International Journal of Hydrogen Energy 30 (2005) 1063 – 1070

www.elsevier.com/locate/ijhydene

Fermentative hydrogen production with Clostridium butyricum

CGS5 isolated from anaerobic sewage sludge
Wen-Ming Chena , Ze-Jing Tsengb , Kuo-Shing Leec , Jo-Shu Changb,∗
a Department of Seafood Science, National Kaohsiung Institute of Marine Technology, Kaohsiung, Taiwan
b Department of Chemical Engineering, National Cheng Kung University, Tainan 701, Taiwan
c Department of Chemical Engineering, Feng Chia University, Taichung, Taiwan

Accepted 30 August 2004

Available online 13 October 2004

Abstract
A Clostridium butyricum strain, isolated from hydrogen-producing sewage sludge, was examined for its ability to produce
H2 from sucrose-based medium under different medium composition, pH, and carbon substrate concentration. The strain,
designated as C. butyricum CGS5, grew and produced hydrogen efficiently on iron-containing medium. Hydrogen started
to evolve when cell growth entered mid-exponential phase and reached maximum production rate at the stationary phase.
The optimal hydrogen production (5.3 l) and hydrogen yield (2.78 mol H2 /mol sucrose) were obtained at an initial sucrose
concentration of 20 g COD/l (17.8 g/l) and a pH of 5.5. However, the CGS5 strain attained its highest hydrogen production
rate (209 ml/h/l) under a medium pH of 6.0. In comparison with pH 5.5, operation at pH 6.0 and 6.5 obtained higher cell
growth rate and cell yield, but resulted in lower total hydrogen production and hydrogen yield. This is most likely due to
rapid conversion of the carbon source into biomass, reducing the formation of hydrogen. Neither hydrogen production nor cell
growth was detected when the strain was cultivated at pH 5.0. A sucrose concentration of 20 g COD/l gave the best hydrogen
fermentation performance, whereas cell growth rate and hydrogen production rate both decreased when sucrose concentration
was elevated to 30 g COD/l, suggesting that substrate inhibition may occur.
䉷 2004 International Association for Hydrogen Energy. Published by Elsevier Ltd. All rights reserved.
Keywords: Biohydrogen; Clostridium butyricum; Strain isolation

1. Introduction (such as Clostridium sp.) or facultative anaerobes (such

Anaerobic digestion of organic substrates to produce
methane and carbon dioxide has been a well-developed
biological treatment for wastewater and waste. Being the
upstream step to methanogenic pathway, acidogenic pro-
cesses produce hydrogen and volatile fatty acids and are
thereby considered an effective and promising means to
produce the clean energy (H2 ) [1]. Fermentative hydrogen
production, achieved by anaerobic acid-forming bacteria
∗ Corresponding author. Fax: +886 6 235 714 6.
E-mail address: changjs@mail.ncku.edu.tw (J.-S. Chang).
as Enterobacter sp.) [1,2], has caught the attention of re-
searchers due to it dual functions of generating hydrogen
and decontaminating organic pollutants in the environment.
The Clostridium sp. (obligate anaerobes) and Enterobacter
sp. (facultative anaerobes) account for the majority of light-
independent fermentative bacteria that have the ability to
produce hydrogen. Numerous investigations of fermenta-
tive hydrogen production with Enterobacter sp. have been
reported [3–7]. However, biohydrogen research using pure
strain of Clostridium sp., which is recognized as an effec-
tive hydrogen producer, has been surprisingly rare. This is
in part due to the difficulty in cultivating strictly anaero-
bic strains [8,9] because they are very sensitive to minute

0360-3199/$30.00 䉷 2004 International Association for Hydrogen Energy. Published by Elsevier Ltd. All rights reserved.
doi:10.1016/j.ijhydene.2004.09.008
1064 W.-M. Chen et al. / International Journal of Hydrogen Energy 30 (2005) 1063 – 1070
amounts of dissolved oxygen. In order to grow Clostridium
sp. for hydrogen production, addition of expensive reduc-
ing agents (such as L-cysteine) may be required, reducing
the feasibility in practical applications. Therefore, some re-
searchers have utilized a mixed culture of Enterobacter sp.
and Clostridium sp. to produce hydrogen under a reducing-
agent-free culture medium and thereby lowering the cost of
H2 production with Clostridium sp. alone [9]. However, be-
cause Clostridium sp. (e.g., Clostridium butyricum) is fre-
quently found in hydrogen-producing bacterial consortia and
is also very effective in producing H2 from organic sub-
strates (esp. carbohydrates), it is still of great value to re-
veal hydrogen production characteristics of Clostridium sp.
in order to maintain or improve hydrogen production per-
formance of a mixed-culture system.
In this work, we isolated hydrogen-producing anaerobes
(identified as Clostridium butyricum strains) from accli-
mated sewage sludge that is very efficient in H2 produc-
tion [10–14]. The pure isolates were cultivated on a less
expensive culture medium for hydrogen production. Effects
of pH control and carbon substrate concentration on hydro-
gen production performance were investigated to identify
the proper conditions for H2 production with the pure iso-
late. The dynamic relationship among cell growth, hydro-
gen evolution and metabolite production was closely mon-
itored and discussed. Characterization of H2 fermentation
features of the isolate is the initial step towards feasibility
assessment of using the domestic hydrogen-producing iso-
lates for bioaugmentation of anaerobic hydrogen-producing
processes.
2. Materials and methods
2.1. Isolation of the bacterial strains and growth conditions
Hydrogen-producing bacterial strains were isolated from
effluent sludge of five anaerobic hydrogen-producing biore-
actors including CSTR, fixed bed, fluidized bed, and a
novel granular sludge bed reactor capable of producing up
to 7.3 l/h/l of H2 from sucrose-based synthetic wastewater
[10–14]. The sludge was pretreated at 80 ◦ C for 30 min,
diluted with sterile distilled water, and then spread onto
CH agar plates incubated under anaerobic condition using
Anaerobic Jar HP11 with Gas Generating Kit BR38 (Ox-
oid). The composition of CH medium was (l−1 ): sucrose,
15 g; yeast extract, 1 g; Na2 HPO4 , 5 g; KH2 PO4 , 1 g; NaCl,
1 g; MgSO4 · 7H2 O, 0.1 g; FeSO4 , 0.025 g; and 2.0 ml of
trace element solution. The trace element solution contained
(g/l) H3 BO3 , 2.86; MnSO4 · 4H2 O, 2.03; 0.08; FeCl3 , 0.1.
The incubation temperature was controlled at 37 ◦ C and the
pH of the medium was adjusted initially to 7.5. The CH
agar plates were prepared by adding 2.0% (w/v) agar to
the CH media. Single colonies obtained on CH agar plates
were re-streaked more than three times to ensure the purity
of the strains. Six strains from pure cultures were obtained
and designated as CGS1, CGS2, CGS3, CGS4, CGS5 and
CGS6.
2.2. Morphological and biochemical test
Morphological examination was observed by a light
microscope (Zeiss Axioskop). Biochemical identification
presented in the Rapid ANA II microtests was determined
by Electronic Code Compendium software version VB.
1.3.97 according to the recommendation of the manufac-
turer (Remel).
2.3. 16S rDNA sequencing and phylogenetic analysis
Amplification and sequence analysis of the 16S rRNA
gene was performed as described previously [15]. The se-
quence was compared with others available in GenBank
and Ribosomal Database Project II. The multiple-sequence
alignment including six hydrogen-producing strains and
their closest relatives were performed using the BioEdit pro-
gram [16]. The phylogenetic reconstruction was inferred by
using the neighbor-joining, UPGMA, maximum-likelihood
and Fitch-Margoliash methods in the BioEdit software
[16]. A bootstrap analysis (confidence values estimated
from 1000 replications of each sequence) was performed
for the neighbor-joining analysis using the CLUSTAL w
1.7 program [17]. A phylogenetic tree was drawn using
the TREEVIEW program [18]. Sequence identities were
calculated using the BioEdit program [16].
2.4. Nucleotide sequence accession numbers
The 16S rDNA sequence reported in this paper has
been deposited in the NCBI nucleotide sequence databases
under accession number as follows: AY540105 (CGS1),
AY540106 (CGS2), AY540107 (CGS3), AY540108 (CGS4),
AY540109 (CGS5) and AY54010 (CGS6).
2.5. Selection of fermentation medium for hydrogen
production
The isolates were pre-cultured on CH medium and
the early-stationary-phase culture was transferred to three
oxygen-free 250-ml serum vials containing 200 ml of three
different media (PM, DM, and DMI) to undergo batch hy-
drogen fermentation. The PM medium, which is deficient in
trace elements, consisted of (g/l) sucrose, 17.8; Na2 HPO4 ,
5; KH2 PO4 , 1; NaCl, 2; MgSO4 , 0.1; (NH4 )2 SO4 , 3; agar,
2; Na2 S · 9H2 O, 0.5; resazurin, 001. The DM medium
comprised (g/l) sucrose, 17.8; NH4 HCO3 , 5.240; NaHCO3 ,
6.72; K2 HPO4 , 0.125; MgCl2 · 6H2 O, 0.1; MnSO4 · 6H2 O,
0.015; Na2 S · 9H2 O, 0.5; resazurin, 001; agar, 2. The DMI
medium is an iron-amended version of DM medium (i.e.,
DM medium plus 0.01 g/l of FeSO4 · 7H2 O). The initial
pH of the medium was 7.5 and the temperature for batch
growth was kept at 37 ◦ C. The cell concentration, pH, and
 W.-M. Chen et al. / International Journal of Hydrogen Energy 30 (2005) 1063 – 1070
biogas production were monitored during the course of the
experiments.
2.6. Hydrogen production in batch fermentors
Thirty milliliters of the pre-culture (on DMI medium)
of C. butyricum CGS5 was inoculated into a 5-l fermen-
tor (Model MDL6C, Mazubishi Inc.; Tokyo, Japan), con-
taining 1.7 and 2.5 l of agar-free DMI medium, respec-
tively, for the runs with and without pH control. For the
experiments investigating the effect of pH, the pH value
was controlled at 6.5, 6.0, 5.5, and 5.0 by automatic addi-
tion of 3 N NaOH. The initial sucrose concentration in the
medium was 17.8 g/l (20 g COD/l). In the other set of ex-
periments, the pH was controlled at 6.5, while the initial
sucrose concentration varied from 5 to 30 g COD/l. In all
cases, the hydrogen fermentation was conducted at a con-
stant temperature of 37 ◦ C. Cell concentration, pH, carbo-
hydrate concentration, and production of biogas and sol-
uble metabolites were determined with respect to culture
time.
2.7. Analytical methods
The gas products (mainly H2 and CO2 ) were analyzed
by gas chromatography (GC) using a thermal conductiv-
ity detector. The carrier gas used was argon and the col-
umn (0.53 mm in inner diameter and 15 m in height) was
packed with Porapak Q. The temperature at the capillary
column was initially 50 ◦ C and was increased to 200 ◦ C at a
rate of 30 ◦ C/min. The temperature at injection was 140 ◦ C.
The volatile fatty acids and ethanol were also detected by
GC using a flame ionization detector. The temperatures at
glass column and injection were 145 and 175 ◦ C, respec-
tively. The carrier gas was N2 and the packing material was
FON (contains polyethylene glycol and 2-nitroterephthalic
acid) obtained from Shimadzu Inc. (Tokyo, Japan). Standard
Methods (APHA, 1995) were used to determine biomass
concentration (in terms of volatile suspended solid; VSS) of
samples taken separately from granular sludge portion and
suspended-cell portion in the reactor. The carbohydrate con-
centration in the effluent was also measured according to
Standard Methods [19].
3. Results and discussion
3.1. Characterization of the hydrogen-producing isolates
Six strains capable of producing hydrogen were isolated
from the anaerobic sludge sources. The isolates only grew
under strict anaerobic conditions. Microscopic examination
showed that they were in rod shape and some with en-
dospores (by spore stain method). Biochemical identifica-
tion by Rapid ANA II microtest system indicates that the
six strains were similar to each other and all belong to
1065
genus Clostridium. The nearly full-length sequences of 16S
rRNA gene (1434 bp) were also determined for the six iso-
lates. Based on the sequence identity of 16S rDNA, CGS1,
CGS2, CGS3, CGS4 and CGS6 resemble to each other with
a 100–99.7% identity, while the sequence of CGS5 is less
similar to that of the rest (99.0% identity). The highest sim-
ilarity values of six strains were obtained towards C. bu-
tyricum ATCC 19398T (99.5–99.3% identity). The similarity
levels towards other Clostridium species were below 97.4%.
The 16S rDNA phylogenic tree was constructed for the
six hydrogen-producing strains and those of the described
Clostridium species were shown in Fig. 1. According to the
results of physiological and 16S rDNA sequence examina-
tions, the strains should belong to C. butyricum. Preliminary
batch tests show that the CGS5 strain possessed superior hy-
drogen production activity over the other five C. butyricum
isolates (data not shown) and thus CGS5 strain was selected
for detailed hydrogen fermentation studies in the rest of this
work.
3.2. Effect of medium composition on hydrogen production
To select for suitable medium composition for hydrogen
fermentation, C. butyricum CGS5 was grown on three me-
dia with different compositions. The liquid media did not
contain expensive reducing agents, but was instead supple-
mented with a dilute concentration of agar (2 g/l) to create
local anaerobic environment in the culture medium. The re-
sults (Table 1) show that C. butyricum CGS5 grew well on
all of the three media. Among them, DMI medium enabled
more efficient cell growth with a growth rate of 0.77 h−1 and
a final concentration of 0.81 g dry cell/l, whereas the strain
grew slower on DM and PM medium (both at ca. 0.48 h−1 )
and also reached a lower cell concentration (0.5–0.6 g dry
cell/l). As for hydrogen production, the iron-enriched DMI
medium was also more favorable with a total H2 production
(VH2 ) of 95 ml, in contrast to a poor hydrogen production for
Fe2+ -excluding DM and MP medium (VH2 = 18 and 5 ml,
respectively) (Table 1). Apparently, supplement of Fe2+ is
extremely crucial to hydrogen production activity of the C.
butyricum CGS5 strain. This is quite reasonable because
Fe2+ is considered an important cofactor of hydrogenase
responsible for hydrogen production in acidogenic bacteria,
such as C. butyricum [20–22]. An iron-carrying complex,
ferrodoxin, is known to mediate electron transfer of reduc-
ing power en route to hydrogenase-catalyzed hydrogen pro-
duction [22,23]. In addition, Table 1 shows that fermentation
with DM medium, a modified version of a medium proposed
for methane fermentation [24], attained better hydrogen pro-
duction efficiency than with PM medium. It is likely that
supplementation of trace element (Mn2+ ) and bicarbonate
ions (acts as an alkali and buffering agent) in DM medium
benefited hydrogen production with the C. butyricum CGS5
strain. The final pH of the cultures all decreased from 7.5 to
4.5–5.0 and the culture with higher H2 production displayed
1066 W.-M. Chen et al. / International Journal of Hydrogen Energy 30 (2005) 1063 – 1070

Fig. 1. Neighbor-joining showing phylogenetic positions of six biohydrogen producing strains and Clostridium species based on 16S rDNA
sequence comparisons. Bacillus aeolius 4-1T was used as an outgroup. Bootstrap values are indicated at nodes. Only bootstrap values > 50%
are shown. Scale bar, 1% sequence dissimilarity (one substitution per 100 nt). Representative sequences in the dendrogram were obtained
from GenBank (accession number in parentheses).

Table 1
Cell growth and hydrogen production of Clostridium butyricum CGS5 on different culture medium

Medium Maximum growth Final cell concentration Final pHa Total hydrogen
rate (h−1 ) (g dry cell/l) production (ml)b

PM 0.48 0.59 5.0 5

DM 0.48 0.51 4.8 18
DMI 0.77 0.81 4.5 95
a Initial pH was 7.5.
bVolume of the medium was 200 ml.
 W.-M. Chen et al. / International Journal of Hydrogen Energy 30 (2005) 1063 – 1070 1067

pH adjustment
1

(g dry cell weight/l)

Cell concentration
120

Sucrose remaining (%)

Cell concentration
(g dry cell weight/l)

1 100
Cell growth
80

60
0.1
Sucrose
40
remaining
0.1
20
(a)
0.01 0

Hydrogen content (%)

Cumulative hydrogen

2.0 50 pH 6.5
H2 content pH=6.5
pH 6.0

concentration (g/l)
production (l)

Residual sucrose
40 15 pH=6.0
1.5 pH 5.5
pH=5.5
30 pH 5.0
pH=5.0
1.0
H2 production 20 10
0.5
10
(b)
0.0 0 5
250
Hydrogen production

200
rate (ml/h/l)

0
150 5
100 Cumulative hydrogen 4
50 production (l)
(c) 3
0
7.5
800
2
Total volatile fatty acid

7.0
(TVFA; mg COD/l)

pH
600 6.5 1
pH

400
200 TVFA
(d)
0 Time (h)
0 5 10 15
Time (h)
Fig. 2. Dynamic behavior of batch fermentation of Clostridium bu-
tyricum CGS5 with DMI medium containing 20 g COD/l (17.8 g/l)
of sucrose as sole carbon source. (a) Profiles cell growth and su-
crose consumption, (b) Profiles of hydrogen evolution and concen-
tration, (c) The profile of hydrogen production rate, and (d) Profiles
of pH and formation of soluble metabolite.
a lower pH. The decrease in pH was apparently due to pro-
duction of acidic metabolites, predominantly consisting of
butyric acid, acetic acid, and propionic acid (Table 3).
3.3. Dynamics of hydrogen fermentation
Although C. butyricum is known as an effective hydrogen
producer [9,25], there is little information regarding hydro-
gen performance of pure strain of C. butyricum in a quanti-
tative fashion. Moreover, the transient relationship between
hydrogen production and cell growth of C. butyricum has
not yet been revealed in detail. Fig. 2 demonstrates the pro-
files of cell growth and hydrogen production during batch
fermentation of C. butyricum CGS5 on DMI medium. The
evolution of H2 appeared to start after the middle-stage of
6.0
5.5 0
5.0 0 10 20 30 40
4.5
20 25 30 35
Fig. 3. Time-course profile of cell growth, sucrose consumption,
and cumulative hydrogen production of Clostridium butyricum
CGS5 cultivated on DMI medium at different pH values (5.0, 5.5.
6.0 and 6.5). The initial sucrose concentration was 20 g COD/l
(17.8 g/l).
exponential growth (14 h) with the maximal hydrogen pro-
duction rate (219 ml/h/l) occurring at 17 h, where cell growth
had entered early stationary phase (Fig. 2a–c). This seems
to imply that generation of hydrogen was not a preferable
event during assimilation of carbon substrate for the gain
of biomass. This may be due to a predominant metabolic
electron flow towards biosynthesis, decreasing the availabil-
ity of electrons for hydrogenase to produce H2 . The hydro-
gen content increased sharply since the onset of hydrogen
production and reached a maximal value of 50% after cul-
tivation for 23 h (Fig. 2b). The pH decreased correspond-
ingly with H2 production and formation of acidic metabo-
lites (expressed by total volatile fatty acid; TVFA), indi-
cating that hydrogen evolution was accompanied by forma-
tion of acidic metabolites (Fig. 2b–d). The hydrogen yield
based on substrate consumption was 0.61 mol H2 /mol su-
crose with a sucrose conversion of ca. 85%. The low hy-
drogen yield was apparently due to predominant conversion
1068 W.-M. Chen et al. / International Journal of Hydrogen Energy 30 (2005) 1063 – 1070

Table 2
Performance of hydrogen fermentation with Clostridium butyricum CGS5 on DMI medium under different pH and initial sucrose concentration

pH Sucrose H2 content Sucrose Hydrogen yield Cell Yield Total H2 Volumetric H2 Specific H2
concentration in biogas conversion (mol H2 /mol (g cell/g production production production rate
(g COD/l) (%) (%) sucrose) sucrose) (l) rate (ml/h/l) (ml/h/g cell)

Maximum Overalla

6.5 62 98 1.16 0.052 2.17 174 116 193

6 50 98 1.46 0.048 2.73 209 149 243
5.5 20 64 99 2.78 0.039 5.25 163 137 214
5 0 3 0 0 0 0 0 0
Not controlled 51 85 0.61 0.061 1.53 146 58 209

5 40 94 1.94 0.036 0.87 154 151 234

5.5 10 46 98 1.85 0.030 1.73 157 145 254
20 64 99 2.91 0.041 5.46 160 141 212
30 57 99 1.73 0.050 4.94 139 127 136
a Overall volumetric H
2 production rate = maximum cumulative hydrogen production (VH2 ,max ) ÷ (time required to reach
VH2 ,max · working volume).

of the carbon substrate into biomass, as only 30% of the
initial sucrose concentration left when H2 started to evolve
at 14 h (Fig. 2a and b). The hydrogen production rate de-
creased from its maximal value of 219–17 ml/h/l when the
pH dropped from 5.6 to 4.6 (Fig. 2c and d). An elevation of
pH from 4.5 to 6.5 seemed to restore hydrogen production
of the culture, suggesting that pH control at an appropriate
level may be required to improve the efficiency of hydrogen
production.
3.4. Effect of pH on hydrogen production
Hydrogen fermentation was conducted at four different
pH values, namely, 6.5, 6.0, 5.5, and 5.0, to identify the op-
timal pH for hydrogen production with C. butyricum CGS5.
For comparison, a control experiment without pH control
was also carried out. The pH range was chosen because op-
timal pH for anaerobic H2 production reported in literature
was essentially within the range of 5.5–6.7 [21,26–30]. The
results show that cell growth and hydrogen production were
strongly inhibited at pH 5.0 (Fig. 3). Operation at pH 5.5
attained the highest total hydrogen production and hydro-
gen yield of 5.3 l and 2.78 mol H2 /mol sucrose, respectively.
However, the best maximal volumetric hydrogen production
rate (0.21 l/h/l) occurred at pH 6.0 (Table 2). In contrast to
the run without pH control, the substrate conversion was
much higher (near 98%) when the culture pH was controlled
at 5.5–6.5. The gas-phase H2 content was in the range of
50–64% for all tests, with the highest value of ca. 64% at
pH 5.5 (Table 2). Although obtaining significantly higher
growth rate than that for pH 5.5, operation at pH 6.0 and
6.5 gave markedly lower hydrogen yield and total hydrogen
production than those obtained for pH 5.5 (Table 2). Fig. 3
indicates that cells grew rapidly at pH 6.0 and 6.5 and su-
crose was completely consumed within 15 h of cultivation,
at which hydrogen production ceased with a total hydrogen
production of 2.73 and 2.17 l, respectively (Table 2). Mean-
while, the biomass concentration reached a maximum value
of 0.9 g/l for pH 6.0 and 1.0 g/l for pH 6.5. However, when
the strain was cultivated at pH 5.5, the rate of cell growth
and sucrose consumption was much slower, and hydrogen
continued to evolve until cultivation for 37 h. The total hy-
drogen production at pH 5.5 was nearly doubled as com-
pared with that obtained at pH 6.0 and 6.5, whereas the fi-
nal cell concentration (0.78 g/l) was lower than that for pH
6.0 and 6.5. Therefore, under batch operations, higher cell
growth efficiency at pH 6.0 and 6.5 led to higher biomass
yields, but lower hydrogen yields (Table 2) since a larger
amount of carbon substrate was converted to biomass, in-
stead of H2 , and the carbon source was quickly exhausted.
However, despite resulting in lower hydrogen yields, pH 6.0
and 6.5 (esp. pH 6.0) gave a slightly highermaximum and
overall hydrogen production rate than those obtained at pH
5.5 (Table 2). The specific hydrogen production rate at pH
6.0 was also 14% higher than that obtained at pH 5.5 (Table
2). Thus, for operations with continual supply of carbon
substrate (e.g., operations at continuous or fed-batch mode),
control at pH 6.0 may be preferable for hydrogen production
due to the potential of attaining higher hydrogen production
rates.
3.5. Effect of sucrose concentration on hydrogen production
Organic loading usually plays a crucial role in the effi-
ciency of anaerobic hydrogen production [31,32]. Table 2
shows the variations in hydrogen fermentation performance
at different concentrations of the carbon source (sucrose).
The results clearly indicate that a sucrose concentration
of 20 g COD/l exhibited the best performance in hydro-
gen production for most of categories compared in Table 2,
 W.-M. Chen et al. / International Journal of Hydrogen Energy 30 (2005) 1063 – 1070 1069

Table 3
Production of soluble metabolites during hydrogen fermentation with Clostridium butyricum CGS5 on DMI medium under different pH and
initial sucrose concentration

pH Sucrose TVFA SMP HAc/SMP HPr/SMP HBu/SMP EtOHl/SMP TVFA/SMP

concentration (mg COD/l) (mg COD/l) (%) (%) (%) (%)
(g COD/l)

6.5 4094 4309 27 30 38 5 0.95

6.0 4270 4543 28 26 40 6 0.94
5.5 20 8635 9926 14 21 52 13 0.87
5.0 445 445 66 24 11 0 1.00
Not controlleda 2823 2823 22 20 58 0 1.00

50 1235 1419 16 11 60 13 0.87

5.5 10 2481 2820 30 22 36 12 0.87
20 8858 10066 14 21 53 12 0.88
30 7394 7469 28 30 41 1 0.99
a Initial pH = 7.5. HAc: acetic acid; HPr: propionic acid; HBu:normal butyric acid; EtOH: ethanol; TVFA = HAc + HPr + HBu; SMP
= TVFA + EtOH.

especially for total hydrogen production (VH2 ) and hydrogen
yield (YH2 ). The VH2 and YH2 both increased as the substrate
concentration increase from 5 to 20 g COD/l, while they de-
creased slightly when the substrate concentration was ele-
vated further to 30 g COD/l. Similar trend was observed for
the dependence of specific growth rate on substrate concen-
tration (data not shown), suggesting that substrate inhibition
may occur when the sucrose concentration was higher than
30 g COD/l. The hydrogen production rates were similar for
the tests with a sucrose concentration of 5–20 g COD/l, but
the rates were slightly lower for operation at 30 g COD/l
(Table 2). The optimal concentration (20 g COD/l) for hydro-
gen production with the C. butyricum CGS5 strain appears
to be identical to that obtained with the anaerobic sewage
sludge [10–14] where the CGS5 strain was isolated. This
seems to indicate that the CGS5 strain belongs to the major
hydrogen-producing bacterial populations in the acclimated
anaerobic sludge that was shown to produce hydrogen very
effectively [12,14].
3.6. Production of soluble metabolites
Results concerning production of soluble products dur-
ing hydrogen fermentation are summarized in Table 3. The
soluble metabolites include butyric acid (HBu), acetic acid
(HAc), propionic acid (HPr), and ethanol (EtOH). The most
abundant product was HBu, which accounted for 36–60%
of total soluble microbial products (SMP). The production
of HAc and HPr were also significant, whereas ethanol was
produced at a lesser amount (Table 3). The EtOH/SMP ratio
was essentially less than 13%. Hence, hydrogen fermenta-
tion was a favorable event in C. butyricum strain because
production of electron-consuming solvents (e.g., ethanol)
was relatively small [33,34]. Comparison between hydro-
gen production (Table 2) and soluble metabolites production
(Table 3) shows that better hydrogen production was accom-
panied by a higher total volatile fatty acid content (TVFA)
and a higher HBu/SMP ratio. This suggests that hydrogen
production with the C. butyricum CGS5 was directed by
acidogenic pathways and was essentially butyrate-type fer-
mentation. The behavior of soluble metabolite production is
quite consistent with that of the original sludge (where the
pure strain was isolated), which produced similar metabo-
lites and HBu was also the predominant soluble product
[10–14].
4. Conclusions
Hydrogen-producing Clostridium butyricum strain was
successfully isolated from anaerobic sludge exhibiting ex-
cellent hydrogen production efficiency. The presence of fer-
ric ions in the culture medium appeared to be critical to the
hydrogen production activity of the pure isolate. The perfor-
mance of hydrogen production was also influenced by the
culture pH and the initial carbon substrate (sucrose) con-
centration. Cell growth and hydrogen production was inhib-
ited at a pH of 5.0. Cell growth was more efficient at pH
6.0–6.5, while the total hydrogen production and hydrogen
yield were higher when the C. butyricum strain was cul-
tivated at pH 5.5. Operation at pH 6.0 attained a slightly
higher hydrogen production rate than that for the other pH
values. Hydrogen production in a batch culture seems to be
limited when cell growth was thriving (e.g., at pH 6.0–6.5),
mainly due to rapid depletion of carbon substrate for the
gain of biomass. Thus, continuous culture may be favor-
able for hydrogen production due to a sufficient supply of
carbon substrate and a controllable cell growth rate via the
adjustment of feeding rate. This study shows that hydrogen
production activity was optimal when the sucrose concen-
tration was 20 g COD/l and the pH was controlled at the
range of 5.5–6.0.
1070 W.-M. Chen et al. / International Journal of Hydrogen Energy 30 (2005) 1063 – 1070
Acknowledgements
The authors gratefully acknowledge the financial support
of National Science Council and Energy Council of Ministry
of Economic Affairs of Taiwan under Grant No. 93-ET-7-
006-001-ET.
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