J. Chin. Inst. Chem. Engrs., Vol. 34, No.

6, 683-687, 2003
Short communication
Sequential Production of Hydrogen and Methane
from Wastewawter Sludge Using Anaerobic
Fermentation
Chi-Chung Wang
[1]
, Chih-Wen Chang
[2]
, Ching-Ping Chu
[3]
, Duu-Jong Lee
[4]

Department of Chemical Engineering, National Taiwan University
Taipei, Taiwan 106, R.O.C.
Bea-Van Chang
[5]

Department of Microbiology, Soochow University
Taipei, Taiwan 111, R.O.C.
AbstractThis study demonstrates the feasibility of sequentially producing both hydrogen
and methane from wastewater sludge using a clostridium strain, as was isolated by Wang et
al. (2003a). Three commonly used pre-treatments were applied to wastewater sludge to in-
crease the hydrogen yield. Then, the waste liquor was externally dosed with methanogenic
bacteria to produce methane. The waste liquor after fermentation of hydrogen produced
more methane than was directly derived without fermentation of hydrogen. The reduction of
nitrogen-containing organic matter is shown to compete with the formation of hydrogen,
yielding ammonium nitrogen (NH
3
-N) in the fermented liquor.
Key Words : Anaerobic fermentation, Hydrogen, Methane, Clostridium, Pre-treatment

INTRODUCTION
Hydrogen is a clean source of energy.
Bio-conversion of biomass to produce hydrogen has
been demonstrated, using the anaerobic fermentation
of high-strength wastewater (Bolliger et al., 1985;
Liu et al., 1995; Ueno et al., 1996; Zhu et al., 1999),
solid waste (Mizuno et al., 2000a; Lay et al., 1999),
and some well-defined compounds in water, such as
molasses (Tanisho and Ishiwata, 1994), glucose
(Kataoka et al., 1997; Lin and Chang, 1999), crystal-
line cellulose (Lay, 2001), peptone (Bai et al., 2001),
and starch (Lay, 2000). Methods for promoting hy-
drogen production have been reported (Tanisho and
Ishiwata, 1995; Tanisho et al., 1998; Mizuno et al.,
2000b; Sparling et al., 1997; Liang et al., 2001). In
the literature, anaerobic fermentation has yielded
around 11 mg-H
2
/g-dried solids (DS) from glucose
solution (Kataoka et al., 1997), and 1.4 mg-H
2
/g-DS
from peptone-containing solution (Liang et al., 2001).
Only a few data on the hydrogen yield from waste-
water sludge have been presented at local confer-
ences, including the data of Huang et al. (2000) and
Cheng et al. (2000).
Recently, Wang et al. (2003a) conducted the first
systematic study of the production of hydrogen from
wastewater sludge, and found a rather high hydrogen
yield from wastewater sludge using a clostridium
strain isolated from the sludge sample. Later, Wang
et al. (2003b) claimed that applying a filtrate to the
sludge could produce more hydrogen than could be
obtained using all of the particles in the sludge. Al-
though these studies successfully established the fea-
sibility of producing hydrogen from wastewater
sludge, the hydrogen formed during the first 16-24 h
of fermentation was consumed in a later stage. Wang
et al. (2003a) blocked the methanogenic pathway
using a pre-treatment. However, much of the pro-
duced hydrogen was still consumed. The pathway for
hydrogen consumption remains unknown, but is of
academic and practical interest.
This study evaluated the feasibility of sequen-
[1]

[2]

[3]

[4]
, To whom all correspondence should be addressed
[5]

684 J. Chin. Inst. Chem. Engrs., Vol. 34, No. 6, 2003

tially producing hydrogen and methane from waste-
water sludge using a clostridium strain under anaero-
bic conditions. Based on test data, a combined proc-
ess, involving two fermenters, is proposed.
Additionally, the possible incorporation of nitrogen
cycles, which compete with the production of hy-
drogen during sludge fermentation, is demonstrated.
MATERIALS AND METHOD
The substrate
Waste activated sludge was extracted from a
wastewater treatment plant of the Presidental Enter-
prise Corp., Taiwan, which daily treats 250 tons of
food-processing wastewater using primary, secon-
dary and tertiary treatments. The pH of the sludge
was about 6.4. The chemical oxygen demand (COD)
for the sludge was 9,600 mg/L (TCOD), as deter-
mined by directly reading a spectrometer (DR/2000,
HACH, U.S.A.). The COD for the filtrate of the
sludge sample after it was filtered through a 0.45 m
membrane was called the soluble COD (SCOD), and
was 465 mg/L for the original sludge. The elemental
composition of the dried samples was C: 34.3%, H:
5.6%, and N: 5.5%, according to an elemental ana-
lyzer (Perkin-Elmer 2400 CHN).
Three pretreatments were applied to the original
sludge to determine their effects on the yield of hy-
drogen. These pretreatments not only released in-
soluble organic matter into water to enhance methane
production (Lee and Mueller, 2001), but also deacti-
vated the methanogenic bacteria in the sludge to
block the pathway of the conversion of hydrogen to
methane. These pretreatments are summarized as
follows.
(1) Acidification: perchloric acid (HClO
4
) was
mixed with the sludge sample for 10 mins to ad-
just the pH of the suspension to 3. Then, the
sample was stored at 4C for 6 h (Jean et al.,
2000).
(2) Sterilization: sludge samples were pasteurized at
121C and 1.2 kgf/cm
2
(HUXLEY AUTOCLAVE,
HL-360) for 30 min.
(3) Freezing and thawing: the sludge was frozen at
17C for 24 h in a freezer and then thawed for
another 12 h in a water bath at 25C (Hung et al.,
1997).
The inoculum
Wang et al. (2003a) isolated the inoculum. The
strain was selected and identified as Clostridium
bifermentans using the polymerase chain reaction
(PCR) and 16S DNA sequence analysis. In some
methane production tests, anaerobe K8 was added to
the samples after bio-hydrogen tests were performed.
K8 was collected from the bottom sediment at a
known site of the Tam-Shui River (near Taipei). This
mixed culture had high methane productivity (Chang
et al., 1996).
Fermentation and tests
Batch fermentation tests were performed in 125
mL serum bottles. In each bottle, 45 mL of substrate,
original or pre-treated, was mixed with 5 mL of seed
bacteria suspension and anaerobically incubated at
35C without stirring or adding any nutrients. The
bottles were capped with butyl rubber stoppers and
wrapped in aluminum foil to prevent photolysis of
the substrate. Gas and liquor samples were collected
at 8, 16, 24, 32, 40, 48, 72, and 96 h of fermentation.
At each time interval, and for each substrate, the gas
compositions of three serum bottles were measured
and their average was reported. After the measure-
ments were made, these samples were abandoned to
prevent the introduction of any possible error associ-
ated with the sampling procedure, such as gas leak-
age.
Tests to determine potential methane production
were performed after 96 h of hydrogen fermentation.
The anaerobe K8 was added to some serum bottles
after the hydrogen fermentation tests were completed.
The gas samples were collected at 24 h intervals up
to 240 h.
A GC-TCD (Shimadzu, GC-8A), equipped with
a stainless column packed with Porapack Q (50/80
mesh) at 70C and a thermal conductivity detector
(TCD), was used to measure the hydrogen and
methane concentrations in the gas phase. The tem-
perature of both the injector and the detector of the
GC was 100C. Nitrogen served as the carrying gas
with a flow rate of 20 mL/min. An integrator
(HP3396 Series II) was used to integrate the peak
area of the effluent curve, and to measure the gase-
ous concentrations. Repeated measurements revealed
that the hydrogen and methane contents thus deter-
mined included maximum relative errors of 15% and
10%, respectively. The hydrogen content in the an-
aerobic glove box was also measured, and was sub-
tracted from the hydrogen concentrations read in the
serum bottles.
The concentrations of ammonia-nitrogen (NH
3
-N)
in the supernatant were measured during the fermen-
tation test. The filtrate samples were extracted by
centrifugating the sludge at 13,500 rpm for 1 min.
The NH
3
-N concentration of the filtrate was meas-
ured spectrophotometrically (425 nm) using a mix-
ture of the filtrate sample with two drops of mineral
stabilizer, three drops of polyvinyl alcohol dispersing
agent, and 1 mL of Nessler agent in a spectropho-
Chi-Chung Wang, Chih-Wen Chang, Ching-Ping Chu, Duu-Jong Lee, and Bea-Van Chang : Sequential Production of 685
Hydrogen and Methane from Wastewawter Sludge Using Anaerobic Fermentation

tometer (DR/2000, HACH, U.S.A.). Standard solu-
tions of NH
3
-N were used for calibration.
RESULTS AND DISCUSSION
Hydrogen production
Figure 1 shows the hydrogen yield for one gram
of dried solid (DS). In contrast to the 66 h time lag
reported by Cheng et al. (2000), this study found
negligible time lag for hydrogen production, perhaps
because the inoculum was directly derived from the
substrate sludge. The hydrogen concentration in the
gas phase yielded an increasing-decreasing curve,
with a peak at about 16-24 h (indicated in Fig. 1 by
the arrow). A specific quantity of produced hydrogen
was, thus, consumed. As Fig. 1 reveals, the spe-
cific hydrogen yield reached 5 g/kg-DS for the
original sludge, much higher than that reported by
Huang et al. (2000) (0.16 g-H
2
/kg-DS). Meanwhile,
sterilization increased the specific hydrogen yield to
about 21.5 g-H
2
/kg-DS. The acidification, freezing
and thawing treatments enhanced the yield to about
9-10 g/kg-DS. This observation shows that the yield
of hydrogen from wastewater sludge is similar to that
from glucose solution if the former is subjected to
suitable pretreatment.
Figure 2 plots the variation in the pH of the solu-
tion during fermentation. Clearly, during hydrogen
production, the solutions pH increased with time
until the yield peaked at 16-24 h, after which time the
pH dropped or did not increase further in the case of
the acidified sludge. The formation of hydrogen
produced a by-product acidic in nature.

Fig. 1. Time course of the hydrogen yield in the gas
phase. Clostridium strain was added at hour 0,
and K8 was added at hour 96. Each data point is
the average of triplicate tests.
Figure 3 plots the time evolution of the concen-
tration of ammonium nitrogen (NH
3
-N) in the sus-
pension. In the case of the original, sterilized and
freeze/thawed sludges, the NH
3
-N concentration in-
creases with the amount of hydrogen produced until
it reached a peak at about 24 h, after which time it
leveled off in the hydrogen consumption phase. The
nitrogen-containing compounds were reduced to
ammonium nitrogen when hydrogen was formed.
The acidified sludge, on the other hand, produced the
least NH
3
-N of all the tests. Hence, although the ni-
trogen cycles competed with hydrogen formation,
they are not preferred in acidic environment.

Fig. 2. Time course of the pH of the suspension.

Fig. 3. Time course of the ammonium nitrogen con-
centration during hydrogen and methane pro-
ducing phases.
Methane production
In the hydrogen fermentation tests, the amount of
methane produced was negligible (0-96 h as shown
in Fig. 4). Hence, the consumption of hydrogen
shown in Fig. 1 was not associated with methane
(
g
/
k
g
-
D
S
)

Time (h)

N
H
3
-
N

(
m
g
/
L
)

200
686 J. Chin. Inst. Chem. Engrs., Vol. 34, No. 6, 2003



Fig. 4. Time course of the methane yield. The anaerobe
K8 was added at hour 96.
production. Since only a small proportion of organic
matter was converted into hydrogen, the potential for
using the exhausted liquor to produce methane was
of interest. Anaerobe K8 was added to the serum bot-
tles after 96 h of hydrogen fermentation.
After K8 was added, as Fig. 4 shows, the amount
of methane accumulated in the serum bottles in-
creased monotonically with time. The amount of
methane produced followed the order freeze/thawed
> original > sterilization >> acidification. Although
the acidification produced hydrogen at a yield two
times that of the original sludge, it produced less
methane. After K8 was added, the solutions pH in-
creased again, corresponding to the formation of
methane. The acidified sludge, however, also did not
undergo an efficient increase in pH, and so generated
least methane.
Sequential process
The data shown in Fig. 4 reveal that the sludge
after hydrogen fermentation was more readily di-
gestable than the unfermented sample. This observa-
tion implies that some products of the fermentation
test promoted methane production. As Wang et al.
(2003b) concluded, fermenting the filtrate of the
sludge alone could produce more hydrogen than fer-
menting all of the sludge (including solids). Hence,
Fig. 5 shows a proposed combined process that
yields hydrogen and methane sequentially. Restated,
the filtrate is first separated from the raw sludge
(with or without pretreatment) and is sent to fer-
menter #1 to produce hydrogen. Then, the waste liq-
uor is mixed with the cake from the solid-liquid
separator and is sent to fermenter #2 to produce
methane. This sequential arrangement effectively
produces hydrogen and methane from wastewater
sludge.
Raw
sludge
Solid-Liquid
Separation
Fermenter
H
2
filtrate
Pretreatment
Fermenter
CH
4
Mixer
Liquor
Cake
Waste
Liquor

Fig. 5. A proposed sequential process that produces
hydrogen and methane from wastewater sludge.
CONCLUSION
The clostridium strain used herein could ferment
wastewater sludge into hydrogen at a much higher
rate than heretofore reported in the literature. Acidi-
fication, sterilization and freezing and thawing could
increase the hydrogen yield. After hydrogen fermen-
tation, adding a methanogenic culture to the fer-
mented liquor accelerated the production of methane,
such that the rate of production was higher than that
of unfermented samples. A combined process that
can produce hydrogen and methane sequentially has,
thus, been proposed. The reduction in the amount of
nitrogen-containing matter can reduce the hydrogen
yield, the effect of which is less profound in an
acidic environment.
ACKNOWLEDGEMENT
Support for this work by the National Science
Council, R.O.C., is gratefully appreciated.
REFERENCES
Bai, M. D., S. S. Cheng, and T. C. Tseng, Biohydrogen Pro-
duced due to Peptone Degradation by Pretreated Seed
Sludge, The IWA Asia-Pacific Regional Conference (Wa-
terQual 2001), Fukuoka, Japan (2001).
Bolliger, R., H. Zrrer, and R. Bachofen, Photoproduction of
Molecular Hydrogen from Wastewater of a Sugar Refinery
by Photosynthetic Bacteria, Appl. Microbiol. Biotechnol.,
23, 147 (1985).
Chang, B. V., J. X. Zheng, and S. Y. Yuan, Effects of Alter-
native Electron Donors, Acceptors and Inhibitors on PCP
Dechlorination in Soil, Chemosphere, 33, 313 (1996).
Cheng, S. S., M. D. Bai, S. M. Chang, K. L. Wu, and W. C.
Chen, Studies on the Feasibility of Hydrogen Production
Hydrolyzed Sludge by Anaerobic Microorganisms, The
25
th
Wastewater Tech. Conference, Yunlin, Taiwan (in
Chinese) (2000).
Chi-Chung Wang, Chih-Wen Chang, Ching-Ping Chu, Duu-Jong Lee, and Bea-Van Chang : Sequential Production of 687
Hydrogen and Methane from Wastewawter Sludge Using Anaerobic Fermentation

Huang, C. S., C. Y. Lin, Y. Y. Tsai, and Y. G. Shieh, Pre-
liminary Study of Anaerobic Hydrogen Production Using
Various Substrates and Incubation Methods, The 25
th

Wastewater Tech. Conference, Yunlin, Taiwan (in Chinese)
(2000).
Hung, W. T., W. H. Feng, I. H. Tsai, D. J. Lee, and S. G.
Hong, Unidirectional Freezing of Waste Activated
Sludge: Radial Freezing Versus Vertical Freezing, Water
Res., 31, 2219 (1997).
Jean, D. S., B. V. Chang, G. S. Liao, G. W. Tsou, and D. J.
Lee, Reduction of Microbial Density Level in Sewage
Sludge through pH Adjustment and Ultrasonic Treat-
ment, Water Sci. Technol., 42(9), 97 (2000).
Kataoka, N., A. Miya, and K. Kiriyama, Studies on Hydro-
gen Production by Continuous Culture System of Hydro-
gen-Producing Anaerobic Bacteria, Water Sci. Technol.,
36(6-7), 41 (1997).
Lay, J. J., Y. J. Lee, and T. Noike, Feasibility of Biological
Hydrogen Production from Organic Fraction of Municipal
Solid Waste, Water Res., 33, 2579 (1999).
Lay, J. J., Modeling and Optimization of Anaerobic Digested
Sludge Converting Starch to Hydrogen, Biotechnol. Bio-
eng., 68, 269 (2000).
Lay, J. J., Biohydrogen Generation by Mesophilic Anaerobic
Fermentation of Microcrystalline Cellulose, Biotechnol.
Bioeng., 74, 280 (2001).
Lee, D. J. and J. A. Mueller, Preliminary Treatments,
Sludge into Biosolids, L. Spinosa and P. A. Vesilind, Ed.,
International Water Association, U.K. (2001).
Liang, T. M., K. L. Wu, and S. S. Cheng, Hydrogen Produc-
tion of Chloroform Inhibited Granular Sludge, The IWA
Asia-Pacific Regional Conference (WaterQual 2001), Fu-
kuoka, Japan (2001).
Lin, C. Y. and R. C. Chang, Hydrogen Production during the
Anaerobic Acidogenic Conversion of Glucose, J. Chem.
Technol. Biotechnol., 74, 498 (1999).
Liu, S. J., W. F. Yang, and P. Q. Zhou, The Research on
Hydrogen Production from the Treatment of Bean Prod-
ucts Wastewater by Immobilized Photosynthetic Bacte-
ria, Environ. Sci., 16, 42 (1995).
Mizuno, O., R. Dinsdale, F. R. Hawkes, D. L. Hawkes, and T.
Noike, Enhancement of Hydrogen Production from Glu-
cose by Nitrogen Gas Sparging, Bioresource Technol., 73,
59 (2000a).
Mizuno, O., T. Ohara, M. Shinya, and T. Noike, Characteris-
tics of Hydrogen Production from Bean Curd Manufactur-
ing Waste by Anaerobic Microflora, Water Sci. Technol.,
42(3-4), 345 (2000b).
Sparling, R., D. Risbey, and H. M. Poggi-Varaldo, Hydrogen
Production from Inhibited Anaerobic Composters, Int. J.
Hydrogen Energy, 22, 563 (1997).
Tanisho, S. and Y. Ishiwata, Continuous Hydrogen Produc-
tion from Molasses by the Bacterium Enterobacter Aero-
genes, Int. J. Hydrogen Energy, 19, 807 (1994).
Tanisho, S. and Y. Ishiwata, Continuous Hydrogen Produc-
tion from Molasses by Fermentation Using Urethane Foam
as a Support of Flocks, Int. J. Hydrogen Energy, 20, 541
(1995).
Tanisho, S., M. Kuromoto, and N. Kadokura, Effect of CO
2

Removal on Hydrogen Production by Fermentation, Int. J.
Hydrogen Energy, 23, 559 (1998).
Ueno, Y., S. Otauka, and M. Morimoto, Hydrogen Produc-
tion from Industrial Wastewater by Anaerobic Microflora
in Chemostat Culture, J. Fermentation Bioeng., 82, 194
(1996).
Wang, C. C., C. W. Chang, C. P. Chu, D. J. Lee, B. V. Chang,
and C. S. Liao, Producing Hydrogen from Wastewater
Sludge by Clostridium Bifermentans, J. Biotechnol., 102,
83 (2003a).
Wang, C. C., C. W. Chang, C. P. Chu, D. J. Lee, B. V. Chang,
C. S. Liao, and J. H. Tay, Using Filtrate to Produce
Bio-Hydrogen by Anaerobic Fermentation, Water Res.,
37, 2789 (2003b).
Zhu, H., T. Suzuki, A. A. Tsygankov, Y. Asada, and J. Mi-
yake, Hydrogen Production from Tofu Wastewater by
Rhodobacter sphaerodies Immobilized in Agar Gels, Int.
J. Hydrogen Energy, 24, 305 (1999).

(Manuscript received Dec. 17, 2002, and accepted Jul. 4, 2003)
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Wang et al. (2003a)