Live Bacillus subtilis natto Promotes Rumen Fermentation by Modulating Rumen Microbiota In Vitro

SIMPLE SUMMARY: Although there is much research on the applications of Bacillus subtilis natto in dairy cows, the regulation of it on rumen microorganisms and the mechanisms of microbiota that affect rumen fermentation is still unclear, such as the mechanism of improving ruminal ammonia nitrogen concentration and the pathway of increasing propionic acid production. In this study, we explored the effects of live and autoclaved B. subtilis natto on rumen microbiota in vitro by 16S rRNA gene sequencing to clarify the ruminal microbial composition and diversity and their underlying mechanisms. ABSTRACT: Previous studies have shown that Bacillus subtilis natto affects rumen fermentation and rumen microbial community structure, which are limited to detect a few microbial abundances using traditional methods. However, the regulation of B. subtilis natto on rumen microorganisms and the mechanisms of microbiota that affect rumen fermentation is still unclear. This study explored the effects of live and autoclaved B. subtilis natto on ruminal microbial composition and diversity in vitro using 16S rRNA gene sequencing and the underlying mechanisms. Rumen fluid was collected, allocated to thirty-six bottles, and divided into three treatments: CTR, blank control group without B. subtilis natto; LBS, CTR with 10(9) cfu of live B. subtilis natto; and ABS, CTR with 10(9) cfu of autoclaved B. subtilis natto. The rumen fluid was collected after 0, 6, 12, and 24 h of fermentation, and pH, ammonia nitrogen (NH(3)-N), microbial protein (MCP), and volatile fatty acids (VFAs) were determined. The diversity and composition of rumen microbiota were assessed by 16S rRNA gene sequencing. The results revealed LBS affected the concentrations of NH(3)-N, MCP, and VFAs (p < 0.05), especially after 12 h, which might be attributed to changes in 18 genera. Whereas ABS only enhanced pH and NH(3)-N concentration compared with the CTR group (p < 0.05), which might be associated with changes in six genera. Supplementation with live B. subtilis natto improved ruminal NH(3)-N and propionate concentrations, indicating that live bacteria were better than autoclaved ones. This study advances our understanding of B. subtilis natto in promoting ruminal fermentation, providing a new perspective for the precise utilization of B. subtilis natto in dairy rations.