Introducing a strategy for producing >1 M ammonia-concentrated fermentation broth via osmotic stress mitigation
Green ammonia (NH3) offers a sustainable alternative to conventional NH3 production, with one approach being the recovery of NH3 from anaerobic digestate. To address the low NH3 concentration in digestate, ammonia fermentation was investigated in a sequencing batch reactor (SBR) using gelatin as a model protein substrate, with its concentration increasing from 2.0 to 22.0 g gelatin-N/L at a hydraulic retention time of 3 days. Initially, as the substrate concentration increased to 10 g gelatin-N/L, NH3 concentration increased from 1.8 to 7.7 g TAN-N/L. However, despite high biomass retention (>20 g VSS/L), highest concentration was limited to 10.8 g TAN-N/L (at 18 g gelatin-N/L). As the nitrogen conversion efficiency declined below 60%, amino acid analysis identified hydrolysis efficiency as the primary bottleneck (91% to 67%). Then, it was speculated that osmotic pressure of 35.0 atm (exceeding seawater level) caused by high NH3 and organic acids (>50 g COD/L) concentrations, was a potential constraint. To alleviate this, the addition of ectoine (1 g/L), known as a compatible solute, was attempted in a batch experiment, which resulted in a 40-50% increase in NH3 concentration. When subsequently applied in the continuous reactor, it enabled NH3 concentration up to 15.7 g TAN-N/L (~1.1 M), which was the highest ever reported. Microbial analysis also revealed a shift from Hathewaya (55.2%) to a more diverse community, including Tepidanaerobacter (46.4%), along with increased abundance of functional genes. Overall, this study shows that osmotic pressure, rather than free NH3 toxicity alone, is a key inhibitory factor in high-concentrated NH3 environments.