Microbial Production Of Xylitol Research Articles

Development of engineered Candida tropicalis strain for efficient corncob-based xylitol-ethanol biorefinery

BackgroundXylitol has a wide range of applications in the pharmaceuticals, cosmetic, food and beverage industry. Microbial xylitol production reduces the risk of contamination and is considered as environment friendly and sustainable compared to the chemical method. In this study, random mutagenesis and genetic engineering approaches were employed to develop Candida tropicalis strains with reduced xylitol dehydrogenase (XDH) activity to eliminate co-substrate requirement for corn cob-based xylitol-ethanol biorefinery.ResultsThe results suggest that when pure xylose (10% w/v) was fermented in bioreactor, the Ethyl methane sulfonate (EMS) mutated strain (C. tropicalis K2M) showed 9.2% and XYL2 heterozygous (XYL2/xyl2Δ::FRT) strain (C. tropicalis K21D) showed 16% improvement in xylitol production compared to parental strain (C. tropicalis K2). Furthermore, 1.5-fold improvement (88.62 g/L to 132 g/L) in xylitol production was achieved by C. tropicalis K21D after Response Surface Methodology (RSM) and one factor at a time (OFAT) applied for media component optimization. Finally, corncob hydrolysate was tested for xylitol production in biorefinery mode, which leads to the production of 32.6 g/L xylitol from hemicellulosic fraction, 32.0 g/L ethanol from cellulosic fraction and 13.0 g/L animal feed.ConclusionsThis work, for the first time, illustrates the potential of C. tropicalis K21D as a microbial cell factory for efficient production of xylitol and ethanol via an integrated biorefinery framework by utilising lignocellulosic biomass with minimum waste generation.

Microbial xylitol production from corn cob using Candida guilliermondii

Xylitol is a polyol that is widely used in the food industries as an alternative sweetener due to some health benefits. The microbial production of xylitol based on corn cobs was potential to be developed due to the abundant availability of corn cobs in Indonesia. In addition, it is an alternative process that is a higher yield and environmentally friendly. This study was conducted to assign the optimum process in the xylitol production based on corn cobs using Candida guilliermondii. The hydrolysis of corn cobs used sulfuric acid 1% at a temperature of 121°C for 60 minutes. The hydrolysate was then added with calcium hydroxide and activated charcoal to remove inhibitors. The design of experimental used was a factorial completely randomized design with three factors included corn cobs hydrolysate concentration (50% and 66%), glucose (0 g l-1 and 5 g l-1), and incubation period (120 h and 168 h). The result showed that concentration of hydrolysate, glucose, and incubation period had an effect on the xylitol produced. The highest xylitol was 144.09 ppm that was produced by the hydrolysate of 66%, glucose of 5 g l-1 and incubation period of 168 h, meanwhile the residual xylose was 2081.87 ppm.

Process development for crystalline xylitol production from corncob biomass by Pichia caribbica

Xylitol, a commodity chemical, is widely used in nutraceutical and pharmaceutical formulations. Microbial xylitol production is a promising alternative to mitigate current industrial practice issues and offers an environment-friendly sustainable conversion route. This study demonstrates a bioprocess for xylitol production from corncob using a mesophilic yeast Pichia caribbica. Corncob was hydrolyzed by dilute acid and steam explosion to recover fermentable xylose and used as the feed for xylitol production. Activated carbon treatment (3% w/v) completely removed the biomass-derived inhibitors furfural and hydroxymethyl furfural from the liquid hydrolysate. The fermenting yeast Pichia caribbica produced 124.1 ± 0.45 g/L xylitol from the detoxified and concentrated corncob hydrolysate with a high yield of 0.80 ± 0.02 g/g. The crystallized xylitol with 96.5% purity demonstrated no harmful effects on the cell line used as a control for the in-vitro toxicity studies. This proof of concept can be applied to help scale up for bio-refinery-based large-scale production of xylitol from corncob biomass.

Microbial xylitol production

Xylitol is pentahydroxy sugar alcohol, existing in very trace amount in fruits and vegetables, and finds varied application in industries like food, pharmaceuticals, confectionaries, etc. and is of prime importance to health. Owing to its trace occurrence in nature and considerable increase in market demand that exceeds availability, alternate production through biotechnological and chemical approach is in process. Biochemical production involves substrates like lignocellulosic biomasses and industrial effluents and is an eco-friendly process with high dependency on physico-chemical parameters. Although the chemical processes are faster, high yielding and economical, they have a great limitation as usage of toxic chemicals and thus need to be regulated and replaced by an environment friendly approach. Microbes play a major role in xylitol production through a biotechnological process towards the development of a sustainable system. Major microbes working on assimilation of xylose for production of xylitol include Candida tropicalis, Candida maltose, Bacillus subtilis, Debaromyces hansenii, etc. The present review reports all probable microbial xylitol production biochemical pathways encompassing diverse bioprocesses involved in uptake and conversion of xylose sugars from agricultural residues and industrial effluents. A comprehensive report on xylitol occurrence and biotechnological production processes with varied substrates has been encompassed. KEY POINTS: • Xylitol from agro-industrial waste • Microbial xylose assimilation.

Process development for detoxification of corncob hydrolysate using activated charcoal for xylitol production

The work describes process development for detoxification of corncob hydrolysate using activated carbon for microbial xylitol production. Activated carbon was used as an adsorbent to completely remove the major fermentation inhibitors obtained during dilute acid and steam explosion of biomass. A xylitol yield of 0.78 g/g was achieved from the detoxified hydrolysate in a 5 L fermenter by Pichia caribbica. The spent carbon was effectively regenerated and used for three consecutive cycles of operation. The successive regeneration and reuse of carbon could reduce the operational cost by ~ 38% and be used as a reference dataset for process scale-up and cost-effective xylitol production.

Optimized Bioconversion of Xylose Derived from Pre-Treated Crop Residues into Xylitol by Using Candida boidinii

Crop residues can serve as low-cost feedstocks for microbial production of xylitol, which offers many advantages over the commonly used chemical process. However, enhancing the efficiency of xylitol fermentation is still a barrier to industrial implementation. In this study, the effects of oxygen transfer rate (OTR) (1.1, 2.1, 3.1 mmol O2/(L × h)) and initial xylose concentration (30, 55, 80 g/L) on xylitol production of Candida boidinii NCAIM Y.01308 on xylose medium were investigated and optimised by response surface methodology, and xylitol fermentations were performed on xylose-rich hydrolysates of wheat bran and rice straw. High values of maximum xylitol yields (58–63%) were achieved at low initial xylose concentration (20–30 g/L) and OTR values (1.1–1.5 mmol O2/(L × h)). The highest value for maximum xylitol productivity (0.96 g/(L × h)) was predicted at 71 g/L initial xylose and 2.7 mmol O2/(L × h) OTR. Maximum xylitol yield and productivity obtained on wheat bran hydrolysate were 60% and 0.58 g/(L × h), respectively. On detoxified and supplemented hydrolysate of rice straw, maximum xylitol yield and productivity of 30% and 0.19 g/(L × h) were achieved. This study revealed the terms affecting the xylitol production by C. boidinii and provided validated models to predict the achievable xylitol yields and productivities under different conditions. Efficient pre-treatments for xylose-rich hydrolysates from rice straw and wheat bran were selected. Fermentation using wheat bran hydrolysate and C. boidinii under optimized condition is proved as a promising method for biotechnological xylitol production.

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.

Biosynthetic strategies to produce xylitol: an economical venture.

Xylitol is a natural five-carbon sugar alcohol with potential for use in food and pharmaceutical industries owing to its insulin-independent metabolic regulation, tooth rehardening, anti-carcinogenic, and anti-inflammatory, as well as osteoporosis and ear infections preventing activities. Chemical and biosynthetic routes using D-xylose, glucose, or biomass hydrolysate as raw materials can produce xylitol. Among these methods, microbial production of xylitol has received significant attention due to its wide substrate availability, easy to operate, and eco-friendly nature, in contrast with high-energy consuming and environmental-polluting chemical method. Though great advances have been made in recent years for the biosynthesis of xylitol from xylose, glucose, and biomass hydrolysate, and the yield and productivity of xylitol are substantially improved by metabolic engineering and optimizing key metabolic pathway parameters, it is still far away from industrial-scale biosynthesis of xylitol. In contrary, the chemical synthesis of xylitol from xylose remains the dominant route. Economic and highly efficient xylitol biosynthetic strategies from an abundantly available raw material (i.e., glucose) by engineered microorganisms are on the hard way to forwarding. However, synthetic biology appears as a novel and promising approach to develop a super yeast strain for industrial production of xylitol from glucose. After a brief overview of chemical-based xylitol production, we critically analyzed and comprehensively summarized the major metabolic strategies used for the enhanced biosynthesis of xylitol in this review. Towards the end, the study is wrapped up with current challenges, concluding remarks, and future prospects for designing an industrial yeast strain for xylitol biosynthesis from glucose.

Preparation of kenaf stem hemicellulosic hydrolysate and its fermentability in microbial production of xylitol by Escherichia coliBL21

Kenaf (Hibiscus cannabinus L.), a potential fibre crop with a desirably high growth rate, could serve as a sustainable feedstock in the production of xylitol. In this work, the extraction of soluble products of kenaf through dilute nitric-acid hydrolysis was elucidated with respect to three parameters, namely temperature, residence time, and acid concentration. The study will assist in evaluating the performance in terms of xylose recovery. The result point out that the maximum xylose yield of 30.7 g per 100 g of dry kenaf was attained from 2% (v/v) HNO3 at 130 °C for 60 min. The detoxified hydrolysate was incorporated as the primary carbon source for subsequent fermentation by recombinant Escherichia coli and the performance of strain on five different semi-synthetic media on xylitol production were evaluated herein. Among these media, batch cultivation in a basal salt medium (BSM) afforded the highest xylitol yield of 0.35 g/g based on xylose consumption, which corresponded to 92.8% substrate utilization after 38 h. Subsequently, fermentation by E. coli in the xylose-based kenaf hydrolysate supplemented with BSM resulting in 6.8 g/L xylitol which corresponding to xylitol yield of 0.38 g/g. These findings suggested that the use of kenaf as the fermentation feedstock could be advantageous for the development of sustainable xylitol production.

Atmospheric and room temperature plasma (ARTP) mutagenesis enables xylitol over-production with yeast Candida tropicalis

Xylitol is a sugar alcohol that is used as a sweetener in food and confections. Industrially, xylitol is manufactured by chemical hydrogenation of d-xylose, which requires expensive separation and purification steps as well as high pressure and temperature. The microbial production of xylitol has been examined as an alternative to the chemical process. In this study, a xylitol over-producing strain is breeded by mutagenesis of a newly isolated yeast Candida tropicalis with a new mutation breeding system named atmospheric and room temperature plasma. The highest yield strain T31 was screened among more than 200 mutants with a xylitol yield of 0.61 g/g, which represents a yield increase of 22%. Furthermore, a two-stage dissolved oxygen supply strategy was used in a fermentation process resulting the maximum xylitol yield 0.79 g/g, which makes it a promising candidate for xylitol production. Further biochemical analysis indicating the relative gene expression and the enzyme activity of xylose reductase were higher in mutants than those in the original strain, which partly explained the high yield of xylitol. Thus, this study provides a new strategy to breed the over-producing strains for the xylitol industry.

Membrane-Based Downstream Processing of Microbial Xylitol Production

Membrane-Based Downstream Processing of Microbial Xylitol Production

Microbial Production of Xylitol from Oil Palm Empty Fruit Bunch Hydrolysate: Effects of Inoculum and pH

Considering its high content of hemicellulose, oil palm empty fruit bunch (EFB) lignocellulosic biomass waste from palm oil processing has the potential to be utilized as the raw material for the production of xylitol, a low calorie, low GI, and anti cariogenic alternative sugar with similar sweetness to sucrose. This research explored the possibility of converting EFB to xylitol via green microbial fermentation, in particular the effects of inoculum and initial pH on the fermentation performance. It was observed that the cell concentration in the inoculum and the initial pH affect cell growth and xylitol production. pH 5 was observed to give the best fermentation performance. Further, the fermentation tended to yield more xylitol at higher initial cell concentration. It was also observed that no growth or fermentation inhibitory compounds were found in the EFB hydrolysate obtained from enzymatic hydrolysis of EFB. Thus it can be used directly as substrate for xylitol fermentation.

Evaluation of some lignocellulosic byproducts of food industry for microbial xylitol production by Candida tropicalis.

Some lignocellulosic food byproducts such as potato peels, wheat bran, barley bran and chestnut shells were evaluated as potential sources of xylose for microbial xylitol production by yeasts. Potential yeast strains were selected after screening xylitol production of some indigenous yeasts in a defined fermentation medium. Candida tropicalis strains gave the highest results with 83.28 and 54.07 g/L xylitol production from 100 g/L xylose. Lignocellulosic materials were exposed to acid hydrolysis at different conditions. Chestnut shells gave the highest xylose yield and the hydrolysate of chestnut shells was used in further experiments in which xylitol productions of two potential C. tropicalis strains were investigated. Combined detoxification method including evaporation, overliming and activated charcoal with the use of threefold concentration and also yeast extract supplementation suggested to be efficient for both growth and product formation in chestnut shell hydrolysate in which 40 % xylitol yield was obtained. It was concluded that detoxified and fortified chestnut shell hydrolysate could be a potential medium for xylitol production.

Dual utilization of NADPH and NADH cofactors enhances xylitol production in engineered Saccharomyces cerevisiae.

Xylitol, a natural sweetener, can be produced by hydrogenation of xylose in hemicelluloses. In microbial processes, utilization of only NADPH cofactor limited commercialization of xylitol biosynthesis. To overcome this drawback, Saccharomyces cerevisiae D452-2 was engineered to express two types of xylose reductase (XR) with either NADPH-dependence or NADH-preference. Engineered S. cerevisiae DWM expressing both the XRs exhibited higher xylitol productivity than the yeast strain expressing NADPH-dependent XR only (DWW) in both batch and glucose-limited fed-batch cultures. Furthermore, the coexpression of S. cerevisiae ZWF1 and ACS1 genes in the DWM strain increased intracellular concentrations of NADPH and NADH and improved maximum xylitol productivity by 17%, relative to that for the DWM strain. Finally, the optimized fed-batch fermentation of S. cerevisiae DWM-ZWF1-ACS1 resulted in 196.2 g/L xylitol concentration, 4.27 g/L h productivity and almost the theoretical yield. Expression of the two types of XR utilizing both NADPH and NADH is a promising strategy to meet the industrial demands for microbial xylitol production.

Microbial Production of Xylitol from Oil Palm Empty Fruit Bunches Hydrolysate: The Effect of Glucose Concentration

Microbial Production of Xylitol from Oil Palm Empty Fruit Bunches Hydrolysate: The Effect of Glucose Concentration

Processes for the Production of Xylitol—A Review

Xylitol is a sugar alcohol that is used as a sweetener for diabetics and also for other purposes (e.g., in chewing gum). Industrially, xylitol is manufactured through a chemical process that has some disadvantages, including a high energy requirement, extensive purification steps, and a high cost of product. The microbial production of xylitol has been examined as an alternative to the chemical process, which on an industrial scale is time-consuming mainly due to sterilization and inoculum development. The enzymatic production of xylitol from lignocellulosics is an attractive and promising alternative to the chemical process, potentially eliminating the major disadvantages of conventional processes. This article reviews the literature on xylitol production processes and identifies ways to further improve xylitol synthesis to compete with the current chemical process.

Crystalline Xylitol Production by a Novel Yeast, Pichia caribbica (HQ222812), and Its Application for Quorum Sensing Inhibition in Gram-Negative Marker Strain Chromobacterium violaceum CV026

Xylitol, a sugar alcohol, is fast gaining ground over other artificial sugar substitutes owing to its advantageous properties. Xylitol is a safer alternative for diabetics because of insulin-independent metabolism. It has beneficial properties suitable to form an important part of odontological formulations. Conventional commercial production of xylitol involves harsh chemical method operating at high temperature and pressure. Thus, microbial production of xylitol is preferred over chemical method, and yeasts have been extensively exploited for this purpose. In the present manuscript, quantitative production of xylitol from D-xylose with the yield of 0.852gm/gm and volumetric productivity of 1.83gm/l/h in crystalline form, using novel yeast Pichia caribbica is reported. Also, a mild, safe procedure for product extraction is described. The ability of xylitol to act as a quorum sensing antagonist in gram-negative marker strain Chromobacterium violaceum CV026 has been demonstrated for the first time.

Effects of overexpression of acetaldehyde dehydrogenase 6 and acetyl-CoA synthetase 1 on xylitol production in recombinant Saccharomyces cerevisiae

Abstract Supply of NAD(P)H is a key factor in microbial production of xylitol from xylose. To facilitate NAD(P)H supplementation, acetaldehyde dehydrogenase 6 ( ALD6 ) and acetyl-CoA synthetase 1 ( ACS1 ) were overexpressed in recombinant Saccharomyces cerevisiae harboring the Pichia stipitis xylose reductase (XR) gene. In-vitro activity analysis confirmed the functional expression of both enzymes. Glucose-limited fed-batch fermentations of Saccharomyces cerevisiae BJ3505:δXR strains overexpressing ACS 1 and/or ALD 6 were performed by feeding 600 g/L glucose in the presence of 100 g/L xylose. Among them, ACS 1 overexpression gave the best result of xylitol production: 91.3 g/L xylitol concentration and 1.76 g/L h xylitol productivity, which were 25% and 11% increase, relative to those of the control and ALD 6-overexpressing strains. Considering the changes of cell growth, ethanol and acetate production, a remarkable enhancement of xylitol production by ACS 1 overexpression seemed be ascribed to energy and NAD(P)H generation through a metabolism from acetaldehyde to acetyl-CoA and TCA cycle.

Microbial production of xylitol from l-arabinose by metabolically engineered Escherichia coli

An Escherichia coli strain, ZUC99(pATX210), which can produce xylitol from L-arabinose at a high yield, has been created by introducing a new bioconversion pathway into the cells. This pathway consists of three enzymes: L-arabinose isomerase (which converts L-arabinose to L-ribulose), D-psicose 3-epimerase (which converts L-ribulose to L-xylulose), and L-xylulose reductase (which converts L-xylulose to xylitol). The genes encoding these enzymes were cloned behind the araBAD promoter in tandem so that they were polycistronically transcribed from the single promoter, like an operon. Expression of the recombinant enzymes in the active form was successfully achieved in the presence of L-arabinose. A xylitol production profile of the recombinant strain was evaluated by shake-flask fermentation. ZUC99(pATX210) produced 2.6 g/l xylitol using 4.2 g/l L-arabinose with a xylitol yield of 0.62 g/g L-arabinose in 36 h. It was determined that utilization of glycerol as a co-substrate significantly improved xylitol production and yield. In the presence of 11.8 g/l glycerol, ZUC99(pATX210) produced 9.7 g/l xylitol from 10.5 g/l L-arabinose with a xylitol yield of 0.92 g/g L-arabinose in 36 h. ZUC99(pATX210) also exhibited efficient conversion in fermentor experiments with 1 l medium containing L-arabinose and glycerol. The strain produced 14.5 g/l xylitol from 15.2 g/l L-arabinose with a xylitol yield of 0.95 g/g L-arabinose in 30 h.

Bioprocess development for microbial xylitol production from agricultural wastes

Bioprocess development for microbial xylitol production from agricultural wastes