海洋固氮菌和解磷菌的分离鉴定及发酵条件优化  

Isolation, characterization and culture optimization of nitrogen-fixing and phosphate-solubilizing bacteria from rhizosphere sediments of Halophila ovalis

作　　者：王聪 凌娟[1] 张燕英[1,3] 林丽云[1,2] 曾思泉 周卫国[1,2] 林显程 尹浩[1] 董俊德 Cong Wang;Juan Ling;Yanying Zhang;Liyun Lin;Siquan Zeng;Weiguo Zhou;Xiancheng Lin;Hao Yin;Junde Dong(Key Laboratory of Tropical Marine Bio-resources and Ecology, South China Sea Institute of Oceanology, Chinese Academy of Sciences, Guangzhou 510260, Guangdong Province, China;University of Chinese Academy of Sciences, Beijing 100049, China;Tropical Marine Biological Research Station in Hainan, Chinese Academy of Sciences, Sanya 572000, Hainan Province, China)

机构地区：[1]中国科学院南海海洋研究所,中国科学院热带海洋生物资源与生态重点实验室,广东广州510260 [2]中国科学院大学,北京100049 [3]中国科学院海南热带海洋生物重点实验站,海南三亚572000

出　　处：《微生物学报》2018年第5期817-829,共13页Acta Microbiologica Sinica

基　　金：中国科学院战略性先导科技专项A(XDA13020300,XDA11020305); 国家自然科学基金(41276113,41276114,41406191,41676163); 广东省公益研究与能力建设专项资金(2015A020216016); 国家“863计划”(2012AA092104,2013AA092901,2013AA092902); 广东省科技计划项目(2014B030301064); 国家重点研发计划(2017YFC0506301); 海南海岸带生态系统修复生物固氮菌剂的关键技术研究(XH2

摘　　要：【目的】从西沙喜盐草根际沉积物中分离纯化得到具有高效固氮能力及解磷能力的菌株。优化其发酵培养条件,研究其制备海洋微生物菌剂的可能性。【方法】从形态学特征、生理生化、16S rDNA及功能基因水平进行鉴定,通过乙炔还原法、钼锑抗显色法检测菌株的固氮酶活性和解磷能力,单因素法和响应面法优化其发酵培养条件,溶血试验和急性毒性实验鉴定菌株的安全性。【结果】结果表明,菌株AZ16属于星箭头菌(Sagittula stellate),革兰氏阴性菌,选择性固氮培养基中菌落呈黄圆形黏稠状,固氮酶活性达34.63 nmol C2H2/(mL·h),最适生长条件为:盐度25‰、pH 7.5、温度33°C、接种量5.0%;菌株XT37为海洋芽孢杆菌(Bacillus sp.),革兰氏阳性菌,选择性固氮培养基中菌落呈深黄色圆形褶皱,植酸酶活性达239.49μg/L,最适合生长条件为:盐度25‰、pH 6.7、温度28°C、接种量5.0%。溶血实验和急性毒性实验证明两株菌属实际无毒级别。【结论】两株菌具有高效的固氮解磷功能,以及抗高盐、强碱等环境的能力,安全无毒,因此有潜力应用于多功能混合微生物菌剂的研制。[Objective] We isolated and purified a nitrogen-fixing bacterium AZ16 and a phosphate-solubilizing bacterium XT37 from the sediment of Halophila ovalis in Xisha, China. Furthermore, we optimized their culture conditions and explored their potential for solid microbial agents. [Methods] The strains were identified by morphological, biochemical and molecular characteristics. The nitrogenase and phosphate solubilizing activities were measured by acetylene reduction method and molybdenum antimony anti-coloring method. The fermentation conditions were optimized by single factor method and response surface method. Safety of the strains was identified by hemolysis test and acute toxicity experiment. [Results] Strain AZ16 is a gram-negative bacterium, belonging to Sagittula stellate, producing yellow, round, sticky colonies on the selective nitrogen fixation medium. The maximum nitrogenase activity was 34.63 nmol C2 H2/（m L·h）. The optimum growth conditions were as follows： salt content 2.5%, p H 7.5, fermentation temperature 33 °C, inocula concentration 5.0%. Strain XT37 showed closest resemblance with Bacillus sp., gram-positive bacteria, developed yellow, round, plicated in the selective nitrogen fixation medium. And the phytase activity was 239.5 μg/L. The optimum conditions were as follows： salt content 2.5%, p H 6.7, fermentation temperature 28 °C, inocula concentration 5.0%. Hemolysis tests and acute toxicity experiments proved that the two strains were in the actual non-toxic level. [Conclusion] Nitrogen-fixing bacterium AZ16 and phosphate-solubilizing bacterium XT37 can resist high salt concentration and can also survive under alkaline condition, hence these strains have the potential for multi-functional mixed microbial agents.

关 键 词：固氮菌 解磷菌 条件优化 海草 微生物菌剂 

分 类 号：Q93-331[生物学—微生物学] TQ920.1[轻工技术与工程—发酵工程]