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R E V I E W P A P E R
Fermentative biohydrogen production:
trends and perspectivesGustavo Davila-Vazquez Sonia Arriaga Felipe Alatriste-Mondragon Antonio de Leon-Rodrguez Luis Manuel Rosales-Colunga Elas Razo-Flores
Received: 15 January 2007 / Accepted: 2 May 2007 / Published online: 6 June 2007
Springer Science+Business Media B.V. 2007
Abstract Biologically produced hydrogen (biohy-
drogen) is a valuable gas that is seen as a future
energy carrier, since its utilization via combustion or
fuel cells produces pure water. Heterotrophic fer-
mentations for biohydrogen production are driven by
a wide variety of microorganisms such as strict
anaerobes, facultative anaerobes and aerobes kept
under anoxic conditions. Substrates such as simple
sugars, starch, cellulose, as well as diverse organic
waste materials can be used for biohydrogen produc-
tion. Various bioreactor types have been used andoperated under batch and continuous conditions;
substantial increases in hydrogen yields have been
achieved through optimum design of the bioreactor
and fermentation conditions. This review explores the
research work carried out in fermentative hydrogen
production using organic compounds as substrates.
The review also presents the state of the art in novel
molecular strategies to improve the hydrogen
production.
Keywords Anaerobic conditions Biohydrogen Biomass Bioreactor Dark fermentation Genemanipulation Hydrogenases Hydrogen production Mixed culture
1 Introduction
A large proportion of the world energy needs are
being covered by fossil fuels, which have led to an
accelerated consumption of these non-renewable
resources. This has resulted in both, the increase in
CO2 concentration in the atmosphere and the rapid
depletion of fossil resources. The former is considered
the main cause of global warming and associated
climate change, whereas the latter will lead to an
energy crisis in the near future (Kapdan and Kargi
2006). For these reasons, large efforts are being
conducted worldwide in order to explore new sus-tainable energy sources that could substitute fossil
fuels. Processes, which produce energy from biomass,
are typical examples of environmentally friendly
technologies as biomass is included in the global
carbon cycle of the biosphere. Large amounts of
biomass are available in the form of organic residues,
such as solid municipal wastes, manure, forest and
agricultural residues. Some of these residues can be
used after minor steps of pre-treatment (usually
G. Davila-Vazquez S. Arriaga F. Alatriste-Mondragon E. Razo-Flores (&)Division de Ciencias Ambientales, Instituto Potosino deInvestigacion Cientfica y Tecnologica, Camino a la PresaSan Jose 2055, Col. Lomas 4a, Seccion, C.P. 78216 SanLuis Potosi, SLP, Mexicoe-mail: [email protected]
A. de Leon-Rodrguez L. M. Rosales-ColungaDivision de Biologa Molecular, Instituto Potosino deInvestigacion Cientfica y Tecnologica, Camino a la PresaSan Jose 2055, Col. Lomas 4a, Seccion, C.P. 78216 SanLuis Potosi, SLP, Mexico
123
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DOI 10.1007/s11157-007-9122-7
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dilution and maceration), while others may require
extensive chemical transformations prior to being
utilized as a raw material for biological energy
production (Claassen et al. 1999). Biological pro-
cesses such as methane and hydrogen production
under anaerobic conditions, and ethanol fermentation
are future oriented technologies that will play a majorrole in the exploitation of energy from biomass.
Using the appropriate microbial mechanisms of
anaerobic digestion, hydrogen (biohydrogen) would
be the desired product of the digestion process while
the organic acids would be the by-products. The
major advantage of energy from hydrogen is the
absence of polluting emissions since the utilization of
hydrogen, either via combustion or via fuel cells,
results in pure water (Claassen et al. 1999).
This mini-review provides an overview of the
state of the art and perspectives of biohydrogenproduction by microorganisms. The review focuses
on the literature published mainly during the years
2005 and 2006 on hydrogen production by hetero-
trophic fermentation (dark hydrogen fermentation).
For a full overview of previous work on this topic
the reader is referred to excellent reviews published
elsewhere (Nandi and Sengupta 1998; Claassen et al.
1999; Hallenbeck and Benemann 2002; Hawkes
et al. 2002; Nath and Das 2004; Kapdan and Kargi
2006).
2 Biohydrogen producing microorganisms
Biohydrogen can be produced by strict and faculta-
tive anaerobes (Clostridia, Micrococci, Methanobac-
teria, Enterobacteria, etc), aerobes (Alcaligenes and
Bacillus) and also by photosynthetic bacteria (Nandi
and Sengupta 1998). Table 1 shows that during the
time period covered by this review, most of the
studies were conducted using mixed cultures, and just
a few of them were pure cultures. Different sources ofinocula were reported (soil, sediment, compost,
aerobic and anaerobic sludges, etc.) and most of
them were heat or acid treated before being used.
These treatments have been used in previous studies
as methods for increasing hydrogen production by
altering the microbial communities present in the
starting mixed population (Cheong and Hansen
2006). The reason for this is that unlike H2-consum-
ing methanogens, H2-producing bacteria are com-
monly tolerant to harsher environmental conditions
(Kawagoshi et al. 2005). For example Clostridium
and Bacillus species tolerate higher temperatures than
H2-consuming methanogens due to the formation of
endospores (Setlow 2000). Also, H2-producing bac-
teria can grow at lower pH than H2-consuming
methanogens (Cheong and Hansen 2006).Various studies have been carried out to identify
the microbial communities present in mixed cultures
used for H2 production (Ueno et al. 2001; Fang et al.
2002; Ueno et al. 2004; Kawagoshi et al. 2005; Kim
et al. 2006). Fang et al. (2002) identified the
microbial species in a granular sludge used for H2production from sucrose. They found that 69.1% of
the microorganisms were Clostridium species and
13.5% were Bacillus/Staphylococcus species. Kaw-
agoshi et al. (2005) studied the effect of both pH and
heat conditioning on different inoculums. In theirstudy they concluded that the highest hydrogen
production was obtained with heat-conditioned anaer-
obic sludge. They also found DNA bands with high
similarity (>95%) to Clostridium tyrobutyricum,
Lactobacillus ferintoshensis, L. paracasei, and Cop-
rothermobacterspp. Kim et al. (2006) indicated that
heat-treatment (908C for 20 min) caused a change in
the microbial community composition of a fresh
culture used to produce H2 from glucose in a
membrane bioreactor. They reported that most of
the species found in the fresh sludge were affiliated tothe Lactobacillus sp. and Bifidobacterium sp.; in
contrast a Clostridium perfringens band was observed
in the heat-treated sludge. When mixed cultures are
used as inocula the predominant species in a biore-
actor depends on operational conditions such as
temperature, pH, substrate, inoculum type, inoculum
pre-treatment, hydrogen partial pressure, etc. Kotay
and Das (2006) studied the H2 production with
glucose and sewage sludge as substrates using a
defined microbial consortium consisting of three
facultative anaerobes, Enterobacter cloacae, Citrob-acter freundii and Bacillus coagulans. They carried
out experiments with the consortium (three species)
and the individual species. E. cloacae produced a
higher yield than the other strains, but similar to the
consortium suggesting that E. cloacae dominated the
consortium. Some studies using pure strains have also
been carried out for H2 production. Escherichia coli
(genetically modified strains), Clostridium butyricum,
C. saccharoperbutylacetonicum, C. thermolacticum,
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Table1
Hydrogenproductionratesandyieldcoefficientsfrompureand
complexsubstratesunderbatch,semi-c
ontinuousandcontinuousoperation
System
Inoculum
Substratea
Volumetric
H2production
rate(mmol
H2/lculture-h)f
H2yield
Culture
conditionsa[HRT
(h),Load,pH,
Temperature
(8C),H2in
biogas(%v/v)]
Reference
Batch
ClostridiumbutyricumCGS5
Sucrose(20gCOD/l)
8.2
2.78molH2/mol
sucrose
,,5.56.0
c,37,64
Chenetal.
(2005)
Clostridium
saccharoperbutylacetonicum
ATCC27021
Crudecheesewhey(ca.
41.4glactose/l)
9.4
2.7m
olH2/mol
lactose
,,6.0
d,30,NRb
Ferchichietal.
(2005)
Escherichiacolistrains
Glucose(4g/l)
NRb
*2m
olH2/mol
glucose
,,7.0,37,NR
Bisaillonetal.
(2006)
Escherichiacolistrains
Formicacid(25mM)
11795
1molH2/mol
formate
,,6.5
d,37,NR
Yoshidaetal.
(2005)
Definedconsortium(1:1:1,and
separatelytested):En
terobacter
cloacaeIIT-BT08,C
itrobacter
freundiiIIT-BTL139
,Bacillus
coagulansIIT-BTS1
Glucose(10g/l)
NR
41.23
mlH2/g
CO
Dremoved
,,6.0
d,37,NR
KotayandDas
(2006)
MesophilicbacteriumH
N001
Starch(20g/l)
59
2molH2/mol
glucose
,,6.0
c,37,NR
Yasudaand
Tanisho
(2006)
Aerobicandanaerobicsludges,
soilandlakesedimen
t(acidand
heatconditioned)
Glucose(20g/l)
NR
1.4m
olH2/mol
glucose
,,6.0
c,35,NR
Kawagoshietal.
(2005)
Aerobicsludge(heatco
nditioned)
Glucose(2g/l)
NR
2.0m
olH2/mol
glucose
,,6.2,d30,87.4
Parketal.(2005)
Soil(heatconditioned)
Organicmatterpresent
infourcarbohydrate-
richwastewaters
6.2
100m
lH2/g
CO
Dremoved
,,6.1,d23,60
VanGinkeletal.
(2005)
Anaerobicsludge(acid
treatment
andacclimatedinaC
STR)
Sucrose(20gCOD/l)
96
1.74molH2/mol
sucrose
,,6.1,d40,45
Wuetal.(2005)
Anaerobicsludge(heat
conditioned)
Glucose(10g/l)
27.2mmolH2/gVSS-
lculture-h
1.75molH2/mol
glucose
,,6.0,d37,40
ZhengandYu
(2005)
Anaerobicsludge
(acidtreatment)
Glucose(*21.3g/l)
4.98.6
0.81.0molH2/mol
hex
ose
,,5.7,c34.5,59
66
Cheongand
Hansen(2006)
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Table1
continued
System
Inoculum
Substratea
Volumetric
H2production
rate(mmol
H2/lculture-h)f
H2yield
Culture
conditionsa[HRT
(h),Load,pH,
Temperature
(8C),H2in
biogas(%v/v)]
Reference
Microflorafromacowdung
compost(heattreatment)
Wheatstrawwastes
(25g/l)
2.7mmolH2/gTVS-
lculture-h
2.7m
molH2/gTVS
,,7.0,d36,52
Fanetal.(2006)
Anaerobicsludge(he
attreated)
Sucrose(10g/l)
8
1.9m
olH2/mol
sucrose
,,5.5,c35,NR
Muetal.(2006a)
Anaerobicsludge(he
attreated)
Sucrose(24.8g/l)
20
3.4m
olH2/mol
sucrose
,,5.5,c34.8,64
Muetal.
(2006b)
Anaerobicsludge(he
attreated)
Glucose(3.76g/l)
9
1.0m
olH2/mol
glucose
,,6.2,d30,66
Salernoetal.
(2006)
Anaerobicsludge(he
attreated)
Glucose(2.82g/l)
NR
0.968molH2/mol
glucose
,,6.2,d25,5772
Ohetal.(2003)
Microflorafromsoil(heat
shocked)
Glucose,sucrose,
molasses,lactate,
potatostarch,
cellulose(each:4g
COD/l)
NR
0.92
molH2/mol
glucose,1.8mol
H2/molsucrose,
0.59molH2/mol
po
tatostarch,e
0.01molH2/mol
lac
tate,0.003mol
H2/molcellulosee
,,6.0,d26,62
Loganetal.
(2002)
Fedbatch
Mixedculture
OFMSW-Semisolid
substrate
14.7mmolH2/
gVSdestroyed
NRb
504,11gVS/
kgwmr
d,6.4,55,
58
Valdez-Vazquez
etal.(2005)
AnaerobicPOMEslu
dge
POME(2.5%w/v)
17.82
NR
24,NR,5.5,60,66
Atifetal.(2005)
Windrowyardwaste
compost
Glucose(2g/l)
7.44
1.75
molH2/mol
glucose
76,NR,5.4,55,NR
Callietal.
(2006)
CSTR
Mixedculture
Sucrose(20gCOD/l)
17
3.5m
olH2/mol
sucrose
12,NR,6.8,35,45.9
Linetal.
(2006b)
Mixedculture
Sucrose(40g/l)
20
1.15
molH2/mol
hexose
12,80g/l-d,5.2,35
,
60
Kyazzeetal.
(2006)
Mixedcultureimmob
ilizedin
siliconegel
Sucrose(30gCOD/l)
612.5
3.86
molH2/mol
sucrose
0.5,NR,6.5,40,44
Wuetal.
(2006a)
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Table1
continued
System
Inoculum
Substratea
Volumetric
H2production
rate(mmol
H2/lculture-h)f
H2yield
Culture
conditionsa[HRT
(h),Load,pH,
Temperature
(8C),H2in
biogas(%v/v)]
Reference
Mixedculture
Xylose(20gCOD/l)
5
1.1m
olH2/mol
xylose
12,NR,7.1,35,32
LinandCheng
(2006)
Mixedculture
Brokenkitchenwastes
(10kgCOD/m3-d)
andcornstarch
(10kgCOD/m3-d)
1.7
NR
96,NR,5.35.6,35,
NR
Chengetal.
(2006)
Mixedculture
Glucose(15gCOD/l)
13.23
1.93molH2/mol
glucose
4.5,80gCOD/l-d,
5.5,37,67
Zhangetal.
(2004)
Dewateredand
thickenedsludge
Glucose(4gCOD/l)
3.47
1.9m
olH2/mol
glucose
10,NR,5.5,35,67
Salernoetal.
(2006)
Mixedculture
Sucrose(20gCOD/l)
15.6
3.6m
olH2/mol
sucrose
12,NR,5.5,35,50
LinandChen
(2006)
Mixedculture
Organicwastewater
(4000mgCOD/l)
4.96
NR
12,NR,4.4,8kg
COD/m3-d,30,
NR
Wangetal.
(2006)
Sewagesludge
Sucrose(20gCOD/l)
52.6
3.43molH2/mol
sucrose
12,NR,6.8,35,50.9
LinandLay
(2005)
Mixedculture
Sucroseandsugarbeet
5.15
1.9m
olH2/mol
hex
ose
15,16kgsugar/m3-
d,5.2,32,NR
Hussyetal.
(2005)
Mixedculture
Glucose(15g/l)
0.115gH2-COD/g
CODFeed
1.38molH2/mol
hex
ose
10,NR,5.5,35,45
Kraemerand
Bagley(2005)
C.thermolacticum(DSM2910)
Lactose(10g/l)
2.58
2.13
molH2/mol
lactose
17.2,NR,7.0,58,55
Colletetal.
(2004)
Seedsludge
Molasses(3000mg
COD/l)
26.13molH2/kg
CODremoved
NR
11.4,27.98kgCOD/
m3reactor-d,4.5,
35,45
Renetal.(2006)
UASB
Mixedculture
Glucose(10g/l)
2.18
2.47molH2/mol
glucose
26.7,NR,4.85.5,
70,NR
Kotsopoulos
etal.(2006)
Mixedculture
Sucroserichwaste
water
5.93
1.61molH2/mol
glucose
12,NR,7,39,NR
MuandYu
(2006)
Mixedculture
Citricacidwastewater
(18kgCOD/l)
1.23
0.84molH2/mol
hex
ose
12,38.4kgCOD/
m3-d,7,35,NR
Yangetal.
(2006)
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Table1
continued
System
Inoculum
Substratea
Volumetric
H2production
rate(mmol
H2/lculture-h)f
H2yield
Culture
conditionsa[HRT
(h),Load,pH,
Temperature
(8C),H2in
biogas(%v/v)]
Reference
Mixedculture
Sucrose(20gCO
D/l)
11.3
1.5mmolH2/mol
sucrose
8,175mmol
sucrose/l-d,6.7,
35,42.4
ChangandLin
(2004)
Mixedculture
Glucose(7.7g/l)
18.4
1.7molH2/mol
glucose
2,NR,6.4,55,36.8
Gavalaetal.
(2006)
(1.3g/l)
19
0.7molH2/mol
glucose
2,NR,4.4,35,29.4
CSTRandUASB
Mixedculture
Starch(10g/l)an
d
xylose(1:1w/w)
4.5
32.9,NR,7,35,68
Camilliand
Pedroni(2005)
4.76
6.7,NR,7,35,68
2.54
20.5,NR,7,35,68
CSTR,UASB,
UFBR
Mixedculture
Glucose(6.86g/l)
37.5
1.6molH2/mol
glucose
12,NR,5.5,60,48
Ohetal.(2004)
TBR
Clostridiumacetobutylicum
(ATCC824)
Glucose(10.5g/l)
8.9
0.9molH2/mol
glucose
0.035,8.3g/l-h,4.9,
30,74
Zhangetal.
(2006)
Mixedculture
Glucose(2g/l)
NR
2.48molH2/mol
glucose
0.5,96kg/m3-d,7.7,
30,NR
Leiteetal.
(2006)
CIGSB
Mixedculture
Sucrose(17.8g/l)
0.298
3.88molH2/mol
sucrose
0.5,NR,6.7,40,4
2
Leeetal.(2006)
PBR
Cowdung
POME(560gC
OD/l)
0.42lbiogas/g
CODdestroyed
NR
37,NR,5,NR,53
56
Vijayaraghavan
andAhmad
(2006)
UACF
Cowdung
Jackfruitpeel(22
.5g
VS/l)
0.72lbiogas/gVS
destroyed
NR
288,NR,5,NR,5
6
Vijayaraghavan
etal.(2006)
MBR
Mixedculture
Glucose(10g/l)
71.4
1.1molH2/mol
glucose
0.79,NR,5.5,37,70
Kimetal.(2006)
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Table1
continued
System
Inoculum
Substratea
Volumetric
H2production
rate(mmol
H2/lculture-h)f
H2
yield
Culture
conditionsa[HRT
(h),Load,pH,
Temperature
(8C),H2in
biogas(%v/v)]
Reference
FBRDTFBR
Mixedculture
Sucrose(20gCOD/l)
50.27
2.10molH2/mol
sucrose
2,NR,6.9,40,40
Wuetal.
(2006b)
95.23
1.22molH2/mol
sucrose
0.5,NR,7,40,35
a
Whenoptimizationtrialswerecarriedout,optimumvaluesarereported
b
NR:Notreported
c
Controlledvalue
d
Initial,notcontrolled
e
Starch,celulose:[(C6H10O5)n]
f
Insomecasesunitconversionsw
eremadeaccordingtotheconditionsreportedbytheauthors.CIGSB:Carrierinducedgranularsludgebed.COD:Chem
icaloxygendemand.
CSTR:Continuousstirredtankreactor.DTFBR:Drafttubefluidizedbedreactor.FBR:Fluidizedbedbioreactor.kgwmr:
Kilogramsofwetmassinthereac
tor.MBR:Membrane
bioreactor.OFMSW:Organicfractionofmunicipalsolidwastes.PBR:Pa
ckedbedreactor.POME:Palmoilmilleffluent.TBR:Tricklingbiofilter.TVS
:totalvolatilesolids.
UACF:Up-flowanaerobiccontact
filter.UASB:Upflowanaerobicsludge
blanket.UFBR:Up-flowfixedbedreactor.VS:volatilesolids.VSS:volatilesuspendedsolids
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and C. acetobutylicum are among the microorganisms
used (Table 1).
3 Biohydrogen producing substrates
The main criteria for substrate selection are: avail-ability, cost, carbohydrate content and biodegradabil-
ity (Kapdan and Kargi 2006). Glucose, sucrose and to
a lesser extent starch and cellulose, have been
extensively studied as carbon substrates for biohy-
drogen production (Table 1). They have been used as
model substrates for research purposes due to their
easy biodegradability and because they can be
present in different carbohydrate-rich wastewaters
and agricultural wastes. Other substrates suitable for
biohydrogen production are protein- and fat-rich
wastes. Although they are less available than carbo-hydrate-rich wastes, they represent potential feeds for
the biological conversion of organic wastes to
hydrogen (Svensson and Karlsson 2005).
A maximum theoretical yield of 12 mol of H2 per
mol of hexose is predicted from the complete
conversion of glucose:
C6H12O6 6H2O ! 12 H2 6CO2 DG00 25 kJ
It should be noted that essentially no energy isobtained from this reaction to allow microbial growth
(Hallenbeck2005). Actual yields in metabolisms that
lead to H2 production are lower compared to the
maximum theoretical yield; ca. 2 and 4 mol of
hydrogen per mol of glucose and sucrose, respec-
tively, are obtained (Table 1). Fermentation of
organic compounds is considered to have an imme-
diate potential for economical hydrogen production,
but only if conversion efficiency could be increased
to 6080% (Benemann 1996). This means that from
each mol of glucose fermented, a minimum of 7 molof hydrogen should be obtained. Nonetheless, due to
thermodynamic constrains, only 4 mol of hydrogen
are released from 1 mol of glucose if acetate is
produced, and 2 mol of hydrogen are obtained when
butyrate or more reduced compounds (lactic acid,
propionic acid, or ethanol) are produced (Hallenbeck
and Benemann 2002; Angenent et al. 2004). As
discussed by Logan (2004), although genetic engi-
neering of bacteria could increase hydrogen recovery,
it is now considered that the maximum conversion
efficiency will still remain below 33%. This so called
fermentation barrier is maintained regardless of the
fermentation system used for H2 production e.g.
batch, semi-continuous or continuous one step-pro-
cesses (Logan 2004).
Another important feature of hydrogen fermenta-tion is volumetric H2 production rate (VHPR). As
suggested by Levin et al. (2004), we agree that in
order to assess the potential for practical application
of biological hydrogen production systems, an effort
should be made in order to report VHPR in
standardized units. By doing this, it would be easier
to calculate and compare the size of a bioreactor
that would be needed to supply hydrogen to a
specific fuel cell for electricity generation. For this
reason an effort was made in this review to report
VHPR in Table 1 in the same units whenever this waspossible.
4 Biohydrogen production in batch, continuous
and semi-continuous systems
Biohydrogen production by dark fermentation is
highly dependent on the process conditions such as
temperature, pH, mineral medium formulation, type
of organic acids produced, hydraulic residence time
(HRT), type of substrate and concentration, hydrogen
partial pressure, and reactor configuration (Table 1).
Temperature is an operational parameter that
affects the growth rate and metabolic activity of
microorganism. Fermentation reactions can be oper-
ated at mesophilic (25408C), thermophilic (40
658C), extreme thermophilic (65808C), or hyper-
thermophilic (>808C) temperatures. Most of the
results presented in Table 1 were obtained under
mesophilic conditions and some under thermophilic
conditions. The effect of temperature on VHPR can
be explained thermodynamically by considering the
changes in Gibbs free energy and in standard
enthalpy of the conversion of glucose to acetate and
assuming a maximum theoretical yield of 4 mol H2per mol glucose (Vazquez-Duhalt 2002):
C6H12O6 2H2O ! 2CH3COOH + 4H2 2CO2
DG 176.1 kJ/mol
DH 90.69 kJ/mol
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The changes in Gibbs free energy and in enthalpy
of the reaction indicate that the reaction can occur
spontaneously and the reaction is endothermic. The
vant Hoff equation can be used to explain the effect
of the temperature on the equilibrium constant (Smith
et al. 2000):
lnK1
K2
DH
R
1
T1
1
T2
1
K1
K2
H2 41 CO2
21 CH3COOH
21
H2 42 CO2
22 CH3COOH
22
2
If temperature increases, the equilibrium kinetic
constant also increase because the reaction is endo-
thermic (DH8 has positive sign, see Eq. 1). Therefore,
increasing the temperature of the glucose fermenta-tion, maintaining reactants concentration constant
(see Eq. 2) would enhance H2 concentration. Two
reports provide support to the previous thermody-
namic considerations. In one of them, Valdez-Vaz-
quez et al. (2005) studied the semi continuous H2production at mesophilic and thermophilic condi-
tions. They found that VHPR was 60% greater at
thermophilic than at mesophilic conditions. The
authors suggested that this behavior may be explained
by the optimal temperature for the enzyme hydrog-
enase (50 and 708C) present in thermophilic Clostri-dia. Also, Wu et al. (2005) reported that VHPR was
greater at 408C than at 308C in batch tests using
immobilized sludge in vinyl acetate copolymer. In
addition, fermentation at temperatures above 378C
may inhibit the activity of hydrogen consumers (Lay
et al. 1999) and suppress lactate-forming bacteria (Oh
et al. 2004) yielding in both cases higher VHPR.
Thermophilic conditions also enhance the efficiency
of pathogens removal (de Mes et al. 2003), which are
present in some residues, allowing the use of these
residues as fertilizers for application on agriculturalsoil.
On the other hand, high temperatures can induce
thermal denaturation of proteins affecting the micro-
organism activity. Lee et al. (2006) studied the effect
of temperature on hydrogen production in a carrier
induced granular sludge bed bioreactor (CIGSB).
They found that increasing the temperature from 35
to 458C may inhibit cell growth or granular sludge
formation due primarily to denaturation of essential
enzymes to paralyze normal metabolic functions or
decreasing in production of extracellular polymeric
substances. Another potential disadvantage of a
thermophilic process is the increased energy costs.
In some of the studies presented in Table 1 the
maximum VHPR was obtained between pH 5.0 and
6.0. However, in other studies the maximum VHPRwas found around pH 7.0 (Lee et al. 2006; Lin and
Cheng 2006; Mu and Yu 2006). Recent studies have
indicated that in order to inhibit methanogenesis,
increase VHPR and enhance stability of continuous
systems, moderate acid pH and high temperatures
should be used (Oh et al. 2004; Atif et al. 2005;
Kotsopoulos et al. 2006). For the operation of batch
systems an optimum initial pH of 5.5 was reported
(Fan et al. 2006; Fang et al. 2006; Mu et al. 2006b;
Mu et al. 2006c). However, the final pH in batch
systems is around 45 regardless of the initial pH.This is due to the production of organic acids, which
decreases the buffering capacity of the medium
resulting in a low final pH. Mu et al. (2006c) found
that volatile fatty acids (VFA) and ethanol formation
was pH dependant. Ethanol production decreased
when pH was decreased from 4.2 to 3.4. On the other
hand, butyrate concentration decreased when pH was
increased from 4.2 to 6.3. It has been reported that
high VHPR is associated with butyrate and acetate
production and that inhibition of hydrogen production
is associated with propionic acid formation (Oh et al.2004; Wang et al. 2006). Therefore, control of pH at
the optimum level (56) is critical. Initial pH also
influences the extent of lag phase in batch hydrogen
production. Some studies reported that low initial pH
in the range of 44.5 causes longer lag periods than
high initial pH levels around 9 (Cai et al. 2004).
However, the yield of hydrogen production decreased
at high initial pH.
The mineral salt composition (MSC) also affects
hydrogen production. Lin and Lay (2005) found an
optimal MSC by using the Taguchi fractional designm et h od a nd v ar y in g t h e p ro p or t io n s o f
MgCl2 6H2O, CoCl2 6H2O, CaCl2 2H2O,NiCl2 6H2O, MnCl2 6H2O, KI, NH4Cl, NaCl,ZnCl2 , F eS O4 7H2O , K Mn SO4 4H2O,CuSO4 5H2O, Na2MoO4 2H2O. The VHPRobtained with the optimal MSC was 66% greater
than the value obtained with conventional acidogenic
n u tr i e nt f o r mu l at i on ( N H4HCO3, K2HPO4,
MgCl2 6H2O, MnSO4 6H2O, FeSO4 7H2O,
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CuSO4 5H2O, CoCl2 5H2O, NaHCO3). Also, theyfound that magnesium, sodium, zinc and iron were
important trace metals affecting VHPR due to the fact
that these elements are needed by bacterial enzyme
cofactors, transport processes and dehydrogenase.
Recent studies reported the effect of sulfate and
ammonia concentrations on VHPR in CSTR systemswith sucrose and glucose as substrate (Lin and Chen
2006; Salerno et al. 2006). Increasing sulfate con-
centration from 0 to 3000 mg/l, at pH 6.7, reduced
VHPR and shifted the metabolic pathway from
butyrate to ethanol fermentation due to the occur-
rence of sulfate reduction. However, increasing the
sulfate concentration to 3000 mg/l, at pH 5.5, raised
the VHPR to 40% and 98.6% of residual sulfate
concentration was found in the effluent, indicating
that the acidic environment was not favorable to
sulfate reducing bacteria and sulfate reduction did notoccur (Lin and Chen 2006). A decrease of 40% on
VHPR and hydrogen yield was observed at ammonia
concentrations of 7.8 g N-NH4/l compared with the
value obtained at 0.8 g N-NH4/l, which was the
optimal ammonia concentration for hydrogen pro-
duction (Salerno et al. 2006).
Hydrogen and VFA can be produced during
exponential and stationary growth phases. However,
various authors have shown that VFA and hydrogen
production are maximal during the exponential
growth phase, and decrease during the stationaryphase due to alcohol production (Lay 2000; Levin
et al. 2004). Hydrogen production in continuous and
discontinuous systems depends on both biomass and
substrate concentrations. Yoshida et al. (2005) stud-
ied the effect of biomass concentration on hydrogen
production. They found that the specific hydrogen
production rate (SHPR) increased 67% by increasing
cell density from 0.41 g/l to 74 g/l.
The maximum hydrogen yield of 4 mol/mol has
not been reached because in nature fermentation
serves to produce biomass and not hydrogen. Also,hydrogen production by fermenting cells is a waste of
energy for the bacterial metabolism, and therefore
elaborated mechanisms exist to recycle the evolved
hydrogen in these cells. Additionally, hydrogen yield
is negatively affected by the partial pressure of the
product. Theoretically, up to 33% of the electrons in
hexose sugars can go to hydrogen when growth is
neglected and at least 66% of the substrate electrons
remain on VFA production.
The most appropriate parameter to analyze con-
tinuous systems is the mass loading rate (L), which is
function of substrate concentration (S) and the
hydraulic retention time (HRT):
L
S
HRT 3
VHPR increase when substrate concentration
increase and HRT diminishes. However, at low
HRT microbial washout might be greater than
microbial growth. Thus, the low concentration of
biomass in the reactor leads to decreased VFA
production and a higher pH. High substrate concen-
trations may result in the accumulation of VFA and
a low pH in the reactor. Accumulation of VFA may
cause hydrogen producing bacteria to switch to
solvent production and cell death, reducing theproduction of hydrogen (Lin and Chang 1999; Wang
et al. 2006; Hawkes et al. 2007). Low pH in the
reactor could inhibit the activity of microorganisms
if they come from non acidic environment. Also, the
proportions of undissociate acids (i.e. acetic, buty-
ric) increases as the pH decreases, the undissociated
forms can pass across the cell membrane, collapsing
the membrane pH gradient, and the cell will
sporulate or die (Hawkes et al. 2007). In addition,
when substrate concentrations increase in batch
systems the partial pressure of hydrogen rises andthe microorganism would switch to alcohol produc-
tion, thus inhibiting hydrogen production (Fan et al.
2006). Park et al. (2005) showed that chemical
scavenging of CO2 increased hydrogen production
by 43% in batch glucose fermentation. Also apply-
ing vacuum, gas sparging or CO2 scavenging may
all be effective methods to increase hydrogen
production (Levin et al. 2004; Valdez-Vazquez
et al. 2006).
For most of the results presented in Table 1,
optimal HRT between 0.5 and 12 h and substrateconcentrations around 20 g/l were reported. Chang
and Lin (2004) studied the effect of HRT on
hydrogen yield, VHPR and SHPR in an up-flow
anaerobic sludge blanket (UASB) reactor fed with
sucrose. They found that hydrogen yield was inde-
pendent of HRT, in the range of 8 to 20 h, but that
VHPR and SHPR were dependent on HRT. Oh et al.
(2004) showed that decreasing the HRT to 4 h and
simultaneously increasing the substrate concentration
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from 6.86 to 20.6 g/l resulted in an increased lactate
concentration, which reduced the VHPR.
The reactor configuration is another parameter that
affects VHPR as is shown in Table 1. The VHPR in
different reactor configurations varied, showing the
best performance with immobilized cell bioreactors.
High cell densities are needed to maximize hydrogenproduction rates. Therefore, major improvements are
expected in systems with biomass retention, e.g. by
immobilized cells. Oh et al. (2004) studied hydrogen
production in a trickling biofilter (TBR) with glucose
as substrate and found a maximum VHPR of
37.5 mmol/l-h. The TBR could maintain a high
biomass density of 1824 g VSS/l, which is higher
than other immobilized systems and significantly
higher than most of the cell suspension reactors like
the continuous stirring tank reactor (CSTR). Re-
cently, fluidized bed reactor (FBR) and draft tubefluidized bed reactor (DTFBR) systems with effluent
recycle and immobilized cells were studied for the
production of hydrogen using sucrose (Lin et al.
2006a; Wu et al. 2006a). A VHPR of 95.23 mmol/l-h
was obtained with the DTFBR, which was 50%
greater than the one obtained with FBR. However,
when using immobilized systems it could be impor-
tant to consider the gas (hydrogen and carbon
dioxide) hold up in the reactor, because if this is
excessive could induce inhibition of microorganism
due to changes of pH.The maximum VHPR that has been obtained is
612.5 mmol/l-h by using a CSTR containing silicone
immobilized sludge (10% v/v) and sucrose as
substrate (Wu et al. 2006a). This VHPR is at least
six times greater than any other VHPR reported
(Table 1). This study demonstrated that an appropri-
ate process design, containing simultaneously gran-
ular, immobilized and freely suspended sludge, had a
pronounced positive effect on hydrogen production.
In the same study, a hydrogen yield of 3.86 mol/mol
was obtained, which is similar to the highest yield of3.88 mol/mol obtained in a CIGSB (Carrier induced
granular sludge) system for fermentation of sucrose
(Lee et al. 2006). Ren et al. (2006) reported a
satisfactory performance of a pilot scale CSTR to
produce hydrogen from molasses. However, CSTR
problems could be present when high dilution rates
(low HRT) are used and washout of the cells is
experienced as the specific biomass growth rate is
proportional to the dilution rate in a steady state
operation. If easily biodegradable substrates (sucrose)
are used for hydrogen production, it could be
important to operate at the optimal dilution rate (near
to wash out) to allow optimal VHPR, small reactor
size and lower capital cost. However, high HRT
should be required when complex substrates (cellu-
lose) are used for the production of hydrogen.Gavala et al. (2006) obtained similar VHPR in
CSTR and UASB reactors for glucose fermentation,
but the hydrogen yield attained in the CSTR was
greater than that obtained with the UASB. Overall,
similar VHPR are obtained by using UASB and
CSTR systems (Table 1) (Chang and Lin 2004;
Gavala et al. 2006; Lin and Chen 2006; Zhang et al.
2006).
Kim et al. (2006) showed that the us e of
membrane bioreactors (MBR) for hydrogen produc-
tion allows advantages, such as high cell density.They used a MBR system with glucose as substrate
and found a maximum VHPR of 71.4 mmol/l-h.
Nevertheless, the use of MBR systems has been
limited to laboratory scale due to high investment
costs. Although immobilized cells and MBR systems
have shown the highest VHPR, it is not easy to
compare different configurations of reactors to draw a
conclusion in regard to what configuration is better,
even under a specific set of conditions. This is due to
the fact that many factors, such as VHPR, hydrogen
yield, long-term stability of the reactor, scale up, etc.,have an impact on the economics of fermentative
hydrogen production. In particular, the VHPR and
hydrogen yield change significantly depending on
experimental conditions including temperature, pH,
substrate concentration, type of substrate and HRT.
5 Molecular approach
Among the few microorganisms genetically modified
reported for biohydrogen production, Escherichiacoli is the most used, because its metabolic pathways
and genomic sequence are known. Also, there are
molecular tools for its manipulation. The metabolic
pathway for biohydrogen production by enterobacte-
ria is shown in Fig. 1. Under anaerobic conditions, a
fraction of pyruvate can be transformed to lactate by
the lactate dehydrogenase (LDH), but most of it is
hydrolyzed by the pyruvate formate liase (PFL) into
acetyl-CoA and formate. PFL cleaves pyruvate only
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when cells grow fermentatively, while pyruvate
dehydrogenase (PDH) decarboxylates pyruvate under
aerobic conditions. Both enzymes are active under
oxygen limiting conditions. The acetyl-CoA is par-tially converted into ethanol and acetate. Formate is
the electron donor in anaerobic metabolism for nitrate
reduction or can be transformed into hydrogen by the
formate-hydrogen lyase complex (FHL). In E. coli
there are three formate dehydrogenases (FDH)
denominated O, N and H. The FDH-H (encoded by
the fdhF gene) forms part of the FHL complex. The
enzymes required for formate metabolism are en-
coded in the formate regulon (Leonhartsberger et al.
2002; Sawers 2005).
The formate regulon includes genes hycB-I, hypA-E, hycA and hypF. HycB-G proteins are the structural
proteins forming the FHL and Hyp proteins are
involved in the maturation of the FHL, whereas
HycA is the negative transcriptional regulator for the
formate regulon. FhlA (encoded by fhlA gene) is the
positive transcriptional regulator for the expression of
fdhFgene (Fig. 2). Then, manipulating these genes it
is possible to control the production and activity of
FHL complex and therefore the biohydrogen produc-
tion (Leonhartsberger et al. 2002). Thus HycA
defective are hydrogen overproducing strains (Pen-
fold et al. 2003; Yoshida et al. 2005). A description offormate regulon has been published elsewhere
(Sawers 2005).
The hydrogenases 1 and 2 and formate dehy-
drogenase N and O also consume formate, but
they do not produce hydrogen. These isoenzymes
are located on the periplasmic space, and they
must be transported by Twin arginine translocation
(Tat) protein system to be active. Whereas HycE
(also known as hydrogenase 3) and FDH-H are
located on cytoplasm and hence are not to be
transported. Thus, Tat defective mutants arehydrogen overproducing strains. Penfold et al.
(2006) reported that mutant strains defective of
Tat transport (DtatC or DtatA-E) showed a hydro-
gen production comparable to E. coli strain
carrying a DhycA allele. However, DtatC DhycA
double mutant strain did not increase hydrogen
production. Thus, it is possible that hydrogen
production by E. coli could be increased by
discarding activities of the uptake hydrogenases,
which recycle a portion of hydrogen, and the
formate hydrogenases N and O, which oxidize theformate without hydrogen production.
Penfold and Macaskie (2004) transformed E. coli
HD701, a hydrogenase-upregulated strain and
FTD701 (a derivative of HD701 that has a deletion
of the tatC gene), with the plasmid pUR400 carrying
Fig. 2 The formate regulon
of E. coli: formate isgenerated by the pfl geneproduct. Genes or operons
positively regulated byformate through the action
of the transcriptionalregulator FhlA aredesignated by + (Modifiedfrom Sawers 2005)
Fig. 1 Metabolic routes of pyruvate and formate in E. coli.
Key reactions in the generation of hydrogen are shown in bold
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the scrregulon to yield E. coli strains, which produce
hydrogen from sucrose, as an alternative to coupling-
in an upstream invertase. The parenteral strains did
not produce hydrogen, whereas recombinant strains
produced 1.27 and 1.38 ml H2/mg dry weight-lculture.
Mishra et al. (2004) overexpressed a [Fe]-hydrog-
enase from Enterobacter cloacae (obtained withdegenerate primers designed from the conserved
zone of hydA gene) in a non-hydrogen producing
E. coli BL21. The resultant recombinant strain
showed the ability to produce hydrogen. Yoshida
et al. (2005) constructed an E. coli strain overex-
pressing FHL by combining hycA inactivation with
fhlA overexpression. With these genetic modifica-
tions, the transcription offdhF (large-subunit formate
dehydrogenase) and hycE(large-subunit hydrogenase
3) increased 6.5- and 7-fold, respectively, and
hydrogen production increased 2.8-fold comparedwith the wild-type strain. The effect of mutations in
uptake hydrogenases, in lactate dehydrogenase gene
(ldhA) and fhlA was studied by Bisaillon et al. (2006).
They reported that each mutation contributed to a
modest increase in hydrogen production and the
effect was synergistic.
As expected, the amino acid sequence of FDH-H
(E.C.1.2.1.2) from E. coli was highly homologous to
the FDH-H sequences reported by NCBI for other
Enterobacteria (Fig. 3). FDH-H sequence identities of
98.5%, 98.3% and 79.4% were calculated for Salmo-nella enterica YP_153159, Salmonella typhimurium
NP_463150, and Erwinia carotovora YP_049356
respectively. The partial sequence available for
Enterobacter aerogenes CAA38512 showed high
homology as well. In addition, the identity with the
FDH-H from the Archaeas Methanocaldococcus
jannaschii NP_248356 and Thermoplasma acidophi-
lum NP_393524 were 58.1% and 54.9%, respectively
and the FDH-H from Photobacterium profundum
ZP_01218756 (belongs to Vibrionaceae family) was
59.8%. The high homology of FDH-H sequenceamong diverse bacteria suggests a high evolutive
conservation of FDH-H.
The hydrogen production by Gram-positive bac-
teria such as Clostridium is shown in Fig. 4. The
pathway for hydrogen production uses two enzymes:
ferredoxin-NAD reductase (FNR) and [Fe]-hydroge-
nase (FR). The overexpression of hydA gene encod-
ing the FR has been used as a strategy to enhance
hydrogen production. For instance, Morimoto et al.
(2005) reported that the hydrogen yield increased 1.7-
times in recombinant Clostridium paraputrificum
overexpressing hydA gene with respect to a wild-
type strain. Harada et al. (2006) proposed to disrupt
thl gene encoding thiolase (THL), which is involved
in the butyrate formation from Clostridium butyri-
cum, like novel molecular strategies to improve thehydrogen production. Since, THL defective mutant
do not uptake NADH, it could be used for the
hydrogen production by the FNR. Nevertheless, at
this time, results on hydrogen production are not
available. Overexpression of FNR could improve
hydrogen production. However, strains overexpress-
ing FNR have not been reported.
6 Economics of biohydrogen production and
perspectives
Even when there are many reports in the literature
about biohydrogen production, only few of them are
related to the economic analyses of the biohydrogen
production. In general, the molar yield of hydrogen
and the cost of the feedstock are the two main barriers
for fermentation technology. The main challenge in
fermentative production of hydrogen is that only 15%
of the energy from the organic source can typically be
obtained in the form of hydrogen (Logan 2004).
Consequently, it is not surprising that mayor effortsare directed to substantially increase the hydrogen
yield. The U.S. DOE program goal for fermentation
technology is to realize yields of 4 and 6 mol
hydrogen per mol of glucose by 2013 and 2018,
respectively, as well as to achieve 3 and 6 months of
continuous operation for the same years (http://
www1.eere.energy.gov/hydrogenandfuelcells/mypp/
). Additionally, some integrated strategies are now
being under development such as the two step
fermentation process (acidogenic + photobiological
or acidogenic + methanogenic processes) or the useof modified microbial fuel cells (de Vrije and
Claassen 2003; Logan and Regan 2006; Ueno et al.
2007). Through these coupled processes more hydro-
gen or energy (in the form of methane) per mol of
substrate are achieved in a second step (Fig. 5).
Hydrogen molar yields may be increased through
metabolic engineering efforts. At this moment the
acceptability of genetically modified microorganisms
is a challenge, since the possible risk of horizontal
Rev Environ Sci Biotechnol (2008) 7:2745 39
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Fig.
3
Multiplealignmentofthe
FDH-HproteinofE.coliwiththeFD
H-Hoftwoarcheas(M.
jannaschiiandT.acidophilum),avibrionales(P.p
rofundum)andother
enterobacteria.Sequencefromlast
linerepresentsthetotalconservedaminoacids
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Fig.
3
continued
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transference of genetic material. However, this can be
ruled out by chromosomal integration and the elim-
ination of plasmids containing antibiotic markers
with available molecular tools (Datsenko and Wanner
2000). Moreover, the improvement of hydrogen
production by gene manipulation is mainly focused
on the disruption of endogenous genes and not
introducing new activities in the microorganisms.
New pathways must be discovered to directly takefull advantage of the 12 mol of H2 available in a mol
of hexose.
de Vrije and Claassen (2003) reported the cost of
hydrogen production using a locally produced ligno-
cellulosic feedstock. The plant was set at a production
capacity of 425 Nm3 H2/h and consisted of a thermo-
bioreactor (95 m3) for hydrogen fermentation fol-
lowed by a photo-bioreactor (300 m3) for the
conversion of acetic acid to hydrogen and CO2.
Economic analysis resulted in an estimated overall
cost of2.74/kg H2. This cost is based on acquisition
of biomass at zero value, zero hydrolysis costs and
excludes personnel costs and costs for civil works, all
of them with potential cost factors. Current estima-
tion for hydrogen production cost is 4/kg H2 or 30/
GJ H2. The estimation is done on the basis of processparameters which seem presently feasible (http://
www.biobasedproducts.nl/UK/5%20Projects/
frame%205%20projecten.htm).
Regarding feedstock costs, commercially pro-
duced food products, such as corn and sugar are not
economical for hydrogen production (Benemann
1996). However, by-products from agricultural crops
or industrial processes with no or low value represent
a valuable resource for energy production. Neverthe-
less, besides hydrogen biological production, other
biofuels (bioethanol, biodiesel, biobutanol, etc.) pro-cesses are being under development (Reisch 2006)
and, eventually, the demand of agricultural by-
products would increase its present low value.
According to the US DOE, for renewable H2 to be
cost competitive with traditional transportation fuels,
the sugar cost must be around $0.05/lb sugar
feedstock and provide a H2 molar yield approaching
10 (http://www1.eere.energy.gov/hydrogenandfuel-
cells/mypp/). Wastewater has a great potential for
economic production of hydrogen; only in the United
States the organic content in wastewater producedannually by humans and animals is equivalent to
0.41 quadrillion British thermal units, or 119.8 terra-
watt h (Logan 2004).
Currently, biologically produced hydrogen is more
expensive than other fuel options. There is no doubt
that many technical and engineering challenges have
to be solved before economic barriers can be
meaningfully considered.
Acknowledgements This work was supported by the Fondo
Mixto San Luis Potos Consejo Nacional de Ciencia yTecnologa (FMSLP-2005-C01-23).
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