Semi-anaerobic microcosms containing different levels of polychlorinated dibenzo-p-dioxins/dibenzofurans (PCDD/Fs) were constructed by seeding with different mass ratios of lake sediment and dioxin-contaminated soil and incubating with organic medium for 1 year. In all microcosms, PCDD/Fs were reduced as a first-order reaction with similar removal rate coefficients, and only trace amounts of less chlorinated congeners were produced as the intermediate and end products. This apparent complete dechlorination of PCDD/Fs seemed to be due to a combination of reductive dechlorination of PCDD/Fs and oxidative degradation of the dechlorinated products. Total cell counting, 16S rRNA gene clone library analyses and quinone profiling showed that the microcosms contained relatively constant total populations with members of the phyla Bacteroidetes, Firmicutes and Proteobacteria (especially "Deltaproteobacteria") as the major constituents, independent of pollution levels. Quantitative real-time PCR with a specific primer set showed that the population density of "Dehalococcoides" and its phylogenetic relatives was highly correlated with the concentration of PCDD/Fs present. Some "Dehalococcoides" strains were isolated from the microcosms by repeated enrichment with chloroaromatics as the terminal electron acceptor. However, these isolates did not match with the major "Dehalococcoides"-related clones directly PCR-amplified. The results of this study suggest that PCDD/Fs in natural environments under given conditions are transformed with similar half-reduction rates independent of their concentrations, and a wide variety of "Dehalococcoides"-related bacteria play the primary role in this process.

Aerobic aromatic-hydrocarbon-degrading bacteria from a semi-anaerobic microbial microcosm that exhibited apparent complete dechlorination of polychlorinated dibenzo-p-dioxins/dibenzofurans (PCDD/Fs) were isolated through enrichment and plating culture procedures with dibenzofuran as the model substrate. By 16S rRNA gene sequence comparisons, these dibenzofuran-degrading isolates were identified as being members of the phyla Actinobacteria, Firmicutes, and Proteobacteria, among which those of the genera Paenibacillus and Rhizobium were most abundant. All of the isolates utilized naphthalene as the sole carbon and energy source and degraded dibenzofuran metabolically or co-metabolically; however, they hardly attacked monochlorinated dibenzofuran and dibenzo-p-dioxin. By PCR cloning and sequencing, genes predicted to encode aromatic-ring-hydroxylating dioxygenase (AhDO) were detected in all test isolates. Real-time quantitative PCR assays with specific primer sets detected approximately 105 copies of the AhDO large subunit genes g−1 wet wt in the microcosm from which the isolates were obtained. This order of the copy number corresponded to approximately 1% of the 16S rRNA gene copies from “Dehalococcoides” and its relatives present as potent dechlorinators. These results suggest that aerobic AhDO-containing bacteria co-exist and play a role in the oxidative degradation of less chlorinated and completely dechlorinated products in the PCDD/F-dechlorinating process, thereby achieving the apparent complete dechlorination of PCDD/Fs.

The reductive dechlorinating abilities for chloroethenes of seven enrichment cultures from polychlorinated-dioxin-dechlorinating microcosm were investigated using culture-independent and -dependent methods. These cultures were constructed and maintained with 1,2,3-trichlorobenzene (1,2,3-TCB) or fthalide as an electron acceptor and hydrogen as an electron donor. Denaturing gradient gel electrophoresis (DGGE) analysis of the amplified fragments targeting the 16S rRNA gene showed one or two major bands, whose nucleotide sequences were then analyzed and were found to suggest that Dehalococcoides was one of the dominant bacteria in all enrichment cultures. The nucleotide sequence data revealed that the identity of the major band was 100% identical to the 16S rRNA gene sequence of the Pinellas subgroup of the Dehalococcoides clusters, that is, strains CBDB1 and FL2. Genetic diagnosis targeting the pceA, tceA, bvcA, vcrA and reductive dehalogenase homologous (rdh) gene was performed to investigate the potential for reductive chloroethene dechlorination of cultures. The required length of PCR-amplified fragments was not observed, suggesting that these cultures are not capable of reductively dechlorinating chloroethenes. However, a culture-dependent test indicated that two cultures, TUT1903 and TUT1952, reductively dechlorinated tetrachloroethene (PCE) to trichloroethene (TCE), although not completely. While, TUT2260 and TUT2264 completely converted PCE to TCE and dichloroethenes, but not further. These results suggest that these TUT cultures might include a novel type of bacteria belonging to the Dehalococcoides group and that currently available information on both the 16S rRNA gene and rdh gene sequences is insufficient to definitively evaluate the potential abilities for reductive dechlorination.

A dechlorinating microbial enrichment culture designated TUT2264 was cultured with tetrachloroethene and then characterized for tetrachloroethene-dechlorination by culture-dependent and -independent methods. The fourth-transferred TUT2264 culture completely dechlorinated tetrachloroethene and trichloroethene, and accumulated more trans-1,2-dichloroethene than cis-1,2-dichloroethene. A real-time PCR analysis revealed that "Dehalococcoides" cells made up only 0.3% of the total. Eight distinct reductive-dehalogenase-homologous genes (rdh) were detected with degenerate primers. Phylogenetic analyses revealed 5 of the 8 RdhAs to be very similar to RdhAs reported previously but not to share 100% identity. Transcriptional levels were quantified as the number of transcripts per rdhA by combining the reverse transcription real-time PCR and exogenous internal reference mRNA methods. TUT2264 responded to all the chloroethenes tested. rdhA4 was transcribed with all chloroethenes except vinyl chloride, whereas rdhA8 was only transcribed on tetrachloroethene. Furthermore, multiple rdhAs were induced to express by a single chloroethene as a growth-supporting or non-supporting substrate. These results suggested that Rdhs are multi-functional and rdhAs are a powerful tool to evaluate the potential of contaminated sites and isolates to dechlorinate chloroethenes.

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IntroductionMicrobial transformation of polychlorinated dibenzop-dioxins/dibenzofurans (PCDD/Fs) has been well documented in connection with natural attenuation and engineered bioremediation of organohalogen pollution (Field and Sierra-Alvarez, 2008;Hiraishi, 2003Hiraishi, , 2008. Previously, we constructed laboratory-scale semi-anaerobic microcosms with sediment contaminated with high concentrations of PCDD/Fs in order to study microorganisms involved in the transformation J. Gen. Appl. Microbiol., 58, 211 224 (2012) Three strains of aerobic chemoorganotrophic naphthalene-degrading bacteria (designated TSY03b T , TSY04, and TSW01) isolated from sediment of a polychlorinated-dioxin-transforming microcosm were characterized. These strains had Gram-negative-stained, rod-shaped cells measuring 0.6 0.9 μm in width and 1.2 3.0 μm in length and were motile by means of peritrichous fl agella. Naphthalene was utilized as the sole carbon and energy source, and the transcription of a putative aromatic-ring hydroxylating gene was inducible by naphthalene. The major component of cellular fatty acids was summed feature 8 (C 18 1 ω7c and/or C 18 1 ω6c), and signifi cant proportions of C 18 0 and C 19 0 cyclo ω8cis were also found. The major respiratory quinone was ubiquinone-10. The G+C content of the DNA was 60.3 60.9 mol%. Phylogenetic analyses by studying sequence information on the housekeeping atpD, dnaK, glnII, gyrB, and recA genes as well as on 16S rRNA genes and the 16S-23S rDNA internal transcribed spacer region revealed that the strains grouped with members of the genus Rhizobium, with Rhizobium selenitireducens as their closest relative but formed a distinct lineage at the species level. This was confi rmed by genomic DNA-DNA hybridization studies. These phenotypic, genotypic, and phylogenetic data strongly suggest that our isolates should be classifi ed under a novel species of the genus Rhizobium. Thus, we propose the name Rhizobium naphthalenivorans sp. nov. to accommodate the novel isolates. The type strain is TSY03b T (= NBRC 107585 T = KCTC 23252 T ).

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