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A high amylase producing Bacillus subtilis WD 161 was used in a co-culture with Clostridium butylicum TISTR 1032 to enhance acetone–butanol–ethanol (ABE) production from starch. The mixed culture of C. butylicum TISTR 1032 and B. subtilis WD 161 without anaerobic pretreatment by reducing agent and N 2 flushing increased amylase activity 10 folds and enhanced ABE production 5.4 and 6.5 folds from soluble starch and cassava starch, respectively, compared to those of the pure culture of Clostridium itself. The medium optimization for ABE production by the mixed culture without anaerobic pretreatment found that cassava starch concentration of 40 g/L, C/N ratio of 4.35 and the mixed nitrogen sources of 265 mM yeast extract and 100 mM NH 4 NO 3 gave the highest ABE production in terms of final concentration and productivity. The benefits of using this high amylase producing aerobic Bacillus in a co-culture with anaerobic Clostridium were not only increasing substrate utilization and ABE production but there was also no requirement to add any costly reducing agent to the medium or flushing with N 2 to ensure anaerobic condition. This thus makes the anaerobic fermentation more economical and cost effective. This co-culture system may contribute greatly to developing industrialized ABE production.