【作者】 张薇；

【导师】 刘云国；

【作者基本信息】 湖南大学 ， 环境工程， 2020， 博士

【摘要】 石油中含有的石油烃和重金属都是持久性污染物,两者都具有很强的毒性特征可致畸、致癌和致突变,对人类健康和生态安全造成了极大的潜在威胁,如何安全有效修复石油污染土壤已经成为环境领域亟待解决的问题之一。国内外的研究人员开展了许多石油污染土壤治理的实验。生物修复技术因具有成本低、不破坏植物生长所需的土壤环境、没有二次污染、操作简单等优势引起了人们的广泛关注。但在研究中发现油污土壤中的高分子链石油烃具有较强的疏水性,容易被吸附在土壤中。这类物质的低水溶性和低生物可利用性使得它们在土壤中不易被生物自然降解。而且在利用微生物修复技术修复石油污染土壤过程中,与石油烃共存的重金属会影响石油烃降解菌的生长和代谢,从而影响其修复效率。同时石油烃由于其疏水的特性,常包裹于菌团的外层抑制其呼吸生长,并阻断了微生物在水中对重金属的作用,严重抑制了微生物的生长,因而也使得土壤中的重金属更加难以去除,给石油污染土壤治理增加了一定难度。近年来环糊精(cyclodextrins CDs)由于其特殊的结构,在石油污染土壤处理领域得到广泛关注。环糊精是由环糊精葡萄糖基转移酶作用于淀粉,形成的一类环状低聚糖。环糊精类化合物的特点是,分子结构中存在一个亲水的外缘和一个疏水的空腔,其疏水的空腔能与一些有机物结合形成主客体包合物。并且由于CDs外侧的亲水性,而使包合物的水溶性大于有机物本身的水溶性,增加了有机物在水中的溶解度。同时,CDs与重金属也有一定的配位作用,能同时去除有机物和重金属,更为可贵的是环糊精本身无毒且自身具有生物降解性,属于环境友好材料,不会产生二次污染。但不足之处是,环糊精仅对部分疏水性有机污染物具有增溶作用,而且它在水中的溶解度较低,这些特点在一定程度上限制了环糊精的广泛应用。基于以上原因我们首先对环糊精进行化学改性,选择水溶性更强且对重金属有配位能力的环糊精衍生物,研究其初始浓度、土壤酸度等对其增溶解吸的影响行为优化其最佳应用条件。通过多次试验制备了甘氨酸-β-环糊精(G-β-CD),红外光谱及XPS分析结果表明G-β-CD在保留原内腔结构的同时还增加了氨基、羧基等基团。亲水性基团如氨基、羧基提高了β-环糊精在水中的溶解性,同时可络合重金属。研究发现在用G-β-CD处理石油污染土壤,在增溶剂G-β-CD浓度为70 g/L、土壤p H值为6.0～7.0、反应温度为30～40℃、土壤的离子浓度为0.25 mol/L、有机质含量为1%时,石油烃与重金属解吸效果均达到较好水平。在以上研究基础上,我们还分析了G-β-CD增溶模式下,石油污染土壤中石油烃的微生物降解行为和土壤重金属形态及其生物有效性等的变化规律,以深入了解石油烃、重金属污染物在土壤环境中的生物学归宿及其生态效应。实验发现G-β-CD处理污染土壤后,土壤中重金属形态也发生了较大的变化,其中Pb、Cu的可交换态、碳酸盐结合态、有机结合态的减少均达到50%以上,氧化物结合态和残渣态含量减少60%左右,表明G-β-CD与重金属形成稳定的可溶性络合物大大降低了土壤对重金属的吸附,从而降低了土壤中重金属的毒性和生物可利用性。而且经G-β-CD处理后的石油污染土壤中多酚氧化酶、过氧化氢酶、脱氢氧化酶以及脂肪酶活性有了显著提升,我们可以认为甘氨酸-β-环糊精能够有效的改良石油烃-重金属复合污染土壤,使其中的生物酶活性增强。实验中发现经过G-β-CD预处理的石油污染土壤,其自身的生物修复能力得到了改善,但还远远没有达到正常土壤水平。因此本文通过石油烃降解菌的驯化、筛选、鉴定得到了一种高效石油烃降解菌,并系统研究了髙效降解菌的降解能力及其影响因素,优化了石油烃降解菌的发酵条件。本研究不仅考虑到选出的最佳条件因素对石油烃降解菌的生长繁殖的影响,还着重考虑了成本低、实用性强和操作步骤简单等实际性和经济性问题。实验结果表明,无机盐培养基里加豆粕粉和玉米淀粉,培养出来的细菌和牛肉膏蛋白胨液体培养基的效果相当,且更经济实惠,适用于现场土壤生物处理的应用。实验最后采用生态毒性试验综合评价该化学-微生物联合修复法的修复效果。利用变性梯度凝胶电泳(PCR-DGGE)技术分析化学-微生物联合修复对土壤微生物多样性的影响。分析结果表明,石油污染物胁迫土壤微生物种群结构,石油污染土壤与清洁土壤细菌群落相似性仅为37.7%,经化学-微生物联合修复后相似性增加至76.2%,说明化学-微生物联合修复后,土壤微生物多样性有了较好的恢复。通过高等植物发芽毒理实验和作物吸收毒理实验观察种子的发芽率、测量幼苗叶片的生理生化指标,评价化学-微生物联合修复后土壤的生态毒性。结果表明种子发芽率及叶片光合色素含量、超氧化物歧化酶(SOD)活性、丙二醛(MDA)含量指标随着化学-微生物联合修复时间的延长而增加,说明土壤生态毒性减弱,但在整个修复的时期土壤都存在着一定的生态毒性。以上结果为G-β-CD-微生物联合修复法的可行性提供了理论依据和技术支持。更多还原

【Abstract】 Petroleum hydrocarbons and heavy metals contained in petroleum are persistent pollutants,both of which have strong toxicity characteristics,can cause teratogenicity,carcinogenicity,mutagenicity,and pose a great potential threat to human health and ecological safety.How to safely and effectively remedy oil-contaminated soil has become one of the urgent problems in the environmental field.Researchers at home and abroad have conducted many experiments on oil-contaminated soil management.Bioremediation technology has attracted more and more attention because of its low cost,no damage to the soil environment required for plant growth,no secondary pollution,and simple operation.However,studies have found that the petroleum chain hydrocarbons in the oily soil have s trong hydrophobicity and are easily adsorbed in the soil.The low water solubility and bioavailability of these substances make them difficult to be bio-degraded in the soil.In the process of repairing petroleum-contaminated soil by microbial remediation technology,heavy metals coexisting with petroleum hydrocarbons will affect the growth and metabolism of petroleum hydrocarbon degrading bacteria,thereby affecting their repair efficiency.In addition,petroleum hydrocarbons are often wrapped in the outer layer of the bacteria group due to their hydrophobic characteristics,and inhibit the respiratory growth and block the effect of microorganisms on the heavy metals in the water,which seriously inhibit the growth of microorganisms,thus making the heavy m etals in the soil more difficult to be removed,and increasing the difficulty of treatment of oil-contaminated soil.In recent years,cyclodextrin has received extensive attention in the field of petroleum contaminated soil treatment due to its special str ucture.Cyclodextrins(CDs)are a class of cyclic oligosaccharides formed by the action of cyclodextrin glucosyltransferase on starch.Cyclodextrin compounds are characterized by the presence of a hydrophilic outer edge and a hydrophobic cavity in the mole cular structure.Its hydrophobic cavity can combine with many organic substances to form host and guest inclusions,and the water solubility of the inclusion compound is greater than the water solubility of the organic matter itself due to the hydrophilici ty of the outer side of the CD,which can increase the solubility of the organic substance in water,and also have a certain coordination effect with heavy metals.The cyclodextrin itself is non-toxic and biodegradable,it is an environmentally friendly material.However,cyclodextrin only has a solubilizing effect on some hydrophobic organic pollutants,and its solubility in water is low,which limits the wide application of cyclodextrin to some extent.Based on the above reasons,we chemically modified cyclodextrin,selected cyclodextrin derivatives with stronger water solubility and coordination ability to heavy metals,studied the effects of initial concentration and soil acidity on their solubility and absorption,and optimized its application condition s.Glycine-β-cyclodextrin(G-β-CD)was prepared through multiple experiments.The infrared spectroscopy and XPS analysis showed that G-β-CD retained the original structure,at the same time,amino groups and carboxyl groups were added.Hydrophilic groups such as amino groups and carboxyl groups increased the solubility of β-cyclodextrin in water and could form complex with heavy metals.The study found that in the treatment of petroleum-contaminated soil with G-β-CD,when the concentration of G-β-CD in the solubilizer was 70 g/L,the p H of the soil was 6.0 ～7.0,the reaction temperature was 30～40°C,and the ion concentration of the soil was 0.25mol/L,the organic matter content was 1%,the desorption effect of petroleum hydrocarbons and heavy metals achieved better results.Based on the previous research,we also analyzed the microbial degradation behavior of petroleum hydrocarbons in petroleum-contaminated soil under G-β-CD solubilization,and the changes of soil heavy metal species and bioavailability to ga in insight into the biological fate of petroleum hydrocarbons,and heavy metals in the soil environment and their ecological effects.It was found that after treatment of contaminated soil with G-β-CD,the form of heavy metals in the soil also changed greatly.The exchangeable fraction,carbonate bound fraction and organic binding fraction of Pb and Cu all decreased by more than 50%.The content of oxidizable and residual state of the matter decreased by about 60%,indicating that G-β-CD formed a stable soluble complex with heavy metals,which greatly reduced the adsorption of heavy metals in the soil,thereby reducing the toxicity and bioavailability of heavy metals in the soil.Moreover,the activities of polyphenol oxidase,catalase,dehydrogenase and lipase in petroleum-contaminated soil treated by G-β-CD were significantly improved.The results suggested that glycine-β-cyclodextrin could effectively amend petroleum hydrocarbon-heavy metal complex contaminated soil,which enhanced the activity of biologic al enzymes.It was found that the bio-remediation of petroleum-contaminated soils pretreated with G-β-CD combined with the soil itself was improved,but it was far from reaching normal soil.Therefore,this paper also strengthened petroleum hydrocarbon deg rading bacteria domestication,screening and identification of a highly efficient petroleum hydrocarbon degrading bacteria,and systematically studied the degradation ability and influencing factors of the degrading bacteria,and optimized the fermentation conditions of petroleum hydrocarbon degrading bacteria.The research not only considered the optimal conditions of the selected factors for the growth and reproduction of petroleum hydrocarbon degrading bacteria,but also the practical and economic problems such as low cost,practicality,and simple operation steps.The results showed that the bacteria cultured by adding soybean meal and corn starch into the inorganic salt medium had the same effect as the beef extract peptone liquid medium,and was more economical and suitable for the application of biological treatment on site.At the end of the experiment,the ecological toxicity test was used to comprehensively evaluate the repair effect of the chemical-microbial combined method.The effect of chemical-microbial combined remediation on soil microbial diversity was analyzed by denaturing gradient gel electrophoresis(PCR-DGGE).The results showed that the similarity of soil microbial population structure of oil-contaminated soil and clean soil was only 37.7%.After chemical-microbial combined remediation,the similarity increased to 76.2%,indicating that soil microbial diversity was well restored after chemical-microbial repair.The germination rate of seeds was observed by higher plant germination toxico logy experiment and crop absorption toxicology experiment,and the physiological and biochemical indexes of seedling leaves were measured to evaluate the ecotoxicity of soil after chemical-microorganism combined repair.The results showed that the seed germination rate,photosynthetic pigment content,superoxide dismutase(SOD)activity and malondialdehyde(MDA)content increased with the prolongation of chemical-microbial combined repair time,indicating that soil ecotoxicity was weakened.But there was some ecotoxicity in the soil during the whole period of restoration.The above results provided theoretical basis and technical support for the feasibility of the G-β-CD-microbial combined repair method.更多还原