Exponential Feeding Research Articles

High-Cell-Density Cultivation of <I>Pseudomonas putida</I> IPT 046 and Medium-Chain-Length Polyhydroxyalkanoate Production From Sugarcane Carbohydrates

We studied high-density cultures of Pseudomonas putida IPT 046 for the production of medium-chain-length polyhydroxyalkanoates (PHAMCL) using an equimolar mixture of glucose and fructose as carbon sources. Kinetics studies of P. putida growth resulted in a maximum specific growth rate of 0.65 h(-1). Limitation and inhibition owing to NH4+ ions were observed, respectively, at 400 and 3500 mg of NH4+/L. The minimum concentration of dissolved oxygen in the broth must be 15% of saturation. Fed-batch strategies for high-cell-density cultivation were proposed. Pulse feed followed by constant feed produced a cell concentration of 32 g/L in 18 h of fermentation and low PHAMCL content. Constant feed produced a cell concentration of 35 g/L, obtained in 27 h of fermentation, with up to 15% PHAMCL. Exponential feed produced a cell concentration of 30 g/L in 20 h of fermentation and low PHAMCL content. Using the last strategy, 21% PHAMCL was produced during a period of 34 h of fed-batch operation, with a final cell concentration of 40 g/L and NH4+ limitation. Using phosphate limitation, 50 g/L cell concentration, 63% PHAMCL and a productivity of 0.8 g/(L x h) were obtained in 42 h of fed-batch operation. The PHAMCL yield factors from consumed carbohydrate for N-limited and P-limited experiments were, respectively, 0.15 and 0.19 g/g.

Effect of Flow Rate Pattern on Glucose‐6‐phosphate Dehydrogenase Synthesis in Fed‐Batch Culture of Recombinant Saccharomycescerevisiae

A strain of genetically modified Saccharomyces cerevisiae (S. cerevisiae) W303 181 was used to improve glucose-6-phosphate dehydrogenase (G6PDH) production in aerobic culture. Fed-batch cultures were carried out in a 5 L fermentor at variable values of the parameter K, namely, 0.2, 0.3, 0.5, 0.7, and 0.8 h(-)(1). The highest G6PDH production (1164 U/L) and specific activity (517 U/g(cell)) were obtained using the following conditions: glucose, 5.0 g/L; adenine, 8 microg/mL; histidine, 8 microg/mL; tryptophan, 8 microg/mL; temperature, 30 degrees C; inoculum, 1.28 g/L; pH, 5.7; agitation, 400 rpm; aeration, 2.2 vvm; and K, 0.2 h(-)(1). The exponential feeding pattern increased cell density (2.14 g/L), enzyme productivity (149.27), and biomass yield (0.18 g(glu)/g(cell)( )(mass)). The level of G6PDH in the genetically modified S. cerevisiae was approximately 4.1-fold higher than that found in a commercial strain.

Expression of recombinant Pseudomonas stutzeri di-heme cytochrome c4 by high-cell-density fed-batch cultivation of Pseudomonas putida

The gene of the di-heme protein cytochrome c 4 from Pseudomonas stutzeri was expressed in Pseudomonas putida. High-yield expression of the protein was achieved by high-cell-density fed-batch cultivation using an exponential glucose feeding strategy. The recombinant cytochrome c 4 protein was purified to apparent homogeneity and analyzed by electronic absorption spectroscopy, nanoflow electrospray ionization time-of-flight mass spectrometry, and electrochemistry. Cyclic voltammograms and UV–vis electronic absorption spectra were indistinguishable from the equivalent data of native P. stutzeri cytochrome c 4. Furthermore, the calculated and experimentally determined molecular masses of recombinant cytochrome c 4 were identical. Biochemical characterization of both wild-type and mutant derivatives of the protein will be greatly enhanced and facilitated by the described high-yield fermentation and rapid isolation procedure.

Perfusion culture of the diatom Nitzschia laevis for ultra-high yield of eicosapentaenoic acid

In the development of high yield of eicosapentaenoic acid (EPA) by the diatom Nitzschia laevis, substrate limitation and toxic metabolite inhibition were the two principal obstacles. In the present study, a perfusion culture was developed to eliminate substrate (glucose) limitation and by-product inhibition simultaneously. Using an exponential feed strategy and manipulating glucose supply rate and by-product removal rate, the optimal glucose concentration in the feed medium was determined to be 50 g l −1. With this feed glucose concentration ( S 0=50 g l −1), the perfusion culture resulted to a cell dry weight concentration of 40 g l −1. Such a high cell density led to an EPA yield of 1112 mg l −1, which is the highest ever reported in microalgal cultures. In addition, the perfusion culture (at S 0=50 g l −1) greatly reduced the amount of wastewater (broth) during cultures. Considering the simple set-up and operation, the proposed perfusion culture is potentially useful for large scale EPA production by N. laevis.

Excretion of human beta-endorphin into culture medium by using outer membrane protein F as a fusion partner in recombinant Escherichia coli.

Escherichia coli BL21 strains were found to excrete a large amount of outer membrane protein F (OmpF) into culture medium during high-cell-density cultivation. From this interesting phenomenon, a novel and efficient OmpF fusion system was developed for the excretion of recombinant proteins by E. coli. The ompF gene of E. coli BL21(DE3) was first knocked out by using the red operon of bacteriophage lambda to construct E. coli MBEL-BL101. For the excretion of human beta-endorphin as a model protein, the beta-endorphin gene was fused to the C terminus of the E. coli ompF gene by using a linker containing the Factor Xa recognition site. To develop a fed-batch culture condition that allows efficient production of OmpF-beta-endorphin fusion protein, three different feeding strategies, an exponential feeding strategy and two pH-stat strategies with defined and complex nutrient feeding solutions, were examined. Among these, the pH-stat feeding strategy with the complex nutrient feeding solution resulted in the highest productivity (0.33 g of protein per liter per h). Under this condition, up to 5.6 g of OmpF-beta-endorphin fusion protein per liter was excreted into culture medium. The fusion protein was purified by anion-exchange chromatography and cleaved by Factor Xa to yield beta-endorphin, which was finally purified by reverse-phase chromatography. From 2.7 liters of culture supernatant, 545.4 mg of beta-endorphin was obtained.

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.

Influence of pH on the xylose reductase activity of Candida guilliermondii during fed-batch xylitol bioproduction

The influence of pH on the xylose reductase (XR) activity was studied. The fed-batch fermentation using exponential feeding rate was employed to produce xylitol. The feeding started when the xylose concentration in the fermenter reached 50 g/l and the pH was adjusted to 2.5, 4.0 or 6.0. The best results for XR activity (0.567 U/mg(protein)) and xylitol volumetric productivity (1.06 g/l.h) were achieved with pH 6.0.

Influence of pH on the xylose reductase activity of Candida guilliermondii during fed-batch xylitol bioproduction.

The influence of pH on the xylose reductase (XR) activity was studied. The fed-batch fermentation using exponential feeding rate was employed to produce xylitol. The feeding started when the xylose concentration in the fermenter reached 50 g/l and the pH was adjusted to 2.5, 4.0 or 6.0. The best results for XR activity (0.567 U/mg(protein)) and xylitol volumetric productivity (1.06 g/l.h) were achieved with pH 6.0.

Introduction of the carbohydrate-activated promoter P(malK) for recombinant protein production.

A production protocol for the use of the malK promoter was established. The protocol includes two phases: an initial fed-batch phase on glucose to reach a high cell density and a fed-batch phase on maltose for production of the desired recombinant protein. It is suggested that this cultivation scheme could be used for all promoters that are catabolite repressed by glucose and where growth and production need to be separated. The specific feature of this system is shown by its ability to control the rate of synthesis of the product protein, ss-galactosidase. In the production phase with a constant feed or an exponential feeding of 0.1 h(-1) it took 4 h longer to reach the maximum specific production rate than with the higher dilution rates of 0.25 h(-1) and 0.4 h(-1), respectively. In the above experiments a dilution rate of 0.3 h(-1) in the growth phase was used. The volumetric production of this system could furthermore be extended to 40 h. All protocol procedures so far tested resulted in the same maximum production rate, but reached in different lengths of time. It is argued that this system is particularly well suited for the production of proteins that have a complex structure and/or need to be produced in a soluble form or to be exported to the periplasm.

Importance of specific growth rate for subtilisin expression in fed-batch cultivation of Bacillus subtilis spoIIG mutant

A feeding strategy was developed for controlling the specific growth rate in a Bacillus subtilis fed-batch operation, and the importance of the specific growth rate was investigated for subtilisin expression. The ratio of the glucose and peptone concentrations in the feeding medium was critical in controlling the specific growth rate, and the appropriate ratio was found to be 1:2. The specific growth rate was well controlled in fed-batch operations including an exponential feeding of a 200 g/liter glucose and 400 g/liter peptone solution. Controlling the value of the specific growth rate had a significant effect on the subtilisin expression, even though the subtilisin expression was only enhanced after the feeding ended. This would appear to be due to cell components, such as sigma factors, produced in the growth phase that influenced the aprE gene expression. The optimal specific growth rate for the maximum expression rate and longest production period was 0.35 h −1.

Cloning, characterization ofPichia etchellsii β-glucosidase II and effect of media composition and feeding strategy on its production in a bioreactor

The cloning and expression of β-glucosidase II, encoded by the genesglu2, from thermotolerant yeastPichia etchellsii intoEscherichia coli is described. Cloning of the 7.3 kbBamHI/SalI yeast insert containingsglu2 in pUC18, which allowed for reverse orientation of the insert, resulted in better enzyme expression. Transformation of this plasmid intoE. coli JM109 resulted in accumulation of the enzyme in periplasmic space. At 50°C, the highest hydrolytic activity of 1686 IU/g protein was obtained on sophorose. Batch and fed-batch techniques were employed for enzyme production in a 14 L bioreactor. Exponential feeding rates were determined from mass balance equations and these were employed to control specific growth rate and in turn maximize cell growth and enzyme production. Media optimization coupled with this strategy resulted in increased enzyme units of 1.2 kU/L at a stabilized growth rate of 0.14 h−1. Increased enzyme production in bioreactor was accompanied by formation of inclusion bodies.

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.

Automated fed-batch culture of Kluyveromyces fragilis based on a novel method for on-line estimation of cell specific growth rate

A corrected feed-forward control strategy was developed for automated substrate feeding in aerobic fed-batch cultures. The control strategy incorporates a novel method for on-line estimation of specific growth rate as a performance indicator of the fed-batch culture. The estimation is based on the measurement of the maximum substrate uptake rate (MSUR) using on-line dissolved oxygen (DO) concentration data and mass balances. It allows the controller to track changes in the specific growth rate to compensate for process disturbances. The control strategy was applied to fed-batch culture of Kluyveromyces fragilis to maximize the volumetric productivity of lactase. A maximum volumetric lactase productivity of 2.98 U/ml h was achieved. The control was shown to be stable throughout the culture period even at the highest cell density of 69 g/l. This technique is applicable to various aerobic systems, especially for microbial cultures showing high specific oxygen uptake rates, since it only requires on-line measurement of DO concentration without the need for off-gas analysis. In a comparison carried out with three other fed-batch control strategies (DO-stat, exponential feeding and exponential feeding with manual feedback control), the corrected feed-forward control strategy exhibited the best performance by achieving the highest volumetric lactase and biomass productivity.

High-level secretory production of human granulocyte-colony stimulating factor by fed-batch culture of recombinant Escherichia coli

Secretory production of human granulocyte colony-stimulating factor fusion protein (hG-CSF) by fed-batch culture of Escherichia coli was investigated in both 2.5-L and 30-L fermentors. To develop a fed-batch culture condition that allows efficient production of hG-CSF, different feeding strategies including pH-stat, exponential and constant feeding were examined. Among these, the constant feeding strategy (0.228 g glucose×min–1) and the exponential feeding that supports a low specific growth rate (µ=0.116 h–1) resulted in the best hG-CSF production. Under these conditions, 4.4 g×L–1 of hG-CSF was produced. The effect of induction time on the protein production was also investigated. For the fed-batch cultures carried out with the pH-stat and exponential feeding strategies, induction at higher cell density (late-exponential phase) resulted in more hG-CSF production compared with induction at lower cell density (early to mid-exponential phase). The constant feeding strategy that supported best hG-CSF production was applied to the scale-up production of hG-CSF in 30 L of fermentor. The maximum dry cell weight and hG-CSF concentration of 51.7 and 4.2 g×L–1, respectively, was obtained.

Enhanced Expression of the Recombinant Lethal Factor of Bacillus anthracis by Fed-Batch Culture

High cell density cultivation has been one of the most effective ways to increase cell as well as the product yields. The structural gene for the 90-kDa lethal factor (LF) isolated from Bacillus anthracis was expressed as fusion protein with 6× histidine residues under the transcriptional regulation of the T5 promoter in Escherichia coli. Various strategies were tried to scale up the expression of the recombinant lethal factor by bioprocess optimization using fed batch culture technique in a 14 litre fermentor. The media, a defined mixture of salts, trace elements, vitamins, etc. along with a specified carbon source was used for the growth. The pH of the media was maintained at 6.8 while the temperature was changed from 37 to 28°C during the cultivation. During the growth and induction phases, the DO was maintained above 20% by automatic control of agitation. The specific growth rate was controlled by utilizing an exponential feeding profile determined from mass balance equations. As a result of control of specific growth rate at two different levels, there was about twenty five fold increase in biomass compared to the biomass in the shake flask. E. coli cells yielded a soluble cytosolic protein with an apparent molecular mass of 90 kDa. The protein was purified to homogeneity using metal chelate affinity chromatography, followed by anion exchange on FPLC using Mono-Q column. In solution, trypsin cleaved protective antigen bound to native and recombinant LF with comparable affinity. The recombinant LF resembled the LF purified from B. anthracis in the macrophage lysis assay, using a murine macrophage cell line J774A.1 sensitive to anthrax toxin. It was possible to achieve a yield of 50 mg of the purified protein from 1 litre culture broth.

An Integrated System for Advanced Monitoring and Control of Fed-Batch Fermevtations of Recombinant E. coli

A system for the on-line monitoring of the major state variables during the production of recombinant proteins with E. coli was developed. Substrate (glucose) and the major by-product (acetate) were measured with a Flow Injection Analysis system, while the carbon dioxide and oxygen transfer rates were calculated from data obtained with exhaust gas analysis. The fermentation culture weight was also continuously assessed with a balance placed below the fermenter, allowing the use of more precise mass-based concentrations. The graphical programming environment LabVIEW was used to acquire and integrate these variables in a supervisor computer, allowing to perform integrated monitoring and control of the process. This framework is illustrated with different experiments for model calibration, state estimation by software sensors and process performance optimisation by exponential feed control.

Improvement of intracellular β-galactosidase production in fed-batch culture of Kluyveromyces fragilis

Four fed-batch control strategies were evaluated to improve the specific lactase activity of Kluyveromyces fragilis. Control strategies tested included DO-stat control, exponential feeding, exponential feeding with manual feedback control and corrected feed-forward control. Each was implemented with standard sensors (i.e., temperature, dissolved oxygen and pH sensors) commonly installed in fermenters. The highest specific activity was obtained using the corrected feed-forward control strategy, a strategy incorporating a novel method for on-line estimation of specific growth rate. The control strategy was able to operate effectively to a final cell density of 69 g dry wt l−1 with a specific lactase activity of 2 U mg−1 cell dry wt.

Fed-batch culture of insect cells with exponential feeding of amino acid and yeastolate solution

Amino acids rather than sugars are the primary limiting substrates for the culture of insect cells in a Grace's medium. When cultures are supplemented with amino acids, the yeastolate components other than the amino acids become the secondary limiting substrates. For the fed-batch culture of insect cells, a solution containing concentrated amino acids and yeastolate was supplied using an exponential feed flow rate calculated from mass balance equations. During the batch period the specific growth rate was 0.02 h−1, whereas during the fed-batch period it was measured as 0.007 and 0.012 h−1 on the basis of the cell numbers and the dry cell weight, respectively. This difference in the specific growth rates in the fed-batch period is caused by an increase in the cell size during this period. Furthermore, in fed-batch cultures, dissolved oxygen was found to be a limiting factor for high cell-density cultures.

Mass production of Rhodopseudomonaspalustris as diet for aquaculture

Three different types of anaerobic fermentations were used for the mass production of the photosynthetic bacterium Rhodopseudomonas palustris as diet for aquaculture. The optimum agitation speed and malate concentration were 300 r.p.m. and 0.2% in the modified MYC medium, respectively. In batch fermentations of R. palustris, the maximum number of viable cells was 1.1×10 10 c.f.u. ml −1 with 2.65 g l −1 of DCW, and the maximum specific growth rate and biomass productivity were estimated to be 0.12 h −1 and 55 mg l −1 h −1, respectively. Crude protein content of R. palustris was about 72–74%. The composition of stearic acid and oleic acid of R. palustris was superior to those of Chlorella and yeasts, while that of other fatty acids tested was not. The amino acid profiles of the protein hydrolysate compared favorably with Food Agricultural Organization (FAO) guidelines. The biomass productivities from fed-batch experiments were found to be 50, 47 and 49 mg l −1 h −1 for linear, exponential, and sigmoidal feeding strategy, respectively. The maximum biomass productivity was found to be 112 mg l −1 h −1 in chemostat. Compared to growth in batch cultures, continuous fermentation yielded two times higher biomass productivity.

Mixed-feed exponential feeding for fed-batch culture of recombinant methylotrophic yeast

A simple, accurate model capable of predicting cell growth and methanol utilization during the mixed substrate fed-batch fermentation of Mut S recombinant Pichia pastoris was developed and was used to design an exponential feeding strategy for mixed substrate fed-batch fermentation at a constant specific growth rate. Mixed substrate feeding has been shown to boost productivity in recombinant fed-batch culture of P. pastoris, while fixed growth rate exponential feeding during fed-batch culture is a useful tool in process optimization and control.