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

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