In this study, a comprehensive approach considering the operational performance, kinetic analysis and microbial ecology was employed to investigate the fermentative biohydrogen (bioH(2)) production from tequila vinasse. Batch fermentations were conducted in a 3-L stirred tank reactor at 35 degrees C and pH 6.5-5.8. The bioH(2) yield of 124 +/- 9 NmL/g VSadded and a peak bioH(2) production rate of 159 +/- 33 NmL/L-h were obtained. Thus, the energy yield and energy production rate were estimated as 1.5 kJ/g VSadded and 48.7 +/- 10.2 kJ/L-d, respectively. The metabolic profile and correlation analysis reflected that bioH(2) was associated with the consumption of lactate and acetate. The lactate-acetate pathway was consistent with the modeling of substrate consumption, product formation and biomass growth accurately described by the modified Gompertz model. Illumina MiSeq high-throughput sequencing revealed that Clostridium beijerinckii, Streptococcus sp. and Acetobacter lovaniensis were the main species at the highest bioH(2) production activity. The results obtained suggested that the lactate-acetate pathway can potentially be used to produce bioH(2) from tequila vinasse.