# Marine Bacteria Associated with Colonization and Alteration of Plastic Polymers
## 2 Materials and Methods
### Bacteria Molecular Identification  
Bacteria adhering to marine sand were detached through ultrasound bath and orbital shaker using a 1:2 ratio (\(w/v\)) of sand:double distilled and autoclaved water. Ultrasound bath was carried out for 1 min \(\times\) 5 times each, at room temperature and orbital shaking was performed overnight at 23 \({}^{\circ}\)C. Detached bacteria in supernatant (200 \(\upmu\)L) were plated in heterotrophic marine agar culture media. Bacteria plates were then cultivated at 23 \({}^{\circ}\)C and individual colonies were successively plated from each plate to obtain the pure colonies. The bacteria pure colonies were then molecularly characterized. When bacteria biomass needed to be increased, pure colonies were cultivated in sterile phosphate-buffered saline (PBS) solution containing tryptone (10 g L\({}^{-1}\)) and yeast extract (5 g L\({}^{-1}\)) at 23 \({}^{\circ}\)C for 24 h. The culture was then centrifuged at 15,000\(\times\)\(g\) for 15 min and the resulting pellet was taken as a source of template DNA. All media and reagents were purchased from Scharlab (Barcelona, Spain). Pure colonies and pellets were homogenized in liquid nitrogen and incubated in 800 \(\mu\)L of DNA isolation solution containing 100 mM Tris-HCl (pH 8.2), 4 M NaCl, 20 mM EDTA, CTAB (2%, \(w/v\)), PVPP (0.1%, \(w/v\)), SDS (0.1%, \(w/v\)) and mercaptoethanol (2%) in a water bath at 65 \({}^{\circ}\)C for 1 h according to the Murray and Thompson procedure [19] with modifications. A volume of chloroform:isoamyl alcohol solution (24:1 \(v/v\)) was added and the samples were gently mixed by inversion at intervals of 20 s. The mixture was then centrifuged for 10 min at 827\(\times\)\(g\) in a Beckman Coulter Allegra X-22R centrifuge (Beckman Coulter Inc., Brea, CA, USA). Successive washings with chloroform:isoamyl alcohol (24:1 \(v/v\)) solution were performed. The supernatant was then placed in a fresh tube and an equal volume of n-propanol (\(-\)20 \({}^{\circ}\)C) was added, mixed gently and centrifuged at 15,000\(\times\)\(g\) for 30 min. The resulting pellet, containing DNA, was washed with ethanol (80% \(v/v\), molecular grade), dried and suspended in sterile deionized water. DNA yields were assessed using a Nanodrop ND-1000 spectrophotometer (NanoDrop Technologies, Wilmington, DE, USA). All samples were in triplicate. Purity DNA was valued by smear absence, migrating on a 0.8% (\(w/v\)) agarose gel. The molecular identification was based on the amplification of the 16S rRNA gene (Table 1), amplified by a polymerase chain reaction in a GenAmp 2400 thermal cycler (PerkinElmer Inc., Waltham, MA, USA) with 30 cycles consisting at 95 \({}^{\circ}\)C for 1 min, 55 \({}^{\circ}\)C for 1 min and 72 \({}^{\circ}\)C for 1.5 min, followed by a final extension step of 5 min at 72 \({}^{\circ}\)C. Each PCR reaction mixture contained 0.5 U Takara Ex TaqMan polymerase (TaKaRa Shuzo Co., Shiga, Japan), 2.5 mM of each dNTP, 10 \(\mu\)L Takara Ex TaqMan PCR buffer with MgCl\({}_{2}\), 10 \(\mu\)M forward and reverse primers (Table 1) and 90-95 ng DNA template. PCR products were visualized with agarose gel electrophoresis. In addition, amplification products ranging from 800 to 1,200 bp were obtained and purified using the QIAEX agarose gel extraction kit (Qiagen Inc., Hilden, Germany). The fragments were then ligated to the pGEM-T-easy cloning vector (Promega, Fitchburg, WI, USA) and cloned in JM109 cells according to the manufacturer's instructions (Promega). Plasmids were isolated using a plasmid purification kit (Qiagen Inc.). The insert in the plasmid was checked by PCR using primers M13F and M13R (Promega). The insert was then sequenced on both strands using an ABI-310 DNA automated sequencer (Applied Biosystems, Foster City, CA, USA) and BigDye Terminator v3.1. Nucleotide sequences were submitted to NCBI GenBank BLAST search and identified through similarity values.