2.2. The experimental apparatus and preparation of the substrates
The experimental apparatus consisted of two glass bottles and
one plastic vessel connected by pipes. In the first bottle (1000 mL),
550 g of a mixture of OP and OMW (165 g of OP and 385 of OMW)
was added in an approximate ratio of 1:2 to obtain a TS concentration
of approximately 10% w/w in the reaction medium (NPbh,
non pretreated mixture before the hydrolysis stage). A first pretreatment
was performed for all the samples before the bioethanol
fermentation, by means of microorganisms, to assist the hydrolysis
of the organic substrates. The OP-OMW mixtures were heated to
120 !C and treated with dilute sulphuric acid until a concentration
of 0.5% v/v H2SO4 (NPah, non pretreated mixture after the hydrolysis
stage) was reached in the mixture. These conditions were maintained
for 30 min. The mixture was allowed to cool naturally to
room temperature, and the pH was adjusted to 7.0 ± 0.2 by adding
NaOH (2 M). According to Sheik et al. [22], the adoption of these
pretreatment conditions optimizes the hydrolysis and breakdown
of the crystalline structure of the cellulose and, consequently, the
release of fermentable sugars. After the different pretreatments
described in the following paragraph, the pH was reported to be
7.0 ± 0.2, which is the optimal pH for the metabolism of S. cerevisiae
[19]. In total, 55 g (10% of the total mass in the system) of the
inoculum containing S. cerevisiae was introduced into the reaction
medium. The bottle was flushed with nitrogen gas for 5 min to
eliminate oxygen from the reaction environment in order to achieve
anaerobic conditions. The second bottle (2000 mL) was used as
the gasholder, and it was initially filled with water at pH ¼ 2 to
prevent CO2 dissolution in water. When fermentation starts, the
produced biogas moves from the first to the second bottle,
replacing the water in the plastic vessel. The three bottles were
agitated by a rotating shaker in a thermostatic room at 30 ± 1 !C at
100 rpm to prevent inhibition by shear stress.