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Heterotrophic enrichments were obtained for samples from all four sites (M0059, M0060, M0063, and M0065) and up to depths of 120 meters below seafloor (mbsf) (Table T1). Heterotrophic enrichments using a polymer mix obtained the highest positive dilution steps (10–4), whereas with a monomer mix the MPN values never exceeded 10–1. Iron- and manganese-reducing organisms could be enriched from all sites at neutral pH (7.5) and different salt concentrations (1‰–30‰). As a general observation, iron and manganese reduction started earlier in the test tubes at salinity ≤10‰ than in the test tubes with higher salinity. This was not observed under nitrate-reducing or heterotrophic conditions. For nitrate-dependent Fe(II)-oxidation conditions, the MPN value was never higher than 10–1, and positive enrichments were obtained only from cores from Sites M0060 and M0065.

Generally, no or only very low methane production was observed in the incubations without the addition of substrates, indicating that substrate availability for the indigenous methanogenic microbial communities was low in all samples (Tables T2 and T3). The only exceptions were one sample from Site M0059 (Sample 347-M0059E-13H-2, 125–130 cm) and two samples from Site M0063 (Samples 347-M0063E-3H-2, 60–65 cm, and 12H-2, 62–67 cm), where methane in the headspace continuously increased over the 12 months of incubation time.

These three samples were also most prominently stimulated by the addition of different substrates, both with simple, monomeric, or complex compounds. Besides well-known substrates for methanogens like acetate, methanol, hydrogen, or TMA (Table T2), more complex compounds from the polymer mix, palmitate, phytane, or hydrocarbons were also degraded. This indicates the presence of a metabolically diverse active community in the sediments, enabling the breakdown of the larger compounds to smaller substrates for the methanogenic organisms at the end of the food chain.

In other sediment samples, only in a few single cases was stimulation of methane production observed. Here, the traditional methanogenic substrates and monomeric compounds seemed to be more favorable, as one would expect. The hydrocarbons tested in the enrichment cultures were the least successful substrates tested, with only one positive sediment sample (347-M0063E-12H-2, 62–67 cm).