‘
Candidatus
Ferrigenium straubiae’ strain KS (KCTC 25982, DSM 118991) is a neutrophilic, Fe(II)-oxidizing bacterium representing up to 98% of the community in culture KS, the most extensively studied mixed culture known for autotrophic nitrate-reducing Fe(II) oxidation. The phylogeny and genome of this bacterium were previously analysed and identified as ‘
Candidatus
Ferrigenium straubiae’. In this study, we report the first-time successful isolation of ‘
Candidatus
Ferrigenium straubiae’ strain KS and its experimental physiological characterization. The bacterium was identified as a non-stalk-forming, rod-shaped and non-halophilic strain with a Gram-negative classification. We characterized its physiology when grown in agarose-stabilized Fe(II)-O
2
gradient tubes where Fe(II) stemming from FeS functions as the electron donor and O
2
as the electron acceptor. It showed growth at temperatures of 20–30 °C (optimal at 25°C) and at pH levels of 6.0–7.5 (optimal at pH 6.5–7.0). The doubling time at 20 °C and pH 6.5 was 16 h. We further optimized the gradient tubes for sustainable culture maintenance using modified Wolfe’s mineral medium (MWMM; 1 g l
−1
NH
4
Cl) supplemented with 7-vitamin solution, SL-10 trace elements, selenite-tungstate solution and selenite-molybdate-nickel-copper-arsenic-vanadium solution (final concentrations of 10 µM Se, 10 µM Mo, 0.1 µM Ni, 0.1 µM Cu, 0.1 µM As and 5 nM V). We also evaluated several Fe(II) sources (with O
2
as electron acceptor), as well as both inorganic and organic substrates for their influence on growth. Although a known member of the denitrifying community in culture KS, the isolated strain ‘
Candidatus
Ferrigenium straubiae’ KS exhibited exclusively microaerobic and autotrophic growth in agarose-stabilized Fe(II)-O
2
gradients, utilizing Fe(II) from FeS as the electron donor.