10 m pore size filters

Cells equivalent to 5 mg DCW were harvested using 10 µm pore size filters (Merck Millipore, Burlington, MA, USA), washed once with 20 mM ammonium carbonate, and immediately suspended in 1 mL of pre-cooled (–30 °C) methanol containing 36 µM piperazine-1,4-bis (2-ethanesulfonic acid) (Dojindo Laboratories, Kumamoto, Japan) and 36 µM l-methionine sulfone (Sigma-Aldrich) as internal standards. The suspension (500 µL) was subjected to cell disruption using 0.5 mm glass beads YGB05 in a multi-bead shocker MB1001C(S) as described for lipid analysis. Subsequently, 150 µL of chloroform and 50 µL of ultrapure water were added and mixed by vortexing for 10 s. After centrifugation at 14,000×g for 5 min at 4 °C, 400 µL of supernatant was collected, mixed with 200 µL of ultrapure water by vortexing for 10 s, and centrifuged at 14,000×g for 5 min at 4 °C. The upper phase was filtered using an Amicon Ultra-0.5 Centrifugal Filter Unit UFC5003BK (Merck Millipore) at 14,000×g for 50 min at 4 °C. The flow-through (300 μL) was dried in a vacuum using an evaporator CEV-3100 (EYELA). Dried samples were resuspended in 20 µL of ultrapure water and analyzed by CE-TOFMS using a G7100 CE and G6224AA liquid chromatograph/mass selective detector (LC/MSD) TOF system (Agilent Technologies) [25 (link), 48 (link)].

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)].