- Email: [email protected]
Screening for basidiomycetous fungi capable of degrading 2,7-dichlorodibenzo-p-dioxin
FEMS Microbiology Letters 213 (2002) 213^217
www.fems-microbiology.org
Screening for basidiomycetous fungi capable of degrading 2,7-dichlorodibenzo-p-dioxin Akira Sato a
a;b;
, Tsuneo Watanabe a , Yoshio Watanabe b , Koichi Harazono a , Takema Fukatsu a
Bioconsortia Program Laboratory, National Institute of Advanced Industrial Science and Technology, Tsukuba Central 6, 1-1-1 Higashi, Tsukuba, Ibaraki 305-8566, Japan b Bioresource Laboratories, Mercian Corporation, 4-9-1, Johnan, Fujisawa, Kanagawa 251-0057, Japan Received 7 May 2002 ; received in revised form 14 June 2002; accepted 14 June 2002 First published online 16 July 2002
Abstract We devised a screening method to obtain basidiomycetous fungi capable of degrading dioxins. About 200 fungal strains were selected from more than 1500 strains by their ability to decolorize Remazol brilliant blue R dye as an indicator. To attempt to eliminate the factor of dioxin sorption by mycelia, we prepared two series of living cultures exposed either long term or short term to 2,7-dichlorodibenzo-pdioxin (2,7-DCDD), and compared the decreases in the levels of this chemical. In only 11 strains was there a significant difference between the two treatments. We chose Panellus stypticus strain 99-334 as a new, effective dioxin degrader, because it gave a close to 100% decrease in 2,7-DCDD levels (from an initial concentration of 10 WM) after 40 days of exposure. The detection of a metabolic intermediate (1-chloro-3,4-dihydroxybenzene) by gas chromatography^mass spectrometry analysis supported the ability of this strain to degrade 2,7DCDD. 8 2002 Federation of European Microbiological Societies. Published by Elsevier Science B.V. All rights reserved. Keywords : Dioxin degradation ; 2,7-Dichlorodibenzo-p-dioxin ; Sorption; White-rot fungi; Panellus stypticus
1. Introduction Several strains of white-rot basidiomycetous fungi, which have the ability to cometabolize polychlorinated dibenzo-p-dioxins (PCDDs) by non-speci¢c lignin-degrading enzymes [1^3], have been evaluated for their potential in the bioremediation of contaminated soil [4^6]. Nevertheless, dioxins tend to remain in the culture despite incubations as long as 1 month, even with Phanerochaete chrysosporium [1,2], a fungus frequently used in bioremediation studies. Previous studies [7,8] suggested the existence of several fungi more active than P. chrysosporium. We therefore anticipated that we would be able to ¢nd e¡ective dioxin-degrading fungi by screening a variety of isolates and natural samples. We investigated a phenomenon in which 2,7-dichlorodibenzo-p-dioxin (2,7-DCDD), a model dioxin compound, was absorbed by fungal biomass but the binding activity
* Corresponding author, at address b. Tel.: +81 (466) 35 15 17; Fax : +81 (466) 35 15 30. E-mail address : [email protected] (A. Sato).
disappeared in autoclaved fungus. In this case, the di¡erence in the reduction of 2,7-DCDD levels between the living and killed fungus can be misinterpreted as degradation. Therefore, it was necessary to establish more appropriate control than killed cells for excluding the factor of sorption. Our aim was to select fungi capable of degrading 2,7DCDD using the new screening method. For the selected fungus, we attempted to detect metabolic intermediates, and performed several brief analyses involving lignin-degrading enzymes and cytochrome P-450 monooxygenase.
2. Materials and methods 2.1. Primary screening method As an initial screening method for selecting dioxin-degrading fungi, we adopted the dye-decolorizing method. The dye Remazol brilliant blue R (RBBR, Sigma Chemical Co., St. Louis, MO, USA) is dramatically decolorized by lignin-degrading fungi [9,10]. The plate medium was composed of double layers. Upper layer: 0.5% malt ex-
0378-1097 / 02 / $22.00 8 2002 Federation of European Microbiological Societies. Published by Elsevier Science B.V. All rights reserved. PII : S 0 3 7 8 - 1 0 9 7 ( 0 2 ) 0 0 8 2 1 - 2
FEMSLE 10573 5-8-02
214
A. Sato et al. / FEMS Microbiology Letters 213 (2002) 213^217
tract (Difco Laboratories, Detroit, MI, USA), 1.0% agar and 1.0% RBBR. Bottom layer: Czapek solution agar (Difco) medium. More than 800 strains, including mainly Basidiomycetes and Deuteromycetes already isolated from rotted woods and soils, were inoculated on to the dye media. In over 700 samples we used direct inoculation from the fruiting body and rotted wood; in these cases methyl 1-(butylcarbamoyl)-2-benzimidazole carbamate (Aldrich Chemical Co. Inc., Milwaukee, WI, USA) was added to the upper layer to a ¢nal concentration of 10 ppm to suppress the growth of mold contaminants. Each dye-decolorizing strain was isolated and then stored on potato dextrose agar (Nissui Pharmaceutical Co. Ltd., Tokyo, Japan) at room temperature. 2.2. Secondary screening method Each dye-decolorizing strain was transferred to four 50-ml glass-stoppered Erlenmeyer £asks, containing 5 ml low-nitrogen medium [11], and preincubated at 25‡C under ambient atmosphere. After 10 days, 20 Wl of 2,7DCDD (AccuStandard, Inc., New Haven, CT, USA) dissolved in N,N-dimethylformamide (DMF) was added to two £asks to a ¢nal 2,7-DCDD concentration of 10 WM (15-day-exposed culture : A). The glass stopper was tightly sealed with Te£on tape after the addition of dioxin. During the 15-day incubation at 25‡C, 250 Wl of 20% glucose solution (¢nal concentration 1%) were added twice, and oxygen was supplied to the headspaces four times. Twenty microliters of DMF, excluding 2,7-DCDD, were added to each of the remaining two £asks, which were then incubated as above (living control culture : B). One day before the end of the incubation period, we added 2,7-DCDD alone to B, and DMF alone to A. The control culture thus became a second experimental culture subjected to shortterm 2,7-DCDD exposure. We then compared the amounts of 2,7-DCDD remaining in A and B. To recover 2,7-DCDD remaining in the culture, 5 ml of concentrated sulfuric acid were added before extraction with n-hexane (three 10-ml portions) [3]. The combined hexane fraction was washed with distilled water, dried under sodium sulfate, and evaporated to 0.1 ml. Anthracene was used as the internal standard. The concentration of 2,7-DCDD was determined by gas chromatography^ mass spectrometry (GC^MS) (TurboMass, Perkin-Elmer Co., Norwalk, CT, USA) in a device ¢tted with an NB-1 capillary column (GL Science Inc., Tokyo, Japan) at 70 eV. The oven temperature was programmed from 100 to 300‡C at 10‡C min31 , with constant temperatures of 100‡C for 2 min and 200‡C for 2 min. 2.3. Detection of metabolic intermediate Detection of the metabolic intermediate of 2,7-DCDD was conducted as described in previous studies [2,3], except that the ¢nal concentration of sodium dithionite as a
reduction agent was 2 mg ml31 . 1-Chloro-3,4-dihydroxybenzene was purchased from Cambridge Isotope Laboratories (Andover, MA, USA). 2.4. Morphological and phylogenetic characterization The morphological features of the dried specimen were compared with a photographic record [12]. The spores were mounted on a microscope slide with a drop of sterilized water and examined at U400. To obtain genomic DNA, 1 g (wet) of mycelial mat was frozen with liquid nitrogen and ground to a ¢ne powder with a mortar and pestle. Genomic DNA was extracted with a FastDNA0 Kit (Bio 101 Inc., Carlsbad, CA, USA) and puri¢ed with a QIAquick PCR Puri¢cation Kit (Qiagen, Hilden, Germany) according to the manufacturers’ protocols. The nuclear rDNA regions of internal transcribed spacers 1 and 2 (ITS 1 and 2) and of the intervening 5.8S gene were ampli¢ed by polymerase chain reaction (PCR) using Taq DNA polymerase (Takara Shuzo Co. Ltd., Shiga, Japan) and the primers ITS-1 and ITS-4 [13]. 2.5. Enzyme assays Activities of the exocellular enzymes involved in lignin degradation in the culture ¢ltrate were measured in 1-ml mixtures at a reaction temperature of 37‡C. The reaction conditions were the same as those used by Kondo et al. [14] for laccase and lignin peroxidase, and by Kuan et al. [15] for manganese peroxidase. The e¡ect of cytochrome P-450 inhibitor was determined by adding piperonyl butoxide (Wako Pure Chemical Industries, Ltd., Osaka, Japan) [16]. Cultures with inhibitor were incubated for 20 min, and then 2,7DCDD (¢nal concentration 10 WM) was added. The cultures were incubated for 15 days and extracted as described above.
3. Results and discussion 3.1. Screening of dioxin-degrading fungi From more than 1500 strains, we selected about 200 that clearly exhibited dye decolorization. The selected strains were subjected to the secondary screening. Relatively large di¡erences between the 15-day culture and the control (20^35%) were found for only 11 strains. These di¡erences were larger than that (ca. 5%) of the known dioxin degrader, Phanerochaete sordida YK-624 [3] (data not shown). When the time course of reduction in 2,7-DCDD levels was determined with 11 active strains, it was found that the most reproducible was strain 99-334 (Fig. 1). The amounts of 2,7-DCDD remaining in long-term-exposed cultures gradually decreased, whereas those in the control
FEMSLE 10573 5-8-02
A. Sato et al. / FEMS Microbiology Letters 213 (2002) 213^217
Fig. 1. Change in concentration of 2,7-DCDD in strain 99-334 cultures exposed to 2,7-DCDD for each incubation day (b) and exposed for 1 day as a living control (a). 2,7-DCDD was added to control cultures 1 day before each sampling. During the 40-day incubation, glucose and oxygen were respectively supplied once a week and twice a week. Bars indicate the maximum and minimum ranges of duplicate £asks.
cultures were almost constant. Remarkably, a 2,7-DCDD level of only a few percent was detected in the culture after 40-day exposure. 3.2. Detection of metabolic intermediates of strain 99-334 Few reports have cited detection of metabolic degrada-
215
tion products as evidence of biodegradation of PCDDs [2,3]. Notably, a compound of diacetyl ester derivative was clearly detected from the 99-334 culture by GC^MS (Fig. 2a) at the same retention time of 13.75 min as the standard reagent of 1-chloro-3,4-dihydroxybenzene (Fig. 2b). We could not detect this compound in the cell-free control that included 2,7-DCDD (Fig. 2c). MS peaks of the compound from the 99-334 culture (percentage relative intensity shown in parentheses) occurred at 230 m/z (3), 228 m/z (Mþ , 9), 188 m/z (4), 186 m/z (15), 146 m/z (31), and 144 m/z (100). This pattern was almost the same as that of the standard reagent (data not shown). These data support the concept that some of the reduction in dioxin levels in the presence of strain 99-334 was caused by degradation. The same intermediate was reported by Valli et al. [2] in 2,7-DCDD degradation by P. chrysosporium. Determination of the yields of the intermediates and CO2 is currently in progress. 3.3. Identi¢cation of strain 99-334 Morphological observation revealed that strain 99-334 was Panellus stypticus and phylogenetic characterization of the ITS region (Fig. 3) supported this (bootstrap = 100%). This strain has 99.4% identity with the other strains of P. stypticus [17], 92.7% identity with Dictyopanus pusillus, and 81.2% identity with Mycena a¡. murina. We isolated this strain from a fruiting body grown on rotted wood in a forest at Agematsu, Nagano, in cen-
Fig. 2. Detection of a metabolic intermediate (1-chloro-3,4-dihydroxybenzene) of 2,7-DCDD in a culture of strain 99-334. Total ion chromatogram of strain 99-334 (a), standard reagent (b) or cell-free control (c) at a GC retention time of 13.00^14.00 min.
FEMSLE 10573 5-8-02
216
A. Sato et al. / FEMS Microbiology Letters 213 (2002) 213^217
tral Japan. P. stypticus is distributed worldwide [17] and is classi¢ed as a white-rot fungus [18]. To our knowledge, our study is the ¢rst to determine that strain 99-334 is a P. stypticus capable of degrading environmentally recalcitrant pollutants. 3.4. Preliminary enzymatic experiments of strain 99-334 A relatively low level of laccase activity (2,6-dimethoxyphenol oxidation activity) was detected at 19^55 nmol ml31 min31 through the 15-day incubation period in the culture ¢ltrate of P. stypticus 99-334 (data not shown). Neither lignin peroxidase nor manganese peroxidase activity was found. In fact, the ¢ltrate alone did not decolorize RBBR. Since none of these exocellular enzymes seems to be associated with the degradation, we suspected the involvement of cytochrome P-450 monooxygenase, which has been noted as a key endocellular enzyme in the oxidation of xenobiotic compounds [19]. We examined the e¡ect of a P-450 inhibitor on the degradation of 2,7DCDD by P. stypticus 99-334. Addition of the inhibitor piperonyl butoxide (PB) reduced the di¡erence in the percentage of 2,7-DCDD remaining between cultures A and B to 12% and 5% at a concentration of 250 WM and 2500 WM, respectively (Table 1). We concluded that cytochrome P-450 plays an important role in dioxin oxidation. Further work should be done to determine the P-450 enzymes and genes involved, and to test the ability of P. stypticus 99-334 to degrade other dioxin congeners.
Table 1 E¡ect of PB on the di¡erence in the percentage of 2,7-DCDD remaining between cultures A and B of P. stypticus PBa (WM)
Di¡erence (%)
0 2.5 25 250 2500
35 35 33 12 5
a 10 Wl of PB dissolved in DMF were added to the culture at the ¢nal concentrations listed in the table.
Acknowledgements We thank Mrs. K. Ikeda, Dr. N. Shinzato, and Mrs. M. Muramatsu for their technical help with the experiments. P. sordida YK-624 was provided by Prof. R. Kondo (Kyushu University, Japan). This study was carried out as part of the Project for Technological Development of Biological Resources in Bioconsortia, an R and D project of the New Industrial Science and Technology Frontiers of the Ministry of Economy, Trade and Industry (METI). This project is run under the centralized management system formed by the National Institute of Advanced Industrial Science and Technology (AIST) and the Japan Bioindustry Associate (JBA), which are entrusted from the New Energy and Industrial Technology Development Organization (NEDO).
References
Fig. 3. Relationships between strain 99-334 and several species of Tricholomataceae, based upon the ITS 1, ITS 2, and 5.8S rDNA sequences. Strains of P. stypticus were reported in Jin et al. [17]. G. adspersum was represented as an outgroup. Bootstrap values from 100 replicates are included. DDBJ, EMBL, and GenBank database accession numbers are: G. adspersum = AJ006685, A. lobata = U66429, O. obscurata = U66448, L. edodes = AF079572, P. tuberregium = AF109975, M. a¡. murina = AF335444, D. pusillus = AF289061, and P. stypticus 4271 = AF289068; 99-334 = AB084488; 4319 = AF289069; 8234 = AF289065; 3342 = AF289066.
[1] Bumpus, J.A., Tien, M., Wright, D. and Aust, S.D. (1985) Oxidation of persistent environmental pollutants by a white rot fungus. Science 228, 1434^1436. [2] Valli, K., Wariishi, H. and Gold, M.H. (1992) Degradation of 2,7dichlorodibenzo-p-dioxin by the lignin-degrading basidiomycete Phanerochaete chrysosporium. J. Bacteriol. 174, 2131^2137. [3] Takada, S., Nakamura, M., Matsueda, T., Kondo, R. and Sakai, K. (1996) Degradation of polychlorinated dibenzo-p-dioxins and polychlorinated dibenzofurans by the white rot fungus Phanerochaete sordida YK-624. Appl. Environ. Microbiol. 62, 4323^4328. [4] Rosenbrock, P., Martens, R., Buscot, F., Zadrazil, F. and Munch, J.C. (1997) Enhancing the mineralization of [U-14 C]dibenzo-p-dioxin in three di¡erent soils by addition of organic substrate or inoculation with white-rot fungi. Appl. Microbiol. Biotechnol. 48, 665^670. [5] Wittich, R.-M. (1998) Degradation of dioxin-like compounds by microorganisms. Appl. Microbiol. Biotechnol. 49, 489^499. [6] Pointing, S.B. (2001) Feasibility of bioremediation by white-rot fungi. Appl. Microbiol. Biotechnol. 57, 20^33. [7] Beaudette, L.A., Davies, S., Fedorak, P.M., Ward, O.P. and Pickard, M.A. (1998) Comparison of gas chromatography and mineralization experiments for measuring loss of selected polychlorinated biphenyl congeners in cultures of white rot fungi. Appl. Environ. Microbiol. 64, 2020^2025. [8] Orth, A.B., Royse, D.J. and Tien, M. (1993) Ubiquity of lignin-degrading peroxidases among various wood-degrading fungi. Appl. Environ. Microbiol. 59, 4017^4023. [9] Pasti, M.B. and Crawford, D.L. (1991) Relationships between the
FEMSLE 10573 5-8-02
A. Sato et al. / FEMS Microbiology Letters 213 (2002) 213^217
[10]
[11] [12]
[13]
abilities of streptomycetes to decolorize three anthron-type dyes and to degrade lignocellulose. Can. J. Microbiol. 37, 902^907. Tachibana, S., Ohkawa, H., Ito, K., Oki, T. and Hirabayashi, T. (1996) Bioremediation by wood-rotting fungi (1): screening of fungi having ability for degradation of dioxins and degradation of 2,7-dichlorodibenzo-p-dioxin by some fungi separated by the screening and by some wood-rotting fungi. J. Jap. Technol. Assoc. Pulp Pap. Ind. 50, 1806^1815 (in Japanese). Tien, M. and Kirk, T.K. (1988) Lignin peroxidase of Phanerochaete chrysosporium. Methods Enzymol. 161, 238^249. Hongo, T. (1987) Tricholomataceae. In: Colored Illustrations of Mushrooms of Japan, Vol. 1 (Imazeki, R. and Hongo, T., Eds.), pp. 94^95. Hoikusha Publishing Co., Osaka (in Japanese). White, T.J., Bruns, T., Lee, S. and Taylor, J. (1990) Ampli¢cation and direct sequencing of fungal ribosomal RNA genes for phylogenetics. In: PCR Protocols (Innis, M.A. et al., Eds.), pp. 315^322. Academic Press, New York.
217
[14] Kondo, R., Harazono, K. and Sakai, K. (1994) Bleaching of hardwood kraft pulp with manganese peroxidase secreted from Phanerochaete sordida YK-624. Appl. Environ. Microbiol. 60, 4359^4363. [15] Kuan, I.-C., Johnson, K.A. and Tien, M. (1993) Kinetic analysis of manganese peroxidase. J. Biol. Chem. 268, 20064^20071. [16] Hiratsuka, N., Wariishi, H. and Tanaka, H. (2001) Degradation of diphenyl ether herbicides by the lignin-degrading basidiomycete Coriolus versicolor. Appl. Microbiol. Biotechnol. 57, 563^571. [17] Jin, J., Hughes, K.W. and Petersen, R.H. (2001) Biogeographical patterns in Panellus stypticus. Mycologia 93, 309^316. [18] Blaich, R. and Esser, K. (1975) Function of enzymes in wood destroying fungi. Arch. Microbiol. 103, 271^277. [19] Masaphy, S., Levanon, D., Henis, Y., Venkateswarlu, K. and Kelly, S.L. (1996) Evidence for cytochrome P-450 and P-450-mediated benzo(a)pyrene hydroxylation in the white rot fungus Phanerochaete chrysosporium. FEMS Microbiol. Lett. 135, 51^55.
FEMSLE 10573 5-8-02
www.fems-microbiology.org
Screening for basidiomycetous fungi capable of degrading 2,7-dichlorodibenzo-p-dioxin Akira Sato a
a;b;
, Tsuneo Watanabe a , Yoshio Watanabe b , Koichi Harazono a , Takema Fukatsu a
Bioconsortia Program Laboratory, National Institute of Advanced Industrial Science and Technology, Tsukuba Central 6, 1-1-1 Higashi, Tsukuba, Ibaraki 305-8566, Japan b Bioresource Laboratories, Mercian Corporation, 4-9-1, Johnan, Fujisawa, Kanagawa 251-0057, Japan Received 7 May 2002 ; received in revised form 14 June 2002; accepted 14 June 2002 First published online 16 July 2002
Abstract We devised a screening method to obtain basidiomycetous fungi capable of degrading dioxins. About 200 fungal strains were selected from more than 1500 strains by their ability to decolorize Remazol brilliant blue R dye as an indicator. To attempt to eliminate the factor of dioxin sorption by mycelia, we prepared two series of living cultures exposed either long term or short term to 2,7-dichlorodibenzo-pdioxin (2,7-DCDD), and compared the decreases in the levels of this chemical. In only 11 strains was there a significant difference between the two treatments. We chose Panellus stypticus strain 99-334 as a new, effective dioxin degrader, because it gave a close to 100% decrease in 2,7-DCDD levels (from an initial concentration of 10 WM) after 40 days of exposure. The detection of a metabolic intermediate (1-chloro-3,4-dihydroxybenzene) by gas chromatography^mass spectrometry analysis supported the ability of this strain to degrade 2,7DCDD. 8 2002 Federation of European Microbiological Societies. Published by Elsevier Science B.V. All rights reserved. Keywords : Dioxin degradation ; 2,7-Dichlorodibenzo-p-dioxin ; Sorption; White-rot fungi; Panellus stypticus
1. Introduction Several strains of white-rot basidiomycetous fungi, which have the ability to cometabolize polychlorinated dibenzo-p-dioxins (PCDDs) by non-speci¢c lignin-degrading enzymes [1^3], have been evaluated for their potential in the bioremediation of contaminated soil [4^6]. Nevertheless, dioxins tend to remain in the culture despite incubations as long as 1 month, even with Phanerochaete chrysosporium [1,2], a fungus frequently used in bioremediation studies. Previous studies [7,8] suggested the existence of several fungi more active than P. chrysosporium. We therefore anticipated that we would be able to ¢nd e¡ective dioxin-degrading fungi by screening a variety of isolates and natural samples. We investigated a phenomenon in which 2,7-dichlorodibenzo-p-dioxin (2,7-DCDD), a model dioxin compound, was absorbed by fungal biomass but the binding activity
* Corresponding author, at address b. Tel.: +81 (466) 35 15 17; Fax : +81 (466) 35 15 30. E-mail address : [email protected] (A. Sato).
disappeared in autoclaved fungus. In this case, the di¡erence in the reduction of 2,7-DCDD levels between the living and killed fungus can be misinterpreted as degradation. Therefore, it was necessary to establish more appropriate control than killed cells for excluding the factor of sorption. Our aim was to select fungi capable of degrading 2,7DCDD using the new screening method. For the selected fungus, we attempted to detect metabolic intermediates, and performed several brief analyses involving lignin-degrading enzymes and cytochrome P-450 monooxygenase.
2. Materials and methods 2.1. Primary screening method As an initial screening method for selecting dioxin-degrading fungi, we adopted the dye-decolorizing method. The dye Remazol brilliant blue R (RBBR, Sigma Chemical Co., St. Louis, MO, USA) is dramatically decolorized by lignin-degrading fungi [9,10]. The plate medium was composed of double layers. Upper layer: 0.5% malt ex-
0378-1097 / 02 / $22.00 8 2002 Federation of European Microbiological Societies. Published by Elsevier Science B.V. All rights reserved. PII : S 0 3 7 8 - 1 0 9 7 ( 0 2 ) 0 0 8 2 1 - 2
FEMSLE 10573 5-8-02
214
A. Sato et al. / FEMS Microbiology Letters 213 (2002) 213^217
tract (Difco Laboratories, Detroit, MI, USA), 1.0% agar and 1.0% RBBR. Bottom layer: Czapek solution agar (Difco) medium. More than 800 strains, including mainly Basidiomycetes and Deuteromycetes already isolated from rotted woods and soils, were inoculated on to the dye media. In over 700 samples we used direct inoculation from the fruiting body and rotted wood; in these cases methyl 1-(butylcarbamoyl)-2-benzimidazole carbamate (Aldrich Chemical Co. Inc., Milwaukee, WI, USA) was added to the upper layer to a ¢nal concentration of 10 ppm to suppress the growth of mold contaminants. Each dye-decolorizing strain was isolated and then stored on potato dextrose agar (Nissui Pharmaceutical Co. Ltd., Tokyo, Japan) at room temperature. 2.2. Secondary screening method Each dye-decolorizing strain was transferred to four 50-ml glass-stoppered Erlenmeyer £asks, containing 5 ml low-nitrogen medium [11], and preincubated at 25‡C under ambient atmosphere. After 10 days, 20 Wl of 2,7DCDD (AccuStandard, Inc., New Haven, CT, USA) dissolved in N,N-dimethylformamide (DMF) was added to two £asks to a ¢nal 2,7-DCDD concentration of 10 WM (15-day-exposed culture : A). The glass stopper was tightly sealed with Te£on tape after the addition of dioxin. During the 15-day incubation at 25‡C, 250 Wl of 20% glucose solution (¢nal concentration 1%) were added twice, and oxygen was supplied to the headspaces four times. Twenty microliters of DMF, excluding 2,7-DCDD, were added to each of the remaining two £asks, which were then incubated as above (living control culture : B). One day before the end of the incubation period, we added 2,7-DCDD alone to B, and DMF alone to A. The control culture thus became a second experimental culture subjected to shortterm 2,7-DCDD exposure. We then compared the amounts of 2,7-DCDD remaining in A and B. To recover 2,7-DCDD remaining in the culture, 5 ml of concentrated sulfuric acid were added before extraction with n-hexane (three 10-ml portions) [3]. The combined hexane fraction was washed with distilled water, dried under sodium sulfate, and evaporated to 0.1 ml. Anthracene was used as the internal standard. The concentration of 2,7-DCDD was determined by gas chromatography^ mass spectrometry (GC^MS) (TurboMass, Perkin-Elmer Co., Norwalk, CT, USA) in a device ¢tted with an NB-1 capillary column (GL Science Inc., Tokyo, Japan) at 70 eV. The oven temperature was programmed from 100 to 300‡C at 10‡C min31 , with constant temperatures of 100‡C for 2 min and 200‡C for 2 min. 2.3. Detection of metabolic intermediate Detection of the metabolic intermediate of 2,7-DCDD was conducted as described in previous studies [2,3], except that the ¢nal concentration of sodium dithionite as a
reduction agent was 2 mg ml31 . 1-Chloro-3,4-dihydroxybenzene was purchased from Cambridge Isotope Laboratories (Andover, MA, USA). 2.4. Morphological and phylogenetic characterization The morphological features of the dried specimen were compared with a photographic record [12]. The spores were mounted on a microscope slide with a drop of sterilized water and examined at U400. To obtain genomic DNA, 1 g (wet) of mycelial mat was frozen with liquid nitrogen and ground to a ¢ne powder with a mortar and pestle. Genomic DNA was extracted with a FastDNA0 Kit (Bio 101 Inc., Carlsbad, CA, USA) and puri¢ed with a QIAquick PCR Puri¢cation Kit (Qiagen, Hilden, Germany) according to the manufacturers’ protocols. The nuclear rDNA regions of internal transcribed spacers 1 and 2 (ITS 1 and 2) and of the intervening 5.8S gene were ampli¢ed by polymerase chain reaction (PCR) using Taq DNA polymerase (Takara Shuzo Co. Ltd., Shiga, Japan) and the primers ITS-1 and ITS-4 [13]. 2.5. Enzyme assays Activities of the exocellular enzymes involved in lignin degradation in the culture ¢ltrate were measured in 1-ml mixtures at a reaction temperature of 37‡C. The reaction conditions were the same as those used by Kondo et al. [14] for laccase and lignin peroxidase, and by Kuan et al. [15] for manganese peroxidase. The e¡ect of cytochrome P-450 inhibitor was determined by adding piperonyl butoxide (Wako Pure Chemical Industries, Ltd., Osaka, Japan) [16]. Cultures with inhibitor were incubated for 20 min, and then 2,7DCDD (¢nal concentration 10 WM) was added. The cultures were incubated for 15 days and extracted as described above.
3. Results and discussion 3.1. Screening of dioxin-degrading fungi From more than 1500 strains, we selected about 200 that clearly exhibited dye decolorization. The selected strains were subjected to the secondary screening. Relatively large di¡erences between the 15-day culture and the control (20^35%) were found for only 11 strains. These di¡erences were larger than that (ca. 5%) of the known dioxin degrader, Phanerochaete sordida YK-624 [3] (data not shown). When the time course of reduction in 2,7-DCDD levels was determined with 11 active strains, it was found that the most reproducible was strain 99-334 (Fig. 1). The amounts of 2,7-DCDD remaining in long-term-exposed cultures gradually decreased, whereas those in the control
FEMSLE 10573 5-8-02
A. Sato et al. / FEMS Microbiology Letters 213 (2002) 213^217
Fig. 1. Change in concentration of 2,7-DCDD in strain 99-334 cultures exposed to 2,7-DCDD for each incubation day (b) and exposed for 1 day as a living control (a). 2,7-DCDD was added to control cultures 1 day before each sampling. During the 40-day incubation, glucose and oxygen were respectively supplied once a week and twice a week. Bars indicate the maximum and minimum ranges of duplicate £asks.
cultures were almost constant. Remarkably, a 2,7-DCDD level of only a few percent was detected in the culture after 40-day exposure. 3.2. Detection of metabolic intermediates of strain 99-334 Few reports have cited detection of metabolic degrada-
215
tion products as evidence of biodegradation of PCDDs [2,3]. Notably, a compound of diacetyl ester derivative was clearly detected from the 99-334 culture by GC^MS (Fig. 2a) at the same retention time of 13.75 min as the standard reagent of 1-chloro-3,4-dihydroxybenzene (Fig. 2b). We could not detect this compound in the cell-free control that included 2,7-DCDD (Fig. 2c). MS peaks of the compound from the 99-334 culture (percentage relative intensity shown in parentheses) occurred at 230 m/z (3), 228 m/z (Mþ , 9), 188 m/z (4), 186 m/z (15), 146 m/z (31), and 144 m/z (100). This pattern was almost the same as that of the standard reagent (data not shown). These data support the concept that some of the reduction in dioxin levels in the presence of strain 99-334 was caused by degradation. The same intermediate was reported by Valli et al. [2] in 2,7-DCDD degradation by P. chrysosporium. Determination of the yields of the intermediates and CO2 is currently in progress. 3.3. Identi¢cation of strain 99-334 Morphological observation revealed that strain 99-334 was Panellus stypticus and phylogenetic characterization of the ITS region (Fig. 3) supported this (bootstrap = 100%). This strain has 99.4% identity with the other strains of P. stypticus [17], 92.7% identity with Dictyopanus pusillus, and 81.2% identity with Mycena a¡. murina. We isolated this strain from a fruiting body grown on rotted wood in a forest at Agematsu, Nagano, in cen-
Fig. 2. Detection of a metabolic intermediate (1-chloro-3,4-dihydroxybenzene) of 2,7-DCDD in a culture of strain 99-334. Total ion chromatogram of strain 99-334 (a), standard reagent (b) or cell-free control (c) at a GC retention time of 13.00^14.00 min.
FEMSLE 10573 5-8-02
216
A. Sato et al. / FEMS Microbiology Letters 213 (2002) 213^217
tral Japan. P. stypticus is distributed worldwide [17] and is classi¢ed as a white-rot fungus [18]. To our knowledge, our study is the ¢rst to determine that strain 99-334 is a P. stypticus capable of degrading environmentally recalcitrant pollutants. 3.4. Preliminary enzymatic experiments of strain 99-334 A relatively low level of laccase activity (2,6-dimethoxyphenol oxidation activity) was detected at 19^55 nmol ml31 min31 through the 15-day incubation period in the culture ¢ltrate of P. stypticus 99-334 (data not shown). Neither lignin peroxidase nor manganese peroxidase activity was found. In fact, the ¢ltrate alone did not decolorize RBBR. Since none of these exocellular enzymes seems to be associated with the degradation, we suspected the involvement of cytochrome P-450 monooxygenase, which has been noted as a key endocellular enzyme in the oxidation of xenobiotic compounds [19]. We examined the e¡ect of a P-450 inhibitor on the degradation of 2,7DCDD by P. stypticus 99-334. Addition of the inhibitor piperonyl butoxide (PB) reduced the di¡erence in the percentage of 2,7-DCDD remaining between cultures A and B to 12% and 5% at a concentration of 250 WM and 2500 WM, respectively (Table 1). We concluded that cytochrome P-450 plays an important role in dioxin oxidation. Further work should be done to determine the P-450 enzymes and genes involved, and to test the ability of P. stypticus 99-334 to degrade other dioxin congeners.
Table 1 E¡ect of PB on the di¡erence in the percentage of 2,7-DCDD remaining between cultures A and B of P. stypticus PBa (WM)
Di¡erence (%)
0 2.5 25 250 2500
35 35 33 12 5
a 10 Wl of PB dissolved in DMF were added to the culture at the ¢nal concentrations listed in the table.
Acknowledgements We thank Mrs. K. Ikeda, Dr. N. Shinzato, and Mrs. M. Muramatsu for their technical help with the experiments. P. sordida YK-624 was provided by Prof. R. Kondo (Kyushu University, Japan). This study was carried out as part of the Project for Technological Development of Biological Resources in Bioconsortia, an R and D project of the New Industrial Science and Technology Frontiers of the Ministry of Economy, Trade and Industry (METI). This project is run under the centralized management system formed by the National Institute of Advanced Industrial Science and Technology (AIST) and the Japan Bioindustry Associate (JBA), which are entrusted from the New Energy and Industrial Technology Development Organization (NEDO).
References
Fig. 3. Relationships between strain 99-334 and several species of Tricholomataceae, based upon the ITS 1, ITS 2, and 5.8S rDNA sequences. Strains of P. stypticus were reported in Jin et al. [17]. G. adspersum was represented as an outgroup. Bootstrap values from 100 replicates are included. DDBJ, EMBL, and GenBank database accession numbers are: G. adspersum = AJ006685, A. lobata = U66429, O. obscurata = U66448, L. edodes = AF079572, P. tuberregium = AF109975, M. a¡. murina = AF335444, D. pusillus = AF289061, and P. stypticus 4271 = AF289068; 99-334 = AB084488; 4319 = AF289069; 8234 = AF289065; 3342 = AF289066.
[1] Bumpus, J.A., Tien, M., Wright, D. and Aust, S.D. (1985) Oxidation of persistent environmental pollutants by a white rot fungus. Science 228, 1434^1436. [2] Valli, K., Wariishi, H. and Gold, M.H. (1992) Degradation of 2,7dichlorodibenzo-p-dioxin by the lignin-degrading basidiomycete Phanerochaete chrysosporium. J. Bacteriol. 174, 2131^2137. [3] Takada, S., Nakamura, M., Matsueda, T., Kondo, R. and Sakai, K. (1996) Degradation of polychlorinated dibenzo-p-dioxins and polychlorinated dibenzofurans by the white rot fungus Phanerochaete sordida YK-624. Appl. Environ. Microbiol. 62, 4323^4328. [4] Rosenbrock, P., Martens, R., Buscot, F., Zadrazil, F. and Munch, J.C. (1997) Enhancing the mineralization of [U-14 C]dibenzo-p-dioxin in three di¡erent soils by addition of organic substrate or inoculation with white-rot fungi. Appl. Microbiol. Biotechnol. 48, 665^670. [5] Wittich, R.-M. (1998) Degradation of dioxin-like compounds by microorganisms. Appl. Microbiol. Biotechnol. 49, 489^499. [6] Pointing, S.B. (2001) Feasibility of bioremediation by white-rot fungi. Appl. Microbiol. Biotechnol. 57, 20^33. [7] Beaudette, L.A., Davies, S., Fedorak, P.M., Ward, O.P. and Pickard, M.A. (1998) Comparison of gas chromatography and mineralization experiments for measuring loss of selected polychlorinated biphenyl congeners in cultures of white rot fungi. Appl. Environ. Microbiol. 64, 2020^2025. [8] Orth, A.B., Royse, D.J. and Tien, M. (1993) Ubiquity of lignin-degrading peroxidases among various wood-degrading fungi. Appl. Environ. Microbiol. 59, 4017^4023. [9] Pasti, M.B. and Crawford, D.L. (1991) Relationships between the
FEMSLE 10573 5-8-02
A. Sato et al. / FEMS Microbiology Letters 213 (2002) 213^217
[10]
[11] [12]
[13]
abilities of streptomycetes to decolorize three anthron-type dyes and to degrade lignocellulose. Can. J. Microbiol. 37, 902^907. Tachibana, S., Ohkawa, H., Ito, K., Oki, T. and Hirabayashi, T. (1996) Bioremediation by wood-rotting fungi (1): screening of fungi having ability for degradation of dioxins and degradation of 2,7-dichlorodibenzo-p-dioxin by some fungi separated by the screening and by some wood-rotting fungi. J. Jap. Technol. Assoc. Pulp Pap. Ind. 50, 1806^1815 (in Japanese). Tien, M. and Kirk, T.K. (1988) Lignin peroxidase of Phanerochaete chrysosporium. Methods Enzymol. 161, 238^249. Hongo, T. (1987) Tricholomataceae. In: Colored Illustrations of Mushrooms of Japan, Vol. 1 (Imazeki, R. and Hongo, T., Eds.), pp. 94^95. Hoikusha Publishing Co., Osaka (in Japanese). White, T.J., Bruns, T., Lee, S. and Taylor, J. (1990) Ampli¢cation and direct sequencing of fungal ribosomal RNA genes for phylogenetics. In: PCR Protocols (Innis, M.A. et al., Eds.), pp. 315^322. Academic Press, New York.
217
[14] Kondo, R., Harazono, K. and Sakai, K. (1994) Bleaching of hardwood kraft pulp with manganese peroxidase secreted from Phanerochaete sordida YK-624. Appl. Environ. Microbiol. 60, 4359^4363. [15] Kuan, I.-C., Johnson, K.A. and Tien, M. (1993) Kinetic analysis of manganese peroxidase. J. Biol. Chem. 268, 20064^20071. [16] Hiratsuka, N., Wariishi, H. and Tanaka, H. (2001) Degradation of diphenyl ether herbicides by the lignin-degrading basidiomycete Coriolus versicolor. Appl. Microbiol. Biotechnol. 57, 563^571. [17] Jin, J., Hughes, K.W. and Petersen, R.H. (2001) Biogeographical patterns in Panellus stypticus. Mycologia 93, 309^316. [18] Blaich, R. and Esser, K. (1975) Function of enzymes in wood destroying fungi. Arch. Microbiol. 103, 271^277. [19] Masaphy, S., Levanon, D., Henis, Y., Venkateswarlu, K. and Kelly, S.L. (1996) Evidence for cytochrome P-450 and P-450-mediated benzo(a)pyrene hydroxylation in the white rot fungus Phanerochaete chrysosporium. FEMS Microbiol. Lett. 135, 51^55.
FEMSLE 10573 5-8-02