【作者】 刘虹；

【导师】 张兰英；

【作者基本信息】 吉林大学 ， 环境工程， 2012， 博士

【摘要】 石油作为一种重要能源，是国民经济发展的重要物质基础。然而，在进行石油开采、储存及生产加工等过程中，由于泄露及不合理排放等原因，使得大量石油烃进入环境，即而通过大气降水、土壤淋溶、地表水渗入等各种途径污染地下水。地下水是人类和其他一切生物的直接供水水源及与地表水发生水力联系的间接水源，对其污染的控制关系到人类的健康与生态的安全。因此，对石油烃污染地下水的修复至关重要。近年来，石油烃污染地下水的原位修复技术得到人们的关注，其中PRB技术因具有较长时间持续原位处理、安装施工方便、运行费用低等诸多优点被广泛使用。PRB技术应用于石油烃污染地下水修复时，大部分选择了生物PRB以达到高效、清洁、廉价的修复目标。然而，在采用生物PRB的实际修复过程中，存在由于地下水温度低、缺乏营养等因素使得微生物活性降低或由于微生物的繁殖、代谢造成PRB介质孔隙堵塞、渗透性下降等因素使得生物PRB不能长期、有效的运行。生物PRB运行的长效性主要取决于高效、持久的微生物降解活性以及PRB保持良好的渗透性。本文以石油烃污染地下水的原位生物PRB修复技术及其长效性为研究对象而展开，从PRB功能菌、填充介质、反应墙体的构建及PRB一旦失活或堵塞采取的技术措施各方面进行研究，确保PRB长期、有效的运行。从功能菌方面，对生物PRB的功能菌株进行紫外诱变，以提高功能菌在PRB长期运行过程中降解的稳定性；从填充介质方面，研究了三种材料在长期运行中的稳定性和对功能菌固定的有效性，添加新型填充材料—电气石作为PRB的活性反应材料，提高了PRB的生物活性和反应介质的渗透性；在确定功能菌和填充介质的基础上，为了保证PRB长期、有效的修复效果，预防PRB运行过程出现堵塞问题，设计、构建两种PRB—多介质组合PRB和复配介质PRB，并结合实际污染场地的水文地质及地下水化学条件进行室内模拟修复，研究、对比了两种PRB修复效果，并对PRB有效运行200d后的生物特性包括微生物数、脱氢酶活性及复配PRB的微生物群落分布进行分析，为了防止反应墙体中微生物的失活，初步研究了采用无机营养盐对微生物进行激活；为了解决PRB一旦发生堵塞而失效的问题，该研究还将在线监测系统应用于PRB运行过程中，对PRB运行过程中的堵塞部位进行在线自动识别并采用超声—高压水冲洗法进行解堵处理。以期为石油烃污染地下水实际修复工程的长期、有效运行提供理论参考依据和技术支持。对功能菌进行紫外诱变的研究中，根据对石油烃的降解效果，选取正突变菌株：S14、S21和S32，三株菌株对石油烃降解率诱变后均有所提高；与原菌相比，诱变菌经10次传代后具有遗传稳定性，在碱性或石油烃浓度较低的环境中能保持对石油烃高效降解的稳定性；在石油烃浓度较低条件下，诱变菌株对石油烃的降解符合一级反应动力学方程，浓度较高时，符合零级反应动力学方程；经紫外诱变后，石油烃降解菌中单菌的菌体及菌落形态未发生变化，仅在菌落所占比例方面有所变化，3种菌株紫外诱变后菌株的9项生理生化特性与原菌株一致。三种填充介质—泥炭、电气石和珍珠岩的特性研究表明，三种介质在与地下水经过长期接触后，无有机物质及重金属元素溶出，少量无机离子（Na+、Mg2+和Ca2+）的溶出使地下水的矿化度和硬度有较少的增加，但不会影响地下水水质，因此，三种材料作为PRB填充介质，在PRB长期运行中均安全、无害，且比较稳定。三种材料不能被微生物分解，具有生物稳定性。三种介质中，泥炭对功能菌固定化效果最好，24h固定化率达到85%以上，电气石和珍珠岩对功能菌也有固定化效果，固定化率为20%—30%。电气石具有辐射中远红外线的特性，在PRB修复石油烃污染地下水的过程中，添加电气石可以调节地下水的pH，增加溶解氧，提高微生物脱氢酶活性，减小水分子团结构，增强水的透过性，一定程度上缓解了PRB的堵塞。构建的两种PRB反应器中，多介质组合PRB按渗透系数K逐层增大的原则，确定3层填充介质为粗砂、电气石、珍珠岩分别以体积比4:1与固定化泥炭混合，复配介质PRB选取粗砂、固定泥炭、电气石，体积比为3：1：1作为的填充介质。结合实际污染场地条件的模拟修复效果表明，两种PRB均能高效修复石油烃污染地下水，在运行的200d内出水中未检出石油烃，石油烃的污染羽随时间逐渐扩大，且石油烃首先出现在反应介质的底层，然后出现在表层，随后扩散至整个介质层。两种PRB对比可以看出，从修复效果来看，复配介质PRB更高效，从渗透性来看，多介质组合PRB的渗透性能较好。复配介质PRB运行200d后，反应介质前端30cm地下水中TPH为0.74mg/L—5.42mg/L，后端30cm地下水中TPH较低且分布较均匀，均低于0.29mg/L。从渗透性方面来说，多介质组合PRB前端0—10cm在反应器运行150d后，渗透系数低于污染场地含水层的渗透系数，10cm—30cm在反应器运行200d后，渗透系数低于污染场地含水层的渗透系数，30cm—50cm、50cm—60cm的渗透系数K在运行的200d内变化微小，远远大于场地的渗透系数。两种PRB运行200d后，对其微生物特性包括微生物数、脱氢酶活性及对复配介质PRB的微生物群落分布的研究表明，地下水中TPH较高的部位，介质中的微生物数量也较多。不同介质中微生物的脱氢酶活性存在差异，但同一介质层的脱氢酶活性随深度的变化趋势大致相同，呈现脱氢酶活性随深度先增加后逐渐降低的趋势。总体来看，微生物数量多的层位，相应地微生物所产生的脱氢酶活性也较高，且具有一定的相关性。200d后复配介质PRB中微生物群落的DGGE图谱分析表明，PRB中微生物群落结构的相似性随着距离的增大而降低，其中B2与B5的相似度最高，达83.1%。微生物群落的Shannon多样性指数在1.78—2.19之间，多样性随距离的增大而增大。结合PRB中TPH分布来看，TPH浓度高的部位，微生物群落较为稳定，多样性较低。反之，TPH浓度低的部位，微生物群落不稳定，多样性较高。PRB运行200d后，PRB6个点位样品中都存在固定化功能菌且它们在整个系统中相对数量占优势。位于PRB的前端，功能菌群的相对数量较高，随着PRB距离的增大，功能菌的相对数量有降低的趋势。Yarrowia sp.菌在运行的200d内在PRB中相对数量均较高且占优势，Pseudomonassp.和Acinetobacter sp.菌在PRB前端相对数量较高，在PRB的后端，相对数量较低，优势不明显。添加无机营养盐对微生物脱氢酶活性有一定的激活效果，未添加无机营养盐的培养液经激活后，微生物脱氢酶活性不但没有继续降低，反而有所提高，激活后第2d微生物脱氢酶活性升高到32μgTTC/（L·h），微生物对石油烃的降解率在激活后也随之提高。采用Diver地下水水位自动监测仪对PRB水位进行在线监测，对PRB堵塞部位进行自动识别，监测结果表明堵塞部位易发生在反应器首段0—15cm内，采用超声—高压水冲洗法进行解堵处理，根据其对微生物脱氢酶活性、微生物降解石油烃效果和渗透系数的影响确定最佳解堵条件为声能密度0.5W/ml、超声时间8min、冲洗水量100ml/min、冲洗时间20min，在该条件下处理后，PRB堵塞部位得到了有效解除。更多还原

【Abstract】 As one of the essential energy sources, petroleum is an important material basisfor the development of national economy. However, a great deal of petroleumhydrocarbons has entered into environment due to leakage, unreasonable recharge andother factors during the petroleum exploitation, reserve and production, whichcontaminated groundwater through atmospheric precipitation, soil leaching, surfacewater leakage and other ways. Groundwater is a direct water supply source for humanand other living things and is an indirect water source connecting with surface water,the contamination of which will affect humans’ health and the safety of ecology.Therefore, the remediation of groundwater contaminated by petroleum hydrocarbonsis significant.In recent years, the remediation of groundwater contaminated by petroleumhydrocarbons has gained considerable attention. Among all the techniques, the PRBtechnique was adopted widely for its long in situ remediation, convenient installationand low cost. When it was applied in the remediation of groundwater contaminated bypetroleum hydrocarbons, the bio-PRB was adopted for its cleanness and cost-effective.However, the microbe activity degradation due to low temperature, nutrientsdeficiency and porosity clogging, permeability reduction caused by microbe growthaffected the long-term efficiency of the PRB. Therefore, the long-term efficiency ofthe PRB depends on the high biodegradation efficiency and hydraulic conductivity.Aiming to the PRB technique and its long-term efficiency for the in-situremediation of groundwater contaminated by petroleum hydrocarbons, the microbes,fillings, the construction of PRB and the techniques for microbe activation andclogging removal were studied. For the microbes, it was mutated by ultravioletmutagenesis to improve stability of biodegradation in PRB. For fillings, the stabilityand the immobilization of three materials were investigated, and the tourmaline wasadded as an active material in the PRB, which had abilities to promote the biological activity and the hydraulic conductivity of PRB. Two biological reactive barriers weredesigned and constructed on the basis of the optimization of bacteria and filling, theywere multi-layer PRB and mixed-media PRB. Combined with the hydrogeology andchemical conditions of the polluted field, the bioremediation simulation by tworeactors operated200days, and the remediation efficiency of two PRBs were studiedand compared. After a200-d operation, the microbes, dehydrogenase activity andmicrobial community of mixed PRB was investigated. Activation by inorganic saltswas also studied once the microbes lost activity. The on-line monitoring system wasapplied in PRB when it was in operation to guard against PRB clogging and identifythe clogging position. Moreover, the ultrasonic combined with high pressure washingtechnique to remove the PRB clogging was studied. It is expected that the study cancontribute to the practical application of PRB in the remediation of groundwatercontaminated by petroleum hydrocarbons.The results of ultraviolet mutagenesis suggested that the biodegradation ofpetroleum hydrocarbons by three positive mutant strains S14, S21and S32was allimproved. The hereditary character of high degradation ability of mutant microbeswas stable and the mutant microbes kept degrading petroleum hydrocarbons whenthey were in basic and low concentration environment. The biodegradation ofpetroleum hydrocarbons by mutant microbes followed first-order kinetics in lowerconcentration while it followed zero-order kinetics in higher concentration. Inaddition, the shape, physiological and biochemical characteristics of the strains andcolony after mutagenesis was the same as before, the only difference was the ratio ofthe colony.The study on the characteristics of three materials peat, tourmaline and perliteindicated that there were no organic substances but a few inorganic substances afterthey contacted with groundwater for a long time, which would increase the mineralcontent and hardness of groundwater but not influence the groundwater quality.Therefore, the material was safe and stable. Meanwhile, they could not bebiodegraded by microbes and had ability of immobilization, the immobilizedefficiency of peat reached85%and it was20%to30%for two others after24hours. The tourmaline had ability to radiate infrared and it adjusted pH, reduced watercluster and promoted hydraulic conductivity and microbial activity during theremediation.Among two PRBs, the multi-layer PRB was designed according to thepermeability coefficient that was increased with the length and the fillings of threelayers were coarse sand, tourmaline and perlite mixed with immobilized peatrespectively, the ratio was4to1for each of them. The filling of mixed-media PRBwas mixed coarse sand, immobilized peat and tourmaline, the ratio was3:1:1. BothPRBs remediated polluted groundwater efficiently and there was no petroleumhydrocarbons detected in the effluent during a200-d operation. With the passage oftime, the polluted plume of petroleum hydrocarbons appeared and diffused gradually,which appeared in the bottom at first and then in the surface layer until it diffused tothe entire media. The contrast of the two PRBs indicated that the mixed-media PRBhad high removal efficiency and the multi-layer PRB was preferable in hydraulicconductivity. In the mixed-media PRB, the THP concentration ranged from0.74mg/Lto5.42mg/L in the fore-end30cm of the reactor, while it reached lower than0.29mg/L in the back-end and distributed averagely after a200-d bioremediation. In themulti-layer PRB, the permeability coefficient in the for-end0to10cm was lower thanthe field after150days, in10cm to30cm it was lower than the field after200days, in30cm to50cm and50cm to60cm of the system, there was little changes and thepermeability coefficient was much greater than the field.After a200-d bioremediation of two PRBs, the investigation of microbes,dehydrogenase activity and microbial community indicated that there were moremicrobes in the position with higher TPH. The dehydrogenase activity was differentin each layer and the change with depth in the same layer was similar, it increased atfirst and then decreased with the depth. In general, the more the microbes, the higherthe dehydrogenase activity, and they were correlated with each other. The atlas ofDGGE showed that the similarity of microbial community in the PRB decreased withlength and the highest reached83.1%between B2and B5. The Shannon diversityindex was from1.78to2.19and the microbial diversity increased with length. Combined with the distribution of TPH, it illustrated that the microbial communityprofiles were more stable and was less diversiform in the position with high THP.Conversely, the microbial community was less stable and was more diversiform in theposition with low THP. The results indicated that the functional immobilizedmicroorganism existed and was dominant in each position of PRB after200-dbioremediation, among which Yarrowia sp. had high band density in each positionwhile Pseudomonas sp. and Acinetobacter sp. had high band density in fore-end ofPRB and low band density in back-end. The microbe could be activated by addinginorganic salts. The dehydrogenase activity improved after it was activated and itreached32μgTTC/（L·h） after2days. Meanwhile, the biodegradation efficiency ofpetroleum hydrocarbons improved.To identify the clogging in the PRB on line, the Diver automatic water levelmonitoring instrument was applied during its operation. The monitoring data showedthat the clogging would probably appear in the fore-end0to15cm of the reactor. Toremove the clogging in the PRB, the ultrasonic combined with high pressure washingtechnique was investigated, ultrasonic density0.5W/ml, ultrasonic time8min,flushing flow100ml/min、flushing time20min were determined according to theinfluence on the microbes, dehydrogenase activity, biodegradation efficiency andpermeability coefficient, under which, the clogging was removed efficiently.更多还原