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

## TL;DR

Researchers developed a stable lab system for a rumen protozoan and found Escherichia bacteria that help it grow.

## Contribution

Established the first stable monoculture system for Entodinium furca monolobum and identified Escherichia spp. as growth-promoting bacteria.

## Key findings

- A stable in vitro monoculture of Ent. furca monolobum was successfully established.
- Escherichia coli and Escherichia fergusonii significantly promoted ciliate proliferation.
- Bacterial community structures differed between monoculture supernatant and protozoa-free rumen fluid.

## Abstract

Entodinium furca monolobum is a common rumen ciliate protozoan, while it remains poorly characterized due to the lack of a stable monoculture system.

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.

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.

This 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.