Fig 1: Extracellular hydrophobic metabolites in mono- or co-culture with different carbon source analyzed by LC-PDA-ESI/MS.Each microorganism was cultured in minimum medium with indicated carbon source for 5 d. Extracellular hydrophobic metabolites in culture supernatant were extracted with acidified ethyl acetate and analyzed by LC-PDA-ESI/MS. Contour maps were constructed from the absorption intensity obtained by LC-PDA analysis. In each monoculture with different carbon source, we see little change in the EHM profiles of B. subtilis. On the other hand, the profiles of A. nidulans are affected by the different carbon sources, which consistent with the previous reports about the regulation of secondary metabolism. EHM in co-culture are similar to those in B. subtilis monoculture regardless of carbon source. The fungal and bacterial cells co-cultured as described above for 5 d in six-well plates. Supernatant of mono- or co-culture was collected by centrifugation at 15,000g for 10 min, and acidified by 1/100 volume of 2 M HCl. Equal volume of ethyl acetate was added to the acidified supernatant, stirred for 1 h and centrifuged at 1,000g for 10 min. The ethyl acetate fraction was collected to other tubes, and lyophilized. Resulting pellet was dissolved in 95% methanol and analyzed by LC-PDA-ESI/MS (LCMS-8030; Shimadzu) equipped with a 150 × 4.6-mm Purospher Star RP-18 column (particle size, 5 μm; Millipore-Merck). The initial mobile phase was solvent A: solvent B = 98:2 (solvent A, 0.1% formic acid; solvent B, acetonitrile), increased to 80% for x min and maintained at that ratio for another 5 min. UV/Vis spectra was monitored by SPD-M30A (Shimadzu). Mass spectra were acquired in the positive mode of LCMS-8030 with the following conditions: capillary voltage, 4.5 kV; detection range, m/z 50–600; desolvation line, 250°C; heat block, 400°C; nebulizer gas, 3 liters/min; drying gas, 15 liters/min.