Influence of different ammonium,lactate and glutamine concentrations on CCO cell growth

In this study the effects of ammonium and lactate on a culture of channel catfish ovary (CCO) cells were examined. We also made investigation on the influence of glutamine, since our previous research revealed that this amino acid stimulated CCO cell growth more than glucose in a concentration-dependent manner. The effect of ammonium in cell culture included the considerable decrease in cell growth rate with eventual growth arrest as well as the retardation of glucose consumption. At ammonium concentrations above 2.5 mM, the cells displayed specific morphological changes. The effect of lactate was different to that of ammonium since the cell growth rate was progressively decreasing with the increase of lactate concentration, whereas the glucose consumption rate remained almost unchanged. Besides that, it was found that lactate was steadily eliminated from the culture medium when its initial concentration was relatively high. The influence of glutamine on CCO cell propagation showed that nutrient requirements of this cell line were mainly dependent on glutamine rather than glucose. The increase in glutamine concentration led to the increase in cell growth rate and consequent ammonia accumulation while the glucose utilization and lactate production were reduced. Without glutamine in culture medium cell growth was arrested. However, the lack of glucose reversed the stimulating effect of glutamine by decreasing cell growth rate and affecting amino acid utilization.     

Metabolic characterization of a L-lysine-producing strain by continuous culture

Continuous culture experiments with the L-producer, Corynebacterium glutamicum, were carried out to characterize the effect of specific growth rate on fermentation yields, specific rates, productivities, and fluxes through the primary metabolism. The specific productivity of L-lysine exhibited a maximum with respect to specific growth rate, with an initial growth-associated behavior up to specific growth rates of about 0.1 h(-1), and a constant specific productivity for specific growth rates in the range of about 0.1 to 0.2 h(-1). The productivity dropped at specific growth rates larger than about 0.2 h(-1). The yield of L-lysine on glucose increased approximately linearly with decreasing specific growth rate over the entire range studied, as did the respiratory quotient. A direct relationship was established between the culture respiratory quotient and the L-lysine yield. By explicitly accounting for glucose used for biomass synthesis, it was shown that the strain synthesizes L-lysine with an intrinsic yield, or efficiency, of about 0.41 mol L-lysine/mol glucose, compared with the theoretical yield of 0.75 mol/mol. Metabolic flux modeling based on the continuous culture data suggests that the production of ATP is not likely to be a limiting factor in L-lysine production, and that a high TCA cycle activity, coupled with a tightly controlled split of metabolite flow at the PEP node, is likely the cause of the large discrepancy between theoretical and actual yields in L-lysine fermentations.     

Effect of the level of the carbon source on the activity of some lytic enzymes released during autolysis of Aspergillus niger

Changes in the activity of -N-acetylglucosaminidase, chitinase, invertase, esterases, glucanases and phosphatases liberated into the culture fluid were followed during the autolytic phase of growth of Aspergillus niger on media with various initial levels of the carbon source. The general pattern was of an accumulation of these lytic enzymes in the culture fluid during autolysis, but some enzymes reached maximum activity and then declined. The initial level of the carbon source affected the enzyme pattern during autolysis. Maximum activity for the various enzymes was always observed either for the lowest initial level of carbon or the highest (3.5 mM glucose, 111 mM glucose). The highest specific activities were those for exopolygalacturonidase (500 mU/mg at 3.45 mM glucose), and for -amylase (about 500 mU/mg at 3.45 mM glucose). Cellulase, chitinase and esterase showed the weakest activity. Acid phosphatase was most active (about 200 mU/mg) at 3.45 mM initial glucose, whereas alkaline phosphatase was most active (45 mU/mg) at 111 mM glucose, both during the autolytic phase of growth.     

Studies of the Acetate Kinase-Phosphotransacetylase and the Butanediol-Forming Systems in Aerobacter aerogenes

Mutants of Aerobacter aerogenes devoid of acetate kinase and phosphotransacetylase activities were isolated by selection for resistance to fluoroacetate on lactate medium. The mutants were used to study the role of the acetate kinase-phosphotransacetylase system in growth on acetate and glucose. Acetate kinase-negative and phosphotransacetylase-negative mutants were unable to grow on acetate minimal medium. Their growth rates on glucose minimal medium were identical with that of the parent strain under aerobic conditions, but lower growth rates were observed in the mutant strains during anaerobic growth on glucose medium. The mutants were unable to incorporate [2-(14)C]-acetate rapidly while growing on glycerol. Variations in acetate kinase and phosphotransacetylase levels during growth on glucose were studied. The specific activities of the enzymes increased approximately fivefold during aerobic growth on glucose in batch culture. The enzyme levels were also studied during anaerobic growth on glucose at constant pH (pH 5.8 and 7.0). Smaller increases in specific activities were found under these conditions. The role of acetate in the induction of the diacetyl (acetoin) reductase was investigated using a mutant deficient in both acetate kinase and phosphotransacetylase. The effect of pH on the induction of this enzyme during growth on glucose under anaerobic conditions was tested. The data support the idea that free acetic acid is the inducer for the enzymes of the butanediol-forming pathway in A. aerogenes.     

A kinetic analysis of hybridoma growth and metabolism in batch and continuous suspension culture: effect of nutrient concentration, dilution rate, and pH

Hybridomas are finding increased use for the production of a wide variety of monoclonal antibodies. Understanding the roles of physiological and environmental factors on the growth and metabolism of mammalian cells is a prerequisite for the development of rational scale-up procedures. An SP2/0-derived mouse hybridoma has been employed in the present work as a model system for hybridoma suspension culture. In preliminary shake flask studies to determine the effect of glucose and glutamine, it was found that the specific growth rate, the glucose and glutamine metabolic quotients, and the cumulative specific antibody production rate were independent of glucose concentration over the range commonly employed in cell cultures. Only the specific rate of glutamine uptake was found to depend on glutamine concentration. The cells were grown in continuous culture at constant pH and oxygen concentration at a variety of dilution rates. Specific substrate consumption rates and product formation rates were determined from the steady state concentrations. The specific glucose uptake rate deviated from the maintenance energy model(1) at low specific growth rates, probably due to changes in the metabolic pathways of the cells. Antibody production was not growth-associated; and higher specific antibody production rates were obtained at lower specific growth rates. The effect of pH on the metabolic quotients was also determined. An optimum in viable cell concentration was obtained between pH 7.1 and 7.4. The viable cell number and viability decreased dramatically at pH 6.8. At pH 7.7 the viable cell concentration initially decreased, but then recovered to values typical of pH 7.1-7.4. Higher specific nutrient consumption rates were found at the extreme pH values; however, glucose consumption was inhibited at low pH. The pH history also influenced the behavior at a given pH. Higher antibody metabolic quotients were obtained at the extreme pH values. Together with the effect of specific growth rate, this suggests higher antibody production under environmental or nutritional stress.     

Control of gene expression from the SUC2 promoter of Saccharomyces cerevisiae with the aid of a glucose analyser

Summary A Saccharomyces cerevisiae strain harbouring the recombinant plasmid pSMF38TMA was cultured in a jar fermentor under the control of glucose concentration. In the recombinant plasmid, the mouse -amylase gene was fused to the S. cerevisiae SUC2 promoter. When glucose concentration in the medium was controlled at 10 g/l, the gene expression was completely repressed. On the other hand, the -amylase was produced and secreted in the medium at a very high level, around 200 mg/l as evaluated from the specific activity of commercially available human salivary amylase, when the glucose was kept at 0.15 g/l. This amount was almost 20-fold that obtained at 10 g/l glucose. The specific growth rate of the yeast in this culture was almost 60% of that attained with 10 g/l glucose. To obtain higher cell growth and productivity, the yeast was at first cultured at 2 g/l glucose and the concentration was then lowered to 0.15 g/l. By this control of the glucose concentration, on-off regulation of gene expression from the SUC promoter could be attained.     

Effects of lactate concentration on hybridoma culture in lactate-controlled fed-batch operation

To investigate the effects of lactate on cell growth and antibody production, a new method of maintaining the lactate concentration constant in a fed-batch culture is described. When the pH was initially adjusted by sodium hydroxide, the specific growth rate decreased and specific death rate increased with an increase of lactate concentration. To investigate whether the inhibition was due to the lactate concentration itself or to the osmotic pressure, the effect of the osmotic pressure adjusted by sodium chloride was compared with that of sodium lactate. When the osmotic pressure was adjusted to same condition as that of sodium lactate using sodium chloride, the specific growth data showed the same degree of growth inhibition. It was thus evident that the inhibition to cell growth was mainly due to osmotic pressure while lactate production from glucose was found to be inhibited by the lactate itself compared with sodium chloride. The specific antibody production rate had a maximum value within a certain range of lactate concentration. Moreover, specific antibody production rate had a unified relationship with the kinetic parameter mu, in spite of the different causes of inhibition by lithium lactate and sodium lactate. A certain "trade-off" relationship between growth and antibody production existed at higher growth rates.     

Influence of pH and malate-glucose ratio on the growth kinetics of Leuconostoc oenos

Summary Growth, substrate utilization and product formation were studied in batch cultures of a Leuconostoc oenos strain. The effect of various culture conditions, i.e. pH-control at different values and various initial concentrations of malate and glucose, on growth and metabolism were investigated. Addition of malate resulted in a marked stimulation of growth, with only a slight increase in final biomass but a high conversion yield of glucose. Under pH control this stimulation was much greater than could be accounted for from changes in pH profile resulting from malate utilization. The specific rate of malate utilization was maximal at pH 4.0 whereas the specific rate of glucose consumption was highest at pH 5.5. During co-metabolism of malic acid and glucose, substrate utilization and product formation agreed with the stoichiometric relationships of the malo-lactic reaction and the heterolactic fermentation of glucose. Offsprint requests to: A. Pareilleux     

Chemostat studies on the regulation of glucose metabolism in Pseudomonas aeruginosa by citrate

The effect of the relative concentrations of citrate and glucose on the regulation of key enzymes of the direct oxidative, phosphorylative, Entner-Doudoroff and pentose-cycle pathways of glucose metabolism in Pseudomonas aeruginosa has been investigated in continuous culture under conditions of NH(4) (+)-limitation. For comparison isocitrate dehydrogenase and aconitase were also assayed. Measurements were made for steady-state and transient conditions and the effect of growth rate was also studied. When cells grew on 75mm-citrate the glucose concentration had to attain 6-8mm before significant induction of enzymes of glucose metabolism occurred; the specific activities increased further as the result of both raising the glucose concentration to 30mm and then subsequently lowering the citrate to 60mm and then to 45mm. The specific activities of the glucose enzymes increased immediately during the transient period between the steady states characteristic of growth on 6mm- and 8mm-glucose, the increase continuing for about two doubling times. The converse experiment of adding increasing citrate concentrations to 45mm-glucose medium revealed an immediate induction of the citrate-transport system, oxidation of citrate following the increase in citrate concentration up to 8mm. Between 8mm- and 16mm-citrate a marked repression of gluconate, glucose 6-phosphate and 6-phosphogluconate dehydrogenases and the Entner-Doudoroff enzymes occurred. Increased growth rate in citrate medium resulted in decreased specific activities of glucose 6-phosphate dehydrogenase and isocitrate dehydrogenase. Increased growth rate in citrate-glucose medium gave decreased specific activities of isocitrate dehydrogenase and aconitase whereas the activities of some of the glucose enzymes decreased initially but then increased at the highest growth rate (0.5h(-1)), at which a marked increase in glucose utilization occurred. These observations accord with the regulation of glucose enzymes by induction with glucose or its metabolites and repression by citrate or its metabolic products.     

A kinetic analysis of hybridoma growth and metabolism in batch and continuous suspension culture: effect of nutrient concentration, dilution rate, and pH. Reprinted from Biotechnology and Bioengineering, Vol. 32, Pp 947-965 (1988)

Hybridomas are finding increased use for the production of a wide variety of monoclonal antibodies. Understanding the roles of physiological and environmental factors on the growth and metabolism of mammalian cells is a prerequisite for the development of rational scale-up procedures. An SP2/0-derived mouse hybridoma has been employed in the present work as a model system for hybridoma suspension culture. In preliminary shake flask studies to determine the effect of glucose and glutaminE, it was found that the specific growth rate, the glucose and glutamine metabolic quotients, and the cumulative specific antibody production rate were independent of glucose concentration over the range commonly employed in cell cultures. Only the specific rate of glutamine uptake was found to depend on glutamine concentration. The cells were grown in continuous culture at constant pH and oxygen concentration at a variety of dilution rates. Specific substrate consumption rates and product formation rates were determined from the steady state concentrations. The specific glucose uptake rate deviated from the maintenance energy model(1) at low specific growth rates, probably due to changes in the metabolic pathways of the cells. Antibody production was not growth-associated; and higher specific antibody production rates were obtained at lower specific growth rates. The effect of pH on the metabolic quotients was also determined. An optimum in viable cell concentration was obtained between pH 7.1 and 7.4. The viable cell number and viability decreased dramatically at pH 6.8. At pH 7.7 the viable cell concentration initially decreased, but then recovered to values typical of pH 7.1-7.4. Higher specific nutrient consumption rates were found at the extreme pH values; however, glucose consumption was inhibited at low pH. The pH history also influenced the behavior at a given pH. Higher antibody metabolic quotients were obtained at the extreme pH values. Together with the effect of specific growth rate, this suggests higher antibody production under environmental or nutritional stress.     

Influence of cytokinins on growth of Phycomyces blakesleeanus and on the activities of the glyoxylate cycle enzymes isocitrate lyase and malate synthase

Treatment of the 1 + strain of Phycomyces blakesleeanus Bgff. with various cytokinins resulted in a stimulation of growth. The magnitude of growth stimulation depended on both the structure of the hormone used and the carbon source in the culture medium. Most of the cytokinin derivatives were active effect in glucose and oleic acid cultures. Benzyladenine (BA) and benzyladenosine stimulated the fungal growth only when oleic acid was the sole carbon source, while they had no effect in glucose cultures within the tested range of concentrations. [¹⁴C]-BA was accumulated by the mycelium of oleic acid cultures. Therefore, differences in BA uptake between glucose and oleic acid cultures could account mainly for the specific growth-promoting effect of BA. In oleic acid cultures isocitrate lyase (EC 4.1.3.1) and malate synthase (EC 4.1.3.2) activities were enhanced by 40 and 34%, respectively, in the presence of BA. A time course of the hormone effect suggests that BA is not involved in induction, but in the regulation of the mentioned enzymes in Phycocmyces. In contrast, acetate when presented as the sole carbon source or after addition to a glucose culture medium, induced isocitrate lyase activity. This enzyme induction was prevented by simultaneous addition of cycloheximide.     

谷氨酰胺对微囊化重组CHO细胞生长代谢及内皮抑素表达的影响

微囊化技术是一种有发展潜力的生物技术,在细胞移植和药物控释等方面具有广泛的应用。然而由于目前微囊化细胞规模化培养技术还不成熟,阻碍了其在临床治疗中的推广与应用。为了了解微囊化重组CHO细胞的生长代谢特性为今后规模化培养优化提供技术参考,考察了主要氮源物质谷氨酰胺对微囊化重组CHO细胞生长代谢及内皮抑素表达的影响。结果显示:当谷氨酰胺起始浓度从2.69mmolL增加到9.05mmolL时最大活细胞密度并没有增高,细胞增殖没有显著差异。当谷氨酰胺起始浓度较低(2.69mmolL)时,葡萄糖的比消耗速率较大;当谷氨酰胺起始浓度增高时(7.91mmolL～9.05mmolL)葡萄糖和谷氨酰胺的比消耗速率增大,但细胞对葡萄糖和谷氨酰胺的利用率降低。谷氨酰胺对产物表达有显著影响,起始浓度为4.97mmolL时的内皮抑素累积浓度最高,达546.36ngmL,过低和过高谷氨酰胺起始浓度下内皮抑素的累积浓度均较低。     

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.     

Isothermic variation of the specific growth rate of Saccharomyces cerevisiae in batch culture

The specific growth rate () of a respiration-deficient mutant of Saccharomyces cerevisiae growing under defined experimental conditions in batch culture (mineral medium plus glucose and vitamins at 25°C) varied from experiment to experiment over a wide range (0.10–0.24 h^-1) and showed a normal distribution. Neither the age of the culture, the history of the inoculum, nor experimental error accounted wholy for the variability of . The variation was positively correlated with the specific rate of glucose transfer and negatively with the specific rate of production of non-fermentative CO₂. The yield decreased with implying higher maintenance requirements in batch culture (4.7 mmoles g^-1 h^-1) than in continuous culture (0.8 mmoles g^-1 h^-1). It was concluded that the strain is capable of establishing any one of several steady states of growth under the same experimental conditions, each steady state displaying some buildin inertia with respect to change. The variations of the specific rates of glucose transfer and non-fermentative CO₂ production, and of the yield appeared to be consequences rather than causes of the variation of . The ultimate causes of the variation of remained unidentified.Part of a doctoral thesis submitted by J. Martinez-Peinado to the University of Navarra Spain     

Effect of glucose analog supplementation on metabolic flux distribution in anaerobic chemostat cultures of Escherichia coli

Previous work in our laboratories investigated the use of methyl alpha-glucoside (alpha-MG), a glucose analog that shares a phosphotransferase system with glucose, to modulate glucose uptake and therefore reduce acetate accumulation. The results of that study showed a significant improvement in batch culture performance and a reduction in acetate excretion without any significant effect on the growth rate in complex medium. The current study investigates the effect of supplementing the culture medium with the glucose analog alpha-MG on the metabolic fluxes of Escherichia coli under anaerobic chemostat conditions at two different dilution rates. Anaerobic chemostat studies utilizing complex media supplemented with glucose or glucose and alpha-MG at dilution rates of 0.1 and 0.4 h(-1), were performed, and the metabolic fluxes were analyzed. It was found that the addition of the glucose analog alpha-MG has an effect on the specific production rate of various extracellular metabolites. This effect is slightly greater at the higher dilution rate of 0.4 h(-1). However, the glucose analog does not cause any major shift in the central metabolic patterns. It was further observed that alpha-MG supplementation does not result in the reduction in specific acetate synthesis rate in anaerobic chemostat cultures. These results emphasize the importance of testing different strategies for metabolic manipulation under the actual operating conditions.     

Biosynthesis of growth hormone in the rat anterior pituitary gland. Stimulation of biosynthesis in vitro by insulin

The effect of insulin on the incorporation of radioactive leucine into growth hormone was investigated by using rat anterior pituitary glands incubated in vitro. A 50% stimulation over control values was observed at insulin concentrations above 2μm (280munits/ml). The effect was specific for growth hormone biosynthesis, over the range 1–5μm-insulin (140–700munits/ml). Lower more physiological concentrations had no significant effect in this system. Above 10μm (1.4 units/ml) total protein synthesis was also increased. The stimulation of growth hormone synthesis could be partially blocked by the addition of actinomycin D, suggesting that RNA synthesis was involved. Insulin was found to stimulate the rate of glucose utilization in a similar way to growth hormone synthesis. 2-Deoxyglucose and phloridzin, which both prevented insulin from stimulating glucose utilization, also prevented the effect of insulin on growth hormone synthesis. If glucose was replaced by fructose in the medium, the effect of insulin on growth hormone synthesis was decreased. We conclude that the rate of utilization of glucose may be an important step in mediating the effect of insulin on growth hormone synthesis.     

Control of L-phenylalanine production by dual feeding of glucose and L-tyrosine

Control of L-phenylalanine production by a recombinant of Escherichia coli AT2471 by means of the dual feeding of glucose and L-tyrosine was investigated. A novel method was developed for on-line monitoring of the maximum glucose uptake rate (MGUR), in which the length of time required for the consumption of added glucose was measured. Accumulation of acetic acid was successfully prevented throughout the whole period of the culture when the glucose concentration was kept below 0.1 g/L by controlling the glucose feeding on the basis of on-line monitoring of the MGUR and the cell concentration with a laser sensor.In a batch culture with glucose feeding, after L-tyrosine was depleted cell growth and the L-phenylalanine production rate decreased along with decreases in the specific enzyme activities of chorismate mutase-p-prephenate dehydratase (CMP) and 3-deoxy-D-arabinoheputulosonate 7-phosphate synthase (DAHP), which are the key enzymes in the L-phenylalanine synthesis pathway. Increasing the L-tyrosine feed rate by an appropriate amount, but not so far as to cause L-tyrosine accumulation in the culture, increased the activities of the enzymes and the specific rates of growth and production while the product yield based on glucose consumption decreased.The average specific rates of growth, production, and MGUR could be expressed as functions of the specific L-tyrosine consumption rate during both the earlier and later periods of L-tyrosine feeding. Estimations of the amount of L-phenylalanine produced, the product yield, and the cost factor by using these functions with several different combinations of two specific L-tyrosine consumption rates for two 10-h periods resulted in a suggested optimum L-tyrosine feeding strategy giving a lower specific L-tyrosine consumption rate in the later period, to suppress cell growth, in comparison to that in the earlier period. During L-tyrosine feeding, the three specific rates (growth, production, and MGUR) could be successfully controlled by adjusting the specific L-tyrosine consumption rate to the predicted value. The cost factor was lowest in this controlled culture, demonstrating experimentally the effectiveness of the strategy. (c) 1996 John Wiley & Sons, Inc.     

Glucose effect on the expression of 150 kDa oxygen-regulated protein in HeLa cells

Chaperones assist in the correct folding of newly synthesised proteins in the endoplasmic reticulum (ER) of cells, this being essential for the translocation of protein molecules to specific subcellular compartments, extracellular matrix or to biological fluids. The biosynthesis of some ER chaperones is regulated by glucose. They are named "glucose-regulated proteins" (GRPs). The function of some GRPs depends on oxygen, a subgroup named "oxygen-regulated proteins" (ORPs). The biosynthesis of ORPs is induced by deprivation of glucose or oxygen. Exposure of HeLa cells to glucose starvation induces the biosynthesis of various GRPs including ORP 150. The expression of ORP 150 is regulated by the concentration of glucose in the culture medium, being induced by a shortage and repressed by a presence of glucose. We have shown that both glucose starvation and transfection of cells with siRNA (specific to ORP 150 mRNA) evoke similar, but quantitatively different, effects. The cells grown for 72 h in a 4.5 mg/ml glucose-containing medium demonstrated low apoptosis (3.7%) whereas in a 0.5 mg/ml glucose-containing medium the apoptosis was increased to 10%. The effect of transfection on apoptosis was distinctly higher with almost 22% of apoptotic cells detected in 72 h cultures. One may conclude that ORP 150 reduces the pro-apoptotic effects of glucose starvation. Such a hypothesis is supported by the observation that the transfection procedure makes HeLa cells resistant to the regulatory effect of glucose on ORP 150 production. The transfected cells do not respond to glucose starvation with an overexpression of ORP 150. It is apparent from our experiments that ORP 150 plays an important role in adaptation of cells to the shortage of glucose and reduces the pro-apoptotic effect of glucose starvation.     

A new process for red pigment production by submerged culture of Monascus purpureus

Formation of red pigment by Monascus purpureus via diauxic growth on glucose and ethanol in submerged culture was optimized based on inoculum preparation and culture medium. A vegetative inoculum was prepared from spores grown on ethanol. The optimized culture medium was low in phosphates, and had an initial pH?of 5.5. The characteristics of Monascus purpureus grown on glucose and on ethanol were compared: the specific consumption rate of glucose (q_G) was higher than the specific consumption rate of ethanol (q_E), whereas the specific growth rate was greatest with ethanol. The specific production rate of red pigment (p_OD) and pigment yield (Y_OD/s) with glucose was twice that with ethanol. A novel fermentation process was developed with M. purpureus initially grown with controlled ethanol formation, and consumption of the latter during pigment formation.