邻苯二甲酸酯类化合物好氧生物降解的实验研究
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摘要
第一章源水及饮用水中PAEs的定性分析
目的：对长江、汉江(武汉段)水源水、饮用水中PAEs的污染状况和深度水处理工艺(臭氧—活性炭)处理后出水中的PAEs化合物进行定性分析。
方法：以武汉自来水厂(宗关水厂、平湖门水厂)的源水、出厂水和管网末梢水为调查对象，分别于2007年、2008年平水期(3月)、丰水期(7月)、枯水期(12月)采集水样，采样量为100～120 L。用XAD-2树脂吸附水中的有机物，用二氯甲烷和丙酮进行洗脱，洗脱液经浓缩后(相对于水中的含量，其浓缩比例为1：5000)，通过GC／MS对有机物的成份进行定性分析。2006年4月在南方某自来水厂的中试基地，将臭氧氧化，活性炭(生物活性炭)吸附工艺与传统水处理工艺(混凝沉淀、加氯消毒)相结合，通过不同的工艺组合对当地源水进行处理。源水和不同组合工艺处理后出水的采集、有机物的浓缩和检测与上面描述的方法相同。
结果：GC／MS结果显示所有水样中都有PAEs检出。武汉两水厂的源水和饮用水中主要为DBP和DEP。同2007年相比，2008年新检出两种PAEs为DMP和BBP。南方某中试基地的源水和不同组合工艺处理后出水中均有DMP和DEP检出。
结论：源水和饮用中的PAEs污染非常普遍，现有的自来水处理技术(常规水处理技术和深度水处理技术)难以将其彻底去除。
第二章高效PAEs降解菌的驯化、筛选及鉴定
目的：驯化活性污泥，从驯化污泥中分离出高效PAEs降解菌，并对其生物种属进行鉴定。
方法：从武汉某印染厂取得活性污泥置于曝气池中，以DMP、DEP和DBP为唯一碳源，采用活性污泥曝气与密度梯度驯化方法，即从第1周开始，等量投加DMP、DEP、DBP作为细菌利用碳源。每周PAEs的投加浓度按10 mg／L、20mg／L、30 mg／L、40 mg／L、60 mg／L、80 mg／L、100 mg／L、140 mg／L依次递增，连续驯化8周。用灭菌采样瓶从曝气池采取污泥样本，用接种环蘸取污泥，在普通营养琼脂平板上分区划线以分离纯化细菌。在250 ml的锥形瓶中加入100 ml无机盐培养液，高压灭菌，加入等量DMP、DEP和DBP(各200 mg)，按等生物量的原则加入分离出的细菌，置于37℃空气浴摇床中(140 rpm)连续降解7天。用二氯甲烷液液萃取各降解液，用GC法检测其浓度。比较各细菌7天降解率的大小，筛选出高效降解细菌。对筛选出的高效PAEs降解菌，进行革兰染色。结合染色结果，用全自动微生物鉴定仪对其进行生化鉴定。用电子显微镜拍摄高效降解菌的透射电镜照片，观察细菌形态学。提取细菌DNA，扩增16s rDNA序列，并进行碱基序列测定。将序列提交Genbank并进行Blast序列比对，通过比对结果进一步鉴定该菌种属。将细菌置于不同的温度和不同的渗透压下(不同的NaCl浓度)培养，以考察该高效降解菌的生长特性。
结果：从印染厂活性污泥曝气池中分离得到7株细菌，命名为菌株L1～L7。菌株L1～L5对DMP、DEP和DBP(200 mg／L)的七天降解率分别为87.5％，99.1％，98.3％；31.9％，10.2％，0.0％；19.1％，14.2％，16.9％；99.0％，98.6％，99.2％与28.9％，20.0％，6.5％。L6、L7菌基本没有降解效果。确定L4为高效PAEs降解菌，对细菌L4进行鉴定，为赤红球菌(Rhodococcus ruber)。赤红球菌L4在12～42℃均有不同程度的生长，最适生长温度为37℃。赤红球菌L4生长的最佳盐浓度≤0.5％，但在盐浓度为0.5～3％时，亦能良好生长，耐受浓度可高达5％。
结论：采用活性污泥曝气与密度梯度驯化方法，筛选出可高效降解PAEs的赤红球菌(Rhodococcus ruber)。该细菌生长温度范围广，能耐受较高的环境渗透压。
第三章赤红球菌L4降解PAEs的特性研究
目的：确定赤红球菌L4降解PAEs的适宜条件(pH值、温度和初始底物浓度)，探索降解动力学，对赤红球菌L4降解相关特性进行初步研究。
方法：通过正交设计(三因素、四水平表)探索赤红球菌L4降解PAEs的最佳温度、pH及初始PAEs浓度。以人工配制的不同初始浓度的PAEs实验水样为降解对象，每天取样检测残留量，以曲线拟合方法获得降解动力学方程。为确定PAEs在水中不同的存在状态对降解效果的影响，分别用直接投加和丙酮(超声)乳化的方法来配制PAEs实验水样(300 mg／L)。为研究赤红球菌L4的环境抵抗特性，将其接种于不同浓度的乙醇、二甲基乙酰胺、正辛烷和甲苯溶液，37℃培养24 h后进行菌落计数以调查其存活率。将赤红球菌L4分别接种于4 mmol／L的苯甲酸钠、苯酚、萘、对硝基酚、邻硝基酚和葡萄糖溶液中，以OD_(600)值作为观测指标，考察该细菌对有机物的利用谱。用油膜法测定PAEs降解液的表面活性。
结果：正交试验显示赤红球菌L4降解PAEs的最佳条件为：pH 7.0～8.0，30～37℃，初始底物浓度≤450 mg／L。赤红球菌L4降解动力学可用方程lnC=—kt+θ’来描述(k为降解反应的速率常数，θ’为积分常数)；当初始底物浓度小于300 mg／L时，其降解半衰期为1.30 d；赤红球菌L4在混合PAEs降解过程中，三种PAEs的降解速率，DMP＞DEP＞DBP。赤红球菌L4对乳化组与非乳化组实验水样5天降解率分别为90.9％和86.9％。赤红球菌L4对10％以下的乙醇和二甲基乙酰胺有较强的耐受能力，对直链烷烃如正辛烷24小时耐受浓度可高达60％，对芳香族化合物如甲苯的耐受能力相对较弱，在4％的甲苯水溶液中仅有少数菌可以存活。赤红球菌L4可分别以苯甲酸钠、苯酚、萘为唯一碳源和能源生长，对苯甲酸钠的利用速度较快，优于葡萄糖，其次为对苯酚和萘的利用，尚未观察到对邻硝基酚、对硝基酚有分解代谢作用。在PAEs的代谢过程中，赤红球菌L4可产生生物表面活性物质，其乳化效果相当于69.2 mg／L的SDS溶液。
结论：赤红球菌L4对PAEs的降解有较强的环境适应性，在最适宜条件下降解效果最好。在最佳降解条件下，赤红球菌L4对不同初始浓度的PAEs的降解可以用一级动力学方程来描述。在降解过程中，较短侧链的PAE被优先利用。赤红球菌L4能产生降低水—油表面张力的表面活性物质，具有乳化能力，有利于生物降解。较宽的利用谱和较强的抗毒性意味着赤红球菌L4在环境污染的生物修复中具有较大的应用潜力。
第四章赤红球菌L4代谢PAEs的机理探讨
目的：应用化学分离、分析技术和分子生物学实验方法探讨赤红球菌L4降解PAEs的机理。
方法：以初始浓度为300 mg／L的DEP实验水样为对象，投加赤红球菌L4进行降解试验，分别于降解24 h，48 h，72 h，96 h时收集降解液，用C18固相萃取柱吸附其中的有机物并用二氯甲烷进行洗脱，洗脱液经浓缩后进行GC／MS检测。用碱裂解法提取细菌质粒，通过琼脂糖凝胶电泳确定其分子量大小。将赤红球菌L4用普通营养肉汤连续传代5次，收集菌体作为对照组；经传代的细菌置于总浓度为300 mg／L的DMP、DEP、DBP混合溶液中培养24 h，收集菌体作为诱导组。用超声波裂解诱导组和对照组细菌，离心后取上清，即为提取的细菌总蛋白(酶)。以不同浓度的小牛血清溶液作为标准系列，用Bradford法测定各组蛋白含量。根据蛋白定量结果，等量投加对照组和诱导组蛋白酶至20μmol／L的邻苯二酚溶液，用分光光度法于260 nm和375 nm处分别测定上述溶液吸光度每分钟的变化值，即可求得邻苯二酚1，2—双加氧酶(C12O)和邻苯二酚2，3—双加氧酶(C23O)的酶活性；用聚丙烯酰胺凝胶电泳对对照组和诱导组细菌总蛋白进行分析。
结果：DEP降解48 h时，其降解液中可以检测到两个中间产物，为邻苯二甲酸单乙酯(monoethyl phthalate)和邻苯二甲酸(phthalate acid)。从赤红球菌L4内提取到3个质粒，其大小分别为65 kb、75 kb和100 kb。酶活性测试说明诱导组细菌的邻苯二酚双加氧酶活性高于肉汤连续传代的对照组(P＜0.05)，其C12O活性为8.78±0.98 U／mg，C23O活性为3.97±0.55 U／mg。聚丙烯酰胺凝胶电泳显示诱导组有两条新增条带。
结论：在降解初始阶段，赤红球菌L4首先水解其中一个酯键，生成邻苯二甲酸单乙酯，然后继续水解另外一个酯键，生成邻苯二甲酸，后者为主要的中间产物。赤红球菌L4体内拥有至少3条较大的质粒；邻苯二酚双加氧酶是赤红球菌L4降解过程中的关键性蛋白酶。赤红球菌L4经诱导后可同时表达C12O和C23O，但C12O活性增加更为明显。
第五章固定化微生物对水中PAEs的降解
目的：比较固定化微生物和游离微生物对水中PAEs的降解效果。
方法：以10％的PVA和0.5％海藻酸钠溶液为包埋剂，以4％的活性炭为吸附剂，以饱和硼酸溶液作为交联剂，采用吸附—包埋法制备固定化微生物小球。配制含DMP、DEP、DBP的实验水样(300mg／L)，以等生物量法，分别将固定化微生物小球或游离细菌投加于其中，然后置于35℃、115 rpm摇床上进行降解试验，测定48 h、72 h的残留量以评价固定化微生物和游离细菌的降解效果。
结果：固定化微生物小球48 h降解率为DMP 72.6％、DEP 70.1％、DBP68.3％；72 h降解率为DMP 79.2％、DEP 94.9％、DBP 72.1％。游离细菌48 h降解率为DMP 48.2％、DEP 41.1％、DBP27.3％；72 h降解率为DMP 72.2％、DEP 71.3％、DBP 58.8％。
结论：以10％的PVA和0.5％海藻酸钠作为包埋剂，以4％的活性炭作为吸附剂可以制备出降解效果良好的固定化微生物小球；固定化微生物对PAEs的降解效果优于游离微生物降解。
第六章PAEs生物降解前后的类雌激素活性的比较
目的：对PAEs混合物生物降解过程中的残留物进行类雌激素活性检测，以评价降解的安全性。
方法：用重组酵母菌雌激素检测系统(Yeast Estrogen Screen，YES)对各样品进行检测。将含有人雌激素受体的重组酵母菌检测液和稀释成一定浓度梯度的DMP、DEP和DBP的单一及混合物在96孔板中培养，以17β—雌二醇作为阳性对照，以CPRG(黄色)作为指示剂，培养3天后，若受试物具有雌激素活性，则溶液由黄色变成红色，红色的深浅代表酶活性强度，用酶标仪在540 nm处予以测定(OD_(540))。用赤红球菌L4对初始总浓度为300 mg／L的DMP、DEP和DBP混合水样进行生物降解，收集降解0 h、24 h、48 h、72 h、96 h后的降解液，用二氯甲烷液液萃取提取其中的有机物。用YES对其进行类雌激素活性检测。
结果：YES试验检测到DEP的类雌激素活性，其强度相当于雌激素(17β—雌二醇)的200万分之一水平，DMP和DBP未检出类雌激素活性。混合PAEs实验水样降解24 h，48 h，72 h，96 h后的残留物的最大类雌激素活性分别为：1.81，1.41，1.25和1.15，低于降解前(0 h)的2.97。
结论：YES试验可以检测DMP，DEP，DBP及其混合物的类雌激素活性，就单一物质而言，DMP、DEP、DBP的类雌激素活性由强至弱依次为DEP＞DMP＞DBP，混合PAEs的类雌激素活性主要由DEP所致；赤红球菌L4降解PAEs的产物的类雌激素活性，随着降解时间的延长，其水平呈逐步下降趋势。
PartⅠPAEs pollution in source water and drinking water by qualitativeinvestigation
Objective: To qualitatively investigate the kinds of PAEs in source water and drinkingwater of the Yangtze River and Hanjiang River (Wuhan section) by GC/MS. Toqualitatively investigate the kinds of PAEs in source water and effluents treated bydifferent advanced water treatment processes including ozone binding biologicalactivated carbon.
Method: The source water and drinking water of Zongguan and Pinghumen waterplants in Wuhan city were collected in March, July and December in the year of 2007and 2008. The volume of each sample was between 100～120 L. The organiccompounds in water were extracted with XAD-2 resin and eluted by acetone anddichloromethane. The eluate was concentrated (1:5000) and qualitatively analyzed byGC/MS. The water samples of a pilot-scale water plant in the south of China werecollected in April, 2006 and the methods of the sample pretreatments were identicalwith the others.
Results: All the water samples have been polluted by PAEs. The main PAEs in sourcewater and drinking water in Wuhan were DBP and DEP.Part of samples had DMP, DOP, and BBP.
Conclusion: The pollution of PAEs in source water and drinking water was universal.The present water treatment processes regardless of conventional or advancedtechniques were hard to remove the PAEs completely.
PartⅡAcclimation, screen and identification for dominant PAEsdegradation bacteria
Objective: To obtain dominant PAEs degradation bacteria and identify it.
Method: Activated sludge was collected at a dyeing plant of Wuhan, an industrial citylocated in central region of China. Eight L were mixed with 24 L of the inorganic saltsolution in an aerated basin. The acclimation process was conducted at roomtemperature. The DMP, DEP and DBP were equally added as sole carbon and energysource. The total concentration of PAEs was increased gradually from 30 to 420 mg/L.After eight weeks of acclimation, the activated sludge was used to inoculate nutrientagar plates under aseptic conditions. The plates were incubated at 37℃and differentcolonies grew after 36 h. The pure clones were obtained by plate streaking repeatedly.Then, each isolated bacterial strain was put into PAEs waste water in order to screendominant bacterium. All the flasks were put in swing bed with speed of 140 rpmunder 37℃. After one week degradation, the dominant PAEs-degrading bacteriumcould be confirmed in term of turbidity and degradation rate. After this screening test,we obtained a high-performance strain named strain L4. The dominant bacterial strainwas identified though analysis with its morphology, physiochemical characteristicsand 16S rDNA sequence. The characteristics of growth including environmentaltemperature and osmotic pressure of strain L4 were studied.
Results: Seven strains have been isolated form the aerated basin, named strain L1～L7. The screen test showed the degradation rates of DMP, DEP and DBP by strainL1～L5 were 87.5%, 99.1%, 98.3%; 31.9%, 10.2%, 0.0%, 19.1%, 14.2%, 16.9%; 99.0%, 98.6%, 99.2% and 28.9%, 20.0%, 6.5%, respectively。L6 and L7 can notutilize DMP, DEP or DBP as sole carbon source. The strain L4 was recognized asdominant biodegradation bacterium. Strain L4 was identified as Rhocococcus ruber.This strain can grow at temperature between 16 and 42℃, but the optimaltemperature was 37℃. The best salinity for strain L4 growth was lower than 0.5%,but it also grew well with salinity of 0.5%～3%.
Conclusion: The Rhodococcus ruber strain L4 had great ability in PAEs degradation.This strain had strong environmental adaptability.
PartⅢThe characteristics of Rhodococcus ruber strain L4about PAEs degradation
Objective: To explore the optimal environmental conditions (pH, temperature andinitial concentration of PAEs) for strain L4 degradaion and to study the kinetics andsome important characteristics related with degradation.
Method: The optimal environmental conditions were explored by orthogonal test(three factors and four levels). The kinetic equations were studied by curve fittingthrough detection the degrading residue of PAEs with different initial concentrations.The PAEs synthetic water samples (300 mg/L) with non-emulsification and completedemulsification were prepared to investigate their effects on PAEs degradation rate.The biosurfactant produced by strain L4 during PAEs degradation was measured bythe repellent size of oil cycle. Frequent aromatic compounds including phenol, 2,4-dinitrophenol, p-nitrophenol, sodium benzoate and naphthalene were prepared withMSM solution (4 retool/L) to explore the utilization spectrum of strain L4. The strainL4 was inoculated to ethanol, acetdimethylamide, octane and toluene solution withdifferent concentrations to investigate the environmental tolerance throughcomparison of survive rate.
Results: The degradation batch tests of DMP, DEP and DBP by the Rhodococcusruber strain L4 showed the optimal pH value, temperature and substrate concentration:pH 7.0～8.0, 30～37℃and PAEs concentration≤450 mg/L. Kinetics studies showedthat the half-life of degradation was about 1.30d when the concentration of PAEsmixture was lower than 300 mg/L. The degradation rates of DMP, DEP and DBP insame system were different, DMP＞DEP＞DBP during degradation process. Littledifference between the above two sample preparations was observed in terms ofultimate degradation rate. Biosurfactant can be produced by strain L4, which candecrease the surface tension between water and oil like 69.2 mg/L SDS solution. Thisstrain can also grow on phenol, sodium benzoate or naphthalene solution as solecarbon source and energy, but cannot utilizing 2, 4-dinitrophenol, p-nitrophenol. Themetabolism velocity of sodium benzoate was faster than that of glucose, phenol,sodium benzoate according to OD_(600) monitoring. Strain L4 can survive in ethanol andacetdimethylamide solution if their concentrations were lower than 10%; alkane waslitter harm to strain L4 as it can tolerate 60% octane solution; strain L4 was fragilewhen tested by 4% toluene as most bacteria were killed after 24 h exposure.Conclusion: Strain L4 had adaptability for various environments, but optimalenvironmental factors were benefit for biodegradation. The kinetics of PAEsdegradation by strain L4 can be described with exponential model under optimalconditions. During degradation, the PAE with shorter chain was prior to be utilized.Production of biosurfactant and mycolic acid by strain L4 were dominance fordegradation. That broad-spectrum utilization of organic compounds andenvironmental tolerance suggested strain L4 can be used as a potential candidate forremedying PAEs containing wastes.
PartⅣThe mechanism of PAEs degradationby Rhodococcus ruber strain L4
Objective: To approach the mechanism of PAEs degradation by strain L4 based on empirical methods of analytical chemistry and molecular biology.
Method: The DEP solution with concentration of 300 mg/L was degraded by strainL4. The intermediate products after 24 h, 48 h, 72 h, 96 h were collected and extractedby C18 columnella, subsequently eluted by dichloromethane and detected by GC/MS.The plasmids of strain L4 were extracted with alkaline lysis method. The MW wasdetermined by agarose gel electrophoresis (AGE). The strain L4 was transferred 5times in nutrient broth culture, which then collected as control group. The abovebacteria were put into 300 mg/L PAEs solution and cultured at 37℃in 24 h, whichthen collected as induction group. The total proteins of strain L4 before and afterPAEs induction were extracted through ultrasound. Their concentrations weremeasured by Bradford method. The activity of C120 and C23O was determined bycolorimetry. The bands of total protein of strain L4 before and after PAEs induction inSDS-PAGE were compared.
Results. Two intermediate products including phthalic acid and monoethyl phthalatewere detected. Three plasmids have been extracted from strain L4, whose MW were65 kb, 75 kb and 100 kb. The activities of C12O and C23O after PAEs induction werehigher than control group (P＜0.05). In induced group, the activity of C12O(8.78±0.98 U/mg) was higher than that of C23O (3.97±0.55 U/mg). The SDS-PAGEshowed two more bands were found in induced group.
Conclusion: Phthalic acid was the main intermediate product during DEP degradation.Strain L4 contains three big plasmids. C12O and C23O were critical enzymes forPAEs degradation.
PartⅤDegradaion of PAEs in water by immobilized bacteria
Objective: To compare the effect of immobilized cells and free cells of strain L4 inPAEs degradation.
Method: The balls with immobilized bacteria were made by 10% PVA, 0.5% sodiumpolymannuronate, with adding 4% activated carbon powder. The balls was made byadding the mixture form injector, and cross linked in saturate boracic acid solution inabout 20 h. The DMP, DEP and DBP mixture (300 mg/L) in water were degraded byimmobilized bacteria and free cells. The removal rates of PAEs were compared.
Results: The degradation rates of PAEs by immobilized bacteria were DMP 72.6%,DEP 70.1%, DBP68.3% in 48 h and 79.2%, DEP 74.9%, DBP 72.1% in 72 h。Thedegradation rates of PAEs by free cells were DMP 48.2%, DEP 41.1%, DBP27.3% in48 h and 72.2%, DEP 71.3%, DBP 58.8% in 72 h。
Conclusion: The immobilized bacteria with good ability in removal of PAEs in watercan be made by 10% PVA, 0.5% sodium polymannuronate and 4% activated carbonpowder. Comparing the degradation effects, the immobilized bacteria were superior tofree cells.
PartⅥEstrogenic activity of PAEs before and after biodegradation
Objective: To evaluate the safety of the residuals of PAEs after degradation bydetermination of estrogenic activity with yeast estrogen screen (YES).
Method: The estrogenic activities of DMP, DEP and DBP or their mixture weredetected by YES. The PAEs in water (300 mg/L) were degraded by strain L4. Theresiduals after 24 h, 48 h, 72 h and 96 h degradation were collected by Liquid-liquidextraction. The estrogenic activity of the residual was detected by YES subsequently.
Results: The estrogenic activity of DEP can be detected, which was like the level of1/2000000 compared to 17β-E2. No obvious estrogenic activity can be found in DMPand DBP. The estrogenic activity of PAEs residues after degradation can be detectedby YES. The biggest activity of PAEs mixture were 1.81, 1.41, 1.25 and 1.15 after 24h, 48 h, 72 h and 96 h degradation, which were lower than the activity of PAEs mixture before degradation (2.97).
Conclusion: The estrogenic activity of DMP, DEP and DBP can be detected by YES.The estrogenic activity of DEP was obvious, and stronger than DMP and DBP. Theestrogenic activity of PAEs mixture was caused by DEP and decreased afterdegradation by strain L4.
目的：对长江、汉江(武汉段)水源水、饮用水中PAEs的污染状况和深度水处理工艺(臭氧—活性炭)处理后出水中的PAEs化合物进行定性分析。
方法：以武汉自来水厂(宗关水厂、平湖门水厂)的源水、出厂水和管网末梢水为调查对象，分别于2007年、2008年平水期(3月)、丰水期(7月)、枯水期(12月)采集水样，采样量为100～120 L。用XAD-2树脂吸附水中的有机物，用二氯甲烷和丙酮进行洗脱，洗脱液经浓缩后(相对于水中的含量，其浓缩比例为1：5000)，通过GC／MS对有机物的成份进行定性分析。2006年4月在南方某自来水厂的中试基地，将臭氧氧化，活性炭(生物活性炭)吸附工艺与传统水处理工艺(混凝沉淀、加氯消毒)相结合，通过不同的工艺组合对当地源水进行处理。源水和不同组合工艺处理后出水的采集、有机物的浓缩和检测与上面描述的方法相同。
结果：GC／MS结果显示所有水样中都有PAEs检出。武汉两水厂的源水和饮用水中主要为DBP和DEP。同2007年相比，2008年新检出两种PAEs为DMP和BBP。南方某中试基地的源水和不同组合工艺处理后出水中均有DMP和DEP检出。
结论：源水和饮用中的PAEs污染非常普遍，现有的自来水处理技术(常规水处理技术和深度水处理技术)难以将其彻底去除。
第二章高效PAEs降解菌的驯化、筛选及鉴定
目的：驯化活性污泥，从驯化污泥中分离出高效PAEs降解菌，并对其生物种属进行鉴定。
方法：从武汉某印染厂取得活性污泥置于曝气池中，以DMP、DEP和DBP为唯一碳源，采用活性污泥曝气与密度梯度驯化方法，即从第1周开始，等量投加DMP、DEP、DBP作为细菌利用碳源。每周PAEs的投加浓度按10 mg／L、20mg／L、30 mg／L、40 mg／L、60 mg／L、80 mg／L、100 mg／L、140 mg／L依次递增，连续驯化8周。用灭菌采样瓶从曝气池采取污泥样本，用接种环蘸取污泥，在普通营养琼脂平板上分区划线以分离纯化细菌。在250 ml的锥形瓶中加入100 ml无机盐培养液，高压灭菌，加入等量DMP、DEP和DBP(各200 mg)，按等生物量的原则加入分离出的细菌，置于37℃空气浴摇床中(140 rpm)连续降解7天。用二氯甲烷液液萃取各降解液，用GC法检测其浓度。比较各细菌7天降解率的大小，筛选出高效降解细菌。对筛选出的高效PAEs降解菌，进行革兰染色。结合染色结果，用全自动微生物鉴定仪对其进行生化鉴定。用电子显微镜拍摄高效降解菌的透射电镜照片，观察细菌形态学。提取细菌DNA，扩增16s rDNA序列，并进行碱基序列测定。将序列提交Genbank并进行Blast序列比对，通过比对结果进一步鉴定该菌种属。将细菌置于不同的温度和不同的渗透压下(不同的NaCl浓度)培养，以考察该高效降解菌的生长特性。
结果：从印染厂活性污泥曝气池中分离得到7株细菌，命名为菌株L1～L7。菌株L1～L5对DMP、DEP和DBP(200 mg／L)的七天降解率分别为87.5％，99.1％，98.3％；31.9％，10.2％，0.0％；19.1％，14.2％，16.9％；99.0％，98.6％，99.2％与28.9％，20.0％，6.5％。L6、L7菌基本没有降解效果。确定L4为高效PAEs降解菌，对细菌L4进行鉴定，为赤红球菌(Rhodococcus ruber)。赤红球菌L4在12～42℃均有不同程度的生长，最适生长温度为37℃。赤红球菌L4生长的最佳盐浓度≤0.5％，但在盐浓度为0.5～3％时，亦能良好生长，耐受浓度可高达5％。
结论：采用活性污泥曝气与密度梯度驯化方法，筛选出可高效降解PAEs的赤红球菌(Rhodococcus ruber)。该细菌生长温度范围广，能耐受较高的环境渗透压。
第三章赤红球菌L4降解PAEs的特性研究
目的：确定赤红球菌L4降解PAEs的适宜条件(pH值、温度和初始底物浓度)，探索降解动力学，对赤红球菌L4降解相关特性进行初步研究。
方法：通过正交设计(三因素、四水平表)探索赤红球菌L4降解PAEs的最佳温度、pH及初始PAEs浓度。以人工配制的不同初始浓度的PAEs实验水样为降解对象，每天取样检测残留量，以曲线拟合方法获得降解动力学方程。为确定PAEs在水中不同的存在状态对降解效果的影响，分别用直接投加和丙酮(超声)乳化的方法来配制PAEs实验水样(300 mg／L)。为研究赤红球菌L4的环境抵抗特性，将其接种于不同浓度的乙醇、二甲基乙酰胺、正辛烷和甲苯溶液，37℃培养24 h后进行菌落计数以调查其存活率。将赤红球菌L4分别接种于4 mmol／L的苯甲酸钠、苯酚、萘、对硝基酚、邻硝基酚和葡萄糖溶液中，以OD_(600)值作为观测指标，考察该细菌对有机物的利用谱。用油膜法测定PAEs降解液的表面活性。
结果：正交试验显示赤红球菌L4降解PAEs的最佳条件为：pH 7.0～8.0，30～37℃，初始底物浓度≤450 mg／L。赤红球菌L4降解动力学可用方程lnC=—kt+θ’来描述(k为降解反应的速率常数，θ’为积分常数)；当初始底物浓度小于300 mg／L时，其降解半衰期为1.30 d；赤红球菌L4在混合PAEs降解过程中，三种PAEs的降解速率，DMP＞DEP＞DBP。赤红球菌L4对乳化组与非乳化组实验水样5天降解率分别为90.9％和86.9％。赤红球菌L4对10％以下的乙醇和二甲基乙酰胺有较强的耐受能力，对直链烷烃如正辛烷24小时耐受浓度可高达60％，对芳香族化合物如甲苯的耐受能力相对较弱，在4％的甲苯水溶液中仅有少数菌可以存活。赤红球菌L4可分别以苯甲酸钠、苯酚、萘为唯一碳源和能源生长，对苯甲酸钠的利用速度较快，优于葡萄糖，其次为对苯酚和萘的利用，尚未观察到对邻硝基酚、对硝基酚有分解代谢作用。在PAEs的代谢过程中，赤红球菌L4可产生生物表面活性物质，其乳化效果相当于69.2 mg／L的SDS溶液。
结论：赤红球菌L4对PAEs的降解有较强的环境适应性，在最适宜条件下降解效果最好。在最佳降解条件下，赤红球菌L4对不同初始浓度的PAEs的降解可以用一级动力学方程来描述。在降解过程中，较短侧链的PAE被优先利用。赤红球菌L4能产生降低水—油表面张力的表面活性物质，具有乳化能力，有利于生物降解。较宽的利用谱和较强的抗毒性意味着赤红球菌L4在环境污染的生物修复中具有较大的应用潜力。
第四章赤红球菌L4代谢PAEs的机理探讨
目的：应用化学分离、分析技术和分子生物学实验方法探讨赤红球菌L4降解PAEs的机理。
方法：以初始浓度为300 mg／L的DEP实验水样为对象，投加赤红球菌L4进行降解试验，分别于降解24 h，48 h，72 h，96 h时收集降解液，用C18固相萃取柱吸附其中的有机物并用二氯甲烷进行洗脱，洗脱液经浓缩后进行GC／MS检测。用碱裂解法提取细菌质粒，通过琼脂糖凝胶电泳确定其分子量大小。将赤红球菌L4用普通营养肉汤连续传代5次，收集菌体作为对照组；经传代的细菌置于总浓度为300 mg／L的DMP、DEP、DBP混合溶液中培养24 h，收集菌体作为诱导组。用超声波裂解诱导组和对照组细菌，离心后取上清，即为提取的细菌总蛋白(酶)。以不同浓度的小牛血清溶液作为标准系列，用Bradford法测定各组蛋白含量。根据蛋白定量结果，等量投加对照组和诱导组蛋白酶至20μmol／L的邻苯二酚溶液，用分光光度法于260 nm和375 nm处分别测定上述溶液吸光度每分钟的变化值，即可求得邻苯二酚1，2—双加氧酶(C12O)和邻苯二酚2，3—双加氧酶(C23O)的酶活性；用聚丙烯酰胺凝胶电泳对对照组和诱导组细菌总蛋白进行分析。
结果：DEP降解48 h时，其降解液中可以检测到两个中间产物，为邻苯二甲酸单乙酯(monoethyl phthalate)和邻苯二甲酸(phthalate acid)。从赤红球菌L4内提取到3个质粒，其大小分别为65 kb、75 kb和100 kb。酶活性测试说明诱导组细菌的邻苯二酚双加氧酶活性高于肉汤连续传代的对照组(P＜0.05)，其C12O活性为8.78±0.98 U／mg，C23O活性为3.97±0.55 U／mg。聚丙烯酰胺凝胶电泳显示诱导组有两条新增条带。
结论：在降解初始阶段，赤红球菌L4首先水解其中一个酯键，生成邻苯二甲酸单乙酯，然后继续水解另外一个酯键，生成邻苯二甲酸，后者为主要的中间产物。赤红球菌L4体内拥有至少3条较大的质粒；邻苯二酚双加氧酶是赤红球菌L4降解过程中的关键性蛋白酶。赤红球菌L4经诱导后可同时表达C12O和C23O，但C12O活性增加更为明显。
第五章固定化微生物对水中PAEs的降解
目的：比较固定化微生物和游离微生物对水中PAEs的降解效果。
方法：以10％的PVA和0.5％海藻酸钠溶液为包埋剂，以4％的活性炭为吸附剂，以饱和硼酸溶液作为交联剂，采用吸附—包埋法制备固定化微生物小球。配制含DMP、DEP、DBP的实验水样(300mg／L)，以等生物量法，分别将固定化微生物小球或游离细菌投加于其中，然后置于35℃、115 rpm摇床上进行降解试验，测定48 h、72 h的残留量以评价固定化微生物和游离细菌的降解效果。
结果：固定化微生物小球48 h降解率为DMP 72.6％、DEP 70.1％、DBP68.3％；72 h降解率为DMP 79.2％、DEP 94.9％、DBP 72.1％。游离细菌48 h降解率为DMP 48.2％、DEP 41.1％、DBP27.3％；72 h降解率为DMP 72.2％、DEP 71.3％、DBP 58.8％。
结论：以10％的PVA和0.5％海藻酸钠作为包埋剂，以4％的活性炭作为吸附剂可以制备出降解效果良好的固定化微生物小球；固定化微生物对PAEs的降解效果优于游离微生物降解。
第六章PAEs生物降解前后的类雌激素活性的比较
目的：对PAEs混合物生物降解过程中的残留物进行类雌激素活性检测，以评价降解的安全性。
方法：用重组酵母菌雌激素检测系统(Yeast Estrogen Screen，YES)对各样品进行检测。将含有人雌激素受体的重组酵母菌检测液和稀释成一定浓度梯度的DMP、DEP和DBP的单一及混合物在96孔板中培养，以17β—雌二醇作为阳性对照，以CPRG(黄色)作为指示剂，培养3天后，若受试物具有雌激素活性，则溶液由黄色变成红色，红色的深浅代表酶活性强度，用酶标仪在540 nm处予以测定(OD_(540))。用赤红球菌L4对初始总浓度为300 mg／L的DMP、DEP和DBP混合水样进行生物降解，收集降解0 h、24 h、48 h、72 h、96 h后的降解液，用二氯甲烷液液萃取提取其中的有机物。用YES对其进行类雌激素活性检测。
结果：YES试验检测到DEP的类雌激素活性，其强度相当于雌激素(17β—雌二醇)的200万分之一水平，DMP和DBP未检出类雌激素活性。混合PAEs实验水样降解24 h，48 h，72 h，96 h后的残留物的最大类雌激素活性分别为：1.81，1.41，1.25和1.15，低于降解前(0 h)的2.97。
结论：YES试验可以检测DMP，DEP，DBP及其混合物的类雌激素活性，就单一物质而言，DMP、DEP、DBP的类雌激素活性由强至弱依次为DEP＞DMP＞DBP，混合PAEs的类雌激素活性主要由DEP所致；赤红球菌L4降解PAEs的产物的类雌激素活性，随着降解时间的延长，其水平呈逐步下降趋势。
PartⅠPAEs pollution in source water and drinking water by qualitativeinvestigation
Objective: To qualitatively investigate the kinds of PAEs in source water and drinkingwater of the Yangtze River and Hanjiang River (Wuhan section) by GC/MS. Toqualitatively investigate the kinds of PAEs in source water and effluents treated bydifferent advanced water treatment processes including ozone binding biologicalactivated carbon.
Method: The source water and drinking water of Zongguan and Pinghumen waterplants in Wuhan city were collected in March, July and December in the year of 2007and 2008. The volume of each sample was between 100～120 L. The organiccompounds in water were extracted with XAD-2 resin and eluted by acetone anddichloromethane. The eluate was concentrated (1:5000) and qualitatively analyzed byGC/MS. The water samples of a pilot-scale water plant in the south of China werecollected in April, 2006 and the methods of the sample pretreatments were identicalwith the others.
Results: All the water samples have been polluted by PAEs. The main PAEs in sourcewater and drinking water in Wuhan were DBP and DEP.Part of samples had DMP, DOP, and BBP.
Conclusion: The pollution of PAEs in source water and drinking water was universal.The present water treatment processes regardless of conventional or advancedtechniques were hard to remove the PAEs completely.
PartⅡAcclimation, screen and identification for dominant PAEsdegradation bacteria
Objective: To obtain dominant PAEs degradation bacteria and identify it.
Method: Activated sludge was collected at a dyeing plant of Wuhan, an industrial citylocated in central region of China. Eight L were mixed with 24 L of the inorganic saltsolution in an aerated basin. The acclimation process was conducted at roomtemperature. The DMP, DEP and DBP were equally added as sole carbon and energysource. The total concentration of PAEs was increased gradually from 30 to 420 mg/L.After eight weeks of acclimation, the activated sludge was used to inoculate nutrientagar plates under aseptic conditions. The plates were incubated at 37℃and differentcolonies grew after 36 h. The pure clones were obtained by plate streaking repeatedly.Then, each isolated bacterial strain was put into PAEs waste water in order to screendominant bacterium. All the flasks were put in swing bed with speed of 140 rpmunder 37℃. After one week degradation, the dominant PAEs-degrading bacteriumcould be confirmed in term of turbidity and degradation rate. After this screening test,we obtained a high-performance strain named strain L4. The dominant bacterial strainwas identified though analysis with its morphology, physiochemical characteristicsand 16S rDNA sequence. The characteristics of growth including environmentaltemperature and osmotic pressure of strain L4 were studied.
Results: Seven strains have been isolated form the aerated basin, named strain L1～L7. The screen test showed the degradation rates of DMP, DEP and DBP by strainL1～L5 were 87.5%, 99.1%, 98.3%; 31.9%, 10.2%, 0.0%, 19.1%, 14.2%, 16.9%; 99.0%, 98.6%, 99.2% and 28.9%, 20.0%, 6.5%, respectively。L6 and L7 can notutilize DMP, DEP or DBP as sole carbon source. The strain L4 was recognized asdominant biodegradation bacterium. Strain L4 was identified as Rhocococcus ruber.This strain can grow at temperature between 16 and 42℃, but the optimaltemperature was 37℃. The best salinity for strain L4 growth was lower than 0.5%,but it also grew well with salinity of 0.5%～3%.
Conclusion: The Rhodococcus ruber strain L4 had great ability in PAEs degradation.This strain had strong environmental adaptability.
PartⅢThe characteristics of Rhodococcus ruber strain L4about PAEs degradation
Objective: To explore the optimal environmental conditions (pH, temperature andinitial concentration of PAEs) for strain L4 degradaion and to study the kinetics andsome important characteristics related with degradation.
Method: The optimal environmental conditions were explored by orthogonal test(three factors and four levels). The kinetic equations were studied by curve fittingthrough detection the degrading residue of PAEs with different initial concentrations.The PAEs synthetic water samples (300 mg/L) with non-emulsification and completedemulsification were prepared to investigate their effects on PAEs degradation rate.The biosurfactant produced by strain L4 during PAEs degradation was measured bythe repellent size of oil cycle. Frequent aromatic compounds including phenol, 2,4-dinitrophenol, p-nitrophenol, sodium benzoate and naphthalene were prepared withMSM solution (4 retool/L) to explore the utilization spectrum of strain L4. The strainL4 was inoculated to ethanol, acetdimethylamide, octane and toluene solution withdifferent concentrations to investigate the environmental tolerance throughcomparison of survive rate.
Results: The degradation batch tests of DMP, DEP and DBP by the Rhodococcusruber strain L4 showed the optimal pH value, temperature and substrate concentration:pH 7.0～8.0, 30～37℃and PAEs concentration≤450 mg/L. Kinetics studies showedthat the half-life of degradation was about 1.30d when the concentration of PAEsmixture was lower than 300 mg/L. The degradation rates of DMP, DEP and DBP insame system were different, DMP＞DEP＞DBP during degradation process. Littledifference between the above two sample preparations was observed in terms ofultimate degradation rate. Biosurfactant can be produced by strain L4, which candecrease the surface tension between water and oil like 69.2 mg/L SDS solution. Thisstrain can also grow on phenol, sodium benzoate or naphthalene solution as solecarbon source and energy, but cannot utilizing 2, 4-dinitrophenol, p-nitrophenol. Themetabolism velocity of sodium benzoate was faster than that of glucose, phenol,sodium benzoate according to OD_(600) monitoring. Strain L4 can survive in ethanol andacetdimethylamide solution if their concentrations were lower than 10%; alkane waslitter harm to strain L4 as it can tolerate 60% octane solution; strain L4 was fragilewhen tested by 4% toluene as most bacteria were killed after 24 h exposure.Conclusion: Strain L4 had adaptability for various environments, but optimalenvironmental factors were benefit for biodegradation. The kinetics of PAEsdegradation by strain L4 can be described with exponential model under optimalconditions. During degradation, the PAE with shorter chain was prior to be utilized.Production of biosurfactant and mycolic acid by strain L4 were dominance fordegradation. That broad-spectrum utilization of organic compounds andenvironmental tolerance suggested strain L4 can be used as a potential candidate forremedying PAEs containing wastes.
PartⅣThe mechanism of PAEs degradationby Rhodococcus ruber strain L4
Objective: To approach the mechanism of PAEs degradation by strain L4 based on empirical methods of analytical chemistry and molecular biology.
Method: The DEP solution with concentration of 300 mg/L was degraded by strainL4. The intermediate products after 24 h, 48 h, 72 h, 96 h were collected and extractedby C18 columnella, subsequently eluted by dichloromethane and detected by GC/MS.The plasmids of strain L4 were extracted with alkaline lysis method. The MW wasdetermined by agarose gel electrophoresis (AGE). The strain L4 was transferred 5times in nutrient broth culture, which then collected as control group. The abovebacteria were put into 300 mg/L PAEs solution and cultured at 37℃in 24 h, whichthen collected as induction group. The total proteins of strain L4 before and afterPAEs induction were extracted through ultrasound. Their concentrations weremeasured by Bradford method. The activity of C120 and C23O was determined bycolorimetry. The bands of total protein of strain L4 before and after PAEs induction inSDS-PAGE were compared.
Results. Two intermediate products including phthalic acid and monoethyl phthalatewere detected. Three plasmids have been extracted from strain L4, whose MW were65 kb, 75 kb and 100 kb. The activities of C12O and C23O after PAEs induction werehigher than control group (P＜0.05). In induced group, the activity of C12O(8.78±0.98 U/mg) was higher than that of C23O (3.97±0.55 U/mg). The SDS-PAGEshowed two more bands were found in induced group.
Conclusion: Phthalic acid was the main intermediate product during DEP degradation.Strain L4 contains three big plasmids. C12O and C23O were critical enzymes forPAEs degradation.
PartⅤDegradaion of PAEs in water by immobilized bacteria
Objective: To compare the effect of immobilized cells and free cells of strain L4 inPAEs degradation.
Method: The balls with immobilized bacteria were made by 10% PVA, 0.5% sodiumpolymannuronate, with adding 4% activated carbon powder. The balls was made byadding the mixture form injector, and cross linked in saturate boracic acid solution inabout 20 h. The DMP, DEP and DBP mixture (300 mg/L) in water were degraded byimmobilized bacteria and free cells. The removal rates of PAEs were compared.
Results: The degradation rates of PAEs by immobilized bacteria were DMP 72.6%,DEP 70.1%, DBP68.3% in 48 h and 79.2%, DEP 74.9%, DBP 72.1% in 72 h。Thedegradation rates of PAEs by free cells were DMP 48.2%, DEP 41.1%, DBP27.3% in48 h and 72.2%, DEP 71.3%, DBP 58.8% in 72 h。
Conclusion: The immobilized bacteria with good ability in removal of PAEs in watercan be made by 10% PVA, 0.5% sodium polymannuronate and 4% activated carbonpowder. Comparing the degradation effects, the immobilized bacteria were superior tofree cells.
PartⅥEstrogenic activity of PAEs before and after biodegradation
Objective: To evaluate the safety of the residuals of PAEs after degradation bydetermination of estrogenic activity with yeast estrogen screen (YES).
Method: The estrogenic activities of DMP, DEP and DBP or their mixture weredetected by YES. The PAEs in water (300 mg/L) were degraded by strain L4. Theresiduals after 24 h, 48 h, 72 h and 96 h degradation were collected by Liquid-liquidextraction. The estrogenic activity of the residual was detected by YES subsequently.
Results: The estrogenic activity of DEP can be detected, which was like the level of1/2000000 compared to 17β-E2. No obvious estrogenic activity can be found in DMPand DBP. The estrogenic activity of PAEs residues after degradation can be detectedby YES. The biggest activity of PAEs mixture were 1.81, 1.41, 1.25 and 1.15 after 24h, 48 h, 72 h and 96 h degradation, which were lower than the activity of PAEs mixture before degradation (2.97).
Conclusion: The estrogenic activity of DMP, DEP and DBP can be detected by YES.The estrogenic activity of DEP was obvious, and stronger than DMP and DBP. Theestrogenic activity of PAEs mixture was caused by DEP and decreased afterdegradation by strain L4.
引文
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