Experimental investigations of multiple steady states in aerobic continuous cultivations of Saccharomyces cerevisiae

The steady-state behavior of a glucose-limited, aerobic, continuous cultivation of Saccharomyces cerevisiae CEN.PK113-7D was investigated around the critical dilution rate. Oxido-reductive steady states were obtained at dilution rates up to 0.09 h(-1) lower than the critical dilution rate by operating the bioreactor as a productostat, where the dilution rate was controlled on the basis of an ethanol measurement. Thus, the experimental investigations revealed that multiple steady states exist in a region of dilution rates below the critical dilution rate. The existence of multiple steady states was attributed to two distinct physiological effects occurring when growth changed from oxidative to oxido-reductive: (i) a decrease in the efficiency of ATP production and utilization (at ethanol concentrations below 3 g/L) and (ii) repression of the oxidative metabolism (at higher ethanol concentrations). The first effect was best observed at low ethanol concentrations, where multiple steady states were observed even when no repression of the oxidative metabolism was evident, i.e., the oxidative capacity was constant. However, at higher ethanol concentrations repression of the oxidative metabolism was observed (the oxidative capacity decreased), and this resulted in a broader range of dilution rates where multiple steady states could be found.     

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.     

Exacting predictions by cybernetic model confirmed experimentally: Steady state multiplicity in the chemostat

We demonstrate strong experimental support for the cybernetic model based on maximizing carbon uptake rate in describing the microorganism's regulatory behavior by verifying exacting predictions of steady state multiplicity in a chemostat. Experiments with a feed mixture of glucose and pyruvate show multiple steady state behavior as predicted by the cybernetic model. When multiplicity occurs at a dilution (growth) rate, it results in hysteretic behavior following switches in dilution rate from above and below. This phenomenon is caused by transient paths leading to different steady states through dynamic maximization of the carbon uptake rate. Thus steady state multiplicity is a manifestation of the nonlinearity arising from cybernetic mechanisms rather than of the nonlinear kinetics. The predicted metabolic multiplicity would extend to intracellular states such as enzyme levels and fluxes to be verified in future experiments. © 2012 American Institute of Chemical Engineers Biotechnol. Prog., 2012     

Metabolic flux analysis of hybridoma continuous culture steady state multiplicity.

Physiological state multiplicity was observed in continuous cultures of the hybridoma cell line ATCC CRL-1606 cultivated in glutamine-limited steady state chemostats. At the same dilution rate (0.04 h-1), two physiologically different cultures were obtained which exhibited similar growth rates and viabilities but drastically different cell concentrations (7.36 x 10(5) and 1.36 x 10(6) cells/mL). Metabolic flux analysis conducted using metabolite and gas exchange rate measurements revealed a more efficient culture for the steady state with the higher cell concentration, as measured by the fraction of pyruvate carbon flux shuttled into the TCA cycle for energy generation. The low-efficiency steady state was achieved after innoculation by growing the cells in a nutrient rich environment, first in batch mode followed by a stepwise increase of the dilution rate to its set point at 0.04 h-1. The high-efficiency steady state was achieved by reducing the dilution rate to progressively lower values to 0.01 h-1 resulting in conditions of stricter nutrient limitation. The high energetic efficiency attained under such conditions was preserved upon increasing the chemostat dilution rate back to 0.04 h-1 with a higher nutrient consumption, resulting in approximate doubling of the steady state cell concentration. This metabolic adaptation is unlikely due to favorable genetic mutations and could be implemented for improving cell culture performance by inducing cellular metabolic shifts to more efficient flux distribution patterns.     

Inhibition Kinetics of Phenol Degradation by Pseudomonas aeruginosa from Continuous Culture and Washout Data

Steady states of a continuous culture with an inhibitory substrate were used to estimate kinetic parameters under substrate limitation (chemostat operation). Pure cultures of an indigenous Pseudomonas aeruginosa were grown in continuous culture on phenol, the sole source of carbon and energy, at dilution rates of 0.010 to 0.20 h^{- 1}. Using different dilution rates, several steady states were investigated and the specific phenol consumption rates were calculated. In addition, phenol degradation was investigated by increasing the dilution rate above the critical dilution rate (washout cultivation). The results showed that the specific phenol consumption rate increased with increased dilution rate at steady state and that the degradation by Pseudomonas aeruginosa can be described by simple substrate inhibition kinetics under substrate limitation but cannot be described by simple substrate inhibition kinetics under washout cultivation. Fitting of the steady-state data from continuous cultivation to various inhibition models resulted in the best fit for the Yano and Koga kinetic inhibition model. The r_{s max} value of 0.278 mg/mg/h obtained from the Yano and Koga equation was comparable to the experimentally calculated r_{s max} value of 0.283 mg/mg/h obtained under washout cultivation.     

Complex responses to culture conditions in Pseudomonas syringae pv. tomato DC3000 continuous cultures: The role of iron in cell growth and virulence factor induction

The growth of a model plant pathogen, Pseudomonas syringae pv. tomato DC3000, was investigated using a chemostat culture system to examine environmentally regulated responses. Using minimal medium with iron as the limiting nutrient, four different types of responses were obtained in a customized continuous culture system: (1) stable steady state, (2) damped oscillation, (3) normal washout due to high dilution rates exceeding the maximum growth rate, and (4) washout at low dilution rates due to negative growth rates. The type of response was determined by a combination of initial cell mass and dilution rate. Stable steady states were obtained with dilution rates ranging from 0.059 to 0.086 h^?1 with an initial cell mass of less than 0.6 OD₆₀₀. Damped oscillations and negative growth rates are unusual observations for bacterial systems. We have observed these responses at values of initial cell mass of 0.9 OD₆₀₀ or higher, or at low dilution rates (<0.05 h^?1) irrespectively of initial cell mass. This response suggests complex dynamics including the possibility of multiple steady states. Iron, which was reported earlier as a growth limiting nutrient in a widely used minimal medium, enhances both growth and virulence factor induction in iron‐supplemented cultures compared to unsupplemented controls. Intracellular iron concentration is correlated to the early induction (6 h) of virulence factors in both batch and chemostat cultures. A reduction in aconitase activity (a TCA cycle enzyme) and ATP levels in iron‐limited chemostat cultures was observed compared to iron‐supplemented chemostat cultures, indicating that iron affects central metabolic pathways. We conclude that DC3000 cultures are particularly dependent on the environment and iron is likely a key nutrient in determining physiology. Biotechnol. Bioeng. 2010;105: 955–964. © 2009 Wiley Periodicals, Inc.     

Cultivation of Saccharomyces cerevisiae in continuous culture

Summary Growth of Saccharomyces cerevisiae was investigated under aerobic conditions in a glucose limited chemostat. The steady state concentrations of cells, glucose and ethanol were measured in dependence of the dilution rate. The growth rate showed a biphasic dependence from the glucose concentration. A shift from respiratory to fermentative metabolism (Crabtree-effect) altering heavily the cell yield and the ethanol yield took place in the range of dilution rates between 0.3 h^-1 and 0.5 h^-1. Therefore the classical theory of continuous cultures is not applicable on aerobic growth of Saccharomyces cerevisiae under glucose limitation without introducing further premises. On the other hand the steady state cell concentration as a function of the dilution rate fits well the theoretically calculated curves, if cells are cultivated under conditions where only fermentation or respiration is possible.     

Long-Term Changes in Chemostat Cultures of Cytophaga johnsonae

Long-term studies with a gliding, heterotrophic bacterium, Cytophaga johnsonae, were conducted in a glucose-limited chemostat at a high and a low dilution rate. To test the stability of the steady state during long-term experiments the following parameters were monitored: optical density, glucose concentration, glucose uptake potential, ATP content of the cells, and plate counts on two different agar media. Biomass remained relatively constant, although the observed changes could have been possible in both directions. During all steady states, glucose uptake showed a stepwise increase and the glucose concentration showed a corresponding decrease. Glucose uptake potential and glucose concentration in the chemostat were inversely proportional. The ATP content of the cells varied up to 33% during the steady state, but did not show a general trend. After long cultivation in all chemostats, plate counts on both agars dropped to values less than 20% of the original steady-state level. These decreases were due to an inability of the cells to grow on agar plates, not to a lack of vitality of the cells in the chemostat. This study showed that even during shorter chemostat runs, e.g., 1 week, changes in important parameters with the steady state must be expected, especially in the uptake potential and the concentration of the limiting substrate.     

Invertase biosynthesis by Saccharomyces carlsbergensis in batch and continuous cultures

The biosynthesis of invertase by Saccharomyces carlsbergensis LAM 1068 was studied in relation to its glucose effect at both unsteady and steady states of growth. Experimental correlations between the dilution rate and invertase specific activity (E/X) in chemostat, cultures led to an optimum for the enzyme synthesis at a particular intermediate growth rate. The value of E/X increased from 1.1 (U/mg biomass) in batch cultures to 13 (U/mg biomass) in chemostat cultures. A mutant strain A3 showed the highest value for E/X = 25 (U/mg biomass) at high dilution rates where glucose repression was observed with the wild strain.     

Validity of the tritiated thymidine method for estimating bacterial growth rates: measurement of isotope dilution during DNA synthesis.

The rate of tritiated thymidine incorporation into DNA was used to estimate bacterial growth rates in aquatic environments. To be accurate, the calculation of growth rates has to include a factor for the dilution of isotope before incorporation. The validity of an isotope dilution analysis to determine this factor was verified in experiments reported here with cultures of a marine bacterium growing in a chemostat. Growth rates calculated from data on chemostat dilution rates and cell density agreed well with rates calculated by tritiated thymidine incorporation into DNA and isotope dilution analysis. With sufficiently high concentrations of exogenous thymidine, de novo synthesis of deoxythymidine monophosphate was inhibited, thereby preventing the endogenous dilution of isotope. The thymidine technique was also shown to be useful for measuring growth rates of mixed suspensions of bacteria growing anaerobically. Thymidine was incorporated into the DNA of a range of marine pseudomonads that were investigated. Three species did not take up thymidine. The common marine cyanobacterium Synechococcus species did not incorporate thymidine into DNA.     

The effect of vitamins and amino acids on glucose uptake in aerobic chemostat cultures of three Saccharomyces cerevisiae strains

In the respiro-fermentative region of aerobic chemostat cultures at steady state, Saccharomyces cerevisiae CBS 8066 produced high concentrations of ethanol with concomitant low levels of residual glucose which followed Monod kinetics. By contrast, very high residual glucose concentrations were observed in cultures of S. cerevisiae strains ATCC 4126 and NRRL Y132 at dilution rates above 60% of the washout dilution rate, resulting in much lower ethanol concentrations, even though clearly glucose-limited at lower dilution rates in the respiratory region. The addition of a vitamin mixture resulted in decreased residual glucose concentrations in respiro-fermentative cultures of all three strains, but the effect was much more pronounced with strains ATCC 4126 and NRRL Y132. Meso-inositol was mainly responsible for this effect, although with strain ATCC 4126 other vitamins as well as an amino acid mixture were also required to minimise the steady-state residual glucose levels. The residual glucose concentration in continuous culture was, therefore, greatly dependent on the growth factor requirements of the particular yeast strain, which apparently increased on increasing the dilution rate into the respiro-fermentative region. The strain differences with respect to growth factor requirements at high dilution rates, which were not evident at low dilution rates, had a profound effect on the kinetics of glucose assimilation in aerobic chemostat culture.     

Growth and accumulation dynamics of poly(3-hydroxyalkanoate) (PHA) in Pseudomonas putida GPo1 cultivated in continuous culture under transient feed conditions

It has been shown that Pseudomonas putida GPo1 is able to grow in continuous culture simultaneously limited by ammonium (N source) and octanoate (C source), and concomitantly accumulate poly([R]-3-hydroxyalkanoate) (PHA). Under such growth conditions the material properties of PHA can be fine-tuned if a second PHA precursor substrate is supplied. To determine the range of dual carbon and nitrogen (C, N)-limited growth conditions, tedious chemostat experiments need to be carried out for each carbon source separately. To determine the growth regime, the C/N ratio of the feed (f) to a chemostat was changed in a stepwise manner at a constant dilution rate of 0.3/h. Dual-(C, N)-limited growth was observed between C(f) /N(f) ≤ 6.4 g/g and C(f) /N(f) >9.5 g/g. In the following, we analyzed alternative approaches, using continuous medium gradients at the same dilution rate, that do not require time consuming establishments of steady states. Different dynamic approaches were selected in which the C(f) /N(f) ratio was changed continuously through a convex increase of C(f) , a convex increase of N(f) , or a linear decrease of C(f) (gradients 1, 2, and 3, respectively). In these experiments, the dual-(C, N)-limited growth regime was between 7.2 and 11.0 g/g for gradient 1, 4.3 and 6.9 g/g for gradient 2, and 5.1 and 8.9 g/g for gradient 3. A mathematical equation was developed that compensated a time delay of the gradient that was caused by the wash-in/wash-out effects of the medium feed.     

Chemostat cultures of yeasts, continuous culture fundamentals and simple unstructured mathematical models.

Fundamental aspects of chemostat cultures are reviewed. Using yeast cultures as examples, it is shown that steady states in chemostats may be predicted quantitatively by combining the correct number of unstructured kinetic models with expressions for existing stoichiometric constraints. The necessary number of such kinetic models corresponds to the number of limiting substrates and increases with the number of different metabolic pathways available to the strain. This is demonstrated by an experimental comparison of yeast growth limited by glucose alone for which metabolism is oxidative, and growth doubly limited by both glucose and oxygen, which occurs according to an oxido-reductive metabolism. The steady state data for such experiments can in principle be predicted based on a minimal amount of information by a simple stoichiometric model. It represents the overall stoichiometry of growth by a superposition of a fully oxidative and a fully reductive growth reaction and uses the concept of "aerobicity" to characterize the relative importance of the two reactions.     

Inhibition kinetics of phenol degradation from unstable steady-state data

Multiplicity of steady states of a continuous culture with an inhibitory substrate was used to estimate kinetic parameters under steady-state conditions. A continuous culture of Pseudomonas cepacia G4, using phenol as the sole source of carbon and energy, was overloaded by increasing the dilution rate above the critical dilution rate. The culture was then stabilized in the inhibitory branch by a proportional controller using the carbon dioxide concentration in the reactor exhaust gas as the controlled variable and the dilution rate as the manipulated variable. By variation of the set point, several unstable steady states in the inhibitory branch were investigated and the specific phenol conversion rates calculated. In addition, phenol degradation was investigated under substrate limitation (chemostat operation).The results show that the phenol degradation by P. cepacia can be described by the same set of inhibition parameters under substrate limitation and under high substrate concentrations in the inhibitory branch. Biomass yield and maintenance coefficients were identical. Fitting of the data to various inhibition models resulted in the best fit for the Yano and Koga equation. The well-known Haldane model, which is most often used to describe substrate inhibition by phenol, gave the poorest fit. The described method allows a precise data estimation under steady-state conditions from the maximum of the biological reaction rate up to high substrate concentrations in the inhibitory branch. Inhibition parameter estimation by controlling unstable steady states may thus be useful in avoiding discrepancies between data generated by batch runs and their application to continuous cultures which have been often described in the literature. (c) 1997 John Wiley & Sons, Inc. Biotechnol Bioeng 54: 567-576, 1997.     

Estimation of steady-state culture characteristics during acceleration-stats with yeasts

Steady-state culture characteristics are usually determined in chemostat cultivations, which are very time-consuming. In contrast, acceleration-stat (A-stat) cultivations in which the dilution rate is continuously changed with a constant acceleration rate are not so time-consuming, especially at high acceleration rates. Therefore, the A-stat could be advantageous to use instead of the chemostat. However, the highest acceleration rate, meaning the fastest A-stat that can be applied for estimating steady-state culture characteristics, is not known yet. Experimental results obtained with Zygosaccharomyces rouxii, an important yeast in soy sauce processes, showed that the culture characteristics during the A-stat with an acceleration rate of 0.001 h(-2) were roughly comparable to those of the chemostat. For higher acceleration rates the deviation between the culture characteristics in the A-stat and those in the chemostat obtained at the same dilution rate generally started to increase. The source of these deviations was examined by simulation for Saccharomyces cerevisiae. The simulations demonstrated that this deviation was not only dependent on the metabolic adaptation rate of the yeast, but also on the rate of change in environmental substrate concentrations during A-stats. From this work, it was concluded that an A-stat with an acceleration rate of 0.001 h(-2) is attractive to be used instead of chemostat whenever a rough estimation of steady-state culture characteristics is acceptable.     

Regulation of ammonia assimilation in ammonia-limited chemostat cultures of Escherichia coli ML 30: evidence of bistability

In a preceding paper evidence of two stationary stable states (bistability) in the specific activity of glutamine synthetase (GS) in ammonia-limited steady-state cultures of Escherichia coli ML 30 at dilution rates (D) about 0.15 h-1 was described (Müller et al. 1977). For better understanding of the regulation mechanisms leading to GS bistability chemostat experiments were performed over a wide range of dilution rates up to D = 0.8 h-1. For each steady state the specific activities of GS and glutamate dehydrogenase (GDH)--the other key enzyme of the two NH3 assimilation routes in E. coli--and in addition the remaining NH3 concentration in the culture liquid were determined. Parallel to GS bistability two states of GDH activity and NH3 concentration are found. The higher state of GS is connected with a lower GDH activity and NH3 concentration. With rising D the GS activities decrease whereas GDH activities and NH3 concentrations increase. Since no adenylation of the GS is detectable GS bistability seems to be regulated on the level of enzyme synthesis like GDH bistability. From the experimental findings a mathematical model is derived based on the bottle neck enzyme theory of growth. It describes the dependence between the specific growth rates on the one hand and the specific enzyme activities and NH3 concentration on the other. It is shown that the specific uptake rate of the limiting NH3 and the specific growth rates, respectively, depend on the simultaneous action of two bottle neck enzymes which are connected by a regulative link.     

Effects of agitation intensity on mycelial morphology and protein production in chemostat cultures of recombinant Aspergillus oryzae

The effects of agitation on fragmentation of a recombinant strain of Aspergillus oryzae and its consequential effects on protein production have been investigated. Constant mass, 5.3-L chemostat cultures at a dilution rate of 0.05 h-1 and a dissolved oxygen level of 75% air saturation, have been conducted at 550, 700, and 1000 rpm. These agitation speeds were chosen to cover a range of specific power inputs (2.2 to 12 kW m-3) from realistic industrial levels to much higher values. The use of a constant mass chemostat linked to a gas blender allowed variation of agitation speed and hence gas hold-up without affecting the dilution rate or the concentration of dissolved oxygen. The morphology of both the freely dispersed mycelia and clumps was characterized using image analysis. Statistical analysis showed that it was possible to obtain steady states with respect to morphology. The mean projected area at each steady state under growing conditions correlated well with the 'energy dissipation/circulation" function, [P/(kD3tc)], where P is the power input, D the impeller diameter, tc the mean circulation time, and k is a geometric constant for a given impeller. Rapid transients of morphological parameters in response to a speed change from 1000 to 550 rpm probably resulted from aggregation. Protein production (alpha-amylase and amyloglucosidase) was found to be independent of agitation speed in the range 550 to 1000 rpm (P/V = 2.2 and 12.6 kW m-3, respectively), although significant changes in mycelial morphology could be measured for similar changes in agitation conditions. This suggests that mycelial morphology does not directly affect protein production (at a constant dilution rate and, therefore, specific growth rate). An understanding of how agitation affects mycelial morphology and productivity would be valuable in optimizing the design and operation of large-scale fungal fermentations for the production of recombinant proteins. Copyright 1999 John Wiley & Sons, Inc.     

Conidiation of Aspergillus niger in continuous culture

Summary Conidiation of Aspergillus niger was studied in carbon-limited and nitrogen-limited chemostat culture. Under citrate-limitation conidiation intensity varied inversely with dilution rate. Conidiophores were less complex than in aerial conidiation and at high dilution rates conidia occasionally developed from modified hyphal tips. Conidiation was difficult to achieve under glucose-limitation. At the low dilution rates that allowed limited conidiation steady state could not be maintained due to onset of autolysis. At higher dilution rates when steady state was readily obtained conidiation did not occur. The maximum yield constants under citrate-limitation and glucose-limitation were respectively 0.145 and 0.4 mg dry weight/mg substrate, while the relative specific maintenance values were 0.045 and 0.018 mg substrate/mg dry weight/h. Under ammonium-limitation with citrate as the carbon source there was no conidiation. When nitrate became the limiting nitrogen source conidiophore initiation occurred but biomass production was low and wash-out occurred at D=0.034 h^-1.     

Continuous culture of Thiorhodaceae

Summary In sulfide limited continuous culture of a marine isolate of Chromatium vinosum, sulfide was undetectable in steady states below dilution rates of 0.06h^-1, that is 1/2 of the maximum specific growth rate. In the same range, sulfur is assumed to attain the role of the growth rate limiting substrate. Furthermore, it could be shown that the rate of sulfur oxidation is a function of the surface area of the sulfur globules rather than of the sulfur concentration. In completely filled chemostats, steady states were obtainable only at dilution rates not exceeding 0.09 h^-1. In the presence of a nitrogen flushed gas phase, steady states were obtained at dilution rates approaching the maximum specific growth rate (0.12h^-1). This phenomenon is ascribed to the particular sulfide tolerance of our strain of Chromatium vinosum. The saturation constant and the inhibition constant (lowest, respectively highest total sulfide concentration at which the specific growth rate is equal to one-half of the maximum specific growth rate in the absence of inhibition) were 0.007 mM and 0.85 mM, respectively.The ecological significance of the data is discussed.Contribution No. 2406 from the Woods Hole Oceanographic Institution.