Lipids of different phytoplankton groups differ in sensitivity to degradation: implications for carbon export

Data were collected on seven cruises on a monthly basis from February to August in 2010 in the northern Adriatic Sea (RV001 (N 45°04.8', E 013°36.6'), SJ107 (N 45°02.9', E 013°19.0'), SJ105 (N 45°02.0', E 0.13°09.3'), SJ103 (N 45°01.0', E 012°59.6'), SJ101 (N 44°59.9', E 012°50.3'), and SJ108 (N 44°48.7', E 012°45.0')). 

A CTD probe (Seabird SBE25, Sea–Bird Electronics Inc., Bellevue, Washington, USA) was used to measure temperature and salinity. Total phosphorus, dissolved inorganic orthophosphates (PO43-), total inorganic nitrogen (TIN), including nitrates (NO3¯), nitrites (NO2¯), and ammonium (NH4+)), were determined by spectrophotometric methods (Parsons et al., 1984) on board and immediately after sampling using Shimadzu UV-Mini 1240 spectrophotometer with 10 cm quartz cuvettes. Organic phosphorus concentration was calculated as the difference between total and inorganic phosphorus concentrations. Subsamples for the determination of chlorophyll a (Chl a) were filtered on Whatman GF/C filters and stored frozen at -20°C until further processing. Chl a concentrations were determined following 3 h extraction in 90% acetone (in the dark, with grinding), on a Turner TD–700 fluorimeter (Parsons et al., 1984).

We preserved 200 mL of seawater with 2% neutralized formaldehyde (final concentration) and performed nano- and microphytoplankton determination and enumeration within one month of sampling. The stored sample was homogenized by gentle shaking, and a subsample was added to the Utermöhl sedimentation chamber (volume: 50 mL; Hydro-Bios Apparatebau, Altenholz, Germany), where it settled for ~30 h. We performed the analysis on a Zeiss Axiovert 200 (Zeiss, Jena, Germany) following the inverted microscope method (Utermöhl, 1958, Hasle 1978). Total phytoplankton included all species counted in the microphytoplankton (20–200 µm) and nanophytoplankton (2-20 µm) groups (Sieburth et al., 1978). Taxa were identified to species level and subsequently grouped to diatoms, dinoflagellates, and nanophytoplankton coccolithophores and phytoflagellates (which included chlorophytes, chrysophytes, cryptophytes and prasinophytes) according to Tomas (1997).

For POC determination, 1 L of seawater was filtered on board through 0.7 mm Whatman GF/F filters precombusted at 450 °C/5h. After filtration, the filters were rinsed with Milli-Q water to remove salts and stored in liquid nitrogen on board and at -80 oC in the laboratory until analysis. POC was analyzed using an SSM–5000A solid sample module connected to a Shimadzu TOC–VCPH carbon analyzer calibrated with glucose (Sugimura and Suzuki, 1988). POC concentrations were corrected based on filter blank measurements. The average filter blank value including the instrument blank value corresponded to 5 mg C L-1. The reproducibility obtained for the glucose standard was 3%.

For particulate lipid analysis, we collected 3 L of seawater prefiltered through a 200 μm stainless steel screen to remove larger particles including microzooplankton. Lipids were collected on through precombusted (450 °C/5h) 47 mm GF/F filters and stored in liquid nitrogen until lipid extraction. It was performed using a modified one-phase solvent mixture of dichloromethane-methanol-water procedure (Bligh and Dyer, 1959; Vrana et al., 2023). In short, in order to assess recoveries in later stages of sample analysis we added 5 µg of standard methyl stearate to the sliced filters together with 10 mL of a one-phase solvent mixture (dichloromethane/methanol/deionized water (1:2:0.8 v/v/v)). This was then subjected to an ultrasonic treatment for three minutes and stored overnight in the refrigerator, afterwards we filtered the extracts through a sinter funnel into a separatory funnel, washed once with a the one-phase solvent mixture (10 mL), once with dichloromethane (5 mL) and 0.73% NaCl (5 mL) (1:1 v/v), and once with 10 mL dichloromethane. The dichloromethane extracts were concentrated by rotary evaporation under a nitrogen atmosphere and kept at -20 °C until measurements were made. To prepare the lipid extracts for analysis, the dichloromethane extracts were evaporated to dryness under nitrogen flow and then dissolved in 20 µL dichloromethane prior to analysis.

Lipid classes were separated on Chromarods SIII and quantified with external calibration using a mixture of standard lipids by a thin-layer chromatograph-flame ionization detector (TLC-FID) Iatroscan Mark-VI (Iatron), using a hydrogen flow of 160 mL min-1 and an air flow of 2000 mL min-1. This method identify eighteen lipid classes: hydrocarbons (HC), steryl esters (SE), fatty acid methyl esters (ME), fatty ketone (KET), triacylglycerols (TG), free fatty acids (FFA), fatty alcohols (ALC), 1,3-diacylglycerols (1,3DG), sterols (ST), 1,2-diacylglycerols (1,2DG), pigments (PIG), monoacylglycerols (MG), three glycolipids (GL) including monogalactosyl-, digalactosyl-, and sulfoquinovosyl- diacylglycerol (MGDG, DGDG, and SQDG, respectively), and three phospholipids (PL) (phosphatidylglycerols (PG), phosphatidylethanolamines (PE), and phosphatidylcholines (PC)). Total lipid concentration is calculated by summing all detected classes. Full details can be found in Gašparović et al. (2015; 2017). In this article we focused on lipid degradation indices trough the lipolysis index (Goutx et al., 2003), which characterize the degree of lipid degradation in seawater. Lipolysis index is calculated as the ratio of the sum of lipid degradation indices (ALC+FFA+MG+DG) to the sum of cell lipids TG, WE, and glyco- and phospho-lipids (Goutx et al., 2003).