Nitrate-reducing enrichment culture with low carbon to nitrate ratio
The obtained 16S rRNA amplicons were prepared in a 50  L final volume containing 5  L of each Illumina Nextera XT index primer (Illumina) and 2X KAPA HotStart ReadyMix (Kapa Biosystems) to index each sample. The PCR thermal steps were as follows: 94 C for 3 min; followed by 8-10 cycles of 98 C for 20 s, 55 C for 30 s and 72 C for 30 s, and then a final 72 C for 5 min. Subsequently, the indexed PCR amplicons were purified with Agencourt AMPure XP beads (Agencourt Bioscience), pooled and diluted to a final loading concentration of 4 pM. DNA paired-end sequencing was performed on the Illumina MiSeq platform with a MiSeq Reagent Kits v3 (600-cycle; Illumina). The sequences were assembled by merging the forward and reverse reads. The OTU picking process was then conducted using the QIIME software (version 1.9.0), with an open-reference method. Taxonomic assignment from the OTUs to related taxa was done using UCLUST against Greengenes database (version 13_8, using a 97% sequence identity threshold).