Rejection and fouling of track-etched microfiltration membranes by Acholeplasma laidlawii: Clues to mycoplasma behavior during “sterile” dead-end filtration

Abstract

Mycoplasma contamination is commonplace during processing of biopharmaceuticals necessitating its sterile filtration. Consequently, we measured the passage of Acholeplasma laidlawii (a small bacterium lacking cell wall like mycoplasma) across track-etched membranes rated at 100 nm and 220 nm that are designated as “sterile filters” and analyzed the entire progression of fouling beginning with pore occlusion before transitioning to a cake. Initial biofouling was quantified to be intermediate blocking before bacteria formed highly compressible cakes. Under identical experimental conditions, phase inversion membranes of the same nominal pore rating rejected A. laidlawii to a significantly greater extent suggesting depth filtration and capture within the polymer matrix in contrast to surface capture by their track-etched counterparts. A facile fluorescence microscopy method to rapidly detect slow-growing A. laidlawii cells was demonstrated under conditions of low retention. Evidence of cake relaxation by depressurizing the filter and subsequent mobilization of individual cells upon re-pressurization is given. Multiple lines of evidence demonstrated the pressure-induced deformation of individual cells resulting in their incomplete rejection by membranes with pore ratings smaller than the bacterium's unstressed diameter: (i) complete retention of rigid silica colloids of similar size, shape, and zeta potential regardless of pressure in contrast to incomplete A. laidlawii removals, (ii) reduced bacterial removal by both membranes with increasing filtration pressure, (iii) 150–600 fold less permeable bacterial cakes at any given pressure compared with silica cakes, and (iv) presence of flattened and irregularly shaped A. laidlawii cells in electron micrographs whereas they were near perfect cocci at atmospheric pressure.