Iron, manganese and sulfate were detected in concentrations of up to 85, 0.1 or 2 μmol cm−3, respectively. The pH was between 8.0 and 8.5 and the in situ water temperature Selleckchem ICG-001 was 14 °C. For incubations established from the Zeebrugge samples, filter-sterilized harbor water (using 0.2-μm membrane filters) served as a medium to mimic in situ conditions. However,

the harbor water naturally contained 2 mM sulfate and sediment microcosms without electron acceptors were therefore impossible to prepare. Basal salts were not added. Dissolved oxygen was removed by nitrogen gassing of 1 L filtered water. All additional manipulations were performed in an anaerobic glove box. To homogenize the sediment sample, a 1/1 mix of sediment and medium was stirred. The slurry was sampled for DNA extraction click here and 20 mL was used to inoculate 40 mL medium in 120-mL serum bottles. These were sealed with butyl rubber stoppers

and aluminum crimp caps. Triplicate microcosms were incubated under a nitrogen headspace at atmospheric pressure at 25 °C. Before inoculation, 2.5 mM ferrihydrite, 1.25 mM manganese dioxide, 1 mM potassium nitrate or 20 mM sodium sulfate was added to the medium. Ferrihydrite was precipitated by neutralization of an FeCl3 solution (Lovley & Phillips, 1986) and manganese dioxide was obtained by oxidation of an MnCl2 solution with KMnO4 (Lovley & Phillips, 1988). To determine indigenous methanogenesis, controls without additional hydrocarbons and electron acceptors were prepared. Controls without hydrocarbons, but with electron acceptors were set up as single incubations. The final hexadecane or ethylbenzene concentrations were 0.1% v/v in 60 mL total liquid volume. To test polyaromatic hydrocarbon (PAH) degradation, 1.6 mg 1-13C-naphthalene or 12C-naphthalene was added to 100 mL medium many containing 20 mL sediment in 120-mL serum bottles sealed with butyl rubber stoppers and aluminum crimp caps. Manganese dioxide was not used in the case of naphthalene.

To examine the activity of anaerobic methanotrophs, the headspace of separate microcosms was flushed with a 1/1 methane–nitrogen mix without additional higher hydrocarbons. Methane and CO2 in headspace samples were analyzed using a GC–FID (+nickel catalyst methanizer, SRI 8610C, SRI Instruments) equipped with a 6-foot Hayesep D column (SRI Instruments) running continuously at 60 °C. Methane and CO2 formation from 12C- and 1-13C-naphthalene was also measured using a Thermo Fisher MAT252 GC–IRMS (Herrmann et al., 2010). The rates were calculated based on the formation of 13CH4 measured in the headspace and subtracted from the of indigenously produced methane. δ13C values are expressed as ‰ vs.