This project assessed the environmental fate and impacts of 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+). Such systems can lower or eliminate environmental risks associated with IHE compounds. The overarching objective of this project was to develop an understanding of the mineralization processes that will enable deployment of microbial strategies for the complete biodegradation of IHE compounds. These strategies could involve natural attenuation, biostimulation and/or bioaugmentation at impacted field sites or enable effective biological treatment of munitions wastewater.

This project was structured around seven scientific tasks: 

1. Identify the microorganisms involved in NTO mineralization and gain insights on their physiology and biochemical pathways to facilitate their application in bioremediation systems. 
2. Investigate the redox cycling of quinone moieties during the respiration of natural organic matter (NOM) as an important mechanism that can contribute to the degradation of insensitive munitions compounds (IMC) and other nitroaromatic compounds in soils. 
3. Demonstrate that DNAN can be mineralized via bioaugmentation with specialized cultures. 
4. Optimize the sequence of redox conditions and treatments to promote the mineralization of both NTO and DNAN, while preventing the accumulation of biotransformation products. 
5. Utilize metagenomic and metatranscriptomic techniques to gain insights on the biodiversity, physiology, and biodegradation pathways of IHE-mineralizing bacteria. 
6. Gain insight into the mechanisms involved in the anaerobic biotransformation of nitroguanidine (NQ). 
7. Translate the knowledge gathered in the previous tasks to develop and optimize bioremediation technologies for the treatment of (waste)water impacted with IHEs.

This study led to improved understanding of the mechanisms driving the microbial transformation of IMC and revealed the involvement of a combination of biotic and abiotic mechanisms in the degradation of these chemicals. This project demonstrated that some microbial systems are able to transform NTO and DNAN into benign mineralized products (CO₂, N₂, NH₄⁺), eliminating concerns about the potential generation of toxic byproducts. Furthermore, this project obtained evidence for the anaerobic biotransformation of NQ.

By providing shifting redox conditions in a sequential anaerobic–aerobic treatment system, full biomineralization of NTO and 3-amino-1,2,4-triazol-5-one (ATO, the product of NTO reduction) to environmentally safe products was attained both in batch and in continuous-flow bioreactors. Furthermore, this study provided multiple lines of evidence to support the occurrence of a novel mechanism of NTO biotransformation entailing NTO respiration by Geobacter anodireducens. The team has also discovered that quinone-respiring bacteria can be implicated in the degradation of a broad spectrum of nitroaromatic compounds beyond NTO. These microorganisms can couple the reduction of quinone moieties in NOM to the abiotic reduction of nitroaromatic compounds by the hydroquinone moieties formed. The link between the microbial respiration of NOM and the chemical reduction of nitroaromatics has important implications for the fate of IMC in the environment and the development of environmental biotechnology processes.

A sulfate-reducing enrichment culture, containing Cupidesulfovibrio oxamicus, was shown to transform NQ into nitrosoguanidine, which is further transformed abiotically to cyanamide and other unidentified products. This is the first description of a microbial community associated with NQ anaerobic biotransformation. The microbial culture utilized lactate as an electron donor and both sulfate and NQ as electron acceptors, and it could not utilize NQ as its sole nitrogen source.

Experiments with synthetic munitions manufacturing wastewater containing NTO and/or DNAN established the feasibility of constructing bacterial consortia with complementary degradation capabilities to achieve the complete biodegradation of these chemicals. This project also developed a down-flow soil/perlite column with anaerobic microniches and aerobic zones, which allowed for the simultaneous biodegradation of NTO and ATO.

The results obtained in this project demonstrate that microbial processes provide promising perspectives for the treatment of munitions manufacturing effluents and for the in situ bioremediation of sites impacted by IMC. This knowledge and insights can be utilized to develop effective strategies to promote the microbial degradation of IMC to environmentally safe end-products. This work also provides valuable insights about the potential contribution of microbially-mediated transformations in the natural attenuation of IMC.

Additional work is needed to further validate the technical and economic feasibility of the proposed environmental biotechnologies and to establish design parameters for full-scale application. The future work should involve testing of real industrial effluents and impacted media at the pilot-scale. Application of the results of this research will ultimately bolster operational capabilities and warfighter preparedness by mitigating the impacts of these chemicals. (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.

Theses and Dissertation

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.