【作者】 邓斌；

【导师】 李卫芬；

【作者基本信息】 浙江大学 ， 动物营养与饲料科学， 2014， 博士

【摘要】 为了降低草鱼养殖水体中的氨氮及亚硝酸盐等污染物,通过定向富集的方法筛选到一株具有良好脱氮性能的施氏假单胞菌(Pseudomonas stutzeri) SC221。用钻60对该菌进行辐射诱变,发现该菌的反硝化脱氮能力从50mg/L提高到了100mg/L。经过辐射诱变后的菌株被命名为SC221-M。随后研究了不同环境条件(不同氮源、碳源、碳氮比和温度)对SC221-M生长、脱氮率及反硝化酶基因表达的影响,结果表明SC221-M的最佳脱氮条件为：氮源为亚硝酸钠、碳源为柠檬酸钠,碳氮比为4-8,温度为20-35℃。环境因素能够显著影响SC221-M的生长及其脱氮率,且脱氮率与反硝化酶基因的表达量之间有一定的相关性。通过Illumina Hiseq2000平台测序和序列组装,发现施氏假单胞菌SC221-M的基因组大小为4545027bp,GC含量为64.01%,共131个scaffold,399个contig。与参考菌株DSM4166的基因组进行比对,发现SC221-M基因组共有60641个SNP,基因区内的同义突变为43396个,非同义突变为10743个。InDel分析显示,基因组中没有插入或缺失突变。SC221-M中的nar、nap、nir、nor及nos反硝化酶相关基因在基因组中呈簇状分布,且SC221-M的NirS和NosZ蛋白序列与DSM4166和A1501的同源性较高,提示施氏假单胞菌SC221-M与A1501和DSM4166之间有着类似的反硝化机制。研究了SC221-M及其复合菌制剂对草鱼养殖水体水化学指标的影响。试验设置了5个不同的试验组,分别在1400L水体中添加OCFU/ml SC221-M、1×105CFU/ml SC221-M、1×105CFU/ml蜡样芽孢杆菌BSC24、1×105CFU/ml的复合菌粉(SC221-M与BSC24等比例混合而成)和3×105CFU/ml的复合菌粉组。经过9d的饲养试验发现,与对照组相比,活菌制剂组水体中的TDS、氨氮、亚硝酸盐、总无机氮、总氮、COD、BOD等水化学指标有不同程度的降低,且复合菌的效果要明显好于单一菌剂。该结果说明有益微生物之间的相互协同作用能有效改良草鱼养殖水体的水质。采集草鱼养殖试验第9d的对照组、BSC24组、SC221-M组和混合菌组的水样,提取细菌总DNA,利用16S rDNA V3引物对各样品进行PCR扩增,再使用Roche454GS FLX Titanium测序平台进行测序,并采用Qiime、Megan、Cytoscape等软件对所测序列进行分析。结果显示,复合菌组的多样性最为丰富。对照组、BSC24组、SC221-M组和复合菌组的细菌都是以变形菌门为主。从UPGMA聚类分析的结果看,各样品均按照不同的处理聚在不同的分支下。结果表明,添加益生菌可显著地改变养殖水体中的微生物群落结构,复合菌组的变化最为明显,水体微生物群落结构的改变与草鱼养殖水体水化学指标的变化相关。更多还原

【Abstract】 In order to reduce the pollutants such as ammonium and nitrite in aquaculture water for grass carp, an isolate of denitrifying bacteria Pseudomonas stutzeri SC221was obtained by directional enrichment. After cobalt-60radiation, the nitrite removal capacity of the strain could be increased from50mg/L to100mg/L. It was named as P. stutzeri SC221-M after such radiation. Following researches on the effect of nitrogen sources, carbon sources, ratio of carbon to nitrogen (C/N) and temperature on the growth, denitrification rate and denitrification gene expression of SC221-M indicated that the best condition for the growth and denitrification of SC221-M was sodium nitrite as nitrogen source, sodium citrate as carbon source, C/N=4-8and at20-35℃. As the results shown, environmental factors could affect the growth and denitrification of SC221-M, and there was certain correlation between denitrification and nirS and nosZ gene expression.Illumina Hiseq2000sequencing platform was used to obtain the complete genome sequence for SC221-M. Based on the assemble result, it was found that the genome size was4,545,027bp with64.01%GC content, the number of scaffold was131with399contig. Comparing with reference strain DSM4166, InDel was not appeared and there were60,641SNPs, including43,396synonymous mutations and10,743non-synonymous mutations in the genome of SC221-M. Through phylogenetic analysis, it was discovered that NirS and NosZ sequences of SC221-M were closely related to those of A1501and DSM4166, indicating that the strains showed a similar denitrification mechanism.The effect of SC221-M and its mixed microbial preparation on hydrochemical indexes of the aquaculture water for grass carp was studied. There were five groups, including addition of0CFU/ml microbial preparation,1×105CFU/ml SC221-M,1×105CFU/ml Bacillus cereus BSC24,1×105CFU/ml mixed preparation (SC221-M: BSC24=1:1) and3×105CFU/ml mixed preparation in15PE buckets containing1400L water. It was found that addition of the microbial preparation could decrease the level of hydrochemical indexes, including TDS, ammonium, nitrite, total inorganic nitrogen, total nitrogen, COD and BOD, and the effect for mixed microbial preparation was better than that for SC221-M or BSC24preparation. The results indicated that the interaction between SC221-M and BSC24played an important role in improving water quality.Water samples were collected from the control and treatment groups after9-day feeding. Bacterial genomic DNA was extracted and the V3region of16S rDNA was amplified by PCR. After the use of Roche454GS FLX Titanium to sequence the PCR products as well as Qiime, Megan and Cytoscapes to analyze DNA sequences, the results revealed that in each group, the most abundant phyla were Proteobacteria. The microbial diversity was increased when adding the mixed preparation and all samples were in different branches in accordance with the treatment through UPGMA cluster analysis. As is shown, microbial community structure in the water was significantly changed after adding microbial preparation, and there was a close relationship between microbial community structure and hydrochemical indexes.更多还原