Comparative metabolic analysis of lactate for CHO cells in glucose and galactose

t-PA producing CHO cells have been shown to undergo a metabolic shift when the culture medium is supplemented with a mixture of glucose and galactose. This metabolic change is characterized by the reincorporation of lactate and its use as an additional carbon source. The aim of this work is to understand lactate metabolism. To do so, Chinese hamster ovary cells were grown in batch cultures in four different conditions consisting in different combinations of glucose and galactose. In experiments supplemented with glucose, only lactate production was observed. Cultures with glucose and galactose consumed glucose first and produced lactate at the same time, after glucose depletion galactose consumption began and lactate uptake was observed. Comparison of the metabolic state of cells with and without the shift by metabolic flux analysis show that the metabolic fluxes distribution changes mostly in the reactions involving pyruvate metabolism. When not enough pyruvate is being produced for cells to support their energy requirements, lactate dehydrogenase complex changes the direction of the reaction yielding pyruvate to feed the TCA cycle. The slow change from high fluxes during glucose consumption to low fluxes in galactose consumption generates intracellular conditions that allow the influx of lactate. Lactate consumption is possible in cell cultures supplemented with glucose and galactose due to the low rates at which galactose is consumed. Evidence suggests that an excessive production and accumulation of pyruvate during glucose consumption leads to lactate production and accumulation inside the cell. Other internal conditions such as a decrease in internal pH, forces the flow of lactate outside the cell. After metabolic shift the intracellular pool of pyruvate, lactate and H⁺ drops permitting the reversal of the monocarboxylate transporter direction, therefore leading to lactate uptake. Metabolic analysis comparing glucose and galactose consumption indicates that after metabolic shift not enough pyruvate is produced to supply energy metabolism and lactate is used for pyruvate synthesis. In addition, MFA indicates that most carbon consumed during low carbon flux is directed towards maintaining energy metabolism.     

Effect of glucose starvation on germ-tube production byCandida albicans

By incubating starved and unstarved yeast cells in synthetic media with a pH of 4.5 or 6.7 at 37°C the effect of a 3 hours' glucose starvation on germ-tube production byCandida albicans was evaluated. In addition the endocellular content of total carbohydrates, glycogen, trehalose and proteins after and before the starvation were dosed. The most interesting result was the overcoming of the pH-regulated dimorphism, thanks to the starvation treatment. Infact the starved cultures produced germtubes indifferently in neutral or acid media, whereas the filamentation of the unstarved cultures was more copious in pH 6.7 medium. The endocellular content of trehalose and protein was unchanged, whereas total carbohydrates and glycogen showed a shortage after the 3 hours' glucose starvation. The possible involvements of these metabolic changes in the regulation of dimorphic transition are discussed.     

Metabolic discrimination of sucrose starvation from<Emphasis Type="Italic">Arabidopsis</Emphasis> cell suspension by<Superscript>1</Superscript>H NMR based metabolomics

Whole cell extracts ofArabidopsis cell cultures maintained on various sucrose concentrations (0,3, and 6%) were analyzed by¹H NMR spectroscopy to determine the comprehensive metabolic change in these cultures during sucrose starvation. The amount of sucrose, glucose, and fructose in the cells decreased to almost nothing after 12 h of culture in medium without sucrose. In contrast, the total free amino acid content of the cells increased as the culture proceeded. Among the free amino acids, phenylalanine and malic acid increased the most, followed by asparagine and alanine, whereas glutamic acid did not change significantly. These results are in agreement with previous studies using HPLC.¹H NMR spectroscopy enabled measurement of changes in the sugar and free amino acid content of whole cell extracts without fractionation and complicated sample preparation. These results indicate that comprehensive metabolic changes in the cells can be determined by a simple, rapid method using whole cell extracts and¹H NMR spectroscopy.     

Effects of pH and trace minerals on long-term starvation of Leuconostoc mesenteroides

Laboratory experiments have definitively shown that exopolymer-producing bacteria have the potential to modify the flow of fluids in oil reservoirs to enhance oil production. Once injected into the reservoir, they will be subjected to a wide range of pH values and to starvation resulting from nutrient depletion. For successful field implementation it is necessary to have a fundamental understanding of these effects on the viability of bacteria. This paper addresses the effects of pH and trace minerals on cell viability of Leuconostoc mesenteroides during carbon source depletion. Two different carbon sources were used to grow cells before transferring the cells to starvation conditions: sucrose and a combination of glucose and fructose. These substrates were chosen because L. mesenteroides produces a significant amount of water-insoluble exopolymers (dextran) under sucrose-fed conditions, which may enhance cell survival under harsh conditions. The effects of dextran on the cell viability were tested at different pH values with and without trace minerals. The rate of cell death followed an exponential-decay law for different values of the solution pH. The optimal solution pH for survival was pH 5, whereas cells died rapidly at pH 3 and below and at pH 13 and above. The sucrose-fed cells showed a greater viability than cells fed glucose and fructose for all pH ranges tested. The results indicated that water-insoluble exopolymers help cells survive for longer periods of time under starvation conditions. The effects of trace minerals on cell culturability were tested at two pH values, 4.5 and 7. For both cases, cells showed a greater culturability (smaller decay rate constant) in the presence of trace minerals than without trace minerals. It was also found that the effects of trace minerals on cell culturability were greater for glucose-fructose-fed cells than for sucrose-fed cells. The Michaelis pH function theory was used for comparing the relationships between the cell decay rate and pH.     

Effect of Nutrient Starvation on the Cellular Composition and Metabolic Capacity of Saccharomyces cerevisiae

This investigation addresses the following question: what are the important factors for maintenance of a high catabolic capacity under various starvation conditions? Saccharomyces cerevisiae was cultured in aerobic batch cultures, and during the diauxic shift cells were transferred and subjected to 24 h of starvation. The following conditions were used: carbon starvation, nitrogen starvation in the presence of glucose or ethanol, and both carbon starvation and nitrogen starvation. During the starvation period changes in biomass composition (including protein, carbohydrate, lipid, and nucleic acid contents), metabolic activity, sugar transport kinetics, and the levels of selected enzymes were recorded. Subsequent to the starvation period the remaining catabolic capacity was measured by addition of 50 mM glucose. The results showed that the glucose transport capacity is a key factor for maintenance of high metabolic capacity in many, but not all, cases. The results for cells starved of carbon, carbon and nitrogen, or nitrogen in the presence of glucose all indicated that the metabolic capacity was indeed controlled by the glucose transport ability, perhaps with some influence of hexokinase, phosphofructokinase, aldolase, and enolase levels. However, it was also demonstrated that there was no such correlation when nitrogen starvation occurred in the presence of ethanol instead of glucose.     

Starvation-Survival Physiological Studies of a Marine Pseudomonas sp

Starved cultures of a marine Pseudomonas sp. showed a 99.9% decrease in viable cell count during the first 25 days of starvation, yet the culture maintained 10 viable cells per ml for over 1 year. The physiological responses of populations of a marine Pseudomonas sp. to nutrient starvation were observed for periods of up to 40 days. At various intervals during starvation, the numbers of total, viable, and respiring cells were determined within the cultures. The ATP content, endogenous respiration rate, uptake rates, and percent respiration for exogenous glucose and glutamate were determined throughout the starvation period to characterize the physiological changes in the cells. It was observed that, after initial adjustment periods, all parameters tested reached stabilized states after 18 to 25 days of starvation. The results indicate that the actively respiring subpopulation, rather than the viable or total cell numbers, is the most appropriate denominator for interpretation of observed activities on an individual cell basis.     

Effect of nutrient starvation on the cellular composition and metabolic capacity of Saccharomyces cerevisiae

This investigation addresses the following question: what are the important factors for maintenance of a high catabolic capacity under various starvation conditions? Saccharomyces cerevisiae was cultured in aerobic batch cultures, and during the diauxic shift cells were transferred and subjected to 24 h of starvation. The following conditions were used: carbon starvation, nitrogen starvation in the presence of glucose or ethanol, and both carbon starvation and nitrogen starvation. During the starvation period changes in biomass composition (including protein, carbohydrate, lipid, and nucleic acid contents), metabolic activity, sugar transport kinetics, and the levels of selected enzymes were recorded. Subsequent to the starvation period the remaining catabolic capacity was measured by addition of 50 mM glucose. The results showed that the glucose transport capacity is a key factor for maintenance of high metabolic capacity in many, but not all, cases. The results for cells starved of carbon, carbon and nitrogen, or nitrogen in the presence of glucose all indicated that the metabolic capacity was indeed controlled by the glucose transport ability, perhaps with some influence of hexokinase, phosphofructokinase, aldolase, and enolase levels. However, it was also demonstrated that there was no such correlation when nitrogen starvation occurred in the presence of ethanol instead of glucose.     

Effects of pH and Trace Minerals on Long-Term Starvation of Leuconostoc mesenteroides

Laboratory experiments have definitively shown that exopolymer-producing bacteria have the potential to modify the flow of fluids in oil reservoirs to enhance oil production. Once injected into the reservoir, they will be subjected to a wide range of pH values and to starvation resulting from nutrient depletion. For successful field implementation it is necessary to have a fundamental understanding of these effects on the viability of bacteria. This paper addresses the effects of pH and trace minerals on cell viability of Leuconostoc mesenteroides during carbon source depletion. Two different carbon sources were used to grow cells before transferring the cells to starvation conditions: sucrose and a combination of glucose and fructose. These substrates were chosen because L. mesenteroides produces a significant amount of water-insoluble exopolymers (dextran) under sucrose-fed conditions, which may enhance cell survival under harsh conditions. The effects of dextran on the cell viability were tested at different pH values with and without trace minerals. The rate of cell death followed an exponential-decay law for different values of the solution pH. The optimal solution pH for survival was pH 5, whereas cells died rapidly at pH 3 and below and at pH 13 and above. The sucrose-fed cells showed a greater viability than cells fed glucose and fructose for all pH ranges tested. The results indicated that water-insoluble exopolymers help cells survive for longer periods of time under starvation conditions. The effects of trace minerals on cell culturability were tested at two pH values, 4.5 and 7. For both cases, cells showed a greater culturability (smaller decay rate constant) in the presence of trace minerals than without trace minerals. It was also found that the effects of trace minerals on cell culturability were greater for glucose-fructose-fed cells than for sucrose-fed cells. The Michaelis pH function theory was used for comparing the relationships between the cell decay rate and pH.     

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.     

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.     

Starvation-Survival Physiological Studies of a Marine Pseudomonas sp.

Starved cultures of a marine Pseudomonas sp. showed a 99.9% decrease in viable cell count during the first 25 days of starvation, yet the culture maintained 10⁵ viable cells per ml for over 1 year. The physiological responses of populations of a marine Pseudomonas sp. to nutrient starvation were observed for periods of up to 40 days. At various intervals during starvation, the numbers of total, viable, and respiring cells were determined within the cultures. The ATP content, endogenous respiration rate, uptake rates, and percent respiration for exogenous glucose and glutamate were determined throughout the starvation period to characterize the physiological changes in the cells. It was observed that, after initial adjustment periods, all parameters tested reached stabilized states after 18 to 25 days of starvation. The results indicate that the actively respiring subpopulation, rather than the viable or total cell numbers, is the most appropriate denominator for interpretation of observed activities on an individual cell basis.     

Changes in Viability, Cell Composition, and Enzyme Levels During Starvation of Continuously Cultured (Ammonia-Limited) Selenomonas ruminantium

Under nitrogen (ammonia)-limited continuous culture conditions, the ruminal anaerobe Selenomonas ruminantium was grown at various dilution rates (D). The proportion of the population that was viable increased with D, being 91% at D = 0.5 h⁻¹. Washed cell suspensions were subjected to long-term nutrient starvation at 39°C. All populations exhibited logarithmic linear declines in viability that were related to the growth rate. Cells grown at D = 0.05, 0.20, and 0.50 lost about 50% viability after 8.1, 4.6, and 3.6 h, respectively. The linear rates of decline in total cell numbers were dramatically less and constant regardless of dilution rate. All major cell constituents declined during starvation, with the rates of decline being greatest with RNA, followed by DNA, carbohydrate, cell dry weight, and protein. The rates of RNA loss increased with cells grown at higher D values, whereas the opposite was observed for rates of carbohydrate losses. The majority of the degraded RNA was not catabolized but was excreted into the suspending buffer. At all D values, S. ruminantium produced mainly lactate and lesser amounts of acetate, propionate, and succinate during growth. With starvation, only small amounts of acetate were produced. Addition of glucose, vitamins, or both to the suspending buffer or starvation in the spent culture medium resulted in greater losses of viability than in buffer alone. Examination of extracts made from starving cells indicated that fructose diphosphate aldolase and lactate dehydrogenase activities remained relatively constant. Both urease and glutamate dehydrogenase activities declined gradually during starvation, whereas glutamine synthetase activity increased slightly. The data indicate that nitrogen (ammonia)-limited S. ruminantium cells have limited survival capacity, but this capacity is greater than that found previously with energy (glucose)-limited cells. Apparently no one cellular constituent serves as a catabolic substrate for endogenous metabolism. Relative to losses in viability, cellular enzymes are stable, indicating that nonviable cells maintain potential metabolic activity and that generalized, nonspecific enzyme degradation is not a major factor contributing to viability loss.     

Effects of pH-variations on the kinetics of growth and energy metabolism in cultured T-lymphoma cells: a microcalorimetric study.

The progression of T-lymphoma cells (CCRF-CEM) growing in suspension has been monitored during long term (12-28 h) batch experiments using microcalorimetry. In parallel with the calorimetric measurements, changes in cell concentration, pH, p(O2) and concentrations of the main energy sources (glucose and glutamine) were determined. The overall metabolic rate per cell (as reflected by the heat production rate per cell, Pcell) and the growth rate decreased with time. These changes could be attributed solely to the decrease in pH of the medium until the total heat production, Q, exceeded 1.2 J per ml (corresponding to an incubation time of 20 h of a batch having an initial cell concentration of 1 x 10(6) cells per ml). The lowering of p(O2) to a level of 0.02 mmol/l or the decrease in concentrations of glucose and glutamine to 7.7 and 1.3 mmol/l, respectively, did not influence Pcell or the growth pattern. No "crowding effect" was observed for the cells in the investigated concentration range (0.6-1.3) x 10(6) cells per ml.     

Optimization of Escherichia coli growth by controlled addition of glucose

During aerobic growth of Escherichia coli (recombinant K-12 and strain B) on protein hydrolysate (L-broth) and a carbon source (glucose), acetic acid is produced via glucose metabolism until the late log phase. At this point, the culture pH starts to increase and the growth rate decreases. In cultures without further glucose supplementation, these changes are associated with the accumulation of ammonia, the utilization of acetic acid, the depletion of amino acids, and the complete depletion of glucose. We hypothesize that, after depletion of the glucose, the bacteria catabolize amino acids for energy and carbon and give off the nitrogen as ammonia. Also contributing to the overall increase in pH is the depletion of the acetic acid produced earlier as it is metabolized upon exhaustion of glucose. However, there is a lag time of about 1 hour after the initial pH increase before the sustained accumulation of ammonia begins. This lag indicates that an unidentified factor, in addition to the increase in ammonia, contributes to the increase in pH. Advantage was taken of the turnaround from acid production to base production as reflected in the culture pH to implement the addition of glucose. In growth experiments during which the pH was controlled in the basic direction by glucose addition, the observed decrease in growth rate was significantly postponed and the pH change in the basic direction was reversed as a result of acid production by the cells from the newly added glucose. Furthermore, coll densities of twice that obtained without glucose feeding were demonstrated. Based on the media cost per unit cell density, the data indicate a 31% cost savings.     

Evidence for new factors in the coordinate regulation of energy metabolism in Escherichia coli. Effects of hypoxia, chloramphenicol succinate, and 2,4-dinitrophenol on glucose utilization, glycogen synthesis, adenylate energy charge, and hexose phosphates during the first two periods of nitrogen starvation.

We studied the effects of decreased aeration, chloramphenicol succinate, and 2,4-dinitrophenol on the cellular rates of glycogen synthesis and glucose utilization and on the cellular concentrations of adenine nucleotides, glucose 6-phosphate, fructose 1,6-diphosphate, and phosphoenolpyruvate during the first two periods of nitrogen starvation of Escherichia coli W4597(K). A quantitative relationship between the changes in the rates and the accompanying changes in the hexose phosphates is demonstrated. However, the relationship for glycogen synthesis is different in different sets of metabolic conditions. We suggest that this difference reflects a change in the steady state level of a previously unknown effector of ADP-glucose synthetase (glucose 1-phosphate adenylyltransferase, EC 2.7.7.27) the rate-limiting enzyme of bacterial glycogen synthesis. We show that the properties of the hypothetical in vivo effector are consistent with the inhibitory effects of ppGpp (guanosine 3'-diphosphate 5'-diphosphate) and pppGpp (guanosine 3'-diphosphate 5'-triphosphate) on this enzyme in vitro. In addition, tetracycline, an inhibitor of the synthesis of these nucleotides, apparently prevents the change in the quantitative relationship. The relationship between glucose utilization and the hexose phosphates is altered at the transition to Period II of nitrogen starvation. We propose that this change reflects the alteration of the cellular steady state level of an unknown effector of the glucose phosphotransferase system. In contrast to the ATP-hexose phosphate system of shared regulatory effects, the specific effects of the unknown effectors allow the rates of glucose utilization and glycogen synthesis to be altered independently of each other and independently of changes in the rate of glycolysis. This independence allows a greater latitude of response for the individual pathways in more severe metabolic stress or in accommodating the metabolic changes necessary for long term survival.     

Computational modeling of nutrient utilization in engineered cartilage

This study presents a mathematical model for simulating cartilaginous culture of chondrocytes seeded in scaffolds and for investigating the effects of glucose and oxygen concentration and pH value on cell metabolic rates. The model can clearly interpret the unexplained experimental observation (Sengers BG, Heywood HK, Lee DA, Oomens CWJ, Bader DL. Nutrient utilization by bovine articular chondrocytes: A combined experimental and theoretical approach. J Biomech Eng. 2005;127:758–766.), which showed that the oxygen concentration within the scaffold may increase instead of continuously decreasing in static cartilaginous culture of chondrocytes. Results from simulation demonstrate that when cells metabolize glucose and form lactate under high glucose concentration conditions, the acidity in the culture environment increases, inhibiting cell metabolic rates in the process. Consequently, the rate of oxygen consumption decreases in later stages of cell culture. As oxygen can be replenished through the free surface of the culture medium, oxygen concentration within the scaffold increases rather than decreases over time in the acidic environment. Different initial glucose concentration yields different results. In low glucose concentration conditions, oxygen concentration basically keeps decreasing with culture time. This is because the pH in the environment does not significantly change because of slower glycolysis rate in low glucose concentration cases, forming less lactic acid. From the simulation results, additional information regarding in vitro culture of chondrocytes is obtained. The correlations between nutrient consumption, lactate secretion, and pH changes during cell culture are also understood and may serve as a reference for in vitro cell culture research of tissue engineering. © 2013 American Institute of Chemical Engineers Biotechnol. Prog., 29: 452–462, 2013     

Carbon starvation can induce energy deprivation and loss of fermentative capacity in Saccharomyces cerevisiae

Seven different strains of Saccharomyces cerevisiae were tested for the ability to maintain their fermentative capacity during 24 h of carbon or nitrogen starvation. Starvation was imposed by transferring cells, exponentially growing in anaerobic batch cultures, to a defined growth medium lacking either a carbon or a nitrogen source. After 24 h of starvation, fermentative capacity was determined by addition of glucose and measurement of the resulting ethanol production rate. The results showed that 24 h of nitrogen starvation reduced the fermentative capacity by 70 to 95%, depending on the strain. Carbon starvation, on the other hand, provoked an almost complete loss of fermentative capacity in all of the strains tested. The absence of ethanol production following carbon starvation occurred even though the cells possessed a substantial glucose transport capacity. In fact, similar uptake capacities were recorded irrespective of whether the cells had been subjected to carbon or nitrogen starvation. Instead, the loss of fermentative capacity observed in carbon-starved cells was almost surely a result of energy deprivation. Carbon starvation drastically reduced the ATP content of the cells to values well below 0.1 micro mol/g, while nitrogen-starved cells still contained approximately 6 micro mol/g after 24 h of treatment. Addition of a small amount of glucose (0.1 g/liter at a cell density of 1.0 g/liter) at the initiation of starvation or use of stationary-phase instead of log-phase cells enabled the cells to preserve their fermentative capacity also during carbon starvation. The prerequisites for successful adaptation to starvation conditions are probably gradual nutrient depletion and access to energy during the adaptation period.     

Carbon Starvation Can Induce Energy Deprivation and Loss of Fermentative Capacity in Saccharomyces cerevisiae

Seven different strains of Saccharomyces cerevisiae were tested for the ability to maintain their fermentative capacity during 24 h of carbon or nitrogen starvation. Starvation was imposed by transferring cells, exponentially growing in anaerobic batch cultures, to a defined growth medium lacking either a carbon or a nitrogen source. After 24 h of starvation, fermentative capacity was determined by addition of glucose and measurement of the resulting ethanol production rate. The results showed that 24 h of nitrogen starvation reduced the fermentative capacity by 70 to 95%, depending on the strain. Carbon starvation, on the other hand, provoked an almost complete loss of fermentative capacity in all of the strains tested. The absence of ethanol production following carbon starvation occurred even though the cells possessed a substantial glucose transport capacity. In fact, similar uptake capacities were recorded irrespective of whether the cells had been subjected to carbon or nitrogen starvation. Instead, the loss of fermentative capacity observed in carbon-starved cells was almost surely a result of energy deprivation. Carbon starvation drastically reduced the ATP content of the cells to values well below 0.1 μmol/g, while nitrogen-starved cells still contained approximately 6 μmol/g after 24 h of treatment. Addition of a small amount of glucose (0.1 g/liter at a cell density of 1.0 g/liter) at the initiation of starvation or use of stationary-phase instead of log-phase cells enabled the cells to preserve their fermentative capacity also during carbon starvation. The prerequisites for successful adaptation to starvation conditions are probably gradual nutrient depletion and access to energy during the adaptation period.