In the present work, a two-stage anaerobic digestion system (TSADS) was newly designed to produce biogas with a greatly reduced H2S content. The role of first (sulfidogenic)-stage digester was not only acidogenesis but also sulfidogenesis (sulfate reduction to H2S), which would minimize the input of H2S-producing source in the followed second (methanogenic)-stage digester. For the coexistence of acidogens and sulfate reducing bacteria (SRB) in the sulfidogenic-stage digester, it was found that pH played a crucial role. The acidogenic activity was not affected within a pH range of 4.5-6.0, while it was important to maintain a pH at 5.5 to achieve a sulfate removal efficiency over 70%. The highest sulfate removal attained was 78% at a hydraulic retention time (HRT) of 5h at pH 5.5 +/- 0.1. The H2S content in the biogas produced in the conventional single-stage digester (SSAD), used as a control, reached 1,650 +/- 25ppm(v). In contrast, the biogas produced in the methanogenic-stage digester of the developed process had an H2S content of 200 +/- 15ppm(v). Microbial analysis, done by the next generation sequencing technique, clearly showed the changes in community under different operating conditions. Desulfovibrio bastinii (4.9%) played a key role in sulfate removal in the sulfidogenic-stage of the TSADS owing to its characteristics of a short doubling time and growth in an acidic environment. Biotechnol. Bioeng. 2017;114: 970-979. (C) 2016 Wiley Periodicals, Inc.