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When the flask content turned milky, the temperature of the system was increased to 95 °C to start the distillation.
The Erlenmeyer flask content was also agitated during 20 min, capped and placed at 25°C (or 15°C, or 45°C) in the dark.
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Inside was a filled stainless-steel flask emblazoned with the words "Gun Club". Today both book and flask (contents intact) remain in Benemann's office, along with other artifacts including a recently acquired leather-bound, untitled book of Gun Club poetry dating to the early 1920s.
The flask contents were heated to 100 °C and left under constant stirring for 24 h.
The flask contents were stirred for 30 min to get good contact between the phases.
A 0.79 mmol solution of sodium borohydride was added to the flask contents and the flask was purged with nitrogen flow to create an inert atmosphere.
The temperatures of the flask contents were measured at the end of the 3 h aeration using a mercury-in-glass thermometer.
Each flask contained 3.2 mL of synthetic sewage and 18 mL of activated sludge to give a final solids concentration of 1500 mg/L (with the exception of the abiotic flask), an appropriate quantity of either PSOA or 3,5-DCP stock solution and reverse osmosis water to give a final flask contents volume of 100 mL.
After inactivation of catalase by heating at 90°C for 15 min, the flask contents were centrifuged and dried at 55°C.
Washing of soiled fabric dipped in respective washing solutions was done by shaking the flask contents at 100 rpm at 37°C for 20 min.
A magnetic agitator (Bellco Glass, Inc., Vineland, NJ, USA) was used to continually mix flask contents at a speed of 225 rpm, and each flask was sealed with a rubber stopper containing apertures for probes, nutrient inlets/outlets, and sample harvesting.
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