Reductive Dechlorination of Chlorinated Ethenes and 1,2-Dichloroethane by “Dehalococcoides ethenogenes” 195

1999 ◽  
Vol 65 (7) ◽  
pp. 3108-3113 ◽  
Author(s):  
Xavier Maymó-Gatell ◽  
Timothy Anguish ◽  
Stephen H. Zinder
Keyword(s):  
Vinyl Chloride ◽  
Growth Medium ◽  
Electron Acceptor ◽  
Reductive Dechlorination ◽  
Electron Acceptors ◽  
Chlorinated Compounds ◽  
Reductive Dehalogenase ◽  
Dehalococcoides Ethenogenes ◽  
Over Time ◽  
Number Of Cells

ABSTRACT “Dehalococcoides ethenogenes” 195 can reductively dechlorinate tetrachloroethene (PCE) completely to ethene (ETH). When PCE-grown strain 195 was transferred (2% [vol/vol] inoculum) into growth medium amended with trichloroethene (TCE),cis-dichloroethene (DCE), 1,1-DCE, or 1,2-dichloroethane (DCA) as an electron acceptor, these chlorinated compounds were consumed at increasing rates over time, which indicated that growth occurred. Moreover, the number of cells increased when TCE, 1,1-DCE, or DCA was present. PCE, TCE, 1,1-DCE, and cis-DCE were converted mainly to vinyl chloride (VC) and then to ETH, while DCA was converted to ca. 99% ETH and 1% VC. cis-DCE was used at lower rates than PCE, TCE, 1,1-DCE, or DCA was used. When PCE-grown cultures were transferred to media containing VC ortrans-DCE, products accumulated slowly, and there was no increase in the rate, which indicated that these two compounds did not support growth. When the intermediates in PCE dechlorination by strain 195 were monitored, TCE was detected first, followed bycis-DCE. After a lag, VC, 1,1-DCE, andtrans-DCE accumulated, which is consistent with the hypothesis that cis-DCE is the precursor of these compounds. Both cis-DCE and 1,1-DCE were eventually consumed, and both of these compounds could be considered intermediates in PCE dechlorination, whereas the small amount oftrans-DCE that was produced persisted. Cultures grown on TCE, 1,1-DCE, or DCA could immediately dechlorinate PCE, which indicated that PCE reductive dehalogenase activity was constitutive when these electron acceptors were used.

scholarly journals Multiple Reductive-Dehalogenase-Homologous Genes Are Simultaneously Transcribed during Dechlorination by Dehalococcoides-Containing Cultures

2005 ◽  
Vol 71 (12) ◽  
pp. 8257-8264 ◽  
Author(s):  
Alison S. Waller ◽  
Rosa Krajmalnik-Brown ◽  
Frank E. Löffler ◽  
Elizabeth A. Edwards
Keyword(s):  
Vinyl Chloride ◽  
Electron Acceptor ◽  
Reductive Dechlorination ◽  
Amino Acid Identity ◽  
Degenerate Primers ◽  
Acid Identity ◽  
Reductive Dehalogenase ◽  
Homologous Genes ◽  
Differential Transcription ◽  
Transcribed Sequences

ABSTRACT Degenerate primers were used to amplify 14 distinct reductive-dehalogenase-homologous (RDH) genes from the Dehalococcoides-containing mixed culture KB1. Most of the corresponding predicted proteins were highly similar (97 to >99% amino acid identity) to previously reported Dehalococcoides reductive dehalogenases. To examine the differential transcription of these RDH genes, KB1 was split into five subcultures amended with either trichloroethene, cis-1,2-dichloroethene, vinyl chloride, 1,2-dichlorethane, or no chlorinated electron acceptor. Total RNA was extracted following the onset of reductive dechlorination, and RDH transcripts were reverse transcribed and amplified using degenerate primers. The results indicate that the transcription of RDH genes requires the presence of a chlorinated electron acceptor, and for all treatments, multiple RDH genes were simultaneously transcribed, with transcripts of two of the genes being present under all four electron-accepting conditions. Two of the transcribed sequences were highly similar to reported vinyl chloride reductase genes, namely, vcrA from Dehalococcoides sp. strain VS and bvcA from Dehalococcoides sp. strain BAV1. These findings suggest that multiple RDH genes are induced by a single chlorinated substrate and that multiple reductive dehalogenases contribute to chloroethene degradation in KB1.

Influence of Different Electron Donors and Acceptors on Dehalorespiration of Tetrachloroethene byDesulfitobacterium frappieri TCE1

1999 ◽  
Vol 65 (12) ◽  
pp. 5212-5221 ◽  
Author(s):  
Jan Gerritse ◽  
Oliver Drzyzga ◽  
Geert Kloetstra ◽  
Mischa Keijmel ◽  
Luit P. Wiersum ◽  
...  
Keyword(s):  
Electron Acceptor ◽  
Reductive Dechlorination ◽  
Electron Acceptors ◽  
Electron Donors ◽  
Strictly Anaerobic ◽  
16S Rrna Sequence ◽  
Selective Enrichment ◽  
Chemostat Cultures ◽  
16S Rrna Sequence Analysis ◽  
Desulfitobacterium Hafniense

ABSTRACT Strain TCE1, a strictly anaerobic bacterium that can grow by reductive dechlorination of tetrachloroethene (PCE) and trichloroethene (TCE), was isolated by selective enrichment from a PCE-dechlorinating chemostat mixed culture. Strain TCE1 is a gram-positive, motile, curved rod-shaped organism that is 2 to 4 by 0.6 to 0.8 μm and has approximately six lateral flagella. The pH and temperature optima for growth are 7.2 and 35°C, respectively. On the basis of a comparative 16S rRNA sequence analysis, this bacterium was identified as a new strain of Desulfitobacterium frappieri, because it exhibited 99.7% relatedness to the D. frappieri type strain, strain PCP-1. Growth with H2, formate,l-lactate, butyrate, crotonate, or ethanol as the electron donor depends on the availability of an external electron acceptor. Pyruvate and serine can also be used fermentatively. Electron donors (except formate and H2) are oxidized to acetate and CO2. When l-lactate is the growth substrate, strain TCE1 can use the following electron acceptors: PCE and TCE (to produce cis-1,2-dichloroethene), sulfite and thiosulfate (to produce sulfide), nitrate (to produce nitrite), and fumarate (to produce succinate). Strain TCE1 is not able to reductively dechlorinate 3-chloro-4-hydroxyphenylacetate. The growth yields of the newly isolated bacterium when PCE is the electron acceptor are similar to those obtained for other dehalorespiring anaerobes (e.g.,Desulfitobacterium sp. strain PCE1 andDesulfitobacterium hafniense) and the maximum specific reductive dechlorination rates are 4 to 16 times higher (up to 1.4 μmol of chloride released · min−1 · mg of protein−1). Dechlorination of PCE and TCE is an inducible process. In PCE-limited chemostat cultures of strain TCE1, dechlorination is strongly inhibited by sulfite but not by other alternative electron acceptors, such as fumarate or nitrate.

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

2004 ◽  
Vol 70 (10) ◽  
pp. 6347-6351 ◽  
Author(s):  
Rosa Krajmalnik-Brown ◽  
Tina Hölscher ◽  
Ivy N. Thomson ◽  
F. Michael Saunders ◽  
Kirsti M. Ritalahti ◽  
...  
Keyword(s):  
Vinyl Chloride ◽  
Reductive Dechlorination ◽  
Complete Sequence ◽  
Mixed Cultures ◽  
Genetic Identification ◽  
Degenerate Primers ◽  
Transcription Analysis ◽  
Reductive Dehalogenase ◽  
Conserved Regions ◽  
Rdase Genes

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.

scholarly journals Comparison of Energy and Growth Yields forDesulfitobacterium dehalogenans during Utilization of Chlorophenol and Various Traditional Electron Acceptors

1998 ◽  
Vol 64 (1) ◽  
pp. 352-355 ◽  
Author(s):  
M. Mackiewicz ◽  
J. Wiegel
Keyword(s):  
Electron Donor ◽  
Electron Acceptor ◽  
Reductive Dechlorination ◽  
Dry Weight ◽  
Electron Acceptors ◽  
Growth Yields ◽  
Donor And Acceptor ◽  
Electron Donor And Acceptor

ABSTRACT Desulfitobacterium dehalogenans grew with formate as the electron donor and 3-chloro-4-hydroxyphenylacetate (3-Cl-4-OHPA) as the electron acceptor, yielding Y X/formate,Y X/2e− , andY X/ATP ranging from 3.2 to 11.3 g of biomass (dry weight)/mol, thus indicating that energy was conserved through reductive dechlorination. Pyruvate was utilized as the electron donor and acceptor, yielding stoichiometric amounts of acetate and lactate, respectively, and a Y X/reduced acceptor of 13.0 g of biomass (dry weight)/mol. The supplementation of pyruvate-containing medium with additional electron acceptors, such as 3-Cl-4-OHPA, nitrate, fumarate, or sulfite, caused pyruvate to be replaced as the electron acceptor and nearly doubled theY X/ATP (Y X/acetate formed). A comparison of the yields for 3-Cl-4-OHPA with those for other traditional electron acceptors indicates that the dehalogenation reaction led to the formation of similar amounts of energy equivalents. The various electron acceptors were used concomitantly with 3-Cl-4-OHPA in nonacclimated cultures, but the utilization rates and amounts utilized differed.

scholarly journals Characterization of Hydrogenase and Reductive Dehalogenase Activities of Dehalococcoides ethenogenes Strain 195

2005 ◽  
Vol 71 (3) ◽  
pp. 1664-1667 ◽  
Author(s):  
Ivonne Nijenhuis ◽  
Stephen H. Zinder
Keyword(s):  
Reductive Dechlorination ◽  
Methyl Viologen ◽  
Hydrogenase Activity ◽  
Atpase Inhibitor ◽  
Benzyl Viologen ◽  
Reductive Dehalogenase ◽  
Cell Extracts ◽  
Dehalococcoides Ethenogenes ◽  
In Cells

ABSTRACT Dehalococcoides ethenogenes strain 195 reductively dechlorinates tetrachloroethene (PCE) and trichloroethene (TCE) to vinyl chloride and ethene using H2 as an electron donor. PCE- and TCE-reductive dehalogenase (RD) activities were mainly membrane associated, whereas only about 20% of the hydrogenase activity was membrane associated. Experiments with methyl viologen (MV) were consistent with a periplasmic location for the RDs or a component feeding electrons to them. The protonophore uncoupler tetrachlorosalicylanilide did not inhibit reductive dechlorination in cells incubated with H2 and PCE and partially restored activity in cells incubated with the ATPase inhibitor N,N′-dicyclohexylcarbodiimide. Benzyl viologen or diquat (E o′ ≈ −360 mV) supported reductive dechlorination of PCE or TCE at rates comparable to MV (−450 mV) in cell extracts.

scholarly journals Expression of Reductive Dehalogenase Genes in Dehalococcoides ethenogenes Strain 195 Growing on Tetrachloroethene, Trichloroethene, or 2,3-Dichlorophenol

2007 ◽  
Vol 73 (14) ◽  
pp. 4439-4445 ◽  
Author(s):  
Jennifer M. Fung ◽  
Robert M. Morris ◽  
Lorenz Adrian ◽  
Stephen H. Zinder
Keyword(s):  
Reductive Dechlorination ◽  
Stop Codon ◽  
Gene Transcript ◽  
High Coverage ◽  
Transcript Levels ◽  
Reductive Dehalogenase ◽  
Dehalococcoides Ethenogenes ◽  
Rd Cells ◽  
Reductive Dehalogenase Genes ◽  
In Cells

ABSTRACT Reductive dehalogenase (RD) gene transcript levels in Dehalococcoides ethenogenes strain 195 were investigated using reverse transcriptase quantitative PCR during growth and reductive dechlorination of tetrachloroethene (PCE), trichloroethene (TCE), or 2,3-dichlorophenol (2,3-DCP). Cells grown with PCE or TCE had high transcript levels (greater than that for rpoB) for tceA, which encodes the TCE RD, pceA, which encodes the PCE RD, and DET0162, which contains a predicted stop codon and is considered nonfunctional. In cells grown with 2,3-DCP, tceA mRNA was less than 1% of that for rpoB, indicating that its transcription was regulated. pceA and DET0162 were the only RD genes with high transcript levels in cells grown with 2,3-DCP. Proteomic analysis of PCE-grown cells detected both PceA and TceA with high peptide coverage but not DET0162, and analysis of 2,3-DCP-grown cells detected PceA with high coverage but not TceA, DET0162, or any other potential RD. Cells grown with PCE or 2,3-DCP were tested for the ability to dechlorinate PCE, TCE, or 2,3-DCP with H2 as the electron donor. 2,3-DCP-grown cells were unable to dechlorinate TCE but dechlorinated PCE to TCE without a lag, and PCE-grown cells dechlorinated 2,3-DCP without a lag. These results show that 2,3-DCP-grown cells do not produce TceA and that DET0162 is transcribed but its translation product is not detectable in cells and are consistent with PceA's being bifunctional, also serving as the 2,3-DCP RD. Chlorophenols naturally occur in soils and are good candidates for the original substrates for PceA.

scholarly journals Complete Detoxification of Vinyl Chloride by an Anaerobic Enrichment Culture and Identification of the Reductively Dechlorinating Population as a Dehalococcoides Species

2003 ◽  
Vol 69 (2) ◽  
pp. 996-1003 ◽  
Author(s):  
Jianzhong He ◽  
Kirsti M. Ritalahti ◽  
Michael R. Aiello ◽  
Frank E. Löffler
Keyword(s):  
Vinyl Chloride ◽  
Electron Donor ◽  
Reductive Dechlorination ◽  
Enrichment Culture ◽  
Electron Acceptors ◽  
Rrna Gene ◽  
Mineral Salts ◽  
Half Saturation Constant ◽  
Dependent Growth ◽  
Shed Light

ABSTRACT A major obstacle in the implementation of the reductive dechlorination process at chloroethene-contaminated sites is the accumulation of the intermediate vinyl chloride (VC), a proven human carcinogen. To shed light on the microbiology involved in the final critical dechlorination step, a sediment-free, nonmethanogenic, VC-dechlorinating enrichment culture was derived from tetrachloroethene (PCE)-to-ethene-dechlorinating microcosms established with material from the chloroethene-contaminated Bachman Road site aquifer in Oscoda, Mich. After 40 consecutive transfers in defined, reduced mineral salts medium amended with VC, the culture lost the ability to use PCE and trichloroethene (TCE) as metabolic electron acceptors. PCE and TCE dechlorination occurred in the presence of VC, presumably in a cometabolic process. Enrichment cultures supplied with lactate or pyruvate as electron donor dechlorinated VC to ethene at rates up to 54 μmol liter−1day−1, and dichloroethenes (DCEs) were dechlorinated at about 50% of this rate. The half-saturation constant (KS ) for VC was 5.8 μM, which was about one-third lower than the concentrations determined for cis-DCE and trans-DCE. Similar VC dechlorination rates were observed at temperatures between 22 and 30°C, and negligible dechlorination occurred at 4 and 35°C. Reductive dechlorination in medium amended with ampicillin was strictly dependent on H2 as electron donor. VC-dechlorinating cultures consumed H2 to threshold concentrations of 0.12 ppm by volume. 16S rRNA gene-based tools identified a Dehalococcoides population, and Dehalococcoides-targeted quantitative real-time PCR confirmed VC-dependent growth of this population. These findings demonstrate that Dehalococcoides populations exist that use DCEs and VC but not PCE or TCE as metabolic electron acceptors.

scholarly journals Fraction of Electrons Consumed in Electron Acceptor Reduction and Hydrogen Thresholds as Indicators of Halorespiratory Physiology

1999 ◽  
Vol 65 (9) ◽  
pp. 4049-4056 ◽  
Author(s):  
Frank E. Löffler ◽  
James M. Tiedje ◽  
Robert A. Sanford
Keyword(s):  
Vinyl Chloride ◽  
Electron Acceptor ◽  
Reductive Dechlorination ◽  
Theoretical Calculations ◽  
Mixed Cultures ◽  
Lower Threshold ◽  
Chlorinated Compound ◽  
Hydrogen Consumption ◽  
Pure Cultures ◽  
Good Agreement

ABSTRACT Measurements of the hydrogen consumption threshold and the tracking of electrons transferred to the chlorinated electron acceptor (fe) reliably detected chlororespiratory physiology in both mixed cultures and pure cultures capable of using tetrachloroethene,cis-1,2-dichloroethene, vinyl chloride, 2-chlorophenol, 3-chlorobenzoate, 3-chloro-4-hydroxybenzoate, or 1,2-dichloropropane as an electron acceptor. Hydrogen was consumed to significantly lower threshold concentrations of less than 0.4 ppmv compared with the values obtained for the same cultures without a chlorinated compound as an electron acceptor. The fe values ranged from 0.63 to 0.7, values which are in good agreement with theoretical calculations based on the thermodynamics of reductive dechlorination as the terminal electron-accepting process. In contrast, a mixed methanogenic culture that cometabolized 3-chlorophenol exhibited a significantly lower fe value, 0.012.

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