Journal of Scientific & Industrial Research
994
Vol. 67, November 2008, pp.994-1016
J SCI IND RES VOL 67 NOVEMBER 2008
Biohydrogen production: molecular aspects
Lemi Türker1* , Selçuk Gümüs1 and Alper Tapan2
1
2
Middle East Technical University, Department of Chemistry, 06531, Ankara, Turkey
Gazi Üniversitesi, Mühendislik Fakültesi, Kimya Mühendislii Bölümü, Maltepe, Ankara, Turkey
Received 15 July 2008; revised 23 September 2008; accepted 13 October 2008
This study reviews biohydrogen systems, molecular and genetic aspects of hydrogen production and technologies of
biohydrogen production. An enormous investment is needed to understand hydrogen-producing mechanisms better in cells of
microorganisms at molecular level on evolution of artificial organisms, which could produce abundant, at least satisfactory,
quantities of hydrogen with a suitable rate of production.
Keywords: Biohydrogen production, Genetic aspects, Molecular aspects
Introduction
Fossil fuel resources are limited and large-scale
consumption of these resources cause an accelerated
release of CO2, which is major cause of global warming
and climatic changes. Among biofuels [bioethanol,
biomethanol, vegetable oils, biodiesel, biogas, biosynthetic gas (bio-syngas), bio-oil, bio-char, FischerTropsch liquids, and biohydrogen], biological hydrogen
production (BHP) processes are more environment
friendly and less energy intensive as compared to thermo
chemical and electrochemical processes 1. Among
alternative energy sources, hydrogen (H2) appears to
be most promising because it burns and produces
environment friendly product, water. In BHP, in algae
and cyanobacteria, solar energy captured by
photosynthetic pathways is converted into chemical
energy through water splitting, which yields oxygen (O2)
and H2.
Reaction between H2 and O2 to form water is a 2eredox reaction. Almost, all life processes derive their
energy from redox reactions, either directly or indirectly.
Photosynthesis, which uses light-driven redox reactions,
constitutes energy storage mechanism not only in higher
plants but also in bacteria. Away from light, bacteria
exploit oxidation of H2, sulfur and other compounds.
Many bacteria obtain energy by oxidation of H2 assisted
*Author for correspondence
Fax: 90-312-2103200; E-mail: [email protected]
by some complex mechanisms. However, principle
mechanism is generation of a transmembrane gradient of
protons, which drives formation of ATP2. In bacteria,
simple reaction between H2 and O2 in a membrane could
have created a transmembrane proton gradient in a
primitive cell. Enzymes involved are embedded in
membrane surrounding cell. Hydrogenase, which
consumes H 2 (H 2 ↔ 2H + + 2e - ), releases protons
(Hydrons) on the outside of cell. Meanwhile, an enzyme,
oxydase facing inwards, reduces oxygen to water (O2 +
4H+ ↔2H2O) takes up protons from the inside of cell.
Consequently, a proton gradient is established across
membrane. Mitchell3 described how gradient could be
exploited to synthesize ATP. Aerobic bacteria use O2 to
oxidize H2 to water, methane to CO2 and so on. On the
other hand, anaerobic bacteria such as Clostridium
pasteurianum produce H2 and acetate from organic matter
by fermentation. In anaerobic environment, H2 is a central
source of reducing power. Fermentative bacteria excrete
H2 as a waste product, while chemolithotropic bacteria
use it as fuel.
BHP using microorganisms offers potential production
of usable H2 from a variety of renewable resources. A
wide range of approaches is provided by biological
systems to generate H 2 , which include direct
biophotolysis, indirect biophotolysis, photo-fermentation
and dark fermentation 1,3-5 . Among three types of
microorganisms available of H 2 generation
(cyanobacteria, anaerobic bacteria, and fermentative
TÜRKER et al: BIOHYDROGEN PRODUCTION: MOLECULAR ASPECTS
bacteria), cyanobacteria directly decompose water to
H2 and O2 in presence of light energy by photosynthesis.
Photosynthetic bacteria use organic substrates like
organic acids. Anaerobic bacteria use organic
substances as sole source of electrons and energy,
converting them into H2. Biohydrogen can be generated
using bacteria such as Clostridia by controlling
temperature, pH, reactor hydraulic retention time (HRT)
and other factors of treatment system. Researchers have
started to investigate H2 production with anaerobic
bacteria since 1980s5,6.
This study reviews biohydrogen systems, molecular
and genetic aspects of hydrogen production and BHP
technologies.
Biohydrogen Systems
Direct Biophotolysis
It is H2 production from water via biological process
converting sunlight into chemical energy.
6H2O + 6CO2
C6H12O6 + 6H2O
hv
hv
995
C6H12O6 + 6O2
12H2 + 6CO2
Cyanobacteria (a blue-green algae) 13 contain
photosynthetic pigments (Chl. a), caratonoids and
phycobiliproteins and can form oxygenic
photosynthesis 14 . Nutritional requirements of
cyanobacteria are based on air, water, some mineral salts
and light15 . Species of cyanobacteria possess many
enzymes directly involved in H 2 metabolism and
production of H 2 . Of these, nitrogenases catalyze
production of H2 as a by-product of nitrogen reduction to
NH3, uptake hydrogenases to catalyze oxidation of H2
(synthesized by the nitrogenase) and bidirectional
hydrogenases synthesize H214. Cyanobacteria based H2
production has been found to be affected by many
factors16-18. Anabaena species and strains produce higher
rates of H219.
Photo Fermentation
2H2O
hv
2H2 + O2
Green algae under anaerobic conditions can either
use H2 as an electron donor in CO2 fixation process or
produce H2. Green micro algae based H2 production
requires several minutes to few hours in anaerobic
incubation and in dark conditions7. During that process
synthesis and/or activation of enzymes including
reversible hydrogenase occur. Hydrogenase converts
H+ to H2. Synthesis of H2 permits sustained electron
flow through electron- transport chain, which assists
synthesis of ATP8. A reversible hydrogenase accepts
electrons directly from reduced ferredoxin to generate
H29. Ferredoxin, photosystem I and II are all involved
in conversion of light into chemical energy as H2
molecule.
Cultures of green algae Chlamydomonas reinhardtii,
deprived of inorganic S exhibit declined photosynthetic
ability, due to need for frequent replacement of H2Ooxidizing protein D1 in PSII10. Under these conditions,
C. Reinhardtii becomes anaerobic in light and
commences to synthesis of H28. Based on this behavior,
some systems for sustained H2 production have been
developed11,12.
Indirect Biophotolysis
Cyanobacteria can also be used for H2 production
under photosynthesis as
Purple non-sulfur bacteria evolve H2 catalyzed by
nitrogenase under nitrogen-deficient conditions using
light energy and organic compounds (organic acids).
C6H12O6 + 6H2O
hv
12H2 + 6CO2
Photoheterotropic bacteria27 (Rhodopseudomonas
capsulata and Rhodospirillum rubrum) have been
investigated extensively for conversion of light energy
into H2 using organic waste compounds20-30. In general,
H2 production rates of photoheterotropic bacteria are
higher when cells are immobilized in or on a solid matrix,
than the cell are free.
Hydrogen Synthesis via Water-Gas Shift Reaction by
Photoheterotropic Bacteria
Some photoheterotropic bacteria belonging to
Rhodospirillaceae family can release H2 and CO2 while
growing in the dark and using CO as sole carbon source
to generate ATP31-33. Net oxidation of CO to CO2 occurs
via water gas shift reaction
CO(g) + H2O(l)
hv
CO2(g) + H2(g)
The reaction is associated with ”Gº =-20 kJ/mol, and
takes place at ambient temperatures and pressures.
Enzyme that binds and oxidizes CO is carbon monoxide:
acceptor oxidoreductase (carbon monoxide
996
J SCI IND RES VOL 67 NOVEMBER 2008
dehydrogenase = CODH7) and is part of membrane bound
enzyme complex33,34. Rubrivivax gelatinosus CBS is a
purple non-sulfur bacterium, which performs water gas
shift reaction in dark having 100% conversion of CO to
near stoichiometric amounts of H235-39.
Dark Fermentation
In dark fermentation, H2 is produced by anaerobic
bacteria (Enterobacter, Bacillus and Clostridium) grown
in dark on carbohydrate-rich substrates 7 between
25-80°C depending on the type of bacteria used. While
direct and indirect photolysis systems produce pure H2,
dark-fermentation processes produce a biogas mixture,
which contains H2 as major component, beside CO2, CH4,
CO and H2S. Glucose, hexose isomers, or polymers
(starch and cellulose) yield different amounts of H2 per
mole of glucose, depending on fermentation pathway
and end-product(s). When acetic acid is end product,
theoretically maximum of 4 moles of H2 per mole of
glucose is obtained.
C6H12O6 + 2H2O
2CH3COOH + 4H2 + 2CO2
If butyrate is end product, maximum theoretical H2
produced per mole of glucose is two.
C6H12O6
CH3CH2CH2COOH + 2H2 + 2CO2
Thus, acetate yield highest theoretical H2 yield.
However, in practice, high H2 productions are associated
with a mixture of acetate and butyrate fermentation
products. Whereas, low H2 yields are associated with
propionate and reduced end products such as lactic acid
and other alcohols. Clostridium pasteurianum, C.
butyricum and C. beijerinkii are known as high H2
producers40.
Molecular Aspects
H2 production by green algae was first reported using
Scenedesmus obliquus in seminal experiments7. Then,
several strains of green algae have been found capable
of producing H241,42. Anaerobic induction and light are
necessary to get highest rates of H 2 production7,43.
However, H2 production is not sustainable in light unless
O 2, which is coproduced by photosynthesis and
inactivates reaction 44, is removed continually from
medium. Electrons for H2 photo reduction are supplied
by photosynthetic electron transport chain, originating
either from oxidation of water by photo system II and/
or from metabolic oxidation of endogenous substrate in
chloroplast via its attendant electron flow to
plastoquinone pool. Also, fermentative algal metabolism
in dark produces H2 but at lower rates7,43. Clamydomonas
reinhardtii, when deprived of sulfate containing
nutrients, can produce H245-49. Activity of photo system
II declines50 to the point where O2 consumption by
respiration is greater than the rate of photosynthetic O2
evolution46,47.
Hydrogenase enzyme is O 2 sensitive. [NiFe]hydrogenase catalyzes H2 oxidation on a graphite
electrode at rates comparable to that of platinum
deposited on an identical electrode51. Hydrogenase is
relatively immune to CO poisoning compared to
platinum. However, electron transfer from active site of
hydrogenase can be a problem because unlike platinum
catalyst, active core of enzyme is deeply buried inside
the protein. Hence, some energy is required to get
electrons to external circuit.
Hydrogenases
Hydrogenases, which catalyze simplest redox-linked
chemical reaction, H2↔2H+ +2e-, can both consume and
produce H2, depending on conditions. Bacterial cells can
get benefit from uptake-activity of hydrogenases through
formation of reducing equivalents required for cell’s
metabolism. On the other hand, bacteria can get rid of
excess electrons (or protons) via H2 production catalyzed
by hydrogenases. Out of known (13) families of
hydrogenases, all but one are involved directly or
indirectly in energy metabolism and either catalyze H2
oxidation (H2-uptake/consumption) linked to energy
conserving reactions or catalyze H+ reduction (H 2
evolution). One family of hydrogenases present in
several autotrophic Protobacteria appears to act as H2sensor. A fourth function for hydrogenases has been
suggested for bidirectional hydrogenases in
cyanobacteria, which may serve to poise redox of
photosynthetic and respiratory electron transport chains.
Biochemical methods used for isolation and biochemical
characterization of hydrogenases are reported51-53.
Presence of oxygen, which interferes or poisons
enzyme, notably [Fe]-hydrogenases, has to be avoided.
A method of measuring an enzyme activity is known as
an essay. For hydrogenases, production of H 2 and
oxidation of H2 are among many types of assays.
Different hydrogenases show significantly different rates
of isotopic exchange reactions with deuterium gas as
TÜRKER et al: BIOHYDROGEN PRODUCTION: MOLECULAR ASPECTS
D2 + 2H2O↔H2 + 2HOD
D2 + H2O ↔HD + HOD
This type of activity has been detected even in dry
samples of hydrogenases2. Similar equilibria exist for
tritium gas. Isotopic exchange reactions have been used
to identify different types of hydrogenases in whole cells
without any purification 54. Kinetic isotope effects
exhibited by hydrogenases provide important clues to
mechanisms involved in enzymes. Reaction cycle of
enzyme depends on several steps, in which H2 atoms
are transferred from site to site. Rates of these transfers
show significant kinetic isotope effects and rates
decrease in the order of H>D>T.
Activation and Activity States
Hydrogenases activity is highly dependent on sample
history. [NiFe] hydrogenases isolated under normal
aerobic conditions do not display activity in H2 exchange
assays even after almost complete removal of O 2.
Whereas, same preparations exhibit some activity when
assayed for H2 evolution or H2 -uptake2. Fernandez et
al 55 interpreted complex activity changes in
hydrogenases (such as those observed from D. gigas) in
terms of interconversions between the states designated
as unready, ready and active states. Unready state is
inactive and prolong treatment with a reducing agent is
required to activate. Ready state is inactive towards H2
and is inactive in assays with electron acceptors of high
redox potential (DCIP). On the other hand, ready state
requires only brief reduction.
Classification of Hydrogenases
Hydrogenases contain some essential transition
metals. In presence of H2 and an electron acceptor,
hydrogenase acts as a H2 -uptake enzyme, whereas in
presence of an electron donor of low potential, it may
use protons from water as electron acceptor and release
H256. Most of the known hydrogenases are iron-sulfur
proteins with two metal atoms at their active site, either
a Ni and / or Fe atom ([NiFe]-hydrogenases57,58) or two
Fe atoms ([NiFe]-hydrogenases 59,60). However, a
different type of hydrogenase exists in some
methanogens 61,62 , which functions as H 2 -forming
methylenetetrahydromethanopterin dehydrogenase
(Hmd). This enzyme contains no Fe-S clusters and no
Ni and it was initially called as “metal free hydrogenase”.
Later, it was renamed as “iron-sulfur-cluster free
hydrogenase” or simply [Fe]-hydrogenase63.
997
[Fe]-Hydrogenases
[Fe]-hydrogenases (Hmd) extensively studied since
its discovery in Methonothermobacter marburgensis61,
is the catalyst of an intermediary step in CO2 reduction
with H2 to methane62,64, that is reduction of methylH4MPT+ (methylenetetrahydromethanopterin] reversibly
to methylene-H4MPT and H+ with H2. Hmd enzyme
differs from [NiFe] and [FeFe]-hydrogenases by primary
and tertiary structures. Additionally, iron required for
its enzymatic activity is not redox active. These
hydrogenases have catalytic properties different from
[NiFe]- and [FeFe]-hydrogenases so that they do not
catalyze 2H+ + 2e- ↔H2 reversible redox reaction.
Activity of Hmd enzyme is associated with an ironcontaining cofactor 65-67 and crystal structure of this
apoenzyme has been established68.
[NiFe]-Hydrogenases
[NiFe]-hydrogenases constitute most numerous class
of hydrogenases. Crystal structures of Desulfovibrio
hydrogenases are known57,58,69-72. Core enzyme consists
of a α, β heterodimer; α-subunit being larger one and
contains bimetallic active site, whereas small β-subunit
possesses Fe-S cluster. Subunits are in extensive
interaction through a large contact surface, forming a
globular heterodimer56. Core, bimetallic NiFe center, is
located in α-subunit and coordinated with S-atoms of 4
cysteine moieties. Also, nonproteinous ligands, one CO
and two CN are coordinated with Fe atom69,73,74 (Fig. 1).
In some cases, ligands SO, CO and CN have been
reported for coordination 58,75 . In NAD-reducing
hydrogenase of Ralstonia eutrapha, active site as
Ni(CN)Fe(CN) 3CO has been reported 76. β-Subunit
contains up to 3 linearly arranged [4Fe-4S] type, cubanelike Fe-S clusters. Their role seems to be conducting
electrons between H2-activating center and physiological
redox site of hydrogenases.
In some bacteria (Desulfomicrobium baculatum77,
Desulfovibrio vulgaris Hidenbrough 77 , etc.,
hydrogenases contain [NiFeSe] core and have been
characterized with presence of 3 [4Fe-4S] clusters
whereas in the case of standard Desulfovibrio [NiFe]hydrogenases a [3Fe-4S] cluster with a relatively high
redox potential exists in between [4Fe-4S] clusters
occupying proximal and distal positions. Some reports
are available on the role of these clusters in gas access
to active site70,79,80.
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J SCI IND RES VOL 67 NOVEMBER 2008
H
Cys68
S
Cys
S
Cys530
S
S
[4Fe 4S]
S
Fe
Ni
65
N
Cys533
CN
CO
Cys
S
S
Fe
Fe
CN
CO
CN
X
CO
CN
C
O
Fig. 1—Schematic structure57 of active site in [NiFe]- and [FeFe]hydrogenases [X: O-2, OH-, OH2, SO, in the reduced
form X: H-]
[FeFe]-Hydrogenases
[FeFe]-hydrogenases are monomeric and contain only
catalytic subunit 56. Some varieties having dimeric,
trimeric and even tetrameric enzymes have also been
reported81,82. In [FeFe]-hydrogenases, active site (Hcluster] consists of a binuclear [FeFe] center bound to a
[4Fe-4S] cluster by means of a bridging cysteine
belonging to protein83. Nonproteneinous ligands CN- and
CO are attached to Fe atoms of binuclear Fe center81,84
(Fig. 1). Two bridging sulfur atoms originating possibly
from a di(thiomethyl)amine molecule coordinates with
Fe atoms82. Fe atom distal to [4Fe-4S] cluster possesses
a vacant coordination site, which is occupied by CO, a
competitive inhibitor in CO-inhibited form of enzyme.
Most hydrogenases are directly or indirectly involved
in energy metabolism. Hydrogenases that are functional
in H2 oxidation (H2-uptake/consumption) are linked to
energy conserving reactions, whereas hydrogenases that
are functional in H + reduction (H 2 production) are
coupled to disposal of excess reducing equivalents
through reoxidation of reduced pyridine nucleotides and
electron carriers. Another family of hydrogenases, which
are found in several autotrophic proteobacteria, appears
to act as H2-sensing component of a complex genetic
relay mechanism controlling expression of other
hydrogenases in these organisms 85-87 . [NiFe]hydrogenases 56 are classified as: I) Uptake [NiFe]hydrogenases; II) Cyanobacterial uptake [NiFe]hydrogenases and H 2 -sensors; III) Bidirectional
heteromultimeric cytoplasmic [NiFe]-hydrogenases; and
IV) H 2 -evolving energy-conserving membraneassociated hydrogenases.
Soluble Hydrogenase
In R. eutropha, energy conservation from H 2 is
mediated by following two different [NiFe]-
hydrogenases that are synthesized coordinately: i) MBH
is bound to cytoplasmic membrane; and ii) A soluble
heterotetramer (SH), a member of heteromultimeric
[NiFe]-hydrogenases, which resides in cytoplasm. Active
site of SH are very different from those of standard
[NiFe]-hydrogenases. It was proposed that SH might
have a (CN)NiFe(CN)3CO active site bound to 4 thiols
of 4 strictly conserved Cys residues in HoxH subunit.
This would make Fe site six coordinated and thus not
reactive during activity cycle of enzyme. Ni site would
be at least five coordinated. Since, H2 cannot readily
react with untreated, aerobic enzyme, it was assumed
that inactive enzyme probably contains an oxygen
species linked to sixth coordination site of Ni88.
Gas Access in Hydrogenases
Recent structural analysis of [NiFe]-hydrogenases has
shown that active site is buried within large subunit
(α-unit) at approx. 30 Å from the surface. In
D. fructosovorans [NiFe]-hydrogenase, a remarkably
extensive network of mainly hydrophobic cavities and
channels was found. This network connects molecular
surface to deeply buried Ni-Fe core70. A very similar
channel structure has been reported in D. gigas [NiFe]hydrogenase80. Based on crystal structure analyses of
D.vulgaris and D. desulfuricans, it has been established
that their [NiFe]-hydrogenase also have similar channels.
Also, most channels are conserved in crystal structure
of D. bacalatum [NiFeSe]- hydrogenase77.
Iron-sulfur (Fe-S) Clusters
Fe-S clusters exist in hydrogenases and also appear
as cofactor in various other enzymes. Several simpler
Fe-S clusters spontaneously assemble into apo form of
Fe-S proteins in reductive aqueous solution with ferrous
iron and sulfide89. Structurally, basic building block of
Fe-S clusters is a Fe ion tetrahedrally coordinated by 4
S ligands (Fig. 1). Simplest cluster is that of one, present
in rubredoxin, in which Fe atom is coordinated by 4
Cys thiol groups (Fig. 1). [2Fe-2S] clusters possess two
inorganic sulfide ligands and 4 Cys thiols. Fe-S clusters
of simple types are mostly found in electron-transfer
proteins such as ferrodoxins or as part of an internal
electron-transfer pathway in larger enzymes. All [NiFe]hydrogenases contain a [4Fe-4S] cluster within 10 Å of
active NiFe site.
Small unit of D.gigas [NiFe]-hydrogenase contains 3
Fe-S clusters (2 [4Fe-4S] clusters and 1 [3Fe-4S] cluster)
and oriented in an almost linear alignment from active
TÜRKER et al: BIOHYDROGEN PRODUCTION: MOLECULAR ASPECTS
site to the surface of protein having an average clusterto- cluster distance of 12 Å57. Rapid electron transfer
over such distances from one center to another, within
proteins, occurs90 and this is partly described as quantum
mechanical tunneling, which depends on the overlap of
wave-functions for two centers. In [NiFe]-hydrogenases,
[4Fe-4S] cluster closest to active site (~10 Å from Ni) is
called proximal cluster. [3Fe-4S] cluster is the cluster to
surface of the molecule and occurs between proximal
and distal cluster. Proximal [4Fe-4S] cluster is involved
in direct electron exchange mechanism with active site
whereas distal [4Fe-4S] cluster is believed to mediate,
through its histidine ligand, electronic exchanges between
hydrogenase and a redox partner. However, involvement
of medial [3Fe-4S] cluster is a matter of debate because
of its high redox potential, which is about 300 mV more
positive than distal and proximal [4Fe-4S]. By the help
of genetic engineering, Pro residue (common in most
[3Fe-4S] proteins] of D. fructosovorans) was replaced
by Cys residue. Concomitantly, [3Fe-4S] cluster was
converted to [4Fe-4S] form91. Modified enzyme was
found to be more sensitive to oxygen but did not show
any increase in electron transfer rate.
Proton Transfer
During heterolytic cleavage of H2 molecule at active
site, a hydride and a proton are formed in the first step92,93.
Then, two electrons of hydride ion are removed in second
step to form a second proton. Since active site is deeply
buried, like electrons also the protons formed have to
be conveyed over a distance of about 15 Å to protein
environment. Reverse of these steps is required for H2
production. Experimental evidences exist for the crucial
role of terminal Ni-bound cycteine ligand as the first
acceptor site in process of proton transfer after
heterolytic cleavage of H2 94,70,72,95-98. Proton transfer in
D. gigas is reported57,99. Role of Mg atom in proton
transfer for [NiFe]-hydrogenase from D. vulgaris was
also proposed58. There are several additional routes
proposed for [NiFe]-hydrogenases and most likely,
proton transfer is not confined to a single route2,57,72,77.
A Deeper Look into Active Site of Hydrogenases
Many [NiFe]-hydrogenases dissolved in aerobic buffer
contain two unpaired electrons, one is located on [3Fe4S] cluster and other in active site. Oxidized proximal
and distal clusters are diamagnetic. By means of EPR
techniques, it has been established that unpaired spin
located in active site is close to nickel atom and at least
999
one of its sulfur ligands. EPR spectra of aerobic [NiFe]hydrogenases are very similar, showing that nickel site
is structurally conserved. Enzyme preparations often
contain two types of inactive enzyme molecules. When
O2 is removed and H2 is provided, then one type of
enzyme molecules shows full activity within minutes
(ready type enzyme]. The other type remains inactive
for prolong periods of time (unready enzyme]. Reduced
enzyme experimentally reoxidized by O2 enriched with
17
O isotope (which has a magnetic nucleus) showed that
an oxygen species ended up close to Ni-based unpaired
spin in the ready as well as in unready state100. It could
be removed only by full reduction and activation of
enzyme.
EPR studies have revealed that Ni sites in ready and
unready enzyme are slightly different. FTIR spectra
indicate that Fe sites in Nir* and Niu* (ready and unready
states, respectively) are very similar. Active site
undergoes reduction by accepting an electron. Upon
reduction of unready enzyme, electron/proton
combination probably takes place at nickel site according
to,
Ni(III)Fe(II) + e- +H+ →(H+)Ni(II)Fe(II)
A shift of CN/CO bands to higher frequencies occurs
in infrared spectrum of Niu-S state. Whereas respective
spectrum for Ni r -S state has bands all shifted to
considerably lower frequencies, indicating a greatly
increased charge density on Fe101,102 as
Ni(III) Fe(II) + e- + H+ →Ni(III)Fe(I)(H+)
Increased electron density on Fe would result in a
large shift (50-100 cm-1) to lower frequencies of CN/
CO bands; protonation of a thiolate ligand would reverse
it largely. In model compounds102, protonation of thiolate
ligand to Fe can increase stretching frequency of CO
bound to Fe, by 40 cm-1. Usually reduction of Fe(II) is
anticipated, however it occurs only at considerably lower
potentials. Reduction of low-spin Fe (II), which is
nonmagnetic, would create an unpaired spin. Its spin
magnetic moment might couple to Ni-based spin,
consequently cancel total magnetism and no EPR signal
could be seen. Added electron and proton both go to Ni
as in the case of Nix*, but then charge density on Fe also
increases due to a better electronic contact between two
metal ions in ready state.
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J SCI IND RES VOL 67 NOVEMBER 2008
For A.vinosum hydrogenase, activation requires not
only reduction but also temperature needs to be elevated.
When enzyme in Ni r* state is reduced at higher
temperatures at about 30°C or higher (but not at 2°C), a
rapid increase in activity is observed and a third EPR
signal of a Ni –based unpaired electron emerges (NiaC*), which is intermediately reduced state. Nia-S is one
–electron reduced state of Ni-Fe site in active enzyme
and Nia-SR stands for the most reduced state.
Nia-S
↔Nia-C* Conversion
In D.gigas hydrogenase, Nia-S ↔ Nia-C* reversible
reaction occurs and pH dependence of potential
accompanied indicates that one electron and one or two
protons are involved in equilibriu103. Nia-C* state
possesses a trivalent nickel104. Hence, Nia-S→Nia-C*
reaction can be written as105
Ni Fe
(II)
(II)
+ e + H → Ni
-
+
(III)
Fe (H2)
(II)
Nia-S→Nia-C* reaction is also H2 driven alone, but
then reverse reaction is extremely slow. To explain
reaction with H2 in absence of mediators, involvement
of a Fe-S cluster has been proposed.
Nia-C* → Nia-SR Conversion
In presence of redox mediators, Nia-C* state can
be further reduced to an EPR-silent state (by increase of
H2 partial pressure) in a reaction requiring one electron
and one proton. Based on assumption that trivalent Ni
exists in Ni a-C* state, probable reaction can be
formulated as
Ni(III)Fe(II)(H2) + e- + H+ → (H-)Ni(II)Fe(II)(H2)
Consistent with two-electron donor nature of H2,
reaction behaved as an n:2 redox reaction. As active site
in Nia-SR state has one electron more than that in NiaC* state, a Fe-S cluster has to be involved in the reaction
with H2.
Ni(III)Fe(II)[4Fe-4S] + H2 → Ni(II)Fe(II) [4Fe-4S] + 2H+
In this process, only proximal and distal clusters can
be involved. When these experiments are performed
under equilibrium conditions, no change of redox states
of Fe-S clusters can be observed. A possible explanation
is to assume that individual enzyme molecules can
exchange electrons. The best suitable site for this is via
distal [4Fe-4S] cluster. It is located on protein surface.
Such an exchange would occur on one-electron basis
having a slower rate than reaction with H2, which is
extremely fast and depends on the rate of diffusion of
H2 into enzyme106. Supposing Nia-C* state is initially
formed with one Fe-S cluster in oxidized state,
Ni(III)Fe(II)(H2)/[Fe-S]P+ / [Fe-S]D+2
Two such molecules could exchange an electron
resulting in one enzyme molecule with two oxidized
[4Fe-4S]+2 clusters and other with two reduced clusters,
Ni(III)Fe(II)(H2)/[Fe-S]P+2 / [Fe-S]D+2 + Ni(III)Fe(II)(H2)/
[Fe-S]P+ / [Fe-S]D+
A simple reaction of former molecule with H2 would
reduce its two clusters again. Final level of reduction of
Fe-S clusters would then only depend on effective redox
potential in the system. An interesting enzyme is H2sensor protein. H2-sensor from Ralstonia eutropa is
reported107. Its active site is highly similar to the one in
standard [NiFe]-hydrogenases. However, its enzymatic
properties are quite different. Sensor enzyme is less
active and is always, even in aerobic solution, in active
Nia-S state. It is also insensitive to O2 and CO and can
be reduced with H2 to Nia-C* state but not further.
Genetic Aspects
Biosynthesis of [NiFe]-hydrogenases
Genes in Proteobacteria that encode H2-uptake
hydrogenases are clustered. These clusters comprise
structural genes (labeled as L for large subunit and S for
small subunit) and accessory genes for maturation and
insertion of metal atoms and ligands (Ni, Fe, CO, and
CN-) at active site of heterodimer. However, in some
microorganisms, hydrogenase gene cluster also
comprises regulatory genes that control expression of
structural genes. Maturation of hydrogenase occurs via
a complex pathway, which involves various (at least 7)
auxiliary proteins that are products of so-called hyp
genes (HypA, HypB, HypC, HypD, HypE, and HypF,
and an endopeptidase). These proteins direct synthesis
and incorporation of metal center into large subunit, and
also control insertion of correct metal, maintain a folding
state of protein for metal addition, and allow necessary
conformational changes of protein. Gene/ protein
designations used for homologous proteins in various
TÜRKER et al: BIOHYDROGEN PRODUCTION: MOLECULAR ASPECTS
microorganisms are reported108,109. Carbamoylphosphate
has been shown to be educt for synthesis of CN ligands
of NiFe metal center110-112, which requires activity of
two hydrogenase maturation proteins that is HypF, a
carbamoyltransferase, and HypE, which receives
carbamoyl moiety to its COOH-terminal cysteine to form
an enzyme-thiocarbamate. HypE dehydrates
S-carbamoyl moiety to yield enzyme thiocyanate, which
then can donate CN moiety to iron113,114. HypE and HypF
form a dynamic complex with HypC and HypD. CN is
transferred to HypC-HypD and then attached to Fe atom
of NiFe site115. It has been proposed that conserved
cysteine residues in HypD protein play a role in
maturation process116.
Biosynthetic route for CO to NiFe active site is
different from that for cyanide117. Products of hupGHIJ
operon have been shown to be involved in maturation
of HupS hydrogenase subunit of Rhizobium
leguminosarum uptake hydrogenase118. Transcriptional
control involves usually one or several two-component
regulatory systems. In response to a specific signal, first
component,
a
sensor
histidine
kinase,
autophosphorylates at a conserved histidine residue and
then transphosphorylates cognate response regulator
transcription factor at a conserved aspartate residue that
activates or represses gene expression when
phosphorylated by sensor kinase119,120. Molecular H2
activates hydrogenase expression in aerobic bacteria
(R. eutropha), in photosynthetic bacteria (R. capsulatus,
R. sphaeroides, R. palustris), or in free-living Rhizobia
(B. japonicum). H 2 -specific regulatory system
comprises a H 2 -sensing regulatory hydrogenase
(HupUV/Hox- BC) and a two-component signal
transduction system, histidine protein kinase HupT/HoxJ,
and response regulator HupR/HoxA. This complex
system has been particularly well studied in R. capsulatus
and R. Eutropha 87,121-125 . In all of these bacteria,
regulatory cascade responding to H2 occurs by detection
of H2 signal by H2-sensor (HupUV/HoxBC) and it is
transmitted to histidine kinase (HupT/HoxJ); it is
transduced by phosphotransfer between histidine kinase
and response regulator (HupR/HoxA) and integrated at
promoter of structural genes of hydrogenase by response
regulator. However, in the absence of H 2-sensor,
hydrogenase synthesis is derepressed, in
R. capsulatus86,123,126, but in B. japonicum, R. eutropha,
and R. Palustris 127-129, there is no synthesis of
membrane-bound uptake hydrogenase. In T.
roseopersicina, components of H2-regulatory system
1001
(HupUV, HupT, and HupR) are present, but expression
of structural hupSL hydrogenase genes is not affected
by the presence or absence of H2130.
Biosynthesis of [FeFe]-Hydrogenases
Accessory genes necessary for biosynthesis of
[FeFe]-hydrogenases have been identified. Two novel
radical S-adenosylmethionine (SAM) proteins were
required for assembly of active site of C. reinhardtii
hydrogenases131. Random insertional mutants having their
hydEF gene inactivated were incapable of assembling
an active [FeFe]-hydrogenase. In C. reinhardtii genome,
hydEF gene is adjacent to another hydrogenase-related
gene, hydG. Their radical-SAM domains contain
conserved motif Cx3- Cx2C, also additional motifs in Cterminal ends that are characteristic of [Fe-S] clusterbinding sites132. Radical SAM proteins generate a radical
species by reductive cleavage of S-adenosylmethionine
through a [Fe-S] center to catalyze reactions involved in
cofactor biosynthesis, metabolism, and synthesis of
deoxyribonucleotides 133. HydF maturation protein
contains at its N-terminal end conserved GTP-binding
motifs. Anaerobically reconstituted HydE and HydG
proteins from Thermotoga maritima are able to cleave
SAM reductively when exposed to reduction by
dithionite, confirming that they are radical SAM
enzymes134 and HydF from T. maritima is a GTPase with
an Fe-S cluster 135. On the other hand, anaerobic
coexpression of C. reinhardtii hydEF, hydG, and hydA1
genes in E. coli resulted in formation of an active HydA1
enzyme131. [Fe-Fe]-hydrogenases with high specific
activities was obtained in Clostridium acetobutylicum
by homologous and heterologous over expression of
hydA gene from C. acetobutylicum, C. reinhardtii, and
S. obliquus, respectively136. Because C. Acetobutylicum
hydE, hydF, and hydG clones are more stable in E. coli
than their C. reinhardtii homologues, an efficient
biosynthetic system has been developed in E. coli by
expression cloning of hydE, hydF, and hydG from
C. acetobutylicum. An active [FeFe]-hydrogenase was
obtained with fully functional maturation proteins and Nterminally deleted C. acetobutylicum HydA and
C. pasteurianum HydA, that is, with catalytic H-clustercontaining domain only137. In accordance with the role
of radical SAM enzymes involved in production of active
[FeFe]- hydrogenases, a mechanistic scheme has been
presented for hydrogenase H-cluster biosynthesis, in
which both CO and cyanide ligands can be derived from
decomposition of a glycine radical138 .
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J SCI IND RES VOL 67 NOVEMBER 2008
Molecular and Genetic Aspects of Technology of
Biohydrogen Production
Numerous microorganisms that can produce H2 by
reactions linked to their energy metabolism use protons
from H2O as electron acceptors to dispose of excess
reducing power in cell and to reoxidize their coenzymes
in the absence of oxygen 139. BHP processes have
advantage of generating H2 not only from a variety of
renewable substrates, but also from organic waste
streams 140,141 . Among various bioprocesses of H 2
production, photo fermentation is favored due to higher
substrate-to- H2 yields and, its ability to trap energy under
a wide range of light spectrum and versatility in sources
of metabolic substrates with promise for waste
stabilization142. In addition, the process can potentially
be driven by solar energy with minimal non-renewable
energy inputs. Economic feasibility of photo
fermentative H2 production systems can be further
improved by utilizing low cost substrates or waste
streams and, by collecting and recycling useful byproducts other than H2143.
Photosynthetic bacteria produce H2 under anaerobic
conditions, in the absence of nitrogen gas, with
illumination and with stressful concentrations of nitrogen
sources. Photo heterotrophic bacteria, such as
Rhodobacter sphaeroides, can grow anaerobically to
produce H2 either from reduced substrates such as
organic acids [purple non-sulfur (PNS) bacteria] or from
reduced S compounds (green and purple sulfur bacteria).
These bacteria use enzyme nitrogenase to catalyze
nitrogen fixation for reduction of molecular nitrogen to
ammonia. Nitrogenase can evolve H2 simultaneously
with nitrogen reduction. Stressful concentrations of
nitrogen are therefore required for H2 evolution144.
Conversion efficiency of light energy to H 2 in
presence of an appropriate substrate and optimum cell
growth conditions is a key factor for economic photo
fermentative biohydrogen production143. Main hurdle,
however, is requirements for large expose area due to
low light efficiency of the process. Design guidelines
for photobioreactors for efficient utilization of light are
still lacking145. Since growth rate of bacteria is a function
of light intensity and substrate concentration, kinetic
models relating the three can be of value in designing
process and in identifying underlying rate-determining
and significant factors. Most photo fermentative
biohydrogen studies have used malic acid as substrate
and R. sphaeroides O.U.001 as microorganisms, under
optimum carbon-to-nitrogen (C/N) ratio146-148 in batch
reactors. Koku et al147 studied growth characteristics of
PNS bacteria and Eroglu et al149 studied dependence of
their growth rate on substrate, while their dependence
on light intensity has been studied by Sasikala et al150.
However, little information has been reported on kinetic
models integrating growth of PNS bacteria with light
utilization and H2 production1,148.
A kinetic model144, developed for photo fermentative
biohydrogen production to predict dynamics of the
process, contains 17 parameters [5 cell growth
parameters (CXm, KS, KI, KXI, KXi), 5 product formation
parameters (CPm, KPS, KPi, Kpl, KPI), values of yield
coefficients for H 2 formation (Y PX ), and malate
consumption (YP, YXS), maximum specific growth rate
(µm), specific malate consumption rate (µSX), specific
product formation (µPX) auto-inhibition constant (KSA)]
to describe cell growth, substrate consumption, and H2
evolution as well as inhibition of the process by biomass,
light intensity, and substrate. Batch experimental results
were used to calibrate and validate model with malic
acid as a model substrate, using Rhodobacter
sphaeroides as a model biomass. Temporal H2 evolution
and cell growth predicted by proposed model agreed well
with experimentally measured data obtained from
published reports, with statistically significant
correlation coefficients exceeding 0.9. Based on
sensitivity analysis performed with validated model, only
6 of 17 parameters were found to be significant. Model
simulations indicated that the range of optimal light
intensity for maximum H2 yield from malate by R.
sphaeroides was 150-250W/m2.
Rhodobacter sphaeroides O.U.001, a purple non-S
bacterium, produces H2 under photoheterotrophic
conditions. In R. sphaeroides, several metabolic
pathways take role in H2 production and consumption.
Total H2 production is limited due to several metabolic
events occurring in cells such as production of poly-3hydroxybutyrate (PHB) or consumption of H 2 by
hydrogenase uptake. Membrane-bound uptake
hydrogenase decreases H2 production efficiency by
catalyzing conversion of molecular H2 to electrons and
protons151. Inactivation of uptake hydrogenase has
resulted in total increase in H2 production152-154. Kars et
al155 worked on manipulation of purple non-S bacterium
R. sphaeroides O.U.001 such that uptake hydrogenase
was inactivated. Yield and rate of H2 production, and
substrate conversion efficiency (SCE) improved in
modified hup-R. sphaeroides O.U.001. Measuring
absorbance at 660 nm at certain time intervals monitored
TÜRKER et al: BIOHYDROGEN PRODUCTION: MOLECULAR ASPECTS
growth of mutant and wild type R. sphaeroides O.U.001.
Wild type cells reached relatively higher absorbance
values (OD660 = 1.90 ± 0.05) compared to hup-mutant
strain (OD660 = 1.71± 0.06). There was no considerable
difference in pH values (7.3-7.8) of mutant and wild type
cells. Significantly higher (20%) H2 accumulated in hupmutant R. sphaeroides O.U.001 when compared to wild
type cells (m = 2.85 l H2/l culture, w = 2.36 l H2/l culture)
under nitrogenase repressed conditions in 60 ml
bioreactors. According to gas chromatography (GC)
analysis, H2 constituted 96-99% (v/v) of overall gas.
Average gas production rates of mutant cells (9.2 ± 0:4
m/l/h) and wild type cells (6.9 ± 0.5 ml/l/h) were
calculated by dividing total volume of gas produced by
volume of culture and by duration of gas production.
SCE, another parameter for comparative analysis of H2
production, is calculated as ratio of actual amount of
produced H2 to theoretical amount. SCE of mutant cells
was 85.2 ± 2%, while that of wild type cells was 70.5 ±
3%. A SCE of 35-57 % for malate was reported for R.
sphaeroides. Directed insertional inactivation of uptake
hydrogenase significantly increased total H2 production
in hup-mutant cells and it did not affect bacterial growth.
High SCE demonstrated that more energy and reducing
equivalents were directed towards nitrogenase enzyme
and therefore more H2 accumulation was achieved.
Hence, results are promising for genetic engineering of
R. Sphaeroides towards enhanced H 2 production
capacity155.
Halobacterium salinarum belongs to halophilic
archaea. Purple membrane (PM) of H. salinarum
contains a retinal transmembrane protein
bacteriorhodopsin (BR), which acts as a light-driven
proton pump (light energy transducing system). Proton
gradient generated is utilized for ATP synthesis by
membrane bound H+ATPase. Studies are required to
elucidate exact mechanism of proton translocation
through BR156,157. H. salinarum lacks both hydrogenase
and nitrogenase or any other system that can reduce
protons into molecular H2. Therefore, packed cells (PC)
of H. salinarum or its PM might be combined with
another system for H2 production158. Zabut et al 159
introduced photobiological H2 production by combined
system of R. sphaeroides O.U.001 and H. salinarum S9
in a column photobioreactor for improvement of
biological H2 production. Photo activities of both PC
and PM fragments of H. salinarum, measured in H2
production medium at 32°C employing two light/dark
cycles, indicated159 that ∆pHmax of light period was 0.08
1003
and that of dark period was 0.10. ∆pHmax values obtained
by PC were higher than those of PM fragments. Stable
and reproducible light/dark responses were obtained for
both PC and PM fragments of H. salinarum in H2
production medium. BR has low photo activity under low
ionic strength and at temperatures more than 30°C160.
H 2 production experiments were conducted under
specified conditions159 and experimental results were
carried out with R. sphaeroides alone, and R. sphaeroides
combined with PC of H. salinarum or PM fragments.
Gas analysis indicated that over 95.0% was H2 and rest
was CO2 in all of experiments. Persistence of pH values
more than 8.0 in culture might limit nitrogenase
productivity and activate uptake hydrogenase, which
preferred slightly alkaline conditions, leading to less H2
evolution161. High initial cell concentrations might cause
fast bacterial growth that might enhance H2 production;
however, high cell density prevented light penetration
through culture 162.
Presence of PC did not significantly affect total gas
production and H 2 production rate compared to the
results obtained with R. sphaeroides when there was no
stirring. Discontinuous stirring of the system for
10 h/day with a magnetic stirrer operated at 300 rpm
created unstable H2 production in the reactors. Stirring
enhanced formation and removal of gas bubbles. Stirring
has been reported to increase conversion efficiency of
lactate to H 2 by Rhodopseudomonas sp. and by
R. Sphaeroides B6 and by R. sphaeroides B5 163.
Cultures of R. Sphaeroides combined with different
concentrations of BR in PC of H. salinarum under
continuous stirring conditions showed that total gas
production was increased from 690 to 1500 ml with
addition of 50 nmol of BR, and H2 production rate was
increased from 11 to 27 ml H2/h/l of culture. Cultures
combined with suspended PC containing 50 nmol of BR
gave best results among others in terms of amount of
total gas production and the rates of H2 production under
continuous stirring conditions. However, an increase of
PC in order to increase amount of BR in the system above
50 nmol had an inverse effect on total gas production
and rate of H2 production. Total gas production decreased
from 1500 to 850 ml and H2 production rate decreased
from 27 to 17 ml H2/h/l of culture by increasing BR
amount from 50 nmol to 150 nmol, attributed to viscosity
increase caused by suspended PC of H. salinarum.
Enhancement of H2 production by R. Sphaeroides
O.U.001 using PC of H. salinarum could be ascribed to
additional protons coming from light induced proton
1004
J SCI IND RES VOL 67 NOVEMBER 2008
pumping of BR. The provided protons were readily used
by nitrogenase of R. sphaeroides O.U.001 under limiting
conditions of nitrogen. Total H2 gas production increased
2.5 times and rate of H2 production enhanced three-fold,
compared to R. sphaeroides only culture within same
experimental period. BR on H2 production has positive
effect on different systems164-168. PC was not vital but
BR in native membrane in its original environment was
still active (continue to pump protons upon illumination).
However, due to low salt content of culture in bioreactor,
photo activity of BR was less compared to natural growth
medium of H. salinarum. Experimental results with
R. sphaeroides and combined systems of
R. Sphaeroides with PM fragments of H. Salinarum
indicated that addition of BR as PM fragments had no
significant effect on H2 production.
BR was found more effective on H2 production, when
R. sphaeroides was combined with PC rather than
R. sphaeroides combined with PM fragments, especially
when this comparison was made between combined
systems containing same amount of BR. On an average,
R. Sphaeroides culture combined with PC of
H. salinarum containing 50 nmol of BR produced 1500
ml H2 with a rate of 0.027 l/l/h, whereas R. sphaeroides
culture combined with PM fragments containing same
amount of BR produced 650 ml H2 with a rate of 0.014
l/l/h culture. This difference could be attributed to higher
photo activity of BR exhibited as in the form of PC,
compared to photo activity of BR as in the form of PM
fragments in Medium II of the study. Medium II (which
was H2 production medium of R. sphaeroides) had low
salt concentration. However, photo activity of BR is
highly affected by salt concentration167,169. Low ionic
strength decreases proton-pumping rate of BR. On the
other hand, two combined systems should be compared
based on orientation of BR, presence of other cell
enclosures (in case of PC) and effect of additional
purification steps (in case of PM fragments) on photo
activity of BR. Overall, combining of R. sphaeroides
with PC of H. salinarum was more efficient for
photobiological H 2 production. Thus, in combined
cultures, continuous stirring, consistency in pH values,
and moderate bacterial density played important roles
in increasing amount of total gas production and rates
of H2 production. It was found that using packed cells
of H. salinarum was better than using PM fragments in
the combined systems. Since a high ionic strength
promotes photo activity of BR, salt tolerant strains of
R. sphaeroides are recommended for future work in
combined systems. Immobilized combined systems are
suggested for future continuous H2 production. Outdoor
and large-scale systems are indicated for further
investigation159.
Most of phototrophic biohydrogen studies were
conducted for pure cultures of 4 PNS (Rhodobacter
sphaeroides 170 , Rhodopseudomonas capsulatus 171 ,
R. palustris143 and Rhodospirillum rubrum172) using
organic substrate as carbon source. Another PNS,
Rubrivivax gelatinosus, can also produce H2 but mainly
using CO as carbon source. Li & Fang173 studied H2
production characteristics of a new strain of
R. gelatinosus, which was isolated from local reservoir
sediment, using various organic substrates. These
characteristics were then correlated with activity of its
nitrogenase, which is responsible to photoheterotrophic
H2 production174, and accumulation of PHB, which may
compete with H2 for electrons175. Results of batch tests
using individual organic substrates showed that
R. gelatinosus L31 was able to produce H 2 from
glucose, sucrose, starch, lactate and malate, however, it
was unable to produce H2 from acetate, propionate,
butyrate, succinate and glutamate. H2 conversion
efficiency is defined as the ratio between actual H2
production and stoichiometric value as
Ca HbOc + (2a-c)H2O
aCO2 + (2a-c+0.5b)H2
Maximum specific H 2 production rates of
R. gelatinosus L31 (193-829 ml/g/h) are higher than
those of other phototrophs using same substrate, except
reported value of 670 ml/g/h by R. palustris P4 using
glucose176. Conversion efficiency (50.5%) for lactate is
higher173 than most reported data (12.4-26.1%) and
comparable to 52.7% by R. capsulatus JP91 177 .
Conversion efficiencies of malate, glucose, sucrose, and
starch are all comparable to reported values.
Starch has been rarely used for phototrophic H2
production. Ike et al 178 has found that although
R. Marinum A-501 could produce H2 from glucose and
sucrose, but could not produce from starch. For
R. gelatinosus173, H2 was produced from starch at a
maximum rate of 12.1 ml/l/h, which is higher than 7.811.3 ml/l/h produced from starch sources such as
cassava, rice, and corn179.
Conversion efficiencies of R. gelatinosus L31 were
50.5% for lactate and 24.6% for malate, both of which
were substantially higher than 7.4-8.8% for three
carbohydrates. R. Gelatinosus L31 could not produce
H2 from acetate, propionate, butyrate, and succinate, even
TÜRKER et al: BIOHYDROGEN PRODUCTION: MOLECULAR ASPECTS
though these organic acids could produce H2 by other
species (R. sphaeroides, R. capsulatus,
Rhodopseudomonas sp., and R. palustris R1). no study
is available on any phototroph capable of producing H2
from glutamate173.
Dark fermentation or acidogenic fermentation of
carbohydrates presents several advantages over photo
fermentation such as a production rate is higher than
those obtained with photobiological processes and
capacity of being run all over the day even during night1.
Main species40 identified for biological H2 production
during acidogenesis of carbohydrates are Enterobacter,
Bacillus and Clostridium. Their metabolism can be
represented by biochemical pathways 180. Thus,
fermentation pathways that produce acetate and butyrate
are mainly responsible for H2 production40,181. On the
other hand, pathways that produce ethanol, lactate and
propionate are unable to produce H2, because they
consume hydrogenated biochemical intermediates like
NADH. Reactions involved in acidogenic fermentation
associated to H2 are mostly presented by assuming a
biomass product equivalent to C4H7O2N as reported182.
H2 is produced as by product during dark fermentation
of glucose and/or sucrose by bacteria for energy
production to grow. Organic acids (VFA) and alcohols
are also formed as by products or intermediates, which
inhibit fermentation by a complex metabolic pathway183.
In dark fermentation, 4 moles of H2 can be produced by
fermentation of glucose with an acetic acid as an end
product5,40. This also results in a net production of 4 mol
of ATP184. However, average yields of H2 using glucose
in mesophilic temperature range are always less than
4 mol-H2/mol-glucose and vary widely185-187. Production
of H2 drops to 2 moles theoretically during production of
butyrate5,40 as
C6H12O6 → CH3CH2CH2COOH + 2CO2 + 2H2
There are no known fermentation pathways that can
achieve conversion efficiency greater than 4 mol H2/mol
hexose (C6H12O6) in dark fermentation188 and as seen in
equations below, propionic acid formation has no
contribution to H2 production:
Homofermentative pathway,
C6H12O6 → 2CH3CHOHCOOH
Heterofermentative pathway,
C6H12O6 →CH3CHOHCOOH + CH3CH2OH + CO2
1005
In addition, formation of propionic acid consumes H2,
therefore it should also be avoided for efficient H 2
production:
C6H12O6 + 2H2 →2CH3CH2COOH + 2H2O
So high H2 yields depend on acetic acid and butyric
acid production, and in contrast to acetic acid and butyric
acid, propionic acid and ethanol are considered as
unfavorable end products41,181,189.
C6H12O6 →CH3CH2CH2COOH + 2H2+ 2CO2
Increased concentrations of VFA and alcohols may
inhibit further production of H2190. During fermentation,
ethanol consumes more electrons from metabolic
reducing power, therefore, it is not desired for H 2
production191. H2 production and fermentation products
in the liquid are related such as during batch and
continuous H 2 production from simulated cheese
processing wastewater via anaerobic fermentation from
mixed microbial communities under mesophilic
conditions, higher H2 yield in biogas was observed when
concentration of ethanol, hexanoic acid, n-butyric acid
in solution is high. On the other hand, propionic acid
concentration was low192. Since butyric acid and acetic
acid are crucial end products for H2 production, ratio of
butyric acid to acetic acid (2.9-4.3) is used as a
performance parameter for dark H2 fermentation studies
using carbohydrates6,193-197.
H2 production is also effected by pH of reaction
environment. Variations in acetate/butyrate ratio are
caused by metabolic alterations due to changes of pH198.
During H2, production fermentation pathway may shift
from VFA producing to alcohol producing when pH was
decreased to 4.5 or below198,199. On the other hand, in
some batch experiments, only a small amount of
methanol was seen in an acidic environment. Increasing
pH also did not show a significant effect on VFA and
alcohol concentrations. However, acetate /butyrate ratio
increased from 0.41 to 1.15, when pH was changed from
4.5 to 7.0. This suggests a shift of fermentation pathway
by pH changes200. pH (5.5-6.0) was considered to be ideal
to avoid both methanogenesis and solventogenesis186 and
could be considered optimum pH range for effective H2
generation. Reactor must be operated at a pH of 6.0 to
facilitate proliferation of acidogenic bacteria for H2
production. Optimum pH for growth of MB
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J SCI IND RES VOL 67 NOVEMBER 2008
(methanogenic bacteria) was between 6.0 and 7.5, while
AB (acidogenic bacteria) functions well below pH
6187,191. Maintenance of pH around 6 resulted in higher
production of H2 compared to near neutral pH190,201-203
and inhibition of methanogenic group of bacteria for
effective H2 yield. If pH was not maintained in desired
range, it could inhibit H2 production or cause a microbial
population shift, resulting in cessation of H 2
production190,204 . Alkalinity (buffering capacity) is
considered as one of the most important factors that is
governed by VFA production and accumulation.
Normally, system alkalinity acts as a buffer to sustain
anaerobic performance in presence of VFA production205.
H2 production by chemical oxygen demand (COD)
removal (mol H2 produced/kg COD removed) changes
with ethanol/acetate ratio in H2 bio-producing reactor
system (HBR). This phenomenon could be related to
complex fermentation mechanism and oxidization/
reduction of nicotinamide adenine dinucleotide (NADH,
Eo’NADH= -320mV). Only pathway in this fermentation
mechanism that can produce H 2 is pyruvate
decarboxylation by ferredoxin and butyric acid, acetic
acid and ethanol are the end products. Pathways are
controlled by NAD+ (oxidation status)/(NADH+ H+)
(reduction status), existing at certain ratio in
microorganisms206. Dynamic equilibrium of oxidation /
reduction of NAD+/NADH + H +, which could be
achieved by mole ratio of ethanol to acetic acid (1:1)
during acetic acid fermentation, plays a major role in
H2 bioproduction. Lower or higher mole ratio of ethanol
to acetic acid than 1:1 destabilizes fermentation leading
to inhibition of H2 production207.
According to Ruzicka model208, main limitation for
producing highest yields of H2 is inhibitory effect of H2
partial pressure. Increase in dissolved H2 concentration
limits transfer of electrons from glucose to H2. NADH
is electron carrier that is involved in transfer of electrons
from pyruvate to H2 (Eo‘= -414 mV)188. Since NADH
has a higher potential than H 2 (NADH, E o’ NADH
= -320mV), dehydrogenation of triose phosphate to
produce 2 mol of H2 can occur only when concentration
of H2 is less than ~6×10-4 atm via oxidation of pyruvate
and ferredoxin can generate another 2 mol of H2 at higher
H2 concentrations up to ~0.3 atm209. Thus, in order to
obtain H2 yields higher than 2 mol-H2/mol glucose,
production of H2 via triose phosphate dehydrogenation
and NADH must be achieved. However, when all NAD+
(oxidized form) is in reduced form (NADH) because it
is unfavourable to transfer electrons from NADH to H2,
flux of glucose through glycolytic pathway and through
phosphoroclastic reaction stops. In order to increase this
glucose flux for maximum ATP production, some bacteria
(C pasteurianum) divert electrons in NADH to butyrate
production, resulting in decrease in H2 yields and
production of 3 mol of ATP. Production of butyrate rather
than acetate allows for NAD+ regeneration, a greater
flux of glucose through bacterial glycolytic pathway, and
a greater overall ATP production rate than what acetate
production alone could sustain.
Energy production with H2 generation is highest when
acetate is produced (4 mol-ATP/mol glucose), but when
H 2 concentrations are high, NAD + can only be
regenerated if compounds other than acetate (butyrate
or butanol) are produced. ATP generation is 3 mol-ATP/
mol-glucose when butyrate is produced. Crabbenbaum
et al210 found that average ATP yield of 3.277 ± 0.02
mol-ATP/mol-glucose, which corresponds to a H2 yield
of 2.7 mol-H2/mol-glucose. Thauer et al188 concluded
that for a HAc: HBu ratio of 0.86 (0.6 mol of acetate
and 0.7 mol of butyrate from 1.0 mol glucose), H2 yield
would be 2.6 mol-H2/mol-glucose at a thermodynamic
efficiency of 85% for biohydrogen production from
glucose. These values for H2 yield are consistent with
results obtained for average HAc:HBu ratio of
0.86 ± 0.14.
Bacterial Communities
H2 producing bacteria may be classified in four
groups5,200 (strictly anaerobes, facultative anaerobes,
aerobes and photosynthetic bacteria). Strictly anaerobic
Clostridium was found most abundant in acidophilic H2
producing sludge in biohydrogen production from rice
slurry. In addition, Clostridia sp. are mainly responsible
for fermenting sugars to H2 at high yields211 producing
acetate, butyrate, and other fermentation end products
as waste products212. Many Clostridium sp., capable of
producing H 2 , include C. acetobutylicum 213 ,
C. butylicum185, C. butyricum214, C. kluyveri215 and
C. pasteurianum 216, some of which are known
acidophilic species. C. acetobutylicum can grow at pH
4.3217 and C. butyricum at pH 4218. Two anaerobic acidtolerant bacteria, C. akagii CK58T and C. acidisoli
CK74T, have been isolated from acidic beech litter and
acidic peat-bog soil, respectively219. Growth of C. akagii
CK58T (pH 3.7-7.1) and C. acidisoli CK74T (pH 3.66.9) on glucose yielded H2, butyrate, lactate, acetate,
formate, and CO 2 . An acidophilic Enterobacter
aerogenes strain HO-39, capable of producing H2 at
TÜRKER et al: BIOHYDROGEN PRODUCTION: MOLECULAR ASPECTS
pH 4.0, has also been isolated 220. Clostridium
pasteurianium, C. butyricum, C. beijerinkii ,
E. aerogenes produce high amount of H 240,142,221 .
C. pasteurianum is an acid producer and produces H2
along with acetate and butyrate189. C. butyricum can be
used as a starch fermenting bacteria for dark
fermentative H2 production by direct starch utilization222224
. Lactobacillus sp. are known to be predominant
microorganisms in batch type bioreactor at high H2 yields
during biohydrogen production from cheese processing
waste water192. For larger scale H2 production, mixtures
of microbial cultures are found cost effective. A mixed
culture of C. butyricum and E. aerogenes gave H2 yield
of 2.0-2.7 mol H2/mol glucose from single-stage H2
production with sweet potato starch 222-224. Several
powerful H2-producing bacterial isolates (Clostridial sp.)
from municipal sewage are also capable of producing
H2 from sugar very efficiently225.
Technological Improvements in Biohydrogen Production
H2 production from wastewater has a great potential
for economical H2 production with high yields226. For
maximizing H2 production in fermentation systems, loss
of H 2 to H 2 -consuming anaerobes, such as
methanogens 227, can be avoided by heat-treating
inoculum to select for spore-formers, such as Clostridia,
for glucose-fed reactors or even heat-treating wastewater
to kill methanogens141,227-229. Low pH can also be used to
minimize growth of methanogens230. Longer hydraulic
retention times (HRT) and lower COD levels contribute
to greater overall efficiency of H2 production in
continuously fed reactors201,229. Other factors (inoculum,
substrate, temperature, nitrogen sparging, and initial start
up) have been examined in an effort to optimize H2
production40,211,231.
Even during H2 production from simple sugars under
optimal conditions, original organic matter (67%) will
remain in solution (COD basis). Typical H2 yields (1-2
mol/mol) result in 80-90% of initial COD remaining in
wastewater as volatile organic acids and solvents (acetic,
propionic, butyric acids and ethanol). One way to recover
remaining organic matter in a useable form for energy
production is to produce methane. Two-stage processes
are already well developed, and could be adapted for
both H 2 and methane production, although these
combined gas processes have not yet been demonstrated
at full scale232. Feasibility of integrating acidogenic
process of H2 generation with anaerobic/methanogenic
process of methane production to utilize residual organic
composition in wastewater was also studied233. Following
1007
equations were used for computing methanogenic
fermentation balance consuming H2 and VFA generated
from primary acidogenic process:
CH3COOH →2H2 + CO2
CH3CH2CH2COOH + 2H2O ↔CH3COOH + 2H2
CH 3CH2CH2CH2COOH + 2H2O
↔2CH3 CH2COOH + CH3COOH + 2H2
4H2 + CO2 →CH4 + 2H2O
Experimental data supported efficacy of integrating
acidogenic H 2 production process with anaerobic
methanogenic process in enhancing substrate
degradation efficiency along with both H2 and CH4
generation as renewable by-products. Integration of
acidogenic and methanogenic processes appeared to be
a feasible option for sustainable H2 production utilizing
wastewater as substrate 234-236. Fluidized-bed reactor
(FBR) and packed-bed reactor (PBR) were developed
to produce H2 and ethanol simultaneously from dark
fermentation of carbohydrate substrates using
polyethylene-octane elastomer immobilized anaerobic
sludge as biocatalyst. Production of two biofuels seemed
to have substrate preference. In FBR, sucrose was
favorable for H2 production, while ethanol production
was better with fructose. However, in PBR, glucose gave
best performance in terms of production rate and yield
of the two biofuels. This difference in substrate
preference could be due to variations in bacterial
population structure resulting from different bioreactor
configuration237.
High H 2 yields that are needed to make process
economical232 can be achieved by H2 production from a
fermentation end product (acetate) by modifying a
microbial fuel cell by applying a small potential to that
generated by bacteria238. H2 yields can be increased in
continuous culture by decreasing H2 partial pressure in
reactor. This can be achieved by stripping H2 from liquid
using N2 sparging210,239 and also by applying a vacuum
to headspace, thereby lowering overall partial pressure
in the system185. It is possible to generate power from
fermentation end products other than H2. For instance,
when propionate end product is fed to microbial fuel
cell (MFC), electricity can be generated with propionate
intermediate with cereal wastewater along with acetate.
However, this reaction would require rapid utilization of
any H 2 produced in the system. Propionate can be
converted to acetate as
1008
J SCI IND RES VOL 67 NOVEMBER 2008
CH3CH2COO- + 3H2O ↔CH3COO+ H+ + HCO3- + 3H2
Under standard conditions, this reaction is not
thermodynamically feasible (∆G=76.1 kJ). Conversion
of propionate to acetate and H 2 is only
thermodynamically possible at H2 concentrations of
10-4 atmospheres (100 ppm). However, H2 could have
remained low in the system due to H2 utilization by
bacteria making conversion of propionate to acetate
possible240. High H2 yields also require novel reactor
technology like a mesophilic unsaturated flow (trickle
bed) reactor, which can achieve high H2 gas recovery
from pretreatment of high carbohydrate containing
wastewaters241. High H2 production rates and stable H2
production can also be achieved at low retention times
in upflow anaerobic sludge blanket reactor when
compared with conventional continuous stirred tank
reactor242.
In H2 production, unstable and low flow rates during
biohydrogen production bring up development of low
energy and efficient purification methods. At this point,
modules containing a polyvinyltrimethylsilane
(PVTMS) membrane are developed for biohydrogen
treatment and efficient separation of CO2 from H2243.
Innovative reactor designs like draft tube fluidized bed
reactor (DTFBR) containing immobilized cell particles
by synthetic polymer (silicone gel, SC) demonstrate an
efficient, stable and reproducible H2-production244.
Among various approaches to increase H2 production
yield from organic wastes, nutrient supplementation
(nitrogen, phosphorus and iron as biostimulants)
improves simultaneous H2 production and pollution
reduction from substrate using thermophilic
fermentation. Anaerobic sequencing batch reactor
(ASBR) fed with nutrient-supplemented POME gave
higher growth and activity of T. Thermosaccharolyticum
than feeding with raw POME. Butyric acid and acetic
acid were main soluble metabolites, which favor H2
production245. Biohydrogen production can be achieved
through bioconversion of syngas by water gas shift
reaction173,247.
CO + H2O ↔ H2 + CO2
R. rubrum, PNS bacterium can catalyze WGS
reaction by reactants other than CO like acetate, malate,
glucose, yeast extract and ammonium246-248, under
anaerobic conditions in a continuous stirred bioreactor
(CSTBR)249. It can consume CO faster than other H2
producing bacteria with high growth rate and cell
concentration 147,174,250-252 . During WGS reaction,
nitrogenase is responsible catalyst for H 2
production143,252. On the other hand, initial substrate
concentrations may inhibit cell growth and H 2
production147,250. Consumption rate of carbon source can
vary for R. rubrum. In the case of CO, 3 folds higher
consumption rates can be achieved than acetate253.
Although feedstocks like starch and cellulose from
crop wastes are abundant for H2 production, rate of
fermentative H2 production may be slow due to substrate
hydrolysis. In the case of starch, even high H2 producers
like C. Butyricum can exhibit low performance by slow
hydrolysis process. In order to eliminate this problem,
starch can be enzymatically hydrolyzed by amylase
producing bacterium Caldimonas taiwanensis On1254.
By utilizing starch pretreatment, even pure cultures that
cannot convert raw starch into H 2 give high H 2
production rates and H2 yields255. In the case of cellulose,
NS culture hydrolyzes carboxymethyl cellulose, and
after that H2 producing bacterial isolates (mainly
Clostridium species) were used to convert cellulose
hyrolysate into H2 energy256.
Conclusions
Biological H 2 production is the most desirable
ultimate target to supply energy demand of mankind.
However, photosynthetic organisms are rather sluggish
to produce H2 and maintenance of optimum conditions
in reactors while production occurs is a delicate task.
Therefore, an enormous investment is needed to
understand H2 producing mechanisms better in cells of
microorganisms at the molecular level in the direction
of evolution of artificial organisms, which could produce
abundant, at least satisfactory, quantities of H2 with a
suitable rate of production.
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