Simplified Laboratory Cultivation of Thiobacillus denitrificans and Sulfurimonas denitrificans with Practical Guidelines for Laboratories Without Advanced Anaerobic Equipment

Chemolithotrophic denitrifying bacteria Thiobacillus denitrificans and Sulfurimonas denitrificans are model organisms for sulfur and nitrogen cycling and are frequently used as model organisms in biogeochemical and applied research. Their cultivation is considered technically demanding, largely due to the assumption that strict anaerobic equipment is required. Here, we provide a simplified, reproducible cultivation workflow designed for laboratories with limited anaerobic infrastructure, with a special focus on establishing growth under practical pseudo-anoxic conditions. Three media were evaluated (modified DSMZ 113, ATCC 152 Thiobacillus broth, and a commercial Thiobacillus broth) in both liquid and solid formats using anaerobic jars and a sealed-bottle "pseudo-anoxic" approach. Consistent growth of both organisms was achieved exclusively in modified DSMZ 113. Importantly, T. denitrificans grew reproducibly not only under anaerobic jar conditions but also under the sealed-bottle pseudo-anoxic setup, demonstrating that successful cultivation is feasible without specialized anaerobic systems. The S. denitrificans grew successfully, but exclusively under anoxic conditions. Additionally, we describe a simple and novel coverslip-on-agar microscopy method for rapid confirmation of small transparent colonies, supporting reliable isolation and contamination control. Overall, our results provide a practical step-by-step cultivation guide, highlighting that T. denitrificans can be robustly cultured even without complying with strict protocol for anaerobic cultivation.