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Sulfur-based denitrification may be a key biogeochemical nitrate (NO 3  − ) removal process in sulfide-rich regions, but it is still poorly understood in natural terrestrial ecosystems. We examined sulfur-driven NO 3  −  reduction using streambank soils in a headwater catchment underlain by marine sedimentary rock in Akita, Japan. In a catchment exhibiting higher sulfide content in streambed sediment, we sampled two adjacent streambank soils of streambank I (two layers) and of streambank II (eight layers). Anaerobic long-term incubation experiments (40 days, using soils of streambank I) and short-term incubation experiments (5 days, using soils of streambank II) were conducted to evaluate variations of N solutes (NO 3  − , NO 2  − , and NH 4  + ), N gases (NO, N 2 O), and the bacterial flora. In both experiments, two treatment solutions containing NO 3  −  (N treatment), and NO 3  −  and S 2 O 3  2−  (N + S treatment) were prepared. In the N + S treatment of the long-term experiment, NO 3  −  concentrations gradually decreased by 98%, with increases in the SO 4  2− , NO 2  − , NO, and N 2 O concentrations and with not increase in the NH 4  + , indicating denitrification had occurred with a high probability. Temporal accumulation of NO 2  −  was observed in the N + S treatment. The stoichiometric ratio of SO 4  2−  production and NO 3  −  depletion rates indicated that denitrification using reduced sulfur occurred even without additional S, indicating inherent S also served as an electron donor for denitrification. In the short-term incubation experiment, S addition was significantly decreased NO 3  −  concentrations and increased NO 2  − , NO, and N 2 O concentrations, especially in some subsoils with higher sulfide contents. Many denitrifying sulfur-oxidizing bacteria ( Thiobacillus denitrificans  and  Sulfuricella denitrificans ) were detected in both streambank I and II, which dominated up to 5% of the entire microbial population, suggesting that these bacteria are widespread in sulfide-rich soil layers in the catchment. We concluded that the catchment with abundant sulfides in the subsoil possessed the potential for sulfur-driven NO 3  −  reduction, which could widely influence N cycling in and NO 3  −  export from the headwater catchment.

Sulfur-Based Denitrification in Streambank Subsoils in a Headwater Catchment Underlain by Marine Sedimentary Rocks in Akita, Japan