Phylogeny of cyclic nitramine-degrading psychrophilic bacteria in marine sediment and their potential role in the natural attenuation of explosives

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Abstract
Previously we reported on in situ mineralization of cyclic nitramine explosives including hexahydro-1,3,5-trinitro-1,3,5-triazine (RDX) and octahydro-1,3,5,7-tetranitro-1,3,5,7-tetrazocine (HMX) in marine sediment from Halifax Harbour. In the present study, we isolated several novel psychrophilic bacteria from the sediment with optimal growth temperature at 10 or 15 °C. Phylogenetic analysis of their 16S rRNA genes identified the isolates as members of the gamma and delta subdivisions of Proteobacteria, Fusobacteria and Clostridiales. The isolates mineralized 3.7–45.2% of RDX (92 μM) in 82 days of incubation at 10 °C under oxygen-limited or anaerobic conditions with the gamma subdivision isolates demonstrating the highest mineralization (45.2% of total C). Removal of RDX by all isolates was accompanied by the formation of all three nitroso derivatives, with the mono nitroso derivative (MNX) being the major one. Isolates of the delta proteobacteria and Fusobacteria removed HMX with concurrent formation of the mononitroso derivative (NO-HMX). Using resting cells of isolates of the gamma subdivision, methylenedinitramine (MEDINA) and 4-nitro-2,4-diazabutanal (NDAB) were detected, suggesting ring-cleavage following denitration of either RDX and/or its initially reduced product, MNX. These results clearly demonstrate that psychrophilic bacteria capable of degrading cyclic nitramines are present in the marine sediment, and might contribute to the in situ biodegradation and natural attenuation of the chemicals.

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