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The temperature-limited fed-batch technique for control of Escherichia coli cultures

by Svensson, Marie

Abstract (Summary)
The objective of this study was to investigate the physiology and productivity in Escherichia coli cultures with emphasis on the temperature-limited fed-batch (TLFB) culture. The TLFB technique controls the oxygen consumption rate of the growing culture by a gradually declining temperature from 37-35 °C down to 20-18 °C. The temperature regulated the DOT around a set-point (30 % air sat.), and all nutrients were in excess. Glucose was fed into the culture continuously, however, high acetate formation was avoided by keeping the glucose at a low, yet excessive, concentration. The biomass productivity was approximately the same in TLFB as in glucose-limited fed-batch (GLFB) cultures, since the specific growth rate and the oxygen consumption rate are limited by the oxygen transfer capacity of the reactor in both techniques. High concentrations of endotoxins were found in the medium of E. coli fed-batch cultures at low specific growth rates (below 0.1 h-1) and severe glucose limitation. In this thesis the TLFB technique was found to suppress the endotoxin release even at low specific growth rates. The repressed release of endotoxins in TLFB cultures was due to the high availability of glucose and not to the low growth rate or the lower temperature. The conclusion was drawn from comparing with the GLFB technique performed at 20 °C, which resulted in high endotoxin release. Extensive release of endotoxin, accompanied with high concentrations of soluble proteins was found in a TLFB culture exposed to a higher energy dissipation rate, 16 kW m-3, instead of 2 kW m-3, due to a higher stirrer speed (1000 instead of 500 rpm). The hypothesis that this is a result of mechanical stress is discussed in context with the common view that cells like E. coli, which are smaller than the Kolmogoroff’s microscale of turbulence, should not be influenced by the turbulence. TLFB cultured cells exhibited more stable cytoplasmic membranes when treated with osmotic shock as compared to the GLFB cultured cells. The concentrations of DNA and soluble proteins in the periplasmic extracts from the TLFB cultured cells were lower than from GLFB cultured cells. In addition, the specific productivity of periplasmic ?-lactamase was higher in the TLFB cultures, suggesting that this technique could be an alternative for protein production. The solubility of a partially aggregated recombinant protein increased in the TLFB compared to the GLFB cultures. However some time after induction, in spite of the gradually declining temperature, the soluble fraction decreased. For obtaining better understanding of the performance of the process and for identifying critical parameters, a mathematical model was developed based on the growth, energy and overflow metabolism at non-limiting nutrient conditions. The temperature-dependent maximum specific glucose and oxygen uptake rates were determined in pH-auxostat cultures for temperatures ranging from 18 to 37 °C. A dynamic simulation model of the TLFB technique was developed and the results were compared to experimental data. The simulation program was also used for sensitivity analysis of some physiological and process parameters to study the impact on biomass concentration and temperature profiles. An effect on the biomass concentration profile but not on the temperature profile was observed when changing the oxygen transfer coefficient. If the maximum specific glucose uptake rate was altered, or if the glucose concentration was permitted to assume other values, the temperature profile but not the biomass concentration profile was influenced. Cell death affected both the biomass concentration profile and the temperature profile.
Bibliographical Information:

Advisor:

School:Kungliga Tekniska högskolan

School Location:Sweden

Source Type:Doctoral Dissertation

Keywords:TECHNOLOGY; Bioengineering; Keywords: endotoxin; Escherichia coli; fed-batch technique; glucose-limitation; mathematical model,

ISBN:91-7178-473-X

Date of Publication:01/01/2006

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