Effect of inoculum-to-substrate ratio on volatile fatty acid production and polyhydroxyalkanoate synthesis during acidogenic fermentation of brewer’s spent grain co-fermented with anaerobic digestion sludge

This study investigated the potential of brewer’s spent grain (BSG) as a sustainable carbon source for polyhydroxyalkanoates (PHA) production through acidogenic fermentation. The effect of inoculum-to-substrate ratio (ISR) on volatile fatty acid (VFA) production was systematically evaluated using anaerobic digestion sludge at ISR values of 0.5, 1.5, 2.5, and 3.5. ISR 0.5 achieved the highest VFA yield of 1.05 mg COD/mg VS, whereas higher ISR conditions resulted in limited accumulation due to enhanced microbial consumption and methane production, with cumulative methane yields of 486–523 mL CH4/g COD at ISR 1.5–3.5 compared to 142 mL CH4/g COD at ISR 0.5. Substrate degradation analysis revealed that carbohydrate-driven acidogenesis dominated the early phase, followed by protein-associated fermentation. Enrichment of PHA-accumulating microorganisms in a sequencing batch reactor (SBR) increased PHA content to approximately 30 wt%. Batch accumulation tests demonstrated that both BSG-derived VFA and acetate supported PHA production, with acetate achieving a higher final PHA content (56.7 wt%) compared to BSG-derived VFA (45.4 wt%). Kinetic analysis using the Richards model showed negligible lag time for BSG-derived VFA, while acetate exhibited a lag phase but higher accumulation rates. These results demonstrate that ISR is a key parameter controlling carbon partitioning and VFA production.