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The Department of Defense (DoD) is concerned about the environmental behavior and impacts of newly introduced insensitive high explosive (IHE) compounds: 3-nitro-1,2,4-triazol-5-one (NTO) and 2,4-dinitroanisole (DNAN). Previous investigations have established that both DNAN and NTO are subject to cometabolic biotransformation (reduction) in soil and waste streams, which generates an array of metabolites of poorly understood toxicity and of increased solubility and mobility. In contrast, the project team has discovered that some microbial systems biodegrade DNAN and NTO to benign mineralized products (CO2, N2, NH4+, NO2-, and/or NO3-). Such systems can lower or eliminate environmental risks associated with IHE compounds.
The overarching objective of this project is to develop an understanding of the mineralization processes that will enable DoD to deploy microbial strategies for the complete biodegradation of IHE compounds. These strategies could involve natural attenuation, biostimulation, and/or bioaugmentation at contaminated field sites or enable effective biological treatment of munitions wastewater.
This research project will identify, isolate, and characterize microorganisms that completely biodegrade NTO and DNAN. The results will be used to develop technologies to utilize and detect these microorganisms. The project team has recently discovered a sequential process in which NTO is completely mineralized to CO2, NH4+, and N2. First, an anaerobic enrichment culture (ECNTO) reduces NTO to 3-amino1,2,4-triazol-5-one (ATO). Second, an aerobic enrichment culture (ECATO) mineralizes ATO as its sole carbon, nitrogen, and energy source. The project will be conducted in five phases:
This project addresses whether biodegradation can prevent the migration of IHE to surface water and groundwater. Bioremediation involving IHE mineralizing bacteria is well suited for testing and training ranges since only relatively simple, non-intrusive treatments are involved to supply inoculum or simple supplements. In addition, this project will investigate the biotreatment of IHE compounds in mainstream wastewater and, if advantageous, in bioreactors treating concentrated side streams. Secondly, the impacts of IHE on the biodegradation of legacy munitions will be evaluated and vice versa. Finally, the project will use advanced metagenomic and metatranscriptomic techniques to better understand IHE compound biodegradation pathways, identify the microbes involved, optimize physiological conditions, and develop methods to monitor the presence and concentration of key IHE mineralizing microorganisms. The results from this research project will provide DoD site managers with molecular tools for the detection and monitoring of the key IHE-mineralizing microorganisms. (Project Completion - 2023)
Madeira, C.L., K.V. Jog, E.T. Vanover, M.D. Brooks, D.K. Taylor, R. Sierra-Alvarez, L.A. Waidner, J.C. Spain, M.J. Krzmarzick, and J.A. Field. 2019. 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). Environmental Science and Technology, 53(21):12648-12656. doi.org/10.1021/acs.est.9b04065.
Madeira, C.L., O. Menezes, D. Park, K.V. Jog, J.K. Hatt, S. Gavazza, M.J. Krzmarzick, R. Sierra-Alvarez, J.C. Spain, K.T. Konstantinidis, and J.A. Field. 2021. Bacteria Make a Living Breathing the Nitroheterocyclic Insensitive Munitions Compound 3-Nitro-1,2,4-Triazol-5-One (NTO). Environmental Science and Technology, 55(9):5806-5814. doi.org/10.1021/acs.est.0c07161.
Menezes, O., C. Owens, E.E. Rios-Valenciana, R. Sierra-Alvarez, J.A. Field, and J.C. Spain. 2022. Designing Bacterial Consortia for the Complete Biodegradation of Insensitive Munitions Compounds in Waste Streams. Biotechnology and Bioengineering, 119(9):2437-2446. doi.org/10.1002/bit.28160.
Menezes, O., K. Kocaman, S. Wong, E.E. Rios-Valenciana, E.J. Baker, J.K. Hatt, J. Zhao, C.L. Madeira, M.J. Krzmarzick, J.C. Spain, R. Sierra-Alvarez, K.T. Konstantinidis, and J.A. Field. 2022. Quinone Moieties Link the Microbial Respiration of Natural Organic Matter to the Chemical Reduction of Diverse Nitroaromatic Compounds. Environmental Science and Technology, 56(13):9387-9397. doi.org/10.1021/acs.est.2c01329.
Rios-Valenciana, E.E., O. Menezes, J. Romero; C. Blubaum, M.J. Krzmarzick, R. Sierra Alvarez, and J.A. Field. 2023. Elucidating the Mechanisms Associated with the Anaerobic Biotransformation of the Emerging Contaminant Nitroguanidine. Water Research, 229: 119496. doi.org/10.1016/j.watres.2022.119496.
Brooks, M. 2020. Community Analysis of 3-amino-1,2,4-triazol-5-one (ATO) and 3-nitro-1,2,4- trizol-5-one (NTO) Degrading Microbial Cultures (Master's Thesis). Oklahoma State University.
Kadoya, W. 2020. The Fate of Nitroaromatic Contaminants in Anaerobic Environments: Formation of Coupling Products Between Reduced Nitroaromatic Intermediates and Covalent Bonding of Aromatic Amines to Humus Model Compounds (Ph.D. Dissertation). University of Arizona.
Madeira, C.L. 2020. Degradation of the Insensitive Munitions Compound 3-nitro-1,2,4-triazol-5- one (NTO) via Sequential Reducing-Oxidizing Conditions (Ph.D. Dissertation). University of Arizona.
Park, D. 2020. Metagenome Analysis of an Enrichment Culture that Degrades the 3-nitro-1,2,4- triazol-5-one (NTO) Explosive (Master's Thesis). Georgia Institute of Technology.
Romero, J.M. 2022. Anaerobic Biotransformation of the Insensitive Munitions Compound Nitroguanidine (Master's Thesis). University of Arizona.