Ichiro Kamei

Degradation of 1,1,1-trichloro-2,2-bis (4-chlorophenyl) ethane (DDT) by brown-rot fungi

Twelve species of brown-rot fungi (BRF) have been investigated for their ability to degrade 1,1,1-trichloro-2,2-bis (4-chlorophenyl) ethane (DDT). In potato dextrose broth (PDB) medium, Gloeophyllum trabeum, Fomitopsis pinicola and Daedalea dickinsii showed a high ability to degrade DDT. 1,1-Dichloro-2,2-bis (4-chlorophenyl) ethane (DDD), 1,1-dichloro-2,2-bis (4-chlorophenyl) ethylene (DDE) and 4,4-dichlorobenzophenone (DBP) were detected as metabolic products of DDT degradation by G. trabeum in PDB medium. The DDT degradation pathway in G. trabeum is proposed, which differs from the proposed pathways in bacteria and other fungi, particularly in the transformation of DDE to DDD. On the other hand, DBP was not detected as a metabolic product of DDT degradation in FeSO(4)-deficient cultures of G. trabeum, whereas DDE and DDD were detected, indicating the involvement of an iron-dependent reaction. Only DBP was detected from DDT, DDE and DDD degradation via a chemical Fenton reaction under conditions similar to those in G. trabeum cultures. These compounds may be directly transformed to DBP via a Fenton reaction.

Phylogenetical approach to isolation of white-rot fungi capable of degrading polychlorinated dibenzo- p -dioxin

A degradation experiment on PCDDs and phylogenetical analyses were carried out on newly isolated 2,7-dichlorodibenzo-p-dioxin (2,7-diCDD)-degrading white-rot fungi, strains BMC3014, BMC9152, and BMC9160. When these fungi were incubated with tri- or tetraCDDs, the substrates were degraded efficiently, and hydroxylated metabolites were detected. On the other hand, 1,3,6,8-tetrachlorodibenzo-p-dioxin was not decreased, and no metabolites were detected. Phylogenetic analysis of internal transcribed spacers (ITSs) containing rRNA gene sequence (ITS-rDNA) clarified that these strains belonged to the genus Phlebia and were closely related to the fungi Phlebia lindtneri, strains MZ-227 and MG-60, which had both been isolated as 2,7-diCDD-degrading fungi in our previous study. Based on this phylogenetical relationship, other Phlebia genera species were used for a degradation experiment on 2,7-diCDD and 1,3,6,8-tetraCDD. Phlebia acerina and Phlebia brevispora degraded 2,7-diCDD about 40 and 80%, respectively, over 14 days of incubation. It became clear that P. brevispora can degrade 1,3,6,8-tetraCDD and transform it to monohydroxy-tetraCDD, monomethoxy-tetraCDD, dimethoxy-tetraCDD, dimethoxy-triCDD, and 3,5-dichlorocatechol in the treatment cultures. In this paper, we could clearly prove for the first time by identifying the metabolites that white-rot fungus P. brevispora could degrade the recalcitrant dioxin, 1,3,6,8-tetraCDD.

Fungal bioconversion of toxic polychlorinated biphenyls by white-rot fungus, Phlebia brevispora

Toxic coplanar polychlorinated biphenyls (Co-PCBs) were used as substrates for a degradation experiment with white-rot fungus, Phlebia brevispora TMIC33929, which is capable of degrading polychlorinated dibenzo-p-dioxins. Eleven PCB congener mixtures (7 mono-ortho- and 4 non-ortho-PCBs) were added to the cultures of P. brevispora and monitored by high resolution gas chromatography and mass spectrometry (HRGC/HRMS). Five PCB congeners, 3,3′,4,4′-tetrachlorobiphenyl, 2,3,3′,4,4′-pentachlorobiphenyl, 2,3′,4,4′,5-pentachlorobiphenyl, 3,3′,4,4′,5-pentachlorobiphenyl, and 2,3′,4,4′,5,5′-hexachlorobiphenyl were degraded by P. brevispora. To investigate the fungal metabolism of PCB, each Co-PCB was treated separately by P. brevispora and the metabolites were analyzed by gas chromatography and mass spectrometry (GC/MS) and identified on the basis of the GC/MS comparison with the authentic compound. Meta-methoxylated metabolite was detected from the culture containing each compound. Additionally, para-dechlorinated and -methoxylated metabolite was also detected from the culture with 2,3,3′,4,4′-pentachlorobiphenyl, 2,3′,4,4′,5-pentachlorobiphenyl, and 2,3′,4,4′,5,5′-hexachlorobiphenyl, which are mono-ortho-PCBs. In this paper, we identified the congener specific degradation of coplanar PCBs by P. brevispora, and clearly proved for the first time by identifying the metabolites that the white-rot fungus, P. brevispora, transformed recalcitrant coplanar PCBs.

Aerobic Mineralization of Hexachlorobenzene by Newly Isolated Pentachloronitrobenzene-Degrading Nocardioides sp. Strain PD653

ABSTRACT A novel aerobic pentachloronitrobenzene-degrading bacterium, Nocardioides sp. strain PD653, was isolated from an enrichment culture in a soil-charcoal perfusion system. The bacterium also degraded hexachlorobenzene, a highly recalcitrant environmental pollutant, accompanying the generation of chloride ions. Liberation of 14CO2 from [U-ring-14C]hexachlorobenzene was detected in a culture of the bacterium and indicates that strain PD653 is able to mineralize hexachlorobenzene under aerobic conditions. The metabolic pathway of hexachlorobenzene is initiated by oxidative dechlorination to produce pentachlorophenol. As further intermediate metabolites, tetrachlorohydroquinone and 2,6-dichlorohydroquinone have been detected. Strain PD653 is the first naturally occurring aerobic bacteria capable of mineralizing hexachlorobenzene.

Metabolism of 4,4′-dichlorobiphenyl by white-rot fungi Phanerochaete chrysosporium and Phanerochaete sp. MZ142

Degradation experiment of model polychlorinated biphenyl (PCB) compound 4,4′-dichlorobiphenyl (4,4′-DCB) and its metabolites by the white-rot fungus Phanerochaete chrysosporium and newly isolated 4,4′-DCB-degrading white-rot fungus strain MZ142 was carried out. Although P. chrysosporium showed higher degradation of 4,4′-DCB in low-nitrogen (LN) medium than that in potato dextrose broth (PDB) medium, Phanerochaete sp. MZ142 showed higher degradation of 4,4′-DCB under PDB medium condition than that in LN medium. The metabolic pathway of 4,4′-DCB was elucidated by the identification of metabolites upon addition of 4,4′-DCB and its metabolic intermediates. 4,4′-DCB was initially metabolized to 2-hydroxy-4,4′-DCB and 3-hydroxy-4,4′-DCB by Phanerochaete sp. MZ142. On the other hand, P. chrysosporium transformed 4,4′-DCB to 3-hydroxy-4,4′-DCB and 4-hydroxy-3,4′-DCB produced via a National Institutes of Health shift of 4-chlorine. 3-Hydroxy-4,4′-DCB was transformed to 3-methoxy-4,4′-DCB; 4-chlorobenzoic acid; 4-chlorobenzaldehyde; and 4-chlorobenzyl alcohol in the culture with Phanerochaete sp. MZ142 or P. chrysosporium. LN medium condition was needed to form 4-chlorobenzoic acid, 4-chlorobenzaldehyde, and 4-chlorobenzyl alcohol from 3-hydroxy-4,4′-DCB, indicating the involvement of secondary metabolism. 2-Hydroxy-4,4′-DCB was not methylated. In this paper, we proved for the first time by characterization of intermediate that hydroxylation of PCB was a key step in the PCB degradation process by white-rot fungi.

Degradation of endosulfan and endosulfan sulfate by white-rot fungus Trametes hirsuta

Endosulfan, an organochlorine insecticide, and its metabolite endosulfan sulfate are persistent in environments and are considered toxic. We investigate the possible nontoxic bioremediation of endosulfan. An endosulfandegrading fungus that does not produce endosulfan sulfate was selected from eight species of white-rot fungi. High degradation of endosulfan and low accumulation of endosulfan sulfate were found in cultures of Trametes hirsuta. A degradation experiment using endosulfan sulfate as the substrate revealed that T. hirsuta is able to further degrade endosulfan sulfate following the oxidative conversion of endosulfan to endosulfan sulfate. Endosulfan and endosulfan sulfate were converted to several metabolites via hydrolytic pathways. In addition, endosulfan dimethylene, previously reported as a metabolite of the soil bacterium Arthrobacter sp., was detected in T. hirsuta culture containing endosulfan sulfate. Our results suggest that T. hirsuta has multiple pathways for the degradation of endosulfan and endosulfan sulfate and thus has great potential for use as a biocatalyst in endosulfan bioremediation.

Biotransformation of dichloro-, trichloro-, and tetrachlorodibenzo-p-dioxin by the white-rot fungus Phlebia lindtneri

The model polychlorinated dibenzo-p-dioxins (PCDDs) 2,7-dichloro-, 2,3,7-trichloro, 1,2,6,7-, 1,2,8,9-, and 1,3,6,8-tetrachlorodibenzo-p-dioxin were used as substrates for a degradation experiment with the white-rot fungus Phlebia lindtneri. 2,7-Dichlorodibenzo-p-dioxin (2,7-diCDD) was biotransformed to hydroxylated diCDD and methoxylated diCDD. With the exception of 1,3,6,8-tetrachlorodibenzo-p-dioxin, the tri- and tetrachlorodibenzo-p-dioxins were biotransformed to hydroxyl and methoxyl compounds by P. lindtneri. The degradation rate of 1,2,6,7-tetrachlorodibenzo-p-dioxin was higher than that of 2,3,7-trichlorodibenzo-p-dioxin and no degradation of 1,3,6,8-tetrachlorodibenzo-p-dioxin was observed. These results indicate that the degradation of these PCDDs depends on the chlorination patterns of the substrates. This is the first report of the hydroxylation and methoxylation of tri- to tetra-CDDs by a fungal strain.

Biodegradation of dieldrin by a soil fungus isolated from a soil with annual endosulfan applications.

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 microM from 14.3 microM during a 10-day incubation at 25 degrees C. Approximately 0.15 microM (9%) of aldrin trans-diol 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 microM (95%) and 0.75 microM (94%), respectively, whereas endosulfan and DDE were degraded by 2.42 microM (80%) and 3.29 microM (79%), respectively, and DDT and heptachlor epoxide were degraded by 6.95 microM (49.3%) and 5.36 microM (67.5%), respectively, compared with the control, which had a concentration of approximately 14 microM. These results suggest that strain DDF could be a candidate for the bioremediation of sites contaminated with various persistent organochlorine pesticides including POPs.

Metabolism of organochlorine pesticide heptachlor and its metabolite heptachlor epoxide by white rot fungi, belonging to genus Phlebia

White rot fungi of the genus Phlebia have demonstrated a high capacity to degrade organic pollutants, including polychlorinated dibenzo-p-dioxins and polychlorinated biphenyls. In this study, we evaluated the ability of 18 white rot fungi species of genus Phlebia to degrade heptachlor and heptachlor epoxide, and described the metabolic pathways by selected white rot fungi. Phlebia tremellosa, Phlebia brevispora and Phlebia acanthocystis removed about 71%, 74% and 90% of heptachlor, respectively, after 14 days of incubation. A large amount of heptachlor epoxide and a small amount of 1-hydroxychlordene and 1-hydroxy-2,3-epoxychlordene were detected as metabolic products of heptachlor from most fungal cultures. The screening of heptachlor epoxide-degrading fungi revealed that several fungi are capable of degrading heptachlor epoxide, which is a recalcitrant metabolite of heptachlor. Phlebia acanthocystis, P. brevispora, Phlebia lindtneri and Phlebia aurea removed about 16%, 16%, 22% and 25% of heptachlor epoxide, respectively, after 14 days of incubation. Heptachlor diol and 1-hydroxy-2,3-epoxychlordene were produced in these fungal cultures as metabolites, suggesting that the hydrolysis and hydroxylation reaction occur in the epoxide ring and in position 1 of heptachlor epoxide, respectively.

Saline-dependent regulation of manganese peroxidase genes in the hypersaline-tolerant white rot fungus Phlebia sp. strain MG-60

ABSTRACT The expression pattern of manganese peroxidases (MnPs) in nitrogen-limited cultures of the saline-tolerant fungus Phlebia sp. strain MG-60 is differentially regulated under hypersaline conditions at the mRNA level. When MG-60 was cultured in nitrogen-limited medium (LNM) containing 3% (wt/vol) sea salts (LN-SSM), higher activity of MnPs was observed than that observed in normal medium (LNM). Sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis demonstrated that two MnP isoenzymes were de novo synthesized in the culture of LN-SSM. Three MnP-encoding genes (MGmnp1, MGmnp2, and MGmnp3) were isolated by reverse transcription (RT)-PCR and rapid amplification of cDNA ends PCR techniques. The corresponding isozymes were identified by peptide mass fingerprinting analysis. MnP isozymes encoded by MGmnp2 and MGmnp3 were observed mainly in LN-SSM. Real-time RT-PCR analysis revealed high levels of MGmnp2 and MGmnp3 transcripts in LN-SSM 48 h after the addition of 2% NaCl. The induction of MnP production and the accumulation of gene transcripts by saline were well correlated in the presence of Mn2+. However, in the absence of Mn2+, there was no clear correlation between mnp transcripts levels and MnP activity, suggesting posttranscriptional regulation by Mn2+.