Characterization of Microbes Capable of Using Vinyl Chloride as a Sole Carbon and Energy Source by Anaerobic Oxidation

ER-1556

Objective

Daughter products from reductive dechlorination of polychlorinated ethenes (including ethene and ethane) are often observed in situ, but the amounts detected are rarely sufficient to explain the disappearance of the chloroethenes, suggesting ethene also may be degraded, or that there may be alternative pathways for chloroethene degradation. For example, vinyl chloride (VC) may undergo direct oxidation to carbon dioxide (CO2) or incomplete oxidation to acetate, which is further metabolized to carbon dioxide and methane; of which none of these products are discernible from other sources. Alternatively, it is possible that VC and ethene are consumed under microaerobic conditions, as a consequence of oxygen diffusing into groundwater at levels too low to detect and consumed at rates too high to accumulate. A number of microcosm studies have provided evidence in support of anaerobic oxidation of VC and ethene. However, it remains unknown what microbes might be performing these activities.

The overall objective of this research project was to culture and ultimately isolate and characterize microbes that are capable of using VC as a sole source of carbon and energy by anaerobic oxidation. The project scope was later expanded to include an evaluation of microbes capable of using ethene as a sole source of carbon and energy by anaerobic oxidation.

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Technical Approach

More than 700 microcosms were prepared with soil and groundwater samples from 11 sites to evaluate the occurrence of anaerobic oxidation of VC and ethene. Isolates from these samples were developed and characterized. Some isolates were subjected to microbiological characterization, including documenting morphology, cultural characteristics, 16S ribosomal ribonucleic acid (rRNA) gene sequence, and through survey of electron donors and acceptors utilized for growth.

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Results

The most important outcome from this effort was the development of a sulfate reducing enrichment culture that grows with ethene as its sole carbon and energy source. Ethene has previously been considered recalcitrant under anaerobic conditions. The evidence supporting ethene oxidation to CO2 from this project includes the disappearance of considerable amounts of ethene without the detection of volatile organic products, such as methane or ethane, by gas chromatography, or of water soluble products like acetate by high performance liquid chromatography (HPLC). Moreover, nearly all of the [14C]ethene added to the enrichment culture was found in the 14CO2 fraction after incubation with the culture; whereas, only small amounts of 14CO2 were detected in abiotic controls with most of the radioactivity remaining as ethene. That sulfate was the electron acceptor was demonstrated by the near stoichiometric accumulation of sulfide in the samples amended with ethene, but not in controls lacking ethene. The production of sulfide and lack of known aerobic ethene oxidizers as major components of the 16S rRNA gene clone library argue against the possibility that ethene oxidation was due to small amounts of oxygen entering the microcosms and cultures.  

The sulfate reducing enrichments were incubated in medium in which, other than trace amounts of vitamins, ethene served as the sole electron donor and organic carbon source, and sulfate was the only added electron acceptor other than CO2. These cultures were transferred multiple times, a condition that requires growth of the biocatalyst. Ethene utilization rates increased over time in the cultures, also consistent with microbial growth. Moreover, microbes were readily apparent in cultures that had consumed ethene, and bacterial numbers, as estimated by quantitative PCR (qPCR), increased greatly relative to cultures lacking ethene or sulfate. Thus, it is clear that the ethene/sulfate couple supported growth of microorganisms in these cultures.

The most numerous phylotype found in the 16S rRNA gene clone library, called MT6, is a member of the Deltaproteobacteria, most closely related to Desulfovirga adipica and several Syntrophobacter spp., organisms that carry out reactions with relatively low thermodynamic yields, and is somewhat more distantly related to Desulfoglaebaalkanexedens, a hydrocarbon utilizer. The MT6 16S rRNA gene is ≤91% identical with those from cultured organisms, and had ≤93% identity with the entire NCBI nr database, demonstrating how unique this phylotype is. While this distance precludes physiological conclusions based on phylotype, it is a reasonable candidate for an organism responsible for ethene oxidation and sulfate reduction.

In light of these and previous results, the possibility of ethene oxidation to CO2 in sulfate reducing zones should be considered. If ethene is reduced to ethane in situ, the ethane can be readily detected by gas chromatography and contribute to mass balance determinations. However, if ethene is oxidized, it joins the large CO2 pool and cannot be accounted for in the mass balance, leading to a mass balance deficit. Presently, there is no way to predict whether this reaction is occurring other than microcosm studies. It is possible that anaerobic ethene oxidation will have a stable isotope fractionation signature as does reduction to ethane. It is also possible that the unique bacterial phylotype associated with this reaction can serve as a biomarker for it, but considerably more study will be needed to support or refute this hypothesis.

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Benefits

Definitive evidence in support of anaerobic bio-oxidation of VC was not found during this project. In a majority of the several hundred microcosms that were monitored (usually for one year or longer), VC was either recalcitrant or underwent reductive dechlorination to ethene. Consumption of VC without accumulation of ethene or ethane did occur in microcosms from one location at one of the sites. However, attempts to link the biodegradation of VC to a specific electron acceptor were unsuccessful, as were attempts to enrich for VC biodegradation by transfers to groundwater or medium. Furthermore, the possibility of oxygen serving as the electron acceptor (via diffusion through the septa) could not be completely ruled out. Consequently, microbes capable of growing anaerobically on VC as the sole source of carbon and energy have yet to be identified.

Several years prior to the start of this project, microcosms from an industrial site exhibited activity consistent with anaerobic bio-oxidation of VC. After consuming several repeat additions of VC under presumptively anaerobic conditions, the rate of VC consumption slowed considerably and the microcosms were set aside. When this project started (six years after the original microcosms were established), an attempt was made to revive the VC biodegradation activity. All attempts to transfer material into defined medium or stimulation of anaerobic VC oxidation activity in these samples failed. The only activity recovered from the groundwater or original microcosms by culturing was the aerobic oxidization of VC. An aerobic VC oxidizer was subsequently isolated and identified as being related to previously described VC oxidizing Mycobacterium spp. This phylotype was also the most numerous in a bacterial 16S rRNA gene clone library, representing 38% of the total clones. The isolation of an aerobic Mycobacterium sp. strain from presumably anaerobic groundwater highlights the fact that groundwater contamination plumes are dynamic, and understandings of the biogeochemical cycles within are not fully understood. Studies that track VC in shallow aquifers show VC mineralization under hypoxic conditions, though this is the first study to identify an organism that may be responsible for these in situ observations. Further studies must be performed to determine whether these strains of Mycobacterium spp. play an important role in in situ VC mineralization.

Despite microcosm evidence that anaerobic VC oxidation occurs within contaminated sites, the organisms involved have so far evaded enrichment and identification in the laboratory. At some of these sites, it is possible that oxygen is below the level of detection but high enough to allow for aerobic VC oxidation. Compound-specific isotope analysis can be used to reduce uncertainty about the fate of biotransformed ethenes, but cannot fully explain the observed VC loss at contaminated sites. To accurately assess the flux of VC at sites where VC is observed to be decreasing without concomitant ethene formation, detection of known VC-oxidizing phylotypes like Mycobacterium spp. within a plume in comparison to non-contaminated groundwater could be indicative of aerobic oxidation of VC at levels of oxygen below detection, as would the ability to enrich aerobic VC oxidizers as demonstrated in this study.

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Points of Contact

Principal Investigator

Dr. David Freedman

Clemson University

Phone: 864-656-5566

Fax: 864-656-0672

Program Manager

Environmental Restoration

SERDP and ESTCP

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