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.

Technical Approach

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:

  • In the first phase of the project, NTO/ATO degraders will be isolated from the enrichment cultures to determine their biochemistry/physiology and develop methods for their application. DNAN can be completely oxidized to CO2 and NO2- by the isolate, Nocardioides sp. JS1661.
  • The second phase of the project will attempt to find and isolate additional DNAN mineralizing bacteria to provide a more diverse set of choices for bioaugmentation and to gain better insight on their distribution and potential for natural attenuation.
  • The third phase will explore the proper sequencing of redox conditions and bioaugmentation to completely biodegrade mixtures of explosive compounds without generating biotransformation products or interfering with the biodegradation of legacy munitions.
  • In the fourth phase, molecular biological techniques (metagenomics, metatranscriptomics, and qPCR) will be utilized to explore biochemical mechanisms and physiology of IHE compound biodegradation and to develop tools to monitor the IHE-degrading bacteria in the field or bioreactors.
  • In the fifth phase, bench-scale experiments will be conducted to demonstrate the proof of concept of utilizing complete biodegradation strategies in soil remediation and in the treatment of synthetic munitions wastewater or contaminated groundwater


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.


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.