Sludge dsrB_gene  Metagenome
Total DNA extractions from sludge samples were performed in triplicate using DNeasy PowerSoil Kit (QIAGEN, Gilden, Germany) following the manufacturers protocol. Triplicate-extracted DNA from each sample was mixed to minimize the potential variation during the DNA extraction process. All the extracted DNAs were stored at  20 C for subsequent Illumina sequencing. The dissimilatory sulfite reductase beta subunit (dsrB) gene of sulphate-reducing bacteria (SRB) encoding a key enzyme in sulfate reduction was was amplified using the primers DSRp2060F: 5- CAA CAT CGT YCA YAC CCA GGG-3 and DSR4R: 5 - GTG TAG CAG TTA CCG CA -3 (Khan et al., 2017). The amplification of the dsrB gene was performed with a final volume of 30 L containing 15 L of PhusionR High-Fidelity PCR Master Mix (New England Biolabs), 0.2 mM of each primer, and 10 ng of template DNA. Amplicons from different samples were purified using PCR purification kit (Solgent, Korea). Amplified DNA was sequenced using the Illumina MiSeq platform (Macrogen, Seoul, South Korea) in 301-bp paired-end format at Macrogen. The raw sequences were demultiplexed to assign to samples, and removed barcode and primer sequence were using the Cutadapt tool. The raw reads were processed using DADA2 with the standard processing steps, quality filtering, denoising, merging, ASV inference, and chimeric sequence removal to obtain the amplicon sequence variants (ASVs). The amplicon sequences variants (ASVs) were also resolved by removing chimeric sequences with a reference database (Silva database) based on the "consensus" method. Subsequent analysis of the richness indices (Chao 1 estimates), diversity indices (Shannon index), and Good's coverage were performed. In addition, QIIME2 was used to evaluate the similarities of bacterial communities between the sludge samples using the unweighted pair group method with arithmetic mean (UPGMA) clustering based on a weighted UniFrac distance matrix. The taxonomic assignment was determined using BLAST against the NCBI SRB-dsrB gene database.