表面活性剂增溶微生物—光催化联合降解污染土壤中的硫丹
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摘要
硫丹是一种广谱高效的有机氯农药,在全世界范围内被广泛使用。因其在环境中残留期长、生物累积性强、对生态系统和人体健康毒害大、可在全球范围内迁移,于2011年4月被新列入持久性有机污染物(POPs)控制清单。硫丹的长期和大量施用使得我国农田土壤中积累了较多的硫丹及其高毒代谢产物硫丹硫酸盐,对农业生态环境和人体健康都构成了较为严重的威胁。随着硫丹被逐步禁止使用,硫丹生产企业关停后的遗留场地及贮存场地土壤的污染问题也日渐显现。我国是《关于持久性有机污染物的斯德哥尔摩公约》的缔约国,有责任和义务控制硫丹的使用,防治硫丹对环境的污染,发展硫丹污染土壤的修复技术和方法。
目前,国内外大多利用微生物降解土壤中的硫丹,但硫丹为疏水性有机氯农药,又可被土壤胶体牢固吸附,生物有效性低,微生物降解效果不佳。同时,降解生成的毒性代谢产物硫丹硫酸盐可在土壤中长期积累,限制了微生物的降解效果和实际应用。因此,微生物降解土壤中硫丹目前还局限在实验室研究阶段。如何增大硫丹在土壤中的溶解能力,提高其生物有效性,在降解硫丹的同时去除高毒代谢产物硫丹硫酸盐,成为发展硫丹污染土壤修复技术、提高修复效率的重要研究内容。另一方面,硫丹对日光稳定,在土壤中难以直接光降解,也未见光催化降解土壤中硫丹的报道,但在光催化剂作用下水相中的硫丹可被彻底降解。因此,采用合适的光催化降解方法去除土壤中的硫丹值得探索。
本文首先利用阴离子表面活性剂十二烷基硫酸钠(SDS)、非离子表面活性剂(Tween80、 Triton X-100)和添加剂Na2SiO3,研究了表面活性剂对硫丹的溶解能力和对污染老化土壤中硫丹的洗脱能力,获得具有最佳洗脱效果的表面活性剂及其组合。然后,从硫丹污染土壤中筛选出可降解硫丹的微生物,并研制获得在可见光区能有效催化降解硫丹的非金属元素掺杂Ti02。在此基础上,以微生物和光催化为降解手段,比较了先洗脱后降解土壤淋出液中的硫丹(先洗脱后降解)、同时洗脱和降解十壤中硫丹(同时洗脱和降解)这两种处理方式对污染土壤中硫丹的去除效果。最后,研究了表面活性剂作用微生物-光催化联合降解污染土壤中硫丹的效果,获得如下研究结果：
1.筛选出可明显增溶和高效洗脱污染老化土壤中硫丹的表面活性剂及其组合。研究发现,a-硫丹在浓度为200～600mg·L-1的单一表面活性剂Tween80、Triton X-100和SDS中的溶解度分别达到9.28～25.70、3.95～8.40和0.88～1.83mg·L-1,溶解度与表面活性剂的临界胶束浓度呈负相关关系；β-硫丹在这3种表面活性剂中的溶解度与a-硫丹相差不大。当非／阴离子混合表面活性剂的质量比大于或等于4：1时,Tween80/SDS和Triton X-100/SDS对硫丹起协同增溶作用,Tween80/SDS对硫丹的增溶能力强于Triton X-100/SDS。表面活性剂对硫丹增溶能力的顺序为Tween80> Tween80/SDS> Triton X-100> Triton X-100/SDS> SDS,添加1000mg·L-1的Na2SiO3可增大表面活性剂及其组合的增溶能力,但不影响增溶能力的大小顺序。
不添加Na2SiO3时,表面活性剂对污染老化土壤中a-、β-硫丹的洗脱率顺序为Tween80/SDS>Tween80> Triton X-100, Triton X-100/SDS在100-500mg·L-1和800-1000mg·L"1时的洗脱率分别低于和高于相应浓度的Triton X-100,加入SDS对提升Triton X-100洗脱土壤中硫丹的能力贡献不大。添加Na2SiO3后,硫丹的洗脱率顺序为Tween80/SDS> Tween80> Triton X-100/SDS> Triton X-100,4种洗脱模式对硫丹的洗脱能力均显著提高,其中a-硫丹的洗脱率分别是不加Na2SiO3的1.17～2.73、1.87～4.02、1.85～6.56和1.87～2.85倍。表面活性剂对污染老化土壤中硫丹的洗脱过程可用4参数2室一级反应动力学模型描述,该过程包括慢速洗脱和快速洗脱两个阶段。添加Na2SiO3可增大快速、慢速洗脱速率常数,降低慢速洗脱百分率。β-硫丹的洗脱速率和洗脱率均低于相应处理的a-硫丹,表明β-硫丹较难从土壤中洗脱。与其它洗脱模式比较,添加Na2Si03的Tween80/SDS能够高效、快速洗脱污染土壤中的硫丹,采用并行解吸法解吸14和20h后,对污染老化土壤中a-、β-硫丹的洗脱率达到最大,分别为91.41%和74.01%。
2.从硫丹污染老化土壤中分离培养获得一株对高浓度硫丹具有耐受性的菌株EB-4,经16S rDNA鉴定确定为苍白杆菌属(Ochrobactrum sp.)。获得苍白杆菌降解洗脱液Eluent1(Tween80+Na2SiO3)、Eluent2(Tween80/SDS)和Eluent3(Tween80/SDS+Na2SiO3)中硫丹的最佳条件：pH为7.5～8.5,温度为35℃,微生物接种量大于10%,葡萄糖添加量1mg·L-1。苍白杆菌对3种洗脱液中硫丹的降解过程可用一级反应动力学模型描述,a-硫丹的半衰期分别为3.83、5.29和4.53d,β-硫丹分别为3.35、4.50和3.79d,β-硫丹的降解速率快于α-硫丹。
先洗脱后降解土壤淋出液中硫丹的结果表明,苍白杆菌在12d内可将土壤淋出液Eluate1、 Eluate2和Eluate3中的硫丹完全降解。同时洗脱和微生物降解土壤中硫丹15d时,Eluent1. Eluent2和Eluent3处理对土壤中α-、β-硫丹的降解率均达到92%以上；苍白杆菌对硫丹的降解速率小于表面活性剂对土壤中硫丹的洗脱速率,微生物降解是同时洗脱和微生物降解污染老化土壤中硫丹的限速步骤。
苍白杆菌以水解和氧化两种方式降解硫丹,代谢产物分别为硫丹二醇和硫丹硫酸盐。其中,硫丹二醇为主要代谢产物,可被苍白杆菌进一步代谢去除,不会在土壤和洗脱液中积累；高毒的硫丹硫酸盐为次要代谢产物,苍白杆菌难以进一步降解,在洗脱液和土壤中均表现出积累性。
3.采用sol-gel法制备N、F元素掺杂的TiO2,通过X射线衍射(XRD)和激光粒度表征,获得粒径范围为310.8～345.6nm的锐钛矿型掺杂TiO2。先洗脱再光催化降解淋出液中硫丹的结果表明,锐钛矿型N、F掺杂Ti02在氙气灯照射下对土壤淋出液中的硫丹具有光催化活性,在强酸和强碱性条件下降解率较高;当光催化剂用量为400mg·L-1时,降解效果最好,洗脱液中的SDS在酸性和碱性条件下对硫丹的光催化降解分别起促进和抑制作用。土壤淋出液中硫丹的光催化降解过程可用一级反应动力学模型进行描述。当用N掺杂Ti02为光催化剂时,土壤淋出液Eluate1’、Eluate2’、和Eluate3’中光降解α-硫丹的半衰期分别为71.7、43.0和58.7min,β-硫丹分别为85.3、51.1和66.5min；当用F掺杂Ti02为光催化剂时,土壤淋出液Eluate1’、 Eluate2’和Eluate3’中光降解α-硫丹的半衰期分别为121.6、138.6和165.0min,β-硫丹分别为135.9、154.0和177.7min。N掺杂Ti02的光催化降解效果好于F掺杂TiO2,β-硫丹的光降解速率明显小于α-硫丹。
同时洗脱和光催化降解土壤中硫丹的结果表明,以N掺杂TiO2为光催化剂,光降解120h时,Eluent1、Eluent2和Eluent3处理中α-硫丹的降解率分别为76.47%、74.81和79.14%,β-硫丹分别为57.79%、57.21%和58.52%；以F掺杂Ti02为光催化剂,a-硫丹的降解率分别为75.65%、73.59%和78.18%,β-硫丹分别为57.12%、56.41%和57.36%。研究发现,硫丹的光催化降解速率明显快于表面活性剂洗脱土壤中硫丹的速率,降解速率和程度取决于表面活性剂对硫丹的洗脱速率和程度。4参数2室一级反应动力学模型可以很好地描述同时洗脱和光催化降解土壤中硫丹的过程,包括快速光降解和慢速光降解两个阶段,后者是整个降解过程的速度控制步骤。由于β-硫丹可被土壤胶体更牢固吸附,α-硫丹的光催化降解速率快于β-硫丹。反应初期不同表面活性剂组合中硫丹的降解率差异显著,顺序为Eluent3> Eluent1> Eluent2,与这3种洗脱液对土壤中硫丹的洗脱率顺序一致。
光催化降解硫丹的主要途径是光氧化,在土壤和表面活性剂上清液中均可检出微量代谢产物硫丹硫酸盐,可通过光氧化作用快速去除；硫丹硫酸盐在土壤中的残留时间比在上清液中长,光催化降解14h可被完全去除,不会在土壤中积累。
4.在表面活性剂洗脱作用下,微生物-光催化联合降解可以缩短反应时间,提高污染土壤中硫丹的降解率,并有效避免硫丹硫酸盐的积累。比较同时洗脱和微生物降解3-9d后再光催化降解对硫丹的去除效果,发现同时洗脱和微生物降解的时间越长,联合降解对硫丹的去除率越高。其中,用Eluent3同时洗脱和微生物降解9d,再经N掺杂Ti02光催化降解480min,总浓度为20mg·kg-1的硫丹污染老化土壤中α-、β-硫丹的去除率分别达到96.57%和91.25%,微生物降解产生的硫丹硫酸盐也可同时去除,可高效修复硫丹污染的老化土壤。
Endosulfan is a widely used organochlorine pesticide (OCP) to control many insects and mites for field crops around the world. Owing to its highly toxic and bioaccumulation to most living organisms, and long-distance transport from its original source in the environment, endosulfan was listed in persistent organic pollutants (POPs) by Stockholm Convention in2011. With the long-term use of endosulfan. more and more endosulfan and its highly toxic metabolite (endosulfan sulfate) have been accumulated in agricultural soil in China, which poses a serious threat to agricultural ecological environment and human health. With being banned to use endosulfan gradually, people begin to concern on soil pollution problem in discarded manufacturing enterprise and storage site. China is a party to the Stockholm Convention on POPs control, and has responsibility and obligation to control the use of endosulfan. to prevent environmental pollution and develop the remedation technology for endosulfan-contaminated soil.
At present, microbial degradation is widely used to remove endosulfan in soil. However, endosulfan is a hydrophobic organochlorine pesticide, and can be solidly adsorbed by soil colloids, which leads to its low bioavailabilitv. and then affact the degradation efficiency of microorganisms. As a toxic metabolite of microbial degradation, endosulfan sulfate can be accumulated in soil for a long time, which may limit the degradation efficiency of microorganisms and practical application of this method. So these studies are still restricted to the laboratory. How to increase the solubility and bioavailability in soil and simultaneously remove endosulfan sulfate becomes an important topic for the development of remediation technology for endosulfan-contaminated soil and improvement the remediation efficiency. On the other hand, endosulfan in soil is difficult to degrade directly by sunlight, and few reports on the photocatalytic degradation of endosulfan in soil can be found. However, it was reported that endosulfan could be degraded thoroughly in aqueous phase by photocatalyst. This research inspires a new idea to degrade endosulfan in the contaminated soil, which is worthy to study using photocatalytic degradation.
In this thesis, we tried to improve the solubility and bioavailability of endosulfan in soil by screening suitable surfactants and their combination. An endosulfan degradading bacterium was isolated from the contaminated aged soil, and was employed to degrade endosulfan in soil with the elution of surfactants. Meanwhile, fluorine and nitrogen-doped TiO2which may effectively catalyze endosulfan and its metabolites to degrade were synthesized by sol-gel method, and were used to degrade endosulfan in soil with the elution of surfactants. With the solubilization of surfactants, we tried to integrate microbial degradation and photocatalytic degradation techniques, and hoped to obtain the combination degradation method for endosulfan in the contaminated soil, and to provide a scientific basis for developing the theory and technology to remedy the contaminated soil. The main results are summarized as follows.
1. Surfactants and their combination which could significantly improve the solubility of endosulfan and elute efficiently the contaminated aged soil were obtained. It could be found that the solubilities of a-endosulfan in Tween80, Triton X-100and SDS (sodium dodecyl sulfate) with the concentration of200～600mg·L'1were9.28～25.70,3.95～8.40and0.88～1.83mg·L-1, respectively, which were negatively correlated with critical micelle concentrations (CMCs) of these surfactants, while the solubilities of β-endosulfan in them showed the similar trend as that of a-endosulfan. When the mass ratio of anionic and nonionic surfactants was not less than4:1, Tween80/SDS and Triton X-100/SDS could synergistically increase the solubility of endosulfan, and the solubilization capacity of the former was stronger than the latter. The solubilization capacities of the surfactants followed a decreasing order of Tween80, Tween80/SDS, Triton X-100, Triton X-100/SDS, and SDS, and those could further increase with the similar trend in the presence of1000mg·L-1Na2SiO3.
The elution percents of α-,β-endosulfan followed a decreasing order of Tween80/SDS, Tween80and Triton X-100in the absence of Na2Si03, while that of Triton X-100/SDS at low concentration (100～500mg·L-1) and high concentration (800～1000mg·L-1) were lower and higher than those by the corresponding concentrations of Triton X-100, respectively. In general, the elution capability of Triton X-100did not obviously improve with the addition of SDS. The elution percents of enduslfan follwed a decreasing order of Tween80/SDS, Tween80, Triton X-100/SDS, and Triton X-100in the presence of Na2SiO3, and those of a-endosulfan were as1.17～2.73,1.87～4.02,1.85～6.56and1.87～2.85times as that in the absence of Na2SiO3, and thus the elution capabilities of the four modes increased evidently. The elution process of endosulfan in contaminated aged soil could be described by a4-parameter biphasic first-order reaction kinetic model, and showed obvious rapid and slow elution phases. The addition of Na2SiO3could increase the rate constants of rapid elution and slow elution, and decrease slow elution percent. Both of the elution percent and elution rate of β-endosulfanl were lower than those of a-endosulfan, which indicated that β-endosulfan in soil was difficult to be eluted. Compared with the other elution modes, endosulfan could be effectively and rapidly eluted by Tween80/SDS in the presence of Na2SiO3. and the elution percents of α-,β-endosulfan in contaminated aged soil reached the maximums (91.41%and74.01%) by parallel desorption for14and20h. respectively.
2. A strain named as EB-4was isolated from the aged soil contaminated by endosulfan, and was identified as Ochrobactrum sp. by16S rDNA sequence analysis. The optimum degradation conditions of this strain to degrade endosulfan in three surfactant solutions were obtained in Eluent1(Tween80+Na2SiO3), Eluent2(Tween80/SDS) and Eluent3(Tween80/SDS+Na2SiO3), i.e., pH7.5～8.5.35℃, the microbial inoculation over10%, and the adding content of glucose1mg·L-1. The degradation process of endosulfan in those surfactant solutions could be described by the first-order reaction kinetic model, and the half-lives of a-endosulfan were3.83,5.29, and4.53d, and those of β-endosulfan were3.35,4.50. and3.79d, respectively. The degradation rate of this strain for β-endosulfan was faster than that for a-endosulfan.
The experimental results showed that endosulfan in Eluate1, Eluate2and Eluate3could be completely degraded in12d for surfactant elution followed with microbial degradation. The degradation percents of endosulfan in soil were more than92%in15d for simultaneous elution and microbial degradation. The microbial degradation rate of endosulfan was lower than the elution rate from the contaminated soil, and the former was the rate determining step in the whole process.
Ochrobactrum sp. can degrade endosulfan with two pathways, i.e.. hydrolysis and oxidation. Hydralysis is the main pathway, and the main metabolic product is endosulfan diol which can be degraded further by this strain, and thus it may not accumulate in soil and eluent. The main metabolite of oxidation is endosulfan sulfate which is difficult to be degraded further, and can be accumulated in soil and eluent.
3. Nitrogen and fluorine doped Anatase TiO2could be synthesized by sol-gel method, and the average particle sizes were in the range of310.8-345.6nm characterized by X-ray diffraction and laser particle size analysis. The results showed that the two doped TiO2had photocatalytic activity for endosulfan in soil eluate under the irradiation of xenon lamp, and high photodegradation rates could be obtained under the conditions of strong acid and strong alkaline for surfactant elution followed with photocatalytic degradation. It was found that the optimum dosage of photocatalyst was400mg·L-1. and SDS in the eluent could promote and inhibit the photodegradation of endosulfan under the acid and alkaline conditions, resepctively. The photocatalytic degradation of endosulfan in soil eluate could be described by the first-order reaction kinetic model. When N-doped TiO2was used to be photocatalyst, the half-lives of a-endosulfan in Eluate1', Eluate2'and Eluate3'were71.7,43.0and58.7min, and those of β-endosulfan were85.3,51.1,66.5min, respectively. When F-doped TiO2was used to be photocatalyst, the half-lives of a-endosulfan in Eluate1', Eluate2'and Eluate3'were121.6,138.6and165.0min, and those of β-endosulfan were135.9,154.0and177.7min, respectively. It indicated that the photocatalytic activity of N-doped TiO2was higher than that of F-doped TiO2, and the photodegradation rate of β-endosulfan was lower than that of a-endosulfan.
For simultaneous elution and photocatalytic degradation, the photodegradation percents of a-endosulfan with the elution of Eluent1-Eluent3using N-doped TiO2were76.47%,74.81%and79.14%at120h irradiation, while.that of β-endosulfan were57.79%,57.21%and58.52%, respectively. The photodegradation percents of a-endosulfan increased to75.65%,73.59%and78.18%using F-doped TiO2at120h irradiation, while that of β-endosulfan increased to57.12%,56.41%and57.36%, respectively. The results also showed that the photodegradation rate of endosulfan was faster than the elution rate from soil, and the surfactant elution was the rate determining step in the whole process. The photodegradation process could be well described by a four-parameter biphasic first-order reaction kinetic model, and showed obvious rapid and slow photodegradation phases, the latter was the rate determining step. The photodegradation rate of β-endosulfan in the contaminated aged soil was lower than that of a-endosulfan, because β-endosulfan would be more strongly adsorbed on soil colloids. The degradation percents of endosulfan in different eluents followed a decreasing order of Eluent3, Eluent1and Eluent2, and were the similar as the variations of the elution percents. Photooxidation is the primary pathway for the photocatalytic degradation of endosulfan, and the degradation product endosulfan sulfate could be detected both in soil and the supernatant, which could be degraded quickly by photooxidation. Endosulfan sulfate in soil could remain longer than in the supernatant, and needed14h to completely remove by photodegradation.
4. With the soblization of surfactants, microbial-photocatalytic combination degradation could shorten the reaction time, and improve the degradation efficiency of endosulfan in the contaminated soil, and thus could effectively avoid the accumulation of endosulfan sulfate. Compared with the removal percents of endosulfan in soil by simultaneous elution and microbial degradation for3-9d followed with photodegradation, it could be found that the longer of the time of simultaneous elution and microbial degradation, the higher the removal percent of endosulfan by combination degradation. Among them, the removal percents of α-,β-endosulfan in contaminated aged soil spiked with20mg·kg-1endosulfan could reach96.57%and91.25%by N-doped TiO2photocatalytic degradation for120min after simultaneous elution using Eluent3and microbial degradation for9d, respectively, and the micariobial metabolite endosulfan sulfate could be also removed well, and thus may effectively remedy the endosulfan-contaminated soil.
目前,国内外大多利用微生物降解土壤中的硫丹,但硫丹为疏水性有机氯农药,又可被土壤胶体牢固吸附,生物有效性低,微生物降解效果不佳。同时,降解生成的毒性代谢产物硫丹硫酸盐可在土壤中长期积累,限制了微生物的降解效果和实际应用。因此,微生物降解土壤中硫丹目前还局限在实验室研究阶段。如何增大硫丹在土壤中的溶解能力,提高其生物有效性,在降解硫丹的同时去除高毒代谢产物硫丹硫酸盐,成为发展硫丹污染土壤修复技术、提高修复效率的重要研究内容。另一方面,硫丹对日光稳定,在土壤中难以直接光降解,也未见光催化降解土壤中硫丹的报道,但在光催化剂作用下水相中的硫丹可被彻底降解。因此,采用合适的光催化降解方法去除土壤中的硫丹值得探索。
本文首先利用阴离子表面活性剂十二烷基硫酸钠(SDS)、非离子表面活性剂(Tween80、 Triton X-100)和添加剂Na2SiO3,研究了表面活性剂对硫丹的溶解能力和对污染老化土壤中硫丹的洗脱能力,获得具有最佳洗脱效果的表面活性剂及其组合。然后,从硫丹污染土壤中筛选出可降解硫丹的微生物,并研制获得在可见光区能有效催化降解硫丹的非金属元素掺杂Ti02。在此基础上,以微生物和光催化为降解手段,比较了先洗脱后降解土壤淋出液中的硫丹(先洗脱后降解)、同时洗脱和降解十壤中硫丹(同时洗脱和降解)这两种处理方式对污染土壤中硫丹的去除效果。最后,研究了表面活性剂作用微生物-光催化联合降解污染土壤中硫丹的效果,获得如下研究结果：
1.筛选出可明显增溶和高效洗脱污染老化土壤中硫丹的表面活性剂及其组合。研究发现,a-硫丹在浓度为200～600mg·L-1的单一表面活性剂Tween80、Triton X-100和SDS中的溶解度分别达到9.28～25.70、3.95～8.40和0.88～1.83mg·L-1,溶解度与表面活性剂的临界胶束浓度呈负相关关系；β-硫丹在这3种表面活性剂中的溶解度与a-硫丹相差不大。当非／阴离子混合表面活性剂的质量比大于或等于4：1时,Tween80/SDS和Triton X-100/SDS对硫丹起协同增溶作用,Tween80/SDS对硫丹的增溶能力强于Triton X-100/SDS。表面活性剂对硫丹增溶能力的顺序为Tween80> Tween80/SDS> Triton X-100> Triton X-100/SDS> SDS,添加1000mg·L-1的Na2SiO3可增大表面活性剂及其组合的增溶能力,但不影响增溶能力的大小顺序。
不添加Na2SiO3时,表面活性剂对污染老化土壤中a-、β-硫丹的洗脱率顺序为Tween80/SDS>Tween80> Triton X-100, Triton X-100/SDS在100-500mg·L-1和800-1000mg·L"1时的洗脱率分别低于和高于相应浓度的Triton X-100,加入SDS对提升Triton X-100洗脱土壤中硫丹的能力贡献不大。添加Na2SiO3后,硫丹的洗脱率顺序为Tween80/SDS> Tween80> Triton X-100/SDS> Triton X-100,4种洗脱模式对硫丹的洗脱能力均显著提高,其中a-硫丹的洗脱率分别是不加Na2SiO3的1.17～2.73、1.87～4.02、1.85～6.56和1.87～2.85倍。表面活性剂对污染老化土壤中硫丹的洗脱过程可用4参数2室一级反应动力学模型描述,该过程包括慢速洗脱和快速洗脱两个阶段。添加Na2SiO3可增大快速、慢速洗脱速率常数,降低慢速洗脱百分率。β-硫丹的洗脱速率和洗脱率均低于相应处理的a-硫丹,表明β-硫丹较难从土壤中洗脱。与其它洗脱模式比较,添加Na2Si03的Tween80/SDS能够高效、快速洗脱污染土壤中的硫丹,采用并行解吸法解吸14和20h后,对污染老化土壤中a-、β-硫丹的洗脱率达到最大,分别为91.41%和74.01%。
2.从硫丹污染老化土壤中分离培养获得一株对高浓度硫丹具有耐受性的菌株EB-4,经16S rDNA鉴定确定为苍白杆菌属(Ochrobactrum sp.)。获得苍白杆菌降解洗脱液Eluent1(Tween80+Na2SiO3)、Eluent2(Tween80/SDS)和Eluent3(Tween80/SDS+Na2SiO3)中硫丹的最佳条件：pH为7.5～8.5,温度为35℃,微生物接种量大于10%,葡萄糖添加量1mg·L-1。苍白杆菌对3种洗脱液中硫丹的降解过程可用一级反应动力学模型描述,a-硫丹的半衰期分别为3.83、5.29和4.53d,β-硫丹分别为3.35、4.50和3.79d,β-硫丹的降解速率快于α-硫丹。
先洗脱后降解土壤淋出液中硫丹的结果表明,苍白杆菌在12d内可将土壤淋出液Eluate1、 Eluate2和Eluate3中的硫丹完全降解。同时洗脱和微生物降解土壤中硫丹15d时,Eluent1. Eluent2和Eluent3处理对土壤中α-、β-硫丹的降解率均达到92%以上；苍白杆菌对硫丹的降解速率小于表面活性剂对土壤中硫丹的洗脱速率,微生物降解是同时洗脱和微生物降解污染老化土壤中硫丹的限速步骤。
苍白杆菌以水解和氧化两种方式降解硫丹,代谢产物分别为硫丹二醇和硫丹硫酸盐。其中,硫丹二醇为主要代谢产物,可被苍白杆菌进一步代谢去除,不会在土壤和洗脱液中积累；高毒的硫丹硫酸盐为次要代谢产物,苍白杆菌难以进一步降解,在洗脱液和土壤中均表现出积累性。
3.采用sol-gel法制备N、F元素掺杂的TiO2,通过X射线衍射(XRD)和激光粒度表征,获得粒径范围为310.8～345.6nm的锐钛矿型掺杂TiO2。先洗脱再光催化降解淋出液中硫丹的结果表明,锐钛矿型N、F掺杂Ti02在氙气灯照射下对土壤淋出液中的硫丹具有光催化活性,在强酸和强碱性条件下降解率较高;当光催化剂用量为400mg·L-1时,降解效果最好,洗脱液中的SDS在酸性和碱性条件下对硫丹的光催化降解分别起促进和抑制作用。土壤淋出液中硫丹的光催化降解过程可用一级反应动力学模型进行描述。当用N掺杂Ti02为光催化剂时,土壤淋出液Eluate1’、Eluate2’、和Eluate3’中光降解α-硫丹的半衰期分别为71.7、43.0和58.7min,β-硫丹分别为85.3、51.1和66.5min；当用F掺杂Ti02为光催化剂时,土壤淋出液Eluate1’、 Eluate2’和Eluate3’中光降解α-硫丹的半衰期分别为121.6、138.6和165.0min,β-硫丹分别为135.9、154.0和177.7min。N掺杂Ti02的光催化降解效果好于F掺杂TiO2,β-硫丹的光降解速率明显小于α-硫丹。
同时洗脱和光催化降解土壤中硫丹的结果表明,以N掺杂TiO2为光催化剂,光降解120h时,Eluent1、Eluent2和Eluent3处理中α-硫丹的降解率分别为76.47%、74.81和79.14%,β-硫丹分别为57.79%、57.21%和58.52%；以F掺杂Ti02为光催化剂,a-硫丹的降解率分别为75.65%、73.59%和78.18%,β-硫丹分别为57.12%、56.41%和57.36%。研究发现,硫丹的光催化降解速率明显快于表面活性剂洗脱土壤中硫丹的速率,降解速率和程度取决于表面活性剂对硫丹的洗脱速率和程度。4参数2室一级反应动力学模型可以很好地描述同时洗脱和光催化降解土壤中硫丹的过程,包括快速光降解和慢速光降解两个阶段,后者是整个降解过程的速度控制步骤。由于β-硫丹可被土壤胶体更牢固吸附,α-硫丹的光催化降解速率快于β-硫丹。反应初期不同表面活性剂组合中硫丹的降解率差异显著,顺序为Eluent3> Eluent1> Eluent2,与这3种洗脱液对土壤中硫丹的洗脱率顺序一致。
光催化降解硫丹的主要途径是光氧化,在土壤和表面活性剂上清液中均可检出微量代谢产物硫丹硫酸盐,可通过光氧化作用快速去除；硫丹硫酸盐在土壤中的残留时间比在上清液中长,光催化降解14h可被完全去除,不会在土壤中积累。
4.在表面活性剂洗脱作用下,微生物-光催化联合降解可以缩短反应时间,提高污染土壤中硫丹的降解率,并有效避免硫丹硫酸盐的积累。比较同时洗脱和微生物降解3-9d后再光催化降解对硫丹的去除效果,发现同时洗脱和微生物降解的时间越长,联合降解对硫丹的去除率越高。其中,用Eluent3同时洗脱和微生物降解9d,再经N掺杂Ti02光催化降解480min,总浓度为20mg·kg-1的硫丹污染老化土壤中α-、β-硫丹的去除率分别达到96.57%和91.25%,微生物降解产生的硫丹硫酸盐也可同时去除,可高效修复硫丹污染的老化土壤。
Endosulfan is a widely used organochlorine pesticide (OCP) to control many insects and mites for field crops around the world. Owing to its highly toxic and bioaccumulation to most living organisms, and long-distance transport from its original source in the environment, endosulfan was listed in persistent organic pollutants (POPs) by Stockholm Convention in2011. With the long-term use of endosulfan. more and more endosulfan and its highly toxic metabolite (endosulfan sulfate) have been accumulated in agricultural soil in China, which poses a serious threat to agricultural ecological environment and human health. With being banned to use endosulfan gradually, people begin to concern on soil pollution problem in discarded manufacturing enterprise and storage site. China is a party to the Stockholm Convention on POPs control, and has responsibility and obligation to control the use of endosulfan. to prevent environmental pollution and develop the remedation technology for endosulfan-contaminated soil.
At present, microbial degradation is widely used to remove endosulfan in soil. However, endosulfan is a hydrophobic organochlorine pesticide, and can be solidly adsorbed by soil colloids, which leads to its low bioavailabilitv. and then affact the degradation efficiency of microorganisms. As a toxic metabolite of microbial degradation, endosulfan sulfate can be accumulated in soil for a long time, which may limit the degradation efficiency of microorganisms and practical application of this method. So these studies are still restricted to the laboratory. How to increase the solubility and bioavailability in soil and simultaneously remove endosulfan sulfate becomes an important topic for the development of remediation technology for endosulfan-contaminated soil and improvement the remediation efficiency. On the other hand, endosulfan in soil is difficult to degrade directly by sunlight, and few reports on the photocatalytic degradation of endosulfan in soil can be found. However, it was reported that endosulfan could be degraded thoroughly in aqueous phase by photocatalyst. This research inspires a new idea to degrade endosulfan in the contaminated soil, which is worthy to study using photocatalytic degradation.
In this thesis, we tried to improve the solubility and bioavailability of endosulfan in soil by screening suitable surfactants and their combination. An endosulfan degradading bacterium was isolated from the contaminated aged soil, and was employed to degrade endosulfan in soil with the elution of surfactants. Meanwhile, fluorine and nitrogen-doped TiO2which may effectively catalyze endosulfan and its metabolites to degrade were synthesized by sol-gel method, and were used to degrade endosulfan in soil with the elution of surfactants. With the solubilization of surfactants, we tried to integrate microbial degradation and photocatalytic degradation techniques, and hoped to obtain the combination degradation method for endosulfan in the contaminated soil, and to provide a scientific basis for developing the theory and technology to remedy the contaminated soil. The main results are summarized as follows.
1. Surfactants and their combination which could significantly improve the solubility of endosulfan and elute efficiently the contaminated aged soil were obtained. It could be found that the solubilities of a-endosulfan in Tween80, Triton X-100and SDS (sodium dodecyl sulfate) with the concentration of200～600mg·L'1were9.28～25.70,3.95～8.40and0.88～1.83mg·L-1, respectively, which were negatively correlated with critical micelle concentrations (CMCs) of these surfactants, while the solubilities of β-endosulfan in them showed the similar trend as that of a-endosulfan. When the mass ratio of anionic and nonionic surfactants was not less than4:1, Tween80/SDS and Triton X-100/SDS could synergistically increase the solubility of endosulfan, and the solubilization capacity of the former was stronger than the latter. The solubilization capacities of the surfactants followed a decreasing order of Tween80, Tween80/SDS, Triton X-100, Triton X-100/SDS, and SDS, and those could further increase with the similar trend in the presence of1000mg·L-1Na2SiO3.
The elution percents of α-,β-endosulfan followed a decreasing order of Tween80/SDS, Tween80and Triton X-100in the absence of Na2Si03, while that of Triton X-100/SDS at low concentration (100～500mg·L-1) and high concentration (800～1000mg·L-1) were lower and higher than those by the corresponding concentrations of Triton X-100, respectively. In general, the elution capability of Triton X-100did not obviously improve with the addition of SDS. The elution percents of enduslfan follwed a decreasing order of Tween80/SDS, Tween80, Triton X-100/SDS, and Triton X-100in the presence of Na2SiO3, and those of a-endosulfan were as1.17～2.73,1.87～4.02,1.85～6.56and1.87～2.85times as that in the absence of Na2SiO3, and thus the elution capabilities of the four modes increased evidently. The elution process of endosulfan in contaminated aged soil could be described by a4-parameter biphasic first-order reaction kinetic model, and showed obvious rapid and slow elution phases. The addition of Na2SiO3could increase the rate constants of rapid elution and slow elution, and decrease slow elution percent. Both of the elution percent and elution rate of β-endosulfanl were lower than those of a-endosulfan, which indicated that β-endosulfan in soil was difficult to be eluted. Compared with the other elution modes, endosulfan could be effectively and rapidly eluted by Tween80/SDS in the presence of Na2SiO3. and the elution percents of α-,β-endosulfan in contaminated aged soil reached the maximums (91.41%and74.01%) by parallel desorption for14and20h. respectively.
2. A strain named as EB-4was isolated from the aged soil contaminated by endosulfan, and was identified as Ochrobactrum sp. by16S rDNA sequence analysis. The optimum degradation conditions of this strain to degrade endosulfan in three surfactant solutions were obtained in Eluent1(Tween80+Na2SiO3), Eluent2(Tween80/SDS) and Eluent3(Tween80/SDS+Na2SiO3), i.e., pH7.5～8.5.35℃, the microbial inoculation over10%, and the adding content of glucose1mg·L-1. The degradation process of endosulfan in those surfactant solutions could be described by the first-order reaction kinetic model, and the half-lives of a-endosulfan were3.83,5.29, and4.53d, and those of β-endosulfan were3.35,4.50. and3.79d, respectively. The degradation rate of this strain for β-endosulfan was faster than that for a-endosulfan.
The experimental results showed that endosulfan in Eluate1, Eluate2and Eluate3could be completely degraded in12d for surfactant elution followed with microbial degradation. The degradation percents of endosulfan in soil were more than92%in15d for simultaneous elution and microbial degradation. The microbial degradation rate of endosulfan was lower than the elution rate from the contaminated soil, and the former was the rate determining step in the whole process.
Ochrobactrum sp. can degrade endosulfan with two pathways, i.e.. hydrolysis and oxidation. Hydralysis is the main pathway, and the main metabolic product is endosulfan diol which can be degraded further by this strain, and thus it may not accumulate in soil and eluent. The main metabolite of oxidation is endosulfan sulfate which is difficult to be degraded further, and can be accumulated in soil and eluent.
3. Nitrogen and fluorine doped Anatase TiO2could be synthesized by sol-gel method, and the average particle sizes were in the range of310.8-345.6nm characterized by X-ray diffraction and laser particle size analysis. The results showed that the two doped TiO2had photocatalytic activity for endosulfan in soil eluate under the irradiation of xenon lamp, and high photodegradation rates could be obtained under the conditions of strong acid and strong alkaline for surfactant elution followed with photocatalytic degradation. It was found that the optimum dosage of photocatalyst was400mg·L-1. and SDS in the eluent could promote and inhibit the photodegradation of endosulfan under the acid and alkaline conditions, resepctively. The photocatalytic degradation of endosulfan in soil eluate could be described by the first-order reaction kinetic model. When N-doped TiO2was used to be photocatalyst, the half-lives of a-endosulfan in Eluate1', Eluate2'and Eluate3'were71.7,43.0and58.7min, and those of β-endosulfan were85.3,51.1,66.5min, respectively. When F-doped TiO2was used to be photocatalyst, the half-lives of a-endosulfan in Eluate1', Eluate2'and Eluate3'were121.6,138.6and165.0min, and those of β-endosulfan were135.9,154.0and177.7min, respectively. It indicated that the photocatalytic activity of N-doped TiO2was higher than that of F-doped TiO2, and the photodegradation rate of β-endosulfan was lower than that of a-endosulfan.
For simultaneous elution and photocatalytic degradation, the photodegradation percents of a-endosulfan with the elution of Eluent1-Eluent3using N-doped TiO2were76.47%,74.81%and79.14%at120h irradiation, while.that of β-endosulfan were57.79%,57.21%and58.52%, respectively. The photodegradation percents of a-endosulfan increased to75.65%,73.59%and78.18%using F-doped TiO2at120h irradiation, while that of β-endosulfan increased to57.12%,56.41%and57.36%, respectively. The results also showed that the photodegradation rate of endosulfan was faster than the elution rate from soil, and the surfactant elution was the rate determining step in the whole process. The photodegradation process could be well described by a four-parameter biphasic first-order reaction kinetic model, and showed obvious rapid and slow photodegradation phases, the latter was the rate determining step. The photodegradation rate of β-endosulfan in the contaminated aged soil was lower than that of a-endosulfan, because β-endosulfan would be more strongly adsorbed on soil colloids. The degradation percents of endosulfan in different eluents followed a decreasing order of Eluent3, Eluent1and Eluent2, and were the similar as the variations of the elution percents. Photooxidation is the primary pathway for the photocatalytic degradation of endosulfan, and the degradation product endosulfan sulfate could be detected both in soil and the supernatant, which could be degraded quickly by photooxidation. Endosulfan sulfate in soil could remain longer than in the supernatant, and needed14h to completely remove by photodegradation.
4. With the soblization of surfactants, microbial-photocatalytic combination degradation could shorten the reaction time, and improve the degradation efficiency of endosulfan in the contaminated soil, and thus could effectively avoid the accumulation of endosulfan sulfate. Compared with the removal percents of endosulfan in soil by simultaneous elution and microbial degradation for3-9d followed with photodegradation, it could be found that the longer of the time of simultaneous elution and microbial degradation, the higher the removal percent of endosulfan by combination degradation. Among them, the removal percents of α-,β-endosulfan in contaminated aged soil spiked with20mg·kg-1endosulfan could reach96.57%and91.25%by N-doped TiO2photocatalytic degradation for120min after simultaneous elution using Eluent3and microbial degradation for9d, respectively, and the micariobial metabolite endosulfan sulfate could be also removed well, and thus may effectively remedy the endosulfan-contaminated soil.
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