结晶紫降解菌株的分离鉴定、降解特性及降解机制研究

发布时间：2018-03-14 13:15

  本文选题：结晶紫　切入点：生物降解　出处：《安徽建筑大学》2015年硕士论文　论文类型：学位论文

【摘要】：结晶紫是一种人工合成的三苯甲烷类染料,常用于纺织、印染、油墨工业、环保、矿业、钢铁、医药和日用化学品的分析。结晶紫含有复杂芳香环结构,因而化学稳定性高、生物可解性低,因而在废水中能够积累导致严重的地表水和地下水污染。环境毒理学表明,结晶紫具有中毒、致癌、致畸和致突变等毒副作用。由结晶紫造成的水源污染和水产品的安全问题已引起了广泛重视并急需解决。生物降解是清除环境污染的有效途径之一,具有高效、安全、成本低、无二次污染等优点。因此筛选高效降解结晶紫的微生物资源,探索其降解的作用机理,充分发挥其降解能力,具有重要的理论意义和应用价值。本课题研究的目标是分离出能够高效降解结晶紫的降解菌株,并对该菌株在各种环境条件下降解结晶紫的特性进行研究,同时探索菌株降解结晶紫的机制,以期为结晶紫染料废水的处理工作提供科学依据。从长期受染料污染的污泥中分离出一株能够对结晶紫降解脱色的菌株SJ-1。根据形态、生理生化和16S r RNA基因序列分析,将菌株SJ-1归属到鞘氨醇单胞菌属(Sphingomonas sp.)。本文探索了降解菌株SJ-1的最适生长条件,菌株SJ-1生长最适温度为30℃,最适生长初始p H为7.0。随着通气量的增加,菌株的生长呈上升趋势。在供试的几种碳氮源中,对蛋白胨和酵母粉的利用为最好。在LB培养基中,SJ-1的生长曲线,0-6 h为延滞期,6-24 h为对数生长期,24 h后为稳定期。对降解菌株降解特性的研究表明,使用LB培养基,菌株SJ-1在3 d可使200mg/L结晶紫几乎完全脱色。该菌株降解结晶紫的适宜温度为25~30℃,适宜p H值为6.0~8.0;通气量对SJ-1影响较小,在缺氧条件下也可较好地降解结晶紫,4 h时通气量较小试验组(装液量≥125 m L)降解率仍大于40%。在对SJ-1结晶紫降解酶的酶学特性的研究中,建立了酶学反应体系:总体积5 m L,PBS 4.85 m L(0.1 mol/L,p H=7.0),结晶紫50μL(终质量浓度200 mg/L),酶液100μL。反应10 min后冰浴终止,用丙酮提取结晶紫残留,根据结晶紫减少量确定酶活。本文中,一个酶活单位是指1 min内去除1μmol结晶紫所需的酶量。实验表明:该结晶紫降解酶在60℃以下酶活损失较慢,p H6.0-8.0时能保持较高的酶活;甲醇和异戊醇对该酶活性影响较小,而二氯甲烷和丙酮对该酶活性影响较大。Zn2+、Ca2+、Fe2+、Cu2+、Co2+等10种金属离子中,Fe2+对结晶紫降解有促进作用,其他金属离子对结晶紫降解酶都有不同程度的抑制作用。酶的诱导和定域实验表明,SJ-1的结晶紫降解酶位于细胞外,属组成型表达酶。根据已报道的三苯甲烷还原酶基因序列设计引物,通过PCR方法从降解菌SJ-1中克隆到了三苯甲烷还原酶,其碱基数为864 bp,编码288个氨基酸序列。通过SJ-1中tmr2基因序列与Gene Bank中同源序列的比较发现,在基因水平上,八株降解菌的三苯甲烷还原酶基因有18个点碱基有差异;相互之间的同源性为98%~100%;在氨基酸水平上,八株降解菌的三苯甲烷还原酶有10个位点有差异,相互之间的同源性为98%~99%,根据基因和氨基酸的差异可以将八株菌分为A、B、C三种亚型。菌株SJ-1降解结晶紫存在三种降解产物:隐性结晶紫、隐性孔雀石绿和苯酚。菌株SJ-1降解结晶紫的途径可能为:第一步可能是结晶紫还原代谢成脂溶性的隐性结晶紫,第二步隐性结晶紫通过脱氨基作用生成隐性孔雀石绿,最后氧化裂解和脱甲基生成苯酚。
[Abstract]:Crystal violet is a kind of artificial synthesis of triphenylmethane dyes used in textile, printing and dyeing, ink industry, environmental protection, mining, steel, chemicals and pharmaceutical analysis. Crystal violet complex containing aromatic ring structure and high chemical stability, biological plausibility is low, so the wastewater can cause serious surface water accumulation and the groundwater pollution. Environmental toxicology showed that crystal violet with toxic, carcinogenic, teratogenic and mutagenic toxicity caused by crystal violet. Water pollution and water product safety issues have attracted wide attention and need to be solved. Biodegradation is one of the effective ways to eliminate environmental pollution has the advantages of high efficiency, safety, cost the advantages of low, no two pollution. Therefore, screening of microbial resources degradation of crystal violet, to explore the mechanism of degradation, give full play to its degradation ability, and has important theoretical significance and application value. The goal of this research is to isolate efficient degradation of crystal violet degradation strains, and to study the strain in various environmental conditions degradation of crystal violet characteristics, and to explore the mechanism of degradation of crystal violet, in order to provide scientific basis for treatment for crystal violet dye wastewater. Isolated strains capable of crystallization UV degradation strain SJ-1. based on morphological from long-term dye contaminated sludge, analysis of physiological and biochemical and 16S r sequence of RNA gene of strain SJ-1 belonging to the genus Sphingomonas (Sphingomonas sp.). This paper explores the optimum growth conditions of degrading strain SJ-1, strain SJ-1, the optimum growth temperature of 30 DEG C, the optimum growth initial p H 7.0. with the increase of gas flux, the growth of bacteria increased. In several carbon and nitrogen sources tested, utilization of peptone and yeast is the best. In the LB medium, SJ-1 鐢熼暱鏇茬嚎,0-6 h涓哄欢婊炴湡,6-24 h涓哄�规暟鐢熼暱鏈�,24 h鍚庝负绋冲畾鏈，

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