A recombinant $\underline{E}$. $\underline{coli}$ HB101(pPAKS2) producing penicillin acylase was cultured in a membrane cell recycle reactor. Cell density could be increased up to 145 g/L without experiencing by-product inhibitions. The main inhibitory by-product was acetic acid, and cell growth ceased when its concentration was above 14 g/L. Glucose limitation and dissolved oxygen control were indispensable conditions for minimizing acetic acid formation. Acetic acid accumulation was less than 0.5 g/l when cells were cultured with medium containing no glucose. $\underline{E}$. $\underline{coli}$ HB101(pPAKS2) was very stable throughout the whole experiment. Growth retardation at high cell density was caused by inhibitory factors such as acetic acid, carbon dioxide, and osmotic pressure rather than by cell-cell interaction. Use of high concentration medium greatly reduced the membrane surface area required. The recycle reactor could be operated in various operational modes including partial bleed and repeated recycle culture. Productivity of repeated recycle system was over ten times higher than that of a simple batch system. Culture time could be shortened by temperature shift during one batch operation.
A mutant $\underline{E}$. $\underline{coli}$ (ATCC 11105) could be also cultured in high cell density. Acetic acid accumulation was less than 0.5 g/L because it was cultured with medium containing no glucose.
From the above fact that glucose limitation and dissolved oxygen control were indispensable conditions for the minimization of acetic acid formation, continuous culture was tried with high concentration media, where glucose could be naturally limited. $\underline{E}$. $\underline{coli}$ HB101(pPAKS2) and $\underline{E}$. $\underline{coli}$ (ATCC 11105) were cultured. Using pure oxygen, high cell density up to 95 g/L was obtained without significant inhibition by the main by-product, acetic acid. The operation was simple and productivity was several times higher than those of conventional batch and continuous culture. Dissolved oxygen level and carbon dioxide concentration were important variables.
