The concentration of sugars (glucose,
maltotriose and dextrins (DP4+)), fermentation by-products (glycerol,
lactic acid) and ethanol was determined using high performance
liquid chromatography (HPLC). Prior to analysis clear
supernatant after centrifugation of fermentation media was diluted
with bidistilled water in a 1:3 v/v ratio and filtered through nylon
syringe filter (0.2 lm). The analysis was performed using Prominence
chromatograph (Shimadzu, Japan) equipped with Rezex
ROA-Organic Acid H + column (300 7.8 mm) (Phenomenex,
USA). Following parameters of HPLC analysis were applied: injection
volume-20 lL, elution temperature-60 C, mobile phase-
0.005 mol L1 H2SO4, mobile phase flow rate-0.6 mL min1. The
analytes were detected using refractive index detector (RID-10A,Shimadzu) at 50 C. Obtained chromatograms were integrated
using external standard method with CHROMAX 10 software
(Pol-Lab, Poland).
On the basis of obtained results following process parameters
were calculated at 24 h time intervals: ethanol production rate
(rp [g L1 h1]) and practical ethanol yield (Yp [%; g kg1 of sugars;
g kg1 of raw material dry matter] on the basis of stoichiometric
reaction where 1 kg of sugars (as glucose) is converted to 511.1 g
of ethanol [30].