Growth of Neocallimastix sp. Strain R1 on Italian Ryegrass Hay: Removal of Neutral Sugars from Plant Cell Walls

The anaerobic fungus Neocallimastix sp. strain R1 was grown for up to 5 days on a medium containing autoclaved Italian ryegrass hay as the carbon source. Culture supernatants and digested cell walls were harvested at 12-h intervals. Supernatants were analyzed for the fermentation products formate and acetate, and residual cell walls were analyzed for dry-matter and neutral-sugar losses. Fungal growth was accompanied by the digestion of plant cell walls and the accumulation of fermentation products in culture media. Dry-matter losses were accounted for by removal of four major neutral sugars (arabinose, galactose, glucose, and xylose) from the plant cell walls. First-order reaction kinetics could be used to describe the loss of each sugar. All cell wall sugars, including arabinose and galactose, which are not fermented by Neocallimastix sp. strain R1 were removed simultaneously. Although the rates of removal of individual sugars were similar, there were significant differences in their extents of removal: the extent of removal of arabinose exceeded that of the other three sugars, and xylose was the least digestible. This study provides the first account of simultaneous (nonpreferential) removal of neutral sugars from plant cell walls by an anaerobic fungus. Although in vitro techniques were used, these results indicate a potentially significant role for the anaerobic fungi as fiber digesters in the rumen.     

Changes in cell wall neutral sugar composition during fruit ripening: a species survey

Non-cellulosic neutral sugar composition of cell walls from seventeen fruit types were analysed during ripening. Galactose was the major non-cellulosic neutral sugar in cell walls of cucurbit and solanaceous fruit, xylose was the predominant non-cellulosic neutral component of berries, and arabinose was the major non-cellulosic component of pome fruits. The major non-cellulosic neutral sugar residue in cell walls of stone fruits varied. In nectarine and peach, plum, and apricot, the major sugar was arabinose, galactose, and xylose, respectively. In 15 of the 17 types of fruit, a net loss of non-cellulosic neutral sugar residues occurred during ripening. No net loss occurred in plums and cucumbers. A net loss of cell wall galactose and/or arabinose occurred in 14 of the types of fruit. Xylose was the major neutral sugar residue lost from walls of apricot during ripening. In general, berry cell walls were comparatively low in galactose and arabinose content.     

Changes in cell wall polysaccharides associated with growth

Changes in the polysaccharide composition of Phaseolus vulgaris, P. aureus, and Zea mays cell walls were studied during the first 28 days of seedling development using a gas chromatographic method for the analysis of neutral sugars. Acid hydrolysis of cell wall material from young tissues liberates rhamnose, fucose, arabinose, xylose, mannose, galactose, and glucose which collectively can account for as much as 70% of the dry weight of the wall. Mature walls in fully expanded tissues of these same plants contain less of these constituents (10%-20% of dry wt). Gross differences are observed between developmental patterns of the cell wall in the various parts of a seedling, such as root, stem, and leaf. The general patterns of wall polysaccharide composition change, however, are similar for analogous organs among the varieties of a species. Small but significant differences in the rates of change in sugar composition were detected between varieties of the same species which exhibited different growth patterns. The cell walls of species which are further removed phylogenetically exhibit even more dissimilar developmental patterns. The results demonstrate the dynamic nature of the cell wall during growth as well as the quantitative and qualitative exactness with which the biosynthesis of plant cell walls is regulated.     

Soluble Cell Wall Polysaccharides Released from Pea Stems by Centrifugation : II. EFFECT OF ETHYLENE

The effect of ethylene on cell wall metabolism in sections excised from etiolated pea stems was studied. Ethylene causes an inhibition of elongation and a pronounced radial expansion of pea internodes as shown by an increase in the fresh weight of excised, 1-cm sections. Cell wall metabolism was studied using centrifugation to remove the cell wall solution from sections. The principal neutral sugars in the cell wall solution extracted with H₂O are arabinose, xylose, galactose, and glucose. Both xylose and glucose decline relative to controls in air within 1 hour of exposure to ethylene. Arabinose and galactose levels are not altered by ethylene until 8 hours of treatment, whereupon they decline in controls in air relative to ethylene treatment. When alcohol-insoluble polymers are fractionated into neutral and acidic polysaccharides, xylose and glucose predominate in the neutral fraction and arabinose and galactose in the acidic fraction. Ethylene depresses the levels of xylose and glucose in the neutral fraction and elevates arabinose and galactose in the acidic fraction. Ethylene treatment does not affect the level of uronic acids extracted with H₂O; however, the level of hydroxyproline-rich proteins in this water-extracted cell wall solution is increased by ethylene. Extraction of sections with CaCl₂ results in an increase in the levels of neutral sugars particularly arabinose. Ethylene depresses the yield of arabinose in calcium-extracted solution relative to controls in air. Similarly, extraction with CaCl₂ increases the yield of extracted hydroxyproline in ethanol-insoluble polymers and ethylene depresses its level relative to controls. Metabolism of uronic acids and neutral sugars and growth in response to ethylene treatment contrast markedly with auxin-induced polysaccharide metabolism and growth. With auxin, sections increase mostly in length not radius, and this growth form is associated with an increase in the levels of xylose, glucose, and uronic acids. With ethylene, on the other hand, stem elongation is suppressed and expansion is promoted, and this growth pattern is associated with a decrease in xylose and glucose in the ethanol-insoluble polysaccharides.     

Degradation of Cell Wall Polysaccharides during Tomato Fruit Ripening

Changes in neutral sugar, uronic acid, and protein content of tomato (Lycopersicon esculentum Mill) cell walls during ripening were characterized. The only components to decline in amount were galactose, arabinose, and galacturonic acid. Isolated cell walls of ripening fruit contained a water-soluble polyuronide, possibly a product of in vivo polygalacturonase action. This polyuronide and the one obtained by incubating walls from mature green fruit with tomato polygalacturonase contained relatively much less neutral sugar than did intact cell walls. The ripening-related decline in galactose and arabinose content appeared to be separate from polyuronide solubilization. In the rin mutant, the postharvest loss of these neutral sugars occurred in the absence of polygalacturonase and polyuronide solubilization. The enzyme(s) responsible for the removal of galactose and arabinose was not identified; a tomato cell wall polysaccharide containing galactose and arabinose (6:1) was not hydrolyzed by tomato β-galactosidase.     

Effect of cocultivation of ruminal chytrid fungi with Methanobrevibacter smithii on lucerne stem degradation and extracellular fungal enzyme activities

Pentoses and hexoses were removed simultaneously from lucerne ( Medicago sativa ) stem by Piromonas communis and Neocallimastix frontalis growing axenically or in coculture with Methanobrevibacter smithii . In monocultures, more arabinose (68–71%) than glucose (52–53%) or xylose (25–30%) was removed. In cocultures, xylose solubilization from cell walls increased for P. communis whereas glucose solubilization increased for both fungi. The rates of sugar removal were monosaccharide-dependent. For both fungi, the specific activities of extracellular hydrolytic enzymes were markedly elevated in methanogenic cocultures.     

中性糖在土壤中的来源与分布特征

糖类(即碳水化合物)是土壤有机质的重要组成部分, 经生物化学降解形成不同结构的单糖。土壤中的中性单糖也叫中性糖, 主要包括木糖、核糖、阿拉伯糖、葡萄糖、半乳糖、甘露糖、岩藻糖和鼠李糖。其中, 植物来源的糖主要为五碳糖, 如木糖和阿拉伯糖; 微生物来源的糖主要包括半乳糖、甘露糖、岩藻糖、鼠李糖等六碳糖。研究中常利用六碳糖和五碳糖的比例指示微生物和植物对土壤有机碳的相对贡献。中性糖是微生物重要的碳源和能量来源, 在团聚体的形成过程中扮演着重要角色。该文整合了近30年土壤中性糖的研究进展, 对比了提取中性糖的常用方法, 分析了不同土地利用类型和不同土壤组分中中性糖的含量、来源和周转特征, 综述了影响中性糖含量和分布的主要环境因素。结果表明, 中性糖在耕地土壤中的绝对含量和相对含量均显著低于针叶林、阔叶林、草地和灌丛4种土地利用类型。(半乳糖+甘露糖)/(阿拉伯糖+木糖)(GM/AX)在不同土地利用间差异不显著, 而(鼠李糖+岩藻糖)/(阿拉伯糖+木糖)(RF/AX)则表明草地土壤中的微生物来源的中性糖含量高于针叶林和耕地。不同密度的土壤组分中, 轻质组分中中性糖的含量比重质组分高, 重质组分中微生物来源的中性糖较多; 就不同粒径(或团聚体)而言, 黏粒(或微团聚体)中微生物来源的中性糖含量更丰富。有关影响土壤中性糖含量和分布的因素的研究, 目前主要集中在人为活动(如耕种和放牧等), 而有关温度、降水等自然环境因素影响的研究较少。     

Chemical Studies on the Cell Walls of Leptospira biflexa Strain Urawa and Treponema pallidum Strain Reiter

The preparation and chemical poperties of the cell walls of Leptospira biflexa Urawa and Treponema pallidum Reiter are described. Both cell walls are composed mainly of polysaccharides and peptidoglycans. The data of chemical analysis indicate that the cell wall of L. biflexa Urawa contains rhamnose, arabinose, xylose, mannose, galactose, glucose and unidentified sugars as neutral sugars, and alanine, glutamic acid, α,ε-diaminopimelic acid, glucosamine and muramic acid as major amino acids and amino sugars. As major chemical constituents of the cell wall of T. pallidum Reiter, rhamnose, arabinose, xylose, mannose, galactose, glucose, alanine, glutamic acid, ornithine, glycine, glucosamine and muramic acid have been detected. The chemical properties of protein and polysaccharide fractions prepared from the cells of T. pallidum Reiter were also partially examined.     

Soluble Cell Wall Polysaccharides Released from Pea Stems by Centrifugation : I. EFFECT OF AUXIN

The metabolism of polysaccharides by pea stem segments treated with and without auxin was investigated using a centrifugation technique for removing solution from the free space of the cell wall. Glucose is the predominant sugar in both the ethanol-soluble and ethanol-insoluble fractions of the cell wall solution extracted with water. In the water-soluble, ethanol-insoluble polysaccharides, arabinose, xylose, galactose, and glucose make up 9.5, 23.8, 23.9, and 39.9%, respectively, of the neutral sugars, while rhamnose, fucose, and mannose are present at concentrations between 0.5 and 2.0%.     

Effect of Auxin on Autolysis of Cell Walls in Azuki Bean Epicotyls

Native cell walls of azuki bean epicotyls incubated in bufferautolytically released neutral sugars, abundant in galactose,and uronic acids. Treatment with 10^–5 M IAA of subapicalor basal epicotyl segments for 3 h did not influence the amountof total neutral sugars released from the cell walls duringautolysis. However, the amount of glucose and xylose releasedfrom subapical cell walls was increased by IAA. Pretreatmentwith IAA of subapical epicotyl segments enhanced the solubilizationof neutral sugars from pectinase-treated cell walls during incubationin buffer at pH 5 to 6. The amount of fucose, xylose, and glucosereleased was specifically increased by IAA. Of the sugar fractionsreleased from pectinase-treated cell walls during autolysisand subsequently separated by gel filtration on a ToyopearlHW-40S column, IAA promoted the release of oligosaccharides,consisting mainly of glucose and xylose. These results suggestthat autolytic degradation of xyloglucans is closely relatedto IAA-induced growth of azuki bean epicotyls. (Received May 19, 1989; Accepted January 5, 1990)     

Fermentation of corn fibre sugars by an engineered xylose utilizing Saccharomyces yeast strain

The ability of a recombinant Saccharomyces yeast strain to ferment the sugars glucose, xylose, arabinose and galactose which are the predominant monosaccharides found in corn fibre hydrolysates has been examined. Saccharomyces strain 1400 (pLNH32) was genetically engineered to ferment xylose by expressing genes encoding a xylose reductase, a xylitol dehydrogenase and a xylulose kinase. The recombinant efficiently fermented xylose alone or in the presence of glucose. Xylose-grown cultures had very little difference in xylitol accumulation, with only 4 to 5g/l accumulating, in aerobic, micro-aerated and anaerobic conditions. Highest production of ethanol with all sugars was achieved under anaerobic conditions. From a mixture of glucose (80g/l) and xylose (40g/l), this strain produced 52g/l ethanol, equivalent to 85% of theoretical yield, in less than 24h. Using a mixture of glucose (31g/l), xylose (15.2g/l), arabinose (10.5g/l) and galactose (2g/l), all of the sugars except arabinose were consumed in 24h with an accumulation of 22g ethanol/l, a 90% yield (excluding the arabinose in the calculation since it is not fermented). Approximately 98% theoretical yield, or 21g ethanol/l, was achieved using an enzymatic hydrolysate of ammonia fibre exploded corn fibre containing an estimated 47.0g mixed sugars/l. In all mixed sugar fermentations, less than 25% arabinose was consumed and converted into arabitol.     

Comparison of carbohydrate substrate preferences in eight species of bifidobacteria

Eight species of bifidobacteria were tested for their abilities to grow on a range of monosaccharides (glucose, arabinose, xylose, galactose and mannose). In contrast to the other sugars, glucose and galactose were utilized by all species and, in general, specific growth rates were highest on these sugars. Different substrate preferences were observed between species when the bacteria were grown in the presence of all five monosaccharides. For example, glucose and xylose were coutilized by Bifidobacterium longum, whereas glucose repressed uptake of all other sugars in B. bifidum and B. catenulatum. Galactose was the preferred substrate with B. pseudolongum. In B. angulatum, glucose and galactose were utilized simultaneously. B. breve did not grow on arabinose when this sugar provided the sole source of energy. However, glucose and arabinose were preferentially taken up during growth on sugar mixtures.     

Effect of IAA on Growth and Soluble Cell Wall Polysaccharides Centrifuged from Pine Hypocotyl Sections

Auxin-induced elongation and cell wall polysaccharide metabolism were studied in excised hypocotyl sections of ponderosa pine (Pinus ponderosa) seedlings. Sections excised from hypocotyls of ponderosa pine elongate in response to the addition of auxin. The neutral sugar composition of the extracellular solution removed from hypocotyl sections by centrifugation was examined. In cell wall solution from freshly excised sections, glucose, galactose, xylose, and arabinose make up more than 90% of the neutral sugars, while rhamnose, fucose, and mannose are relatively minor components. The neutral sugar composition of the polysaccharides of the pine cell wall solution is both qualitatively and quantitatively similar to that of pea. Following auxin treatment of pine hypocotyls, the neutral sugar composition of the cell wall changes; glucose, xylose, rhamnose, and fucose increase by nearly 2-fold relative to controls in buffer without auxin. These changes in neutral sugars in response to auxin treatment are similar to those found in pea, with the exception that in pea, rhamnose levels decline in response to auxin treatment.     

Fermentation of Maize Bran, Oat Bran, and Wheat Bran by Bacteroides ovatus V975

Bacteroides ovatus is a Gram-negative obligate anaerobe that was isolated from the human colon and is capable of utilizing xylan. The objective of this study was to evaluate the ability of B. ovatus V975 to digest maize bran, oat bran, and wheat bran as well as the isolated cell walls from each bran source. Strain V975 was incubated in basal medium that contained either 0.1 or 0.3 g of each bran or each bran cell wall for 0, 24, 48, and 72 h. Acetate and succinate were the main products detected from each fermentation; however, less of each end product was produced from the isolated cell walls than from each bran. More of the oat bran was digested (in vitro dry matter disappearance = 74.8%) during the 72 h incubation than any other bran source. While each bran contained arabinose and xylose, more glucose, galactose, and mannose were utilized by strain V975 during the 72-h incubation than either pentose sugar. Compared with each bran, the bran cell walls had lower concentrations of most sugars, and more glucose than any other sugar was utilized by strain V975. These results suggest that strain V975 preferentially utilizes glucose, galactose, and mannose in each bran, while glucose is the main sugar fermented in bran cell walls. Received: 19 June 1997 / Accepted: 31 July 1997     

Enzymic analysis of cell wall structure in apple fruit cortical tissue

Galactanase from Phytophthora infestans and an arabinosidase isoenzyme from Sclerotinia fructigena attacked the cortical cell walls of apple fruits liberating galactose and arabinose residues, respectively. Other arabinosidase isoenzymes from S. fructigena attacked cell walls very slowly. A S. fructigena polygalacturonase isoenzyme liberated half of the uronic acid residues with few associated neutral residues, while a second polygalacturonase isoenzyme released more uronic acid with a substantial proportion of arabinose and galactose and lesser amounts of xylose, rhamnose and glucose; reaction products of this enzyme could be further degraded by the first isoenzyme to give high MW fragments, rich in arabinose with most of the xylose, rhamnose and glucose, and low MW fragments rich in galactose and uronic acid. Endoglucanase from Trichoderma viride released a small proportion of the glucose residues from cell walls together with uronic acid, arabinose, xylose and galactose; more extensive degradation occurred if walls were pre-treated with the second polygalacturonase isoenzyme. Endoglucanase reaction products were separated into a high MW fraction, rich in arabinose, and lower MW fractions rich in galactose and glucose residues. The high MW polygalacturonase and endoglucanase products could be degraded with an arabinosidase isoenzyme to release about 75% of their arabinose. Cell walls from ripe fruit showed similar susceptibility to arabinosidase and galactanase to those from unripe apples. Cell walls from fruit, ripened detached from the tree were more susceptible to degradation by polygalacturonase than walls from unripe fruit or fruit ripened on the tree. Endoglucanase released less carbohydrate from ripe fruit cell walls than from unripe fruit cell walls.     

Characterization of sugar mixtures utilization by an Escherichia coli mutant devoid of the phosphotransferase system

Due to catabolite repression in microorganisms, sugar mixtures cannot be metabolized in a rapid and efficient manner. Therefore, the development of mutant strains that avoid this regulatory system is of special interest to fermentation processes. In the present study, the utilization of sugar mixtures by an Escherichia coli mutant strain devoid of the phosphotransferase system (PTS) was characterized. This mutant can transport glucose (PTS- Glucose+ phenotype) by a non-PTS mechanism as rapidly as its wild-type parental strain. In cultures grown in minimal medium supplemented with glucose-xylose or glucose-arabinose mixtures, glucose repressed arabinose- or xylose-utilization in the wild-type strain. However, under the same culture conditions with the PTS- Glucose+ mutant, glucose and arabinose were co-metabolized, but glucose still exerted a partial repressive effect on xylose consumption. In cultures growing with a triple mixture of glucose-arabinose-xylose, the wild-type strain sequentially utilized glucose, arabinose and finally, xylose. In contrast, the PTS- Glucose+ strain co-metabolized glucose and arabinose, whereas xylose was utilized after glucose-arabinose depletion. As a result of glucose-arabinose co-metabolism, the PTS- Glucose+ strain consumed the total amount of sugars contained in the culture medium 16% faster than the wild-type strain. [14C]-Xylose uptake experiments showed that in the PTS- Glucose+ strain, galactose permease increases xylose transport capacity and the observed partial repression of xylose utilization depends on the presence of intracellular glucose.     

Deletion of methylglyoxal synthase gene (<Emphasis Type="Italic">mgsA</Emphasis>) increased sugar co-metabolism in ethanol-producing <Emphasis Type="Italic">Escherichia coli</Emphasis>

The use of lignocellulose as a source of sugars for bioproducts requires the development of biocatalysts that maximize product yields by fermenting mixtures of hexose and pentose sugars to completion. In this study, we implicate mgsA encoding methylglyoxal synthase (and methylglyoxal) in the modulation of sugar metabolism. Deletion of this gene (strain LY168) resulted in the co-metabolism of glucose and xylose, and accelerated the metabolism of a 5-sugar mixture (mannose, glucose, arabinose, xylose and galactose) to ethanol.     

Hypocotyl growth of Pinus pinaster seedlings. Changes in osmotic potential and cell wall composition

The changes in osmotic potential and cell wall composition of hypocotyl cell walls from different hypocotyl regions were investigated during growth of etiolated seedlings of Pinus pinaster Aiton. The osmotic potential in the subapical 5 mm part was minimum when hypocotyl growth rate was low, and increased when the fast growth phase began. The main non-cellulosic sugars of the cell wall from pine hypocotyl were arabinose, galactose, xylose, glucose and uronic acids, although their relative proportions were different from those found for angiosperm cell walls. Non-cellulosic glucose was the sugar showing the most important changes during hypocotyl growth as well as along the hypocotyl, suggesting that a glucose-rich polysaccharide is involved in a very active turnover during growth. A partial degradation of a xyloglucan during growth is suggested.     

Carbohydrate utilization patterns and substrate preferences in Bacteroides thetaiotaomicron

Specific growth rates of Bacteroides thetaiotaomicron NCTC 10582 with either glucose, arabinose, mannose, galactose or xylose as sole carbon sources were 0.42/h, 0.10/h, 0.38/h, 0.38/h and 0.16/h respectively, suggesting that hexose metabolism was energetically more efficient than pentose fermentation in this bacterium. Batch culture experiments to determine whether carbohydrate utilization was controlled by substrate-induced regulatory mechanisms demonstrated that mannose inhibited uptake of glucose, galactose and arabinose, but had less effect on xylose. Arabinose and xylose were preferentially utilized at high dilution rates (D > 0.26/h) in carbon-limited continuous cultures grown on mixtures of arabinose, xylose, galactose and glucose. When mannose was also present, xylose was co-assimilated at all dilution rates. Under nitrogen-limited conditions, however, mannose repressed uptake of all sugars, showing that its effect on xylose utilization was strongly concentration dependent. Studies with individual D-ZU-14C]-labelled substrates showed that transport systems for glucose, galactose, xylose and mannose were inducible. Measurements to determine incorporation of these sugars into trichloroacetic acid-precipitable material indicated that glucose and mannose were the principal precursor monosaccharides. Xylose was only incorporated into intracellular macromolecules when it served as growth substrate. Phosphoenolpyruvate:phosphotransferase systems were not detected in preliminary experiments to elucidate the mechanisms of sugar uptake, and studies with inhibitors of carbohydrate transport showed no consistent pattern of inhibition with glucose, galactose, xylose and mannose. These results indicate the existence of a variety of different systems involved in sugar transport in B. thetaiotaomicron.     

Mechanical properties and polysaccharide nature of the cell walls of maize root

Changes in mechanical properties and chemical nature of the cell walls of the different zones along elongating maize ( Zea mays L. cv. LG 11) roots were analyzed and the following results were obtained. (1) The apical region 2 to 5 mm from the tip of 15 mm long roots showed rapid elongation whereas the region 8–10 mm from the tip showed very little growth. (2) The minimum stress-relaxation time (To) and the mean stress-relaxation rate (R) of the cell wall were small whereas the maximum stress-relaxation time (Tm) was large in the region where cell elongation was optimum. The To and R increased and the Tm decreased gradually towards the base of the root. (3) The amounts of non-cellulosic polysaccharides of the cell wall were highest in the region 1.5–2.5 mm from the tip, decreasing until 5 mm from the tip, and then increasing towards the base. However, the proportion of this fraction in the total cell wall polysaccharides was highest in the extreme tip (cap and meristem, 0–1 mm) and decreased towards the base. (4) Major neutral sugars constituting the non-cellulosic polysaccharides of the cell wall were xylose, arabinose, galactose and glucose, with minor amounts of rhamnosc. mannose and fucose. The 1–15 mm region was on the whole rich in glucose and xylose and contained arabinose to a lesser extent. However, the chemical nature in the apical region, (0–2 mm, was rather special, being rich in galactose and fucose. (5) The cell wall of maize roots contained, as a whole, only little pectic substances but was high in hemicellulose 1 (rich in xylose, arabinose and glucose) and hemicellulose 2 (rich in glucose and xylose). (6) It appeared that in the elongating region (apical 2 to 5 mm) the cell elongation rate (CET) showed a rather good correlation with the parameters of mechanical properties (To, Tm and R) and with neutral sugar compositions in the non-cellulosic polysaccharides.