【作者】 陈光村；

【导师】 陈英旭； 陈新才；

【作者基本信息】 浙江大学 ， 微生物学， 2011， 博士

【摘要】 土壤重金属污染是全球广泛关注的环境问题,地球上99%以上的微生物都是以生物膜的形式存在,与游离态微生物相比,微生物生物膜在土壤重金属的分子形态、生物有效性和迁移转化过程中发挥着更为重要的作用。本论文以高铜锌耐性微生物恶臭假单胞菌CZ1为研究对象,采用原子力显微镜(AFM)、激光共聚焦显微镜(CLSM)、透射电镜(TEM)、X射线吸收精细结构谱(XAFS)和X射线荧光光谱(XRF)等微结构分析技术系统研究了重金属与非饱和生物膜之间的相互作用,从分子水平上初步阐明了非饱和生物膜及其组分在调控重金属耐性和吸收过程中的作用机制,旨在为土壤重金属污染风险评价和修复控制提供理论依据。研究成果主要包括：揭示了不同培养条件下非饱和生物膜中大分子组成和表面特性的变化规律。通过聚酯膜模拟培养恶臭假单胞菌非饱和生物膜,发现生物膜胞内外多糖、蛋白和核酸等大分子物质的组成受营养条件的调控,且胞外多聚物(EPS)更易受到影响。高温、低pH和一定强度的渗透压胁迫能显著促进恶臭假单胞菌CZ1分泌更多的胞外多聚物。随着生物膜的成熟,生物膜表面由光滑变粗糙,细胞表面布满直径15-50nm的椭球形胞外多聚物。在高渗透压胁迫下生物膜中细胞紧密粘合在一起,表明胞外多聚物的合成和生物膜中细胞间的相互协作也是生物膜应对环境胁迫的重要机制。弄清了重金属对游离态微生物、饱和及非饱和生物膜的毒性效应机制。生物膜对铜、锌、铅、铁、锰、镍等重金属都具有很高的抗性和富集能力。与其游离态细胞相比,恶臭假单胞菌生物膜对铜锌的抗性增加了2-8倍,且生物膜中细胞只有在长时间暴露于重金属时才会被逐渐杀死。SEM、 CLSM和AFM的研究结果表明,生物膜的表面形态、空间结构及胞外多聚物都与其高重金属抗性相关。阐明了非饱和生物膜对铜吸收、转运和累积的时空变化规律。XRF分析表明,生物膜中元素的空间分布规律存在显著的空间异质性,生物膜空气界面层是金属元素最主要的分布区域。非饱和生物膜对铜的吸收、转运和累积过程是一个快速主动的过程。生物膜从培养基中以柠檬酸铜的形式吸收铜,运输到生物膜的各个部位,并在靠近空气界面层的约40μm厚的小区域内以类似磷酸铜的形式沉积于胞外,从而保护培养基界面层细胞免受铜的毒害。明确了铜在非饱和生物膜及其不同组分上的分布特征、结合部位及其结合的分子形态。生物膜的EPS是铜的主要分布区,约60-67%的铜分布在EPS中,而其蒴状EPS中铜占到42.3-44.7%。铜胁迫下,胞内蛋白、胞外核酸和胞外酸性多糖的含量显著增加,红外光谱中相应的酰胺Ⅰ带、磷酰基和羧基峰也发生了显著变化,表明这些大分子物质在铜络合中发挥着重要作用。XAFS研究也表明,在生物膜的微生物细胞内、细胞壁和胞外多聚物中,铜分别主要以与巯基、磷酸基和羧基结合的类似形态存在。揭示了胞外多聚物中胞外核酸也能参与铜的络合。随机扩增多态性DNA(RAPD)分析表明,铜胁迫下生物膜的胞外核酸中某些条带含量显著增加,同时也有新特异条带出现。XAFS分析表明与胞内核酸相比,在胞外核酸中与铜配位的氧原子数更少,键长也更短。更多还原

【Abstract】 Heavy metal pollution in soil is one of the global environmental issues. More than99%of microbes live as biofilm, which would paly more important role in the species, bioavailability and transportation processes of heavy metals in soil compared with plan tonic microbes. In the present study, a high heavy metal-resistant rhizobacterium Pseudomonas putida CZ1was studied. The macromolecular composition, surface morphology, spatial structure, metal resistance and accumulation, temporal and spatial distribution of metal elements, as well as the Cu speciation of unsaturated Pseudomonas putida CZ1biofilm were investigated using atomic force microscope (AFM), confocal laser scanning microscope (CLSM), transmission electron microscopy (TEM), X-ray absorption spectroscopy (XAS), X-ray fluorescence microprobe (XRF) combined with conventional physical-chemical methods. These results illustrated the molecular mechanisms of metal resistance, accumulation and transportation within the unsaturated biofilms, which could provide theory basis for risk evaluation and bioremediation of heavy metal in contaminated soils.It was found that the protein, carbohydrate and DNA in the cellular and extracellular fractions of biofilm were regulated by nutritional factors, while the extracellular polymeric substances (EPS) were more easily influenced. Low pH, high temperature and appropriate osmotic stress (120mM NaCl) distinctly stimulated EPS production, and the main component enhanced was extracellular protein. With the aging of biofilm, the surfaces as well as the valleys among cells were studded with15-50nm spheroid-like EPS. Cells in the biofilm adhere tightly together to maintain a hydrated microenvironment under high osmotic (328mM NaCl) stress. These results indicated the variations of EPS composition and the cooperation of cells in biofilms is important for the survival of Pseudomonas putida CZ1from environmental stresses in unsaturated environments such as rhizosphere.P. putida CZ1biofilm has high resistant and accumulation capacity for Cu, Zn, Pb, Fe, Mn, and Ni. It was found that biofilm cultures were2to8times more resistant to Cu and Zn than the planktonic bacteria, and bacterial cells in the biofilm were killed by metals only for long-term exposure. Furtherly, AFM, SEM and CLSM analyses revealed that the spatial structure as well as the EPS of biofilm was responsible for the high metal resistance of biofilm.The spatial and temporal distribution of metals in unsaturated Pseudomonas putida CZ1biofilms was determined using synchrotron-based X-ray fluorescence microscopy (XRF). It was found that Fe, Mn and Ca were primarily located near the air-biofilm interface of biofilms. The sorption of copper by biofilm was rapid, with copper being found throughout the biofilm after only1h of exposure. Copper initially colocalized with Fe and Mn element layers in the biofilm, and then precipitated as copper phosphate in a40μm thick layer near the air-biofilm interface when exposed for12h. The results indicate the heterogeneity of metal distribution within the biofilm is one of the most important mechanisms for high metal resistance and accumulation.Within the biofilm,60-67%of copper were located in the extracellular fraction of biofilms, with44.7-42.3%in the capsular EPS. Enhanced production of cellular proteins, extracellular DNA and alginate were found for Cu stressed biofilm. Additionally, with Cu treatment, the variations of absorption band of amide I,>P=O phosphodiester functional groups and carboxylate group in the FTIR spectra of cells and EPS were observed as well. These results indicated the important role of cellular protein, extracellular DNA and alginate in Cu binding of biofilm. Moreover, XAFS study furtherly revealed that Cu was primarilly bound with hydrosulfide, phosphate and carboxyl like ligands within the cell, cell wall and extracellular polymeric matrix, respectively.Extracellular DNA was also found to play a role in Cu binding. It was found that the composition of extracellular DNA was distinctly changed for Cu stress as revealed by RAPD analyses, and Cu was bound with oxygen ligands in the extracellular DNA with less oxygen and shorter radial distance compared with cellular DNA.更多还原