Cell growth phase of Skeletonema marinoi growth and aggregation under future ocean acidification conditions from UCSB MSI Passow Laboratory from 2009 to 2010 (OA - Ocean Acidification and Aggregation project)

<p><strong>Series 3: <em>Skeletonema marinoi</em> growth and aggregation under future ocean acidification conditions<br />
&nbsp; - Phase 1: Cell Growth Phase<br />
&nbsp; - Experiments 1 and 2</strong></p>

<p><strong>METHODS</strong><br />
<strong>2.1 General set up:</strong> The experiment consisted of two treatments (Present and Future) representing different pCO2 conditions and two sequential experimental phases: <strong>the cell growth phase</strong> and <strong>the aggregation phase</strong>. The experiment was sequentially replicated. The two replicates are going to be referred as Experiment 1 and Experiment 2.<br />
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<strong>2.2 Cell growth phase:</strong> During the cell growth phase, the effect of OA was tested on a culture of S. marinoi that was incubated for five days, in 5 l transparent bags. Two replicate bags were used for each treatment. The pH was measured daily immediately after collection in each bag, while the TA samples were collected for later analysis in one sample per treatment at the beginning and at the end of the cell growth phase. The algal cells were counted, sized and their instantaneous in vivo chlorophyll fluorescence (Ft) and quantum yield (Qy) were measured daily in the morning. The concentration of bacteria and TEP were measured at t 0 ,t2.8 and t4.6. The nutrients concentration was sampled at t2.8 and t4.6. The dry weight, particulate carbon, hydrogen and nitrogen were measured at t4.6. After 5 days the cultures in the two bags per treatment were pooled, the carbonate system was readjusted to the starting conditions and subsequently they were inoculated into cylindrical rolling tanks.</p>

<p><strong>2.3 Aggregation phase:</strong> The aggregation phase consisted in the incubation of the cultures into cylindrical tanks on rolling tables (Edmondson, 1989). A total of 8 tanks (4 replicates per treatment) were incubated over the rolling table for two days in the darkness in the environmental room at 15°C. Solid body rotation is established in these rolling tanks within less than three hours (Ploug, Terbrüggen, Kaufmann, Wolf- Gladrow, &amp; Passow, 2010) and the sinking of particles through the water column was simulated and aggregation promoted. After 38 hours of incubation the aggregates sinking velocity, number and size were measured. The algal cell number, bacterial cell number, TEP, dry weight, particulate carbon, hydrogen and nitrogen were evaluated, both for the aggregated fraction and for the surrounding water. The carbonate system was characterized by measuring pH in all the tanks and TA in one tank per treatment.</p>

<strong>系列3：<em>海洋骨条藻（Skeletonema marinoi）</em>在未来海洋酸化条件下的生长与聚集<br />&nbsp; — 第一阶段：细胞生长阶段<br />&nbsp; — 实验1与实验2</strong><br /><p><strong>实验方法</strong><br /><strong>2.1 实验总体设置：</strong>本实验设置代表不同二氧化碳分压（pCO₂）条件的“当前”与“未来”两组处理，包含两个连续实验阶段：<strong>细胞生长阶段</strong>与<strong>聚集阶段</strong>。实验将依次重复开展，两次重复实验分别记为实验1与实验2。</p><p><strong>2.2 细胞生长阶段：</strong>细胞生长阶段中，本实验针对海洋骨条藻（S. marinoi）培养液开展了为期5天的培养，以探究海洋酸化（Ocean Acidification, OA）对其生长的影响。实验采用5升透明培养袋，每组处理设置两个重复培养袋。每日采样后立即测定各培养袋的pH值；于细胞生长生长阶段的起始与结束时刻，每组处理各采集1份总碱度（Total Alkalinity, TA）样品用于后续分析。每日清晨对藻细胞进行计数、粒径测量，并测定其活体瞬时叶绿素荧光强度（Ft）与量子产率（Qy）。分别于t₀、t₂.₈及t₄.₆时刻测定细菌与透明胞外聚合物（Transparent Exopolymer Particles, TEP）浓度；于t₂.₈及t₄.₆时刻采集营养盐浓度样品。于t₄.₆时刻测定干重、颗粒碳、氢与氮含量。培养5天后，将每组处理的两个培养袋中的培养液混合，将碳酸盐体系重新调整至初始条件，随后接种至圆柱型滚动培养罐中。</p><p><strong>2.3 聚集生长阶段：</strong>聚集生长阶段将培养液置于滚动台（Edmondson, 1989）上的圆柱型培养罐中进行培养。本实验共设置8个培养罐（每组处理4个重复），于15℃的环境室黑暗条件下培养2天。此类滚动培养罐可在3小时内建立刚体旋转（solid body rotation）（Ploug, Terbrüggen, Kaufmann, Wolf-Gladrow, & Passow, 2010），从而模拟颗粒物在水层中的沉降过程并促进聚集。培养38小时后，测定聚集物的沉降速度、数量与粒径。分别针对聚集组分与周围水体，测定藻细胞数量、细菌数量、透明胞外聚合物（TEP）浓度、干重、颗粒碳、氢与氮含量。通过测定所有培养罐的pH值与每组处理1个培养罐的总碱度（TA），表征碳酸盐体系特征。</p>