Methodology from Shen, Y., et al. (2017), Bioavailable dissolved organic matter and biological hot spots during austral winter in Antarctic waters, J. Geophys. Res. Oceans, 121, doi:10.1002/2016JC012301.

Study Area and Sample Collection

Sampling was conducted as part of the Antarctic Marine Living Resources (AMLR) Program aboard the RVIB Nathaniel B. Palmer off the Antarctic Peninsula and South Shetland Islands (SSI) during August 2012 (Figure 1). The sampling areas are characterized by the confluence of different water masses originating from the Antarctic Circumpolar Current, Bellingshausen Sea, and Weddell Sea. A total of 110 seawater samples were collected from discrete depths (5, 10, 15, 50, 75, 100, 200, 750 m) at 25 stations within the historic AMLR research area, extending from the southern Drake Passage to the Bransfield Strait (Figure 1). A rosette sampler system with 24, 12 L bottles and a Seabird Conductivity-Temperature-Depth (CTD) instrument was used for water collections. Water samples for chlorophyll-a (chl-a) were filtered (GF/F; 0.7 mm pore size; Whatman) immediately following collection. Samples for POC measurements were collected mostly at 5 and 200 m, filtered (GF/F; Whatman; precombusted at 4508C for 5 h), and stored frozen until analysis. Water samples for analyses of DOC and amino acids were stored frozen (2808C) in 60 mL high density polyethylene screw-cap bottles immediately after collection. Hydrographic data were obtained from the CTD sensors and were used to determine the depth of upper mixed layer and to identify water masses (Table 1).

Chemical Analyses

The filters for chl-a determinations were extracted in 7 mL of methanol for 24 h, centrifuged, and measured for fluorescence using an acidification module in a Turner Trilogy fluorometer. Readings were calibrated with a 5-point calibration curve using a chl-a standard obtained from Sigma, the concentration of which was determined using a Lambda-18 spectrophotometer. Samples (GF/F filters) for POC analysis were treated with 10% v/v hydrochloric acid (HCl), dried at 608C, and analyzed using an Exeter Analytical CEC 440HA elemental analyzer.

Water samples for DOC and amino acid measurements were filtered through 0.2 mm pore size membranes (SuporVR -200, Life Sciences). The Supor membranes were cleaned with methanol and then rinsed thoroughly with Milli-Q UV-Plus water before use. The DOC samples were acidified to pH 2–3 with 2 mol L21 HCl. Concentrations of DOC were determined by high-temperature combustion using a Shimadzu total organic carbon TOC-V analyzer equipped with an autosampler. Milli-Q UV-Plus water and seawater reference standards were injected every sixth sample [Benner and Strom, 1993]. Blanks (Milli-Q water) were negligible and the measured concentrations of reference standards were within the reported range (41–44 mmol L21). The coefficient of variation among four injections of a given DOC sample was typically 61.1%.

The D-enantiomer and L-enantiomer of amino acids were analyzed using an Agilent 1260 ultrahigh-performance liquid chromatography (UPLC) system equipped with a fluorescence detector (excitation: 330 nm; emission: 450 nm) [Shen et al., 2015]. Amino acids were determined in all samples that were filtered through Supor membranes (0.2 mm pore size) and in a subset of unfiltered samples as total dissolved amino acids (TDAA) and total particulate amino acids (TPAA), respectively. Hydrolysis and derivatization followed the procedures described by Kaiser and Benner [2005]. Briefly, water samples (100 mL) were dried and hydrolyzed using a vapor-phase technique with 6 mol L21 HCl at 1508C for 32.5 min. Amino acid enantiomers were derivatized with o-phthaldialdehyde and N-isobutyryl-L-cysteine and were separated on a Poroshell 120 EC-C18 (4.6 3 100 mm, 2.7 mm particles) column. A linear binary gradient was used starting with 100% potassium di-hydrogen phosphate (KH2PO4; 48 mmol L21, pH56.25) to 61% KH2PO4 and 39% methanol:acetonitrile (13:1, v/v) at 13.3 min, 46% KH2PO4 at 19.2 min, 40% KH2PO4 at 21.3 min, and 20% KH2PO4 at 22 min. Eighteen amino acids were included in the analysis: asparagine1aspartic acid (Asx), glutamine1glutamic acid (Glx), serine (Ser), histidine (His), glycine (Gly), threonine (Thr), b-alanine (b-Ala), arginine (Arg), alanine (Ala), c-aminobutyric acid (c-Aba), tyrosine (Tyr), valine (Val), phenylalanine (Phe), isoleucine (Ile), leucine (Leu), and lysine (Lys). Acid-catalyzed racemization was corrected according to Kaiser and Benner [2005]. This method has a limit of quantification of 0.5 nmol L21 for individual amino acids. D-enantiomers of Asx, Glx, Ser, and Ala are reported in this study.

Concentrations of TDAA and TPAA were determined as the total concentrations of the eighteen dissolved and particulate amino acids, respectively. DOC-normalized yields of TDAA were calculated as the percentage contributions of amino acid carbon to the total DOC, using equation (1) and being reported in units of %DOC:

TDAA (%DOC) = ( [TDAA-C] / [DOC] ) x 100

where [DOC] and [TDAA-C] are the concentrations of bulk DOC and carbon measured in the total dissolved amino acids, respectively. This calculation excluded the two nonprotein amino acids (b-Ala and c-Aba) that are thought to be byproducts of decomposition [Cowie and Hedges, 1994].

DOC_aminoAcids.csv