828 Biotechnol. Prog.

2003, 19, 828−832

Hydrogen Production with Immobilized Sewage Sludge in

Three-Phase Fluidized-Bed Bioreactors
Shu-Yii Wu,† Chi-Num Lin,† and Jo-Shu Chang*,‡
Department of Chemical Engineering, Feng Chia University, Box 25-102, Taichung 407, Taiwan, and
Department of Chemical Engineering, National Cheng Kung University, Tainan 701, Taiwan

Municipal sewage sludge was immobilized with a modified alginate gel entrapment
method, and the immobilized cells were used to produce hydrogen gas in a three-
phase fluidized bed. The hydrogen-producing fluidized beds were operated at different
liquid velocity (U0) and hydraulic retention time (HRT). The results show that in
response to operating liquid velocities, the fluidized-bed system had three flow regimes,
namely, plug flow, slug flow, and free bubbling. Pressure fluctuation analysis was
used to analyze the hydrodynamic properties in this three-phase fluidized bed when
it was under a steady-state production of biogas. With a steady-state biogas production
rate (Ug) of 0.196 mL/s/L, a transition state occurred at a liquid velocity (U0) of 0.85
cm/s. As U0 < 0.85 cm/s, the system was basically a nonhomogeneous fluidized bed,
whereas the bed became homogeneous when U0 was higher than 0.85 cm/s. The
fluidized bed can be stably carried out at high loading rates (HRT as low as 2 h).
Hydrogen fermentation results show that the maximal hydrogen production rate was
0.93 L/h/L and the best yield (YH2/sucrose) was 2.67 mol H2/mol sucrose.

Introduction more complicated mixed-culture systems. There have

Hydrogen has become a promising energy source
because it is clean and has high conversion efficiency (Das
and Veziroglu, 2001). Production of hydrogen gas via
biological means is pollutant-free, needs low energy costs,
and is thereby considered a potential alternative to
conventional physical/chemical methods for H2 produc-
tion. Biohydrogen production can be achieved by algae
and cyanobacteria through photolysis of water and by
anaerobic or photosynthetic bacteria through fermenta-
tive conversion of organic substrates in the presence or
absence of light (Das and Veziroglu, 2001). In general,
hydrogen fermentation with anaerobic bacteria (dark
fermentation) has a higher H2 production rate, but
hydrogen production with photosynthetic bacteria (light
fermentation) possesses a higher theoretical conversion
(Das and Veziroglu, 2001). Hydrogen production by
photolysis of water is normally very slow and cannot be
conducted under the light, since the hydrogenase that
catalyzes electron transfer for H2 formation is severely
inhibited by O2, the major product of photosynthesis
(Miyake, 1998).
For better retention of biomass in bioreactors using a
continuous mode, immobilized-cell systems are frequently
used. Although application of cell immobilization for
wastewater treatment started as early as the 1930s
(Miller et al., 1981), not until the 1960s were immobilized
cells on moving particles such as the fluidized-bed
bioreactor (FBB) utilized. Three-phase fluidized-bed biore-
actors (TPFBB), though used in the food, chemical, and
pharmaceutical industries (Fan, 1989), are relatively less
used in wastewater treatment, which normally involves
* To whom correspondence should be addressed. Fax: +886-6-
2344496. E-mail: changjs@mail.ncku.edu.tw
† Feng Chia University.
‡ National Cheng Kung University.
10.1021/bp0201354 CCC: $25.00 © 2003 American Chemical Society and American Institute of Chemical Engineers
Published on Web 05/14/2003
been examples of successful commercial-scale TPFBB
processes. Nevertheless, most TPFBB applications have
still been limited to bench-scale models in the laboratory
because of the difficulty in the bioreactor design and in
locating optimal operating and control parameters, pri-
marily as a result of complicated biochemical and ecologi-
cal characteristics of the microorganisms. Thus, there is
still a great demand for further research on TPFBB.
Utilizing organic substance in wastewater to produce
hydrogen gas with anaerobic bacteria has high economi-
cal potential and has been the focus of investigation by
many researchers in the past decades (Das and Veziroglu,
2001). In many cases, the conventional CSTR process was
used for hydrogen fermentation, but its hydrogen produc-
tion performance was restricted considerably by low
dilution rates due to the low specific growth rates of
hydrogen producers (Chen et al., 2001). Hence, retention
of hydrogen-producing bacteria in continuous cultures
needs to be enhanced in order to achieve a higher
hydrogen production rate. Cell immobilization techniques
have been applied to improve cell retention and were
shown to be suitable for continuous hydrogen production
(Zhu et al., 1999; Kumar and Das, 2001; Yokoi et al.,
1997). Most hydrogen fermentation associated with im-
mobilized cells was conducted in packed-bed reactors
(Palazzi et al., 2000; Rackman et al., 1998; Yokoi et al.,
1997; Kumar and Das, 2001), which often suffer inef-
ficient mass transfer even though they are relatively
inexpensive and easy to operate. However, for im-
mobilized cells created by entrapment methods, mass
transfer efficiency is often a limiting factor. Generation
of gaseous products from hydrogen fermentation further
raises the importance of mass transfer efficiency for the
immobilized-cell system. As a result, three-phase fluid-
ized beds are preferable compared to the pack-bed
reactors in biohydrogen production. Unfortunately, re-
Biotechnol. Prog., 2003, Vol. 19, No. 3 829

ports regarding the use of fluidized beds for hydrogen

fermentation have been quite rare. This study is a novel
attempt to use three-phase fluidized beds to produce H2
from sucrose with immobilized hydrogen-producing sludge.
The hydrodynamic patterns and biohydrogen production
behavior were investigated to evaluate the feasibility of
using the fluidized beds for practical hydrogen-producing
bioprocesses.

Materials and Methods

Hydrogen-Producing Sludge. The anaerobic seed
sludge was obtained from a local wastewater treatment
plant in central Taiwan. The seed sludge was subject to
acidic pretreatment (the pH was adjusted to ca. 3.0 by
HCl for 24 h and readjusted to 7.0 by NaOH) to enhance
its hydrogen productivity (Chen et al., 2002). The acid-
treated sludge acclimated in the CSTR reactor at a
hydraulic retention time of 8 h, and the sludge in the
effluent was used to prepare immobilized cells.
Cell Immobilization. Detailed procedures of cell
immobilization with an acrylic latex plus silicone (ALSC)
method were described in our recent work (Wu et al.,
2002). In brief, hydrogen-producing sludge of ca. 3 g/L
was mixed with 75% (w/v) of acrylic latex/silicon (DAP,
Inc., Baltimore, MD), supplemented by a small amount
(adjustable) of sodium alginate and activated carbon. The
mixture was dropped into concentrated CaCl2 solution
to form insoluble particles 3.0-4.0 mm in diameter.
Medium Composition. The limiting carbon substrate
used for hydrogen fermentation was sucrose (Sigma). The
medium contained 20 g COD/L of sucrose and sufficient
inorganic and mineral compounds (Chang et al., 2002).
Setup and Operation of Three-Phase Fluidized
Bed. Figure 1 describes the experimental rig of the three-
phase fluidized bed, which was composed of a column 8
cm in diameter and 120 cm in height and a total working
volume of 10 L (including a 4-L buffer tank; device no.
6). The static bed height of the immobilized-cell particles
was 40 cm. The medium (device no. 4) was fed from the
bottom entrance into the bed loaded with immobilized
cells. The effluent of the reactor was introduced to a gas- Figure 1. Schematic description of the three-phase fluidized-
liquid separator (device no. 5), where the gaseous and bed bioreactor.
soluble products were collected separately. The temper-
ature in the bed was controlled at 35 °C throughout the the bioreactor was operated in a batch mode. The
operations. The pH in the reactor was not controlled but temperature was rapidly elevated to 70 °C, which was
was in the range of 5.8-6.8 during the course of experi- maintained for ca. 30 min. After that, the temperature
ments. was cooled naturally to 35 °C, and the reactor was
To determine the hydrodynamic properties in the switched to a continuous mode.
fluidized bed, 10 L of medium was fed into the substrate Analytical Methods. The pressure fluctuation analy-
tank with the adjustment of liquid flow rate (U0) to sis was conducted by the Pasco data acquisition analysis
fluidize the bed materials. When a steady state was system (model CI-7500, Pasco Scientific, CA). The biogas
reached, the hydraulic retention time (HRT) was con- composition was analyzed by gas chromatography (GC-
trolled at 2 h and the U0 was changed from 0.2 to 1.1 14A, Shimadzu, Japan) equipped with a TCD detector;
cm/s. During operation, pressure fluctuation data were analysis of soluble metabolites was also conducted by gas
collected and analyzed to identify the hydrodynamics of chromatography with a FID detector (Chang et al., 2002).
the bed. The quantity of total biogas production was measured
For the hydrogen fermentation experiments, the me- by a gas flow meter (type TG1) purchased from Ritter
dium substrate was continuously fed into the bioreactor Inc. (Germany).
with a shift down of HRT (from 6 to 1 h). The biogas
(primarily consisting of CO2 and H2) production from Results and Discussion
hydrogen fermentation was monitored by a gas flow Physical and Fluidization Properties of ALSC
meter. The compositions of gaseous and soluble products Immobilized Cells. For hydrogen fermentation, the
were also analyzed at designated time intervals. strength and stability of the immobilized cells play an
If the hydrogen-producing activity in the culture important role in the successful operation of the three-
declined during long-term operations, thermal treatment phase fluidized-bed bioreactors. The immobilized cells
was performed to recover the hydrogen production per- (ALSC cells) were developed in our laboratory (Wu et al.,
formance. The procedures of thermal treatment are as 2002). The physical characteristics of the ALSC cells are
follows: the influent and effluent were switched off, and indicated in Table 1. Our recent investigation (Wu et al.,
830 Biotechnol. Prog., 2003, Vol. 19, No. 3

Figure 2. The effect of liquid superficial velocity on the

pressure drop of the bed containing ALSC immobilized cells.
Static bed height ) 40 cm, dp ) 3-4 mm, Fp ) 1.11 g/mL, temp
) 35 °C.

Table 1. Physical and Fluidization Properties of the

ALSC Immobilized Cells
property value
diameter of particle (dp) 3-4 mm
density of particle (Fp) 1.11 g/mL
liquid hold-up (l) 0.4 mL/mL
minimum liquid fluidization velocity (Umfl) 1.06 cm/s
initial biomass loading 0.667 g VSS/L
static bed height 40 cm

2002) showed that the ALSC cells exhibited excellent

stability and durability in hydrogen production during
long-term repeated batch operations. Therefore, the
ALSC cells were selected as the bed material and
hydrogen producer in the three-phase fluidized-bed biore-
actor.
The minimum liquid fluidization velocity (Umfl) of the
ALSC cells (static bed height of 40 cm) was calculated
from the abscissa of the point from which the pressure
drop remained constant. Figure 2 shows the effect of
liquid superficial velocity on pressure drop for ALSC cells
at 35 °C. It can be observed from Figure 2 that the value
of Umfl was ca. 1.06 cm/s.
Characterization of Flow Regimes of the Fluid-
ized Bed. A three-phase fluidized-bed bioreactor typi-
cally has three flow regimes: coalescence plug flow, free
bubbling flow, and slug flow (Fan, 1989). As shown in
Figure 3, when steady-state biogas production rate (Ug)
was 0.196 mL/s/L and the liquid velocity (U0) was 0.43 Figure 3. Different flow regimes in the three-phase fluidized
cm/s, small bubbles formed and filled the space of the bed. (a) Photographs of the flow patterns. (b) Pressure fluctua-
bed; finally, a plug flow pattern was formed in the tion analysis: (1) plug flow, U0 ) 0.43 cm/s, Ug ) 0.196 mL/s/L;
fluidized-bed bioreactor (Figure 3a-1). When U0 was (2) slug flow, U0 ) 0.85 cm/s, Ug ) 0.196 mL/s/L; (3) free
bubbling, U0 ) 1.1 cm/s, Ug ) 0.196 mL/s/L. (c) Typical pressure
increased to 0.85 cm/s, the bed expansion also increased, time series in the fluidized bed (U0 ) 0.85 cm/s, Ug ) 0.196 mL/
the solid concentration changed with axial direction and s/L).
the slug flow pattern occurred as shown in (Figure 3a-
2). When U0 was further increased to 1.06 cm/s, the fluctuation signals (typical signals shown in Figure 3c)
minimum liquid fluidization velocity was reached (Figure appear from different states (Fan, 1989). The pressure
2); the bubbles homogeneously filled the space of the fluctuation signals were transformed into amplitude
reactor, indicating the free bubbling regime in the system versus frequency plots as shown in Figure 3b. There were
(Figure 3a-3). three natural frequencies from the fluidized beds (Figure
The developing state from fixed bed to fluidized bed 3b-1, 3b-2, and 3b-3) located in peaks of 60, 180, and 200
was classified in three phases, namely, fixed bed, non- Hz, which were most likely caused by the liquid pump
homogeneous fluidized bed, and homogeneous bed (Fan, and distributor. When the reactor showed plug flow, no
1989). Figure 3a suggests that a nonhomogeneous state extra peaks appeared, while the small gas bubbles still
took place when Ug ) 0.196 mL/s/L and U0 < 0.85 cm/s, stuck to the surface of the immobilized-cell particles. The
while a homogeneous state occurred as U0 > 0.85 cm/s. buoyant forces of the bubbles from immobilized cells were
At U0 ) 0.85 cm/s, there was a transition state. not enough to escape the adhesion forces between the
A pressure fluctuation analysis was conducted to bubbles and the particles. When the flow pattern showed
further support the observation, as different pressure slug flow, several additional peaks appeared between 60
Biotechnol. Prog., 2003, Vol. 19, No. 3 831

and 250 Hz (Figure 3b-2). This may occur because the

bubbles formed in the liquid phase had smaller bubble
coalescence with each other and the bubble diameter was
larger than one-third of the diameter of the column,
allowing formation of a slugging pattern. When the
system was in free bubbling phase, the bubbles were
homogeneous and scattered. The Fourier spectra of the
pressure signals also showed several extra peaks (Figure
3b-3). The extra fluctuation signals seem to reflect that
damping behavior occurred when the free bubbles burst
into the surface of the immobilized-cell particles. Unlike
the observation for slug flow, no sharp peaks occurred
in pressure fluctuation signals for free bubbling. This
confirms the observation that the phase changed from
plug flow to slug flow and then to free bubbling, as shown
in Figure 3a.
In this work, the hydrodynamics of bioreactors was
closely related to the production of biogas by hydrogen
fermentation. The three phases (solid/liquid/gas) in the
bed were affected by the formation of biogas bubbles and
their rising in the bed, combined with bubble coalescence,
as well as splitting and slipping velocity between the
bubble and liquid phases. The interaction of the above
factors appeared to determine the fluidization regimes
of the bioreactor.
The produced hydrogen gas was liberated from the
interior of the immobilized cell particles through the
micro- and/or mesopores; the hydrogen gas was formed
on the surface of the particles, as shown in Figure 3a-3.
The mechanism of how mass transfer around the im-
mobilized cells affected the hydrogen production process
is quite complicated. Many physical and chemical factors,
such as particle size, pore size, substrate concentration,
etc., are expected to be involved. Most importantly,
accumulation of gas bubbles on the surface of particles
may hinder the transport of substrate into the im- Figure 4. HRT-dependent profiles of biogas production rate,
mobilized cells and needs to be prevented by a suitable soluble metabolite production, and substrate conversion yield
pore size of the particles and by appropriate bioreactor in the three-phase fluidized bed containing ALSC immobilized
design and operation. Further studies are still required cells.
to understand the detailed mechanism.
Table 2. Effect of Hydraulic Retention Time (HRT) on
Dependence of Hydrogen Production on HRT in Performance of Hydrogen Production in Fluidized Beds
the Three-Phase Fluidized Bed. Using sucrose as the
limiting carbon substrate, the HRT-dependent perfor- volumetric hydrogen substrate
mance of hydrogen production of the fluidized-bed biore- medium production conversion H2 content
HRT feeding rate rate yield (mol H2/ in biogas
actor is shown in Figure 4. It shows that the hydrogen (h) (mL/min) (L/h/L) mol sucrose) (%)
production rate increased from 0.15 to 0.51 L/h/L as the
HRT was shifted down from 6 to 2 h. The hydrogen 6 4 0.147 1.27 23.6
4 6 0.235 1.35 24.0
production rate notably increased nearly 2-fold when the 2 12 0.511 1.47 28.1
HRT decreased from 4 to 2 h (Table 2). The result shows 1 24 0.084 0.126 7.10
that the kinetics of hydrogen fermentation in the fluid- 2a 12 0.925 2.67 38.0
ized beds was predominantly controlled by the rate of a After thermal treatment.
substrate loading. For HRT of 2-6 h, the substrate
conversion yield (YH2/sucrose) was nearly constant (1.36 ( crease along with the decrease in H2, it is likely that some
0.07 mol H2/mol sucrose) and the H2 content in the biogas non-hydrogen producers started to dominate the culture
was also fairly stable at 25.2 ( 1.91% (Table 2), indicating at high loading rates (i.e., low HRTs) and converted the
that the immobilized-cell culture consisted of a stable carbon substrate to CO2 without H2 production. This idea
bacterial population, which converted the substrate via was supported by the restoration of hydrogen production
a steady metabolic flux. In all experiments, no methane performance after the culture was subject to a thermal
was detected, and the sucrose concentration in the treatment to inactivate the non-hydrogen-producing popu-
effluent was less than 0.2 g COD/L, indicating a substrate lations (Figure 4). Thus, the result in Figure 4 also
utilization efficiency of over 90%. suggests the existence of an optimal HRT of 2 h for stable
However, when HRT changed from 2 to 1 h, a different and efficient hydrogen fermentation.
HRT dependence of hydrogen production was observed. Enhancement of Hydrogen Production via Ther-
Although the substrate was fed at a faster rate, the mal Treatment. To prove that the decline of hydrogen
hydrogen production rate decreased dramatically (Figure production rate at HRT of 1 h (Figure 4) was due to
4). This may be due to an overload of medium causing a domination of non-hydrogen producers in the culture, a
substrate inhibition effect on hydrogen producers or an thermal treatment was conducted after operation at HRT
improper food to microorganism (F/M) ratio resulting in of 1 h for 24 h. Thermal treatment was found to be
inefficient hydrogen fermentation. As CO2 did not de- effective in acclimation of hydrogen-producing popula-
832
tions from seed sludge (Lin et al., 2000), because hydro-
gen producers in anaerobic cultures are often spore-
forming bacteria, such as Clostridium sp. (Das and
Veziroglu, 2001), which are resistant to elevated tem-
peratures. In this study, the thermal treatment was
carried out by turning the operation to batch mode and
heating the culture to 70 °C for 30 min. The thermal
treatment was expected to inactivate or kill the non-
hydrogen-producing cells (such as methane-forming bac-
teria and non-spore-forming acidogenic bacteria), while
the thermal-resistant hydrogen-producing cells would
survive. As expected, the results show that after thermal
treatment, the hydrogen production rate returns to its
original level of 0.53 L/h/L in a short period and soon
increased to a maximal rate of 0.93 L/h/L (Figure 4).
Meanwhile, the substrate conversion yield and H2 content
also increased significantly to ca. 2.67 mol H2/mol sucrose
and 38% (Table 2), respectively. Thus, the thermal
treatment was apparently very effective in eliminating
the competition of non-hydrogen-producing cells in the
culture for the substrate and thus allowed re-establish-
ment of efficient hydrogen-producing population. Utiliz-
ing fixed-bed bioreactors for hydrogen production, our
recent study (Chang et al., 2002) also observed a similar
effect of quick thermal treatment on the recovery of
hydrogen production performance. Thus, the thermal
treatment strategy may be a solution to maintain stable
hydrogen production during operations at low HRTs.
In comparable hydrogen-producing systems, the opti-
mal HRT varied violently from 1 to 13.5 h, depending on
the reactor configuration and the substrate used (Chang
et al., 2002; Liu and Fang, 2002; Yu et al., 2002). The
maximal hydrogen production rate (0.93 L/h/L) obtained
in our fluidized-bed reactor was about 5-fold higher than
that obtained from an upflow anaerobic reactor (Yu et
al., 2002) and was also much higher than that from a
CSTR system with granular sludge (Liu and Fang, 2002).
However, a fixed-bed system (Chang et al., 2002) appears
to show a slightly better hydrogen generation rate (up
to 1.3 L/h/L) than that observed in this study.
Soluble Metabolites from Hydrogen Fermenta-
tion in Three-Phase Fluidized Beds. Figure 4 shows
that the soluble metabolites of the immobilized cells
during hydrogen fermentation were mainly butyric acid
(HBu), followed by acetic acid and propionic acid. A lesser
amount of ethanol was also observed. The profile of
hydrogen production rate tended to follow the trend of
butyric acid formation (Figure 4), suggesting that the
primary hydrogen producers in the culture were HBu-
producing bacteria. As the acid products dominated the
soluble metabolites and the production of solvents (e.g.,
ethanol) was insignificant, the production of H2 in the
culture was metabolically favorable (Yan et al., 1988).
Conclusions
This study demonstrates a novel approach of using a
fluidized-bed bioreactor to produce hydrogen gas from
sucrose-limiting medium. Containing ALSC immobilized
cells as the bed material, the fluidized beds were able to
stably produce H2 at a HRT of 1-6 h with a maximal
steady-state rate of 0.93 L/h/L and an optimal yield of
2.67 mol H2/mol sucrose. The pressure fluctuation analy-
sis was shown to easily monitor the flow patterns of
fluidized beds and thus may become a powerful tool to
control the stability of the system. The fluidized-bed
bioreactor holds the advantage of being flexible to operate
and easy to scale up. The immobilized cells were stable
mechanically and had a high level of hydrogen-producing
activity. Therefore, the fluidized-bed system used in this
Biotechnol. Prog., 2003, Vol. 19, No. 3
study seems to have the potential to be practically applied
in large-scale biohydrogen production from organic wastes.
Acknowledgment
The authors acknowledge the financial support by
National Science Council of Taiwan, R.O.C (grant NSC-
88-2211-E-035-020) and by Feng Chia University (grant
FCU-89-J040).
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Accepted for publication March 11, 2003.
BP0201354