3-Nitro-1,2,4-triazol-5-one (NTO)
is one of the main ingredients
of many insensitive munitions, which are being used as replacements
for conventional explosives. As its use becomes widespread, more research
is needed to assess its environmental fate. Previous studies have
shown that NTO is biologically reduced to 3-amino-1,2,4-triazol-5-one
(ATO). However, the final degradation products of ATO are still unknown.
We have studied the aerobic degradation of ATO by enrichment cultures
derived from the soil. After multiple transfers, ATO degradation was
monitored in closed bottles through measurements of inorganic carbon
and nitrogen species. The results indicate that the members of the
enrichment culture utilize ATO as the sole source of carbon and nitrogen.
As ATO was mineralized to CO2, N2, and NH4
+, microbial growth was observed in the culture.
Co-substrates addition did not increase the ATO degradation rate.
Quantitative polymerase chain reaction analysis revealed that the
organisms that enriched using ATO as carbon and nitrogen source were Terrimonas spp., Ramlibacter-related spp., Mesorhizobium spp., Hydrogenophaga spp., Ralstonia spp., Pseudomonas spp., Ectothiorhodospiraceae, and Sphingopyxis. This is the first study to report the complete mineralization of
ATO by soil microorganisms, expanding our understanding of natural
attenuation and bioremediation of the explosive NTO.
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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