*3.5. The 16S rDNA Identification of Bacterial Isolates*

Genomic DNA was extracted from cell pellets of single colony bacterial cultures in marine broth 2216. The Qiagen DNeasy Blood and Tissue kit was used (QIAGEN, Valencia, CA, USA). Specifically, overnight culture containing approximately 1–3 × 109 bacterial cells were transferred to a 1.5-mL Eppendorf tube on ice and centrifuged (13,000× *g*, 1 min). Prior to the addition of RNase buffer (1.5 μL), the cells were lysed with lysis buffer (300 μL, 65 ◦C, 15 min), incubated (37 ◦C, 15 min), and centrifuged. The resulting supernatant from centrifugation (13,000× *g*, 1 min) was thereafter treated with genomic-binding buffer (0.5 mL), centrifuged (10,000 rpm, 2 min), and passed through a DNA-binding column. Washing the column with genomic wash buffer (1 mL) and three times with 1 mL of 75% ethanol followed by dry spinning afforded purified DNA on the column. The resulting DNA was eluted from the column with milliQ water.

Reacting a mixture containing 10 × Qiagen buffer (2.5 μL), MgCl2 (0.5 μL), d'NTPS (1 μL), 2.5 μL of primer 27 F (5 -AGAGTTTGATCCTGGCTCAG-3 ), and 2.5 μL of primer 1492 R (5 -TACGGYTACCTTGTTACGACTT-3 ), DMSO (0.75 μL) and Taq polymerase (0.1 μL) gave the polymerase chain reaction (PCR) product of the 16S rDNA. Sterile Milli Q water was added to adjust the final volume of the PCR mixture to 25 μL. Thermal cycling was performed with an Eppendorf Mastercycler AG 22331 (EPPENDORF, Hamburg, Germany). The sample utilized the standard initial denaturation step (95 ◦C, 5 min; 95 ◦C, 30 s) followed by annealing (40 ◦C, 1 min). Procedurally, the thermal profile used was 30 cycles. This profile consisted of 1 min of primer annealing at 55 ◦C, 1 min of extension at 72 ◦C, and 1 min of denaturation at 95 ◦C. A final extension step consisting of 10 min at 72 ◦C was also included. PCR products were detected by agarose gel electrophoresis and visualized by UV fluorescence after ethidium bromide staining. The PCR products generated by the 27 F and 1492 R primers were approximately 1 kb in size and purified using the Qiagen genomic DNA as specified by the manufacturer. Sequencing of the final DNA extracts was done by LGC Genomics (Germany).
