Abstract: [Objective] To characterize the microbial structure of litters at different depths in dairy cow fermentation bed, and to assess the application effect of fermentation bed in dairy cow raising. [Method] The number of colonies of three commonly observed pathogenic bacteria in litters with or without fermentation bed was compared. During a two-year monitoring period, the incidence of limb-hoof disease, milk yield, milk somatic cell count （SCC） of the dairy cows raised with or without application of fermentation bed were determined and compared. In addition, the total number of bacteria in raw milk produced by dairy cows using varied litter supplements （rice husk and dried cow feces residue, wood sawdust, rice husk powder) of fermentation bed were evaluated. After two-year period of application, the samples of litters at surface layer （at 5 cm）, middle layer （at 25 cm） and deep layer （at 45-50 cm） were collected and subjected to examination of basic physical and chemical indicators. The distributive characteristics of bacteria and fungi of litters at different depths in fermentation bed were analyzed by using 16S rDNA and ITS1 based high-throughput sequencing methods, respectively. The metabolic functions of the microbial community of the fermentation bed were predicted by bioinformatics software. [Result] The number of colonies of Escherichia coli, Streptococci and Klebsiella in litters with fermentation bed was lower than that in litters without fermentation bed. During the two-year monitoring period, the dairy cows raised with application of fermentation bed had lower incidence of limb-hoof disease, significantly （P<0.05） higher average milk yield, and reduced average SCC in raw milk compared with their counterparts without application of fermentation bed. The lowest total number of bacteria in raw milk was observed in the dairy cows using rice husk and dried cow feces residue as litter supplements. The basic physical and chemical parameters （temperature, water content, pH value, and concentrations of ammonia and hydrogen sulfide） of the fermentation bed with rice husk and dried cow feces residue as litter supplements still met the production needs after two-year continuous and stable application. The micro-ecological community of the fermentation bed was dominated by bacterial flora. Bacteroidetes and Firmicutes were mostly distributed in the middle and deep layers, and Actinobacteria were mostly distributed in the surface layer. The samples of litters at different depths in fermentation bed had similar fungal community composition, and most of the fungi were distributed in the deep layer after fermentation. KEGG pathway enrichment analysis demonstrated that the bacterial community had a greater impact on the entire micro-ecosystem of the fermentation bed. The bacterial metabolism in the litters was the most active, and there were still a large number of fungal genera to be further annotated and explained. [Conclusion] The health status of dairy cows and the quality of their raw milk have been improved by applying the fermentation bed technology.

Key words: fermentation bed, dairy cow raising, microbial community, high-throughput sequencing, raw milk