MIR spectroscopic analysis on sugar metabolic and ethanol productive kinetics of suspension TBY-2 and rice cells pre-cultured in various media

The influence of sugars in pre-cultivation media suspended plant cells on the kinetics of the sugar uptake and the ethanol production was studied by mid-infrared spectroscopy using a Fourier transform infrared spectrometer (FT-IR) equipped with an attenuate total reflection accessory (ATR). We performed the plant cell cultivation with Nicotiana tabacum cv. Bright Yellow No.2 (TBY-2) cells and Oryza sativa L., Japonica, cv. Nipponbare (rice) cells, respectively, in pre-culture and culture media, which had various types of glucose, fructose, sucrose or glucose–fructose mixtures. The results confirmed the kinetic differences between the TBY-2 cells and rice cells. These results suggested that the TBY-2 cells consumed sugar before growth and the rice cells consumed sugar after growth, moreover, the ethanol content increased just after cell growth was activated based on the non-dimensional cultivation time for the cell growth behavior.     

Sugar uptake analysis of suspension Arabidopsis, tobacco, and rice cells in various media using an FT-IR/ATR method

The kinetic behavior of the sugar uptake phenomena of a suspension of Arabidopsis cells was investigated by mid-infrared spectroscopy using Fourier transform infrared spectrometers and attenuated total reflection techniques. The kinetic behavior of the cell growth was also studied and the growth and the sugar uptake behaviors were discussed for three typical plant cells (Arabidopsis, TBY-2, and rice cells). The cell growth rate and the lag period were influenced by not only the types of the plant cells, but also the sugar species used as the carbon source. The characteristics of the sugar uptake behavior were clarified based on the difference in the three types of plant cells. The cell growth and the sugar uptake progressed at approximately the same time in the TBY-2 cells. In the rice cells, the sugar uptake rate was relatively lower than that of the others. On the other hand, the sugar uptake of the Arabidopsis cells started before the cell growth. Furthermore, glucose as the carbon source of the Arabidopsis cell cultivation seems to significantly influence the sugar metabolism. Glucose had a significant influence on the sugar metabolism of the other sugar under the conditions for the mixture of glucose and the other sugar. The characteristics of the sugar uptake phenomena based on the cell growth stage was typical for each plant cell except for some sugars, such as galactose and trehalose, and the behavior of the total sugar uptake had not changed. These results suggested that the cell growth and the sugar uptake in the plant cell cultivation processes may be controlled by the combined supply of the sugar species as the carbon source. The detailed data for plant cell cultivation using each sugar obtained in this study would be useful for bioscience research and for cultivation process control using various sugars, for example, purified or sugar mixtures formed from biomass materials.     

Sugar metabolic analysis of suspensions of plant cells using an FT-IR/ATR method

A simple, rapid and accurate evaluation of the sugar uptake rate of suspended plant cells from culture media was developed with the predicted sugar contents measured by mid-infrared spectroscopy using a Fourier transform infrared (FT-IR) spectrometer equipped with an attenuated total reflectance (ATR) accessory. We performed plant cell cultivation with Nicotiana tabacum cv. Bright Yellow No.2 (TBY-2) in culture media, which had various combinations of glucose, fructose and sucrose concentrations at the initial stage, and measured simultaneously each sugar content in the medium by the FT-IR/ATR method. By applying a logistic function to the predicted sugar contents and cell density in the medium during cultivation, the specific sugar uptake rates by the suspended TBY-2 cells were easily and continuously obtained. Thus the kinetic sugar uptake phenomena by the TBY-2 cells were well confirmed overall using the developed method. Additionally it was found that the fraction of sucrose of the initial total sugar content might kinetically affect the sugar uptake process and cell growth. Also, the relationship between the nondimensional cell density and sucrose content could be classified into three groups on the basis of the initial fraction of sucrose.     

Metabolic Changes in Trichomonas gallinae Resulting from Growth in Various Carbohydrates*†

SYNOPSIS. The influence of the type of growth carbohydrate on the subsequent metabolic activity of Trichomonas gallinae was investigated. Washed suspensions of cells collected from CPL-glucose, CPL-maltose, CPL-galactose, and CPL-glucose-maltose media were examined in the Warburg respirometer for their ability to utilize glucose, maltose, and galactose. Comparisons of the metabolic parameters of substrate consumption, changes in glycogen content, and CO₂ and H₂ production were made. The pattern of utilization of the sugars, both qualitatively and quantitatively, depended upon the type of carbohydrate in the CPL medium used to culture the cells and upon the time of exposure of the cells to a particular sugar in the medium.     

Uptake patterns and transport enhancements in cultures of hamster cells deprived of carbohydrates.

Dense cell cultures of the hamster lines, NIL, and polyoma transformed NIL were exposed to culture media containing various sugars (or no sugar). Various responses to these culture conditions were observed as changes in the uptake of galactose and its subsequent metabolism. Cells deprived of sugar have higher uptake rates for galactose and markedly different accumulation products from identical cells treated with sugar. A persistent increase in the transport of the amino acid, cycloleucine, was also observed as a response to culture conditions devoid of sugar     

A defect in carbon catabolite repression associated with uncontrollable and excessive maltose uptake

Summary The previously isolated recessive mutant allele hex2-3 of Saccharomyces cerevisiae caused a defect in carbon catabolite repression of maltase, invertase, malate dehydrogenase, and respiration but at the same time led to an extreme sensitivity to maltose (Zimmermann and Scheel, 1977; Entian and Zimmermann, 1980). Addition of maltose to a growing culture of a hex2-3 mutant resulted within 60 to 90 min in an inhibition of growth, glycolysis, and de novo protein synthesis. This was not accompanied by any abnormal levels of glycolysis metabolites or glycolytic enzyme activities. However, inhibitory effects coincided with a dramatic increase in intracellular glucose up to 150 mM relative to cell water as opposed to 2.5 mM in wild-type cells. This abnormal behavior is interpreted as a result of an uncontrolled maltose uptake in hex2 mutants, which in combination with increasing maltase activity results in an accumulation of intracellular glucose. Obviously the amount of available glucose surpassed glycolytic capacity in hex2 mutants.Properties of mutant alleles hex2 and hex1 (see Entian and Zimmermann, 1980) clearly show, that specific gene functions are involved in adapting the rate of sugar uptake into the cell to the actual glycolytic capacity.     

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.     

Enzymes of the ascorbate biosynthesis and ascorbate-glutathione cycle in cultured cells of tobacco Bright Yellow 2

The ascorbate (ASC) and glutathione (GSH) metabolisms were studied in cultured Nicotiana tabacum cv. Bright Yellow 2 (TBY-2) cells. TBY-2 cells were found to be endowed with L-galactono-γ-lactone dehydrogenase (GLDH) (EC 1.3.2.3), an enzyme that converts L-galactono-γ-lactone into ASC. Cellular fractionation of TBY-2 protoplasts indicated that this enzyme is exclusively localised in mitochondria and associated to the membrane fractions. During the growth cycle of TBY-2 cell culture, GLDH transiently increased, reaching the maximum value on the third day of culture, at the beginning of the exponential phase, when the cell proliferative activity was also higher. Similar behaviour has been observed for ASC and GSH contents. The activities of ascorbate peroxidase (APX) (EC 1.11.1.11), ascorbate-free radical reductase (AFRR) (EC 1.6.5.4), dehydroascorbic acid reductase (DHAR) (EC 1.8.5.1) and glutathione reductase (GR) (EC 1.6.4.2) also transiently raised. However, the scale of the increases varied being about 4-fold for APX and AFRR, 2-fold for DHAR and more than 11-fold for GR. The behaviour of the ASC and GSH recycling enzymes allowed TBY-2 cells to maintain both dehydroascorbic acid and glutathione disulphide at low levels, even under conditions of high ASC and GSH utilisation. The relationship between the ASC and GSH metabolisms during the growth cycle of TBY-2 cell suspension cultures is also discussed.     

Respiration-Dependent Utilization of Sugars in Yeasts: a Determinant Role for Sugar Transporters

In many yeast species, including Kluyveromyces lactis, growth on certain sugars (such as galactose, raffinose, and maltose) occurs only under respiratory conditions. If respiration is blocked by inhibitors, mutation, or anaerobiosis, growth does not take place. This apparent dependence on respiration for the utilization of certain sugars has often been suspected to be associated with the mechanism of the sugar uptake step. We hypothesized that in many yeast species, the permease activities for these sugars are not sufficient to ensure the high substrate flow that is necessary for fermentative growth. By introducing additional sugar permease genes, we have obtained K. lactis strains that were capable of growing on galactose and raffinose in the absence of respiration. High dosages of both the permease and maltase genes were indeed necessary for K. lactis cells to grow on maltose in the absence of respiration. These results strongly suggest that the sugar uptake step is the major bottleneck in the fermentative assimilation of certain sugars in K. lactis and probably in many other yeasts.     

Substrate inhibition kinetics of <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis> in fed-batch cultures operated at constant glucose and maltose concentration levels

Fed-batch culture is the mode of operation of choice in industrial baker’s yeast fermentation. The particular mode of culture, operated at stable glucose and maltose concentration levels, was employed in this work in order to estimate important kinetic parameters in a process mostly described in the literature as batch or continuous culture. This way, the effects of a continuously falling sugar level during a batch process were avoided and therefore the effects of various (stable) sugar levels on growth kinetics were evaluated. Comparing the kinetics of growth and the inhibition by the substrate in cultures grown on glucose, which is the preferential sugar source for Saccharomyces cerevisiae, and maltose, the most common sugar source in industrial media for baker’s yeast production, a milder inhibition effect by the substrate in maltose-grown cells was observed, as well as a higher yield coefficient. The observed sugar inhibition effect in glucostat cultures was taken into account in modeling substrate inhibition kinetics. The inhibition coefficient K _i increased with increasing sugar concentration levels, but it appeared to be unaffected by the type of substrate and almost equal for both substrates at elevated concentration levels.     

Comparative studies of glucose-fed and glucose-starved hamster cell cultures: responses in galactose metabolism.

The metabolic flow of trace amounts of D-[14C]-galactose was followed in cultures of transformed and untransformed hamster cells over a period ranging from five minutes to two hours. The results of chromatographic and enzymatic analyses of the soluble pools are described. Non-glycolytic cells(previously deprived of sugar periods of up to 24 hours) convert D-galactose to galactose-1-phosphate and uridine diphosphoglucuronic acid in 10 to 20 minutes. In the same short assay time, glycolytic cells which have been maintained for 24 hours in media containing glucose or galactose convert D-galactose to uridine diphsphogalactose and uridine diphosphoglucose (ratio 1.4:1). Long term diprivation of sugar also results in 3- to 4-fold increases in the uptake of galactose. In addition, the incorporation of galactose label into chloroformethanol soluble material appears to be influenced by the culture conditions of the untransformed cells while incorporation in the transformed cells appears unaffected. When cycloheximide is included in the maintenance medium for extended periods, the non-glycolytic cells also show increases in galactose uptake rates but the glucose-fed, glycolytic cells llose uptake ability. UDPhexose is the main galactose metabolic peak in the soluble pools of the cycloheximide-treated, glycolytic and the cycloheximide-treated, non-glycolytic cells. The results of these experiments suggests that uptake of galactose and its subsequent metabolism are under separate control.     

Influence of 2-deoxy-D-glucose upon growth and invertase activity of carrot (Daucus carota L.) cell suspension cultures

Carrot (Daucus carota L.) cell suspension cultures grew well when provided with glucose, fructose, sucrose or raffinose. Galactose and melibiose supported less growth unless supplemented with glucose or fructose. In combination with ten different sugar mixtures, 2-deoxy-D-glucose (dGlc) inhibited culture growth. Inhibitory effects of dGlc were more marked with fructose, melibiose, raffinose or mixtures of these sugars in the culture medium. The presence of glucose or galactose reduced the inhibitory effects of dGlc on culture growth. Experiments with radioactive labelled sugars demonstrated that dGLc uptake was greater in the presence of fructose than glucose, and that growth inhibition of dGlc coincided with its uptake. Reduced protein content was also associated with the inhibitory effects of dGlc. Cultured cells contained lower levels of invertase (EC 3.2.1.26) activity during the active phase of culture growth (up to 25 days after subculture) than when growth had peaked and subsequently declined. Acid and alkaline invertase activities were not greatly reduced by exogenous hexoses. Invertase activity was greatest during periods of low protein content in all cultures and was inhibited by dGlc during the latter phases of the culture period. Free intracellular sugars throughout the culture period consisted mainly of glucose and fructose.     

VARIATIONS WITHIN SINGLE CELL CLONES OF TOBACCO TISSUE CULTURES

Comparative studies were made utilizing two series of secondary clones (single cell clones derived from single cell clones H 196 and H 241) of hybrid tobacco (Nicotiana tabacum ♂ × Nicotiana glutinosa ♀ ) tissue grown in vitro. Secondary clones derived from a single parent varied in color, consistency, the ability to grow, and rate of growth with various carbohydrates and growth-promoting substances. The growth of the secondary clones generally resembled that of the parent clone from which derived. Many of the 23 secondary clones of H 196 grew satisfactorily on media supplemented with sucrose, dextrose, levulose, or maltose; lactose, galactose, and xylose were unsatisfactory supplements. Similarly, the series of 30 secondary clones isolated from H 241 grew well on some media but poorly on others. Growth generally decreased when α-napthaleneacetic acid or 2,4-dichlorophenoxyacetic acid was omitted from the basal coconut milk medium. Growth decreased considerably when coconut milk was omitted from the basal medium. The optimum sugar concentration was 1/2 to 1 per cent.     

Comparison of productivity and quality of bacterial nanocellulose synthesized using culture media based on seven sugars from biomass

Komagataeibacter xylinus ATCC 23770 was statically cultivated in eight culture media based on different carbon sources, viz. seven biomass-derived sugars and one sugar mixture. The productivity and quality of the bacterial nanocellulose (BNC) produced in the different media were compared. Highest volumetric productivity, yield on consumed sugar, viscometric degree of polymerization (DP_v, 4350–4400) and thermal stability were achieved using media based on glucose or maltose. Growth in media based on xylose, mannose or galactose resulted in lower volumetric productivity and DP_v, but in larger fibril diameter and higher crystallinity (76–78%). Growth in medium based on a synthetic sugar mixture resembling the composition of a lignocellulosic hydrolysate promoted BNC productivity and yield, but decreased fibril diameter, DP_v, crystallinity and thermal stability. This work shows that volumetric productivity, yield and properties of BNC are highly affected by the carbon source, and indicates how industrially relevant sugar mixtures would affect these characteristics.     

Variation in the fermentative pattern of some Saccharomyces species

Summary The acquisition of the ability to ferment galactose, maltose, sucrose, raffinose and melibiose by yeast strains of the genus Saccharomyces was investigated. During cultivation in selective media 11 strains belonging to 5 species gained the ability to ferment one or several of these sugars. De-adaptation was not usually observed after cultivation in glucose medium, indicating that the saltants are stable in this medium.     

Carbohydrate utilization affects Lactobacillus delbrueckii subsp. lactis 313 cell-enveloped-associated proteinase production

The effect of different sugars (glucose, glycerol, maltose, galactose and lactose) on cell-membrane-associated proteinase production by Lactobacillus delbrueckii subsp. lactis 313 (LDL 313) was investigated. The experimental results showed that aside glycerol and galactose, all the other sugars supported high growth levels of LDL 313, with glucose displaying the maximum biomass concentration of 0.85 mg/mL dry cell weight for cells harvested at the mid-exponential phase of ??12 h after inoculation. The specific proteinase yield, a measure of the rate of proteinase production relative to cell wall biosynthesis, was used to evaluate the preferential degree of proteinase metabolism as induced by the consumption of different sugar substrates by LDL 313. It was found that maltose displayed the highest specific proteinase yield of 12.59 U/mg sugar consumed. Further, molecular differences were observed in the SDS electrophoretic profile of cell surface proteins generated for the different carbon substrates. This is a preliminary study which supports the inference that different sugars stimulate the production of different cell-surface proteins with a significant effect on cell proteinase activity.     

beta-D-phosphogalactoside galactohydrolase of Streptococcus faecalis and the inhibition of its synthesis by glucose.

The lactose hydrolysing system of Streptococcus faecalis is described. It is closely related to that one of the group N streptocci as it consists of a beta-D-phosphogalactoside galactohydrolase (beta-Pgal). The uptake of methyl-beta-D-thiogalactoside (TMG), lactose, and glucose is maintained by the phosphoenolpyruvate-dependent phosphotransferase system (PTS) but the uptake of galactose is not. The induction time is 6--7 min. Inducers are lactose and galactose but not isopropyl-beta-D-galactoside (IPTG) and TMG. In the presence of glucose, mannose, and maltose no induction of beta-Pgal occurs but pyruvate and glycerol allow induction. The competitive inhibition of uptake of TMG by glucose suggests inducer exclusion by this sugar. TMG accumulates in the cells exclusively as a derivative.     

Growth and metabolism of Penicillium lilacinum Thom. with reference to the effects of riboflavin and nicotinic acid

Summary Growth and metabolism of Penicillium lilacinum were followed over a period of incubation of 18 days on a high sugar-salts medium favourable for fat formation with or without the addition of riboflavin or nicotinic acid to the growth medium. The high sugar content in the culture media helped rapid uptake and vigorous growth. Nicotinic acid and to a less extent riboflavin, enhanced sugar and nitrogen absorption and the rate of building up of cellular material in consequence. Nitrogenous compounds have been released from the mycelial cells into the external media before growth started to decline; the release being earlier and more rapid in the presence of nicotinic acid. It is suggested that the release of nitrogenous compounds in this case is not purely due to autolysis, and that nicotinic acid affected this process by increasing cell permeability. Both riboflavin and nicotinic acid accelerated the accumulation of carbohydrates and fat in the mycelium. Fat formation became active only when the nitrogen content of the culture media dropped to a very low value and the building of nitrogenous compounds almost stopped. The inverse relationship between synthesis of fat and of complicated nitrogenous compounds was quite clear under the present experimental conditions and was not affected by either riboflavin or nicotinic acid.     

Coregulation of beta-galactoside uptake and hydrolysis by the hyperthermophilic bacterium Thermotoga neapolitana

Regulation of the beta-galactoside transport system in response to growth substrates in the extremely thermophilic anaerobic bacterium Thermotoga neapolitana was studied with the nonmetabolizable analog methyl-beta-D-thiogalactopyranoside (TMG) as the transport substrate. T. neapolitana cells grown on galactose or lactose accumulated TMG against a concentration gradient in an intracellular free sugar pool that was exchangeable with external galactose or lactose and showed induced levels of beta-galactosidase. Cells grown on glucose, maltose, or galactose plus glucose showed no capacity to accumulate TMG, though these cells carried out active transport of the nonmetabolizable glucose analog 2-deoxy-D-glucose. Glucose neither inhibited TMG uptake nor caused efflux of preaccumulated TMG; rather, glucose promoted TMG uptake by supplying metabolic energy. These data show that beta-D-galactosides are taken up by T. neapolitana via an active transport system that can be induced by galactose or lactose and repressed by glucose but which is not inhibited by glucose. Thus, the phenomenon of catabolite repression is present in T. neapolitana with respect to systems catalyzing both the transport and hydrolysis of beta-D-galactosides, but inducer exclusion and inducer expulsion, mechanisms that regulate permease activity, are not present. Regulation is manifest at the level of synthesis of the beta-galactoside transport system but not in the activity of the system.