Bioresource Technology 97 (2006) 1302–1307

Kinetic modeling of batch hydrogen production process

by mixed anaerobic cultures
Yang Mu, Gang Wang, Han-Qing Yu *

School of Chemistry, University of Science & Technology of China, Hefei, Anhui 230026, China

Received 7 April 2005; received in revised form 18 May 2005; accepted 26 May 2005
Available online 1 August 2005

Abstract

The kinetics of batch anaerobic hydrogen production by mixed anaerobic cultures was systemically investigated in this study.
Unstructured models were used to describe the substrate utilization, biomass growth and product formation in the hydrogen pro-
duction process. The relationship between the substrate, biomass and products were also evaluated. Experimental results show that
the Michaelis–Menten equation, Logistic model and modified Gompertz equation all could be adopted to respectively describe the
kinetics of substrate utilization, biomass growth and product formation. Furthermore, the relationship between the acidogenic prod-
ucts and biomass was simulated by Luedeking–Piret model very well. The experimental results suggest that the formation of hydro-
gen and the main aqueous products, i.e., butyrate and acetate, was all growth-associated.
 2005 Elsevier Ltd. All rights reserved.

Keywords: Anaerobic; Hydrogen; Kinetics; Mixed cultures; Model

1. Introduction substrate (Taguchi et al., 1995). Hydrogen was produced

Hydrogen is a clean source of energy. Combustion of
hydrogen produces no greenhouse gases, and has a high-
energy yield of 122 kJ/g, which is 2.75-fold greater than
that of hydrocarbon fuels. Thus, using hydrogen as a
clean energy source seems to be promising (Nath and
Das, 2004). The feasibility of applying fermentation of
organic wastes to produce hydrogen has been widely
demonstrated at various laboratories (Yu et al., 2002;
Hussy et al., 2003; Nath and Das, 2004). Both pure
cultures such as Clostridium sp. and mixed cultures of
anaerobic bacteria have been used to convert various
carbohydrates to hydrogen in previous studies (Taguchi
et al., 1992; Ueno et al., 1995; Lay, 2001). For example,
a pure strain of Clostridium could produce hydrogen in a
continuous culture at pH 6.0 when xylose was used as a
*
Corresponding author. Fax: +86 551 360 1592.
E-mail address: hqyu@ustc.edu.cn (H.-Q. Yu).
at a rate of 1600 ml-H2/l/day and a yield of 2.14 mol-
H2/mol-hexose when a mixed culture was employed to
ferment a 0.75% solution of soluble starch (Lay, 2000).
Kinetic models could be used to describe relationship
among the principal state variables and to explain the
behavior of fermentation quantitatively. In addition, it
can provide useful information for the analysis, design
and operation of a fermentation process. Kinetic models
are normally divided into two classes: structured and
unstructured one. Structured models take metabolic
pathways into consideration and are generally compli-
cated. A structured model for acetone–butanol fermen-
tations was established by Votruba et al. (1985). In
this model the biochemical and physiological aspects
of growth and metabolite synthesis were considered
simultaneously, and the key fermentation rates were ex-
pressed and evaluated with regard to substrate con-
sumption and end-product inhibitory effects. On the
other hand, the unstructured kinetic models are much
simpler than the structured ones (Ŏzilgen, 1998). In

0960-8524/$ - see front matter  2005 Elsevier Ltd. All rights reserved.
doi:10.1016/j.biortech.2005.05.014
 Y. Mu et al. / Bioresource Technology 97 (2006) 1302–1307
the unstructured kinetic models microorganisms are
usually considered to be a component or reactant in
the system. The unstructured kinetic models are the
most frequently employed for modeling microbial sys-
tems because they are simple, but are good enough for
technical purposes (Chen et al., 2001; Hu et al., 2002;
Fujikawa et al., 2004).
During the anaerobic hydrogen production from or-
ganic wastes, in addition to hydrogen, carbon dioxide,
various volatile fatty acids (VFA), and sometimes alco-
hols, are simultaneously produced. Thus, this is a com-
plex multiproduct process. Depending on operating
conditions, the distribution of the acidogenic prod-
ucts varies substantially. Furthermore, some acidogenic
products, e.g., acetate, butyrate, hydrogen, carbon
dioxide, may form more complex long-chain fatty acids
or alcohols as the hydrogen is consumed (Yu et al.,
2004). In this case, it is almost impossible to establish
structured models to describe such a multiproduct pro-
cess, but unstructured models might be a better option.
However, few studies have been carried out on unstruc-
tured models to describe the kinetics of biological
hydrogen production by mixed anaerobic cultures.
Therefore, in this study unstructured models were
used to describe the kinetics of biological hydrogen pro-
duction from sucrose by mixed anaerobic cultures in a
batch mode, including substrate utilization, biomass
growth and product formation. In addition, relationship
between substrate, biomass and products was also eval-
uated. The results from this study were expected to be
helpful for understanding the behavior of anaerobic
hydrogen production process.
2. Methods
2.1. Hydrogen-producing sludge
The anaerobic microflora used in this study was ob-
tained from a full-scale upflow anaerobic sludge blanket
reactor treating citrate-producing wastewater located
Benpu, China. Prior to use, the seed sludge was heated
at 102 C for 90 min to inactivate the hydrogentrophic
methanogens and to enrich the hydrogen-producing
bacteria as described by van Ginkel et al. (2001).
2.2. Experiments
Hydrogen production experiments were carried out in
a 5-l fermentor (Baoxin Biotech Ltd., Shanghai). One
liter hydrogen-producing sludge with volatile suspended
solids (VSS) of 15.2 g l1 and 3 ml of nutrients solution
were added to the fermentor. The working volume of
the fermentor was adjusted to 3.0 l with distilled water.
The solution in the fermentor was composed as follows
(unit in mg l1): NH4HCO3 2025; K2HPO4 Æ 3H2O 800;
1303
CaCl2 50; MgCl2 Æ 6H2O 100; FeCl2 25; NaCl 10; CoCl2 Æ
6H2O 5; MnCl2 Æ 4H2O 5; AlCl3 2.5; (NH4)6Mo7O24 15;
H3BO4 5; NiCl2 Æ 6H2O 5; CuCl2 Æ 5H2O 5; ZnCl2 5. Prior
to operation, the fermentor was purged with nitrogen gas
for 10 min to ensure anaerobic condition.
The pH of the mixed liquor was kept at constant by
feeding 4 M NaOH or 2 M HCl solutions via respective
peristaltic pumps. The agitation rate in the fermentor
was kept at 120 rpm. A biogas sampling port was in-
stalled between the meter and the reactor to allow direct
biogas sampling with a syringe. A 10-ml liquor sample
was taken from reactor at each given interval and was
analyzed. In this trail, the pH was chosen as 5.5, which
was optimum for hydrogen production by mixed an-
aerobic cultures obtained by many previous studies
(van Ginkel et al., 2001; Fan et al., 2004). The tempera-
ture and substrate concentration was 35.0 C and
30 g l1, respectively. Moreover, the trial was replicated
at least three times and results averaged.
2.3. Analytical methods
The amount of biogas produced was recorded using
water-replace equipment. The biogas contents were ana-
lyzed by a gas chromatograph (Lunan, Model SP-
6800A) equipped with a thermal conductivity detector
and a 1.5 m stainless-steel column packed with 5 Å
molecular sieve. The temperatures of injector, detector
and column were kept at 100 C, 105 C and 60 C,
respectively. Argon was used as carrier gas at a flow rate
of 30 ml min1. The concentrations of VFA, including
acetate, propionate, and butyrate in the effluent were
determined by second gas chromatograph (Agilent,
Model 6890NT) equipped with a flame ionization detec-
tor and a 30 m · 0.25 mm · 0.25 lm fused-silica capil-
lary column (DB-FFAP). The liquor samples were
first centrifuged at 12000 rpm for 5 min, and were then
acidified by formic acid and filtrated through 0.2 lm
membrane and finally measured for free acids. The
temperatures of the injector and detector were 250 C
and 300 C, respectively. The initial temperature of oven
was 70 C for 3 min followed with a ramp of
20 C min1 for 5.5 min and to final temperature of
180 C for 3 min. Nitrogen was used as carrier gas with
a flow rate of 2.6 ml min1. Sucrose concentration was
measured using enthrone–sulfuric acid method (Koeher,
1952). VSS concentration was measured according to
the Standard Methods (APHA, 1995).
3. Results and discussion
3.1. Kinetics of substrate utilization
The Michaelis–Menten equation was used to model
the substrate degradation as follows (Wu et al., 2002):
1304 Y. Mu et al. / Bioresource Technology 97 (2006) 1302–1307

vm S where X0 (g l1) is the initial microbial concentration (g-

v¼
km þ S
where v (g g-VSS1 h1) is the specific substrate degra-
dation rate; vm (g g-VSS1 h1) is the maximum specific
substrate degradation rate; km (g l1) is the dissociation
constant; and S (g l1) is the substrate concentration.
Linearization of Eq. (1) gives:
1 km 1 1 volume. Fig. 2 shows the relationship between the
¼ þ
v vm S vm
Plotting 1/v against 1/S, a straight line was obtained
with an intercept of 1/vm and a slope of km/vm. This plot
is shown in Fig. 1, from which vm and km were estimated
as 0.28 g g-VSS1 h1 and 13.5 g l1, respectively. The
regression line had a correlation coefficient of 0.963,
suggesting the applicability of Eq. (1). The km value
represents the substrate concentration required to
achieve 50% of the maximum specific substrate degrada-
tion rate. This item could be used for adjusting the most
appropriate substrate concentration in the feed.
ð1Þ
VSS l1).
Although the predictive power of Eq. (4) may be lim-
ited since it does not involve a substrate term (Panikov,
1995), the logistic model is a fair approximation of the
microbial growth curve for the batch hydrogen produc-
tion experiments in this study, due to constant values of
the initial substrate concentrations and the inoculation
ð2Þ
microbial concentration and cultivation time, including
both experimental data and fitted curve obtained based
on the logistic model. Correspondingly, the logistic
model parameters of kc and Xmax were estimated as
0.07 h1 and 9.46 g-VSS l1, respectively. The R2 value,
as a measure of the goodness of the fit, was about 0.954,
indicating excellent agreement of the model to the exper-
imental data. Moreover, the measured Xmax, 9.14 g-
VSS l1, was close to the estimated value by the Logistic
model, 9.46 g-VSS l1.
3.3. Kinetics of product formation

3.2. Kinetics of microbial growth In this study, the desirable gaseous product from the
acidogenesis of sucrose was hydrogen, whereas the main
The Logistic model (Eq. (3)) was adapted to describe aqueous products were butyrate and acetate. Therefore,
the kinetics of the microbial growth (Fujikawa et al., the formation kinetics of hydrogen, butyrate and acetate
2004): were focused. A modified Gompertz equation was em-
  ployed to model the formation of product (van Ginkel
dX X
¼ kcX 1  ð3Þ et al., 2001; Lin and Lay, 2004):
dt X max   
Rmax;i  e
where kc (h1) is the apparent specific growth rate; X (g- P i ¼ P max;i exp  exp ðki  tÞ þ 1 ð5Þ
P max;i
VSS l1) is the microbial concentration; and Xmax (g l1)
is the maximum microbial concentration. Integration of where i represents hydrogen, butyrate and acetate
Eq. (3) gives the following equation for microbial respectively; Pi is the product i formed per liter of reac-
concentration: tor volume at fermentation time t; Pmax,i is the potential
maximum product formed per liter of reactor volume;
X 0 expðk c tÞ Rmax,i is the maximum rate of product formed; is the
X ¼ ð4Þ
1  ðX 0 =X max Þð1  expðk c tÞÞ lag time to exponential product formed.

24 10

9
Microbial concentration

18
1/v (g-VSS h g-1)

(g-VSS l-1)

12 2
7 R = 0.954
y = 48.3x+3.58
2
R = 0.963
6
6

5
0.0 0.1 0.2 0.3 0.4 0 20 40 60 80 100
1/S (l g-1) Fermentation time (h)

Fig. 1. Linearized Michaelis–Menten equation plot for the degrad- Fig. 2. Logistic model plot for the growth of hydrogen-producing
ation of sucrose. microorganisms.
 Y. Mu et al. / Bioresource Technology 97 (2006) 1302–1307 1305

The fitted curves are shown in Fig. 3 and the param- 3.4. Relationship between biomass and products
eters estimated for the formation of various products
are summarized in Table 1. All correlation coefficients The relationship between biomass and the products
of non-linear analysis by Eq. (5) was over 0.990, sug- for the anaerobic hydrogen production by mixed anaer-
gesting that the modified Gompertz equation was able obic cultures could be simulated by the Luedeking–Piret
to adequately describe the formation of various prod- model:
ucts for anaerobic hydrogen production by mixed dP i dX
cultures. ¼ ai þ bi X ð6Þ
dt dt
where ai is the growth-associated formation coefficient
of product i; bi is non-growth-associated formation coef-
5000 ficient of product i.
Hydrogen This model was originally proposed for describing the
4000 lactic acid production by Lactobacillus delbrucckii
(Luedeking and Piret, 1959). The first term of Eq. (6),
P (ml l-1)

3000
i.e., ai dX/dt, is referred as the growth-associated forma-
2000 tion rate of product i. This implies that the growing cells
produce the product in constant proportion of their
1000 P = 4335*exp{-exp[201*e/4335*(9.3-t)+1]}
2
growth. Non-growth associated product formation term
R = 0.999 (biX) shows that all the microorganisms produce the
0
product i in a constant proportion of their concentra-
tion, regardless of the growth phase. Eq. (6) could be
10
Butyrate changed into:
8 dP i dX
¼ ai þ bi
X dt X dt
P (mg l-1)

6
or
4
dP i
¼ ai l þ b i ð7Þ
2 P = 9.30*exp{-exp[0.51*e/9.30*(8.5-t)+1]} X dt
2
R = 0.997 where dPi/dt/X is the specific formation rate of product i;
0
and l is the specific growth rate of the microorganisms.
1.0 A straight line could be obtained with an intercept of
Acetate bi and a slope of ai, if plotting dPi/dt/X against l. The
0.8 fitted plots are shown in Fig. 4 for the three products
(hydrogen, butyrate and acetate). The high correlation
P (mg l-1)

0.6
coefficients indicate that the Luedeking–Piret model
0.4 could properly describe the relationship between bio-
mass and product in the anaerobic hydrogen production
0.2 P = 0.92*exp{-exp[0.043*e/0.92*(9.5-t)+1]} process. The estimated values of a and b, indicating the
2
R = 0.999 dependence of product formation and biomass concen-
0.0
0 20 40 60 80 100 tration, are shown in Table 2. These values could be
Fermentation time (h) used to predict the productivity in the bioreactor design.
Since all b values were zero in Table 2, the formation of
Fig. 3. Modified Gompertz equation plot for the formation of
acidogenic products.
hydrogen, butyrate and acetate in the anaerobic hydro-
gen production was associate-growth.

3.5. Relationship between substrate and products

Table 1
Calculated results using the modified Gompertz equation for the Since the formation of product was associate-growth
formation of acidogenic products for the anaerobic hydrogen production by mixed anaer-
Product Pmax (g l1) Rmax (g l1 h1) k (h) R2 obic cultures, the correlation between product i and sub-
Hydrogen 4335 a
201b
9.4 0.999 strate could be expressed as Eqs. (8) and (9) (Yang et al.,
Butyrate 9.3 0.51 8.5 0.997 1988):
Acetate 0.92 0.043 9.5 0.999
a
Unit in ml l1.
dP i dS
¼ Y P i ð8Þ
b
Unit in ml l1 h1. dt dt
1306 Y. Mu et al. / Bioresource Technology 97 (2006) 1302–1307

30 5000
Hydrogen Hydrogen
4000
(ml g-VSS h )
-1 -1

20
dP/dt/X

P (ml l )
3000

-1
10 2000
y = 759x
2
R = 0.834 1000 y = 143x
2
0 R = 0.998
0
0.10
Butyrate 10
0.08 Butyrate
(g g-VSS h )
-1 -1

8
dP/dt/X

0.06

P (g l )
6

-1
0.04
y = 2.45x
4
0.02 2
R = 0.890
y = 0.35x
0.00 2 2
R = 0.995
0
0.008
Acetate
1.0
0.006
(g g-VSS h )
-1 -1

Acetate
dP/dt/X

0.8
0.004
0.6
P (g l )
-1

0.002 y = 0.15x
2
R = 0.964 0.4
0.000
0.00 0.01 0.02 0.03 0.04 0.2 y = 0.03x
2
-1 R = 0.999
µ (h ) 0.0
0 10 20 30
Fig. 4. Luedeking–Piret model plot for the relationship between the
-1
products and microorganisms. S0-S (g l )

Fig. 5. Plot for the relationship between the products and sucrose.
Table 2
Calculated results using the Luedeking–Piret model for the relation-
ship between the acidogenic products and biomass
Product a (g g-VSS1) b (g g-VSS1 h1) R2
a b
Table 3
Hydrogen 759 0 0.834 Calculated results from Eq. (8) for the relationship between the
Butyrate 2.45 0 0.890 products and substrate
Acetate 0.15 0 0.964
Product YP (g g1) R2
a
Unit in ml g-VSS1.
b
Unit in ml g-VSS1 h1. Hydrogen 143a 0.998
Butyrate 0.35 0.995
Acetate 0.03 0.999
a
Unit in ml g1.
dP i ¼ Y P i dS
Z Pi Z S
dP i ¼ Y P i dS ð9Þ
0 S0
successfully. The yields of hydrogen, butyrate and
acetate were estimated as 143 ml g1, 0.35 g g1 and
P i ¼ Y P i ðS 0  SÞ
0.030 g g1, respectively.
where Y P i is the yield of product i.
Solution of Eq. (9) is plotted in Fig. 5 and the esti-
mated YP values for hydrogen, butyrate and acetate 4. Conclusions
are summarized in Table 3. The high correlation coeffi-
cients in Table 3 suggest that the relationship between With the experimental results of batch hydrogen pro-
products and substrate could be simulated by Eq. (8) duction by mixed anaerobic cultures, unstructured mod-
 Y. Mu et al. / Bioresource Technology 97 (2006) 1302–1307
els were used to describe the substrate utilization, bio-
mass growth and product formation in this process.
The Michaelis–Menten equation, Logistic model and
modified Gompertz equation could respectively describe
the kinetics of substrate utilization, biomass growth and
product formation very well, while Luedeking–Piret
model was able to simulate the relationship between aci-
dogenic products and biomass. The experimental results
also suggest that the formation of hydrogen, butyrate
and acetate was all growth-associated.
Acknowledgements
The authors wish to thank the Natural Science Foun-
dation of China (Grant No. 20377037), the National
Basic Research Program of China (Grant No.
2004CB719602), and the Trans-Century Training Pro-
gram Foundation for the Talents, Ministry of Educa-
tion, China, for the financial support of this study.
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