Tansol Park

Rumen methanogens and mitigation of methane emission by anti-methanogenic compounds and substances

Methanogenic archaea reside primarily in the rumen and the lower segments of the intestines of ruminants, where they utilize the reducing equivalents derived from rumen fermentation to reduce carbon dioxide, formic acid, or methylamines to methane (CH4). Research on methanogens in the rumen has attracted great interest in the last decade because CH4 emission from ruminants contributes to global greenhouse gas emission and represents a loss of feed energy. Some DNA-based phylogenetic studies have depicted a diverse and dynamic community of methanogens in the rumen. In the past decade, researchers have focused on elucidating the underpinning that determines and affects the diversity, composition, structure, and dynamics of methanogen community of the rumen. Concurrently, many researchers have attempted to develop and evaluate interventions to mitigate enteric CH4 emission. Although much work has been done using plant secondary metabolites, other approaches such as using nitrate and 3-nitrooxy propanol have also yielded promising results. Most of these antimethanogenic compounds or substances often show inconsistent results among studies and also lead to adverse effects on feed intake and digestion and other aspects of rumen fermentation when fed at doses high enough to achieve effective mitigation. This review provides a brief overview of the rumen methanogens and then an appraisal of most of the antimethanogenic compounds and substances that have been evaluated both in vitro and in vivo. Knowledge gaps and future research needs are also discussed with a focus on methanogens and methane mitigation.

Inhibition of the Rumen Ciliate Entodinium caudatum by Antibiotics

Axenic cultures of free-living aerobic ciliates, such as Tetrahymena thermophila and Paramecium aurelia, have been established and routinely used in laboratory research, greatly facilitating, or enabling characterization of their metabolism, physiology, and ecology. Ruminal protozoa are anaerobic ciliates, and they play important roles in feed digestion and fermentation. Although, repeatedly attempted, no laboratory-maintainable axenic culture of ruminal ciliates has been established. When axenic ciliate cultures are developed, antibiotics are required to eliminate the accompanying bacteria. Ruminal ciliates gradually lose viability upon antibiotic treatments, and the resultant axenic cultures can only last for short periods of time. The objective of this study was to evaluate eight antibiotics that have been evaluated in developing axenic cultures of ruminal ciliates, for their toxicity to Entodinium caudatum, which is the most predominant ruminal ciliate species. Scanning and transmission electron microscopy (TEM) showed that the antibiotics damaged both the cell surface and nuclei of E. caudatum and increased accumulation of intracellular glycogen. Combinations of the three least toxic antibiotics failed to eliminate the bacteria that are present in the E. caudatum culture. The combination of ampicillin, carbenicillin, streptomycin, and oxytetracycline was able to eliminate all the bacteria, but the resultant axenic E. caudatum culture gradually lost viability. Adding the bacterial fraction (live) separated from an untreated E. caudatum culture reversed the viability decline and recovered the growth of the treated E. caudatum culture, whereas feeding nine strains of live bacteria isolated from E. caudatum cells, either individually or in combination, could not. Nutritional and metabolic dependence on its associated bacteria, accompanied with direct and indirect inhibition by antibiotics, makes it difficult to establish an axenic culture of E. caudatum. Monoxenic or polyxenic cultures of E. caudatum could be developed if the essential symbiotic partner(s) can be identified.

Do Ruminal Ciliates Select Their Preys and Prokaryotic Symbionts

Ruminal ciliates both preys on and form symbiotic relationships with other members of the ruminal microbiota for their survival. However, it remains elusive if they have selectivity over their preys or symbionts. In the present study, we investigated the above selectivity by identifying and comparing the free-living prokaryotes (FLP) and the ciliate-associated prokaryotes (CAP) using Illumina MiSeq sequencing of 16S rRNA gene amplicons. We used single ciliates cells of both monocultures of Entodinium caudatum and Epidinium caudatum and eight different ciliate genera isolated from fresh rumen fluid of dairy cows. Irrespective of the source (laboratory monocultures vs. fresh isolates) of the single ciliate cells, the CAP significantly differed from the FLP in microbiota community profiles. Many bacterial taxa were either enriched or almost exclusively found in the CAP across most of the ciliate genera. A number of bacteria were also found for the first time as ruminal bacteria in the CAP. However, no clear difference was found in methanogens between the CAP and the FLP, which was confirmed using methanogen-specific qPCR. These results suggest that ruminal ciliates probably select their preys and symbionts, the latter of which has rarely been found among the free-living ruminal prokaryotes. The bacteria enriched or exclusively found in the CAP can be target bacteria to detect and localize using specific probes designed from their 16S rRNA sequences, to characterize using single-cell genomics, or to isolate using new media designed based on genomic information.

— Invited Review — Metagenomic investigation of gastrointestinal microbiome in cattle

The gastrointestinal (GI) tract, including the rumen and the other intestinal segments of cattle, harbors a diverse, complex, and dynamic microbiome that drives feed digestion and fermentation in cattle, determining feed efficiency and output of pollutants. This microbiome also plays an important role in affecting host health. Research has been conducted for more than a century to understand the microbiome and its relationship to feed efficiency and host health. The traditional cultivation-based research elucidated some of the major metabolism, but studies using molecular biology techniques conducted from late 1980’s to the late early 2000’s greatly expanded our view of the diversity of the rumen and intestinal microbiome of cattle. Recently, metagenomics has been the primary technology to characterize the GI microbiome and its relationship with host nutrition and health. This review addresses the main methods/techniques in current use, the knowledge gained, and some of the challenges that remain. Most of the primers used in quantitative real-time polymerase chain reaction quantification and diversity analysis using metagenomics of ruminal bacteria, archaea, fungi, and protozoa were also compiled.

Draft Macronuclear Genome Sequence of the Ruminal Ciliate Entodinium caudatum

The transcriptionally active macronucleus of a ruminal ciliate, Entodinium caudatum MZG-1, was sequenced using the Illumina MiSeq and Oxford Nanopore MinION platforms. This is the first draft macronuclear genome sequence of a ruminal protozoon, and the genomic information will provide useful insight into the metabolism, physiology, and ecology of ruminal ciliates. ABSTRACT The transcriptionally active macronucleus of a ruminal ciliate, Entodinium caudatum MZG-1, was sequenced using the Illumina MiSeq and Oxford Nanopore MinION platforms. This is the first draft macronuclear genome sequence of a ruminal protozoon, and the genomic information will provide useful insight into the metabolism, physiology, and ecology of ruminal ciliates.

Aerobic cultivation of anaerobic rumen protozoa, Entodinium caudatum and Epidinium caudatum

Rumen protozoa, primarily ciliates, are one of the important groups of strictly anaerobic microbes living in the rumen. Despite their ubiquitous occurrence in the rumen and significant contribution to host animals, it is still poorly understood why they live only in the rumen and similar environment. Because rumen protozoa require strict anaerobic conditions to sustain their viability and grow, only a few laboratories equipped with protozoology expertise and anaerobic facilities can grow rumen protozoa in laboratory. Also for the same reason, only a few species have been grown and maintained as laboratory cultures for research. Prompted by a recent study, we hypothesized that anaerobic rumen protozoa could also be cultivated aerobically if antioxidants were included in the media. Indeed, our experiments showed that the cultures of both Entodinium caudatum and Epidinium caudatum, two major rumen protozoal species, could be cultured successfully in aerobic media supplemented with ascorbic acid, glutathione and α-ketoglutarate as antioxidants. Anaerobic fermentation was maintained through the fermentation characteristics and microbial populations were altered to some extent under aerobic conditions. The antioxidants also enhanced the revival of cryopreserved stock cultures of both rumen protozoal species. The results of this study may facilitate and promote future research in which rumen protozoa need to be cultured in laboratory.

Corrigendum: Inhibition of the Rumen Ciliate Entodinium caudatum by Antibiotics

[This corrects the article on p. 1189 in vol. 8, PMID: 28702015.].