Abstract
BACKGROUND: Feed shortage is an important factor limiting livestock production in the world. To effectively utilize natural woody plant resources, we used wilting and microbial additives to prepare an anaerobic fermentation feed of mulberry, and used PacBio single-molecule real-time (SMRT) sequencing technology to analyse the "enzyme-bacteria synergy" and fermentation mechanism. RESULTS: The fresh branches and leaves of mulberry have high levels of moisture and nutrients, and also contain a diverse range of epiphytic microorganisms. After ensiling, the microbial diversity decreased markedly, and the dominant bacteria rapidly shifted from Gram-negative Proteobacteria to Gram-positive Firmicutes. Lactic acid bacteria (LAB) emerged as the dominant microbial population, resulting in increased in the proportion of the carbohydrate metabolism and decreased in the proportion of the amino acid and "global and overview map" (GOM) metabolism categories. The combination of cellulase and LAB exhibited a synergistic effect, through which cellulases such as glycanase, pectinase, and carboxymethyl cellulase decomposed cellulose and hemicellulose into sugars. LAB converted these sugars into lactic acid through the glycolytic pathway, thereby improving the microbial community structure, metabolism and fermentation quality of mulberry silage. The GOM, carbohydrate metabolism, and amino acid metabolism were the main microbial metabolic categories during ensiling. The presence of LAB had an important effect on the microbial community and metabolic pathways during silage fermentation. A "co-occurrence microbial network" formed with LAB, effectively inhibiting the growth of harmful microorganisms, and dominating the anaerobic fermentation process. CONCLUSIONS: In summary, PacBio SMRT was used to accurately analyse the microbial network information and regulatory mechanism of anaerobic fermentation, which provided a scientific basis for the study of woody silage fermentation theory. This study reveals for the first time the main principle of the enzyme-bacteria synergy in a woody silage fermentation system, which provides technical support for the development and utilization of woody feed resources, and achieves sustainable livestock production.