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.     

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 initial pH on biological synthesis of xylitol using xylose-rich hydrolysate

Sugarcane bagasse, an agricultural residue plentiful in Brazil, was utilized for xylitol production by a biotechnological process. Am edium fermentation prepared with this xylose-rich biomass at an oxygen transfer volmetric coefficient of 10/h¹ and different initial pH value was inoculated with cells of Candida guilliermondii FTI 20037. The maximum values of xylitol and cell volumetric productivities (Q _p=0.56 g/[L·h] and Q _p=0.11 g/[g·h]), xylitol yield factor (Y _p/s=0.79 g/g), and xylose uptake rate (qs=0.197 g/[g·h]) wereattained atp H 7.0 without further pH control. The results show that the yeast performance was influeced by the pH, an im portant bioengineering prameter in this fermentation process.     

Effect of Initial Cell Concentration on Ethanol Production by Flocculent Saccharomyces cerevisiae with Xylose-Fermenting Ability

Different initial cell concentrations of a recombinant flocculent Saccharomyces cerevisiae MA-R4 were evaluated for their effects on xylose fermentation and glucose–xylose cofermentation. A high initial cell concentration greatly increased both the substrate utilization and ethanol production rates. During xylose fermentation, the highest rates of xylose consumption (2.58 g/L h) and ethanol production (0.83 g/L h) were obtained at an initial cell concentration of 13.1 g/L. During cofermentation, the highest rates of glucose consumption (14.4 g/L h), xylose consumption (2.79 g/L h), and ethanol production (6.68 g/L h) were obtained at an initial cell concentration of 12.7 g/L. However, a high initial cell density had no positive effect on the maximum ethanol concentration and ethanol yield mainly due to the increased amount of by-products including xylitol. The ethanol yield remained almost constant (0.34 g/g) throughout xylose fermentation (initial cell concentration range, 1.81–13.1 g/L), while it was slightly lower at high initial cell concentrations (9.87 and 12.7 g/L) during cofermentation. The determination of the appropriate initial cell concentration is necessary for the improvement of substrate utilization and ethanol yield.     

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.     

High-yield fermentation of pentoses into lactic acid

Lactobacillus species capable of fermenting glucose are generally incapable of utilizing xylose for growth or fermentation. In this study, a novel aspect of a well-known Lactobacillus strain, L. casei subsp. rhamnous (ATCC 10863), was uncovered: it can ferment xylose as efficiently as glucose. This strain is a registered organism, extremely stable on long-term operation. Fermentation by this strain is characterized by an initial lag phase lasting 24–72 h before xylose consumption takes place. The yield (grams/gram) of lactic acid from xylose is in excess of 80% with initial volumetric productivity of 0.38 g/(L-h). Acetic acid is the primary byproduct formed at the level of about 10% of the lactic acid. In addition to xylose, it can ferment all other minor sugars in hemicellulose except arabinose. Subjected to mixed sugar fermentation, this strain consumes glucose first, then mannose, followed by almost simultaneous utilization of xylose and galactose. It shows high tolerance for lactic acid as well as extraneous toxins. It can ferment the mixed sugars present in acid-treated hydrolysate of softwood, giving yields similar to that of pure sugar but at a slower rate. Author to whom all correspondence and reprint requests should be addressed.     

The combined effects of acetic acid, formic acid, and hydroquinone on Debaryomyces hansenii physiology

The combined effects of inhibitors present in lignocellulosic hydrolysates was studied using a multivariate statistical approach. Acetic acid (0–6 g/L), formic acid (0–4.6 g/L) and hydroquinone (0–3 g/L) were tested as model inhibitors in synthetic media containing a mixture of glucose, xylose, and arabinose simulating concentrated hemicellulosic hydrolysates. Inhibitors were consumed sequentially (acetic acid, formic acid, and hydroquinone), alongside to the monosaccharides (glucose, xylose, and arabinose). Xylitol was always the main metabolic product. Additionally, glycerol, ethanol, and arabitol were also obtained. The inhibitory action of acetic acid on growth, on glucose consumption and on all product formation rates was found to be significant (p≤0.05), as well as formic acid inhibition on xylose consumption and biomass production. Hydroquinone negatively affected biomass productivity and yield, but it significantly increased xylose consumption and xylitol productivity. Hydroquinone interactions, either with acetic or formic acid or with both, are also statistically signficant. Hydroquinone seems to partially lessen the acetic acid and amplify formic acid effects. The results clearly indicate that the interaction effects play an important role on the xylitol bioprocess.     

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.     

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.     

Effects of Oxygen Limitation on Xylose Fermentation, Intracellular Metabolites, and Key Enzymes of Neurospora crassa AS3.1602

The effects of oxygen limitation on xylose fermentation of Neurospora crassa AS3.1602 were studied using batch cultures. The maximum yield of ethanol was 0.34 g/g at oxygen transfer rate (OTR) of 8.4 mmol/L·h. The maximum yield of xylitol was 0.33 g/g at OTR of 5.1 mmol/L·h. Oxygen limitation greatly affected mycelia growth and xylitol and ethanol productions. The specific growth rate (μ) decreased 82% from 0.045 to 0.008 h⁻¹ when OTR changed from 12.6 to 8.4 mmol/L·h. Intracellular metabolites of the pentose phosphate pathway, glycolysis, and tricarboxylic acid cycle were determined at various OTRs. Concentrations of most intracellular metabolites decreased with the increase in oxygen limitation. Intracellular enzyme activities of xylose reductase, xylitol dehydrogenase, and xylulokinase, the first three enzymes in xylose metabolic pathway, decreased with the increase in oxygen limitation, resulting in the decreased xylose uptake rate. Under all tested conditions, transaldolase and transketolase activities always maintained at low levels, indicating a great control on xylose metabolism. The enzyme of glucose-6-phosphate dehydrogenase played a major role in NADPH regeneration, and its activity decreased remarkably with the increase in oxygen limitation.     

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.     

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).     

Yields from glucose, xylose, and paper sludge hydrolysate during hydrogen production by the extreme thermophile Caldicellulosiruptor saccharolyticus

This study addressed the utilization of an industrial waste stream, paper sludge, as a renewable cheap feedstock for the fermentative production of hydrogen by the extreme thermophile Caldicellulosiruptor saccharolyticus. Hydrogen, acetate, and lactate were produced in medium in which paper sludge hydrolysate was added as the sole carbon and energy source and in control medium with the same concentration of analytical grade glucose and xylose. The hydrogen yield was dependent on lactate formation and varied between 50 and 94% of the theoretical maximum. The carbon balance in the medium with glucose and xylose was virtually 100%. The carbon balance was not complete in the paper sludge medium because the measurement of biomass was impaired owing to interfering components in the paper sludge hydrolysate. Nevertheless, >85% of the carbon could be accounted for in the products acetate and lactate. The maximal volumetric hydrogen production rate was 5 to 6 mmol/(L·h), which was lower than the production rate in media with glucose, xylose, or a combination of these sugars (9–11 mmol/[L·h]). The reduced hydrogen production rate suggests the presence of inhibiting components in paper sludge hydrolysate.     

Biotechnological Production of Xylitol: Enhancement of Monosaccharide Production by Post-Hydrolysis of Dilute Acid Sugarcane Hydrolysate

Dilute-acid hydrolysis pretreatment of sugarcane bagasse resulted in release of 48% (18.4 g/L) of the xylan in the hemicellulose fraction into the hydrolysate as monomeric xylose. In order to enhance the recuperation of this monomer, a post-hydrolysis stage consisted of thermal treatment was carried out. This treatment resulted in an increase in xylose release of 62% (23.5 g/L) of the hemicellulose fraction. Original and post-hydrolysates were concentrated to the same levels of monomeric xylose in the fermentor feed. During the fermentation process, cellular growth was observed to be higher in the post-hydrolysate (3.5 g/L, Y _x/s?=?0.075 g cells/g xylose) than in the original hydrolysate (2.9 g/L, Y _x/s?=?0.068 g cells/g xylose). The post-treated hydrolysate required less concentration of sugars resulting in a lower concentration of fermentation inhibitors, which were formed primarily in the dilute acid hydrolysis step. Post-hydrolysis step led to a high xylose–xylitol conversion efficiency of 76% (0.7 g xylitol/g xylose) and volumetric productivity of 0.68 g xylitol/L h when compared to 71% (0.65 g xylitol/g xylose and productivity of 0.61 g xylitol/L h) for the original hemicellulosic hydrolysate.     

Effect of starting xylose concentration on the microaerobic metabolism of Debaryomyces hansenii: the use of carbon material balances

Xylitol production by Debaryomyces hansenii NRRL Y-7426 was performed on synthetic medium varying the initial xylose concentration between 50 and 300 g/L. The experimental results of these tests were used to investigate the effect of substrate level on xylose consumption by this yeast. Satisfactory values of product yield on substrate (0.74–0.83 g/g) as well as volumetric productivity (0.481–0.694 g/L·h) were obtained over a wide range of xylose levels (90–200 g/L), while a worsening of kinetic parameters took place at higher concentration, likely due to a substrate inhibition phenomenon. The metabolic behavior of D. hansenii was studied, under these conditions, through a carbon material balance to estimate the fractions of xylose consumed by the cell for different activities (xylitol production, biomass growth, and respiration) during the lag, exponential, and stationary phases.     

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.     

Optimization of Brewery's spent grain dilute-acid hydrolysis for the production of pentose-rich culture media

Dilute-acid hydrolysis of brewery's spent grain to obtain a pentose-rich fermentable hydrolysate was investigated. The influence of operational conditions on polysaccharide hydrolysis was assessed by the combined severity parameter (CS) in the range of 1.39–3.06. When the CS increased, the pentose sugars concentration increased to a maximum at a CS of 1.94, whereas the maximum glucose concentration was obtained for a CS of 2.65. The concentrations of furfural, hydroxymethylfurfural (HMF), as well as formic and levulinic acids and total phenolic compounds increased with severity. Optimum hydrolysis conditions were found at a CS of 1.94 with >95% of feedstock pentose sugars recovered in the monomeric form, together with a low content of furfural, HMF, acetic and formic acids, and total phenolic compounds. This hydrolysate containing glucose, xylose, and arabinose (ratio 10∶67∶32) was further supplemented with inorganic salts and vitamins and readily fermented by the yeast Debaryomyces hansenii CCMI 941 without any previous detoxification stage. The yeast was able to consume all sugars furfural, HMF, and acetic acid with high biomass yield, 0.68C-mol/C-mol, and productivity, 0.92 g/(L·h). Detoxification with activated charcoal resulted in a similar biomass yield and a slight increase in the volumetric productivity (11%).     

Production of Astaxanthin from Cellulosic Biomass Sugars by Mutants of the Yeast <Emphasis Type="Italic">Phaffia rhodozyma</Emphasis>

Astaxanthin is a potential high-value coproduct in an ethanol biorefinery. Three mutant strains of the astaxanthin-producing yeast Phaffia rhodozyma, which were derived from the parent strain ATCC 24202 (UCD 67-210) and designated JTM166, JTM185, and SSM19, were tested for their capability of utilizing the major sugars that can be generated from cellulosic biomass, including glucose, xylose, and arabinose, for astaxanthin production. While all three strains were capable of metabolizing these sugars, individually and in mixtures, JTM185 demonstrated the greatest sugar utilization and astaxanthin production. Astaxanthin yield by this strain (milligrams astaxanthin per gram of sugar consumed) was highest for xylose, followed by arabinose and then glucose. The kinetics of sugar utilization by strain JTM185 was studied in fermenters using mixtures of glucose, xylose, and arabinose at varied concentrations. It was found that glucose was utilized preferentially, followed by xylose, and lastly, arabinose. Astaxanthin yield was significantly affected by sugar concentrations. Highest yields were observed with sugar mixtures containing the highest concentrations of xylose and arabinose. Hydrolysates produced from sugarcane bagasse and barley straw pretreated by the soaking in aqueous ammonia method and hydrolyzed with the commercial cellulase preparation, Accellerase™ 1000, were used for astaxanthin production by the mutant strain JTM185. The organism was capable of metabolizing all of the sugars present in the hydrolysates from both biomass sources and produced similar amounts of astaxanthin from both hydrolysates, although these amounts were lower when compared to yields obtained with reagent grade sugars.     

Cofermentation of glucose,xylose, and arabinose by mixed cultures of two genetically engineeredZymomonas mobilis strains

Cofermentation of xylose and arabinose, in addition to glucose, is critical for complete bioconversion of lignocellulosic biomass, such as agricultural residues and herbaceous energy crops, to ethanol. A factorial design experiment was used to evaluate the cofermentation of glucose, xylose, and arabinose with mixed cultures of two genetically engineeredZymomonas mobilis strains (one ferments xylose and the other arabinose). The pH range studied was 5.0-6.0, and the temperature range was 30-37°C The individual sugar concentrations used were 30 g/L glucose, 30 g/L xylose, and 20 g/L arabinose. The optimal cofermentation conditions obtained by data analysis, using Design Expert software, were pH 5.85 and temperature 31.5°C. The cofermentation process yield at optimal conditions was 72.5% of theoritical maximum. The results showed that neither the arabinose strain nor arabinose affected the performance of the xylose strain; however, both xylose strain and xylose had a significant effect on the performance of the arabinose strain. Although cofermentation of all three sugars is achieved by the mixed cultures, there is a preferential order of sugar utilization. Glucose is used rapidly, then xylose, followed by arabinose.     

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.