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.     

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.     

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.     

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.     

PVA-Hydrogel Entrapped <Emphasis Type="Italic">Candida Guilliermondii</Emphasis> for Xylitol Production from Sugarcane Hemicellulose Hydrolysate

Viable cells of Candida guilliermondii were immobilized by inclusion into polyvinyl alcohol (PVA) hydrogel using the freezing–thawing method. Entrapment experiments were planned according to a 2³ full factorial design, using the PVA concentration (80, 100, and 120 g L⁻¹), the freezing temperature (−10, −15, and −20 °C), and the number of freezing-thawing cycles (one, three, and five) as the independent variables, integrated with three additional tests to estimate the errors. The effectiveness of the immobilization procedure was checked in Erlenmeyer flasks as the pellet capability to catalyze the xylose-to-xylitol bioconversion of a medium based on sugarcane bagasse hemicellulosic hydrolysate. To this purpose, the yield of xylitol on consumed xylose, xylitol volumetric productivity, and cell retention yield were selected as the response variables. Cell pellets were then used to perform the same bioconversion in a stirred tank reactor operated at 400 rpm, 30 °C, and 1.04 vvm air flowrate. At the end of fermentation, a maximum xylitol concentration of 28.7 g L⁻¹, a xylitol yield on consumed xylose of 0.49 g g⁻¹ and a xylitol volumetric productivity of 0.24 g L⁻¹ h⁻¹ were obtained.     

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.     

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.     

Influence of carbon and nitrogen sources and temperature on hyperproduction of a thermotolerant β-glucosidase from synthetic medium by Kluyveromyces marxianus

The effect of carbon source and its concentration, inoculum size, yeast extract concentration, nitrogen source, pH of the fermentation medium, and fermentation temperature on β-glucosidase production by Kluyveromyces marxianus in shake-flask culture was investigated. These were the independent variables that directly regulated the specific growth and β-glucosidase production rate. The highest product yield, specific product yield, and productivity of β-glucosidase occurred in the medium (pH 5.5) inoculated with 10% (v/v) inoculum of the culture. Cellobiose (20 g/L) significantly improved β-glucosidase production measured as product yield (Y _P/S ) and volumetric productivity (Q _P ) followed by sucrose, lactose, and xylose. The highest levels of productivity (144 IU/[L·h]) of β-glucosidase occurred on cellobiose in the presence of CSL at 35°C and are significantly higher than the values reported by other researchers on almost all other organisms. The thermodynamics and kinetics of β-glucosidase production and its deactivation are also reported. The enzyme was substantially stable at 60°C and may find application in some industrial processes.     

Novel Isolates for Biological Detoxification of Lignocellulosic Hydrolysate

In this paper, two new strians, Issatchenkia occidentalis (Lj-3, CCTCC M 2006097) and Issatchenkia orienalis (S-7, CCTCC M 2006098), isolated from different environments on solid media, were used in the detoxification process of the hemicellulosic hydrolysate of sugarcane bagasse. High-pressure liquid chromatography elution curve of UV-absorption compounds represented by acetic acid, furfural, and guaiacol (toxic compounds found in the hemicellulosic hydrolysate) showed that several chromatographic peaks were evidently diminished for the case of detoxified hydrolysate with isolate strains compared to the high peaks resulted for no detoxified hydrolysate. It was clear that these inhibitors were degraded by the two new isolates during their cultivation process. Fermentation results for the biodetoxified hydrolysate showed an increase in xylitol productivity (Q _p) by 1.97 and 1.95 times (2.03 and 2.01 g l⁻¹ h⁻¹) and in xylitol yield (Y _p) by 1.72 and 1.65 times (0.93 and 0.89 g xylitol per gram xylose) for hydrolysate treated with S-7 and Lj-3, respectively, in comparison with no detoxified hydrolysate (1.03 g l⁻¹ h⁻¹ and 0.54 g xylitol per gram xylose). This present work demonstrated the importance of Issatchenkia yeast in providing an effective biological detoxification approach to remove inhibitors and improve hydrolysate fermentability, leading to a high xylitol productivity and yield.     

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.     

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.     

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 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).     

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.     

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.     

The Fed-Batch Production of a Thermophilic 2-Deoxyribose-5-Phosphate Aldolase (DERA) in <Emphasis Type="Italic">Escherichia coli</Emphasis> by Exponential Feeding Strategy Control

2-Deoxyribose-5-phosphate aldolase (DERA) catalyzes a sequential aldol reaction useful in synthetic chemistry. In this work, the effect of a feeding strategy on the production of a thermophilic DERA was investigated in fed-batch cultures of recombinant Escherichia coli BL21 (pET303-DERA008). The predetermined specific growth rate (μ _set) was evaluated at 0.20, 0.15, and 0.10 h⁻¹, respectively. The DERA concentration and volumetric productivity were associated with μ _set. The cells synthesized the enzyme most efficiently at μ _set = 0.15 h⁻¹. The maximum enzyme concentration (5.12 g/L) and total volumetric productivity (0.256 g L⁻¹ h⁻¹) obtained were over 10 and five times higher than that from traditional batch cultures. Furthermore, the acetate concentration remained at a relatively low level, less than 0.4 g/L, under this condition which would not inhibit cell growth and target protein expression. Thus, a specific growth rate control strategy has been successfully applied to induce fed-batch cultures for the maximal production of the thermophilic 2-deoxyribose-5-phosphate aldolase.     

High-Cell-Density cultivation of pseudomonas putida IPT 046 and medium-chain-length polyhydroxyalkanoate production from sugarcane carbohydrates

We studied high-density cultures of Pseudomonas putida IPT 046 for the production of medium-chain-length polyhydroxyalkanoates (PHA_MCL) using an equimolar mixture of glucose and fructose as carbon sources. Kinetics studies of P. putida growth resulted in a maximum specific growth rate of 0.65h⁻¹. Limitation and inhibition owing to NH₄ ⁺ ions were observed, respectively, at 400 and 3500 mg of NH₄ ⁺/L. The minimum concentration of dissolved oxygen in the broth must be 15% of saturation. Fed-batch strategies for high-cell-density cultivation were proposed. Pulse feed followed by constant feed produced a cell concentration of 32 g/L in 18 h of fermentation and low PHA_MCL content. Constant feed produced a cell concentration of 35 g/L, obtained in 27 h of fermentation, with up to 15% PHA_MCL. Exponential feed produced a cell concentration of 30 g/L in 20 h of fermentation and low PHA_MCL content. Using the last strategy, 21% PHA_MCL was produced during a period of 34 h of fed-batch operation, with a final cell concentration of 40 g/L and NH₄ ⁺ limitation. Using phosphate limitation, 50 g/L cell concentration, 63% PHA_MCL and a productivity of 0.8 g/(L·h) were obtained in 42 h of fed-batch operation. The PHA_MCL yield factors from consumed carbohydrate for N-limited and P-limited experiments were, respectively, 0.15 and 0.19 g/g.     

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.