Environmental Sponge Microbiota Characterization

Each environmental sponge sample collected for microbiota characterization was homogenized with 50 ml of phosphate buffer containing 0.9% NaCl in a stomacher, for 7 min at 230 rpm. Fifty milliliters of the homogenate were transferred to a sterile 50 ml conical tube and centrifuged at 11,000×g and 4 °C for 20 min (Beckman Coulter, Avanti J-26 XPI) [34 (link)]. After centrifugation, supernatants were discarded and pellets were stored at − 80 °C until DNA extraction. DNA was extracted from approximately 0.25 g of each sample using DNeasy PowerSoil DNA extraction kit (QIAGEN) following manufacturer’s protocol. Approximately 0.25 g of a sterile sponge was also sampled and used as a negative control to confirm the absence of microbial DNA contaminants on the sterile sponge. DNA extracted from the sponge was processed following the same protocol as described below for other samples. The concentration of DNA in each test sample and in the control sample was determined both spectrophotometrically using NanoDrop One (Thermo Scientific) and fluorometrically using Qubit 3 (Invitrogen) and Qubit dsDNA High Sensitivity Assay Kit. DNA samples were stored at − 80 °C until further use.