【作者】 赵文强；

【导师】 黄巧云； 蔡鹏；

【作者基本信息】 华中农业大学 ， 土壤学， 2013， 博士

【摘要】 全球畜牧养殖业每年产生1010t-1011t的粪便排泄物,其中我国数量为3×109t。这些废弃物中含有大量肠道病原菌,以大肠杆菌、猪链球菌和沙门氏菌数量最多,若未经有效处理就将其排放到土壤或作为有机肥施入到农田,便会严重污染环境并威胁人类健康。近年来,世界各国报道了多起肠道病原菌污染问题,例如2005年我国四川的猪链球菌感染事件以及2011年欧洲的大肠杆菌中毒(毒黄瓜)事件,引起数千人染病或死亡。病原菌进入土壤后,可吸附在土壤颗粒上或随水流进行运移,该过程深刻影响其感染活性和扩散范围。因此,有必要详细研究病原菌与土壤的界面作用和代谢活性机制,以减缓类似事件的爆发,这对于预测病原菌在土壤环境中的分布规律和制定污染土壤修复方案具有重要理论和实际意义。本文以大肠杆菌(Escherichia coli)、猪链球菌(Streptococcus suis)以及多种土壤组分(粘土矿物、地带性和非地带性土壤)为材料,综合运用等温吸附、土柱实验、扫描电子显微镜、微量热、电位滴定、傅里叶变换衰减全反射红外光谱等技术,考察了菌株种类、溶液条件(pH和离子浓度)、土壤类型、有机质含量和胞外聚合物对病原菌吸附的影响,获得了细菌与不同土壤颗粒间的吸附能力、运移距离、表面形貌、代谢活性和官能团变化等信息。同时,借助zeta电位仪、PATH法(Particle adhesion to hydrocarbon)、比表面仪、X射线衍射和土壤物理化学性质测定方法,系统测定了表面电荷、疏水性、比表面积、矿物组成、阳离子交换量、有机质、质地、电导率等性质,用以全面解释互作机制。此外,运用经典的DLVO胶体稳定理论(Classic Derjaguin-Landau-Verwey-Overbeek theory)和统计学手段(一元线性回归、偏相关和多元逐步回归)分析细菌与土壤组分间的作用能信息和显著性影响因素,揭示了吸附过程中的主导作用力及其贡献程度。获得的主要结果如下：(1)探明了病原菌在粘土矿物表面的吸附行为。二种病原菌在蒙脱石和高岭石上的吸附等温线符合Freundlich方程(R2>0.91),猪链球菌在矿物上吸附的分配系数是大肠杆菌的2倍-8倍,蒙脱石对细菌的吸附量(0.63mL g-1-5.07mLg-1)大于高岭石(0.58mL g-1-1.23mL g-1)。扫描电镜图片显示,大肠杆菌呈杆状,长度大于1μm,猪链球菌粒径略小于大肠杆菌,呈卵圆形。提高溶液pH值(4.0-9.0)或降低离子浓度(20mmol L-1-1mmol L-1)能使细胞和矿物表面负电荷量逐渐增多,静电斥力变大,从而导致吸附量不断减少。DLVO理论计算的作用能数据表明,斥力能障值在该溶液条件下不断升高(1.4快T-408.1kT),与吸附趋势相吻合。各体系吸附量与对应能障值呈显著负相关(pH:Y=-0.031×X+13.4,R2=0.469,P<0.01;离子浓度：Y=-0.004×X+2.7,R2=0.354, P<0.05), DLVO作用力(静电斥力和范德华引力)显著影响着吸附过程。高离子浓度下(20mmol L-1-100mmol L-1),猪链球菌在2种矿物上的吸附量均出现显著下降,偏离了DLVO理论的预测结果,由细菌胞外聚合物与矿物间的空间位阻作用(非DLVO作用力)引起。CaCl2对胶粒表面扩散双电层的压缩作用更强,降低了互作体系间的能障值(0.8kT-52.7kT),且能在细菌和矿物之间形成多价阳离子桥,对吸附量的促进作用强于KCl。(2)揭示了病原菌在不同粒径土壤颗粒中的吸附、运移与代谢活性规律。土壤颗粒对病原菌吸附能力的大致顺序为：粘粒(<2gm)>粉粒(2μm-48μm)>砂粒(48μm-250μm),去有机质颗粒(9.9×1010cells g-1-59.4×1010cells g-1)>含有机质颗粒(7.8×1010cells g-1-43.9×1010cells g-1)。土壤颗粒表面的zeta电位与比表面积大小与该趋势一致,可从长程的扩散双电层作用力和表面位点角度解释吸附行为,短程疏水作用力和阳离子交换量不能作为土壤-病原菌相互作用的预测指标。微量热参数表明,相比对照体系(只含细菌和培养基),加入粉粒和砂粒后,猪链球菌生长的时间-功率曲线峰值PH相对于自由态细菌(289.6μW)升高了8.1%-27.1%,对应出峰时间PT值(391.0min-408.7min)早于对照体系(424.7min),总体代谢活性增强；粘粒体系PH值下降了11.4%-23.2%,出峰时间较晚(441.7min-464.7min),猪链球菌代谢活性减弱。大肠杆菌在去有机质粘粒上的PH值(119.6μW)小于对照体系(147.2μW),出峰时间更晚(313.5min>281.9min),活性受到抑制,其它5种土壤颗粒均促进大肠杆菌的代谢活性。扫描电镜直接证实了病原菌在砂粒和粉粒表面的吸附,细胞分布较为分散,有利于细胞对土壤颗粒表面吸附的营养物质进行充分分解,促进细菌的代谢活性。粘粒紧密包裹覆盖在细胞上,外表面难以观察到清晰的吸附态细菌,阻碍了细菌生长空间及其与外界营养物质和代谢产物的利用和交换,抑制其生长和代谢过程。猪链球菌能运移到20cm深土层,比大肠杆菌运移距离更远(10cm),物理阻塞对病原菌运移的影响力高于吸附作用。(3)阐明了病原菌在土壤胶体颗粒上的作用力机制。猪链球菌在红壤胶体表面吸附的分配系数(Kf)是大肠杆菌的4.5倍-6.1倍,细菌在去有机质胶体表面吸附的Kf值为含有机质胶体的2.4倍-3.2倍。比表面积越大或表面负电荷量越少,细菌吸附能力越强,大肠杆菌表面电荷密度较高(541.1μC cm-2),进一步增强与土壤胶体间的静电斥力。猪链球菌和大肠杆菌zeta电位与对应吸附量可分别拟合指数和线性方程,吸附态细菌位于距土壤胶体表面90nm-100nm处的次级小能位置。随着溶液体系pH降低(9.0--4.0)或KCl离子强度增大(1mmol L-1-10mmol L-1),细菌与红壤胶体互作能障降低(354.6kT-0.3kT),次级小能引力增强(-0.020kT--0.536kT),分隔距离不断缩短(111nm-16nm).此时细菌吸附量持续增大,与能障值呈显著负相关(P<0.05),吸附机制符合DLVO理论,疏水作用力影响不显著。高离子强度下(50mmol L-1-100mmol L-1),猪链球菌在去有机质和含有机质颗粒上的吸附量分别降低了3.4%和5.6%,细胞表面蛋白质和土壤有机质共同增强了空间位阻排斥力。(4)探讨了土壤性质与病原菌吸附能力间的相关性及贡献程度。一元线性回归结果表明,土壤溶液pH(P<0.01)和电导率EC(P=0.033)与猪链球菌分配系数呈显著负相关,能分别解释81.9%和38.4%的吸附过程。大肠杆菌分配系数仅与溶液电导率呈显著正相关(P<0.01),R2值高达0.923。偏相关分析发现,在排除其它因素的间接影响后,pH(P=0.013)和电导率(P=0.034)分别是猪链球菌和大肠杆菌吸附的决定性因素。pH显著促进猪链球菌的吸附量(P<0.05),而电导率此时无显著影响(P>0.05),对吸附能力有极微弱的促进作用(偏相关系数为0.298),与一元线性回归相矛盾。这是因为pH和有机质对吸附能力的抑制作用较大(偏相关系数分别为-0.952和-0.735),从而屏蔽了电导率对土壤的微弱影响,偏相关更能反映单独的某种土壤性质对细菌吸附的真实作用。土壤有机质、粘粒含量、比表面积、阳离子交换量与分配系数无显著相关(P>0.05),仅能单独解释30%以下的吸附行为。扫描电镜手段显示病原菌主要吸附于土壤团聚体的外表面,无法吸附在团聚体内部的小尺寸颗粒表面。球面-平板DLVO理论计算的能障值(Energy barrier, EB)与大肠杆菌分配系数Ks呈显著负相关：Ks=-0.057×EB+22.6(R2=0.577,P<0.01,n=10),但该模型无法解释猪链球菌在土壤颗粒上的吸附行为。通过多元逐步回归手段可得到病原菌分配系数与土壤性质的相关性方程,猪链球菌：Ks=-45.93×pH.1.31×CEC+389.75(R2=0.929,P<0.01)；大肠杆菌：Ks=0.24×EC+2.005(R2.=0.932,P<0.01).该方程拟合值与实测值较为接近,相差数值小于14.3mL g-1,可初步用来预测细菌在土壤表面的吸附能力。(5)明确了胞外聚合物对病原菌表面性质及吸附能力的影响。采用阳离子交换树脂(CER)去除细胞表面的胞外聚合物(Extracellular polymeric substances, EPS),红外光谱数据显示,CER处理后的去EPS细胞在3500cm-1-1000cm-1波数范围内的吸收峰均有明显降低、消失或偏移,表面相应的蛋白质、多糖和脂类物质含量被去除。大肠杆菌细胞壁表面含有羟基、羧基、酰胺基、磷酸基、酯基、醛基、巯基等基团,官能团种类比猪链球菌更为丰富。去除EPS后,2种细菌表面电荷密度和总位点浓度分别降低了7%-17%和3%-7%。离子强度从1mmol L-1上升到100mmolL-1,细菌表面负电荷量逐渐减少。电荷量大小顺序为：含EPS猪链球菌<去EPS猪链球菌<去EPS大肠杆菌<含EPS大肠杆菌。4种细菌表面疏水性范围为3%-43%,去除EPS后,猪链球菌疏水性上升了约5%,大肠杆菌疏水性平均减少了11%。含EPS和去EPS细胞表面性质受官能团种类和EPS组成影响。含EPS猪链球菌在黄棕壤上吸附的分配系数Ks值最大(49.8mL g-1),其次是去EPS猪链球菌(16.1mLg-1)、去EPS大肠杆菌(8.2mL g-1)和含EPS大肠杆菌(8.0mLg-1)。1mmol L-1-60mmol L-1离子强度下的吸附趋势符合球面-平板DLVO模型,吸附量(Y)与斥力能障值(X)呈显著线性负相关：Y=-0.0064×X+2.99(R2=0.602, P<0.01)。当离子强度从60mmol L-1上升到100mmol L-1时,含EPS猪链球菌在土壤颗粒上的吸附量下降了16.0%,而去EPS猪链球菌的吸附量逐渐增大,这表明猪链球菌与土壤间的空间位阻排斥力来源于细胞表面的EPS组分。更多还原

【Abstract】 The world annually produces1010-1011tons of animal manure, whereas the production is3×109tons in China. These wastes contain large amount of enteric pathogens, the most of which are Escherichia coli, Streptococcus suis and Salmonella enterica. The fecal wastes are partly applied to the soil as a source of plant nutrients. If they are not managed properly, the risk of contaminating environment and threatening human health will be increased severely. In recent years, many countries over the world have reported the problems of enteric pathogenic pollution. For instance, the large outbreaks of Streptococcus suis in China and Escherichia coli in Europe (poisonous cucumber) suddenly appreared in2005and2011, respectively, which caused several thousand people infected or died. After pathogens enter into the soil, they can adsorb on soil particles or be transported with water flow, which remarkably affects their infection activity and the extent of spreading. Therefore, there is a need to comprehensively investigate the mechanisms of interfacial interactions and metabolic activities between pathogens and soils, so as to decrease the exposure of similar incidents. Such knowledge is also of theoretical and practical importance in predicting the pathogen distribution in soil environments and developing plans for the remediation of contaminated soils. The test materials include Escherichia coli, Streptococcus suis and various soil components (clay minerals, zonal and azonal soil types). Adsorption isotherm, soil column experiment, scanning electron microscope, micro-calorimetric, potentiometric titration and attenuated total reflection fourier transform infrared spectroscopy techniques were utilized to study the effects of bacterial strain, solution condition (pH and ionic strength), soil type, organic matter content and extracellular polymeric substances on pathogen adsorption. The information of adsorption capacity, transport distance, surface appearance, metabolic activity and the change of functional groups between bacteria and different soil particles were obtained. Meanwhile, zeta potential analyzer, particle adhesion to hydrocarbon test, specific-surface-area-measuring equipment, X-ray diffraction and soil physicochemical determining methods were employed to systematically evaluate the surface charge, hydrophobicity, specific surface area, mineral constituent, cation exchange capacity, organic matter, soil texture and electric conductivity. These properties were used to explain the overall interaction mechanisms. Additionally, the information of interaction energies and significant impact factors between bacteria and soil components were analyzed by applying the classic Derjaguin-Landau-Verwey-Overbeek theory and statistical tools (simple linear regression, partial correlation and multiple stepwise regression analysis). The major findings were summarized as follows:(1) Adsorption behaviors of pathogens on clay minerals were investigated. The adsorption isotherms of two pathogenic strains on montmorillonite and kaolinite conformed to the Freundlich equation (R2>0.97). The partition coeffients of S. suis were2-8times as high as those of E. coli. More bacterial cells were found to be adsorbed by montmorillonite (0.63mL g-1-5.07mL g-1) than by kaolinite (0.58mL g-1-1.23mL g-1). Scanning electron microscope images indicate that E. coli is a rod-shaped strain of>1μm-long length, while the size of S. suis is shorter than E. coli and exhibit ovoid shape. Increasing solution pH (4.0-9.0) or decreasing ionic concentrations (20mmol L-1-1mmol L-1) result in the increase of negative charges on bacterial and mineral surfaces, which enlarges the electrostatic repulsions and leads to the reduction of adsorption amount. The interaction energy data calculated by DLVO theory suggest that repulsive energy barriers increased continuously (1.4kT-408.1kT) under these solution conditions, aggreeing with the adsorption trends. Adsorption amounts of each system are significantly negatively correlated with corresponding energy barriers (pH:Y=-0.031×X+13.4, R2=0.469, P<0.01; ionic concentration:Y=-0.004×X+2.7, R2=0.354, P<0.05). DLVO-type forces (electrostatic repulsion and van der Waals attraction) significantly affect the adsorption processes. At higher ionic concentrations (20mmol L-1-100mmol L-1), the adsorption of S. suis on two clay minerals both decreased significantly, which deviated from the results predicted by DLVO theory. This phenomenon was caused by the steric hindrance (non-DLVO force) between the extracellular polymeric substances and clay minerals. CaCl2could efficiently compress the diffuse double layer outside the colloidal surfaces, decrease the energy barriers (0.8kT-52.7kT) among the interaction systems, and form multivalent-cation-bridge between bacteria and minerals. These effects induced the result that CaCl2was more effective than KCl in enhancing adsorption amount.(2) The adsorption, transport and metabolic activity phenomena of pathogens on soil particles of different sizes were elucidated. The adsorption capacities of pathogens on soil particles generally followed the order:clay (<2μm)> silt (2μm-48μm)> sand (48μm-250μm), inorganic particle (9.9×1010cells g-1-59.4×1010cells g-1)> organic particle (7.8×1010cells g-1-43.9×1010cells g-1). The zeta potential and specific surface area values of soil particles were consistent with the adsorption trends. The long-range diffuse double layer interaction force and surface available site contributed to the adsorption behaviors. However, the short-range hydrophobic force and cation exchange capacity values could not be considered as the parameters for predicting the soil-pathogen interactions. Micro-calorimetric data showed that the PH values of power-time curves in the systems of silts and sands increased by8.1%-27.1%than those in the system of free S. suis (289.6μW). The corresponding PT values occurred earlier (391.0min-408.7min) than the control experiment (424.7min). Metabolic activities of S. suis were enhanced. As a result of the decreasing PH (11.4%-23.2%) and larger PT values (441.7min-464.7min), the metabolic activities of the S. suis-clay systems were inhibited. The PH value of E. coli-inorganic clay system (119.6μW) was lower than that of control experiment (147.2μW), and its PT value occurred later (313.5min>281.9min), which restrained the acivity of E. coli. The other five soil particle types all promoted E. coli’s metabolic activities. Scanning electron microscope directly confirmed that pathogens adsorbed on silt and sand surfaces, and the cells were dispersedly distributed. Thus, the adsorbed nutrient substances could help the cells to decompose nutrients more sufficiently, which promoted bacterial metabolic activities. The cell surfaces were covered with clays tightly, and the soil outside surfaces did not show adsorbed cells distinctly. This phenomenon restrained the bacterial utilization and exchange processes of external nutrient materials and metabolites, as well as their growth spaces. So the clay particles repressed bacterial growth and metabolic activities.5. suis was able to transport to the20cm-depth soil layer, which was deeper than E. coli (10cm-depth). Physical straining had greater influence on pathogen transport than adsorption behavior.(3) The interaction force mechanisms of pathogen adsorption on soil colloidal particles were clarified. The partition coefficients (Kf) of S. suis adsorption on soil colloids were4.5-6.4times as large as those of E. coli, while the Kf values of bacterial adsorption on inorganic colloids were2.4-3.2times as large as those on organic colloids. The larger the specific surface areas and the fewer negative charges on cell or soil colloidal surface, the greater adsorption capacity of bacteria. The surface charge density of E. coli was higher, further strengthening the electrostatic repulsions between E. coli cells and soil colloids. The correlations between the zeta potentials of S. suis and E. coli and the corresponding adsorption amount fitted exponential and linear equations, respectively. Pathogens adsorbed in the secondary energy minima at separation distances of90nm-100nm away from the soil colloids. With the decrease of pH (9.0-4.0) or the increase of KCl concentrations (1mmol L-1-10mmol L-1), the interaction energy barriers between the cells and soil colloids reduced (354.6kT-0.3kT). The attractive secondary energy minima increased (-0.020kT--0.536kT), and the separation distances decreased (111nm-16nm) constantly. Under these conditions, bacterial adsorption amount increased continuously, which were significantly negatively correlated with the energy barriers (P<0.05) and consistent with DLVO theory. Hydrophobic force had a negligible impact on cell adsorption. At higher ionic strengths (50mmol L-1-100mmol L"1), S. suis adsorption on inorganic and organic colloids decreased by3.4%and5.6%, respectively. Cell surface proteins and soil organic matter both enchanced the steric repulsions.(4) The correlations and contribution rates between soil properties and pathogen adsorption capacities were analyzed. Simple linear regression results show that soil solution pH (P<0.01) and electric conductivity (P=0.033) were significantly negatively correlated with the partition coefficients of S. suis, which could explain81.9%and38.4%of the adsorption processes. The partition coefficients of E. coli were only significantly correlated (positive) with solution electric conductivity, with R2values of0.923. Partial correlation analysis found that after excluding the indirect effects of other factors, pH (P=0.013) and electric conductivity{P=0.034) were the determinant factors of S. suis and E. coli adsorption, respectively. Solution pH increased S. suis adsorption significantly (P <0.05), while electric conductivity did not show a significant effect-(P>0.05) and had a little positive influence (partial correlation coefficient0.298). These data did not agree with the simple linear regression analysis. Because pH (partial correlation coefficient-0.952) and organic matter (partial correlation coefficient-0.735) had great suppressive impacts on the adsorption capacities, they could mask the weak effect of electric conductivity. Thereby, partial correlation was able to reflect the real influence of a certain soil property on bacterial adsorption. Soil organic matter, clay content, specific surface area and cation exchange capacity had insignificant impacts on partition coefficients (P>0.05), which could only explain less than30%of the adsorption behavior. Scanning electron microscope technique suggests that pathogens mainly adsorbed on the external surfaces of soil aggregates. Bacteria could not adsorb on the internal small-size particle surfaces of aggregates. The partition coefficients of E. coli were significantly negatively correlated with the corresponding energy barriers calculated by sphere-plate DLVO theory:Ks=-0.057×EB+22.6(R2=0.577, P<0.01, n=10). However, this model could not explain the adsorption behaviors of S. suis on soil particles. Correlation equations between pathogen partition coefficients and soil properties were obtained by applying multiple stepwise regression method. The equations were shown below:S. suis-Ks=-45.93×pH-1.31×CEC+389.75(R2=0.929, P<0.01); E. coli-Ks=0.24×EC+2.005(R2-0.932, P<0.01). The partition coefficients calculated by the two equations were comparable with the measured values, with discrepant values less than14.3mL g-1. These models were able to initially predict the adsorption capacities of pathogens on soil surfaces.(5) The effects of extracellular polymeric substances (EPS) on pathogenic suface properties and adsorption capacities were interpreted. Cation exchange resin (CER) was employed to remove the EPS on cell surface. Infrared spectrum data show that the absorption peaks of EPS-removed cells between3500cm-1and1000cm-1reduced, disappeared or deviated after the treatment of CER, indicating the corresponding amounts of proteins, polysaccharides and lipid materials were removed. The cell wall surface of E. coli contains hydroxyl, carboxyl, amido, phosphate, ester, aldehyde and sulphydryl groups, which was more plentiful than the functional group types of S. suis. After the removal of EPS, the surface charge densities and total site concentrations of two bacterial strains reduced by7%-17%and3%-7%, respectively. When the ionic strength increased from1mmol L-1to100mmol L"1, the negative charges on bacterial surface decreased constantly, following the order of EPS-left S. suis<EPS-removed S. suis<EPS-removed E. coli<EPS-left E. coli. The hydrophobicities of four bacterial types ranged from3%to43%. After the removal of EPS, the hydrophobicities of S. suis increased by-5%and those of E. coli decreased by-11%on average. The surface properties of EPS-left and EPS-removed bacterial were affected by the species of functional groups and EPS components. The Ks value (partition coefficient) of EPS-left S. suis adsorption on Yellow-Brown soil was the greatest (49.8mL g-1), followed by EPS-removed S. suis (16.1mL g-1), EPS-removed E. coli (8.2mL g-1), and EPS-left E. coli (8.0mL g’1). The adsorption trend at IS1mmol L-1-60mmol L-1was in agreement with the sphere-plate DLVO model. The adsorption amounts (Y) were significantly negatively correlated with the repulsive energy barriers (X):Y=-0.0064×X+2.99(R2=0.602, P<0.01). At IS ranging from60mmol L-1to100mmol L-1, the adsorption of EPS-left S. suis on soil particles reduced by16.0%, while that of EPS-removed S. suis increased gradually. This phenomenon indicates that the steric hindrance between S. suis and soil derived from the EPS constituents on cell surface.更多还原