Pore-scale mass transfer heterogeneity shapes nutrient accessibility and functional assembly in porous microbial ecosystems.

Porous ecosystems represent ubiquitous microbial habitats across natural settings including soil, gut tract, and food matrices, where microscale spatial architecture critically shapes microbial colonization and interactions. Yet, the mechanisms of how pore-scale physical constraints influence microbial community assembly and metabolic performance remain poorly understood. Here, we employed a microfluidic platform with tunable inter-pillar spacings, coupled with a multi-omics approach including in situ imaging, exometabolomics, metagenomics, and metatranscriptomics, to investigate how pore-size modulates microbial community dynamics. Comparing representative small (50 μm) and large (150 μm) pore-sizes, we found that larger pore-sizes promoted greater biomass accumulation and significantly enhanced exometabolite production, particularly of amino acids. Microscopy and quantitative assays revealed that 150 μm pores facilitated more efficient substrate degradation, especially of carbohydrates. Taxonomic profiling showed that increasing pore-size reduced community evenness while enhancing richness, selectively enriching carbohydrate-degrading and amino acid-producing taxa, and promoting more complex, positively correlated co-occurrence networks. Metatranscriptomic analysis further demonstrated that larger pore-size significantly upregulated key functional genes involved in substrate degradation, amino acid biosynthesis, and stress response pathways. Fluorescent tracer assays revealed pronounced mass transfer heterogeneity, where smaller pores exhibited prolonged solute persistence and steeper chemical gradients, ultimately restricting substrate availability and microbial activity. Collectively, our results reveal that alleviation of microscale spatial constraints enhances nutrient accessibility, metabolic function, and community organization in porous ecosystems, underscoring the pivotal role of physical microstructure in regulating both the taxonomic composition and functional capacity of microbial ecosystems.