Sediment Core Incubations
When the O2 concentrations in all sediment cores had decreased below 0.2 mg/L, ca two weeks after closing them, several incubations of the sediment cores were done over the course of 8 weeks to determine fluxes of PO43-, NH4+, O2, CH4 (methane), and H2S (hydrogen sulfide). The method is based on differences in solute concentrations at the start and end of the incubation. The flux Jx (mmol m-2 d-1) equals (CE - CS) ⋅ h / t, where CE and CS are end and start concentration in μmol/L, h is the core water column height in m, and t is incubation time in days.
Sampling of solutes in the core water was done from the middle of the column using a syringe with a silicone tubing. Samples for PO43- and NH4+ were filtered through a 0.45 µm polyethersulfone (PES) syringe filter into heat-treated (water filled at 60°C for 24 h) PP-tubes, and analyzed using method 2 mentioned in the section Chemical Analyses below. All water samples were stored at 4 °C before analysis. Water for CH4 analysis was transferred without filtering to 12 mL exetainer vials and any biological activity was stopped with addition of 100 µL 7M zinc chloride, to prevent microbial methane production or oxidation while awaiting analysis. Details on all chemical analyses are provided in a separate paragraph, below.
Termination
The experiment was terminated 8 weeks after closing the cores. Water was collected for nutrient analysis, as described above, and for H2S by siphoning and overflowing core water into winkler flasks that were then fixated with 4 mL zinc acetate. The samples were stored at 4 °C until analysis. A counting of benthic fauna, especially large worms, could not be performed due to the decay of the animals.
The top 2 cm of the sediment surface was sliced. A portion of the wet slice was weighed and dried to establish water content and organic matter content using mass loss on ignition (LOI) at 520 °C for 6 h. The remaining wet slice was transferred to 50 mL PP-tubes and carefully bubbled with nitrogen via 0.40 mm syringe needles to purge oxygen and preserve anoxic conditions. pH was measured, and aliquots were centrifuged at 4000 rpm for 5 minutes before supernatant porewater was analyzed for dissolved PO43- using method 1 mentioned in the section Chemical Analyses below.
Chemical Analyses
Watercolumn pH, oxygen, and salinity were measured with a multimeter (HACH Hq40d). To determine CH4 concentration in incubation samples, headspace-gas chromatography was done, following the method by Sithersingh and Snow (2012). A headspace was created in the Exetainer vial containing the samples by replacing 2 mL of sample with nitrogen gas using Ø 0.4 mm needles pushed through the septum of the lid. After 24 h equilibration at room temperature, 1 mL headspace gas was injected into a Shimadzu 8A gas chromatograph (GC) equipped with a flame ionization detector (FID). We made repeated injections of 1 mL standard gas containing 50 ppm methane (Air Liquide, France), to calibrate the instrument. Additionally, a solubility coefficient for methane in water with salinity of 6 was used to calculate the concentration of CH4 in the water.