JOURNALoF BIOSCIENCE AND BIOENGINEERING

Vol. 91, No. 1, 58-63. 2001

Microbial Hydrogen Production from Sweet Potato Starch Residue

HARUHIKO YOKOI,* AKIO SAITSU, HIROYUKI UCHIDA, JUN HIROSE, SACHIO HAYASHI,
AND YOSHIYUKI TAKASAKI
Department of Applied Chemistry, Faculty of Engineering, Miyazaki University, 1-1 Nishi, Gakuen-kibanadai,
Miyazaki 889-2192, Japan
Received 7 August 2000/Accepted 20 October 2000

Clostridium butyricum could produce hydrogen from a sweet potato starch residue upon supplementation of
nitrogen sources. A repeated batch culture using a mixed culture of C. butyricum and Enterobacter aerogenes
produced hydrogen with a yield of 2.4 mol Hz/mol glucose under a controlled culture pH of 5.25 in a medium
consisting of the sweet potato starch residue and 0 . 1 ~ Polypepton without addition of any reducing agents.
Rhodobacter sp. M-19 produced hydrogen from the supernatant of the culture broth obtained in the re-
peated batch culture containing C. butyricum and E. aerogenes when 50 pg/1 NazMoO4.2H20 and 20 mg/1
EDTA were added to the supernatant and it was cultured under a controlled culture pH of 7.5. A high yield of
hydrogen of 7.0 mol H z / m o l glucose from the starch remaining in the starch residue was attained in two-step
repeated batch cultures containing C. butyricum and E. aerogenes, and by Rhodobacter sp. M-19.
[Key words: hydrogen, hydrogen production, sweet potato starch, starch residue]

The annual yield of sweet potatoes for the production
of starch is about 2.1 × 105 tons in Kagoshima prefecture
in the southern part of Japan. About 8 × 104 tons of a
sweet potato starch residue which contains about 80%
water is also produced as a by-product of starch
manufacture. The sweet potato starch residue contains
starch, fibers consisting of cellulose and hemicellulose,
ash, moisture and other substances. The starch residue
was formerly used for citric acid fermentation by Asper-
gillus niger (1). However, at present, most of the starch
residue is discarded since the amount of citric acid pro-
duced domestically has decreased. Therefore, new
methods for converting the starch residue to other useful
substances are greatly desired.
Recently, microbial hydrogen production by anaerobic
bacteria (2-11) and photosynthetic microorganisms (12-
19) has been extensively investigated since hydrogen is
considered to be a future clean source of energy. Among
the anaerobic bacteria, Clostridium butyricum produces
hydrogen from starch at a high yield of about 2 mol H2/
mol glucose (11). Although C. butyricum is suitable for
high-yield hydrogen production from starch, the addi-
tion of an expensive reducing agent such as L-cysteine is
required since the bacterium is very sensitive to slight
amounts of oxygen in the culture medium. In our previ-
ous study, it was found that a mixed culture of C.
butyricum and Enterobacter aerogenes removed oxygen
from the reactor and produced hydrogen from starch
with a high yield of more than 2 mol HE/mOl glucose
without the need to add any reducing agents to the medi-
um (11). However, there have been few reports on hydro-
gen production by a mixed culture from actual organic
waste which contains starch except for a study on the
photoproduction of hydrogen from raw starch-rich algal
biomasses by a halophilic bacterial community (14). Fur-
thermore, we previously reported that hydrogen produc-
tion of about 6.6 mol H2/mol glucose could be attained
from starch using a mixed culture of C. butyricum and
Rhodobacter sp. M-19, since the photosynthetic bacteri-
um produced hydrogen from organic acids produced by
* Corresponding author, e-mail: yokoi@cc.miyazaki-u.ac.jp
58
C. butyricum (20). If the starch remaining in the sweet
potato residue is available for hydrogen production by a
mixed culture of C. butyricum and E. aerogenes, and a
culture broth which contains organic acids is also availa-
ble for hydrogen production by the photosynthetic bac-
terium, the starch residue can be utilized as a raw mate-
rial for effective hydrogen production. In this study, a
method for high-yield hydrogen production from the
sweet potato starch residue by C. butyricum, E. aero-
genes and Rhodobacter sp. M-19 was established.
MATERIALS AND METHODS
Microorganisms and preparation of cell suspensions
C. butyricum IFO13949 was precultured anaerobically at
36°C for 18 h in a GP medium (pH 6.5) consisting of
1.0% glucose, 2.0% Polypepton, 0.2% KHEPO4, 0.05%
yeast extract, 0.05% Mg2SO4.7H20 and 0.1% L-cys-
teine-HC1.H20, a reducing agent. After centrifugation
of 8 ml of the precultured broth at 8500 x g for 10 rain,
harvested cells were washed with physiological saline and
suspended in 8 ml of the saline to give a cell suspension
of C. butyricum (0.75 g dry cells//). E. aerogenes strain
HO-39 (10) was precultured aerobically at 30°C for 12 h
in a modified GP medium (pH 6.5) which contained no
L-cysteine. HCI. H20. A cell suspension of E. aerogenes
(4.8 g dry cells//) was prepared in the same manner as
that of C. butyricum. Rhodobacter sp. M-19 (20) was
cultured anaerobically in an R medium at 30°C under il-
lumination at 5000 Ix using an incandescent lamp (type
K-RD40W, National Electric Co., Tokyo). The R medi-
um was composed of 50 mM butyric acid, 2.8 mg/l H3BO4,
0.75 mg/1 NaEMoO4.2H20, 0.24 mg/l ZnSO4.7H20,
2.1 mg/l MnSO4.4H20, 0.04 mg/l Cu(NO3)2-3H20,
50 mg/I CaC12, 20 mg/l EDTA, 11.8 mg/l FeSO4-7H20,
200 mg/l MgESO4.7H20, 1.0 g/l yeast extract, 0.15 pg/l
biotin, 0.15 l~g/l vitamin B1, 0.15 pg/l p-aminobenzoic
acid, 0.15pg/l nicotinic acid, O.15pg/1 nicotinamide,
1.25g/l (NH4)2SO4 and 100mM sodium phosphate
buffer (pH 6.5). A cell suspension of Rhodobacter sp.
M-19 (2.5 g dry cells//) was also prepared in the same
manner.
Vot. 91, 2001
Hydrogen production by C. butyricum and E. aer-
ogenes in batch cultures For hydrogen production by
a monoculture of C. butyricum in a batch culture, 8 ml
of a cell suspension and 192 ml of a starch residue medi-
um containing 0.1% L-Cysteine.HC1.H20 were put in a
250-ml stirred reactor. In the case of a mixed culture of
C. butyricum and E. aerogenes in a batch culture, 11 ml
of the cell suspension of C. butyricum, 1 ml of that of
E. aerogenes and 188 ml of the starch residue medium
containing no L-cysteine.HCl.H20 were put in the
stirred reactor. After replacement of the gas phase of the
reactors with argon, the cells were cultured anaerobically
at 37°C at a controlled pH of 5.25 with stirring. Gas
evolved was collected and the volume was measured in
an inverted measuring cylinder over a 20% NaOH solu-
tion.
Hydrogen production in a repeated batch culture by
C. butyricum and E. aerogenes After the completion
of a batch culture of C. butyricum and E. aerogenes in
200ml of the starch residue medium without L-cys-
teine.HCI.H20 in the 250-ml stirred reactor which was
carried out anaerobically at 37°C at a controlled pH of
5.25 with stirring, 100 ml of the culture broth was drawn
out using a peristaltic pump. Then, 100 ml of the fresh
starch residue medium was added to the remaining 100
ml of the culture broth in the reactor and cultured for
a further 24 h. In this way, a repeated batch culture was
performed at 24-h intervals.
Hydrogen production in a repeated batch culture by
Rhodobacter sp. M-19 The culture broth obtained in
the repeated batch culture of C. butyricum and E. aer-
ogenes was centrifuged at 8500xg for 15 min. After cen-
trifugation, 50 pg/l Na2MoO4.2H20 and 20 mg/l EDTA
were added to the supernatant to prepare a culture medi-
um (medium S) for Rhodobacter sp. M-19. Two ml of
a cell suspension of Rhodobacter sp. M-19 and 48 ml
of the medium S were put in a glass tube reactor (i.d.
2 × 20 cm), and the head space of the reactor was re-
placed with argon gas. After carrying out a batch cul-
ture at 35°C for 5 d with stirring under illumination at
50001x by the incandescent lamp, 40 ml of the culture
broth was drawn out using a peristaltic pump. Then,
40 ml of the medium S were poured into the reactor and
cultivation was continued for a further 5 d. A repeated
batch culture was performed at 5-d intervals.
Sweet potato starch residue The sweet potato
starch residue used in this study is a dried residuum
which was obtained after extraction of the starch from
sweet potatoes and drying in a starch-manufacturing
process. The dried sweet potato starch residue contained
49.0% starch, 14.2% moisture, 5.2% ash and other com-
ponents. Most of the other components were proteins
(about 3%), fats (less than 1%) and fibers which originat-
ed from the peels and tubers of the sweet potatoes and
consisted of cellulose, hemicellulose, pectin substances
and lignin.
Analytical methods Analyses of NH4 +, acetic acid,
butyric acid, lactic acid and starch in the culture broth
and hydrogen in the evolved gas were performed as
described previously (20).
RESULTS AND DISCUSSION
Hydrogen production from a sweet potato starch
residue by a monoculture of C. butyricum At first,
hydrogen production by a monoculture of C. butyricum
HYDROGEN PRODUCTIONFROM STARCH RESIDUE 59
was performed without pH control in starch residue me-
dia to evaluate whether the bacterium could produce
hydrogen from the starch remaining in the sweet potato
starch residue as a carbon source. Although 124mi of
hydrogen was produced by C. butyricum in a basal
starch residue medium consisting of 0.5% sweet potato
starch residue, 1.0% Polypepton, 0.2% KH2PO4, 0.05%
MgSO4-7H20, 0.05% yeast extract and 0.1% L-cys-
teine-HC1. H20, the bacterium could not produce hydro-
gen from the starch residue not supplemented with any
other medium components.
To identify the medium components necessary for
hydrogen production from the starch residue by C.
butyricum, hydrogen evolution was observed in media
which contained 0.5% starch residue, 0.1% L-cys-
teine. HCI. H20 and one of each of the medium compo-
nents of the basal starch residue medium. Hydrogen was
not produced from the starch residue supplemented with
0.2% KH2PO4 or 0.05% MgSO4.7H20. However, the rel-
ative hydrogen production from the starch residue sup-
plemented with 1.0% Polypepton or 0.05% yeast extract
was 103% and 20% that in the basal starch residue medi-
um, respectively, indicating that addition of a nitrogen
source to the starch residue is essential for hydrogen pro-
duction (data not shown).
To determine the optimum nitrogen source for hydro-
gen production, the bacterium was cultured in media
consisting of 0.5% sweet potato starch residue, 0.1% L-
cysteine-HCI-H20 and 1.0% Polypepton, casamino
acids, urea, (NH4)2SO4 or NH4C1. Among the nitrogen
sources, the highest level of hydrogen production was
obtained with Polypepton. However, hydrogen was not
produced in the media to which urea, (NH4)2SO4 or NH4CI
was added (data not shown).
Organic acids such as acetic, butyric and lactic acids
are produced as by-products concomitant with hydrogen
evolution from sugars by C. butyricum (20). Since ac-
cumulation of the organic acids causes a decrease in the
culture pH and inhibition of hydrogen-producing activ-
ity of the bacterium, pH control of the culture is neces-
sary. Figure 1 shows the effect of controlling the culture
pH on hydrogen evolution from 0.5% sweet potato
starch residue supplemented with 1.0% Polypepton by
the bacterium in batch cultures. Hydrogen evolution was
high in the pH region from 5.0 to 5.5 and the optimum
pH was 5.25.
When hydrogen is produced from sweet potato starch
residue, it is desired to minimize the amount of Polypep-
200
150
~ 100
~ 50
0 i i
4 5 6 7
pH
FIG. 1. Effect of culture pH on hydrogen production by C.
butyricum from sweet potato starch residue.
60 YOKOIET AL. J. BloSCi.BIOENO.,

200 6 i0o
, (A)
5 80
150
60
,...,
100
40
o 50
2 20

0 I I I I I
l
4 0 50 100 150 200 250
Time (h)
-A- 100
--A- (B)
80
~2
_,>
o 60

0 I I I
0 0.2 0.4 0.6 0.8 1.0
Polypeptone (%)
FIG. 2. Effectof the concentration of Polypepton added to the
sweet potato starch residue on hydrogen evolution (O), NH4+ pro-
duction in culture broth (O) and hydrogen yield (A).
ton added to the medium. The effects of Polypeptone
concentration on hydrogen evolution, hydrogen yield
and NH4 + production by the bacterium in a batch cul-
ture at pH 5.25 in a medium consisting of 0.5% sweet
potato starch residue, Polypepton and 0.1% L-cys-
teine. HCI. H20 are shown in Fig. 2. The hydrogen yield
(tool H2/mol glucose) represents the molar ratio of
hydrogen evolved to glucose contained in the starch of
the sweet potato starch residue added to the medium.
The hydrogen yield is not, however, a molar ratio of the
hydrogen evolved to the net glucose contained in the
starch of the sweet potato starch residue consumed in
the culture. Almost equal amounts of hydrogen and high
hydrogen yields of about 2.4 tool H2/mol glucose were
obtained at concentrations of Polypepton above 0.1%.
However, hydrogen evolution and hydrogen yield
decreased markedly when the concentrations was less
than 0.05%. NH4 + concentration in the culture broth
increased with increasing Polypepton concentration. There-
fore, the addition of 0.1% Polypepton to the starch
residue was necessary for hydrogen production by the
bacterium.
H2 production from a sweet potato starch residue by a
mixed culture of C. butyricum and E. aerogenes
Since the hydrogen-producing ability of C. butyricum is
markedly inhibited by a slight amount of oxygen, the
addition of a reducing agent such as L-cysteine. HCI. H20
to the culture medium is an absolute requirement. The
use of reducing agents which are relatively expensive is
40
~ 20
1.2
0~
0 50 100 150 200 250
Time (h)
FIG. 3. Hydrogen production from sweetpotato starch residue in
repeated batch cultures by a monoculture of C. butyricum (A) and by
a mixed culture of C. butyricum and E. aerogenes (B).
uneconomical in a hydrogen production process. The
authors previously reported that a mixed culture of C.
butyricum and E. aerogenes could produce hydrogen
from starch at a high yield of more than 2 mol H2/mol
glucose without the need to add any reducing agents to
the medium since E. aerogenes, a facultative anaerobe,
removed oxygen and generated anaerobic conditions in
the reactor (11). Therefore, the production of hydrogen
from a sweet potato starch residue by a mixed culture of
C. butyricum and E. aerogenes was examined. Table 1
shows the parameters relating to hydrogen production
from 0.5 to 1.0% sweet potato starch residue supple-
mented with 0.1% Polypepton with pH control at 5.25
by a monoculture of C. butyricum and by a mixed cul-
ture of C. butyricum and E. aerogenes in batch cultures.
The monoculture was performed in the presence of 0.1%
L-cysteine.HCl. H20. The volume of hydrogen evolved
increased with increasing starch residue concentration in
the medium for both cultures. Although hydrogen could
be produced by the mixed culture without the addition
of a reducing agent, hydrogen production and hydrogen
yield for the mixed culture were less than those for the
monoculture. It is known that C. butyricum and E. aer-
ogenes produce hydrogen from glucose at yields of
about 2 and 1 mol H2/mol glucose, respectively (9, 10). It
appears that the hydrogen yield for the mixed culture
might be lower since C. butyricum was not predominant

TABLE 1. Hydrogen production from starch residue by monoculture of C. butyricum and by mixed culture of C. butyricum and E. aerogenes
Monoculture of C. butyricum Mixed culture of C. butyricum and E. aerogenes
Starch residue
(%) Hydrogen evolveda Hydrogen yield Hydrogen evolved Hydrogen yield
(ml) (mol H2/mol glucose) (ml) (mol H2/mol glucose)
0.5 151 2.4 105 1.7
0.75 203 2.2 136 1.5
1.0 268 2.2 N.T. b N.T.
a Volumes of hydrogen evolved from 200 ml of a culture medium.
b Not tested.
VOL. 91, 2001
and the number of E. aerogenes cells was high in the
mixed culture.
Figure 3 shows a comparison of the hydrogen produc-
tion in repeated batch cultures by C. butyricum in the
presence of 0.1% T.-cysteine. HC1. H20 and by the mixed
culture of C. butyricum and E. aerogenes in the absence
of z-cysteine-HC1.H20. The repeated batch cultures
were performed by drawing out 100ml of the culture
broth from the reactor and adding 100 mi of the fresh
medium consisting of 0.5% sweet potato starch residue
and 0.1% Polypepton at 24-h intervals. In the case of
the monoculture of C. butyricum, stable hydrogen pro-
duction was observed from the first batch culture. The
average volume of hydrogen evolved per batch culture
and the average hydrogen yield were about 75 ml and
2.4 mol H2/mol glucose, respectively. Although the vol-
umes of hydrogen evolved in the early batch cultures by
the mixed culture of C. butyricum and E. aerogenes
were less than those by the monoculture, hydrogen pro-
duction by the mixed culture increased with increasing
numbers of batch cultures. After the fourth batch cul-
ture, an average volume of hydrogen evolved of about
76 ml/batch culture and an average hydrogen yield of
2.4 mol H2/mol glucose were obtained for the mixed cul-
ture in the absence of the reducing agent. E. aerogenes
cannot utilize the starch in the starch residue for its
growth and hydrogen production, but C. butyricum
produces hydrogen from the starch since it has the ability
to produce amylases. Therefore, to survive in the starch
residue medium, E. aerogenes must utilize sugars such as
glucose and maltose which are generated in the degrada-
tion of the starch by amylases from C. butyricum.
Amylase-producing C. butyricum might grow more easi-
ly in the starch residue medium than E. aerogenes not
producing amylase. Therefore, it appears that hydrogen
production proceeded in a manner similar to that in the
monoculture of C. butyricum and the hydrogen yield
increased to a level equal to that of the monoculture since
C. butyricum in the mixed culture became gradually
dominant by repeating the batch cultures.
Next, the effect of starch residue concentration on
hydrogen production by the mixed culture in repeated
batch cultures was determined. The repeated batch cul-
tures were performed with media consisting of 0.5 to 2%
sweet potato starch residue and 0.1% Polypepton with
pH control at 5.25. As shown in Table 2, hydrogen pro-
duction increased with increasing concentration of the
starch residue. A stable and high hydrogen yield of 2.3
to 2.4 mol H2/mol glucose was obtained up to 2.0% of
the starch residue, and 1.4 to 2.0 mM NH4 + was present
in the culture broth.
From these results, it was shown that high-yield hydro-
TABLE 2. Effectof starch residue concentration on hydrogen
production by mixed culture of C. butyricum and E.
aerogenes in repeated batch cultures
Starch Hydrogen Hydrogen yield NI-h+
residue
(%) evolved
(ml) a (mol H2/mol glucose) (mM)
0.5 76 2.4 1.5
0.75 116 2.4 1.4
1.0 147 2.3 1.5
1.5 211 2.3 2.0
2.0 295 2.4 2.0
a Averagevolumesof hydrogenevolvedfrom 100ml of the medium
per batch culture.
HYDROGEN PRODUCTION FROM STARCH RESIDUE 61
60
_~ 50 •
~ 4o
~ ao
~ 2o
~ 10
I0 I00 I000
NaxMo04"2H20 (/~g//)
FIG. 4. Effect of Na2MoO4o2H20 concentration on hydrogen
production by Rhodobacter sp. M-19in the supernatant of the culture
broth obtained in a repeated batch culture by C. butyricum and E.
aerogenes in a sweet potato starch residue medium.
gen production of 2.4mol H2/mol glucose from the
starch remaining in the sweet potato starch residue in the
absence of reducing agents could be attained by the addi-
tion of 0.1% Polypepton to the starch residue and by
using a mixed culture of C. butyricum and E. aerogenes in
repeated batch cultures at a controlled pH of 5.25. The
procedure necessary to produce hydrogen from the sweet
potato starch residue by the mixed culture developed in
this study would be applicable to other organic wastes
which contain starch.
Hydrogen production by Rhodobacter sp. M-19 To
increase the amount of hydrogen produced from the
potato starch residue, Rhodobacter sp. M-19 was cul-
tured in the supernatant of the culture broth obtained in
a repeated batch culture of C. butyricum and E. aero-
genes. At first, hydrogen production by Rhodobacter sp.
M-19 was performed without controlling the pH of the
supernatant to determine whether the photosynthetic bac-
terium could produce hydrogen from organic acids such
as acetic, butyric and lactic acids in the supernatant.
Although hydrogen was produced by Rhodobacter sp.
M-19 in the supernatant supplemented with all of the
medium components of the R medium except for butyric
acid, the bacterium could not produce hydrogen in the
supernatant not supplemented with any other medium
200 7
6
4 "'~'"~
tO0 ~'~
o 2~'6
~50
0
6 7 8 9
pH
FIG. 5. Effect of culture pH on hydrogen evolution (o) and
hydrogen yield (o) by Rhodobacter sp. M-19 in the supernatant of
the culture broth obtained in a repeated batch culture by C. butyricum
and E. aerogenesin a sweet potato starch residue medium.
62 YOKOIET AL.
~ 150 I
~5
10
~"~ 6
~ ~ 4
0 III II I)llll II I I I I l l l l l
0 100 200 300 400
Time (h)
FIG. 6. Hydrogen production in repeated batch cultures by
Rhodobacter sp. M-19in the supernatant of the culture broth obtained
in a repeated batch culture by C. butyricum and E. aerogenesin a
sweet potato starch residue medium.
components. It was found that Na2MoO4.2H20 was ab-
solutely necessary for hydrogen production from the
supernatant by the bacterium among the medium compo-
nents tested (data not shown). Figure 4 shows the effect
of the amount of Na2MoO4.2H20 added to the super-
natant on hydrogen production by Rhodobacter sp. M-
19 in batch cultures. The optimum concentration of
Na2MoO4.2H20 for the hydrogen production was high-
er than 20 pg/l. Furthermore, hydrogen production was
slightly promoted by the addition of 10 to 2 0 m g / l
EDTA together with Na2MoO4-2H20 to the supernatant
(data not shown).
Next, the optimum culture conditions for hydrogen
production by Rhodobacter sp. M-19 in the supernatant
supplemented with 50 pg/l Na2MoO4.2H20 and 20 mg/l
EDTA were determined. Figure 5 shows the effect of the
culture pH on hydrogen production with pH control in
batch cultures. Hydrogen evolved shown in the figure
means the volumes of hydrogen produced by Rhodobac-
ter sp. M-19 in the supernatant and it does not contain
the volumes of hydrogen produced by the mixed culture
of C. butyricum and E. aerogenes in the repeated batch
cultures. The hydrogen yield (tool HE/mOl glucose) repre-
sents the molar ratio of hydrogen evolved by Rhodobac-
ter sp. M-19 in the supernatant to glucose contained in
all of the starch of the sweet potato starch residue added
to the starch residue medium used for hydrogen produc-
tion by the mixed culture of C. butyricum and E. aer-
ogenes in the repeated batch cultures. Therefore, hydro-
gen yields obtained by the mixed culture of C. butyri-
cure and E. aerogenes in the repeated batch cultures are
not added to the hydrogen yields in the figure. The opti-
mum culture pH for hydrogen production was about 7.5
to 8.0.
Furthermore, continuous hydrogen production by
Rhodobacter sp. M-19 in a repeated batch culture was
carried out. A medium prepared by adding 5 0 p g / /
Na2MoO4.2H20 and 20 mg/! EDTA to the supernatant
J. Bzoscl. BzOESG.,
of a culture broth obtained in a repeated batch culture
of C. butyricum and E. aerogenes was used. The culture
pH was controlled at 7.5. The repeated batch culture was
performed by repeating batch cultures at 5-d intervals.
As shown in Fig. 6, stable hydrogen production was ob-
served for more than 30d and the average hydrogen
yield was about 4.6 mol H2/mol glucose. Therefore, it
was determined that continuous hydrogen production at
a total hydrogen yield of 7 tool HE/mOl glucose could be
attained in two-step repeated batch cultures by a mixed
culture of C. butyricum and E. aerogenes (2.4 mol H2/
mol glucose) and by Rhodobacter sp. M-19 from the
starch remaining in the starch residue. Although it is
known theoretically that 12 mol of hydrogen are pro-
duced per 1 mol of glucose, the actual hydrogen yield is
lower than 12 mol H2/mol glucose since a portion of glu-
cose is utilized for cell synthesis. It has been reported
that high-yield hydrogen production of 7 mol H2/mol
glucose could be attained from glucose by immobilized
I[ IIIlll II1[I cells of C. butyricum and Rhodobacter spheroids (21).
500 600 700 In comparison, the hydrogen yield in this study is sig-
nificant since hydrogen was produced from the starch in
the sweet potato starch residue, an organic waste, in a
long-term continuous culture. The two-step repeated
batch cultures by a mixed culture of C. butyricum and
E. aerogenes and by Rhodobacter sp. M-19 developed in
this study would be applicable to hydrogen production
from other organic wastes containing starch and sugars.
ACKNOWLEDGMENTS
This study was performed as part of the project entitled "High
and Ecological Utilization of Regional Carbohydrates", through
Special Coordination Funds for Promoting Science and Technology
(Leading Research Utilizing Potential of Regional Science and Tech-
nology) of the Science and Technology Agency of the Japanese
Government, 1999.
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