Nitrogenase Activity (Acetylene Reduction) of an Iron-Oxidizing Leptospirillum Strain Cultured as a Pioneer Microbe from a Recent Volcanic Deposit on Miyake-Jima, Japan

The genus Leptospirillum is known to dominate acid mine drainage and bioleaching systems. In this paper, we describe the isolation of iron-oxidizing bacteria closely related to Leptospirillum ferrooxidans from an acidic volcanic ash deposit on the island of Miyake (Miyake-jima), Japan. We further show the nitrogenase activity (acetylene reduction) for one (strain C2-1) of the isolates. The deposit harbored 1.2 x 10(8) total direct count (g dry weight)(-1) and 7.1 x 10(5) (most-probable-number, MPN) (g dry weight)(-1) of iron-oxidizer. A ferrous iron-limited, aerobic chemostat culture using Leptospirillum HH medium (pH 1.8) was performed to select and isolate the Leptospirillum group. Nine isolates were regarded as pure cultures based on uniform colony morphology on Fe2+-containing silica gel plates and absence of growth on 100-fold diluted nutrient broth plates. Six strains examined further shared 99.9-100% identity in 16S rRNA gene sequence with each other. Homology-based searches showed that all of the strains belonged to the Leptospirillum ferrooxidans clade. Strain C2-1 grown in ammonium sulfate-free Leptospirillum HH medium (pH 1.8) showed an initial rate of acetylene reduction of 58 mu mol h(-1) (g cell carbon)(-1).