Identification of Hexahydro-1,3,5-trinitro-1,3,5-triazine-Degrading Microorganisms via ¹⁵N-Stable Isotope Probing

This study reported the application of 15N-stable isotope probing (SIP) to identify active hexahydro-1,3,5-trinitro-1,3,5-triazine (RDX)-utilizing microorganisms in groundwater microcosms. Fifteen 16S rRNA gene sequences were derived from the 15N-DNA fraction (contributed from active microorganisms capable of using RDX as a nitrogen source) of microcosms receiving cheese whey. The 16S rRNA gene sequences belonged to Actinobacteria (two clones), alpha-Proteobacteria (seven clones), and gamma-Proteobacteria (six clones). Except for five sequences with high similarity to two known RDX degraders (Enterobacter cloacae and Pseudomonas fluorescens I-C), our results suggested that phylogenetically diverse microorganisms were capable of using RDX as a nitrogen source. Six sequences of the xplA gene (a known RDX-degrading catabolic gene) were detected from the 15N-DNA fraction. The xplA gene sequences were 96-99% similar to the xplA gene of Rhodococcus sp. DN22(a known RDX utilizer), suggesting that other RDX utilizers might contain xplA-like genes. Twenty-five 16S rRNA gene sequences recovered from the unenriched, RDX-contaminated groundwater clustered differently from those obtained from the 15N-DNA fraction of the cheese-whey-amended microcosm. Our results suggested that active RDX utilizers can be stimulated by nutrient source additions even if they are present at low densities, and that use of 15N-SIP can identifythese functional members of the microbial community.