Dataset: Isotopic composition of phospholipid-linked fatty acids (PLFAs)
Deployment: Plum_Island

The three ponds are located in the high marsh (1.43-1.46 m above North American Vertical Datum of 1988) of the Plum Island Ecosystems – Long Term Ecological Research (PIE-LTER) site. Surface sediments were collected weekly June 25-August 13 and November 11-25, 2014. Cores (5 cm diameter x 2 cm deep) were collected from three 1 m² quadrats placed at random locations along two crisscrossing transects in each pond. Sediments were combined in combusted glass vials to form composite samples and stored (-80 ℃) until analysis. Lipid biomarker compounds were extracted using a modified Bligh & Dyer (1959) method (Spivak and Reeve 2015). Sediments were extracted with a methanol : chloroform : phosphate buffer saline mixture (2:1:0.8, v:v:v) using a microwave-accelerated reaction system (MARS6); samples were heated to 80°C for 10 min with continuous stirring. Samples were then partitioned and the organic phase removed. The total lipid extract was concentrated under N₂ and samples were eluted on silica gel columns with chloroform, acetone (F1/2), and methanol (F3) (Guckert et al. 1985). The F3 (phospholipids) was dried under N₂ and saponified with 0.5 M NaOH at 70°C for 4 h. Saponified samples were acidified and extracted 3 times with hexane. The extract was methylated with acidic methanol (95:5 methanol:HCl) and heated overnight at 70°C to form fatty acid methyl esters (FAME). Samples were analyzed with an Agilent 7890 gas chromatograph with the effluent split ~70:30 between a 5975C mass spectrometer and a flame ionization detector. Compounds were separated on an Agilent DB-5 ms column (60 m, 0.25 mm inner diameter, 0.25 μm film). FAME concentrations were quantified using methyl heneicosanoate as an internal standard. FAs are designated A:BωC, where A is the number of carbon atoms, B is the number of double bonds, and C is the position of the first double bond from the aliphatic ‘ω’ end of the molecule. Iso- and anteiso refer to whether the methyl group of branched compounds is attached to the penultimate or antepenultimate carbon atom. Stable carbon isotope ratios of FAMEs were determined by the WHOI Organic Mass Spectrometry Facility with a Hewlett-Packard 6890 GC coupled to a DeltaPlus isotope-ratio-monitoring gas chromatography-mass spectrometer (IRM-GCMS) via a GCCIII combustion interface held at 850 °C with a constant oxygen trickle. Isotopic values of phospholipid-linked fatty acids (PLFAs) were derived from the isotopic composition of FAMEs and corrected for the δ¹³C of the carbon added during methylation using a mass balance approach as well as for a -3‰ fractionation during lipid synthesis (Bouillon and Boschker 2006; Hayes 2001). The δ¹³C of PLFA subclasses was calculated as concentration weighted averages (Spivak and Ossolinski 2016).