【作者】 杨丹丹；

【导师】 陈敏；

【作者基本信息】 杭州师范大学 ， 遗传学， 2012， 硕士

【摘要】 从岱山盐场采集土样和水样,利用选择性培养基分离可培养嗜盐细菌和嗜盐古菌,探讨盐田环境可培养嗜盐菌的多样性及产酶特性。共获得了181株嗜盐菌菌株,采用真细菌和古生菌两对引物扩增其16S rRNA基因序列,并用限制性内切酶Hinf I进行ARDRA (Amplified rDNA Restriction Analysis)多态性分析,可分为21个不同的操作分类单元(Operational Taxonomic Unit, OTU),其中嗜盐细菌有12个OTUs,嗜盐古菌有9个OTUs,选取具有不同酶切图谱的代表菌株进行克隆测序,Blast比对及系统发育分析将嗜盐细菌归于7个不同的属,分别为嗜盐单胞菌属(Halomonas),假单胞菌属(Pseudomonas),海杆菌属(Marinobacte),产微球茎菌属(Microbulbifer), Idiomarina属,喜盐芽孢杆菌属(Halobacillus)釉Piscibacillus属；其中嗜盐单胞菌属的菌株数占优势,是嗜盐细菌总数的46.77%；嗜盐古菌归于4个不同的属,包括盐盒菌属(Haloarcula),盐红菌属(Halorubrum),盐富饶菌属(Haloferax)和盐陆生菌属(Haloterrigena),其中盐盒菌属的菌株数占优势,是嗜盐古菌总数的49.12%。研究结果表明岱山盐场具有较为丰富的嗜盐菌多样性。对分离到的181株嗜盐菌进行产功能酶活性筛选,共得到62株产酶菌株,其中39株产淀粉酶,20株产蛋白酶,39株产脂肪酶。有3株菌株可以同时产淀粉酶、蛋白酶和脂肪酶,18株菌株同时产淀粉酶和脂肪酶,7株菌株同时产淀粉酶和蛋白酶,5株菌株同时产脂肪酶与蛋白酶。盐单胞菌属产酶菌株数最多,其次是喜盐芽孢杆菌属和盐盒菌属。对产酶菌株的酶活性分析结果表明,岱山盐田蕴含丰富的产功能酶嗜盐菌资源。选取了一株具较高产酶活性的极端嗜盐菌DSFD111进行菌种鉴定。培养1周后菌落呈红色,圆形,边缘整齐,上表面光滑有突起,直径3mm左右。显微镜下菌体细胞为球状,革兰氏染色阴性,不产芽胞,含有类脂粒。该菌株生长NaCl耐受范围为18%～35%,最适生长NaCl浓度为25%；pH耐受范围为6～8,最适为7；生长温度范围为25℃～50℃,最适生长温度为37℃。结合形态学特征分析、生理生化试验及其16S rRNA基因测序,鉴定为Haloarcula属的成员,将其命名为Haloarcula sp. DSFD111.对Haloarcula sp. DSFD111所产淀粉酶的酶学性质进行研究,结果表明,该酶在20℃～60℃范围内均有催化活性,其中37℃时活性最强；在pH6～8的范围内具有催化活性,pH为7时活性最强。在37℃～50℃以及pH6～8范围内酶稳定性良好。对该菌株的产酶条件进行了初步研究,菌株的酶活力大小与其生长基本呈正相关。单因素实验表明,摇瓶发酵产生淀粉酶的最佳条件为：25%NaCl,1%可溶性淀粉(碳源),1%细菌蛋白胨(氮源),pH7.0,37℃振荡培养60～90h。本研究结果表明,岱山盐田环境中不仅具有较为丰富的嗜盐菌多样性,同时是筛选产酶菌株的重要资源库,为今后进一步开发利用盐田环境中的嗜盐菌资源提供了理论依据。更多还原

【Abstract】 This study was aimed at investigating the diversity of culturable halophiles in Daishan Saltworks, a hypersaline environment, and discovering new strains with enzyme-producing activities. A total of181strains were isolated from soil and saline water collected from the Daishan Saltworks by culture-dependent method based on selective media. Specific primers were used to amplify16S rRNA gene of bacteria and archaea. The operation taxonomy units (OTUs) were determined by ARDRA method and the representative strain of every OTU was sequenced. The phylogenetic position of all the isolated strains was determined by16S rRNA sequencing. The results showed the isolated181strains displayed21operational taxonomic units (OTUs), of which12OTUs belonged to halophilic bacteria and the others were haloarchaea. Phylogenetic analysis indicated that there were7genera presented among the halophilic bacteria group, include Halomonas, Pseudomonas, Marinobacte, Microbulbifer, Idiomarina, Halobacillus and Piscibacillus, the dominant halophilic strains were Halomonas, which accounted for46.77%in halophilic bacteria. There were4genera among the haloarchaea group, include Haloarcula, Halorubrum, Haloferax, Haloterrigena, the dominant halophilic strains were Haloarcula, which accounted for49.12%in haloarchaea respectively. All the results indicated an abundant diversity of halophiles in Daishan Saltworks.Amylase, protease, lipase and cellulase from181strains were screened. The result showed that39strains have amylase activity,20strains have protease activity, and39strains have lipase activity. Additionally, there are3strains can produce three enzymes, besides18strains produce amylase and lipase,7strains produce amylase and protease,5strains produce lipase and protease. Most of enzymes producing strains belong to Halomonas, then Haloferax and Haloarcula. It was indicated that there are abundant resource of halophilic microorganism and enzyme in Daishan Saltworks.On the basis of the hamology and phylogenetic analysis of16S rDNA, it was considered that DSFS111which can produce amylase and lipase was related to the genera Haloarcula. Colonies on agar plates containing25%(w/v) total salts were red, smooth-faced, elevated and round, diameter3mm after a week. This microbe could grow in medium containing18-35%NaCl, optimal salinity at25%, the tolerance temperature between25℃to50℃, and optimal temperature at37℃, pH at6.0-8.0while optimally at7. Cells of the strain are round and Gram-negative, no spores and containing lipid particles.Enzyme characteristics of DSFD111were investigated in this study. The results shown that the amylase activity was in20℃to60℃range, with the highest activity at37℃, and in pH6to8range, with the strongest activity pH in7. The amylase appears good thermal stability between37℃to50℃and pH stability between pH6-8.Moreover, the condition of producing enzyme was detected, the result shown that the activity of amylase was positive correlated with the growing status. Single factor experiment proved that the optimum fermenting condition of Haloarcula sp. DSFD111producting amylase is:25%concentration of NaCl,1%soluble starch as carbon source,1%bacterial peptone as nitrogen source, pH7.0,37℃shaking culture for60-90h. This provided a favorable scientific basis in the the Soltworks environment to seek high activity amylase strains.It was indicated that there are abundant resource of halophilic microorganism and enzyme in Daishan Solar Saltern.更多还原