High Xylose Research Articles

Improvement of xylitol production by Candida guilliermondii FTI 20037 previously adapted to rice straw hemicellulosic hydrolysate.

To evaluate a simple and economical technique to improve xylitol production using concentrated xylose solutions prepared from rice straw hemicellulosic hydrolysate. Experiments were carried out with rice straw hemicellulosic hydrolysate containing 90 g l-1 xylose, with and without the addition of nutrients, using the yeast Candida guilliermondii previously grown on the hydrolysate (adapted cells) or on semi-defined medium (unadapted cells). By this method, the yield of xylitol increased from 17 g l-1 to 50 g l-1, and xylose consumption increased from 52% to 83%, after 120 h of fermentation. The xylitol production rates were very close to that (0.42 g l-1 h-1) attained in a medium simulating hydrolysate sugars. Yeast strain adaptation to the hydrolysate showed to be a suitable method to alleviate the inhibitory effects of the toxic compounds. Adapted cells of Candida guilliermondii can efficiently produce xylitol from hydrolysate with high xylose concentrations. Yeast adaptation helps the bioconversion process in hydrolysate made from lignocellulosic materials. This low-cost technique provides an alternative to the detoxification methods used for removal of inhibitory compounds. In addition, the use of adapted inocula makes it possible to schedule a series of batch cultures so that the whole plant can be operated almost continuously with a concomitant reduction in the overall operation time.

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 x 2H2O, and 20.0 g/L of rice bran at 35 degrees 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 x 10(-2)M (xylose) and 9.5 x 10(-3) mM (NADPH); for XD, 1.6 x 10(-1)M (xylitol) and 9.9 x 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 Vmax, some interference on the overall xylitol production by the yeast could be expected.

Effects of environmental conditions on xylose reductase and xylitol dehydrogenase production by Candida guilliermondii.

The effects of environmental conditions, namely initial pH (2.5-7.0) and temperature (25 and 35 degrees C), on xylose reductase and xylitol dehydrogenase levels, as well as on xylitol production, were evaluated. Although the fermentative parameter values increased with an increase in pH and temperature (the maximum Yp/s and Qp were 0.75 g/g and 0.95 g/[L.h], respectively, both attained at pH 6.0, 35 degrees C), the highest xylose reductase activities (nearly 900 IU/mg of protein) were observed at an initial pH varying from 4.0 to 6.0. Xylitol dehydrogenase was favored by an increase in both initial pH and temperature of the medium. The highest xylitol dehydrogenase specific activity was attained at pH 6.5 and 35 degrees C (577 IU/mg of protein).

Degradability and digestibility of full-fat soybeans treated with different sugar and heat combinations

Two experiments were conducted to study the effects of different combinations of sugars and heating time and temperature on the ruminal undegradable protein fraction and DM digestibility of full-fat soybeans. In the first experiment, the influence of sugar addition before heating (none, soaked in either a high fructose corn syrup [HFCS] solution, lignosulfonate or molasses), micronization temperatures (100, 110, 120, and 130 °C), and temperature holding times (0, 30, 60, and 120 min) was examined in a 4 × 4 × 4 factorial arrangement of treatments. In the second experiment, the effect of heating in a sugar solution was studied in a 4 × 2 × 4 factorial design with four sugar treatments (none, soaked either in fructose, molasses or a high xylose product), two soaking temperatures (100 and 120 °C), and four soaking times (0, 60, 120, and 240 min). In vitro dry matter digestibility (IVDMD), in vitro ruminal undegradable protein (RUP), and in sacco RUP were measured. In the first experiment, increased temperatures and heating to 120 min resulted in an increased RUP fraction of full-fat soybeans with no effect on DM digestibility, relative to when no sugar or fructose was added. There was a decrease in digestibility with increased temperature when lignosulfonate or molasses was added and with increased heating time when lignosulfonate was added. In the second experiment where soaking in sugar and heating were done simultaneously, lignosulfonate increased the RUP of soybeans without affecting its digestibility but values were numerically lower than when soaking and micronizing were conducted in two different steps as performed in the first experiment. Key words: protein, degradability, digestibility, micronization

Structure and physicochemical properties of barley non-starch polysaccharides—II. Alkaliextractable β-glucans and arabinoxylans

Three fractions containing hemicellulosic material were obtained by sequential extraction of barley residue (left after removal of water-extractable polysaccharides) with saturated barium hydroxide [Ba(OH) 2 fraction], distilled water [ Ba(OH) 2 H 2O fraction], and 1 m sodium hydroxide [NaOH fraction]. The yields of the fractions were 1.6, 1.7, and 2.6% (w/w), respectively, of the dry barley grist. The Ba(OH) 2 fraction contained mainly arabinose and xylose, 35.8% and 60.9%, respectively. The Ba(OH) 2 H 2O fraction in addition to 26.7% Ara and 36.6% Xyl contained also 34.8% Glc. The NaOH fraction was composed of 14.2% Ara, 44.0% Xyl, and 40.9% Glc. The Ba(OH) 2 H 2O and NaOH extracts were further fractionated by stepwise (NH 4) 2SO 4 precipitation into several subfractions with varying amounts of β-glucans and arabinoxylans. β-Glucans in Ba(OH) 2 H 2O and NaOH fractions were characterized by high ratios of β-(1→4) β-(1→3) linkages, large amounts of contiguously linked β-(1→4) segments, and high ratios of cellotriosyl/cellotetraosyl units. The alkali-extractable arabinoxylans, especially those NaOH-extractable, were characterized by a very low degree of substitution, high xylose arabinose ratio, and a small content of doubly substituted xylose residues. Some populations of arabinoxylans displayed structural features that would enable them to self-associate or to interact with β-glucans.

Xylitol production by immobilized recombinant Saccharomyces cerevisiae in a continuous packed‐bed bioreactor

Continuous xylitol production with two different immobilized recombinant Saccharomyces cerevisiae strains (H475 and S641), expressing low and high xylose reductase (XR) activities, was investigated in a lab-scale packed-bed bioreactor. The effect of hydraulic residence time (HRT; 1.3–11.3 h), substrate/cosubstrate ratio (0.5 and 1), recycling ratio (0, 5, and 10), and aeration (anaerobic and oxygen limited conditions) were studied. The cells were immobilized by gel entrapment using Ca-alginate as support and the beads were treated with Al3+ to improve their mechanical strength. Xylose was converted to xylitol using glucose as cosubstrate for regeneration of NAD(P)H required in xylitol formation and for generation of maintenance energy. The stability of the recombinant strains after 15 days of continuous operation was evaluated by XR activity and plasmid retention analyses. Under anaerobic conditions the volumetric xylitol productivity increased with decreasing HRT with both strains. With a recycling ratio of 10, volumetric productivities as high as 3.44 and 5.80 g/L · h were obtained with the low XR strain at HRT 1.3 h and with the high XR strain at HRT 2.6 h, respectively. However, the highest overall xylitol yields on xylose and on cosubstrate were reached at higher HRTs. Lowering the xylose/cosubstrate ratio from 1 to 0.5 increased the overall yield of xylitol on xylose, but the productivity and the xylitol yield on cosubstrate decreased. Under oxygen limited conditions the effect of the recycling ratio on production parameters was masked by other factors, such as an accumulation of free cells in the bioreactor and severe genetic instability of the high XR strain. Under anaerobic conditions the instability was less severe, causing a decrease in XR activity from 0.15 to 0.10 and from 3.18 to 1.49 U/mg with the low and high XR strains, respectively. At the end of the fermentation, the fraction of plasmid bearing cells in the beads was close to 100% for the low XR strain; however, it was significantly lower for the high XR strain, particularly for cells from the interior of the beads. © 1996 John Wiley & Sons, Inc.

Xylitol production by immobilized recombinantSaccharomyces cerevisiae in a continuous packed-bed bioreactor

Continuous xylitol production with two different immobilized recombinant Saccharomyces cerevisiae strains (H475 and S641), expressing low and high xylose reductase (XR) activities, was investigated in a lab-scale packed-bed bioreactor. The effect of hydraulic residence time (HRT; 1.3-11.3 h), substrate/cosubstrate ratio (0.5 and 1), recycling ratio (0, 5, and 10), and aeration (anaerobic and oxygen limited conditions) were studied. The cells were immobilized by gel entrapment using Ca-alginate as support and the beads were treated with Al(3+) to improve their mechanical strength. Xylose was converted to xylitol using glucose as cosubstrate for regeneration of NAD(P)H required in xylitol formation and for generation of maintenance energy. The stability of the recombinant strains after 15 days of continuous operation was evaluated by XR activity and plasmid retention analyses. Under anaerobic conditions the volumetric xylitol productivity increased with decreasing HRT with both strains. With a recycling ratio of 10, volumetric productivities as high as 3.44 and 5.80 g/L . h were obtained with the low XR strain at HRT 1.3 h and with the high XR strain at HRT 2.6 h, respectively. However, the highest overall xylitol yields on xylose and on cosubstrate were reached at higher HRTs. Lowering the xylose/cosubstrate ratio from 1 to 0.5 increased the overall yield of xylitol on xylose, but the productivity and the xylitol yield on cosubstrate decreased. Under oxygen limited conditions the effect of the recycling ratio on production parameters was masked by other factors, such as an accumulation of free cells in the bioreactor and severe genetic instability of the high XR strain. Under anaerobic conditions the instability was less severe, causing a decrease in XR activity from 0.15 to 0.10 and from 3.18 to 1.49 U/mg with the low and high XR strains, respectively. At the end of the fermentation, the fraction of plasmid bearing cells in the beads was close to 100% for the low XR strain; however, it was significantly lower for the high XR strain, particularly for cells from the interior of the beads.

Studies on nutrient requirements and cost-effective supplements for ethanol production by recombinant E. coli.

This article describes a systematic study of the nutritional requirements of a patented recombinant ethanologenic Escherichia coli (11303:pLO1297) and provides cost-effective formulations that are compatible with the production of fuel ethanol in fermentations of lignocellulosic prehydrolysate characterized by high xylose conversion efficiency. A complex and nutrient-rich laboratory medium, Luria broth (LB), provided the benchmark with respect to fermentation performance standard. Xylose fermentation performance was assessed in terms of the target values for operational process parameters established by the US National Renewable Energy Laboratory (NREL)-final ethanol concentration (25 g/L), xylose-to-ethanol conversion efficiency (90%), and volumetric productivity (0.52 g/L.h). Biomass prehydrolysates that are rich in xylose also contain acetic acid, and in anticipation of a need to reduce acetic acid toxicity, the fermentors were operated with a pH control set-point of 7.0 Growth and fermentation in the minimal defined salts (DS) medium was only about 15% compared to the reference medium. Amendment of the minimal medium containing 6 wt% xylose with both vitamins and amino acids resulted in improved growth, but the volume productivity (0.59 g/L.h) was still only about 54% of that with LB (1.1g/L.h). Formulations directed at cost reduction through the use of less expensive commercial complex nutritional supplements were within 90% of the NREL process target with respect to yield and provided a productivity at about 80% of the LB medium, but were not economical. Corn steep liquor (CSL) at about 7-8 g/L was shown to be a complete source of nutritional requirements and supported a fermentation performance approaching that of LB. At a cost of CSL of $50/t(dry wt), the economic impact of using this amount CSL as the sole nutritional supplement in a cellulosic ethanol plant was estimated to be about 4 cents/gal of ethanol.

Dual mechanisms of xylose uptake in the thermophilic bacteriumThermoanaerobacter thermohydrosulfuricus

Thermoanaerobacter thermohydrosulfuricus Rt8.B1 catabolized xylose by the pentose phosphate pathway, and xylose isomerase and xylulokinase were inducible. The uptake of xylose was by two low-affinity, inducible systems. Both systems were resistant to the protonophore, tetrachlorosalicylanilide, the F1F0-ATPase inhibitor, N,N-dicyclohexylcarboiimide, and the sodium/proton antiporter, monensin. The high capacity system (100 nmol min−1 (mg protein)−1) was only expressed when the bacterium was grown with a high concentration of xylose (50 mM). It took more than 60 mM xylose to saturate the high capacity system. When T. thermohydrosulfuricus was grown with a low concentration of xylose (5 mM), xylose uptake was saturated by as little as 10 mM xylose (18 nmol min−1 (mg protein)−1). Cells grown with 50 mM xylose could not transport glucose, and high capacity xylose transport was not inhibited by glucose or non-metabolizable glucose analogues. Cells grown with 5 mM xylose transported glucose at a rapid rate (30 nmol min−1 (mg protein)−1), and low capacity xylose uptake was competitively inhibited by either glucose or 2-deoxy-glucose. Because the glucose uptake of cells grown on 5 mM xylose was competitively inhibited by xylose, it appeared that the low capacity xylose uptake system was a glucose/xylose carrier.

Simultaneous fermentation and isomerization of xylose to ethanol at high xylose concentration

Simultaneous fermentation and isomerization of xylose to ethanol at high xylose concentration

Chemical compositions of the nitrogen containing gum tragacanth exudates from Asiatic Astragalus species, grown in North America

The gum exudates from four Asiatic Astragalus species, grown for the purpose of assessment in the USA, have been characterized chemically. The present analytical data extend those available previously for eight species, and confirm earlier indications that the gums from different Astragalus species vary widely in their chemical composition, physico-chemical parameters, solubility and amino acid composition. All species have high hydroxyproline and proline contents; some have high histidine and serine contents. In comparison with the lower viscosity exudates, the higher viscosity gums have high galacturonate, xylose, fucose and methoxyl contents and low arabinose and nitrogen contents. The data suggest that California and Arizona are suitable locations for gum tragacanth production.

A novel, highly thermostable d-xylanase

We have recently established that heat-treated aerobic sewage sludge is an excellent source of industrially useful microorganisms. 1 Samples of such sludge were grown on xylan media in enrichment cultivations at 65°C and we succeeded in isolating and identifying an extremely thermophilic Bacillus stearothermophilus- like strain which produces a highly thermostable xylanase (1,4-β- d-xylan xylanohydrolase, EC 3.2.1.8) which has a catalytic temperature optimum of 78°C at pH 7.5. The production of the xylanase is not directly associated to the cellular growth; larchwood xylan is a better inducer than oat-spelts xylan. No xylanase is obtained in presence of trace amounts of starch. The enzyme, when assayed in culture broth supernatants shows 65%of the optimum activity at the supraoptimum temperature of 90°C. It has a broad pH range of 5.5–8.5 and a half-life of 15 h at 75°C, whereas at 68°C it remains stable for more than 5 days. Kinetic analysis indicates that xylan as substrate stabilizes the enzyme against thermal inactivation. Estimated by the yield of reducing sugars, the enzyme degrades larchwood xylan to almost 50%. With thin-layer chromatography the degradation products can be identified as xylotriose, xylobiose and xylose, xylose appearing only after prolonged incubation periods. No other neutral xylanase with a similar high thermal stability and high degree of xylan digestion has been so far described. Due to its stability against thermal denaturation and the high xylose productivity, the enzyme could find technical applications such as large-sacle bioconversion processes at high temperature of xylan, a major component of wastes from the timber and farming industries .

Comparative evaluation of ethanol production by xylose-fermenting yeasts presented high xylose concentrations

Three strains ofPichiastipitis and three ofCandidashehatae were compared withPachysolentannophilus in their abilities to ferment xylose at concentrations as high as 200 g/L when subjected to both aerobic and microaerophilic conditions. Evaluations based on accumulated ethanol concentrations, ethanol productivities, xylose consumption, and ethanol and xylitol yields were determined from batch culture time courses. Of the strains considered,P.stipitis NRRL Y-7124 seemed most promising since it was able to utilize all but 7 g/L of 150 g/L xylose supplied aerobically to produce 52 g/L ethanol at a yield of 0.39 g per gram xylose (76% of theoretical yield) and at a rate comparable to the fastest shown byC.shehatae NRRL Y-12878. For all strains tested, fermentation results from aerobic cultures were more favorable than those from microaerophilic cultures.

Comparison between maize root cells and their respective regenerating protoplasts: wall polysaccharides

The cell-wall polysaccharides from different parts of maize roots have been analysed. The arabinose, galactose and mannose contents are influenced by cell differentiation, whereas xylose, rhamnose and uronic-acid contents are not. In cap cells, the pectin content is low but rhamnose and fucose are present in larger quantities. The cell-wall polysaccharides from cells of the elongation zone and their respective regenerating protoplasts were also analysed. The walls of the protoplasts contained higher xylose and mannose levels and a much lower level of cellulose than the cells from which they were derived.

Changes in the lignin and hemicellulose concentrations of ten varieties of temperate grasses with increasing maturity

AbstractThe lignin and total hemicellulose concentrations of ten varieties of temperate grasses (five species) were determined with increasing maturity. A strong correlation existed between the lignin and hemicellulose concentrations. Lignin and hemicellulose concentrations tended to be higher in cocksfoot and timothy than in perennial ryegrass and diploid varieties also had higher lignin and hemicellulose concentrations than their tetraploid counterparts. The hemicellulose fractions were further separated into their linear and branched components whose compositions were determined. The varieties with higher lignin concentrations showed a higher linear:branched ratio and the ‘linear’ components also had higher xylose: arabinose ratios. These findings were consistent for both leaf and stem tissue although the effects from stem tissue were more marked.

Suitability of monosaccharides as markers for particle identification in carbonate sediments*†

ABSTRACTThe identification of the remains of organisms contributing to carbonate sediments by means of scanning electron‐microscopy is limited to particles of the 2–20 μm size class. Mineralogy and the content of Mg, Sr and trace elements alone are usually insufficient to solve the problem of identification, especially in the differentiation between algal and coral aragonite. The organic matrix of calcareous organisms consists of stable biopolymers such as polysaccharides and glycoproteins which are intimately associated with the carbonate skeleton. Analysis of these hydrolysed compounds gives rise to characteristic arrays of monosaccharides which provide independent criteria for producer identification. The calcareous green algae Halimeda, Penicillus and Udotea show high xylose and low fucose levels. Xylose and fucose levels are elevated in the red algae Amphiroa but only fucose is prominent in the brown algae Padina. The corals Oculina, Porites, Millipora and Montastrea are relatively rich in fucose and show little or no xylose. In the bivalves Arca, Codakia and in Argopecten mannose may be characteristic. Analysis of artificial and natural sediments demonstrates that coral and algal aragonite can be distinguished on the basis of the total sugar concentration and respective xylose and fucose levels. The applicability of the technique in comparison to geochemical and mineralogical methods has been demonstrated for surface sediments from varying water depths of Harrington Sound, Bermuda.