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

2000 ◽  
Vol 66 (8) ◽  
pp. 3535-3542 ◽  
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.

Substrate interactions during aerobic biodegradation of methane, ethene, vinyl chloride and 1,2-dichloroethenes

2001 ◽  
Vol 43 (5) ◽  
pp. 333-340 ◽  
Author(s):  
D. L. Freedman ◽  
A. S. Danko ◽  
M. F. Verce
Keyword(s):  
Vinyl Chloride ◽  
Natural Attenuation ◽  
Enrichment Culture ◽  
Active Role ◽  
Aerobic Biodegradation ◽  
Aerobic Conditions ◽  
Lag Period ◽  
Anaerobic Zone ◽  
Set Up ◽  
Cis And Trans

Intrinsic biodegradation of trichloroethene and 1,1,1-trichloroethane in groundwater at a Superfund site in California has been observed. An anaerobic zone exists in the area closest to the source location, yielding the expected complement of reductive dechlorination daughter products, including cis-1,2-dichloroethene (cis-DCE) and vinyl chloride (VC). Significant levels of methane and ethene were also generated in the anaerobic zone. The groundwater returns to aerobic conditions downgradient of the source, with methane, ethene, VC, and several other compounds still present. Attenuation of VC in the aerobic zone suggests that it is being biodegraded. In this study microcosms were used to evaluate the role of methane and ethene as primary substrates for aerobic biodegradation of VC. Biodegradation of VC was fastest in the bottles containing ethene, with 40 μmol of VC consumed over a 150 day period, compared to approximately 15–20 μmol with methane or a mixture of methane and ethene. VC did not noticeably inhibit ethene biodegradation but did slow the rate of methane use. Methane inhibited ethene metabolism, which apparently caused a reduction in VC biodegradation when methane was present with ethene. These results suggest that ethene plays an important role during in situ natural attenuation of VC under aerobic conditions. Microcosms were also set up with VC alone. Following a 75 day lag period, VC consumption began and subsequent additions were consumed without a lag, suggesting the presence of organisms capable of using VC as a growth substrate. After providing VC alone for nearly 400 days, aliquots of the enrichment culture were used to evaluate its ability to biodegrade cis- and trans-DCE. Both compounds were readily consumed, although addition of VC as the primary substrate was needed to sustain biodegradation of repeated additions. This result suggests that organisms capable of using VC as a sole substrate may play an active role in aerobic natural attenuation of DCEs.

scholarly journals Characterization of the Community Structure of a Dechlorinating Mixed Culture and Comparisons of Gene Expression in Planktonic and Biofloc-Associated “Dehalococcoides” and Methanospirillum Species

2008 ◽  
Vol 74 (21) ◽  
pp. 6709-6719 ◽  
Author(s):  
Annette R. Rowe ◽  
Brendan J. Lazar ◽  
Robert M. Morris ◽  
Ruth E. Richardson
Keyword(s):  
Gene Expression ◽  
16S Rrna ◽  
16S Rrna Gene ◽  
Population Distribution ◽  
Enrichment Culture ◽  
Expression Profiles ◽  
Expression Patterns ◽  
Rrna Gene ◽  
Hydrogenase Gene

ABSTRACT This study sought to characterize bacterial and archaeal populations in a perchloroethene- and butyrate-fed enrichment culture containing hydrogen-consuming “Dehalococcoides ethenogenes” strain 195 and a Methanospirillum hungatei strain. Phylogenetic characterization of this microbial community was done via 16S rRNA gene clone library and gradient gel electrophoresis analyses. Fluorescence in situ hybridization was used to quantify populations of “Dehalococcoides” and Archaea and to examine the colocalization of these two groups within culture bioflocs. A technique for enrichment of planktonic and biofloc-associated biomass was developed and used to assess differences in population distribution and gene expression patterns following provision of substrate. On a per-milliliter-of-culture basis, most D. ethenogenes genes (the hydrogenase gene hupL; the highly expressed gene for an oxidoreductase of unknown function, fdhA; the RNA polymerase subunit gene rpoB; and the 16S rRNA gene) showed no statistical difference in expression between planktonic and biofloc enrichments at either time point studied (1 to 2 and 6 h postfeeding). Normalization of transcripts to ribosome (16S rRNA) levels supported that planktonic and biofloc-associated D. ethenogenes had similar gene expression profiles, with one notable exception; planktonic D. ethenogenes showed higher expression of tceA relative to biofloc-associated cells at 6 h postfeeding. These trends were compared to those for the hydrogen-consuming methanogen in the culture, M. hungatei. The vast majority of M. hungatei cells, ribosomes (16S rRNA), and transcripts of the hydrogenase gene mvrD and the housekeeping gene rpoE were observed in the biofloc enrichments. This suggests that, unlike the comparable activity of D. ethenogenes from both enrichments, planktonic M. hungatei is responsible for only a small fraction of the hydrogenotrophic methanogenesis in this culture.

scholarly journals Biodegradation of Chloromethane by Pseudomonas aeruginosa Strain NB1 under Nitrate-Reducing and Aerobic Conditions

2004 ◽  
Vol 70 (8) ◽  
pp. 4629-4634 ◽  
Author(s):  
David L. Freedman ◽  
Meghna Swamy ◽  
Nathan C. Bell ◽  
Mathew F. Verce
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Suspended Solids ◽  
Specific Growth ◽  
Oxygen Demand ◽  
Sole Source ◽  
Maximum Specific Growth Rate ◽  
Utilization Rate ◽  
Aerobic Conditions ◽  
Nitrate Consumption ◽  
Metabolic Products

ABSTRACT Pseudomonas aeruginosa strain NB1 uses chloromethane (CM) as its sole source of carbon and energy under nitrate-reducing and aerobic conditions. The observed yield of NB1 was 0.20 (±0.06) (mean ± standard deviation) and 0.28 (±0.01) mg of total suspended solids (TSS) mg of CM−1 under anoxic and aerobic conditions, respectively. The stoichiometry of nitrate consumption was 0.75 (±0.10) electron equivalents (eeq) of NO3 − per eeq of CM, which is consistent with the yield when it is expressed on an eeq basis. Nitrate was stoichiometrically converted to dinitrogen (0.51 ± 0.05 mol of N2 per mol of NO3 −). The stoichiometry of oxygen use with CM (0.85 ± 0.21 eeq of O2 per eeq of CM) was also consistent with the aerobic yield. Stoichiometric release of chloride and minimal accumulation of soluble metabolic products (measured as chemical oxygen demand) following CM consumption, under anoxic and aerobic conditions, indicated complete biodegradation of CM. Acetylene did not inhibit CM use under aerobic conditions, implying that a monooxygenase was not involved in initiating aerobic CM metabolism. Under anoxic conditions, the maximum specific CM utilization rate (k) for NB1 was 5.01 (±0.06) μmol of CM mg of TSS−1 day−1, the maximum specific growth rate (μmax) was 0.0506 day−1, and the Monod half-saturation coefficient (Ks ) was 0.067 (±0.004) μM. Under aerobic conditions, the values for k, μ max , and Ks were 10.7 (±0.11) μmol of CM mg of TSS−1 day−1, 0.145 day−1, and 0.93 (±0.042) μM, respectively, indicating that NB1 used CM faster under aerobic conditions. Strain NB1 also grew on methanol, ethanol, and acetate under denitrifying and aerobic conditions, but not on methane, formate, or dichloromethane.

scholarly journals Involvement of Linear Plasmids in Aerobic Biodegradation of Vinyl Chloride

2004 ◽  
Vol 70 (10) ◽  
pp. 6092-6097 ◽  
Author(s):  
Anthony S. Danko ◽  
Meizhong Luo ◽  
Christopher E. Bagwell ◽  
Robin L. Brigmon ◽  
David L. Freedman
Keyword(s):  
Ethylene Oxide ◽  
Vinyl Chloride ◽  
Suspended Solids ◽  
Total Suspended Solids ◽  
Sole Source ◽  
Luria Bertani ◽  
Aerobic Biodegradation ◽  
Linear Plasmids ◽  
Hazardous Waste Sites ◽  
Luria Bertani Broth

ABSTRACT Pseudomonas putida strain AJ and Ochrobactrum strain TD were isolated from hazardous waste sites based on their ability to use vinyl chloride (VC) as the sole source of carbon and energy under aerobic conditions. Strains AJ and TD also use ethene and ethylene oxide as growth substrates. Strain AJ contained a linear megaplasmid (approximately 260 kb) when grown on VC or ethene, but it contained no circular plasmids. While strain AJ was growing on ethylene oxide, it was observed to contain a 100-kb linear plasmid, and its ability to use VC as a substrate was retained. The linear plasmids in strain AJ were cured, and the ability of strain AJ to consume VC, ethene, and ethylene oxide was lost following growth on a rich substrate (Luria-Bertani broth) through at least three transfers. Strain TD contained three linear plasmids, ranging in size from approximately 90 kb to 320 kb, when growing on VC or ethene. As with strain AJ, the linear plasmids in strain TD were cured following growth on Luria-Bertani broth and its ability to consume VC and ethene was lost. Further analysis of these linear plasmids may help reveal the pathway for VC biodegradation in strains AJ and TD and explain why this process occurs at many but not all sites where groundwater is contaminated with chloroethenes. Metabolism of VC and ethene by strains AJ and TD is initiated by an alkene monooxygenase. Their yields during growth on VC (0.15 to 0.20 mg of total suspended solids per mg of VC) are similar to the yields reported for other isolates (i.e., Mycobacterium sp., Nocardioides sp., and Pseudomonas sp.).

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

2004 ◽  
Vol 70 (9) ◽  
pp. 5538-5545 ◽  
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.

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 Characterization of a Dehalobacter Coculture That Dechlorinates 1,2-Dichloroethane to Ethene and Identification of the Putative Reductive Dehalogenase Gene

2009 ◽  
Vol 75 (9) ◽  
pp. 2684-2693 ◽  
Author(s):  
Ariel Grostern ◽  
Elizabeth A. Edwards
Keyword(s):  
Enrichment Culture ◽  
Growth Yield ◽  
Individual Species ◽  
Rrna Gene ◽  
Degenerate Primers ◽  
Reductive Dehalogenase ◽  
Gene Copies ◽  
Parent Culture ◽  
Dehalogenase Gene

ABSTRACT Dehalobacter and “Dehalococcoides” spp. were previously shown to be involved in the biotransformation of 1,1,2-trichloroethane (1,1,2-TCA) and 1,2-dichloroethane (1,2-DCA) to ethene in a mixed anaerobic enrichment culture. Here we report the further enrichment and characterization of a Dehalobacter sp. from this mixed culture in coculture with an Acetobacterium sp. Through a series of serial transfers and dilutions with acetate, H2, and 1,2-DCA, a stable coculture of Acetobacterium and Dehalobacter spp. was obtained, where Dehalobacter grew during dechlorination. The isolated Acetobacterium strain did not dechlorinate 1,2-DCA. Quantitative PCR with specific primers showed that Dehalobacter cells did not grow in the absence of a chlorinated electron acceptor and that the growth yield with 1,2-DCA was 6.9 (±0.7) × 107 16S rRNA gene copies/μmol 1,2-DCA degraded. PCR with degenerate primers targeting reductive dehalogenase genes detected three distinct Dehalobacter/Desulfitobacterium-type sequences in the mixed-parent culture, but only one of these was present in the 1,2-DCA-H2 coculture. Reverse transcriptase PCR revealed the transcription of this dehalogenase gene specifically during the dechlorination of 1,2-DCA. The 1,2-DCA-H2 coculture could dechlorinate 1,2-DCA but not 1,1,2-TCA, nor could it dechlorinate chlorinated ethenes. As a collective, the genus Dehalobacter has been show to dechlorinate many diverse compounds, but individual species seem to each have a narrow substrate range.

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

2003 ◽  
Vol 69 (2) ◽  
pp. 953-959 ◽  
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.

Sign in / Sign up

Export Citation Format

Share Document