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).

本研究以韩国丽水市石油与化工行业污水处理厂采集的3份活性污泥（activated sludge）样品（编号1、2、3）为实验材料，开展富集分析。将原始活性污泥悬浮于300 mL 4℃的限定性最小培养基（defined minimal media, DMM）中，加入经灼烧灭菌的3 mm直径玻璃珠，于200 rpm、4℃条件下振荡均质2 h。通过550×g离心5 min得到均匀浆液，再以3940×g离心15 min收集细胞沉淀。使用300 mL DMM结合差速离心步骤对细胞沉淀进行2次洗涤。将收集得到的细胞置于100 mL DMM中，于25℃、200 rpm条件下预培养24 h；随后取20 mL等分培养液转移至装有20片乙醇灭菌的低密度聚乙烯（LDPE）或聚丙烯（PP）薄膜（规格：5 mm宽×30 mm长×50~100 μm厚）的培养瓶内。定时收集培养后的污泥样品用于宏基因组分析（metagenomic analysis）。实验分别在富集培养起始阶段（0时刻）以及每2周更换一次富集培养基的节点（第2、4、6、8周）制备样品，整个培养周期总计8周。使用PowerSoil DNA提取试剂盒，提取预培养污泥培养基（0时刻）及第2至8周富集培养基中的总DNA。采用Herculase II Fusion DNA聚合酶Nextera XT索引试剂盒V2版构建V3-V4区域文库，随后参照《16S宏基因组测序文库制备方案》（部件号15044223 Rev. B），通过MiSeq（2×301双端测序）平台（Illumina，美国加利福尼亚州圣地亚哥）完成16S宏基因组测序。