Poly(3-hydroxybutyrate) production from ricotta whey by Cupriavidus necator: effects of coffee oil and oxidative stress

Abstract In this study, ricotta cheese exhausted whey (RCEW), an abundant dairy by-product, was evaluated as a low-cost substrate for poly(3-hydroxybutyrate) (PHB) production by Cupriavidus necator DSM 428 through an integrated process strategy combining substrate conditioning, enzymatic lactose hydrolysis, oxidative stress modulation, and cultivation under controlled bioreactor conditions. Shake-flask experiments demonstrated that RCEW supported efficient biomass formation (1.60 g L⁻ 1 ) and high intracellular PHB accumulation (71.4% of dry cell weight, DCW), while supplementation with coffee oil extract increased biomass up to 4.41 g L⁻ 1 and PHB titer to 2.87 g L⁻ 1 . Controlled oxidative stress further enhanced polymer accumulation, with hydrogen peroxide addition (5.0 mmol L⁻ 1 at 24 h) promoting intracellular PHB contents up to 80.8% DCW. Cultivation in a stirred-tank bioreactor confirmed process robustness, and fed-batch cultivation resulted in PHB concentrations up to 3.85 g L⁻ 1 (77.9% DCW) using a chlorine-free downstream recovery protocol. The recovered polymer consisted exclusively of 3-hydroxybutyrate units and exhibited a high–molecular weight (876 kDa). Overall, these results demonstrate the feasibility of producing high-quality PHB from RCEW using a non-recombinant C. necator strain, supporting the development of sustainable bioprocesses for dairy by-product valorization. Key points Thermally conditioned ricotta cheese exhausted whey supports efficient PHB production by C. necator Coffee oil supplementation enhances biomass formation whereas oxidative stress promotes PHB accumulation Fed-batch cultivation and chlorine-free recovery support process efficiency and sustainability