Ultrafiltration of yeast cells – the effects of pH and thermal stress on membrane fouling and biomolecule transmission

This study investigates the impact of heat and alkaline pretreatment on crossflow ultrafiltration (UF) for yeast cell clarification, protein clearance and the recovery of two small molecule pharmaceutical compounds from a whole cell biotransformation reaction. Flow cytometry indicated extensive cell rupture for untreated yeast cells during UF, leading to a severe decline in permeate flux and reduced transmission of the two alkaloids, oripavine (297 Da) and nororipavine (283 Da). The exposure of yeast cells to pH 12 or to a temperature of 80 o C prior to filtration, however, led to significantly less cell rupture during UF, suggesting greater resistance to shear stress. At a volumetric concentration factor of 1.5, either pretreatment enhanced UF flux by approximately three-fold relative to the untreated broth. Microscopy revealed a dense membrane fouling layer consisting of ruptured cells for untreated broth, leading to complete protein rejection. In contrast, pH 12 resulted in a fouling cake comprised of a bilayer of intact cells and slightly lower protein rejection (98.1 ± 0.6%). Heat pretreatment led to the lowest protein rejection (87 ± 2%). The transmission of the two alkaloids followed similar trends with the lowest transmission for the untreated broth. No selectivity was observed between the alkaloids. These heat or alkaline pretreatments also increased yeast cell permeability, leading to accumulation of 9% or 22% of alkaloids within the cells, respectively. While these pretreatments enhance UF flux and alkaloid transmission, intracellular drug accumulation suggests that an additional diafiltration step is necessary to wash the accumulated product from within cells.