Ultrasonication-induced Metabolic Pathway Shifts and Reduced Electron Carrier Washout with Biomass Enhanced Hydrogen Yield in a Continuous Stirred Tank Reactor

Biohydrogen is pivotal for hydrogen economy, providing a renewable, carbon -neutral source of hydrogen from organic waste and biomass. It addresses environmental concerns and supports circular economy principles. However, challenges like managing metabolic pathways and dissolved hydrogen hinder attainment of higher hydrogen yields. This study explored the impact of organic loading rates (OLR) and ultrasonication on glucose fermentation by Clostridium pasteurianum for biohydrogen production. Results showed ultrasonication, particularly at 10 g/L.d OLR, increased hydrogen yield from 1.04 to 1.28 molH 2/mol-glucose. Higher OLR boosted biogas production, and at 10 g/L.d OLR, ultrasonication improved hydrogen purity from 46% to 53%, enhancing yield by 13%. C. pasteurianum shifted metabolic pathways under higher OLR, increasing lactate production to address redox imbalance. However, ultrasonication negatively affected lactate and acetate reutilisation, redirecting carbon flux from lactate to butyrate, increasing hydrogen yield. Combining ultrasonication and increased OLR enhanced glycolysis carbon flux by 14.5% and reduced biomass carbon flux by 10.5%. Hydrogen yield, influenced by glycolysis carbon flux, reached 82% under ultrasonication and increased OLR. Ultrasonication mitigated inhibitory effects on electron carrier molecule re -oxidation, emphasising the need to balance biomass, dissolved hydrogen concentration, and OLR for optimal hydrogen recovery. Despite decreased acetate/butyrate ratio and unchanged theoretical hydrogen production with ultrasonication, hydrogen yield increased. This likely resulted from reduced electron carrier molecule wastage with biomass, influenced by higher biomass concentration and ultrasonication. The study underscored preventing electron carrier molecule washout for higher hydrogen production and recommends using batch reactors over continuous stirred tank reactors in dark fermentation operations.