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RESEARCHARTICLE

Copyright copy 2007 American Scientific PublishersAll rights reservedPrinted in the United States of America

Journal ofNanoscience and Nanotechnology

Vol 7 2696ndash2708 2007

Cyanobacteria as Bioreactors for theSynthesis of Au Ag Pd and Pt Nanoparticles

via an Enzyme-Mediated Route

Roberta Brayner1lowast Heacutelegravene Barberousse2 Miryana Hemadi3 Chakib Djedjat2 Claude Yeacutepreacutemian2Thibaud Coradin3lowast Jacques Livage34 Fernand Fieacutevet1 and Alain Couteacute21Interfaces Traitements Organisation et Dynamique des Systegravemes (ITODYS) UMR-CNRS 7086Universiteacute Paris 7-Denis Diderot case 7090 2 place Jussieu 75251 Paris Cedex 05 France

2Deacutepartement RDDM USM 505 Museacuteum National drsquoHistoire Naturelle case 39 57 rue Cuvier 75005 Paris France3Chimie de la Matiegravere Condenseacutee de Paris UMR-CNRS 7574-Universiteacute Paris 6-Pierre et Marie Curie

4 Place Jussieu 75252 Paris Cedex France4Collegravege de France 11 place Marcelin-Berthelot 75005 Paris France

Common Anabaena Calothrix and Leptolyngbya cyanobacteria are shown to form Au Ag Pdand Pt nanoparticles of well-controlled size These nanoparticles are synthesized intra-cellularlyand naturally released in the culture medium where they are stabilized by algal polysaccharidesallowing their easy recovery The size of the recovered particles as well as the reaction yield isshown to depend on the cyanobacteria genus Investigations of nanoparticle formation indicatethat the intracellular nitrogenase enzyme is responsible for the metal reduction but that the cellularenvironment is involved in the colloid growth process

Keywords Cyanobacteria Nanoparticles Nitrogenase Enzymatic Reduction

1 INTRODUCTION

Recent concern for sustainable development urgeschemists to find new synthetic routes with limited eco-logical impact involving natural renewable resources Inthe field of nanomaterials a number of living organismsare already well-known to elaborate nanostructuredcomposites1ndash4 However inorganic elements that biomin-eralizing organisms have learned to deal with are of verylimited diversity5 In order to take profit from living organ-isms capability to elaborate materials many studies havebeen devoted to the use of cell extracts or biomimeticmolecules to control non biogenic inorganic phase forma-tion6ndash13 Alternatively it should be possible to use the syn-thetic capabilities of living cells for the design of newnanomaterials14ndash17 Thus bacteria fungi and plant cellswere recently used for the intra- and extracellular synthesisof silver and gold nanoparticles but with a limited controlof particle size and morphology18ndash26

In this context cyanobacteria appear as very promis-ing organisms for designing metal-based nanomaterialsThese widespread photosynthetic prokaryotes are well-known to be able to uptake a number of heavy-metal

lowastAuthors to whom correspondence should be addressed

ions27ndash28 a property that have been widely used for thetreatment of domestic and industrial effluents29 Moreovercyanobacteria are assumed to be predominant atmosphericN2-fixing microorganisms The reduction of atmosphericdinitrogen gas (N2) to ammonia is catalyzed by the nitro-genase enzyme It is therefore interesting to evaluate theability of this enzyme to reduce metal salts and controlnanoparticle growth

The interest of this approach was recently demonstratedby Lengke et al who showed that Au nanoparticles couldbe synthesized by the filamentous Plectonema boryanumcyanobacteria with variation in particle morphology withgold salt30 In this work we demonstrate that threeother filamentous cyanobacteria strains ie AnabaenaCalothrix and Leptolyngbya have the capability to formAu nanoparticles of well-controlled size Moreover Agas well as Pd and Pt nanoparticles both of which havenever been reported to be synthesized by living organismsbefore could be formed intra-cellularly The colloids werenaturally released in the culture medium embedded in apoly-saccharidic network that limit their aggregation andallow their easy recovery TEM studies allow the localiza-tion of particle formation in specific cells where nitroge-nase is present In vitro ability of this enzyme to reduce

2696 J Nanosci Nanotechnol 2007 Vol 7 No 8 1533-4880200772696013 doi101166jnn2007600

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RESEARCHARTICLE

Brayner et al Cyanobacteria as Bioreactors for the Synthesis of Au Ag Pd and Pt Nanoparticles

metal salts was evaluated leading to the formation ofnanoparticles whose with shapes and dimensions differfrom in vivo colloids This therefore suggests that nitro-genase is responsible for the reduction process but thatthe nanoparticle growth is also controlled by cyanobacteriacellular environment

2 EXPERIMENTAL DETAILS

21 Micro-Algae Description and Culture

The epilithic cyanobacteria Calothrix pulvinata strainALCP 745A (MNHN culture collection) was isolated froma sample of a black soiling developing on a building nearGenegraveve (Switzerland) Anabaena flos-aquae strain ALCPB24 came from MNHN Culture Collection Leptolyng-bya foveolarum strain ALCP 671B (MNHN culture col-lection) was isolated from a sample of a black soilingdeveloping on a wall in Strasbourg (France)

Morphologically the three cyanobacteria genera usedfor our experimentation are composed of one linear seriesof vegetative cells Moreover Calothrix possesses one het-erocyt at its basal apex (heterocyt has a thick wall andthe cell inside appears yellowish and empty Its peculiarphysiology is atmospheric dinitrogen gas (N2) fixation toammonia that is catalysed by nitrogenase enzyme gen-erated by the cell itself) On the other hand Anabaenamay have a lot of heterocyts dispersed all along thetrichome and finally Leptolyngbya has none Calothrix tri-chome is embedded in a gelatinous sheath and its diam-eter is decreasing from the base to the apex Anabaenaand Leptolyngbya are isodiametric In addition in thiswork Anabaena was grown deprived of nitrogen sourcein order to favor high production of heterocyts Finally thegenus Leptolyngbya which fixes nitrogen anaerobically31

presents only vegetative cells and consequently it has lessnitrogenase than Calothrix or Anabaena

All cyanobacteria were grown in 250 ml Erlenmeyerflasks each containing 150 ml of sterile Boldrsquos Basalmedium Calothrix Anabaena and Leptolyngba weregrown for one month 20 days and 6 days respec-tively before addition of metal salts (HAuCl4 AgNO3Pd(NO32 and H2PtCl6) in concentrations from 10minus6 M to10minus3 M

22 In vitro Enzymatic Reduction of Metallic Salts

Au Ag Pd and Pt metallic nanoparticles were synthesizedalso by in vitro enzymatic reduction of their metallic saltsby nitrogenase The general procedure involves addition ofone unit of nitrogenase in Boldrsquos Basal medium containing1 g middotLminus1 of Calothrix micro-algae released polysaccharides(RPS) extracted following published procedures32 to sim-ulate in vivo conditions The reduction was observed afteraddition of 10minus3 M of metallic salts under vigorous stirringat 25 C during 20 minutes It was checked that no signif-icant reduction occurs when only the RPS was present

23 Cyanobacteria and NanoparticleCharacterizations

Photonic microscopy of cyanobacteria was performed witha Nikon EFD3 interferential contrast microscope Themaximum quantum efficiency of photosystem II (FvFm)that corresponds to the photosynthetic activity of thesemicro-algae was measured using a Heinz Walz PAMfluorimeter

TEM measurements were performed with a HitachiH-700 operating at 75 kV For TEM studies cyanobacteriawere fixed in a mixture containing 25 of glutaraldehyde10 of acrolein and 01 of ruthenium red in a phos-phate Soumlrengen Buffer (01 M pH 74) Dehydration wasthen achieved in a series of ethanol baths and the sam-ples were processed for flat embedding in Epon 812 resinUltrathin sections were made using a Reicherd E YoungUltracut ultramicrotome (Leica) Sections were contrastedwith ethanolic uranyl acetate before visualization

For scanning electron microscopy (SEM) the biomasswas coated with carbon in a Balzers Union SCD 40sputter-coater and analyzed on a Cambridge Stereoscan120 instrument at an accelerating voltage of 20 kV X-rayenergy dispersive spectrometry (EDX) was performedusing a (EDXD) EDAX system equipped with a super ultrathin window (SUTW) connected to a JEOL JSM 6100scanning electron microscope operating at 25 kV Atomiccompositions () were obtained with Genesis software

X-ray powder diffraction (XRD) patterns were recordedusing a CuK radiation The diffractometer was calibratedusing a standard Si sample The counting time was 30 s perstep of 005 2 The mean crystallite size was estimatedusing the Scherrer equation

X-ray photoelectron spectroscopy (XPS) was performedin a Thermo VG Scientific Sigma Probe spectrometerequipped with a monochromatic AlK X-ray source(14866 eV) was used at a spot size of 400 m The passenergy was set at 100 and 40 eV for the survey and thehigh-resolution spectra respectively The step size was1 eV for the survey spectrum and 02 eV for the high-resolution spectra Charge compensation was achievedwith a flood gun of 6 eV electrons using standard proce-dures for this spectrometer The surface composition wasdetermined using the manufacturerrsquos sensitivity factors

UV-Visible-NIR spectra were recorded on a Cary 5Espectrophotometer in transmission mode

3 RESULTS

31 Gold

Before reaction for all experiments the culture mediumadded with (i) [Au3+ = 10minus3 M presents a transparent-yellow colored solution (ii) [Au3+ = 10minus4 M presents aweak transparent-yellow colored solution and finally (ii)[Au3+= 10minus6 M presents a transparent-uncolored solutionbecause Au3+ concentration is very weak All experiments

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RESEARCHARTICLE

Cyanobacteria as Bioreactors for the Synthesis of Au Ag Pd and Pt Nanoparticles Brayner et al

Fig 1 (a) Conical flasks showing Calothrix in different culture [Au3+]concentration medium photonic micrograph of (b) Calothrix in sterileBoldrsquos Basal medium vegetative cells (VC) heterocyt (H) gelatinoussheath (SH) (c) Calothrix grown in [Au3+= 10minus3 M solution (d) a shortfragment of Anabaena trichome in sterile medium deprived of nitrogensource (e) Anabaena grown in [Au3+ = 10minus3 M solution (f) Leptolyn-gbya in sterile Boldrsquos Basal medium and (g) Leptolyngbya grown in[Au3+= 10minus4 M solution

were carried out until observation of color modification ofthe biomass andor of the culture medium

With Calothrix grown in culture medium containing[Au3+= 10minus3 M the solution color remained transparent-yellow after 48 h (Fig 1(a)) and photonic microscopyshowed that heterocyt color changed from yellow-greento purple-blue (Figs 1(b) and (c)) Vegetative cells werebleached (Fig 1(c)) and an increase of cell-wall thicknessand sheath production was also observed In this caseAu3+ concentration may be very close to the lethal doseleading to photosynthetic activity decrease and bleachingof vegetative cells In flasks containing Calothrix with[Au3+= 10minus4 M the culture medium color start to changefrom transparent-yellow to transparent-purple after a fewhours (Fig 1(a)) while in the presence of [Au+3 =10minus6 M the medium remained transparent-uncolored over48 h (Fig 1(a)) In both last cases the cyanobacteria pre-sented their normal blue-green color which means thatthey were alive

Figure 2(a) shows the main features of Calothrix asseen under TEM On very thin sections of Calothrix tri-chomes grown in diverse [Au3+] concentrations used inour experiment it was possible to observe from TEManalysis (Table 1) a lot of spherical well-shaped 54 nm(standard deviation () equal to 06) dark dots on thelamellae inside the heterocyt (Fig 2(b)) and also in veg-etative cells specifically on the thylacoids (Fig 2(c)) Toprecise the nature of these dark dots Calothrix sampleswere lyophilized and characterized by X-ray energy dis-persive spectrometry (EDS Fig 3(a)) X-ray photoelectron

Fig 2 TEM micrographs of cyanobacteria thin sections (a) Calothrixin sterile Boldrsquos Basal medium vegetative cell (VC) and heterocyt cell(H) gelatinous sheath (SH) cell wall (CW) (b) Calothrix grown in a10minus4 M Au3+ concentration (c) Thylacoids (TH) in Calothrix vegeta-tive cell (10minus4 M Au3+ concentration) (d) vegetative cell of Anabaena(10minus4 M Au concentration) (e) heterocyt of Anabaena (10minus4 M Au con-centration) and (f) vegetative cell of Leptolyngbya (10minus4 M Au3+ con-centration) The dark dots observed on Figures 2(b)ndash(f) represent Aumetallic nanoparticles (NP)

Table I Particle sizes obtained by TEM measurements

Micro-algae Element Particle size (nm)

Calothrix Au 54plusmn06Anabaena Au 120plusmn07Leptolyngbya Au 60plusmn06Ex-Calothrix Released-Au 57plusmn08Ex-Anabaena Released-Au 250plusmn32Ex-Leptolyngbya Released-Au 62plusmn08Calothrix Ag 150plusmn08Anabaena Ag 400plusmn42Ex-Calothrix Released-Ag 100plusmn12Ex-Anabaena Released-Ag 252plusmn25Calothrix Pd 35plusmn03Calothrix Pt 32plusmn03RPS-nitrogenase Au 400 nm (edge length)RPS-nitrogenase Ag 400plusmn40RPS-nitrogenase Pd 100plusmn12

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RESEARCHARTICLE

Brayner et al Cyanobacteria as Bioreactors for the Synthesis of Au Ag Pd and Pt Nanoparticles

(a)

(b)

(c)

20 30 40 50 60 70

Inte

nsity

(a

u)

2θ

Au0

NaCl

Au4f72(879 eV)

Binding Energy (eV)

Au4f52(842 eV)

Fig 3 Calothrix grown in [Au3+ = 10minus4 M after lyophilization (a)EDS analysis (b) XPS core-level Au4f region and (c) XRD patterns ofCalothrix

spectroscopy (XPS Fig 3(b)) and X-ray diffraction (XRDFig 3(c))

The chemical composition of Calothrix was quantifiedby EDS measurements which allow a limit of electronpenetration of about 3 m (Fig 3(a)) Samples were notcoated with gold but with carbon to allow Au quantifica-tion All samples presented C O Na P Cl K Ca Feand Au peaks Atomic percentages of Na P Cl K Ca

and Fe corresponding to algae nutriments varied between2 and 9 at while Au varied between 50 at and 15 atin Calothrix grown in 10minus3 M and 10minus4 M Au3+ concen-trations respectively These results confirm the presence ofAu inside the biomass with a rate of reproducibility about = 14 XPS analysis was used to better characterize thechemical structure of nanoparticles (Fig 3(b)) C1s O1sN1s Na1s K1s P2p Cl2p and Au4f peaks were observedin all samples but we focused our attention only on Au4fcore-level region It is important to know that XPS analy-sis is only a surface characterization technique allowing alimit of electron penetration of about 10 nm Atomic per-centages of algae nutriments (Na K P Cl) were very closeto the ones obtained by EDS measurements Au4f regionof this sample (Fig 3(b)) was fitted with two componentscentered at 842 and 879 eV assigned to metallic Au(842 eV for Au4f72 and 879 eV for Au4f52 electrons)33

To confirm the presence of metallic Au XRD measure-ments were carried out (Fig 3(c)) An amorphous organicphase was observed due to the presence of cyanobacteriabiomass Two structures were identified one face-centeredcubic (fcc) metallic Au and one fcc NaCl Au mean crys-tallite size calculation from (111) line compared to par-ticle size obtained from TEM are quite similar (sim5 nm)indicating that each particle is a single crystal Accordingto XPS TEM and XRD analysis we can finally confirmthat the dark dots observed inside the cells of Calothrixcorrespond to Au metallic nanoparticles

Figure 4(d) allows the investigation of Au nanoparticlerelease mechanism In this micrograph intercellular con-tact between adjacent cells of Calothrix filament wasobserved and it is probably maintained by means of fineplasmodesma-like structures (PS) which traverse the sep-tum and connect the plasma membranes of the two cellsSimilar structures are present in the septum between vege-tative cells and heterocyts Here we can observe the pres-ence of well-shaped 54 nm Au nanoparticles inside theseintercytoplasmic connections and outside vegetative cellsmore specifically surrounded by Calothrix mucilaginoussheath composed by exo-polysaccharides (SH)

Finally TEM examination (Table I) of the transparent-purple Calothrix culture medium showed spherical nano-particles with a narrow size distribution (= 57 nm =08) without agglomeration probably due to the presenceof released polysaccharides that promote nanoparticle sta-bilization (Fig 4(a)) Corresponding UV-Visible spectrumof this solution is presented in Fig 4(b) showing a broadband at max = 577 nm corresponding to the surface plas-mon resonance of Au nanoparticles33ndash34

For Anabaena color modification of the culture mediumtook only five minutes This behavior was assigned to thehigher number of heterocyts that produce more nitro-genase than Calothrix one For all Au3+ concentrations(from 10minus3 M to 10minus6 M) the culture medium changedfrom transparent yellow to transparent pink-purple due to

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Cyanobacteria as Bioreactors for the Synthesis of Au Ag Pd and Pt Nanoparticles Brayner et al

0

005

01

015

02

400 450 500 550 600 650 700 750 800

Leptolyngbya-Au 10-4MCalothrix-Au 10-4MAnabaena-Au 10-4M

Abs

orba

nce

Wavelength (nm)

537

577

562

10 nm

(a)

(c)

(b)

(d)

10 nm

Exo-polysaccharide (SH)

VC

PS

2 microm

Fig 4 (a) TEM micrograph of Au nanoparticles released in Calothrix culture medium (b) UV-Visible spectra of Au nanoparticles released in allcyanobacteria surrounding medium (c) TEM micrograph of Au nanoparticles released in Anabaena culture medium and (d) TEM micrograph of Aunanoparticle release mechanism using genus Calothrix (thin section)

the formation of Au metallic nanoparticles released bythe genus Anabaena These results were confirmed byUV-visible measurements that present a narrow surfaceplasmon resonance band at max = 537 nm (Fig 4(b))and biomass XPS measurements (Fig 5(a)) Photonicmicroscopy showed that heterocyt colour changed fromyellow-green to purple-blue (Figs 1(d) and (e)) The veg-etative cells of Anabaena presented a blue-green colourcharacteristic of a normal photosynthetic activity but thecell wall color changed from blue-green to pink-purple(Fig 1(e)) Contrary to the genus Calothrix it is important

to note that the genus Anabaena withstands even 10minus3 MAu3+ concentration without bleaching of vegetative cellsIn this case the lethal dose was not reached FinallyAu nanoparticles with particle size distribution equal to12 nm ( = 07 Table I) were observed on the thylakoids(Fig 2(d)) Moreover most part of these nanoparticles wasformed inside the heterocyts where nitrogenase concen-tration is very high (Fig 2(e)) In this case coalescencephenomenon was also observed leading to an increase ofparticle size distribution from 12 nm to 25 nm ( = 12Fig 2(e) Table I) This behavior was also observed for

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Brayner et al Cyanobacteria as Bioreactors for the Synthesis of Au Ag Pd and Pt Nanoparticles

(a)

(b)

Fig 5 TEM micrographs of cyanobacteria subcultures after contactavec 10minus4 M of Au3+ solution Formation of Au metallic nanoparticles(a) inside genus Calothrix (b) inside genus Anabaena

Au nanoparticles released in the surrounding medium thatpresents a broad size distribution of about 25 nm ( = 32Fig 4(c) Table I)

For Leptolyngbya color modification of the culturemedium was also noted It took a lot of time (more than100 hours) This behavior can be explained because thegenus Leptolyngbya does not possess heterocyts and fixesnitrogen only anaerobically (it is extremely sensitive tooxygen) and only in the dark31 With Leptolyngbya grownin [Au3+ = 10minus3 M the culture medium color remainedyellow In this case Au3+ concentration may be veryclose to the lethal dose similarly to Calothrix so that adecrease of photosynthetic activity and bleaching of vege-tative cells were also observed Consequently the metallicAu nanoparticles produced remained inside the cells andwere not released in the surrounding medium On the other

hand when incubated with 10minus4 M Au3+ concentrationphotonic microscopy showed that vegetative cells colorchanged from green to pinkish (Figs 1(f) and (g)) and theculture medium color changed from transparent yellow totransparent purple UV-visible spectroscopy measurementsconfirm the formation of Au metallic nanoparticles thatpresent a weak and broad surface plasmon resonance bandat max = 562 nm (Fig 4(b)) Finally for 10minus6 M Au3+

concentration the vegetative cells presented a blue-greencolor characteristic of a normal photosynthetic activityFigure 2(f) shows a thin section of Au nanoparticlesformed on Leptolyngbya thylakoids Contrary to the genusAnabaena the non-heterocyteous genus Leptolyngbya pro-duces nitrogenase only in the dark consequently thereduction reaction was very low but the particle size dis-tribution was equal to 6 nm ( = 06 Table I) similarly toCalothrix No coalescence phenomenon was observed

The best performances for Au nanoparticles productionand release were obtained with Calothrix and Anabaenacyanobacteria In order to evaluate the possibility to designa cell-based continuous bioreactor part of the cells thatwere in contact with 10minus4 M concentration of Au3+

solutions was isolated in a new sterile Boldrsquos Basal

0 20 40 60 80 100 120

020

025

030

035

040

045

050

055

Addition of HAuCl4

Fv

Fm

Time (days)

Calothrix

0 20 40 60 80 100

015

020

025

030

035

040

045 Addition of HAuCl4

Fv

Fm

Time (days)

Anabaena

(a)

(b)

Fig 6 Photosynthetic activities (a) Calothrix (Au3+ addition after20 days subcultures) (b) Anabaena (Au3+ addition after 40 dayssubcultures)

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Cyanobacteria as Bioreactors for the Synthesis of Au Ag Pd and Pt Nanoparticles Brayner et al

medium and the same concentration of Au3+ solution wasadded to 20 days of Calothrix and Anabaena subculturesThe formation and release of Au metallic nanoparticleswas observed (Fig 5) confirming the possibility to makea continuous cyanobacteria bioreactor The photosyntheticactivities of Calothrix and Anabaena after addition of Ausalts (10minus4 M) in the culture medium were measured usinga PAM fluorimeter (Fig 6) After addition of Au saltsa progressive decrease of photosynthetic activities wasobserved In both cases (Figs 6(a) and (b)) the minimumFV Fm was observed after 10 days of incubation in the pres-ence of Au nanoparticles This behavior was maybe dueto the presence of Au salts on thylakoids (photosynthe-sis sites) and their posterior reduction After reduction thethylakoids were not completely recovered by Au nanopar-ticles and the photosynthetic activities may increase againThese micro-algae remain alive more than three months inthe presence of Au nanoparticles

32 Silver

It is well known that Ag ions and Ag-based compoundsare highly toxic to microorganisms35ndash36 showing strongbiocidal effects on many species of bacteria includingE Coli37 Cyanobacteria have Gram-negative cell wallsuch as E coli so in this work biocidal tests with Ag+

salts were carried out with Calothrix and Anabaena micro-algae The possibility to produce also Ag metallic nanopar-ticles with controlled size and shape inside cyanobacteriacells was also studied Before reaction the culture mediumadded with [Ag+ = 10minus4 M presents a transparent-uncolored solution The experiments were carried out untilobservation of biomass andor culture medium color mod-ification (i) 24 hours for genus Calothrix (ii) 30 min-utes for genus Anabaena and (iii) more than 72 hoursfor genus Leptolyngbya For all cyanobacteria the culturemedium color changed from transparent-uncolored to redPhotonic microscopy showed that heterocyt and vegetativecell colors changed from blue-green to orange-red and yel-low (Fig 7(a)) Thin sections of Anabaena trichomes showspherical 40 nm Ag nanoparticles with a broad size dis-tribution = 42 (Fig 7(b) Table 1) On the other handgenus Calothrix presents well-shaped 15 nm with a narrowsize distribution ( = 08) confirming that Calothrix tri-chome led to the formation of well-controlled size metallicnanoparticles (Table I)

Figure 8(a) presents UV-visible spectra of culture mediaafter color modification For all samples a broad plasmonband was observed with maxima centered between 420ndash450 nm which may be attributed to inter-particle cou-pling effects (multipolar resonance) This behavior wasconfirmed by TEM micrographs of released Ag nanopar-ticles stabilized by micro-algae exo-polysaccharides in thesurrounding medium (Figs 8(b) and (c) Table I) Thesemicro-algae remain alive about one month in the presence

Fig 7 (a) Photonic micrograph of Anabaena grown in [Ag+ =10minus4 M (b) TEM micrograph of Anabaena grown in [Ag+ = 10minus4 Mand (c) TEM micrograph of Calothrix thin sections grown in [Ag+ =10minus4 M

of Ag nanoparticles No biocidal effect was observed dur-ing this time It was observed that released Ag nanopar-ticles by Anabaena trichome present about 25 nm insize with narrow size distribution such as Au nanopar-ticles released by the same micro-algae showing thatcyanobacteria wall may have a particle size limitation dur-ing release process (Table I)

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Brayner et al Cyanobacteria as Bioreactors for the Synthesis of Au Ag Pd and Pt Nanoparticles

0

005

01

015

02

025

03

035

04

300 400 500 600 700 800

Leptolyngbya-Ag 10-4MCalothrix-Ag 10-4MAnabaena-Ag 10-4M

Abs

orba

nce

Wavelength (nm)

100 nm

(a)

(b)

(c)

Fig 8 (a) UV-Visible spectra of Ag nanoparticles released in allcyanobacteria TEM micrographs of Ag nanoparticles released in(b) Anabaena and (c) Calothrix culture medium

33 Palladium and Platinum

Until now only Au and Ag metallic nanoparticles weresynthesized using living cells such as fungi and bacteria23

In order to extend the significance of our approach wehave evaluated the ability of cyanobacteria to reducePd2+ and Pt4+ ions as Pt Pd and PdO nanoparticles

Calothrix-Pd

Anabaena-Pd

Calothrix-Pt

Anabaena-Pt

(a)

(b)

(c)

(d)

Binding energy (eV)

Binding energy (eV)

Binding energy (eV)

Binding energy (eV)

Fig 9 XPS core-level (a) Pd3d52 region (b) Pd3d52 region (c) Pt4f72

region and (d) Pt4f72 region

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Cyanobacteria as Bioreactors for the Synthesis of Au Ag Pd and Pt Nanoparticles Brayner et al

Table II Measured binding energies (BE) of Pd3d32 and Pt4f72 insidecyanobacteria cells and their chemical state concentrations

Chemical stateSample BE (eV) concentrations ()

Calothrix-Pd 3345 Pd0 3893364 Pd2+ 611

Anabaena-Pd 3351 Pd0 2793370 Pd2+ 721

Calothrix-Pt 710 Pt0 90734 Pt2+ 665758 Pt4+ 245

Anabaena-Pt 715 Pt0 427737 Pt2+ 435761 Pt4+ 138

have interesting properties for several catalytic pro-cess (i) hydrogenation of alkenes38 (ii) oxidation ofvolatile organic compounds (VOC)39 and (iii) combustionreactions40

Fig 10 TEM micrographs of Calothrix thin sections grown in(a) [Pd2+= 10minus4 M (b) [Pt4+= 10minus4 M

Before reaction the culture media added with [Pd2+=10minus4 M and [Pt4+ = 10minus4 M present both a transparent-yellow colored solutions After salt addition color mod-ification was observed after (i) 30 minutes for generaCalothrix and Anabaena in the presence of Pd salts and(ii) about fifteen days for both micro-algae in the presenceof Pt salts For all cases the culture medium color slightlychanges from transparent-yellow to brownish-yellow Noheterocyt or vegetative cell color change was observedby photonic microscopy analyses only an increase ofmucilaginous sheath was visible The presence of Pd andPt metallic nanoparticles was confirmed by XPS analysis(Fig 9) Table II shows the binding energies (BE) observedafter peaks decomposition Here only Pd352 and Pt4f72

regions will be discussed For cyanobacteria-Pd samplesPd3d52 region was fitted with two components centered at3345 and 3364 eV for Calothrix and 3351 and 3370 eVfor Anabaena (Figs 9(a) and (b) Table II) The first onewas assigned to metallic Pd and the second one to Pd2+ inan oxygen-containing environment (PdO)41ndash42 Pd nanopar-ticles are not stable in air and a surface passivation mayoccur It was observed that genus Calothrix (389 ofPd0) is more resistant to Pd surface passivation than genusAnabaena (279 of Pd0) (Table II) For cyanobacteria-Ptsamples Pt4f72 region was fitted with three componentscentered at 710 734 and 758 eV for genus Calothrix and715 737 and 761 eV for genus Anabaena (Figs 9(c)and (d) and Table II) The first peak was attributed tometallic Pt4143 The last two peaks were attributed to Pt2+

and Pt4 in the coordination sphere of H2PtCl4 and H2PtCl6respectively In this case only one part of H2PtCl6 wascompletely reduced The best results were also obtainedwith genus Calothrix (Table II) Figure 10 shows Calothrixtrichomes thin sections grown in [Pd2+ = 10minus4 M and[Pt4+= 10minus4 M In both cases well-shaped nanoparticleswith a very narrow size distribution (( = 35 nm =03) for Pd nanoparticles and ( = 32 nm = 03) forPt nanoparticles) were observed (Table I)

34 In vitro Enzymatic Reduction of Metallic Salts

The results obtained in vivo led to conclude that reductionoccurs inside cyanobacteria heterocyts and vegetative cellsfollowed by a partial release of metallic nanoparticlesin the culture medium Because these cells specificallycontain nitrogenase a reducing enzyme we hypothesizedthat it could be responsible for nanoparticle formation Totry to confirm this hypothesis in vitro enzymatic reduc-tion of metallic salts was carried out in a Boldrsquos basalmedium using nitrogenase enzyme (NAD[P]H) as reducingagent and Calothrix released polysaccharide (RPS) to sim-ulate in vivo conditions Solution color modification wasobtained after (i) 10 minutes for Au and Ag (ii) 2 hours forPd and (iii) 72 hours for Pt Moreover for Pt only a partialreduction was observed Figure 11 show TEM micrographs

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RESEARCHARTICLE

Brayner et al Cyanobacteria as Bioreactors for the Synthesis of Au Ag Pd and Pt Nanoparticles

Fig 11 TEM micrographs obtained after in vitro enzymatic reductionof (a) Au3+ (b) Ag+ and (c) Pd2+

of Au Ag and Pd nanoparticles obtained by in vitro enzy-matic reduction Figure 11(a) presents TEM image of Aunanoparticles which contained 60 equilateral triangularnanoplates of about 400 nm in edge length and 40hexagonal plates of 70ndash150 nm in size (Table I) On theother hand for Ag and Pd spherical nanoparticles of about40 and 10 nm size respectively were synthesized by thismethod (Figs 11(b) and (c) Table I) In these last twocases morphological polysaccharide modifications were

observed Ag nanoparticles were surrounded by lamel-lae polysaccharide while Pd nanoparticles were alignedinside polysaccharide nanowires In all cases nanoparti-cles obtained by in vitro enzymatic reduction differ dras-tically than that obtained by in vivo reduction

4 DISCUSSION

41 Intra-Cellular Nanoparticle FormationThe Role of Nitrogenase

The formation of Au and Ag nanoparticles by living organ-isms has already been widely reported18ndash26 either extra-or intracellularly Although precise mechanisms have notbeen fully elucidated it was proposed that the reductionoccurs via interactions with cellular molecules such asorganic sulfurs sugars or proteins However at this timethe possibility for an enzyme-mediated reduction reactionwas never suggested In the case of cyanobacteria thishypothesis had to be envisioned since these cells possestwo class of reducing enzymes ie nitrogenase and hydro-genase enzymes Cyanobacteria can fix elemental nitrogenfrom the atmosphere thanks to the action of nitrogenasewhich is mainly located inside the heterocyts (Calothrixand Anabaena) However this enzyme is known to be irre-versibly inhibited upon exposure to molecular oxygen44ndash45

which should be in disagreement with observations of N2-fixation in vegetative cells sites of production of molecularoxygen via photosynthesis Thus an explanation could bethat temporal separation protects nitrogenase from oxygenas for example the occurrence of N2-fixation during dark-ness phases (Leptolyngbya)3244ndash45 It was demonstratedthat the species Anabaena variabilis produces two differ-ent nitrogenases one which functions only in heterocytunder either anaerobic or aerobic conditions and the otherone that operates in both heterocyts and vegetative cellsbut only under anaerobic conditions It has been proposedthat the latter is regulated by environmental conditions andthe former is developmentally regulated46 Hydrogenase isable to reduce amongst others electron acceptors hydro-gen ions to molecular hydrogen so that it may also beinvolved in the observed reaction Indeed the presenceof ferredoxins on the thylakoids works as a link betweenhydrogenase and different electron donors and acceptorsin both photosynthetic and non-photosynthetic systemsso that it seems that both nitrogenase and hydrogenaseenzymes are susceptible to act as reducing agents to pro-mote the reduction of some metallic salts

12

N2 +3H++3eminusnitrogenaseminusminusminusminusminusrarr NH3E

0 =minus310 eV

2H++2eminushydrogenaseminusminusminusminusminusrarr H2E

0 = 0 eV

Au3++3eminus rarr Au0E0 = 1002 eV

Ag++ eminus rarr Ag0E0 = 080 eV

J Nanosci Nanotechnol 7 2696ndash2708 2007 2705

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RESEARCHARTICLE

Cyanobacteria as Bioreactors for the Synthesis of Au Ag Pd and Pt Nanoparticles Brayner et al

Pd2++2eminus rarr Pd0E0 = 092 eV

PtCl2minus6 +2eminus rarr PtCl2minus

4 +2ClminusE0 = 073 eV

PtCl2minus4 +2eminus rarr Pt0 +4ClminusE0 = 073 eV

However in this study Leptolyngbya was selected becauseit does not posses heterocyt and fixes nitrogen only anaer-obically and in the dark In this case it was possible toobserve that the reduction reaction was far less efficientthan for the two other cyanobacteria and occurs only indarkness thus only in the presence of nitrogenase In con-trast the genus Anabaena presents several heterocyts thatproduce high concentration of nitrogenase In this case thereaction was very fast (about 5ndash10 minutes) for all concen-trations of Ag+ and Au3+ As a consequence these ionshad not completely penetrated inside the cells and mostpart of Au and Ag nanoparticles was formed near the cellwalls and were bigger than that formed inside Calothrixand Leptolyngbya (Figs 2(d e) 5(b) and 7(b)) Due to thefast reaction nucleation and growth steps were probablynot separated so that coalescence of Au and Ag nanopar-ticles may have occurred The genus Calothrix appears asan intermediate case in terms of nitrogenase content com-bining short reaction time and small un-aggregated col-loids All these data indicate that nitrogenase is specificallyinvolved in the reduction process

This assumption is strengthened by our in vitro test ofnitrogenase ability to reduce noble metal salts Au Ag Pdand to a lesser extent Pt nanoparticles could be obtainedthrough this approach with reaction times that follow atrend similar to what was observed for cyanobacteria Thissimilarity is particularly clear for platinum where a partialreduction leaving un-reacted Pt4+ and partially-reducedPt2+ ions in the solution was observed both in vivo andin vitro However it is interesting to note that the sizeand morphology of nanoparticles obtained in vitro stronglydiffer from those obtained within the cyanobacteria indi-cating the cellular environment of the enzyme play a sig-nificant role in particle growth control In this contextour studies with Calothrix Anabaena and Leptolyngbyacyanobacteria provide some clues about nitrogenase local-ization The enzyme may be present on the fibrous layersinside the heterocyt and also on the thylakoids inside thevegetative cells However since no metallic nanoparti-cles were observed in intrathylakoidal or intrafibrous layerspaces nitrogenase is only located on the cell membranesOn the basis of membrane-controlled biomineralizationprocesses5 the possibility for these membranes to play arole in nanoparticle growth control should not be put aside

42 Extra-Cellular Nanoparticle ReleaseThe Role of Released Poly-Saccharides

After their intra-cellular synthesis metallic nanoparticlesare released in the culture medium via inter-cellular canal-like intercytoplasmic connections (Fig 4(d)) TEM studies

indicate that the released particles are embedded in apoly-saccharide network Such production of extracel-lular polysaccharides by cyanobacteria is already well-documented they are negatively charged and rich in uronicacids exhibit metal-binding capacities47 They may thusbind toxic metals and be used in the treatment of toxicwaste waters48 In the natural environment these polysac-charides could act as natural chelators binding cationsand concentrating essential trace elements present at sub-marginal concentrations in oligotrophic environments49 Inthis work these poly-saccharides prevent the aggregationof released nanoparticles in most cases One exceptionis for Au nanoparticles formed by Anabaena whose sizeincreases when going from intra- to extra-cellular mediaprobably as a consequence of the fast kinetics of reductionmentioned above

More information on Au colloidal solutions obtainedafter Au nanoparticles release by the cyanobacteria canbe obtained form UV-Visible absorption spectra Theseabsorption bands correspond to the surface plasmon reso-nance of Au metallic nanoparticles According to absorp-tion calculation based on Mie theory34 spherical colloidalAu (red colored) would show max at 530 nm Au col-loidal solution released by the genus Anabaena presentsa pink-purple color the sample exhibiting a narrow bandat max = 537 nm close the calculated value On theother hand Au colloidal solutions released by the genusCalothrix and by the genus Leptolyngbya present both apurple-blue color and a broad bands at max = 577 nm andmax = 562 nm respectively This increase of the plasmonbandwidth may be attributed to the physical dimensionof the nanoparticles (sim6 nm)50 The same behavior wasobserved for Ag nanoparticles According to Mie theory34

spherical colloidal Ag (yellow colored) would show max at410 nm Ag colloidal solution released by genera Calothrixand Anabaena present both a red color and they showa broad plasmon resonance band that may be attributedto interparticle coupling (Fig 8(a)) The red-shift of theabsorption band observed in both cases may be attributedto the presence of adsorbed polysaccharides in the Au andAg surfaces (this behavior was also observed for adsorbedproteins)3351

Another important result obtained by UV-Visible anal-ysis was a qualitative release yield of Au nanoparticlesin the surrounding medium For the same Au3+ concen-tration (10minus4 M) absorption intensities vary from 001 to012 Au colloidal solutions released by Anabaena andCalothrix present a very close final Au metallic concen-tration (012 au) The worst result was obtained with thegenus Leptolyngbya (001 au) On the other hand for Agnanoparticles released by all cyanobacteria the absorptionintensities were not very different showing that all Ag+

salts were reduced to Ag0 probably due to the lower redoxpotential and hence the easier reduction of Ag+Ag0 com-pared to Au3+Au0

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Brayner et al Cyanobacteria as Bioreactors for the Synthesis of Au Ag Pd and Pt Nanoparticles

5 CONCLUSION

Production strategies for metallic nanoparticles usingenvironmental-friendly routes using renewable naturalresources have great potential in the next future Ourwork demonstrates that common cyanobacteria strainshave the ability to synthesize Au Ag but also Pd andPt nanoparticles intra-cellularly This process appearsenzyme-mediated as the efficiency of the reduction processcould be related to the presence of nitrogenase We alsoshow that the size of the particle and the reaction yielddepend on the selected strain so that it should be possiblein a near future to identify other cyanobacteria genera withoptimal properties Finally first attempts to use cyanobac-teria as recyclable bioreactors demonstrate that the cellsrecover their full activity after a first metal salt additionand reduction and efforts are in progress through encap-sulation approaches to design continuous flow reactors

Moreover metallic nanoparticles produced insidecyanobacteria cells were released in the culture mediumsurrounded by stabilizing poly-saccharides and can be the-refore used as-synthesized in homogeneous catalysis52ndash53

and other applications such as chemical and biosens-ing54ndash55 biolabeling56ndash57 and photonics58ndash59 The nanopar-ticles may be immobilized in matrices or shaped as thinfilms for optoelectronic applications this being impossibleto achieve if the nanoparticles would have remained insidethe biomass The possibility to extend our approach tooxide andor sulfur nanoparticles elaboration is currentlyunder study

On a more general level this work is a new indicationthat the biological activities of living organisms optimizedthrough evolution to perform specific tasks can be divertedto perform other functions that may answer some of thecurrent challenges in synthetic chemistry

References and Notes

1 C M Zaremba A M Belcher M Fritz Y Li S Mann P KHansma D E Morse J S Speck and G D Stucky Chem Mater8 679 (1996)

2 X W Su and F Z Cui Mater Sci Eng C 7 19 (1999)3 J F Banfield S A Welch H Zhang T T Ebert and R L Penn

Science 289 751 (2000)4 (a) R Wetherbee S Crawford and P Mulvaney Biomineralization

1 89 (2000) (b) J C Weaver L I Pietrasanta N Hedin B FChmelka P K Hansma and D E Morse J Struct Biol 144 271(2003)

5 (a) H A Lowenstam and S Weiner Biomineralization Oxford Uni-versity Press Oxford (1989) (b) J Webb and R J P Williams(Eds) Biomineralization VCH Weinheim (1989) (c) E Baueuer-lein (Ed) Biomineralization Wiley-VCH Weinheim (2000)

6 T Douglas and V T Stark Inorg Chem 39 1828 (2000)7 R Wahl M Mertig J Raff S Selenska-Pobell and W Pompe Adv

Mater 13 736 (2001)8 J He T Kunitake and A Nakao Chem Mater 15 4401 (2003)9 R Brayner T Coradin F Fieacutevet-Vincent J Livage and F Fieacutevet

New J Chem 29 681 (2005)10 C A Mirkin Inorg Chem 38 2258 (2000)

11 (a) M G Warner and J E Hutchinson Nat Mater 2 272 (2003)(b) M Sarakaya C Tamerler A K Y K Schulten and F BabeyxNat Mater 2 577 (2003) (c) B D Reiss C Mao D J SolisK S Ryan T Thomson and A M Belcher Nano Lett 4 1127(2004)

12 B M Rabatic R C Claussen and S I Stupp Chem Mater 175877 (2005)

13 S S Shankar A Rai B Ankawar A Singh A Ahmad andM Sastry Nature 3 482 (2004)

14 C T Dameron R N Reese R K Mehra A R Kortan P J CarrollM L Steigerwald L E Brus and D R Winge Nature 338 596(1989)

15 T Klaus-Joerger J Joerger E Olsson and C G Granqvist TrendsBiotechnol 19 15 (2001)

16 L Q Wu and G F Payne Trends Biotechnol 22 593 (2004)17 T J Beveridge M N Hughes H Lee K T Leung R K Poole

I Savaidis S Silver and J T Trevors Adv Microb Physiol 38177 (1997)

18 G Southam and T J Beveridge Geochim Cosmochim Acta 584527 (1994)

19 G Southam and T J Beveridge Geochim Cosmochim Acta 604369 (1996)

20 K Kashefi J M Tor K P Nevin and D R Lovley Appl EnvironMicrobiol 67 3275 (2001)

21 P Mukherjee A Ahmad D Mandal S Senapati S R Saikar M IKhan R Ramani R Parischa P V Ajaykumar M Alam M Sastryand R Kumar Angew Chem Int Ed 40 3585 (2001)

22 B Nair and T Pradeep Cryst Growth Des 2 293 (2002)23 P Mukherjee S Senapati D Mandal A Ahmad M I Khan

R Kumar and M Sastry Chem Bio Chem 5 461 (2002)24 A Ahmad S Senapati M I Khan R Kumar and M Sastry

Langmuir 19 3550 (2003)25 S S Shankar A Ahmad R Pasricha and M Sastry J Mater

Chem 13 1822 (2003)26 K C Bhainsa and S F DrsquoSouza Colloids Surf B 47 160 (2006)27 H Kuepper and P M H Kroneck Metal Ions in Biological Systems

44 97 (2005)28 S Silver and L T Phung Ann Rev Microb 50 753 (1996)29 R S Prakasham and S V Ramakrishna J Sci Industr Res 57 258

(1998)30 M F Lengke M E Fleet and G Southam Langmuir 22 2780

(2006)31 B Bergman J Gallon A N Rai and L Stal FEMS Microbiol

Rev 19 139 (1997)32 C Bertocchi L Navarini A Cezagraverio and M Anastasio Carbohydr

Polym 12 127 (1990)33 R Brayner T Coradin M-J Vaulay C Mangeney J Livage and

F Fieacutevet Coll Surf A 256 191 (2005)34 J A Creighton and D G Eadon J Chem Soc Faraday Trans 87

3881 (1991)35 R M Slawson M I Van Dyke H Lee and J T Trevors Plasmid

27 72 (1992)36 G J Zhao and S E Stevens Biometals 11 27 (1998)37 I Sadi and B Salopek-Sondi J Coll Interf Sci 275 177 (2004)38 (a) R Brayner G Viau G M da Cruz F Fieacutevet-Vincent F Fieacutevet

and F Bozon-Verduraz Catal Today 57 187 (2000) (b) R BraynerG Viau and F Bozon-Verduraz J Mol Catal 182ndash183 227 (2002)

39 R Brayner D S Cunha and F Bozon-Verduraz Catal Today 78419 (2003)

40 L M Bronstein D M Chernyshov R Karlinsey J W ZwanzigerV G Matveeva E M Sulman G N Demidenko H P Hentze andM Antonietti Chem Mater 15 2623 (2003) (b) J R Gonzalez-Velasco J A Botas R Ferret M P Gonzales-Marcos J L Marcand M A Gutieacuterrez-Ortiz Catal Today 59 395 (2000)

41 D Briggs and M P Seah Practical Surface Analysis Auger andX-ray Photoelectron Spectroscopy Wiley (1990) Vol 1

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RESEARCHARTICLE

Cyanobacteria as Bioreactors for the Synthesis of Au Ag Pd and Pt Nanoparticles Brayner et al

0

005

01

015

02

400 450 500 550 600 650 700 750 800

Leptolyngbya-Au 10-4MCalothrix-Au 10-4MAnabaena-Au 10-4M

Abs

orba

nce

Wavelength (nm)

537

577

562

10 nm

(a)

(c)

(b)

(d)

10 nm

Exo-polysaccharide (SH)

VC

PS

2 microm

Fig 4 (a) TEM micrograph of Au nanoparticles released in Calothrix culture medium (b) UV-Visible spectra of Au nanoparticles released in allcyanobacteria surrounding medium (c) TEM micrograph of Au nanoparticles released in Anabaena culture medium and (d) TEM micrograph of Aunanoparticle release mechanism using genus Calothrix (thin section)

the formation of Au metallic nanoparticles released bythe genus Anabaena These results were confirmed byUV-visible measurements that present a narrow surfaceplasmon resonance band at max = 537 nm (Fig 4(b))and biomass XPS measurements (Fig 5(a)) Photonicmicroscopy showed that heterocyt colour changed fromyellow-green to purple-blue (Figs 1(d) and (e)) The veg-etative cells of Anabaena presented a blue-green colourcharacteristic of a normal photosynthetic activity but thecell wall color changed from blue-green to pink-purple(Fig 1(e)) Contrary to the genus Calothrix it is important

to note that the genus Anabaena withstands even 10minus3 MAu3+ concentration without bleaching of vegetative cellsIn this case the lethal dose was not reached FinallyAu nanoparticles with particle size distribution equal to12 nm ( = 07 Table I) were observed on the thylakoids(Fig 2(d)) Moreover most part of these nanoparticles wasformed inside the heterocyts where nitrogenase concen-tration is very high (Fig 2(e)) In this case coalescencephenomenon was also observed leading to an increase ofparticle size distribution from 12 nm to 25 nm ( = 12Fig 2(e) Table I) This behavior was also observed for

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Brayner et al Cyanobacteria as Bioreactors for the Synthesis of Au Ag Pd and Pt Nanoparticles

(a)

(b)

Fig 5 TEM micrographs of cyanobacteria subcultures after contactavec 10minus4 M of Au3+ solution Formation of Au metallic nanoparticles(a) inside genus Calothrix (b) inside genus Anabaena

Au nanoparticles released in the surrounding medium thatpresents a broad size distribution of about 25 nm ( = 32Fig 4(c) Table I)

For Leptolyngbya color modification of the culturemedium was also noted It took a lot of time (more than100 hours) This behavior can be explained because thegenus Leptolyngbya does not possess heterocyts and fixesnitrogen only anaerobically (it is extremely sensitive tooxygen) and only in the dark31 With Leptolyngbya grownin [Au3+ = 10minus3 M the culture medium color remainedyellow In this case Au3+ concentration may be veryclose to the lethal dose similarly to Calothrix so that adecrease of photosynthetic activity and bleaching of vege-tative cells were also observed Consequently the metallicAu nanoparticles produced remained inside the cells andwere not released in the surrounding medium On the other

hand when incubated with 10minus4 M Au3+ concentrationphotonic microscopy showed that vegetative cells colorchanged from green to pinkish (Figs 1(f) and (g)) and theculture medium color changed from transparent yellow totransparent purple UV-visible spectroscopy measurementsconfirm the formation of Au metallic nanoparticles thatpresent a weak and broad surface plasmon resonance bandat max = 562 nm (Fig 4(b)) Finally for 10minus6 M Au3+

concentration the vegetative cells presented a blue-greencolor characteristic of a normal photosynthetic activityFigure 2(f) shows a thin section of Au nanoparticlesformed on Leptolyngbya thylakoids Contrary to the genusAnabaena the non-heterocyteous genus Leptolyngbya pro-duces nitrogenase only in the dark consequently thereduction reaction was very low but the particle size dis-tribution was equal to 6 nm ( = 06 Table I) similarly toCalothrix No coalescence phenomenon was observed

The best performances for Au nanoparticles productionand release were obtained with Calothrix and Anabaenacyanobacteria In order to evaluate the possibility to designa cell-based continuous bioreactor part of the cells thatwere in contact with 10minus4 M concentration of Au3+

solutions was isolated in a new sterile Boldrsquos Basal

0 20 40 60 80 100 120

020

025

030

035

040

045

050

055

Addition of HAuCl4

Fv

Fm

Time (days)

Calothrix

0 20 40 60 80 100

015

020

025

030

035

040

045 Addition of HAuCl4

Fv

Fm

Time (days)

Anabaena

(a)

(b)

Fig 6 Photosynthetic activities (a) Calothrix (Au3+ addition after20 days subcultures) (b) Anabaena (Au3+ addition after 40 dayssubcultures)

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Cyanobacteria as Bioreactors for the Synthesis of Au Ag Pd and Pt Nanoparticles Brayner et al

medium and the same concentration of Au3+ solution wasadded to 20 days of Calothrix and Anabaena subculturesThe formation and release of Au metallic nanoparticleswas observed (Fig 5) confirming the possibility to makea continuous cyanobacteria bioreactor The photosyntheticactivities of Calothrix and Anabaena after addition of Ausalts (10minus4 M) in the culture medium were measured usinga PAM fluorimeter (Fig 6) After addition of Au saltsa progressive decrease of photosynthetic activities wasobserved In both cases (Figs 6(a) and (b)) the minimumFV Fm was observed after 10 days of incubation in the pres-ence of Au nanoparticles This behavior was maybe dueto the presence of Au salts on thylakoids (photosynthe-sis sites) and their posterior reduction After reduction thethylakoids were not completely recovered by Au nanopar-ticles and the photosynthetic activities may increase againThese micro-algae remain alive more than three months inthe presence of Au nanoparticles

32 Silver

It is well known that Ag ions and Ag-based compoundsare highly toxic to microorganisms35ndash36 showing strongbiocidal effects on many species of bacteria includingE Coli37 Cyanobacteria have Gram-negative cell wallsuch as E coli so in this work biocidal tests with Ag+

salts were carried out with Calothrix and Anabaena micro-algae The possibility to produce also Ag metallic nanopar-ticles with controlled size and shape inside cyanobacteriacells was also studied Before reaction the culture mediumadded with [Ag+ = 10minus4 M presents a transparent-uncolored solution The experiments were carried out untilobservation of biomass andor culture medium color mod-ification (i) 24 hours for genus Calothrix (ii) 30 min-utes for genus Anabaena and (iii) more than 72 hoursfor genus Leptolyngbya For all cyanobacteria the culturemedium color changed from transparent-uncolored to redPhotonic microscopy showed that heterocyt and vegetativecell colors changed from blue-green to orange-red and yel-low (Fig 7(a)) Thin sections of Anabaena trichomes showspherical 40 nm Ag nanoparticles with a broad size dis-tribution = 42 (Fig 7(b) Table 1) On the other handgenus Calothrix presents well-shaped 15 nm with a narrowsize distribution ( = 08) confirming that Calothrix tri-chome led to the formation of well-controlled size metallicnanoparticles (Table I)

Figure 8(a) presents UV-visible spectra of culture mediaafter color modification For all samples a broad plasmonband was observed with maxima centered between 420ndash450 nm which may be attributed to inter-particle cou-pling effects (multipolar resonance) This behavior wasconfirmed by TEM micrographs of released Ag nanopar-ticles stabilized by micro-algae exo-polysaccharides in thesurrounding medium (Figs 8(b) and (c) Table I) Thesemicro-algae remain alive about one month in the presence

Fig 7 (a) Photonic micrograph of Anabaena grown in [Ag+ =10minus4 M (b) TEM micrograph of Anabaena grown in [Ag+ = 10minus4 Mand (c) TEM micrograph of Calothrix thin sections grown in [Ag+ =10minus4 M

of Ag nanoparticles No biocidal effect was observed dur-ing this time It was observed that released Ag nanopar-ticles by Anabaena trichome present about 25 nm insize with narrow size distribution such as Au nanopar-ticles released by the same micro-algae showing thatcyanobacteria wall may have a particle size limitation dur-ing release process (Table I)

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Brayner et al Cyanobacteria as Bioreactors for the Synthesis of Au Ag Pd and Pt Nanoparticles

0

005

01

015

02

025

03

035

04

300 400 500 600 700 800

Leptolyngbya-Ag 10-4MCalothrix-Ag 10-4MAnabaena-Ag 10-4M

Abs

orba

nce

Wavelength (nm)

100 nm

(a)

(b)

(c)

Fig 8 (a) UV-Visible spectra of Ag nanoparticles released in allcyanobacteria TEM micrographs of Ag nanoparticles released in(b) Anabaena and (c) Calothrix culture medium

33 Palladium and Platinum

Until now only Au and Ag metallic nanoparticles weresynthesized using living cells such as fungi and bacteria23

In order to extend the significance of our approach wehave evaluated the ability of cyanobacteria to reducePd2+ and Pt4+ ions as Pt Pd and PdO nanoparticles

Calothrix-Pd

Anabaena-Pd

Calothrix-Pt

Anabaena-Pt

(a)

(b)

(c)

(d)

Binding energy (eV)

Binding energy (eV)

Binding energy (eV)

Binding energy (eV)

Fig 9 XPS core-level (a) Pd3d52 region (b) Pd3d52 region (c) Pt4f72

region and (d) Pt4f72 region

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Table II Measured binding energies (BE) of Pd3d32 and Pt4f72 insidecyanobacteria cells and their chemical state concentrations

Chemical stateSample BE (eV) concentrations ()

Calothrix-Pd 3345 Pd0 3893364 Pd2+ 611

Anabaena-Pd 3351 Pd0 2793370 Pd2+ 721

Calothrix-Pt 710 Pt0 90734 Pt2+ 665758 Pt4+ 245

Anabaena-Pt 715 Pt0 427737 Pt2+ 435761 Pt4+ 138

have interesting properties for several catalytic pro-cess (i) hydrogenation of alkenes38 (ii) oxidation ofvolatile organic compounds (VOC)39 and (iii) combustionreactions40

Fig 10 TEM micrographs of Calothrix thin sections grown in(a) [Pd2+= 10minus4 M (b) [Pt4+= 10minus4 M

Before reaction the culture media added with [Pd2+=10minus4 M and [Pt4+ = 10minus4 M present both a transparent-yellow colored solutions After salt addition color mod-ification was observed after (i) 30 minutes for generaCalothrix and Anabaena in the presence of Pd salts and(ii) about fifteen days for both micro-algae in the presenceof Pt salts For all cases the culture medium color slightlychanges from transparent-yellow to brownish-yellow Noheterocyt or vegetative cell color change was observedby photonic microscopy analyses only an increase ofmucilaginous sheath was visible The presence of Pd andPt metallic nanoparticles was confirmed by XPS analysis(Fig 9) Table II shows the binding energies (BE) observedafter peaks decomposition Here only Pd352 and Pt4f72

regions will be discussed For cyanobacteria-Pd samplesPd3d52 region was fitted with two components centered at3345 and 3364 eV for Calothrix and 3351 and 3370 eVfor Anabaena (Figs 9(a) and (b) Table II) The first onewas assigned to metallic Pd and the second one to Pd2+ inan oxygen-containing environment (PdO)41ndash42 Pd nanopar-ticles are not stable in air and a surface passivation mayoccur It was observed that genus Calothrix (389 ofPd0) is more resistant to Pd surface passivation than genusAnabaena (279 of Pd0) (Table II) For cyanobacteria-Ptsamples Pt4f72 region was fitted with three componentscentered at 710 734 and 758 eV for genus Calothrix and715 737 and 761 eV for genus Anabaena (Figs 9(c)and (d) and Table II) The first peak was attributed tometallic Pt4143 The last two peaks were attributed to Pt2+

and Pt4 in the coordination sphere of H2PtCl4 and H2PtCl6respectively In this case only one part of H2PtCl6 wascompletely reduced The best results were also obtainedwith genus Calothrix (Table II) Figure 10 shows Calothrixtrichomes thin sections grown in [Pd2+ = 10minus4 M and[Pt4+= 10minus4 M In both cases well-shaped nanoparticleswith a very narrow size distribution (( = 35 nm =03) for Pd nanoparticles and ( = 32 nm = 03) forPt nanoparticles) were observed (Table I)

34 In vitro Enzymatic Reduction of Metallic Salts

The results obtained in vivo led to conclude that reductionoccurs inside cyanobacteria heterocyts and vegetative cellsfollowed by a partial release of metallic nanoparticlesin the culture medium Because these cells specificallycontain nitrogenase a reducing enzyme we hypothesizedthat it could be responsible for nanoparticle formation Totry to confirm this hypothesis in vitro enzymatic reduc-tion of metallic salts was carried out in a Boldrsquos basalmedium using nitrogenase enzyme (NAD[P]H) as reducingagent and Calothrix released polysaccharide (RPS) to sim-ulate in vivo conditions Solution color modification wasobtained after (i) 10 minutes for Au and Ag (ii) 2 hours forPd and (iii) 72 hours for Pt Moreover for Pt only a partialreduction was observed Figure 11 show TEM micrographs

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Brayner et al Cyanobacteria as Bioreactors for the Synthesis of Au Ag Pd and Pt Nanoparticles

Fig 11 TEM micrographs obtained after in vitro enzymatic reductionof (a) Au3+ (b) Ag+ and (c) Pd2+

of Au Ag and Pd nanoparticles obtained by in vitro enzy-matic reduction Figure 11(a) presents TEM image of Aunanoparticles which contained 60 equilateral triangularnanoplates of about 400 nm in edge length and 40hexagonal plates of 70ndash150 nm in size (Table I) On theother hand for Ag and Pd spherical nanoparticles of about40 and 10 nm size respectively were synthesized by thismethod (Figs 11(b) and (c) Table I) In these last twocases morphological polysaccharide modifications were

observed Ag nanoparticles were surrounded by lamel-lae polysaccharide while Pd nanoparticles were alignedinside polysaccharide nanowires In all cases nanoparti-cles obtained by in vitro enzymatic reduction differ dras-tically than that obtained by in vivo reduction

4 DISCUSSION

41 Intra-Cellular Nanoparticle FormationThe Role of Nitrogenase

The formation of Au and Ag nanoparticles by living organ-isms has already been widely reported18ndash26 either extra-or intracellularly Although precise mechanisms have notbeen fully elucidated it was proposed that the reductionoccurs via interactions with cellular molecules such asorganic sulfurs sugars or proteins However at this timethe possibility for an enzyme-mediated reduction reactionwas never suggested In the case of cyanobacteria thishypothesis had to be envisioned since these cells possestwo class of reducing enzymes ie nitrogenase and hydro-genase enzymes Cyanobacteria can fix elemental nitrogenfrom the atmosphere thanks to the action of nitrogenasewhich is mainly located inside the heterocyts (Calothrixand Anabaena) However this enzyme is known to be irre-versibly inhibited upon exposure to molecular oxygen44ndash45

which should be in disagreement with observations of N2-fixation in vegetative cells sites of production of molecularoxygen via photosynthesis Thus an explanation could bethat temporal separation protects nitrogenase from oxygenas for example the occurrence of N2-fixation during dark-ness phases (Leptolyngbya)3244ndash45 It was demonstratedthat the species Anabaena variabilis produces two differ-ent nitrogenases one which functions only in heterocytunder either anaerobic or aerobic conditions and the otherone that operates in both heterocyts and vegetative cellsbut only under anaerobic conditions It has been proposedthat the latter is regulated by environmental conditions andthe former is developmentally regulated46 Hydrogenase isable to reduce amongst others electron acceptors hydro-gen ions to molecular hydrogen so that it may also beinvolved in the observed reaction Indeed the presenceof ferredoxins on the thylakoids works as a link betweenhydrogenase and different electron donors and acceptorsin both photosynthetic and non-photosynthetic systemsso that it seems that both nitrogenase and hydrogenaseenzymes are susceptible to act as reducing agents to pro-mote the reduction of some metallic salts

12

N2 +3H++3eminusnitrogenaseminusminusminusminusminusrarr NH3E

0 =minus310 eV

2H++2eminushydrogenaseminusminusminusminusminusrarr H2E

0 = 0 eV

Au3++3eminus rarr Au0E0 = 1002 eV

Ag++ eminus rarr Ag0E0 = 080 eV

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RESEARCHARTICLE

Cyanobacteria as Bioreactors for the Synthesis of Au Ag Pd and Pt Nanoparticles Brayner et al

Pd2++2eminus rarr Pd0E0 = 092 eV

PtCl2minus6 +2eminus rarr PtCl2minus

4 +2ClminusE0 = 073 eV

PtCl2minus4 +2eminus rarr Pt0 +4ClminusE0 = 073 eV

However in this study Leptolyngbya was selected becauseit does not posses heterocyt and fixes nitrogen only anaer-obically and in the dark In this case it was possible toobserve that the reduction reaction was far less efficientthan for the two other cyanobacteria and occurs only indarkness thus only in the presence of nitrogenase In con-trast the genus Anabaena presents several heterocyts thatproduce high concentration of nitrogenase In this case thereaction was very fast (about 5ndash10 minutes) for all concen-trations of Ag+ and Au3+ As a consequence these ionshad not completely penetrated inside the cells and mostpart of Au and Ag nanoparticles was formed near the cellwalls and were bigger than that formed inside Calothrixand Leptolyngbya (Figs 2(d e) 5(b) and 7(b)) Due to thefast reaction nucleation and growth steps were probablynot separated so that coalescence of Au and Ag nanopar-ticles may have occurred The genus Calothrix appears asan intermediate case in terms of nitrogenase content com-bining short reaction time and small un-aggregated col-loids All these data indicate that nitrogenase is specificallyinvolved in the reduction process

This assumption is strengthened by our in vitro test ofnitrogenase ability to reduce noble metal salts Au Ag Pdand to a lesser extent Pt nanoparticles could be obtainedthrough this approach with reaction times that follow atrend similar to what was observed for cyanobacteria Thissimilarity is particularly clear for platinum where a partialreduction leaving un-reacted Pt4+ and partially-reducedPt2+ ions in the solution was observed both in vivo andin vitro However it is interesting to note that the sizeand morphology of nanoparticles obtained in vitro stronglydiffer from those obtained within the cyanobacteria indi-cating the cellular environment of the enzyme play a sig-nificant role in particle growth control In this contextour studies with Calothrix Anabaena and Leptolyngbyacyanobacteria provide some clues about nitrogenase local-ization The enzyme may be present on the fibrous layersinside the heterocyt and also on the thylakoids inside thevegetative cells However since no metallic nanoparti-cles were observed in intrathylakoidal or intrafibrous layerspaces nitrogenase is only located on the cell membranesOn the basis of membrane-controlled biomineralizationprocesses5 the possibility for these membranes to play arole in nanoparticle growth control should not be put aside

42 Extra-Cellular Nanoparticle ReleaseThe Role of Released Poly-Saccharides

After their intra-cellular synthesis metallic nanoparticlesare released in the culture medium via inter-cellular canal-like intercytoplasmic connections (Fig 4(d)) TEM studies

indicate that the released particles are embedded in apoly-saccharide network Such production of extracel-lular polysaccharides by cyanobacteria is already well-documented they are negatively charged and rich in uronicacids exhibit metal-binding capacities47 They may thusbind toxic metals and be used in the treatment of toxicwaste waters48 In the natural environment these polysac-charides could act as natural chelators binding cationsand concentrating essential trace elements present at sub-marginal concentrations in oligotrophic environments49 Inthis work these poly-saccharides prevent the aggregationof released nanoparticles in most cases One exceptionis for Au nanoparticles formed by Anabaena whose sizeincreases when going from intra- to extra-cellular mediaprobably as a consequence of the fast kinetics of reductionmentioned above

More information on Au colloidal solutions obtainedafter Au nanoparticles release by the cyanobacteria canbe obtained form UV-Visible absorption spectra Theseabsorption bands correspond to the surface plasmon reso-nance of Au metallic nanoparticles According to absorp-tion calculation based on Mie theory34 spherical colloidalAu (red colored) would show max at 530 nm Au col-loidal solution released by the genus Anabaena presentsa pink-purple color the sample exhibiting a narrow bandat max = 537 nm close the calculated value On theother hand Au colloidal solutions released by the genusCalothrix and by the genus Leptolyngbya present both apurple-blue color and a broad bands at max = 577 nm andmax = 562 nm respectively This increase of the plasmonbandwidth may be attributed to the physical dimensionof the nanoparticles (sim6 nm)50 The same behavior wasobserved for Ag nanoparticles According to Mie theory34

spherical colloidal Ag (yellow colored) would show max at410 nm Ag colloidal solution released by genera Calothrixand Anabaena present both a red color and they showa broad plasmon resonance band that may be attributedto interparticle coupling (Fig 8(a)) The red-shift of theabsorption band observed in both cases may be attributedto the presence of adsorbed polysaccharides in the Au andAg surfaces (this behavior was also observed for adsorbedproteins)3351

Another important result obtained by UV-Visible anal-ysis was a qualitative release yield of Au nanoparticlesin the surrounding medium For the same Au3+ concen-tration (10minus4 M) absorption intensities vary from 001 to012 Au colloidal solutions released by Anabaena andCalothrix present a very close final Au metallic concen-tration (012 au) The worst result was obtained with thegenus Leptolyngbya (001 au) On the other hand for Agnanoparticles released by all cyanobacteria the absorptionintensities were not very different showing that all Ag+

salts were reduced to Ag0 probably due to the lower redoxpotential and hence the easier reduction of Ag+Ag0 com-pared to Au3+Au0

2706 J Nanosci Nanotechnol 7 2696ndash2708 2007

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Brayner et al Cyanobacteria as Bioreactors for the Synthesis of Au Ag Pd and Pt Nanoparticles

5 CONCLUSION

Production strategies for metallic nanoparticles usingenvironmental-friendly routes using renewable naturalresources have great potential in the next future Ourwork demonstrates that common cyanobacteria strainshave the ability to synthesize Au Ag but also Pd andPt nanoparticles intra-cellularly This process appearsenzyme-mediated as the efficiency of the reduction processcould be related to the presence of nitrogenase We alsoshow that the size of the particle and the reaction yielddepend on the selected strain so that it should be possiblein a near future to identify other cyanobacteria genera withoptimal properties Finally first attempts to use cyanobac-teria as recyclable bioreactors demonstrate that the cellsrecover their full activity after a first metal salt additionand reduction and efforts are in progress through encap-sulation approaches to design continuous flow reactors

Moreover metallic nanoparticles produced insidecyanobacteria cells were released in the culture mediumsurrounded by stabilizing poly-saccharides and can be the-refore used as-synthesized in homogeneous catalysis52ndash53

and other applications such as chemical and biosens-ing54ndash55 biolabeling56ndash57 and photonics58ndash59 The nanopar-ticles may be immobilized in matrices or shaped as thinfilms for optoelectronic applications this being impossibleto achieve if the nanoparticles would have remained insidethe biomass The possibility to extend our approach tooxide andor sulfur nanoparticles elaboration is currentlyunder study

On a more general level this work is a new indicationthat the biological activities of living organisms optimizedthrough evolution to perform specific tasks can be divertedto perform other functions that may answer some of thecurrent challenges in synthetic chemistry

References and Notes

1 C M Zaremba A M Belcher M Fritz Y Li S Mann P KHansma D E Morse J S Speck and G D Stucky Chem Mater8 679 (1996)

2 X W Su and F Z Cui Mater Sci Eng C 7 19 (1999)3 J F Banfield S A Welch H Zhang T T Ebert and R L Penn

Science 289 751 (2000)4 (a) R Wetherbee S Crawford and P Mulvaney Biomineralization

1 89 (2000) (b) J C Weaver L I Pietrasanta N Hedin B FChmelka P K Hansma and D E Morse J Struct Biol 144 271(2003)

5 (a) H A Lowenstam and S Weiner Biomineralization Oxford Uni-versity Press Oxford (1989) (b) J Webb and R J P Williams(Eds) Biomineralization VCH Weinheim (1989) (c) E Baueuer-lein (Ed) Biomineralization Wiley-VCH Weinheim (2000)

6 T Douglas and V T Stark Inorg Chem 39 1828 (2000)7 R Wahl M Mertig J Raff S Selenska-Pobell and W Pompe Adv

Mater 13 736 (2001)8 J He T Kunitake and A Nakao Chem Mater 15 4401 (2003)9 R Brayner T Coradin F Fieacutevet-Vincent J Livage and F Fieacutevet

New J Chem 29 681 (2005)10 C A Mirkin Inorg Chem 38 2258 (2000)

11 (a) M G Warner and J E Hutchinson Nat Mater 2 272 (2003)(b) M Sarakaya C Tamerler A K Y K Schulten and F BabeyxNat Mater 2 577 (2003) (c) B D Reiss C Mao D J SolisK S Ryan T Thomson and A M Belcher Nano Lett 4 1127(2004)

12 B M Rabatic R C Claussen and S I Stupp Chem Mater 175877 (2005)

13 S S Shankar A Rai B Ankawar A Singh A Ahmad andM Sastry Nature 3 482 (2004)

14 C T Dameron R N Reese R K Mehra A R Kortan P J CarrollM L Steigerwald L E Brus and D R Winge Nature 338 596(1989)

15 T Klaus-Joerger J Joerger E Olsson and C G Granqvist TrendsBiotechnol 19 15 (2001)

16 L Q Wu and G F Payne Trends Biotechnol 22 593 (2004)17 T J Beveridge M N Hughes H Lee K T Leung R K Poole

I Savaidis S Silver and J T Trevors Adv Microb Physiol 38177 (1997)

18 G Southam and T J Beveridge Geochim Cosmochim Acta 584527 (1994)

19 G Southam and T J Beveridge Geochim Cosmochim Acta 604369 (1996)

20 K Kashefi J M Tor K P Nevin and D R Lovley Appl EnvironMicrobiol 67 3275 (2001)

21 P Mukherjee A Ahmad D Mandal S Senapati S R Saikar M IKhan R Ramani R Parischa P V Ajaykumar M Alam M Sastryand R Kumar Angew Chem Int Ed 40 3585 (2001)

22 B Nair and T Pradeep Cryst Growth Des 2 293 (2002)23 P Mukherjee S Senapati D Mandal A Ahmad M I Khan

R Kumar and M Sastry Chem Bio Chem 5 461 (2002)24 A Ahmad S Senapati M I Khan R Kumar and M Sastry

Langmuir 19 3550 (2003)25 S S Shankar A Ahmad R Pasricha and M Sastry J Mater

Chem 13 1822 (2003)26 K C Bhainsa and S F DrsquoSouza Colloids Surf B 47 160 (2006)27 H Kuepper and P M H Kroneck Metal Ions in Biological Systems

44 97 (2005)28 S Silver and L T Phung Ann Rev Microb 50 753 (1996)29 R S Prakasham and S V Ramakrishna J Sci Industr Res 57 258

(1998)30 M F Lengke M E Fleet and G Southam Langmuir 22 2780

(2006)31 B Bergman J Gallon A N Rai and L Stal FEMS Microbiol

Rev 19 139 (1997)32 C Bertocchi L Navarini A Cezagraverio and M Anastasio Carbohydr

Polym 12 127 (1990)33 R Brayner T Coradin M-J Vaulay C Mangeney J Livage and

F Fieacutevet Coll Surf A 256 191 (2005)34 J A Creighton and D G Eadon J Chem Soc Faraday Trans 87

3881 (1991)35 R M Slawson M I Van Dyke H Lee and J T Trevors Plasmid

27 72 (1992)36 G J Zhao and S E Stevens Biometals 11 27 (1998)37 I Sadi and B Salopek-Sondi J Coll Interf Sci 275 177 (2004)38 (a) R Brayner G Viau G M da Cruz F Fieacutevet-Vincent F Fieacutevet

and F Bozon-Verduraz Catal Today 57 187 (2000) (b) R BraynerG Viau and F Bozon-Verduraz J Mol Catal 182ndash183 227 (2002)

39 R Brayner D S Cunha and F Bozon-Verduraz Catal Today 78419 (2003)

40 L M Bronstein D M Chernyshov R Karlinsey J W ZwanzigerV G Matveeva E M Sulman G N Demidenko H P Hentze andM Antonietti Chem Mater 15 2623 (2003) (b) J R Gonzalez-Velasco J A Botas R Ferret M P Gonzales-Marcos J L Marcand M A Gutieacuterrez-Ortiz Catal Today 59 395 (2000)

41 D Briggs and M P Seah Practical Surface Analysis Auger andX-ray Photoelectron Spectroscopy Wiley (1990) Vol 1

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Brayner et al Cyanobacteria as Bioreactors for the Synthesis of Au Ag Pd and Pt Nanoparticles

(a)

(b)

Fig 5 TEM micrographs of cyanobacteria subcultures after contactavec 10minus4 M of Au3+ solution Formation of Au metallic nanoparticles(a) inside genus Calothrix (b) inside genus Anabaena

Au nanoparticles released in the surrounding medium thatpresents a broad size distribution of about 25 nm ( = 32Fig 4(c) Table I)

For Leptolyngbya color modification of the culturemedium was also noted It took a lot of time (more than100 hours) This behavior can be explained because thegenus Leptolyngbya does not possess heterocyts and fixesnitrogen only anaerobically (it is extremely sensitive tooxygen) and only in the dark31 With Leptolyngbya grownin [Au3+ = 10minus3 M the culture medium color remainedyellow In this case Au3+ concentration may be veryclose to the lethal dose similarly to Calothrix so that adecrease of photosynthetic activity and bleaching of vege-tative cells were also observed Consequently the metallicAu nanoparticles produced remained inside the cells andwere not released in the surrounding medium On the other

hand when incubated with 10minus4 M Au3+ concentrationphotonic microscopy showed that vegetative cells colorchanged from green to pinkish (Figs 1(f) and (g)) and theculture medium color changed from transparent yellow totransparent purple UV-visible spectroscopy measurementsconfirm the formation of Au metallic nanoparticles thatpresent a weak and broad surface plasmon resonance bandat max = 562 nm (Fig 4(b)) Finally for 10minus6 M Au3+

concentration the vegetative cells presented a blue-greencolor characteristic of a normal photosynthetic activityFigure 2(f) shows a thin section of Au nanoparticlesformed on Leptolyngbya thylakoids Contrary to the genusAnabaena the non-heterocyteous genus Leptolyngbya pro-duces nitrogenase only in the dark consequently thereduction reaction was very low but the particle size dis-tribution was equal to 6 nm ( = 06 Table I) similarly toCalothrix No coalescence phenomenon was observed

The best performances for Au nanoparticles productionand release were obtained with Calothrix and Anabaenacyanobacteria In order to evaluate the possibility to designa cell-based continuous bioreactor part of the cells thatwere in contact with 10minus4 M concentration of Au3+

solutions was isolated in a new sterile Boldrsquos Basal

0 20 40 60 80 100 120

020

025

030

035

040

045

050

055

Addition of HAuCl4

Fv

Fm

Time (days)

Calothrix

0 20 40 60 80 100

015

020

025

030

035

040

045 Addition of HAuCl4

Fv

Fm

Time (days)

Anabaena

(a)

(b)

Fig 6 Photosynthetic activities (a) Calothrix (Au3+ addition after20 days subcultures) (b) Anabaena (Au3+ addition after 40 dayssubcultures)

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Cyanobacteria as Bioreactors for the Synthesis of Au Ag Pd and Pt Nanoparticles Brayner et al

medium and the same concentration of Au3+ solution wasadded to 20 days of Calothrix and Anabaena subculturesThe formation and release of Au metallic nanoparticleswas observed (Fig 5) confirming the possibility to makea continuous cyanobacteria bioreactor The photosyntheticactivities of Calothrix and Anabaena after addition of Ausalts (10minus4 M) in the culture medium were measured usinga PAM fluorimeter (Fig 6) After addition of Au saltsa progressive decrease of photosynthetic activities wasobserved In both cases (Figs 6(a) and (b)) the minimumFV Fm was observed after 10 days of incubation in the pres-ence of Au nanoparticles This behavior was maybe dueto the presence of Au salts on thylakoids (photosynthe-sis sites) and their posterior reduction After reduction thethylakoids were not completely recovered by Au nanopar-ticles and the photosynthetic activities may increase againThese micro-algae remain alive more than three months inthe presence of Au nanoparticles

32 Silver

It is well known that Ag ions and Ag-based compoundsare highly toxic to microorganisms35ndash36 showing strongbiocidal effects on many species of bacteria includingE Coli37 Cyanobacteria have Gram-negative cell wallsuch as E coli so in this work biocidal tests with Ag+

salts were carried out with Calothrix and Anabaena micro-algae The possibility to produce also Ag metallic nanopar-ticles with controlled size and shape inside cyanobacteriacells was also studied Before reaction the culture mediumadded with [Ag+ = 10minus4 M presents a transparent-uncolored solution The experiments were carried out untilobservation of biomass andor culture medium color mod-ification (i) 24 hours for genus Calothrix (ii) 30 min-utes for genus Anabaena and (iii) more than 72 hoursfor genus Leptolyngbya For all cyanobacteria the culturemedium color changed from transparent-uncolored to redPhotonic microscopy showed that heterocyt and vegetativecell colors changed from blue-green to orange-red and yel-low (Fig 7(a)) Thin sections of Anabaena trichomes showspherical 40 nm Ag nanoparticles with a broad size dis-tribution = 42 (Fig 7(b) Table 1) On the other handgenus Calothrix presents well-shaped 15 nm with a narrowsize distribution ( = 08) confirming that Calothrix tri-chome led to the formation of well-controlled size metallicnanoparticles (Table I)

Figure 8(a) presents UV-visible spectra of culture mediaafter color modification For all samples a broad plasmonband was observed with maxima centered between 420ndash450 nm which may be attributed to inter-particle cou-pling effects (multipolar resonance) This behavior wasconfirmed by TEM micrographs of released Ag nanopar-ticles stabilized by micro-algae exo-polysaccharides in thesurrounding medium (Figs 8(b) and (c) Table I) Thesemicro-algae remain alive about one month in the presence

Fig 7 (a) Photonic micrograph of Anabaena grown in [Ag+ =10minus4 M (b) TEM micrograph of Anabaena grown in [Ag+ = 10minus4 Mand (c) TEM micrograph of Calothrix thin sections grown in [Ag+ =10minus4 M

of Ag nanoparticles No biocidal effect was observed dur-ing this time It was observed that released Ag nanopar-ticles by Anabaena trichome present about 25 nm insize with narrow size distribution such as Au nanopar-ticles released by the same micro-algae showing thatcyanobacteria wall may have a particle size limitation dur-ing release process (Table I)

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Brayner et al Cyanobacteria as Bioreactors for the Synthesis of Au Ag Pd and Pt Nanoparticles

0

005

01

015

02

025

03

035

04

300 400 500 600 700 800

Leptolyngbya-Ag 10-4MCalothrix-Ag 10-4MAnabaena-Ag 10-4M

Abs

orba

nce

Wavelength (nm)

100 nm

(a)

(b)

(c)

Fig 8 (a) UV-Visible spectra of Ag nanoparticles released in allcyanobacteria TEM micrographs of Ag nanoparticles released in(b) Anabaena and (c) Calothrix culture medium

33 Palladium and Platinum

Until now only Au and Ag metallic nanoparticles weresynthesized using living cells such as fungi and bacteria23

In order to extend the significance of our approach wehave evaluated the ability of cyanobacteria to reducePd2+ and Pt4+ ions as Pt Pd and PdO nanoparticles

Calothrix-Pd

Anabaena-Pd

Calothrix-Pt

Anabaena-Pt

(a)

(b)

(c)

(d)

Binding energy (eV)

Binding energy (eV)

Binding energy (eV)

Binding energy (eV)

Fig 9 XPS core-level (a) Pd3d52 region (b) Pd3d52 region (c) Pt4f72

region and (d) Pt4f72 region

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Table II Measured binding energies (BE) of Pd3d32 and Pt4f72 insidecyanobacteria cells and their chemical state concentrations

Chemical stateSample BE (eV) concentrations ()

Calothrix-Pd 3345 Pd0 3893364 Pd2+ 611

Anabaena-Pd 3351 Pd0 2793370 Pd2+ 721

Calothrix-Pt 710 Pt0 90734 Pt2+ 665758 Pt4+ 245

Anabaena-Pt 715 Pt0 427737 Pt2+ 435761 Pt4+ 138

have interesting properties for several catalytic pro-cess (i) hydrogenation of alkenes38 (ii) oxidation ofvolatile organic compounds (VOC)39 and (iii) combustionreactions40

Fig 10 TEM micrographs of Calothrix thin sections grown in(a) [Pd2+= 10minus4 M (b) [Pt4+= 10minus4 M

Before reaction the culture media added with [Pd2+=10minus4 M and [Pt4+ = 10minus4 M present both a transparent-yellow colored solutions After salt addition color mod-ification was observed after (i) 30 minutes for generaCalothrix and Anabaena in the presence of Pd salts and(ii) about fifteen days for both micro-algae in the presenceof Pt salts For all cases the culture medium color slightlychanges from transparent-yellow to brownish-yellow Noheterocyt or vegetative cell color change was observedby photonic microscopy analyses only an increase ofmucilaginous sheath was visible The presence of Pd andPt metallic nanoparticles was confirmed by XPS analysis(Fig 9) Table II shows the binding energies (BE) observedafter peaks decomposition Here only Pd352 and Pt4f72

regions will be discussed For cyanobacteria-Pd samplesPd3d52 region was fitted with two components centered at3345 and 3364 eV for Calothrix and 3351 and 3370 eVfor Anabaena (Figs 9(a) and (b) Table II) The first onewas assigned to metallic Pd and the second one to Pd2+ inan oxygen-containing environment (PdO)41ndash42 Pd nanopar-ticles are not stable in air and a surface passivation mayoccur It was observed that genus Calothrix (389 ofPd0) is more resistant to Pd surface passivation than genusAnabaena (279 of Pd0) (Table II) For cyanobacteria-Ptsamples Pt4f72 region was fitted with three componentscentered at 710 734 and 758 eV for genus Calothrix and715 737 and 761 eV for genus Anabaena (Figs 9(c)and (d) and Table II) The first peak was attributed tometallic Pt4143 The last two peaks were attributed to Pt2+

and Pt4 in the coordination sphere of H2PtCl4 and H2PtCl6respectively In this case only one part of H2PtCl6 wascompletely reduced The best results were also obtainedwith genus Calothrix (Table II) Figure 10 shows Calothrixtrichomes thin sections grown in [Pd2+ = 10minus4 M and[Pt4+= 10minus4 M In both cases well-shaped nanoparticleswith a very narrow size distribution (( = 35 nm =03) for Pd nanoparticles and ( = 32 nm = 03) forPt nanoparticles) were observed (Table I)

34 In vitro Enzymatic Reduction of Metallic Salts

The results obtained in vivo led to conclude that reductionoccurs inside cyanobacteria heterocyts and vegetative cellsfollowed by a partial release of metallic nanoparticlesin the culture medium Because these cells specificallycontain nitrogenase a reducing enzyme we hypothesizedthat it could be responsible for nanoparticle formation Totry to confirm this hypothesis in vitro enzymatic reduc-tion of metallic salts was carried out in a Boldrsquos basalmedium using nitrogenase enzyme (NAD[P]H) as reducingagent and Calothrix released polysaccharide (RPS) to sim-ulate in vivo conditions Solution color modification wasobtained after (i) 10 minutes for Au and Ag (ii) 2 hours forPd and (iii) 72 hours for Pt Moreover for Pt only a partialreduction was observed Figure 11 show TEM micrographs

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Brayner et al Cyanobacteria as Bioreactors for the Synthesis of Au Ag Pd and Pt Nanoparticles

Fig 11 TEM micrographs obtained after in vitro enzymatic reductionof (a) Au3+ (b) Ag+ and (c) Pd2+

of Au Ag and Pd nanoparticles obtained by in vitro enzy-matic reduction Figure 11(a) presents TEM image of Aunanoparticles which contained 60 equilateral triangularnanoplates of about 400 nm in edge length and 40hexagonal plates of 70ndash150 nm in size (Table I) On theother hand for Ag and Pd spherical nanoparticles of about40 and 10 nm size respectively were synthesized by thismethod (Figs 11(b) and (c) Table I) In these last twocases morphological polysaccharide modifications were

observed Ag nanoparticles were surrounded by lamel-lae polysaccharide while Pd nanoparticles were alignedinside polysaccharide nanowires In all cases nanoparti-cles obtained by in vitro enzymatic reduction differ dras-tically than that obtained by in vivo reduction

4 DISCUSSION

41 Intra-Cellular Nanoparticle FormationThe Role of Nitrogenase

The formation of Au and Ag nanoparticles by living organ-isms has already been widely reported18ndash26 either extra-or intracellularly Although precise mechanisms have notbeen fully elucidated it was proposed that the reductionoccurs via interactions with cellular molecules such asorganic sulfurs sugars or proteins However at this timethe possibility for an enzyme-mediated reduction reactionwas never suggested In the case of cyanobacteria thishypothesis had to be envisioned since these cells possestwo class of reducing enzymes ie nitrogenase and hydro-genase enzymes Cyanobacteria can fix elemental nitrogenfrom the atmosphere thanks to the action of nitrogenasewhich is mainly located inside the heterocyts (Calothrixand Anabaena) However this enzyme is known to be irre-versibly inhibited upon exposure to molecular oxygen44ndash45

which should be in disagreement with observations of N2-fixation in vegetative cells sites of production of molecularoxygen via photosynthesis Thus an explanation could bethat temporal separation protects nitrogenase from oxygenas for example the occurrence of N2-fixation during dark-ness phases (Leptolyngbya)3244ndash45 It was demonstratedthat the species Anabaena variabilis produces two differ-ent nitrogenases one which functions only in heterocytunder either anaerobic or aerobic conditions and the otherone that operates in both heterocyts and vegetative cellsbut only under anaerobic conditions It has been proposedthat the latter is regulated by environmental conditions andthe former is developmentally regulated46 Hydrogenase isable to reduce amongst others electron acceptors hydro-gen ions to molecular hydrogen so that it may also beinvolved in the observed reaction Indeed the presenceof ferredoxins on the thylakoids works as a link betweenhydrogenase and different electron donors and acceptorsin both photosynthetic and non-photosynthetic systemsso that it seems that both nitrogenase and hydrogenaseenzymes are susceptible to act as reducing agents to pro-mote the reduction of some metallic salts

12

N2 +3H++3eminusnitrogenaseminusminusminusminusminusrarr NH3E

0 =minus310 eV

2H++2eminushydrogenaseminusminusminusminusminusrarr H2E

0 = 0 eV

Au3++3eminus rarr Au0E0 = 1002 eV

Ag++ eminus rarr Ag0E0 = 080 eV

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Cyanobacteria as Bioreactors for the Synthesis of Au Ag Pd and Pt Nanoparticles Brayner et al

Pd2++2eminus rarr Pd0E0 = 092 eV

PtCl2minus6 +2eminus rarr PtCl2minus

4 +2ClminusE0 = 073 eV

PtCl2minus4 +2eminus rarr Pt0 +4ClminusE0 = 073 eV

However in this study Leptolyngbya was selected becauseit does not posses heterocyt and fixes nitrogen only anaer-obically and in the dark In this case it was possible toobserve that the reduction reaction was far less efficientthan for the two other cyanobacteria and occurs only indarkness thus only in the presence of nitrogenase In con-trast the genus Anabaena presents several heterocyts thatproduce high concentration of nitrogenase In this case thereaction was very fast (about 5ndash10 minutes) for all concen-trations of Ag+ and Au3+ As a consequence these ionshad not completely penetrated inside the cells and mostpart of Au and Ag nanoparticles was formed near the cellwalls and were bigger than that formed inside Calothrixand Leptolyngbya (Figs 2(d e) 5(b) and 7(b)) Due to thefast reaction nucleation and growth steps were probablynot separated so that coalescence of Au and Ag nanopar-ticles may have occurred The genus Calothrix appears asan intermediate case in terms of nitrogenase content com-bining short reaction time and small un-aggregated col-loids All these data indicate that nitrogenase is specificallyinvolved in the reduction process

This assumption is strengthened by our in vitro test ofnitrogenase ability to reduce noble metal salts Au Ag Pdand to a lesser extent Pt nanoparticles could be obtainedthrough this approach with reaction times that follow atrend similar to what was observed for cyanobacteria Thissimilarity is particularly clear for platinum where a partialreduction leaving un-reacted Pt4+ and partially-reducedPt2+ ions in the solution was observed both in vivo andin vitro However it is interesting to note that the sizeand morphology of nanoparticles obtained in vitro stronglydiffer from those obtained within the cyanobacteria indi-cating the cellular environment of the enzyme play a sig-nificant role in particle growth control In this contextour studies with Calothrix Anabaena and Leptolyngbyacyanobacteria provide some clues about nitrogenase local-ization The enzyme may be present on the fibrous layersinside the heterocyt and also on the thylakoids inside thevegetative cells However since no metallic nanoparti-cles were observed in intrathylakoidal or intrafibrous layerspaces nitrogenase is only located on the cell membranesOn the basis of membrane-controlled biomineralizationprocesses5 the possibility for these membranes to play arole in nanoparticle growth control should not be put aside

42 Extra-Cellular Nanoparticle ReleaseThe Role of Released Poly-Saccharides

After their intra-cellular synthesis metallic nanoparticlesare released in the culture medium via inter-cellular canal-like intercytoplasmic connections (Fig 4(d)) TEM studies

indicate that the released particles are embedded in apoly-saccharide network Such production of extracel-lular polysaccharides by cyanobacteria is already well-documented they are negatively charged and rich in uronicacids exhibit metal-binding capacities47 They may thusbind toxic metals and be used in the treatment of toxicwaste waters48 In the natural environment these polysac-charides could act as natural chelators binding cationsand concentrating essential trace elements present at sub-marginal concentrations in oligotrophic environments49 Inthis work these poly-saccharides prevent the aggregationof released nanoparticles in most cases One exceptionis for Au nanoparticles formed by Anabaena whose sizeincreases when going from intra- to extra-cellular mediaprobably as a consequence of the fast kinetics of reductionmentioned above

More information on Au colloidal solutions obtainedafter Au nanoparticles release by the cyanobacteria canbe obtained form UV-Visible absorption spectra Theseabsorption bands correspond to the surface plasmon reso-nance of Au metallic nanoparticles According to absorp-tion calculation based on Mie theory34 spherical colloidalAu (red colored) would show max at 530 nm Au col-loidal solution released by the genus Anabaena presentsa pink-purple color the sample exhibiting a narrow bandat max = 537 nm close the calculated value On theother hand Au colloidal solutions released by the genusCalothrix and by the genus Leptolyngbya present both apurple-blue color and a broad bands at max = 577 nm andmax = 562 nm respectively This increase of the plasmonbandwidth may be attributed to the physical dimensionof the nanoparticles (sim6 nm)50 The same behavior wasobserved for Ag nanoparticles According to Mie theory34

spherical colloidal Ag (yellow colored) would show max at410 nm Ag colloidal solution released by genera Calothrixand Anabaena present both a red color and they showa broad plasmon resonance band that may be attributedto interparticle coupling (Fig 8(a)) The red-shift of theabsorption band observed in both cases may be attributedto the presence of adsorbed polysaccharides in the Au andAg surfaces (this behavior was also observed for adsorbedproteins)3351

Another important result obtained by UV-Visible anal-ysis was a qualitative release yield of Au nanoparticlesin the surrounding medium For the same Au3+ concen-tration (10minus4 M) absorption intensities vary from 001 to012 Au colloidal solutions released by Anabaena andCalothrix present a very close final Au metallic concen-tration (012 au) The worst result was obtained with thegenus Leptolyngbya (001 au) On the other hand for Agnanoparticles released by all cyanobacteria the absorptionintensities were not very different showing that all Ag+

salts were reduced to Ag0 probably due to the lower redoxpotential and hence the easier reduction of Ag+Ag0 com-pared to Au3+Au0

2706 J Nanosci Nanotechnol 7 2696ndash2708 2007

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Copyright American Scientific Publishers

RESEARCHARTICLE

Brayner et al Cyanobacteria as Bioreactors for the Synthesis of Au Ag Pd and Pt Nanoparticles

5 CONCLUSION

Production strategies for metallic nanoparticles usingenvironmental-friendly routes using renewable naturalresources have great potential in the next future Ourwork demonstrates that common cyanobacteria strainshave the ability to synthesize Au Ag but also Pd andPt nanoparticles intra-cellularly This process appearsenzyme-mediated as the efficiency of the reduction processcould be related to the presence of nitrogenase We alsoshow that the size of the particle and the reaction yielddepend on the selected strain so that it should be possiblein a near future to identify other cyanobacteria genera withoptimal properties Finally first attempts to use cyanobac-teria as recyclable bioreactors demonstrate that the cellsrecover their full activity after a first metal salt additionand reduction and efforts are in progress through encap-sulation approaches to design continuous flow reactors

Moreover metallic nanoparticles produced insidecyanobacteria cells were released in the culture mediumsurrounded by stabilizing poly-saccharides and can be the-refore used as-synthesized in homogeneous catalysis52ndash53

and other applications such as chemical and biosens-ing54ndash55 biolabeling56ndash57 and photonics58ndash59 The nanopar-ticles may be immobilized in matrices or shaped as thinfilms for optoelectronic applications this being impossibleto achieve if the nanoparticles would have remained insidethe biomass The possibility to extend our approach tooxide andor sulfur nanoparticles elaboration is currentlyunder study

On a more general level this work is a new indicationthat the biological activities of living organisms optimizedthrough evolution to perform specific tasks can be divertedto perform other functions that may answer some of thecurrent challenges in synthetic chemistry

References and Notes

1 C M Zaremba A M Belcher M Fritz Y Li S Mann P KHansma D E Morse J S Speck and G D Stucky Chem Mater8 679 (1996)

2 X W Su and F Z Cui Mater Sci Eng C 7 19 (1999)3 J F Banfield S A Welch H Zhang T T Ebert and R L Penn

Science 289 751 (2000)4 (a) R Wetherbee S Crawford and P Mulvaney Biomineralization

1 89 (2000) (b) J C Weaver L I Pietrasanta N Hedin B FChmelka P K Hansma and D E Morse J Struct Biol 144 271(2003)

5 (a) H A Lowenstam and S Weiner Biomineralization Oxford Uni-versity Press Oxford (1989) (b) J Webb and R J P Williams(Eds) Biomineralization VCH Weinheim (1989) (c) E Baueuer-lein (Ed) Biomineralization Wiley-VCH Weinheim (2000)

6 T Douglas and V T Stark Inorg Chem 39 1828 (2000)7 R Wahl M Mertig J Raff S Selenska-Pobell and W Pompe Adv

Mater 13 736 (2001)8 J He T Kunitake and A Nakao Chem Mater 15 4401 (2003)9 R Brayner T Coradin F Fieacutevet-Vincent J Livage and F Fieacutevet

New J Chem 29 681 (2005)10 C A Mirkin Inorg Chem 38 2258 (2000)

11 (a) M G Warner and J E Hutchinson Nat Mater 2 272 (2003)(b) M Sarakaya C Tamerler A K Y K Schulten and F BabeyxNat Mater 2 577 (2003) (c) B D Reiss C Mao D J SolisK S Ryan T Thomson and A M Belcher Nano Lett 4 1127(2004)

12 B M Rabatic R C Claussen and S I Stupp Chem Mater 175877 (2005)

13 S S Shankar A Rai B Ankawar A Singh A Ahmad andM Sastry Nature 3 482 (2004)

14 C T Dameron R N Reese R K Mehra A R Kortan P J CarrollM L Steigerwald L E Brus and D R Winge Nature 338 596(1989)

15 T Klaus-Joerger J Joerger E Olsson and C G Granqvist TrendsBiotechnol 19 15 (2001)

16 L Q Wu and G F Payne Trends Biotechnol 22 593 (2004)17 T J Beveridge M N Hughes H Lee K T Leung R K Poole

I Savaidis S Silver and J T Trevors Adv Microb Physiol 38177 (1997)

18 G Southam and T J Beveridge Geochim Cosmochim Acta 584527 (1994)

19 G Southam and T J Beveridge Geochim Cosmochim Acta 604369 (1996)

20 K Kashefi J M Tor K P Nevin and D R Lovley Appl EnvironMicrobiol 67 3275 (2001)

21 P Mukherjee A Ahmad D Mandal S Senapati S R Saikar M IKhan R Ramani R Parischa P V Ajaykumar M Alam M Sastryand R Kumar Angew Chem Int Ed 40 3585 (2001)

22 B Nair and T Pradeep Cryst Growth Des 2 293 (2002)23 P Mukherjee S Senapati D Mandal A Ahmad M I Khan

R Kumar and M Sastry Chem Bio Chem 5 461 (2002)24 A Ahmad S Senapati M I Khan R Kumar and M Sastry

Langmuir 19 3550 (2003)25 S S Shankar A Ahmad R Pasricha and M Sastry J Mater

Chem 13 1822 (2003)26 K C Bhainsa and S F DrsquoSouza Colloids Surf B 47 160 (2006)27 H Kuepper and P M H Kroneck Metal Ions in Biological Systems

44 97 (2005)28 S Silver and L T Phung Ann Rev Microb 50 753 (1996)29 R S Prakasham and S V Ramakrishna J Sci Industr Res 57 258

(1998)30 M F Lengke M E Fleet and G Southam Langmuir 22 2780

(2006)31 B Bergman J Gallon A N Rai and L Stal FEMS Microbiol

Rev 19 139 (1997)32 C Bertocchi L Navarini A Cezagraverio and M Anastasio Carbohydr

Polym 12 127 (1990)33 R Brayner T Coradin M-J Vaulay C Mangeney J Livage and

F Fieacutevet Coll Surf A 256 191 (2005)34 J A Creighton and D G Eadon J Chem Soc Faraday Trans 87

3881 (1991)35 R M Slawson M I Van Dyke H Lee and J T Trevors Plasmid

27 72 (1992)36 G J Zhao and S E Stevens Biometals 11 27 (1998)37 I Sadi and B Salopek-Sondi J Coll Interf Sci 275 177 (2004)38 (a) R Brayner G Viau G M da Cruz F Fieacutevet-Vincent F Fieacutevet

and F Bozon-Verduraz Catal Today 57 187 (2000) (b) R BraynerG Viau and F Bozon-Verduraz J Mol Catal 182ndash183 227 (2002)

39 R Brayner D S Cunha and F Bozon-Verduraz Catal Today 78419 (2003)

40 L M Bronstein D M Chernyshov R Karlinsey J W ZwanzigerV G Matveeva E M Sulman G N Demidenko H P Hentze andM Antonietti Chem Mater 15 2623 (2003) (b) J R Gonzalez-Velasco J A Botas R Ferret M P Gonzales-Marcos J L Marcand M A Gutieacuterrez-Ortiz Catal Today 59 395 (2000)

41 D Briggs and M P Seah Practical Surface Analysis Auger andX-ray Photoelectron Spectroscopy Wiley (1990) Vol 1

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RESEARCHARTICLE

Cyanobacteria as Bioreactors for the Synthesis of Au Ag Pd and Pt Nanoparticles Brayner et al

medium and the same concentration of Au3+ solution wasadded to 20 days of Calothrix and Anabaena subculturesThe formation and release of Au metallic nanoparticleswas observed (Fig 5) confirming the possibility to makea continuous cyanobacteria bioreactor The photosyntheticactivities of Calothrix and Anabaena after addition of Ausalts (10minus4 M) in the culture medium were measured usinga PAM fluorimeter (Fig 6) After addition of Au saltsa progressive decrease of photosynthetic activities wasobserved In both cases (Figs 6(a) and (b)) the minimumFV Fm was observed after 10 days of incubation in the pres-ence of Au nanoparticles This behavior was maybe dueto the presence of Au salts on thylakoids (photosynthe-sis sites) and their posterior reduction After reduction thethylakoids were not completely recovered by Au nanopar-ticles and the photosynthetic activities may increase againThese micro-algae remain alive more than three months inthe presence of Au nanoparticles

32 Silver

It is well known that Ag ions and Ag-based compoundsare highly toxic to microorganisms35ndash36 showing strongbiocidal effects on many species of bacteria includingE Coli37 Cyanobacteria have Gram-negative cell wallsuch as E coli so in this work biocidal tests with Ag+

salts were carried out with Calothrix and Anabaena micro-algae The possibility to produce also Ag metallic nanopar-ticles with controlled size and shape inside cyanobacteriacells was also studied Before reaction the culture mediumadded with [Ag+ = 10minus4 M presents a transparent-uncolored solution The experiments were carried out untilobservation of biomass andor culture medium color mod-ification (i) 24 hours for genus Calothrix (ii) 30 min-utes for genus Anabaena and (iii) more than 72 hoursfor genus Leptolyngbya For all cyanobacteria the culturemedium color changed from transparent-uncolored to redPhotonic microscopy showed that heterocyt and vegetativecell colors changed from blue-green to orange-red and yel-low (Fig 7(a)) Thin sections of Anabaena trichomes showspherical 40 nm Ag nanoparticles with a broad size dis-tribution = 42 (Fig 7(b) Table 1) On the other handgenus Calothrix presents well-shaped 15 nm with a narrowsize distribution ( = 08) confirming that Calothrix tri-chome led to the formation of well-controlled size metallicnanoparticles (Table I)

Figure 8(a) presents UV-visible spectra of culture mediaafter color modification For all samples a broad plasmonband was observed with maxima centered between 420ndash450 nm which may be attributed to inter-particle cou-pling effects (multipolar resonance) This behavior wasconfirmed by TEM micrographs of released Ag nanopar-ticles stabilized by micro-algae exo-polysaccharides in thesurrounding medium (Figs 8(b) and (c) Table I) Thesemicro-algae remain alive about one month in the presence

Fig 7 (a) Photonic micrograph of Anabaena grown in [Ag+ =10minus4 M (b) TEM micrograph of Anabaena grown in [Ag+ = 10minus4 Mand (c) TEM micrograph of Calothrix thin sections grown in [Ag+ =10minus4 M

of Ag nanoparticles No biocidal effect was observed dur-ing this time It was observed that released Ag nanopar-ticles by Anabaena trichome present about 25 nm insize with narrow size distribution such as Au nanopar-ticles released by the same micro-algae showing thatcyanobacteria wall may have a particle size limitation dur-ing release process (Table I)

2702 J Nanosci Nanotechnol 7 2696ndash2708 2007

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RESEARCHARTICLE

Brayner et al Cyanobacteria as Bioreactors for the Synthesis of Au Ag Pd and Pt Nanoparticles

0

005

01

015

02

025

03

035

04

300 400 500 600 700 800

Leptolyngbya-Ag 10-4MCalothrix-Ag 10-4MAnabaena-Ag 10-4M

Abs

orba

nce

Wavelength (nm)

100 nm

(a)

(b)

(c)

Fig 8 (a) UV-Visible spectra of Ag nanoparticles released in allcyanobacteria TEM micrographs of Ag nanoparticles released in(b) Anabaena and (c) Calothrix culture medium

33 Palladium and Platinum

Until now only Au and Ag metallic nanoparticles weresynthesized using living cells such as fungi and bacteria23

In order to extend the significance of our approach wehave evaluated the ability of cyanobacteria to reducePd2+ and Pt4+ ions as Pt Pd and PdO nanoparticles

Calothrix-Pd

Anabaena-Pd

Calothrix-Pt

Anabaena-Pt

(a)

(b)

(c)

(d)

Binding energy (eV)

Binding energy (eV)

Binding energy (eV)

Binding energy (eV)

Fig 9 XPS core-level (a) Pd3d52 region (b) Pd3d52 region (c) Pt4f72

region and (d) Pt4f72 region

J Nanosci Nanotechnol 7 2696ndash2708 2007 2703

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Cyanobacteria as Bioreactors for the Synthesis of Au Ag Pd and Pt Nanoparticles Brayner et al

Table II Measured binding energies (BE) of Pd3d32 and Pt4f72 insidecyanobacteria cells and their chemical state concentrations

Chemical stateSample BE (eV) concentrations ()

Calothrix-Pd 3345 Pd0 3893364 Pd2+ 611

Anabaena-Pd 3351 Pd0 2793370 Pd2+ 721

Calothrix-Pt 710 Pt0 90734 Pt2+ 665758 Pt4+ 245

Anabaena-Pt 715 Pt0 427737 Pt2+ 435761 Pt4+ 138

have interesting properties for several catalytic pro-cess (i) hydrogenation of alkenes38 (ii) oxidation ofvolatile organic compounds (VOC)39 and (iii) combustionreactions40

Fig 10 TEM micrographs of Calothrix thin sections grown in(a) [Pd2+= 10minus4 M (b) [Pt4+= 10minus4 M

Before reaction the culture media added with [Pd2+=10minus4 M and [Pt4+ = 10minus4 M present both a transparent-yellow colored solutions After salt addition color mod-ification was observed after (i) 30 minutes for generaCalothrix and Anabaena in the presence of Pd salts and(ii) about fifteen days for both micro-algae in the presenceof Pt salts For all cases the culture medium color slightlychanges from transparent-yellow to brownish-yellow Noheterocyt or vegetative cell color change was observedby photonic microscopy analyses only an increase ofmucilaginous sheath was visible The presence of Pd andPt metallic nanoparticles was confirmed by XPS analysis(Fig 9) Table II shows the binding energies (BE) observedafter peaks decomposition Here only Pd352 and Pt4f72

regions will be discussed For cyanobacteria-Pd samplesPd3d52 region was fitted with two components centered at3345 and 3364 eV for Calothrix and 3351 and 3370 eVfor Anabaena (Figs 9(a) and (b) Table II) The first onewas assigned to metallic Pd and the second one to Pd2+ inan oxygen-containing environment (PdO)41ndash42 Pd nanopar-ticles are not stable in air and a surface passivation mayoccur It was observed that genus Calothrix (389 ofPd0) is more resistant to Pd surface passivation than genusAnabaena (279 of Pd0) (Table II) For cyanobacteria-Ptsamples Pt4f72 region was fitted with three componentscentered at 710 734 and 758 eV for genus Calothrix and715 737 and 761 eV for genus Anabaena (Figs 9(c)and (d) and Table II) The first peak was attributed tometallic Pt4143 The last two peaks were attributed to Pt2+

and Pt4 in the coordination sphere of H2PtCl4 and H2PtCl6respectively In this case only one part of H2PtCl6 wascompletely reduced The best results were also obtainedwith genus Calothrix (Table II) Figure 10 shows Calothrixtrichomes thin sections grown in [Pd2+ = 10minus4 M and[Pt4+= 10minus4 M In both cases well-shaped nanoparticleswith a very narrow size distribution (( = 35 nm =03) for Pd nanoparticles and ( = 32 nm = 03) forPt nanoparticles) were observed (Table I)

34 In vitro Enzymatic Reduction of Metallic Salts

The results obtained in vivo led to conclude that reductionoccurs inside cyanobacteria heterocyts and vegetative cellsfollowed by a partial release of metallic nanoparticlesin the culture medium Because these cells specificallycontain nitrogenase a reducing enzyme we hypothesizedthat it could be responsible for nanoparticle formation Totry to confirm this hypothesis in vitro enzymatic reduc-tion of metallic salts was carried out in a Boldrsquos basalmedium using nitrogenase enzyme (NAD[P]H) as reducingagent and Calothrix released polysaccharide (RPS) to sim-ulate in vivo conditions Solution color modification wasobtained after (i) 10 minutes for Au and Ag (ii) 2 hours forPd and (iii) 72 hours for Pt Moreover for Pt only a partialreduction was observed Figure 11 show TEM micrographs

2704 J Nanosci Nanotechnol 7 2696ndash2708 2007

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RESEARCHARTICLE

Brayner et al Cyanobacteria as Bioreactors for the Synthesis of Au Ag Pd and Pt Nanoparticles

Fig 11 TEM micrographs obtained after in vitro enzymatic reductionof (a) Au3+ (b) Ag+ and (c) Pd2+

of Au Ag and Pd nanoparticles obtained by in vitro enzy-matic reduction Figure 11(a) presents TEM image of Aunanoparticles which contained 60 equilateral triangularnanoplates of about 400 nm in edge length and 40hexagonal plates of 70ndash150 nm in size (Table I) On theother hand for Ag and Pd spherical nanoparticles of about40 and 10 nm size respectively were synthesized by thismethod (Figs 11(b) and (c) Table I) In these last twocases morphological polysaccharide modifications were

observed Ag nanoparticles were surrounded by lamel-lae polysaccharide while Pd nanoparticles were alignedinside polysaccharide nanowires In all cases nanoparti-cles obtained by in vitro enzymatic reduction differ dras-tically than that obtained by in vivo reduction

4 DISCUSSION

41 Intra-Cellular Nanoparticle FormationThe Role of Nitrogenase

The formation of Au and Ag nanoparticles by living organ-isms has already been widely reported18ndash26 either extra-or intracellularly Although precise mechanisms have notbeen fully elucidated it was proposed that the reductionoccurs via interactions with cellular molecules such asorganic sulfurs sugars or proteins However at this timethe possibility for an enzyme-mediated reduction reactionwas never suggested In the case of cyanobacteria thishypothesis had to be envisioned since these cells possestwo class of reducing enzymes ie nitrogenase and hydro-genase enzymes Cyanobacteria can fix elemental nitrogenfrom the atmosphere thanks to the action of nitrogenasewhich is mainly located inside the heterocyts (Calothrixand Anabaena) However this enzyme is known to be irre-versibly inhibited upon exposure to molecular oxygen44ndash45

which should be in disagreement with observations of N2-fixation in vegetative cells sites of production of molecularoxygen via photosynthesis Thus an explanation could bethat temporal separation protects nitrogenase from oxygenas for example the occurrence of N2-fixation during dark-ness phases (Leptolyngbya)3244ndash45 It was demonstratedthat the species Anabaena variabilis produces two differ-ent nitrogenases one which functions only in heterocytunder either anaerobic or aerobic conditions and the otherone that operates in both heterocyts and vegetative cellsbut only under anaerobic conditions It has been proposedthat the latter is regulated by environmental conditions andthe former is developmentally regulated46 Hydrogenase isable to reduce amongst others electron acceptors hydro-gen ions to molecular hydrogen so that it may also beinvolved in the observed reaction Indeed the presenceof ferredoxins on the thylakoids works as a link betweenhydrogenase and different electron donors and acceptorsin both photosynthetic and non-photosynthetic systemsso that it seems that both nitrogenase and hydrogenaseenzymes are susceptible to act as reducing agents to pro-mote the reduction of some metallic salts

12

N2 +3H++3eminusnitrogenaseminusminusminusminusminusrarr NH3E

0 =minus310 eV

2H++2eminushydrogenaseminusminusminusminusminusrarr H2E

0 = 0 eV

Au3++3eminus rarr Au0E0 = 1002 eV

Ag++ eminus rarr Ag0E0 = 080 eV

J Nanosci Nanotechnol 7 2696ndash2708 2007 2705

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RESEARCHARTICLE

Cyanobacteria as Bioreactors for the Synthesis of Au Ag Pd and Pt Nanoparticles Brayner et al

Pd2++2eminus rarr Pd0E0 = 092 eV

PtCl2minus6 +2eminus rarr PtCl2minus

4 +2ClminusE0 = 073 eV

PtCl2minus4 +2eminus rarr Pt0 +4ClminusE0 = 073 eV

However in this study Leptolyngbya was selected becauseit does not posses heterocyt and fixes nitrogen only anaer-obically and in the dark In this case it was possible toobserve that the reduction reaction was far less efficientthan for the two other cyanobacteria and occurs only indarkness thus only in the presence of nitrogenase In con-trast the genus Anabaena presents several heterocyts thatproduce high concentration of nitrogenase In this case thereaction was very fast (about 5ndash10 minutes) for all concen-trations of Ag+ and Au3+ As a consequence these ionshad not completely penetrated inside the cells and mostpart of Au and Ag nanoparticles was formed near the cellwalls and were bigger than that formed inside Calothrixand Leptolyngbya (Figs 2(d e) 5(b) and 7(b)) Due to thefast reaction nucleation and growth steps were probablynot separated so that coalescence of Au and Ag nanopar-ticles may have occurred The genus Calothrix appears asan intermediate case in terms of nitrogenase content com-bining short reaction time and small un-aggregated col-loids All these data indicate that nitrogenase is specificallyinvolved in the reduction process

This assumption is strengthened by our in vitro test ofnitrogenase ability to reduce noble metal salts Au Ag Pdand to a lesser extent Pt nanoparticles could be obtainedthrough this approach with reaction times that follow atrend similar to what was observed for cyanobacteria Thissimilarity is particularly clear for platinum where a partialreduction leaving un-reacted Pt4+ and partially-reducedPt2+ ions in the solution was observed both in vivo andin vitro However it is interesting to note that the sizeand morphology of nanoparticles obtained in vitro stronglydiffer from those obtained within the cyanobacteria indi-cating the cellular environment of the enzyme play a sig-nificant role in particle growth control In this contextour studies with Calothrix Anabaena and Leptolyngbyacyanobacteria provide some clues about nitrogenase local-ization The enzyme may be present on the fibrous layersinside the heterocyt and also on the thylakoids inside thevegetative cells However since no metallic nanoparti-cles were observed in intrathylakoidal or intrafibrous layerspaces nitrogenase is only located on the cell membranesOn the basis of membrane-controlled biomineralizationprocesses5 the possibility for these membranes to play arole in nanoparticle growth control should not be put aside

42 Extra-Cellular Nanoparticle ReleaseThe Role of Released Poly-Saccharides

After their intra-cellular synthesis metallic nanoparticlesare released in the culture medium via inter-cellular canal-like intercytoplasmic connections (Fig 4(d)) TEM studies

indicate that the released particles are embedded in apoly-saccharide network Such production of extracel-lular polysaccharides by cyanobacteria is already well-documented they are negatively charged and rich in uronicacids exhibit metal-binding capacities47 They may thusbind toxic metals and be used in the treatment of toxicwaste waters48 In the natural environment these polysac-charides could act as natural chelators binding cationsand concentrating essential trace elements present at sub-marginal concentrations in oligotrophic environments49 Inthis work these poly-saccharides prevent the aggregationof released nanoparticles in most cases One exceptionis for Au nanoparticles formed by Anabaena whose sizeincreases when going from intra- to extra-cellular mediaprobably as a consequence of the fast kinetics of reductionmentioned above

More information on Au colloidal solutions obtainedafter Au nanoparticles release by the cyanobacteria canbe obtained form UV-Visible absorption spectra Theseabsorption bands correspond to the surface plasmon reso-nance of Au metallic nanoparticles According to absorp-tion calculation based on Mie theory34 spherical colloidalAu (red colored) would show max at 530 nm Au col-loidal solution released by the genus Anabaena presentsa pink-purple color the sample exhibiting a narrow bandat max = 537 nm close the calculated value On theother hand Au colloidal solutions released by the genusCalothrix and by the genus Leptolyngbya present both apurple-blue color and a broad bands at max = 577 nm andmax = 562 nm respectively This increase of the plasmonbandwidth may be attributed to the physical dimensionof the nanoparticles (sim6 nm)50 The same behavior wasobserved for Ag nanoparticles According to Mie theory34

spherical colloidal Ag (yellow colored) would show max at410 nm Ag colloidal solution released by genera Calothrixand Anabaena present both a red color and they showa broad plasmon resonance band that may be attributedto interparticle coupling (Fig 8(a)) The red-shift of theabsorption band observed in both cases may be attributedto the presence of adsorbed polysaccharides in the Au andAg surfaces (this behavior was also observed for adsorbedproteins)3351

Another important result obtained by UV-Visible anal-ysis was a qualitative release yield of Au nanoparticlesin the surrounding medium For the same Au3+ concen-tration (10minus4 M) absorption intensities vary from 001 to012 Au colloidal solutions released by Anabaena andCalothrix present a very close final Au metallic concen-tration (012 au) The worst result was obtained with thegenus Leptolyngbya (001 au) On the other hand for Agnanoparticles released by all cyanobacteria the absorptionintensities were not very different showing that all Ag+

salts were reduced to Ag0 probably due to the lower redoxpotential and hence the easier reduction of Ag+Ag0 com-pared to Au3+Au0

2706 J Nanosci Nanotechnol 7 2696ndash2708 2007

Delivered by Publishing Technology to Florida State University - TallahasseeIP 14620120822 On Fri 26 Sep 2014 103634

Copyright American Scientific Publishers

RESEARCHARTICLE

Brayner et al Cyanobacteria as Bioreactors for the Synthesis of Au Ag Pd and Pt Nanoparticles

5 CONCLUSION

Production strategies for metallic nanoparticles usingenvironmental-friendly routes using renewable naturalresources have great potential in the next future Ourwork demonstrates that common cyanobacteria strainshave the ability to synthesize Au Ag but also Pd andPt nanoparticles intra-cellularly This process appearsenzyme-mediated as the efficiency of the reduction processcould be related to the presence of nitrogenase We alsoshow that the size of the particle and the reaction yielddepend on the selected strain so that it should be possiblein a near future to identify other cyanobacteria genera withoptimal properties Finally first attempts to use cyanobac-teria as recyclable bioreactors demonstrate that the cellsrecover their full activity after a first metal salt additionand reduction and efforts are in progress through encap-sulation approaches to design continuous flow reactors

Moreover metallic nanoparticles produced insidecyanobacteria cells were released in the culture mediumsurrounded by stabilizing poly-saccharides and can be the-refore used as-synthesized in homogeneous catalysis52ndash53

and other applications such as chemical and biosens-ing54ndash55 biolabeling56ndash57 and photonics58ndash59 The nanopar-ticles may be immobilized in matrices or shaped as thinfilms for optoelectronic applications this being impossibleto achieve if the nanoparticles would have remained insidethe biomass The possibility to extend our approach tooxide andor sulfur nanoparticles elaboration is currentlyunder study

On a more general level this work is a new indicationthat the biological activities of living organisms optimizedthrough evolution to perform specific tasks can be divertedto perform other functions that may answer some of thecurrent challenges in synthetic chemistry

References and Notes

1 C M Zaremba A M Belcher M Fritz Y Li S Mann P KHansma D E Morse J S Speck and G D Stucky Chem Mater8 679 (1996)

2 X W Su and F Z Cui Mater Sci Eng C 7 19 (1999)3 J F Banfield S A Welch H Zhang T T Ebert and R L Penn

Science 289 751 (2000)4 (a) R Wetherbee S Crawford and P Mulvaney Biomineralization

1 89 (2000) (b) J C Weaver L I Pietrasanta N Hedin B FChmelka P K Hansma and D E Morse J Struct Biol 144 271(2003)

5 (a) H A Lowenstam and S Weiner Biomineralization Oxford Uni-versity Press Oxford (1989) (b) J Webb and R J P Williams(Eds) Biomineralization VCH Weinheim (1989) (c) E Baueuer-lein (Ed) Biomineralization Wiley-VCH Weinheim (2000)

6 T Douglas and V T Stark Inorg Chem 39 1828 (2000)7 R Wahl M Mertig J Raff S Selenska-Pobell and W Pompe Adv

Mater 13 736 (2001)8 J He T Kunitake and A Nakao Chem Mater 15 4401 (2003)9 R Brayner T Coradin F Fieacutevet-Vincent J Livage and F Fieacutevet

New J Chem 29 681 (2005)10 C A Mirkin Inorg Chem 38 2258 (2000)

11 (a) M G Warner and J E Hutchinson Nat Mater 2 272 (2003)(b) M Sarakaya C Tamerler A K Y K Schulten and F BabeyxNat Mater 2 577 (2003) (c) B D Reiss C Mao D J SolisK S Ryan T Thomson and A M Belcher Nano Lett 4 1127(2004)

12 B M Rabatic R C Claussen and S I Stupp Chem Mater 175877 (2005)

13 S S Shankar A Rai B Ankawar A Singh A Ahmad andM Sastry Nature 3 482 (2004)

14 C T Dameron R N Reese R K Mehra A R Kortan P J CarrollM L Steigerwald L E Brus and D R Winge Nature 338 596(1989)

15 T Klaus-Joerger J Joerger E Olsson and C G Granqvist TrendsBiotechnol 19 15 (2001)

16 L Q Wu and G F Payne Trends Biotechnol 22 593 (2004)17 T J Beveridge M N Hughes H Lee K T Leung R K Poole

I Savaidis S Silver and J T Trevors Adv Microb Physiol 38177 (1997)

18 G Southam and T J Beveridge Geochim Cosmochim Acta 584527 (1994)

19 G Southam and T J Beveridge Geochim Cosmochim Acta 604369 (1996)

20 K Kashefi J M Tor K P Nevin and D R Lovley Appl EnvironMicrobiol 67 3275 (2001)

21 P Mukherjee A Ahmad D Mandal S Senapati S R Saikar M IKhan R Ramani R Parischa P V Ajaykumar M Alam M Sastryand R Kumar Angew Chem Int Ed 40 3585 (2001)

22 B Nair and T Pradeep Cryst Growth Des 2 293 (2002)23 P Mukherjee S Senapati D Mandal A Ahmad M I Khan

R Kumar and M Sastry Chem Bio Chem 5 461 (2002)24 A Ahmad S Senapati M I Khan R Kumar and M Sastry

Langmuir 19 3550 (2003)25 S S Shankar A Ahmad R Pasricha and M Sastry J Mater

Chem 13 1822 (2003)26 K C Bhainsa and S F DrsquoSouza Colloids Surf B 47 160 (2006)27 H Kuepper and P M H Kroneck Metal Ions in Biological Systems

44 97 (2005)28 S Silver and L T Phung Ann Rev Microb 50 753 (1996)29 R S Prakasham and S V Ramakrishna J Sci Industr Res 57 258

(1998)30 M F Lengke M E Fleet and G Southam Langmuir 22 2780

(2006)31 B Bergman J Gallon A N Rai and L Stal FEMS Microbiol

Rev 19 139 (1997)32 C Bertocchi L Navarini A Cezagraverio and M Anastasio Carbohydr

Polym 12 127 (1990)33 R Brayner T Coradin M-J Vaulay C Mangeney J Livage and

F Fieacutevet Coll Surf A 256 191 (2005)34 J A Creighton and D G Eadon J Chem Soc Faraday Trans 87

3881 (1991)35 R M Slawson M I Van Dyke H Lee and J T Trevors Plasmid

27 72 (1992)36 G J Zhao and S E Stevens Biometals 11 27 (1998)37 I Sadi and B Salopek-Sondi J Coll Interf Sci 275 177 (2004)38 (a) R Brayner G Viau G M da Cruz F Fieacutevet-Vincent F Fieacutevet

and F Bozon-Verduraz Catal Today 57 187 (2000) (b) R BraynerG Viau and F Bozon-Verduraz J Mol Catal 182ndash183 227 (2002)

39 R Brayner D S Cunha and F Bozon-Verduraz Catal Today 78419 (2003)

40 L M Bronstein D M Chernyshov R Karlinsey J W ZwanzigerV G Matveeva E M Sulman G N Demidenko H P Hentze andM Antonietti Chem Mater 15 2623 (2003) (b) J R Gonzalez-Velasco J A Botas R Ferret M P Gonzales-Marcos J L Marcand M A Gutieacuterrez-Ortiz Catal Today 59 395 (2000)

41 D Briggs and M P Seah Practical Surface Analysis Auger andX-ray Photoelectron Spectroscopy Wiley (1990) Vol 1

J Nanosci Nanotechnol 7 2696ndash2708 2007 2707

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RESEARCHARTICLE

Brayner et al Cyanobacteria as Bioreactors for the Synthesis of Au Ag Pd and Pt Nanoparticles

0

005

01

015

02

025

03

035

04

300 400 500 600 700 800

Leptolyngbya-Ag 10-4MCalothrix-Ag 10-4MAnabaena-Ag 10-4M

Abs

orba

nce

Wavelength (nm)

100 nm

(a)

(b)

(c)

Fig 8 (a) UV-Visible spectra of Ag nanoparticles released in allcyanobacteria TEM micrographs of Ag nanoparticles released in(b) Anabaena and (c) Calothrix culture medium

33 Palladium and Platinum

Until now only Au and Ag metallic nanoparticles weresynthesized using living cells such as fungi and bacteria23

In order to extend the significance of our approach wehave evaluated the ability of cyanobacteria to reducePd2+ and Pt4+ ions as Pt Pd and PdO nanoparticles

Calothrix-Pd

Anabaena-Pd

Calothrix-Pt

Anabaena-Pt

(a)

(b)

(c)

(d)

Binding energy (eV)

Binding energy (eV)

Binding energy (eV)

Binding energy (eV)

Fig 9 XPS core-level (a) Pd3d52 region (b) Pd3d52 region (c) Pt4f72

region and (d) Pt4f72 region

J Nanosci Nanotechnol 7 2696ndash2708 2007 2703

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RESEARCHARTICLE

Cyanobacteria as Bioreactors for the Synthesis of Au Ag Pd and Pt Nanoparticles Brayner et al

Table II Measured binding energies (BE) of Pd3d32 and Pt4f72 insidecyanobacteria cells and their chemical state concentrations

Chemical stateSample BE (eV) concentrations ()

Calothrix-Pd 3345 Pd0 3893364 Pd2+ 611

Anabaena-Pd 3351 Pd0 2793370 Pd2+ 721

Calothrix-Pt 710 Pt0 90734 Pt2+ 665758 Pt4+ 245

Anabaena-Pt 715 Pt0 427737 Pt2+ 435761 Pt4+ 138

have interesting properties for several catalytic pro-cess (i) hydrogenation of alkenes38 (ii) oxidation ofvolatile organic compounds (VOC)39 and (iii) combustionreactions40

Fig 10 TEM micrographs of Calothrix thin sections grown in(a) [Pd2+= 10minus4 M (b) [Pt4+= 10minus4 M

Before reaction the culture media added with [Pd2+=10minus4 M and [Pt4+ = 10minus4 M present both a transparent-yellow colored solutions After salt addition color mod-ification was observed after (i) 30 minutes for generaCalothrix and Anabaena in the presence of Pd salts and(ii) about fifteen days for both micro-algae in the presenceof Pt salts For all cases the culture medium color slightlychanges from transparent-yellow to brownish-yellow Noheterocyt or vegetative cell color change was observedby photonic microscopy analyses only an increase ofmucilaginous sheath was visible The presence of Pd andPt metallic nanoparticles was confirmed by XPS analysis(Fig 9) Table II shows the binding energies (BE) observedafter peaks decomposition Here only Pd352 and Pt4f72

regions will be discussed For cyanobacteria-Pd samplesPd3d52 region was fitted with two components centered at3345 and 3364 eV for Calothrix and 3351 and 3370 eVfor Anabaena (Figs 9(a) and (b) Table II) The first onewas assigned to metallic Pd and the second one to Pd2+ inan oxygen-containing environment (PdO)41ndash42 Pd nanopar-ticles are not stable in air and a surface passivation mayoccur It was observed that genus Calothrix (389 ofPd0) is more resistant to Pd surface passivation than genusAnabaena (279 of Pd0) (Table II) For cyanobacteria-Ptsamples Pt4f72 region was fitted with three componentscentered at 710 734 and 758 eV for genus Calothrix and715 737 and 761 eV for genus Anabaena (Figs 9(c)and (d) and Table II) The first peak was attributed tometallic Pt4143 The last two peaks were attributed to Pt2+

and Pt4 in the coordination sphere of H2PtCl4 and H2PtCl6respectively In this case only one part of H2PtCl6 wascompletely reduced The best results were also obtainedwith genus Calothrix (Table II) Figure 10 shows Calothrixtrichomes thin sections grown in [Pd2+ = 10minus4 M and[Pt4+= 10minus4 M In both cases well-shaped nanoparticleswith a very narrow size distribution (( = 35 nm =03) for Pd nanoparticles and ( = 32 nm = 03) forPt nanoparticles) were observed (Table I)

34 In vitro Enzymatic Reduction of Metallic Salts

The results obtained in vivo led to conclude that reductionoccurs inside cyanobacteria heterocyts and vegetative cellsfollowed by a partial release of metallic nanoparticlesin the culture medium Because these cells specificallycontain nitrogenase a reducing enzyme we hypothesizedthat it could be responsible for nanoparticle formation Totry to confirm this hypothesis in vitro enzymatic reduc-tion of metallic salts was carried out in a Boldrsquos basalmedium using nitrogenase enzyme (NAD[P]H) as reducingagent and Calothrix released polysaccharide (RPS) to sim-ulate in vivo conditions Solution color modification wasobtained after (i) 10 minutes for Au and Ag (ii) 2 hours forPd and (iii) 72 hours for Pt Moreover for Pt only a partialreduction was observed Figure 11 show TEM micrographs

2704 J Nanosci Nanotechnol 7 2696ndash2708 2007

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RESEARCHARTICLE

Brayner et al Cyanobacteria as Bioreactors for the Synthesis of Au Ag Pd and Pt Nanoparticles

Fig 11 TEM micrographs obtained after in vitro enzymatic reductionof (a) Au3+ (b) Ag+ and (c) Pd2+

of Au Ag and Pd nanoparticles obtained by in vitro enzy-matic reduction Figure 11(a) presents TEM image of Aunanoparticles which contained 60 equilateral triangularnanoplates of about 400 nm in edge length and 40hexagonal plates of 70ndash150 nm in size (Table I) On theother hand for Ag and Pd spherical nanoparticles of about40 and 10 nm size respectively were synthesized by thismethod (Figs 11(b) and (c) Table I) In these last twocases morphological polysaccharide modifications were

observed Ag nanoparticles were surrounded by lamel-lae polysaccharide while Pd nanoparticles were alignedinside polysaccharide nanowires In all cases nanoparti-cles obtained by in vitro enzymatic reduction differ dras-tically than that obtained by in vivo reduction

4 DISCUSSION

41 Intra-Cellular Nanoparticle FormationThe Role of Nitrogenase

The formation of Au and Ag nanoparticles by living organ-isms has already been widely reported18ndash26 either extra-or intracellularly Although precise mechanisms have notbeen fully elucidated it was proposed that the reductionoccurs via interactions with cellular molecules such asorganic sulfurs sugars or proteins However at this timethe possibility for an enzyme-mediated reduction reactionwas never suggested In the case of cyanobacteria thishypothesis had to be envisioned since these cells possestwo class of reducing enzymes ie nitrogenase and hydro-genase enzymes Cyanobacteria can fix elemental nitrogenfrom the atmosphere thanks to the action of nitrogenasewhich is mainly located inside the heterocyts (Calothrixand Anabaena) However this enzyme is known to be irre-versibly inhibited upon exposure to molecular oxygen44ndash45

which should be in disagreement with observations of N2-fixation in vegetative cells sites of production of molecularoxygen via photosynthesis Thus an explanation could bethat temporal separation protects nitrogenase from oxygenas for example the occurrence of N2-fixation during dark-ness phases (Leptolyngbya)3244ndash45 It was demonstratedthat the species Anabaena variabilis produces two differ-ent nitrogenases one which functions only in heterocytunder either anaerobic or aerobic conditions and the otherone that operates in both heterocyts and vegetative cellsbut only under anaerobic conditions It has been proposedthat the latter is regulated by environmental conditions andthe former is developmentally regulated46 Hydrogenase isable to reduce amongst others electron acceptors hydro-gen ions to molecular hydrogen so that it may also beinvolved in the observed reaction Indeed the presenceof ferredoxins on the thylakoids works as a link betweenhydrogenase and different electron donors and acceptorsin both photosynthetic and non-photosynthetic systemsso that it seems that both nitrogenase and hydrogenaseenzymes are susceptible to act as reducing agents to pro-mote the reduction of some metallic salts

12

N2 +3H++3eminusnitrogenaseminusminusminusminusminusrarr NH3E

0 =minus310 eV

2H++2eminushydrogenaseminusminusminusminusminusrarr H2E

0 = 0 eV

Au3++3eminus rarr Au0E0 = 1002 eV

Ag++ eminus rarr Ag0E0 = 080 eV

J Nanosci Nanotechnol 7 2696ndash2708 2007 2705

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RESEARCHARTICLE

Cyanobacteria as Bioreactors for the Synthesis of Au Ag Pd and Pt Nanoparticles Brayner et al

Pd2++2eminus rarr Pd0E0 = 092 eV

PtCl2minus6 +2eminus rarr PtCl2minus

4 +2ClminusE0 = 073 eV

PtCl2minus4 +2eminus rarr Pt0 +4ClminusE0 = 073 eV

However in this study Leptolyngbya was selected becauseit does not posses heterocyt and fixes nitrogen only anaer-obically and in the dark In this case it was possible toobserve that the reduction reaction was far less efficientthan for the two other cyanobacteria and occurs only indarkness thus only in the presence of nitrogenase In con-trast the genus Anabaena presents several heterocyts thatproduce high concentration of nitrogenase In this case thereaction was very fast (about 5ndash10 minutes) for all concen-trations of Ag+ and Au3+ As a consequence these ionshad not completely penetrated inside the cells and mostpart of Au and Ag nanoparticles was formed near the cellwalls and were bigger than that formed inside Calothrixand Leptolyngbya (Figs 2(d e) 5(b) and 7(b)) Due to thefast reaction nucleation and growth steps were probablynot separated so that coalescence of Au and Ag nanopar-ticles may have occurred The genus Calothrix appears asan intermediate case in terms of nitrogenase content com-bining short reaction time and small un-aggregated col-loids All these data indicate that nitrogenase is specificallyinvolved in the reduction process

This assumption is strengthened by our in vitro test ofnitrogenase ability to reduce noble metal salts Au Ag Pdand to a lesser extent Pt nanoparticles could be obtainedthrough this approach with reaction times that follow atrend similar to what was observed for cyanobacteria Thissimilarity is particularly clear for platinum where a partialreduction leaving un-reacted Pt4+ and partially-reducedPt2+ ions in the solution was observed both in vivo andin vitro However it is interesting to note that the sizeand morphology of nanoparticles obtained in vitro stronglydiffer from those obtained within the cyanobacteria indi-cating the cellular environment of the enzyme play a sig-nificant role in particle growth control In this contextour studies with Calothrix Anabaena and Leptolyngbyacyanobacteria provide some clues about nitrogenase local-ization The enzyme may be present on the fibrous layersinside the heterocyt and also on the thylakoids inside thevegetative cells However since no metallic nanoparti-cles were observed in intrathylakoidal or intrafibrous layerspaces nitrogenase is only located on the cell membranesOn the basis of membrane-controlled biomineralizationprocesses5 the possibility for these membranes to play arole in nanoparticle growth control should not be put aside

42 Extra-Cellular Nanoparticle ReleaseThe Role of Released Poly-Saccharides

After their intra-cellular synthesis metallic nanoparticlesare released in the culture medium via inter-cellular canal-like intercytoplasmic connections (Fig 4(d)) TEM studies

indicate that the released particles are embedded in apoly-saccharide network Such production of extracel-lular polysaccharides by cyanobacteria is already well-documented they are negatively charged and rich in uronicacids exhibit metal-binding capacities47 They may thusbind toxic metals and be used in the treatment of toxicwaste waters48 In the natural environment these polysac-charides could act as natural chelators binding cationsand concentrating essential trace elements present at sub-marginal concentrations in oligotrophic environments49 Inthis work these poly-saccharides prevent the aggregationof released nanoparticles in most cases One exceptionis for Au nanoparticles formed by Anabaena whose sizeincreases when going from intra- to extra-cellular mediaprobably as a consequence of the fast kinetics of reductionmentioned above

More information on Au colloidal solutions obtainedafter Au nanoparticles release by the cyanobacteria canbe obtained form UV-Visible absorption spectra Theseabsorption bands correspond to the surface plasmon reso-nance of Au metallic nanoparticles According to absorp-tion calculation based on Mie theory34 spherical colloidalAu (red colored) would show max at 530 nm Au col-loidal solution released by the genus Anabaena presentsa pink-purple color the sample exhibiting a narrow bandat max = 537 nm close the calculated value On theother hand Au colloidal solutions released by the genusCalothrix and by the genus Leptolyngbya present both apurple-blue color and a broad bands at max = 577 nm andmax = 562 nm respectively This increase of the plasmonbandwidth may be attributed to the physical dimensionof the nanoparticles (sim6 nm)50 The same behavior wasobserved for Ag nanoparticles According to Mie theory34

spherical colloidal Ag (yellow colored) would show max at410 nm Ag colloidal solution released by genera Calothrixand Anabaena present both a red color and they showa broad plasmon resonance band that may be attributedto interparticle coupling (Fig 8(a)) The red-shift of theabsorption band observed in both cases may be attributedto the presence of adsorbed polysaccharides in the Au andAg surfaces (this behavior was also observed for adsorbedproteins)3351

Another important result obtained by UV-Visible anal-ysis was a qualitative release yield of Au nanoparticlesin the surrounding medium For the same Au3+ concen-tration (10minus4 M) absorption intensities vary from 001 to012 Au colloidal solutions released by Anabaena andCalothrix present a very close final Au metallic concen-tration (012 au) The worst result was obtained with thegenus Leptolyngbya (001 au) On the other hand for Agnanoparticles released by all cyanobacteria the absorptionintensities were not very different showing that all Ag+

salts were reduced to Ag0 probably due to the lower redoxpotential and hence the easier reduction of Ag+Ag0 com-pared to Au3+Au0

2706 J Nanosci Nanotechnol 7 2696ndash2708 2007

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RESEARCHARTICLE

Brayner et al Cyanobacteria as Bioreactors for the Synthesis of Au Ag Pd and Pt Nanoparticles

5 CONCLUSION

Production strategies for metallic nanoparticles usingenvironmental-friendly routes using renewable naturalresources have great potential in the next future Ourwork demonstrates that common cyanobacteria strainshave the ability to synthesize Au Ag but also Pd andPt nanoparticles intra-cellularly This process appearsenzyme-mediated as the efficiency of the reduction processcould be related to the presence of nitrogenase We alsoshow that the size of the particle and the reaction yielddepend on the selected strain so that it should be possiblein a near future to identify other cyanobacteria genera withoptimal properties Finally first attempts to use cyanobac-teria as recyclable bioreactors demonstrate that the cellsrecover their full activity after a first metal salt additionand reduction and efforts are in progress through encap-sulation approaches to design continuous flow reactors

Moreover metallic nanoparticles produced insidecyanobacteria cells were released in the culture mediumsurrounded by stabilizing poly-saccharides and can be the-refore used as-synthesized in homogeneous catalysis52ndash53

and other applications such as chemical and biosens-ing54ndash55 biolabeling56ndash57 and photonics58ndash59 The nanopar-ticles may be immobilized in matrices or shaped as thinfilms for optoelectronic applications this being impossibleto achieve if the nanoparticles would have remained insidethe biomass The possibility to extend our approach tooxide andor sulfur nanoparticles elaboration is currentlyunder study

On a more general level this work is a new indicationthat the biological activities of living organisms optimizedthrough evolution to perform specific tasks can be divertedto perform other functions that may answer some of thecurrent challenges in synthetic chemistry

References and Notes

1 C M Zaremba A M Belcher M Fritz Y Li S Mann P KHansma D E Morse J S Speck and G D Stucky Chem Mater8 679 (1996)

2 X W Su and F Z Cui Mater Sci Eng C 7 19 (1999)3 J F Banfield S A Welch H Zhang T T Ebert and R L Penn

Science 289 751 (2000)4 (a) R Wetherbee S Crawford and P Mulvaney Biomineralization

1 89 (2000) (b) J C Weaver L I Pietrasanta N Hedin B FChmelka P K Hansma and D E Morse J Struct Biol 144 271(2003)

5 (a) H A Lowenstam and S Weiner Biomineralization Oxford Uni-versity Press Oxford (1989) (b) J Webb and R J P Williams(Eds) Biomineralization VCH Weinheim (1989) (c) E Baueuer-lein (Ed) Biomineralization Wiley-VCH Weinheim (2000)

6 T Douglas and V T Stark Inorg Chem 39 1828 (2000)7 R Wahl M Mertig J Raff S Selenska-Pobell and W Pompe Adv

Mater 13 736 (2001)8 J He T Kunitake and A Nakao Chem Mater 15 4401 (2003)9 R Brayner T Coradin F Fieacutevet-Vincent J Livage and F Fieacutevet

New J Chem 29 681 (2005)10 C A Mirkin Inorg Chem 38 2258 (2000)

11 (a) M G Warner and J E Hutchinson Nat Mater 2 272 (2003)(b) M Sarakaya C Tamerler A K Y K Schulten and F BabeyxNat Mater 2 577 (2003) (c) B D Reiss C Mao D J SolisK S Ryan T Thomson and A M Belcher Nano Lett 4 1127(2004)

12 B M Rabatic R C Claussen and S I Stupp Chem Mater 175877 (2005)

13 S S Shankar A Rai B Ankawar A Singh A Ahmad andM Sastry Nature 3 482 (2004)

14 C T Dameron R N Reese R K Mehra A R Kortan P J CarrollM L Steigerwald L E Brus and D R Winge Nature 338 596(1989)

15 T Klaus-Joerger J Joerger E Olsson and C G Granqvist TrendsBiotechnol 19 15 (2001)

16 L Q Wu and G F Payne Trends Biotechnol 22 593 (2004)17 T J Beveridge M N Hughes H Lee K T Leung R K Poole

I Savaidis S Silver and J T Trevors Adv Microb Physiol 38177 (1997)

18 G Southam and T J Beveridge Geochim Cosmochim Acta 584527 (1994)

19 G Southam and T J Beveridge Geochim Cosmochim Acta 604369 (1996)

20 K Kashefi J M Tor K P Nevin and D R Lovley Appl EnvironMicrobiol 67 3275 (2001)

21 P Mukherjee A Ahmad D Mandal S Senapati S R Saikar M IKhan R Ramani R Parischa P V Ajaykumar M Alam M Sastryand R Kumar Angew Chem Int Ed 40 3585 (2001)

22 B Nair and T Pradeep Cryst Growth Des 2 293 (2002)23 P Mukherjee S Senapati D Mandal A Ahmad M I Khan

R Kumar and M Sastry Chem Bio Chem 5 461 (2002)24 A Ahmad S Senapati M I Khan R Kumar and M Sastry

Langmuir 19 3550 (2003)25 S S Shankar A Ahmad R Pasricha and M Sastry J Mater

Chem 13 1822 (2003)26 K C Bhainsa and S F DrsquoSouza Colloids Surf B 47 160 (2006)27 H Kuepper and P M H Kroneck Metal Ions in Biological Systems

44 97 (2005)28 S Silver and L T Phung Ann Rev Microb 50 753 (1996)29 R S Prakasham and S V Ramakrishna J Sci Industr Res 57 258

(1998)30 M F Lengke M E Fleet and G Southam Langmuir 22 2780

(2006)31 B Bergman J Gallon A N Rai and L Stal FEMS Microbiol

Rev 19 139 (1997)32 C Bertocchi L Navarini A Cezagraverio and M Anastasio Carbohydr

Polym 12 127 (1990)33 R Brayner T Coradin M-J Vaulay C Mangeney J Livage and

F Fieacutevet Coll Surf A 256 191 (2005)34 J A Creighton and D G Eadon J Chem Soc Faraday Trans 87

3881 (1991)35 R M Slawson M I Van Dyke H Lee and J T Trevors Plasmid

27 72 (1992)36 G J Zhao and S E Stevens Biometals 11 27 (1998)37 I Sadi and B Salopek-Sondi J Coll Interf Sci 275 177 (2004)38 (a) R Brayner G Viau G M da Cruz F Fieacutevet-Vincent F Fieacutevet

and F Bozon-Verduraz Catal Today 57 187 (2000) (b) R BraynerG Viau and F Bozon-Verduraz J Mol Catal 182ndash183 227 (2002)

39 R Brayner D S Cunha and F Bozon-Verduraz Catal Today 78419 (2003)

40 L M Bronstein D M Chernyshov R Karlinsey J W ZwanzigerV G Matveeva E M Sulman G N Demidenko H P Hentze andM Antonietti Chem Mater 15 2623 (2003) (b) J R Gonzalez-Velasco J A Botas R Ferret M P Gonzales-Marcos J L Marcand M A Gutieacuterrez-Ortiz Catal Today 59 395 (2000)

41 D Briggs and M P Seah Practical Surface Analysis Auger andX-ray Photoelectron Spectroscopy Wiley (1990) Vol 1

J Nanosci Nanotechnol 7 2696ndash2708 2007 2707

Delivered by Publishing Technology to Florida State University - TallahasseeIP 14620120822 On Fri 26 Sep 2014 103634

Copyright American Scientific Publishers

RESEARCHARTICLE

Cyanobacteria as Bioreactors for the Synthesis of Au Ag Pd and Pt Nanoparticles Brayner et al

Table II Measured binding energies (BE) of Pd3d32 and Pt4f72 insidecyanobacteria cells and their chemical state concentrations

Chemical stateSample BE (eV) concentrations ()

Calothrix-Pd 3345 Pd0 3893364 Pd2+ 611

Anabaena-Pd 3351 Pd0 2793370 Pd2+ 721

Calothrix-Pt 710 Pt0 90734 Pt2+ 665758 Pt4+ 245

Anabaena-Pt 715 Pt0 427737 Pt2+ 435761 Pt4+ 138

have interesting properties for several catalytic pro-cess (i) hydrogenation of alkenes38 (ii) oxidation ofvolatile organic compounds (VOC)39 and (iii) combustionreactions40

Fig 10 TEM micrographs of Calothrix thin sections grown in(a) [Pd2+= 10minus4 M (b) [Pt4+= 10minus4 M

Before reaction the culture media added with [Pd2+=10minus4 M and [Pt4+ = 10minus4 M present both a transparent-yellow colored solutions After salt addition color mod-ification was observed after (i) 30 minutes for generaCalothrix and Anabaena in the presence of Pd salts and(ii) about fifteen days for both micro-algae in the presenceof Pt salts For all cases the culture medium color slightlychanges from transparent-yellow to brownish-yellow Noheterocyt or vegetative cell color change was observedby photonic microscopy analyses only an increase ofmucilaginous sheath was visible The presence of Pd andPt metallic nanoparticles was confirmed by XPS analysis(Fig 9) Table II shows the binding energies (BE) observedafter peaks decomposition Here only Pd352 and Pt4f72

regions will be discussed For cyanobacteria-Pd samplesPd3d52 region was fitted with two components centered at3345 and 3364 eV for Calothrix and 3351 and 3370 eVfor Anabaena (Figs 9(a) and (b) Table II) The first onewas assigned to metallic Pd and the second one to Pd2+ inan oxygen-containing environment (PdO)41ndash42 Pd nanopar-ticles are not stable in air and a surface passivation mayoccur It was observed that genus Calothrix (389 ofPd0) is more resistant to Pd surface passivation than genusAnabaena (279 of Pd0) (Table II) For cyanobacteria-Ptsamples Pt4f72 region was fitted with three componentscentered at 710 734 and 758 eV for genus Calothrix and715 737 and 761 eV for genus Anabaena (Figs 9(c)and (d) and Table II) The first peak was attributed tometallic Pt4143 The last two peaks were attributed to Pt2+

and Pt4 in the coordination sphere of H2PtCl4 and H2PtCl6respectively In this case only one part of H2PtCl6 wascompletely reduced The best results were also obtainedwith genus Calothrix (Table II) Figure 10 shows Calothrixtrichomes thin sections grown in [Pd2+ = 10minus4 M and[Pt4+= 10minus4 M In both cases well-shaped nanoparticleswith a very narrow size distribution (( = 35 nm =03) for Pd nanoparticles and ( = 32 nm = 03) forPt nanoparticles) were observed (Table I)

34 In vitro Enzymatic Reduction of Metallic Salts

The results obtained in vivo led to conclude that reductionoccurs inside cyanobacteria heterocyts and vegetative cellsfollowed by a partial release of metallic nanoparticlesin the culture medium Because these cells specificallycontain nitrogenase a reducing enzyme we hypothesizedthat it could be responsible for nanoparticle formation Totry to confirm this hypothesis in vitro enzymatic reduc-tion of metallic salts was carried out in a Boldrsquos basalmedium using nitrogenase enzyme (NAD[P]H) as reducingagent and Calothrix released polysaccharide (RPS) to sim-ulate in vivo conditions Solution color modification wasobtained after (i) 10 minutes for Au and Ag (ii) 2 hours forPd and (iii) 72 hours for Pt Moreover for Pt only a partialreduction was observed Figure 11 show TEM micrographs

2704 J Nanosci Nanotechnol 7 2696ndash2708 2007

Delivered by Publishing Technology to Florida State University - TallahasseeIP 14620120822 On Fri 26 Sep 2014 103634

Copyright American Scientific Publishers

RESEARCHARTICLE

Brayner et al Cyanobacteria as Bioreactors for the Synthesis of Au Ag Pd and Pt Nanoparticles

Fig 11 TEM micrographs obtained after in vitro enzymatic reductionof (a) Au3+ (b) Ag+ and (c) Pd2+

of Au Ag and Pd nanoparticles obtained by in vitro enzy-matic reduction Figure 11(a) presents TEM image of Aunanoparticles which contained 60 equilateral triangularnanoplates of about 400 nm in edge length and 40hexagonal plates of 70ndash150 nm in size (Table I) On theother hand for Ag and Pd spherical nanoparticles of about40 and 10 nm size respectively were synthesized by thismethod (Figs 11(b) and (c) Table I) In these last twocases morphological polysaccharide modifications were

observed Ag nanoparticles were surrounded by lamel-lae polysaccharide while Pd nanoparticles were alignedinside polysaccharide nanowires In all cases nanoparti-cles obtained by in vitro enzymatic reduction differ dras-tically than that obtained by in vivo reduction

4 DISCUSSION

41 Intra-Cellular Nanoparticle FormationThe Role of Nitrogenase

The formation of Au and Ag nanoparticles by living organ-isms has already been widely reported18ndash26 either extra-or intracellularly Although precise mechanisms have notbeen fully elucidated it was proposed that the reductionoccurs via interactions with cellular molecules such asorganic sulfurs sugars or proteins However at this timethe possibility for an enzyme-mediated reduction reactionwas never suggested In the case of cyanobacteria thishypothesis had to be envisioned since these cells possestwo class of reducing enzymes ie nitrogenase and hydro-genase enzymes Cyanobacteria can fix elemental nitrogenfrom the atmosphere thanks to the action of nitrogenasewhich is mainly located inside the heterocyts (Calothrixand Anabaena) However this enzyme is known to be irre-versibly inhibited upon exposure to molecular oxygen44ndash45

which should be in disagreement with observations of N2-fixation in vegetative cells sites of production of molecularoxygen via photosynthesis Thus an explanation could bethat temporal separation protects nitrogenase from oxygenas for example the occurrence of N2-fixation during dark-ness phases (Leptolyngbya)3244ndash45 It was demonstratedthat the species Anabaena variabilis produces two differ-ent nitrogenases one which functions only in heterocytunder either anaerobic or aerobic conditions and the otherone that operates in both heterocyts and vegetative cellsbut only under anaerobic conditions It has been proposedthat the latter is regulated by environmental conditions andthe former is developmentally regulated46 Hydrogenase isable to reduce amongst others electron acceptors hydro-gen ions to molecular hydrogen so that it may also beinvolved in the observed reaction Indeed the presenceof ferredoxins on the thylakoids works as a link betweenhydrogenase and different electron donors and acceptorsin both photosynthetic and non-photosynthetic systemsso that it seems that both nitrogenase and hydrogenaseenzymes are susceptible to act as reducing agents to pro-mote the reduction of some metallic salts

12

N2 +3H++3eminusnitrogenaseminusminusminusminusminusrarr NH3E

0 =minus310 eV

2H++2eminushydrogenaseminusminusminusminusminusrarr H2E

0 = 0 eV

Au3++3eminus rarr Au0E0 = 1002 eV

Ag++ eminus rarr Ag0E0 = 080 eV

J Nanosci Nanotechnol 7 2696ndash2708 2007 2705

Delivered by Publishing Technology to Florida State University - TallahasseeIP 14620120822 On Fri 26 Sep 2014 103634

Copyright American Scientific Publishers

RESEARCHARTICLE

Cyanobacteria as Bioreactors for the Synthesis of Au Ag Pd and Pt Nanoparticles Brayner et al

Pd2++2eminus rarr Pd0E0 = 092 eV

PtCl2minus6 +2eminus rarr PtCl2minus

4 +2ClminusE0 = 073 eV

PtCl2minus4 +2eminus rarr Pt0 +4ClminusE0 = 073 eV

However in this study Leptolyngbya was selected becauseit does not posses heterocyt and fixes nitrogen only anaer-obically and in the dark In this case it was possible toobserve that the reduction reaction was far less efficientthan for the two other cyanobacteria and occurs only indarkness thus only in the presence of nitrogenase In con-trast the genus Anabaena presents several heterocyts thatproduce high concentration of nitrogenase In this case thereaction was very fast (about 5ndash10 minutes) for all concen-trations of Ag+ and Au3+ As a consequence these ionshad not completely penetrated inside the cells and mostpart of Au and Ag nanoparticles was formed near the cellwalls and were bigger than that formed inside Calothrixand Leptolyngbya (Figs 2(d e) 5(b) and 7(b)) Due to thefast reaction nucleation and growth steps were probablynot separated so that coalescence of Au and Ag nanopar-ticles may have occurred The genus Calothrix appears asan intermediate case in terms of nitrogenase content com-bining short reaction time and small un-aggregated col-loids All these data indicate that nitrogenase is specificallyinvolved in the reduction process

This assumption is strengthened by our in vitro test ofnitrogenase ability to reduce noble metal salts Au Ag Pdand to a lesser extent Pt nanoparticles could be obtainedthrough this approach with reaction times that follow atrend similar to what was observed for cyanobacteria Thissimilarity is particularly clear for platinum where a partialreduction leaving un-reacted Pt4+ and partially-reducedPt2+ ions in the solution was observed both in vivo andin vitro However it is interesting to note that the sizeand morphology of nanoparticles obtained in vitro stronglydiffer from those obtained within the cyanobacteria indi-cating the cellular environment of the enzyme play a sig-nificant role in particle growth control In this contextour studies with Calothrix Anabaena and Leptolyngbyacyanobacteria provide some clues about nitrogenase local-ization The enzyme may be present on the fibrous layersinside the heterocyt and also on the thylakoids inside thevegetative cells However since no metallic nanoparti-cles were observed in intrathylakoidal or intrafibrous layerspaces nitrogenase is only located on the cell membranesOn the basis of membrane-controlled biomineralizationprocesses5 the possibility for these membranes to play arole in nanoparticle growth control should not be put aside

42 Extra-Cellular Nanoparticle ReleaseThe Role of Released Poly-Saccharides

After their intra-cellular synthesis metallic nanoparticlesare released in the culture medium via inter-cellular canal-like intercytoplasmic connections (Fig 4(d)) TEM studies

indicate that the released particles are embedded in apoly-saccharide network Such production of extracel-lular polysaccharides by cyanobacteria is already well-documented they are negatively charged and rich in uronicacids exhibit metal-binding capacities47 They may thusbind toxic metals and be used in the treatment of toxicwaste waters48 In the natural environment these polysac-charides could act as natural chelators binding cationsand concentrating essential trace elements present at sub-marginal concentrations in oligotrophic environments49 Inthis work these poly-saccharides prevent the aggregationof released nanoparticles in most cases One exceptionis for Au nanoparticles formed by Anabaena whose sizeincreases when going from intra- to extra-cellular mediaprobably as a consequence of the fast kinetics of reductionmentioned above

More information on Au colloidal solutions obtainedafter Au nanoparticles release by the cyanobacteria canbe obtained form UV-Visible absorption spectra Theseabsorption bands correspond to the surface plasmon reso-nance of Au metallic nanoparticles According to absorp-tion calculation based on Mie theory34 spherical colloidalAu (red colored) would show max at 530 nm Au col-loidal solution released by the genus Anabaena presentsa pink-purple color the sample exhibiting a narrow bandat max = 537 nm close the calculated value On theother hand Au colloidal solutions released by the genusCalothrix and by the genus Leptolyngbya present both apurple-blue color and a broad bands at max = 577 nm andmax = 562 nm respectively This increase of the plasmonbandwidth may be attributed to the physical dimensionof the nanoparticles (sim6 nm)50 The same behavior wasobserved for Ag nanoparticles According to Mie theory34

spherical colloidal Ag (yellow colored) would show max at410 nm Ag colloidal solution released by genera Calothrixand Anabaena present both a red color and they showa broad plasmon resonance band that may be attributedto interparticle coupling (Fig 8(a)) The red-shift of theabsorption band observed in both cases may be attributedto the presence of adsorbed polysaccharides in the Au andAg surfaces (this behavior was also observed for adsorbedproteins)3351

Another important result obtained by UV-Visible anal-ysis was a qualitative release yield of Au nanoparticlesin the surrounding medium For the same Au3+ concen-tration (10minus4 M) absorption intensities vary from 001 to012 Au colloidal solutions released by Anabaena andCalothrix present a very close final Au metallic concen-tration (012 au) The worst result was obtained with thegenus Leptolyngbya (001 au) On the other hand for Agnanoparticles released by all cyanobacteria the absorptionintensities were not very different showing that all Ag+

salts were reduced to Ag0 probably due to the lower redoxpotential and hence the easier reduction of Ag+Ag0 com-pared to Au3+Au0

2706 J Nanosci Nanotechnol 7 2696ndash2708 2007

Delivered by Publishing Technology to Florida State University - TallahasseeIP 14620120822 On Fri 26 Sep 2014 103634

Copyright American Scientific Publishers

RESEARCHARTICLE

Brayner et al Cyanobacteria as Bioreactors for the Synthesis of Au Ag Pd and Pt Nanoparticles

5 CONCLUSION

Production strategies for metallic nanoparticles usingenvironmental-friendly routes using renewable naturalresources have great potential in the next future Ourwork demonstrates that common cyanobacteria strainshave the ability to synthesize Au Ag but also Pd andPt nanoparticles intra-cellularly This process appearsenzyme-mediated as the efficiency of the reduction processcould be related to the presence of nitrogenase We alsoshow that the size of the particle and the reaction yielddepend on the selected strain so that it should be possiblein a near future to identify other cyanobacteria genera withoptimal properties Finally first attempts to use cyanobac-teria as recyclable bioreactors demonstrate that the cellsrecover their full activity after a first metal salt additionand reduction and efforts are in progress through encap-sulation approaches to design continuous flow reactors

Moreover metallic nanoparticles produced insidecyanobacteria cells were released in the culture mediumsurrounded by stabilizing poly-saccharides and can be the-refore used as-synthesized in homogeneous catalysis52ndash53

and other applications such as chemical and biosens-ing54ndash55 biolabeling56ndash57 and photonics58ndash59 The nanopar-ticles may be immobilized in matrices or shaped as thinfilms for optoelectronic applications this being impossibleto achieve if the nanoparticles would have remained insidethe biomass The possibility to extend our approach tooxide andor sulfur nanoparticles elaboration is currentlyunder study

On a more general level this work is a new indicationthat the biological activities of living organisms optimizedthrough evolution to perform specific tasks can be divertedto perform other functions that may answer some of thecurrent challenges in synthetic chemistry

References and Notes

1 C M Zaremba A M Belcher M Fritz Y Li S Mann P KHansma D E Morse J S Speck and G D Stucky Chem Mater8 679 (1996)

2 X W Su and F Z Cui Mater Sci Eng C 7 19 (1999)3 J F Banfield S A Welch H Zhang T T Ebert and R L Penn

Science 289 751 (2000)4 (a) R Wetherbee S Crawford and P Mulvaney Biomineralization

1 89 (2000) (b) J C Weaver L I Pietrasanta N Hedin B FChmelka P K Hansma and D E Morse J Struct Biol 144 271(2003)

5 (a) H A Lowenstam and S Weiner Biomineralization Oxford Uni-versity Press Oxford (1989) (b) J Webb and R J P Williams(Eds) Biomineralization VCH Weinheim (1989) (c) E Baueuer-lein (Ed) Biomineralization Wiley-VCH Weinheim (2000)

6 T Douglas and V T Stark Inorg Chem 39 1828 (2000)7 R Wahl M Mertig J Raff S Selenska-Pobell and W Pompe Adv

Mater 13 736 (2001)8 J He T Kunitake and A Nakao Chem Mater 15 4401 (2003)9 R Brayner T Coradin F Fieacutevet-Vincent J Livage and F Fieacutevet

New J Chem 29 681 (2005)10 C A Mirkin Inorg Chem 38 2258 (2000)

11 (a) M G Warner and J E Hutchinson Nat Mater 2 272 (2003)(b) M Sarakaya C Tamerler A K Y K Schulten and F BabeyxNat Mater 2 577 (2003) (c) B D Reiss C Mao D J SolisK S Ryan T Thomson and A M Belcher Nano Lett 4 1127(2004)

12 B M Rabatic R C Claussen and S I Stupp Chem Mater 175877 (2005)

13 S S Shankar A Rai B Ankawar A Singh A Ahmad andM Sastry Nature 3 482 (2004)

14 C T Dameron R N Reese R K Mehra A R Kortan P J CarrollM L Steigerwald L E Brus and D R Winge Nature 338 596(1989)

15 T Klaus-Joerger J Joerger E Olsson and C G Granqvist TrendsBiotechnol 19 15 (2001)

16 L Q Wu and G F Payne Trends Biotechnol 22 593 (2004)17 T J Beveridge M N Hughes H Lee K T Leung R K Poole

I Savaidis S Silver and J T Trevors Adv Microb Physiol 38177 (1997)

18 G Southam and T J Beveridge Geochim Cosmochim Acta 584527 (1994)

19 G Southam and T J Beveridge Geochim Cosmochim Acta 604369 (1996)

20 K Kashefi J M Tor K P Nevin and D R Lovley Appl EnvironMicrobiol 67 3275 (2001)

21 P Mukherjee A Ahmad D Mandal S Senapati S R Saikar M IKhan R Ramani R Parischa P V Ajaykumar M Alam M Sastryand R Kumar Angew Chem Int Ed 40 3585 (2001)

22 B Nair and T Pradeep Cryst Growth Des 2 293 (2002)23 P Mukherjee S Senapati D Mandal A Ahmad M I Khan

R Kumar and M Sastry Chem Bio Chem 5 461 (2002)24 A Ahmad S Senapati M I Khan R Kumar and M Sastry

Langmuir 19 3550 (2003)25 S S Shankar A Ahmad R Pasricha and M Sastry J Mater

Chem 13 1822 (2003)26 K C Bhainsa and S F DrsquoSouza Colloids Surf B 47 160 (2006)27 H Kuepper and P M H Kroneck Metal Ions in Biological Systems

44 97 (2005)28 S Silver and L T Phung Ann Rev Microb 50 753 (1996)29 R S Prakasham and S V Ramakrishna J Sci Industr Res 57 258

(1998)30 M F Lengke M E Fleet and G Southam Langmuir 22 2780

(2006)31 B Bergman J Gallon A N Rai and L Stal FEMS Microbiol

Rev 19 139 (1997)32 C Bertocchi L Navarini A Cezagraverio and M Anastasio Carbohydr

Polym 12 127 (1990)33 R Brayner T Coradin M-J Vaulay C Mangeney J Livage and

F Fieacutevet Coll Surf A 256 191 (2005)34 J A Creighton and D G Eadon J Chem Soc Faraday Trans 87

3881 (1991)35 R M Slawson M I Van Dyke H Lee and J T Trevors Plasmid

27 72 (1992)36 G J Zhao and S E Stevens Biometals 11 27 (1998)37 I Sadi and B Salopek-Sondi J Coll Interf Sci 275 177 (2004)38 (a) R Brayner G Viau G M da Cruz F Fieacutevet-Vincent F Fieacutevet

and F Bozon-Verduraz Catal Today 57 187 (2000) (b) R BraynerG Viau and F Bozon-Verduraz J Mol Catal 182ndash183 227 (2002)

39 R Brayner D S Cunha and F Bozon-Verduraz Catal Today 78419 (2003)

40 L M Bronstein D M Chernyshov R Karlinsey J W ZwanzigerV G Matveeva E M Sulman G N Demidenko H P Hentze andM Antonietti Chem Mater 15 2623 (2003) (b) J R Gonzalez-Velasco J A Botas R Ferret M P Gonzales-Marcos J L Marcand M A Gutieacuterrez-Ortiz Catal Today 59 395 (2000)

41 D Briggs and M P Seah Practical Surface Analysis Auger andX-ray Photoelectron Spectroscopy Wiley (1990) Vol 1

J Nanosci Nanotechnol 7 2696ndash2708 2007 2707

Delivered by Publishing Technology to Florida State University - TallahasseeIP 14620120822 On Fri 26 Sep 2014 103634

Copyright American Scientific Publishers

RESEARCHARTICLE

Brayner et al Cyanobacteria as Bioreactors for the Synthesis of Au Ag Pd and Pt Nanoparticles

Fig 11 TEM micrographs obtained after in vitro enzymatic reductionof (a) Au3+ (b) Ag+ and (c) Pd2+

of Au Ag and Pd nanoparticles obtained by in vitro enzy-matic reduction Figure 11(a) presents TEM image of Aunanoparticles which contained 60 equilateral triangularnanoplates of about 400 nm in edge length and 40hexagonal plates of 70ndash150 nm in size (Table I) On theother hand for Ag and Pd spherical nanoparticles of about40 and 10 nm size respectively were synthesized by thismethod (Figs 11(b) and (c) Table I) In these last twocases morphological polysaccharide modifications were

observed Ag nanoparticles were surrounded by lamel-lae polysaccharide while Pd nanoparticles were alignedinside polysaccharide nanowires In all cases nanoparti-cles obtained by in vitro enzymatic reduction differ dras-tically than that obtained by in vivo reduction

4 DISCUSSION

41 Intra-Cellular Nanoparticle FormationThe Role of Nitrogenase

The formation of Au and Ag nanoparticles by living organ-isms has already been widely reported18ndash26 either extra-or intracellularly Although precise mechanisms have notbeen fully elucidated it was proposed that the reductionoccurs via interactions with cellular molecules such asorganic sulfurs sugars or proteins However at this timethe possibility for an enzyme-mediated reduction reactionwas never suggested In the case of cyanobacteria thishypothesis had to be envisioned since these cells possestwo class of reducing enzymes ie nitrogenase and hydro-genase enzymes Cyanobacteria can fix elemental nitrogenfrom the atmosphere thanks to the action of nitrogenasewhich is mainly located inside the heterocyts (Calothrixand Anabaena) However this enzyme is known to be irre-versibly inhibited upon exposure to molecular oxygen44ndash45

which should be in disagreement with observations of N2-fixation in vegetative cells sites of production of molecularoxygen via photosynthesis Thus an explanation could bethat temporal separation protects nitrogenase from oxygenas for example the occurrence of N2-fixation during dark-ness phases (Leptolyngbya)3244ndash45 It was demonstratedthat the species Anabaena variabilis produces two differ-ent nitrogenases one which functions only in heterocytunder either anaerobic or aerobic conditions and the otherone that operates in both heterocyts and vegetative cellsbut only under anaerobic conditions It has been proposedthat the latter is regulated by environmental conditions andthe former is developmentally regulated46 Hydrogenase isable to reduce amongst others electron acceptors hydro-gen ions to molecular hydrogen so that it may also beinvolved in the observed reaction Indeed the presenceof ferredoxins on the thylakoids works as a link betweenhydrogenase and different electron donors and acceptorsin both photosynthetic and non-photosynthetic systemsso that it seems that both nitrogenase and hydrogenaseenzymes are susceptible to act as reducing agents to pro-mote the reduction of some metallic salts

12

N2 +3H++3eminusnitrogenaseminusminusminusminusminusrarr NH3E

0 =minus310 eV

2H++2eminushydrogenaseminusminusminusminusminusrarr H2E

0 = 0 eV

Au3++3eminus rarr Au0E0 = 1002 eV

Ag++ eminus rarr Ag0E0 = 080 eV

J Nanosci Nanotechnol 7 2696ndash2708 2007 2705

Delivered by Publishing Technology to Florida State University - TallahasseeIP 14620120822 On Fri 26 Sep 2014 103634

Copyright American Scientific Publishers

RESEARCHARTICLE

Cyanobacteria as Bioreactors for the Synthesis of Au Ag Pd and Pt Nanoparticles Brayner et al

Pd2++2eminus rarr Pd0E0 = 092 eV

PtCl2minus6 +2eminus rarr PtCl2minus

4 +2ClminusE0 = 073 eV

PtCl2minus4 +2eminus rarr Pt0 +4ClminusE0 = 073 eV

However in this study Leptolyngbya was selected becauseit does not posses heterocyt and fixes nitrogen only anaer-obically and in the dark In this case it was possible toobserve that the reduction reaction was far less efficientthan for the two other cyanobacteria and occurs only indarkness thus only in the presence of nitrogenase In con-trast the genus Anabaena presents several heterocyts thatproduce high concentration of nitrogenase In this case thereaction was very fast (about 5ndash10 minutes) for all concen-trations of Ag+ and Au3+ As a consequence these ionshad not completely penetrated inside the cells and mostpart of Au and Ag nanoparticles was formed near the cellwalls and were bigger than that formed inside Calothrixand Leptolyngbya (Figs 2(d e) 5(b) and 7(b)) Due to thefast reaction nucleation and growth steps were probablynot separated so that coalescence of Au and Ag nanopar-ticles may have occurred The genus Calothrix appears asan intermediate case in terms of nitrogenase content com-bining short reaction time and small un-aggregated col-loids All these data indicate that nitrogenase is specificallyinvolved in the reduction process

This assumption is strengthened by our in vitro test ofnitrogenase ability to reduce noble metal salts Au Ag Pdand to a lesser extent Pt nanoparticles could be obtainedthrough this approach with reaction times that follow atrend similar to what was observed for cyanobacteria Thissimilarity is particularly clear for platinum where a partialreduction leaving un-reacted Pt4+ and partially-reducedPt2+ ions in the solution was observed both in vivo andin vitro However it is interesting to note that the sizeand morphology of nanoparticles obtained in vitro stronglydiffer from those obtained within the cyanobacteria indi-cating the cellular environment of the enzyme play a sig-nificant role in particle growth control In this contextour studies with Calothrix Anabaena and Leptolyngbyacyanobacteria provide some clues about nitrogenase local-ization The enzyme may be present on the fibrous layersinside the heterocyt and also on the thylakoids inside thevegetative cells However since no metallic nanoparti-cles were observed in intrathylakoidal or intrafibrous layerspaces nitrogenase is only located on the cell membranesOn the basis of membrane-controlled biomineralizationprocesses5 the possibility for these membranes to play arole in nanoparticle growth control should not be put aside

42 Extra-Cellular Nanoparticle ReleaseThe Role of Released Poly-Saccharides

After their intra-cellular synthesis metallic nanoparticlesare released in the culture medium via inter-cellular canal-like intercytoplasmic connections (Fig 4(d)) TEM studies

indicate that the released particles are embedded in apoly-saccharide network Such production of extracel-lular polysaccharides by cyanobacteria is already well-documented they are negatively charged and rich in uronicacids exhibit metal-binding capacities47 They may thusbind toxic metals and be used in the treatment of toxicwaste waters48 In the natural environment these polysac-charides could act as natural chelators binding cationsand concentrating essential trace elements present at sub-marginal concentrations in oligotrophic environments49 Inthis work these poly-saccharides prevent the aggregationof released nanoparticles in most cases One exceptionis for Au nanoparticles formed by Anabaena whose sizeincreases when going from intra- to extra-cellular mediaprobably as a consequence of the fast kinetics of reductionmentioned above

More information on Au colloidal solutions obtainedafter Au nanoparticles release by the cyanobacteria canbe obtained form UV-Visible absorption spectra Theseabsorption bands correspond to the surface plasmon reso-nance of Au metallic nanoparticles According to absorp-tion calculation based on Mie theory34 spherical colloidalAu (red colored) would show max at 530 nm Au col-loidal solution released by the genus Anabaena presentsa pink-purple color the sample exhibiting a narrow bandat max = 537 nm close the calculated value On theother hand Au colloidal solutions released by the genusCalothrix and by the genus Leptolyngbya present both apurple-blue color and a broad bands at max = 577 nm andmax = 562 nm respectively This increase of the plasmonbandwidth may be attributed to the physical dimensionof the nanoparticles (sim6 nm)50 The same behavior wasobserved for Ag nanoparticles According to Mie theory34

spherical colloidal Ag (yellow colored) would show max at410 nm Ag colloidal solution released by genera Calothrixand Anabaena present both a red color and they showa broad plasmon resonance band that may be attributedto interparticle coupling (Fig 8(a)) The red-shift of theabsorption band observed in both cases may be attributedto the presence of adsorbed polysaccharides in the Au andAg surfaces (this behavior was also observed for adsorbedproteins)3351

Another important result obtained by UV-Visible anal-ysis was a qualitative release yield of Au nanoparticlesin the surrounding medium For the same Au3+ concen-tration (10minus4 M) absorption intensities vary from 001 to012 Au colloidal solutions released by Anabaena andCalothrix present a very close final Au metallic concen-tration (012 au) The worst result was obtained with thegenus Leptolyngbya (001 au) On the other hand for Agnanoparticles released by all cyanobacteria the absorptionintensities were not very different showing that all Ag+

salts were reduced to Ag0 probably due to the lower redoxpotential and hence the easier reduction of Ag+Ag0 com-pared to Au3+Au0

2706 J Nanosci Nanotechnol 7 2696ndash2708 2007

Delivered by Publishing Technology to Florida State University - TallahasseeIP 14620120822 On Fri 26 Sep 2014 103634

Copyright American Scientific Publishers

RESEARCHARTICLE

Brayner et al Cyanobacteria as Bioreactors for the Synthesis of Au Ag Pd and Pt Nanoparticles

5 CONCLUSION

Production strategies for metallic nanoparticles usingenvironmental-friendly routes using renewable naturalresources have great potential in the next future Ourwork demonstrates that common cyanobacteria strainshave the ability to synthesize Au Ag but also Pd andPt nanoparticles intra-cellularly This process appearsenzyme-mediated as the efficiency of the reduction processcould be related to the presence of nitrogenase We alsoshow that the size of the particle and the reaction yielddepend on the selected strain so that it should be possiblein a near future to identify other cyanobacteria genera withoptimal properties Finally first attempts to use cyanobac-teria as recyclable bioreactors demonstrate that the cellsrecover their full activity after a first metal salt additionand reduction and efforts are in progress through encap-sulation approaches to design continuous flow reactors

Moreover metallic nanoparticles produced insidecyanobacteria cells were released in the culture mediumsurrounded by stabilizing poly-saccharides and can be the-refore used as-synthesized in homogeneous catalysis52ndash53

and other applications such as chemical and biosens-ing54ndash55 biolabeling56ndash57 and photonics58ndash59 The nanopar-ticles may be immobilized in matrices or shaped as thinfilms for optoelectronic applications this being impossibleto achieve if the nanoparticles would have remained insidethe biomass The possibility to extend our approach tooxide andor sulfur nanoparticles elaboration is currentlyunder study

On a more general level this work is a new indicationthat the biological activities of living organisms optimizedthrough evolution to perform specific tasks can be divertedto perform other functions that may answer some of thecurrent challenges in synthetic chemistry

References and Notes

1 C M Zaremba A M Belcher M Fritz Y Li S Mann P KHansma D E Morse J S Speck and G D Stucky Chem Mater8 679 (1996)

2 X W Su and F Z Cui Mater Sci Eng C 7 19 (1999)3 J F Banfield S A Welch H Zhang T T Ebert and R L Penn

Science 289 751 (2000)4 (a) R Wetherbee S Crawford and P Mulvaney Biomineralization

1 89 (2000) (b) J C Weaver L I Pietrasanta N Hedin B FChmelka P K Hansma and D E Morse J Struct Biol 144 271(2003)

5 (a) H A Lowenstam and S Weiner Biomineralization Oxford Uni-versity Press Oxford (1989) (b) J Webb and R J P Williams(Eds) Biomineralization VCH Weinheim (1989) (c) E Baueuer-lein (Ed) Biomineralization Wiley-VCH Weinheim (2000)

6 T Douglas and V T Stark Inorg Chem 39 1828 (2000)7 R Wahl M Mertig J Raff S Selenska-Pobell and W Pompe Adv

Mater 13 736 (2001)8 J He T Kunitake and A Nakao Chem Mater 15 4401 (2003)9 R Brayner T Coradin F Fieacutevet-Vincent J Livage and F Fieacutevet

New J Chem 29 681 (2005)10 C A Mirkin Inorg Chem 38 2258 (2000)

11 (a) M G Warner and J E Hutchinson Nat Mater 2 272 (2003)(b) M Sarakaya C Tamerler A K Y K Schulten and F BabeyxNat Mater 2 577 (2003) (c) B D Reiss C Mao D J SolisK S Ryan T Thomson and A M Belcher Nano Lett 4 1127(2004)

12 B M Rabatic R C Claussen and S I Stupp Chem Mater 175877 (2005)

13 S S Shankar A Rai B Ankawar A Singh A Ahmad andM Sastry Nature 3 482 (2004)

14 C T Dameron R N Reese R K Mehra A R Kortan P J CarrollM L Steigerwald L E Brus and D R Winge Nature 338 596(1989)

15 T Klaus-Joerger J Joerger E Olsson and C G Granqvist TrendsBiotechnol 19 15 (2001)

16 L Q Wu and G F Payne Trends Biotechnol 22 593 (2004)17 T J Beveridge M N Hughes H Lee K T Leung R K Poole

I Savaidis S Silver and J T Trevors Adv Microb Physiol 38177 (1997)

18 G Southam and T J Beveridge Geochim Cosmochim Acta 584527 (1994)

19 G Southam and T J Beveridge Geochim Cosmochim Acta 604369 (1996)

20 K Kashefi J M Tor K P Nevin and D R Lovley Appl EnvironMicrobiol 67 3275 (2001)

21 P Mukherjee A Ahmad D Mandal S Senapati S R Saikar M IKhan R Ramani R Parischa P V Ajaykumar M Alam M Sastryand R Kumar Angew Chem Int Ed 40 3585 (2001)

22 B Nair and T Pradeep Cryst Growth Des 2 293 (2002)23 P Mukherjee S Senapati D Mandal A Ahmad M I Khan

R Kumar and M Sastry Chem Bio Chem 5 461 (2002)24 A Ahmad S Senapati M I Khan R Kumar and M Sastry

Langmuir 19 3550 (2003)25 S S Shankar A Ahmad R Pasricha and M Sastry J Mater

Chem 13 1822 (2003)26 K C Bhainsa and S F DrsquoSouza Colloids Surf B 47 160 (2006)27 H Kuepper and P M H Kroneck Metal Ions in Biological Systems

44 97 (2005)28 S Silver and L T Phung Ann Rev Microb 50 753 (1996)29 R S Prakasham and S V Ramakrishna J Sci Industr Res 57 258

(1998)30 M F Lengke M E Fleet and G Southam Langmuir 22 2780

(2006)31 B Bergman J Gallon A N Rai and L Stal FEMS Microbiol

Rev 19 139 (1997)32 C Bertocchi L Navarini A Cezagraverio and M Anastasio Carbohydr

Polym 12 127 (1990)33 R Brayner T Coradin M-J Vaulay C Mangeney J Livage and

F Fieacutevet Coll Surf A 256 191 (2005)34 J A Creighton and D G Eadon J Chem Soc Faraday Trans 87

3881 (1991)35 R M Slawson M I Van Dyke H Lee and J T Trevors Plasmid

27 72 (1992)36 G J Zhao and S E Stevens Biometals 11 27 (1998)37 I Sadi and B Salopek-Sondi J Coll Interf Sci 275 177 (2004)38 (a) R Brayner G Viau G M da Cruz F Fieacutevet-Vincent F Fieacutevet

and F Bozon-Verduraz Catal Today 57 187 (2000) (b) R BraynerG Viau and F Bozon-Verduraz J Mol Catal 182ndash183 227 (2002)

39 R Brayner D S Cunha and F Bozon-Verduraz Catal Today 78419 (2003)

40 L M Bronstein D M Chernyshov R Karlinsey J W ZwanzigerV G Matveeva E M Sulman G N Demidenko H P Hentze andM Antonietti Chem Mater 15 2623 (2003) (b) J R Gonzalez-Velasco J A Botas R Ferret M P Gonzales-Marcos J L Marcand M A Gutieacuterrez-Ortiz Catal Today 59 395 (2000)

41 D Briggs and M P Seah Practical Surface Analysis Auger andX-ray Photoelectron Spectroscopy Wiley (1990) Vol 1

J Nanosci Nanotechnol 7 2696ndash2708 2007 2707

Delivered by Publishing Technology to Florida State University - TallahasseeIP 14620120822 On Fri 26 Sep 2014 103634

Copyright American Scientific Publishers

RESEARCHARTICLE

Cyanobacteria as Bioreactors for the Synthesis of Au Ag Pd and Pt Nanoparticles Brayner et al

Pd2++2eminus rarr Pd0E0 = 092 eV

PtCl2minus6 +2eminus rarr PtCl2minus

4 +2ClminusE0 = 073 eV

PtCl2minus4 +2eminus rarr Pt0 +4ClminusE0 = 073 eV

However in this study Leptolyngbya was selected becauseit does not posses heterocyt and fixes nitrogen only anaer-obically and in the dark In this case it was possible toobserve that the reduction reaction was far less efficientthan for the two other cyanobacteria and occurs only indarkness thus only in the presence of nitrogenase In con-trast the genus Anabaena presents several heterocyts thatproduce high concentration of nitrogenase In this case thereaction was very fast (about 5ndash10 minutes) for all concen-trations of Ag+ and Au3+ As a consequence these ionshad not completely penetrated inside the cells and mostpart of Au and Ag nanoparticles was formed near the cellwalls and were bigger than that formed inside Calothrixand Leptolyngbya (Figs 2(d e) 5(b) and 7(b)) Due to thefast reaction nucleation and growth steps were probablynot separated so that coalescence of Au and Ag nanopar-ticles may have occurred The genus Calothrix appears asan intermediate case in terms of nitrogenase content com-bining short reaction time and small un-aggregated col-loids All these data indicate that nitrogenase is specificallyinvolved in the reduction process

This assumption is strengthened by our in vitro test ofnitrogenase ability to reduce noble metal salts Au Ag Pdand to a lesser extent Pt nanoparticles could be obtainedthrough this approach with reaction times that follow atrend similar to what was observed for cyanobacteria Thissimilarity is particularly clear for platinum where a partialreduction leaving un-reacted Pt4+ and partially-reducedPt2+ ions in the solution was observed both in vivo andin vitro However it is interesting to note that the sizeand morphology of nanoparticles obtained in vitro stronglydiffer from those obtained within the cyanobacteria indi-cating the cellular environment of the enzyme play a sig-nificant role in particle growth control In this contextour studies with Calothrix Anabaena and Leptolyngbyacyanobacteria provide some clues about nitrogenase local-ization The enzyme may be present on the fibrous layersinside the heterocyt and also on the thylakoids inside thevegetative cells However since no metallic nanoparti-cles were observed in intrathylakoidal or intrafibrous layerspaces nitrogenase is only located on the cell membranesOn the basis of membrane-controlled biomineralizationprocesses5 the possibility for these membranes to play arole in nanoparticle growth control should not be put aside

42 Extra-Cellular Nanoparticle ReleaseThe Role of Released Poly-Saccharides

After their intra-cellular synthesis metallic nanoparticlesare released in the culture medium via inter-cellular canal-like intercytoplasmic connections (Fig 4(d)) TEM studies

indicate that the released particles are embedded in apoly-saccharide network Such production of extracel-lular polysaccharides by cyanobacteria is already well-documented they are negatively charged and rich in uronicacids exhibit metal-binding capacities47 They may thusbind toxic metals and be used in the treatment of toxicwaste waters48 In the natural environment these polysac-charides could act as natural chelators binding cationsand concentrating essential trace elements present at sub-marginal concentrations in oligotrophic environments49 Inthis work these poly-saccharides prevent the aggregationof released nanoparticles in most cases One exceptionis for Au nanoparticles formed by Anabaena whose sizeincreases when going from intra- to extra-cellular mediaprobably as a consequence of the fast kinetics of reductionmentioned above

More information on Au colloidal solutions obtainedafter Au nanoparticles release by the cyanobacteria canbe obtained form UV-Visible absorption spectra Theseabsorption bands correspond to the surface plasmon reso-nance of Au metallic nanoparticles According to absorp-tion calculation based on Mie theory34 spherical colloidalAu (red colored) would show max at 530 nm Au col-loidal solution released by the genus Anabaena presentsa pink-purple color the sample exhibiting a narrow bandat max = 537 nm close the calculated value On theother hand Au colloidal solutions released by the genusCalothrix and by the genus Leptolyngbya present both apurple-blue color and a broad bands at max = 577 nm andmax = 562 nm respectively This increase of the plasmonbandwidth may be attributed to the physical dimensionof the nanoparticles (sim6 nm)50 The same behavior wasobserved for Ag nanoparticles According to Mie theory34

spherical colloidal Ag (yellow colored) would show max at410 nm Ag colloidal solution released by genera Calothrixand Anabaena present both a red color and they showa broad plasmon resonance band that may be attributedto interparticle coupling (Fig 8(a)) The red-shift of theabsorption band observed in both cases may be attributedto the presence of adsorbed polysaccharides in the Au andAg surfaces (this behavior was also observed for adsorbedproteins)3351

Another important result obtained by UV-Visible anal-ysis was a qualitative release yield of Au nanoparticlesin the surrounding medium For the same Au3+ concen-tration (10minus4 M) absorption intensities vary from 001 to012 Au colloidal solutions released by Anabaena andCalothrix present a very close final Au metallic concen-tration (012 au) The worst result was obtained with thegenus Leptolyngbya (001 au) On the other hand for Agnanoparticles released by all cyanobacteria the absorptionintensities were not very different showing that all Ag+

salts were reduced to Ag0 probably due to the lower redoxpotential and hence the easier reduction of Ag+Ag0 com-pared to Au3+Au0

2706 J Nanosci Nanotechnol 7 2696ndash2708 2007

Delivered by Publishing Technology to Florida State University - TallahasseeIP 14620120822 On Fri 26 Sep 2014 103634

Copyright American Scientific Publishers

RESEARCHARTICLE

Brayner et al Cyanobacteria as Bioreactors for the Synthesis of Au Ag Pd and Pt Nanoparticles

5 CONCLUSION

Production strategies for metallic nanoparticles usingenvironmental-friendly routes using renewable naturalresources have great potential in the next future Ourwork demonstrates that common cyanobacteria strainshave the ability to synthesize Au Ag but also Pd andPt nanoparticles intra-cellularly This process appearsenzyme-mediated as the efficiency of the reduction processcould be related to the presence of nitrogenase We alsoshow that the size of the particle and the reaction yielddepend on the selected strain so that it should be possiblein a near future to identify other cyanobacteria genera withoptimal properties Finally first attempts to use cyanobac-teria as recyclable bioreactors demonstrate that the cellsrecover their full activity after a first metal salt additionand reduction and efforts are in progress through encap-sulation approaches to design continuous flow reactors

Moreover metallic nanoparticles produced insidecyanobacteria cells were released in the culture mediumsurrounded by stabilizing poly-saccharides and can be the-refore used as-synthesized in homogeneous catalysis52ndash53

and other applications such as chemical and biosens-ing54ndash55 biolabeling56ndash57 and photonics58ndash59 The nanopar-ticles may be immobilized in matrices or shaped as thinfilms for optoelectronic applications this being impossibleto achieve if the nanoparticles would have remained insidethe biomass The possibility to extend our approach tooxide andor sulfur nanoparticles elaboration is currentlyunder study

On a more general level this work is a new indicationthat the biological activities of living organisms optimizedthrough evolution to perform specific tasks can be divertedto perform other functions that may answer some of thecurrent challenges in synthetic chemistry

References and Notes

1 C M Zaremba A M Belcher M Fritz Y Li S Mann P KHansma D E Morse J S Speck and G D Stucky Chem Mater8 679 (1996)

2 X W Su and F Z Cui Mater Sci Eng C 7 19 (1999)3 J F Banfield S A Welch H Zhang T T Ebert and R L Penn

Science 289 751 (2000)4 (a) R Wetherbee S Crawford and P Mulvaney Biomineralization

1 89 (2000) (b) J C Weaver L I Pietrasanta N Hedin B FChmelka P K Hansma and D E Morse J Struct Biol 144 271(2003)

5 (a) H A Lowenstam and S Weiner Biomineralization Oxford Uni-versity Press Oxford (1989) (b) J Webb and R J P Williams(Eds) Biomineralization VCH Weinheim (1989) (c) E Baueuer-lein (Ed) Biomineralization Wiley-VCH Weinheim (2000)

6 T Douglas and V T Stark Inorg Chem 39 1828 (2000)7 R Wahl M Mertig J Raff S Selenska-Pobell and W Pompe Adv

Mater 13 736 (2001)8 J He T Kunitake and A Nakao Chem Mater 15 4401 (2003)9 R Brayner T Coradin F Fieacutevet-Vincent J Livage and F Fieacutevet

New J Chem 29 681 (2005)10 C A Mirkin Inorg Chem 38 2258 (2000)

11 (a) M G Warner and J E Hutchinson Nat Mater 2 272 (2003)(b) M Sarakaya C Tamerler A K Y K Schulten and F BabeyxNat Mater 2 577 (2003) (c) B D Reiss C Mao D J SolisK S Ryan T Thomson and A M Belcher Nano Lett 4 1127(2004)

12 B M Rabatic R C Claussen and S I Stupp Chem Mater 175877 (2005)

13 S S Shankar A Rai B Ankawar A Singh A Ahmad andM Sastry Nature 3 482 (2004)

14 C T Dameron R N Reese R K Mehra A R Kortan P J CarrollM L Steigerwald L E Brus and D R Winge Nature 338 596(1989)

15 T Klaus-Joerger J Joerger E Olsson and C G Granqvist TrendsBiotechnol 19 15 (2001)

16 L Q Wu and G F Payne Trends Biotechnol 22 593 (2004)17 T J Beveridge M N Hughes H Lee K T Leung R K Poole

I Savaidis S Silver and J T Trevors Adv Microb Physiol 38177 (1997)

18 G Southam and T J Beveridge Geochim Cosmochim Acta 584527 (1994)

19 G Southam and T J Beveridge Geochim Cosmochim Acta 604369 (1996)

20 K Kashefi J M Tor K P Nevin and D R Lovley Appl EnvironMicrobiol 67 3275 (2001)

21 P Mukherjee A Ahmad D Mandal S Senapati S R Saikar M IKhan R Ramani R Parischa P V Ajaykumar M Alam M Sastryand R Kumar Angew Chem Int Ed 40 3585 (2001)

22 B Nair and T Pradeep Cryst Growth Des 2 293 (2002)23 P Mukherjee S Senapati D Mandal A Ahmad M I Khan

R Kumar and M Sastry Chem Bio Chem 5 461 (2002)24 A Ahmad S Senapati M I Khan R Kumar and M Sastry

Langmuir 19 3550 (2003)25 S S Shankar A Ahmad R Pasricha and M Sastry J Mater

Chem 13 1822 (2003)26 K C Bhainsa and S F DrsquoSouza Colloids Surf B 47 160 (2006)27 H Kuepper and P M H Kroneck Metal Ions in Biological Systems

44 97 (2005)28 S Silver and L T Phung Ann Rev Microb 50 753 (1996)29 R S Prakasham and S V Ramakrishna J Sci Industr Res 57 258

(1998)30 M F Lengke M E Fleet and G Southam Langmuir 22 2780

(2006)31 B Bergman J Gallon A N Rai and L Stal FEMS Microbiol

Rev 19 139 (1997)32 C Bertocchi L Navarini A Cezagraverio and M Anastasio Carbohydr

Polym 12 127 (1990)33 R Brayner T Coradin M-J Vaulay C Mangeney J Livage and

F Fieacutevet Coll Surf A 256 191 (2005)34 J A Creighton and D G Eadon J Chem Soc Faraday Trans 87

3881 (1991)35 R M Slawson M I Van Dyke H Lee and J T Trevors Plasmid

27 72 (1992)36 G J Zhao and S E Stevens Biometals 11 27 (1998)37 I Sadi and B Salopek-Sondi J Coll Interf Sci 275 177 (2004)38 (a) R Brayner G Viau G M da Cruz F Fieacutevet-Vincent F Fieacutevet

and F Bozon-Verduraz Catal Today 57 187 (2000) (b) R BraynerG Viau and F Bozon-Verduraz J Mol Catal 182ndash183 227 (2002)

39 R Brayner D S Cunha and F Bozon-Verduraz Catal Today 78419 (2003)

40 L M Bronstein D M Chernyshov R Karlinsey J W ZwanzigerV G Matveeva E M Sulman G N Demidenko H P Hentze andM Antonietti Chem Mater 15 2623 (2003) (b) J R Gonzalez-Velasco J A Botas R Ferret M P Gonzales-Marcos J L Marcand M A Gutieacuterrez-Ortiz Catal Today 59 395 (2000)

41 D Briggs and M P Seah Practical Surface Analysis Auger andX-ray Photoelectron Spectroscopy Wiley (1990) Vol 1

J Nanosci Nanotechnol 7 2696ndash2708 2007 2707

Delivered by Publishing Technology to Florida State University - TallahasseeIP 14620120822 On Fri 26 Sep 2014 103634

Copyright American Scientific Publishers

RESEARCHARTICLE

Brayner et al Cyanobacteria as Bioreactors for the Synthesis of Au Ag Pd and Pt Nanoparticles

5 CONCLUSION

Production strategies for metallic nanoparticles usingenvironmental-friendly routes using renewable naturalresources have great potential in the next future Ourwork demonstrates that common cyanobacteria strainshave the ability to synthesize Au Ag but also Pd andPt nanoparticles intra-cellularly This process appearsenzyme-mediated as the efficiency of the reduction processcould be related to the presence of nitrogenase We alsoshow that the size of the particle and the reaction yielddepend on the selected strain so that it should be possiblein a near future to identify other cyanobacteria genera withoptimal properties Finally first attempts to use cyanobac-teria as recyclable bioreactors demonstrate that the cellsrecover their full activity after a first metal salt additionand reduction and efforts are in progress through encap-sulation approaches to design continuous flow reactors

Moreover metallic nanoparticles produced insidecyanobacteria cells were released in the culture mediumsurrounded by stabilizing poly-saccharides and can be the-refore used as-synthesized in homogeneous catalysis52ndash53

and other applications such as chemical and biosens-ing54ndash55 biolabeling56ndash57 and photonics58ndash59 The nanopar-ticles may be immobilized in matrices or shaped as thinfilms for optoelectronic applications this being impossibleto achieve if the nanoparticles would have remained insidethe biomass The possibility to extend our approach tooxide andor sulfur nanoparticles elaboration is currentlyunder study

On a more general level this work is a new indicationthat the biological activities of living organisms optimizedthrough evolution to perform specific tasks can be divertedto perform other functions that may answer some of thecurrent challenges in synthetic chemistry

References and Notes

1 C M Zaremba A M Belcher M Fritz Y Li S Mann P KHansma D E Morse J S Speck and G D Stucky Chem Mater8 679 (1996)

2 X W Su and F Z Cui Mater Sci Eng C 7 19 (1999)3 J F Banfield S A Welch H Zhang T T Ebert and R L Penn

Science 289 751 (2000)4 (a) R Wetherbee S Crawford and P Mulvaney Biomineralization

1 89 (2000) (b) J C Weaver L I Pietrasanta N Hedin B FChmelka P K Hansma and D E Morse J Struct Biol 144 271(2003)

5 (a) H A Lowenstam and S Weiner Biomineralization Oxford Uni-versity Press Oxford (1989) (b) J Webb and R J P Williams(Eds) Biomineralization VCH Weinheim (1989) (c) E Baueuer-lein (Ed) Biomineralization Wiley-VCH Weinheim (2000)

6 T Douglas and V T Stark Inorg Chem 39 1828 (2000)7 R Wahl M Mertig J Raff S Selenska-Pobell and W Pompe Adv

Mater 13 736 (2001)8 J He T Kunitake and A Nakao Chem Mater 15 4401 (2003)9 R Brayner T Coradin F Fieacutevet-Vincent J Livage and F Fieacutevet

New J Chem 29 681 (2005)10 C A Mirkin Inorg Chem 38 2258 (2000)

11 (a) M G Warner and J E Hutchinson Nat Mater 2 272 (2003)(b) M Sarakaya C Tamerler A K Y K Schulten and F BabeyxNat Mater 2 577 (2003) (c) B D Reiss C Mao D J SolisK S Ryan T Thomson and A M Belcher Nano Lett 4 1127(2004)

12 B M Rabatic R C Claussen and S I Stupp Chem Mater 175877 (2005)

13 S S Shankar A Rai B Ankawar A Singh A Ahmad andM Sastry Nature 3 482 (2004)

14 C T Dameron R N Reese R K Mehra A R Kortan P J CarrollM L Steigerwald L E Brus and D R Winge Nature 338 596(1989)

15 T Klaus-Joerger J Joerger E Olsson and C G Granqvist TrendsBiotechnol 19 15 (2001)

16 L Q Wu and G F Payne Trends Biotechnol 22 593 (2004)17 T J Beveridge M N Hughes H Lee K T Leung R K Poole

I Savaidis S Silver and J T Trevors Adv Microb Physiol 38177 (1997)

18 G Southam and T J Beveridge Geochim Cosmochim Acta 584527 (1994)

19 G Southam and T J Beveridge Geochim Cosmochim Acta 604369 (1996)

20 K Kashefi J M Tor K P Nevin and D R Lovley Appl EnvironMicrobiol 67 3275 (2001)

21 P Mukherjee A Ahmad D Mandal S Senapati S R Saikar M IKhan R Ramani R Parischa P V Ajaykumar M Alam M Sastryand R Kumar Angew Chem Int Ed 40 3585 (2001)

22 B Nair and T Pradeep Cryst Growth Des 2 293 (2002)23 P Mukherjee S Senapati D Mandal A Ahmad M I Khan

R Kumar and M Sastry Chem Bio Chem 5 461 (2002)24 A Ahmad S Senapati M I Khan R Kumar and M Sastry

Langmuir 19 3550 (2003)25 S S Shankar A Ahmad R Pasricha and M Sastry J Mater

Chem 13 1822 (2003)26 K C Bhainsa and S F DrsquoSouza Colloids Surf B 47 160 (2006)27 H Kuepper and P M H Kroneck Metal Ions in Biological Systems

44 97 (2005)28 S Silver and L T Phung Ann Rev Microb 50 753 (1996)29 R S Prakasham and S V Ramakrishna J Sci Industr Res 57 258

(1998)30 M F Lengke M E Fleet and G Southam Langmuir 22 2780

(2006)31 B Bergman J Gallon A N Rai and L Stal FEMS Microbiol

Rev 19 139 (1997)32 C Bertocchi L Navarini A Cezagraverio and M Anastasio Carbohydr

Polym 12 127 (1990)33 R Brayner T Coradin M-J Vaulay C Mangeney J Livage and

F Fieacutevet Coll Surf A 256 191 (2005)34 J A Creighton and D G Eadon J Chem Soc Faraday Trans 87

3881 (1991)35 R M Slawson M I Van Dyke H Lee and J T Trevors Plasmid

27 72 (1992)36 G J Zhao and S E Stevens Biometals 11 27 (1998)37 I Sadi and B Salopek-Sondi J Coll Interf Sci 275 177 (2004)38 (a) R Brayner G Viau G M da Cruz F Fieacutevet-Vincent F Fieacutevet

and F Bozon-Verduraz Catal Today 57 187 (2000) (b) R BraynerG Viau and F Bozon-Verduraz J Mol Catal 182ndash183 227 (2002)

39 R Brayner D S Cunha and F Bozon-Verduraz Catal Today 78419 (2003)

40 L M Bronstein D M Chernyshov R Karlinsey J W ZwanzigerV G Matveeva E M Sulman G N Demidenko H P Hentze andM Antonietti Chem Mater 15 2623 (2003) (b) J R Gonzalez-Velasco J A Botas R Ferret M P Gonzales-Marcos J L Marcand M A Gutieacuterrez-Ortiz Catal Today 59 395 (2000)

41 D Briggs and M P Seah Practical Surface Analysis Auger andX-ray Photoelectron Spectroscopy Wiley (1990) Vol 1

J Nanosci Nanotechnol 7 2696ndash2708 2007 2707

Delivered by Publishing Technology to Florida State University - TallahasseeIP 14620120822 On Fri 26 Sep 2014 103634

Copyright American Scientific Publishers

RESEARCHARTICLE

Cyanobacteria as Bioreactors for the Synthesis of Au Ag Pd and Pt Nanoparticles Brayner et al

42 G B Hoflund H A E Hagelin J F Weaver and G N SalaitaAppl Surf Sci 205 102 (2003)

43 J M Ramallo-Lopez G F Santori L Giovanetti M L CasellaO A Ferretti and F G Requejo J Phys Chem B 107 11441(2003)

44 I Berman-Frank P Lundgren Y B Chen H Kuumlpper Z KolberB Bergman and P Falkowski Science 294 1534 (2001)

45 I Berman-Frank P Lundgren and P Falkowski Res Microbiol154 157 (2003)

46 T Thiel E M Lyons J C Erker and A Ernst Proc Natl AcadSci USA 92 9358 (1995)

47 D Kaplan D Christiaen and S M Arad Algal BiotechnologyElsevier Applied Science (1987)

48 W J Oswald Algae and Human Affairs Cambridge UniversityPress Cambridge (1998)

49 W Lange and J Canad Microbiol 22 1181 (1976)50 D Pan E Towe and S Kennerly Appl Phys Lett 73 1937 (1998)

51 J J Storhoff A A Lazarides R C Mucic C A Mirkin R LLetsinger and G C Schatz J Am Chem Soc 122 4640 (2000)

52 M Date Y Ichihashi T Yamashija A Chiorino F Boccuzzi andM Haruta Catal Today 72 89 (2002)

53 C J Zhong and M Maye Adv Mater 13 1507 (2001)54 L P J Marques I Cavaco J P Pinheiro V Ribeiro and G N M

Ferreira Clin Chem Lab Med 41 475 (2003)55 M Zayats A B Kharitonov S P Pogorelova O Lioubashevski

E Katz and I Willner J Am Chem Soc 125 16006 (2003)56 W Baschong and N G Wringley J Elec Microsc Tech 14 313

(1990)57 W Jahn J Struc Biol 127 106 (1999)58 S A Maier M L Brongersma P G Kik S Meltzer A A G

Requicha and H A Atwater Adv Mater 13 1501 (2001)59 J P Novak L C Brousseau F W Vance R C Johnson B I

Lemon J T Hupp and D L Feldheim J Am Chem Soc 12212029 (2000)

Received 21 July 2006 RevisedAccepted 11 November 2006

2708 J Nanosci Nanotechnol 7 2696ndash2708 2007