2.3. Fermentation experiment variants
2.3.1. Direct conversion of raw material to ethanol without
pretreatment (Variant I)
Waste bread samples (30 g dry matter basis) were slurred in ca
150 mL of distilled water preheated to 35 C in a 300 mL conical
flasks. The pH of the slurry was adjusted to 4.5 using 1 mol L1
H2SO4 solution. GSHE (2 mL per 1 kg of raw material dry matter)
and proteolytic (Neutrase 0.8 L, 0.875 mL kg1 of raw material
dry matter) preparations were added and the total weight of the
slurry was adjusted to 200 g with distilled water resulting in a
raw material loading of 150 g of dry matter per kilogram of fermentation
medium (the experiments were conducted at relatively
low raw material loading because used raw material showed the
high water binding capacity and application of higher substrate
loading resulted in obtaining solid like consistence of the fermentation
medium what was observed earlier [11,27]). Then the waste
bread slurry was inoculated with 2 mL of yeast suspension giving
initial yeast concentration of 1 g kg1. The flasks were sealed with
rubber stoppers with fermentation tube filled with glycerol and a
sampling port as described by Kim et al. [28] and were placed in
a WNB7-45 water bath shaker (Memmert, Germany) at 35 C with
agitation speed of 150 rpm (2.5 Hz) for 72 h.