Cultivation
Effluent oil well water was collected from several wells around the Illinois oil basin and transported to the laboratory (West Lafayette, IN, USA) the same day. For cultivation of the oil well microbiome, 9 ml of effluent water was mixed in Hungate tubes with either 1 ml of sterile 40% (w/v) corn syrup or 1 ml of sterile 40% (w/v) molasses in an anaerobic chamber (PLOS, Grand Rapids, MI, USA) with an atmosphere of 85% N2, 10% CO2, & 5% H2. Cultures that were treated with additional non-essential micronutrient supplements [final concentration of 0.5 g/L potassium nitrate, 0.5 g/L sodium molybdate, 0.25 g/L potassium monophosphate and 0.25 g/L potassium diphosphate, or 0.25 g/L sodium chloride and 0.25 g/L potassium chloride] – solutions were sterile filtered and equilibrated overnight in the anaerobic chamber. After nutrient solutions were added, cultures were thoroughly mixed and incubated at 27 ˚C. Pressure measurements were taken every 24 hours with a pressure gauge (APG, Logan, UT, USA),59 1 ml of culture was removed for microbiome analysis and pH monitoring by pH test strips (pH2-8, MilliporeSigma, St. Louis, MO, USA). After sampling, the cultures were then vented to a gauge pressure of 0 and replaced in the incubator; cultures were monitored and sampled for 7-10 days.