沼泽红假单胞菌（NS-04）的分离、鉴定及其在暗纹东方鲀养殖上的应用

【作者】 吴向华；

【导师】 杨启银；

【作者基本信息】 南京师范大学 ， 微生物学， 2004， 硕士

【摘要】 本文以从水塘污泥中分离出一株红假单胞菌（Rhodopseudomonas）NS-04菌株为材料，对它进行了初步的菌种鉴定、营养成分分析、发酵条件的优化，并研究了不同浓度的NS-04菌株培养液在暗纹东方鲍养殖中的作用。 通过富集培养水塘污泥筛选光合细菌，经电镜观察形态学特征、活细胞扫描以及碳源利用试验了解菌株的生理生化特征，并通过16S rRNA的同源性比较，了解序列保守性。结果表明NS-04菌株革兰氏反应阴性，单个细胞为杆状至卵形，菌体大小为0.6-0.9×1.2-2.0μm，二分分裂，极生或亚极生鞭毛，片层状光合内膜，位于细胞质膜之下并与之平行，菌落为棕红色，光照厌氧和黑暗好氧条件下均可生长，菌体富含类胡萝卜素和细菌叶绿素a，能利用多种碳源，酵母膏对其生长有明显刺激作用，16S rRNA基因的分子系统学分析表明，该菌株与GenBank中的沼泽红假单胞菌菌株的同源性为97％，初步确定筛选出的NS-04菌株为沼泽红假单胞菌（Rhodopseudomonas palustris）。 通过对菌株的营养成分的分析可知，蛋白质、脂肪、灰分、可溶性糖类分别占细胞干重的61.24％、8.11％、7.5％和5.73％，类胡萝卜素占细胞干重的4.24mg／g，富含维生素B₂、B₆和烟酸，氨基酸比值达92.38％，必需氨基酸总含量达40.92％，必需氨基酸与非必需氨基酸比值为79.52％，符合FAO／WHO对饲料的理想模式，是一种比较优质的饲料蛋白源。 菌株的发酵条件优化试验表明，比较理想的发酵培养基配方（g／L）为：黄豆芽100，苹果酸1.5，乙酸钠2，微量元素1，pH 7.0。最适发酵条件；接种量1％，装液量为80mL／250mL，40001x光照下，30℃好氧培养36h后转入厌氧培养24h。在此条件下生物量的活菌数可达到1.4×10⁹cfu／mL，符合农业部的标准。 将光合细菌应用于特种水产养殖上，初步研究了施加不同浓度光合细菌在暗纹东方鲀养殖中的效果。试验表明添加光合细菌浓度在50μg／L以上时能有效的改善养殖水环境，使DO浓度增加，COD、BOD₅、氨态氮、亚 南京师范大学20(冈届硕士学位论文 沼泽红假单胞菌（NS一04）的分离、鉴定及其在暗纹东方纯养殖上的应用研究硝基盐浓度下降并保持在正常范围，与不投加光合细菌的对照池相比，鱼苗成活率增加，体长体重增加，减轻了病原微生物的感染。更多还原

【Abstract】 This article elaborately studied the identification, analysis and evaluation of nutrient components the optimum fermentation of Photosynthetic bacteria NS-04 which isolated from the sludge of the pond in Nanjing. In addition, the effect of different concentrations of strain NS-04 on the aquaculture on Takif u gu obscurus is also studied .Photosynthetic bacteria were screened by enrichment culture from the sludge. Transmission electron microstcope, tests of using different carbon sources of PSB and of absorption of live cell suspension were used to see the biological character of the PSB. In addition, we also analysed the sequence of 16S rRNA of the PSB to know the homology. The strain NS-04 screened from the sludge is a Gram-negative bacterium. Cell is ovoid or rod in shape, 0.6-0.9 um wide and 1.2-2.0 um long, multriplication by binary fission. The photosynthetic membrance was lamellar type, which is under and parallel to intro-cytoplasmic membrance. The clone appeared brown with major pigments being Bchll a and carotenoids. PSB NS-04 can grow both under anaerobic condition in the light and aerobic condition in the dark. The growth of cells can also prompted by yeast extract. Phylotenetic tree of 16S rRNA sequences indicated that the similarity of strain NS-04 and standard bacterium of Rhodopseudomonas palustris in GenBank is 97%. According to Bergey’s Manual of Systematic Bacteriology, strain NS-04 was identified as R. palustris.The nutrient components of strain NS-04 were analysised. The result indicated that the content of protein, crude fat, ash and soluble carbohydrate in dried bacteria were 61.24%, 8.11%, 7.5% and 5.73% respectively. The content of carotenoids and amino acid was 4.24mg/g and 92.38%. In addition VitaminB2 VitaminB6 and nicotinic acid were abound in PSB. In all, the R.palustris is a good resource of protein in feedingstuff industry.To obtain the high level of cfu/mL, the media components and cultivationconditions were investigated. The tests showed the optimum fermentation medium was consisted of soybean sprout 100, malic acid 1.5, NaAc 2, trace elements 1, pH 7.0. The incubata condition is: inoculation 1%, 80mL/250mL flask, 40001x, 30C in aerobiotic condition for 36h and then in anaerobic condition for 24h In this condition the titre was 1.4 X 10 cfu/mL and it measured up to the national standard of PSB.As far as the effect of the PSB on the aquiculture the effect of different concentrations of PSB on the Takifu.gu obscurus were studied. The experiment showed that the PSB can improve the water quality and make the numerical value or the concentration of DO, COD, BOD,N-NH3 and NO2 decrease when that of the PSB is over 50 u g/L.And the livability, weight and incidence of a disease decreased compared with the control.更多还原