Persistent low PHB accumulation during pilot-scale gas fermentation in Cupriavidus necator

Climate change and food security are tightly linked global challenges. Gas fermentation offers a sustainable route to convert CO₂ into microbial single-cell protein (SCP), with hydrogen-oxidizing bacteria (HOB) emerging as efficient biomass producers via the Calvin-Benson-Bassham cycle. While SCP can replace protein-rich feed components, intracellular polyhydroxybutyrate (PHB) accumulation redirects carbon and reducing power away from biomass and protein synthesis, reducing process efficiency. With this aim, a low amount of PHB is needed to focus on protein production rather than PHB accumulation. This study shows that the PHB formation in Cupriavidus necator H16 during autotrophic gas fermentation is low under non-limiting conditions. Three optimized 48-hour fermentations were analyzed using two quantification methods: GC-FID and HPLC-UV. Both approaches yielded comparable results, but HPLC-UV proved superior in speed, safety, and compliance with green chemistry principles. Time-course analysis revealed consistently low PHB levels (∼6 % CDW) under optimized conditions. These findings demonstrate that tailored process control can suppress PHB accumulation in wild-type strains, improving carbon conversion toward SCP. This approach enhances energy efficiency and supports SCP's potential as a climate-resilient protein source for food and feed applications.