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Bioresource Technology 82 (2002) 8793

Eect of pH on hydrogen production from glucose


by a mixed culture
Herbert H.P. Fang *, Hong Liu
Centre for Environmental Engineering Research, Department of Civil Engineering, University of Hong Kong, Pokfulam Road, Hong Kong
Received 6 March 2001; received in revised form 15 June 2001; accepted 7 July 2001

Abstract
The eect of pH on the conversion of glucose to hydrogen by a mixed culture of fermentative bacteria was evaluated. At 36 C,
six hours hydraulic retention, over 90% of glucose was degraded at pH ranging 4.07.0, producing biogas and an euent comprising
mostly fatty acids. At the optimal pH of 5.5, the biogas comprised 64  2% of hydrogen with a yield of 2:1  0:1 mol-H2 /molglucose and a specic production rate of 4:6  0:4 l-H2 /(g-VSS day). The euent was composed of acetate (15.334.1%) and
butyrate (31.245.6%), plus smaller quantities of other volatile fatty acids and alcohols. The diversity of microbial communities
increased with pH, based on 16S rDNA analysis by denaturing gradient gel electrophoresis (DGGE). 2002 Published by Elsevier
Science Ltd.
Keywords: Acidication; Anaerobic; Fermentation; Glucose; Hydrogen; pH

1. Introduction
Organic pollutants are converted into methane in the
conventional anaerobic treatment of wastewater (Hulsho Pol and Lettinga, 1986; Fang and Liu, 2000) and
solid wastes (Iglesias et al., 1998, 2000). The process can
be divided into two distinct stages: acidication and
methane production. Each stage is carried out by a
number of microorganisms through syntrophic interactions. Acidication produces hydrogen as a by-product,
which in turn is used as an electron donor by many
methanogens at the second stage of the process. However, hydrogen itself is of high commercial value. It can
be used as a raw material in a variety of industrial applications (Kirk et al., 1985), as well as a clean energy
source for fuel cells (Hart, 1997). It might be feasible to
harvest hydrogen at the acidication stage of anaerobic
treatment, leaving the remaining acidication products
for further methanogenic treatment (Mizuno et al.,
2000a).
Microorganisms are capable of producing hydrogen
via either fermentation (Fumiaki et al., 1996; Yokoi et al.,
1997) or photosynthesis (Lichtl et al., 1997; Hansel and

Corresponding author. Tel.: +852-2859-2660; fax: +852-2559-5337.


E-mail address: hrechef@hkucc.hku.hk (H.H.P. Fang).

Lindblad, 1998; Matsunaga et al., 2000). The former is


generally preferred, because it does not rely on the
availability of light sources and the transparency of the
mixed liquor (Hart, 1997). Production of hydrogen by
fermentation has been studied for a large group of pure
fermentative bacteria, such as Clostridia (Heyndrickx
et al., 1991; Fumiaki et al., 1993) and Enterobacteria
(Rachman et al., 1997, 1998; Kumar and Das, 2000).
However, studies of hydrogen production by mixed
cultures have attracted research attention only recently.
Batch experiments have been conducted to produce
hydrogen from solid wastes (Lay et al., 1999; Mizuno
et al., 2000b) and cellulose wastewater (Ueno et al.,
1995; Lay and Noike, 1999), and continuous experiments from glucose (Nakamura et al., 1993; Majizat
et al., 1997) and sugary wastewater (Ueno et al., 1996).
Results so far indicated that the control of pH is crucial
to the hydrogen production, due to the eects of pH on
the hydrogenase activity (Dabrock et al., 1992) and/or
on the metabolism pathways (Lay, 2000). But, the
reported optimal pH value for hydrogen production is
conicting, varying from pH 9.0 for batch fermentation
of sucrose (Lee et al., 1999) to pH 4.04.5 and pH 4.7
5.7, respectively, for the continuous fermentation of
sucrose (Ren et al., 1995) and starch (Lay, 2000).
This study was thus conducted to investigate the
eects of pH on the continuous production of hydrogen

0960-8524/02/$ - see front matter 2002 Published by Elsevier Science Ltd.


PII: S 0 9 6 0 - 8 5 2 4 ( 0 1 ) 0 0 1 1 0 - 9

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H.H.P. Fang, H. Liu / Bioresource Technology 82 (2002) 8793

by mixed culture in a fermentor and on the responsible


microbial communities. Glucose was used as the model
substrate for carbohydrates.

2. Methods
2.1. Seed sludge
The seed sludge of mixed culture was taken from a
continuous stirred tank reactor, which produced hydrogen by fermentation of sucrose at 36 C and pH
5:0  0:5. The reactor, which had been operated for six
months, contained 1.0 g-VSS/l and produced a biogas
comprising 50% hydrogen. Degrading each gram of
sucrose produced 0.15 l of hydrogen.
2.2. Experiments of hydrogen production
Hydrogen production experiments were conducted in
a three liter fermentor (Biostat B, B. Braun Biotech), as
illustrated in Fig. 1, at 36 C and 6 h hydraulic retention.
The feed solution was composed of 7000 mg/l of glucose,
plus the following nutrients (in mg/l): NaHCO3 1000;
NH4 Cl 500; KH2 PO4 250; K2 HPO4 250; MgSO4  7H2 O
320; FeCl3 50; NiSO4 32; CaCl2 50; Na2 BO7  H2 O 7;
NH4 6 Mo7 O24  4H2 O 14; ZnCl2 23; CoCl  6H2 O 21;
CuCl2  2H2 O 10; MnCl2  4H2 O 30.
The fermentor was stirred at a constant 200 rpm to
ensure a thorough mixing and to facilitate rapid diusion of hydrogen. However, the eect of mixing was not
studied. The pH of the mixed liquor was controlled
automatically by feeding NaOH (6 M) and HCl (4 M)
solutions via respective peristaltic pumps. A level probe
was used to control the mixed liquor volume at 1.7 l.
The fermentor was kept in the dark by wrapping with
aluminium foil to prevent the growth of photosynthetic
bacteria. The pH of the mixed liquor in the fermentor

Fig. 1. Experimental setup.

was adjusted stepwise from 4.0 to 7.0 with 0.5 increments. At each pH level, steady state was reached within
14 days, judging from the constant glucose degradation,
hydrogen production, and euent quality. Five sets of
thorough analyses were conducted on the reactor performance during days 1421, before increasing the pH
to the next level.
An additional 50 ml of the original seed sludge was
added into the fermentor each time that the pH was
adjusted.
2.3. Analyses
The amount of biogas produced was recorded daily
using the water displacement method. The contents of
hydrogen, carbon dioxide and methane in the biogas
were analyzed by a gas chromatograph (GC) (Hewlett
Packard 5890 II) equipped with a thermal conductivity
detector and a 2 m  2 mm (inside diameter) stainlesssteel column packed with Porapak N (80100 mesh).
Injector, detector and column temperatures were kept at
57, 180 and 50 C, respectively.
The concentrations of volatile fatty acids (VFA) and
alcohols in the euent were analyzed by a second GC of
the same model equipped with a ame ionization detector and a 10 m  0:53 mm HP-FFAP fused-silica
capillary column. The VFA analyzed included acetate,
propionate, butyrate, i-butyrate, valerate, i-valerate and
caproate, whereas alcohols included methanol, ethanol,
propanol and butanol. Euent samples were rst ltered through a 0.2 lm membrane, acidied by formic
acid and measured for free acids. The initial temperature
of the column was 70 C for 3 min followed with a ramp
of 10 C/min and a nal temperature of 180 C for 4.5
min. The temperatures of the injector and detector were
200 and 250 C, respectively. Helium was used as the
carrier gas at a ow rate of 25 ml/min.
A total organic carbon (TOC) analyzer (TOC-5000A,
Shimazu) was used to measure the organic content in the
feed solution and the euent. The concentration of
glucose in both inuent and euent was measured using
the phenolsulfuric acid method (Herbert et al., 1971).
The biomass was measured by volatile suspended solid
(VSS) according to the Standard Methods (APHA,
1992).
The morphology of hydrogen-producing bacteria in
this study was analyzed using a scanning electron microscope (SEM) (Cambridge Stereoscan 360). A 50 ll
sludge sample was diluted to 50 ml with a 0.1 M, pH 7.2
phosphate-buer solution. About 25 ml of the diluted
sample was ltered through a 0.2 lm nucleopore membrane. The sludge on the membrane was then xed in
the phosphate-buer solution with 2.5% glutaraldehyde
for 2 h. The xed sample was dehydrated stepwise in a
graded series of water/ethanol solutions, and criticalpoint dried with carbon dioxide (Fang et al., 1994).

H.H.P. Fang, H. Liu / Bioresource Technology 82 (2002) 8793

Lastly, the dried sample was sputter coated with gold


prior to SEM observation.
In order to analyze the complexity of the hydrogenproducing microbial communities, the DNA in the
sludge sampled at each pH level was extracted, the 16S
rDNA fragments were amplied by polymerase chain
reaction (PCR), and separated by denaturing gradient
gel electrophoresis (DGGE). For DNA extraction, each
sludge sample was washed with a pH 7.4 phosphatebuer, followed by centrifugation at 4000 rpm for 10
min. The total community DNA from the sludge was
extracted in three steps: cell lysis, phenol/chloroform/
isoamyl-alcohol extraction, and precipitation by isopropanol and ethanol (Liu et al., 1997). The extracted
DNA was used as the template in PCR amplication
(Muyzer et al., 1995) with an annealing temperature of
54 C (Zhang and Fang, 2000). One set of primers was
used for PCR amplication: 968F (bacteria domain
specic forward primer, E. coli position 968-984) with
GC-clamp (Muyzer et al., 1995) and 1392R (universal
reverse primer, E. coli position 1392-1406) (Ferris et al.,
1996). The DGGE was performed following the method
of Muyzer et al. (1993). A 6% (w/v) acrylamide solution
was used to cast a gel with denaturant gradients ranging
4060%. Electrophoresis was conducted in a 1xTAE
buer solution at 200 V and 60 C for 5 h. The 16S
rDNA bands on the gel were then stained with silver
nitrate (Riesner et al., 1989).

89

(a)

(b)

(c)

(d)

3. Results and discussion


3.1. Production of hydrogen
Wastewater containing 7000 mg/l of glucose was
treated in all experiments at 36 C, 6 h hydraulic retention and pH varying from 4.0 to 7.0. Five sets of
extensive analysis were conducted at each pH level under steady-state condition. Fig. 2 illustrates the pH effects on: (a) glucose conversion, (b) biogas content, (c)
hydrogen yield, and (d) specic hydrogen production
rate. Fig. 2(a) illustrates that glucose degradation increased from 90:3  1:0% at pH 4.0 to 99:3  0:9% at
pH 5.5, and remained nearly constant (98.899.5%) for
pH ranging 5.57.0. Fig. 2(b) illustrates that the biogas
comprised mostly hydrogen and carbon dioxide. The
hydrogen content increased from 40  2% at pH 4.0 to
64  2% at pH 5.5, in correspondence to the glucose
degradation. Further increase of pH drastically lowered
the hydrogen content. At pH 7.0, the biogas comprised
only 35  1% of hydrogen. The carbon dioxide content
in biogas followed an opposite trend of hydrogen. The
biogas was free of methane at pH 5.5 or lower, due to
the suppression of methanogenic activity under acidic
condition. But considerable quantities of methane were
produced as pH further increased, due to the bioactivity

Fig. 2. pH eects on (a) glucose degradation, (b) biogas composition,


(c) hydrogen yield, and (d) specic hydrogen production rate.

of hydrogenotrophic methanogens. The methane content increased from 3  1% at pH 6.0 to 9  1% at pH


7.0, accompanied by the decrease of hydrogen content.
Fig. 2(c) illustrates that the hydrogen yield reached
the optimum at pH 5.5. Table 1 lists the hydrogen yields
in the literature from glucose at similar pH for comparison. It shows that the maximum yield of 2:1  0:1
mol-H2 /mol-glucose observed in this study was substantially higher than the yields reported for other mixed
cultures. Treating a wastewater of concentrated glucose
(18720 mg/l) at 35 C, pH 5.7 and 6 h hydraulic retention using a mixed culture, Lin and Chang (1999) reported a yield of 1.7 mol-H2 /mol-glucose. The low yield
is likely due to the poor degradation of glucose (81.7%)

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H.H.P. Fang, H. Liu / Bioresource Technology 82 (2002) 8793

Table 1
Comparison of hydrogen yield using glucose as substrate
Microorganism

pH

Mixed culture
Mixed culture
Mixed culture
E. aerogenes
E. cloacae
C. butyricum
a

Hydrogen yield (mol-H2 /mol-glucose)


a

5.5
5.7
Unspecied
5.56.0
5.06.0
6.7

Reference
This study
Lin and Chang (1999)
Roychowdhury et al. (1988)
Tanisho et al. (1989)
Kumar and Das (2000)
Kataoka et al. (1997)

2:1  0:1
1.7
0.7
1.0
2.2
1.42.3

Mean  S.D. n 5.

resulting from the high concentration of glucose in the


wastewater. Another study using a mixed culture reported a low yield of 0.7 mol-H2 /mol-glucose (Roychowdhury et al., 1988); the reason is unknown because
little information regarding the experimental condition
was provided.
The hydrogen yield found in this study is also higher
than the 1.0 mol-H2 /mol-glucose reported for the pure
culture of Enterobacter aerogenes (Tanisho et al., 1989),
but comparable to the yields of two other pure
cultures, i.e., 2.2 mol-H2 /mol-glucose for E. cloacae
(Kumar and Das, 2000) and 1.42.3 mol-H2 /mol-glucose for Clostridium butyricum (Kataoka et al., 1997).
Results of this study and those reported for the pure
cultures seem to suggest that the maximum hydrogen
yield by glucose fermentation is about 2.12.3 mol-H2 /
mol-glucose.
Fig. 2(d) illustrates that the specic hydrogen production rate ranged 4.55.1 l-H2 /(g-VSS day) at pH 4.5
5.5. At pH 5.5, the specic hydrogen production rate
was 4:6  0:4 l-H2 /(g-VSS day).

According to the stoichiometry of the two following


reactions:

3.2. Production of VFA and alcohols

3.3. Carbon balance and sludge yield

Table 2 lists the distribution of the key VFA and


alcohols in the euents at various pH. It shows that
butyrate and acetate were the two most abundant species in the euent. Increase of pH from 4.0 to 7.0 resulted in the decrease of butyrate but in the increase of
acetate. At pH 4.06.0, the euent contained mostly
butyrate (41.432.4% on carbon basis), followed by acetate (15.3029.5%). At pH 6.5 and 7.0, acetate (33.1
34.1%) and butyrate (31.531.2%) became about equally
abundant.

Carbon in the inuent was converted into biomass,


carbon dioxide in the biogas, plus those in the euent.
Table 3 summarizes the overall carbon mass balance.
The TOCs of inuent were calculated from the glucose
concentration, whereas those of the euent were measured. The inorganic carbon (IC) in the inuent was
attributed to the added NaHCO3 , and the IC content in
the euent was the sum of CO2 (aq), HCO3 and CO23 ,
calculated from the partial pressures of carbon dioxide,
according to Henry's law, and the dissociation constants

C6 H12 O6 2H2 O ! 2CH3 COOH 4H2 2CO2


C6 H12 O6 ! CH3 CH2 2 COOH 2H2 2CO2

1
2

converting 1 mole of glucose into acetate would produce


4 moles of hydrogen. Whereas converting one mole
of glucose into butyrate would produce only 2 moles of
hydrogen. The yield of 2.1 mol-H2 /mol-glucose observed
in this study reects the observation that most of the
glucose was converted to butyrate, instead of acetate.
Ethanol (4.610.1%) was the third most abundant of
all in the euent; the maximum production of ethanol
occurred at pH 5.06.0. Other VFA and alcohols in the
euent included caproate (0.55.8%) and propionate
(0.915.9%), plus minute but detectable amounts of
methanol, propanol, butanol, i-butyrate, i-valerate,
valerate and i-caproate. Production of propionate was
suppressed at low pH as observed by others (Inanc et al.,
1999), but it increased drastically at pH 6.5 and 7.0,
accompanied by the increased production of methane.

Table 2
Product distribution (on carbon basis) in euent
pH

TOC (C-mg/l)

Glucose (%)

Butyrate (%)

Acetate (%)

Ethanol (%)

Lactate (%)

Caproate (%)

Propionate (%)

4.0
4.5
5.0
5.5
6.0
6.5
7.0

1312
1452
1356
1337
1317
1487
1281

20.7
7.2
6.8
1.5
1.1
1.4
2.7

41.4
45.6
38.3
35.1
32.4
31.5
31.2

15.3
23.1
23.4
29.2
29.5
33.1
34.1

6.5
6.9
9.0
9.8
10.1
5.9
4.6

2.4
3.1
3.8
4.1
4.6
2.8
2.0

1.4
4.0
5.8
2.4
0.5
0.8
2.5

1.2
1.0
0.9
1.2
2.9
4.9
15.9

H.H.P. Fang, H. Liu / Bioresource Technology 82 (2002) 8793

91

Table 3
Carbon balance for each liter of inuent
pH

Inuent

Euent

Biogas

TOC (mg)

IC (mg)

TOC (mg)

IC (mg)

CO2 (mg)

CH4 (mg)

4.0
4.5
5.0
5.5
6.0
6.5
7.0

2816
2834
2803
2846
2867
2859
2866

143
152
150
170
151
150
154

1312
1451
1355
1336
1317
1486
1281

170
136
137
113
181
330
827

753
687
632
479
516
384
257

0
0
0
0
37
39
38

Biomass (mg)

Recovery (%)

522
615
705
785
795
815
835

93.1
96.8
95.8
90.0
94.3
101.5
107.2

Fig. 3. Morphologies of hydrogen-producing bacteria at (a) pH 4.5, and (b) pH 5.5.

of H2 CO3 and HCO3 . The carbon content in the biomass was calculated assuming the composition of
C5 H7 O2 N. Table 3 shows that the overall carbon balance was 90.0107%.
Table 3 also shows that the sludge yield increased
with pH. Degrading one gram of glucose produced 0.15
g of VSS at pH 4.0, 0.21 g VSS at the optimal pH of 5.5
and 0.22 g VSS at pH 7.0.
3.4. Microbiology
Sludges sampled at pH 4.05.5 were creamy white,
and the color was darkened with the increase of pH, due
to the increased suldogenic activity. At pH > 6:0, sulfate-reducing bacteria converted the sulfate into sulde
which reacted with metals forming dark precipitates.
Fig. 3 illustrates that hydrogen-producing bacteria at (a)
pH 4.5 and (b) pH 5.5 were mostly composed of bacilli
of various lengths, plus some diplobacilli and streptobacilli.
Fig. 4 illustrates the DGGE proles of the 16S rDNA
gene fragment amplied from the sludges sampled from
pH 4.0 to 7.0. Each band on the DGGE prole corre-

Fig. 4. DGGE proles for hydrogen-producing communities.

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H.H.P. Fang, H. Liu / Bioresource Technology 82 (2002) 8793

sponds to a gene fragment of unique 16S rDNA


sequences, and thus representing a specic species in the
microbial community. The staining intensity of a band
represents the relative abundance of the corresponding
microbial species (Zhang and Fang, 2000). The DGGE
proles in Fig. 4 clearly show that the microbial community changed with pH. The number of bands increased with pH, from 6 at pH 4.0 to 14 at pH 7.0. The
increase of bands at higher pH was likely due to the
presence of methanogens, as evidenced by the increased
methane production in the biogas. Overall, there are 24
detectable bands found in the seven sludge samples. Of
which, only four bands were common in all sludges.
Further analysis is needed to reveal the complexity and
population dynamics of the microbial communities.

Acknowledgements
The authors wish to thank the Hong Kong Research
Grant Council for the nancial support of this project
(HKU7004/98E), and Mr. Tong Zhang for his technical
assistance.

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