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 CO2-fixation on the concurrent usage of organic substrate(s). Potentially, this could reveal which substrates out of a pool of dissolved organic carbon (DOC) 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 (EA-IRMS) of biomass and gas chromatography-mass spectrometry (GC-MS) 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. 13C-Incorporation 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.