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The nitroheterocyclic 3-nitro-1,2,4-triazol-5-one (NTO) is an ingredient of insensitive explosives increasingly used by the military, becoming an emergent environmental pollutant. Cometabolic biotransformation of NTO occurs in mixed microbial cultures in soils and sludges with excess electron-donating substrates. Herein, we present the unusual energy-yielding metabolic process of NTO respiration, in which the NTO reduction to 3-amino-1,2,4-triazol-5-one (ATO) is linked to the anoxic acetate oxidation to CO2 by a culture enriched from municipal anaerobic digester sludge. Cell growth was observed simultaneously with NTO reduction, whereas the culture was unable to grow in the presence of acetate only. Extremely low concentrations (0.06 mg L–1) of the uncoupler carbonyl cyanide m-chlorophenyl hydrazone inhibited NTO reduction, indicating that the process was linked to respiration. The ultimate evidence of NTO respiration was adenosine triphosphate production due to simultaneous exposure to NTO and acetate. Metagenome sequencing revealed that the main microorganisms (and relative abundances) were Geobacter anodireducens (89.3%) and Thauera sp. (5.5%). This study is the first description of a nitroheterocyclic compound being reduced by anaerobic respiration, shedding light on creative microbial processes that enable bacteria to make a living reducing NTO.

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