Macromolecular data of diatoms exposed to Ocean Acidification - Mesocosm Experiments at Davis Station, Antarctica, 2014-2015

Synchrotron based FTIR macromolecule profiles of 5 diatom species from the AAS_4026 ocean acidification project. Data represent the peak areas for wavenumbers related to key macromolecules. For details on methods see Duncan et al. (2021) New Phytologist. Experimental design and mesocosm set upMesocosm set up and conditions were as described previously (Deppeler et al., 2018; Hancock et al., 2018). Briefly, a near-shore, natural Antarctic microbial community was collected from an ice-free area among broken fast ice approximately 1km offshore from Davis Station, Antarctica (68° 35ʹ S, 77° 58ʹ E) on 19 November 2014. This community was incubated in 6 x 650L polyurethane tanks (mesocosms) across a gradient of fCO2 levels (343, 506, 634, 953, 1140 and 1641 μatm; denoted M1 – M6). These fCO2 levels corresponded to pH values ranging from 8.17 to 7.57. Temperature was maintained at 0.0 °C ± 0.5 °C and the mesocosms were stirred continuously by a central auger (15 r.p.m.) for gentle mixing and covered with an air-tight lid. Irradiance was initially kept low (0.8 ± 0.2 μmol photons m-2s-1), while cell physiology was left to acclimate to increasing fCO2 levels (over 5 days). When target fCO2 levels were reached in all six mesocosms, light was gradually increased (days 5-8) to 89 ± 16 μmol photons m-2s-1 on a 19 h:5 h light:dark cycle, to mimic current natural conditions. To generate the gradient in carbonate chemistry, filtered seawater saturated with CO2 was added to five of the mesocosms. Daily measurements were taken to monitor pH and dissolved inorganic carbon (DIC). For details of fCO2 manipulations, analytical procedures and calculations see Deppeler et al., (2018). Samples for physiological and macromolecular measurements in this study were taken on day 18, at the end of the incubation period (Deppeler et al., 2018). Cell volumeCell volume was determined for selected taxa from M1 and M6 via light microscopy. Cells were imaged on a calibrated microscope (Nikon Eclipse Ci-L, Japan) and length, width and height (24-77 cells per taxa) determined using ImageJ software (Schneider et al., 2012). Biovolume was then calculated according to the cell morphology and corresponding equations described by Hillebrand et al (1999). Macromolecular content by FTIR The macromolecular composition of the selected diatom taxa sampled from all six mesocosms on day 18 was determined using Synchrotron based FTIR microspectroscopy on formalin-fixed (2% v/v final concentration) cells. Measurements were made on hydrated cells and processed according to previous studies (Sackett et al. 2103; 2014; Sheehan et al. 2020). Briefly, fixed cells were loaded directly onto a micro-compression cell with a 0.3 mm thick CaF2 window. Spectral data of individual cells (between 15-49 cells per taxon per mesocosm) were collected in transmission mode, using the Infrared Microspectroscopy Beamline at the Australian Synchrotron, Melbourne, in November 2015. Spectra were acquired over the measurement range 4000− 800 cm−1 with a Vertex 80v FTIR spectrometer (Bruker Optics) in conjunction with an IR microscope (Hyperion 2000, Bruker) fitted with a mercury cadmium telluride detector cooled with liquid nitrogen. Co-added interferograms (n = 64) were collected at a wavenumber resolution of 6 cm−1s. To allow for measurements of individual cells, all measurements were made in transmission mode, using a measuring area aperture size of 5 × 5 µm. Spectral acquisition and instrument control were achieved using Opus 6.5 software (Bruker). Normalised spectra of biologically relevant regions revealed absorbance bands representative of key macromolecules were selected. Specifically, the amide II (~1540 cm-1), Free Amino Acid (~1452 cm-1), Carboxylates (~1375 cm-1), Ester carbonyl from lipids (~1745 cm-1) and Saturated Fatty Acids (~2920 cm-1) bands were selected. Infra-red spectral data were analysed using custom made scripts in R (R Development Core Team 2018). The regions of 3050-2800, 1770-1100 cm-1, which contain the major biological were selected for analysis. Spectral data were smoothed (4 pts either side) and second derivative (3rd order polynomial) transformed using the Savitzky-Golay algorithm from the prospectr package in R (Stevens and Ramirez-Lopez, 2014) and then normalised using the method of Single Normal Variate (SNV). Macromolecular content for individual taxon was estimated based on integrating the area under each assigned peak, providing metabolite content according to the Beer-Lambert Law, which assumes a direct relationship between absorbance and relative analyte concentration (Wagner et al., 2010). Integrated peak areas provide relative changes in macromolecular content between samples. Because of the differences in absorption properties of macromolecules, peak areas can only be used as relative measure within compounds.

本数据集为AAS_4026海洋酸化项目中5种硅藻的同步辐射傅里叶变换红外（Synchrotron-based FTIR）大分子谱图。数据为与关键大分子相关的波数对应的峰面积。实验方法细节详见Duncan等人（2021）发表于《新植物学家（New Phytologist）》的研究。

## 实验设计与中型受控生态系统（mesocosm）构建
中型受控生态系统的构建与实验条件详见此前研究（Deppeler等，2018；Hancock等，2018）。简要而言，研究样本采集于2014年11月19日，采自南极洲戴维斯站（68°35′S，77°58′E）附近离岸约1km的破碎固定冰间无冰区域的近岸天然南极微生物群落。该微生物群落被接种于6个650L的聚氨酯中型受控生态系统（mesocosms）中，设置了梯度化的二氧化碳分压（fCO2）水平（343、506、634、953、1140及1641 μatm，分别标记为M1至M6）。上述fCO2水平对应的pH值范围为8.17至7.57。
实验温度维持在0.0℃±0.5℃，中型生态系统通过中央螺旋搅拌器（转速15转/分钟）持续温和搅拌，并加盖气密盖。光照初始维持在较低水平（0.8±0.2 μmol光子·m⁻²·s⁻¹），使细胞生理适应逐步升高的fCO2水平，适应周期为5天。当6个中型生态系统均达到目标fCO2水平后，于第5至8天将光照逐步提升至89±16 μmol光子·m⁻²·s⁻¹，采用19小时光照:5小时黑暗的光周期，以模拟当前自然环境条件。
为构建碳酸盐化学梯度，向其中5个中型生态系统中添加了二氧化碳饱和的过滤海水。每日进行监测以记录pH值与溶解无机碳（DIC）水平。fCO2调控、分析流程与计算方法的细节详见Deppeler等（2018）的研究。本研究中用于生理与大分子检测的样本采集于培养周期结束时的第18天（Deppeler等，2018）。

## 细胞体积测定
本研究通过光学显微镜测定了M1与M6组中选定类群的细胞体积。使用经过校准的显微镜（Nikon Eclipse Ci-L，日本）对细胞成像，通过ImageJ软件（Schneider等，2012）测定细胞的长、宽、高（每个类群测定24~77个细胞）。随后根据细胞形态与Hillebrand等（1999）提出的对应公式计算细胞生物体积。

## 傅里叶变换红外（FTIR）大分子含量检测
本研究通过同步辐射傅里叶变换红外显微光谱技术，对第18天从全部6个中型生态系统中采集的、经福尔马林固定（终浓度2%体积比）的选定硅藻类群细胞进行大分子组成分析。检测采用水合细胞进行，实验流程参照此前研究（Sackett等，2013、2014；Sheehan等，2020）。
简要而言，固定后的细胞直接加载于带有0.3mm厚氟化钙（CaF₂）窗片的微压缩样品池中。2015年11月，在澳大利亚墨尔本同步辐射装置的红外显微光谱光束线站，以透射模式采集单个细胞的光谱数据（每个类群在每个中型生态系统中采集15~49个细胞）。采用Vertex 80v型傅里叶变换红外光谱仪（Bruker Optics）搭配Hyperion 2000型红外显微镜（Bruker）进行光谱采集，该显微镜配备液氮冷却的汞镉碲（MCT）探测器，光谱采集范围为4000~800 cm⁻¹。以6 cm⁻¹的波数分辨率采集共叠加干涉图（n=64）。为实现单个细胞的检测，所有测量均采用透射模式，测量区域孔径尺寸为5×5 μm。光谱采集与仪器控制通过Opus 6.5软件（Bruker）完成。
选取具有生物学意义的归一化光谱区域，提取代表关键大分子的吸收峰。具体选取的吸收峰包括：酰胺II带（~1540 cm⁻¹）、游离氨基酸带（~1452 cm⁻¹）、羧酸根带（~1375 cm⁻¹）、脂质酯羰基带（~1745 cm⁻¹）以及饱和脂肪酸带（~2920 cm⁻¹）。
红外光谱数据通过R语言中自定义脚本进行分析（R开发核心团队，2018）。选取包含主要生物组分的3050~2800 cm⁻¹与1770~1100 cm⁻¹区域进行分析。光谱数据经平滑处理（两侧各4个点）与二阶导数（三阶多项式）变换，变换采用R语言prospectr包中的Savitzky-Golay算法（Stevens与Ramirez-Lopez，2014），随后通过单变量归一化（SNV）方法进行归一化。
单个类群的大分子含量通过积分每个指定峰下的面积进行估算，基于比尔-朗伯定律（Beer-Lambert Law）计算代谢物含量，该定律假设吸光度与分析物相对浓度呈直接线性关系（Wagner等，2010）。积分峰面积可反映样本间大分子含量的相对变化。由于不同大分子的吸收特性存在差异，峰面积仅可用于同一化合物类别的相对定量。