Intrinsic waste component synergy: calcium-rich eggshell waste modulates fungal-bacterial microbiome toward selectively medium-chain fatty acid production.

The valorization of waste streams into medium-chain fatty acids (MCFAs) through fungi-bacteria synergy is often hindered by substrate competition and distinct ecological niches. This study demonstrates that eggshell waste acts as a bioregulator to optimize this interaction for caproate production. At a 20 g/L dosage, eggshells facilitated high caproate production (22.3 ± 1.3 gCOD/L) driven by in-situ ethanol supply (11.3 ± 1.9 gCOD/L). The amendment established stable micro-niches, significantly enriching yeasts (Wickerhamomyces, Candida, and Issatchenkia, 69.2%) and chain-elongating bacteria (CEB, Caproiciproducens, and Clostridium_sensu_stricto_12, 10.2%), while metagenomics confirmed upregulated glycolysis and reverse β-oxidation pathways. Additionally, yeast synergy with CEB via ethanol cross-feeding in a sugar-rich environment can be disrupted under the sugar-depleted phase. The coculture experiments unveiled that 8 g/L Ca2+ alleviates fungi-bacteria conflict and promotes CEB functionality. This study presents a waste valorization strategy, leveraging intrinsic waste synergies to optimize fungal-bacterial interactions and drive endogenous ethanol-based caproate production.