Meta-omics analysis revealed structural and metabolic shifts of acidification communities after feeding with thermal hydrolysis pretreated food waste
Engineering environmental microbiomes enables carbon upcycling from organic solid waste into valuable products, such as volatile fatty acids (VFAs). This approach leverages the broad metabolic potential of natural communities to process highly heterogeneous substrates. However, solid waste conversion is often constrained by microbial hydrolysis, which limits the breakdown of particulate substrates into soluble compounds available for fermentation. Top-down engineering enhancements, such as thermal hydrolysis pretreatment (THP), can improve feedstock solubility but do not consistently increase total acid yields and may instead shift acid profiles. This highlights our limited understanding of microbiome responses to feedstock-level engineering controls. In this study, we applied genome-resolved multi-omics analysis to link VFA production performance with molecular mechanisms in a top-down engineered microbiome fed with thermally hydrolysed food waste. THP reduced total VFA yield (p = 0.003), accompanied by decreased Shannon diversity (p = 0.03), acid-production potential (DNA; log2FC = -1.3), and acid-production activity (mRNA; log2FC = -0.2). Propionate remained the dominant product with and without THP, consistent with the predominance of the Prevotella genus; however, THP selectively enriched two Prevotella species with elevated expression of ATP-generating steps in propionate production. THP also increased butyrate and valerate fractions (p < 0.001), together with upregulated reverse {beta}-oxidation for chain elongation driven by CAG-791 sp900320025 and Megasphaera sp000417505. Overall, our results provide performance- and molecular-level evidence that THP applied to complex feedstocks didnt increase total VFA yields, but reshaped microbiome structure and function, maintained propionate dominance, and promoted chain elongation.