Experimental Study of Biodegradation of Ethanol and Toluene Vapors

growth on ethanol, 20-48 hours for growth on benzyl alcohol) to harvest the cells for
bioreaction studies. Inoculation and transfer cultures are occasionally checked for
contamination by Gram staining. Contamination was never observed.
3.3 Analytical Methods
3.3.1 Measurement of Biomass Concentration
The biomass concentrations were measured as optical density at a wavelength of
620 nm (OD 620 ) using a spectrophotometer (Milton Roy, 1001 Plus). The
spectrophotometer had been calibrated using known dry weights of Pseudomonas
putida (ATCC 23973) suspended in culture media. The absorbance was related to dry
weight using a pre-determined calibration curve (See Figure B-3 in Appendix B). The
calibration curve was developed by using the dry weight method described below.
A high biomass concentration was achieved in a shake flask with 250-mL of
media following the method in Section 3.2.3. 100 mL of this broth (broth a) was then
transferred to centrifuge tubes and centrifuged at 9900 rpm for 15 minutes to allow the
microorganisms to settle to the bottom of the tube. The supernatant was then decanted.
A few drops of distilled water were used to rinse the tube wall, followed by mixing
using a vortex mixer. The procedures of centrifuging, decanting and rinsing were
repeated two times. Finally, this solution was transferred to pre-weighed aluminum
dishes and placed into a vacuum oven that was operated at a temperature of 65 o C and a
pressure of –22 in Hg (gauge pressure) for 24 hours. Before the dishes were weighed,
these samples were put in a dessicator for 1 hour to bring the dishes to equilibrium with
room temperature. The difference in weight between the dried dish with biomass and
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