【作者】 崔岱宗；

【导师】 赵敏；

【作者基本信息】 东北林业大学 ， 微生物学， 2012， 博士

【摘要】 偶氮染料是目前种类最多的一大类染料。在生产和应用的过程中,约有10%-15%的染料未经处理即被排放到环境中去并可能严重影响接触者的健康。因此,偶氮废水必需在排放前进行无害化处理。目前,已有多种处理偶氮废水的方法,其中微生物脱色法被认为是最为有效且对环境无害的方法。本研究的目的是从长期受到偶氮染料污染的环境中筛选分离出具有高效降解偶氮染料能力的菌株,考察其对偶氮染料的脱色特性,并研究各种因素对偶氮染料脱色的影响,为相关菌株的工业应用提供技术参数。研究在好氧条件下和厌氧条件下染料的降解机制,深入探讨偶氮还原酶的作用机理。培育出对环境无害、抗逆性强、对偶氮染料具有较高降解效率的混合菌群,并比较好氧和厌氧降解菌群结构的异同。本论文具体的研究结果如下：(1)本研究在污染的土壤样品中成功的分离到一株可在厌氧条件下降解偶氮染料的菌株Y3。经鉴定后,发现该菌株属于肺炎克雷伯氏菌。菌株Y3可在16h内完全降解100mg/L的甲基红,表明其具有很高的偶氮降解能力。对菌株Y3的染料降解特性的研究显示,该菌株可在25-45℃、pH值为4-9及盐度为1%-4%的条件下有效降解偶氮染料。该菌株可以以甲基红为唯一碳源及能源物质进行生长并使其降解。菌株Y3可对多种不同结构的偶氮染料进行降解,并能耐受较高的染料浓度。植物毒理实验表明经Y3处理后的偶氮废水毒性大大降低,然而,紫外—可见光谱分析显示菌株Y3并不能使偶氮染料完全矿化,而是使染料降解为无色的中间产物。(2)采用与厌氧筛选相似的实验方法,本研究从染料废水中分离了一株好氧偶氮降解菌。染料降解实验表明,该菌株可在好氧条件下,有效降解不同结构的偶氮染料,且对各种理化因素均具有良好的耐受性。经鉴定该菌株为大肠杆菌,并将其定名为E.coli CD-2。对该菌株细胞内的偶氮还原酶进行纯化,经质谱鉴定后,发现该酶的序列与大肠杆菌K12醌还原酶的序列具有较高的同源性。酶活检测显示,该还原酶同时具有偶氮还原酶及醌还原酶的活性。结合之前的其它研究成果,本研究提出观点：细菌细胞中可能并不存在专门降解偶氮染料的特异性还原酶。偶氮键的断裂可能是由一些原本在细菌细胞内催化重要生化反应的还原酶非特异性催化的。然而,打开偶氮键并不是这类酶在细胞中的单一功能,甚至不是主要功能。本研究对该酶的性质做了较为深入的研究,结果表明,该酶在细胞外仍具有良好的偶氮染料降解能力,且在较宽范围的pH、温度及盐度等理化条件下维持良好的稳定性。以上特性使该酶有很大潜力应用于日后的偶氮染料降解工业用酶的开发中。对酶的降解产物进行高效液相色谱分析后表明,该酶可以通过NADH所提供的H离子打开偶氮键,使染料脱色,形成的中间代谢产物则可能需要细胞内其它酶的参与从而达到彻底降解。(3)本研究测定了在好氧条件下,蒽醌类化合物对菌株CD-2降解偶氮染料效率的影响。结果表明,蒽醌介体可以大幅提高菌株CD-2的好氧偶氮降解效率。基于此,本研究认为,好氧体系中存在蒽醌介体时,菌株对偶氮染料的降解可能不再是一个特异性的酶促反应。蒽醌介体可以加速电子从还原剂传递到偶氮染料的速度,从而使偶氮染料的降解速率加快。虽然介体可以显著提高细菌细胞对偶氮染料的降解速率,但当细胞破碎后,介体并不再直接对偶氮染料的降解起促进作用。该结果表明,细菌完整的细胞膜阻碍了偶氮染料与细胞内还原酶接触的机会,而蒽醌介体起到了电子的跨膜传递作用。因此,在有介体存在的情况下,偶氮还原速率得到提高。而当细胞破碎后,细胞质中的还原酶可以与染料直接接触,葸醌介体的电子传递作用即不再重要。(4)本研究利用不断加强偶氮染料筛选强度的方法,成功的从环境中培育出可分别在好氧和厌氧条件下降解偶氮染料的混合菌群,通过对混合菌群降解特性的研究发现,在相同条件下,混合菌群降解偶氮染料的效果要好于纯菌株。厌氧菌群的降解效果略好于好氧菌群。利用传统的平板划线分离的方法对两个混合菌群中的菌株进行分离,结果表明,在菌群中分离的13株细菌均为肠杆菌科的兼性厌氧细菌。对两个菌群的PCR-DGGE分析显示,随着甲基红浓度的逐渐升高,混合菌群的结构出现明显变化。当菌群稳定时,对DGGE条带进行测序分析后表明,好氧菌群中所包含的降解菌分别属于克雷伯氏属、布丘菌属和芽孢杆菌属；厌氧菌群中的主要细菌则属于克雷伯氏属、埃希菌属、芽孢杆菌属和梭菌属。更多还原

【Abstract】 Azo dyes are the largest and most versatile class of dyes. During the dyeing process, upto15%of the dyestuffs are discharged into the environment. These dyes are toxic and threatening people’s health seriously. Thus, industrial wastewater containing azo dyes must be treated before it is released into the environment. Many new processes for azo dye decolorization have been developed. One promising strategy is to use microbes to decolorize azo dyes. The biodegradation of azo dyes is considered to be an environmentally friendly option.The purpose of this study was to isolate azo dye degrading bacteria from dye-contaminating environment. The decolorizing characteristics of azo dye were investigated and effects of various factors on the decoloration were studied, which offered the technology parameters for the industrial application of related strains. In addition, the mechanism of azo dye decolorization by bacteria was explored through azoreductase and redox mediator studies. Moreover, two environmentally friendly consortia which could decolorize azo dyes under aerobic and anaerobic conditions, respectively, were enriched. Similarities and differences of microbial stuctures between the two consortia were compared by using PCR-DGGE method. The main results are as follows:(1) In this study, we isolated and characterized a new strain of Klebsiella sp. Y3, that was capable of decolorizing azo dyes under anaerobic conditions. The strain Y3could decolorize100mg/L Methyl Red completely within16h, indicating that it had high ablility to decolorize azo dyes. The effects of physico-chemical parameters, including the temperature, pH and salinity of the culture medium on the Methyl Red degradation by the strain were determined. The results indicated that strain Y3exhibited a good decolorization ability in the ranges of pH from4-9, temperature from25-45℃and salinity from1%-4%. A50%Methyl Red decolorization was observed within64h without an additional carbon source. The strain could decolorize a broad spectrum of azo dyes with different structures. Strain Y3could tolerate and degrade azo dyes at high concentrations. An almost complete mineralization of Methyl Red and Congo Red at the concentration of800mg/L was observed within48h. The phytotoxicity tests showed that the degradation products were less toxic to plant seeds compared with the control Methyl Red, indicating that Methyl Red could be detoxicated by strain Y3. UV-visible analysis suggested that all the azo dyes were decolorized completely. However, the appearance of the new peak in the UV spectra indicated the formation of other metabolites.(2) Using the similar method with anaerobic bacteria isolation, we found a new strain which could degrade azo dyes effectively under aerobic conditions. Identification of this isolate by16S rDNA technique revealed that the strain clustered within Escherichia coli. Various environmental factors influencing dye degradation were investigated, the results showed that the strain could decolorize different azo dyes effectively at high salt concentration and over a wide range of pH. This degradation potential increased the applicability of this strain for the azo dye removal.An oxygen-insensitive intracellular enzyme that was responsible for the decolorization of azo dyes was purified from strain CD-2by ion-exchange and molecular exclusion chromatography. Protein identification indicated that the enzyme had high sequence homology with Escherichia coli K12quinone reductase, and the enzyme was proved to have both azoreductase and quinone reductase activity. According to these, we concluded that several kinds of enzymes which played important roles in bacterial cells could also reduce azo compounds as their secondary activities.The purified enzyme could efficiently decolorize Methyl Red outside cells and was relatively stable over wide ranges of pH, temperature and salinity. Therefore, this enzyme might have great potential for industrial use. The HPLC analysis showed that the purified enzyme could catalyze the reductive cleavage of the azo bond of Methyl Red in the presence of NADH as electron donor. The metabolites of Methyl Red might be degraded by other enzymes.(3) In present study, quinone-mediated decolorization of different azo dyes by strain CD-2under aerobic conditions was investigated. The results showed that reduction rates of different azo dyes by strain CD-2were greatly increased in the presence of quinone compouds as redox mediators. Based on these, we concluded that the aerobic dye decolorization reaction was unspecific when the system contained redox mediators. The quinone compouds could accelerate electron transmembrane reaction. Thus, the decolorization rate was enhanced.Although the whole cells incubated with quinones could significantly increase the rate of decolorization of azo dyes, the quinone compounds did not promote azoreductase activity directly. It was likely because that the reduction of azo dyes was limited by the permeation of the dyes through the cell membrane, and when the decolorization system contained redox mediators, the quinones were enzymatically reduced first, and the azo dyes were subsequently reduced in a purely chemical redox reaction. However, when the cell membrane was broken up, the crude cell extracts could reduce azo dyes directly outside the cell and it did not need quinones to transfer H ions through the cell membrane.(4) In this study, two consortia which could decolorize different azo dyes under aerobic and anaerobic conditions, respectively, were finally enriched. Both of the two consortia could decolorize different azo dyes effectively in a short time, and tolerate Methyl Red with high concentrations. Azo dye decolorization rate was significantly higher with the use of consortia compared to that with the use of individual strains. Thirteen strains were isolated from the two consortia and each isolate was identified by16S rDNA sequencing. The results revealed that all of the isolated strains were facultative anaerobic bacteria of the family Enterobacteriaceae. To provide further insight into the microbial diversity of the bacteria consortia under aerobic and anaerobic conditions, PCR-DGGE analysis was performed. PCR-DGGE profiles revealed that the microbial community changed significantly with varying initial concentrations of Methyl Red. Phylogenetic analysis indicated that microbial populations in the aerobic compartment belong to Klebsiella, Buttiauxella and Bacillus, whereas Klebsiella, Escherichia, Bacillus and Clostridium were present in the anaerobic compartment.更多还原