Appl Microbiol Biotechnol (2002) 58:224228

DOI 10.1007/s002530100814

O R I G I N A L PA P E R

Chin-Chao Chen Chiu-Yue Lin Min-Cheng Lin

Acidbase enrichment enhances anaerobic hydrogen production process

Received: 10 June 2001 / Received revision: 22 July 2001 / Accepted: 17 August 2001 / Published online: 30 November 2001
Springer-Verlag 2001

Abstract This study offers a novel and quick enrichment technology that can be used as a preliminary method to obtain a hydrogen-producing species from the biological sludge produced by wastewater treatment. The
influences of acidbase enrichment (by sludge pH adjustment) on the anaerobic hydrogen-producing microorganisms were investigated using serum bottle assays.
The enrichment pH values were controlled at 3, 4, 5, 7,
10, 11 and 12 with 1 N hydrochloric acid and 1 N sodium hydroxide. For each enrichment pH, the cultivation
pH values were controlled at 5, 6 and 7. Based on the experimental results, hydrogen accumulation from sludge
with acid or base enrichment is higher than that of the
control. The hydrogen-production potential of the sludge
with acid or base enrichment is 200 and 333 times enhanced, compared with the control, when the enrichment
pH is 10 and 3, respectively. The enhancement is due
to a shortening of the micro-organisms lag-time which
occurs at a proper cultivation-pH level.

to heat or harmful chemicals including acid and base and

cannot be destroyed easily (Brock et al. 1994). In the acidogenic phase of anaerobically digesting organic wastes
such as sewage sludge, hydrogen gas is produced. In an
anaerobic digester, hydrogen-utilizing methanogenic
bacteria are present and will consume the hydrogen produced. At pH values lower than 6.3 or higher than 7.8,
the methanogenesis rate decreases or stops (Van Haandel
and Lettinga 1994). Consequently, using a low or high
pH environment to prevent hydrogen reduction and to
obtain dominant microbes for hydrogen production from
sludge seems to be a feasible method.
This study aimed to investigate the efficiencies of
acidbase enrichment (by adjusting sludge pH) and to
find a suitable incubation pH level (by adjusting substrate pH) in the cultivation of anaerobic hydrogen-producing micro-organisms.

Materials and methods

Introduction
Anaerobic digestion of organic waste produces methane
that can be used as an energy source. However, methane
and its combustion product, carbon dioxide, are both
greenhouse gases. This leads to focusing more efforts on
the development of clean energy sources. Hydrogen is a
promising energy alternative and can be produced in the
acidogenesis process during anaerobic degradation of organics (Christopher 1996; Sparling et al. 1997).
Clostridium is an important anaerobic hydrogen-producing micro-organism (Kataoka et al. 1997; Reimann et
al. 1996). Clostridia are Gram-positive, spore-forming,
rod-shaped bacteria. Endospores might be very resistant
C.-C. Chen () C.-Y. Lin M.-C. Lin
Graduate Institute of Civil and Hydraulic Engineering,
Feng Chia University, P.O. Box 25-123, Taichung,
Taiwan 407, Republic of China
e-mail: p8600082@knight.fcu.edu.tw
Fax: +886-4-24519746

Seed sludge
The sludge was obtained from the sludge-drying bed at the
Li-Ming municipal sewage treatment plant (Taichung, Taiwan). The
collected sludge was screened with a No. 8 mesh (diam. 2.35 mm).
The pH, volatile suspended solids (VSS, to express the biomass
concentrations) and total solids (TS) concentrations of the seed
sludge were pH 6.81, 33,280 mg l1and 65,130 mg l1, respectively.
Medium composition
The substrate glucose concentration was 20,000 mg chemical oxygen demand (COD) l1. The substrate contained sufficient inorganics (Endo et al. 1982; mg l1): 5,240 NH4HCO3, 125 K2HPO4,
100 MgCl26H2O, 15 MnSO46H2O, 25 FeSO47H2O, 5 CuSO4
5H2O, 125 CoCl25H2O and 6,720 NaHCO3.
Experimental procedure
Enrichment
pH adjustment was conducted with 1 N hydrochloric acid and 1 N
sodium hydroxide. Six enrichment values were designed at pH 3,

225
4, 5, 10, 11 and 12. A blank without pH adjustment was also prepared. After pH adjustment, the seed sludge, including the blank,
was incubated in the dark at 351 C for 24 h.
Cultivation
For each enrichment pH value, the enriched seed sludge was cultivated at pH values of 5, 6 and 7. During cultivation the hydrogen
production was monitored. These hydrogen-production experiments were performed in serum vials with a working volume of
100 ml, using a batch test. The vials were initially gassed with argon and then the pH-enriched seed sludge (35 ml) and substrate
(35 ml) were added. The vials were placed in a reciprocal waterbath shaker (Reciprocation: 3.0 cm150 strokes min1), with the
temperature controlled at 351 C. The contents were drawn out at
6, 12, 18, 24, 36, 48 and 72 h, using a syringe to determine the
volatile fatty acids (VFA) content and concentration. The total gas
production and its composition were determined to measure the
hydrogen production. Each experimental condition was carried out
in triplicate.
Analyses
Hydrogen gas and liquid VFA were determined with a Shimadzu
(Japan) GC-14A gas chromatograph equipped either with a thermal conductivity detector (stainless column at 55 C, injection
temperature 90 C, Ar as carrier gas, with Porapak Q packing,
Shimadzu C-R3A Chromatopac integrator) or a flame ionization
detector (glass column at 145 C, injection temperature 175 C, N2
as carrier gas, with FON packing, Shimadzu C-R6A Chromatopac
integrator). VSS and TS were measured according to the procedures in the APHA standard methods (APHA 1995).

Results
Acid enrichment (pH 35)
In the acid enrichment, the enrichment values were pH 3,
4 and 5. As an example, Fig 1 shows the time course of
hydrogen production in the acid enrichment (pH 3). In
cultivation at pH 6 and 7, a marked hydrogen production
was observed. However, no hydrogen production was
determined for the blank and for cultivation at pH 5.
These results indicated that cultivation pH affected hydrogen production. Table 1 lists the hydrogen evolutions
after 72 h incubation and the ratio of hydrogen evolution
to that of the blank after acid and base enrichments at
various levels of cultivation pH. The ratios ranged over
0.3333 and acid enrichment produced higher ratios. For
the experiments with acid or base enrichment, most of
Table 1 Hydrogen evolution
(ml) from acid or base enrichment, after 72 h incubation.
Data are given as meanSD,
n=3, with (ratio of hydrogen
evolution to that of the blank)
in parentheses

Cultivation pH

Blank (8.45)
5
6
7

Fig. 1 Hydrogen evolution in the acid enrichment experiment (enrichment at pH 3). mL Millilitres

them produced no methane. Only the cultivations with

enrichment at pH 5 and pH 12 had methane production
(1.2 ml, 13.5 ml, respectively) when cultivated at pH 7.
However, these amounts were rather small, compared
with that of the blank (72 ml). These results revealed
that, with the acid and base enrichments, the methane
evolution was significantly inhibited.
As Table 1 reveals, for acid enrichment (except for
cultivation at pH 5), the hydrogen evolution after 72 h
digestion at various levels of enrichment pH was in the
order: pH 3>pH 4>pH 5>blank. This result reveals that
the hydrogen production of acid enrichment was higher
than that of the blank. Moreover, the effect of cultivation
pH on hydrogen evolution was pH 7>pH 6>pH 5. The
pH 7 cultivation enhanced the micro-organisms to produce hydrogen gas from 0.24 ml (blank) to 80, 58.2 and
33.9 ml at enrichments pH 3, 4 and 5, respectively. This
implies that pH 7 was the best cultivation value of the
tested pH range. The hydrogen production potential of
acid enrichment (compared to the blank) was enhanced
from 0.3 times (at enrichment pH 3, cultivation pH 5) to
333 times (at enrichment pH 3, cultivation pH 7).
Base enrichment (pH 1012)
Comparing the hydrogen evolution values of each enrichment pH at each cultivation pH (Table 1), it is shown
that the cultivation pH effects on hydrogen production

Enrichment pH
3

Blank (6.81) 10

11

12

0.060.01
(0.3)
62.16.5
(259)
80.07.8
(333)

55.85.3
(233)
58.25.4
(243)

11.61.0
(48)
35.03.4
(146)
33.93.1
(141)

0.240.02
0

12.11.2
(50)
13.31.1
(55)

0.110.01
(0.5)
45.54.3
(190)
9.60.8
(40)

0.90.08
(4)
28.62.5
(119)
19.71.7
(82)

4.00.3
(17)
47.94.5
(200)
46.34.3
(193)

226
Table 2 Characteristics of acidbase enrichment vial contents (mg
chemical oxygen demand l1). Data are given as meanSD, n=3.
HAc Acetic acid, HBu normal butyric acid, HPr propionic acid,
HVa normal valeric acid
HAc

HPr

HBu

Blank (cultivation pH 7)
71078
1560159
2690264

HVa

HBu/HAc

2630225

3.78

Acid enrichment (enrichment pH 3, cultivation pH 7)

53035
51058
5250375
0
9.88
Base enrichment (enrichment pH 10, cultivation pH 6)
68065
75061
4400398
0
6.46

Fig. 2 Butyric acid concentration of acidbase enrichment (blank,

pH 6.81). COD chemical oxygen demand, n-HBu normal butyric
acid

was in the order: pH 7= pH 6>pH 5. This implies that

pH 7 and pH 6 were the best cultivation levels for enhancing hydrogen production. The pH 7 cultivation level
enhanced the hydrogen production by micro-organisms
from 0.24 ml (blank) to 46.3, 9.6 and 19.7 ml at enrichment pH 10, 11 and 12, respectively. The hydrogen production potential for base enrichment was enhanced
0.5 times (at enrichment pH 11, cultivation pH 5) to
200 times (at enrichment pH 10, cultivation pH 6), compared with the blank (Table 1).
VFA concentrations after enrichment
Table 2 lists the acid and base enrichment vial content
characteristics. With no enrichment, the intermediate anaerobic digestion products were acetic (HAc), propionic
(HPr), butyric (HBu) and valeric (HVa) acids and their
concentrations ranged over 7102,690 mg COD l1. HBu
and HVa were major component (about 35% for each).
In base or acid enrichment, the intermediate products
were HAc, HPr and HBu, with HBu as the major component (7085%). This indicates that, after enrichment
treatment, the HBu concentrations increased markedly.
Figure 2 shows the HBu concentrations after 72 h digestion. The HBu production was observed to be cultivation
pH-dependent. Cultivation at pH 6 and pH 7 produced

higher HBu concentrations. These VFAs might result

from the decrease in pH within the vials. For cultivation,
the pH was adjusted before starting the cultivation. The
final pH values after 72 h incubation ranged over
4.04.8, 4.64.9 and 5.96.6 for cultivation at pH 5, 6
and 7, respectively.

Discussion
Enhancement of hydrogen production after enrichment
Table 1 reveals that, in the acid and base enrichments,
the hydrogen evolution was dependent on both enhancement pH and cultivation pH. This might be because, during the enrichments, the hydrogen-utilizing methanogens
were killed or inhibited, but clostridia were not. The bacterial endospore has a complex, multilayered structure
and differs structurally from the vegetative cell. These
endospores are very resistant to heat, drying, radiation,
acids and chemical disinfectants; and they cannot be destroyed easily, even by harsh chemicals (Brock et al.
1994). The control of pH is fundamental to the maintenance of optimal bacterial growth and/or conversion processes in anaerobic microbial systems (Stronach et al.
1986). Some investigators have obtained hydrogen production with pure cultures of clostridia at pH ranges
of 6.07.0 (Kataoka et al. 1997; Reimann et al. 1996;
Taguchi et al. 1995). Therefore, at the proper cultivationpH (pH 67), the clostridia were alive and produced a
large amount of hydrogen. Based on the experimental results, by properly adjusting the cultivation-pH value, the
optimum conditions for acidogenesis and hydrogen production during the anaerobic degradation of organics
may be obtained.

Hydrogen production rate of the enrichments

A hydrogen production rate (HPR) exhibits the hydrogen production ability of biomass and can be used to
compare the hydrogen production efficiency of the acid
and base enrichments. Figure 3 shows the HPR for acid
and base enrichments at various levels of cultivation pH.
The ranking of HPR after 72 h digestion, at various
levels of enrichment pH, was in the order: acid enrichment (pH 35) > base enrichment (pH 1012) > blank
(pH 6.81). Moreover, the cultivation-pH effect on HPR
was in the order: pH 7>pH 6>pH 5 for acid enrichment
and pH 6>pH 7>pH 5 for base enrichment. The highest
HPR values were 1.6 ml g VSS h1 for acid enrichment
(pH 3, cultivation pH 7) and 0.730.75 ml g VSS h1 for
base enrichment (pH 1012, cultivation pH 6). The different HPR values might suggest that they involve different sludge micro-organisms after acid or base enrichment. These HPR values are comparable to the reported
hydrogen production rates of 0.116.8 ml g VSS h1
from cultivated sludge (Lay et al. 1999).

227
Table 3 Modified Gompertz equation parameters (see text for details). Data insufficient for simulation
Enrichment
pH
3
4
5
Fig. 3 Hydrogen production rate of acidbase enrichment (blank,
pH 6.81). g VSS/h Grams of volatile suspended solids per hour

10

Variation of VFA concentrations during enrichment

11

The formation of hydrogen is accompanied with VFA or

solvent production during an anaerobic digestion process. Therefore, the distribution of VFA concentrations
and their fractions is a useful indicator for monitoring
hydrogen production.
The data in Figs. 2 and 3 show that, generally, the
HPR markedly increased at high HBu concentrations.
The high HBu concentrations reveal that the reaction
was a butyrate fermentation type. Clostridium species
are, therefore, considered to be the dominant organisms
in the vial, because these organisms are responsible for
butyrate fermentation (Dinopoulou et al. 1988; Yokoi et
al. 1997). The HPR (Fig. 3) and the HBu concentrations
(Fig.2) show that their levels were generally correlated.
This result is consistent with the biochemical pathway of
the anaerobic degradation of organics.
The roles of acetate and butyrate in the anaerobic
degradation of organics have been described. The available hydrogen from glucose degradation during fermentation has been determined, using the butyrate/acetate ratio (Nandi and Sengupta 1998; Ueno et al. 1995). C. butyricum is reported to form butyrate and acetate at a concentration ratio of about 2:1 (White 1995). The butyrate/acetate concentration ratio has been reported to be
0.75 for Butyribacterium methylotrophicum (Annous et
al. 1996). In this paper, we also observed a relationship
between acetate and butyrate. Table 2 reveals that the
HBu/HAc ratio was 3.78 with no pH enrichment, but
was 9.88 and 6.46 in acid and base enrichments, respectively. This indicates that, for higher HBu/HAc ratios, a
higher HPR value was obtained.
However, in the metabolic pathways of C. acetobutylicum, there was no HPr formation when the microorganisms anaerobically degraded glucose (Girbal et al.
1995; White 1995). The differences between our results
and former reports result from pure culture versus mixed
culture. The micro-organisms in mixed culture had some
symbiotic nature or syntrophic interactions that produced
HPr. Another speculation is that, after acid or base enrichment, the surviving or dominant micro-organisms are
micro-organisms other than C. acetobutylicum. More in-

12

Cultivation
pH

P (ml)

Rm (ml h1)

(h)

5
6
7
5
6
7
5
6
7
5
6
7
5
6
7
5
6
7

61.5
81.6

56.1
60.3
11.8
35.1
34.1
3.9
47.6
47.0

45.1
39.1
0.8
28.4
20.0

5.1
3.2

5.1
2.2
0.7
2.4
2.7
0.9
3.7
2.1

2.8
2.4
0.1
2.4
1.0

5.8
1.9

3.9
0.6
10.0
2.3
2.7
15.6
3.6
3.4

3.1
3.1
12.4
5.6
1.3

0.999
0.992

0.999
0.977
0.999
1.0
1.0
1.0
0.998
0.998

0.991
0.993
0.984
0.995
0.996

depth investigations on the identification of the microorganisms, using denaturing-gradient gel electrophoretic
analysis of PCR-amplified genes coding for 16S rRNA
gene might solve this problem.
Kinetic analysis
The modified Gompertz equation (Eq. 1) has been used
to describe the progress of cumulative hydrogen production obtained from a batch experiment (Lay et al. 1999;
Lee et al. 1999; Onodera et al. 1999). Using the cumulative hydrogen production data obtained from batch experiments to fit the modified Gompertz equation, the results are listed in Table 3. The correlation coefficient values ranged over0.9771.0. This indicates that the modified Gompertz equation could also be used to estimate
the hydrogen production potential, maximum hydrogen
production rate and lag-phase time.
(1)
H(t) is the cumulative hydrogen production (ml), P is the
hydrogen production potential (ml), Rm is the maximum
hydrogen production rate (ml h1), e is 2.71828..., is
the lag-phase time (h) and t is time (h).
Table 3 reveals that the maximum hydrogen production potential (P) was 81.6 ml (at enrichment pH 3, cultivation pH 7) and 47.6 ml (at enrichment pH 10, cultivation pH 6) at acid and base enrichment, respectively.
These calculated values are consistent with our experimental data (80 ml, 47.9 ml; Table 1).
For acid enrichment of pH 3, 4 and 5, it was observed
that, when the P value was high, both the Rm and values were low. However, for base enrichment of pH 10,
11 and 12, when the P value was high, the Rm value was

228

high, but the value was medium. These results might

suggest that the sludge micro-organisms possess a range
of responsive capacities for different adverse circumstances. Thus, they can display a certain characteristic,
such as P, Rm and , or even others, i.e. the difference in
hydrogen production (acid enrichment > base enrichment, as listed in Table 1) and the difference in VFA production (as Table 2 shows).
For enrichment at pH 3, where the hydrogen production enhancement was the most obvious, the at cultivation pH 6 was higher than that at pH 7 (5.8 h, 1.9 h, respectively). Because of the value, it is known that under the condition of cultivation at pH 6, the micro-organisms took more time to modify their physiological state
to adapt to a new environment (Onodera et al. 1999).
This indicates that a proper cultivation-pH level could
shorten the lag-phase time and be useful in acclimating
anaerobic micro-organisms for producing hydrogen. For
enrichment pH 4 at cultivation pH 7, a small negative
value was observed. This might result from the fact that,
in this environmental condition, the micro-organisms did
not have an obvious lag-phase time.
Acknowledgements The authors would like to thank the National
Science Council of the Republic of China for financially supporting this manuscript under Contract No. NSC 88-2211-E-035-020.
Part of this paper was presented at Biotechnology 2000, the
World Congress on Biotechnology, 38 Sept. 2000, in Berlin,
Germany.

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