Table 2_Establishment of a stable monoculture system for Entodinium furca monolobum and isolation of Escherichia spp. as growth-promoting bacteria.docx

IntroductionEntodinium furca monolobum is a common rumen ciliate protozoan, while it remains poorly characterized due to the lack of a stable monoculture system.
MethodsIn 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.
ResultsA 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.
DiscussionThis work not only overcomes the long-standing challenge of maintaining Ent. furca monolobumin 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.