Nitrogen, carbon, and sulfur isotopic change during heterotrophic (Pseudomonas aureofaciens) and autotrophic (Thiobacillus denitrificans) denitrification reactions

• Two types of denitrification experiments using two bacterial strains were done.
• In heterotrophic denitrification (HD) δ13CDIC changed with the increase of DIC.
• In autotrophic denitrification (AD) fractionation of δ13CDIC and δ34SSO4 occurred.
• Combined δ15NNO3, δ13CDIC, and δ34SSO4 measurements help to separate HD from AD.

In batch culture experiments, we examined the isotopic change of nitrogen in nitrate (δ15NNO3), carbon in dissolved inorganic carbon (δ13CDIC), and sulfur in sulfate (δ34SSO4) during heterotrophic and autotrophic denitrification of two bacterial strains (Pseudomonas aureofaciens and Thiobacillus denitrificans). Heterotrophic denitrification (HD) experiments were conducted with trisodium citrate as electron donor, and autotrophic denitrification (AD) experiments were carried out with iron disulfide (FeS2) as electron donor. For heterotrophic denitrification experiments, a complete nitrate reduction was accomplished, however bacterial denitrification with T. denitrificans is a slow process in which, after seventy days nitrate was reduced to 40% of the initial concentration by denitrification. In the HD experiment, systematic change of δ13CDIC (from − 7.7‰ to − 12.2‰) with increase of DIC was observed during denitrification (enrichment factor εN was − 4.7‰), suggesting the contribution of C of trisodium citrate (δ13C = − 12.4‰). No SO42 − and δ34SSO4 changes were observed. In the AD experiment, clear fractionation of δ13CDIC during DIC consumption (εC = − 7.8‰) and δ34SSO4 during sulfur use of FeS2–S (around 2‰), were confirmed through denitrification (εN = − 12.5‰). Different pattern in isotopic change between HD and AD obtained on laboratory-scale are useful to recognize the type of denitrification occurring in the field.