All raw data for Fig 1.

Metabolic cross-feeding networks are central to shaping microbial community dynamics in environments ranging from the rhizosphere, gut, and marine carbon cycling. Yet cross-feeding has predominantly been viewed by examining exchanged small metabolites. In contrast, the role of extracellular polymeric substance (EPS)—a complex mixture of proteins, polysaccharides, DNA, and humic-like compounds—in cross-feeding remains poorly understood, mainly due to technical challenges in measuring their secretion relative to small metabolites. Using chitin-degrading microbes as a model system, we used a bicarbonate-buffered bioreactor coupled with elemental analysis, which allowed us to quantify both EPS and small metabolite secretion. This revealed that ~25% of carbon exuded by a chitin degrader is in the form of EPS. EPS was produced at similar levels across marine chitin-degrading isolates and seawater communities, underscoring its importance relative to small metabolites. Notably, different sources of EPS were found to select for distinct and diverse microbial communities. Combining in vitro enzyme assays and untargeted metabolomics, we show that EPS undergoes sequential degradation—from large oligomers to smaller, broadly accessible monomers. This sequential breakdown creates a temporal succession of metabolic niches, potentially fueling a shift from specialist species degrading complex substrates to a more diverse community of generalists using simpler monomers. By identifying EPS as a major and dynamic contributor to cross-feeding networks, our findings reveal a hidden layer of complexity in how microbial communities assemble and function across ecosystems.