Optimized batch fermentation conditions for enhanced specific hydrogen yield of Bacillus safensis TH2

Biohydrogen (BioH₂), a sustainable energy source derived from diverse carbon substrates, is limited by low yields and undefined optimal fermentation conditions. This study isolated Bacillus tequilensis TH1 and Bacillus safensis TH2 from anaerobic sludge of a full scale digester, both exhibiting robust amylase, protease, cellulase, and lipase activities. B. tequilensis TH1 achieved cumulative hydrogen yields of 188.5 mL-H/gVS from starch, 145.8 mL-H/gVS from glucose, and 121.3 mL-H/gVS from kitchen food waste. B. safensis TH2 outperformed TH1, yielding 272.7 mL-H/gVS from starch, 237.3 mL-H/gVS from glucose, and 276.8 mL-H/gVS from kitchen food waste. Optimized conditions for B. safensis TH2 (2% substrate, 1.5 OD600 inoculum, pH 5.5, 40°C) increased daily hydrogen yield to 35.7 mL/d and cumulative production to 373.98 mL-H/gVS from kitchen food waste, with a specific hydrogen production rate (SHPR) of 13.2 mL/g·d. This optimization improved cumulative hydrogen by 11.3% and SHPR by 18.1%, achieving a peak hydrogen yield of 1.32 mol H₂/mol hexose. These strains' ability to convert diverse substrates into BioH₂ highlights their potential for large-scale production, advancing sustainable energy solutions.