Xylitol Production Rate Research Articles

Evaluation of Xylose-Utilising Yeasts for Xylitol Production from Second-Generation Ethanol Vinasse and Effect of Agitation Intensity in Flask-Scale Xylitol Production

This study aimed to select a yeast strain that effectively utilises xylose to produce xylitol from the vinasse of ethanol broth obtained from the fermentation of sugarcane bagasse hydrolysate. Eleven strains of xylose-fermenting yeasts were evaluated for their abilities to utilise xylose and produce xylitol. Two strains that showed outstanding performance in the semi-defined xylose medium were selected for further testing with a vinasse medium. Candida guilliermondii TISTR 5068 showed a superior xylitol production of 7.03 ± 0.08 g/L with the xylitol yield of 0.70 g/gxylose when cultured in bagasse-based ethanol vinasse. The strain was further tested for its xylitol production performance when cultured at four different agitation intensities. Excessive agitation resulted in a rapid xylitol production rate but caused xylitol consumption once the xylose was depleted. Moderate agitation resulted in the highest xylitol yield of 0.79 g/gxylose. The results of this study have provided important information for the development of the xylitol production process using waste streams from cellulosic ethanol production.

Sugarcane Bagasse-Based Ethanol Production and Utilization of Its Vinasse for Xylitol Production as an Approach in Integrated Biorefinery

Biorefinery of sugarcane bagasse into ethanol and xylitol was investigated in this study. Ethanol fermentation of sugarcane bagasse hydrolysate was carried out by Saccharomyces cerevisiae. After ethanol distillation, the vinasse containing xylose was used to produce xylitol through fermentation by Candida guilliermondii TISTR 5068. During the ethanol fermentation, it was not necessary to supplement a nitrogen source to the hydrolysate. Approximately 50 g/L of bioethanol was produced after 36 h of fermentation. The vinasse was successfully used to produce xylitol. Supplementing the vinasse with 1 g/L of yeast extract improved xylitol production 1.4-fold. Cultivating the yeast with 10% controlled dissolved oxygen resulted in the best xylitol production and yields of 10.2 ± 1.12 g/L and 0.74 ± 0.04 g/g after 60 h fermentation. Supplementing the vinasse with low fraction of molasses to improve xylitol production did not yield a positive result. The supplementation caused decreases of up to 34% in xylitol production rate, 24% in concentration, and 24% in yield.

Stepwise metabolic engineering of Candida tropicalis for efficient xylitol production from xylose mother liquor

BackgroundCommercial xylose purification produces xylose mother liquor (XML) as a major byproduct, which has become an inexpensive and abundant carbon source. A portion of this XML has been used to produce low-value-added products such as caramel but the remainder often ends up as an organic pollutant. This has become an issue of industrial concern. In this study, a uracil-deficient Candida tropicalis strain was engineered to efficiently convert XML to the commercially useful product xylitol.ResultsThe xylitol dehydrogenase gene was deleted to block the conversion of xylitol to xylulose. Then, an NADPH regeneration system was added through heterologous expression of the Yarrowia lipolytica genes encoding 6-phosphate-gluconic acid dehydrogenase and 6-phosphate-glucose dehydrogenase. After process optimization, the engineered strain, C. tropicalis XZX-B4ZG, produced 97.10 g L− 1 xylitol in 120 h from 300 g L− 1 XML in a 5-L fermenter. The xylitol production rate was 0.82 g L− 1 h− 1 and the conversion rate was 92.40 %.ConclusionsIn conclusion, this study performed a combination of metabolic engineering and process optimizing in C. tropicalis to enhance xylitol production from XML. The use of C. tropicalis XZX-B4ZG, therefore, provided a convenient method to transform the industrial by-product XML into the useful material xylitol.

Overexpression of Mutant Galactose Permease (ScGal2_N376F) Effective for Utilization of Glucose/Xylose or Glucose/ Galactose Mixture by Engineered Kluyveromyces marxianus.

Mutant sugar transporter ScGAL2-N376F was overexpressed in Kluyveromyces marxianus for efficient utilization of xylose, which is one of the main components of cellulosic biomass. K. marxianus ScGal2_N376F, the ScGAL2-N376F-overexpressing strain, exhibited 47.04 g/l of xylose consumption and 26.55 g/l of xylitol production, as compared to the parental strain (24.68 g/l and 7.03 g/l, respectively) when xylose was used as the sole carbon source. When a mixture of glucose and xylose was used as the carbon source, xylose consumption and xylitol production rates were improved by 195% and 360%, respectively, by K. marxianus ScGal2_N376F. Moreover, the glucose consumption rate was improved by 27% as compared to that in the parental strain. Overexpression of both wild-type ScGAL2 and mutant ScGAL2-N376F showed 48% and 52% enhanced sugar consumption and ethanol production rates, respectively, when a mixture of glucose and galactose was used as the carbon source, which is the main component of marine biomass. As shown in this study, ScGAL2-N376F overexpression can be applied for the efficient production of biofuels or biochemicals from cellulosic or marine biomass.

Furfural and glucose can enhance conversion of xylose to xylitol by Candida magnoliae TISTR 5663

Xylitol production from xylose by the yeast Candida magnoliae TISTR 5663 was enhanced by supplementing the fermentation medium with furfural (300mg/L) and glucose (3g/L with an initial mass ratio of glucose to xylose of 1:10) together under oxygen limiting conditions. In the presence of furfural and glucose, the final concentration of xylitol was unaffected relative to control cultures but the xylitol yield on xylose increased by about 5%. Supplementation of the culture medium with glucose alone at an initial concentration of 3g/L, stimulated the volumetric and specific rates of xylose consumption and the rate of xylitol production from xylose. In a culture medium containing 30g/L xylose, 300mg/L furfural and 3g/L glucose, the volumetric production rate of xylitol was 1.04g/L h and the specific production rate was 0.169g/g h. In the absence of furfural and glucose, the volumetric production rate of xylitol was ∼35% lower and the specific production rate was nearly 30% lower. In view of these results, xylose-containing lignocellulosic hydrolysates contaminated with furfural can be effectively used for producing xylitol by fermentation so long as the glucose-to-xylose mass ratio in the hydrolysate does not exceed 1:10 and the furfural concentration is ≤300mg/L.

Evaluation of cotton stalk hydrolysate for xylitol production

ABSTRACTCotton stalk is a widely distributed and abundant lignocellulosic waste found in Turkey. Because of its rich xylose content, it can be a promising source for the production of xylitol. Xylitol can be produced by chemical or biotechnological methods. Because the biotechnological method is a simple process with great substrate specificity and low energy requirements, it is more of an economic alternative for the xylitol production. This study aimed to use cotton stalk for the production of xylitol with Candida tropicalis Kuen 1022. For this purpose, the combined effects of different oxygen concentration, inoculum level and substrate concentration were investigated to obtain high xylitol yield and volumetric xylitol production rate. Candida tropicalis Kuen 1022 afforded different concentrations of xylitol depending on xylose concentration, inoculum level, and oxygen concentration. The optimum xylose, yeast concentration, and airflow rate for cotton stalk hydrolysate were found as 10.41 g L−1, 0.99 g L−1, and 1.02 vvm, respectively, and under these conditions, xylitol yield and volumetric xylitol production rate were obtained as 36% and 0.06 g L−1 hr−1, respectively. The results of this study show that cotton stalk can serve as a potential renewable source for the production of xylitol.

Optimized Production of Xylitol from Xylose Using a Hyper-Acidophilic Candida tropicalis.

The yeast Candida tropicalis DSM 7524 produces xylitol, a natural, low-calorie sweetener, by fermentation of xylose. In order to increase xylitol production rate during the submerged fermentation process, some parameters-substrate (xylose) concentration, pH, aeration rate, temperature and fermentation strategy-have been optimized. The maximum xylitol yield reached at 60–80 g/L initial xylose concentration, pH 5.5 at 37 °C was 83.66% (w/w) on consumed xylose in microaerophilic conditions (kLa = 2·h−1). Scaling up on 3 L fermenter, with a fed-batch strategy, the best xylitol yield was 86.84% (w/w), against a 90% of theoretical yield. The hyper-acidophilic behaviour of C. tropicalis makes this strain particularly promising for industrial application, due to the possibility to work in non-sterile conditions.

Production of Xylitol from D-Xylose by Overexpression of Xylose Reductase in Osmotolerant Yeast Candida glycerinogenes WL2002-5.

Efficient bioconversion of D-xylose into various biochemicals is critical for the developing lignocelluloses application. In this study, we compared D-xylose utilization in Candida glycerinogenes WL2002-5 transformants expressing xylose reductase (XYL1) in D-xylose metabolism. C. glycerinogenes WL2002-5 expressing XYL1 from Schefferomyces stipitis can produce xylitol. Xylitol production by the recombinant strains was evaluated using a xylitol fermentation medium with glucose as a co-substrate. As glucose was found to be an insufficient co-substrate, various carbon sources were screened for efficient cofactor regeneration, and glycerol was found to be the best co-substrate. The effects of glycerol on the xylitol production rate by a xylose reductase gene (XYL1)-overexpressed mutant of C. glycerinogenes WL2002-5 were investigated. The XYL1-overexpressed mutant produced xylitol from D-xylose using glycerol as a co-substrate for cell growth and NAD (P) H regeneration: 100 g/L D-xylose was completely converted into xylitol when at least 20 g/L glycerol was used as a co-substrate. XYL1 overexpressed mutant grown on glycerol as co-substrate accumulated 2.1-fold increased xylitol concentration over those cells grown on glucose as co-substrate. XYL1 overexpressed mutant produced xylitol with a volumetric productivity of 0.83 g/L/h, and a xylitol yield of 98 % xylose. Recombinant yeast strains obtained in this study are promising candidates for xylitol production. This is the first report of XYL1 gene overexpression of C. glycerinogenes WL2002-5 for enhancing the efficiency of xylitol production.

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.

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%).

Xylitol production from olive-pruning debris by sulphuric acid hydrolysis and fermentation with Candida tropicalis

AbstractThe debris of olive pruning is a renewable, low-cost and widely available agricultural waste. Its biochemical conversion by hydrolysis and fermentation was undertaken in the present study. Diluted acid hydrolysis was conducted in a heterogeneous stirred tank reactor at 90°C and at a low sulphuric acid concentration (0.0–1.0 N) for 300 min. To increase thed-xylose/d-glucose ratio into the hydrolysate, in another experiment amorphous cellulose and extracts were removed by means of a pretreatment in an extruder with 1 N H2SO4at 70°C before the acid hydrolysis. The fermentation of hydrolysates was performed under microaerobic conditions in a batch bioreactor at 30°C and pH 5 withCandida tropicalisNBRC 0618. The controlled fermentation parameters included maximum specific growth rate, biomass productivity, rate of the specific substrate uptake, rates of specific ethanol and xylitol production, and overall yield of ethanol and xylitol. In the presence of 1.0 N H2SO4, the fermentation of the pretreated hydrolysate led to specific xylitol production rates and overall xylitol yield (0.1 g g-1 h for t=25 h; 0.49 g g-1, respectively) higher than those achieved without pretreatment (0.03 g g-1 h for t=25 h; 0.39 g g-1, respectively). Under these conditions, 53 g xylitol kg-1of dry olive-pruning debris was obtained from the pretreated culture, whereas without pretreatment 70 g ethanol and 34 g xylitol were recovered.

Application of Response Surface Methodology for Optimization of Xylitol Production from Lignocellulosic Hydrolysate in a Fluidized Bed Reactor

AbstractThis work aims to contribute to the development of a viable technology for the production of xylitol from sugarcane bagasse hemicellulose hydrolysate, using a fluidized bed bioreactor with yeast cells of Candida guilliermondii FTI 20037. To evaluate the technical viability of this biotechnological process, simple batch fermentations were realized according to a 23 factorial design with three center points. Furthermore, the influence of the variables fluidization flux, hydrolysate concentration factor and air flow on process yield and volumetric productivity were evaluated. The results obtained showed that only an increase in fluidization flux had a positive influence on process yield and volumetric productivity. This is due to better oxygen transfer to the inside of the cell, leading to increased xylose consumption and xylitol production rates.

Effect of Furfural, Vanillin and Syringaldehyde on Candida guilliermondii Growth and Xylitol Biosynthesis

Xylitol is a five-carbon sugar alcohol with established commercial use as an alternative sweetener and can be produced from hemicellulose hydrolysate. However, there are difficulties with microbiological growth and xylitol biosynthesis on hydrolysate because of the inhibitors formed from hydrolysis of hemicellulose. This research focused on the effect of furfural, vanillin, and syringaldehyde on growth of Candida guilliermondii and xylitol accumulation from xylose in a semi-synthetic medium in microwell plate and bioreactor cultivations. All three compounds reduced specific growth rate, increased lag time, and reduced xylitol production rate. In general, increasing concentration of inhibitor increased the severity of inhibition, except in the case of 0.5 g vanillin per liter, which resulted in a faster late batch phase growth rate and increased biomass yield. At concentrations of 1 g/l or higher, furfural was the least inhibitory to growth, followed by syringaldehyde. Vanillin most severely reduced specific growth rate. All three inhibitors reduced xylitol production rate approximately to the same degree.

Enhancement of xylitol productivity and yield using a xylitol dehydrogenase gene-disrupted mutant of Candida tropicalis under fully aerobic conditions

The effects of glycerol and the oxygen transfer rate on the xylitol production rate by a xylitol dehydrogenase gene (XYL2)-disrupted mutant of Candida tropicalis were investigated. The mutant produced xylitol near the almost yield of 100% from D: -xylose using glycerol as a co-substrate for cell growth and NADPH regeneration: 50 g D: -xylose l(-1) was completely converted into xylitol when at least 20 g glycerol l(-1) was used as a co-substrate. The xylitol production rate increased with the O(2) transfer rate until saturation and it was not necessary to control the dissolved O(2) tension precisely. Under the optimum conditions, the volumetric productivity and xylitol yield were 3.2 g l(-1) h(-1) and 97% (w/w), respectively.

Increased xylitol production rate during long-term cell recycle fermentation of Candida tropicalis.

Long-term cell recycle fermentations of Candida tropicalis were performed over 14 rounds of fermentation. The average xylitol concentrations, fermentation times, volumetric productivities and product yields for 14 rounds were 105 g l(-1), 333 h, 4.4 g l(-1) h(-1) and 78%, respectively, in complex medium; and 110 g l(-1), 284 h, 5.4 g l(-1) h(-1) and 81%, respectively, in a chemically defined medium. These productivities were 1.7 and 2.4 times those with batch fermentation in the complex and chemically defined media, respectively. The xylitol yield from xylose with cell recycle fermentation using the chemically defined medium was 81% (w/w), which was 7% greater than the xylitol yield with batch fermentation (74%); both modes of fermentation gave the same yield using the complex medium. These results suggest that the chemically defined medium is more suitable for production of xylitol than complex medium.

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.

Xylitol production by Ca-alginate entrapped cells: comparison of different fermentation systems

Candida guilliermondii cells were entrapped in Ca-alginate beads and used for xylitol production from concentrated sugarcane bagasse hemicellulosic hydrolysate in three different fermentation systems, namely 125-ml Erlenmeyer flasks (EF), 2.4-l stirred tank reactor (STR) and 2.4-l basket-type stirred tank reactor (BSTR). The EF system provided a xylitol production of 21.0 g P l −1, a product yield based on xylose consumption of 0.54 g P g S −1, and an overall production rate of 0.44 g P l −1 h −1 after 48 h of fermentation. By the STR system, 23.5 g P l −1 was produced after 60 h of fermentation, corresponding to a yield of 0.58 g P g S −1 and a production rate of 0.39 g P l −1 h −1. As the average volume of the beads decreased by 10.7% ( p<0.05) during this fermentation, a basket was fitted into the reactor vessel to prevent the beads abrasion and thus maintain their integrity. However, the xylitol yield (0.46 g P g S −1), production rate (0.21 g P l −1 h −1) and concentration (15.0 g P l −1) attained by this system were affected by mass-transfer limitations.

Optimization of fed-batch fermentation for xylitol production by Candida tropicalis.

Xylitol, a functional sweetener, was produced from xylose by biological conversion using Candida tropicalis ATCC 13803. Based on a two-substrate fermentation using glucose for cell growth and xylose for xylitol production, fed-batch fermentations were undertaken to increase the final xylitol concentration. The effects of xylose and xylitol on xylitol production rate were studied to determine the optimum concentrations for fed-batch fermentation. Xylose concentration in the medium (100 g l(-1)) and less than 200 g l(-1) total xylose plus xylitol concentration were determined as optimum for maximum xylitol production rate and xylitol yield. Increasing the concentrations of xylose and xylitol decreased the rate and yield of xylitol production and the specific cell growth rate, probably because of an increase in osmotic stress that would interfere with xylose transport, xylitol flux to secretion to cell metabolism. The feeding rate of xylose solution during the fed-batch mode of operation was determined by using the mass balance equations and kinetic parameters involved in the equations in order to increase final xylitol concentration without affecting xylitol and productivity. The optimized fed-batch fermentation resulted in 187 g l(-1) xylitol concentration, 0.75 g xylitol g xylose(-1) xylitol yield and 3.9 g xylitol l(-1) h(-1) volumetric productivity.

Growth model and prediction of oxygen transfer rate for xylitol production from d-xylose by C. guilliermondii

The K L a values for optimum xylitol productivity using Candida guilliermondii with initial cell concentrations of 1.0 and 4.0 g/l and an initial d-xylose concentration of 50 g/l were predicted as 20 and 100 h −1, respectively, by means of growth, substrate and oxygen uptake simulations. The highest xylitol production rate (1.52 g/l h) was experimentally obtained with an initial cell concentration of 4 g/l and K L a 100 h −1. Kinetic parameters were estimated for Monod, Contois and Tessier growth models using a non-linear method. The Contois model was chosen as the most suitable due to the agreement with data reported in literature for yeasts and statistical confidence of its parameters. The specific oxygen uptake rate (SOUR) was determined by the dynamic oxygen balance. It had a maximum value of 153 mg O 2/g cell h after 2 h growth. The choice of the best K L a value was made through dissolved oxygen concentration simulations for fermentations carried out with two different inoculum sizes, resulting in a maximum volumetric productivity of 1.52 g/l h, for an initial cell concentration of 4 g/l, employing a K L a value of 100 h −1.

Use of Immobilized Candida Yeast Cells for Xylitol Production from Sugarcane Bagasse Hydrolysate

Candida guilliermondii cells were immobilized in Ca-alginate beads and used for xylitol production from concentrated sugarcane bagasse hydrolysate. A full factorial design was employed to determine whether variations in the immobilization conditions would have any effects on the beads, chemical stability and on the xylitol production rates. Duplicate fermentation runs were carried out in 125-mL Erlenmeyer flasks maintained in a rotatory shaker at 30 degrees C and 200 rpm for 72 h. Samples were periodically analyzed to monitor xylose and acetic acid consumption, xylitol production, free cell growth, and bead solubilization. Concentrations of sodium alginate at 20.0 g/L and calcium chloride at 11.0 g/L and bead curing time of 24 h represented the most appropriate immobilization conditions within the range of conditions tested.