 1994; Pasti-Grigsby et al., 1994a), as shown in Figure 4. metabolites of crystal violet, N,N,”,”,N”-pentamethSpadaro et al. (1992) demonstrated that P. chrysospoylpararosaniline, N,N,”,”-tetramethylpararosaniline, rium is capable of mineralizing a variety of azo dyes and N,”,N”-trimethylpararosaniline, were formed by without sulfo groups and azo dye Disperse orange 3 with sequential N-demethylations of the parent compound. a nitro group. From this evidence, it was suggested that fungal ligniDecolorization of the effluent from a pigment plant by nase may catalyze N-demethylation reactions. However, another white-rot fungus,Pycnoporus cinnabarinus, has since a nitrogen-rich medium was used, crystal violet been reported (Schiephake et al., 1993). P. cinnabarinus degradation by P. chrysosporium may have involved a rapidly decolorized and clarified the wastewater samples nonligninolytic enzyme system. passed through a packed-bed bioreactor. Cripps et al. (1990) added an entirely new family of Pasti-Grigsby et al. (1994b) examined a number of azo dyes, the azo dyes, to the long list of organic compounds dyes for their potential use as substrates for assaying attacked by P. chrysosporium. Azo dyes, the largest class lignin peroxidases and manganese peroxidases of whiteof commercially produced dyes, are not readily degraded rot fungi. They found that the novel dyes 3,bdimethylby microorganisms (Kulla et al., 1984; Wuhrmann et al., 4-hydroxyazobenzene-4‘-sulfonic acid and Orange I both 1980). Acid azo dyes are characterized by the presence served well for specific assays of Mn(I1) peroxidase, as of a chromophoric azo group whose nitrogen atoms are did 3,5-difluoro-4-hydroxybenzene-4’-sulfonic acid and linked to sp2-hybridized carbon atoms of the aromatic Orange I1 for specific assays of lignin peroxidases. These ring, which may also carry sulfonic acid groups. These dyes therefore are potentially useful for both enzyme compounds are used for dyeing and printing natural and work and isolation of specific types of peroxidase musynthetic fibers, leather, furs, and paper and for other tants. coloring purposes (Hunger et al., 1985). Sulfo and azo Capalash and Shrama (1992) found that both the groups are not naturally occurring; thus, sulfonated azo ligninases of P. chrysosporium and absorption biomass dyes are recalcitrant to biodegradation. The dyes rewere responsible for decolorizing8 out of 18 commercially ported (Cripps et al., 1990) to be degraded by the fungus include Azure B [3-(dimethylamino)-7-(methylamino)- used dyes. From 40 to 73% decolorization was detected in 5-day-old cultures after 72 h of treatment. In their phenothiazin-5-ium chloride], Tropaeolin 0 [4-[(2,4-didescription of both ligninase- and horseradish peroxidasehydroxypheny1)azolbenzenesulfonic acid], Orange I1 [4-[(2catalyzed desulfonations of 3,5-dimethyl-4-hydroxybenhydroxy-l-naphthyl)azolbenzenesulfonic acid], and Congo Red [3,3’-(1,l’-biphenyl-4,4’-diazo)bis(4-amino-l-naphtha-zenesulfonic acid and 3,5-dimethy1-4-aminobenzenesulfonic acid, Muralikrishna and Renganathan (1993)postulated lenesulfonic acid)]. the formation of 2,6-dimethyl-l,4-benzoquinone and sulWe have shown that the susceptibility of azo dyes to fide anion. Further research is needed to confirm sulfide degradation by P. chrysosporium and Streptomyces speformation in these reactions. cies can be increased by attaching guaiacyl substituents New dye structures that P. chrysosporium can degrade similar to structures found in lignin (Paszczynski et al., are continually being found. Recent reports have de1991b). We demonstrated that P. chrysosporium was scribed the degradation of methylene blue by a crude able to degrade azo dyes in concentrations up to 300 ppm extracellular medium of P. chrysosporium Wing and (Paszczynski et al., 1991a), whereas only about 50 ppm Neb, 1991) and the aerobic biodegradation of the exotic was degraded by several strains of Streptomyces (Pasti dye Rose Bengal (tetrachlorotetraiodofluorescein) (Gogna et al., 1991). Our general observation was that degradet al., 1991). ability depended on the substitution pattern of the Lignocellulosic Materials aromatic ring, with lignin-like structures improving biodegradability. Recently, we confirmed the mineralizaIn Europe and Scandinavia, most chemical wood pulp tion of sulfonated azo dyes by P. chrysosporium using is produced by the sulfate pulping process, during which U-ringJ4C-labeled compounds, including both dyes and most of the wood‘s lignin is partially degraded and free sulfanilic acid. In shaken culture the fungus was dissolved from the cellulose fiber. The residual lignin able to release up to 35% of the label from certain dyes (about 50 kg/metric ton of pulp) is removed in variety of as I4CO2after 21 days of incubation. We again observed bleaching sequences. For example, when a six-step bleaching and washing process is applied, it uses chlothat very specific changes in the molecular structure of rine, chlorine dioxide (usually twice), and hypochlorite azo dyes enhanced their biodegradability (Paszczynski et al., 1992; Pasti-Grigsby et al., 1992). Dyes with for bleaching, followed by two alkali extractions (Lindstrom et al., 1981). Chlorine bleaching produces waste naphthalene rings were also degraded, and those with

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Figure 4. Degradation pathway of azo dyes (Goszczynski et al., 1994).

streams containing low concentrations of many chlorinated compounds. In the United States, the primary pulping process involves dissolution of the lignin from wood a t high temperatures using solutions of NaOH and NazS (the Kraft process). Pulps produced by Kraft pulping are bleached in part to remove residual lignin. The degradability of toxic chlorinated Kraft bleach mill effluents containing high molecular weight chlorolignin and low molecular weight chlorinated organic compounds was investigated using white-rot fungi. Bleach plant effluent from the first alkali extraction stage after chlorine bleaching was decolorized by P. chrysosporium. The degradation showed no selectivity with regard to molecular weight, and partial dechlorination of the polymers was observed. Ligninolytic culture conditions were required for the degradation of bleach plant products (Sundman et al., 1980; Eaton et al., 1980). A number of larger scale bioprocesses, including continuous treatments, have been developed for the fungal treatment of bleach plant effluents. Different reactor designs have been developed, including the mycelial color removal (MyCoR) and related MYCOPOR processes. These have been assessed using P. chrysosporium as the biocatalyst (Campbell et al., 1982; Messner et al., 1990). EMuent color reductions of up to 80% have been observed in 2 days of operation (Chang et al., 1983). Another whiterot fungus, Coriolus versicolor, was also used successfully for this purpose (Archibald et al., 1990). The fungal and bacterial enzymes with potential for use in wastewater treatment in the pulp and paper industry include white-rot fungal peroxidases and oxidases, as discussed by Hakulinen (1988). High molecular weight chlorolignins ( M W 7 30 000) from bleach plant effluents were depolymerized and decolorized in the

presence of Mn3+stabilized with organic acids or manganese peroxidase, MnZT,and hydrogen peroxide (Lackner et al., 1991). Ferrer et al. (1991) immobilized a type I11 lignin peroxidase from Chrysonilia sitophila and used it to remove color from Kraft effluent. The fungal enzyme was only slightly more active than immobilized horseradish peroxidase and about half as active as whole fungal cultures. Only about 30% of the color was removed in 72 h by the immobilized system. This system appears to be significantly less promising than similar Phanerochaete processes. The authors cited numerous additional papers concerning the use of peroxidases and laccases to remove color from pulp mill wastes. Some aspects of larger scale aerobic and anaerobic biological treatment of bleached pulp effluents were discussed by Boman et al. (1988). A recent review (Bajpai and Bajpai, 1994) compared the ability of different organisms to decolorize pulp and paper wastewater. Two white-fot fungi, P. chrysosporium and C. versicolor, seem to be suitable for the efficient degradation of these effluents, but requirements for high oxygen tension and a growth substrate constrain the practical application of fungal decolorization.

Nitro-Substituted Compounds Nitro-substituted compounds belong to a larger group of explosive chemicals that also encompasses nitrate esters and nitro amines, along with derivatives of chloric acid, perchloric acid, azides, and other compounds. The toxicity and mutagenicity of nitroaromatics and their recalcitrance to biodegradation underlie concerns about their environmental fate (Kaplan, 1992). It has been shown that P. chrysosporium is able to degrade nitroarenes. The biodegradation of 2,4,6-trinitrotoluene (TNT)

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by the fungus was first reported in 1990 by Fernando et al. When a low concentration of TNT (1.3 mgL) was used, approximately 35% of [l*C]TNT was mineralized in liquid culture. Only about 7% was mineralized in soil supplemented with fungal-colonized corncobs after 18 days of incubation. When higher concentrations of TNT were used (100 mg/L, 10000 mgkg in soil), approximately 85% of the TNT was removed; however, only 19% was mineralized (Fernando et al., 1990). In a succeeding report, the same group showed that the fungus was able to degrade both TNT and the nonaromatic explosive royal demolition explosive (RDX, hexahydro-,1,3,5-trinitro1,3,5-triazine) in contaminated water and soil (Fernando and Aust, 1991). The authors suggested that treatments utilizing P. chrysosporium might be effective and economical methods for the remediation of TNT- and RDXcontaminated water and soil (Lebron et al., 1992). Spiker et al. (1992) showed that TNT, RDX, and HMX (octahydro-1,3,4,7-tetranitro-l,3,5,7-tetrazocine) had inhibitory effects on the degradation of TNT. P. chrysosporium was able to degrade TNT at up to 100 ppm, but growth was seriously inhibited. Concentrations in the range of 20 ppm of TNT did not inhibit fungal growth (Spiker et al., 1992). The negative effect of increasing concentrations of TNT on its degradation by Phanerochaete chrysosporium was confirmed recently (Michels and Gottschalk, 1994). 2-(Hydroxyamino)-4,6-dinitrotoluene and its isomer 4-(hydroxyamino)-2,6-dinitrotoluene, early intermediates formed in TNT degradation, were shown to inhibit the veratryl alcohol oxidase activity of lignin peroxidase. Increases in the concentration of veratryl alcohol in the reaction mixture prevented the loss of peroxidase activity. The initial TNT degradation products and the inhibitory effect of 4-(hydroxyamino)2,6-dinitrotoluene on the ligninase H8 veratryl alcohol oxidative reaction were confirmed by Bumpus and Tatarko (1994). Biotreatment of red water, a hazardous waste stream from explosives manufacture, was also investigated on the laboratory scale with Phanerochaete. HPLC analyses showed decreases in the concentration of 2,4,6-trinitrobenzenesulfonic acid and removal of TNT and DNT isomers (2,4-dinitrotoluene and 2,6-dinitrotoluene). Pretreatment with ultraviolet light profoundly increased the observed biodegradation rates (Tsai, 1991). Other research showed that biotreatment of pink water, another waste stream associated with munitions processing, with the white-rot fungus might be a cost-effective alternative to carbon absorption. A n experiment was described in which P. chrysosporium immobilized on a rotating biological contactor was able to remove 99.5% of TNT at 120-175 ppm and RDX at 25 ppm from contaminated water (Sublette et al., 1992). Very recent research has shown that the plasma membrane redox system of P. chrysosporium may be involved in the reduction of TNT. The rate of TNT, 2-AmDNT (2-amino-4,6-dinitrotoluene), and 4-AmDNT (4-amino-2,6-dinitrotoluene) reduction correlated directly with mycelial mass. Toxicity was inversely related to the amount of fungal hyphae present (Stahl and Aust, 1993a)b). However, mycelia (Stahl and Aust, 1993a) were added at the rate of 18 g/L (dry Wuvol), at TNT concentrations of 10-150 mg/L. It would appear that, to treat contaminated soils, additions of dry mycelia would exceed the weight of soil treated at these mycelia: TNT ratios. Under ligninolytic conditions, P. chrysosporium mineralized 2,kdinitrotoluene (2,4-DNT). The pathway proposed for the degradation for 2,4-DNT (Figure 5) involved an initial reduction of one nitro group to an aromatic amine, making the pollutant susceptible to


c

OH

Figure 5. Proposed pathway for the degradation of 2,4dinitrotoluene, modified from Valli et al. (1992a).

peroxidase attack. The product, 4-nitro-1,2-benzoquinone, underwent reduction and methylation, generating additional substrates for peroxidases. A second nitro group was removed by alternative action of ligninase or Mn(I1) peroxidase on 4-nitro-1,2-hydroquinone or 1,2dimethoxy-4-nihbenzene, respectively (Valli et al., 1992a).

Modified Polymers Aqamand and Sandermann (1985,1986) demonstrated that P. chrysosporium not only will degrade pure lignin but will also simultaneously degrade chloroaniline bound to lignin. The metabolites formed from wheat lignin containing covalently bound 4-chloroaniline and 3,4dichloroaniline were mineralized as readily as chloroaniline-free lignins. This suggested that the fungal ligninolytic system recognized and degraded both natural lignins and those modified by the incorporation of xenobiotic contaminants. Turnover times in soil for humic acids are estimated to range from hundreds to thousands of years (Stout et al., 19811, but Haider and Martin (1988), in their investigation of the degradation of humic acid polymers by P. chrysosporium, found that the fungus degraded humic acids, as well as xenobiotic molecules bound to humic acids, and that the favored culture conditions for degradation were similar to those required for lignin degradation. Manganese peroxidase seemed to play a major role in the initial breakdown and decolorization of high molecular weight chlorolignin in bleach plant eMuents (Lockner et al., 1991). Although polystyrene [poly(l-phenylethylene)]is highly resistant to microbial attack (Kaplan et al., 19791, fungal biodegradation of lignopolystyrene graft copolymers has been reported (Milstein et al., 1992). White-rot basidiomycetes, including P. chrysosporium, were able to degrade LPS (lignin-polystyrene) graft copolymers. In tests with copolymerization products containing 10, 32,

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and 50% lignin, after 3 weeks of incubation P. chrysosporium degraded 40% of the polystyrene and 80% of the lignin from LPS containing 32% lignin. Only about 4% of pure polystyrene was degraded by the fungus in a parallel experiment. Production of lignin peroxidases and Mn(I1) peroxidases was observed during these experiments (Milstein et al., 1992). Polyethylene containing 6% starch was incubated with P. chrysosporium in another study (Lee et al., 1991). The fungus did not degrade polyethylene, but this may have been due to the high nitrogen content of the medium used, which is known to inhibit the ligninolytic system of most whiterot fungi. P. chrysosporium may also be able to transform other naturally occurring polymers such, as lignite and subbituminous coals (Wondrack et al., 1989). Incubation of a water-soluble fraction of subbituminous or lignite coal with a partly purified preparation of ligninase and manganese peroxidase resulted in substantial depolymerization of the coal, although no releases of monomeric compounds were observed. The oxidation of thianthrene heterocyclic sulfur compounds reported to be part of the coal and petroleum macrostructure was also reported (Schreiner et al., 1988). Ligninase oxidized thianthrene to thianthrene monosulfoxide.

Other Compounds In a report on the metabolism of cyanide, Shah et al. (1991) showed that P. chrysosporium was able to mineralize [14C]KCN to 14C02 and that lignin peroxidase oxidized cyanide to cyano radical in the presence of hydrogen peroxide. Spores were found to be more sensitive to cyanide than were mycelial cells. The degradation of a fluorinated organic compound by P. chrysosporium was recently described by Abernethy and Walker (1993). The degradation of hydramethylenon, (HMN, an amidohydrazone-type insecticide) by P. chrysosporium yielded two major breakdown products, p-(trifluoromethy1)cinnamic acid and p-(trifluoromethy1)benzoic acid, and a tentative pathway for HMN breakdown was described. The oxidation of halides (iodine, bromide, and chloride) by lignin peroxidases and their subsequent reduction by EDTA or HzOz was demonstrated recently (Shah and Aust, 1993).

Utility of Phanerochaete chrysosporium in Bioremediation The biodegradative range of Phanerochaete chrysosporium has yet to be widely applied to environmental restoration at contaminated sites. The activities of whiterot fungi appear to be too complex and unpredictable for practitioners of environmental restoration to be confident of success in the field. This is particularly true when inoculating from liquid culture into soil. More work is needed to develop reliable application methods and to understand degradation mechanisms, as well as the physiological and enzymatic regulation involved in the mineralization of particular pollutants. It is unfortunate that a microbial system of such amazing versatility and promise has not yet shown convincing utility within the bioremediation industry. The degradation by P. chrysosporium of compounds like DDT (Bumpus and Aust, 1987b) is too incomplete even under ideal laboratory conditions to move this technology into the field until conditions conducive to complete removal of the contaminant are found. At present, only about 4% of the DDT in medium is mineralized and only 50% transformed in 30 days, starting at a rather low concentration of 1-2 ppm. Environmentally undesirable products like DDE and

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DDB are formed, and it is unclear whether these biotransformation products can be degraded sufficiently for acceptable environmental application (Bumpus et al., 1993). For nitroaromatic compounds like trinitrotoluene (TNT), the situation is similar. It is conceivable that white-rot fungal systems might yet be developed to treat munitions compounds in soil and water, but numerous problems still require attention before this can happen. Conditions must be found that eliminate significant accumulations of potentially toxic and mutagenic biotransformation products like aminotoluenes or compounds such as (hydroxyamino)nitrotoluenes(Michels and Gottschalk, 1994), which inactivate ligninases. Also, these systems should be shown to degrade all the components in the complex mixtures of munitions residues and the products of their environmental weathering, which are found, often at very high concentrations (10-50000 ppm or more), in decades-old munitions dumping grounds (Spiker et al., 1992). Systems also should not require the addition of massive amounts of lignocellulosiccarriers andor mycelia material to overcome toxicity problems (Stahl and Aust, 1993a,b). Some uses of white-rot fungi do appear to be ready for industrial application. The almost 2-decade-old technology of decolorizing pulp mill bleach effluents by fungi (Campbell et al., 1982; Chang et al., 1983; Messner et al., 1990; Archibald et al., 1990; Ander, 1990) is overdue for use in solving some of the environmental problems of the paper industry. The “rediscoveres” of the whiterot fungi as degraders of tough environmental pollutants have not always given these earlier pioneering studies proper recognition. Unfortunately, the pulp and paper industry is one that has been slow to employ new technologies in its long-established process streams with their associated heavy capital investment. Thus, the industry probably will fully adopt new bleaching techniques that replace the use of chlorine (e.g., oxygen-based systems) before it turns to the white-rot fungi to solve the problems created by chlorination or to replace the pulping process itself with the long sought biological pulping alternative (Boman et al., 1988). White-rot fungi have been evaluated for their potential in papermaking and as additives to wood pulp (Johnson and Carlson, 1978). Up to 10% of fungal mycelia might be incorporated into wood fiber without serious deleterious effects on paper strength. Studies indicate that fungal pretreatment of coarse thermomechanical pulp (TMC) with P. chrysosporium in the presence of glucose can substantially reduce the energy requirements for secondary refining of TMP (Bar-lev et al., 1982). A review of the biological treatment of lignocellulosic materials can be found in the text by Coughlan and Collaco (1991). On the basis of treatability studies done under conditions that approximate those at hazardous waste sites, and studies done under conditions that might be reasonably reproduced under industrial constraints, there do appear to be niches for Phanerochaete chrysosporium and other white-rot fungi in the hazardous waste treatment industry. However, most of the highly publicized potential of this fungus is as yet unrealized. This is true in part because research into xenobiotic degradation by white-rot fungi has focused primarily on P. chrysosporium, a microorganism that requires low nitrogen for the onset of lignin degradation and special culture conditions (often static). Not only do other white-rot fungi need to be screened for the ability to degrade pollutants under a wide range of environmental conditions (Field et al., 19931, but genetic manipulation of P. chrysosporium toward the creation of a ligninase-overproducingmutant or secondary metabolism-deregulated strains could also


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be beneficial for future applications of this organism in the commercial remediation of pollutants (Orth et al., 1991; Tien and Myer, 1990; Kakar, 1994). Furthermore, optimization studies show that biodegradation rates increase manyfold when P. chrysosporium is immobilized. In one packed-bed reactor (1800 cm2), the fungus was able to reduce the 2-chlorophenol concentration from 500 ppm at the inlet to 0.1 ppm at the outlet of the reactor (Lewandowski et al., 1990). This is a further indication of the need for improved reactor designs for hazardous waste treatment using white-rot fungi (Venkatadri et al., 1992). Some of the methods in which the irradiation of recalcitrant compounds (e.g., the PCBs and chlorinated dioxins) is combined with fungal treatments seem especially promising (Tsai, 1991; Katayama and Matsumura, 1991). These novel approaches deserve increased support for testing through the scale-up and development stages. The treatment of soils contaminated by wastes of the wood preservatives industry, particularly pentachlorophenol (PCP) and creosote, with strains of Phanerochaete (not necessarily P. chrysosporium) is on the verge of real utility in the field (Lamar et al.,1989,1990;Lamar and Dietrich, 1990; Fungal Treatment Bulletin, 1993). There would be some poetic justice in using a woodrotting fungus to destroy chemicals originally produced to prevent the decay of wood products that are exposed to natural microbial populations. Some problems remain before the large-scale use of white-rot fungi to treat PCP can take place, including the formation in some soils of significant amounts of pentachloroanisole, which probably retains significant toxicity, and the incomplete removal of PCP when initial concentrations are high. Work to date, however, indicates that there is reason to expect these problems to be overcome through the use of the proper fungal strains andlor proper modifications of process physiological conditions. Finally, scientists are beginning to use what they have learned during almost 20 years of studying the ligninolytic system of Phanerochaete chrysosporium to design biodegradability in chemicals like plastics (Milstein et al., 1992) and textile dyes (Pasti et al., 1991; PastiGrigsby et al., 1992; Paszczynski et al., 1991a-1992). It is highly appropriate that the knowledge gained in spending millioins of dollars for basic research into ligninolysis by Phanerochaete chrysosporium can finally be used in this manner.

Acknowledgment We thank Connie Bollinger for editorial assistance. Our work on biodegradable azo dyes was supported by Grant R818356-01-0 from the U.S.Environmental Protection Agency, Ofice of Exploratory Research.

Literature Cited Abernethy, G. A.; Walker, J. R. L. Degradation of the insecticide Hydramethylnon by Phanerochaete chrysosporium. Biodegradation 1993,4, 131-139. Ander, P. The use of white-rot fungi and their enzymes for biopulping and biobleaching. In Advances in biological treatment of lignocellulosic materials; Coughlan, M. P., Collaco, M. T. A., Eds.; Elsevier: New York, 1990; pp 287-298. Archibald, F.; Paice, M. G.; Jurasek, L. Decolorization of kraft bleach effluent lignins. In Biotechnology in pulp and paper manufacture, applications and fundamental investigations, Proceedings of the 4th International Conference on Biotechnology in Pulp and Paper Industry; Kirk, T. K., Chang, H.-m., Eds.; Butterworth-Heinemann: Boston, 1990; pp 253262. Arjmand, M.; Sandermann, H., Jr. Mineralization of chloroanilinekgnin conjugates and of free chloroanilines by the

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Abstract published in Advance ACS Abstracts, March 1,1995.

Potential for Bioremediation of Xenobiotic Compounds by the White

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