AbstractA safe microorganism (class 1), Paenibacillus polymyxa, was used for 2,3-butanediol and acetoin production, which could make the fermentation process cheaper and less complex. It showed a broad substrate spectrum, such as mannose, galactose, cellobiose, glycerol, the mixture of glucose and xylose, and the mixture of glucose and cellobiose. In addition, the strain can utilize highly concentrated glucose that was obtained by enzymatic hydrolysis of ionic liquid-pretreated cellulose. With a 15% initial cellulose consistency, the final glucose concentration was 109.5 g/L with 65.7% glucose yield. Without any treatment, the hydrolysate was successfully used to produce 2,3-butanediol and acetoin with a yield of 81.7% and a productivity of 0.7 g/(L·h) by Paenibacillus polymyxa. Higher concentration and higher productivity with relatively high yield, compared with previous works by acid hydrolysis, of 2,3-butanediol and acetoin were achieved. All these novel improvements offer significant opportunities to further decrease the cost of large-scale 2,3-butanediol and acetoin production.