Regulation of the lipopolysaccharide-specific sialyltransferase activity of gonococci by the growth state of the bacteria, but not by carbon source, catabolite repression or oxygen supply

The enzyme sialyltransferase (STase) of Neisseria gonorrhoeae is a major pathogenicitiy determinant. Using a refined method for assaying the STase activity, the Km for CMP-NANA was shown to be 14 +/– 2 M, higher than that reported previously. Rates of sialylation by Nonidet extracts, prepared under conditions that optimise solubilisation of the membrane-bound enzyme, were 6 to 20 nmol of NANA transferred from CMP-¹⁴C-NANA onto isolated lipopolysaccharide/min./mg of extracted protein, far higher than the previously reported rates of less than 1 nmol of NANA transferred/min./mg of extracted protein. Gonococci grew more slowly with lactate or pyruvate than with glucose as the carbon source. Although growth with a mixture of limiting concentrations of both glucose and lactate was biphasic, diauxic growth was also found in the control culture supplied with glucose alone. The growth rate in the presence of lactate alone was slower than with glucose. The growth rate increased slightly relative to the glucose culture when both substrates were available; lactate was consumed more rapidly than glucose. Higher STase activities were found in bacteria harvested in the exponential than in the stationary phase of aerobic growth: the activity in aerated cultures was higher than those of oxygen-limited or anaerobic cultures. Similar STase activities were found in bacteria that had been grown with glucose, lactate or pyruvate as the carbon and energy source. Sialyltransferase synthesis is essentially constitutive: it is not regulated by glucose repression or by induction by lactate or anaerobiosis.     

Schistosoma mansoni: effects of in vitro serotonin (5-HT) on aerobic and anaerobic carbohydrate metabolism

The effect of Serotonin on carbohydrate metabolism, excreted end products, and adenine nucleotide pools in Schistosoma mansoni was determined following 60 min in vitro incubations under air (= 21% O2) and anaerobic (95% N2:5% CO2) conditions. In the presence of 0.25 mM Serotonin, glucose uptake increased by 82-84% and lactate excretion increased by 77-78%; levels of excreted lactate were significantly higher under aerobic than under anaerobic conditions. The tissue pools of glucose, hexosephosphates, fructose 1,6-bisphosphate, pyruvate, and lactate were significantly increased under anaerobic conditions compared to air incubation; the presence of Serotonin decreased tissue glucose pools and increased the size of the pyruvate and lactate tissue pools. The glycolytic carbon pool was significantly greater under anaerobic than under aerobic conditions, irrespective of Serotonin. Serotonin increased adenosine 5'-diphosphate and adenosine 5'-monophosphate levels under aerobic conditions; neither Serotonin nor gas phase significantly affected total adenine nucleotide levels or the adenylate energy charge. Serotonin increased energy requirements by S. mansoni due to increased muscle contractions; demand was met by enhanced rates of carbohydrate metabolism. Irrespective of gas phase, 74-78% of available carbohydrate was converted to lactate. In the presence of Serotonin, conversion of glucose to lactate was reduced to 63-67%. In view of the requirements by S. mansoni for an abundant supply of glycoprotein and glycolipid precursors for surface membrane renewal, it is suggested that carbohydrate (glucose and glycogen) that was not converted to lactate may have been incorporated into biosynthetic processes leading to membrane synthesis.     

Anaerobic and aerobic continuous cultures of<Emphasis Type="Italic"> Saccharomyces cerevisiae</Emphasis>: comparison of plasmid stability and<Emphasis Type="Italic"> EXG1</Emphasis> gene expression

Two bioreactor continuous cultures, at anaerobic and aerobic conditions, were carried out using a recombinant Saccharomyces cerevisiae strain that over-expresses the homologous gene EXG1. This recombinant system was used to study the effect of dissolved oxygen concentration on plasmid stability and gene over-expression. Bioreactor cultures were operated at two dilution rates (0.14 and 0.03 h^–1) to investigate the effect of other process parameters on EXG1 expression. Both cultures suffered severe plasmid instability during the first 16 generations. Segregational plasmid loss rate for the aerobic culture was two-fold that of the anaerobic operation. In spite of this fact, exo--glucanase activity at aerobic conditions was 12-fold that of the anaerobic culture. This maximal activity (30 U ml^–1) was attained at the lowest dilution rate when biomass reached its greatest value and glucose concentration was zero.     

Induced pluripotent stem cells can utilize lactate as a metabolic substrate to support proliferation

Human-induced pluripotent stem cells (iPSCs) hold the promise to improve cell-based therapies. Yet, to meet rising demands and become clinically impactful, sufficient high-quality iPSCs in quantity must be generated, a task that exceeds current capabilities. In this study, K3 iPSCs cultures were examined using parallel-labeling metabolic flux analysis (¹³C-MFA) to quantify intracellular fluxes at relevant bioprocessing stages: glucose concentrations representative of initial media concentrations and high lactate concentrations representative of fed-batch culture conditions, prior to and after bolus glucose feeds. The glucose and lactate concentrations are also representative of concentrations that might be encountered at different locations within 3D cell aggregates. Furthermore, a novel method was developed to allow the isotopic tracer [U-¹³C₃] lactate to be used in the ¹³C-MFA model. The results indicated that high extracellular lactate concentrations decreased glucose consumption and lactate production, while glucose concentrations alone did not affect rates of aerobic glycolysis. Moreover, for the high lactate cultures, lactate was used as a metabolic substrate to support oxidative mitochondrial metabolism. These results demonstrate that iPSCs have metabolic flexibility and possess the capacity to metabolize lactate to support exponential growth, and that high lactate concentrations alone do not adversely impact iPSC proliferation.     

Effect of storage conditions on detection of mycoplasma in biopharmaceutical products

Mycoplasma contamination affects many different aspects of cell culturing, resulting in unreliable experimental results and potentially harmful biological products. Therefore, the specificity, sensitivity, and reliability of detecting mycoplasma contamination are important aspects of quality control in biotechnological products. In this study, Mycoplasma hyorhinis was adopted as a model strain to evaluate the effects of storage on the viability of Mycoplasma species in cell culture samples. Medium X was compared with conventional media 243 and 988 for the ability to detect M. hyorhinis. The 10¹ CFU/ml of M. hyorhinis was inoculated into medium X prepared using the same lots of components and preserved for 7 d, 1 mo, and 2 mo. M. hyorhinis grew readily and typically on agar plates prepared within 1 mo. The viable mycoplasmas in samples containing different initial titers (10¹ and 10⁶ CFU/ml) after storage at 4° C and −30° C were analyzed. During storage, viable organisms were found with little or no reduction in titers after storage for 8 wk at −30° C under aerobic and anaerobic conditions. A reduction in titers of 3 log10 occurred after 4 wk storage for high-dose cultures (10⁶ CFU/ml) at 4° C. The titers of viable organisms were diminished over 8 wk at 4° C under aerobic and anaerobic conditions.     

Studies on Oxidation-Reduction Potentials (ORP) of Microbial Cultures

Aerobacter aerogenes No. 505 isolated from soil by Uyeda produced l-valine extracellularly by an aerobic shaking culture. Under anaerobic conditions the production of this amino acid was inhibited while lactic acid as well as a small amount of alanine were produced. The changes in ORP during the incubation under both conditions were investigated. When l-valine was the main product under aerobic conditions the ORP showed a constant value (rH 8.0) from 16 to 40 hr after inoculation. But when lactic acid was the main product and alanine was produced as the only amino acid under anaerobic conditions, the ORP drifted to rH 0 (zero). The phenomenon of the conversion of fermentation was shown clearly by the ORP of the culture broth.

The endpotentiai of lactic acid fermentation by Rhizopus G-36 was rH 13 to 14 when measured in the presence of trace amounts of redox dye mixtures. Without dyes, the rH was 18 to 22 and this fungal culture was slower in reaching endpotentials than bacterial cultures. It was postulated that the amount of redox substances exhibiting electromotive activity was not sufficient in this culture.

rH value 13 to 14 was not obtained under such conditions that lactic acid was not produced; that is in a medium with higher concentration of the nitrogen source in the presence of Fe²⁺ and Zn²⁺, or in a medium containing acetate in place of glucose as the carbon source.

Mycelia of Rhizopus G-36 after 36 hr-culture produced lactic acid even in the absence of oxygen. But unexpectedly, the ORP under anaerobic secondary culture was exactly the same as that in the aerobic shaking culture (rH 13.2).

A method for homogenization of the culture without secondary oxidation was improved. The ORP of anaerobically homogenized cultures was rH 11, and was thought to be due to the activities of all redox systems in the mycelium.

The respiration system of this strain was switched from cytochrome system to flavin system at the point of change in KGN-sensitivity. The ORP of this strain may be influenced by respiration through the flavin system.     

Effects of oxygen on invertase expression in continuous culture of recombinant Saccharomyces cerevisiae containing the SUC2 gene

The yeast SUC2 gene, cloned on a multicopy plasmid pRB58, was used to study the effect of oxygen on the invertase expression of the recombinant Saccharomyces cerevisiae. Glucose repression was not the only factor affecting the invertase expression. The results obtained from the single-stage continuous cultures under microaerobic conditions showed that invertase expression was also strongly dependent on oxygen availability, and moving from anaerobic to aerobic conditions led to a five-fold increase in specific invertase activity. However, the cell yields under anaerobic conditions were quite low compared to those under aerobic conditions. These opposite effects of oxygen on cell growth and gene expression offer a strategy for maximizing invertase productivity by a two-stage continuous culture. The first stage was operated at a low level of glucose, around 100 mg/l, under aerobic conditions in order to obtain a high yield of yeast biomass, and the second stage maintained anaerobic conditions with residual glucose levels of 50 mg/l to derepress and fully induce invertase expression. The two-stage continuous culture resulted in a 2.5-fold increase in invertase productivity over that of a single-stage continuous culture. Received: 28 July 1998 / Received revision: 22 September 1998 / Accepted: 7 November 1998     

Injury to primary cultures of rat heart endothelial cells by hypoxia and glucose deprivation

Summary Primary cultures of rat heart endothelial cells were subjected to simulated conditions of ischemia: hypoxia and glucose deprivation for 4 and 24 hr. Cellular injury was evaluated by measuring changes in viability, total protein, cellular morphology, and leakage of cytoplasmic enzymes from the cells into the culture medium. Deprivation of oxygen and glucose for 4 or 24 hr did not lethally injure the cells as noted by no change in cell viability, morphology, and total protein when compared to controls. However, reversible or nonlethal cellular injury was produced as reflected by a significant release of lactate dehydro-genase (LDH) from the cells into the medium after treatment with hypoxia and glucose deprivation for 4 or 24 hr. When the cultures were deprived of glucose, but were oxygenated, cellular injury was not evident after 24 hr. Deprivation of oxygen but not glucose resulted in significant loss of LDH after 4 or 24 hr. When the cultures were allowed to recover after oxygen and glucose deprivation in complete medium containing 1000 mg glucose per l and a normal atmosphere of 20% O₂, they had levels of LDH leakage comparable to those of control cultures. This study was supported by Research Grant HL 18647 from the National Heart, Lung, and Blood Institute and by a National Chicano Council on Higher Education Post-Doctoral Fellowship awarded to D. Acosta from the Ford Foundation. Additional support was provided to D. Acosta by a Faculty Research Assignment Award from the University of Texas Research Institute.     

Relationship between the production of spirosomes and anaerobic glycolysis activity in Escherichia coli B

The effects of culture conditions (aerobic or anaerobic) and glucose in the medium on the production of spirosomes in Escherichia coli B were studied by SDS-PAGE and electron microscopy. The Mr of the spirosome of E. coli B was estimated to be 97,000. Electron microscopy revealed that the amount of spirosomes derived from anaerobic cultures was about eightfold larger than that from aerobic cultures. In SDS-PAGE, the bands of spirosome protein derived from anaerobic cultures were more intense than those derived from aerobic cultures, either in peptone water or in Davis-Mingioli's minimal medium. With increased glucose concentration under aerobic conditions, the intensity of the band of spirosome protein was similar to that observed under anaerobic conditions in basal media. These results suggest that spirosome production by E. coli B is related to its anaerobic glycolysis activity.     

Physiological and biochemical role of the butanediol pathway in Aerobacter (Enterobacter) aerogenes.

Aerobacter (Enterobacter) aerogenes wild type and three mutants deficient in the formation of acetoin and 2,3-butanediol were grown in a glucose minimal medium. Culture densities, pH, and diacetyl, acetoin, and 2,3-butanediol levels were recorded. The pH in wild-type cultures dropped from 7.0 to 5.8, remained constant while acetoin and 2,3-butanediol were formed, and increased to pH 6.5 after exhaustion of the carbon source. More 2,3-butanediol than acetoin was formed initially, but after glucose exhaustion reoxidation to acetoin occurred. The three mutants differed from the wild type in yielding acid cultures (pH below 4.5). The wild type and one of the mutants were grown exponentially under aerobic and anaerobic conditions with the pH fixed at 7.0, 5.8, and 5.0, respectively. Growth rates decreased with decreasing pH values. Aerobically, this effect was weak, and the two strains were affected to the same degree. Under anaerobic conditions, the growth rates were markedly inhibited at a low pH, and the mutant was slightly more affected than the wild type. Levels of alcohol dehydrogenase were low under all conditions, indicating that the enzyme plays no role during exponential growth. The levels of diacetyl (acetoin) reductase, lactate dehydrogenase, and phosphotransacetylase were independent of the pH during aerobic growth of the two strains. Under anaerobic conditions, the formation of diacetyl (acetoin) reductase was pH dependent, with much higher levels of the enzyme at pH 5.0 than at pH 7.0. Lactate dehydrogenase and phosphotransacetylase revealed the same pattern of pH-dependent formation in the mutant, but not in the wild type.     

Energy metabolism of some representatives of the Haemophilus group

The purpose of this investigation was to characterize the carbohydrate catabolism and the constellation of the respiratory chain components of Haemophilus influenzae RAMC 18 Bensted, H. parainfluenzae 1 Fleming, H. parainfluenzae 429 Pittman and H. aegyptius 180a Pittman. These strains represent several physiological types with respect to respiratory quinones and glucose catabolism.On addition of glucose or lactate to the complex growth medium a remarkable increase in cell mass was observed. Depending on the growth rate, carbohydrate degradation varied with the strains examined so that at the end of the exponential growth phase only small amounts of the supplements could be demonstrated.All strains were found to possess functional enzymes of Embden-Meyerhof-Parnas-, Entner-Doudoroff-pathways, hexosemonophosphate shunt, tricarboxylic acid cycle and gluconeogenesis with an extremely high activity of malate dehydrogenase.The concentration of cytochromes varied according to culture conditions. The cytochromes a₁, d, o and b+c were found to occur under aerobic conditions. In cells grown anaerobically in the presence of fumarate cytochromes a₁ and d could not be demonstrated. Under aerobic conditions preparations of H. parainfluenzae 1 Fleming exhibited an -maximum at 558 nm, whereas under anaerobic culture conditions with fumarate as terminal electron acceptor an -maximum at 552 nm occurred, suggesting different roles of b and c type cytochromes in aerobic and anaerobic electron transport to fumarate, respectively.     

Kinetics of biphasic growth of yeast in continuous and fed-batch cultures

The influence of dilution rate on the production of biomass, ethanol, and invertase in an aerobic culture of Saccharomyces carlsbergensis was studied in a glucose-limited chemostat culture. A kinetic model was developed to analyze the biphasic growth of yeast on both the glucose remaining and the ethanol produced in the culture. The model assumes a double effect where glucose regulates the flux of glucose catabolism (respiration and aerobic fermentation) and the ethanol utilization in yeast cells. The model could successfully demonstrate the experimental results of a chemostat culture featuring the monotonic decrease of biomass concentration with an increase of dilution rate higher than 0.2 hr^?1 as well as the maximum ethanol concentration at a particular dilution rate around 0.5 hr^?1. Some supplementary data were collected from an ethanol-limited aerobic chemostat culture and a glucose-limited anaerobic chemostat culture to use in the model calculation. Some parametric constants of cell growth, ethanol production, and invertase formation were determined in batch cultures under aerobic and anaerobic states as summarized in a table in comparison with the chemostat data. Using the constants, a prediction of the optimal control of a glucose fed-batch yeast culture was conducted in connection with an experiment for harvesting a high yield of yeast cells with high invertase activity.     

Effects of oxygen on the growth and metabolism of Actinomyces viscosus

Abstract Actinomyces viscosus is a predominant microorganism in dental plaque. It is, just as the oral Streptococcus spp., a saccharolytic and aero-tolerant organism. We have investigated the effects of oxygen on the growth and metabolism of A. viscosus . To this end A. viscosus Ut 2 was grown in a glucose limited chemostat culture on a chemically defined medium ( D = 0.2 h⁻¹) with exposure to variable amounts of oxygen. The Y_glucose increased from 62.5 g · mol⁻¹ under anaerobic conditions to 149 g · mol⁻¹ under aerobic conditions, while, concomitantly, the carbon recovery from acidic fermentation products decreased from 75% to 7%. Addition of [¹⁴C]glucose to the chemostat showed that the glucose, which was not converted to acidic fermentation products, was instead converted to carbon dioxide or used for the production of biomass. Under aerobic and anaerobic conditions identical cytochrome spectra, containing only two cytochrome b -type absorption bands, were found. It was concluded that electron transport phosphorylation probably occurs both under aerobic and anaerobic conditions. Anaerobically, fumarate served as the electron acceptor, while the high growth yields observed under aerobic conditions are likely to be explained by citric acid cycle activity coupled to electron transport phosphorylation.     

Quantitative on-line monitoring of hippocampus glucose and lactate metabolism in organotypic cultures using biosensor technology

Quantitative glucose and lactate metabolism was assessed in continuously perfused organotypic hippocampal slices under control conditions and during exposure to glutamate and drugs that interfere with aerobic and anaerobic metabolism. On-line detection was possible with a system based on slow perfusion rates, a half-open (medium/air interface) tissue chamber and a flow injection analytic system equipped with biosensors for glucose and lactate. Under basal conditions about 50% of consumed glucose was converted to lactate in hippocampal slice cultures. Using medium containing lactate (5 mm) instead of glucose (5 mm) significant lactate uptake was observed, but this uptake was less than the net uptake of lactate equivalents in glucose-containing medium. Glucose deprivation experiments suggested lactate efflux from glycogen stores. The effects of drugs compromising or stimulating energy metabolism, i.e. 2-deoxyglucose, 3-nitropropionic acid, alpha-cyano-4-hydroxycinnamate, l-glutamate, d-asparate, ouabain and monensin, were tested in this flow system. The data show that maintaining Na+ and K+ gradients consumed much of the energy but do not support the hypothesis that l-glutamate stimulates glycolysis in hippocampal slice cultures.     

Reduction of cell injury in hypoxic cultures of rat myocardial cells by methylprednisolone

Summary An in vitro model to study myocardial cell injury was developed with primary monolayer cultures of rat myocardial cells. Two important conditions associated with myocardial ischemia were simulated by depriving the cultures of oxygen and glucose for a specified period of time. Cellular injury caused by hypoxia and glucose deprivation resulted in significant leakage of lactate dehydrogenase (LDH) from the cells into the culture medium. The cells were not lethally injured by treatments as reflected by a lack of change in cell viability and protein content when compared to controls. Pretreatment of cultures with methylprednisolone for 24 hr provided protection to the cells when challenged by hypoxia and glucose deprivation. Methylprednisolone exhibited a dose-response effect in reducing LDH leakage in cultures, which were subsequently deprived of oxygen and glucose for 4 hr. Similar pretreatment with hydrocortisone had no effect in limiting cellular injury in hypoxic and glucose-deprived cultures. The research was supported by Grant HL 18647 from the National Heart, Lung, and Blood Institute and by a National Chicano Council on Higher Education Post-Doctoral Fellowship awarded to D. Acosta from the Ford Foundation. Additional support was provided to D. Acosta by a Faculty Research Assignment Award from the University of Texas Research Institute.     

Lactate production in the perfused rat liver

1. In aerobic conditions the isolated perfused liver from well-fed rats rapidly formed lactate from endogenous glycogen until the lactate concentration in the perfusion medium reached about 2mm (i.e. the concentration of lactate in blood in vivo) and then production ceased. Pyruvate was formed in proportion to the lactate, the [lactate]/[pyruvate] ratio remaining between 8 and 15. 2. The addition of 5mm- or 10mm-glucose did not affect lactate production, but 20mm- and 40mm-glucose greatly increased lactate production. This effect of high glucose concentration can be accounted for by the activity of glucokinase. 3. The perfused liver released glucose into the medium until the concentration was about 6mm. When 5mm- or 10mm-glucose was added to the medium much less glucose was released. 4. At high glucose concentrations (40mm) more glucose was taken up than lactate and pyruvate were produced; the excess of glucose was probably converted into glycogen. 5. In anaerobic conditions, livers of well-fed rats produced lactate at relatively high rates (2.5mumol/min per g wet wt.). Glucose was also rapidly released, at an initial rate of 3.2mumol/min per g wet wt. Both lactate and glucose production ceased when the liver glycogen was depleted. 6. Addition of 20mm-glucose increased the rate of anaerobic production of lactate. 7. d-Fructose also increased anaerobic production of lactate. In the presence of 20mm-fructose some glucose was formed anaerobically from fructose. 8. In the perfused liver from starved rats the rate of lactate formation was very low and the increase after addition of glucose and fructose was slight. 9. The glycolytic capacity of the liver from well-fed rats is equivalent to its capacity for fatty acid synthesis and it is pointed out that hepatic glycolysis (producing acetyl-CoA in aerobic conditions) is not primarily an energy-providing process but part of the mechanism converting carbohydrate into fat.     

Culture of Plasmodium falciparum: the role of pH, glucose, and lactate

Yields of P. falciparum in intraerythrocytic in vitro cultures were maximized when extracellular pH was maintained between 7.2 and 7.45, and extracellular lactate was kept below 12 mM. Host erythrocytes metabolized 4.6 +/- 1.5 microM glucose/10(9) RBC/24 hr and produced 7.9 +/- 1.8 microM lactate/10(9) RBC/24 hr. Asynchronous parasite cultures used 122 +/- 34 microM glucose/10(9) parasitized RBC/24 hr and produced 143 +/- 47 microM lactate/10(9) parasitized RBC/24 hr. Synchronous cultures that were 80 to 100% ring forms after 24 hr in culture exhibited significantly lower glycolysis per 10(9) parasitized RBC than cultures that were 0 to 25% ring forms after 24 hr. The percent of glucose utilization accounted for by lactate production by parasites was significantly less than that of uninfected erythrocytes. These optimum ranges and metabolic rates can be used in the development of parasite culture techniques.     

Physiological properties of Cellulomonas fermentans,a mesophilic cellulolytic bacterium

Summary The fermentation of cellobiose, glucose and cellulose MN 300 by Cellulomonas fermentans was studied. The molar growth yields (i.e. grams of cells per mole of hexose equivalent) were similar on cellobiose and cellulose at low sugar consumption levels (47.8 and 46.5 respectively), but was lower on glucose (38.0). The occurrence of cellobiose phosphorylase activity, detected in cellobiose- and cellulose-grown cells, might explain this result. The specific growth rates measured in cultures on cellobiose, glucose and cellulose were 0.055 h^-1, 0.040 h^-1 and 0.013 h^-1 respectively. Growth inhibition was observed, and a drop in Y_H occurred after relatively low but different quantities of hexose were consumed (2.2 mM, 5 mM and 8 mM hexose equivalent with cellulose, glucose and cellobiose respectively), which coincided with a change in the fermentative metabolism from a typical mixed acid metabolism (1 ethanol, 1 acetate and 2 formate synthesized by consumed hexose) to a more ethanolic fermentation. When growth ceased in cellulose cultures, consumption of cellulose continued, as did production of ethanol.Molar growth yields of C. fermentans were similar in anaerobic and aerobic cellobiose cultures (47.8 g/mol and 42.2 g/mol respectively). Specific growth rates were also quite similar under both culture conditions (0.055±0.013 h^-1 and 0.070±0.007 h^-1 respectively). Aerobic metabolism was studied using ¹⁴C glucose. During the exponential growth phase, acetate, succinate and nonidentified compound(s) accumulated in the supernatant, but no ¹⁴CO₂ was produced. During the stationary phase, acetate was oxidized and ¹⁴CO₂ produced, but without any further biomass synthesis. It seems that a blocking of metabolite oxidation may have occurred in C. fermentans except in the case of acetate, but acetate oxidation was apparently not coupled with production of energy utilizable in biosynthesis.     

Growth characterizations of a human monocytic leukemia cell line in a serum-free medium

A clone, AH-01S, derived from a human monocytic leukemia cell line, THP-1, grew rapidly in a serum-free medium containing insulin, transferrin, ethanolamine, and sodium selenite. In batch culture using the serum-free medium, the AH-01S cells proliferated at a specific growth rate (μ) of 0.30 to 0.50 (1/day) from a cell concentration of 1 × 10⁴ cells/ml to 1.6 × 10⁶ cells/ml, an increase of 160 times. A higher cell concentration of 0.45 × 10⁷ cells/ml (cell volume ratio was 0.5%) was obtained in spinner flask culture using the serum-free medium. A mean specific growth rate 0.50 (1/day) was also observed in a culture in a fully instrumented cell culture fermentor. However, μ decreased drastically after the cell concentration reached 1.5 × 10⁶ cells/ml. Analyses of medium composition during cultivation revealed that under lower cell concentration, l-glutamine was the main carbon source while glucose was converted to lactate almost stoichiometrically, and that the production of lactate from glucose decreased at higher cell concentrations. To obtain cultures of 1 × 10⁹ cells, 1,200 to 1,300 mg of a carbon source (glucose) and 400 to 500 of amino acids were consumed during high cell concentration cultivation of the AH-01S cells in the serum-free medium.