The Processes And Mechanisms Of Bacteria Biofilm Formation In The Precence Of Soil Minerals

Microbial biofilms,sessile microbial communities enveloped in a self-produced extracellular polymeric substance(EPS),are ubiquitous in the natural environment.Biofilm formation is of great medical,industrial and environmental significance.Bacteria take advantages through forming biofilm such as quorum sensing,adhesion on a surface,commensalism,avoiding predation,preventing from drought,reducing cytotoxicity of pollutants etc.The micro-and nano-minerals in soil have large specific surface area,high charge density and the surfaces of these minerals are rich in ion,water and other nutrients.The interactions between soil minerals and microorganisms play significant roles in biogeochemical interface formation,structure regulation and functions etc.However,the study between soil minerals and bacteria biofilm formation still remains poorly understood.In this paper,the common minerals and microorganisms in the soil were selected as the study materials.The traditional chemical and microbiological methods,combining with laser confocal microscopy,atomic force microscopy,attenuated total reflection fourier transform infrared spectroscopy,were introduced to study the differences between planktonic and biofilm cells and the effect of typical soil minerals on soil bacteria biofilm formation.The major results were summarized as follows:(1)The differences between planktonic cells and biofilm cells of the gram-positive bacteria(Bacillus subtilis)and the gram-negative bacteria(Pseudomonas putida)in the soil were analyzed.The results showed that the biofilm cycle of P.putida was 24 h and the biofilm cycle of B.subtilis was 72 h.Once the biofilm formed,the size of the cells became smaller and the shape of cells changed from rod to ellipsoid,and the hydrophobic of cells were enhanced.Besides,the cell surface charges and electronegativity were both increased.The types of cell surface functional groups were not changed after biofilm formation,but the total amounts of surface functional groups were incresed.(2)We investigated the effects of montmorillonite,kaolinite,and goethite on the biofilm formation of the representative soil bacteria Bacillus subtilis.The bacterial biofilm formation in goethite was found to be impaired in the initial 24 h but burst at 48 h in the liquid–air interface.Confocal laser scanning microscopy showed that the biofilm in goethite was matured with several layers.Live/Dead staining showed that cells had the highest death rate of 60% after 4 h of contact with goethite,followed by kaolinite and montmorillonite.Atomic force microscopy showed that the interaction between goethite and bacteria may injure bacterial cells by puncturing cell wall,leading to the swarming of bacteria toward the liquid–air interface.Additionally,the expressions of abrB and sinR,key players in regulating the biofilm formation,were upregulated at 24 h and downregulated at 48 h in goethite,indicating the initial adaptation of the cells to minerals.The physical and chemical properties of minerals are the key factors on biofilm formation.(3)The mechanism of the effects of different concentrations of typical soil metal oxides(goethite,boehmite and dinitrite)on the growth and biofilm of P.putida was clarified.The growth of P.putida and the formation of biofilm were promoted when the concentration of goethite is 1 ~ 100 mg L-1,while the growth of bacteria and the formation of biofilm were inhibited when the concentration of goethite is 100 ~ 500 mg L-1.The boehmite promoted the growth of bacteria and biofilm formation when its concentration was less than 20 mg L-1,while it inhibitted the growth of P.putida and biofilm formation at 20 ~ 500 mg L-1.Different concentrations of birnessite had no effects on the growth of P.putida,but birnessite inhibited the formation of bacterial biofilm.Atomic force microscopy images showed that goethite could pierce the cell membrane of P.putida and damaged the cells and boehmite could induce cells dissolved.The interactions between birnessite and P.putida made the cell size smaller and the surface folds increased.The interactions between soil metal oxides and bacteria play a essential role on bacteria growth and biofilm formation.EPS in biofilm could significantly improve the tolerance of P.putida to minerals.