Bacterial community structure changes in activated sludge samples enriched by polyethylene and polypropylene

Samples of 3 activated sludges (samples 1, 2, and 3) obtained from wastewater treatment plants in petroleum and chemical industries in Yeosu, Korea were used in enrichment analysis. The primary sludges were suspended in 300 mL defined minimal media at 4 degree C and homogenized by shaking with ignited glass beads (diameter, 3 mm) at 200 rpm and 4 degree C for 2 h. Uniform slurries were obtained by centrifugation at 550 x g for 5 min and cell pellets were then obtained by centrifugation at 3,940 x g for 15 min. The cell pellets were washed 2 times more with 300 mL DMM and the differential centrifugation procedure. The harvested cells were conditioned in 100 mL DMM at 25 degree C and 200 rpm for 24 h and 20 mL aliquots were transferred to culture bottles containing 20 slices of ethanol-sterilized LDPE or PP films (5 mm W x 30 mm L x 50 to 100 micrometer thickness). The incubated sludge samples were timely collected for metagenomic analysis. The samples were prepared at the start of enrichment (0 time) and when the enriched media were replaced every 2 weeks (at 2, 4, 6, and 8 weeks)for a total period of 8 weeks.Total DNA samples from conditioned sludge media (0 time) and enriched media between 2 and 8 weeks were prepared using PowerSoil DNA isolation kits. 16S metagenomic sequencing of the V3-V4 region library prepared using Herculase II Fusion DNA Polymerase Nextera XT Index V2 kit was performed according to the protocol of 16S Metagenomic Sequencing Library Preparation Part No., 15044223 Rev. B by MiSeq (2x301 paired-end) platform (Illumina, San Diego, CA, USA).