Quantification of multiple environmental controls on lipid biomarkers in common marine diatoms and dinoflagellates

In the monocultures of two algal species, i.e., Phaeodactylum tricornutum (Bacillariophyceae; strain MACC/B254) and Prorocentrum minimum (Dinophyceae; strain HYESL63), we investigated responses of lipid biomarkers (sterols and fatty acids (FAs)) to different temperatures (12, 18 and 24℃), nitrogen and phosphorus concentrations and their molar ratios (N:P ratios) of 10:1, 24:1 and 63:1.

Algal cells were counted daily with an improved Neubauer hemacytometer (Glaswarenfabrik Karl Hecht GmbH) under a microscope (Olympus CX41). To analyze particulate organic carbon (POC), sterols and FAs, algal cells at steady-state conditions were harvested on pre-combusted GF/F filters (Whatman) after filtering 15-30 mL of cultures depending on cell density in the culture flask and the parameters to be determined. Samples were kept at − 80℃ after filtration.

POC was determined by an elemental analyzer (Thermo Flash 2000) (Sharp 1974, https://doi.org/10.4319/lo.1974.19.6.0984). Sterols and FAs were analyzed according to the methods in Eglinton et al. (1996 https://doi.org/10.1021/ac9508513), Galy et al. (2011, https://doi.org/10.1016/j.epsl.2011.02.003) and Zhao et al. (2006, https://doi.org/10.1016/j.orggeochem.2005.08.022). The trimethylsilyl ether derivatives of sterols and fatty acid methyl esters (FAMEs) were analyzed in a gas chromatograph (Agilent Technologies 8890A) equipped with a flame ionization detector, and a HP-1 column (50 m, 0.32 mm i.d., 0.17 μm film; Agilent J&W) and a SP-2560 column (100 m, 0.25 mm i.d., 0.20 μm film; Supelco) for sterol and FAME analysis, respectively.

The identification of sterols was performed by gas chromatography-mass spectrometry (GC-MS) analysis at 70 eV using an Agilent 7890B GC (HP-5MS column; 30 m, 0.25 mm i.d., 0.25 μm film; Agilent J&W) connected to an Agilent MSD 5977B mass selective detector (ion source temperature 230℃). Sterols were identified be comparison of the mass spectra of their trimethylsilyl ether derivatives to those of published GC-MS values (Lisboa et al. 1982, https://doi.org/10.1016/0305-0491(82)90281-4; Taipale et al. 2016, https://doi.org/10.3389/fpls.2016.00212), based on the molecular ion and prominent ions. The following sterols were identified: brassicasterol/epi-brassicasterol,and dinosterol. FAs were identified with reference to the standard Supelco 37 component FAME mixture. C-normalized (on a per POC basis; μg mg C-1) and per-cell (pg cell-1) contents of sterols and FAs were presented, and FA proportions (% of total fatty acids (TFAs)) were also reported in the dataset.

本研究以两种单培养藻类为研究对象，即三角褐指藻（Phaeodactylum tricornutum，硅藻门Bacillariophyceae；菌株MACC/B254）与海洋原甲藻（Prorocentrum minimum，甲藻门Dinophyceae；菌株HYESL63），探究了脂质生物标志物（甾醇与脂肪酸（fatty acids，FAs））对不同温度（12、18、24℃）、氮磷浓度及其摩尔比（N:P比值，10:1、24:1、63:1）的响应规律。

采用改良牛鲍计数板（improved Neubauer hemacytometer，Glaswarenfabrik Karl Hecht GmbH）在显微镜（Olympus CX41）下每日计数藻类细胞数量。为分析颗粒有机碳（particulate organic carbon，POC）、甾醇与脂肪酸含量，待培养至稳态后，根据培养瓶内细胞密度及待测参数，过滤15~30 mL藻液，将藻细胞收集于预灼烧后的GF/F滤膜（Whatman）上。过滤完成后，样品置于-80℃环境保存。

颗粒有机碳采用元素分析仪（Thermo Flash 2000）进行测定（Sharp 1974，https://doi.org/10.4319/lo.1974.19.6.0984）。甾醇与脂肪酸的分析参照Eglinton等（1996，https://doi.org/10.1021/ac9508513）、Galy等（2011，https://doi.org/10.1016/j.epsl.2011.02.003）及Zhao等（2006，https://doi.org/10.1016/j.orggeochem.2005.08.022）的方法。将甾醇与脂肪酸甲酯（fatty acid methyl esters，FAMEs）衍生为三甲基硅醚衍生物后，分别使用配备氢火焰离子化检测器（flame ionization detector）的气相色谱仪（Agilent Technologies 8890A）进行分析：甾醇分析采用HP-1色谱柱（HP-1 column，50 m，内径0.32 mm，膜厚0.17 μm；Agilent J&W），脂肪酸甲酯分析采用SP-2560色谱柱（SP-2560 column，100 m，内径0.25 mm，膜厚0.20 μm；Supelco）。

甾醇的定性分析采用气相色谱-质谱联用仪（gas chromatography-mass spectrometry，GC-MS）完成：在70 eV电离能量下，使用连接有Agilent MSD 5977B质谱选择性检测器（mass selective detector，离子源温度230℃）的Agilent 7890B气相色谱仪（HP-5MS色谱柱，HP-5MS column，30 m，内径0.25 mm，膜厚0.25 μm；Agilent J&W）进行检测。基于分子离子峰与特征离子峰，通过将三甲基硅醚衍生物的质谱与已发表的GC-MS质谱数据（Lisboa等，1982，https://doi.org/10.1016/0305-0491(82)90281-4；Taipale等，2016，https://doi.org/10.3389/fpls.2016.00212）进行比对，完成甾醇的定性。本研究鉴定出的甾醇包括：菜籽甾醇/表菜籽甾醇（brassicasterol/epi-brassicasterol）以及甲藻甾醇（dinosterol）。脂肪酸的定性参照Supelco 37组分脂肪酸甲酯标准混合品完成。本数据集同时给出了甾醇与脂肪酸的碳标准化含量（以每单位POC计，单位：μg mg C⁻¹）、单细胞含量（pg cell⁻¹），以及脂肪酸占总脂肪酸（total fatty acids，TFAs）的比例（%）。