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Effect of heating rate on pDNA production by E. coli

Paper ID Volume ID Publish Year Pages File Format Full-Text
3289 162 2013 9 PDF Available
Title
Effect of heating rate on pDNA production by E. coli
Abstract

•The effect of different heating rates on pDNA production was investigated in high cell-density cultivations.•Heat induction resulted in lower biomass synthesis, whereas glucose and acetate were accumulated.•pDNA yield and productivity were maximum at heating rates between 0.05 and 0.10 °C, whereas supercoiling was not affected.•On-line capacitance and conductivity measurements, allowed estimation of growth rate, acetate and biomass concentrations.•The results are useful for the design of improved cultivation strategies for pDNA production.

The effects of heating rate (HR) on the performance of two-phase (batch followed by fed-batch) high cell-density cultivations (HCDC) of E. coli DH5α for the production of plasmid DNA (pDNA) were investigated. Optimal temperatures for the HCDC, as selected from shake flask experiments at constant temperatures between 30 and 45 °C, were 35 °C for biomass accumulation in the batch phase and 42 °C for inducing pDNA replication during the fed-batch. In HCDC the temperature was increased at HR of 0.025, 0.05, 0.10 and 0.25 °C/min and the performance of the cultivations were compared to a HCDC run at constant temperature (35 °C). Compared to constant 35 °C, heat-induced HCDC accumulated up to 50% less biomass within the same cultivation time and acetate and glucose accumulated to high concentrations. The overall specific productivity (QP) and average pDNA yield (Yp/x) in HCDC at 35 °C were 0.22 ± 0.02 mg/g h and 5.3 ± 0.00 mg/g, respectively. Such parameters were maximum at a HR of 0.05 °C/min, reaching 0.56 ± 0.06 mg/g h and 9.3 ± 0.6 mg/g, respectively. At HR above 0.5 °C/min, Yp/x remained relatively constant, whereas QP tended to decrease. The supercoiled pDNA fraction remained around 80% at all HR. Bioreactors were equipped with a capacitance/conductivity probe. In all cases biomass concentration correlated closely with the capacitance signal and acetate and glucose accumulation was accompanied by an increase in the conductivity signal. Thus, it was possible to calculate acetate and biomass concentrations, as well as μ, from online capacitance and conductivity signals using estimators. Altogether, in this study it was shown that it is possible to maximize pDNA productivity by choosing an appropriate HR and that relevant parameters can be estimated by capacitance/conductivity signals, which are useful for better process control and development.

Keywords
pDNA vaccine; Temperature induction; Acetate; E. coli; High cell-density; Capacitance sensor
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Publisher
Database: Elsevier - ScienceDirect
Journal: Biochemical Engineering Journal - Volume 79, 15 October 2013, Pages 230–238
Authors
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Subjects
Physical Sciences and Engineering Chemical Engineering Bioengineering
Get Full-Text Now
Don't Miss Today's Special Offer
Price was $35.95
You save - $31
Price after discount Only $4.95
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Full-text PDF Download
Online Support
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