Lower methane emissions were associated with higher abundance of ruminal Prevotella in a cohort of Colombian buffalos

Abstract

Background: Ruminants burp massive amounts of methane into the atmosphere and significantly contribute to the deposition of greenhouse gases and the consequent global warming. It is therefore urgent to devise strategies to mitigate ruminant's methane emissions to alleviate climate change. Ruminal methanogenesis is accomplished by a series of methanogen archaea in the phylum Euryarchaeota, which piggyback into carbohydrate fermentation by utilizing residual hydrogen to produce methane. Abundance of methanogens, therefore, is expected to affect methane production. Furthermore, availability of hydrogen produced by cellulolytic bacteria acting upstream of methanogens is a rate-limiting factor for methane production. The aim of our study was to identify microbes associated with the production of methane which would constitute the basis for the design of mitigation strategies.

Results: Moderate differences in the abundance of methanogens were observed between groups. In addition, we present three lines of evidence suggesting an apparent higher abundance of a consortium of Prevotella species in animals with lower methane emissions. First, taxonomic classification revealed increased abundance of at least 29 species of Prevotella. Second, metagenome assembly identified increased abundance of Prevotella ruminicola and another species of Prevotella. Third, metabolic profiling of predicted proteins uncovered 25 enzymes with homology to Prevotella proteins more abundant in the low methane emissions group.

Conclusions: We propose that higher abundance of ruminal Prevotella increases the production of propionic acid and, in doing so, reduces the amount of hydrogen available for methanogenesis. However, further experimentation is required to ascertain the role of Prevotella on methane production and its potential to act as a methane production mitigator.

Keywords: Methane emissions; Methanogenesis; Prevotella abundance; Propionic acid synthesis.

Fig. 1

Bioinformatics pipeline used for data analysis. Twelve buffaloes were included in each group. After quality control, individual sequences were taxonomically classified with Kraken2 and functionally analyzed with HUMAnN2. Combined assembly was conducted with SPAdes and assembled contigs were annotated with Prokka. In parallel, binning of contigs was conducted with MetaBAT2 and such bins were phylogenetically analyzed with MAGpy. De novo assembly of proteins was carried out with PLASS. Protein sequences annotated with Prokka or assembled with PLASS were consolidated and clustered with Linclust to determine a set of non-redundant representative sequences, which were aligned against several protein databases using Diamond. All statistical comparisons were conducted with LEfSe. A description of all command lines used is in the bitbucket repository (https://github.com/buffGenomic/PipelineColBuff)

Fig. 2

Characterization of the rumen microbiome in buffalo cohorts. a Principal coordinate analysis plot. Permanova analysis between groups showed a non-significant p-value. Taxonomic classification of sequences at the phylum (b), order (c), family (d), and genus (e). Prevotella or upper taxa containing it are in pink colors in panels b-e

Fig. 3

Hierarchical clustering of the 100 most abundant taxa using Bray-Curtis dissimilarities and the hclust method. Green bars on the right side of the heatmap indicate Prevotella species. Red bars indicate Ruminococcus species. Blue bars indicate Butyrivibrio species

Fig. 4

Statistical comparison of taxa. a Linear discriminant analysis results. LEfSe was run with default parameters. b Box plots presenting the relative abundance of 12 Prevotella species in samples associated with high or low methane emissions

Fig. 5

Metagenome assembled genomes (MAGs). a Phylogenetic tree generated by MAGpy. b Relative contribution of each sample to each putative genome

Fig. 6

Summary of bacterial protein identification. a Hierarchical clustering of the 100 most abundant proteins delineated two clusters of samples (see upper dendrogram). b Relative abundance of proteins in animals with high or low methane emissions. Proteins with homology to Prevotella proteins are highlighted with green squares