Recovery of recombinant Escherichia coli by chitosan-conjugated magnetite

Magnetic separation of a recombinant Escherichia coli harboring the beta-ga lactosidase gene was investigated. We prepared a chitosan-conjugated magnet ite (chitosan-mag) that disperses well in aqueous solution. After adding it to a cell suspension, it can recover various microorganisms including E. c oli as the precipitant within only 1 min. Over 90% of E. coli cells were re covered in a wide pH range from 3.0 to 7.0 and the adsorption mechanism is considered to be mainly due to the electrostatic force arising from the neg ative surface potential of microorganisms. An E. coli cell suspension obtai ned after 10 h cultivation was subjected to magnetic separation using 2.0 g /l of chitosanmag at room temperature for 30 min. Almost all cells were sep arated and the recombinant protein, beta-galactosidase, was recovered at th e same level that was achieved by centrifugation (10 000xg, 10 min). When v arious amounts of chitosan-mag were added to the E. coli cell suspension, a n equilibrium condition was attained after 10 min. The relationship between the equilibrium cell concentration in the supernatant and the equilibrium adsorbed amount of the cell per chitosan-mag was expressed by a Freundlich- type adsorption equation. The recycling of chitosan-mag was also investigat ed. When the used chitosan-mag was exposed to alkaline conditions from pH 1 0 to pH 13 (60 degrees C, 60 min), about 90% of the cells were recovered ev en after the third separation. Thus, magnetic separation was found to be ap plicable to separation of cells from a culture broth and the chitosan-mag w as reusable. (C) 1999 Elsevier Science S.A. All rights reserved.