深海沉积物高效脱氮菌筛选分离及其脱氮特性研究

Isolation and characterization of high efficient denitrifying bacteria from deep sea sediments

摘要:

[背景] 深海海域具有高压、低温、无光等环境条件，蕴含着丰富而独特的微生物资源。[目的] 从深海沉积物中定向分离、筛选脱氮效率高的好氧脱氮菌株资源，并揭示其脱氮特性，为开发水体脱氮微生物技术提供物质基础。[方法] 以东太平洋、南大西洋、西南印度洋共10个站位的深海沉积物为研究材料，在28℃下使用无机氮源连续进行两轮富集培养，然后定性筛选可以脱除氨氮、亚硝态氮和硝态氮的菌株，并通过形态学和16S rRNA基因序列分析进行初步分类鉴定；对优选得到的功能菌株，分别采用以氨氮、亚硝态氮、硝态氮为唯一氮源的培养基定量研究其生长和脱氮性能。[结果] 从10份大洋深海沉积物样品中共分离得到49株好氧反硝化菌，其中3株在有氧条件下反硝化效率较高，分别命名为Pseudomonas sp.G111、Pseudomonas sp.G112和Dietzia maris W023a，其中菌株G111和G112与模式菌株博岑假单胞菌Pseudomonas bauzanensis BZ93^T的16S rRNA基因序列相似度为99.2%，菌株W023a与模式菌株海洋迪茨氏菌Dietzia maris ATCC35013^T的16S rRNA基因序列相似度为99.9%。菌株G111、G112和W023a培养48 h后，对氨氮的脱除率分别为98.0%、85.2%和97.6%；对亚硝态氮的脱除率分别为71.9%、67.5%和34.7%；对硝态氮的脱除率分别为66.0%、52.6%和56.3%。菌株G111、G112和W023a均为异养硝化-好氧反硝化菌，可通过好氧反硝化作用将亚硝态氮和硝态氮还原为含氮气体，也可通过异养硝化-好氧反硝化作用将氨氮转化为含氮气体。[结论] 从深海沉积物中分离筛选得到3株高效好氧反硝化菌，所获得的菌株在水体净化、污水处理、生态系统修复等领域具有应用潜力。

Abstract:

[Background] With the environmental features of high pressure, low temperature and no light, deep sea areas contain abundant and characteristic microbial resources. [Objective] This study aims to isolate and screen aerobic denitrification bacteria strains with high denitrification efficiency from deep sea sediments, and reveal their denitrification characteristics, so as to provide material basis for the development of water denitrification microtechnology. [Methods] In this paper, deep-sea sediments from 10 stations in the East Pacific Ocean, South Atlantic Ocean and Southwest Indian Ocean were used as research materials. Two consecutive rounds of enrichment culture were carried out with inorganic nitrogen at 28℃. The strains capable of removing ammonia nitrogen, nitrite nitrogen and nitrate nitrogen were isolated, purified and screened by their qualitative denitrification capacities. The obtained strains were preliminary identified and classified through morphological and 16S rRNA gene sequence analysis. The growth and denitrification performance of the obtained strains were quantitatively studied in the media with ammonia nitrogen, nitrite nitrogen and nitrate nitrogen as the only nitrogen source, respectively. [Results] A total of 49 aerobic denitrifying bacteria were isolated from 10 deep-sea sediment samples, of which 3 strains, designated Pseudomonas sp. G111, Pseudomonas sp. G112 and Dietzia maris W023a, showed high denitrifying efficiency under aerobic conditions. Strains G111 and G112 shared 16S rRNA gene similarities of 99.2% with Pseudomonas bauzanensis BZ93^T. The 16S rRNA gene similarity between strain W023a and Dietzia maris ATCC 35013^T was 99.9%. After strains G111, G112 and W023a were cultured for 48 hours, their removal rates of ammonia nitrogen were 98.0%, 85.2% and 97.6%, respectively; their removal rates of nitrite nitrogen were 71.9%, 67.5% and 34.7%, respectively; their removal rates of nitrate nitrogen were 66.0%, 52.6% and 34.7%, respectively. Strains G111, G112 and W023a are three heterotrophic nitrifying-aerobic denitrifying bacteria. These strains could reduce nitrite or nitrate to nitrogenous gases by using aerobic denitrification, and convert ammonia nitrogen to nitrogenous gases by using heterotrophic nitrification-aerobic denitrification. [Conclusion] In this study, three strains of highly efficient aerobic denitrifying bacteria were isolated from deep-sea sediments, and the obtained strains have application potential in water purification, sewage treatment, ecosystem restoration.