Oscillatory behavior of Saccharomyces cerevisiae in continuous culture: I. Effects of pH and nitrogen levels

The appearance of sustained oscillations in bioreactor variables (biomass and nutrient concentrations) in continuous cultures of Saccharomyces cerevisiae indicates the complex nature of microbial systems, the inadequacy of current growth kinetic models, and the difficulties which may arise in bioprocess control and optimization. In this study we investigate continuous bioreactor behavior over a range of operating conditions (dilution rate, feed glucose concentration, feed ammonium concentration, dissolved oxygen, and pH) to determine the process requirements which lead to oscillatory behavior. We present new results which indicate that high feed ammonium concentrations may eliminate oscillations and that under oscillatory conditions ammonium levels are generally low and oscillatory as well. The effects of pH are complex and oscillations were only observed at pH values 5.5 and 6.5; no oscillations were observed at a pH of 4.5. Under our nominal operating conditions (feed glucose concentration 10 g/L, dilution rate 0.145 h(-1), feed ammonium concentration 0.0303M, dissolved oxygen level 50%, pH 5.5, and T = 30 degrees C) we found two possible final bioreactor states depending on the transient used to reach the nominal operating conditions. One of the states was oscillatory and characteristic of oxidative metabolism and the other was nonoscillatory and fermentative.     

Autonomous oscillations in Saccharomyces cerevisiae during batch cultures on trehalose

We report that autonomous oscillations, which usually happen in aerobic glucose-limited continuous cultures of yeast at low dilution rate, were also observed in trehalose discontinuous cultures of Saccharomyces cerevisiae. This unexpected oscillatory behaviour was therefore examined using fast Fourier transformation of online gas measurements. This robust mathematical analysis underlined the existence of two types of oscillation. The first was found to be linked to the cell cycle because (a) the periodicity corresponded to a fraction of the generation time and (b) the oscillations were accompanied by a transient increase in the budding index, mobilization of storage carbohydrates, and fermentative activity. Moreover, these oscillations occurred in a range of specific growth rates between 0.04 and 0.15 h(-1). All these criteria were consistent with the cell-cycle-related metabolic oscillations observed in the same range of growth rates in glucose-limited continuous cultures. The second type were short-period respiratory oscillations, independent of the specific growth rate. Both types of oscillation were found to take place consecutively and/or simultaneously during batch culture on trehalose. In addition, mobilization of intracellular trehalose emerged as a key parameter for the sustainability of these autonomous oscillations as they were no longer observed in a mutant defective in neutral trehalase activity. We propose that batch culture on trehalose may be an excellent device for further investigation of the molecular mechanisms that underlie autonomous oscillations in yeast.     

Continuous photoautotrophic cultures of the eukaryotic alga Chlorella vulgaris can exhibit stable oscillatory dynamics

Sustained oscillations in cell concentration, average per cell DNA content, and average cell size were found in continuous photoautotrophic cultures of Chlorella vulgaris at low dilution rates (0.1/day). The period of oscillation was approximately 10 days. DNA histograms determined by flow cytometry exhibited reproducible pattern through consecutive oscillations. At the maximum cell concentration during an oscillation, the DNA histograms showed that the majority of the cells were not replicating their chromosomes, and most of the culture was comprised of single cells in G0/G1 phase. The cells then initiated DNA replication; however, because of the long generation time, the cell concentration decreased to a minimum, and at the same time the average per cell DNA content reached its maximum value. At this point the cells began to divide, and the cell concentration increased until it reached its maximum value at the beginning of the next oscillation. Calculations based on the supplied nutrients and comparison to biomass generation showed that the oscillatory behavior in continuous photoautotrophic cultures of C. vulgaris was not due to nutrient limitation, but most likely was due to the secretion of compounds that alter cell cycle kinetics. The oscillatory behavior disappeared when the dilution rate was increased to 0.3/day and the culture reached a stable steady state.     

初始条件对高浓度乙醇连续发酵过程的影响及振荡行为提高发酵效率的机理分析

前期实验在稀释速率为0.027h-1的高浓度乙醇连续发酵过程中,发现了一种长周期、宽振幅的参数振荡现象。本实验进一步考察了不同稀释速率下的连续发酵过程,发现在稀释速率为0.04h-1条件下,也能出现类似的振荡现象;在稀释速率为0.027h-1或0.04h-1的条件下,改变系统的初始状态可以得到振荡和稳态两种不同的发酵过程。比较振荡和稳态过程的实验数据后,发现在稀释速率为0.04h-1的条件下,与稳态过程相比,振荡过程的平均残糖浓度降低了14.8%,平均乙醇浓度提高了12.6%,平均设备生产强度提高了12.3%。进一步分析表明:与稳态过程相比,振荡过程动力学行为不仅存在滞后,而且在相同残糖和乙醇浓度条件下,所对应的平均比生长速率提高了53.8%。     

Transient behavior of a continuous stirred tank biological reactor utilizing phenol as an inhibitory substrate

Transient experiments were conducted on a Pseudomomas utilizing phenol in a continuous culture by disturbing the influent substrate concentration and dilution rate. Two stable steady states existed for some ranges of the parameters. Highly damped oscillations were observed in approaching a new high conversion steady state or in returning to a new high conversion steady state following a small disturbance. When a large disturbance was applied there was a smooth (overdamped) approach to a new low conversion steady state. The observed oscillatory behavior for small disturbances was predicted by a modified Powell-Ierusalemskii bottleneck model, but could not be predicted by a Monod-Haldane model; neither model was accurate for predicting the effect of large disturbances. A constant wall growth factor was used to account for microbial film activity, and the existence of two stable states was directly due to the presence of the film.     

Nature and significance of oscillatory behavior during solvent production by Clostridium acetobutylicum in continuous culture

The concurrent production of acids and solvents and the production of acetone during continuous culture in a product-limited chemostat indicated that the culture contained a mixture of acid- and solvent-producing cells. Periodic oscillations in the yield of end products and the specific growth rate of the culture were ob served during undisturbed continuous culture at a constant dilution rate. The increased specific growth rate was associated with an increased acid yield and an increase in the rate of cell division and the proportion of short rods. The decreased specific growth rate was as sociated with an increase in the solvent yield and a decrease in the rate of cell division, resulting in the production of elongated rods. It is proposed that the oscillatory behavior observed during continuous culture is an inherent characteristic related to the shift from primary to secondary metabolism. A major consequence of the oscillation of the specific rates of growth and division in cultures containing acid- and solvent-producing cells is that it precludes the attainment of a true steady state during continuous culture.     

Response of continuous cultures to stimuli in glucose feed rate and dilution rate

The response of continuous cultures of yeast was investigated following step disturbances in glucose feed rate and dilution rate. The responses of the culture to the stimuli were oscillatory. The oscillatory responses were explained in terms of cell synchrony which was induced by the step change. An understanding of continuous cultures to stimuli was made possible with an appreciation of the inherently oscillatory events occurring in the single cell cycle between one mitosis and the next. Step changes in glucose feed rate and dilution rate induced a partial synchrony, which enabled the inherently oscillatory behavior of the individual cells to be made observable in the culture as a whole.     

Induction and elimination of oscillations in continuous cultures of Saccharomyces cerevisiae

Continuous cultures of Saccharomyces cerevisiae are known to exhibit oscillatory behavior in the oxidative region. Important findings of a series of experiments conducted to identify the causes for initiation of and the means for elimination of oscillations in these cultures are reported in this paper. These oscillations are seen to be connected to the growth kinetics of the microorganism and are induced at very low glucose concentrations and at dissolved oxygen (DO) levels that are neither high nor low (DO values between 20 and 78% air saturation at a dilution rate of 0.2 h(-1) and pH of 5.5 at 30 degrees C). The oscillatory behavior is encountered over a range of dilution rates (0.09-0.25 h(-1) at 30 degrees C for pH = 5.5 and DO = 50% air saturation). The oscillations can be eliminated by raising the DO level above a critical value or by lowering the DO level below a critical value.     

Continuous cultivation of fission yeast: Classical and flow microfluorometry observations

The fission yeast Schizosaccharomyces pombe was grown in glucose-limited medium in a steady-state continuous flow reactor. Changes in mean cell protein and RNA contents with growth rate are consistent with earlier observations under different conditions. Flow microfluorometry measurements of the frequency functions of DNA at different dilution rates show changes in coordination of DNA synthesis and cell separation. Shifting from batch growth to small dilution rates results in unusual cell aggregation which leads to multiple steady states at identical operating conditions.     

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.     

Free intracellular amino acid pools during autonomous oscillations in Saccharomyces cerevisiae

In the present work dynamic changes of free intracellular amino acid pools during autonomous oscillations of Saccharomyces cerevisiae were quantified in glucose-limited continuous cultivations. At a dilution rate of D = 0.22 h(-1) cyclic changes with a period of 120 min were found for many variables such as carbon dioxide production rate, dissolved oxygen, pH, biomass content, and various metabolite concentrations. On the basis of the observed dynamic patterns, free intracellular amino acids were classified to show oscillatory, stationary, or chaotic behavior. Amino acid pools such as serine, alanine, valine, leucine, or lysine were subjected to clear oscillations with a frequency of 120 min, identical to that of other described cultivation variables, indicating that there is a direct correlation between the periodic changes of amino acid concentrations and the metabolic oscillations on the cellular level. The oscillations of these amino acids were unequally phase-delayed and had different amplitudes of oscillation. Accordingly, they exhibited different patterns in phase plane plots vs. intracellular trehalose. Despite the complex and marked metabolic changes during oscillation, selected intracellular amino acids such as histidine, threonine, isoleucine, or arginine remained about constant. Concentrations of glutamate and glutamine showed a chaotic behavior. However, the ratio of glutamate to glutamine concentration was found to be oscillatory, with a period of 60 min and a corresponding figure eight-shaped pattern in a plot vs. trehalose concentration. Considering the described diversity, it can be concluded that the observed periodic changes are neither just the consequence of low or high rates of protein biosynthesis/degradation nor correlated to changing cell volumes during oscillation. The ratio between doubling time (189 min) and period of oscillation of intracellular amino acids (120 min) was 1:6. The fact that there is a close relationship between doubling time and period of oscillation underlines that the described autonomous oscillations are cell-cycle-associated.     

Model simulations of continuous rotifer cultures

Derived from the Monod-model and regulating principles a regulation model of the rotifer development in chemostats was developed. The model was validated in continuous cultures of Brachionus angularis both in steady-states, when undisturbed, and in transient-states after perturbations by step changes of dilution rate or input substrate concentration. Simulations of the simple model monotonically approached steady-states, but cultures show overshoots and damped oscillations before reaching this state. After introducing time-lags into the model it depends on the size of the time lag if model rotifer densities reach stable steady-state values (at low time lags) or stable limit cycles with periodic oscillations (at high time lags). At even higher time lags chaotic conditions occur in the model with final extinction of the rotifers.     

Dynamics of Predator‐Prey Interactions in Continuous Cultures

This paper studies the behavior of a general unstructured kinetic model for continuous bioreactors involving interactions between predator, prey and a limiting substrate. The analysis carried out in this paper shows how closed analytical conditions for arbitrary growth rates can be derived that describe the conditions for the existence of the interacting species in an oscillatory behavior. It is also demonstrated how practical diagrams in terms of operating and kinetic parameters can be constructed that classify the different behavior predicted by the model. Applications of these general results to a number of experimentally validated models have revealed that the saturation model always predicts hard oscillations for a certain range of dilution rates, for any values of model parameters. Bifurcation diagrams in the operating parameter space permitted the delineation of regions of hard oscillations, regions of static coexistence, regions of predator washout and regions of total washout. The analysis of the double saturation model has proven its ability to predict two Hopf points. Hard oscillations are therefore expected within the dilution rates corresponding to the two Hopf points. Practical diagrams were also constructed to delineate the boundaries separating hard oscillations from static coexistence and washout conditions.     

Observed quasi-steady kinetics of yeast cell growth and ethanol formation under very high gravity fermentation condition

Using a generalSaccharomyces cerevisiae as a model strain, continuous ethanol fermentation was carried out in a stirred tank bioreactor with a working volume of 1,500 mL. Three different gravity media containing glucose of 120, 200 and 280 g/L, respectively, supplemented with 5 g/L yeast extract and 3 g/L peptone, were fed into the fermentor at different dilution rates. Although complete steady states developed for low gravity medium containing 120 g/L glucose, quasi-steady states and oscillations of the fermented parameters, including residual glucose, ethanol and biomass were observed when high gravity medium containing 200 g/L glucose and very high gravity medium containing 280 g/L glucose were fed at the designated dilution rate of 0.027 h⁻¹. The observed quasi-steady states that incorporated these steady states, quasi-steady states and oscillations were proposed as these oscillations were of relatively short periods of time and their averages fluctuated up and down almost symmetrically. The continuous kinetic models that combined both the substrate and product inhibitions were developed and correlated for these observed quasi-steady states.     

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.     

Transitional states in a chemostat culture of Candida utilis

Transient states of the chemostat Candida utilis 1668-3-37 culture were studied when its growth was limited by ethanol and an abrupt acidification of the medium from pH 5.0 to 2.2 was done or when the dilution rate was rapidly changed from D = 0.1 to 0.3 h-1 and back to 0.07 h-1. The pH shock was found to cause stronger oscillations in a number of parameters (the weight of dry biomass, the content of residual ethanol, the content of RNA in the cells) than a change in the dilution rate. In the latter case the population density changed more smoothly than the content of RNA did. DNA content remained at one and the same level in all of the experiments. All of the oscillations were observed only in the first generation after a shock; there upon, the culture remained for a long time (7 to 10 generations) in a very stable state typical of chemostat cultures. The oscillations induced by the unfavourable pH of the medium were compared with those caused by an abrupt change in the dilution rate. The pH shock brought about multiple damping oscillations of the parameters whereas a change in the dilution rate resulted, most often, in a merely one oscillation.     

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.     

Recombinant protein synthesis and plasmid instability in continuous cultures of Escherichia coli JM103 harboring a high copy number plasmid

Escherichia coli JM103[pUC8] was employed as a model to investigate the behavior of a recombinant microbial system harboring a plasmid at high copy numbers. Experiments with batch and continuous cultures of recombinant and plasmid-free cells were conducted in a well-controlled bio-reactor. In batch experiments, plasmid copy number varied typically from an average of 500 during the exponential growth phase to as high as 1250 during the stationary phase. While the segregational plasmid instability was negligible in batch experiments, severe segregational instability occurred in continuous experiments conducted over a range of dilution rates, resulting in complete loss of plasmid-bearing cells from the continuous cultures within few residence times after transition to continuous operation. The profound differences in the specific growth rates and mass yields of the plasmid-free and plasmid-bearing cells resulting from the extra metabolic burden on the plasmid-bearing cells mainly due to excessive plasmid DNA content was the major cause for the plasmid instability. Plasmid multirnerization was detected in batch and continuous cultures and was found to have significant influence on the effective copy number and was partially responsible for the severe segregational instability in continuous cultures. A quasi-steady state representative of plasmid-bearing cells was established in the initial portion of each continuous culture experiment. Due to the profound growth rate differential between the two types of cells, transients of considerable duration were observed in each continuous culture experiment (initiated with a pure culture of plasmid bearing cells) following the slow accumulation of plasmid-free cells near the end of the quasi-steady state. Significant variations in various culture parameters (including a rapid decline in the plasmid-bearing fraction of the total cell population) occurred during this period, leading ultimately to a steady state for a culture dominated entirely by plasmid-free cells. In continuous cultures, plasmid copy number during the quasi-steady states increased with decreasing dilution rate from 50 (at 0.409 h(-1)) to 941 (at 0.233 h(-1)). Production of the plasmid-encoded protein (beta-lactamase) in these experiments was maximized at an intermediate dilution rate, corresponding to an optimum copy number of about 450. A similar optimum copy number was observed in batch cultures. Significant excretion of beta-lactamase was observed at both low and high dilution rates.     

Continuous fermentation of undetoxified dilute acid lignocellulose hydrolysate by Saccharomyces cerevisiae ATCC 96581 using cell recirculation

Saccharomyces cerevisiae ATCC 96581 was cultivated in a chemostat reactor with undetoxified dilute acid softwood hydrolysate as the only carbon and energy source. The effects of nutrient addition, dilution rate, cell recirculation, and microaerobicity were investigated. Fermentation of unsupplemented dilute acid lignocellulose hydrolysate at D = 0.10 h(-1) in an anaerobic continuous reactor led to washout. Addition of ammonium sulfate or yeast extract was insufficient for obtaining steady state. In contrast, dilute acid lignocellulose hydrolysate supplemented with complete mineral medium, except for the carbon and energy source, was fermentable under anaerobic steady-state conditions at dilution rates up to 0.14 h(-1). Under these conditions, washout occurred at D = 0.15 h(-1). This was preceded by a drop in fermentative capacity and a very high specific ethanol production rate. Growth at all different dilution rates tested resulted in residual sugar in the chemostat. Cell recirculation (90%), achieved by cross-flow filtration, increased the sugar conversion rate from 92% to 99% at D = 0.10 h(-1). Nutrient addition clearly improved the long-term ethanol productivity in the recirculation cultures. Application of microaerobic conditions on the nutrient-supplemented recirculation cultures resulted in a higher production of biomass, a higher cellular protein content, and improved fermentative capacity, which further improves the robustness of fermentation of undetoxified lignocellulose hydrolysate.     

State selection in coupled identical biochemical systems with coexisting stable states

This work examines state selection for coupled biochemical systems with coexisting stable states. For biochemically identical biochemical systems, different coupled systems are examined for the coexistence of (a) one steady state and one oscillatory state or (b) two oscillatory states. For case (a), it is revealed that state selection is always governed by two key factors: the values of kinetic parameters and the coupling strength. When the coupling strength is small, the coupled systems remain in the basin of attraction of their original states. When it is sufficiently large, all coupled systems are always entrained, independently of their original states. Furthermore, for the entrainment, which of the two coexisting states is selected depends sensitively on the activity of recycling enzyme (one of kinetic parameters). It is shown that this is because changing the activity of recycling enzyme alters the size of basin of attraction of each state. When both systems in the same oscillatory state are coupled, an additional factor, namely phase shift between two oscillations, may also affect state selection, and coupling may cause the systems to select either the original oscillatory state or the coexisting steady state. In addition to the features of case (a), case (b) also supports quasiperiodic oscillations and synchronisation of two periodic oscillations. Implications of the results for understanding state selection during the evolution of coupled biochemical systems with coexisting stable states are discussed.