Screening And Identification Of Xylose-Fermenting Funci And Study Of Their Metabolic Characteristics For Ethanol Production

Xylose is the principal component of lignocellulose and the second abundant sugar in nature. It is very important for lignocellulose ethanol production to isolate xylose-fermenting microbes with high ethanol production capabilities. In this paper,207 strains of xylose-fermenting yeast were screened from soil samples collected from orchards, rice-fields, vegetable fields, sugar cane plantations, forest, sugar factories, livestock pens, dumps and composts in subtropical areas of China by the enriching and purifying method using xylose as sole carbon source. Two strains, XY-19 and GX-15, with higher ethanol production capabilities were selected according to the capabilities of ethanol fermentation from xylose and glucose. XY-19 was identified as Candida tropicalis according to the morphological characteristics and the 18S rDNA and the nuclear ribosomal internal transcribed spacer (ITS) sequences and designated as C. tropicalis XY-19. GX-15 was identified as Kluyveromyces marxianus according to the morphological characteristics and the D1/D2 domain of the large-subunit (LSU) rDNA sequence and designated as K. marxianus GX-15.The optimum temperature and pH for ethanol fermentation by XY-19 were 32℃and 4.0, respectively. The results of kinetics study showed that the ethanol production capability of XY-19 was similar to Saccharomyces cerevisiae Angel, a commercial ethanol producer popularly used in China. Theμmax, Vs, Vp and YP/S of XY-19 were 0.11 h-1,2.12 g/1-h,1.11 g/1-h and 0.47 g/g glucose, respectively. As other xylose-fermenting yeasts, XY-19 catalyzed xylose through the xylose reductase (XR)-xylitol dehydrogenase (XDH) pathway. The XR of XY-19 used NADPH as cofactor, while the XDH used NAD+ as cofactor. The ratio of XR activity to XDH activity of XY-19 was 3.4. Xylose could induce high level expression of XDH of XY-19, while glucose might not induce expression of it. XY-19 utilized xylose under the aerobic condition. The rate of xylose utilization by XY-19 was lower than that of glucose utilization. The velocity of glucose consumption (Vs)under the aerobic condition was 3.02 g/1·h, while Vs of xylose consumption was 0.73 g/1-h. Xylose utilization was suppressed by glucose. XY-19 accumulated large amount of xylitol during ethanol fermentation from xylose. The ethanol tolerance of XY-19 was lower than S. cerevisiae. XY-19 could not grow in YPD broth containing ethanol over 7%(v/v). Four mutant strains (G3-13, G3-18, G3-57 and G3-60) of XY-19 which could grow in YPD broth containing 12%(v/v) of ethanol were obtained by genome shuffling for four cycles and screening with xylose-ethanol gradient plates. The ethanol tolerances of the 4 mutant strains were 71% higher than that of wild-type strain of XY-19.The optimum temperature and pH for ethanol fermentation by GX-15 were 40℃and 4.0, respectively. GX-15 could not grow in YPD broth containing ethanol over 9%(v/v). The results of kinetics study showed that theμmax, Vs VP and YP/S of GX-15 were 0.11 h-1,1.78 g/1-h,0.76 g/1-h and 0.43 g/g glucose, respectively. XY-19 could not grow in YPD broth containing ethanol over 7% (v/v). Four mutant strains of GX-15 which have higher ethanol production capability than the wild-type strain GX-15 were obtained by multiple mutating alternately treated with ultraviolet irradiation and nitrosoguanidine for two cycles and screening with 2,3,5-triphenyltetrazolium chloride (TTC) agar plates. When fermenting glucose, theμmax, Vs and Vp of GX-UN120, one of the 4 mutant strains, were 38%,18.2% and 26.3% higher than that of GX-15, respectively. The ethanol tolerance of GX-UN120 was improved from 9% to 11%.One filamentous fungus strain, XE-1, which could grow and utilized xylose under anaerobic condition was screened from soil samples by the anaerobic enriching and anaerobic purifying method using xylose as sole carbon source. XE-1 was identified as a species of Pestalotiopsis according to the morphological characteristics and the sequences of 18S rDNA, ITS and the D1/D2 domain of the LSU rDNA and designated as Pestalotiopsis sp. XE-1. XE-1 could ferment xylose, arabinose, glucose, fructose, mannose, galactose, cellobiose, maltose, sucrose and starch to produce ethanol. The optimum growth temperature and ethanol fermentation temperature of XE-1 were 30℃and the optimum ethanol fermentation pH was 6.5. XE-1 could tolerance 6%(v/v) of ethanol, grow and produce ethanol in 100 g/1 of xylose. The xylose tolerance of XE-1 was higher than other reported xylose-fermenting microbes. XE-1 also catalyzed xylose through the XR-XDH metabolic pathway, but the coenzyme specificity of its XR was different from other xylose-fermenting fungi. The XR of XE-1 used either NADPH or NADH as its coenzyme and the XR activity specific for NADH was 68% higher than that specific for NADPH. XE-1 was the second microbe which XR activity specific for NADH was higher than that specific for NADPH. The ratio of XR activity to XDH activity of XE-1 was 0.43 (specific for NADPH) or 1.13 (specific for NADH). Both xylose and glucose could induce the expression of XDH of XE-1. XE-1 did not accumulate any xylitol and could ferment xylose to produce ethanol under anaerobic condition due to its dual coenzyme specificity and the lower ratio of XR activity to XDH activity. Such xylose-fermenting fungi were seldom reported up to day.Although XE-1 could ferment xylose to produce ethanol under anaerobic condition, oxygen affected the ethanol fermentation from xylose obviously. Under semi-aerobic and aerobic conditions, XE-1 consumed xylose (20 g/1) completely at 84 h and 96 h, respectively. Under micro-aerobic and anaerobic conditions, XE-1 utilized xylose slowly. The concentration of xylose had decreased about 75% from the initial concentration under micro-aerobic condition and only about 48% under anaerobic condition after 120 h cultivation. XE-1 reached its maximum ethanol concentration of 4.1 g/1 at 84 h under semi-aerobic condition. Under aerobic, micro-aerobic and anaerobic conditions, the maximum ethanol concentrations were 80%,78% and 41% of those under semi-aerobic conditions, respectively. The major fermentation end-product of xylose fermentation of XE-1 was acetate with no xylitol accumulation. Acetic acid produced in xylose fermenting process resulted in the decrease of pH. Under pH-uncontrolled condition, the biomass, xylose consumption rate and ethanol yield were significantly lower than those under pH-controlled condition.