(651f) Process Optimization of Semi-Continuous Methanotroph Fermentation for Sustainable Single-Cell Protein Production Via Biogas and Digestate Valorization

Methanotrophs—methane-oxidizing bacteria—offer a promising dual solution to global environmental and resource challenges by reducing greenhouse gas emissions and enabling sustainable production of protein-rich biomass. This study systematically optimizes semi-continuous fermentation for enhanced single-cell protein (SCP) production using a co-culture of Methylophilus methylotrophus and Methylosinus sporium. A structured, six-phase optimization framework was employed to refine key operational parameters, including hydraulic retention time (HRT), pH, mixing speed, and temperature, using an ammonium mineral salts medium with high-purity methane and air. Subsequent phases transitioned to more sustainable inputs by replacing synthetic methane with biogas and substituting commercial nitrogen sources with digestate, thus incorporating waste-derived feedstocks into the process. Process stability was ensured through daily monitoring of pH, methane uptake, optical density (OD600), and dry biomass, with system adjustments made only after achieving steady-state conditions (<20% variability across replicates). Protein content and microbial performance were assessed using modified Lowry assays and genomic profiling. The results demonstrate the scalability and flexibility of methanotroph-based SCP production systems, highlighting their potential for industrial applications. By integrating biogas and agricultural byproducts, this approach supports circular bioeconomy principles and presents a viable pathway for producing nutrient-dense biomass for food, feed, and biomanufacturing sectors while reducing dependence on synthetic resources.