Compound specific isotope analysis of amino acids from Escanaba Trough sediments, off the coast of Northern California, USA, from May-June 2022.

SEDIMENT COLLECTION. Samples were collected aboard the R/V Thomas Thompson via push coring using the ROV Jason. Once recovered shipboard, pore waters were extracted via rhizon samplers, cores were subsectioned into one to five cm slices, the exterior sediment which was in contact with the core liner was removed, and remaining sediment was stored frozen in pre-combusted glass jars.

COMPOUND SPECIFIC ISOTOPE ANALYSIS OF AMINO ACIDS: Amino acids were purified prior to compound specific isotope analysis of amino acids (CSI-AA) according to protocols of the McCarthy lab at the University of California, Santa Cruz (McCarthy and others, 2004). Sediments were subject to acid hydrolysis by adding 6N HCl to each sample, evacuating the headspace with N2 gas, and heating at 110 degrees Celsius for 20 hours. Samples were then mixed, centrifuged at 2200 revolutions per minute for five minutes (min), and supernatant pipetted off to new vial. This process (mixing, centrifuging, and removal of supernatant) was repeated with 1 mL 6N HCl and four 1 milliliter rinses of MilliQ H2O. The supernatant from all HCl and MilliQ H2O rinses were combined and dried under N2 gas at 80 degrees Celsius. Samples were redissolved in 0.1N HCl and a known quantity of Norleucine internal standard was added to each sample. Samples were filtered with pre-combusted glass wool followed by 1.0 micrometer glass fiber filters to remove large particles and then AA were purified using cation-exchange chromatography with Bio-Rad AG50WX8 resin (200-400 mesh) according to Takano and others (2010). Samples were dried under N2 gas at 80 degrees Celsius, redissolved in 0.2N HCl, flushed with N2 gas, and then heated at 110 degrees Celsius for 5 min to re-protonate the amino acids. Trifluoroacetyl isopropyl ester derivatives were prepared after Silfer and others (1991). A final purification step was completed via liquid-liquid extraction using chloroform and a phosphate buffer following Ueda and others (1989). Samples were dried under N2 gas at room temperature, rederivatized, and then stored at 20 degrees Celsius until analysis. Derivatized samples were analyzed for d13C-amino acid and d15N-amino acid isotope values by a Thermo Trace 1310 gas chromatograph coupled to an IsolinkII (Thermo combustion reactor 1000 degrees Celsius)/ConfloIV and Thermo Delta V Plus isotope ratio mass spectrometer. Samples were dried under N2 gas, redissolved in ethyl acetate, and injected in triplicate unless otherwise noted. Amino acids were separated for d15N analyses using a BPX5 column (60 meter x 0.32 millimeter, 1 micrometer film thickness; SGE Analytical Science, Trajan, Austin, TX, USA) and for d13C analyses using a DB-5 column (50 meter x 0.32 millimeter, 0.52 micrometer film thickness; Agilent Technologies, Santa Clara, CA, USA). The injector temperature was 250 degrees Celsius with He column flow rate of 2 mL/min. The GC temperature program for nitrogen isotope analysis was: initial temp = 70 degrees Celsius hold for 1 min; ramp 1=10 degrees Celsius/min to 185 degrees Celsius, hold for 2 min; ramp 2 = 2 degrees Celsius/min to 200 degrees Celsius, hold for 10 min; ramp 3 = 30 degrees Celsius/min to 300 degrees Celsius, hold for 6 min. The GC temperature program for carbon isotope analysis was: initial temp = 75 degrees Celsius hold for 2 min; ramp 1 = 4 degrees Celsius/min to 90 degrees Celsius, hold for 4 min; ramp 2 = 4 degrees Celsius/min to 185 degrees Celsius, hold for 5 min; ramp 3 = 10 degrees Celsius/min to 250 degrees Celsius, hold for 2 min; ramp 4 = 20 degrees Celsius/min to 300 degrees Celsius, hold for 5 min.