bioaugmentation petroleum-contaminated soil
Total genomic DNA was extracted from 0.25 g of soil sample using the DNeasy PowerSoil Pro Kit (Qiagen, Hilden, Germany) according to the manufacturer's instructions, with an additional bead-beating step at 30 Hz for 5 min. The V3-V4 hypervariable region of the bacterial 16S rRNA gene was amplified using primers 341F (CCTACGGGNGGCWGCAG) and 805R (GACTACHVGGGTATCTAATCC), which included Illumina adapter overhang sequences. PCR reactions were performed in a 25 L volume containing 12.5 L of 2 KAPA HiFi HotStart ReadyMix (Roche), 0.2 M of each primer, and 10 ng of template DNA. The thermal cycling protocol consisted of initial denaturation at 95C for 3 min, followed by 25 cycles of 95C for 30 s, 55C for 30 s, and 72C for 30 s, with a final extension at 72C for 5 min. Amplicons were purified using AMPure XP magnetic beads (Beckman Coulter) at a 0.8 ratio. Subsequently, dual-index barcodes were attached via a second round of PCR (8 cycles) using the Nextera XT Index Kit (Illumina), followed by another AMPure XP clean-up (0.7). The final libraries were quantified using the Qubit 4.0 Fluorometer (Invitrogen) and assessed for size distribution on an Agilent 4200 TapeStation. Libraries were normalized to 4 nM, pooled, denatured with NaOH, and sequenced on the Illumina NovaSeq 6000 platform using a 2  250 bp paired-end (PE250) configuration. Extraction blanks and PCR negative controls were included throughout to monitor potential contamination.