2.3 Small scale cultivations
Y. lipolytica strains were inoculated from a YPD agar plate to an
initial OD600 of 0.2 into 2.5 mL YPG medium (10 g/L
yeast extract, 10 g/L peptone, 40 g/L glycerol) in 24 well-plate
(EnzyScreen). The plate was incubated at 28°C and 300 rpm for 22 hours.
The plate was centrifuged at 3,500 g for 5 min at 20°C, the medium was
removed and the cells were resuspended in 1.25 mL production medium (50
g/L glycerol, 5 g/L yeast extract, 4 g/L
KH2PO4, 1.5 g/L MgSO4,
0.2 g/L NaCl, 0.265 g/L CaCl2.2H2O, 2
mL/L trace elements solution: 4.5 g/L
CaCl2.2H2O, 4.5 g/L
ZnSO4.7H2O, 3 g/L
FeSO4.7H2O, 1 g/L
H3BO3, 1 g/L
MnCl2.4H2O, 0.4 g/L
Na2MoO4.2H2O, 0.3 g/L
CoCl2.6H2O, 0.1 g/L
CuSO4.5H2O, 0.1 g/L KI, 15 g/L EDTA).
The plate was incubated at 28°C and 300 rpm for 28 h. Each strain was
cultivated in triplicate.