Droplet microfluidic iX-seq platform enables discovery of gut bacterial cross-feeding between Phascolarctobacterium faecium and Eubacterium limosum

Background The human gut microbiota comprises hundreds of bacterial species that form a dynamic metabolic network, in which the exchange of microbial metabolites, known as cross-feeding, is integral to community function and host health. However, defining and isolating specific cross-feeding interactions remains challenging due to the limitations of conventional culture-based approaches. Droplet microfluidics enables high-throughput screening and isolation of bacteria from complex communities by encapsulating cells in water-in-oil droplets, allowing for parallel cultivation of thousands of stochastically contained microcultures. In this study, we developed a droplet microfluidics-based screening platform integrated with 16S rRNA gene amplicon sequencing, collectively termed iX-seq, to identify novel cross-feeding interactions directly from a human fecal sample. Results Using iX-seq, we identified Phascolarctobacterium faecium and Eubacterium limosum as potential unidirectional cross-feeders, wherein the growth of P. faecium was obligately supported by E. limosum in droplet co-cultures. The growth of P. faecium was modestly affected when co-cultured in vitro but notably required E. limosum co-inoculation for intestinal colonization in germ-free mice. Metabolomic profiling implicated 2-oxoglutarate (2-OG), an intermediate in P. faecium ’s succinate metabolic pathway, as the key differentiating metabolite between E. limosum -inoculated mouse and control mouse feces. Supplementation of 2-OG in monoculture increased P. faecium growth in a concentration-dependent manner, confirming its supportive role. Conclusions Our results reveal a previously unrecognized, potential 2-OG-mediated cross-feeding relationship between P. faecium and E. limosum , offering new insight into gut microbial metabolic dependencies. By enabling high-throughput, culture-based screening directly from fecal samples, the iX-seq approach demonstrates a practical framework for studying cross-feeding interactions within the gut microbiota and represents a potential paradigm shift in how complex microbial communities are experimentally investigated.