In the biotechnological desulfurization process under haloalkaline conditions, dihydrogen sulfide (H 2 S) is removed from sour gas and oxidized to elemental sulfur (S 8 ) by sulfide-oxidizing bacteria. Besides S 8 , the byproducts sulfate (SO 4 2- ) and thiosulfate (S 2 O 3 2- ) are formed, which consume caustic and form a waste stream. The aim of this study was to increase selectivity toward S 8 by a new process line-up for biological gas desulfurization, applying two bioreactors with different substrate conditions (i.e., sulfidic and microaerophilic), instead of one (i.e., microaerophilic). A 111-day continuous test, mimicking full scale operation, demonstrated that S 8 formation was 96.6% on a molar H 2 S supply basis; selectivity for SO 4 2- and S 2 O 3 2- were 1.4 and 2.0% respectively. The selectivity for S 8 formation in a control experiment with the conventional 1-bioreactor line-up was 75.6 mol %. At start-up, the new process line-up immediately achieved lower SO 4 2- and S 2 O 3 2- formations compared to the 1-bioreactor line-up. When the microbial community adapted over time, it was observed that SO 4 2- formation further decreased. In addition, chemical formation of S 2 O 3 2- was reduced due to biologically mediated removal of sulfide from the process solution in the anaerobic bioreactor. The increased selectivity for S 8 formation will result in 90% reduction in caustic consumption and waste stream formation compared to the 1-bioreactor line-up.