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

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.

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

Applied and Environmental Microbiology ◽

10.1128/aem.64.1.352-355.1998 ◽

1998 ◽

Vol 64 (1) ◽

pp. 352-355 ◽

Cited By ~ 20

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.

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Characterization of a Highly Enriched Microbial Consortium Reductively Dechlorinating 2,3-Dichlorophenol and 2,4,6-Trichlorophenol and the Corresponding cprA Genes from River Sediment

Polish Journal of Microbiology ◽

10.5604/17331331.1215613 ◽

2016 ◽

Vol 65 (3) ◽

pp. 341-352 ◽

Cited By ~ 1

Author(s):

Wael S. El-Sayed

Keyword(s):

Electron Donor ◽

Reductive Dechlorination ◽

River Sediment ◽

Microbial Consortium ◽

Denaturing Gradient Gel Electrophoresis ◽

Bacterial Species ◽

Rrna Gene ◽

Gradient Gel Electrophoresis ◽

Dominant Bacteria

Anaerobic reductive dechlorination of 2,3-dichlorophenol (2,3DCP) and 2,4,6-trichlorophenol (2,4,6TCP) was investigated in microcosms from River Nile sediment. A stable sediment-free anaerobic microbial consortium reductively dechlorinating 2,3DCP and 2,4,6TCP was established. Defined sediment-free cultures showing stable dechlorination were restricted to ortho chlorine when enriched with hydrogen as the electron donor, acetate as the carbon source, and either 2,3-DCP or 2,4,6-TCP as electron acceptors. When acetate, formate, or pyruvate were used as electron donors, dechlorination activity was lost. Only lactate can replace dihydrogen as an electron donor. However, the dechlorination potential was decreased after successive transfers. To reveal chlororespiring species, the microbial community structure of chlorophenol-reductive dechlorinating enrichment cultures was analyzed by PCR-denaturing gradient gel electrophoresis (DGGE) of 16S rRNA gene fragments. Eight dominant bacteria were detected in the dechlorinating microcosms including members of the genera Citrobacter, Geobacter, Pseudomonas, Desulfitobacterium, Desulfovibrio, and Clostridium. Highly enriched dechlorinating cultures were dominated by four bacterial species belonging to the genera Pseudomonas, Desulfitobacterium, and Clostridium. Desulfitobacterium represented the major fraction in DGGE profiles indicating its importance in dechlorination activity, which was further confirmed by its absence resulting in complete loss of dechlorination. Reductive dechlorination was confirmed by the stoichiometric dechlorination of 2,3DCP and 2,4,6TCP to metabolites with less chloride groups and by the detection of chlorophenol RD cprA gene fragments in dechlorinating cultures. PCR amplified cprA gene fragments were cloned and sequenced and found to cluster with the cprA/pceA type genes of Dehalobacter restrictus.

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Complete Genome Sequence of Dehalococcoides mccartyi Strain WBC-2, Capable of Anaerobic Reductive Dechlorination of Vinyl Chloride

Genome Announcements ◽

10.1128/genomea.01375-16 ◽

2016 ◽

Vol 4 (6) ◽

Cited By ~ 1

Author(s):

Olivia Molenda ◽

Shuiquan Tang ◽

Elizabeth A. Edwards

Keyword(s):

Vinyl Chloride ◽

Genome Sequence ◽

Complete Genome Sequence ◽

Reductive Dechlorination ◽

Complete Genome ◽

Microbial Consortium ◽

Enrichment Culture ◽

Metagenomic Sequencing ◽

The West ◽

Dehalococcoides Mccartyi

Dehalococcoides mccartyi strain WBC-2 dechlorinates carcinogen vinyl chloride to ethene in the West Branch Canal Creek (WBC-2) microbial consortium used for bioaugmentation. We assembled and closed the complete genome sequence of this prokaryote using metagenomic sequencing from an enrichment culture.

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Characterization of an Isolate That Uses Vinyl Chloride as a Growth Substrate under Aerobic Conditions

Applied and Environmental Microbiology ◽

10.1128/aem.66.8.3535-3542.2000 ◽

2000 ◽

Vol 66 (8) ◽

pp. 3535-3542 ◽

Cited By ~ 63

Author(s):

Matthew F. Verce ◽

Ricky L. Ulrich ◽

David L. Freedman

Keyword(s):

Vinyl Chloride ◽

Suspended Solids ◽

Specific Growth ◽

Enrichment Culture ◽

Initial Step ◽

Aerobic Biodegradation ◽

Utilization Rate ◽

Rrna Gene ◽

Donor Source

ABSTRACT An aerobic enrichment culture was developed by using vinyl chloride (VC) as the sole organic carbon and electron donor source. VC concentrations as high as 7.3 mM were biodegraded without apparent inhibition. VC use did not occur when nitrate was provided as the electron acceptor. A gram-negative, rod-shaped, motile isolate was obtained from the enrichment culture and identified based on biochemical characteristics and the sequence of its 16S rRNA gene asPseudomonas aeruginosa, designated strain MF1. The observed yield of MF1 when it was grown on VC was 0.20 mg of total suspended solids (TSS)/mg of VC. Ethene, acetate, glyoxylate, and glycolate also served as growth substrates, while ethane, chloroacetate, glycolaldehyde, and phenol did not. Stoichiometric release of chloride and minimal accumulation of soluble metabolites following VC consumption indicated that the predominant fate for VC is mineralization and incorporation into cell material. MF1 resumed consumption of VC after at least 24 days when none was provided, unlike various mycobacteria that lost their VC-degrading ability after brief periods in the absence of VC. When deprived of oxygen for 2.5 days, MF1 did not regain the ability to grow on VC, and a portion of the VC was transformed into VC-epoxide. Acetylene inhibited VC consumption by MF1, suggesting the involvement of a monooxygenase in the initial step of VC metabolism. The maximum specific VC utilization rate for MF1 was 0.41 μmol of VC/mg of TSS/day, the maximum specific growth rate was 0.0048/day, and the Monod half-saturation coefficient was 0.26 μM. A higher yield and faster kinetics occurred when MF1 grew on ethene. When grown on ethene, MF1 was able to switch to VC as a substrate without a lag. It therefore appears feasible to grow MF1 on a nontoxic substrate and then apply it to environments that do not exhibit a capacity for aerobic biodegradation of VC.

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

Applied and Environmental Microbiology ◽

10.1128/aem.70.9.5538-5545.2004 ◽

2004 ◽

Vol 70 (9) ◽

pp. 5538-5545 ◽

Cited By ~ 159

Author(s):

Melanie Duhamel ◽

Kaiguo Mo ◽

Elizabeth A. Edwards

Keyword(s):

16S Rrna ◽

16S Rrna Gene ◽

Vinyl Chloride ◽

Denaturing Gradient Gel Electrophoresis ◽

Enrichment Culture ◽

Phylogenetic Analyses ◽

Rrna Gene ◽

Sufficient Information ◽

Gene Copies ◽

Gradient Gel Electrophoresis

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) × 108 16S rRNA gene copies/μmol 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) × 108 16S rRNA gene copies/μmol 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-H2 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.

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Anaerobic transformation of tetrachloroethane, perchloroethylene, and their mixtures by mixed-cultures enriched from contaminated soils and sediments

Water Science & Technology ◽

10.2166/wst.2005.0539 ◽

2005 ◽

Vol 52 (1-2) ◽

pp. 357-362 ◽

Cited By ~ 10

Author(s):

F. Aulenta ◽

A. Fina ◽

M. Potalivo ◽

M. Petrangeli Papini ◽

S. Rossetti ◽

...

Keyword(s):

Vinyl Chloride ◽

Electron Donor ◽

Electron Acceptor ◽

Contaminated Soils ◽

Enrichment Culture ◽

Mixed Cultures ◽

Soil Enrichment ◽

Soils And Sediments

The focus of this research was to investigate the anaerobic transformation of tetrachloroethane (TeCA), perchloroethylene (PCE), and their mixtures by mixed cultures enriched from contaminated soils or sediments. Batch transformation studies were conducted using TeCA (60 μM), PCE (60 μM), or TeCA + PCE (each added at 60 μM) as electron acceptor(s) and H2+acetate (each added at 3 mM) or butyrate (3 mM) as electron donor(s). A Dehalococcoides spp.-containing, sediment-enrichment dechlorinated PCE rapidly to ethene (ETH) but slowly and incompletely dechlorinated TeCA. Moreover, when present in mixture with PCE, TeCA disrupted the ability of Dehalococcoides to dechlorinate vinyl chloride. In contrast, the soil-enrichment culture was able to completely dechlorinate TeCA and PCE to ETH, both when added as single contaminants and when added as a mixture.

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

Applied and Environmental Microbiology ◽

10.1128/aem.65.7.3108-3113.1999 ◽

1999 ◽

Vol 65 (7) ◽

pp. 3108-3113 ◽

Cited By ~ 222

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.

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Anaerobic biodegradation of chloroform and dichloromethane with a Dehalobacter enrichment culture

Applied and Environmental Microbiology ◽

10.1128/aem.01970-21 ◽

2021 ◽

Author(s):

Hao Wang ◽

Rong Yu ◽

Jennifer Webb ◽

Peter Dollar ◽

David L. Freedman

Keyword(s):

Carbon Dioxide ◽

Organic Acids ◽

Electron Donor ◽

Contaminated Soil ◽

Reductive Dechlorination ◽

Enrichment Culture ◽

Reducing Power ◽

Anaerobic Conditions ◽

Aliphatic Compounds ◽

Oxidative Pathway

Chloroform (CF) and dichloromethane (DCM) are among the more commonly identified chlorinated aliphatic compounds found in contaminated soil and groundwater. Complete dechlorination of CF has been reported under anaerobic conditions by microbes that respire CF to DCM and others that biodegrade DCM. The objectives of this study were to ascertain if a commercially available bioaugmentation enrichment culture (KB-1® Plus CF) uses an oxidative or fermentative pathway for biodegradation of DCM; and to determine if the products from DCM biodegradation can support organohalide respiration of CF to DCM in the absence of an exogenous electron donor. In various treatments with the KB-1 ® Plus CF culture to which 14 C-CF was added, the predominant product was 14 CO 2 , indicating that oxidation is the predominant pathway for DCM. Recovery of 14 C-DCM when biodegradation was still in progress confirmed that CF first undergoes reductive dechlorination to DCM. 14 C-labeled organic acids, including acetate and propionate, were also recovered, suggesting that synthesis of organic acids provides a sink for the electron equivalents from oxidation of DCM. When the biomass was washed to remove organic acids from prior additions of exogenous electron donor and only CF and DCM were added, the culture completely dechlorinated CF. The total amount of DCM added was not sufficient to provide the electron equivalents needed to reduce CF to DCM. Thus, the additional reducing power came via the DCM generated from CF reduction. Nevertheless, the rate of CF consumption was considerably slower in comparison to treatments that received an exogenous electron donor. IMPORTANCE Chloroform (CF) and dichloromethane (DCM) are among the more commonly identified chlorinated aliphatic compounds found in contaminated soil and groundwater. One way to address this problem is to add microbes to the subsurface that can biodegrade these compounds. While microbes are known that can accomplish this task, less is known about the pathways used under anaerobic conditions. Some use an oxidative pathway, resulting mainly in carbon dioxide. Others use a fermentative pathway, resulting in formation of organic acids. In this study, a commercially available bioaugmentation enrichment culture (KB-1 ® Plus CF) was evaluated using carbon-14 labelled chloroform. The main product formed was carbon dioxide, indicating the use of an oxidative pathway. The reducing power gained from oxidation was shown to support reductive dechlorination of CF to DCM. The results demonstrate the potential to achieve full dechlorination of CF and DCM to nonhazardous products that are difficult to identify in the field.

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Anaerobic biodegradation of chloroform and dichloromethane with a Dehalobacter enrichment culture

10.1101/2021.07.26.453918 ◽

2021 ◽

Author(s):

Hao Wang ◽

Rong Yu ◽

Jennifer Webb ◽

Peter Dollar ◽

David L Freedman

Keyword(s):

Organic Acids ◽

Electron Donor ◽

Reductive Dechlorination ◽

Enrichment Culture ◽

Reducing Power ◽

Anaerobic Biodegradation ◽

Anaerobic Conditions ◽

Aliphatic Compounds ◽

Predominant Product ◽

Complete Dechlorination

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Growth of a Dehalococcoides-Like Microorganism on Vinyl Chloride and cis-Dichloroethene as Electron Acceptors as Determined by Competitive PCR

Applied and Environmental Microbiology ◽

10.1128/aem.69.2.953-959.2003 ◽

2003 ◽

Vol 69 (2) ◽

pp. 953-959 ◽

Cited By ~ 175

Author(s):

Alison M. Cupples ◽

Alfred M. Spormann ◽

Perry L. McCarty

Keyword(s):

Vinyl Chloride ◽

Electron Donor ◽

Growth Yield ◽

Kinetic Studies ◽

Maximum Growth ◽

Reductive Dehalogenation ◽

Utilization Rate ◽

Rrna Gene ◽

Competitive Pcr ◽

Average Growth

ABSTRACT A competitive PCR (cPCR) assay targeting 16S ribosomal DNA was developed to enumerate growth of a Dehalococcoides-like microorganism, bacterium VS, from a mixed culture catalyzing the reductive dehalogenation of cis-1,2-dichloroethene (cDCE) and vinyl chloride (VC), with hydrogen being used as an electron donor. The growth of bacterium VS was found to be coupled to the dehalogenation of VC and cDCE, suggesting unique metabolic capabilities. The average growth yield was (5.2 ± 1.5) × 108 copies of the 16S rRNA gene/μmol of Cl− (number of samples, 10), with VC being used as the electron acceptor and hydrogen as the electron donor. The maximum VC utilization rate (q̂) was determined to be 7.8 × 10−10 μmol of Cl− (copy−1 day−1), indicating a maximum growth rate of 0.4 day−1. These average growth yield and q̂ values agree well with values found previously for dechlorinating cultures. Decay coefficients were determined with growth (0.05 day−1) and no-growth (0.09 day−1) conditions. An important limitation of this cPCR assay was its inability to discriminate between active and inactive cells. This is an essential consideration for kinetic studies.

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