2011 International Conference on Alternative Energy in Developing Countries and Emerging Economies
- 27 -
immersing them in a water bath at 85°C for 10 minutes.
After that, all the test tubes were immediately put into
an ice bath for 10 minutes in order to break the spores.
Then the inoculum was acclimated as described below.
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
that contained sufficient inorganic nutrients to support
microbial growth 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 6500 mg/l and it
was used to acclimate the heat treated microorganisms.
TABLE I
N
UTRIENT BROTH FORMULA FOR CULTIVATION OF MICROORGANISMS
IN GLUCOSE BASED SUBSTRATE
.
Ingredients
Concentration
(g/l)
Peptic digest of animal tissue
5.00
Sodium chloride
5.00
Beef extract
1.50
Yeast extract
1.50
800 ml of heat treated mixed bacteria were added to
a 2 liter plastic bottle that was wrapped with aluminum
foil to prevent the entrance of light in order to facilitate
dark fermentation. 500 ml of synthetic glucose-based
substrate was added to the bottle to promote growth.
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 daily filled and drawn at 500 ml/day for
15 days and then starch wastewater was daily fed at the
same rate for 30 days. Thus, the hydraulic retention
time (HRT) for fill-and-draw of wastewater was 2.6
days (HRT = 1300 ml/500 ml-day). The heat treated
mixed bacteria were acclimated at ambient temperature
(30±1°C). Microbial growth was monitored by
observing the volume of biogas production. The
acclimated bacteria were used as a parent culture.
B. Batch Experiments for Hydrogen Production
Two sets of batch experiments were carried out to
determine the effects of the ratio of cell concentrations
to initial starch wastewater concentration on bio-
hydrogen production from starch wastewater by
fermentation in the dark. H
2
production was evaluated
using two different ratios of the biomass and substrate
concentration (Cell:COD) achieved by means of
variations in the biomass content. The ratio of
Cell:COD was 4 g cell/g COD for Batch set 1 and it
was 0.26 g cell/g COD for Batch set 2. The Cell:COD
in each batch set was controlled by similar methods.
Serum bottles were used as test bioreactors.
Experiments were carried out in 120 ml serum bottles
by batch tests. These bioreactors, equipped with a
separate gas collection system that was based on
displacement of water by exiting gases (using 0.05 M
sulfuric acid substitute water), were placed in a shaking
water-bath with temperature controlled at 35±1° C. The
serum bottles were wrapped with aluminum foil to
prevent exposure to light in order to conduct dark
fermentation. The acclimated heat treated bacteria,
which were used as the parent culture, were added to
the test bottles. The initial starch wastewater
concentration was 25000 mg COD/l; that is the normal
wastewater concentration from the plant. The initial pH
of the starch wastewater for both Batch sets 1 and 2 was
adjusted to 4, 4.5, 5, 5.5, 6, 6.5 and 7 using 10 M HCl
or 10 M NaOH before addition to the test bottles. This
pH adjusted wastewater was then added to the test
bottles. The total volume in each test bottle was 100 ml,
the sum of the volume of biomass slurry and added
wastewater. In the Batch set 1, the fermentation
medium included 80 ml of heat treated biomass slurry
and 20 ml of wastewater yielding a cell mass at
approximately 20000 mgMLVSS/l and yielding a
Cell:COD of 4 g cell/gCOD. This ratio of Cell:COD
ratio was kept constant for all test bottles for Batch
set 1. In the Batch set 2,
the fermentation medium
included 20 ml of heat treated biomass slurry and 80 ml
of starch wastewater yielding a cell mass at
approximately 5000 mgMLVSS/l and yielding a Cell :
COD of 0.26 g cell/gCOD. This ratio of Cell:COD ratio
was kept constant for all test bottles for Batch set 2.
Heat treated seed sludge and distilled water served as
the control test. The conditions were: initial COD
(25000 mg/l), MLVSS of the heat treated biomass
(20000 mgMLVSS/l for Batch set 1 and 5000
mgMLVSS/l for Batch set 2), Cell:COD (0.4 g cell/g
COD for Batch set 1 and 0.26 g cell/g COD for Batch
set 2) and temperature (35
o
C).
Each experiment started after a preliminary
operation for 5 minutes in order to minimize the effects
of environmental changes and gas phase differences.
The total gas production was recorded (at 35° C) and
collected every 3 hours at the beginning of the
experiment and then was collected every 6 hours
depending on the rate of biogas produced. The
percentage of H
2
and CH
4
present was then determined
by a Shimadzu GC-14B gas chromatograph equipped
with a thermal conductivity detector (TCD). Argon gas
was used as carrier and Molecular Sieve 13X was used
as a column. Temperatures of the injection port, oven,
and TCD were 70
0
C, 60
0
C and 120
0
C, respectively.
During the bioreaction period, liquid samples (5 ml)
were withdrawn periodically and analyzed for COD
according to the procedure of Standard Methods [11].
The study lasted about 120 hours, by which time the
biogas production normally had leveled off. Based on
the measurements, the accumulated hydrogen
production was calculated.
The hydrogen gas volumes
were then adjusted to standard temperature (0° C) and
pressure (760 mm Hg) (STP). All experiments were
conducted in duplicate and the results were calculated
using the mean of the experimental values. Cumulative
