E Vargas

J C Denardin

R Lavín

P Mascaró

Corinne Chanéac

Thibaud Coradin

C Chaneac

Magnetization analysis of oriented chains of hexagonal cobalt nanoplates

HAL is

I. INTRODUCTION

In recent years, controllable synthesis of low-dimensional metallic materials with special geometries has attracted interest due to their diverse potential applications in biomedicine and as novel material for engineering and electronic devices [1]. Nano and submicronparticles with high aspect ratio, particularly of Fe, Co and Ni elements, have been studied because controlling the shape and morphology of particles is an effective method for tuning the magnetic properties [2]. In this line, several studies have focused on the simple and large-scale synthesis of regular and periodic magnetic structures [3]. Recently there were various reports in the literature that describe the dispersion of ferromagnetic particles in polymeric matrices by in-situ polymerization, melt compounding and solution blending [4][5][6]. This latter method appears to be an attractive route because an external magnetic field provides the possibility of controlling the organization of the particles in the matrix.

In this work, large scale and high quality single-crystalline hexagonal cobalt nanoplates have been obtained by a simple chemical reduction method at room conditions without complex apparatus [7]. These particles were incorporated in a polyethylene matrix by a solution blending method. A complex structure formed by chains of spherical agglomerates of Co platelets forms spontaneously but the direction of these chains can be controlled by solidification of the solution of nanoparticles in the presence of a magnetic field. The easy axis of the Co stacks is oriented along the field, in the direction of the chains length. The collective magnetic properties of this material have been studied by hysteresis loops and using first-order reversal curves (FORC) analysis.

II. EXPERIMENTAL METHODS

All reagents were analytical grade and used without further purification. Cobalt nanoplates were synthesized by our previous reported procedure [7]. In brief, 50 uL of cobalt chloride (0.4M) and 15 mL of cetyltrimethylammonium bromide (4 mM) were mixed in typical glass vial on ultrasonic bath. Then, 100 uL of hydrazine was dropped in the reaction mixture and the pH was adjusted to 10 using ammonia solutions. The general redox reaction could be expressed as:

2Co 2+ + N 2 H 4 + 4OH -→ 2Co + N 2 + 4H 2 O (1)
The final product was purified by magnetic decantation, washed several times and dried.

The morphological and structural characterization of the samples was carried out by scanning electron microscopy (SEM), in a Zeiss EVO MA10 and transmission electron microscopy (TEM), in a 120 kV Tecnai Spirit G2. The hysteresis loops was measured at room temperature on a vibrating sample magnetometer (VSM). The FORC curves were measured in a homemade Alternating Gradient Force Magnetometer (AGFM).

The nancomposite film was produced by solution blending method. First, the Co nanoplates were dispersed into 1 mL of toluene. After that, 100 uL of black colloidal system was incorporated into the dilution of polyethylene-toluene (10% wt). Finally, the mixture was supported on a square glass and vacuum dried under an external magnetic field of 2500 Oe.

Micromagnetic simulations were performed using the 3D OOMMF package [START_REF] Donahue | OOMMF User's Guide[END_REF]. Under this frame, the ferromagnetic system is divided into cubic cells with a uniform magnetization inside each cell. For our simulations we use the typical Cobalt parameters: saturation magnetization M s = 1400x10 3 A/m, exchange stiffness constant A = 30 J/m, and a mesh size of 3 nm × 3 nm × 5 nm, where spins are free to rotate in three dimensions. In all the cases the damping constant is 0.5. It was observed that the nanoparticles tend to agglomerate and to form long chains (see Figure 1 (a)) due to their strong remanent magnetization and dipolar interactions. In fact, the aspect ratio of the nanoparticles and the strong magnetic anisotropy make them very sensitive to an external magnetic field and it is also possible to artificially generate arrays of magnetic chains by solidification of the solution of nanoparticles in the presence of magnetic field in wax or other polymers. Using this approach we generated nanocomposite films of Co nanoplates and polyethylene that were prepared in the presence of external field (oriented chains) and without field (not-oriented chains).

III. RESULTS AND DISCUSSION

The magnetic properties of the samples were characterized with hysteresis loops as shown in Figure 2 (a) for not-oriented chains and oriented chains with field. The samples that formed micro chains were measured with the field parallel and perpendicular to the chains.

From Figure 2 (a) one can see that the disordered Co nanoplates (not-oriented chains) have a coercivity of 280 Oe and a remanent magnetization of 0.2. Figure 2 (a) shows that there is a clear anisotropy, or easy magnetization axis, in the direction of the chains aligned by the external field. This means that it is easier to reach saturation when the field is applied in the direction of the chains than in the perpendicular direction. The coercivity values of the sample measured parallel and perpendicular to the chains are 150 Oe and 230 Oe respectively.

In order to infer the magnetic behavior of these hexagonal nanoplates and how the arrays of nanoplates would tend to agglomerate we have performed micromagnetic simulations, obtaining the coercivity and remanence of an isolated nanoplate with a diameter of 300 nm, with the magnetic field applied in different angles θ with the z-axis (the out of plane axis). One can see that the coercivity is strongly dependent on the out-ofplane orientation of the nanoplates with respect to the external magnetic field, varying from 306 to 915 Oe. The in-plane remanence of the nanoplate is more than two times higher than the out-of-plane remanence. This means that the individual nanoplates will tend to form agglomerates due to their strong in-plane remanent field, stacking individual hexagonal nanoplates to generate almost spherical agglomerates, as observed in the SEM image of Figure 1. When these agglomerates of nanoplates are dried in presence of the magnetic field, their easy axis (the xy plane) will be oriented in the direction of the field, and consequently along the length of the chains, and the z-axis of the nanoplates can be oriented randomly in any direction perpendicular to the length of the chains.

In order to understand in more details the mechanism of magnetization reversion on these systems we performed a more detailed magnetic analysis using the technique of FORC diagrams [START_REF] Pike | [END_REF][10][11][12]. This technique is already being used to study different aspects of magnetic systems [12][13][14].

Figure 3 shows the FORCs (a to c) and the corresponding diagrams (d to f) for the samples with not-oriented chains, and the oriented chains measured parallel and perpendicular to the field. The FORCs resemble the hysteresis curves of Figure 2 with the difference that now the complete internal area of the curves are filled with reversal curves. From Figure 3 it is worth noting that the FORCs for the not-oriented chains and the oriented chains measured in perpendicular direction do not present any visible difference (Figure 3 (a) and (c)). On the other hand, the curve for oriented chains measured parallel is clearly different, being more vertical.

The diagrams of Figure 3 (d to f) show the distribution of coercivity fields (H c ) and interaction fields (H i ) [13,[START_REF] Bertotti | Hysteresis in Magnetism[END_REF], and allow one to perform a more detailed analysis of the magnetization reversal mechanisms that take place in these three configurations of the nanoplate arrays. The diagram for the not-oriented chains (Figure 3 (d)) shows two distinct areas (sometimes called magnetic phases) that are more intense. One of this areas is centered at H c = 0, that is attributed to the reversible magnetization contribution. This contribution is usually associated to the presence of a low coercivity reversible magnetic phase, and in this case could represent the contribution of the Co nanoplates that are in chains perpendicular to the field, or that have their hard axis (z plane) in the field direction.

This reversible magnetization component can be observed in the FORC curves of figures 3 (a) as the positive slope at the beginning of each FORC curve.

The other region of the diagram is a small spot centered in H c =0.28 kOe, which represents the irreversible magnetic phase due to the nanoplates or arrays of nanoplates that reverse completely their magnetization with the external field, reaching a new equilibrium magnetization. One can also observe that this region presents a broad distribution of H c , as a consequence of a distribution of nanoparticle size and/or a distribution of orientation of the nanoparticles with respect to the external field. The effect of interaction among the nanoparticles can be appreciated by the width of the distributions in the vertical axis, the H i distribution.

The diagram obtained for the oriented chains measured parallel to the field (see Figure 3(e))

shows only one region of irreversible magnetization centered at H c = 0.25 kOe. The size of this magnetic phase is smaller than in the disordered sample (Figure 3 (d)), both in H c and in H i . As the physical size distribution of the nanoparticles is the same in both samples, this difference in the value of H c distribution could be attributed to the smaller distribution of easy axis (xy planes) in the direction of the chains, as compared to the not-oriented sample.

The fact that the reversible magnetic phase is not present is another indication of the bistability of the oriented chains generated by the external field in the sample preparation process.

When the oriented arrays of nanoparticles are measured in the perpendicular direction the FORCs are very similar to the ones obtained for the disordered sample (see Figure 3 From the previous results one can conclude that when the oriented chains are formed, very few nanoplates are oriented with their hard axis (z-axis) in the direction of the chains, resulting in a more vertical magnetization curve, with a small coercive field. When the magnetization curve is measured perpendicular to the chains, the magnetization reversal occurs through nanoplates that are oriented at different angles θ, increasing the coercivity of the system.

IV. CONCLUSION

Based on the analysis of the different magnetic cooperative mechanisms, one can identify different magnetic entities that are present in the here-described Cobalt nanoplates systems.

The simplest one is composed by the individual magnetic hexagonal Co platelets. The magnetic behavior of these individual hexagons cannot be observed by macroscopic measuring techniques as their strong magnetic anisotropy and aspect ratio induce their agglomeration in almost spherical particles. The more complex structure is the chain of spherical agglomerates, that also forms spontaneously but whose direction can be controlled by solidification of the solution of nanoparticles in the presence of magnetic field in a polymer. The easy axis of the Co stacks are oriented along the field, in the direction of the chains length. Micromagnetic simulations and the careful analysis of the magnetization curves and the FORC diagrams are particularly useful to investigate the collective magnetic properties of these systems.

FIGURE CAPTIONS:

Figure 1 (

 1 Figure 1(a), shows the SEM image and reveals micron-long chains formed by the lineal organization of small particles. The magnified image (inset) shows particles with irregular shape and faceted surfaces, suggesting the existence of agglomerated nanoplates. To visualize details, a dilute colloidal solution was used to prepare TEM samples, as shown in Figure 1(b). This image shows perfect hexagonal particles. The size distribution was determined by processing 387 particles in several micrographs and has been adjusted by Gauss distribution, obtaining an average size about 300 nm. The insert of Figure 1(b) corresponds to the selected area electron diffraction (SAED) and shows the spots of mono crystalline hexagonal particle in 0001 hcp axis zone.

Figure 2 (

 2 Figure 2 (b) shows the coercivity and remanence from simulated curves as a function of angle θ between the z-axis and the external magnetic field. Because the easy axis is in the xy plane of the nanoplate, it is expected that when θ decreases the coercivity increases, as shown in Figure 2 (b). One can see that the coercivity is strongly dependent on the out-of-

  (a) and (c)). However, the diagrams show distinct features, as shown in Figure3(d) and (f). The main difference is the presence of two small spots superimposed in the main irreversible magnetic phase. The first spot is in centered at the same H c value of Figure3(e), and the second spot spreads over a high field region, giving in average the same H c distribution than in Figure3 (d). The H i distribution is smaller than in Figure3(d) and the reversible component appears again in this diagram, as a consequence of the fact that the z-axis of the nanoplates are oriented randomly in any direction perpendicular to the length of the chains.

Figure 1 :

 1 Figure 1: (a) Panoramic and magnified SEM images of chains of agglomerated nanoplates.

Figure 2 :

 2 Figure 2: (a) Magnetization curves normalized by M s of Co chains in polymer, and

Figure 3 :

 3 Figure 3: FORCs measured for the three configurations: (a) not-oriented chains, (b) oriented chains with the external field parallel and (c) perpendicular to the chains. The corresponding FORC diagrams for the three configurations are shown in (d to f).

ACKNOWLEDGMENTS

The support from Fondecyt grants 1110252 and 11110130; Millennium Science Nucleus, Basic and Applied Magnetism Grant N°P10-061-F, AFOSR FA9550-11-1-0347 and CONICYT BASAL CEDENNA FB0807, is gratefully acknowledged.


Si Amar Dahoumane

Claude Yéprémian

Chakib Djédiat

Alain Couté

Fernand Fiévet

Thibaud Coradin
email: thibaud.coradin@upmc.fr

Dr Roberta Brayner
email: roberta.brayner@univ-paris-diderot.fr

A Global Approach of the Mechanism Involved in the Biosynthesis of Gold Colloids using Micro-algae

Keywords: Gold Nanoparticles, Gold Colloids, Biosynthesis, Thylakoids, NP stabilization, Polysaccharides, Micro-algae

The use of micro-algae for the production of noble metal nanoparticles has drawn much attention recently. This paper aims to address some questions raised by our earlier publications and some recent reports from other groups, among which the biological pathways involved in the bioreduction of noble metal cations into nanoparticles and the design of stable colloids. TEM micrographs, taken at the early stage of contact between cells and salt solutions, show undoubtedly that the biomineralization process occurs within the thylakoidal membranes, which are the organelles responsible for photosynthesis. We strongly believe that the available enzymatic machinery (respiratory enzymes and their cofactors) are the key-molecules that allow such reduction, promoting therefore the formation of nanoparticles. In addition, by comparing the characteristics of gold colloids made by polysaccharides producing and non-producing micro-algae strains, we demonstrate that the stability of those colloids is ensured predominantly by those biopolymers. These macrobiomolecules control partly the size and the shape of NPs.

Introduction

Scientists have been exploiting the exceptional diversity of micro-organisms, such as bacteria, yeast, fungi and micro-algae, to fabricate a wide variety of functional nanomaterials. [START_REF] Lloyd | Biotechnological Synthesis of Functional Nanomaterials[END_REF][START_REF] Narayanan | Biological Synthesis of Metal Nanoparticles by Microbes[END_REF] For instance, cellcontaining bacteria cultures or their supernatants can promote the synthesis of noble metal nanoparticles (NPs), such as gold (Au), [START_REF] Gericke | Biological Synthesis of Metal Nanoparticles[END_REF][START_REF] Deplanche | Biorecovery of Gold by Escherichia coli and Desulfovibrio desulfuricans[END_REF][START_REF] Kalishwaralal | Biological Synthesis of Gold Nanocubes from Bacillus licheniformis[END_REF][START_REF] Kashefi | Reductive Precipitation of Gold by Dissimilatory Fe(III)-reducing Bacteria and Archaea[END_REF][START_REF] Husseiny | Biosynthesis of Gold Nanoparticles using Pseudomonas aeruginosa[END_REF][START_REF] He | Biological Synthesis of Gold Nanowires Using Extract of Rhodopseudomonas capsulata[END_REF] silver (Ag), [START_REF] Klaus | Silver-based crystalline nanoparticles, microbially fabricated[END_REF][START_REF] Parikh | Extracellular Synthesis of Crystalline Silver Nanoparticles and Molecular Evidence of Silver Resistance from Morganella sp.: towards Understanding Biochemical Synthesis Mechanism[END_REF][START_REF] Kalimuthu | Biosynthesis of Silver Nanocrystals by Bacillus licheniformis[END_REF] palladium (Pd) [START_REF] Coker | Microbial Engineering of Nanoheterostructures: Biological Synthesis of a Magnetically Recoverable Palladium Nanocatalyst[END_REF] and platinum (Pt), [START_REF] Konishi | Bioreductive Deposition of Platinum Nanoparticles on the Bacterium Shewanella algae[END_REF][START_REF] Riddin | Two Different Hydrogenase Enzymes from Sulphate-reducing Bacteria Are Responsible for the Bioreductive Mechanism of Platinum into Nanoparticles[END_REF] or bimetallic Ag-Au nanoparticles. [START_REF] Nair | Coalescence of Nanoclusters and Formation of Submicron Crystallites Assisted by Lactobacillus Strains[END_REF] Bacteria can also promote the biosynthesis of a variety of metal oxides nano-objects, such as magnetite (Fe 3 O 4 ), [START_REF] Lovley | Anaerobic Production of Magnetic by a Dissimilatory Iron-reducing Microorganism[END_REF] greigite (Fe 3 S 4 ), [START_REF] Bazylnski | Controlled Biomineralization of Magnetite (Fe 3 O 4 ) and Greigite (Fe 3 S 4 ) in a Magnetotactic Bacterium[END_REF] titania (TiO 2 ) [START_REF] Jha | Synthesis of TiO 2 Nanoparticles Using Microorganisms[END_REF] and zinc oxide (ZnO). [START_REF] Prasad | ZnO Nanoparticles: Synthesis and Adsorption Study[END_REF] The same cultures can be involved in the bioproduction of different chalcogenides, such as cadmium sulfide (CdS), [START_REF] Sweeney | Bacterial Biosynthesis of Cadmium Sulfide Nanocrystals[END_REF][START_REF] Bai | Biosynthesis of Cadmium Sulfide Nanoparticles by Photosynthetic Bacteria Rhodopseudomonas palustris[END_REF][START_REF] Kowshik | Microbial Synthesis of Semiconducteur CdS Nanoparticles, Their Characterization, and Their Use in The Fabrication in an Ideal Diode[END_REF] lead sulfide (PbS), [START_REF] Gong | Microbiological Synthesis of Nanophase PbS by Desulfotomaculum sp[END_REF] elemental selenium nanospheres and zinc selenide (ZnSe), [START_REF] Pearce | Microbial Manufacture of Chalcogenide-based Nanoparticles via the Reduction of Selenite using Veillonella atypica: an in situ EXAFS Study[END_REF] and photoactive arsenic sulfide nanotubes (AsS). [START_REF] Lee | Biogenic Formation of Photoactive Arsenic-sulfide Nanotubes by Shewanella sp[END_REF] Additionally, different fungus and yeast strains can be used to initiate the biosynthesis of noble metal nanoparticles, such as gold [START_REF] Mukherjee | Bioreduction of AuCl 4 -Ions by the Fungus Verticillium sp. and Surface Trapping of the Gold Nanoparticles Formed[END_REF][START_REF] Mukherjee | Extracellular Synthesis of Gold Nanoparticles by the Fungus Fusarium oxysporum[END_REF][START_REF] Pimprikar | Influence of Biomass and Gold Salt Concentration on Nanoparticle Synthesis by the Tropical Marine Yeast Yarrowia lipolytica NCIM 3589[END_REF] and silver, [START_REF] Mukherjee | Fungus-Mediated Synthesis of Silver Nanoparticles and Their Immobilization in the Mycelial Matrix: A Novel Biological Approach to Nanoparticle Synthesis[END_REF][START_REF] Duran | Mechanistic Aspects of Biosynthesis of Silver Nanoparticles by Several Fusarium oxysporum Strains[END_REF][START_REF] Kowshik | Extracellular Synthesis of Silver Nanoparticles by a Silver-tolerant Yeast Strain MKY3[END_REF] different oxide nano-objects, such as magnetite, [START_REF] Bharde | Extracellular Biosynthesis of Magnetite using Fungi[END_REF] zirconia (ZrO), [START_REF] Bansal | Biosynthesis of Zirconia Nanoparticles using the Fungus Fusarium oxysporum[END_REF] barium titanate (BaTiO 3 ), [START_REF] Bansal | Room-Temperature Biosynthesis of Ferroelectric Barium Titanate Nanoparticles[END_REF] antimony trioxide (Sb 2 O 3 ), [START_REF] Jha | A Green Low-cost Biosynthesis of Sb 2 O 3 Nanoparticles[END_REF] titania and silica, [START_REF] Bansal | Fungus-mediated Biosynthesis of Silica and Titania Particles[END_REF] and chalcogenide nanomaterials, such as CdS, [START_REF] Kowshik | Microbial Synthesis of Semiconducteur CdS Nanoparticles, Their Characterization, and Their Use in The Fabrication in an Ideal Diode[END_REF][START_REF] Sanghi | A Facile Green Extracellular Biosynthesis of CdS Nanoparticles by Immobilized Fungus[END_REF][START_REF] Ahmad | Enzyme Mediated Extracellular Synthesis of CdS Nanoparticles by the Fungus, Fusarium oxysporum[END_REF][START_REF] Krumov | Accumulation of CdS Nanoparticles by Yeasts in a Fed-batch Bioprocess[END_REF][START_REF] Williams | Efficient Production of Microbially Synthesized Cadmium Sulfide Quantum semiconductor crystallites[END_REF] and CdSe. [START_REF] Kumar | Extracellular Biosynthesis of CdSe Quantum Dots by the Fungus Fusarium oxysporum[END_REF] Similarly, there have been several reports dealing with the use of algal resources for the biosynthesis of noble metal nanomaterials. For instance, biomass extracted from seaweed, [START_REF] Mata | Gold(III) Biosorption and Bioreduction with the Brown Alga Fucus vesiculosus[END_REF][START_REF] Singaravelu | A Novel Extracellular Synthesis of Monodisperse Gold Nanoparticles using Marine alga, Sargassum wightii Greville[END_REF][START_REF] Aarthi Rajathi | Biosynthesis of Antibacterial Gold Nanoparticles Using Brown Alga, Stoechospermum marginatum (kutzing)[END_REF][START_REF] Liu | Optimization of High-Yield Biological Synthesis of Single-Crystalline Gold Nanoplates[END_REF][START_REF] Greene | Interaction of Gold(I) and Gold(III) Complexes with Algal Biomass[END_REF] microalgae [START_REF] Greene | Interaction of Gold(I) and Gold(III) Complexes with Algal Biomass[END_REF][START_REF] Senapati | Intracellular Synthesis of Gold Nanoparticles Using Alga Tetraselmis kochinensis[END_REF][START_REF] Chakraborty | Biorecovery of Gold using Cyanobacteria and an Eukaryotic Alga with Special Reference to Nanogold Formationa Novel Phenomenon[END_REF] and cyanobacteria [START_REF] Parial | Gold Nanorod Production by Cyanobacteria -A Green Chemistry Approach[END_REF][START_REF] Lengke | Morphology of Gold Nanoparticles Synthesized by Filamentous Cyanobacteria from Gold(I)-Thiosulfate and Gold(III)-Chloride Complexes[END_REF] has been tested successfully in the bioreduction of gold cations into their metallic nanoscaled-counterparts. Cyanobacterial biomasses can also be used to carry out the bioproduction of Ag, Pt and Pd nanoparticles. [START_REF] Lengke | Synthesis of Platinum Nanoparticles by Reaction of Filamentous Cyanobacteria with Platinum(IV)-Chloride Complex[END_REF][START_REF] Lengke | Biosynthesis of Silver Nanoparticles by Filamentous Cyanobacteria from a Silver(I) Nitrate Complex[END_REF][START_REF] Lengke | Synthesis of Palladium Nanoparticles by Reaction of Filamentous Cyanobacterial Biomass with a Palladium(II) Chloride Complex[END_REF] Brayner's group was the first team to ever show the ability of living cultures of cyanobacteria to perform the biosynthesis of stable colloids made of Ag, Au, Pt and Pd nanoparticles. [START_REF] Brayner | Cyanobacteria as Bioreactors for the Synthesis of Au, Ag, Pd, and Pt Nanoparticles via an Enzyme-Mediated Route[END_REF] Since then, the reduction of gold cations into gold nanoparticles by living diatoms [START_REF] Schröfel | Biosynthesis of Gold Nanoparticles Using Diatoms -Silica-Gold and EPS-Gold Bionanocomposite Formation[END_REF] and Chlorella vulgaris cultures [START_REF] Luangpipat | Gold Nanoparticles Produced in a Microalga[END_REF] has been reported. Dahoumane et al. have demonstrated the ability of several strains of fresh-water green micro-algae living cultures to produce very stable gold colloids. [START_REF] Dahoumane | Species Selection for the Design of Gold Nanobioreactor by Photosynthetic Organisms[END_REF] The introduction of chloro-auric acid solutions into the cultures triggers the biosorption by the cells of these cations, followed by their reduction into metallic gold, leading, therefore, to the subsequent intracellular formation of the NPs. Finally these NPs are released into culture media. In other words, each cell acts as a microbioreactor and the whole culture as a bioreactor. Importantly, Sicard et al. have demonstrated that micro-algae keep their reductive ability when encapsulated within sol-gel based materials. [START_REF] Sicard | Nano-gold Biosynthesis by Silica-encapsulated Microalgae: A "living" Bio-hybrid Material[END_REF] Moreover, Dahoumane et al. have shown that micro-algae can adapt to the toxicity of gold cations and handle larger amounts of these cations. [START_REF] Dahoumane | Recycling and Adaptation of Klebsormidium flaccidum Microalgae for the Sustained Production of Gold Nanoparticles[END_REF] More recently, Dahoumane et al. have used living cultures of Chlamydomonas reinhardtii, a unicellular fresh-water green micro-alga, to perform the synthesis of bimetallic Ag-Au alloy colloids with a very good stoichiometrical control over NPs composition comparable to those chemically made. [START_REF] Dahoumane | Stoichiometrically Controlled Production of Bimetallic Gold-Silver Alloy Colloids Using Micro-alga Cultures[END_REF] A schematic mechanism was proposed to account for the bioformation of gold colloids after the introduction of gold cations into living micro-algae cultures. [START_REF] Dahoumane | Species Selection for the Design of Gold Nanobioreactor by Photosynthetic Organisms[END_REF] It was demonstrated, using optical microscopy, that the NP synthesis is intracellular, and suggested, using TEM imaging, that this process occurs within the thylakoids, which constitutes the 1 st level of NP size and shape control. This study also brought to mind the potential role played by respiratory enzymes and their cofactors in the reduction of gold cations into metallic counterparts, and the role of cell-produced polysaccharides (PS) in NP stabilization. PS capping the NPs constitutes the 2 nd level of NP size and shape control by preventing NP growth or coalescence. Recently, Shabnam et al. have used thylakoid suspensions, isolated from aquatic and terrestrial plants, to carry out the synthesis of Au-NPs, starting from Au(III) complexes. They have evidenced that these organelles have the ability to reduce Au 3+ and generate Au-NPs through a light-dependent process. [START_REF] Shabnam | Photosynthetic Electron Transport System Promotes Synthesis of Au-Nanoparticles[END_REF] We report in this paper, first, on the localization of the first produced Au-NPs within the cells, using TEM pictures, after the introduction of chloro-auric acid solution into a living culture of Cosmarium impressulum (Ci), a unicellular fresh water green micro-alga. Second, wishing to learn more about the role played by the PS in the control of NP size and shape, and colloid stability over time, we compare exhaustively the characteristics of NPs produced by two different micro-algal strains, Kirshneriella lunaris (Kl), a unicellular moon-shaped fresh water green micro-alga known to produce PS, and a non-producing one, Euglena gracilis (EgM). Third, we demonstrate the ability of PS, harvested from an old Ci culture, to stabilize chemically generated Au-NPs. Finally, we provide with the whole picture of the biological process underlying gold colloid design.

Materials and methods

Micro-algal strains description and culture

Three photosynthetic organisms with distinct structural or physiological features were selected: Cosmarium impressulum (Ci) and Kirchneriella lunaris (Kl), two planktonic single-celled eukaryotic green algae coated with EPS; and Euglena gracilis (EgM), a single-celled eukaryotic euglenoid without EPS. Ci (ALCP #15), Kl (ALCP#92) and EgM (ALCP #217) came from MNHN Culture Collection. Ci and Kl were grown in 250-mL Erlenmeyer flasks, in sterile Bold's basal (BB) medium whose pH was adjusted to 7 using 1 M NaOH solution and buffered with 3.5 mM phosphate buffer at a controlled temperature of 20.0 ± 1.0 °C and luminosity (30-60 lmol m -2 s -1 PPF) and under ambient CO 2 conditions. EgM was grown in 250-mL Erlenmeyer flasks, in Mineral (M) medium at a controlled temperature of 20.0 ± 1.0 °C and luminosity (70-100 lmol m -2 s -1 PPF) under ambient CO 2 conditions. The pH of the medium was adjusted to 3.6 using 1 M HCl solution.

Before addition of gold salts, the culture was transferred (10 % (v/v) of inoculum) into the culture medium, and grown for 2 weeks. Ci culture was used to determine the organelle responsible of the gold cation reduction into metallic gold, leading hence to the formation of GNPs. To do so, 10.0 mL of HAuCl 4 aqueous solution, at an initial concentration of 2.5 × 10 -3 M, were introduced into Ci culture to obtain a final concentration of 2.5 × 10 -4 M. One hour later, an amount of Ci cells were fixed and imaged using TEM, following the procedure described below.

To study the role played by PS in the stabilization of gold colloids, two micro-algal species were chosen: Kl which is known to be coated in a PS layer, and EgM which does not produce any PS. To each of these cultures, 10.0 mL of HAuCl 4 aqueous solution, at an initial concentration of 1.0 × 10 -3 M, were added to obtain a final concentration of 1.0 × 10 -4 M.

PS extraction

PS were isolated from an aged Ci culture of more than 7 months, of an initial volume of 200 mL, which appeared viscous and gelatinous, following a procedure adapted from Bertocchi et al. [START_REF] Bertocchi | Polysaccharides from Cyanobacteria[END_REF] In order to dissolve the PS, the cell biomass was mixed with H 2 O and boiled for one hour, and then centrifuged. Three volumes of ethanol were added to the isolated supernatant containing PS, which was then purified using a dialysis membrane, and finally lyophilized to harvest the PS. The yield was 25 mg for 100 mL of the initial cell culture.

Stability of gold colloids

The evolution of surface plasmon resonance (SPR) band intensity of the obtained colloids, after the addition of gold salt solutions into Kl and EgM cultures, was monitored using a Cary 5E spectrophotometer. ~2 mL of the colloids were scanned between 400 and 800 nm, at different dates.

Chlorophyll a measurements

The chlorophyll a was extracted from 1 mL of unicellular algal culture in 9 mL of acetone, according to a protocol published by Ninfa et al. [START_REF] Ninfa | Fundamental Laboratory Approches for Biochemistry and Biotechnology[END_REF] After 1 min of vortex, the mixture was heated at 37 °C for 3 min followed by centrifugation. The evolution of chlorophyll a band, centered at 663 nm, was followed by UV-Vis spectroscopy, using a Cary 5E spectrophotometer.

Cell preparation for TEM observation

Biomass transmission electron microscopy (TEM) imaging was performed with a Hitachi H-700 operating at 75 kV equipped with a Hamatsu camera. To determine where the first GNPs appear within the cells, Ci cells were fixed, one hour (h+1) after their contact with gold cations, with a mixture containing 2.5 % of glutaraldehyde, 1.0 % of picric acid in a phosphate Sörensen Buffer (0.1 M, pH 7.4). Dehydration was then achieved in a series of ethanol baths, and the samples were processed for flat embedding in Spurr resin. Ultrathin sections were made using a Reicherd E Young Ultracut ultramicrotome (Leica). Sections were contrasted with ethanolic uranyl acetate before visualization.

Photonics

Optical microscopy was performed using Primo Star optical microscopy from Zeiss.

Results and Discussion

TEM localization of Au-NPs place of birth

To determine with certainty the place of birth of the very first Au-NPs within the cells, we proceeded to the fixation of Ci cells an hour (h+1) after chloro-auric acid solution had been introduced into the culture to get a final concentration of 2.5 × 10 -4 M. Figure 1 displays an optical image of a whole Ci cell (a-1) and culture (a-2) before the addition of Au(III) solution. Five days (D+5) after the introduction of the above mentioned solution into the culture, both cells (b-1) and culture (b-2) turned from green to purple evidencing respectively the intracellular reduction of gold cations into metallic gold ,the subsequent formation of Au-NPs, and the release of these latter into culture media, leading to the design of stable gold colloids. Figure (c) represents a micrograph of a whole Ci cell fixed one hour (h+1) after the gold salts solution were added to the culture. One can distinguish clearly all cell compartments, in the two lobes or hemistomates, separated by the isthmus. From outside to inside, we can easily recognize the cell wall (CW), the plasmic membrane (PM) and the periplasmic space between CW and PM, several vacuoles (V), the nucleus (N) located in the central region of the cell, and its nuclear membrane (NM), several thylakoids (Th) some of which are surrounding the pyrenoid (P). This latter is known to be the cell stock organelle. One can also notice black spots which may be dust grains or artefacts due to sample preparation for TEM imaging. Same sample TEM pictures at a higher magnification (Fig. 2-a and 2-b), taken at h+1, evidence the presence of the NPs, in their vast majority within the thylakoids or in their vicinity. A few of them are visible at the intracellular membranes and cytoplasmic membrane. Some of these nano-objects are lined up along the thylakoidal membranes. All these Au-NPs are uniformly sphere-shaped. It seems that the intracellular growth of NPs favors, exclusively, the round-shaped morphology. However, their size varies from a few to several nanometers. The smallest NPs are hardly recognizable while the biggest are of tens of nm in diameter. This discrepancy in size may be due to the ongoing process of NP formation: the oldest ones having underwent growth and gained in size are outer NPs that are most likely to be in close contact each other and then to coalesce than the inner ones, leading therefore to bigger NPs. The ultrastructure of a thylakoid confirmed the above-mentioned findings (Fig. 3). This picture depicts undoubtedly the space-repartition of Au-NPs within the chloroplasts according to size, the smaller ones found at the inner part of those organelles, in the space between the thylakoidal membranes, whereas the biggest nano-objects are located at the outer space and in the vicinity of these photosynthesis responsible organelles. This result corroborates that the reduction of gold cations into the metallic gold, that is to say the biosynthesis of Au-NPs, occurs within the chloroplast, and the structure of the thylakoids and the space between the thylakoidal membranes play a key role in the control of NP dimensions. This is the first time that the role played by thylakoids during the bioformation of noble metal NPs is demonstrated. This corroborates the studies done by Zhang et al. [START_REF] Zhang | Biosynthesis of Gold Nanoparticles Using Chloroplasts[END_REF] and Shabnam et al. [START_REF] Shabnam | Photosynthetic Electron Transport System Promotes Synthesis of Au-Nanoparticles[END_REF] which showed that isolated chloroplasts, the photosynthetic organelles of algae and plants, could be used to synthesize nanomaterials. This also confirms what we stated in our earlier publications by suggesting that the thylakoids are the place of birth of Au-NPs, after the reduction of gold cations into metallic gold. 54,57

Role of EPS in the stabilization of Au-NPs, and the control of their size and shape

In the previous section, we have demonstrated that the reduction of Au(III) into Au(0) occurs within the cells, more precisely within the thylakoidal membranes, leading therefore to the bioformation of Au-NPs. This intracellular synthesis constitutes the first level of NP size and shape control. In the following section, we explore the influence of the presence of PS or not on gold colloid characteristics, such as the shape and the size of the NPs, and colloid stability over time. To do so, we chose two different unicellular algal strains, Kirchneriella lunaris, Kl, known to produce extracellular matrices, ECM or PS, and Euglena gracilis, EgM, not known to produce such biopolymers. We added the same amount of HAuCl 4 solution, at a final concentration of 10 -4 M, to both cultures.

In the case of EgM (Fig. 4 a-1), one day (D+1) after the cells were brought into contact with Au(III), the culture turned straight away from green (D+0) to dark purple which confirms a rapid gold cation reduction into metallic gold and the release of the as-produced Au-NPs into culture media (CM). However, this color became lighter with time as displayed by the picture taken almost two weeks later (D+13). This colloid was not stable and the Au-NPs eventually started to sediment. On the other hand, Kl culture (Fig. 4 b-1) exhibited a quite different behavior. Initially green (D+0), this culture turned into light red (D+1) and, as the process of NP release continued gradually, this color became darker with time (D+14).

The trend in color change, due to NP intracellular biosynthesis and release into culture media, was monitored, for both cultures, using UV-Vis spectroscopy. Both species displayed the characteristic surface plasmon resonance (SPR) band of spheric Au-NPs, located at ~540 and ~520 nm for EgM (Fig. 4 a-2) and Kl (Fig. 4 b-2) cultures respectively. However, the intensity of the SPR band decreased with time for EgM culture while it increased for the Kl one. This trend was confirmed by plotting the maximum of SPR band intensity vs. time, Abs max = f(t). In the case of EgM (Fig. 4 a-3), the NP release reached its maximum one day after the cells have been brought into contact with chloro-auric acid solution. After that time, the intensity tended to drop down quasi-linearly. On the contrary, the SPR intensity of Kl sample (Fig. 4 b-3) increases gradually with time and plateaus approximately nine days after the addition of HAuCl 4 to the culture. It is important to notice that, for both samples, there is a good agreement between the evolution of the macroscopic aspect and UV-Vis measurements. To study the size and the shape of the Au-NPs made by each strain, TEM images were performed on droplets taken from each sample. EgM sample (Fig. 5 a-1) shows three distinguishable NP populations, smaller and medium objects which seem to be spherical and imprisoned within an organic network, and bigger round-shaped objects. NPs made by Kl are more uniformly shaped and sized (Fig. 5 b-1). These objects appear to be all sphere shaped with a diameter of a few nanometers (~ 5nm). The presence of the organic matter in the case of EgM, due likely to the massive cell death triggered by the toxicity of gold cations, did not prevent the NPs from aggregation and sedimentation. This cell death was confirmed by the evolution of chlorophyll a intensity, monitored using UV-Vis spectroscopy measurements. For both strains (Fig. 5 a-2 and b-2), the trend was similar. The addition of chloro-auric acid solution led to a huge cell death. However, after a while, both species recovered and grow up significantly. It is important to notice that, in the case of Kl (Fig. 5 b-2), the use of acetone to prepare the sample for chlorophyll a measurements did not alter the stability of the gold colloid. Indeed, Au-NPs remained stable and their SPR band was still visible evidencing a strong anchoring of PS into NP surfaces. This was not the case of EgM (Fig. 5 a-2). If the released organic matter from cells, most likely proteins, was involved in the capping of the NPs and the stabilization of the colloids, one would expect the same behavior for both strains, the PS producing species and the non-producing one. This was not the case. It is why we believe those biopolymers are the predominantly macrobiomolecules involved in the stabilization of the colloids by avoiding the aggregation, the growth and the sedimentation of the NPs.

Euglena gracilis (EgM)

Kirchneriella lunaris (Kl) To investigate the role played by PS in the stabilization of gold colloids, we extracted these biopolymers from a very old Ci culture. This strain is known to produce huge amounts of PS. Seven months after its launch with an initial volume of 200 mL, the volume shrunk and the cells formed a green and very gelatinous mass. This pasty aspect provides an idea of its richness of PS. 50 mg of PS were collected making the yield at 0.25 mg/mL. After that, we compared the stability of chemicallymade Au-NPs. In this experiment, two glass vials were filled with 10 mL of BB culture medium containing Au(III) at a final concentration of 10 -4 M. To the first vial (Fig. 6 a-1) were added PS at a mass concentration of 0.25 mg/mL while the second one was kept (Fig.

Conclusion and perspectives

This work aimed to contribute to a better understanding of the biological pathways involved in the bioformation of gold colloids after living micro-algal cultures were put into contact with Au(III) solutions by elucidating the cell organelles responsible for the reduction of such cations and the biomolecule assuring the stabilization of the colloids. Micrographs at the early stages of this contact have demonstrated that gold cations migrate into the photosynthetic organelles, i.e. chloroplasts or thylakoids, where they are reduced into their metallic counterparts, leading therefore to the production of Au-NPs. The fact that NP formation occurs within the thylakoids constitutes the first level of shape and size control. In the presence of PS in appropriate amounts, these NPs will form stable colloid after being released into culture media. Even if we do not exclude the contribution of the intracellular organic matter, we can claim that PS are the predominant macrobiomolecules responsible for the colloidal stability. This constitutes the second and last level of NP shape and size control by hindering their merging and their growth.

We have summarized our findings regarding the most likely mechanism of gold colloid design using living algal cultures in However, several questions remain unanswered and a thorough investigation should be implemented in order to understand the following issues: (i) what incites living micro-algal cells to reduce noble metal cations into metallic entities while, in the case of iron cations for instance, the cells promote the synthesis of oxides? [START_REF] Brayner | Intracellular Biosynthesis of Superparamagnetic 2-lines Ferri-hydrite Nanoparticles Using Euglena gracilis Microalgae[END_REF][START_REF] Brayner | Photosynthetic Microorganism-mediated Synthesis of Akaganeite (beta-FeOOH) Nanorods[END_REF][START_REF] Dahoumane | Design of Magnetic Akaganeite-Cyanobacteria Hybrid Biofilms[END_REF] (ii) What explains the difference in shape and size between these nano-oxides and their noble-metal counterparts? (iii) What explains the fact that oxides NPs are kept within the cells whereas noble metal NPs are released into culture media? Is this related to any evolutionary process? (iv) What molecules and/or biomolecules are the electron donors in the case of noble metal cations reduction? We suggested in a previous paper that NADP(H) is the most likely molecule to fulfill this role as it is involved in such processes in several biochemical pathways. [START_REF] Dahoumane | Species Selection for the Design of Gold Nanobioreactor by Photosynthetic Organisms[END_REF] (v) Is this reduction process light-driven, as suggested recently? [START_REF] Shabnam | Photosynthetic Electron Transport System Promotes Synthesis of Au-Nanoparticles[END_REF] Or isolated chloroplasts do not behave the same way when they are parts within the cells?

Figure 1 :

 1 Figure 1: Optical image of a single cell of Cosmarium impressulum, Ci (a-1), and a digital picture of Ci culture (a-2) before addition of HAuCl 4 solution. (b-1) displays an optical image of a single Ci cell, and (b-2) a digital image of Ci culture, five days (D+5) after HAuCl 4 solution introduction. (c) a micrograph of a whole cell of Ci taken one hour (h+1) after HAuCl 4 solution introduction.

Figure 2 :

 2 Figure 2: TEM micrographs at higher magnification of (a) thylakoids surrounding the pyrenoid, and (b) a thylakoid located close to the cell wall.

Figure 3 :

 3 Figure 3: Ultrastructure of a region of thylakoid showing Au-NPs of different sizes.

Figure 4 :

 4 Figure 4: Evolution of the macroscopic aspect of EgM (a-1) and Kl (b-1) cultures respectively. (a-2) and (b-2): Evolution of the SPR band for EgM and Kl respectively. (a-3) and (b-3): Kinetics of NP release into culture media for EgM and Kl respectively.

Figure 5 :

 5 Figure 5: Micrographs of Au-NPs made by EgM (a-1) and Kl (b-1); and evolution of the cell viability of EgM (a-2) and Kl (b-2).

  6 b-1) PS free. The addition of hydrazine(10 -4 M) under a vigorous magnetic stirring to each vial triggered a color change for both vials, the first one containing PS became purplish (Fig.6a-2) while the second (Fig.6b-2), PSfree, became blue. The color change is the evidence of Au-NPs apparition in both vials. However, one day (D+1) after the magnetic stirring had been turned off, no remarkable change was noticed for the first vial (Fig.6a-3) whereas the second vial became transparent (Fig.6b-3) due to Au-NPs sedimentation with the formation of a visible layer at the bottom of the vial and a deposit on the upper part of the glass wall, at the interface between the liquid and the air. This is another proof that the stabilization of micro-algal-made Au-NPs is made possible by the presence of the available PS within and/or at the surface of the cells.

Figure 6 :

 6 Figure 6: Digital images demonstrating the role played by micro-algal extracted PS in the stability of gold colloids. (a-1): HAuCl 4 solution at 10 -4 M containing PS at 0.25 mg/mL; (a-2): a few minutes after addition of hydrazine under vigorous stirring; and (a-3) one day later (D+1) after the stirring had been turned off. (b-1): HAuCl 4 solution at 10 -4 M without PS; (b-2): a few minutes after addition of hydrazine under vigorous stirring; and (b-3) one day later (D+1) after the stirring had been turned off.

Figure 7 :

 7 (i) Addition of Au(III) aqueous solution into a healthy algal culture whose cells are known to produce PS; (ii) internalization of Au(III) by the cells through an osmotic process; (iii) intracellular reduction of Au(III) into Au(0) within the thylakoidal membranes taking profit from the available enzymatic machinery; (iv) growth of Au-NPs after the merging of Au atoms; (v) diffusion of Au-NPs from the chloroplasts into the cytoplasmic membrane and cell wall; (vi) encapsulation of Au-NPs within PS-based networks at the cell wall; (vii) release of the as-protected Au-NPs into culture media; (viii) elaboration of stable colloids.

Figure 7 :

 7 Figure 7: Global picture of the schematic mechanism involved in the bioproduction of stable gold colloids through a micro-algal mediated route.