Reaction engineering analysis of L-lysine transport by Corynebacterium glutamicum

To identify potential L-lysine export limitations by Corynebacterium glutamicum in the L-lysine production process, the excretion of L-lysine was studied in continuous and fed-batch operated stirred tank reactors. A structured biochemical model of the L-lysine excretion mechanism was used to determine the activity of the export carrier and to calculate a cell-specific concentration of the export carrier. For the biochemical characterization of this specific carrier concentration a standardized L-lysine efflux test was developed. Carrier activity, cell-specific carrier concentration, and the specific L-lysine export rate were identified as a function of pH value and L-lysine concentration in the reactors. Also, the correlation of these parameters to the metabolic state of C. glutamicum was determined. The pH value in the reactor governs the carrier activity (maximum at pH 6.5) and the specific carrier concentration (maximum at pH 8.0). The specific L-lysine export rate, as the product of carrier activity and specific carrier concentration, revealed a maximum at pH 7.0. Decreasing L-lysine productivities also correlated with decreasing specific carrier concentrations. The L-lysine concentration in the reactor had no effect on the specific carrier concentration but strongly inhibited the carrier activity. The specific export rate was reduced to 50% at 400 mM L-lysine compared to the specific export rate at 80 mM L-lysine. (c) 1996 John Wiley & Sons, Inc.     

Indirect estimation of cell mass and substrate concentration using a computer-coupled mass spectrometer

On-line estimation of cell mass and substrate concentration based on exhaust gas analysis was developed. The O₂, CO₂, H₂O, and N₂ contents at the inlet and outlet of fermentor, analyzed by a computer-coupled quadrupole mass spectrometer, were used to calculate the oxygen uptake rate and carbon dioxide evolution rate, and these rates were further used to evaluate cell mass and substrate concentration in a recombinant Escherichia coli fermentation. Cell mass, glucose concentration, specific growth rate, and specific consumption rate of glucose were well estimated by this method; the oxygen uptake rate gave more accurate estimates for these state variables than did the carbon dioxide evolution rate.     

Relationship between substrate concentration,growth rate,and respiration rate of Escherichia coli in continuous culture

Summary Using a continuous flow technique the relationship between growth rate and substrate concentration was investigated with glucose as the limiting factor of a culture of Escherichia coli. Graphical and numerical analysis of the experimental data demonstrated that the application of the Michaelis-Menten equation produced erroneous results, whereas, the constants obtained from the Teissier equation were in agreement with the experimental data. On this basis, new equations defining the steady state cell and substrate concentration in continuous flow cultures were developed and tested against experimental data.Comparison of the specific growth rates, substrate uptake rates and oxygen consumption rates demonstrated that all were directly proportional to each other and could be related to each other by mathematical equations. Specifically it was shown that as the growth rate increased from 0.06 to k _m =0.76 the substrate uptake rate increased from 134 to 1420 mg glucose per gram cell weight per hour and the oxygen consumption rate increased from 48.6 to 505 mg O₂ per gram cell weight per hour. Independent of the growth rate 37% of the carbohydrate consumed were oxidized. The yield factor varied from 0.44 at low growth rates to 0.54 at high growth rates. Analysis of the growth rate-substrate uptake rate relationship indicated that a minimum substrate uptake rate of 55 mg glucose per gram cell weight per hour existed below which cell reproduction would cease. This was supported by the fact that steady state conditions could not be maintained in the culture at D values below 0.02 when the substrate supply rate decreased below 45 mg glucose per gram cell weight per hour.Material contained in this paper was submitted as a thesis in partial fulfillment of the requirements for the Ph. D. degree of Dr. R. S. Lipe.     

A mechanistic model of the aerobic growth of Saccharomyces cerevisiae.

A two-stage deterministic model of the growth of Saccharomyces cerevisiae is presented. The cell cycle of this organism was used to suggest the basic model structure. The model represents the preparatory processes of substrate uptake and conversion separately from replication and division. The regulation of the fraction of the culture devoted to each of these broad areas of metabolism, and the overall growth rate, is related to the nature and availability of the energy substrate. The simulation of respiration and glycolysis is achieved by including two alternative energy producing pathways. The regulation of these pathways is described in terms of the postulated primary regulation of the proportion of the culture required for substrate uptake and conversion, and the overall kinetic constants for each pathway. This regulation is dictated primarily by the growth rate rather than the nature or concentration of the energy substrate. The model successfully describes both batch and continuous growth of S. cerevisiae under conditons of glucose limitation and oxygen excess. A preliminary assessment indicates that adjustment of the relevant parameters will allow the model to describe the growth of S. cerevisiae on other sugars and under oxygen limitation. Similarly the model could be expected to describe the growth characteristics of other yeast species.     

Studies on the kinetics and mechanism of BOD exertion in dilute systems

The mechanisms and kinetic course of BOD exertion were compared in both open and closed systems. Two open reactors, a simulated stream device, and an open stirred reactor were employed, and the closed systems consisted of standard BOD bottles and 2.4-liter vessels. In the closed systems, both quiescent and stirred conditions of incubation were examined. Biological solids concentration, bacteria and protozoa concentration, substrate analysis, and chemical oxygen demand as well as biochemical oxygen utilization were employed to assess the performance of these systems. Oxygen uptake rate constants were observed to increase with increasing concentration o carbon source, thus militating against irect use of the usual dilution technique for predicting rate of deoxygenation in receiving streams. The relationship between specific O₂ uptake rate and substrate concentration approximated a hyperbolic function similar to the Mono relationship for specific growth rate and substrate concentration. A technique using an open stirred reactor than the standard BOD bottle dilution technique is recommended.     

Bioreactor scale-up and oxygen transfer rate in microbial processes: An overview

In aerobic bioprocesses, oxygen is a key substrate; due to its low solubility in broths (aqueous solutions), a continuous supply is needed. The oxygen transfer rate (OTR) must be known, and if possible predicted to achieve an optimum design operation and scale-up of bioreactors. Many studies have been conducted to enhance the efficiency of oxygen transfer. The dissolved oxygen concentration in a suspension of aerobic microorganisms depends on the rate of oxygen transfer from the gas phase to the liquid, on the rate at which oxygen is transported into the cells (where it is consumed), and on the oxygen uptake rate (OUR) by the microorganism for growth, maintenance and production.     

Modelling of hexadecane degradation in continuous-flow cultures

Microorganisms of Wadden Sea sediments are able to degrade hydrocarbons in suspensions. (Berthe-Corti, L., Bruns, A., Hulsch, R., 1997. J. Microb. Methods 29, 129-137) have observed in continuous culture experiments that the growth rate of microorganisms increases roughly proportional to the dilution rate. The growth rate is nearly independent of the oxygen saturation down to about 0.5%. Even at very low oxygen supply, corresponding to an oxygen saturation far below 0.1%, growth takes place at a reduced rate. In this paper, a model is presented which can reproduce the results of these experiments. The model treats the following processes, selection of the active fraction of microorganisms growing on hexadecane, uptake of hexadecane and transformation into palmitate as a first metabolic step, synthesis of biomass, respiration and exudation. The processes are regulated by the substrate concentration, the internal palmitate quota, the exudates' concentration and an inhibiting factor. For the experiments under very low oxygen conditions, the observed growth with reduced O(2)-consumption and CO(2)-production is modelled by assuming an anoxic metabolic pathway.     

Analysis of a continuous, aerobic, fixed-film bioreactor. II. Dynamic behavior

The dynamic analysis of a continuous, aerobic, fixed-film bioreactor has been performed. Rigorous mathematical models have been developed for a fluidized-bed fermentor with biofilm growth. The transient performance of the reactor is appraised in terms of outlet penicillin concentration for constant, as well as variable carbon substrate feed rates. The effect of the reactor oxygen transfer capacity is elucidated for those cases employing substrate feeding strategies. The results show that penicillin production in a continuous, fixed-film bioreactor reaches a maximum with processing time, but subsequently decreases as cell mass accumulates and substrate deficiencies occur. The maximum production level can be maintained for increased operating times if the substrate supply is continuously increased. The duration of this prolonged production is a direct function of the rate of increase and the operating time at which the increase is initiated. The oxygen transfer capacity of the reactor was found to be important to the effectiveness of a feeding strategy.     

Continuous cultures limited by a gaseous substrate: Development of a simple, unstructured mathematical model and experimental verification with Methanobacterium thermoautotrophicum

This article presents a simple, unstructured mathematical model describing microbial growth in continuous culture limited by a gaseous substrate. The model predicts constant gas conversion rates and a decreasing biomass concentration with increasing dilution rate. It has been found that the parameters influencing growth are primarily the gas transfer rate and the dilution rate. Furthermore, it is shown that, for correct simulation of growth, the influence of gaseous substrate consumption on the effective gas flow through the system has to be taken into account.Continuous cultures of Methanobacterium thermoautotrophicum were performed at three different gassing rates. In addition to the measurement of the rates of biomass production, product formation, and substrate consumption, microbial heat dissipation was assessed using a reaction calorimeter. For the on-line measurement of the concentration of the growth-limiting substrate, H(2), a specially developed probe has been used. Experimental data from continuous cultures were in good agreement with the model simulations. An increase in gassing rate enhanced gaseous substrate consumption and methane production rates. However, the biomass yield as well as the specific conversion rates remained constant, irrespective of the gassing rate. It was found that growth performance in continuous culture limited by a gaseous substrate is substantially different from "classic" continuous culture in which the limiting substrate is provided by the liquid feed. In this report, the differences between both continuous culture systems are discussed.     

The use of model-fitting in the interpretation of 'dual' uptake isotherms

Abstract. Published data of the concentration dependence of the uptake rate (uptake isotherms) of K⁺ Na⁺, Cl^?, SO2^?₄, and L-lysine in barley roots, and glucose and 3-O-methylglucose in potato tuber tissue, were re-examined. In as much as these isotherms yield non-linear, concave upward Eadie-Hofstee plots, they might have been termed ‘dual’ isotherms. In addition, all these isotherms have been considered to display discontinuous transitions in gradient. The following models that yield continuous isotherms were fitted to the isotherms: (1) the sum of a Michaelis-Menten term and a linear term; (2) the sum of two Michaelis-Menten terms; (3) the sum of two Michaelis-Menten terms and a linear one. Goodness of fit was judged from: (i) the weighted mean square of deviates; (ii) the standard errors of the kinetic parameters; (iii) the algebraic significance of the terms; (iv) a Rankits plot of the residuals; (v) a Runs test on the residuals. For the precise and detailed isotherms of SO^2? uptake, only model (3) gave a fit that was satisfactory in all respects. There appeared to be no reason to consider these isotherms as multiphasic. The same conclusion was reached for the L-lysine uptake isotherms. For the other isotherms the results were less conclusive. Thai for K⁺ and Na+ could, at any rate, be described satisfactorily by a continuous model, the best fit being obtained with model (2). The uptake isotherms of Cl^? and 3-0-methylglucose could be best described by model (2), and that of glucose by model (3), only the result of the Runs test being unsatisfactory. It is concluded that there is hardly any evidence that the presumed ‘jumps’ or discontinuities or inflections in the gradient of uptake isotherms are not due to experimental error in the data. It is suggested that many uptake isotherms may be described by model (3), although the reason for this is still incompletely understood.     

Control Mechanisms Operative in a Natural Microbial Population Selected for Its Ability to Degrade L-Lysine. III. Effects of Carbohydrates in Continuous-Flow Systems Under Shock Load Conditions

Two naturally selected microbial populations were maintained under continuousflow conditions with glucose or magnesium growth-limiting. The reactors were subjected to shock loads by changing the influent substrate from L-lysine to a mixture of L-lysine and glucose, L-lysine and fructose, or L-lysine and ribose. During the subsequent transient state, the following parameters were examined: lysine chemical oxygen demand (COD), carbohydrate COD, total COD, biological solids concentration, cell protein, enzymatic capability (lysine-degrading enzymes), and the rate of lysine removal. The carbohydrate was then removed from the influent and the same parameters were examined until a new steady state was established. In all cases, glucose and fructose caused a significant repression of the synthesis of lysine-degrading enzymes, resulting in a decrease in the enzymatic capability of the cells. In the carbon-limited reactor, the faster the flow rate, the greater was the repression, whereas, in the magnesium-limited reactor, the slower the flow rate, the greater was the repression. The introduction of ribose into the reactors caused an initial increase in lysine enzymatic capability followed by a slight repression when ribose degradation started.     

A new model of uptake of multiple sugars by S. cerevisiae

Carrying on from our work with yeast in batch culture with mixed substrates of glucose and fructose, a model developed previously was modified to incorporate firstly the use of sucrose as substrate and secondly the ability to simulate continuous culture. Experiments using sucrose as the limiting substrate were simulated based on sugar uptake, biomass and ethanol production, carbon dioxide production and oxygen consumption. The new sugar uptake models were able to successfully simulate these experiments. On the basis of these experiments there is a strong suggestion that a proportion of the sucrose utilised is transported into the cell rather than hydrolysed prior to entry. Continuous culture experiments were also simulated by adapting the modelling program into the format of a multiple differential equation problem solver called SPEEDUP.     

Kinetic studies on hybridoma cells immobilized in fixed bed reactors

Cultures with immobilized hybridoma cells were performed in fixed bed systems. "Steady state" values for volume-specific substrate uptake and metabolite production rates were determined at various perfusion rates and superficial flow velocities of the medium within the carrier matrix. Data from fixed bed volumes between 50 and 600 ml did not show any difference. The volume-specific glutamine and glucose uptake rate turned out to be independent of the superficial flow velocity, but decreased with decreasing glutamine and glucose concentration. The volume-specific oxygen uptake rate increased with increasing superficial flow velocity and substrate concentration, respectively. A similar behavior was observed for the ratio between oxygen and glucose uptake rate. The production rate for monoclonal antibodies was neither affected by the substrate concentration nor by the superficial flow velocity. The metabolic parameters of the immobilized cells were put into kinetic equations and compared to those of suspended cells. It could be concluded that the metabolism of the immobilized cells is determined by the oxygen supply within the macroporous carriers. (c) 1997 John Wiley & Sons, Inc. Biotechnol Bioeng 55: 535-541, 1997.     

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.     

Dual substrate removal by an axenic bacterial culture

A pure bacterial culture capable of utilizing either L-lysine or 2-chlorophenol (2-CP) as sole carbon source was isolated and used in continuous culture experiments to determine its response to dual substrate limitation by those two compounds. Dilution rate and feed composition were each set at three levels in a two factorial experimental design. The total chemical oxygen demand (COD) of the feed was fixed at 225 mg/L and its composition was varied by changing the ratio of lysine to 2-CP. The effects of the two independent variables (dilution rate and feed composition) on the concentrations of cells, lysine, COD, and dissolved organic carbon (DOC) in the reactors were systematic whereas the effects on the 2-CP concentration were less predictable. The concentrations of the two substrates responded to the two independent variables in a complex interactive manner which is not explained by existing models for dual, substitutable substrates. Rather, the results suggested that the prediction of the fate of a single organic component in a reactor receiving a multicomponent feed is a very difficult task.     

K+--induced changes of oxygen uptake by neuronal enriched and glial enriched fractions from mouse brain cortex.

Neuronal and glial enriched fractions were incubated in a medium with 10mM pyruvate, 5mM fumarate and 0.9mM 5'-AMP and the effect of increased external K+ concentrations was studied upon oxygen uptake. A concentration of 65 mM K+ had a different effect on the oxygen consumption of glial and neuronal perikarya. The rate of oxygen uptake by glia was stimulated by 52.81% whilst an insignificant decrease of 15.79% occurred in the neurones. The highest rate of oxygen uptake by incubated cells was estimated in the presence of the substrate system containing pyruvate, fumarate and 5'-AMP. The significance of components in the substrate system for a high rate of oxygen uptake by cells was also tested with 6.2 mM K+ and 65 mM K+.     

Kinetics of biodegradation of p-xylene and naphthalene and oxygen transfer in a novel airlift immobilized bioreactor

The scope of this study included the biodegradation performance and the rate of oxygen transfer in a pilot-scale immobilized soil bioreactor system (ISBR) of 10-L working volume. The ISBR was inoculated with an acclimatized population of contaminant degrading microorganisms. Immobilization of microorganisms on a non-woven polyester textile developed the active biofilm, thereby obtaining biodegradation rates of 81 mg/L x h and 40 mg/L x h for p-xylene and naphthalene, respectively. Monod kinetic model was found to be suitable to correlate the experimental data obtained during the course of batch and continuous operations. Oxygen uptake and transfer rates were determined during the batch biodegradation process. The dynamic gassing-out method was used to determine the oxygen uptake rate (OUR) and volumetric oxygen mass transfer, K(L) a. The maximum volumetric OUR of 255 mg O(2)/L x h occurred approximately at 720-722 h after inoculation, when the dry weight of biomass concentration was 0.67 g/L.     

Continuous cultivation of Saccharomyces cerevisiae. I. Growth on ethanol under steady-state conditions

Growth of Saccharomyces cerevisiae LBG H 1022 on ethanol under steady-state conditions was studied. As a cultivation device, an aerated Chemap fermentor combined with continuously working gas analyzers for oxygen and carbon dioxide was used. Dry matter, substrate concentration, yield, specific oxygen uptake, specific carbon dioxide release, and respiration quotient, as well as nitrogen, carbon, phosphorus, hydrogen, and protein content of the cells were measured in dependence on the dilution rate. Cell size distribution, as a function of the specific growth rate, was determined with the aid of a Celloscope 202. A fair agreement with the theory of continuous culture for all metabolic curves could be established. An increased turnover rate resulted from the addition of glutamic acid to the synthetic growth medium. The primary effect of this supplement could be a rise in the flow rate of the tricarboxylic acid cycle.     

Respirometric evaluation and modeling of glucose utilization by Escherichia coli under aerobic and mesophilic cultivation conditions

The study presents a mechanistic model for the evaluation of glucose utilization by Escherichia coli under aerobic and mesophilic growth conditions. In the first step, the experimental data was derived from batch respirometric experiments conducted at 37 degrees C, using two different initial substrate to microorganism (S(0)/X(0)) ratios of 15.0 and 1.3 mgCOD/mgSS. Acetate generation, glycogen formation and oxygen uptake rate profile were monitored together with glucose uptake and biomass increase throughout the experiments. The oxygen uptake rate (OUR) exhibited a typical profile accounting for growth on glucose, acetate and glycogen. No acetate formation (overflow) was detected at low initial S(0)/X(0) ratio. In the second step, the effect of culture history developed under long-term growth limiting conditions on the kinetics of glucose utilization by the same culture was evaluated in a sequencing batch reactor (SBR). The system was operated at cyclic steady state with a constant mean cell residence time of 5 days. The kinetic response of E.coli culture was followed by similar measurements within a complete cycle. Model calibration for the SBR system showed that E. coli culture regulated its growth metabolism by decreasing the maximum growth rate (lower microH) together with an increase of substrate affinity (lower K(S)) as compared to uncontrolled growth conditions. The continuous low rate operation of SBR system induced a significant biochemical substrate storage capability as glycogen in parallel to growth, which persisted throughout the operation. The acetate overflow was observed again as an important mechanism to be accounted for in the evaluation of process kinetics.