2-level full factorial design was used in order to identify
important parameters in the screening analysis. The factors were
inoculation rate of A.sojae, T.harzianum and S.cerevisiae, ranging for
each between 2 and 20 % (w/v), aeration (vented or sealed) and
agitation speed (0e200 rpm). Total of 40 experiments were conducted
with 8 center points. Since after aerobic growth, large
amount ofmycelial masswas formed which made the totalmycelial
mass addition into the fermentation flask impossible, inoculation
rates of microorganisms were expressed as percentage (% w/v). In
fact these were kept in a broad range in order to catch any possible
effect on bioethanol production. After aseptic inoculation of the
mycelial mass from aerobic fermentation, plastic paraffin film was
used to seal the flasks which provided strictly anaerobic conditions,
whereas vented flasks allowed small amounts of gases (O2 and CO2)
to pass in and out through a silicone tube packed tightly with
cotton.