2-Keto-gluconic acid (2KGA) was produced in a semi-continuous process using Pseudomonas fluorescens AR4 and rice starch hydrolysate (RSH). The bacterium was cultured in medium with an initial glucose concentration of 170 g/L supplied as RSH. Once the glucose level had dropped to 20 g/L, 60% of the culture volume was replaced with fresh medium containing 190 g/L of glucose in the form of RSH. After an additional two cycles of growth and media replacement, a total of 476.88 g/L of glucose was consumed and 444.96 g/L of 2KGA was produced. A total productivity of 6.74 g/L and a yield of 0.93 g/g were obtained. These findings suggest that P. fluorescens AR4 is suitable for the production of commercially acceptable levels of 2KGA in semi-continuous culture. (C) 2012 Elsevier Ltd. All rights reserved.