At each sampling time-point, 5 mL culture sample was withdrawn

from the fermentation vessel and immediately applied to a 0.8 µm Supor 800 filter (Pall) placed on a magnetic filtration funnel (Pall) attached to a vacuum filtration manifold. On the filter disc, the cells were subsequently washed twice with one volume 2.63% (w/v) NaCl solution each, and immediately after the second washing step, the Inhibitors,research,lifescience,medical filter was transferred to a 50 mL tube containing 25 mL 60% methanol solution pre-chilled on an ethanol bath at −23 °C. The whole procedure from sampling to quenching of metabolism was completed within 10–15 s. The samples of each sampling point were collected on the ethanol bath at Inhibitors,research,lifescience,medical −23 °C, subsequently snap-frozen in liquid nitrogen and

stored at −80 °C until metabolite extraction. 3.3. Metabolite Extraction Samples stored at −80 °C were completely thawed on an ethanol bath at −23 °C. An internal standard mix was added to each 25 mL sample (biomass from 5 mL sample on filter in 25 mL 60% methanol solution) yielding final concentrations of 3.34 mM D3-alanine, 312.5 µM D4-succinate, 1.67 µM D8-valine, 62.5 µM 13C6-glucose, 0.416 µM 13C10, 15N5-adenosine monophosphate and 1.04 µM 13C1-α-ketoisocaproic acid). This standard mix included compounds to be used as Inhibitors,research,lifescience,medical internal standards in different analytical methods for metabolites with different chemical properties (organic acids, phosphometabolites, sugars). Samples were then subjected to three cycles of freezing on liquid Inhibitors,research,lifescience,medical nitrogen and thawing at −23 °C on the ethanol bath, found to be sufficient for reaching a maximum of compound extraction into the 60% methanol, and thereafter centrifuged for 5 min at −9 °C and 6000 × g. Supernatants were transferred to new tubes pre-chilled at −23 °C and then divided into aliquots á 6 mL in 15 mL screw cap tubes for analysis using different metabolite profiling methods. Samples were frozen at −80 °C and subsequently subjected to solvent

evaporation on a freeze-dryer for 24 h. The freeze-dried samples were stored at −80 °C Inhibitors,research,lifescience,medical until MS analysis. 3.4. Metabolite Derivatization with Methyl Chloroformate (MCF) and GC-MS Analysis Dried extract samples were dissolved in a solvent mixture consisting of 380 µL 1 M NaOH, 333 µL 100% MeOH, and 67 µL pyridine following a modified protocol of Villas-Boas et al. [40]. 20 µL 1 mM D5-glutamate Non-specific serine/threonine protein kinase was added to each sample as an analytical internal standard, and the dissolved sample was then transferred to a silanized 5 mL glass tube. While vortex PCI-32765 mw mixing, the following steps were performed for derivatization with MCF, extraction with chloroform, and stopping the reaction with sodium hydrogencarbonate: 40 µL MCF added, 30 s vortex mixing, 40 µL MCF added, 30 s vortex mixing, 400 µL chloroform added, 10 s vortex mixing, 400 µL 50 mM NaHCO3 added, 10 s vortex mixing. The chloroform phase was dried with anhydrous Na2SO4 prior to GC-MS analysis.