Zobell marine broth 2216

The bacterial strain V. splendidus LGP32 was inoculated in Zobell Marine Broth 2216 (HiMedia Laboratories, Mumbai, India) and allowed to grow for 12–16 h at 25°C. Before use, the culture was refreshed by diluting it to 1:500 and further grown for 1–3 h. The concentration of bacteria was estimated by turbidimetry at 600 nm (Biospectrometer Basic, Eppendorf, Hamburg, Germany) based on a standard curve relating absorbance values to colony-forming units (CFUs).

Bacterial biofilm formed on corroded metal pieces from underwater hull of a fishing vessel in Goa, India, was harvested and used for the study. Each metal piece was
washed three times with sterile phosphate buffer solution to remove any loosely attached bacteria. The remaining sessile bacteria were scraped off with sterile scalpel and then transferred to 100 ml sterile glass container filled with Zobell marine broth 2216 (Himedia, India). The bottle was incubated at 27 °C for 48 h. After successful growth, bacterial colonies were purified and subcultured in Luria–bertani broth for further investigation. D. marinisediminis GSR3 (KR303707) isolated from the same ship hull was used as test microorganism. This bacterium was cultivated in 15 ml screw cap tube containing Postgate’s B medium (5 ml 70 % sodium lactate, 0.5 g KH₂PO₄, 1.0 g NH₄Cl, 1.0 g Na₂SO₄, 2.0 g MgSO₄·7H₂O, 1.0 g yeast extract, 0.1 g CaCl₂·2H₂O, 0.5 g FeSO₄·7H₂O and 20 g NaCl in 1L of distilled water) (pH 7.5–8.0) and sodium thioglycolate (0.1 g) and vitamin C (0.1 g) as reducing agents.