Enrichment cultures are investigated for the bioremediation potential of tetrachloroethene (PCE). This process is based on anaerobic respiration in which chlorinated compounds are used as electron acceptors (organohalide respiration, OHR). The key enzyme in OHR is the reductive dehalogenase (RdhA). SL2, a culture dechlorinating PCE to harmless ethene, was shown to have a small number of OHR bacteria, mainly Sulfurospirillum spp. and Dehalococcoides spp. (1). Aim: We aim at a better understanding of the functional diversity of rdhA genes in the SL2 culture and their dynamic during PCE degradation. Methods: By applying different frequencies of culture transfer, 3 sub-cultures were obtained from the SL2 enrichment showing different degradation patterns. The degradation profile was followed by gas chromatography and chloride titration. DNA and RNA were also extracted, PCR and qPCR was performed for the detection of rdhA genes belonging to Sulfurospirillum. Besides, a T-RFLP method was developed to amplify all identified rdhA genes and distinguish them using a capillary sequencer. Results: An initial SL2 sub-culture fed on PCE (designated SL2-PCEb) and containing different Sulfurospirillum spp. produced cis-dichloroethene (cis-DCE) as end-product and showed a clear degradation pattern in two steps. From SL2-PCEb, 2 other sub-cultures were obtained, designated SL2-PCEc and SL2-TCE, by feeding them with PCE and trichloroethene (TCE), respectively. While the former only dechlorinated to TCE, the latter has kept the potential to dechlorinate PCE to cis-DCE. Three rdhA genes were identified in SL2 sub-cultures, two of them similar to pceA of S. multivorans (Smu-pceA) and S. halorespirans (Sha-pceA), and a third one (Sul-rdhA2) sharing 62% identity with the other two. The dedicated T-RFLP analysis of the three sub-cultures showed the interplay of all three rdhA genes, and allowed identifying which reductive dehalogenase was involved in each dechlorination step. Conclusions: An apparent functional redundancy in the reductive dehalogenase genes was observed in SL2 sub-cultures, however specific dechlorination activities could be associated with Smu-pceA and Sha-pceA, clearly showing that despite a strong sequence similarity those two reductive dehalogenases have different substrate specificities. So far no activity could be identified for Sul-rdhA2. Reference: (1) Maillard et al., Biodegradation, 2011, 22:949.