DOI: 10.5285/bb4b1e74-d5af-4a73-9174-62a01807c641
title: Fatty acid composition of 32 cold-water algal strains cultured under different conditions to support the interpretation of in situ algal FA data from the MOSAiC expedition 2019/2020

description: A seasonal cycle of the FA composition of particulate organic matter from surface waters, Chlorophyll-a maximum layer and bottom sea ice, sampled during the MOSAiC expedition in the Central Arctic Ocean (2019-2020), suggests the importance of phylogenetic and environmental drivers. To improve our understanding of these different drivers, we conducted culture experiments with 32 cold-water algal strains where temperature, light intensity, and nutrient supply were manipulated individually or in combination. The culture experiments were carried out at the Culture Collection of Algae and Protozoa (CCAP; Oban, Scotland), the Roscoff Culture Collection (RCC; Roscoff, France) and the Alfred-Wegener-Institute-Helmholtz-Centre for Polar and Marine Research (AWI; Bremerhaven, Germany). The strains were part of the culture collections, had been isolated in the Arctic (25 strains), Southern Ocean (2 strains) or North Atlantic (5 strains), and included diatoms, chlorophytes, haptophytes, cryptophytes, chrysophytes, dinoflagellates and cyanobacteria. Some of the species are Arctic sea ice diatoms (e.g. Nitzschia frigida, Attheya spp.) or pelagic diatoms (e.g. Thalassiosira gravida), while others are non-diatom species that are becoming increasingly prominent in the Arctic, e.g. the coccolithophore Emiliania huxleyi (synonym Gephyrocapsa huxleyi), the prymnesiophyte Phaeocystis pouchetii, the chlorophyte Micromonas spp. and the cyanobacterium Synechococcus spp.. The experiments can be divided into three groups: First, those that tested a low light-low temperature setting, second, those that tested a low light-low temperature and a higher light-higher temperature setting and, third, those that tested the effect of nutrient (nitrate, phosphate and silicate) shortage in combination with low and high light intensity. The first set of experiments was conducted with all 32 strains, the second set with all strains grown at CCAP and AWI, and the third set focuses on the keystone under-ice diatom Melosira arctica. The experiments were run for 4-7 weeks to accumulate sufficient biomass for biomarker extractions (FA and sterols), C:N analysis and light-microscopy of cell size and cell concentration. At the end of the experiments, the algae were filtered onto GF/F filters and deep frozen until analysis. This dataset comprises the results from the FA analysis only (sterol and C:N analysis are separate). The separation of the lipid biomarkers was carried out at the University of Plymouth. After addition of internal standards for FA and sterols, the filters were saponified with KOH. Thereafter, non-saponifiable lipids (sterols) were extracted with hexane and purified by open column chromatography on silica gel. FA were obtained by adding concentrated HCl to the saponified solution and re-extracted with hexane. Further steps of the FA analysis were carried out at the AWI. Here samples were converted into fatty acid methyl esters (FAME) and analysed using an Agilent 6890N gas chromatograph with FID detector. The Clarity chromatography software system (DataApex, Czech Republic) was used for chromatogram data evaluation. FAME were quantified via the internal standard, Tricosanoic acid methyl ester (23:0) (Supelco, Germany) to provide the total amount of FA (TFA) per filter. These FA datasets of cultured algae are presented in a manuscript together with the FA pattern seen in sea ice- and water column POM in the CAO during the MOSAiC expedition and in previously published data from Arctic shelf regions. The manuscript focusses mainly on two important long-chain omega-3 FA (eicosapentaenoic acid and docosahexaenoic acid) that are considered essential for the nutrition of higher trophic levels, including humans, and their production to decline with global temperature rise.

Contributions by KS were funded by the UK's Natural Environment Research Council MOSAiC Thematic project SYM-PEL: 'Quantifying the contribution of sympagic versus pelagic diatoms to Arctic food webs and biogeochemical fluxes: application of source-specific highly branched isoprenoid biomarkers'/ (NE/S002502/1). CRM was funded by the NERC National Capability Services and Facilities Programme (NE/R017050/1).
description: Microalgae cultures: 
In 2022, we conducted FA analysis on 32 cold-water strains that were isolated in the Arctic (25 strains), Southern Ocean (2 strains) or North Atlantic (5 strains), and included diatoms, chlorophytes, haptophytes, cryptophytes, chrysophytes, dinoflagellates and cyanobacteria. These strains were either obtained from commercial culture collections (CCAP or RCC) or provided by the AWI. The CCAP cultures were inoculated in 50 ml Erlenmeyer conical flasks with 25 ml of F/2 medium and added 60 micromol L-1 silicate for diatoms (Guillard and Ryther 1962). The algal strains were grown for four weeks at either low temperature - low light intensity (3-4 degrees Celsius, ~10 micromol m-2 s-1) or higher temperature - higher light intensity (8 degrees Celsius, 20 micromol m-2 s-1) and a light:dark cycle of 12:12 hrs. 
The culturing conditions at RCC resembled those at CCAP, but only for the low temperature - low light intensity (3-4d degrees Celsius, 10 micromol m-2 s-1). The algae growth rates were not monitored, but with these culture conditions and time span, the strains usually reach late exponential-early stationary growth (C. Rad-Menendez, I. Probert, pers. comm.). From each strain, two 5 ml technical replicates were filtered via a vacuum pump (-20 kPA) onto pre-combusted (12 h, 450 degrees Celsius) 25 mm Whatman GF/F filters, freeze dried and stored in aluminium foil at -20 degrees Celsius until FA analysis. 
Note: Three of the strains from CCAP (CCAP-1023/3, CCAP-1029/29, CCAP-1029/30) were obtained and analysed as 'Fragilariopsis sp.' but subsequently identified as 'Grammonema sp.'. 

Experiments with Melosira arctica: 
The experiments with Melosira arctica were carried out at the AWI in 2021 and 2022, using a strain that was isolated in the Central Arctic Ocean in 2015 (79.56 degrees N, 4.84 degrees W; strain PS93.1_030). The strain was grown as semi-continuous batch cultures under 6 different environmental conditions including the species' natural range of temperature, light and nutrient availability (Spilling and Markager 2008, Fernandez-Mendez et al. 2014). Therefore, low temperatures (0-1 dgrees Celsius) were combined with high and low light intensity (10 and 100 micromol m-2 s-2), each with high and low nutrient supply (details below), and higher temperatures (3 and 6 degrees Celsius) with high light and nutrient availability. Experiments were performed with 4 biological replicates in sterile 1-L Schott bottles in temperature-controlled rooms, with bottles at 3 and 6 degrees Celsius being immersed in water-filled aquaria for additional temperature stability. Day light lamps (Biolux T8, 6500K, Osram) provided continuous light and irradiance levels were adjusted with a black mesh fabric and measured using a 4 pi spherical sensor (Li-Cor) and data logger (ULM-500, Walz). Cells were cultivated in 0.2 micrometer sterile-filtered Arctic seawater (salinity 33.5), with or without added macronutrients, vitamins and trace metals according to F/2 or F/20 media (Guillard and Ryther, 1962). Initial nutrient concentrations in set-ups with F/20 media were 10.4 +/-0.24 micromol L-1 nitrate-and-nitrite, 18.1 +/-1.36 micromol L-1 silicate and 1.2 +/- 0.19 micromol L-1 phosphate. In set-ups without added medium, initial nutrient concentrations were 1.8+/-0.07 micromol L-1 nitrate-and-nitrite, 14.4 +/-2.3 micromol L-1 silicate and 0.4 +/-0.01 micromol L-1 phosphate. To minimize changes in carbonate chemistry and to remain close to natural population densities, cultures were diluted every 1-2 weeks with Arctic seawater, with or without medium. Sufficient algal biomass for subsequent lipid analysis was grown after 4 weeks for cultures that received media, and after 7 weeks for those without media. At the end of the experiment, 2 technical replicates were taken from each biological replicate, filtered onto pre-combusted (12 h, 450 degrees Celsius) 25 mm Whatman GF/F filters, freeze dried and stored in aluminium foil at -20 degrees Celsius until FA analysis. 
Note: Three of the strains from CCAP (CCAP-1023/3, CCAP-1029/29, CCAP-1029/30) were obtained and analysed as 'Fragilariopsis sp.' but subsequently identified as 'Grammonema sp.'. 

Experiments with Melosira arctica: 
The experiments with Melosira arctica were carried out at the AWI in 2021 and 2022, using a strain that was isolated in the Central Arctic Ocean in 2015 (79.56 degrees N, 4.84 degrees W; strain PS93.1_030). The strain was grown as semi-continuous batch cultures under 6 different environmental conditions including the species' natural range of temperature, light and nutrient availability (Spilling and Markager 2008, Fernandez-Mendez et al. 2014). Therefore, low temperatures (0-1 dgrees Celsius) were combined with high and low light intensity (10 and 100 micromol m-2 s-2), each with high and low nutrient supply (details below), and higher temperatures (3 and 6 degrees Celsius) with high light and nutrient availability. Experiments were performed with 4 biological replicates in sterile 1-L Schott bottles in temperature-controlled rooms, with bottles at 3 and 6 degrees Celsius being immersed in water-filled aquaria for additional temperature stability. Day light lamps (Biolux T8, 6500K, Osram) provided continuous light and irradiance levels were adjusted with a black mesh fabric and measured using a 4 pi spherical sensor (Li-Cor) and data logger (ULM-500, Walz). Cells were cultivated in 0.2 micrometerm sterile-filtered Arctic seawater (salinity 33.5), with or without added macronutrients, vitamins and trace metals according to F/2 or F/20 media (Guillard and Ryther, 1962). Initial nutrient concentrations in set-ups with F/20 media were 10.4 +/-0.24 micromol L-1 nitrate-and-nitrite, 18.1 +/-1.36 micromol L-1 silicate and 1.2 +/- 0.19 micromol L-1 phosphate. In set-ups without added medium, initial nutrient concentrations were 1.8+/-0.07 micromol L-1 nitrate-and-nitrite, 14.4 +/-2.3 micromol L-1 silicate and 0.4 +/-0.01 micromol L-1 phosphate. To minimize changes in carbonate chemistry and to remain close to natural population densities, cultures were diluted every 1-2 weeks with Arctic seawater, with or without medium. Sufficient algal biomass for subsequent lipid analysis was grown after 4 weeks for cultures that received media, and after 7 weeks for those without media. At the end of the experiment, 2 technical replicates were taken from each biological replicate, filtered onto pre-combusted (12 h, 450 degrees Celsius) 25 mm Whatman GF/F filters, freeze dried and stored in aluminium foil at -20 degrees Celsius until FA analysis. All dilutions and final sampling were conducted under sterile conditions, using a laminar flow hood.

Fatty acid analysis: The separation of the lipid biomarkers was carried out at the University of Plymouth. After addition of internal standards for FA and sterols, the filters were saponified with KOH. Thereafter, non-saponifiable lipids (sterols) were extracted with hexane and purified by open column chromatography on silica gel. FA were obtained by adding concentrated HCl to the saponified solution and re-extracted with hexane. Further steps of the FA analysis were carried out at the AWI. Here samples were converted into fatty acid methyl esters (FAME) and analysed using an Agilent 6890N gas chromatograph with FID detector. The Clarity chromatography software system (DataApex, Czech Republic) was used for chromatogram data evaluation. FAME were quantified via the internal standard, Tricosanoic acid methyl ester (23:0) (Supelco, Germany) to provide the total amount of FA (TFA) per filter. These TFA quantities per filter can be normalised to the volume of filtered seawater. Additionally, we provide the mass percentage composition of the TFA, considering 48 individual FA.

Data details:
TFA quantities per filter can be normalised to the volume of filtered seawater. Additionally, we provide the mass percentage composition of the TFA, considering 48 individual FA. The FA are presented in shorthand notation, i.e., A:B(n-x), where: A indicates the number of carbon atoms in the straight fatty acid chain, B represents the number of double bonds present, n represents the terminal methyl group and x denotes the position of the first double bond from the terminal end. The nomenclature of the fatty acids following IUPAC rules is provided.
description: Fatty acid analysis: FAME were quantified using an Agilent 6890N gas chromatograph (Agilent Technologies, USA) with a DB-FFAP capillary column (60 m, 0.25 mm I.D., 0.25 micrometer film thickness, Agilent Technologies, USA) supplied with a splitless injector and a flame ionization detector using temperature programming.

Chromatogram data evaluation: Clarity chromatography software system (version 8.8.0, DataApex, Czech Republic)
description: The fatty acid profiles were compared to several commercial- and self-produced standards (e.g. Arctic algae standard, Bacteria standard, Calanus spp. standard), and fatty acid peaks were identified accordingly. In a few cases, samples were also analysed with the mass spectrometer and peaks were identified via (1) the mass of the compound, (2) the retention time of the compound and (3) the equivalent chain length method. Unusual peaks were also compared with published FA profiles from related species (e.g. Leblond et al. 2006 for cold-adapted dinoflagellates).