Engineered Microbial Consortium for the Efficient Conversion of Biomass to Biofuels Page: 39
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CHAPTER 3
MATERIALS AND METHODS
3.1 Determination of the Ethanol Tolerance of Purified Cellulosomes
3.1.1 Bacterial Strain and Media
C. cellulolyticum (ATCC 35319) was purchased from the American Type Culture
Collection. The culture was first grown in C. cellulolyticum medium (ATCC1368) using glucose
as the carbon source. Medium preparation involved the addition of resazurin to indicate for the
presence of oxygen. The medium was boiled under pressure, with the removal of oxygen using a
vacuum pump installed. At the point at which the resazurin changed color from blue to pink, the
boiling was stopped and the medium allowed to cool down to room temperature. The medium was
tranfered into the Coy Anaerobic chamber where 10 ml each were dispensed into sterile serum
bottles. The bottles were sealed to prevent any introduction of oxygen and the medium sterilized
using standard autoclave conditions. Thereafter, the C. cellulolyticum culture was aseptically
transferred into the 10 ml medium, gassed for 1 minute using 90% Hydrogen and 10% carbon gas
mix. The culture was incubated for 24 hours in 37C shaking incubator at 225 revolutions per
minute. After 24 hours, they were routinely transferred into Clostridium cellulolyticum medium
(ATCC medium 1368) containing microcrystalline cellulose as carbon source and incubated for
72 hours under the same conditions.
3.1.2 Cellulosome Purification
Cultures were purified according to the methods of Gal et al. [181] as described. Cells were
cultivated anaerobically in two 500-ml flasks at 37C and 160 r.p.m. in C. cellulolyticum medium.
After 5 days of growth, cells and residual cellulose were harvested by centrifugation at 13000 g39
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Anieto, Ugochukwu Obiakornobi. Engineered Microbial Consortium for the Efficient Conversion of Biomass to Biofuels, dissertation, August 2014; Denton, Texas. (https://digital.library.unt.edu/ark:/67531/metadc699973/m1/50/?rotate=270: accessed July 17, 2024), University of North Texas Libraries, UNT Digital Library, https://digital.library.unt.edu; .