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.     

Analysis of CHO cells metabolic redistribution in a glutamate-based defined medium in continuous culture.

The effect of glutamine replacement by glutamate and the balance between glutamate and glucose metabolism on the redistribution of t-PA-producing recombinant CHO cells metabolism is studied in a series of glucose shift down and shift up experiments in continuous culture. These experiments reveal the existence of multiple steady states, and experimental data are used to perform metabolic flux analysis to gain a better insight into cellular metabolism and its redistribution. Regulation of glucose feed rate promotes a higher efficiency of glucose and nitrogen source utilization, with lower production of metabolic byproducts, but this reduces t-PA specific production rate. This reduction under glucose limitation can be attributed to the fact that the cells are forced to efficiently utilize the carbon and energy source for growth, impairing the production of dispensable metabolites. It is, therefore, the combination of growth rate and carbon and energy source availability that determines the level of t-PA production in continuous culture.     

On describing microbial growth kinetics from continuous culture data: Some general considerations,observations, and concepts

Analysis of continuous culture methodology suggests that this potentially powerful tool for kinetic analysis can be improved by minimizing several inherent shortcomings. Medium background substrates — organic carbon, phosphate, and manganese — were shown to dominate kinetic observations at concentrations below chemical detection methods. Reactor wall growth, culture size distribution changes, sample removal-induced steady state perturbations, and limiting substrate leakage from organisms are treated in terms of kinetic measurement errors. Large variations in maximal growth rates and substrate uptake rates found are attributed to experimental protocol-induced transient states. Relationships are presented for correcting limiting substrate concentrations for lability during sampling, contamination with unreacted medium, and background substrate effects. Analytical procedures are discussed for improved measurement of limiting substrate kinetics involving enzymes, isotopes, and material balance manipulation. Relaxation methods as applied to continuous culture are introduced as a means for isolating separate rate constants describing net substrate transport and for evaluating cellular metabolite leakage. Low velocity growth, multiple substrate metabolism, and endogenous metabolism are discussed along with measurements showing that 1-month generation times for aquatic microorganisms can be quite normal and that the kinetics are compatible withμg/liter limiting substrate concentrations. The concept of regarding growth kinetics as the sum of several net accumulation processes is suggested.     

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.     

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.     

Multiplicity and stability analysis of microorganisms in continuous culture: effects of metabolic overflow and growth inhibition

Metabolic overflow (enhanced uptake of substrate and secretion of intermediates) is a phenomenon often observed for cells grown under substrate excess. Growth inhibition by substrate and/or product is also normally found for this kind of culture. An effort is made in this work to analyze the dynamic behavior of a continuous culture subject to metabolic overflow and growth inhibition by substrate and/or product. Analysis of a model system shows that in a certain range of operating conditions three nonwashout steady state solutions are possible. Local stability analysis indicates that only two of them are stable thus leading to multiplicity and hysteresis. Further analysis of the intrinsic effects of different terms describing the metabolic overflow and growth inhibitions reveals that for the model system and the parameters considered, the combined effects of product inhibition and an enhanced formation rate of product under substrate excess cause the multiplicity and hysteresis. Growth inhibition by substrate and/or an enhanced substrate uptake appear not to be necessary conditions. The combined effects of enhanced product formation and product inhibition can also lead to unusual dynamic behavior such as a prolonged time period to reach a steady state, oscillatory transition from one steady state to another, and sustained oscillations. Using the occurrence of multiplicity and oscillation as criteria, the operating regime of a continuous culture can be divided into four domains: one with multiplicity and oscillation, one with unique steady state but possible oscillatory behavior, the other two with unique and stable steady state. The model predictions are in accordance with recent experimental results. The results presented in this work may be used as guidelines for choosing proper operating conditions of similar culture systems to avoid undesired instability and multiplicity. Copyright 1998 John Wiley & Sons, Inc.     

Model for the feedback control system of bacterial growth. II. Growth in continuous culture

A mathematical model is developed that describes substrate limited bacterial growth in a continuous culture and that is based upon the conceptual framework elaborated in a previous paper for describing the feedback control system of cell growth [S. Bleecken, (1988). J. theor. Biol. 133, 37.] Central to the theory are the ideas that the limiting substrate is converted into low molecular weight building blocks of macromolecular synthesis which again are converted into biomass (RNA and protein) and that the rates of RNA and protein synthesis are controlled by the intracellular concentration of building blocks. It is shown that a continuous culture can be simulated by two interconnected feedback control systems the actuating signals of which are limiting substrate concentration and the intracellular concentration of building blocks, respectively. Three types of steady-states are found to appear in a continuous culture, besides the well-known stable steady-state of the whole culture there exist two batchlike steady-states of the biotic part of the culture which are metastable. The model is used to analyse the steady-states and their stability properties as well as the dynamic responses of biomass, RNA, protein, building block and substrate concentrations to changes in environmental conditions. Especially the inoculation of a continuous culture and the effects of step changes in dilution rate, inlet substrate concentration and growth temperature are studied in detail. Relations between the growth behaviour of a single cell and that of a continuous culture are derived. The RNA to protein ratio is introduced as a rough measure of the physiological state of cells and it is shown that a cell reacts to environmental changes with a simple pattern of basic responses in growth rate and physiological state. There are reasons to assume that the model presented is the minimal version of a structured model of bacterial growth and represents an optimum compromise between biological relevance and mathematical practicability.     

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.     

Some effects of growth conditions on steady state and heat shock induced htpG gene expression in continuous cultures of Escherichia coli

Most of the data concerning heat shock gene expression reported in the literature are derived from batch culture experiments under substrate and nutrient sufficient conditions. Here, the effects of dilution rate and medium composition on the steady state and heat shock induced htpG gene expression have been investigated in continuous cultures of Escherichia coli, using a chromosomal htpG-lacZ gene fusion. During steady state growth temperature dependent patterns of the relative htpG expression were found to be largely similar, irrespective of the growth condition. However, nitrogen-limited growth resulted in a markedly reduced specific steady state htpG expression as compared to growth under carbon limitation or in complex medium, correlating qualitatively with the total cellular protein content. During heat shock, tight temperature controlled expression was evident. While the relative heat shock induced expression was largely identical at various dilution rates in a given growth medium, significantly different response patterns were observed in the three growth media at any give dilution rate. From these results a clearly temperature regulated htpG expression during both, steady and transient state growth in continuous culture is evident, which is further significantly affected by the growth condition used.     

Multiplying steady-state culture in multi-reactor system

Cultivation of microorganisms in batch experiments is fast and economical but the conditions therein change constantly, rendering quantitative data interpretation difficult. By using chemostat with controlled environmental conditions the physiological state of microorganisms is fixed; however, the unavoidable stabilization phase makes continuous methods resource consuming. Material can be spared by using micro scale devices, which however have limited analysis and process control capabilities. Described herein are a method and a system combining the high throughput of batch with the controlled environment of continuous cultivations. Microorganisms were prepared in one bioreactor followed by culture distribution into a network of bioreactors and continuation of independent steady state experiments therein. Accelerostat cultivation with statistical analysis of growth parameters demonstrated non-compromised physiological state following distribution, thus the method effectively multiplied steady state culture of microorganisms. The theoretical efficiency of the system was evaluated in inhibitory compound analysis using repeated chemostat to chemostat transfers.     

Continuous culture of Thiobacillus ferrooxidans on a zinc sulfide concentrate

A zinc sulfide concentrate was leached microbiologically by Thiobacillus ferrooxidans in a continuous stirred tank reactor. A model was developed to predict, the leaching kinetics when the bacterial growth rate was not limited by any substrate other than the zinc concentrate, and it was modified to explain the observed results. Stable steady sates were obtained over a range of dilution rates from 0.0171 to 0.1038 hr^?1. Because a solid substrate was used, the specific growth rate of the bacaeria was not a unique function of the subastrate concentration, and conventional contnuous culture theory based on the Monod equation did not apply to this system. The leaching rates and bacterial growth rates were first order in mineral surface area cocentration.     

The production and growth characteristics of yeast and mycelial forms of Candida albicans in continuous culture.

The growth characteristics of Candida albicans CM145,348 have been examined under aerobic conditions in continuous culture. At different steady states the environment was controlled with respect to the concentrations of dissolved oxygen, carbon and nitrogen, the pH, and the temperature. Dry matter, substrate concentration, yield, specific oxygen uptake, specific carbon dioxide release and respiration quotient were examined as a function of the dilution rate. The morphology depended on the carbon source. Maltose produced a mycelial morphology, whereas with lactate a yeast culture was obtained. With fructose or glucose as a carbon source a mixed morphology of yeast, pseudo-mycelial and mycelial forms was produced. A larger number of different growth conditions were examined in batch culture but a mixed morphology was always obtained.     

The renaissance of continuous culture in the post-genomics age

The development of continuous culture techniques 60 years ago and the subsequent formulation of theory and the diversification of experimental systems revolutionised microbiology and heralded a unique period of innovative research. Then, progressively, molecular biology and thence genomics and related high-information-density omics technologies took centre stage and microbial growth physiology in general faded from educational programmes and research funding priorities alike. However, there has been a gathering appreciation over the past decade that if the claims of systems biology are going to be realised, they will have to be based on rigorously controlled and reproducible microbial and cell growth platforms. This revival of continuous culture will be long lasting because its recognition as the growth system of choice is firmly established. The purpose of this review, therefore, is to remind microbiologists, particularly those new to continuous culture approaches, of the legacy of what I call the first age of continuous culture, and to explore a selection of researches that are using these techniques in this post-genomics age. The review looks at the impact of continuous culture across a comprehensive range of microbiological research and development. The ability to establish (quasi-) steady state conditions is a frequently stated advantage of continuous cultures thereby allowing environmental parameters to be manipulated without causing concomitant changes in the specific growth rate. However, the use of continuous cultures also enables the critical study of specified transition states and chemical, physical or biological perturbations. Such dynamic analyses enhance our understanding of microbial ecology and microbial pathology for example, and offer a wider scope for innovative drug discovery; they also can inform the optimization of batch and fed-batch operations that are characterized by sequential transitions states.     

Cybernetic modeling and regulation of metabolic pathways in multiple steady states of hybridoma cells

Hybridoma cells utilize a pair of complementary and partially substitutable substrates, glucose and glutamine, for growth. It has been shown that cellular metabolism shifts under different culture conditions. When those cultures at different metabolic states are switched to a continuous mode, they reach different steady states under the same operating conditions. A cybernetic model was constructed to describe the complementary and partial substitutable nature of substrate utilization. The model successfully predicted the metabolic shift and multiple steady-state behavior. The results are consistent with the experimental observation that the history of the culture affects the resulting steady state.     

A mathematical model for the continuous culture of microorganisms utilizing inhibitory substrates

A mathematical model is presented for both batch and continuous cultures of microorganisms utilizing inhibitory substrates. The key feature of the model is the use of a inhibition function to relate substrate concentration and specific growth rate. Simulation studies show that the primary result of inhibition by substrate in a batch culture is an increase in the lag time whereas in continuous culture inhibition by substrate may result in process instability. The model should be of value in investigations of the stability of biological processes used for the treatment of certain industrial wastes such as those containing phenols, thiocyanates, nitrates, ammonia, volatile acids, etc., which are known to be inhibitory to many of the organisms metabolizing them.     

Heat shock gene expression in continuous cultures of Escherichia coli.

Temperature inducible systems for the controlled expression of recombinant genes are finding increasing industrial applications. These involve either short or long term exposure of the process culture to superoptimum temperatures. It is well known that bacteria respond to a sudden increase in their environmental temperature with an immediate transient increase in the synthesis rates of specific heat shock proteins. The use of continuous flow processes for the production of recombinant proteins would allow higher productivity and smaller scale bioreactors. However, the induction patterns of heat shock proteins in continuous culture after defined heat shocks are not well defined despite a large amount of information which is now available concerning heat shock protein induction in batch cultures. An overview of this information is presented to enable a better understanding of the response in continuous cultures. The latter was investigated by monitoring the transient expression of a representative heat shock gene, htpG, in E. coli in continuous culture. The relative magnitude of the response was found to be both temperature and exposure time dependent, but growth rate independent. Changing medium composition resulted in both different steady and transient state expression levels.     

Maximization of steady-state bacterial production in a chemostat with pH and substrate control.

This analytical study deals with the steady-state behavior and control of microbial growth in continuous cultures. A second order Haldane-Monod model of continuous cultures is used as a basis for study of the effects of the adjustment of pH by the addition of acidic (or basic) materials. The treatment of a hydrogen ion concentration, in addition to substrate and microbial concentrations as state variables, results in a third order system of equations describing the process. The analysis of the system in equilibrium yields several admissible steady states, that is, steady states which satisfy all constraints. An optimal control problem is formulated and subsequently solved to maximize steady-state microbial production.     

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.     

The dynamics of continuous microbial culture described by cell age distribution and concentration of one substrate

A mathematical model has been considered in which the known equation of McKendrick and Von Foerster for cell age distribution is combined with that for substrate concentration. The dependence of cell division rate on cell age has been taken as a step function. The interrelation between culture parameters describing the substrate consumption and cell division has been found. The shape of cell age distribution as well as the values of substrate and cell concentrations in steady and transient states have been investigated. Stationary regimes at the initial culture state synchronized by ages have been found to be established as damped oscillations and age waves. Under definite conditions the transition from one steady growth regime to another includes sharp single-time age synchronization of the culture.     

Kinetic effects of simultaneous inhibition by substrate and product

The starting point for the present investigations was the finding that increasing influent concentrations from 10 to 380 mmol/L glucose decreased the attainable growth rate of an acidogenic population in continuous culture from 0.52 to 0.05 h(-1) To account for this phenomenon, a new kinetic model is developed that combines substrate and product inhibition. Both effects are connected through the product yield, giving rise to a complex dependency of the growth rate on the substrate concentration. As a main feature, the maximum attainable growth rate decreases almost hyperbolically above some optimal substrate concentration in the influent. Furthermore, under certain conditions the kinetic model predicts the existence of three steady states: a high-conversion and a low-conversion state that are both stable and a metastable intermediate state. The latter states from the multiple-steady-state region are to be avoided, and eventual transitions to these states may have important consequences for the stability and the operation of such reaction systems. Substrate as well as product inhibition is reported for Propionibacterium freundenreichii and recently could be demonstrated for the above-mentioned acidogenic population. The proposed model allows optimization of anaerobic wastewater treatment processes and is applicable also to other fermentations.