Metagenomic analysis of denitrifying wastewater enrichment cultures able to transform the explosive, 3-nitro-1,2,4-triazol-5-one (NTO)

The Department of Defense (DoD) criteria for newer energetics dictates that munition constituents achieve improved energetic performance with reduced sensitivity to external unplanned stimuli. Nitrotriazolone (3-nitro-1,2,4-triazol-5-one) or NTO is a new energetic compound that meets these requirements and was recently fielded as a component of the insensitive munitions IMX-101 and IMX-104. The goal of this study was to assess the biotransformation potential of NTO in a wastewater sludge. Removal of NTO microcosms was investigated in conjunction with heterotrophic and autotrophic denitrifying growth conditions by a microbial consortium from a wastewater treatment plant. Microcosms were supplemented with molasses, methanol, or thiosulfate. Cultures were passaged twice by transferring 10% of the culture volume to fresh media on days 11 and 21. Rates of NTO removal were 18.71, 9.04, and 4.34mg/L/day while rates of nitrate removal were 20.08, 21.58, and 24.84mg/L/day respectively, for molasses, methanol, or thiosulfate. Metagenomic analysis revealed that Proteobacteria and Firmicutes were the major phyla in the microbial communities. In molasses supplemented cultures, the community profile at the family level changed over time with Pseudomonadaceae the most abundant (67.4%) at day 11, Clostridiaceae (65.7%) at day 21, and Sporolactobacillaceae (35.4%) and Clostridiaceae (41.0%) at day 29. Pseudomonadaceae was the dominant family in methanol and thiosulfate supplemented cultures from day 21 to 29 with 76.6% and 81.6% relative abundance, respectively. In conclusion, we demonstrated the removal of NTO under both autotrophic and heterotrophic denitrifying conditions. Metagenomic sequencing showed that Proteobacteria was the dominant phylum while Pseudomonadaceae was the most abundant family.