Response Of Bacterial Biofilms To Natural And Engineered Nanoparticles

More than 90% of the microorganisms in the soil exist as biofilms.Biofilms can gain competitive advantages through the production of extracellular polymers,such as the enhancement of quorum-sensing effects among microorganisms in biofilms,the colonization of cells on the surface,and the reduction of cytotoxicity of contaminants.In soil,engineered nanoparticles?NPs?released from industrial production and naturally occurring nanoparticles in soil have an important role in the formation,structural regulation,and function of soil biofilms,due to their strong surface activity.However,current understanding regarding the interaction between biofilms and NPs in the soil is still limited.In particular,knowledge about these processes and mechanisms at the molecular level is superficial.Therefore,exogenous engineering NPs-zinc oxide?ZnO?,soil nano-mineral hematite?hematite?and representative soil bacteria-Pseudomonas putida were selected in this project.Chemical methods,microbiological and molecular biology techniques as well as modern instruments analytical methods such as confocal laser scanning microscopy?CLSM?,transmission electron microscopy?TEM?,scanning electron microscopy?SEM?,Quartz Crystal Microbalance with Dissipation?QCM-D?,microcalorimetry?ITC?,structured light illumination super-resolution microscopy?SIM?were utilized to analyze the interaction mechanism between different types of NPs and bacteria.In addition,the molecular mechanisms responsible for the formation,structure,and function of bacterial biofilms in response to NPs exposure have also been explored.The major results were summarized as follows:?1?The mechanism of cytotoxicity of hematite nanoparticles mediated by surface-bound humic acid?HA?on Pseudomonas putida was elucidated.Pure hematite NPs could inhibit the bacterial growth with a median lethal concentration(24 h LC₅₀)of23.58 mg L^-1,while surface-bound HA could significantly mitigate the toxicity of hematite with the greatest LC₅₀of 4774.23 mg L^-1,which were corroborated by the transcriptional regulation of Pseudomonas putida cell activity-related genes.Co-precipitation experiment and transmission electron microscopy observation revealed that surface-bound HA prevented the adhesion of hematite to the cells and limited cell internalization.Compared to hematite NPs,the generation of intracellular reactive oxygen species?ROS?and the expression of oxidative stress genes were significantly inhibited in hematite surface-bound HA systems.The prevention of adhesion and inhibition of ROS production could account for the HA-mitigated nanotoxicity.Interfacial interactions between hematite and bacteria were also evaluated on the basis of the Derjaguin-Landau-Verwey-Overbeek?DLVO?theory,suggesting that the more adhesive conditions resulted in greater toxicity and the more repulsive conditions resulted in a reduced toxicity of the model microorganism.?2?The response mechanism of engineered nanoparticle?ZnO NPs?and the concentration effect of NPs were revealed during the development of bacterial biofilms.NPs within low concentration threshold(0.5³0 mg L^-1)significantly promote cell growth and biofilm formation,while high concentration NPs(>30 mg L^-1)significantly inhibit biofilm formation.Confocal laser scanning microscope?CLSM?analysis reveal that 0.5 mg L^-1 of Zn O NPs-coated substratum promotes biofilm colonization and biomass production,but biofilm colonization and biomass production of 250 mg L^-1 of ZnO NPs-coated substrate surface was 11 times lower than that of the control substrate without NPs.An increase in the protein and sugar contents of the biofilm matrix corroborated the low concentration stimulation effect of ZnO NPs.Both changes in cell growth and biofilm formation in the presence of NPs are supported by changes in expression of genes associated with oxidative stress as well as biofilm development.0.5mg L^-1 of ZnO NPs stimulates the expression of quorum sensing,lipopolysaccharide biosynthesis,and antibiotic resistance genes;High-concentration exposure of Pseudomonas putida cells to 500 mg L^-1 of ZnO NPs induced genotoxicity and upregulated antioxidant genes.?3?The effects of nanoparticles?Zn O NPs?on the morphology and structure of bacterial biofilms were investigated.The results of QCM-D showed that the exposure of ZnO NPs decreased the thickness and viscoelasticity as well as increased the roughness of the bacterial biofilm;and the ZnO NPs could accelerate the disintegration of mature biofilm cells.After the NPs were applied,the concentration of cell surface functional groups in the biofilm significantly decreased,and the structure of the secondary protein on the cell surface also changed.After adsorbed on the surface of quartz crystal,Pseudomonas putida can form a stable three-dimensional bacterial cluster structure,and the cells inside the biofilm are closely arranged.However,ZnO NPs inhibit the cells from expanding in the third dimension,and cells on the surface of NPs are scattered and disorderly arranged,making it difficult to form a mature biofilm.