Substrate-dependent CO₂ fixation in heterotrophic bacteria revealed by stable isotope labelling

Virtually all heterotrophs incorporate carbon dioxide by anaplerotic fixation. Little explored, however, is the interdependency of pathways and rates of CO₂fixation on the concurrent usage of organic substrate(s). Potentially, this could reveal which substrates out of a pool of dissolved organic carbon are utilised by environmental microorganisms. To explore this possibility, Bacillus subtilis W23 was grown in a minimal medium with normalised amounts of either glucose, lactate or malate as only organic substrates, each together with 1 g/L NaH¹³CO₃. Incorporation of H¹³CO₃^- was traced by elemental analysis-isotope ratio mass spectrometry of biomass and gas chromatography-mass spectrometry of protein-derived amino acids. Until the late logarithmic phase, ¹³C incorporation into the tricarboxylic acid cycle increased with time and occurred via [4-¹³C]oxaloacetate formed by carboxylation of pyruvate. The levels of ¹³C incorporation were highest for growth on glucose and lowest on malate. Incorporation of ¹³C into gluconeogenesis products was mainly detected in the lactate and malate experiment, whereas glucose down-regulated this path. A proof-of-principle study with a natural groundwater community confirmed the ability to determine incorporation from H¹³CO₃^- by natural communities leading to specific labelling patterns. This underlines the potential of the labelling approach to characterise carbon sources of heterotrophic microorganisms in their natural environments.