Photosynthetic response of sea ice algae to low iron

Fast repetition rate fluorometer (FRRF) study of sea ice algae in low iron conditions. Algae were grown in an ice tank and the measurements were made at the end with a Chelsea Instruments FRRF. Materials and Methods (see the download document for original formatting and formulas)1. Ice tank incubationThe polar pennate diatom Fragilariopsis cylindrus, isolated from Antarctic pack ice in 2015 (Davis station, East Antarctica) was incubated in a purpose designed ice tank (Island Research, Tasmania). The ice tank, which was constructed of titanium to minimise dissolved Fe, was placed into a freezer (–20 degrees C), and the ice thickness and temperature gradient controlled by interaction between a basal heater and the adjustable ambient freezer temperature (see Kennedy et al., 2012). This enabled an ice thickness of approximately 5.5 cm to be maintained during the experiment. The diatom F. cylindrus was incubated in Aquil media (Price et al. 1989) buffered with ethylenediaminetetraacetic acid (EDTA) at 150 micro mol photons m−2 s−1 (PAR), a salinity of 35, and a Fe concentration of 400 nM, where the concentration of total inorganic forms of Fe (Fe') was 1.54 nM, this being continuously supplied to the medium and the exact value calculated using the software Visual MINTEQ, ver. 3.1 (https://vminteq.lwr.kth.se). Before a freezing cycle started, the seawater temperature was maintained at 2.5 degrees C, and a sample was obtained to assess the original physiological state of the algae (Day−5, hereafter). After obtaining the sample, the seawater temperature was set to −1.8 degrees C to initiate ice formation in the ice tank. Once ice had formed at Day−2, the under-ice seawater was partially replaced with ultrapure water to reduce the salinity down to 35, because the salinity had increased (to approximately 38) as a result of brine rejection from the ice. After a 2-day acclimation to the new salinity, ice samples were obtained every 5 days for 20 days (i.e., Days 0, 5, 10, 15, and 20). To minimize the heterogeneity among ice cores, ice samples were randomly collected from the tank chamber with a trace metal-free hand drill (2 cm in diameter) from randomly annotated grids on the ice surface, following normal random sampling numbers generated by the software R (https://www.r-project.org/). To assess the effects of melting and high light exposure, the ice was melted at 2.5 degrees C for 2 days. After the ice had completely melted, the seawater was exposed to a high light level, which was adjusted to represent the likely summer light intensity at the surface in ice-edge regions (800 micro mol photons m−2 s−1; MODIS Aqua), Seawater samples were obtained both after the melting and light exposure events (Melt and Light, respectively, hereafter). 2. Fast repetition rate (FRR) fluorometryTo monitor the photophysiology of F. cylindrus during the freezing and melting processes, variable chlorophyll a fluorescence (ChlF) measurements were conducted using a bench-top Fast Repetition Rate fluorometer (FRRf) (FastOcean Act2Run Systems, Chelsea Technologies) with Act2Run software (Chelsea Technologies). Ice samples were directly thawed at 2 degrees C in the dark for 30 min, and the slushily melted ice samples were placed in a quartz tube and their fluorescence (ChlF) was measured. A single turnover protocol was applied for the ChlF measurements; 100 flashlets with 1 micro second duration at a wavelength 450 nm and 2 micro second intervals for excitation of reaction centres of photosystem II (PSII, hereafter), and 20 flashlets with 1 μs duration and 100 micro second intervals for relaxation. Eighteen light steps were applied to generate a rapid light curve (RLC) from 0 to 1800 μmol photons m−2 s−1, taking less than 5 min to complete one RLC. At each light step (~15 s), at least five induction and relaxation curves were averaged to obtain ChlF yields, described in Table, after calibrating the ChlF yields with filtered seawater. According to the models proposed by Kolber et al. (1998), photosynthetic parameters of chlorophyll a (chl a) induction and relaxation curves were calculated based on the ChlF yields as shown in Table. Electron transport rate though the reaction centres of PSII (RCII) (ETRRCII) was calculated as per the equation detailed in the download document.

低铁条件下海冰藻类的快速重复率荧光计（Fast repetition rate fluorometer，FRRF）研究。实验所用藻类于冰箱中培养，实验结束后采用Chelsea Instruments品牌的FRRF完成测量。
材料与方法（原始格式与公式详见下载文档）
1. 冰箱培养实验
本研究于2015年从南极东部戴维斯站的南极浮冰中分离得到极地羽纹硅藻圆柱拟脆杆藻（*Fragilariopsis cylindrus*，后文简称F. cylindrus），将其置于塔斯马尼亚Island Research公司定制的冰箱中培养。该冰箱以钛合金制造以最大限度降低溶解态铁含量，随后被移入-20℃的低温冰柜中，冰厚与温度梯度通过底部加热器与可调环境冰柜温度的协同调控实现（详见Kennedy等，2012），实验期间可维持约5.5cm的冰厚。
圆柱拟脆杆藻培养于Aquil培养基（Price等，1989）中，以乙二胺四乙酸（ethylenediaminetetraacetic acid，EDTA）进行缓冲，光照条件为150 μmol光子·m⁻²·s⁻¹（光合有效辐射，PAR），盐度35，总铁浓度400 nM，其中总无机铁形态（Fe'）浓度为1.54 nM。铁元素持续供给至培养基中，其精确浓度通过Visual MINTEQ 3.1版本软件（https://vminteq.lwr.kth.se）计算得到。
在结冰周期启动前，海水温度维持在2.5℃，并采集样本评估藻类的原始生理状态（记为Day -5，后文简称）。采样完成后，将海水温度设置为-1.8℃以启动冰箱内的结冰过程。至Day -2时冰层形成，此时因冰体排盐作用导致冰下海水盐度升高至约38，故采用超纯水部分置换冰下海水，将盐度回调至35。在新盐度下驯化2天后，每隔5天采集冰样本，持续20天（即Day 0、5、10、15和20）。
为尽量降低冰芯样本的异质性，采用无痕金属手持钻（直径2cm）从冰表面随机标注的网格中随机采集冰样，随机采样编号通过软件R（https://www.r-project.org/）生成。为评估融化与高光暴露的影响，将冰层置于2.5℃下融化2天。冰层完全融化后，将海水暴露于高光强度下，该光照强度设置为模拟冰缘区域表层夏季光照强度（800 μmol光子·m⁻²·s⁻¹；MODIS Aqua）。分别在融化与光照暴露后采集海水样本（后文分别记为Melt组与Light组）。
2. 快速重复率荧光光度法
为监测圆柱拟脆杆藻在结冰与融化过程中的光生理特性，采用台式快速重复率荧光计（Fast Repetition Rate fluorometer，FRRf）（FastOcean Act2Run Systems，Chelsea Technologies）搭配Act2Run软件（Chelsea Technologies）完成可变叶绿素a荧光（variable chlorophyll a fluorescence，ChlF）测量。
冰样本于黑暗中2℃下解冻30分钟，将半融的冰样品置于石英管中进行荧光（ChlF）测量。本实验采用单周转方案开展ChlF测量：以波长450nm、时长1μs的100个微闪激发光系统II（photosystem II，PSII，后文简称）的反应中心，微闪间隔为2μs；随后以时长1μs、间隔100μs的20个微闪完成弛豫过程。
设置18个光照梯度以生成0至1800 μmol光子·m⁻²·s⁻¹的快速光曲线（RLC），单条快速光曲线的测量时长不足5分钟。每个光照梯度持续约15秒，对至少5条诱导与弛豫曲线取平均值以得到ChlF产量；采用过滤海水对ChlF产量进行校准后，相关参数详见附表。根据Kolber等（1998）提出的模型，基于ChlF产量计算叶绿素a诱导与弛豫曲线的光合参数。光系统II反应中心（RCII）的电子传递速率（ETR_RCII）按照下载文档中详述的公式计算得到。