Evaluation of inoculum of Candida guilliermondii grown in presence of glucose on xylose reductase and xylitol dehydrogenase activities and xylitol production during batch fermentation of sugarcane bagasse hydrolysate

The effect of glucose on xylose-xylitol metabolism in fermentation medium consisting of sugarcane bagasse hydrolysate was evaluated by employing an inoculum of Candida guilliermondii grown in synthetic media containing, as carbon sources, glucose (30 g/L), xylose (30 g/L), or a mixture of glucose (2 g/L) and xylose (30 g/L). The inoculum medium containing glucose promoted a 2.5-fold increase in xylose reductase activity (0.582 IU/mg_prot) and a 2-fold increase in xylitol dehydrogenase activity (0.203 IU/mg_prot) when compared with an inoculum-grown medium containing only xylose. The improvement in enzyme activities resulted in higher values of xylitol yield (0.56 g/g) and productivity (0.46 g/[L·h]) after 48 h of fermentation.     

Effects of environmental conditions on xylose reductase and xylitol dehydrogenase production by Candida guilliermondii

The effects of environmental conditions, namely initial pH (2.5–7.0) and temperature (25 and 35°C), on xylose reductase and xylitol dehydrogenase levels, as well as on xylitol production, were evaluated. Although the fermentative parameter values increased with an increase in pH and temperature (the maximum Y_P/s and Q _p were 0.75 g/g and 0.95 g/[L·h], respectively, both attained at pH 6.0, 35°C), the highest xylose reductase activities (nearly 900 1U/mg of protein) were observed at an initial pH varying from 4.0 to 6.0. Xylitol dehydrogenase was favored by an increase in both initial pH and temperature of the medium. The highest xylitol dehydrogenase specific activity was attained at pH 6.5 and 35°C (577 1U/mg of protein).     

Xylose reductase and xylitol dehydrogenase activities of Candida guilliermondii as a function of different treatments of sugarcane bagasse hemicellulosic hydrolysate employing experimental design

The sugarcane bagasse hydrolysate, which is rich in xylose, can be used as culture medium for Candida guilliermondii in xylitol production. However, the hydrolysate obtained from bagasse by acid hydrolysis at 120°C for 20 min has by-products (acetic acid and furfural, among others), which are toxic to the yeast over certain concentrations. So, the hydrolysate must be pretreated before using in fermentation. The pretreatment variables considered were: adsorption time (15,37.5, and 60 min), type of acid used (H2So4 and H3Po4), hydrolysate concentration (original, twofold, and fourfold. concentrated), and active charcoal (0.5, 1.75 and 3.0%). The suitability of the pretreatment was followed by measuring the xylose reductase (XR) and xylitol dehydrogenase (XD) activity of yeast grown in each treated hydrolysate. The response surface methodology (2⁴ full factorial design with a centered face) indicated that the hydrolysate might be concentrated fourfold and the pH adjusted to 7.0 with CaO, followed by reduction to 5.5 with H₃PO₄. After that it was treated with active charcoal (3.0%) by 60 min. This pretreated hydrolysate attained the high XR/XD ratio of 4.5.     

Xylose reductase activity of Candida guilliermondii during xylitol production by fed-batch fermentation

Xylose reductase activity of Candida guilliermondii FTI 20037 was evaluated during xylitol production by fed-batch fermentation of sugarcane bagasse hydrolysate. A 2^4-1 fractional factorial design was used to select process variables. The xylose concentrations in the feeding solution (S _F ) and in the fermentor (S ₀), the pH, and the aeration rate were selected for optimization of this process, which will be undertaken in the near future. The best experimental result was achieved at S _F =45 g/L, S ₀=40 g/L, pH controlled at 6.0, and aeration rate of 1.2 vvm. Under these conditions, the xylose reductase activity was 0.81 U/mg of protein and xylitol production was 26.3 g/L, corresponding to a volumetric productivity of 0.55 g/(L·h) and a xylose xylitol yield factor of 0.68 g/g.     

Effect of the oxygen transfer coefficient on xylitol production from sugarcane bagasse hydrolysate by continuous stirred-tank reactor fermentation

The effect of the oxygen transfer coefficient on the production of xylitol by biocon version of xylose present in sugarcane bagasse hemicellulosic hydrolysate using the yeast Candiada guilliermondii was investigated. Continuous cultivation was carried out in a 1.25-L fermentor at 30°C, pH 5.5, 300 rpm, and a dilution rate of 0.03/h, using oxygen transfer coefficients of 10,20, and 30/h. The results showed that the microbial xylitol production (11 g/L) increased by 108% with the decrease in the oxygen volumetric transfer coefficient from 30 to 20/h. The maximum values of xylitol productivity (0.7g/[L…h]) and yield (0.58 g/g) were obtained at k _L a 20/h.     

Influence of oxygen availability on cell growth and xylitol production by Candida guilliermondii

Oxygen availability is the most important environmental parameter in the production of xylitol by yeasts, directly affecting yields and volumetric productivity. This work evaluated the cell behavior in fermentations carried out with different dissolved oxygen concentrations (0.5–30.0% of saturation), as well as a limited oxygen restriction (0% of saturation), at several oxygen volumetric transfer coefficients (12 ≤ k _L a ≤ 70 h⁻¹). These experiments allowed us to establish the specific oxygen uptake rate limits to ensure high yields and volumetric productivity. When oxygen availability was limited, the specific oxygen uptake rate values were between 12 and 26 mg of O₂/of g cell·h, resulting in a yield of 0.71 g of xylitol/xylose consumed, and 0.85 g/[L·h] for the volumetric productivity. According to the results, the effective control of the specific oxygen uptake rate makes it possible to establish complete control over this fermentative process, for both cell growth and xylitol production.     

Aspects of xylitol formation in sugarcane bagasse hydrolysate by Candida guillie rmondii in the presence of tetracycline

The biocon version of xylose intoxylitol using pH values of 4.0, 5.5 and 7.0 and tetracycline concentrations of 20 and 40 mg/L was carried out to verify the influence of these parameters on Candida guilliermondii metabolism for xylitol production. Experiments were performed with sugarcane bagasse hemicellulosi chydrolysate (48.5 g/L of xylose) in 125-mL Erlenmeyer flasks, at 30°C, 200 rpm, during 88 h. The results demostrated that the bioconversion of xylose into xylitol was significantly influenced by the pH. On the other hand, in media containing 20 or 40 mg/L of tetracycline, this bioconversion was not significantly affected. The best results of xylitol production were obtained in hemicellulosic hydrolysate without tetracycline, at pH 7.0 In these conditions, the maxim um specific growth rate was 0.014/h and the yield factor of xylitol and volumetric productivity were 0.85g/g and 0.70g/L/h respectively. Xylitol and cell growth occureed simultaneously.     

Oxygen uptake rate in production of xylitol by Candida guilliermondii with different aeration rates and initial xylose concentrations

The global oxygen uptake rate (OUR) and specific oxygen uptake rates (SOUR) were determined for different values of the volumetric oxygen mass transfer coefficient (15, 43, and 108 h⁻¹), and for varying initial xylose concentrations (50, 100, 150, and 200 g/L) in shaking flasks. The initial cell concentration was 4.0 g/L, and there was only significant growth in the fermentation with the highest oxygen availability. In this condition, OUR increased proportionally to cell growth, reaching maximum values from 2.1 to 2.5 g of O₂/(L·h) in the stationary phase when the initial substrate concentration was raised from 50 to 200 g/L, respectively. SOUR showed different behavior, growing to a maximum value coinciding with the beginning of the exponential growth phase, after which point it decreased. The maximum SOUR values varied from 265 to 370 mg of O₂/(g of cell·h), indicating the interdependence of this parameter and the substrate concentration. Although the volumetric productivity dropped slightly from 1.55 to 1.18 g of xylitol/(L·h), the strain producing capacity (γ _P/X ) rose from 9 to 20.6 g/g when the initial substrate concentration was increased from 50 to 200 g/L. As for the xylitol yield over xylose consumed (γ _P/S ), there was no significant variation, resulting in a mean value of 0.76 g/g. The results are of interest in establishing a strategy for controlling the dynamic oxygen supply to maximize volumetric productivity.     

Kinetics of xylitol fermentation by Candida guilliermondii grown on rice straw hemicellulosic hydrolysate

The fermentation kinetics for the conversion of rice straw hemicellulosic hydrolysate to xylitol by the yeast Candida guilliermondii was evaluated under batch conditions. The fermentation was accomplished in a 1 L working volume stirred-tank reactor with aeration of 1.3 vvm and agitation of 300 rpm (k_La=15/h). The maximum specific rate of xylitol formation (0.12 g/g) was achieved when the specific growth rate was lowered to 1/5 of its highest value. From analysis of the fermentation kinetics, a linear correlation between specific growth rate (μ_x) and specific rate of xylitol formation (q_p) was evident. Based on the Gaden model, this bioprocess was classified as growth-associated production and the relationship between μ_x and q_p can be described by the equation q_p=6.31μ_x.     

Activity of xylose reductase from Candida mogii grown in media containing different concentrations of rice straw hydrolysate

Xylose reductase (XR) activity was evaluated in extracts of Candida mogii grown in media containing different concentrations of rice straw hydrolysate. Results of X Ractivity were compared to xylitol production and a similar behavior was observed for these parameters. Highest values of specific production and productivity were found for xylose reductase (35 U/g of cell and 0.97 U/[g of cell·h], respectively) and for xylitol (5.63 g/g of cell and 0.13 g/[g of cell·h]) in fermentation conducted in medium containing 49.2 g of xylose/L. The maximum value of XR:XD ratio (1.82) was also calculated under this initial xylose concentration with 60 h of fermentation.     

Changing flux of xylose metabolites by altering expression of xylose reductase and xylitol dehydrogenase in recombinant Saccharomyces cerevisiae

We changed the fluxes of xylose metabolites in recombinant Saccharomyces cerevisiae by manipulating expression of Pichia stipitis genes (XYL1 and XYL2) coding for xylose reductase (XR) and xylitol dehydrogenase (XDH), respectively. XYL1 copy number was kept constant by integrating it into the chromosome. Copy numbers of XYL2 were varied either by integrating XYL2 into the chromosome or by transforming cells with XYL2 in a multicopy vector. Genes in all three constructs were under control of the strong constitutive glyceraldehyde-3-phosphate dehydrogenase promoter. Enzymatic activity of XR and XDH in the recombinant strains increased with the copy number of XYL1 and XYL2. XR activity was not detected in the parent but was present at a nearly constant level in all of the transformants. XDH activity increased 12-fold when XYL2 was on a multicopy vector compared with when it was present in an integrated single copy. Product formation during xylose fermentation was affected by XDH activity and by aeration in recombinant S. cerevisiae. Higher XDH activity and more aeration resulted in less xylitol and more xylulose accumulation during xylose fermentation. Secretion of xylulose by strains with multicopy XYL2 and elevated XDH supports the hypothesis that d-xylulokinase limits metabolic flux in recombinant S. cerevisiae.     

Xylose reductase production by candida guilliermondii

The effect of pH, time of fermentation, and xylose and glucose concentration on xylitol production, cell growth, xylose reductase (XR), and xylitol dehydrogenase (XD) activities ofCandida guilliermondii FTI 20037 were determined. For attaining XR and XD activities of 129-2190 U/mg of protein and 24-917 U/mg of protein, respectively, the cited parameters could vary as follows: initial pH: 3.0-5.0; xylose: 15-60 g/L; glucose: 0-5 g/L; and fermentation time: 12-24 h. Moreover, the high XR and XD activities occurred when the xylitol production by the yeast was less than 19.0 g/L.     

Maximizing the xylitol production from sugar cane bagasse hydrolysate by controlling the aeration rate

Batch fermentations of sugar cane bagasse hemicellulosic hydrolysate treated for removing the inhibitors of the fermentation were performed byCandida guilliermondii FTI20037 for xylitol production. The fermentative parameters agitation and aeration rate were studied aiming the maximization of xylitol production from this agroindustrial residue. The maximal xylitol volumetric productivity (0.87 g/L h) and yield (0.67 g/g) were attained at 400/min and 0.45 v.v.m. (K_La 27/h). According to the results, a suitable control of the oxygen input permitting the xylitol formation from sugar cane bagasse hydrolysate is required for the development of an efficient fermentation process for large-scale applications.     

Sugarcane bagasse as raw material and immobilization support for xylitol production

Xylose-to-xylitol bioconversion was performed utilizing Candida guillier-mondii immobilized in sugarcane bagasse and cultured in Erlenmeyer flasks using sugarcane bagasse hydrolysate as the source of xylose. Fermentations were carried out according to a factorial design, and the independent variables considered were treatment, average diameter, and amount of bagasse used as support for cell immobilization. By increasing the amount of support, the xylitol yield decreased, whereas the biomass yield increased. The diameter of the support did not influence xylitol production, and treatment of the bagasse with hexamethylene diamine prior to fermentation resulted in the highest amount of immobilized cells.     

Application of factorial design to the study of xylitol production from eucalyptus hemicellulosic hydrolysate

This study deals with the bioconversion of xylose into xylitol by Candida guilliermondii FTI 20037 using eucalyptus hemicellulosic hydrolysate obtained by acid hydrolysis. The influence of various parameters (ammonium sulfate, rice bran, pH, and xylose concentration) on the production of xylitol was evaluated. The experiments were based on multivariate statistical concepts, with the application of factorial design techniques to identify the most important variables in the process. The levels of these variables were quantified by the response surface methodology, which permitted the establishment of a significant mathematical model with a coefficient determination of R ²=0.92. The best results (xylitol=10.0 g/L, yield factor=0.2 g/g, and productivity=0.1 g/[L·h]) were attained with hydrolysate containing ammonium sulfate (1.1 g/L), rice bran (5.0 g/L), and xylose (initial concentration of 60.0 g/L), after 72 h of fermentation. The pH of fermentation was adjusted to 8.0 and the inoculum level utilized was 3 g/L.     

Xylitol production from wood hydrolyzates by entrapped Debaryomyces hansenii and Candida guilliermondii cells

Debaryomyces hansenii cells were entrapped in Ca-alginate beads and used for producing xylitol from wood hydrolyzates. Batch experiments showed that bioconversion was severely hindered when Ca-alginate beads were hardened with Al³⁺ solutions. As an alternative to Al³⁺ hardening, the improvements in both mechanical stability of bioparticles and fermenting ability of the immobilized system derived from using increased concentrations of sodium alginate were assessed. The best results were obtained using a 4% (w/v) Na-alginate solution in the gelification step. This concentration was selected to perform continuous fermentations in a packed-bed reactor using raw or charcoal-treated hydrolyzates (15.5 g of xylose/L) with two different yeasts: Candida guilliermondii and Debaryomyces hansenii. With a final cell concentration of about 50 g of cells/L (0.075 g of cells/g of beads), the volumetric productivities reached with these yeasts in media made from charcoal-treated hydrolyzates were 0.58 and 0.91 g/L·h, respectively.     

Xylitol production from hardwood hemicellulose hydrolysates by Pachysolen tannophilus, Debaryomyces hansenii, and Candida guilliermondii

Three different yeasts, Pachysolen tannophilus, Debaryomyces hansenii, and Candida guilliermondii, were evaluated to ferment xylose solutions prepared from hardwood hemicellulose hydrolysates, among which P. tannophilus proved to be the most promising microorganism. However, the presence of both lignin-derived compounds (LDC) and acetic acid rendered a poor fermentation. To enhance the fermentation kinetics, different treatments to purify the hydrolysates were studied, including overliming, charcoal adsorption for LDC removal, and evaporation for acetic acid and furfural stripping. Under the best operating conditions assayed, 39.5g/L of xylitol were achieved after 96 h of fermentation, which corresponds to a volumetric productivity of 0.41 g/L·h and a yield of product on consumed substrate of 0.63 g _p /g_S.     

Purification and properties of xylitol dehydrogenase from the xylose-fermenting yeastCandida shehatae

Xylitol dehydrogenase (EC1.1.1.9) from xylose-grown cells ofCandida shehatae was purified 215-fold by sequential chromatography on NAD-C8 affinity, Superose-12, and Cibacron blue columns, and a single band was observed by SDS gel electrophoresis. The purified enzyme had a native molecular weight of 82 kDa and a denatured molecular weight of 40 kDa following SDS gel electrophoresis, indicating that it was composed of two subunits. Alcohol dehydrogenase copurified on the NAD-C8 but was substantially removed by Superose-12 and was not detected following Cibacron blue chromatography. The kinetic properties of the C.shehatae xylitol dehydrogenase differed considerably from those described previously for thePachysolen tannophilus enzyme. The K_m of the C.shehatae enzyme for xylitol was 3.8 times smaller, whereas the K_m for xylulose was 1.7-fold bigger. These factors could account for the lower xylitol production by C.shehatae.     

Use of immobilized Candida yeast cells for xylitol production from sugarcane bagasse hydrolysate

Applied Biochemistry and Biotechnology - Candida guilliermondii cells were immobilized in Ca-alginate beads and used for xylitol production from concentrated sugarcane bagasse hydrolysate. A full...     

Optimization of acid hydrolysis of sugarcane bagasse and investigations on its fermentability for the production of xylitol by Candida guilliermondii

The dilute-acid hydrolysis of sugarcane bagasse was optimized using a statistical experimental design resulting in hydrolysates containing 57.25 g/L of xylose, which were fermented with a high inoculum concentration (10 g/L of the yeast Candida guilliermondii IM/UFRJ 50088). The addition of urea reduced the time of conversion (t _C) to 75 h (without nitrogen source addition t _C>127 h), and, consequently, improving the rates of xylitol bioproduction. Fermentator experiments, using the optimized conditions, resulted in enhanced conversion rates, reducing t _C to 30 h. The stability of the yeast in the hydrolysate was also verified in a 480-h cultivation.