METAGENOME ANALYSIS OF AN ENRICHMENT CULTURE THAT DEGRADES THE 3-NITRO-1,2,4-TRIAZOL-5-ONE (NTO) EXPLOSIVE

3-Nitro-1,2,4-triazol-5-one (NTO) is an essential component of newly introduced insensitive high explosive (IHE) compounds as it has several advantages for this purpose namely, low shock sensitivity and easy synthesis. With increasing use by the military, it is important to understand the fate of NTO in the environment and its biodegradation process. Previous studies have shown that NTO is firstly reduced to 3-amino-1,2,4-triazol-5-one (ATO) under anaerobic conditions, and subsequently, ATO is fully mineralized to NH4+, CO2, and N2 by a microbial consortium aerobically. To provide new insights into the degradation processes, we perform shotgun metagenome sequencing of one NTO degrading enrichment culture and three ATO degrading enrichment cultures; the ATO cultures were derived from one another using dilution to extinction in order to reduce the diversity of the consortium. Two metagenome-assembled genomes (MAGs) were recovered from the NTO metagenome that made up >95% of the total culture and carried putative nitroreductases that might be involved in NTO reduction. In addition, the presence of extracellular electron transfer systems in the most abundant MAG (a Geobacter sp., making up 89.3% of the total) suggested that these systems might be also involved in NTO reduction. The ATO metagenomes revealed seven MAGs that were consistently observed and -more or less- evenly distributed in the three consortia. Functional annotation of these MAGs and flux balanced analysis revealed several auxotrophies in amino acids and metabolite exchange, respectively, which probably underlie the interactions among the several MAGs during ATO mineralization. Collectively, the findings reported here provide the genetic basis for NTO biodegradation and have implications for NTO bioremediation efforts in the future.M.S