【作者】 方临川；

【导师】 黄巧云；

【作者基本信息】 华中农业大学 ， 土壤学， 2011， 博士

【摘要】 重金属在土壤中的活性和生物有效性受到多种因素的制约,特别是各种有机胶体、无机矿物以及有机无机复合体对重金属离子的吸附、络合、氧化还原等。因此,重金属与土壤中各种固相组分的相互作用一直是土壤学和环境化学等众多领域科学工作者研究的热点。本文以自然界中常见的细菌、细菌胞外聚合物及粘土矿物为材料、以宏观吸附实验为基础,结合现代仪器分析手段如等温滴定微量热技术(ITC)、傅立叶变换红外光谱(FTIR)、X-射线吸收精细结构(XAFS)技术等系统研究了重金属铜和镉在细菌、胞外聚合物(EPS)、矿物等单一组分和细菌-矿物、胞外聚合物-矿物等二元复合体表面的结合机制。主要结果如下。1.阐明了钝顶螺旋藻(Spirulina platensis 439)、苏云金芽孢杆菌(Bacillus thuringiensis)、大肠杆菌(Escherichia coli)等自然界几种常见细菌的细胞壁表面官能团对Cu(Ⅱ)和Cd(Ⅱ)吸附的贡献。三种细菌对Cu(Ⅱ)和Cd(Ⅱ)吸附能力的大小顺序为：钝顶螺旋藻>苏云金芽孢杆菌>大肠杆菌。细菌表面羧基酯化后,蓝细菌、苏云金芽孢杆菌和大肠杆菌对Cu(Ⅱ)和Cd(Ⅱ)吸附能力分别下降45.6%～55.5%、72.3%～75.3%和8.2%～22.3%,表明羧基在蓝细菌和苏云金芽孢杆菌吸附Cu(Ⅱ)和Cd(Ⅱ)的过程中的贡献远大于其在大肠杆菌中的贡献。电位滴定分析进一步证实,Cu(Ⅱ)和Cd(Ⅱ)主要和苏云金芽孢杆菌表面的羧基结合,而大肠杆菌表面的磷酰基团是其吸附金属离子的主要位点。NH4NO3和EDTA对蓝细菌表面吸附态铜的解吸率分别为53.7%和72.7%,吸附态镉的解吸率分别为58.0%和80.7%,这表明离子交换和表面络合是钝顶螺旋藻吸附铜和镉的主要机理。XAFS分析结果阐明羧酸铜的双五元螯合环结构是Cu(Ⅱ)在蓝细菌表面的主要形态。2.首次联用ITC和XAFS技术、结合等温吸附和动力学模型拟合,阐明了枯草芽胞杆菌(Bacillus subtilis)表面游离态EPS对Cu(Ⅱ)的吸附机制。结果表明EPS对Cu(Ⅱ)的吸附是个快速过程,在开始10 min内,吸附量可以达到最大吸附量的95%以上,60 min内吸附达到平衡。离子强度的增加显著降低Cu(Ⅱ)在EPS表面的吸附量和吸附速率,准二级动力学方程、Elovich方程和颗粒内扩散方程均能较好拟合不同离子强度下EPS对Cu(Ⅱ)的吸附,拟合结果显示,化学吸附及吸附初期的内部扩散作用对EPS吸附铜有着重要的影响。铜在枯草芽胞杆菌和EPS表面的配位形态非常类似,主要与羧基结合形成内圈络合物。铜在枯草芽胞杆菌表面形成单五元螯合环和双五元螯合环两种结构,而在EPS表面则以单五元螯合环为主。EPS中的高亲和力位点与铜的吸附热比低亲和力位点的更大,是五元螯合铜环形成的主要部位。3.查明了细胞表面固定态EPS对细菌吸附铜和镉的影响机制。枯草芽胞杆菌(Bacillus subtilis)和恶臭假单胞菌(Pseudomonas putida)表面官能团总的浓度分别为2.89×10-3和1.85×10-3molg-1。去除EPS后,两种细菌表面官能团的数量分别减少62.3%和38.9%。去除EPS后的枯草芽胞杆菌对Cu(Ⅱ)和Cd(Ⅱ)的最大吸附量分别下降37.8%和51.4%；去除EPS后的恶臭假单胞菌体对Cu(Ⅱ)和Cd(Ⅱ)的吸附量分别下降25.4%和9.7%,表明EPS的去除显著降低细菌表面吸附位点的数量及吸附重金属的能力,EPS对枯草芽胞杆菌吸附重金属的影响更大。去除EPS前后的枯草芽胞杆菌、恶臭假单胞菌对质子的吸附行为,都可以用三位点非静电络合模型进行拟合,且羧基、磷酸基及羟基是细菌表面的三种主要官能团。去除EPS前后的细菌细胞壁的组成和化学特性没有发生改变,铜和镉主要与细菌表面的羧基、磷酸基团配位。去除EPS前后菌体对质子和金属离子吸附行为的相似性暗示,金属离子在细菌和生物膜上的吸附行为可以用相同的模型进行描述。4.揭示了细菌和矿物的相互作用对细菌-矿物复合体表面吸附位点及重金属吸附行为的影响机制。结果表明,细菌、矿物等单一组分对Cu(Ⅱ)的吸附能力大小顺序为：苏云金芽孢杆菌(28.15 mgg-1)>恶臭假单胞菌(20.70 mg g-1)>蒙脱石(14.30mg g-1)>高岭石(9.43 mg g-1)>针铁矿(3.78 mg g-1)。苏云金芽孢杆菌,恶臭假单胞菌与蒙脱石形成复合体后,其表面位点浓度升高1.94%-6.20%,吸附能力增加16.4%-30.6%；而与针铁矿形成复合体后,其表面位点浓度和吸附能力分别减少6.26%和19.6%。这表明细菌与矿物的相互作用机制决定细菌-矿物复合体的表面活性位点浓度。蒙脱石-细菌间松散结合的相互作用能增加复合体表面的吸附位点,而针铁矿-细菌间紧密结合的相互作用则掩蔽一些反应位点。苏云金芽孢杆菌(革兰氏阳性)对细菌-矿物复合体吸附行为的影响程度远大于对恶臭假单胞菌(革兰氏阴性)的影响。Cu(Ⅱ)在细菌及细菌-矿物复合体表面的吸附是吸热过程,而在矿物表面的吸附为放热反应。首次获得了金属离子在细菌-矿物复合体表面吸附的热力学参数,Cu(Ⅱ)在复合体表面吸附的焓变和熵变分别为-0.78-6.14kJ mol和32.96-58.89 J mol-1 K-1,意味着Cu(Ⅱ)主要与细菌-矿物复合体表面的羧基和磷酸基形成内圈络合物。5.首次探讨了EPS-矿物复合体对铜的吸附行为。EPS与蒙脱石混合后,表面位点浓度增加5.2%,吸附Cu(Ⅱ)的能力提高13.9%；与之相反,EPS-针铁矿复合体的表面位点浓度降低8.5%,铜吸附量下降19.1%。说明EPS与蒙脱石形成复合体的过程可能创造出了一些新的重金属吸附位点,而与针铁矿复合过程中则掩蔽了一些反应位点。ITC分析结果表明,Cu(Ⅱ)在EPS及EPS-矿物复合体表面吸附的焓变(△H)为19.34-24.11 kJ mol-1,熵变为99.53-121.98 J mol-1K-1。EPS表面位点与Cu(Ⅱ)的结合热是复合体吸附Cu(Ⅱ)过程中热量的主要来源。说明Cu(Ⅱ)在EPS-矿物复合体表面主要与羧基和磷酸基团配位形成内圈络合物。6.获得了EPS与针铁矿的相互作用的分子机制。结果表明,针铁矿对EPS中各组分的吸附量大小顺序为：EPS-C (27.57 mgg-1)> EPS-N (10.27 mgg-1)>EPS-P (6.32 mg g-1)。EPS各组分与针铁矿吸附的亲和力及分配系数的大小顺序则为：EPS-P>EPS-N>EPS-C,表明EPS中的含P组分与针铁矿的结合最牢固,且被优先吸附。FTIR结果显示,EPS与针铁矿吸附后,>P=O双键振动吸收峰消失,新增了P-O-Fe的伸缩振动特征吸收峰,说明EPS中的磷酸基团与针铁矿表面羟基配位形成内圈络合物,而且这些磷酸基团主要来自EPS蛋白质和核酸中的磷酰基。XAFS结果表明,低pH时(pH 3),磷酸基团仅有一个去质子化的含氧阴离子直接与针铁矿表面的FeOH1/2-基团结合形成单齿络合物；高pH时(pH 9),磷酸盐基团中有2个含氧阴离子与针铁矿表面的2个FeOH1/2-基团结合形成双齿络合物。体系pH由低到高时(3-9),吸附产物构型由单基配位向双基配位过渡。体系pH通过影响溶液中EPS-P中磷酸根质子解离和缔合,是导致吸附产物构型变化的重要原因。更多还原

【Abstract】 The mobility, speciation, transport and bioavailability of toxic metallic cations in the soil environment depend largely on their interactions (adsorption, complexation and redox) with inorganic and organic surfaces, principally microorganisms, minerals and their composites, respectively. Therefore, the interactions of heavy metals and soil components are always the hot spots studied by environmental chemistry and soil science investigators. In our studies, the common bacteria, extracellular polymeric substances (EPS) and minerals were used as model materials. The binding characteristics of Cu(Ⅱ) and Cd(Ⅱ) by the individual components (bacteria, EPS, minerals) and binary composites (bacteria-mineral composites, EPS-mineral composites) were investigated using a combination of chemical modifications, batch adsorption experiments, isothermal titration calorimetry (ITC), Fourier transform infrared spectroscopy (FTIR), X-ray absorption fine structure (XAFS) spectroscopy. The main results were listed as following:1. The effect of functional groups on Cu(Ⅱ) and Cd(Ⅱ) adsorption on bacteria (Bacillus thuringiensis, Escherichia coli, Cyanobacterium Spirulina platensis) were studied. The order of Cu(Ⅱ) and Cd(Ⅱ) adsorption capacity was S. platensis> B. thuringiensis> E. coli. Esterified cells resulted in the reduction in the binding of Cu(Ⅱ) and Cd(Ⅱ) on S. platensis, B. thuringiensisand E. coli by 45.6%-55.5%,72.3%-75.3% and 8.2%-22.3%, respectively, which demonstrated that the carboxyl groups on S. platensis and B. thuringiensis surfaces play a more important role in the binding of metal ions than that on E. coli. Potentiometric titration results provide the further evidences that the carboxyl groups on B. thuringiensis surfaces was the major ligands responsible for the binding of Cu(Ⅱ) and Cd(Ⅱ). As for E. coli, it is likely that the phosphate groups were important for the binding of metal ions. A percentage of 53.7% and 72.7% of adsorbed Cu(Ⅱ) on S. platensis surfaces was desorbed by NH4NO3 and EDTA, respectively. The percent desorption of Cd(Ⅱ) by NH4NO3 and EDTA was 58.0% and 80.7%. This results indicated that Ion exchange and complexation are the dominating mechanisms for Cu(Ⅱ) and Cd(Ⅱ) adsorption on S. platensis surfaces. XAFS analysis provided further evidence for the inner-sphere complexation of Cu by carboxyl ligands and showed that Cu was complexed by two 5-membered chelate rings on S. platensis surface.2. The binding characteristics of Cu(Ⅱ) by the soluble EPS of Bacillus subtilis were investigated using a combination of XAFS, ITC and batch adsorption experiments. The results showed that the adsorption kinetics was rapid and 95% of biosorption capacity was achieved in the first 10 min of contact and then attained adsorption equilibrium in 60 min. The adsorption capacity and rate decreased with the increase of ion strength. The kinetics experiments demonstrated that Second-order equation, Intra-particle diffusion and Elovich equation all provide the good fit to the experimental data. The fitting results indicated that a chemical reaction mechanism and intra-particle diffusion in the first time play an important role in Cu(Ⅱ) adsorption. Furthermore, XAFS analysis provided further evidence on the inner-sphere complexation of Cu by carboxyl ligands for the adsorption of Cu(II) on EPS (B. subtilis) and B. subtilis surface. Cu(Ⅱ) is complexed by one or two 5-membered chelate rings on B. subtilis surface and is complexed by one 5-membered chelate on EPS (B. subtilis). The larger adsorption heat was observed for the incteraction between Cu(Ⅱ) and the high-affinity sites on the EPS. Moreover, the high-affinity sites on the EPS were the major place for the formation of 5-membered chelate rings.3. The role of bound EPS in Cu(Ⅱ) and Cd(Ⅱ) adsorption by Bacillus subtilis and Pseudomonas putida was investigated. The total site concentrations on untreated B. subtilis and P. putida surface were 2.89×10-3 and 1.85×10-3mol g-1 and decreased by 62.3%and 38.9%, respectively, after EPS molecules were removed by CER, suggesting that the removing of EPS from bacterial cells can significantly reduce the site concentrations on bacterial surfaces. As compared with untreated bacteria, Cd(Ⅱ) adsorption decreased by 51.4% and 9.7%, respectively on EPS-free B. subtilis and P. putida. Cu(Ⅱ) adsorption decreased by 37.8% and 25.4%, respectively on EPS-free B. subtilis and P. putida. These results indicated that the absence of EPS on bacteria may significantly reduce the concentration of binding sites and Cu(Ⅱ) adsorption capacity, especially for Gram-positive B. subtilis. Surface complexation modeling of titration data showed the similar pKa values of functional groups (carboxyl, phosphate and hydroxyl) between untreated and EPS-free bacteria. A three site non-electrostatic surface complexation modeling of titration data showed the similar pKa values of functional groups (carboxyl, phosphate and hydroxyl) between untreated and EPS-free bacteria. FTIR spectra also showed that no significant difference in peak positions was observed between untreated and EPS-free bacteria and carboxyl and phosphate groups were responsible for Cd adsorption on bacterial cells. Our study suggested that generalized model could be used to quantify the bacteria-metal adsorption behavior in geologic systems.4. Impact of bacteria and mineral types on the surface sites and adsorption behaviors were revealed in this study. As for individual components, the order of Cu(Ⅱ) adsorption capacity was B. thuringiensis (28.15 mg g-1)> P. putida (20.70 mg g-1) montmorillonite (14.30 mg g-1)> goethite (9.43 mg g-1)> kaolinite (3.78 mg g-1). The B. thuringiensis- and P. putida-montmorillonite mixture have more adsorption sites (1.94%～6.20%) and bound 16.4%-30.6%% larger amount of Cu(Ⅱ) than that predicted by their individual components. However, the bacteria-goethite composites have less adsorption sites (6.26%) and bound 19.6% less amount of Cu(Ⅱ) than that predicted by their individual components that predicted by their individual components. The different changes in the concentration of surface sites between montmorillonite- and goethite-bacteria composites suggest that the strength of interaction between bacteria and minerals affects the concentration of reactive sites on their composite surfaces. Our results demonstrated that the interaction of montmorillonite with bacteria increased the reactive sites and resulted in greater adsorption of Cu(Ⅱ) on their composites, while goethite-bacteria composite decreased surface sites and adsorption capacity for Cu(Ⅱ). XAFS analysis showed that the adsorption of Cu(Ⅱ) on bacteria and their composites with minerals was an endothermic reaction, while that on minerals was exothermic. The enthalpy changes (△Hads) from endothermic (6.14 kJ mol-1) to slightly exothermic (-0.78 kJ mol-1) suggested that Cu(Ⅱ) is complexed with the anionic oxygen ligands on the surface of bacteria-mineral composites. Large entropies (32.96-58.89 J mol-1 K-1) of Cu(Ⅱ) adsorption onto bacteria-mineral composites demonstrated the formation of inner-sphere complexes in the presence of bacteria. The thermodynamic data obtained in this study are the first to investigate the binding mechanism in terms of calorimetric determinations, which implied that Cu(Ⅱ) mainly bound to the carboxyl and phosphoryl groups as inner-sphere complexes on bacteria and mineral-bacteria composites.5. The adsorption of Cu(Ⅱ) by EPS extracted from Pseudomonas putida, minerals and their composites were investigated. The EPS-montmorillonite mixture have 5.2% more adsorption sites and bound 13.9% larger amount of Cu(Ⅱ) than that predicted by their individual components, However, the bacteria-goethite composites have 8.5% less adsorption sites and bound 19.1% less amount of Cu(Ⅱ) than that predicted by their individual components that predicted by their individual components. Our results presented that the interaction of montmorillonite with EPS increased the reactive sites and resulted in greater adsorption of Cu(Ⅱ) on their composites, while goethite-EPS composite decreased surface sites and adsorption capacity for Cu(Ⅱ). The△Hads values of the adsorption of Cu(Ⅱ) on EPS and mineral-EPS composites were in the range of 19.34～24.11 kJ mol-1. The measured△Sads values for Cu(Ⅱ) adsorption on EPS and mineral-EPS composites (99.53～121.98 J mol-1 K-1) indicated that Cu(Ⅱ) mainly interacts with carboxyl and phosphoryl groups as inner-sphere complexes on EPS molecules or their composites with minerals. The thermodynamic data obtained in this study are the first to investigate the binding mechanism in terms of calorimetric determinations, which implied that Cu(II) mainly interacts with carboxyl and phosphoryl groups as inner-sphere complexes on EPS molecules or their composites with minerals.6. The interactive molecular of goethite with extracellular polymeric substances (EPS) isolated from P. putida was investigated. The adsorption isotherms of EPS on goethite conformed to the Langmuir equation and the amount of EPS-C,-N and-P adsorbed followed the order:EPS-C (27.57 mg g-1)> EPS-N (10.27 mg g-1)> EPS-P (6.32 mg g-1). However, the adsorption energy constant (K) and distribution coefficient (Kd) of EPS on goethite were in the sequence of EPS-P> EPS-N> EPS-C, indicating that P-containing moieties was adsorbed strongly and preferentially than EPS-N and EPS-C. Emergence of the new band and the disappearance stretching vibration of PO2 are consistent with inner-sphere complexation of EPS phosphate groups (deriving principally from phosphodiesters of nucleic acids and proteins) at goethite surface hydroxyls. XAFS studies demonstrated that phosphate can form monodentate or bidentate inner-sphere complexes with goethite surface sites and the structure of complexes is sensitive to changes in pH. The two different inner-sphere structure may occur for the adhesion of EPS to a-FeOOH surface.1) The phosphate groups of EPS can form a monodentate inner-sphere complex, where one oxygen of the anion binds directly the Fe atom of a FeOH1/2-group, releasing the attached OH-.2) A bidentate inner-sphere complex, where two oxygens of the anion bind two Fe atoms of two adjacent FeOH1/2-groups. The phosphate groups of EPS. Solution pH is an important factor affecting the complexes structures due to the influence of pH on the deprotonation or protonation of the phosphate groups of EPS.更多还原