Agilent 1200 pump

A specimen of Phorbas sp. (voucher number 07G-26) was collected by hand from Gageo Island, South Korea, in 2007. Optical rotations were measured on a JASCO P-1010 polarimeter (Jasco, Easton, MD, USA). IR spectra were recorded using a JASCO FT/IR 4100 spectrometer (Jasco, Easton, MD, USA), and ultraviolet (UV) spectra using a Varian Cary 50 UV-Visible spectrophotometer (Agilent, Santa Clara, CA, USA). High-resolution (HR)-electrospray ionization (ESI) mass spectra were obtained using a SCIEX X500R mass spectrometer (Sciex, Framingham, MA, USA). The NMR spectra were recorded on a Varian VNMRS 500 NMR spectrometer (Varian, Palo Alto, CA, USA) operating at 500 (¹H) or 125 MHz (¹³C), respectively, with chemical shifts given in ppm using a methanol-d4 solution concerning residual solvent peaks at 3.30 and 49.0 ppm. Semi-preparative liquid chromatography was performed using an Agilent 1200 pump (Agilent, Santa Clara, CA, USA) and an RI detector. Vacuum column chromatography was performed using RP-18 silica gel 60 (Merck, Darmstadt, Germany).

Acetic acid (or acetate depending on the pH of the culture medium) was the sole carbon source added to the culture media used. The decrease in acetic acid concentration during growth was analyzed using the LCK 365 kit for organic acids from Hach Lange or by high-performance liquid chromatography (HPLC). For HPLC analysis, 2 mL samples of the culture medium were taken every 24 h [29 (link)]. Prior to injection, cell-free supernatant was obtained by centrifugation (13,500× g, Eppendorf Centrifuge 5424, 10 min, 4 °C), and subsequently filtered over a 0.2 µm cellulose acetate (CA) membrane. The filtered aqueous samples were analyzed with an Agilent 1200 HPLC equipped with an Agilent 1200 pump, a refractive index detector, and a standard ultraviolet detector. Samples were separated over a Bio-Rad organic acid column (Aminex HPX-87H) which was maintained at 60 °C. An eluent of 5 mM sulfuric acid was used, with a flow rate of 0.55 mL/min. The elution of acetic acid was followed at 210 nm. Calibration curves of acetic acid were prepared for accurate quantification and were based on a minimum of five data points within the range of 1–20 g/L acetic acid with an excellent linear fit (R² > 0.99).