Disentangling the Complexity of the Rumen Microbial Diversity Through Fractionation Using a Sucrose Density Gradient.

Ruth Hernández,Alejandro Caro-Quintero,Cesar Vargas-Garcia,Alejandro Reyes,Hugo Jimenez

doi:10.3389/fmicb.2021.664754

Abstract

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.

Highlights

Ruminants contribute to food security by providing adequate protein and energy to the human population (Matthews et al, 2018)
The Sucrose Density Gradient Allows for the Separation of the Microbial Community by Size
We developed a methodology to disentangle the complex composition of the ruminal microbial community using sucrosebased fractionation of microbial cells

Summary

Introduction

Ruminants contribute to food security by providing adequate protein and energy to the human population (Matthews et al, 2018) These animals have developed a unique ability to convert plant cell wall carbohydrates into meat and milk, mostly due to the complex and not completely characterized symbiotic microbiota in their rumen (Morgavi et al, 2013; Ribeiro et al, 2016). Many studies conducted in the rumen have tried to understand the numbers, composition, and function of the ruminal microbial community, mostly using conventional culture-based methods (Morgavi et al, 2013; Ribeiro et al, 2016) These types of methods have only allowed for the isolation and cultivation of about 15% of the estimated bacterial species potentially present in the rumen (Ribeiro et al, 2016). For most of the ruminal microorganisms, little is known about their functional role, and many of them have not been taxonomically classified at the genus or species level (Henderson et al, 2015; Puniya et al, 2015)

Methods

Results

Conclusion