2.5.2. Two-stage fermentation The continuous ethanol fermentation of thick juice was carried out using a two-stage tower shaped reactor (Fig. 1). The reactors were sterilized with NaClO solution (20 mg L1, overnight) and washed with sterilized tap water for one day. Each of the reactors was filled by 450 cm3 of inoculum, and then the thick juice and water were fed to initiate the continuous ethanol fermentation [17]. Thick juice and tap water containing mineral nutrients were continuously and separately fed into the first reactor (R1) at feeding rates of f11 and f12, respectively. The fermented mash overflowed from the top of R1 into the bottom of the second reactor (R2), and thick juicewas simultaneously fed into R2 at a feeding rate of f2. The sugar concentration in R1 (S1) and R2 (S2) were kept at 140 g L1 and 190 g L1, respectively, assuming no sugar was consumed. The dilution rate of R1 (D1) was kept at 0.2 h1. S1, S2 and D1 were calculated as follows: