IMIS - Marine Onderzoeksgroepen

Protocols:
Total organic carbon
(TOC) was determined on homogenized and finely powdered sediments using a Carlo Erba Elemental Analyser NA-1500 after removal of carbonate by in situ acidification with 30% HCl in silver sample cups (Nieuwenhuize et al, 1994).
Mineral surface area
was measured using the multi-point Brunauer, Emmett and Teller (BET) method on a Quantachrome NOVA 3000 surface area analyser. Prior to analysis, samples were desalted by rinsing them with aceton and distilled water (10:90) and with distilled water alone, centrifuged (2862 G, 10 min) and freezedried, after which the organic matter on the surfaces was removed by heating at 350°C for 12 h.
Total hydrolysable amino acids
(THAA) were determined following Dauwe & Middelburg (1998): duplicate samples of 0.1 g freezedried sediment were hydrolysed (6N HCl, 110°C, 20 h, under N2 atmosphere). To measure total concentrations, 0.1 ml of the hydrolyzate was added to 2 ml potassium borate buffer (pH 10) and neutralised with 0.1 ml of 6N NaOH. The solutions were vortexed and left standing at room temperature for 1 hour, after which they were vortexed again to set free any ammonium present in the mixture. Fluorescence derivatives were obtained by adding 0.2 to 0.4 ml of the solutions and an equal amount of ortho-phthaldialdehyde (OPA) reagens to 2 ml phosphate buffer (pH 8) in a cuvette and vortexing the solution. After 5 minutes, total concentrations were determined by measuring the fluorescence in a spectrofluorometer (excitation wavelength: 340 nm; emission wavelength: 455 nm). By comparison with a standard amino acid mixture (Sigma), these measurements were then converted to concentrations. Individual amino acids were determined by reverse phase HPLC after Fitznar et al. (1999).
Enzymatically hydrolysable amino acids
(EHAA) were extracted in duplicate samples following the method described by Mayer et al. (1995) and Dauwe et al. (1999b): 0.1 g of freezedried sediment was poisoned by adding a bacterial inhibitor (0.1M sodium arsenate and 0.1mM pentachlorophenol in a pH 8 sodium phosphate buffer) and this mixture was shaken on an orbit shaker. After 1 hour 0.1 ml proteinase K solution (1.0 mg ml-1) was added and the samples were shaken again at room temperature for 6 hours. Subsequently the samples were centrifuged to remove remaining parcticulate material, 75 µl TCA was added to 750µl of the supernatant to stop the reaction and precipitate macromolecules, after which samples were centrifuged again. In this way, only the bioavailable fraction (oligo-peptides with chains containing < 7-15 amino acids and monomers) is left (Mayer, 1995). Finally, 750 µl of the supernatant was hydrolysed (12N HCl, 110°C, 20 h, under a N2 atmosphere) and measured as is described for THAA. Blank-assays, where the adding of the proteinase K solution was replaced by adding the same amount of phosphate buffer, account for a possible degradation of the enzyme, but they also represent readily available oligo- and monomeric amino acids already present in the sediment. To calculate EHAA, which is the pool only attributable to proteinase k, these blanks were subtracted from the results with the enzyme (Dauwe et al., 1999b).