BACKGROUND: A great amount of wastewater with high contents of chemical oxygen demand (COD) are produced by ethanol production. It would be useful to utilize distillery wastewater to produce L-lactic acid, which could be a high additional value byproduct of ethanol production. The fermentation process of L-lactic acid production by a newly isolated Enterococcus hawaiiensis CICIM-CU B0114 is reported for the first time. RESULTS: The strain 'produced 569 L-1 of L-lactic acid after cultivation for 48 h in optimized medium consisting of (g L-1) 80 glucose, 10 peptone, 10 yeast extract, 1.5 Na2HPO4 and 0.2 MgSO4. E. hawaiiensis CICIM-CU B0114 was isolated and purified by subculture for growing and producing L-lactic acid in distillery wastewater of very high gravity (VHG) from ethanol fermentation. L-lactic acid fermentation was further studied with distillery wastewater substrate in 7 L and 15 L fermentors. The results showed that L-lactic acid concentrations of 52 g L-1 and 689 L-1 was achieved in 7 L and 15 L fermentors with the initial sugar concentrations of 67 g L-1 and 87 g L-1, respectively. CONCLUSION: The production of L-lactic acid by the newly isolated E. hawaiiensis CICIM-CU B0114 was carried out and the fermentation medium was optimized by orthogonal experimental design. This new strain holds the promise of L-lactic acid production utilizing distillery wastewater from VHG ethanol fermentation. (C) 2010 Society of Chemical Industry