Multi-stage synthetic microbial consortia outperform single-stage augmentation by remodeling metabolism and mediating...

Anaerobic digestion (AD) of food waste often suffers from low methane yield and volatile fatty acids (VFAs) accumulation, primarily due to inefficiencies or imbalances within the native microbial community. To address these metabolic and ecological limitations, we constructed two synthetic microbial communities (SynComs) using a function-driven strategy: a methanogen-only consortium (SynCom-J) and a multi-stage consortium comprising hydrolytic, acidogenic, and methanogenic members (SynCom-YSJ). Both SynComs were introduced into semi-continuous reactors that already harbored a metabolically complete native microbiome, serving as bioaugmentation agents. When fed daily with partially hydrolyzed feedstock containing residual macromolecular organics and short-chain VFAs, SynCom-YSJ consistently outperformed SynCom-J during the entire hydraulic retention time. Compared to the non-bioaugmented control under identical operating conditions, SynCom-YSJ increased methane yield by 22% (vs. 8% for SynCom-J) and nearly eliminated the start-up lag phase, while both consortia reduced propionate accumulation by 1.6-fold. Successful colonization of the SynComs reshaped the AD microenvironment-characterized by elevated acetate, reduced propionate, and a moderate, non-inhibitory increase in total ammonia nitrogen-thereby imposing deterministic selection on the resident community. Metagenomic analysis revealed that SynCom-YSJ triggered broader metabolic reprogramming, upregulating genes involved in hydrolysis, acidogenesis, interspecies electron transfer, energy metabolism, and acetoclastic/hydrogenotrophic methanogenesis. Notably, a trade-off between microbial network stability and process performance emerged: SynCom-J promoted a more robust network, whereas SynCom-YSJ formed a more complex and high-efficiency network that prioritized methane yield. This study demonstrates that coordinated multi-stage bioaugmentation optimizes methanogenesis through targeted metabolic remodeling and provides an ecology-informed design principle for engineering SynComs that balance system performance with stability. These findings highlight the potential of multi-stage bioaugmentation to enhance both functional robustness and system resilience in food waste AD.