Phylogenetic characterization of a polychlorinated-dioxin- dechlorinating microbial community by use of microcosm studies

Abstract

Microcosms capable of reductive dechlorination of polychlorinated dibenzo-p-dioxins/dibenzofurans (PCDD/Fs) were constructed in glass bottles by seeding them with a polluted river sediment and incubating them anaerobically with an organic medium. All of the PCDD/F congeners detected were equally reduced without the accumulation of significant amounts of less-chlorinated congeners as the intermediate or end products. Alternatively, large amounts of catechol and salicylic acid were produced in the upper aqueous phase. Thus, the dechlorination of PCDD/Fs and the oxidative degradation of the dechlorinated products seemed to take place simultaneously in the microcosm. Denaturing gel gradient electrophoresis and clone library analyses of PCR-amplified 16S rRNA genes from the microcosm showed that members of the phyla Firmicutes, Proteobacteria, and Bacteroidetes predominated. A significant number of Chloroflexi clones were also detected. Quantitative real-time PCR with specific primer sets showed that the 16S rRNA genes of a putative dechlorinator, "Dehalococcoides," and its relatives accounted for 0.1% of the total rRNA gene copies of the microcosm. Most of the clones thus obtained formed a cluster distinct from the typical "Dehalococcoides" group. Quinone profiling indicated that ubiquinones accounted for 18 to 25% of the total quinone content, suggesting the coexistence and activity of ubiquinone-containing aerobic bacteria. These results suggest that the apparent complete dechlorination of PCDD/Fs found in the microcosm was due to a combination of the dechlorinating activity of the "Dehalococcoides"-like organisms and the oxidative degradation of the dechlorinated products by aerobic bacteria with aromatic hydrocarbon dioxygenases.

FIG. 1.

Changes in the concentrations of PCDD/Fs and lower chlorinated congeners (1-3CDD/Fs) (a) and cumulative amounts of catechol and salicylic acid produced (b) during 1 year of incubation of the microcosm. Closed circles, PCDD/Fs; open circles, 1-3CDD/Fs; closed triangles, catechol; open triangles, salicylic acid. The data show the averages with standard deviations (shown by error bars) of three different determinations. A regression equation and a correlation coefficient for the relationships between the concentration of PCDD/Fs and time are shown in panel a.

FIG. 2.

Changes in DGGE patterns of PCR-amplified 16S rRNA gene fragments during the startup operation of the microcosm. The DGGE bands designated A to H were purified and subjected to sequence analysis (see Table 2).

FIG. 3.

Neighbor-joining distance matrix tree of 16S rRNA gene clones detected from the microcosm and their phylogenetic relatives. The sequence of Aquifex pyrophilus was used as an outgroup to root the tree. Scale = 10% base substitution (K_nuc). The clones obtained in this study are shown in boldface, and those with TSA and TSB numbers were obtained from the microcosm on days 140 and 300, respectively. The asterisks indicate those clones obtained previously from dechlorinating mixed cultures and chlorinated-compound-polluted environments (5, 21, 62, 67). The figures in parentheses following the species and clone names are DDBJ/EMBL/GenBank accession numbers. Bootstrap values (>600) with 1,000 resamplings are shown at branching points.

FIG. 4.

Changes in total bacterial and “Dehalococcoides” populations during 1 year of incubation of the microcosm. (a) Total counts by EtBr staining (solid line) and the “Dehalococcoides” population assumed based on the relative rRNA gene copies (broken line); (b) the ratio of “Dehalococcoides” rRNA gene copies to the total rRNA gene copies as measured by real-time PCR using a specific primer set of DHC793f and DHC946r. The data show the geometric means with standard deviations (shown by error bars) of three different determinations.

FIG. 5.

Neighbor-joining distance matrix tree of the 16S rRNA gene clones amplified with two different “Dehalococcoides”-specific primer sets, DHC793f and DHC946r (a) and DHC66f and DHC180r (b). The clones obtained in this study are shown in boldface. The sequence of Aquifex pyrophilus was used as an outgroup to root the tree. Scale = 2% (a) or 5% (b) base substitution (K_nuc). The figures in parentheses following the species and clone names are DDBJ/EMBL/GenBank accession numbers. Bootstrap values (>600) with 1,000 resamplings are shown at branching points.