From raw microalgae to bioplastics: Conversion of Chlorella vulgaris starch granules into thermoplastic starch.

Microalgae are emerging as a promising feedstock for bioplastics, with Chlorella vulgaris yielding significant amounts of starch. This polysaccharide is convertible into thermoplastic starch (TPS), a biodegradable plastic of industrial relevance. In this study, we developed a pilot-scale protocol for extracting and purifying starch from 430 g (dry weight - DW) of starch-enriched Chlorella vulgaris biomass. More than 200 gDW of starch were recovered, with an extraction yield and starch purity degree reaching 98 and 87 %, respectively. We have characterized this extracted starch and processed it into TPS using twin-screw extrusion and injection molding. Microalgal starch showed similar properties to those of native plant starch, but with smaller granules. We compared the mechanical properties of microalgal TPS with two controls, namely a commercial TPS and a TPS prepared from commercial potato starch granules. TPS prepared from microalgal starch showed a softer and more ductile behavior compared to the reference materials. This study demonstrates the feasibility of recovering high-purity microalgal starch at pilot scale with high yields, and highlights the potential of microalgal starch for the production of TPS using industrially relevant processes.

Chiral nematic nanocomposites with pitch gradient elaborated by filtration and ultraviolet curing of cellulose nanocrystal suspensions.

An innovative method combining frontal filtration with ultraviolet (UV) curing has been implemented to design cellulosic nanocomposite films with controlled anisotropic textures from nanometric to micrometric length scales. Namely, an aqueous suspension containing poly (ethylene glycol) diacrylate polymer (PEGDA) as a photocurable polymer and cellulose nanocrystals (CNCs) at a 70/30 mass ratio was processed by frontal filtration, followed by in-situ UV-curing in a dedicated cell. This procedure allowed designing nanocomposite films with highly oriented and densely-packed CNCs, homogeneously distributed in a PEGDA matrix over a thickness of ca. 500 µm. The nanocomposite films were investigated with small-angle X-ray scattering (SAXS), by raster-scanning along their height with a 25 µm vertically-collimated X-ray beam. The CNCs exhibited a high degree of orientation, with their director aligned parallel to the membrane surface, combined with an increase in the degree of alignment as concentration increased towards the membrane surface. Scanning electron microscopy images of fractured films showed the presence of regularly spaced bands lying perpendicular to the applied transmembrane pressure, highlighting the presence of a chiral nematic (cholesteric) organization of the CNCs with a pitch gradient that increased from the membrane surface to the bulk.

Manufacturing of starch-based materials using ultrasonic compression moulding (UCM): toward a structural application.

An experimental study of the ultrasonic compression moulding (UCM) to manufacture biobased composites made of semicrystalline starch powders and softwood fibres is described. The main objective was to assess the potential of using this fast and economical processing technique to elaborate a 100% biobased composite which might substitute more usual polymer materials for structural applications. The starch powder was chosen as raw material for the matrix while the reinforcement was made of softwood fibres. Tablets made of starch only and composite beams were processed under different conditions and characterised by several techniques. Three types of starch powders and two types of fibres were used as raw materials. A morphological and crystalline analysis was carried out by scanning electron microscopy (SEM) and X-ray diffraction (XRD). The native semicrystalline structure of starch granules was not totally preserved so as to obtain a homogeneous material. Diametral compression tests on tablets were performed to improve the processing route and obtain the materials with the best properties. Bending tests were used on composite beams to quantify the mechanical properties and study the effects of the processing parameters. The optimum processing parameters were defined and allowed obtaining a matrix for which the flexural strength reached 21 MPa. Mechanical properties were improved when fibres were added into the matrix: three-points bending tests showed a Young's modulus of 6 GPa, a flexural strength of 75 MPa and a flexural strain at break of 6% for a bulk density of 1.25. Considering these results, UCM appears to be a promising process to design a 100% biobased composite with mechanical properties comparable to those of classical discontinuous fibre composites.

Ultrafine heat-induced structural perturbations of bone mineral at the individual nanocrystal level.

The nanoscale characteristics of the mineral phase in bone tissue such as nanocrystal size, organization, structure and composition have been identified as potential markers of bone quality. However, such characterization remains challenging since it requires combining structural analysis and imaging modalities with nanoscale precision. In this paper, we report the first application of automated crystal orientation mapping using transmission electron microscopy (ACOM-TEM) to the structural analysis of bone mineral at the individual nanocrystal level. By controlling the nanocrystal growth of a cortical bovine bone model artificially heated up to 1000 °C, we highlight the potential of this technique. We thus show that the combination of sample mapping by scanning and the crystallographic information derived from the collected electron diffraction patterns provides a more rigorous analysis of the mineral nanostructure than standard TEM. In particular, we demonstrate that nanocrystal orientation maps yield valuable information for dimensional analysis. Furthermore, we show that ACOM-TEM has sufficient sensitivity to distinguish between phases with close crystal structures and we address unresolved questions regarding the existence of a hexagonal to monoclinic phase transition induced by heating. This first study therefore opens new perspectives in bone characterization at the nanoscale, a daunting challenge in the biomedical and archaeological fields, which could also prove particularly useful to study the mineral characteristics of tissue grown at the interface with biomaterials implants. STATEMENT OF SIGNIFICANCE: In this paper, we propose a new approach to assess the mineral properties of bone at the individual nanocrystal level, a major challenge for decades. We use a modified Transmission Electron Microscopy acquisition mode to perform an Automated Crystal Orientation Mapping (ACOM-TEM) by analyzing electron diffraction patterns. We tune the mineral nanocrystal size by heating a model bovine bone system and show that this method allows precisely assessing the mineral nanocrystal size, orientation and crystallographic phase. ACOM-TEM therefore has sufficient sensitivity to solve problems that couldn't be answered using X-ray diffraction. We thus revisit the fine mechanisms of bone nanocrystal growth upon heating, a process currently used for bone graft manufacturing, also of practical interest for forensic science and archaeology.

Biodistribution and preliminary toxicity studies of nanoparticles made of Biotransesterified ß-cyclodextrins and PEGylated phospholipids.

BACKGROUND: The modification of ß-cyclodextrins (ßCDs) by grafting alkyl chains on the primary and/or secondary face yields derivatives (ßCD-C10) able to self-organize under nanoprecipitating conditions into nanoparticles (ßCD-C10-NP) potentially useful for drug delivery. The co-nanoprecipitation of ßCD-C10 with polyethylene glycol (PEG) chains yields PEGylated NPs (ßCD-C10-PEG-NP) with potentially improved stealthiness. The objectives of the present study were to characterize the in vivo biodistribution of ßCD-C10-PEG-NP with PEG chain length of 2000 and 5000Da using nuclear imaging, and to preliminarily evaluate the in vivo acute and extended acute toxicity of the most suitable system. RESEARCH DESIGN AND METHODS: The in vivo and ex vivo biodistribution features of naked and decorated nanoparticles were investigated over time following intravenous injection of 125I-radiolabeled nanoparticles to mice. The potential toxicity of PEGylated ßCD-C10 nanosuspensions was evaluated in a preliminary in vivo toxicity study involving blood assays and tissue histology following repeated intraperitoneal injections of nanoparticles to healthy mice. RESULTS: The results indicated that ßCD-C10-PEG5000-NP presented increased stealthiness with decreased in vivo elimination and increased blood kinetics without inducing blood, kidney, spleen, and liver acute and extended acute toxicity. CONCLUSIONS: ßCD-C10-PEG5000-NPs are stealth and safe systems with potential for drug delivery.

Micro-mechanics of electrostatically stabilized suspensions of cellulose nanofibrils under steady state shear flow.

In this study, we characterized and modeled the rheology of TEMPO-oxidized cellulose nanofibril (NFC) aqueous suspensions with electrostatically stabilized and unflocculated nanofibrous structures. These colloidal suspensions of slender and wavy nanofibers exhibited a yield stress and a shear thinning behavior at low and high shear rates, respectively. Both the shear yield stress and the consistency of these suspensions were power-law functions of the NFC volume fraction. We developed an original multiscale model for the prediction of the rheology of these suspensions. At the nanoscale, the suspensions were described as concentrated systems where NFCs interacted with the Newtonian suspending fluid through Brownian motion and long range fluid-NFC hydrodynamic interactions, as well as with each other through short range hydrodynamic and repulsive colloidal interaction forces. These forces were estimated using both the experimental results and 3D networks of NFCs that were numerically generated to mimic the nanostructures of NFC suspensions under shear flow. They were in good agreement with theoretical and measured forces for model colloidal systems. The model showed the primary role played by short range hydrodynamic and colloidal interactions on the rheology of NFC suspensions. At low shear rates, the origin of the yield stress of NFC suspensions was attributed to the combined contribution of repulsive colloidal interactions and the topology of the entangled NFC networks in the suspensions. At high shear rates, both concurrent colloidal and short (in some cases long) range hydrodynamic interactions could be at the origin of the shear thinning behavior of NFC suspensions.

Influence of the acid type in the production of chitosan films reinforced with bacterial nanocellulose.

Chitosan films reinforced with bacterial cellulose (BC) nanoribbons were studied to understand the influence of acid (acetic and lactic acids) on the reinforcing effect. For both acids, the maximum concentration of the reinforcing constituent was 5wt% with respect to the dry weight of chitosan. The infrared spectra, mechanical properties, morphology and antimicrobial activity of the films were analyzed. The results showed a difference between the acids in their behavior and effect on the reinforcement, with a tensile strength of 12.3MPa for the acetic acid films and 3.3MPa for the lactic acid films. Additionally, the bacterial inhibition tests were shown to be positive for the lactic acid films and negative for the acetic acid films. Therefore, exchanging the acid used in these films may be desirable for certain applications.

Silica encapsulation by miniemulsion polymerization: distribution and localization of the silica particles in droplets and latex particles.

The impact of including hydrophobically modified silica on the morphology of miniemulsified monomer mixtures and that of the resulting polymer particles was investigated, with emphasis placed on the distribution and localization of the inorganic phase. Silica nanoparticles with diameters of 20 and 78 nm were first modified with γ-methacryloxypropyl trimethoxysilane (γ-MPS) to favor their dispersion in methyl methacrylate (MMA)/n-butyl acrylate (BuA) and mixtures of varying MMA to BuA weight ratios. The monomer-silica dispersions were then emulsified by ultrasonication, and the resulting silica-loaded droplets were examined using cryo-transmission electron microscopy (cryo-TEM). This represents the first time such silica-loaded nanodroplets were examined in this way. The results of the cryo-TEM show that whereas the silica particles could easily be dispersed in MMA or a mixture of MMA and BuA to produce stable dispersions, the emulsification step promotes the (re)localization of the silica at the oil-water interfaces. It was also shown that not all droplets are equal; some droplets and particles contain no silica whereas others contain many silica particles. After the subsequent polymerization step, the silica was buried inside the latex particles.

Raster microdiffraction with synchrotron radiation of hydrated biopolymers with nanometre step-resolution: case study of starch granules.

X-ray radiation damage propagation is explored for hydrated starch granules in order to reduce the step resolution in raster-microdiffraction experiments to the nanometre range. Radiation damage was induced by synchrotron radiation microbeams of 5, 1 and 0.3 µm size with ∼0.1 nm wavelength in B-type potato, Canna edulis and Phajus grandifolius starch granules. A total loss of crystallinity of granules immersed in water was found at a dose of ∼1.3 photons nm(-3). The temperature dependence of radiation damage suggests that primary radiation damage prevails up to about 120 K while secondary radiation damage becomes effective at higher temperatures. Primary radiation damage remains confined to the beam track at 100 K. Propagation of radiation damage beyond the beam track at room temperature is assumed to be due to reactive species generated principally by water radiolysis induced by photoelectrons. By careful dose selection during data collection, raster scans with 500 nm step-resolution could be performed for granules immersed in water.

Biodistribution of intravenously administered amphiphilic beta-cyclodextrin nanospheres.

Amphiphilic beta-cyclodextrin (betaCDa) nanospheres (mean diameter 90-110 nm) prepared by the solvent displacement method were developed as a colloidal drug delivery system. In order to survey the fate of these nanoparticles, the amphiphilic beta-cyclodextrin was first iodinated by a two-step procedure involving iodination of the primary face followed by an acylation of the secondary face. After radiolabeling of this derivative with (125)I, nanospheres made of betaCDa/betaCDa (125)I were formulated. After a single intravenous injection of labeled nanoparticles in mice, the organ distribution was analyzed from 10 min to 6 days. A rapid clearance of (125)I-labeled betaCDa nanospheres from the blood circulation to the mononuclear phagocyte system was visualized by non-invasive planar imaging study. Radioactivity measurements in organs showed that the nanospheres mainly concentrated in the liver and the spleen where 28 and 24% of the radioactivity per gram of organ was, respectively, found 10 min after injection. At the opposite, the blood activity was low at that time and become negligible thereafter. Finally, the fact that no particular sign of toxicity is observed in injected animals should be emphasized since it is the first report on intravenous administration of betaCDa nanoparticles.

Miscellaneous nanoaggregates made of beta-CD esters synthesised by an enzymatic pathway.

Various beta-cyclodextrin (beta-CD) fatty esters with different chain lengths (C4-C14) were synthesised by transesterification of beta-cyclodextrin by vinyl fatty ester using thermolysin in DMSO. For each cyclodextrin derivatives, two batches of synthesis were realized. The ability of these derivatives to form nano-organized systems was investigated through the solvent displacement technique. During the formulation step, the effects of the initial concentration of beta-CD fatty esters in the organic phase and that of the final volume of the aqueous non-solvent phase were studied. Except for the beta-CD C4 ester, the transesterified beta-CD derivatives led to measurable nanoparticles. Cryo-electron microscopy images showed a significant morphological variability. Spherical, rod-like or more irregularly-shaped nano-objects were observed with either matricial or lamellar structures. A statistical analysis by a two-way ANOVA was computed for each class of beta-cyclodextrin esters in order to determine the effects of batch and formulation on the final size of nanoparticles.

Structural aspects in semicrystalline samples of the mannan II family.

A series of samples having the mannan II character were prepared by either (i) desincrusting stems of Acetabularia crenulata, or (ii) acetylating these stems, followed by dissolution and recrystallization under deacetylation conditions, or (iii) recrystallizing at low temperature the alkali soluble fraction of ivory nut mannan. The samples were characterized by transmission electron microscopy, X-ray and electron diffraction analysis together with (13)C CP/MAS NMR spectroscopy. Whereas the A. crenulata stems consisted of a mixture of mannan I and mannan II, the recrystallized samples were all of the hydrated mannan II family and occurred in a ribbonlike morphology where the mannan chains were organized with their molecular axis perpendicular to the ribbon long axis. The recrystallized ivory nut mannan samples presented X-ray and electron diffraction diagrams, together with (13)C solid-state NMR spectra recorded at 95% RH, different from those of recrystallized A. crenulata recorded under the same RH conditions. They corresponded therefore to a new allomorph of the mannan II family. Despite this difference, when the recrystallized samples were in an aqueous environment, they revealed an additional well-defined perhydrated phase, which showed the same (13)C solid-state NMR spectrum for both samples. As this phase, which gave 6-band NMR spectra with narrow line-width and low T1, had no counterpart in X-ray diffraction, it was attributed to specific amorphous segments of mannan chains, gaining some mobility when swollen in water. When the samples were totally dried, their NMR spectra lost their resolution, thus indicating the role played by water for the structural organization of the crystalline and amorphous components of mannan II.

Split crystallization during debranching of maltodextrins at high concentration by isoamylase.

Debranching and crystallization occurring during the enzymatic treatment of 25% (w/v) aqueous solutions of maltodextrins by isoamylase at 52 degrees C were studied. The morphology as well as the crystal and molecular structures of the precipitates formed at different stages of the reaction were characterized. Two types of resulting products, differing in terms of structure and morphology, were evidenced. A loose B-type network, containing linear and branched chains of highest molecular weight, was mainly formed during the first 12 h of reaction, whereas aggregates of A-type lamellar crystals, made of short linear chains, were predominantly obtained between 12 and 48 h. The aggregation behavior as a function of temperature and molecular weight distribution of such substrates was discussed and compared to that of related starch products.

Long-term shelf stability of amphiphilic beta-cyclodextrin nanosphere suspensions monitored by dynamic light scattering and cryo-transmission electron microscopy.

Amphiphilic beta-cyclodextrins (betaCDa) were synthesized by statistically grafting hexanoyl carbon chains on the secondary hydroxyl functions of the betaCD glucopyranosyl units. The obtained derivative was used to prepare submicronic colloidal nanosphere suspensions using a nano-precipitation method. The fresh suspensions contained particles with a diameter ranging from 60-100 nm. Taking into account that the physical stability of colloidal systems remains one of the major problems which can restrict their use in pharmaceutical particulate carrier formulations, the long-term stability of the aqueous nano-dispersions was investigated. Two complementary characterization methods, namely dynamic light scattering and cryo-transmission electron microscopy, were used to control the size distribution and morphology of the nanospheres during storage. The zeta potential was measured as well. An unexpected good physical stability of the suspensions after 3 year storage at room temperature was observed. This behaviour appears to be related to the small size and structural organization of the nanoparticles. The mean diameters determined from light scattering experiments are consistent with those measured from electron micrographs. The slight difference between the values obtained by both methods is discussed.

Biosynthesis of (1-->3)-beta-D-glucan (callose) by detergent extracts of a microsomal fraction from Arabidopsis thaliana.

The aim of this work was to develop a biochemical approach to study (1-->3)-beta-D-glucan (callose) biosynthesis using suspension cultures of Arabidopsis thaliana. Optimal conditions for in vitro synthesis of callose corresponded to an assay mixture containing 50 mM Mops buffer, pH 6.8, 1 mM UDP-glucose, 8 mM Ca2+ and 20 mM cellobiose. The enzyme was Ca2+-dependent, and addition of Mg2+ to the reaction mixture did not favour cellulose biosynthesis. Enzyme kinetics suggested the existence of positive homotropic cooperativity of (1-->3)-beta-D-glucan synthase for the substrate UDP-glucose, in agreement with the hypothesis that callose synthase consists of a multimeric complex containing several catalytic subunits. Detergents belonging to different families were tested for their ability to extract and preserve membrane-bound (1-->3)-beta-D-glucan synthase activity. Cryo-transmission electron microscopy experiments showed that n-octyl-beta-D-glucopyranoside allowed the production of micelle-like structures, whereas vesicles were obtained with Chaps and Zwittergent 3-12. The morphology and size of the (1-->3)-beta-D-glucans synthesized in vitro by fractions obtained with different detergents were affected by the nature of the detergent tested. These data suggest that the general organization of the glucan synthase complexes and the properties of the in vitro products are influenced by the detergent used for protein extraction. The reaction products synthesized by different detergent extracts were characterized by infrared spectroscopy, methylation analysis, 13C-NMR spectroscopy, electron microscopy and X-ray diffraction. These products were identified as linear (1-->3)-beta-D-glucans having a degree of polymerization higher than 100, a microfibrillar structure, and a low degree of crystallinity.

Structural data on the intra-crystalline swelling of beta-chitin.

The intra-crystalline swelling of the highly crystalline beta-chitin from Tevnia jerichonana was investigated by X-ray crystallography and Fourier transform infrared (FTIR) spectroscopy, using hydrogenated and deuterated hydrochloric acids as swelling agents. Three levels of swelling were identified that could be defined as inter- and intra-sheet swelling. A moderate and reversible swelling in water and methanol gave crystalline beta-chitin cystallosolvates, namely dihydrate and methanolate, respectively. In these, an inter-sheet swelling was observed, corresponding to an expansion of only the b parameter of the unit cell of beta-chitin. Under these swelling conditions, the use of deuterated reagents had no effect on the amide N&z.sbnd;H⋯O&z.dbnd6;C hydrogen bonds that hold the structure of beta-chitin together, but only induced a partial and reversible deuteration of the chitin hydroxymethyl groups. A more severe swelling - but still reversible - occurred with 6 N HCl or DCl, which converted the crystals of beta-chitin into a paracrystalline gel-like product resulting from inter-sheet+intra-sheet swelling. With this acid strength, the deuteration pattern indicated that a fraction of the amide hydrogen bonds was broken and became susceptible to an irreversible deuteration. A very severe and irreversible swelling occurred with 8 N HCl or DCl. In that case, the inter- and intra-sheet swelling was extensive to the point where all memory of the parallel-chain beta-chitin was lost. In addition, this swelling was accompanied by a drastic and rapid depolymerization. The treatment with 8 N HCl led invariably to crystalline alpha-chitin when the samples were neutralized.

Ultrastructural aspects of phytoglycogen from cryo-transmission electron microscopy and quasi-elastic light scattering data.

Phytoglycogen particles extracted from the sugary maize mutant su 1 and dispersed in water were studied using transmission electron microscopy (TEM) and light scattering. Dried specimens were either negatively stained with uranyl acetate or shadowed with W/Ta. Frozen-hydrated unstained particles embedded in a thin film of vitreous ice were also observed using cryo-TEM. The particles exhibited a spheroidal shape, with a diameter ranging from 30 to 100 nm. Some of them presented a multilobular morphology and appeared to be formed by smaller subunits, 20-30 nm in diameter, resembling the described beta-particles for animal glycogen. The diameter of stained and ice-embedded particles was measured from electron micrographs. The corresponding size distribution histograms showed that the average weight diameter of ice-embedded particles was higher than that of stained ones. In the latter case, a shrinkage of the particle was believed to occur during the drying process. Light scattering experiments confirmed the diameter of ice-embedded particles and indicated that they could be considered as uniformly dense spheroidal objects.

Single crystals of inulin.

Lamellar crystals of inulin were grown by crystallizing sharp fractions of low molecular weight inulin from dilute aqueous ethanol solutions. The crystals were analyzed using three-dimensional electron diffraction and X-ray powder diagrams. Two crystalline polymorphs were observed, depending on the hydration conditions: a hydrated form which indexed on an orthorhombic unit cell, with space group P2(1)2(1)2(1) and with cell dimensions of a = 1.670 nm, b = 0.980 nm and c (chain axis) = 1.47 nm, together with a pseudo-hexagonal semi-hydrated form with unit cell parameters a = 1.670 nm, b = 0.965 nm and c (chain axis) = 1.44 nm. These parameters, together with the density data, indicate that inulin crystallizes along a pseudo-hexagonal six-fold symmetry with an advance per monomer of 0.24 nm. The difference between the hydrated and the semi-hydrated unit cells does not seem to correspond to any change in the conformation of inulin, but rather to a variation in water content.