We investigated the distribution and activity of chloroethene-degrading microorganisms and associated functional genes during reductive dehalogenation of tetrachloroethene to ethene in a laboratory continuousflow column. Using real-time PCR, we quantified "Dehalococcoides" species 16S rRNA and chloroethenereductive dehalogenase (RDase) genes (pceA, tceA, vcrA, and bvcA) in nucleic acid extracts from different sections of the column. Dehalococcoides 16S rRNA gene copies were highest at the inflow port [(3.6 ؎ 0.6) ؋ 10 6 (mean ؎ standard deviation) per gram soil] where the electron donor and acceptor were introduced into the column. The highest transcript numbers for tceA, vcrA, and bvcA were detected 5 to 10 cm from the column inflow. bvcA was the most highly expressed of all RDase genes and the only vinyl chloride reductase-encoding transcript detectable close to the column outflow. Interestingly, no expression of pceA was detected in the column, despite the presence of the genes in the microbial community throughout the column. By comparing the 16S rRNA gene copy numbers to the sum of all four RDase genes, we found that 50% of the Dehalococcoides population in the first part of the column did not contain either one of the known chloroethene RDase genes. Analysis of 16S rRNA gene clone libraries from both ends of the flow column revealed a microbial community dominated by members of Firmicutes and Actinobacteria. Higher clone sequence diversity was observed near the column outflow. The results presented have implications for our understanding of the ecophysiology of reductively dehalogenating Dehalococcoides spp. and their role in bioremediation of chloroethenes.Tetrachloroethene (PCE) and trichloroethene (TCE) are the most-abundant groundwater contaminants in the United States (32). In situ bioremediation is a promising technology for the removal of these chlorinated solvents from contaminated aquifers (6,23,29). Of particular interest for bioremediation are microorganisms of the genus "Dehalococcoides" (1,7,10,11,13,15,31). In addition to other recalcitrant chloroorganic pollutants, Dehalococcoides spp. reductively dechlorinate PCE, TCE, cis-dichloroethene (cDCE), and vinyl chloride (VC) to ethene. While some microbial species other than Dehalococcoides spp. degrade chlorinated solvents, reductive dechlorination of PCE past cDCE has been linked exclusively to members of the genus Dehalococcoides (11,31,36,45).The reduction of chloroethenes by Dehalococcoides spp. is mediated by reductive dehalogenase (RDase) enzymes. While many RDase genes have been identified, only a few have been characterized for their function. Known RDase genes involved in chloroethene reduction are pceA, encoding PCE reductases from Dehalococcoides ethenogenes strain 195 (DET0318; GenBank accession no. NC_002936) (28) and Dehalococcoides sp. strain CBDB1 (cbdB_A1588; GenBank accession no. NC_007356) (8); tceA, encoding TCE reductases from D. ethenogenes strain 195 (DET0079; GenBank accession no. NC_002936) (27) and Dehalococcoides sp. s...

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A model was developed to describe toxicity from high concentrations of chlorinated aliphatic hydrocarbons (CAHs) on reductively dechlorinating cultures under batch-growth conditions. A reductively dechlorinating anaerobic Evanite subculture (EV-cDCE) was fed trichloroethene (TCE) and excess electron donor to accumulate cis-1,2-dichloroethene (cDCE) in batch-fed reactors. A second Point Mugu (PM) culture was also studied in the cDCE accumulating batch-fed experiment, as well as in a time- and concentration-dependent cDCE exposure experiment. Both cultures accumulated cDCE to concentrations ranging from 9,000 to 12,000 microM before cDCE production from TCE ceased. Exposure to approximately 3,000 and 6,000 microM cDCE concentrations for 5 days during continuous TCE dechlorination exhibited greater loss in activity proportional to both time and concentration of exposure than simple endogenous decay. Various inhibition models were analyzed for the two cultures, including the previously proposed Haldane inhibition model and a maximum threshold inhibition model, but neither adequately fit all experimental observations. A concentration-dependent toxicity model is proposed, which simulated all the experimental observations well. The toxicity model incorporates CAH toxicity terms that directly increase the cell decay coefficient in proportion with CAH concentrations. We also consider previously proposed models relating toxicity to partitioning in the cell wall (K(M/B)), proportional to octanol-water partitioning (K(OW)) coefficients. A reanalysis of previously reported modeling of batch tests using the Haldane model of Yu and Semprini, could be fit equally well using the toxicity model presented here, combined with toxicity proportioned to cell wall partitioning. A companion paper extends the experimental analysis and our modeling approach to a completely mixed reactor and a fixed film reactor.

A model that was used to describe toxicity from high concentrations of chlorinated aliphatic hydrocarbons (CAHs) on reductively dechlorinating cultures in batch reactors (Sabalowsky and Semprini (in press)) was extended here to simulate observations in continuous flow suspended and attached growth reactors. The reductively dechlorinating anaerobic Evanite subculture (EV-cDCE) was fed trichloroethene (TCE) and excess electron donor to accumulate cis-1,2-dichloroethene (cDCE) in a continuous flow stirred tank reactor (CFSTR); and an attached growth recirculating packed column (RPC). A concentration-dependent toxicity model used to simulate the results of batch reactors in part I (Sabalowsky and Semprini (in press) Biotechnol Bioeng) also simulated well the observations for the CFSTR and RPC growth modes. The toxicity model incorporates cDCE and TCE toxicity coefficients that directly increase the cell decay coefficient in proportion with cDCE and TCE concentrations. Simulated estimates of the cDCE and TCE toxicity coefficients indicate reductively dechlorinating cells are most sensitive to high concentrations of cDCE and TCE in batch-fed growth, followed by CFSTR, with attached growth being least sensitive. The greater toxicity of TCE than cDCE, and ratio of the modeled toxicity coefficients, agrees with previously proposed models relating toxicity to partitioning in the cell wall (K(M/B)), proportional to octanol-water partitioning (K(OW)) coefficients.