Mechanisms white rot fungi use to degrade pollutants - ACS Publications


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MECHANISMS WHITE ROT FUNGI

USE TO DEGRADE POLLUTANTS D A V I D

P. B A R R

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S T E V E N

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Environ S a Techno1 , Vol 28. No 2, 1994

0013-936X/94/0927~78A$045010

1994 American Chemical Society

n recent years the possibility of using white rot fungi for bioremediation strategies has initiated considerable research effort in academic, industrial, and government institutions. The interest in this subject arises from the ability of white rot fungi to degrade an extremely diverse range of very persistent or toxic environmental pollutants. This ability sets the use of white rot fungi apart from many of the existing methods of bioremediation. Perhaps the easiest way to understand the nonspecific ability of these fungi to degrade pollutants is to c o n s i d e r t h e i r e c o l o g i c a l “niche.” White rot fungi are those organisms that are able to degrade lignin, the structural polymer found in woody plants. Lignin is a very complex three-dimensional polymer consisting of nonrepeating phenyl propanoid units linked by various carbon-carbon and ether bonds (Figure 1)(1). The stereo irregularity of lignin makes it very resistant to attack by enzymes. In addition, it is impossible for lignin to be absorbed and degraded by intracellular enzymes. The enzymatic degradation of lignin is further complicated by the chiral carbons in this polymer that exist in both the L and D configurations. Thus, the white rot fungi have developed very nonspecific mechanisms for degrading lignin. This review discusses the degradation of pollutants by these fungi from a mechanistic standpoint.

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Advantages of white rot fungi Most of these mechanisms depend on a group of heme-containing peroxidases secreted into the extracellular environment of these fungi. These enzymes, known as lignin peroxidases (Lip) and manganesedependent peroxidases (MnP), are produced by the fungi in response to low levels of key sources of carbon, nitrogen, or sulfur nutrients (2,31.These conditions are often referred to as ligninolytic. The purification and characterization of these enzymes have been extensively reviewed recently (4, 5). The fungi contain enzymes that produce extracellular hydrogen peroxide from molecular oxygen. It has been reported that the fungi produce oxidase enzymes that utilize glucose, glyoxal, methyl glyoxal, and other products of cellulose and lignin degradation as substrates for the production of H,O, (6, 7). Interestingly, these fungi do not use lignin

as a carbon source for growth; instead, they degrade the lignin to obtain the cellulose that is toward the interior of the wood fiber ( 8 ) . The same unique, nonspecific mechanisms that give these fungi the ability to degrade lignin also allow them to degrade a wide range of pollutants to carbon dioxide-(see box1 (9-171. Along with their ability to degrade these chemicals, white rot fungi possess a number of advantages not associated with other bioremediation systems. Because key components of the white rot fungi lignin-degrading system are

extracellular, the fungi can degrade very insoluble chemicals such as lignin or many of the hazardous environmental pollutants. Many of the pollutants in a hazardous waste site are toxic to the organisms that may be employed to degrade them. For example, cyanide is known to be a potent inhibitor of respiratory oxidase enzymes: thus, uptake of cyanide by bacteria inhibits growth. Yet, in order to metabolize cyanide, bacteria must take up the pollutant because the enzymes are located inside the cell. As a result, cyanide concentrations as low as 4 ppm could inhibit microbial growth in a municipal sewage treatment system (18). However, the extracellular system of the white rot fungi enables the fungi to tolerate considerably higher concentrations of a toxic pollutant such as cyanide. For example, cyanide was found to be quite

toxic to spores of the white rot fungus Phonerochaete chrysosporium (50% inhibition of glucose metabolism occurred at 2.6 ppm). This toxicity was due to the absence of LIP, which rapidly metabolize cyanide. However, ligninolytic, 6-day-old cultures could tolerate considerably higher cyanide concentrations (i.e., 50% inhibition of glucose metabolism occurred at 182 ppm), further supporting the idea that the Lip protect this fungus from cyanide (9). Because of this increased tolerance, the inhibition of cyanide mineralization by P. chrysosporium was not e v i d e n t u n t i l concentrations reached 260 ppm cyanide. It was also found that complexing cyanide with metals enhanced its mineralization by the fungus [19). The enhancement of mineralization by iron, for example, follows the observation that potassium ferricyanide is membrane impermeable and presents very little toxicity to the fungus.This result is relevant because cyanide in soil may indeed be complexed with metals such as iron. Interestingly, we found that the mineralization rate of cyanide was linear when 3000 ppm were added to the cultures of P. chrysosporium in soil (19). The very nonspecific nature of the mechanisms used by these fungi allows them to degrade even complex mixtures of pollutants, such as creosote and Aroclor, all the way to carbon dioxide (20,21).In contrast, a consortium of bacteria may be needed to successfully and completely degrade these same mixtures. For example, Abramowicz et al. (221 have found that anaerobic bacteria in Hudson River sediment can effectively dehalogenate polychlorinated biphenyls (PCBs) to their monochloro congeners. However, aerobic bacteria are then required to degrade these monochlor i n a t e d b i p h e n y l s t o carbon dioxide. Another advantage of white rot fungi is that they do not require preconditioning to a particular pollutant. Because the degrading system of white rot fungi is induced by nutrient deprivation, limiting the nutrient source (i.e,, glucose or ammonia) can initiate degradation. Furthermore, repression of enzyme synthesis does not occur when the concentration of a chemical is reduced to a level that is ineffective for enzyme induction. Because the induction of the degradative enzymes is not dependent on the presence of the chemical, the fungi can Environ. Sci. Technol., Vol. 28, No. 2, 1994 79 A

1

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of these mechanisms will be neces,L

sary for the successful application

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of this technology. It is important to realize that the first step in the degradation of many chemicals by white rot fungi often involves the formation of a highly reactive free radical intermediate (13, 24). Free radicals are formed anytime one electron is removed or added to the ground state of a chemical. Such free radicals are very reactive and will rapidly give up or abstract an electron from another chemical. Thus, free radical reactions often occur as chain reactions in which many different radicals are generated subsequent to the formation of the initial radical species. This free radical process provides some basis for the nonspecific nature of white rot fungi. Using free radical reactions for the degradation of lignin is quite logical because the lignin polymer is synthesized by a free radical mechanism. In this process, plant peroxidases catalyze the one-electron oxidation of phenolic compounds, forming phenoxy1 radicals that subsequently polymerize to form lignin (25).It seems reasonable to use a similar mechanism for lignin degradation. The free radical reactions catalyzed by the peroxidases from white rot fungi also appear to be involved in the degradation of many pollutants.

Pollutant oxidation mechanisms Peroxidases, including Lip and MnP, use hydrogen peroxide to proeffectively degrade very low con- white rot fungi in soil (23).The lig- mote the one-electron oxidation of centrations of a pollutant to nonde- nin-degrading system allows the chemicals to free radicals. In the tectable or nearly nondetectable fungi to access the limited carbon “resting state,” the heme iron of the peroxidase is in the ferric state. Hysource from such substrates. Other levels (10). In fact, the rates of pollutant deg- soil microbes are less able to utilize drogen peroxide oxidizes the ferric radation are proportional to the these substrates. Therefore, the enzyme by two electrons to a form concentration of the chemical. This fungi maintain a competitive ad- of the enzyme known as compound is related to the pseudo-first-order vantage. In addition, white rot fungi I (cpd I), a ferry1 (Fe IV) Ic-porphyrin kinetics that are observed for the can be grown in liquid culture (9). cation radical. A chemical can then free radical mechanisms used by Thus, it appears there is promise for be oxidized by one electron to a radthese fungi (these mechanisms will the white rot fungi technology in ical, and compound I can be rebe discussed later). Thus, the time both soil and liquid reactor systems. duced by one electron to compound to achieve decontamination is a Soil may also be treated in situ, for ll (cpd II). A subsequent oxidation more useful criterion than is the rate surface contamination, for example. of another molecule by compound Although there are many advan- ll returns the peroxidase to its ferric of degradation, and this will be tages to this technology, the com- resting state (26). Figure 2 summachemical- and condition-specific. The effective decontamination of plexity of the mechanisms used by rizes the catalytic cycle of peroxivery low concentrations of chemi- white rot fungi has slowed its emer- dases. Many structurally diverse cals is important because govern- gence as a bioremediation strategy. chemicals. including pollutants ment regulatory agencies are con- These mechanisms remain largely such as benzo(a1pyrene (271,dioxtinually lowering the maximum unknown to many researchers try- ins (28) and cyanides (131, are dipermissible levels of these hazard- ing to apply the technology, which rectly oxidized in vitro by the lignin has led to some discouraging re- peroxidases. This is possible beous environmental pollutants. Very inexpensive growth sub- sults. However, several of the mech- cause the peroxidases themselves strates such as corn cobs or other anisms used by these fimgi to de- are very nonspecific. The chemical crop residues, wood chips, or sur- grade chemicals have recently been is not required to bind to the enzyme; instead, oxidation occurs plus grains can be used to cultivate elucidated. A clear understanding 80 A Envimn. Sci. Technol.. Val. 28. No.2,1994

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Environmentalpollutants degraded by the white rot fungus Phanerochaefe chrysosporiud Polycyclic aromatic compounds Benzo[a]pyrene Pyrene Anthracene Chrysene Chlorinated aromatic compounds Pentachlorophenol 4-Chloroaniline 2,4,5-Trichlorophenoxyacetic acid Polychlorinated biphenyls Dioxin Pesticides DDT [1.1,1 -trichloro-2,2-bis(4-chlorophenyl)ethane] Lindane Chlordane Toxaphene Dyes Crystal violet Azure blue Munitions TNT (2,4,6-trinitrotoluene) RDX (cyclotrimethylenetrinitroamine) HMX (cyclotetramethylenetetranitramine)

Others Cyanides Azide Aminotriazole Carbon tetrachloride *This is a partial listing that is r ponum.

cent studies have provided evidence for the in vivo oxidation of pollutants such as polycyclic aromatic hydrocarbons and 2,4,5trichlorophenol by lignin peroxidases (31,32). In many cases, however, it appears that chemicals are not directly accessible to the heme of Lip. and thus direct oxidation does not occur. The addition of a chemical that is oxidized to a free radical by LiP can result in the subsequent oxidation of chemicals that are not directly oxidized by the enzyme. This p d s s can be regarded as indirect oxidation. Interestingly, the white rot fungus P. chrysosporium produces veratryl alcohol (3,4-dimethoxybenzyl alcohol) from either lignin or glucose (33. 34). Veratryl alcohol is an excellent substrate for LiP and is oxidized to a cation radical (35).which C M then oxidize other chemicals that are not directly oxidized by Lip. There has been much debate about veratryl alcohol serving as an electron mediator for such oxidations. In the absence of a substrate, cpd n of a lignin peroxidase can react with H,O, to generate a catalytically less active compound III (cpd III) form of the enzyme (36). Many researchers believe that the reaction of cpd Il with H,O, prevails in the presence of chemicals that are poor Lip substrates and that veratryl alcohol prevents the formation of cpd

III (36).

through simple electron transfer. The active intermediates of LiP (Le., compound I and compound n) have considerably higher reduction potentials than do other peroxidases, extending the number of chemicals that can be oxidized. It has been reported that the reduction potential of Lip-cpd I is 250 mV higher than that of horseradish peroxidase (29). The oxidative reactions following the direct reaction

of a chemical with LiP often result in carbon-carbon bond cleavages, benzylic alcohol oxidations, demethylations, hydroxylations, and dimerizations (4, 24, 30). For instance, methoxybenzenes are often converted to quinones with the concomitant release of methanol. F'yrene is converted to a quinone via hydroxylation (27).Phenols can be dimerized by reaction with LiP or MnP (271.In addition, several re-

However. this does not adequately explain the kinetics that am observed when certain chemicals am added to LiP reaction mixtures. For example, organic acids were only oxidized in the presence of veratcyl alcohol (37, 38).Concomitant with the decarboxylation of these acids was the inhibition of veratryl aldehyde formation from the alcohol and the formation of organic acid anion radicals. If veratryl alcohol was merely protecting LiP from inactivation (convmion to cpd III), we would expect to see a time-dependent loss in the oxidation of these acids in the absence of veratryl alcohol. However, this was not the case. Other researchers have concluded that the inhibition of product formation (Le., veratryl aldehyde) is due to the redox cycling of the veratryl alcohol cation radical back to veratryl alcohol upon reaction of this radical with the organic acid (37-41). The increased ability of the fungus to achieve lignin oxidation using a low molecular weight, freely diffusible oxidant such as the Emrimn. Sci. Technol., Vol. 28. No. 2, IS94 81 A

veratryl alcohol cation radical gives this theory merit. Chemicals that have been found to be indirectly oxidized by this LiP-dependent process include lignin (4.21, the herbi1,2,4-triazole(43), and cide 3-&0 various organic acids (37,38). More recently it has been found that the fungus may use the veratryl alcohol cation radical to produce molecular oxygen from H,O, (44). Our laboratory results demonstrated that H,O, can be oxidized to the superoxide anion radical (O,*) by the veratryl alcohol cation radical. Subsequent dismutation of' , 0 resulted in the evolution of oxygen (details will be discussed later).

tryl alcohol cation radical and is dependent on Lip. As stated previously, the veratryl alcohol cation radical is an oxidizing species. Our unpublished laboratory results indicate a reduction potential for this radical of about 1.3 V. This radical can effectively oxidize certain chemicals by one electron. In some cases the radical formed from this oxidation can result in reduction. Initial studies concerning this reductive mechanism were performed using ethylenediamine tetracetic acid (EDTA). Although EDTA is often thought to be a metal chelator, the four carboxyl groups i n this molecule make it a good reductant for the veratryl alcohol cation radical. The chemistry of EDTA with regard to its reductive behavior has been discussed (48). We found that the veratryl alcohol cation radical could decarboxylate EDTA to an anion radical. In this process the veratryl alcohol cation radical is reduced back to veratryl alcohol (38). Direct evidence for EDTA radicals was obtained through the use of electron paramagnetic resonance (EPR) spin trapping experiments with a reaction mixture containing Lip, H,O,, veratryl alcohol, and EDTA. The EDTA radicals effectively reduced other chemicals such as nitroblue tetrazolium, tetranitromethane, and molecular oxygen

Degradation of oxidized pollutants Many of the pollutants that are degraded to CO, by white rot fungi are already highly oxidized. Examples include polychlorinated biphenyls (451, chlorinated phenols (461, and nitroaromatic munitions (12).In fact, the highly oxidized state of these chemicals makes them very resistant to other forms of microbial degradation. It was found that the Lip were involved in the degradation of these pollutants. because the rates and extents of degradation were significantly lower when the fungus was cultured with sufficient nutrients. These results have puzzled researchers for several years. It does not seem logical that an organism using an oxidative mechanismsuch as that catalyzed by a peroxidase-could effectively degrade these chemicals, which are already highly oxidized. Thus, it seemed that the chemicals must first be reduced before further conversion could take place. Our laboratory found that one of the major metabolites formed during l,l-bis(4-chloropheny1)-2,~,2trichloroethane (DDT) degradation by P. chrysosporium was the reductive dechlorination product 1,1bis(4-chlorophenyl)-2,2-dichloroeth a n e (DDD) (45). The a m i n o congeners of the explosive 2,4,6-trinitrotoluene (TNT) were also found in cultures of P. chrysosporium ) (47). Pentachlorophenol (F"provides another example of a highly oxidized pollutant that is degraded to CO, by white rot fungi. Therefore, it appears that the fungi do have some mechanism or mechanisms for reducing these chemicals. W-catalyzed chemical reduction One mechanism we discovered for reduction also involves the vera82 A Envimn. sci. Technol., Vd. 28, NO.2,1994

(38). The general scheme for the LiP-dependent reductive pathway of P. chrysospon'um is presented in Figure 3. More interestingly, it was found that carbon tetrachloride (CClJ could be dechlorinated using this Lip-dependent mechanism (49). Thus, it appears that the higbly oxidized chlorinated pollutants might be metabolized (oxidized) by the fungus through this LiP-dependent reductive pathway. Since EDTA is not produced by the *us, the question mom as to what might be the physiological electron donor if this reductive pathway was in fact used by the fungus.It is known that organic acids are commonly secreted extracellularly by many filamentous fungi including white rot fungi (50). Oxalic acid is secreted by P. chrysospon'um (39, 40, 51). In fact, it was produced in millimolar concentrations when the funguswes cultured in liquid under low-nitrogen conditions (40). The maximum oxalate concentration was attained the day prior to the detection of Lip activity in the cultures. As the LiP activity in the cultures increased, the concentration of oxalate decreased. This suggested that Lip might be involved in the metabolism of oxalate in vivo. In addition, oxalate was rapidly mineralized by nutrient nitrogen limited cultures of P. chry-

+

co*

sosporium (40). In vitro experiments using purified Lip demonstrated that oxalate could indeed serve as the electron donor for the LiP-dependent reductive pathway of P. chrysosporium. For instance, Popp et al. (39)reported the consumption of oxygen in reaction mixtures containing LiP, H,O,, veratryl alcohol, and oxalate. They concluded that the oxygen consumption arose from the reaction of the carbon dioxide anion radical with molecular oxygen. Thus, it appeared that the veratryl alcohol cation radical. generated via oxidation by Lip, oxidized oxalate to yield CO, and the carbon dioxide anion radical [CO,’-). The C0,’then reduced molecular oxygen to superoxide (Oz-). These investigators used EPR spin trapping experiments to provide direct evidence for the CO,’ radical and 0,’ in these reaction mixtures (39). In a separate study, Akamatsu et al. (37)demonstrated the liberation of “CO, in reaction mixtures containin Lip. H,O,, veratryl alcohol, and [’ 8Cl oxalate. Analogous experiments performed by Shah et al. (38) using EDTA further supported the idea that oxalate could function as the physiological electron donor for the LiP-dependent reductive pathway. In addition, oxalate can inhibit the formation of veratryl aldehyde in reaction mixtures containing Lip, H,O,, and veratryl alcohol (37).Presumably, this inhibition of veratryl aldehyde formation was the result of the reaction of the veratryl alcohol cation radical with oxalate, which reduced the cation radical back to veratryl alcohol. We detected the reduction of various electron acceptors via the reductive pathway using oxalate as the electron donor. In fact, the trichloromethy1 radical was detected in reaction mixtures containing LiP,H,O,, veratryl alcohol, oxalate, and carbon tetrachloride using the EPR spin trapping technique (unpublished data). Manganese peroxidase catalyzed reductions Recently, a mechanism for reduction using the M n p from P. chrysosporium has been elucidated. MnP typically oxidize Mn+’ to MII+~;the Mn” is then used to oxidize chemicals (52).However, in the presence of hydroquinones and Mncz, MnP has been found to catalyze reductions (53).It appears that h h + 3can oxidize hydroquinones to their corresponding semiquinone radicals,

which can then act as reducing agents. Thus, in the process of MnPdependent reduction, a hydroquinone is converted to a benzoquinone via two separate single electron oxidation s t e p s (see scheme in Figure 4). P. chrysosporium also produces an extracellular quinone reductase; however its role has not been conclusively established (54).Results from our laboratory suggest that the function of this quinone reductase might involve the maintenance of a hydroquinone pool for MnP-catalyzed reductions. Evidence for this hypothesis was obtained from reaction mixtures containing MnP, H,O,, Mn+’, and benzoquinone being unable to reduce oxidized cytochrome c. However, upon the addition of the quinone reductase, cytochrome c was reduced. Therefore, it was concluded that the quinone reductase was required to reduce benzoquinone to hydroquinone so that the reductive pathway shown in Figure 4 could function. It has also been reported that P. chrysosporium produces a number of quinones during the degradation of lignin or when grown on glucose (55).Because the fungus also excretes a quinone reductase, we believe that an MnP-dependent reductive mechanism using hydroquinones as the electron donors might be important in the degradation of lignin as well as environmental pollutants by these fungi. Plasma membrane potential A number of highly oxidized

chemicals are reduced by cultures of P. chrysosporium under conditions where the peroxidases do not exist. For example, TNT disappeared during the first and second days following starting cultures with spores (47).The Lip and MnP are not produced until about day three by these same cultures (56). Thus, it appears that the fungus had some sort of mechanism for converting TNT independent of Lip or MnP. The TNT metabolites found on day two were primarily the reduced products: 2-amino a n d 4-amino dinitrotoluenes. It was also found that these amino products could be obtained from TNT when the fungus was cultured with high levels of ammonia where the fungus is not ligninolytic, further evidence that the mechanism was independent of Lip or MnF’. The predominant pathway that has been reported for the bioconversion of TNT involves the reduction of TNT to the amino dinitrotoluene congeners. Organisms such as bacteria and mammals have intracellular nitroreductases which accomp l i s h t h e s e r e d u c t i o n s (57). However, despite extensive efforts, no such nitroreductase has ever been found for the white rot fungi. An alternative method of reduction used by many microbes, including filamentous fungi, involves the maintenance of a proton gradient across the plasma membrane. Recently, it has been reported that several redox active dyes could be reduced by various fungi using such a membrane potential (58).The

The roposed mechanism for the reduction of cytochrome c by &P Mn(ll)

MnP + H202

Mn(lll)

I

\

Envlron. Sd. Techd.. Val. 2%, No. 2,1994 89 A

ability of these dyes to be reduced by various fungi was related to the reduction potential of the dye. Results from our laboratory demonstrated that a plasma-membrane-dep e n d e n t r e d o x s y s t e m of P. chrysosporium was responsible for the reduction of TNT to the amino congeners (47). In this investigation,the reduction of TNT was found to depend on the presence of live intact mycelia. It was also found that the rate of TNT reduction by mycelia correlated with the rate of proton pumping. In many previous studies it has been observed that the fungus has the ability to conIml the pH of its environment, usually by lowering it to about 4.5. It now appears that the fungus does this through proton excretion. In addition, if a pollutant such as TNT is present, it can be reduced via the same mechanism. The mechanism apparently responsible for TNT reduction by P . chrysosporium is shown in Figure 5. Through reduction such as this, the fungi are able to lower the toxicity of TNT, which is important when high concentrations of the chemical are present. In a separate report, the detoxification and metabolism of TNT by P. chrysosporium was discussed (59). It appeared first that mono and diamino congeners of TNT were formed. Following these reductions, the amino congeners were oxidized by MnP. The evolution of CO, corresponded to the production of Lip by the fungus. We believe that the membrane potential of P. chrysosporium may also be involved in the reduction of other chemicals, and an investigation of this possibility is under way. Production of active oxygen species Earlier work by Haemmerli et al. (60) demonstrated that at pH 3.0the veratryl alcohol cation radical can react with molecular oxygen to produce superoxide (0,’). This reaction resulted in a multitude of products being formed from veratryl alcohol, including two quinones and two-ring opened lactones. It also became apparent that the Lipdependent oxidation of veratryl alcohol (VA) to its cation radical (VAS+)might be important in regulating the oxygen concentration in the environment surrounding the fungus. This idea stems from the observation that H,O, and oxalate, both of which are excreted by the fungus, can react with the veratryl alcohol cation radical (44). In one 04 A

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case, oxygen is produced via reaction 2 when H,O, reacts with the cation radical.

tensively researched prior to the discovery of Lip. Evidence for the production of 02-, hydroxyl radical (.OH) and H,O, by intact cultures of VA” + H,O, + VA + 0,’ + the fungus was obtained during this 2H+ (1) period. For example, it was found 0,’ + HO,. + H+ -) H,O, + 0, ( 2 ) that lignin degradation could be inhibited by superoxide dismutase as Net reaction = 2 VA’+ + 2H,O, + well as various scavengers of the hy2VA + 0, + 2H+ + H,O, droxyl radical (64).The evolution of Alternatively, if oxalate reacts ethylene gas hom the .OH-depenwith the cation radical, oxygen is dent oxidation of a-keto-Lthiobutyric acid was found to be 100-fold consumed in reaction 4. higher in nitrogen-starved cultures (Le., ligninolytic) as opposed to niVA’+ + oxalate + VA + trogen-sufficient cultures of P. chryCO,+CO,’ (3) sosporium (65). These results sugCO,’ + 0, + co, + 0,gest that lignin peroxidases might (4) be involved in .OH production by Net reaction = V A ” + oxalate + this fungus. 0, + VA + 2C0, + 0,’ Recently we reported that puriIn vitro experiments demon- fied Lip can catalyze the production strated that increasing the concen- of .OH (40). As was mentioned eartration of H,O, inhibited LiP-depen- lier, oxygen C M be reduced to 0,’ d e n t oxygen c o n s u m p t i o n i n by the Lip-catalyzedreductive pathreaction mixtures containing oxa- way. The 0,’ produced in this prolate and veratryl alcohol. In addi- cess can be used to reduce chelated tion, increasing the concentration of ferric iron to the ferrous form. The oxalate decreased Lip-dependent reaction of ferrous iron with H,O, oxygen production. Thus, it ap- (which is also produced by the funpeared that the relative concentra- gus) results in the production of tions of H,O, and oxalate deter- .OH. Initial evidence for this mechm i n e d w h e t h e r o x y g e n w a s anism was obtained from experiproduced or consumed. These reac- ments in which ferric iron reductions may have physiological rele- tion was monitored in reaction vance, as the oxygen concentration mixtures containing Lip, H,O,, vermarkedly influences the degrada- atryl alcohol, oxalate, or EDTA ustion of chemicals by this fungus.For ing the ferrous iron specific chelator example, numerous researchers 1,lO-phenanthroline. Direct evihave observed increased rates and dence for the .OH was obtained usextents of lignin degradation when ing EPR spin trapping experiments. they used 100% 0, rather than air The proposed pathway for .OH radto flush cultures (61-63). ical production by Lip is presented The involvement of activated ox- in Figure 6. ygen in lignin degradation was exThe use of the hydroxyl radical as

FIGURE 5

Proposed mechanism for TNT reduction by white roi

I

intracellular

7 7

, TNT

\;xfracel~ar AmDNTa CH3

CH3

NH,

NO,

I

an oxidizing agent for the treatment of hazardous wastes has received much attention i n recent years. Methods of .OH production include addition of ferrous iron directly to H 0, (Le., Fenton's reagent) (661, pkotolysis of H,O, (67).and photolysis of titanium dioxide (68). It appears that the fungus has its own method of generating Fenton's reagent. This may have significance with regard to the degradation of halogenated highly oxidized pollutants, as the .OH has a reduction potential of about 1.5 V (69).It has been reported that PCBs as well as chlorinated phenols are hydroxylated by Fenton's reagent (66, 70). Concomitant with the hydroxylation of these compounds was the release of chloride. Therefore, it appeared that the .OH reacted with these compounds through electrophilic addition. It is possible that .OH could be involved in the degradation of several pollutants by this fungus, for it is known that .OH reacts with most organic molecules at diffusion limited rates (71). Fuhm considerations The mechanisms described here make the white rot fungi technology unique among more established methods of bioremediation (Le,, mixed microbial communities). Bacteria, for example, rely on various enzymes including monooxygenases, dioxygenases, nitroreductases, haloreductases, esterases, and cytochrome P-450~to degrade chemicals (57.72-76).These are enzymatic conversions that follow normal Michaelis-Menton type kinetics and occur intracellularly. Frequently these enzymes must be induced by the pollutant for degradation to occur.Thus,bacterial cultures are often selected by using various growth enrichment techniques (77).Following the isolation of the appropriate microbes. the system must be preconditioned to the pollutant. In contrast, the white rot fungi

use a very nonspecific, free radical mediated process to degrade chemicals, which often follows pseudofirst-order kinetics. Although this process of pollutant degradation requires enzymes (e.g., Lip and Mnp), the same group of enzymes appears to be able to catalyze a wide variety of oxidations and reductions, as well as to produce highly reactive oxygen species. More important, these reactions occur in the extracellular environment of the fungi. The complexity of such mechanisms makes the design of a bioremediation strategy quite different from that of existing technologies. The growth of the fungi and production of Lip and MnP are not the only factors to be considered. The production of H,O,, veratryl alcohol, and various electron donors [e.g., organic acids and hydroquinones), which are involved in the free radical process, are equally important. In fact, Kurek a n d Odier ( 4 2 ) showed that increasing the Lip concentration, by adding enzyme to cultures, actually dermased the rate of lignin degradation after a certain point. It is possible that the addition of exogenous Lip could have decreased the amount of electron mediators such as the veratryl alcohol cation radical. This could occur if the excess Lip oxidized the veratryl alcohol cation radical to its corresponding aldehyde. With the current level of knowledge of the mechanisms employed by the fungi, we may be able to manipulate the culture conditions so as to facilitate the degradation of certain chemicals. For example, TNT requires reduction before further degradation can occur. The reduction rate of TNT by white rot fungi is proportional to the mycelial mass, is faster at higher pH, and does not require Lip or M n p (47). Therefore, growing the fungus initially at a high pH and under high nutrient conditions will facilitate the reduction of TNT because myce-

lial growth is greater under these conditions. When all of the TNT has been converted to the amino congeners, the system should be nutrient deficient to induce the M n p and Lip that are required for the further degradation of TNT to CO,. It should not be assumed, however, that the white rot fungi will always be successful when applied as an axenic culture to a particular site. It is likely that using these fungi in conjunction with other microbes will optimize the decontamination of pollutants at many sites. For instance, many bacteria possess the enzymes required to rapidly convert various aromatic pollutants to b-ketoadipate, from which they obtain energy. However, several of these pollutants may be toxic to bacteria. Filamentous fungi do not always degrade pollutants to gain energy. Instead, it appears that the conversion of pollutants by fungi is often a detoxification mechanism (78).These two factors (i.e., detoxification by fungi and rapid assimilation by bacteria) may create a synergistic environment that could greatly facilitate the degradation of hazardous chemicals. A more specific example of fungi assisting in bacterial degradation involves highly chlorinated phenols. Although bacteria may possess the dioxygenase enzymes required to catalyze ring cleavage, the negative inductive effect of the chlorines on these phenols makes them very poor substrates for the bacterial enzymes (79).In some cases these highly chlorinated phenols can even act as suicide inhibitors of bacterial dioxygenases (79). If white rot fungiwere used, reductive dechlorination of the phenol via the free radical, mediated, or membrane-dependent mechanism could take place. This would reduce the number of chlorines on the phenol and make it a better substrate for bacterial enzymes. It is also known that white rot

FIGURE 6

Proposed pathway for the productionof 'OH by lignin peroxidase

LiPW

L%d)

36+ . : 3c

co2+ c0;cop

1 :

F&

Fe(")

Oxalate VA = verawl alcohol, VA- = veratryl alcohol cation radical The cmbon dioxide anen

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fungi have the ability to methylate phenolic pollutants, thus reducing their toxicity (80).Lamar et al. (81) have shown that white rot fungi converted pentachlorophenol to pentachloroanisole in soils, which appears to be the initial step in the metabolism of this pollutant (81).In fact, many filamentous fungi appear to methylate chlorinated phenols. This is thought to be a detoxification mechanism, as the chlorinated phenols are inhibitors of mitochondrial electron transport (80). In some cases, white rot fungi may antagonize the growth of other microbes. Not only can these fungi grow under nutrient-deficient conditions, which do not promote bacterial growth, but they also produce highly reactive oxygen species. In addition to degrading pollutants, oxygen radicals such as .OH can cause oxidative damage to other microbes. White rot fungi can adjust the pH of their environment, usually lowering it. Thus, bacteria that have a pH optimum different fiom these fungi may not grow well after white rot fund have been introduced. Manv of the subiects discussed above may explain 'what appear to be anomalies concerning the degradation of environmental pollutants by white rot fungi. Kohler et al. (82) concluded that the lignin peroxidases were not involved in the degradation of DDT by the white rot fungus P. chrysosporiurn. This conclusion was made because DDT disappeared even under nutrient nitrogen-sufficient conditions (Le., when the Lip are not produced). We now know, however, that reductive dechlorination of DDT occurs in nonligninolytic cultures of the fungus (21).Disappearance of DDT occurs but DDT mineralization does not. Thus, when one assays only for the disappearance of DDT, it appears that DDT is metabolized under nonligninolytic conditions. The same example applies to TNT as it is first reduced by the membrane potential under nonligninolytic conditions. Therefore, TNT disappears under nonligninolytic conditions, but mineralization is much greater under ligninolytic conditions (59). As another example, Spiker et al. (83)concluded that P. chrysosporium would not be useful for the bioremediation of niwoaromatic ex-

-

plosives such as TNT because TNT could be toxic to the fungus. The toxicity studies in their report were performed by adding 0-100 ppm TNT to either spores or 11 mg dry weight of mycelia in 50 mL of malt 88 A Envimn. sci. Technol.. Vol. 28,No. 2,1944

extract broth. Using these conditions, it was conclided that TNT concentrations as low as 20 ppm were toxic to the fungus. However, at this time. the initial step of reducing TNT by the cell surface membrane potential of the fungus was not known (47).We now realize that this could be a mechanism for TNT detoxification and that the rate of reduction and detoxification would he proportional to the mycelial mass. Subsequently, we demonstrated that the toxicity of TNT to the fungus was inversely proportional to the mycelial mass ( 4 9 ) . When 360 mg dry weight mycelia were used in 150 mL of liquid culture, concentrations of TNT as high as 1000 pprn were not toxic to the fungus. In contrast. when only 5.5 mg of mycelia were used, 1000 pprn TNT was found to be quite toxic to the fungus. Therefore, it appears that effective biodegradation may be attained simply by changing the conditions. In conclusion, the interest in the use of white rot fungi for bioremediation appears to be continually growing Although much has been learned in recent years about the mechanisms used by these fungi, continued research is warranted. The ability of these fungi to degrade such a wide variety ofenvironmentally persistent pollutants makes their potential use an extremely valuable tool with regard to the hazardous waste problem. However, only through our understanding of the unique mechanisms used by these fungi we be to this technology successfully. Acknowledgments This research was su ported in part by NIH grant ES04922 !om the National Institute of Environmental Health Sciences. The authors acknowledge the many postdoctoral fellows, graduate students,technicians,and students who actually conducted this research and published the primary articles. They thank Terri Maughan for typing the manuscript. References (1) Sarkanen, K. V.: Ludwig, C. H. In

Lignins: Occurrence, Formation and Structure: Wiley-Interscience: New York, 1971: pp. 1-18. (2) Tien. M.: Kirk, T. K. Proc. NaiJ. Acad. Sci. USA 1984,81,2280-84.

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phys. 1984,234,35342, M. CRC f i t . Rev. Microbiol.

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David P. Barr (I] is a doctoral candidate in the Deportment of Chemistryand Biochemistry at Utah State University. He holds a B.S. degree in biology from Western Stote College of Colorado. His research interests include free radical chemistry involved in biological processes, specifically free radical reactions catalyzed by peroxidases. Steven D. Aust (r) is a professor

in the Department of Chemistry and Biochemistry at Utah State University and director of an NIEHS Superfund Basic Research and Training grant program. He is also executive vice-president for research at Intech One-Eighty Corporation, which obtained on exclusive license from the university to market the white rot fungi technology. His research interests are in free radicals in biology.

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7 perspectives and recommendations from the Chemrawn VI1 conference, Chemical Research Applied to World Needs, and addresses the problem of protecting the earth's atmosphere from the damage caused by human activity. It addresses the problem from the many economic, political, and technical perspectives that must be considered by the policy makers involved today. Viewpoints of science and industry, as well as developing countries, are presented, and the theme of international cooperation is emphasized. The book serves as an introduction to the subject for members of the general public and is a useful resource for environmental scientists, workers in industry, and government and international organizations involved with public policy decisions. John W. Birks, University of Colorado, Editor Jack G. Culvert, National Center for Atmospheric Research, Editor Robert E. Severs, University of Colorado, Editor 180 pages (1993) Clothbound: ISBN 0-8412-2532-X-$34.95 Paperbound: ISBN 0-8412-2533-8-$24.95

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