A non-sterile and buffer-free 2-keto-gluconic acid (2KGA) production method was proposed by using resting cells from an industrial strain of Pseudomonas fluorescens AR4. 2KGA achieved a maximum production performance of 195.00 g/L, which corresponded to 3.05 g/L h of total productivity and 1.07 g/g of yield under optimal conditions, including resting cell aged 20 h, a cell concentration of 3.0 g/L, a reaction temperature of 30 degrees C, a glucose concentration of 181.82 g/L, and a reaction volume of 20 mL during the 64-h bioconversion process. P. fluorescens AR4 resting cells showed stable bioconversion activity after reuse 3 times or storage for less than 28 d at 4 degrees C. These findings suggested that the proposed non-sterile and buffer-free bioconversion of glucose to 2KGA using P. fluorescens AR4 resting cells had remarkable advantages, including ease handling and low cost, and they are a potent application for industrial production of 2KGA. (C) 2015 Elsevier Ltd. All rights reserved.