REACTOR 2 BED
FastDNA SPIN Kit for soil (MP Biomedicals, Irvine, CA) was used for DNA extraction using approximately 0.5 g from each sample according to the manufacturers instructions. The quantity and quality of the extracted genomic DNA was determined using a NanoDrop spectrophotometer (NanoDrop Technologies and gel electrophoresis on 1,8% agarose). Extracted DNAs were stored at 20C until further use. The universal primers 515F and 806R, targeting the V4 region of the 16S rRNA gene were used (341F (5-GTGYCAGCMGCCGCGGTAA-3) and 802R (5-GGACTACNVGGGTWTCTA- 3)) (Yu et al., 2005). The PCR was performed in a single-step 30 cycle PCR using the HotStarTaq Plus Master Mix Kit (Qiagen, USA) under the following conditions: 94C for 5 min, followed by 30 cycles of 94C for 30 sec, 55C for 30 sec and 72C for 1.5 min and a nal elongation step at 72C for 10 min. The expected-size PCR products were puried in 1,0% agarose gel using a commercial kit (ZR Zymoclean TM Gel DNA Recovery Kit, USA). Adapter and barcodes for Ion Torrent sequencing were included in the primers following a previous strategy (Claesson et al., 2009). Sequencing of the 16S rRNA gene amplicon libraries was carried out using the Illumina Novaseq6000 PE 250 in the genomic plataform at the Bymycell Inova Simples Ltda (Ribero Petro, So Paulo state, Brazil).