Microorganisms, such as bacteria are used extensively in industrial processes to manufacture biopharmaceuticals, vaccine components, plasmid DNAs, vaccine DNAs and many specialty chemicals, including amino acids. Bacteria used in industrial processes are typically grown in liquid medium supplemented with glucose as a source of carbon. Large amounts of glucose are required in industrial processes to grow bacteria to the high densities desired for maximizing volumetric productivity, for producing specialty chemicals and for maintenance of the bacteria. Bacteria take up and assimilate metabolites at a high rate. The flux of metabolites can be so high that it overwhelms one or more of the biochemical reactions in the central carbon pathways of the bacteria as the concentration of certain metabolites rise. The bacteria dispose of the high concentrations of metabolites by utilizing one or more “overflow” pathways.
E. coli tend to secrete acetate when oxygen becomes scarce during high cell density fermentations. The root cause is the need for the cell to dispose of electrons to an acceptor other than O2. The proximate cause is intracellular accumulation of acetyl-CoA, a product of glycolysis, which in the presence of O2 is normally burned by the TCA cycle. However, when O2 is low, the TCA cycle cannot metabolize acetyl-CoA efficiently, so it builds up in the cell. Excess acetyl-CoA is converted to acetate which is excreted along with other organic acids to remove electrons from the cell. This provides a viable but slow metabolic solution for E. coli in the wild, but in industrial fermentors as cell density is driven to higher levels, excess acetate can accumulate to toxic levels.
This problem has traditionally been addressed by fed batch fermentation wherein the carbon source is metered into the culture so the cell growth is limited by carbon starvation to levels commensurate with the available O2. Despite metering the carbon source, oxygen utilization eventually becomes limiting at high cell densities. In this situation the fermentor environment also becomes heterogeneous due to the lack of perfect mixing, and the O2 level varies from place to place in pockets. Although acetate excreted in a low O2 region can be taken back up by cells that are carried to regions with more O2, this may be metabolically inefficient and, eventually, as more cells accumulate, the acetate level inexorably climbs even with fed batch technology. As the organic acid levels rise beyond the buffering capacity of the media, pH is maintained by titration with mineral bases such as NaOH which not only require additional equipment and attention from plant personnel, but can also increase the complexity of downstream product purification as well as the salt burden in the fermentor waste stream. A fermentation regimen that increases specific product formation by minimizing over flow metabolism would represent a significant improvement to the art.