Fed-batch Culture System Research Articles

Bioprocess development of the production of the mutant P-219-L human d-amino acid oxidase for high soluble fraction expression in recombinant Escherichia coli

In order to examine the structure–activity relationship and the substrate specificity of human d-amino acid oxidase (h.DAO), a single amino acid mutation had been established as proline-219-luecine (P-219-L). The gene encoding mutant h.DAO has been cloned and expressed in Escherichia coli BL21 (DE3). It was observed that the host cell was negatively affected by the expressed mutant h.DAO, resulting in a remarkable decrease in the cell growth and consequently the amount of the produced enzyme. To overcome this problem, we investigated several factors that may affect the cell growth rate and the mutant h.DAO production such as optimization of the glucose concentration as a main carbon source and the yeast extract concentration as a main nitrogen source, optimization of dissolved oxygen (DO%) concentration and the addition of benzyl alcohol (BA, which can artificially induce a strong heat shock response at low temperature), to enhance the production of natively folded soluble fraction of the recombinant protein. These parameters were tested on both shake flask level and fed-batch bioreactor level. The Western blot analysis and the enzyme activity assay indicated the higher level of the mutant expression towards enhancement of the conditions by using our designed approach. The specific activity (which was used as an indicator for the level of the desired protein produced = U/mg protein) and the OD 600 nm of the host cells (which was used as an indicator for the cell growth), reached to be 0.061 U/mg protein and 3.44, respectively upon using fed-batch culture system containing the optimized medium composition (15 g/l glucose and 5 g/l yeast extract). While upon using the shake flask level, these values were 0.032 and 1.1, respectively. Enhancement of the cell growth and the enzyme production was noticed after DO% optimization upon using 500 rpm agitation speed and 1.8 v.v.m. (volume volume minute) aeration. The specific activity for the mutant enzyme and the OD 600 nm of the host cells reached to be 0.14 U/mg protein and 7.1, respectively. Finally upon using the optimized culture composition (15 g/l glucose and 5 g/l yeast extract), optimized DO% (using 500 rpm agitation speed and 1.8 v.v.m.) and 0.1 mM BA at the fed-batch bioreactor level, the specific activity and the OD 600 nm of the host cells increased significantly to be 0.21 U/mg protein and 11.3, respectively at 24 h culture. These results indicate the importance of our approaches to overproducing mutant h.DAO in soluble form in E. coli.

Improvement of isopropanol production by metabolically engineered Escherichia coli using gas stripping

To improve isopropanol production by metabolically engineered Escherichia coli strain TA76, the optimization of fermentation conditions and isopropanol removal by gas stripping were performed. Isopropanol is one of the simplest secondary alcohols, and it can be dehydrated to yield propylene, which is currently derived from petroleum as a monomer for making polypropylene. Initially, using a pH-controlled fed-batch culture with the intermittent addition of glucose, strain TA76 produced 667 mM (40.1g/L) of isopropanol after 60 h, representing 73.2% (mol isopropanol/mol glucose) of the theoretical maximum yield. Because the accumulation of isopropanol drastically reduced production yields, a gas stripping recovery method was incorporated into the fed-batch culture system. Using this approach, strain TA76 produced 2378 mM (143 g/L) of isopropanol after 240 h with a yield of 67.4% (mol/mol). To our knowledge, this titer represents the highest level of isopropanol production by E. coli to date and suggests that strain TA76 has a great potential for commercial fermentative isopropanol production.

Using a fed-batch culture strategy to enhance rAAV production in the baculovirus/insect cell system

Recombinant adeno-associated virus (rAAV) is one of the most promising vectors for human gene therapy. However, the production systems that are currently available have a limited capacity and cannot provide sufficient quantities of rAAV for preclinical or clinical trials. Many novel methods for improving rAAV production have been developed, but few researchers have focused on the culture process. In this study, we use a fed-batch culture system to enhance rAAV yield in the baculovirus/insect cell system. When the insect cells were co-infected with MOI=5 of Bac-GFP at a ratio of 1:9:9 (Bac-GFP: Bac-Rep: Bac-VP), the fed-batch culture achieved optimal rAAV yields. In batch culture, the optimal cell density for producing rAAV was found to be 1x10(6) cells/ml, and the highest rAAV yield (1.22x10(8) IVP/ml, 122 IVP/cell) occurred at day 5 post-infection. In the fed-batch culture, rAAV yield reached 2.13x10(8) IVP/ml at day 4 post-infection, and the highest rAAV yield was 2.40x10(8) IVP/ml (240 IVP/cell) at day 5 post-infection. The cost of the batch and fed-batch cultures is similar; however, the rAAV yield was 2.6-fold higher in the fed-batch culture system compared with that in the batch culture system. Therefore, here we demonstrated an economical and efficient strategy for rAAV production.

Optimal control for nonlinear dynamical system of microbial fed-batch culture

In fed-batch culture of glycerol bio-dissimilation to 1, 3-propanediol (1, 3-PD), the aim of adding glycerol is to obtain as much 1, 3-PD as possible. So a proper feeding rate is required during the process. Taking the concentration of 1, 3-PD at the terminal time as the performance index and the feeding rate of glycerol as the control function, we propose an optimal control model subject to a nonlinear dynamical system and constraints of continuous state and non-stationary control. A computational approach is constructed to seek the solution of the above model in two aspects. On the one hand we transcribe the optimal control model into an unconstrained one based on the penalty functions and an extension of the state space; on the other hand, by approximating the control function with simple functions, we transform the unconstrained optimal control problem into a sequence of nonlinear programming problems, which can be solved using gradient-based optimization techniques. The convergence analysis of this approximation is also investigated. Numerical results show that, by employing the optimal control policy, the concentration of 1, 3-PD at the terminal time can be increased considerably.

Modelling and optimal control for nonlinear multistage dynamical system of microbial fed-batch culture

In this paper, we propose a new controlled multistage system to formulate the fed-batch culture process of glycerol bio-dissimilation to 1,3-propanediol (1,3-PD) by regarding the feeding rate of glycerol as a control function. Compared with the previous systems, this system doesn't take the feeding process as an impulsive form, but a time-continuous process, which is much closer to the actual culture process. Some properties of the above dynamical system are then proved. To maximize the concentration of 1,3-PD at the terminal time, we develop an optimal control model subject to our proposed controlled multistage system and continuous state inequality constraints. The existence of optimal control is proved by bounded variation theory. Through the discretization of the control space, the control function is approximated by piecewise constant functions. In this way, the optimal control model is approximated by a sequence of parameter optimization problems. The convergence analysis of this approximation is also investigated. Furthermore, a global optimization algorithm is constructed on the basis of the above descretization concept and an improved Particle Swarm Optimization (PSO) algorithm. Numerical results show that, by employing the optimal control policy, the concentration of 1,3-PD at the terminal time can be increased considerably.

Vector measure as controls for explicit nonlinear impulsive system of fed-batch culture

In this paper, we consider an optimal control problem of microbial fermentation process in which glycerol is converted to 1,3-propanediol by Klebsiella pneumoniae in fed-batch culture. During the period of reaction, the variation of pH value is monitored to determine glycerol replenishment quantity, guaranteeing that microorganism can always keep growing fast under enough nutrition. Every time pH value is lower than seven, the quantity of glycerol added is such that pH value returns seven again. Glycerol is poured into reactor at discrete time instant and the quantity is controllable. The problem is to determine for each discrete time instant the glycerol quantity to add and maximize the final concentration of 1,3-propanediol. We present a controlled explicit nonlinear impulsive dynamical system of fed-batch culture with state independent vector measures as controls and study the existence, uniqueness, boundedness, continuous dependence and Gâteaux differentiability of its solution with respect to controls. We then propose a multiple objective programming model and demonstrate the regularity of cost functionals and weak compactness of admissible control set. Finally we discuss the existence of optimal control and implement a hybrid particle swarm optimization algorithm to solve the model optimally. Computational results are presented on a numerical example.

Expression of a hemA Gene from Agrobacterium radiobacter in a Rare Codon Optimizing Escherichia coli for Improving 5-aminolevulinate Production

The 5-aminolevulinate (ALA) synthase gene (hemA) from Agrobacterium radiobacter zju-0121, which was cloned previously in our laboratory, contains several rare codons. To enhance the expression of this gene, Escherichia coli Rosetta(DE3), which is a rare codon optimizer strain, was picked out as the host to construct an efficient recombinant strain. Cell extracts of the recombinant E. coli were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis under the appropriate conditions. The results indicated that the activity of ALA synthase expressed in Rosetta(DE3)/pET-28a(+)-hemA was about 20% higher than that in E. coli BL21(DE3). Then the effects of precursors (glycine and succinate) and glucose, which is an inhibitor for ALA dehydratase as well as the carbon sources for cell growth, on the production of 5-aminolevulinate were investigated. Based on an optimal fed-batch culture system described in our previous work, up to 6.5 g/l (50 mM) ALA was produced in a 15-l fermenter.

Effect of Constant Glucose Feeding on the Production of Exopolysaccharides by Tremella fuciformis Spores

A fed-batch culture system with constant feeding (glucose 80 g L(-1), 0.25 ml min(-1)) was used to study the influence of glucose on cell dry weight and exopolysaccharides production from submerged Tremella fuciformis spores in a 5-L stirred-tank bioreactor. The results showed that high levels of cell mass (9.80 g L(-1)) and exopolysaccharides production (3.12 g L(-1)) in fed-batch fermentation were obtained after 1 h of feeding, where the specific growth rate (mu) and exopolysaccharides yield on substrate consumed (YP/S) were 0.267 d(-1) and 0.14 g g(-1). Unlike batch fermentation, maximal cell mass and exopolysaccharides production merely reached 7.11 and 2.08 g L(-1); the specific growth rate (mu) and exopolysaccharides yield on substrate consumed (YP/S) were 0.194 d(-1) and 0.093 g g(-1), respectively. It is concluded that the synthesis of exopolysaccharides can be promoted effectively when feeding glucose at a late exponential phase.

PARAMETERS IDENTIFICATION AND STABILITY OF THE IMPULSIVE SYSTEM OF MICROORGANISM FERMENTATION

The nonlinear impulsive system of microorganism fed-batch culture is presented in this note.Then the identification model (PDP) is given for some sensitive parameters.The relationship between the parameters and the solution of the impulsive system is discussed,and the identifiability of the PDP is deduced.At last the optimal solution of PDP is derived by using the constructive algorithm and the stability of impulsive system for fed-batch fermentation is considered.

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 4.1 g/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 6 h was regulated at pH 5.9, after then at pH 6.2, and the ALA yield was as high as 6.6 g/l via fed-batch fermentation.

Controlling the sucrose concentration increases Coenzyme Q10 production in fed-batch culture of Agrobacterium tumefaciens

The production yield of Coenzyme Q(10) (CoQ(10)) from the sucrose consumed by Agrobacterium tumefaciens KCCM 10413 decreased, and high levels of exopolysaccharide (EPS) accumulated after switching from batch culture to fed-batch culture. Therefore, we examined the effect of sucrose concentration on the fermentation profile by A. tumefaciens. In the continuous fed-batch culture with the sucrose concentration maintained constantly at 10, 20, 30, and 40 g l(-1), the dry cell weight (DCW), specific CoQ(10) content, CoQ(10) production, and the production yield of CoQ(10) from the sucrose consumed increased, whereas EPS production decreased as maintained sucrose concentration decreased. The pH-stat fed-batch culture system was adapted for CoQ(10) production to minimize the concentration of the carbon source and osmotic stress from sucrose. Using the pH-stat fed-batch culture system, the DCW, specific CoQ(10) content, CoQ(10) production, and the product yield of CoQ(10) from the sucrose consumed increased by 22.6, 13.7, 39.3, and 39.3%, respectively, whereas EPS production decreased by 30.7% compared to those of fed-batch culture in the previous report (Ha SJ, Kim SY, Seo JH, Oh DK, Lee JK, Appl Microbiol Biotechnol, 74:974-980, 2007). The pH-stat fed-batch culture system was scaled up to a pilot scale (300 l), and the CoQ(10) production results obtained (626.5 mg l(-1) of CoQ(10) and 9.25 mg g DCW(-1) of specific CoQ(10) content) were similar to those obtained at the laboratory scale. Thus, an efficient and highly competitive process for microbial CoQ(10) production is available.

Nonlinear hybrid kinetic system of microbial bioconversion in fed-batch culture

In this paper, the nonlinear kinetic system of fed-batch fermentation is investigated in the process of glycerol bio-dissimilation to 1,3-propanediol by Klebsiella pneumoniae. Taking into account the discontinuity of the process of adding glycerol and alkali and kinetics of microbial bioconversion in fed-batch culture, we present a nonlinear hybrid kinetic system of fed-batch fermentation in terms of the kinetic system of batch culture and continuous culture, which is well suitable for the factual fed-batch fermentation. Then we discuss the existence, uniqueness and boundedness of solutions to the hybrid system as well as the relative Lipschitz continuity and differentiability of solutions with respect to the initial-state-control pairs. Finally a numerical example is employed to carry out numerical simulation to the hybrid system. The results of numerical simulation reveal that the errors between experimental and simulated values by means of the hybrid system of fed-batch culture are less than those using the previous impulsive system.

Production of Alkaline Protease with Teredinobacter turnirae in Controlled Fed-batch Fermentation

By using our previously optimized media and a fed-batch operation controlled by LabVIEW Software, the key parameter for a high production of alkaline protease using the marine bacterium, Teredinobacter turnirae, was to maintain a low concentration of C and N-sources ( < 2 g sucrose l(-1) and < 0.2 g NH4C l l(-1)) using an appropriate fed-batch culture system. A maximum protease activity of 8250 U ml(-1 )was thus achieved.

Nonlinear impulsive system of fed-batch culture in fermentative production and its properties☆

Abstract In this study, the nonlinear dynamical system of fed-batch fermentation is investigated in the process of bio-dissimilation of glycerol to 1,3-propanediol by Klebsiella pneumoniae . Considering the abrupt increase of glycerol in fed-batch culture, this paper proposes a nonlinear impulsive system of the culture process, which is fit for formulating the factual fermentation better than the continuous models in being. We study the questions of existence and properties of mild solutions for the system and the continuous dependence of solutions on initial values and the controllable variable. Finally, the numerical simulations show that the errors between experimental and computational values using the impulsive system are less than those using the previous continuous system.

Nonlinear impulsive system of microbial production in fed-batch culture and its optimal control

In this study the optimal control of fed-batch glycerol fermentation is investigated based on an impulsive dynamical system. Considering the sudden increase of the glycerol and alkali in fed-batch culture of biodissimilation of glycerol to 1,3-propanediol, this paper proposes a nonlinear impulsive system of fed-batch culture. The existence, uniqueness and regularity properties of piecewise solution for the system are proved. In view of the controllability of volumes of glycerol added to the reactor instantaneously, the paper constructs an optimal control model based on the nonlinear impulsive system and the existence of the optimal control is obtained. The control variables here are the moments and the sizes of jumps in the states at the discrete instants and the objective is to maximize the productivity of 1,3-propanediol over one cycle.

Identification of genes whose expressions are enhanced or reduced in baker's yeast during fed-batch culture process using molasses medium by DNA microarray analysis

Genes whose expression levels are enhanced or reduced during the cultivation process that uses cane molasses in baker's yeast production were identified in this study. The results showed that baker's yeast grown in molasses medium had higher fermentation ability and stress tolerance compared with baker's yeast grown in synthetic medium. Molasses apparently provided not only sugar as a carbon source but also provided functional components that enhanced or reduced expression of genes involved in fermentation ability and stress tolerance. To identify the genes whose expression is enhanced or reduced during cultivation in molasses medium, DNA microarray analysis was then used to compare the gene expression profile of cells grown in molasses with that of cells grown in synthetic medium. To simulate the commercial baker's yeast production process, cells were cultivated using a fed-batch culture system. In molasses medium, genes involved in the synthesis or uptake of vitamins (e.g., biotin, pyridoxine and thiamine) showed enhanced expression, suggesting that vitamin concentrations in molasses medium were lower than those in synthetic medium. Genes involved in formate dehydrogenase and maltose assimilation showed enhanced expression in molasses medium. In contrast, genes involved in iron utilization (e.g., siderophore, iron transporter and ferroxidase) showed enhanced expression in synthetic medium, suggesting that iron starvation occurred. The genes involved in the metabolism of amino acids also showed enhanced expression in synthetic medium. This identification of genes provides information that will help improve the baker's yeast production process.

Nonlinear dynamical systems of bio-dissimilation of glycerol to 1,3-propanediol and their optimal controls

In this study simplified mathematical modelsof the nonlinear dynamic systems of dissimilation of glycerol to1,3-propanediol by {\it Klebsiella pneumoniae} in continuous,batch and fed-batch cultures are investigated. Considering bigerrors between the experimental results and computational valuesin the existing models, the parameter identification models for these systems are established. The properties of the solutions for the nonlinear dynamic systems are discussed and theidentifiability of the parameters is proved. In view of the sudden increase of the glyceroland alkali in fed-batch culture, this paper proposes a nonlinearimpulsive system of fed-batch culture. The existence, uniquenessand regularity properties of piecewise solution for the system areproved. Based on the nonlinear impulsive system, the paperconstructs an optimal control model in view of the controllabilityof volumes of glycerol added to the reactor instantaneously, andthe existence of the optimal control is obtained.

A Growth Kinetics Model of &lt;I&gt;Euglena gracilis&lt;/I&gt; in Fed-batch Mixotrophic Culture

Mixotrophic culture of Euglena gracilis was carried out using a fed-batch culture system. Glucose and L-glutamic acid were added every 24 hours for 6 days to make the concentrations in the culture medium return to their initial values. We proposed a mixotrophic growth kinetics model to correlate a specific growth rate with a specific rate of energy consumption. In the model, the specific growth rate was proportional to the (1/n)th power of the specific energy consumption rate, and a corresponding yield parameter and a maintenance factor depended on light intensity. The specific growth rate in mixotrophic culture was correlated with incident light intensity by an equation that combined pseudo-photoautotrophic growth rate comparable to that of the modified Aiba model with heterotrophic growth rate. The optimum light intensity that produced the attainable maximum growth rate in mixotrophic culture could be estimated by the equation.

Enhancement and stabilization of desulfurization activity of Rhodococcus erythropolis KA2-5-1 by feeding ethanol and sulfur components

We developed a fed-batch culture system fed with ethanol and restricted amounts of sulfur compounds to enhance and stabilize the desulfurizing activity in bacterial cells. In this system using dibenzothiophene (DBT) as the sole sulfur source, a desulfurizing bacterium Rhodococcus erythropolis KA2-5-1 cultivated with small amounts of sulfur showed stable desulfurizing activity and a low rate of growth. However, the cells cultured with excess amounts of sulfur showed unstable activity and a high growth rate. DBT had disadvantages as a sulfur source for cultivation because it is immiscible with water and toxic to cells. We then investigated water-soluble sulfur compounds for use as the sole sulfur source for the cultivation of R. erythropolis KA2-5-1 with desulfurizing activity, and found 2-aminoethanesulfonic acid to be the most effective. When 2-aminoethanesulfonic acid was used instead of DBT as the sole sulfur source in the fed-batch fermentation system, R. erythropolis KA2-5-1 showed the highest desulfurizing activity, 111 mmol of 2-HBP/kg-cells/h, a high growth rate (μ = 0.37/h) and a final cell concentration of 20.0 g-dry cells/ l during 89 h of cultivation. The production levels of the desulfurizing enzymes in the bacterial cells cultivated with DBT or 2-aminoethanesulfonic acid were evaluated by immunoblot analysis with specific antisera, indicating that the same quantity of desulfurizing enzymes was expressed in both cases.

Effects of growth medium composition, iron sources and atmospheric oxygen concentrations on production of luciferase-bacterial magnetic particle complex by a recombinant Magnetospirillum magneticum AMB-1

Growth conditions for mass production of luciferase-bacterial magnetic particles (BMPs) by a recombinant Magnetospirillum magneticum AMB-1 were investigated in a pH-regulated fed-batch culture system. Enrichment of growth medium with L-cysteine, yeast extract and polypeptone enhanced both bacterial growth and BMP production. The presence of L-cysteine in the medium was useful for induction of cell growth. Strict anaerobic conditions led to a prolonged lag phase and limited the final cell density. Trace oxygen enhanced cell growth with increasing BMP production. As iron sources, ferrous sulfate and ferric gallate dramatically enhanced BMP yield as compared with ferric quinate, an iron chelate conventionally used. The optimized conditions increased cell density to 0.59 ± 0.03 g cell dry weight/liter and BMP production to 14.8 ± 0.5 mg dry weight/liter in fed-batch culture for four days.