Young Scholar Presentation: Towards a Better Understanding of the Metabolism, Physiology and Ecology of Rumen Protozoa: New Insights from Culturomics and Genomics

Rumen ciliates contribute to the digestion of feed, including fiber, and to the physiological stability of the rumen environment. Many researchers have attempted to establish axenic cultures of ruminal protozoa using antibiotics, but no maintainable axenic culture was ever established. Thus, the first study investigated the toxicity of antibiotics to Entodinium caudatum. We concluded that antibiotics are directly toxic to Ent. caudatum and indirectly inhibitory by killing the prokaryotes that are essential for its survival. This species may have to be cultured as monoxenic or multixenic cultures.

The rumen microbiome consists of various guilds of microbes, both prokaryotes and eukaryotes. The interaction between ruminal protozoa and amino acid-fermenting bacteria (AAFB) has important implications to nitrogen utilization inefficiency in ruminants. In our study, washed cells of Entodinium caudatum and AAFB served as the inocula, and they were cultured as single cultures or co-cultures of both. Mutualistic interaction was noted between Ent. caudatum and AAFB, but their interaction might not enhance ammoniagenesis under the experimental conditions.

The prokaryotes associated with ruminal protozoa were analyzed by identifying and comparing the free-living prokaryotes (FLP) and those recovered together with washed single ruminal protozoal cells collected from monocultures and fresh rumen fluid. Protozoa-associated prokaryotes (PAP) and FLP were identified and compared between the two fractions. The community of PAP significantly differed from that of FLP. Across different ruminal protozoa and irrespective of sources, PAP-specific bacteria were found, and some of them may be true symbionts and essential for the survival of ruminal protozoa.

Finally, we sequenced the macronuclear genome to help understand Ent. caudatum as a model species of ruminal protozoa. The draft macronuclear genome of Ent. caudatum revealed many nanochromosomes. More than 13,490 protein-coding genes were predicted and annotated. The draft genome provided genomic evidence on the substrate spectrum, metabolic pathways, and physiological features.

Collectively, using multipronged approaches, the findings in these studies shine new lights on the metabolism, physiology, and ecology of ruminal protozoa. The approaches can help future studies on ruminal protozoa in general and their roles in ruminal functions.