The primary objective of this project was to combine state-of-the-art analytical techniques, molecular approaches, and biogeochemical studies to enhance understanding of the biodegradation of hexahydro-1,3,5-trinitro-1,3,5-triazine (RDX) in groundwater aquifers.

The following research was conducted:

1. Key indicator metabolites of aerobic and anaerobic RDX biodegradation were analyzed in groundwater from multiple contaminated sites using specialized analytical approaches. New preservation methods were developed and evaluated to ensure that the analyte concentrations detected in the laboratory accurately represented those in the site groundwater.
2. Laboratory microcosm and mesocosm studies were conducted to better understand the influence of important geochemical variables on the rates, extents, and pathways of RDX biodegradation.
3. Stable isotope probing (SIP) methods were developed and employed using ¹³C- and ¹⁵N-labeled RDX to better understand the microbial communities involved in RDX biodegradation in groundwater environments.
4. Compound-specific stable isotope analysis (CSIA) methods were developed and tested to quantify fractionation factors for C and N in RDX during biodegradation via aerobic and anaerobic pathways using pure cultures.

A mesocosm study was conducted with samples from Dahlgren, Virginia, to evaluate the degradation of RDX under different electron-accepting conditions and to evaluate the responsible organisms using SIP with ¹³C-RDX and ¹⁵N-RDX. RDX degradation was not observed under NO₃^--reducing conditions (or aerobic conditions in a preliminary study with site samples). RDX degradation with high accumulation (and slow degradation) of nitroso-metabolites was observed with Mn(IV) and Fe(III) as the predominant electron acceptors. Conversely, more rapid RDX degradation was documented in microcosms under both sulfate-reducing and methanogenic conditions, with low and transient accumulation of nitroso metabolites (10% and 40% of the added RDX at a maximum, respectively), and with MNX as the primary metabolite. The data indicate that sequential nitro group reduction was the major RDX degradation pathway under Mn(IV)- and Fe(III)-reducing conditions; whereas, a pathway involving denitration or ring cleavage likely dominated under sulfate-reducing and methanogenic conditions.

Microcosms were prepared from each of the electron acceptor mesocosms and incubated with either ¹³C-labeled RDX or ring-, nitro-, or fully-labeled ¹⁵N-RDX. RDX degradation was monitored in each microcosm, and SIP was conducted when degradation of RDX and intermediates was complete or nearing completion. Using the ¹³C-DNA fractions from the microcosms that had received ¹³C-labeled RDX as templates for cloning and sequencing, identities of active bacteria capable of using RDX and/or RDX intermediates as a carbon source were determined.

The data suggest that, from a remediation perspective, sulfate-reducing or methanogenic conditions are desirable for RDX treatment in order to avoid the long-term accumulation of nitroso-intermediates. The selection of carbon substrate added during biostimulation may be one means of driving the degradative processes in a favorable direction. Use of emulsified vegetable oils has been shown to generate good sulfate-reducing and methanogenic conditions, and also serves as a long-term source of short chain organic acid that may favor the growth of preferred degradative genera. The data also suggest, surprisingly, that some groups of bacteria, such as Desulfosporosinus, can play an important role in RDX degradation under a variety of different dominant electron-accepting conditions. One area for future study is better understanding of the roles of Desulfosporosinus and Pseudomonas (and closely-related species) during in situ RDX degradation.

The SIP analyses conducted during this project reveal that the organisms capable of using carbon and/or nitrogen from RDX or its degradation intermediates under anoxic conditions are much more diverse than previously thought based on studies with pure cultures. Moreover, the studies show that bacteria can degrade the nitramine under a variety of different geochemical conditions and that the organisms involved as well as the dominant pathways utilized may vary depending on those conditions. Interestingly, despite a wealth of research on aerobic RDX biodegradation, the team was unable to stimulate aerobic degradation at the sites evaluated for SIP analysis.

During the final task in this project, gas-chromatography isotope-ratio mass spectrometry (GC-IRMS) methods were developed to quantify stable isotope ratios of both nitrogen (d¹⁵N) and carbon (d¹³C) in RDX. These CSIA methods were subsequently used to assess stable isotope enrichment during aerobic and anaerobic degradation of RDX by eleven different pure cultures that degrade the nitramine through the known aerobic pathway and multiple anaerobic pathways. There were clear distinctions in the stable isotope fractionation which occurred via aerobic and anaerobic RDX degradation pathways.

The results of this project provide new insights into the biodegradation of RDX in groundwater, including the detection of new groups of organisms involved in this process under varying geochemical conditions and quantification of stable isotope fractionation of both carbon and nitrogen during both aerobic and anaerobic degradation. The SIP and CSIA techniques developed are expected to have application at Department of Defense (DoD) sites for documenting in situ RDX biodegradation as a natural attenuation process or during bioremediation efforts. Moreover, the CSIA data for RDX indicate that N stable isotope ratio analysis can be useful for documenting both aerobic and anaerobic biodegradation of RDX in field samples, and potentially for discerning the two processes. In addition, C stable isotope analysis can be useful for documenting anaerobic RDX biodegradation. Finally, a new technique was developed that provides effective preservation for most of the key intermediates from both the aerobic and anaerobic biodegradation of RDX in groundwater. This method can be applied at DoD and other sites to ensure that labile degradation products of RDX are preserved in groundwater samples between collection and laboratory analysis.