Periodic Reporting for period 1 - RUMIC (Prebiotic Functional Enhancement of Rumen Microbiomes) Reporting period: 2019-05-01 to 2021-04-30 Summary of the context and overall objectives of the project The research project entitled RUMIC: Prebiotic Functional Enhancements of Rumen Microbiomes investigates how prebiotics act upon the rumen microbiome of food-producing animals to determine how we can use prebiotics to address the worlds growing demand for sustainable food production.In recent years research has shown that the microbiome - the community of microorganisms in animal and human digestive systems - is an integral component of animal health and production. Yet, our investigation of animal microbiomes and how they are affected by diet and supplements is still in its infancy. If we wish to move towards a greener economy, we must rethink our livestock production to include microbiomes and focus on sustainable and secure food production.One approach is the application of prebiotics, “a substrate that is selectively utilised by host microorganisms conferring a health benefit” (Gibson 2018). Prebiotics function by modulating an animal’s microbiome towards improved food digestion and increase milk and meat production. They can also confer added health benefits such as antipathogenic and anti-inflammatory effects. Another added benefit of prebiotics is that they can be sourced from agricultural residues or sustainable sources such as seaweeds and, therefore, add economic value to agricultural waste streams. Prebiotics are a promising tool to increase animal health, performance, and productivity sustainably. The RUMIC project aims to investigate how prebiotics affect rumen microbiomes and their function in animals, to provide evidence-based research tools to study the direct effect of prebiotics and help address the worlds growing demand for food production. Work performed from the beginning of the project to the end of the period covered by the report and main results achieved so far In the first phase the RUMIC project investigated whether glycans found in human and livestock diets could be fluorescently labelled and whether the produced fluorescently labelled polysaccharides (FLA-PS) could be used to visualise selective glycan metabolism in rumen isolates and complex rumen microbiomes.Dr. Greta Reintjes, in collaboration with the Abbott Lab of the Lethbridge Research and Development Center, Agriculture and Agri-Food Canada, successfully labelled two prominent dietary glycans, yeast -mannan (YM) and rhamnogalacturonan-II (RGII), and incubated these with the model gut Bacteroidetes thetaiotaomicron VPI-5482 (B.theta). We could show, for the first time, that FLA-PS can be used to visualise the selective glycan metabolism of B.theta. In addition, we could demonstrate the differential uptake of FLA-PS by individual cells in a population through a combination of super-resolution structured illumination microscopy and flow cytometry. Furthermore, using genetically modified B.theta strains, we could also show that the FLA-PS uptake was highly specific and dependent on the presence of polysaccharide utilization loci (PULs).This investigation of the RUMIC project highlighted that FLA-PS are a novel functional tool that can be applied to rapidly assign metabolic phenotypes in rumen microorganisms and can be used to decipher glycan-microbial interactions in microbiomes. These findings were published in a short communication in The ISME Journal. Hehemann, J. H., Reintjes, G., Klassen, L., Smith, A. D., Ndeh, D., Arnosti, C., Amann, R. and Abbott, D. W. (2019) 'Single cell fluorescence imaging of glycan uptake by intestinal bacteria', ISME J, 13(7), pp. 1883-1889. https://doi.org/10.1038/s41396-019-0406-z Dr. Reintjes also investigated whether the novel FLA-PS could be used to identify individual cell physiologies in bovine B.theta strains. The bovine B.theta isolates consume YM using the same set of proteins encoded on PULs as the model B.theta strain. Compellingly, however, the bovine B.theta strains showed two distinct growth phenotypes on YM for which there were no distinct genotypic explanations. Using FLA-PS, Dr. Reintjes could visualise the metabolism of YM by individual bovine B.theta strains and identify strain-level variability in the carbohydrate utilisation systems of the two foraging phenotypes. Furthermore, this new next-generation physiology approach combined with "omics" approaches enabled her to characterise microbial adaptations to a prebiotic (YM) in the rumen ecosystem. These findings were published in the journal Microbiome. Klassen, L., Reintjes, G., Tingley, J. P., Jones, D. R., Hehemann, J. H., Smith, A. D., Schwinghamer, T. D., Arnosti, C., Jin, L., Alexander, T. W., Amundsen, C., Thomas, D., Amann, R., McAllister, T. A. and Abbott, D. W. (2021) 'Quantifying fluorescent glycan uptake to elucidate strain-level variability in foraging behaviors of rumen bacteria', Microbiome, 9(1), pp. 23. https://doi.org/10.1186/s40168-020-00975-xFurther preliminary work performed within the RUMIC project includes a culture-independent analysis of prebiotic enhancements on glycan-microbial interactions in simplified artificial and complex rumen systems. The two investigations were performed at the Lethbridge Research and Development Center using a rumen simulated technique (RUSITEC), an in vitro artificial rumen system that simulates the microbial rumen processes in a regulated environment, and live animal feed trials.These investigations applied the novel approach of targeting cell-specific physiologies using FLA-PS to illuminate the functional potential encoded within each microbial community. Preliminary results demonstrate that both artificial and native rumen microbiomes respond rapidly to the addition of prebiotics and that there is a functional adaptation to the presence of specific prebiotics from agricultural and seaweed sources. The data analysis of these investigations is still in progress and will be finalized in the project's final phase.Finally, Dr. Reintjes has successfully performed the functional sorting of metabolically active cells from a complex microbial community, as proposed in the RUMIC project. This novel approach enables a targeted analysis of specific metabolic capacities and the discovery of novel microorganisms, pathways, and enzymes with specific functional abilities. The first functional sorting of metabolically active rumen microorganisms confirms that fluorescence-activated cell sorting using FLA-PS is possible and that the sorted microbes can be directly identified using 16S rRNA sequencing and metagenomics. The data analysis of this investigation is still in progress and will be finalized in the final phase of the project. Progress beyond the state of the art and expected potential impact (including the socio-economic impact and the wider societal implications of the project so far) RUMIC is a transatlantic collaboration between research labs in Germany and Canada, which has ushered in a next-generation physiological approach to study prebiotic metabolism in complex microbial communities. The scientific discoveries of RUMIC have provided direct evidence of prebiotic effects in livestock and increasing our knowledge of how prebiotics are metabolised within the rumen microbiome. The RUMIC project will continue to investigate how rumen microbiomes respond to prebiotic and help provide evidence-based tools to study the effect of prebiotics on feed efficiency, aiming to enhance animal health and enable a more sustainable and secure food production. RUMIC: Prebiotic Functional Enhancements of Rumen Microbiomes
