鳗草根际固氮菌的分离鉴定及培养条件的筛选

为了解鳗草(Zostera marina)根际微生物的共生固氮功能,并分离得到对海草有潜在促生效果的功能微生物,以山东荣城天鹅湖鳗草为研究对象,采用选择性固氮培养基从其根际分离得到了两株具较高固氮酶活性的菌株(3A和4G),从形态学、生理生化特性、16S rDNA和固氮基因nifH等方面对菌株进行筛选和鉴定,探讨菌株的最佳培养条件并获得了菌株典型生长曲线。结果表明,菌株3A为海旋菌(Thalassospira),革兰氏阳性菌,菌落圆形桔黄色,可利用的碳源:D-甘露糖,D-松三糖,L-鼠李糖等,最佳培养条件:盐度2.585%,pH 8.18,温度31.49℃;菌株4G为芽孢杆菌(Bacillus),革兰氏阴性菌,菌落圆形乳白色,可利用的碳源:D-纤维二糖,D-葡萄糖,D-麦芽糖,蔗糖,D-甘露醇,D-海藻糖等,最佳培养条件:盐度2.920%,pH 7.99,温度37.27℃。利用乙炔还原法测定二者的固氮酶活性,分别为252.21 nmol C_2H_4/(mL·h)和196.31 nmol C_2H_4/(mL·h)。研究证实两株菌株具有良好的固氮性能,可用于海草固氮肥的研制,在海草床生态系统恢复研究领域具有潜在的应用前景。

Isolation and characterization of nitrogen-fixing bacteria in the rhiz-osphere of Zostera marina and optimization of its culture conditions

Seagrass beds are highly productive ecosystems that provide essential habitat and food for various ma-rine species. However, seagrasses have been declining worldwide for decades because of human activities and global climate change. The conservation and restoration of seagrasses has become extremely urgent, especially in China. Recent studies showed that, in the rhizosphere of seagrasses, the organic carbon exudates from plant resi-dues and roots are an important carbon source for Azotobacter. Nitrogen fixation by Azotobacter could provide nitrogen for the growth of the plants, thereby promoting the restoration of seagrasses. Therefore, investigating N2 fixation by Azotobacter in the seagrass rhizosphere is essential since it might play an important role in the nitrogen cycle of the seagrass bed ecosystem. In this study, the growth-promoting activities of the rhizobacteria of eelgrass Zostera marina was investigated by isolating two nitrogen-fixing strains (3A and 4G) from the rhizosphere sedi-ments of Z. marina by using N-free medium. Based on the morphology, physiological and biochemical characte-ristics, sequence analysis of 16S rDNA, and the nitrogenase structural gene nifH, we identified the 3A bacterium as Thalassospira sp. and 4G bacterium as Bacillus sp.. The optimal culture conditions for the two Rhizobium strains were established, and typical growth curves for the two trains were obtained. In particular, strain 3A was a gram-positive bacterium and had an orange yellow-colored circular colony. In addition, it used D-mannose, D-melezitose, and L-rhamnose as carbon sources. The optimal growth temperature, pH, and salinity of 3A were 31.49℃, 8.18, and 2.585%, respectively. In contrast, strain 4G was gram-negative bacteria and had milky circular colony. It could use a series of hydrocarbons as carbon sources, such as D-cellobiose, D-glucose, D-maltose, su-crose, D-mannitol, and D-trehalose. Its optimal growth temperature, pH, and salinity were 37.27℃, 7.99, and 2.920%, respectively. Previous studies have shown that Thalassospira sp. can promote seagrass growth and in-crease the content of carotene, soluble sugar, and other substances, indicating that this species is a kind of growth-promoting bacteria. Nitrogen-fixing Bacillus sp. is a typical plant growth-promoting bacterium with strong stress resistance that can produce beneficial plant growth-promoting and antibacterial substances. The nitrogen fixation rates of 3A and 4G were 252.21 nmol C2H4/(mL·h) and 196.31 nmol C2H4/(mL·h), as revealed by the acetylene reduction method. Comparison with the nitrogen-fixing bacteria reported in other studies revealed that the two strains isolated in this study showed good nitrogen fixation ability. Under suitable growth conditions, the two bacterial strains exhibited logarithmic phase proliferation. Accurate positioning of the bacterial growth loga-rithmic phase and elucidating the growth characteristics of the bacterial strains could provide basic data for the production of bacterial fertilizers in the future. The bacterial strains obtained in this study might be applied in the future development and application of microbial inoculants and seagrass Azotobacter fertilizers, thereby benefit-ting the recovery of Z. marina beds in the future.