The following chapter:

Use of Fungi by J. W. BENNET, K. G. WUNCH, AND B. D. FAISON

Has been taken from the book:

Manual of Environmental Microbiology Second Edition Editor in Chief Christon J. Hurst

ASM Press Washington, D.C. 2002.

Useof Fungiin Biodeadation

l. \7. BENNETI K. G.\yUNCH, AND B. D. FAISON

87

In nature, fungi do much of the dirty work. Th"y are par- Stramenopila, and four protist phyla. ln this classification, ticularly efficient at degrading the major plant polymers, the kingdom Fungi encompassesfour phyla: Chytridiomy- cellulose and lignin, but they also decomposea huge array cota, Zygomycota, Ascomycota, and Basidiomycota (18, of other organic molecules including waxes, rubber, feath- 26). The Stramenopila encompassesthree phyia' Oomy- ers, insect cuticles, and animal flesh. Although industrial cota, Hyphochytriomycota, and Labyrinthulornycota. From microbiologists regularly hamess fungal metabolism for the perspectiveof researchon fungal degradation,most of brewing, baking, cheesepreparation, and for production of the speciesof interest are in the kingdom Fungi. antibiotics, commercial enzymes, and a number of com- In filamentous forms, the individual thread-like cells are modity chemicals, fungi are best known for their dirry called hyphae. A fungal colony, or porrion of a colony, work. They spoil our foods, blight our crops, rot our build- composed of many hyphae together is called a mycelium. ings, contaminate our petri dishes, and cause some rather The filamentous/mycelial growth form poses problems in loathsome diseases.Paradoxically, despite this notoriety, determining the sizeof a single organism and in measuring "thal- the use of fungi in has been limited com- the growth of fungi. In the older literarure, the term pared to that of bacteria. Here we present a brief intro- lus" is often used to describe macroscopic mycelial forma- duction to fungal taxonomy and mycological techniques, tions. introduce methods for isolating fungi and for growing them Fungal taxonomy is based on reproducrive morphology, in the laboratory, define some important terms, review ex, which consists of both meiotic and mitotic spore-bearing amples of the successfulapplications of fungal organisms structures. Both sexual and asexual spores are typically and enzymesfor biodegradation, and point out the advan- made in vast numbers. Many fungi have more than one tages and disadvantagesof fungi as agents of bioremedia- morphologically distinct spore rype ar different phasesin tion. their life cycles. Further complicating rhis fungal pleomor- phism is the fact that some fungi exist as either yeasror filamentous forms depending on the environmental milieu, A LITTLETAXONOMY a phenomenon called dimorphism, best known from med- Like rnany other higher-order raxonornic units, the term ically important species. "fungus" is difficult to define. It embracesa large group of Like other eukaryotes,fungi have nuclei, mitochondria, nonphotosynthetic lower eukaryotesonce consideredpart B0S ribosomes,and chromosomes.Fungal cells may be hap- of the plant kingdom and later afforded srarusin their own loid or diploid; the nuclei within a mycelium rnay all be "Fifth kingdom, the Kingdom," on the basisof their char- genetically identical (monokaryotic) or may be a mixture acteristic absorptive mode of nutrition (102, 117, 168). of different genetic types (heterokaryotic). Basidiomycetes taditionally, the Myxomycora, or slirne molds, and the often have a special form of heterokaryon called a dikar- "true Eumycota, or fungi," comprised the two major sub- yon. Although rnany fungi are microscopic,the best-known divisions within this fungal kingdom. A plasmodium or speciesform macroscopicfruiting structuressuch as mush- pseudoplasmodiumcharacterized the Myxomycota, a group rooms and truffles. Fungi are ubiquitous in terrestrial en- rvhich included well-known senera such as Dict^tosielium vironments, and many fungi are capable of growing in and Physarum,while in the Eumycora the assimilative environments hostile ro mosr other forms of life (97). For phase was usually {ilamentous or yeasr-like.More recenrly, example, fungi are the only eukaryotesthat include mem- classificationbased on evolutionary relationships(i.e., phy- bers with thermophilic (60 to 62'C) oprimal growrh tem- logenetic classification) has led to a realization rhar the peratures(158). organismsthat have traditionally been called fungi, on the In summary, the broadly understood concept of fungi basis of shared nutritional modes and morphological char- comprisesa polyphyletic group of eukaryotic, heterotrophic acters, do not represenr a monophyletic lineage. Derived organisms that absorb their food. Mycology texts such as primarily from data on small-ribosomal-subunit(rDNA) se- those by Alexopoulos, Mims, and Blackwell (10), Ross quence analysis,the assemblageof traditional fungi is noiv ( 143), Moore-Landecker(124), and Carlile and Watkinson placed in three groups: the kingdom Fungi, rhe kingdom (10) are helpful introductions ro this economically impor' 960 87. Use of Funsi in Biodesradation I 961 rant group. Another useful tool is Ainsw,orth6 Blsbi''s Moreover, dormant ftrngal sp()resmay prrlduce nlllnerous Dictionaryof ilrc Fungi (83). General trrx.rnonricprinciples, colonies ri'hile thri'u'ing nonsporulating colonies may not as rvell as a guicle to the sometimes arcllne principles of be recoveredat all. ln licluidshake culture, many filamen- fungal nomenclature, which are govcrned by the lnterna- tous ftrngi form pellets, thus rnaking direct turbidity assays tional Code of Botanical Nomenclature, are presented by impractical and making dry weights the most cornmonly Havyksworth(79). The last volumes (in two parts) of the used measure.in batcl'rculture, the synthesisof m:rny fun- classicseries, The Fungl: an AduancedTreatise,provide com- gal procluctsand en:ymes is not correlated with growth but prehensivetaxonomic coverage(2,3), rvhile the hrst vol- is triggereclby the lin-rit:rtionof an essentialnutrient. The "trophophase" "idiophase," umes (4-0) give an almost encyclopedic revierv of the terms and roughly comparable classicalmycological literature. Anotl-rer useful resource is to bacterial log phase and stationary phase, respectively, the multivolume seriesentitled The Mycota, an Encyclopedia havc been used to describe {ilamentous growth. Both of Fungi (50). Volume VII addressesfungal classilication secondary-metabolite production and the ligninolytic- and taxonomy (120). enzyme production are correlated u'itl-r idiophase. Enrichment cultures arc a classicalmicrobiological tech- nique, commonly used ftrr hnding a specific microbe to de- grade a certain toxic . Enrichment cultures favor the ACQUtStilON,CARE, AND FEEDING growth of a particular speciesbasecl on its nutritional re- OF FUNGI quirements. h-r the most colnmon application of this Filamentous fungi have more described speciesthan any method, aliquots of rvater, soil, leaf , or other mixed other group of microorganisms,with :rbotrt 80,000 already inocula arc placed into a medium containing the targeted named and approximately 1,800 new species published cornpound as the sole carbon source.Only organismswith each year. As with bacteria, it is believed that only a small degradativeability will grow. In liquid cttlture, competition proportion of extant species are known tt-r science. The for the substratewill leacl to enrichment of the microbial total number of fungal species,both knorvn and unknorvn, strain that is able to grorv fastest.On petri plates, colonies has been estimatedat more than 1.5 million (80, BZ). representing many species are usually isolated; these are About 170,000 pure strains of fungi are maintained in then subcultured and tested further. With ferv exceptions, culture collections internationally, representingan esti- this approach leads to the isolation of bacteria. In general, mated 7,000 different species(Bi ). Information about these fungi are slower growing and produce fewer propagulesthan resources can be accessed through the World l)irectory of do bacteria. In addition, fungi are lesslikely than bacteria CoLlectionsof Cukure.s of Microorganisms(156). One of the to have the capracity to use xenobirttics as sole carbon oldest and largest fungal collections is the Centrarrlburear sources.Many fungi need a supplemental carbon source to voor Schemmelculturesin the Netherlands. The major col- sustain growth, i.e., their tlegradative potential is cometa- lection in the United States is the Atnerican Type Culture bolic. Collection (98). In addition, the collection at the Thus, the key to successftrlisolation of fungi for xeno- Northern Regional Research Laboratory of the U.S. De- biotic clegradatic-rnis trvofcrld:(i) the recognition that fungi partment of Agriculture in Peoria, lll., housesa large nLrm- are easily outgrown by bacteria and (ii) the recognition ber of economically important fungi, ancl the Forest that they produce many potent biodcgradativeenzymes ca- Products Laboratory in Madison, Wis., holds a major col- pable of degrading toxic pollutants yet do not use these lection of wood-rotting species.Major culture collections breakdon'nproJucts to sustaingrowth. To successfullyiso- maintain \Veb sites with information about holdings and late fungi with potential for bioremediation, it is necessary instructions about ordering. As with bacterial and viral to impose imaginative enrichment conditions, including strains, a fee is usua[y charged for obtaining cultures. the careful selection of supplementarycarbon sourcesand ln the laboratory, fungi and bacteria are treated simi- bacteriostatic agents. larly. They can be grown on agar rnedia in petri plates or Serving as a manual to both methods and references, in liquid broths. Depending on the nutritional require- volume 4 of Method.sin Microbiologl Q4) is specilically de- ments of the individual species,either a complex or defined voted to distinguishing mycological and bacteriological medium is used. Many fungi prefer media rvith acidic pH. perspectives.Specialized techniques for collecting, isolat' On petri plates, molds are readily distinguished from most ing, cultivating, manipulating, and preserving fungi are bacteria becausethev usuallv form drv aerial colonies that given. lndividual chapters are devoted to soil fungi, air may be brightly colored due to the pigmentation of their s:rrnpling,and aquatic organisms.Although severaldecirdes spores.With the naked eye, yeastcolonies are nor so easily old, this book is still a wonderful resource.See also Tech' distinguished from bacteria, but under the light micro- niquesin MicrobictlEcology (7 4), Mycology Guidebook (155), scope,their large size,compared to any run-of-the-mill pro- and chapters 6, 35, and 49 for guidance on the special karyote, is easy to discern. Recipes for common and handling of fungi. Other valuable referencesconcentrating specialty media for cultivating fungi are available in My- on applied mycology are Arora et al. (13), Smith and Berry cology Guidpbook ( 155 ) and Handbook of Microbiologlcalltl.e- (151,l5/) and Smith et al. (153). dia (15). ln isolating fungi from nature, antibiotics, such as streptomycin, or other bacterial growth inhibitors, such as rose bengal, are frequently added to media, thereby selec- tivcly enhancing the growth of fungi (119). DEFINITIONSAND DISTINCTIONS When isolating cuiturable microorganismsfrom natural Biodegradation, mineralization, bioremediation, biodeter- substrates,it is relativcly easy tc-robtain a colony count ioration, biotransformation, cometabolism, and bioaccu' basedon the unicells of typical bacteria. Sampling for fungi mulation are terrns not ah,vaysused with appropriate is much harder due to their {ilarnentous growth habit. sensitivity to their subtle diff-erencesin meaning. The spe- There may be a large massof fungal m1'celiumpresent, but cial role played by fungi in nature and in the human ex- the methods adapted frr-rmbacteriology may not detect it. ploitation of their often unique metabolism can be clarihed 962 T BIOTRANSFORMATIONAND BIODECRADATION by thinking about the distinctions implied in this ecolog- A second meaning of cometabolism is to describe the ical lexicon. degradation of a given compound by the combined "Biodegradation" efforts is the biologically mediated break- of severalorganisms pooling their biochemical resourcesfor down of chemical compounds. It is an umbrella term, en- mutual efforts(41). compassing most of the other jargon addressedin this section, and generally implies a seriesof biochemical re- actions. \Vhen biodegradation is complere, the processis THE FUNGALWAY OF DEGRADATION "mineralization," called i.e., the total breakdown of organic The organismsknown as fungi, encompassingboth Fungi molecules into water, CO2, and/or other inorganic end and Stramenopila, share a unique nutritional strategy,i.e., producb. Not all authors are careful to disringuishbetrveen their cells secreteextracellular enzymeswhich break down degreesof biodegradation; some use the term to describe potential food sources,which are rhen absorbedback into almost any biologically mediated change in a substrare,and the fungal colony. This way of life means that any discus- others use it to describe mineralization (9). sion of fungal biodegradation must cover "Biotransformation," an extraordinarv a rvord often used synonymously amount of catalytic capability. The decomposition "bioconversion," of lig- vi'ith usually refers tc-ra single step in a nocellulose is probably the single mosr imporranr degra- biochemical pathway, in which a molecule (the precursor) dative event in the Earth's carbon cycle. The utilization is catalytically converted into a different molecule (the and transformation of the dead remains of other organisms product). Many biotransformations in sequenceconstitute is essentialto the Earth'seconomy. An enormousecological a metabolic pathway. Industrial processesfrequently incor- literature exists on the role of fungi as primary and sec- "cycles" porate biotransformation reactions.A famousexample is in ondary decomposersin these classic of nature (see, the manufacture of steroids for birth control pills (141). for example,references 8, 3I,36, and 60). When ecologistsdescribe biotransformation, the environ- From the human perspective, the power of fungal en- mental status of the transformed product is a primary con- zymes is Janus-faced. Molds destroy more food than any sideration. Is it more water soluble and thus more easily other group of microorganisms.They damagestanding tim- excreted by the cell? Is it less toxic/ Is it more hazardous ber, finished wood products, fibers, and a wide range of than its precursor?\7hen relatively innocuous precursors noncellulosic products such as plastics, fuels, paints, glues, are converted into more toxic products, the processis drugs, and other human arrifacrs (48, "activation." l4Z). On the other called The metabolic activation of mercury is hand, many of the oldest biotechnological pracrices are a il'ell-kno\,vnexample of a biotransformation with malign also basedon fungal catalytic power: baking, brewing, wine environmental imnact. fermentation, production of certain cheeses,and "Biodeterioration" "bioremec-liation" the koji and are the two as- processare ancient examplesof the way humans have em- pects of biodegradation rvith an anthropomorphic empha- ployed fungi for their own benefit. In the 20th century, sis. Biodeterioration is the breakdorvn of economicallv numerous hydrolytic enzymesinvolved in the degradation useful substances.Often the term is used narrowly to refer of relatively simple biopolymers such as starch and protern to the deterioration of substancesthat are normally resis- have been purilied, characterized,and utilized within in- tant to biological attack such as metals,plastics, drugs, cos- dustrial settings. These include fungal amylases, gluco- metics, paintings, sculpture, wood products, electrical amylases,lipases, pectinases, and proteases(see references equipment, fuels and oils, and other economically valuable 19 and 72 for reviews). Fungal cellulasesprovide a good objects (147). example of the contrasting faces of a single enzymatic ca- in bioremediation, biological systemsare used to trans- pability. During World War ll, researchby the U.S. Army form and/or degradetoxic compounds or otherwise render on the rnicrobial destruction of military clothing and tents them harmless. Bioremediation can involve indigenous led to the characterization of the cellulolytic mold Tricho- microbial populations with or withour nurrienr zupple- derma reesei.Continuing research on T. reeseiidentified a mentation, or it can involve inoculation of exogenous complete set of cellulase enzymes required for the break- organisms into the site. When exogenous organisms are down of cellulose to glucose (128, 139). These "bioaugmenrarion." enzymes added, the process is called In either now prornise the potenrial of converting waste cellulosics case, the goal is to disarm noxious chemicals without the into foods for our burgeoning population and have been fornration of ncw toxins. the subject of intense (51,173). "Bioaccumulation," molecular biology research sometimes used loosely synony- Although cellulase-producedglucose "biosorption," is not yet economi- mously with is the concentration of sub- cally competitive, anorher traditional fungal process rs: stanceswithout any metabolic transformarion. Both living mushroom cultivation on lignocellulose(34, 154). These and dead cells may be involved. Bioaccumulation tech- and some other examples of economically advantageous niques can be used to concentrate metals such as copper, uses of fungal biodegradation are displayed in Th- lead, silver, uranium, and certain radionuclidesfrom aqu.- ble 1. ous environments,anJ the resultantloaded can he Fungi are also good at bioaccumulation of metals. Many recycled or contained. species can adsorb "cornetabolism" cadmium, copper, lead, mercury, and The term is used in two senses.Usuallv. zinc clnto their rnycelium and spores.Sometimes the walls it describesthe situation where an organism is able to bio- of dead fungi bind better than living ones. Systemsusing transform a substratebut is un:rble ro qrow on it. As defined Rhiloprzsarrhizus have "Co-metabolisrn been developed for treating uranium by Horvath, referstoany oxidarion of sub- and thorium (161). Spent fungal biomass from industrial stancesrvithout utilization of the energy derived from the fermentations is an availableresource for the concentration oxidation to supporr microbial growrh" (BB).The phenom- of heavy-rnetalconramination (62, 63, I44). "cooxidation" "qratuitous" enon has also been called and "fortuitous" What about fungal degradation of pollutants and toxic or merabolisrn.Many biochemistsdislike the ? In some cases, traditional methods are beine term (67, 89). Nevertheless,it hasbecone well entrenchetl adopted for contemporary needs.For example, composting in the literature. has been usedto rrear borh pesricides(59) and munitions BZ. Use of Fungi in Biodegradation t 963

TABLE 1 Econonricallybenehcial examples of fungal degradation

Process Substrate Species Rerrresentative citations

Composting Straw, rnAnure,agricultural waste, Consortia of bacteria and fungi, usually zl, 55 bark uncharacterized

Mushroom cultivation Lignocellulose, anirnal manure Agaricus bisporus 34,84 Strarv, sawdust Pleurotus ostreatus(oyster mushroom ) r54 Wood logs Itntinus odoides ( shiitake ) 154

Single-cell protein Alkanes Yeasts,e.g., Candi&t tropicalis 42,100 production Brewery wastes,molasses Saccharomyces cerevisiae, S. carlsbergensis rr4 Sulfite rvasteliquid Candidn wilis, P aecilom)ces varioti rr4

Solid-rvaste treatrnenr Sludge/ Consortia of bacteria and fungi, usually 7'7 lO uncharacterized Pulp and paper mill e{lluent Coriolus ver sicolor, Phrtnerochaete 106,107 chrysosporium

\Tastervater treatment Distillery waste Yeasts, especially Candi&t utilis 6l Kraft bleaching effluent P hnnero chne te chry so s p orium 49 Tannery effluent A-spergllas, P enicillium rz7

wastes(169). There is also rather a lot of descriptive bio- decaying speciesagainst a surprisingly largc battery of en- chemistry concerning the ability of various fungi and their vironmental contaminants (79, 58). Recent advances in enzymes to biotransform pesticides (see reference 138 for the use of fungi in environmental remediation and bio- enzymesancl reference 150 for lists of speci{rccompounds technology have been summarized by Paszczynskiand and organisms),but, to date, the most sophisticatedfungal Crarvford (137). approachesto environmental clean-up have grown out of prior research on degradation of petroleum hydrocarbons (16, 37, 66) and on the adaptation of researchon fungal PHANEROCHAETE CH RYSOSPORIUM treatment of lignocellulytic wastes in the pulp and paper Phanerochaetechrysosponum is a higher basidiomycete be- industry(i06, 107, 109). longing to the white rot group of fungi. P. chrysosporiumis The ability to grow on petroleum hydrocarbonsis rvide- the best studied of the ligninolytic fungi, a group whose spreadamong the fungi (14,33,66). Jet crashescaused by natural habitat is forest litter and rotting wood. White rot blocked fuel lines due to the growth of resi- fungi '!Var Cladosporium are unique among eukaryotes in having evolved non- nne, frrst reported during World Il, are among the specific mechanismsfor degrading lignin. more dramatic negativeconsequcnces of the ability of fungi Lignin is unlike many natural polymers in that it con- to thrive in extreme habitats (113). Considerable infor- sists of irregular phenylpropanoid units linked by nonhy- mation is available about the mycological flora associated drolyzablecarbon-carbon and ether bonds. Lignin contains with marine petroleum spills (33). On the industrial side, chiral carbons in both the L and n configuration, and this the years of researchon single-cell protein, instituted with stereo irregularity renders it still more resistant to attack the goal of turning petroleum hydrocarbonsinto feed, have by most microorganisms.Nevertheless, many extracellular paid off in the study of the enzymatic mechanismsused by ligninolytic enzymesproduced by white rot fungi can cat- yeastsand other microorganisms in the biodegradation of alyze the breakdown of lignin. petroleum wastes for environmental remediation (100, Under conditions of nitrogen, sulfur, and/or carbon 140). Cytochrome P-450s are mixed-function oxidases deprivation, P. chrysosporium produces families of lignino- (monooxygenases),derived from a superfamily of genes, lytic enzymes (54, 68, 160) including lignin peroxidase which are involved in many stepsof petroleum degradation (LiP) (EC 1.11.1.12)(160) and manganese-dependentper- and in the biotransformation of a variety of environmental oxidase(MnP) (EC 1.11.1.13)(69). The peroxidasesuse pollutants ( 166). Both detoxification and activation are as- hydrogen peroxide generated by glyoxal oxidase, glucose sociated with the action of P-450. Fungal monooxygenases oxidase, and cellobiose oxidase to promote the oxidation are more similar to mammalian than to bacterial cyto- of lignin to free radicals that then undergo spontaneous chromes; Sariaslani(146) has presenteda particularly thor- reactions with oxygen or water, which leads to depolyrner- ough review of theseenzymes, and Kellner et al. ( 10i ) have ization. The depolymerization of lignin by nonspecific discussedtheir use in bioremediation. Extensive biochem- extracellular peroxidases is sometimes called enzymatic ical and genetic data are available for severalyeasts; there combustion (108). Both LiP and MnP are encoded by fam- is also a large literature surrounding the aseptatefilamen- ilies of structurally related genesthat have been cloned and tous speciesCunninghamelln elegans (33, 66). sequenced(for reviews,see references7, 25,40, and 87). Similarly, and to an even greater extent, research on These genes are differentially regulated in response [o a pulp , such as the decolorizationof effluent variety of environmental signals,especially starvation (91). from kraft pulp mills, and the subsequentmushrooming of During the 1980s it became apparent that P. chryso- research on rvhite rot fungi have shown the power of rvoocl- sporium, in addition to degrading lignin, is capable of 964 I BIOTRANSFORMATIONAND BIODECRADATION degrading a wide variety of xenobiotics. Polyaromatic conditions many contaminants are only partially broken hydrocarbons, chlorinated phenols, nitroaromatics, dyes, down. In some cases,envlronmentally undesirable break- and many other environmental toxins have been biotrans- down products are formed. In addition, many theoretical formed or mineralized by P. chrysosporium,sometimes in questions remain about the biochemical activities of P. complex mixtures of xenobiotics (Table 2). The ability to chrysosponumin nature and the relationship between the degrade such a broad spectrum of highly toxic and gener- enzymesof lignin degradation and the rnechanismsof xe- ally recalcitrant substratesis unusual for a single species.It nobiotic removal. In practice, the application of P. chry- is often assumedthat this broad-spectrumxenobiotic bio- sosporiumto contaminated habitats has been hampered by degradation is effected by the same extracellular enzymes the fact that its natural habitat is not soil. Notwithstand- used in lignin degradation. In addition, a variety of other ing, the successesof P. chrysosporium in laboratory biodeg- factors are thought to contribute, such as intracellular en- radation studies ensure that intensive research on this zymes (e.g., reductase,methyltransferases, and cytochrome specieswill continue. These successeshave also stimulated "plasma P-450 oxygenases), membrane potentials," and quests for other filamentous fungi with the porential for bioabsorption onto mycelia (17, 99, 163, 164). Postulated degrading xenobiotics. mechanisms used by the white rot fungi to degradepollut- ants have been summarizedby Barr and Aust (17). In the laboratory, most of the successfulmineralization experiments have been conducted under ligninolytic con- wooD ROTS,LTTTER ROTS, AND OTHER ditions using rvhole ceils in liquid culture. Moreover, pu- FILAMENTOUSFUNGI ri{ied preparations of lignin peroxidases are capable of There are more than 1,500 different speciesof white rot oxidizing a variety of the xenobiotics known to be miner- fungi. In addition, there are thousandsof orher fungal spe- alized by whole cell cultures of P. chrysosponum(e.g.,7,(- cies loosely categorizedas brorvn rots, dry rots, litter rors, dinitrotoluene, lignite, polyaromatics,pentachlorophenols, soft rots, mycorrhiza, terricolous, and so fcrrth. Most of and dichlorodibenzo-p-dioxin, pyrene), although they are these specieshave never been studied in the laboratory and not involved in others (".g., DDT [1,1,1-trichloro-2,2- represent a large potenrial for biodegradation research.In bis(4-chlorophenyl)ethanel)(85, 86, 110). On the other recent years,several groups have done comparative studies hand, many questions remain unanswered. In one study, of white rot fungi with the expectarion of linding better the disappearance of 2,4,6-trinitrotoluene (TNT) began lignin-degradingsysrems (45, 46,136). Others have taken prior to the onset of ligninase activity, while secondary advantage of this resource by screening isolates from products were formed after the appearance of LiP and MnP culture collections and natural habitars against a variety (78). In another study, P. chrysosporiumremoved phenan- of pollutants. Many new specieswith bioremediarion po- threne under both ligninolytic and nonligninolytic condi- tential have been identilied (58, 118, 170). In a num- tions (43). ber of cases,these fungi show more practical promise than To recapitulate, among filamentous fungi, P. chrysospor- P. chrysosporiumsince their growrh strategies offer better izm has become a model system for studying xenobiotic sustainability in natural habits; for example, they do not degradation. A critical assessmentof its potential as a bio- require constant additions of wood or orher substrates(73). remediation tool has been given by Paszczynskiand Craw- ln addition, their enzymaric reperroires offer fresh ap- ford (131), who point out rhar the field applications have proaches to xenobiotic degradation. In the survey by been unpredictable and that even under ideal laboratory Gramss et al. (71), 58 speciesfrom differenr physioecol-

TABLE 2 Examplesof xenobiotics degradedor transformedby P. chrysosporium

Type of compound Exarnples Reference(s) Aromatic hydrocarbons Benzo[a]pyrene 75 Phenanthrene 27,r75,157 Pyrene 76

Chlorinate..l organics Alkyl halide insecticides 103 Atrazine rz6 Chloroanilines ll DDT 28,110 Pentachlorophenols lll, 172 Tiichlorophenol 99 Polychlorinated biphenyls, Aroclor 44, 47 Polychlorinated dibenzo-p-dioxins 70,76,164 Trichlorophenoxyacetic acid r45

Nitrocen aromatics 2,4-Dinitrotoluene t62 TNT <'7 Hexahydro-1,3,5-trinitro- 1,3,5 -triazine 56

Miscellaneous Dyes 39,130, 134,135 BZ. Use of Fungi in Biodegradation I 965 ogical groups (wood degrading, woocl and straw degrading, a fluoroquinoloneanribioric (167).No doubt many orher terricolous, ectomycorrhizal, and mitosporic) were grown interesting brown rot specieswill be found to demonstrare in liquid cultures and tested against a battery of polyaro- activity against a spectrum of xenobiotics. matic hydrocarbons. On average, wood-degrading species rvere best at metabolizing polyaromatic hydrocarbons, but competent fungi were found in all five groups.Polyaromatic MYCOREMEDIATION hydrocarbon conversion was correlated rvith the produc- Many rvorkers divide bioremediation strategiesinto three tion of MnP, peroxidase,and laccase(71). general categories: (i) the target compo,,.rd is used as a Two particular white rot speciesthat have receivedcon- carbon source, (ii) the rarger compound is enzymatically siderable attention are Pleurotus ostreatusand Tiametesuer- attacked but is not usedas a carbon source(cometabolism), sicolor.They are both efficient at mineralizing polyaromaric and (iii) the target compound is not metabolizedat all but hydrocarbons (20, 165) and at degrading polychlorinated is taken up and concenrrated within the organism (bioac- biphenyls (174).P. ostreatu.sis better able to colonize soils cumulation). Although fungi parricipare in all three srrat- than P. chrysosporium(129), and although it is a successful egies,they are often more proficient at cometabolism and lignin-clegrading species,it does not exhibit LiP acriviry bioaccumulation than at using xenobiotics as sole carbon (77). P. ostre&tusdoes, however, produce several laccase sources. isoenzymesencoded by families of laccasegenes (64, 65). The attributes that distinguish filamentous fungi from Laccase (p-diphenol-dicrxygen oxidoreducrase other life-forms determine why they are good biodegraders. "blue [EC 1.10.3.21)is a member of the copper oxidase"family First, the mycelial growrh habit gives a comperirive advan- ( 104, 159, 172). Laccasesoxiclize subsrrares by one-elecrron tage over single cells such as bacteria and yeasts,especially transfer steps and are active against lignin model corn- with respect to the colonization of ir-rsolublesubstrates. pounds in the presenceof mediators(93, 116). Many phe- Fungi can rapidly ramify through subsrrares,literally di- nols and chlorophenols are transformed by these enzymes gesting their way along by secreting a battery of extracel- to radicals that subsequently undergo spontaneous poly- lular degradative enzymes.Hyphal penetrarion provides a merizations. Laccase-mediator combinations also show mechanical adjunct ro rhe chemical breakdown effected by activity against acenaphthene, acenapthylene, and the secretedenzymes. The high surface-to-cellratio char- anthracene (94,95). a purified laccaseisolated from T. acteristic of filaments maximizes both mechanical and uersicolorcould oxidize a variety of polyaromatic hydrocar- enzymatic contact with the environment. Second, the bons in vitro, including anthracene, benzo[a]pyrene,fluor- extracellular nature of the degradative enzvmes enables anthene, and chrysene (115). In addition to laccase,an fungi to tolerate higher .o.,...,i.rtions of toxic chemicals enzyme with properries of both LiP and MnP has been than would be possible if these compounds had to be isolated from species of Pleurotus and Bjerknndera. This brought into the cell. "third In addition, insoluble compounds type" of lignin peroxidaseshares catalytic properties that cannot crossa cell membrane are susceptibleto attack. of MnP (eflicient oxidation of MnZ* ro Mnr*) and LiP Fungi even solubilize low-rank coal, a parricularly persis- (oxidarion of lignin model dimers) and has a high affinity tent, irregular, and complex polymeric substrate,although for substituted hydroquinones (171, I47). they do so slowly (33,53,132). Finally, since the relevant A representarivelist of filamentous fungi with the abil- enzymesare usually induced by nutritional signalsindepen- ity to degradexenobiotics is presentedin Table 3. Cell-free dent of the target compound during secondarymetabolism, studieswith enzymesfrom some of these specieshave been they can act independently of the concentration of the conducted. For example, a cell-free sysremof the MnP of substrate, and their frequently nonspecific nature means the white rot fungus Nematoloma frowardii is capable of that they can act on chemically diverse substrates. mineralizing pentachlorophenol, carechol, and pyrene.The Among filamentous fungi, Phanerochaetechrysosporium ratio of MnP activity to rhe concentration of reduced glu- has emerged as the model sysrem for studying xenobiotic tathione affectsthe extent of mineralizarion(85). degradation.A great deal remains ro be learned about the Brown rot fungi degradethe cellulose and hemicellulose fundamentals of how this lvhite rot fungus mineralizespol- components of rvood, leaving a residue of modified lignin lutants; not surprisingly,even lessis known about the deg- that is dark brown. Although they are enormously destruc- radative mechanisms used by fungi in general. Oxidative tive of rvood produc$, the mechanism of wood decay by enzymes play a major role, but organic acids and chelators brown rot fungi has received far less attention than that of excreted by the fungus also contribute to the process.Many wood decay by white ror fungi (173).lt is believed that of the toxic chemicals mineralized by fungi are already the early steps in brown rot decay are nonenzymaric. Early highly oxidized. The ability of fungi to lower the pH of studies demonsrrate that the cellulose in wood can be de- their environment appearsto be involved in the reduction polymerized by Fenton's reagenr [H2O2 plus Fe(ll)], and of some of thesecompounds (17). most contemporary models invoke a Fenton-type extracel- What about the futurel Various brown rots, litrer rors, lular system that produceshydroxyl radicals or-other pow- aquatic fungi, anaerobic fungi, and mycorrhizal fungi, in erful oxidants thar then attack the wood components (72). conjunction with pollutant-tolerant plants, all provide op- Different mechanisms proposed for hydroxyl radical pro- portunities for new research. Genetic engineering is an- duction by brorvn ror fungi have been reviewed by Hyde other frontier. Fungal genesfor degradative enzymescan be and Wood (90). In studies on Gloeophyllumnabeum, 4,5- added to bacteria; alternatively, comperent fungi can be dimethoxycatechol and 2,5-dimerhoxyhydroquinone have modilied to grow in an extended range of environments. been implicated as electron transport carriers to Fenton's For example, several groups have investigated the recom- reactions ( 133). The ability of brown rors to degradexeno- binant expressionof fungal peroxidasesin order to facilitate biotics is a relatively new avenue of research.Cirrent stud- large-scalecommercial production of these enzymes.LiP ies have implicared two species of Gloeophyllum in the and MnP have been produced in an Aspergillusniger host degradatit-rn,rf chlorophenols (52, 149), and G . trabeumis and in the insect bacr-rlovirusexpression system, although active against polyethylene glycol (105) and ciprofloxacin, the LiP was not active (35, 96). Another development is 966 T BIOTRANSFORMATIONAND BIODECRADATION

TABLE 3 Representativexenobiotic-degrading filamentous fungi

Group Species Substrate(s) Reference(s)

White rot fungi Agroc.^tbeaegarita Benz[a]anthracene 118 Agrocybe praecox Phenanthrene, pyrene 71 Clitocybula duseni Lignite 86 Coriolopsis gallica Anthracene, phenanthrene, pyrene r37 Dichomitus squabns Benz[a]anthracene 118 Doedoela quercina Benz[a]anthracene 118 Ganodtrma applarwtum Benz[c]anthracene i18 Hypholoma fascicukne Anthracene, fluoranthene, pyrene 7r Kuehner omyc e s munbili s Anthracene, fl uoranthene, 7r phenanthrene, pyrene Itntinus edodes Benz[a]anthracene 118 Itnzites betulina Anthracene, phenanthrene 7I Nemntoloma frowardii Dinitrotoluene and trinitrotoluene, 85, 96, 149 lignite coal, pentachlorophenol Pleurotus dryinus, P. eryngii, Benz[a]anthracene 118 P. fuscul^otns,P. flabellntus, P. pulmonnius, P. sajor-caju P1 cnopor u s cinnabannu s Dibenzofuran 97,118 Str opharia r ugo s o annulntn Anthracene, fluoranthene, 71,119 phenanthrene, pyrene Trametes hirsuta. Textile dyes 1

Mycorrhizal fungi Morchella conica Anthracene, fluoranthene, 7r phenanthrene Tylospcno fibrilnsa Fluorobiphenyl 73

Others Agaricus bisporus Anthracene, fluoranthene, 7r phenanthene, pyrene Copinus comatus Anthracene, fluoranthene, 7r phenanthrene, pyrene Crinipellis stipinna Pyrene r17 Glaeophyllum striatum Dichlorophenol 52 G. trabeum Pentachlorophenol r49 Marasmiellus tToJonus Benzo[a]pyrene l7r M. rotuln Pyrene ll7

that of large-scaleDNA sequencing.As this review is being degradation are more likely to occur through the combined completed, the genomic analysisof white rot fungi is being effects of many organisms.For example, cocultures of the initiated. The U.S. Department of Energy is using a whole- bacterium Stencttrophomonnsmaltophilia and the fungus Pen- shotgun approach to sequence the genome of P. chrys- icillium j anthinellum degrade high-molecular-weight poly- osponum (D. Cullen, personal communication). The cyclic aromatic hydrocarbons more efficiently than does availability of DNA sequencedata for the model white rot either microorganism alone (73), fungus, coupled with the capacity to build DNA microar- Judicious combinations of chemical and physical pro- rays for transcriptional profrling and gene discovery, will cesseswith biological schemesalso offer promise. Filamen- provide powerful tools for identifying genes for hitherto tous fungi, yeasts,and nonphotosynthetic bacteria are the undiscovered degradative enzymes from other filamentous workhorsesof biological degradation.Therefore, it is ironic "green" fungi. that the popular presshas chosen the word to de- As we get better at recognizing what can and cannot scribe environmentally friendly technologies such as bio- be done with bioremediation, we will create a menu of remediation. Decidedly not green in color (except for a few choices using a broad range of organisms. In some situa- spore types) and most certainly underappreciated(even by tions, bioconcentration of a is the best we can most microbiologists), the fungi possessthe most varied do. ln others, the nonspecificity of the white rot fungi is and most efficient battery of depolymerizing enzymesof all ideally suited to treating low concentrations of mixed decomposers.When joined with their bacterial brethren in wastesin a nutrient-deficient habitat. ln vet others. anaer- cooperative catabolism, fungal-bacterial consortia will fos- obic bacteria are clearly the best candidates. Microbiolo- ter the ecological recovery of contaminated habitats world. gists know that pure cultures are rare in nature. Common u'ide. Filamentous fungi will alwaysbe major playersin the "greening" sensetells us that in the real world, complete pathways of of toxic \r'astesites and other polluted habitats. 87. Use of Fungi in Biodegradation I 967

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