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.

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光谱（Synchrotron-based Fourier Transform Infrared, FTIR）大分子谱。数据代表与关键大分子相关的波数对应的峰面积。方法详情参见Duncan等人（2021）发表于《New Phytologist》的研究。

实验设计与围隔培养设置
围隔培养的设置及条件如先前所述（Deppeler等人，2018；Hancock等人，2018）。简要来说，2014年11月19日，从南极洲戴维斯站（Davis Station）近海约1公里处的破碎固定冰间无冰区采集了近岸天然南极微生物群落（坐标：68°35′S，77°58′E）。该群落被培养在6个650升聚氨酯水箱（围隔培养系统）中，CO₂分压（partial pressure of CO₂, fCO₂）梯度为343、506、634、953、1140和1641微大气压（分别记为M1至M6）。这些fCO₂水平对应的pH值范围为8.17至7.57。温度维持在0.0°C±0.5°C，围隔培养系统通过中央螺旋桨（15转/分钟）持续搅拌以实现温和混合，并加盖密封。初始辐照度保持较低水平（0.8±0.2 μmol光子/m²/s），同时让细胞生理适应fCO₂水平的升高（为期5天）。当所有6个围隔培养系统达到目标fCO₂水平后，在第5至8天将光照逐渐增加至89±16 μmol光子/m²/s，并采用19小时光照：5小时黑暗的周期，以模拟当前自然条件。为构建碳酸盐化学梯度，向其中5个围隔培养系统中添加经CO₂饱和的过滤海水。每日测量以监测pH值和溶解无机碳（dissolved inorganic carbon, DIC）。fCO₂调控、分析流程及计算的详情参见Deppeler等人（2018）。本研究中用于生理和大分子测量的样本在培养期结束时（第18天）采集（Deppeler等人，2018）。

细胞体积
通过光学显微镜测定M1和M6中选定类群的细胞体积。在已校准显微镜（尼康Eclipse Ci-L，日本）下对细胞成像，并使用ImageJ软件（Schneider等人，2012）测定细胞的长度、宽度和高度（每个类群24至77个细胞）。随后根据细胞形态及Hillebrand等人（1999）所述的相应公式计算生物体积。

基于FTIR的大分子含量分析
对第18天从所有6个围隔培养系统中采集的选定硅藻类群，采用同步辐射傅里叶变换红外显微光谱对福尔马林固定（终浓度2% v/v）的细胞进行大分子组成测定。测量在水合细胞上进行，并根据先前研究（Sackett等人，2013；2014；Sheehan等人，2020）处理数据。简要来说，固定细胞直接加载到带有0.3 mm厚氟化钙（CaF₂）窗口的微压缩池中。2015年11月，在墨尔本澳大利亚同步辐射装置的红外显微光谱光束线（Infrared Microspectroscopy Beamline）上，以透射模式采集单个细胞的光谱数据（每个围隔培养系统中每个类群15至49个细胞）。使用Vertex 80v FTIR光谱仪（布鲁克光学）结合配备液氮冷却碲镉汞检测器的红外显微镜（Hyperion 2000，布鲁克），在4000至800 cm⁻¹的测量范围内采集光谱。采集64次共加干涉图，波数分辨率为6 cm⁻¹。为实现单个细胞测量，所有测量均以透射模式进行，测量区域孔径大小为5×5 µm。光谱采集和仪器控制通过Opus 6.5软件（布鲁克）实现。选择生物相关区域的归一化光谱中代表关键大分子的吸收带。具体而言，选择了酰胺II（~1540 cm⁻¹）、游离氨基酸（~1452 cm⁻¹）、羧酸盐（~1375 cm⁻¹）、脂质中的酯羰基（~1745 cm⁻¹）和饱和脂肪酸（~2920 cm⁻¹）吸收带。红外光谱数据通过R语言（R Development Core Team 2018）中的自定义脚本进行分析。选择包含主要生物信号的3050-2800 cm⁻¹和1770-1100 cm⁻¹区域进行分析。光谱数据经平滑处理（两侧各4点），并使用R语言prospectr包中的Savitzky-Golay算法进行二阶导数（三阶多项式）变换（Stevens和Ramirez-Lopez，2014），随后采用单变量归一化（Single Normal Variate, SNV）方法归一化。单个类群的大分子含量通过积分每个指定峰下的面积估算，根据比尔-朗伯定律（Beer-Lambert Law）提供代谢物含量，该定律假设吸光度与相对分析物浓度呈直接关系（Wagner等人，2010）。积分峰面积反映样本间大分子含量的相对变化。由于大分子吸收特性的差异，峰面积仅可作为化合物内的相对测量指标。