舟山群岛海域沉积物厌氧氨氧化细菌多样性

Diversity of anaerobic ammonium oxidizing bacteria in marine sediments from the Zhoushan Islands

通过构建16S rRNA基因文库和克隆测序研究了舟山海域沉积物中厌氧氨氧化细菌(AAOB)的多样性。从5个克隆文库中共获得297条16S rRNA基因序列,包含16个操作分类单元(OTUs)。离岸距离较近的3个站具有相似的群落结构,且与离岸较远的2个站具有明显差异。系统发育结果显示,Scalindua属是该海域AAOB的优势类群,95.3%的序列与Scalindua属AAOB具有较近的亲缘关系;1条序列与Kuenenia属具有较近的亲缘关系;此外还有15条序列与已知的AAOB相似性较低。相关性分析表明沉积物有机碳含量与多样性指数具有显著正相关,可能是该海域AAOB多样性变化的重要影响因子。

Anaerobic ammonium oxidation（ anammox） is an important process regulating the balance of marine nitrogen and ecosystem health,particularly under anoxic conditions. The Zhoushan Islands are located east of the Changjiang river estuary,and collect a high load of anthropogenic nitrogen,which leads to severe eutrophication and seasonal hypoxia.Therefore,bacteria that mediate the anammox process are of major interest in this area. Although the importance of anammox-mediating bacteria is known,few studies on these bacteria have been conducted in the East China Sea. To the best of our knowledge,this study is the first to report the diversity,community composition,and distribution of anammox bacteria in the Zhoushan Islands. Field surveys were conducted in June 2012; triplicate surface sediment samples were collected at each site and stored in sterile plastic bags at-80℃ for subsequent DNA extraction and molecular analysis. Total genomic DNA was extracted using the Fast DNA SPIN Kit for soil. Environmental DNA extracted from sediment samples was used as the template for PCR amplification of anammox 16 S rRNA genes using primers Amx368f—Amx820r. The purified fragments were cloned and sequenced for phylogenetic and statistical analyses. In total,297 sequences belonging to16 operational taxonomic units（ OTUs） were obtained from five 16 S rRNA gene libraries. The biodiversity of anammox bacteria was examined using rarefaction analysis of the 16 S rRNA genes,the Chao1 estimator,and Shannon index calculations. EZ3-1,EZ3-3,and EZ1-5 exhibited higher diversity than EZ1-3 and EZ3-5. A significant positive correlation between Shannon index and organic carbon content indicate that sediment organic carbon content plays an important role inmodulating anammox bacterial diversity in the Zhoushan Island area. Weighted Uni Frac PCo A analysis of the 16 S rRNA genes demonstrated spatial heterogeneity in the community composition of anammox bacteria; the anammox bacteria in the study area could be divided into three distinct groups. EZ3-1,EZ3-3,and EZ1-3 exhibited similar community composition,while EZ1-5 and EZ3-5 clustered separately. The composition might be affected by distance from land and water depth.Phylogenetic analysis indicated that anammox bacterial communities were dominated by the genus Scalindua（ 283 of 297sequences）. The Scalindua cluster comprised of 245 Scalindua sequences with 95. 4% —98. 5% sequence similarity to Candidatus Scalindua brodae,and 36 Scalindua sequences with 94.5% —97.6% sequence identity to the 16 S rRNA gene of Candidatus Scalindua wagneri. In addition,two Scalindua sequences that grouped in the Scalindua cluster were distantly related to known Scalindua species,indicating that they might represent unidentified species of Scalindua. One sequence recovered from the EZ3-5 library was closely related to genus Kuenenia,which is traditionally considered an anammox bacterial genus present in freshwater or low-salinity environments. Our results suggest that members of the genus Kuenenia may possess the ability to survive in high-salinity marine environments. Additionally,two clusters of unknown sequences（ unknown cluster I and II） were not classified under any known anammox bacterial genus,but were most closely related to16 S rRNA gene sequences recovered from arctic sediment and mangrove sediment,respectively.