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I N T E R N AT I O N A L J O U R N A L O F H Y D R O G E N E N E R G Y 3 3 ( 2 0 0 8 ) 1 5 2 2 – 1 5 3 1
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Process performance evaluation ofintermittent–continuous stirred tank reactor for anaerobichydrogen fermentation with kitchen waste
Shiue-Lin Li, Shih-Chiang Kuo, Jian-Sheng Lin, Ze-Kun Lee,Yu-Hsuan Wang, Sheng-Shung Cheng�
Department of Environmental Engineering, National Cheng Kung University, No. 1, University Road, Tainan 701, Taiwan, ROC
a r t i c l e i n f o
Article history:
Received 11 July 2007
Accepted 28 September 2007
Available online 3 December 2007
Keywords:
I-CSTR
Kitchen waste
TRFLP
Anaerobic batch test
nt matter & 2007 Internae.2007.09.049
hor. Tel.: +886 6 2757575xscheng@mail.ncku.edu.t
a b s t r a c t
A 3-L laboratory scale hydrogen fermentor with fill-and-draw operation defined as
intermittent–continuous stirred tank reactor (I-CSTR) was established. There were four
operational periods included in this study. In run 1 during the acclimation period, corn
starch was added as the auxiliary substrate. The feedstocks of the next two periods were
corn starch and kitchen mixture, the Taipei kitchen waste and the Kaohsiung kitchen
waste, respectively. Compared between these two periods, run 3 fed with Kaohsiung
kitchen waste gives a higher hydrogen producing rate of 27 mmol/L/day than run 2. At run 4
that loading rate was increased and hydraulic retention time was decreased. The
maximum hydrogen producing rate of 118 mmol/L/day was found. By the detection of
terminal restriction fragment length polymorphism (TRFLP) method, some species of
Clostridium such as Clostridium stercorarium, Clostridium thermolacticum, Clostridium aldrichii,
Clostridium cellobioparum, Clostridium termitidis (cluster III) and Clostridium formicoaceticum
(cluster XI) were presented in all the operational periods. Furthermore, the anaerobic batch
test for the substrate utilizing efficiency of the sludge taken from the fermentor was also
detected. In the test, the maximum hydrogen producing rate of 48 mmol/L/day was
detected. The VSS and the total carbohydrate removal were 35% and 66%, respectively.
Organic nitrogen had only a little fraction converted to ammonia, but the oil and grease
were not degraded.
& 2007 International Association for Hydrogen Energy. Published by Elsevier Ltd. All rights
reserved.
1. Introduction
Hydrogen is an ideal alternative fuel because of its clean,
sustainable and renewable characteristics. Hydrogen has the
energy yield of 122 kJ/g that is 2.75 times higher than
hydrocarbon fuels and could be directly used to produce
electricity through fuel cells [1,2]. Biological hydrogenation
studies of single substrate such as glucose, sucrose or starch
have been extensively performed [3–5]. Researches of hydro-
tional Association for Hy
65827; fax: +886 6 2752790w (S.-S. Cheng).
genation from food waste have also been reported. Lay
et al. [6] discovered the optimal nutrient condition as Fe2þ¼
132 mg/L, NHþ4 ¼ 537 mg/L and PO3�4 ¼ 1331 mg/L for high
solid hydrogen fermentation, and the specific hydrogen
production rate were 20 mmol/gTVS/day. Some of the special
bioreactor designs was applied in kitchen waste fermentation
for a higher efficiency. For example, Han and Shin [7] developed
a leaching-bed bioreactor for hydrogenation, and the max-
imum hydrogen production rate was 138.5 mmol/L/day. Shin
drogen Energy. Published by Elsevier Ltd. All rights reserved.
.
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and Yuon [8] developed a thermophilic hydrogen fermentor
and the hydrogen producing rate of 39.5 mmol/L/day has been
reported. Angelidaki et al. [9] developed a two-stage process
combining hydrogen and methane production, and the
methane was re-sparging back to the hydrogen fermentor.
The hydrogen production rate was increased to 138 mmol/L/
day via the methane sparging devices.
In Taiwan, the kitchen waste has been collected and
classified by the collecting systems of the local environmental
protection administrations since 2003, and the amount of it is
increasing. According to the statistic data, the amount of
kitchen waste collected in 319 towns in Taiwan amounted to
1600 tons per day, and the recycling percentage was about
33.5%. The government in Taiwan also announced that the
recycling percentage will increase to 75% in 2020. As the
promotion of the policy, the kitchen waste has become the
largest amount and nutritive waste in Taiwan. Since kitchen
waste is the sustainable feedstock for hydrogen fermentation,
it is more suitable for truly continuous bio-energy production
process.
Therefore, the aim of this study was to investigate the
composition of different kitchen waste resourses and hydro-
genation potential in the anaerobic process. Terminal restric-
tion fragment length polymorphism (TRFLP), a novel molecular
method, was applied in this study for bio-dynamic monitoring.
The batch test in this study was to confirm the hydrogenation
mechanism and the biodegradability between the complex
substrates: carbohydrate, protein and fat. All the information
obtained from this study could be used to assess the feasibility
in practical applications.
2. Materials and methods
2.1. Feedstock sources
There were two sources of kitchen waste in this study from
Taipei City and Kaohsiung City in Taiwan. The kitchen waste
Fig. 1 – Schematic diagram of I-CSTR anaerobi
from Taipei was collected from the household waste and part of
the vegetable market waste. These wastes were collected as the
composting material for fertilizing application. The composition
of Taipei kitchen waste was considered as some rare and
cellulose-like materials. The kitchen waste from Kaohsiung was
collected from the restaurant. Compared with that in Taipei, the
composition of kitchen waste in Kaohsiung was supposed as
more cooked materials and easily degradable compounds.
2.2. Operation of intermittent–continuous stirred tankreactor
An intermittent–continuous stirred tank reactor (I-CSTR) as
shown in Fig. 1 was used in this study to enrich hydrogen
producing bacteria. The total volume of the CSTR is 3 L. A
complete-mix condition was achieved with a mechanic mixer
at an agitating speed of 160 rpm. The screw type of baffle was
set to increase the turbulence during operation. The amount
of gases produced in the I-CSTR due to fermentation was
measured with a wet-gas flow meter. The I-CSTR and the off-
gas flow meter were kept in an air-bath incubator and the
temperature was maintained at 35� 1 �C. The agitation rate
was 160 rpm. The collected kitchen waste was stored at 4 �C in
a refrigerator at all time and fed into the I-CSTR using a
peristaltic pump with fill-and-draw operation. The fill and
draw operated with 750-mL influent and 750-mL effluent
every cycle. In order to provide sufficient alkalinity required
for neutralizing the acids produced in the I-CSTR, the pH
controller (Suntex pH/ORP controller PC-330) was set to
maintain pH value between 5.3 and 5.6. The seeding micro-
organisms for the I-CSTR were obtained from 3-ton pilot scale
acidogenesis tank in Hong-Gin kitchen waste composting
plant. The amount of seeding microorganisms in the start-up
phase was about 40 g dry weight in the fermentor. Every run
was operated at least for 20 days to get the sufficient samples
for statistic evaluation. Different feedstock and hydraulic
retention time (HRT) controlled are listed in Table 1.
c hydrogen fermentor with kitchen waste.
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Table 1 – Kitchen waste characteristic
Food wastecharacteristics
Units (g/L)
Taipeiðn ¼ 4Þ
Kaohsiungðn ¼ 7Þ
TS 50� 16 55� 10
TVS 40� 15 50� 10
SS 39:9� 11:8 42� 9
VSS 36:7� 12:5 41� 9
Total COD 82� 28 106� 30
Soluble COD 30:5� 10 35� 1:2
Total carbohydrate 5:3� 2:6 15� 5
Soluble carbohydrate 0:7� 0:4 4� 3
Oil and grease 7:6� 2 11� 4
NHþ4 –N 0:3� 0:2 0:2� 0:1
Total org-N 1:6� 0:5 2:5� 0:6
Soluble org-N 0:6� 0:3 0:8� 0:4
pH 4:5� 0:1 4:5� 0:2
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2.3. Analytical methods
The composition of biogas in the headspace was analyzed
using a gas chromatograph (China GC 8900, Taipei, Taiwan)
equipped with a thermal conductivity detector (TCD). A 2 m
stainless-steel column packed with Hayesep Q (60/80 mesh)
was installed in a 60 �C oven. The operational temperatures of
the injection port, the oven and the detector were all set at
60 �C. Nitrogen was used as the carrier gas at a flow rate of
15 mL/min. Concentrations of volatile fatty acids (VFAs) were
determined using an ion chromatography (Dionex DX-120,
California, USA) equipped with an anion IonPac ICE-ASI
column, an AMMA-ICE II suppressor and a conductivity
detector. One millimolar heptafluorobutylic acid was used
as the eluent at a flow rate of 0.8 mL/min. Five millimolar
Tetrabutylammonium hydroxide was used as the regenerant.
The carbohydrate was analyzed using the phenol–sulfuric
acid method [10]. The pH, NHþ4 –N, chemical oxygen demand
(COD), total solids (TS), total volatile solids (TVS), suspended
solids (SS), volatile suspended solids (VSS) and oil and grease
were measured according to Standard Methods [11].
2.4. DNA extraction
A modification of the sodium dodecyl sulfate (SDS)-based
extraction method was used to obtain genomic DNA from the
activated sludge samples [12]. Briefly, 1-mL samples of
biomass taken from the fermentor were centrifuged in
1.5-mL microtest tubes at 13,000 rpm for 5 min. The super-
natant was removed, and resuspended with phosphate buffer
(100 mM NaH2PO4, pH 8). This procedure was repeated for
three times for cleaning up the pellet. After cleaning up, the
pellets were mixed and resuspended with 300mL phosphate
buffer and 300mL of lysis buffer (100 mM NaCl, 500 mM Tris,
10%SDS, pH 8). The mixture was incubated at 70 �C for 5 min.
After the incubation, the mixture was centrifuged at
13,000 rpm for 3 min. Then, the DNA from the supernatant
and the sediments were extracted following the phenol–
chloroform–isoamyl alcohol procedure, respectively. The
extracted DNA was stored at �20 �C for later use in TRFLP.
2.5. Polymerase chain reaction
The polymerase chain reaction (PCR) was performed on the
extracted DNA in order to amplify the 16S ribosomal DNA (16S
rDNA) genes. The forward primer of EUB 0338 (S-D-Bact-0338-
a-S-18, bacterial primer) with a 6-carboxy-fluorescine label
and the reverse primer of 1392r (S-*-Univ-1392-a-A-15, uni-
versal primer) were used for PCR reactions [13]. The PCR
reaction mixture contained 1� PCR buffer, 0.2 mM of deox-
ynucleoside triphosphate (2.5 mM each), 0:2mM each of
forward and reverse primers, 0:5mL of the DNA template
and 2.5 U of Taq DNA polymerase (Takara Shuzo Co., Otsu,
Japan) in a final volume of 30mL. Amplification was performed
using an automated thermal cycler (RoboCycler, Stratagene,
La Jolla, USA). PCR reactions were performed as follows: 30
cycles of denaturation (1 min at 94 �C), annealing (1 min at
55 �C) and extension (1.5 min at 72 �C), followed by a final
extension of 10 min. The products were checked by applying
to a 2% agarose gel.
2.6. Terminal restriction fragment polymorphism
The purified fluorescently labeled PCR products were digested
with the restriction enzyme of MseI (Promega, Madison, WI,
USA) for 3 h at 37 �C [13]. The fluorescently labeled terminal
restriction fragments obtained after digestion were analyzed
for gel electrophoresis in the Nucleic Acid Analysis and
Synthesis Core Laboratory at the National Cheng Kung
University in Tainan, Taiwan, to determine the size of
fragments obtained from each sample. The lengths of the
terminal fragments were obtained by electrophoresis with an
ABI Prism 377 automated sequencer (Perkin-Elmer Corp.,
Wellesley, MA).
3. Results and discussion
3.1. Characterization of kitchen waste
The characteristics of kitchen waste are listed in Table 1.
These data were collected and averaged from several
analyses. Large amount of standard deviation was shown as
the great variety of kitchen waste. Generally speaking, the
characteristics of kitchen waste in Taipei have higher variety
than that in Kaohsiung. The kitchen waste in Kaohsiung
revealed higher COD concentration, 35,000 mg/L, than that in
Taipei. Furthermore, the carbohydrate, 15,000 mg/L, the oil
and the grease, 11,000 mg/L, and the organic nitrogen of
2500 mgN/L of the kitchen waste in Kaohsiung were all higher
than the data in Taipei. The kitchen waste in Taipei had a
significant difference between TS and SS concentrations of
10.1 g/L, and that in Kaohsiung had the difference in
concentrations of 13 g/L. This phenomenon was caused by
high salt concentration of special diet customs in Taiwan. In
addition, high concentration of fat and organic nitrogen
caused by diet custom was also detected. Because of the hot
weather in Taiwan, kitchen waste can easily self-acidify in
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the storage tank. That is why the low concentration of
carbohydrate and the high concentration of ammonia pro-
vided by protein cleavage were detected. High concentration
of soluble COD showed the VFA produced from carbohydrate
and protein acidification.
The COD distribution of two different resources was also
calculated. The oil and grease were considered to be the
experience formula of triolein as C57H104O6 [14]. The solid
protein and the soluble organic nitrogen were considered to
be the experience formula as C16H24O5N4 [15]. After the
electron distribution calculation, the COD recovery reached
90%. Thus, the calculated results were credible and are
presented in Fig. 2. Compared with these two resources, the
fraction of soluble organic nitrogen was 7% and similar in two
different sources. The kitchen waste from Kaohsiung has 12%
solid carbohydrate fraction, larger than that in Taipei. The
kitchen waste in Taipei was mainly composed by the solid
protein while that in Kaohsiung was by the oil and grease.
However, oil and grease accounted for a great quantity in the
two sources. Oil and grease were considered to be the hard
degradable but electronphilic compounds in the kitchen
waste. Greasy compound degradation would be a challenge
in hydrogen fermentation process study.
3.2. Process performance evaluation of differentoperational periods of hydrogen fermentor
The operational parameters and reactor performance of the
anaerobic hydrogen fermentor in different experimental runs
are summarized in Table 2. In all the operation periods, run 4
had the greatest hydrogen production rate due to the highest
volumetric loading rate of 60 mmol/L/day. But run 1 had the
greatest hydrogen production yield of 2 mmol/gCOD, because
there was corn starch added as auxiliary substrate, and corn
starch can be easily converted to hydrogen than real kitchen
Fig. 2 – Electron distribution of kitchen waste from (A) Taipei
City and (B) Kaohsiung City in terms of COD.
waste. Run 2 had the worst hydrogen producing rate and
yield, and there was also some methane production in run 2.
In all the runs the MLVSS removal rate was over 45%, and it
contributed a lot in waste reducing and hydrolysis. The COD
removal rate was about 17–25% in all the operational periods
due to the oil and grease removal. The oil and grease were not
supposed to be degraded but stacked on the wall of the
reactor. There was the largest amount of ammonium
production at run 3. In a dark fermentation, acetate and
butyrate were the major by-products [16]. In all the opera-
tional periods, butyrate concentration was greater than
acetate except in run 1. The lactate was the major VFA in
the collected kitchen waste. During the operation, the lactate
would be degraded in the fermentor. The lactate degrading
mechanism and how it affected hydrogen production are still
unknown in this study.
3.3. Process performance evaluation of the Taipei kitchenwaste fermentation
The fermentor starts with HRT 4 days, loading rate as
20 gCOD/L/day (Fig. 3). The pH value was controlled well at
5.5. In the first 20 days, corn starch was added as the auxiliary
substrate. The kitchen waste loading rate was 10 gCOD/L/day,
and corn starch loading rate was also 10 gCOD/L/day. It made
the high concentration of carbohydrate of about 40 g/L. In this
operation period, the hydrogen producing rate was about
39 mmol/L/day. There was no methane production in run 1.
The hydrogen concentration and producing rate remained
stable in the first 20 days. The total COD concentration
removal was 20–40%. The effluent MLVSS concentration was
about 18 g/L and had 50% removal. There was no significant
ammonia production or organic nitrogen degradation de-
tected. The effluent oil and grease concentration were
2000 mg/L and had 60% removal. At run 2, the operational
HRT and loading rate were the same as run 1, but the corn
starch, the auxiliary substrate, was removed. The kitchen
waste was all loaded with 20 kgCOD/m3/day, and influent
carbohydrate was about 4000–5000 mg/L. The carbohydrate
removal was significantly lower than run 1. After the 20-day
operation in run 2, the carbohydrate removal could only reach
50%. At day 30, the peak hydrogen producing rate was
detected because of the higher carbohydrate concentration
kitchen waste fed in the fermentor. The different influent
analysis data are not shown in this figure. Besides, the
average hydrogen producing rate was only 0.03 mmol/L/day.
The methane production was 0.62 mmol/L/day. Total biogas
consisted mainly of carbon dioxide and the production rate
was significantly lower than that in run 1. The COD removal
and fat removal were the same as that in run 1. There was still
no obvious ammonia produced nor organic nitrogen degrada-
tion detected, and the oil and grease maintained 60%
degradation. There are several possible reasons giving an
explanation to the lower hydrogen production rate observed
at full kitchen waste loading condition. The possible explana-
tions include (1) lack of degradable carbohydrate in influent
carbohydrate, (2) high ammonia concentration inhibition,
(3) high oil and grease concentration inhibition of hydrogen
production. Sung and Liu [17] indicated ammonia inhibition
phenomena in anaerobic system. Lalman and Bagley [18] also
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Table 2 – Operational parameters and performance indicators in this study
Operational parameter Unit Run 1 Run 2 Run 3 Run 4
Loading gCOD/L/day 20� 3 20� 4 26� 7 50� 16Feedstock – CSa/TpKb TpK KhKc KhKc
HRT day 4 4 4 2
Performance indicator
Hydrogen production rate mmol/L/day 40� 4 0:04� 0:12 28� 10 60� 20
Hydrogen production yield mmol/gCOD 2� 0:6 1:2� 0:1d 1� 0:1 1:2� 0:04
Hydrogen percentage in biogas % 36� 4 1:5� 1 33� 6 35� 5
VSS removal % 57� 9 45� 8 46� 7 48� 2
COD removal % 26� 8 17� 8 21� 12 33� 3
Total carbohydrate removal % 86� 5 40� 13 66� 8 41� 7
Soluble carbohydrate removal % 91� 8 59� 11 69� 19 70� 16
Organic nitrogen conversion % 15� 10 69� 6 29� 11 69� 8
Oil and grease removal % 46� 33 53� 15 63� 8 55� 26
Ammonia production mgN/L 80� 10 300� 100 600� 400 558� 90
Acetate production mg/L 2870� 240 500� 150 970� 0 1620� 400
Propionate production mg/L �90� 11 250� 150 �25� 139 210� 50
Butyrate production mg/L 2190� 140 500� 193 2287� 120 2200� 830
Lactate removal mg/L 4700� 800 5600� 1000 8120� 900 6800� 530
a CS, corn starch.b TpK, Taipei kitchen waste.c KhK, Kaohsiung kitchen waste.d Unit: mmol/gCOD.
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indicated that oil and grease existence would affect mass
transfer in anaerobic process. Thus, ammonia and fat might
be the inhibition factors of anaerobic hydrogen production.
3.4. Process performance evaluation of Kaohsiung Citykitchen waste fermentation
The kitchen waste in Kaohsiung was selected as the better
substrate for hydrogen fermentation in run 3. The character-
istics of kitchen waste in Kaohsiung with higher COD
concentration and high variation are listed in the previous
section. The time course profile of biogas production and
water quality is presented in Fig. 4. The operational condi-
tions were still controlled at pH 5.5, 35 �C, 4-day HRT. During
the operation, the hydrogen concentration was maintained at
33% but there was no methane production, and the hydrogen
production rate reached 28 mmol/L/day significantly higher
than run 2. The carbohydrate removal was about 66% and of
30% total organic nitrogen was converted to ammonia. The
kitchen waste collected from Kaohsiung seemed greasier
than that collected from Taipei, but the hydrogen production
rate was not inhibited by the high concentration of oil and
grease. The ammonia concentration in kitchen waste in
Kaohsiung was slightly lower than that in Taipei. In run 3,
the ammonia production was 300 mg/L which was higher
than run 2 but did not make the hydrogen production
performance worse. The major problem might be the
composition of carbohydrate in different resources of feed-
stock. The carbohydrate concentration in kitchen waste in
Kaohsiung was three-fold more than that in Taipei. Though
the fraction of cellulose or starch contained in the two
resources could not be confirmed in this study, one believed
that materials collected from restaurant would have much
cooked material and more starch-like carbohydrates. Thus,
the better performance was achieved because of the easily
degradable composition.
3.5. Performance evaluation of shortened HRT
Semi-continuous operation would cause insufficient provi-
sion of substrate in every cycle of the operation. Shortening
the frequency of feeding substrate, which means shortening
the HRT, would provide the sufficient substrate in the
appropriate dilution rate. The time course of biogas produc-
tion during runs 3 and 4 is presented in Fig. 5. In run 4, the
frequency of feeding was increased to two times per day, and
the HRTwas shortened to 2 days. The volumetric loading rate
was also increased to 50 gCOD/L/day. During the 20-day
operation, the hydrogen concentration was held at 35%, and
the hydrogen production rate was increased along with the
increase of the loading rate. The maximum hydrogen
production rate of 118 mmol/L/day was observed at loading
rate up to 50 gCOD/L/day. The effects of shortening HRTwould
not only provide sufficient substrate but also disadvantaged
the longer generation time species, such as methanogenic
bacteria. In the engineering opinion, the reactor designed for
shortenening HRT would save the landscape.
3.6. Bio-community monitoring by TRFLP method
The dynamics of hydrogen producing bacterium commu-
nities was tracked using the 16S rDNA-based TRFLP method.
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Fig. 3 – Changes in (A) biogas, (B) MLSS/MLVSS, (C) total COD
and soluble COD, (D) total carbohydrate and soluble
carbohydrate, (E) soluble organic nitrogen and ammonia, (F)
oil and grease, (G) ORP and pH control from run 1 (the first 20
days) to run 2.
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Fig. 6 indicates 16S rDNA-based TRFLP electropherograms for
the mixed liquor samples taken from the fermentor operated
at different operational periods of runs 2–4. In run 2, terminal
fragment length (TFL) signatures of 231 and 256 were found in
the fermentor. As the operation went on, the TFL of 231
disappear in the periods of runs 2 and 3. The TFL of 503 and
520 were found in all the operational periods, and the TFL of
520 was the dominant at all conditions operated in this study.
Referring to the expected TFL signatures summarized by
Duangmanee et al. [13], the 16S rDNA-based TFL signature, as
shown in Fig. 5, appeared in the samples taken from the
fermentor suggesting that Clostridium clusters I and II lineage
ðTFL ¼ 231Þwere shifted following the operation in runs 3 and
4. The Clostridium stercorarium, and Clostridium thermolacticum
which belonged to Clostridium cluster III ðTFL ¼ 256Þ were
presented in all the operations, but were not the dominant
bacteria. The TFL of 503 indicated some kind of Clostridium
genus such as Clostridium aldrichii, Clostridium cellobioparum,
Clostridium termitidis (cluster III) and Clostridium formicoaceti-
cum (cluster XI). These species of Clostridium were referred to
the cellulose degrading bacteria in anaerobic condition. Many
Clostridium species were capable of producing acetic acid,
butyric acid and hydrogen from carbohydrate fermentation
[19]. In addition, several studies surveyed microbial commu-
nities of anaerobic hydrogen producing systems, using
traditional isolation/identification [20] and/or molecular
methods [21], and demonstrated that Clostridium species were
dominant bacterial groups and their presence in correlation
with hydrogen production as well. Though the TFL of 520
presented as the most dominant signature in the electro-
pherograms, it has not been identified. The database of
hydrogen producing bacterium might be developed by the
cloning sequencing in the future study. Besides, there were a
lot of peaks which were not identified in the database; these
peaks also presented that the community was diverse in
kitchen waste hydrogen fermentor.
3.7. Degrading efficiency batch test by fresh cooked food assubstrate
In order to confirm the biodegradability of the biomass, the
sludge was taken out from the fermentor during run 4
operation for the 2-L bottle batch test, and the cooked food
contained 46% of rice, 38% of vegetable and 16% of meat was
ground as the substrate used in the batch test. As the batch
experiment got started, the sludge concentration was con-
trolled at 10 g/L, and the substrate ground from lunch box was
controlled at 20.4 g/L in the 2-L bottle. The medium pH was
adjusted to 5.5 using potassium hydroxide and hydrochloric
acid. The bottle was capped tightly with rubber septum
stoppers and flushed with oxygen free nitrogen gas. The
drainage method was applied to the biogas measurement.
The bottle was incubated with a magnetic stir rotational rate
of 120 rpm and a temperature of 35� 1 �C. The results of the
accumulative biogas production and the substrates and by-
products profile are presented in Fig. 7. In the first 14 h, the
hydrogen was rapidly produced, and the production rate
reached 2.36 mmol/L/h. During the 48-h operation, the VSS
decreased slightly from 22.9 to 15.1 g/L, and the removal rate
was about 35%, which was worse than continuous operation.
The total carbohydrate removal rate was about 66%, and this
result was better than the results in run 4. Only a small
amount of organic nitrogen was converted to ammonia. From
beginning to end, the oil and grease were kept at the
concentration of 5.5 g/L, and that gave the evidence for the
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Fig. 4 – Changes in (A) biogas, (B) MLSS/MLVSS, (C) total COD and soluble COD, (D) total carbohydrate and soluble
carbohydrate, (E) soluble organic nitrogen and ammonia, (F) oil and grease, (G) ORP and pH control in run 3.
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poor degradability of the biomass in the hydrogen fermentor.
The acetate production was about 590 mg/L and 1333 mg/L of
butyrate. Lactate especially became the major by-product in
the batch test and the result is quite different from the results
from continuous operation. The possible explanations in-
clude: (1) the pH value was not adjusted in the batch test, thus
affecting the performance, (2) the difference between sub-
strate sources, fresh ground foods and kitchen waste caused
the different results. Especially, the result of lactate produc-
tion might explain the composition of the kitchen waste we
collected which contained large amount of lactate.
Fig. 5 – Variation of biogas production rate in mesophilic
hydrogen fermentor feed with kitchen waste from run 3
(40–60 days) to run 4.
Fig. 6 – TRFLP analysis based on 16S rDNA of the PCR-amplifie
sludge during the operation period A of runs 2–4.
4. Conclusions
Nutritive composition of kitchen waste is highly feasible for
anaerobic fermentation with hydrogen production. Based on
our results, batch test, morphology observation and molecu-
lar method detection, abundant and potential hydrogen
producing bacterium existed in the acidogenesis biological
system. To get sufficient degradable substrate, shorter HRT
and higher organic loading rate are suggested to be applied
for the hydrogenation process performance.
We had provided a feasibility study to apply kitchen waste
hydrogenation in semi-continuous process. The I-CSTR
system treating kitchen waste produced nearly 35% H2 and
60% of CO2 in the gas phase, while butyric acid accounted for
the majority of the VFAs. The best performance of hydrogen
producing rate was presented in run 4 at 60 mmol/L/day, even
though the higher producing rate at 120 mmol/L/day was
presented at day 76 under the relative high loading. Com-
pared with the hydrogen yield, the highest yield of 2 mmol/
gCOD in run 1 was caused by the high fraction of corn starch
addition. In the past studies, the carbohydrate was the major
substrate responsible for hydrogen fermentation. However,
kitchen waste collection system must be more completed and
more efficient to prevent carbohydrates getting self-acidified
during the storage and transportation process.
Previous attempts showed that the thermal treatment of
the sludge and acid/base acclimation could be applied in
screening the hydrogen producing bacteria. But in this study,
there was no pretreatment of the sludge taken from the pilot
scale acidogenesis tank. It is believed that, if the environ-
mental condition can be controlled well, the hydrogen
producing system can be established and the methanogenic
d HPB fragment. The total DNA extracted from suspended
ARTICLE IN PRESS
Fig. 7 – Time course profile of (A) biogas, (B) solid, (C) carbohydrate, (D) VFA, (E) organic nitrogen, (F) oil and grease, (G) pH and
ORP value during 48-h biodegradation time in the mixture item.
I N T E R N AT I O N A L J O U R N A L O F H Y D R O G E N E N E R G Y 3 3 ( 2 0 0 8 ) 1 5 2 2 – 1 5 3 11530
bacteria or other hydrogen consuming bacteria will be
restricted in the system. Though the fermentor was con-
trolled at pH 5.5, it could not inhibit methanogenic bacteria
effectively. Previous study mentioned that a dilution rate of
0:075 h�1 was fairly enough to cause the complete washout of
methanogenic bacteria [3]. In this study, the operational
condition at dilution rate of 0:25 d�1 was large enough to
inhibit the methane production. It is suggested that applying
sufficient easily degradable substrate at the appropriate
dilution rate to the fermentor advantages the hydrogen
producing bacterium.
Two-stage digester of acidogenesis tank and methanogenesis
tank has been developed for many years. Some of the re-
searches have shown the possible scheme of hydrogen–methane
ARTICLE IN PRESS
I N T E R N A T I O N A L J O U R N A L O F H Y D R O G E N E N E R G Y 3 3 ( 2 0 0 8 ) 1 5 2 2 – 1 5 3 1 1531
two-stage bio-energy recovery system [22]. National Institute
of Advanced Industrial Science and Technology (AIST) in
Japan developed the world’s first biogas plant based on two-
stage fermentation process in 2004 [23]. By the two-stage
process, the high rate and high efficiency of energy recovery
were emphasized. In the future, acidogenesis bioreactor
reformed to hydrogen fermentor should be considered as
the recovery of energy.
Acknowledgments
The authors would like to express their gratitude for the
financial support provided by the National Science Council,
ROC (NSC 94-2211-E-006-033), and the ITRT CESH projects.
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