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Analysis of oxygen transport enhancement by functionalized magnetic nanoparticles (FMP) in biopolymer production processes
New Biotechnology · Volume 25S · September 2009
ABSTRACTS
2.6.034
2.6.035
Analysis of oxygen transport enhancement by functionalized magnetic nanoparticles (FMP) in biopolymer production processes
Use of yeast extract to achieve recombinant protein expression under lacUV5 promoter without addition of any specific inducer: possible industrial applications
F. Ataíde ∗ , F. Freitas, M.A. Miranda Reis, R. Oliveira
S. Padmanabhan ∗ , R. Nair, S. Soorapaneni, P. Salvi, P. Kotwal, V. Raiker, S. Banerjee
REQUIMTE, Departamento de Química, Faculdade de Ciências e Tecnologia, Universidade Nova de Lisboa, Portugal
The central goal of this study is to investigate if functionalized magnetic nanoparticles (FMP) can be utilized to enhance oxygen transport in highly viscous non-Newtonian microbial fermentation systems for biopolymer production and which operational variables are critical for this enhancement. FMP have a magnetic core (easy recovery by magnetic filtration) and a hydrophobic coating (usually surfactants or polymers that provide colloidal stability to the particle). In fermentations, when the biopolymer is extracellular, the broth apparent viscosity increases exponentially with the accumulation of polymer in the liquid phase. The main consequence is that the oxygen transfer becomes a severe limitation factor of the biological transformations. The hypothesis investigated here, is if the use of FMP may extend the time window of effective oxygen supply and therewith the productivity, yield and final polymer concentration. The use of FMP in Escherichia coli cultivations was shown to enhance oxygen transport by 40—60% but the underlying mechanisms for transfer enhancement are not yet fully understood. Silica-coated FMPs were applied to Pseudomonas oleovorans fedbatch cultures that produce an extracellular polysaccharide (EPS), using crude glycerol byproduct from the biodiesel industry as substrate. At the final stage of the culture, apparent viscosity can be as high as 1600 cP at low shear rates. Oxygen transport experiments in water/FMP/air dispersions and EPS solutions/FMP/air dispersions were performed. The experimental set-up consisted of a 2 l stirred tank bioreactor equipped with a dissolved oxygen electrode and an off-gas oxygen analyzer. Experiments were started by N2 sparging to strip out all the dissolved oxygen. Then, aeration was applied and the dynamics of oxygen concentration in the liquid and exhaust gas were recorded. In addition, to mimic fermentation conditions, and consequently collect more accurate mass transfer coefficient data, it was used the oxidation of sodium sulfite method to simulate oxygen consumption by cells. Also, dynamic surface tension measurements were collected to establish the surface activity of the FMP dispersions used. Several experiments were conducted changing EPS concentration (0—10 g/L), aeration rate (0.5—4 vvm), stirrer speed (200—600 rpm) and particles loading (0.5—5% (v/v)). The collected data was analyzed by several models. The most likely enhancement mechanisms are the increase of interfacial area by either inhibiting bubble coalescence or facilitating bubble breakup due to surface tension reduction. The results with EPS solutions suggest that random Brownian motion, causing convection in the surrounding fluid, is also a determinant mechanism in highly viscous fermentation broths.
Lupin Limited, Pune, India
T7 RNA polymerase of inducible expression systems under the control of T7lacUV5 promoter is commonly used to produce a wide variety of recombinant proteins in E. coli. We demonstrate a simple and cost-effective auto inducible media for hyper-expression of proteins in E. coli under this promoter system. High content of yeast extract, a regular component of complex fermentation media for bacterial production of recombinant proteins, is usually used for achieving high cell density in fermentation processes. Yeast extract at high concentration was found to stimulate the expression of T7 RNA polymerase in BL21 (DE3) cells while such an effect was not seen in BL21A1 cells. A systematic study on the effect of varying concentrations of yeast extract indicated several folds higher expression of genes namely, human granulocyte colony stimulating factor (rhGCSF), human interferon alpha 2b (rhIFN␣2b) and staphylokinase (rSAK) in BL21(DE3) cells as estimated by ELISA and activity assays. In all the cases, no specific inducer like IPTG or lactose was added. Additional investigations on the inducible component of the yeast extract and time of induction showed potential use of this component for high-level expression of recombinant proteins without induction. Inclusion of cAMP inhibitors did not affect the expression of proteins ruling out the possibility of cAMP dependent derepression. Yeast extract preparations are often rich in amino acids, peptides, nucleotides and carbohydrates and we report herein for the first time that lactose, a well-known inducer for lacUV5 promoter, is present as one of the contaminants in certain commercial preparations of yeast extract (∼0.004% lactose/gm of yeast extract) and this concentration of lactose is sufficient to induce expression of proteins under this promoter. It is also interesting to note that various brands of yeast extract showed variable levels of protein expression possibly due to differences in ratio of lactose to glucose (lacUV5 repressor) content as tested by methylamine hydrochloride method. Such an auto induction method is free from plasmid loss and offers easy and efficient screening of recombinant clones for higher expression resulting in a cost-effective fermentation process as compared to the conventional IPTG induction. This paper highlights the easy scalability of use of the present media component for large scale manufacturing in biotechnology industry. doi:10.1016/j.nbt.2009.06.141
doi:10.1016/j.nbt.2009.06.140
www.elsevier.com/locate/nbt S203
ABSTRACTS
2.6.034
2.6.035
Analysis of oxygen transport enhancement by functionalized magnetic nanoparticles (FMP) in biopolymer production processes
Use of yeast extract to achieve recombinant protein expression under lacUV5 promoter without addition of any specific inducer: possible industrial applications
F. Ataíde ∗ , F. Freitas, M.A. Miranda Reis, R. Oliveira
S. Padmanabhan ∗ , R. Nair, S. Soorapaneni, P. Salvi, P. Kotwal, V. Raiker, S. Banerjee
REQUIMTE, Departamento de Química, Faculdade de Ciências e Tecnologia, Universidade Nova de Lisboa, Portugal
The central goal of this study is to investigate if functionalized magnetic nanoparticles (FMP) can be utilized to enhance oxygen transport in highly viscous non-Newtonian microbial fermentation systems for biopolymer production and which operational variables are critical for this enhancement. FMP have a magnetic core (easy recovery by magnetic filtration) and a hydrophobic coating (usually surfactants or polymers that provide colloidal stability to the particle). In fermentations, when the biopolymer is extracellular, the broth apparent viscosity increases exponentially with the accumulation of polymer in the liquid phase. The main consequence is that the oxygen transfer becomes a severe limitation factor of the biological transformations. The hypothesis investigated here, is if the use of FMP may extend the time window of effective oxygen supply and therewith the productivity, yield and final polymer concentration. The use of FMP in Escherichia coli cultivations was shown to enhance oxygen transport by 40—60% but the underlying mechanisms for transfer enhancement are not yet fully understood. Silica-coated FMPs were applied to Pseudomonas oleovorans fedbatch cultures that produce an extracellular polysaccharide (EPS), using crude glycerol byproduct from the biodiesel industry as substrate. At the final stage of the culture, apparent viscosity can be as high as 1600 cP at low shear rates. Oxygen transport experiments in water/FMP/air dispersions and EPS solutions/FMP/air dispersions were performed. The experimental set-up consisted of a 2 l stirred tank bioreactor equipped with a dissolved oxygen electrode and an off-gas oxygen analyzer. Experiments were started by N2 sparging to strip out all the dissolved oxygen. Then, aeration was applied and the dynamics of oxygen concentration in the liquid and exhaust gas were recorded. In addition, to mimic fermentation conditions, and consequently collect more accurate mass transfer coefficient data, it was used the oxidation of sodium sulfite method to simulate oxygen consumption by cells. Also, dynamic surface tension measurements were collected to establish the surface activity of the FMP dispersions used. Several experiments were conducted changing EPS concentration (0—10 g/L), aeration rate (0.5—4 vvm), stirrer speed (200—600 rpm) and particles loading (0.5—5% (v/v)). The collected data was analyzed by several models. The most likely enhancement mechanisms are the increase of interfacial area by either inhibiting bubble coalescence or facilitating bubble breakup due to surface tension reduction. The results with EPS solutions suggest that random Brownian motion, causing convection in the surrounding fluid, is also a determinant mechanism in highly viscous fermentation broths.
Lupin Limited, Pune, India
T7 RNA polymerase of inducible expression systems under the control of T7lacUV5 promoter is commonly used to produce a wide variety of recombinant proteins in E. coli. We demonstrate a simple and cost-effective auto inducible media for hyper-expression of proteins in E. coli under this promoter system. High content of yeast extract, a regular component of complex fermentation media for bacterial production of recombinant proteins, is usually used for achieving high cell density in fermentation processes. Yeast extract at high concentration was found to stimulate the expression of T7 RNA polymerase in BL21 (DE3) cells while such an effect was not seen in BL21A1 cells. A systematic study on the effect of varying concentrations of yeast extract indicated several folds higher expression of genes namely, human granulocyte colony stimulating factor (rhGCSF), human interferon alpha 2b (rhIFN␣2b) and staphylokinase (rSAK) in BL21(DE3) cells as estimated by ELISA and activity assays. In all the cases, no specific inducer like IPTG or lactose was added. Additional investigations on the inducible component of the yeast extract and time of induction showed potential use of this component for high-level expression of recombinant proteins without induction. Inclusion of cAMP inhibitors did not affect the expression of proteins ruling out the possibility of cAMP dependent derepression. Yeast extract preparations are often rich in amino acids, peptides, nucleotides and carbohydrates and we report herein for the first time that lactose, a well-known inducer for lacUV5 promoter, is present as one of the contaminants in certain commercial preparations of yeast extract (∼0.004% lactose/gm of yeast extract) and this concentration of lactose is sufficient to induce expression of proteins under this promoter. It is also interesting to note that various brands of yeast extract showed variable levels of protein expression possibly due to differences in ratio of lactose to glucose (lacUV5 repressor) content as tested by methylamine hydrochloride method. Such an auto induction method is free from plasmid loss and offers easy and efficient screening of recombinant clones for higher expression resulting in a cost-effective fermentation process as compared to the conventional IPTG induction. This paper highlights the easy scalability of use of the present media component for large scale manufacturing in biotechnology industry. doi:10.1016/j.nbt.2009.06.141
doi:10.1016/j.nbt.2009.06.140
www.elsevier.com/locate/nbt S203