【作者】 丁娜；

【导师】 徐建明；

【作者基本信息】 浙江大学 ， 土壤学， 2012， 博士

【摘要】 多氯联苯(Polychlorinated biphenyls)是环境中普遍存在的具有代表性的一类重要持久性有机污染物(Persistant organic pollutants, POPs),在土壤环境中不断积累,严重危胁着土壤的生产和生态功能、农产品质量和人类健康。防治和修复多氯联苯污染、保障土壤环境和农产品安全、以实现土壤资源的可持续利用是当前全球关注的焦点。植物修复是利用植物-微生物在根际环境降解有机污染物的复合生物修复技术,是目前最具潜力的土壤生物修复技术之一。研究植物修复多氯联苯的根际响应机制及其在根际微域中的降解作用机理,将为针对性地拟定经济高效的土壤PCBs污染防治和修复的实用技术提供基础理论依据。本论文以多氯联苯为目标污染物,首先优化、确立了土壤环境中PCBs的提取-净化-分离的分析方法,并在此基础上采用多隔层根箱法和磷脂脂肪酸(PLFAs)技术开展了离根表不同距离微域中PCBs降解效应的研究；从根际微生物、根系分泌物角度研究了黑麦草修复PCBs污染的根际响应机制；采用溶液培养的手段收集黑麦草根系分泌物,研究了模拟根际环境条件下PCBs的降解以及微生物的响应；最后,就黑麦草对PCBs的吸收、积累和体内运输以及植物体内解毒酶的响应进行了探讨。主要研究结果如下：(1)以Aroclor1242为目标污染物,分别对土壤样品中的分析条件进行了优化,研究了萃取溶剂、温度、时间等萃取条件对多氯联苯回收效率的影响,建立了超声萃取-气相色谱质谱法测定土壤中多氯联苯的分析方法。采用正己烷/丙酮(v/v=1/1)混合液作为萃取溶剂,萃取最佳温度为35℃,萃取时间为30 min,在此条件下多氯联苯Aroclor1242的平均加标回收率在81.4-90.2%之间,相对标准偏差为2.8-3.8%。采用湿法填柱、20ml正己烷作为淋洗溶剂,有效去除了萃取液中的干扰物质。优化了测定土壤中PCBs的色谱、质谱条件,最终实现了Aroclor1242不同同系物之间的有效分离。(2)采用改进的多隔层三室根箱法,通过尼龙撩网插片的控制,对根际土壤根室,距离根表1 mm、2 mm、3 mm、4 mm和5 mm的近根际,以及>5 mm远根际的微域进行划分。Aroclor 1242的根际消减行为存在距离根表不同距离远近的差异,在8mg/kg(低浓度)和16mg/kg(高浓度)两个污染水平下均呈现出一致的趋势,即近根际>根室>远根际；Aroclor1242的最大消减水平发生在距离根表3mm处,而不是距离根表最近的根室和1、2 mm处,表现出根际效应的污染物消减特性,有别于传统植物营养学中发现的营养因子根际梯度递减效应；不同氯含量的PCBs表现出不同的消减效率,研究发现低氯联苯,尤其是二氯联苯表现出较高的降解效率。作为土壤中重要活性物质DOC在不同PCBs污染水平下有不同的响应,高污染水平下DOC含量明显低于对照和低浓度污染水平。(3)借助土壤学现代发展起来的微生物鉴定技术-PLFAs (Phospholipid fatty acids)技术,围绕PCBs根际特异消减行为的土壤微生物学作用机制,系统深入研究了根际微域中特异性根分泌物诱导的土壤微生物群落结构的微空间演变特征。研究发现,受到黑麦草生长及根系分泌物的影响,微生物群落结构在毫米级微域内发生规律性变化,其在诱导PCBs快速消减方面起着决定性的作用。在种植物的处理中,单个PLFA i16:0,16:0 N alcohol,18:0(10Me), i16:1, a15:0, i14:1,14：020H,18：1ω9c,a17：0,14：0 3OH,i14：0,a16：0,16：1ω5c与Aroclor1242的消减呈显著正相关(p<0.05)。对不同PCBs污染浓度水平下微生物群落结构的研究表明,16 mg/kg高浓度污染水平下,真菌种群受到激发,真菌PLFAs较对照、低浓度处理显著增加(p<0.05),这可能是Aroclor1242作为碳源,被真菌利用所致。单体PLFA i16:1在黑麦草处理和对照处理中对Aroclor 1242的消减都有较大的贡献,这一发现解释了在未种植物处理中Aroclor1242消减的原因之一,进一步证明在受PCBs污染的自然土壤中,某些微生物对多氯联苯的自然消减发挥着重要的作用。(4)以邻位和对位取代氯的PCB7(24-CB)为研究对象,研究了其在黑麦草根际环境中24-CB的降解、黑麦草体内的运输以及植物体内抗氧化系统(SOD,POD)的响应。45天的盆栽实验结果表明,在两种土壤中,黑麦草根际对24-CB在土壤中的消减均有显著的促进作用。由初始浓度的30±0.5mg/kg分别减少到3.43 mg/kg和2.09 mg/kg。植株根部和地上部分也均有24-CB的检出,并且根部24-CB富集量远远高于植株地上部分。说明从土壤吸收到植株体内的24-CB主要富集在植物根部,只有少量的24-CB能够转移到地上部分。植物吸收不是土壤中PCBs减少的主要原因。24-CB胁迫显著影响黑麦草体内活性氧清除系统,在有机质含量不同的两种土壤中表现有所差异。淋溶土处理中,POD活性较对照处理显著增加,说明在适度的24-CB胁迫下,黑麦草可激发其自身的防御体系；松软土中与未受24-CB胁迫的对照比较,POD在污染胁迫下没有显著变化。SOD活性在两种土壤处理中也表现出不同的响应。因此土壤类型差异,尤其是有机质也是影响24-CB生物有效性、最终影响到植物对其的修复效率的重要因素之一。(5)采用水培法收集黑麦草根系分泌物,将其添加在2345-CB污染的土壤中,模拟根际环境条件,研究了根系分泌物对2345-CB污染土壤的修复效果。根系分泌物的引入激发了土壤中微生物群落结构的明显变化,好氧微生物受到了抑制,而厌氧微生物,特别是甲烷氧化菌和硫酸盐还原菌大量繁殖,进而影响到2345-CB的降解速率和降解途径。根系分泌物的添加在短期内显著提高了2345-CB的消减速率,随着培养时间的延长,根系分泌物对其消减的影响逐渐减弱。此外研究发现,在2345-CB的消减途径中,主要表现为还原脱氯作用,发生在间位(meta-)和对位(para-)位点的脱氯,从而导致了邻位234-CB,245-CB,25-CB的大量积累。更多还原

【Abstract】 Polychlorinated biphenyls (PCBs), a class of typical POPs (Persistent Organic Pollutants), are widely accumulated in the soil environment. Soil contamination with PCBs poses a great threat to the production and ecological function of soil, the food quality and human health due to their widespread occurrence, persistence in soil ecosystems, carcinogenic, mutagenic and not easy degradable properties. Phytoremediation is a relatively novel approach to remove organic contaminants from soils by plants, rhizosphere microorganisms and root exudates. Investigations of the environmental behavior of PCBs in soils, including the residue, dissipation and the controlling key factors, seem urgent and effective.Firstly, a method for detecting PCBs residue in soils from the extraction, cleanup to the final detection, was modified and investigated. Based on the fast, accurate, high sensitivity method, the degradation of PCBs in the rhizosphere of the ryegrass and the corresponding responses, including soil enzyme and microbial structure were studied. A special-designed rhizobox and a novel method of phospholipid fatty acids (PLFAs) were used in this research. Rhizo environment by the addition of root exudates was simulated and dissipation of PCBs in soils was studied. The adsorption, transfer and accumulation of PCBs from soils to plants and the detoxification enzymes in ryegrass to 24-CB were investigated in the study. The main conclusions are as following:(1) The ultrasonic extraction was used for the extraction of PCBs residue in soil sample. The influence factors of solvents, temperature, and time on the extraction efficiency of were discussed. The results indicated that the optimal analytical conditions of solvent, temperature, and time were hexane-acetone (v:v=1:1),35℃and 30min respectively. The spike standard recoveries rates were between 81.4 and 90.2%, relative standard deviation was 2.8 to 3.8%. Florisil column (wet and dry filled column) for purification and separation were used. The results demonstrated that wet filled column was useful and effective. And the optimal parameters for GC-MS were modified and established.(2) A special-designed rhizobox experiment was conducted in the greenhouse. The ryegrass seedlings were grown for 135 days in soils spiked with Aroclor 1242 at concentrations of 8 and 16mg/kg soil to investigate rhizosphere effects on the dissipation of PCBs. The soil in the rhizobox was divided into six separate compartments at various distances from the root surface. Changes in Aroclor 1242 concentration with increasing distance from the root compartment of rhizobox were then assessed. In the treatment of 8 mg/kg, the Aroclor 1242 degradation gradients observed in the rhizobox of planted treatments appeared to be highest at the 3 mm zone layer, then at 2,4,1 and 5 mm sequentially. Thus the greatest decrease in Aroclor 1242 concentration could be achieved here because 3 mm zone represents balance between microbial activities and root exudates availability. The trend observed in the high spiked level was similar. The dissipation gradient followed the order:near rhizosphere>root compartment>far-rhizosphere soils zones. The results also showed that different congeners with different chlorine atoms had the various dissipation rates and the PCBs with two chlorine atoms appeared high degradation rates.(3) Further investigations were conducted using phospholipid fatty acids (PLFAs) profiles to follow the millimeter spatial response of the soil microbial community with the purpose to illustrate the mechanism of dissipation gradients of Aroclor 1242 in the rhizosphere of ryegrass (Lolium perenne L). The highest concentration of total PLFAs also occurred at 3 mm from the root zone in planted soils. Bacteria (cyl7:0,16:0), gram positive bacteria (a15:0, i16:1, a17:0) and actinomycete (18:2ω6,9c) were significantly higher in planted soils than in unplanted soils. Furthermore, individual PLFAs (i16:0,16:0 N alcohol,18:0(10Me), i16:1, a15:0, i14:1,14:02OH,18:1ω9c, a17:0,14:03OH, i14:0, a16:0,16:lω5c) were strongly correlated with the Aroclor 1242 degradation rates (%) (p<0.05) in planted treatments, whereas individual PLFAs i16:1,12:03OH,15:0, a15:0 had significant correlations with the Aroclor 1242 degradation rates (%) (p<0.05) in unplanted soils. In particular, individual PLFAs i16:1 had strong correlations with Aroclor 1242 degradation both in treatments with and without ryegrass.(4) PCB 7 (24-CB) was selected as the target pollutant, the dissipation rate in rhizosphere soils, the adsorption, transfer and accumulation of 24-CB from soils to plants, and the detoxification enzymes to in ryegrass 24-CB were conducted in this research. Two different soils which contain different DOM content were selected in this experiment. The results showed that ryegrass was able to absorb 24-CB from soils, and only very small amount could be transferred to the overground part. We found that the transfer of 24-CB in plants was quite limited. The 24-CB by ryegrass was mainly accumulated in root. The uptake and accumulation of 24-CB was not the main reason which induced the dissipation of 24-CB concentration. The physiological response related to the 24-CB metabolism in ryegrass was also investigated. We found that SOD and POD appeared different responses in two types of soils. We speculated that DOM played the key role on the 24-CB of bioavailability in this study, further impacted the detoxification enzymes in ryegrass.(5) The remediation effects and mechanisms of root exudates on rhizo-remediation in 2345-CB-spiked soils were studied in a simulated rhizo environment. The addition of root exudates improved the fast dissipation of 2345-CB at the very beginning of the experiment. Then the dissipation rates became quite slow with the increase of the time. The PLFAs profile indicated that the aerobic microbes were inhibited while the anaerobic microbes were stimulated, especially the methane oxidizing bacteria and sulfate reducing bacteria biomass. Our reports indicated that ortho-, meta-, para-dechlorination exhibited different sequence and optima. Chlorines at the position of meta-and para-were removed, result to the accumulation of 234-CB, 245-CB,25-CB.更多还原