Oscillations in continuous cultures of budding yeast: a segregated parameter analysis

Sustained oscillations have been observed in continuous cultures of Saccharomyces cerevisiae. These oscillations appear spontaneously under aerobic conditions and may constitute a severe limitation for process control. We have found that oscillations arise only in a well defined range of dilution rates and dissolved oxygen values. The period of the oscillations is related, but not equal, to the mass doubling time, and shows a relation ship with both the parent cells and daughter cells generation times. At high dilution rates two oscillatory regimens, with different periods, are observed. The analysis of the budding index shows a marked degree of synchronization of the culture, however significant differences, both in phase and in amplitude, are ob served if the budding index of parent cells and of daughter cells are considered separately. The complex changes of the cell population are clearly demonstrated by the continuous and periodic modification of both cell volume distributions and protein distributions. Ethanol is always accumulated before the drop of dissolved oxygen concentration and one of the peaks of budding index. We propose a model that explains the insurgence of these oscillation as a consequence of changes in cell cycle parameters due to alternate growth in glucose and in ethanol.     

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.     

Cell cycle of Chlorella vulgaris can deviate from the synchronous binary division model

Conditioned medium from high density Chlorella vugaris cultures was freeze dried extracted into ethanol. The ethanol was volatilized and the resulting powder then introduced with C. vulgaris photoautotrophic cultures which underwent asynchronous DNA replication forming cells with two, three, four, and six autospores instead of 2 n characteristic of synchronous DNA replication. These cells were blocked at the division stage of the cell cycle.     

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.     

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.     

Multiple stable states and hysteresis in continuous, oscillating cultures of budding yeast

The conditions that precede the onset of autonomous oscillations in continuous yeast cultures were studied in three different types of experiments. It was found that the final state of the culture depended on the protocol used to start up the reactor. Batch cultures, switched to continuous operation at different stages of the batch growth curve, all exhibited similar dynamics-ethanol depletion followed by autonomous oscillations. Small perturbations of the distribution of states in the reactor, achieved by addition of externally grown cells, were able to quench the oscillatory dynamics. Reaching the desired operating point by slow dilution rate changes gave rise to different final states, two oscillatory states and one steady state, depending on the rate of change in dilution rate. The multiplicity of stable states at a single operating point is not explained by any current distributed model and points toward a segregated mechanism of these oscillations.     

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%。     

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.     

Oxygen, pH value, and carbon source induced changes of the mode of oscillation in synchronous continuous culture of Saccharomyces cerevisiae

Oscillations of measured process parameters occur in continuous cultures of Saccharomyces cerevisiae owing to a partial synchronization of budding. Intentional changes of the oxygen concentration, pH value, and carbon source cause effects on the period length similar to those known from variations of the dilution rate. The generation times of parent and daughter cells frequently differ in synchronous culture. To analyze the oscillation the term mode IJ of oscillation is used, which is defined as the ratio IJ of the generation times of parent and daughter cells. When the dissolved oxygen concentration was reduced to zero, the mode of oscillation changed within two periods from mode 12 to mode 11, caused by a decrease of the generation time of daughter cells and an increase of that of the parent cells. When the pH value was slowly reduced from 5.0 to 3.9, a change from mode 112 to mode 13 was observed. Mode 13, representing one parent and three daughter cell populations (the start of budding of each of the three being delayed by one period), denotes an elongated generation time of the daughter cells compared to mode 112, marked by one parent and two different daughter cell classes. When the carbon source galactose was replaced by glucose a mode change from mode 12 to mode 11 was observed. This alteration of the mode was found to be dependent on the status of the cell cycle at the time when the carbon source is changed. The population distribution in batch cultures with glucose or galactose as a substrate was analysed by dyeing the DNA and counting the bud scars. Galactose provoked higher growth rates for the older cells. According to the model for stationary synchronous growth parameters like DO, pH value or the type of carbon source can be varied within a certain range without effecting the period length. If the variation imposes a certain stress, the culture switches to a new mode. These kinds of parameters therefore provide selective measures to influence the period lengths and the modes of oscillation.     

微生物细胞连续培养过程中振荡和混沌行为的研究进展

本文对微生物细胞连续培养过程中产生振荡现象的条件,影响振荡特征（振幅和周期）的因素,振荡机理的研究和振荡行为的应用等进行了全面的论述,并指出了振荡研究的发展方向。     

Oscillatory behavior of Saccharomyces cerevisiae in continuous culture: II. Analysis of cell synchronization and metabolism

Sustained oscillations of biomass, ethanol, and ammonium concentrations, specific growth rate, and specific uptake rates of ethanol, ammonium, and oxygen were found in continuous cultures of Saccharomyces cerevisiae under controlled dissolved oxygen (DO), pH, and temperature conditions. The period of oscillations was approximately 2.5-3 h at a pH of 5.5 and 2-2.5 h at a pH of 6.5. Oscillations were observed only under conditions of low carbon (glucose below the minimum detectable level), nitrogen nutrient (ammonium concentration varied between 0.00001 and 0.0015M), and ethanol concentration (0.002-0.085 g/L) in the bioreactor.The oscillatory behavior at pH 5.5 was also characterized by partially synchronized cell growth and reproduction. Not only did the total percentage of budding cells oscillate with the same period as observed for the global biomass and nutrient concentrations, but the peaks in the individual subpopulations of initial budding, middle budding, and late budding cells appeared sequentially during the oscillation period. This provides strong evidence of the hypothesis that variations in metabolism during different periods in the cell cycle of a partially synchronized cell population are responsible for the observed oscillatory bioreactor behavior.The specific nutrient uptake rates for ammonium and oxygen as well as the net specific ethanol uptake rate oscillated with the same period as the biomass oscillations. These results show a dramatic increase in the ammonium and oxygen consumption rates prior to the initial budding of the synchronized subpopulation and a decrease in these rates during the late budding phase. At a pH of 5.5, the late budding phase is characterized by high specific ethanol productivity; however, the ethanol productivity lags the late budding phase at a pH pf 6.5. The observed time-varying metabolism in the oscillatory operating regime appears to be the result of the metabolic changes which occur during the cell cycle. Models which can predict the oscillatory biomass concentration and nutrient levels in this regime must be capable of predicting the concentrations and metabolic rates of the subpopulations as well.     

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.     

Effects of uncoupling of mitochondrial energy conservation on the ultradian clock-driven oscillations in Saccharomyces cerevisiae continuous culture

Protonophores have several different perturbative effects on dissolved O2 concentrations in continuous cultures of Saccharomyces cerevisiae. As well as uncoupling energy conservation from mitochondrial electron transport in vivo, they reset ultradian clock-driven respiratory oscillations and produce cell cycle effects. Thus, additions at low concentration (1.25 microM) of either m-chlorocarbonyl-cyanide phenylhydrazone (CCCP) or 5-chloro-3-t-butyl-2-chloro-4(1)-nitrosalicylanilide (S13) led to phase resetting of the 48 min ultradian clock-driven respiratory oscillations. At 2.5 microM CCCP or 4 microM S13, transient inhibition of oscillatory respiration (for 5 h) preceded synchronisation of the cell division cycle seen as a slow (9 h period) wave that enveloped the 48 min oscillation. At still higher concentrations of CCCP (5 microM), the cell division cycle was prolonged by about 7 h, and during this phase, the respiratory oscillation became undetectable. The significance of these observations with respect to the time-keeping functions of the ultradian clock is discussed.     

Phosphorylation of the D1 photosystem II reaction center protein is controlled by an endogenous circadian rhythm

The light dependence of D1 phosphorylation is unique to higher plants, being constitutive in cyanobacteria and algae. In a photoautotrophic higher plant, Spirodela oligorrhiza, grown in greenhouse conditions under natural diurnal cycles of solar irradiation, the ratio of phosphorylated versus total D1 protein (D1-P index: [D1-P]/[D1] + [D1-P]) of photosystem II is shown to undergo reproducible diurnal oscillation. These oscillations were clearly out of phase with the period of maximum in light intensity. The timing of the D1-P index maximum was not affected by changes in temperature, the amount of D1 kinase activity present in the thylakoid membranes, the rate of D1 protein synthesis, or photoinhibition. However, when the dark period in a normal diurnal cycle was cut short artificially by transferring plants to continuous light conditions, the D1-P index timing shifted and reached a maximum within 4 to 5 h of light illumination. The resultant diurnal oscillation persisted for at least two cycles in continuous light, suggesting that the rhythm is endogenous (circadian) and is entrained by an external signal.     

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.     

Involvement of a cell size control mechanism in the induction and maintenance of oscillations in continuous cultures of budding yeast

Spontaneous oscillations occur in glucose-limited continuous cultures of Saccharomyces cerevisiae under aerobic conditions. The oscillatory behavior is detectable as a periodic change of many bioparameters such as dissolved oxygen, ethanol production, biomass concentration, as well as cellular content of storage carbohydrates and is associated to a marked synchronization of the yeast population. These oscillations may be related to a periodic accumulation of ethanol produced by yeast in the culture medium.The addition of ethanol to oscillating yeast cultures supports this hypothesis: indeed, no effect was observed if ethanol was added when already present in the medium, while a marked phase oscillation shift was obtained when ethanol was added at any other time. Moreover, the addition of ethanol to a nonoscillating culture triggers new oscillations. An accurate analysis performed at the level of nonoscillating yeast populations perturbed by addition of ethanol showed that both the growth rate and the protein content required for cell division increased in the presence of mixed substrate (i.e., ethanol plus limiting glucose). A marked synchronization of the yeast population occurred when the added ethanol was exhausted and the culture resumed growth only on limiting glucose. A decrease of protein content required for cell division was also apparent. These experimental findings support a new model for spontaneous oscillations in yeast cultures in which the alternative growth on limiting glucose and limiting glucose plus ethanol modifies the critical protein content required for cell division.     

酒精酵母在连续发酵中的振荡行为研究

初步分析酒精酵母在连续发酵中的振荡行为的产生条件及产生机理。通过改变稀释率、pH值、溶氧和进料葡萄糖浓度等条件 ,观察不同操作条件对酒精酵母菌生长和代谢行为的影响。在 10～ 15 g/L的较低葡萄糖浓度 ,0 .10～ 0 .2 0h-1的较低稀释率 ,以及 70 %左右的适度的溶氧浓度等发酵条件下 ,酒精酵母会出现同步的代谢振荡现象。一定条件下 ,菌体浓度处于振荡状态 ,残余葡萄糖浓度不可测或在很低水平振荡 ,这些发现预示着控制机制的新发展。     

Cell cycle- and growth phase-dependent variations in size distribution, antibody productivity, and oxygen demand in hybridoma cultures

Simultaneous determination of cell size and DNA content of hybridomas (HB-32) revealed a direct correlation between average cell volume and progression through the cell cycle. Pseudocontinuous experiments showed that G(1) cells, as estimated from cell size measurements, secreted monoclonal antibody at rates higher than those of cells in other stages of interphase and mitosis. Similarly, fed-batch and batch experiments suggested that specific oxygen uptake rate (qO(2)) is also a function of cell cycle, being minimum for cells in G(0) and G(1) phase. In batch cultures, HB-32 showed a rapid decrease in oxygen uptake rate (OUR) just prior to reaching maximum cell concentration. The OUR steadily increased from 0.01-0.05 to 0.5-0.7 mmol O(2)/L h as the cells went from the lag to the midexponential phase. The qO(2) increased from 0.3 x 10(-10)-0.9 x 10(-10) mmol O(2)/cell h at inoculation to 3.3 x 10(-10)-3.7 x 10(-10) mmol O(2)/cell h during the early exponential phase where it remained relatively constant. Several hours before maximum cell concentration was reached, OUR and qO(2) rapidly decreased to levels below those observed at inoculation. The time at which the shift in OUR and qO(2) occurred and the onset of decrease in the average cell size corresponded to the time of glutamine depletion. Based on monitoring OUR on-line in batch cultures, glutamine was supplemented, resulting in increased cell concentration, extension of culture viability, and increased MAb concentration.     

Parameter oscillation attenuation and mechanism exploration for continuous VHG ethanol fermentation

A bioreactor system composed of a stirred tank and three tubular bioreactors in series was established, and continuous ethanol fermentation was carried out using a general Saccharomyces cerevisiae strain and a very high gravity medium containing 280 g L(-1) glucose, supplemented with 5 g L(-1) yeast extract and 3 g L(-1) peptone. Sustainable oscillations of glucose, ethanol, and biomass were observed when the tank was operated at the dilution rate of 0.027 h(-1), which significantly affected ethanol fermentation performance of the system. After the tubular bioreactors were packed with 1/2' Intalox ceramic saddles, the oscillations were attenuated and quasi-steady states were achieved. Residence time distributions were studied for the packed bioreactors by the step input response technique using xylose as a tracer, which was added into the medium at a concentration of 20 g L(-1), indicating that the backmixing alleviation assumed for the packed tubular bioreactors could not be established, and its contribution to the oscillation attenuation could not be verified. Furthermore, the role of the packing's yeast cell immobilization in the oscillation attenuation was investigated by packing the tubular bioreactors with packings with significant difference in yeast cell immobilization effects, and the experimental results revealed that only the Intalox ceramic saddles and wood chips with moderate yeast cell immobilization effects could attenuate the oscillations, and correspondingly, improved the ethanol fermentation performance of the system, while the porous polyurethane particles with good yeast cell immobilization effect could not. And the viability analysis for the immobilized yeast cells illustrated that the extremely lower yeast cell viability within the tubular bioreactors packed with the porous polyurethane particles could be the reason for their inefficiency, while the yeast cells loosely immobilized onto the surfaces of the Intalox ceramic saddles and wood chips could be renewed during the fermentation, guaranteeing their viability and making them more efficient in attenuating the oscillations. The packing Raschig rings without yeast cell immobilization effect did not affect the oscillatory behavior of the tubular bioreactors, further supporting the role of the yeast cell immobilization in the oscillation attenuation.     

High-density photoautotrophic algal cultures: design, construction, and operation of a novel photobioreactor system

A photobioreactor system has been designed, constructed and implemented to achieve high photosynthetic rates in high-density photoautotrophic algal cell suspensions. This unit is designed for efficient oxygen and biomass production rates, and it also can be used for the production of secreted products. A fiber-optic based optical transmission system that is coupled to an internal light distribution system illuminates the culture volume uniformly, at light intensities of 1.7 mW/cm(2) over a specific surface area of 3.2 cm(2)/cm(3). Uniform light distribution is achieved throughout the reactor without interfering with the flow pattern required to keep the cells in suspension. An on-line ultrafiltration unit exchanges spent with fresh medium, and its use results in very high cell densities, up to 10(9) cells/mL [3% (w/v)] for eukaryotic green alga chlorella vulgaris. DNA histograms obtained form flow cytometric analysis reveal that on-line ultrafiltration influences the growth pattern. Prior to ultrafiltration the cells seem to have at a particular point in the cell cycle where they contain multiple chromosomal equivalents. Following ultrafiltration, these cells divide, and the new cells are committed to division so that cell growth resumes. The Prototype photobioreactor system was operated both in batch and in continuous mode for over 2 months. The measured oxygen production rate of 4-6 mmol/L culture h under continuous operation is consistent with the predicted performance of the unit for the provided light intensity.