Impact of acetate on CO₂ fixation pathways in thermophilic and hydrogenotrophic bacteria

The bacterial-type Wood-Ljungdahl (WL) pathway and reductive tricarboxylic acid (rTCA) cycle are the dominant chemolithotrophic CO₂ fixation pathways in bacteria inhabiting aphotic geothermal and deep-sea hydrothermal ecosystems. However, the activity of these bacterial metabolic systems in ecosystems with available organic carbons remains unclear. Here, we examined the impact of extracellular acetate on the CO₂-fixation pathways of three thermophilic hydrogen-oxidizing and non-acetogenic bacteria using ¹³C tracer-based metabolomics. Under chemolithoautotrophic conditions, Thermodesulfatator indicus and Hydrogenobacter thermophilus fixed CO₂ through the WL pathway and rTCA cycle, respectively, whereas Thermovibrio ammonificans, which has been suggested to operate both of these pathways, exhibited significant CO₂ fixation through only the rTCA cycle. Under chemolithomixotrophic conditions with acetate, H. thermophilus and T. ammonificans assimilated both CO₂ and acetate via the rTCA cycle. In contrast, acetate suppressed CO₂ fixation through the WL pathway in T. indicus and was used as the primary carbon source under chemolithomixotrophic conditions. These results suggest that the contribution of the WL pathway for CO₂ fixation might be overestimated in ecosystems where acetate is available. Moreover, the present findings indicate that simultaneous CO₂ fixation through both the WL pathway and rTCA cycle in a cell, which has been proposed as a possible metabolic strategy for CO₂-fixation in ancestral life, is not advantageous in extant microorganisms.