International Journal of Hydrogen Energy 29 (2004) 1355 – 1363

www.elsevier.com/locate/ijhydene

Hydrogen production from food waste in anaerobic mesophilic

and thermophilic acidogenesis
Hang-Sik Shina;∗ , Jong-Ho Younb , Sang-Hyoun Kima
a Departmentof Civil and Environmental Engineering, Korea Advanced Institute of Science and Technology, 373-1, Guseong-dong,
Yuseong-gu, Daejeon, 305-701, South Korea
b Department of Environmental Information and Engineering, Shinsung College, 49, Duckma-ri, Jungmi-myun, Dangjin-gun,

Chungnam, 343-861, South Korea

Accepted 23 September 2003

Abstract
Hydrogen production from food waste by the mesophilic and thermophilic acidogenic culture acclimated with food waste
at 5 days HRT for the e1ect of pH and volatile solid (VS) concentrations was evaluated. The biogas produced from the
thermophilic acidogenic culture was free of methane at all tested pH and VS concentrations, but methane was detected from the
mesophilic acidogenic culture at all tested pH. The amount of hydrogen production from the thermophilic acidogenic culture
was much higher than that from the mesophilic culture at all tested pH because of the methane free condition and negligible
propionate production. Increase of VS concentrations from 3 to 10 g VS l−1 resulted in the increase of quantity and quality of
hydrogen production. The maximum hydrogen content was 69% (v/v) at 10 g VS l−1 . The hydrogen yield was in the range
of 0.9 –1:8 mol-H2 =mol-hexose and peaked at 6 g VS l−1 . Normal butyrate was the main acid product, and the percentages
of butyrate, acetate and propionate at tested VS concentrations were 54 – 60%, 22–31% and 0.3–1%, respectively. Hydrogen
producing microorganisms of Thermoanaerobacterium thermosaccharolytium and Desulfotomaculum geothermicum were
detected from the thermophilic acidogenic culture, while Thermotogales strain and Bacillus species were detected from the
mesophilic acidogenic culture by PCR-DGGE analysis.
? 2003 International Association for Hydrogen Energy. Published by Elsevier Ltd. All rights reserved.

Keywords: Hydrogen; Volatile fatty acids; Food waste; PCR-DGGE; Mesophilic; Thermophilic culture; Thermoanaerobacterium
thermosaccharolytium

1. Introduction either for methane production by methanogenesis or a read-

Hydrogen is a promising alternative to fossil fuels due
to its clean and high-energy yield. Anaerobic acidi@cation
of organic wastes produces various volatile fatty acids
(VFA), H2 , CO2 and other intermediates. The reactions
involved in hydrogen production are rapid and do not re-
quire solar radiation, making them useful for treating large
quantities of organic wastes. Not only hydrogen gas itself
is a bene@cial energy source, but also VFA can be used
∗ Corresponding author. Tel.: +82-42-869-3613;
fax: +82-42-869-3610.
E-mail address: hangshin@kaist.ac.kr (H.-S. Shin).
ily biodegradable carbon source for biological nutrients re-
moval [1–3]. Therefore, the harvest of hydrogen at the acid-
i@cation stage of anaerobic treatment, leaving the remaining
acidi@cation products such as acetate and butyrate for fur-
ther methane production or external carbon source for bio-
logical nutrients treatment process is a great challenge for its
economic aspect. Acidi@cation of organic wastes, however,
needs hydraulic retention time (HRT) longer than 3 days in
which hydrogen consumers such as methanogenesis could
be proliferated. Because of this reason, most researches on
hydrogen production have been carried out under inhibitory
condition of hydrogen consumers. In order to inactivate hy-
drogen consumers, inocula were cultivated with pure che-
micals such as glucose or sucrose at short HRT and/or low

0360-3199/$ 30.00 ? 2003 International Association for Hydrogen Energy. Published by Elsevier Ltd. All rights reserved.
doi:10.1016/j.ijhydene.2003.09.011
1356 H.-S. Shin et al. / International Journal of Hydrogen Energy 29 (2004) 1355 – 1363
pH [4–7], or preheated to harvest spore-forming anaerobic
bacteria [6]. Continuous production of hydrogen was also
tried at short HRT to prevent the growth of hydrogen con-
sumers [8,9]. However, there have been no studies on con-
tinuous hydrogen production at enough HRT from organic
solid wastes. To date, the majority of research has been di-
rected at expensive pure substrates or to a much lesser de-
gree solid waste or wastewaters, however, for a truly sus-
tainable production process and to meet the demand for re-
newable energy, more sustainable feed stocks will have to be
utilized [10]. Food waste, a carbohydrate-rich organic solid
waste, was used in this study as a substrate for hydrogen
production. As the generation of food waste amounted to
11; 577 tons per day in Korea, which was 25.4% of munici-
pal solid waste [11], its management causes much concern.
Therefore, the aim of this study was to investigate the
feasibility of hydrogen production from food waste. The
mesophilic and thermophilic acidogenic culture, accli-
mated with food waste at 5 days HRT which was enough
HRT for acidifying food waste, was used as seed microor-
ganisms. This study focused on the ability of hydrogen
production from the thermophilic acidogenic culture, and
the mesophilic acidogenic culture was also examined for
comparison. The responsible microorganisms for hydro-
gen production were examined by denaturing gradient gel
electrophoresis (DGGE) of the polymerase chain reaction
(PCR)—ampli@ed V3 region of 16S rDNA.
2. Materials and methods
2.1. Feedstock
Food waste collected from a dining hall was ground after
sorting out animal bones and clamshells. It was mixed with
deionized (DI) water (food waste:DI water=1 : 3), and then
sieved with a screen (No.4, 4:75 mm ID). Table 1 shows
the characteristics of food waste.
2.2. Seed microorganisms
Two kinds of seed microorganisms, mesophilic and ther-
mophilic acidogenic culture, were taken from two identical
5 l continuous stirred acidogenic reactors which were oper-
ated at 35 ± 1◦ C and 55 ± 1◦ C, respectively. Both reactors
were fed semi-continuously with food waste. The reactors
were operated at 3 g VS l−1 day−1 , 5 days HRT and pH
5:6 ± 0:2 for one and half months at steady state. Table 2
shows the characteristics of the two seed microorganisms
and the average operating parameter values in the mesophilic
and thermophilic reactor at steady state.
2.3. Experimental apparatus and procedure
The production of hydrogen and VFA from food waste
by the mesophilic and thermophilic acidogenic culture was
Table 1
Characteristics of food waste
Item Unit Value
pH 5.8
TS g l−1 67.8
VS g l−1 63.7
VS/TS 0.94
Total carbohydrate g l−1 25.5
Carbon, C % TS 51.2
Hydrogen, H % TS 7.7
Oxygen, O % TS 38.3
Nitrogen, N % TS 2.8
Sulfur, S % TS 0.7
C/N 18.3
studied in 715 ml serum bottles. The mesophilic and ther-
mophilic culture taken from each reactor were settled for
1 h, and the supernatant was then removed. The settled cul-
tures were used as seed microorganisms. The seed microor-
ganisms were washed 10 times with the anaerobic medium
to remove VFA. The anaerobic medium of phosphate bu1er,
mineral salts and trace metals was made according to Shel-
ton and Tiedge [12]. The bottles were @lled with 250 ml
of anaerobic medium. Food waste was added as a substrate
along with 2:5 g NaHCO3 as a bu1er. The bottles were @lled
to the 400 ml mark using nano-pure water. The desired each
initial pH was adjusted by 2 N KOH and 2 N HCl, and then
50 ml of seed microorganisms was added. The desired each
initial pH was adjusted again after the bottles were @lled to
the 500 ml level using nano-pure water. The bottles were
Nushed with N2 and capped tightly before being put on the
shaking incubators with 100 rpm at 35±1◦ C for mesophilic
and 55 ± 1◦ C for thermophilic condition. Control bottles for
both mesophilic and thermophilic were also prepared with-
out addition of substrate. To maintain the desired each pH
during the test period, 1:0 ml was taken from the serum bot-
tles and the pH was measured using semi-micro pH elec-
trode (Corning, USA). The desired each pH was adjusted
by adding 2 N KOH or 2 N HCl with a syringe.
2.4. Analyses
The biogas produced was measured using glass syringes,
and gas composition was analyzed using a gas chromato-
graph (Gow Mac series 580, USA) with a thermal conduc-
tivity detector and two columns. The methane and carbon
dioxide were detected with a column packed with porapak
Q (80/100 mesh), and the hydrogen was detected with a col-
umn packed with molecular sieve 5A. The temperatures of
injector, detector and column were kept at 80◦ C, 90◦ C and
50◦ C. Helium was used as a carrier gas. VFA was quan-
ti@ed by a high-performance liquid chromatography (Spec-
trasystem P2000, USA) with an ultraviolet (210 nm) detec-
tor and an Aminex HPX-97H (300×7:8 mm2 ) column after
 H.-S. Shin et al. / International Journal of Hydrogen Energy 29 (2004) 1355 – 1363
Table 2
Characteristics of the seed microorganisms and the average operating parameter values from the mesophilic and thermophilic acidogenic
reactors at steady state
Seed microorganism
TSS VSS pH Alkalinity
(mg l−1 ) (mg l−1 ) (mg l−1 as CaCO3 )
T 6408 6280 5.5 1300
M 8725 8470 5.6 1900
T : Thermophilic acidogenic reactor; M : Mesophilic acidogenic reactor; TVFA : total VFA, HAc: acetate, HPr : propionate, n-HBu:
n-butyrate.
pretreatment with 0:45 m membrane @lter. H2 SO4 of
0:005 M was used as a mobile phase. Carbohydrate was
measured using the calorimetric ferric-cyanide method
[13]. Measurements of total solid (TS), volatile solid (VS),
volatile suspended solid (VSS) and pH were performed
according to the Standard Methods [14].
In order to identify the hydrogen producing microorgan-
isms, DNAs from the mesophilic and thermophilic acido-
genic culture were extracted by using the Ultraclean Soil
DNA Kit (Cat # 12800-50; Mo Bio Laboratory Inc., USA).
The Ultraclean Soil DNA Kit was more e1ective than other
methods such as Phenol/chloroform method etc. for DNA
extraction and puri@cation in this study [15]. The 16S rDNA
fragments were ampli@ed by PCR. The region correspond-
ing to positions 357 and 518 in the 16S rDNA of Escherichia
coli was PCR-ampli@ed using the forward primer EUB357f
(5 -CCTACGGGAGGCAGCAG-3 ) with a GC clamp
(5 -CGCCCGCCGCGCCCCGCGCCCGGCCCGCCGCCC
CCG CCCC-3 ) at the 5 end to stabilize the melting
behavior of the DNA fragments and the reverse primer
UNIV518r (5 -ATTACCGCGGCTGCTGG-3 ). PCR am-
pli@cation was conducted in an automated thermal cycler
(MWG-Bio TECH, Germany) using the protocol; that is,
initial denaturation for 4 min at 94◦ C and 30 cycles of
denaturation for 40 s at 94◦ C, annealing for 40 s at 55◦ C,
extension for 1 min at 72◦ C, followed by a @nal extension
for 8 min at 72◦ C. PCR mixtures had a @nal volume of
50 l which contained 5 l of 10 × PCR bu1er, 0:8 mM
MgSO4 , 0:5 mM of each primer, 0:1 mM dNTP, 25 pg
template and 1U polymerase. PCR products were elec-
trophoresed on 2% (wt/vol) agarose gel in 1 × TAE for
30 min for 50 V, and then checked with ethidium bromide
staining to con@rm the amplication. DGGE was carried
out using the Dcode Universal Mutation Detection System
(BioRad, USA) in accordance with the manufacturer’s in-
structions. PCR products were electrophoresed in 1 × TAE
bu1er for 480 min at 70 V and 60◦ C on polyacrylamide gel
(7.5%) containing a linear gradient ranging from 40% to
60% denaturant. After electrophoresis, polyacrylamide gel
was stained with ethidium bromide for 30 min, and then
visualized on UV transilluminator. Most of the bands were
1357
Average operating parameters
Gas content (%) VFA(mg l−1 as COD)
H2 CH4 TVFA HAc HPr n-HBu
54 0 7800 1200 250 5300
3 17 7500 2570 1560 2300
excised from DGGE polyacrylamide gel for 16S rDNA se-
quencing. DNA fragments from the bands excised were pu-
ri@ed using a QIAEX IIGel Extraction Kit (Qiagen, USA),
and then PCR-ampli@ed with the forward primer EUB357f
without a GC clamp and the reverse primer UNIV518r.
After PCR ampli@cation, PCR products were puri@ed us-
ing MultiScreen Vacuum Manifold (MILLIPORE com.,
USA). All the strands of the puri@ed PCR products were
sequenced with primers EUB357f by ABI PRISM Big Ter-
minator Cycle Sequencing Kit (Applied Biosystems, USA)
in accordance with the manufacturer’s instructions. Search
of the GenBank database was conducted using the BLAST
program [16].
3. Results and discussion
3.1. E9ect of pH on hydrogen production
The @rst set of batch experiments was performed at
2 g VS l−1 and pH 4.5, 5.5 and 6.5 for the mesophilic
and thermophilic acidogenic culture. During the tests, each
pH could be adjusted in the range of 4.1– 4.5, 5.3–5.5 and
6.3– 6.8 for the mesophilic test, and 4.2– 4.6, 5.5 –5.8 and
6.4 – 6.8 for the thermophilic test by adding 2 N KOH or
2 N HCl with a syringe. Fig. 1 shows the cumulative hy-
drogen production from the mesophilic and thermophilic
acidogenic culture at tested pH.
The cumulative hydrogen production data were @tted to
the modi@ed Gompertz equation [6] by using the “@t curve”
function in Sigma Plot 2001 version. All of the correlation
coeVcients, R2 , were greater than 0.97 indicating the per-
fect @t to the experimental data. The longer lag-phase of the
thermophilic test than that of the mesophilic test could be
explained by the fact that the thermophilic and mesophilic
seed microorganisms were exposed to a room temperature
(≈ 20◦ C) during the setting time of the tests, thus the ac-
tivity of the thermophilic seed microorganism was more af-
fected by the low temperature than the mesophilic seed mi-
croorganism. Since the mesophilic and thermophilic seed
microorganims were taken from the reactors which were
1358 H.-S. Shin et al. / International Journal of Hydrogen Energy 29 (2004) 1355 – 1363
50
Cumulative hydrogen production (mL)
40
30
20
10
0
0 20
Fig. 1. E1ect of pH on hydrogen production from the mesophilic and thermophilic acidogenic culture.
operated at pH 5:6 ± 0:2, the lag-phase times of pH 4.5
and 6.5 from both mesophilic and thermophilic culture were
longer than the corresponding values of pH 5.5. Table 3
illustrates the distribution of key VFA, cumulative hydrogen
production and headspace gas content with incubation time
at pH 5.5 from the mesophilic and thermophilic acidogenic
culture. Table 4 shows the results from the mesophilic and
thermophilic culture at pH 4.5, 5.5 and 6.5.
From the thermophilic test, butyrate was the main acid
product, while propionate was negligible. The shift of pH
from 4.5 to 6.5 resulted in the decrease of butyrate and hy-
drogen content, while acetate and carbon dioxide gas content
were increased. No methane was detected through out the
test period at all tested pH. In the case of the mesophilic test,
propionate was one of the major acid products and increased
as pH increased. Methane was detected from the incubation
time of 15, 20 and 50 h at pH 6.5, 5.5 and 4.5, respectively,
which almost coincided with the incubation time of max-
imum cumulative hydrogen production of each test. Total
hydrogen production from the thermophilic test was greater
than that from the mesophilic test. The hydrogen produc-
tion reached the maximum at pH 4.5, but the lag-phase time
was the longest among the tested pH for both mesophilic
and thermophilic tests. The yield of hydrogen from the ther-
mophilic test was much higher than that from the mesophilic
test, and reached the maximum of 0:9 mol-H2 =mol-hexose
at pH 4.5. This result was di1erent from the reported op-
timum pH for glucose [4], and high-strength rice winery
wastewater fermentation [9].
Hydrogen gas could be produced if surplus electrons form
in the reaction, and then reduce protons by hydrogenase.
The formation of acetate and butyrate accompanies reduc-
ing power, whereas the formation of lactate, propionate con-
sumed reducing equivalents [17]. It was also reported that
Thermo. pH 4.5
Thermo. pH 5.5
Thermo. pH 6.5
Meso. pH 4.5
Meso. pH 5.5
Meso. pH 6.5
40 60 80 100 120 140 160
Incubation time (hours)
molecular hydrogen was produced during the production of
acetate and butyrate, while hydrogen wass consumed dur-
ing the production of propionate [5,18]. Therefore, lower
hydrogen production from the mesophilic test than the ther-
mophilic test might be related with the higher production
of propionate that consumed hydrogen, and methanogenesis
which converted hydrogen to methane.
3.2. E9ect of VS concentrations on hydrogen production
The second set of batch experiments was performed at
3, 6, 8 and 10 g VS l−1 with the thermophilic acidogenic
culture at pH 5.5. The pH 5.5 was thought to be better to
evaluate hydrogen production on VS concentions because it
could reduce lag-phase time and be less inNuenced by high
VS concentration than pH 4.5. The pH could be adjusted at
5:6 ± 0:2 during the test period. Fig. 2 shows the cumula-
tive hydrogen production at each VS concentration from the
thermophilic acidogenic culture at pH 5.5.
All the correlation coeVcients, R2 , were greater than 0.98.
Table 5 shows the analysis from the thermophilic acidogenic
culture at tested VS concentrations.
The hydrogen production and content were increased
as VS concentration increased, and the maximum hydro-
gen content of 69% occurred at 10 g VS l−1 . The biogas
was free of methane at all tested VS concentrations, and
the concentrations of butyrate, acetate and propionate
were 54–60 × 22–31%, and 0.3–1% (c/c), respectively.
The speci@c hydrogen production potentials were from
46.1 to 91:5 ml g VS−1 , and reached the maximum of
91:5 ml g VS−1 at 6 g VS l−1 . The speci@c hydrogen pro-
duction rates ranged from 12 to 19 ml g VS S−1 h−1 , and
the maximum appeared at 10 g VS l−1 . The speci@c hy-
drogen production potentials were similar to the @ndings
 H.-S. Shin et al. / International Journal of Hydrogen Energy 29 (2004) 1355 – 1363 1359

Table 3
Analysis from the mesophilic and thermophilic acidogenic culture at pH 5.5

Time Thermophilic acidogenic culture Mesophilic acidogenic culture

(h)
HAc HPr n-Hbu Cumulative Headspace HAc HPr n-Hbu Cumulative Headspace
(mg l−1 ) (mg l−1 ) (mg l−1 ) H2 (ml) gas content (%) (mg l−1 ) (mg l−1 ) (mg l−1 ) H2 (ml) gas content (%)

H2 CO2 N2 CH4 H2 CO2 N2 CH4

10 0 0 51 0 0 38 62 0 164 130 268 2 1 53 46 0

30 116 1 365 21 15 49 36 0 224 265 299 2.5 0 59 39 2
54 103 8 593 30 15 60 25 0 286 305 294 2.5 0 60 37 3
85 95 12 793 38 17 58 25 0 343 319 278 2.5 0 60 35 5
134 137 0 898 43 21 57 22 0 408 365 294 2.5 0 60 34 6

Table 4
Analysis from the mesophilic and thermophilic acidogenic culture at tested pH

pH H2 H2 H2 yield SHPP SHPR Lag-phase VFA (mg l−1 as COD)

(ml) (%) (mol-H2 / (ml H2 g VS−1 ) ml H2 g VS S−1 h−1 ) time (h)
mol-hexose) TVFA HAc HPr n-HBu

4.5 46.3 23 0.9 46.3 3.0 23.0 994 65 0 925

T 5.5 40.1 21 0.8 40.1 2.9 11.7 1120 137 0 898
6.5 28.4 14 0.6 28.4 2.5 14.9 992 254 0 651

4.5 5.0 4 0.1 5.0 0.4 3.6 1152 185 141 628
M 5.5 2.5 1 0.05 2.5 0.7 0.5 1337 408 365 294
6.5 1.3 0.5 0.03 1.3 0.3 0.1 1286 524 460 177

T : Thermophilic acidogenic culture; M: Meosphilic acidogenic culture; SHPP : Speci@c hydrogen production potential; SHPR : Speici@c
hydrogen production rate.

400
3gVS
6gVS
Cumulative hydrogen production (mL)

8gVS
10gVS
300

200

100

0
0 50 100 150 200 250 300
Time (hours)

Fig. 2. E1ect of VS concentrations on hydrogen production from the thermophilic acidogenic culture at pH 5.5.
1360 H.-S. Shin et al. / International Journal of Hydrogen Energy 29 (2004) 1355 – 1363

Table 5
Analysis using the thermophilic acidogenic culture at tested VS concentrations

VS H2 H2 H2 yield SHPP SHPR Lag-phase VFA (mg l−1 as COD)

(g l−1 ) (ml) (%) (mol-H2 / (ml H2 g VS−1 ) (ml H2 g VS S−1 h−1 ) time (h)
mol-hexose) TVFA HAc HPr n-HBu

3 69 23 0.9 46.1 13.7 10.7 1310 375 4 730

6 274 55 1.8 91.5 12.0 17.4 3625 490 9 1315
8 297 64 1.5 74.1 12.0 12.4 5245 847 10 1633
10 350 69 1.4 70.1 19.0 18.0 5580 808 36 2237

Table 6
Comparison of hydrogen yield obtained in this study to those cited in the literature

Acclimated condition of seed sludges (Substrate) Hydrogen production

HRT Temp. (o C) pH pH H2 yield Reference

(mol-H2 /mol-hexose)

5 day 55 ± 1 5:6 ± 0:2 Food waste 5.5 1.80 This study

10 h 35 5:0 ± 0:2 Rice bran 6.0 1.29 [21]
12 h 60 6.8 Sugar wastewater 6.8 2.59 [20]
3 day 60 6.8 Sugar wastewater 6.8 1.91 [20]
10 h 35 4.5 –5.0 Glucose 6.0 1.43 [8]
6h 35 ± 1 5.7 Glucose 5.7 1.70 [7]

of Okamoto et al. [19] in which the maximum hydrogen
production potential was 96 ml g VS−1 from rice with 4%
TS. Table 6 compares the hydrogen yield of this study with
those found in the literature.
The maximum hydrogen yield of 1:8 mol-H2 =mol-hexose
obtained in this study was comparable with the yield of
rice bran [21], sugar wastewater [20] and glucose [8,7].
Ueno et al. [20] successfully performed continuous hydro-
gen production from sugary wastewater with an yield of
1:91 mol-H2 =mol-hexose at 3 days HRT, 60◦ C and pH 6.8.
The biogas was composed of 64% hydrogen, 36% carbon
dioxide and less than 0.13% methane. From the results, it
was suggested that the thermophilic acidogenic condition
inactivating methanogenesis resulted in high yield of hydro-
gen from food waste.
3.3. PCR-DGGE analysis
The microbial community in the mesophilic and ther-
mophilic acidogenic culture was analyzed and compared by
PCR-DGGE analysis targeted at eubacterial 16S rDNA, and
the DGGE pro@les are shown in Fig. 3.
The major bands in the DGGE gels were excised and pu-
ri@ed to determine the sequence. The results of the sequence
aVliation determined by the BLAST are shown in Table 7.
The number of bands detected from the mesophilic acido-
genic culture was greater than that from the thermophilic
acidogenic culture. This is comparable with the result of
Lapara et al. [22] who suggested that elevated temperature
could reduce species diversity.
Thermoanaerobacterium thermosaccharolyticum (band
B-1) and Desulfotomaculum geothermicum (bands B-5,
B-6), which were known as hydrogen producing bacteria,
appeared from the thermophilic acidogenic culture.
T. thermosaccharolyticum is a thermophilic saccha-
rolytic microorganism which can produce large amount
of hydrogen from carbohydrates [23]. Ueno et al. [17]
studied hydrogen production by thermophilic anaerobic mi-
croNora enriched from sludge compost by using an arti@cial
medium containing cellulose powder. Under all applied
culture conditions in batch and chemostat tests, microor-
ganisms closely related to T. thermosaccharolyticum were
isolated and detected with strong intensity by PCR-DGGE
analysis. Ueno et al. [17] suggested that T. thermosac-
charolyticum involved in acetate/butyrate fermentation led
to hydrogen production. According to the characteristic
study of T. thermosaccharolyticum [24], the maximum
growth of T. thermosaccharolyticum appeared at pH 5 to
6, and the optimum temperature was 60◦ C. The production
yield of hydrogen from T. thermosaccharolyticum was
2:4 mol-H2 =mol-glucose, a nearly equivalent hydrogen pro-
duction ability to those of Clostridium butyricum which
had hydrogen production yield of 2:4 mol-H2 =mol-hexose.
D. geothermicum was a thermophilic, fatty acid-degrading,
sulfate-reducing bacterium [25]. Thermodesulfobacteria
class. nov. ferment pyruvate, and principal fermentation
 H.-S. Shin et al. / International Journal of Hydrogen Energy 29 (2004) 1355 – 1363 1361

Table 7
AVliation of DGGE fragments determined by their 16S rDNA
sequence

Band AVliation Similaritya Accession

no. (%) no.

A-1 Bacteroidales str.. 93 AB078832

A-2 Prevotella sp. 87 AF537212
A-3 TM7 phylum sp. 90 AF385506
A-4 Thermotogales str. 87 AJ431248
A-5 Bacillus sp. 87 AY178858
A-6 Rhizosphere soil bacterium 88 AJ252680
A-7 Eubacterium sp. 83 AF287761
A-8 Arctic sea ice bacterium 87 AF468442
A-9 P. nigrescens 89 AF414844
A-10 P. bryantii 89 AY189149
A-11 Bacteroidales str. 82 AF481205
B-1 T. thermosaccharolytium 92 M59119
B-2 TM7 phylum sp. 90 AF385506
B-3 Arctic sea ice bacterium 87 AF468442
B-4 P. nigrescens 89 AF414844
B-5 D. geothermicum 98 AJ294428
B-6 D. geothermicum 98 AJ294428
a Percentage similarity to the closest relative according to the

BLAST comparison.

produced acetate, CO2 and H2 from the fermentation of glu-

Fig. 3. DGGE pro@les of the PCR-ampli@ed 16S rDNA extracted
from the mesophilic and thermophilic acidogenic culture. (A)
Mesophilic acidogenic culture, and (B) thermophilic acidogenic
culture.
cose, was detected [28]. A band aVliated with the Bacillus
species (band A-5) that produced H2 , was detected [29], and
the Bacteroidales strain (band A-1, A-11) which produced
succinate, acetate, lactate, formate, or propionate from the
fermentation of carbohydrate or peptone, was also detected
[30]. Bands each aVliated with the TM7 phylum species
(band A-3, B-2) [31], Arctic sea ice bacterium (band A-8,
B-3) [32], Rhizosphere soil bacterium (band A-6) [33]
which were not related to hydrogen production, were de-
tected. TM7 phylum species, Arctic sea ice bacterium and
P. nigrescens, were detected from both thermophilic and
mesophilic culture.

end products were acetate, CO2 , and hydrogen. Sulfate 4. Conclusions

and thiosulfate were used as electron acceptor for growth,
while lactate and pyruvate as electron donors for growth
[26]. The DGGE-PCR analysis from the thermophilic aci-
dogenic culture indicated that T. thermosaccharolyticum
and Desulfotomaculum geothermicum were the microor-
ganisms involved in hydrogen producing acetate/butyrate
fermentation.
From the mesophilic acidogenic culture, bands A-2, A-9
and A-10 which were closely related to Prevotella species,
Prevotella nigrescens and P. bryantii, were detected. Pre-
votella species produced acetic, isobutyric, isovaleric and
succinic acids as metabolic end products from the fer-
mentation of glucose, sucrose and starch [27]. A band
aVliated with the Thermotogales strain (band A-4) that
Thermophilic acidogenesis acclimated with food waste
at 5 days HRT which was enough HRT for the acidi@-
cation of the waste showed e1ective hydrogen produc-
tion. The thermophilic condition had inhibitory e1ect on
methane and propionate production. The higher hydrogen
production from the thermophilic acidogenic culture than
the mesophilic acidogenic culture was caused by free of
methane and negligible propionate which were hydrogen
consumers. Increase of VS concentrations resulted in the
increase of quantity and quality of hydrogen production.
Hydrogen producing microorganisms of T. thermosaccha-
rolytium and D. geothermicum were detected from the ther-
mophilic acidogenic culture, while Thermotogales strain
1362 H.-S. Shin et al. / International Journal of Hydrogen Energy 29 (2004) 1355 – 1363
and Bacillus species were detected from the mesophilic aci-
dogenic culture by PCR-DGGE analysis. The results of this
study showed the feasibility of hydrogen production from
carbohydrate-rich organic solid wastes such as food waste
at enough HRT for the wastes acidi@cation by thermophilic
acidogenesis.
Acknowledgements
This work was supported by grant No. M1-0203-00-0063
from the National Research Laboratory Program of the Ko-
rean Ministry of Science and Technology.
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