Sediment properties, benthic biogenic compounds, benthic fauna density and biomasses, and benthic diffusive and total fluxes from three stations (Faro, Creek, Isla D) in Potter Cove, Antarctic

For the determination of sediment properties and biogenic sediment compounds, sediment was sampled with 3.6 cm diameter cores in five replicates by SCUBA divers. Sediment subsamples were taken with cut-off syringes (cross-sectional area = 1.65 cm²) and sliced in 1 cm intervals down to 5 cm sediment depth. Each interval was analyzed for various parameters including median grain size, porosity, photosynthetic pigments, total carbon, total organic carbon and total nitrogen. Sediment samples for photosynthetic pigments were stored at -80°C. Sediment samples of other parameters were stored at -20°C. The median grain size was determined with a Malvern Mastersizer 2000G, hydro version 5.40. Sediment porosity was determined after drying sediment samples over minimum two days at 105°C. The sediment porosity was calculated following Burdrige (2006, Geochemistry of marine sediments, Princeton University Press). Chlorophyll a (Chl a), phaeophytin (Phaeo) and fucoxanthin (Fuco) pigment concentrations were determined by HPLC The total carbon (TC) and total nitrogen (TN) was measured by combustion using an ELTRA CS2000The total organic carbon (TOC) was measured using the same method, but after acidifying the sample (3 ml of 10 M HCl).

For prokaryotic density determination, five replicate sediment sub-samples were taken with cut-off syringes (cross-sectional area = 1.65 cm²), sliced at 1 cm intervals down to 5 cm sediment depth, fixed in a 2% formaldehyde/seawater filtered solution (9 ml) and stored at 4°C. The acridine-orange-direct-count method after Hobbie (1977, https://www.ncbi.nlm.nih.gov/pmc/articles/PMC170856/) was used to stain prokaryotes in the sub-samples and subsequently counted with a microscope (Axioskop 50, Zeiss) under UV-light (CQ-HXP-120, LEj, Germany). For each sample, single cells were counted on two replicate filters and for 30 random grids per filter (dilution factor 4000). Prokaryotic biomass was estimated by the determination of the mean prokaryotic cell volume in the first two centimetres with a "New Portion" grid (Graticules Ltd, Tonbridge, UK), converted into biomass using a conversion factor of 3.0 x 10-13 g C pm-3 after Børsheim et al. (1990, https://www.ncbi.nlm.nih.gov/pmc/articles/PMC183343/) and multiplied with the prokaryotic density.
For the determination of meiofauna density, biomass and identification of meiofauna taxa, five sediment samples were taken with small sediment cores (Ø 3.6 cm). Sediment samples of the first five centimeters were stored in filtered seawater buffered 4% formaldehyde solution at 4°C till the start of the analyses. The samples were sieved over a 1 mm and 32 µm mesh. The sample was then centrifuged three times in a colloidal silica solution (Ludox TM-50) with a density of 1.18 g cm-3 and stained with Rose Bengal after Heip et al. (1985, The Ecology of marine nematodes, Oceanography and Marine Biology - An Annual Review; vol. 23). Afterwards, the taxa were identified and counted. Calcifying organisms (except nematodes and polychaetes) were acidified prior the total organic carbon content of single taxa were analyzed with a FLASH 2000 NC Elemental Analyzer (0.01% detection limit)
The benthic macrofauna was sampled by using a Van Veen grab (530 cm² surface area). At each station, four recovered sediment samples were sieved over a 1 mm mesh, stored in seawater buffered 4% formaldehyde and stained with Rose Bengal after Heip et al. (1985). In the laboratory, the taxa were identified to the lowest possible taxonomic level (at least family level), counted, weighted, and the Shannon-Wiener diversity index (H') calculated in Primer v6.0. Ash-free dry weight (AFDW) was determined by subtracting the ash weight (after combustion at 500°C) from the dry weight (dried for 48 h at 60°C). AFDW was converted into carbon by assuming that 50% of the AFDW is carbon after Wijsman et al. (1999, http://www.jstor.org/stable/24849594). The Van Veen grab sampling results in a strong underestimation of the density of the important Antarctic bivalve Laternula elliptica. Therefore, two rows of eight grids (45 cm x 45 cm) were randomly placed on the seafloor by scuba divers and photos were taken (Nikon D750 with a rectilinear Nikon 16-35 mm lens in a Nauticam underwater housing and two Inon Z-240 strobes). The photos were used to count siphons of L. elliptica to determine the density and to measure the siphon width (maximum distance between outer edges of the two siphons of one individual) at the three sites. Assuming a linear relationship between siphon width and AFDW, a conversion from the siphon width to estimated biomass of L. elliptica was performed. The calculation of the conversion relationship of the siphon width to AFDW was performed on data from the same L. elliptica population. The community bioturbation potential was calculated following formula Queiros et al. (2013, doi:10.1002/ece3.769).

Three transparent and three black chambers (inner diameter 19 cm, height 33 cm) were carefully pushed into the sediment at each station by SCUBA divers, who took special care to not disturb the sediment surface during the procedure. About 15 cm of sediment and 18 cm of overlying water was enclosed. During the incubation, which lasted 20-22 h and encompassed light and dark hours, cross-shaped stirrers powered by a 12 V lead-acid battery kept the overlying water homogenous. HOBO Pendant loggers (Onset, Bourne, USA) were placed both in situ and on land to record the amount of radiation (150-1200 nm) during the incubation with a temporal resolution of 5 minutes. The enclosed overlying water in the chambers was sampled through valves attached to the chamber lids at the start and end of the chamber incubation, using gas-tight glass syringes. The water samples were kept at in situ temperature and in darkness until further processing, which took place within 1.5 h after the samples were taken.
Subsamples were taken to either determine the oxygen concentration, the concentration of dissolved inorganic carbon (DIC) and the concentration of phosphate, ammonium, nitrite, nitrate, and sulfate. Winkler titration was used to immediately determine the oxygen concentration in the water sample in technical duplicates. For DIC analyses technical triplicates were poisoned with HgCl2 and stored at 4°C until measurement after 6 months. DIC samples were analyzed using an autosampler (Techlab, Spark Basic Marathon) with a digital conductivity measuring cell (VWR, digital conductivity meter, Germany). For nutrient analyses technical triplicates were filtered through a GF/F filter and stored at -20°C until analysis. The samples were analyzed with an autosampler (CFA SAN-plus, Skalar Analytical B.V., Netherlands) for ammonium, phosphate, nitrite and [nitrate + nitrite] concentrations. The nitrate concentration was determined by subtracting the nitrite concentration from the [nitrate + nitrite] concentration. The resulting total fluxes were calculated following Glud et al. (2008, doi:10.1080/17451000801888726).

High-resolution in situ oxygen profiles were measured using a microprofiler. The microsensors were driven from the water phase into the sediment with a spatial resolution of 100 µm and a temporal resolution of 30 seconds. On the profiler electronic unit, three custom made electrochemical O2 microsensors after Revsbech (1989, doi:10.4319/lo.1989.34.2.0474) were mounted and calibrated before deployment in oxygen saturated and oxygen depleted water. The microprofiler was programmed, so microsensors penetrated the SWI around noon at the same or the following day after the deployment. Running average smoothed profiles (https://doi.pangaea.de/10.1594/PANGAEA.885472) were used to calculate the diffusive oxygen uptake (DOU) over the SWI using Fick's first law.
For the calculation of the diffusive flux of sulfate, DIC, and nutrients, sediment was sampled with cores (10cm diameter) with predrilled holes at 1 cm depth intervals that were sealed with diffusion-tight tape. The porewater was extracted using Rhizons (type: core solution sampler, Rhizosphere Research Products, filter pore diameter of 0.1 mm) connected to 10 mL Luer lock syringes. The Rhizons were horizontally inserted into the cores and by creating a permanent vacuum in the syringes, porewater was extracted. The first drops were used to rinse the syringe and then discarded. The extracted pore water was split for sulfate analyses (sample fixed in 5% ZnAc, stored at 4°C), DIC analyses (sample fixed in HgCl2, stored at 4°C) and nutrient analyses (frozen at -20°C). DIC and nutrients were analyzed as described above. Sulfate was analyzed by using non-suppressed ion chromatography with the Methrom 761 Compact IC equipped with a Metrosep A SUPP 5 column (Methrom, Herisau, Switzerland). From the resulting depth profiles, diffusive fluxes were calculated using the same formula as for the DOU calculation, but with Ds of the specific molecule.

为测定沉积物性质与生源沉积物组分，研究人员采用直径3.6 cm的柱状样开展5次重复采样，采样由水肺潜水员完成。采用截尾注射器（横截面积=1.65 cm²）获取沉积物分样，将沉积物按1 cm间隔切割至5 cm深度。对每个间隔样品开展多项参数分析，包括粒径中值（median grain size）、孔隙度（porosity）、光合色素（photosynthetic pigments）、总碳（total carbon, TC）、总有机碳（total organic carbon, TOC）和总氮（total nitrogen, TN）。光合色素相关沉积物样品保存于-80℃，其余参数的沉积物样品保存于-20℃。粒径中值采用马尔文Mastersizer 2000G激光粒度仪（Hydro 5.40版本）测定。沉积物孔隙度通过将样品置于105℃烘干至少2天后测定，孔隙度计算参照Burdridge（2006，《海洋沉积物地球化学》，普林斯顿大学出版社）的方法。叶绿素a（Chlorophyll a, Chl a）、脱镁叶绿素（Phaeophytin, Phaeo）和岩藻黄质（Fucoxanthin, Fuco）的色素浓度采用高效液相色谱（High Performance Liquid Chromatography, HPLC）测定。总碳（TC）与总氮（TN）采用ELTRA CS2000型元素分析仪通过燃烧法测定；总有机碳（TOC）采用相同方法测定，但需预先用3 mL 10 M盐酸（HCl）对样品进行酸化处理。

为测定原核生物（prokaryote）密度，采用截尾注射器（横截面积=1.65 cm²）获取5份重复沉积物分样，按1 cm间隔切割至5 cm沉积物深度，随后将样品置于9 mL 2%甲醛过滤海水溶液中固定，并保存于4℃。采用Hobbie（1977，https://www.ncbi.nlm.nih.gov/pmc/articles/PMC170856/）提出的吖啶橙直接计数法（acridine-orange-direct-count method）对分样中的原核生物进行染色，随后使用蔡司（Zeiss）Axioskop 50型显微镜配合德国LEj公司CQ-HXP-120型紫外光源进行计数。每个样品在两张重复滤膜上随机选取30个网格进行单细胞计数（稀释倍数4000）。原核生物生物量通过以下方式估算：采用英国Tonbridge市Graticules有限公司的“New Portion”计数网格，测定沉积物表层0~2 cm的原核生物平均细胞体积，参照Børsheim等（1990，https://www.ncbi.nlm.nih.gov/pmc/articles/PMC183343/）提出的转换系数3.0×10^-13 g C·pm^-3将细胞体积转换为生物量，再乘以原核生物密度得到总生物量。

为测定小型底栖生物（meiofauna）密度、生物量并鉴定其类群，采用直径Ø3.6 cm的小型柱状样采集5份沉积物样品。表层0~5 cm的沉积物样品保存于4%甲醛缓冲过滤海水中，置于4℃直至分析开始。样品先后经1 mm和32 μm孔径网筛过筛，随后参照Heip等（1985，《海洋线虫生态学》，《海洋学与海洋生物学年度评论》第23卷）的方法，将样品置于密度为1.18 g·cm^-3的胶体二氧化硅溶液（Ludox TM-50）中离心三次，并用玫瑰红（Rose Bengal）染色，之后对类群进行鉴定与计数。除线虫和多毛类外的钙化生物需预先酸化，随后采用FLASH 2000 NC型元素分析仪测定单个类群的总有机碳含量（检测限0.01%）。

底栖大型生物（benthic macrofauna）采用Van Veen采泥器（Van Veen grab，表面积530 cm²）采集。每个站位采集的4份沉积物样品经1 mm孔径网筛过筛后，参照Heip等（1985）的方法，保存于4%甲醛缓冲海水中并用玫瑰红染色。实验室中，将类群鉴定至尽可能低的分类阶元（至少至科水平），并进行计数、称重，采用Primer v6.0软件计算香农-威纳多样性指数（Shannon-Wiener diversity index, H'）。烧失量干重（Ash-free dry weight, AFDW）通过以下方式测定：将样品置于60℃烘干48 h得到干重，再减去500℃灼烧后的灰分重量。参照Wijsman等（1999，http://www.jstor.org/stable/24849594）的研究，假设烧失量干重的50%为碳，将其转换为有机碳含量。

但Van Veen采泥器会严重低估南极重要双壳类类群椭圆侧筋蛤（Laternula elliptica）的种群密度。因此，研究人员由水肺潜水员在海底随机布设两排共8个45 cm×45 cm的样方，并使用搭载Nauticam水下摄影罩的尼康（Nikon）D750相机搭配尼克尔16-35 mm定焦镜头，配合两台Inon Z-240型闪光灯进行拍照。通过照片计数椭圆侧筋蛤的虹吸管数量以确定其种群密度，并测量三个站位个体的虹吸管宽度（单个个体两根虹吸管外边缘的最大间距）。假设虹吸管宽度与烧失量干重呈线性关系，据此建立虹吸管宽度到椭圆侧筋蛤估算生物量的转换关系，该转换关系的计算基于同一椭圆侧筋蛤种群的实测数据。群落生物扰动潜力参照Queiros等（2013，doi:10.1002/ece3.769）提出的公式进行计算。

每个站位由水肺潜水员小心将3个透明及3个黑色的培养舱（内径19 cm，高33 cm）推入沉积物中，操作过程中特别注意避免扰动沉积物表层。每个培养舱将约15 cm厚的沉积物与18 cm厚的上覆水密闭其中。培养时长为20~22 h，涵盖光照与黑暗时段，期间由12 V铅酸蓄电池驱动的十字形搅拌器维持上覆水混合均匀。HOBO Pendant型数据记录仪（美国Bourne市Onset公司）分别置于原位与陆地，记录培养期间的辐射量（150~1200 nm），时间分辨率为5分钟。培养开始与结束时，通过连接培养舱盖的阀门使用气密玻璃注射器采集舱内密闭的上覆水样品。水样置于原位温度与黑暗环境中保存，采样后1.5 h内完成后续处理。

分样分别用于测定溶解氧浓度、溶解无机碳（Dissolved Inorganic Carbon, DIC）浓度以及磷酸盐、铵盐、亚硝酸盐、硝酸盐和硫酸盐浓度。采用温克勒滴定法（Winkler titration）对两份平行水样立即测定溶解氧浓度。溶解无机碳分析的三份平行水样加入氯化汞（HgCl2）固定，置于4℃保存6个月后进行测定，采用自动进样器（Techlab公司Spark Basic Marathon型）搭配德国VWR公司数字电导池（数字电导仪）完成分析。营养盐分析的三份平行水样经GF/F滤膜过滤后，置于-20℃保存直至分析，采用荷兰Skalar Analytical B.V.公司CFA SAN-plus型自动进样器分析铵盐、磷酸盐、亚硝酸盐以及[硝酸盐+亚硝酸盐]浓度。硝酸盐浓度通过[硝酸盐+亚硝酸盐]浓度减去亚硝酸盐浓度得到。总通量的计算参照Glud等（2008，doi:10.1080/17451000801888726）的方法。

采用微型剖面仪（microprofiler）原位测定高分辨率溶解氧剖面。微型传感器从水相插入沉积物，空间分辨率为100 μm，时间分辨率为30 s。剖面仪电子单元上搭载3支定制化电化学氧微型传感器，参照Revsbech（1989，doi:10.4319/lo.1989.34.2.0474）的方法制作，部署前在饱和氧与无氧水中进行校准。剖面仪预设程序，使微型传感器于部署当日或次日正午时分穿过沉积物-水界面（sediment-water interface, SWI）。采用滑动平均平滑后的剖面（https://doi.pangaea.de/10.1594/PANGAEA.885472），通过菲克第一定律（Fick's first law）计算沉积物-水界面的扩散性耗氧量（Diffusive Oxygen Uptake, DOU）。

为计算硫酸盐、溶解无机碳与营养盐的扩散通量，采用直径10 cm的柱状样采集沉积物，样管预先按1 cm深度间隔钻孔，并用防扩散胶带密封。采用连接10 mL鲁尔锁注射器（Luer lock syringes）的Rhizons型孔隙水采样器（型号：core solution sampler，Rhizosphere Research Products公司，滤膜孔径0.1 mm）抽取孔隙水：将Rhizons采样器水平插入柱状样管，通过注射器持续抽真空以抽取孔隙水，首滴样品用于冲洗注射器后弃去。抽取的孔隙水分装为三份：硫酸盐分析样品用5%乙酸锌（ZnAc）固定，保存于4℃；溶解无机碳分析样品用氯化汞固定，保存于4℃；营养盐分析样品冷冻保存于-20℃。溶解无机碳与营养盐的分析方法如前文所述，硫酸盐采用非抑制型离子色谱法分析，仪器为瑞士Herisau市Methrom公司的761 Compact IC型离子色谱仪，搭配Metrosep A SUPP 5色谱柱。根据得到的深度剖面，采用与扩散性耗氧量计算相同的公式计算扩散通量，仅将参数替换为对应分子的扩散系数（Ds）。