The substrate solution used in the cross-flow filtra- tion as the feed, contained either catechol or phenol. Catechol at a concentration of 2 mol/ml was dis- solved in 0.033 M of Tris–HCl buffer at pH 7.6. The catechol solution was fed to the membrane unit at var- ious cross-flow rates in which the cross-flow rate was altered in order to obtain a desired flux rate, ranging from 8.52 × 10−4 to 46.02 × 10−4 ml/cm2 s. In each experiment, 1l solution was used without recycling the permeate or retantate streams Due to the absence of any particulate matter in the solution, steady state conditions were reached almost immediately, without any significant reduction in the permeate flux. At the end of each experiment, 300 ml solution was perme- ated per 1 l of feed. Catechol 1,2-dioxygenase activity of the immobilized enzyme was measured after the flux reached to a steady state. Measurement of the im- mobilized enzyme activity was based on the amount of degraded catechol which caused an increase in ab- sorbancy at 260 nm by an amount of 0.56 absorbance units per ml of the permeate [22]. No enzyme was detected in the retantate stream as a result of break up of the enzyme from the membrane.
Phenol solutions were prepared at various phe- nol concentrations, ranging from 200 to 950 mg/l in Tris–HCl buffer at pH 7.6. Phenol concentrations were measured by direct photometric method (4-amino an- tipyrine) [22]. The substrate solution was fed in to the cross-flow membrane filtration module at a constant transmembrane pressure of 4.2 bar to obtain flux rate of 4.60 × 10−3 ml/cm2 s. The amount of phenol in the permeate was measured after the flux reached to a steady state.