Feed Glucose Concentration Research Articles

Glucose Repression in Saccharomyces cerevisiae Is Related to the Glucose Concentration Rather Than the Glucose Flux

Glucose plays an important regulatory role in the yeast Saccharomyces cerevisiae, which is mostly reflected at the transcriptional level by glucose repression. The signal that initiates glucose repression is unknown, but data indicate that it is located at or above the level of glucose 6-phosphate, suggesting the involvement of either the intracellular or extracellular glucose concentration or the glucose flux in triggering glucose repression. We have investigated the role of the glucose flux and the extracellular glucose concentration in glucose repression by growing the cells in continuous culture under nitrogen limitation. By a step-wise increase in the glucose feed concentration, the glucose flux and extracellular glucose concentrations were modulated in an accurate way. Furthermore, the glucose flux and glucose concentrations were modulated independently of each other by increasing the dilution rate or by the use of fructose as a substrate. Using these approaches we demonstrate that glucose repression is related to the extracellular (or intracellular) glucose concentration rather than the glucose flux. At external glucose concentrations lower than 14 mM, glucose repression of SUC2 gene transcription was not triggered, whereas glucose repression of this gene was activated when the glucose concentration exceeded 18 mM. A comparable effect was observed for the glucose-repressible carbon source fructose.

Blood metabolite concentrations in late pregnant ewes as indicators of nutritional status

AbstractIn a feeding experiment β-hydroxybutyrate (BHB), glucose, albumin, total protein, globulin and urea concentrations in the plasma of twin-bearing ewes were analysed. Mature Suffolk cross ewes were given either formic acid (FA)-treated grass silage or grasslmolassed sugar-beet pulp (MSBP) silage in late pregnancy. The experiment commenced on day 91 of pregnancy and the dietary treatments were FA-treated silage (Tl), FA-treated silage + soya-bean meal (SBM) (T2), MSBP silage (T3), MSBP silage + SBM (T4), FA-treated silage + MSBP (T5), FA-treated silage + MSBP + SBM (T6) or FA-treated silage + 150 g crude protein (CP) per kg concentrate (T7). SBM was given only in the last 22 days of pregnancy aiming for a total CP intake of 220 g per ewe per day. Blood samples were collected by jugular venipuncture from each ewe 3 h following consumption of the morning dietary allowance on days 121, 128, 135 and 142 of pregnancy. Daily metabolizable energy (ME) intakes of 6·8, 11·4, 9·6, 12·8, 10·5, 13·7 and 14·7 (s.e. 0·58) MJ per ewe were recorded for Tl to T7 respectively over the last 3 weeks of pregnancy. Respective CP intakes of 72, 213, 110, 225, 109, 215 and 175 (s.e. 5·64) g per ewe were recorded for Tl to T7 respectively over the last 3 weeks of pregnancy. BHB concentrations (mmol/l) on day 121 of pregnancy of 1·18, 1·25, 0·52, 0·52, 0·56, 0·39 and 0·45 (s.e. 0·17), on day 128 of pregnancy of 1·17, 0·94, 0·52, 0·51, 0·72, 0·62 and 0·39 (s.e. 0·20), on day 135 of pregnancy of 1·53, 0·68, 0·68, 0·66, 0·71, 0·62 and 0·46 (s.e. 0·20) and on day 142 of pregnancy of 1·43, 0·60, 0·62, 0·56, 0·62, 0·56 and 0·63 (s.e. 0·20) were recorded for Tl to T7 respectively. There was a quadratic relationship between plasma BHB concentration and ME intake on days 121 (R2 = 0·538,P< 0·001), 128 (R2 = 0·324,P< 0·001), 135 (R2 = 0·429,P< 0·001)) and 142 (R2 = 0·344,P< 0·002) of pregnancy. There was a positive relationship between plasma glucose concentration and ME intake on day 222 (R2 = 0·208,P< 0·002), 228 (R2 = 0·203,P< 0·05), and 135 (R2 = 0·160,P< 0·02) of pregnancy. Albumin concentrations (gll) on day 128 of pregnancy of 21·8, 21·7, 23·6, 22·9, 22·5, 22·9 and 24·3 (s.e. 0-75), on day 135 of 20·9, 23·6, 24·2, 24·1, 22·4, 24·1 and 23·8 (s.e. 0·75), and on day 142 of 16·9, 22·6, 20·7, 22·2, 20·4, 22·7 and 21·1 (s.e. 1·05) were recorded for Tl to T7 respectively. Plasma albumin concentrations were significantly affected by SBM supplementation (P< 0·05). Despite the lower than generally recommended energy concentrations in T2 to T7, the concentrations of plasma BHB were within the normal range for healthy sheep.

Experimental optimization of fed-batch culture for poly-β-hydroxybutyric acid production

The optimal feed rate profiles of glucose and ammonium hydroxide were calculated using a proposed model, and implemented for the production of poly-beta-hydroxybutyric acid (PHB) by Alcaligenes eutrophus. By implementing these optimal feed rates with a high glucose feed concentration of 700 g/L and an ammonium hydroxide concentration of 7%(w/w), it was possible to achieve a high final cell concentration of 141 g/L and a high PHB concentration of 105 g/L in 40 h of fed-batch operation. The PHB productivity was as high as 2.63 g/(L hr). (c) 1997 John Wiley & Sons, Inc. Biotechnol Bioeng 56: 697-705, 1997.

Identification of key patterns in the metabolism of hybridoma cells in culture

In transformed cell lines, glucose and glutamine transport into cells as well as glycolysis and glutaminolysis pathways occur at high rates. As a consequence, cells in culture show a poor exploitation of these resources and produce excessive amounts of lactic acid and ammonium. In this work, a murine hybridoma cell line (KB-26.5) was cultivated in batch cultures with different concentrations of oxamate, an specific inhibitor of lactate dehydrogenase. The results show that the conversion of pyruvate into lactate is absolutely necessary for cell survival as a way to reoxidize the large amounts of NADH produced in the rapid glycolysis; moreover, this metabolic analysis was completed with a continuous culture at changing inlet concentrations of glucose and glutamine. Steady-state viable cell, total cell, glucose, lactate, ammonium glutamine, and other amino acids were measured over a wide range of glucose and glutamine feed concentrations. The results show the metabolic deregulation existing in hybridoma cells and make it possible to calculate the minimal specific requirements for glucose (76.8 nmol 10 6 cells −1 h −1) and glutamine (30.72 nmol 10 6 cells −1 h −1) to maintain a cell population with minimal excretion of partly oxidized undesirable end products and a minimal increase in osmolarity. An explanation of these metabolic patterns is discussed in terms of NADH regeneration.

Production of Ethanol by Continuous Fermentation and Liquid-Liquid Extraction

A continuous pilot plant was constructed for fermentation production of ethanol, using liquid–liquid extraction to remove the product and with recycle of the fermented broth raffinate. The plant was operated for up to 18 days with feed glucose concentrations in the range 10·0–45·8% (w/w). The solvent was n-dodecanol and immobilised yeast was used to overcome the problem of emulsification. The concentration of by-products in the fermented broth had no adverse effect on the rate of ethanol production. A mathematical model to predict the time required for achievement of 99% of the steady-state by-products concentrations was shown to be in good agreement with the experimentally determined concentration of the main by-product, glycerol. At a feed glucose concentration of 45·8% (w/w), the aqueous purge was equivalent to 2·8 m3 of effluent per m3 of ethanol produced and represented a 78% reduction in the volume of the aqueous purge compared with using a feed containing 10% (w/w) glucose.

Quantification of small enthalpic differences in anaerobic microbial metabolism—a calorimetry-supported approach

A calorimetry-supported approach to the measurement of small enthalpic differences in microbial metabolism is presented. Anaerobic fermentations with the bacterium Zymomonas mobilis and the yeast Saccharomyces cerevisiae were carried out in a fermenter-calorimeter at a constant dilution rate ( D = 0.15 h −1). The glucose concentration of the feed was increased stepwise from 10 to 80 g l −1. Salts, vitamins and trace elements were present in excess. Different steady state conditions were analysed. For Z. mobilis, specific production rates for ethanol, carbon dioxide and heat were three times higher than those measured for S. cerevisiae. If the glucose feed concentration exceeded 20 g l −1 during the S. cerevisiae fermentations, glucose could be measured in the fermenter. Further increasing glucose concentrations of the feed caused higher specific ethanol, carbon dioxide and heat production rates, whereas the biomass yield Y X S decreased remarkably. This result can be explained as the Crabtree effect under anaerobic conditions and interpreted as metabolic uncoupling of anabolic biomass formation and catabolic energy substrate consumption. A futile cycle contributing to the heat production is considered to provide S. cerevisiae with a regulatory mechanism in the control of the flux of metabolites through the glycolytic pathway.

Stability analysis of continuous culture in diauxic growth

The stability of steady states and dynamic behaviors in a diauxic growth process were studied in a continuous culture with a mixed substrate. The stability of steady states was analytically discussed based on the eigenvalues using the authors' previously proposed diauxic model. The number and stability of steady states appeared to vary with operational conditions such as dilution rate and feed substrate concentrations. The steady states can be classified into three categories: (i) a steady state where both substrates were consumed, (ii) a steady state where only one substrate was consumed and (iii) washout. To achieve a stable steady state where both substrates are consumed, the manipulated variables such as feed substrate concentration and dilution rate can be selected within a comparatively large area of values: dilution rate (0 to 0.7 h −1) against feed maltose concentration (0 to 1 g/ l) in the case of feed glucose concentration (0.3 g/ l) and against feed glucose concentration (0 to 1 g/ l) in the case of feed maltose concentration (0.7 g/ l). In the experiment involving a step change from a batch culture to a continuous culture, not only the dilution rate but also the amount of inducible enzyme in the cells was closely connected to the consumption of the two substrates in a continuous culture. The dynamic behaviors of cell concentration and substrate concentrations in the case of large step changes in feed concentrations were also examined under several operational conditions.

Continuous fermentation with product recovery by in‐situ extraction

AbstractThe productivity of fermentations is often limited by end product inhibition. This can be avoided by continuous removal of the inhibiting product from the broth. Such in‐situ separation can be conveniently accomplished by liquid‐liquid extraction. As an example, the continuous fermentation of ethanol by the thermophilic, anaerobic bacterium Clostridium thermohydrosulfuricum is investigated in a 20‐1 fermenter with simultaneous in‐situ extraction by oleyl alcohol as organic solvent. Continuous fermentations with and without in‐situextraction were carried out with systematic variation of the independent operating conditions, viz. feed glucose concentration, broth flow rate and solvent flow rate. The experimental results of 18 steady states are reported. They show that in‐situ extractions doubles the yield, selectivity and space‐time yield of ethanol in comparison to fermentations without in‐situ extraction. A biomodel elucidates the influence of feed glucose concentration, broth flow rate and solvent flow rate on the productivity of the fermentation process. Finally, a cost model was developed for the investigated fermentation which allows economic evaluation of the results of experiments and simulation. A sensitivity study elucidates the economic limits and advantages of fermentation with in‐situ extraction compared to a common fermentation without product separation.

The Electrocatalytic Hydrogenation of Glucose: II . Raney Nickel Powder Flow‐Through Reactor Model

A computer model which simulates the operation of a flow‐through Raney nickel powder electrocatalytic hydrogenation reactor for the synthesis of sorbitol from glucose with simultaneous evolution has been developed. The model utilizes porous electrode theory, considers both mass‐transfer and surface kinetics effects, and contains no adjustable parameters. Hydrogen evolution on Raney nickel is described by a Volmer‐Heyrovský rate expression. The rate equation for glucose hydrogenation is identical to that for the chemical catalytic synthesis of sorbitol with pressurized gas. For constant‐current reactor operation, computed sorbitol current efficiencies match well with experimental data for a range of current densities (0.0053 to 0.021 A/cm2) and glucose feed concentrations (0.4 to 1.6M), with an average error of 8.8%. Calculations show that a large fraction of adsorbed hydrogen on the nickel cathode surface is produced by the oxidation of electrogenerated via the backward Heyrovský reaction. According to the model, significantly higher sorbitol current efficiencies can be achieved by pulsing the current to the reactor.

Production of acetone-butanol-ethanol by Clostridium acetobutylicum using a spin filter perfusion bioreactor

The performance of a custom-built cell perfusion bioreactor was investigated in the biosynthesis of acetone, butanol and ethanol by Clostridium acetobutylicum. In a continuous flow operating mode with total cell retention, i.e., when only the cell-free spent medium was removed, steady-state concentrations of acetone, butanol, ethanol, butyric acid, acetic acid and residual glucose and increasing biomass concentration were observed up to 49 g l −1 of glucose in the feed stream. The steady-state concentrations of acetone, butanol, ethanol, butyric acid and acetic acid were independent of the dilution rate and a function of the feed glucose concentration. Under these conditions a complete utilization of glucose with a maximum volumetric productivity of 1.14 g l −1 h −1 of total solvents was obtained. Increasing the feed glucose concentration to 58 and 63 g l −1 in total cell retention mode led to sustained undampened oscillations resulting from product inhibition. These oscillations were eliminated when a full harvest stream of cell containing spent medium was withdrawn in addition to the cell-free filtrate stream.

Optimal feeding policy for recombinant protein production via the yeast saccharomyces cerevisiae in fed-batch culture

Optimisation of the glucose feed rate and feed concentration for the production of a recombinant protein (β-galactosidase) with respect to two different objective functions is addressed in this work. The performance indices optimised were recombinant protein productivity and overall production cost, respectively. The optimisation was based on a model developed and validated independently. The design of a suboptimal feeding policy which is more practical and easier to implement was confirmed experimentally. Sensitivity analyses of the model parameters allowed the effect of disturbances on recombinant protein productivity to be assessed.

Fuzzy control of baker's yeast fed-batch bioprocess: A robustness study

The purpose of the investigation was to determine if, based on the monitoring of the residual substrate concentration, fuzzy control is an effective way of controlling a fermentation process. Various simulations were made using a knowledge model, first to evaluate the performance of the control for the biomass concentration, and then to analyze how it reacted in the presence of disturbances. Results showed high final productivity and biomass concentration. Despite different disturbances, as the feeding glucose concentration changed or the temporary substrate feed-rate increased, the fuzzy controller provided process stability and robustness near the set-point. Moreover, these properties were maintained in the case of noisy measurements.

Maximizing the production of acetone—butanol in an alginate bead fluidized bed reactor using clostridium acetobutylicum

AbstractLow volumetric solvent productivities are one of the characteristics of an acetone‐butanol fermentation by C. acetobutylicum. A calcium alginate immobilized continuous culture was used in a novel gas‐sparged reactor to strip the solvents from the aqueous phase and reduce their toxicity. A dilution rate of 0.07 h−1 was found to give maximum solvent productivity at 0.58 g dm−3 h−1, although at 0.12 h−1 the productivity was slightly lower. In order to increase glucose uptake by the culture, feed glucose concentrations were increased over time to attempt to acclimatize the culture. This resulted in a productivity as high as 0.72 g dm−3 h−1 although this production rate was found to be unstable.

Performance of a cross-linked immobilized cell reactor with Zymomonas mobilis using synthetic and complex media for ethanol production

The performance of ethanolic bacterium, Zymomonas mobilis, ATCC 31821, cross-linked to glutaraldehyde-treated, gelatin-coated spherical glass beads in an immobilized cell reactor (ICR) was evaluated for feed glucose concentrations of 150 and 200 g l −1. A synthetic medium, containing inorganic nitrogen and salts, and a complex medium, containing yeast extract, were both used. Two acid-base buffers, succinate and acetate, were added to the medium to regulate the pH of the broth in the ICR. The use of acetate in the broth increased the maximum volumetric ethanol productivity to 19 g l −1 h −1 compared to 16 gl −1 h −1 without acetate. Adding acetate to the medium increased the reactor operating life by decreasing cell growth rate. The reactors were operated from 34–57 days. The ICR fermentations conducted with the synthetic medium gave a comparable maximum volumetric ethanol productivity of 16 gl −1 h −1. Axial temperature distributions of 4–6°C were measured during the fermentation.

Physiological Effects of γ-Linolenic Acid and Its Production by Fungal Cultivation

As a n-6 essential fatty acid, γ-linolenic acid (GLA) has been shown to be more effective than linoleic acid. Its effects cover anti-atherosclerosis including hypocholesterolic and anti-thrombogenic effect, anti-cancer, higher alcohol oxidation, anti-inflammatory effect and others. These effects are generally attributed to by-passing Δ6-desaturase reaction.Using some species in genus Mortierella, lipid accumulative fungi, attempt at increase in lipid and GLA productivity has been made changing carbon and nitrogen source, pH, temperature and others. Decane as carbon source resulted in higher polar lipid content in spite of lower growth than glucose. Cultivation at higher glucose concentration (>100 g/L), originally intended to get higher cell mass per unit volume of culture medium, resulted in higher lipid content. The optimum culture conditions for lipid accumulation were lower both in pH and temperature than those for growth, which lead to our proposal of 2 tanks continuous system to separate growth from lipid accumulation phase. The maximum lipid productivity of 1.1 g/L·h and GLA productivity of 106 mg/L·h were obtained in a continuous operation at 0.15-1 of dilution rate and 100 g/L of feed glucose concentration.

Kinetics and regulation of α-tyrosinase synthesis in continuous culture of Escherichia intermedia

Studies on synthesis of α-tyrosinase in E. intermedia were conducted in continuous culture with glucose as a carbon source and tyrosine as an inducer. Dynamic responses in the specific activity of the α-tyrosinase, RNA content in cells, cell and glucose concentrations were monitored. The specific synthesis rate of α-tyrosinase and its corresponding ribosomal efficiency, defined as the enzyme synthesis rate per unit weight of RNA, were estimated to study the kinetics and regulation of the enzyme synthesis in steady-state culture. Sustained oscillation in α-tyrosinase activity was observed for continuous culture at low dilution rates. This might be due to the interacting effect of the induction by tyrosine and of the repression by phenol formed endogenously from tyrosine assimilation on α-tyrosinase protein synthesis. The specific rate of the enzyme synthesis was found to be proportional to the specific growth rate of the cell. The ribosomal efficiency for enzyme synthesis increased linearly with the specific growth rate. Much more glucose metabolism might be required to achieve a high rate and efficient protein synthesis for extremely fast growing cells and, thus, resulted in a lower cellular yield. Synthesis of α-tyrosinase was repressed by glucose metabolism. Increasing the feeding glucose concentration led to a lowering in ribosomal efficiency and slight increase in RNA content in cells. The glucose effect on ribosomal efficiency was further elucidated by adding stimulator or inhibitor to regulate gene transcription.

Enhanced spore production ofBacillus thuringiensis by fed-batch culture

Fed-batch culture was carried out to increase cell mass followed by batch culture for spore production ofbacillus thuringiensis. High cell mass obtained by increasing the feeding glucose concentration in constant fed-batch culture which supported fast cell growth resulted in good sporulation during subsequent batch culture, and the maximum cell mass of 72.6 g/L and spore concentration of 1.25×1010 spores/mL could be obtained.

Glucose‐limited chemostat culture of chinese hamster ovary cells producing recombinant human interferon‐γ

A Chinese hamster ovary (CHO) cell line expressing recombinant human interferon-gamma (IFN-gamma) was grown under glucose limitation in a chemostate at a constant dilution rate of 0.015 h(-1) with glucose feed concentrations of 2.75 mM and 4.25 mM. The changes in cell concentration that accompanied changes in the glucose feed concentration indicated that the cells were glucose-limited. The cell yield on glucose remained constant, but there was a decline in residual glucose concentration and a reduced lactate yield from glucose in the latter stages of the culture. The consumption rates for many of the essential amino acids were increased later in the culture. The volumetric rate of interferon-gamma production was maintained throughout the course of this culture, indicating that IFN-gamma expression was stable under these conditions. However, the specific rate of IFN-gamma production was significantly lower at the higher glucose feed concentration. Under glucose limitation, the proportion of fully glycosylated IFN-gamma produced by these cells was less than that produced in the early stages of batch cultures. The proportion of fully glycosylated IFN-gamma increased during transient periods of glucose excess, suggesting that the culture environment influences the glycosylation of IFN-gamma.

Physiology of myeloma cells grown in glucose-limited chemostat cultures.

A recombinant myeloma NS1-derived clone was grown in chemostat cultures in Dulbecco's MEM/Ham's F12 (1:1) medium containing various concentrations of glucose, at a dilution rate of 0.028 h-1. Serum-supplemented cultures were virtually glucose-limited at a large range of glucose feed concentrations (0.7-5 mM). True glucose-limited cultures, however, were only established at low glucose supply levels to 1.3 mM at a maximum. In cultures obtained at higher glucose concentrations methionine was shown to be the growth-limiting compound. The pattern derived for serum-free chemostat cultures was similar, except that growth yields on glucose were much lower. Glucose was shown to be the growth-limiting substrate in cultures fed with media containing less than 4.5 mM glucose. Upon supplying glucose at higher concentrations such cultures presumably run into methionine and/or tryptophan limitation.

Measurement and regulation of the culture reduction state in Clostridium acetobutylicum

With a constant glucose feed concentration, the change in the continuous culture dillution rate resulted in an altered fermentation profile and the cellular NADH content. The cultures growing at high dillution rates demonstrated an oxidative metabolism low NADH and butanol concentrations. The low specific NADH flourescence (F/X) at high butanol production rates suggested that a rapid regeneration of NADH to NAD is essential for a high solventogenic culture activity. The culture florescence and butanol concentration remained constant in the solventogenic dilution rate range of D = 0.05-0.2 h(-1) with an inverse relationship between the specific flourescence (F/X) and the specific butanol production rate, q(B). Flourometric NADH observations were confirmed by enzymatic NADH determination. The stiochiometric "Fermentation Equation" was used to check the experimental data consistency and to investigate the role of the available biosynthetic and reduction energy on the culture metabolic activities under different growth conditions. The butanol concentration in the broth was stabilized in a fed-batch process when the culture NADH fluorescence was being controlled through the addition of fresh medium.