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of 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 l1 resulted in the increase of quantity and quality of
hydrogen production. The maximum hydrogen content was 69% (v/v) at 10 g VS l1 . The hydrogen yield was in the range
of 0.9 1:8 mol-H2 =mol-hexose and peaked at 6 g VS l1 . Normal butyrate was the main acid product, and the percentages
of butyrate, acetate and propionate at tested VS concentrations were 54 60%, 2231% and 0.31%, 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
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 require 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
either for methane production by methanogenesis or a readily biodegradable carbon source for biological nutrients removal [13]. Therefore, the harvest of hydrogen at the acidi@cation stage of anaerobic treatment, leaving the remaining
acidi@cation products such as acetate and butyrate for further methane production or external carbon source for biological 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 hydrogen consumers, inocula were cultivated with pure chemicals 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
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H.-S. Shin et al. / International Journal of Hydrogen Energy 29 (2004) 1355 1363
Table 1
Characteristics of food waste
Item
pH
TS
VS
VS/TS
Total carbohydrate
Carbon, C
Hydrogen, H
Oxygen, O
Nitrogen, N
Sulfur, S
C/N
Unit
g l1
g l1
g l1
% TS
% TS
% TS
% TS
% TS
Value
5.8
67.8
63.7
0.94
25.5
51.2
7.7
38.3
2.8
0.7
18.3
studied in 715 ml serum bottles. The mesophilic and thermophilic culture taken from each reactor were settled for
1 h, and the supernatant was then removed. The settled cultures were used as seed microorganisms. The seed microorganisms 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 Shelton 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 351 C for mesophilic
and 55 1 C for thermophilic condition. Control bottles for
both mesophilic and thermophilic were also prepared without addition of substrate. To maintain the desired each pH
during the test period, 1:0 ml was taken from the serum bottles and the pH was measured using semi-micro pH electrode (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 chromatograph (Gow Mac series 580, USA) with a thermal conductivity 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 column 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 quanti@ed by a high-performance liquid chromatography (Spectrasystem P2000, USA) with an ultraviolet (210 nm) detector and an Aminex HPX-97H (3007:8 mm2 ) column after
H.-S. Shin et al. / International Journal of Hydrogen Energy 29 (2004) 1355 1363
1357
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
T
M
TSS
(mg l1 )
VSS
(mg l1 )
pH
6408
8725
6280
8470
5.5
5.6
Alkalinity
(mg l1 as CaCO3 )
VFA(mg l1 as COD)
H2
CH4
TVFA
HAc
HPr
n-HBu
1300
1900
54
3
0
17
7800
7500
1200
2570
250
1560
5300
2300
T : Thermophilic acidogenic reactor; M : Mesophilic acidogenic reactor; TVFA : total VFA, HAc: acetate, HPr : propionate, n-HBu:
n-butyrate.
excised from DGGE polyacrylamide gel for 16S rDNA sequencing. DNA fragments from the bands excised were puri@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 using MultiScreen Vacuum Manifold (MILLIPORE com.,
USA). All the strands of the puri@ed PCR products were
sequenced with primers EUB357f by ABI PRISM Big Terminator Cycle Sequencing Kit (Applied Biosystems, USA)
in accordance with the manufacturers 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 l1 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, 5.35.5 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 hydrogen 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 perfect @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 activity of the thermophilic seed microorganism was more affected by the low temperature than the mesophilic seed microorganism. 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
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
30
20
10
0
0
20
40
60
80
100
120
140
160
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
(h)
HAc
HPr
n-Hbu
Cumulative Headspace
gas content (%)
(mg l1 ) (mg l1 ) (mg l1 ) H2 (ml)
H2 CO2 N2 CH4
10
30
54
85
134
0
116
103
95
137
0
1
8
12
0
51
365
593
793
898
0
21
30
38
43
0
15
15
17
21
38
49
60
58
57
62
36
25
25
22
0
0
0
0
0
H2 CO2 N2 CH4
164
224
286
343
408
130
265
305
319
365
268
299
294
278
294
2
2.5
2.5
2.5
2.5
1
0
0
0
0
53
59
60
60
60
46
39
37
35
34
0
2
3
5
6
Table 4
Analysis from the mesophilic and thermophilic acidogenic culture at tested pH
pH
H2
(ml)
H2
(%)
H2 yield
(mol-H2 /
mol-hexose)
SHPP
(ml H2 g VS1 )
SHPR
ml H2 g VS S1 h1 )
Lag-phase
time (h)
HAc
23
21
14
0.9
0.8
0.6
46.3
40.1
28.4
3.0
2.9
2.5
23.0
11.7
14.9
994
1120
992
65
137
254
0
0
0
925
898
651
0.1
0.05
0.03
5.0
2.5
1.3
0.4
0.7
0.3
3.6
0.5
0.1
1152
1337
1286
185
408
524
141
365
460
628
294
177
4.5
5.5
6.5
46.3
40.1
28.4
4.5
5.5
6.5
5.0
2.5
1.3
4
1
0.5
HPr
n-HBu
T : Thermophilic acidogenic culture; M: Meosphilic acidogenic culture; SHPP : Speci@c hydrogen production potential; SHPR : Speici@c
hydrogen production rate.
400
3gVS
6gVS
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
(g l1 )
H2
(ml)
H2
(%)
H2 yield
(mol-H2 /
mol-hexose)
SHPP
(ml H2 g VS1 )
SHPR
(ml H2 g VS S1 h1 )
Lag-phase
time (h)
HAc
HPr
n-HBu
3
6
8
10
69
274
297
350
23
55
64
69
0.9
1.8
1.5
1.4
46.1
91.5
74.1
70.1
13.7
12.0
12.0
19.0
10.7
17.4
12.4
18.0
1310
3625
5245
5580
375
490
847
808
4
9
10
36
730
1315
1633
2237
Table 6
Comparison of hydrogen yield obtained in this study to those cited in the literature
Acclimated condition of seed sludges
HRT
Temp. (o C)
pH
5 day
10 h
12 h
3 day
10 h
6h
55 1
35
60
60
35
35 1
5:6 0:2
5:0 0:2
6.8
6.8
4.5 5.0
5.7
(Substrate)
Hydrogen production
Food waste
Rice bran
Sugar wastewater
Sugar wastewater
Glucose
Glucose
pH
H2 yield
(mol-H2 /mol-hexose)
Reference
5.5
6.0
6.8
6.8
6.0
5.7
1.80
1.29
2.59
1.91
1.43
1.70
This study
[21]
[20]
[20]
[8]
[7]
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
no.
AVliation
Similaritya
(%)
Accession
no.
A-1
A-2
A-3
A-4
A-5
A-6
A-7
A-8
A-9
A-10
A-11
B-1
B-2
B-3
B-4
B-5
B-6
Bacteroidales str..
Prevotella sp.
TM7 phylum sp.
Thermotogales str.
Bacillus sp.
Rhizosphere soil bacterium
Eubacterium sp.
Arctic sea ice bacterium
P. nigrescens
P. bryantii
Bacteroidales str.
T. thermosaccharolytium
TM7 phylum sp.
Arctic sea ice bacterium
P. nigrescens
D. geothermicum
D. geothermicum
93
87
90
87
87
88
83
87
89
89
82
92
90
87
89
98
98
AB078832
AF537212
AF385506
AJ431248
AY178858
AJ252680
AF287761
AF468442
AF414844
AY189149
AF481205
M59119
AF385506
AF468442
AF414844
AJ294428
AJ294428
produced acetate, CO2 and H2 from the fermentation of glucose, 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 detected. TM7 phylum species, Arctic sea ice bacterium and
P. nigrescens, were detected from both thermophilic and
mesophilic culture.
4. Conclusions
Thermophilic acidogenesis acclimated with food waste
at 5 days HRT which was enough HRT for the acidi@cation of the waste showed e1ective hydrogen production. 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. thermosaccharolytium and D. geothermicum were detected from the thermophilic 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 acidogenic 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 Korean Ministry of Science and Technology.
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