DNA extraction, PCR, Sequencing & analysis.
To evaluate the composition of the native oil well, ~1L of effluent water was prefiltered with Glass Fiber Filters (1.2 μm pore, MilliporeSigma) and then biomass was concentrated on Express™ PLUS Membrane Filters (0.22 μm pore, MilliporeSigma) similar to previous work.60 DNA was then extracted from the microbial biomass on the second filter using the DNeasy PowerFecal Kit (QIAGEN, Germantown, MD, USA) along with the FastPrep 24 5G homogenizer (MP Biomedicals, Santa Ana, CA, USA) as per instructed by the manufacturers. All DNA was stored at − 20 °C until further analysis. DNA concentration and quality were assessed on a NanoPhotometer (Implen NP80, Los Angeles, CA, USA). The microbial community composition of oil well microcosms was assessed by DNA extracted from a 1-ml aliquot of the cultures as mentioned above. The 1 ml aliquots were centrifuged at 10,000g for 7 min to pellet the cells. After removing the supernatant, the microbial DNA was extracted with the same DNeasy PowerFecal kit as noted above.
As in previous microcosm studies,61 the microbial community was then assessed by amplifying and sequencing the V4-V5 region of the 16S rRNA gene from the extracted DNA (primers: 515f - forward, 5′ GTGYCAGCMGCCGCGGTAA 3′; and 926r - reverse 5′ CCGYCAATTYMTTTRAGTTT 3′).62 16S rRNA gene PCRs were set up using Phusion Polymerase MasterMix (Thermo Fisher Scientific, Waltham, MA, USA) as follows PCR-grade water (22 μl), Phusion mastermix (25 μl), forward primer (10 μM, 1.0 μl), reverse primer (10 μM, 1.0 μl), and template DNA (1.0 μl) in a total reaction volume of 50 μl; for samples that had very low template concentration up to 5 μl of DNA template was used and the water volume reduced to 17 μl. PCR amplification conditions were set to 98 °C for 0.5 min; 35 cycles of 98 °C for 15 s, 50 °C for 30 s, 72 °C for 60 s; and 72 °C for 10 min, and then a 4 °C hold. The resulting amplicons were confirmed by gel electrophoresis, purified using the Clean and Concentrator kit (Zymo Research Irvine, CA, USA), and then labeled using unique 8-bp tagged i5 (i509-i516) index forward primers and 8-bp tagged i7 (i713-i724) index reverse primers as suggested by the manufacturer (Illumina San Diego, CA, USA). Pooled amplicons were then multiplexed and sequenced via 2 × 250-bp paired reads on an Illumina MiSeq at the Purdue Genomics facilities. Sequences were analyzed using the QIIME2 pipeline63 which quality-filtered, joined paired reads, checked for chimeras, and denoised the data (via DADA264). The operational taxonomic units (OTUs) were assigned using the SILVA SSU database (SILVA 138)65for assigning taxonomy to the representative OTUs. All alpha and beta diversity metrics were calculated using QIIME’s built in analysis tools.