东海单细胞固氮生物的类群组成及其固氮贡献

Biological nitrogen fixation is an important part of the marine nitrogen cycle as it provides a source of new nitrogen that can support biological carbon export and sequestration. Recent studies have shown that small-sized unicellular diazotrophs (e.g., UCYN-A, UCYN-B, UCYN-C, and Proteobacteria) play an important role in global marine nitrogen fixation, in addition to the filamentous cyanobacteria Trichodesmium. Moreover, the contribution of unicellular diazotrophs to nitrogen fixation can be equal to or greater than that of Trichodesmium in some warm seas. Nitrogen is deficient and nitrogen fixation rate is high on the middle and outer shelf of the East China Sea (ECS) controlled by oligotrophic Kuroshio. Previous studies have demonstrated that seasonal changes of nitrogen fixation rate (spring > autumn > summer and winter) clearly differed from the Trichodesmium abundance (summer > autumn > spring and winter) in the ECS. Therefore, it is hard to explain this finding by the present Trichodesmium abundance and its contribution to nitrogen fixation. We speculate that unicellular diazotrophs make an important contribution to nitrogen fixation..To examine the group composition and spatiotemporal distribution patterns of unicellular diazotrophs and their dominance in diazotrophic community in the ECS, the methods of high-throughput nitrogenase (nifH) sequencing, quantitative Polymerase Chain Reaction (qPCR), Reverse Transcription qPCR (RT-qPCR), and fluorescence microscopical counting will be applied comprehensively. To ascertain the contribution of unicellular diazotrophs to nitrogen fixation, size-fractioned (10 μm) nitrogen fixation rate will be determined through an improved 15N-isotope dilution method. On the basis of above-mentioned works, main controlling factors of nitrogen fixation in different seasons in the ECS will be revealed in combination with environmental conditions. The project findings will clarify the composition and distribution of unicellular diazotrophs and their contribution to nitrogen fixation in the ECS. Furthermore, it can provide a better understanding on the regional nitrogen cycle.

生物固氮是海洋新生氮的重要来源，深刻影响海洋氮循环。近年研究证实，单细胞固氮生物是除束毛藻之外全球海洋生物固氮的重要贡献者，在部分高温海区其固氮量贡献等同甚至超过了束毛藻。受黑潮影响，东海中、外陆架氮较缺乏，固氮速率较高。以往研究发现，东海固氮速率呈春＞秋＞夏冬季，但束毛藻丰度呈夏＞秋＞春冬季。因此，仅根据束毛藻丰度及其固氮贡献尚无法完整解释该现象，推测单细胞固氮生物可能是另一个重要的固氮贡献者。本项目拟运用nifH基因高通量测序、qPCR、RT-qPCR和荧光显微镜检等方法，探明东海单细胞固氮生物的类群组成、时空分布及其在固氮生物群落中的优势度；采用改进的15N同位素示踪法测定分级固氮速率，估算单细胞固氮生物的固氮量贡献；在此基础上，结合环境因子，揭示不同季节单细胞固氮生物对东海固氮作用的影响机制。研究成果将较全面地了解东海单细胞固氮生物组成及其固氮贡献，对深入认识东海氮循环有重要意义。

生物固氮是海洋新生氮的重要来源，深刻影响海洋氮循环。受黑潮影响，东海中、外陆架氮较缺乏，固氮速率较高。但目前，对东海生物固氮的来源仍知之甚少，缺乏单细胞固氮生物对东海固氮贡献的认识。为此，本项目研究了东海的固氮生物组成及其固氮速率。显微镜检发现，2021年春、秋季束毛藻丰度分别为27.34×103、1532.2×103 trichomes m−2，胞内植生藻/眉藻丰度分别为24.93×103、22.44×103 heterocysts m−2；2013年夏季东海束毛藻、胞内植生藻/眉藻丰度分别为10.41×106 trichomes m−2、2129×103 heterocysts m−2。nifH基因高通量测序发现，2021年春、秋季，东海固氮生物以变形菌、蓝藻（含UCYN-A、束毛藻、眉藻等）、厚壁菌为主，秋季固氮生物多样性显著高于春季；2016年夏、冬季，长江口及邻近海域固氮生物以异养的变形菌和厚壁菌为主，束毛藻、UCYN-A等蓝藻占比较低，长江口缺氧水体的固氮生物多样性指数显著高于非缺氧水体，表明缺氧形成可能有利于固氮生物的生长及固氮作用。基于15N2充气法，2013年夏季黄东海平均固氮速率为81.7 μmol N m−2 d−1，最高可达511.8 μmol N m−2 d−1；2014年夏季长江口及邻近陆架固氮速率平均值为24.3 μmol N m–2 d–1，根据10 μm分级固氮速率数据，单细胞固氮生物的固氮量贡献约为40%。基于15N2溶解法，2021年春季东海固氮速率为9.42 μmol N m–2 d–1，单细胞固氮生物的固氮量贡献为61%；秋季在计入、不计0.1%光强所在水层固氮时，固氮速率分别为104.64、51.19 μmol N m–2 d–1，其中单细胞固氮生物的固氮量贡献分别为72%、71%。空间尺度上，丝状固氮蓝藻丰度、固氮速率在黑潮、台湾暖流海域较高，在长江冲淡水、东海近岸海域较低；单细胞固氮生物的固氮贡献在长江口较高，在东海东南部较低。相关性分析显示，夏季固氮速率与束毛藻和胞内植生藻丰度、温度、盐度显著正相关，而与硝酸盐和氮磷比显著负相关。项目研究成果初步发现了黑潮入侵对固氮生物组成及其固氮的调控作用，阐明了单细胞固氮生物的固氮量贡献，对深入认识东海的氮循环有重要意义。