Open-loop control of specific growth rate in fed-batch cultures of recombinant E.coli

Summary A computer-based algorithm was used for the open-loop control of specific growth rate in fed-batch cultures of recombinant E.coli.. The control of nutrient feed rate to an exponential trajectory resulted in growth of the culture at a constant specific growth rate. Stable specific growth rates between 0.08 and 0.4 h^–1 were achieved.     

Profile control of the specific growth rate in fed-batch experiments

Summary The aim of this paper is to apply a computer control scheme to a laboratory scale fermentor so that the specific growth rate in a baker's yeast fed-batch culture, which cannot be measured directly, will follow as accurately as possible the desired profile specified in advance. Using an extended Kalman filter and programmed controller/feedback compensator (PF) system proposed previously, profile control of the specific growth rate () was achieved experimentally in a baker's yeast fed-batch culture. Also, bang-bang type profile control of minimized the proportion of budding cells, which have a strong correlation with the fermentative activity in bread-making.     

Inferential control of the specific growth rate in fed-batch cultivation processes

Inferential control algorithms and systems for set-point control of the specific growth rate in fed-batch cultivation processes are presented. Realization of the proposed control systems does not require mathematical model and a priori knowledge of the culture of microorganisms under control. Feed-back signal in PID control loops of the investigated systems is based on measurements of the O₂ concentration in exhaust gas and the air supply rate, however, measurements of the other technological parameters related to the culture growth dynamics can also be applied.     

Calorimetric control of the specific growth rate during fed-batch cultures of Saccharomyces cerevisiae

The specific growth rate of a Saccharomyces cerevisiae strain with glucose as limiting C-source was estimated from the measured heat flow produced by the cells. For the cultivation a standard 30 l laboratory bioreactor was used, which was extended in such a way that heat balancing is possible. The feed rate was adjusted by a feedforward/feedback controller such that the specific growth rate was kept on the desired set-point value. On the basis of experimental investigations it was demonstrated that the specific growth rate can be controlled at a given set point value below the critical value to prevent the production of growth-inhibitory ethanol due to the Crabtree effect. With this control strategy high biomass concentrations of more than 110 g l(-1) can be obtained.     

Influences of yeast extract on specific cellular yield of Ovine growth hormone during fed-batch fermentation of E. coli

In most cases of E. coli high cell density fermentation process, maximizing cell concentration helps in increasing the volumetric productivity of recombinant proteins usually at the cost of lower specific cellular protein yield. In this report, we describe a process for maintaining the specific cellular yield of Ovine growth hormone (oGH) from E. coli by optimal feeding of yeast extract during high cell density fermentation process. Recombinant oGH was produced as inclusion bodies in Escherichia coli. Specific cellular yield of recombinant oGH was maintained by feeding yeast extract along with glucose during fed-batch fermentation. Glucose to yeast extract ratio of 0.75 was found to be optimum for maintaining the specific cellular oGH yield of 66 mg/g of E. coli cells. Continuous feeding of yeast extract along with glucose helped in reducing acetic acid secretion and promoted higher cell growth during fed-batch fermentation. High cell growth of E. coli and high specific yield of recombinant oGH thus helped in achieving high volumetric productivity of the expressed protein. A maximum of 2 g/l of ovine growth hormone was expressed as inclusion bodies in 12 h of fed-batch fermentation.     

Adaptive control of specific growth rate based on proton production in anaerobic fed-batch culture

Summary This paper develops a practical and useful computer control scheme to control the specific growth rate as accurately as possible by measuring the released protons in anaerobic alcohol fermentation. In the case of current study, most of the released protons are due to the uptake of cationic ammonium ion via the conversion: NH₄ ⁺ NH₃(cell) + H⁺. Correlating the Proton Production (PP) and the Proton Production Rate (PPR) with specific growth rate () proved PP as a better measured variable. Using a simple adaptive control algorithm, was successfully controlled in a Zymomonas mobilis fed-batch culture.     

Constant specific growth rate in fed-batch cultivation of Bordetella pertussis using adaptive control

Monitoring and control of production processes for biopharmaceuticals have become standard requirements to support consistency and quality. In this paper, a constant specific growth rate in fed-batch cultivation of Bordetella pertussis is achieved by a newly designed specific growth rate controller. The performance of standard control methods is limited because of the time-varying characteristics due to the exponentially increasing biomass and volume. To cope with the changing dynamics, a stable model reference adaptive controller is designed which adapts the controller settings as volume and biomass increase. An important asset of the design is that dissolved oxygen is the only required online measurement. An original design without considering the dissolved oxygen dynamics resulted experimentally in oscillatory behaviour. Hence, in contrast to common believes, it is essential to include dissolved oxygen dynamics. The robustness of this novel design was tested in simulation. The validity of the design was confirmed by laboratory experiments for small-scale production of B. pertussis. The controller was able to regulate the specific growth rate at the desired set point, even during a long fed-batch cultivation time with exponentially increasing demands for substrates and oxygen.     

A pseudo-exponential feeding method for control of specific growth rate in fed-batch cultures

A simple feeding method for controlling specific growth rate in fed-batch culture was developed. This method applies a constant feed rate using a concentrate reservoir and two mixing chambers in series to simulate the exponential feeding. Fed-batch cultures with Escherichia coli showed that the present feeding method could sustain the cells growing at predetermined specific growth rates, where the time length for exponential growth was dependent on the magnitude of the growth rate. The present feeding method is convenient to operate, requires no computerized control equipments, and thus could expect an extensive application in fed-batch culture.     

Simple control of specific growth rate in biotechnological fed-batch processes based on enhanced online measurements of biomass

Reliable control of the specific growth rate (μ) in fed-batch fermentations depends on the availability of accurate online estimations of the controlled variable. Due to difficulties in measuring biomass, μ is typically estimated using reference models relating measurements of substrate consumption or oxygen uptake rate to biomass growth. However, as culture conditions vary, these models are adapted dynamically, resulting in complex algorithms that lack the necessary robustness for industrial applicability. A simpler approach is presented where biomass is monitored using dielectric spectroscopy. The measurements are subjected to online balances and reconciled in real time against metabolite concentrations and off-gas composition. The reconciled biomass values serve to estimate the growth rate and a simple control scheme is implemented to maintain the desired value of μ. The methodology is developed with the yeast Kluyveromyces marxianus, tested for disturbance rejection and validated with two other strains. It is applicable to other cellular systems with minor modifications.     

Expression of recombinant epidermal growth factor inE. coli

Epidermal growth factor (EGF) known as a urgastrone is a powerful mitogen with a wide variety of possibilities for medical usages. A mature EGF coding region was isolated from human prepro-EGF sequence by a conventional PCR and cloned into pQE vector in which the gene product was supposed to be expressed with 6×His tag for the subsequent purification. The recombinant mature EGF was expressed in M15[Rep4], anEscherichia coli host strain, in amount of 30–40% of total proteins present inE. coli extract by the addition of isopropylthio-β-galactopyranoside (IPTG). The recombinant EGF purified using a Ni²⁺-NTA affinity column chromatography was active in its ability to induce phosphorylation on tyrosine residues of several substrate proteins when murine NIH3T3 and human MRC-5 fibroblast cells were stimulated with it. This work may provide the basic technology and information for the production of recombinant EGF.     

Optimal nonsingular control of fed-batch fermentation

Presented is a new simple method for multidimensional optimization of fed-batch fermentations based on the use of the orthogonal collocation technique. Considered is the problem of determination of optimal programs for fermentor temperature, substrate concentration in feed, feeding profile, and process duration. By reformulation of the state and control variables is obtained a nonsingular form of the optimization problem which has considerable advantage over the singular case since a complicated procedure for determination of switching times for feeding is avoided. The approximation of the state variables by Lagrange polynomials enables simple incorporation of split boundary conditions in the approximation, and the use of orthogonal collocations provides stability for integration of state and costate variables. The interpolation points are selected to obtain highest accuracy for approximation of the objective functional by the Radau-Lobatto formula. The control variables are determined by optimization of the Hamiltonian at the collocation points with the DFP method. Constraints are imposed on state and control variables.The method is applied for a homogeneous model of fermentation with volume, substrate, biomass, and product concentrations as the state variables. Computer study shows considerable simplicity of the method, its high accuracy for low order of approximation, and efficient convergence.     

Recursive on-line estimation of the specific growth rate from off-gas analysis for the adaptive control of fed-batch processes

A recursive estimation algorithm for the specific growth rate of aerobic fed-batch processes was developed. The estimate (t) is independent of the state estimates X(t) of the process. This improves the stability of the closed loop system. The performance of the nonlinear control system was tested on fermentations with streptomyces tendae with great success. The methodology of the nonlinear control system is very general. Thus, it can be applied to substrate control problems of any other aerobic fed-batch process.     

Generic model control of the specific growth rate in recombinant Escherichia coli cultivations

Generic model control is shown to be a powerful tool for keeping a microbial cultivation process close to its predetermined (optimized) control profile. This is demonstrated at the example of the green fluorescent protein expressed in genetically modified Escherichia coli host cells. It is shown that the process can be run very closely to a predefined complex profile of the specific cell growth rate mu(t). Controlling the experiments at many different growth conditions is a straightforward way of effectively collecting the data necessary for optimization of recombinant protein production systems. Although the process dynamics is rather complex, the model for the controller can be kept quite simple. The control technique, used here for specific growth rate control, is quite universal and can be applied for different biotechnological processes as well.     

Nonlinear estimation of specific growth rate for aerobic fermentation processes

The specific growth rate of the biomass, a very important parameter of almost every fermentation process, cannot be measured directly or estimated from related variables, as the concentrations of biomass, substrates, or products, due to the lack of reliable and cheap sensors. In this article a stable adaptive estimator of the specific growth rate is designed for those aerobic processes where the measurement of the oxygen uptake rate is available on-line. This particular approach can be applied also for other reaction rates if the model of the process satisfies some very general assumptions, which make the dynamics of the measured reaction rate a nonlinear function only of two unknown parameters, the specific growth rate and its time derivative. With respect to a previous similar approach, the new estimator has one additional parameter and a different nonlinear structure. From the analysis of the dynamics of the estimation error, a tuning criterion is derived, by which the two different algorithms can be compared under similar conditions. Simulation results show a good performance of both estimators for various kind of processes and disturbances. (c) 1995 John Wiley & Sons, Inc.     

Nonlinear observation of specific growth rate in aerobic fermentation processes

Simple nonlinear observers for the on-line estimation of the specific growth rate from presently attainable real-time measurements are presented. The proposed observers do not assume or require any model for the specific growth rate and they are very successful in accurately estimating this parameter. Moreover, they are very easy to implement and to calibrate. Indeed, due to the particular structure of their gain, their tuning is reduced to the calibration of a single parameter. Simulation results obtained under different operating conditions are given in order to highlight the performances of the proposed estimators.     

On-line control of fed-batch fermentation of dilute-acid hydrolyzates

Dilute-acid hydrolyzates from lignocellulose are, to a varying degree, inhibitory to yeast. In the present work, dilute-acid hydrolyzates from spruce, birch, and forest residue, as well as synthetic model media, were fermented by Saccharomyces cerevisiae in fed-batch cultures. A control strategy based on on-line measurement of carbon dioxide evolution (CER) was used to control the substrate feed rate in a lab scale bioreactor. The control strategy was based solely on the ratio between the relative increase in CER and the relative increase in feed rate. Severely inhibiting hydrolyzates could be fermented without detoxification and the time required for fermentation of moderately inhibiting hydrolyzates was also reduced. The feed rate approached a limiting value for inhibiting media, with a corresponding pseudo steady-state value for CER. However, a slow decrease of CER with time was found for media containing high amounts of 5-hydroxymethyl furfural (HMF). The success of the control strategy is explained by the conversion of furfural and HMF by the yeast during fed-batch operation. The hydrolyzates contained between 1.4 and 5 g/l of furfural and between 2.4 and 6.5 g/l of HMF. A high conversion of furfural was obtained (between 65-95%) at the end of the feeding phase, but the conversion of HMF was considerably lower (between 12-40%).     

Effect of operating parameters on specific production rate of a cloned-gene product and performance of recombinant fermentation process

A two-stage continuous system in combination with a temperature-sensitive expression system were used as model systems to maximize the productivity of a cloned gene and minimize the problem associated with the plasmid instability for a high-expression recombinant. In order to optimize the two-stage fermentation process, the effects of such operational variables as temperature and dilution rate on productivity of cloned gene were studied using the model systems and a recombinant, Escherichia coli K12 DeltaH1 Deltatrp/pPLc23trp A1. When the expression of cloned gene is induced by raising the operating temperature above 38 degrees C, a significant decrease in the colony-forming-units (CFU) of the plasmid-harboring cell was observed, and the decrease was related to the product concentration. In order to describe this phenomenon, a new kinetic parameter related to the metabolic stress (metabolic stress factor) was introduced. It is defined as the ratio of the rate of change of pheno-type from colony-forming to non-colony-forming cells to the product accumulation per unit cell mass. At a fixed temperature of 40 degrees C, the varying dilution rate D in the range of 0.35-0.90 h(-1) did not affect the metabolic stress factor significantly. At a fixed dilution rate of D = 0.35 h(-1), this factor remained practically constant up to 41 degrees C but increased rapidly beyond 41 degrees C. The effects of temperature and dilution rate in the second stage on the specific production rate were also studied while maintaining the apparent specific growth rate (mu(2) (app)) of the second stage constant at or near mu(2) (app) = 0.26 h(-1). Under a constant dilution rate, D(2) = 0.35 h(-1), the maximum specific production rate obtained was about q(p, max) = 38 units TrpA/mg cell/h at 41 degrees C. At a constant temperature, T(2) = 40 degrees C, specific production rate increased with decreasing dilution rate with in the dilution rate range of D(2) = 0.35-0.90 h(-1). Based on the results of our study, the optimal operating conditions found were dilution rate D(2) = 0.35 h(-1) and operating temperature T(2) = 41 degrees C at the apparent specific growth rate of 0.26 h(-1). Under the optimal operating conditions, about threefold increase in productivity was achieved compared to the best batch culture result. In addition, the fermentation period could be extended for more than 100 h.     

Translational control inE. coli: The case of threonyl-tRNA synthetase

Genetic studies have shown that expression of theE. coli threonyl-tRNA synthetase (thrS) gene is negatively auto-regulated at the translational level. A region called the operator, located 110 nucleotides downstream of the 5 end of the mRNA and between 10 and 50bp upstream of the translational initiation codon in thethrS gene, is directly involved in that control. The conformation of anin vitro RNA fragment extending over thethrS regulatory region has been investigated with chemical and enzymatic probes. The operator locus displays structural similarities to the anti-codon arm of threonyl tRNA. The conformation of 3 constitutent mutants containing single base changes in the operator region shows that replacement of a base in the anti-codon-like loop does not induce any conformational change, suggesting that the residue concerned is directly involved in regulation. However mutation in or close to the anti-codon-like stem results in a partial or complete rearrangement of the structure of the operator region. Further experiments indicate that there is a clear correlation between the way the synthetase recognises each operator, causing translational repression, and threonyl-tRNA.