BACKGROUND: Acetoin is an important biorefinery platform chemical with many industrial uses. It is mainly produced by chemical synthesis or mesophilic fermentation with high costs. RESULTS: A thermophilic Bacillus strain was isolated to efficiently produce acetoin from maize hydrolysate at 50 degrees C. The fermentation medium was statistically optimized by Plackett-Burman design. Operational conditions were then optimized using a 4.2 L fermenter conducted at 50 degrees C without sterilization. Oxygen enriched air (65% oxygen) accelerated substrate consumption but did not increase product titer. In comparison with 600 rpm, agitation speed controlled at 400 rpm yielded less acetoin but more 2,3-butanediol in the final products. A two-stage pH control strategy (pH first at 6.6-7.1 then at 7.7-8.0) was applied to further increase the yield of acetoin. Finally, 50.8 g L-1 of acetoin and 32.1 g L-1 of 2,3-butanediol were obtained from 251 g L-1 of degermed maize flour (dry weight). The conversion yield from sugar to the 4-carbon compounds (acetoin and 2,3-butanediol) was 94.4% of the theoretical value. CONCLUSION: This is the first report of non-sterilized fermentative production of acetoin. Using degermed maize hydrolysate as the main feedstock, this work sets a new titer record of acetoin fermentation among known thermophilic producing systems. (C) 2017 Society of Chemical Industry