Characterization of the Community Structure of a Dechlorinating Mixed Culture and Comparisons of Gene Expression in Planktonic and Biofloc-Associated “Dehalococcoides” and Methanospirillum Species

Anaerobic dechlorination of chlorinated organic compounds is an important mechanism for the remediation of common groundwater pollutants (11, 58). It is now accepted that members of the “Dehalococcoides” group play a crucial role in the remediation of compounds such as chloroethenes, chlorobenzenes, chloroalkanes, chlorophenols, dioxins, and polychlorinated biphenyls, in some cases dechlorinating these compounds to nontoxic end products (1, 2, 6, 16, 18, 26, 32, 35). While researchers have been able to isolate and perform pure culture studies of these organisms, there is significant evidence that reductive dechlorination in environmental systems and in the most robust laboratory cultures is the work of microbial consortia (4, 11). All cultured representatives of the Dehalococcoides group require hydrogen as an electron donor (often supplied by syntrophic fermentation) and a halogenated organic as an electron acceptor. In addition, Dehalococcoides grow robustly in mixed cultures, likely due to currently undetermined growth factors from other community members (11, 18, 31, 34, 44). Though reductive dechlorination is an energetically favorable process under syntrophic conditions with low hydrogen partial pressures (19, 57, 58), other, less favorable metabolic reactions such as methanogenesis and acetogenesis often occur in these communities, especially when excess hydrogen or a donor fermented at high hydrogen partial pressures is available (19, 20, 24, 38). Many methanogens depend on acetate and/or H2, which are both utilized by Dehalococcoides. This suggests that competition for resources is an important interaction within dechlorinating microbial communities containing both methanogenic and Dehalococcoides populations. Several studies have looked at dechlorinating microbial communities derived from both enrichment cultures and environmental systems (8, 12, 17, 21, 22, 28, 30, 33, 43, 54, 55, 64). Several distinct lineages of microorganisms, representing a variety of metabolic capabilities, are commonly found in these consortia, supporting the potential complexity of community dynamics. The D2 enrichment culture, which has been studied previously (19, 20, 46, 48, 52, 53), is derived from the same consortium from which “Dehalococcoides ethenogenes” strain 195 was isolated (13, 14, 44, 45). In this study, the phylogenetic community structure of the D2 enrichment culture including D. ethenogenes was assessed from phylogenetic analysis of bacterial and archaeal 16S rRNA gene libraries created from community DNA. Within this heterogeneous enrichment culture, two distinct cellular attachment phases were observed: planktonic cells (individual suspended cells) and cells associated with bioflocs (suspended cell aggregates). In the D2 culture, bioflocs (typically 10 to 100 μm in diameter) tended to contain multiple species and form around mineral precipitates from the medium (see Movie S1 in the supplemental material for a three-dimensional [3D] micrograph). Planktonic and biofloc-associated growth forms are common in environmental microbial communities (i.e., activated sludge, marine, sediments, and groundwater) (5, 10, 41, 60). In this study, a technique for physical enrichment of these two cell attachment phases via low-speed centrifugation was developed. Fluorescence in situ hybridization (FISH) with 16S rRNA-targeting probes was used to estimate the distribution of D. ethenogenes populations between plankton and bioflocs and to examine colocalization of D. ethenogenes and methanogenic Archaea within the bioflocs. Potential differences in gene expression between the two attachment forms were determined for both D. ethenogenes and the hydrogenotrophic methanogen present in the culture, Methanospirillum hungatei, using quantitative reverse transcription-PCR. This method was also used to compare expression of housekeeping and hydrogenase genes between these organisms. Understanding the distribution and difference in gene expression of the two D. ethenogenes cell attachment phases not only is important for elucidating the ecology of these organisms; it also has implications for the use of DNA and RNA as bioindicators of Dehalococcoides activity. A groundwater sample, while easier and less expensive to obtain, would predominantly sample planktonic Dehalococcoides. Therefore, it is important to establish whether the populations and activities of the planktonic phase reflect those of the community as a whole.