Unveiling the Dual Role of Eggshell as a pH Buffer and Calcium-Driven Promoter for Enhanced Caproate Production via Two-Stage Anaerobic Fermentation

Converting food waste into medium-chain carboxylic acids (MCCAs) via chain elongation (CE) is a promising valorization route, but it is constrained by the high cost of external electron donors (EDs) and acidification-induced instability. This study proposes an eggshell-mediated two-stage strategy to drive high-titer caproate production using endogenous EDs. Initially, eggshell was found to shift the metabolic pathway from ethanol/lactate accumulation to butyrate synthesis, a promotion identified to be driven by both pH buffering and calcium-mediated biostimulation (with significant enhancement observed at Ca 2+ concentrations of 1.5–3.0 g/L). Based on this, a two-stage process was developed: an acidogenic stage to accumulate high concentrations of endogenous ethanol and lactate, followed by an eggshell-amended stage for CE. This strategy achieved a remarkable caproate titer of 8.20 gCOD/L. Metagenomic analysis revealed that eggshell addition reshaped the microbiome, selectively enriching functional guilds, including Caproicibacterium amylolyticum and Ruminococcaceae bacterium BL-6. This metabolic shift was underpinned by the enhanced genetic potential of substrate transport systems (e.g., sugar/peptide transporters) and the energy-conserving Rnf complex. Crucially, the metabolic network was redirected to enhance the reverse β-oxidation (RBO) pathway while suppressing the tricarboxylic acid (TCA) cycle. This study demonstrates that eggshell acts as a multifunctional regulator, providing a cost-effective solution for sustainable MCCA recovery from complex organic waste by modulating interkingdom microbial interactions.