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doi:10.2204/iodp.proc.331.203.2016

Results and conclusions

The results for the microcalorimetric activity measurements of Expedition 331 samples are shown in Tables T1 and T2. Microbial activity can be calculated as the difference between total and chemical heat output. The data show considerable activity at 90°C, which is partly attributed to microbial activity (50% for aerobic conditions). At 25°C, significant activity could only be detected for aerobic conditions. Overall, microcalorimetric measurements reveal activity of thermophilic microorganisms in the Expedition 331 samples.

Table T3 gives an overview of enrichment cultures and isolates; details are described in the following text. Overall, enhanced microbial activity at 90°C was mirrored by the enrichment of thermophilic sulfate reducers in agreement with geochemical data (Aoyama et al., 2014). The cultures we obtained from hydrothermally influenced, deeply buried marine sediments at 12°C were not significantly different from those of cold and temperate deeply buried marine sediments. We were not able to cultivate Archaea, for example, despite the fact that they are broadly found with 16S rRNA gene approaches (Yanagawa et al., 2014).

Thermophiles

The first attempts to cultivate thermophiles at 70° and 80°C in liquid media under aerobic and anaerobic conditions with monomer or polymer solutions as a carbon source were unsuccessful; no growth was observed after 3 months of incubation. In a second enrichment attempt, incubation temperature was decreased to 60°C under anoxic conditions. Over an incubation period of 10 months, nearly all inoculated tubes showed activity by formation of black sulfide precipitation. We interpreted the formation of this precipitation as a sign of an ongoing sulfate reduction, because in control tubes with autoclaved or no inoculums, no formation of sulfide precipitation was observed. However, subculturing remained unsuccessful.

Aerobic litho(hetero)trophs

No (stable) sulfide- or iron-oxidizing enrichment culture at 12°C was obtained from the investigated depth intervals in the three cores. For microaerophilic Fe(II) oxidizers, only culture tubes inoculated with samples from Sites C0013 (0.2 and 0.3 mbsf), C0014 (2.1 mbsf), and C0017 (6.4 and 10.9 mbsf) showed a sharp band of Fe(III) precipitation, which indicates a microbially enhanced Fe(II) oxidation, in contrast to the fuzzy band in the chemical control tubes. Similar observations were made for the enriched aerobic sulfide oxidizers. None of these enrichment cultures could be successfully maintained. On the other hand, stable Mn(II)-oxidizing enrichment cultures were obtained from all core depth intervals. In all of these enrichment cultures, a sharp brown band 1–2 cm beneath the top of the agar in the gradient tubes developed over 3–4 weeks. In the chemical controls, only a weak and fuzzy band was observed. These putative Mn(II)-oxidizing bacteria could successfully be transferred to agar plates with the polymer mix as the carbon and energy source. The 16S rRNA gene of five isolates was partially sequenced (320–430 bp), and the isolates were affiliated to Bacillus oceani (91%), whereas two isolates were affiliated to Bacillus aquimaris (92%). The obtained Mn(II)-oxidizing cultures could grow on agar plates in the presence of 5 mM Mn(II) but without an additional carbon source.

Aerobe and anaerobe heterotrophic mesophiles

Aerobe heterotrophic mesophiles were successfully cultivated from all samples at 12°C with monomer and polymer as the carbon source on agar plates. From Site C0013, two isolates were obtained affiliated to Halobacillus litoralis (92%) and one to Bacillus niacini (95%); both are species originally isolated from deep-sea samples. Two additional isolates from Site C0013 are affiliated to Marinobacter salsuginis (90%) and Marinobacter hydrocarbonoclasticus (93%). From Hole C0017, we obtained two isolates affiliated to Shewanella benthica (89%), three isolates affiliated to Cytophaga fermentans (92%), and one isolate affiliated to Idiomarina spp. (92%) and Psychrobacter spp. (95%). None of the obtained isolates could grow at 60°C.

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