Escherichia coli expressing NAD-dependent xylitol-4-dehydrogenase (XDH) from Pantoea ananatis and growing on glucose or glycerol converts xylitol to the rare sugar l-xylulose. Although blocking potential l-xylulose consumption (l-xylulosekinase, lyxK) or co-expression of the glycerol facilitator (glpF) did not significantly affect l-xylulose formation, co-expressing XDH with water-forming NADH oxidase (NOX) from Streptococcus pneumoniae increased l-xylulose formation in shake flasks when glycerol was the carbon source. Controlled batch processes at the 1L scale demonstrated that the final equilibrium l-xylulose/xylitol ratio was correlated to the intracellular NAD+/NADH ratio, with 69% conversion of xylitol to l-xylulose and a yield of 0.88g l-xylulose/g xylitol consumed attained for MG1655/pZE12-xdh/pCS27-nox growing on glycerol. NADH oxidase was less effective at improving l-xylulose formation in the bioreactor than in shake flasks, likely as a result of an intrinsic maximum NAD+/NADH and l-xylulose/xylitol equilibrium ratio being attained. Intermittently feeding carbon source was ineffective at increasing the final l-xylulose concentration because introduction of carbon source was accompanied by a reduction in NAD+/NADH ratio. A batch process using 12g/L glycerol and 22g/L xylitol generated over 14g/L l-xylulose after 80h, corresponding to 65% conversion and a yield of 0.89g l-xylulose/g xylitol consumed.