A phenomenological model to represent the kinetics of growth by <Emphasis Type="Italic">Corynebacterium glutamicum</Emphasis> for lysine production

Corynebacterium glutamicum is commonly used for lysine production. In the last decade, several metabolic engineering approaches have been successfully applied to C. glutamicum. However, only few studies have been focused on the kinetics of growth and lysine production. Here, we present a phenomenological model that captures the growth and lysine production during different phases of fermentation at various initial dextrose concentrations. The model invokes control coefficients to capture the dynamics of lysine and trehalose synthesis. The analysis indicated that maximum lysine productivity can be obtained using 72 g/L of initial dextrose concentration in the media, while growth was optimum at 27 g/L of dextrose concentration. The predictive capability was demonstrated through a two-stage fermentation strategy to enhance the productivity of lysine by 1.5 times of the maximum obtained in the batch fermentation. Two-stage fermentation indicated that the kinetic model could be further extended to predict the optimal feeding strategy for fed-batch fermentation.     

Bacterial cellulose production by fed-batch fermentation in molasses medium

Batch and fed-batch fermentations for bacterial cellulose (BC) production using molasses as a carbon source by Acetobacter xylinum BPR2001 were carried out in a jar fermentor. For improvement of BC production, molasses was subjected to H2SO4-heat treatment. The maximum BC concentration by this treated molasses increased 76%, and the specific growth rate increased 2-fold compared with that by untreated molasses. In batch fermentation, when the initial sugar concentrations of H2SO4-heat-treated molasses were varied from 20 to 70 g/L, the highest value of maximum BC concentration of 5.3 g/L was observed at 20 g/L. BC production in intermittent fed-batch (IFB) fermentation was conducted referring to the data in batch fermentation, and the highest BC production of 7.82 g/L was obtained when 0.2 L of molasses medium was added five times. When continuous fed-batch (CFB) fermentations were conducted, maximum BC concentration was obtained with a feeding rate of 6.3 g-sugar/h, which was derived from the optimal IFB experiment.     

Optimal production of glutathione by controlling the specific growth rate of yeast in fed-batch culture

The optimal of the specific growth rate was obtained with simple mathematical model in a yeast fed-batch cultures. The model was based on the mass balance around the fed-batch system and the relationship between the specific growth rate, mu, and the specific production rate of glutathione, rho(G). The optimal profile of mu was calculated as a bang-bang type, That is mu, should start from the maximum value, mu(max) and should be kept at mu(max); then mu should be switched to mu(c), which gives a maximum value of rho(G). It was proven from the maximum principle that switching was needed only once, with the switching time from mu(max) to mu(c) depending on the final required glutathione content. Finally, this ideal profile of mu for the maximum production of glutathione was realized by manipulating the substrates feed rate in the fed-batch culture. Using the extended Kalman filter and a programmed-controller/feedback-compensator (PF) system, mu could be controlled at the optimal profile obtained. As a result, the maximum production of glutathione was accomplished fairly successfully. However, further improvement in the controller performance for mu is desired. The control strategy employed here can be applied to other batch reaction processes.     

Growth kinetics and astaxanthin production of Phaffia rhodozyma on glycerol as a carbon source during batch fermentation

Glycerol was studied as a substrate for astaxanthin by Phaffia rhodozyma PR 190. With co-utilisation of yeast extract and peptone, the maximum specific growth rate was 0.24 ± 0.02 h–1. Astaxanthin percentage in total pigment is constant (0.78 mg/g) and its yield from glycerol is always 0.97 mg/g. The yield of biomass from glycerol alone is 0.50 ± 0.02 g/g. The specific rate of astaxanthin production versus the cell growth rate reached a maximum for an optimal specific growth rate of 0.075 h–1. For this optimal value, the maximum specific astaxanthin production rate is 0.09 ± 0.01 mg/g.h. The best astaxanthin results were : 33.7 mg/l, 0.2 mg/l.h and 1.8 mg/g yeast after a fermentation term of 168 hours. Our results suggest a strategy of astaxanthin production in fed batch culture or chemostat at a growth rate of 0.075 h–1. © Rapid Science Ltd. 1998     

Modelling and optimization of environmental conditions for kefiran production by Lactobacillus kefiranofaciens

A mathematical model for kefiran production by Lactobacillus kefiranofaciens was established, in which the effects of pH, substrate and product on cell growth, exopolysaccharide formation and substrate assimilation were considered. The model gave a good representation both of the formation of exopolysaccharides (which are not only attached to cells but also released into the medium) and of the time courses of the production of galactose and glucose in the medium (which are produced and consumed by the cells). Since pH and both lactose and lactic acid concentrations differently affected production and growth activity, the model included the effects of pH and the concentrations of lactose and lactic acid. Based on the mathematical model, an optimal pH profile for the maximum production of kefiran in batch culture was obtained. In this study, a simplified optimization method was developed, in which the optimal pH profile was determined at a particular final fermentation time. This was based on the principle that, at a certain time, switching from the maximum specific growth rate to the critical one (which yields the maximum specific production rate) results in maximum production. Maximum kefiran production was obtained, which was 20% higher than that obtained in the constant-pH control fermentation. A genetic algorithm (GA) was also applied to obtain the optimal pH profile; and it was found that practically the same solution was obtained using the GA.     

Productivity improvement of recombinant <Emphasis Type="Italic">Escherichia coli</Emphasis> fermentation via robust optimization

A nonlinear model of a recombinant Escherichia coli producing porcine growth hormone (pGH) fermentation was developed. The model was used to calculate a glucose feeding and temperature strategy to optimize the production of pGH. Simulations showed that the implementation of optimal feed and temperature profiles was sensitive to the maximum specific growth rate, and a mismatch could result in excessive acetate production and a significant reduction in pGH yield. An optimization algorithm was thus developed, using feedback control, to counter the effects of uncertainty in the specific growth rate and thus determine an optimal operating strategy for pGH production. This policy was experimentally implemented in a 10 L fermenter and resulted in a 125% increase in productivity over the previous best experimental result with this system—in spite of significant plant-model mismatch.     

An on-line physiological state recognition system for the lysine fermentation process based on a metabolic reaction model

A metabolic reaction model was developed for the lysine fermentation process by Corynebacterium glutamicum AJ-3462 to estimate the physiological state of the cells-that is, the growth and production activity, and the flux distribution of metabolites-from on-line measurable rates only. First, the extended Kalman filter was applied to eliminate noise in the measured rates. Then, using the metabolic reaction model, the lysine production rate and flux distribution were calculated. The estimation results allowed the physiological state of lysine production to be recognized, and an appropriate measure corresponding to the estimated state, such as intermittent addition of glucose and/or leucine, to be taken to maintain a high level of lysine productivity in batch culture. Finally, application of the recognition system enabled lysine to be produced from glucose at a higher yield than that from glucose- or leucine-limited exponential fed-batch cultures. (c) 1997 John Wiley & Sons, Inc. Biotechnol Bioeng 55: 170-181, 1997.     

Optimal substrate feeding policy for a fed batch fermentation with substrate and product inhibition kinetics

The optimal substrate feeding policy for the fed batch fermentation which is governed by product and substrate inhibited kinetics is presented. The conjunction point between nonsingular and singular arcs and the feeding policy along the singular arc are derived analytically in terms of the concentrations of substrate and product and the liquid volume. Thus, it is possible to determine the feeding rate by monitoring the state variables (i.e., closed loop control). As a specific example, an optimization study of the fed batch fermentation for ethanol production by Saccharomyces cerevisiae is presented. It is shown that the optimal feeding patterns are heavily dependent upon the initial conditions. The point selectivity provides the guideline for predicting the optimal feeding patterns and explaining the results of rigorous mathematical analysis.     

Optimal pH control of batch processes for production of curdlan by Agrobacterium species

We sought an optimal pH profile to maximize curdlan production in a batch fermentation of Agrobacterium species. The optimal pH profile was calculated using a gradient iteration algorithm based on the minimum principle of Pontryagin. The model equations describing cell growth and curdlan production were developed as functions of pH, sucrose concentration, and ammonium concentration, since the specific rates of cell growth and curdlan production were highly influenced by those parameters. The pH profile provided the strategy to shift the culture pH from the optimal growth condition (pH 7.0) to the optimal production one (pH 5.5) at the time of ammonium exhaustion. By applying the optimal pH profile in the batch process, we obtained significant improvement in curdlan production (64 g L⁻¹) compared to that of constant pH operation (36 g L⁻¹). Received 24 November 1998/ Accepted in revised form 17 June 1999     

Enhancement of Candida rugosa lipase production by using different control fed-batch operational strategies

Simulation studies have predicted that maximum lipase activity is reached with fed-batch operation strategies. In this work, two different fed-batch operational strategies have been studied: constant substrate feeding rate and specific growth rate control. A constant substrate feeding rate strategy showed that maximum aqueous lipolytic activity (55 U/mL) was reached at low substrate feeding rates, whereas lipase tends to accumulate inside the cell at higher rates of substrate addition. In the second fed-batch strategy studied, a feedback control strategy has been developed based on the estimation of state variables (X and mu) from the measurement of indirect variables such as CER by means of mass spectrometry techniques. An on-off controller was then used to maintain the specific growth rate at the desired value by adjusting the substrate feeding rate. A constant specific growth rate strategy gave higher final levels of aqueous lipolytic activity (117 U/mL) at low specific growth rates. At higher specific growth rates the enzyme remained accumulated inside the cell, as was observed with a constant feeding fed-batch strategy. With a constant specific growth rate strategy, lipase production by Candida rugosa was enhanced 10-fold compared to a batch operation. Purification studies have demonstrated that lipolytic and esterasic specific activity ratios of Candida rugosa isoenzymes can be modified by using different operational conditions. These studies have also showed that the isoenzymes obtained in a controlled growth rate strategy are around three- to four-fold more active than those obtained in a constant feeding rate strategy.     

毕赤酵母高密度发酵工艺的研究

高密度发酵是毕赤酵母提高蛋白表达量的一种重要策略,发酵工艺是高密度发酵的一个重要因素。采用下列措施均可以有效地提高表达水平：调节基础培养基,采用变pH和变温发酵,提高DO,选择最适的诱导前菌体密度和比生长速率并降低甘油初始浓度和采用分段式指数流加进行调控。选择合适的甲醇补料策略：甲醇限制补料（MLFB）、氧气限制补料（OLFB）、甲醇不限制补料（MNLFB）和温度限制补料（TLFB）。采用两种方式调控补料：诱导阶段菌体生长时,甲醇比消耗速率(qMeOH)为0.02-0.03gg-1h-1,而菌体不生长时,qMeOH采用较高值。     

A novel feeding strategy for enhanced plasmid stability and protein production in recombinant yeast fedbatch fermentation

A novel feeding strategy in fedbatch recombinant yeast fermentation was developed to achieve high plasmid stability and protein productivity for fermentation using low-cost rich (non-selective) media. In batch fermentations with a recombinant yeast, Saccharomyces cerevisiae, which carried the plasmid pSXR125 for the production of beta-galactosidase, it was found that the fraction of plasmid-carrying cells decreased during the exponential growth phase but increased during the stationary phase. This fraction increase in the stationary phase was attributed to the death rate difference between the plasmid-free and plasmid-carrying cells caused by glucose starvation in the stationary phase. Plasmid-free cells grew faster than plasmid-carrying cells when there were plenty of growth substrate, but they also lysed or died faster upon the depletion of the growth substrate. Thus, pulse additions of the growth substrate (glucose) at appropriate time intervals allowing for significant starvation period between two consecutive feedings during fedbatch fermentation should have positive effects on stabilizing plasmid and enhancing protein production. A selective medium was used to grow cells in the initial batch fermentation, which was then followed with pulse feeding of concentrated non-selective media in fedbatch fermentation. Both experimental data and model simulation show that the periodic glucose starvation feeding strategy can maintain a stable plasmid-carrying cell fraction and a stable specific productivity of the recombinant protein, even with a non-selective medium feed for a long operation period. On the contrary, without glucose starvation, the fraction of plasmid-carrying cells and the specific productivity continue to drop during the fedbatch fermentation, which would greatly reduce the product yield and limit the duration that the fermentation can be effectively operated. The new feeding strategy would allow the economic use of a rich, non-selective medium in high cell density recombinant fedbatch fermentation. This new feeding strategy can be easily implemented with a simple IBM-PC based control system, which monitors either glucose or cell concentration in the fermentation broth.     

The role of amino acids in the amplification and quality of DNA vectors for industrial applications

In this study, we have demonstrated that the type and feeding regimen of amino acids have a significant impact on the quality as well as the quantity of DNA vectors produced. Nutrient pool and factorial design experiments were carried out in order to identify the amino acids involved in increased biomass and induction of plasmid amplification. Leucine, glycine, and histidine were responsible for increased biomass and leucine starvation in the presence of histidine was implicated in plasmid amplification. Supercoiling of the plasmid was optimized using a dual feeding strategy. As a result of this, a fed-batch fermentation strategy for the production of a 6.9 kb plasmid, pSVß, in Escherichia coli DH5α was developed. In batch fermentation, a maximum plasmid yield of 39.4 mg/L equivalent to 11.3 mg/g dry cell weight (DCW) was achieved with casein hydrolysate limitation. About 90% of plasmid was in the supercoiled (SC) form after 31 hr of fermentation but only remained so for a short period, leading to a very brief window for harvesting cells at scale. Subsequently, a fed-batch fermentation using a dual feeding strategy was employed. A mean maximum plasmid yield of 44 mg/L equivalent to 9.1 mg plasmid/g DCW was achieved. After 25 hr, 90% of plasmid was in the SC form and remained at this level for the remaining 10 hr of the fermentation, allowing adequate time for the harvesting of cells without the loss of supercoiling of product. This study emphasized that optimizing fermentation strategy and identifying the essential nutrients are beneficial for bioprocessing of plasmid DNA for therapeutic applications.     

Optimization of conditions and cell feeding procedures for alcohol fermentation

Alcohol fermentation was studied with an emphasis on the separation of cell growth and alcohol production stages. Experiments were conducted to establish the optimal conditions for alcohol production in batch fermentations and to simulate continuous fermentations with cell feeding at various stages. It was found that the glucose concentration should be kept under 10% (w/v), and the temperature should be between 40 and 42.5 degrees C for maximum specific alcohol productivity. If the cell concentration is increased, a decrease in specific alcohol productivity is observed. Higher cell concentrations are needed for higher final alcohol concentrations. Among the cell feeding procedures into alcohol production stages, a cocurrent design was found to be better than recycle and countercurrent designs.     

Multivariable control of alcohol concentrations in the production of polyhydroxyalkanoates (PHAs) by Paracoccus denitrificans

A novel multivariable control strategy is developed for alcohol (ethanol and n-pentanol) concentrations in the production of poly(3-hydroxybutyrate-co-3-hydroxyvalerate), P(HB-co-HV), a biodegradable polymer by Paracoccus denitrificans ATCC 1774. This controller, which is developed to control the mole fraction of P(HB-co-HV), consists of two parts: one is for ethanol concentration control and the other is for mole fraction control, based on the concept of metabolic flux distribution control. A simple metabolic reaction (MR) model is constructed for flux distribution analysis. The relationship between mole ratio of specific consumption rate of the two alcohols (ethanol and n-pentanol) and the mole fraction of 3HV units in the polymer is linear. This result suggests that the split ratio at a branched point of 3-ketovaleryl-CoA in the P(HB-co-HV) synthetic pathway is constant for several fermentation conditions. When the mole fraction of 3HV units has a target value, the feed rate of n-pentanol becomes a function of the feed rate of ethanol and the set value of 3HV, based on the MR model. The mole fraction of 3HV units successfully reached the target value using this strategy. The mole fraction control strategy is combined with an optimal production strategy based on the optimal profile of the specific growth rate. The combined strategy is realized using multivariable controllers and P(3HB-co-3HV) production is maximized with a given value of mole fraction of 3HV units at the final step of fermentation.     

Control of L-phenylalanine production by dual feeding of glucose and L-tyrosine

Control of L-phenylalanine production by a recombinant of Escherichia coli AT2471 by means of the dual feeding of glucose and L-tyrosine was investigated. A novel method was developed for on-line monitoring of the maximum glucose uptake rate (MGUR), in which the length of time required for the consumption of added glucose was measured. Accumulation of acetic acid was successfully prevented throughout the whole period of the culture when the glucose concentration was kept below 0.1 g/L by controlling the glucose feeding on the basis of on-line monitoring of the MGUR and the cell concentration with a laser sensor.In a batch culture with glucose feeding, after L-tyrosine was depleted cell growth and the L-phenylalanine production rate decreased along with decreases in the specific enzyme activities of chorismate mutase-p-prephenate dehydratase (CMP) and 3-deoxy-D-arabinoheputulosonate 7-phosphate synthase (DAHP), which are the key enzymes in the L-phenylalanine synthesis pathway. Increasing the L-tyrosine feed rate by an appropriate amount, but not so far as to cause L-tyrosine accumulation in the culture, increased the activities of the enzymes and the specific rates of growth and production while the product yield based on glucose consumption decreased.The average specific rates of growth, production, and MGUR could be expressed as functions of the specific L-tyrosine consumption rate during both the earlier and later periods of L-tyrosine feeding. Estimations of the amount of L-phenylalanine produced, the product yield, and the cost factor by using these functions with several different combinations of two specific L-tyrosine consumption rates for two 10-h periods resulted in a suggested optimum L-tyrosine feeding strategy giving a lower specific L-tyrosine consumption rate in the later period, to suppress cell growth, in comparison to that in the earlier period. During L-tyrosine feeding, the three specific rates (growth, production, and MGUR) could be successfully controlled by adjusting the specific L-tyrosine consumption rate to the predicted value. The cost factor was lowest in this controlled culture, demonstrating experimentally the effectiveness of the strategy. (c) 1996 John Wiley & Sons, Inc.     

Improving ethanol production and viability of Saccharomyces cerevisiae by a vitamin feeding strategy during fed-batch process

Several bottlenecks in the alcoholic fermentation process must be overcome to reach a very high and competitive performance of bioethanol production by the yeast Saccharomyces cerevisiae. In this paper, a nutritional strategy is described that allowed S. cerevisiae to produce a final ethanol titre of 19% (v/v) ethanol in 45 h in a fed-batch culture at 30 degrees C. This performance was achieved by implementing exponential feeding of vitamins throughout the fermentation process. In comparison to an initial addition of a vitamin cocktail, an increase in the amount of vitamins and an exponential vitamin feeding strategy improved the final ethanol titre from 126 g l(-1) to 135 g l(-1) and 147 g l(-1), respectively. A maximum instantaneous productivity of 9.5 g l(-1) h(-1) was reached in the best fermentation. These performances resulted from improvements in growth, the specific ethanol production rate, and the concentration of viable cells in response to the nutritional strategy.     

Enhancement of 5-aminolevulinate production with recombinant Escherichia coli using batch and fed-batch culture system

5-Aminolevulinate (ALA) production with recombinant Escherichia coli Rosetta (DE3)/pET28a(+)-hemA was studied. In batch fermentation, the addition of glucose and glycine was effective to improve ALA production. Then the fed-batch fermentation was conducted with continuous feeding of precursors. When the concentrations of succinic acid and glycine were 7.0 g/l and 4.0 g/l, respectively, in the feeding, the ALA yield reached 4.1g/l. But the molar yield (ALA/glycine) was decreased in the fed-batch fermentation compared to batch fermentation. And it was found that the pH control during fed-batch cultivation was very important for the cell growth and ALA production. A two-stage pH value controlling strategy was suggested, in which, the pH value in the first 6h was regulated at pH 5.9, after then at pH 6.2, and the ALA yield was as high as 6.6g/l via fed-batch fermentation.     

Model aided dynamic process analysis and optimization for the nourseothricin fermentation

The relation between product formation and growth kinetics could be characterized by two facts: the specific product formation rate depends on the ageing of the population and on the specific growth rate. These relation was formulated and quantified by a mathematical model, which was fitted to experimental data of a representative fermentation run und used to predict an optimal fermentation mode. In the result of this discussion cyclic fed batch fermentation was found to be optimal.     

General characteristics of optimal feed rate profiles for various fed-batch fermentation processes

General Characteristics of the optimal feed rate profiles have been deduced for various fed-batch fermentation processes by analyzing singular controls and singular arcs. The optimal control sequences depend on the shapes of the specific growth and product formation rates, mu andpi, and the initial conditions. For fed-batch processes described by four mass balance equations, the most general optimal control sequence consists of a period of maximum feed rate, a period of minimum feed rate (a batch period), a period of singular feed rate (variable and intermediate), and a batch period. Degenerate sequences in which one or more periods are missing can result with a particular set of initial conditions. If the fermentation time is not critical, the singular control maximizes the net yield of product and only when the time is also important, it balances a trade off between the yield of product and the specific growth rate which dictates the fermentation time. With the sequence of optimal control known, the optimal feed rate profile determination is reduced to a problem of determining switching times.