International Journal of Hydrogen Energy 27 (2002) 1399 – 1405

www.elsevier.com/locate/ijhydene

Hydrogen production of Enterobacter aerogenes altered by

extracellular and intracellular redox states
Y. Nakashimada, M.A. Rachman, T. Kakizono, N. Nishio∗
Department of Molecular Biotechnology, Graduate School of Advanced Sciences of Matter, Hiroshima University, Kagamiyama 1-3-1,
Higashi-Hiroshima 739-8530, Japan

Received 21 March 2002; received in revised form 21 May 2002; accepted 15 June 2002

Abstract
Enterobacter aerogenes HU-101, tested for its hydrogen production in batch cultures on various substrates, produced the
highest amount of hydrogen when the substrate was glycerol. The yield of hydrogen is a function of the degree to which
the substrates are reduced. To examine the e6ect of intracellular redox state on hydrogen yield, glucose-limiting chemostat
cultures were carried out at various pH using strain HU-101 and its mutant AY-2. For both strains, the molar yield and
the production rate of hydrogen and the hydrogenase activity in the cell-free extract were optimal at the culture pH of 6.3.
The highest NADH/NAD ratio in both strains was also observed at pH 6.3, at which the ratio in AY-2 was more than
two-fold that of HU-101. Furthermore, NAD(P)H-dependent hydrogen formation was observed in the cell-free extract of
AY-2, and hydrogenase activity was found not in the cytoplasmic but in the cell membrane fraction, suggesting that a high
intracellular redox state, that is a high NADH/NAD ratio, would accelerate hydrogen production by driving membrane-bound
NAD(P)H-dependent hydrogenase.
? 2002 International Association for Hydrogen Energy. Published by Elsevier Science Ltd. All rights reserved.

Keywords: Enterobacter aerogenes; Hydrogen production; Redox state; Culture pH; Hydrogenase

1. Introduction regulated such that either oxidized or reduced products are

Hydrogen gas (H2 ), which is expected to become a clean
alternative energy, can be generated by either photosynthetic
microorganisms [1,2] or fermentative anaerobes [3]. Of the
latter, Clostridium species have received wide attention for
the production of either solvents (butanol and acetone) or
acids (butyrate and acetate) as well as for their production
of H2 [4]. We have been interested in H2 production by
Enterobacter aerogenes [5,6] because E. aerogenes, unlike
clostridia, exhibits uninhibited growth in an atmosphere of
100% H2 . The H2 evolution rate of E. aerogenes was also
higher than those of photosynthetic microorganisms [7]. E.
aerogenes carries out mixed fermentation of glucose to form
both oxidized products (organic acids) and reduced prod-
ucts (alcohols). In contrast, the metabolism of clostridia is
∗ Corresponding author. Tel.: +81-824-247760; fax: +81-824-
247046.
E-mail address: nnishio@hiroshima-u.ac.jp (N. Nishio).
formed.
One of the important factors in determining the diver-
sity of fermentation products is the intracellular redox state.
Nicotinamide adenine dinucleotide coenzymes (NADH and
NADPH) play an important role in this state. Their actions
in numerous anabolic and catabolic reactions range widely
throughout the biological system [8]. It is noteworthy that
the NADH/NAD ratio decreased signiGcantly when cells of
Klebsiella aerogenes, Escherichia coli, and Staphylococcus
albus were switched from anaerobic to aerobic conditions
[9]. Regarding the H2 production in clostridia under acido-
genic conditions, NADH is reoxidized via its production of
H2 : ferredoxin oxidoreductase and hydrogenase [10,11]. The
activity of the former enzyme is regulated by the ratios of
NADH/NAD and acetyl-CoA/CoA, and all excess reducing
powers are disposed as H2 [12]. On the other hand, in facul-
tative anaerobes such as the genera Enterobacter, Klebsiella
and Bacillus, NADH is usually used as the reductant for
the production of 2,3-butanediol, ethanol and lactate from

0360-3199/02/$ 22.00 ? 2002 International Association for Hydrogen Energy. Published by Elsevier Science Ltd. All rights reserved.
PII: S 0 3 6 0 - 3 1 9 9 ( 0 2 ) 0 0 1 2 8 - 3
1400 Y. Nakashimada et al. / International Journal of Hydrogen Energy 27 (2002) 1399 – 1405
pyruvate, but it is not used for H2 production. In these bacte-
ria, H2 was produced by hydrogenase from formate, which is
generated by splitting pyruvate via pyruvate–formate lyase.
The intracellular redox state can be a6ected by various
environmental factors such as substrates [13], culture pH
[10], electron mediators [14], and the nature of the electron
acceptor [15]. To optimize H2 production by E. aerogenes,
it is important to know systematically the cellular responses
to these environmental conditions. In this study, the e6ects
of carbon sources possessing various redox levels on H2
production by E. aerogenes HU-101 were examined and,
subsequently, so were the e6ects of di6erent culture pH. The
intracellular redox state was analyzed using E. aerogenes
HU-101 and its higher H2 -yielding mutant, AY-2 [5] on
glucose medium.
2. Materials and methods
2.1. Microorganisms and culture conditions
The microorganisms used were the HU-101 strain
of E. aerogenes, isolated in our laboratory, and its
double-resistance mutant AY-2, which is resistant to both
allyl alcohol and to the proton suicide method [5]. Cultures
were maintained at −80◦ C with 15% glycerol.
In preculture and batch experiments, a complex medium
[5] was used for the modiGcation of carbon sources. For
continuous culture, the minimal medium of Koesnandar et
al. [16] was modiGed by using two-fold concentrations of
salts and ammonium sulfate. A modiGed Hungate technique
in combination with serum bottles (ca. 125 ml, containing
50 ml culture medium) was used for both precultures and
batch cultures [17]. The precultures were each inoculated
with a portion of the storage culture into medium contain-
ing 10 g=l glucose. Batch cultures were inoculated with 2%
(v/v) preculture broth containing the corresponding carbon
source of either glucose, fructose, xylose, mannitol, sorbitol,
glycerol, or potassium gluconate.
Chemostat cultures were inoculated with 5.0% of precul-
ture broth into a 1-l fermenter containing 500 ml minimal
medium with 0:1 ml A-nol (Able Co., Tokyo, Japan) as
antifoam. The culture was stirred continuously with a mag-
netic stirrer and the temperature was kept at 37◦ C. Contin-
uous cultivation was started by feeding sterilized medium
at a dilution rate of 0:2 h−1 with a peristaltic pump. These
experiments were performed at various culture pH values,
which were monitored by a pH electrode and maintained by
adding either 9:0 N of KOH or 3:0 N of HCl.
2.2. Analysis
The cell growth was followed by measuring optical den-
sity (OD) at 680 nm; 1 OD was estimated as 0:74 g dry cell
mass/l. Ethanol and 2,3-butanediol were analyzed by gas
chromatography (GC 14 A, Shimadzu, Kyoto, Japan) with
a Lame ionization detector. Gas production was measured
periodically by the displacement of saturated aqueous NaCl
in a graduate cylinder. The compositions of CO2 and H2
were determined by way of gas chromatography (GC 8A,
Shimadzu) with a thermal conductivity detector [18]. Lac-
tate, acetate, formate, pyruvate and acetoin were measured
by HPLC [19]. D-glucose was determined by a glucose an-
alyzer (Flow injection analyzer equipped with glucose oxi-
dase, Bio Flow-3, New Oji Paper, Tokyo, Japan).
2.3. Preparation of cell-free extracts
Cells grown on 15 g=l of glucose-feeding medium were
harvested by centrifugation at 12; 000 × g for 10 min at
4◦ C. The harvested cells were washed twice with 100 mM
phosphate bu6er (pH 7.0). The washed cells were resus-
pended in the same bu6er as used for hydrogenase assay or
in 0:1 M MOPS (pH 7.0) bu6er containing 20 mM MgSO4 ,
0:01 mM EDTA, and 0:2 mM DTT for analyzing the activ-
ities of other enzymes. Cells were sonicated (8 min; 45 s
bursts with 1 min cooling on ice). The supernatant was col-
lected from the cell lysate by centrifugation at 12; 000 × g
for 10 min at 4◦ C to remove unbroken cells and cell debris.
At each step, the extracts were maintained under anaerobic
conditions, and the cell-free extract obtained was used as
the enzyme source without further puriGcation. Cytoplasmic
and membrane fractions were obtained from the cell-free
extract by an ultracentrifuge (OptimaTM series TL 100 ultra-
centrifuge, Beckman Instruments Inc., Palo Alto, CA, USA)
at 100; 000 rpm for 1 h at 4◦ C.
2.4. Enzyme assays
The decrease of absorbance was monitored by a spec-
trophotometer (UV 1600, Shimadzu) with a temperature-
controlled water jacket for measuring enzyme activities. All
procedures were performed anaerobically.
Hydrogenase (EC 1.18.3.1) activity was measured as the
decrease in absorbance of dithionite-reduced methyl violo-
gen (MV) (604 = 13:9 mM−1 cm−1 ) in a rubber-stoppered
cuvette (ca. 4 ml) at 25◦ C containing 0:1 M HEPES bu6er
(pH 8.0), 1:5 mM reduced MV, and cell-free extracts [20].
Lactate dehydrogenase (EC 1.1.1.27, LDH) activity was
assayed by the rate of NADH oxidation at 30◦ C (340 =
6:3 mM−1 cm−1 ). The reaction mixture contained 0:1 M
imidazole-HCl, pH 6.7 (1:0 ml), 10 mM NADH (1:0 ml),
0:25 mM sodium pyruvate (0:8 ml) and cell-free extracts
(0:2 ml) [21]. Alcohol dehydrogenase (EC 1.1.1.1, ADH)
activity was measured by the rate of NAD reduction at
25◦ C (340 nm). Cell-free extracts (0:2 ml) were Grst added
with 0:1 M Tris-HCl, pH 8.5 (1:0 ml) and 0:5 mM NAD
(0:8 ml), and then mixed with 0:5 mM ethanol (1:0 ml)
[22]. 2,3-Butanediol dehydrogenase (1.1.1.76, BDDH) ac-
tivity was analyzed similar to the ADH assay except that the
substrate was replaced by 0:5 mM 2,3-butanediol.
 Y. Nakashimada et al. / International Journal of Hydrogen Energy 27 (2002) 1399 – 1405 1401

The protein concentration in each cell-free extract was
determined by a bicinchoninic acid protein assay reagent
kit at 562 nm and 37◦ C (Pierce Biotechnology, Rockford,
IL, USA) [23]. The enzyme activity in each extract was ex-
pressed as the amount of enzyme catalyzing the conversion
of 1 mol substrate min−1 . All assays were carried out in
triplicate with appropriate controls. The replicates di6ered
by less than 10%.
2.5. Assay for NADH, NAD and NAD(P)H-dependent
H2 evolution
NADH and NAD were assayed by Luorescence spec-
trophotometry (Model FP 770, Jasco, Tokyo, Japan) with
excitation and emission wavelengths of 350 and 550 nm,
respectively, and with slit widths of 10 nm. NADH was as-
sayed with 1:5 mmol of pyruvate in 1:0 ml of 50 mM tri-
ethanolamine hydrochloride bu6er (pH 7.4) containing the
extract. The reaction was started by adding 2:0 g of LDH.
NAD was assayed with 0.5% of ethanol in 1:0 ml of 50 mM
triethanolamine hydrochloride bu6er (pH 7.4) containing
the extract. The reaction was started by adding 12 g of
ADH [24]. All enzymes for the assays were supplied by
Boehringer (Mannheim, Germany).
NAD(P)H-dependent H2 evolution was assayed in small
vials (ca. 5 ml) using 250 mM Tris-HCl bu6er (pH 6.3)
and cell-free extract with 0.1–4 mg protein. The assays were
started by injecting an anaerobic NAD(P)H solution and
incubated at 37◦ C [25]. H2 was measured periodically by
gas chromatography (GC 8A, Shimadzu) with a thermal
conductivity detector [18].
3. Results and discussion
3.1. E:ect of redox state of substrates on H2 yield
E. aerogenes HU-101 produced H2 with various carbon
sources (Table 1). On the basis of weight, the H2 yields
on fructose and galactose were similar to that on glucose.
However, the H2 yields on mannitol and sorbitol were
2.5-fold higher than that on glucose, but the H2 yield on
glycerol was higher still: 3.3-fold that of glucose. On the
other hand, the H2 yield on potassium gluconate was much
lower than that on glucose. The e6ect of carbon sources on
ethanol yield was similar to their e6ect on H2 yield, while
their e6ect on acetate yield ran counter to this trend. The
carbon sources did not a6ect the lactate yield, but butane-
diol and acetoin yields decreased drastically on glycerol.
One of the reasons for the di6erences in H2 productivity
at di6erent carbon sources might be their redox states. To
clarify the relationship between H2 production and carbon
source, the available electrons per carbon for each carbon
source (Cave ) should be deGned as follows:
Cave = (degree of reduction in compound)=
(number of carbons in one mole of compound);
where the degree of reduction in compound was calculated
as C = 4; O = −2; H = 1. Using this equation, for example,
Cave of glucose= 24
6
=4 and Cave of mannitol= 266
=4:33. Since
Cave implies the redox state of carbon in each compound,
it can be used as the parameter by which to compare each
redox state of compounds, even if the number of carbon
atoms were di6erent. In Fig. 1, the relationship between Cave
and product yields per mole carbon was postulated. H2 and
ethanol yields increased linearly with Cave , while the acetate
yield decreased with Cave . This clearly indicated that the
redox state of carbon sources directly a6ected H2 production
by E. aerogenes.
3.2. E:ects of culture pH on enzyme activity, intracellular
redox state, and H2 production in continuous culture
It was considered that the intracellular redox state had to
be high under the conditions where H2 yield was high, even
if the same substrates were used. Tanisho et al. [26] found
that cell growth and H2 production were dependent on
the culture pH in batch cultures of E. aerogenes E.82005,
and that the apparent optimal pH levels for cell-mass

Table 1
Yields of products from various carbon sources by E. aerogenes HU-101

Substrate Cave Yield (mmol/g substrate)

H2 CO2 Ethanol Acetate Butanediol Acetoin Lactate

Gluconate 3.67 1.44 4.85 0.86 2.69 1.59 0.11 1.35

Glucose 4.00 1.97 8.25 2.59 0.81 2.66 0.10 1.99
Fructose 4.00 2.17 8.02 2.73 1.32 2.46 0.15 1.53
Galactose 4.00 1.90 7.87 2.65 1.02 2.61 0.12 1.28
Sorbitol 4.33 4.96 8.29 5.80 0.74 1.27 0.03 1.08
Mannitol 4.33 5.20 7.68 5.30 0.37 1.43 0.07 2.15
Glycerol 4.67 6.69 7.59 7.05 0.17 0.15 0.00 1.95

Culture conditions: substrate, 10 g=l; culture time, 14 h. Experimental values represent the average of duplicate cultures.
1402 Y. Nakashimada et al. / International Journal of Hydrogen Energy 27 (2002) 1399 – 1405

0.25 because the pH dependency of H2 production from glucose

was found in a preliminary experiment with batch cultures
(data not shown). Therefore, for the e6ects of culture pH on
0.20 enzyme activities and redox state, the metabolite production
H2 yield (mol/mol-C)

of a chemostat culture was examined at di6erent culture pH

0.15
0.10
0.05
0
3.5 3.8
Fig. 1. E6ect of Cave on yields of metabolites per mole carbon of
substrates for E. aerogenes HU-101. The way to calculate Cave is
described in Results and Discussion. Symbols: yield of
, H2 ;
,
lactate; , acetate;  2,3- butanediol; and , ethanol.
productivity and H2 evolution rate were 7.0 and 5.8, respec-
tively. It can be suggested that the pH dependency of H2
evolution could be explained by both the intracellular redox
state and related enzyme activities. A similar e6ect was ex-
pected for strain HU-101 and for its double mutant (AY-2),
levels and glucose as the limiting factor.
3.2.1. Growth and product diversity
As shown in Fig. 2, cell growth increased linearly from
pH 5.0 to 7.0 for the strains HU-101 and AY-2, with com-
plete consumption of glucose. As for H2 production, strain
AY-2 exhibited a strong pH dependence on the H2 molar
yield and H2 production rate, with pH 6.3 as the optimum.
In contrast, the culture pH had only a slight e6ect on strain
HU-101. In terms of metabolite formation, pH dependen-
4.1 4.4 4.7 5.0 cies of the metabolism for alcohols and acids were clearly
reproduced in chemostat culture. However, strain AY-2 pro-
Cave
duced smaller amounts of the alcohols and acids than strain
HU-101 at the pH range examined [5].
3.2.2. Enzyme activity
To study whether some of the related enzyme activities
depended on the culture pH, we investigated the activities
of hydrogenase, ADH, BDDH and LDH in pH-controlled
chemostat culture. As shown in Fig. 3, hydrogenase activity
proGles against culture pH were found to be very similar
to that of H2 -yield proGles, especially for strain AY-2. The
hydrogenase activity of strain AY-2 was 1.5-fold higher than
that of HU-101 at culture pH 6.3. However, LDH activity of
H2 production rate Glucose remained

1.3 10
Products (mM) (mol/mol glucose) Cell mass (g/l)

(A) (B)
1.1
(mM)

0.9 5

0.7
0.5 0
1.6 3
30
1.2
H2 yield

20
(mM/h)

0.8
0.4 10

0.0 0
80

60

40

20

0
4.5 5.5 6.5 7.5 4.5 5.5 6.5 7.5
Culture pH

Fig. 2. E6ect of pH control on growth, H2 yield and metabolite production for wild-type HU-101 (A) and double mutant AY-2 (B) of E.
aerogenes in continuous culture. Culture conditions: glucose feeding at 83:3 mM and dilution rate of 0:2 h−1 . Symbols:
, cell mass; , •
glucose remained; 4, yield of H2 , production rate of H2 ;
, lactate; ×, acetate; c, ethanol; and , 2,3-butanediol.
 Y. Nakashimada et al. / International Journal of Hydrogen Energy 27 (2002) 1399 – 1405 1403

500 80
Specific enzyme activity

NADH (µmol/mg protein)

(mU/mg protein)

400
60

300
40
200

100
20

0
150 (A) 0
Specific enzyme activity
(mU/mg protein)

4
100

NADH/NAD ratio
50

0
4.5 5.5 6.5 7.5
1
Culture pH

Fig. 3. E6ect of culture pH on activities of hydrogenase, LDH, (B)

ADH, and BDDH in glucose-limited chemostat culture. Chemostat
cultures with 0:2 h−1 of dilution rate were carried out for wild-type
HU-101 (closed symbol) and mutant AY-2 (open symbol) of E.
aerogenes. Symbols: ; , hydrogenase; 4; , LDH;
; ,
ADH; and ;
; BDDH. • Fig. 4. The e6ect of culture pH on NADH/NAD ratio and
mutant AY-2 was 60 –70% lower than that of HU-101 for
the pH range between 5.0 and 7.3, which may be the result of
mutation by the proton suicide method [5]. In addition, the
ADH and BDDH activities of strain HU-101 were markedly
decreased at higher culture pH levels, which agreed well
with the lower production of alcohols at the neutral pH.
These enzyme activities of strain AY-2 were found to be
negligible above pH 5.5, which could have been derived
from the allyl alcohol resistance [27]. It seems very interest-
ing that the higher H2 -producing mutant AY-2 showed more
pH- dependence for H2 production yield than strain HU-101
did, and the same proGle corresponded well with the hydro-
genase activity proGle against culture pH for strain AY-2.
3.2.3. Intracellular redox state
As can be seen in Fig. 4, the culture pH value could
be used to manipulate the steady-state values of NADH
concentrations and NADH/NAD ratios, and the pH giv-
ing maximum NADH values was consistent with that giv-
ing maximum H2 yield. Moreover, although the amounts of
NADH between the two strains were nearly identical at each
0
5.0 5.3 5.7 6.0 6.3 6.7 7.0 7.3
pH
amount of NADH in cell extract of E. aerogenes HU-101 and
AY-2. Organisms were grown anaerobically on chemostat culture
(D = 0:2 h−1 ; 37◦ C, pH control). Symbols: (A) , NADH for
AY-2,
, NADH for HU-101; and (B) , NADH/NAD ratio for
AY-2,
, NADH/NAD ratio for HU-101.
measured pH, the maximum NADH/NAD ratio of AY-2 was
3.0-fold higher than that of HU-101 at pH 6.3. It is known
that the energy source and pH of the culture were inLuenced
by the NADH/NAD ratio in Enterococcus faecalis NCTC
775 [28], and that the NADH/NAD ratio a6ected the dis-
tribution of carbon Lux through the metabolites’ routes in
anaerobic conditions.
3.3. NADH and NADPH supported H2 formation on
extract of E. aerogenes
Provided that the H2 generation is derived from the pyru-
vate metabolism to ethanol and acetate formations in glu-
cose fermentation, the maximum molar H2 yield should not
exceed the sum of ethanol and acetate produced. As can be
determined from the data shown in Fig. 2, for strain HU-101
at pH 6.3, the H2 yield of 0.80 was nearly the same as the
1404 Y. Nakashimada et al. / International Journal of Hydrogen Energy 27 (2002) 1399 – 1405
1.0
H 2 production (µmol/mg protein)
0.8
0.6
0.4
0.2
0.0
0.0 0.2 0.4 0.6
Amount of reductant (mM)
Fig. 5. NADH- and NADPH-dependent H2 evolution in cell-free
extract from E. aerogenes AY-2. Experiments were carried out in
vials (without pH control (initial pH = 6:3), 37◦ C, anaerobically).
H2 evolution after 48 h reaction was determined by average values
in at least three di6erent experiments. Symbols: , H2 evolution
using NADH as a reductant;
, H2 evolution using NADPH as a
reductant; and
, without reductant as a substrate.
sum (0.72) of ethanol (0.51) and acetate (0.21), while for
strain AY-2, the H2 yield of 1.48 was signiGcantly greater
than the sum (0.45) of ethanol (0.31) and acetate (0.14).
As suggested in a previous study, H2 should be generated
from excess NADH via ferredoxin-NADH oxidoreductase,
resulting in reduced forms of ferredoxin and the possibility
of an NADH pathway for H2 production by thermodynamic
calibration [26]. With the assumption of a high NADH/NAD
ratio and a suPcient inter-membrane pH gradient, H2 could
be generated from NADH as a thermodynamically favor-
able reaction. In our chemostat culture experiment, a culture
medium with high H2 evolution capability at pH 6.3 exhib-
ited extremely low redox potential at −540 mV (data not
shown), which is much lower than those of strict anaerobes
such as methanogens, at −420 mV. Therefore, in vitro en-
zymatic evolution of H2 from NADH was studied.
After a reaction time of 48 h, both NADH and NADPH
supported H2 formation on extract of E. aerogenes (Fig. 5).
NADH was four-fold more active than NADPH at the ap-
plied pH, while no gas was produced in the absence of any
reductant and/or in the absence of E. aerogenes cell extracts
(control). For NADH, the yield of H2 production ranged
from 0.56 to 0:9 mol=mol, while the yield of NADPH to H2
was only 0.13 to 0:19 mol=mol. The highest speciGc activ-
ity on H2 production was 0:019 mol=mg protein h when
0:7 mM NADH was used as a substrate (data not shown).
To learn the localization of NAD(P)H-dependent hydro-
genase, we measured the H2 evolution from NADH in cy-
toplasmic and cell membrane fractions of AY-2 (Table 2).
Table 2
NADH-dependent H2 evolution and hydrogenase measured using
cell-free extract from vial fermentation without pH control at 37◦ C
Localization NADH-dependent H2 Hydrogenase
evolution ( mol=ml) activity (U-MV/ml)
Cytoplasmic 0.03 ND
Cell membrane 0.26 0.17
Cell-free extract 0.35 0.17
Cytoplasmic and membrane fractions were prepared by ultracen-
trifuge (105; 000 × g for 1 h, 4◦ C). Reaction time for H2 evolu-
tion: 48 h. Experimental values represent the average of duplicate
reactions. ND: not detected.
0.8 1.0 The result clearly demonstrated that the hydrogenase was
localized in the cell membrane. It can be speculated that
when the intracellular redox state is high, and thus when
the NADH/NAD ratio is high, a portion of the NADH is
oxidized on the inside surface of the cell membrane, while
protons are reduced on the outside surface by means of a
membrane-bound hydrogenase; this has been reported for
• Klebsiella pneumoniae, a close relative of E. aerogenes [29].
To study NADH-dependent H2 evolution in more detail,
puriGcation of membrane-bound hydrogenase and its char-
acterization will be needed. Such experiments are now in
progress.
Acknowledgements
This work was supported in part by a Grant-in-Aid for Sci-
entiGc Research (No. 13750738) from the Ministry of Edu-
cation, Culture, Sports, Science and Technology of Japan.
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