16S rRNA sequence of acid producing bacteria grown on carbon steel coupon in the presence of nitrate-3
Genomic DNA was extracted from APB consortia grown on carbon steel coupons in the presence or absence of nitrate for three weeks. The samples (1 mL) were centrifuged at 17,000 X g for 5 min to pellet the cells. DNA was isolated from the pellet using the FastDNA extraction kit for soil, quantified with a Qubit fluorimeter. Sequencing was performed by targeting the V3-V4 region of 16S rRNA genes. The 16S rRNA genes were amplified through two-step PCR reactions with nonbarcoded universal 16S rRNA forward primer (319F, ACTCCTACGGGAGGCAGCAG) and reverse primer (806R, GGACTACHVGGGTWTCTAAT) for 25 cycles followed by amplicon purification and PCR using sequencing adaptors and barcodes. The purified PCR product (100 ng) was sequenced using Ilumina Miseq (PE300) platform to generate clusters.