Bioresource Technology 102 (2011) 8524–8533

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

Bioreactor and process design for biohydrogen production

Kuan-Yeow Show a, Duu-Jong Lee b,⇑, Jo-Shu Chang c
a
Department of Environmental Engineering, Faculty of Engineering and Green Technology, Universiti Tunku Abdul Rahman, Jalan University, Bandar Barat,
31900 Kampar, Perak, Malaysia
b
Department of Chemical Engineering, National Taiwan University of Science and Technology, Taipei 106, Taiwan
c
Department of Chemical Engineering, National Cheng Kung University, Tainan 701, Taiwan

a r t i c l e i n f o a b s t r a c t

Article history: Biohydrogen is regarded as an attractive future clean energy carrier due to its high energy content and
Received 28 November 2010 environmental-friendly conversion. It has the potential for renewable biofuel to replace current hydrogen
Received in revised form 16 April 2011 production which rely heavily on fossil fuels. While biohydrogen production is still in the early stage of
Accepted 18 April 2011
development, there have been a variety of laboratory- and pilot-scale systems developed with promising
Available online 30 April 2011
potential. This work presents a review of advances in bioreactor and bioprocess design for biohydrogen
production. The state-of-the art of biohydrogen production is discussed emphasizing on production path-
Keywords:
ways, factors affecting biohydrogen production, as well as bioreactor configuration and operation. Chal-
Biohydrogen
Biophotolysis
lenges and prospects of biohydrogen production are also outlined.
Bioreactor Ó 2011 Elsevier Ltd. All rights reserved.
Dark fermentation
Photo-fermentation

1. Introduction While attractive features of biohydrogen production have been

There have been growing concerns of greenhouse gas emissions
and other pressing environmental issues over the use of fossil fuels.
Hydrogen fuel is a promising alternative to conventional fossil
fuels because it has the potential to eradicate all the environmental
problems that the fossil fuels would create. Many scientists per-
ceive hydrogen gas as the future fuel because of its non-polluting
features and its use in highly efficient fuel cells for electricity.
However, a major doubt over the use of hydrogen as a clean energy
alternative is the way it is produced. The current hydrogen gas pro-
duction is unfriendly as it is being generated from fossil fuels
through thermo-chemical processes, such as hydrocarbon reform-
ing, coal gasification and partial oxidation of heavier hydrocarbons.
Biohydrogen production by microorganisms has attracted
increasing global attention, owing to its potential for inexhaustible,
low-cost and renewable source of clean energy. Studies on biohy-
drogen production have been focusing on biophotolysis of water
using algae and cyanobacteria, photo-decomposition of organic
compounds by photosynthetic bacteria, and dark fermentation
from organic compounds with anaerobes. Anaerobic hydrogen fer-
mentation appears to be most favorable since hydrogen could be
generated at higher rates. Moreover, the process can be carried
out on various organic wastes and wastewaters enriched with car-
bohydrates, thereby achieving sustainable low cost biohydrogen
production with concomitant waste purification.
⇑ Corresponding author.
E-mail address: djlee@ntu.edu.tw (D.-J. Lee).
demonstrated in laboratory studies, the technology is yet to com-
pete with commercial hydrogen production processes from fossil
fuels in terms of cost, efficiency and reliability (Lee et al., 2010).
Thus, improving the efficiency of biohydrogen production poses a
major challenge to many researchers. This review provides an
overview of the state-of-the-art and perspectives of biohydrogen
production research. The review focuses on biohydrogen produc-
tion pathways, major factors affecting biohydrogen production,
as well as bioreactor configuration and operation. Challenges and
prospects of biohydrogen production are also outlined.
2. Hydrogen production pathways
Biohydrogen can be generated via different pathways which can
be broadly categorized into two distinct groups, viz. light-depen-
dent and dark fermentative processes. Light-depended processes
include photolysis and photo-fermentation, whereas dark fermen-
tation is the major light independent process.
2.1. Photolysis
Hydrogen can be produced directly via water-splitting process
through the photosynthetic capability of algae and cyanobacteria.
The biohydrogen production occurs via direct absorption of light
and transfer of electrons to hydrogenases and/or nitrogenases en-
zymes. Under anaerobic or excessive energy conditions, the excess
electrons are released by microorganisms using hydrogenase
enzyme which converts the hydrogen ions to hydrogen gas (Turner

0960-8524/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.biortech.2011.04.055
 K.-Y. Show et al. / Bioresource Technology 102 (2011) 8524–8533
et al., 2008). The protons and electrons extracted via the
water-splitting reactions are recombined by chloroplast hydroge-
nase to form molecular hydrogen gas (Hankamer et al., 2007).
In the water-splitting process, oxygen is also produced which in
turn suppresses hydrogen production (Kapdan and Kargi, 2006). To
prevent oxygen buildup leading to hydrogen suppression, research
work has been carried out to identify or engineer less oxygen-sen-
sitive algae and bacteria, separate the hydrogen and oxygen se-
quence, or change the ratio of photosynthesis to respiration
process (US DOE, 2007). Sulfate has been found effective in sup-
pressing oxygen generation, but the hydrogen production is also
inhibited (Turner et al., 2008).
With the major substrate being water which is abundant, direct
photolysis via water-splitting demonstrates promising future.
However, there are challenges to be dealt with. Current problems
such as the need of large surface area to collect light and difficulty
in achieving continuous hydrogen production under aerobic condi-
tions are to be addressed (US DOE, 2007).
Biohydrogen can also be produced through some microorgan-
isms like algae that can directly produce hydrogen under certain
conditions. It has been reported that sulfur-deficient green algae
whose energy was derived from light under anaerobic conditions
could induce the hydrogenase reactions to produce hydrogen
photo-synthetically (Laurinavichene et al., 2008). Under the sul-
fur-deprivation condition, the algae consume large amounts of cel-
lular starch and protein. Apparently, these catabolic reactions help
to sustain indirectly the hydrogen production process.
Photolysis biohydrogen production is deemed feasible if the
efficiency of photon conversion can be improved via large-scale al-
gal bioreactors. Engineering approach to regulate light inputs in
improving the algae photon conversion efficiency can be optimized
through the algal bioreactor operation. Substantial increases in
light conversion efficiency from about 5% to 15% had been reported
(Laurinavichene et al., 2008). Attaining 10–13% efficiency is feasi-
ble by using molecular approach to engineer the microorganisms
to better utilize the light energy (Turner et al., 2008).
2.2. Photo-fermentation
Unlike the photolysis process in which hydrogen is produced by
cyanobacteria and/or green algae directly or indirectly from water,
purple photosynthetic microbes are able to generate hydrogen
from organic substrates through photo-fermentation. Despite the
relatively lower yields of hydrogen from photosynthetic bacteria,
the fermentative pathway is a promising process of biohydrogen
production, due mainly to its higher rate of hydrogen evolution
in the absence of any light source (Wang et al., 2010a). The process
can be versatile in terms of types of feed to the microbes (Redwood
et al., 2009).
In contrast to green algae, the photosynthetic purple bacteria are
unable to split water by their simple photosynthetic structure.
However, these bacteria are able to use simple organic acids or even
dihydrogensulfide as electron donor derived from the organic sub-
strates under anaerobic conditions (Akkerman et al., 2003). The elec-
trons extracted from the organics are pumped through electron
carriers, during which protons are driven across the membrane.
An electrochemical gradient is developed and the ATP synthase syn-
thesize adenosine triphosphate (ATP) from adenosine diphosphate
(ADP) by using the energy generated from protons moving down
the gradient. The surplus ATP energy can be used to transport the
electrons further to ferredoxin – an electron carrier in the chloro-
plasts. When molecular nitrogen is deficient, the ATP synthase can,
again with the extra ATP energy, reduce protons into hydrogen gas
using the electrons derived from the ferredoxin (Akkerman et al.,
2003). In this manner, the organic acids derived from the substrates
can be ultimately transformed into hydrogen and carbon dioxide.
8525
2.3. Dark fermentation
Dark fermentation under anaerobic conditions seems to be the
most favorable among the bioproduction processes. The fermenta-
tion can be carried out at higher rates and lower cost using various
organic substrates and wastewaters (Hallenbeck and Ghosh, 2009).
Dark fermentation uses primarily anaerobic bacteria, although
some algae are also used, on carbohydrate rich substrates grown
without the need of light energy (Kapdan and Kargi, 2006).
Molecular hydrogen formation is generally realized via two
pathways in the presence of specific coenzymes, i.e. either by for-
mic acid decomposition route or by the re-oxidization of nicotin-
amide adenine dinucleotide (NADH) route (Eqs. (1) and (2))
2þ þ
NADH þ Hþ þ 2Fd !2Hþ þ NADþ þ 2Fd ð1Þ
þ 2þ
2Fd þ 2Hþ ! 2Fd þ H2 ð2Þ
hydrogenase
The Embden–Meyerhof or glycolytic pathway is undoubtedly
the most common route for glucose degradation to pyruvate,
which is found in all major of microorganisms and functions in
the presence or absence of oxygen (Prescott et al., 2002). In this
pathway, glucose is converted into pyruvate associated with the
conversion of NADH from NAD+ via anaerobic glycolysis, which
could be represented by Eq. (3):
C6 H12 O6 þ 2NADþ ! 2CH3 COCOOH þ 2NADH þ 2Hþ ð3Þ
The disposal of electrons via pyruvate-ferredoxin oxidoreduc-
tase or NADH-ferredoxin oxidoreductase and hydrogenase might
be affected by the corresponding NADH and acetyl-CoA levels as
well as environmental conditions. As a result, the oxidation–reduc-
tion state has to be balanced through the NADH consumption to
form some reduced compounds, i.e. lactate, ethanol and butanol,
resulting in a lowered hydrogen yield.
In the recent development, metabolic engineering has received
increasing attention in improving biohydrogen production.
Improvements in hydrogen yields by existing pathways has been
attempted by increasing the flux through gene knockouts of com-
peting pathways or increased homologous expression of enzymes
involved in the hydrogen-generating pathways (Hallenbeck and
Ghosh, 2009). To further elucidate a better understanding in
improving biohydrogen production, major factors affecting hydro-
gen production such as operating conditions, feedstock and reactor
configuration will be systematically reviewed and extensively dis-
cussed in the following sections.
3. Factors affecting biohydrogen production
Inevitably, performance of hydrogen-producing bioreactor sys-
tems and operation are dictated by various factors. Factors associ-
ated with environmental conditions, process operating conditions,
and chemical conditions are reviewed in this section.
3.1. Culture pH
System pH has been recognized to be one of the most important
environmental factors affecting metabolic pathways and the yield
of biohydrogen. Comparative studies with respect to the effect of
pH on continuous hydrogen production revealed that the optimum
pH range to achieve the maximum hydrogen yield or specific
hydrogen production rate was found between 5.2 and 6.0 in most
studies using pure or mixed cultures of bacteria (Oh et al., 2004a;
Zhang et al., 2008a). Fang and Liu (2002) investigated the effect of
pH on hydrogen production from glucose in a CSTR over the range
of 4.0–7.0 and found the optimum hydrogen yield occurring at a
8526 K.-Y. Show et al. / Bioresource Technology 102 (2011) 8524–8533
pH of 5.5. Chang and Lin (2004) observed hydrogen production
from glucose using continuous cultures under identical conditions
except pH values, and concluded that higher hydrogen yield and
production rate are attained at a pH of 5.7 compared with pH
6.4. Ren et al. (2009) emphasized the maximum hydrogen yield
was achieved only when microbial reactions followed an ethanol
fermentation type that occurred at a pH of about 4.5.
Venkata Mohan (2009) reported that the initial pH values of
5.5–7.5 may represent the optimum and acceptable ranges of pH
for hydrogen production, whereas the hydrogen yield may be shar-
ply dropped at pH out of the optimum range. While system pH be-
low 6 had shown negative influence on the substrate degradation
efficiency due to the inhibition of methanogens, increase in feed
pH has resulted in suppressed hydrogen production. pH control
could stimulate the microorganisms to achieve maximum hydro-
gen production ability because the activity of hydrogenase was
inhibited at low or high pH during fermentation (Venkata Mohan,
2009).
3.2. Hydraulic retention time
Hydraulic retention time (HRT) could be used as a tool to se-
lect microbial populations whose growth rates are able to catch
up with mechanical dilution created by continuous volumetric
flow. Typical specific growth rate of methane-producing bacteria
is about 0.0167–0.02 h1, which is much lower than that of
hydrogen-producing bacteria of about 0.172 h1 (Lo et al.,
2009a). Therefore, hydrogen-producing bacteria can be retained
while washing out methane-producing bacteria by regulating
HRT. Lo et al. (2009a) found that the methane concentration ran-
ged between 0.0011 and 0.0058 mol l1 at low dilution rates
(D = 0.002–0.0167 h1) was hardly measureable at higher dilu-
tions (D > 0.075 h1), indicating negligible methanogenic activity
at high dilution rates. This means that HRT is capable of inhibit-
ing or terminating methanogenesis in hydrogen production via
anaerobic fermentation.
Zhang et al. (2006a) found that shortening the HRT from 8 h to
6 h would reduce microbial diversity associated with inhibition of
propionate production without affecting the existence of dominant
species, leading to an increase in the hydrogen yield. Similar find-
ing was also reported by other researchers (Hussy et al., 2003).
These results provided evidence that hydrogen yield, a function
of the microbial populations, can be affected by HRT (Zhang
et al., 2006a). This might explain the view of HRT-dependent
hydrogen yield obtained by some other researchers in the similar
systems (Fan et al., 2006; Venkata Mohan, 2009).
3.3. Hydrogen partial pressure
The dissolved hydrogen concentration in the liquid phase, con-
tributing to the hydrogen partial pressure, is one of the key factors
affecting microbial pathways in fermentative hydrogen produc-
tion. Hydrogen production is less favorable as the hydrogen partial
pressure rises (Tiwari et al., 2006).
Thus, it is important to remove excess hydrogen from the sys-
tem to maintain hydrogen production. Several studies have re-
vealed that reducing hydrogen partial pressure can considerably
enhance hydrogen production. Many strategies of removal or sep-
arating excess hydrogen gas have been developed to avoid the neg-
ative effect of the hydrogen accumulation in the gas phase. Mizuno
et al. (2000) found an increase in the hydrogen yield from 0.85 to
1.43 mol mol1 hexose when the reactor is sparged with nitrogen
at 15 times hydrogen produced rate. Hussy et al. (2003) also re-
ported a 1.5 times enhanced hydrogen yield from 1.3 to
1.9 mol mol1 hexose by sparging the reactor with nitrogen to re-
duce hydrogen in the off-gas from 50% to 7%.
3.4. Nutrients
In hydrogen fermentation processes, nitrogen, phosphate and
other inorganic trace minerals are necessary supplements for car-
bohydrate-based feedstocks in order to obtain optimal cell cultiva-
tion and hydrogen production. Previous study indicated that
organic nitrogen seems to be more favorable for hydrogen evolu-
tion compared with inorganic one (Yokoi et al., 2001). The highest
level of H2 production from sweet potato starch residue by a re-
peated batch culture containing C. butyricum IFO13949 when
1.0% polypepton was added as nitrogen source was reported (Yokoi
et al., 2001). In contrast, addition of urea, (NH4)2SO4 or NH4Cl re-
sulted in the absence of hydrogen evolution by the same culture.
Phosphate is one of the important inorganic nutrients required
for optimal hydrogen production (Lin and Lay, 2004a). Excess
phosphate may favor VFAs and hydrogen production over solvent
production, so phosphate supplementation may be needed with
carbohydrate-rich feeds.
Lin and Lay (2004b) examined the effect of trace elements
including Mg, Na, Zn, Fe, K, I, Co, NHþ 4 , Mn, Ni, Cu, Mo and Ca on
hydrogen production by a C. pasteurianum-predominant culture.
The authors found elements Mg, Na, Zn and Fe being important
supplements and proposed an optimum nutrient formulation con-
taining (mg l1) MgCl26H2O 120, NaCl 1000, ZnCl2 0.5 and FeS-
O47H2O 3 for the optimal hydrogen yield. However, less effect of
NHþ 4 and a lower value of optimum iron concentration indicated
here are inconsistent with those values recommended in other
studies (Lin and Lay, 2004c; Wang and Wan, 2009).
Iron is a key component in the enzymatic activity of hydrogen
production. Biological formation and consumption of molecular
hydrogen (H2) are catalyzed by hydrogenases, of which three phy-
logenetically unrelated types are known: [NiFe]-hydrogenases,
[FeFe]-hydrogenases, and [Fe]-hydrogenase (Shima et al., 2008).
Among these classes, [FeFe]- and [NiFe]-hydrogenases are the ma-
jor enzymes; most of hydrogenases found in microorganisms are
belong to one of these two enzymes except [Fe]-hydrogenase that
were discovered in some methanogens. The mostly monomeric
[FeFe]-hydrogenases are more involved in H2 evolution, and dis-
play high sensitivity to oxygen (O2) and carbon monoxide (CO)
(Bleijlevens et al. 2004; Frey, 2002). [NiFe]-hydrogenases, com-
posed of two subunits, are involved in H2 oxidation, but also cata-
lyze reversible reactions (Adams and Hall, 1979; Nishihara et al.,
1997). These enzymes are less active than [FeFe]-hydrogenases
by 10–102-fold, but much more tolerant to O2 or CO in a reversible
manner (Frey, 2002). This property is a significant advantage for
the biotechnological application of hydrogenase in biohydrogen
production, since it is not necessary to protect the production pro-
cess from O2.
3.5. Temperature
Microbes are capable of producing hydrogen in a temperature
range of 15–85 °C (Kanai et al., 2005), but laboratory-scale studies
from a review indicated that about 73% studies were carried out
with mesophilic cultures (Li and Fang, 2007). Chang and Lin
(2004) studied the hydrogen production capability of a mixed cul-
ture under varying temperatures from 15 to 34 °C and found that
hydrogen yield and specific hydrogen production rate increased
with temperature, achieving respective maximum values of
359 mmol l1 d1 and 1.42 mol H2 mol1 glucose at 30–34 °C and
28–32 °C, respectively. However, both values obtained at the ambi-
ent temperatures are much lower compared with the correspond-
ing values of 574 mmol l1 d1 and 1.70 mol H2 mol1 glucose at a
consistent temperature of 35 ± 1 °C. Minnan et al. (2005) also re-
ported that increasing temperatures from 25 °C to 35–36 °C im-
proved the hydrogen production activity.
 K.-Y. Show et al. / Bioresource Technology 102 (2011) 8524–8533
Studies using mesophilic cultures (Lee et al., 2006) seemed to
indicate that, although hydrogen-producing bacteria are able to
perform at ambient temperature conditions, increasing tempera-
ture in the mesophilic regime always improves the hydrogen pro-
duction, while further increasing culture temperature beyond
mesophilic range may cause a decrease in hydrogen production,
presumably dependent on physiological characteristics of micro-
bial cultures.
3.6. Substrate concentration
The effect of substrate concentration on hydrogen production,
however, has been a point of debate. In studies concerning the ef-
fect of substrate concentration, Kim et al. (2006) found hydrogen
yield to increase with increasing glucose concentration from 10
to 35 g l1 at a HRT of 12 h, whereas Kyazze et al. (2006) examined
continuous hydrogen production at 12 h HRT on 10–50 g l1 su-
crose and found that the hydrogen yield decreased from
1.7 ± 0.2 mol H2 mol1 hexose added at 10 g l1 sucrose to
0.8 ± 0.1 mol H2 mol1 hexose added at 50 g l1. A decrease in
hydrogen yield could occur at HRTs of 2.5 h and 10 h when the
CSTR feeding strength increased from 10 g glucose l1 to 40 g glu-
cose l1 (Van Ginkel and Logan, 2005). These studies indicate that
besides substrate concentration, other operating conditions such
as HRT and composition of microbial cultures also affect continu-
ous hydrogen production. As iron is a key component in the enzy-
matic activity of biohydrogen production, the observed decrease of
the hydrogen yield in continuous cultures when increasing the or-
ganic loading rate could also be due to limitation (starvation) in
iron compound. At high glucose inlet concentrations, the limiting
factor may not be glucose but iron in the substrate.
A wide range of substrate concentration ranging from 2.5 to
120 g hexose l1 had been reported (Zhang et al., 2007a). With a
high feeding sucrose concentration of up to 30–40 g COD l1 the
granular sludge-based continuously stirred anaerobic bioreactor
produced hydrogen more efficiently at a short HRT of 0.5 h, with
the highest volumetric rate of 15 l h1 l1 and an optimal yield of
ca. 3.5 mol H2 mol1 sucrose (Wu et al., 2006). Although the opti-
mum hydrogen yield was obtained at a glucose concentration of
10 g l1 in granule sludge or biofilm sludge reactors, Zhang et al.
(2007a) noted that hydrogen production was almost kept consis-
tent at a substrate concentration ranging from 10 to 30 g l1, but
sharp decrease in hydrogen yield took place at glucose concentra-
tions larger than 40 g l1.
3.7. Seed culture
Clostridium and Enterobacter are most widely used as inocu-
lums for fermentative hydrogen production (Wang and Wan
2009; Zhang et al., 2006b, 2008b). Most of the studies using pure
cultures of bacteria for fermentative hydrogen production were
conducted in batch mode and used glucose as substrate (Wang
and Wan, 2009). Mixed cultures of bacteria from anaerobic sludge,
municipal sewage sludge, compost and soil had been widely used
as inoculums for fermentative hydrogen production (Li and Fang,
2007). Fermentative hydrogen production processes using mixed
cultures are more practical than those using pure cultures, as the
former are simpler to operate and easier to control, and may have
a broader source of feedstock (Li and Fang, 2007). In addition, there
are also several other potential merits of using microbial consortia
instead of pure cultures. Practical hydrogen fermentations will
have to be carried out under non-sterile conditions using readily
available feedstocks with minimal pretreatment. Mixed consortia
would be able to address these issues as they have been selected
for growth and are robust under non-sterile conditions (Zhang
et al., 2007b, 2008b). As microbial enriched consortia, they are also
8527
able to support a wide variety of metabolite activities, and they are
potentially more resilient to changes in environmental conditions
(Hallenbeck and Ghosh, 2009).
3.8. Feedstock
Simple sugars such as glucose, sucrose and lactose are readily
biodegradable and are thus preferred as model substrates for
hydrogen production. However, the costs for pure carbohydrate
sources are high for practical-scale hydrogen production, which
can only be viable when based on renewable and low cost sources.
As reported by numerous studies of biohydrogen fermentative pro-
cesses, carbohydrates are the main source of hydrogen. Thus,
wastes and biomass rich in sugars and/or complex carbohydrates
turn out to be the most suitable feedstocks for biohydrogen gener-
ation (Lo et al., 2008, 2009a; Luo et al., 2011; Ntaikou et al., 2010).
Some complex substrates are not ideal for fermentative hydro-
gen production due to their complex structures. The main technical
difficulty for practical hydrogen production is the process stability
and the short duration of the hydrogen production from wastes
and complex substrates. Studies have indicated that it might be
uneconomical to recover hydrogen from waste such as wastewater
sludge (Okamoto et al., 2000; Van Ginkel et al., 2005). The hydro-
gen yields obtained were rather low.
Complex substrates, however, after being pretreated by some
methods, they can be easily used by hydrogen-producing bacteria
(Luo et al., 2010). For example, Zhang et al. (2007b) reported that
the hydrogen yield from cornstalk wastes after acidification pre-
treatment was much larger than that from cornstalk wastes with-
out any pretreatment. Complex solid wastes, such as organic
residues, food processing, mixed wastes, digester sludge, and mu-
nicipal wastes have also been tested as feedstocks for fermentative
hydrogen production (Juang et al., 2011; Kim and Lee, 2010; Ntai-
kou et al., 2010). Apart from carbohydrates, such wastes usually
have quite high contents of proteins and fats, and thus their bio-
transformation to hydrogen is comparatively lower than those ob-
tained from carbohydrate based wastewaters. Biotransformation of
wastes and wastewater towards hydrogen can be considered quite
appealing from both the environmental and the economical stand-
point (Ntaikou et al., 2010).
4. Bioreactor configuration and operation
4.1. Photo-bioreactors
Design of a photo-bioreactor depends on microbiological pro-
cesses associated with microalgae, diatoms or cyanobacteria.
While these photo-heterotrophic bacteria differ in photochemical
efficiency, absorption coefficient and size, the light regime includ-
ing light and dark cycles is assumed to be much more determining
than biological factors (Akkerman et al., 2003). Hence the produc-
tivity of a photo-bioreactor is light-dependent, and a large surface
to volume ratio is a prerequisite for a productive photo-bioreactor
for optimal light exposure of the algae. Provisions for thermal con-
trol and monitoring flow rates, pH, and dissolved oxygen, sulfur,
and hydrogen are essential. Technical development is now moving
towards devising gas-tight systems, microalgae culturing as well as
computer-controlled system for monitoring and automatic nutri-
ent delivery and culture dilution.
Photo-bioreactors have been designed to achieve an economi-
cal, rapid multiplication and high density of the microalgae culture
(Evens et al., 2000). Different photo-bioreactor designs including
flat plate, tubular, pond or pool-type, have been investigated (Akk-
erman et al., 2003). Detailed cost analyses must be conducted on
minimizing the materials and operating costs, and also for optimiz-
8528 K.-Y. Show et al. / Bioresource Technology 102 (2011) 8524–8533
ing the yield and gas collection. Critical parameters in the cost
analyses include the light environment, the climate and land space,
reactor construction materials, mechanism of culture mixing, reac-
tor maintenance and long-term operational stability with maximal
gas production. Adequate light supply is vital and light limitations
must be kept to a minimum. Light conversion efficiencies are low
(theoretically not more than 10%) and tend to decrease at higher
light intensities because of light saturation effect (Akkerman
et al., 2003). Hence for efficient biohydrogen production, it is crit-
ical to dilute the light and distribute it over the reactor volume, and
mix the culture at high rates, so that cells are exposed to the light
only for a short time.
The exact configuration of the bioreactor also needs to be estab-
lished for the most effective use of light and surface area. Biomass
mixing is hence significant to ensure uniform dispersion of nutri-
ents and illumination of the culture, and to prevent sedimentation
of culture. Modular design of experimental system should be al-
lowed for possible scale-up. Such commercial scale should achieve
sustainable gas output and high hydrogen yields with compact
configuration. Trapping and removal of hydrogen gas in the system
are also important design consideration for photo-bioreactors. Gi-
ven the current advancement in photo-biohydrogen production,
technical and economic strategies for cycling of the microalgae
between sulfur-deprivation and supply must be developed
(Laurinavichene et al., 2008).
4.2. Dark fermentation bioreactors
Studies on batch, semi-continuous and continuous hydrogen-
producing bioreactors have been conducted. Batch hydrogen fer-
mentation normally brings about lower hydrogen production rates
in comparison with its semi-continuous or continuous counterpart.
From the engineering’s standpoint, continuous hydrogen produc-
tion is preferred. Besides the extensively studied Continuous Stir-
red Tank Reactor (CSTR), numerous biohydrogen bioreactor
processes such as Anaerobic Sequencing Batch Reactor (ASBR),
Membrane Bioreactor (MBR), Fixed-bed Bioreactor, Fluidized-bed
Bioreactor and Upflow Anaerobic Sludge Blanket (UASB) Bioreactor
have been developed with high production yields and output.
4.2.1. Continuous stirred tank reactor
Continuous stirred tank reactor (CSTR) are commonly used for
continuous hydrogen production (Younesi et al., 2008; Ding
et al., 2010). In a CSTR, hydrogen-producing microbes are com-
pletely-mixed and suspended in the reactor liquor from the mixing
pattern. Under such hydrodynamics, a good substrate-microbes
contact and mass transfer can be accomplished. On the other hand,
the CSTR is unable to maintain high levels of fermentative biomass
because of the rapidly mixed operating pattern. Biomass washout
may occur at short hydraulic retention times (HRTs), thus the
hydrogen production rates are considerably restricted. Depending
on the HRT, volatile suspended solids retained in the bioreactor
normally ranged between 1 and 4 g VSS l1 (Show et al., 2007,
2010; Zhang et al., 2006a). The highest CSTR hydrogen production
rate of 1.12 l h1 l1 was reported in fermentation of sucrose with a
mixed hydrogen-producing culture.
Fang et al. (2002) demonstrated that hydrogen-producing aci-
dogenic sludge could agglutinate into granules in a well-mixed
CSTR treating a synthetic sucrose-base wastewater at 26 °C and a
pH of 5.5. Operated at a HRT of 6 h, formation of the granular
sludge significantly enhanced retention of biomass (up to
20 g l1) with hydrogen production rate of up to 0.54 l h1 l1.
The CSTR system examined by Wu et al. (2006) was initially seeded
with silicone-immobilized sludge at 40 °C and pH 6.6 ± 0.2, and
reactor performance was examined at a HRT of 0.5–6 h and an
influent sucrose concentration of 10–40 g COD l1. It was noted
that self-flocculated granular sludge occurred at a HRT of 0.5 h
and reached a concentration of up to 35.4 g VSS l1, which in turns
resulted in a significant increase in hydrogen production rate up to
15 l h1 l1. In another CSTR study with granular sludge fermenting
glucose wastewater (10 g l1) at a pH of 5.5 and 37 °C, Zhang et al.
(2007c) reported a maximum hydrogen yield of 1.81 mol H2 mol1
glucose and a maximum hydrogen production rate of 3.20 l h1 l1
at a HRT of 0.5 h. Show et al. (2007) and Zhang et al. (2007d) found
that formation of granular sludge significantly increased overall
reactor biomass to as much as 16.0 g VSS l1, which enabled CSTR
to operate at an OLR of up to 20 g glucose h1 l1 and hence en-
hanced performance in H2 production.
4.2.2. Anaerobic sequencing batch reactor
The influence of substrate loading rate on fermentative hydro-
gen production was studied in biofilm configured sequencing
batch reactor using chemical wastewater as substrate (Vijaya
Bhaskar et al., 2008). Reactor was operated with selectively en-
riched anaerobic mixed microflora at different organic loading
rates after adjusting the feed to a pH of 6.0 to provide suitable envi-
ronment for acidogenic bacterial function. Variation in hydrogen
production yield from 6.064 to 13.44 mol H2 kg1 COD d1 was ob-
served with change in organic loading rate from 6.3 to
7.9 kg COD m3 d1 (Vijaya Bhaskar et al., 2008). Increase in the or-
ganic loading rate showed marked reduction in COD removal effi-
ciency from 22.6% to 17.2%.
In another study using a sequencing batch reactor (Buitron and
Carvajal, 2010), the effect of the temperature, the hydraulic reten-
tion time (HRT), and initial substrate concentration on hydrogen
production from Tequila vinasse was examined. When 25 °C and
12 h HRT were applied, insignificant quantity of biogas was pro-
duced. Using a longer HRT of 24 h and temperatures of 25–35 °C,
biogas containing hydrogen was produced. A maximum volumetric
hydrogen production rate of 50.5 ml H2 h1 l1 and an average
hydrogen content in the biogas of 29.2% were obtained when the
reactor was fed with 3 g COD l1 at 35 °C and 12 h HRT.
4.2.3. Membrane bioreactor
Use of a membrane bioreactor (MBR) to control biomass con-
centration was reported (Oh et al., 2004b). Operated with a HRT
of 3.3 h, the biomass concentration increased from 2.2 g l1 in a
control reactor (no membrane chemostat) to 5.8 g l1 in an anaer-
obic MBR by controlling sludge retention time (SRT) at 12 h. Under
such operating conditions, the hydrogen production rate increased
from 0.50 to 0.64 l h1 l1. Increasing the SRT would further en-
hance biomass retention which favors substrate utilization, but re-
sults in a decrease in hydrogen production rate. In a review by Li
and Fang (2007) on studies of hydrogen production by MBR, hydro-
gen production rates in a range of 0.25–0.69 l h1 l1 was reported.
It was concluded, however, that MBR did not exhibit superiority
other high-rate hydrogen production systems. Membrane fouling
and high operating cost also limit the use of MBR process in biohy-
drogen fermentation.
4.2.4. Fixed-bed bioreactor
A fixed-bed bioreactor is operated with support carriers packed
within the tank. The hydraulic mixing regime is less turbulent
comparing with the CSTR, this results in higher mass transfer resis-
tance along with lowered rates of substrate conversion and hydro-
gen production. High hydrogen yield could not be maintained
consistently in a fixed-bed reactor, because pH gradient distribu-
tion along the reactor column caused a heterogeneous distribution
of microbial activity. To overcome the mass transfer resistance and
pH heterogeneous distribution, recirculation flow was recom-
mended. Fixed-bed reactors with tubular, tapered or rhomboid
shape configurations operated at a HRT of 1.08 h were reported.
 K.-Y. Show et al. / Bioresource Technology 102 (2011) 8524–8533
The study reviewed that the rhomboid bioreactor with conver-
gent–divergent configuration has a maximum hydrogen produc-
tion rate of 1.60 l h1 l1 as compared with tapered reactor
(1.46 l h1 l1) and tubular reactor (1.40 l h1 l1). The superiority
in the production rate could be attributed to higher turbulent mix-
ing favoring mass transfer and low gas hold-up. It was observed
that both hydrogen production and substrate conversion rates in-
creased with slurry recycling ratio.
Support carriers play an important role in biomass retention
and hydrogen production in fixed-bed reactors. Loofah sponge, ex-
panded clay, and activated carbon had been used to support
growth of sewage sludge culture (Lee et al., 2006; Lo, 2009b). It
was found that loofah sponge is inefficient for biomass immobili-
zation, while other carriers exhibit better biomass yields. Activated
carbon is a better choice for support carriers, which resulted in the
maximum hydrogen production rate of 1.32 l h1 l1 by the fixed-
bed bioreactor operated at a HRT of 1 h and a sucrose concentra-
tion of 20 g l1.
4.2.5. Fluidized-bed bioreactor
In an examination on immobilized sewage sludge, Lin et al.
(2009) found the microbial culture could produce hydrogen effi-
ciently in a 3-phase fluidized-bed reactor at a HRT ranging from
2 to 6 h with a maximal steady-state rate of 1.82 l h1 l1 and an
optimal yield of 4.26 mol H2 mol1 sucrose. A much higher hydro-
gen production rate of 2.36 l h1 l1 was recorded by Zhang et al.
(2007d) who examined biofilm culture growing on granulated acti-
vated carbon in an anaerobic fluidized-bed reactor. Operated at
HRTs of 0.5–4 h and influent glucose concentrations of 10–
30 g l1, a culture pH of 4.0 was found to be most favorable for
hydrogen production. At that operating pH, biofilm sludge concen-
tration up to 21.5 g VSS l1 was retained. The findings suggested
superiority of fluidized-bed in biohydrogen production in compar-
ison with the fixed-bed counterpart.
4.2.6. Upflow anaerobic sludge blanket bioreactor
Upflow Anaerobic Sludge Blanket (UASB) bioreactor has been
used in biohydrogen research because of its good treatment effi-
ciency and capability in retaining high biomass concentration. It
was found that hydrogen yield was HRT-dependent and stabilized
at 1.5 mol H2 mol1 sucrose at HRT of 8–20 h (Chang and Lin,
2004). The yields decreased drastically at a HRT of 4 or 24 h. At a
HRT of 8 h, the maximum hydrogen production and specific hydro-
gen production rates were recorded at 0.25 l h1 l1 and
53.5 mmol H2 g1 VSS d1, respectively. Biomass retention reached
the maximum level of 7.2 g VSS l1 at a HRT of 24 h, but decreased
to 5.0 g l1 at the optimum HRT of 8 h (Table 1).
Anaerobic granular sludge bed bioreactors were supplemented
with activated carbon carriers and combined with distributors in-
stalled at different locations to investigate the effect of distributor/
carrier on biohydrogen production efficiency (Lo et al., 2009b). The
results show that plastic net stimulated the substrate/microorgan-
isms contact and sludge granulation, thereby leading to a much
better hydrogen production performance when compared with
those obtained from traditional CSTR. The highest hydrogen pro-
duction rate (7.89 l h1 l1) and yield (3.42 mol H2 mol1 sucrose)
were obtained when two pieces of plastic nets were installed at
both 4 cm and 16 cm from the bottom of bioreactor without carrier
addition operated at a HRT of 0.5 h. Addition of carriers led to sig-
nificant improvement on the hydrogen production efficiency at a
longer HRT (1–4 h) when compared with the carrier-absent
system.
A coupled hydrodynamics-reaction kinetics model was formu-
lated from computational fluid dynamics code to simulate a gas–li-
quid–solid three-phase expanded granular sludge bed (EGSB)
reactor for the first time (Wang et al., 2010b). The hydrodynamics
8529
model is used to formulate prediction of the flow field and the
reaction kinetics model then portrays the reaction conversion pro-
cess. The coupled model is verified and used to simulate the behav-
ior of the EGSB reactor for biohydrogen production. The coupled
model also demonstrates a qualitative relationship between
hydrodynamics and biohydrogen production.
4.3. Microbial electrolysis cells
Studies of microbial electrolysis cells or microbial electro-
hydrogenesis cells (MECs) for biohydrogen production through
electro-hydrogenesis have been reported recently (Call and Logan,
2008; Wang et al., 2011). The MEC is a modified microbial fuel cell,
in which organic substrates are converted into biohydrogen. In
contrast to microbial fuel cells, a MEC operates with a supply of
an external voltage rather than generated by it. The energy supply
is needed since the substrate conversion is not spontaneous under
standard conditions (Call and Logan, 2008). Anaerobic microorgan-
isms in a MEC decompose organic substances and transfer elec-
trons to the anode. After traveling through an external load, the
electrons combine at the cathode, with protons and oxygen form-
ing water. Hydrogen gas production occurs at the cathode via reac-
tion of hydrogen ion with electrons. MECs can be operated with
versatility of microbial community and substrates. In addition to
decomposing fermentation metabolites as substrate, glucose or
glucose-containing substrates such as cellulose can also be con-
verted into hydrogen (Call and Logan, 2008).
Initial design of most MEC systems used flat electrode. Flat elec-
trode designs, however, have limited surface area for the exoelec-
trogens causing the membranes to increase the ohmic resistances.
Alternative design uses a graphite brush for the exoelectrogen sub-
strate (anode) without the need for membrane separator. This de-
sign managed to decrease Vapp from 1.0 V using a gas diffusion
membrane and 0.5 V with a Nafion membrane to 0.4 V in the mem-
braneless design. It was reported the efficiency is a function of the
lower heating value of the hydrogen divided by the lower heating
value of the organic material plus the electrical energy provided
(Call and Logan, 2008). The efficiency was raised from 23% with a
gas diffusion membrane and 53% with a Nafion membrane to
76% in the membraneless reactor. Under these conditions, a hydro-
gen production rate of 3.12 m3 m3 reactor per day can be
achieved (Call and Logan, 2008). However, the methane production
rates also increased to an average of 3.5% methane in the gaseous
product. To control the methanogenesis, strategies involving inter-
mittent draining and air exposure or in situ air-sparging have been
proposed (Call and Logan, 2008). However, these strategies will
likely increase operation and maintenance requirements leading
to more costly systems. In addition to methane suppression, con-
tinuous operation, decreasing the pH, operating under carbon lim-
ited conditions, increasing the microorganisms tolerance to
impurities, and examining other feedstocks are all issues to be
addressed.
MEC can be incorporated into the second stage of a two-stage
hybrid bioreactor system which will be discussed in the following
section. In the second stage reaction, electricity applied to a MEC
provides the energy needed in converting organic acids to hydro-
gen (Call and Logan, 2008; Wang et al., 2011). In a MEC, the micro-
bial consortia accomplish dark fermentation in situ, and the
resulting fermentation metabolites are used by the electrogenic
microorganisms. The electrogenic community oxidizes their sub-
strate using the anode as terminal electron acceptor.
Achieving satisfactory efficiency of MEC with nominal electrical
inputs, however, is still a major challenge. Hydrogen production
rates are still substantially lower than those derived from dark fer-
mentations at relatively high voltages (800 mV). There remain sub-
stantial challenges to the practical implementation of this
8530 K.-Y. Show et al. / Bioresource Technology 102 (2011) 8524–8533

Table 1
Biohydrogen production performance of some dark fermentation systems.

Process Optimal HRTa (h) HPRb (L/L h) SHPRc (mmol/g-VSS h) Biomass (g-VSS/L) Ref.
Fixed-bed 1 1.32 3.73 15.8 Lee et al. (2006)
Tricking biofilter 4 1.07 1.99 24 Oh et al. (2004a)
AFBR 2 1.82 – – Lin et al. (2009)
AFBR 1 2.36 4.9 21.5 Zhang et al. (2007d)
Packed-bed 0.5 7.41 12.82 25.8 Lee et al. (2006)
Packed-bed 10 0.25 3.70 3.02 Palazzi et al. (2000)
UASB 8 0.28 2.47 5.06 Chang and Lin (2004)
UASB 26.7 0.05 – – Kotsopoulos et al. (2006)
CSTR 6 0.54 1.30 20 Fang et al. (2002)
CSTR 0.5 15.09 19.27 35.84 Wu et al. (2006)
CSTR 0.5 3.20 8.31 16.03 Zhang et al. (2007c)
Column reactor 0.25 7.49 – 35 Zhang et al. (2008c)
a
On the basis of hydrogen production rate.
b
Hydrogen production rate.
c
Specific hydrogen production rate.

technology. These include the replacement of expensive platinum
electrode, the high current densities and the reduction of the elec-
trical input requirement.
4.4. Hybrid bioreactors
A basic principle of the hybrid two-stage bioreactors is that bio-
fermentation of substrate to hydrogen and organic acids takes
place in a conventional reactor in a first stage of process, and addi-
tional gaseous energy in the form of methane or hydrogen is ex-
tracted from in the second stage (Koutrouli et al., 2009). In
optimizing gas production, a different reactor for the second stage
operated under different conditions (such as higher pH and longer
HRT) is applied (Hwang et al., 2011).
An attempt for maximizing the overall energy extraction in the
second stage is the utilization of photo-fermentation (Kapdan and
Kargi, 2006) or fuel cell (Garcia-Pena et al., 2009). This can be
achieved by recovering additional hydrogen from the metabolites
of the dark fermentation. Non-sulfur photo-synthetic microbes
capable of converting organic acids to hydrogen were cultivated
in the system (Redwood et al., 2009). Theoretically, complete con-
version of organics into hydrogen via photo-fermentations is pos-
sible owing to the fact that hydrogen production is driven by
ATP-dependent nitrogenase. The required ATP is formed with the
energy derived from the sunlight.
The feasibility of hybrid two-stage photo-fermentation system
has been demonstrated in which the second stage was actually
fed with effluent from a hydrogen-producing reactor, although
hydrogen yields were well below the hypothetical values (Nath
et al., 2008). On the other hand, a combination of dark and
photo-fermentation in a hybrid two-stage system can improve
the overall yield of hydrogen (Nath and Das, 2006). The synergy
of the hybrid system lies in the maximal utilization of the substrate
due to improved thermodynamics. In the first stage, the biomass is
fermented to acetate, carbon dioxide and hydrogen in a thermo-
philic dark fermentation. In second stage photobioreactor, acetate
is converted to hydrogen and carbon dioxide (Nath and Das,
2006). The combination could be expected to reach as close to
the theoretical maximum production of 12 mol of hydrogen per
mol glucose equivalent as possible. While the research so far have
indicated encouraging findings, the main technical barrier to the
practical application of photo-fermentation lies with the low pho-
tosynthetic efficiencies with concomitant low hydrogen yields un-
der moderate to high light intensities.
In another approach, MECs are incorporated into the second
stage, in which electricity applied to a microbial fuel cell provides
the energy needed in converting organic acids to hydrogen (Call
and Logan, 2008; Wang et al., 2011). In principle, a second stage
MEC could produce 12 H2/glucose with only a small input of
energy.
It has been reported that the hybrid two-stage system has al-
ready been scaled up to pilot plant stage (Ueno et al., 2007). Such
a hybrid system offers several merits over conventional methano-
genesis, including an effective solubilization of substrates and in-
creased tolerance to high OLRs. Successful operation of such
systems using actual wastes has also been documented, including
a pilot-scale fermentation of kitchen waste generating relatively
high production rates of 5.4 m3 H2 m3 d1 and 6.1 m3 CH4
m3 d1 while achieving COD removal of 80% (Ueno et al., 2007).
The superior performance of this hybrid system is illustrated from
the methane yields which were two times better than a single-
stage process (Ueno et al., 2007).
4.5. Multi-stage bioreactors
Multi-stage bioreactors involving three or even four stages
(Fig. 1) have been proposed to maximize the hydrogen production
from the substrate (US DOE, 2007; Wang et al., 2011). Sunlight is
first filtered through the first stage direct photolysis reactor where
the visible light is utilized by blue green algae, and the unfiltered

H2 Biomass feed

Water Photo- Dark Microbial

Photolysis Voltage
fermentation fermentation electrolysis supply

Organic acids

Fig. 1. Multi-stage bioreactor system. Adapted from US DOE (2007).

 K.-Y. Show et al. / Bioresource Technology 102 (2011) 8524–8533
infrared light is used by photo-synthetic microbes in the second
stage photo-fermentative reactor. The effluent from the second
stage photo-fermentation together with the biomass feedstock is
fed into a third stage dark fermentation reactor where the bacteria
convert the substrate into hydrogen and organic acids. As the efflu-
ent is enriched with organic acids, a supply of external organic
acids for the photo-fermentative process can be eliminated. The
forth stage is the use of a MEC to produce hydrogen. The MEC uses
the organic acids generated from the dark fermentation under
light-independent process. It thus can be operated during the night
or low light condition (US DOE, 2007).
Integration of multiple processes produces significant chal-
lenges for the reactor engineering, system design, process control,
and operation and maintenance. The major challenges with the
coproduction of hydrogen and oxygen from photolytic hydrogen
production include photosynthetic and respiration capacity ratio,
co-culture balance, and concentration and processing of cell bio-
mass (Holladay et al., 2009).
The increasing interest on hythane, a highly efficient and ultra
clean burning alternative fuel (a mixture of hydrogen and meth-
ane) which is probably the most promising biogas for industrial
applications, has led many researchers to attempt hydrogen pro-
duction by anaerobic digestion of biomass in hybrid or multi-stage
bioreactors. An experimental work optimizing a two phase ther-
mophilic anaerobic digestion process for bio-hythane production
treating source collected organic fraction of municipal solid waste
was carried out (Cavinato et al., 2009). An hydrogen rich biogas
production of 0.06 m3 gas/kgTVS was reached in the fermentative
reactor, showing a 65% CO2 content. The methanogenic phase
shown constant stability parameters (pH value 8, VFA
255 mg COD/l, ammonia 1150 mg N/l) and a specific gas produc-
tion of 0.73 m3/kgTVS.
5. Challenges and prospects
The relatively low hydrogen yield and production rate are two
common challenges for the biological hydrogen-producing sys-
tems. Enhancement in hydrogen yield may be possible by using
suitable microbial strain, process modification, efficient bioreactor
design and also genetic and molecular engineering technique, to
redirect metabolic pathway. Some integrated strategies are now
being developed such as the two-step fermentation process (acido-
genic + photobiological or acidogenic + methanogenic processes)
or the use of modified microbial fuel cells (de Vrije and Claasen,
2003; Ueno et al., 2007). Applying genetic and metabolic engineer-
ing techniques to improve the hydrogen yields of those microbial
cultures with higher hydrogen production rates might also be an-
other feasible option (Chittibabu et al., 2006; Nath and Das, 2006).
Apart from that more easily degradable substance, green wastes
will, most probably, to a large extent be the targeted feedstocks for
hydrogen fermentation because of their abundance. Lignocellulose
is the most abundant renewable natural resource and substrate
available for conversion to fuels. There is great potential in the
use of green waste biomass as a renewable source of energy via
microbial breakdown to sugars that can then be converted to bio-
hydrogen (Lo et al., 2008, 2009a).
There has been a growing resistance against the use of energy
crops as feedstocks for biofuels generation. This backlash was cen-
tered on the food-vs-fuel debate, with the main arguments that
crops that could support human dietary needs are diverted to the
production of biofuels thus inflates food prices. As an answer to
those issues, the production of second generation biofuels is pro-
posed, i.e. biofuels produced by feedstocks that are not competitive
to edible crops such as wastes and residues (Juang et al., 2011; Kim
and Lee, 2010; Ntaikou et al., 2010).
8531
Technical challenges in achieving practical applications of bio-
hydrogen include lowering the cost of hydrogen production, deliv-
ery, storage, conversion, and end use applications. Economics
would strongly favor large-scale hydrogen production systems.
The problem with the dark hydrogen fermentation is that in the
dark, fermentative bacteria produce only relatively small amounts
of hydrogen, typically less than 30% stoichiometrically. Economic
feasibility will not be sustainable at these yield level. Currently,
less than 10% of the algae photosynthetic capacity was utilized
for biohydrogen production. Research is underway in improving
further biohydrogen production capacity using molecular engi-
neering approach. With technology advancement, biohydrogen
production may offer a sustainable alternative energy resource in
future.
6. Conclusions
Extensive research in the past two decades have reviewed
promising prospect of biohydrogen production. There have been
substantial improvement and development in both the yield and
volumetric production rates of hydrogen fermentations. For realis-
tic applications that make economic sense, hydrogen yields and
production rates must at least surpass considerably the present
achievements. Technological breakthrough must be sought after
to extract most of hydrogen from substrate, if not all. Investigation
addressing this challenge should be one of focuses of future re-
search. Scaling up of the process to full-scale systems will further
refine the technology for future commercialization.
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