Comparative structural studies of lignin-carbohydrate complexes from Digitaria decumbens (pangola grass) before and after chlorite delignification

Lignin-carbohydrate complexes (LCCs) were extracted with methyl sulphoxide from cellulase-digested stem cell-walls of mature pangola grass. Comparative studies, using gel filtration, g.l.c., and spectroscopy, were conducted concurrently on digested cell-walls which had also been treated with sodium chlorite. All the LCCs contained hemicellulose and protein with low contents of hydroxyproline. Gel filtration showed markedly increased polydispersity of the LCCs from chlorite-treated residues. Chlorite treatment caused loss of arabinose and galactose, with a concomitant increase in the proportion of xylose degradable by sodium metaperiodate. The LCCs from untreated cell-walls comprised polysaccharide, esterified p-coumaric acid, and a water-insoluble, lignified “hemicellulose A” component (6% carbohydrate) having a low xylose-to-arabinose ratio and a high proportion of hexoses. These components were liberated with alkali, as were, unexpectedly, some free xylose and arabinose. Chlorite-treated walls gave LCCs with ester bonds still present, but p-coumaric acid was degraded to lignin-like products, and a ”hemicellulose A“ xylan (94% carbohydrate) was isolated. The structural relationship of the components in the LCCs from untreated material was complex, and appeared to involve monomer and oligomer pentose units, diesterified at least, in bridging structures with p-coumaric acid. Xylose reducing-units also appeared to be liberated by alkali, suggesting esterification at C-1 in some xylan in the LCC. Thus, chlorite delignification causes significant structural changes in the polysaccharides and non-carbohydrate components of the cell walls, and it is suggested that chlorite treatment be omitted when in situ structural data on plant cell-walls are being sought.     

Composition of cell wall preparations of rice bran and germ

Cell walls of petrol-defatted non-waxy IR32 rice bran and germ were prepared by protein removal with 0.5% SDS—0.6% β-mercaptoethanol, heating the residue to 80°, and destarching with Bacillus licheniformis α-amylase. A waxy rice, IR29, had a similar cell wall composition as IR32. Principal wall sugars were arabinose, xylose, and glucose. The 0.5 M sodium or potassium hydroxide and 8 M urea preferentially extracted arabinose-, xylose- and uronic acid-rich polysaccharides but 6 M sodium hydroxide—0.81 M boric acid extracted mannose-rich polysaccharides. DEAE-cellulose BO₃^3? chromatography of the 0.5 M sodium hydroxide extracts gave fractions of similar arabinose— xylose ratios. Proteins in the cell wall preparations had only 0.4–1.6% hydroxyproline, and were bound mainly to polysaccharides, based on disc gel electrophoresis. The preparations were autofluorescent in UV and rich in phenols, mainly ferulic acid. The cell wall preparations and their 8 M urea fractions had a softening effect on defatted waxy starch aqueous gel at 0.2–2% of the starch.     

Water-soluble polysaccharides of Opuntia ficus-indica cv “Burbank's Spineless”

Carbohydrate-containing polymers have been extracted with water from the fleshy, lobed stems of Opuntia ficus-indica cv “Burbank's Spineless”. By ion exchange chromatography, the material was separated into one neutral and two acidic fractions. Each fraction was separated in two by gel filtration. The neutral fractions consisted of two glucans and a glycoprotein, containing arabinose and galactose. All four acidic fractions contained galacturonic acid, arabinose, rhamnose, galactose and xylose in different proportions. The cell wall structure of O. ficus-indica is discussed.     

Carbohydrate composition and physical properties of tobacco phosphodiesterase

Some physical and chemical properties of phosphodiesterase from cultured tobacco cells were studied. The enzyme contained ca 50% carbohydrate consisting of residues of arabinose, glucose, glucosamine, galactose, mannose and xylose. Analyses showed that the enzyme had a sedimentation coefficient, s_20,w of 16 S, a Stokes'radius of 5.7 nm and a calculated specific volume of 0.66 ml/g. The MW of the enzyme was calculated to be 300 000 from these values.     

Characterization and fouling properties of exopolysaccharide produced by Klebsiella oxytoca

Klebsiella oxytoca produced a type of exopolysaccharide (EPS) with the average molecular weight (Mw) of 116,018 Da and the average size of 260 nm. The EPS monosaccharide components contained rhamnose, fucose, arabinose, xylose, mannose, galactose and glucose and the molar ratio among them was 0.033:0.0411:0.0147:0.0051:0.2393:0.0986:0.1304. Typical EPS absorption peaks in FT-IR spectrum and pseudoplastic properties were also revealed. The polyvinylidenefluoride (PVDF) membrane showed a relatively larger flux decline resulted from the EPS fouling. The EPS filtration was dominated by more than one mechanism at the beginning phase and mainly by the cake formation at the later phase for both membranes. The pore blocking resistance had a predominant contribution to the filtration resistance and the cake resistance played a secondary role for both the membranes. The EPS adsorption resulted in a weak membrane fouling. The PVDF membrane exhibited a larger adsorption resistance than the polypropylene (PP) membrane.     

Arabinogalactans in a purified allergen preparation from pollen of timothy (Phleum pratense)

The purified allergen preparation representing a certain fraction of an aqueous timothy pollen extractcontained ca. 20% carbohydrate, mainly as arabinose (7%) and galactose (13%). The protein content was 63%. Fractionation on DEAE-Sephadex and Sephadex G-100 gave one neutral and two acidic fractions, all containing protein, arabinose and galactose. The structure of the carbohydrate moiety was investigated by methylation analysis, periodate oxidation and enzyme incubation. The acidic fraction contained (1→6)-linked galactose residues, some being substituted on O-3 with arabinose. The neutral fraction consisted of a more extensively branched arabinogalactan with longer side chains of (1→3)- and (1→5)-linked arabinose. The arabinose was present mainly as α-l-arabinofuranosyl residues. Alkaline degradation and subsequent fractionation indicated the presence of a covalent linkage between hydroxyproline and arabinose. Periodate oxidation or incubation with α-l-arabinofuranosidase did not affect the allergenic activity of the extract.     

Analytical and structural features of the gum exudate from Combretum hartmannianum schweinf.

The gum exudate from Combretum hartmannianum is water-soluble, forms very viscous solutions, and contains galactose (22%), arabinose (43%), mannose (10%), xylose (6%), rhamnose (4%), glucuronic acid (6%), 4-O-methylglucuronic acid (2%), and galacturonic acid (7%). The acidic components produced on hydrolysis of the gum were 6-O-(β-D-glucopyranosyluronic acid)-D-galactose, and two saccharides that had the same chromatographic mobility, and contained mannose and galacturonic acid, and galactose and 4-O-methylglucuronic acid, respectively. Methylation and methanolysis of the gum indicated the presence of terminal uronic acid, rhamnose, xylose, galactose, arabinofuranose, and arabinopyranose. Controlled, acid hydrolysis indicated the presence of (1→3)-linked arabinopyranose side-chains and (1→6)-linked galactose residues. C. hartmannianum gum, when subjected to two Smith-degradations, yielded Polysaccharides I and II, both of which contained galactose, arabinose, and mannose. Insufficient crude gum was available for a complete structural study, but the molecule was shown to contain long, sparsely branched chains of (1→6)-linked galactose residues, to which are attached (1→3)-linked arabinose and (1→3)-linked mannose side-chains.     

Solution structures of ferrihaem in some dipolar aprotic solvents and their binary aqueous mixtures

1. Conductivity and u.v. and visible spectroscopic techniques were used to investigate the solution structure of the prosthetic group of the ferric haemoproteins (ferrihaem) in dimethyl sulphoxide, NN-dimethylacetamide, NN-dimethylformamide and sulpholane, and certain of their aqueous mixtures. 2. In neutral or acid dimethyl sulphoxide, chlorohaemin is monomeric and completely dissociated into Cl⁻ion and a ferrihaem species with dimethyl sulphoxide molecules in the fifth and sixth co-ordination positions on iron. 3. In neutral NN-dimethylacetamide and NN-dimethylformamide chlorohaemin is monomeric but is largely undissociated, giving different spectra from that of chlorohaemin in dimethyl sulphoxide. On acidification, dissociation occurs and the dimethyl sulphoxide type of spectrum results. 4. Studies in a fourth solvent, sulpholane, indicate that solvent co-ordinating power (ligand strength) rather than bulk dielectric constant is responsible for dissociation of chlorohaemin. 5. In neutral dimethyl sulphoxide–water mixtures chlorohaemin remains monomeric and completely dissociated, and spectra are independent of mixture composition, except at high water concentrations, when precipitation occurs. In alkaline dimethyl sulphoxide–water mixtures, where the complete solvent mixture range is accessible, ferrihaem is polymeric (probably dimeric) and spectra are dependent on solvent composition. A quantitative analysis indicates that the spectral changes are due to replacement by water of one molecule of co-ordinated dimethyl sulphoxide per ferrihaem aggregate, and do not involve a two-molecule replacement as has been suggested for the alkaline pyridine–water system.     

Composition of ethanol-insoluble polysaccharides in water extracts of ripening tomatoes

The carbohydrate composition of the 80% ethanol-insoluble polysaccharides (EIP) from water extracts of ‘Rutgers,’ rin (ripening inhibitor) and nor (non-ripening) tomatoes has been determined. The amount of EIP extracted from ‘Rutgers’ fruit increased from 0.34 to 0.61 mg/g fr. wt during ripening little change occurred in rin or nor fruit. The carbohydrate composition (μg/g fr. wt) of EIP from mature green fruit was: galacturonic acid (48); rhamnose (3); arabinose (20); xylose (48); mannose (31); glucose (139); galactose (51). The most obvious changes that accompanied ripening were a 7.4-fold and 4-fold increase in galacturonic acid and rhamnose content, respectively. These changes were attenuated in the ripening mutants. EIP was fractionated into three major peaks by using DEAE-cellulose ion exchange chromatography. The first peak, which was not retained by the column, contained predominantly glucose and mannose, with lower amounts of galacturonic acid and galactose. The two retained peaks which eluted at 0.1 and 0.2 M sodium chloride contained primarily galacturonic acid, xylose, galactose and arabinose. The galacturonic acid content of these two fractions increased substantially during ripening, whereas the other components decreased. No changes were evident in the ripening mutants. No increase in water-soluble polysaccharides high in galactose content was observed during ripening.     

Production of carbohydrates by the marine diatom Chaetoceros affinis var. willei (Gran) Hustedt. II. Preliminary investigation of the extracellular polysaccharide

The isolation of an extracellular polysaccharide from cultures of Chaetoceros affinis var. willei (Gran) Hustedt is described. The polysaccharide behaved as a homogeneous, polyanionic compound in free-boundary electrophoresis at both pH 2 and 7. It contained sulphur, presumably as sulphate half ester groups (8.7% of SO₂Na), and the following monosaccharides were tentatively identified: rhamnose, fucose, arabinose, and galactose, with the two former constituting 63% of the polysaccharide preparation. The main cellular polysaccharide was a glucan and could be extracted from the cells by dilute acid. The remaining material gave, after hydrolysis, a complex mixture of monosaccharides with rhamnose as the major component. It is concluded that the extracellular polysaccharide is probably excreted from healthy cells.     

Structural investigations on the lignin–carbohydrate complexes of Lolium perenne

1. Lignin-carbohydrate complexes isolated from leaf blade, leaf sheath and stem tissue of ryegrass by extraction with dimethyl sulphoxide were examined by fractionation procedures. Although the complexes are heterogeneous, heterogeneity is shown only in the ratio of the individual monosaccharide residues and not in the ratio of lignin to carbohydrate. 2. The molecular weight of the complexes is high (>/=150000), but chemical modification by alkaline hydrolysis, borohydride reduction or lead tetra-acetate oxidation does not drastically decrease it. Low-molecular-weight fragments released by alkaline treatment were shown to contain acetic acid, ferulic acid and p-coumaric acid. 3. On the basis of the chemical stability of the complexes, it is postulated that at least three types of bonding may be present between lignin and carbohydrate, namely one cleaved on borohydride reduction, another cleaved by alkali and a linkage resistant to alkali. 4. The carbohydrate portion of the complexes is composed of beta-(1-->4)-linked d-glucose residues (cellulose) and beta-(1-->4)-linked chains of xylose residues. Side chains involving arabinose and galactose residues are linked to C-3 of some of the xylose residues. 5. How the components of the complexes are held together is not certain, but it is suggested that the phenolic acids may act as cross-linking agents.     

Apple fruit pectic substances

1. The pectic substances of apple have been extracted and separated into a pure pectinic acid and a neutral arabinan–galactan complex by precipitation of the acidic component with ethanol and with cetylpyridinium chloride. 2. The composition of the fractions has been determined. The pectinic acid contained galacturonic acid, arabinose, galactose, rhamnose, xylose and several trace sugars. 3. Transelimination degradation of the pectinic acid gave rise to two components completely separable by zone electrophoresis and by Sephadex gel filtration. Analysis of these components confirmed that the pectinic acid molecules contained long chains of esterified galacturonosyl residues, but showed in addition that more neutral portions containing a high proportion of arabinofuranose residues were attached to them. 4. The identification of rhamnose, galactose and xylose in aldobiouronic acids obtained from a partial hydrolysate of pectinic acid has shown that these sugars are covalently linked in the molecule, and it is suggested that the galacturonosyl-(1→2)-rhamnose link is a general feature of pectinic acid structure. 5. The possible biological significance of pectinic acid structure has been discussed. 6. The arabinan–galactan complex contained nearly equal quantities of arabinose and galactose residues and some of its physical properties have been investigated.     

Comparing the xylose reductase/xylitol dehydrogenase and xylose isomerase pathways in arabinose and xylose fermenting Saccharomyces cerevisiae strains

Background

Ethanolic fermentation of lignocellulosic biomass is a sustainable option for the production of bioethanol. This process would greatly benefit from recombinant Saccharomyces cerevisiae strains also able to ferment, besides the hexose sugar fraction, the pentose sugars, arabinose and xylose. Different pathways can be introduced in S. cerevisiae to provide arabinose and xylose utilisation. In this study, the bacterial arabinose isomerase pathway was combined with two different xylose utilisation pathways: the xylose reductase/xylitol dehydrogenase and xylose isomerase pathways, respectively, in genetically identical strains. The strains were compared with respect to aerobic growth in arabinose and xylose batch culture and in anaerobic batch fermentation of a mixture of glucose, arabinose and xylose.

Results

The specific aerobic arabinose growth rate was identical, 0.03 h^-1, for the xylose reductase/xylitol dehydrogenase and xylose isomerase strain. The xylose reductase/xylitol dehydrogenase strain displayed higher aerobic growth rate on xylose, 0.14 h^-1, and higher specific xylose consumption rate in anaerobic batch fermentation, 0.09 g (g cells)^-1 h^-1 than the xylose isomerase strain, which only reached 0.03 h^-1 and 0.02 g (g cells)^-1h^-1, respectively. Whereas the xylose reductase/xylitol dehydrogenase strain produced higher ethanol yield on total sugars, 0.23 g g^-1 compared with 0.18 g g^-1 for the xylose isomerase strain, the xylose isomerase strain achieved higher ethanol yield on consumed sugars, 0.41 g g^-1 compared with 0.32 g g^-1 for the xylose reductase/xylitol dehydrogenase strain. Anaerobic fermentation of a mixture of glucose, arabinose and xylose resulted in higher final ethanol concentration, 14.7 g l^-1 for the xylose reductase/xylitol dehydrogenase strain compared with 11.8 g l^-1 for the xylose isomerase strain, and in higher specific ethanol productivity, 0.024 g (g cells)^-1 h^-1 compared with 0.01 g (g cells)^-1 h^-1 for the xylose reductase/xylitol dehydrogenase strain and the xylose isomerase strain, respectively.

Conclusion

The combination of the xylose reductase/xylitol dehydrogenase pathway and the bacterial arabinose isomerase pathway resulted in both higher pentose sugar uptake and higher overall ethanol production than the combination of the xylose isomerase pathway and the bacterial arabinose isomerase pathway. Moreover, the flux through the bacterial arabinose pathway did not increase when combined with the xylose isomerase pathway. This suggests that the low activity of the bacterial arabinose pathway cannot be ascribed to arabitol formation via the xylose reductase enzyme.     

Water-soluble polysaccharides of antarctic and cultured Phormidium species of the cyanophyceae

Hot water extraction of a Phormidium species from Antarctica and of a sample of Phormïdium foveolarum which had been cultured axenically led to the isolation of a water-soluble polysaccharide from both materials. Acidic hydrolysis of each gave a similar pattern of monosaccharides comprising arabinose, xylose, rhamnose, fucose, galactose, mannose and glucose, and both contained uronic acid. All attempts by a variety of methods to fractionate the Antarctic polysaccharide into more than a single entity were unsuccessful. Periodate oxidation, partial hydrolysis and methylation studies on this polysaccharide supported a highly branched molecule with 1,3-linked glucose and 1,4-linked galactose as dominant features.     

Comparison of cell wall compositions of a desert xerophyte and a related mesophyte

A comparison was made of the cell wall compositions of stem internode tissues from two members of the Chenopodiaceae. Cell walls from Anabasis syriaca (a desert xerophyte) contained non-cellulosic polysaccharides rich in arabinose, xylose and galacturonic acid. The non-cellulosic polysaccharides from cell walls of Spinacia oleracea (a mesophyte) were rich in glucose. Anabasis syriaca cell walls contained relatively more cellulose and lignin than those of Spinacia oleracea.     

Efficiencies of designed enzyme combinations in releasing arabinose and xylose from wheat arabinoxylan in an industrial ethanol fermentation residue

The generation of a fermentable hydrolysate from arabinoxylan is an important prerequisite for utilization of wheat hemicellulose in production of ethanol or other value added products. This study examined the individual and combined efficiencies of four selected, commercial, multicomponent enzyme preparations Celluclast 1.5 L (from Trichoderma reesei), Finizym (from Aspergillus niger), Ultraflo L (from Humicola insolens), and Viscozyme L (from Aspergillus aculeatus) in catalyzing arabinose and xylose release from water-soluble wheat arabinoxylan in an industrial fermentation residue (still bottoms) in lab scale experiments. Different reaction conditions, i.e. enzyme dosage, reaction time, pH, and temperature, were evaluated in response surface and ternary mixture designs. Ultraflo L treatment gave optimal arabinose release: treatment (6 h, 60 °C, pH 6) with this enzyme preparation liberated up to 46% by weight (wt.%) of the theoretically maximal arabinose yield from the substrate. Celluclast 1.5 L was superior to the other enzyme preparations in releasing xylose and catalyzed release of up to 25 wt.% of the theoretical maximum xylose yield (6 h, 60 °C, pH 4). Prolonged treatment for 24 h with a 50:50 mixture of Celluclast 1.5 L and Ultraflo L at 50 °C, pH 5 exhibited a synergistic effect in xylose release and 62 wt.% of the theoretically maximal xylose yield was achieved. Addition of pure β-xylosidase from T. reesei to the Ultraflo L preparation released the same amounts of xylose from the substrate as the 50:50 mixture of Celluclast 1.5 L and Ultraflo L. The data thus signified that the synergistic effect in xylose release between Celluclast 1.5 L and Ultraflo L is the result of a three-step interaction mechanism involving α-l-arabinofuranosidase and different xylan degrading enzyme activities in the two enzyme preparations.     

Techno-economic evaluation of stillage treatment with anaerobic digestion in a softwood-to-ethanol process

Background

Cost-effective fermentation of lignocellulosic hydrolysate to ethanol by Saccharomyces cerevisiae requires efficient mixed sugar utilization. Notably, the rate and yield of xylose and arabinose co-fermentation to ethanol must be enhanced.

Results

Evolutionary engineering was used to improve the simultaneous conversion of xylose and arabinose to ethanol in a recombinant industrial Saccharomyces cerevisiae strain carrying the heterologous genes for xylose and arabinose utilization pathways integrated in the genome. The evolved strain TMB3130 displayed an increased consumption rate of xylose and arabinose under aerobic and anaerobic conditions. Improved anaerobic ethanol production was achieved at the expense of xylitol and glycerol but arabinose was almost stoichiometrically converted to arabitol. Further characterization of the strain indicated that the selection pressure during prolonged continuous culture in xylose and arabinose medium resulted in the improved transport of xylose and arabinose as well as increased levels of the enzymes from the introduced fungal xylose pathway. No mutation was found in any of the genes from the pentose converting pathways.

Conclusion

To the best of our knowledge, this is the first report that characterizes the molecular mechanisms for improved mixed-pentose utilization obtained by evolutionary engineering of a recombinant S. cerevisiae strain. Increased transport of pentoses and increased activities of xylose converting enzymes contributed to the improved phenotype.     

Purification,properties, and mode of action of hemicellulase II produced by Ceratocystis paradoxa

An extra-cellular endo-hemicellulase (HC-II) from a culture isolate of the fungal plant pathogen Ceratocystis paradoxa (CP₂) was purified 147-fold by ammonium sulphate precipitation, DEAE-Sephadex chromatography, iso-electric focusing at pH 3–10, and gel-permeation chromatography. The resulting enzyme preparation, which contained traces of invertase, gave a single protein-band on disc electrophoresis at pH 8.4, and was active towards sucrose, hemicellulose, and carboxymethylcellulose (CMC). HC-II randomly degraded hemicelluloses from several different sources, to xylose and to arabinose-xylose and xylose oligosaccharides of d.p. 3–6 and 2–5, respectively, and also produced a degraded hemicellulose which precipitated from the digest solution. The precipitated hemicellulose contained less arabinose and uronic acid than the original hemicellulose. When redissolved by alkali-treatment, it was susceptible to further degradation by hemicellulases HC-I and HC-II. CMC was degraded by HC-II, mainly to D-glucose and cellobiose, with trace amounts of unidentified higher oligosaccharides, while cellobiose remained unattacked. Xylotriose (Xyl₃) was the lowest homologue of the xylose oligosaccharides attacked by HC-II at a significant rate, yielding xylobiose [Xyl₂; β-D-Xylp-(1→4)-D-Xyl] and xylose. AraXyl₃AraXyl₅ were mainly hydrolysed to AraXyl₂, xylose, and Xyl₂ or Xyl₃. HC-II had a temperature optimum of 80°, and was stable for 1 h at temperatures up to 70°. The pH optimum was 5.1, and HC-II was stable between pH 5–10. The K_m was 0.267 mg of hemicellulose B/ml. The effects of mercury(II) ions and high concentrations of xylose on the activity of HC-II were also investigated.     

Continuous Ethanol Fermentation of Pretreated Lignocellulosic Biomasses,Waste Biomasses,Molasses and Syrup Using the Anaerobic,Thermophilic Bacterium Thermoanaerobacter italicus Pentocrobe 411

Lignocellosic ethanol production is now at a stage where commercial or semi-commercial plants are coming online and, provided cost effective production can be achieved, lignocellulosic ethanol will become an important part of the world bio economy. However, challenges are still to be overcome throughout the process and particularly for the fermentation of the complex sugar mixtures resulting from the hydrolysis of hemicellulose. Here we describe the continuous fermentation of glucose, xylose and arabinose from non-detoxified pretreated wheat straw, birch, corn cob, sugar cane bagasse, cardboard, mixed bio waste, oil palm empty fruit bunch and frond, sugar cane syrup and sugar cane molasses using the anaerobic, thermophilic bacterium Thermoanaerobacter Pentocrobe 411. All fermentations resulted in close to maximum theoretical ethanol yields of 0.47–0.49 g/g (based on glucose, xylose, and arabinose), volumetric ethanol productivities of 1.2–2.7 g/L/h and a total sugar conversion of 90–99% including glucose, xylose and arabinose. The results solidify the potential of Thermoanaerobacter strains as candidates for lignocellulose bioconversion.