【作者】 曾晓希；

【导师】 刘学端； 汤建新；

【作者基本信息】 中南大学 ， 生物工程， 2010， 博士

【摘要】 随着重金属污染日趋严重,污染治理已成备受关注的焦点。生物修复是颇具潜力的治理方法,应用从污染区分离筛选的本土微生物能有效提高生物修复的效率。因此,研究本土抗重金属微生物对修复重金属污染有重要意义。本研究首先通过驯化、筛选和分离,从株洲冶炼厂的土壤中获得一批抗镉微生物。其中抗性最高的细菌和真菌分别是E1和M1,E1能在18 mmol/L的固体培养基和10 mmol/L的液体培养基中生长,M1能80 mmol/L的固体培养基和40 mmol/L的液体培养基中生长。抗镉菌株E1是革兰氏阴性两端钝圆的短杆菌,有端生鞭毛,大小约0.5-1.0μm×2.0-3.0μm。在无镉培养基中产生黄绿或蓝绿色色素,在镉浓度高于1 mmol/L的培养基中一般不产生色素。菌株E1的生理生化特性与假单胞菌很相似。16S rDNA序列同源性分析表明菌株E1与GenBank中的铜绿假单胞菌(Pesudomonas aeruginosa)的同源性最高达98%。可将E1初步鉴定为铜绿假单胞菌。菌株M1菌丝体呈白色或淡紫色,扫描电镜分析表明气生菌丝有隔和分枝,分生孢子细长,长在气生菌丝上,孢子呈椭圆形。通过18S rDNA、ITS和β-微管蛋白基因序列的分析,表明该菌与淡紫拟青霉菌(Paecilomyces lilacinus)同源性达99%以上,将M1初步鉴定为淡紫拟青霉菌。对抗镉菌株E1和M1的抗性和生长条件进行了研究,结果表明,它们不仅具有抗镉的能力,而且还对其它重金属Zn、Mn、Gu、Pb和Co以及多种抗生素具有抗性。菌株E1和M1的适宜生长和抗镉(2mmol/L)条件为温度20-35℃,pH6-8,装液量80-100 mL (250 mL摇瓶)。但是E1的生长和抗镉最适pH值为7,而M1的为6,它们的最适温度是30℃。50mg/L的Zn和Mn能促进或不因影响菌株的生长,而50 mg／L而Pb和Co对两个菌株的生长和抗镉都有抑制作用。其次,利用分子生物学技术初步研究了P.aeruginosa E1的抗镉机理。采用煮沸法、SDS法和试剂盒提取P.aeruginosa E1的质粒,没有得到结果；采用SDS、吖啶橙和苯甲酸钠培养消除质粒后测试菌株的抗镉性能,结果发现质粒消除前后的抗镉性能没有明显差异。表明该菌株内没有质粒,抗性基因存在染色体上。利用RT-PCR技术,对P.aeruginosa E1中相关的基因在镉胁迫下的表达差异进行了研究,以了解菌株的抗镉机理。在镉胁迫1h后czcA, czcB和czcC上调表达量达到最大,然后回落到一个相对稳定的范围。czcD却在加入镉离子10min后表达量上调到最高值,达15.67倍,然后在1-4h内保持相对稳定的范围。czcD的表达量一般高于其他三个基因,在抗镉中表现出很重要的作用。在镉胁迫下,基因cysM的表达较cysK强,主要表现为上调表达,而cysK的表达差异较小,表明镉胁迫下由cysM编码的O-乙酰丝氨酸裂解酶B在半胱氨酸的合成中起主要作用。mgt体系对镉离子的转运调控反应比较快,主要在镉胁迫的前期起作用；znu转运体系的表达上调比较缓和而持久。初步认为镉胁迫下菌株E1是通过外排镉离子,增加半胱氨酸的合成来适应环境的,znu和mgt体系“轮流”负责镉离子的内流转运。同时,对抗镉P.aeruginosa E1和P.lilacinus M1的镉吸附作用进行了初步探讨。表明抗镉菌株E1的活细菌和灭活细菌都对水环境中的镉有明显去除作用,吸附24h后,活细菌的去镉效果比灭活细菌好,吸附率分别为43.3%和22.6%。对M1在100mg/L镉离子培养基生长过程中的吸附作用进行了研究。在生长对数期,菌株对镉离子的吸附率从8.09%增加到68.8%,该菌株在培养期3-4天的时候吸附效率最高。利用生物陶粒固定微生物进行了镉吸附试验, E1和M1最大吸附率可达42.36%和64.9%,摇瓶和反应器吸附试验均表明固定菌体后的生物陶粒能有效的吸附溶液中的镉离子；红外光谱分析表明E1对镉离子的吸附主要与C-O、C=O、C-H、O-H、C-N、N-H以及磷酸功能基团有关,M1主要与C=O、C-H、N-H等有关；能谱分析证实固定E1和M1的陶粒可以较好的吸附溶液中的镉；M1菌丝体对镉的吸附可能是通过分泌大量胞外多糖类物质来实现的。此外,从污染土壤中还分离到一株抗汞菌株D2,通过分析它的16SrDNA序列,将其鉴定为P. aeruginosa。菌株D2能生长在60 mg/L Hg2+的固体培养基中,但在60mg/L Hg2+的液体培养基中不能繁殖。对菌株D2的抗性进行了研究,结果表明它对其他的重金属Cu、Co、Mn、Zn、Cd和Pb有抗性,对抗生素也具有一定抗性。质粒分析表明该菌株内可能没有质粒,抗性基因存在染色体上。通过PCR获得基因merART序列,它们都与R. Metallidurans CH34菌株的质粒和转座子Tn501等的相应基因有很高的相似性,可能是汞的长期胁迫下,抗汞基因发生横向转移的结果。更多还原

【Abstract】 Heavy metal pollution is increasing with metals exploitation and application. Bioremediation plays a role in pollution treatment. Using the microbe isolated from indigenous soils can improve the efficiency. So the study of the indigenous heavy-metal-resistant strains is significant for bioremediation.Some strains resisted to high concentration of cadmium were isolated by acclimation of concentration gradient from the polluted soils of the smeltery in Hunan province, China. Strains marked E1 and M1 with higher resistance were selected in the following experiments. Strain E1 could survive in 18 mmol/L Cd2+ solid medium and 10 mmol/L liquid medium. M1 could survive in 80 mmol/L Cd2+solid medium and 40 mmol/L liquid medium. E1 presents Gram-negative, rod-shaped with polar flagellum, and the size is (0.5-1.0)μm×(2.0-3.0)μm. It produced green or blue pigment, and the pigment disappeared in the absence of cadmium. Based on the characterizations of the physiological and biochemical characteristics 16S rDNA sequence analysis, strain E1 was identified as Pseudomonas aeruginosa.On Czapek and PDA solid medium, strain M1 appeared white density mycelia and the bottom of plates showed yellow at the first 3-5 day. But after then the mycelia gradually became yellow or brown. Scanning electron microscope observation showed, the aerial mycelia had septate and branches, slender conidiophores grew out from aerial mycelia and conidia present oval. The strain M1 was identified as Paecilomyces lilacinus based on the morphologic characteristics and the sequences analysis of 18s rRNA, ITS region andβ-tubulin.The strains could resist not only to cadmium but also to other metals including Cu, Co, Mn, Pb and Zn. P.aeruginosa E1 and P.lilacinus M1 have broad-spectrum resistance to heavy metals and antibiotics. The growth conditions of two strains were similar. The suitable of conditions were 20-35℃, pH 6-8,80-100 mL medium in 250 mL shaker. But the optimum initial pH in medium was 7 and 6 for E1 and M1.50 mg/L Zn and Mn had some help to the growth. While 50 mg/L Pb and Co would restrain the growths of two strains in cadmium and no cadmium mediums.The plasmid did not be extracted from P.aeruginosa E1 by the methods of boiling, SDS and plasmid kit. SDS, acridine and sodium benzoate were singlely added into medium to eliminate plasmid. The resistance ability to cadmium of the cultures performed plasmid eliminations were tested and did not present obviously difference. It was demonstrated that the strain has not plasmid and the resistant gene maybe locate on chromosome. The response of strain E1 to cadmium ion was investigated based on the genes expression profiling examined by using Real-time PCR. Exposure to cadmium for 1 h, the gene czcA,czcB and czcC expression reached the highest level, and then declined to a stable range. czcD gene expression was up-regulated 15.67-fold after exposure to cadmium for 10 min. With the fast and high up-regulation expression, czcD gene was thought as more important in the response of strain E1 to cadmium stress than czcABC. Exposure to cadmium cysM gene expression was present more up-regulation than cysK. It showed that o-acetylserine sulphydrylase-B coded by the cysM played a role in cysteine biosynthesis to resist cadmium. The response of mgt gene to cadmium press was fast and the gene expression up regulated mainly in earlier stage, while the up-regulation of znu was gently and lasting.Both living and non-living cells of P.aeruginosa E1 can remove Cd from solution. After being treated for 24 h, the cadmium removal rates of the living cell and non-living cell reached 43.3% and 22.6%, respectively. It suggested that the biosorption ability of the living cell was better than the non-living cell. During the log phase, the cadmium removal by P.lilacinus M1 displayed a rising from 8.09%to 68.8%and reached a plateau phase on day 4. The biosorption amount also presented a increasing trend, from 17.98 mg/g on day 1 to 24.23 mg/g on day 3, and subsequently following a minimal decreasing. The result showed that the better cadmium adsorption by the fungus occurred on after culturing for 3-4 day.P.aeruginosa E1 and P.lilacinus M1 were single immobilized on bio-ceramic to adsorb cadmium ion from solution. The biosorptions both of batch and reactor confirmed that immobilized microbes could remove cadmium. The removal rates were 42.36% and 64.9% for P.aeruginosa E1 and M1. The EDX analysis confirmed the above result. Moreover,extracellular polysaccharide was presumed as the main role in M1 biosorption based on EDX analysis. FTIR spectrum suggested that biosorption of cadmium ion by biomass of strain E1 mainly related to C=O, C-O, C-H, O-H, C-N, N-H groups and phosphate functional groups, M1 related to C=O、C-H、N-H. The biosorption reactions occurred between cadmium ion and the above groups of biomass.Strain D2 resistance to high concentration mercury ion was isolated. Based on analysis of 16S rDNA the strain was identified as P. aeruginosa. It can grow in the soil medium containing 60 mg/L Hg2+. But when the concentration of mercuric ion in liquid medium reaches 60 mg/L, the strain hardly grows. Besides mercury, strain D2 can tolerant to other heavy metals Cu, Co, Mn, Zn, Cd, and Pb. Antibiotics disc tests demonstrated it could resistant to the 12 antibiotics. The resistance to mercuric ion of strain D2 was related to chromosome. The merRT and merA gene sequences were obtained. Sequence alignment demonstrated that the mer gene was high similar to R. Metallidurans CH34, Tn501 and so on.更多还原