15850

international journal of hydrogen energy 38 (2013) 15849

e15855

Table 2 e Specic growth rate at different feeding

substrate concentrations.
Substrate
(g total sugar/L)
20
30
40
60
100

t (h)

dX/dt
(g VSS/h)

X (g VSS)

33
27
23
27
73

0.16
0.25
0.57
0.50
0.28

1.11
1.12
2.04
2.12
4.29

m(h

0.14
0.22
0.28
0.24
0.05

FeSO4.7H 2O (25 mg/L), CuSO 4.5H 2O (5 mg/L) and CoCl 2.5H 2O

(0.125 mg/L) as per the Endo composition
[31].
Fig. 1 e The VSS versus time at different feeding substrate
concentrations.

2.2.

Experimental procedure and conditions

2.

Materials and methods

Initially, the enrichment reactor (2 L) was charged with 200 ml

seed sludge and 1800 mL substrate. The reactor was operated
with inlet substrate concentration of 20 g total sugar/L, temperature of 35 C, pH 5.5 and HRT of 8 h. The efuent of the
enrichment reactor was taken for kinetic studies in a batch
mode where the biomass concentration was stable around
3.5
0.2 g VSS/L. Subsequently, the batch reactor (2 L) was
lled with 360 ml sludge and 1440 ml substrate. The initial pH
was adjusted to 5.5
0.02 by 3N HCl and temperature was
controlled at 35 C. Argon gas was purged into the bioreactor
to attain anaerobic condition. The various initial substrate
concentrations studied were 20, 30, 40, 60 and 100 g total
sugar/L, followed by the experiments on pH versus microbial
growth at substrate concentration of 40 g total sugar/L where
pH was varied from 5 to 7 with a regular increment of 0.5. The
VSS, gas composition, sugar concentration and metabolites
were then detected at a regular time intervals.

2.1.

Seed sludge and chemicals

2.3.

biohydrogen production model in a modied Gompertz

equation [12,13,16,25e 28] and modied Monod equation
[13,26,29,30]. However, designing a continuous system using
modied Gompertz equation is tough. A narrow range of initial
substrate concentrations and pH values have been considered
in the earlier reports on reaction kinetics.
There are few reports on the use of mixed culture in a broad
range of the initial substrate concentrations and pH values for
anaerobic hydrogen production. However, most of the anaerobic hydrogen production systems used mixed cultures for its
cost-effectiveness. Therefore, the aim of this study was to nd
a suitable model and accompanying kinetic parameters for
scaling up of a batch anaerobic hydrogen production reactor.

The activated sludge from the Li-Ming Municipal Sewage

Treatment Plant in Taichung, Taiwan was used as seed. The
collected sludge was screened with a No. 8 mesh (dia. 2.35 mm)
and heated at 100 C for 1 h (to inhibit the hydrogen consuming
methanogens). The characteristics of the seed sludge were pH
6.7, Alkalinity 2.1 g COD/L as CaCO 3, TCOD 25.9 g/L, SCOD 8.9 g/
L, T-Carbohydrate 5.7 g/L, S-Carbohydrate, 2.4 g/L and NH
3e N
912 mg/L. The substrate used was high organic content
wastewater from brewery plant and its characteristics were pH
3.41, ORP 287, S-COD 760e 860 g/L, S-Carbohydrate 610 e 670 g/
L and NH 3e N 5 g/L. The nutrient supplement used was prepared with NH 4HCO3 (5240 mg/L), NaHCO 3 (4000 mg/L), K 2HPO4
(125 mg/L), MgCl.6H 2O (100 mg/L), MnSO 4.6H 2O (15 mg/L),

Analysis

During the hydrogen production experiments, the parameters

monitored were pH, ORP (oxidation
e reduction potential),
alkalinity, volatile fatty acid (VFA) distribution and gas production. The Standard Methods
[32] analytical procedures
were used to determine the chemical oxygen demand (COD)
and volatile suspended solid (VSS). Ethanol and soluble microbial product (SMP) were analyzed by a gas chromatograph
(SHIMADZU GC-14 Gas Chromatograph) with a ame ionizaC; injection temperature,
tion detector (glass column, 145
175 C; carrier gas, N 2; packing, FON 10%). Gas composition
was analyzed by a gas chromatograph (CHINA Chromatograph 8700T) with a thermal conductivity detector (TCD)
C; carrier gas, Argon;
(column 55 C; injection temperature, 90

Table 1 e Hydrogen production and yield performances at different feeding substrate concentrations.
Substrate
(g total sugar/L)
20
30
40
60
100

Carbohydrate
degradation (
)
91.1
97.6
89.3
93.2
87.2

Total biogas
production (L)
11.8
22.3
25.8
54.5
50.0

Total H 2
production (L)
6.3
11.8
14.6
28.3
27.4

Hydrogen yield, HY
(mol H 2/mol hexose)
1.39
1.61
1.41
2.00
1.40

-1)
specific growth rate (h

20

40

60

80

100

Substrate concentration
g TS/L)
(

packing, Porapak Q, mesh 80/100) [33,34]. Anthrone method

was used to measure the hexose concentration
[35].

3.

Results and discussion

3.1.
Effect of the substrate concentration and model
establishment
The VSS versus time at a different feeding substrate
concentration is shown in
Fig. 1. The feeding substrate concentrations were controlled at 20, 30, 40, 60 and 100 g total
sugar/L. As shown in Fig. 1, the lag time decreased with the
increase in feeding substrate concentration from 20 to 40 g
total sugar/L and further increased with increase in feeding
substrate concentration from 40 to 100 g total sugar/L. A lower
feeding substrate concentration resulted in slower cell growth
which can be attributed to insufcient availability of the carbon source. However, the bacteria suffered at a high substrate
concentration of 40 g total sugar/L. The increase in lag at
higher substrate concentrations beyond 40 g total sugar/L
could be attributed to the phenomenon of bacterial readaptation at high substrate concentrations.
As shown in Fig. 1 the bacterial growth increased with increase in substrate concentration up to 60 g total sugar/L. A
maximum of VSS 5.5 g/L was attained at 60 g total sugar/L and
thereafter did not vary for any further increase in substrate
concentration. The hydrogen production and yield performances at different feeding substrate concentration have

been shown in Table 1 . The carbohydrate degradation ranged

from 87.2 to 97.6
. The peak hydrogen production of 28.3 L
and hydrogen yield of 2.00 mol H 2/mol hexose were obtained
at feeding substrate concentration of 60 g total sugar/L. In this
study, peak of accumulation of hydrogen is not an indicator of
optimal condition as experiments were performed at different
initial substrate loadings. Hence, specic bacterial growth rate
was used for modeling the biohydrogen production based on
the assumption of growth associated product formation.
Specic bacterial growth rate at a different feeding substrate concentrations were set up in
Table 2 based on the
experimental data in
Fig. 1. The experimental data of specic
growth rate shown in
Fig. 2 indicates that the inhibition effect
was raised beyond the initial substrate concentration of 40 g
total sugar/L. Therefore, the general inhibition models of
Haldane, Aiba and Teissier were used in this study for curve
tting of bacterial growth rate. In order to establish the bacterial growth rate model, three inhibition- models were used
to t the experimental data which were collected as shown in
Table 2 . The regression results are shown in the
Fig. 2 and
Table 3 by Haldane Model ( equation (1) ) [36], Aiba Model
(equation (2) ) [37] and Teissier Model ( equation (3) ) [38]. It was