Overflow Metabolism Research Articles

Characterization of the metabolic burden on Escherichia coli DH1 cells imposed by the presence of a plasmid containing a gene therapy sequence

The presence of a plasmid, containing gene sequences for DNA immunotherapy that are not expressed in microbial culture, imposed a degradation in bioreactor performance in cultures of the host E. coli strain. Significant decreases in growth rate (24%) and biomass yield (7%) and a corresponding increase in overflow metabolism were observed in a strain containing a therapeutic sequence (a hepatitis B antigen under the control of a CMV promotor). The observed increase in overflow metabolism was incorporated into a Metabolic Flux Analysis (MFA) model (as acetate secretion). Metabolic flux analysis revealed an increase in TCA cycle flux, consistent with an increased respiration rate observed in plasmid-containing cells. These effects are thought to result from increased ATP synthesis requirements (24%) arising from the expression of the Kanr plasmid marker gene whose product accounted for 18% of the cell protein of the plasmid-containing strain. These factors will necessitate significantly higher aeration and agitation rates or lower nutrient feed rates in high-density cultures than would be expected for plasmid-free cultures.

On-line multi-analyzer monitoring of biomass, glucose and acetate for growth rate control of a Vibrio cholerae fed-batch cultivation

In situ near-infrared (NIR) spectroscopy and in-line electronic nose (EN) mapping were used to monitor and control a cholera-toxin producing Vibrio cholerae fed-batch cultivation carried out with a laboratory method as well as with a production method. Prediction models for biomass, glucose and acetate using NIR spectroscopy were developed based on spectral identification and partial-least squares (PLS) regression resulting in high correlation to reference data (standard errors of prediction for biomass, glucose and acetate were 0.20 g l −1, 0.26 g l −1 and 0.28 g l −1). A compensation algorithm for aerated bioreactor disturbances was integrated in the model computation, which in particular improved the prediction by the biomass model. First, the NIR data were applied together with EN in-line data selected by principal component analysis (PCA) for generating a trajectory representation of the fed-batch cultivation. A correlation between the culture progression and EN signals was demonstrated, which proved to be beneficial in monitoring the culture quality. It was shown that a deviation from a normal cultivation behavior could easily be recognized and that the trajectory was able to alarm a bacterial contamination. Second, the NIR data indicated the potential of predicting the concentration of formed cholera toxin with a model prediction error of 0.020 g l −1. Third, the on-line biomass prediction based on the NIR model was used to control the overflow metabolism acetate formation of the V. cholerae culture. The controller compared actual specific growth rate as estimated from the prediction with the critical acetate formation growth rate, and from that difference adjusted the glucose feed rate.

Optimization of dissimilation of glycerol to 1,3-propanediol by Klebsiella pneumoniae in one- and two-stage anaerobic cultures

In this study, the optimal conditions of batch and continuous glycerol fermentations by Klebsiella pneumoniae were investigated using the volumetric productivity of 1,3-propanediol (1,3-PD) as an optimization target based on a mathematical model that considers the growth kinetics of multiple inhibitions and the metabolic overflow of substrate consumption and product formation. For batch culture with a given inoculation of 0.1 g biomass l−1, the optimal initial glycerol concentration was found to be 960 mmol l−1, which lead to the highest volumetric productivity (52.6 mmol l−1 h−1) of 1,3-propanediol. For continuous fermentations, the optimal dilution rate and initial glycerol concentration in feed were 0.29 h−1 and 731 mmol l−1, respectively. The corresponding productivity was 114 mmol l−1 h−1 that was more than twice the productivity of an optimal batch culture. A comparison between experimental and computational results of the concentration, yield and productivity of 1,3-propanediol showed that most of the continuous fermentation data were lower than or approached to the calculated values. Stability analysis of continuous cultivations indicated that two regions of multiple states occurred at relatively high concentrations of initial glycerol in feed. One of them approached to the wash-out line. A two-stage continuous process was proposed, in which the first stage was operated at the optimal conditions and the second one was used to consume the residual glycerol in the first one. Model analysis showed that the dilution rate should be much higher in the second stage than in the first one. In terms of the concentration, yield and productivity of 1,3-propanediol, a two-step bioprocess of two bioreactors in series appeared to be more favorable for 1,3-propanediol production than a single bioreactor system with the same volume.

Optimization of dissimilation of glycerol to 1,3-propanediol by Klebsiella pneumoniae in one- and two-stage anaerobic cultures

In this study, the optimal conditions of batch and continuous glycerol fermentations by Klebsiella pneumoniae were investigated using the volumetric productivity of 1,3-propanediol (1,3-PD) as an optimization target based on a mathematical model that considers the growth kinetics of multiple inhibitions and the metabolic overflow of substrate consumption and product formation. For batch culture with a given inoculation of 0.1 g biomass l −1, the optimal initial glycerol concentration was found to be 960 mmol l −1, which lead to the highest volumetric productivity (52.6 mmol l −1 h −1) of 1,3-propanediol. For continuous fermentations, the optimal dilution rate and initial glycerol concentration in feed were 0.29 h −1 and 731 mmol l −1, respectively. The corresponding productivity was 114 mmol l −1 h −1 that was more than twice the productivity of an optimal batch culture. A comparison between experimental and computational results of the concentration, yield and productivity of 1,3-propanediol showed that most of the continuous fermentation data were lower than or approached to the calculated values. Stability analysis of continuous cultivations indicated that two regions of multiple states occurred at relatively high concentrations of initial glycerol in feed. One of them approached to the wash-out line. A two-stage continuous process was proposed, in which the first stage was operated at the optimal conditions and the second one was used to consume the residual glycerol in the first one. Model analysis showed that the dilution rate should be much higher in the second stage than in the first one. In terms of the concentration, yield and productivity of 1,3-propanediol, a two-step bioprocess of two bioreactors in series appeared to be more favorable for 1,3-propanediol production than a single bioreactor system with the same volume.

Macromolecular composition of unsaturated Pseudomonas aeruginosa biofilms with time and carbon source

Bacteria in nature grow mostly as biofilms, surface-associated cells enveloped by hydrated extracellular polymeric substances (EPS) of bacterial origin. The composite of EPS and biofilm cells is measured when quantifying bacterial biomass from natural samples. However, little is known regarding the relative magnitude of the EPS and cellular fractions of biofilm biomass, particularly in unsaturated systems such as soil and food surfaces. In this study, we examined the cellular and extracellular fractions of Pseudomonas aeruginosa biofilms for DNA, protein and carbohydrate content. Biofilms were cultured in a model laboratory system that simulates the nutrient gradients and poorly mixed nature of unsaturated systems. We found that unsaturated biofilms exhibited two growth phases – an initial rapid phase and a second phase of slower or negligible development. However, no lag phase was observed for either carbon source. Cellular DNA accumulated linearly with biomass whereas cellular protein and carbohydrates accumulated exponentially with biomass. High levels of carbohydrate, protein and DNA were observed in the EPS of all samples, representing as much as 50% of these macromolecules in the biofilm. Most EPS accumulated during the second phase of growth, when cellular DNA increased only slightly. However, for biofilms cultured on a poorly bioavailable carbon source, EPS DNA decreased during the second phase, suggesting that EPS may affect bacterial survival under nutrient-limited conditions. Whether a product of overflow metabolism or cell lysis, EPS is a significant component of unsaturated biofilm biomass that probably impacts on bacterial ecology.

Responses of the central metabolism in Escherichia coli to phosphoglucose isomerase and glucose-6-phosphate dehydrogenase knockouts.

The responses of Escherichia coli central carbon metabolism to knockout mutations in phosphoglucose isomerase and glucose-6-phosphate (G6P) dehydrogenase genes were investigated by using glucose- and ammonia-limited chemostats. The metabolic network structures and intracellular carbon fluxes in the wild type and in the knockout mutants were characterized by using the complementary methods of flux ratio analysis and metabolic flux analysis based on [U-(13)C]glucose labeling and two-dimensional nuclear magnetic resonance (NMR) spectroscopy of cellular amino acids, glycerol, and glucose. Disruption of phosphoglucose isomerase resulted in use of the pentose phosphate pathway as the primary route of glucose catabolism, while flux rerouting via the Embden-Meyerhof-Parnas pathway and the nonoxidative branch of the pentose phosphate pathway compensated for the G6P dehydrogenase deficiency. Furthermore, additional, unexpected flux responses to the knockout mutations were observed. Most prominently, the glyoxylate shunt was found to be active in phosphoglucose isomerase-deficient E. coli. The Entner-Doudoroff pathway also contributed to a minor fraction of the glucose catabolism in this mutant strain. Moreover, although knockout of G6P dehydrogenase had no significant influence on the central metabolism under glucose-limited conditions, this mutation resulted in extensive overflow metabolism and extremely low tricarboxylic acid cycle fluxes under ammonia limitation conditions.

Knockout of the high-coupling cytochrome aa3 oxidase reduces TCA cycle fluxes in Bacillus subtilis

The metabolic impact of electron rerouting in the respiratory chain of Bacillus subtilis was quantitatively assessed during batch growth of quinol oxidase mutants by 13C-tracer experiments. While disruption of the low-coupling cytochrome bd oxidase was without any apparent phenotype, deletion of the high-coupling cytochrome aa 3 oxidase caused a severe reduction of tricarboxylic acid cycle fluxes and increased overflow metabolism. Since the product-corrected biomass yields were identical in mutants and parent, the results show that efficient ATP generation is not overly important for exponential growth of B. subtilis in batch culture.

Theoretical analysis of effects of metabolic overflow and time delay on the performance and dynamic behavior of a two-stage fermentation process

The performance and dynamic behavior of a two-stage fermentation process are studied for continuous cultivation of microorganisms with metabolic overflow, the growth of which is subject to inhibitions of substrate and product. Metabolic overflow of product and its inhibition of cell growth lead to multiplicity, in which a steady state with higher biomass or product concentrations can be obtained by operating the bioreactor under certain conditions. For instance, a high density cell cultivation should be carried out by increasing the initial concentration of substrate in feed at a constant dilution rate. In contrast, a high yield of target product to substrate could be obtained by decreasing the initial concentration of substrate in feed. If a time delay between biomass formation and the change of the operation conditions is higher than a critical value, persistent periodical oscillations would occur. The critical time delay is dependent on the operating conditions, i.e. the initial substrate concentration and dilution rate. It is very large when the initial concentration of substrate is relatively low or high at a constant dilution rate. A two-stage fermentation process in terms of dynamic behavior is more efficient than one stage fermentation in steady state. The bioreactor performance, e.g. substrate consumption, product concentration, yield of product from substrate, and productivity, can be improved by using self-sustained oscillations in a system of two bioreactors in series.

Bacillus subtilis Metabolism and Energetics in Carbon-Limited and Excess-Carbon Chemostat Culture

The energetic efficiency of microbial growth is significantly reduced in cultures growing under glucose excess compared to cultures growing under glucose limitation, but the magnitude to which different energy-dissipating processes contribute to the reduced efficiency is currently not well understood. We introduce here a new concept for balancing the total cellular energy flux that is based on the conversion of energy and carbon fluxes into energy equivalents, and we apply this concept to glucose-, ammonia-, and phosphate-limited chemostat cultures of riboflavin-producing Bacillus subtilis. Based on [U-(13)C(6)]glucose-labeling experiments and metabolic flux analysis, the total energy flux in slow-growing, glucose-limited B. subtilis is almost exclusively partitioned in maintenance metabolism and biomass formation. In excess-glucose cultures, in contrast, uncoupling of anabolism and catabolism is primarily achieved by overflow metabolism, while two quantified futile enzyme cycles and metabolic shifts to energetically less efficient pathways are negligible. In most cultures, about 20% of the total energy flux could not be assigned to a particular energy-consuming process and thus are probably dissipated by processes such as ion leakage that are not being considered at present. In contrast to glucose- or ammonia-limited cultures, metabolic flux analysis revealed low tricarboxylic acid (TCA) cycle fluxes in phosphate-limited B. subtilis, which is consistent with CcpA-dependent catabolite repression of the cycle and/or transcriptional activation of genes involved in overflow metabolism in the presence of excess glucose. ATP-dependent control of in vivo enzyme activity appears to be irrelevant for the observed differences in TCA cycle fluxes.

CMA: integration of fluid dynamics and microbial kinetics in modelling of large-scale fermentations

Transport limitation is regarded as one of the major phenomena leading to process yield reduction in large-scale fermentations. Knowledge of both the fluid dynamics and the microbial kinetics is needed for understanding and describing situations in large-scale production bioreactors. Microbial kinetics of Escherichia coli including flow metabolism was determined in lab-scale batch and fed-batch experiments. The effect of high substrate fluctuations on metabolism was quantified in scale-down experiments. This knowledge was incorporated into a flow model based on the compartment model approach (CMA). The flow model was verified by mixing time experiments on aerated reactors mixed with multiple impellers at different regimes with liquid volumes 8–22 m 3. The integral model, predicting local glucose, acetate and biomass concentrations in different parts of the reactor, was compared to three large-scale fermentations performed in two different reactors. If lab-scale kinetics was used, the biomass prediction overestimated the biomass concentration. Lab-scale kinetics modified by the results of scale-down experiments incorporating the effect of substrate fluctuations gave a rather satisfying description of biomass concentration. Glucose gradients in different parts of the reactor and acetate produced as a result of overflow metabolism were predicted on a qualitative level. The simulations show that at present the decisive factor for a successful integration of fluid dynamics and microbial kinetics is the kinetics.

A biochemically structured model for Saccharomyces cerevisiae

A biochemically structured model for the aerobic growth of Saccharomyces cerevisiae on glucose and ethanol is presented. The model focuses on the pyruvate and acetaldehyde branch points where overflow metabolism occurs when the growth changes from oxidative to oxido-reductive. The model is designed to describe the onset of aerobic alcoholic fermentation during steady-state as well as under dynamical conditions, by triggering an increase in the glycolytic flux using a key signalling component which is assumed to be closely related to acetaldehyde. An investigation of the modelled process dynamics in a continuous cultivation revealed multiple steady states in a region of dilution rates around the transition between oxidative and oxido-reductive growth. A bifurcation analysis using the two external variables, the dilution rate, D, and the inlet concentration of glucose, S f, as parameters, showed that a fold bifurcation occurs close to the critical dilution rate resulting in multiple steady-states. The region of dilution rates within which multiple steady states may occur depends strongly on the substrate feed concentration. Consequently a single steady state may prevail at low feed concentrations, whereas multiple steady states may occur over a relatively wide range of dilution rates at higher feed concentrations.

Avoiding acetate accumulation in Escherichia coli cultures using feedback control of glucose feeding.

An automated glucose feeding strategy that avoids acetate accumulation in cultivations of Escherichia coli is discussed. We have previously described how a probing technique makes it possible to detect and avoid overflow metabolism using a dissolved oxygen sensor. In this article these ideas are extended with a safety net that guarantees that aerobic conditions are maintained. The method is generally applicable, as no strain-specific information is needed and the only sensor required is a standard dissolved oxygen probe. It also gives the highest feed rate possible with respect to limitations from overflow metabolism and oxygen transfer, thus maximizing bioreactor productivity. The strategy was implemented on three different laboratory-scale platforms and fed-batch cultivations under different operating conditions were performed with three recombinant strains, E. coli K-12 UL635, E. coli BL21(DE3), and E. coli K-12 UL634. In spite of disturbances from antifoam and induction of recombinant protein production, the method reproducibly gave low concentrations of acetate and glucose. The ability to obtain favorable cultivation conditions independently of strain and operating conditions makes the presented strategy a useful tool, especially in situations where it is important to get good results on the first attempt.

Physiological responses to mixing in large scale bioreactors

Escherichia coli fed-batch cultivations at 22 m 3 scale were compared to corresponding laboratory scale processes and cultivations using a scale-down reactor furnished with a high-glucose concentration zone to mimic the conditions in a feed zone of the large bioreactor. Formate accumulated in the large reactor, indicating the existence of oxygen limitation zones. It is suggested that the reduced biomass yield at large scale partly is due to repeated production/re-assimilation of acetate from overflow metabolism and mixed acid fermentation products due to local moving zones with oxygen limitation. The conditions that generated mixed-acid fermentation in the scale-down reactor also induced a number of stress responses, monitored by analysis of mRNA of selected stress induced genes. The stress responses were relaxed when the cells returned to the substrate limited and oxygen sufficient compartment of the reactor. Corresponding analysis in the large reactor showed that the concentration of mRNA of four stress induced genes was lowest at the sampling port most distant from the feed zone. It is assumed that repeated induction/relaxation of stress responses in a large bioreactor may contribute to altered physiological properties of the cells grown in large-scale bioreactor. Flow cytometric analysis revealed reduced damage with respect to cytoplasmic membrane potential and integrity in cells grown in the dynamic environments of the large scale reactor and the scale-down reactor.

Citrate efflux in glucose-limited and glucose-sufficient chemostat culture of Penicillium simplicissium.

Citrate excretion by Penicillium simplicissimum was investigated in a chemostat. Carbon-limited grown P. simplicissimum did not excrete no citrate. Citrate was excreted, however, when growth was nitrogen-limited. Further effects of nitrogen-limitation were a slightly increased rate of glucose and oxygen consumption. This behaviour is typical for a so-called 'overflow metabolism', i.e. the uncoupling of anabolism from catabolism under conditions of carbon excess. Still more citrate was excreted by nitrogen-limited P. simplicissimum when (i) the extracellular osmolarity was increased from 0.2 to 1.5 osm kg(-1) or (ii) when the pH was increased from 4 to 7; or (iii) when the extracellular potassium concentration was lowered from 6 to 0.5 mM. These results were interpreted in terms of a higher energy-consumption under these conditions.

Exopolysaccharide production by the epipelic diatom Cylindrotheca closterium: effects of nutrient conditions.

During the stationary phase of a batch culture of the epipelic diatom Cylindrotheca closterium, accumulation of exopolysaccharides and intracellular carbohydrates was observed. When nitrogen was added to the culture in the stationary phase, growth was resumed and the accumulation of exopolysaccharides was delayed. This indicated that nitrogen depletion caused cessation of growth, and stimulated exopolysaccharide accumulation. Exopolysaccharide accumulation was also stimulated when cells were either resuspended in medium lacking N or P, or when they were inoculated in medium with low concentrations of N or P. Growth was not immediately affected by low N or P concentrations. S depletion only resulted in exopolysaccharide accumulation when growth was affected. Si or Fe depletion did not stimulate exopolysaccharide accumulation, even when growth rates were lowered. Apparently, stimulation of exopolysaccharide accumulation is dependent on the type of nutrient depletion. Intracellular storage carbohydrates did not accumulate when cells were incubated at low N or P concentrations. Cells grown with ammonium as nitrogen source produced more carbohydrates (both extracellular and intracellular) than cells grown with nitrate as nitrogen source, indicating that both exopolysaccharides and intracellular carbohydrates accumulated as a result of overflow metabolism.

Role of CcpA in regulation of the central pathways of carbon catabolism in Bacillus subtilis.

The Bacillus subtilis two-dimensional (2D) protein index contains almost all glycolytic and tricarboxylic acid (TCA) cycle enzymes, among them the most abundant housekeeping proteins of growing cells. Therefore, a comprehensive study on the regulation of glycolysis and the TCA cycle was initiated. Whereas expression of genes encoding the upper and lower parts of glycolysis (pgi, pfk, fbaA, and pykA) is not affected by the glucose supply, there is an activation of the glycolytic gap gene and the pgk operon by glucose. This activation seems to be dependent on the global regulator CcpA, as shown by 2D polyacrylamide gel electrophoresis analysis as well as by transcriptional analysis. Furthermore, a high glucose concentration stimulates production and excretion of organic acids (overflow metabolism) in the wild type but not in the ccpA mutant. Finally, CcpA is involved in strong glucose repression of almost all TCA cycle genes. In addition to TCA cycle and glycolytic enzymes, the levels of many other proteins are affected by the ccpA mutation. Our data suggest (i) that ccpA mutants are unable to activate glycolysis or carbon overflow metabolism and (ii) that CcpA might be a key regulator molecule, controlling a superregulon of glucose catabolism.

Adaptive responses of Ralstonia eutropha to feast and famine conditions analysed by flow cytometry

Results obtained by flow cytometry allow conclusions to be drawn about how the physiological states of Ralstonia eutropha JMP134 are connected with survival strategies under distinct growth conditions. During both feast and famine conditions the cells were found to proceed through sharply separated phases of life. Two sources of carbon and energy, one poor (0.02% phenol) and one rich (0.2% pyruvate and 0.1% yeast extract) were chosen to study the cellular responses. Despite the major differences in carbon source, when growth stages of the bacteria on the two substrates were characterised in batch growth, only minor differences were found in the time course of the membrane potential related fluorescence intensity (MPRFI). This also applied to the rRNA content and the size-correlated forward scatter (FSC) signal of the cells, both of which increased to high levels during the (early) exponential growth phase. On the rich medium, DNA synthesis initially occurred in an uncoupled manner, then a high rate of PHB formation followed when nutrients began to be limiting. Under famine conditions, the cellular responses were much more complex. PHB was synthesised, then DNA synthesis occurred in a ‘eukaryotic’ mode, to be succeeded by renewed PHB synthesis. To obtain defined cell physiological states, the chemostat technique was used in addition to batch experiments. The results obtained clearly indicated that key events in cell physiology, including initiation of DNA replication and overflow metabolism, occurred in a hierarchically ordered manner and were tightly correlated with changes in the environmental conditions of the bacterial cells.

Modelling of aerobic growth of Saccharomyces cerevisiae in a pH-auxostat

A model for growth and overflow metabolism of Saccharomyces cerevisiae was applied to simulate continuous cultivations in a pH-auxostat. The concentrations of glucose, biomass and ethanol are controlled by the flow ratio r between fresh medium and titrant solution, both of which are pH-regulated. A critical value of r could be derived, below which the culture becomes substrate depleted, resulting in a stop-flow condition with retained biomass but without growth. At r-values slightly above the critical value the pH-auxostat is substrate limited and unstable. Further increase of the r value results in a stable continuous culture growing at μmax. Thus, the pH-auxostat complements the chemostat in the growth range at or close to μmax. Even at μmax conditions, the ethanol concentration can be controlled at a low level.

Towards a high-yield bioconversion of ferulic acid to vanillin

Natural vanillin is of high interest in the flavor market. Microbial routes to vanillin have so far not been economical as the medium concentrations achieved have been well below 1 g l−1. We have now screened microbial isolates from nature and known strains for their ability to convert eugenol or ferulic acid into vanillin. Ferulic acid, in contrast to the rather toxic eugenol, was found to be an excellent precursor for the conversion to vanillin, as doses of several g l−1 could be fed. One of the isolated microbes, later identified as Pseudomonas putida, very efficiently converted ferulic acid to vanillic acid. As vanillin was oxidized faster than ferulic acid, accumulation of vanillin as an intermediate was not observed. A completely different metabolic flux was observed with Streptomyces setonii. During the metabolism of ferulic acid, this strain accumulated vanillic acid only to a level of around 200 mg l−1 and then started to accumulate vanillin as the principal metabolic overflow product. In shake-flask experiments, vanillin concentrations of up to 6.4 g l−1 were achieved with a molar yield of 68%. This high level now forms the basis for an economical microbial production of vanillin that can be used for flavoring purposes.

Modeling of overflow metabolism in batch and fed-batch cultures of Escherichia coli.

A dynamic model of glucose overflow metabolism in batch and fed-batch cultivations of Escherichia coli W3110 under fully aerobic conditions is presented. Simulation based on the model describes cell growth, respiration, and acetate formation as well as acetate reconsumption during batch cultures, the transition of batch to fed-batch culture, and fed-batch cultures. E. coli excreted acetate only when specific glucose uptake exceeded a critical rate corresponding to a maximum respiration rate. In batch cultures where the glucose uptake was unlimited, the overflow acetate made up to 9. 0 +/- 1.0% carbon/carbon of the glucose consumed. The applicability of the model to dynamic situations was tested by challenging the model with glucose and acetate pulses added during the fed-batch part of the cultures. In the presence of a glucose feed, E. coli utilized acetate 3 times faster than in the absence of glucose. The cells showed no significant difference in maximum specific uptake rate of endogenous acetate produced by glucose overflow and exogenous acetate added to the culture, the value being 0.12-0.18 g g-1 h-1 during the entire fed-batch culture period. Acetate inhibited the specific growth rate according to a noncompetitive model, with the inhibition constant (ki) being 9 g of acetate/L. This was due to the reduced rate of glucose uptake rather than the reduced yield of biomass.