Redox dye mix a

Phenotype Microarrays (Biolog, Hayward, CA, USA) were used for the characterization of P. ganghwense C2.2 (DSM 109767) with regard to the usage pf carbon (PM1 plates) and nitrogen (PM3 plates) sources, osmolyte requirements (PM9 plates), and pH tolerance (PM10 plates). Synthetic seawater minimal medium (ASW) [44 (link)] without NH₄Cl was the basal medium for the microarrays, supplemented with: 0.2% (w/v) urea for PM1; 2% (w/v) glycerol for PM3; 0.2% (w/v) urea, 2% (w/v) glycerol, and no NaCl for PM9; and 0.2% (w/v) urea, 2% (w/v) glycerol, 2% (w/v) NaCl, and pH adjusted to 7 before inoculation for PM10. For the PM array inoculation, the strain was grown on ASW supplemented with 1 g/L yeast extract and 5 g/L peptone (designated as “marine peptone”—MP) agar [7 (link)] for 24 h at 20 °C. Transmittance values were adjusted to 65%, and Biolog Redox Dye Mix A (#74221) was added, with a dilution factor of 100. A working volume of 100 µL was used in each well. Plates were incubated at 20 °C, and the optical density at 590 nm (OD₅₉₀) was measured at 24 h intervals using the Biolog Microstation Reader (Biolog, Hayward, CA, USA). For data analysis, the OD₅₉₀ values measured at the time of inoculation were subtracted from each of the following readings. Heat maps were generated using a custom python script.

The carbon utilization profile of strain S5.2 was assessed using the 96-well PM1 and PM2A plates (Biolog, USA). In brief, the overnight cultured bacterial colonies were inoculated into IF-0a GN/GP base inoculating fluid (Biolog, USA) to reach 85% transmittance (T) according to the manufacturer’s protocol. Aliquots (100 µl) of cell suspension and 1× Biolog redox dye mix A were inoculated into each well of the plates respectively, followed by incubation at 28 °C. The utilization and growth of 192 different carbon substrates from the plates were monitored for 48 h with readings taken at 15 min intervals.
The kinetic information was recorded and quantified using OmniLog OL_FM_12 kinetic software (Biolog, USA) followed by data analysis (Bochner et al. 2001 (link)). In the event of bacterial growth, photographic readings of colour intensity resulted in dye reduction were represented in OmniLog units (OU) (Khatri et al. 2013 (link)). The threshold for positive bacterial growth was established at 100 OU, calculated with the subtraction of maximum growth value with the first reading (0 h).