Water-in-oil emulsion fermentation as a platform for poly(3-hydroxybutyrate-co-3-hydroxyhexanoate) production from...

Water-in-oil (W/O) emulsion fermentation was proposed and investigated for producing poly(3-hydroxybutyrate-co-3-hydroxyhexanoate) (PHBH) using Cupriavidus necator Re2058/pCB113. Model emulsions composed of deionized_water/palm_oil/Span_80 were first studied to evaluate emulsion stability. Two stability regimes were identified: Regime I, a shear-dependent regime where sufficient shear to generate fine droplets, and Regime II, a surfactant-activity-dependent regime, where emulsion stability became insensitive to shear and instead negatively correlated with surfactant concentration, suggesting altered interfacial organization at high surfactant levels. When culture solution was used as the aqueous phase, the emulsions primarily exhibited Regime I behavior, with stability governed by homogenization speed. At high shear (13,000 rpm), small, uniform droplets (ca. 10 μm) were generated and remained stable during cultivation, whereas lower shear led to coalescence and phase separation. Nitrogen source concentration greatly affected emulsion fermentation. At 5 g/L NH₄Cl, cell dry weight (CDW) reached 3.6 ± 0.8 g/L and PHBH concentration peaked at 1.7 ± 0.5 g/L. Reducing NH₄Cl to 1.5 g/L significantly improved performance, yielding CDW of 15.2 ± 1.1 g/L, PHBH concentration of 13.7 ± 2.8 g/L, PHBH content of 90 ± 10 wt%, and a 3-hydroxyhexanoate molar ratio of 9 ± 1 mol% at 72 h. Overall, these findings demonstrate that W/O emulsions can support microbial bioplastic production and provide a framework for exploiting emulsion systems to integrate hydrophobic feedstocks into sustainable PHBH biosynthesis.