Characterization of a Defined 2,3,5,6-Tetrachlorobiphenyl- ortho -Dechlorinating Microbial Community by Comparative Sequence Analysis of Genes Coding for 16S rRNA

Abstract

Defined microbial communities were developed by combining selective enrichment with molecular monitoring of total community genes coding for 16S rRNAs (16S rDNAs) to identify potential polychlorinated biphenyl (PCB)-dechlorinating anaerobes that orthodechlorinate 2,3,5,6-tetrachlorobiphenyl. In enrichment cultures that contained a defined estuarine medium, three fatty acids, and sterile sediment, a Clostridium sp. was predominant in the absence of added PCB, but undescribed species in the δ subgroup of the class Proteobacteria, the low-G+C gram-positive subgroup, the Thermotogales subgroup, and a single species with sequence similarity to the deeply branching speciesDehalococcoides ethenogenes were more predominant during active dechlorination of the PCB. Species with high sequence similarities to Methanomicrobiales andMethanosarcinales archaeal subgroups were predominant in both dechlorinating and nondechlorinating enrichment cultures. Deletion of sediment from PCB-dechlorinating enrichment cultures reduced the rate of dechlorination and the diversity of the community. Substitution of sodium acetate for the mixture of three fatty acids increased the rate of dechlorination, further reduced the community diversity, and caused a shift in the predominant species that included restriction fragment length polymorphism patterns not previously detected. Although PCB-dechlorinating cultures were methanogenic, inhibition of methanogenesis and elimination of the archaeal community by addition of bromoethanesulfonic acid only slightly inhibited dechlorination, indicating that the archaea were not required for ortho dechlorination of the congener. Deletion of Clostridium spp. from the community profile by addition of vancomycin only slightly reduced dechlorination. However, addition of sodium molybdate, an inhibitor of sulfate reduction, inhibited dechlorination and deleted selected species from the community profiles of the class Bacteria. With the exception of one 16S rDNA sequence that had the highest sequence similarity to the obligate perchloroethylene-dechlorinating Dehalococcoides, the 16S rDNA sequences associated with PCB ortho dechlorination had high sequence similarities to the δ, low-G+C gram-positive, andThermotogales subgroups, which all include sulfur-, sulfate-, and/or iron(III)-respiring bacterial species.

Identification of a microorganism that links its growth to the reductive dechlorination of 2,3,5,6‐chlorobiphenyl

“…Therefore, it was possible that the dechlorinating microorganisms and the methanogens might compete for the acetate or the methanogens might dechlorinate the PCB. Previously it was demonstrated that sediment‐containing precursors to the ortho ‐dechlorinating culture would dechlorinate 2,3,5,6‐CB in the absence of methanogenesis when 3 mM bromoethanesulphonate (BES) and 5 mM sodium acetate were added (Pulliam Holoman et al ., 1998). Here, in contrast, amendment of the sediment‐free ortho ‐dechlorinating culture with 1–3 mM BES and 5 mM acetate resulted in a reduction of dechlorinating activity below detectable levels.…”

Soil-specific limitations for access and analysis of soil microbial communities by metagenomics

“…For example, anaerobic dechlorinators of polychlorinated biphenyls (PCBs) are present at a low density in the environment, which did not allow detection by classical methods. Their identification required successive enrichments combined with the monitoring of dechlorination activity and 16S rRNA gene patterns (Pulliam Holoman et al , 1998). This might also be relevant for microorganisms producing natural products such as enzymes or antibiotics.…”

Site‐specific microbial communities in three PCB‐impacted sediments are associated with different in situ dechlorinating activities

“…The sample and buffers were mixed by hand prior to 30 s of bead beating at speed '4.5' using a FastPrep120 (Q-Biogene, CA). Nucleic acid extraction and purification were performed using a phenol/chloroform-based protocol (Pulliam Holoman et al, 1998).…”