Showing papers on "Dehalococcoides published in 2004"

Molecular Identification of the Catabolic Vinyl Chloride Reductase from Dehalococcoides sp. Strain VS and Its Environmental Distribution

Abstract: Reductive dehalogenation of vinyl chloride (VC) to ethene is the key step in complete anaerobic degradation of chlorinated ethenes. VC-reductive dehalogenase was partially purified from a highly enriched culture of the VC-respiring Dehalococcoides sp. strain VS. The enzyme reduced VC and all dichloroethene (DCE) isomers, but not tetrachloroethene (PCE) or trichloroethene (TCE), at high rates. By using reversed genetics, the corresponding gene (vcrA) was isolated and characterized. Based on the predicted amino acid sequence, VC reductase is a novel member of the family of corrinoid/iron-sulfur cluster containing reductive dehalogenases. The vcrA gene was found to be cotranscribed with vcrB, encoding a small hydrophobic protein presumably acting as membrane anchor for VC reductase, and vcrC, encoding a protein with similarity to transcriptional regulators of the NosR/NirI family. The vcrAB genes were subsequently found to be present and expressed in other cultures containing VC-respiring Dehalococcoides organisms and could be detected in water samples from a field site contaminated with chlorinated ethenes. Therefore, the vcrA gene identified here may be a useful molecular target for evaluating, predicting, and monitoring in situ reductive VC dehalogenation.

Dehalococcoides ethenogenes strain 195 reductively dechlorinates diverse chlorinated aromatic pollutants.

Abstract: Dehalococcoides ethenogenes strain 195 dechlorinates tetrachloroethene to vinyl chloride and ethene, and its genome has been found to contain up to 17 putative dehalogenase gene homologues, suggesting diverse dehalogenation ability. We amended pure or mixed cultures containing D. ethenogenes strain 195 with 1,2,3,4-tetrachlorodibenzo-p-dioxin, 2,3,7,8-tetrachlorodibenzo-p-dioxin, 2,3-dichlorodibenzo-p-dioxin, 1,2,3,4-tetrachloro-dibenzofuran, 2,3,4,5,6-pentachlorobiphenyl, 1,2,3,4-tetrachloronaphthalene, various chlorobenzenes, or a mixture of 2-, 3-, and 4-chlorophenol to determine the dehalogenation ability. D. ethenogenes strain 195 dechlorinated 1,2,3,4-tetrachlorodibenzo-p-dioxin to a mixture of 1,2,4-trichlorodibenzo-p-dioxin and 1,3-dichlorodibenzo-p-dioxin. 2,3,4,5,6- Pentachlorobiphenyl was dechlorinated to 2,3,4,6- and/or 2,3,5,6-tetrachlorobiphenyl and 2,4,6-trichlorobiphenyl. 1,2,3,4-Tetrachloronaphthalene was dechlorinated primarily to an unidentified dichloro-naphthalene congener. The predom...

Genetic Identification of a Putative Vinyl Chloride Reductase in Dehalococcoides sp. Strain BAV1

Abstract: Dehalococcoides sp. strain BAV1 couples growth with the reductive dechlorination of vinyl chloride (VC) to ethene. Degenerate primers targeting conserved regions in reductive dehalogenase (RDase) genes were designed and used to PCR amplify putative RDase genes from strain BAV1. Seven unique RDase gene fragments were identified. Transcription analysis of VC-grown BAV1 cultures suggested that bvcA was involved in VC reductive dechlorination, and the complete sequence of bvcA was obtained. bvcA was absent in Dehalococcoides isolates that failed to respire VC, yet was detected in four of eight VC-respiring mixed cultures.

Characterization of a Highly Enriched Dehalococcoides-Containing Culture That Grows on Vinyl Chloride and Trichloroethene

Abstract: A highly enriched culture that reductively dechlorinates trichloroethene (TCE), cis-1,2-dichloroethene (cDCE), and vinyl chloride (VC) to ethene without methanogenesis is described. The Dehalococcoides strain in this enrichment culture had a yield of (5.6 +/- 1.4) x 10(8) 16S rRNA gene copies/micromol of Cl(-) when grown on VC and hydrogen. Unlike the other VC-degrading cultures described in the literature, strains VS and BAV1, this culture maintained the ability to grow on TCE with a yield of (3.6 +/- 1.3) x 10(8) 16S rRNA gene copies/micromol of Cl(-). The yields on an electron-equivalent basis measured for the culture grown on TCE and on VC were not significantly different, indicating that both substrates supported growth equally well. PCR followed by denaturing gradient gel electrophoresis, cloning, and phylogenetic analyses revealed that this culture contained one Dehalococcoides 16S rRNA gene sequence, designated KB-1/VC, that was identical (over 1,386 bp) to the sequences of previously described organisms FL2 and CBDB1. A second Dehalococcoides sequence found in separate KB-1 enrichment cultures maintained on cDCE, TCE, and tetrachloroethene was no longer present in the VC-H(2) enrichment culture. This second Dehalococcoides sequence was identical to that of BAV1. As neither FL2 nor CBDB1 can dechlorinate VC to ethene in a growth-related fashion, it is clear that current 16S rRNA gene-based analyses do not provide sufficient information to distinguish between metabolically diverse members of the Dehalococcoides group.

Multiple Nonidentical Reductive-Dehalogenase-Homologous Genes Are Common in Dehalococcoides

Abstract: Degenerate primers were used to amplify large fragments of reductive-dehalogenase-homologous (RDH) genes from genomic DNA of two Dehalococcoides populations, the chlorobenzene- and dioxin-dechlorinating strain CBDB1 and the trichloroethene-dechlorinating strain FL2. The amplicons (1,350 to 1,495 bp) corresponded to nearly complete open reading frames of known reductive dehalogenase genes and short fragments (approximately 90 bp) of genes encoding putative membrane-anchoring proteins. Cloning and restriction analysis revealed the presence of at least 14 different RDH genes in each strain. All amplified RDH genes showed sequence similarity with known reductive dehalogenase genes over the whole length of the sequence and shared all characteristics described for reductive dehalogenases. Deduced amino acid sequences of seven RDH genes from strain CBDB1 were 98.5 to 100% identical to seven different RDH genes from strain FL2, suggesting that both strains have an overlapping substrate range. All RDH genes identified in strains CBDB1 and FL2 were related to the RDH genes present in the genomes of Dehalococcoides ethenogenes strain 195 and Dehalococcoides sp. strain BAV1; however, sequence identity did not exceed 94.4 and 93.1%, respectively. The presence of RDH genes in strains CBDB1, FL2, and BAV1 that have no orthologs in strain 195 suggests that these strains possess dechlorination activities not present in strain 195. Comparative sequence analysis identified consensus sequences for cobalamin binding in deduced amino acid sequences of seven RDH genes. In conclusion, this study demonstrates that the presence of multiple nonidentical RDH genes is characteristic of Dehalococcoides strains.

Comparative evaluation of chloroethene dechlorination to ethene by Dehalococcoides-like microorganisms.

Abstract: Reductive dehalogenation of tetrachloroethene (PCE), trichloroethene (TCE), cis-1,2-dichloroethene (DCE), and vinyl chloride (VC) was examined in four cultures containing Dehalococcoides-like microorganisms. Dechlorination and growth kinetics were compared using a Monod growth-rate model for multiple electron acceptor usage with competition. Included were the Victoria mixed culture containing Dehalococcoides species strain VS (from Victoria, TX), the mixed culture KB-1/VC (from southern Ontario), the Pinellas mixed culture (from Pinellas, FL), and D. ethenogenes strain 195. All cultures, with the exception of D. ethenogenes strain 195, grew with VC as catabolic electron acceptor. A dilution method was developed that allows a valid comparison to be made of dehalogenating kinetics between different mixed cultures. Using this procedure, maximum growth rates on VC were found to be similar for strain VS and KB-1/VC (0.42-0.49 +/- 0.02 d(-1)) but slower for the Pinellas culture (0.28 +/- 0.01 d(-1)). The 16S rRNA gene sequences were determined to ensure that no cross contamination between cultures had occurred. Following enrichment of the VC dechlorinating microorganisms on VC, the cultures were amended with DCE, TCE, or PCE. The three mixed cultures failed to dechlorinate PCE or did so very slowly. However, the dilution technique indicated that all experienced growth on TCE and DCE as well as on VC. Maximum growth rates on DCE alone were quite similar (0.43-0.46 d(-1)), while the Pinellas culture grew faster on TCE alone (0.49 d(-1)) than did the other two mixed cultures (0.33-0.35 d(-1)). Half-velocity and inhibition constants for growth on TCE were also determined for the three mixed cultures; both constants were found to be essentially equal and the same for the different cultures, varying between only 8.6 and 10.5 microM. The ability of the strain VS, KB-1/VC, and Pinellas cultures to utilize TCE rapidly with conversion to ethene is quite different from that of any other reported microorganism. It was separately confirmed with more traditional cell-counting techniques that strain VS coupled TCE, as well as DCE and VC, utilization with growth. This is the first report of an organism obtaining energy for growth through every step in the reduction of TCE to ethene. Also, as suggested by the dilution technique, the dehalogenating organisms in the KB-1/VC and Pinellas cultures appear to obtain growth from TCE utilization as well. Such ability to grow while dehalogenating TCE to ethene will be an important advantage for their use in bioaugmentation.

Anaerobic microbial reductive dechlorination of tetrachloroethene to predominately trans-1,2-dichloroethene.

Abstract: While most sites and all characterized PCE and TCE dechlorinating anaerobic bacteria produce cis-DCE as the major DCE isomer, significant amounts of trans-DCE are found in the environment. We have obtained microcosms from some sites and enrichment cultures that produce more trans-DCE than cis-DCE. These cultures reductively dechlorinated PCE and TCE to trans-DCE and cis-DCE simultaneously and in a ratio of 3(±0.5):1 that was stable through serial transfers with a variety of electron donors and occurred in both methanogenic and nonmethanogenic enrichments. Two sediment-free, nonmethanogenic enrichment cultures produced trans-DCE at rates of up to 2.5 μmol L-1 day-1. Dehalococcoides populations were detected in both trans-DCE producing cultures by their 16S rRNA gene sequences, and trans-DCE was produced in the presence of ampicillin. Because trans-DCE can be the major product from PCE and TCE microbial dechlorination, high fractions of trans-DCE at chloroethene-contaminated sites are not necessarily from s...

Populations implicated in anaerobic reductive dechlorination of 1,2-dichloropropane in highly enriched bacterial communities.

Abstract: 1,2-Dichloropropane (1,2-D), a widespread groundwater contaminant, can be reductively dechlorinated to propene by anaerobic bacteria. To shed light on the populations involved in the detoxification process, a comprehensive 16S rRNA gene-based bacterial community analysis of two enrichment cultures derived from geographically distinct locations was performed. Analysis of terminal restriction fragments, amplicons obtained with dechlorinator-specific PCR primers, and enumeration with quantitative real-time PCR as well as screening clone libraries all implied that Dehalococcoides populations were involved in 1,2-D dechlorination in both enrichment cultures. Physiological traits (e.g., dechlorination in the presence of ampicillin and a requirement for hydrogen as the electron donor) supported the involvement of Dehalococcoides populations in the dechlorination process. These findings expand the spectrum of chloroorganic compounds used by Dehalococcoides species as growth-supporting electron acceptors. The combined molecular approach allowed a comparison between different 16S rRNA gene-based approaches for the detection of Dehalococcoides populations.

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

Abstract: 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 micromol 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.

Dehalococcoides isolate for bioremediation

Abstract: A bacterium, designated BAV 1 belonging to the Pinellas group within the Dehalococcoides cluster has been discovered. BAV 1 is able to completely metabolize chloroethenes to non-toxic products. The isolate is able to grow with dichloroethenes and vinyl chloride (VC) as electron acceptors, reductively dechlorinating dichloroethenes and VC to ethene and inorganic chloride under anaerobic conditions. The BAV1 isolate is useful for bioremediation approaches for cleaning contaminated subsurface environments and restoring drinking water reservoirs.

Methods and reagents for quantitative analysis of Dehalococcoides species

Abstract: The invention sets forth specific probes and primer pairs for such quantitative analysis and methods of obtaining template DNA from the Dehalococcoides species of interest. These techniques are useful in a bioremediation process to monitor and control the dechlorination of chlorinated hydrocarbons.

Molecular characterization of key enzymes involved in dehalorespiration with tetrachloroethene

Abstract: Chloroethenes, and most particularly tetra- (PCE) and trichloroethene (TCE) are major groundwater pollutants due to their extensive industrial use as solvents since the 1920s. The strong electronegativity of the chlorines renders them very stable under aerobic conditions. However, biodegradation of chloroethenes under anaerobic conditions has been shown to be a promising strategy for remediation of chloroethene-contaminated sites. To date around fifteen bacterial strains have been isolated with the property of using chloroethenes as terminal electron acceptor in a process called dehalorespiration. Anaerobic dehalorespiring bacteria show an unequal chloroethene substrate range and an unequal extent of dechlorination. While most of the dehalorespiring bacteria dechlorinate PCE and TCE to cis-1,2-dichloroethene cis-1,2-DCE) and belong to the phyla Firmicutes, δ- and e-Proteobacteria, a few strains of the genus Dehalococcoides affiliated with the phylum Chloroflexi and are able to dechlorinate cis-1,2-DCE and vinyl chloride (VC) to the non-toxic ethene. Dehalobacter restrictus and Dehalococcoides isolates were found to be completely restricted to dehalorespiration which gave rise to some basic evolutionary questions. Identification of the key enzyme in the dechlorination reaction, the reductive dehalogenase, has revealed a new class of enzymes containing a corrinoid and two ironsulfur clusters as cofactors. At the beginning of this thesis, nine chloroethene reductive dehalogenases have been characterized on biochemical level, while only little information was available on molecular level. Therefore, the overall goal of this thesis was to characterize on a molecular level the reductive dehalogenases involved in tetrachloroethene dehalorespiration and to get some indications on the evolution of this novel anaerobic respiration process. Starting from the N-terminal sequence of the PCE reductive dehalogenase (PceA) of Dehalobacter restrictus and from a conserved amino acid stretch found in two already sequenced reductive dehalogenases, a degenerate PCR approach allowed the isolation of the gene encoding PceA. Comparison with unpublished data from Desulfitobacterium sp. strain PCE-S showed 100% sequence identity. The full sequence of the pceAB gene of strain PCE-S helped to isolate the corresponding gene cluster from D. restrictus and Desulfitobacterium hafniense strain TCE1, which has also been shown to contain an identical N-terminal sequence. Sequence analysis confirmed the presence of a Twin-Arginine Translocation (Tat) signal peptide, which is involved in the incorporation of the reductive dehalogense into the cytoplasmic membrane. Detailed analysis of the iron-sulfur cluster binding motifs present in PceA of D. restrictus and the chlorophenol reductive dehalogenase (CprA) of Desulfitobacterium dehalogenans revealed differences in the second motif, which may explain results obtained by EPR spectroscopy, namely the presence of two [4Fe-4S] clusters in the former enzyme and the presence of one [3Fe-4S] and one [4Fe-4S] cluster in the latter one. Structure breaking residues such as glycine and proline are present at the two extremities of the ten amino acid stretch separating the first and second ironbinding cysteine residues of the second motif in PceA, but not in CprA. This primary structure probably allows the formation of a loop in the tertiary structure and the participation of the first cyteine as a ligand in a [4Fe-4S] cluster. In both new sequences, the presence of a short gene (pceB) encoding a hydrophobic protein with three conserved trans-membrane α-helices was confirmed, indicating a possible role in anchoring the catalytic unit of the reductive dehalogenase into the membrane. The complete sequence identity observed in the newly isolated reductive dehalogenases raised the question of a possible horizontal gene transfer between Dehalobacter restrictus and Desulfitobacterium hafniense strain TCE1. Therefore, the flanking regions of the reductive dehalogenase genes (pceAB) in Desulfitobacterium hafniense strain TCE1 and Dehalobacter restrictus were investigated. This study revealed the presence of a composite transposon (named Tn-Dha1) in strain TCE1 bordered with two identical insertion sequences (ISDha1, including the transposase gene tnpA1) and containing six open reading frames: the already characterized pceAB, two genes (pceCT) related to members of the o-chlorophenol reductive dehalogenase gene cluster of Desulfitobacterium dehalogenans, and two possibly truncated genes with homology to another transposase (tnpA2) and to a subunit of the Tat machinery (tatA), respectively. In contrast, only the pceABCT gene cluster (i.e. without the transposon structure and the other two genes) was present in Dehalobacter restrictus, indicating that the genes encoding the key enzymes for the dechlorination activity are stably integrated into the genome. A detailed investigation of Tn-Dha1 by PCR and Southern blot analysis indicated that Tn-Dha1 may form various circular molecules, an indication for an active mobile genetic element. A model for the transposition of Tn-Dha1 was proposed, in which the transposon may excise from the chromosome and circularize, forming an unstable structure with two abutted ISDha1. The strong promoter formed by the junction of both IS would lead to high expression of the transposase, which in turn reacts with the circular element by either re-integrating it in the chromosome or excising one or both ISDha1 from that element. The resulting structures would be single IS, IS tandems and circular molecules with one or no remaining IS, both latter structures being dead-end products of the transposition event. The hypothesis of mobile reductive dehalogenase genes was also investigated using a genomic approach in preliminary sequence data (released by The Institute for Genome Research, TIGR) of the genome of Dehalococcoides ethenogenes, a dehalorespiring bacterium capable to completely dechlorinate PCE to ethene. The genome was shown to contain the extraordinary number of eighteen different copies of reductive dehalogenase genes, including the well characterized tceA. A genomic signature of D. ethenogenes was obtained by calculating the frequency of 4-letter DNA words along the genome and was graphically represented. Local disruptions of the genomic signature in certain segments of the genome were highlighted, corresponding to DNA, which may have been acquired by horizontal gene transfer, so-called original regions. It revealed that from the eighteen putative reductive dehalogenase genes present in the genome of D. ethenogenes, fifteen were located in original regions. Moreover, several genes encoding for recombinases (transposase, integrase) were found within these original regions, strongly indicating that these may have been acquired horizontally. 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. Therefore, 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. Detection of cytochromes 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 study, although further investigations are needed, indicated that a c-type cytochrome may be involved in the direct electron transfer to the PCE reductive dehalogenase of D. hafniense strain TCE1. At present, numerous sequences of reductive dehalogenase genes have been reported and deposited on sequence databases, revealing the great interest shown for this new anaerobic respiration pathway. While several degenerate PCR approaches have led to the isolation of 22 mostly partial genes, analysis of preliminary genome sequence data from Dehalococcoides ethenogenes and Desulfitobacterium hafniense strain DCB-2 has revealed 18 and 6 sequences, respectively. Sequence alignment and homology analysis of the 66 reductive dehalogenases genes available in August 2004 revealed four main clusters, two corresponding to chlorophenol and chloroethene reductive dehalogenases found in the phylum Firmicutes, one with sequences mostly isolated from e-Proteobacteria, and one containing most of the genes isolated from the genus Dehalococcoides. Hence, the reductive dehalogenases appear to be rather conserved wihtin phylogenetic groups, indicating a relatively ancient enzyme class. Reductive dehalogenases show some features such as the presence of a Tat signal peptide and iron-sulfur clusters that are common to most of terminal reductases. However, the presence of a corrinoid at the catalytic center and of several specific conserved amino acid stretches makes them a new class of anaerobic reductases. Finally, the strong variation in the topology of the dehalorespiration chain and the variable presence and involvement of different electron transferring components such as quinones and cytochromes in dehalorespiring bacteria indicate that reductive dehalogenases may have been integrated into existing respiration chains rather than that dehalorespiration has evolved as a whole.

Physiological and enzymatic studies of respiration in Dehalococcoides species strain CBDB1

Abstract: The dehalorespiring anaerobe Dehalococcoides sp. strain CBDB1 used HCB and PeCB as electron acceptors in an energy-conserving process with hydrogen as electron donor. Previous attempts to grow Dehalococcoides sp. strain CBDB1 with HCB or PeCB as electron acceptors failed if these compounds were provided as solutions in hexadecane. However, Dehalococcoides sp. strain CBDB1 was able to grow with HCB or PeCB when added in crystalline form directly to cultures. Growth of Dehalococcoides sp. strain CBDB1 by dehalorespiration resulted in a growth yield (Y) of 2.1±0.24 g protein/mol Cl released with HCB as electron acceptor; with PeCB, the growth yield was 2.9±0.15 g/mol Cl. HCB was reductively dechlorinated to PeCB, which was converted to a mixture of 1,2,3,5and 1,2,4,5-TeCB. Formation of 1,2,3,4-TeCB was not detected. The final end-products of HCB and PeCB dechlorination were 1,3,5-TCB, 1,3and 1,4-DCB, which were formed in a ratio of about 3:2:5. As reported previously, Dehalococcoides sp. strain CBDB1 converted 1,2,3,5-TeCB exclusively to 1,3,5-TCB, and 1,2,4,5-TeCB exclusively to 1,2,4-TCB. The organism therefore catalyses two different pathways to dechlorinate highly chlorinated benzenes. In the route leading to 1,3,5-TCB, only doubly flanked chlorine substituents were removed, while in the route leading to 1,3-and 1,4-DCB via 1,2,4TCB singly flanked chlorine substituents were also removed. Reductive dehalogenase activity measurements using whole cells pregrown with different chlorobenzene congeners as electron acceptors indicated that different reductive d ehalogenases might be induced by the different electron acceptors. This is the first demonstration of dehalorespiration with a pure culture depending on PeCB/HCB.

Anaerobic transformation of chlorinated dioxins by microorganisms

Abstract: Mixed bacterial cultures catalyze diverse chlorodioxin-dehalogenation pathways. Some of these pathways lead to relatively harmless end products, which can undergo further biological degradation e.g. by aerobic bacteria. However, the possible formation of highly toxic products is a critical problem for a bioremediation approach but also for untreated sites where such dechlorination reactions can occur. Bioaugmentation with suitable pure or mixed cultures is promising. This has recently been demonstrated in a tetrachloroethene-contaminated groundwater using a Dehalococcoidescontaining inoculum that almost completely converted tetrachloroethene to ethene without accumulation of the toxic intermediate vinyl chloride. With Dehalococcoides sp. strain CBDB1 the first bacterium is now known, that grows by dehalorespiration with dioxins. Learning from the physiology and biochemistry of this bacterium will help us to understand the role of these bacteria in the environment and to predict the fate of dioxin pollution.

Electron donor and acceptor utilization by halorespiring bacteria

Abstract: Chlorinated ethenes are widespread soil and groundwater pollutants. Over the last 2 decades a lot of effort has been made to understand the degradation mechanisms for these pollutants. In the early eighties reduction of tetrachloroethene (PCE) was observed in anaerobic soil samples, which was shown to be mediated by microorganisms. The first microorganism able to couple the anaerobic reduction of PCE to growth in a process called halorespiration (alternative terms are chlororespiration, chloridogenesis or dehalorespiration) was isolated in 1993. Since then, about 15 bacteria able to reduce PCE metabolically have been isolated. This thesis describes research on different aspects influencing the reductive dechlorination of chlorinated ethenes by anaerobic halorespiring bacteria.A new halorespiring bacterium is described in chapter 1. This bacterium, Sulfurospirillum halorespirans PCE-M2, was isolated from a polluted soil near Rotterdam harbor. Strain PCE-M2 is a metabolically versatile bacterium able to use a variety of electron acceptors and electron donors. This new strain is closely related to Dehalospirillum multivorans, but more detailed studies indicated that strain PCE-M2 belongs to the genus Sulfurospirillum, It also appeared that Dehalospirillum multivorans had to be included in this genus. Consequently, it was reclassified to Sulfurospirillum multivorans.Members of the genus Sulfurospirillum were originally known for their sulphur, selenate and arsenate respiring properties. Therefore, we screened a number of halorespiring and related bacteria for their metal reducing properties (Chapter 2). It was shown that the reduction of metals such as ferric iron, manganese, selenate and arsenate is a common property amongst halorespiring bacteria. We also investigated the quinone reducing and oxidizing abilities. AU tested bacteria are able to reduce AQDS7 a quinone-bearing humic acid analogue. Some of the tested bacteria (Desulfitobacterium hafniense DP7, Sulfurospirillum barnesii, S. deleyianum and S. arsenophilum) are also able to oxidize AEbQDS coupled to nitrate reduction.The influence of some alternative electron acceptors on the reductive dechlorination is discussed in chapter 3. Sulfurospirillum halorespirans preferably reduces nitrate (to ammonium) and then PCE. In contrast, Sulfurospirillum multivorans reduces nitrate only to nitrite, and PCE reduction is blocked irreversibly in the presence of nitrate. In Desulfitobacterium frappieri TCEl, PCE and nitrate are reduced simultaneously in excess of electron donor. Under electron donor limitation PCE reduction was inhibited (Gerritse Bt al., AppI. Environ. Microbiol. 1999, 65, 5212-5221). The influence of nitrate on the reduction of chlorinated ethenes by halorespiring bacteria differs between species and may also depend on the availability of electron donor. Sulphate, which is not used as electron acceptor by chlorinated ethenes respiring bacteria is often found at polluted sites. We have tested the influence of sulphate on halorespiring bacteria (Chapter 3). It appeared that sulphate does not influence these microorganisms. Sulphite however, a possible electron acceptor for Desulfitobacterium species, inhibits the reduction of PCE. This inhibition may be the result of a chemical interaction between sulphite and cobalamine containing dehalogenases. We also studied the adaptation of Sulfurospirillum halorespirans PCE-M2 to different alternative electron acceptors (Chapter 3). Both nitrate and arsenate are reduced by cells pre-grown on PCE, nitrate, arsenate and selenate. This indicates that the enzymes responsible for the reduction of nitrate and arsenate are constitutiveiy present in S, halorespirans. In contrast, PCE and selenate are only reduced by cells pre-grown on PCE or selenate respectively.Halorespiring bacteria have a high affinity for hydrogen (H2). H2 may even be the most important electron donor for these organisms in natural environments. We have studied H2^thresh0ld concentrations in pure cultures of halorespiring bacteria (Chapter 4). H2-threshold values between 0.05 and 0.08 nM under PCE-reducing and nitrate-reducing conditions were measured. Furthermore, we measured H2 concentrations at a field site polluted with chlorinated ethenes. PCE and trichloroethene (TCE) reduction can occur at H2 concentrations below 1 nM. However, for the reduction of lower chlorinated ethenes a higher H2 concentration seems to be required.Accumulation of cis-l,2-dichloroethene (cis-l,2-DCE) and vinyl chloride (VC) under anaerobic conditions is often observed. The enrichment of two cultures (DCE-I and DCE-2) able to reduce VC at relative high rates is described in chapter 5. Cis-l,2-DCE is reduced at approximately 20-30 fold lower rates than VC. Our results suggest that these two enrichment cultures are able to gain energy from the reduction of lower chlorinated ethenes. When we performed these studies, no microorganisms had been isolated able to grow by the reduction of VC. However, recently He et al. (Nature. 2003, 424, 62-65) isolated Dehalococcoides strain BAVl, which is able to couple the reduction of DCE and VC to ethene to growth.Finally, the results obtained are combined with available literature data to obtain a state-of-the-art on chlorinated ethenes respiring microorganisms, the influence of alternative electron acceptors on these microorganisms and the role of H3 and H?-threshold values in halorespiration.