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Marine aquaculture and fishery industries produce millions of tonnes of waste every year. According to FAO, global fish production reached about 179 million tonnes in 2018 and the 70 wt% of the processed seafood was discharged as waste. This waste is often just dumped in the sea or disposed in landfill in developing countries while in developed countries their disposal can be costly. Shells, in particular, represent the main marine waste but they also harbour useful chemicals such as chitin, protein and calcium carbonate. So, turning cast off shells into new materials, applying the concept of a circular economy, would benefit both the environment and the economy. Mussel shells, for example, are mainly built up of single crystals of calcite and aragonite in an external and an internal layer, respectively. The recovery of these biomineral building units offers the possibility to overcome the synthetic difficulties in replicating the ability of organisms to act as crystal shapers and morphology modifiers. Mollusk shells are a massive source of biogenic calcium carbonate (bCC) that can potentially substitute ground and precipitated calcium carbonate. These materials can be used as filler in polymeric matrices after a surface coating with hydrophobic molecules. bCC may also be transformed into a new added-value material such as apatite with cytocompatible and osteoinductive properties for biomedical applications or it may be converted into nano-crystalline and amorphous calcium carbonate for applications that span from medical to material science. bCC can also be extracted from crab and shrimp shells individually or in combination with other molecules such as proteins and chitin for potential applications as dye adsorbents. Therefore, this thesis aimed to valorise seafood wastes from different calcifying marine organisms by recovering the biomaterials they are composed of and using them for different applications exploiting their unique features as biominerals.

Waste seashells from aquaculture are a massive source of biogenic calcium carbonate (bCC) that can be a potential substitute for ground calcium carbonate and precipitated calcium carbonate. These last materials find several applications in industry after a surface coating with hydrophobic molecules, with stearate as the most used. Here, we investigate for the first time the capability of aqueous stearate dispersions to coat bCC powders from seashells of market-relevant mollusc aquaculture species, namely the oyster Crassostrea gigas, the scallop Pecten jacobaeus, and the clam Chamelea gallina. The chemical-physical features of bCC were extensively characterized by different analytical techniques. The results of stearate adsorption experiments showed that the oyster shell powder, which is the bCC with a higher content of the organic matrix, showed the highest adsorption capability (about 23 wt % compared to 10 wt % of geogenic calcite). These results agree with the mechanism proposed in the literature in which stearate adsorption mainly involves the formation of calcium stearate micelles in the dispersion before the physical adsorption. The coated bCC from oyster shells was also tested as fillers in an ethylene vinyl acetate compound used for the preparation of shoe soles. The obtained compound showed better mechanical performance than the one prepared using ground calcium. In conclusion, we can state that bCC can replace ground and precipitated calcium carbonate and has a higher stearate adsorbing capability. Moreover, they represent an environmentally friendly and sustainable source of calcium carbonate that organisms produce by high biological control over composition, polymorphism, and crystal texture. These features can be exploited for applications in fields where calcium carbonate with selected features is required.

Nanocrystalline calcium carbonate (CaCO) and amorphous CaCO (ACC) are materials of increasing technological interest. Nowadays, they are mainly synthetically produced by wet reactions using CaCO reagents in the presence of stabilizers. However, it has recently been discovered that ACC can be produced by ball milling calcite. Calcite and/or aragonite are the mineral phases of mollusk shells, which are formed from ACC precursors. Here, we investigated the possibility to convert, on a potentially industrial scale, the biogenic CaCO (bCC) from waste mollusk seashells into nanocrystalline CaCO and ACC. Waste seashells from the aquaculture species, namely oysters (Crassostrea gigas, low-Mg calcite), scallops (Pecten jacobaeus, medium-Mg calcite), and clams (Chamelea gallina, aragonite) were used. The ball milling process was carried out by using different dispersing solvents and potential ACC stabilizers. Structural, morphological, and spectroscopic characterization techniques were used. The results showed that the mechanochemical process produced a reduction of the crystalline domain sizes and formation of ACC domains, which coexisted in microsized aggregates. Interestingly, bCC behaved differently from the geogenic CaCO (gCC), and upon long milling times (24 h), the ACC reconverted into crystalline phases. The aging in diverse environments of mechanochemically treated bCC produced a mixture of calcite and aragonite in a species-specific mass ratio, while the ACC from gCC converted only into calcite. In conclusion, this research showed that bCC can produce nanocrystalline CaCO and ACC composites or mixtures having species-specific features. These materials can enlarge the already wide fields of applications of CaCO, which span from medical to material science.

Seashell wastes from aquaculture and canning industries represent an important environmental issue nowadays [1]. Shells are made of calcium carbonate (CaCO3) with a low content of proteins and polysaccharides (1-5 wt.%). The valorization of this waste by using it as a raw material for the production of calcium phosphates may have a positive impact both environmental and economic, thus contributing to the sustainability of this important sector. In some biomedical industries, there is a growing demand for calcium phosphate (apatite) crystals including nanosized, micron-sized, and larger sizes. This work is devoted to producing functional apatite nanocrystals, eg. doped with luminescent lanthanide (Ln3+) ions [2,3], using oyster shells (Mg-calcite, 5 wt.% Mg) from the species Crassostrea gigas as a Ca source. Experiments were performed by a one-pot hydrothermal method using KH2PO4 as a P reagent, a P/CaCO3 ratio of 0.6 (stoichiometric respect to hydroxyapatite), and either Eu3+ or Tb3+ (10and 20 mM). Characterization by XRD, FTIR, Raman, and ICP revealed the full transformation of biogenic CaCO3 particles into doped apatite. It was obtained at 160 ºC with (Ca+Ln)/P ratios 1.72 and 1.68 when adding Eu3+ and Tb3+ (10 mM) and 1.88 and 1.99 when the lanthanide concentration in the solution increased to 20 mM. In both cases, nanocrystals displayed needleor plate-like morphologies and polydisperse size distribution. Luminescence characterization of the nanoparticles showed different luminescence spectra depending on the doping ion. They displayed excitation and emission wavelengths of 395nm and 616 nm for the Eu3+-, and 372 and 543 nm for the Tb3+-doped samples. The relative luminescence intensities correlated well with their Ln3+ content while luminescence lifetimes (up to 1600 ¿s) were higher for Tb3+-doped apatites. Overall, the nanoparticles showed notable luminescent behavior and could find application as luminescent probes for bioimaging or nanophosphors for the elect

Fishery industry waste seashells represent an important environmental issue and imply the loss of potentially useful biomaterials [1]. They are composite materials made of CaCO3 and an organic matrix (1-5 wt.%) formed mainly of proteins and polysaccharides. Fabrication of functional calcium phosphates (eg. doped nanoapatites) using this type of biogenic CaCO3 as a calcium source may partially alleviate the ecological problem and be an alternative source of biocompatible materials intended for biomedical uses. In this work, the one-pot hydrothermal method [2] has been used in the preparation of biocompatible Mg2+, Mn2+, and Co2+-doped apatites. These divalent transition metal ions fulfill different roles in skeletal metabolism and may stimulate bone tissue regeneration [3-5]. The experiments were performed in a hydrothermal multitube set-up as well as in an autoclave, using oyster shell calcium carbonate particles from the species Crassostrea gigas, the KH2PO4 as a P reagent (P/CaCO3 0,6), and temperatures from 25ºC to 200ºC. Full transformation of CaCO3 was obtained at 160 ºC, yielding platy-shaped apatite nanoparticles doped with either 0.22 mol% Mg2+, 0.012 mol%Mn2+, or 0.16 mol% Co2+, and sizes within the range 75-90 nm. All samples showed a high cytocompatibility/biocompatibility when incubated with human mesenchymal stem cells for 1 and 3 days, and a small decrease in cell viability after 7 days of incubation in a dose-dependent concentration. Overall, the method was demonstrated to be promising in the preparation of doped biocompatible apatite nanocrystals with osteogenic features, and the biogenic CaCO3 a huge and unexplored calcium source for the preparation of apatite-based biomaterials. Acknowledgements: Grant ref. PCI2020-112108 is funded by MCIN/AEI/10.13039/501100011033 (Spain) and the European Union "NextGenerationEU"/PRTR". PCI2020-112108 is part of the project CASEAWA of ERA-NET Cofund BlueBio H2020.

Waste seashells from the fishery industry represent an important environmental issue. Its valorization by transforming the CaCO3 into apatite micro/nanoparticles may have a positive environmental impact besides an economic profitability since the global apatite market is expected to grow at 6.5% to around USD 3.086,05 million by 2027. Many of methods to transform biogenic CaCO3 into apatite particles are two-step processes involving calcination to CaO at 900-1000 °C followed by titration with a phosphate reagent (typically H3PO4). Here we present a one-step hydrothermal method using oyster shells Crassostrea gigas as model raw material. Shells were treated with NaClO 5% v/v, crushed, milled, and sieved (Ø<45 ¿m mesh), before submitting to hydrothermal conversion. Firstly, we explored the influences of KH2PO4 and K2HPO4, P/Ca molar ratios (0.24, 0.6, and 0.96), and temperature (25-200°C) in the transformation process. The minimum temperatures to obtain full transformation when using KH2PO4 and K2HPO4 for P/Ca molar ratios 0.6 (stoichiometric) were 160 ºC and 120ºC respectively, while for P/Ca ratios 0.96, they were 120 ºC and 80 ºC. The precipitates (Mg2+-doped carbonated-apatite micro/nanoparticles) are the result of a dissolution/re-precipitation mechanism driven by pH variation. Secondly, we explored the influence of osteogenic ions Mg2+, Mn2+, and Co2+ at P/Ca molar ratios 0.6. Full transformation of CaCO3 was obtained at 160 ºC, yielding platy-shaped apatite nanoparticles doped with either 0.22 mol% Mg2+, 0.012 mol% Mn2+, or 0.16 mol% Co2+, and sizes within the range 75-90 nm. Compared to the previous two-step processes, the one reported here is straightforward, one-pot, easily to scale up, and can be operated at relatively low temperatures without any pH adjustment.

Biominerals are inorganic/organic hybrid solids formed by organisms. In mollusk shells calcium carbonate is the main component, where it represents more than 95 wt.%. These biominerals are characterized by a shape, morphology, and composition that differentiate them from their geogenic and synthetic counterparts [1]. Mollusk shells are a waste by-product from aquaculture whose disposal represents an environmental and economic issue but they also are a renewable and cheap alternative source for biogenic calcium carbonate [2]. In this context, the presented research regards the valorization of waste mussel shells from the species Mytilus galloprovincialis in order to recover biomineral building units using a simple, sustainable, and environmentally friendly procedure [3]. Mussel shell consists of three crystalline layers (Fig. 1A): the inner layer of aragonite nacre, the outer layer of calcite fibrous prisms and the myostracum [4]. In order to separate the different layers, different treatments have been applied. They were: a bleaching process with NaClO solution to remove the outer organic periostracum layer; a thermal treatment for the mechanical separation of nacre; an acidic treatment with a CH3COOH solution to remove the myostracum. The nacre layer was then separated into almost single aragonite tablets by a bleaching and sonication process (Fig. 1B) while the prismatic layer was crashed by mortar and pestle, sieved at 600 ¿m and treated with NaClO solution to obtain single fibrous crystals of calcite (Fig. 1C). The disassembled calcite fibers were stirred in water to form spherical aegagropilae-like aggregates of fibers (Fig. 1D) having a potential application for gas adsorption.

We have developed a straightforward, one-pot, low-temperature hydrothermal method to transform oyster shell waste particles (bCCP) from the species Crassostrea gigas (Mg-calcite, 5 wt% Mg) into hydroxyapatite (HA) micro/nanoparticles. The influence of the P reagents (HPO, KHPO, and KHPO), P/bCCP molar ratios (0.24, 0.6, and 0.96), digestion temperatures (25-200 °C), and digestion times (1 week-2 months) on the transformation process was thoroughly investigated. At 1 week, the minimum temperature to yield the full transformation significantly reduced from 160 °C to 120 °C when using KHPO instead of KHPO at a P/bCCP ratio of 0.6, and even to 80 °C at a P/bCCP ratio of 0.96. The transformation took place via a dissolution-reprecipitation mechanism driven by the favorable balance between HA precipitation and bCCP dissolution, due to the lower solubility product of HA than that of calcite at any of the tested temperatures. Both the bCCP and the derived HA particles were cytocompatible for MG-63 human osteosarcoma cells and m17.ASC murine mesenchymal stem cells, and additionally, they promoted the osteogenic differentiation of m17.ASC, especially the HA particles. Because of their physicochemical features and biological compatibility, both particles could be useful osteoinductive platforms for translational applications in bone tissue engineering.

Control over the shape and morphology of single crystals is a theme of great interest in fundamental science and for technological application. Many synthetic strategies to achieve this goal are inspired by biomineralization processes. Indeed, organisms are able to produce crystals with high fidelity in shape and morphology utilizing macromolecules that act as modifiers. An alternative strategy can be the recovery of crystals from biomineralization products, in this case, seashells. In particular, waste mussel shells from aquaculture are considered. They are mainly built up of single crystals of calcite fibers and aragonite tablets forming an outer and an inner layer, respectively. A simple mechanochemical treatment has been developed to separate and recover these two typologies of single crystals. The characterization of these single crystals showed peculiar properties with respect to the calcium carbonate from quarry or synthesis. We exploited these biomaterials in the water remediation field using them as substrate adsorbing dyes. We found that these substrates show a high capability of adsorption for anionic dye, such as Eosin Y, but a low capability of adsorption for cationic dyes, such as Blue Methylene. The adsorption was reversible at pH 5.6. This application represents just an example of the potential use of these biogenic single crystals. We also envision potential applications as reinforcing fillers and optical devices.