【作者】 李可；

【导师】 郑天凌；

【作者基本信息】 厦门大学 ， 微生物学， 2007， 博士

【摘要】 本论文针对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,基于微生态理论和芽孢杆菌作为微生态制剂的优良特性,从对虾养殖环境获得一批产芽孢细菌,并加以鉴定;以胞外消化酶为指标分离筛选可高效降解水产养殖残余饵料淀粉、蛋白及脂肪作用的若干株产芽孢功能菌,通过酶活性测定,确定一株地衣芽孢杆菌S-40作为潜在益生菌进行了对虾养殖实验;研究了对虾养殖环境的细菌群落组成,主要研究结果如下:1.构建了对虾养殖环境细菌16SrDNA文库,研究其细菌群落组成。结果表明:对虾肠道细菌分属于变形细菌(Proteobacteria)和厚壁菌类群(Firmicute),Proteobacteria占据了绝对优势,为98.7%,其中γ-Proteobacteria为97.4%;养殖水体菌群主要由变形细菌(24.1%)和拟杆菌门(17.7%)组成,其中α-Proteobacteria所占比例为21.5%;养殖环境表层沉积物的细菌多样性要丰富得多,有变形细菌(52.3%),拟杆菌门(Bacteroidetes,9.3%),浮霉状菌(Planctomycete,4.7%),厚壁菌类群(Firmicute,1.9%)和放线菌门(Actinobacteria,0.9%)。此外,水样和沉积物中蕴含着数量丰富的单细胞藻类,它们的比例分别高达55.7%和23.4%。2.从对虾养殖环境(对虾肠道和虾池表层沉积物)分离筛选出191株产芽孢细菌,细胞形态和RFLP分型后,对其中70株菌进行了生理生化以及分子生物学方法鉴定。结果显示它们分属于短芽孢杆菌属(2株)、芽孢乳杆菌属(2株)、类芽孢杆菌属(4株)和芽孢杆菌属(44株)。其中芽孢杆菌属中,我们鉴定得到13个种,包括环状芽孢杆菌、迟缓芽孢杆菌、巨大芽孢杆菌、球形芽孢杆菌、花园芽孢杆菌、蕈状芽孢杆菌、泛酸芽孢杆菌、短小芽孢杆菌,坚强芽孢杆菌、纺锤芽孢杆菌、地衣芽孢杆菌、凝结芽孢杆菌和枯草芽孢杆菌。3.利用选择性培养基,从前期工作得到的产芽孢细菌中分别筛选出9株具有较高淀粉酶、蛋白酶和脂肪酶活性的菌株,对其发酵条件以及粗酶液性质进行了初步的研究。结果表明多株菌在一定的温度、pH及盐度区间内均有较强的酶活力,一株地衣芽孢杆菌S-40由于其良好而稳定的消化酶活性,作为养殖水体添加剂进行对虾养殖实验。4.应用从对虾养殖环境筛选的一株地衣芽孢杆菌S-40,作为潜在的微生态制剂菌株,分别以10~3cfu/mL,10~4cfu/mL,10~5cfu/mL三种不同浓度添加到凡纳滨对虾养殖水体中,测定了饲养40天后对虾消化道的总菌和弧菌数及多项免疫指标。结果显示:与对照组对虾相比,所有实验组对虾消化道的弧菌数有显著下降,而总菌数没有呈现显著性差异;所有实验组中对虾血淋巴上清液的血细胞数,酚氧化酶活性(PO)均有显著性上升,超氧化物歧化酶(SOD)仅在10~5cfu/ml时有显著上升,菌剂的加入对对虾血淋巴上清液的的蛋白浓度并无显著影响。5.本研究选用微孔淀粉为吸附剂吸附地衣芽孢杆菌S-40菌株芽孢,通过多因素分析,确定最佳的吸附包埋条件:pH7.5,温度30℃,吸附时间1.5h。更多还原

【Abstract】 The probiotics recently used in aquaculture have the deficiency of short effect time and no obvious dominance in the environment.This study aimed at exploring the application potential of spore-producing bacteria to shrimp aquaculture based on the microecological theory and the good characteristics of Bacillus used as probiotics in the previous works.Many spore-producing bacteria were therefore isolated from shrimp aquaculture environment and shrimp intestinal.Their abilities to remove the feeding remains in the aquaculture were evaluated by determining the activity of producing extracellular digestive enzymes（amylase,protease and lipase）.Then,a potential probiotic Bacillus licheniformis strain S-40 was tested as an additive for improving the shrimp aquaculture.In addition,the bacterial compositions of shrimp aquaculture systems were investigated in the study.The main results were as follows:1.The bacterial compositions in the shrimp culturing environment were characterized by analyzing three 16S rDNA clone libraries constructed from the intestines of shrimp,water and surface sediment of the shrimp pond respectively. The RFLP analysis and sequence alignment of 16SrDNA libraries showed that intestinal microflora in shrimp belonged to Proteobacteria（98.7%）and Firmicute（1.3%）phylogenetically,of whichγ-Proteobacteria was dominant （97.4%）compared toα-Proteobacteria（1.3%）;The bacteria in shrimp pond water mainly consisted of Proteobacteria（24.1%）and Bacteroidetes（17.7%）,withα-Proteobacteria 21.5%;The bacterial diversity was much higher in the surface sediment of the shrimp pond with the proportion of 52.3%Proteobacteria,9.3% Bacteroidetes,4.7%Planctomycete,1.9%Firmicute and 0.9%Actino-bacteria. However,it deserved to mention that in the water and sediment sample, approximately 55.7%and 23.4%of representative sequences were PCR-amplified from unicellular algae,probably derived from the organelle.2.One hundred and ninety-one spore-producing bacteria were isolated from shrimp intestines and the sediments of shrimp pond.Based on their morphology and results of RFLP analysis,70 of them were chosen for physiological and biochemical tests as well as 16S rRNA identification.The results indicated that these strains were grouped into four genera Brevibacillus,Sporolactobacillus, Paenibacillus,Bacillus.Thirteen species of genus Bacillus were found out, including B.circulans,B.lentus,B.megaterium,B.sphaericus,B.horis,B.mycoides, B.pantothenticus,B.pumilis,B.firmus,B.fusiformis,B.licheniformis,B.coagulans, B.subtilis.3.Nine strains,with high efficiency of producing amylase,protease and lipase,had been screened out from those spore-producing bacteria by selective media.Their conditions of enzyme-producing were optimized and the activities of crude enzymes were determined.The results showed that most of them had high enzyme activities in the certain condition of temperature,pH value and salinity.Among them,one strain,designated as Bacillus licheniformis S-40,was chosen as potential probiotic for shrimp culture experiment according to its high and stable activities of digestive enzymes.4.An experiment was conducted with the strain Bacillus licheniformis S-40 as a supplement,to investigate its effects on the intestinal microflora,and the immune response of the white shrimp,Litopenaeus vannamei.Shrimp were fed in four tanks for 40 days with different water conditions,control（non-supplemented）,and water supplemented with B.licheniformis S-40 at 10³ cfu·mL^-1,10⁴ cfu·mL^-1,and 10⁵ cfu·mL^-1respectively.The results indicated that compared to the control group, the total bacterial counts in the shrimp intestine remained constant,while Vibrio sp.numbers decreased significantly,and the total haemocyte count（THCs）and PO activity in the shrimps increased significantly,SOD activity increased significantly only when water was supplemented with B.licheniformis S-40 at 10⁵ cfu·mL^-1.However,the protein contents were not remarkably different between the experimental and control groups.5.Finally,the spores of B.licheniformis S-40 were embedded in microporous starch to make microcapsule.By analyzing multiple factors,the optimal embedding conditions were estimated as follows:pH 7.5,temperature 30℃,absorption time 1.5h.更多还原