Bioethanol and xylitol production of selected xylose fermenting yeast

Abstract

One hundred and twenty-nine xylose-utilization yeasts were isolated from 60 herbivore animal fecal samples in Thailand and 3 soils sample in Japan. These yeast isolates were divided into ten groups based on their phenotype and genotype of D1/D2 region of large-subunit ribosomal RNA gene. Ninety-three ascomycetous yeasts were identified as Candida (62 isolates), Sporopachydermia (9 isolates), Kluyveromyces (3 isolates), Meyerozyma (3 isolates), Zygoascus (1 isolate), Barnettozyma (3 isolates), Pichia (4 isolates), Issatchenkia (2 isolates), Geotrichum (14 isolates) and as Cyberlindnera (2 isolates). Twentysix basidiomycetous yeasts isolates were identified as Trichosporon. Candida sp. ELP19 and Geotrichum sp. (14 isolates) were identified as the novel specie based on their differential phenotype and D1/D2 region of large subunit ribosomal RNA gene showing the similarity 94 to 97%. In the xylose fermentation, 117 isolates could ferment xylose (4%) to ethanol (0.01-2.388 g/l after 24 h.) while 95 isolates could ferment xylose to xylitol (0.03-29.47 g/l after 24 h). Zygoascus meyerae E23 produced the highest ethanol (3.61 g/l after 72h). C. tropicalis A26 produced the highest xylitol (21.30 g/l) which corresponded to 0.53 g xylitol/g xylose after 24 h. Thus, C. tropicalis A26 was selected to optimize for xylitol production. This strain produced highest xylitol production in shake flask with 42.517g/l when xylose and peptone were 80 g/l and 40 g/l, respectively. Statistical regression model predicted the maximum xylitol production at 45.83 g/l. In 5L stirred fermenter under the optimized condition, the highest xylitol production and xylitol yield were 71.59 g/l and 0.89 g/g xylose, respectively. Then characterization of xylose reductase gene (xyl1) of both strains E23 and A26 was carried out. Deduced amino acids sequence of the cloned xylose reductase gene of Z. meyerae E23 showed 58.96% identity and 73.88% similarity to xylose reductase of Ogataea siamensis (ACN78427) while C. tropicalis A26 xylose reductase gene showed 98.42% identity and 98.95% similarity to C. tropicalis (ABX60132C). Moreover, deduce amino acids of both strains showed tetra-amino acid motif (Ile-Pro-Lys-Ser) which is conserved among NADPHdependent xylose reductase.