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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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