substrate
The extracted metagenomic DNA was used to amplify the hypervariable region V3-V4 of the 16S ribosomal RNA gene. The conserved regions V3 and V4 (470 bp) of the 16S rRNA gene were amplified using 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 (Satari et al., 2020). The following primers were used: 341F (5 CCTAYGGGRBGCASCAG 3) and 806R (5 GGACTAC-NNGGGTATCTAAT 3). The amplification was carried out using the KAPA HiFi HotStart Read-yMix PCR kit (KK2602). The 16S rRNA amplicons were mixed with Illumina sequencing bar-coded adaptors (Nextera XT index kit v2, FC-131-2001), and libraries were normalized and merged. The pools with indexed amplicons were loaded onto the MiSeq reagent cartridge v3 (MS-102-3003) and spiked with 10 % PhiX control to improve the sequencing quality. Sequencing was conducted using paired-end 2X250pb or 2  300pb cycle runs on an Illumina MiSeq device