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 7 ( 2 0 1 2 ) 1 5 6 3 2 e1 5 6 4 2

Available online at www.sciencedirect.com

journal homepage: www.elsevier.com/locate/he

Fermentative hydrogen production from wastewaters: A

review and prognosis
Chiu-Yue Lin a,b,c,*, Chyi-How Lay a,b, Biswarup Sen a,b,c, Chen-Yeon Chu b,c,d,
Gopalakrishnan Kumar a, Chin-Chao Chen e, Jo-Shu Chang f,g,h
a

Department of Environmental Engineering and Science, Feng Chia University, 40724, Taiwan
Green Energy Development Center, Feng Chia University, 40724, Taiwan
c
Master Program of Green Energy Science and Technology, Feng Chia University, 40724, Taiwan
d
Department of Chemical Engineering, Feng Chia University, 40724, Taiwan
e
Environmental Resources Laboratory, Department of Landscape Architecture, Chung Chou University of Science and Technology,
51022, Taiwan
f
Department of Chemical Engineering, National Cheng Kung University, 70101, Taiwan
g
Center for Bioscience and Biotechnology, National Cheng Kung University, 70101, Taiwan
h
Research Center for Energy Technology and Strategy, National Cheng Kung University, 70101, Taiwan
b

article info

abstract

Article history:

Biohydrogen is a promising candidate which can replace a part of our fossil fuels need in

Received 3 December 2011

day-to-day life due its perceived environmental benefits and availability through dark

Received in revised form

fermentation of organic substrates. Moreover, advances in biohydrogen production tech-

9 February 2012

nologies based on organic wastewater conversion could solve the issues related to food

Accepted 13 February 2012

security, climate change, energy security and clean development in the future. An evalu-

Available online 16 March 2012

ation of studies reported on biohydrogen production from different wastewaters will be of

immense importance in economizing production technologies. Here we have reviewed

Keywords:

biohydrogen production yields and rates from different wastewaters using sludges and

Dark fermentation

microbial consortiums and evaluated the feasibility of biohydrogen production from

Biohydrogen

unexplored wastewaters and development of integrated bioenergy process. Biohydrogen

Wastewaters

production has been observed in the range of substrate concentration 0.25e160 g COD/L,

Integrated bioenergy process

pH 4e8, temperature 23e60
C, HRT 0.5e72 h with various types of reactor configuration.
The most efficient hydrogen production has been obtained at an organic loading rate (OLR)
320 g COD/L/d, substrate concentration 40 g COD/L, HRT 3 h, pH 5.5e6.0, temperature 35
C
in a continuously-stirred tank reactor system using mixed cultures and fed with condensed
molasses fermentation soluble wastewater. The net energy efficiency analysis showed
vinasse wastewater has the highest positive net energy gain followed by glycerin wastewater and domestic sewage as 140.39, 68.65, 51.84 kJ/g COD feedstock with the hydrogen
yield (HY) of 10 mmol/g COD respectively.
Copyright 2012, Hydrogen Energy Publications, LLC. Published by Elsevier Ltd. All rights
reserved.

* Corresponding author. Feng Chia University, Department of Environmental Engineering and Science, 100 Wenhwa Road, Seatwen,
Taichung 40724, Taiwan. Tel.: 886 4 24517250x6200; fax: 886 4 35072114.
E-mail address: [email protected] (C.-Y. Lin).
0360-3199/$ e see front matter Copyright 2012, Hydrogen Energy Publications, LLC. Published by Elsevier Ltd. All rights reserved.
doi:10.1016/j.ijhydene.2012.02.072

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 7 ( 2 0 1 2 ) 1 5 6 3 2 e1 5 6 4 2

1.

Introduction

The drastic increase in oil prices and growing environmental

awareness are leading to new developments in the fuel
markets around the world. The best known developments are
in the field of biofuels. Biofuels have the potential to replace
a part of our need for fossil fuels, especially in the transport
sector because of their assumed unlimited availability and
perceived environmental benefits. Constantly increasing
demand for energy requires search for new sources and
methods for generation. Utilization of organic wastes for
biological hydrogen, biohydrogen, production is a new and
promising approach to meet the increasing need for energy.
The US Department of Energy Hydrogen program in the
United States estimates that contribution of hydrogen to total
energy market will be 8e10% by 2025 [1]. The US Department
of Energy issued a national hydrogen energy road map in
November 2002 which explored a series of activities that must
be undertaken at the public, private, state, federal, industrial,
and academic levels to advance hydrogen potentials as the
countrys dominant energy carrier.
Several issues have been raised on biofuels-based energy
supply; for example, concerns on the use of land, water,
pesticides and whether the net CO2 balance of the production
of biofuels is positive or negative. Ethical concerns raised by
the use of biofuels are about food vs. fuel, and it is questionable whether land should be put for fuel or food production
when many people are dying of starvation. Most developed
countries in the world have recognized the pivotal role that
hydrogen may play in the future and thus experts are advocating the concept of a hydrogen economy [2].
In recent years a great deal of attention is being paid to the
utilization of hydrogen as alternative and ecofriendly fuel
throughout the world, because it does not have carbon, sulfur
or nitrogen that cause pollution during combustion [3]. Among
various substrates rich in organic content, wastewaters have
attained a considerable attention due to the advantages such
as high organic loading possibilities, low nutrient requirements and positive net energy gain. Thus the exploration of
wastewater as substrate for biohydrogen production with
concurrent wastewater treatment is an attractive and effective way of tapping clean energy from renewable sources in
a sustainable approach. This provides dual environmental
benefits in the direction of wastewater treatment along with
sustainable bioenergy generation. Currently the worldwide
production of hydrogen is mainly from natural gas (40%),
crude oil (30%), coal (18%) and water electrolysis (4%). Biohydrogen could be a more renewable and sustainable alternative fuel than the other biofuels, however, owing to its
present low production rate and yield, it has still to reach the
commercial scale to contribute significantly to the hydrogen
supply globally.
The microbial conversion of wastewaters to biohydrogen
by anaerobic fermentation is a complex series of biochemical
reactions manifested by diverse group of selective bacteria [4].
A large number of microorganisms belonging to different
taxonomic and physiological groups can produce biohydrogen
from wastewaters however with different yields and rates.
The integration of dark fermentation and photo fermentation

15633

of wastewaters in two stages is a very efficient technology to

effectively convert wastewaters into biohydrogen with net
energy gain and no generation of acids in the effluent. This
review deals with the biohydrogen production from different
wastewaters and factors influencing the rates and yields.
Future research needed toward developing a sustainable biohydrogen technology from wastewaters and prognosis is also
discussed.

2.
Optimal process parameters for
biohydrogen production from wastewaters
Table 1 indicates the key process and performance parameters of fermentative biohydrogen production in 31 cases of
real wastewaters. The range and optimum values of key
process parameters including substrate concentration, pH,
temperature, hydraulic retention time (HRT), reactor type and
seed sludge are summarized. The performance parameters
were biohydrogen production yield (HY, defined as the biohydrogen production per loading substrate (mol-H2/g-COD))
and biohydrogen production rate (HPR, defined as the biohydrogen production per working volume per day (L/L/d)).
More than 30 different types of wastewaters listed in this
review could produce biohydrogen with a HY range of
0.5e25 mmol-H2/g-COD and a HPR range of 0.03e9.5 L/L/d.

2.1.

Type of inoculum and pretreatment

Pretreatment of naturally occurring mixed microflora by

thermal or acid/alkali treatment enriches the biohydrogen
producers and inactivates non-sporulating hydrogen
consumers like methanogens [5,6]. In thermal treatment
process, the production of biohydrogen mainly depends on
the duration of thermal treatment of sludge. However,
a recent report [7] mentioned that during the thermal treatment, a certain amount of methane was still produced indicating the presence of some heat-resistant methanogens in
sludge. As for the acid treatment method, the pretreated
sludge showed a smaller amount of biohydrogen production
with respect to substrate removal than the heat treatment
method [8]. However, acid pretreatment shows high efficiency
in the removal of methanogens. Therefore, the main disadvantage for thermal treatment is the presence of methanogens, whereas low efficiencies in substrate removal and
biohydrogen production are the major drawbacks of acid
treatment. An appropriate combination of thermal and acid
treatments could lead to better performance in biohydrogen
production [7,9]. Numerous sources have been used as the
seed inoculum for anaerobic biohydrogen fermentation of
wastewaters.
Both mixed cultures and pure cultures have been extensively used for fermentation of wastewaters. Pure cultures of
Clostridium species, such as Clostridium butyricum, Clostridium
acetobutylicum, Clostridium acetobutyricum, Clostridium beijerinckii,
Clostridium
thermolacticum,
Clostridium
saccharoperbutylacetonicum, Clostridium pasteurianum etc., are highly
efficient in the conversion of carbohydrates to acetate, butyrate, hydrogen, carbon dioxide and organic solvents [10].
However, wastewaters always contain a mixed population of

Wastewaters

Potato processing wastewater

Preserved fruit soaking solution
Probiotic wastewater
Vinasse wastewater
Dairy wastewater
Distillery wastewater
Cheese processing wastewater
Cheese whey wastewater
Coffee drink manufacturing
wastewater
Condensed molasses
fermentation solubles
Olive pulp water
Purified terephthalic acid
Sugarbeet wastewater
Sugary wastewater
Citric acid wastewater
Coffee drink
manufacturing wastewater
Rice winery wastewater

Seed
sludge

Substrate conc. (g COD/L)

Range
studied

Optimal

Temp. (
C)

pH

HRT (h)

Range Optimal Range Optimal Range Optimal

studied
studied
studied

HY (mol H2/mol HPR References

hexose)
(L/L/d)

Batch
Batch
Batch
Batch
Batch

Soil
AS
SS
AS
AM

9
2e12
1.324
8.92
2.75

e
6.05
e
e
e

6.1
4e8
4.5e7.5
6.0
6.0i

e
5.95
5.5
e
e

23
25e45
30e55
30
29

e
36
45
e
e

e
e
e
e
e

e
e
e
e
e

4.08 mmol/g COD

6.11 mmol/g COD
12.41 mmol/g COD
0.24
1.25 mmol/g COD

2.16
8.58
0.34
e
e

[4]
[26]
[44]
[7]
[45]

Batch

Co-culture1

10e160

50

7.0i

35

1.78

1.92

[11]

Batch

AS

10e160

40

4.0e8.0i

6.0i

35

1.5

2.39

[46]

Batch

Soil

6.5

6.1

23

6.94 mmol/g COD

0.24

[4]

Batch
Batch
Batch
Batch
Batch
Batch

10
0.25
0.25
1.67
68.1
100

e
e
e
e
e
e

5.2e7.0
5.5
5.5
6.0
6.8
5.0e8.5

e
e
e
e
e
5.5

26e39
25
25
30
35
30e55

e
e
e
e
e
37

e
e
e
e
e
e

e
e
e
e
e
e

2.76
6.01
6.03
0.51
0.54
1.30

1.56
0.16
0.19
e
0.07
8.27

[12]
[21]
[21]
[7]
[47]
[10]

Batch
Batch
Batch
Batch
ASBR
ASBR
CSTR
CSTR
CSTR

Co-culture2
ADS
ADS
AS
ADS
C. bytyricum
EB6
Soil
AS
ADS
ADS
AM
ADS
ADS
ADS
AS

20
1.24e6.2
2e8
0.25
2.4e4.7
9.6
5.0e7.0
21e47
20

e
3.72
5.0
e
4.7
e
7.0
47
e

6.1
4.0e8.0i
4.5e7.0
5.5
4.56e6.28
5.2e7.0
4.79
5.5
5.5

e
6.0i
5.5
e
e
e
e
e
e

23
35
37
25
28
28
35e38
55
35

e
e
e
e
e
e
e
e
e

e
e
e
e
24
24
12e24
24e84
6e12

e
e
e
e
e
e
24
84
6

5.71 mmol/g COD

3.72
1.8
24.97 mmol/g COD
e
e
3.21 mmol/g COD
22.00 mmol/g COD
0.20

5.04
e
e
0.60
0.03
5.15
1.00
1.5
0.34

[4]
[20]
[48]
[21]
[49]
[8]
[50]
[22]
[51]

CSTR

AS

40

5.5

35

3e24

0.9

9.50

[24]

CSTR
CSTR
CSTR
CSTR

ADS
ADS
ADS
Sludge
compost
AB
AS

17.8e19.6
4.0
10
31.85

19.6
e
e
e

4.8e5.0
6.0
5.2
6.8

4.9
e
e
e

35
35
32
60

e
e
e
e

7.5e30
6
14.2
0.5e72

7.5
e
e
0.5

2.8
19.29 mmol/g COD
1.7
2.52

0.48
0.79
e
4.85

[52]
[53]
[54]
[55]

5.0e19.2
20

19.2
e

7.0
5.5

e
e

35e38
35

e
e

8e48
4e8

12
4

0.84
0.96

0.72
4.64

[56]
[51]

AS

14e36

34

4.5e6.0

5.5

20e55

55

2e24

2.14

3.81

[57]

UASB
UASB
Upflow
reactor

mmol/g COD
mmol/g COD
mmol/g COD
mmol/g COD

Co-culture 1, co-culture of Clostirium sporosphaeroides F52 and C. pasteurianum F40; AS, Anaerobic sewage sludge; AM, Anaerobic mixed microflora; AB, Anaerobic bacteria; ADS, anaerobic digest sludge;
Co-culture 2, co-culture of C. freundii 01, E. aerogens E10 and R. palustric P2. i stands for initial pH.

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 7 ( 2 0 1 2 ) 1 5 6 3 2 e1 5 6 4 2

Apple processing wastewater

Brewery wastewater
Cattle wastewater
Cereal wastewater
Chemical wastewater
and domestic
sewage wastewater
Condensed molasses
fermentation solubles
Condensed molasses
fermentation solubles
Confectionery processing
wastewater
Distillery effluent
Domestic sewage
Glycerin wastewater
Lagoon wastewater
Olive mill wastewater
Palm oil effluent

Culture
type

15634

Table 1 e Literature review of anaerobic biohydrogen production processes using wastewater as feedstock.

15635

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 7 ( 2 0 1 2 ) 1 5 6 3 2 e1 5 6 4 2

2.2.

Organic loading rate

It is known that higher substrate concentrations could

enhance biohydrogen production efficiency, but substrate or

product inhibitions would occur when the substrate/organic

loading exceeds a threshold level. Moreover, there is no set
optimal substrate concentration for the fermentation of
various substrates to produce biohydrogen [20]. There are
several articles in the literature that demonstrates the effect
of substrate concentration and the optimum conditions on the
anaerobic fermentation of wastewater and solid waste for
biohydrogen production (Table 1). Most studies reported biohydrogen production from wastewaters and solid wastes at
substrate concentrations lower than 40 g COD/L. It is also
observed that higher HYs were obtained at lower substrate
concentrations (Fig. 1). The maximum HY of 25 mmol/g COD
(612.5 mL/g COD) was reported from vinasse with a very low
wastewater concentration (0.25 g COD/L) in batch model
system [21]. Fig. 2 shows that HY and HPR decreased with
increasing HRT. Higher HYs (>245 mL/g COD) and HPRs (>3 L/
L/d) were observed at shorter HRTs (<10 h). However, Azbar
et al. [22] using cheese whey wastewater (47 g COD/L) in
a CSTR reported high HY of 22 mmol/g COD (539 mL/g COD) at
a longer HRT of 3.5 days.
Biohydrogen production rate in a continuous system is OLR
dependent which can be controlled either by increasing
substrate concentration or shorting HRT. As illustrated in Figs.
1b and 3b, there is a general trend that increasing substrate
concentration and OLR leads to an increase in HPR in
continuous systems fed with wastewaters. Lin and Lay [23]
mentioned that the highest HPR value of 362 L/L/d was obtained at a HRT of 0.5 h (OLR 1920 g COD/L/d) using 40 g COD/L

30

HY (mmol/g COD)

25

20

15

10

0
10

HPR (L/L/d)

microorganisms, have complex composition of organic matter

and variable oxygen content, which could either compete with
biohydrogen producers for the carbon sources or inhibit their
growth. Therefore, mixed culture systems seem to be more
effective for the desired purpose. Hsiao et al. [11] utilized the
co-culture of two isolated strains (i.e., Clostridium sporosphaeroides F52 and C. pasteurianum F40) to produce biohydrogen. In this co-culture system, the C. sporosphaeroides F52
strain first degraded the carbohydrate and glutamate in the
wastewater, and then C. pasteurianum F40 used the degraded
hexose to produce biohydrogen at a high production rate.
Vatsala et al. [12] reported that using a co-culture of Citrobacter
freundii 01, Enterobacter aerogenes E10 and Rhodopseudomonas
palustris P2 to ferment distillery effluent could enhance biohydrogen productivity significantly. Yokoi et al. [13] isolated
an acid tolerant E. aerogenes, strain HO-39, which was able to
grow and produce biohydrogen at low pH of 4.5. Cultures of
high biohydrogen yielding strict anaerobe C. butyricum and
oxygen consuming E. aerogenes were combined in a continuous fermentation process. This resulted in fermentation
with no need for an expensive reducing agent since the
presence of E. aerogenes was sufficient to rapidly restore
anaerobic conditions in the fermentor upon short oxygen
exposures. Enrichment cultures of the microflora are prepared
by forced aeration of the sludge or by heat treatment which
inhibits the activity of the hydrogen consumers while the
spore-forming anaerobic bacteria survive [7]. Additionally, in
continuous fermentations higher dilution rates are used to
wash out the slow growing methanogens and select for the
acid-producing bacteria.
More recently, some thermophilic bacteria (e.g., Thermotoga
neapolitana, Thermotoga elfii, and Caldicellulosiruptor saccharolyticus) have been found to have the ability to produce biohydrogen through dark fermentation [14e16]. Several new
biohydrogen-producing microorganisms (e.g. Ca. saccharolyticus, Gloeocapsa alpicola, Rubrivivax gelatinous and T. elfii)
have been reported since the past decade; however, there is no
significant improvement in biohydrogen production efficiency. On the other hand many unidentified mixed anaerobic
bacteria have been used to produce biohydrogen from
wastewaters and from renewable raw materials [17,18]. Using
mixed cultures in anaerobic fermentative biohydrogen
process has the advantage of being simpler to operate and
easier to control [19]. Mixed cultures can be obtained from
a large variety of sources, including the soil. The soil can
simply be thermally treated to selectively target the
biohydrogen-producing bacteria. Mixed cultures are also
recognized as an ideal system for biohydrogen production
from wastewaters. Mixed cultures can be used in anaerobic or
micro-aerobic conditions and also tend to be more robust and
are less easily contaminated. The feed source intended for use
in mixed cultures can be very complex, as the mixed microbial
community could have the ability to adapt to a variety of
carbon sources and nutrients from a domestic or industrial
waste stream for biohydrogen production.

0
0

20

40

60

80

100

120

Substrate concentration (g COD/L)

Fig. 1 e Relationship between substrate concentration and

biohydrogen production performance using wastewater
feedstock: (a) hydrogen production yield (HY) and (b)
hydrogen production rate (HPR) based on the data listed in
Table 1 (C, batch mode; B, continuous mode).

15636

25

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 7 ( 2 0 1 2 ) 1 5 6 3 2 e1 5 6 4 2

25

20

HY (mmol/g COD)

HY (mmol/g COD)

20

15

10

15

10

12
0

10
0

10
8

HPR (l/l/d)

HPR (l/l/d)

0
0

10

15

20

25

30 80

85

HRT (h)

Fig. 2 e Relationship between hydraulic retention time

(HRT) and biohydrogen production performance using
wastewater feedstock: (a) hydrogen production yield (HY)
and (b) hydrogen production rate (HPR) based on the data
listed in Table 1.

of sucrose as feedstock in a CSABR (Continuously-Stirred

Anaerobic Bioreactor) system. High HPR of 8.58 L/L/d and
8.27 L/L/d from brewery wastewater (6.05 g COD/L) and palm
oil effluent (100 g COD/L), respectively in batch systems have
been reported [10]. The maximum HPR was obtained in CSTR
system using condensed molasses fermentation solubles
wastewater (40 g COD/L) as feedstock at a HRT of 3 h and an
OLR of 320 g COD/L/d [24]. In contrary to HPR, it has been
observed that at high OLR there is a decrease in HY. High OLR
results in low HY probably because of the metabolic shift to
solventogenic phase which is unfavorable for biohydrogen
production (Fig. 3a). Wu et al. [25] had reported that high OLR
lead to the production of propionate and ethanol.

2.3.

pH

The control of pH is crucial to the dark fermentative biohydrogen production, due to its effect on the hydrogenase
activity and on the metabolic pathways. When the pH of the
fermentation medium is too low, either metabolic activity of
the hydrogen producing bacterial population would be
inhibited or there would be a switch in metabolic pathway
resulting in cessation of biohydrogen generation. Table 1 lists

100

200

300

400

500 1400

1600

OLR (g COD/l/d)

Fig. 3 e Relationship between organic loading rate (OLR)

and biohydrogen production performance using
wastewater feedstock: (a) hydrogen production yield (HY)
and (b) hydrogen production rate (HPR) based on the data
listed in Table 1.

studies on dark fermentation using wastewaters at slightly

acidic environment (<pH 7.0) on batch and continuous
systems. Typically, the maximum HY and HPR values were
obtained when the pH was in a range of 5.5 and 6.0. For
example, while using vinasse for batch biohydrogen production, the maximum HY (25 mmol/g COD) was obtained at an
initial pH of 5.5 (Table 1 and Fig. 4). Azbar et al. [22] reported
the highest HY (22 mmol/g COD) in a CSTR system fed with
cheese processing wastewater at a pH of 5.5. The maximum
HPR was also obtained at a constant pH of 5.5 in a CSTR system
using condensed molasses fermentation solubles (40 g COD/L)
feedstock [24]. The other high HPR values of 8.3e8.6 L/L/d were
obtained at an initial pH of 6.05 using brewery wastewater [26]
and at pH 5.5 using palm oil effluent [10]. From literature it is
quite evident that regardless of batch or continuous culture,
the best operational pH for dark fermentative biohydrogen
production from wastewaters is within the range of 5.5e6.0.

2.4.

Cultivation temperature

Fermentative biohydrogen production by mixed cultures has

been performed mostly under mesophilic (20e40
C) and
thermophilic (50e60
C) conditions, while there are few

15637

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 7 ( 2 0 1 2 ) 1 5 6 3 2 e1 5 6 4 2

30

30

20

15

10

15

10

0
10

HPR (L/L/d)

HPR (L/L/d)

20

0
10

0
4.5

5.0

5.5

6.0

6.5

7.0

7.5

pH

Fig. 4 e Relationship between pH and biohydrogen

production performance using wastewater feedstock: (a)
hydrogen production yield (HY) and (b) hydrogen
production rate (HPR) based on the data listed in Table 1
(C, batch mode; B, continuous mode).

studies that were carried out under extreme thermophilic

(65e75
C) conditions. Fig. 5 shows that the used cultivation
temperatures ranged from 23 to 60
C and within this range it
was observed that HY and HPR increased along with the
increase in temperature in both batch and continuous
systems. The maximum HY was observed when dark
fermentation of vinasse was carried out at 25
C in batch mode
operation [21]. The possible reason could be that at low
substrate concentration, the substrate got completely converted to biohydrogen. In contrast, the maximum HPR was
obtained at 35
C during condensed molasses solubles
fermentation in CSTR system [24]. While developing biohydrogen production technology, it is very important that the
system is operated at lower temperatures that would not only
have positive energy gain but also safe during maintenance
and monitoring. However, for certain wastewaters like textile
industry effluent with a temperature around 70e80
C the
biohydrogen production system might need to be operated at
thermophilic conditions.

2.5.

25

HY (mmol/g COD)

HY (mmol/g COD)

25

Reactor configuration

Several types of reactors have been studied to generate biohydrogen efficiently. However, each reactor type has its own
benefits and drawbacks. Batch reactors are easy to operate but
less efficient, therefore are used mainly in laboratory tests.
Upflow anaerobic sludge blanket (UASB) bioreactor has been
used extensively in laboratory or pilot scale studies [27]. The

0
20

30

40

50

60

70

Temperature ( oC)

Fig. 5 e Relationship between temperature and

biohydrogen production performance using wastewater
feedstock: (a) hydrogen production yield (HY) and (b)
hydrogen production rate (HPR) based on the data listed in
Table 1 (C, batch mode; B, continuous mode).

UASB reactor is effective in treating organic wastes and converting them into biohydrogen. Fixed bed reactors are also
shown to produce biohydrogen efficiently [28], but there are
several problems associated with fixed-bed reactors, such as
localized populations differing over the length of the reactor,
channeling due to inefficient mixing, and incomplete
conversion of substrates due to poor mass transfer efficiency.
There are also some very efficient hydrogen producing bioreactors associated with formation of self-flocculating granular
sludge or matrix entrapped immobilized cells, such as carrierinduced granular sludge bed reactor (CIGSB) [29], CSABR with
silicone immobilized cells [25] and agitated granular sludge
bed reactor (AGSBR) [23]. These reactors produced biohydrogen at an extremely high rate due primarily to retaining
very high cell density in the bioreactor at a high dilution rate,
but still need to overcome the problems related to inefficient
mixing [30] and stability of functional granules.
On the other hand, bioreactors with suspended bacterial
populations are generally not able to operate at a high dilution
rate due to problems of biomass wash-out, thereby having
lower biohydrogen production rates when compared with
granular sludge or immobilized cells-based bioreactors. Biohydrogen production has been conducted using chemostats
and continuously stirred reactors [24] or an anaerobic
sequencing batch reactor (AnSBR) process, which is one of the
novel and promising high-rate anaerobic processes.
Sequencing batch reactor offers distinct advantages when
compared with continuous processes, including a high degree

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of process flexibility and no requirement for a separate clarifier [31]. Each of these types of reactors has been shown to be
capable of producing biohydrogen. However, due to the high
solids content that restricts flow, the best reactor type for
these experiments is the one utilizing suspended cultures in
a well mixing environment.
As shown in Table 1, there are 13 cases using continuousfeeding systems such as ASBR (2 cases), CSTR (8 cases),
UASB (2 cases) and upflow system (1 case) for fermenting
wastewater to produce biohydrogen. In addition, three types
of high-rate biohydrogen production systems (CSABR, CIGSBR
and AGSBR) were also developed [25,29,30]. A 400 L pilot
reactor of AGSBR type for hydrogen production was built by
Green Energy Development Center (GEDC), Feng Chia
University in Taiwan. A synthetic wastewater (sucrose-based)
and a fermentation wastewater (condensed molasses solubles) were examined for their biohydrogen production efficiency in this pilot system [23,32]. A HPR of 15.6 L/L/d was
obtained with sucrose-based synthetic wastewater at pH 6.0,
35
C, and an OLR of 240 g COD/L/d. For the fermentation
wastewater, a HPR of 1.5 L/L-d was obtained with an initial
cultivation pH 6.7, 37
C cultivation temperature and substrate
concentration of 40 g COD/L.

3.
Feasibility of fermentative biohydrogen
production from unexplored/new wastewaters
There are various kinds of wastewaters that remain unexplored for biohydrogen production process, such as oil
industry wastewaters having low pH [33]. Moreover, the
concept of combined wastewaters could also lead to a new
path for biohydrogen production. For example, combining
a carbohydrate-rich wastewater with a wastewater containing
high content of nitrogen source could practically achieve
a higher biohydrogen yield [34]. Apart from combination of
two types of wastewaters, a combination of solid organic
wastes and wastewater could also be a novel approach for
biohydrogen production.

In addition to fermentative hydrogen production, various

species of Clostridium, in particular C. acetobutylicum, can
produce 1-butanol (biobutanol), whose energy content (27 MJ/
L) is similar to that of gasoline (32 MJ/L) and turns out to be
a promising biofuel that can completely replace gasoline or
mix with gasoline at any ratio for transportation. Therefore,
using an appropriate seed culture and fermentation conditions, biohydrogen and biobutanol could be produced simultaneously utilizing organic wastewaters as substrate. On the
other hand, methane is another interesting biofuel produced
from anaerobic fermentation technology. A two-stage anaerobic process producing both biohydrogen and methane from
organic waste materials has been proposed [40]. In this twostage process, acidogenic bacteria in the first stage convert
substrates such as carbohydrates to biohydrogen, carbon
dioxide and fatty acids. The gaseous products exit the reactor
and the volatile fatty acids (VFAs) enter the second stage
where they are further converted to methane and carbon
dioxide by methanogens. The overall products of the process
are biohydrogen, methane and carbon dioxide.
Autotrophic algae can convert solar radiations and carbon
dioxide to produce energy to support their growth [38].
Therefore, the carbon dioxide produced during dark fermentation, photo fermentation as well as anaerobic digestion
(methane production) could be captured by algae culture for
their growth. Lo et al. [36] have conducted a proof-of-concept
study by combining dark and photo fermentation processes
with microalgae culture system to completely remove the
carbon dioxide generated from the fermentation processes.
Fig. 6 describes the scheme of the development of an
advanced integration bioenergy producing process, which
combines pretreatment, dark biohydrogen production,
anaerobic digestion, and carbon dioxide capture technologies
for converting non-food feedstock into a variety of bioenergy
(biohydrogen, biobutanol and methane) without any carbon
dioxide emission. This process completely conforms to the
concept of green energy production, being an eco-friendly,
economic, and sustainable source of energy.

4.
Advanced integrated bioenergy
production systems using wastewaters
To increase the bioenergy production efficiency, a two-stage
fermentation process, such as dark fermentation with
subsequent photo fermentation [35e37] or dark fermentation
with subsequent anaerobic digestion (i.e., methane formation)
have been proposed [38]. Combination of dark fermentation
and photo fermentation could lead to maximum theoretical
yield of 12 mol hydrogen per mole of glucose. This process is
investigated in the HYVOLUTION project within the European
Unions Sixth Framework Program for Research and Technological Development [39]. The aim of HYVOLUTION was to
deliver prototypes of process modules, which are needed to
produce biohydrogen of high quality in a bioprocess fed on
multiple biomass feedstocks. The combination of photoheterotrophic bacteria with thermophilic fermentation
process achieved 75% conversion efficiency of the maximum
theoretical HY (i.e., 9 mol hydrogen per mole glucose).

Fig. 6 e A scheme of integrated bioenergy production

processes using wastewater feedstock.

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5.

Net energy efficiency analysis

EN

In order to develop an energy efficient biohydrogen production system, it is often necessary to make an evaluation of the
Net Energy Gain (NEG). Such analysis is an integral feature of
energy economics which is calculated as the difference
between the energy input to harvest an energy source and the
amount of energy gained from such harvest. Many dark
fermentation studies have been operated at more than
ambient temperatures, in order to get high yield, without
considering net energy gain [41].
The net energy gain during dark fermentation of wastewaters is indirectly proportional to the cultivation temperature. Most dark fermentation studies have reported HY in
terms of moles biohydrogen produced per mole feedstock
used. The net energy gain defined above can be estimated
from the reported yields from the following equation [41]:

YCkVLHV MWH2 =MWf 103  VrW cr Tf  Ta
VC

where, Y is moles biohydrogen produced per mole feedstock, C

is the COD concentration of the feedstock [g COD/L], k is the
COD equivalent of the feedstock (g feedstock/g COD), V is the
liquid volume in the reactor [L], MW is the molecular weight,
LHV is the lower heating value of hydrogen [120,000 kJ/kg], Tf is
fermentation temperature, Ta is ambient temperature, rw is
the density of water [1 kg/L]; cp is the specific heat of water
[4.2 kJ/kg K]. In our calculation Ta was set equal to the standard
ambient temperature of 25
C. In this review we have calculated the net energy gain for dark fermentation of all types of
wastewaters at their operational temperature. Generally, the
operations performed at ambient temperatures have positive
energy gain because not much energy is used to increase the
operating temperature. Based on the net energy gain calculation, we found vinasse (140.39 kJ/g COD) followed by glycerin

HY (mmol/g COD feedstock)

0

10

15

20

25

30

140.39

25

Vinasse WW
Sugary WW

-0.15

60

2.97

32

Sugarbeet WW

55

Rice winery WW

0.58

35

Purified terephthalic acid

22.01

37

Probiotic WW

-4.47

35

Preserved fruit soaking solution

-3.25
3.26

23

Potato processing WW

37

Palm oil effluent

Olive pulp WW

0.06

35

2.86

35

Olive mill WW

-0.18

30

Lagoon WW

-6.91
51.84

25

Domestic sewage

39

Distillery effluent

-2.01

23

Confectionery processing WW

4.25

Coffee drink WW

35

1.62

CMS

35

0.71
68.65

25

Clycerin WW

0.61

37

Citric acid WW
Chemical WW and DSW

29

-4.28
55

cheese whey WW

3.49

38

cheese processing WW

-1.63

29

Cereal WW

-5.81
45

Cattle WW

-60.47

36

Brewery WW

-6.17

23

Apple processing WW

5.81

10 20 30 40 50 60 70 80 90 100 -50

Temperature (oC)

(1)

50

100

150

Net energy gain (kJ/g COD feedstock)

Fig. 7 e Net energy efficiency analysis of various wastewaters.

200

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wastewater (68.65 kJ/g COD) and domestic sewage (51.84 kJ/g

COD) has the highest positive net energy gains, with the HY of
10 mmol/g COD (Fig. 7).

6.
Concluding remarks and future
perspectives
Biohydrogen is a promising energy-carrier which can be converted into electricity via fuel cell with high efficiency. The
clean characteristics make biohydrogen play an important
role in solving global climate change problems and thus
hydrogen economy is proposed. The environment-friendly
green biohydrogen can be obtained by dark fermentation
using non-food feedstock and waste organic materials. To
integrate the up- and down-stream technologies of biohydrogen and to evaluate the feasibility of the green hydrogen
economy, declaring hydrogen society scenario and constructing a model of feasible biohydrogen energy technology
development roadmap are the key steps to accelerate the
realization of hydrogen economy.
The most feasible commercial process for the biohydrogen
generation technologies could be a wastewater on-site
system, such as the factories and the communities, enabling
stable and sufficient supply of high-organic content wastes as
the feedstock for biohydrogen production. Integrating the
biohydrogen process to the conventional wastewater treatment process has many advantages such as improving environmental compatibility of the wastewater treatment process
and lowering the wastewater treatment cost by generating
clean and valuable bioenergy products. The biohydrogen
produced during wastewater treatment can be fed into the
boiler to reduce the fossil fuel loading or can be converted to
electricity by fuel cell to supply the power for the factory.
Moreover, the carbon dioxide produced could be captured and
re-utilized to gain additional benefits for the factory resulting
in the reduction of capital investment.
Another biohydrogen energy application has been suggested by Chu et al. [42] who established a feasible model of
a biohydrogen energy-based sustainable house. The
biohydrogen-based house confirms the concept of sustainable
green energy design by performing the stages of energy
production, storage, distribution control, load applications,
and recycling and reuse. In order to increase the efficiency of
total energy recovery and to reduce the COD of organic
effluent for discharging into a community sewer system, Chu
et al. [43] coupled an anaerobic digester to a dark biohydrogen
fermentation process to produce methane using the effluent
of dark fermentation as the substrate at the sustainable green
energy house. There are also several alternative feedstocks for
bioenergy production in the sustainable green energy houses,
such as kitchen waste, convenient store dairy waste, fruit and
vegetable market waste, tofu factory waste and sewage
sludge. A support system in a green energy sustainable house
includes: (1) a biohydrogen/methane chamber, (2) biohydrogen storage/methane tanks, (3) a biohydrogen supply
system, (4) fuel cells, (5) a power distribution panel and (6)
building power load. A biohydrogen filling facility must be
added if hydrogen fuel cell vehicles are used in a future
hydrogen energy society. The biohydrogen/methane

production chamber should contain a substrate tank,

a nutrient tank, a biohydrogen production fermentor, a gasliquid separator, a biohydrogen purification device, and
a methane fermentor [43]. This biohydrogen-based sustainable green energy house demonstrates a feasible solution to
the development of a self-sustainable community utilizing
biohydrogen and methane as the major energy sources.

Acknowledgments
The authors gratefully acknowledge the financial support by
Taiwans Bureau of Energy (grant no. 99-D0204-3), Taiwans
National Science Council (NSC-99-2221-E-035 -024 -MY3, NSC99-2221-E-035 -025 -MY3, NSC-99-2632-E-035 -001 -MY3), Feng
Chia University (FCU-10G27101) and APEC Research Center for
Advanced Biohydrogen Technology.

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