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Two novel, thermophilic piezophiles, capable of chemolithoautotrophic growth, are successfully cultivated and isolated from a black smoker chimney at the TAG field (Mid Atlantic Ridge: MAR) by using a piezophilic cultivation technique. Both strains (strains 106 and 108) represent dominant cultivated populations of the microbial communities in the chimney surface habitat. Strain 106 represents typically thin, long spiral cells under the piezophilic growth condition but short bent cells under the non-piezophilic condition. It is a strictly chemolithoautotrophic gammaproteobacterium using reduced sulfur compounds as the electron donors, and nitrate and O2 as the electron acceptors. Based on the 16S rRNA gene sequence, strain 106 would represent a novel genus of the previously uncultivated group (Symbiont Group I; a potentially novel family) within theGammaproteobacteria, and ‘Thioprofundum lithotrophica’ gen. nov., sp. nov. is proposed. Strain 108 is a short, oval rod at any of the growth pressures. It is a facultative chemoautotroph, capable of both chemolithoautotrophic growth with H2 and S oxidations and organotrophic growth with complex organics or organic acids using nitrate and O2 as the electron acceptors. The chemolithoautotrophic growth is strictly piezophilic and under the organotrophic growth condition, it grows at conventional pressures (0.1 MPa). Strain 108 is phylogenetically distinctive from any of the previously described genera of the familyRhodobacteraceaewithin theAlphaproteobacteria, and ‘Piezobacter thermophilus’ gen. nov., sp. nov. is proposed. The piezophilic cultivation technique can be a powerful tool to isolate and characterize the previously uncultivated phylotypes in the deep-sea hydrothermal vent environments.