A surface seawater sample was collected using
a Rosette sampler with CTD (conductivity–temperature–depth) from a coastal region of the Yellow Sea (33°59.827′N, 123°0.123′E), China, in July 2008. The temperature and salinity of the seawater at the time of sampling were 25.96 °C and 31.2 p.p.t., respectively. One hundred microlitres of the 10- and 102-fold-diluted seawater (diluted in 0.9% w/v saline) was spread onto triplicate marine 2216E agar (MA, Difco) plates and incubated at 28 °C for 7 days. Individual colonies appearing on the plates were picked off and purified by successive streaking and restreaking on plates of MA. Nineteen cultures were purified and identified using 16S rRNA gene sequences and morphological characteristics. Working cultures were maintained
at 28 °C on MA plates, and stocks were kept as suspensions in sterile 0.9% (w/v) saline supplemented with 15% (v/v) glycerol at −70 °C. One of learn more the isolates, designated WH169T, did not correspond with any of the taxa included as a validly published bacterial name, and has been characterized using a polyphasic approach. Aestuariibacter salexigens DSM 15300T obtained from Deutsche Sammlung von Mikroorganismen und Zellkulturen (DSMZ, Braunschweig, Germany) was used as the reference strain. Standard protocols, including those of Gram staining, catalase and oxidase activities, degradation of casein, starch, gelatin, Tween 80 and urea, endospore formation and nitrate reduction, were used (Tindall et al., 2007). The range of salinity supporting the growth of the strain was tested using two Cyclopamine mouse kinds of media, which showed similar results. (1) MB was used as the basal medium. The various salinities (0.5–10% w/v) contained in MB were adjusted by addition of NaCl (at increments of 1% w/v). The salinity in the media was determined using a portable refractometer (Opticon Series FG100sa, Jinan, China); (2) synthetic marine 2216E broth [5 g Bacto peptone, 1 g yeast extract and 0.1 g FePO4 in 1 L of artificial seawater (ASW; Lyman
& Fleming, 1940) minus NaCl (ASWN−)] with slight modifications (Na+ in ASWN− was replaced by appropriate K+, ASWN−K+) was used as the basal medium. Growth in various concentrations of NaCl (0–15% w/v) was assessed in appropriately modified synthetic marine 2216E find more broth. The requirement for sea salts was tested in synthetic 2216E broth without ASW, but containing 3% NaCl (w/v). Inoculated media were incubated at 28 °C for 2 or 14 (in broth that contained 0% and >11% NaCl) days. The temperature range for growth was determined by incubating cultures at 4–50 °C for 2 or 14 (at 4 and 50 °C) days in MB. Growth at pH 4–11 was determined in MB with citrate/phosphate buffer or Tris/HCl buffer (Breznak & Costilow, 1994). Growth was determined in five replicates by an increase in OD600 nm, as measured spectrophotometrically.