11:45 AM - 12:00 PM
[BBG02-10] Isolation of the first thermophilic and actively nitrogen-fixing bacteria in the deep branching phylum Aquificae
Keywords:Aquificales, nitrogen fixation, geothermal spring, thermophile, chemosynthetic bacteria
Thermophilic nitrogen-fixing bacteria have been suggested to occur in hydrothermal vents and terrestrial hot springs from molecular and functional-based analyses of prokaryotic communities. Nif genes, biomarkers for nitrogen-fixing bacteria, already have been found in some chemosynthetic thermophilic isolates, too. Although nitrogen-fixing ability was observed in methanogenic archaea at 92ºC, active nitrogen fixation in thermophilic bacteria more than 70ºC has not been demonstrated yet.
In this study, we isolated two novel Aquificae, a deeply branching bacterial phylum, into axenic culture under nitrogen-fixing conditions from chemosynthetic microbial communities at 70-77ºC in sulfidic alkaline hot springs (Nakabusa, Nagano, Japan). Phylogenetic analysis based on 16S rRNA gene classified both strains within the genus Hydrogenobacter; strain 1-6 showed 98.7% nt identity to Hydrogenobacter subterraneus HGP1 and strain 2-18 had 97.6% identity to H. hydrogenophilus DSM 2913. Both isolated strains contained nifH gene sequences, encoding a key enzyme component of nitrogen fixation, with 96.5% and 97.4% amino acid identity to Hydrogenobacter thermophilus TK-6. Nitrogenase activities were confirmed in both strains incubated at 70ºC using the acetylene reduction test. Both strains showed nitrogen-gas-dependent growth under lowered aerobic conditions with approximately up to 10% oxygen using CO2 as a sole carbon source and N2 as sole nitrogen source with H2 or thiosulfate as electron donors at 70ºC.
This is the first demonstration of active nitrogen-fixation in thermophilic bacteria (more than 70ºC) and in the phylum Aquificae. The potential impact of nitrogen fixation in thermophilic chemosynthetic bacteria will be discussed with respect to life on earth before the appearance of photosynthesis.
In this study, we isolated two novel Aquificae, a deeply branching bacterial phylum, into axenic culture under nitrogen-fixing conditions from chemosynthetic microbial communities at 70-77ºC in sulfidic alkaline hot springs (Nakabusa, Nagano, Japan). Phylogenetic analysis based on 16S rRNA gene classified both strains within the genus Hydrogenobacter; strain 1-6 showed 98.7% nt identity to Hydrogenobacter subterraneus HGP1 and strain 2-18 had 97.6% identity to H. hydrogenophilus DSM 2913. Both isolated strains contained nifH gene sequences, encoding a key enzyme component of nitrogen fixation, with 96.5% and 97.4% amino acid identity to Hydrogenobacter thermophilus TK-6. Nitrogenase activities were confirmed in both strains incubated at 70ºC using the acetylene reduction test. Both strains showed nitrogen-gas-dependent growth under lowered aerobic conditions with approximately up to 10% oxygen using CO2 as a sole carbon source and N2 as sole nitrogen source with H2 or thiosulfate as electron donors at 70ºC.
This is the first demonstration of active nitrogen-fixation in thermophilic bacteria (more than 70ºC) and in the phylum Aquificae. The potential impact of nitrogen fixation in thermophilic chemosynthetic bacteria will be discussed with respect to life on earth before the appearance of photosynthesis.