Yeast growth and lipid production
Yeast growth on cellobiose and cellodextrins was performed
in a 40-well microplate. A single colony from
a fresh YPD plate was transferred into 5 mL of defined
medium containing 10 g/L of glucose and pre-cultured
until the mid-exponential phase. The cells were then harvested,
washed, suspended in sterile water and used to
inoculate 200 μL YNBcasa media containing 5 g/L cellobiose
or cellodextrins in the microplate, achieving an initial
OD600 of 0.1. This culture was grown in a microplate reader (Spectrostar Omega, BMG Labtech, Germany) at
30°C with continuous shaking (150 rpm) and automatic
OD600 recording. Similarly, yeast growth on cellobiose
was also performed on 30 ml defined medium containing
10 g/L cellobiose in 250 mL Erlenmeyer flasks.
For lipid production a fresh yeast culture in exponential
phase was used to inoculate 50 mL defined medium
containing 50 g/L Avicel in Erlenmeyer flasks, achieving
an initial OD600 of 1.0. Celluclast 1.5L (60 FPU/mL, gift
from Novozymes, Denmark) was added (7.5 U/g cellulose)
and growth was pursued for 5 days (30°C, 150 rpm).
Samples were taken at regular intervals to determine
concentrations of biomass, glucose, cellobiose and citric
acid. In parallel, two control experiments were conducted
under the same conditions, with or without the addition
of extra β-glucosidase (810 IU/mL Novozyme 188, gift
from Novozyme, Denmark) at 12.0 IU/g cellulose as recommended
[54].