Integrated Strategy for Sustainable Outdoor Photo-Fermentative Biohydrogen Production: Sequential Optimization, Repeated-Batch Operation, Modular Bioreactor Simulation, and Biomass Valorization

This study developed an integrated strategy for outdoor photo-fermentative biohydrogen production by Rhodopseudomonas pentothenatexigens KKU-SN1/1 through low-cost medium optimization, repeated-batch operation, modular bioreactor simulation, and biomass valorization. A sequential experimental design combining one-factor-at-a-time screening and a 2 × 2 factorial experiment was conducted under high-temperature conditions (40 °C). Headspace gas purging was unnecessary, while trace element supplementation increased cumulative biohydrogen production 2.3-fold, from 601 to 1356 mL-H2/L. The vinegar-yeast extract medium achieved a cumulative biohydrogen production of 1832 mL-H2/L and reduced the estimated substrate cost by 93.6% compared with the conventional formulation. Outdoor repeated-batch operation with 20% fermentation broth reuse retained 83.9% of the Cycle 1 biohydrogen production in the subsequent cycle. Experimental outdoor data were then used to simulate a modular clustered bioreactor system, which projected quasi-continuous hydrogen production over 68 days, with average and peak production rates of 60 and 84 mL-H2/L/d, respectively. The residual biomass also showed potential for valorization, containing 66% protein, 0.37% carotenoids, 1.11% bacteriochlorophyll, and 0.01% coenzyme Q10. The integrated approach combines low-cost medium formulation, repeated-batch operation, modular bioreactor simulation, and biomass valorization, providing a framework for resource-efficient outdoor biohydrogen production within a circular bioeconomy framework.