| 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. |
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