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.