Influence of fructose and oxygen gradients on fed-batch recombinant protein production using Bacillus megaterium

摘要

Bacillus megaterium expressing a variant of the green fluorescent protein (GFP) was used to investigate the influence of gradients in dissolved oxygen (DO) and fructose concentration on biomass and product formation. For this purpose, several fed-batch experiments with DO as feed control parameter were performed using suboptimal and optimal adjusted control parameters. In a first approach, suboptimal DO-feeding was reached by varying randomly the controller output between 10 and 20% of the maximal pumping capacity. This led to fluctuations in DO between 20 and 80% and to substrate gradients around 3 g/L. GFP formation thereby was decreased. In a second approach, an optimal feeding profile was realized using a Pi-control with a constant controller output of 20% was used. Thereby no gradients in substrate concentration, but low-amplitude and high-frequency oscillations in the DO concentration were obtained, leading to increased GFP formation. In a final experimental approach, large-scale conditions were emulated in a two-compartment scale-down system consisting of a 3.7-L stirred tank reactor and a 0.7-L non-stirred vessel. DO-based feeding was applied with the measurement in the 3.7-L bioreactor and feeding in the small vessel. This setup led to afairly constant gradient of 20% between both vessels, which also resulted in a decreased GFP productivity.