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.

Fig. 1

Restriction maps of and PCR cloning strategies for DNA regions containing AhDO genes from Paenibacillus sp. strain TSY30 and Rhizobium sp. strain TSY03b. (a) Arrangement of ORFs on strain TSY30 DNA, (b) restriction sites and PCR-targeted regions of strain TSY30 DNA with used primer names (see Table S1), (c) arrangement of ORFs on strain TSY03b DNA, (d) restriction sites and PCR-targeted regions of strain TSY03b DNA with used primer names (see Table S1). Large and small subunits of AhDO genes are shown by black boxes and other genes by grey boxes.

Fig. 2

Growth of Paenibacillus sp. strain TSY30 and Rhizobium sp. strain TSY03b by degrading aromatic compounds as the substrate. (a) Degradation of naphthalene and production of salicylic acid (SA) by strain TSY30; (b) degradation of DF and production of SA by strain TSY30; (c) degradation of DD in the presence of DF by strain TSY30; (d) degradation of naphthalene and production of SA by strain TSY03b; (e) degradation of DF in the presence of naphthalene by strain TSY03b; (f) degradation of biphenyl in the presence of naphthalene by strain TSY03b. Symbols: closed circles, growth (OD₆₆₀); open circles, concentration of naphthalene; open squares, DF; closed squares, DD; open triangles, biphenyl; closed triangles, SA.

Fig. 3

Neighbor-joining distance matrix tree of AhDOa proteins of the AHD isolates and related Rieske-type dioxygenases based on deduced amino acid sequences (scale = 10% sequence dissimilarity). The sequence of Pseudomonas resinovorans CA10 CarA is used as an outgroup to root the tree. The DDBJ/EMBL/GenBank accession numbers for the sequences are shown behind organism names. The sequences determined in this study are shown in bold. Branching points supported by >800 bootstrap values with 1,000 resamplings are shown by closed circles. Clusters I, II, and III show the Toluene/Benzoate, Biphenyl, and Naphthalene dioxygenase families, respectively, based on the Batie classification system (3, 42, 46).