Gill, N.K.; (2008) Design and characterisation of parallel miniature bioreactors for bioprocess optimisation and scale-up. Doctoral thesis , University of London.

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Abstract

The establishment of a high productivity microbial fermentation process requires the experimental investigation of many interacting variables. In order to speed up this procedure a novel miniature stirred bioreactor system is described which enables parallel operation of 4-16 independently controlled fermentations. Each miniature bioreactor is of standard geometry (100 mL maximum working volume) and is fitted with a magnetically driven six-blade miniature turbine impeller (dj = 20 mm, dj/dj = 1/3) operating in the range 100 - 2000 rpm. Aeration is achieved via a sintered sparger at flow rates in the range of 0 - 2 vvm. Continuous on-line monitoring of each bioreactor is possible using miniature pH, dissolved oxygen and temperature probes, while PC-based software enables independent bioreactor control and real-time visualisation of parameters monitored on-line. Initial characterisation of the bioreactor involved quantification of the volumetric oxygen mass transfer coefficient as a function of agitation and aeration rates. The maximum kLa value obtained was 0.11 s" The reproducibility of E. coli TOP10 pQR239 and B. subtilis ATCC6633 fermentations was shown in four parallel fermentations of each organism. For E. coli (1000 rpm, 1 vvm) the maximum specific growth rate, umax, was 0.68 0.01 h"1 and the final biomass concentration obtained, Xr,nai, was 3.8 0.05 g.L"1. Similarly for B. subtilis (1500 rpm, 1 vmm) umax was 0.45 0.01 h"1 and Xrinai was 9.0 0.06 g.L"1. Biomass growth kinetics increased with increases in agitation and aeration rates and the implementation of gas blending for control of DOT levels enabled umax and Xfmai values as high as 0.93 h"1 and 8.1 g.L"1 respectively to be achieved. The value of the miniature bioreactor design for high throughput experimentation was further demonstrated when Design of Experiments (DoE) techniques were employed to assess three variables temperature, pH and inducer concentration, for the optimisation of CHMO expression in E. coli TOP 10 pQR239. The optimised regression model derived from the results of 20 fermentations concluded that only temperature and inducer concentration had a significant influence, predicting a maximum specific CHMO activity of 105.9 U.g"1 at 37.1 C and 0.11 %w/v. This was in good agreement with the experimentally determined results at these conditions. In order to enable the predictive scale-up of miniature bioreactor results, the engineering characterisation of the miniature turbine impeller predicted a Power number of 3.5 based on experimental ungassed power consumption measurements. As a result of the numerous literature correlation relating kLa, gassed power per unit volume and superficial gas velocity being designed for much larger scale bioreactors, a similar correlation has been specifically derived for the miniature bioreactor scale. Constant ki,a has been shown to be the most reliable basis for predictive scale-up of fermentation results from the miniature bioreactor to conventional laboratory scale. This was confirmed over a range of kLa values (0.04-0.11 s"1), with good agreement between final biomass concentrations and maximum specific growth rates. In addition successful scale-up of the DoE results for optimum CHMO expression in E. coli at a constant kLa value of 0.04 s"1 yielded final biomass concentrations of 4.9 g.L"1 and 5.1 g.L"1 respectively in the miniature bioreactor and the 2 L vessel, and the CHMO activity obtained was 105.9 U.g"1 and 105.2 U.g"1 respectively. Finally, alternative on-line methods for monitoring cell growth within the miniature bioreactors without the need for repeated sampling have been described. The application of a novel optical density probe for monitoring biomass growth kinetics on-line has shown that comparable results for calculated maximum specific growth rates were obtained from off-line and on-line OD measurements 0.67 and 0.68 h"1 respectively. Thermal profiling techniques were also investigated as an alternative means for monitoring cell growth based on the natural heat generated by a microbial culture. Initial results showed that the heat generated during E. coli TOP 10 pQR239 fermentations followed the same pattern as the off-line growth curve. The maximum specific growth rates calculated from off-line and on-line thermal data were also in good agreement, 0.66 0 0.04 h"1 and 0.69 0 0.05 h"1 respectively. In summary the miniature bioreactor system designed and evaluated here provides a useful tool for the rapid optimisation and scale-up of microbial fermentation processes.

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