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 LÀ1 H2SO4 solution. GSHE (2 mL per 1 kg of raw material dry matter) and proteolytic (Neutrase 0.8 L, 0.875 mL kgÀ1 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). Then the waste bread slurry was inoculated with 2 mL of yeast suspension giving initial yeast concentration of 1 g kgÀ1. The flasks were sealed with rubber stoppers with fermentation tube filled with glycerol and a sampling port as described by Kim et al. 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.