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Thin Solid Films j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / t s f
Design of magnetic akaganeite-cyanobacteria hybrid biofilms Si Amar Dahoumane a, Chakib Djediat b, Claude Yéprémian b, Alain Couté b, Fernand Fiévet a, Roberta Brayner a,⁎ a b
Université Paris Diderot (Paris 7), CNRS, UMR 7086, Interfaces, Traitements, Organisation et Dynamique des Systèmes (ITODYS), 15 rue Jean de Baïf, F-75205 Paris Cedex 13, France Muséum National d'Histoire Naturelle (MNHN), Département RDDM, USM 505, 57 rue Cuvier, F-75005 Paris, France
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Article history: Received 5 November 2009 Received in revised form 22 March 2010 Accepted 1 April 2010 Available online xxxx Keywords: Akaganeite Cyanobacteria Biofilm
a b s t r a c t Common Anabaena cyanobacteria are shown to form intra-cellularly akaganeite β-FeOOH nanorods of wellcontrolled size and unusual morphology at room temperature. High-resolution transmission electron microscopy showed that these nanorods present a complex arrangement of pores forming a spongelike structure. These hybrid akaganeite-cyanobacteria were used to form “one-pot” hybrid biofilms. The hybrid biofilm presents higher coercivity (Hc = 44.6 kA m− 1 (560 Oe)) when compared to lyophilized akaganeitecyanobacteria powder (Hc = 0.8 kA m− 1 (10 Oe)) due to the quasi-assembly of the cells on the glass substrate compared to the lyophilized randomly akaganeite-cyanobacteria powder. © 2010 Elsevier B.V. All rights reserved.
1. Introduction The development of reliable, eco-friendly processes for the synthesis of nanomaterials urges chemists to find synthetic routes with limited ecological impact, involving natural, renewable resources [1]. Indeed, there are innumerable examples of living organisms that synthesize exquisite organized inorganic structure under mild pH, pressure, and temperature conditions, such as magnetotatic bacteria (Fe3O4 nanoparticles), cyanobacteria (Au, Ag, Pd, and Pt), diatoms (siliceous materials), etc. [2–12]. Iron was probably a much more common ion in primordial anoxic oceans than today's oxygen-rich surface waters. Thus, early cells most likely had an abundant supply of Fe2+ ions and became dependent on iron as a cofactor for many enzymes [13]. When iron is relatively abundant, some algae can store it within protein aggregates known as phytoferritin. Some small cyanobacteria, certain dinoflagelates, and also some diatoms are known to harvest iron ions at low concentrations in seawater by producing surface iron-binding organic molecules known as siderophores [14]. The present work is focused on the design of magnetic akaganeite-cyanobacteria hybrid biofilms. Anabaena flos-aquae can produce intracellulary akaganeite (β-FeOOH) nanorods by a mechanism involving iron–siderophore complex formation. Akaganeite has been the subject of numerous studies since its first synthesis by Bohm in 1928, due to its sorption, ionexchange, and catalytic properties [15–19]. Moreover, the β-FeOOH phase is widely used as a precursor in the preparation of ferromagnetic materials such as maghemite (γ-Fe2O3) [20]. In this work we
⁎ Corresponding author. E-mail address:
[email protected] (R. Brayner).
showed that akaganeite-cyanobacteria biofilms presents higher coercivity (Hc = 44.6 kA m− 1 (560 Oe)) when compared to lyophilized akaganeite-cyanobacteria material (Hc = 0.8 kA m− 1 (10 Oe)) due to the quasi-assembly of the cells on the glass substrate compared to the lyophilized randomly akaganeite-cyanobacteria powder.
2. Materials and methods 2.1. Photosynthetic microorganism's description and culture Anabaena flos-aquae planktonic cyanobacteria, strain ALCP B24, came from MNHN Culture Collection. This microorganism was grown in 250 ml Erlenmeyer flasks, in sterile Bold's basal medium and buffered with 3.5 mM phosphate buffer at a controlled temperature of 20.0 ± 0.5 °C and luminosity (30–60 µmol m− 2 s− 1 Photosynthetic Photon Flux) under ambient CO2 conditions. The pH of the medium was adjusted to 7.0 using 1 M NaOH solution. Before addition of iron salts in concentrations ranging from 10− 3 M to 10− 1 M, the culture was transferred (20% (v/v) of inoculum) into the culture medium, and grown for 4 weeks. After synthesis of intracelluary akaganeite nanorods, the biofilms were prepared by addition of glass substrate in the culture medium containing the hybrid akaganeite-cyanobacteria microorganisms. The biofilms were grown also for 4 weeks. Morphologically, Anabaena flos-aquae strain used for our experimentation is composed of one linear series of vegetative cells. Anabaena flos-aquae may have a lot of heterocyts (heterocyt has a thick wall and its interior appears yellowish and empty. Its peculiar physiology is atmospheric dinitrogen gas (N2) fixation to ammonia that is catalyzed by nitrogenase enzyme generated by the cell itself) dispersed all along the trichome.
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2.2. Cyanobacteria and nanoparticle characterization Optical microscopy was performed with a Nikon EFD3 interferential contrast microscope. The photosynthetic activity of the microalgae was measured using the pulsed amplitude modulation (PAM) method with a Handy PEA (Hansatech instruments) fluorimeter. This method uses the saturation pulse method, in which a phytoplankton sample is subjected to a short beam of light that saturates the photosystem II (PS II) reaction centers of the active chlorophyll molecules. This process suppresses photochemical quenching, which might otherwise reduce the maximum fluorescence yield. A ratio of variable over maximal fluorescence (Fv/Fm) can then be calculated which approximates the potential quantum yield of PS II. Biomass transmission electron microscopy (TEM) imaging was performed with a Hitachi H-700 operating at 75 kV equipped with a Hamatsu camera. For TEM studies, the cyanobacteria were fixed with a mixture containing 2.5% of glutaraldehyde, 1.0% of acrolein and 0.1% of ruthenium red in a phosphate Sörengen 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 a Spurr resin. Ultrathin sections were made using a Reicherd E Young Ultracut ultramicrotome (Leica). Sections were contrasted with ethanolic uranyl acetate before visualization. X-ray powder diffraction (XRD) patterns were recorded using X'Pert PRO (PANalytical) diffractometer with CoKα radiation. The diffractometer was calibrated using a standard Si sample. Magnetic measurements were performed using a Quantum
Design MPMS-5S superconducting quantum interference devices (SQUID) magnetometer in the temperature range 4–300 K. 3. Results and discussion 3.1. Cyanobacteria and β-FeOOH akaganeite characterizations Photonic microscopy images and the photosynthetic activity of Anabaena flos-aquae cyanobacteria are shown in Fig. 1. Before addition of a mixed FeCl3·4H2O and FeSO4(NH4)2SO4·6H2O ([Fe2+; Fe3+] = 10− 2 M), the culture medium presents a transparent yellow-colored solution due to the presence of iron salts. After 1 week, the solution underwent a color change to clear brown. This color change was also observed on vegetative cells (Fig. 1a and c). All vegetative cells color turned from blue-green to brown. The photosynthetic activity before and after addition of a mixed iron salts ([Fe2+; Fe3+] = 10− 2 M), was measured using a PAM fluorimeter (Fig. 1b and d). Before addition of iron salts, Anabaena flos-aquae presents a stable photosynthetic activity for more than 2 months (Fig. 1b). On the other hand, after addition of iron salts, an increase of the photosynthetic activity was observed between 5 and 15 days, with Fv/Fm ratios being higher than Anabaena flos-aquae in the absence of iron salts (Fig. 1d). This behavior may be explained by the algae adaptation that allows them to harvest and store iron when it is available. XRD patterns of lyophilized Anabaena flos-aquae after reaction with iron salts indicate the presence of β-FeOOH akaganeite tetragonal structure, together with an amorphous phase attributed to the presence of algae biomass
Fig. 1. Photonic microscopy images of (a) Anabaena flos-aquae before iron salts addition; (b) after iron salts addition [Fe2+, Fe3+] = 10− 2 M. Photosynthetic activities of (c) Anabaena flos-aquae before iron salts addition; (d) after iron salts addition (salt addition at T = 0).
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(Fig. 2). TEM ultrathin sections of this microorganism are shown in Fig. 3. Fig. 3a presents a vegetative cell thin section, before iron salts addition, showing the chloroplast and the cell wall. After reaction with the iron salts, dark β-FeOOH akaganeite nanorods were observed inside the cells (Fig. 3b). In addition high-resolution transmission electron microscopy (HRTEM) micrograph shows that these nanorods present a complex arrangement of pores forming a spongelike structure. Inside the cells, no preferential orientation of these nanorods was observed. The akaganeite formation mechanism was suggested following the strategy II. In the case of Anabaena flosaquae strain, iron-limited cells are able to use a wide variety of chelated/bound iron sources involving ligands such as ethylenediamine di(o-hydroxyphenyl acetic acid) and desferrioxamine B. It was shown that the rate of iron uptake was a function of the chelator strength [21]. Concisely, under oxic conditions, iron is generally in the 3+ oxidation state and forms various insoluble minerals. To obtain iron from such minerals, cells produce iron-binding siderophores that bind iron and transport it into the cell. The microorganisms can consume part of these complexes, and the excess can precipitate to form β-FeOOH akaganeite nanorods. The siderofores can also play a role in formation of biofilms. It was shown that a siderofore produced by Pseudomonas aeruginosa bacteria can firmly bound to the bacterial cell surface and simultaneously bind covalently to TiO2 and Fe3+ oxide [22,23]. In our case, the siderofores produced by Anabaena flos-aquae may also bind to the cyanobacterial cell surface and simultaneously bind covalently to SiO2 glass surface to form strong adherent biofilms. 3.2. Hybrid akaganeite-cyanobacteria biofilm formation Bacteria, cyanobacteria and microalgae are commonly encountered colonizers of buildings façades. The adhesion process is mediated by properties of the coating such as roughness and porosity, but microorganism and substrate physico-chemical properties are also believed to intervene [24–26]. In vitro adhesion experiments with some microalgae showed (i) the longest the initial contact time of cells with model surfaces, the strongest the adhesion strength and (ii) the adhesion strength of algae depend on the culture age, adhesion of old-cultures cells being weaker than that of young-culture cells [27]. In the literature, hybrid biofilms were formed after numerous steps. Consequently, it is more difficult to control nanoparticle size
Fig. 3. TEM micrograph thin sections of (a) Anabaena vegetative cell before iron salts addition; (b) Anabaena vegetative cell with akaganeite nanorods inside the cell; (c) HRTEM image of Anabaena-based akaganeite nanorods.
Fig. 2. XRD patterns of hybrid akaganeite-Anabaena powder after lyophilization. Asterisk symbol (*) corresponds to algal biomass, in particular cellulose.
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and shape before their organization on cell surfaces to form these hybrid biofilms. For example, Saraf et al. obtained highly selective, electrically conductive monolayer of Au nanoparticles on live bacteria but their biofilm was “constructed” after various steps: (i) fabrication of Au nanoparticles, (ii) functionalization of Au nanoparticle with lysine (iii) bacteria adhesion on glass; (iv) affinity between the chosen bacteria and lysine; (v) contact with the immobilized bacteria and Aulysine solution for 12 h [28]. In our study the cyanobacteria capacity to form biofilms naturally was used to design a “one-pot” hybrid akaganeite-cyanobacteria biofilm. The culture containing akaganeiteAnabaena flos-aquae after 15 days of incubation (a young-culture) was used to form the hybrid biofilms (Scheme 1). Glass substrates, after washing, were added to the akaganeite-cyanobacteria culture medium for more than 15 days. After this time the biofilms formed were then washed and dried to clear up the cell that were not adhered on the glass substrates. SEM images of hybrid biofilms are shown in Fig. 4a and b. As we can see, the surface was not completely recovered after 15 days of incubation (Fig. 4a). On the other hand, after formation of akaganeite nanorods intracellulary followed by adhesion on glass substrate, the cell morphology retains the same shape (Fig. 4b). 3.3. Preliminary magnetic results Akaganeite exhibits usually an anti-ferromagnetic ordering with Néel temperature lying in the range 270–296 K [29]. It has been recently observed for different anti-ferromagnetic nanoparticles, a non-zero magnetization below the bulk Néel temperature [30–32]. The magnetic behavior observed at low temperature may be due to surface effects owing to the large surface/volume ratio of these nanoparticles. Superparamagnetism behavior is due to an incomplete magnetic compensation between anti-ferromagnetically coupled magnetic sublattices, in agreement with Néel model; the overall moment of each particle being due to uncompensated surface spins
Fig. 4. (a) SEM image of akaganeite-Anabaena biofilm formed; (b) SEM image of one filament of akaganeite-Anabaena hybrid material; (c) normalized magnetization (M/MS) vs. magnetic field (T = 5 K).
Scheme 1. Biofilm formation.
[33]. The particle spins lattice could reverse coherently and randomly under thermal activation and the resulting moment of the uncompensated surface spins would fluctuate above a blocking temperature as expected for superparamagnetic particles. Magnetization curves at 5 K of lyophilized akaganeite-cyanobacteria powder and of akaganeite-cyanobacteria hybrid biofilm are shown in Fig. 4c. At 5 K, lyophilized akaganeite-cyanobacteria powder presents a coercive field equal to 0.8 kA m− 1 (10 Oe), whereas akaganeite-cyanobacteria hybrid biofilm presents Hc equal to 44.6 kA m− 1 (560 Oe). Usually, larger and less linear magnetizations (as a function of Hc) are found
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Scheme 2. Akaganeite nanorods orientation: after lyophilization and after biofilm.
for smaller anti-ferromagnetic spherical particles [30]. Moreover, for anisotropic shaped particles, this increase is related to their aspect ratio and/or their relative orientation. Here, the aspect ratio was the same for both samples. On the other hand, the nanorods orientation was slightly different (Scheme 2). The lyophilized powder presents a randomly nanorods 3D orientation, while the hybrid biofilm present a 2D quasi-assembly of nanorods inside the cells on glass substrate. It is important to know that these nanorods are not completely oriented in the same direction but the slight orientation difference may explain the coercivity increase. 4. Conclusion Here we show that hybrid akaganeite-cyanobacteria are able to form, “one-pot” hybrid biofilms. The hybrid biofilm presents higher coercivity (Hc = 44.6 kA m− 1) when compared to lyophilized akaganeite-cyanobacteria powder (Hc = 0.8 kA m− 1) due to the quasi-assembly of the cells on the glass substrate compared to the lyophilized randomly akaganeite-cyanobacteria powder. The akaganeite nanorods orientation was slightly different. The lyophilized powder presents a randomly nanorods 3D orientation, while the hybrid biofilm present a 2D quasi-assembly of nanorods inside the cells on glass substrate. The biofilm orientation may explain the coercivity increase from 0.8 to 44.6 kA m− 1. References [1] J.A. Dahl, B.L.S. Maddux, J.E. Hutchison, Chem. Rev. 107 (2007) 2269. [2] S. Mann, Biomineralization, Oxford University Press, Oxford, 2001. [3] S. Mann, Angew. Chem. Int. Ed. 47 (2008) 5306.
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