Effect of heating rate on pDNA production by E. Coli

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 DH5 alpha 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 degrees C, were 35 degrees C for biomass accumulation in the batch phase and 42 degrees 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 degrees C/min and the performance of the cultivations were compared to a HCDC run at constant temperature (35 degrees C). Compared to constant 35 degrees 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 (Q(P)) and average pDNA yield (Y-p/x) in HCDC at 35 degrees 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 degrees C/min, reaching 0.56 +/- 0.06 mg/g h and 9.3 +/- 0.6 mg/g, respectively. At HR above 0.5 degrees C/min, Y-p/x remained relatively constant, whereas Q(P) tended to decrease. The supercolied 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 mu, 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. (C) 2013 Elsevier B.V. All rights reserved.