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基于微生物全细胞传感器(组)的土壤镉、铅、砷 生物可利用度检测和评价研究
Alternative TitleDetection and assessment of bioavailable cadmium, lead, and arsenic in soil based on miocrobial whole-cell sensor (group)
侯启会
Subtype博士
Thesis Advisor庄国强
2014-05
Degree Grantor中国科学院研究生院
Place of Conferral北京
Degree Discipline环境科学
Keyword镉/铅/砷 生物可利用度 微生物全细胞传感器(组) 长期不同施肥 化学固定 Cd/pb/as Bioavailability Microbial Whole-cell Sensor (Group) Long-term Different Fertilization Chemical Immobilization
Abstract    随着社会经济的发展,我国土壤重金属污染形势日益严峻,开展重金属污染治理势在必行。对土壤重金属污染水平和风险进行合理、准确地检测和评估是开展重金属污染防治工作的重要前提。
     重金属生物可利用度是对重金属毒性和环境风险进行真实、合理评估的重要参数。目前,重金属生物可利用度检测方法主要为化学方法,例如,Tessier和BCR-SEPs连续提取法等。化学方法具有高灵敏度和高准确性的特点,但由于其只对重金属生物可利用度进行模拟,并非检测真实的重金属生物可利用度,因此不具有反映重金属真实环境风险的能力。微生物全细胞传感器由宿主细胞和可响应化学诱导物的重组质粒组成,因微生物细胞的易操作、繁殖力强、易储存、稳定性高等特点,微生物全细胞传感器已发展成为一种经济、便捷、快速的重金属生物可利用度检测和评估的工具。
     然而,由于重金属调控蛋白CadC、ZntR、ArsR对强毒性重金属Cd、Pb、As识别的低特异性,导致以这几种调控蛋白基因为识别元件的微生物全细胞传感器对Cd、Pb、As检测的低特异性。当利用这些微生物全细胞传感器对复合Cd、Pb、As污染情况下的某一重金属进行检测时,会受到其它重金属的干扰,进而高估目标重金属的污染水平。因此,开发一种可以提高微生物全细胞传感器检测特异性的方法是十分必要的。土壤环境条件如pH、有机质、无机盐和阳离子总量的变化会影响土壤重金属的形态、移动性和生物可利用度,进而影响重金属在土壤环境的生态毒性。因此,阐明某些环境行为下,土壤因子变化对重金属生物可利用的影响特征也是十分有意义的。基于这两方面的科学问题,本研究开展了以下工作:
1. 通过构建Cd、Pb、As微生物全细胞传感器组实现了复合重金属污染物Cd、Pb、As生物可利用度的特异检测。首先,分别以重金属抗性调控基因cadC、zntR、arsR及相应启动子为感应元件构建了检测Cd、Pb、As的Escherichia coli全细胞传感器pcadCluc、pzntRluc、parsRluc;然后,通过分析这3种传感器对Cd、Pb、As的特异性将3种传感器分为两组:组1为传感器pcadCluc和pzntRluc (两种传感器可被Cd 和Pb诱导),组2为传感器parsRluc (该传感器可被As和Cd诱导);然后对两组传感器进行复合诱导,进而利用数学软件建立了可对复合Cd/Pb/As污染物进行回归分析的二元线性方程组;最后利用该传感器组模型对复合重金属污染土壤样品的重金属生物可利用度进行了检测。检测结果表明,在Cd/Pb/As复合污染的土壤样品中,基于微生物全细胞传感器组的Cd、Pb和As生物可利用度分别只有基于单一传感器的生物可利用度的29.64%-69.79%、8.35%-15.95%和52.94%-60.94%。因此,微生物全细胞传感器组的方法极大提高了对复合污染环境中重金属生物可利用度检测的特异性和准确性。
2. 利用Cd微生物全细胞传感器pcadCluc比较评价了磷酸盐(K2HPO4,DKP) 和硅酸盐 (Na2SiO3· 9H2O,SS) 对土壤中Cd (450 mg/kg) 的固定效果。结果表明,在最大固定剂添加剂量下(2:1,P/Cd或Si/Cd,摩尔比),SS处理分别将水提取Cd总量、水提取Cd生物可利用度、水土悬浊液Cd生物可利用度从56.47 mg/kg、 42.11 mg/kg和206.72 mg/kg降到了16.63 mg/kg、15.90 mg/kg和67.57 mg/kg。与DKP相比,SS分别提高了25.68%、19.5%和9.54%对水提取Cd总量、水提取Cd生物可利用度、水土悬浊液Cd生物可利用度的固定效率。因此,本研究认为,在化学性质类似的土壤环境中,SS对Cd的固定效率要高于DKP。固定剂处理后土壤pH的变化差异和两种固定剂在土壤中的溶解度差异可能是导致两者对Cd固定效果差异的主要原因。不同水平下Cd检测结果表明,水提取Cd生物可利用度只有水土悬浊液Cd生物可利用度的16.13%-35.41%。因此,水土悬浊液能反映更高的Cd生物可利用度,通过检测该水平的Cd可以避免过高评价固定剂的固定效率。
3. 利用As微生物全细胞传感器parsRluc和化学BCR-SEPs连续提取法评价了长期不同施肥 (N、NP、NPK、M、NPK+M) 对农业土壤As生物可利用度的影响。BCR-SEPs分析结果表明,在M和NPK+M处理的土壤中,弱酸提取态As (F1) 分别是对照土壤 (CK,无施肥处理) 的2-7倍和2-5倍,可还原态As (F2)分别是对照组土壤的1.5-2.5倍和1.5-2.3倍;传感器parsRluc分析结果表明,在M和NPK+M处理的土壤中,生物可利用As分别是对照土壤的7倍和9倍,土壤有机碳的增加可能是M和NPK+M处理土壤As生物可利用度明显增高的主要原因。比较两种方法的检测结果发现,传感器所测定As分别只占BCR-SEPs测定F1组分 和 F2组分 的18.46%–85.17 %和18.46%–85.17%,因此本研究认为BCR-SEPs法所测定As可能高估了土壤中真实的As生物可利用度。
Other Abstract    With the development of the economic, heavy metals pollution in soil have become a more and more serious environment problem, and the remediation of soil polluted by heavy metals is imperative. Furthermore, a rational and accuracy method for monitoring the content and assessing the risk of heavy metals in soil is significantly important for soil remediation.
    Bioavailability of heavy metals in soil and sediment is an important term to evaluate environmental risk. At present, the main method for monitoring the bioavailble heavy metals in soil is the chemical sequential extraction, such as Tessier and BCR-SEPs. The chemical sequential extraction may have high sensitivity and high veracity with using the precise measure instruments, but the chemical sequential extraction can’t reflect the actual toxicity of heavy metals because it can’t reflect the actual bioavailability of heavy metals. The microbial whole-cell sensor is comprised of the host cell and recombinant plasmid who can response to the chemical inducers. Compared with the chemical sequential extraction, whole-cell sensor is a more cheaper, faster, convenient and accurate tool to detect the heavy metals in soil and water because of the trait of microbial cell, more importantly, it can refect the actual bioavailability of heavy metals.
    However, because of the non-specific recognition of the regulatory protein CadC, ZntR, ArsR to the highly toxic heavy metal cadmium (Cd), lead (Pb) and arsenic (As), the microbial whole-cell sensors based on these regulatory proteins will be interfered when they are employed to detect the bioavailability of Cd, Pb and As in co-polluted environment. Therefore, a optimized method for realizing the specific detection of Cd, Pb and As based on microbial whole-cell sensor is required. In addition, the change of soil factors such as pH, organic matter, inorganic salt, and cation charge and so on may lead to the variation of bioavailable heavy metals, so assessing the bioavailability of heavy metals in soil with different factors is extremely neccessary.
    Consequently, the following work was carried out:
1. In this study, we proposed a strategy for the specific detection of bioavailable Cd, Pb, and As in co-polluted environment based on microbial whole-cell sensor groups. First, three conventional Escherichia coli whole-cell sensors for Cd, Pb, and As detection were constructed respectively based on the regulatory gene (containing promoter) cadC, zntR, and arsR, and respectively termed as pcadCluc, pzntRluc and parsRluc. Then, we tested the specificity of each sensor to Cd, Pb, and As solution. The results suggested that the sensors can be classified as two groups according to their respondings to Cd, Pb, and As. The group 1 (pcadCluc and pzntRluc) could be induced by a Cd/Pb mix, and the group 2 (parsRluc) could be induced by a Cd/As mix. Based on diviation responses of each sensor to mixed metal solution, two binary linear equations for each sensor group were built to calculate the specific concentration in mixed Cd/Pb/As metal solution. Finally, by the sensor groups and mathematic calculation, each of the bioavailable Cd, Pb, and As in co-polluted mining soil was accurately quantified. In contrast, conventional method with a single sensor overestimated the bioavailability of each metal. In detail, the bioavailability of Cd, Pb, and As in the co-polluted mining soil based on sensor groups was respectively only 29.64%-69.79%, 8.35%-15.59%, and 52.94%-60.94% of the values based on a single whole-cell sensor.
2. In this study, the E. coli whole-cell sensor pcadCluc for Cd detection was employed to estimate Cd bioavailability in soils (Cd=450 mg/kg) spiked with different contents of dipotassium phosphate (DKP, K2HPO4) or sodium silicate (SS, Na2SiO3·9H2O). Total Cd in soil-water extracts (TSWE) was determined with ICP-MS, and the bioavailable Cd in soil-water extracts (BSWE) , bioavailable Cd in soil-water suspensions (BSWS) were measured with the whole-cell sensor pcadCluc. Results showed that spiked SS (Si:Cd=2:1, mol/mol ) reduced the different forms of Cd (TSWE, BSWE and BSWS) from 56.47 mg/kg, 42.11 mg/kg, and 206.72 mg/kg to 16.63 mg/kg, 15.90 mg/kg, and 67.57 mg/kg, respectively. In other words, SS had 25.68%, 19.5%, and 9.54% better immobilizing effciency to Cd, respectively, compared with DKP. All the results supported SS was more effcient than DKP in immobilizing Cd in soil, and soil pH and solubility of the immobilizing agents in soil may have been the major factors affecting their immobilizing effciency to Cd. In addition, the total and bioavailable Cd in soil-water extracts was only 16.13%-35.41% of the sensor contact assay-determined Cd (BSWS), which indicated that the whole-cell sensor-based contact assay was more practical in assessing the risk of Cd in soil after immobilization since it would not overrate the immobilizing capacity of the agents.
3. Both chemical sequential extraction, Community Bureau of Reference recommended sequential extraction procedures (BCR-SEPs) and E. coli whole-cell sensor parsRluc for As detection, were employed to assess the impact of different long-term fertilization regimes containing N, NP, NPK, M (manure), and NPK+M treatments on the bioavailability of arsenic (As) in soil. Results showed, per the BCR-SEPs analysis, the application of M andM+NPK led to a significant (P<0.01) increase of acid soluble As (2-7 times and 2-5 times, respectively) and reducible As (1.5-2.5 times and 1.5-2.3 times, respectively) compared with the no fertilization treated soil (CK). In addition, direct contact assay of E. coli whole-cell sensor parsRluc with soil particles also supported that bioavailable As in manure-fertilized (M and M+NPK) soil was significantly higher (P<0.01) than that in CK soil (7 times and 9 times, respectively). Organic carbon may be the major factor governing the increase of bioavailable As. More significantly, E. coli whole-cell sensor parsRluc-determined As was only 18.46%-85.17% of acid soluble As and 20.68%-90.1% of reducible As based on BCR-SEPs. In conclusion, NKP fertilization was recommended as a more suitable regime in As-polluted soil especially with high As concentration, and this E. coli whole-cell sensor was a more realistic tool to assess the bioavailability of As in soil since it would not overrate the risk of As in the soil.
Document Type学位论文
Identifierhttp://ir.rcees.ac.cn/handle/311016/13471
Collection中国科学院环境生物技术重点实验室
Recommended Citation
GB/T 7714
侯启会. 基于微生物全细胞传感器(组)的土壤镉、铅、砷 生物可利用度检测和评价研究[D]. 北京. 中国科学院研究生院,2014.
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