Fresh CM was chosen as an inoculum source due to its high microbial diversity and capacity to supply essential nutrients, making it beneficial for establishing mixed anaerobic cultures . Unlike AD and AS, which are often pre-adapted to anaerobic digestion, CM introduces a broader microbial community that could adapt to CO-rich conditions. We hypothesized that inoculating with fresh CM in a CO-fed TBR system would drive shifts in microbial community composition, enhancing CO utilization as the primary carbon and energy source. We analyzed microbial communities in the inoculum and biofilm and sump from TBRs at two steady states using 16S/18S rRNA gene sequencing. These results provided new insights into how inoculum source diversity impacts biofilm establishment and microbial community dynamics in TBRs, informing strategies for optimizing CO conversion and methane production in continuous bioprocessing systems.