Aerobic biodegradation of emerging azole contaminants by return activated sludge and enrichment cultures.

Azoles are an emerging class of contaminants with a growing ubiquitous presence in the environment. This study investigates the aerobic microbial degradation of four azoles, pyrazole (PA), 1,2,4-triazole (TA), benzotriazole (BTA) and 5-methylbenzotriazole (5-MBTA), with return activated sludge and microbial enrichment cultures. Slow degradation of PA was observed in the presence of glucose and NH4+ with a peak degradation rate of 0.5 mg d-1 gVSS-1. TA was found to be highly persistent, with no significant degradation observed in 6-8 months under any incubation condition. In contrast, the benzotriazoles were readily degraded at faster rates in all incubation conditions. The degradation rates observed for BTA and 5-MBTA, when provided as the sole substrates, were 8.1 and 16.5 mg d-1 gVSS-1, respectively. Two enrichment cultures, one degrading BTA and the other degrading 5-MBTA, were developed from the activated sludge. Mass balance studies revealed complete mineralization of 5-MBTA and partial breakdown of BTA by the enrichment cultures. Nocardioides sp. and Pandoraea pnomenusa were the most abundant bacteria in the BTA and 5-MBTA degrading enrichment cultures, respectively. The research shows large differences in the biodegradability of various azoles, ranging from complete mineralization of 5-MBTA to complete persistence for TA.

Bacteria Make a Living Breathing the Nitroheterocyclic Insensitive Munitions Compound 3-Nitro-1,2,4-triazol-5-one (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.

Rapid biotransformation of the insensitive munitions compound, 3-nitro-1,2,4-triazol-5-one (NTO), by wastewater sludge.

As the use of the new insensitive munitions compound 3-nitro-1,2,4-triazol-5-one (NTO) increases, wastewaters, runoff and groundwater containing NTO will be generated. Little is known about the fate of NTO during biological wastewater treatment. The objective of this study was to explore the ability of wastewater sludges to promote the biotransformation of NTO. Three different sludges, i.e., anaerobic granular sludge, anaerobic digested sludge, and return activated sludge, were used to study the biotransformation of NTO under anaerobic conditions. Three different electron donor amendments were compared- hydrogen, ethanol, and acetate. Mixed microbial communities in each of the three sludge sources were effective in the reductive biotransformation of NTO. 3-amino-1,2,4-triazol-5-one (ATO) was observed as the major product of NTO biotransformation. The highest maximum specific rate of NTO reduction, about 120 mg NTO/g volatile suspended solids/d, was observed in anaerobic granular sludge with hydrogen or ethanol supplied as electron donors. NTO biotransformation to ATO by anaerobic digested sludge was also studied under denitrifying conditions. In this case, reduction of NTO started only after complete denitrification of added nitrate. An important implication of this paper is that sludge from wastewater treatment plants can rapidly and readily reduce NTO to ATO.

Microbial Enrichment Culture Responsible for the Complete Oxidative Biodegradation of 3-Amino-1,2,4-triazol-5-one (ATO), the Reduced Daughter Product of the Insensitive Munitions Compound 3-Nitro-1,2,4-triazol-5-one (NTO).

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.