This study investigated the impacts of thiosulfate (S2O32-) as well as chemically synthesized and biogenic elemental sulfur (S-0) on the rates of sulfur-based denitrification in batch bioassays. The use of S2O32- resulted in the highest denitrification rate (52.5 mg N-NO3-/Ld), whereas up to 10 times slower nitrate (NO3-) removal was observed with S-0. Biogenic S-0 was tested for the first time as electron donor for chemolithotrophic denitrification, resulting in 1.7-fold faster NO3- removal than that achieved with chemically synthesized S-0. The effects of increasing concentrations of S2O32- and chemically synthesized S-0 on denitrification were evaluated testing three different sulfur-to-nitrogen (S/N) molar ratios (1.8, 3.5 and 5.1) on a pure culture of Thiobacillus denitrificans and a mixotrophic enrichment dominated by Thiobacillus thioparus. S2O32- concentrations exceeding 2.2 g/L inhibited the activity of T. denitrificans, whereas a stimulatory effect was observed on mixotrophic denitrification. The increase in S-0 concentration slightly enhanced denitrification by both microbial cultures due to the low solubility of chemically synthesized S-0. The temperature dependence of the thiosulfate-driven denitrification by T. denitrificans was investigated to further optimizethe process and modeled by the Arrhenius equation with an apparent activation energy E-a of 76.6 kJ/mol and a temperature coefficient Q(10) of 3.0. (C) 2016 Elsevier Ltd. All rights reserved.