Bacterial Adhesion and Motility at Oil-Water Interfaces

Degradation of hydrocarbons by bacteria is one of the most important processes in oil spill cleanup. In attempt to increase the rate of biodegradation, chemical dispersants have been deployed in many oil spill scenarios to increase the surface area per unit volume available to bacteria. Biofilm formation is one of the important pathways in degradation of oil by bacteria. Because adhesion of bacteria to surface is one of the important steps in biofilm formation. It is important to study what factors affect the bacterial adhesion on oil/water interfaces. Adhesion of bacteria on solid surfaces is widely studied but surprisingly, how bacteria adhere on oil/water interface, and the effect of surfactants and bacterial motility on adhesion of bacteria on oil/water interface is not well studied. First, we designed and fabricated a microfluidic device to produce denser monodispersed oil in water emulsion. We developed a method to be able to capture 3D images of bacteria adhering to oil droplets with minimal number of cells attached to imaging chamber and with minimizing the wetting (~ 180° contact angle) of oil droplet to the imaging chamber. We developed tracking algorithms to visualize the cells adhering on the droplet and to calculate the contact angle that each bacterium makes to the droplet surface. In the first part of the project, we studied the effect of surfactant chemistries (anionic [dioctyl sodium sulfosuccinate, dicyclohexyl sodium sulfosuccinate, dibutyl sodium sulfosuccinate], cationic [cetyltrimethylammonium bromide], and nonionic [Tween 20]) and surfactant concentration on adhesion of nonmotile Marinobacter hydrocarbonoclasticus SP17 on dodecane droplets. Secondly, we found that motile bacteria Halomonas titanicae adhering to dodecane droplets were able to move the droplets in aqueous suspension. We explored the physics of droplet rotation driven by bacteria. Droplets rotate in clockwise direction when viewed from the liquid side, due to symmetry-breaking hydrodynamic interactions of bacteria with the surface. We examined the effect of droplet size on angular speed of droplets. We further investigated the effect of surfactant concentration and interfacial affinity of bacteria (by using three different bacteria species Escherichia coli, Shewanella haliotis, and Halomonas titanicae) on droplet rotation. Thirdly, we investigated the effect of bacterial motility on adhesion of bacteria on hexadecane droplets. Here, we show that bacterial motility enhances adhesion to surfactant-decorated oil droplets dispersed in artificial sea water. Motile Halomonas titanicae adhered to hexadecane droplets stabilized with dioctyl sodium sulfosuccinate (DOSS) more rapidly and at greater surface densities compared to nonmotile H. titanicae, whose flagellar motion was arrested through addition of a proton decoupler. Increasing the concentration of DOSS reduced the surface density of both motile and nonmotile bacteria as a result of the reduced interfacial tension. Finally, we investigated the effect of concentration of anionic surfactant dioctyl sodium sulfosuccinate (DOSS) and calcium chloride on aggregation of nonmotile Marinobacter hydrocarbonoclasticus and Halomonas titanicae in synthetic seawater. Bacteria aggregation may occur due to environmental stresses as a protective mechanism or it can occur as a first step towards biofilm formation and subsequent biodegradation. There are two physical mechanisms known in aggregation of bacteria: (1) aggregation by depletion attraction, and (2) aggregation by bridging attraction due to EPS or polymers. In our study, we found that aggregation (size and number density) increases with increase in DOSS concentration and calcium chloride concentration. Motile Halomonas titanicae showed higher aggregation compared to nonmotile bacteria. Together, we studied bacteria motility and adhesion interactions on cell-solid surface, on cell-liquid interfaces, and on cell/cell interfaces. Broadly, this research contributes to the fields of bioremediation and antifouling.