** AWARDS ISSUED PRIOR TO JANUARY 20, 2025, WERE FUNDED UNDER PREVIOUS ADMINISTRATIONS AND MAY NOT REFLECT THE PRIORITIES AND POLICIES OF THE CURRENT ADMINISTRATION.** RUMINANTS (COWS, SHEEP, GOATS) ARE UNIQUE BECAUSE MOST OF THE PROTEIN THEY DIGEST COMES FROM MICROBES GROWING IN THE FORESTOMACH (RUMEN). INCREASING THIS MICROBIAL PROTEIN IS A PERENNIAL GOAL OF RUMINANT NUTRITION, BUT PROGRESS HAS STALLED BECAUSE MICROBES GROW WITH POOR EFFICIENCY AND FOR REASONS THAT ARE UNCLEAR. IN FACT, WE DO NOT KNOW HOW INEFFICIENT MICROBES REALLY ARE; IT IS UNCERTAIN HOW MUCH CELLULAR ENERGY (ATP) THEY GENERATE DURING METABOLISM AND HAVE AVAILABLE FOR GROWTH. OUR OVERARCHING HYPOTHESIS IS THAT MICROBES GROW WITH POOR EFFICIENCY BECAUSE THEY DIRECT ATP ENERGY AWAY FROM GROWTH AND TOWARDS ENERGY SINKS. WE FURTHER HYPOTHESIZE THAT MICROBES GENERATE MORE ATP ENERGY DURING METABOLISM THAN PREVIOUSLY REALIZED, AND THUS THEY GROW WITH EVEN POORER EFFICIENCY THAN THOUGHT. TO TEST THESE HYPOTHESES, WE PROPOSE THE SPECIFIC AIMS OF 1) DETERMINING HOW MUCH ATP ENERGY MICROBES EXPEND ON TWO ENERGY SINKS (MAINTENANCE AND GLYCOGEN SYNTHESIS) 2) DETERMINING HOW MUCH ATP ENERGYCELLS YIELD DURING METABOLISM OF CARBOHYDRATE (GLUCOSE). WE WILL USE EXPERIMENTS SPANNING FROM LABORATORY CULTURES OF BACTERIA TO DAIRY COWS, AND WE EXPECT OUR RESULTS TO SHOW BOTH ENERGY SINKS (MAINTENANCE, GLYCOGEN SYNTHESIS) ARE LARGE AND IMPORTANT TO DECREASING EFFICIENCY. HOWEVER, WE EXPECT THE EXACT IMPORTANCE OF EACH SINK VARIES WITH THE ANIMAL DIET AND MICROBIAL GROUP. WE FURTHER EXPECT OUR RESULTS TO SHOW THAT MICROBES GENERATE UP TO 50% MORE ATP ENERGY DURING METABOLISM OF GLUCOSE THAN PREVIOUSLY RECOGNIZED. WE ANTICIPATE THE IMPACT OF THIS PROJECT WILL BE TO REVEAL MECHANISMS BEHIND POOR GROWTH EFFICIENCY, GUIDE STRATEGIES TO INCREASE MICROBIAL EFFICIENCY, AND REDUCE FEEDING OF PROTEIN TO RUMINANTS. WE ANTICIPATE THAT WE COULD IMPROVE EFFICIENCY BY AT LEAST 5%, REDUCING FEED COSTS FOR THE US DAIRY INDUSTRY BY $122 MILLION/YR.