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.
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