Acetylene Fuels TCE Reductive Dechlorination by Defined Dehalococcoides/Pelobacter Consortia

Abstract

Acetylene (C₂H₂) can be generated in contaminated groundwater sites as a consequence of chemical degradation of trichloroethene (TCE) by in situ minerals, and C₂H₂ is known to inhibit bacterial dechlorination. In this study, we show that while high C₂H₂ (1.3 mM) concentrations reversibly inhibit reductive dechlorination of TCE by Dehalococcoides mccartyi isolates as well as enrichment cultures containing D. mccartyi sp., low C₂H₂ (0.4 mM) concentrations do not inhibit growth or metabolism of D. mccartyi. Cocultures of Pelobacter SFB93, a C₂H_2-fermenting bacterium, with D. mccartyi strain 195 or with D. mccartyi strain BAV1 were actively sustained by providing acetylene as the electron donor and carbon source while TCE or cis-DCE served as the electron acceptor. Inhibition by acetylene of reductive dechlorination and methanogenesis in the enrichment culture ANAS was observed, and the inhibition was removed by adding Pelobacter SFB93 into the consortium. Transcriptomic analysis of D. mccartyi strain 195 showed genes encoding for reductive dehalogenases (e.g., tceA) were not affected during the C₂H₂-inhibition, while genes encoding for ATP synthase, biosynthesis, and Hym hydrogenase were downregulated during C₂H₂ inhibition, consistent with the physiological observation of lower cell yields and reduced dechlorination rates in strain 195. These results will help facilitate the optimization of TCE-bioremediation at contaminated sites containing both TCE and C₂H₂.

Graphical Abstract

INTRODUCTION

Groundwater contamination by trichloroethene (TCE) poses a serious threat to human health and can lead to the generation of vinyl chloride (VC), a known human carcinogen¹ via abiotic and biotic reduction processes.^2,3 Dehalococcoides mccartyi is the only known species of bacteria that can completely dechlorinate TCE to the benign product ethene (C₂H₄).⁴ D. mccartyi is a strict hydrogenotroph characterized by specific requirements for an exogenous supply of key compounds, including hydrogen (H₂), acetate, corrinoids, biotin, and thiamine, of all which are usually provided by other microbial members of anaerobic food webs within dechlorinating communities.^5–8 D. mccartyi has also been studied with respect to its genetic regulation, physiological characteristics and metabolic interactions with other microbial species.^5,9–12 D. mccartyi strain 195 (strain 195) can use TCE and cis-dichloroethene (cis-DCE) as terminal electron acceptors for the oxidation of H₂ to form VC, but VC is further reduced to ethene cometabolically by strain 195, and is not linked to energy generation.⁵ In contrast, D. mccartyi strain BAV1 can conserve energy by metabolically dechlorinating both cis-DCE and trans-DCE to VC, and VC to ethene.¹⁰

Acetylene (C₂H₂) can be generated at TCE-contaminated sites as a consequence of the abiotic degradation of TCE by zerovalent iron or by in situ reaction with other minerals such as iron sulfide (FeS)- or Co (II)-containing sulfides.^13–15 Acetylene inhibits a variety of microbial processes including fermentation,¹⁶ methanogenesis,¹⁷ methane oxidation,¹⁸ nitrogen fixation (as a competitive inhibitor of nitrogenase¹⁹), and both nitrification and denitrification²⁰ by interaction with the metal components of enzymes such as hydrogenases, oxidoreductases, and monooxygenases.^21,22 Therefore, acetylene has commonly been used in experiments measuring nitrogen fixation (acetylene reduction assay) and denitrification (acetylene blockage of N₂O reductase assay).²³ Acetylene has also been shown to inhibit biotic reductive dechlorination of TCE to DCE and VC in laboratory experiments, and thus can cause TCE accumulation at groundwater remediation sites.²⁴ The levels of acetylene inhibition on microbial processes are strongly dependent on its concentration.²⁴

However, acetylene can be used as a substrate by anaerobic bacteria in estuarine sediments and enrichment cultures.^25,26 For example, Pelobacter acetylenicus can metabolize acetylene via acetylene hydratase and acetaldehyde dismutatse enzymes generating H₂, ethanol, and acetate as end products.^27–30 H₂ and acetate formed from acetylene fermentation could therefore potentially serve as the electron donor and carbon source to fuel reductive dechlorination by D. mccartyi. Desulfovibrio vulgaris Hildenborough and Syntrophomonas wolfei have been shown to serve as successful syntrophic partners with D. mccartyi to support its reductive dechlorination as well as its growth via interspecies H₂ transfer.^8,31

In this study, we hypothesized that the inhibitory effect of acetylene on reductive dechlorination could be removed by adding a Pelobacter strain to D. mccartyi-containing cultures performing TCE-respiration. Moreover, we expected that the fermentation products of acetylene metabolism would provide D. mccartyi with electron donors needed to carry out reductive dechlorination. The proposed ecological interactions between D. mccartyi strains and Pelobacter strain SFB93 are illustrated in Figure 1. In order to test this hypothesis, we systematically evaluated (i) the inhibition effect of TCE on Pelobacter strain SFB93; (ii) acetylene inhibition on two D. mccartyi strains and a dechlorinating enrichment; and (iii) cell growth in cocultures of Pelobacter strain SFB93 with D. mccartyi strain 195 or BAV1 using acetylene as electron donor with TCE or VC as electron acceptor.

Figure 1.

Ecological interactions between A) D. mccartyi strain 195 and Pelobacter SFB93; B) D. mccartyi strain BAV1 and Pelobacter SFB93. Solid lines indicate the substrate is used for energy production. Dotted lines indicate the substrate is used for biomass production.

MATERIALS AND METHODS

Bacterial Cultures and Growth Conditions.

D. mccartyi strain 195 (strain 195) was grown in defined medium with H₂/ CO₂ headspace (80:20), 0.6 mM TCE as electron acceptor, and 2 mM acetate as carbon source.⁶ D. mccartyi strain BAV1 (BAV1) was grown in the same defined medium with H₂/CO₂ headspace, 1.2 mM cis-DCE as electron acceptor, and 2 mM acetate as carbon source. An isolate from San Francisco Bay, Pelobacter strain SFB93 (SFB93), was grown in modified artificial bay water medium (ABW) with N₂ headspace and acetylene (0.1–1.5%) as electron donor and the carbon source.³⁰ Bacterial cocultures of strains 195 and SFB93 (10% vol/vol inoculation of each bacterium) were initially established in 60 mL serum bottles containing 25 mL ABW medium with TCE supplied at a liquid concentration of 0.1 mM (corresponding to 3 μmol TCE per bottle), 0.1 mM acetylene (corresponding to 0.15 mL gas), and N₂ headspace incubated at 34 °C without agitation. The cocultures were subsequently transferred (5% vol/vol inoculation) to generate subcultures after 20 μmol TCE was reduced to VC and 36 μmol acetylene was depleted. After three subcultures, we measured reductive dechlorination performance and cell growth of the coculture with multiple feedings of TCE and acetylene. In order to establish the coculture of BAV1 and SFB93, two consecutive additions of acetylene (88.1 μmol in total) were first amended to SFB93 in 20 mL carbonate media³² with N₂ headspace. After acetylene was consumed, BAV1 (2.5 mL 10% inoculation) and 13.5 ± 1.7 μmol VC (0.4 mM) were inoculated to develop the coculture with subsequent amendment of 22.8 μmol acetylene (0.8 mM) and incubation at 28 °C without agitation.

The dechlorinating enrichment culture used in this study (ANAS) has been maintained in our laboratory for over 15 years in a continuously stirred semibatch fed reactor amended with 25 mM lactate and 0.1 mM TCE as previously described.^33,34 Two distinct D. mccartyi strains have been isolated from ANAS: (1) strain ANAS1 which contains the TCE reductive dehalogenase gene tceA, and (2) strain ANAS2 which contains the VC reductive dehalogenase gene vcrA.³⁵ The community structure and dechlorination performance of ANAS has been previously described.^33,34,36

Chemical Analysis.

Chloroethenes, ethene, and acetylene were measured by FID-gas chromatography using 100 μL headspace samples, and hydrogen was measured by RGD-gas chromatography using 300 μL headspace samples.^33,37 The mass of each compound was calculated based on gas–liquid equilibrium by using Henry’s law constants at 34 and 28 °C according to mass (μmol/bottle) = C_l×V_l+C_g×V_g

DNA Extraction and Cell Number Quantification.

Liquid samples (1.5 mL) were collected for cell density measurements and cells were harvested by centrifugation (21 000g, 10 min at 4 °C). Genomic DNA was extracted from cell pellets using Qiagen DNeasy Blood and Tissue Kit according to the manufacturer’s instructions for Gram-positive bacteria. qPCR using SYBR Green-based detection reagents was applied to quantify gene copy numbers of strain 195 with D. mccartyi tceA gene primers (forward primer 5′-ATCCAGATTATGACCCTGGTGAA-3′ and reverse primer 5′-GCG-GCATATATTAGGGCATCTT-3′), as previously described.³⁸

RNA Preparation and Transcriptome Analysis.

For the transcriptomic data, 5 mL acetylene (corresponding to 1.3 mM in liquid phase) were added to strain 195 cultures on day 5 during mid log growth phase when 50% of TCE was degraded. Cultures were sampled on day 7 when control bottles without acetylene exhibited late exponential growth (around 75% of 78 μmol TCE was dechlorinated, i.e., ~20 μmol TCE remained) and no further TCE degradation was evident in experimental bottles. In order to collect sufficient material for transcriptomic microarray analysis, 60 bottles of acetylene-inhibited strain 195 cultures and 18 control bottles (strain 195, without acetylene) were inoculated and grown from triplicate bottles (160 mL serum bottles, with H₂/CO₂ headspace) of the isolate. For each sampling, triplicate subsamples of cells from 20 bottles were collected by vacuum filtration on day 6 from the experimental and control groups [300 mL culture per filter, 0.2-μm autoclaved GVWP filter (Durapore membrane, Millipore, Billerica, MA)]. Each filter was placed in a 2 mL orange-cap microcentrifuge tube, frozen with liquid nitrogen and stored at −80 °C until further processing. RNA extraction and preparation were described previously.⁸

Transcriptomic Microarray Analysis.

Our use of the Affymetrix GeneChip microarray has been reported previously.³⁵ Briefly, the chip contains 4,744 probe sets that represent more than 98% of the ORFs from four published Dehalococcoides genomes (strain 195, VS, BAV1, and CBDB1). cDNA was synthesized from 9 μg RNA, then each cDNA sample was fragmented, labeled, and hybridized to each array. All procedures were performed with minimal modifications to the protocols in section 3 of the Affymetrix GeneChip Expression Analysis Technical Manual (Affymetrix, Santa Clara, CA http://www.affymetrix.com). Microarray data analysis methods were previously described.^31,34

RESULTS

Inhibitory Effects of Acetylene and TCE on Isolates.

We evaluated the effects of TCE on growth of Pelobacter strain SFB93 and observed no inhibition of growth at any of the tested concentrations (0.1–0.5 mM). Unsurprisingly, there was no evidence of TCE consumption by strain SFB93 during these incubations (data not shown). No inhibition of strain 195 dechlorination was observed by C₂H₂ concentrations <0.4 mM while partial inhibition (58%) occurred at 0.8 mM acetylene, and complete inhibition at 1.3 mM (Figure S1 of the Supporting Information, SI). In order to determine whether the inhibition of TCE dechlorination was reversible, actively dechlorinating strain 195 was amended with 1.3 mM acetylene. After 4 days, a subset of these cultures were amended with Pelobacter strain SFB93 cells. Acetylene was consumed within 3 days in the SFB93-amended samples (data not shown), and TCE reduction resumed at a rate of 63% of the initial no-acetylene control with all TCE reduced by the end of the experiment (Figure 2). In contrast, in the culture without Pelobacter amendment no additional TCE was removed, demonstrating that acetylene inhibition of TCE dechlorination by strain 195 is reversible.

Figure 2.

C₂H₂ inhibition on TCE reduction by D. mccartyi strain 195. Symbols represent the mean of biological triplicates, and error bars are standard deviations. Absence of bars indicates error was smaller than the symbol.

Acetylene Fuels Dechlorination by D. mccartyi Strains.

An anaerobic coculture consisting of strain 195 and strain SFB93 was established with acetylene serving as the electron donor and carbon source (Figure 3). While strain 195 metabolically reduces TCE to VC using H₂ as electron donor, strain SFB93 ferments acetylene to ethanol, acetate, and H₂.^26,30 In dechlorinating communities, ethanol is rapidly oxidized to acetate and H₂, thereby providing additional electron donors and carbon sources to dechlorinators, sulfatereducers, and methanogens. Complete fermentation of 1 mol acetylene can generate 1 mol hydrogen, which then can serve as electron donor to reduce 0.5 mol TCE to VC. In the SFB93/195 coculture, 26.2 μmol of TCE was reduced and 25.0 ± 2.4 μmol VC accumulated as the major reduction product, with trivial amounts of cis-DCE and ethene (Figure 3). Acetylene was readily consumed within 2 days of each amendment. Aqueous H₂ concentrations ranged from 114 to 217 nM during TCE-dechlorination, well above the H₂ threshold for reductive dechlorination.³⁹ By the end of the experiment, strain 195 cell concentrations reached 3.3 ± 0.1 × 10⁷ cell mL⁻¹, with a cell yield of 3.7 ± 0.3 × 10⁷ cells μmol⁻¹ Cl⁻ released, demonstrating that acetylene fermenting strain SFB93 can provide the H₂ and acetate required to support the growth of dechlorinating strain 195.

Figure 3.

Coculture D. mccartyi strain 195 grown with Pelobacter SFB93. A) TCE reduction and VC production; B) C₂H₂ and H₂ concentrations; and C) cell numbers of strain 195. Arrows indicate TCE A) and C₂H₂ B) amendments to the coculture. Symbols represent the mean of biological triplicates, and error bars are standard deviations. Absence of bars indicate error was smaller than the symbol.

We also explored the coculture growth of D. mccartyi strain BAV1 with strain SFB93 on VC and acetylene. Unlike strain 195, BAV1 gains energy from dechlorination of VC to ethene.¹⁰ However, similar to strain 195, BAV1 does not transform acetylene and cannot use it as an electron donor for reductive dechlorination (Figure 4 A). When BAV1 was cocultured with SFB93, acetylene was consumed within 1 day (data not shown), and VC was completely reduced to ethene with a reduction rate of 0.9 ± 0.1 μmol day⁻¹ (Figure 4B).

Figure 4.

VC reduction by A) D. mccartyi strain BAV1 with C₂H₂ as the sole electron donor and N₂ headspace; B) coculture of D. mccartyi strain BAV1 and Pelobacter strain SFB93 with C₂H₂ as sole electron donor and N₂ headspace. Symbols represent the mean of biological triplicates, and error bars are standard deviations. Absence of bars indicates that the error was smaller than the symbol.

Effect of Acetylene on D. mccartyi-Containing Enrichment Cultures.

A methanogenic reductive-dechlorinating enrichment culture (ANAS) was selected to examine the inhibition of acetylene on the culture’s capacity for reductive dechlorination. During these experiments, ANAS was reducing TCE primarily to VC rather than to ethene. When ANAS was amended with acetylene in the absence of any other electron donor (e.g., lactate), only a small amount of TCE dechlorination (34%) to DCE was observed (Figure 5A) with no consumption of acetylene (Figure 5B). In contrast, when SFB93 was added to the ANAS culture, acetylene was completely consumed and had to be reamended twice to provide sufficient electron donors for reduction of TCE to VC and ethene (Figure 5C,D). No methane production occurred in the absence of strain SFB93 (Figure 5A), although significant methanogenesis occurred in its presence (Figure 5C). Acetylene is a known inhibitor of methanogenesis,¹⁷ but this inhibition can be reversed by the presence of active populations of acetylene-fermenting bacteria, as was recently demonstrated with anaerobic freshwater lake sediments.³⁰

Figure 5.

Reductive dechlorinating enrichment culture ANAS with 0.3 mM C₂H₂ as sole electron donor and carbon source. A) and B) ANAS without Pelobacter amendment; C) and D) ANAS with Pelobacter strain SFB 93 amendment. Arrows indicate C₂H₂ reamendment. Symbols represent the mean of biological triplicates, and error bars are standard deviations. Absence of bars indicates error was smaller than the symbol.

We also tested the acetylene inhibition effects on the ANAS enrichment when lactate was supplied as external electron donor. This was done with both low (0.3 mM) and high (1.0 mM) acetylene amendments. In the 0.3 mM acetylene amendment, no inhibition of TCE reduction or methane production was observed (Figure S2), while with 1.0 mM acetylene, the TCE reduction rate decreased by 57%. In both cases, acetylene was not consumed throughout the experiment (Figure S3A,B). However, when strain SFB93 was amended to ANAS, complete reductive dechlorination was observed and acetylene was consumed and required replenishment (Figure S3C,D), but no methane production was observed confirming the inhibition of acetylene to methanogenesis. The results demonstrate acetylene was efficient at blocking methanogenesis in lactate-fed cultures of ANAS when cocultured with SFB93, but not in cultures that were solely fed acetylene (Figure 5).

Transcriptomic Analysis of Acetylene Inhibition on D. mccartyi Strain 195.

The effects of acetylene on D. mccartyi strain 195 gene expression were analyzed to better understand the mechanism(s) of its inhibition. Transcriptomic analysis showed that 298 genes were significantly (i.e., ≥2-fold change) down regulated, while 173 genes were significantly up-regulated in the presence of acetylene (Figure 6). The short-term exposure to acetylene did not change the expression pattern of most genes encoding for dehalogenases (Table S1), with the exception of DET1545 whose expression under both conditions was quite low (<700). However, down-regulated genes include ATP synthase (DET0558–0565), protontranslocating NADH-quinone oxidoreductase (DET0928–0933), Hym [Fe]-hydrogenase (DET0145–0148), ribosome proteins (DET0473–0493), and genes related to biosynthesis (Table S1), suggesting an interruption of the electron transport chain. The transcriptomic results agree with the lower cell yields observed in experimental bottles (0.8 ± 0.2 × 10⁸ cells/mL) compared to controls with no acetylene exposure (1.6 ± 0.3 × 10⁸ cells/mL) (two-tailed student’s t test, P = 0.03). Upregulated gene expressions were observed in a subset of the genes encoding for nitrogen regulation and transport (DET 1124–1125, DET1154–1158), ferrous iron transport protein (DET0095–0097, DET1503–1505), phosphate ABC transporters (DET 0138–0142), and a membrane-bounded molybdopterin oxidoreductase (DET0102–0103).

Figure 6.

Microarray signal intensities of transcripts from strain 195 (x-axis) versus C₂H₂ amended strain 195 (y-axis). Colored points represent statistically significant differential transcription: average intensity> 200, p < 0.05, more than 2-fold difference, genes significantly up-regulated (red, +) or down-regulated (green, −) in C₂H₂ amended strain 195 versus strain 195. All measurements are averages from three biological replicates.

DISCUSSION

In this study, we found that acetylene can inhibit reductive dechlorination in D. mccartyi-containing cultures, but only when applied at relatively high concentrations (>0.4 mM). Moreover, that inhibition was eliminated when acetylene-consuming Pelobacter strain SFB93 was present (Table 1). One proposed mechanism of acetylene inhibition on reductive dechlorination is that acetylene directly inhibits reductive dehalogenase required for TCE and VC reduction. However, no differential gene expressions of reductive dehalogenase tceA were observed in acetylene inhibited cells compared to the control bottles. This unchanged transcriptomic profile of reductive dehalogenase was also observed during sulfide inhibition experiments with strain 195 (unpublished data), and during comparisons of strain 195 while in exponential and stationary phase growth.¹² This finding suggests the transcriptional control and cell growth become decoupled when cells are exposed to the inhibitors, making it problematic to use reductive dehalogenase (RDase) expression levels as accurate biomarkers to indicate the physiological activity of D. mccartyi under field conditions.

Table 1.

C₂H₂ Inhibition Effects on D. mccartyi-Containing Cultures

cultures	e−donor	e− acceptor conc. (mM)	C2H2 aqueous conc. (mM)	Pelobacter strain SFB 93	C2H2 inhibition on RDa
strain 195	H2	0.3 (TCE)	0–1.3	no	yes, >0.8 mM
strain 195	H2	0.3 (TCE)	1.3	yes	no
strain 195/SFB 93		0.25 (TCE)	0.1 (×3 doses)	yes	no
strain BAV1/SFB 93		0.3 (VC)	0.4	yes	no
ANAS		0.14 (TCE)	0.3	no	yes
ANAS		0.14 (TCE)	0.3	yes	no
ANAS	lactate	0.14 (TCE)	1.0	no	yes
ANAS	lactate	0.14 (TCE)	1.0	yes	no

^aRD: reductive dechlorination.

Acetylene inhibition of hydrogenase activity has been suggested to be responsible for the inhibited growth of many hydrogenase containing microorganisms, such as cyanobacteria and nitrogen fixing bacteria. There are other biological functions of hydrogenases. For example, bidirectional hydrogenases can control excess reducing equivalents, especially in photosynthetic microorganisms (e.g., cyanobacteria). Hydrogenases may also be involved in membrane-linked energy conservation through the generation of a transmembrane proton motive force in nitrogen-fixing bacteria.⁴⁰ For example, acetylene is known to inhibit Fe/Ni-containing hydrogenases²¹ and the same down-regulation pattern of [Fe]-hydrogenase (Hym) was observed in this study. In fact, genes involved in energy conversion by the electron transport chain (e.g., ATP synthase, proton-translocating NADH-quinone oxidoreductase) were also significantly down-regulated in acetylene inhibited strain 195, which agrees with the physiological observations of lower cell yields and reduced dechlorination rates of strain 195 during acetylene inhibition. Acetylene inhibited TCE dechlorination by D. mccartyi strain 195 and the ANAS enrichment culture at levels above 0.3 mM (Table 1), at concentrations similar to that previously reported (0.2 mM) for inhibiting a VC-dechlorinating consortium.²⁴

Phylogenetic analysis of 16S rRNA genes showed that Pelobacter strain SFB93 has 96% similarity to Pelobacter acetylenicus, and 97% sequence similarity to strain P. carbolinicus, which are able to grow by fermentation with acetate and hydrogen as end products.^30,41,42 Strain SFB93 also contains acetylene hydratase (AH), the enzyme capable of metabolizing acetylene.^30,43 In the coculture studies, we demonstrated that strain SFB93 can ferment acetylene to hydrogen to support TCE reduction to VC by strain 195 and VC reduction to nontoxic end-product ethene by strain BAV1 as well as acetate to support cell growth. Specifically, the dechlorination rate (0.6 μmol TCE·d⁻¹) of the cocultured 195/ SFB93 was similar to cocultured strain 195 with Desulfovibrio desulfuricans (0.4 μmol d⁻¹; He et al. 2007), and comparable to other sustainable growing D. mccartyi- containing syntrophic cocultures (2.1–11 μmol d⁻¹).^8,31 The cell yield of strain 195 (3.7 ± 0.3 × 10⁷ cells μmol⁻¹ Cl⁻ released) was comparable to previous D. mccartyi-containing coculture studies: 3.3 × 10⁷ to 4.2 × 10⁸ cells μmol⁻¹ Cl⁻ released.⁸ During our coculture experiments, H₂ concentrations remained at levels between 100 to 210 nM (Figure 3B) indicating that the H₂ generation rate was at about the same level as its consumption rate, and that the growth of the two species was strictly coupled via interspecies hydrogen transfer.

No reductive dechlorination, methanogenesis, or hydrogen production was observed in ANAS when acetylene was present as the only potential electron donor. This was likely due to the absence of Pelobacter in the microbial enrichment.^36,37 By introducing strain SFB93, acetylene promoted the complete reduction of TCE to VC and methane was produced, demonstrating that the fermentation of acetylene by Pelobacter strain SFB93 to hydrogen and acetate supports the reductive dechlorination and other processes in the enrichment (e.g., methanogenesis).

Previous studies showed that acetylene can inhibit methanogens at relatively low concentrations of 42–48 μM.^24,44 In our study, we found methanogenesis was inhibited at 300 μM acetylene, but that inhibition was also entirely eliminated in the presence of strain SFB93 (Figure 5C, Figure S3), demonstrating that acetylene inhibition on methanogenesis can also be removed by Pelobacter fermentation. However, at higher acetylene concentrations (~1 mM), methanogenesis was inhibited even with Pelobacter present (Figure S3C), indicating that the methanogens in ANAS are more sensitive to acetylene inhibition than D. mccartyi strains.

While acetylene is known to inhibit biological reductive dehalogenation of TCE, we have now demonstrated that through the intersession of acetylene-fermenting bacteria this inhibition can be eliminated. Moreover, under these conditions acetylene can also serve as a carbon source and hydrogen donor to fuel microbial dehalogenation of TCE and its daughter products to yield a desired, innocuous end-product (ethene). We suggest that a future focus upon acetylene and the bacteria that ferment it may help further our understanding of the present constraints encountered in bioremediation efforts of subsurface environments contaminated with compounds like TCE that lead to a buildup of more toxic VC. Such investigations could lead to practical solution to this widespread and vexing environmental contamination problem.