Effect of nitrogen sources on xylitol production from D-xylose by Candida Sp. L-102

Summary The production of extracellular xylitol from D-xylose by an efficient xylitol-producing yeast, Candida sp. L-102, was studied in shake flask cultures with different nitrogen sources in the basic salt medium. Maximum xylitol production was obtained with urea as the nitrogen source. A final concentration of 100 g/L of xylitol from 114 g/L D-xylose was obtained from the yeast with an indicated yield of 87.7% (based on D-xylose consumed). The average specific xylitol production rate of 0.46 g/g.h was achieved within 65 hours of incubation using 0.3% urea.     

一株高效利用木糖的酵母菌的分离及鉴定

从256个自然试样中筛选到1株高效转化D-木糖为木糖醇的酵母菌株441-28—1。初始木糖质量浓度为90g/L的条件下,24h内的木糖利用效率为3．0g／（L·h）。通过高效液相分析,菌株441-28—1的主要代谢产物为木糖醇。在初始木糖质量浓度为65g/L的条件下,摇瓶分批发酵,木糖醇生成速率达1．1g／（L·h）,木糖醇转化率为70％。经过形态、生理生化特征测定,以及ITS序列分析（GenBank的登记号为EU121523）,将441-28—1菌株鉴定为热带假丝酵母（Candida tropicalis）。Candida tropicalis（热带假丝酵母）已保存于中国高校工业微生物资源数据平台,保藏编号CICIM Y0092。     

Increase of xylitol production rate by controlling redox potential in Candida parapsilosis

The effect of redox potential on xylitol production by Candida parapsilosis was investigated. The redox potential was found to be useful for monitoring the dissolved oxygen (DO) level in culture media, especially when the DO level was low. An increase in the agitation speed in a 5 L fermentor resulted in an increased culture redox potential as well as enhanced cell growth. Production of xylitol was maximized at a redox potential of 100 mV. As the initial cell concentration increased from 8 g/L to 30 g/L, the volumetric productivity of xylitol increased from 1.38 g/L. h to 4.62 g/L. h. A two-stage xylitol production strategy was devised, with stage 1 involving rapid production of cells under well-aerated conditions, and stage 2 involving cultivation with reduced aeration such that the culture redox potential was 100 mV. Using this technique, a final xylitol concentration of 180 g/L was obtained from a culture medium totally containing 254.5 g/L xylose in a 3,000 L pilot scale fermentor after 77 h fermentation. The volumetric productivity of xylitol during the fermentation was 2.34 g/L. h.     

Xylitol production by <Emphasis Type="Italic">Candida tropicalis</Emphasis> in a chemically defined medium

A chemically defined medium that included urea (5 g l(-1)) as a nitrogen source and various vitamins was substituted for a complex medium containing yeast extract (10 g l(-1)) in the production of xylitol by Candida tropicalis. In a fed-batch culture with the chemically defined medium, 237 g xylitol l(-1) was produced from 270 g xylose l(-1) after 120 h. The volumetric rate of xylitol production and the xylitol yield from xylose were 2 g l(-1) h(-1) and 89%, respectively. These values were about 5% lower and 4% higher, respectively, than those obtained using the complex medium. These results indicate that xylitol can be produced effectively in a chemically defined medium.     

Biotechnological production of xylitol in a three-phase fluidized bed bioreactor with immobilized yeast cells in Ca-alginate beads

Cells of Candida guilliermondii entrapped in Ca-alginate beads were used for xylitol production, from concentrated hemicellulose hydrolyzate of sugarcane bagasse, in a fluidized bed bioreactor (FBR). The maximum xylitol concentration 28.9 g xylitol/L was obtained at a high aeration rate of 600 mL/min after 70 h of fermentation, indicating that the use of high aeration rate in this system is favored for better oxygen transfer into the immobilized cells. The specific xylitol productivity and the xylitol yield were of 0.4 g xylitol/L.h and 0.58 g xylitol/g xylose respectively. The immobilization efficiency at the end of the fermentation was of 65 %. After 90 h of fermentation xylitol productivity and yield decreased to 0.25 g xylitol/L.h and 0.47 g xylitol/g xylose respectively, indicating the beginning of xylitol consumption by the yeast. The use of FBR system with immobilized cells presented high xylitol yield and productivity.     

Microbial production of xylitol from D-xylose and sugarcane bagasse hemicellulose using newly isolated thermotolerant yeast Debaryomyces hansenii

A thermotolerant yeast capable of fermenting xylose to xylitol at 40°C was isolated and identified as a strain of Debaryomyces hansenii by ITS sequencing. This paper reports the production of xylitol from D-xylose and sugarcane bagasse hemicellulose by free and Ca-alginate immobilized cells of D. hansenii. The efficiency of free and immobilized cells were compared for xylitol production from D-xylose and hemicellulose in batch culture at 40°C. The maximum xylitol produced by free cells was 68.6 g/L from 100 g/L of xylose, with a yield of 0.76 g/g and volumetric productivity 0.44 g/L/h. The yield of xylitol and volumetric productivity were 0.69 g/g and 0.28 g/L/h respectively from hemicellulosic hydrolysate of sugarcane bagasse after detoxification with activated charcoal and ion exchange resins. The Ca-alginate immobilized D. hansenii cells produced 73.8 g of xylitol from 100 g/L of xylose with a yield of 0.82 g/g and volumetric productivity of 0.46 g/L/h and were reused for five batches with steady bioconversion rates and yields.     

Xylitol production is increased by expression of codon-optimized Neurospora crassa xylose reductase gene in Candida tropicalis

Xylose reductase (XR) is the first enzyme in D: -xylose metabolism, catalyzing the reduction of D: -xylose to xylitol. Formation of XR in the yeast Candida tropicalis is significantly repressed in cells grown on medium that contains glucose as carbon and energy source, because of the repressive effect of glucose. This is one reason why glucose is not a suitable co-substrate for cell growth in industrial xylitol production. XR from the ascomycete Neurospora crassa (NcXR) has high catalytic efficiency; however, NcXR is not expressed in C. tropicalis because of difference in codon usage between the two species. In this study, NcXR codons were changed to those preferred in C. tropicalis. This codon-optimized NcXR gene (termed NXRG) was placed under control of a constitutive glyceraldehyde-3-phosphate dehydrogenase (GAPDH) promoter derived from C. tropicalis, and integrated into the genome of xylitol dehydrogenase gene (XYL2)-disrupted C. tropicalis. High expression level of NXRG was confirmed by determining XR activity in cells grown on glucose medium. The resulting recombinant strain, LNG2, showed high XR activity (2.86 U (mg of protein)(-1)), whereas parent strain BSXDH-3 showed no activity. In xylitol fermentation using glucose as a co-substrate with xylose, LNG2 showed xylitol production rate 1.44 g L(-1) h(-1) and xylitol yield of 96% at 44 h, which were 73 and 62%, respectively, higher than corresponding values for BSXDH-3 (rate 0.83 g L(-1) h(-1); yield 59%).     

Cloning and characterization of the xyl1 gene, encoding an NADH-preferring xylose reductase from Candida parapsilosis, and its functional expression in Candida tropicalis

Xylose reductase (XR) is a key enzyme in D-xylose metabolism, catalyzing the reduction of D-xylose to xylitol. An NADH-preferring XR was purified to homogeneity from Candida parapsilosis KFCC-10875, and the xyl1 gene encoding a 324-amino-acid polypeptide with a molecular mass of 36,629 Da was subsequently isolated using internal amino acid sequences and 5' and 3' rapid amplification of cDNA ends. The C. parapsilosis XR showed high catalytic efficiency (kcat/Km = 1.46 s(-1) mM(-1)) for D-xylose and showed unusual coenzyme specificity, with greater catalytic efficiency with NADH (kcat/Km = 1.39 x 10(4) s(-1) mM(-1)) than with NADPH (kcat/Km = 1.27 x 10(2) s(-1) mM(-1)), unlike all other aldose reductases characterized. Studies of initial velocity and product inhibition suggest that the reaction proceeds via a sequentially ordered Bi Bi mechanism, which is typical of XRs. Candida tropicalis KFCC-10960 has been reported to have the highest xylitol production yield and rate. It has been suggested, however, that NADPH-dependent XRs, including the XR of C. tropicalis, are limited by the coenzyme availability and thus limit the production of xylitol. The C. parapsilosis xyl1 gene was placed under the control of an alcohol dehydrogenase promoter and integrated into the genome of C. tropicalis. The resulting recombinant yeast, C. tropicalis BN-1, showed higher yield and productivity (by 5 and 25%, respectively) than the wild strain and lower production of by-products, thus facilitating the purification process. The XRs partially purified from C. tropicalis BN-1 exhibited dual coenzyme specificity for both NADH and NADPH, indicating the functional expression of the C. parapsilosis xyl1 gene in C. tropicalis BN-1. This is the first report of the cloning of an xyl1 gene encoding an NADH-preferring XR and its functional expression in C. tropicalis, a yeast currently used for industrial production of xylitol.     

假丝酵母发酵玉米芯半纤维素水解液生产木糖醇

采用一株驯化过的假丝酵母(Candida sp.)直接发酵经过简单脱毒处理的玉米芯半纤维素水解液生产木糖醇。确定了水解液的最适浓缩倍数在3.0～3.72的范围内。利用正交实验,确定了摇瓶分批发酵工艺条件的最适组合为：摇床转速180r/min,起始C/N为50,起始pH 5.5,接种量5% (体积比)。在此基础上,重点研究了在发酵罐中通气量对酵母发酵玉米芯水解液生产木糖醇的影响。结果表明采用先高后低的分段通气发酵在木糖醇得率方面明显优于恒定通气发酵;其中,在0～24h,3.75 L/min;24～108h,1.25 L/min的分段通气条件下(装液量为2.5L),木糖醇得率(木糖醇/木糖,g/g) 达到0.75 g/g。该结果将有助于建立一种高效的、大规模的利用玉米芯半纤维素水解液发酵生产木糖醇的工艺。     

Formation of xylitol in Candida guilliermondii 2581 culture

The yeast strain Candida guilliermondii 2581 was chosen for its ability to produce xylitol in media with high concentrations of xylose. The rate of xylitol production at a xylose concentration of 150 g/l is 1.25 g/l per h; the concentration of xylitol after three days of cultivation is 90 g/l; and the relative xylitol yield is 0.6 g per g substrate consumed. The growth conditions were found that resulted in the maximum relative xylitol yield with complete consumption of the sugar: xylose concentration, 150 g/l; pH 6.0; and shaking at 60 rpm. It was shown that the growth under conditions of limited aeration favors the reduction of xylose.     

Isolation and identification of xylitol-producing yeasts from agricultural residues

Selected yeast strains isolated from corn silage and viticulture residues were screened for their capacities to convert D-xylose into xylitol A conventional TLC was adapted for easy determination of xylose and xylitol in the culture supernatant solutions. This technique is suitable for the first steps of a screening program to select xylitol-producing yeasts from natural environments. Candida tropicalis ASM III (NRRL Y-27290), isolated from corn silage, appears to be a promising strain for xylitol production with a high yield (0.88 g xylitol per g of xylose consumed).     

Enhancement of xylitol production in Candida tropicalis by co-expression of two genes involved in pentose phosphate pathway

The yeast Candida tropicalis produces xylitol, a natural, low-calorie sweetener whose metabolism does not require insulin, by catalytic activity of NADPH-dependent xylose reductase. The oxidative pentose phosphate pathway (PPP) is a major basis for NADPH biosynthesis in C. tropicalis. In order to increase xylitol production rate, xylitol dehydrogenase gene (XYL2)disrupted C. tropicalis strain BSXDH-3 was engineered to co-express zwf and gnd genes which, respectively encodes glucose-6-phosphate dehydrogenase (G6PDH) and 6-phosphogluconate dehydrogenase (6-PGDH), under the control of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) promoter. NADPH-dependent xylitol production was higher in the engineered strain, termed "PP", than in BSXDH-3. In fermentation experiments using glycerol as a co-substrate with xylose, strain PP showed volumetric xylitol productivity of 1.25 g l(-1) h(-1), 21% higher than the rate (1.04 g l(-1) h(-1)) in BSXDH-3. This is the first report of increased metabolic flux toward PPP in C. tropicalis for NADPH regeneration and enhanced xylitol production.     

The influence of aeration and hemicellulosic sugars on xylitol production by Candida tropicalis

The influence of other hemicellulosic sugars (arabinose, galactose, mannose and glucose), oxygen limitation, and initial xylose concentration on the fermentation of xylose to xylitol was investigated using experimental design methodology. Oxygen limitation and initial xylose concentration had considerable influences on xylitol production by Canadida tropicalis ATCC 96745. Under semiaerobic conditions, the maximum xylitol yield was 0.62 g/g substrate, while under aerobic conditions, the maximum volumetric productivity was 0.90 g/l h. In the presence of glucose, xylose utilization was strongly repressed and sequential sugar utilization was observed. Ethanol produced from the glucose caused 50% reduction in xylitol yield when its concentration exceeded 30 g/l. When complex synthetic hemicellulosic sugars were fermented, glucose was initially consumed followed by a simultaneous uptake of the other sugars. The maximum xylitol yield (0.84 g/g) and volumetric productivity (0.49 g/l h) were obtained for substrates containing high arabinose and low glucose and mannose contents.     

Specific features of fermentation of D-xylose and D-glucose by xylose-assimilating yeasts

The ability to assimilate D-glucose and D-xylose was studied in 21 yeast species of the following genera: Candida, Kluyveromyces, Pachysolen, Pichia, and Torulopsis. All the cultures fermented D-glucose with the formation of ethanol. During the assimilation of D-xylose, ethanol was produced by P. stipitis and C. shehatae, whereas xylitol was produced by C. didensiae, C. intermediae, C. parapsilosis, C. silvanorum, C. tropicalis, K. fragilis, K. marxianus, P. guillermondii, and T. molishiama. The yeast P. tannophilus produced comparable amounts of both alcohols. The possible use of xylose-assimilating yeasts for the production of xylitol and ethanol is discussed.     

酵母发酵蔗渣半纤维素水解物生产木糖酶

采用二次正交旋转组合设计研究了蔗渣半纤维素水解过程中硫酸浓度与液 固比对木糖收率的影响。回归分析表明 ,这两个因素与木糖的收率之间存在显著的回归关系。通过回归方程优化水解条件 ,当硫酸浓度 2 .4g L ,液 固 =6 .2 ,在蒸汽压力 2 .5× 10 4Pa的条件下水解 2 .5h ,10 0g蔗渣可水解生成木糖约 2 4g。大孔树脂吸附层析处理蔗渣半纤维素水解物 ,能有效地减少其中的酵母生长抑制物含量 ,显著改善水解物的发酵性能。用大孔树脂在pH 2条件下处理过的蔗渣半纤维素水解物作基质 ,含木糖 2 0 0g L ,产木糖醇酵母菌株CandidatropicalisAS2 .1776发酵 110h耗完基质中的木糖 ,生成木糖醇 12 7g L ,产物转化率 0 .6 4(木糖醇g 木糖g) ,产物生成速率 1.15g L·h .     

Production of xylitol from Candida tropicalis by using an oxidation-reduction potential-stat controlled fermentation

An on-line device, ORP (oxidation-reduction potential)-stat, was used to control glucose-feeding for enhancing xylitol conversion from D-xylose during an oxygen-limited fermentation by Candida tropicalis. The fermentation was carried out in a 5 l jar fermenter. After glucose in the medium was depleted, a switching to a limited aeration and feeding glucose controlled by ORP-stat was performed. The maximum xylitol yield was obtained under a condition at an ORP of -180 mV and at an aeration rate of 0.2 l min(-1).     

Xylitol Production by a Culture ofCandida guilliermondii2581

The yeast strain Candida guilliermondii2581 was chosen for its ability to produce xylitol in media with high concentrations of xylose. The rate of xylitol production at a xylose concentration of 150 g/l is 1.25 g/l per h; the concentration of xylitol after three days of cultivation is 90 g/l; and the relative xylitol yield is 0.6 g per g substrate consumed. The growth conditions were found that resulted in the maximum relative xylitol yield with complete consumption of the sugar: xylose concentration, 150 g/l; pH 6.0; and shaking at 60 rpm. It was shown that the growth under conditions of limited aeration favors the reduction of xylose.     

Production of xylitol from Candida tropicalis by using an oxidation-reduction potential-stat controlled fermentation

An on-line device, ORP (oxidation-reduction potential)-stat, was used to control glucose-feeding for enhancing xylitol conversion from D-xylose during an oxygen-limited fermentation by Candida tropicalis. The fermentation was carried out in a 5 l jar fermenter. After glucose in the medium was depleted, a switching to a limited aeration and feeding glucose controlled by ORP-stat was performed. The maximum xylitol yield was obtained under a condition at an ORP of - 180 mV and at an aeration rate of 0.2 l min(-1).     

Screening of facultative anaerobic bacteria utilizing D-xylose for xylitol production

Seventeen cultures belonging to three genera of facultative bacteria ( Serratia, Cellulomonas, and Corynebacterium) were screened for the production of xylitol, a sugar alcohol used as a sweetener in the pharmaceutical and food industries. The bacterial strains that utilized D-xylose for growth were investigated for xylitol production. A chromogenic assay of both solid and liquid cultures showed that ten of the 17 bacteria screened could grow on D-xylose and produce detectable quantities of xylitol during 24-96 h of fermentation. Among the screened cultures, Corynebacterium sp. B-4247 produced the highest amount of xylitol. In addition, the ten bacterial cultures that initially produced xylitol were studied for the effect of the environmental factors, such as temperature, concentration of D-xylose and aeration, on xylitol production.     

Influence of temperature and pH on xylitol production from xylose by Debaryomyces hansenii

The production of xylitol from concentrated synthetic xylose solutions (S(o) = 130-135 g/L) by Debaryomyces hansenii was investigated at different pH and temperature values. At optimum starting pH (pH(o) = 5.5), T = 24 degrees C, and relatively low starting biomass levels (0.5-0.6 g(x)/L), 88% of xylose was utilized for xylitol production, the rest being preferentially fermented to ethanol (10%). Under these conditions, nearly 70% of initial carbon was recovered as xylitol, corresponding to final xylitol concentration of 91.9 g(P)/L, product yield on substrate of 0.81 g(P)/g(S), and maximum volumetric and specific productivities of 1.86 g(P)/L x h and 1.43 g(P)/g(x) x h, respectively. At higher and lower pH(o) values, respiration also became important, consuming up to 32% of xylose, while negligible amounts were utilized for cell growth (0.8-1.8%). The same approach extended to the effect of temperature on the metabolism of this yeast at pH(o) = 5.5 and higher biomass levels (1.4-3.0 g(x)/L) revealed that, at temperatures ranging from 32-37 degrees C, xylose was nearly completely consumed to produce xylitol, reaching a maximum volumetric productivity of 4.67 g(P)/L x h at 35 degrees C. Similarly, both respiration and ethanol fermentation became significant either at higher or at lower temperatures. Finally, to elucidate the kinetic mechanisms of both xylitol production and thermal inactivation of the system, the related thermodynamic parameters were estimated from the experimental data with the Arrhenius model: activation enthalpy and entropy were 57.7 kJ/mol and -0.152 kJ/mol x K for xylitol production and 187.3 kJ/mol and 0.054 kJ/mol x K for thermal inactivation, respectively.