固定化微球菌降解废水中邻苯二甲酸酯的研究
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摘要
邻苯二甲酸酯（Phthalic Acid Esters，简称PAEs）属于典型的持久性有机污染物，具有内分泌干扰作用。本论文选择邻苯二甲酸二丁酯（Di-n-butyl phthalate，简称DBP）为代表性污染物，从选育高效降解微生物入手，研究了DBP的生物降解特性，探讨了高效微生物的固定化，考察了生物强化技术去除废水中DBP的特性，利用末端限制性片段长度多态性（T-RFLP）方法监测了微生物群落结构的动态变化。选题具有重要的理论意义和应用价值。论文工作取得以下主要研究成果：（1）分离获得一株DBP高效降解菌，经鉴定为微球菌（Micrococcus sp.）。该菌株能够以DBP为唯一碳源和能源生长。葡萄糖的存在不影响DBP的降解，低浓度DBP的存在对葡萄糖的降解趋势也不产生影响，但高浓度DBP会对葡萄糖的降解产生抑制。微量金属离子的加入会促进菌株Micrococcus sp.的生长，并促进DBP的降解。GC/MS分析结果表明，DBP的生物降解途径为：邻苯二甲酸二丁酯首先水解成邻苯二甲酸单丁酯，然后变成邻苯二甲酸及相应的醇，最后降解为CO2和H2O。
（2）研究了微生物固定化方法，重点探讨了克服PVA固定化微生物颗粒水溶膨胀性问题。综合比较固定化微生物颗粒的生物活性、机械性能以及化学稳定性，PVA-SA载体是包埋固定化微生物的适宜材料。优化了PVA固定化微生物的适宜条件，在该条件下制备的固定化微生物，膨胀率为15%，相对生物活性为61%。
（3）利用PVA为载体固定化Micrococcus sp.，结果发现，与游离微生物相比，固定化微生物的最佳pH和温度范围有所拓宽，显示出固定化微生物的优势。比较了固定化微生物的不同保藏方法，发现真空冷藏是保藏固定化微生物的较适宜方式。固定化微生物颗粒使用30d后，其膨胀率在20%以内，相对生物活性在90%以上。固定化Micrococcus sp.菌降解DBP的过程可以用Haldane抑制动力学方程描述。
（4）在活性污泥处理系统中引入固定化高效降解菌Micrococcus sp.可以加速反应器的启动。当DBP初始浓度为50mg/L时，固定化细胞对DBP的强化去除作用不明显；当DBP初始浓度在100~500mg/L范围内，引入固定化高效降解菌可以显著提高反应器中DBP的去除。T-RFLP分析结果表明，固定化高效降解菌存在于反应器运行的整个运行过程中，投加高效降解菌会影响活性污泥中微生物的群落结构。
Phthalic acid esters (PAEs for short) are global toxic organic pollutants, belonging to atypical persistent organic pollutants (POPs), they are endocrine-disrupting chemicals. PAEshave been widely existed in the environment, and their pollutions are increasing annually inour country, which may cause serious environmental contamination and ecological risk.Microbial degradation is a key route for PAEs removal from the environment. In this paper,Di-n-butyl phthalate (DBP for short) was selected as a representative PAEs, firstly, ahigh-efficient microorganism capable of degrading DBP was isolated and identified, then thecharacteristics and mechanism of DBP biodegradation was investigated, the microbialimmobilization method using PVA as carrier was optimized, finally, the DBP removal fromwastewater by bioaugmentated systems was studied, and the dynamics of microbialcommunity structure was analyzed by Terminal Restriction Fragment Length Polymorphism(T-RFLP). The results have an important theoretic and practical significance for the removalof recalcitrant pollutants from wastewater.
The main conclusions obtained from this study are as follows:
(1) A high-efficient stain capable of degrading DBP was isolated and identified asMicrococcus sp., which can use DBP as sole carbon and energy source. DBP as high as500mg/L did not inhibit the growth of Micrococcus sp. The strain can use DBP and glucosesimultaneously. The degradation of DBP was not affected by the existence of glucose, DBP atlow concentration did not influence the degradation trend of glucose; however, DBP at highconcentration inhibited the glucose degradation at some degree. The addition of trace metallicelements could stimulate the bacterial growth and enhance DBP degradation. The results ofgas chromatography-mass spectrometry (GC/MS) analysis indicated the pathway of DBPdegradation was as follows: DBP was hydrolyzed to mono-butyl phthalate, then to phthalicacid, and finally to be completely degraded to carbon dioxide.
（2）The microbial immobilization methods using polyvinyl alcohol (PVA) were studied,focusing on overcoming the swelling performance of immobilized cell beads. Based on thecomprehensive comparison of biologic activity, mechanical strength and chemical stability,PVA-SA was a suitable carrier for microbial immobilization. During immobilization processusing PVA as carrier, after cross-linking in saturated boric acid, the resulted immobilized cell beads were stored at4℃for24h, the swelling ratio of immobilized cell beads couldsignificantly decreased, and the biological activity of the beads did not obviously affected.The immobilization conditions using PVA as carrier were optimized, and the swelling ratioand relative biological activity of the immobilized cells prepared at this condition was15%and61%, respectively.
（3）The microbial cells of Micrococcus sp. were entrapped by PVA carrier, the resultsindicated that after immobilization, the immobilized cells showed wider range of optimal pHand temperature in comparison with the free cells, suggesting the advantages of immobilizedmicrobial cells. The different methods for the storage of the immobilized cells was studied, itwas found that vacuum storage was a suitable storage method, the immobilized cells beadscould retain90%of its initial activity after12months in vacuum storage at4℃. The physicalcharacteristics and microbial activity of the beads changed with time, and stabilized finally.The swelling ratio and relative biological activity of the immobilized beads was less than20%and more than90%, respectively after30days application. The DBP degradation process byimmobilized Micrococcus sp. could be described by the Haldane inhibition kinetics equation.
（4）The introduction of immobilized Micrococcus sp. into the activated sludge processcould speed up the start-up of the reactor. When the initial DBP concentration was50mg/L,the effect of the bioaumentation on the removal rate of DBP was not obvious; however, whenthe initial DBP concentration increased to the range of100-500mg/L, the introduction of theimmobilized cells could significantly enhanced the removal rate of DBP. The analyticalresults of T-RFLP indicated that the immobilized Micrococcus sp. could exist in the wholeoperational process. The introduction of the immobilized Micrococcus sp. had a significantimpact on microbial community structure of the activated sludge system.
（2）研究了微生物固定化方法，重点探讨了克服PVA固定化微生物颗粒水溶膨胀性问题。综合比较固定化微生物颗粒的生物活性、机械性能以及化学稳定性，PVA-SA载体是包埋固定化微生物的适宜材料。优化了PVA固定化微生物的适宜条件，在该条件下制备的固定化微生物，膨胀率为15%，相对生物活性为61%。
（3）利用PVA为载体固定化Micrococcus sp.，结果发现，与游离微生物相比，固定化微生物的最佳pH和温度范围有所拓宽，显示出固定化微生物的优势。比较了固定化微生物的不同保藏方法，发现真空冷藏是保藏固定化微生物的较适宜方式。固定化微生物颗粒使用30d后，其膨胀率在20%以内，相对生物活性在90%以上。固定化Micrococcus sp.菌降解DBP的过程可以用Haldane抑制动力学方程描述。
（4）在活性污泥处理系统中引入固定化高效降解菌Micrococcus sp.可以加速反应器的启动。当DBP初始浓度为50mg/L时，固定化细胞对DBP的强化去除作用不明显；当DBP初始浓度在100~500mg/L范围内，引入固定化高效降解菌可以显著提高反应器中DBP的去除。T-RFLP分析结果表明，固定化高效降解菌存在于反应器运行的整个运行过程中，投加高效降解菌会影响活性污泥中微生物的群落结构。
Phthalic acid esters (PAEs for short) are global toxic organic pollutants, belonging to atypical persistent organic pollutants (POPs), they are endocrine-disrupting chemicals. PAEshave been widely existed in the environment, and their pollutions are increasing annually inour country, which may cause serious environmental contamination and ecological risk.Microbial degradation is a key route for PAEs removal from the environment. In this paper,Di-n-butyl phthalate (DBP for short) was selected as a representative PAEs, firstly, ahigh-efficient microorganism capable of degrading DBP was isolated and identified, then thecharacteristics and mechanism of DBP biodegradation was investigated, the microbialimmobilization method using PVA as carrier was optimized, finally, the DBP removal fromwastewater by bioaugmentated systems was studied, and the dynamics of microbialcommunity structure was analyzed by Terminal Restriction Fragment Length Polymorphism(T-RFLP). The results have an important theoretic and practical significance for the removalof recalcitrant pollutants from wastewater.
The main conclusions obtained from this study are as follows:
(1) A high-efficient stain capable of degrading DBP was isolated and identified asMicrococcus sp., which can use DBP as sole carbon and energy source. DBP as high as500mg/L did not inhibit the growth of Micrococcus sp. The strain can use DBP and glucosesimultaneously. The degradation of DBP was not affected by the existence of glucose, DBP atlow concentration did not influence the degradation trend of glucose; however, DBP at highconcentration inhibited the glucose degradation at some degree. The addition of trace metallicelements could stimulate the bacterial growth and enhance DBP degradation. The results ofgas chromatography-mass spectrometry (GC/MS) analysis indicated the pathway of DBPdegradation was as follows: DBP was hydrolyzed to mono-butyl phthalate, then to phthalicacid, and finally to be completely degraded to carbon dioxide.
（2）The microbial immobilization methods using polyvinyl alcohol (PVA) were studied,focusing on overcoming the swelling performance of immobilized cell beads. Based on thecomprehensive comparison of biologic activity, mechanical strength and chemical stability,PVA-SA was a suitable carrier for microbial immobilization. During immobilization processusing PVA as carrier, after cross-linking in saturated boric acid, the resulted immobilized cell beads were stored at4℃for24h, the swelling ratio of immobilized cell beads couldsignificantly decreased, and the biological activity of the beads did not obviously affected.The immobilization conditions using PVA as carrier were optimized, and the swelling ratioand relative biological activity of the immobilized cells prepared at this condition was15%and61%, respectively.
（3）The microbial cells of Micrococcus sp. were entrapped by PVA carrier, the resultsindicated that after immobilization, the immobilized cells showed wider range of optimal pHand temperature in comparison with the free cells, suggesting the advantages of immobilizedmicrobial cells. The different methods for the storage of the immobilized cells was studied, itwas found that vacuum storage was a suitable storage method, the immobilized cells beadscould retain90%of its initial activity after12months in vacuum storage at4℃. The physicalcharacteristics and microbial activity of the beads changed with time, and stabilized finally.The swelling ratio and relative biological activity of the immobilized beads was less than20%and more than90%, respectively after30days application. The DBP degradation process byimmobilized Micrococcus sp. could be described by the Haldane inhibition kinetics equation.
（4）The introduction of immobilized Micrococcus sp. into the activated sludge processcould speed up the start-up of the reactor. When the initial DBP concentration was50mg/L,the effect of the bioaumentation on the removal rate of DBP was not obvious; however, whenthe initial DBP concentration increased to the range of100-500mg/L, the introduction of theimmobilized cells could significantly enhanced the removal rate of DBP. The analyticalresults of T-RFLP indicated that the immobilized Micrococcus sp. could exist in the wholeoperational process. The introduction of the immobilized Micrococcus sp. had a significantimpact on microbial community structure of the activated sludge system.
引文
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