The largest known bacteria, Thiomargarita spp., have yet to be isolated in pure culture, but their large size allows for individual cells to be monitored in time course experiments or to be individually sorted for omics-based investigations. Here we investigated the metabolism of individual cells of Thiomargarita spp. by using a novel application of a tetrazolium-based dye that measures oxidoreductase activity. When coupled with microscopy, staining of the cells with a tetrazolium-formazan dye allows metabolic responses in Thiomargarita spp. to be to be tracked in the absence of observable cell division. Additionally, the metabolic activity of Thiomargarita sp. cells can be differentiated from the metabolism of other microbes in specimens that contain adherent bacteria. The results of our redox dye-based assay suggest that Thiomargarita is the most metabolically versatile under anoxic conditions, where it appears to express cellular oxidoreductase activity in response to the electron donors succinate, acetate, citrate, formate, thiosulfate, H2, and H2S. Under hypoxic conditions, formazan staining results suggest the metabolism of succinate and likely acetate, citrate, and H2S. Cells incubated under oxic conditions showed the weakest formazan staining response, and then only to H2S, citrate, and perhaps succinate. These results provide experimental validation of recent genomic studies of Candidatus Thiomargarita nelsonii that suggest metabolic plasticity and mixotrophic metabolism. The cellular oxidoreductase response of bacteria attached to the exterior of Thiomargarita also supports the possibility of trophic interactions between these largest of known bacteria and attached epibionts.

Carbonate rocks at marine methane seeps are commonly colonized by sulfur-oxidizing bacteria that co-occur with etch pits that suggest active dissolution. We show that sulfur-oxidizing bacteria are abundant on the surface of an exemplar seep carbonate collected from Del Mar East Methane Seep Field, USA. We then used bioreactors containing aragonite mineral coupons that simulate certain seep conditions to investigate plausible in situ rates of carbonate dissolution associated with sulfur-oxidizing bacteria. Bioreactors inoculated with a sulfur-oxidizing bacterial strain, Celeribacter baekdonensis LH4, growing on aragonite coupons induced dissolution rates in sulfidic, heterotrophic, and abiotic conditions of 1773.97 (±324.35), 152.81 (±123.27), and 272.99 (±249.96) μmol CaCO3 • cm−2 • yr−1, respectively. Steep gradients in pH were also measured within carbonate-attached biofilms using pH-sensitive fluorophores. Together, these results show that the production of acidic microenvironments in biofilms of sulfur-oxidizing bacteria are capable of dissolving carbonate rocks, even under well-buffered marine conditions. Our results support the hypothesis that authigenic carbonate rock dissolution driven by lithotrophic sulfur-oxidation constitutes a previously unknown carbon flux from the rock reservoir to the ocean and atmosphere.

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