Identification of novel perchloroethene-respiring microorganisms in anoxic river sediment by RNA-based stable isotope probing.

The halogenated compound tetrachloroethene (perchloroethene, PCE) is a persistent contaminant of aquifers, soils and sediments. Although a number of microorganisms are known to reductively dechlorinate PCE by dehalorespiration, their diversity and community structure especially in pristine environments remain elusive. In this study, we report on the detection of a novel group of dehalorespiring bacteria that reductively dechlorinate PCE to cis-dichloroethene by RNA-based stable isotope probing. Pristine river sediment was incubated at 15 degrees C with PCE at low aqueous concentration. Upon formation of dechlorination products, the microbial community was probed with (13)C-labelled acetate as electron donor and carbon source. Terminal restriction fragment length polymorphism (T-RFLP) analysis of density-separated 16S rRNA revealed a predominantly (13)C-labelled bacterial population only in the microcosm with PCE in high-density gradient fractions, whereas in the control without PCE Bacteria-specific rRNA was restricted to light gradient fractions. By cloning and sequence analysis of 16S rRNA, the predominant population was identified as a novel group of bacteria within the phylum Chloroflexi. These microorganisms, designated Lahn Cluster (LC), were only distantly related to cultivated dehalorespiring Dehalococcoides spp. (92-94% sequence identity). Minor clone groups detected (13)C-labelled and thus, potentially involved in PCE dehalorespiration, were related to beta-proteobacterial Dechloromonas spp., and delta-Proteobacteria (Geobacteraceae, Desulfobacteraceae, Desulfobulbaceae). In contrast, clones from an ethene-producing microcosm incubated at 20 degrees C grouped with known Dehalococcoides spp. Our results show that stable isotope probing allows targeting dehalorespiring bacteria as functional guild, and to identify novel PCE-respiring populations previously not recognized.