A set of flasks was utilized to simulate a discrete countercurrent bioreactor. An enzymatic hydrolysis of penicillin G to 6-aminopenicillanic acid with immobilized penicillin acylase in a cloud point system was carried out with the discrete countercurrent operational mode. Byproduct phenylacetic acid is effectively transported with the auxiliary phase (coacervate phase), while product 6-aminopenicillanic acid and reactant penicillin G are effectively transported with the aqueous phase, where the enzymatic hydrolysis of penicillin G takes place. The experimentally results show that the product concentration and product yield are improved from the batch operational mode of about 38 MM and 50% to the corresponding countercurrent operational mode of above 50 mM and 65%, respectively. However, experimentally examination of the effect of operational parameters, such as feed point location, volume ratio of auxiliary phase to aqueous phase and initial reactant concentration, indicates that the achievement of a high product concentration and a high product yield at the same time is difficult by a simple utilization of the continuous countercurrent operational mode. This can be contributed to the facts that the enzymatic hydrolysis favors at a relatively higher pH, but the extraction of PAA only takes place at a relatively lower pH. (c) 2007 Elsevier Inc. All rights reserved.