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.