Experimental Study of Biodegradation of Ethanol and Toluene Vapors

alcohol) was inoculated with two loops 1 of bacteria from a previously cultivated agar
plate. The shake flask was placed on a rotary shaker at 200 rpm and maintained at room
temperature (normally 25-30 o C) until the bacteria achieved their exponential phase of
growth (12 to 24 hours depending on the age of the stored bacteria). This broth served
as the inoculum source for all bioremediation experimental runs containing substrate,
and was also used to propagate the bacteria on fresh agar plates.
3.2.2 Agar Plates
The agar plates were made by adding 3 grams of Bacto-agar to 100 mL of
distilled water. These ingredients were stirred and heated to a temperature just below
the mixtures’ boiling point or until the solution became clear, then sterilized for 30
minutes at 121 o C. Under the biofilter cabinet hood, benzyl alcohol was added into the
warm agar solution, and this agar solution with 0.1%(w/v) benzyl alcohol was poured
into Petri dishes to make six agar plates. To propagate bacteria on fresh agar, several
loops of broth solution from the shake flask (see the above section) were aseptically
removed and streaked on agar plates. The sealed agar plates were then placed in the
CO 2 incubator at 30 o C for 24 hours. These agar plates were renewed every two months
to ensure that a fresh source of microbes was always available.
3.2.3 Inoculum
Initially 250 mL of medium is sterilized in a shake flask and then a certain
amount of benzyl alcohol or ethanol was added into the flask under the biofilter cabinet.
Two loops of bacteria taken from the agar plate were then added into the flask. Finally
the flask was put on the shaker and agitated at 180 rpm at room temperature (25 o C) for
a period of time that is within the log phase (predetermined to be 10-16 hours for
1 A circular metal ring shaped instrument used for scooping bacteria from an agar plate.
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