Study of rumen bacterial diversity using a sucrose density gradient to separate it by size and density. In this way, it is possible to enriched in the fractions of the gradient, the low abundant bacteria in the rumen and performs shotgun sequencing which will facilitate to know the function of these bacterial groups.

The ruminal microbial community is an important element in the study of livestock productivity, health, and climate impact. Despite the historical and current efforts to characterize this microbial diversity much of their members remains unidentified, making it challenging to associate microbial groups with functions. Here we present a low-cost methodology for ruminal sample treatment that separates the microbial community based on cell size, allowing for identification of subtle compositional changes. In brief, the sample is centrifuged through a series of sucrose density gradients, where cells remain in the fraction with the closest density. From each fraction, DNA is extracted and 16S rRNA gene amplicons are sequenced. We tested our methodology on four animals under two different conditions, fasting and post-feeding. Each fraction was examined by confocal microscopy showing that the same sucrose fraction consistently separated similar cell-sized microorganisms independent of the animal or treatment. The microbial composition analysis using metabarcoding showed that our methodology detected low abundance bacterial families and subtle family and population changes between fasting and post-feeding treatments that could not be observed by bulk DNA analysis. In conclusion, the sucrose-based method is a powerful low-cost approximation to untwine, enrich, and potentially isolate uncharacterized members of the ruminal microbiome.