Fitzgerald, K. S. | Search bioRxiv

bioRxiv2026

Demonstration of an Integrated Process for Manure-Based Nitrogen Recovery Using the Biopolymer Cyanophycin

The trend towards concentrated animal feeding operations (CAFOs) has served to concentrate not only livestock animals but the waste they produce to comparatively smaller areas. The point-source nature of this waste is an opportunity for the recovery and valorization of the nitrogen therein. Such a process would be viable on small to intermediate scales and require minimal inputs at the CAFO. In this study, we demonstrate the potential of the biopolymer cyanophycin to serve as a medium for manure-nitrogen recovery. In the first step, genetically modified strains of Escherichia coli produce intracellular cyanophycin from mock manure hydrolysates. Next, cyanophycin is recovered from microbial biomass via acid solubilization and base precipitation using electrochemically generated acids and bases. Finally, to improve both the yield and recoverable fraction of cyanophycin produced, we leverage the tunability of our genetically engineered system to probe the impacts of cyanophycin synthetase solubility, N-domain activity, and cyanophycin molecular weight on cyanophycin recoverability. Collectively, this work serves as a proof of concept for nitrogen recovery from agricultural waste, aligning with global sustainability initiatives.

bioengineering↗

bioRxiv2026

High-Yield Recovery of Reactive Nitrogen as Cyanophycin by Engineering Acinetobacter baylyi ADP1 under Wastewater-Relevant Conditions

Municipal wastewater constitutes a major reservoir of unutilized reactive nitrogen, representing a significant opportunity for biological valorization. The biopolymer cyanophycin is promising as a means of nitrogen capture and recovery, but current production strategies are not optimized for the physicochemical constraints of municipal wastewater systems. Here, we engineered the naturally competent soil bacterium Acinetobacter baylyi ADP1 ISx to synthesize cyanophycin from carbon and nitrogen sources prevalent in municipal wastewater and over a range of wastewater-relevant temperatures. To overcome the recurring problem of arginine availability limiting cyanophycin synthesis, we engineered an arginine-producing strain (AP1) which accumulated cyanophycin when grown on acetate and ammonium (19% CDW), nitrate (9% CDW), or urea (29% CDW) and without arginine supplementation. During this work, we observed that conditions associated with reduced cell fitness correlated with increased intracellular cyanophycin content. As temperature strongly influences cell growth but cannot be realistically modulated in wastewater contexts, we investigated the potential of induced fructose-auxotrophy to modulate cell growth independently from temperature. This intervention, accomplished with a single knockout ({Delta}gap), expanded the effective range of cyanophycin accumulation from 12 {degrees}C up to 30 {degrees}C. Collectively, these results establish the relevance of arginine-producing strains for cyanophycin biosynthesis and position A. baylyi as a promising chassis for continued development under real-world wastewater conditions.

bioengineering↗