Abundance and activity of vinyl chloride (VC)-oxidizing bacteria in a dilute groundwater VC plume biostimulated with oxygen and ethene.

Clean-up of vinyl chloride (VC)-contaminated groundwater could be enhanced by stimulating aerobic VC-oxidizing bacterial populations (e.g., methanotrophs) with amendments such as molecular oxygen. In addition, ethene gas injection could further stimulate a different group of aerobic ethene- and VC-oxidizing bacteria called "etheneotrophs." We estimated the abundance and activity of these different VC-oxidizing bacteria in portions of a dilute groundwater VC plume subjected to oxygen and ethene biostimulation. Pyrosequencing of 16S rRNA genes, amplified from community DNA extracted from five groundwater monitoring wells, revealed that Proteobacteria dominated the microbial community. Among the Proteobacteria, methanotroph relative abundance was 6.00 % (well RB52I), 2.81 % (well RB46D), 56.3 % (well RB58I), 23.8 % (well RB63I), and 2.57 % (well RB64I). Reverse transcription qPCR (RT-qPCR) analysis was used to determined methanotroph and etheneotroph functional gene expression from selected monitoring wells. Resulting transcript per gene ratios for methanotroph functional genes (pmoA and mmoX) were 0.013 (RB46D), 0.017 (RB63I), 0.112 (RB64I), and 0.004 (RB46D), 0.239 (RB63I), and 0.199 (RB64I), respectively. Transcript per gene ratios for etheneotroph functional genes (etnC and etnE) were 0.37 (RB46D), 0.81 (RB63I), 5.85 (RB64I), and 0.38 (RB46D), 0.67 (RB63I), and 2.28 (RB64I), respectively. When considered along with geochemical and contaminant data from these wells, our RT-qPCR results suggest that methanotrophs and etheneotrophs were participating in VC cometabolism. We conclude that these molecular diagnostic techniques could be helpful to site managers interested in documenting the effectiveness of VC bioremediation strategies.

Microbial community dynamics during acetate biostimulation of RDX-contaminated groundwater.

Biostimulation of groundwater microbial communities (e.g., with carbon sources) is a common approach to achieving in situ bioremediation of organic pollutants (e.g., explosives). We monitored a field-scale approach to remediate the explosive RDX (hexahydro-1,3,5-trinitro-1,3,5-triazine) in an aquifer near the Iowa Army Ammunition Plant in Middletown, IA. The purpose of the study was to gain insight into the effect of biostimulation on the microbial community. Biostimulation with acetate led to the onset of RDX reduction at the site, which was most apparent in monitoring well MW309. Based on previous laboratory experiments, we hypothesized that RDX degradation and metabolite production would correspond to enrichment of one or more Fe(III)-reducing bacterial species. Community DNA from MW309 was analyzed with 454 pyrosequencing and terminal restriction fragment length polymorphism. Production of RDX metabolites corresponded to a microbial community shift from primarily Fe(III)-reducing Betaproteobacteria to a community dominated by Fe(III)-reducing Deltaproteobacteria (Geobacteraceae in particular) and Bacteroidetes taxa. This data provides a firsthand field-scale microbial ecology context to in situ RDX bioremediation using modern sequencing techniques that will inform future biostimulation applications.

A quantitative PCR assay for aerobic, vinyl chloride- and ethene-assimilating microorganisms in groundwater.

Vinyl chloride (VC) is a known human carcinogen that is primarily formed in groundwater via incomplete anaerobic dechlorination of chloroethenes. Aerobic, ethene-degrading bacteria (etheneotrophs), which are capable of both fortuitous and growth-linked VC oxidation, could be important in natural attenuation of VC plumes that escape anaerobic treatment. In this work, we developed a quantitative, real-time PCR (qPCR) assay for etheneotrophs in groundwater. We designed and tested degenerate qPCR primers for two functional genes involved in aerobic, growth-coupled VC- and ethene-oxidation (etnC and etnE). Primer specificity to these target genes was tested by comparison to nucleotide sequence databases, PCR analysis of template DNA extracted from isolates and environmental samples, and sequencing of qPCR products obtained from VC-contaminated groundwater. The assay was made quantitative by constructing standard curves (threshold cycle vs log gene copy number) with DNA amplified from Mycobacterium strain JS60, an etheneotrophic isolate. Analysis of groundwater samples from three different VC-contaminated sites revealed that etnC abundance ranged from 1.6 × 10(3) - 1.0 × 10(5) copies/L groundwater while etnE abundance ranged from 4.3 × 10(3) - 6.3 × 10(5) copies/L groundwater. Our data suggest this novel environmental measurement method will be useful for supporting VC bioremediation strategies, assisting in site closure, and conducting microbial ecology studies involving etheneotrophs.

Association of missense mutations in epoxyalkane coenzyme M transferase with adaptation of Mycobacterium sp. strain JS623 to growth on vinyl chloride.

Vinyl chloride (VC) is a toxic groundwater pollutant associated with plastic manufacture and chlorinated solvent use. Aerobic bacteria that grow on VC as a carbon and energy source can evolve in the laboratory from bacteria that grow on ethene, but the genetic changes involved are unknown. We investigated VC adaptation in two variants (JS623-E and JS623-T) of the ethene-oxidizing Mycobacterium strain JS623. Missense mutations in the EtnE gene developed at two positions (W243 and R257) in cultures exposed to VC but not in cultures maintained on ethene. Epoxyalkane-coenzyme M transferase (EaCoMT) activities in cell extracts of JS623-E and JS623-T (150 and 645 nmol/min/mg protein, respectively) were higher than that of wild-type JS623 (74 nmol/min/mg protein), and in both variant cultures epoxyethane no longer accumulated during growth on ethene. The heterologous expression of two variant etnE alleles (W243G [etnE1] and R257L [etnE2]) from strain JS623 in Mycobacterium smegmatis showed that they had 42 to 59% higher activities than the wild type. Recombinant JS623 cultures containing mutant EtnE genes cloned in the vector pMV261 adapted to growth on VC more rapidly than the wild-type JS623 strain, with incubation times of 60 days (wild type), 1 day (pMVetnE1), and 35 days (pMVetnE2). The JS623(pMVetnE) culture did not adapt to VC after more than 60 days of incubation. Adaptation to VC in strain JS623 is consistently associated with two particular missense mutations in the etnE gene that lead to higher EaCoMT activity. This is the first report to pinpoint a genetic change associated with the transition from cometabolic to growth-linked VC oxidation in bacteria.

Proteomic analysis of ethene-enriched groundwater microcosms from a vinyl chloride-contaminated site.

Contamination of groundwater with vinyl chloride (VC), a known human carcinogen, is a common environmental problem at plastics manufacturing, dry cleaning, and military sites. At many sites, there is the potential to cleanup VC groundwater plumes with aerobic VC-oxidizing microorganisms (e.g., methanotrophs, etheneotrophs, and VC-assimilating bacteria). Environmental biotechnologies that reveal the presence and activity of VC-oxidizing bacteria in contaminated groundwater samples would provide valuable lines of evidence that bioremediation of VC is occurring at a site. We applied targeted shotgun mass spectrometry-based proteomic methods to ethene-enriched groundwater microcosms from a VC-contaminated site. Polypeptides from the enzymes alkene monooxygenase (EtnC) and epoxyalkane:CoM transferase (EtnE), both of which are expressed by aerobic etheneotrophs and VC-assimilating bacteria, were identified in 7 of the 14 samples analyzed. Bioinformatic analysis revealed that 2 EtnC and 5 EtnE peptides were unique to deduced EtnC and EtnE sequences from two different cultivated strains. In addition, several partial EtnE genes sequenced from microcosms matched with observed EtnE peptides. Our results have revealed broader etheneotroph functional gene diversity and demonstrate the feasibility, speed, and accuracy of applying a targeted metaproteomics approach to identifying protein biomarkers from etheneotrophs in complex environmental samples.

Adaptation of aerobic, ethene-assimilating Mycobacterium strains to vinyl chloride as a growth substrate.

Contamination of drinking water source zones by vinyl chloride (VC), a known human carcinogen and common groundwater contaminant, poses a public health risk. Bioremediation applications involving aerobic, VC-assimilating bacteria could be useful in alleviating environmental VC cancer risk, but their evolution and activity in the environment are poorly understood. In this study, adaptation of ethene-assimilating Mycobacterium strains JS622, JS623, JS624, and JS625 to VC as a growth substrate was investigated to test the hypothesis that VC-assimilating bacteria arise from naturally occurring ethene-assimilating bacteria. VC consumption in the absence of microbial growth was initially observed in cultures grown in both ethene and 1/10-strength trypticase soy agar + 1% (w/v) glucose. After extended incubations (55-476 days), all strains commenced growth-coupled VC consumption patterns. VC-adapted cultures grown on 20 mM acetate subsequently retained their ability to assimilate VC. Three independent purity check methods (streak plates, 16S rRNA gene sequencing, and repetitive extragenic palindromic polymerase chain reaction) verified culture purity prior to and following VC adaptation. Overall, our results suggest that ethene-assimilating mycobacteria have a widespread ability to adapt to VC as a growth substrate.