N Lopez Ferber

D Pham

• Q Falcoz

• A Meffre

N Tessier-Doyen

• A Nzihou

• V Goetz

N Lopez Ferber
email: n.lopezferber@gmail.com

Development of a Thermal Energy Storage Pressed Plate Ceramic Based on Municipal Waste Incinerator Bottom Ash and Waste Clay

Keywords: Thermal energy storage material, Waste-based ceramic, Sintering, Municipal waste incinerator bottom ashes

Pressed plates ceramics made of gross-milled bottom ashes and waste clay, were made using technologies available in the building bricks and tiles industry, to ease production upscaling at low-cost. These sintered ceramics are intended for use as a high-temperature thermal energy storage material. They represent an alternative to the waste-based petrurgic ceramics previously developed for this application. Post-treated incinerator bottom ashes from a commercial incinerator were collected, characterized and processed to form ceramic materials, using clay as a binder. Ashes were milled, dried, and mixed with various amounts of an illitic clay (produced as washing mud by a quartz quarry in proportions from 20 to 70% dry weight) prior to uniaxial pressing (12 × 5 × 1 cm slabs) and firing at various temperatures, ranging from 1050 to 1125 °C. The sintered samples have been characterized in terms of volumic mass, mechanical strength, thermal capacity and thermal conductivity. Their mineral structure has also been studied. The resulting sintered ceramics exhibit relatively high mechanical resistance and low thermal conductivity, along with moderate volumic mass. These properties allow envisioning the use as filler material for thermocline thermal storage systems (structured beds), and could be interesting for further work regarding applications in the construction field (bricks, tiles, pavements…).

Introduction

Municipal waste incineration is a common technique for household waste (usually high carbon content) treatment. It consists in the combustion of this waste at high temperature (900-1200 °C), therefore reducing their volume by up to 90%, decreasing land use compared to direct household wastes landfilling [START_REF]Management of Bottom Ash from WTE Plants-An Overview of Management Options and Treatment Methods[END_REF][START_REF] Amorce | Etat des lieux de la gestion des mâchefers en France[END_REF]. Most incinerators recover the heat generated to produce industrial steam or electricity. Considered as renewable energy, it represents around 1.7 TWh e in 2015 in France (comparable to biogas, or solid biomass) [START_REF]Chiffres clés des énergies renouvelables -Édition 2016[END_REF].

The main solid byproduct of incineration is Municipal Waste Incineration Bottom Ash (MWIBA), representing around 25% of the total input weight, the others being fly ash, air pollution control residues and dusts [START_REF]Waste-to-Energy Stateof-the-Art-Report[END_REF]. MWIBA is a complex material, with composition varying in time and space, as revealed by the literature (Table 1).

MWIBA is typically classified as "non-dangerous waste" and is often landfilled (most of the 12 Mt produced annually in Europe is landfilled [START_REF]Management of Bottom Ash from WTE Plants-An Overview of Management Options and Treatment Methods[END_REF][START_REF] Amorce | Etat des lieux de la gestion des mâchefers en France[END_REF]). Other countries extensively use it although some countries extensively use it as a filler for road sub-base construction. For example, around 80% of the 3 Mt of bottom ashes generated yearly in France are used as road sub-base filler [START_REF] Amorce | Etat des lieux de la gestion des mâchefers en France[END_REF] after several treatment steps involving milling, sieving, ferrous metals removal (magnetic separation) and non-ferrous metals removal (Eddy currents), and ageing (stabilization of leaching properties through calcium oxide carbonation) [START_REF] Sétra | Acceptabilité environnementale de matériaux alternatifs en technique routière -Les mâchefers d'incinération de déchets non dangereux (MIDND)[END_REF].

Being mostly a mixture of mineral compounds, namely alumina, hematite, silica and alkali oxides in the form of a heterogeneous gravel, MWIBA can be compatible with ceramic materials production [START_REF] Bethanis | Properties and microstructure of sintered incinerator bottom ash[END_REF][START_REF] Rambaldi | The recycling of MSWI bottom ash in silicate based ceramic[END_REF]. It can represent a new valorization path providing new feedstock for ceramic industry and saving resources and environmental impacts related to clay and sand extraction. An interesting application calls for high temperature storage systems, using ceramics as sensible thermal storage material in thermocline packed bed or structured bed systems [START_REF] Lopez Ferber | Flexibility and robustness of a hightemperature air / ceramic thermocline heat storage pilot[END_REF][START_REF] Zanganeh | High-temperature thermal energy storage for concentrated solar power with air as heat transfer fluid[END_REF]. Some authors worked on such waste-based ceramics, using molten asbestos containing wastes [START_REF] Py | Recycled material for sensible heat based thermal energy storage to be used in concentrated solar thermal power plants[END_REF], metallurgical slags [START_REF] Dejean | Valorisation de laitiers sidérurgiques comme matériaux de stockage thermique pour procédés énergétiques durables[END_REF] or coal fly ash [START_REF] Kere | Applicability of thermal energy storage recycled ceramics to high temperature and compressed air operating conditions[END_REF], mostly using the petrurgic method implying casting and controlled cooling from molten state, a technique that is not considered as easily transferable to industry and massproduction. Sintering alumino-silicate industrial byproducts appears to be preferable, due to the higher production capabilities of the sintering production pathway [START_REF]Document de référence sur les meilleures techniques disponibles -Fabrication des céramiques[END_REF].

Research projects have been carried out regarding the sintering behavior of MWIBA, but being focused on the sintering or the crystallization behavior of materials containing variable proportions of MWIBA rather than industrial production considerations; the processes and materials they proposed might not be suited for mass-production at reasonable cost (micronic wet-milling [START_REF] Bethanis | Properties and microstructure of sintered incinerator bottom ash[END_REF], casting at molten state or sinter-crystallization from a glass [START_REF] Schabbach | Integrated approach to establish the sinter-crystallization ability of glasses from secondary raw material[END_REF], valuable additives like high-grade kaolin [START_REF] Taurino | New fired bricks based on municipal solid waste incinerator bottom ash[END_REF] and so on).

In this work, we have chosen another perspective aiming to develop a 100% waste-based ceramic, using production protocols as simple and widely available as possible. The strategy chosen focuses on the simplicity of the production process, because the amount of materials required for sensible heat storage technology could be important, in both the sectors of industrial energetic efficiency and concentrated solar power techniques [22], implying that the developed material should be compatible with mass-production at low cost. This process is based on the uniaxial compression shaping technique, used for example in the tile industry (and in most of the literature regarding MWIBA use as ceramic production feedstock). The ceramics obtained should exhibit properties suitable for sensible heat thermal storage up to 1000 °C, under air, in a thermocline thermal storage system. Hence, the desired properties of the resulting material are as follows:

• Maximal volumetric thermal capacity (ρ x Cp), implying maximizing densification during sintering. This is especially important for thermal storage applications, since it would increase thermal storage capacity per unit volume. • High thermal conductivity, that is beneficial for storage behavior in thermocline air/ceramic storage systems, ensuring that the Biot number is significantly lower than 1 (heat transfer between the air and the solid does not generate a significant inta-particle thermal gradient). This could also be achieved by decreasing the size of the particle, at the cost of increased pressure drop. • Satisfying mechanical strength, measured as compressive strength (> 20 MPa). A high compressive strength for a thermal storage material will allow increasing the weight of the bed without damaging the pieces. • Thermal stability over a large range of temperatures that can be encountered in thermal energy storage (up to 800-1000 °C), under air • Satisfying thermomechanical behavior, measured as the Coefficient of Thermal Expansion (CTE). This CTE should be as low as possible, or at least present predictable variations with temperature, to avoid increasing mechanical stress during use.

This paper will mostly focus on the influence of the clay to bottom ash ratio in the formulation and firing temperature on the resulting properties of the sintered samples.

Materials and Methods

Materials Processing

Sampling 450 kg of an aged MWIBA have been collected in the maturation platform of a commercial incinerator in southern France using coning and quartering method as defined by the UIPAC [START_REF] Iupac | Compendium of Chemical Terminology-Gold Book[END_REF], after post-treatment process (magnetic removal of ferrous and non-ferrous metals, ageing). The bottom ashes complied with the requirements for homologation as "MWIBA gravel 0-40 mm" as defined in the Setra guide for MWIBA management [START_REF] Sétra | Acceptabilité environnementale de matériaux alternatifs en technique routière -Les mâchefers d'incinération de déchets non dangereux (MIDND)[END_REF]. Like most MWIBA, it consists in a mixture of ashes, sand, glassy material, metallic and ceramic fragments and residual unburnt organic compounds [START_REF] Becquart | Monotonic aspects of the mechanical behaviour of bottom ash from municipal solid waste incineration and its potential use for road construction[END_REF].

Milling

The particle size distribution (PSD) of as-collected MWIBA was not compatible with shaping and firing protocols, so the MWIBA had to be milled. The goal being to assess the feasibility of MWIBA ceramics production in building bricks and tile industry, we disregarded extensive wet milling techniques. These techniques, although relevant to produce thin and homogeneous powders, seemed too complicated and not easily transferable to industrial settings, so an easier method has been favored. MWIBA has been dried at 105 °C for one day, before being milled with a tungsten jaw mill. MWIBA has been milled with three passes inside the jaw mill, the jaw being enclosed between each pass, from a 10 mm gap to a 1 mm gap. The powder obtained has then be sieved at 500 µm and the passing fraction has then been used for the trials. Milling several hundred grams with this technique requires approximately 45 s, and can be up-scaled reasonably easily due to the great availability of jaw mills in the mineral industries and the fact that these milling devices easily allow continuous production.

Shaping

Shaping the green bodies before firing being an important step for any applications, adding a binding agent to the MWIBA was considered, in order to give the mixture high enough plasticity to undergo shaping protocols. Thus, a wasted clay was used as binder. This clay is a by-product of a quartz producing company located in southern France, with a generation rate of 100 t/day. It is a fine powder (d 50 = 16 µm) of an illitic clay, obtained by flocculation/ decantation of a washing mud. For these trials, we used a hydraulic press, to produce 12 × 5 × 1 cm slabs, after adding water to the powders in a rotary plate device (granulation). The added clay represents 20, 30, 40 or 70% of the formulation dry weight.

Firing

After being dried at 105 °C for 12 h, green bodies have been fired in electrical furnaces, with a heating rate of 5°C/ min from room temperature to 500 °C, then 2°C/min from 500 °C to the maximum temperature, with isothermal firing duration of 2 h. The furnaces are not equipped with an active cooling system, implying cooling durations of about 10 h. The different stages of the process are illustrated by the Fig. 1.

Raw Material Characterization

Chemical composition of the raw materials has been characterized by ICP-AES. Samples have been milled and sieved at 40 µm. The clay and the MWIBA have been dissolved into a mix of nitric and fluorhydric acid into a micro-wave oven (Anton Paar) under heat. The samples have been heated up to 240 °C and 60 bars in ten minutes, with 40 min dwell time. After total dissolution, the samples have been characterized using a Perkin-Elmer Optima 8300 ICP device.

PSD of the powders have been measured using a Malvern Mastersizer 3000 granulometer, on carefully dried powders, for a particle size range of 0-1000 µm.

Loss On Ignition has been measured for the raw powders by comparing the mass of a sample, before and after being heated up to 1000 °C for 2 h. This characteristic of the powders also had been confirmed with TG-DSC (Labsys Evo from Setaram, with 3L/h air, and a heating rate of 5°C/min from room temperature to 1100 °C).

The mineral phases present in the raw materials have been identified with X-Ray Diffraction, using a Philips PANalytical X'pert Pro MPD device (copper emitter, K α = 1543 Å), from 2θ = 10° to 2θ = 80°.

Characterization of the Sintered Samples

The goal being to assess the potential of these ceramics for thermal storage at high temperature, the main properties investigated are as follows:

-Volumic mass, expressed in kg/m 3 or in relative density, has been measured with the Archimede method [START_REF]Archimedes' Principle[END_REF], comparing dry and immersed sample apparent weight. -Thermal capacity, expressed in J/(g.K), has been measured by a modular DSC (Q200 from TA), with a 50 ml/ min nitrogen flow, at room temperature and 390 °C, 390 °C being close to the average temperature of use. -Thermal diffusivity has been measured with a Laser Flash Analysis device from Netzsch (LFA 457) from room temperature to 1000 °C, with a heating ramp of 20°/min. The knowledge of the variations of specific heat with temperature allowed calculating the thermal conductivity of these samples. -Thermal stability in the temperature range of 0-1000 °C is of highest importance. The behavior of the sintered samples under thermal cycling has been investigated with DTA and TMA (Setsys device from Setaram). The coefficient of thermal expansion should also been as low as possible, or at least predictable so to avoid generating strains inside a bed of these materials.

-Compressive strength of the ceramics will set the limit to the system's size. Therefore, high compressive strength can be beneficial to this material. It has been measured by submitting samples to an uniaxial force, using a hydraulic press and increasing the load until sample's rupture, on small cylindrical samples. -The mineral phases present in the sintered samples have been identified with X-Ray Diffraction, using a Philips PANalytical X'pert Pro MPD device (copper emitter, K α = 1543 Å), from 2θ = 10° to 2θ = 80°.

Results and Discussion

Raw material Characterization

Composition and Particle Size Distribution

Both the clay and MWIBA are mostly composed of silica.

The clay has higher proportion of alumina and potassium oxides, whereas the MWIBA contains more sodium oxides (Table 2). These last oxides are known for their relevance as fluxing agents, and so might facilitate sintering.

The composition is on part with other MWIBA studied in the scientific literature, although this MWIBA in particular is richer in silicon oxide than most of the samples. Its "lower than usual" content in alumina could be explained by differences in waste-sorting policies.

The clay was not milled, and exhibits a Gaussian-like particle size distribution, centered on its d 50 of 16 µm. The milled MWIBA exhibits a completely different reparation of particle size, with an over-representation of coarse particles (> 100 µm), mixed with smaller particles. The d 50 of the milled MWIBA is 166 µm, superior of that's of the clay by an order of magnitude (Fig. 2).

This situation, where one of the powder is much coarser and with a sharper distribution and the other one is smaller with a broader PSD is favorable to maximal mixture quality and compacity of the green bodies, the smaller particles being able to fill the porosity between the coarser ones. A difference of particle size of around 1:10 is usually described as relevant for increasing green bodies compacity [START_REF] Rahaman | Ceramic Processing[END_REF]. The two powders comply with this requirement (Fig. 2), increasing the maximal compacity and homogeneity achievable for the green bodies.

Raw Materials Mineralogy

Both clay and MWIBA powders have been characterized in terms of mineral composition. The clay exhibits typical illitic clay phases, such as illite and kaolinite, associated with quartz and K-feldspath, (presumably microcline). The MWIBA powder exhibits typical quartz and calcite phases, associated with wollastonite and pyrite (Fig. 3). Pyrite is not usually reported in the literature as being present in bottom ashes, the iron being mostly present as oxide (hematite).

Raw Materials Thermal Behavior

MWIBA has been milled in a tungsten carbide ring mill and sieved at 50 µm to obtain a fine powder suitable for DSC-TG analysis. This sample exhibits a thermal behavior similar to those studied in literature (Fig. 4), namely:

• An endothermic phase between room temperature and around 250 °C, associated with mass loss, which is most often attributed to the vaporization of residual, bound and hydration water. • An exothermic phase between 300 and 500 °C, associated with mass loss. These peaks are presumably due to the combustion of unburnt organic matter and the release of occluded gases [START_REF] Bethanis | Properties and microstructure of sintered incinerator bottom ash[END_REF]. • A sharp endothermic peak around 700 °C, associated with a sharp weight loss. This is typically associated with calcite decarbonation (CaCO 3 → CaO + CO 2 ). The usual temperature for calcite decarbonation is around 900 °C, but this phenomenon appears to occur at lower temperature when calcite is found in MWIBA [14, 27, 28] • Above 1000 °C, the thermal behavior is more difficult to interpret, although partial melting can be observed, and sintering and crystallization phenomena occurs. At high temperature, some weight losses can be attributed to alkali metal sulfates dissociation [START_REF] Bethanis | Properties and microstructure of sintered incinerator bottom ash[END_REF].

The weight loss is higher than noticed during sintering tests, which is presumably due to concentration of volatile residual organic matter into the small-sized particles.

The clay exhibits very common thermal behaviour, with an endothermic peak around 100-150 °C associated with bounded water removal, followed by a sharp weight loss between 450 and 600 °C associated to kaolinite deshydroxylation and a 980 °C exothermic peak related to structural transformation of illite to mullite. This clay starts vitrifying at 1250 °C, a temperature where it reaches its maximum shrinkage (7%) (Information provided by supplier).

Sintered Samples Properties

Sixteen batches have been fired by combining four temperatures (1050, 1075, 1100 and 1125 °C), with four clay contents (20%, 30%, 40% and 70% dry weight), to investigate the influence of firing temperature and clay content on fired samples properties. From there, they will be named as follows: XXXX-YY with XXXX the firing temperature [°C] and YY the clay content [% dry weight].

Volumic Mass and Loss on Ignition

The volumic mass of fired samples has been measured with Archimede's method. The samples fired at 1050 and 1075 °C showed poor cohesion; indicating insufficient grain-bounding during firing. They also exhibited the lowest volumic mass. The samples fired at 1100 and 1125 °C are much more cohesive (which will be reflected by their compressive strength), and presented higher volumic mass (Table 3), thus have been selected for further characterizations. However, their losses on ignition are also superior, especially for the MWIBA-rich formulations, reaching up to 14% for the 1125-20 sample, which also exhibited significant closed porosity (Fig. 5). This value is unusually high, and could be related not only to combustion/pyrolysis/dissociation of certain compounds, but also to losses of material (dusts) during firing. This sample also exhibited traces of partial melting. The samples exhibiting the highest densities had tendencies to creep, but the dimensional changes are still reasonable. Shrinkage was not considered as crucial as other properties, and was not properly investigated, although it is estimated in the range of 5-12% depending on the sample. Only the samples fired at 1100 and 1125 °C had their properties further investigated due to their superior volumic mass, but also their superior cohesion.

Compressive Strength

Compressive strength of the samples fired at 1125 °C greatly exceeded those of samples fired at 1100 °C. Along with superior volumic mass, it revealed that an optimal firing temperature was around 1125 °C. The maximal compressive strength was reached for the 1125-20 sample, with around 65 MPa. Compressive strength was then reduced when increasing the clay content, although the 1100-70 and 1125-70 samples exhibited high compressive strength (even after being fired at 1100 °C). The difference between 1100 and 70 and 1125-70 compressive strength was significantly lesser than between 1100 and 20 and 1125-20 samples (Fig. 6). This could be explained by the fact that, clay and MWIBA powders being chemically different, the formed phases during firing might be different (in nature or prevalence), resulting in different properties. Another explanation relates to the fact that the benefit of having such different granulometry for the powders is conditioned by a balance between fines (clay) and coarse (mwiba) particles.

Thermal Properties

The volumetric specific heat is the product of volumetric mass and specific heat. The samples fired at 1125 °C exhibit higher densities, but the 1100-70 sample exhibit higher specific heat. However, the highest volumetric specific heat is obtained for 1125-20 sample. It is interesting to notice that the gain in volumetric specific heat between firing at 1100 and 1125 °C is more important for the 20% wt clay than for the other formulations (Fig. 7). The fact that this sample shows the higher compressive strength and the second density indicates that its operational conditions enabled satisfying sintering and interparticulate void filling. Thermal conductivity is low for every sample, not one reaching 1 W/(mK). Again, the samples sintered at 1125 °C exhibit higher properties than those sintered at 1100 °C. The gain in thermal conductivity is less important the higher the clay content.

Our goal being to use mainly MWIBA to produce the best thermal storage ceramic achievable, we choose to focus on 20 and 30% clay content formulation, with 1125 °C firing temperature. These ceramics are required to withstand thermal cycling without mineralogical modifications, and reasonably low thermal expansion, so their behavior when cycled from room temperature to 1000 °C has been investigated.

Thermal Stability

This ceramic material is supposed to store and deliver heat over long periods, and will undergo thermal cycling, up to 1000 °C, mostly under air. It should therefore be thermally stable between room temperature and 1000 °C, regarding both mineralogy and thermal expansion. Even if the samples have been fired at temperatures exceeding 1000 °C, it is necessary to verify that there will be no significant changes in structure and properties during use. The best samples (1125-20 and 1125-30) have been thermally cycled three times from 30 to 1000 °C under air, with 10°C/min heating ramp and 30 min isotherm in a TMA/TG-ATD device (TMA Setsys, from Setaram, TG-ATD Labsys from Setaram), to detect any transformations that might occur. It appears that, even if the heat flow signal varies and present hysteresis between the heating and the cooling phases, the curves are almost identical from one cycle to another, so it is reasonable to conclude that the sample did not undergo significant irreversible transformations (Fig. 8). The clear endothermic behavior can be attributed either to an increase in specific heat, or to a reversible phase change, which did not affect the thermomechanical behavior, thus not introducing concerning weaknesses when the samples are cycled.

The thermal behavior of the samples has been investigated with TMA, to monitor thermal expansion coefficient as a function of temperature. It appeared that 1125-20 and 1125-30 samples exhibited the exact same thermal dilatation coefficient for a given temperature (Fig. 9), reaching a value of 8 × 10 -6 /K at 1000 °C, similar to alumina [START_REF] Auerkari | Mechanical and physical properties of engineering alumina ceramics[END_REF]. The endothermic events detected on DTA, if attributed to phase transformation, did not generate disruptions in the variations of CTE.

Mineralogy

Samples fired at 1100 and 1125 °C, with 20, 30, 40% dry weight clay have been characterized in terms of mineral composition (Fig. 10). Overall, all samples exihibited the presence of the same mineral phases, namely Quartz, Clinoenstatite, Wollastonite and Albite. No new phases appeared depending on the clay ratio nor the firing temperature. Some samples exhibited low-level noise under 20°, typical linked to glassy phases. The XRD patterns of the samples reveal some interesting points:

• After firing at 1100 °C, the same mineral phases have been detected, with 20,30 and 40% wt clay samples, namely quartz, wollastonite, clinoenstatite and albite.

However, increasing the clay content seems to increase the signal for albite (perhaps favored by the higher alumina content of the clay) and quartz, while decreasing it seems to favor wollastonite (perhaps favored by the higher calcium content of MWIBA) and clinoenstatite (favored by the presence of magnesia, which is not contained into the clay). The fact that albite melts in these temperature ranges (1100-1120 °C) [START_REF] Anovitz | Dry melting of high albite[END_REF] can explain the higher density of samples sintered at 1125 °C, the resulting viscous phase allowing viscous flow sintering. However, the fact that Albite can show high thermal expansion with increasing temperature [START_REF] Anovitz | Dry melting of high albite[END_REF] could be partly responsible for the formation of closed porosity in the bulk.

Conclusions

Municipal Waste Incinerator Bottom Ashes have been milled and mixed with various amounts of a wasted illictic clay, to explore the technical feasibility of ceramic sintering, to be used for thermal energy storage material production.

The trials revealed the feasibility of pressed plates production, using only widely available production techniques. The samples fired at 1125 °C exhibited the best properties, whether on the mechanical or thermal characteristics, the samples containing 20, 40 and 70% wt clay reaching over 40 MPa for compressive strength, as well as exhibiting the highests thermal volumetric storage capacity. XRD analysis revealed that all samples fired at 1100 and 1125 °C contained the same mineral phases, the variations of properties could therefore be explained by variations of the relative proportions of those mineral phases, and quality of sintering (especially regarding porosity filling during firing).

The samples have been cycled thermally between room temperature and 1000 °C, and did not exhibit significant modification of their properties, allowing considering them reasonably stables.

However, the visible high level of closed porosity of the samples limits the volumetric heat capacity, and should therefore be addressed to increase their potential as thermal energy storage materials.

-Decreasing the particle size of MWIBA powder could be interesting, as it would allow higher specific surface of the powder, thus favor grain-bounding during firing. -Replacing the clay by another one exhibiting lower densification temperature could be an option, as in our case the MWIBA starts melting long before the maximum clay densification. -An optimization of powders granulometries prior to mixing could be relevant, using a Funk strategy. This consists in mixing powders with different particle size distribution, to reach an uniform repartition of particle size in a broad particle size range. This approach, currently used for concrete production [START_REF] Radix | Self-Compacting concrete: the role of the particle size distribution[END_REF] as well as refractory cement production [START_REF] Kalpakli | Effects of particle size distribution on the refractory properties and corrosion mechanism of ultra-low cement castables[END_REF] could greatly improve green bodies compacity, favoring densification and reducing bulk porosity after firing (as well as linear shrinkage).

Although this work focused on the development of a thermal energy storage ceramic to be used in thermocline storage system, the high compressive strength, associated with limited thermal conductivity and average volumic mass could be compatible with the requirements for building purposes. Regarding these applications however, more work would be required to limit shrinkage and creeping during firing operations, as well as investigating water absorption, frost resistance and pollutant leaching behavior over time.

Fig. 1

 1 Fig. 1 Process steps. From left to right: raw MWIBA, milled MWIBA, granulated powder mix, green body, fired sample

Fig. 2 aFig. 3 a

 23 Fig. 2 a Particle size distribution of milled MWIBA (open circle) and clay (filled circle), b cumulative passing fractions of milled MWIBA (open circle) and clay (filled circle)

Fig. 4

 4 Fig. 4 Thermal behaviour (red) and weight loss (black) up of MWIBA from room temperature to 1100 °C. (Color figure online)

Fig. 5 Fig. 6

 56 Fig. 5 Sintered plates samples (40 and 20% wt clay on the left and right respectively) fired at 1125 °C. The closed porosity of the 1125-20 sample is obvious

Fig. 7 aFig. 8

 78 Fig. 7 a Thermal volumetric capacity at room temperature (squares) and 390 °C (dots), for samples fired at 1100 and 1125 °C, using 20% (filled blue circle), 30% (filled red circle), 40% (filled black circle) and 70% (filled brown circle) wt clay. b Thermal conductivity at

  The behavior of MgO during firing might need further investigation because, although being present at low concentrations in MWIBA, MgOcontained mineral phases are ubiquitous in works regarding MWIBA, be it clinoenstatite (in the present work) or diopside [11, 27, 30]. • After firing at 1125 °C, the peaks on the XRD pattern are similar. No new phase appeared, although the peaks are slightly narrower, which could indicate better crystallization. The fact that the low-level noise at low angles is lower for the samples fired at 1125 °C could imply better crystallization as well. The superior properties of the 1125 °C samples are therefore not related to the crystallization of new phases, but to crystal growth, densification and relative proportions of the different mineral phases.

Fig. 9 Fig. 10 a

 910 Fig. 9 Thermal expansion as a function of temperature, for 1125-20 (filled blue circle) and 1125-30 (filled red circle) samples. (Color figure online)

Table 1

 1 Composition of several MWIBA samples studied in the literature

	Reference	Vu et al. [5] Bethanis et al. [6]	Barbieri et al. [7]	Schabbach et al. [8]	Aloisi et al. [9] Appendino et al. [10]	Bourtsalas et al. [11]	Barbieri et al. [12]
	Analysis	ICP AES	ICP AES	XRF	ICP	XRF	AAS	XRD	ICP
	Compound (% dry weight)
	SiO 2 CaO	31.1 10.0	40.1-42.1 18.4-20.0	46.7 26.3	30.3-35.1 23.1-26.1	40.9 12.2	42.5-40.9 16.4-12.2	30.4 26.8	45.1 18.8
	Al 2 O 3 Na 2 O MgO	2.57 2.10 0.28	9.92-14.2 2.60-2.75 1.79-1.90	6.86 4.62 2.22	13.0-13.6 1.94-2.44 2.83-3.73	13.6 10.9 3.1	17.6-13.6 12.9-10.9 2.3-3.1	11.5 3.32 2.64	9.73 4.64 2.20
	Fe 2 O 3 TiO 2 P 2 O 5 SO 3 K 2 O CrOx	4.36 0.83 2.07 0.60 0.86 0.90	6.20-6.60 1.00-1.14 1.34-1.44 -0.86-0.92 -	4.69 0.77 -2.18 0.89 -	10.0-11.5 1.07-1.71 1.96-1.90 -0.94-1.45 -	5.7 1.2 --1.8 -	5.7-5.7 2.5-1.2 --1.8-1.8 -	13.1 1.39 2.18 3.05 1.33 -	3.84 0.93 --1.29 -
	MnO	-	0.08	-	0.18-0.17	-	0.1-0.1	0.31	-
	LOI (% dry wt)	5.21	-	11	-	-	-	-	-

Table 2

 2 Chemical composition of clay and MWIBA (ICP-AES)

	Compound (%wt)	Clay	MWIBA
	Al 2 O 3 SiO 2 CaO	27.1 53.7 2.3	6.2 58.7 5.3
	Fe 2 O 3 K 2 O MgO	5.9 5.2 -	6.0 1.8 2.1
	Na 2 O SO x LOI (1000 °C) (% dry wt)	0.8 -4.9	7.7 1.0 5.2
	d 50 (µm)	16	166 (milled)

Table 3

 3 Fired density and LOI of the sintered samples

	Sample	Relative Density (g/cm 3 )	LOI (% dry weight) (%)
	1050-20	1.56	-2
	1050-30	1.69	-3
	1050-40	1.65	-3
	1050-70	1.77	-3
	1075-20	1.65	-3
	1075-30	1.74	-4
	1075-40	1.58	-5
	1075-70	1.83	-4
	1100-20	1.72	-5
	1100-30	1.73	-2
	1100-40	1.60	-3
	1100-70	1.83	-3
	1125-20	2.14	-14
	1125-30	2.02	-3
	1125-40	1.80	-6
	1125-70	1.82	-4

Acknowledgements This work was supported by the Programme "Investissements d'avenir" (Investment for the Future) of the Agence Nationale de la Recherche (National Agency for Research) of the French State under award number ANR-10-LABX-22-01-SOLSTICE. The author would also like to thank Eco-Tech Ceram for their scientific, material and financial support and the SPCTS CNRS UMR 7315 for their support.


Jianming Yang
email: jianming.yang@unistra.fr

Patrice Twardowski

Philippe Gérard

Joël Fontaine

Design of a large field-of-view see-through near to eye display with two geometrical waveguides

A novel waveguide near to eye display (WGNED), with new in-coupling and propagation subsystems, is proposed for the first time to enlarge the vertical field-ofview (FOV) and the vertical size of the eye box. Two waveguides are stacked, one is for in-coupling and the other for out-coupling. A freeform prism is used to correct the aberrations. These components are combined together to form the WGNED. We have simulated such a system; as a result, we show that it achieves a FOV of 30° Horizontal (H) ×60° Vertical (V) and an eye box of about 15 mm (H) ×12 mm (V). The modulation transfer function (MTF) of the system is larger than 0.3 at 33 lp/mm and the distortion is smaller than 5%.

According to the combiner type, NEDs can be separated into two families [16]: one is free space combiner that has no capability to expand pupil size, the other is waveguide combiner which can expand pupil size without increasing Lagrange invariant. Therefore, the use of waveguide can reduce the design difficulties and probably is the most promising approach. Waveguide technology can be classified into two types: geometrical waveguides and holographic waveguides. Both of them consist of input-coupling and output-coupling subsystem. Lumus company employs a reflective mirror for the input-coupling optics and an array of partially reflective mirrors (PRMA) for the outputcoupling optics [18]. The PRMA is specially designed to have the right reflectance for different wavelengths and angles. Optinvent company uses small cascaded mirrors as out-coupling subsystem because the focus point of eye is far from the mirrors [19]. Microsoft [2], Konica Minolta [20], Sony and BAE Systems [21,22] corporations have used diffractive optical elements (DOEs) or holographic optical elements (HOEs) to develop their WGNED. However, having large FOV in both horizontal and especially in vertical direction is a difficult problem in WGNEDs. Horizontally, the FOV is limited by three parameters: the total internal reflection angle of the material of the waveguide, the expanded pupil size and the distance between each reflection inside the waveguide. Normally, horizontal FOV reaches around 20° inside the waveguide and 30° in air. As light from a pupil propagates along the waveguide divergently, vertical FOV is greatly dependent on the pupil size in this direction in order to reach the eye. It is about 9° with a pupil size of 8 mm from the equation proposed in [14]. In order to enlarge the FOV vertically, Amitai [14] proposed a twodimensional aperture expanding method using cascaded mirrors. Using the same concept Eisen et al. [23] proposed a system with three holographic gratings for in-coupling light into the waveguide and expanding the eye box along orthogonal directions. Microsoft corporation has also adopted this concept to develop HoloLens [2]. However, the FOV does not exceed 40°. Cheng [15] proposed a two-pupil design by combining the freeform prism and waveguide together to address this problem. Using this method, Han [24] designs a similar system with a 18° (H) × 24° (V) FOV. In this paper we introduce a new method that can increase the vertical FOV to 60° and enlarge the vertical size of the eye box to 12 mm. Using cascaded mirrors as Lumus waveguide type, the horizontal size of the eye box can be expanded to 15 mm. The total FOV is then 30° (H) x 60° (V) with a total eye box of 15 mm (H) × 12 mm (V), which is, to our knowledge, the largest FOV among the published WGNEDs.

Fig. 1 shows the structure of the system and the ray path inside. Two waveguides are stacked up with a small air gap so that light can propagate inside the two waveguides independently. The light emitted by the micro-display is coupled into the first waveguide by the in-coupling subsystem. Light propagates inside the first waveguide until reaching a cylindrical mirror at the end of the waveguide. In the area near the mirror, the two waveguides are combined together. Then, the light reflected by the cylindrical mirror is coupled into the second waveguide. Using existing method such as hologram or cascaded mirrors, the light is coupled out from the second waveguide. The in-coupling system consists of a prism with four different optical freeform surfaces (from S2 to S5) and a lens made of a freeform surface (S1) and a plane surface. This complex prism gives more freedom to correct aberrations. Moreover, it can be fabricated in one piece. As the FOV in horizontal direction is limited to 30° in air, the total light section is small; as a consequence, S2 and S4 surfaces can be separated. The in-coupling pupil has a rectangular shape with a size of 1.9 mm (H) × 12 mm (V) which acts as a stop aperture and then greatly reduces the stray light. Pupil size is enlarged by adding vignetting along z direction as shown in Fig. 1

(b) and (c).

The center O of the in-coupling pupil, shown in Fig. 2(a), is the center of the cylindrical mirror and also the center of the cylindrical edge of the air gap. After the reflection, the chief ray of each field will go backward and converge to a point. The associated propagating length is the radius of the cylindrical mirror. The position of the eye should be near this point, so that each field can be seen as shown in Fig. 1(c). The focal property of light after the in-coupling pupil is different in zx plane and yx plane because of the cylindrical mirror which is shown in Fig. 2. We suppose that the radius of the mirror is R. In the case of paraxial approximation, rays converge to the focal plane of the cylindrical mirror which is located at R/2 from the center in zx plane as shown in Fig. 2(a). Then rays are reflected by the cylindrical mirror and become a parallel beam that is coupled into the second waveguide. However, as the cylindrical mirror has no optical power in yx plane, the light should be a parallel beam after the in-coupling pupil as shown in Fig. 2(b). To compensate the large optical power caused by the cylindrical mirror, we use a freeform prism and a lens to correct aberrations, in which all the surfaces are XY polynomials. The optical power in yx plane is mainly given by the mirror S3 (Fig. 1(a)). The advantage of this design is a large pupil size (12 mm) in z direction with high image quality. Using the in-coupling system mentioned above, the beam then becomes parallel in both directions after the reflection from the cylindrical mirror. Finally, it is coupled out to the user's eye. For coupling the light from the first waveguide to the second one, the length L of the transition region (filled area with points on Fig. 3) and the thickness of the second waveguide d2 are interdependent; both are critical. As shown in Fig. 3, the red arrow lines correspond to the propagating field with minimum tilt angle (α) and the black arrow lines correspond to the field with maximum tilt angle (β). In the following we call minimum (resp. maximum) field, the field whose chief ray has the minimum (resp. maximum) tilt angle. As the air gap is very small, it is not represented in the figure. We suppose that the refractive indices of the two waveguides and the transition region are n1 and n2 respectively. For each field, part of the rays cannot be coupled into the second waveguide. As example it is the case for the chief ray of the maximum field reflected inside the grey area and the chief ray of the minimum field reflected inside the red area. These rays are reflected back to the first waveguide, which limits the coupling efficiency.

The side lengths of the filled area along the waveguide are l1 for the minimum field and l2 for the maximum field. They are given by:

( )         - - = ) ( sin ) sin( tan 2 2 2 1 2 2 1 1 1    n n Ln d l (1) ( )         - - = 2 2 2 1 2 2 1 2 ) ( sin ) sin( tan 2 d n n Ln l    (2)
Fig. 3. The ray path of coupling from the first waveguide to the second waveguide.

In the design process we first fix d1. Lengths l1 and l2 should be zero if all the light from the first waveguide is coupled into the second waveguide. The limit value of the length L of the transition region is obtained by setting l1=0:

) sin( ) ( sin 1 2 2 1 2 2 1   n n n d L - = . ( 3
)
The minimum thickness of the second waveguide d2 is obtained when l2=0:

) ( sin ) sin( 2 2 1 2 2 1 2   n n Ln d - = . (4)
Combining ( 3) and ( 4) we get the minimum value for d2:

) ( sin ) sin( ) ( sin ) sin( 2 2 1 2 2 2 2 1 2 2 1 min 2     n n n n d d - - = (5)
The relations between d2 with respect to other parameters can be found in Ref [25]. It would be interesting to reduce the thickness of the second waveguide to decrease the weight and size. This introduces some additional coupling losses. In the following text, the transition region and the two waveguides are assumed to be made with the same material and the thicknesses verify d1<d2 in order to avoid high losses. By setting l1= l2=0, we get the critical values for α and β respectively:

      = L d 1 arctan '  (6)       = L d 2 arctan '  (7)
Three situations occur for the coupling from the first waveguide to the second one. In the first one, the field angle lies inside the interval [α, α'] and l1 the side length of the red area along the waveguide is given by Eq(1). In the second situation, when the field angle lies inside the interval [α', β'] there is no coupling loss. When the field angle lies inside the interval [β', β], l2 the side length of the shaded area along the waveguide is determined by Eq (2). In order to calculate the length of the transition region, as the first case we can set l1=l2, then we obtain:

2 ) cot( ) cot( 2 1   d d L + = . ( 8
)
On Fig. 3 W1 and W2 are the lengths of the waveguide covered by the minimum and the maximum fields. The coupling efficiencies are different if l1= l2. To obtain the same coupling efficiency, we need:

2 2 1 1 W l W l = . (9)
In this second case, the length of the transition region is given by:

2 1 2 1 1 2 ) cot( ) cot( W W d W d W L + + =   . ( 10
)
As an example, using PMMA (n= 1.49), if we set d1=1.5 mm, d2=2 mm, we obtain the different parameters shown in table 1 for the two cases. The maximum value of β'-α' which represents the range without loss is 8.2°. The value of β'-α' for the two cases are 7.9° and 8° respectively. The differences from the maximum value are very small. The second case offers a better coupling uniformity. Actual efficiency for a specific field could be higher depending of the amount of light falling into the filled regions (Fig. 3). The efficiency for the vertical fields is uniform because the center of the incoupling pupil O is also the center of the cylindrical mirror and the center of the cylindrical edge of the air gap (Fig. 2(a)). Therefore, each vertical field has the same coupling efficiency.

We have designed a system with the presented method and the parameters described above using Zemax. A 0.61 in OLED microdisplay with 852×600 pixels is used as image source. The pixel size is 15 μm which corresponds to a Nyquist frequency of 33 lp/mm. The horizontal FOV is set to 20°. The vertical FOV is 60°. The MTF of the 12 object fields are plotted in Fig. 4(a). They are evaluated with a full rectangular pupil size of 1.9 mm (H) × 12 mm (V). All the values are above 0.3 at 33 lp/mm, which is sufficient for a visual system. The vertical and horizontal magnifications are different and in a ratio of 1.6. Fig. 5 shows the grid distortion of the system with respect to the central field. The maximum distortion is 4.5% at the corner. The performance of the system is summarized in the table 2. To estimate the value of the eye box in the horizontal direction a Lumus waveguide type out-coupling system has been used. The weight in table 2 takes into account all the optical components. Figures 4 and5 show that a good image quality can be expected for the whole FOV. A foreseen difficulty comes from fabrication and testing since the freeform prism has a complex shape. The current way to realize it, is plastic molding injection. A prototype fabrication is still a high cost operation.

In conclusion, we proposed a new polychromatic compact WNED system design by combining a freeform prism and two waveguides. The two waveguides have a cylindrical edge and are separated by a small air gap. The coupling from the first waveguide to the second one has been investigated and optimized. As a result, we obtain an increased FOV of 30° (H)×60° (V) and a very large eye box of about 15 mm (H) × 12 mm (V) allowing large eye movements.

Fig. 1 .

 1 Fig. 1. The whole structure and optical path of the proposed system from different points of view: (a) yx view, (b)zx view, (c)zy view.

Fig. 2 .

 2 Fig. 2. Schematic for the designed system. (a) Ray path in zx plane. The dashed rays are the rays that are coupled into the second waveguide. (b) Ray path in yx plane. The air gap is magnified for clarity.

Fig. 4 .

 4 Fig. 4. MTF curves of the system with the 12 evaluated fields in object angle mode. T and S represent the tangential and sagittal MTFs of each field separately.

Fig. 5 .

 5 Fig. 5. The grid distortion of the system.

Table 1 .

 1 The value of the parameters in two cases (case 1: the lengths l1 and l2 are equal; case 2: the same coupling efficiency)

	Parameters	Case1 Case2
	L (mm)	2.3	2.2
	α (°)	28	28
	β (°)	48	48
	α' (°)	33.0	34.3
	β' (°)	40.9	42.3
	l1 (mm)	1.02	1.25
	l2 (mm)	1.02	0.8
	Coupling efficiency for the minimum field	74.3% 68.6%
	Coupling efficiency for the maximum field	59.7% 68.6%

Table 2 .

 2 Performance of the system

	Parameters	Value
	Field-of-view (H×V)	30° × 60°
	In-coupling pupil (H×V mm)	1.9×12
	Eye box (H×V mm)	≈15×12
	System MTF	> 0.3 at 33 lp/mm
	Distortion	< 4.5%
	Monocular weight (g)	11
	Magnification ratio (V/H)	1.6

Acknowledgment. Portions of this work were presented at the SPIE Photonics Europe Conference in 2016, Photonics and Digital Technologies for Imaging Applications IV, 989605.


Carl Safi
email: carl.safi@ensiacet.fr

Michaël Charton

Alina Violeta Ursu

Céline Laroche

Bachar Zebib

Pierre-Yves Pontalier

Carlos Vaca-Garcia

Michael Charton

Release of hydro-soluble microalgal proteins using mechanical and chemical treatments

Keywords:

à la diffusion de documents scientifiques de niveau recherche, publiés ou non, émanant des établissements d'enseignement et de recherche français ou étrangers, des laboratoires publics ou privés.

Introduction

In the 9th century AD the Kanem Empire in Chad discovered the benefits of the cyanobacterium Arthrospira platensis and used it as food (called dihé) for human consumption [START_REF] Barsanti | Algae: Anatomy, Biochemistry, and Biotechnology[END_REF]. Later on in the 14th century AD, the Aztecs harvested the same species from Lake Texcoco and used it to make a sort of cake called tecuilatl. They also used these microorganisms as fodder, fertilisers and remedies. Nowadays, additional species are being industrially and profitably marketed worldwide for the same purposes.

The microalgal industry has grown rapidly over the last decade. Primarily, this is due to the capacity of these micro-organisms to produce lipids suitable for the biodiesel industry, and to grow in a wide variety of geographical and environmental locations, thus precluding competition with arable lands as well as intensive deforestation. Therefore, the major part of microalgal studies has concentrated on enhancing this bioenergy production to the detriment of other high-value biomolecules, but forgetting ancient history and the other advantages of these species.

Today the microalgal bioenergy industry is struggling to find a place in the market due to its uncompetitive cost and its overall unsustainable production [START_REF] Jorquera | Comparative energy life-cycle analyses of microalgal biomass production in open ponds and photobioreactors[END_REF][START_REF] Lam | Microalgae biofuels: a critical review of issues, problems and the way forward[END_REF][START_REF] Singh | A critical review of biochemical conversion, sustainability and life cycle assessment of algal biofuels[END_REF][START_REF] Stephenson | Life-cycle assessment of potential algal biodiesel production in the United Kingdom: a comparison of raceways and air-lift tubular bioreactors[END_REF][START_REF] Yang | Life-cycle analysis on biodiesel production from microalgae: water footprint and nutrients balance[END_REF] sometimes leaving negative footprints on the environment, and public opinion.

Microalgae were originally considered as an important source of protein, a major fraction of their composition; on a dry weight basis the Cyanobacterium Arthrospira platensis is composed of 50-70% proteins [START_REF] Batista | Rheological characterization of coloured oil-in-water food emulsions with lutein and phycocyanin added to the oil and aqueous phases[END_REF][START_REF] Chronakis | The behaviour of protein preparations from blue-green algae (Spirulina platensis strain Pacifica) at the air/water interface, Colloids Surf[END_REF], the Chlorophycea Chlorella vulgaris 38-58% [START_REF] Batista | Comparison of microalgal biomass profiles as novel functional ingredient for food products[END_REF][START_REF] Safi | Influence of microalgae cell wall characteristics on protein extractability and determination of nitrogen-to-protein conversion factors[END_REF][START_REF] Tokuşoglu | Biomass nutrient profiles of three microalgae: Spirulina platensis, Chlorella vulgaris, and Isochrisis galbana[END_REF], the Eustigmatophyceae Nannochloropsis oculata 22-37% [START_REF] Rebolloso-Fuentes | Biomass nutrient profiles of the microalga Nannochloropsis[END_REF], the Chlorophyceae Haematococcus pluvialis 45-50% [START_REF] Batista | Rheological characterization of coloured oil-in-water food emulsions with lutein and phycocyanin added to the oil and aqueous phases[END_REF], and the Rhodophyta Porphyridium cruentum 8-56% protein [START_REF] Becker | Microalgae: Biotechnology and Microbiology[END_REF][START_REF] Safi | Evaluation of the protein quality of Porphyridium cruentum[END_REF]. They have a profile composed of a set of essential and non essential amino acids [START_REF] Safi | Influence of microalgae cell wall characteristics on protein extractability and determination of nitrogen-to-protein conversion factors[END_REF], with relatively similar ratios between species and generally unaffected by growth phase and light conditions [START_REF] Barsanti | Algae: Anatomy, Biochemistry, and Biotechnology[END_REF]. To the best of our knowledge, studies on microalgal proteins have generally either concentrated on finding and proposing the nitrogen to protein conversion factor [START_REF] Safi | Influence of microalgae cell wall characteristics on protein extractability and determination of nitrogen-to-protein conversion factors[END_REF][START_REF] Diniz | Gross chemical profile and calculation of nitrogen-to-protein conversion factors for five tropical seaweeds[END_REF][START_REF] Lopez | Protein measurements of microalgal and cyanobacterial biomass[END_REF][START_REF] Lourenço | Distribution of intracellular nitrogen in marine microalgae: calculation of new nitrogen-to-protein conversion factors[END_REF][START_REF] Lourenço | Distribution of intracellular nitrogen in marine microalgae: basis for the calculation of specific nitrogen-to-protein conversion factors[END_REF], in order to avoid incorrect estimations of microalgal total protein content, or focused on determining the best method for protein quantification using colorimetric techniques [START_REF] Barbarino | An evaluation of methods for extraction and quantification of protein from marine macro-and microalgae[END_REF][START_REF] Servaites | A dye binding method for measurement of total protein in microalgae[END_REF][START_REF] Slocombe | A rapid and general method for measurement of protein in micro-algal biomass[END_REF]. However, for some species such as the green microalgae C. vulgaris, N. oculata and H. pluvialis, maximising the recovery of proteins requires a unit cell disruption operation to overcome the barrier of their rigid cell wall and release the intracellular biomolecules. Thus, many cell disruption methods were used to break the cell wall of these microalgae, such as bead milling, ultrasonication, microwaves, enzymatic treatment and high-pressure homogenization [START_REF] Fleurence | The enzymatic degradation of algal cell walls: a useful approach for improving protein accessibility?[END_REF][START_REF] Gouveia | Microalgae as a raw material for biofuels production[END_REF][START_REF] Lee | Comparison of several methods for effective lipid extraction from microalgae[END_REF][START_REF] Mendes-Pinto | Evaluation of different cell disruption processes on encysted cells of Haematococcus pluvialis: effects on astaxanthin recovery and implications for bio-availability[END_REF][START_REF] Zheng | Disruption of Chlorella vulgaris cells for the release of biodiesel-producing lipids: a comparison of grinding, ultrasonication, bead milling, enzymatic lysis, and microwaves[END_REF]. Conversely, fragile cell walled microalgae such as P. cruentum and A. platensis require milder techniques to enhance recovery.

The main objective of this study is to evaluate the effect of two different cell disruption techniques on aqueous phase protein extractability, in five microalgae with different cell wall characteristics, while simultaneously evaluating and comparing the profile of amino-acids subsequent to these two cell disruption methods.

Materials and methods

Microalgae

The selected microalgae were supplied as frozen paste from Alpha Biotech (Asserac, France): the Cyanobacteria Arthrospira platensis (strain PCC 8005), two different Chlorophyceae Chlorella vulgaris (strain SAG 211-19), and Haematococcus pluvialis (unknown strain), one Rhodophyta Porphyridium centum (strain UTEX 161), and the Eustigmatophyceae Nannochloropsis oculata (unknown strain).

Each microalga was cultivated on a different culture media; Hemerick media was used for P. cruentum, Sueoka media for C. vulgaris, Basal media for H. pluvialis, Conway media for N. oculata and Zarrouk media for A. platensis. All were grown in batch mode in an indoor tubular Air-Lift PhotoBioReactor (PBR, 10 L) at 25 °C, inoculated from a prior culture in a flat panel Air-Lift PBR (1 L). Culture homogenization was by sterile air injection at the bottom of the PBR. The pH and temperature were recorded using a pH/temperature probe (Mettler Toledo SG 3253 sensor) monitored by LabVIEW acquisition software. The pH was regulated at 7.5 with CO 2 bubbling. Microalgae were harvested during the exponential growth phase, concentrated by centrifugation, and the biomass which contained 20% dry weight, was then frozen.

Chemicals

The Lowry kit ((prepared mixture of Lowry reagent plus bovine standard albumin (BSA) standards and 2 N Folin-Ciocalteu reagent)) was purchased from Thermo Fisher Scientific. NaOH and HCl 37% were purchased from Sigma Aldrich and used as received.

High-pressure cell disruptor

A "TS Haiva series, 2.2-kW" homogenizer from Constant Systems Limited (Northants, UK), was used. For each experiment, a biomass concentration of 2% dry weight (0.5 g of freeze dried cells dispersed in 25 mL distilled water) was passed through the machine twice at a pressure of 2700 bar.

Alkaline treatment

Mother solutions were prepared with approximately 500 mL of ultrapure water and some drops of 2 N NaOH to adjust to pH 12. A sample of 1 g of freeze-dried biomass was added to 50 mL of mother solution and the mixture heated at 40 °C with stirring for 1 h. Separation of the solid-liquid mixture was conducted by centrifugation at 5000 g for 10 min. Samples of the supernatant were taken for protein analysis by the Lowry colorimetric method and for amino acid analysis.

Lowry method

The procedure involves reacting proteins with cupric sulphate and tartrate in an alkaline solution, leading to the formation of tetradentate copper protein complexes. The addition of the Folin-Ciocalteu reagent leads to the oxidation of the peptide bonds by forming molybdenum blue with the copper ions. Therefore, a calibration curve was prepared using a BSA concentration range from 0 to 1500 μg mL -1 . In order to measure the protein content, 0.2 mL of each standard or samples containing the crude protein extract were taken, and then 1 mL of modified Lowry reagent was added to each sample, which was then vortexed and incubated for exactly 10 min at room temperature. After incubation, 0.1 mL of Folin-Ciocalteu Reagent (1 N) was added and the sample again vortexed and incubated for exactly 30 min at room temperature. The blue colour solution absorbance was then measured at 750 nm with a UV-1800 Shimadzu spectrophotometer, previously zeroed with a blank sample containing all the reagents minus the extract.

Elemental analysis

The total nitrogen was evaluated by LCC (Laboratoire de Chimie de Coordination, Toulouse-France) using a PerkinElmer 2400 series II elemental analyser. The samples of 2 mg were placed in thin capsules and then heated to 925 °C using pure oxygen as the combustion gas, and pure helium as the carrier gas. The percentage nitrogen was evaluated and converted into protein percentage using the conversion factors obtained for each microalga in another study [START_REF] Safi | Influence of microalgae cell wall characteristics on protein extractability and determination of nitrogen-to-protein conversion factors[END_REF].

Amino acid analysis

The biomass amino acid composition was determined using a well known standard method (Moore and Stein 1948). The samples were hydrolysed with 6 N HCl at 103 °C for 24 h., and the hydrolysed material was then adjusted to pH 2.2 with 6 N NaOH and stabilised with a pH 2.2 citrate buffer solution. The final solution was then filtered over a 0.45 μm PTFE membrane to remove any residual solids remaining in the solution. The analysis was performed using a Biochrom Ltd 32 + (Cambridge, UK) amino acid analyser, equipped with a high pressure PEEK "column + pre-column" (size, 200 × 4.6 mm) packed with Ultropac cation exchange resin containing sodium. The separation of amino acids was carried out by elution with loading buffers (flow rate 25 mL.h -1) at different pH's. After reaction with ninhydrin (flow rate 35 mL.h -1), amino acids were detected with a UV detector at a 570 nm wavelength, except for proline, where detection was at 440 nm. Ammonia was added to compensate for the value of some less resistant amino acids, broken down by the strong acid hydrolysis.

Statistical analysis

Three experiments were conducted separately for each microalga. Measurements of three replicates for each sample were repeatable at maximum ±5% of the respective mean values.

Results

The total protein content of crude microalgae was determined from the value of total nitrogen obtained through elemental analysis, and the conversion factor found for each crude microalga in a separate study (Safi et al. 2012b). In all cases, the total protein content was high and consistent with literature values, ranging from 49 to 58% dry weight (Table 1). The fraction of hydro-soluble proteins released into water after both cell disruption techniques is presented in Fig. 1, after quantification by the Lowry method in cases where the mechanical method recovered more proteins compared to the alkaline treatment. The hydro-soluble protein fraction of total proteins present in the microalgae was also evaluated, and all these results are shown in Table 1.

The amino acid profile was first determined for the crude microalgae, with Aspartic acid being the highest member for P. cruentum, A. platensis and C. vulgaris and Alanine for N. oculata and H. pluvialis (Table 2). In addition, this profile was evaluated alkaline treatment, and here Aspartic acid was the highest for P. cruentum, Alanine for A. platensis, C. vulgaris and H. pluvialis, and Proline for N. oculata (Table 3). However, with high-pressure homogenization Proline was the highest for P. cruentum, A. platensis and N. oculata and Alanine for C. vulgaris and H. pluvialis (Table 4). Furthermore, the percentages of essential and non-essential amino acids before and after both cell disruption treatments, were also evaluated and are shown in Table 5. The proportion of non-essential amino acids was higher than that of essential amino acids for all the microalgae after both treatments. Nevertheless, it can be seen (Table 5) that the proportion of non-essential amino acids was much higher with high-pressure homogenization than with the alkaline treatment.

Discussion

This study used two different cell wall treatments on five different microalgae followed by quantification of the proteins [START_REF] Lowry | Protein measurement with the folin phenol reagent[END_REF] released in the aqueous phase, and then assessed the amino acid profile of these proteins for each treatment. The characteristics of the microalgal cell walls play an important role in the release of these biomolecules. Nonetheless, regardless of cell wall characteristics we have shown that at the 95% confidence level using three replicates for each microalga, all the latter have statistically equivalent protein values (Table 1). It should however be noted that the total nitrogen estimation includes other nitrogenous compounds, such as intracellular inorganic materials [START_REF] Lourenço | Distribution of intracellular nitrogen in marine microalgae: basis for the calculation of specific nitrogen-to-protein conversion factors[END_REF] pigments, nucleic acids, glucosamine and amines that could account for about 10% of the overall nitrogen content in microalgae [START_REF] Batista | Rheological characterization of coloured oil-in-water food emulsions with lutein and phycocyanin added to the oil and aqueous phases[END_REF][START_REF] Becker | Microalgae in human and animal nutrition, Handbook of Microalgal Culture[END_REF].

After conducting both cell wall treatments, the highest content of hydro-soluble proteins in the extract was from P. cruentum which has a pseudo-cell wall composed of exopolysaccharide mucilages [START_REF] Arad | Effect of nitrogen on polysaccharide production in a Porphyridium sp[END_REF][START_REF] Geresh | The extracellular polysaccharides of the red microalgae: chemistry and rheology[END_REF][START_REF] Geresh | Sulfation of extracellular polysaccharides of red microalgae: preparation, characterization and properties[END_REF] making it very fragile and offering very little resistance any treatment. Conversely, the lowest microalgae protein content in this study was obtained from H. pluvialis, known for its cell wall composed of cellulose and sporopollenin, which is remarkably resistant to chemical and mechanical treatment [START_REF] Safi | Influence of microalgae cell wall characteristics on protein extractability and determination of nitrogen-to-protein conversion factors[END_REF][START_REF] Mendes-Pinto | Evaluation of different cell disruption processes on encysted cells of Haematococcus pluvialis: effects on astaxanthin recovery and implications for bio-availability[END_REF]. Moreover, if we observe the decrease in protein recovery, we can see that this mirrors the increasing rigidity of the cell walls (Table 1) in all the microalgae. Nonetheless, compared to alkaline treatment, mechanical treatment gave more aqueous phase protein recovery for all the microalgae, with the lowest increase reserved for the fragile cell walled microalgae; 11% and 14% calculated for A. platensis and P. cruentum respectively. Indeed, both of these offer very little resistance to cell disruption treatment, and this small increase in protein recovery suggests more effective disruption of protein aggregates by high-pressure homogenization, leading to better solubilisation of hydro-soluble proteins in the aqueous phase. Similarly, a higher increase in protein recovery for the rigid cell walled microalgae was also detected, with 18%, 33% and 36% for C. vulgaris, H. pluvialis and N. oculata respectively. Here, the mechanical treatment applied in this study, is more effective at breaking the cell walls and protein aggregates, allowing more protein to be solubilised. Furthermore, the alkaline treatment does have an effect on protein recovery, because the chemical action acts in synergy with the mechanical characteristics of the cell wall (Safi et al. 2012b). Similarly, as mentioned earlier, the sporopollenin contained in the most rigid cell wall (H. pluvialis) is known to be extremely resistant to chemical agents [START_REF] Hagen | Ultrastructural and chemical changes in the cell wall of Haematococcus pluvialis (Volvocales, Chlorophyta) during aplanospore formation[END_REF]. But for cellulose-rich cell walls, such as in C. vulgaris and N. oculata, the sodium hydroxide is able to penetrate the cellulose microcrystalline structure to form alcoholates in a process similar to mercerisation, and can also dissolve the hemicelluloses attached to the cellulose. Partial permeation of this kind of cell wall can therefore occur by alkaline action, favouring solubilisation of cell wall proteins but making it difficult to recover cytoplasmic and chloroplastic proteins. Finally, A. platensis is a gram-negative cyanobacteria with a thin cell wall rich in amino sugars cross-linked with oligopeptide chains. Under alkaline conditions, the former are labile by deamidation of the N-acetylglucosamine while the latter are soluble. Therefore the cell wall becomes highly permeable allowing alkaline extraction of proteins by penetration of the cytoplasmic and chloroplastic space, enhancing protein recovery. The proteins' amino acid profile was also evaluated by analysing the crude microalgae (Table 2), the alkaline treatment protein extracts (Table 3) and the high-pressure homogenization extracts (Table 4). The proportion of essential and non-essential amino acids was also evaluated (Table 5), and showed that the percentage of non-essential amino acids derived from both treatments was higher than essential amino acids. This suggests that non-essential amino acids are more concentrated inside the cell wall barrier, and also that it is not the same proteins being released in the aqueous phase when comparing both treatments. However, compared to the alkaline treatment, high-pressure homogenization increased the percentage of non-essential amino acids for the fragile cell walled species from 20% to 26% for A. platensis and P. cruentum respectively. Similarly, for the rigid cell walled green species, they increased by 7%, 10% and 12% for N. oculata, H. pluvialis and C. vulgaris respectively. Moreover, for the latter species it is noteworthy that after alkaline treatment, the proportions of essential to nonessential amino acids was statistically the same compared with those for the crude fragile microalgae, and this was not the case after mechanical treatment of the same species. However, from the literature, few studies have distinguished between cell wall and intracellular amino acids of microalgae. It has been reported for instance, that after isolating and purifying the cell wall of C. vulgaris from the cytoplasmic medium, this contained peptides rather than proteins, although the amino acid profile was limited to their detection without quantifying the proportions [START_REF] Punnett | The amino acid composition of algal cell[END_REF].

In conclusion, it has been noticed that after both treatments, essential and non-essential amino acids were present but in different ratios, suggesting that the quality and quantity of proteins in the extract depends on the effectiveness of the cell disruption method, and also on the structural morphology of each microalgal cell wall. Therefore, mechanical treatment is more effective than chemical treatment due to its capacity to disrupt the cell walls and protein aggregates. And the logical next step will be to conduct high performance liquid chromatography in order to identify the type of proteins released after cell disruption.

At present, the FAO and WHO recommend microalgal proteins for human consumption because they contain all the necessary amino acids, however, the reported presence of toxins in microalgae [START_REF] Heussner | Toxin content and cytotoxicity of algal dietary supplements[END_REF], reopens the debate on this biomass as a supplementary food product. Notwithstanding, microalgal technology is still in its infancy and has a promising future in tomorrow's food industry, although additional clarification is required to include microalgae in the daily food intake.

Fig. 1 .

 1 Fig. 1. Protein % d.w of biomass in water after of five microalgae by a mechanical and chemical method. Results are based on 3 replicates for 3 experiments ± SD (n = 9).

Table 1

 1 Different protein contents in crude microalgae calculated according to the following equation: Proportion of hydro-soluble protein in total protein for different microalgae.

	Chemical treatment	High-pressure homogenization

TOTAL : Total protein in microalgae = N EA × NTP. a N EA : Total nitrogen % (d.w) obtained by elemental analysis. b NTP: Nitrogen-to-protein conversion factors of Safi et al. (2012b) for each microalga. c P Lowry : Hydro-soluble protein % (d.w) at pH 12 and 40 °C and by high-pressure homogenization calculated using the Lowry method.

Table 2

 2 Results of total amino acids of 5 microalgae expressed in g per 100 g of algal protein representing 3 replicates for 3 experiments ± SD (n = 9).

	Freeze-dried microalgae amino acid composition
	Amino acids	P. cruentum	A. platensis	C. vulgaris	N. oculata	H. pluvialis
	Aspartic acid	12.41 ± 0.45	13.10 ± 0.11	11.20 ± 0.02	10.13 ± 0.05	9.76 ± 0.10
	Threonine	6.91 ± 0.25	6.83 ± 0.10	6.24 ± 0.01	6.55 ± 0.03	5.75 ± 0.06
	Serine	8.98 ± 0.29	7.59 ± 0.02	7.97 ± 0.04	7.23 ± 0.01	7.31 ± 0.05
	Glutamic acid	9.04 ± 0.29	11.64 ± 0.09	9.30 ± 0.01	11.41 ± 0.02	10.44 ± 0.11
	Glycine	7.59 ± 0.28	8.60 ± 0.06	8.81 ± 0.01	9.97 ± 0.01	9.98 ± 0.09
	Alanine	7.39 ± 3.67	10.99 ± 0.08	11.17 ± 0.03	12.11 ± 0.01	12.44 ± 0.12
	Cysteine	0.37 ± 0.01	0.20 ± 0.02	0.20 ± 0.01	0.22 ± 0.01	0.25 ± 0.01
	Valine	2.76 ± 0.10	3.17 ± 0.02	3.17 ± 0.01	3.65 ± 0.02	3.67 ± 0.04
	Methionine	3.08 ± 0.11	1.91 ± 0.02	0.66 ± 0.01	1.66 ± 0.01	0.71 ± 0.01
	Isoleucine	5.81 ± 0.24	0.13 ± 0.01	0.09 ± 0.01	0.13 ± 0.01	4.99 ± 0.04
	Leucine	6.46 ± 0.21	7.79 ± 0.02	7.68 ± 0.02	8.99 ± 0.05	8.92 ± 0.10
	Tyrosine	4.90 ± 0.18	5.35 ± 0.05	8.63 ± 0.01	3.76 ± 0.02	3.08 ± 0.04
	Phenylalanine	5.54 ± 0.20	5.34 ± 0.04	5.96 ± 0.01	5.59 ± 0.01	5.42 ± 0.07
	Histidine	1.22 ± 0.04	1.00 ± 0.01	1.29 ± 0.01	1.04 ± 0.01	0.99 ± 0.01
	Lysine	6.09 ± 0.21	5.65 ± 0.62	6.99 ± 0.07	6.32 ± 0.01	6.31 ± 0.08
	Arginine	8.62 ± 0.29	8.52 ± 0.07	7.57 ± 0.03	6.58 ± 0.02	6.73 ± 0.08
	Proline	2.80 ± 0.17	2.16 ± 0.05	3.04 ± 0.07	4.65 ± 0.07	3.24 ± 1.15

Table 4

 4 Results of total amino acids of 5 microalgae expressed in g per 100 g of algal protein representing 3 replicates for 3 experiments ± SD (n = 9).

	Amino acid composition in the protein extract after high-pressure homogenization
	Amino acids	P. cruentum	A. platensis	C. vulgaris	N. oculata	H. pluvialis
	Aspartic acid	12.15 ± 0.14	8.67 ± 0.02	7.47 ± 0.01	7.27 ± 0.34	8.26 ± 0.10
	Threonine	3.39 ± 0.03	4.66 ± 0.25	5.13 ± 0.02	4.45 ± 0.26	4.36 ± 0.09
	Serine	5.57 ± 0.07	5.00 ± 0.03	5.46 ± 0.01	4.43 ± 0.05	5.59 ± 0.12
	Glutamic acid	9.76 ± 0.51	13.85 ± 0.05	10.23 ± 0.06	11.51 ± 0.12	11.41 ± 0.11
	Glycine	6.72 ± 0.09	7.67 ± 0.01	10.07 ± 0.05	9.15 ± 0.13	10.62 ± 0.09
	Alanine	11.69 ± 0.02	10.10 ± 0.04	16.93 ± 0.11	10.28 ± 0.04	17.05 ± 0.14
	Cysteine	1.21 ± 0.05	0.42 ± 0.01	0.39 ± 0.01	0.74 ± 0.20	0.87 ± 0.03
	Valine	7.06 ± 0.20	5.63 ± 0.02	6.19 ± 0.05	5.36 ± 0.02	5.33 ± 0.10
	Methionine	4.34 ± 0.10	1.09 ± 0.01	1.05 ± 0.02	1.43 ± 0.11	0.92 ± 0.41
	Isoleucine	3.91 ± 0.06	4.14 ± 0.04	3.19 ± 0.03	3.05 ± 0.26	2.52 ± 0.33
	Leucine	5.59 ± 0.06	7.13 ± 0.01	7.03 ± 0.04	7.14 ± 0.07	5.10 ± 0.17
	Tyrosine	1.74 ± 0.07	2.27 ± 0.03	2.31 ± 0.01	1.09 ± 0.10	1.35 ± 0.02
	Phenylalanine	2.01 ± 0.07	2.90 ± 0.04	2.74 ± 0.03	3.07 ± 0.08	2.31 ± 0.02
	Histidine	0.92 ± 0.02	1.15 ± 0.01	0.98 ± 0.02	1.48 ± 0.03	0.96 ± 0.01
	Lysine	3.13 ± 0.04	3.12 ± 0.04	4.95 ± 0.04	3.37 ± 0.18	3.61 ± 0.04
	Arginine	4.26 ± 0.02	4.06 ± 0.03	3.89 ± 0.03	3.20 ± 0.27	5.92 ± 0.05
	Proline	16.52 ± 0.80	18.11 ± 0.17	11.98 ± 0.15	22.93 ± 0.47	13.79 ± 0.19

Table 3

 3 Results of total amino acids of 5 microalgae expressed in g per 100 g of algal protein representing 3 replicates for 3 experiments ± SD (n = 9).

	Protein extract amino acid composition after chemical treatment
	Amino acids	P. cruentum	A. platensis	C. vulgaris	N. oculata	H. pluvialis
	Aspartic acid	11.65 ± 0.02	11.27 ± 0.02	7.52 ± 0.34	5.02 ± 0.04	7.30 ± 0.09
	Threonine	5.88 ± 0.01	6.05 ± 0.05	4.60 ± 0.23	3.57 ± 0.05	4.92 ± 0.02
	Serine	8.15 ± 0.03	7.92 ± 0.03	7.42 ± 0.33	4.38 ± 0.09	6.32 ± 0.02
	Glutamic acid	9.85 ± 0.01	12.71 ± 0.02	12.85 ± 0.63	25.42 ± 0.30	15.13 ± 0.03
	Glycine	8.35 ± 0.01	9.19 ± 0.02	10.77 ± 0.52	9.89 ± 0.14	12.28 ± 0.02
	Alanine	11.38 ± 0.02	11.95 ± 0.02	18.58 ± 0.92	15.77 ± 0.20	21.34 ± 0.04
	Cysteine	0.30 ± 0.01	0.22 ± 0.01	0.27 ± 0.02	0.42 ± 0.01	0.45 ± 0.01
	Valine	3.43 ± 0.01	3.62 ± 0.01	4.04 ± 0.20	2.66 ± 0.04	3.13 ± 0.01
	Methionine	2.58 ± 0.01	1.86 ± 0.01	1.42 ± 0.06	1.38 ± 0.03	1.16 ± 0.01
	Isoleucine	5.81 ± 0.02	0.14 ± 0.01	2.60 ± 0.07	1.21 ± 0.01	3.42 ± 0.02
	Leucine	7.94 ± 0.01	8.75 ± 0.02	7.90 ± 0.40	4.62 ± 0.06	5.58 ± 0.03
	Tyrosine	4.00 ± 0.01	4.74 ± 0.01	2.83 ± 0.13	2.42 ± 0.09	2.13 ± 0.02
	Phenylalanine	4.50 ± 0.01	4.65 ± 0.01	3.03 ± 0.16	1.84 ± 0.03	2.94 ± 0.01
	Histidine	0.86 ± 0.03	0.81 ± 0.01	0.59 ± 0.03	0.32 ± 0.01	0.48 ± 0.01
	Lysine	6.08 ± 0.01	6.88 ± 0.01	6.81 ± 0.33	2.64 ± 0.10	3.88 ± 0.01
	Arginine	7.21 ± 0.03	7.46 ± 0.01	6.46 ± 2.96	2.20 ± 0.11	7.68 ± 0.01
	Proline	2.03 ± 0.02	2.03 ± 0.09	2.33 ± 0.14	16.21 ± 0.44	1.80 ± 0.04