Influence of inhibitory compounds and minor sugars on xylitol production by <Emphasis Type="Italic">Debaryomyces hansenii</Emphasis>

To obtain in-depth information on the overall metabolic behavior of the new good xylitol producer Debaryomyces hansenii UFV-170, batch bioconversions were carried out using semisynthetic media with compositions simulating those of typical acidic hemicellulose hydrolysates of sugarcane bagasse. For this purpose, we used media containing glucose (4.3–6.5 g/L), xylose (60.1–92.1 g/L), or arabinose (5.9–9.2 g/L), or binary or ternary mixtures of them in either the presence or absence of typical inhibitors of acidic hydrolysates, such as furfural (1.0–5.0 g/L), hydroxymethylfurfural (0.01–0.30 g/L), acetic acid (0.5–3.0 g/L), and vanillin (0.5–3.0 g/L). D. hansenii exhibited a good tolerance to high sugar concentrations as well as to the presence of inhibiting compounds in the fermentation media. It was able to produce xylitol only from xylose, arabitol from arabinose, and no glucitol from glucose. Arabinose metabolization was incomplete, while ethanol was mainly produced from glucose and, to a lesser less extent, from xylose and arabinose. The results suggest potential application of this strain in xyloseto-xylitol bioconversion from complex xylose media from lignocellulosic materials.     

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.     

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.     

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.     

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.     

Complete bioconversion of hemicellulosic sugars from agricultural residues into lactic acid by Lactobacillus pentosus

On the basis of previous knowledge, different agroindustrial wastes were submitted to dilute-acid hydrolysis with H2SO4 to obtain hemicellulosic sugars and then employed for lactic acid production by Lactobacillus pentosus. Toxic compounds released from lignin did not affect lactic acid fermentation when hydrolysates from trimming vine shoots, barley bran husks, or corncobs were employed as carbon source, and complete bioconversion of hemicellulosic sugars was achieved. Nevertheless, Eucalyptus globulus hydrolysates had to be submitted to a detoxification process with activated charcoal. Maximum lactic acid concentration (33 g/L) was reached employing barley bran hydrolysates, whereas corncobs, trimming vine shoots, and detoxified E. globulus hydrolysates yielded 26, 24, and 14.5 g/L of lactic acid, respectively. The maximum product yield from pentoses (0.76 g/g) was achieved using hydrolysates from trimming vine shoots, followed by hydrolysates from detoxified E. globulus (0.70 g/g), barley bran (0.57 g/g), and corncob (0.53 g/g). These results confirm that L. pentosus can be employed to ferment hemicellulosic sugars (mainly xylose, glucose, and arabinose) from acid hydrolysates of most agricultural residues without appreciable substrate inhibition.     

Modeling of the hydrolysis of sugar cane bagasse with hydrochloric acid

Sugar cane bagasse was hydrolyzed under different concentrations of hydrochloric acid (2–6%), reaction times (0–300 min), and temperatures (100–128°C). Sugars obtained (xylose, glucose, arabinose, and glucose) and deg-radation products (furfural and acetic acid) were determined. Based on the Saeman model and the two-fraction model, kinetic parameters for predicting these compounds in the hydrolysates were developed. The influence of temperature was studied using the Arrhenius equation. The optimal conditions selected were 128°C, 2% HCl, and 51.1 min. Using these conditions, 22.6g xylose/L, 3.31 garabinose/L, 3.77 g glucose/L, 3.59 g acetic acid/L, and 1.54 g furfural/L were obtained.     

Production of Xylitol from D-Xylose byDebaryomyces hansenii

Xylitol, a naturally occurring five-carbon sugar alcohol, can be produced from D-xylose through microbial hydrogenation. Xylitol has found increasing use in the food industries, especially in confectionary. It is the only so-called “second-generation polyol sweeteners” that is allowed to have the specific health claims in some world markets. In this study, the effect of cell density on the xylitol production by the yeastDebaryomyces hansenii NRRL Υ-7426 from D-xylose under microaerobic conditions was examined. The rate of xylitol production increased with increasing yeast cell density to 3 g/L. Beyond this amount there was no increase in the xylitol production with increasing cell density. The optimal pH range for xylitol production was between 4.5 and 5.5. The optimal temperature was between 28 and 37°C, and the optimal shaking speed was 300 rpm. The rate of xylitol production increased linearly with increasing initial xylose concentration. A high concentration of xylose (279 g/L) was converted rapidly and efficiently to produce xylitol with a product concentration of 221 g/L was reached after 48 h of incubation under optimum conditions.

     

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.     

Characterization of a Recombinant Flocculent Saccharomyces cerevisiae Strain that Co-ferments Glucose and Xylose: I. Influence of the Ratio of Glucose/Xylose on Ethanol Production

Glucose/xylose mixtures (90 g/L total sugar) were evaluated for their effect on ethanol fermentation by a recombinant flocculent Saccharomyces cerevisiae, MA-R4. Glucose was utilized faster than xylose at any ratio of glucose/xylose, although MA-R4 can simultaneously co-ferment both sugars. A high percentage of glucose can increase cell biomass production and therefore increase the rate of glucose utilization (1.224 g glucose/g biomass/h maximum) and ethanol formation (0.493 g ethanol/g biomass/h maximum). However, the best ratio of glucose/xylose for the highest xylose consumption rate (0.209 g xylose/g biomass/h) was 2:3. Ethanol concentration and yield increased and by-product (xylitol, glycerol, and acetic acid) concentration decreased as the proportion of glucose increased. The maximum ethanol concentration was 41.6 and 21.9 g/L after 72 h of fermentation with 90 g/L glucose and 90 g/L xylose, respectively, while the ethanol yield was 0.454 and 0.335 g/g in 90 g/L glucose and 90 g/L xylose media, respectively. High ethanol yield when a high percentage of glucose is available is likely due to decreased production of by-products, such as glycerol and acetic acid. These results suggest that ethanol selectivity is increased when a higher proportion of glucose is available and reduced when a higher proportion of xylose is available.     

Effect of detoxification of dilute-acid corn fiber hydrolysate on xylitol production

Four different detoxification methods were evaluated for the production of xylitol from corn fiber dilute-acid hydrolysate using Candida tropicalis. Although C. tropicalis could ferment the dilute partially neutralized hydrolysate to xylitol in low yields (0.1 g/g), it could not ferment the concentrated hydrolysate. Overliming, calcium hydroxide neutralization, neutralization combined with activated charcoal, and overliming combined with activated charcoal methods were used to improve the fermentation of the concentrated hydrolysates. The partial neutralization combined with activated charcoal treatment was the most effective method with respect to xylitol yield and productivity. The highest xylitol yield (0.4 g of xylitol/g of xylose) was obtained for the highest concentration of hydrolysate (three times the original) that had been treated with calcium hydroxide and activated charcoal. The corresponding productivity was 0.23 g/(L x h). Overliming caused reduction in xylitol yield.     

Comparative ethanol productivities of different Zymomonas recombinants fermenting oat hull hydrolysate

Iogen Corporation of Ottawa, Canada, has recently built a 50 t/d biomass-to-ethanol demonstration plant adjacent to its enzyme production facility. Iogen has partnered with the University of Toronto to test the C6/C5 cofermentation performance characteristics of National Renewable Energy Laboratory's metabolically engineered Zymomonas mobilis using its biomass hydrolysates. In this study, the biomass feedstock was an agricultural waste, namely oat hulls, which was hydrolyzed in a proprietary two-stage process involving pretreatment with dilute sulfuric acid at 200–250°C, followed by cellulase hydrolysis. The oat hull hydrolysate (OHH) contained glucose, xylose, and arabinose in a mass ratio of about 8:3:0.5. Fermentation media, prepared from diluted hydrolysate, were nutritionally amended with 2.5 mL/L of corn steep liquor (50% solids) and 1.2 g/L of diammonium phosphate. The estimated cost for large-scale ethanol production using this minimal level of nutrient supplementation was 4.4c/gal of ethanol. This work examined the growth and fermentation performance of xyloseutilizing, tetracycline-resistant, plasmid-bearing, patented, recombinant Z. mobilis cultures: CP4:pZB5, ZM4:pZB5, 39676:pZB4L, and a hardwood prehydrolysate-adapted variant of 39676:pZB4L (designated asthe “adapted” strain). In pH-stat batch fermentations with unconditioned OHH containing 6% (w/v) glucose, 3% xylose, and 0.75% acetic acid, rec Zm ZM4:pZB5 gave the best performance with a fermentation time of 30h, followed by CP4:pZB5 at 48h, with corresponding volumetric productivities of 1.4 and 0.89 g/(L·h), respectively. Based on the available glucose and xylose, the process ethanol yield for both strains was 0.47 g/g (92% conversion efficiency). At 48 h, the process yield for rec Zm 39676:pZB4L and the adapted strain was 0.32 and 0.34 g/g, respectively. None of the test strains was able to fermentarabinose. Acetic acid tolerance appeared to be a major determining factor in cofermentation performance.     

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.     

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.     

Regulation of Xylanase Biosynthesis in Bacillus cereus BSA1

Microbial xylanases have a promising biotechnological potential to be used in industries. In this study, regulation of xylanase production was examined in Bacillus cereus BSA1. Xylanase production was induced by xylan. The enzyme production further increased in the presence of xylose and arabinose in very low concentration with addition of xylan (0.5% up to 6.02 U/ml). Addition of glucose (about 0.1%) to the media supplemented with xylan repressed xylanase production. Even higher concentration (>0.1%) of xylose and arabinose repressed xylanase biosynthesis. Glucose-mediated repression was partially relived by addition of cyclic adenosine monophosphate. Chemical like 2-4-dinitrophenol, which can inhibit adenosine triphosphate synthesis in cell, repressed xylanase synthesis and it suggested xylanase synthesis to be an energy dependent process.     

Determination of the compositions of polysaccharides from Chinese herbs by capillary zone electrophoresis with amperometric detection

In this paper, capillary zone electrophoresis with amperometric detection (CZE-AD) was applied to determine the compositions of hetero-polysaccharides from Chinese herbs, Angelica sinensis and flax by analyzing their hydrolyzed monosaccharides: fucose, galactose, glucose, arabinose, rhamnose and xylose. Under the selected optimum conditions, the six monosaccharides could be perfectly separated within 25 min and showed significant current responses at copper electrodes. The linear ranges of the six monosaccharides were all from 5.0 x 10(-6) to 2.0 x 10(-4) mol L(-1) and their detection limits were lower or near 1.0 x 10(-6) mol L(-1) (S/N = 3). Experiments showed that the Angelica sinensis polysaccharide was composed of fucose, galactose, glucose, arabinose, rhamnose and xylose (mole ratio 1.0:13.6:15.0:8.7:21.3:3.7), and the flax polysaccharide was composed of galactose, glucose and arabinose (mole ratio 1.0:4.98:1.1). The purity of these polysaccharides leached by the introduced leaching method was 98.3 and 97.6%, respectively. Analyzing polysaccharides by this method has some merits of speed, simple instrumentation and operation, high sensitivity and high reproducibility.     

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 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.