2.3. Pretreatment methodology
OP includes high contents of lignocellulosic materials (cellulose,
lignin, and hemicellulose), and polymers constituted by pentose
and hexose sugars (mainly xylose and glucose). Glucose is known to
be converted to ethanol by the S. cerevisiae strain. Four groups of
tests were performed using the OP and OMW in the quantities
reported in the above paragraph. The groups all included a hydrolysis
stage with 0.5H2SO4 v/v and heating at 120 C for 30 min, as
previously mentioned. One group was not subjected to any further
pretreatment (NPah test) in order to evaluate the efficacy of each the
other pretreatments. The other tests included an ultrasonic pretreatment
(UP), a basic pretreatment (BP) and a calcium carbonate
pretreatment (PCa), which consisted of adding calcium carbonate
(5 g/L) to the reaction medium. All of the test groups were conducted
simultaneously in triplicate. The BP consisted of adding
NaOH (3 M) until a pH ¼ 12 was reached and then keeping the
sample for 24 h. The pH was then lowered to pH ¼ 7.0, and the
solution was inoculated with S. cerevisiae. PCa was obtained by
adding 5 g/L CaCO3 to the reaction medium in order to reduce the
concentration of the inhibiting polyphenols. Battista et al. [23]
demonstrated that this type of pretreatment assists the formation
of micro-flocs, which are able to absorb and capture colloidal
substances, including polyphenols. After adding CaCO3, the pH was
brought to 5.5 before inoculation with S. cerevisiae. The ultrasonic
pretreatment (UP) was performed in an ultrasonic machine (Ultrasonic
Cleaner model CP823). Before the inoculation with S. cerevisiae,
the reaction medium was put into a glass bottle which was
placed in the ultrasonic machine under the following conditions:
30 !C atmospheric pressure, and a power of 1.8 kW for 30 min. In
this case, the aim was to determine whether this pretreatment was
able to break down large molecules and convert them into smaller
ones that are more easily digested by microorganisms. At the end of
the UP, S. cerevisiae were introduced into the reaction medium in
order to permit fermentation.