Model compound studies

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 in vestigated using experimental design methodology. Oxygen limitation and initial xylose concentration had strong influences on xylitol production by Candida tropicalis ATCC 96745. Under semiaerobic conditions, xylitol yield was highest (0.62 g/g), whereas under aerobic conditions volumetric productivity was highest (0.90g/[L·h]). In the presence of glucose, xylose utilization was strongly repressed and sequential sugar utilization was observed. Ethanol produced from the glucose caused a 50% reduction in xylitol yield when the ethanol con centration 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 highest 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.     

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.     

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.     

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.     

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.     

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.     

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.     

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 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).     

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.     

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.     

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.     

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.     

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-controlled Biosurfactant Production in a Bench Scale Bioreactor

Rhamnolipids have been pointed out as promising biosurfactants. The most studied microorganisms for the aerobic production of these molecules are the bacteria of the genus Pseudomonas. The aim of this work was to produce a rhamnolipid-type biosurfactant in a bench-scale bioreactor by one strain of Pseudomonas aeruginosa isolated from oil environments. To study the microorganism growth and production dependency on oxygen, a nondispersive oxygenation device was developed, and a programmable logic controller (PLC) was used to set the dissolved oxygen (DO) concentration. Using the data stored in a computer and the predetermined characteristics of the oxygenation device, it was possible to evaluate the oxygen uptake rate (OUR) and the specific OUR (SOUR) of this microorganism. These rates, obtained for some different DO concentrations, were then compared to the bacterial growth, to the carbon source consumption, and to the rhamnolipid and other virulence factors production. The SOUR presented an initial value of about 60.0 mgO₂/g_DW h. Then, when the exponential growth phase begins, there is a rise in this rate. After that, the SOUR reduces to about 20.0 mgO₂/g_DW h. The carbon source consumption is linear during the whole process.     

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.     

Characterization of a high-productivity recombinant strain of Zymomonas mobilis for ethanol production from glucose/xylose mixtures

The fermentation characteristics of a recombinant strain of Zymomonas mobilis ZM4(pZB5) capable of converting both glucose and xylose to ethanol have been further investigated. Previous studies have shown that the strain ZM4(pZB5) was capable of converting a mixture o 65 g/L of glucose and 65 g/L of xylose to 62 g/L of ethanol in 48 h with an overall yield of 0.46 g/g. Higher sugar concentrations (e.g., 75/75 g/L) resulted in incomplete xylose utilization (80 h). In the present study, further kinetic evaluations at high sugar levels are reported. Acetate inhibition studies and evaluation of temperature and pH effects indicated increased maximum specific uptake rates of glucose and xylose under stressed conditions with increased metabolic uncoupling. A high-productivity system was developed that involved a membrane bioreactor with cell recycling. At sugar concentrations of approx 50/50 g/L of glucose/xylose, an ethanol concentration of 50 g/L, an ethanol productivity of approx 5 g/(L·h), and a yield (Y _p/s) of 0.50 g/g were achieved. Decreases in cell viability were found in this system after attainment of an initial steady state (40–60 h); a slow bleed of concentrated cells may be required to overcome this problem.     

Preliminary kinetic characterization of xylose reductase and xylitol dehydrogenase extracted from Candida guilliermondii FTI 20037 cultivated in sugarcane bagasse hydrolysate for xylitol production

Candida guilliermondii FTI 20037 was cultured in sugarcane bagasse hydrolysate supplemented with 2.0 g/L of (NH₄)₂SO₄, 0.1 g/L of CaCl₂·2H₂O, and 20.0 g/L of rice bran at 35°C; pH 4.0; agitation of 300 rpm; and aeration of 0.4, 0.6, or 0.8 vvm. The high xylitol production (20.0 g/L) and xylose reductase (XR) activity (658.8 U/mg of protein) occurred at an aeration of 0.4 vvm. Under this condition, the xylitol dehydrogenase (XD) activity was low. The apparent K _M for XR and XD against substrates and cofactors were as follows: for XR, 6.4×10⁻² M (xylose) and 9.5×10⁻³ mM (NADPH); for XD, 1.6×10⁻¹ M (xylitol) and 9.9×10⁻² mM (NAD+). Because XR requires about 10-fold less xylose and cofactor than XD for the condition in which the reaction rate is half of the V _max, some interference on the overall xylitol production by the yeast could be expected.     

Production of xylitol byCandida mogii from rice straw hydrolysate

The influence of aeration level, initial pH, initial cell concentration, and fermentation time on the xylitol production from rice straw hemicellulose hydrolysate byCandida mogii was studied. A multifactorial experimental design was adopted to evaluate this influence. A statistical analysis of the results showed that the aeration level and the initial pH had significant effects on yield factor, volumetric productivity, and xylose consumption. For the latter, fermentation time was also a significant variable. Based on the response surface methodology, models for the range investigated were proposed. The maximum values for the yield factor (Y_p/s) and volumetric productivity (Q_p) were, respectively, 0.71 g/g and 0.46 g(Lh).