Xylonic acid is a promising compound for the substitution of gluconic acid. Gluconobacter oxydans DSM 2343 has proven to be a highly potent biocatalyst for the conversion of xylose to xylonic acid. In the present study, different nitrogen sources for the growth of G. oxydans and subsequent xylonic acid production were investigated for the first time with minimal medium. Application of 0.32 g/L glutamate supplemented with 0.15 g/L ammonium sulfate as a cheap nitrogen source enabled a xylonic acid productivity of 2.92 g/(Lh) which is similar to findings involving a complex medium (3.20 g/(Lh)). The study further investigated the impact of the xylose source on the growth and production of G. oxydans. Dose-response curves confirmed that G. oxydans is mainly insensitive towards the main inhibitory compounds, acetate and hydroxymethylfurfural, up to a concentration of 5 g/L and 2.5 g/L, respectively. However, batch investigations indicated that substitution of 25 % of the pure xylose with hemicellulosic xylose resulted in a xylonic acid yield of 90 % compared to the control approach without hemicellulosic xylose. The feeding of hemicellulosic xylose in a pulsed fed-batch mode even enabled the use of 50 g/L demonstrating that the proper selection of a feeding strategy for the hemicellulosic xylose greatly improves the production of xylonic acid.