BACKGROUND: 2,3-Butanediol (2,3-BD) has a wide range of applications in chiral molecular synthesis, biofuel additives, and in food flavor additive manufacturing. Fermentation is a favorable method for 2,3-BD production. However, it requires much time and produces several NADH related byproducts which compete with 2,3-BD production. Bacillus subtilis has an excellent ability for 2,3-BD production by biocatalysis. However, its production is limited by low intracellular NADH and the reversible property of acetoin reductase (AR/2,3-BDH). The whole cell biocatalyst process with two different NADH regeneration systems was designed for efficient production of 2,3-BD in B. subtilis 168. RESULTS: Formate dehydrogenase and glucose dehydrogenase for NADH regeneration were successfully co-expressed with acetoin reductase in B. subtilis 168. After optimization of biocatalyst bioconversion conditions, B. subtilis 168/pMA5-bdhA-HpaII-fdh yielded 74.5 g L-1 of 2, 3-BD with 9.3 g L-1 h(-1) productivity by fed batch and 115.4 g of 2,3-BD was achieved using same batch bacterium by three repeated batch bioconversions. On the other hand, 63.7 g L-1 of 2, 3-BD was produced with 7.92 g L-1 h(-1) productivity by B. subtilis 168/pMA5-bdhA-HpaII-gdh. To our knowledge, the volume productivity obtained here is the highest ever reported for biocatalysis. CONCLUSION: A higher productivity of 2,3-BD from acetoin was achieved by whole cell biocatalysis with NADH regeneration systems in B. subtilis 168. This approach can be applied for NADH related bio-based chemicals production to improve titer, yield and productivity. (C) 2017 Society of Chemical Industry