Role of Acidophilic Methanotrophs in Long Term Natural Attenuation of VOCs in Low pH Aquifers

Dr. Paul Hatzinger | CB&I Federal Services



The main objective of this project was to determine the extent to which methanotrophs may contribute to the biodegradation of chlorinated volatile organic compounds (cVOCs) in low pH groundwater aquifers. The objectives included (1) determining whether methanotrophs exist in acidic groundwater aquifers and are capable of degrading methane and cVOCs, with a specific focus on trichloroethene (TCE); and (2) to identity the key methanotrophs and/or methane-oxidizing genes present in acidic groundwater systems using advanced molecular techniques, including stable isotope probing (SIP). The role of methanotrophs in biodegrading cVOCs in low pH groundwater has received little study as has the potential to enhance their activity with methane or other amendments. 

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Technical Approach

Aquifer samples were obtained from three Department of Defense (DoD) sites (including two different aquifer formations at one site) with low pH groundwater. Microcosms were initially prepared from each site to determine whether the addition of methane and air into the samples stimulated native methanotrophs to biodegrade TCE. Ethane and ethene were also tested as co-metabolic substrates at one of the sites. Degradation rates were determined where applicable, and samples from active microcosms were added to mineral salts media with methane as a sole substrate to enrich and isolate native acidophilic methanotrophs. Enrichments with ethane or ethene were also prepared from some samples. Characterization of the methanotrophic communities in the acidic aquifers was conducted using stable isotope probing (SIP) and probing for pmoA and mmoX genes in samples, which code for the two common enzymes (particulate methane monooxygenase; pMMO and soluble methane monooxygenase; sMMO) used by methanotrophs to convert methane to methanol (and also to oxidize cVOCs). For SIP analysis, 13C-methane (13CH4) was added to active bottles, and organisms that degraded the 13CH4 were identified based upon 13C enrichment in DNA. Finally, a column study was performed using site materials from one location to assess methane and cVOC degradation in a more realistic flow-through system.

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Methane consumption and degradation of cVOCs was observed in acidic aquifer samples collected from all three DoD sites. Interestingly, at one site where samples were collected from two different aquifer formations, methane and cVOC degradation was observed in one formation but not the other, possibly due to the anaerobic conditions in the latter formation where oxygen loss was evident (probably via abiotic oxidation of reduced minerals), but methane was not consumed. Methanotrophic enrichment cultures capable of degrading TCE and several other cVOCs at pH 4 were isolated from two of the three sites. Cultures capable of degrading cVOCs at pH 4 after growth on ethane or ethene also were enriched. Although degradation of only a small subset of chlorinated ethenes and ethanes by the various enrichment cultures was evaluated, the data indicate differences in the cVOCs degraded among the acidophilic enrichment cultures and between some of these cultures and the more widely studied neutrophilic methanotrophs. Further study is warranted to better understand the selectivity of the presumptive sMMO or pMMO enzymes catalyzing reactions under acidic conditions.

Stable isotope probing (SIP) of active microcosms from the two sites tested showed that a variety of methanotrophs incorporated 13C into their DNA (from added 13CH4).  A total of thirty-five 16S rRNA clones were derived and identified. The clones clustered largely in the Alphaproteobacteria and Gammaproteobacteria but were not closely related to known acidophilic methanotrophs. The results were unexpected but interesting, suggesting that many methanotrophs were present in the acidic groundwater and that they were more phylogenetically diverse than previously reported. Future work is needed to isolate these acidophilic methanotrophs to better understand their abundance and role in biodegradation of cVOCs and other contaminants at acidic groundwater sites.

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Overall, this project showed that a diversity of acidophilic methanotrophs were present in low-pH groundwater aquifers, and that these organisms can potentially be important in degradation of TCE and other cVOCs. There is very little research on this group of organisms in low pH groundwater, their potential contribution to natural attenuation of cVOCs, or the possibility of enhancing their activity. In that, reductive dechlorination does not generally occur at low pH, aerobic co-metabolism may be particularly important as a remedial mechanism for cVOCs. The presence of organisms capable of growing at low pH on ethane or ethene and degrading cVOCs was also indicated based on microcosm and enrichment culture data. The potential for these co-metabolic substrates (which are often formed during reductive dechlorination of cVOCs) to stimulate also has received little study to date.

Based on the initial results of this project, there are several areas the researchers believe warrant additional investigation including (1) measuring the activity and degradation kinetics of acidophilic methanotrophs at low methane and cVOC concentrations as may be observed in dilute cVOC plumes that are problematic for DoD; (2) assessing the potential for acidophilic methanotrophs from groundwater to biodegrade cVOCs after growth on substrates other than methane (e.g., small fatty acids, alcohols), as several methanotrophs recently been described that are facultative; (3) further assessing the suite of cVOCs that may be susceptible to degradation by acidophilic methanotrophs, to include chlorinated methanes, ethanes, ethenes, and whether activity is culture-specific; (4) evaluating key factors contributing to differences in cVOC degradation among sites and/or strains (e.g., effect of dissolved metals or other co-contaminants, extent of substrate inhibition, pH optima and extremes); and (5) more clearly identifying the types of MMO enzymes that are active at low pH, determining how they differ from those of neutrophilic bacteria, and developing appropriate primer sets to detect and quantify them via qPCR as a measure of degradative potential in acidic aquifers.

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Points of Contact

Principal Investigator

Dr. Paul Hatzinger

APTIM Federal Services

Program Manager

Environmental Restoration