This project seeks to demonstrate proof-of-concept that synthetic waste stream byproduct ammonium nitrate solution (ANSOL) generated from energetic material production can undergo significant bioenzymatic remediation and be converted into useful industrial feedstock such as amino acids and some of their non-natural analogs. This would provide benefits across all services by converting a costly waste stream into a valuable, reliable source of useful materials while also minimizing hazardous waste management. The project team plans undertaking a two-year research program with four goals that include:

(i) Conversion of nitrate in ANSOL directly into ammonia by the coupled activity of nitrate reductase and nitrite reductase to yield an enriched ammonia solution for subsequent downstream processing.

(ii) Synthesis of aspartic acid by aspartate ammonia lyase or tyrosine by tyrosine phenol lyase using ammonia from (i).

(iii) Utilizing nanoparticle (NP) display to significantly increase both enzyme stability and the efficiency of the coupled enzymatic processes of (i) and (ii) by accessing enzymatic channeling phenomena in self-assembled (NP)-enzyme bioconjugates within a one-pot direct reaction scheme. This format is designed toward eventually producing even further products in a similar manner.

(iv) Detailed reaction parameters for (i) - (iii) including conversion rates, enzymatic efficiency, potential inhibitory effects of other ANSOL components, required level of ANSOL dilutions for efficient reactions, and chemistries for enzymatic immobilization and long-term stability on NPs.

Enzymes will be cloned and expressed at the U.S. Naval Research Laboratory. Enzymes will be self assembled to NPs using established bioconjugation chemistries to form NP-enzyme clusters and then utilized in assays to confirm specific conversions and their efficiency. ANSOL or a reconstituted ANSOL mixture will be used as substrate in these reactions and diluted as needed. Assay progress will be monitored spectrophotometrically and with a dedicated ultra-performance liquid chromatography – mass spectrometry system. Enzymatic reaction parameters will be estimated using the Michaelis-Menten formalism and conversion efficiencies measured. Efficiency will be iteratively optimized by varying enzyme ratios and reaction conditions as needed.

This research will provide critical conceptual proof that NP-immobilized enzymatic catalysis and the principles of synthetic biology can be exploited to convert a significant percentage of ANSOL into useful products in a manner based in the principles of green chemistry. This research will also provide a strong foundation for implementing subsequent pilot scale-up studies if warranted. This can help convert a significant amount of hazardous waste into a continuous resource for production of useful commodities in a manner that does not itself contribute to waste.