For the development of repeated batch fermentation and cel recycling continuous fermentation processes with high throughput of xylitol, Candida tropicalis mutants with high flocculating activity were screened by random mutagenesis with methanesulfonic acid ethylester(EMS). Among the mutants screened, BS2 mutant showed the highest flocculating activity and better growth and xylitol production. Using the BS2 mutant, repeated batch and fed batch cultures were carried out in flask and jar fermenter. In repeated batch cultures using a 250㎖ flask, the average volumetric productivity of 0.98g/ℓ·h was obtained with the xylitol yield of 0.63g/g while the volumetric productivity was 0.93g/ℓ·h with the xylitol yield of 0.61g/g in a single batch culture. In repeated batch cultures using a 2.5L jar fermenter, the average volumetric productivity of 1.58g/ℓ·h was obtained under oxygen limited condition with the xylitol yield of 0.67g/g. In repeated-fed batch culture using a 2.5L jar fermenter, the volumetric productivity of 2.07g/ℓ·h was obtained with the xylitol yield of 0.79g/g. These results showed that the volumetric productivity could be increased by 84.8% in repeated fed batch cultures compared with that in single batch cultures. It was also found that flocculating activity of BS2 mutant was affected by culture pH with an optimum pH 3.
For further enhancement of xylitol yield by disruption of xylitol dehydrogenase (XDH; EC 1.1.1.9), a partial genomic sequence of XYL2 encoding XDH was cloned by the polymerase chain reaction(PCR) from Candida tropicalis using the DNA sequence of XYL2 of Pichia stipitis. The sequence of the PCR fragment encoded a protein with homology of 88.2% to the XDH of P.stipitis. Using this partial genomic clone of XYL2, XDH defective mutant of C.tropicalis will be developed by disruption of XYL2.
