Transient response of continuous cultures to changes in temperature

The purpose of this experimental study was to examine the transient response of a chemostat-type continuous culture of Escherichia coli B to step changes in temperature by following transient limiting substrate concentration and calculating from it the transient growth rate. The transient response to step changes of temperature was tested for four different situations. In the first two cases, temperature was shifted down from 37 to 27°C., and 37 to 32°C. In the last two, it was shifted up from 32 to 37°C., and 27 to 37°C. When the temperature was shifted up, the growth rate increased rather rapidly to its transient maximum value and then decreased slowly until it, settled back into the steady-state value. On the other hand, when the temperature was shifted down, the growth rate decreased relatively rapidly to its transient minimum and then it slowly increased and returned gradually to the steady-state value. The magnitude of the transients was less than would be expected if the transient growth rates followed an Arrhenius function.     

Application of a noninhibitory growth model to predict the transient response in a chemostat

A method of adapting a kinetic model based on steady-state chemostat data to predict the transient performance of a chemostat culture is presented. The proposal provides for a time delay which can be considered equivalent to a period of reduced activity of the organism subsequent to the introduction of a step change in operating conditions. The adapted kinetic model gives substantially better performance in predicting the transient response of an experimental system than the unmodified kinetic model.     

The response of a nitrogen-limited chemostat culture of Escherichia coli B/r to pH and dilution rate steps

The effects of four pH steps and one dilution rate step are described for an ammonia-nitrogen-limited continuous culture of Escherichia coli B/r. Two of the pH steps, 6.06-5.49 and 5.96-5.60, led to prolonged transients in both cell density and rate-limiting nutrient concentration. The other two pH steps, 6.20-5.96 and 5.60-6.20, had almost no effect on the culture. The dilution rate step led to a sharp transition in the steady-state external pyruvate concentration. Monitoring of the external pyruvate concentration for the pH 5.96-5.60 step revealed that the transient phase continued long after the cell density and rate-limiting nutrient concentration returned to steady-state values. The implications for industrial and laboratory fermentations are discussed.     

Kinetic studies of the lactic acid fermentation in batch and continuous cultures

The fermentation kinetics of the homofermentative organism Lactobacillus delbrueckii in a glucose-yeast extract medium is studied in both batch and continuous culture under conditions of controlled pH. From a graphical analysis of the batch data, a mathematical model of the process is derived which relates bacterial growth, glucose utilization, and lactic acid formation. The parameters in the model represent the activity of the organism and are a function of pH, having a maximum value at about 5.90. In a continuous stirred tank fermentor (CSTF), the effect of pH, feed concentration, and residence time is observed. The feed medium is a constant ratio of two parts glucose to one part yeast extract plus added mineral salts. An approximate prediction of the steady-state behavior of the CSTF can be made using a method based on the kinetic model derived for the batch case. In making step changes from one steady state to another, the transient response is observed. Using the kinetic model to simulate the transient period, the calculated behavior qualitatively predicts the observed response.     

The relationship between kinetics of substrate-limited transitions and steady-state growth in continuous cultures ofAquaspirillum autotrophicum limited by pyruvate

Heterotrophic growth at steady state and during transient states caused by the sudden change of the concentration of the limiting factor in the feed medium was investigated experimentally for continuous cultures ofAquaspirillum autotrophicum limited by pyruvate. A model for describing the growth at steady state was selected from three unstructured models after statistical tests of the data. This model postulates that the growth yield increases linearly with the growth rate. Growth during transitions where the substrate remained limiting at all times was fitted with first-order kinetics. Theoretical predictions of these kinetics were derived from the unstructured models used to describe steady state. The predicted rate coefficients of the transients were compared to the experimental coefficients. It appeared that the model which best described steady-state growth also provided the best predictions for growth during the transient state. It is a widespread opinion that unstructured models are adequate to describe growth under steady-state conditions but not to predict transitions in continuous culture. However, for the particular case studied here, no higher degree of complexity was required to describe transitions, provided the growth of the culture was always limited by the substrate.     

Dynamics of mixed cultures of Lactobacillus plantarum and Propionibacterium shermanii

The mixed culture of Lactobacillus plantarum and Propionibacterium shermanii grown anaerobically in glucose minimal medium exhibits features typical of a commensal interaction even though a number of complicating factors, such as a large maintenance requirement of L. plantarum and inhibition of growth of P. shermanii at low pH, are present. A simple mathematical model of the system is presented and is shown to reproduce rather well some of the features of the continuous mixed culture system in both steady-state and transient situations.     

Response of a continuous anaerobic culture to periodic variation of the feeding mash concentration

The authors studied the influence of periodic variations (one cycle per 24 hr) of the feeding mash concentration of a continuous anaerobic culture of Saccharomyces cerevisiae in sugarcane molasses media at constant dilution rate. It was observed that the average yield coefficients during the transient state were practically equal to the yield coefficient obtained during steady-state experiments. It was also observed, in each experiment, that the average specific growth rate during the transient state was equal to the dilution rate.     

Mechanistic model of the growth of Streptococcus cremoris HP at super optimal temperatures

The kinetic response of Streptococcus cremoris HP to growth at super optimal temperatures is reported. The response to a step increase in temperature was shown to be transient and to result from an increased metabolic rate caused by the raised temperature combined with thermal deactivation of the cell mass present. The catabolic and anabolic activities of the cell were shown to decay at different rates resulting in an accumulation of cells capable of catabolism (energy production) but unable to reproduce. The proposed mechanism was confirmed by independent estimates of the catabolic and anabolic activities of the culture. A mathematical model based on the proposed mechanism and incorporating simultaneous exponential growth, thermal death, and catabolic uncoupling of anabolically inactive cells was developed. Experimental evaluation of the model indicated the presence of a delay in deactivation of metabolic activity in response to a temperature transient. After the inclusion of this delay in death, it was confirmed that the model was capable of prediction of the balanced growth and transient response of this organism to changes in growth temperature. The delay in death was shown to be of major significance to the control of a simulated cheddar cheese fermentation.     

A comparative analysis of thermal blood perfusion measurement techniques

The object of this study was to devise a unified method for comparing different thermal techniques for the estimation of blood perfusion rates and to perform a comparison for several common techniques. The approach used was to develop analytical models for the temperature response for all combinations of five power deposition geometries (spherical, one- and two-dimensional cylindrical, and one- and two-dimensional Gaussian) and three transient heating techniques (temperature pulse-decay, temperature step function, and constant-power heat-up) plus one steady-state heating technique. The transient models were used to determine the range of times (the time window) when a significant portion of the transient temperature response was due to blood perfusion. This time window was defined to begin when the difference between the conduction-only and the conduction-plus-blood flow transient temperature (or power) responses exceeded a specified value, and to end when the conduction-plus-blood flow transient temperature (or power) reached a specified fraction of its steady-state value. The results are summarized in dimensionless plots showing the size of the time windows for each of the transient perfusion estimation techniques. Several conclusions were drawn, in particular: (a) low perfusions are difficult to estimate because of the dominance of conduction, (b) large heated regions are better suited for estimation of low perfusions, (c) noninvasive heating techniques are superior because they have the potential to minimize conduction effects, and (d) none of the transient techniques appears to be clearly superior to the others.     

Physiological changes ofCandida utilis in transient state during continuous cultivation

In a continuous culture ofCandida utilis, the air supply was interrupted for 15 min at 1-d intervals after the steady state had been reached. Analysis of morphology and physiology of the cell showed that after this intervention the indicators of the physiological state changed their values with different time delays and needed different times to resume their steady-state values. Another consequence of the interrupted aeration was a higher degree of the culture synchronization. The possibility to bring about a transient (i. e. unsteady) state offers a tool for a directed control of mutual proportion of intracellular components.     

Acclimation of the respiration/photosynthesis ratio to temperature: insights from a model

Based on short-term experiments, many plant growth models – including those used in global change research – assume that an increase in temperature stimulates plant respiration (R) more than photosynthesis (P), leading to an increase in the R/P ratio. Longer-term experiments, however, have demonstrated that R/P is relatively insensitive to growth temperature. We show that both types of temperature response may be reconciled within a simple substrate-based model of plant acclimation to temperature, in which respiration is effectively limited by the supply of carbohydrates fixed through photosynthesis. The short-term, positive temperature response of R/P reflects the transient dynamics of the nonstructural carbohydrate and protein pools; the insensitivity of R/P to temperature on longer time-scales reflects the steady-state behaviour of these pools. Thus the substrate approach may provide a basis for predicting plant respiration responses to temperature that is more robust than the current modelling paradigm based on the extrapolation of results from short-term experiments. The present model predicts that the acclimated R/P depends mainly on the internal allocation of carbohydrates to protein synthesis, a better understanding of which is therefore required to underpin the wider use of a constant R/P as an alternative modelling paradigm in global change research.     

Periodic operation of a continuous culture of Baker's yeast

The possibility of enhancing the biomass productivity of a continuous culture of Saccharomyces cerevisiae growing on a glucose-limited medium is addressed. An unstructured Monod-type model is first identified using steady-state data. The culture is subjected to step changes in dilution rate, and it is seen that the Monod model is unable to predict even qualitatively the dynamic response of the culture. Incorporation of a time delay allows significant improvement in the transient fit. It is found that the culture has a time lag of about 3 h in adapting its growth rate. Cycling the dilution rate with a period of 3 h leads to substantial improvement in the average biomass productivity.     

Induction of heat shock proteins during initiation of solvent formation inClostridium acetobutylicum

Summary The response to stresses produced by changes in the fermentation conditions ofClostridium acetobutylicum in continuous culture was determined under acid- and solvent-producing conditions. Using a phosphate-limited chemostat it was found that specificheatshockproteins (hsp 73, hsp 72 [Dnak], hsp 67 [GroEL], hsp 17 and hsp 14) were synthesized at elevated levels during the shift from acid to solvent formation. The induction of these stress proteins was observed before acetone and butanol were detected in the medium and was therefore not a response to these solvents present in the medium. Simultaneously with the induction of hsps, changes in the synthesis rates of other cellular proteins were observed. Synthesis of proteins associated with the acid production phase decreased and of proteins correlated with the solvent production phase increased. Some hsps, including the DnaK- and GroEL-similar proteins, hsp 73 and hsp 21, were also induced by a change in the growth rate and/or the pH. The analysis of the general regulation of the heat shock response inC. acetobutylicum revealed that the induction of at least 15 hsps after a temperature up-shift was transient and that two temporal classes of hsps could be distinguished. The synthesis of one group of hsps reached a maximum after 6 min and another around 11 min after the temperature upshift and returned to steady-state levels 30 to 40 min after the shock.     

The kinetics of stomatal responses to VPD in Vicia faba: electrophysiological and water relations models

Abstract. An Ohm's law analogy is frequently employed to calculate parameters of leaf gas exchange. For example, resistance to water vapour loss is calculated as the quotient of vapour pressure difference (VPD) and vapour loss by transpiration. In the present research, this electrical analogy was extended. Steady-state transpiration as a function of VPD, assayed in leaflets of Vicia faba using gas exchange techniques, was compared with steady-state K⁺ current magnitude as a function of voltage in isolated guard cell protoplasts of Vicia faba, assayed using the patch clamping technique in the whole cell configuration. An electrophysiological model originally developed to explain the kinetics of current changes following step changes in voltage across a cell membrane was used to fit the kinetics of transpiration changes following step changes in VPD applied to leaflets of Vicia faba. Following step increases in VPD, transpiration exhibited an initial increase, reflecting the increased driving force for water loss and, for large step increases in VPD, a transient decrease in stomatal resistance. Transpiration subsequently declined, reflecting stomatal closure. By analogy to electrophysiological responses, it is hypothesized that the humidity parameter that is sensed by guard cells is VPD. Two models based on epidermal water relations were also applied to transpiration kinetics. In the first model, the transient increase in transpiration following a step increase in VPD was attributed partially to an increase in the Physical driving force (VPD) and partially to a transient decrease in stomatal resistance resulting from reduced epidermal backpressure. In the second model, the transient decrease in stomatal resistance was attributed to a direct response of the guard cells to VPD. Both models based on water relations gave good fits of the data, emphasizing the need for further study regarding the metabolic nature of the guard cell response to humidity.     

The response by microorganisms to steady state growth in controlled concentrations of oxygen and glucose. I. Candida utilis

Dissolved oxygen and glucose concentrations have been independently maintained at various concentrations for extended periods during growth of Candida utilis in continuous culture. Simultaneous observations of cytochrome concentration, growth rate, rates of uptake of oxygen and glucose and rates of production of CO₂, ethanol and acid have been made during steady states at various levels of oxygen and glucose. There is an inverse relationship between dissolved oxygen and cytochrome and between glucose cocentration and cytochrome. Studies of the transient state following a step change from high to low dissolved oxygen show that there is a lag of about 10 hr during which there is no change in the above parameters. This is followed by rapid oscillatory changes in cytochrome content and a change to a more fermentative metabolism.     

Production of gramicidin S in batch and continuous culture

A mathematical model for the production of gramicidin S in batch and continuous culture is proposed. It is based on the division of the age of a cell into two phases—an immature and a mature one. A nongrowth associated product, such as an antibiotic, is assumed to be produced when the organism is in the older of these two phases, the mature state. The parameters describing the model were evaluated from batch and single stage transient continuous culture of Bacillus brevis, which produces the antibiotic gramicidin S. The predictive value of the model was studied in steady-state single stage continuous culture and in a transient two stage system. Good agreement between the theoretical curves and the experimental results was found in the transient response of both the first and second stage systems, although at high dilution rates (0.34 hr^?1) in the first stage, deviations from the predicted response were observed in the second stage. These may have been due to chemostat instability at dilution rates close to washout, lags in cell growth, and a metabolic lag on going from stage one to stage two.     

Transient responses of hybridoma cells to nutrient additions in continuous culture: I. Glucose pulse and step changes

Glucose and glutamine are the main nutrients used by mammalian cells in culture. Each provides unique biosynthetic precursors but are complementary for production of other metabolites and energy. The transient and steady-state responses of hybridoma growth and metabolism to glucose pulse and step changes have been examined. Metabolic quotients are reported for oxygen, glucose, lactate, ammonia, glutamine, alanine, and other amino acids. The glucose consumption rate increased by 100-200% immediately after glucose was added to the reactor, and the increased glycolytic ATP production appears to be responsible for the concurrent rapid decrease in the oxygen consumption rate. The effects on glutamine consumption were delayed, probably due to buffering by the TCA cycle and interrelated pathways. A period of increased biosynthetic activity, as evidenced by an increase in the estimated specific ATP production rate and lower by-product yields from glutamine, preceded the increase in cell concentration after the glucose step change. The biosynthetic yield of cells from ATP was calculated, and it was estimated that maintenance accounted for about 60% of the energy used by the cells at a specific growth rate of 0.66 day(-1). The estimated 22% ATP production due to glycoysis was twice as great as that before the step change.     

Pre-tRNA turnover catalyzed by the yeast nuclear RNase P holoenzyme is limited by product release

Ribonuclease P (RNase P) is a ribonucleoprotein that catalyzes the 5′ maturation of precursor transfer RNA in the presence of magnesium ions. The bacterial RNase P holoenzyme consists of one catalytically active RNA component and a single essential but catalytically inactive protein. In contrast, yeast nuclear RNase P is more complex with one RNA subunit and nine protein subunits. We have devised an affinity purification protocol to gently and rapidly purify intact yeast nuclear RNase P holoenzyme for transient kinetic studies. In pre-steady-state kinetic studies under saturating substrate concentrations, we observed an initial burst of tRNA formation followed by a slower, linear, steady-state turnover, with the burst amplitude equal to the concentration of the holoenzyme used in the reaction. These data indicate that the rate-limiting step in turnover occurs after pre-tRNA cleavage, such as mature tRNA release. Additionally, the steady-state rate constants demonstrate a large dependence on temperature that results in nonlinear Arrhenius plots, suggesting that a kinetically important conformational change occurs during catalysis. Finally, deletion of the 3′ trailer in pre-tRNA has little or no effect on the steady-state kinetic rate constants. These data suggest that, despite marked differences in subunit composition, the minimal kinetic mechanism for cleavage of pre-tRNA catalyzed by yeast nuclear RNase P holoenzyme is similar to that of the bacterial RNase P holoenzyme.