【作者】 王晓颖；

【导师】 郑天凌；

【作者基本信息】 厦门大学 ， 微生物学， 2007， 硕士

【摘要】 本论文针对当前水产养殖现行使用的微生物制剂作用时效短,在使用过程中优势地位不明显等缺陷,基于微生态理论,在对虾蟹混养池天然生态系主要细菌类群调查的基础上,以胞外酶为指标分离筛选能高效降解水产养殖残余饵料蛋白、淀粉作用的功能菌,同时结合PCR-DGGE等分子生态技术,对虾池原位环境中优势功能菌群进行研究。研究取得以下主要结果:1.利用平板计数方法对养殖环境中功能微生物进行了调查,结果表明,在整个养殖期内,新旧养殖池水体和底质中可培养异养菌数均呈现较大的波动幅度,其它功能菌与异养菌相似,在整个养殖周期也呈现较大的波动性。新池水体的功能菌数量要高于旧池,而旧池表层沉积物的功能菌数量要高于新池,一般底泥环境的微生物数量要比水体环境数量高1-2个数量级。统计分析表明,养殖环境中的淀粉降解菌菌数、有机磷降解菌菌数、油脂降解菌菌数、弧菌数等与可培养异养细菌菌数之间呈现着明显的正相关,而几丁质降解菌菌数、无机磷溶解菌菌数、纤维素降解菌菌数以及硫氧化细菌菌数与可培养异养菌菌数之间则无明显的相关关系。2.利用选择性培养基从养殖环境中分离并筛选出6株具有较高淀粉酶和蛋白酶活性的菌株,对其发酵条件以及粗酶液性质进行了初步的研究。其中菌株SL-9、S-32与OP-8具有较高的淀粉酶活性,最适作用温度均为50℃,最适作用pH值分别为9.5,8.0,9.0;而菌株NOPL-2、SL-1以及O-19具有较高的蛋白酶活性,最适作用温度分别为60℃,50℃,40℃,最适作用pH值分别为8.5,7.0,8.5,几株菌均在碱性条件下具有较高的胞外酶活性。菌株OP-8和NOPL-2所产生的酶蛋白对热不敏感,另外四株菌所产生的酶蛋白对热敏感。3.克隆6株菌的16S rRNA基因并测序,系统发育分析6株细菌分属于芽孢杆菌、沙雷氏菌属、节杆菌属以及假单胞菌属。4.菌株SL-9、SL-1以及OP-19还具有较高的脂肪酶活性;NOPL-2还具有淀粉酶和磷酸酯酶活性;OP-8和S-32还具有蛋白酶活性等多种其它的胞外酶活性,以上结果表明这些功能菌株并不只是分泌单一的胞外酶,它们具有多种复合酶系,可为进一步筛选优良的复合酶微生态制剂候选菌株奠定基础。5.提取沉积物样品和功能菌的总DNA,利用通用引物对其V3可变区的16Sr RNA基因进行PCR扩增,将扩增所得的长约200 bp的PCR产物进行梯度凝胶电泳(DGGE),最后对两者的电泳图谱进行比较,将迁移至相同位置的条带切胶回收测序,结果显示纯菌的序列与原位环境总DNA的序列的系统发育学地位相距较远,由此推测筛选获得的功能菌在原位环境中可能并未占据优势地位,可能是它们更适合于所提供的培养基才得以被培养和分离。更多还原

【Abstract】 There is an urgent need in aquaculture to develop microbial control strategies, since disease outbreaks are recognized as important constraints to aquaculture production and trade and since antibiotic resistance has become a matter of growing concern.In attempt to bioremedy aquaculture shrimp farming environments, one of the alternatives to antimicrobials in disease control could be the use of probiotic bacteria as microbial control agents. Being lack of validity, pertinene and obvious dominance of probiotic bacteria exsiting in the shrimp farming, this study is conducted to isolate and screen some functional microbes which can degrade the protein and starch of remaining feeds in the shrimp and crab mix-culturing environment by their extracellular enzyme producing activity on the basis of the indigenous bacterial flora in shrimp and crab mix-culturing ponds having been investigated, and those predominant functional microbes were further studied using a culture-independent method of 16S rDNA PCR-DGGE. The main results are as the follows:1. The number of culturable heterotrophic bacteria including amylase-degrading bacteria(AB), organic phosphate-degrading bacteria (OPB),inorgan phosphate-solublzing bacteria (IPB) , chitin-degrading bacteria(CLB) ,lipin-degrading bacteria(LB), cellulose-degrading bacteria (CB),sulfur-oxidizing bacteria (SOB) and Vibrio (VB) in the water and sediments of a new-built and a fifteen-year old shrimp and crab mix-culturing pond in the East Sea of Quan Zhou were estimated by using the plate counting methods from October 2003 to May 2004. The correlation between culturable heterotrophic bacteria and other functional microbes was studied. The number of culturable heterotrophic bacteria showed high fluctuation in the water as well as in sediments of culturing ponds in the whole farming periods. The same was the case for other functional microbes and Vibrio. The number of functional microbes in the water of the old culturing ponds was larger than that of the new ponds,but the number of functional microbes in the sediments of the new culturing ponds was larger than that of the old one. In both of the culturing ponds, the number of functional microbes in the sediments was larger than that of the water by two or one order of magnitude. Statistical analysis indicated that AB, OPB, LB and VB were positively correlated with HB, while CLB, CB and SOB had no obvious correlation with HB.2. The producing conditions and the properties of extracellular amylase and protease of the six strains of marine bacteria isolated from the sediments of shrimp and crab mix-culturing ponds by the selected media were studied. Among the six strains obtained, Pseudomonas sp. strain SL-9, Arthrobacter sp. strain S-32 and Bacillus sp. strain OP -8 had higher amylase activity. The optimum temperature of the amylase activity of the three strains is all 50℃, and optimum pH of each strain is respectively 9.5, 8.0, 9.0. The other three strains of bacteria including Bacillus sp. strain NOPL-2, Serratia sp. strain SL-1 and Serratia sp. strain O-19 showed higher protease activity. The optimum temperature and pH of the protease activity of the three strains are respectively 60℃, 50℃, 40℃and pH 8.5, 7.0, 8.5. With the exception of OP-8 and NOPL-2, the enzyme protein of the other four strains is sensitive to heat.3. The 16S rRNA gene of the six marine bacteria strains were cloned and sequenced. The phylogenetic analysis demonstrated that the six marine bacteria strains belong to genera Bacillus, Pseudomona, Arthrobacter and Serratia, respectively.4. Pseudomonas sp. strain SL-9, Serratia sp. strain SL-1 and Serratia sp. strain O-19 can also elaborate lipase activity; Bacillus sp. strain NOPL-2 can produce amylase and phosphatease; Bacillus sp. strain OP-8 and Serratia sp. strain S-32 can also excrete protease. The results indicated that all strains could produce several extracellular enzymes and should have lay a foundation for the further screening of the probiotics candidate strains of complex enzymes to some extent.5. Total community DNA and strains DNA were extracted and puried.The V3 variable region of 16S rRNA gene was then amplified by PCR with bacterial primers and analyzed by denaturing gradient gel electrophoresis(DGGE), Comparaison of the DGGE profilings of the isolated strains and the samples showed that the isolated bacteria are not the predominant species in the community. Probably due to the fact that the strains best adapted to the enriched environmental conditions were isolated and cultured.更多还原