title: KM10
design: Microbial samples, specifically for this research, were also collected at each well. Autoclaved tubing (C-Flex, 0.635 cm inner diameter, Cole Parmer, Vernon Hills, IL) was attached to the sterilized spigot or the pump outlet. The sample was collected by filtering one liter of groundwater through a 0.22 m filter (SterivexTM-GP Sterile Vented Filter Unit, Millipore Sigma, St. Louis, MO). Smaller volumes were filtered at sites with turbid water. A sterile, 10 mL syringe filled with air was used to remove excess water from the filter unit before capping with Luer Lock plugs. The capped filter cartridge was labeled and placed in a 50 mL conical centrifuge tube (Falcon, Thermo Fisher Scientific, Waltham, MA) for storage and transport. Each filter was then placed in a plastic bag and stored on ice before returning to the lab. Filters were stored at -20C until further processing. The microbial DNA was extracted from the Sterivex filters after thawing on ice and was isolated using the DNeasy PowerWater Sterivex Kit (Qiagen, Germantown, MD) according to the manufacturers instructions. Briefly, cells were lysed by vortexing, heating, and bead beating. Inhibitors were removed, and DNA was eluted from the filter prior to storage at -20C until further processing. The isolated DNA (1-50 ng/L, 20 L) was shipped to Argonne National Laboratory, Environmental Sample Preparation and Sequencing Facility (ESPSF).The v4 region of the prokaryotic 16S rRNA gene (515F-806R) was amplified to prepare the amplicon libraries. Each PCR reaction mixture contained 9.5 L of MO BIO PCR DNA-free water, 12.5 L of QuantaBios AccuStart II PCR ToughMix (1x final concentration), 1 L of Golay barcode tagged forward primer (200 pM final concentration), 1 L reverse primer (200 pM final concentration), and 1 L of template DNA. The PCR reaction was initiated at 94C for 3 minutes to denature the DNA, then 35 cycles at 94C for 45 seconds, then 50C for 60 seconds, and then 72C for 90 seconds, with a final extension time of 10 minutes at 72C. The amplicon libraries were quantified using PicoGreen (Invitrogen, Waltham, MA) and a plate reader (Infinite 200 PRO, Tecan, Switzerland). The amplicons were pooled into a single tube, with each amplicon represented in equimolar amounts. The pooled samples were cleaned using AMPure XP Beads (Beckman Coulter, Brea, CA) and quantified with a fluorimeter (Qubit, Invitrogen, Waltham, MA). The pool was then diluted to 2nM, denatured, and diluted to a final concentration of 6.75 pM with a 10% PhiX spike. Amplicons were sequenced on a 151bp x 12bp x 151bp Illumina MiSeq run.17,18