Benthic anammox and dentrification rates measured in a slurry incubation experiments at four different stations in the Arabian Sea off Pakistan during the METEOR cruise M74/2 in 2007

Vertical distributions of benthic denitrification and anammox rates within the sediment were estimated from slurry incubation experiments. Rates were used to calculate the contribution of anammox and denitrification to the total N-loss. Briefly, MUC sediment cores were sliced in 2 cm intervals and the sediment was diluted and incubated with degassed bottom water in a gas tight bag. After pre-incubating the bags for 2 h, 15N-labeled substrates were injected into the bags and the slurries were thoroughly mixed. Incubations were performed in the dark at in situ temperatures. The N2 isotope ratio (28N2, 29N2, and 30N2) was determined by gas chromatography-isotopic ratio mass spectrometry (VG Optima, Micromass) and calculated according to Kuypers et al. (2005) and Holtappels et al. (2011), respectively.Furthermore, total organic carbon and nitrogen concentrations were measured of core sediment layers corresponding to those used for rate measurements. Concentrations of organic carbon and nitrogen were determined by combustion/gas chromatography (Carlo Erba NA-1500 CNS analyzer) of dried sediment samples after acidification. The same sediment layer were also used to extract nucleic acids. The concentrations of the DNA in the samples were measured spectrophotometrically with a NanoDrop instrument (Thermo Fisher Scientific Inc.). The biomarker functional gene nirS, encoding the cd1-containing nitrite reductase, for both denitrifiers and marine anammox bacteria were quantified with real-time PCR, using the primers cd3aF/R3cd (5'-GTSAACGTSAAGGARACSGG-3' (Michotey et al., 2000)/5'-GASTTCGGRTGSGTCTTGA-3'; Throback et al., 2004) and Scnir372F/Scnir845R (5'-TGTAGCCAGCATTGTAGCGT-3'/5'-TCAAGCCAGACCCATTTGCT-3'; Lam et al., 2009).

本研究通过泥浆培养实验，估算了沉积物内底栖反硝化（benthic denitrification）与厌氧氨氧化（anammox）速率的垂向分布格局。基于上述速率，可计算厌氧氨氧化与反硝化对总氮流失（total N-loss）的贡献占比。简要而言，将MUC沉积物岩芯以2 cm为间隔进行分层切片，将沉积物稀释后与脱气底层水一同置于气密袋中进行培养。待预培养2小时后，向袋内注入15N标记底物，随后充分混匀泥浆体系。培养全程保持避光，并维持原位培养温度。采用气相色谱-同位素比值质谱法（gas chromatography-isotopic ratio mass spectrometry，型号VG Optima，Micromass公司）测定N₂同位素比值（²⁸N₂、²⁹N₂与³⁰N₂），并分别参照Kuypers等（2005）与Holtappels等（2011）的方法进行计算。此外，针对速率测定所用的沉积物岩芯层位，同步测定了总有机碳与总氮浓度。有机碳与总氮浓度的测定采用酸化预处理后的干燥沉积物样品，通过燃烧-气相色谱法（Carlo Erba NA-1500 CNS元素分析仪）完成。上述同一沉积物层位同时用于核酸提取，样品中DNA浓度采用NanoDrop仪器（Thermo Fisher Scientific Inc.）通过分光光度法进行测定。针对反硝化菌与海洋厌氧氨氧化菌的生物标志物功能基因nirS（编码含cd1结构域的亚硝酸还原酶），采用实时荧光定量PCR进行定量检测，所用引物对为cd3aF/R3cd（5'-GTSAACGTSAAGGARACSGG-3'，Michotey等，2000；5'-GASTTCGGRTGSGTCTTGA-3'，Throback等，2004）与Scnir372F/Scnir845R（5'-TGTAGCCAGCATTGTAGCGT-3'；5'-TCAAGCCAGACCCATTTGCT-3'，Lam等，2009）。