Xylitol Production Research Articles

Immobilization of recombinant Escherichia coli on multi-walled carbon nanotubes for xylitol production

E. coli has been engineered to produce xylitol, but the production faces bottlenecks in terms of production yield and cell viability. In this study, recombinant E. coli (rE. coli) was immobilized on untreated and treated multiwalled carbon nanotubes (MWCNTs) for xylitol production. The immobilized rE. coli on untreated MWCNTs gave the highest xylitol production (5.47 g L−1) and a productivity of 0.22 g L−1 h−1. The doubling time for the immobilized cells increased up to 20.40 h and was higher than that of free cells (3.67 h). Cell lysis of the immobilized cells was reduced by up to 73 %, and plasmid stability improved by up to 17 % compared to those of free cells. Xylitol production using the optimum parameters (pH 7.4, 0.005 mM and 29 °C) achieved a xylitol production and productivity of 6.33 g L−1 and 0.26 g L−1 h−1, respectively. A seven-cycle repeated batch fermentation was carried out for up to 168 h, which showed maximum xylitol production of 7.36 g L−1 during the third cycle. Hence, this new adsorption immobilization system using MWCNTs is an alternative to improve the production of xylitol.

PH-Neutralization, Redox-Balanced Process with Coupled Formate Dehydrogenase and Glucose Dehydrogenase Supports Efficient Xylitol Production in Pure Water.

Enzymatic production of xylitol is a promising alternative to the chemical hydrogenation process. However, it encounters problems that are largely due to protein susceptibility to environmental factors. In this study, to develop a robust, practical enzymatic process for xylitol production, a coupled enzyme system consisting of formate dehydrogenase (FDH), glucose dehydrogenase (GDH), and xylose reductase (XR) was constructed, wherein the alkaline product produced by FDH and the acidic product produced by GDH could neutralize each other during cofactor regeneration. After optimization of conditions, a pH-neutralization, redox-balanced process was developed that could be carried out in pure water requiring no pH regulation. As a result, a xylitol production of 273.6 g/L that is much higher than those yet reported was obtained from 2 M xylose in 24 h, with a relatively high productivity of 11.4 g/(L h). The strategy demonstrated here can be adapted for the production of other NADH-consuming products.

Fermentation of hemicellulose liquor from Brewer's spent grain using Scheffersomyces stipitis and Pachysolen tannophilus for production of 2G ethanol and xylitol

AbstractBeer production generates brewer's spent grain as a byproduct. This biomass contains lignin, cellulose, and hemicellulose, and can be used for the production of second‐generation (2G) ethanol and xylitol. For this to be economically viable, fermentable fractions must be used, allowing the conversion of cellulose and hemicellulose to ethanol. This study evaluated the fermentation of hemicellulose liquor from brewer's spent grain (HLBS) in aerobic and oxygen‐ limited conditions using Scheffersomyces stipitis and Pachysolen tannophilus. Results were compared with data obtained in a complex medium (YPX) and a minimal medium (MMX). In aerobic conditions, for S. stipitis in the HLBS medium, μ max = 0.12 h−1, Y ethanol/xylose = 0.14 and selectivity for ethanol over xylitol (S ethanol/xylitol) 3.4 gethanol g−1 xylitol were obtained. Under oxygen‐limited conditions, Y ethanol/xylose = 0.22 and S ethanol/xylitol = 1.17 gethanol g−1 xylitol were obtained. For P. tannophilus, under aerobic conditions, the HLBS medium gave μ max = 0.08 h−1, and Y ethanol/xylose = 0.005, with selectivity for ethanol over xylitol (S ethanol/xylitol) of 0.12 gethanol g−1 xylitol. In oxygen‐limited conditions, Y ethanol/xylose = 0.09 and S ethanol/xylitol = 0.20 gethanol g−1 xylitol were obtained. This is because the metabolism of these aerobic yeasts directs the carbon flow to cell growth, while with oxygen limitation it is directed to metabolite production. The HLBS medium was promising for production in the context of a biorefinery. When comparing the two yeasts, S. stipitis presented better ethanol production and P. tannophilus better xylitol production under the conditions that were studied. © 2019 Society of Chemical Industry and John Wiley & Sons, Ltd

Isolation of xylose-assimilating yeasts and optimization of xylitol production by a new Meyerozyma guilliermondii strain.

Production of xylitol from lignocellulosic biomass is of interest to modern biorefineries, because this biomass should be processed into a spectrum of chemicals (bio-based products) and not only energy. The isolation of new yeast strains capable of efficiently converting xylose into xylitol and withstanding inhibitors released from biomass hydrolysis can contribute to making its production feasible in biorefineries. Forty-three out of 128 yeast strains isolated from the gut of Passalidae beetles were capable of assimilating xylose as the sole carbon source. Meyerozyma guilliermondii UFV-1 was selected due to its ability to grow and ferment D-xylose in a synthetic medium. This yeast assimilated the broad range of sugars present in lignocellulosic biomass hydrolysates, such as xylose, raffinose, cellobiose, rhamnose, arabinose, and glucose. Its optimum growth conditions were pH 8.0 and a temperature of 30 °C. In concentrations of 0.07 mol/Lacetic acid, 0.05 mol/L 5-hydroximethylfurfural, and 0.04 mol/L furfural, M. guilliermondii UFV-1 did not grow. Maximum xylitol production in aerobiosis and hypoxia were 51.88 and 27.73 g/L, respectively. Under aerobic condition, xylose concentration and agitation rate were the factors which were statistically significant, while only the agitation rate was significant in hypoxia. We fitted a response surface (RS) that estimated the best agitation rate (113.33 rpm) and xylose concentration (90 g/L) for maximum xylitol production in aerobiosis. Therefore, M. guilliermondii UFV-1 displays potential for being used for xylitol production in biorefineries.

Evaluation of Simultaneous Saccharification and Fermentation of Oil Palm Empty Fruit Bunches for Xylitol Production

The biological process route of xylitol production from lignocellulosic materials, via enzymatic hydrolysis which is followed by fermentation, offers a more sustainable or greener process than the chemical process route. Both the enzymatic hydrolysis and the fermentation processes are conducted at moderate process condition and thus require less energy and chemicals. However, the process proceeds slower than the chemical one. In order to improve process performance, the enzymatic hydrolysis and the fermentation processes can be integrated as Simultaneous Saccharification and Fermentation (SSF) configuration. This paper discusses the evaluation of SSF configuration on xylitol production from Oil Palm Empty Fruit Bunches (OPEFB). To integrate two processes which have different optimum temperature, the performance of each process at various temperature was first evaluated. Later, SSF was evaluated at various hydrolysis and fermentation time at each optimum temperature. SSF showed better process performance than the separated hydrolysis and fermentation processes. The best result was obtained from configuration with 72 hours of prior hydrolysis followed by simultaneous hydrolysis and fermentation, giving yield of 0.08 g-xylitol/g-OPEFB.

Xylitol Production from Agricultural Wastes by Candida tropicalis

Xylan produced various agricultural residues including wheat (Furat, Abugraib and Abaa), Papyrus and Sunflower stalks in different ways, including the use of diluted acid, dilute base and self-degradation. The results showed that the acidic method in the production of xylan from various agricultural residues compared with other methods was superior, the highest quantity of xylan 187.6 µg.ml-1 was obtained from the agricultural waste of Papyrus, while it was 157.6, 157.6, 161.6 and 161.3 µg.ml-1 of wheat category of furat, wheat Abu Ghraib, wheat Abaa and sunflower stalks respectively, based on the results obtained, the xylan produced by the acidic method of the different agricultural residues was selected to determine the optimal carboon source for production of xylanase using bacteria Bacillus subtilis strain RS1 locally isolated. After the production of xylitol, the descriptive diagnosis was performed using an HPLC device, depending on the time of the 38.4 minute time lapse reaction of the standard Xylitol and compared with the time of the production of Candida tropicalis, the amount of the processed xylitol was 8.3 µg.ml-1, the calculated xylitol was compared standard xylitol

Xylitol Production: Identification and Comparison of New Producing Yeasts.

Xylitol is a sugar alcohol with five carbons that can be used in the pharmaceutical and food industries. It is industrially produced by chemical route; however, a more economical and environmentally friendly production process is of interest. In this context, this study aimed to select wild yeasts able to produce xylitol and compare their performance in sugarcane bagasse hydrolysate. For this, 960 yeast strains, isolated from soil, wood, and insects have been prospected and selected for the ability to grow on defined medium containing xylose as the sole carbon source. A total of 42 yeasts was selected and their profile of sugar consumption and metabolite production were analyzed in microscale fermentation. The six best xylose-consuming strains were molecularly identified as Meyerozyma spp. The fermentative kinetics comparisons on defined medium and on sugarcane bagasse hydrolysate showed physiological differences among these strains. Production yields vary from YP/S = 0.25 g/g to YP/S = 0.34 g/g in defined medium and from YP/S = 0.41 g/g to YP/S = 0.60 g/g in the hydrolysate. Then, the xylitol production performance of the best xylose-consuming strain obtained in the screening, which was named M. guilliermondii B12, was compared with the previously reported xylitol producing yeasts M. guilliermondii A3, Spathaspora sp. JA1, and Wickerhamomyces anomalus 740 in sugarcane bagasse hydrolysate under oxygen-limited conditions. All the yeasts were able to metabolize xylose, but W. anomalus 740 showed the highest xylitol production yield, reaching a maximum of 0.83 g xylitol/g of xylose in hydrolysate. The screening strategy allowed identification of a new M. guilliermondii strain that efficiently grows in xylose even in hydrolysate with a high content of acetic acid (~6 g/L). In addition, this study reports, for the first time, a high-efficient xylitol producing strain of W. anomalus, which achieved, to the best of our knowledge, one of the highest xylitol production yields in hydrolysate reported in the literature.

Xylose-Enriched Ethanol Fermentation Stillage from Sweet Sorghum for Xylitol and Astaxanthin Production

Developing integrated biorefineries requires the generation of high-value co-products produced alongside cellulosic ethanol. Most industrial yeast strains produce ethanol at high titers, but the small profit margins for generating ethanol require that additional high-value chemicals be generated to improve revenue. The aim of this research was to boost xylose utilization and conversion to high-value co-products that can be generated in an integrated biorefinery. Pretreated sweet sorghum bagasse (SSB) was hydrolyzed in sweet sorghum juice (SSJ) followed by ethanol fermentation. Ethanol was removed from the fermentation broth by evaporation to generate a stillage media enriched in xylose. Candida mogii NRRL Y-17032 could easily grow in non-detoxified stillage media, but a high xylitol yield of 0.55 g xylitol/g xylose consumed was achieved after recovered cells were resuspended in synthetic media containing supplemented xylose. Phaffia rhodozyma ATCC 74219 could be cultivated in non-detoxified stillage media, but astaxanthin generation was increased 4-fold (from 17.5 to 71.7 mg/L) in detoxified media. Future processing strategies to boost product output should focus on a two-step process where the stillage media is used as the growth stage, and a synthetic media for the production stage utilizing xylose generated from SSB through selective hemicellulase enzymes.

Acid pretreatment optimization for xylose production from Agave tequilana Weber var. azul, Agave americana var. oaxacensis, Agave karwinskii, and Agave potatorum bagasses using a Box-Behnken design

There is a growing interest in the use of lignocellulosic biomass for biofuel production leading to by-products with high added value such as xylitol, a sweetener obtained from chemical synthesis or fermentative pathways, characterized by high degree of sweetness, recommended for people with diabetes, making it a product of great interest in the industrial market. The objective of this study was to use the bagasse of four agave plants from Mexico: Agave tequilana Weber var. azul (AT), Agave Americana var. oaxacensis (AA), Agave karwinskii (AK), and Agave potatorum (AP) to obtain xylose as an alternative for xylitol or ethanol production. The dilute acid pretreatment was optimized to obtain the maximum concentration of xylose and the minimum concentration of acetic acid, using a Box-Behnken design, where H2SO4 concentration, hydrolysis time, and liquid-to-solid ratio were evaluated. Xylose concentrations of 23.18 g/L (AT), 27.63 g/L (AA), 31.8 g/L (AK), and 24.42 g/L (AP) were achieved. It was also observed that by using more concentrated H2SO4 solutions, higher concentrations of xylose were obtained, but also high concentrations of acetic acid (1.02 g/L in AT, 2.3 g/L in AA, 3.46 g/L in AK, and 2.01 g/L in AP). This study shows that AT, AA, AK, and AP are a promising alternative feedstock for bioethanol or xylitol production using xylose as substrate.

Combined Cell Surface Display of β-d-Glucosidase (BGL), Maltose Transporter (MAL11), and Overexpression of Cytosolic Xylose Reductase (XR) in Saccharomyces cerevisiae Enhance Cellobiose/Xylose Coutilization for Xylitol Bioproduction from Lignocellulosic Biomass.

Xylitol is a highly valuable commodity chemicalused extensively in the food and pharmaceutical industries. The production of xylitol from d-xylose involves a costly and polluting catalytic hydrogenation process. Biotechnological production from lignocellulosic biomass by micro-organisms like yeasts is a promising option. In this study, xylitol is produced from lignocellulosic biomass by a recombinant strain of Saccharomyces cerevisiae (S. cerevisiae) (YPH499-SsXR-AaBGL) expressing cytosolic xylose reductase (Scheffersomyces stipitis xylose reductase [SsXR]), along with a β-d-glucosidase (Aspergillus aculeatus β-glucosidase 1 [AaBGL]) displayed on the cell surface. The simultaneous cofermentation of cellobiose/xylose by this strain leads to an ≈2.5-fold increase in Yxylitol/xylose (=0.54) compared to the use of a glucose/xylose mixture as a substrate. Further improvement in the xylose uptake by the cell is achieved by a broad evaluation of several homologous and heterologous transporters. Homologous maltose transporter (ScMAL11) shows the best performance in xylose transport and is used to generate the strain YPH499-XR-ScMAL11-BGL with a significantly improved xylitol production capacity from cellobiose/xylose coutilization. This report constitutes a promising proof of concept to further scale up the biorefinery industrial production of xylitol from lignocellulose by combining cell surface and metabolic engineering in S. cerevisiae.

Metabolic flux analysis for metabolome data validation of naturally xylose-fermenting yeasts

BackgroundEfficient xylose fermentation still demands knowledge regarding xylose catabolism. In this study, metabolic flux analysis (MFA) and metabolomics were used to improve our understanding of xylose metabolism. Thus, a stoichiometric model was constructed to simulate the intracellular carbon flux and used to validate the metabolome data collected within xylose catabolic pathways of non-Saccharomyces xylose utilizing yeasts.ResultsA metabolic flux model was constructed using xylose fermentation data from yeasts Scheffersomyces stipitis, Spathaspora arborariae, and Spathaspora passalidarum. In total, 39 intracellular metabolic reactions rates were utilized validating the measurements of 11 intracellular metabolites, acquired by mass spectrometry. Among them, 80% of total metabolites were confirmed with a correlation above 90% when compared to the stoichiometric model. Among the intracellular metabolites, fructose-6-phosphate, glucose-6-phosphate, ribulose-5-phosphate, and malate are validated in the three studied yeasts. However, the metabolites phosphoenolpyruvate and pyruvate could not be confirmed in any yeast. Finally, the three yeasts had the metabolic fluxes from xylose to ethanol compared. Xylose catabolism occurs at twice-higher flux rates in S. stipitis than S. passalidarum and S. arborariae. Besides, S. passalidarum present 1.5 times high flux rate in the xylose reductase reaction NADH-dependent than other two yeasts.ConclusionsThis study demonstrated a novel strategy for metabolome data validation and brought insights about naturally xylose-fermenting yeasts. S. stipitis and S. passalidarum showed respectively three and twice higher flux rates of XR with NADH cofactor, reducing the xylitol production when compared to S. arborariae. Besides then, the higher flux rates directed to pentose phosphate pathway (PPP) and glycolysis pathways resulted in better ethanol production in S. stipitis and S. passalidarum when compared to S. arborariae.

Efficient production of xylitol by the integration of multiple copies of xylose reductase gene and the deletion of Embden-Meyerhof-Parnas pathway-associated genes to enhance NADPH regeneration in Escherichia coli.

Cofactor supply is a rate-limiting step in the bioconversion of xylose to xylitol. Strain WZ04 was first constructed by a novel simultaneous deletion-insertion strategy, replacing ptsG, xylAB and ptsF in wild-type Escherichia coli W3110 with three mutated xylose reductase genes (xr) from Neurospora crassa. Then, the pfkA, pfkB, pgi and/or sthA genes were deleted and replaced by xr to investigate the influence of carbon flux toward the pentose phosphate pathway and/or transhydrogenase activity on NADPH generation. The deletion of pfkA/pfkB significantly improved NADPH supply, but minimally influenced cell growth. The effects of insertion position and copy number of xr were examined by a quantitative real-time PCR and a shake-flask fermentation experiment. In a fed-batch fermentation experiment with a 15-L bioreactor, strain WZ51 produced 131.6gL-1 xylitol from hemicellulosic hydrolysate (xylitol productivity: 2.09gL-1h-1). This study provided a potential approach for industrial-scale production of xylitol from hemicellulosic hydrolysate.

Improved upstream processing for detoxification and recovery of xylitol produced from corncob

This work deals with the development of an improved process for xylitol production from corn cob hydrolysate by biotechnological routes emphasizing the detoxification of corncob acid hydrolysate. The acid hydrolysate obtained by acid hydrolysis of corn cob was concentrated and detoxified by activated charcoal, membrane process and ion exchange resin process. The resultant partially purified corncob hydrolysate was used in fermentation. The fermentation of acid hydrolysate containing 56.5 g/L xylose was carried out in a 14 L fermenter at pH 4.5 for 48 h with 150 rpm stirring rate at 30 °C. A xylitol yield of 62% was achieved from the partially purified acid hydrolysate medium during fermentation using Candida tropicalis MTCC 6192. The purity of xylitol was increased to 92–94% upon downstream processing of carbonation, subsequently ion exchange process and activated charcoal.

Xylitol Production From Byproducts Generated During Sequential Acid-/Alkali-Pretreatment of Empty Palm Fruit Bunch Fiber by an Adapted Candida tropicalis

Xylose is a pentose sugar with the potential to convert a variety of valuable chemical products. In this study, acidic pretreatment wastewater generated during a sequential acid-/alkali-pretreatment process was recycled to increase the hydrolyzed hemicellulose fraction from empty palm fruit bunch fiber (EPFBF), a lignocellulosic biomass. The xylose in the reused wastewater was subjected to overliming and an activated charcoal column was used to remove inhibitory compounds, for xylitol fermentation using the adapted C. tropicalis strain. The cell growth and xylose uptake rates in the adapted strain were 1.7- and 5-fold higher, respectively, compared to the wild-type strain. During batch fermentation using the adapted yeast strain and the post-pretreated xylose solution, 35.2 ± 0.8 g/L xylitol was obtained within 61 h for a production yield of 0.44 g xylitol/g xylose. These results indicate that xylose in the byproducts produced in the bioethanol process could be recovered for production of xylitol.

Sugarcane Syrup Improves Xylitol Bioproduction from Sugarcane Bagasse and Straw Hemicellulosic Hydrolysate

To evaluate the supplementation of the sugarcane bagasse and straw hemicellulosic hydrolysate with sugarcane syrup, which is a sucrose-rich co-product, for xylitol production by Candida guilliermondii FTI 20037. Recently, we demonstrated that sucrose supplementation to sugarcane straw hemicellulosic hydrolysate favoured this bioprocess. A mixture 1:1 of sugarcane bagasse and straw was submitted to dilute-acid hydrolysis with H2SO4 to obtain the hemicellulosic hydrolysate, which was concentrated, detoxified and autoclaved. Three sugarcane syrup concentrations (0.83, 2.50 and 3.33% v/v) were added to the hemicellulosic hydrolysate, which was also supplemented with rice bran extract, (NH4)2SO4, CaCl2·2H2O. Next, a full factorial design was performed to assess the replacement of conventional nutrients by sugarcane syrup. Fermentations were performed in 125 mL Erlenmeyer flasks containing 50 mL of fermentation medium, for 48 h at 30 °C and 200 rpm. Improvements of 16.85% and 12.24% in xylose uptake rate (1.04 ± 0.01 g L−1 h−1) and xylitol volumetric productivity (0.55 ± 0.02 g L−1 h−1), respectively, were achieved with syrup supplementation (2.50% v/v), which led to a final xylitol titer of 26.19 ± 0.02 g L−1. Conventionally employed nutrients (rice bran extract, (NH4)2SO4 and CaCl2·2H2O) did not have significant influence on xylitol production when sugarcane syrup was added to the fermentation medium. Sugarcane syrup as a nutritional supplement improved xylitol production from a mixture of bagasse and straw, becoming an alternative to reduce costs in the formulation of the fermentation medium and contributing to the integration of this bioprocess in a sugarcane biorefinery.

In-Vitro and In-Silico Characterization of Xylose Reductase from Emericella nidulans

Background: Lignocellulosic residues generated by various anthropogenic activities can be a potential raw material for many commercial products such as biofuels, organic acids and nutraceuticals including xylitol. Xylitol is a low-calorie nutritive sweetener for diabetic patients. Microbial production of xylitol can be helpful in overcoming the drawbacks of traditional chemical production process and lowring cost of production. Objective: Designing efficient production process needs the characterization of required enzyme/s. Hence current work was focused on in-vitro and in-silico characterization of xylose reductase from Emericella nidulans. Methods: Xylose reductase from one of the hyper-producer isolates, Emericella nidulans Xlt-11 was used for in-vitro characterization. For in-silico characterization, XR sequence (Accession No: Q5BGA7) was used. Results: Xylose reductase from various microorganisms has been studied but the quest for better enzymes, their stability at higher temperature and pH still continues. Xylose reductase from Emericella nidulans Xlt-11 was found NADH dependent and utilizes xylose as its sole substrate for xylitol production. In comparison to whole cells, enzyme exhibited higher enzyme activity at lower cofactor concentration and could tolerate higher substrate concentration. Thermal deactivation profile showed that whole cell catalysts were more stable than enzyme at higher temperature. In-silico analysis of XR sequence from Emericella nidulans (Accession No: Q5BGA7) suggested that the structure was dominated by random coiling. Enzyme sequences have conserved active site with net negative charge and PI value in acidic pH range. Conclusion: Current investigation supported the enzyme’s specific application i.e. bioconversion of xylose to xylitol due to its higher selectivity. In-silico analysis may provide significant structural and physiological information for modifications and improved stability.

Optimised fractionation of wheat bran for arabinose biopurification and xylitol fermentation by Ogataea zsoltii within a biorefinery process

Wheat bran is an agro-industrial by-product having huge potential for value-added utilisation in biorefinery processes. In this study, a two-step, dilute-sulphuric-acid fractionation process was developed to gain xylose-rich and arabinose-enriched hydrolysates separately. In the first step the reaction time and acid concentration were optimised by statistical evaluation. Depending on the parameters of the optimisation, two process conditions resulting in high arabinose (62–74%) but low xylose yields (41–45%) were determined. Arabinose biopurification of the obtained hydrolysates by Ogataea zsoltii resulted in pure arabinose solutions (arabinose purity of 91–94%). The second acidic hydrolysis resulted in xylose-rich hydrolysates containing 20 g/L xylose. The effect of pH and aeration on the xylitol fermentation by Ogataea zsoltii was investigated. Xylitol fermentation on the xylose-rich hydrolysate under the best conditions resulted in a xylitol yield of 53% in the bioreactor. Hence, an integrated process for arabinose and xylitol production from wheat bran was invented.

Isolation and Characterization of the Stress-Tolerant Candida tropicalis YHJ1 and Evaluation of Its Xylose Reductase for Xylitol Production From Acid Pre-treatment Wastewater.

A stress-tolerant yeast was isolated from honey using acid hydrolysate generated from sequential acid-/alkali-pretreatment of empty palm fruit bunch fiber (EPFBF). The isolated yeast was identified molecularly, taxonomically, and morphologically as Candida tropicalis YHJ1, and analyzed for application in xylitol production. The isolated yeast showed stress tolerance toward various chemical reagents and could grow with up to 600 g/L xylose in the culture medium. This yeast also had a broad carbohydrate utilization spectrum, and its xylitol yield was greatest in medium supplemented with xylose as the sole carbon source. In batch fermentation for xylitol production, the yeast could convert xylose prepared from acidic EPFBF pretreatment wastewater into xylitol. Interestingly, C. tropicalis YHJ1 xylose reductase, containing a Ser279 residue, exhibited more effective xylitol conversion compared to orthologous Candida enzymes containing Leu279 or Asn279; this improvement was associated with NADPH binding, as predicted through homologous structure modeling and enzyme kinetic analysis. Taken together, these results show a novel stress-tolerant yeast strain that may be applicable to xylitol production from toxic lignocellulosic byproducts.

Production of bioalcohols and antioxidant compounds by acid hydrolysis of lignocellulosic wastes and fermentation of hydrolysates with Hansenula polymorpha.

The effect of the H2SO4 concentration in the hydrolysis of sunflower-stalk waste, at 95ºC and using a liquid/solid relation of 20, was studied. In a later stage, the hydrolysates were fermented at different temperatures with the aim of ethanol and xylitol production. A total conversion of the hemicellulose at the acid concentration of 0.5mol/L was achieved; whereas an acid concentration of 2.5mol/L was needed to reach the maximum value in the conversion of the cellulose fraction. The analysis of the hydrolysis kinetics has enabled to determine the apparent reaction order, which was 1.3. The hydrolysates from hydrolysis process with H2SO4 0.5mol/L, once detoxified, were fermented at pH 5.5, temperatures 30, 40, and 50ºC with the yeast Hansenula polymorpha (ATCC 34438), resulting in a sequential uptake of sugars. In relation to ethanol and xylitol yields, the best results were observed at 50°C( =0.11g/g; =0.12g/g). Instantaneous xylitol yields were higher than in ethanol, at the three temperatures essayed. Different phenolic compounds were analyzed in the hydrolysates; hydroxytyrosol was the most abundant (3.79mg/L). The recovery of these compounds entails the elimination of inhibitors in the fermentation process and the production of high value-added antioxidant products.

Xylitol Production by Xylose Reductase over producing Recombinant Escherichia coli M15

Xylitol is a functional sugar alcohol widely used as a sugar substitute. Chemical route of xylitol production is highly expensive, whereas microbial sources offer lower yield and conversion efficiency. In the present investigation, xylose reductase (XR) gene from Candida tropicalis GRA1 was cloned in pQE30Xa vector and transformed into E. coli M15. The resultant recombinant strain produced xylitol from xylose that harboured Xr gene transcribed under the control of lac operon. Such transcribed Xr protein converts xylose into xylitol using NADPH as a co-factor. Xylitol production was achieved by the recombinant E. coli M15 pQE30XrCt under shake-flask fermentation (2.1 g.l-1 in 24 h) with xylose as the sole substrate in the medium. Addition of co-substrate, glucose in the growth medium enhanced the xylitol production to the tune of 3.4g.l-1. Furthermore, the addition of co-substrate glycerol along with xylose enhanced a xylitol yield of 6.4g.l-1.