2.3. Biotransformation experiments: liquid culture inoculum was prepared by pre-culturing the fungal strains in Petri dishes containing malt extract solid medium (MEA: glucose 20 g/L, malt extract 20 g/L, agar 20 g/L, peptone 2 g/L). The fungus was inoculated as a conidial suspension (1 × 10^6 conidia/mL) in a 50 mL conical flask containing 40 mL of malt extract liquid medium. The conical flasks were placed in a constant temperature shaker at 25°C and incubated with oscillation at 110 rpm. After 2 days of preincubation, a DMSO solution with 500 mM substrate was added to give a starting substrate concentration (c0) of 1-5 mM. Three biological replicates were set up for each substrate. After addition of substrate, the experiment continued for 3 days, during which time 1 mL was sampled at predetermined time intervals (typically 24, 48, and 72 h.) After each sampling, the sample was extracted with 500 μL of ethyl acetate (EtOAc), and the organic phase was dried over anhydrous Na2SO4 and analyzed by GC/MS. In some cases (see Section 2.4), the reduction products were separated. Before each set of biotransformation experiments, initial biomass and pH were determined. At the end of the experiment, these parameters were evaluated again. The liquid medium was separated from the biomass by filtration for pH measurements; mycelium was dried at 60°C for 24 h and the dry weight of the biomass was determined. The yield of the product 2-(thiophen-2-yl)ethanol was 72% (3.7 mg) and the yield of the by-product methyl 2-(thiophen-2-yl)acetate was 96% (24.6 mg).1H NMR (400 MHz, CDCl3, TMS): δ = 7.20 (m, 1H, heteroaromatic hydrogens), 6.99 (m, 1H, heteroarylhydrogen), 6.90 (m, 1H, heteroaromatic hydrogen), 3.85 (t, 2H, J=6.2 Hz, CH2OH), 3.02 (t, 2H, J=6.2 Hz, CH2CH2OH). 13C NMR (100 MHz, CDCl3, TMS): δ=140.5, 127.0, 125.8, 124.0, 63.4, 33.3. GC/MS: tR= 9.47 min, m/z 128 (M+, 30%), 110 (5%), 97 (100%).
