2,2-Bis(hydroxymethyl)butyric acid (BHMB) is an important multifunctional chemical for the emerging bio-based polymer industry. It can be produced from trimethylolpropane (TMP) by selective oxidation using growing cells of Corynebacterium sp. ATCC 21245. However, this process is limited by the low volumetric productivity and low concentration of the final product. In the present study, we performed sequential batch operation with cell recycling in media containing glycerol, acetic acid, and increasing concentrations of yeast extract. This approach enhanced the conversion of 10 and 15 g/L IMP to 11.0 and 16.3 g/L BHMB at rates of 0.50 and 0.20 g/L.h, respectively. Applying a cell bleeding strategy resulted in an overall 10-fold improvement in productivity. The consequently prolonged biocatalyst viability resulted in a quantitative conversion of 20 g/L TMP to 22.3 g/L BHMB and a yield of 1.10 g(BHMB)/g(TMP) (100% molar yield). This work facilitates further studies of the selective oxidation on other industrially important polyols.