Biotechnological production of xylitol from agroindustrial residues

Batch, fed-batch, and semicontinuous fermentation processes were used for the production of xylitol from sugarcane bagasse hemicellulosic hydrolysate. The best results were achieved by the semicontinuous fermentation process: a xylitol yield of 0.79 g/g with an efficiency of 86% and a volumetric productivity of 0.66 g/L/h.     

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).     

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 aeration rate on production of xylitol from corncob hemicellulose hydrolysate

The effects of different aeration conditions on xylitol production from corncob hemicellulose hydrolysate by Candida sp. ZU04 were investigated. Batch fermentations were carried out in a 3.7-L fermentor at 30°C, pH5.5, and agitation of 300 rpm. It was found that the two-phase aeration process was more effective than the one-phase aeration process in xylitol production. In the first 24h of the aerobic phase, a high aeration rate was applied, glucose was soon consumed, and biomass increased quickly. In the second fermentation phase, aeration rate was reduced and an improved xylitol yield was obtained. The maximum xylitol yield (0.76 g/g) was obtained with an aeration rate of 1.5 vvm (KLa of 37 h⁻¹) for the first 24 h and 0.3 vvm (KLa of 6 h⁻¹) from 24 to 96 h.     

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.     

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.     

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.     

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.     

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.     

The effect of cell density on the production of xylitol fromd-xylose by yeast

The rate of xylitol production from D-xylose increased with increasing yeast cell density. The optimal temperature for xylitol production is 36‡ C, and the optimal pH range is from 4.0 to 6.0. At high initial yeast cell concentration of 26 mg/mL, 210 g/L of xylitol was produced from 260 g/L of D-xylose after 96 h of incubation with an indicated yield of 81% of the theoretical value.     

Factors that affect the biosynthesis of xylitol by xylose-fermenting yeasts

Xylitol is a sweetener with important technological properties like anticariogenicity, low caloric value, and negative dissolution heat. Because it can be used successfully in food formulations and pharmaceutical industries, its production is in great demand. Xylitol can be obtained by microbiological process, since many yeasts and filamentous fungi synthesize the xylose reductase enzyme, which catalyses the xylose reduction into xylitol as the first step in the xylose metabolism. The xylitol production by biotechnological means has several economic advantages in comparison with the conventional process based on the chemical reduction of xylose. The efficiency and the productivity of this fermentation chiefly depends upon the microorganism and the process conditions employed. In this mini-review, the most significant upstream parameters on xylitol production by biotechnological process are described.     

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.     

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.     

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.     

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.     

Cellulose production based on hemicellulose hydrolysate from steam-pretreated willow

The production cost of cellulolytic enzymes is a major contributor to the high cost of ethanol production from lignocellulosics using enzymatic hydrolysis. The aim of the present study was to investigate the cellulolytic enzyme production ofTrichoderma reesei Rut C 30, which is known as a good cellulase secreting micro-organism, using willow as the carbon source. The willow, which is a fast-growing energy crop in Sweden, was impregnated with 1–4% SO₂ and steam-pretreated for 5 min at 206°C. The pretreated willow was washed and the wash water, which contains several soluble sugars from the hemicellulose, was supplemented with fibrous pretreated willow and used for enzyme production. In addition to sugars, the liquid contains degradation products such as acetic acid, furfural, and 5-hydroxy-methylfurfural, which are inhibitory for microorganisms. The results showed that 50% of the cellulose can be replaced with sugars from the wash water. The highest enzyme activity, 1.79 FPU/mL and yield, 133 FPU/g carbohydrate, was obtained at pH 6.0 using 20 g/L carbon source concentration. At lower pHs, a total lack of growth and enzyme production was observed, which probably could be explained by furfural inhibition.     

The production and properties of a new xylose reductase from fungus

Neurospora crassa XI was found to ferment xylose and glucose simultaneously. Xylose was the appropriate inducer for the production of xylose reductase that had two isoenzymes designated as EI and EII. Both EI and EII, which were purified by affinity chromatography, had NADPH-dependent xylose reductase activities. EII also had NADH-dependent activity, and EI is the only xylose reductase found so far without any NADH-dependent activity. EI and EII had MWs of 30 kDa and 27 kDa, and pIs of 5.6 and 5.2, respectively. The specifities of EI and EII against triose, pentoses, and hexoses were studied. The K_ms against xylose for EI and EII were 2.3 mM and 1.1 mM respectively, which were much lower than those of the xylose reductase from yeast.     

The effect of lactic acid on fuel ethanol production byZymomonas

Efficient utilization of the pentosan fraction of hemicellulose from lignocellulosic feedstocks offers an opportunity to increase the yield and to reduce the cost of producing fuel ethanol. During prehydrolysis (acid hydrolysis or autohydrolysis of hemicellulose), acetic acid is formed as a consequence of the deacetylation of the acetylated moiety of hemicellulose. Recombinant Escherichia coli B (ATCC 11303), carrying the plasmid pLO1297 with pyruvate decarboxylase and alcohol dehydrogenase II genes from Zymomonas mobilis (CP4), converts xylose to ethanol with a product yield that approaches theoretical maximum. Although other pentose-utilizing microorganisms are inhibited by acetic acid, the recombinant E. coli displays a high tolerance for acetic acid. In xylose fermentations with a synthetic medium (Luria broth), where the pH was controlled at 7, neither yield nor productivity was affected by the addition of 10.7 g/L acetic acid. Nutrient-supplemented, hardwood (aspen) hemicellulose hydrolysate (40.7 g/L xylose) was completely fermented to ethanol (16.3 g/L) in 98 h. When the acetic acid concentration was reduced from 5.6 to 0.8 g/L, the fermentation time decreased to 58 h. Overliming, with Ca(OH)₂ to pH 10, followed by neutralization to pH 7 with sulfuric acid and removal of insolubles, resulted in a twofold increase in volumetric productivity. The maximum productivity was 0.93 g/L/h. The xylose-to-ethanol conversion efficiency and productivity in Ca(OH)₂-treated hardwood prehydrolysate, fortified with only mineral salts, were 94% and 0.26 g/L/h, respectively. The recombinant E. coli exhibits a xylose-to-ethanol conversion efficiency that is superior to that of other pentose-utilizing yeasts currently being investigated for the production of fuel ethanol from lignocellulosic materials.     

Pretreatment of bagasse by UCT-Solvent for the enzymatic hydrolysis

A thermochemical pretreatment of bagasse for the enzymatic hydrolysis has been carried out, in which pretreatment bagasse was autoclaved with binary solvent, composed of Water and organic solvent having upper critical temperature (UCT) on the mutual solubility curve. The pretreatment was named “UCT-solvent pretreatment.” The hydrophobic decomposition products from lignin and hemicellulose, that dissolved in organic phase at room temperature, could be easily separated from the solid and sugars in the aqueous phase. By using UCT-solvent instead of only water, the sugar recoveries from bagasse through the pretreatment and the enzymatic hydrolysis were much improved. There exists an optimal mixing ratio between organic solvent and water to maximize the effect of the pretreatment for enzymatic hydrolysis. The optimal ratio can be explained by the competitive effect between the ability of water as a reagent for the hydrolysis and the ability of solvent for the extraction of the decomposition product, and furthermore by the competitive effect between affinities of the solvent to hydrophilic hemicellulose and hydrophobic lignin. Decomposition of hemicellulose at lower temperature than 190°C was decreased, and hence the degradation of xylose during the pretreatment decreased. These favorable effects of UCT-solvent pretreatment are significantly attributed to the formation of the homogeneous single phase of organic solvent and water at high temperature and the phase separation at room temperature.     

Biosolubilization and liquid fuel production from coal

Recently, several microorganisms have been shown to be capable of directly solubilizing low-rank coals. This bioextract has a high molecular weight and is water soluble, but is not useful as a liquid fuel. This paper presents the results of studies to biologically solubilize coal and convert the solubilized coal into more useful compounds. Preliminary experiments have been conducted to isolate cultures for the serial biological conversion of coal into liquid fuels. Coal particles have been solubilized employing an isolate from the surface of Arkansas lignite. Natural inocula, such as sheep rumen and sewage sludge, are then employed in developing cultures for converting the bioextract into fuels. This paper presents preliminary results of experiments in coal solubilization and bioextract conversion.