Table Of ContentKimetal.MicrobialCellFactories2012,11:2
http://www.microbialcellfactories.com/content/11/1/2
RESEARCH Open Access
Improved production of biohydrogen in light-
powered Escherichia coli by co-expression of
proteorhodopsin and heterologous hydrogenase
Jaoon YH Kim1, Byung Hoon Jo2, Younghwa Jo1 and Hyung Joon Cha1,2*
Abstract
Background: Solar energy is the ultimate energy source on the Earth. The conversion of solar energy into fuels
and energy sources can be an ideal solution to address energy problems. The recent discovery of proteorhodopsin
in uncultured marine g-proteobacteria has made it possible to construct recombinant Escherichia coli with the
function of light-driven proton pumps. Protons that translocate across membranes by proteorhodopsin generate a
proton motive force for ATP synthesis by ATPase. Excess protons can also be substrates for hydrogen (H )
2
production by hydrogenase in the periplasmic space. In the present work, we investigated the effect of the co-
expression of proteorhodopsin and hydrogenase on H production yield under light conditions.
2
Results: Recombinant E. coli BL21(DE3) co-expressing proteorhodopsin and [NiFe]-hydrogenase from
Hydrogenovibrio marinus produced ~1.3-fold more H in the presence of exogenous retinal than in the absence of
2
retinal under light conditions (70 μmole photon/(m2·s)). We also observed the synergistic effect of proteorhodopsin
with endogenous retinal on H production (~1.3-fold more) with a dual plasmid system compared to the strain
2
with a single plasmid for the sole expression of hydrogenase. The increase of light intensity from 70 to 130 μmole
photon/(m2·s) led to an increase (~1.8-fold) in H production from 287.3 to 525.7 mL H /L-culture in the culture of
2 2
recombinant E. coli co-expressing hydrogenase and proteorhodopsin in conjunction with endogenous retinal. The
conversion efficiency of light energy to H achieved in this study was ~3.4%.
2
Conclusion: Here, we report for the first time the potential application of proteorhodopsin for the production of
biohydrogen, a promising alternative fuel. We showed that H production was enhanced by the co-expression of
2
proteorhodopsin and [NiFe]-hydrogenase in recombinant E. coli BL21(DE3) in a light intensity-dependent manner.
These results demonstrate that E. coli can be applied as light-powered cell factories for biohydrogen production by
introducing proteorhodopsin.
Keywords: biohydrogen, Escherichia coli, proteorhodopsin, light-driven proton pump, light-powered cell factory
Background energy [1]. Among various renewable energy sources,
Since the Industrial Revolution, energy consumption has solar energy is the most abundant and ultimate source.
increased exponentially and most energy has been The total amount of solar energy absorbed by the
derived from fossil fuels. Currently, we still depend on Earth’s surface is 1.74 × 105 terawatts (TW) [2], which
fossil fuels for more than 80 percent of our demands for is a tremendous amount compared to the world’s energy
electricity, transportation, and industries, although con- consumption (~13 TW) [1]. Thus, the conversion of
cerns about the exhaustion of fossil fuels and global solar energy to fuels may constitue the most sustainable
warming have led to increased attention to renewable way to solve the energy crisis.
In the field of biotechnology, the photosynthetic pro-
cess in algae and cyanobacteria has been actively investi-
*Correspondence:[email protected] gated for the conversion of solar energy to useful
1DepartmentofChemicalEngineering,PohangUniversityofScienceand
biofuels [3-5]. Photosynthesis requires a highly complex
Technology,Pohang790-784,Korea
Fulllistofauthorinformationisavailableattheendofthearticle
©2012Kimetal;licenseeBioMedCentralLtd.ThisisanOpenAccessarticledistributedunderthetermsoftheCreativeCommons
AttributionLicense(http://creativecommons.org/licenses/by/2.0),whichpermitsunrestricteduse,distribution,andreproductionin
anymedium,providedtheoriginalworkisproperlycited.
Kimetal.MicrobialCellFactories2012,11:2 Page2of7
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photosystem composed of numerous proteins and including 6 genes (crtE, B, I, Y, b-diox, and pR), that are
photosynthetic enzymes, such as Rubisco [1]. In addi- required for the functional heterologous expression of
tion, many challenges still remain for engineering photo- proteorhodopsin in E. coli (Figure 1). The recombinant
synthetic microorganisms [1,6]. Recently, a new type of E. coli BL21(DE3) harboring pACYC-RDS was cultured,
rhodopsin, called proteorhodopsin, was discovered in and protein expression was induced under exposure to
the metagenome of uncultured marine g-proteobacteria 70 μmol photon/(m2·s) light. From the harvested cell
[7]. Proteorhodopsin can be heterologously expressed in pellet, we observed that the cells expressing proteorho-
Escherichia coli to possess proton-pumping activity [7], dopsin with endogenous retinal have a distinctively red-
which is different from bacteriorhodopsin found in halo- dish color compared to wild-type cells (Figure 2A).
bacteria [1,8]. This property of proteorhodopsin enables In addition, we confirmed that the membrane fraction,
the investigation of its impact on cellular energy and including recombinant proteorhodopsin (generated
phototrophy [8]. There have also been reports of the by the expression of a single pR gene), absorbs light at a
enhancement of cell viability or growth via light-driven specific wavelength of 520 nm in the presence of exo-
proton pumping by proteorhodopsin under nutrient- genous retinal, indicating the functional expression of
limited conditions [9-11]. However, there have been no recombinant proteorhodopsin in E. coli (Figure 2B).
substantial applications in biofuel production using pro-
teorhodopsin, although this potential has been men- Co-expression effect of proteorhodopsin and
tioned recently [1]. hydrogenase on H production
2
Hydrogen (H ) has been recognized as one promising After confirmation of proteorhodopsin function in
2
alternative energy source to fossil fuels. It does not emit recombinant E. coli, we investigated the effect of co-
carbon dioxide during combustion and can be easily expressing proteorhodopsin and H. marinus [NiFe]-
converted to electricity using fuel cells. In addition, it hydrogenase on H production. We used two kinds of
2
has a higher energy density than other energy sources. expression systems: a single plasmid system of
Although the current production of H mainly depends pET-HmH/pR (without endogenous retinal) and a dual
2
on thermochemical methods using fossil fuels [12], bio- plasmid system of pET-HmH and pACYC-RDS (with
logical approaches have been actively investigated to endogenous retinal) (Figure 1). E. coli BL21(DE3) trans-
generate H in a more sustainable manner [13-17]. formed with pET-HmH/pR or cotransformed with pET-
2
Among them, photobiological H production has HmH and pACYC-RDS was cultured in 125 mL serum
2
attracted great attention due to its eco-friendly proper- bottles under exposure to 70 μmol photon/(m2·s) light.
ties, such as its usage of solar energy and carbon assimi- We found that E. coli with pET-HmH/pR produced
lation. Nevertheless, there are still many obstacles to more H after retinal addition under light than the cells
2
overcome, including slow cell growth, the low conver- without retinal (Figure 3A). This result indicates that
sion efficiency of light to H , the inhibitory effect of the gained function of recombinant proteorhodopsin by
2
oxygen on hydrogenase activity, and others [16,17]. the addition of retinal has a synergistic effect on H pro-
2
E. coli has been used widely as a cell factory for many duction with the heterologous expression of hydroge-
types of bio-products (including biofuels), but it cannot nase. A negative control strain containing the parent
utilize light energy. Therefore, constructing E. coli cap-
able of absorbing light energy and converting it to other
biofuels through the introduction of proteorhodopsin
might increase biofuel production efficiency. It has been
shown that protons generated by rhodopsin can migrate
along the membrane surface [18] and thus, they can act
as substrates of hydrogenase for H evolution. Thus, in
2
the present work, for the first time (to our knowledge),
Figure1Plasmidmapsfortheexpressionofproteorhodopsin
we introduced proteorhodopsin into E. coli, generating inE.coli.ErwiniauredovoracrtE,B,I,Y(forb-carotenesynthesis),
bacteria capable of utilizing light and investigated its mouseb-dioxgene(forconversionofb-carotenetoretinal),andpR
effect on H production yield using the previously con- genecodingproteorhodopsinwereclonedintopACYC-Duet1
2
structed recombinant E. coli expressing Hydrogenovibrio vectortoconstructpACYC-RDS.Allofthegeneswereamplified
usingtheprimersinTable1anddigestedusingrestrictionenzymes
marinus-originated [NiFe]-hydrogenase [15].
forcloningintopACYC-Duet1.pET-HmH/pRwasconstructedby
cloningasinglepRgeneintopET-HmH,whichexpressesH.marinus
Results [NiFe]-hydrogenase,toinvestigatethefunctionofproteorhodopsin
Functional expression of proteorhodopsin in E. coli withexogenousretinal.pACYC-pR(withoutcrtE,B,I,Y,b-diox)was
E. coli does not have an intrinsic ability to absorb light alsoconstructedtomeasuretheabsorptionspectrumofE.coliwith
membranesexpressingproteorhodopsin(Figure2B).
energy. We constructed a plasmid, pACYC-RDS
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(A) (A)
10
s
e
ottl
m b 8
u
er
s
L)mL 6
n (m125
productiomedium / 4
ogen 0 mL 2 ppEETT--H21mbH/pR-Retinal
Hydrin 10 0 pET-HmH/pR+Retinal
0 10 20 30 40
Time (h) after induction
(B)
14
(BB))
s
e
ottl12
b
m
n (mL)125 mL seru108
productiomedium / 46
Hydrogen in 100 mL 02 ppEETT--HHmmHH & pACYC-RDS
0 10 20 30 40
Time (h ) after induction
Figure 3 Co-expression effect of proteorhodopsin and
Figure 2 Functional heterologous expression of hydrogenaseonH2production.(A)Co-expressioneffectof
proteorhodopsininE.coli.(A)Color-shiftbyexpressionof proteorhodopsinwithexogenousretinalandhydrogenaseonH
2
proteorhodopsin.Leftsample:E.coliBL21(DE3)/pACYC-RDS production.RecombinantE.coliBL21(DE3)withasingleplasmid,
(proteorhodopsinexpression&retinalsynthesis),rightsample:wild- pET-HmH/pR,weregrownwithorwithoutretinalunderalight
typeE.coliBL21(DE3).(B)Absorptionspectraofthemembrane intensityof70μmolephoton/(m2·s).E.coliharboringtheparent
ofE.coliexpressingproteorhodopsinalone.Membranefractions pET-21bvectorwasusedasanegativecontrol.(B)Co-expression
in50mMTris-Cl(pH8.0)and5mMMgCl2weremixedwith20μM effectofproteorhodopsinwithendogenousretinaland
all-trans-retinal.Spectraweremeasuredevery10minfor30min. hydrogenaseonH production.RecombinantE.coliBL21(DE3)
2
withtwoplasmids,pET-HmHandpACYC-RDS,weregrownunder
thesamelightconditions.H productionwasmeasuredusingGCas
2
describedintheMethods.Eachvalueanditserrorbarsrepresent
vector (pET-21b) did not produce H2 under the same themeanoftwoindependentculturesandthestandarddeviation,
conditions. Using the dual plasmid system for endo- respectively.
genous retinal biosynthesis, we also observed similar
results (Figure 3B). The BL21(DE3) strain with the
dual plasmid system (pET-HmH and pACYC-RDS) culture. We increased light intensity from 70 μmole
also produced ~1.3-fold more H compared to the photon/(m2·s) to 130 μmole photon/(m2·s) at the middle
2
strain expressing hydrogenase (pET-HmH) alone. part of culture bottles by changing the light source from
Although the strain harboring two plasmids showed two 20 W fluorescent lamps to two 30 W fluorescent
lower H production than the strain expressing only lamps. We observed that the cells cultured at a light
2
hydrogenase at 12 h, its production rapidly increased intensity of 130 μmole photon/(m2·s) produced more H
2
and surpassed the H production of the hydrogenase- than those cultured at 70 μmole photon/(m2·s) (Figure
2
only strain after 19 h (Figure 3B). 4). At 24 h after induction, the cells grown under 130
μmole/(m2·s) light produced 184 ± 8.9 mL H while
2
Light intensity effect on H production by co-expression cells grown under 70 μmole photon/(m2·s) light pro-
2
of proteorhodopsin and hydrogenase duced 100.5 ± 0.8 mL H , corresponding to yields of
2
To investigate the effect of light energy on H produc- 525.7 ± 25.4 mL H /L-culture and 287.3 ± 2.1 mL H /
2 2 2
tion by the co-expression of proteorhodopsin and L-culture, respectively. A production rate of 21.9 mL
hydrogenase, light intensity was changed during the H /(L-culture·h) was achieved from the culture for 24 h
2
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600 200
70 mol photon/(m2.s)
d 500 130 mol photon/(m2.s)
drogen production yielL H/ L culture)2 234000000 51100500 H production (mL)2
Hy(m 100
0 0
0 13 24
Time (h) after induction
Figure4TheeffectoflightintensityonH productionbyco-
2
expressionofproteorhodopsinandhydrogenase.Recombinant
E.coliBL21(DE3)withtwoplasmids,pET-HmHandpACYC-RDS,
weregrownin500mLsealedserumbottlescontaining350mLM9
medium.Lightintensitywasadjustedto70μmolephoton/(m2·s)or Figure 5 Schematic diagram of H2 production by the co-
130μmolephoton/(m2·s).H productionwasmeasuredusingGC. expressionofproteorhodopsinand[NiFe]-hydrogenaseinE.
Eachvalueanderrorbarsre2presentthemeanoftwoindependent coli.Proteorhodopsintransportsprotonsacrossthemembraneby
culturesandthestandarddeviation. absorptionoflightenergy.Protonsaretransferredtotheactivesite
inthelargesubunit(L)of[NiFe]-hydrogenaseintheperiplasmand
reducedtoH bytheadditionofanelectron,whichistransferred
2
thorough[Fe-S]clustersinthesmallsubunitof[NiFe]-hydrogenase
at a light intensity of 130 μmole photon/(m2·s). Cell (S)andb-typecytochrome(Cytb,encodedbyhyaCinE.coli)inthe
innermembrane.Abbreviations:OM,outermembrane;PS,
growth was similar between the two cultures under dif-
periplasmicspace;IM,innermembrane.
ferent light conditions (data not shown). This indicated
that improved H production (0.0835 L H , equivalent
2 2
to 0.902 kJ) was derived from enhanced light energy (60 function, gained with exogenous retinal, provided a
μmole photon/(m2·s) for 24 h, equivalent to 26.579 kJ). synergistic effect on H production through the sup-
2
Thus, we might determine that the conversion efficiency plementation of protons. Similarly to the single plas-
of light energy to H was ~3.4% in the present work mid system, we also observed a positive effect of
2
using recombinant E. coli BL21 expressing both proteor- proteorhodopsin using a dual plasmid system. H pro-
2
hodopsin and H. marinus [NiFe]-hydrogenase. duction levels of cells co-expressing hydrogenase, pro-
teorhodopsin, and retinal synthesis proteins was ~1.3-
Discussion fold higher at the final time point than that of the cells
E. coli is a chemotroph that cannot utilize light energy. expressing only hydrogenase. It is noteworthy that H
2
Bacteriorhodopsins, including proteorhodopsin, are the production from the strain co-expressing proteorho-
simplest molecules that enable microorganisms to uti- dopsin and hydrogenase quickly surpassed H produc-
2
lize solar energy to create a proton gradient and gener- tion from the strain expressing only hydrogenase at a
ate ATP. In addition, protons translocated by rhodopsin late phase. This retarded H production profile of the
2
can migrate along the membrane [18] and might be dual plasmid system might be attributed to the meta-
substrates of hydrogenase for the evolution of H (Fig- bolic burden caused by the over-expression of multiple
2
ure 5). Thus, we tried to convert light energy to H by proteins required for the biosynthesis of retinal and
2
exploiting the light-driven proton-pumping function of proteorhodopsin.
proteorhodopsin in recombinant E. coli expressing Because the function of proteorhodopsin is driven by
hydrogenase. the absorption of light energy, light intensity can be a
We observed functional expression of proteorhodop- key factor for H production. We found that the H
2 2
sin in the recombinant E. coli BL21(DE3) and thus production from cells co-expressing hydrogenase and
investigated the effect of co-expressing proteorhodop- proteorhodopsin with endogenous retinal is strongly
sin and H. marinus [NiFe]-hydrogenase on H produc- dependent on light intensity. Increasing the light inten-
2
tion. Using a single plasmid system expressing both sity from 70 μmole photon/(m2·s) to 130 μmole photon/
hydrogenase and proteorhodopsin without biosynthesis (m2·s) increased H production yield ~1.8-fold. However,
2
of endogenous retinal, we found that H production cell growth was not different in the two cultures under
2
was improved by ~29% in the presence of added ret- different light conditions. This tendency was consistent
inal under light. This indicates that proteorhodopsin with a previous report that proteorhodopsin contributes
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to cell growth only under nutrient-limited conditions NH Cl, 0.5 g/L NaCl, and 1 mg/L thiamine supple-
4
[9]. Thus, this indicates that H production was mented with 240.73 mg/L MgSO , 11.09 mg/L CaCl )
2 4 2
improved by the absorption of enhanced light energy including 5 g/L casamino acids and 5 g/L glucose. 0.1
through the proton-pumping function of proteorhodop- M of FeSO and 1 M of NiSO solutions were added
4 4
sin. In the present study, it seems that the reduction to M9 medium at a concentration of 30 μM for the
from proton to H mediated by hydrogenase generates functional expression of H. marinus [NiFe]-hydroge-
2
additional proton gradient, driving proteorhodopsin to nase. All cultures were maintained under normal aero-
pump protons, the substrates for hydrogenase. When we bic or micro-aerobic conditions [14]. Cells were grown
calculated the production rate per culture volume (21.9 in serum bottles (125 or 500 mL; Wheaton, USA)
mL H /(L-culture·h)) and the conversion efficiency of sealed with rubber stoppers and aluminum capping at
2
light energy to H (~3.4%), the levels achieved in this 37°C in an air-shaking incubator (Jeiotech, Korea) at a
2
study were comparable to the results of photobiological gyration rate of 200-230 rpm. For the assessment of
hydrogen production in previous studies: H production the functional activity of proteorhodopsin, cells were
2
rate in green algae, 0.048-4.48 mL H /(L-culture·h), cya- irradiated by 20 W or 30 W fluorescent lamps. The
2
nobacteria, 4.03-13 mL H /(L-culture·h), photosynthetic light intensity (400-700 nm) at a given location in the
2
bacteria, 7.6-131 mL H /(L-culture·h), and light conver- culture was measured using a light meter (Apogee,
2
sion efficiency in photoautotrophs, 3-10% with removal USA) in units of μmol photon/(m2·s).
of O or 1-2%, and photoheterotrophs, 0.308-9.23%)
2
[17,19]. Although cell growth was mainly supported by Plasmid construction
exogenous nutrients in this system, these results are For the expression of proteorhodopsin in E. coli BL21
quite meaningful for the development of an E. coli sys- (DE3), four genes (Erwinia uredovora crt E, B, I, Y; Gen-
tem that can utilize light energy as a supplementary bank: D90087) for b-carotene synthesis and b-diox gene
source. for the conversion of b-carotene to retinal (mouse b-
carotene-15,15’-dioxygenase; Genbank: AF271298) were
Conclusions obtained from pORANGE and b-plasmid (a kind gift
Here, we demonstrated the substantial application of from Dr. J. von Lintig), respectively [20]. pR gene coding
proteorhodopsin for light-driven biohydrogen produc- for proteorhodopsin (Genbank: AF279106) was also
tion in a recombinant E. coli system. E. coli engineered obtained from BAC clone EBAC31A08 (a kind gift from
to express H. marinus [NiFe]-hydrogenase and proteor- Dr. E. Delong) [7]. All six genes above were amplified
hodopsin produced more H with the existence of ret- by polymerase chain reaction (PCR) using the primers
2
inal under light conditions. Engineered strains also in Table 1 and cloned into the pACYCDuet-1 (Novagen)
produced more H as light intensity increased, although vector to construct pACYC-RDS (Figure 1), which is
2
there was no difference in cell growth. These results compatible with the pET vector. For the expression of
suggest that our system works for converting light H. marinus [NiFe]-hydrogenase genes, the pET-HmH
energy to H via the cooperation of proteorhodopsin vector was used [15]. To analyze the effect of functional
2
and hydrogenase. In addition, engineering E. coli as proteorhodopsin with retinal, the proteorhodopsin gene
light-powered cell factories could provide a solution for (pR), without the genes for retinal synthesis, was cloned
developing potential strategies for photobiological H into the pET-HmH vector to construct pET-HmH/pR
2
production. (Figure 1).
Methods Measurement of In vivo H production
2
Bacterial strains and culture conditions H gas produced in cell culture was obtained from the
2
E. coli Top10 (Invitrogen, USA) was used for the headspace of a sealed serum bottle (125 or 500 mL).
manipulation and cloning of target genes. E. coli BL21 Usually, 20-100 μL of the gas sample was analyzed using
(DE3) (Novagen, USA) was used for expression of a gas chromatograph (GC; Younglin Instrument, Korea)
recombinant proteins. We used LB medium including equipped with a carboxen-1010 PLOT column (0.53
proper antibiotics (50 μg/mL ampicillin and 30 μg/mL mm × 30 m, Supelco, USA) and pulsed discharge detec-
chloramphenicol) for genetic manipulation. For protein tor (Valco Instrument, USA). Elution was performed
expression, 1 mM (as a final concentration) of isopro- using helium as a carrier gas at a flow rate of 10 mL/
pyl-b-D-thiogalactopyranoside (IPTG; BioBasic, min, and the temperatures of the injector, detector, and
Canada) was added to each culture medium. For in oven were set to 130, 250, and 100°C, respectively. The
vivo H production, cells were grown in M9 minimal H concentration in the gas sample was calculated using
2 2
medium (6 g/L Na HPO , 3 g/L KH PO , 1 g/L a standard curve. The H amount was determined based
2 4 2 4 2
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Table 1Primer sequences for amplification ofgenes related tob-carotene synthesis,retinal synthesis, and
proteorhodopsin
Primername Sequence(5’®3’)(___:ribosomebindingsite,:___restrictionsite)
crtEforward GCCCATGGATGACGGTCTGCGCAAAAAAACACG
crtEreverse GCGAATTCTTAACTGACGGCAGCGAGTTTTTTG
crtBforward CCGAATTCAAGGAGATATACCAATGAATAATCCGTCGTTACTCAATCATGC
crtBreverse CGGTCGACCTAGAGCGGGCGCTGCCAGAG
crtIforward CCGTCGACAAGGAGATATACAAATGAAACCAACTACGGTAATTG
crtIreverse CGAAGCTTTCATATCAGATCCTCCAGCATC
crtYforward CCAAGCTTGAAGGAGATATACCAATGCAACCGCACTATGATCTGATTCTC
crtYreverse GCCTTAAGTTAGCGATGAGTCGTCATAATGGC
b-dioxforward GGAGATCTAAGGAGATATACATATGGAGATAATATTTGGCCAGAATAAG
b-dioxreverse CCGGTACCTTAAAGACTTGAGCCACCATGACCC
pRforward CCGGTACCAAGGAGATATACAAATGGGTAAATTATTACTGATATTAGGTAG
CCGCGGCCGCAAGGAGATATACAAATGGGTAAATTATTACTGATATTAGGTAG
pRreverse GCCTCGAGTTAAGCATTAGAAGATTCTTTAACAGCAAC
on the H concentration and gas volume of headspace Acknowledgements
2
(including expanded volume). ThisworkwassupportedbytheManpowerDevelopmentProgramfor
MarineEnergyfundedbytheMinistryofLand,TransportandMaritime
Affairs,KoreaandtheNationalResearchFoundationGrant(NRF-2009-
Measurement of absorption spectra of proteorhodopsin 0093214)andtheBrainKorea21ProgramfundedbytheMinistryof
The absorption spectrum of cells expressing proteorho- Education,ScienceandTechnology,Korea.WethankDr.Johannesvon
Lintig(CaseWesternReserveUniv.)andDr.EdwardDeLong(MIT)fortheir
dopsin was measured using spectrophotometry [7]. To
giftsofplasmids.
prepare crude membrane fractions, recombinant E. coli
wereharvestedbycentrifugationat4°Cand4,000rpmfor Authordetails
1DepartmentofChemicalEngineering,PohangUniversityofScienceand
15min.Thecellpelletwasresuspendedin50mMTris-Cl Technology,Pohang790-784,Korea.2SchoolofInterdisciplinaryBioscience
(pH6.8)anddisruptedwithasonicdismembrator(Fisher andBioengineering,PohangUniversityofScienceandTechnology,Pohang
Scientific,USA)for10minat50%power (5secpulseon 790-784,Korea.
and2 sec pulse off). The disrupted cell suspension(total Authors’contributions
celllysate)wascentrifugedat4°Cand10,000gfor20min. JYHKandHJCdesignedresearch.JYHK,BHJ,andYJperformedandanalyzed
Theresultantsupernatant(crudeextract)wascentrifuged biohydrogenproductioninrecombinantE.coli.JYHKandHJCwrotethe
paper.Allauthorshavereadandapprovedthefinalversionofthe
at 4°C and 120,000 g for 120 min. The pellet was resus-
manuscript.
pended in 50 mM Tris-Cl (pH 8.0) and 5 mM MgCl ,
2
whichisregardedasthecrudemembrane.Theabsorption Competinginterests
Theauthorsdeclarethattheyhavenocompetinginterests.
spectrumwas measured at spectrum mode using a spec-
trophotometer(Shimadzu,Japan)aftertheaddition of20 Received:22December2011 Accepted:4January2012
μMofall-trans-retinal(Sigma). Published:4January2012
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