Bioresource Technology 80 (2001) 227231

Eect of iron concentration on hydrogen fermentation

Young Joon Lee a, Takashi Miyahara b, Tatsuya Noike c,*
a

Water Quality Research Department, National Institute of Environmental Research, Seoul 122-040, Republic of Korea
b
Department of Systems Engineering, Faculty of Engineering, Shizuoka University, Shizuoka 432-8561, Japan
c
Department of Civil Engineering, Graduate School of Engineering, Tohoku University, Sendai 980-8579, Japan
Received 3 October 2000; received in revised form 22 February 2001; accepted 5 March 2001

Abstract
The eect of the iron concentration in the external environment on hydrogen production was studied using sucrose solution and
the mixed microorganisms from a soybean-meal silo. The iron concentration ranged from 0 to 4000 mg FeCl2 l 1 . The temperature
was maintained at 37C. The maximum specic hydrogen production rate was found to be 24:0 ml g 1 VSS h 1 at
4000 mg FeCl2 l 1 . The specic production rate of butyrate increased with increasing iron concentration from 0 to 20 mg FeCl2 l 1 ,
and decreased with increasing iron concentration from 20 to 4000 mg FeCl2 l 1 . The maximum specic production rates of ethanol
682 mg g 1 VSS h 1 and butanol 47:0 mg g 1 VSS h 1 were obtained at iron concentrations of 5 and 3 mg FeCl2 l 1 , respectively. The maximum hydrogen production yield of 131:9 ml g 1 sucrose was obtained at the iron concentration of
800 mg FeCl2 l 1 . The maximum yields of acetate 389:3 mg g 1 sucrose), propionate 37:8 mg g 1 sucrose), and butyrate (196.5
mg g 1 sucros) were obtained at iron concentrations of 3, 200 and 200 mg FeCl2 l 1 , respectively. The sucrose degradation eciencies were close to 1.0 when iron concentrations were between 200 and 800 mg FeCl2 l 1 . The maximum biomass production
yield was 0:283 g VSS g 1 sucrose at an iron concentration of 3000 mg FeCl2 l 1 . 2001 Elsevier Science Ltd. All rights reserved.
Keywords: Hydrogen fermentation; Hydrogen production; Iron; Solvents; Sucrose; Volatile fatty acids

1. Introduction
Methane and hydrogen are important gaseous fuels
produced by the anaerobic fermentation of organic
waste (Brosseau et al., 1982). Within the environment of
a microbial food chain, organic fractions of a waste are
degraded by several groups of anaerobic bacteria.
During the rst step of the food chain, organic polymers
are hydrolyzed by extracellular enzymes of hydrolytic
bacteria to carbohydrates, amino acids, long chain fatty
acids, and other low molecular weight compounds.
These products are fermented by acidogenic bacteria
leading to volatile fatty acids (VFAs), and hydrogen.
VFAs and hydrogen are further converted by syntrophic
acetogenic bacteria and methanogenic bacteria to
methane.
Hydrogen gas produced by hydrogen-producing
bacteria (Holmes and Freischel, 1978; Taguchi et al.,
1996) is converted into methane or hydrogen sulde by
hydrogen-consuming bacteria, such as methanogenic
bacteria, and sulfate-reducing bacteria in the anaerobic

Corresponding author.

environment. Both from an economic and environmental standpoint, hydrogen is more attractive than
methane as an energy source for replacing conventional
fossil fuels (Roychowdhury et al., 1988; Taguchi et al.,
1994; Ueno et al., 1996; Sparling et al., 1997) and eorts
have been directed toward production of hydrogen
rather than methane (Taguchi et al., 1992, 1996; Ueno
et al., 1995; Lay et al., 1999).
Kim and Zeikus (1985) reported that the metabolism
shift of the culture, Clostridium acetobutylicum, from
acidogenesis to solventogenesis was accompanied by
decreases in the specic hydrogen production rate and
the specic activity of in vivo hydrogenase. C. acetobutylicum breaks down hexose to acetyl-CoA to produce 2
mol of NADH and 2 mol of reduced ferredoxin.
NADHferredoxin reductase oxidizes NADH to NAD
(Kim and Zeikus, 1985). For the saccharolytic Clostridia, hydrogen evolution via a hydrogenase is a major
route through which the cells dispose excess electrons
produced from the oxidative breakdown of carbohydrates (Chen and Mortenson, 1974). Hydrogenase
(E.C.1.12.7.1) present in anaerobic bacteria oxidizes
reduced ferredoxin to produce molecular hydrogen
(Adams et al., 1980). Ferredoxin from C. pasteurianum

0960-8524/01/$ - see front matter 2001 Elsevier Science Ltd. All rights reserved.
PII: S 0 9 6 0 - 8 5 2 4 ( 0 1 ) 0 0 0 6 7 - 0

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Y.J. Lee et al. / Bioresource Technology 80 (2001) 227231

is an ironsulfur protein with a molecular weight 6000

containing 8 non-heme iron and 8 acid-labile sulfur atoms. This protein functions primarily as an electron
carrier and is involved in pyruvate oxidation to acetylCoA and carbon dioxide as well as proton reduction to
molecular hydrogen (Schoenheit et al., 1979). Chen and
Mortenson (1974) have demonstrated that the hydrogenase (E.C.1.12.7.1), which has a molecular weight of
60,500, contains about 12 iron atoms and 12 acid-labile
sulfur groups per molecule. There have been some
studies on how the external iron concentration aects
fermentation by anaerobic non- photosynthetic bacteria,
and it has been reported that the in vivo activity of the
hydrogenase decreases with iron depletion (Junelles
et al., 1988; Dabrock et al., 1992; Peguin and Soucaille,
1995). However these researches mainly focused on
solvent production and little work has been performed
on hydrogen production.
The purpose of this study was to investigate the eect
of iron concentration on hydrogen production by an
anaerobic non-photosynthetic microora.
2. Methods
2.1. Seed sludge
The seed sludge was taken from a ve-liter laboratory
scale digester with a working volume of three liters,
operated at 37C with a hydraulic retention time (HRT)
of 10 h. The chemostat reactor was conducted by introducing a sucrose solution, one liter of which contained sucrose, 10 g; NH4 HCO3 ; 1 g; KH2 PO4 , 0.5 g;
MgSO4
7H2 O; 50 mg; NaCl; 5 mg; Na2 MoO4
2H2 O,
5 mg; CaCl2
H2 O, 5 mg; MnSO4
7H2 O, 8 mg;
FeSO4
7H2 O, 1.4 mg. The pH was adjusted to 6.0
throughout the experiment. The seed sludge cultivated
in the digester was originally obtained from soybean
meal stored in a silo which had exploded owing to the
accumulation of biologically produced hydrogen. The
average hydrogen proportion of the biogas, the hydrogen production rate, and MLVSS of the digester sludge
were 50.6%, 10:0 l g 1 VSS d 1 and 1:3 g l 1 , respectively.

KOH and HCl solutions. All operations were conducted

under an atmosphere of 80% N2 and 20% CO2 to ensure
anaerobic conditions. The biogas production was determined by glass syringes of 1050 ml using the Owen
method (Owen et al., 1979). Each experiment was performed once.
2.3. Analytical methods
Gas samples (1.0 ml) were taken from the bottles
using a pressure-lock gas tight syringe (MS-GAN 100,
Ito). The proportion of hydrogen was determined by a
gas chromatograph (Shimadzu 8A) equipped with a
thermal conductivity detector (TCD). The column was
packed with Porapak Q (50/80 mesh). Nitrogen was
used as the carrier gas at a ow rate of 30 ml min 1 . The
temperatures of column and detector block were maintained at 100. The proportions of methane and carbon
dioxide were measured by a second GC of the same
model as above equipped with a TCD. The column was
packed with Porapak T (50/80 mesh). Helium was used
as the carrier gas at a ow rate of 30 ml min 1 . The
temperatures of column and detector were maintained at
100C. The concentrations of the VFAs were determined using a third GC of the same model equipped
with a ame ionization detector (FID) and a two meter
glass column packed with Unisole F-200 (30/60 mesh).
The temperatures of the column and detector were
145C and 170C, respectively. The concentrations of
solvents were measured by a fourth GC of the same
model equipped with an FID and a two meter glass
column packed with Gaskuropack 54 (60/80 mesh). The
temperatures of the column and detector were 185C
and 200C, respectively. Helium was used as the carrier
gas for the determinations of VFAs and solvents at a
ow rate of 30 ml min 1 . pH was determined by a TOA
pH meter equipped with a GST-5425C probe. The
concentrations of volatile suspended solids (VSS) were
determined according to the procedures described in
Standard Methods (APHA, 1995). Carbohydrate concentration was measured by the phenolsulfuric acid
method using glucose as a standard (Dubios et al.,
1956).

2.2. Experimental mixtures, apparatus and procedure

3. Results

The experiments were conducted in glass bottles at

37C. One liter of the medium contained NH4 HCO3 ;
2 g; KH2 PO4 ; 1g; MgSO4
7H2 O; 100 mg; NaCl; 10 mg;
Na2 MoO4
2H2 O;10 mg; CaCl2
2H2 O; 10 mg; MnSO4

7H2 O; 15 mg. Twenty ml of the seed sludge, 20 ml of the
medium, 20 ml of the sucrose solution 40 g l 1 , and 20 ml
of the FeCl2 solution (ranging from 0 to 16 g FeCl2 l 1
were added to each glass bottle. The initial pH value of
each experimental mixture was controlled to 6.0 with

3.1. Eect of iron on fermentation characteristics

Hydrogen, acetate, butyrate, ethanol and butanol
were produced by the fermentation of sucrose at iron
concentrations of 0 and 80 mg FeCl2 l 1 . Acetate was
the major aqueous product at the iron concentration of
0 mg FeCl2 l 1 . Hydrogen fermentation at the iron
concentration of 0 mg FeCl2 l 1 ceased at 25 h after
inoculation. The amount of hydrogen production at the

Y.J. Lee et al. / Bioresource Technology 80 (2001) 227231

iron concentration of 80 mg FeCl2 l 1 was about ve

times higher than that at 0 mg FeCl2 l 1 . The hydrogen
production at the iron concentration of 80 mg FeCl2 l 1
ceased at 37 h after inoculation. The hydrogen fermentation at an iron concentration of 800 mg FeCl2 l 1
produced many kinds of metabolites, hydrogen, acetate,
i-butyrate, n-butyrate, valerate, ethanol, butanol, and
propanol. The hydrogen fermentation at an iron concentration of 800 mg FeCl2 l 1 ceased at 154 h after
inoculation. A large amount of hydrogen (104.9 ml) was
produced under this condition. It was obvious that
production of metabolites, including hydrogen, increased with increasing iron concentration. The nal pH
values of the experimental mixtures were around 4.2
under the three iron concentrations (0, 80 and
800 mg FeCl2 l 1 .
3.2. Kinetic parameters
In order to determine the specic hydrogen production yield (ml g 1 sucrose) and rate ml g 1 VSS h 1 ,
each cumulative hydrogen production data was used to
estimate the three parameters of the modied Gompertz
equation (Lay et al., 1999). This was found to be a
suitable equation for describing the progress of cumulative hydrogen production obtained from a batch experiment.



Rm e
k t 1 ;
1
Ht P exp
exp
P
where H t is the cumulative hydrogen production, P is
hydrogen production potential (ml), Rm is the maximum
hydrogen production rate ml h 1 , and k is the lagphase time (h). The specic hydrogen production yield
(ml g 1 sucrose) was obtained by dividing P by sucrose
consumed while the specic hydrogen production rate
ml g 1 VSS h 1 was calculated by dividing Rm by the
dry biomass weight. The three parameters, P; Rm and k

229

were non-linearly evaluated using the function of `solver' in Microsoft Excel version 5.0 for Macintosh by
converging the residual sum of squares (RSS) between
the experiment and the estimation to a minimum value.
Table 1 summarizes the values of the parameters.
The specic hydrogen production rate increased from
1.1 to 14:7 ml g 1 VSS h 1 with increasing iron concentration from 0 to 400 mg FeCl2 l 1 . At iron concentrations above 400 mg FeCl2 l 1 , the specic
hydrogen production rate slightly increased with increasing iron concentration. The maximum specic hydrogen production rate was 24:0 ml g 1 VSS h 1 at an
iron concentration of 4000 mg FeCl2 l 1 . The specic
production rate of acetate decreased with increasing iron
concentration from 0 to 400 mg FeCl2 l 1 . At iron
concentrations above 400 mg FeCl2 l 1 , the specic
production rate of acetate slightly decreased with increasing iron concentration. The maximum specic
production rate of acetate was 0:25 g g 1 VSS h 1 at an
iron concentration of 0 mg FeCl2 l 1 . However, the
specic production rate of butyrate increased with increasing iron concentration from 0 to 20 mg FeCl2 l 1 ,
and decreased with increasing iron concentration from
20 to 4000 mg FeCl2 l 1 . The maximum specic production rate of butyrate was 72 mg g 1 VSS h 1 at an
iron concentration of 20 mg FeCl2 l 1 . The maximum
specic production rates of ethanol (682 mg g 1 VSS h 1 )
and butanol 47:0 mg g 1 VSS h 1 were obtained at
iron concentration of 5 and 3 mg FeCl2 l 1 , respectively. The specic production rate of propanol was low
and not inuenced by iron concentration.
The specic production yields of hydrogen, VFAs
and solvents (Table 2), were calculated by dividing the
amounts of hydrogen, VFAs and solvents produced by
the amount of sucrose consumed. When the iron concentrations were less than 800 mg FeCl2 l 1 , the hydrogen production yields were increased with increasing
iron concentration. The maximum hydrogen production

Table 1
Kinetic parameters on hydrogen fermentation calculated from non-linear regression of Eq. (1)
Iron (mg l 1 )

P (ml H2 )

Rm (ml h 1 )

k (h)

R2

RSS

0
3
5
10
20
40
80
200
400
800
1600
3000
4000

1.14
5.73
3.49
5.92
5.52
6.26
5.64
69.97
86.25
99.74
80.41
79.01
87.49

0.10
0.44
0.29
0.48
0.47
0.49
0.43
1.31
1.93
2.17
2.60
4.34
4.96

0.55
0.00
0.00
0.00
0.03
0.00
0.04
5.91
3.39
3.03
6.71
18.04
19.02

0.995
0.990
0.990
0.994
0.995
0.993
0.994
0.997
0.994
0.990
0.992
1.000
0.999

0.01
0.41
0.14
0.30
0.22
0.37
0.24
23.45
74.47
163.87
93.43
4.56
11.90

P hydrogen production potential (ml). Rm the maximum hydrogen production rate (ml h 1 ). k the lag-phase time (h). R2 correlation
coecient. RSS the residual sum of squares (RSS).

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Y.J. Lee et al. / Bioresource Technology 80 (2001) 227231

Table 2
Eect of the iron concentration on the specic production yields
Iron (mg l
0
3
5
10
20
40
80
200
400
800
1600
3000
4000

H2 ml g
2.0
11.5
6.5
11.2
10.0
13.5
7.5
92.3
113.5
131.9
112.2
114.7
122.7

sucrose)

Products (mg g

Acetate

Propionate

Butyrate

Ethanol

Propanol

Butanol

260.6
389.3
318.6
337.1
282.9
339.4
206.9
176.1
127.5
134.9
95.0
136.9
105.9

6.9
7.1
6.9
8.8
7.8
17.4
12.2
37.8
18.5
15.5
10.0
26.7
20.9

32.9
38.1
34.9
39.1
33.8
47.3
25.7
196.5
142.4
131.7
86.3
46.9
23.2

4.6
5.8
6.4
6.4
5.8
8.9
4.4
43.3
104.4
103.2
83.1
76.6
73.9

3.6
1.9
3.3
3.9
3.5
4.7
2.5
2.5
2.4
2.3
3.0
2.0
1.5

2.3
4.1
2.3
3.2
3.2
5.1
4.4
106.0
92.3
67.6
101.0
4.1
3.6

sucrose)

yield of 131:9 ml g 1 sucrose was obtained at the iron

concentration of 800 mg FeCl2 l 1 . The acetate production yield decreased with increasing iron concentration. The propionate production yield and butyrate
production yield increased with increasing the iron
concentration when the iron concentration was less than
200 mg FeCl2 l 1 . The butyrate production yield also
decreased with increasing iron concentration from 200
to 4000 mg FeCl2 l 1 . The maximum production yields
of acetate 389:3 mg g 1 sucrose), propionate
37:8 mg g 1 sucrose), and butyrate 196:5 mg g 1 sucrose) were obtained at iron concentrations of 3, 200
and 200 mg FeCl2 l 1 , respectively. The butanol production yield increased with increasing iron concentration when iron concentrations were less than
200 mg FeCl2 l 1 . The ethanol production yield increased from 4.4 to 104:4 mg g 1 sucrose with increasing iron concentration from 80 to 400 mg FeCl2 l 1 , and
decreased with increasing iron concentrations greater
than 400 mg FeCl2 l 1 . The propanol production yields
were below 4:7 mg g 1 sucrose at all iron concentrations.
3.3. Eect of iron on sucrose conversion into biomass
The sucrose degradation eciency was estimated by
dividing the amount of sucrose consumed by the
amount of initial sucrose. The biomass production yield
was estimated by dividing the amount of biomass produced by the amount of sucrose consumed. The soluble
carbohydrate concentration and VSS were measured at
the end of each batch experiment for the calculation of
the amount of sucrose consumed and the amount of
biomass produced, respectively. Individual batch experiments were observed until the hydrogen production
from each bottle stopped. The sucrose degradation efciency increased with increasing iron concentration.
The sucrose degradation eciencies were close to 1.0
when iron concentrations of the experimental mixtures

were between 200 and 800 mg FeCl2 l 1 . At iron concentrations below 3000 mg FeCl2 l 1 , the biomass production yield increased with increasing iron. The
maximum
biomass
production
yield
was
0:283 g VSS g 1 sucrose at an iron concentration of
3000 mg FeCl2 l 1 .
4. Discussion
Schoenheit et al. (1979) demonstrated that growth of
C. pasteurianum in batch cultures was growth limited
when the iron concentration of the medium was less
than 10 lmol l 1 . Dabrock et al. (1992) reported that
iron concentrations up to 10 lmol l 1 were growth
limiting for C. pasteurianum but they also reported that
iron limitation did not cause a decrease of hydrogen
production. Junelles et al. (1988) demonstrated that iron
limitation caused a decrease of hydrogenase specic
activity, a decrease of acid production, and an increase
of solvent production. This experiment was conducted
with iron concentrations of 0.7 and 36 lmol l 1 . When
the iron supply was non-limiting (iron concentration of
above 25 lmol l 1 , the metabolism of C. acetobutylicum was normally acidogenic and molecular hydrogen
was one of the metabolites (Peguin and Soucaille, 1995).
In our study, the iron concentrations of experimental
mixtures were changed from 1.26 to 31:6 lmol l 1 , and
the maximum specic hydrogen production rate and
yield were at iron concentrations of 31.6 and 6.31 mmol
l 1 , respectively. These iron concentrations were much
higher than the 10 and 25 lmol l 1 below which the
growth of Clostridia was limited by the iron concentration (Schoenheit et al., 1979; Dabrock et al., 1992; Peguin and Soucaille, 1995), and derived from experiments
which did not include the extremely high range of the
iron concentrations. It may be concluded that the eect
of the iron concentration on the hydrogen production
rate of Clostridia may be caused by not only the iron

Y.J. Lee et al. / Bioresource Technology 80 (2001) 227231

concentration of the external as limiting factor for the

growth but also other environmental conditions which
iron concentration aected. The seed sludge that we
used contained hydrogen-producing bacteria as well as
bacteria which do not have hydrogen production ability
since the seed sludge was mixed anaerobic microorganisms. Changes in the iron concentration may have affected the relationship between these anaerobic bacteria.
5. Conclusions
The eects of iron concentration on the production of
hydrogen, VFAs and solvents were investigated by
batch experiments. The following conclusions could be
drawn.
1. The maximum specic hydrogen production rate was
24:0 ml g 1 VSS h 1 at iron concentration of
4000 mg FeCl2 l 1 . The specic production rate of
butyrate increased with increasing iron concentration
from 0 to 20 mg FeCl2 l 1 , and decreased with increasing
iron
concentration
from
20
to
4000 mg FeCl2 l 1 . The maximum specic production rates of ethanol 682 mg g 1 VSS h 1 and butanol 47:0 mg g 1 VSS h 1 were obtained at iron
concentration of 5 and 3 mg FeCl2 l 1 , respectively.
2. The maximum hydrogen production yield of 131.9 ml
g 1 sucrose was obtained at the iron concentration of
800 mg FeCl2 l 1 . The maximum production yields
of acetate 389:3 mg g 1 sucrose), propionate
37:8 mg g 1 sucrose), and butyrate 196:5 mg g 1
sucrose) were obtained at iron concentrations of 3,
200 and 200 mg FeCl2 l 1 , respectively.
3. The sucrose degradation eciencies were close to 1.0
when iron concentrations were between 200 and
800 mg FeCl2 l 1 . The maximum biomass production yield was 0:283 g VSS g 1 sucrose at an iron
concentration of 3000 mg FeCl2 l 1 .
Acknowledgements
This work was supported by the CREST (Core Research for Evolutional Science and Technology) foundation of Japan Science and Technology (JST).
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