Prof. Christof Holliger, Katia Szynalski, Julien Maillard, Laure Prat
Swiss National Science Foundation
May 2002 – April 2005
Prof. W. Hagen, TU Delft, The Netherlands ; Prof. J. Fontecilla, IBS/LCCP, Grenoble, France; Prof. P.J. Deschavanne and Dr. C. Dufraigne, INSERM, Paris, France.
The goal of this project was two-fold. On the one hand the biodiversity of chloroethene-dechlorination was investigated with special emphasis on DCE and VC dechlorination, and on the other hand, the genetics and the reaction mechanism of PCE reductive dehalogenase was studied.
The objectives of sub-project A were
- to check the enrichments for the presence of already known PCE-dehalorespiring bacteria,
- to characterize the bacterial and archaeal communities with molecular fingerprinting techniques,
- to characterize the eco-physiology of the enrichments, and
- to isolate and characterize the chloroethene-dechlorinating bacteria.
The main objective of sub-project B was to characterize the unique biochemistry of PCE reductive dechlorination in more detail by a molecular biology approach and by biochemical investigations applying advanced experimental techniques and spectroscopic methods. More specifically, the objectives were
- to study in detail the genetic information in the neighborhood of pceA and pceB,
- to characterize the reaction mechanism involved in the reductive dechlorination, and
- to elucidate the natural direct electron donor of the PCE reductive dehalogenase
The distribution of chloroethene-dehalorespiring bacteria such as Dehalobacter restictus, Sulfurospirillum multivorans, Desulfuromonas choroethenica and Desulfitobacterium spp. reductively dechlorinating tetrachloroethene (PCE) and trichloroethene (TCE) to cis-1,2-dichloroethene (DCE) and Dehalococcoides spp. dechlorinating PCE completely to ethene, was examined in diverse environmental samples and enrichment cultures thereof amended with different chloroethenes and mixtures of substrates. PCR detection based on 16S rRNA gene sequences demonstrated that the genera Dehalococcoides and Desulfitobacterium were particularly widespread in the environment. In addition, it was demonstrated by PCR product band intensities and Real-Time PCR quantification that members of the two genera increased in numbers in many enrichment cultures on PCE. The genus Dehalococcoides seemed to play a key role in complete reductive dechlorination to ethene, but it could not always be linked to this activity. It was not detected in some enrichments where dechlorination to VC or ethene was found and it was frequently detected in enrichments where dechlorination stopped at DCE. This suggests that other bacteria than Dehalococcoides-like populations were involved in complete dechlorination to ethene, possibly members of the genus Desulfitobacterium. Terminal restriction fragment length polymorphism analysis indicated that the Dehalococcoides-like populations involved in the enrichment cultures belong to the Cornell and Pinellas subgroups with a predominance of the Pinellas-like populations. The terminal fragment of the type strain Dehalococcoides ethenogenes strain 195 was never observed. The Desulfitobacterium-like populations detected belong probably to the species Desulfitobacterium hafniense. Dehalobacter restrictus-like populations were not as widespread and were probably only involved in dechlorination in some selected enrichments. Sulfurospirillum multivorans and Desulfuromonas chloroethenica were almost never detected and seem not to play a key role in chloroethene dechlorination.
Two enrichment cultures from two different inocula, one dechlorinating PCE (23 SL) and the other dechlorinating DCE to ethene (2 SO), were tested under eleven conditions with different electron donors and with and without chloroethenes during six to nine weeks of incubation. The enrichment culture 2 SO was able to reductively dechlorinate DCE and VC to ethene, but also PCE, with hydrogen or ethanol as electron donors. Homoacetogenesis was not occurring in these cultures and methanogenesis was only observed after depletion of DCE or in the absence of a chloroethene. The enrichment 23 SL dechlorinated PCE most efficiently to VC with molecular hydrogen as electron donor, and only very slowly with lactate. TCE was the first dechlorination product accumulating in the cultures, and only after almost complete depletion of PCE, TCE was further dechlorinated. Analysis of the 16S rRNA genes from these enrichments by clone libraries, terminal restriction fragment length polymorphism (T-RFLP) and Real-Time PCR indicated that members closely related to the genus Desulfitobacterium reductively dechlorinated DCE to VC in enrichment 2 SO, whereas members that affiliated with the uncultured Dehalococcoides-like clone Pinellas completely dechlorinated VC to ethene. Members of Dehalococcoides spp. were also present in the enrichment 23 SL, probably involved in DCE dechlorination, but another population producing a terminal restriction fragment of 255 bp was more predominant and seemed to play a key role in the incomplete reductive dechlorination of PCE to DCE. Sequencing and cloning of 16S rRNA genes of this latter culture revealed that a Sulfurospirillum-like population is producing this terminal restriction fragment of 255 bp.
Three substrate mixtures (mix-1: formate and acetate; mix-2: propionate, butyrate and ethanol; mix-3: pyruvate and lactate) were tested with different chloroethenes in enrichment cultures inoculated with either a mixture of anaerobic digester sludge from wastewater treatment plants, a sample of a full-scale PCE-dechlorinating bioreactor, or samples from two chloroethene-contaminated aquifers. Dechlorination of tetrachloroethene (PCE) and trichloroethene (TCE) to cis-1,2-dichloroethene (DCE) was obtained quite easily without significant lag phase with all four inocula and with all three substrate mixtures. In some of the enrichments, the dechlorination continued to VC and ethene, particularly in enrichments fed with mix-2 and mix-3, but only after lag periods of several weeks to months. This suggests that the reductive dechlorination of DCE and VC is a hydrogen-dependent dechlorination reaction since propionate, butyrate, ethanol, lactate and pyruvate can be considered as secondary donors of hydrogen. However, in enrichments inoculated with the anaerobic digester sludge mixture, complete dechlorination only occurred when fed with mix-1 indicating that the physiological needs are not the same for all DCE-dechlorinating microbial communities.
First results of the reverse PCR approach indicated that a gene cluster containing the PCE reductive dehalogenase genes (pceAB) was present on a circular transposon element in Desulfitobacterium hafniense strain TCE1, a bacterium that shares 98% sequence homology for pceAB with D. restrictus. Further investigation revealed that a linear copy of this transposon was present in the genome of D. hafniense strain TCE1, comprising the pceABCT gene cluster surrounded by two identical copies of ISDha1, the insertion sequence already found in the circular element. It showed the typical features of a composite transposon, called Tn-Dha1, including a 8bp duplication at the insertion site in the genome. Characterization by PCR of the junction between both inverted repeats (IR junction) of ISDha1 in the circular form of the transposon suggested that the circular element may be a dead-end product of the transposition. A model for the possible transposition events of Tn-Dha1 was proposed. For comparison purposes, the flanking regions around pceAB in D. restrictus were also isolated, indicating a different situation: the pceABCT gene cluster was present but both ISDha1 copies were not, revealing that the transposon structure is missing in D. restrictus, although a truncated transposase-like gene is also found in the direct upstream region of pceA.
A side activity in this project in which the genome of another dehalorespiring bacterium, Dehalococcoides ethenogenes strain 195, was studied using a bioinformatic approach, indicated that most of the numerous reductive dehalogenase copies (eighteen in total) present in D. ethenogenes were located in portions of the genome that showed an original signature compared to the background signature of the complete genome. This study indicated that the genes for reductive dehalogenases may have been acquired by the microorganism through horizontal gene transfer.
The complete electron transport chain leading the electrons to the reductive dehalogenase has not yet been characterized for any of dehalorespiring bacteria and the direct electron-donor has not yet been elucidated for any of reductive dehalogenases. As a first approach, the presence of cytochromes in cells of Desulfitobacterium hafniense strain TCE1 was investigated with regard to the presence or absence of PCE in the growth medium. Desulfitobacterium hafniense strain TCE1 was grown with different combinations of electron donors and acceptors. Hydrogen or lactate was supplied as electron donor, while tetrachloroethene (PCE) or fumarate served as terminal electron acceptors. Detection of cytochromes in different cell fractions using a sensitive detection method based on chemiluminescence revealed a strongly enhanced signal in the membrane fractions of strain TCE1 cells grown on PCE instead of fumarate as terminal electron acceptor. Western blot analysis revealed the presence of a 45 kDa protein in membrane fraction, corresponding most probably to a c-type cytochrome. UV-visible spectroscopy confirmed the presence of c-type cytochromes in membrane fractions. This indicated that a c-type cytochrome may be involved in the direct electron transfer to the PCE reductive dehalogenase of D. hafniense strain TCE1, and that the cytochrome c is induced in the presence of the chlorinated electron acceptor.