biohydrogen production: molecular aspects

994 J SCI IND RES VOL 67 NOVEMBER 2008Journal of Scientific & Industrial ResearchVol. 67, November 2008, pp.994-1016

*Author for correspondenceFax: 90-312-2103200; E-mail: [email protected]

Biohydrogen production: molecular aspects

Lemi Türker1* , Selçuk Gümüs1 and Alper Tapan2

1Middle East Technical University, Department of Chemistry, 06531, Ankara, Turkey2Gazi 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 ofbiohydrogen production. An enormous investment is needed to understand hydrogen-producing mechanisms better in cells ofmicroorganisms 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-scaleconsumption of these resources cause an acceleratedrelease of CO2, which is major cause of global warmingand climatic changes. Among biofuels [bioethanol,biomethanol, vegetable oils, biodiesel, biogas, bio-synthetic gas (bio-syngas), bio-oil, bio-char, Fischer-Tropsch liquids, and biohydrogen], biological hydrogenproduction (BHP) processes are more environmentfriendly and less energy intensive as compared to thermochemical and electrochemical processes1. Amongalternative energy sources, hydrogen (H2) appears tobe most promising because it burns and producesenvironment friendly product, water. In BHP, in algaeand cyanobacteria, solar energy captured byphotosynthetic pathways is converted into chemicalenergy through water splitting, which yields oxygen (O2)and H2.

Reaction between H2 and O2 to form water is a 2e-

redox reaction. Almost, all life processes derive theirenergy from redox reactions, either directly or indirectly.Photosynthesis, which uses light-driven redox reactions,constitutes energy storage mechanism not only in higherplants but also in bacteria. Away from light, bacteriaexploit oxidation of H2, sulfur and other compounds.Many bacteria obtain energy by oxidation of H2 assisted

by some complex mechanisms. However, principlemechanism is generation of a transmembrane gradient ofprotons, which drives formation of ATP2. In bacteria,simple reaction between H2 and O2 in a membrane couldhave created a transmembrane proton gradient in aprimitive cell. Enzymes involved are embedded inmembrane surrounding cell. Hydrogenase, whichconsumes H2 (H2↔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 acrossmembrane. Mitchell3 described how gradient could beexploited to synthesize ATP. Aerobic bacteria use O2 tooxidize H2 to water, methane to CO2 and so on. On theother hand, anaerobic bacteria such as Clostridium

pasteurianum produce H2 and acetate from organic matterby fermentation. In anaerobic environment, H2 is a centralsource of reducing power. Fermentative bacteria excreteH2 as a waste product, while chemolithotropic bacteriause it as fuel.

BHP using microorganisms offers potential productionof usable H2 from a variety of renewable resources. Awide range of approaches is provided by biologicalsystems to generate H2, which include directbiophotolysis, indirect biophotolysis, photo-fermentationand dark fermentation1,3-5. Among three types ofmicroorganisms available of H2 generation(cyanobacteria, anaerobic bacteria, and fermentative

TÜRKER et al: BIOHYDROGEN PRODUCTION: MOLECULAR ASPECTS 995

bacteria), cyanobacteria directly decompose water toH2 and O2 in presence of light energy by photosynthesis.Photosynthetic bacteria use organic substrates likeorganic acids. Anaerobic bacteria use organicsubstances as sole source of electrons and energy,converting them into H2. Biohydrogen can be generatedusing bacteria such as Clostridia by controllingtemperature, pH, reactor hydraulic retention time (HRT)and other factors of treatment system. Researchers havestarted to investigate H2 production with anaerobicbacteria since 1980s5,6.

This study reviews biohydrogen systems, molecularand genetic aspects of hydrogen production and BHPtechnologies.

Biohydrogen SystemsDirect Biophotolysis

It is H2 production from water via biological processconverting sunlight into chemical energy.

2H2O 2H2 + O2hv

Green algae under anaerobic conditions can eitheruse H

2 as an electron donor in CO

2 fixation process or

produce H2. Green micro algae based H

2 production

requires several minutes to few hours in anaerobicincubation and in dark conditions7. During that processsynthesis and/or activation of enzymes includingreversible hydrogenase occur. Hydrogenase convertsH+ to H

2. Synthesis of H

2 permits sustained electron

flow through electron- transport chain, which assistssynthesis of ATP8. A reversible hydrogenase acceptselectrons directly from reduced ferredoxin to generateH

29. 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 photosyntheticability, due to need for frequent replacement of H

2O-

oxidizing protein D1 in PSII10. Under these conditions,C. Reinhardtii becomes anaerobic in light andcommences to synthesis of H

28. 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

6H2O + 6CO2 hv C6H12O6 + 6O2

C6H12O6 + 6H2O 12H2 + 6CO2hv

Cyanobacteria (a blue-green algae)13 containphotosynthetic pigments (Chl. a), caratonoids andphycobiliproteins and can form oxygenicphotosynthesis14. Nutritional requirements ofcyanobacteria are based on air, water, some mineral saltsand light15. Species of cyanobacteria possess manyenzymes directly involved in H

2 metabolism and

production of H2. Of these, nitrogenases catalyze

production of H2 as a by-product of nitrogen reduction to

NH3, uptake hydrogenases to catalyze oxidation of H

2

(synthesized by the nitrogenase) and bidirectionalhydrogenases synthesize H

214. Cyanobacteria based H

2

production has been found to be affected by manyfactors16-18. Anabaena species and strains produce higherrates of H

219.

Photo Fermentation

Purple non-sulfur bacteria evolve H2 catalyzed by

nitrogenase under nitrogen-deficient conditions usinglight energy and organic compounds (organic acids).

C6H12O6 + 6H2O 12H2 + 6CO2hv

Photoheterotropic bacteria27 (Rhodopseudomonas

capsulata and Rhodospirillum rubrum) have beeninvestigated extensively for conversion of light energyinto H

2 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 toRhodospirillaceae family can release H

2 and CO

2 while

growing in the dark and using CO as sole carbon sourceto generate ATP31-33. Net oxidation of CO to CO

2 occurs

via water gas shift reaction

CO(g) + H2O(l) hv CO2(g) + H2(g)

The reaction is associated with ”Gº =-20 kJ/mol, andtakes 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 boundenzyme complex33,34. Rubrivivax gelatinosus CBS is apurple non-sulfur bacterium, which performs water gasshift reaction in dark having 100% conversion of CO tonear stoichiometric amounts of H2

35-39.

Dark Fermentation

In dark fermentation, H2 is produced by anaerobicbacteria (Enterobacter, Bacillus and Clostridium) grownin dark on carbohydrate-rich substrates7 between25-80°C depending on the type of bacteria used. Whiledirect 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 permole of glucose, depending on fermentation pathwayand end-product(s). When acetic acid is end product,theoretically maximum of 4 moles of H2 per mole ofglucose 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 associatedwith a mixture of acetate and butyrate fermentationproducts. Whereas, low H2 yields are associated withpropionate and reduced end products such as lactic acidand 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 usingScenedesmus obliquus in seminal experiments7. Then,several strains of green algae have been found capableof producing H2

41,42. Anaerobic induction and light arenecessary to get highest rates of H2 production7,43.However, H2 production is not sustainable in light unlessO2, which is coproduced by photosynthesis andinactivates reaction44, is removed continually frommedium. Electrons for H2 photo reduction are suppliedby photosynthetic electron transport chain, originatingeither from oxidation of water by photo system II and/

or from metabolic oxidation of endogenous substrate inchloroplast via its attendant electron flow toplastoquinone pool. Also, fermentative algal metabolismin dark produces H2 but at lower rates7,43. Clamydomonas

reinhardtii, when deprived of sulfate containingnutrients, can produce H2

45-49. Activity of photo systemII declines50 to the point where O2 consumption byrespiration is greater than the rate of photosynthetic O2

evolution46,47.

Hydrogenase enzyme is O2 sensitive. [NiFe]-hydrogenase catalyzes H2 oxidation on a graphiteelectrode at rates comparable to that of platinumdeposited on an identical electrode51. Hydrogenase isrelatively immune to CO poisoning compared toplatinum. However, electron transfer from active site ofhydrogenase can be a problem because unlike platinumcatalyst, active core of enzyme is deeply buried insidethe protein. Hence, some energy is required to getelectrons to external circuit.

Hydrogenases

Hydrogenases, which catalyze simplest redox-linkedchemical reaction, H2↔2H+ +2e-, can both consume andproduce H2, depending on conditions. Bacterial cells canget benefit from uptake-activity of hydrogenases throughformation of reducing equivalents required for cell’smetabolism. On the other hand, bacteria can get rid ofexcess electrons (or protons) via H2 production catalyzedby hydrogenases. Out of known (13) families ofhydrogenases, all but one are involved directly orindirectly in energy metabolism and either catalyze H2

oxidation (H2-uptake/consumption) linked to energyconserving reactions or catalyze H+ reduction (H2

evolution). One family of hydrogenases present inseveral autotrophic Protobacteria appears to act as H2-sensor. A fourth function for hydrogenases has beensuggested for bidirectional hydrogenases incyanobacteria, which may serve to poise redox ofphotosynthetic and respiratory electron transport chains.Biochemical methods used for isolation and biochemicalcharacterization of hydrogenases are reported51-53.

Presence of oxygen, which interferes or poisonsenzyme, notably [Fe]-hydrogenases, has to be avoided.A method of measuring an enzyme activity is known asan essay. For hydrogenases, production of H2 andoxidation of H2 are among many types of assays.Different hydrogenases show significantly different ratesof isotopic exchange reactions with deuterium gas as


D2 + 2H2O↔H2 + 2HODD

2 + H

2O ↔HD + HOD

This type of activity has been detected even in drysamples of hydrogenases2. Similar equilibria exist fortritium gas. Isotopic exchange reactions have been usedto identify different types of hydrogenases in whole cellswithout any purification54. Kinetic isotope effectsexhibited by hydrogenases provide important clues tomechanisms involved in enzymes. Reaction cycle ofenzyme depends on several steps, in which H2 atomsare transferred from site to site. Rates of these transfersshow significant kinetic isotope effects and ratesdecrease in the order of H>D>T.

Activation and Activity States

Hydrogenases activity is highly dependent on samplehistory. [NiFe] hydrogenases isolated under normalaerobic conditions do not display activity in H2 exchangeassays even after almost complete removal of O2.Whereas, same preparations exhibit some activity whenassayed for H2 evolution or H2 -uptake2. Fernandez et

al55 interpreted complex activity changes inhydrogenases (such as those observed from D. gigas) interms of interconversions between the states designatedas unready, ready and active states. Unready state isinactive and prolong treatment with a reducing agent isrequired to activate. Ready state is inactive towards H2

and is inactive in assays with electron acceptors of highredox potential (DCIP). On the other hand, ready staterequires only brief reduction.

Classification of Hydrogenases

Hydrogenases contain some essential transitionmetals. In presence of H2 and an electron acceptor,hydrogenase acts as a H2 -uptake enzyme, whereas inpresence of an electron donor of low potential, it mayuse protons from water as electron acceptor and releaseH2

56. Most of the known hydrogenases are iron-sulfurproteins with two metal atoms at their active site, eithera Ni and / or Fe atom ([NiFe]-hydrogenases57,58) or twoFe atoms ([NiFe]-hydrogenases59,60). However, adifferent type of hydrogenase exists in somemethanogens61,62, which functions as H2-formingmethylenetetrahydromethanopterin dehydrogenase(Hmd). This enzyme contains no Fe-S clusters and noNi and it was initially called as “metal free hydrogenase”.Later, it was renamed as “iron-sulfur-cluster freehydrogenase” or simply [Fe]-hydrogenase63.

[Fe]-Hydrogenases

[Fe]-hydrogenases (Hmd) extensively studied sinceits discovery in Methonothermobacter marburgensis61,is the catalyst of an intermediary step in CO2 reductionwith H2 to methane62,64, that is reduction of methyl-H4MPT+ (methylenetetrahydromethanopterin] reversiblyto methylene-H4MPT and H+ with H2. Hmd enzymediffers from [NiFe] and [FeFe]-hydrogenases by primaryand tertiary structures. Additionally, iron required forits enzymatic activity is not redox active. Thesehydrogenases have catalytic properties different from[NiFe]- and [FeFe]-hydrogenases so that they do notcatalyze 2H+ + 2e- ↔H2 reversible redox reaction.Activity of Hmd enzyme is associated with an iron-containing cofactor65-67 and crystal structure of thisapoenzyme has been established68.

[NiFe]-Hydrogenases

[NiFe]-hydrogenases constitute most numerous classof hydrogenases. Crystal structures of Desulfovibrio

hydrogenases are known57,58,69-72. Core enzyme consistsof a α, β heterodimer; α-subunit being larger one andcontains bimetallic active site, whereas small β-subunitpossesses Fe-S cluster. Subunits are in extensiveinteraction through a large contact surface, forming aglobular heterodimer56. Core, bimetallic NiFe center, islocated in α-subunit and coordinated with S-atoms of 4cysteine moieties. Also, nonproteinous ligands, one COand two CN are coordinated with Fe atom69,73,74 (Fig. 1).In some cases, ligands SO, CO and CN have beenreported for coordination58,75. In NAD-reducinghydrogenase of Ralstonia eutrapha, active site asNi(CN)Fe(CN)3CO has been reported76. β-Subunitcontains up to 3 linearly arranged [4Fe-4S] type, cubane-like Fe-S clusters. Their role seems to be conductingelectrons between H2-activating center and physiologicalredox site of hydrogenases.

In some bacteria (Desulfomicrobium baculatum77,Desulfovibrio vulgaris Hidenbrough77, etc.,hydrogenases contain [NiFeSe] core and have beencharacterized with presence of 3 [4Fe-4S] clusterswhereas in the case of standard Desulfovibrio [NiFe]-hydrogenases a [3Fe-4S] cluster with a relatively highredox potential exists in between [4Fe-4S] clustersoccupying proximal and distal positions. Some reportsare available on the role of these clusters in gas accessto active site70,79,80.


[FeFe]-Hydrogenases

[FeFe]-hydrogenases are monomeric and contain onlycatalytic subunit56. Some varieties having dimeric,trimeric and even tetrameric enzymes have also beenreported81,82. In [FeFe]-hydrogenases, active site (H-cluster] consists of a binuclear [FeFe] center bound to a[4Fe-4S] cluster by means of a bridging cysteinebelonging to protein83. Nonproteneinous ligands CN- andCO are attached to Fe atoms of binuclear Fe center81,84

(Fig. 1). Two bridging sulfur atoms originating possiblyfrom a di(thiomethyl)amine molecule coordinates withFe atoms82. Fe atom distal to [4Fe-4S] cluster possessesa vacant coordination site, which is occupied by CO, acompetitive inhibitor in CO-inhibited form of enzyme.Most hydrogenases are directly or indirectly involvedin energy metabolism. Hydrogenases that are functionalin H2 oxidation (H2-uptake/consumption) are linked toenergy conserving reactions, whereas hydrogenases thatare functional in H+ reduction (H2 production) arecoupled to disposal of excess reducing equivalentsthrough reoxidation of reduced pyridine nucleotides andelectron carriers. Another family of hydrogenases, whichare found in several autotrophic proteobacteria, appearsto act as H2-sensing component of a complex geneticrelay mechanism controlling expression of otherhydrogenases in these organisms85-87. [NiFe]-hydrogenases56 are classified as: I) Uptake [NiFe]-hydrogenases; II) Cyanobacterial uptake [NiFe]-hydrogenases and H2-sensors; III) Bidirectionalheteromultimeric cytoplasmic [NiFe]-hydrogenases; andIV) H2-evolving energy-conserving membrane-associated hydrogenases.

Soluble Hydrogenase

In R. eutropha, energy conservation from H2 ismediated by following two different [NiFe]-

hydrogenases that are synthesized coordinately: i) MBHis bound to cytoplasmic membrane; and ii) A solubleheterotetramer (SH), a member of heteromultimeric[NiFe]-hydrogenases, which resides in cytoplasm. Activesite of SH are very different from those of standard[NiFe]-hydrogenases. It was proposed that SH mighthave a (CN)NiFe(CN)3CO active site bound to 4 thiolsof 4 strictly conserved Cys residues in HoxH subunit.This would make Fe site six coordinated and thus notreactive during activity cycle of enzyme. Ni site wouldbe at least five coordinated. Since, H2 cannot readilyreact with untreated, aerobic enzyme, it was assumedthat inactive enzyme probably contains an oxygenspecies linked to sixth coordination site of Ni88.

Gas Access in Hydrogenases

Recent structural analysis of [NiFe]-hydrogenases hasshown that active site is buried within large subunit(α-unit) at approx. 30 Å from the surface. InD. fructosovorans [NiFe]-hydrogenase, a remarkablyextensive network of mainly hydrophobic cavities andchannels was found. This network connects molecularsurface to deeply buried Ni-Fe core70. A very similarchannel structure has been reported in D. gigas [NiFe]-hydrogenase80. Based on crystal structure analyses ofD.vulgaris and D. desulfuricans, it has been establishedthat their [NiFe]-hydrogenase also have similar channels.Also, most channels are conserved in crystal structureof D. bacalatum [NiFeSe]- hydrogenase77.

Iron-sulfur (Fe-S) Clusters

Fe-S clusters exist in hydrogenases and also appearas cofactor in various other enzymes. Several simplerFe-S clusters spontaneously assemble into apo form ofFe-S proteins in reductive aqueous solution with ferrousiron and sulfide89. Structurally, basic building block ofFe-S clusters is a Fe ion tetrahedrally coordinated by 4S ligands (Fig. 1). Simplest cluster is that of one, presentin rubredoxin, in which Fe atom is coordinated by 4Cys thiol groups (Fig. 1). [2Fe-2S] clusters possess twoinorganic sulfide ligands and 4 Cys thiols. Fe-S clustersof simple types are mostly found in electron-transferproteins such as ferrodoxins or as part of an internalelectron-transfer pathway in larger enzymes. All [NiFe]-hydrogenases contain a [4Fe-4S] cluster within 10 Å ofactive NiFe site.

Small unit of D.gigas [NiFe]-hydrogenase contains 3Fe-S clusters (2 [4Fe-4S] clusters and 1 [3Fe-4S] cluster)and oriented in an almost linear alignment from active

CysS

Cys

CONi

S

Fe

S

CN

CN

X

Cys

SCys65

530

68

533

N

H

O

COFe

S

Fe

S

CNC

SCys

CN

[4Fe 4S]

CO

Fig. 1—Schematic structure57 of active site in [NiFe]- and [FeFe]-hydrogenases [X: O-2, OH-, OH

2, SO, in the reduced

form X: H-]


site to the surface of protein having an average cluster-to- cluster distance of 12 Å57. Rapid electron transferover such distances from one center to another, withinproteins, occurs90 and this is partly described as quantummechanical tunneling, which depends on the overlap ofwave-functions for two centers. In [NiFe]-hydrogenases,[4Fe-4S] cluster closest to active site (~10 Å from Ni) iscalled proximal cluster. [3Fe-4S] cluster is the cluster tosurface of the molecule and occurs between proximaland distal cluster. Proximal [4Fe-4S] cluster is involvedin direct electron exchange mechanism with active sitewhereas distal [4Fe-4S] cluster is believed to mediate,through its histidine ligand, electronic exchanges betweenhydrogenase and a redox partner. However, involvementof medial [3Fe-4S] cluster is a matter of debate becauseof its high redox potential, which is about 300 mV morepositive than distal and proximal [4Fe-4S]. By the helpof genetic engineering, Pro residue (common in most[3Fe-4S] proteins] of D. fructosovorans) was replacedby Cys residue. Concomitantly, [3Fe-4S] cluster wasconverted to [4Fe-4S] form91. Modified enzyme wasfound to be more sensitive to oxygen but did not showany increase in electron transfer rate.

Proton Transfer

During heterolytic cleavage of H2 molecule at activesite, a hydride and a proton are formed in the first step92,93.Then, two electrons of hydride ion are removed in secondstep to form a second proton. Since active site is deeplyburied, like electrons also the protons formed have tobe conveyed over a distance of about 15 Å to proteinenvironment. Reverse of these steps is required for H2

production. Experimental evidences exist for the crucialrole of terminal Ni-bound cycteine ligand as the firstacceptor site in process of proton transfer afterheterolytic cleavage of H2

94,70,72,95-98. Proton transfer inD. gigas is reported57,99. Role of Mg atom in protontransfer for [NiFe]-hydrogenase from D. vulgaris wasalso proposed58. There are several additional routesproposed 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 buffercontain two unpaired electrons, one is located on [3Fe-4S] cluster and other in active site. Oxidized proximaland distal clusters are diamagnetic. By means of EPRtechniques, it has been established that unpaired spinlocated in active site is close to nickel atom and at least

one of its sulfur ligands. EPR spectra of aerobic [NiFe]-hydrogenases are very similar, showing that nickel siteis structurally conserved. Enzyme preparations oftencontain two types of inactive enzyme molecules. WhenO2 is removed and H2 is provided, then one type ofenzyme molecules shows full activity within minutes(ready type enzyme]. The other type remains inactivefor prolong periods of time (unready enzyme]. Reducedenzyme experimentally reoxidized by O2 enriched with17O isotope (which has a magnetic nucleus) showed thatan oxygen species ended up close to Ni-based unpairedspin in the ready as well as in unready state100. It couldbe removed only by full reduction and activation ofenzyme.

EPR studies have revealed that Ni sites in ready andunready enzyme are slightly different. FTIR spectraindicate that Fe sites in Nir* and Niu* (ready and unreadystates, respectively) are very similar. Active siteundergoes reduction by accepting an electron. Uponreduction of unready enzyme, electron/protoncombination probably takes place at nickel site accordingto,

Ni(III)Fe(II) + e- +H+ →(H+)Ni(II)Fe(II)

A shift of CN/CO bands to higher frequencies occursin infrared spectrum of Niu-S state. Whereas respectivespectrum for Nir-S state has bands all shifted toconsiderably lower frequencies, indicating a greatlyincreased 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 alarge shift (50-100 cm-1) to lower frequencies of CN/CO bands; protonation of a thiolate ligand would reverseit largely. In model compounds102, protonation of thiolateligand to Fe can increase stretching frequency of CObound to Fe, by 40 cm-1. Usually reduction of Fe(II) isanticipated, however it occurs only at considerably lowerpotentials. Reduction of low-spin Fe (II), which isnonmagnetic, would create an unpaired spin. Its spinmagnetic moment might couple to Ni-based spin,consequently cancel total magnetism and no EPR signalcould be seen. Added electron and proton both go to Nias in the case of Nix*, but then charge density on Fe alsoincreases due to a better electronic contact between twometal ions in ready state.


For A.vinosum hydrogenase, activation requires notonly reduction but also temperature needs to be elevated.When enzyme in Nir* state is reduced at highertemperatures at about 30°C or higher (but not at 2°C), arapid increase in activity is observed and a third EPRsignal of a Ni –based unpaired electron emerges (Nia-C*), which is intermediately reduced state. Nia-S is one–electron reduced state of Ni-Fe site in active enzymeand Nia-SR stands for the most reduced state.

Nia-S ↔Ni

a-C* Conversion

In D.gigas hydrogenase, Nia-S ↔ Ni

a-C* reversible

reaction occurs and pH dependence of potentialaccompanied indicates that one electron and one or twoprotons are involved in equilibriu103. Nia-C* statepossesses a trivalent nickel104. Hence, Nia-S→Nia-C*reaction can be written as105

Ni(II)Fe(II) + e- + H+ → Ni(III)Fe(II)(H2)

Nia-S→Nia-C* reaction is also H2 driven alone, butthen reverse reaction is extremely slow. To explainreaction with H2 in absence of mediators, involvementof a Fe-S cluster has been proposed.

Nia-C* → Ni

a-SR Conversion

In presence of redox mediators, Nia-C* state canbe further reduced to an EPR-silent state (by increase ofH2 partial pressure) in a reaction requiring one electronand one proton. Based on assumption that trivalent Niexists in Nia-C* state, probable reaction can beformulated 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 sitein Nia-SR state has one electron more than that in Nia-C* state, a Fe-S cluster has to be involved in the reactionwith H2.

Ni(III)Fe(II)[4Fe-4S] + H2 → Ni(II)Fe(II) [4Fe-4S] + 2H+

In this process, only proximal and distal clusters canbe involved. When these experiments are performedunder equilibrium conditions, no change of redox statesof Fe-S clusters can be observed. A possible explanationis to assume that individual enzyme molecules can

exchange electrons. The best suitable site for this is viadistal [4Fe-4S] cluster. It is located on protein surface.Such an exchange would occur on one-electron basishaving a slower rate than reaction with H2, which isextremely fast and depends on the rate of diffusion ofH2 into enzyme106. Supposing Nia-C* state is initiallyformed 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 electronresulting 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 wouldreduce its two clusters again. Final level of reduction ofFe-S clusters would then only depend on effective redoxpotential in the system. An interesting enzyme is H2-sensor protein. H2-sensor from Ralstonia eutropa isreported107. Its active site is highly similar to the one instandard [NiFe]-hydrogenases. However, its enzymaticproperties are quite different. Sensor enzyme is lessactive and is always, even in aerobic solution, in activeNia-S state. It is also insensitive to O2 and CO and canbe reduced with H2 to Nia-C* state but not further.

Genetic AspectsBiosynthesis of [NiFe]-hydrogenases

Genes in Proteobacteria that encode H2-uptakehydrogenases are clustered. These clusters comprisestructural genes (labeled as L for large subunit and S forsmall subunit) and accessory genes for maturation andinsertion of metal atoms and ligands (Ni, Fe, CO, andCN-) at active site of heterodimer. However, in somemicroorganisms, hydrogenase gene cluster alsocomprises regulatory genes that control expression ofstructural genes. Maturation of hydrogenase occurs viaa 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 synthesisand incorporation of metal center into large subunit, andalso control insertion of correct metal, maintain a foldingstate of protein for metal addition, and allow necessaryconformational changes of protein. Gene/ proteindesignations used for homologous proteins in various


microorganisms are reported108,109. Carbamoylphosphatehas been shown to be educt for synthesis of CN ligandsof NiFe metal center110-112, which requires activity oftwo hydrogenase maturation proteins that is HypF, acarbamoyltransferase, and HypE, which receivescarbamoyl moiety to its COOH-terminal cysteine to forman enzyme-thiocarbamate. HypE dehydratesS-carbamoyl moiety to yield enzyme thiocyanate, whichthen can donate CN moiety to iron113,114. HypE and HypFform a dynamic complex with HypC and HypD. CN istransferred to HypC-HypD and then attached to Fe atomof NiFe site115. It has been proposed that conservedcysteine residues in HypD protein play a role inmaturation process116.

Biosynthetic route for CO to NiFe active site isdifferent from that for cyanide117. Products of hupGHIJ

operon have been shown to be involved in maturationof HupS hydrogenase subunit of Rhizobium

leguminosarum uptake hydrogenase118. Transcriptionalcontrol involves usually one or several two-componentregulatory systems. In response to a specific signal, firstcomponent, a sensor histidine kinase,autophosphorylates at a conserved histidine residue andthen transphosphorylates cognate response regulatortranscription factor at a conserved aspartate residue thatactivates or represses gene expression whenphosphorylated 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). H2-specific regulatory systemcomprises a H2-sensing regulatory hydrogenase(HupUV/Hox- BC) and a two-component signaltransduction system, histidine protein kinase HupT/HoxJ,and response regulator HupR/HoxA. This complexsystem has been particularly well studied in R. capsulatus

and R. Eutropha87,121-125. In all of these bacteria,regulatory cascade responding to H2 occurs by detectionof H2 signal by H2-sensor (HupUV/HoxBC) and it istransmitted to histidine kinase (HupT/HoxJ); it istransduced by phosphotransfer between histidine kinaseand response regulator (HupR/HoxA) and integrated atpromoter of structural genes of hydrogenase by responseregulator. However, in the absence of H2-sensor,hydrogenase synthesis is derepressed, inR. capsulatus86,123,126, but in B. japonicum, R. eutropha,and R. Palustris127-129, there is no synthesis ofmembrane-bound uptake hydrogenase. In T.

roseopersicina, components of H2-regulatory system

(HupUV, HupT, and HupR) are present, but expressionof structural hupSL hydrogenase genes is not affectedby the presence or absence of H2

130.

Biosynthesis of [FeFe]-Hydrogenases

Accessory genes necessary for biosynthesis of[FeFe]-hydrogenases have been identified. Two novelradical S-adenosylmethionine (SAM) proteins wererequired for assembly of active site of C. reinhardtii

hydrogenases131. Random insertional mutants having theirhydEF gene inactivated were incapable of assemblingan active [FeFe]-hydrogenase. In C. reinhardtii genome,hydEF gene is adjacent to another hydrogenase-relatedgene, hydG. Their radical-SAM domains containconserved motif Cx3- Cx2C, also additional motifs in C-terminal ends that are characteristic of [Fe-S] cluster-binding sites132. Radical SAM proteins generate a radicalspecies by reductive cleavage of S-adenosylmethioninethrough a [Fe-S] center to catalyze reactions involved incofactor biosynthesis, metabolism, and synthesis ofdeoxyribonucleotides133. HydF maturation proteincontains at its N-terminal end conserved GTP-bindingmotifs. Anaerobically reconstituted HydE and HydGproteins from Thermotoga maritima are able to cleaveSAM reductively when exposed to reduction bydithionite, confirming that they are radical SAMenzymes134 and HydF from T. maritima is a GTPase withan Fe-S cluster135. On the other hand, anaerobiccoexpression of C. reinhardtii hydEF, hydG, and hydA1

genes in E. coli resulted in formation of an active HydA1enzyme131. [Fe-Fe]-hydrogenases with high specificactivities was obtained in Clostridium acetobutylicum

by homologous and heterologous over expression ofhydA gene from C. acetobutylicum, C. reinhardtii, andS. obliquus, respectively136. Because C. Acetobutylicum

hydE, hydF, and hydG clones are more stable in E. coli

than their C. reinhardtii homologues, an efficientbiosynthetic system has been developed in E. coli byexpression cloning of hydE, hydF, and hydG fromC. acetobutylicum. An active [FeFe]-hydrogenase wasobtained with fully functional maturation proteins and N-terminally deleted C. acetobutylicum HydA andC. pasteurianum HydA, that is, with catalytic H-cluster-containing domain only137. In accordance with the roleof radical SAM enzymes involved in production of active[FeFe]- hydrogenases, a mechanistic scheme has beenpresented for hydrogenase H-cluster biosynthesis, inwhich both CO and cyanide ligands can be derived fromdecomposition of a glycine radical138 .


Molecular and Genetic Aspects of Technology of

Biohydrogen Production

Numerous microorganisms that can produce H2 byreactions linked to their energy metabolism use protonsfrom H2O as electron acceptors to dispose of excessreducing power in cell and to reoxidize their coenzymesin the absence of oxygen139. BHP processes haveadvantage of generating H2 not only from a variety ofrenewable substrates, but also from organic wastestreams140,141. Among various bioprocesses of H2

production, photo fermentation is favored due to highersubstrate-to- H2 yields and, its ability to trap energy undera wide range of light spectrum and versatility in sourcesof metabolic substrates with promise for wastestabilization142. In addition, the process can potentiallybe driven by solar energy with minimal non-renewableenergy inputs. Economic feasibility of photofermentative H2 production systems can be furtherimproved by utilizing low cost substrates or wastestreams and, by collecting and recycling useful by-products other than H2

143.Photosynthetic bacteria produce H2 under anaerobic

conditions, in the absence of nitrogen gas, withillumination and with stressful concentrations of nitrogensources. Photo heterotrophic bacteria, such asRhodobacter sphaeroides, can grow anaerobically toproduce H2 either from reduced substrates such asorganic acids [purple non-sulfur (PNS) bacteria] or fromreduced S compounds (green and purple sulfur bacteria).These bacteria use enzyme nitrogenase to catalyzenitrogen fixation for reduction of molecular nitrogen toammonia. Nitrogenase can evolve H2 simultaneouslywith nitrogen reduction. Stressful concentrations ofnitrogen are therefore required for H2 evolution144.

Conversion efficiency of light energy to H2 inpresence of an appropriate substrate and optimum cellgrowth conditions is a key factor for economic photofermentative biohydrogen production143. Main hurdle,however, is requirements for large expose area due tolow light efficiency of the process. Design guidelinesfor photobioreactors for efficient utilization of light arestill lacking145. Since growth rate of bacteria is a functionof light intensity and substrate concentration, kineticmodels relating the three can be of value in designingprocess and in identifying underlying rate-determiningand significant factors. Most photo fermentativebiohydrogen studies have used malic acid as substrateand R. sphaeroides O.U.001 as microorganisms, underoptimum carbon-to-nitrogen (C/N) ratio146-148 in batch

reactors. Koku et al147 studied growth characteristics ofPNS bacteria and Eroglu et al149 studied dependence oftheir growth rate on substrate, while their dependenceon light intensity has been studied by Sasikala et al150.However, little information has been reported on kineticmodels integrating growth of PNS bacteria with lightutilization and H2 production1,148.

A kinetic model144, developed for photo fermentativebiohydrogen production to predict dynamics of theprocess, contains 17 parameters [5 cell growthparameters (CXm, KS, KI, KXI, KXi), 5 product formationparameters (CPm, KPS, KPi, Kpl, KPI), values of yieldcoefficients for H2 formation (YPX), and malateconsumption (YP, YXS), maximum specific growth rate(µm), specific malate consumption rate (µSX), specificproduct 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 resultswere used to calibrate and validate model with malicacid as a model substrate, using Rhodobacter

sphaeroides as a model biomass. Temporal H2 evolutionand cell growth predicted by proposed model agreed wellwith experimentally measured data obtained frompublished reports, with statistically significantcorrelation coefficients exceeding 0.9. Based onsensitivity analysis performed with validated model, only6 of 17 parameters were found to be significant. Modelsimulations indicated that the range of optimal lightintensity 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 photoheterotrophicconditions. In R. sphaeroides, several metabolicpathways take role in H2 production and consumption.Total H2 production is limited due to several metabolicevents occurring in cells such as production of poly-3-hydroxybutyrate (PHB) or consumption of H2 byhydrogenase uptake. Membrane-bound uptakehydrogenase decreases H2 production efficiency bycatalyzing conversion of molecular H2 to electrons andprotons151. Inactivation of uptake hydrogenase hasresulted in total increase in H2 production152-154. Kars et

al155 worked on manipulation of purple non-S bacteriumR. sphaeroides O.U.001 such that uptake hydrogenasewas inactivated. Yield and rate of H2 production, andsubstrate conversion efficiency (SCE) improved inmodified hup-R. sphaeroides O.U.001. Measuringabsorbance at 660 nm at certain time intervals monitored


growth of mutant and wild type R. sphaeroides O.U.001.Wild type cells reached relatively higher absorbancevalues (OD660 = 1.90 ± 0.05) compared to hup-mutantstrain (OD660 = 1.71± 0.06). There was no considerabledifference in pH values (7.3-7.8) of mutant and wild typecells. Significantly higher (20%) H2 accumulated in hup-mutant R. sphaeroides O.U.001 when compared to wildtype cells (m = 2.85 l H2/l culture, w = 2.36 l H2/l culture)under nitrogenase repressed conditions in 60 mlbioreactors. According to gas chromatography (GC)analysis, H2 constituted 96-99% (v/v) of overall gas.Average gas production rates of mutant cells (9.2 ± 0:4m/l/h) and wild type cells (6.9 ± 0.5 ml/l/h) werecalculated by dividing total volume of gas produced byvolume of culture and by duration of gas production.SCE, another parameter for comparative analysis of H2

production, is calculated as ratio of actual amount ofproduced H2 to theoretical amount. SCE of mutant cellswas 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 uptakehydrogenase significantly increased total H2 productionin hup-mutant cells and it did not affect bacterial growth.High SCE demonstrated that more energy and reducingequivalents were directed towards nitrogenase enzymeand therefore more H2 accumulation was achieved.Hence, results are promising for genetic engineering ofR. Sphaeroides towards enhanced H2 productioncapacity155.

Halobacterium salinarum belongs to halophilicarchaea. Purple membrane (PM) of H. salinarum

contains a retinal transmembrane proteinbacteriorhodopsin (BR), which acts as a light-drivenproton pump (light energy transducing system). Protongradient generated is utilized for ATP synthesis bymembrane bound H+ATPase. Studies are required toelucidate exact mechanism of proton translocationthrough BR156,157. H. salinarum lacks both hydrogenaseand nitrogenase or any other system that can reduceprotons into molecular H2. Therefore, packed cells (PC)of H. salinarum or its PM might be combined withanother system for H2 production158. Zabut et al159

introduced photobiological H2 production by combinedsystem of R. sphaeroides O.U.001 and H. salinarum S9in a column photobioreactor for improvement ofbiological H2 production. Photo activities of both PCand PM fragments of H. salinarum, measured in H2

production medium at 32°C employing two light/darkcycles, indicated159 that ∆pHmax of light period was 0.08

and that of dark period was 0.10. ∆pHmax values obtainedby PC were higher than those of PM fragments. Stableand reproducible light/dark responses were obtained forboth PC and PM fragments of H. salinarum in H2

production medium. BR has low photo activity under lowionic strength and at temperatures more than 30°C160.H2 production experiments were conducted underspecified conditions159 and experimental results werecarried 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 restwas CO2 in all of experiments. Persistence of pH valuesmore than 8.0 in culture might limit nitrogenaseproductivity and activate uptake hydrogenase, whichpreferred slightly alkaline conditions, leading to less H2

evolution161. High initial cell concentrations might causefast bacterial growth that might enhance H2 production;however, high cell density prevented light penetrationthrough culture 162.

Presence of PC did not significantly affect total gasproduction and H2 production rate compared to theresults obtained with R. sphaeroides when there was nostirring. Discontinuous stirring of the system for10 h/day with a magnetic stirrer operated at 300 rpmcreated unstable H2 production in the reactors. Stirringenhanced formation and removal of gas bubbles. Stirringhas been reported to increase conversion efficiency oflactate to H2 by Rhodopseudomonas sp. and byR. Sphaeroides B6 and by R. sphaeroides B5163.Cultures of R. Sphaeroides combined with differentconcentrations of BR in PC of H. salinarum undercontinuous stirring conditions showed that total gasproduction was increased from 690 to 1500 ml withaddition of 50 nmol of BR, and H2 production rate wasincreased from 11 to 27 ml H2/h/l of culture. Culturescombined with suspended PC containing 50 nmol of BRgave best results among others in terms of amount oftotal gas production and the rates of H2 production undercontinuous stirring conditions. However, an increase ofPC in order to increase amount of BR in the system above50 nmol had an inverse effect on total gas productionand rate of H2 production. Total gas production decreasedfrom 1500 to 850 ml and H2 production rate decreasedfrom 27 to 17 ml H2/h/l of culture by increasing BRamount from 50 nmol to 150 nmol, attributed to viscosityincrease 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 toadditional protons coming from light induced proton


pumping of BR. The provided protons were readily usedby nitrogenase of R. sphaeroides O.U.001 under limitingconditions of nitrogen. Total H2 gas production increased2.5 times and rate of H2 production enhanced three-fold,compared to R. sphaeroides only culture within sameexperimental period. BR on H2 production has positiveeffect on different systems164-168. PC was not vital butBR in native membrane in its original environment wasstill 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 growthmedium of H. salinarum. Experimental results withR. sphaeroides and combined systems ofR. Sphaeroides with PM fragments of H. Salinarum

indicated that addition of BR as PM fragments had nosignificant effect on H2 production.

BR was found more effective on H2 production, whenR. sphaeroides was combined with PC rather thanR. sphaeroides combined with PM fragments, especiallywhen this comparison was made between combinedsystems containing same amount of BR. On an average,R. Sphaeroides culture combined with PC ofH. salinarum containing 50 nmol of BR produced 1500ml H2 with a rate of 0.027 l/l/h, whereas R. sphaeroides

culture combined with PM fragments containing sameamount of BR produced 650 ml H2 with a rate of 0.014l/l/h culture. This difference could be attributed to higherphoto activity of BR exhibited as in the form of PC,compared to photo activity of BR as in the form of PMfragments in Medium II of the study. Medium II (whichwas H2 production medium of R. sphaeroides) had lowsalt concentration. However, photo activity of BR ishighly affected by salt concentration167,169. Low ionicstrength decreases proton-pumping rate of BR. On theother hand, two combined systems should be comparedbased on orientation of BR, presence of other cellenclosures (in case of PC) and effect of additionalpurification steps (in case of PM fragments) on photoactivity of BR. Overall, combining of R. sphaeroides

with PC of H. salinarum was more efficient forphotobiological H2 production. Thus, in combinedcultures, continuous stirring, consistency in pH values,and moderate bacterial density played important rolesin increasing amount of total gas production and ratesof H2 production. It was found that using packed cellsof H. salinarum was better than using PM fragments inthe combined systems. Since a high ionic strengthpromotes photo activity of BR, salt tolerant strains ofR. sphaeroides are recommended for future work in

combined systems. Immobilized combined systems aresuggested for future continuous H2 production. Outdoorand large-scale systems are indicated for furtherinvestigation159.

Most of phototrophic biohydrogen studies wereconducted for pure cultures of 4 PNS (Rhodobacter

sphaeroides170, Rhodopseudomonas capsulatus171,R. palustris143 and Rhodospirillum rubrum172) usingorganic substrate as carbon source. Another PNS,Rubrivivax gelatinosus, can also produce H2 but mainlyusing CO as carbon source. Li & Fang173 studied H2

production characteristics of a new strain ofR. gelatinosus, which was isolated from local reservoirsediment, using various organic substrates. Thesecharacteristics were then correlated with activity of itsnitrogenase, which is responsible to photoheterotrophicH2 production174, and accumulation of PHB, which maycompete with H2 for electrons175. Results of batch testsusing individual organic substrates showed thatR. gelatinosus L31 was able to produce H2 fromglucose, sucrose, starch, lactate and malate, however, itwas unable to produce H2 from acetate, propionate,butyrate, succinate and glutamate. H2 conversionefficiency is defined as the ratio between actual H2

production and stoichiometric value as

CaHbOc + (2a-c)H2O aCO2 + (2a-c+0.5b)H2

Maximum specific H2 production rates ofR. gelatinosus L31 (193-829 ml/g/h) are higher thanthose of other phototrophs using same substrate, exceptreported value of 670 ml/g/h by R. palustris P4 usingglucose176. Conversion efficiency (50.5%) for lactate ishigher173 than most reported data (12.4-26.1%) andcomparable to 52.7% by R. capsulatus JP91177.Conversion efficiencies of malate, glucose, sucrose, andstarch are all comparable to reported values.

Starch has been rarely used for phototrophic H2

production. Ike et al178 has found that althoughR. Marinum A-501 could produce H2 from glucose andsucrose, but could not produce from starch. ForR. gelatinosus173, H2 was produced from starch at amaximum rate of 12.1 ml/l/h, which is higher than 7.8-11.3 ml/l/h produced from starch sources such ascassava, rice, and corn179.

Conversion efficiencies of R. gelatinosus L31 were50.5% for lactate and 24.6% for malate, both of whichwere substantially higher than 7.4-8.8% for threecarbohydrates. R. Gelatinosus L31 could not produceH2 from acetate, propionate, butyrate, and succinate, even


though these organic acids could produce H2 by otherspecies (R. sphaeroides, R. capsulatus,Rhodopseudomonas sp., and R. palustris R1). no studyis available on any phototroph capable of producing H2

from glutamate173.Dark fermentation or acidogenic fermentation of

carbohydrates presents several advantages over photofermentation such as a production rate is higher thanthose obtained with photobiological processes andcapacity of being run all over the day even during night1.Main species40 identified for biological H2 productionduring acidogenesis of carbohydrates are Enterobacter,Bacillus and Clostridium. Their metabolism can berepresented by biochemical pathways180. Thus,fermentation pathways that produce acetate and butyrateare mainly responsible for H2 production40,181. On theother hand, pathways that produce ethanol, lactate andpropionate are unable to produce H2, because theyconsume hydrogenated biochemical intermediates likeNADH. Reactions involved in acidogenic fermentationassociated to H2 are mostly presented by assuming abiomass product equivalent to C4H7O2N as reported182.H2 is produced as by product during dark fermentationof glucose and/or sucrose by bacteria for energyproduction to grow. Organic acids (VFA) and alcoholsare also formed as by products or intermediates, whichinhibit fermentation by a complex metabolic pathway183.In dark fermentation, 4 moles of H2 can be produced byfermentation of glucose with an acetic acid as an endproduct5,40. This also results in a net production of 4 molof ATP184. However, average yields of H2 using glucosein mesophilic temperature range are always less than4 mol-H2/mol-glucose and vary widely185-187. Productionof H2 drops to 2 moles theoretically during production ofbutyrate5,40 as

C6H12O6 → CH3CH2CH2COOH + 2CO2 + 2H2

There are no known fermentation pathways that canachieve conversion efficiency greater than 4 mol H2/molhexose (C6H12O6) in dark fermentation188 and as seen inequations below, propionic acid formation has nocontribution to H2 production:

Homofermentative pathway,C

6H

12O

6 → 2CH

3CHOHCOOH

Heterofermentative pathway,

C6H12O6 →CH3CHOHCOOH + CH3CH2OH + CO2

In addition, formation of propionic acid consumes H2,therefore it should also be avoided for efficient H2

production:

C6H12O6 + 2H2 →2CH3CH2COOH + 2H2O

So high H2 yields depend on acetic acid and butyricacid production, and in contrast to acetic acid and butyricacid, propionic acid and ethanol are considered asunfavorable end products41,181,189.

C6H

12O

6 →CH

3CH

2CH

2COOH + 2H

2+ 2CO

2

Increased concentrations of VFA and alcohols mayinhibit further production of H2

190. During fermentation,ethanol consumes more electrons from metabolicreducing power, therefore, it is not desired for H2

production191. H2 production and fermentation productsin the liquid are related such as during batch andcontinuous H2 production from simulated cheeseprocessing wastewater via anaerobic fermentation frommixed microbial communities under mesophilicconditions, higher H2 yield in biogas was observed whenconcentration of ethanol, hexanoic acid, n-butyric acidin solution is high. On the other hand, propionic acidconcentration was low192. Since butyric acid and aceticacid are crucial end products for H2 production, ratio ofbutyric acid to acetic acid (2.9-4.3) is used as aperformance parameter for dark H2 fermentation studiesusing carbohydrates6,193-197.

H2 production is also effected by pH of reactionenvironment. Variations in acetate/butyrate ratio arecaused by metabolic alterations due to changes of pH198.During H2, production fermentation pathway may shiftfrom VFA producing to alcohol producing when pH wasdecreased to 4.5 or below198,199. On the other hand, insome batch experiments, only a small amount ofmethanol was seen in an acidic environment. IncreasingpH also did not show a significant effect on VFA andalcohol concentrations. However, acetate /butyrate ratioincreased from 0.41 to 1.15, when pH was changed from4.5 to 7.0. This suggests a shift of fermentation pathwayby pH changes200. pH (5.5-6.0) was considered to be idealto avoid both methanogenesis and solventogenesis186 andcould be considered optimum pH range for effective H2

generation. Reactor must be operated at a pH of 6.0 tofacilitate proliferation of acidogenic bacteria for H2

production. Optimum pH for growth of MB


(methanogenic bacteria) was between 6.0 and 7.5, whileAB (acidogenic bacteria) functions well below pH6187,191. Maintenance of pH around 6 resulted in higherproduction of H2 compared to near neutral pH190,201-203

and inhibition of methanogenic group of bacteria foreffective H2 yield. If pH was not maintained in desiredrange, it could inhibit H2 production or cause a microbialpopulation shift, resulting in cessation of H2

production190,204. Alkalinity (buffering capacity) isconsidered as one of the most important factors that isgoverned by VFA production and accumulation.Normally, system alkalinity acts as a buffer to sustainanaerobic performance in presence of VFA production205.

H2 production by chemical oxygen demand (COD)removal (mol H2 produced/kg COD removed) changeswith ethanol/acetate ratio in H2 bio-producing reactorsystem (HBR). This phenomenon could be related tocomplex fermentation mechanism and oxidization/reduction of nicotinamide adenine dinucleotide (NADH,Eo’

NADH= -320mV). Only pathway in this fermentationmechanism that can produce H2 is pyruvatedecarboxylation by ferredoxin and butyric acid, aceticacid and ethanol are the end products. Pathways arecontrolled by NAD+ (oxidation status)/(NADH+ H+)(reduction status), existing at certain ratio inmicroorganisms206. Dynamic equilibrium of oxidation /reduction of NAD+/NADH + H+, which could beachieved by mole ratio of ethanol to acetic acid (1:1)during acetic acid fermentation, plays a major role inH2 bioproduction. Lower or higher mole ratio of ethanolto acetic acid than 1:1 destabilizes fermentation leadingto inhibition of H2 production207.

According to Ruzicka model208, main limitation forproducing highest yields of H2 is inhibitory effect of H2

partial pressure. Increase in dissolved H2 concentrationlimits transfer of electrons from glucose to H2. NADHis electron carrier that is involved in transfer of electronsfrom pyruvate to H2 (E

o‘= -414 mV)188. Since NADHhas a higher potential than H2 (NADH, Eo’

NADH

= -320mV), dehydrogenation of triose phosphate toproduce 2 mol of H2 can occur only when concentrationof H2 is less than ~6×10-4 atm via oxidation of pyruvateand ferredoxin can generate another 2 mol of H2 at higherH2 concentrations up to ~0.3 atm209. Thus, in order toobtain H2 yields higher than 2 mol-H2/mol glucose,production of H2 via triose phosphate dehydrogenationand NADH must be achieved. However, when all NAD+

(oxidized form) is in reduced form (NADH) because itis unfavourable to transfer electrons from NADH to H2,

flux of glucose through glycolytic pathway and throughphosphoroclastic reaction stops. In order to increase thisglucose flux for maximum ATP production, some bacteria(C pasteurianum) divert electrons in NADH to butyrateproduction, resulting in decrease in H2 yields andproduction of 3 mol of ATP. Production of butyrate ratherthan acetate allows for NAD+ regeneration, a greaterflux of glucose through bacterial glycolytic pathway, anda greater overall ATP production rate than what acetateproduction alone could sustain.

Energy production with H2 generation is highest whenacetate is produced (4 mol-ATP/mol glucose), but whenH2 concentrations are high, NAD+ can only beregenerated if compounds other than acetate (butyrateor butanol) are produced. ATP generation is 3 mol-ATP/mol-glucose when butyrate is produced. Crabbenbaumet al210 found that average ATP yield of 3.277 ± 0.02mol-ATP/mol-glucose, which corresponds to a H2 yieldof 2.7 mol-H2/mol-glucose. Thauer et al188 concludedthat for a HAc: HBu ratio of 0.86 (0.6 mol of acetateand 0.7 mol of butyrate from 1.0 mol glucose), H2 yieldwould be 2.6 mol-H2/mol-glucose at a thermodynamicefficiency of 85% for biohydrogen production fromglucose. These values for H2 yield are consistent withresults obtained for average HAc:HBu ratio of0.86 ± 0.14.

Bacterial Communities

H2 producing bacteria may be classified in fourgroups5,200 (strictly anaerobes, facultative anaerobes,aerobes and photosynthetic bacteria). Strictly anaerobicClostridium was found most abundant in acidophilic H2

producing sludge in biohydrogen production from riceslurry. In addition, Clostridia sp. are mainly responsiblefor fermenting sugars to H2 at high yields211 producingacetate, butyrate, and other fermentation end productsas waste products212. Many Clostridium sp., capable ofproducing H2, include C. acetobutylicum213,C. butylicum185, C. butyricum214, C. kluyveri215 andC. pasteurianum216, some of which are knownacidophilic species. C. acetobutylicum can grow at pH4.3217 and C. butyricum at pH 4218. Two anaerobic acid-tolerant bacteria, C. akagii CK58T and C. acidisoli

CK74T, have been isolated from acidic beech litter andacidic peat-bog soil, respectively219. Growth of C. akagii

CK58T (pH 3.7-7.1) and C. acidisoli CK74T (pH 3.6-6.9) on glucose yielded H2, butyrate, lactate, acetate,formate, and CO2. An acidophilic Enterobacter

aerogenes strain HO-39, capable of producing H2 at


pH 4.0, has also been isolated220. Clostridium

pasteurianium, C. butyricum, C. beijerinkii ,

E. aerogenes produce high amount of H240,142,221.

C. pasteurianum is an acid producer and produces H2

along with acetate and butyrate189. C. butyricum can beused as a starch fermenting bacteria for darkfermentative H2 production by direct starch utilization222-

224. Lactobacillus sp. are known to be predominantmicroorganisms in batch type bioreactor at high H2 yieldsduring biohydrogen production from cheese processingwaste water192. For larger scale H2 production, mixturesof microbial cultures are found cost effective. A mixedculture of C. butyricum and E. aerogenes gave H2 yieldof 2.0-2.7 mol H2/mol glucose from single-stage H2

production with sweet potato starch222-224. Severalpowerful H2-producing bacterial isolates (Clostridial sp.)from municipal sewage are also capable of producingH2 from sugar very efficiently225.

Technological Improvements in Biohydrogen Production

H2 production from wastewater has a great potentialfor economical H2 production with high yields226. Formaximizing H2 production in fermentation systems, lossof H2 to H2-consuming anaerobes, such asmethanogens227, can be avoided by heat-treatinginoculum to select for spore-formers, such as Clostridia,for glucose-fed reactors or even heat-treating wastewaterto kill methanogens141,227-229. Low pH can also be used tominimize growth of methanogens230. Longer hydraulicretention times (HRT) and lower COD levels contributeto greater overall efficiency of H2 production incontinuously fed reactors201,229. Other factors (inoculum,substrate, temperature, nitrogen sparging, and initial startup) 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%) willremain in solution (COD basis). Typical H2 yields (1-2mol/mol) result in 80-90% of initial COD remaining inwastewater as volatile organic acids and solvents (acetic,propionic, butyric acids and ethanol). One way to recoverremaining organic matter in a useable form for energyproduction is to produce methane. Two-stage processesare already well developed, and could be adapted forboth H2 and methane production, although thesecombined gas processes have not yet been demonstratedat full scale232. Feasibility of integrating acidogenicprocess of H2 generation with anaerobic/methanogenicprocess of methane production to utilize residual organiccomposition in wastewater was also studied233. Following

equations were used for computing methanogenicfermentation balance consuming H2 and VFA generatedfrom primary acidogenic process:

CH3COOH →2H2 + CO2

CH3CH2CH2COOH + 2H2O ↔CH3COOH + 2H2

CH3CH2CH2CH2COOH + 2H2O ↔2CH3 CH2COOH + CH3COOH + 2H2

4H2 + CO

2 →CH

4 + 2H

2O

Experimental data supported efficacy of integratingacidogenic H2 production process with anaerobicmethanogenic process in enhancing substratedegradation efficiency along with both H2 and CH4

generation as renewable by-products. Integration ofacidogenic and methanogenic processes appeared to bea feasible option for sustainable H2 production utilizingwastewater as substrate234-236. Fluidized-bed reactor(FBR) and packed-bed reactor (PBR) were developedto produce H2 and ethanol simultaneously from darkfermentation of carbohydrate substrates usingpolyethylene-octane elastomer immobilized anaerobicsludge as biocatalyst. Production of two biofuels seemedto have substrate preference. In FBR, sucrose wasfavorable for H2 production, while ethanol productionwas better with fructose. However, in PBR, glucose gavebest performance in terms of production rate and yieldof the two biofuels. This difference in substratepreference could be due to variations in bacterialpopulation structure resulting from different bioreactorconfiguration237.

High H2 yields that are needed to make processeconomical232 can be achieved by H2 production from afermentation end product (acetate) by modifying amicrobial fuel cell by applying a small potential to thatgenerated by bacteria238. H2 yields can be increased incontinuous culture by decreasing H2 partial pressure inreactor. This can be achieved by stripping H2 from liquidusing N2 sparging210,239 and also by applying a vacuumto headspace, thereby lowering overall partial pressurein the system185. It is possible to generate power fromfermentation end products other than H2. For instance,when propionate end product is fed to microbial fuelcell (MFC), electricity can be generated with propionateintermediate with cereal wastewater along with acetate.However, this reaction would require rapid utilization ofany H2 produced in the system. Propionate can beconverted to acetate as


CH3CH2COO- + 3H2O ↔CH3COO-

+ H+ + HCO3- + 3H2

Under standard conditions, this reaction is notthermodynamically feasible (∆G=76.1 kJ). Conversionof propionate to acetate and H2 is onlythermodynamically possible at H2 concentrations of10-4 atmospheres (100 ppm). However, H2 could haveremained low in the system due to H2 utilization bybacteria making conversion of propionate to acetatepossible240. High H2 yields also require novel reactortechnology like a mesophilic unsaturated flow (tricklebed) reactor, which can achieve high H2 gas recoveryfrom pretreatment of high carbohydrate containingwastewaters241. High H2 production rates and stable H2

production can also be achieved at low retention timesin upflow anaerobic sludge blanket reactor whencompared with conventional continuous stirred tankreactor242.

In H2 production, unstable and low flow rates duringbiohydrogen production bring up development of lowenergy and efficient purification methods. At this point,modules containing a polyvinyltrimethylsilane(PVTMS) membrane are developed for biohydrogentreatment and efficient separation of CO2 from H2

243.Innovative reactor designs like draft tube fluidized bedreactor (DTFBR) containing immobilized cell particlesby synthetic polymer (silicone gel, SC) demonstrate anefficient, stable and reproducible H2-production244.

Among various approaches to increase H2 productionyield from organic wastes, nutrient supplementation(nitrogen, phosphorus and iron as biostimulants)improves simultaneous H2 production and pollutionreduction from substrate using thermophilicfermentation. Anaerobic sequencing batch reactor(ASBR) fed with nutrient-supplemented POME gavehigher growth and activity of T. Thermosaccharolyticum

than feeding with raw POME. Butyric acid and aceticacid were main soluble metabolites, which favor H2

production245. Biohydrogen production can be achievedthrough bioconversion of syngas by water gas shiftreaction173,247.

CO + H2O ↔ H2 + CO2

R. rubrum, PNS bacterium can catalyze WGSreaction by reactants other than CO like acetate, malate,glucose, yeast extract and ammonium246-248, underanaerobic conditions in a continuous stirred bioreactor

(CSTBR)249. It can consume CO faster than other H2

producing bacteria with high growth rate and cellconcentration147,174,250-252. During WGS reaction,nitrogenase is responsible catalyst for H2

production143,252. On the other hand, initial substrateconcentrations may inhibit cell growth and H2

production147,250. Consumption rate of carbon source canvary for R. rubrum. In the case of CO, 3 folds higherconsumption rates can be achieved than acetate253.

Although feedstocks like starch and cellulose fromcrop wastes are abundant for H2 production, rate offermentative H2 production may be slow due to substratehydrolysis. In the case of starch, even high H2 producerslike C. Butyricum can exhibit low performance by slowhydrolysis process. In order to eliminate this problem,starch can be enzymatically hydrolyzed by amylaseproducing bacterium Caldimonas taiwanensis On1254.By utilizing starch pretreatment, even pure cultures thatcannot convert raw starch into H2 give high H2

production rates and H2 yields255. In the case of cellulose,NS culture hydrolyzes carboxymethyl cellulose, andafter that H2 producing bacterial isolates (mainlyClostridium species) were used to convert cellulosehyrolysate into H2 energy256.

Conclusions

Biological H2 production is the most desirableultimate target to supply energy demand of mankind.However, photosynthetic organisms are rather sluggishto produce H2 and maintenance of optimum conditionsin reactors while production occurs is a delicate task.Therefore, an enormous investment is needed tounderstand H2 producing mechanisms better in cells ofmicroorganisms at the molecular level in the directionof evolution of artificial organisms, which could produceabundant, at least satisfactory, quantities of H2 with asuitable rate of production.

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