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Ocean crust of young age is generally rich in reduced substances such as Fe(II) and sulfur (Bach and Edwards, 2003). Hydrogen was found to be produced from the ocean crust by abiotic basalt-water reactions at low temperature (Stevens and Mckinley, 1995, 2000). These reduced substances in the ocean crust can serve as electron donors for endolithic microorganisms. Meanwhile, seawater is oxic and contains oxygen, nitrate, and sulfate that are preferably utilized as electron acceptors by microorganisms. The circulation of seawater through the upper ocean crust can therefore energize potential endolithic communities in which chemolithoautotrophs utilizing Fe(II), sulfur, and hydrogen play a role as primary producers. This type of crustal biosphere is most likely to exist on young ridge flanks (Fisher et al., 2003; Lever et al., 2013; McCarthy et al., 2011). The North Pond site located on the western flank of the Mid-Atlantic Ridge is one of ideal study sites to examine the ridge flank crustal biosphere (see the “Expedition 336 summary” chapter [Expedition 336 Scientists, 2012a]).

The results of several cultivation studies targeting endolithic or crustal fluid microorganisms have been reported previously. The results from experiments using basaltic core samples collected during Ocean Drilling Program (ODP) Leg 187 at Southeast Indian Ridge (maximum penetration was 374 meters below seafloor [mbsf]) presented the methane production and Fe(III) reduction in enrichment cultures, indicating the existence of active hydrogenotrophic methanogens and Fe(III)-reducing microorganisms within the cores (Lysnes et al., 2004). Cultivation of seafloor basalts collected at low- and high-temperature fluid vent systems along the Mid-Atlantic Ridge detected diverse bacteria belonging to the classes Alphaproteobacteria, Betaproteobacteria, Gammaproteobacteria, Actinobacteria, and Bacilli, and some of the isolates that were assigned to the genus Bacillus were shown to grow on a medium specific for manganese oxidizers (Rathsack et al., 2009). In a study of an Integrated Ocean Drilling Program (IODP) site on the Juan de Fuca Ridge flank, in situ colonization systems deployed in the borehole for 4 years revealed the presence of active and cultivable Fe(II)-oxidizing bacteria belonging to eight genera, including gammaproteobacterial Alcanivorax, Marinobacter, and Halomonas as the predominant genera; however, all cultivated bacteria that utilized low-organic compounds for their growth were not proven to be facultative chemolithoautotrophs in that study (Smith et al., 2011). The existence of methanogens and sulfate reducers in the subseafloor basalt was indicated by a combination of molecular phylogenetic, isotopic, and cultivation analyses using cores collected at the Juan de Fuca Ridge flank during IODP Expedition 301 (Lever et al., 2013). Ocean crust is also inferred to provide habitats for fungi; it has been used to examine fossilized fungi-like filamentous or spherical structures found in thin sections of basaltic cores collected during ODP legs (Ivarsson et al., 2012; Schumann et al., 2004).

Although many microorganisms have been previously enriched or isolated from ocean crust as mentioned above, most of those seem to be heterotrophs. We therefore tried to cultivate chemolithoautotrophic microorganisms from cores of basalt and overlying sediments retrieved by the R/V JOIDES Resolution from a North Pond site during IODP Expedition 336.