Bioresource Technology 101 (2010) S48S52

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Bioresource Technology
journal homepage: www.elsevier.com/locate/biortech

Fermentative hydrogen production from tofu-processing waste and anaerobic

digester sludge using microbial consortium
Mi-Sun Kim a,*, Dong-Yeol Lee b
a
b

Bioenergy Research Center, Korea Institute of Energy Research, Daejeon 305-343, Republic of Korea
National Institute for Environmental Studies, Research Center for Material Cycles and Waste Management, 16-2 Onogawa, Tsukuba, Ibaraki 305-8506, Japan

a r t i c l e

i n f o

Article history:
Received 2 December 2008
Received in revised form 10 March 2009
Accepted 12 March 2009
Available online 25 April 2009
Keywords:
Biohydrogen
Pre-treatment
Thermophilic fermentation
Tofu-processing waste
Anaerobic digester sludge

a b s t r a c t
The combination of tofu-processing waste and anaerobic digester sludge was studied for its fermentative
H2 production capacity in batch and continuous modes using a thermophilic mixed culture. Heat-treatment (110 C, 30 min) in the presence of 0.5% HCl increased the soluble carbohydrate content of the tofu
waste from 2.2 to 10.4 g/l. Anaerobic digester sludge was added to the tofu waste for supplementary
nutrients with the optimal mixing ratio of 20% (v/v) under batch conditions. In continuous experiments,
the effects of HRT(hydraulic retention time) and pH were investigated for the ranges of 26 h and 5.06.0,
respectively. The maximal H2 production rate (12.0 l H2/l/day) and yield (2.3 mol H2/mol glucose equivalent) were obtained at HRT 4 h and pH 5.5 while maintaining the head space gas at 5060% (v/v) of H2
without CH4. This study indicates that the combination of tofu-processing waste and digester sludge can
be considered to be one of the most promising forms of organic waste for continuous H2 production.
2009 Elsevier Ltd. All rights reserved.

1. Introduction
Among the various microbial hydrogen (H2) production technologies, photosynthetic and fermentative processes are the most
commonly used. Photosynthetic H2 production is carried out by
photosynthetic microorganisms under anaerobic conditions,
whereas the fermentative process involves facultative or obligate
anaerobes. Fermentative H2 production generally exhibits higher
cell-growth and H2 production rates than the photosynthetic process, and is well known as a cutting edge technology. The economic
feasibility of fermentative H2 production largely depends on the
availability of organic waste/wastewater as raw material as well
as the H2 productivity of that material (Benemann, 1998). However, so far, most studies of biohydrogen production have been
limited to the use of pure carbohydrates such as glucose, sucrose
and starch (Lin and Chang, 1999; Lay, 2000; Nguyen et al., 2008).
Soy bean curd (tofu) processing is a traditional oriental food.
The typical process consists of soy bean grinding, cooking (boiling),
ltration, protein coagulation, preservation, and packaging (Chai
et al., 1999). During processing (especially in the ltration step),
up to 30% of the soy bean is lost, becoming waste (in amounts of
about 8  105 tons per year in Korea and Japan). However, only a
small percentage of tofu waste is utilized as nutritious feed for
livestock, the remainder being incinerated and/or reclaimed as

* Corresponding author. Tel.: +82 42 860 3554; fax: +82 42 860 3739.
E-mail address: bmmskim@kier.re.kr (M.-S. Kim).
0960-8524/$ - see front matter 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.biortech.2009.03.040

industrial waste, thereby contributing to serious pollution problems (Chai et al., 1999; Mizuno et al., 2000; Noike et al., 2005).
There have been some efforts to use tofu-processing waste in
the form of organic substrates for H2 production. Noike et al.
(2002) investigated H2 fermentation from tofu waste using Clostridium acetobutylicum under mesophilic conditions, but the H2
production could not be maintained longer than 5 days, due to
the signicant inhibitory effect of lactate-producing bacteria. After
heat-treatment of the tofu waste at 70 C for 30 min, however, a
relatively stable H2 production of 1.2 l H2/l/day could be achieved,
using a co-culture of three different facultative anaerobic bacteria
(Noike et al., 2005). However, the H2 production yield was very
low: 0.52 mol H2/mol hexose. It should also be noted that it can
be difcult to use pure cultures for industrial applications, considering the problem of contamination by various H2 utilizers such as
methane (CH4)-producing bacteria, and sulfate-reducing bacteria.
In the present study, we investigated the feasibility of high-rate
H2 production from a combination of tofu-processing waste and
anaerobic digester sludge, with an inoculum of mixed cells. In order to enhance the solubilization of organic substrates from tofu
waste, various pre-treatments including heat-treatment, acid/alkali treatment, homogenization and ultrasonication were examined
alone or in combination. Anaerobic digester sludge was added to
the tofu waste in the form of nutrient supplementation. The fermentation was conducted under a thermophilic condition at
60 C, since at high temperatures, H2 is less soluble, and lactateforming bacteria, which inhibit H2-forming bacteria, can be
repressed. The bioreactor was operated at short retention times

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M.-S. Kim, D.-Y. Lee / Bioresource Technology 101 (2010) S48S52

of 26 h, and the H2 production performance of the tofu waste was

compared with those of other organic wastes.
2. Methods
2.1. Feedstocks
Tofu-processing waste was obtained from a local tofu company
(Daejeon, Korea) as a by-product of the ltration of boiled (95
105 C) bean curd. The tofu waste was stored at 30 C before subsequent use. Anaerobic digester sludge was collected from the second anaerobic digester at the local municipal wastewater
treatment facility (Daejeon, Korea). The sludge was sieved through
a 1 mm mesh, and stored at 4 C before use. The characteristics of
the tofu-processing waste and digester sludge are listed in Table 1.
2.2. Pre-treatment of tofu-processing waste
The stored tofu waste was thawed, diluted with tap water at a
1:4 ratio (w/w), and homogenized using a blender (Model
HGBRWTG4, Waring Commercial Co., Ltd., USA) for 1 min. Before
H2 fermentation, the tofu waste was further treated using various
pre-treatment methods alone or in combination. The tofu waste
was heated at 120130 C using a cylindrical 304 stainless steel
column (inner diameter, 13 cm; length, 30 cm; working volume,
1.5 l) equipped with an electric heating cover, a temperature controller, a temperature sensor, a pressure gauge, and a relief valve.
During the thermal treatment, the waste was continuously mixed
at 100 rpm using a Teon magnetic bar. As an acid/alkali treatment, HCl and NaOH were added to the thermal reactor above
mentioned, at ratios of 0.1% (w/v) and 0.5% (w/v) of tofu waste.
The tofu waste was also treated using an ultrasonic processor
(VCX 750, Sonics and Materials, Inc., USA) or a homogenizer
(HMG2400, Misung Scientic Co., Korea).
2.3. Inoculum and H2 fermentation
An inoculum for the H2 fermentation was obtained from a 5 l
CSTR (continuous-stirred tank reactor) operated for the production
of H2 at 60 C, pH 5.5, and an HRT (hydraulic retention time) of
10.3 h for 3 months. The biomass concentration and volumetric
H2 production rate were 1.11 g VSS (volatile suspended solid)/l
and 4.5 l H2/l/day, respectively. It was transferred anaerobically
by a sterile hypodermic disposable syringe and inoculated at the
ratio of 10% (v/v).

Batch and continuous H2 fermentations were conducted in a

1.5 l CSTR (working volume, 590 ml), made of Pyrex glass and
equipped with a water jacket. The temperature was maintained
at 60 C and the pH at 5.5 by the automatic addition of 2.0 N NaOH.
For the continuous experiments, initially the reactor was operated
at HRTs of 126 h with a synthetic medium containing 1% (w/v)
glucose. After the reactor had attained a steady state with respect
to the glucose removal efciency (95%) and the H2 production rate
(8.0 l H2/l/day), the mixture of tofu waste and digester sludge began to be added. The mixture was stored at 4 C and purged continuously with argon gas (99.999%). The mixing ratio of the waste
mixture to the synthetic medium was gradually increased from
20% to 100% (v/v) while maintaining the HRT at 6 h.
2.4. Analytical methods
The volume of biogas produced was measured by the water displacement method. The H2 and CH4 contents in the biogas were
analyzed using a gas chromatograph (Model 14-B, Shimadzu Co.,
Japan) equipped with a thermal conductivity detector. A stainless
steel column packed with a Molecular Sieve 5A (80/100 mesh; Alltech, Deereld, USA) was used for H2, and one packed with a Hayesep Q (80/100 mesh; Alltech) was used for CH4. VS (volatile solid),
VSS and TS (total solid) were measured according to the analytical
procedures listed in the standard methods (APHA, 1995).
Liquid samples were centrifuged at 10,000 rpm for 2 min, ltered through a 0.45 lm membrane and analyzed for various components. The protein concentration was determined by the Lowry
method using a protein assay kit (Bio-Rad, USA). Bovine serum
albumin was used as a standard. The carbohydrate content was
measured by the dinitrosalicylic acid method using glucose as a
standard (Miller, 1959). The COD (chemical oxygen demand), TN
2
2
(total nitrogen), TP (total phosphorus), NH
3 , SO4 , and PO4 concentrations were assayed by colorimetric methods according to
the manufacturers instructions (DR-2000; Hach Co., USA). Various
organic acids and ethanol were analyzed using a high-performance
liquid chromatograph (Model VP, Shimadzu Co., Japan) equipped
with a sulfonated divinyl benzene-styrene copolymer column
(300 mm  7.8 mm, Aminex HPX-87H, BioRad, USA). A photometric detector (216 nm) was used to quantify the organic acids, a
refractive index detector being used to quantify the ethanol. The
organic acids analyzed included succinate, lactate, formate, acetate, propionate, n-butyrate, and iso-valerate. An aqueous solution
of 10 mM H2SO4 was used as an eluting solution at 0.6 ml/min, and
the column was maintained at 30 C.

Table 1
Characteristics of digester sludge and tofu-processing waste used in this study.
Raw tofu-processing waste
TS (%, w/w)
VS (%, w/w)
Water (%, w/w)
Ash (%, w/w)
TS (g/l)
VS (g/l)
pH
Soluble componentb
COD (g/l)
Carbohydrate (g/l)
Protein (g/l)
TN (mg/l)
TP (mg/l)
NH3+ (mg/l)
SO2
4 (mg/l)
PO2
4 (mg/l)
a
b

Diluted tofu wastea

Pre-treated tofu waste (0.5% HCl, 110 C, 30 min)

Digester sludge waste

4.9

23.5 3.5
22.4 3.2
1.0

18.7 3.7
10.8 4.3
6.8

37.3 1.8
10.4 2.5
1.19 0.41
640 46
334 41
859 119
1081 83
577 60

0.5 0.1
0.28 0.27
0.01 0.01
415 41
56 17
414 77
46 11
57 2

22.8 0.2
21.2 0.7
77.2 0.2
1.6 0.6

5.5 0.8
2.4 0.7

Diluted with tap water in 1:4 ratio (w/w) and homogenized using blender for 1 min.
Centrifuged at 10,000 rpm for 2 min and ltered through 0.45 lm membrane.

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15

3.1. Effect of various pre-treatments on solubilization of organic

substances from tofu waste

12

In this study, various pre-treatments including ultrasonication,

homogenization, acid/alkali treatment, and heat-treatment were
attempted in order to enhance the solubilization of the organic
substances from the tofu waste. It has been reported that carbohydrate is the major substrate released from tofu waste used in H2
production (Mizuno et al., 2000; Noike et al., 2005). Therefore,
the soluble carbohydrate concentration was selected as the key
factor in evaluating the efciency of the pre-treatment methods.
Time-course proles of the soluble carbohydrate content during
the pre-treatments of tofu waste using ultrasonication and homogenization were measured. Prior to the pre-treatments, the tofu
waste was diluted with tap water at a 1:4 ratio (w/w). When the
tofu waste was disrupted by a homogenizer, the carbohydrate concentration gradually increased from 2.9 g/l to 5.4 g/l With sonication treatment, only 2.7 g carbohydrate/l was obtained after
120 min. For the control, without any pre-treatment, no appreciable variation with time was observed. Fig. 1 illustrates the effects of
HCl and NaOH treatments on the carbohydrate solubilization from
the tofu waste. HCl and NaOH were added at ratios of 0.1% (w/v)
and 0.5% (w/v) of the tofu waste, respectively. When the tofu waste
was solubilized at 0.1% (w/v) HCl, a lower carbohydrate content
(3.1 g/l) was observed compared with that for the homogenization treatment. However, upon increasing the HCl concentration
to 0.5% (w/v) of the tofu waste, the carbohydrate content increased
signicantly with time, a high value of 8.0 g carbohydrate/l being
obtained after 120 min. After treatments with 0.1% and 0.5% HCl,
the pH of the tofu waste decreased from 4.9 to 3.1 and 1.3, respectively. Further HCl addition was not attempted, considering the
cost of HCl and the problem of pH control for subsequent H2 fermentation. With the NaOH treatment at 0.10.5% (w/v), the solubilized carbohydrate concentrations were as low as 3.03.4 g/l.
Although higher NaOH concentrations, up to 4.0% (w/v), were employed, the solubilization efciencies of the carbohydrate (6.3 g/l)
were not improved compared with that of HCl at 0.5%. This result
indicated that HCl-acidication is more effective than NaOH-alkalization for solubilization of organic substances from tofu waste.
Fig. 2 shows the effect of the thermal treatments with and without
0.5% (w/v) HCl on the carbohydrate solubilization from the tofu

Soluble carbohydrate (g/L)

12

0
0

20

40

60

80

100

120

140

Time (min)
Fig. 1. Effects of HCl and NaOH treatments on solubilization of carbohydrate from
tofu-processing waste. Symbols: control (d), HCl (0.1%, s; 0.5%, j; w/v), NaOH
(0.1%, h; 0.5%, N; w/v).

Soluble carbohydrate (g/L)

3. Results and discussion

0
0

20

40

60

80

100

120

140

Temperature ( C)
Fig. 2. Effect of heat-treatment with (s) and without (d) 0.5% (w/v) HCl on
solubilization of carbohydrate from tofu-processing waste.

waste. The temperature was varied from 20 C (room temperature)

to 130 C, and the reaction time was xed at 30 min. Without HCl,
the soluble carbohydrate concentration increased gradually from
1.9 to 6.9 g/l with increasing temperatures to 110 C. At the high
temperature of 130 C, the carbohydrate concentration was similar
to that at 110 C. In the presence of 0.5% (w/v) HCl, a similar temperature-dependence prole was observed to that without HCl, but
there was a considerably higher carbohydrate concentration for
each temperature. The maximal carbohydrate concentration
(10.811.3 g/l) was obtained at 110130 C. Heat-treatments in
combination with other treatments (0.5% NaOH, ultrasonication
and homogenization) were no better than with 0.5% HCl in respect
to the carbohydrate concentration (data not shown). Table 1 shows
the detailed characteristics of the tofu waste treated at 110 C with
0.5% (w/v) HCl. Noike et al. (2005) and Mizuno et al. (2000) reported that the soluble carbohydrate concentration could be increased from 2.7 to 5.0 g/l after heat-treatment of tofuprocessing waste at 70 C for 30 min.
3.2. Effect of digester sludge addition on H2 fermentation of tofu waste
Fig. 3 shows time-course proles of the batch H2 fermentation
using tofu waste plus anaerobic digester sludge at 60 C. The tofu
waste was prepared according to the pre-treatment conditions
(0.5% HCl, 110 C, 30 min) optimized above. The digester sludge
was added to the tofu waste as supplementary nutrients at three
different mixing ratios (1:9, 2:8 and 3:7 by volume). The pH was
maintained at 5.5. The results presented in Fig. 4 indicate that, with
the appropriate mixing of tofu waste and digester sludge, co-fermentation could improve the H2 production better than could fermentation using tofu waste alone. The impact of the cofermentation was, however, dependent on the mixing ratio of the
sludge. The fastest H2 production rate (36 ml H2/h) and the highest
conversion yield (1.7 mol H2/mol glucose equivalent consumed)
were observed from the mixture at the volumetric sludge-totofu-waste ratio of 2:8. The waste mixture containing the sludge
at a 3:7 (v/v) ratio produced a lower H2 production (27 ml H2/h),
which might have been due to the reduced carbohydrate content
in this mixture (see also Table 1). The carbohydrate removal efciency was in the range of 7682% regardless of the mixing ratio
of the sludge.
In this study, the digester sludge might have provided nutrients
necessary for the growth of H2-producing bacteria. Micronutrients
such as minerals and vitamins have been reported to be important

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M.-S. Kim, D.-Y. Lee / Bioresource Technology 101 (2010) S48S52

1000

HRT (hr)
6

H2 production (L H2/L/day)

H2 (mL)

600

400

200

Carbohydrate removal (%)

Soluble carbohydrate (g/L)

12

10
8

20

50

80

100

100

100

12

(A)

100
80
60
40
20

(B)

Waste content(%)

16

800

10

15

20

Time (day)

Fig. 4. Continuous H2 fermentation using mixture of tofu waste and digester sludge
at mixing ratio (v/v) of 2:8. (A) H2 production (B) Carbohydrate removal.

0
0

20

40

60

80

Time (hr)
Fig. 3. Effect of digester sludge addition on fermentative H2 production from tofu
waste. Symbols: tofu waste alone (d), combination of digester sludge and tofu
waste at mixing ratios (v/v) of 1:9 (s), 2:8 j, 3:7 (h). The temperature and pH were
maintained at 60 C and 5.5, respectively.

for the growth and metabolic activity of H2-producing bacteria (Li

and Fang, 2007; Zhu et al., 2008), and could possibly have been
present in the digester sludge, though their composition and concentrations were not analyzed in this study. It has been reported by
previous research that when digester sludge alone was used, H2
production was negligible, which might be due to the low carbohydrate content and biodegradability nature of digester sludge (Kim
et al., 2004). It was expected that H2-forming bacteria and H2-consuming bacteria (especially hydrogenotrophic methanogens) coexisted in the digester sludge. However, throughout the
fermentation, no CH4 was detected in the biogas, which fact might
presumably be related to the acidic (pH 5.5) and thermophilic
(60 C) experimental conditions of the bioreactor (Oh et al.,
2004). Kim et al. (2004) reported that sewage sludge enhanced
H2 production from food waste and that the optimal mixing ratio
of the sewage sludge to the food waste was 13% on a VS basis.
3.3. Continuous H2 fermentation using mixture of tofu waste and
digester sludge
Fig. 4 shows the results of the continuous H2 fermentation from
the combination of tofu waste and digester sludge. Initially the
reactor was operated at an HRT of 126 h with a synthetic medium
containing 1% (w/v) glucose (see Section 2 for details), subsequently receiving the mixture of tofu waste and digester sludge
as a feedstock for H2 production. The temperature and pH were

maintained at 60 C and 5.5. When the volumetric mixing ratio

of the waste mixture to the synthetic medium was increased gradually from 20% to 100% at an HRT of 6 h, the volumetric H2 production rate was almost maintained at 8 l H2/l/day, but the
carbohydrate removal efciency decreased gradually from 95% to
76%. When the HRT was further decreased to 4 h with 100% waste
mixture, the maximal H2 production rate (12.0 l H2/l/day) and yield
(2.3 mol H2/mol glucose equivalent removed or 170 ml H2/g VS
added) could be obtained after 16 days, though the carbohydrate
removal efciency had slightly decreased by 12% compared with
that at 6 h HRT. However, at the very short retention time of 2 h,
the reactor became unstable, presumably due to the high loading
rate resulted in the wash-out of cells from the reactor and the volumetric H2 production rate decreased below 15% of that of the HRT
at 4.0 h. No further operation at this HRT was attempted. In the
course of the normal fermentation, the H2 content in the biogas
was in the range of 5357% (v/v), and the soluble protein in the
feed was almost consumed (removal efciency, 9496%). Mizuno
et al. (2000) reported that, during a mesophilic H2 fermentation
of tofu-processing waste, carbohydrate was rapidly degraded, protein degradation hardly being observed. Moreover, it has been reported that thermophilic ora showed higher biodegradation
activity for peptone and could tolerate signicantly more ammonia
inhibition than mesophilic ora (Gallert et al., 1998). During the
present H2 production, complex organic substances were hydrolyzed into various organic acids (acetate, n-butyrate, lactate, succinate, and propionate) and ethanol. Among the organic acids,
acetate and n-butyrate were detected as the major soluble metabolites, but trace levels of lactate, succinate, propionate, and ethanol
were also detected (data not shown).
The effect of pH on the H2 production was investigated at 5.0,
5.5, and 6.0 (data not shown). The HRT was xed at 6 h. The optimal pH was 5.5, and the carbohydrate removal efciency and volumetric H2 production rate were 76% and 8.1 l H2/l/day,
respectively. At a higher or lower pH, the H2 production rate fell

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M.-S. Kim, D.-Y. Lee / Bioresource Technology 101 (2010) S48S52

slightly by 6% and 16%, respectively, compared with that at pH 5.5.

The H2 content in the biogas was in the range of 5658% (v/v), and
no CH4 whatsoever was detected, regardless of the pH.
Hydrogen fermentation using organic waste/wastewater has already been reported in the literature. Noike et al. (2005) investigated continuous H2 production from tofu-processing waste,
reporting an H2 production rate of 1.2 l H2/l/day and an H2 conversion yield of 0.52 mol H2/mol hexose. Yu et al. (2002) reported 3.8 l
H2/l/day and 2.1 mol H2/mol hexose from rice winery wastewater
in the continuous mode. Under batch conditions, values of 69 ml
H2/g VS (beer lees biomass; Fan et al., 2006), 92 ml H2/g VS (food
waste; Shin et al., 2004), 112 ml H2/g VS (ternary mixture of food
waste, waste activated sludge and primary sludge; Zhu et al.,
2008), 150 ml H2/g VS (cornstalk waste; Zhang et al., 2007),
180 ml H2/g VS (organic municipal solid waste; Lay et al., 1999)
have been reported. Direct comparison of H2 yields on biomass
and/or maximum H2 production rates among various organic
wastes is difcult, since results vary greatly according to the pretreatment and fermentation conditions. Nevertheless, we can conclude that the combination of tofu-processing waste and anaerobic
digester sludge should be considered to be one of most promising
organic wastes for continuous H2 production, considering its high
H2 productivity (12.0 l H2/l/day) and yield (2.3 mol H2/mol glucose
equivalent or 170 ml H2/g VS).
Anaerobic fermentation from the pretreated tofu-processing
waste using the mixed microora produced a high yield of H2 for
a 2.3 mol H2/mol glucose equivalent at 60 C. This is the highest
yield reported among the fermentative H2 production studies using
organic wastes. A few speculations merit attention in discussion of
the process optimization and post-treatment to maximize H2 production from organic materials. First, our experiment requires an
elevated temperature of 60 C compared to the mesophiles
(30 C) to obtain the reported results. However, given that the
tofu-processing procedures requires 95100 C, it appears that
there is no real additional cost incurred in the fermentation process. Second, the present CSTR system would be connected with
a membrane ltration unit to retain more biomass in the reactor
and expect to lower the HRT to 2 h and increase the H2 production
rate in further research at this lab. Finally, the main liquid-state
metabolites during the fermentation were acetic and butyric acids
indicating that the thermophilic operation provided the favorable
condition for H2 production. Further researches for H2 production
is necessary in order to develop ways in which post-treatment processes, such as photo-fermentation to produce H2, microbial fuel
cells for electricity, and CH4 fermentation, can be connected to
maximize the energy-efciency potential of organic wastes.
4. Conclusions
The combination of tofu-processing waste and anaerobic digester sludge was studied for its thermophilic H2 production capacity
at 60 C. A high-soluble carbohydrate concentration of 10.4 g/l was
obtained with thermochemical pre-treatment at 0.5% (w/v) HCl
and 110 C. The digester sludge enhanced the H2 production from
the tofu waste, and the optimal sludge-to-tofu-waste mixing ratio
was 20% on a volume basis. With continuous fermentation of the
mixture of tofu waste and digester sludge, the maximal H2 produc-

tion rate of 12.0 l H2/l/day and H2 production yield of 2.3 mol H2/
mol glucose equivalent were obtained, which are comparable to
or higher than those with several industrial organic wastes. In
short, the results of this study indicate that thermophilic H2 fermentation using a mixture of tofu-processing waste and digester
sludge can be considered to be one of the most promising processes of continuous H2 production.
Acknowledgements
This research was conducted for the Hydrogen Energy R&D Center, a 21st Century Frontier R&D Program, and was funded by the
Ministry of Education, Science and Technology of Korea. We thank
JT Park(Korea Institute of Energy Research) and MS Kang(Woorhitech Co.) for the technical assistance and valuable discussions.
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