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Assessment of chitinase production: optimization and scale-up
New Biotechnology · Volume 29S · September 2012
study of process dynamics, a scale-down simulator of industrial installation is developed in TU-Berlin. It consists of interrelated stirred tank (STR) and plug flow (PFR) reactors where the PFR part is additionally equipped with pH and DO sensors. Nevertheless, there is still lack of on-line measurements of main process variables and parameters. A multi disciplinary team including biotechnologies from TU-Berlin and bioengineers from ISER-BAS was formed to solve this problem. The bioengineers developed a new original approach for on-line monitoring of inhomogeneous dynamics in industrial bioreactors including Cascade Software Sensors System. For this system design, a simple process model with time-varying yield coefficients is derived. Such coefficients cover the unmodeled process dynamics. For the cascade system an original tuning procedure is proposed. Data obtained in TU-Berlin including two experiments with B. subtilis are used for approbation of theoretical results. The advantages of the new approach for monitoring system design are: (1) adaptiveness, proven by tuning on the basis of one set of experimental data and verification using the other one; (2) user-friendly tuning algorithm (realized as software package in MATLAB environment) that allows by arbitrary fixing of one parameter, the optimal values of the others to be calculated automatically. From expert point of view the obtained new measurements are good enough for monitoring of process dynamics and could be applied for process intensification and control design. http://dx.doi.org/10.1016/j.nbt.2012.08.214 Poster 1.4.11 DoE based screening system for development of upscalable bioreactor cultivation for glutathione-enriched yeasts E. Lorenz, K. Neumann, S.L. Riedel ∗ , U. Stahl, J. Bader Research and Teaching Institute for Brewing in Berlin, Research Institute for Microbiology, Berlin, Germany The development of production processes is a time consuming and expensive procedure in biotechnology. Therefore a high throughput system (HTS) for screening and process optimization with integrated methodology of design of experiment (DoE) was developed to speed up the process development. We applied the methodology of DoE for the establishment of our yeast screening platform technology including the optimization of media components as amino acids, salts, vitamins, C-, Nor S-sources. The obtained results from milliliter reaction vessels were scaled up and verified with shake flask experiments and fermentations in 5 L bioreactors. For these experiments the applied yeast should be improved regarding its intracellular glutathione (L-␥-glutamyl-Lcysteinylglycine, GSH) content. GSH is responsible for the cellular redox balance, inactivation of toxic substances and reactive oxygen species (ROS) in yeasts as well as in mammals. Furthermore, it can help to improve the processing properties of baked goods in food industry. Duration of development could be drastically reduced by the application of the developed screening system. Due to usage of DoE an increase of GSH-content up to 2% of cell dry weight (CDW)
could be achieved. An improvement of space-time-yield (STY) for GSH and CDW was achieved in bioreactor processes. In sum, this research project presents an upscalable screening system for yeast cells. All determined data were statistical reliable due to the usage of the methodology of DoE. Keywords: High throughput system (HTS); Design of experiment (DoE); Glutathione; Small shaking bioreactor; Upscalable screening system http://dx.doi.org/10.1016/j.nbt.2012.08.215 Poster 1.4.12 Assessment of chitinase production: optimization and scale-up F. Moscoso 1 , M.A. Fernández de Dios 1 , M.C. Sieiro 2 , M.A. Longo 1 , M.A. Sanromán 1,∗ 1
Department of Chemical Engineering, University of Vigo, Isaac Newton Building, Campus As Lagoas, Marcosende 36310 Vigo, Spain 2 Department of Functional Biology and Health Sciencies, Microbiology Area, University of Vigo, Lagoas - Marcosende, 36310 Vigo, Spain Chitinases (E.C. 3.2.1.14) are the enzymes that can hydrolyze the -(1 → 4)-linkages of chitin. These enzymes are present in a wide range of organisms such as fungi, insects, crustaceans, bacteria and plants. Expression of chitinase enzymes in mesophilic hosts such as E. coli capable of effective secretion of heterologous proteins into the growth medium reduces the cost of production and simplifies downstream processing. In this work, a chitinase from a Lactococcus strain expressed in E. coli was used. In order to improve growth and/or favor enzyme secretion, the medium composition as well as culture conditions (i.e., pH, temperature, and aeration) was studied in shake flask cultures. Moreover, the efficiency of chitinase production was assessed using a logistic and Luedeking and Pirettype models proposed previously for other bioprocesses and the scaled-up to a 5 L-bioreactor was approached. Finally, a comparative study about the effect of the inducer used (isopropyl-beta-thio galactopyranoside (IPTG) or lactose) revealed that similar results can be obtained by addition of the lactose (15 mM), reducing the cost of the process with the same efficiency. Keywords: Chitinase; Lactococcus strain; Scaling-up Acknowledgement: This work was financed by Xunta de Galicia, Spain, project 10PXIB310278PR. http://dx.doi.org/10.1016/j.nbt.2012.08.216
www.elsevier.com/locate/nbt S77
study of process dynamics, a scale-down simulator of industrial installation is developed in TU-Berlin. It consists of interrelated stirred tank (STR) and plug flow (PFR) reactors where the PFR part is additionally equipped with pH and DO sensors. Nevertheless, there is still lack of on-line measurements of main process variables and parameters. A multi disciplinary team including biotechnologies from TU-Berlin and bioengineers from ISER-BAS was formed to solve this problem. The bioengineers developed a new original approach for on-line monitoring of inhomogeneous dynamics in industrial bioreactors including Cascade Software Sensors System. For this system design, a simple process model with time-varying yield coefficients is derived. Such coefficients cover the unmodeled process dynamics. For the cascade system an original tuning procedure is proposed. Data obtained in TU-Berlin including two experiments with B. subtilis are used for approbation of theoretical results. The advantages of the new approach for monitoring system design are: (1) adaptiveness, proven by tuning on the basis of one set of experimental data and verification using the other one; (2) user-friendly tuning algorithm (realized as software package in MATLAB environment) that allows by arbitrary fixing of one parameter, the optimal values of the others to be calculated automatically. From expert point of view the obtained new measurements are good enough for monitoring of process dynamics and could be applied for process intensification and control design. http://dx.doi.org/10.1016/j.nbt.2012.08.214 Poster 1.4.11 DoE based screening system for development of upscalable bioreactor cultivation for glutathione-enriched yeasts E. Lorenz, K. Neumann, S.L. Riedel ∗ , U. Stahl, J. Bader Research and Teaching Institute for Brewing in Berlin, Research Institute for Microbiology, Berlin, Germany The development of production processes is a time consuming and expensive procedure in biotechnology. Therefore a high throughput system (HTS) for screening and process optimization with integrated methodology of design of experiment (DoE) was developed to speed up the process development. We applied the methodology of DoE for the establishment of our yeast screening platform technology including the optimization of media components as amino acids, salts, vitamins, C-, Nor S-sources. The obtained results from milliliter reaction vessels were scaled up and verified with shake flask experiments and fermentations in 5 L bioreactors. For these experiments the applied yeast should be improved regarding its intracellular glutathione (L-␥-glutamyl-Lcysteinylglycine, GSH) content. GSH is responsible for the cellular redox balance, inactivation of toxic substances and reactive oxygen species (ROS) in yeasts as well as in mammals. Furthermore, it can help to improve the processing properties of baked goods in food industry. Duration of development could be drastically reduced by the application of the developed screening system. Due to usage of DoE an increase of GSH-content up to 2% of cell dry weight (CDW)
could be achieved. An improvement of space-time-yield (STY) for GSH and CDW was achieved in bioreactor processes. In sum, this research project presents an upscalable screening system for yeast cells. All determined data were statistical reliable due to the usage of the methodology of DoE. Keywords: High throughput system (HTS); Design of experiment (DoE); Glutathione; Small shaking bioreactor; Upscalable screening system http://dx.doi.org/10.1016/j.nbt.2012.08.215 Poster 1.4.12 Assessment of chitinase production: optimization and scale-up F. Moscoso 1 , M.A. Fernández de Dios 1 , M.C. Sieiro 2 , M.A. Longo 1 , M.A. Sanromán 1,∗ 1
Department of Chemical Engineering, University of Vigo, Isaac Newton Building, Campus As Lagoas, Marcosende 36310 Vigo, Spain 2 Department of Functional Biology and Health Sciencies, Microbiology Area, University of Vigo, Lagoas - Marcosende, 36310 Vigo, Spain Chitinases (E.C. 3.2.1.14) are the enzymes that can hydrolyze the -(1 → 4)-linkages of chitin. These enzymes are present in a wide range of organisms such as fungi, insects, crustaceans, bacteria and plants. Expression of chitinase enzymes in mesophilic hosts such as E. coli capable of effective secretion of heterologous proteins into the growth medium reduces the cost of production and simplifies downstream processing. In this work, a chitinase from a Lactococcus strain expressed in E. coli was used. In order to improve growth and/or favor enzyme secretion, the medium composition as well as culture conditions (i.e., pH, temperature, and aeration) was studied in shake flask cultures. Moreover, the efficiency of chitinase production was assessed using a logistic and Luedeking and Pirettype models proposed previously for other bioprocesses and the scaled-up to a 5 L-bioreactor was approached. Finally, a comparative study about the effect of the inducer used (isopropyl-beta-thio galactopyranoside (IPTG) or lactose) revealed that similar results can be obtained by addition of the lactose (15 mM), reducing the cost of the process with the same efficiency. Keywords: Chitinase; Lactococcus strain; Scaling-up Acknowledgement: This work was financed by Xunta de Galicia, Spain, project 10PXIB310278PR. http://dx.doi.org/10.1016/j.nbt.2012.08.216
www.elsevier.com/locate/nbt S77