Decolorization of azo dye by the white-rot basidiomycete Phanerochaete sordida and by its manganese peroxidase

.ICW.NAL OF BIOSCIENCF AND BIOENGINEERING Vol. 95,No.5.455459.2003

Decolorization of Azo Dye by the White-Rot Basidiomycete Phanerochaete sordida and by Its Manganese Peroxidase KOICHI HARAZONO,‘” YOSHIO WATANABE,’ AND KAZUNORI NAKAMURA’ Institute.fbr Biological Resources and Functions, National Institute qfAdvanced Industrial Science and Technology (MST), Central 6, 1-1-l Higashi, Tsukuba, Ibaraki 305-8566. Japan’and Bioresource Laboratories, Mercian Corporation, 1808 Nakaizumi, Iwata 438-0078. Japan2 Received 21 October 2002/Accepted 15 January 2003

We investigated the decolorization of an azo-reactive dye, Reactive Red 120, by a white-rot basidiomycete, Phanerochaete sordida strain YK-624. In liquid culture of I! sordida in a medium containing 3% malt extract and 200 mgll of the dye, the dye was 90.6% decolorized after 7 d. Manganese peroxidase (MnP) activity was detected during the decolorization process. The dye could be decolorized by purified MnP of Z? sordida in the presence of Mn(I1) and Tween 80. The involvement of lipid peroxidation during decolorization with MnP was considered. With shaking, the dye could be decolorized without the addition of hydrogen peroxide. The decolorization did not occur under anaerobic conditions, suggesting that dye decolorization by MnP is influenced by dissolved oxygen. Since catalase did not inhibit the decolorization with MnP, we inferred that the MnP catalytic cycle would be promoted by hydroperoxides formed from the decomposition of malonate or from lipid peroxidation. [Key words: degradation, textile dye, white-rot fungi, Phanerochaete sordida, manganese peroxidase, peroxidation]

Azo dyes account for most textile dyestuffs produced and are the most commonly used synthetic dyes in the textile, food, papermaking, and cosmetic industries (1). The release of azo dyes into the environment in effluent from dye-utilizing industries has become a major concern in wastewater treatment, since some azo dyes or their metabolites may be mutagens or carcinogens (2). White-rot basidiomycetes are well known not only as decomposers of lignin, but also for their ability to degrade a wide variety of organopollutants (3). Decolorization of azo, anthraquinone, heterocyclic, triphenylmethane, and polymeric dyes (4, 5) and partial mineralization of azo dyes (6, 7) by the white-rot fungus Phanerochaete chrysosporium have been reported. J? chrysosporium produces extracellular lignin peroxidase (LIP) and manganese peroxidase (MnP) during the degradation of lignin or xenobiotic compounds. LIP directly catalyzes the oxidation of non-phenolic aromatic compounds (8). MnP oxidizes Mn(II) to Mn(III), which is responsible for the oxidation of many phenolic compounds (9). LIP has higher oxidation potentials than MnP. Preferential degradation of different sulfonated azo dyes either by MnP and Mn(I1) or by LIP was demonstrated ( IO). Some azo dyes are not oxidized by MnP (11). Previous researchers have proposed a mechanism for the degradation of azo dyes with simple structures (12, 13). Peroxidases or Mn(III)-malonate complex oxidize the phenolic

lipid

ring of the dyes with two electrons to produce a carbonium ion on the carbon bearing the azo linkage. Water attacks the carbonium ion to cause hydrolytic cleavage of the azo linkage. The decolorization rate is dependent on the chemical structure of the azo dye ( 10). The white-rot fungus Phanerochaete sordida strain YK624 was isolated from decayed wood obtained from a forest; it has a remarkable ability to bleach kraft pulp (14). It has been found to secrete MnP, which is presumed to partake in the biodegradation of lignin residues (14). No LIP or lactase activity has been detected under a variety of culture conditions (15). We investigated the decolorization of the dye by the white-rot fungus l? sordida. We selected Reactive Red 120 as the model azo dye. To our knowledge, Reactive Red 120 has not been investigated for degradation by white-rot fungi before. It has a structure consisting ofazo, anthrazine, naphthalene, and sulfonated groups (Fig. 1). We found that it produces MnP during the decolorization of the azo dye, and we then characterized the decolorization with MnP. MATERIALS

AND METHODS

Evaluation of decolorization of the dye Reactive Red 120 (ICN Biomedicals, Aurora, OH, USA) was used in this study. Dye decolorization was measured photometrically at visible wavelengths (400,450,500,550,600, and 650 nm) by using a Beckman DU640 scanning spectrophotometer (Beckman Coulter, Fullerton, CA, USA). COD was calculated as the sum of absorbances at each wavelength. Color removal (%) was calculated as the extent of de-

* Corresponding author. e-mail: [email protected] phone/fax: +81-(0)298-6 l-68 12 455

456

HARAZONO

j. ~IOX’I.

ET AL.

Na03S -SO

Na03S -SO

3Na

t~lOhN~i.,

3Na

FIG. I. Structure of Reactive Red 120. crease from the initial value of COD. I-1 sordida YK-624 Strain, media, and culture conditions (ATCC 90872) was maintained on potato dextrose agar (Difco Laboratories, Detroit, MI, USA) at 4°C. For liquid culture, IOO-ml flasks containing 10 ml of medium containing 3% malt extract and the dye (200 mg/l) were inoculated with three plugs of actively growing fungus and incubated at 30°C statically. The dye (500, 750 and 1000 mgll) was also treated by J! sordida for 7 d. MnP and lactase activiMeasurement of enzyme activities ties were assayed by monitoring the oxidation of 2,6-dimethoxyphenol at 469 nm (9, 14). LiP activity was assayed by monitoring the oxidation of veratryl alcohol at 3 10 nm (8). One unit (U) of enzyme activity was defined as I pmol of oxidative compound produced in 1 min. MnP was prepared from Treatment of the dye with MnP liquid cultures of R sordida YK-624 as previously reported (16, 17). The enzyme was purified on DEAE-Sepharose CL-6B (50x 100 mm) and MONO-Q (lo/lo) (Amersham Biosciences, Piscataway, NJ, USA) columns to obtain a single protein (RZ value is 3.0). Low-molecular-mass compounds were removed from the enzyme solution by subjecting to PD-10 column (Sephadex G-25; Amersham Biosciences) before use. The dye (500 mg/l) was treated in a reaction mixture (5 ml) containing MnP (0.1 U/ml), 0.1 mM MnSO,, 0.1% Tween 80, and 50 mM malonate (pH 4.5). The oxidation of glucose (5 mM) by glucose oxidase (0.5 U) (from Aspergillus niger; Wako Pure Chemical Industries, Tokyo) was used to supply H,O, continuously to the reaction mixture. H,O, and tertbutyl hydroperoxide were obtained from Wako Pure Chemical lndustries and Aldrich Chemical Company (Milwaukee, WI, USA) respectively. Tubes including reaction mixtures were shaken at 150 rpm at 37°C. Triton X-100, Nonidet NP-40, SDS, Tween 20, and Tween 85 were also used as surfactants instead of Tween 80. The inhibitor Inhibitor test for decolorization with MnP test used 5 ml of reaction mixture containing 500 mg/l of the dye, 0.1 U/ml of MnP, 0.5 mM MnSO,, 1.O% Tween 80, and 50 mM malonate (pH 4.5). Concentrations of EDTA and NaN,, as inhibitors were adjusted to 2.0 mM each. Acetate (100 mM), which is not a good chelator for Mn(II1) stabilization, was used instead of malonate as the good Mn(II1) chelator (9). To keep the conditions anaerobic, the reaction mixture prior to the addition of MnP was boiled and cooled under N, atmosphere. It was then transferred gently to a test tube and N, gas was used to purge the headspace of the tube. To verify the anaerobic conditions, resazurin (O.Ol%), which is colorized by 0, contamination, was added to the tube containing the reaction mixture (18). Detection of fatty acids Mycelia of p sordida were harvested by paper filtration, frozen, and then dried under reduced pressure. Supematants filtered p sordida cultures were also dried under reduced pressure. The dried samples were directly transmethylated with 50% H,SO, in methanol at 60°C for 6 h. The resultant fatty acid methyl esters were extracted with chloroform and then analyzed with a TurboMass gas chromatography mass spectrometer (Perkin-Elmer, Norwalk, CT, USA) equipped with a 0.25 x30-mm Neutra Bond-l capillary column (GL Science, Tokyo) operating at an ionization voltage of 70 eV, with a scan range of

50-650 Da.

RESULTS

Decolorization of the dye in liquid culture of I! sordida We studied the ability of I? sordidu to decolorize Reactive Red 120 in liquid culture. The culture supematant was analyzed daily for 7 d for COD and enzyme activities. Color disappeared rapidly on day 3 (Fig. 2). No increase in absorbance by a new chromophoric product transformed from the dye was observed. Fungal biomass production increased up to a maximum of about 0.04 g/10 ml at day 4, after which time the culture entered idiophase. MnP activity was detected, but LIP and lactase activities were not detected during the 7-d incubation. The culture showed maximum MnP activity on day 2. MnP activity was not detected in liquid culture without the dye (results not shown), which suggests that MnP production was induced by the dye. When 200, 500, 750 and 1000 mgll of dye were also treated for 7 d, p sordida decreased COD by 90.7%, 89.4%, 85.6% and 75.9%. Adsorption of the dye to mycelia after incubation for 7 d was not found on all conditions. Effect of Mn(I1) and Tween 80 concentrations on decolWe examined whether orization of the dye with MnP Reactive Red 120 could be decolorized with MnP from p sordida. A precipitate by polymerization or aggregation of the dye was not formed during all experiments. First we determined the optimum Mn(I1) concentration for decolorization of the dye with MnP in the presence of 1 .O% Tween 80, 0.5 U glucose oxidase. and 50 mM sodium malonate at 37°C. ?he dye was not decolorized in the absence of

Time (d)

FIG. 2. Decolorization 01‘200 mg// Reactive Red 120 by I! .~or&lu. circles, color removal; triangles, MnP activity; squares, mycelial dry weight. Experiments were carried out in triplicate with error bars representing&S.D, Symbols:

VOL. 95,2003

DYE DECOLORIZATION BY MANGANESE PEROXIDASE

457

c

0

5

10

15

20

25

Time (h) FIG. 3. Effect of H,O, addition on decolorization of Reactive Red 120 by the MnP reaction. Reaction mixtures contained 0.1 U/ml MnP, 0.5 mM Mn(II), 1.O% Tween 80, and 50 mM malonate. Initial H,O, concentrations (mM): closed circles, 0 (no MnP); closed triangles, 0; closed squares, 0.02; closed diamonds, 0.05; open circles, 0.1; open triangles, 0.2; open squares. 0.5.

Mn(I1). In the presence of 0.05, 0.1, 0.5, and 1 .O mM Mn(II), the COD was decreased by 32.4%, 42.7%, 75.3%, and 74.1% in 24 h. Higher concentrations of Mn(I1) resulted in increases in decolorization rate. The optimum concentration of Mn(I1) for decolorization with MnP was 0.5 mM. We also determined the effect of Tween 80 concentration on decolorization of the dye with MnP in the presence of 0.5 mM Mn(II), 0.5 U glucose oxidase, and 50 mM sodium malonate. In the presence of O%, O.l%, 0.5%, 1.0% and 2.0% Tween 80, the COD was decreased by 8.58%, 25.3%, 60.0%, 78.5% and 82.0% in 24 h. Higher concentrations of Tween 80 resulted in significant increases in decolorization rate. In the presence of 2.0% Tween 80, the COD was decreased by 90.5% in 48 h. Effect of H,O, and dissolved oxygen on decolorization In the above experiments, glucose of the dye with MnP oxidase was used as the H,O,-producing enzyme. We also evaluated the effect of H20, addition on the time course of decolorization of Reactive Red 120 with MnP (Fig. 3). H,O, was used without the addition of glucose and glucose oxidase. The addition of H,O, increased the rate of decolorization. The addition of tert-butyl hydroperoxide (2.0 mM) also increased the decolorizing rate (data not shown). In the shaking procedure, the dye could also be decolorized without the addition of H20Z. In cases of no addition of H,O, or glucose oxidase, the rate of decolorization during incubation without shaking was markedly lower than that in the shaking procedure (Fig. 4). Decolorization did not occur under anaerobic conditions without the addition of H,O,. These results suggest that dye decolorization by MnP is influenced by dissolved oxygen. In the presence of various surfactants (1 .O%), the dye was decolorized with MnP without the addition of H,O, (Fig. 5). When Tween 80 was used, the dye was decolorized efficiently. It was not decolorized in the presence of Triton X100, Nonidet NP-40, SDS, or Tween 20. The rate of decolorization in the presence of Tween 85 was much lower than that in the presence of Tween 80. Effect of inhibitors and other factors on decoloriza-

0 0

20

40

60

80

Time (h) FIG. 4. Effect of dissolved oxygen on decolorization of Reactive Red 120 by the MnP reaction. Reaction mixtures contained 0.1 U/ml MnP, 0.5 mM Mn(II), 1.O% Tween 80, and 50 mM malonate. Symbols: circles, agitation under aerobic conditions; triangles, agitation under anaerobic conditions; squares, stationary under aerobic conditions.

1

0

20

40

60

80

Time (h) FIG. 5. Decolorization of Reactive Red 120 by the MnP reaction in presence of various surfactants (1 .O%). Reaction mixtures contained 0.1 U/ml MnP, 0.5 mM Mn(II), and 50 mM malonate. Symbols: closed circles, Tween 80; closed triangles, Tween 85; closed squares, Tween 20; closed diamonds, Nonidet NP-40; open circles, Triton X-100; open triangles, SDS.

tion of the dye with MnP We examined the effects of inhibitors on decolorization. When azide, which inhibits heme-containing enzymes, was added to the reaction mixture, decolorization was inhibited completely. The addition of EDTA, which is a strong metal chelator, also inhibited decolorization. When acetate was used instead of malonate as a Mn(III)-chelator, decolorization did not occur. The addition of catalase, which degrades Hz02, did not affect decolorization with MnP. Detection of fatty acids To see the presence of an unsaturated lipid in l? sordida cultures, analysis for fatty acids was done by GC-MS. When we used H,SO,-methanol to analyze for fatty acids, we detected linoleic acid methyl ester by GC-MS. Linoleic acid methyl ester could be detected in samples from I? sordida mycelia grown for incubation times from 2 d to 7 d, but not supernatants of p sordida cul-

458

HARAZONO ET AL.

tures. DISCUSSION We studied the use of p sordida strain YK-624 to decolorize azo dye. The fungus could decolorize Reactive Red 120 during incubation in malt extract medium, decolorizing most of the dye by 3 d (Fig. 2). The culture showed maximum MnP activity just before the maximum decolorization was reached. MnP of Pleurotus eryngii shows manganese-independent activity for decolorization of textile dyes (19). However, MnP of 19 sordida needed Mn(I1) for decolorization of Reactive Red 120. The dye could not be effectively decolorized by MnP reaction without Tween 80. Tween 80 (polyoxyethylene sorbitan monooleate) is an anionic surfactant made from an unsaturated fatty acid, oleic acid. It has been reported that the addition of Tween 80 to I! chrysosporium culture improved the degradation of phenanthrene (20). Tween 80 plus MnP caused the lipid peroxidation of oleic acid (20). MnP was [ 1-(4also able to mineralize non-phenolic compounds ethoxy-3-methoxyphenyl)-2-phenoxypropane-1,3-diol and methylated synthetic guaiacyl lignin] in the presence of an unsaturated lipid (21). The oxygen-centered radicals produced during lipid peroxidation are known to trigger xenobiotic cooxidations (22). Kapich et al. also showed that a non-phenolic lignin model dimer was oxidized by peroxyl radicals formed from arachidonic acid (23). Lipid hydroperoxides formed by MnP or autoxidation are transformed to peroxyl radicals by MnP (Fig. 6). The surfactants other than Tween 80 and Tween 85 (polyoxyethylene sorbitan trioleate) did not contribute to decolorization with MnP. It may be that the involvement of lipid peroxidation enhances the oxidation potential of the MnP system, allowing it to oxidize the dye. Thus, we predicted that a mediator would participate in decolorization with MnP in a I? sordida culture. Methylation of the mycelium with methanol and H,SO, allowed linoleic acid methyl ester to be detected. Since linoleic acid methyl ester was not detected in the supematant, we consider that the mediator is derived from a cell membrane lipid. Thus, lipid peroxidation may contribute to in vivo degradation by MnP. MnP needs hydroperoxides containing H,O, for the initiation of Mn(I1) oxidation (Fig. 6). The addition of H,O, to the reaction mixture increased the decolorization rate. Decolorization with MnP also occurred without the addition of ROOH

ROH + Hz0

MnP

ROOG> ROOH

+ 2H+

,

Compound [Fe(lV)=O]

COOH-CH,-COOH + Mn(II1) H++ Mn( 11) + COOH-CH;+CO,+

(1)

COOH-CH;+

(2)

0,

+

COOH-CH,OO’

COOH-CH,OO’+ Mn(I1) + H’ + COOH-CH,OOH + Mn(II1)

(3)

COOH-CH,OO’+ 0, + COOH-COOH+O,‘~

(4)

+ 1~’

COOH-COOH + Mn(II1) _ -+ CO,+CO,‘-+ Mn(I1) coZ’-+oZ

-+ co,+o,‘~ 02’- + Mn(II) + 2H’ + *H&J+ Mn(III)

(5) (6) (7)

A trace amount of Mn(lI1) formed by autoxidation ot Mn(I1) initiates the reaction cascade. In this study, the lag period was confirmed during the initiation of dye decolorization with MnP without the addition of H-0, (Fig. 3). It has been reported that a single addition of s&i amounts of Mn(II1) shortened the lag period considerably, indicating that Mn(II1) is able to initiate the action of MnP and therefore its own formation (24). The decolorizing rate in the absence of H,O, is thought to be dependent on the initial Mn(II1) formation rate. Hydroperoxides composed of hydroperoxy acetic acid and H,O, and formed in these reactions can be used by MnP as co-substrates (Eqs. 3 and 7). Catalase did not inhibit decolorization during treatment with MnP. This result may indicate that MnP could use hydroperoxides except H,O, as co-substrates. The dye was not decolorized in the absence of malonate. On the other hand, Watanabe et al. have proposed the transfer of two electrons to native MnP from lipid hydroperoxides produced by the MnP reaction or autoxidation (25). These views suggest that the MnP catalytic cycle could be promoted by hydroperoxides formed from the decomposition of malonate or from lipid peroxidation. In conclusion, we have established a system for the decolorization of an azo dye with MnP and no addition of exogenous HZO,. The method improves the decolorization of the dye under the control of dissolved oxygen. For the practical treatment of wastewater containing azo dyes, it is important to screen for a replacement for Tween 80, since the release of synthetic surfactants into the environment has become a concern (26). The isolation and determination of the compounds that function during in vivo decolorization by the fungus is now in progress.

ACKNOWLEDGMENTS

L&R-. II

k

H,O,. Since the dye was not decolorized under anaerobic conditions, dissolved oxygen may be a factor in the initiation of decolorization by MnP. The MnP cycle is promoted by the reaction between dissolved oxygen, malonate. and manganese ions (24). The main reactions possibly involved in the oxidation of malonate by MnP are proposed as follows (24):

ROO

FIG. 6. Scheme for MnP catalytic cycle. ROOHs indicate hydroperoxides. Compounds I and II indicate oxidized states of MnP (9).

This study was carried out as part of a project on industrial technology research for the New Energy and Industrial Technology Development Organization (NEDO) at the National Institute of Advanced Industrial Science and Technology (AIST), Japan.

VOL.. 95.2003

DYEDECOLORlZATlONBYMANGANESEPEROXIDASE REFERENCES

1. Zollinger,

2.

3.

4.

5.

6.

7.

8. 9.

10.

Il.

12.

13.

14.

H.: Color chemistry-syntheses, properties and applications of organic dyes and pigments, p. 92-102. VCH Publishers, New York (1987). McCann, J. and Ames, B. N.: Detection of carcinogens as mutagens in the Salmonellulmicrosome test. Assay of 300 chemicals: discussion. Proc. Natl. Acad. Sci. USA, 73, 950954 (1975). Bumpus, J. A,, Tien, M., Wright, D., and Aust, S. D.: Oxidation of persistent environmental pollutants by a white rot fungus. Science, 228, 143441436 (1985). Glenn, J. and Gold, M. H.: Decolorization of several polymeric dyes by the lignin-degrading basidiomycete Phanerochaete chrysosporium. Appl. Environ. Microbial., 45, 17411747 (1983). Rodriguez, E., Pickard, M.A., and Vazquez-Duhalt, R.: Industrial dye decolorization by laccases from hgninolytic fungi. Curr. Microbial., 38, 27-32 (1999). Spadaro, J. T., Gold, M. H., and Renganathan, V.: Degradation of azo dyes by the lignin-degrading fungus Phanerochaete chtysosporium. Appl. Environ. Microbial., 58, 23972401 (I 992). Paszczynski, A., Pasti-Grigsby, M. B., Goszczynski, S., Crawford, R.L., and Crawford, D.L.: Mineralization of sulfonated azo dyes and sulfanilic acid by Phanerochaete chrysosporium and Streptomyces chromojiiscus. Appl. Environ. Microbial., 58, 3598-3604 (1992). Tien, M. and Kirk, T.: Lignin peroxidase of Phanerochaete chrysosporium. Methods Enzymol., 161,238-249 (1988). Wariishi, H., Valli, K., and Gold, M. H.: Manganese (11) oxidation by manganese peroxidase from the basidiomycete Phanerochaete chrysosporium. Kinetic mechanism and role of chelators. J. Biol. Chem., 267,23688-23695 (1992). Pasti-Grigsby, M. B., Paszczynski, A., Goszczynski, S., Crawford, D. L., and Crawford, R. L.: influence of aromatic substitution patterns on azo dye degradability by Streptomyces spp. and Phanerochaete chrysosporium. Appl. Environ. Microbial., 58, 3605-3613 (1992). Heinfling, A., Ruiz-Duefias, F. J., Martinez, M. J., Bergbauer, M., Szewzyk, U., and Martinez, A. T.: A study on reducing substrates of manganese-oxidizing peroxidases from Pleurotus eryngii and Bjerkundera adusta. FEBS Len, 428, 141-146 (1998). Spadaro, J. T. and Renganathan, V.: Peroxidase-catalyzed oxidation of azo dyes: mechanism of disperse yellow 3 degradation Arch. Biochem. Biophys., 312, 301-307 (1994). Goszczynski, S., Paszczynski, A., Pasti-Grigsby, M. B., Crawford, R.L., and Crawford, D.L.: New pathway for degradation of sulfonated azo dyes by microbial peroxidases of Phanerochaete ch ysosporium and Streptomyces chromofuscus. J. Bacterial., 176, 1339-1347 (1994). Hirai, H., Kondo, R., and Sakai, K.: Screening of lignin-de-

15.

16.

17.

18.

19.

20.

21.

22.

23.

24.

25.

26.

459

grading fungi and hgninolytic enzyme activities during biological bleaching of kraft pulp. Mokuzai Gakkaishi, 40, 980986 (1994). Riittimann-Johnson, C., Cullen, D., and Lamar, R. T.: Manganese peroxidases of the white rot fungus Phanerochaete sordidu. Appl. Environ. Microbial., 60, 599-605 (1994). Kondo, R., Harazono, K., and Sakai, K.: Bleaching of hardwood krati pulp with manganese peroxidase secreted from Phanerochaete sordidu YK-624. Appl. Environ. Microbiol., 60,43594363 (1994). Harazono, K., Kondo, R., and Sakai, K.: Bleaching of hardwood kraft pulp with manganese peroxidase secreted from Phanerochaete sordida YK-624 without addition of MnSO,. Appl. Environ. Microbial., 62, 913-917 (1996). Brune, A., Miambi, E., and Breznak, J. A.: Roles of oxygen and the intestinal microflora in the metabolism of lignin-derived phenylpropanoids and other monoaromatic compounds by termites. Appl. Environ. Microbial., 61,2688-2695 (1995). Heinfling, A., Martinez, M. J., Martinez, A. T., Bergbauer, M., and Szewzyk, U.: Transformation of industrial dyes by manganese peroxidases from Bjerkandera adusta and Pleurotus eryngii in a manganese-independent reaction. Appl. Environ. Microbial., 64,2788-2793 (1998). Moen, M. A. and Hammel, K. E.: Lipid peroxidation by the manganese peroxidase of Phanerochaete chrysosporium is the basis for phenanthrene oxidation by the intact fungus. Appl. Environ. Microbial., 60, 19561961 (1994). Bao, W., Fukushima, Y., Jensen, K. H., Jr., Moen, M. A., and Hammel, K. E.: Oxidative degradation of non-phenolic lignin during lipid peroxidation by fungal manganese peroxidase. FEBS Lett., 354,297-300 (1994). Lawrence, J., Marnett, L. J., Gregory, A., Reed, G. A., and Johnson, J. T.: Prostaglandin synthetase dependent benzo(a) pyrene oxidation: products of the oxidation and inhibition of their formation by antioxidants. Biochem. Biophys. Res. Commun., 79, 569-576 (1977). Kapich, A. N., Jensen, K. A., and Hammel, K. E.: Peroxyl radicals are potential agents of lignin biodegradation. FEBS Len, 461, 115-119 (1999). Hofrichter, M., Ziegenhagen, D., Vares, T., Friedrich, M., Jiiger, M. G., Fritsche, W., and Hatakka, A.: Oxidative decomposition of malonic acid as basis for the action of manganese peroxidase in the absence of hydrogen peroxide. FEBS Len, 434,362-366 (1998). Watanabe, T., Katayama, S., Enoki, M., Honda, Y., and Kuwahara, M.: Formation of acyl radical in lipid peroxidation of linoleic acid by manganese-dependent peroxidase from Ceriporiopsis subvermispora and Bjerkandera adusta. Eur. J. Biochem., 267,42224231 (2000). Mann, R. M. and Bidwell, J. R.: The acute toxicity of agricultural surfactants to the tadpoles of four Australian and two exotic frogs. Environ. Pollut., 114, 195-205 (2001).