Isolation and Functional Gene Analyses of Aromatic-Hydrocarbon-Degrading Bacteria from a Polychlorinated-Dioxin-Dechlorinating Process

Abstract

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.

Characterization of Rhizobium naphthalenivorans sp. nov. with special emphasis on aromatic compound degradation and multilocus sequence analysis of housekeeping genes

“…The organisms used in this study were strains TSY03b T , TSY04, and TSW01, all of which were isolated from the sediment slurry taken from a laboratory-scale semi-anaerobic PCDD/ F-transforming microcosm (Kaiya et al, 2012). For comparison, Rhizobium radiobacter strain NBRC 13532 T and Rhizobium selenitireducens strain LMG 24075 T , which were obtained from the NITE Biological Resource Center (NBRC), Kisarazu, Japan, and the LMG Bacteria Collection, Gent, Belgium, respectively, were used.…”

“…An AhDO large subunit (AhDOa) gene was PCR-amplifi ed by using a primer set of PAH2 and PAH5r and sequenced as described previously (Kaiya et al, 2012). Substrate-induced transcription of AhDOa genes was studied by a combination of reverse transcription-PCR and real-time quantitative PCR (qPCR).…”

“…One of the possible explanations for this phenomenon was that anaerobic dehalorespiring bacteria and aerobic aromatic-hydrocarbon-degrading bacteria coexisted and played roles in reductive dechlorination of PCDD/Fs and oxidative degradation of fewer chlorinated and dechlorinated products, respectively. Indeed, the microcosms yielded high population densities of Dehalococcoides and its relatives within the phylum Chlorofl exi as potent dechlorinators and lower levels of aromatic-hydrocarbon-degrading bacteria, as revealed by culture-independent molecular approaches and cultivation methods Kaiya et al, 2012;Yoshida et al, 2005).…”

“…From one of the PCDD/F-transforming microcosms, we have successfully isolated a number of aromatichydrocarbon-degrading bacteria, among which those affi liated with the genus Rhizobium by 16S rRNA gene sequence comparisons formed one of the most abundant taxonomic groups detected (Kaiya et al, 2012). While limited information has been available on the potential degradation of aromatic hydrocarbon by members of the genus Rhizobium (Ben Said et al, 2008;Sanchez et al 2005), our Rhizobium isolates were able to utilize naphthalene as the sole carbon and energy source and to degrade biphenyl and dibenzofuran co-metabolically.…”

“…While limited information has been available on the potential degradation of aromatic hydrocarbon by members of the genus Rhizobium (Ben Said et al, 2008;Sanchez et al 2005), our Rhizobium isolates were able to utilize naphthalene as the sole carbon and energy source and to degrade biphenyl and dibenzofuran co-metabolically. A genomic DNA region covering a gene encoding putative aromatic-ring-hydroxylating oxygenase (AhDO) in one of the isolates was cloned and identifi ed (Kaiya et al, 2012).…”