【作者】 Sardar Alam Cheema；

【导师】 陈英旭；

【作者基本信息】 浙江大学 ， 环境工程， 2009， 博士

【摘要】 过去几个世纪以来，人口的迅速增长、矿产的开采、工业化进程的加快等原因，使得土壤环境遭受了严重污染。多环芳烃（Polycyclic AromaticHydrocarbons，PAHs）是一类广泛分布于土壤环境中的持久性有机污染物，对全球的农产品质量和人体健康构成威胁。因此，对被PAHs污染的土壤进行修复势在必行。相比较传统修复技术而言，植物修复技术是一种极具潜力的土壤有机污染物修复技术，成本低，且对环境友好。实验室研究表明，植物修复技术可以显著促进土壤中PAHs的去除，但较为深入的机制并没有得到很好的阐述。虽然植物修复技术相对于其他修复技术具有许多优势之处，但是，由于PAHs较低的水溶性以及土壤颗粒对其具有强吸附性，土壤植物修复的效率始终受到限制。在本论文中，我们考察了不同植物及其组合对PAHs的去除效率，研究了PAHs在高羊茅根际的去除机理，同时，通过添加表面活性剂强化修复PAHs污染土壤实验，研究了表面活性剂对PAHs去除效率的影响，本论文的相关试验结果可为植物修复技术在PAHs污染土壤中的实际应用提供理论基础。本研究成果可归纳如下。（1）考察了四种植物（高羊茅，黑麦草，紫花苜蓿和甘蓝型油菜）单一盆栽种植或组合种植对PAHs添加污染土壤的植物修复效率。经过65天的植物生长后，测定植物生物量、脱氢酶等参数。结果表明，所添加浓度下PAHs对所有植物的生长均具有一定的抑制作用，其中对苜蓿生长抑制作用最为明显，相对于无PAHs添加对照植物生物量减少35％，油菜对PAHs表现出最大的耐性。相对于无植物对照，种植植物后土壤中水溶性酚显著提高，含量达到3.71-5.63 ug g^-1，分别比对照高出1.05-1.68倍。植物盆栽处理也提高了土壤的脱氢酶活性。相对于无植物对照，植物处理强化了土壤中PAHs的去除。各植物对PAHs的去除率有所差异，油菜处理PAHs去除率最高（菲和芘去除率分别为98％和86％），其次是黑麦草和高羊茅，苜蓿去除率最低，菲和芘去除率分别为97％和79.8％。植物组合盆栽处理进一步提高了这种强化效果，高羊茅和油菜组合处理PAHs去除率最高，菲和芘去除率分别达到99.1％和95.7％。相关分析表明，不同的植物组合处理中土壤中的生物活性（脱氢酶活性和水溶性酚总量）与土壤中残余的菲、芘量之间具有非常好的负相关关系（P＜0.01）。本节结果表明，采用这四种植物对PAHs污染土壤进行植物修复是可行的，不同植物组合栽培可进一步提高植物修复能力。（2）添加不同浓度梯度的PAHs(菲，11-344 mg kg^-1，芘15-335 mg kg^-1)，较细致地考察了高羊茅对PAHs污染土壤的植物修复潜力。通过分析植物生物量、土壤微生物计数、脱氢酶活性、水溶性酚总量，微生物群落结构等参数，探讨了高羊茅修复土壤PAHs的可能机理。结果表明，较低浓度PAHs不影响植物的生物量，但是随着浓度升高，可观察到生物量明显降低，最高浓度下植物生物量减少至无PAHs添加对照的53.5％（茎）和29.7％（根）。相对于无植物对照，植物处理显著提高了微生物量，水溶性酚类物质量，脱氢酶活性，PAHs去除率显著提高。进一步分析PCR-DGGE结果，植物处理Shannon指数值为3.65-3.79，显著高于无植物处理（3.51-3.68），表明植物处理增加了土壤中的微生物群落结构多样性；同时本研究结果也显示，随着PAHs浓度升高，微生物群落结构多样性降低。植物处理65天后，土壤中菲的去除率为91.70-97.78％，芘的去除率为70.80-89.97％，相对于无植物处理，菲和芘去除率分别提高了1.88-3.19％和8.85-20.69％。综合以上结果分析，土壤中PAHs的去除可能是源于根际土壤生物活性的提高，从而使得PAHs的去除率显著提高。（3）研究了高羊茅对菲和芘的吸收和累积，以考察其对根际PAHs去除效率的贡献。结果表明，对于菲和芘初始浓度分别为199.97 mg kg^-1和199.34 mg kg^-1的污染土壤，植物盆栽处理65天后，根吸收因子（Root concentration factors，RCFs）分别为0.33-0.46和0.56-1.21，茎吸收因子（shoot concentration factors，SCFs）分别为0.15-0.22和0.017-0.083。茎中的菲、芘累积主要来源于根部；尽管茎部也会从大气中吸收菲、芘，但从对照处理可见，菲、芘并没有从茎部向根部迁移。通过植物吸收累积途径去除的PAHs量占PAHs总去除量的0.10-1.42％（菲）和0.18-2.04％（芘）。由此说明，植物根际土壤中PAHs的主要去除途径是生物去除而不是植物吸收累积。本文的结果也表明，生长于PAHs污染土壤中的植物体中会累积一定量的污染物，因此，对于修复后的植物或生长于污染场地的农作物需妥善处理，以防止污染物进入食物链。（4）采用盆栽试验方法，研究了添加非离子表面活性剂（Tween 80、Triton x-100），生物表面活性剂（大豆卵磷脂）以及随意甲基化-β-环糊精（RAMEB）四种表面活性剂对高羊茅修复PAHs（芘）污染土壤的影响。芘初始浓度为243 mgkg^-1，不同表面活性剂添加浓度设置5个梯度，分别为0，200，600，1000和1500mg kg^-1，同时，设置无植物、不添加表面活性剂及灭菌等对照处理，盆栽60天后收获植物进行植物生物量测试和PAHs残留分析。结果显示，相对于不添加表面活性剂对照，4种表明活性剂添加后均显著提高了植物生物量。在不遮光灭菌和遮光灭菌对照处理情况下PAHs损失量分别为3.9％和3.2％，证明根际土壤中的PAHs去除主要是生物降解作用，光解与挥发作用较小。相对于不添加表面活性剂对照，所有的表面活性剂均显著提高了芘的去除率。在种植植物情况下，RAMEB添加后芘的去除率强化作用最为明显，去除率为无RAMEB添加对照的1.40-1.80倍，其次是Triton x-10，芘去除率为无Triton x-10添加对照的1.34-1.66倍。表面活性剂强化PAHs的去除主要是因为增加了PAHs在土壤中的微生物可利用性。研究表明，添加表面活性剂能够提高PAHs污染土壤的植物修复效率。更多还原

【Abstract】 Over the past centuries, rapid growth of population, mining, industrialization, etc., have significantly contributed to extensive soil contamination. Polycyclic aromatic hydrocarbons （PAHs）, a class of persistent organic pollutants （POPs）, are widely distributed in the soil, water and air environment. Soil contamination with PAHs pose great threat worldwide to the agricultural food quality and human health and calls for an immediate action to remediate the contaminated sites. The prospect of phytoremediation for soil organic contaminants is an attractive cost effective alternative to traditional engineering approaches. Many studies have demonstrated that plants have potential to enhance the dissipation of PAHs when compared to unplanted controls. However, the basic mechanisms involved are not well elucidated. Although using plants for remediation of persistent contaminants have advantages over other methods, efficiency of phytoremediation of PAH contaminated soil is always limited by the poor water solubility and strong adsorption of PAHs to soil particles, resulting in lower biodegradation rates and longer time to achieve the standards. The use of surfactants to enhance the apparent aqueous solubility, desorption of organic compounds from solids and microbial bioavailability of PAHs in soil solution has been well documented. However, there is a lack of experimental data to elucidate the effect of surfactants on the phytoremediation efficiency of PAHs.In this dissertation, we examined the PAH removal in the presence of plants to contribute to the technology for field phytoremediation in practice. The PAH dissipation mechanisms were investigated to determine if phytodegradation, especially rhizodegradation plays a role in PAH removal. In addition, the effect of surfactants on the phytoremediation efficiency was also addressed. The present study can be summarized as follows.The capability of four plant species （Festuca arundinacea, Lolium perenne, Medicago sativa and Brassica napus） was investigated for phytoremediation of PAH contaminated soil. The plants were grown alone and in combination to see the effect of combined plantation on phytoremediation process. After 65 day of plant growth, plants were harvested and plant biomass, dehydrogenase activity, water-soluble phenolic （WSP） compounds and residual concentrations of phenanthrene and pyrene were determined. The results showed that PAHs had an inhibitive effect on the biomass yield of all plants. The greatest reduction in biomass was observed in Medicago sativa, which produced approximately 35% of the biomass of control. Brassica napus was the most resistant to the presence of PAHs. Significant higher WSP compounds were observed in planted soils than unplanted controls in PAHs polluted soils. WSP compounds of the PAH polluted soils were 3.71-5.63 ug vanillic acid g^-1 of soil, which were 1.05-1.68 times higher than in uncontaminated soil (i.e. 3.06-4.03 ug vanillic acid g^-1 soil). Similarly, higher dehydrogenase activities were observed in the planted soil compared to unplanted soil. The results also indicated higher rates of PAHs loss in planted soils （i.e., with a rhizosphere） than in unplanted control soils. This effect was especially marked with the combined plant cultivation. Rape seed, tall fescue and rye grass were statistically at par in degradation of PAHs; however, the degradation rates were different. Rape seed displayed the highest PAHs degradation rate （98% of phenanthrene and 86% of pyrene） followed by rye grass and tall fescue whereas alfalfa showed the lowest degradation rate, i.e. 97 % of phenanthrene and 79.8% of pyrene. Among combined plant cultivation, the combination of tall fescue and rape seed had the highest removal rate of PAHs, i.e. 99.1% for phenanthrene and 95.7% for pyrene. The correlation analysis indicated a significant inverse relation （P < 0.01） between biological activities （dehydrogenase activity and water-soluble phenolic compounds） and residual concentrations of phenanthrene and pyrene in soils planted with different plant combinations. Phytoremediation with tall fescue, alfalfa, and rape seed could be a feasible choice for PAHs contaminated soils. Moreover, the combined plant cultivation has potential to enhance the process.Tall fescue （Festuca arundinacea） was grown in soil artificially contaminated with varying concentrations of phenanthrene (11-344 mg kg^-1) and pyrene (15-335 mg kg^-1) to evaluate its phytoremediation potential for PAHs contaminated soil. After 65 day of tall fescue growth, plant biomass, microbial viable counts, dehydrogenase activity, water soluble phenolic compounds, bacterial diversity by using polymerase chain reaction-denaturing gradient gel electrophoresis （PCR-DGGE） and PAHs residual concentrations were determined. The results showed that target PAHs （phenanthrene and pyrene） did not affect plant biomass at lower concentrations but a reduced biomass （only 53.46% of shoot and 29.74 % of root compared to control） was observed at higher concentrations. Higher biological activities （microbial viable counts, water soluble phenolic compounds, dehydrogenase activity） and PAHs degradation rates were detected in planted soils than those of unplanted controls. Shannon diversity index for planted soils were 3.65-3.79 which were significantly higher compared to unplanted soils i.e. 3.51-3.68 suggesting that presence of plants increased the bacterial diversity. It is also evident from the results that increasing levels of PAHs reduced the bacterial diversity. After harvest, 91.70-97.78% of phenanthrene and 70.80-89.97% of pyrene degraded in planted soils which were 1.88-3.19% and 8.85-20.69% larger than those in corresponding unplanted soils. This enhanced dissipation of target PAHs in planted soils might be derived from increased biological activities in the rhizosphere.Uptake, accumulation and translocation of phenanthrene and pyrene were comparatively investigated to assess the uptake and accumulation contribution in plant promoted degradation of PAHs. Root concentration factors （RCFs） of phenanthrene and pyrene for plants grown in contaminated soils with initial phenanthrene of 199.97 mg kg^-1 and pyrene of 199.34 mg kg^-1 were 0.33-0.46 and 0.56-1.21, whereas the shoot concentration factors （SCFs） of these compounds were 0.15-0.22 and 0.017-0.0837, respectively. Alfalfa exhibited the highest root concentrations of phenanthrene (8.17 mg kg^-1) and pyrene (105.8 mg kg^-1), followed by rape seed which accumulated 5.33mg kg^-1 of phenanthrene and 70.48 mg ^-1 of pyrene, while tall fescue contained the lowest portion of these compounds (phenanthrene of 1.6 mg kg^-1 and pyrene of 25.62 mg kg^-1). The contributions of plant off-take of these chemicals to the plant promoted dissipation were only 0.10-1.42% for phenanthrene and 0.18-2.04% for pyrene. Plants promoted the biodegradation of PAHs mainly were the predominant contribution to the remediation enhancement for soil phenanthrene and pyrene in the presence of vegetation.In order to enhance the phytoremediation process, a study was conducted to evaluate the effects of two non ionic surfactants （Tween 80 and triton x-100） a biosurfactant （Soya Lecithin） and randomly methylated-β-cyclodextrins （RAMEB） on the removal of pyrene from soil cultivated with tall fescue （Festuca arundinacea）. Soils with pyrene concentration of about 243 mg kg^-1 grown with the tall fescue and were individually amended with 0, 200, 600, 1000 and 1500 mg kg^-1 of tween 80, triton x-100, biosurfactant, and RAMEB. Unplanted （with and without surfactants） and sterile microcosms （covered and uncovered） were prepared as the controls. Plant biomass dehydrogenase activity and pyrene concentrations were quantified after 60 d of plant growth. The results indicated that all surfactants have significant greater yields of plant biomass compared to unamended soil. Results of dehydrogenase activities showed that with the application of all surfactants, significant higher microbial activities were recorded; however, some surfactants （triton x 100 and RAMEB） inhibited the microbial activities at higher concentration levels. Only 3.9% and 3.2% of pyrene was disappeared in the uncovered and covered abiotic sterile controls, indicating that the removal mechanism of PAHs in soil was mainly due to microbial degradation of the PAHs in soil. In the planted treatment receiving no surfactant amendment, the removal rate of pyrene was 45% which is significantly higher than that of corresponding unplanted control soil, suggesting that the cultivation of tall fescue alone could significantly improve the overall removal of pyrene in soil. All surfactants had significantly higher rates of PAH degradation compared to the unamended planted soil. Overall RAMEB displayed the highest degradation rates （1.4-1.8 times higher than unamended planted control） followed by the triton X-100 （1.34-1.66 times higher than unamended planted control）. The positive effect of surfactants on pyrene removal could probably due to their capacities to enhance bioavailability in soil. This study suggests that the addition of surfactants could facilitate phytoremediation of PAH contaminated soil.更多还原