Application of Stable Isotope Probing to Identify RDX-degrading Bacteria in Groundwater

Hexahydro-1,3,5-trinitro-1,3,5-triazine (RDX) is soluble, nonvolatile cyclic nitramine explosive. Long-term manufacturing and various applications of RDX have resulted in RDX contamination in soil and groundwater. RDX is a possible human carcinogen; therefore, occurrence of RDX in groundwater has raised a public health concern. As RDX is biodegradable; bioremediation of RDX-contaminated groundwater has been recognized as a feasible cleanup technology. Several RDX-degrading isolates are known to have ability to utilize RDX as carbon and/or nitrogen source. However, little is known about these isolates and their roles in the natural or engineered systems during RDX degradation, or about RDX-degrading microbial communities in responding to engineered interventions. Stable isotope probing (SIP) is a powerful culture independent method that can identify functional active bacteria in various environmental samples. In this study, we applied SIP with ^(13)C-labeled or one of the ring-, nitro- and fully-labeled ^(15)N-RDX to identify microorganisms capable of utilizing RDX and its metabolites as carbon and/or nitrogen sources in groundwater microcosms, and to associate active RDX-degrading microbial communities in responding to engineered interventions. Derived sequences from ^(13)C-DNA were clustered in Bacteroidia, Clostridia, alpha-, beta- and delta-Proteobacteria, and Spirochaetes, which were different from previously described RDX degraders. Cheese whey amendment stimulated RDX biotransformation, altered the types of RDX-degrading bacteria, and decreased microbial community diversity. Derived sequences from ^(15)N-DNA were grouped in Clostridia, beta-Proteobacteria and Spirochaetes. In comparison to the results among ^(13)C-SIP and ^(15)N-SIP studies with presence of cheese whey, derived sequences were clustered in gamma-Proteobacteria and Bacilli. The combination of these findings suggested that RDX-degrading microorganisms in groundwater are more phylogenetically diverse than what has been inferred from studies with RDX-degrading isolates. RDX biodegradation was observed when amended microcosms with different electron acceptors: Mn(IV), Fe(III), sulfate and CO_(2) (from added succinate). Derived clones from different electron-accepting conditions were identified, which were grouped in alpha-, gamma-Proteobacteria, and Clostridia. A real-time PCR assay targeted catabolic xenB gene was validated and tested with soil and groundwater samples. The presence of xenB gene would indicate that indigenous of microbial population with xenB gene are present, which can be used to estimate the potential of natural attenuation of RDX