? PROJECT 3 The production of butyrate from gut microbiomes results from interactions between microbes in anaerobic food webs. Identifying butyrogenic combinations of microbes, environmental conditions and fermentable fibers will underlie strategies for manipulating the microbiomes in BMT patients to provide therapeutic concentrations of butyrate. Hypotheses: 1) Maximal production of SCFAs from fermentable fibers requires the combination of a primary fiber degraders, secondary fermenters and hydrogen-consuming microbes. 2) The ratio of butyrate to total SCFAs is dependent on the taxonomic membership of communities, which is selected by the concentrations of H2, bile acids, pH and turnover time of the environment. Approaches: We will use covariation analysis of microbiomes from a healthy human cohort to identify butyrogenic combinations of fermentable fibers, physical conditions and microbes. We will test these predictions by assembling synthetic communities of the identified microbes and measuring butyrate production in vitro under various conditions, including the removal of hydrogen by hydrogenotrophic microbes. We will also select co-evolved butyrogenic communities from fecal inocula using multiple passages through media containing fermentable fiber as the primary carbon and energy source. Once transported into an intestinal epithelial cell, butyrate can be oxidized by mitochondria. It and can also stimulate mitochondrial biogenesis through Peroxisome Proliferator-Activated Receptors (PPARs) located on the nucleus. Understanding the interaction between a butyrogenic microbiome and epithelial cells will provide a mechanistic explanation of the temporal dynamics between butyrate production and host cell respiration. Hypothesis: In vitro and in vivo respiration and mitochondrial biogenesis in colonic epithelial cells will be stimulated by butyrate. Approaches: Respiration and PPAR activation will be measured in enteroids exposed to butyrate under varying environmental conditions. For in vivo estimates of respiration, mice at six weeks of age will be placed on a Western diet supplemented with a fermentable fiber(s) or accessible starch. GI tissues will be harvested at intervals up to 12 weeks and succinate dehydrogenase and cytochrome oxidase activities will be measured to quantify the capacity for cellular respiration. Cell respiration and mucosal O2 concentrations will be tracked in germ-free mice treated 5- aminosalicylic acid, a PPAR agonist, to distinguish between mitochondrial biosynthesis and butyrate oxidation. Mice colonized with synthetic communities of microbes to be used to test the impact of microbiomes of different butyrogenic capacity on respiration.