Collaborators
Prof. Christof Holliger, Katia Szynalski, Julien Maillard
Funding agency
Swiss National Science Foundation
Project period
May 1999 – April 2002
Collaborations
Prof. W. Hagen, TU Delft, The Netherlands ; Prof. J. Fontecilla, IBS/LCCP, Grenoble, France
Objectives
This project investigated two quite different topics involved in biotransformation of tetrachloroethene into harmless products. One topic concerned the enrichment and characterization of chloroethene-dechlorinating consortia, the other the genetic and biochemical characterization of the tetrachloroethene reductive dehalogenase of Dehalobacter restrictus.
Research on the first topic put special emphasis on dechlorination of dichloroethene (DCE) and vinyl chloride (VC) and investigated three main objectives, namely (i) the investigation of the composition of tetrachloroethene (PCE)-dechlorinating microbial communities of environmental samples, (ii) the enrichment of dechlorinating activity on PCE, TCE, DCE and VC and following the population dynamics, and (iii) the isolation and characterization of DCE-and VC-dechlorinating microorganisms.
Research on the second topic that started only in the middle of the project period concentrated on the isolation and characterization of the PCE reductive dehalogenase genes of Dehalobacter restrictus.
Results
Environmental sample from 10 different locations have been collected and used as inocula for enrichments on tetrachloroethene (PCE). The electron donor supplied was a mixture of seven compounds (formate, acetate, propionate, butyrate, ethanol, lactate, pyruvate) and was added once a week over a period of several months. PCE dechlorination was followed by measuring the increase in chloride in the specially designed low-chloride content medium. PCE dechlorination was found in all enrichment cultures. However, the patterns of dechlorination were different. In nine of the twenty-three tested samples dechlorination stopped at cis-1,2-dichloroethene, in seven at vinyl chloride, and in seven complete dechlorination was found. Dechlorination rates calculated from the chloride production was quite similar in the majority of the enrichment cultures and were about 150 μM per day. Only one culture dechlorinated at significantly higher rates (250 μM per day). The dechlorination activity could be successfully transferred.
A detailed analysis of original environmental samples and samples of the enrichments with PCR primers specific for the detection of the genus Dehalococcoides revealed that in five samples where dechlorination to vinyl chloride or ethene has been found this genus was really present. More interestingly, in nine enrichments where dechlorination beyond dichloroethene occurred Dehalococcoides was not detected.
In addition, enrichment cultures with four different inocula have been set in on all different chloroethenes with three different substrate mixtures. The identity of the bacteria involved in the dechlorination activity in the enrichments was investigated by the use of molecular probes specific for already known chloroethene-dechlorinating bacteria such as Dehalococcoides sp., Dehalobacter restrictus, Desulfuromonas sp., Dehalospirillum multivorans and Desulfitobacterium sp. In none of the samples Dehalospirillum multivorans was detected, Desulfitobacterium sp. and Dehalobacter restrictus were found in enrichments of three inocula, Dehalococcoides sp. in enrichments of all four.
The comparison of the sequence of a 1.1kb PCR product obtained from genomic DNA of Dehalobacter restrictus with the sequence of the pceA gene of Desulfitobacterium sp. strain PCE-S revealed that they were almost identical. Based on this information, PCR primers were designed allowing the amplification of the genes pceA and pceB of D. restrictus. The sequences of both genes were almost identical with the sequences of strain PCE-S. PCR with the same primers on Desulfitobacterium frappieri strain TCE1 gave also a product containing the two genes pceA and pceB with again almost identical sequences. A recently published sequence of the reductive dehalogenase of another Desulfitobacterium strain isolated in Japan was also almost identical with the other three isolated from three different strains from two different genera. We have apparently discovered here a gene family that the different bacteria might have acquired by horizontal gene transfer.