hydrogen production by photosynthetic green algae -...
TRANSCRIPT
lane
t sent to;t for theiitted for
withoutrnicating
Hydrogen production by photosynthetic green algae
Maria L Ghirardi, National Renewable Energy Laboratory, Golden, CO 80401, USA
E-mail: [email protected]
Received 29 March 2006; revised 28 June 2006
IndianJournal of Biochemistry & BiophysicsVol.43, August 2006, pp. 201-210
Minireview
Oxygenic photosynthetic organisms such as cyanobacteria, green algae and diatoms are capable of absorbing light andstoring up to 10-13% of its energy into the H-H bond of hydrogen gas. This process, which takes advantage of thephotosynthetic apparatus of these organisms to convert sunlight into chemical energy, could conceivably be harnessed forproduction of significant amounts of energy from a renewable resource, water. The harnessed energy could then be coupledto a fuel cell for electricity generation and recycling of water molecules. In this review, current biochemical understandingof this reaction in green algae, and some of the major challenges facing the development of future commercial algalphotobiological systems for H2 production have been discussed.
Keywords: Green algae, Hydrogen production, Algal hydrogenases
IntroductionThe photoproduction of H2 by green algae is an
alternative pathway to utilize the reductants generatedfrom photosynthetic water oxidation, as shown inFig. 1. The discovery of this pathway was done byHans Gaffron and his co-workers in the lateI930s/early 1940s, and their research has beenreviewed recently':". Normally, photosyntheticallygenerated reductants in green algae are used for CO2
fixation into starch" which is then mobilizedaccording to the energy requirements of the organism.However, under dark, anaerobic conditions, the CO2
fixation pathway is inactivated and a new enzymaticsystem, consisting of [FeFe]-hydrogenase enzymes is
starcn • Iaer7FNR/C02 fixation
reductants
anaer~HydrogenaseIH2production
expressed. It is proposed that the role of this system isto prevent accumulation of reductants uponsubsequent exposure of the organisms to light and topoise the organism at an appropriate redox lever':".
An additional source of reductants for algalhydrogenases includes glycolytic degradation ofstored starch", as shown in Fig. l. The contributionfrom this source varies widely from organism-to-organism and is dependent on physiological andenvironmental conditions'"!". As such, an optimal Hz-producing green alga should be able to utilize bothwater oxidation. and starch degradation as comple-mentary sources of reductants. Although green algaealso produce H2 fermentatively in the dark'"!', therates of this reaction are orders of magnitude, lowerthan the light-induced reactions.
The potential light conversion efficiency of algalhydrogen production is about 10-13%, based on (a)the 43-45% amount of energy present in the portion ofsolar spectrum that is absorbed by the chlorophyll(Chl)-containing organisms 12, and (b) the amount ofenergy stored in a mole H2 molecules 13. In a regionexposed to high light intensity, such as the U.S.Southwest (l kW 1m2), this conversion efficiencytranslates into a 100 km vs 100 km area (about 4,500square miles or 0.12% of the U. S. land area) neededto produce enough energy to completely displacegasoline use by the U.S. transportation sector (236million cars)!". This estimate underscores the promiseof the technology to generate H2 in an efficient andrenewable manner.
H20
Y.02+ 2H+
Fig. I-Pathways from photosynthetic water oxidation to CO2fixation (through the ferredoxin/NADP oxidoreductase or FNR),under aerobic conditions or to H2 production (through thehydrogenase enzyme), under anaerobic conditions [An -alternativepathway that provides reductants to the photosynthetic electronIransport chain through starch degradation is also indicated]
-
202 INDIAN J. BIOCHEM. BIOPHYS., VOL. 43, AUGUST 2006
Other photosynthetic organisms also have thecapability of photoproducing H2 from waterI5,16,
although in some of them (such as cyanobacteria), thereaction is catalyzed by a different class of enzymes,[NiFe ]-hydrogenases. These organisms are! also beingconsidered for future applied systems. Thus, althoughpromising, photosynthetic systems are not ready to beexploited for commercial uses yet. The majorchallenge facing these systems is the extreme sensi-tivity of H2 metabolism to O2, an obligatory by-product of photosynthetic water oxidation. This sensi-tivity occurs at four levels: (a) gene transcription,(b) [FeFe]-hydrogenase maturation, (c) activity of thehydrogenase catalytic site, and (d) competition forphotosynthetic reductants with other physiologicalpathways. The next sections address in more detaileach of these areas, followed by a description of tworecently described algal systems based on physio-logical manipulation of the algae that currently photo-produce H2 gas sustainably.
Green algal hydrogenases
i) Catalytic site and enzyme mechanismHydrogenases are classified as [FeFe], [NiFe] or
FeS-cluster-free (Fe-only), according to the metalcomposition of their catalytic site. The catalytic site ofalgal hydrogenases consists of an H-cluster,containing a 4Fe4S center, linked by a cysteineresidue to a uruque 2Fe2S cluster (Fig. 2). This
e·Ferredoxit
Fig. 2-Schematic drawing of algal [FeFe]-hydrogenase enzymestructure showing relative location of the [4Fe4S] and [2Fe2S]components of Il-cluster, the proposed proton pathway (dashedline) and computationally-modeled O2 pathways (full lines) [Thediffusion of H2 gas has been proposed to occur by multiplepathways. See text for more information]
characteristic composition places them in the categoryof [Fefel-hydrogenases'v'", which are also found inclostridia, desulfovibria, Thermatoga sp., the protistTrichomonas sp. and anaerobic fungi. The [FeFeJ-hydrogenases have the highest turnover rates (6,000-9,000 S-I) of all hydrogenases'', and are usuallyinvolved in Hy-production, in contrast with the mostlyHy-uprake [NiFe]-enzymes. Interestingly, the presenceof an open-reading-frame (ORF) has been reported inEnterobacter cloacae, with high homology to theH-cluster-binding C' -end of other [FeFe]-hydro-genases 19. Moreover, when expressed in E. coli(without the co-expression of assembly genes, seesection (iii) below), the respective gene productexhibited H2 production activity. If, indeed thisE.. cloacae ORF encodes a hydrogenase enzyme, itscatalytic site has different characteristics from thoseof "the usual [FeFe]-hydrogenases, since one of thefour conserved cysteine residues that bind theH-cluster is absent in the putative E. cloacae hydro-genase. In order to explain how an H-cluster can beassembled in the latter, Tosatto et al.20did homologymodels of the putative E. cloacae hydrogenase andassumed that the fourth cysteine ligand is provided bya non-conserved cysteine residue Cys 56. Althoughintriguing, their work will have to be validated bymore specific biochemical and biophysical assays.
The 2Fe2S cluster of [FeFe ]-hydrogenases bindsCO, CN and a dithiomethylamine species (however,see Nicolet et al" for an alternative proposedligand."). The presence of CO and CN ligands, whichare also found in [NiFe]-hydrogenases is believed toimpart the catalytic center with a higher electron-accepting characteristic", facilitating electron trans-port to a bound H+ (see arrows in Fig. 2). Hydrogenproduction involves double reduction of the protonbound to the distal iron, followed by recombination ofresulting R with another H+, bound perhaps to thebridging dithiomethylamine ligand, as suggested bywork with theoretical studies and model compounds"
The direct donation of electrons to the hydrogenaseoccurs through the intermediate [2Fe2S]-containingprotein ferredoxin. In green algae, ferredoxin shuttleselectrons between the photosynthetic electrontransport chain (that generates the electrons fromwater) and the hydrogenase (see also Fig. 1). Reducedferredoxin is believed to dock 10 the hydrogenase nearthe 4Fe4S center and to transfer electrons to thehydrogenase in two consecutive reactions. Thetransfer of protons to the catalytic site of the hydro-
genaci:sur:cys
sitepatlmohycenzUSll
simaccsugtheare
1whidistbotl[Nisentdenhydair"hypN-tlWOI
reasto (unddiffstru
ii) G1
enciiderdes}monencltargdifflthatTA"5' -1.187Adointnandscaf
categoryound in~ protist[FeFe]-(6,000-usually
~mostlyiresenceorted in
to theI-hydro-E. colires, seeproducted this.me, itsn those
of thend thehydro-can be
mologyise andided byIthoughited byiys.
; bindsiwever,oposed. whicheved toectron-
trans-drogenprotonition ofto the
:ted byunds23.
genasetaininghuttleslectron; fromeducedse nearto the. Thehydro-
GHIRARDI: HYDROGEN PRODUCTION BY GREEN ALGAE
genase.. on the other hand, is proposed to involveacidic residues located between the H-cluster and thesurface" of the enzyme and possibly ending on acysteine residue, next to the catalytic siteIS•25,26.
The pathway by which H2 gas leaves the catalyticsite has been a matter of debate. A putative H2 gaspathway was initially identified using either amolecular dynamics approach", a prediction ofhydrophobic cavities using static structures of the
2127 h c . d ..enzyme' or a searc lor xenon-occupie cavitiesusing crystallographic methods'". However, recentsimulations based on molecular dynamics and solventaccessibility maps of the CpI [FeFe]-hydrogenase2s.29
suggest that hydrogen diffuses more freely throughthe protein structure, although two major pathwaysare mainly utilized [see section (iv) below].
The H-cluster is irreversibly inactivated by O2,which binds to the empty coordination shell of thedistal Fe, but is reversibly inhibited by CO. In contrast,both O2 and CO, in some cases reversibly inactivate the[NiFe] metallo-cluster of [NiFe]-hydrogenases. Indeed,sensitivity of algal hydrogenases to O2 has beendemonstrated to be higher than that of other [FeFe]-hydrogenases, and it is on the order of a few seconds inair3o. Although this extreme sensitivity washypothesized to be due to the lack of a more extensiveN-terminal accessory cluster domain, mutagenesiswork demonstrated otherwise". Although the structuralreason for the higher sensitivity of algal hydrogenasestoO2 is not known, efforts are underway to gain furtherunderstanding of the energetics of H2 and O2diffusion" and to obtain and solve the X-ray crystalstructure of the algal [FeFe]-hydrogenases.
ii)Gene transcriptionTwo algal hydrogenase genes HydAl and HydA2,
encoding for two distinct proteins have beenidentified in Chlamydomonas reinhardti32.33, Scene-desmus obiiquus34
•35, Chiarella fusca'" and Chlamydo-··37 Th . Imonas moewusu:': e two proteins are nuc ear
encoded and contain putative transit sequences thattarget them to the chloroplasr'". There are otherdifferences between the HydAl and HydA2 genesthat may affect their transcription - a characteristicTATA box is present 24 bp upstream from the5'-UTR in HydA2, but it is located much further up,187 bp from the 5'-UTR region in HydA133,3s.Additionally, they contain a different number ofintrons (8 in HydAl and 10 in HydA2, see Fig. 3),and their sequences are contained in differentscaffolds, 10 and 12, respectively.
203
z 6
HydA1
5'UTR158 bp
COding sequence: 1491 bp 3' UTR747bp
l
HydA2
Coding sequerce:1515 bp
Fig. 3-Structure of the genes encoding for the algalhydrogenases RydAl and HydA2 [The coding sequences areshown, respectively in blue and yellow, and each exon isidentified by a number. Introns are indicated by lines above thecoding sequence and they are also identified by numbers. The S'and 3' UTRs are shown by fi lied boxes are each end of the codi ngsequence. TP represents the transit peptide]
Very little is known about the transcriptionalregulation of either the algal or of other [FeFe]-hydro-genase genes. However, anaerobicity plays a majorrole, as observed in the recent studies32.33. Thesereports showed that the accumulation of hydrogenasetranscripts occurred only upon anaerobic treatment ofthe algal cultures, either through dark incubation orsulfur deprivation in the light. Identification of pro-moter region (128 to 21 bp from the transcriptioninitiation site) responsible for anaerobic expression ofthe HydAl gene was described recently'". However,other factors might also contribute at least as modu-lators of gene expression under anaerobic conditions'".
The Hj-production capabilities of two C. reinh-ardtii mutants that are unable to accumulate starch(due to a defective isoamylase gene) have beencharacterized in a recent study?'. These mutantscannot sustain hydrogenase gene expression undersulfur deprivation or dark anaerobic conditions,suggesting that either the energy level31 or metaboliteflux conditions of the cells42 play a major role inregulation of algal [FeFe]-hydrogenase genes. Itwould be interesting to find out, whether a two-component regulatory PAS system (the 2-componentsignal-recognition module named for the initially-identified members Per, ARNT and Sim proteins (seerefs in Reinelt et al.43) is also involved in sensing O2
levels and regulating hydrogenase gene transcription,
..-
204 INDIAN J. BIOCHEM. BIOPHYS., VOL. 43, AUGUST 2006
as has been described in other systems that synthesize[NiFe]-hydrogenases I6,44. No evidence is available yetfor the operation of such a system in organisms thatcontain [FeFe [-hydrogenases.
Hi) Enzyme assemblyThe assembly and maturation of catalytic H-cluster
of [FeFe]-hydrogenases requires 3 genes, in contrastwith the 7 accessory proteins required for theassembly of [NiFe]-hydrogenases (reviewed in Bocket al.45). This is an interesting observation, since thetwo types of hydrogenases have a unifying feature,the existence of CN and/or CO ligands to the activesite Fe. Yet, the maturation process of two types ofenzymes utilizes not only different enzymes, but verydifferent chemical mechanisms'f:". The maturation of[NiFe]-hydrogenases is mostly an aerobic process,while anaerobicity is a sine qua non for the expressionand catalytic function of accessory proteins respon-sible for the maturation of [Fef'el-hydrogenases'".
Two genes, HydEF and HydG that encode forproteins that function in maturation of [FeFe]-hydrogenases have been reported in the alga C.reinhardtii by Posewitz et .a". They screened alibrary of insertional C. reinhardtii for mutants unableto photoproduce H2 and among the selected clones,identified a: mutant, hydEF-l that was disrupted in theHydEF gene. The ability to photoproduce H2 wasrestored, when the mutant was complemented with afull copy of the HydEF gene. This result directlylinked the Hrnon-producing phenotype to the HydEFgene function. Upstream from the HydEF gene in C.reinhardtii is another gene HydG, which is orienteddivergently with respect to the HydEF gene andpossibly regulated by the same promoter'f".
The hydEF-l mutant transcribed both HydAl andHydA2 genes upon anaerobic induction, synthesizesboth the HydAl and HydA2 proteins (of slightlyhigher molecular weight than in the wild-type), butexhibited no Hy-production activity. It was proposedthat' the protein expressed by the mutant was inactiveand possibly not mature. This hypothesis wasconfirmed by experiments in which the hydrogenasestructural gene HydAl was co-expressed in E. coliwith the two assembly genes HydEF and HydG,yielding an active algal hydrogenase.". The generalityof the assembly mechanism among differentorganisms has been recently demonstrated''", and inmost other biological systems the maturation proteinsare encoded by three separate genes: HydE, HydF andHydG. The authors expressed [FeFe]-hydrogenases
from C. reinhardtii, C. pasteurianum or C. acetobu-tylicum using the E. coli system and maturation genesfrom C. acetobutylicum.
Homologues of the HydEF and HydG genes wereidentified in all organisms containing [FeFe]-hydrogenases. In all instances, with the exception ofgreen algae, HydEF is represented as two separategenes HydE and HydF, and in some cases the three
. hi . I 45 46 H dF .genes occur WIt In a SIng e operon ' . y . containsmotifs that are characteristic of GTPases, while bothHydE and HydG show signature motifs of RadicalSAM proteins", This unique class of proteins isinvolved in reactions that are initiated by thegeneration of the 5' -deoxyadenosyl radical, followingthe reductive cleavage of S-adenosyl methionine. The5' -deoxyadenosyl radical utilizes different substratesto perform a variety of anaerobic metabolic functions,such as cofactor biosynthesis", synthesis of biornole-cules'", insertion of sulfur into organic substrates'",etc. Recently, the HydE and HydG from Thermatogamaritima were demonstrated to have Radical SAMproperties in vitro"; and HydF was shown to be aGTPase in vitro as we1l52
. Although the specific rolesof HydE, HydF and HydG gene products in thematuration of the [FeFe]-hydrogenase H-clusters isnot clear, potential reaction mechanisms and subs-trates have been postulated recently''v". Thus, furtherexperiments are required to deconvolute the entirematuration process.
iv) O2 inhibition of hydrogenase activityInactivation of [FeFe]-hydrogenases by O2 is
believed to occur by irreversible binding of O2 to theH-cluster, possibly including .the distal Fe of the[2Fe2S]-center, the site of H2 catalysis, which alsobinds one of the protons [see section (i) above]. Thefinal oxygen species present in the inactivated stateenzyme is not yet known. However, it is clear that thecatalytic site of the enzyme is embedded into theprotein structure, and for inactivation, it is necessaryfor O2 gas to diffuse into the protein structure. Currentefforts to prevent O2 inactivation of the [FeFe]-hydrogenase involve two different approaches. Thefirst one, proposes to use random mutagenesis togenerate multiple mutants of the enzyme, followed byscreening for increase in O2 tolerance. This approachrequires the availability of high-throughput screeningassays to handle the large number of colonies to beexamined, such as the chemochromic methoddescribed previously T'". The second method is basedon (a) identifying the pathways by which O2 gains
acce:muta
Reto tIof sC. p,moledemcout cSincereportionacavitithe althe restroruis indrestricmolecmutajspecifO2 dilthat tlgas dirandoicules.possibwithoipathw
v) ComThe
reducr,(FNR)ferredcenzymunderCalvinCO2 fiother IT
observedepriverentiallhydrog'instead.tolerantt~e hy,concernof fernhydrogethat the
ttobu-genes
were~eFe]-on ofparatethree
ntains: bothadical.ns IS
I theiwing:. The:tratestions,mole-Ites50,atogaSAMbe aroles
1 theers issubs-irtherentire
)2 is:0 thef the
also. Thestate
at the) thessaryirrenteFe]-
Theis toed byroacheningto beethodrasedgains
GHIRARDI: HYDROGEN PRODUCTION BY GREEN ALGAE 205
access to the catalytic site, and (b) using site-directedmutagenesis to reduce accessibility".
Recent advances using the second method have ledto the identification of two pathways, consistingof separate cavities for O2 gas diffusion in theC. pasteurianum CpI [FeFe]-hydrogenase, based onmolecular dynamic simulations28,29. The studies alsodemonstrated that in contrast with O2, H2 gas diffusesout of the catalytic' site through multiple pathways.Since these results apparently contradicted previousreports26,27, they were reinforced by a new computa-tional method that allows one to map transientcavities based on the dynamics of the protein itself, inthe absence of gas molecule/". This method confirmsthe results from the molecular dynamics simulations,strongly suggesting that the diffusion of O2 in theory,is indeed restricted to only two pathways, and that thisrestriction is primarily based on the size of the O2
molecule. This conclusion is directing site-directedmutagenesis studies aimed at changing the nature ofspecific amino acid residues, located in the identifiedO2 diffusion pathways. Finally, it is important to notethat the use of additional approaches to identify O2
gas diffusion pathways has also confirmed the morerandomized H2 diffusion behavior of H2 gas mole-cules. These results strengthen the hypothesis that it ispossible to modify O2 access to the catalytic sitewithout concomitantly affecting H2 gas diffusionpathways and properties38.
v) Competition for reductantsThe normal fate of photosynthetically generated
reductants is the ferredoxin/NAOPH oxidoreductase(FNR) that generates NAOPH from reducedferredoxin for other metabolic pathways, such as theenzymes of the CO2 fixation Calvin cycle. However,under anaerobic conditions, the main enzyme of theCalvin cycle, Rubisco, is inactivated " and thus theCO2 fixation does not compete for reductants withother metabolic pathways. Rubisco inactivation is alsoobserved when anaerobiosis is induced by sulfurdeprivatiorr'". As a result, ferredoxin will prefe-rentially reduce other enzymes such as the [FeFe]-hydrogenases and H2-production activity is observed,instead. However, in a system driven by an Ortolerant hydrogenase, competition between FNR andthe hydrogenase for reductants may be a majorconcern. This concern is based on the higher affinityof ferredoxin for the FNR (0.4 !!M60) than forhydrogenase (10-35 !!M30,61,62),although it is possiblethat the relative levels of FNR and hydrogenase could
reduce the competition. Future investigations willhave to address this potential drain of reductants awayfrom H2 production.
Physiological manipulation of green algaeThe engineering of [FeFe]-hydrogenases for O2
tolerance is a long-term approach for addressing thecurrent lack of continuity of algal Hrproduction.However, a short-term solution has become availablethat allows sustainable H2-production as a pure gas,but at limited rates and lower light conversionefficiency'". It is based on the partial inactivation ofphotosynthetic O2 evolution by depriving algalcultures of sulfate nutrients. As shown earlier, sulfatedeprivation prevents the normal repair of the light-damaged 01 protein ,of photosystem II (PSII)64,leading to the accumulation of inactive centers thatare unable to reduce QB 65 and thus are incapable ofmaintaining high photosynthetic electron flow toferredoxin. Sulfate deprivation also .leads to an initialover-accumulation of starch, followed by the
.11- ••.•••• pz;" •• ::::: -·-:481' ..- ~~ ..\,. ~~
Fig. 4-Two sets of photobioreactor systems used to monitorcontinuous H2 production by sulfur-deprived cultures ofChlamydomonas reinhardtii [The algal cultures are initiallygrown photosynthetically in suspension under controlled pH in thephotobioreactors shown in the center of the photograph. Thecultures are grown in the chemos tat mode, and are continuouslyflushed with medium containing limiting amounts of sulfate.Under these conditions, they do not produce Hb but accumulatestarch. The amount of dissolved O2 and redox potential of thesuspensions \ is continuously monitored through the electrodesshown in each side of the reactors. The two photobioreactors atthe edge contain cultures that were initially deprived of sulfateand that are actively producing H2. The cultures in those reactorsare being continuously replaced by fresh cultures from the twofirst reactors, thus ensuring the uninterrupted Hz production (seetext). Hydrogen .gas is accumulated from the ouput medium in aglass cylinder under water (not shown)]
206 INDIAN}. BIOCHEM. BIOPHYS., VOL. 43, AUGUST 2006
anaerobiosis in the culture vessels66.67
. Anaerobiosisoccurs because sulfate deprivation has a only veryslight effect on the respiratory activity of the cells63. Itleads to a decrease in the levels of Rubisco ",induction of hydrogenase expression and activity, andcatabolic metabolism of starch and proteins66
,68,69.\ Thesubsequent sustained production of H2 gas wasdemonstrated to depend on two separate sources ofphotosynthetic reductants: (a) those originated by theresidual water oxidation activity of the cultures, and(b) those released upon by oxidation of starch andproteins and transferred to the photosynthetic electrontransport chain at the level of the intermediate
. plastoquinone.The rate of H2 production under sulfate deprivation
has been shown to be only about 1/lOlh of the capacityof the photosynthetic apparatus to process electrons'",It has been proposed that prolonged anaerobiosisleads to down-regulation of electron transport, due tonon-dissipation of proton gradient":" that results inincreased, non-productive cyclic electron trans-port72
,73. Indeed, a 3-4 times increase in the rate of H2photoproduction was reported with a C. reinhardtiimutant that is blocked in state transitions and thusunable to perform cyclic electron transfer".
Although H2-production activity lasts only 3-4 daysunder sulfur deprivation, processes have beendeveloped to sustain it beyond that period of time.One approach consisted in running cycles of minus-sulfate (5 days) land plus-sulfate (2 days) to allow thecultures to temporarily recover their photosyntheticactivity, and to re-accumulate their storage of starchand protein": A second approach utilized theconcept of physical separation of photosyntheticOz-production and anaerobic, H2-production phases".This was achieved using two separate photobio-reactors, as shown in Fig. 4. The culture in firstphotobioreactor is grown photosynthetically, underlimiting sulfate concentration in a chemostat mode.The cells are used to continuously replace the cells ina second photobioreactor, under anaerobic H2-
production conditions. This system produced H2 gasuninterruptedly for a period of 6 months 76.
Whenever algal suspensions are used for H2production under sulfate deprivation, there is alimitation on the maximum cell density that can beachieved. To circumvent this problem, flagella-lessalgal cultures were immobilized onto glass fibers andsubmitted to sulfate deprivation". The same numberof cells were concentrated into a much smaller
Table I-Light conversion efficiency calculations of the Hz-producing capabilities of sulfur-deprived cultures
[Assumptions: 1 ml H2 contains 33 umoles H2 (at NREL, suspension cultures) or 44.633 umoles H2 (in Pushchino, Russia, immobilizedcultures); l umole H2 contains 0.237 J energy (at 298°K and 1 atrn); I IlE of 560 nm light contains 0.214 Joules of energy;
1 cm2 = 10-4 m2]
S. No. Parameters Suspension cultures Immobilized algae(at NREL, in CO) (in Pushchino)
1 Reactor dimensions (in ern) 22.86 x I 1.4 x 5 20 x 10 x 0.82 Illuminated surface 2 x 261 cm2 = 522 cm2 200 cm2
(0.0522 nl) (0.02 m2)3 rnl H2 producedlreactor (Vav) during time of 2.5 mllh 0.7 ml/h with partial
operation (3-4 days for suspension cultures and pressure of 0.1-0.0 I atm23 days for immobilized cultures)
4 umoles H2 producedlreactor (Vavx 33 urnoles/ 82.5 umoles/h 31.22 urnoles/hml in CO, 44.6llmoles/ml in Puschino)
5 Energy content of H2 produced by the reactor 19.55 J 6.681during a 1 h period [(4) x 0.237]
6 Flux of incident light on all illuminated surfaces 200 IlE m·2 s' 120 IlE m·2 S·I
of reactor7 Energy of total incident light, as above 42.81 m·2 s' 25.68 J m·2 s'
[(6) x 0.2 14111lE]8 Energy of incident light per m2 during a period of 154,080 11m2 92,448 J/m2
1 h [[(7) x 3,600 s/h]9 Energy of incident light on illuminated surfaces 8.043 x 103 J 1.85 X 103 J
during a I h period [(8) x (2)]10 Efficiency of incident light energy conversion 0.24% 0.36%
into H2 [(5)/(9)]
volume, bAlthoughChI) decrelight comincreasedcell imrnproductioncycling ofsulfate, wmethods. Idata showcontinuousand the li~reflect thsaerobic, su
A prorrmethod hpermeasethat is res]cytosol in!ferencetephotosyntlhave beenconditionsmutants wexpressiona mutant ilto the lev,normallywould bethe sulfate
ConclusioPhotobi
the potentenergy in!solar effieindirectchallengesbiologicalwill requlaboratoriethat the v..impact theprocesses.with H2-pcally linkcharge-co:productiorthis area b
Y3-4 daysavebeenj of time.of minus-allow the
osyntheticof starchized theisyntheticphases 76.
photobio-~ in firstIy, underat mode.e cells inibic Hrd H2 gas
for H2re is a.t can be~ella-Iessbers andnumbersmaller
mobilized5Y;
al1 atm
GHIRARDI: HYDROGEN PRODUCTION BY GREEN ALGAE 207 .
volume, but with the same illuminated surface area.Although the specific rate of H2 production (per mgChi) decreased by 20-25% by cell immobilization, thelight conversion efficiency of the system into· H2increased by about 50%, as shown in Table 1. Thecell immobilization prolongs the phase of H2production from 3-4 to 21 days, and facilitates thecycling of the cultures from minus-sulfate to plus-sulfate, without requiring expensive cell-harvestingmethods. Finally, it is important to emphasize that thedata shown in Table 1 have been obtained undercontinuous illumination at non-saturating intensities,and the light conversion efficiencies presented do notreflect the light utilization that occurs during theaerobic, sulfur-replete photosynthetic phase.
A promising off-shoot of the sulfate-deprivationmethod has been reported recently". A sulfatepermease gene has been identified in C. reinhardtiithat is responsible for the uptake of sulfate from thecytosol into the chloroplast'". By using mRNA inter-ference technology, algal mutants with decreasedphotosynthetic activity and normal respiration rateshave been generated under photoheterotrophic growthconditions'". As mRNA interference generatesmutants with different degrees of attenuation of geneexpression, it would be possible to actually select fora mutant in which photosynthesis is already depressedto the level of respiration. Such a mutant would benormally submitted to anaerobic conditions andwould be expected to photoproduce H2 gas withoutthe sulfate deprivation requirement.
ConclusionsPhotobiological H2-production technologies have
the potential to convert up to 10% of the sunlightenergy into H2, clearly competing with the currentsolar efficiencies of photoelectrochemical and otherindirect Hrproduction methods. However, thechallenges required to convert an interestingbiological observation into a commercial applicationwill require the concerted efforts from researchlaboratories worldwide. Finally, it is important to notethat the work on photobiological systems could alsoimpact the development of biomimetic Hy-productionprocesses. The latter are based on synthetic catalystswith H2-producing capability, electrically or chemi-cally linked to a photochemical light-absorbing,charge-conversion complex for artificial solar H2production. Interesting work is being conducted inthis area by many groups81,82 and references therein.
AcknowledgementsI thank Dr. Paul King, NREL for useful
suggestions and revision of the manuscript, and Drs.Michael Seibert (NREL) and Anatoly Tsygankov(Russian Academy of Sciences, Pushchino, Russia)for help with the calculations in Table 1. I acknow-ledge the U.S. DOE's Hydrogen, Fuel Cells andInfrastructure Technologies Program and the Officeof Science for financial support.
References1 Homann P (2003) Hydrogen metabolism of green algae,
discovery and early research - A tribute to Hans Gaffron andhis coworkers. Photosyuth Res 76, 93-103
2 Melis A & Happe T (2004) Trails of green alga hydrogenresearch - from Hans Gaffron to new frontiers. PhotosyntliRes 80, 401-409
3 Schulz R (1996) Hydrogenases and hydrogen production ineukaryotic organisms and cyanobacteria. J Mar Biotecluiol 4,16-22
4 Appel J & Schulz R (1998) Hydrogen metabolism inorganisms with oxygenic photosynthesis: Hydrogenases asimportant regulatory devices for a proper redox poising? JPhotocheui Photobiol B 47, I-II
5 Happe T, Hernscherneier A, Winkler M & Kaminski A(2002) Hydrogenases in green algae: Do they save thealgae's life and solve our energy problems? Trends Plant Sci7,246-250
6 Healey F P (1970) The mechanism of hydrogen evolution byChlamydomonas moewusii. Plaut Physiol45, 153-159
7 Stuart T S & Gaffron H (1971) The kinetics of hydrogenphotoproduction by adapted Scenedesmus. Planta 100, 228-243
8 Gfeller R P & Gibbs M (1984) Fermentative metabolism ofChlamydomonas reinliardtii. I. Analysis of fermentativeproducts from starch in dark and light. Plant Physiol 75,212-218
9 Randt C & Senger H (1985) Participation of the twophotosystems in light dependent hydrogen evolution inScenedesnius obliquus. Photochem PhOIObiol42, 553-557
10 Schnackenberg J, Ikemoto H & Miyachi S (1995) Relation-ship between oxygen-evolution and hydrogen-evolution in aChlorococcum strain with high COrtolerance. J PhotochemPhotobiol B 28, 171-174
11 Ohta S, Miyamoto K & Miura Y (1987) Hydrogen evolutionas a consumption mode of reducing equivalents in greenalgal fermentation. Plant Physiol 83, 1022-1026
12 Blankenship R E (2002) Molecular Mechanisms of Photo-synthesis, Blackwell Science, London
13 Akkerman I, Janssen J, Rocha J & Wijffels R H (2002)Photobiological hydrogen production, photochemical. effi-ciency and bioreactor design. Int J Hydrogen Energy 27,1195-1208
14 Ghirardi M L, Maness P C & Seibert M (2006)Photobiological methods of renewable hydrogen production
·0208 INDIAN J. BIOCHEM. BIOPHYS., VOL. 43, AUGUST 2006
in "Solar Generation of Hydrogen", Springer Verlag (inPress)
15 Brand J J, Wright J N & Lien S (1989) Hydrogen productionby eukaryotic algae. Biotechnol Bioeng 33, 1482-1488
16 Vignais PM, Billoud B & Meyer J (2001) Classification andphylogeny of hydrogenases. FEMS Microbiol Rev 25,455-50 I
17 Adams M W W (1990) The structure and mechanism of iron-hydrogenases. Biocliim Biophys Acta 1020, 115-145
18 Frey M (2003) Hydrogenases, hydrogen-activating enzymes.Chembiochem 3, 153-160
19 Mishra J, Kumar N, Ghosh A K & Das D (2002) Isolationand molecular characterization of hydrogenase gene from ahigh rate of hydrogen-producing bacterial strain Entero-bacter cloacae IlT-BT 08. lnt J Hydrogen Energy 27, 1475-1479
20 Tosatto S C E, Giacomelli G M, Valle G & Costantini P(2006) Functional insights from the structural modeling of asmall Fe-hydrogenase. Biocheni Biophys Res COIllIllIIII399,277-283
21 Nicolet Y, Piras C, Legrand P, Hatchikian E C & Fontecilla-Camps J C (1999) Desulfovibrio desulfuricans iron hydro-genase, the structure shows unusual coordination to an activesite Fe binuclear center. Structure 7, 13-23
22 Volbeda A, Charon M H, Piras C, Hatchikian E C, Frey M &Fonteci lla-Camps J C (1995) Crystal structure of the nickel-iron hydrogenase from Desulfovibrio gigas. Nature 373,580-587
23 Li H & Rauschfuss B (2002) Iron carbonyl sulfides formal-dehyde and amines condense to given the proposed azadi-thiolate cofactor of the Fe-only hydrogenaes. } Am Cheui Soc124, 726-727
24 Horner D S, Heil B, Happe T & Embley T M (2002) Ironhydrogenases-ancient enzymes in modern eukaryotes.Trends Biochem Sci 27, 148-153
25 Peters J W, Lanzilotta W N, Lemon B J & Seefeldt L C(1998) X-ray structure of the Fe-only hydrogenase (Cpl)from Clostridium pasteurianuni to 18 A resolution. Science282, 1853-1858
26 Nicolet Y, deLacey A L, Vernede X, Fernandez V M,Hatchikian E C & Fontecilla-Camps J C (2001) Crystallo-graphic and FTIR spectroscopic evidence of changes in Fecoordination upon reduction of the active site of the Fe-onlyhydrogenase from Desulfovibrio desulfuricans. } Am ChemSoc 123, 1596-1601
27 Montet Y, Amara P, Volbeda A, Vernede X, Hatchikian E C,Field M J, Frey M & Fontecilla-Carnps J C (1997) Gasaccess to the active site of Ni-Fe hydrogenases probed by X-ray crystallography and molecular dynamics. Nat Struct Biol4,523-526
28 Cohen J, Kim K, Posewitz M, Ghirardi M L, Schulten K,Seibert M & King P (2005a) Molecular dynamics andexperimental investigation of H2 and O2 diffusion in [Fe]-hydrogenase. Biocliem Soc Trans 33, 80-82
29 Cohen J, Kim K, King P, Seibert M & Schulten K (2005b)Finding gas diffusion pathways in proteins, O2 and H2 gas
transport in Cpl [FeFe]-hydrogenase and the role of packingdefects. Structure 13, 1-9
30 King P W, Posewitz M C, Ghirardi M L & Seibert M (2006)Functional studies of the [FeFe] hydrogenase maturation inan Escherichia coli biosynthetic system. J Bacterial188,2163-2172
31 Ghirardi M L, King P W, Posewitz M C, Maness P C,Fedorov A, Kim K, Cohen J, Schulten K & Seibert M(2005a) Approaches to developing biological Hz-photopro-ducing organisms and processes. Biochem Soc Trans 33,70-72
32 Happe T & Kaminski A (2002) Differential regulation of the[Fe]-hydrogenase during anaerobic adaptation in the greenalga Chlamydomonas reinliardtii. Eur J Biochem 269, 1-11
33 Forestier M, King P, Zhang L, Posewitz M, Schwarzer S,Happe T, Ghirardi M L & Seibert M (2003) Expression oftwo [Fe]-hydrogenases in Chlamydomonas reinliardtii underanaerobic conditions. Eur J Biochem 270, 2750-2758
34 Wiinschiers R, Slangier K, Senger H & Schulz R (2001)Molecular evidence for a [Fe]-hydrogenase in the green algaScenedesmus obliquus. Curr Microbiol 42,353-360
35 Florin L, Tsokogou A & Happe T (2001) A novel type of[Fe]-hydrogenase in the green alga Scenedesnius obliquus islinked to the photosynthetic electron transport chain. J BioiClient 276,6125-6130
36 Winkler M, Heil B, Hei B & Happe T (2002) Isolation andmolecular characterization of the [Fe]-hydrogenase from theunicellular green alga Chlorella fusca. Biochim Biophys Acta1576,330-334
37 Winkler M, Maeurer C, Hemschemeier A & Happe T (2004)The isolation of green algal strains with outstandingHj-productivity in Biohydrogen III (Miyake J, Igarashi Y &Roegner M, eds.), pp. 103-116, Elsevier Science, Oxford
38 Ghirardi M L, King P, Kosourov S, Forestier M, Zhang L &Seibert M (2005b) Development of algal systems forhydrogen photoproduction - addressing the hydrogenaseoxygen-sensitivity problem in Artificial Photosynthesis(Collings C, ed), Wiley- VCH Verlag Weinheim, Germany
39 Stirnberg M, Happe T ((2004» Identification of a cis-actingelement controlling anaerobic expression of the HydA genefrom Chlamydomonas reinhardtii Biohydrogen 1Il (MiyakeJ, Igarashi Y & Roegner M, eds), pp. 117-127, ElsevierScience, Oxford
40 Posewitz M C, King P W, Smolinski S L, Smith II, R D,Ginley A R, Ghirardi M L & Seibert M (2005) Identificationof genes required for hydrogenase activity In Chlaniydo-monas reinhardtii. Biochem Soc Trans 33, 102-104
41 Posewitz M C, Smolinski S L, Kanakagiri S, Melis A.Seibert M & Ghirardi M L (2004a) Hydrogen photopro-duction is attenuated by disruption of an isoamylase gene inChlamydomonas reinhardtii. Plant Cell 16, 2151-2163
42 Melis A, Seibert M & Ghirardi M L (2006) Hydrogen fuelproduction in transgenic microalgae (in Press)
43 Reinelt S, Hoffman E, Gerharz T, Bott M & Madden 0 R(2003) The structure of the peri plasmic ligand-bindingdomain of the sensor kinase CitA reveals the firstextracellular PAS domain. J Biol Che1ll278, 39189-39196
44 FriedrhydrohydroTrailS
45 BockMatur
46 Pose",M&Svade:an act
47 SofiaMillerlinkinwith Inew aAcids
48 AllenJ ncortiron-IT270,2
49 MartirMutat:adeno:Chem
50 JarrettBiol1:
51 RubacBioch:only Ituaritii
52 BrazzrFontecproteiran iror
53 Petersradicalhydro]
54 Seiber!DevelcidentifOrto\(234, PI
55 Seiber!apparamicroo6,277,~
56 Seiber!nnprovBioHYieds), PI
57 Seibertbiohyda chem
58 Gravesphotos)sustain.
GHIRARDT: HYDROGEN PRODUCTION BY GREEN ALGAE 209
de of packing44 Friedrich B, Buhrke T, Burgdorf T & Lenz 0 (2005) A
hydrogen-sensing multi protein complex controls aerobichydrogen metabolism in Ralstonia eutropha. Biochetn SocTrails 33, 97-101oertM (2006)
maturation inl45 Bock A, King P W, Blokesch M & Posewitz M C (2006)J Bacterial Maturation of Hydrogenases. Adv Microb Physiol (in Press)
Maness P C,\{ Seibert MHrphotopro-)c Trans 33,
ulation of thein the green1269, 1-11
46 Posewitz M C, King P W, Smolinski S L, Zhang L, SeibertM & Ghirardi M L (2004b) Discovery of two novel radicalS-adenosylmethionine proteins required for the assembly ofan active [Fe] hydrogenase. J Bioi Cheni 279, 25711-25720
47 Sofia H J, Chen G, Hetzler B G, Reyes-Spindola J F &Miller N E (2001) Radical SAM a novel protein superfamilylinking unresolved steps in familiar biosynthetic pathwayswith radical mechanisms, functional characterization usingnew analysis and information visualization methods. NucleicAcids Res 29, /097-1106
48 Allen R M, Chatterjee R, Ludden P W & Shah V K (1995)Incorporation of iron and sulfur from NifB cofactor into theiron-molybdenum cofactor of dinitrogenase. J Bioi Chem270, 26890-26896
a cis-acti ngHydA geneIII (Miyakett, Elsevier
55 Seibert M, Flynn T & Benson D (2001a) Method andapparatus for rapid biohydrogen phenotypic screening ofmicroorganisms using a chemochrornic sensor. US Patent #6,277,589
56 Seibert M, Flynn T & Ghirardi ML (2001b) Strategies forimproving oxygen tolerance of algal hydrogen production inBiollydrogen I (Miyake J, Matsunaga T & San Pietro A,eds), pp. 65-76, Pergamon Press, Amsterdam
57 Seibert M, Flynn T & Benson D (2002) System for rapidbiohydrogen phenotypic screening of microorganisms usinga chernochromic sensor. US Patent # 6,448,068
58 Graves D A, Tevault C V & Greenbaum E (1989) Control ofphotosynthetic reductant, the role of light and temperature onsustained hydrogen photoevolution by Chlamydomonas sp in
lchwarzer S,ixpression ofhardtii under2758
rlz R (2001)ie green alga 149160
Martinez-Gomez N C, Roberts M & Downs D M (2004)Mutational analysis of ThiH a member of the radical S-adenosylmethionine (AdoMet) protein superfamily. J BioiChem 279, 40505-40510
Jarrett J T (2005) Biotin synthase, Enzyme or reactant? CheniBioi 12, 409-410
Rubach J K, Brazzolotto X, Gaillard J & Fontecave M (2005)Biochemical characterization of the HydE and HydG iron-only hydrogenase maturation enzymes from Thennatogamaritime. FEBS Left 579, 5055-5060
Brazzolotto X, Rubach J, Gaillard J, Gambarelli S, Atta M &Fontecave M (2006) The [Fe-Fe]-hydrogenase maturationprotein HydF from Thermatoga maritima is a GTPase withan iron-sulfur cluster. J Bioi Che111281, 769-774
ovel type ofs obliquus is.hain. J Bioi I 50
solation and I 51ase from theliopliys Acta
ipe T (2004) 1 52outstanding
garashi Y &Oxford
Zhang L & 1 53.ystems fornydrogenaseotosynthesisGermany
Peters J W, Szilagyi R K, Naumov A & Douglas T (2006) Aradical solution for the biosynthesis of the H-clustet ofhydrogenase. FEBS Left 580,363-367
54 Seibert M, Flynn T, Benson D, Tracy E & Ghirardi M (1998)Development of selection/screening procedures for rapididentification of Hj-producing algal mutants with increasedOy-tolerance in Biohydrogen (Zaborsky 0 R, ed), pp. 227-234, Plenum Publishing Corporation, New York
ith II, R D,ientificarionChlamydo-)4
, Melis A,1 photopro-lase gene in2163
drogen fuel
.adden D Rand-binding
the first9-39196
an anoxic carbon-dioxide-containing atmosphere. PhotochemPhotobiol 50, 571-576
59 White A L & Melis A (2006) Biochemistry of hydrogenmetabolism in Chlamydomonas reiuhardtii wild type and aRubisco-less mutant. J lnt Hydrogen Energy 31,455-464
60 Jacquot J P, Stein M, Suzuki A, Liottet S, Sandoz G &Miginiac-Maslow M (1997) Residue Glu-91 of Chlamydo-monas reinhardtii ferredoxin is essential for electron transferto ferredoxin-thioredoxin reductase. FEBS Lett 400,293-296
61 Roessler P & Lien S (1984) Purification of hydrogenase fromChlamydomonas reiuhardtii. Plant Physiol 75, 705-709
62 Happe T & Naber J D (1993) Isolation characterization andN-tenninal amino acid sequence of hydrogenase from greenalga Chlamydomonas reinhardtii. Eur J Biochem 214,475-481
63 Melis A, Zhang L, Forestier M, Ghirardi M L & Seibert M(2000) Sustained photobiological hydrogen gas productionupon reversible inactivation of oxygen evolution in the greenalga Chlamydomonas reinhardtii. Plant Physiol 122, 127-135
64 Melis A, Seibert M & Happe T (2004) Genomics of greenalgal hydrogen research. Photosyntli Res 82, 277-288 andrefs therei n
65 Wykoff D D, Davies J P, Melis A & Grossman A R (1998)The regulation of photosynthetic electron transport duringnutrient deprivation in Chlamydomonas reinhardtii. PlantPhysioll77,129-139
66 Kosourov S, Tsygankov A, Seibert M & Ghirardi M L(2002) Sustained hydrogen photoproduction by Chlamydo-monas reinhardtii - Effects of culture parameters. BiotechnolBioeng 78, 731-740
67 Kosourov S, Seibert M & Ghirardi M L (2003) Effects ofextracellular pH on the metabolic pathways in sulfur-deprived Hz-producing Chlamydomonas reinhardtii cultures.Planl Cell Physiol 44, 146-155
68 Zhang L, Happe T & Melis A (2002) Biochemical andmorphological characterization of sulfur deprived and Hz-producing Chlamydomonas reinhardtii. (green algae) Planta214, 552-561
69 Tsygankov A, Kosourov S, Seibert M & Ghirardi M L(2002) Hydrogen photoproduction under continuous illumi-nation by sulfur-deprived synchronous Chlamydomonasreinhardtii cultures. J lnt Hydrogen Energy 27, 1239-1244
70 Lee J W & Greenbaum E (2003) A new oxygen sensitivityand its potential application in photosynthetic H2 production.Appl Biochem Bioeng 105-108,303-313
71 De Vitry C, Oyuang Y. Finazzi G. Wollman FA & Kallas T(2004), The chloroplast Rieske iron-sulfur protein - at thecrossroad of electron transport and signal transduction. J BioiClietn 2?9, 44621-44627
72 Finazzi G & Rappaport F (1998) In vivo characterization ofthe electrochemical proton gradient generated in darkness ingreen algae and its kinetic effects on cytochrome b6fturnover. Biochem 38, 9999-10005
73 Finazzi G, Furia A, Barbagallo R P & Forti G (1999) Statetransitions cyclic and linear electron transport and
210 INDIAN J. BIOCHEM. BIOPHYS., VOL. 43, AUGUST 2006
photophosphorylation in Chlamydomonas reinhardtii.Biochim Biophys Acta 1413, 117-129
74 Kruse 0, Rupprecht J, Bader K-P, Thomas-Hall S, Schenk PM, Finazzi G & Hankamer B (2005) Improved photo-biological H2 production in engineered green algal cells. JBioI Chem 280,34170-34177
75 Ghirardi M L, Zhang L, Lee J W, Flynn T, Seibert M,Greenbaum E & Melis A (2000) Sustained photobiologicalhydrogen gas production upon reversible inactivation ofoxygen evolution in the green alga Chlamydomonasreinhardtii. Trends Biotechnol 8, 506-511
76 Fedorov A S, Kosourov S, Ghirardi M L & Seibert M (2005)Continuous hydrogen photoproduction by Chlamydomonasreinhardtii using a novel two-stage sulfate-limited chemostatsystem. Appl Biochem BiotechnolI21-124, 403-412
77 Laurinavichene T V, Fedorov A S, Ghirardi M L, Seibert M& Tsygankov A A (2006) Demonstration of sustainedhydrogen photoproduction by immobilized sulfur-deprivedChlamydomonas reinhardtii cells. Intern J Hydrogen Energy31,659-667
78 Melis A & Chen H-C (2005) Chloroplast sulfate transport ingreen algae, genes proteins and effects. Photosytuh Res 86,299-30
79 Chen H-C & Melis A (2004) Localization and functionof SulP a nuclear-encoded chloroplast sulfate per-mease 111 Chlamydomonas reinhardtii. Planta 220,198-210
80 Chen H-C, Newton A J & Melis A (2005) Role of SulP anuclear-encoded chloroplast sulfate permease in sulfate
.transport and Hz evolution in Chlamydomonas reinhardtii.Photosyntli Res 84, 289-296
8l Harnmarstrom L, Sun L, Akerrnark B & Styring S (2001)A biomimetic approach to artificial photosynthesis,Ru(lI-polypyridine photo-sensitisers linked to tyrosineand manganese electron donors. Spectrochim Acta 37A,2145-2160
82 Moore T A, Moore A L & Gust D (2002) The design andsynthesis of artificial photosynthetic antennas reactioncentres and membranes. Phil Trans R Soc London B 357,1481-1498
IndianVol. 4:
X-raymodelimponbiologavailalthe 31'Highaspectstructua largwouldprogradetermstructuatomicappliesearlier'ACORsecondstrandsACOR
Dynpowerf
E-mail:t