【作者】 张艳；

【导师】 王印庚；

【作者基本信息】 中国海洋大学 ， 水产养殖， 2004， 硕士

【摘要】 本文主要研究了3株有益菌在工厂化大菱鲆养殖中的应用及效果评价。经过菌种的富集、分离、筛选等步骤获得所要的有益菌，采用API-系统和16S rRNA序列分析的方法对其进行了菌种鉴定；将有益菌扩大培养，采用生物膜固定化的方法应用于水循环系统中，对其净化效果进行了检测；同时还进行有益菌的生物安全性临床实验。 在虾池中取底泥作为筛选细菌的材料，通过选择性培养基的富集、分离和筛选，共得到了硝化细菌4株，分别为X1、X2、X3和X4；亚硝化细菌6株，Y1、Y2、Y3、Y4、Y5、Y6；氨氧化细菌3株，分别为A1、A2和A3。对这三类细菌进行NH₄⁺-N和NO₂^--N的去除实验对比，结果发现，硝化细菌中以X3的硝化作用最强，可达31.9％，而X1、X2和X4的NO₂^--N去除率分别为25.1％、26.2％、19.3％；亚硝化菌中Y1的NH₄⁺-N去除能力最强，可达77.4％，其余的Y2、Y3、Y4、Y5及Y6的去除率分别为70.2％、70.7％、70.8％、70.2％、56.1％；氨氧化细菌中A3的去除氨氮能力最强，可达25.3％，而A1、A2的氨氮去除率分别为7.37％和12.5％。 在细菌对大菱鲆生存的安全性实验中，选取孵化后25d左右的大菱鲆仔鱼作为实验材料，将X3、Y1、A3、J3、J7和J8六株菌的菌液加入到养殖大菱鲆仔鱼的玻璃缸中，使菌液浓度达到1×10⁷CFU／mL左右，结果显示加入有益菌的实验组与对照组中鱼苗的生存情况无明显差异，证明这几株有益菌对大菱鲆无毒害作用。 将筛选好的3株有益菌X3、Y1、A3采用API系统和16S rRNA相结合的方法进行了鉴定。菌株X3为革兰氏阳性，细菌呈短杆形或近球形，在构建系统发育树中，与Rhodococcus phenolicus相似性很高，达98％，故把菌株X3确定为红球菌属（Rhodococcus sp．）。菌株Y1革兰氏染色为阴性，细菌球形，属于盐单胞菌属（Hatomonas sP.），菌株A3革兰氏染色阴性，细菌短杆形，属于亚硝化单胞菌属（解trosomonas Sp.）。 将筛选好的X3、YI、A3以及另外3株有机降解菌J3、J7和J8按照一定的比例混合，进行扩大培养，得到500L的大批量菌液，转移至挂膜容器中，再进行培养，成功获得lm3左右的菌液，达到挂膜所需要的数量。选取一种纤维滤料作为细菌载体，加入到培养好细菌的容器中，定期补充营养，适时检测温度、pH值、溶解氧的变化，保证细菌处于最适的生长条件。2一3周后得到驯化成熟的生物膜片。 将制备好的生物膜片放入大菱虾养殖的废水中，对其进行净化效果检测，结果发现氨氮浓度由初始的0.300mg/L降为0.06om留L，去除率为80%;亚硝酸氮浓度由o.ol6m叭增到0.052mg/L;硝酸氮由初始的o.36om叭增到0.39om叭;而养殖废水的COD也有很大的降解效果，由4.42m留L降到2.17m叭，降解率可达50.9%。 本实验还现场进行了单株菌X3、YI、A3对大菱鲜养殖废水的净化效果检测。其结果与实验室中筛选细菌时进行的检测效果有一定的差异，实验室中Yl的氨氮去除率可达77.4%，A3氨氮的去除率达25.3%，X3的亚硝氮的去除率达31，9%，而养殖现场本实验中这三株菌的净化效果分别为42.3%、44.1%和14.9%。更多还原

【Abstract】 This paper mainly studied on the application of three strains of probiotics in turbot culture system. The experimental targets included enrichment, isolation, selection and identification for the probiotic bacteria. By using biofilm which was build from the probiotic microbes, the changes of NOi’-N and NH4+-N in a recirculating culture system were monitored. While the security of these probiotic microbes to turbot culture had also been examined in the present study.The probiotic microbes were originally isolated from the mud in shrimp ponds, in which enrichment and selection were carried out by using selective culture medium. Eventually, 4 strains of nitrobacteria were obtained, numbered XI, X2, X3 and X4; 6 strains of nitrite bacteria were gained, numbered Yl, Y2, Y3, Y4, Y5 and Y6; 3 strains of ammonia-oxidation bacteria were acquired, numbered Al, A2, A3. Comparing to the degradation rate of NO2~-N and NH4+-N, the result indicated that X3, Yl and A3 were the best, whose degradation rate were 31.9%, 77.4% and 25.3%. The degradation rate of the rest 3 strains of nitrobacteria were 25.1%, 26.2%, 19.3%; Y2, Y3, Y4, Y5 and Y6 were 72.5%, 70.7%, 70.8%, 70.3%, 56.2%; Al, A2 were 7.37% and 12.5%, respectively.Two methods for bacterial identification were adopted, namely API-system and 16S rRNA gene screening analysis. The strain X3 was Gram-positive, short bacilliform or spherical, belonging to Rhodococcus sp.; the strain Yl was Gram-negative, spherical, belonging to Halomonas sp.; while the strain A3 was Gram-negative, short bacilliform, belonging to Nitrosomonas sp..The security of the probiotic microbes in a culture system was estimated, in which turbot larvae aging 25 days were respectively immersed into six strains ofprobiotics (X3. Y1. A3. J3. J7and J8, their final working concentration about 107CFU/mL). Compared the death of tested fish to the control group, there were no difference, indicating the strains of probiotics were safe for the cultured turbot.X3, Yl, A3, J3, J7 and J8 were cultured and then mixed to get 500L bacterial liquid in a big container. The growth of the microbes for some days allowed the volume added up to 1M3. A kind of fiber material as a carrier was then transferred to the container, allowing the microbes adhered onto it. During the experiment, regularly supplying nutrition, examining the changes of water quality such as temperature, pH, and dissolved oxygen, to assure the good conditions for the bacterial growth. After 2-3 weeks, biofilms with numerous bacteria was established.Putting the biofilm into wastewater derived from turbot culture tanks, and then the changes of NH4+-N , NCV-N and NO3N were examined. The results indicated that NH4+-N lowered from 0.300mg/L to 0.060mg/L, the degradation rate being 80%; NO2"-N and NO3"-N increased from 0.016mg/L to 0.052mg/L, 0.360mg/L to 0.390mg/L, respectively. While the COD of wastewater changed from 4.42mg/L to 2.17mg/L, the degradation rate was 50.9%.Purifying efficiency of 3 bacterial strains (Yl, A3 and X3) on wastewater from turbot culture tanks were examined on site. The experiment showed that there were some difference between site testing and laboratory testing, The degradation for NH4+-N acted by Yl and A3, they were tested as 42.3%, 44.1%, individually. While the degradation for NC>2~-N acted by X3, it was tested as 14.9%. However, the degradation by Yl, A3 and X3 in laboratory, were 77.4%, 25.3% and 31.4%, respectively.更多还原