【作者】 安永磊；

【导师】 张兰英； 刘娜；

【作者基本信息】 吉林大学 ， 环境工程， 2012， 博士

【摘要】 硝基苯是一种普遍的、高毒性的、地下水中常见的环境有机污染物，已被许多国家列为环境优先控制污染物。硝基苯进入地下水后，可对生态环境造成危害，破坏地下水资源，并通过饮用水或食物链进入人体，造成多种疾病，因此硝基苯污染地下水亟待有效修复。在众多的地下水修复技术中，原位生物修复技术被认为是最具有发展潜力的绿色地下水修复技术。众所周知，硝基苯在好氧环境下或厌氧环境下均可被相应的功能微生物降解。因此，本研究结合实际硝基苯污染地下水的场地特征及微生物群落特点，室内模拟研究了强化自然生物修复和强化人工生物原位修复硝基苯污染地下水效果，并利用变性梯度凝胶电泳（PolymeraseChain Reaction-Denaturing Gradient Gel Electrophoresis, PCR-DGGE）技术和实时荧光定量PCR（Real Time-Fluorescence Quantitative–PCR, RT-FQ-PCR）技术研究了地下水微生物群落结构与功能。首先，采样分析了实际硝基苯污染地下水微生物群落特性和地下水水化学因子，并用典范对应分析（Canonical CorrespondenceAnalysis, CCA）和相关性分析研究了环境因子与微生物群落间的关系。结果表明，硝基苯污染地下水中存在一定丰度的生物量，包括细菌、放线菌、酵母菌、霉菌以及真菌等，随着采样点不同微生物丰度存在着较大的差异性；地下水中微生物群落保持良好的多样性，个别物种保持一定的优势度，优势物种包括Acidovorax sp.、Flectobacillus lacus、Pseudomonas corrugata、Rhizobium sp.；在地下水水平方向不同位置，微生物群落结构保持较高的相似性，同时也存在一定的差异；硝基苯污染地下水中微生物含有硝基苯降解关键基因nbzA，除S2、S8、S9三个取样点的nbzA基因丰度较高外，其它各采样点的nbzA基因丰度基本保持同一个水平；nbzA基因的存在亦说明了该地下水中含有硝基苯降解菌。CCA分析结果表明，硝基苯污染地下水中的微生物空间分布与总氮（TN）、硝基苯呈明显的相关性。相关性分析结果表明，可培养微生物丰度与总有机碳（TOC）、pH、SO₄^2-和硝基苯等因子呈正相关关系，与F^-、Cl^-、NO_<sup>2-以及Cd呈负相关关系；nbzA基因丰度与F^-、Cl^-、硝基苯呈正相关关系，与TOC、pH、NO_<sup>2-、SO₄^2-、Cd呈负相关关系。其次，研究了强化自然生物原位修复硝基苯污染地下水的修复效果及微生物群落结构。激活剂筛选结果表明，乳糖磷酸盐、蛋白胨、牛肉膏对硝基苯污染地下水中的土著微生物具有一定的激活作用，刺激了微生物的生长，硝基苯得到了有效降解和去除。室内模拟激活土著微生物修复硝基苯污染地下水结果表明，自然条件下（未激活）模拟地下水下游硝基苯浓度比原地下水降低了17.20%；乳糖磷酸盐激活后，硝基苯去除率达到62.05%；蛋白胨激活后，硝基苯去除率为74.87%；牛肉膏激活后，硝基苯去除率达到71.27%。乳糖磷酸盐、蛋白胨和牛肉膏激活后，地下水微生物脱氢酶活性、生物量以及生物多样性均得到明显的提高；微生物群落结构发生了较大的变化，优势物种种类也发生了改变，但仍保持一定的同源性；硝基苯降解菌（携带nbzA基因）的相对丰度和生物量均得到明显的提高。以上研究结果表明，投加乳糖磷酸盐、蛋白胨和牛肉膏确实激活了硝基苯污染地下水中的土著微生物，尤其是硝基苯降解菌，并对地下水中硝基苯产生了有效的生物降解作用，激活剂的激活效率顺序为：蛋白胨>牛肉膏>乳糖磷酸盐，但是投加生物激活剂不能将地下水中的硝基苯彻底去除。然后，在采用强化人工生物修复硝基苯污染地下水之前，先从实际硝基苯污染地下水中筛选鉴定硝基苯高效降解菌，并对降解菌的降解特性、nbzA基因表达量及降解动力学进行了研究。结果表明，从硝基苯污染的地下水中筛选出一株以硝基苯为唯一碳源和氮源好氧硝基苯降解菌，命名为ZG菌，该菌为革兰氏阴性短杆细菌，初步鉴定为恶臭假单胞菌，含有硝基还原酶基因（nbzA），降解硝基苯遵循半还原代谢途径；ZG菌具有一定的环境变化耐冲击能力，在温度10³0℃、pH4⁹、硝基苯初始浓度≤600mg/L的范围内，ZG菌均可有效地降解硝基苯，并且在20℃、pH为7、底物浓度≤300mg/L时，其降解效果最好，硝基苯降解率达到99%以上；随着环境条件（温度和pH）的变化，ZG菌降解硝基苯的关键功能基因nbzA的相对表达量亦随之变化，其变化趋势基本与硝基苯去除效果一致；地下水中铁和锰对ZG菌降解硝基苯效果以及nbzA基因相对表达量有着显著的影响，当Fe²⁺为低浓度（<5mg/L）时，对nbzA表达基本无影响，高浓度（>25mg/L）时，nbzA表达明显受到抑制；Mn²⁺浓度在0¹5mg/L内，nbzA基因表达量持续上升，说明一定浓度的Mn²⁺对nbzA基因表达具有促进作用；在实际硝基苯污染地下水环境中，ZG菌的nbzA基因相对表达量要明显高于无机盐培养基，说明该地下水没有抑制nbzA表达的因子，相反可能含有促进因子。动力学研究表明，在初始硝基苯浓度300mg/L以内，ZG菌降解硝基苯符合一级动力学，其半衰期为常数ln（1/2）/K，硝基苯浓度越高，其降解速率越大，否则相反。最后，在室内模拟研究了强化人工生物修复技术——投菌法原位修复硝基苯污染地下水效果及微生物群落结构与功能，投菌修复模式分为两种，单一投菌模式和曝气-投菌模式。结果表明，单一投菌修复模式下，修复后地下水硝基苯去除率可达到66.31%，硝基苯残留浓度为10.39mg/L；曝气-投菌修复以后，地下水硝基苯去除率达到89.92%，硝基苯残留浓度低于4.16mg/L；投加ZG菌后，地下水溶解氧浓度比未投菌平均降低了0.85mg/L，说明ZG消耗了地下水中的溶解氧，并且曝气供氧是投菌修复硝基苯污染地下水的关键性因素；ZG菌投加入地下水系统后，可向地下水各个方向迁移，尤其是沿地下水流向迁移趋势最大，水平迁移速率约为0.08m/d，但受水流冲刷和环境营养条件所限制，最终ZG菌在地下水中的生存量基本稳定在1.0×10⁵¹.8×10⁵cfu/mL；向硝基苯污染地下水单一曝气后，微生物脱氢酶活性提高了76.25%；单一投加ZG菌后，脱氢酶活性提高了82.50%；曝气同时投加ZG菌，微生物脱氢酶活性提高了138.75%³56.25%。PCR-DGGE结果表明，单一曝气明显改变了硝基苯污染地下水微生物群落结构，与天然地下水微生物群落结构相似性仅为42.6%，曝气激活了ZG菌和其它微生物并成为优势物种；单一投菌基本对地下水微生物群落结构影响不大，投菌前后微生物群落结构相似性可达61%；曝气-投菌修复后地下水微生物分布较为相似，并且大量检测到ZG菌的存在，这进一步说明ZG菌能够在地下水中各向迁移并在地下水中保持一定优势度，并且ZG菌的大量投加并没有明显表现出对其它物种的促进或抑制作用。RT-FQ-PCR结果显示，无论是曝气还是投加ZG菌，均能提高地下水微生物nbzA基因的相对丰度和相对表达量，并且硝基苯浓度可能是影响nbzA基因丰度和表达量的关键因素。总之，原位生物修复技术包括强化自然生物修复技术和强化人工生物修复技术，是一种经济、可行、有效、绿色的硝基苯污染地下水修复技术。在实际修复过程中，可根据实际情况选择直接强化人工生物修复技术或者采用先强化自然生物修复降低硝基苯浓度后强化人工生物修复的策略。更多还原

【Abstract】 As a common, high toxicity environmental organic pollutant, nitrobenzene hasbeen detected frequently in organic contaminated groundwater and has been listed asthe environmental priority pollutant by many countries. Nitrobenzene in groundwaterwould result in the damage of ecological environment, destruction of groundwaterresources, and cause human diseases through drinking water or food chain. Based onthese reasons, it is significant to remediate nitrobenzene-contaminated groundwatereffectively. Among all the remediation technologies for groundwater, in-situbioremediation technology is considered as the most potential and green remediationtechnology. As known to all, nitrobenzene can be biodegrade by the correspondingfunctional microorganisms both in aerobic or anaerobic condition. Therefore, thisresearch firstly investigate the microbial community characteristics in actualnitrobenzene-contaminated groundwater, and then study the biostimulation andbioaugmentation methods to bioremediate nitrobenzene-contaminated groundwater inlaboratory. Also PCR-DGGE and RT-FQ-PCR technology were applied to researchthe varieties of microbial community structure and function in groundwater.First of all, the microbial community characteristics and water chemical factorsof actual nitrobenzene-contaminated groundwater were investigated, and therelationships between microbial community characteristics and water chemical factorswere studied by CCA and correlation analysis. The results showed that a measuredamount of microorganisms including bacteria, actinomycetes, mould, yeast and fungi,were determined in nitrobenzene-contaminated groundwater and large differences ofbiomass existed among the sampling points. The microbial communities maintained agood diversity and the species including Acidovorax sp., Flectobacillus Lacus,Pseudomonas. Corrugata, Rhizobium sp., took large degrees of dominance in groundwater microbial communities. The similarities among all the microbialcommunities from different sampling points were high but some differences were stillexisted. As the key gene to degrade nitrobenzene according to the partial reductivepathway, gene nbzA were detected in all groundwater samples and the relativeabundances of nbzA were especially high in sample S2, S8and S9, which indicatedthat there existed nitrobenzene-degrading microbes in nitrobenzene-contaminatedgroundwater. The results of CCA showed that obvious correlation were maintainedbetween the distributions of microorganisms and TN, NB. Correlation analysisshowed that the abundances of cultured microbes were positively correlated with thevariables of TOC, pH, SO42-and NB, and negatively correlated with the variables of F-,Cl-, NO2-and Cd. The relative abundances of gene nbzA were positively correlatedwith F-, Cl-and NB, and negatively correlated with TOC, pH, NO2-, SO42-and Cd.Secondly, the removal effects of nitrobenzene and the changes of microbialcommunity in groundwater were investigated detailed. The results of screeningbiostimulants showed that lactose-phosphate, peptone, beef extract could enhance thegrowth of indigenous microbes, respectively, and the nitrobenzene in groundwaterwas removed effectively. The results of simulated experiments of in-situbioremediation by biostimulation showed that the removal rate of nitrobenzene ingroundwater could be17.20%under natural condition without adding anybiostimulant,62.05%with lactose phosphate,74.87%with peptone and71.27%withbeef extract. After biostimulation, the microbial dehydrogenase activities, biomassand biodiversities were all increased, microbial community structures changed butstill maintained some homology and the dominant species changed synchronously.The relative abundances of gene nbzA and biomass of nitrobenzene-degradingmicrobes （containing gene nbzA） were improved obviously. Above research resultsdemonstrated that adding lactose-phosphate, peptone and beef extract tonitrobenzene-contaminated groundwater could actually activate the indigenousmicroorganisms, especially nitrobenzene-degrading microbes that had biodegradednitrobenzene in groundwater effectively. The order of biostimulation effects waslactose-phosphate> peptone> beef extract. However, only biostimulation could not remove nitrobenzene completely and a measured amount of nitrobenzene was existedin groundwater.Then, nitrobenzene-degrading bacteria were screened from actualnitrobenzene-contaminated groundwater before the experiments of simulatedbioremediation by bioaugmentation. The identification, degradation characteristics,level of nbzA gene expression and degradation kinetics of nitrobenzene-degradingbacteria was also investigated. The results showed that one strain of nitrobenzene-degrading bacterium named as ZG was screened successfully. The ZG strain wasgram-negative short rod bacterium and could utilize nitrobenzene as the sole carbonsource and nitrogen source. The results of16S rDNA sequence analysis showed thatZG strain was most probable Pseudomonas putida. In addition, ZG strain containedgene nbzA encoded nitroreductase and biodegraded nitrobenzene according to thepartial reductive pathway. ZG strain showed a relatively strong bearing capabilityagainst environmental variety. When temperature was maintained in the range of10³0℃, pH4⁹, initial concentration of nitrobenzene less than600mg/L, ZG straincould biodegrade nitrobenzene effectively. The best condition for ZG strain degradingnitrobenzene was temperature20℃, pH7, initial concentration of nitrobenzene lessthan300mg/L,then the removal rate of nitrobenzene could be more than99%. Therelative expression of gene nbzA changed with the variety of environmentalconditions （temperature and pH） and the variation trends were same. The relativeexpression of gene nbzA and removal effects of nitrobenzene was influenced by ironand manganese significantly. When the concentration of Fe²⁺was less than5mg/L,there was nearly no influence on the removal effects of nitrobenzene and the relativeexpression of gene nbzA. If the concentration of Fe²⁺was more than25mg/L, theretook the opposite results. When the concentration of Mn²⁺was rising from0mg/L to15mg/L, the removal effects of nitrobenzene and the relative expression of gene nbzAalso increased continuously, which indicated that Mn²⁺in the range of0¹5mg/Lwould enhance the relative expression of gene nbzA of ZG strain. In the actualnitrobenzene-contaminated groundwater, the relative expression of gene nbzA of ZGstrain was higher that in mineral medium, which suggested that there would be no inhibiting factor for the nbzA gene expression. On the contrary, there may be somepromoting factors. The results of degrading-kinetics showed that it corresponded tothe first-order kinetics for ZG strain to degrade nitrobenzene and the half-life was ln（1/2）/K, which indicated that nitrobenzene concentration is higher then the removaleffect is better.Finally, laboratory-scale study on in-situ bioremediation of nitrobenzene-contaminated groundwater by bioaugmentation was carried out and the remediationeffects and microbial communities were investigated detailed. Two modes ofbioaugmentation including adding ZG strain to groundwater solely and combinedaeration and adding ZG strain, were operated respectively. The results showed that theremoval rate of nitrobenzene in groundwater could reach66.31%and the residualnitrobenzene was about10.39mg/L after bioremediation with the mode of adding ZGsolely. While the removal rate of nitrobenzene could reach89.92%and the residualnitrobenzene was below4.16mg/L after bioremediation with the mode of combinedaeration and adding ZG strain. After adding ZG strain, the concentrations of dissolvedoxygen in groundwater were0.85mg/L less on average than that before adding ZGstrain, which meant that ZG strain consumed dissolved oxygen which was the keyfactor for in-situ bioremediation with bioaugmentation. ZG strain could migrate torandom direction, especially along the groundwater flow direction, the horizontalmigration rate could reach0.08m/d. The final concentration of ZG strain ingroundwater was about1.0×10⁵¹.8×10⁵cfu/mL because of groundwater scouringand limited microbial environmental nutrition condition. Compared with the naturalgroundwater, the microbial dehydrogenase activity in groundwater was increased by76.25%with single aeration,82.50%with single adding ZG strain,138.75%³56.25%with combined aeration and adding ZG strain. PCR-DGGE results showedthat the microbial community structure had been changed significantly by singleaeration, and the similarity of microbial community structure between before and aftersingle aeration was only42.6%. After single aeration, in indigenous ZG strain andother microbial species were activated and became the dominant species. There waslittle changes for groundwater microbial communities structure with adding ZG strain along, and the similarity of microbial community structure between before and aftersingle adding ZG strain could reach up to61%. After remediation by combinedaeration and adding ZG strain, the microbial distribution in whole simulatedgroundwater system was similar and a measured abundance of ZG strain was detected,which also indicated that ZG strain actually had migrated and kept certain abundancein groundwater. What’s more, there was no obvious promoting or inhabiting effect tothe other species in groundwater with adding large number of ZG strain. TheRT-FQ-PCR results showed that both aeration and adding ZG strain could improve therelative abundance of gene nbzA and its relative expressions. Moreover, theconcentration of nitrobenzene may be the key factor to affect the relative abundanceand expression of gene nbzA.In conclusion, in-situ bioremediation technology including biostimulation andbioaugmentation was an economic, practical, efficient and green remediationtechnology for nitrobenzene-contaminated groundwater. In the actual remediationprocess, according to the actual situation of contaminated site, it could choosebioaugmentation method directly or choose the sequencing batch craft of combinedbiostimulation and bioaugmentation.更多还原