Oxic enrichment culture refreshed with air Round 3
In preparation for DNA extraction, Fe(III) (hydr)oxides were removed from cultures by washing them with a 0.3 M ammonium oxalate solution with the pH adjusted to 3 with oxalic acid (Nicomrat et al., 2006). DNA was then extracted from remaining biomass using MoBio Powerbiofilm DNA isolation kits (MoBio Laboratories, Inc., Carlesbad, CA), according to the manufacturers instructions. Partial 16S rRNA gene sequencing was performed using the extracted DNA at Molecular Research, LP (Shallowater, TX). PCR was performed with 515F and 806R (Escherichia coli position) primers using the HotStar Taq Plus Master Mix Kit (Qiagen USA, Valencia, CA) with 3 min melting at 94, and 28 cycles of 30 sec 94, 40 sec 53, and 1 min 72, and then a 5 min elongation step at 72. The samples were then pooled and an Illumina DNA library was prepared from Ampure XP bead-purified samples. The amplicons were sequenced by Illumina MiSeq (San Diego, CA) using the manufacturers instructions.