Establishment of a stable monoculture system for Entodinium furca monolobum and isolation of Escherichia spp. as growth-promoting bacteria

Introduction Entodinium furca monolobum is a common rumen ciliate protozoan, while it remains poorly characterized due to the lack of a stable monoculture system. Methods In the present study, we established a stable in vitro monoculture of Ent. furca monolobum using a modified SP medium with inoculum derived from Holstein cows through filtration-based enrichment and serial dilution. We isolated bacteria from the monoculture supernatant and ciliate-associated fractions, and evaluated candidate isolates in co-culture assays. We also profiled bacterial communities by amplicon sequencing. Results A stable in vitro monoculture of Ent. furca monolobum was established. Eight bacterial species were isolated from the monoculture supernatant and four from the ciliate-associated community. Escherichia coli and Escherichia fergusonii were consistently detected in both fractions and significantly promoted ciliate proliferation, with E. coli showing a stronger effect ( p < 0.05). Community profiling showed distinct bacterial structures between monoculture supernatant and protozoa-free rumen fluid, and Gammaproteobacteria dominated in E. coli -supplemented cultures. Discussion This work not only overcomes the long-standing challenge of maintaining Ent. furca monolobum in vitro culture, but also provides a methodological framework for developing defined monoxenic systems. The stable monoculture system and identification of growth-promoting Escherichia spp. provide a methodological basis for mechanistic studies of protozoan–bacterial interactions in the rumen.