Detection and quantitative estimation of Dehalococcoides spp. in a dechlorinating bioreactor by a combination of fluorescent in situ hybridisation (FISH) and kinetic analysis

The unique capacity of Dehalococcoides ethenogenes of completely dechlorinating the common groundwater pollutant tetrachloroethene (PCE) to the harmless ethene makes this microorganism very attractive for application in natural or engineered bioremediation systems. In this study, the qualitative and quantitative determination of Dehalococcoides spp. in a lab-scale bioreactor was performed based on the combination of fluorescent in situ hybridisation (FISH) for specific detection, and kinetic batch tests at non-limiting hydrogen and PCE concentration for quantitative determination. The dechlorinating bioreactor was operated at a high and constant PCE loading rate of 255 mumol PCE [g volatile suspended solids (VSS)](-1) day(-1). Pale coccoid cells resembling the distinctive morphotype of D. ethenogenes were present in the microbial culture. These cocci hybridised with both eubacterial probes and the Dhe1259t probe recently designed for detecting Dehalococcoides spp. Positive hybridisation was also observed when the DHC1377 reverse primer was used as a specific probe and applied to the dechlorinating microbial consortium. The maximum dechlorination rate obtained under non-limiting hydrogen and PCE concentrations was 3.22 +/- 0.08 mmol Cl- l(-1) day(-1). From the specific activity of D. ethenogenes [i.e. 0.055 +/- 0.008 mmol Cl- (mg VSS)(-1) day(-1)], as reported from pure culture study, this observed maximum rate corresponded to a concentration of this bacterium in the mixed liquor of the bioreactor of 59.0+/-10.4 mg VSS.l(-1) (41.5+/-11.2% of overall VSS). This calculated relative abundance of D. ethenogenes was in agreement with the percentage of methanol (in terms of reducing equivalents) channeled to reductive dechlorination (approximately 30%) supporting the assumption that most reductive dechlorination was actually due to this microorganism.