Production of xylitol by recombinant Saccharomyces cerevisiae containing xylose reductase gene in repeated fed-batch and cell-recycle fermentations

Xylitol is a well-known sugar substitute with low-calorie and anti-cariogenic characteristics. An effort of biological production of xylitol from xylose was made in repeated fed-batch and cell-recycle fermentations of recombinant Saccharomyces cerevisiae BJ3505/δXR harboring the xylose reductase gene from Pichia stipitis. Batch fermentation with 20 g/l xylose and 18 g/l glucose resulted in 9.52 g/l dry cell mass, 20.1 g/l xylitol concentration and approximately 100% conversion yield. Repeated fed-batch operation to remove 10% of culture broth and to supplement an equal volume of 200 g/l xylose was designed to improve xylitol production. In spite of a sudden drop of cell concentration, an increase in dry cell mass led to high accumulation of xylitol at 48.7 g/l. To overcome loss of xylitol-producing biocatalysts in repeated fed-batch fermentation, cell-recycle equipment of hollow fiber membrane was implemented into a xylitol production system. Cell-recycle operation maintained concentration of the recombinant cells high inside a bioreactor. Final dry cell mass of 22.0 g/l, 116 g/l xylitol concentration, 2.34 g/l h overall xylitol productivity were obtained in cell-recycle fermentation supplemented with xylose and yeast extract solution, which were equivalent to 2.3-, 5.8- and 3.8-fold increases compared with the corresponding values of batch-type xylitol production parameters.     

Increase of xylitol yield by feeding xylose and glucose in Candida tropicalis

Candida tropicalis, a strain isolated from the sludge of a factory manufacturing xylose, produced a high xylitol concentration of 131 g/l from 150 g/l xylose at 45 h in a flask. Above 150 g/l xylose, however, volumetric xylitol production rates decreased because of a lag period in cell growth. In fed-batch culture, the volumetric production rate and xylitol yield from xylose varied substantially with the controlled xylose concentration and were maximum at a controlled xylose concentration of 60 g/l. To increase the xylitol yield from xylose, feeding experiments using different ratios of xylose and glucose were carried out in a fermentor. The maximum xylitol yield from 300 g/l xylose was 91% at a glucose/xylose feeding ratio of 15%, while the maximum volumetric production rate of xylitol was 3.98 g l⁻¹ h⁻¹ at a glucose/xylose feeding ratio of 20%. Xylitol production was found to decrease markedly as its concentration rose above 250 g/l. In order to accumulate xylitol to 250 g/l, 270 g/l xylose was added in total, at a glucose/xylose feeding ratio of 15%. Under these conditions, a final xylitol production of 251 g/l, which corresponded to a yield of 93%, was obtained from 270 g/l xylose in 55 h. Received: 20 April 1998 / Received revision: 29 May 1998 / Accepted: 19 June 1998     

Xylose fermentation by Candida shehatae and Pichia stipitis: effects of pH,temperature and substrate concentration

The effects of temperature, pH and xylose concentration on the fermentation parameters of Candida shehatae and Pichia stipitis were evaluated. The optimum pH was in the region of pH 4–5.5, with an optimum fermentation temperature of 30°C. Maximum fermentation rates were reached at 50 g l⁻¹ xylose. A maximum volumetric ethanol productivity of about 0.9 g (l h)⁻¹ was obtained with both yeast strains. The ethanol yield of C. shehatae decreased considerably when cultivated above 30°C or when the xylose concentration was increased. Xylitol accumulated concomitantly. Xylitol production by P. stipitis was observed only during cultivation at 36°C. Whereas the ethanol yield of C. shehatae was usually about 75% of the theoretical maximum, it was 85–90% with P. stipitis.     

The influence of aeration and hemicellulosic sugars on xylitol production by Candida tropicalis

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 investigated using experimental design methodology. Oxygen limitation and initial xylose concentration had considerable influences on xylitol production by Canadida tropicalis ATCC 96745. Under semiaerobic conditions, the maximum xylitol yield was 0.62 g/g substrate, while under aerobic conditions, the maximum volumetric productivity was 0.90 g/l h. In the presence of glucose, xylose utilization was strongly repressed and sequential sugar utilization was observed. Ethanol produced from the glucose caused 50% reduction in xylitol yield when its concentration 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 maximum 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.     

Characterization of two-substrate fermentation processes for xylitol production using recombinant Saccharomyces cerevisiae containing xylose reductase gene

Fermentation characteristics of recombinant Saccharomyces cerevisiae containing a xylose reductase gene from Pichia stipitis were investigated in an attempt to convert xylose to xylitol, a natural five-carbon sugar alcohol used as a sweetener. Xylitol was produced with a maximum yield of 0.95 g g⁻¹ xylitol xylose consumed in the presence of glucose used as a co-substrate for co-factor regeneration. Addition of glucose caused inhibition of xylose transport and accumulation of ethanol. Such problems were solved by adopting glucose-limited fed-batch fermentations where a high ratio of xylose to glucose was maintained during the bioconversion phase. The optimized two-substrate fed-batch fermentation carried out with S. cerevisiae EH13.15:pY2XR at 30°C resulted in 105.2 g l⁻¹ xylitol concentration with 1.69 g l⁻¹ h⁻¹ productivity.     

Kinetic modeling of Candida shehatae ATCC 22984 on xylose and glucose for ethanol production

Candida shehatae ATCC 22984, a xylose-fermenting yeast, showed an ability to produce ethanol in both glucose and xylose medium. Maximum ethanol produced by the yeast was 48.8?g/L in xylose and 52.6?g/L in glucose medium with ethanol yields that varied between 0.3 and 0.4?g/g depended on initial sugar concentrations. Xylitol was a coproduct of ethanol production using xylose as substrate, and glycerol was detected in both glucose and xylose media. Kinetic model equations indicated that growth, substrate consumption, and product formation of C. shehatae were governed by substrate limitation and inhibition by ethanol. The model suggested that cell growth was totally inhibited at 40?g/L of ethanol and ethanol production capacity of the yeast was 52?g/L, which were in good agreement with experimental results. The developed model could be used to explain C. shehatae fermentation in glucose and xylose media from 20 to 170?g/L sugar concentrations.     

Production of xylitol from concentrated wood hydrolysates by Debaryomyces hansenii: Effect of the initial cell concentration

Summary Eucalyptus globulus wood hydrolysates were concentrated by vacuum evaporation to increase their xylose contents, treated with activated charcoal, supplemented with nutrients and used as culture media for xylitol production by Debaryomyces hansenii NRRL Y-7426. The susceptibility of hydrolysates to fermentation was strongly dependent on the initial cell concentration: media containing 58–78 g xylose/l were hardly consumed in batch experiments starting with 16 g cells/l, whereas 39–41 g xylitol/l were achieved in fermentations carried out with similar concentration of the carbon source and initial cell concentrations of 50–80 g/l).     

The behavior of key enzymes of xylose metabolism on the xylitol production by <Emphasis Type="Italic">Candida guilliermondii</Emphasis> grown in hemicellulosic hydrolysate

A variety of raw materials have been used in fermentation process. This study shows the use of rice straw hemicellulosic hydrolysate, as the only source of nutrient, to produce high added-value products. In the present work, the activity of the enzymes xylose reductase (XR); xylitol dehydrogenase (XD); and glucose-6-phosphate dehydrogenase (G6PD) during cultivation of Candida guilliermondii on rice straw hemicellulosic hydrolysate was measured and correlated with xylitol production under different pH values (around 4.5 and 7.5) and initial xylose concentration (around 30 and 70 g l(-1)). Independent of the pH value and xylose concentration evaluated, the title of XD remained constant. On the other hand, the volumetric activity of G6PD increased whereas the level of XR decreased when the initial xylose concentration was increased from 30 to 70 g l(-1). The highest values of xylitol productivity (Q (P) approximately 0.40 g l(-1)) and yield factor (Y (P/S) approximately 0.60 g g(-1)) were reached at highest G6PD/XR ratio and lowest XR/XD ratio. These results suggest that NADPH concentrations influence the formation of xylitol more than the activity ratios of the enzymes XR and XD. Thus, an optimal rate between G6PD and XR must be reached in order to optimize the xylitol production.     

L-lactate production from xylose employingLactococcus lactis IO-1

Summary The growth, substrate utilisation and L-lactate production ofLactococcus lactis IO-1 were examined on xylose, and glucose and xylose media. The yield of lactate on xylose was 0.47 g lactate/g xylose at an initial xylose concentration of 51.2 g/l and the _max was 0.72 h^–1. Xylose cultures were more susceptible to lactate inhibition than were glucose cultures but showed similar kinetic behaviour. The organism was capable of complete sugar utilisation when grown on a mixture of 20 g/l xylose and 20 g/l glucose and synthesised 0.66 g lactate/g sugar.     

Fumaric acid production from xylose by immobilized Rhizopus arrhizus cells

Summary The production of fumaric acid by immobilized Rhizopus arrhizus TKK 204-1-1a mycelium was optimized in batch fermentations using statistical experimental design and empiric modelling. The maximum fumaric acid concentration was obtained at a xylose concentration of about 6% and a carbon:nitrogen ratio of about 160. In repeated batch fermentations with immobilized cells the highest volumetric productivity of fumaric acid reached was 87 mg/l per hour when the initial xylose concentration was 10%, the C:N ratio 160 and the residence time 1.75 days. The maximum product concentration was 16.4 g/l when the initial xylose concentration was 10%, the C:N ratio 160 and the residence time 10.25 days. The maximum yield from initial xylose (6.47%) was 23.7% with a product concentration of 15.3 g/l and volumetric productivity of 71 mg/l per hour at a residence time of 9 days and a C:N ratio of 188.3. Immobilization could increase the fumaric acid concentration to a level 3.4 times higher than that produced by free cells.     

Ethanol production from corn cob hydrolysates by <Emphasis Type="Italic">Escherichia coli</Emphasis> KO11

Corn cob hydrolysates, with xylose as the dominant sugar, were fermented to ethanol by recombinant Escherichia coli KO11. When inoculum was grown on LB medium containing glucose, fermentation of the hydrolysate was completed in 163 h and ethanol yield was 0.50 g ethanol/g sugar. When inoculum was grown on xylose, ethanol yield dropped, but fermentation was faster (113 h). Hydrolysate containing 72.0 g/l xylose and supplemented with 20.0 g/l rice bran was readily fermented, producing 36.0 g/l ethanol within 70 h. Maximum ethanol concentrations were not higher for fermentations using higher cellular concentration inocula. A simulation of an industrial process integrating pentose fermentation by E. coli and hexose fermentation by yeast was carried out. At the first step, E. coli fermented the hydrolysate containing 85.0 g/l xylose, producing 40.0 g/l ethanol in 94 h. Baker's yeast and sucrose (150.0 g/l) were then added to the spent fermentation broth. After 8 h of yeast fermentation, the ethanol concentration reached 104.0 g/l. This two-stage fermentation can render the bioconversion of lignocellulose to ethanol more attractive due to increased final alcohol concentration. Journal of Industrial Microbiology & Biotechnology (2002) 29, 124–128 doi:10.1038/sj.jim.7000287 Received 20 February 2002/ Accepted in revised form 04 June 2002     

Fed-batch Cultivation of Mucor indicus in Dilute-acid Lignocellulosic Hydrolyzate for Ethanol Production

Mucor indicus fermented dilute-acid lignocellulosic hydrolyzates to ethanol in fed-batch cultivation with complete hexose utilization and partial uptake of xylose. The fungus was tolerant to the inhibitors present in the hydrolyzates. It grew in media containing furfural (1 g/l), hydroxymethylfurfural (1 g/l), vanillin (1 g/l), or acetic acid (7 g/l), but did not germinate directly in the hydrolyzate. However, with fed-batch methodology, after initial growth of M. indicus in 500 ml enzymatic wheat hydrolyzate, lignocellulosic hydrolyzate was fermented with feeding rates 55 and 100 ml/h. The fungus consumed more than 46% of the initial xylose, while less than half of this xylose was excreted in the form of xylitol. The ethanol yield was 0.43 g/g total consumed sugar, and reached the maximum concentration of 19.6 g ethanol/l at the end of feeding phase. Filamentous growth, which is regarded as the main obstacle to large-scale cultivation of M. indicus, was avoided in the fed-batch experiments.     

Optimization of fed-batch fermentation for xylitol production by Candida tropicalis

Xylitol, a functional sweetener, was produced from xylose by biological conversion using Candida tropicalis ATCC 13803. Based on a two-substrate fermentation using glucose for cell growth and xylose for xylitol production, fed-batch fermentations were undertaken to increase the final xylitol concentration. The effects of xylose and xylitol on xylitol production rate were studied to determine the optimum concentrations for fed-batch fermentation. Xylose concentration in the medium (100 g l⁻¹) and less than 200 g l⁻¹ total xylose plus xylitol concentration were determined as optimum for maximum xylitol production rate and xylitol yield. Increasing the concentrations of xylose and xylitol decreased the rate and yield of xylitol production and the specific cell growth rate, probably because of an increase in osmotic stress that would interfere with xylose transport, xylitol flux to secretion to cell metabolism. The feeding rate of xylose solution during the fed-batch mode of operation was determined by using the mass balance equations and kinetic parameters involved in the equations in order to increase final xylitol concentration without affecting xylitol and productivity. The optimized fed-batch fermentation resulted in 187 g l⁻¹ xylitol concentration, 0.75 g xylitol g xylose⁻¹ xylitol yield and 3.9 g xylitol l⁻¹ h⁻¹ volumetric productivity. Journal of Industrial Microbiology & Biotechnology (2002) 29, 16–19 doi:10.1038/sj.jim.7000257 Received 15 October 2001/ Accepted in revised form 30 March 2002     

Improved ethanol production from xylose with glucose isomerase and Saccharomyces cerevisiae using the respiratory inhibitor azide

Summary Ethanol was produced from xylose, using the enzyme glucose isomerase (xylose isomerase) and Saccharomyces cerevisiae. The influence of aeration, pH, enzyme concentration, cell mass and the concentration of the respiratory inhibitor sodium azide on the production of ethanol and the formation of by-products was investigated. Anaerobic conditions at pH 6.0, 10 g/l enzyme, 75 g/l dry weight cell mass and 4.6 mM sodium azide were found to be optimal. Under these conditions theoretical yields of ethanol were obtained from 42 g/l xylose within 24 hours.In a fed-batch culture, 62 g/l ethanol was produced from 127 g/l xylose with a yield of 0.49 and a productivity of 1.35 g/l·h.     

Alcoholic fermentation of D-xylose by immobilizedPichia stipitis yeast

Summary Pichia stipitis NRRL Y-7124 yeast cells were for the first time immobilized both in agar gel beads and on fine nylon net for ethanol fermentation on D-xylose, in order to investigate the possibility of using the biocatalyst for improved utilization of the biomass pentose fraction. With free cells the initial xylose level affected little ethanol production, with a maximum of 22 g/l ethanol obtained in 5 days on 5% and of 40 g/l in 8 days on 10% xylose, and an average volumetric productivity of about 0.22 g/lh. The maximum ethanol concentration of 19.5% on 5% xylose with the nylon net attached cells in a continuous packed-bed column reactor was obtained with 35 h residence time. The volumetric productivities of 0.56 g/lh at 19.5 g/l ethanol and 1.0 g/lh at 15.0 g/l ethanol were markedly higher than those obtained with free cells. The stability of the immobilized biocatalyst was excellent. The same reactor could be used for at least 80 days without significant activity loss.     

Effects of culture conditions on the co-fermentation of a glucose and xylose mixture to ethanol by a mutant of Saccharomyces diastaticus associated with Pichia stipitis

Substrates that contain hexose as well as pentose sugars can form an interesting substrate for the production of ethanol. Pichia stipitis and a respiratory-deficient mutant of Saccharomyces diastaticus were used to convert such a substrate into ethanol under continuous culture conditions. With a sugar mixture (glucose 70%/xylose 30%) at 50 g/l, the xylose was entirely consumed when the dilution rate (D) did not exceed 0.006 h^–1 whereas the glucose was entirely consumed whatever the D. The study of influence of initial substrate concentration (S₀) was performed at D = 0.015 h^–1. Under these conditions the substrate was entirely consumed when its initial concentration did not exceed 20 g/l. With S₀ = 80 g/l the residual xylose concentration reached 20.5 g/l. At low D or at low S₀, P. stipitis was the dominant species in the fermentor. Increasing the D or S₀ resulted in the wash-out of P. stipitis mainly because of its low ethanol tolerance. Correspondence to: J. P. Delgenes     

树干毕赤酵母酒精发酵碳元素代谢流向分析

实验对树干毕赤酵母（Pichia stipitis）进行了4个阶段共400 h连续恒化培养,在不同阶段以30.0 g/L葡萄糖作为基本碳源,添加30.0或15.0 g/L的木糖,通过控制温度（35±1）℃,进气量100～150mL/min,搅拌转速250～300 r/min。4个阶段共建立4个连续培养的"稳态"。对碳元素进行物料衡算发现,四个阶段碳元素回收率分别为118.0 %、105.6 %、113.5 %和94.7 %。对4个近似"稳态"的碳元素的代谢流向进行分析发现：将近50.0 %左右碳元素流向产物酒精,其次是CO2和酵母细胞;木糖醇浓度与流入底物中木糖浓度有直接关系,在相同发酵条件下流入的木糖浓度越大代谢生成木糖醇浓度也越高;实验所采用的通气条件更适合底物为30.0 g/L葡萄糖和30.0 g/L木糖混合液的连续发酵。     

Itaconic acid production by immobilizedAspergillus terreus from xylose and glucose

Summary Aspergillus terreus NRRC 1960 spores were entrapped in calcium alginate gel beads or alternotely the fungal mycelium was immobilized either on Celite R-626 or in agar gel cubes, and the biocatalyst was employed both in repeated batch and in continuous column reactors to produce itaconic acid from D-xylose or D-glucose. The highest itaconic acid yield obtained in a submerged culture batch fermentation was 54.5% based on total initial glucose (55 g/l) with a volumetric productivity of 0.32 g/l h, and 44.8% from xylose (67 g/l) with a productivity of 0.20 g/l h. In a repeated batch fermentation mycelium immobilized in agar gel had a productivity of 0.112 g/l h, and mycelium grown from spores immobilized in calcium alginate gel 0.06 g/l h, both from xylose (60 g/l). With the best immobilized biocatalyst system used employing Celite R-626 as a carrier, volumetric productivities of 1.2 g/l h from glucose and 0.56 g/l h from xylose (both at 60 g/l) were obtained in continuous column operation for more than 2 weeks.     

Influence of the toxic compounds present in brewer's spent grain hemicellulosic hydrolysate on xylose-to-xylitol bioconversion by Candida guilliermondii

Fermentation media containing different concentrations of toxic compounds were prepared from brewer's spent grain (BSG) hemicellulosic hydrolysate, and used for xylose-to-xylitol bioconversion by Candida guilliermondii. Such fermentation media were composed of the hydrolysate in the following ways: raw (RH); concentrated four-fold (CH); concentrated and treated with activated charcoal (TCH); raw supplemented with sugars until a concentration four-fold higher (SRH); concentrated and subsequently diluted but supplemented with sugars until a concentration four-fold higher (SDCH). All media presented an initial xylose concentration of 85 g/l, except RH, which contained 23 g/l xylose. Fermentation results revealed that the sugars supplementation to raw hydrolysate favored the xylitol production. Nevertheless, xylitol production from CH was negatively affected due to the high concentration of toxic compounds present in the medium. The hydrolysate treatment with activated charcoal partially removed the toxic compounds, and the xylitol production was higher than in CH, but not so efficient as in SRH. It was thus concluded that to obtain an efficient xylose-to-xylitol bioconversion from BSG hydrolysate, the sugars concentration must be increased, but the toxic compounds concentration must be reduced to the same level present in the raw hydrolysate.     

Characterization of recombinantE. coli ATCC 11303 (pLOI 297) in the conversion of cellulose and xylose to ethanol

This work describes the characterization of recombinantEsherichia coli ATCC 11303 (pLOI 297) in the production of ethanol from cellulose and xylose. We have examined the fermentation of glucose and xylose, both individually and in mixtures, and the selectivity of ethanol production under various conditions of operation. Xylose metabolism was strongly inhibited by the presence of glucose. Ethanol was a strong inhibitor of both glucose and xylose fermentations; the maximum ethanol levels achieved at 37°C and 42°C were about 50 g/l and 25 g/l respectively. Simmultaneous sacharification and fermentation of cellulose with recombinantE. coli and exogenous cellulose showed a high ethanol yield (84% of theoretical) in the hydrolysis regime of pH 5.0 and 37°C. The selectivity of organic acid formation relative to that of ethanol increased at extreme levels of initial glucose concentration; production of succinic and acetic acids increased at low levels of glucose ( <1 g/l), and lactic acid production increased when initial glucose was higher than 100 g/l.