Plastics derived from fossil resources represent a major environmental concern. At the same time, wastewater treatment plants generate large amounts of residual biomass that can serve as substrates for the production of valuable biopolymers. This study evaluated polyhydroxybutyrate (PHB) production by Paracoccus denitrificans using hydrolysates of residual microalgae–bacteria biomass generated during piggery wastewater treatment. The effects of hydrolysis method, hydrolysate concentration, nutrient availability and aeration conditions in biomass growth and PHB accumulation were assessed. Bacterial growth did not occur on thermo-alkaline hydrolysates. In contrast, 10% thermo-acid hydrolysate supported the highest PHB levels, yielding 0.75 g/L biomass with 8.3% PHB in closed reactors. Ultrasound-assisted enzymatic hydrolysate diluted to 20% resulted in similar biomass concentrations but lower PHB content (3.6%) in identical conditions. Bacteria consumed multiple carbon and nitrogen sources without clear preference. Glucose addition (1 g/L, C/N = 6.5) during cultivation in 10% thermo-acid hydrolysate increased biomass to 0.77 g/L achieving 17.5% of PHB content. Aeration enhanced biomass production and specific growth rate but negatively affected PHB accumulation, as supported by flux balance analysis. Overall, these results confirm that thermo-acid hydrolysates from waste microalgal biomass are suitable substrates for PHB production and identify key operational parameters to improve biopolymer yields.
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