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AIMS The purpose of this study was to characterize the prokaryotic community and putative microbial interactions between H2 -producing bacteria (HPB) and non-HPB using an anaerobic sequencing batch biofilm reactor (AnSBBR) fed with tequila vinasses in co-digestion with acid hydrolysates of Agave tequilana var. azul bagasse (ATAB). METHODS AND RESULTS The AnSBBR was operated for H2 production to correlate changes in physicochemical and biological variables by Principal Components Analysis (PCA). Results indicated that H2 yield was supported by Ethanoligenens harbinense and Clostridium tyrobutyricum through the PFOR pathway. However, only E. harbinense was able to compete for sugars against non-HPB. Competitive exclusion associated with competition for sugars, depletion of essential trace elements, bacteriocin production, and resistance to inhibitory compounds could be carried out by non-HPB, increasing their relative abundances during the dark fermentation (DF) process. CONCLUSIONS The global scenario obtained by PCA correlated the decrease of H2 production with the lactate:acetate molar ratio in the influent. At the beginning of co-digestion, this ratio had the minimum value considered for a net gain of ATP. This fact could cause the reduction of the relative abundance of C. tyrobutyricum. SIGNIFICANCE and Impact of the Study This is the first study that demonstrated the feasibility of H2 production by Clostridiales from acid hydrolysates of ATAB in co-digestion with tequila vinasses.