重金属在土壤—微生物界面相互作用的分子机制
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摘要
土壤微生物是土壤中的活性胶体，它们比表面大带电荷，代谢活动旺盛。受到重金属污染的土壤，往往富集多种耐重金属的真菌和细菌，微生物可通过多种作用方式影响土壤重金属的活性。本论文从受重金属污染的土壤中筛选到一株具有较高铜锌耐性的微生物，采用电镜、能谱、红外光谱和X-射线吸收光谱等现代分析技术并结合传统的物理化学方法，阐明了恶臭假单胞菌CZ1对Cu、Zn的吸附行为及其结合的分子形态，并初步探讨了微生物-矿物-重金属相互作用机制，旨在为重金属污染土壤的风险评价和生物修复提供理论依据。通过研究取得了以下主要结果：
从浙江诸暨哩浦铜矿废弃矿区铜耐性植物海州香薷根际土壤中分离到一株具有较高铜锌耐性的微生物，编号为CZ1，根据形态学观察、生理生化特性和16S rDNA序列同源性比对，鉴定为Pseudomonas putida。CZ1可分别耐受3 mM Cu或5 mM Zn，对氨苄青霉素具有抗性，而对卡那霉素无抗性。重金属耐性实验发现固体培养基中最低抑制浓度小于液体培养基中最低抑制浓度，而且Cu的毒性要大于Zn的毒性。在含有27.7mg／L Cu或26.0mg／L Zn的无机盐液体培养基中，CZ1对Cu和Zn的去除能力可分别达到87.2％和99.8％。尽管较低浓度的重金属会对微生物细胞造成一定程度的损伤，但CZ1依然表现出了较高的铜锌富集能力。
溶液pH对CZ1吸附Cu~(2+)、Zn~(2+)的能力影响较大，活菌体和死菌体吸附Cu~(2+)的最佳pH分别为5.0和4.5，而吸附Zn~(2+)的最佳pH均为5.0；在最佳吸附条件下，CZ1对Cu~(2+)、Zn~(2+)的吸附量随着起始浓度的增加而增加，但去除效率却随之下降；CZ1对Cu~(2+)、Zn~(2+)的吸附等温线均符合Langmuir等温吸附方程。活菌体对Cu~(2+)、Zn~(2+)的吸附能力远大于死菌体，并且包括主动吸收和被动吸附两个过程，其中活菌体通过主动吸收方式结合的Cu~(2+)、Zn~(2+)含量占整个吸附量的40～50％，而被动吸附则为50～60％。解吸实验表明，0.1M盐酸对死菌体中Cu~(2+)、Zn~(2+)的解吸效率分别为95.3％和83.8％，而活菌体仅为72.5％和45.6％，进一步说明活菌体对Cu~(2+)、Zn~(2+)的吸附存在着主动吸收的过程。在0.01 M KNO_3溶液中，Cu~(2+)、Zn~(2+)吸附体系均表现出了明显的颗粒物浓度(C_p)效应，不同C_p下的所有吸附等温线可以用Freundlich型C_p效应等温式进行统一描述，分别为q_(eq)=2.553·C_p~(-0.7106)·C_(eq)~(0.8971)和q_(eq)=2.412·C_p~(-0.8305)·C_(eq)~(0.6504)。通过
Soil microorganisms are the active colloids in the soil, with charges, high specific surface areas and metabolic activities. Many tolerant fungi and bacteria will exist in the soil after polluted by heavy metals, they could influence the species, bioavailability and transportation processes of heavy metals through varies actions. In this paper, a heavy metal-tolerant strain, Pseudomonas putida CZ1, was isolated from the contaminated soils. The actions of biosorption and molecular species of heavy metal binding, the interaction mechanisms of microorganism-clay-metal were investigated using Electron microscopy (EM), Energy dispersive spectroscopy (EDS), Fourier transform infrared spectroscopy (FTIR) and X-ray absorption spectroscopy (XAS) combined with conventional physical-chemical methods, which could provide theory basis for risk evaluation and bioremediation of heavy metal contaminated soils. The main results were as follows:
A strain was isolated from the rhizosphere of Elsholtzia splendens obtained from the heavy-metal-contaminated soil in the north-central of Zhejiang province of China. Based on 16S ribosomal DNA sequencing, the microorganism was closely related to Pseudomonas putida. It exhibited high minimal inhibitory concentration (MIC) values (about 3 mM Cu and 5 mM Zn) for metals and antibiotic resistance to ampicillin, but not to kanamycin. Based on the results of heavy metal toxicity screening, inhibitory concentrations in solid media were lower than those in liquid media. Moreover, it was found that the toxicity of copper was higher than that of zinc. Pseudomonas putida CZ1 was capable of removing about 87.2% of Cu and 99.8% of Zn during the active growth cycle with a specific biosorption capacity of 27.7 and 26.0 mg/L, respectively. Although copper and zinc under low concentration slightly damage the surfaces of some cells, it was still demonstrated to have higher biosorption capacities of copper and zinc.
It was found that the optimum pH for Zn(II) removal by living and nonliving cells was 5.0, while it was 5.0 and 4.5, respectively for Cu(II) removal. At the optimal conditions, metal ion biosorption was increased as the initial metal concentration increased but the removal efficency decreased. The adsorption data with respect to both metals provide an excellent fit to the Langmuir isotherm. The binding capacity of living cells is significantly higher than that of nonliving cells at tested conditions. It demonstrated that about 40-50%
从浙江诸暨哩浦铜矿废弃矿区铜耐性植物海州香薷根际土壤中分离到一株具有较高铜锌耐性的微生物，编号为CZ1，根据形态学观察、生理生化特性和16S rDNA序列同源性比对，鉴定为Pseudomonas putida。CZ1可分别耐受3 mM Cu或5 mM Zn，对氨苄青霉素具有抗性，而对卡那霉素无抗性。重金属耐性实验发现固体培养基中最低抑制浓度小于液体培养基中最低抑制浓度，而且Cu的毒性要大于Zn的毒性。在含有27.7mg／L Cu或26.0mg／L Zn的无机盐液体培养基中，CZ1对Cu和Zn的去除能力可分别达到87.2％和99.8％。尽管较低浓度的重金属会对微生物细胞造成一定程度的损伤，但CZ1依然表现出了较高的铜锌富集能力。
溶液pH对CZ1吸附Cu~(2+)、Zn~(2+)的能力影响较大，活菌体和死菌体吸附Cu~(2+)的最佳pH分别为5.0和4.5，而吸附Zn~(2+)的最佳pH均为5.0；在最佳吸附条件下，CZ1对Cu~(2+)、Zn~(2+)的吸附量随着起始浓度的增加而增加，但去除效率却随之下降；CZ1对Cu~(2+)、Zn~(2+)的吸附等温线均符合Langmuir等温吸附方程。活菌体对Cu~(2+)、Zn~(2+)的吸附能力远大于死菌体，并且包括主动吸收和被动吸附两个过程，其中活菌体通过主动吸收方式结合的Cu~(2+)、Zn~(2+)含量占整个吸附量的40～50％，而被动吸附则为50～60％。解吸实验表明，0.1M盐酸对死菌体中Cu~(2+)、Zn~(2+)的解吸效率分别为95.3％和83.8％，而活菌体仅为72.5％和45.6％，进一步说明活菌体对Cu~(2+)、Zn~(2+)的吸附存在着主动吸收的过程。在0.01 M KNO_3溶液中，Cu~(2+)、Zn~(2+)吸附体系均表现出了明显的颗粒物浓度(C_p)效应，不同C_p下的所有吸附等温线可以用Freundlich型C_p效应等温式进行统一描述，分别为q_(eq)=2.553·C_p~(-0.7106)·C_(eq)~(0.8971)和q_(eq)=2.412·C_p~(-0.8305)·C_(eq)~(0.6504)。通过
Soil microorganisms are the active colloids in the soil, with charges, high specific surface areas and metabolic activities. Many tolerant fungi and bacteria will exist in the soil after polluted by heavy metals, they could influence the species, bioavailability and transportation processes of heavy metals through varies actions. In this paper, a heavy metal-tolerant strain, Pseudomonas putida CZ1, was isolated from the contaminated soils. The actions of biosorption and molecular species of heavy metal binding, the interaction mechanisms of microorganism-clay-metal were investigated using Electron microscopy (EM), Energy dispersive spectroscopy (EDS), Fourier transform infrared spectroscopy (FTIR) and X-ray absorption spectroscopy (XAS) combined with conventional physical-chemical methods, which could provide theory basis for risk evaluation and bioremediation of heavy metal contaminated soils. The main results were as follows:
A strain was isolated from the rhizosphere of Elsholtzia splendens obtained from the heavy-metal-contaminated soil in the north-central of Zhejiang province of China. Based on 16S ribosomal DNA sequencing, the microorganism was closely related to Pseudomonas putida. It exhibited high minimal inhibitory concentration (MIC) values (about 3 mM Cu and 5 mM Zn) for metals and antibiotic resistance to ampicillin, but not to kanamycin. Based on the results of heavy metal toxicity screening, inhibitory concentrations in solid media were lower than those in liquid media. Moreover, it was found that the toxicity of copper was higher than that of zinc. Pseudomonas putida CZ1 was capable of removing about 87.2% of Cu and 99.8% of Zn during the active growth cycle with a specific biosorption capacity of 27.7 and 26.0 mg/L, respectively. Although copper and zinc under low concentration slightly damage the surfaces of some cells, it was still demonstrated to have higher biosorption capacities of copper and zinc.
It was found that the optimum pH for Zn(II) removal by living and nonliving cells was 5.0, while it was 5.0 and 4.5, respectively for Cu(II) removal. At the optimal conditions, metal ion biosorption was increased as the initial metal concentration increased but the removal efficency decreased. The adsorption data with respect to both metals provide an excellent fit to the Langmuir isotherm. The binding capacity of living cells is significantly higher than that of nonliving cells at tested conditions. It demonstrated that about 40-50%
引文
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