fermentative hydrogen production with clostridium butyricum

8/8/2019 Fermentative Hydrogen Production With Clostridium Butyricum

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

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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, Taiwanb 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 (2.78mol 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.

2004 International Association for Hydrogen Energy. Published by Elsevier Ltd. All rights reserved.

Keywords: Biohydrogen; Clostridium butyricum; Strain isolation

1. Introduction

Anaerobic digestion of organic substrates to produce

methane and carbon dioxide has been a well-developedbiological 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: [email protected] (J.-S. Chang).

0360-3199/$30.00 2004 International Association for Hydrogen Energy. Published by Elsevier Ltd. All rights reserved.

doi:10.1016/j.ijhydene.2004.09.008

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

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

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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 lessexpensive 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 7.3l/h/l of H2 from sucrose-based synthetic wastewater

[10–14]. The sludge was pretreated at 80 ◦C for 30min,

diluted with sterile distilled water, and then spread onto

CH agar plates incubated under anaerobic condition usingAnaerobic Jar HP11 with Gas Generating Kit BR38 (Ox-

oid). The composition of CH medium was (l−1): sucrose,

15 g; yeast extract, 1 g; Na2HPO4, 5 g; KH2PO4, 1 g; NaCl,

1 g; MgSO4 · 7H2O, 0.1 g; FeSO4, 0.025g; and 2.0ml of

trace element solution. The trace element solution contained

(g/l) H3BO3, 2.86; MnSO4 · 4H2O, 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 andtheir 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 threedifferent 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; Na2HPO4,

5; KH2PO4, 1; NaCl, 2; MgSO4, 0.1; (NH4)2SO4, 3; agar,

2; Na2S · 9H2O, 0.5; resazurin, 001. The DM medium

comprised (g/l) sucrose, 17.8; NH4HCO3, 5.240; NaHCO3,

6.72; K2HPO4, 0.125; MgCl2 · 6H2O, 0.1; MnSO4 · 6H2O,

0.015; Na2S · 9H2O, 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 · 7H2O). 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

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W.-M. Chen et al. / International Journal of Hydrogen Energy 30 (2005) 1063 – 1070 1065

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

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 0.81g 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 (0.5–0.6g dry

cell/l). As for hydrogen production, the iron-enriched DMI

medium was also more favorable with a total H2 production

(V H2 ) of 95 ml, in contrast to a poor hydrogen production for

Fe2+-excluding DM and MP medium (V H2 = 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

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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

aInitial pH was 7.5.b

Volume of the medium was 200 ml.




C e l l c o

n c e n t r a t i o n

( g d r y c

e l l w e i g h t / l )

0.01

0.1

1

S u c r o s e r e m a i n i n g ( % )

0

20

40

60

80

100

120

C u m u l a t i v e h y d r o g e n

p r o d u c t i o n ( l )

0.0

0.5

1.0

1.5

2.0

H y d r o g e n c o n t e n t ( % )

0

10

20

30

40

50

H y d r o g e n p r o d

u c t i o n

r a t e ( m l / h

/ l )

0

50

100

150

200

250

Time (h)

0 5 10 15 20 25 30 35

p H

4.5

5.0

5.5

6.0

6.5

7.0

7.5

T o t a l v o l a t i l e f a t t y a c i d

( T V F A ; m g C O D / l )

0

200

400

600

800

Sucroseremaining

Cell growth

H2 content

H2 production

pH

TVFA

pH adjustment

(a)

(b)

(c)

(d)

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

C e

l l c

o n c e n

t r a t i o n

( g d r y

c e

l l w e

i g h t / l )

0.1

1

pH=6.5

pH=6.0

pH=5.5

pH=5.0

R e s

i d u a

l s u c r o s e

c o n c e n

t r a t i o n

( g / l )

0

5

10

15

pH 6.5

pH 6.0

pH 5.5

pH 5.0

Time (h)

0 10 20 30 40

C u m u

l a t i v e

h y

d r o g e n

p r o

d u c

t i o n

( l )

0

1

2

3

4

5

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

(17.8g/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 metabolicelectron 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

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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

aOverall volumetric H2 production rate = maximum cumulative hydrogen production (V H2,max) ÷ (time required to reach

V H2,max · working volume).

of the carbon substrate into biomass, as only 30% of the

initial sucrose concentration left when H2 started to evolve

at 14h (Fig. 2a and b). The hydrogen production rate de-

creased from its maximal value of 219–17ml/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 (0.78g/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 productiondue 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,

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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

aInitial 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 (V H2) and hydrogen

yield (Y H2 ). The V H2 and Y H2 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 30g 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 (20g 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. Thesoluble 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.

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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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