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

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、接种量5.0%；菌株XT37为海洋芽孢杆菌（Bacillussp.），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、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。

关键词:固氮菌;解磷菌;条件优化;海草;微生物菌剂

Abstract:

[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 C₂H₂/(mL·h). The optimum growth conditions were as follows: salt content 2.5%, pH 7.5, fermentation temperature 33 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%, pH 6.7, fermentation temperature 28 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.

Key words:nitrogen-fixing bacteria;phosphate-solubilizing bacteria;optimization of conditions;seagrass;microbial agents