Study On The Regulation Of Escherichia Coli K12 Biofilm Development By Quorum-Quenching Of AI-2 Through TiO₂ Photoexcitation

With the rapid development of nanotechnology, artificial nanomaterials and their products have been widely applied and released into the environment by various means. In recent years, the potential environment and health risks caused by nanomaterials have attracted public attentions increasingly. The biological safety and ecotoxicological effects of artificial nanomaterials have gradually become hot topics of environmental science. Nano-TiO₂ has been used in many fields because of its inherent stability and excellent photocatalytic property. A great deal of studies have indicated that reactive oxygen species（ROS） produced by nano-TiO₂ photocatalysis can damage cell membrane and inhibit the growth of bacteria. However, major studies related to the environmental effects of nano-TiO₂ foucus on the acute toxicity of photoexcitation to individual cell. Studies about the influence of non-toxic stress on the behaviors of microbial population have not been reported yet. Therefore, in this study nano-Ti O₂ has been carried out as a research object to investigate its influences to the group behaviours of Escherichia coli K12 under the weak photoexcitation. Furthermore, the non-toxic stress mechanism of nano-Ti O₂ photoexcitation to the biofilm development has been elucidated from a perspective of bacterial quorum sensing. Finally, the following conclusions have been acquired in this study:（1） TiO₂-alone（concentrations with 50, 100 or 200 mg/L） and UV-alone（light intensities with 150, 300 or 450 μW/cm2） treatment had no significant inhibiton on the proliferation of E. coli K12 cells. However, the combination of TiO₂ and UV strongly reduced the biofilm biomass of E. coli K12 during the early stage of growth. When reaching the stable stage of growth, the biofilm gradually returned to the normal phenotype. The result showed that nano-TiO₂ photoexcitation delayed the origination of biofilm formation process rather than inhibit the biofilm development of E. coli K12 completely.（2） According to the experimental results, the optimum photocatalytic condition was determined as the TiO₂ concentration of 100 mg/L and the UV intensity of 150 μW/cm2. Under this experimental condition, E. coli K12 cells were labelled by green fluorescent protein（GFP）, and the three dimensional microstructure of biofilm was reconsituted. The result indicated that TiO₂ photocatalysis seriously hindered the normal development of E. coli K12 biofilm, which had caused the decrease of biofilm biomass significantly and the formation of the loose biofilm structure.（3） The activity of quorum-sensing signal AI-2 was related to the formation of E. coli K12 biofilm. During the first 10-h incubation, the increase of AI-2 activity was impacted by TiO₂ photoexcitation with the inhibition of biofilm developmen. Since the productivity of AI-2 secreted by E. coli K12 had increased gradually with the raise of cell density, AI-2 molecules still accumulated and eventually triggered the quorum sensing system of E. coli K12.（4） Nano-TiO₂ photoexcitation could produce two types of ROS, such as O₂?- and ?OH. Though low-level ROS failed to induce obvious cytotoxicity, they still showed an ability to degrade AI-2 molecules. Addition of AI-2 precursor, DPD, could remarkably restore biofilm development of E. coli K12 stressed by the TiO₂ photoexcitation. Moreover, the expressions of biofilm-formation genes, motA and rcsB which were regulated positively by AI-2 signal, had been significantly suppressed by TiO₂ photoexcitation. These results above clearly showed that the photoexcited TiO₂ could contribute to a retarded development of E. coli K12 biofilm through quenching AI-2 signals.