Measurement of acetate during the production of recombinant proteins with E. coli

摘要

Acetate is known to be the major by-product during fermentation of recombinant E. coli.Among the consequences of that production, the more important are the decrease of the biomassyield for a given carbon source, the inhibition of the growth when acetate is present at highconcentrations (typically 10 g/L) and the decrease of the production of recombinant proteins. Forthese reasons, its accurate and fast measurement is a very important issue when trying to achievehigh productivities in this kind of processes.A method based on Flow Injection Analysis (FIA) was then adapted and optimized for theon-line monitoring of fermentations of recombinant E. coli.The method is based on the pre-acidification of the sample with sulfuric acid followed by thediffusion of the acetate into a stream containing an acid-base indicator through a hydrophobicmembrane. The decrease in the absorbance of the acid-base indicator at 560 nm is proportionalto the concentration of acetate and is measured with a photometer for that wavelength.The composition of the indicator was studied in order to achieve a compromise betweenstability and sensitivity of the method. Several solutions are proposed, depending on theconcentration range of acetate to be measured. It was possible to achieve linearity until 10 g/L ofacetate with a sensibility of less than 0.1 g/L.The correlation between acetate measured with FIA and with other methods (HPLC andenzymatic kit from R-Biopharm) is also acceptable, the differences never exceeding 20%.The method is very reproducible, being the averaged relative standard deviations around 2%for 20 replicates of the same sample. The method is also very fast, being the sampling rate of 30hˉ¹.