堆肥中新菌的分离鉴定及其铬还原特性研究

发布时间：2018-01-08 23:15

本文关键词：堆肥中新菌的分离鉴定及其铬还原特性研究　出处：《上海海洋大学》2015年硕士论文　论文类型：学位论文

【摘要】：微生物是地球上分布最为广泛的生命形式,其物种多样性非常丰富。据估计,目前人类发现的微生物物种仅占微生物物种总数的10%左右,大量的微生物物种还没有被发现。微生物广泛存在于人类的生活环境中,通过发现微生物新物种及其功能,为人类探究大自然奥秘和利用微生物造福人类提供更多的机会。铬作为工业生产中广泛应用的一种重金属,其六价离子对细胞具有很高的毒性。传统治理铬污染的方式成本高、效果差,而微生物法治理铬污染作为一种新兴的方法,克服了上述缺点,提供了新的解决办法。目前人类已经发现了一些具有铬还原功能的微生物,但是大量尚未被发现的微生物物种资源吸引着人类去进行更多更深入的研究。本研究从两个不同采样来源的堆肥土壤中分离出两个疑似新菌,运用多相分类学方法,对菌株进行了系统鉴定,并对得到的新菌GSS04T进行了铬还原特性的研究。主要结论如下:(1)从大宝山矿区堆肥中分离得到了菌株GSS04T。GSS04T能形成直径为1-2 mm的浅黄色、圆形、表面光滑、微微向上隆起的不透明菌落。GSS04T为兼性厌氧的革兰氏阳性菌,在无氧条件下生长相对缓慢。菌体细胞宽度为0.8μm,长度为2.6-3.0μm,有鞭毛,能产生椭圆的内生孢子。在LB液体培养基中,GSS04T的生长温度范围为15-50℃,最适生长温度为30-37℃;生长p H范围为5.5-10.0,最适生长p H为7.0;可以在0-3%Na Cl浓度范围内生长,在1.0%的盐浓度下生长最好。GSS04T的呼吸醌主要为甲基萘醌7(MK-7),主要脂肪酸为C15:0(42.9%)和anteiso-C15:0(24.1%)。GSS04T主要极性脂包括双磷脂酰甘油(DPG)、磷脂酰甲基乙醇胺(PME)、磷脂酰甘油(PG)、磷脂酰乙醇胺(PE)以及三种未识别出的磷脂和两种未知脂质。GSS04T的DNA G+C含量为41.1 mol%。GSS04T在进化树中的位置支持其属于芽孢杆菌属的结论。在形态特征、生理生化特征和分子生物学特征数据的基础上,GSS04T被认定为芽孢杆菌属的一个新种,命名为Bacillus dabaoshanensis。典型菌株GSS04T保藏于中国典型培养物保藏中心(保藏号为CCTCC AB 2013260T)和韩国典型培养物保藏中心(保藏号为KCTC 33191T)。(2)从广东省东莞市堆肥中分离得到了菌株GSS05T。GSS05T能形成直径为0.5-1 mm的浅黄色、圆形、表面光滑、无隆起的不透明菌落。GSS05T为革兰氏阳性菌,菌体细胞宽度为0.3-0.8μm,长度为1.1-2.6μm,具有运动性,有周生鞭毛,能产生椭圆的内生孢子。在LB液体培养基中,GSS05T生长温度为15-50℃,最适生长温度为30-37℃,该菌能够在5.5-10.0的p H条件下生长,p H=7.5为其最佳生长Ph,可以在0-3%Na Cl浓度范围内生长,在1.0%的盐浓度下生长最好。生理鉴定结果表明,GSS05T氧化酶实验呈现阳性,过氧化氢酶实验为阴性。该菌可以将酪蛋白进行水解,可以产生D-岩藻糖和糖原。GSS05T的细胞壁肽聚糖类型为A4β,呼吸醌主要类型为甲基萘醌7(MK-7),这些结果与其模式菌的主要呼吸醌相同,并且符合鸟氨酸芽孢杆菌属的特征,从另一个方面证明GSS05T属于鸟氨酸芽孢杆菌属。GSS05T的主要脂肪酸为C15:0(38.1%)和anteiso-C15:0(31.1%)。其在进化树中的位置支持将GSS05T归为鸟氨酸芽孢杆菌属的结论。通过GSS05T与其模式菌的生理生化特征进行对比,可以将GSS05T与其他同属菌株分别开来。在形态特征、生理生化特征和分子生物学特征数据的基础上,GSS05T被认定为鸟氨酸芽孢杆菌属的一个新种,命名为Ornithinibacillus composti。典型菌株GSS05T保藏在位于武汉的中国典型培养物保藏中心(保藏号为CCTCC AB 2013261T)和位于韩国的韩国典型培养物保藏中心(保藏号为KCTC 33192T)。(3)GSS04T在有氧和厌氧条件下,均具有还原六价铬离子的能力,而GSS05T没有这些特性。GSS04T的六价铬离子最小抑制浓度为600 mg l-1。GSS04T最佳铬还原条件为37℃,p H=7,初始六价铬离子浓度为100 mg l-1。在最佳铬还原条件下,GSS04T可以在120h内将100 mg l-1六价铬离子完全还原。在铬还原过程中,添加乙酸钠、蔗糖、丙酮酸、乳酸、草酸钠、葡萄糖作为电子供体,有利于GSS04T铬还原速率的提高,含氧阴离子SO42-、NO3-、PO43-的添加则对GSS04T铬还原过程没有促进或者抑制作用。GSS04T和GSS05T的发现增加了对微生物物种多样性的了解,丰富了对堆肥中微生物资源的认识。GSS04T铬还原特性的发现和研究,在理论方面为进一步探究微生物铬还原原理提供研究基础,同时在环境治理应用方面,为铬污染治理提供更多的铬还原微生物选择。
[Abstract]:Microorganisms are the most widely distributed on earth life forms, the species diversity is very rich. It is estimated that the microbial species found in human microbial species accounted for only about 10% of the total, a large number of microbial species has not been found. Microorganisms widely exists in the human living environment, through the discovery of new microbial species and its function. For human being to explore the mystery of nature and the use of microorganisms to provide more opportunities for the benefit of mankind. A kind of heavy metal chromium is widely used in industrial production, the six valence ions have high toxicity to the cells. The cost of traditional way of high chromium pollution control, poor effect, and microbial treatment of chromium pollution as a new method. To overcome these shortcomings, provides a new solution. At present people have found some with chromium to restore the function of microorganisms, but has not yet been found a large number of micro Biological species resources attracted humans to make further research. This study from two different sampling sources in compost soil isolated two new suspected bacteria, using polyphasic taxonomy methods, the strains are systematically identified, and the new strain GSS04T obtained were studied. The main conclusions of the chromium reduction characteristics are as follows: (1) the strain GSS04T.GSS04T can form 1-2 diameter mm light yellow, round, smooth surface separated from the compost Baoshan mine, gram positive bacteria slightly upward uplift of the opaque colony.GSS04T is a facultative anaerobic, relatively slow growth under anaerobic conditions. The cell width is 0.8 m. The length of 2.6-3.0 m, a flagellum, can produce oval endospores. In LB liquid medium, growth temperature range of GSS04T to 15-50 DEG C, the optimum growth temperature is 30-37 DEG C; the growth of P H is in the range of 5.5-10.0, the optimum growth p H is 7; 0-3%Na can grow in the concentration range of Cl, the best.GSS04T growth at 1% salt concentration under the main respiratory quinone menadione (MK-7), 7 major fatty acids were C15:0 (42.9%) and anteiso-C15:0 (24.1%).GSS04T polar lipids including cardiolipin phosphatidylcholine (DPG). Methyl diethanolamine (PME), phosphatidyl glycerol (PG), phosphatidylethanolamine (PE) and DNA G+C content of three kinds of unrecognized phospholipid and two unknown lipid.GSS04T was 41.1 mol%.GSS04T in the evolutionary tree in position to support its belonging to the genus Bacillus. In conclusion the morphological characteristics, physiological and biochemical basis the characteristics and characteristics of molecular biology data, GSS04T was identified as a new species of the genus bacillus, named Bacillus dabaoshanensis. GSS04T typical strains preserved in China type culture collection (CCTCC AB accession number 2013260T) and South Korea type culture collection (Center for the preservation of KCTC 33191T). (2) the strain GSS05T.GSS05T can form 0.5-1 diameter mm light yellow, round, smooth surface isolated from Guangdong city of Dongguan Province in the compost, no uplift of the opaque colony.GSS05T gram positive bacteria, the cell width is 0.3-0.8 ~ m, length of 1.1-2.6 m. With the movement, a week flagella, can produce oval endospores. In LB liquid medium, GSS05T growth temperature is 15-50 DEG C, the optimum growth temperature of 30-37 DEG C, the bacteria can grow in 5.5-10.0 P H P H=7.5 conditions, the best growth Ph, can grow in 0-3%Na the concentration range of Cl, the best growth in the 1% salt. The results showed that the physiological identification, GSS05T oxidase test positive, catalase test was negative. The bacteria can be casein hydrolysis, cell wall peptidoglycan can produce D- fucose sugar and raw.GSS05T Type A4 beta, the main types of respiratory quinone menadione (MK-7 7), these results mainly respiratory and pattern bacteria and accord with the characteristics of ornithine quinone, bacillus, from another aspect that GSS05T belongs to the main fatty acids ornithine Bacillus.GSS05T for C15:0 (38.1%) and anteiso-C15:0 (31.1% in the evolutionary tree). The position will support GSS05T belongs to the genus Bacillus. Conclusion ornithine by comparing the physiological and biochemical characteristics of GSS05T and its pattern of bacteria, can be GSS05T and other strains belong to separate. In the morphological characteristics, physiological and biochemical characteristics and molecular biological features of the data, GSS05T was identified as a new species of the genus bacillus ornithine, named as Ornithinibacillus composti. typical strains preserved in GSS05T culture collection center in Wuhan (China typical preservation number is CCTCC AB 2013261T) In South Korea and South Korea type culture collection (KCTC accession number 33192T). (3) GSS04T under aerobic and anaerobic conditions, have the ability to restore six chromium ions, but not GSS05T six chromium ion minimum inhibitory concentration of these properties of.GSS04T 600 mg l-1.GSS04T the best conditions for the reduction of chromium 37 C, P H=7, six valence chromium ion concentration of 100 mg l-1. reduction in the initial optimal chromium conditions, GSS04T in 120h 100 mg L-1 six chromium ion. In the complete reduction of chromium reduction process, adding sodium acetate, sucrose, pyruvic acid, lactic acid, sodium oxalate, glucose as electron donor to GSS04T the reduction rate of chromium increased, oxygen anion SO42-, NO3-, PO43- of GSS04T added chromium reduction process did not promote or inhibit.GSS04T and GSS05T are found to increase the diversity of microbial species in the solution enriched in the compost. The recognition of material resources, the discovery and research of Cr reduction characteristics in.GSS04T, provide theoretical basis for further exploring the principle of microbial chromium reduction in theory, and provide more chromium reduction microbial selection for environmental remediation.

【学位授予单位】：上海海洋大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：S141.4;X172

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