Disentangling The Complexity Of The Rumen Microbial Diversity Through Fractionation Using A Sucrose Density Gradient

The ruminal microbial community is an important element in health, nutrition, livestock productivity, and climate impact. Despite the historic and current efforts to characterize this microbial diversity, many of its members remain unidentified, making it challenging to associate microbial groups with functions. Here we present a low-cost methodology for rumen sample treatment that separates the microbial community based on cell size, allowing for the identification of subtle compositional changes. In brief, the sample is centrifuged through a series of sucrose density gradients, and cells migrate to their corresponding density fraction. 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. Microbial composition analysis using metabarcoding showed that our methodology detected low abundance bacterial families 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.