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.