Fed-batch Approach Research Articles

Production of the short peptide surfactant DAMP4 from glucose or sucrose in high cell density cultures of Escherichia coli BL21(DE3).

BackgroundPeptides are increasingly used in industry as highly functional materials. Bacterial production of recombinant peptides has the potential to provide large amounts of renewable and low cost peptides, however, achieving high product titers from Chemically Defined Media (CDM) supplemented with simple sugars remains challenging.ResultsIn this work, the short peptide surfactant, DAMP4, was used as a model peptide to investigate production in Escherichia coli BL21(DE3), a classical strain used for protein production. Under the same fermentation conditions, switching production of DAMP4 from rich complex media to CDM resulted in a reduction in yield that could be attributed to the reduction in final cell density more so than a significant reduction in specific productivity. To maximize product titer, cell density at induction was maximized using a fed-batch approach. In fed-batch DAMP4 product titer increased 9-fold compared to batch, while maintaining 60% specific productivity. Under the fed-batch conditions, the final product titer of DAMP4 reached more than 7 g/L which is the highest titer of DAMP4 reported to date. To investigate production from sucrose, sucrose metabolism was engineered into BL21(DE3) using a simple plasmid approach. Using this strain, growth and DAMP4 production characteristics obtained from CDM supplemented with sucrose were similar to those obtained when culturing the parent strain on CDM supplemented with glucose.ConclusionsProduction of a model peptide was increased to several grams per liter using a CDM medium with either glucose or sucrose feedstock. It is hoped that this work will contribute cost reduction for production of designer peptide surfactants to facilitate their commercial application.Electronic supplementary materialThe online version of this article (doi:10.1186/s12934-014-0099-y) contains supplementary material, which is available to authorized users.

Kinetics of enzymatic hydrolysis of olive oil in batch and fed-batch systems.

This work reports experimental data, kinetic modeling, and simulations of enzyme-catalyzed hydrolysis of olive oil. This reaction was performed in batch system and an ordered-sequential Bi Bi model was used to model the kinetic mechanism. A fed-batch system was proposed and experimental data were obtained and compared to the simulated values. The kinetic model used was able to correlate the experimental data, in which a satisfactory agreement between the experimental data and modeling results was obtained under different enzyme concentration and initial free water content. Therefore, the modeling allowed a better understanding of the reaction kinetics and affords a fed-batch simulation for this system. From the results obtained, it was observed that the fed-batch approach showed to be more advantageous when compared to the conventional batch system.

Improving adeno‐associated vector yield in high density insect cell cultures

Recombinant adeno-associated virus (rAAV) are the most promising vectors for gene therapy. However, large-scale rAAV production remains a challenge for the translation of rAAV-based therapeutic strategies to the clinic. The baculovirus expression vector system (BEVS) has been engineered to produce high rAAV titers in serum-free suspension cultures of insect cells. The typical approach of rAAV production in BEVS has been based on a synchronous infection with three baculoviruses at high multiplicity of infection (MOI) [>3 plaque forming units (pfu)/cell]. An alternative approach is to co-infect at low MOI (0.1 pfu/cell). Both strategies (high and low MOI) were compared at a cell density of 1.0 x 10(6) cells/ml in shake-flask experiments. To increase the rAAV titer, a low MOI combined with an initial cell density at infection of 5.0 x 10(6) cells/ml, in fed-batch mode, was evaluated. Subsequently, the production strategy was validated in 3-l bioreactor runs. An increase of 210% in the rAAV titer (4.7 x 10(11) enhanced transduction units/l) was observed when using low MOI, an effect primarily caused by the increase in cell density. The fed-batch approach resulted in a seven-fold increase of rAAV yield. Controlled operations in bioreactor contributed to further increase the rAAV yield (2.8 x 10(14) vector genomes/l) by 25% in comparison to the shake flask results. This high yield production process using low MOIs and a feeding strategy successfully addresses several limitations of current rAAV production in insect cells and contributes to position the BEVS system as one of the most efficient for large-scale manufacturing of rAAV vectors.

The First 200-L Scale Asymmetric Baeyer−Villiger Oxidation Using a Whole-Cell Biocatalyst

Biocatalytic Baeyer−Villiger oxidations using oxygen as an environmentally friendly oxidant in aqueous media have been shown to proceed with excellent stereo- and enantioselectivity for a large number of substrates at laboratory scale. These are good starting boundary conditions for process research and development compared to systems with reactive oxidants and flammable organic solvents. In this paper we discuss some of the considerations required to scale up a whole-cell biocatalytic oxidation from the laboratory to pilot-plant (200 L) scale. Issues for fermentation, bioconversion, and product recovery are discussed, supported by data from pilot-plant and scale-down experimentation. A simple fed-batch approach has been used.

An aqueous–organic two-phase bioprocess for efficient production of the natural aroma chemicals 2-phenylethanol and 2-phenylethylacetate with yeast

The natural aroma chemicals 2-phenylethanol (2-PE) and 2-phenylethylacetate (2-PEAc) are of high industrial relevance and can be produced from L-phenylalanine in a yeast-based process with growth-associated product formation. Due to product inhibition, in situ product removal is mandatory to obtain economically interesting concentrations. A fed-batch approach using polypropylene glycol 1200 as in situ extractant and the precursor in a saturated concentration led to the highest 2-PE productivity reported for a bioprocess so far. With Kluyveromyces marxianus CBS 600, 26.5 g/l 2-PE and 6.1 g/l 2-PEAc in the organic phase were obtained, corresponding to space-time yields of 0.33 and 0.08 g/l h, respectively.

A comparison between batch and fed-batch simultaneous saccharification and fermentation of steam pretreated spruce

In order to improve the process economy it is important to use as high dry matter content as possible in simultaneous saccharification and fermentation (SSF). However, too high dry matter content often gives rise to severe inhibition of the yeast metabolism, due to the increased levels of toxic compounds. The aim of the present work was to increase the fibrous content in SSF of steam pretreated spruce to 10% by adapting the yeast to the inhibitory substrate and by using a fed-batch process. Both batch and fed-batch approaches were evaluated. The fed-batch experiments were started with a batch fermentation containing 6% dry matter. Fibrous slurry from the pretreatment was then added four times during the first 24 h giving a final dry matter content corresponding to 10%. The yeast used in the fermentation was produced aerobically on the hemicellulose hydrolysate obtained from the pretreatment. SSF batch and fed-batch experiments with a cell mass concentration of 2, 3 and 5 g/L were carried out. When adapted yeast was used, the available hexoses were completely converted within 72 h and the final ethanol concentrations reached 40–44 g/L. No major differences in performance between batch and fed-batch were seen, but the ethanol productivity during the first 24 h was higher in the fed-batch SSF experiments, particularly during the experiments with a cell mass concentration of 2 and 3 g/L.

Optimisation of media and cultivation conditions for L(+)(S)-lactic acid production by Lactobacillus casei NRRL B-441.

Process variables and concentration of carbon in media were optimised for lactic acid production by Lactobacillus casei NRRL B-441. Lactic acid yield was inversely proportional to initial glucose concentration within the experimental area (80-160 g l(-1)). The highest lactic acid concentration in batch fermentation, 118.6 g l(-1), was obtained with 160 g 1(-1) glucose. The maximum volumetric productivity, 4.4 g 1(-1) h(-1) at 15 h, was achieved at an initial glucose concentration of 100 g l(-1). Similar lactic acid concentrations were reached with a fedbatch approach using growing cells, in which case the fermentation time was much shorter. Statistical experimental design and response surface methodology were used for optimising the process variables. The temperature and pH optima for lactic acid production were 35 degrees C, pH 6.3. Malt sprout extract supplemented with yeast extract (4 g l(-1)) appeared to be an economical alternative to yeast extract alone (22 g l(-1)) although the fermentation time was a little longer. The results demonstrated both the separation of the growth and lactic acid production phases and lactic acid production by non-growing cells without any nutrient supplements. Resting L. casei cells converted 120 g l(-1) glucose to lactic acid with 100% yield and a maximum volumetric productivity of 3.5 g l(-1) h(-1).

Biodegradation of phenol at high initial concentrations in two-phase partitioning batch and fed-batch bioreactors

A two-phase organic-aqueous system was used to degrade phenol in both batch and fed-batch culture. The solvent, which contained the phenol and partitioned it into the aqueous phase, was systematically selected based on volatility, solubility in the aqueous phase, partition coefficient for phenol, biocompatibility, and cost. The two-phase partitioning bioreactor used 500 mL of 2-undecanone loaded with high concentrations of phenol to deliver the xenobiotic to Pseudomonas putida ATCC 11172 in the 1-L aqueous phase, at subinhibitory levels. The initial concentrations of phenol selected for the aqueous phase were predicted using the experimentally determined partition coefficient for this ternary system of 47.6. This system was initially observed to degrade 4 g of phenol in just over 48 h in batch culture. Further loading of the organic phase in subsequent experiments demonstrated that the system was capable of degrading 10 g of phenol to completion in approximately 72 h. The higher levels of phenol in the system caused a modest increase in the duration of the lag phase, but did not lead to complete inhibition or cell death. The use of a fed-batch approach allowed the system to ultimately consume 28 g of phenol in approximately 165 h, without experiencing substrate toxicity. In this system, phenol delivery to the aqueous phase is demand based, and is directly related to the metabolic activity of the cells. This system permits high loading of phenol without the corresponding substrate inhibition commonly seen in conventional bioreactors.

Optimization of cell yield of Candida krusei SO1 and Saccharomyces sp. LK3G cultured in sorghum hydrolysate

The fed-batch approach to the production of single-cell protein (SCP) from synthetic glucose medium or sorghum hydrolysate by Candida krusei SO1 and Saccharomyces sp. LK3G was studied in aerobic conditions. The results obtained with a synthetic glucose showed better growth parameters under fed-batch conditions than under batch conditions. Biomass yield ( Y x s ) was increased by as much as 64% for Candida krusei and for Saccharomyces sp. LK3G by as much as 70%. Volumetric cell productivity was also improved for both yeasts: 0.75 g/l.h in fed-batch conditions against 0.68 g/l.h in batch conditions for Candida krusei SO1 and 0.63 g/l.h against 0.56 g/l.h for Saccharomyces sp. LK3G. Chemical analyses of yeast cells grown on either synthetic medium or on sorghum hydrolysate showed that the fed-batch process did not alter the nutritional quality of either yeast strain: in these conditions their protein content (47–50%) and their amino acid profiles compared favourably with the Food and Agricultural Organization references. Furthermore, both yeast strains were good sources of mineral salts.

High production of 1,3-propanediol from glycerol by Clostridium butyricum VPI 3266 in a simply controlled fed-batch system

A simple fed-batch system which controls substrate feeding by measuring the CO2 produced during the fermentation, was developped. This Fed-batch approach allowed high production of 1,3-propanediol from glycerol by Clostridium butyricum by avoiding substrate inhibition phenomena. 65 g/l of 1,3-propanediol was produced with a productivity of 1.21 g/l.h and a yield of 0.56. The concentration of 1,3-propanediol obtained and the productivity were significantly higher than those reached in batch culture.

Monoclonal antibody process development using medium concentrates.

A fed-batch process using concentrated medium was evaluated for its ability to improve cell culture longevity and final monoclonal antibody (MAb) titers for two monoclonal antibody producing cell lines. It was found to result in up to 7-fold increases in final antibody titers compared to batch culture controls. Although the development cell line specific fed-batch protocols is critical to the development of cost-efficient large-scale production processes, the use of complete medium concentrates provided us with a quick and simple method for producing large quantities of antibodies in the early stages of process development, thus accelerating early work on purification process development, analytical development, biochemical characterization, and safety studies. Insights gained from the concentrated medium fed-batch approach were valuable for the development of refined, cell line specific feeding strategies yielding final MAb titers on the order of 1-2 g/L. Process development data on the effects of inhibitory growth byproducts, medium osmolarity, and the mode of nutrient feed addition on culture longevity and MAb production and information on culture metabolic behavior were successfully incorporated in the development of the optimized fed-batch protocols.

Fed-batch fermentation of glucose to acetate by an improved strain ofClostridium thermoaceticum

The fed-batch approach to the production of acetate from glucose by an improved strain ofClostridium thermoaceticum resulted in better performance than the batch fermentation, especially in media containing an excess (3X) of nutrients and trace salts. At pH 6.6, 46 g/l acetic acid was produced in 192 hours with 93% substrate utilization. In contrast, batch fermentation under similar conditions resulted in a maximum of 35 g/l acetic acid with less than 82% substrate utilization.

A simple device for fed‐batch control in alcoholic fermentation

Biotechnology and BioengineeringVolume 29, Issue 6 p. 775-777 Communications to the EditorFree Access A simple device for fed-batch control in alcoholic fermentation M. Mota, M. Mota University of Porto, PortugalSearch for more papers by this authorJ. M. Besle, J. M. Besle Institut National des Sciences Appliquées, UA CNRS 544, Avenue Rangueil, 31077 Toulouse Cédex, FranceSearch for more papers by this authorP. Strehaiano, P. Strehaiano Institut National des Sciences Appliquées, UA CNRS 544, Avenue Rangueil, 31077 Toulouse Cédex, FranceSearch for more papers by this authorG. Goma, Corresponding Author G. Goma Institut National des Sciences Appliquées, UA CNRS 544, Avenue Rangueil, 31077 Toulouse Cédex, FranceInstitut National des Sciences Appliquées, UA CNRS 544, Avenue Rangueil, 31077 Toulouse Cédex, FranceSearch for more papers by this author M. Mota, M. Mota University of Porto, PortugalSearch for more papers by this authorJ. M. Besle, J. M. Besle Institut National des Sciences Appliquées, UA CNRS 544, Avenue Rangueil, 31077 Toulouse Cédex, FranceSearch for more papers by this authorP. Strehaiano, P. Strehaiano Institut National des Sciences Appliquées, UA CNRS 544, Avenue Rangueil, 31077 Toulouse Cédex, FranceSearch for more papers by this authorG. Goma, Corresponding Author G. Goma Institut National des Sciences Appliquées, UA CNRS 544, Avenue Rangueil, 31077 Toulouse Cédex, FranceInstitut National des Sciences Appliquées, UA CNRS 544, Avenue Rangueil, 31077 Toulouse Cédex, FranceSearch for more papers by this author First published: 20 April 1987 https://doi.org/10.1002/bit.260290619Citations: 11AboutPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL References 1 M. Mota, P. Strehaiano, and G. Goma, J. Inst. Brew., 90, 359 (1984). 2 W. Woherer and M. Roehr, Biotechnol. Bioeng., 23, 567 (1981). 3 K. Dairaku, E. Izumoto, H. Morikawa, S. Shioya, and T. Takamatsu, J. Ferment. Technol., 61, 189 (1983). 4 R. Moletta and G. Albagnac, Biotechnol. Lett., 4, 319 (1982). 5 M. Novak, P. Strehaiano, M. Moreno, and G. Goma, Biotechnol. Bioeng., 23, 201 (1981). 6 R. W. Silman, Biotechnol. Bioeng., 26, 247 (1984). Citing Literature Volume29, Issue620 April 1987Pages 775-777 ReferencesRelatedInformation

Butanediol production from lignocellulosic substrates by Klebsiella pneumoniae grown in sequential co-culture with Clostridium thermocellum

A sequential co-culture approach was investigated for the conversion of lignocellulosic substrates to butanediol and ethanol. Growth of Clostridium thermocellum on solka floc and aspenwood xylan resulted in the release of extracellular endoglucanase and xylanase enzymes into the culture medium. Low levels of fermentation products were formed and unutilized sugars accumulated in the medium. Inoculation of Klebsiella pneumoniae as a sequential culture resulted in the rapid utilization of the accumulated sugars and the formation of additional fermentation products, including butanediol, ethanol, and acetoin. This approach was applicable to the use of mixed cellulose and hemicellulose substrates, including steam-exploded aspenwood. Further improvement in solvent production from steam-exploded substrates could be obtained by using a fed-batch approach to circumvent the problem of inhibitors associated with the natural substrates.