【作者】 曹媛媛；

【导师】 黄巧云；

【作者基本信息】 华中农业大学 ， 土壤学， 2013， 博士

【摘要】 细菌胞外聚合物(EPS)是细菌生长代谢过程中分泌的生物大分子聚合物的总称。EPS与土壤固相的界面作用深刻影响着生物膜的形成、细胞的迁移、矿物的溶解和生物成矿、土壤的形成等诸多地球化学过程,其在重金属等污染物富集中的重要作用也日益受到关注。本文以土壤中常见细菌的EPS和典型矿物、采集并提取的地带性土壤(棕壤)各粒级土壤颗粒为供试材料,基于化学分析方法,综合运用DLVO理论、等温滴定量热(ITC)、扫描电镜(SEM)、傅立叶红外光谱(FTIR)和X-射线吸收精细结构谱(XAFS)等手段,研究了EPS在土壤颗粒和矿物表面的吸附行为和作用机理。表征了6种细菌EPS生物化学性质,考查了它们在针铁矿表面的吸附及其对Cu(Ⅱ)的吸附能力,从统计学角度分析了EPS生物化学性质与吸附的相关性,获得如下主要结果：(1)比较了枯草芽孢杆菌EPS在土壤不同粘土矿物和氧化物表面的吸附行为和作用机理。EPS主要组分为多糖和蛋白质,含量分别为437.6±11.8mg g-1和249.1±3.3mg g-1,并含有少量的核酸(9.9±0.2mg g-1)。EPS含有的官能团分别是羧基(pKa=4.58±0.33)、磷酸基(pKa=6.49±0.18)、氨基或羟基(pKa=9.11±0.41)。蒙脱石、高岭石和针铁矿对EPS-C、-N和-P的等温吸附趋势均能较好地拟合Langmuir方程。蒙脱石对EPS-C的吸附量分别是针铁矿和高岭石4.8倍和8.9倍,对EPS-N的吸附量分别是针铁矿和高岭石8.9倍和16.2倍。针铁矿对EPS-P的吸附量大约是蒙脱石和高岭石的5倍。因此,含有EPS-C和-N的多糖和蛋白质等组分主要被蒙脱石优先吸附,而含有EPS-P的核酸组分则优先吸附于针铁矿表面。EPS在针铁矿表面的吸附亲和力最高,高岭石次之,蒙脱石最小。EPS在矿物表面的吸附随着pH的降低和离子强度的升高而升高,吸附与EPS和矿物表面zeta电位密切相关,静电力主导EPS在矿物表面的吸附。结合FTIR结果可知,除了静电力之外,氢键参与了EPS在蒙脱石和高岭石表面的吸附,配位交换也是针铁矿选择吸附EPS组分的作用力之一。(2)联用DLVO理论、ITC、SEM、FTIR和XAFS技术,结合化学平衡吸附,揭示了恶臭假单胞菌EPS与粘土矿物和氧化物的界面作用机理。恶臭假单胞菌EPS中多糖、蛋白质和核酸的含量分别为532.2±7.0mg g-1、152.2±1.8mg g-1和9.2±0.2mg g-1。随着pH3.0升高到9.0,蒙脱石、高岭石和针铁矿对EPS各元素的吸附百分比均呈下降趋势,这一现象符合DLVO理论,表明静电力主导了EPS在矿物表面的吸附。同时,DLVO理论作用势能估算结果表明,其它引力作用如氢键、疏水作用和共价键也参与了EPS在蒙脱石(pH≥3.0)、高岭石(pH≥5.0)和针铁矿(pH≥9.0)表面的吸附。SEM结果显示,体系pH为7.0时,相比EPS-蒙脱石和EPS-高岭石复合体,EPS-针铁矿复合体结构更加紧密,这与针铁矿等温吸附EPS-C、-N和-P的拟合亲和力参数K均高于蒙脱石和高岭石的现象一致。EPS在矿物表面的吸附是一个放热过程(△Had=-0.02～-12.34Jg-1),这种负吸附焓变是由除静电力外的多种作用力(如氢键、疏水作用和共价键)共同参与作用的结果。FTIR结果证实了EPS在粘土矿物表面吸附过程中有氢键的形成,针铁矿吸附EPS时形成了P-OFe配位键。XAFS明确了EPS中磷酸基团与针铁矿表面内圈络合物的配位信息,结果表明,pn较低时磷酸基团与针铁矿表面羟基为单齿配位,随着pH升高时逐渐转变为双齿配位。(3)考查了不同土壤颗粒对EPS的吸附及离子强度对吸附的影响。恶臭假单胞菌EPS在土壤颗粒表面的吸附可用Langmuir方程定量描述。EPS主要吸附在土壤粘粒表面,有机质的存在阻碍了EPS在土壤颗粒表面的吸附。来源于EPS中蛋白质等分子的EPS-C和-N组分主要被在土壤中的粘粒优先吸附,其中对EPS-N的选择性更强,粉粒和砂粒优先吸附含EPS-C的组分,如多糖等分子。体系pH为6.0时,随着NaCl和CaCl2浓度从0mM增加到30mM, EPS-C和_N在土壤颗粒表面的吸附百分比显著增加,继续增加离子浓度,吸附增加趋势趋于平缓。然而,土壤颗粒对EPS-P的吸附百分比随着离子浓度的增加呈下降的趋势。DLVO理论能够解释土壤粒级和有机质对EPS吸附的影响,以及EPS-C和-N吸附随离子强度变化的趋势,但不能解释离子强度对EPS-P在土壤颗粒表面吸附的影响,这说明EPS-P在土壤颗粒表面的吸附机制不同于EPS-C和_N。(4)全面表征了不同来源EPS的生物化学性质,查明了决定EPS在矿物表面吸附及对重金属固定的关键因子。6种细菌EPS中,多糖和蛋白质含量合计占EPS总质量的65%-86%,多糖与蛋白质含量比在0.14-6.81之间：官能团总量在7.61±0.39mmol g-1至34.78±0.38mmol g-1范围内,平均为23.5±0.66mmol g-1;平均分子量在2.19×104g m01-1至3.51×105g mol-1之间；表面均带负电荷,等效直径在525.9-1701nm范围内。EPS在针铁矿表面的吸附都可用Langmuir模型来描述,与其它5种EPS相比S. suis EPS在针铁矿表面的吸附吸附量较大,并呈不同的吸附趋势。聚合物分子间的作用可能使S. suis EPS在针铁矿表面发生多层吸附。EPS在针铁矿表面的吸附与EPS的大分子组成密切相关,尤其是蛋白质含量决定了EPS在针铁矿表面的吸附量。不同细菌EPS对Cu(Ⅱ)的吸附差异非常明显,吸附量最高可达830.6mg g-1。EPS团聚颗粒的等效粒径是影响EPS对Cu(Ⅱ)的重要因素。更多还原

【Abstract】 Growth and metabolism of bacteria are accompanied by the production of extracellular polymeric substances (EPS), which are complex mixtures of various biomacromolecules. The interactions of EPS with soil solid interface have significant effects on many geochemical processes such as biofilm formation, the migration of bacterial cells, mineral dissolution, biomineralization and forming of soil. The important role of EPS in heavy metals accumulation in soil or aquatic environments has also received growing concern. The adsorption and binding mechanism of EPS from bacteria on Brown soil particles or minerals such as montmorillonite, kaolinite and goethite were studied using batch adsorption experiments, Derjaguin-Landau-Verwey-Overbeek (DLVO) theory, isothermal titration calorimetry (ITC), scanning electron microscopy (TEM), fourier transform infrared spectrumscopy (FTIR) and X-ray Absorption Fine Structure (XAFS). Biochemical properties of EPS from six bacteria and their correlation with adsorption on goethite and adsorption for Cu(Ⅱ) were statistically investigated. The major results were summarized as follows:(1) EPS from Bacillus subtilis are composed dominantly of polysaccharides (437.6±11.8mg g-1) and proteins (249.1±3.3mg g-1), with nucleic acids (9.9±0.2mg g-1) as minor constituents. EPS contain three functional groups which correspond to carboxyl (pKa=4.58±0.33), phosphoryl (pKa=6.49±0.18) and amino or hydroxyl (pKa=9.11±0.41). The adsorption of EPS-C,-N and-P on montmorillonite, kaolinite and goethite conform to Langmuir equation quite well. The amount of EPS-C adsorption on montmorillonite was4.8and8.9-fold greater than that on goethite and kaolinite, respectively. EPS-N adsorption on montmorillonite was also far greater than that on goethite (8.9-fold) and kaolinite (16.2-fold). However, EPS-P adsorbed by goethite was about5-times greater than that by montmorillonite and kaolinite. Therefore, EPS-C and-N constituents (from proteins) are mainly adsorbed by montmorillonite and EPS-P constituents (from nucleic acids) are predominantly adsorbed by goethite. Goethite shows the highest affinity to EPS among the examined clay minerals and iron oxide. An increase in the concentration of cations and/or a decrease in the pH favored EPS adsorption on minerals. Combining with the FTIR result, hydrogen bonding and the electrostatic interaction are the main forces governing the adsorption of EPS on clay minerals. Besides these interaction forces, chemical bonding interactions (ligand exchange) also contribute to selective fractionation of EPS adsorption on goethite.(2) The binding characteristics of EPS from Pseudomonas putida on clay mineral and oxide were studied using a combination of DLVO theory, ITC, SEM, FTIR, XAFS and batch adsorption experiments. Extracted EPS from P. putida consist of polysaccharides (532.2±7.0mg g-1), proteins (152.2±1.8mg g-1) and nucleic acids (9.2±0.2mg g-1). A marked decrease in the adsorption of EPS on montmorillonite, kaolinite and goethite was observed with the increase of pH from3.0to9.0. This phenomenon conforms to DLVO theory and showed that electrostatic forces dominate the adsorption of EPS on mineral surfaces. Hydrogen bond, hydrophobic and covalent may also participate in the adsorption of EPS on montmorillonite (pH≥3.0), kaolinite (pH≥5.0) and goethite (pH≥9.0). SEM showed more firm structrue for EPS-goethite complex than EPS-montmorillonite and EPS-kaolinite complexes at pH7.0, which was in accordance with the higher K value of goethite of adsorption EPS-C,-N and-P than that of montmorillonite and kaolinite. Adsorption of EPS on minerals surfaces were exothermic (AHad=-0.02～-12.34J g-1), and governed by combination of forces such as hydrogen bond, hydrophobic and covalent interaction in addition to electrostatic force. The hydrogen bond between EPS and clay minerals were confirmed by FTIR, as well as P-OFe coordination bond between EPS and goethite. XAFS showed that phosphate groups of EPS can manly form monodentate inner-sphere complexes with Fe centers on the goethite surface at low pH, while gradually changed to the bidentate inner-sphere complexes as pH rising.(3) The adsorption of EPS from P. putida on soil particles could be quantitatively described with Langmuir isotherm equation. EPS are mainly adsorbed by fine soil particles, however organic matter hinder the adsorption of EPS on soil particles. EPS-N moieties mainly from proteins are adsorbed preferentially on clay, and EPS-C moieties predominantly from polysaccharides are adsorbed selectively by silt and sand. The mass fraction of EPS-C, and-N adsorbed on soil particles increased in the presence of0-30mM Na+or Ca2+and remained constant or increased slightly with continuously increasing ionic concentration at pH6.0. In contrast, the adsorption of EPS-P on soil particles was on the decline with the increase of NaCl and CaCl2. DLVO theory can explain the effects of size fraction and organic matter on the absorption of EPS, also predict the adsorption of EPS-C and-N trend with ionic strength. However this theory failed to explain the influence of ionic concentration on adsorption of EPS-P, indicated that the binding mechanism of EPS-P on soil particles was different to EPS-C and-N.(4) The biochemical properties of EPS extracted from different bacteria were characterized in order to charigy the key factors controlling EPS adsorption on minerals and heavy metal binding. Polysaccharides and protein accounted for65%-86%of EPS composition, and the ratios of polysaccharides and protein were0.14-6.81. The total function group contents were from7.61±0.39mmol g-1to34.78±0.38mmol g-1, with an average of23.5±0.66mmol g-1. Mw values of six EPS were from2.19×104g mol"1to3.51×105g mol-1. The equivalent diameter of EPS was in the range of525.9to1701nm, with negatively charged surface. Adsorption of EPS on goethite can be well described by Langmuir model. The adsorbed amount of EPS from S. suis on goethite was far greater than the other EPS, and showed a different trend of adsorption, indicating that polymeric interaction promoted multilayer adsorption of EPS from S. suis on goethite. Adsorption of EPS on goetihte showed a significant correlation with the macromolecular composition of EPS, especially the content of protein. Adsorption of Cu(Ⅱ) by EPS differed markedly, and Cu(II) adsorption capacity was up to830.6mg g-1. The equivalent diameter of EPS was the key factor affecting adsorption of Cu(Ⅱ).更多还原