2011 International Conference on Alternative Energy in Developing Countries and Emerging Economies
- 94 -
then enriching for H
2
producing bacteria. The
pretreatment methods performed were as follow:
C. Heat-shock pretreatment
The heat-shock pretreatment was conducted by
immersing test tubes that contained the original sludge
in a water bath at 85
0
C for 10 min. After that, all the test
tubes were immediately put into an ice bath for 10
minutes.
D. Acid pretreatment
The acid pretreatment was conducted by adjusting the
pH of the sludge to 3.0 with 10 N HCl and maintaining
it at that level for 24 hrs before readjusting it to pH 7
with the addition of 10 N NaOH.
E. Base pretreatment
The alkaline pretreatment was conducted by adjusting
the pH of the sludge to 11.0 with 10 N NaOH and
maintaining it at that level for 24 hrs before readjusting
it to pH 7 with the addition of 10 N HCl.
F. Chloroform pretreatment
The chloroform pretreatment was carried out by
adding chloroform to the sludge at a concentration of 2%
by volume and maintaining it for 24 hrs before use.
G. Freezing and thawing pretreatment
The freezing and thawing pretreatment was carried
out by freezing the sludge at -20
0
C for 24 hrs followed
by 5 hrs thaw at room temperature (30
0
C).
The acid, base, and chloroform pretreatments were all
conducted at room temperature of about 30
0
C. These
pretreated mixed bacteria prepared by various methods
were used separately as inocula.
H. Test Bioreactors and Operation
Five reactors were constructed from two-liter capacity
polyethylene terephthalate (PET) plastic bottles with 1.3
liter working volume. Each was equipped with an outlet
port for gas venting. Gases were vented through a tube
in the rubber cap that was connected to a tube containing
water. The reactors were wrapped with aluminum foil to
prevent exposure to light in order to conduct dark
fermentation.
Glucose solution was prepared by adding 5.0 gram of
glucose to 1 liter of distilled water. The nutrient solution
was prepared by adding 13.0 gram of Nutrient Broth
(which has the formulation shown in Table 1) to 1 liter
of distilled water. The synthetic glucose substrate
contained sufficient inorganic nutrients to support
microbial growth and was prepared by mixing the
glucose solution with the Nutrient Broth solution at a
ratio of 1:1 by volume. The COD of this synthetic
glucose-based substrate was approximately 6800 mg/l
and it was used to add into the experimental bioreactors
that contained the pretreated bacteria.
TABLE
I
N
UTRIENT BROTH FORMULA FOR CULTIVATION OF MICROORGANISMS
IN GLUCOSE BASED SUBSTRATE
.
Separate samples of 800 ml of pretreated mixed
bacteria, prepared by the five different methods
described above, were added to one of the test bottles
that was wrapped with aluminum foil. 500 ml of
synthetic glucose-based substrate was added to all the
bottles. The total working volume was 1300 ml. Gas
generation began on the first day of fermentation and the
produced gas was vented through a tube in the rubber
cap of the plastic bottle into a tube containing water.
After the first 2 days, the synthetic glucose-based
substrate was filled daily and drawn at 500 ml/day for 15
days. At two hours after feeding the substrate, gas
sampling was begun from the sampling port on the tube
to determine the content of H
2
and CH
4
.
At day 15,
glucose-based substrate commingled with brewery
wastewater was fed to the reactors for 5 days. Then,
brewery wastewater alone with a strength of
approximately 6000 mg/l was fed daily at the same rate
up to 50 days. A COD concentration of wastewater of
6000 mg/l was recommended as the appropriate
concentration for brewery wastewater to produce H
2
[13]. The experiments were conducted in a semi-
continuous mode. The hydraulic retention time (HRT)
for fill-and-draw of wastewater was 2.6 days (HRT =
1300 ml/500 ml-day). Experiments with the various
pretreated mixed bacteria were performed at the original
pH of the wastewater from the treatment plant (pH 6.3)
in order to keep the original condition of brewery
wastewater and hydrogen production is reported to
proceed without methane production in the reactor in the
pH range 4.5
–
6.7 [12]. The tests were performed at
ambient temperature (30±1°C).
pH was not artificially
controlled during the operation. The pH of the mixed
liquor in all reactors was measured everyday. Gas was
sampled at two hours after feeding the brewery
wastewater to determine the content of H
2
and CH
4
. In
order to achieve the objective of the project, the content
of H
2
and CH
4
during the bioreaction time was used to
estimate the feasibility of H
2
productivity.
The percentage of H
2
and CH
4
in the gas produced
was analyzed by using a GC (Model Shimadzu GC14 B)
equipped with a thermal conductivity detector (TCD)
and column packed with molecular sieve 13 X. Total
Kjeldahl Nitrogen (TKN) and total phosphate (TPO
4
3-
)
of brewery wastewater were measured according to the
Standard Methods [19].
III. R
ESULTS AND DISCUSSION
Figs 1a, 2a and 3a illustrate the effect of heat, acid,
and base pretreatment performed on bacteria on the
percentage of H
2
they produce, respectively while Figs
1b, 2b and 3b illustrate the effect of those three
pretreatment methods on the percentage of CH
4
from the
Ingredients
Concentration (g/l)
Peptic digest of animal tissue
5.00
Sodium chloride
5.00
Beef extract
1.50
Yeast extract
1.50
