2.6.1. Quantification of Farnesol

Levels of intra- and extracellular cellular farnesol were quantified following an HPLC procedure. This compound was quantified from both culture broths (=culture filtrates) and mycelia through the following steps. (i) Extraction of farnesol from culture broths: First, 2 mL of broth were extracted with 400 μL of ethyl acetate, mixed and centrifuged for 10 min at 4000 rpm. Supernatant was collected and extracted twice again following the same procedure. Finally, the extract was evaporated in a SpeedVac vacuum concentrator (Thermo Fisher) and resuspended in 50 μL acetonitrile. (ii) Extraction of farnesol from mycelia: Mycelia were frozen at −80 °C, freeze-dried, and pestled to powder in a mortar. Mycelia (50 mg each) were resuspended into 1 mL of Tris-HCl 50 mM, pH 6.8 buffer, extracted with 200 μL of ethyl acetate and processed as described above. (iii) HPLC quantification: A measure of 20 μL of the concentrated extracts was applied to a reversed-phase C18 column of 150 mm × 4.6 mm i.d., 4 μm (octadecyl-silane (ODS)) (YMC Europe GmbH) and eluted with a 1 mL/min flux of mobile phase (90% water/10% acetonitrile at zero time, performing a gradient to reach 100% acetonitrile in 40 min, then returned to 10% acetonitrile at 50 min). Using these conditions, farnesol eluted at 34.7 min and quantification was carried out by comparison of the peak area with a standard curve. For determination of the specific production of farnesol, the total protein concentration from culture supernatants was quantified as described by Bradford (1976) [25].