Biodegradation of Dieldrin by a Soil Fungus Isolated from a Soil with

Jul 21, 2010 - An aerobic dieldrin-degrading fungus, Mucor racemosus strain DDF, was isolated from a soil to which endosulfan had been annually applie...
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Environ. Sci. Technol. 2010, 44, 6343–6349

Biodegradation of Dieldrin by a Soil Fungus Isolated from a Soil with Annual Endosulfan Applications R Y O T A K A T A O K A , † K A Z U H I R O T A K A G I , * ,† I C H I R O K A M E I , †,‡ H I R O M A S A K I Y O T A , § AND YUUKI SATO§ Organochemicals Division, National Institute for Agro-Environmental Sciences, 3-1-3, Kannondai, Tsukuba-shi, Ibaraki, 305-8604, Japan, Division of Forest Science, Department of Biological Production and Environmental Science, Miyazaki University, 1-1, Gakuen Kibanadai-nishi, Miyazaki-shi, 889-2192, Japan, and Laboratory of Applied Bioorganic Chemistry, Department of Applied Bioorganic Chemistry, Division of Bioscience & Biotechnology for Future Bioindustries, Graduate School of Agricultural Science, Tohoku University, Aoba-ku, Sendai 981-8555, Japan

Received January 5, 2010. Revised manuscript received June 29, 2010. Accepted July 7, 2010.

An aerobic dieldrin-degrading fungus, Mucor racemosus strain DDF, was isolated from a soil to which endosulfan had been annually applied for more than 10 years until 2008. Strain DDF degraded dieldrin to 1.01 µM from 14.3 µM during a 10day incubation at 25 °C. Approximately 0.15 µM (9%) of aldrin transdiol was generated from the dieldrin degradation after a 1-day incubation. The degradation of dieldrin by strain DDF was detected over a broad range of pH and concentrations of glucose and nitrogen sources. Extracellular fluid without mycelia also degraded dieldrin. Strain DDF degraded not only dieldrin but also heptachlor, heptachlor epoxide, endosulfan, endosulfan sulfate, DDT, and DDE. Endosulfan sulfate and heptachlor were degraded by 0.64 µM (95%) and 0.75 µM (94%), respectively, whereas endosulfan and DDE were degraded by 2.42 µM (80%) and 3.29 µM (79%), respectively, and DDT and heptachlor epoxide were degraded by 6.95 µM (49.3%) and 5.36 µM (67.5%), respectively, compared with the control, which had a concentration of approximately 14 µM. These results suggest that strain DDF could be a candidate for the bioremediation of sites contaminated with various persistent organochlorine pesticides including POPs.

Introduction Organochlorine insecticides, including dieldrin, are synthetic chemicals, some of which have been extensively used for controlling diseases of humans and domestic animals carried by insects and mites. They have also been used against insect pests that greatly damage agricultural crops. Due to their efficiency as insecticides, these compounds were originally considered to be a boon to agriculture and medical entomology. However, their use has been prohibited in many countries since the 1970s because of their biological magnification, high toxicity, and long persistence in the environ* Corresponding author tel/fax: +81-29-838-8325; [email protected]. † National Institute for Agro-Environmental Sciences. ‡ Miyazaki University. § Tohoku University. 10.1021/es1000227

 2010 American Chemical Society

Published on Web 07/21/2010

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ment. Organochlorine insecticides are still found in the environment more than 30 years after their prohibition (1, 2). In Japan, dieldrin concentrations at problem levels exceeding 0.02 mg/L have been detected in cucumbers. Dieldrin was also detected in the soil at a maximum concentration of 2.6 mg/kg dry soil (2). Therefore, contamination with organochlorine insecticides is still a serious environmental problem and an efficient remediation method is required. Microbial degradation is a cost-effective and efficient way to remediate contaminated environments. To date, the degradation of organochlorine pesticides using bacteria (3, 4) and fungi (5) has been reported. The filamentous fungi such as white-rot fungi have been reported to be capable of degrading a variety of environmentally persistent organic pollutants, including hexachlorocyclohexane (HCH) (14), DDT (15), and endosulfan (16). Matsumura and Boush (6) isolated dieldrin-degrading Trichoderma viride from soil that had been heavily contaminated with various insecticides. They also suggested that 6,7-trans-dihydroxydihydroaldrin (aldrin trans-diol) might be a major product on the basis of an identical Rf value to an authentic control. Moreover, Wedemeyer (7) described the conversion of dieldrin in vitro by Aerobacter aerogenes to a compound chromatographically similar to 6,7-trans-dihydroxydihydroaldrin. Aldrin transdiol (LD50; 1250 mg/kg) showed a much lower toxicity to mice than dieldrin (LD50; 65 mg/kg) (24). Therefore, it is important to distinguish between the toxicity of dieldrin and its metabolites. However, limited information is available about metabolites from the aerobic biodegradation of dieldrin (6, 8). Therefore, there is still an urgent need to develop an effective bioremediation method for aerobic zones polluted with organochlorine insecticides. The objective of this study was to find a soil fungus that can degrade dieldrin. This was investigated by screening a range of fungi for their ability to degrade dieldrin from soils to which endosulfan had been applied annually.

Experimental Section Soils. Three soil samples collected from different agricultural sites (two soils from Kagoshima and one soil from Ehime) having a history of repeated endosulfan applications were used for the isolation of dieldrin-degrading fungi. The basic properties of the three types of soils were determined and are shown in Table S1 of the Supporting Information. Chemicals. Organochlorine insecticides used included dieldrin ((1aR,2aR,6aR,7aR)-3,4,5,6,9,9-hexachloro-1a,2, 2a,3,6,6a,7,7a-octahydro-2β,7β:3R,6R-dimethanonaphth[2,3b]oxirene), heptachlor (1,4,5,6,7,8,8-heptachloro-3a,4,7,7atetrahydro-4,7-methano-1H-indene), heptachlor epoxide (1,2,,4,5,6,7,8,8-octachloro-2,3,3a,4,7,7a-hexahydro-4,7methano-1H-indene), endosulfan (6,7,8,9,10,10-hexachloro1,5,5a,6,9,9a-hexahydro-6,9-methano-2,4,3-benzodioxathiepin 3-oxide), endosulfan sulfate (6,7,8,9,10,10hexachloro-1,5,5a,6,9,9a-hexahydro-6,9-methano-2,4,3benzodioxathiepin 3,3-dioxide), DDT (1,1,1-trichloro-2,2bis(4-chlorophenyl)ethane), and DDE (1,1-dichloro-2,2bis(4-chlorophenyl)ethane). (()-Aldrin trans-diol ((()1,2,3,4,10,10-hexachloro-1,4,4aβ,5,6,7,8,8aβ-octahydro1β,4β:5R,8R-dimethanonaphthalene-6β,7R-diol) was synthesized from aldrin according to Bedford and Harrod’s (9) procedure with slight modification (Figure S1, Supporting Information). Epoxidation of aldrin with m-chloroperbenzoic acid afforded dieldrin. The epoxy ring of dieldrin was then opened using acidic conditions [(i) conc. H2SO4, CH3CO2H-H2O, reflux; (ii) (CH3CO)2O, reflux], and the VOL. 44, NO. 16, 2010 / ENVIRONMENTAL SCIENCE & TECHNOLOGY

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resulting diacetate was hydrolyzed (K2CO3, CH3OH) to give aldrin trans-diol. Microorganisms and Isolation of Fungi from Soil. Thirty-five strains of Trichoderma spp., isolated from raw wood cultivated with Lentinula edodes (Berk.) Pegler, were provided by K. Yokota, Tokyo University of Agriculture. One gram of each soil was mixed with 20 mL of sterile distilled water using a Universal Homogenizer (Nihon Seiki Seisakusho, Tokyo, Japan), and then diluted to 10-2, 10-3, and 10-4 dilutions. Aliquots of 100 µL of the resulting soil solutions were then placed onto a plate of Martin agar medium (10) including chloramphenicol (0.25 g L-1). The plates were then incubated at 25 °C for 5 days. The fungal colonies were classified into several groups according to their morphology, growth rate, and color, and representatives of each group were picked up with a sterile needle and subcultured on potato dextrose agar (PDA; Difco Laboratories, Detroit, MI) plates. Degradation Experiments. All isolated fungi were grown on PDA agar medium in Petri dishes at 25 °C. Fungal disks (6 mm diameter) were taken from the margin of fungal colonies grown for 14 days. Each fungal disk was transferred into 10 mL of a modified Czapek-Dox (MCD) liquid medium (glucose 10 g; MgSO4 · 7H2O 0.5 g; NaNO3 2.0 g; FeSO4 · 4H2O 0.01 g; K2HPO4 1.0 g; Bacto Yeast extract (Difco) 0.5 g per liter) in a sterilized 100-mL Erlenmeyer flask. After preincubation for 7 days, 50 µL of dieldrin in acetone was added to each inoculated flask (final concentration: 13.2 µM). To prevent the volatilization of dieldrin, the flask was sealed with a glass stopper (23). The cultures were incubated statically for 14 days at 25 °C under dark conditions. As a control, the cultures were killed by autoclaving (121 °C, 15 min) after 7 days of preincubation. The cultures were homogenized with 15 mL of acetone, and the residual biomass was removed by centrifugation at 3000g for 10 min. An aliquot of 1 mL of supernatant was transferred into a test tube, and extracted with 5 mL of hexane. Dieldrin was analyzed by gas chromatography with a 63Ni electron capture detector (GC/ ECD). The GC/ECD was performed on an HP 6890 GC system linked to an HP 5890 detector and a 15 m column (HP50+, J&W Scientific, Folson, CA). The oven temperature was programmed to increase from 150 to 200 at 20 °C/min and 200 to 280 at 20 °C/min. The recovery rate of dieldrin in the control cultures was found to be 90-100%, which indicated that the extraction was efficient, and the analysis deviation was small. In the screening assay, the replication of samples and controls was one and three, respectively. After screening, the time course degradation experiment for dieldrin-degrading fungus was performed in triplicate. Experiments investigating the degradation of heptachlor, heptachlor epoxide, DDT, DDE, endosulfan, and endosulfan sulfate by dieldrin-degrading fungus were also performed as described above. Identification of the Dieldrin-Degrading Fungus. To identify the dieldrin-degrading fungal species, DNA was extracted from pure cultures of fungus using a FastDNA Kit as described by the manufacturer (Q-BIOgene). The internal transcribed spacer (ITS) region of rDNA was amplified using an ITS1-ITS4 primers set (11). DNA sequences were determined using an ABI 3130 Genetic Analyzer with a reaction kit (Big Dye Terminator v.1.1 Cycle Sequencing Kit, Applied Biosystems) following the manufacturer’s manual. The DNA sequence was compared with the sequences of known species in the GenBank database. All sequence data, including newly obtained and retrieved sequences, were aligned with the computer program ClustalX (available at http://bips. u-strasbg.fr/fr/Documentation/ClustalX/). Distance-based phylogenetic trees were generated by the neighbor-joining method (12) with the model of Jukes and Cantor (13). The topology of phylogenetic trees was evaluated by bootstrap 6344

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resampling (1000 replicates). Clustal W provided by the DNA Data Bank of Japan (available at http://www.ddbj.nig.ac.jp/ Welcome-j.html) was used for the analyses. Detection of the Metabolite in Dieldrin Degradation. The dieldrin-degrading fungus was grown in 500 mL of MCD medium containing 13.2 µM dieldrin for 5 days. The whole culture was homogenized and separated into a mycelial fraction and a fluid fraction by centrifugation. The fluid fraction was acidified to pH 2.0 with 0.1 N HCl and extracted three times with ethyl acetate. The mycelial fraction was also extracted three times with ethyl acetate. The ethyl acetate extracts were mixed, dried with anhydrous sodium sulfate, and evaporated to a state of dryness. The concentrate was resuspended into acetone/hexane (1:1) and was derivatized with bis(trimethylsilyl) trifluoroacetamide (BSTFA). Aldrin trans-diol in the BSTFA-derivatized concentrate was detected by GC/ECD. The oven temperature was programmed to increase from 150 to 200 at 20 °C/min and 200 to 280 at 20 °C/min. The inlet temperature was set at 180 °C. GC/MS analysis for confirmation of aldrin trans-diol was performed with a HP6890 series high-resolution gas chromatograph (HRGC) with a 60-m column (DB5-MS, J&W Scientific, Folson, CA) interfaced to a high-resolution mass selective detector (HRMS) (Micromass Autospec-Ultima) to detect low concentrations of the material. The oven temperature was programmed to increase from 140 to 200 at 10 °C/min, 200 to 270 at 2 °C/min, and 270 to 300 at 30 °C/min. The inlet temperature was set at 180 °C. Effect of pH and Carbon and Nitrogen Source Concentrations on the Degradation of Dieldrin. The dieldrindegrading strain DDF fungus was grown on PDA agar medium at 25 °C. The biodegradation potential of the fungus was assessed at various glucose and nitrogen source concentrations in the MCD liquid culture after 10 days’ incubation. The pH of the MCD medium was adjusted by using 0.1 N HCl or 0.1 N NaOH to obtain pH values of 4.0, 6.0, and 8.0. Glucose concentrations varied from 0.1 to 10% and nitrogen source concentrations varied from 0.01 to 2.0 fold. The nitrogen source was added as yeast and sodium nitrate. Heatkilled controls were prepared by autoclaving cultures that had been preincubated using the same conditions and over the same time period as described above. Effect of Extracellular Fluid of the Dieldrin-Degrading Fungus on the Degradation of Dieldrin. The fungal disks of the dieldrin-degrading fungus (6 mm diameter) were transferred into 1 L of MCD liquid medium. After incubation for 14 days, the spent broth culture was filtered through a 0.45µm pore size membrane filter (Whatman, Wallingford, United Kingdom) to prepare extracellular fluid. The extracellular fluid was concentrated by ultrafiltration with a 1-kDa cutoff membrane filter (Millipore corp., Billerica, MA) at 4 °C. The ultrafiltration concentrate was precipitated with ammonium sulfate (30-80% saturation). Each precipitate was collected by centrifugation at 15,000g for 30 min at 4 °C and dissolved in 15 mL of 50 mM phosphate buffer (pH 7.2). The ultrafiltrate and the ammonium sulfate fraction of the concentrate were filtered through a 0.2-µm pore size membrane filter (Whatman, Wallingford, United Kingdom). Ten mL of the filtrate was supplemented with 50 µL of dieldrin (final concentration: 13.2 µM). Degradation assays were performed on the filtrate for 5 days at 25 °C in sterilized 100-mL Erlenmeyer flasks. Degradation assays were examined twice for reproducibility.

Results Degradation Experiment. The degradation experiment was performed using 35 strains of Trichoderma spp. and 36 strains of soil isolates. Among Trichoderma spp., Trichoderma sp. strain 93155 was capable of degrading dieldrin with 19.7% degradation after 14 days of incubation. However, a fungus, isolated from soil A (Kagoshima Table S1), contaminated

FIGURE 1. Time course of degradation of dieldrin by strain DDF. Solid circles indicate the concentration of remaining dieldrin and open triangles indicate the hyphal growth as dry weight. Error bars indicate ( SE for triplicate samples. with endosulfan, degraded dieldrin by 95.8% compared with 13.2 µM in the initial concentration. It was named strain DDF. Another fungal strain with the ability to degrade dieldrin but having a lower degradation capability of 43.3% was also isolated. A further experiment was performed using strain DDF. In the time course degradation experiment, strain DDF degraded dieldrin by more than 90% over 10 days at 25 °C (Figure 1). Identification of the Dieldrin-Degrading Fungus. The ITS rRNA sequence of strain DDF (471 nucleotides, GenBank accession no. AB536702) was compared with those of the fungal sequences in the GenBank. Strain DDF exhibited a high sequence similarity to those of Mucoraceae fungi as shown by the constructed phylogenetic dendrogram (Figure 2). The highest sequence identity (100%) was found with Mucor racemosus f. racemosus (GenBank accession no. AY213659). Strain DDF was designated as Mucor racemosus strain DDF. Detection of Metabolite in Dieldrin Degradation. In the ethyl acetate extracts of culture from strain DDF, a different

peak with dieldrin was detected at the retention time of 7.39 min by GC/ECD. This peak was assigned as aldrin trans-diol on the basis of coelution with an authentic standard. The time courses of dieldrin degradation and aldrin trans-diol production by strain DDF are shown in Figure 3a. When dieldrin was added to the MCD cultures, it rapidly decreased with accompanying growth of strain DDF and aldrin transdiol was produced. In contrast, production of aldrin transdiol was not detected in the autoclaved control culture. The concentration of aldrin trans-diol was highest (0.15 µM) in the 1-day culture after adding dieldrin (Figure 3a). The full scan mass spectrum of aldrin trans-diol derivatized with BSTFA is shown in Figure 3b. It has a molecular ion peak at m/z 542, and remarkable fragment ions were detected at m/z 506.8763 and m/z 414.8248. Aldrin transdiol as a metabolite was confirmed by monitoring of the chlorination pattern using the selective ion monitoring (SIM) mode of HRGC-HRMS. The mass spectra m/z 414.8248, 416.8241, 506.8763, and 508.8708 of aldrin trans-diol were selected. A lock-mass m/z (430.9728) from perfluorokerosene (PFK) was used for drift correction. The relative intensity of the target fragment ions of the metabolite was 39.3 (414.8248), 70.2 (416.8241), 100 (506.8763), and 54.0 (508.8708). The metabolite was detected at the retention time of 27.95 min, and its retention time and relative intensity of chlorination pattern matched those of the standard (Figure 3c). Degradation of Other Organochlorine Pesticides by Strain DDF. Heptachlor epoxide, heptachlor, endosulfan, endosulfan sulfate, DDT, and DDE were used to examine the degradation capability of strain DDF. Strain DDF degraded all of the compounds (Table 1). Endosulfan sulfate and heptachlor were degraded by 2.42 and 3.29 µM, respectively, whereas endosulfan and DDE were degraded by 2.42 and 3.29 µM, respectively. DDT and heptachlor epoxide were degraded by 6.95 and5.36 µM, respectively, compared with the control concentration of approximately 14 µM. Effect of pH, and Carbon and Nitrogen Concentrations in the Medium on the Degradation of Dieldrin. The degradation of dieldrin was investigated at pH 4.0, 6.0, and 8.0, and at various carbon and nitrogen source concentra-

FIGURE 2. Phylogenetic relationships of strain DDF isolated in this study and related species. The phylogenetic tree of ITS region sequences was generated by the neighbor-joining method. The tree was tested for support by performing bootstrap resampling (1000 replicates). The bootstrap values are given at branch, and accession numbers of each sequence employed are in parentheses. VOL. 44, NO. 16, 2010 / ENVIRONMENTAL SCIENCE & TECHNOLOGY

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FIGURE 3. (a) Time courses for disappearance of 13.2 µM of dieldrin and formation of aldrin trans-diol. Values represent the means of triplicate incubations. Dieldrin-degradation and aldrin trans-diol generation are represented in solid circles and squares, respectively, and open circles and open triangles indicate concentration of dieldrin and aldrin trans-diol in the control culture, respectively. (b) Mass spectra of aldrin trans-diol derivatized with BSTFA. The base ion peak m/z 73 was omitted because its abundance is too high. Monitoring ions were selected for the comparatively high intensity chlorination pattern. Chemical structure of aldrin trans-diol derivatized with BSTFA is also indicated. (c) Retention time of aldrin trans-diol derivatized with BSTFA detected by GC/MS analysis with a HRMS-HRGC, and the set of individual intensity of the target ion based on the chlorination pattern. tions. Approximately 90% of dieldrin was degraded at all three pH values (Figure 4a). With regard to the effect of carbon concentration, the maximum degradation was 89.8% at an added glucose concentration of 1.0%. No significant differ6346

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ences were observed at added glucose concentrations of 0.5%, 1.0%, and 2.0%, in contrast with lower degradation at concentrations of 0.1% and 10% (Figure 4b). Dieldrin was degraded over the range of added nitrogen concentrations,

TABLE 1. Degradation of Organochlorine Pesticides by Strain DDF

dieldrin R endosulfan β endosulfan endosulfan sulfate heptachlor heptachlor epoxide DDT DDE

DDF µM ((S.D.)

control µM ((S.D.)

% degradation

1.05 ( 0.06 2.42 ( 0.24 2.78 ( 0.33 0.64 ( 0.21 0.75 ( 0.13 5.36 ( 0.62 6.95 ( 0.87 3.29 ( 0.42

14.3 ( 0.57 13.8 ( 0.73 13.2 ( 0.57 13.2 ( 0.24 13.3 ( 1.76 16.5 ( 0.77 13.7 ( 1.13 15.7 ( 2.15

92.7 82.5 78.9 95.2 94.4 67.5 49.3 79.0

although the degradation varied with nitrogen concentrations (Figure 4c). However, there were no significant differences among0.5-,1-,and2-foldvariationsinnitrogenconcentrations. Effect of the Extracellular Fluid of Strain DDF on the Degradation of Dieldrin. Initially, the extracellular fluid of the spent broth culture was used for the dieldrin degradation experiments. However, the degradation was not reproducible (data not shown). Therefore, the extracellular fluid was

FIGURE 5. Degradation of dieldrin by the extracellular fluid from strain DDF. Filtrates of ammonium sulfate fraction of extracellular fluid of strain DDF were incubated with 13.2 µM of dieldrin for 5 days. Solid bars indicate the remaining dieldrin in the cultures of the ammonium sulfate fractions. Open bar indicates the control incubation, which represents the remaining dieldrin in the phosphate buffer. Values represent the means of duplicate incubations. concentrated by ultrafiltration, and the ultrafiltrate was further fractionated with ammonium sulfate. The ultrafiltrate degraded dieldrin by 46% (mean value of duplicates) compared with the control. As well, degradation of dieldrin was observed in the fraction corresponding to 55-75% saturation of ammonium sulfate, whereas almost no degradation was observed in the fraction corresponding to 35-45% saturation. The highest degradation was obtained in the 55% fraction (Figure 5).

Discussion

FIGURE 4. Effect of pH, and carbon and nitrogen source concentrations in the medium on the degradation of dieldrin. Cultures were incubated with 13.2 µM of dieldrin for 14 days at various pH values (a) and various concentrations of glucose (b) and nitrogen (c) sources with either living strain DDF (solid bars) or autoclaved controls (open bars). Values represent the means of triplicate incubations; error bars indicate ( SE of the means.

In the present study, a new aerobic dieldrin-degrading fungus, Mucor racemosus strain DDF, was isolated from a soil with a history of repeated endosulfan applications. The dieldrindegrading capability of strain DDF was greater than that of any previously reported microorganisms. Degradation of dieldrin was reported by Kennedy et al. (17), who described how Phanerochaete chrysosporium was able to carry out the epoxidation of aldrin to dieldrin, but that further degradation was very slow. Other microbial degradation by fungi such as Trichoderma, Fusarium, and Penicillium spp. also often leads to the formation of an epoxide ring structure, which tends to be more stable than the parent compound (18). Therefore, dieldrin is more stable than aldrin in soil, and no microorganisms have been reported to degrade dieldrin by more than 30%. Anderson et al. (5) reported that Mucor alternans (a synonym of M. circinelloides) degraded dieldrin by 1.81 µM from 2.6 µM. Thus, strain DDF degrades dieldrin more rapidly and to a much greater extent than previously reported microorganisms. In the present study, we isolated the strain DDF fungus from soil A to which endosulfan had been annually applied. Endosulfan is a structural analog with dieldrin in part of the cyclodiene structure. Endosulfan sulfate, a metabolite of endosulfan, was accumulated at concentrations of 1.0 and 0.53 mg kg-1 in soil A and soil B, respectively. Matsumoto et al. (4) reported that the bacterial consortia in uncontaminated soil have considerable potential for aerobic degradation of dieldrin. They demonstrated that enrichment culture using 1,2-epoxycyclohexane, a dieldrin analog, showed high degradation activity toward dieldrin. Previous studies (6, 19) made extensive use of soil heavily contaminated with dieldrin to search for aerobic dieldrin-degrading microorganisms. However, no microorganisms capable of strongly degrading dieldrin were isolated. This study indicates that the enVOL. 44, NO. 16, 2010 / ENVIRONMENTAL SCIENCE & TECHNOLOGY

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dosulfan contaminated soil A has potential as a promising source for the isolation of degraders of organochlorine insecticides. In the case of remediation, different sites will have various pH values, but strain DDF could degrade dieldrin under a wide range of pH (Figure 4a). It is also advantageous that strain DDF is capable of rapid growth in soil compared with white-rot fungi. Moreover, strain DDF degraded not only dieldrin but also DDT, DDE, heptachlor, heptachlor epoxide, and endosulfan and its metabolite endosulfan sulfate. Anderson et al. (5) reported that M. alternans degraded not only dieldrin but also DDT. However, microorganisms able to degrade heptachlor epoxide have never been previously isolated. Thus, strain DDF has potential as a candidate for bioremediation. Strain DDF degraded dieldrin under a wide range of carbon and nitrogen source concentrations. As shown in Figure 1, hyphal growth and a high degradation rate of dieldrin occurred in parallel. At low concentrations of nitrogen or carbon sources, a low degradation capability of strain DDF was observed (Figures 3b, c). The hyphal growth was poor at the low concentrations of glucose and nitrogen probably from nutrient deficiency, and at 10% glucose due to high osmotic pressure (data not shown). These results raise the possibility that strain DDF needs a suitable growth situation to degrade dieldrin. Kamei et al. (23) reported that Phlebia sp. YK543 degraded 39.1% of dieldrin and generated 9-hydroxydieldrin as a metabolite. They suggest hydroxylation by fungal monooxygenase such as P450 might be involved in the transformation of dieldrin. On the other hand, white-rot fungi possess an extracellular oxidative enzymatic system that degrades wood lignin (20). Three ligninolytic enzymes, manganese peroxidase (MnP), lignin peroxidase (LiP), and laccase, also catalyze the degradation of organic pollutants (21). These ligninolytic enzyme systems of the best-studied model organism, P. chrysosporium, are triggered in response to N, C, or S limitation (16, 22). In the present study, 9-hydroxydieldrin or other metabolites could not be detected in the culture of DDF. In addition, the degradation of dieldrin was also observed at high nitrogen source concentrations. Therefore, the mechanism of dieldrin degradation by DDF might be different from that above. However, Bzalel et al. (26) showed that extracellular epoxide hydrolase of Pleurotus ostreatus was involved in the degradation of phenanthrene. Aspergillus niger also produce epoxide hydrolase (25). This enzyme involves in the reaction proceeds opening of the epoxide leading to the formation of the trans-diol. Thus we consider that the enzyme such as epoxide hydrolase might be involved in the transformation of dieldrin to aldrin trans-diol. Wedemeyer (7) used GC/ECD to describe how Aerobacter aerogenes converted dieldrin in vitro to a compound chromatographically similar to aldrin trans-diol. In the present study, we also confirmed that dieldrin converted to aldrin trans-diol. In the time course experiment, the concentration of generated aldrin trans-diol was 0.15 µM and reached a maximum after 1 day of incubation with a subsequent gradual decrease (Figure 3a). During this period, 13.2 µM of initial dieldrin decreased to 12.1 µM, and approximately 9% of aldrin trans-diol was generated from the degraded dieldrin. In a preliminary study, aldrin trans-diol disappeared when used as the initial substrate (data not shown). Thus, we consider that aldrin trans-diol is not the ultimate end product. On the basis of the result that strain DDF degraded aldrin transdiol, the possibility that disappearance of the metabolite is more rapid than its generation is higher than the possibility that bioconversion of dieldrin into aldrin trans-diol is a minor pathway. 6348

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Acknowledgments This work was supported by a grant from the Ministry of Agriculture, Forestry and Fisheries of Japan (Research project for ensuring food safety from farm to table PO-2215). We thank Dr. Kenji Yokota for providing Trichoderma spp. We also thank Dr. Akio Iwasaki (Kowa Research Institute, and Kowa Co., Ltd) for his critical suggestions in preparing this article.

Supporting Information Available One figure and one table. This material is available free of charge via the Internet at http://pubs.acs.org.

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