Temperature tolerance of western Baltic Sea Fucus vesiculosus - growth, photosynthesis and survival

Fucus vesiculosus L. (Phaeophyceae) is the most abundant and hence ecologically most important primary producer, carbon sink and habitat provider in the western Baltic Sea.
All F. vesiculosus L. specimens were collected on 23 April 2014 from a depth of 0.2-1 m in the non-tidal Kiel Fjord, western Baltic Sea (54°27'N; 10°12'E), where this species forms dense and almost monospecific stands on stones. After sampling the algal thalli were stored in a refrigerator box with water from the sampling site, transported to Bremerhaven and stored at 10 °C for one day in filtered seawater. Experiments were conducted with vegetative apical tips (6.7±0.5 cm length), the actively growing region of F. vesiculosus, which were randomly selected and cut from 144 different individuals prior to the experiments. These tips were acclimated to laboratory conditions for three days in filtered seawater at 10 °C before the start of the experiment. Furthermore, 30 additional vegetative apices were freeze-dried to document the initial biochemical status of F. vesiculosus in its native habitat.
A temperature gradient was installed in a walk-in constant cooling chamber (15 °C) in nine water baths (5, 10, 15, 20, 24, 26, 27, 28 and 29 °C ± 0.1 °C) which were tempered by thermostats (5, 10 and 15 °C: Huber Variostat CC + Pilot ONE, Peter Huber Kältemaschinen GmbH, Offenburg, Germany; 20 and 28 °C: Haake DC3, Thermo Fisher Scientific Inc., Waltham, USA; 24, 26, 27 and 29 °C: Haake DC10). Every temperature treatment consisted of four 2 L glass beakers (n = 4). In each beaker four F. vesiculosus apices were grown in 2 µm-filtered North Sea water diluted with demineralized water in a ratio of 1:1 and enriched with nutrients after Provasoli (1968; 1/10 enrichment), leading to a salinity of about 15.6 which equaled habitat conditions. The algae were exposed to an irradiance of 130 µmol photons m-2 s-1 ±10 % (Powerstar HGI-TS 150 W, OSRAM GmbH, Bad Homburg, Germany) measured at the top of the beaker under a 16:8 h L:D cycle. The media in the beakers was changed every third or fourth day and aerated with artificial air containing 380 ppm CO2 (gas mixing device; HTK Hamburg GmbH, Hamburg, Germany). Before the experiment, the algae were acclimated to the final temperatures in steps of 5 °C for 2 days each, beginning at 10 °C. After 21 days exposure time, three out of four samples per replicate were freeze-dried for further biochemical analyses, and afterwards the thermostats were turned off to reduce the temperature to 16±0.4 °C for another 10 days permitting growth under post-culture conditions.

墨角藻（Fucus vesiculosus L.）隶属于褐藻纲（Phaeophyceae），是波罗的海西部海域中生物量最为丰富、因此在生态功能上最为重要的初级生产者、碳汇及栖息地提供者。
所有墨角藻样本均于2014年4月23日采自波罗的海西部无潮汐影响的基尔峡湾（54°27'N; 10°12'E）水深0.2~1 m的区域，该区域内该物种在石块表面形成密集且近乎单优的群落。采样完成后，藻体被置于装有采样现场海水的冷藏箱中保存，转运至不来梅港后，于过滤海水中于10 ℃条件下保存1天。
实验采用墨角藻的活跃生长区域——营养生长顶端组织（长度为6.7±0.5 cm）开展，该组织从144个不同个体中随机选取并切割获取。正式实验前，将其置于10 ℃的过滤海水中驯化培养3天以适应实验室环境。此外，额外选取30个营养体顶端组织进行冷冻干燥，以记录墨角藻在原生栖息地中的初始生化状态。
本研究在一间步入式恒温冷却室（设定温度15 ℃）内搭建了温度梯度系统，包含9个恒温水浴槽（温度分别为5、10、15、20、24、26、27、28和29 ℃，控温精度±0.1 ℃），各水槽分别通过恒温器控温：5、10和15 ℃采用Huber Variostat CC + Pilot ONE恒温器（Peter Huber Kältemaschinen GmbH，奥芬堡，德国）；20和28 ℃采用Haake DC3恒温器（赛默飞世尔科技公司，Thermo Fisher Scientific Inc.，沃尔瑟姆，美国）；24、26、27和29 ℃采用Haake DC10恒温器。每个温度处理设置4个2 L玻璃烧杯作为重复（n=4）。每个烧杯中放入4个墨角藻顶端组织，培养于经2 µm过滤的北海海水与去离子水按1:1比例混合的培养液中，并按照Provasoli（1968）的配方添加1/10浓度的营养盐，最终培养液盐度约为15.6，与采样栖息地的盐度条件一致。
藻体置于光照强度为130 µmol photons m⁻² s⁻¹（波动幅度±10%）的光照条件下培养，光源采用Powerstar HGI-TS 150 W灯（欧司朗有限公司，OSRAM GmbH，巴特洪堡，德国），光照周期设置为16:8 h的光暗比，光照强度在烧杯液面处测定。烧杯内培养液每3~4天更换一次，培养液通入含380 ppm CO₂的人工空气（气体混合装置：HTK Hamburg GmbH，汉堡，德国）。实验正式开始前，藻体以每2天升温5 ℃的梯度逐步驯化至目标温度，起始温度为10 ℃。
经过21天的暴露培养后，每个重复的4个样本中选取3个进行冷冻干燥，用于后续生化分析；随后关闭恒温器，将温度降至16±0.4 ℃，继续培养10天，使藻体在后续培养条件下恢复生长。