Pollutant Degradation by White Rot Fungi

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Pollutant Degradation by White Rot Fungi. David P. Barr* and Steven D. Aust* ..J".,. Contents. I. Introduction. 49. II. The Lignin-Degrading System. 50. A. Lignin ...
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Pollutant Degradation by White Rot Fungi David P. Barr* and Steven D. Aust* ..J".,

Contents I. Introduction II. The Lignin-Degrading System A. Lignin Peroxidases and Manganese-Dependent Peroxidases B. Hydrogen Peroxide Generating Enzymes C. Other Components III. Mechanisms of Pollutant Degradation A. Direct and Indirect LiP-Dependent Oxidation B. The LiP-Dependent Reductive Pathway.................................... C. Pollutant Detoxification Summary Acknowledgments References

49 50 52 52 53 57 57 59 62 66 67 67

.

Pollutant Degradation

51

Table 1. Examples of Environmental Pollutants Degraded by Phanerochaete chrysosporium 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, l-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 From Barr and Aust (1994c).

Schoemaker (1990), and Tien (1987). Two of the initial findings in the field of lignin degradation by white rot fungi were that H202 is produced by these fungi (Koenigs 1974) and that lignin degradation seems to occur in response to nutrient limitation (Kirk et al. 1978). The fact that extracellular H202 was produced led many researchers to investigate a possible role of oxygen radicals, such as the highly reactive hydroxyl radical, in lignin degradation (Forney et al. 1982). Although some evidence was obtained to support this hypothesis in the early 1980s, the discovery in 1984 of a group of extracellular heme proteins produced by white rot fungi provided the key "----" for elucidating their ligninolytic ability and the role of H202• The heme proteins were demonstrated to have peroxidase activity and thus were named lignin peroxidases (LiP) (Tien and Kirk 1983). Later, it was found

52

D.P. Barr and S.D. Aust

that some of these peroxidases require manganese (II) for activity, and therefore these were called manganese-dependent peroxidases (MnP) (Glenn and Gold 1985). Because peroxidases require H202 for activity, extensive research has also been performed to elucidate the mechanism by which white rot fungi produce H202. To date, two intracellular glucose oxidases (Kelley and Reddy 1986) as well as an extracellular glyoxal oxidase (Kersten 1990) have been found and are thought to provide the H202used by the peroxidases to degrade chemicals.

/-

A. Lignin Peroxidases and Manganese-Dependent Peroxidases Lignin peroxidases and manganese-dependent peroxidases are produced by white rot fungi in response to nutrient limitation and are similar to other peroxidases in that they are activated by H202(Tien 1987). The heme iron in the LiP or MnP is in the ferric state, often referred to as the "resting" or "native" state. Hydrogen peroxide oxidizes the heme by two electrons to a r>. form of the enzyme known as compound I. The two-electron oxidized heme of compound I is considered to be a ferryl-z-porphyrin cation radical and is highly reactive toward a variety of reducing chemicals. Compound I oxidizes such reducing chemicals to free radicals, which results in the formation of compound II. Compound II can then react with another chemical and be reduced back to the resting enzyme. The catalytic cycle of LiP is shown in Fig. 1. Manganese-dependent peroxidase differs from LiP in that Mn+2 serves as the reducing agent for compound I and compound II. The Mn+3 produced during the cycle can diffuse away and promote the subsequent oxidation of other chemicals. This process (i.e., the LiP or MnP catalytic cycle) is thought to be the method by which the fungus initiates the oxidation and subsequent degradation of lignin and several environmental pollutants. The details of this process with regard to pollutant degradation will be discussed later. For a more extensive discussion of LiP and MnP, readers should refer to several excellent reviews (Tien 1987; Kirk and Farrell 1987; Cai and Tien 1993). B. Hydrogen Peroxide Generating Enzymes Two major enzymes, glucose oxidase and glyoxal oxidase, are thought to be responsible for H202production and have been isolated from white rot fungi (Kelley and Reddy 1986; Kersten and Kirk 1987). Both of these enzymes utilize their respective substrates to reduce molecular oxygen to H202.In addition, the activity of these enzymes is also induced by nutrient limitation. Kelley and Reddy (1986) have isolated an intracellular glucose-Ioxidase from Phanerochaete chrysosporium and have suggested that the H202 produced by this enzyme diffuses to the extracellular environment. Howevo'l more ""] an ext ra cellular oxidase 9xi~MVI whiT~

nOXal

j utilizes two carbon aldehydes, a-hydroxy carbonyl and o-dicarbonyl compounds, as substrates for H202 production was discovered (Kersten and

Y'">

Pollutant Degradation



'.

53 HP2

G'"

H20

RH

o

GL

0*

Compound II

Compound 1



RH

Fig. 1. The peroxidase catalytic cycle. Iron is in the form of protoporphyrin IX. (From Barr and Aust 1994c. Reprinted with permission from Environ Sci Technol 28:78A-87A. © 1994 American Chemical Society.)

Kirk 1987). The fact that this enzyme was found in the extracellular environment and that its substrates are common products of lignin breakdown was particularly intriguing. Glyoxal oxidase is able to utilize a broad range of substrates, such as glyoxal, formaldehyde, glycoaldehyde, and methyl glyoxal. It was also reported that the two substrates that provided the greatest activity, methyl glyoxal and glyoxal, were found in the extracellular fluid of cultures of P. chrysosporium grown on glucose (Kersten and Kirk 1987). Thus, it appears that the fungus produces substrates for this enzyme from glucose, even when it is nutrient limited. C. Other Components

'---./

1. Veratryl Alcohol. Due to the extremely complex structure of lignin, the idea of free-radical charge transfer or electron-mediated oxidation has received considerable attention (Harvey et al. 1986). This theory suggests that small organic molecules are oxidized to free radicals by LiP. These radicals then diffuse into the lignin matrix and promote the further oxidation of the polymer. Therefore, the role of LiP in lignin degradation would be considered that of an initiator of a free-radical chain reaction.

54

D.P. Barr and S.D. Aust

Veratryl alcohol (3,4-dimethoxybenzyl alcohol) is a compound synthesized by the white rot fungus P. chrysosporium (Shimada et al. 1981) and has been the focus of extensive research. For example, the oxidation of veratryl alcohol to veratryl aldehyde is the standard assay for determining the activity of LiP (Tuisel et al. 1990). The oxidation of veratryl alcohol proceeds via two separate one-electron steps involving reactions with compound I and compound II. The intermediate one-electron oxidized product is known as the veratryl alcohol cation radical (Harvey et al. 1986; Gilardi et al. 1990). Many researchers believe that the cation radical can diffuse away from the active site and co-oxidize other chemicals, such as lignin. Thus, the production of veratryl alcohol by the fungus would be important for electron mediation. This seems likely when one considers the oxidative ability that the fungus would gain by using a freely diffusible oxidant, such as the veratryl alcohol cation radical. In addition, veratryl alcohol is an excellent substrate for LiP; therefore, without mediation, it should inhibit the LiP-dependent degradation of . other chemicals by the fungus. However, a number of studies suggest that exactly the opposite might be expected. The addition of veratryl alcohol to cultures actually enhances LiP activity (Kirk et al. 1986) and the degradation of lignin (Faison and Kirk 1985). This phenomenon has been so widely recognized that it has become common practice to add veratryl alcohol to liquid cultures of the fungus when studying the degradation of chemicals or when producing LiP and MnP to be used for in vitro investigations. For example, the oxidation of organopollutants, such as azo, triphenylmethane, heterocyclic, and polymeric dyes (Ollika et al. 1993), 1,2,4-aminotriazole (Tuisel et al. 1992), and various polycyclic aromatic hydrocarbons (Haemmerli et al. 1986) by LiP was shown to be markedly increased when veratryl alcohol was added to the reaction mixture. Veratryl alcohol also seems to have a protective effect on the activity of LiP. As with other peroxidases, compound II of LiP can react with H202 to form a catalytically inactive form of the enzyme known as compound III (Cai and Tien 1990; Wariishi and Gold 1989, 1990). This frequently occurs when a substrate reacts more readily with compound I of LiP than with compound II. In such situations, the formation of compound III occurs in a manner that is dependent on the concentration of H202• Phenols provide the most intensively studied example of this phenomenon (Harvey and Palmer 1990), but compound III formation has also been observed during the oxidation of other chemicals (Haemmerli et al. 1986). On the other hand, veratryl alcohol reacts readily with compound II of LiP and returns it to the resting ferric state (Tien et al. 1984). Therefore, it is believed that the role of veratryl alcohol in vivo is to prevent the formation of inactive r>. compound III during the oxidation of chemicals that are poor substrates fer ~QmfQ1JnQ II ~W~rii~hi~nQ GQIQ r,

l~~~l'

Results from our laboratory have suggested yet another role for veratryl

Pollutant Degradation

55

alcohol during LiP catalysis. The formation of LiP compound III is likely to occur to some degree during the degradation of chemicals by the fungus. For example, compound III can be formed by the complexation of superoxide (Oi) with resting ferric enzyme (Yamazaki and Piette 1963). Previous studies have shown that Oi- is produced in liquid cultures of P. chrysospor. ium (Faison and Kirk 1983) as well as during steady-state LiP turnover (Barr et al. 1993). It was also demonstrated that superoxide dismutase (SOD) prevented the formation of compound III during the oxidation of veratryl alcohol by LiP (Barr and Aust 1994a). This was thought to occur because Oi- is produced from the reaction of H202 with the veratryl alcohol cation radical (Barr et al. 1993). Therefore, it would be advantageous for the fungus to have a mechanism whereby inactive compound III could be converted back to active enzyme. This type of mechanism was found to occur with the compound III form of horseradish peroxidase (HRP). Nakajima et al. (1991) demonstrated that HRP compound III could react with certain phenoxyl radicals and, in the process, be converted back to the resting ferric enzyme. This was thought to be an oxidative conversion in which an electron was abstracted from compound III (which exists as a ferric-superoxo complex) by the radical. Thus, the radical is reduced back to the parent compound and one mole of O2 is liberated per mole of compound III. Findings from our laboratory suggest that certain methoxybenzene cation radicals formed during LiP catalysis, including that of veratryl alcohol, are able to convert LiP compound III back to the ferric enzyme by this mechanism (Barr and Aust 1994b). The reaction for the oxidative conversion of LiP compound III back to ferric LiP via the veratryl alcohol cation radical (VA +) is illustrated below (the O2 comes from the superoxo portion of LiP-compound III). LiP-compound III

+ VA

+ ---+

ferric-LiP

+ VA + O2,

Therefore, for these reasons (i.e., electron mediation, protection against compound III formation, and reactivation of compound III), it seems that the fungus produces veratryl alcohol from glucose, even when under nutrient-deficient conditions.

----./

2. Oxalate. It has been known for some time that certain organic acids are produced and excreted by several basidiomycetes (Takao 1965). It was found that P. chrysosporium produces and excretes oxalate (Barr et al. 1992; Kuan and Tien 1993). The production of oxalate by these fungi is thought to be important for pollutant degradation for two reasons. The first involves the manganese-dependent peroxidases. Certain organic chelators are able to influence the reactivity of transition metals, often by lowering their redox potential. Kuan and Tien (1993) demonstrated that micro-

D.P. Barr and S.D. Aust

56

molar concentrations of oxalate significantly increased the oxidation rate of Mn+2 to Mn+3 by MnP. Also, organic acids, such as oxalate, may stabilize trivalent manganese and allow it to diffuse away from the enzyme and promote the further oxidation of chemicals (Glenn et al. 1986). Another role of oxalate in the degradation of chemicals is the LiPdependent reductive pathway, which involves veratryl alcohol as an electron mediator. This pathway can result in the reduction of highly oxidized pollutants as well as the production of the hydroxyl radical COH). The details of LiP-dependent reduction and ·OH radical production are discussed with regard to specific pollutants later. 3. Quinones and Quinone Reductases. During lignin breakdown, a variety of products are liberated, including quinones and hydroquinones (Schoemaker 1990). The fate of these quinones has been the topic of some ~.

MnP+Hp2 Mn(II)



Mn(HI)

HQ.

CBQRase+ Cellobiose

Q

Cyt.c.;

Cyt.cral

Fig. 2. Proposed mechanism for reduction of chemicals by MnP. H2Q indicates hydroquinones, HQ· their corresponding semiquinones, and Q their corresponding 9uinone~. {From Chun et al. 1993. Reprinted with permission from Academic

O

Press.)

/"

Pollutant Degradation

57

research, primarily due to the discovery of an enzyme known as cellobiose quinone reductase. This enzyme is produced by several white rot fungi, including P. chrysosporium, and catalyzes the cellobiose-dependent twoelectron reduction of quinones to hydro qui nones (Westermark and Eriksson 1974). It is found in the extracellular fluid of liquid cultures of the fungus, but a conclusive role for the enzyme has not yet been established. One possible role involves the MnP. Trivalent manganese serves as an excellent oxidant for a variety of hydroquinones (Chung et al. 1993). The semiquinone radicals produced by this oxidation effectively reduce other more oxidized chemicals. The quinone formed by this process is reduced back to the hydroquinone by cellobiose quinone reductase. Thus, highly oxidized pollutants, which must first be reduced before further metabolism can take place, might undergo this type of MnP-dependent reduction (Chung et al. 1993). A scheme for MnP-dependent reduction using ferricytochrome C as the electron acceptor is presented in Fig. 2. ------./

III. Mechanisms of Pollutant Degradation A. Direct and Indirect LiP-Dependent Oxidation

\...J

A unique feature of the LiP compared with other peroxidases is its relatively high redox potential (Kersten et al. 1990). Thus, chemicals having high redox potentials that are not oxidized by other peroxidases are oxidized by LiP. Polycyclic aromatic hydrocarbons (PAH), the primary constituent found in coal tar and creosote, provide an example of pollutants that are directly oxidized by LiP (Haemmerli et al. 1986). Aust and coworkers (1989) demonstrated that, in a period of 30 d, phenylnaphthalene, benzo [a] fluorene, l-rnethylpyrene, and benz[a]anthracene were all degraded to near nondetectable levels by ligninolytic cultures of P. chrysosporium. In another study using ligninolytic cultures of P. chrysosporium and 14Clabeled phenanthrenes, 6070 of the chemical was converted to carbon dioxide (i.e., mineralized) in 30 d (Bumpus 1989). Mineralization continued after 30 d when supplemental glucose was added to the cultures. Hammel et al. (1986) reported that both benzo[a]anthracene and dibenzodioxin are oxidized by purified LiP. The oxidation of a variety of other PAHs by LiP was also investigated. The relative reactivity of LiP toward these chemicals correlated with their ionization potentials (i.e., the energy required to remove one electron). Cyanides are also effectively oxidized by LiP, which is unique because cyanide is a potent inhibitor of most heme-containing enzymes (Knowles 1976). The ability of LiP to oxidize cyanide allows the fungus to effectively degrade the pollutant to CO2, Shah and Aust (1993) found that the rate of cyanide mineralization was linear with respect to its concentration up to 2 mM when added to ligninolytic liquid culture of P. chrysosporium, and 30% of an initial 2-mM cyanide concentration was converted to CO2 in 3 d.

D.P. Barr and S.D. Aust

58

Up to 45070 conversion to CO2 occurred in 3 d when 200 ItM cyanide was used. The fungus was also able to mineralize cyanide in soil. Cyanide was converted to the cyanyl radical when added to a reaction mixture containing purified LiP and H202• Therefore, this conversion is likely the first step in cyanide metabolism by the fungus. Various dyes have been extensively studied and found to be directly oxidized by LiP (Cripps et al. 1990). The decolorization of azo, triphenyl methane, and heterocyclic and polymeric dyes occurs rapidly when they are added to ligninolytic cultures of P. chrysosporium. For example, Bumpus and Brock (1988) found that the triphenyl methane dye, crystal violet, was 99% decolorized in 24 hr when added to ligninolytic cultures of P. chrysosporium. Ollika et al. (1993) found that several azo, triphenylmethane, heterocyclic, and polymeric dyes were extensively decolorized by purified LiP. However, the decolorization was markedly increased by the addition of veratryl alcohol to the reaction mixture, which brings up an important point. As mentioned earlier, veratryl alcohol appears to increase the rate and extent of chemical degradation by white rot fungi. One reason for this may be that these chemicals (e.g., dyes, PAH, etc.) are efficient substrates for compound I of LiP but not compound II. In this case, if veratryl alcohol were not present, compound II would react with H202 to form the inactive compound III. Thus, the presence of veratryl alcohol ensures that compound II is reduced to the resting ferric enzyme. In addition, the veratryl alcohol cation radical produced during the reduction of compound II can convert any compound III that is formed back to resting ferric enzyme. In other cases, veratryl alcohol may serve as an electron mediator to facilitate the oxidation of pollutants. Oxidation of veratryl alcohol to its cation radical is catalyzed by compound I and/or compound II. Electron transfer between the veratryl alcohol cation radical and the pollutant would then occur, resulting in oxidation of the pollutant. This type of LiPdependent reaction, termed indirect oxidation, seems to be the way in which pollutants, such as the herbicide aminotriazole, are initially metabolized (Tuisel et al. 1992). Tuisel et al. (1992) found that aminotriazole was effective at inhibiting veratryl alcohol oxidation by LiP. The inhibition appeared to be competitive with respect to veratryl alcohol, but very little binding of aminotriazole to LiP was observed. In addition, the presence of veratryl alcohol was almost an absolute requirement for the oxidation of aminotriazole by LiP. Therefore, it became apparent that veratryl alcohol may be acting as an electron mediator for the oxidation of aminotriazole. In this situation, the veratryl alcohol cation radical would be reduced back to veratryl alcohol, and it would appear as if veratryl alcohol oxidation were being inhibited (i.e., because no veratryl aldehyde formation was observed). The following reaction scheme depicts this mechanism of indirect oxidation

Oy liP (VA and V!' + nr~v~rntrylnlMk~l dt\~ ~hMt~Mf~J!Ml.and1~ and AT

+

indicate aminotriazole and the aminotriazole radical).

"

/\

r>.

PollutantDegradation

"

VA

LiP, H202:>

VA'+

VA+

+ AT

> VA + AT+

59

Also, aminotriazole alone did not prevent inactivation (i.e., compound III formation) of LiP when LiP was incubated with H202 and aminotriazole. This would indicate that veratryl alcohol was also affecting aminotriazole oxidation by protecting against compound III formation. B. The LiP-Dependent Reduction Pathway

'----'"

,------,'

One of the most puzzling anomalies that has been observed with regard to pollutant degradation by white rot fungi is that certain chemicals that are already very electron deficient are oxidized to CO2 by ligninolytic cultures of the fungus. In addition, these chemicals, which include 1,1bis(4-chlorophenol)-2,2,2-trichloroethane (DDT), carbon tetrachloride, and 2,4,6-trinitrotoluene (TNT), are not oxidized by purified LiP. Therefore, it seems that such pollutants should first be reduced before oxidations by LiP can occur. 1. Reduction of Highly Oxidized Chemicals. A mechanism has recently been elucidated whereby LiP can catalyze reductions using secondary metabolites that are produced by the fungus (Shah et al. 1992; Barr et al. 1992). The veratryl alcohol cation radical readily reacts with oxalate to produce the carboxylate anion radical. The veratryl alcohol cation radical is reduced back to veratryl alcohol (Fig. 3). Thus, veratryl alcohol acts as an electron mediator for the oxidation of oxalate. Ethylene diamine tetraacetic acid (EDTA) is also an efficient reductant for the veratryl alcohol cation radical and is capable of supporting the LiP-dependent reductive pathway. Shah et al. (1992) initially used EDTA to demonstrate the LiPdependent reductive pathway, whereas oxalate is thought to be the physiological reductant (Barr et al. 1992). Carbon dioxide is evolved from the oxidation of oxalate, which makes the reaction quite favorable thermodynamically. The other product, the carboxylate anion radical (CO;), is a powerful reducing agent with a reduction potential of approximately - 1.9 V (Simic 1990). The primary evidence for this pathway was provided by using electron paramagnetic resonance (EPR) spin-trapping techniques. In a reaction mixture containing LiP, H202, veratryl alcohol, oxalate, and the spin trap 5,5-dimethyl-l-pyrroline-N-oxide (DMPO), the COi- radical adduct spectrum was observed (Popp et al. 1990). When any of the reactants was omitted, the CO;- was not observed. Additional evidence was obtained when [14C] carbon dioxide was liberated from the same reaction mixture using radiolabeled oxalate (Akamatsu et al. 1990). Once again, the evolu-

D.P. Barr and S.D. Aust

60 CHl)l-{

CH20H

LiP/HP2

> OCH3

OCH3 OCH3

OCH3

Cation radical

Veratryl alcohol

,~

Organic acid radical

Organic acid

Decarboxylated organic acid

Electron acceptor

+C02

Reduced electron acceptor

Fig. 3. Mechanism for LiP-dependent reduction of chemicals. Organic acids shown to serve as reductants for the veratryl alcohol cation radical include EDT A and ~ oxalate (which is secreted by the fungus). Electron acceptors studied include cyto-

~~[[~~~mm[WI 1m 1 ~i\iliiij~1iil[~[~~~[~m~,

cnromc C n1\r~~

and carbon tetrachloride. (From Barr and Aust 1994c. Reprinted with permission from Environ Sci Technol 28:78A-87 A. © 1994 American Chemical Society.)

Pollutant Degradation

"



~.

61

tion of CO2 required that all reactants (i.e., LiP, HzOz, veratryl alcohol, and oxalate) be present. The CO;- radical produced is able to reduce a variety of chemicals, including molecular oxygen, ferricytochrome C, nitroblue tetrazolium, and ferric iron (Shah et al. 1992). More importantly, the CO;- radical can reductively dechlorinate carbon tetrachloride as well as other halogenated alkanes. Using the LiP-reductive reaction mixture (i.e., LiP, H202, veratryl alcohol, and oxalate) described above, Shah et al. (1993) were able to detect the trichloromethyl radical when phenyl-t-butylnitrone (PBN) was used as the spin trap. In addition, unpublished results from our laboratory have shown that carbon tetrachloride can be mineralized by P. chrysosporium, but only when cultured under ligninolytic conditions. Therefore, it appears that LiP may even be required for the metabolism of highly oxidized pollutants, such as carbon tetrachloride. 2. LiP-Dependent Production of 'OH. As stated earlier, it was known that white rot fungi produced extracellular H202 even before the lignin peroxidases were discovered (Koenigs 1974). Therefore, researchers thought that lignin degradation occurred due to extracellular production of active oxygen species, such as the hydroxyl radical ('OH). In support of this theory, Forney et al. (1982) reported that 'OH was produced by cultures of P. chrysosporium, In a separate report, it was found that lignin degradation could be inhibited by superoxide dismutase as well as various scavengers of 'OH, which further implicated a role of oxygen radicals in lignin degradation (Faison and Kirk 1985). Interestingly, Forney et al. (1982) demonstrated that the evolution of ethylene gas from the 'Ol-l-dependent oxidation of o-keto-v-thiobutyric acid was lOO-foldhigher in ligninolytic cultures (i.e., nitrogen limited) than in nonligninolytic cultures. These results suggested that the lignin peroxidases may be involved in the production of 'OH by the fungus. Since then, it has been found that 'OH can be produced by the LiP-dependent reductive pathway (Barr et al. 1992). In the absence of another electron acceptor, the carboxylate anion radical will reduce molecular oxygen to the superoxide anion radical (0;-). The 0;- produced by the LiP-dependent reductive pathway can then reduce ferric iron to ferrous iron. Hydrogen peroxide then readily reacts with chelated ferrous iron to produce the ·OH. The EPR spectrum of the 'OH radical spin adduct with DMPO was detected in a reaction mixture containing LiP, H202, veratryl alcohol, and EDT A, which was used as both the reductant for the veratryl alcohol cation radical and the iron chelator (Barr et al. 1992). Because iron reduction was also observed when oxalate replaced EDT A as the reductant for the veratryl alcohol cation radical, it was concluded that oxalate could be used for 'OH production by the fungus. The pathway for the LiPdependent production of 'OH is shown in Fig. 4. The production of extracellular 'OH by white rot fungi is extremely significant with regard to the degradation of recalcitrant environmental

D.P. Barr and S.D. Aust

62

,·"x xm,+m,'X:' )«,m'X'

OH OR

2

LiP L"

;P,."

CO

'

VA

VA"

Oxalate

,Po""

+

H,0,

Fig. 4. Proposed pathway for production of . OH by lignin peroxidase. EDTA can replace oxalate in the proposed scheme. VA stands for veratryl alcohol, while the VA cation radical is represented by VA· "). (From Barr et al. 1992. Reprinted with permission from Academic Press.)

pollutants. In fact, extensive research has been performed to develop nonbiological methods for 'OR production that could be used to degrade pollutants. Methods such as the addition of Fenton reagent (i.e., ferrous iron and R202) (Barbeni et al. 1987) and the photolysis of titanium dioxide (Jessem~ ing et al. 1991) effectively produce 'OR, which can then oxidize pollutants. However, these methods are expensive and therefore not economically feasible for many hazardous waste problems. The reason why the 'OR has received so much attention regarding the degradation of environmental pollutants is due to its incredible oxidizing ability. The reduction potential of 'OR is approximately 2.3 V (Buettner 1993), allowing it to react favorably with most organic molecules at diffusion-limited rates. For example, the 'OR produced via photolysis of R202 was able to react with several chlorobenzenes at a rate of 109 M-1 sec-1 (Kochany and Bolton 1992). In addition, various polychlorinated biphenyls (PCB) and polychlorinated phenols were readily hydroxylated by the 'OR with the concomitant release of chloride (Sedlak and Andren 1991; Barbeni et al. 1987). Because PCBs are mineralized by the fungus (Bumpus and Aust 1987b), it is possible that the fungus uses the 'OR to reduce the number of chlorine atoms on such pollutants, making them less recalcitrant to the fungus as well as bacteria. C. Pollutant Detoxification Many organopollutants are resistant to microbial degradation due to their toxicity to the organism being employed to degrade them. For example, chlorinated phenols, such as pentachlorophenol (PCP), are potent inhibitors of oxidative phosphorylation and thus are quite toxic to a multitude of organisms (Haggblom et al. 1988). These chemicals have been extensively used in the past as fungicides in treating railroad ties, poles, and fence posts, making them a relatively ubiquitous pollutant in both rural and urban areas. White rot fungi are capable of degrading chlorinated phenols to CO2, but it appears that the first step of metabolism involves detoxification. r>.

1. Methylation of Phenolic Compounds. Various organisms, including white rot fungi, are known to methylate phenolic compounds, which is

Pollutant Degradation

J

"---"

~

63

generally thought to be a detoxification mechanism. Extensive studies have demonstrated that white rot fungi have the ability to methylate a wide variety of phenolics (Harper et al. 1989, 1990). The primary methyl donors appear to be S-adenosylmethionine (SAM) and methyl chloride, which is synthesized by these fungi from SAM (Harper and Hamilton 1988). In addition, it has been reported that chloromethane is liberated by white rot fungi during secondary metabolism (Harper et al. 1989). The importance of methylation in lignin degradation may be related to the fact that phenolic compounds are released during lignin breakdown (Schoemaker 1990). As mentioned earlier, phenols are not efficient substrates for compound II of LiP, and thus inactive compound III forms during the oxidation of phenols. However, aromatic compounds that have been o-methylated, such as veratryl alcohol, are very efficiently oxidized by LiP compound II. Therefore, by methylating phenolic compounds, the fungus would achieve a greater degree of lignin degradation. Also, chlorinated phenolics, such as 2,4,5-dichlorophenol and PCP, are mineralized by white rot fungi, and the first step of this metabolism appears to be methylation (Joshi and Gold 1993; Mileski et al. 1988). This has relevance, because the methylated products (e.g., pentachloroanisle) are known to be less toxic than the phenolic compounds (Ruckdeschel and Rener 1986). It would also be expected that the methylated congeners would be more readily oxidized by LiP. In a field study reported by Kirk et al. (1992), it was found that approximately 90070of the PCP at a site contaminated with a commercial wood preservative was degraded by white rot fungi in about 6 weeks. Between 9% and 14% of the PCP was extracted as pentachloroanisole. The remaining fraction was thought to have been either mineralized or converted to soilbound metabolites. Therefore, it seems that white rot fungi use methylation as a mechanism to detoxify pollutants such as PCP. Following detoxification, the fungi can use LiP to effectively metabolize these pollutants further. 2. Plasma-Membrane-Dependent Reduction of Chemicals. It has been observed that the disappearance of certain highly oxidized pollutants, such as TNT and DDT, occurred in cultures of white rot fungus prior to production of the LiP (Fernando et al. 1990; Bumpus and Aust 1987a). In addition, the disappearance, but not mineralization, of DDT occurred even in nonligninolytic cultures (Kohler et al. 1988). It appeared that these fungi had a mechanism for metabolizing these compounds that was independent of the LiP or MnP. It is reasonable to assume that such a mechanism might exist because TNT and DDT are not oxidized by LiP or MnP in vitro. The initial clue to this mechanism was provided by Sollod et al. (1992), who demonstrated that a plasma-membrane redox system is present in several fungi. Several redox active dyes were effectively reduced using this membrane-dependent system. The fungi accomplish this by generating a

64

D.P. Barr and S.D. Aust

proton gradient across the plasma membrane, which in turn produces an electromotive potential capable of reducing chemicals. Because chemicals such as DDT and TNT are highly oxidized, they seem to be likely candidates for such a membrane-dependent redox system. Trinitrotoluene (TNT) is an explosive that has been produced and disposed of in mass quantities since the early 1900s (Nay et al. 1974). The contamination of soils, groundwater, and streams by TNT poses a toxicity threat to wildlife as well as to the general public. Trinitrotoluene has been shown to cause liver damage and anemia in humans and is quite toxic to fish (Kaplan and Kaplan 1982). In addition, it has been found to inhibit microbial growth. For example, TNT was found to significantly inhibit growth in a municipal sewage sludge reactor (Schott et al. 1943). Thus, the use of bioremediation as a strategy for ridding the environment of TNT waste has been considered problematic. However, the white rot fungus P. chrysosporium has been demonstrated to degrade TNT to CO2 (Fernando et al. 1990). Effective degradation was observed in both soil and liquid cultures. The disappearance of TNT was also observed in nonligninolytic cultures. In fact, the initial rate of TNT disappearance was the same in nonligninolytic cultures as in ligninolytic cultures (Stahl and Aust 1993b). The disappearance of TNT in both cultures began on day 2 and was accompanied by the appearance of the 2- and 4-amino dinitrotoluene congeners. Stahl and Aust demonstrated that the initial disappearance of TNT was due to a plasma-membrane-dependent redox system of P. chrysosporium (Stahl and Aust 1993a) (Fig. 5). Several lines of evidence were provided to support this theory. The reduction of TNT appeared to depend on the presence of live intact mycelia. When the mycelia were submitted to conditions that would disrupt the membrane (i.e., grinding with glass beads or freeze/ thawing), TNT reduction to the amino congeners was not observed. Chemicals that disrupt membrane potential gradients, such as dinitrophenol and sodium azide, as well as alternate electron acceptors (i.e., redox active dyes), were able to inhibit TNT reduction. The rate of TNT reduction by the fungus was proportional to the mycelial mass, which explains why reduction occurs very efficiently under nutrient-sufficient conditions. The rate of TNT reduction was related to the rate of proton excretion by the fungus. In has been known for some time that white rot fungi often lower the pH of their environment (Barr and Aust 1994c). It appears that this is accomplished by excreting protons via the membrane-dependent redox system. Therefore, at high pH values, the fungus excretes protons and lowers the pH to its growth optimum (approximately 4.5). The rate of proton excretion is also greater at high pH. Correspondingly, the rate of TNT reduction is also faster at high pH. Stahl and Aust (1993a) studied both the rates of proton excretion and TNT reduction by mycelia from P. chryso-

sporium betwe en pH 40Q QU~M ~Ug fvvnij

llii~~m

'n~114fIfl~lm

highly correlated. As the initial pH of the incubation mixture was increased the relative rates of TNT reduction and proton excretion increased to the same degree.

.~

r>;

Pollutant Degradation

65 Intracellular

Plasma Membrane

Extracellular

TNT

~

AmDNT

~N¢NO' ~N¢NO' ~N¢~ CH,

CH,

CH3

~I ~

N02

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Fig. 5. Proposed mechanism for TNT reduction by white rot fungi. (AmDNT stands for aminodinitrotoluene). (From Barr and Aust 1994c. Reprinted with permission from Environ Sci TechnoI28:78A-87A. © 1994 American Chemical Society.)

'----"

The conversion of TNT to its amino congeners is a significant step in detoxifying TNT so that further metabolism of the compound can occur. It was also demonstrated that the toxicity of TNT to P. chrysosporium was inversely related to mycelial mass (Stahl and Aust 1993b). For example, when 360-mg dry-weight mycelia were subjected to a TNT concentration of 1000 ppm, no toxicity was observed. In contrast, if only 5-mg mycelia were used, this concentration was quite toxic. Therefore, if the fungus is grown such that a sufficient mycelial mass is obtained, even high concentrations of TNT can be converted to the amino congeners. The amino congeners are oxidized by MnP and are eventually converted to CO2 (Stahl and Aust 1993b). Considerable research efforts have been made to understand the mechanisms used by white rot fungi. However, the majority of this work has been performed in the laboratory, where conditions can be easily controlled. In the field, environmental factors, such as temperature, moisture, pH, and competition for growth substrates by other microbes, are not easily con-

66

D.P. Barr and S.D. Aust

trolled. This will inevitably present challenges in applying white rot fungi for site decontamination. At the present time, there is a need for researchers to use the knowledge obtained in the laboratory to design feasible strategies for larger-scale decontamination projects using white rot fungi. When difficulties arise in applying the fungus at a particular site, it is necessary to consider many of the factors that were reviewed above before concluding that the application is not feasible. In the past, a lack of knowledge concerning the biochemical mechanisms used by white rot fungi has prevented their successful application in the field. However, the knowledge gained in recent years will definitely have a positive effect on white rot fungus technology in the near future. Summary The white rot fungi technology is very different from other more well-established methods of bioremediation (e.g., bacterial systems). The differences are primarily due to the mechanisms discussed previously. The unusual mechanisms used by the fungi provide them with several advantages for pollutant degradation, but the complexity of these mechanisms has also made the technology slow to emerge as a viable method of bioremediation. One distinct advantage that white rot fungi have over bacterial systems is that they do not require preconditioning to a particular pollutant. Bacteria must be preexposed to a pollutant to allow the enzymes that degrade the pollutant to be induced. The pollutant must also be present in a significant concentration, otherwise induction of enzyme synthesis will not occur. Therefore, there is a finite level to which pollutants can be degraded by bacteria. In contrast, the degradative enzymes of white rot fungi are induced by nutrient limitation. Thus, cultivate the fungus on a nutrient that is limited in something, and the degradative process will be initiated. Also, because the induction of the lignin-degrading system is not dependent on the chemical, pollutants are degraded to near-nondetectable levels by white rot fungi. Another unique feature of pollutant degradation by white rot fungi involves kinetics. The process of chemical conversion by these fungi occurs via a free-radical process, and thus the degradation of chemicals often follows pseudo-first-order kinetics. In fact, in several studies, it has been found that the rate of mineralization or disappearance of a pollutant is proportional to the concentration of the pollutant. This makes the time required to achieve decontamination more important than the rate of degradation. Because the metabolism of chemicals by bacteria involves mostly enzymatic conversions, pollutant degradation often follows MichaelisMenton-type kinetics. Therefore, Km values of various degradative enzymes with

=r= to the rOllutant

must be

cqru;i~~II~whiR

Wi~~'mlill

for bioremediation. Considering this, the solubility of a pollutant or a mixture of pollutants might also present a problem for bacterial degrada-

~

~

Pollutant Degradation

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67

tion. In contrast, using a nonspecific free-radical-based mechanism, the fungi are able to degrade insoluble complex mixtures of pollutants, such as creosote (Aust and Bumpus 1989) and Arochlor (Bumpus and Aust 1987b). Inexpensive nutrient sources, such as sawdust, wood chips, surplus grains, and agricultural wastes, can be used to effectively cultivate white rot fungi. Thus, depending on the geographical region, the fungus might be grown in the U.S. on sawdust in the northwest or ground corn cobs in the midwest ern U.S. in order to degrade chemicals. Such growth substrates are not readily utilized by other microorganisms, which would likely allow the fungus to establish itself among the indigenous microbial flora. The fungus may also antagonize microbial growth. In addition to being able to grow under nutrient limitation, the fungi also produce oxygen radicals such as the 'OH, which is capable of oxidizing biomolecules, such as proteins and DNA, that could result in the death of other microbes. Using the plasmamembrane-dependent redox system, the fungus is able to adjust the pH of its surrounding environment. Thus, microbes with pH optimums that differ from that of the fungus might not grow well after the fungus has been introduced. Perhaps the most significant feature of pollutant degradation by white rot fungi is that it occurs extracellularly. This allows the lignin-degrading system to generate very potent oxidizing species (i.e., the veratryl alcohol cation radical and the 'OH), which would be quite toxic if produced inside the cell. Also, toxic pollutants need not be internalized for metabolism, providing another advantage. In conclusion, the extreme nonspecificity of the mechanisms described here makes the white rot fungi an attractive solution to many of our evergrowing hazardous waste problems. However, only through our understanding and continued research efforts, with regard to these mechanisms, will we be able to successfully design bioremediation strategies employing the white rot fungi. Acknowledgments This work was supported in part by NIH Grant Number ES04922. The authors wish to extend their gratitude to Terri Maughan for her expert secretarial services in the preparation of this review. References

~

Akamatsu Y, Ma DB, Higuchi T, Shimada M (1990) A novel enzymatic decarboxylation of oxalic acid by the lignin peroxidase system of white-rot fungus Phanerochaete chrysosporium. Fed Eur Bioi Socs 269:261-263. Aust SD, Bumpus JA (1989) Biological mineralization of constituents of coal tar by the white rot fungi. In: Proceedings of the Symposium on Biological Processing of Coal and Coal-Derived Substances, EPRI ER-6572, pp 4-49-4-63.

D.P. Barr and S.D. Aust

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Aust SD (1993) The fungus among us: Use of white rot fungi to biodegrade environmental pollutants. Environ Hlth Perspect 101:232-233. Barbeni M, Minero C, Pellizetti E (1987) Chemical degradation of chlorophenols with Fenton's reagent. Chemosphere 16:2225-2237. Barr DP, Shah MM, Grover TA, Aust SD (1992) Production of hydroxyl radical by lignin peroxidase from Phanerochaete chrysosporium. Arch Biochem Biophys 298:480-485. Barr DP, Shah MM, Aust SD (1993) Veratryl alcohol dependent production of molecular oxygen by lignin peroxidase. J Bioi Chern 268:241-244. Barr DP, Aust SD (1994a) Effect of superoxide and superoxide dismutase on lignin peroxidase. Arch Biochem Biophys 311:378-382. Barr DP, Aust SD (1994b) Conversion of lignin peroxidase compound III to active enzyme. Arch Biochem Biophys 312:511-515. Barr DP, Aust SD (1994c) Mechanisms white rot fungi use to degrade environmental pollutants. Environ Sci TechnoI28:78A-87A. Boominathan K, and Reddy, CA (1992) Fungal degradation of lignin: Biotechnological applications. In: Arora DK, Elander RP, Mukerji KG, (eds) Handbook of ~ Applied Mycology. Marcel DekkerInc, New York, vol 4, pp 763-821. Buettner GR (1993) The pecking order of free radicals and antioxidants: Lipid peroxidation, a-tocopherol and ascorbate. Arch Biochem Biophys 300:535-543. Bumpus JA, Aust SD (1987a) Biodegradation of DDT [1,1, I-trichloro-2,2-bis(4-chlorophenyl) ethane] by the white rot fungus Phanerochaete chrysosporium. Appl Environ MicrobioI53:2001-2oo8. Bumpus JA, Aust SD (1987b) Mineralization of recalcitrant environmental pollutants by a white rot fungus. In: Proceedings of the National Conference on Hazardous Wastes and Hazardous Materials. Lib Congr Cat No 87-80469, pp 146-151. Bumpus JA, Brock BJ (1988) Biodegradation of crystal violet by the white rot fungus Phanerochaete chrysosporium. Appl Environ Microbiol 54: 1143-1150. Bumpus JA (1989) Biodegradation of polycyclic aromatic hydrocarbons by Phanerochaete chrysosporium. Appl Environ MicrobioI55:154-158. Cai D, Tien M (1990) Characterization of the oxycomplex of lignin peroxidases from Phanerochaete chrysosporium: Equilibrium and kinetics studies. Biochemistry 29:2085-2091. Cai D, Tien M (1993) Lignin-degrading peroxidases of Phanerochaete chrysosporium. J Biotechnol 30:79-90. Chung N, Shah MM, Grover TA, Aust SD (1993) Reductive activity of a manganese-dependent peroxidase from Phanerochaete chrysosporium. Arch Biochem Biophys 306:70-75. Coulter C, Kennedy JT, McRoberts WC, Harper DB (1993) Purification and properties of an S-adenosylmethionine 2,4-disubstituted phenol o-methyl transferase from Phanerochaete chrysosporium. Appl Environ Microbiol 59:706-711. Crawford R (1981) In: Lignin Biodegradation and Transformation. Wiley, New York. Cripps C, Bumpus JA, Aust SD (1990) Biodegradation of azo and heterocyclic dyes by Phanerochaete chrysosporium. Appl Environ Microbiol 56:1114-1118. r>; Faison BD, Kirk TK (1983) Relationship between lignin degradation and production of reduced oxygen species by Phanerochaete chrysosporium. Appl Environ Mi-

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Pollutant Degradation

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