Mixed-carbon co-feeding of formate, acetate, and propionate enables tunable polyhydroxyalkanoate biosynthesis

The utilization of waste-derived volatile fatty acids (VFAs) as carbon sources for polyhydroxyalkanoate (PHAs) biosynthesis offers a promising route toward sustainable bioplastics. However, reliance on single VFAs often limits product diversity and structural tunability. In this study, we developed and optimized a co-feeding strategy using a ternary mixture of formate, acetate, and propionate in Cupriavidus necator to enable tailored PHAs production. Using response surface methodology for multi-objective optimization, a VFA ratio (formate:acetate:propionate = 0:0.38:0.62) was identified that balances PHA titer (1.33 ± 0.13 g/L) with a 3-hydroxyvalerate (3-HV) content of 62.02% (in mol). Formate exerted no beneficial effect on the biosynthesis of PHA or 3-HV under mixed substrate conditions. Fermentation kinetic analysis further revealed that acetate primarily promoted cell growth and PHAs backbone synthesis, whereas propionate enhanced 3-HV formation while competitively inhibiting acetate uptake. The resulting copolymer exhibited improved thermal processability, as evidenced by a lower glass transition temperature (46.81 °C ± 0.01) and a reduced primary melting temperature (81.05 °C ± 0.14). Collectively, this work provides a viable strategy for producing tunable bioplastics from renewable mixed VFAs, advancing circular bio-manufacturing.