SEM based morphology of Emiliania huxleyi, 2011-2012 (E Hux Response to pCO2 project)
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<p><strong>Culturing</strong>:</p> <p>Cultures of <em>E. huxleyi </em>Strain CCMP2668, were innoculated at low cell density into media prepared from autoclaved filtered seawater with nutrient amendments based on F/2 medium kit from the National Center for Marine Algae and Microbiota, with a 1:25 reduction in nutrient additions. These were allowed to acclimate for approximately five generations, until cell density neared levels likely to significantly change the pH/pCO2. Daily dilutions of cultures with pre-equilibrated media kept cell density low (<1x10<sup>5</sup> cells/ml), ensured cells remained in exponential growth phase and prevented excessive drawdown of nutrients and CO<sub>2</sub>. Cell density was determined by flow cytometry (Model) and each flask was diluted with media that was continuously sparged with air containing 400, 750 or 1000 ppm CO2. Air mixtures were created using CO2 free air (Powerex air compressor, and Twin Towers CO2 scrubber) and pure CO2 (Airgas) combined using a system of mass flow controllers (Sierra Instruments) and verified using a non-dispersive infrared CO2 sensor (Licor 820). Cultures were maintained in 1 liter polycarbonate flasks at 15<sup>0 </sup>C under a 12/12 light dark cycle. Replicates (n=5) were placed in Plexiglas chambers which were supplied with a flow of the appropriate air mixture for each treatment. Preliminary experiments showed that gas exchange across the air/water surface significantly helped to maintain the target pCO2 in cultures without the mechanical disturbance of bubbling. Sedimentation was minimized by gentle mixing of the cultures by rotation of the bottles twice a day, during sampling and dilution. Cell densities ranged between about 30,000 cells/ml after dilutions to 80,000 cells/ml on the following day. The culture volume that was removed was used for analyses, and replaced with pre-equilibrated media. Cultures were maintained in this fashion for about 8 days. Since the first dilution occurred on day 4 after innoculation, this gives a total of 12 to 14 days in culture at experimental conditions. This experiment was carried out twice, in 2011 and 2012. </p> <p><strong>SEM</strong>:</p> <p>On days 1 and 8, a few small volume of culture from each replicate was dropped onto SEM stubs and allowed to dry. The stubs were sputter coated with Palladium gold for approximately three minutes (2012) or one minute (2011). Five images of each replicate with three or more cells in each image were taken at 5000x magnification (Smith et al. 2012) using an FEI Quanta 450 Scanning Electron Microscope. To compose each image the field was zoomed out to about 200x magnification to reduce bias in finding clusters of three or more cells. When a cluster of three of more cells was found, the magnification was changed to 5000x magnification and the image was focused and captured.</p> <p>Parameters including cell size, coccolith size, coccoliths per cell, and percentage of malformed coccoliths were measured from the SEM images. Cell size (control: n= 235, moderate: n= 234, high: n= 200) and coccolith size (control: n= 75, moderate: n= 72, high: n= 67) were calculated using the free hand tool in Image J. To determine the number of coccoliths per cell and the percentage of malformed coccoliths, images were loaded into Windows Photo Viewer (control: n= 1453, moderate: n= 1409, high: n= 1215). Coccoliths malfomation was assessed by the scheme of DeBodt et al, 2010. </p> <p><strong>Carbonate chemsitry: (THIS DATASET)</strong></p> <p>pH was measured photometrically using 1 cm cuvettes, m-cresol dye and an Agilent 5480 UV-VIS spectrophotometer (+/- 0.02). Alkalinity was measured by gran titration using a Titrando 888, and 0.1 N HCl titrant, in a temperature controlled titration vessel (+/1 5 ueq/kg). Other parameters were calculated with CO2sys. pCO2 conditions were the same in the two experiments with the exception that the moderate concentration had slightly more elevated pCO2 in 2011 (662 ppm compared to 602 ppm). (When day 1 is included for 2012, there is no significant difference; the difference in the Moderate treatment is present when only day 12 and 14 are included.)</p> <p><strong>Related dataset: </strong><a href=\"http://www.bco-dmo.org/dataset/520651\" target=\"_blank\">Emiliania huxleyi carbonate data</a></p>
**培养方法**： 将赫氏球石藻（*Emiliania huxleyi*）CCMP2668菌株以低细胞密度接种至培养基中，该培养基以高压灭菌过滤海水为基底，添加美国国家海洋藻类与微生物培养中心（National Center for Marine Algae and Microbiota）出品的F/2培养基试剂盒配套营养成分，且营养添加量缩减为原配方的1/25。将培养物驯化约5代，直至细胞密度达到可显著改变pH/pCO₂的水平。每日使用预平衡培养基稀释培养物，维持细胞密度低于1×10⁵ cells/ml，以保证细胞始终处于指数生长期，并避免营养物质与CO₂的过度消耗。细胞密度通过流式细胞术（型号未标注）测定，每个培养瓶使用持续通入含400、750或1000 ppm CO₂的空气的培养基进行稀释。空气混合气由无CO₂空气（Powerex空气压缩机、双塔CO₂洗涤器）与纯CO₂（Airgas）通过质量流量控制器（Sierra Instruments）混合配制，并采用非色散红外CO₂传感器（Licor 820）进行浓度验证。培养物置于1升聚碳酸酯培养瓶中，在15℃、12/12小时光暗周期下培养。设置5个生物学重复（n=5），将其置于有机玻璃培养舱中，每个处理组通入对应配比的空气混合气。预实验结果显示，气液界面的气体交换可在无需鼓泡机械扰动的前提下，有效维持培养物的目标pCO₂水平。通过每日取样与稀释时每日两次旋转培养瓶进行轻柔混匀，以减少细胞沉降。稀释后细胞密度约为3×10⁴ cells/ml，次日可增长至8×10⁴ cells/ml。取出的培养体积用于后续分析，并以预平衡培养基补足体积。以此方式维持培养约8天。由于首次稀释于接种后第4天进行，因此实验条件下的总培养时长为12~14天。本实验分别于2011年与2012年重复开展两次。 **扫描电子显微镜（Scanning Electron Microscope, SEM）分析**： 分别于第1天和第8天，从每个重复培养物中取少量样本滴加至SEM载样台，自然晾干。2012年的载样台采用钯金溅射镀膜约3分钟，2011年则镀膜1分钟。使用FEI Quanta 450型扫描电子显微镜，以5000倍放大倍率拍摄每个重复样本的5张图像（每张图像包含至少3个细胞）（Smith et al. 2012）。为避免筛选细胞团时的偏倚，首先将视野缩放至约200倍以定位包含3个及以上细胞的集群，随后切换至5000倍放大倍率进行对焦与成像。 从SEM图像中测量多项参数：细胞大小、球石（coccolith）大小、每个细胞的球石数量以及畸形球石比例。细胞大小（对照组n=235，中等浓度组n=234，高浓度组n=200）与球石大小（对照组n=75，中等浓度组n=72，高浓度组n=67）通过Image J软件的自由手绘工具计算获得。统计每个细胞的球石数量与畸形球石比例时，将图像导入Windows照片查看器（对照组n=1453，中等浓度组n=1409，高浓度组n=1215）。畸形球石的判定参考DeBodt等人2010年提出的标准。 **碳酸盐化学（本数据集）**： 采用1cm比色皿、间甲酚紫染料与安捷伦5480型紫外-可见分光光度计，通过光度法测定pH值（测量误差±0.02）。碱度通过Gran滴定法测定：使用Titrando 888滴定仪、0.1N HCl滴定液，在控温滴定槽中完成操作（测量误差±5 μeq/kg）。其余参数通过CO2sys软件计算得到。两次实验的pCO₂处理条件基本一致，仅2011年中等浓度组的pCO₂略高（662 ppm，2012年为602 ppm）。（若包含2012年的第1天样本，则两组无显著差异；仅统计第12和14天样本时，中等浓度组存在上述差异。） **相关数据集**：<a href="http://www.bco-dmo.org/dataset/520651" target="_blank">赫氏球石藻碳酸盐数据</a>
