Document Type dissertation Author Name Abu-Lail, Nehal Ibrahim URN etd-0918103-153031 Title The Effect of Biopolymer Properties on Bacterial Adhesion: an Atomic Force Microscopy (AFM) Study Degree PhD Department Chemical Engineering Advisors Terri A. Camesano, Advisor Robert Thompson, Committee Member William Clark, Committee Member John Bergendahl, Committee Member Keywords Bacterial Adhesion Ionic Strength Polarity Lipopolysaccharides Heterogeneity Elasticity FJC Steric Interactions AFM Date of Presentation/Defense 2003-09-12 Availability unrestricted
The effect of bacterial surface biopolymers on bacterial adhesion to surfaces was studied through experiments and modeling. Atomic Force Microscopy (AFM) provided the tool to measure the interaction forces between different bacterial cells and silicon nitride tips under different chemical conditions at a nanoscopic level. Two bacterial strains were considered: Pseudomonas putida KT2442 and Escherichia coli K-12 JM109. This study addressed the following issues: 1) the effect of solution ionic strength and solvent polarity on adhesion between Pseudomonas putida KT2442 and the silicon nitride AFM tip, 2) role of heterogeneity of bacterial surface biopolymers on bacterial adhesion, 3) role of lipopolysaccharides (LPS) on adhesion at three different scales: continuous, batch, and nanoscale, and 4) nature of interactions between E. coli JM109 and a model surface (silicon nitride tip).
To address the first issue, formamide, water, and methanol were used to investigate the effect of polarity on surface characteristics of biopolymers on the bacterial surface while a range of salt concentrations between that of water to 1 M KCl were used to study the effect of ionic strength. The adhesion increased with decreasing polarity of the solvent, indicating that the polymers on the bacterial surface are hydrophilic in nature. The adhesion was slightly affected by ionic strength variations up to a concentration of 0.1 M KCl; this may have been due to the fact that the ionic concentration in the solution did not counterbalance the ionic concentration in the biopolymer brush on the bacterial surface. However, a dramatic increase in the adhesion magnitude was observed when the salt concentration increased above 0.1 M KCl. This transition in adhesion with ionic strength from a low to high value induced a transition in the elasticity of the bacterial surface biopolymers. The biopolymer brush layer did change from rigid to soft with increasing the ionic strength. The elasticity was quantified mainly by the use of the freely jointed chain (FJC) model.
Our interest in investigating the role of heterogeneity on adhesion developed from the results of the first study. The bacterial surface polymers were thought to be different in their chemical and physical nature since they were found to span a range of segment lengths. Analyzing the adhesion forces for P. putida KT2442 showed that the bacterial surface is heterogeneous. The heterogeneity was evident on the same cell surface and between different cells from the same population.
To resolve the third issue, approximately, 80% of the surface LPS of E. coli K-12 JM109 were removed by treating the cells with 100 mM ethylenediaminetetraacetic acid (EDTA). The effect of LPS removal on the adhesion of the cells to the silicon nitride tip was studied in water and phosphate buffered silane (PBS). The adhesion results from the AFM experiments were compared to batch retention experiments with glass as the substratum and column attachment experiments with columns packed with quartz sand. LPS controlled bacterial adhesion to the different surfaces in the study at three scales: batch, continuous, and nano-scale.
Finally, the nature of interactions between E. coli JM109 and a model surface (silicon nitride tip) were investigated in solvents of varying polarity (formamide, water, and methanol). The Young’s modulus of elasticity for the bacterial surface was estimated by fitting of the Hertzian model to the force-indentation curves. Young’s modulus values increased as the solvent polarity decreased, indicating a stiffer bacterial surface in lower polarity solvents. The average adhesion force in each solvent was negatively correlated with the dielectric constant of the solvent, suggesting hydrophilic biopolymers. Specific and non-specific interaction forces between the AFM tip and the biopolymers were further characterized by applying a Poisson statistical analysis to the discrete adhesion data. The specific and non-specific interaction forces were the highest in methanol (-4 and -1.48 nN respectively). These values are in accordance with the high adhesion magnitude values measured with AFM in methanol. The results of my different studies emphasized the important role of AFM in studying biological interactions to different surfaces and in characterizing bacterial surface biopolymers.
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