Valorisation of glycerine pitch into bioflocculant extracellular polymeric substances ( EPS ): Influence of organic loading rate and C/N ratio

Abstract BACKGROUND Glycerine pitch (GP), a carbon‐rich byproduct of biodiesel production, is commonly disposed of through energy‐intensive routes, contributing to indirect CO 2 emissions. Valorising GP into extracellular polymeric substances (EPS) offers a biological pathway for carbon utilisation; however, the effects of operational conditions on EPS production from GP remain underexplored. This study evaluated how the organic loading rate (OLR) and carbon‐to‐nitrogen (C/N) ratio influence EPS yield and composition using a mixed microbial culture in a sequencing batch reactor (SBR). OLRs of 350 to 1050 mg C/L·d and C/N ratios of 5 to 28 were investigated. RESULTS The highest EPS yield (588 mg VS EPS/g VS biomass) was achieved at an OLR of 700 mg C/L·d and a C/N ratio of 28, representing a 78% improvement over a previously reported GP‐based EPS production system using the same inoculum and substrate. Lower OLRs favoured EPS production due to prolonged starvation phases, while elevated C/N ratios induced nitrogen limitation and redirected carbon from biomass growth towards EPS synthesis. Variations in EPS composition were primarily driven by changes in protein content, whereas polysaccharides remained relatively stable. The extracted EPS, with a polysaccharide‐to‐protein ratio of 0.7, achieved 72% flocculation activity at 30 mg EPS/L in a kaolin suspension. CONCLUSION The study demonstrates the feasibility of valorising GP into functional bioflocculant EPS and provides operational guidance for optimising OLR and C/N ratio of enhanced EPS yield and tailoring its composition for sustainable wastewater treatment. © 2026 Society of Chemical Industry (SCI).