Open Archive Toulouse Archive Ouverte (OATAO) OATAO is an open access repository that collects the work of Toulouse researchers and makes it freely available over the web where possible. This is an author-deposited version published in: http://oatao.univ-toulouse.fr/ Eprints ID: 5910 To link to this article: DOI:10.1016/J.MEMSCI.2008.09.049 URL: http://dx.doi.org/10.1016/J.MEMSCI.2008.09.049 To cite this version: Lebleu, Nathalie and Roques, Christine and Aimar, Pierre and Causserand, Christel (2009) Role of the cell-wall structure in the retention of bacteria by microfiltration membranes. Journal of Membrane Science, vol. 326 (n°1). pp. 178-185. ISSN 0376-7388 Any correspondence concerning this service should be sent to the repository administrator: [email protected] Role of the cell•wall structure in the retention of bacteria by microfiltration membranes Nathalie Lebleu a, Christine Roques b, Pierre Aimar a, Christel Causserand a,∗ a Laboratoire de Génie Chimique, Université Paul Sabatier, 31062 Toulouse Cedex 9, France b LU 49, Adhésion Bactérienne et Formation de Biofilms, Université Paul Sabatier, 31062 Toulouse Cedex 9, France a b s t r a c t This experimental study investigates the retention of bacteria by porous membranes. The transfer of bacteria larger than the nominal pore size of microfiltration track•etched membranes has been studied for several kinds of bacterial strains. This unexpected transfer does not correlate to the hydrophobicity, neither to the surface charge of the microorganism, as suggested in previous reports. We conclude that, in our conditions, the kind of bacteria (Gram•positive or Gram•negative) is finally the most important parameter. As the distinction between those two types of bacteria is related to the cell•wall structure, we provide an experimental evidence, via the action of an antibiotic, that the cell•wall flexibility triggers the transfer of the bacteria through artificial membranes, when the pores are smaller in size than the cell. 1. Introduction a strong effect on the retention predicted by considering only a size exclusion mechanism. For instance, Herath et al. [9] have The retention of microorganisms is one of the key advantages shown that the rejection of MS2 and Q␤ (25 nm) by microfiltra• of using membrane filtration for the production of drinking water. tion membranes of 0.05 ␮m mean diameter was larger for pH Ultrafiltration and microfiltration are known as efficient processes values close to their isoelectric point where bacteriophages are not to remove bacteria and their selectivity is dominated by a sieving charged anymore and thus are more likely to aggregate and to be effect [1,2]. Nevertheless, several studies report leaks of bacteria rejected. through membranes with a nominal pore size smaller than the For bacterial cells, electrostatic interactions influence was also bacteria size [3–7]. Assuming the presence of a small number of reported in case of deep filtration on granular bed. A low ionic abnormally large pores as compared to the average pore rating strength or a high negative charge of the microorganism are some• [3,4,8], is not sufficient to justify those results. In most cases the times recommended to facilitate the bacteria transport through a diameter of such “defects” remains smaller than the cell size. In this porous medium [10,11]. However, in membrane processes, electro• context, we may assume that the biological nature of the filtered static interactions appear early at the entrance of the particles into particles is likely to induce specific behaviour and so to modify the pore, and thus the transposition of those last results to explain the expected transfer mechanisms. Therefore, the role of the cell the unexpected leakage is not immediate. properties, including their surface properties or their mechani• In addition, the transfer of bacteria could be also originated from cal stiffness has been questioned in the present study, so as to the mechanical properties of the bacterial cell•wall. However, only understand to which extend this could explain the abnormal leaks a few publications can be found on the effect of these properties on observed through filtration membranes. bacterial behaviour during filtration experiments. In this context In cases of filtration of biological particles such as viruses or papers already published on yeast and red blood cells have to be bacteriophages, several authors have pointed out that, due to their considered, since these are numerous, and meant to exhibit similar physico•chemical properties, electrostatic interactions may have behaviours. The deformability of biological particles during filtration experiments has already been invoked to describe the properties ∗ Corresponding author. Tel.: +33 5 61 55 86 90; fax: +33 5 61 55 61 39. of filtration cakes resulting from the build up of retained cells E•mail address: [email protected]•tlse.fr (C. Causserand). at the membrane surface. Zydney et al. [12] study the extent of cellular blockage of membranes pores and the properties of a cake al. [14] have shown that this volume reduction comes with the cell composed of red blood cells which are known for being highly deformation which magnitude is determined by cell mechanical deformable. Thereby, the cell bed is compressible which leads these properties such as cell•wall flexibility. authors to develop relationships between hydraulic permeability, From this literature survey it appears that if the changes in porosity and compressive pressure based on experimental data shape and size of bacteria under stress have been well considered to obtained by filtration and centrifugation and including pore block• explain deviation from ideality in filtration, less attention has beeen age effect. Then, they incorporate those relationships into a model paid so far to the consequences of this phenomenon on the leakage for predicting the cross•flow permeation flux, assuming that the of bacteria through micro or ultrafiltration membranes. The objec• hydraulic resistance provided by cross•flow filtration of red blood tives of the present paper are to show the ability of various bacteria cells was equivalent to that observed in an unstirred filtration to pass through a membrane characterized by a nominal pore size system. smaller than the cell size at rest and to explore the possible causes Numerous filtration studies of yeast suspensions have been pub• for this transfer considering operating conditions (feed concen• lished, in which the hydraulic resistance of cell deposits is evaluated tration and transmembrane pressure) and bacteria characteristics, as a function of the operating pressure. They conclude that the com• such as size, shape, and surface charge or hydrophobicity, but also pressibility of the cells should be invoked to explain the observed the cell•wall structure and flexibility. For this last purpose, we discrepancy between experimental data and the data calculated focused upon the fundamental distinction between Gram•positive using the classical Kozeny•Carman model. In this context, Meireles and Gram•negative bacteria related to their cell•wall structure. As et al. [13] propose a numerical approach for modeling the hydraulic an experimental evidence, the cell•wall structure and flexibility resistance of cakes obtained by dead•end filtration of baker’s yeasts of a Gram•positive bacteria was modified by using a biochemical (Saccharomyces cerevisiae). These authors use a simplified version treatment. of the models developed by Smith et al. [14–16] which take the mechanical properties of the cells obtained by compression exper• 2. Materials and methods iments and the osmotic equilibrium into account. In a wider sense, several authors have made the assumption that, 2.1. Experimental setup and procedure in terms of deformability, bacterial cells behave as yeasts. Then, by assuming bacteria deformation at constant volume, Hwang et al. Filtration experiments were performed using the setup shown [17] develop a model to evaluate the effects of operating condi• in Fig. 1, which consists of a 50 mL dead•end filtration stirred cell tions on the filtration performance based on Lu et al. [18] study of (Model 8050, Amicon) connected to a pressurised tank contain• calcium•alginate gel particles. Their simulated results are in good ing the bacterial suspension. The pressure on the permeate side agreement with experimental data obtained in cross•flow filtration was atmospheric under all conditions. The transmembrane pres• of Pseudomonas species. sure was set by a pressure reducing valve located on the feed side. Concerning the study of biological cell filtrability through mem• The filtration cell contained a microfiltration membrane with an brane pores smaller than their size, most publications focus on red effective area of 1.34 × 10−3 m2. This setup has a small size which blood cells [19–21]. Scanning electron microscopy observations of allows an easy disinfection and manipulations under laminar air the transfer of bovine red blood cell [22] through microfiltration flow. membranes of average pore diameter of 0.3 and 0.4 ␮m reveal a Prior to the experiment, the membrane was disinfected by soak• considerable deformation of the cell enabling it to penetrate in ing in a solution of sodium hypochlorite at 200 ppm for 20 min pores much smaller than it. This ability results from the high flex• and then thoroughly rinsed with sterile distilled water. The filtra• ibility of their bilipidic membrane. However, bacteria present a tion cell was soaked in a more concentrated solution of sodium more complex cell•wall structure, composed not only of a lipidic hypochlorite (1000 ppm) for 30 min. All other pieces of equipment bilayer but also of a cross•linked polymer network responsible were sterilized (20 min under 120 ◦C) and kept under laminar air for mechanical strength and stiffness of their cell•wall which are flow to prevent contamination. larger than those of red blood cells. This is well reflected by the Each experiment was performed at room temperature in three Young modulus: the one of the red blood cells is in the range steps.