本文選題：結(jié)晶紫　切入點(diǎn)：生物降解　出處：《安徽建筑大學(xué)》2015年碩士論文　論文類型：學(xué)位論文

【摘要】：結(jié)晶紫是一種人工合成的三苯甲烷類染料,常用于紡織、印染、油墨工業(yè)、環(huán)保、礦業(yè)、鋼鐵、醫(yī)藥和日用化學(xué)品的分析。結(jié)晶紫含有復(fù)雜芳香環(huán)結(jié)構(gòu),因而化學(xué)穩(wěn)定性高、生物可解性低,因而在廢水中能夠積累導(dǎo)致嚴(yán)重的地表水和地下水污染。環(huán)境毒理學(xué)表明,結(jié)晶紫具有中毒、致癌、致畸和致突變等毒副作用。由結(jié)晶紫造成的水源污染和水產(chǎn)品的安全問題已引起了廣泛重視并急需解決。生物降解是清除環(huán)境污染的有效途徑之一,具有高效、安全、成本低、無二次污染等優(yōu)點(diǎn)。因此篩選高效降解結(jié)晶紫的微生物資源,探索其降解的作用機(jī)理,充分發(fā)揮其降解能力,具有重要的理論意義和應(yīng)用價(jià)值。本課題研究的目標(biāo)是分離出能夠高效降解結(jié)晶紫的降解菌株,并對(duì)該菌株在各種環(huán)境條件下降解結(jié)晶紫的特性進(jìn)行研究,同時(shí)探索菌株降解結(jié)晶紫的機(jī)制,以期為結(jié)晶紫染料廢水的處理工作提供科學(xué)依據(jù)。從長期受染料污染的污泥中分離出一株能夠?qū)Y(jié)晶紫降解脫色的菌株SJ-1。根據(jù)形態(tài)、生理生化和16S r RNA基因序列分析,將菌株SJ-1歸屬到鞘氨醇單胞菌屬(Sphingomonas sp.)。本文探索了降解菌株SJ-1的最適生長條件,菌株SJ-1生長最適溫度為30℃,最適生長初始p H為7.0。隨著通氣量的增加,菌株的生長呈上升趨勢(shì)。在供試的幾種碳氮源中,對(duì)蛋白胨和酵母粉的利用為最好。在LB培養(yǎng)基中,SJ-1的生長曲線,0-6 h為延滯期,6-24 h為對(duì)數(shù)生長期,24 h后為穩(wěn)定期。對(duì)降解菌株降解特性的研究表明,使用LB培養(yǎng)基,菌株SJ-1在3 d可使200mg/L結(jié)晶紫幾乎完全脫色。該菌株降解結(jié)晶紫的適宜溫度為25~30℃,適宜p H值為6.0~8.0;通氣量對(duì)SJ-1影響較小,在缺氧條件下也可較好地降解結(jié)晶紫,4 h時(shí)通氣量較小試驗(yàn)組(裝液量≥125 m L)降解率仍大于40%。在對(duì)SJ-1結(jié)晶紫降解酶的酶學(xué)特性的研究中,建立了酶學(xué)反應(yīng)體系:總體積5 m L,PBS 4.85 m L(0.1 mol/L,p H=7.0),結(jié)晶紫50μL(終質(zhì)量濃度200 mg/L),酶液100μL。反應(yīng)10 min后冰浴終止,用丙酮提取結(jié)晶紫殘留,根據(jù)結(jié)晶紫減少量確定酶活。本文中,一個(gè)酶活單位是指1 min內(nèi)去除1μmol結(jié)晶紫所需的酶量。實(shí)驗(yàn)表明:該結(jié)晶紫降解酶在60℃以下酶活損失較慢,p H6.0-8.0時(shí)能保持較高的酶活;甲醇和異戊醇對(duì)該酶活性影響較小,而二氯甲烷和丙酮對(duì)該酶活性影響較大。Zn2+、Ca2+、Fe2+、Cu2+、Co2+等10種金屬離子中,Fe2+對(duì)結(jié)晶紫降解有促進(jìn)作用,其他金屬離子對(duì)結(jié)晶紫降解酶都有不同程度的抑制作用。酶的誘導(dǎo)和定域?qū)嶒?yàn)表明,SJ-1的結(jié)晶紫降解酶位于細(xì)胞外,屬組成型表達(dá)酶。根據(jù)已報(bào)道的三苯甲烷還原酶基因序列設(shè)計(jì)引物,通過PCR方法從降解菌SJ-1中克隆到了三苯甲烷還原酶,其堿基數(shù)為864 bp,編碼288個(gè)氨基酸序列。通過SJ-1中tmr2基因序列與Gene Bank中同源序列的比較發(fā)現(xiàn),在基因水平上,八株降解菌的三苯甲烷還原酶基因有18個(gè)點(diǎn)堿基有差異;相互之間的同源性為98%~100%;在氨基酸水平上,八株降解菌的三苯甲烷還原酶有10個(gè)位點(diǎn)有差異,相互之間的同源性為98%~99%,根據(jù)基因和氨基酸的差異可以將八株菌分為A、B、C三種亞型。菌株SJ-1降解結(jié)晶紫存在三種降解產(chǎn)物:隱性結(jié)晶紫、隱性孔雀石綠和苯酚。菌株SJ-1降解結(jié)晶紫的途徑可能為:第一步可能是結(jié)晶紫還原代謝成脂溶性的隱性結(jié)晶紫,第二步隱性結(jié)晶紫通過脫氨基作用生成隱性孔雀石綠,最后氧化裂解和脫甲基生成苯酚。
[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涓哄歡婊炴湡,6-24 h涓哄鏁扮敓闀挎湡,24 h鍚庝負(fù)紼沖畾鏈，

本文編號(hào)：1611369