In vivo 13C Metabolite Labeling with CO2

To perform in vivo ¹³C labeling of newly synthesized metabolites using radiolabeled CO₂, cells were harvested on day 1.5 of semi-continuous culture using 10 µm pore size filters (Merck Millipore) and resuspended in MB 12 N medium containing 2% (w/v) sea salt and 25 mM NaH¹³CO₃ (Cambridge Isotope Laboratories, Tewksbury, MA, USA). After incubation under white fluorescent lamps at 250 μmol photons m⁻² s⁻¹ and shaking at 100 rpm, cells were harvested and the intracellular metabolites were analyzed as described for the metabolome analysis. The ¹³C labeling ratio was calculated as described in a previous report [25 (link), 48 (link)].