Fermentations were performed in an external recycle bioreactor using CO2 and D-glucose at feed concentrations of 20 and 40 g L . Severe biofilm formation prevented kinetic analysis of suspended cell ('chemostat') fermentation, while perlite packing enhanced the volumetric productivity by increasing the amount of immobilised cells.The highest productivity of 6.35 g L h was achieved at a dilution rate of 0.56 h . A constant succinic acid yield of 0.69±0.02 g/(g of glucose consumed) was obtained and found to be independent of the dilution rate, transient state and extent of biofilm buildup -approximately 53% of the carbon that formed phosphoenolpyruvate ended up as succinate. Byproduct analysis indicated that pyruvate oxidation proceeded solely via the formate-lyase pathway. Cell growth and corresponding biofilm formation were rapid at dilution rates higher than 0.35 h when the product concentrations were low (succinic acid<10 g L ), while minimal growth was observed at succinic acid concentrations above this threshold.

BackgroundSuccinic acid (SA) has become a prominent biobased platform chemical with global production quantities increasing annually. Numerous genetically modified E. coli strains have been developed with the main aim of increasing the SA yield of the organic carbon source. In this study, a promising SA-producing strain, E. coli KJ134 [Biotechnol. Bioeng. 101:881–893, 2008], from the Department of Microbiology and Cell Science of the University of Florida was evaluated under continuous and batch conditions using D-glucose and CO2 in a mineral salt medium. Production characteristics entailing growth and maintenance rates, growth termination points and metabolic flux distributions under growth and non-growth conditions were determined.ResultsThe culture remained stable for weeks under continuous conditions. Under growth conditions the redox requirements of the reductive tricarboxylic acid (TCA) cycle was solely balanced by acetic acid (AcA) production via the pyruvate dehydrogenase route resulting in a molar ratio of SA:AcA of two. A maximum growth rate of 0.22 h-1 was obtained, while complete growth inhibition occurred at a SA concentration of 18 g L-1. Batch culture revealed that high-yield succinate production (via oxidative TCA or glyoxylate redox balancing) occurred under non-growth conditions where a SA:AcA molar ratio of up to five was attained, with a final SA yield of 0.94 g g-1. Growth termination of the batch culture was in agreement with that of the continuous culture. The maximum maintenance production rate of SA under batch conditions was found to be 0.6 g g-1 h-1. This is twice the maintenance rate observed in the continuous runs.ConclusionsThe study revealed that the metabolic flux of E. coli KJ134 differs significantly for growth and non-growth conditions, with non-growth conditions resulting in higher SA:AcA ratios and SA yields. Bioreaction characteristics entailing growth and maintenance rates, as well as growth termination markers will guide future fermentor designs and improvements.