土壤矿物介导下细菌生物膜形成过程及机制

发布时间：2017-12-28 02:09

本文关键词：土壤矿物介导下细菌生物膜形成过程及机制　出处：《华中农业大学》2017年博士论文　论文类型：学位论文

【摘要】：生物膜作为细菌存在于自然界生物体或非生物体表面的重要形态,其在医学、工业以及农业上的影响和作用受到广泛的重视。作为地球上最普遍的一种生命形式,生物膜可通过形成的胞外聚合物(EPS)使微生物获得竞争性优势,如加强了生物膜内微生物之间群体感应效应、帮助细胞在表面定殖和互养共生、保护细胞防止被捕食、抵抗干旱、减少污染物对细胞的毒性等。土壤中微米和纳米级矿物由于比表面积大、电荷密度高,表面富含离子、水分及其它营养物质,常与微生物结合在一起,在土壤生物地球化学界面的形成、结构调控、活性和功能发挥等方面具有重要的作用。然而,到目前为止,从生物膜角度研究细菌与土壤矿物相互作用还比较少见。本文以土壤中常见的矿物和微生物为供试材料,借助激光共聚焦显微镜(CLSM)、原子力显微镜(AFM)、衰减全反射傅里叶变换红外光谱(ATR-FTIR)和q-PCR等现代仪器分析技术,研究了土著细菌游离态细胞与生物膜细胞表面性质的差异,以及土壤典型矿物对细菌生物膜形成的影响,获得如下主要结果:(1)明晰了土著细菌游离态细胞与生物膜细胞之间的表面性质差异。恶臭假单胞菌成膜速度快,生物膜周期为24 h,而枯草芽孢杆菌的生物膜周期为72 h;细菌在形成生物膜后,细胞的尺寸变小、细胞形状由杆状变为椭球形,细胞的疏水性增强,表面带电量增多、电负性增强;与游离态细胞相比,生物膜细胞表面官能团的种类不发生改变,但是表面官能团的浓度会增加。(2)探明了土壤中典型的粘土矿物(蒙脱石和高岭石)以及铁氧化物(针铁矿)对土壤中常见的细菌(枯草芽孢杆菌)生物膜形成的影响机制。实验结果表明,在针铁矿体系中,生物膜在48 h时生物量达到最大值,而蒙脱石和高岭石体系中生物膜生物量在60 h达到最大。激光共聚焦显微镜观察到48 h时针铁矿体系中形成了致密、具有多层细胞的生物膜。细菌与矿物作用4 h后,针铁矿体系中细胞失活率高达60%,其次是高岭石和蒙脱石。原子力显微镜结果显示细菌与针铁矿之间发生紧密吸附,针状的针铁矿可以刺破细胞壁,细胞感应到不利环境,通过调控枯草芽孢杆菌生物膜形成的两个主要基因abrB和sinR,使细胞倾向于向气液表面运动并聚集形成生物膜。矿物的理化性质是矿物影响细菌形成生物膜的主导因素。(3)明确了不同浓度的典型土壤金属氧化物(针铁矿、勃母石和水钠锰矿)对恶臭假单胞菌生长及生物膜形成的影响机制。当针铁矿浓度为1~100 mg L-1时,可以促进恶臭假单胞菌的生长和生物膜的形成,而针铁矿浓度为100~500 mg L-1时,会抑制细菌的生长和生物膜的形成;当勃姆石浓度低于20 mg L-1时可促进细菌的生长和生物膜的形成,20~500 mg L-1勃姆石会抑制恶臭假单胞菌的生长和生物膜的形成;不同浓度的水钠锰矿对恶臭假单胞菌的生长没有影响,但是会抑制细菌生物膜的形成。原子力显微镜图像显示,针铁矿表面细菌的细胞膜被刺破,细胞完整性受损;勃姆石表面细胞发生了溶解;水钠锰矿表面细胞粒径变小、表面褶皱增加。不同土壤金属氧化物与细菌相互作用的强度和沉降能力是影响恶臭假单胞菌生物膜形成的关键因素。生物膜中的EPS可以显著的提高恶臭假单胞菌对矿物的耐受能力。
[Abstract]:Biofilm as an important form of bacteria exists on the surface of natural organisms or non organisms, and its influence and function in medicine, industry and agriculture has been widely valued. As one of the most common form of life on earth, biological membrane can be formed by extracellular polymers (EPS) enable the microorganism to gain a competitive advantage, such as strengthen the biofilm microbial community, help cells in the induction effect of surface colonization and syntrophic symbiosis, protect cells to prevent predation, drought resistance, reduce on the cell toxicity of pollutants. The soil in the micron and nano minerals due to large surface area, high charge density, the surface is rich in ion and water and other nutrients, and microbes together in formation, soil biogeochemical interface structure, activity and function regulation play an important role. However, so far, it is rare to study the interaction between bacteria and soil minerals from the biofilm angle. In this paper, the soil minerals and microorganisms in common as tested materials, using laser scanning confocal microscope (CLSM), atomic force microscopy (AFM), attenuated total reflection Fourier transform infrared spectroscopy (ATR-FTIR) and q-PCR and other modern instrument analysis technology, studied the indigenous bacterial free surface properties of cells and cell membrane biological state difference well, the influence of soil minerals on the typical bacterial biofilm formation, the main results are as follows: (1) clear differences between the surface properties of indigenous bacteria free cells and biofilm cells. Pseudomonas putida film speed, biofilm cycle is 24 h, and the biofilm cycle of Bacillus subtilis was 72 h; bacteria in biofilm formation, cell size, cell shape by baculovirus to ellipsoid, the cell hydrophobicity enhancement, surface charge quantity and electronegativity enhanced; compared with free cells, types of biofilm cells and surface functional groups are not changed, but the concentration of surface functional groups will increase. (2) the influence mechanism of typical clay minerals (montmorillonite and kaolinite) and iron oxide (goethite) on biofilm formation of soil bacteria (Bacillus subtilis) was explored. The experimental results showed that in the goethite system, the biomass reached the maximum value at 48 h, while the biomass of montmorillonite and kaolinite reached the maximum at 60 h. A compact, multi-layer cell membrane was formed in the 48 h clockwise iron ore system by laser confocal microscopy. After 4 h of the bacteria and minerals, the cell deactivation rate of the goethite system was 60%, followed by kaolinite and montmorillonite. Atomic force microscopy results show close adsorption between bacteria and goethite, acicular goethite can pierce the cell wall, cell induction to the adverse environment, two major genes abrB and sinR formed by regulating the Bacillus subtilis biofilm, biofilm formation in the cells tend to gas-liquid surface movement and aggregation. The physicochemical properties of minerals are the dominant factors that affect the formation of biofilms by minerals. (3) the influence mechanism of typical soil metal oxides of different concentrations (goethite, boomite and sodium manganese ore) on the growth and biofilm formation of Pseudomonas putida was clarified. When the concentration of 1~100 mg L-1 goethite, can promote the growth and biofilm formation of Pseudomonas putida, and goethite concentration was 100~500 mg L-1, the growth and biofilm formation will be inhibited; when the formation of boehmite concentration lower than 20 mg L-1 can promote bacterial growth and biofilm. The formation, growth and biofilm 20~500 mg L-1 boehmite can inhibit Pseudomonas putida; did not affect the growth of birnessite with different concentration on Pseudomonas putida, but can inhibit the formation of bacterial biofilm. Atomic force microscopy images show that the cell membrane of bacteria on the surface of the iron needle was punctured, the damaged cell integrity; the dissolution of boehmite surface cells; sodium manganese cell surface has smaller particle size, surface fold increase. The strength and settling capacity of the interaction of different soil metal oxides and bacteria is the key factor affecting the formation of Pseudomonas aeruginosa biofilm. The EPS in the biofilm can significantly improve the tolerance of Pseudomonas stinosa to mineral.
【学位授予单位】：华中农业大学
【学位级别】：博士
【学位授予年份】：2017
【分类号】：S154.3;S153.6

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