Nitrogen and carbon processes in the South Indian Ocean and the French Southern and Antarctic Lands

Our data, as part of the OISO (Ocean Indien Service d'Observation) campaign, contributes to a better understanding of the physical and biological factors controlling N2 fixation in the Southern Indian Ocean and the French Southern and Antarctic lands during Austral summer January and February 2017.
We measured N2 and C fixation as well as NH4+ and NO3- assimilation in 3-6 replicates per station. Additionally, we measured diagnostic pigment concentrations to evaluate phtosynthetic community composition.
For pigment analysis 4L water was filtered through 25mm Whatman GF/F filters (pressure drop <10kPa). Samples were stored at -80°C until analysis. Pigments were analysed using High Performance Liquid Chromatography (HPLC). Pigment concentration were calculated according to Kilias et al (2013, doi:10.1111/jpy.12109).
N2 fixation experiments were carried out in three to six replicates for each station. Incubations were done in pre-acid washed polycarbonate bottles on deck with ambient light conditions. All polycarbonate incubation bottles were rinsed with deionized water, and seawater prior to incubation. We used the combination of the bubble approach (Montoya et al., 1996) and the dissolution method (Mohr et al., 2010, doi:10.1371/journal.pone.0012583) proposed by Klawonn et al. (2015, doi:10.3389/fmicb.2015.00769). Bottles were filled up to capacity to avoid air contamination. Incubations were initialized by adding a 10 ml 15-15N gas bubble. Bottles were gently rocked for 15 minutes. Finally, the remaining bubble was removed to avoid equilibration between gas and aqueous phase. after 24 hours a water subsample was taken to a 12 ml exetainer and preserved with 100 µl HgCl2 solution for later determination of exact 15N-15N concentration. Natural 15N2 was determined using Membrane Inlet Mass Spectrometry (MIMS; GAM200, IPI) for each station. Analysis of 15N2 incorporated was carried out by the Isotopic Laboratory at the UC Davis, California campus.
We used stable isotope tracers (15N) to measure dissolved inorganic nitrogen (DIN) assimilation rates. Experiments were initiated by adding a known concentration of 0.05 of K15NO3 and 15NH4Cl for oligotrophic waters of the IO and 0.625 µmol L-1 for HNLC regions in the ACC and PF (Knap et al., 1994, Waite et al., 2007, doi:10.1016/j.dsr2.2006.12.010) to one litre polycarbonate bottles. For C assimilation experiments, we added 20 µmol L-1 of NaH13CO3 to one of each of N2 fixation, NH4+ and NO3- assimilation experiment bottles. For incubation, we followed the same procedure as for N2 fixation experiments.
Findings reveal that N2 fixation occurs throughout the whole sampling area up to 55°S latitude. In addition, variations of N2 fiaxation rates between replicates were relatively high indicating a great heterogeneity of the French Southern and Antarctic waters.

References:
Montoya 1996: Montoya, Joseph P., et al. "A Simple, High-Precision, High-Sensitivity Tracer Assay for N (inf2) Fixation." Applied and environmental microbiology 62.3 (1996): 986-993.
Knap et al 1994: Knap, A., Michaels, A., Close, A., Ducklow, H. & Dickson, A. 1994. Protocols for the Joint Global Ocean Flux Study (JGOFS) Core Measurements, JGOFS, Reprint of the IOC Manuals and Guides No. 29. UNESCO, 19, 1.

本数据集作为印度洋观测服务（Ocean Indien Service d'Observation, OISO）科考航次的组成部分，旨在加深对2017年南半球夏季（1月至2月）南印度洋及法属南部和南极领地海域中调控固氮作用（N₂ fixation）的物理与生物因子的认知。
本研究为每个站位设置3至6个平行样，测定了固氮作用、固碳作用以及铵态氮（NH₄⁺）、硝态氮（NO₃⁻）的同化速率。此外，本研究通过检测诊断性色素浓度，评估光合群落的组成结构。
色素分析步骤为：取4升水样，经25mm Whatman GF/F滤膜过滤（过滤压降小于10kPa）。样品保存于-80℃环境直至上机分析。色素检测采用高效液相色谱法（High Performance Liquid Chromatography, HPLC），浓度计算参照Kilias等（2013，doi:10.1111/jpy.12109）的方法。
固氮作用实验为每个站位设置3至6个平行样。培养实验在经酸洗预处理的聚碳酸酯瓶中进行，全程置于甲板上的自然光环境中。所有培养瓶均先用去离子水、再用现场海水润洗后方可使用。本研究采用Klawonn等（2015，doi:10.3389/fmicb.2015.00769）提出的气泡法（Montoya等，1996）与溶解法（Mohr等，2010，doi:10.1371/journal.pone.0012583）联用方案。将培养瓶完全注满以避免空气混入。通过注入10毫升¹⁵N₂-¹⁵N气泡启动培养实验，随后将培养瓶轻柔摇晃15分钟。最后移除瓶内剩余气泡，防止气相与水相发生同位素平衡。培养24小时后，取12毫升水样转移至Exetainer瓶中，加入100微升氯化汞溶液保存，用于后续精确测定¹⁵N₂-¹⁵N浓度。每个站位的天然¹⁵N₂丰度均采用膜进样质谱法（Membrane Inlet Mass Spectrometry, MIMS；GAM200，IPI）测定。固氮结合的¹⁵N₂同位素分析由美国加州大学戴维斯分校同位素实验室完成。
本研究采用稳定同位素示踪剂（¹⁵N）测定溶解态无机氮（Dissolved Inorganic Nitrogen, DIN）的同化速率。对于印度洋寡营养海域，向1升聚碳酸酯瓶中加入终浓度为0.05 μmol·L⁻¹的K¹⁵NO₃与¹⁵NH₄Cl；对于南极绕极流（Antarctic Circumpolar Current, ACC）与极锋区（Polar Front, PF）的高营养盐低叶绿素（High Nutrient Low Chlorophyll, HNLC）海域，则加入终浓度为0.625 μmol·L⁻¹的上述同位素示踪剂（Knap等，1994；Waite等，2007，doi:10.1016/j.dsr2.2006.12.010）。固碳同化实验则分别向固氮作用、NH₄⁺同化以及NO₃⁻同化实验所用的培养瓶中加入20 μmol·L⁻¹的NaH¹³CO₃。培养流程与固氮作用实验保持一致。
研究结果显示，在整个采样区域直至南纬55°的海域均存在固氮作用。此外，平行样之间的固氮速率差异相对较大，表明法属南部和南极领地海域存在显著的水文异质性。
参考文献：
Montoya等1996：Montoya, Joseph P., 等. 《一种简便、高精度、高灵敏度的N₂固氮作用示踪检测方法》，《应用与环境微生物学》62.3（1996）：986-993。
Knap等1994：Knap, A., Michaels, A., Close, A., Ducklow, H. & Dickson, A. 1994. 《联合全球海洋通量研究（JGOFS）核心测量规程》，JGOFS，重印于《IOC手册与指南第29号》，联合国教科文组织，19, 1。