Increased Hydrogen Production by Genetic Engineering of Escherichia coli - Genetic Engineering: Recent Developments in Applications - page 13

g/L tryptone, 5 g/L yeast extract, 12 g/L K 2 HPO 4 , 3 g/L KH 2 PO 4 ; pH 7.6) or in
M9 salts medium (Sigma-Aldrich) containing trace elements [35], [36]. When
required, kanamycin was added at a concentration of 30 µg/ml. For studies re-
quiring plasmid-directed gene expression, 1 mM IPTG was added to the cultures
1 hour following inoculation of the fermentation medium, and induction was
performed for approximately 4 hours.
Fermentations were carried out in 37 ml serum vials (Wheaton) containing
15 ml of TYP or M9 medium. Following the addition of medium, the vials were
sealed using silicone stoppers and aluminum crimp seals (Supelco), and auto-
claved. Prior to inoculation, the headspace was evacuated and flushed with Argon
(ultra high purity, Airgas). Supplements included in TYP or M9 medium were:
0.5% or 1% (w/v) glucose; 1 µM sodium molybdate; 1 µM sodium selenite; 50
µM nickel sulfate. Approximately 1.3% (v/v) of inoculum culture, prepared under
aerobic conditions in TYP, was used to inoculate fermentation medium. Specific
hydrogen yields were determined at approximately 5 hours, and 17 hours in TYP
and M9 medium, respectively. Fermentations in 10 ml of M9 medium containing
0.5% glucose (w/v) were run for 17 hours to determine the molar yields of hydro-
gen. The concentrations of all products generated from glucose were determined
for strains cultured anaerobically for 34 hours in 15 ml of M9 medium contain-
ing 1.0% glucose (w/v) to allow adequate time for the consumption of all of the
glucose contained in the medium. All experiments were repeated six times with
at least duplicate fermentations contained within each experiment. The A600 for
strains cultured anaerobically in M9 medium was measured by removing samples
anoxically with a syringe in intervals for 34 hours.
Analytical Methods
The gas mixture from culture headspace was withdrawn using gastight syringes
(Hamilton), and monitored for hydrogen formation. Hydrogen was detected us-
ing a Shimadzu GC-17A gas chromatograph (GC) equipped with a thermal con-
ductivity detector (TCD), a 60/80 Molecular Sieve 5A column (Supelco), with
argon as the carrier gas. The Clarity software package (DataApex) was used to
analyze the chromatograms.
The formation of fermentation products were detected by high performance
liquid chromatography (HPLC) using an Aminex HPX-87H ion exchange col-
umn (Bio-Rad; 7.8×300 mm) and a Varian ProStar 410 liquid chromatography
instrument equipped with UV absorbance and refractive index detectors. Culture
supernatant was filtered through 0.45 µM cellulose acetate filters, and 40 µL of
sample was injected. The column was eluted isocratically with 5 mM H2SO4 at
a flow rate of 0.5 ml/minute. Data was analyzed using the Star Chromatography