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Bio-fermentation technology has been successfully developed for ensiling rice straw; however, its effects on the particle-attached microbial community remains unknown. Therefore, rice straw (RS) and bio-fermented rice straw (BFRS) were used as substrates for in vitro rumen fermentation to investigate the effect of bio-fermentation on particle-attached microbial community, as well as their effects on gas and methane production, fermentation products, and fiber degradation. Our results have shown that total gas production, fiber degradation, and in vitro fermentation products were significantly higher (p < 0.05) for the BFRS than the RS, while methane concentration in total gas volume was significantly lower (p < 0.05) for the BFRS than RS. Linear discriminant effect size (LefSe) analysis revealed that the relative abundance of the phyla Bacteroidetes, Fibrobacteres, Proteobacteria, and Lantisphaerae, as well as the genera Fibrobacter, Saccharofermentans, and [Eubacterium] ruminantium groups in the tightly attached bacterial community, was significantly higher (p < 0.05) for the BFRS than the RS, whereas other microbial communities did not change. Thus, bio-fermentation altered the tightly attached bacterial community, thereby improving gas production, fiber degradation, and fermentation products. Furthermore, bio-fermentation reduced methane concentration in total gas volume without affecting the archaeal community. [ABSTRACT FROM AUTHOR]