孔雀石绿降解菌株的分离鉴定、降解特性及其降解机制研究

发布时间：2018-03-11 22:02

  本文选题：孔雀石绿　切入点：生物降解　出处：《安徽建筑大学》2017年硕士论文　论文类型：学位论文

【摘要】：三苯甲烷类染料是继偶氮、蒽酮类染料之后,世界上使用最为广泛的第三大类染料。广泛应用于造纸业、纺织业、医药业、水产养殖业等。孔雀石绿化学官能团三苯甲基化学稳定性高,生物可降解性低,具有高毒、高残留、致癌和致突变等毒副作用,其排放到环境中对人类和其他生物的健康构成很大的威胁。生物法由于其经济、环保、操作简单等优点被认为是降解此类废水最为有效的方法,目前已分离筛选出了包括细菌、真菌、酵母菌等多种降解菌。但寻找能够更为高效、快速降解孔雀石绿菌种,研究它们的生长降解特性仍具有重要的意义。本课题从某印染厂下水道污泥中分离出一株能高效降解孔雀石绿的菌株,并对该菌株的生理生化、生长降解特性、酶学特征以及其降解途径等方面进行研究,以期为三苯甲烷类废水的处理提供了菌株资源和理论依据。采用富集培养的方式,从长时间受染料污染的污泥中分离出一株细菌菌株,能够较好对孔雀石绿染料进行降解脱色,并将其命名为KL-1。根据对菌株的形态及其生理生化特征作研究分析,将菌株KL-1归属到假单胞菌属(Pseudomonas sp.)。本文研究了菌株KL-1的最适生长条件,最适生长温度为30℃;最适生长p H为7.0;随着装液量的增加,菌株生长呈下降趋势;该菌株可利用碳源较少,在供试碳源中,仅在添加了葡萄糖的的无机盐培养基中生长较好,在以蛋白胨和酵母粉作为碳源的培养基中生长状况最好;在供试在氮源中,菌株在无机氮源中均能较好生长,有机氮源蛋白胨和酵母粉较无机氮源对菌株生长的支持作用更为明显;KL-1生长曲线,0-2h迟滞期,3-6h对数生长期,6h后进入稳定期。在菌株KL-1最适降解条件实验中,研究表明,在LB培养基中,菌株KL-1在6h内,可以使得100mg/L的孔雀石绿几乎完全被降解脱色;降解最适温度为30℃;菌株在p H7.0~14.0范围均能很好的降解孔雀石绿,降解脱色率均能达到72%以上;装液量对KL-1降解孔雀石绿影响较大,脱色率随着装液量的增加而减少。孔雀石绿的脱色率随孔雀石绿的初始浓度的升高而降低,当初始质量浓度≤200mg/L,4 h时,孔雀石绿脱色率达到80%以上;降解谱实验显示,菌株KL-1有较为广泛的降解谱,5d内使LB中20mg/L的偶氮类染料甲基红,三苯甲烷类染料结晶紫、维多利亚蓝的降解率达到90%以上;溴百里香酚蓝、甲基橙、碱性品红、苏丹Ⅲ的降解率也达到56%以上。酶学特性实验研究表明:建立酶活反应体系,结果表明菌株KL-1孔雀石绿降解酶主要位于细胞外,且属于组成型表达酶;最适酶促反应温度为30℃;最适反应p H为7.0;金属离子Fe~(2+)和Mg~(2+),对该降解酶有少许促进作用,其相对酶活力分别提高了5.26%和3.17%。Ag+、Hg~(2+)、Fe3+、Ca~(2+)、Zn~(2+)、Mn~(2+)、Cu~(2+)金属离子均对该降解酶有抑制作用;有机溶剂均对降解酶有不同程度的抑制作用,其中该酶在甲醇和乙醇中相对较为稳定,相对酶活力仅减少了10%左右;有机溶剂二氯甲烷、三氯甲烷和丙酮对其抑制均较大,其相对酶活力均减少了50%以上。降解途径实验表明,该菌株在降解孔雀石绿的过程中,可能的降解产物有两种:隐性孔雀石绿和苯酚。KL-1降解孔雀石绿的可能途径为:第一步可能被还原代谢成为脂溶性的隐性孔雀石绿,经过进一步的降解,最终氧化裂解和脱甲基生成苯酚。
[Abstract]:Triphenylmethane dye is the azo dyes, after authrone, the world's most widely used third kinds of dyes. Widely used in papermaking industry, textile industry, pharmaceutical industry, aquaculture. The government can group three malachite green methyl benzene high chemical stability, low biodegradability, high toxic, high residue, carcinogenic mutagenesis and other side effects, its emissions to pose a great threat to human health and other biological environment. Biological method because of its economic and environmental protection, simple operation and is considered to be the most effective method for the degradation of this kind of wastewater, has been isolated including bacteria, fungi, yeast, etc. but the search for bacteria. It is more efficient, rapid degradation of Malachite Green strain, still has an important significance to study the growth characteristics of degradation of them. From the sewer sludge in a dyeing plant isolated high drop The solution of malachite green strains, the strain on the physiological and biochemical characteristics, growth and degradation, on Enzymatic Characteristics and the degradation pathway, provide strain resources and theoretical basis in order to triphenylmethane wastewater. By enrichment culture, isolation of a bacterium by dye pollution from long time in the sludge, can be used for decolorization and degradation of malachite green dye, and named it KL-1. analysis based on morphological and physiological and biochemical characteristics of the strain, the strain KL-1 belong to Pseudomonas sp. (Pseudomonas sp.). The optimum growth conditions of strain KL-1 were studied in this paper. The optimum temperature is 30 C; the optimum growth of P H was 7; with the increase of liquid volume, the growth of strains decreased; the strain can use less carbon, in the tested carbon sources, glucose was added only in the inorganic salt medium students Long good, in order to peptone and yeast powder as the carbon source in the medium in the best condition of growth; in the tested nitrogen, strains could grow better in inorganic nitrogen, organic nitrogen source peptone and yeast than inorganic nitrogen sources to support the growth of strains was more obvious; the growth of KL-1 0-2h curve, lag phase, logarithmic growth phase of 3-6h, 6h into the stable. Studies show that under the optimum conditions of strain KL-1 degradation experiment, and in the LB medium, KL-1 strain in 6h, can make the malachite green 100mg/L was almost completely degradation; the degradation of the optimum temperature is 30 DEG C; strain P H7.0~14.0 was a good degradation of malachite green, the decolorization rate could reach more than 72%; a large amount of liquid on KL-1 degradation of malachite green, the decolorization rate decreases with the increase of liquid volume. The rate of decolorization of malachite green with Malachite Green initial concentration increased and decreased 浣，

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