Carbon Source Requirements for Exopolysaccharide Production by Lactobacillus casei CG11 and Partial Structure Analysis of the Polymer

Exopolysaccharide production by Lactobacillus casei CG11 was studied in basal minimum medium containing various carbon sources (galactose, glucose, lactose, sucrose, maltose, melibiose) at concentrations of 2, 5, 10, and 20 g/liter. L. casei CG11 produced exopolysaccharides in basal minimum medium containing each of the sugars tested; lactose and galactose were the poorest carbon sources, and glucose was by far the most efficient carbon source. Sugar concentrations had a marked effect on polymer yield. Plasmid-cured Muc^- derivatives grew better in the presence of glucose and attained slightly higher populations than the wild-type strain. The values obtained with lactose were considerably lower for both growth and exopolysaccharide yield. The level of specific polymer production per cell obtained with glucose was distinctively lower for Muc^- derivatives than for the Muc⁺ strain. The polymer produced by L. casei CG11 in the presence of glucose was different from that formed in the presence of lactose. The polysaccharide produced by L. casei CG11 in basal minimum medium containing 20 g of glucose per liter had an intrinsic viscosity of 1.13 dl/g. It was rich in glucose (76%), which was present mostly as 2- or 3-linked residues along with some 2,3 doubly substituted glucose units, and in rhamnose (21%), which was present as 2-linked or terminal rhamnose; traces of mannose and galactose were also present.     

Production of d-Mannitol and Glycerol by Yeasts

D-Mannitol has not so far been known as a major product of sugar metabolism by yeasts. Three yeast strains, a newly isolated yeast from soy-sauce mash, Torulopsis versatilis, and T. anomala, were found to be good mannitol producers. Under optimal conditions, the isolate produced mannitol at good yield of 30% of the sugar consumed. Glucose, fructose, mannose, galactose, maltose, glycerol, and xylitol were suitable substrates for mannitol formation. High concentrations of yeast extract, Casamino Acids, NaCl, and KCl in media affected significantly the mannitol yield, whereas high levels of inorganic phosphate did not show any detrimental effect.     

Succinic acid production by <Emphasis Type="Italic">Actinobacillus succinogenes</Emphasis> from batch fermentation of mixed sugars

Succinic acid production from the monosaccharides xylose, arabinose, glucose, mannose and galactose was studied using the bacterium Actinobacillus succinogenes. In Duran bottle cultures, containing 10 g/L of each of sugar, succinic acid was produced from all sugars except for galactose. The highest succinate yield, 0.56 g/g, was obtained with glucose, whereas the succinate yield was 0.42, 0.38 and 0.44 g/g for xylose, mannose and arabinose, respectively. The specific succinate productivity was 0.7 g/g h for glucose, but below 0.2 g/g h for the other sugars. Batch bioreactor fermentations were carried out using a sugar mixture of the five sugars giving a total concentration of 50 g/L, mimicking the distribution of sugars in spent sulfite liquor (SSL) from Eucalyptus which is rich in xylose. In this mixture, an almost complete conversion of all sugars (except galactose) was achieved resulting in a final succinate concentration of 21.8–26.8 g/L and a total yield of 0.59–0.68 g/g. There was evidence of co-consumption of glucose and xylose, whereas mannose was consumed after glucose. The main by-products were acetate 0.14–0.20 g/g and formate 0.08–0.13 g/g. NADH balance calculations suggested that NADH required for succinate production was not met solely from formate and acetate production, but other means of NADH production was necessary. Results from mixed sugar fermentations were verified using SSL as substrate resulting in a succinate yield of 0.60 g/g. In addition, it was found that CO₂ sparging could replace carbonate supply in the form of MgCO₃ without affecting the succinate yield.     

Influence of transport energization on the growth yield of Escherichia coli.

The growth yields of Escherichia coli on glucose, lactose, galactose, maltose, maltotriose, and maltohexaose were estimated under anaerobic conditions in the absence of electron acceptors. The yields on these substrates exhibited significant differences when measured in carbon-limited chemostats at similar growth rates and compared in terms of grams (dry weight) of cells produced per mole of hexose utilized. Maltohexaose was the most efficiently utilized substrate, and galactose was the least efficiently utilized under these conditions. All these sugars were known to be metabolized to glucose 6-phosphate and produced the same pattern of fermentation products. The differences in growth yields were ascribed to differences in energy costs for transport and phosphorylation of these sugars. A formalized treatment of these factors in determining growth yields was established and used to obtain values for the cost of transport and hence the energy-coupling stoichiometries for the transport of substrates via proton symport and binding-protein-dependent mechanisms in vivo. By this approach, the proton-lactose stoichiometry was found to be 1.1 to 1.8 H+ per lactose, equivalent to approximately 0.5 ATP used per lactose transported. The cost of transporting maltose via a binding-protein-dependent mechanism was considerably higher, being over 1 to 1.2 ATP per maltose or maltodextrin transported. The formalized treatment also permitted estimation of the net ATP yield from the metabolism of these sugars; it was calculated that the growth yield data were consistent with the production of 2.8 to 3.2 ATP in the metabolism of glucose 6-phosphate to fermentation products.     

Influence of carbon source on α-amylase production by Aspergillus oryzae

The influence of the carbon source on alpha-amylase production by Aspergillus oryzae was quantified in carbon-limited chemostat cultures. The following carbon sources were investigated: maltose, maltodextrin (different chain lengths), glucose, fructose, galactose, sucrose, glycerol, mannitol and acetate. A. oryzae did not grow on galactose as the sole carbon source, but galactose was co-metabolized together with glucose. Relative to that on low glucose concentration (below 10 mg/l), productivity was found to be higher during growth on maltose and maltodextrins, whereas it was lower during growth on sucrose, fructose, glycerol, mannitol and acetate. During growth on acetate there was no production of alpha-amylase, whereas addition of small amounts of glucose resulted in alpha-amylase production. A possible induction by alpha-methyl-D-glucoside during growth on glucose was also investigated, but this compound was not found to be a better inducer of a-amylase production than glucose. The results strongly indicate that besides acting as a repressor via the CreA protein, glucose acts as an inducer.     

Catabolite inhibition: a general phenomenon in the control of carbohydrate utilization

When Escherichia coli is grown in synthetic medium with radioactive galactose or lactose as the carbon source, the addition of glucose rapidly inhibited utilization of the radioactive substrate, whether the formation of (14)CO(2) or acid-insoluble products was measured. The inhibition was reversed after the removal of glucose. Experiments with mutants blocked in subsequent steps of galactose and lactose metabolism demonstrated that the inhibition occurs prior to the formation of the first metabolic product. The utilization of a variety of sugars, including maltose, lactose, mannose, galactose, l-arabinose, xylose, and glycerol was inhibited by glucose. Of a number of carbohydrates tested as potential inhibitors, only glucose and, to a lesser extent, glucose-6-phosphate (G-6-P) were capable of inhibiting the utilization of all of the substrates. Glucose did not inhibit G-6-P utilization but G-6-P inhibited glucose utilization. With all substrates, except glycerol, there was a delay before the onset of inhibition by G-6-P. We conclude that E. coli has a general regulatory mechanism, termed catabolite inhibition, which controls the activity of early reactions in carbohydrate metabolism, allowing certain substrates to be utilized preferentially.     

Various Hexoses and di-hexoses Differently Influence Growth,Morphology and Pigment Synthesis in Transformed Root Cultures of Red Beet (Beta vulgaris)

Red beet hairy root cultures, obtained after genetic transformation with Agrobacterium rhizogenes, are completely heterotrophic and synthesize betalaines (BNs). Upon subjecting the hairy roots to treatments containing different sugars (3% w/v) it was found that sucrose was rapidly utilized, followed by maltose, and a very limited use of glucose, but the other hexoses – fructose, lactose, xylose and galactose or glycerol totally suppressed both growth and BN synthesis. No habituation or adaptability to maltose or glucose occurred, evidenced by the lack of growth upon re-culture in respective medium. Glycerol, was not taken up alone, but was utilized to a considerable extent in the presence of low levels of sucrose for growth only but not BN synthesis. Red beet hairy root culture did not exogenously hydrolyse sucrose to hexoses, as there were only traces of reducing sugar present in the medium soon after inoculation, without an increase later, confirmed by HPLC. There was an increase in medium osmolarity in the presence of fructose indicating the exudation of certain compounds from the roots. Red beet hairy roots appear useful as a model system to study sugar metabolism/signalling due to their sensitivity to different sugars that may directly link to morphological changes and BN synthesis.     

Chemoattractant effects of sugars and their related compounds on black abalone Haliotis discus

The chemoattractant properties of sugars and their related compounds were statistically estimated on the basis of an exploratory behavior of black abalone Haliotis discus. Six monosaccharides, three disaccharides, six sugar alcohols, six glycosides and two artificial sweeteners were tested. The active compounds were: glucose and galactose (monosaccharides), maltose (disaccharides), sorbitol, mannitol, maltitol, dulcitol and erythritol (sugar alcohols), all the glycosides, and saccharin (artificial sweeteners). Maltose, dulcitol and particularly phyllodukin showed the highest activity. The chemoattractant properties of maltose and phyllodukin increased as concentrations increased. The activity of phyllodulcin was higher at all concentrations tested than that of maltose.     

Utilization of organic substrates during mixotrophic and heterotrophic cultivation of algae

Pure cultures ofChlorella pyrenoidosa (82) andScenedesmus obliquus (125) were grown in the nutrient medium according to Benson in the presence of 0·05m sugars or 0·025m sodium salts of organic acids. The density of culture was measured throughout the course of growth. Satisfactory heterotrophic sources of nutrition forChlorella pyrenoidosa appear to be galactose, glucose and acetate, whereasScenedesmus utilizes glucose, cellobiose and acetate. The growth ofChlorella in the light is enhanced by galactose, glucose, fructose, cellobiose and maltose, that ofScenedesmus by glucose, fructose, cellobiose, galactose, maltose, acetate and pyruvate. Soluble starch suppresses growth of both cultures. The role of the substrates is discussed. It follows from the results that the growth-promoting sugars and organic acids can act not only as a source of carbon during general carbon shortage but also as ergastic material. The mechanism of utilization of some organic substrates will be taken up in a subsequent paper.     

Carbohydrate utilization by Trichomonas gallinae: effects on growth and metabolic activity under nongrowth conditions

Trichomonas gallinae used 13 of 29 carbohydrates for growth. Quantitative relationships between final populations, acid production, and cellular glycogen contents varied depending on the substrate. The effect of growth on different carbohydrates on the subsequent utilization of carbohydrates by cells under nongrowth conditions was studied by measuring carbohydrate uptake, changes in cellular glycogen content, and gas production. Two major utilization patterns were found. Cells grown on maltose or starch used these substrates well, but cells grown on other sugars did not. All cells used glucose, fructose, galactose, and mannose, but cells grown on maltose or starch did not use them as well as cells grown on other sugars. All cells used ribose slightly but not xylose or arabinose. Turanose, a disaccharide yielding high populations in growth medium, was not used under nongrowth conditions.     

The Growth of Acer pseudoplatanus Cells in a Synthetic Liquid Medium: Response to the Carbohydrate, Nitrogenous and Growth Hormone Constituents

A synthetic culture medium which supports a high level of growth of a scrially propagated cell suspension culture of Acer pseudoplatanus is described. The sucrose of this medium can be effectively replaced by glucose or fructose or a mixture of glucose and fructose or galactose or maltose or soluble starch. When the carbohydrate is glucose or fructose no other sugars appear in the culture medium in significant amounts. Glucose is absorbed in greater quantity than fructose from an equimolar mixture of these sugars. When sucrose is supplied both glucose and fructose appear in the medium. Glucose appears in maltose medium, and maltose and glucose in soluble starch medium. Under the standard conditions of culture, media containing 2 % sucrose or 2 % glucose become depleted of sugar before the 25th day of incubation. Enhanced yield of the cultures can be obtained by raising the initial sucrose concentration to 6 %. – A supply of nitrate is essential for maximum yield and healthy growth. Growth, in the presence of nitrate, is significantly enhanced by a supply of urea. Addition of casein hydrolysate or of a mixture of amino acids enhances growth in the presence of nitrate and urea and particularly when nitrate is omitted. – When kinetin is omitted or incorporated at the standard level (0.25 mg/I), 2,4-dichlorophenoxyacetic acid (2,4-D) at 1.0 mg/l is essential for continuation of growth at a high level. It cannot be replaced by indol-3yl-acetic acid (IAA). 1-naphthaleneacetic acid (NAA) at 10 mg/l permits of a low level of growth with abnormal aggregation. When the level of kinetin is raised to 10 mg/l a high level of growth occurs in the absence of added auxin but the cultures become brown and tend to show increasing aggregation on subculture.     

The effects of various carbohydrate diets on Aedes aegypti infected with Dirofilaria immitis.

Aedes aegypti infected with Dirofilaria immitis and uninfected mosquitoes were maintained on various carbohydrate diets (glucose, galactose, fructose, sucrose, trehalose, maltose, and melibiose). The value of each of these sugars in supporting survival of adult A. aegypti, and in supporting egg production, viability of eggs, and development of third-stage larvae of D. immitis in A. aegypti was analyzed. Fructose, glucose, maltose, sucrose, and trehalose provided the strongest support for survival of adult male, and infected and uninfected adult female A. aegypti. Galactose and melibiose provided the least support for survival of all groups of mosquitoes. The mean number of eggs laid per uninfected adult female A. aegypti was greatest when mosquitoes were maintained on glucose, melibiose, maltose, fructose, sucrose, and trehalose. The same was true for female mosquitoes infected with D. immitis; except for melibiose which provided poor support for egg production. In both Dirofilaria-infected and in uninfected mosquitoes, galactose supported the production of low mean numbers of eggs per adult female A. aegypti. High percentages of eggs laid by uninfected and by infected female mosquitoes fed glucose, melibiose, maltose, sucrose, and trehalose hatched. While galactose supported a high percentage of hatching in eggs laid by uninfected A. aegypti, a much lower percentage of eggs laid by infected female mosquitoes maintained on this same carbohydrate hatched. The lowest percentages of eggs that hatched were from among those laid by infected and by uninfected females fed fructose. The highest mean number of D. immitis larvae (L₃) were recovered from adult A. aegypti fed glucose, maltose, fructose, and sucrose; the second best sugar in this regard was trehalose. The lowest mean number of D. immitis larvae were isolated from female A. aegypti fed galactose and melibiose.     

Gamma-radiation induced modifications in substrate specificity of glucose dehydrogenase and carbon source utilization pattern of phosphate-solubilizing <Emphasis Type="Italic">Pantoea</Emphasis> strains

Glucose, maltose, and mannose as sole carbon sources, induced synthesis of glucose dehydrogenase (GDH) in three strains of Pantoea with specific activities from 0.14 to 0.6 U/mg proteins. Utilization of lactose indicated that the enzyme belongs to GDH type B isozyme. Of mutant clones, developed through radiation mutagenesis, P2-M2 utilized ribose with GDH specific activity of 0.57 U/mg protein, P4-M3 grown on glucose gave 1.5 U/mg protein and P4-M5 had high activities, when grown on galactose, maltose, and lactose. Clones P3-M2 and P2-M5 had versatile utilization of sugars and released higher amounts of P from tri-calcium phosphate and can be efficiently used for biofertilization.     

Cross-adapted sugar responses in the mouse taste cell

1. Intracellular recordings of mouse taste cell responses were made using a glass micro-electrode filled with Procion yellow dye solution. 2. Six sugars (sucrose, maltose, lactose, glucose, galactose and fructose) produced the depolarization responses. 3. Gustatory cross adaptation between sugars was determined. When the taste cell was pre-adapted with one of the six sugars, the other five sugars, cross adapted, produced depolarization, hyperpolarization or null responses. 4. From these observations, it is suggested that there are multiple sugar receptor sites on the receptor membrane of the mouse taste cell.     

Impairment by hexoses of the utilization of maltose by Saccharomyces cerevisiae1

The effect of hexoses with different transport and phosphorylation systems on the utilization of maltose by a galactose constitutive mutant of Saccharomyces cerevisiae has been studied. Galactose, mannose and fructose inhibit both the entrance of maltose in the cells and the phosphorylation of the glucose generated by intracellular hydrolysis of maltose. Transport of maltose is less affected than glucose phosphorylation and, once inside the cell, maltose is hydrolysed and the sparing glucose subsequently excreted. In addition to the well known inactivating effect of glucose, we have found that galactose inactivates the maltose transporter and that this inactivation is enhanced by maltose, which fails to inactivate the system by itself. As reported for glucose, inactivation by galactose involves proteolysis. Other strains of yeast with inducible pathways for both galactose and maltose behave similarly to the galactose constitutive mutant, with some minor changes. The use of maltose as a source of intracellular glucose has allowed to find the existence of mutual interferences in the utilization of hexoses by yeast at the phosphorylation step, that otherwise would have remained unnoticed.     

Growth of the yeast Kluyveromyces marxianus CBS 6556 on different sugar combinations as sole carbon and energy source

The yeast Kluyveromyces marxianus has been pointed out as a promising microorganism for a variety of industrial bioprocesses. Although genetic tools have been developed for this yeast and different potential applications have been investigated, quantitative physiological studies have rarely been reported. Here, we report and discuss the growth, substrate consumption, metabolite formation, and respiratory parameters of K. marxianus CBS 6556 during aerobic batch bioreactor cultivations, using a defined medium with different sugars as sole carbon and energy source, at 30 and 37 °C. Cultivations were carried out both on single sugars and on binary sugar mixtures. Carbon balances closed within 95 to 101 % in all experiments. Biomass and CO₂ were the main products of cell metabolism, whereas by-products were always present in very low proportion (<3 % of the carbon consumed), as long as full aerobiosis was guaranteed. On all sugars tested as sole carbon and energy source (glucose, fructose, sucrose, lactose, and galactose), the maximum specific growth rate remained between 0.39 and 0.49 h^?1, except for galactose at 37 °C, which only supported growth at 0.31 h^?1. Different growth behaviors were observed on the binary sugar mixtures investigated (glucose and lactose, glucose and galactose, lactose and galactose, glucose and fructose, galactose and fructose, fructose and lactose), and the observations were in agreement with previously published data on the sugar transport systems in K. marxianus. We conclude that K. marxianus CBS 6556 does not present any special nutritional requirements; grows well in the range of 30 to 37 °C on different sugars; is capable of growing on sugar mixtures in a shorter period of time than Saccharomyces cerevisiae, which is interesting from an industrial point of view; and deviates tiny amounts of carbon towards metabolite formation, as long as full aerobiosis is maintained.     

Production of a New Acidic Polysaccharide,Succinoglucan by Alcaligenes faecalis var. myxogenes

A slimy non-spore-forming bacterium strain 10C3 isolated from soil was motile with peritrichous flagella and named Alcaligenes faecalis var. myxogenes. Studies were made on the conditions necessary for maximal production of a new acidic succinoglucan polysaccharide by this strain in shaken cultures. Much production was observed with sucrose, glucose, xylose, galactose, cellobiose, maltose, fructose, mannose and rhamnose. The yield was greatest with sucrose and decreased in order with the above sugars from about 36 to 23 per cent. The most suitable medium contained 4 per cent sugar, 0.5 per cent yeast extract and one per cent calcium carbonate in tap water. The optimum temperature was 28°C.     

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.     

High-Affinity Maltose Binding and Transport by the Thermophilic Anaerobe Thermoanaerobacter ethanolicus 39E

Thermoanaerobacter ethanolicus is a gram-positive thermophile that produces considerable amounts of ethanol from soluble sugars and polymeric substrates, including starch. Growth on maltose, a product of starch hydrolysis, was associated with the production of a prominent membrane-associated protein that had an apparent molecular weight of 43,800 and was not detected in cells grown on xylose or glucose. Filter-binding assays revealed that cell membranes bound maltose with high affinity. Metabolic labeling of T. ethanolicus maltose-grown cells with [¹⁴C]palmitic acid showed that this protein was posttranslationally acylated. A maltose-binding protein was purified by using an amylose resin affinity column, and the binding constant was 270 nM. Since maltase activity was found only in the cytosol of fractionated cells and unlabeled glucose did not compete with radiolabeled maltose for uptake in whole cells, it appeared that maltose was transported intact. In whole-cell transport assays, the affinity for maltose was approximately 40 nM. Maltotriose and α-trehalose competitively inhibited maltose uptake in transport assays, whereas glucose, cellobiose, and a range of disaccharides had little effect. Based on these results, it appears that T. ethanolicus possesses a high-affinity, ABC type transport system that is specific for maltose, maltotriose, and α-trehalose.     

Influence of cosubstrate concentration on xylose conversion by recombinant, XYL1-expressing Saccharomyces cerevisiae: a comparison of different sugars and ethanol as cosubstrates.

Conversion of xylose to xylitol by recombinant Saccharomyces cerevisiae expressing the XYL1 gene, encoding xylose reductase, was investigated by using different cosubstrates as generators of reduced cofactors. The effect of a pulse addition of the cosubstrate on xylose conversion in cosubstrate-limited fed-batch cultivation was studied. Glucose, mannose, and fructose, which are transported with high affinity by the same transport system as is xylose, inhibited xylose conversion by 99, 77, and 78%, respectively, reflecting competitive inhibition of xylose transport. Pulse addition of maltose, which is transported by a specific transport system, did not inhibit xylose conversion. Pulse addition of galactose, which is also transported by a specific transporter, inhibited xylose conversion by 51%, in accordance with noncompetitive inhibition between the galactose and glucose/ xylose transport systems. Pulse addition of ethanol inhibited xylose conversion by 15%, explained by inhibition of xylose transport through interference with the hydrophobic regions of the cell membrane. The xylitol yields on the different cosubstrates varied widely. Galactose gave the highest xylitol yield, 5.6 times higher than that for glucose. The difference in redox metabolism of glucose and galactose was suggested to enhance the availability of reduced cofactors for xylose reduction with galactose. The differences in xylitol yield observed between some of the other sugars may also reflect differences in redox metabolism. With all cosubstrates, the xylitol yield was higher under cosubstrate limitation than with cosubstrate excess.