This project is a two-phase effort to investigate the roll of acidophilic methanotrophs in natural attenuation of chlorinated volatile organic compounds (cVOCs) in low pH aquifers. During the initial proof-of-concept phase, 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 key methanotrophs and/or methane-oxidizing genes present in acidic groundwater systems using advanced molecular techniques, including stable isotope probing (SIP). Overall, this initial effort showed that a diversity of unique acidophilic methanotrophs were present in two low-pH groundwater aquifers that were evaluated, and that these organisms can potentially be important in degradation of TCE and other cVOCs. Phase 2 of this project expands upon the previous studies, with a key objective of further defining the role and importance of acidophilic methanotrophs in biodegrading cVOCs in acidic groundwater aquifers.

There are several areas that warrant further investigation including (1) measuring the activity and degradation kinetics of acidophilic methanotrophs at low methane and cVOC concentrations as may be observed in dilute plumes that are problematic for the 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 from acidic bog environments have recently been described that are facultative; (3) assessing the suite of cVOCs (including chlorinated methanes, ethanes, and ethenes) that may be susceptible to degradation by acidophilic methanotrophs, and whether the degradation activity is culture-specific; (4) evaluating relevant methane and TCE degradation kinetics and key factors contributing to differences in TCE degradation rates (e.g., substrate inhibition, pH); and (5) further evaluating the types of acidophilic methanotrophs and enzymes involved in degradation of cVOCs via current molecular approaches. A range of different experiments with acidic aquifer samples and pure cultures of acidophilic methanotrophs will be conducted to answer these key questions.

Schematic Diagram

Acidophilic methanotrophs may be important in the long term attenuation of chlorinated solvent plumes in low pH groundwater aquifers, such as those that occur along much of the eastern United States and are an impediment to anaerobic bioremediation at numerous DoD sites. However, the occurrence and importance of this newly discovered group of organisms and their potential role in the degradation of cVOCs is largely unstudied. Demonstration, assessment, and quantification of methanotrophic cVOC degradation in acidic aquifer environments could be critical for understanding and optimizing long term natural attenuation of dilute cVOC plumes at a large number of DoD sites. Because anaerobic treatment options (i.e., biostimulation or bioaugmentation for reductive dechlorination) at low pH sites are largely ineffective, an improved understanding of aerobic cometabolic degradation under these conditions is important. Moreover, because aerobic degradation of chlorinated ethenes via cometabolism produces no readily identified daughter products, this process is difficult to assess in situ, and may play a much more important role in natural attenuation than currently thought. (Anticipated Phase II Completion - 2024)

Hwangbo, M., Y. Shao, P.B. Hatzinger, and K-H Chu. 2023. Acidophilic Methanotrophs: Occurrence, Diversity, and Possible Bioremediation Applications. Environmental Microbiology Reports, 1-17. doi.org/10.1111/1758-2229.13156.

Theses and Dissertation

Groome, E. 2022. Application of a 14C-Assay to Assess Methanotrophic Biodegradation of TCE in Low pH Groundwater. (M.S. Thesis). Clemson University, Clemson, South Carolina. tigerprints.clemson.edu/all_theses/3848.

Szwast, N.A. 2021. Evaluation of Methanotrophic Biodegradation of TCE at Low pH. (M.S. Thesis). Clemson University, Clemson, South Carolina. tigerprints.clemson.edu/all_theses/3625.