Microbial synthesis structures organic compound composition in anaerobic digestion

Anaerobic digestion (AD) is a cornerstone technology for sustainable waste treatment and renewable energy recovery, yet its complex microbe-metabolite interactions remain poorly understood. Here, we combined high-resolution molecular profiling and microbial community sequencing in a three-month study across seven full-scale digesters to resolve dissolved organic matter (DOM) and microbiome dynamics. A total of 28 925 DOM molecules, including a conserved core of 1154 metabolites, were identified. By disentangling metabolic pathways, we observed complex transformation patterns that extend beyond simple substrate breakdown. Molecules within a mass window (183.57-390.81 m/z) exhibited high persistence, strong microbial associations, and distinct transformation trajectories. Within this mass window, microbial community composition and feedstock input, together explained ~30.1%-43.4% of the observed spatiotemporal variation. In each digester, 1260-2108 molecules were closely associated with microbial metabolism, forming 7.77-24.52 microbe-metabolite associations on average. The accumulation and turnover of these microbial metabolites were strongly linked to methane production and system performance, highlighting microbial processing of DOM as a significant factor shaping microbe-metabolite interactions. This perspective emphasizes the importance of microbe-metabolite interplay in AD, providing a conceptual framework for predictive monitoring and optimization of engineered biotechnologies.