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During ethanol production, the ratio of various sources of starch-hydrolyzing enzymes significantly influences hydrolysis efficiency in a very high gravity (VHG) system. In this study, the enzyme matching approach was proposed to optimize liquefaction and saccharification yield of cassava root and cassava starch. For fresh cassava root, the synergistic effect of using Techzyme Q-Add enzyme (93 °C pH 5.6) for liquefaction and GC 147 enzyme (61.5 °C pH 4.2) for saccharification achieved the highest yield among all enzyme-matching designs. At 25% solid, highest total reducing sugar (TRS) yields from sequential liquefaction and saccharification of cassava starch, fresh cassava, and dry cassava were 87.9%, 85.1%, and 70.0% corresponding to 286.8, 249.4, and 241.1 g/L, respectively. For VHG ethanol fermentation by Saccharomyces cerevisiae TISTR 5606, separate liquefaction and simultaneous saccharification and fermentation (SLSSF) gave a significantly higher ethanol concentration compared with separate liquefaction and saccharification and fermentation (SLSF). From a 35% solid SLSSF system, the highest ethanol produced at 30 °C was 27.3 g/L from 72 h, respectively. The results suggested that SLSSF could effectively shorten the time course of the whole process for liquefaction, saccharification, and fermentation from 74 h to only 26 h for similar ethanol production yields. [ABSTRACT FROM AUTHOR]