Exploring the Capability of New Wild-Type Strain Cupriavidus oxalaticus USM2A2 to Synthesize P(3HB-co-4HB)

Poly(3-hydroxybutyrate-co-4-hydroxybutyrate), [P(3HB-co-4HB)], has emerged as a valuable biopolymer due to its biocompatibility and tuneable mechanical properties, making it relevant for medical applications such as soft tissue engineering or biomedical devices. This study explores the capability of a newly isolated wild-type strain, Cupriavidus oxalaticus USM2A2, to synthesize this copolymer. Exploring various structurally related and unrelated carbon sources enabled C. oxalaticus USM2A2 to synthesize P(3HB) and P(3HB-co-4HB). Co-supplementation of oleic acid (0.56 wt% C) with 1,4-butanediol (0.28 wt% C) achieved a maximum biomass of 2.09 g/L, PHA contents up to 41 wt% with trace amounts of 4HB. The 4HB incorporation was significantly enhanced under sole 4HB substrates and mixed-alkanediols (1,4-butanediol with 1,6-hexanediol) conditions, resulting in 4HB contents ranging from 25 mol% to 64 mol%. The structure and composition of the synthesized P(3HB-co-4HB) were analysed by 1H NMR, while 13C NMR analysis revealed four distinct dyad signals consistent with random distribution of 3HB and 4HB units (D = 0.94). DSC analysis further validated these results, showing a shift from a single Tm of 170.27 °C in the homopolymer to dual melting endotherms (158.04 °C and 45.69 °C) and a single Tg (– 25.33 °C) in the copolymer. Molecular weight analysis revealed Mw of 1213 kDa (PDI 1.5) for the P(3HB) homopolymer and 874 kDa (PDI 1.6) for the P(3HB-co-37% 4HB) copolymer. This study represents the first report of P(3HB-co-4HB) production by C. oxalaticus USM2A2, demonstrating its potential as a novel copolymer producer. These findings identify substrate selection and feeding strategies as key factors for optimizing copolymer production, composition, and biomass.