Experimental Study of Biodegradation of Ethanol and Toluene Vapors

the original dish was used to calculate the original broth biomass concentration
(milligrams per liter of broth). Another 100 mL of the same broth (broth a) was
subjected to a number of dilutions and analyzed for optical density. These dilutions
provided a range of known concentrations with measured optical densities and were
used to plot the dry-weight calibration curve for Pseudomonas putida. Samples with
optical densities exceeding the range of the calibration curve were diluted until their
optical densities fell within the calibrated range.
3.3.2 Analyses of Organic Chemicals
Prior to measuring the substrate concentrations, samples from bioremediation
experiments were centrifuged at 2500 rpm for 5 min to let the bacteria settle to the
bottom of the tube, then the supernatant was mixed with an equal volume of an aqueous
solution of 1-butanol, the internal standard, to measure substrate concentrations in the
liquid samples.
Ethanol, toluene and benzyl alcohol concentrations in the liquid were analyzed
using a GC (HP 5890) equipped with a Flame Ionizing Detector (FID) and capillary
column (PTE TM 5 Fused Silica Capillary Column, 30 m, 0.25 mm ID, Supelco, Inc., PA,
USA). The temperatures of the injector and detector were 210 ºC and 250 ºC,
respectively. The oven temperature was ramped from 45 ºC to 150 ºC at the rate of
25 o C/min. 1-Butanol was used as the internal standard for all liquid sample analyses.
Ethanol, toluene and benzyl alcohol standards were prepared as aqueous solutions for
the calibration of the GC analyses. The calibration curves are shown in the Appendix B
(See Figures B-4 to B-6).
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