A physiological and enzymatic study of Debaryomyces hansenii growth on xylose- and oxygen-limited chemostats

Abstract

The effect of changing growth rate and oxygen transfer rate (OTR) on Debaryomyces hansenii physiology was studied using xylose-limited and oxygen-limited chemostat cultures, respectively, and complemented with enzymatic assays. Under xylose-limited chemostat (oxygen-excess), neither ethanol nor xylitol was produced over the entire range of dilution rate (D). The maximal volumetric biomass productivity was 2.5 g l-1 h-1 at D=0.25 h-1 and cell yield was constant at all values of D. The respiratory rates and xylose consumption rate increased linearly with growth rate but, above 0.17 h-1, oxygen consumption rate had a steeper increase compared to carbon dioxide production rate. Enzymatic analysis of xylose metabolism suggests that internal fluxes are redirected as a function of growth rate. For values of D up to 0.17 h-1, the xylose reductase (XR) titre is lower than the xylitol dehydrogenase (XDH) titre, whereas above 0.17 h-1 XR activity is about twice that of XDH and the NADPH-producing enzymes sharply increase their titres indicating an internal metabolic flux shift to meet higher NADPH metabolic requirements. Moreover, the enzymes around the pyruvate node also exhibited different patterns if D was above or below 0.17 h-1. Under oxygen-limited chemostat (xylose-excess) the metabolism changed drastically and, due to oxidative phosphorylation limitation, cell yield decreased to 0.16 g g-1 for an OTR of 1.4 mmol l-1 h-1 and xylitol became the major extracellular product along with minor amounts of glycerol. The enzymatic analysis revealed that isocitrate dehydrogenase is not regulated by oxygen, whereas XR, XDH and the NADPH-producing enzymes changed their levels according to oxygen availability.