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).