2.1 Bacterial strains and culture conditions
Artificial seawater (Sw) medium was composed of (l−1): 0.15 g NH4Cl, 0.2 g KH2PO4, 0.5 g KCl, 18.0 g NaCl, 2.6 g MgCl2·6H2O, 0.15 g CaCl2·2H2O, 2.5 g NaHCO3, 1% (vol/vol) of filter-sterilized vitamin solution, as described by Wolin et al. (1963), and 1.0 ml of a trace element solution, as used by Touzel and Albagnac (1983), lacking NaCl and supplemented with 4.0 mg/l of Na2SeO3·5H2O. SPYSw medium was prepared by adding 10 mM sodium sulfate, 10 mM sodium pyruvate, and 0.01% (wt/vol) yeast extract (Bacto Yeast Extract, Becton Dickinson) to Sw medium. YPSw medium was prepared by adding 0.2% (wt/vol) yeast extract and 0.2% (wt/vol) Polypepton (Nihon Pharmaceutical) to Sw medium. NYPSw medium was prepared by adding 10 mM sodium nitrate to YPSw medium. YSw medium was prepared by adding 0.1% (wt/vol) yeast extract to Sw medium.
The medium was dispensed by 20 ml into each 50-ml serum bottle. Dissolved air in SPYSw, NYPSw, and YSw media was removed by flushing with N2:CO2 (4:1 [vol/vol]), and dissolved air in YPSw media was removed by flushing with H2:CO2 (4:1) at an approximate pressure of 0.15 MPa. The bottles were sealed with butyl rubber stoppers. The pH of the medium was adjusted to 7.0 with 10 mM NaHCO3.
P. bellariivorans F2T (Holmes et al. , 2007) was obtained with JCM accession number of JCM 13498T from the Japan Collection of Microorganisms of the RIKEN Bioresource Center (RIKEN-BRC JCM).