Amino acid concentration and isotopic composition in sedimentary 13C-labeling incubation of sediment core HE483/25-2

Amino acids derivatization and analysis: Sediment slurries were centrifuged to separate the sediments from the medium and the sediments were freeze-dried and homogenized. 50 mg of samples spiked with the internal reference standard norleucine were decalcified and acid hydrolyzed (6M, 110℃) for 15 h. The hydrolysate was centrifuged to remove the sediments and subsequently defatted with hexane/ DCM mixture (V/V, 2:1) three times. The purification and derivatization of amino acids were performed by following the protocols described by Takano et al.53 and Chikaraishi et al.54, respectively. In brief, the hydrolysates were evaporated under N2 gas to dryness and purified using Dowex 50WX8 200 400 mesh cation exchange resin to eliminate the matrix effects. The purified amino acids were then isopropylated with a mixture of isopropanol and acetyl chloride (4/1, v/v) at 100 ℃ for 2 h. The solution was then evaporated to dryness and followed 3 times with DCM addition and evaporation to remove any remaining reagent. The AA isopropyl esters were then acylated using a mixture of pivaloyl chloride and DCM (1/1, v/v) at 100 ℃ for 2 h to obtain pivaloyl-isopropyl ester (Pv/AA/iPr). The Pv/AA/iPr solution was then evaporated to dryness and followed 3 times DCM addition and evaporation to remove any remaining reagent. Liquid-liquid extraction was performed by using MiliQ water and a hexane/DCM mixture (2/1, v/v). The Pv/AA/iPr were stored frozen (-20℃) and dissolved in ethyl acetate before analysis.