Effect of heating rate on pDNA production by E. coli

Abstract

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±0.02mg/g h and 5.3±0.00mg/g, respectively. Such parameters were maximum at a HR of 0.05°C/min, reaching 0.56±0.06mg/g h and 9.3±0.6mg/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.