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

Yujia Wang¹Congjie Hua¹Yang Xiao¹Jinying He^1,2Bingqi Chen¹Beibei Li¹Dongsong Li¹Tingyu Zheng¹Kai Liu¹Jie Xiong²Wei Miao²Jinmei Feng^1*

• ¹Jianghan University, Wuhan, China
• ²Institute of Hydrobiology Chinese Academy of Sciences, Wuhan, China

Entodinium furca monolobum, a common rumen ciliate protozoan belonging to the order Entodiniomorphida, remains poorly characterized due to the absence of a stable monoculture system. In this study, we established a stable in vitro monoculture of Ent. furca monolobum using a modified SP medium with inoculum derived from Holstein cows. From this system, eight bacterial species were isolated from the culture supernatant and four from the ciliate-associated community. Notably, Escherichia coli and Escherichia fergusonii were consistently present in both fractions, and they were subsequently selected for co-culture experiment. Both E. coli and E. fergusonii significantly promoted ciliate proliferation, with E. coli being the more effective promoter (P < 0.05). Amplicon sequencing revealed a distinct bacterial community structure in the monoculture supernatant compared to protozoa-free rumen fluid. Moreover, Gammaproteobacteria was found to be the dominant class in E. coli–supplemented cultures. This study presents the stable in vitro monoculture of Ent. furca monolobum and the characterization of its associated bacterial community. By identifying E. coli and E. fergusonii as growth-promoting partners, our work provides a methodological breakthrough that enables in-depth investigation of protozoan–bacterial interactions and their ecological roles in the rumen.