Pyruvate metabolism is a key step in the central metabolism of Escherichia coli. Under microaerobic condition, converting pyruvate into acetyl-coenzyme A is mainly achieved through pyruvate formate-lyase (Pfl). The reaction through pyruvate oxidase converts pyruvate directly into acetate in Escherichia coli and it contributes to pyruvate catabolism under microaerobic condition. It was reported that Pfl protein is an oxygen-radical protein, and the yfiD gene product can reactivate the inactive Pfl protein in the presence of oxygen by replacing the fragmented part of Pfl. Since pyruvate is an important metabolic regulation node, disrupting yfiD and poxB which affect the pyruvate metabolism should affect the metabolic patterns in the cell. To quantitate the contribution of YfiD and PoxB proteins in metabolism, well-controlled chemostat cultures were constructed for the E. coli yfiD mutant, the yfiD arcA double mutant, the poxB mutant and the poxB arcA double mutant strains using glucose as a sole carbon source under microaerobic conditions. To study the effect of oxygen availability on the activity of PoxB, two different oxygen levels were used for the chemostat cultures of the poxB mutant and poxB arcA double mutant strains. The metabolic flux distributions were studied and compared with those in the wild type E. coli and the arcA mutant strains based on C-13 labeling experiments. In the current work, the effect of deleting the yfiD or poxB genes in the wild type and the arcA mutant strains on pyruvate metabolism and TCA cycle flux distributions will be discussed.