Helsinki University of Technology, Department of Chemical Technology

The aim of this study was to develop a biotechnological production process for xylitol. The xylitol production characteristics of Candida millerii, Candida guilliermondii and Candida tropicalis were compared. C. tropicalis was the best xylitol producer. A volumetric productivity of 5.7 g xylitol L h was achieved with 69 % yield from D-xylose on a mineral medium with a modified repeated fed batch production method. The xylitol production mechanism was confirmed by chemostat cultivation studies. Xylitol production is a consequence of an accumulation of intracellular NADH under oxygen limitation. NADH inhibits xylitol dehydrogenase enzyme (XDH) resulting in xylitol excretion out of the cell. The intracellular metabolic fluxes were studied by in vitro enzyme assays and more theoretically by Metabolic Flux Analysis (MFA). The determined enzyme activities revealed that different metabolic pathways are operating simultaneously enabling substrate cycling to maintain ATP turnover and redox balance under oxygen limited conditions. It was possible to calculate the ATP and NAD(P)H turnover by MFA. Connection between the cofactor dependency of xylose reductase (XR) and redox metabolite production was illustrated by a novel experimental set-up using an ascending xylose feed rate and descending oxygen consumption rate profiles simultaneously. This indicated that C. tropicalis, which XR is dual dependent is more responsive in respect to xylitol production under oxygen limited conditions than C. guilliermondii, which XR is only NADPH dependent. The role of additional NADH on xylitol flux was further confirmed by formate feeding. Formate is catabolised to NADH and CO2 in the cell. C. tropicalis resulted in increased xylose uptake and xylitol production compared to C. guilliermondii. XR was purified from C.guilliermondii and its characteristics was studied. C. milleri was able to produce minor amounts of xylitol from xylose in the presence of glucose.