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南海深海氮循环微生物的原位培养与多样性分析
王蕾,王丽萍,董纯明,李小义,李登峰,邵宗泽
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(宁波大学海洋学院,浙江 宁波 315211;自然资源部第三海洋研究所、海洋生物遗传资源重点实验室,福建 厦门 361005)
摘要:
借助自主研发的深海水体原位定植培养系统,在南海3 300 m的深海水体中进行了氮循环微生物的原位培养.通过向富集仓中投加缓释肥(铵盐、硝酸盐和尿素)进行了为期17个月的原位富集.结合高通量测序技术和分离培养的方法,对深海原位富集物及实验室二次富集物进行了微生物多样性分析.16S rRNA基因高通量测序结果表明,在南海原位富集样品中,细菌以变形菌门丰度最高,富集仓内脱脂棉附着介质和仓内水样中的最优势属分别为希瓦氏菌属(Shewanella)和科韦尔氏菌属(Colwellia);古菌以奇古菌门为主,其中氨氧化古菌(AOA)所占比例很高.通过平板培养分离到17株细菌,主要包括盐单胞菌属(Halomonas)、海杆菌属(Marinobacter)和亚硫酸盐杆菌属(Sulfitobacter)等.通过不同起始氮源(氨氮、硝酸盐、亚硝酸盐)和培养温度(28℃和10℃),对原位富集样品进行实验室二次富集,获得了5个富集菌群.分析发现,它们均具有较好的脱氮效果,可以产生N2O或N2;这些菌群均以盐单胞菌属占绝对优势,其次为海源菌属(Idiomarina)或海杆菌属,其中盐单胞菌和海源菌成功获得分离.单菌功能验证表明,分离获得的盐单胞菌具有好氧反硝化能力,其在原位环境下反硝化活性尚有待确认.
关键词:  海洋生物学  氮循环  原位富集  微生物多样性  好氧反硝化  深海
DOI:10.3969/J.ISSN.2095-4972.2019.01.001
基金项目:国家自然科学基金资助项目(41506152);科技部深海生物863计划资助项目(2012AA092102);科技部海洋微生物资源共享服务子平台运行与服务支撑资助项目(NIMR-2018-9)
Deep sea in-situ cultivation and diversity analysis of microorganism involved in nitrogen cycling in the South China Sea
WANG Lei,WANG Li-ping,DONG Chun-ming,LI Xiao-yi,LI Deng-feng,SHAO Zong-ze
(School of Marine Sciences, Ningbo University, Ningbo 315211, China;Key laboratory of Marine Biogenetic Resources, Third Institute of Oceanography, Ministry of Natural Resources, Xiamen 361005, China)
Abstract:
In this study, an in-situ enrichment in depth of 3 300 m in the South China Sea was newly developed for 17 months by adding slowly releasing ammonium, nitrate and urea into a circulation chamber of a deep-sea water incubation system. The response of microbial community was analyzed combining with Miseq high-throughput sequencing technology and the lab seperation. Our results showed that γ-Proteobacteria is the most abundant in the bacterial population. The bacterial composition on genus level differed obviously in the cotton samples and water samples in the circulation chamber, where Shewanella spp. and Colwellia spp. are the main members, respectively. Thaumarchaeota dominated in the archaea domain with a high proportion of the ammonia oxidizing archaea (AOA). Culture results showed that a total of 17 bacterial strains were isolated from the in-situ samples, including Halomonas, Marinobacter,Sulfitobacter and so on.The in-situ enrichment in the South China Sea was further selectively enriched in laboratory under different initial nitrogen sources (ammonia, nitrate, nitrite) and temperatures (28℃ and 10℃). A total of five secondary enrichments were obtained. The enriched consortia exhibited a strong nitrogen removal capacity, by releasing N2O or N2 as the end products, indicating their denitrification ability. Bacterial community analysis of the secondary enrichments showed that Halomonas became the dominant bacteria, followed by Marinobacter and Idiomarina. Among them Halomonas and Idiomarina were successfully isolated and Halomonas was further confirmed of aerobic denitrification ability.
Key words:  marine biology  nitrogen cycle  in-situ enrichment culture  microbial diversity  aerobic denitrification  deep sea

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