Relating Carbon and Nitrogen Isotope Effects to Reaction Mechanisms during Aerobic or Anaerobic Degradation of RDX (Hexahydro-1,3,5-Trinitro-1,3,5-Triazine) by Pure Bacterial Cultures

ABSTRACT

Kinetic isotopic fractionation of carbon and nitrogen during RDX (hexahydro-1,3,5-trinitro-1,3,5-triazine) biodegradation was investigated with pure bacterial cultures under aerobic and anaerobic conditions. Relatively large bulk enrichments in ¹⁵ N were observed during biodegradation of RDX via anaerobic ring cleavage (ε ¹⁵ N = −12.7‰ ± 0.8‰) and anaerobic nitro reduction (ε ¹⁵ N = −9.9‰ ± 0.7‰), in comparison to smaller effects during biodegradation via aerobic denitration (ε ¹⁵ N = −2.4‰ ± 0.2‰). ¹³ C enrichment was negligible during aerobic RDX biodegradation (ε ¹³ C = −0.8‰ ± 0.5‰) but larger during anaerobic degradation (ε ¹³ C = −4.0‰ ± 0.8‰), with modest variability among genera. Dual-isotope ε ¹³ C/ε ¹⁵ N analyses indicated that the three biodegradation pathways could be distinguished isotopically from each other and from abiotic degradation mechanisms. Compared to the initial RDX bulk δ ¹⁵ N value of +9‰, δ ¹⁵ N values of the NO ₂ ⁻ released from RDX ranged from −7‰ to +2‰ during aerobic biodegradation and from −42‰ to −24‰ during anaerobic biodegradation. Numerical reaction models indicated that N isotope effects of NO ₂ ⁻ production were much larger than, but systematically related to, the bulk RDX N isotope effects with different bacteria. Apparent intrinsic ε ¹⁵ N-NO ₂ ⁻ values were consistent with an initial denitration pathway in the aerobic experiments and more complex processes of NO ₂ ⁻ formation associated with anaerobic ring cleavage. These results indicate the potential for isotopic analysis of residual RDX for the differentiation of degradation pathways and indicate that further efforts to examine the isotopic composition of potential RDX degradation products (e.g., NO _x ) in the environment are warranted.

IMPORTANCE This work provides the first systematic evaluation of the isotopic fractionation of carbon and nitrogen in the organic explosive RDX during degradation by different pathways. It also provides data on the isotopic effects observed in the nitrite produced during RDX biodegradation. Both of these results could lead to better understanding of the fate of RDX in the environment and help improve monitoring and remediation technologies.