A microbial consortium for the bioremediation of sulfate-rich wastewater originating from an edible oil industry
axonomic composition of the microbial consortium colonizing the bioreactors was studied by next-generation sequencing (NGS) of 16S rRNA amplicons. Briefly, metagenomic DNA was extracted using the Power Soil DNA isolation kit (MO BIO Laboratories, Carlsbad, CA, USA), and the resulting DNA was quantified using the QUBIT dsDNA HS-high sensitivity kit (Invitrogen, CA, USA). The conserved regions V3 and V4 (459 bp) of the 16S rRNA gene were then amplified using forward and reverse primers: 5-TCG TCG GCA GCG TCA GAT GTG TAT AAG AGA CAG CCT ACG GGN GGC WGC AG - 3 and 5- GTC TCG TGG GCT CGG AGA TGT GTA TAA GAG ACA GGA CTA CHV GGG TAT CTA ATC C -3, respectively [24]. Amplification was carried out using the KAPA HiFi HotStart ReadyMix PCR kit (KK2602) and the following PCR cycle: initial denaturation at 95 C for 3 min; 25 cycles of amplification (30 s at 95 C, 30 s at 55 C, 30 s at 72 C); and 5 min of extension at 72 C. Amplicons were mixed with Illumina sequencing adaptors and dual-index barcodes (Nextera XT index kit v2, FC-131-2001). Libraries were normalized and merged before the sequencing. Then, the pool containing indexed amplicons was loaded onto the MiSeq reagent cartridge v3 (MS-102-3003), spiked with 10% PhiX control to enhance the quality of the sequencing. Finally, paired-end sequencing (2300 bp) was carried out on the Illumina MiSeq sequencing system.