Screening the ruminal microbiota of dairy cows using culturomics to identify ammonia utilizing bacteria to improve rumen nitrogen metabolism

Improving the efficiency of nitrogen utilization is a critical challenge in ruminant production, with current nitrogen utilization efficiency (NUE) averaging only 25%. This inefficiency not only limits productivity but also contributes to environmental pollution. This thesis focuses on the isolation, functional characterization, and application of rumen bacteria, particularly ammonia-utilizing strains, to improve NUE in ruminants. By integrating culturomics, in vitro screening, and in vivo studies, this research aims to develop innovative microbial solutions for sustainable livestock production. In Chapter 3, culturomics was applied to isolate and identify rumen bacteria from dairy cows. Using 15 culture conditions with different media and gas environments, 203 strains were isolated, representing 7.62% of the operational taxonomic units (OTUs) in the rumen microbiota. Among these, 8 potential novel species were identified. Strains such as Limosilactobacillus, Ligilactobacillus, and Bacillus were highlighted for their probiotic potential. This chapter demonstrates the utility of culturomics in expanding the rumen microbial library and identifying functional strains. In Chapter 4, ammonia-utilizing bacteria were screened from the isolated strains. Among the 115 evaluated strains, Ligilactobacillus agilis W70 exhibited the highest ammonia utilization capacity, assimilating 32.09% of ammonium sulfate in tube screening tests. In vitro fermentation experiments confirmed its functionality, with L. agilis W70 significantly reducing ammonia nitrogen (NH3N) (P < 0.01) and increasing microbial protein (MCP) synthesis (P < 0.01). Genomic analysis revealed the presence of gdhA and glnA genes, which encode key enzymes involved in ammonia assimilation. In Chapter 5, the dose-dependent effects of L. agilis W70 were evaluated in vitro using diets with different rumen degradable protein (RDP) levels. The results showed that at 1×1010 cfu/mL, L. agilis W70 enhanced MCP production (P < 0.05) and reduced the acetate-to-propionate ratio (P < 0.01) in low RDP diets. These findings establish the optimal dosage and dietary conditions for applying L. agilis W70 to maximize fermentation efficiency and nitrogen utilization. In Chapter 6, the in vivo effects of L. agilis W70 supplementation were tested in lactating Holstein cows under low and high dietary protein levels. Supplementation improved NUE (P < 0.01), feed conversion efficiency (P < 0.01), reduced NH3N concentration and increased OTU 60 Solibacillus silvestris, OTU 28 Prevotella NA, and OUT 152 Bacillus psychrosaccharolyticus in the rumen (P < 0.01), but not affect the milk production. Although 17% CP diets increased milk yield (P < 0.05), they also elevated nitrogen excretion, emphasizing the importance of balancing dietary protein with microbial additives. This thesis demonstrates that integrating culturomics and functional microbial supplementation offers a promising strategy for addressing the inefficiencies in ruminant nitrogen metabolism. The findings provide a foundation for developing sustainable feed additives, such as L. agilis W70, to enhance productivity while mitigating environmental impacts. Future research should focus on long-term validation, environmental impact assessments, and scalability to ensure widespread adoption in the livestock industry.