Higher-order microbial interactions revealed by comparative metabolic modeling of synthetic communities with varying species composition.

Understanding how microbial interactions scale with community complexity is key to microbiome engineering and ecological theory. This study investigates emergent metabolic behaviors in controlled synthetic anaerobic communities of two, three, or four species: cellulolytic bacterium (), a hydrogenotrophic methanogen (), an acetoclastic methanogen (), and a sulfate-reducing bacterium (), representing core metabolic guilds in cellulose degradation and carbon conversion. We applied a systems biology framework combining proteogenomics, stoichiometric flux modeling, and SMETANA (Species Metabolic Coupling Analysis) to quantify syntrophic cooperation and competition across configurations. Cooperation peaked in tri-cultures and declined nonlinearly in more complex assemblies. Species roles shifted contextually. was the dominant donor, adjusting cellulase and hydrogenase expression by partner. became fully metabolite-dependent while enhancing methanogenesis. improved syntrophic efficiency via redox and hydrogen turnover. In contrast, 's metabolic centrality declined despite higher CH₄ output, suggesting interaction strength depends more on compatibility than richness. Reduced interactions in the four-species community stemmed from a single configuration and need further validation. This study moves beyond descriptive work by quantitatively resolving how metabolic networks rewire across defined communities. By characterizing context-dependent flux shifts at multiple layers, we provide a framework for interpreting and engineering stable, functionally interdependent microbial ecosystems.