This limited scope project sought to demonstrate proof-of-concept that waste stream byproduct ammonium nitrate solution (ANSOL) can undergo bioenzymatic remediation and be upcycled into useful industrial feedstocks such as amino acids and downstream products.

This two-year research program aimed to address four technical objectives:

Objective i: Conversion of nitrate in ANSOL directly into ammonia.

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

Objective iii: Utilizing quantum dots to increase both enzyme stability and especially the efficiency of coupled enzymatic processes.

Objective iv: Detailed reaction parameters for (i) – (iii).

Several enzymes were examined for their biocatalytic potential for various applications; many were examined utilizing quantum dots toward informing efforts of remediation/upcycling enzymes. Enzymes were produced at the U.S. Naval Research Laboratory or acquired commercially. The activity of the enzymes nitrate reductase, nitrite reductase, aspartate ammonia lyase, and tyrosine phenol lyase were examined towards the end goals of ANSOL remediation and upcycling to produce amino acid(s). Assay progress was monitored spectrophotometrically and with a dedicated liquid chromatography/mass spectrometry system.

This research provided critical conceptual proof that enzymatic catalysis and the principles of synthetic biology can be exploited to convert nitrate into the valuable amino acid, aspartic acid. In summary:

• Nitrate reductase can convert nitrate to nitrite and does not lose significant activity when attached to quantum dot nanoparticles.
• Nitrite reductase can convert nitrite to ammonia.
  • Aspartate ammonia lyase (AspB) can convert ammonia and fumarate to aspartic acid even with high concentrations of nitrate present. AspB can work in high percentages of ANSOL.
  • Tyrosine phenol lyase can convert ammonia, pyruvate, and phenol to tyrosine even with high concentrations of nitrate present.
  • Nitrate can be enzymatically remediated to ammonia, which can then be enzymatically upcycled to aspartic acid.

Overall, this research could 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 and could work as a stand-alone solution or in conjunction with other methods.

Detailed Understanding of Reaction Parameters