# Investigation of structural and mechanical properties of BioCaCO3-LDPE composites

Serife Yalcin, Daniel Chateigner, Loïc Le Pluart, Stéphanie Gascoin, Sophie Eve

► To cite this version:

Serife Yalcin, Daniel Chateigner, Loïc Le Pluart, Stéphanie Gascoin, Sophie Eve. Investigation of structural and mechanical properties of BioCaCO3-LDPE composites. Metals and Materials International, 2019, 1 (2), pp.29-43. 10.33263/Materials12.029043. hal-02472107

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Submitted on 30 May 2024

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# Investigation of structural and mechanical properties of BioCaCO₃-LDPE composites

Serife Yalcın <fnref n="1" />*, Daniel Chateigner <fnref n="2" /> ID, Loïc Le Pluart <fnref n="3" /> ID, Stéphanie Gascoin <fnref n="2" /> ID,
Sophie Eve <fnref n="2" /> ID

**Abstract:** The three different Mollusk shells, *Pecten maximus*, *Crepidula fornicata* and *Crassostrea gigas*, were studied and compared with synthetic and commercial powders. All samples were analysed by X-ray diffraction, Quantitative phase analysis, and quantitative line broadening (microstructure) analysis using the Combined Analysis method. LDPE-CaCO3 composites were prepared in a twin screw extruder in the composition range of 0–10.8 filler content. Ultimate Mechanical properties of dog-bone type injection molded tensile specimens (ISO-527-2-5A) were measured. Results are showing that the biogenic calcium carbonate is less efficient in improving polyethylene stiffness than the synthetic ones, independently of its crystalline form, to use stearic acid coating allows an improvement of the matrix stiffening. The yield strength is unchanged whatever the kind of filler used, which makes shell spares valid for reuse in polymer industry.

**Keywords:** Mollusk Shell, Combined Analysis, CaCO3 filler, Coating, Mechanical properties.

© 2019 by the authors. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (http://creativecommons.org/licenses/by/4.0/).

## 1. Introduction

Mollusk shells that have been subject of numerous studies are high performance organic/inorganic enhancing bio-composite materials [1-3]. They exhibit excellent mechanical properties, thanks to their large resilience to crack propagation [4]. Since mineral calcium carbonate is hard and brittle and organic layers are soft materials [4], their combination inspired the

development of high performance ceramic composites with an improved resistance to crack propagation [5-6]. Some researches have expressed that biogenic crystals have structural differences from their fully mineral counterparts that potentially has important influence on some properties of [7-9].

<footnote n="1">¹ Harran University, Faculty of Arts and Science, Department of Physics, Osmanbey Campus, 63300, Sanliurfa, Turkey</footnote>

<footnote n="2">² Normandie Université, Ecole Nationale Supérieure d'Ingénieurs de Caen (ENSICAEN), Université de Caen Normandie (UNICAEN), Centre National de la Recherche Scientifique (CNRS), Institut Universitaire de Technologie (IUT)- Caen, Laboratoire de CRISTALlographie et Sciences des MATériaux (CRISMAT), 6, Bd M. Juin, 14050, Caen, France</footnote>

<footnote n="3">³ Normandie Université, ENSICAEN, UNICAEN, CNRS, Centre National de Recherche Technologique (CNRT), Laboratoire de Chimie Moléculaire et Thio-organique (LCMT), 6, Bd du Maréchal Juin 14050, Caen Cedex, France</footnote>

<footnote>* Correspondence: serifeyalcin@harran.edu.tr; Scopus ID: 17347094700</footnote>
# Investigation of structural and mechanical properties of BioCaCO₃-LDPE composites

Serife Yalcın <fnref n="1" />*, Daniel Chateigner <fnref n="2" /> ID, Loïc Le Pluart <fnref n="3" /> ID, Stéphanie Gascoin <fnref n="2" /> ID,
Sophie Eve <fnref n="2" /> ID

**Abstract:** The three different Mollusk shells, *Pecten maximus*, *Crepidula fornicata* and *Crassostrea gigas*, were studied and compared with synthetic and commercial powders. All samples were analysed by X-ray diffraction, Quantitative phase analysis, and quantitative line broadening (microstructure) analysis using the Combined Analysis method. LDPE-CaCO3 composites were prepared in a twin screw extruder in the composition range of 0–10.8 filler content. Ultimate Mechanical properties of dog-bone type injection molded tensile specimens (ISO-527-2-5A) were measured. Results are showing that the biogenic calcium carbonate is less efficient in improving polyethylene stiffness than the synthetic ones, independently of its crystalline form, to use stearic acid coating allows an improvement of the matrix stiffening. The yield strength is unchanged whatever the kind of filler used, which makes shell spares valid for reuse in polymer industry.

**Keywords:** Mollusk Shell, Combined Analysis, CaCO3 filler, Coating, Mechanical properties.

© 2019 by the authors. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (http://creativecommons.org/licenses/by/4.0/).

## 1. Introduction

Mollusk shells that have been subject of numerous studies are high performance organic/inorganic enhancing bio-composite materials [1-3]. They exhibit excellent mechanical properties, thanks to their large resilience to crack propagation [4]. Since mineral calcium carbonate is hard and brittle and organic layers are soft materials [4], their combination inspired the

development of high performance ceramic composites with an improved resistance to crack propagation [5-6]. Some researches have expressed that biogenic crystals have structural differences from their fully mineral counterparts that potentially has important influence on some properties of [7-9].

<footnote n="1">¹ Harran University, Faculty of Arts and Science, Department of Physics, Osmanbey Campus, 63300, Sanliurfa, Turkey</footnote>

<footnote n="2">² Normandie Université, Ecole Nationale Supérieure d'Ingénieurs de Caen (ENSICAEN), Université de Caen Normandie (UNICAEN), Centre National de la Recherche Scientifique (CNRS), Institut Universitaire de Technologie (IUT)- Caen, Laboratoire de CRISTALlographie et Sciences des MATériaux (CRISMAT), 6, Bd M. Juin, 14050, Caen, France</footnote>

<footnote n="3">³ Normandie Université, ENSICAEN, UNICAEN, CNRS, Centre National de Recherche Technologique (CNRT), Laboratoire de Chimie Moléculaire et Thio-organique (LCMT), 6, Bd du Maréchal Juin 14050, Caen Cedex, France</footnote>

<footnote>* Correspondence: serifeyalcin@harran.edu.tr; Scopus ID: 17347094700</footnote>
Mollusk shells are composite biomaterials composed of 95 to 99 wt% of calcium carbonate, the remaining components forming the organic matrix [10-11]. They represent nowadays large amounts of spare sea shells discarded to the environment, after harvest and marketing, from aquaculture, farming, fishing, food, pearl, and canning industries [12-13]. These solid wastes, always associated to some organics, can participate to water and even decompose in the marine environment [14]. Consequently a reuse of shell spares would advantageously contribute to environmental issues. For instance, dispersion of nanofillers in a polymer matrix is assisted by strong interactions, enabling the preparation of polymer nanocomposites with higher loading of nanofillers [15].

Lazzeri et al. (2005) reported that an addition of precipitated calcium carbonate (average particle size 70 nm) to HDPE rises both Young modulus and yield stress, and decreases the impact strength, but that a stearic acid compatibilization treatment negatively affects these, compared to uncoated composites [16]. However, Gai et al. (2005) reported that the PP mechanical properties using

powder fillers (average particle size 5.2 µm) are larger than those of PP materials produced using the original untreated powders [17]. On another hand González et al. (2006) incorporated sea shell wastes in various proportions (average particle size 78 µm) to PP and HDPE matrices and did not notice significant modification of the overall mechanical and rheological properties, except for an increase of the Young's Modulus [18]. A good agreement in montmorillonite/ polymer nanocomposites is shown between experimental measurements and predictions of tensile strength [19].

The purpose of this study is to examine the influence of ground biogenic sea shells addition to low density polyethylene (LDPE) on the mechanical properties and matrix crystallinity, by comparison with composites containing synthetic calcite and aragonite CaCO₃ particles. Ground shells are from three different mollusk species from Gastropoda and Bivalvia to test different CaCO₃ microstructures from broadly expended taxa. Powders are controlled with particle sizes under 1 µm to increase the specific filler/polymer interface in the composite.

## 2. Materials and Methods

### 2.1. Materials

Low density Polyethylene Flexirene MR50 (Polimeri Europa) and commercially available Calcium Chloride dehydrate, Potassium Hydrogen Carbonate and Calcium Carbonate (Sigma Aldrich, 10 µm mean particle size) were used. The filler particles are obtained by grinding non-biogenic powders, and biogenic mollusk sea shells from three different species: i) the gastropod *Crepidula fornicata*, the most abundant parasite along the ocean French coasts made of aragonitic CaCO₃ layers; ii) the edible bivalves *Pecten maximus*, the largest scallop made of calcitic layers and; iii) *Crassostrea gigas*, the most expanded oyster species over the world also made of calcitic layers. All samples were picked from dead animals on the Channel sea coast, Sword beach (49°17′52″ N, 0°17′58″ W). Stearic acid (Fluka AG) was used to avoid agglomeration during the grinding stage and potentially improve the dispersion state of calcium carbonate particles in the polymer matrix.

### 2.2. Fillers elaboration

Biogenic CaCO₃ fillers were obtained by powderising mollusk shells. Complete shells were introduced for grinding in laboratory with Retsch planetary ball milling machine (Retsch, Haan, Germany). The resulting powders are then composed of an average of the microstructures (aragonitic crossed lamellaes, foliated calcite, columnar, lath-type and rod type fibrous prismatic, chalky lenses, lamellar, parallel lamellar) composing the whole shell. To grind samples, we used different milling conditions because of the varying hardness between samples and to obtain the same mean particle sizes (as controlled using different grinding times and conditions). We ground each shell different grinding rotation speed and time because thickness and hardness of shells is different. The eventually remaining inside soft tissues were removed carefully and washed with pure water.
Mollusk shells are composite biomaterials composed of 95 to 99 wt% of calcium carbonate, the remaining components forming the organic matrix [10-11]. They represent nowadays large amounts of spare sea shells discarded to the environment, after harvest and marketing, from aquaculture, farming, fishing, food, pearl, and canning industries [12-13]. These solid wastes, always associated to some organics, can participate to water and even decompose in the marine environment [14]. Consequently a reuse of shell spares would advantageously contribute to environmental issues. For instance, dispersion of nanofillers in a polymer matrix is assisted by strong interactions, enabling the preparation of polymer nanocomposites with higher loading of nanofillers [15].

Lazzeri et al. (2005) reported that an addition of precipitated calcium carbonate (average particle size 70 nm) to HDPE rises both Young modulus and yield stress, and decreases the impact strength, but that a stearic acid compatibilization treatment negatively affects these, compared to uncoated composites [16]. However, Gai et al. (2005) reported that the PP mechanical properties using

powder fillers (average particle size 5.2 µm) are larger than those of PP materials produced using the original untreated powders [17]. On another hand González et al. (2006) incorporated sea shell wastes in various proportions (average particle size 78 µm) to PP and HDPE matrices and did not notice significant modification of the overall mechanical and rheological properties, except for an increase of the Young's Modulus [18]. A good agreement in montmorillonite/ polymer nanocomposites is shown between experimental measurements and predictions of tensile strength [19].

The purpose of this study is to examine the influence of ground biogenic sea shells addition to low density polyethylene (LDPE) on the mechanical properties and matrix crystallinity, by comparison with composites containing synthetic calcite and aragonite CaCO₃ particles. Ground shells are from three different mollusk species from Gastropoda and Bivalvia to test different CaCO₃ microstructures from broadly expended taxa. Powders are controlled with particle sizes under 1 µm to increase the specific filler/polymer interface in the composite.

## 2. Materials and Methods

### 2.1. Materials

Low density Polyethylene Flexirene MR50 (Polimeri Europa) and commercially available Calcium Chloride dehydrate, Potassium Hydrogen Carbonate and Calcium Carbonate (Sigma Aldrich, 10 µm mean particle size) were used. The filler particles are obtained by grinding non-biogenic powders, and biogenic mollusk sea shells from three different species: i) the gastropod *Crepidula fornicata*, the most abundant parasite along the ocean French coasts made of aragonitic CaCO₃ layers; ii) the edible bivalves *Pecten maximus*, the largest scallop made of calcitic layers and; iii) *Crassostrea gigas*, the most expanded oyster species over the world also made of calcitic layers. All samples were picked from dead animals on the Channel sea coast, Sword beach (49°17′52″ N, 0°17′58″ W). Stearic acid (Fluka AG) was used to avoid agglomeration during the grinding stage and potentially improve the dispersion state of calcium carbonate particles in the polymer matrix.

### 2.2. Fillers elaboration

Biogenic CaCO₃ fillers were obtained by powderising mollusk shells. Complete shells were introduced for grinding in laboratory with Retsch planetary ball milling machine (Retsch, Haan, Germany). The resulting powders are then composed of an average of the microstructures (aragonitic crossed lamellaes, foliated calcite, columnar, lath-type and rod type fibrous prismatic, chalky lenses, lamellar, parallel lamellar) composing the whole shell. To grind samples, we used different milling conditions because of the varying hardness between samples and to obtain the same mean particle sizes (as controlled using different grinding times and conditions). We ground each shell different grinding rotation speed and time because thickness and hardness of shells is different. The eventually remaining inside soft tissues were removed carefully and washed with pure water.
Purely mineral CaCO₃ fillers had also to go through some grinding in order to obtain similar grain sizes as in the case of biogenic grains. In this case we used a mixture of 10g of calcium carbonate, 50 ml of distilled water and 2 wt% of polyacrylic acid in the ball milling machine. The wet mixture obtained after two days of grinding at 300 rpm, 5 repetitions, 38 minutes, and then 300 rpm, 15 repetitions, 38 minutes, was dried at 80°C for one day in air.

The metastable synthetic aragonite calcium carbonate polymorph was prepared by precipitation according to the method of Lucas et al (2000) [20], at a temperature of 80°C to help aragonite formation [21]:

$$2 KHCO_3 + CaCl_2 \rightarrow CaCO_3 + CO_2 + 2 KCl + H_2O (1)$$

After cooling down to room temperature, aragonite precipitates were washed with distilled water, and dried for one day at 120 °C.

Stearic acid fillers coating was obtained in the following way. After dissolution of 0,4 g of NaOH in 200 ml of distilled water, 1g of stearic acid was added under magnetic stirring at 250 rpm during 1h 45 min at a temperature of 80 °C. Then, 10 g of CaCO₃ was added to the solution for 2h. After filtration, the powder was washed with hot water to eliminate remaining pure stearic acid, before drying at 120°C for one day.

## 2.3. Composites elaboration

Whatever the powders (biogenic or not, coated or not, calcite or aragonite) used to elaborate the composites, Ground Calcium Carbonate (GCC) powders and low density Polyethylene (LDPE) were dried under vacuum conditions for a minimum of 24h before processing. Processing experiments are performed with a 15 cm³ DSM Xplore (Geleen, Netherlands) corotating twin screw µ-extruder. The barrel temperature is set at 200°C and the screw speed at 100 rpm. LDPE is melt blended with 10 of GCC during 2 min. Then, the blend is injected and molded with an Xplore 10 cm³ injection unit in a mold whose temperature is set at 30°C to obtain normalized tensile testing dog bone samples (ISO-527-2-5A) [22-23].

## 2.4. Samples Characterization

Sample's morphology and particle sizes were examined by Scanning Electron Microscopy (SEM, Carl ZEISS SUPRA 55) in secondary electrons mode. We used an applied voltage of 3kV, a 30 µm diaphragm aperture and a working distance of 4-10 mm, to avoid samples' charging as much as possible. Observations were performed on non-coated samples when this was possible. However, peculiarly for GCC-PE composites in which PE prevents any reasonable electrical conduction, carbon-sputtered sample surfaces were studied. We examined shell microstructures using fractured shell cross sections. The terminology of shell microstructures is usually based on the morphology of sub-units visible with a SEM. Shell microstructures were described using the terminology of Carter and Clark (1985), but emphasise that these definitions only represent a terminology (the names are convenient, brief summaries of observed morphologies), not necessarily a statement of homology. The 'first-order' and 'second-order' (prisms, lamellae), was used to describe increasingly fine microstructural elements with morphological distinction [24]. For instance, simple crossed lamellar structure is composed of 'first-order lamellae' (approx. 10 µm thick), each of which is composed of 'second-order lamellae' (1 µm in thickness). The shell reference frame is defined by the growth (G), margin (M) and normal (N) directions. More details of macroscopic shell frames and microstructural types can be found elsewhere [25].

All samples were analysed by X-ray diffraction using a D8 Advance Vario1 Bruker diffractometer equipped with a front Johansson Ge(111) monochromator and a LynxEye™ detector (using a detection window of 3°). We used the Cu Kα₁ radiation (λ = 1.54060 Å). The patterns were measured at room temperature in a 2θ range from 10° to 110° (0.0105° increment). The instrument aberrations were calibrated using the LaB₆ srmb standard from NIST. Quantitative phase analysis, quantitative line broadening (microstructure) analysis and unit-cell parameters were refined using the Combined Analysis method [26], using an enhanced Rietveld-like fit [27] with
Purely mineral CaCO₃ fillers had also to go through some grinding in order to obtain similar grain sizes as in the case of biogenic grains. In this case we used a mixture of 10g of calcium carbonate, 50 ml of distilled water and 2 wt% of polyacrylic acid in the ball milling machine. The wet mixture obtained after two days of grinding at 300 rpm, 5 repetitions, 38 minutes, and then 300 rpm, 15 repetitions, 38 minutes, was dried at 80°C for one day in air.

The metastable synthetic aragonite calcium carbonate polymorph was prepared by precipitation according to the method of Lucas et al (2000) [20], at a temperature of 80°C to help aragonite formation [21]:

$$2 KHCO_3 + CaCl_2 \rightarrow CaCO_3 + CO_2 + 2 KCl + H_2O (1)$$

After cooling down to room temperature, aragonite precipitates were washed with distilled water, and dried for one day at 120 °C.

Stearic acid fillers coating was obtained in the following way. After dissolution of 0,4 g of NaOH in 200 ml of distilled water, 1g of stearic acid was added under magnetic stirring at 250 rpm during 1h 45 min at a temperature of 80 °C. Then, 10 g of CaCO₃ was added to the solution for 2h. After filtration, the powder was washed with hot water to eliminate remaining pure stearic acid, before drying at 120°C for one day.

## 2.3. Composites elaboration

Whatever the powders (biogenic or not, coated or not, calcite or aragonite) used to elaborate the composites, Ground Calcium Carbonate (GCC) powders and low density Polyethylene (LDPE) were dried under vacuum conditions for a minimum of 24h before processing. Processing experiments are performed with a 15 cm³ DSM Xplore (Geleen, Netherlands) corotating twin screw µ-extruder. The barrel temperature is set at 200°C and the screw speed at 100 rpm. LDPE is melt blended with 10 of GCC during 2 min. Then, the blend is injected and molded with an Xplore 10 cm³ injection unit in a mold whose temperature is set at 30°C to obtain normalized tensile testing dog bone samples (ISO-527-2-5A) [22-23].

## 2.4. Samples Characterization

Sample's morphology and particle sizes were examined by Scanning Electron Microscopy (SEM, Carl ZEISS SUPRA 55) in secondary electrons mode. We used an applied voltage of 3kV, a 30 µm diaphragm aperture and a working distance of 4-10 mm, to avoid samples' charging as much as possible. Observations were performed on non-coated samples when this was possible. However, peculiarly for GCC-PE composites in which PE prevents any reasonable electrical conduction, carbon-sputtered sample surfaces were studied. We examined shell microstructures using fractured shell cross sections. The terminology of shell microstructures is usually based on the morphology of sub-units visible with a SEM. Shell microstructures were described using the terminology of Carter and Clark (1985), but emphasise that these definitions only represent a terminology (the names are convenient, brief summaries of observed morphologies), not necessarily a statement of homology. The 'first-order' and 'second-order' (prisms, lamellae), was used to describe increasingly fine microstructural elements with morphological distinction [24]. For instance, simple crossed lamellar structure is composed of 'first-order lamellae' (approx. 10 µm thick), each of which is composed of 'second-order lamellae' (1 µm in thickness). The shell reference frame is defined by the growth (G), margin (M) and normal (N) directions. More details of macroscopic shell frames and microstructural types can be found elsewhere [25].

All samples were analysed by X-ray diffraction using a D8 Advance Vario1 Bruker diffractometer equipped with a front Johansson Ge(111) monochromator and a LynxEye™ detector (using a detection window of 3°). We used the Cu Kα₁ radiation (λ = 1.54060 Å). The patterns were measured at room temperature in a 2θ range from 10° to 110° (0.0105° increment). The instrument aberrations were calibrated using the LaB₆ srmb standard from NIST. Quantitative phase analysis, quantitative line broadening (microstructure) analysis and unit-cell parameters were refined using the Combined Analysis method [26], using an enhanced Rietveld-like fit [27] with
the help of the MAUD (Materials Analysis Using
Diffraction) software [28]. No attempt was made
to refine atomic positions during the fits in our
case of ground shells and powders, such
parameters being best approached on textured
layers [29]. Aragonite and Calcite initial structures
used in the fits were taken from the
Crystallography Open Database [30], entries n°
2100187 and 4502441 [31-32], with the respective
space groups and unit-cell parameters (Pmcn:
a=4.96Å, b=7.97Å, c=5.74Å and R-3c:H:
a=4.99Å, c=17.05Å). When possible the mean
crystallite shapes were refined within Combined
Analysis using the Popa model [33].

**3. Results and Discussion**

**3.1. Microstructure and structure analyses
using SEM and XRD**

**3.1.1. Biogenic CaCO<sub>3</sub>**

A cross sectional view of a *Pecten maximus* flat
valve (Figure 1a), taken at 2/3 of the shell radius
(far from the myostracum) illustrates the complex
architecture of the Shell. From the Inner (bottom)
to the Outer (top) sides of the valve, a stacking of
all calcitic Complex Crossed Lamellar (ICoCL),
Intermediate Irregular Prisms (IIP), and Complex
Crossed Lamellar (CoCL) layers are observed, with
varying thicknesses along the growth direction
(horizontal). The ICoCL layers are made of
bladelike lamellaes, typically 10 micrometers long,
1 µm wide and 0.1 µm thick (Figure 1b), with their
largest surface approximately parallel to the (G,M)
plane. The lamellaes group into bundles of several
hundreds which intersect each others at angles of
around 120° (Figure 1c). The number of IIP layers
varies from place to place in the shell with a
general trend to lower or even disappear close to
the juvenile growth stage. Also, at some places and
more often at adult stage, some lamellae intercalate
between some irregular prism layers (Figure 1a,
between the two top most IIP layers, zoomed in
Figure 1d). The prisms are indeed very irregular in
shape (Figure 1e), neither made of subunits as in
columnar nacre of *Turbo undulatus* [26] nor forming
well delineated paralepiped as in calcite prisms of
*Atrina serrata* [35]. Except just below the adductor
muscle, a location which we tried to remove as
much as possible for our analyses, the mineral part

Thermogravimetric analyses (TGA) were
performed using a Perkin Elmer TGA 7 analyzer
with a heating rate of 20°C.min⁻¹ from 50 to 900
°C under nitrogen flow (80 ml/min). The value of
the residue at 550°C (i.e. before CaCO₃
decarbonation) was used in order to determine the
mineral content of the processed composite
materials.

Crystallinity of the composites has been
studied with Differential Scanning Calorimetry
(DSC) using a Perkin Elmer DSC 4000 analyser,
calibrated with indium, with a heating rate of
10°C.min⁻¹ from 20 to 250 °C under nitrogen flow.

of all the shell layers is calcite (Supplementary
material Figure S1), with a refined value of
99.8(3)%. The refined cell parameters of this
calcite are a=4.99(5)Å and c=17.08(2)Å,
representing 0.03% and 0.16% of relative cell
distortion along the two main axes respectively.
Such weak levels of cell distortions are commonly
observed in biogenic calcite layers [32] when the
layers are powderized. The mean coherent size
domains after grinding are refined as roughly
equisized (56(3) nm) prisms, i.e. the individual
first-order lamellae of the ICoCL and OCoCL
have been reduced to few crystallites along their
thickness and typically 10 along their widths.

*Crepidula fornicata* cross sections reveal three
different layers, all of the crossed lamellar
microstructure (Figure 2a). The Outer and Inner
Comarginal Crossed Lamellar layers (OCCL and
ICCL resp.) sandwich an Intermediate Radial one
(IRCL). All first-order and second-order lamellaes
(Figure 2b) for these three layers are of similar
shapes and sizes as those observed in other
gastropods like *Charonia lampas* [29], with several
micrometers long laths and typically submicronic
sizes in the other two directions. As usual in
Crossed Lamellar layers, the major mineral part is
aragonite, with only 1.7(3)% of calcite
(Supplementary material Figure S2). The refined
unit-cell parameters of aragonite are a=4.96 (2)Å,
b= 7.96 (4)Å and c= 5.75(3)Å, *i.e.* no cell
distortion along a, and only 0.05% distortions
along b and c axes compared to non biogenic
the help of the MAUD (Materials Analysis Using
Diffraction) software [28]. No attempt was made
to refine atomic positions during the fits in our
case of ground shells and powders, such
parameters being best approached on textured
layers [29]. Aragonite and Calcite initial structures
used in the fits were taken from the
Crystallography Open Database [30], entries n°
2100187 and 4502441 [31-32], with the respective
space groups and unit-cell parameters (Pmcn:
a=4.96Å, b=7.97Å, c=5.74Å and R-3c:H:
a=4.99Å, c=17.05Å). When possible the mean
crystallite shapes were refined within Combined
Analysis using the Popa model [33].

**3. Results and Discussion**

**3.1. Microstructure and structure analyses
using SEM and XRD**

**3.1.1. Biogenic CaCO<sub>3</sub>**

A cross sectional view of a *Pecten maximus* flat
valve (Figure 1a), taken at 2/3 of the shell radius
(far from the myostracum) illustrates the complex
architecture of the Shell. From the Inner (bottom)
to the Outer (top) sides of the valve, a stacking of
all calcitic Complex Crossed Lamellar (ICoCL),
Intermediate Irregular Prisms (IIP), and Complex
Crossed Lamellar (CoCL) layers are observed, with
varying thicknesses along the growth direction
(horizontal). The ICoCL layers are made of
bladelike lamellaes, typically 10 micrometers long,
1 µm wide and 0.1 µm thick (Figure 1b), with their
largest surface approximately parallel to the (G,M)
plane. The lamellaes group into bundles of several
hundreds which intersect each others at angles of
around 120° (Figure 1c). The number of IIP layers
varies from place to place in the shell with a
general trend to lower or even disappear close to
the juvenile growth stage. Also, at some places and
more often at adult stage, some lamellae intercalate
between some irregular prism layers (Figure 1a,
between the two top most IIP layers, zoomed in
Figure 1d). The prisms are indeed very irregular in
shape (Figure 1e), neither made of subunits as in
columnar nacre of *Turbo undulatus* [26] nor forming
well delineated paralepiped as in calcite prisms of
*Atrina serrata* [35]. Except just below the adductor
muscle, a location which we tried to remove as
much as possible for our analyses, the mineral part

Thermogravimetric analyses (TGA) were
performed using a Perkin Elmer TGA 7 analyzer
with a heating rate of 20°C.min⁻¹ from 50 to 900
°C under nitrogen flow (80 ml/min). The value of
the residue at 550°C (i.e. before CaCO₃
decarbonation) was used in order to determine the
mineral content of the processed composite
materials.

Crystallinity of the composites has been
studied with Differential Scanning Calorimetry
(DSC) using a Perkin Elmer DSC 4000 analyser,
calibrated with indium, with a heating rate of
10°C.min⁻¹ from 20 to 250 °C under nitrogen flow.

of all the shell layers is calcite (Supplementary
material Figure S1), with a refined value of
99.8(3)%. The refined cell parameters of this
calcite are a=4.99(5)Å and c=17.08(2)Å,
representing 0.03% and 0.16% of relative cell
distortion along the two main axes respectively.
Such weak levels of cell distortions are commonly
observed in biogenic calcite layers [32] when the
layers are powderized. The mean coherent size
domains after grinding are refined as roughly
equisized (56(3) nm) prisms, i.e. the individual
first-order lamellae of the ICoCL and OCoCL
have been reduced to few crystallites along their
thickness and typically 10 along their widths.

*Crepidula fornicata* cross sections reveal three
different layers, all of the crossed lamellar
microstructure (Figure 2a). The Outer and Inner
Comarginal Crossed Lamellar layers (OCCL and
ICCL resp.) sandwich an Intermediate Radial one
(IRCL). All first-order and second-order lamellaes
(Figure 2b) for these three layers are of similar
shapes and sizes as those observed in other
gastropods like *Charonia lampas* [29], with several
micrometers long laths and typically submicronic
sizes in the other two directions. As usual in
Crossed Lamellar layers, the major mineral part is
aragonite, with only 1.7(3)% of calcite
(Supplementary material Figure S2). The refined
unit-cell parameters of aragonite are a=4.96 (2)Å,
b= 7.96 (4)Å and c= 5.75(3)Å, *i.e.* no cell
distortion along a, and only 0.05% distortions
along b and c axes compared to non biogenic
aragonite. Since all crossed lamellar layers do not exhibit the same level of cell distortions [2], we observe here the average distortion coming from all three layers cumulated to the powderizing effect, both tending to distortions isotropization. The mean crystallites after grinding are revealed elongated along their c-axes (70.6(6) nm) and quasi isotropic along the other two main directions

(32.4(9) nm and 31.0(9) nm along **a** and **b** axes resp.). As previously for the calcite layers of *Pecten maximus*, several crystallites are necessary to build up the elongated laths seen in SEM images, but a full Combined Analysis including a quantitative description of the layers' textures are required to give more reliable descriptions [29].

<figure><img src="image_1.png" /><figcaption>**Figure 1.** SEM observation of a cross section of the flat valve of *Pecten maximus*. a) global view at approximately 2/3rd of the total shell along **G**. b) zoom on first-order lamellae of the OCoCL top layer. c) Intersected bundles of first-order lamellae. d) One IIP layer sandwiched between the ICoCL and OCoCL layers at 1/5th of the total shell. e) zoom on the irregular prisms of the IIP layer.</figcaption></figure>

<figure><img src="image_2.png" /><figcaption>**Figure 2.** SEM observation of a cross section of the *Crepidula fornicata* shell. a) global view at approximately 2/3rd of the total shell along **G** (Outer an Inner sides of the shell are at top and bottom resp. **G** is horizontal, **N** vertical. b) zoom on first- and second-order lamellae of the ICCL layer.</figcaption></figure>

The second, fully calcitic bivalve *Crassostrea gigas* which we undertook in this study (Figure 3), is made of several microstructural layers, mainly foliated calcites (FC) located in the inner parts of the Shell, and an Outer Prismatic Calcite (OPC).

This complex stacking incorporates at least four different types of microstructures including chalky-like [35], prisms [36], granular and semi-nacres [37], all containing only calcite as mineral (Supplementary material Figure S3). With refined
aragonite. Since all crossed lamellar layers do not exhibit the same level of cell distortions [2], we observe here the average distortion coming from all three layers cumulated to the powderizing effect, both tending to distortions isotropization. The mean crystallites after grinding are revealed elongated along their c-axes (70.6(6) nm) and quasi isotropic along the other two main directions

(32.4(9) nm and 31.0(9) nm along **a** and **b** axes resp.). As previously for the calcite layers of *Pecten maximus*, several crystallites are necessary to build up the elongated laths seen in SEM images, but a full Combined Analysis including a quantitative description of the layers' textures are required to give more reliable descriptions [29].

<figure><img src="image_2.png" /><figcaption>**Figure 1.** SEM observation of a cross section of the flat valve of *Pecten maximus*. a) global view at approximately 2/3rd of the total shell along **G**. b) zoom on first-order lamellae of the OCoCL top layer. c) Intersected bundles of first-order lamellae. d) One IIP layer sandwiched between the ICoCL and OCoCL layers at 1/5th of the total shell. e) zoom on the irregular prisms of the IIP layer.</figcaption></figure>

<figure><img src="image_3.png" /><figcaption>**Figure 2.** SEM observation of a cross section of the *Crepidula fornicata* shell. a) global view at approximately 2/3rd of the total shell along **G** (Outer an Inner sides of the shell are at top and bottom resp. **G** is horizontal, **N** vertical. b) zoom on first- and second-order lamellae of the ICCL layer.</figcaption></figure>

The second, fully calcitic bivalve *Crassostrea gigas* which we undertook in this study (Figure 3), is made of several microstructural layers, mainly foliated calcites (FC) located in the inner parts of the Shell, and an Outer Prismatic Calcite (OPC).

This complex stacking incorporates at least four different types of microstructures including chalky-like [35], prisms [36], granular and semi-nacres [37], all containing only calcite as mineral (Supplementary material Figure S3). With refined
cell parameters of $a=4.99(1)Å$ and $c=17.05(6)Å$, the 0.03% and 0.01% relative distortions are again very small, and probably averages of more pronounced ones existing in the original biogenic textured layers. Indeed, strong textures have been observed in *Crassostrea gigas* layers [38 and 39], without analysed consequences on cell distortions.

The mean crystal sizes after grinding are also roughly isotropic (58(2) nm and 52(3) nm along *c* and *a* axes resp.), and much smaller than the grain sizes visible in SEM images. Only the platelets (Figure 3c) of semi nacre might be made of a single crystallite along their thickness.

<figure><img src="image_3.png" /><figcaption>**Figure 3.** SEM observation of a cross section of the flat valve of *Crassostrea gigas* shell. **a)** Complex architecture made of several microstructural layers, foliated and prismatic calcites. **b)** outermost layers (Outside is at top, **G** is horizontal, **N** vertical), showing a foliated calcite layer and the outer prisms. **c)** zoom on semi nacre tablets, **d)** outer prisms, **e)** Foliated calcite and **f)** chalky calcite.</figcaption></figure>

The three selected shell species represent large variations in microstructural types, i.e. distant organic matrices, including two different calcium carbonate polymorphs (aragonite and calcite) exhibiting very different elastic behaviours, and for the calcite polymorph different microstructural elements (prisms, tablets, laths). Biogenic calcite and aragonite might offer different bondings to the polymer matrix via their organic elements, and together with their mineral constituents result in different plastic and elastic characteristics of the CaCO$_3$-PE composites [40].

### 3.1.2. Non-biogenic CaCO$_3$

#### 3.1.3. Ground and Stearic Acid coated samples

Sample grinding can affect not only crystal and grain sizes, but also introduce microstrains and cell distortions. In the case of commercial calcite, our grinding conditions revealed a crystallite mean

size reduction (Supplementary Figure S4c and d) by typically 15 times after grinding (150(2) nm). On the other hand, the microstrain level and cell distortion are insignificantly modified (3.7(4) $10^{-4}$ r.m.s., $a = 4.99(6)$ Å and $c = 17.06(3)$ Å respectively). The mean crystallite shapes are also modified under grinding (Supplementary Figure S4b and c insets), from roughly spherical in the as received calcite powder to more rhomb-like in the ground sample. Such shape modification associated to the crystallite size decrease indicates that some gliding system has been activated during grinding, linked to the rhombohedral planes of calcite. Consequently, the only effect that grinding operates on calcite crystals is to decrease their mean sizes via crystalline gliding activation, hereby increasing the coating specific surface, without any significant increase in internal energy via defect creation and cell distortions. The aspect of calcite
cell parameters of a=4.99(1)Å and c=17.05(6)Å,
the 0.03% and 0.01% relative distortions are again
very small, and probably averages of more
pronounced ones existing in the original biogenic
textured layers. Indeed, strong textures have been
observed in *Crassostrea gigas* layers [38 and 39],
without analysed consequences on cell distortions.

The mean crystal sizes after grinding are also
roughly isotropic (58(2) nm and 52(3) nm along **c**
and **a** axes resp.), and much smaller than the grain
sizes visible in SEM images. Only the platelets
(Figure 3c) of semi nacre might be made of a single
crystallite along their thickness.

<figure><img src="image_4.png" /><figcaption>**Figure 3.** SEM observation of a cross section of the flat valve of *Crassostrea gigas* shell. **a)** Complex architecture made of several microstructural layers, foliated and prismatic calcites. **b)** outermost layers (Outside is at top, **G** is horizontal, **N** vertical), showing a foliated calcite layer and the outer prisms. **c)** zoom on semi nacre tablets, **d)** outer prisms, **e)** Foliated calcite and **f)** chalky calcite.</figcaption></figure>

The three selected shell species represent
large variations in microstructural types, i.e. distant
organic matrices, including two different calcium
carbonate polymorphs (aragonite and calcite)
exhibiting very different elastic behaviours, and for
the calcite polymorph different microstructural
elements (prisms, tablets, laths). Biogenic calcite
and aragonite might offer different bondings to the
polymer matrix via their organic elements, and
together with their mineral constituents result in
different plastic and elastic characteristics of the
CaCO₃-PE composites [40].

3.1.2. Non-biogenic CaCO₃

3.1.3. Ground and Stearic Acid coated
samples

Sample grinding can affect not only crystal
and grain sizes, but also introduce microstrains and
cell distortions. In the case of commercial calcite,
our grinding conditions revealed a crystallite mean

size reduction (Supplementary Figure S4c and d)
by typically 15 times after grinding (150(2) nm).
On the other hand, the microstrain level and cell
distortion are insignificantly modified (3.7(4) 10⁻⁴
r.m.s., a = 4.99(6) Å and c = 17.06(3) Å
respectively). The mean crystallite shapes are also
modified under grinding (Supplementary Figure S4
b and c insets), from roughly spherical in the as
received calcite powder to more rhomb-like in the
ground sample. Such shape modification
associated to the crystallite size decrease indicates
that some gliding system has been activated during
grinding, linked to the rhombohedral planes of
calcite. Consequently, the only effect that grinding
operates on calcite crystals is to decrease their
mean sizes via crystalline gliding activation, hereby
increasing the coating specific surface, without any
significant increase in internal energy via defect
creation and cell distortions. The aspect of calcite
after grinding can be seen on Supplementary
Figure S5b.

A very similar process occurs while grinding
shell layers (Supplementary Figure S1, 2 and 3),
although initial crystals formed various and
different habits. Ground biogenic calcite layers
(Supplementary Figure S1 and 3) also exhibit
rhomb-like calcite crystals, i.e. inter- and intra-
crystalline molecules do not hinder intrinsic
mineral deformation processes in such layers. In
these shell powders we recognize initial
microstructural elements present in the shells, e.g.
lamellae of *C. fornicata* (Supplementary Figure S2
inset) and *Pecten maximus* (Supplementary Figure
S1 inset) or foliae of *C. gigas* (Supplementary Figure
S3 inset), together with more roundish and smaller
grains due to grinding indicating that this latter did
not affect all initial crystals.

Ground calcium carbonate particles usually
exhibit strong tendency to aggregate, which would
prevent good dispersion within the final
composite. Therefore, a suitable surfactant for the
surface treatment of these particles is necessary, to
hindер aggregates formation. As in previous works
we chose Stearic Acid (SA) to ensure full coverage
of the particles, whether biogenic or not [41-42].
Once SA coated, ground calcite indeed appears
more homogeneously distributed, with a lower
level of aggregation than the uncoated calcite
(Supplementary Figure S6).

Synthesized aragonite and commercial calcite
powders exhibit needle-like (Supplementary Figure
S4a) and rhombic (Supplementary Figure S5a)
grains respectively. Such crystal shapes are usual
for these two polytypes of calcium carbonate.
Aragonite exhibits average grain lengths and
widths varying in the 10-55 µm and 0.5-3.5 µm
ranges respectively. The mean crystallite sizes of
calcite as determined from XRD (Supplementary
Figure S4c) is 2400(200) nm with a low level of
microstrains (3.4(1) 10⁻⁴ r.m.s.) and cell parameter
distortions (a = 4.99(4) Å and c = 17.06(2) Å).

**3.2. Composite materials**

**3.2.1. SEM characterisation of filler
dispersion in composite materials**

The dispersion and spatial distribution of
fillers in the composites were investigated by SEM
on plastically fractured composites samples after

tensile testing. From Figure 4, it can be concluded
that the composites processing does not
significantly decresase nor increase the calcite
aggregates size since the particles observable are
similar in size and aspect to those of the
Supplementary Figures S5 and 6. On the other
hand aragonite needles seem to have been
fractured during the processing since their average
length is strongly reduced. Moreover it appears
clearly that the interfacial interactions between
uncoated particles and the polyethylene matrix are
weak. No embedded particles are detectable
suggesting the failure if the composites originated
from their interface with the matrix during
polyethylene plastic deformation. Because of lack
of chemical bonding between matrix–filler and
high stress in front of agglomerated particles,
cracks propagate easily [43]. No polymer residue
can be evidenced at the surface of these particles
either. Therefore it can be concluded that the
adherence level of the matrix to these uncoated
synthetic particles is very low. Regarding biogenic
fillers, the analysis on SEM pictures is more
difficult due to the heterogeneous shapes of the
CaCO₃ crystals. Biogenic calcite from oyster shells
seem to show very low compatibility with the
apolar polyethylene matrix since no residue is
observed on aggregates present in the fractured
region and that some decohesion zones are visible
on partially embedded particles. Concerning
biogenic aragonite, the adhesion level seems
slightly higher since *C. Fornicata* particles remained
embedded with polyethylene residue at their
surface.

Figure 5 illustrates the influence of the stearic
acid coating in the case of *Crepidula fornicata* in the
polyethylene matrix, which is representative of all
the characterised composites. The compatibility of
the fillers with the apolar matrix has been
improved since the CaCO3 particles remain
embedded in the matrix.

**3.2.2. Composite properties**

The mechanical properties of filled polymers
depend on the filler mechanical properties and the
quality of their interface with the polymer matrix,
but they are also strongly related to the crystallinity
of the matrix and to the filler content [44]. Table 1
reports the crystallinity values obtained from DSC
after grinding can be seen on Supplementary
Figure S5b.

A very similar process occurs while grinding
shell layers (Supplementary Figure S1, 2 and 3),
although initial crystals formed various and
different habits. Ground biogenic calcite layers
(Supplementary Figure S1 and 3) also exhibit
rhomb-like calcite crystals, i.e. inter- and intra-
crystalline molecules do not hinder intrinsic
mineral deformation processes in such layers. In
these shell powders we recognize initial
microstructural elements present in the shells, e.g.
lamellae of *C. fornicata* (Supplementary Figure S2
inset) and *Pecten maximus* (Supplementary Figure
S1 inset) or foliae of *C. gigas* (Supplementary Figure
S3 inset), together with more roundish and smaller
grains due to grinding indicating that this latter did
not affect all initial crystals.

Ground calcium carbonate particles usually
exhibit strong tendency to aggregate, which would
prevent good dispersion within the final
composite. Therefore, a suitable surfactant for the
surface treatment of these particles is necessary, to
hindер aggregates formation. As in previous works
we chose Stearic Acid (SA) to ensure full coverage
of the particles, whether biogenic or not [41-42].
Once SA coated, ground calcite indeed appears
more homogeneously distributed, with a lower
level of aggregation than the uncoated calcite
(Supplementary Figure S6).

Synthesized aragonite and commercial calcite
powders exhibit needle-like (Supplementary Figure
S4a) and rhombic (Supplementary Figure S5a)
grains respectively. Such crystal shapes are usual
for these two polytypes of calcium carbonate.
Aragonite exhibits average grain lengths and
widths varying in the 10-55 µm and 0.5-3.5 µm
ranges respectively. The mean crystallite sizes of
calcite as determined from XRD (Supplementary
Figure S4c) is 2400(200) nm with a low level of
microstrains (3.4(1) 10⁻⁴ r.m.s.) and cell parameter
distortions (a = 4.99(4) Å and c = 17.06(2) Å).

**3.2. Composite materials**

**3.2.1. SEM characterisation of filler
dispersion in composite materials**

The dispersion and spatial distribution of
fillers in the composites were investigated by SEM
on plastically fractured composites samples after

tensile testing. From Figure 4, it can be concluded
that the composites processing does not
significantly decresase nor increase the calcite
aggregates size since the particles observable are
similar in size and aspect to those of the
Supplementary Figures S5 and 6. On the other
hand aragonite needles seem to have been
fractured during the processing since their average
length is strongly reduced. Moreover it appears
clearly that the interfacial interactions between
uncoated particles and the polyethylene matrix are
weak. No embedded particles are detectable
suggesting the failure if the composites originated
from their interface with the matrix during
polyethylene plastic deformation. Because of lack
of chemical bonding between matrix–filler and
high stress in front of agglomerated particles,
cracks propagate easily [43]. No polymer residue
can be evidenced at the surface of these particles
either. Therefore it can be concluded that the
adherence level of the matrix to these uncoated
synthetic particles is very low. Regarding biogenic
fillers, the analysis on SEM pictures is more
difficult due to the heterogeneous shapes of the
CaCO₃ crystals. Biogenic calcite from oyster shells
seem to show very low compatibility with the
apolar polyethylene matrix since no residue is
observed on aggregates present in the fractured
region and that some decohesion zones are visible
on partially embedded particles. Concerning
biogenic aragonite, the adhesion level seems
slightly higher since *C. Fornicata* particles remained
embedded with polyethylene residue at their
surface.

Figure 5 illustrates the influence of the stearic
acid coating in the case of *Crepidula fornicata* in the
polyethylene matrix, which is representative of all
the characterised composites. The compatibility of
the fillers with the apolar matrix has been
improved since the CaCO₃ particles remain
embedded in the matrix.

**3.2.2. Composite properties**

The mechanical properties of filled polymers
depend on the filler mechanical properties and the
quality of their interface with the polymer matrix,
but they are also strongly related to the crystallinity
of the matrix and to the filler content [44]. Table 1
reports the crystallinity values obtained from DSC
measurements, the filler content deduced from TGA analysis as well as the stiffness of the filled polymers (G* modulus obtained from DMA in torsion) and their ultimate tensile properties determined by tensile testing.

All the filled materials contain approximately 10 wt% of filler as initially planned except PE-C. Gigas, PE-C. Fornicata and PE-Calcite-SA which are significantly less filled. This has been taken into account to evaluate the matrix crystallinity. The matrix crystallinity is slightly lowered for all the filled systems except those containing synthetic aragonite. For the biogenic $CaCO_3$ containing composites, no direct link between the $CaCO_3$ nature and the crystallinity of the matrix can be established. The SA coating does not seem to influence significantly the matrix crystallinity either. These slight changes in polyethylene crystallinity do not overcome the effect of filler incorporation on the polymer mechanical properties. Due to their intrinsic stiffness most fillers contribute to an improvement of the polymer matrix G* modulus. According to the data reported in Table 1, it seems that biogenic calcium carbonate is less efficient in improving polyethylene stiffness than the synthetic ones, independently of its crystalline form. This might be attributed to the greater tendency to form

aggregates of the grinded mollusk shells and their lesser morphological homogeneity. Using stearic acid coating allows an improvement of the matrix stiffening, probabling by avoiding excessive aggregation and allows reaching modulus values which are of the same order as those obtained with synthtic calcite or aragonite. Regarding the yield strength, it is unchanged whatever the kind of filler used, which suggests that interfacial interactions are rather poor in those composites. This is confirmed by the strain at break values which are always lower than for pure polyethylene. This embrittlement is probably caused by earlier crack propagation initiated at the filler/polymer interfaces due to stress concentration and poor interfacial bonding.

The bonding to the polymer matrix which could have been expected with these various biogenic $CaCO_3$ forms due to the organic elements they contain is not significantly different from one species to another from a mechanical point of view. However these biogenic fillers are not detrimental to the polymers mechanical properties and allow reaching the same properties than traditionnally filled polymers. This opens the possibility to use these marine industrial wastes in the polymer compounding industry.

<figure><img src="image_4.png" /><figcaption>**Figure 4.** SEM pictured of fractured PE-Ca$CO_3$ Tensile Specimens a) PE-calcite b) PE-aragonite c) PE-C. gigas d) PE-C. Fornicata</figcaption></figure>
measurements, the filler content deduced from TGA analysis as well as the stiffness of the filled polymers (G* modulus obtained from DMA in torsion) and their ultimate tensile properties determined by tensile testing.

All the filled materials contain approximately 10 wt% of filler as initially planned except PE-C. Gigas, PE-C. Fornicata and PE-Calcite-SA which are significantly less filled. This has been taken into account to evaluate the matrix crystallinity. The matrix crystallinity is slightly lowered for all the filled systems except those containing synthetic aragonite. For the biogenic $CaCO_3$ containing composites, no direct link between the $CaCO_3$ nature and the crystallinity of the matrix can be established. The SA coating does not seem to influence significantly the matrix crystallinity either. These slight changes in polyethylene crystallinity do not overcome the effect of filler incorporation on the polymer mechanical properties. Due to their intrinsic stiffness most fillers contribute to an improvement of the polymer matrix G* modulus. According to the data reported in Table 1, it seems that biogenic calcium carbonate is less efficient in improving polyethylene stiffness than the synthetic ones, independently of its crystalline form. This might be attributed to the greater tendency to form

aggregates of the grinded mollusk shells and their lesser morphological homogeneity. Using stearic acid coating allows an improvement of the matrix stiffening, probabling by avoiding excessive aggregation and allows reaching modulus values which are of the same order as those obtained with synthtic calcite or aragonite. Regarding the yield strength, it is unchanged whatever the kind of filler used, which suggests that interfacial interactions are rather poor in those composites. This is confirmed by the strain at break values which are always lower than for pure polyethylene. This embrittlement is probably caused by earlier crack propagation initiated at the filler/polymer interfaces due to stress concentration and poor interfacial bonding.

The bonding to the polymer matrix which could have been expected with these various biogenic $CaCO_3$ forms due to the organic elements they contain is not significantly different from one species to another from a mechanical point of view. However these biogenic fillers are not detrimental to the polymers mechanical properties and allow reaching the same properties than traditionnally filled polymers. This opens the possibility to use these marine industrial wastes in the polymer compounding industry.

<figure><img src="image_5.png" /><figcaption>**Figure 4.** SEM pictured of fractured PE-CaCO₃ Tensile Specimens a) PE-calcite b) PE-aragonite c) PE-C. gigas d) PE-C. Fornicata</figcaption></figure>
<figure><img src="image_5.png" /></figure>

<figure><img src="image_6.png" /><figcaption>Figure 5. SEM pictures of PE-C. Fornicata coated with SA.</figcaption></figure>

<table><caption>Table 1. The shear modulus, young modulus and yield stress of the composite materials</caption><thead><tr><th></th><th>Filler content (wt%)</th><th>Matrix Crystallinity (%)</th><th>G* (MPa)</th><th>σ<sub>y</sub> (MPa) (±0,5 MPa)</th><th>ε<sub>r</sub> (%) (±80 %)</th></tr></thead><tbody><tr><td>PE</td><td>0</td><td>52</td><td>2,8</td><td>16</td><td>1100</td></tr><tr><td>PE-Calcite</td><td>9,6</td><td>48</td><td>3,2</td><td>16</td><td>720</td></tr><tr><td>PE-Calcite-SA</td><td>7,7</td><td>48</td><td>3,1</td><td>15</td><td>720</td></tr><tr><td>PE-Aragonite</td><td>10,3</td><td>51</td><td>3,45</td><td>15</td><td>910</td></tr><tr><td>PE-Aragonite-SA</td><td>9,3</td><td>53</td><td>2,6</td><td>16</td><td>930</td></tr><tr><td>PE-C. Fornicata</td><td>8,6</td><td>49</td><td>2,8</td><td>16</td><td>670</td></tr><tr><td>PE-C. Fornicata-SA</td><td>9,5</td><td>49</td><td>3</td><td>15</td><td>740</td></tr><tr><td>PE-C. Gigas</td><td>6,5</td><td>52</td><td>2,8</td><td>16</td><td>730</td></tr><tr><td>PE-C. Gigas-SA</td><td>9,3</td><td>50</td><td>3,2</td><td>15</td><td>830</td></tr><tr><td>PE-P. Maximus</td><td>10,8</td><td>47</td><td>3</td><td>16</td><td>680</td></tr><tr><td>PE-P. Maximus-SA</td><td>9,7</td><td>50</td><td>3,2</td><td>16</td><td>760</td></tr></tbody></table>

## 4. Conclusions

In this study, shell structures of *Crepidula fornicata*, *Crassostrea gigas* and *Pecten maximus* have been characterized by SEM and XRD and their potential use as polymer fillers investigated.

Lamellar and columnar shell microstructures were observed in *Pecten maximus* and *Crepidula fornicata* via scanning electron microscopy. Lamellar laths are observed in the Scallop and in the *Crepidula* species while *Crassostrea gigas* structure exhibits foliated, lamellar, columnar and chalky microstructural morphologies, very different from the microstructures of the two other species.

When these biogenic sources were incorporated in a polymer matrix after grinding, they gave rise to similar impacts on the polymer structural and mechanical properties as the synthetic mineral calcium carbonates when aggregation is limited by using a stearic acid treatment. Even if a significant effect of the presence of organic molecules in these biogenic fillers has been detected, no detrimental effects have been observed. Biogenic calcium carbonate sources, which are usually considered as wastes from several industries can therefore be used as fillers in the compounding industry and lead to composite materials with properties equivalent to classically filled polymers.
<figure><img src="image_6.png" /><figcaption>Figure 5. SEM pictures of PE-C. Fornicata coated with SA.</figcaption></figure>

<table><caption>Table 1. The shear modulus, young modulus and yield stress of the composite materials</caption><thead><tr><th></th><th>Filler content (wt%)</th><th>Matrix Crystallinity (%)</th><th>G* (MPa)</th><th>σ<sub>y</sub> (MPa) (±0,5 MPa)</th><th>ε<sub>r</sub> (%) (±80 %)</th></tr></thead><tbody><tr><td>PE</td><td>0</td><td>52</td><td>2,8</td><td>16</td><td>1100</td></tr><tr><td>PE-Calcite</td><td>9,6</td><td>48</td><td>3,2</td><td>16</td><td>720</td></tr><tr><td>PE-Calcite-SA</td><td>7,7</td><td>48</td><td>3,1</td><td>15</td><td>720</td></tr><tr><td>PE-Aragonite</td><td>10,3</td><td>51</td><td>3,45</td><td>15</td><td>910</td></tr><tr><td>PE-Aragonite-SA</td><td>9,3</td><td>53</td><td>2,6</td><td>16</td><td>930</td></tr><tr><td>PE-C. Fornicata</td><td>8,6</td><td>49</td><td>2,8</td><td>16</td><td>670</td></tr><tr><td>PE-C. Fornicata-SA</td><td>9,5</td><td>49</td><td>3</td><td>15</td><td>740</td></tr><tr><td>PE-C. Gigas</td><td>6,5</td><td>52</td><td>2,8</td><td>16</td><td>730</td></tr><tr><td>PE-C. Gigas-SA</td><td>9,3</td><td>50</td><td>3,2</td><td>15</td><td>830</td></tr><tr><td>PE-P. Maximus</td><td>10,8</td><td>47</td><td>3</td><td>16</td><td>680</td></tr><tr><td>PE-P. Maximus-SA</td><td>9,7</td><td>50</td><td>3,2</td><td>16</td><td>760</td></tr></tbody></table>

## 4. Conclusions

In this study, shell structures of *Crepidula fornicata*, *Crassostrea gigas* and *Pecten maximus* have been characterized by SEM and XRD and their potential use as polymer fillers investigated.

Lamellar and columnar shell microstructures were observed in *Pecten maximus* and *Crepidula fornicata* via scanning electron microscopy. Lamellar laths are observed in the Scallop and in the *Crepidula* species while *Crassostrea gigas* structure exhibits foliated, lamellar, columnar and chalky microstructural morphologies, very different from the microstructures of the two other species.

When these biogenic sources were incorporated in a polymer matrix after grinding, they gave rise to similar impacts on the polymer structural and mechanical properties as the synthetic mineral calcium carbonates when aggregation is limited by using a stearic acid treatment. Even if a significant effect of the presence of organic molecules in these biogenic fillers has been detected, no detrimental effects have been observed. Biogenic calcium carbonate sources, which are usually considered as wastes from several industries can therefore be used as fillers in the compounding industry and lead to composite materials with properties equivalent to classically filled polymers.
<figure><img src="image_7.png" /></figure>

## Funding

This research received no external funding.

## Conflicts of Interest

The authors declare no conflict of interest.

## Acknowledgement

This study was performed in "Laboratoire de Chimie Moléculaire et Thio-organique" (CNRS UMR 6507) and "Laboratoire de CRISTallographie et Science des MATériaux » (CNRS UMR 6508). Authors wish to thank TUBITAK 2219 post-doc research fellowship for support and also gratefully thank Clément Paul for precious discussions.

## References

1. Dauphin, Y.; Luquet, G.; Salome, M.; Bellot-Gurlet, L.; Cuif, J.P. Structure and composition of Unio pictorum shell: arguments for the diversity of the nacroprismatic arrangement in molluscs. *J. Microsc.* **2018** *Volume 270, Issue 2*, pp. 156-169.
https://doi.org/10.1111/jmi.12669

2. Chateigner, D.; Ouhenia, S.; Krauss, C.; Belkhir, M.; Morales, M. Structural distortion of biogenic aragonite in strongly textured mollusk shell layers. *Nucl. Instrum. Methods Phys. Res. B* **2010**, *Volume 268, Issues 3-4*, pp. 341-345.
https://doi.org/10.1016/j.nimb.2009.07.007

3. Chateigner, D.; Ouhenia, S.; Krauss, C.; Hedegaard, C.; Gil, O.; Morales, M.; Lutterotti, L.; Rousseau, M.; Lopez, E. Voyaging around nacre with the X-ray shuttle: From bio-mineralisation to prosthetics via mollusk phylogeny. *Mat. Sci. Eng. A-Struct.* **2010**, *Volume 528, Issue 1*, pp. 37-51.
https://doi.org/10.1016/j.msea.2010.07.032

4. Yang, W.; Zhang, G.P.; Zhu, X.F.; Li, X.W.; Meyers, M.A. Structure and mechanical properties of *Saxidomus purpuratus* biological shells. *J. Mech. Behav. Biomed. Mater.* **2011**, *Volume 4, Issue 7*, pp. 1514-1530.
https://doi.org/10.1016/j.jmbbm.2011.05.021

5. Neves, N.M.; Mano, J.F. Structure/Mechanical behaviour relationships in crossed-lamellar sea shells. *Mater. Sci. Eng. C* **2005** *Volume 25, Issue 2*, pp. 113-118

6. Lee, S.W.; Kim, G.H.; Choi, C.S. Characteristic crystal orientation of folia in oyster shell, *Crassostrea gigas*. *Mater. Sci. Eng. C* **2008**, *Volume 28, Issue 2*, pp. 258-263

7. Pokroy, B.; Fitch, A.N.; Zolotoyabko, E. The Microstructure of Biogenic Calcite: A View by High-Resolution Synchrotron Powder Diffraction. *Adv. Mater.* **2006**, *Volume 18, Issue 18*, pp. 2363-2368.
https://doi.org/10.1002/adma.200600714

8. Kunitake, M.E.; Mangano, L.M.; Peloquin, J.M.; Baker, S.P.; Stroff, L.A. Evaluation of strengthening

mechanisms in calcite single crystals from mollusc shells. *Acta Biomater.* **2013** *Volume 9, Issue 2*, pp. 5353-5359

9. Lee, S.W.; Jang, Y.N.; Ryu, K.W.; Chae, S.C.; Lee, Y.H.; Jeon C.W. Mechanical characteristics and morphological effect of complex crossed structure in biomaterials: fracture mechanics and microstructure of chalky layer in oyster shell. *Micron* **2011** *Volume 42, Issue 1*, 60-70

10. Marin, F.; Luquet, G. Molluscan shell proteins. *C. R. Palevol.* **2004**, *Volume 3, Issues 6-7*, pp. 469-492.
https://doi.org/10.1016/j.crpv.2004.07.009

11. Takeuchi, T.; Sarashina, I.; Iijima, M.; Endo, K. In vitro regulation of CaCO₃ crystal polymorphism by the highly acidic molluscan shell protein Aspein. *FEBS Lett.* **2008**, *Volume 582, Issue 5*, pp. 591-596.
https://doi.org/10.1016/j.febslet.2008.01.026

12. Morris, J.P.; Backeljau, T.; Chapelle, G. Shells from aquaculture: a valuable biomaterial, not a nuisance waste product. *Rev. Aquacult.* **2019**, *Volume 11, Issue 1*, pp. 42-57.
https://doi.org/10.1111/raq.12225

13. Fombuena, V.; Bernardi, L.; Fenollar, O.; Boronat, T.; Balart, R. Characterization of green composites from biobased epoxy matrices and bio-fillers derived from seashell wastes. *Mater. Design.* **2014**, *Volume 57*, 168-174.
https://doi.org/10.1016/j.matdes.2013.12.032

14. Samant, S.; Naik, M.M.; Vaingankar, D.C.; Mujawar, S.Y.; Parab, P.; Meena, S.N. Chapter 1: Biodegradation of fish waste by seaweed associated bacteria and application of seafood waste for ethanol production. *Advances in Biological Science Research, A Practical Approach* **2019**, Academic Press, 149-158

15. Sharma, S.K.; Pujari, P.K. Role of free volume characteristics of polymer matrix in bulk physical properties of polymer nanocomposites: A review of positron annihilation lifetime studies. *Prog. Polym. Sci.* **2017**, *Volume 75*, pp. 31-47
<figure><img src="image_7.png" /></figure>

## Funding

This research received no external funding.

## Conflicts of Interest

The authors declare no conflict of interest.

## Acknowledgement

This study was performed in "Laboratoire de Chimie Moléculaire et Thio-organique" (CNRS UMR 6507) and "Laboratoire de CRISTallographie et Science des MATériaux » (CNRS UMR 6508). Authors wish to thank TUBITAK 2219 post-doc research fellowship for support and also gratefully thank Clément Paul for precious discussions.

## References

1. Dauphin, Y.; Luquet, G.; Salome, M.; Bellot-Gurlet, L.; Cuif, J.P. Structure and composition of Unio pictorum shell: arguments for the diversity of the nacroprismatic arrangement in molluscs. *J. Microsc.* **2018** *Volume 270, Issue 2*, pp. 156-169.
https://doi.org/10.1111/jmi.12669

2. Chateigner, D.; Ouhenia, S.; Krauss, C.; Belkhir, M.; Morales, M. Structural distortion of biogenic aragonite in strongly textured mollusk shell layers. *Nucl. Instrum. Methods Phys. Res. B* **2010**, *Volume 268, Issues 3-4*, pp. 341-345.
https://doi.org/10.1016/j.nimb.2009.07.007

3. Chateigner, D.; Ouhenia, S.; Krauss, C.; Hedegaard, C.; Gil, O.; Morales, M.; Lutterotti, L.; Rousseau, M.; Lopez, E. Voyaging around nacre with the X-ray shuttle: From bio-mineralisation to prosthetics via mollusk phylogeny. *Mat. Sci. Eng. A-Struct.* **2010**, *Volume 528, Issue 1*, pp. 37-51.
https://doi.org/10.1016/j.msea.2010.07.032

4. Yang, W.; Zhang, G.P.; Zhu, X.F.; Li, X.W.; Meyers, M.A. Structure and mechanical properties of *Saxidomus purpuratus* biological shells. *J. Mech. Behav. Biomed. Mater.* **2011**, *Volume 4, Issue 7*, pp. 1514-1530.
https://doi.org/10.1016/j.jmbbm.2011.05.021

5. Neves, N.M.; Mano, J.F. Structure/Mechanical behaviour relationships in crossed-lamellar sea shells. *Mater. Sci. Eng. C* **2005** *Volume 25, Issue 2*, pp. 113-118

6. Lee, S.W.; Kim, G.H.; Choi, C.S. Characteristic crystal orientation of folia in oyster shell, *Crassostrea gigas*. *Mater. Sci. Eng. C* **2008**, *Volume 28, Issue 2*, pp. 258-263

7. Pokroy, B.; Fitch, A.N.; Zolotoyabko, E. The Microstructure of Biogenic Calcite: A View by High-Resolution Synchrotron Powder Diffraction. *Adv. Mater.* **2006**, *Volume 18, Issue 18*, pp. 2363-2368.
https://doi.org/10.1002/adma.200600714

8. Kunitake, M.E.; Mangano, L.M.; Peloquin, J.M.; Baker, S.P.; Stroff, L.A. Evaluation of strengthening

mechanisms in calcite single crystals from mollusc shells. *Acta Biomater.* **2013** *Volume 9, Issue 2*, pp. 5353-5359

9. Lee, S.W.; Jang, Y.N.; Ryu, K.W.; Chae, S.C.; Lee, Y.H.; Jeon C.W. Mechanical characteristics and morphological effect of complex crossed structure in biomaterials: fracture mechanics and microstructure of chalky layer in oyster shell. *Micron* **2011** *Volume 42, Issue 1*, 60-70

10. Marin, F.; Luquet, G. Molluscan shell proteins. *C. R. Palevol.* **2004**, *Volume 3, Issues 6-7*, pp. 469-492.
https://doi.org/10.1016/j.crpv.2004.07.009

11. Takeuchi, T.; Sarashina, I.; Iijima, M.; Endo, K. In vitro regulation of CaCO₃ crystal polymorphism by the highly acidic molluscan shell protein Aspein. *FEBS Lett.* **2008**, *Volume 582, Issue 5*, pp. 591-596.
https://doi.org/10.1016/j.febslet.2008.01.026

12. Morris, J.P.; Backeljau, T.; Chapelle, G. Shells from aquaculture: a valuable biomaterial, not a nuisance waste product. *Rev. Aquacult.* **2019**, *Volume 11, Issue 1*, pp. 42-57.
https://doi.org/10.1111/raq.12225

13. Fombuena, V.; Bernardi, L.; Fenollar, O.; Boronat, T.; Balart, R. Characterization of green composites from biobased epoxy matrices and bio-fillers derived from seashell wastes. *Mater. Design.* **2014**, *Volume 57*, 168-174.
https://doi.org/10.1016/j.matdes.2013.12.032

14. Samant, S.; Naik, M.M.; Vaingankar, D.C.; Mujawar, S.Y.; Parab, P.; Meena, S.N. Chapter 1: Biodegradation of fish waste by seaweed associated bacteria and application of seafood waste for ethanol production. *Advances in Biological Science Research, A Practical Approach* **2019**, Academic Press, 149-158

15. Sharma, S.K.; Pujari, P.K. Role of free volume characteristics of polymer matrix in bulk physical properties of polymer nanocomposites: A review of positron annihilation lifetime studies. *Prog. Polym. Sci.* **2017**, *Volume 75*, pp. 31-47
16. Lazzeri, A.; Zebarjad, S.M.; Pracella, M.; Cavalier, K.; Rosa, R. Filler toughening of plastics. Part 1- The effect of surface interactions on physico-mechanical properties and rheological behavior of ultrafine CaCO₃/HDPE nanocomposites. *Polymer* **2005**, *Volume 46, Issue 3*, pp. 827-844

17. Gai, G.S.; Yang, Y.F.; Fan, S.M.; Cai, Z.F. Preparation and properties of composite mineral powders. *Powder Technol. 2005*, *Volume 153, Issue 3*, pp. 153-158

18. González, J.; Albano, C.; Candal, M.V.; Ichazo, M.; Hernández, M.; Mayz, A.A.; Martínez, A. Characterization of composites based in polyolefins and seashells. *Rev. Fac. Ing. Univ. Centr. Ven. 2006*, *Volume 21, Issue 3*, pp. 89-104

19. Zare, Y.; Rhee, K.Y. Modeling the roles of carbon nanotubes and interphase dimensions in the conductivity of nanocomposites. *Appl. Clay Sci.* **2017**, *Volume 150, Issue 15*, pp. 102562. https://doi.org/10.1016/j.rinp.2019.102562

20. Lucas, A.; Gaudé, J.; Carel, C.; Michel, J.-F.; Cathelineau, G. A synthetic aragonite-based ceramic as a bone graft substitute and substrate for antibiotics. *Int. J. Inorg. Mater.* **2001**, *Volume 3*, Issue 1, 87-94

21. Chakrabarty, D.; Mahapatra, S. Aragonite crystals with unconventional morphologies. *J. Mater. Chem.* **1999**, *Volume 9, Issue 11*, pp. 2953-2957. https://doi.org/10.1039/a905407c

22. Dobircau, L.; Delpouve, N.; Herbinet, R.; Domenek, S.; Le Pluart, L.; Delbreilh, L.; Ducruet, V.; Dargent, E. Molecular mobility and physical ageing of plasticized poly(lactide). *Polym. Eng. Sci.* **2015**, *Volume 55, Issue 4*, pp. 858-865. https://doi.org/10.1002/pen.23952

23. White, J.L.; Kim, E.K. (Eds.) "Twin Screw Extrusion: Technology and Principles", Hanser Publishers, New York, **1991**

24. Carter, J.G.; Clark, G.R. Classification and phylogenetic significance of molluscan shell microstructure. In: Molluscs, Notes for a Short Course. University of Tennessee, Department of Geological Sciences, Studies in Geology Publishers, Volume 13; Bottjer, D.J.; Hickman, C.S.; Ward, P.D.; Broadhead, T.W. (Eds.) **1985**, pp. 50-71

25. Chateigner, D.; Hedegaard, C.; Wenk, H.R. Mollusc shell microstructures and crystallographic textures. *J. Struct. Geol.* **2000**, *Volume 22, Issue 11*, pp. 1723-1735

26. Chateigner, D. (Ed.), "Combined Analysis", Wiley-ISTE Publishers, **2010**.

27. Rietveld, H.M. A profile refinement method for nuclear and magnetic structures. *J. Appl. Cryst.* **1969**, *Volume 2*, pp. 65-71. https://doi.org/10.1107/S0021889869006558

28. Lutterotti, L.; Bortolotti, M. Object oriented programming and fast computation techniques in Maud, a program for powder diffraction analysis written in java. *IUCr: Com. Powd. Diff. Newsletter* **2003**, *Volume 1*, pp. 43-50

29. Ouhenia, S.; Chateigner, D.; Belkhir, M.A.; Guilmeau, E.; Krauss, C. Synthesis of Calcium Carbonate Polymorphs in the Presence of Polyacrylic Acid. *J. Cryst. Growth.* **2008**, *Volume 310, Issue 11*, pp. 2832-2841

30. Grazulis, S.; Chateigner, D.; Downs, R.T.; Yokochi, A.F.T.; Quiros, M.; Lutterotti, L.; Manakova, E.; Butkus, J.; Moeck, P.; Le Bail, A. Crystallography Open Database- an open- Access collection of crystal structures. *J. Appl. Cryst.* **2009**, *Volume 42, Issue 4*, pp. 726-729

31. Caspi, E.N.; Pokroy, B.; Lee, P.L.; Quintana, J.P.; Zolotoyabko, E. On the structure of aragonite. *Acta Cryst.* **B 2005**, *Volume 61*, pp. 129-132

32. Popa N.C. The (hkl) dependence of diffraction-line broadening caused by strain and size for all Laue groups in Rietveld refinement, *J. Appl. Cryst.* 1998, *Volume 31*, 176-180

33. Esteban-Delgado, F.J.; Harper, E.M.; Checa, A.G.; Rodriguez-Navarro, A.B. Origin and expansion of foliated microstructure in pteriomorph bivalves. *Biol. Bull.* **2008**, *Volume 214, Issue 2*, pp. 153-165. https://doi.org/10.2307/25066672

34. MacDonald, J.; Freer, A.; Cusack, M. Alignment of Crystallographic c-Axis throughout the Four Distinct Microstructural Layers of the Oyster *Crassostrea gigas*. *Cryst. Growth Des.* **2010**, *Volume 10, Issue 3*, pp. 1243-1246. https://doi.org/10.1021/cg901263p

35. Checa, G.; Esteban-Delgado, F.J.; Ramírez-Rico, J. Crystallographic reorganization of the calcitic prismatic layers of oysters *J. Struct. Biol.* **2009**, *Volume 167, Issue 3*, 261-270

36. Taylor, P.D.; Weedon, M.J. Skeletal ultrastructure and phylogeny of cyclostome bryozoans. *Zool. J. Linnean Soc.* **2000**, *Volume 128, Issue 4*, pp. 337-399. https://doi.org/10.1111/j.1096-3642.2000.tb01521.x

37. England, J.; Cusack, M.; Dalbeck, P.; Perez-Huerta, A. Comparison of the Crystallographic Structure of Semi Nacre and Nacre by Electron Backscatter Diffraction. *Cryst. Growth Des.* **2007**, *Volume 7, Issue 2*, pp. 307-310. https://doi.org/10.1021/cg060374p

38. Chateigner, D.; Morales, M.; Harper, E.M. QTA of prismatic calcite layers of some bivalves, a link to trichite ancestral. *Mater. Sci. Forum* **2002**, *Volume 408-412*, 1687-1692

39. Misra, R.D.K; Nerikar, P.; Bertrand, K.; Murphy, D. Some aspects of surface deformation and fracture of 5-
16. Lazzeri, A.; Zebarjad, S.M.; Pracella, M.; Cavalier, K.; Rosa, R. Filler toughening of plastics. Part 1- The effect of surface interactions on physico-mechanical properties and rheological behavior of ultrafine CaCO₃/HDPE nanocomposites. *Polymer* **2005**, *Volume 46, Issue 3*, pp. 827-844

17. Gai, G.S.; Yang, Y.F.; Fan, S.M.; Cai, Z.F. Preparation and properties of composite mineral powders. *Powder Technol. 2005*, *Volume 153, Issue 3*, pp. 153-158

18. González, J.; Albano, C.; Candal, M.V.; Ichazo, M.; Hernández, M.; Mayz, A.A.; Martínez, A. Characterization of composites based in polyolefins and seashells. *Rev. Fac. Ing. Univ. Centr. Ven. 2006*, *Volume 21, Issue 3*, pp. 89-104

19. Zare, Y.; Rhee, K.Y. Modeling the roles of carbon nanotubes and interphase dimensions in the conductivity of nanocomposites. *Appl. Clay Sci.* **2017**, *Volume 150, Issue 15*, pp. 102562. https://doi.org/10.1016/j.rinp.2019.102562

20. Lucas, A.; Gaudé, J.; Carel, C.; Michel, J.-F.; Cathelineau, G. A synthetic aragonite-based ceramic as a bone graft substitute and substrate for antibiotics. *Int. J. Inorg. Mater.* **2001**, *Volume 3*, Issue 1, 87-94

21. Chakrabarty, D.; Mahapatra, S. Aragonite crystals with unconventional morphologies. *J. Mater. Chem.* **1999**, *Volume 9, Issue 11*, pp. 2953-2957. https://doi.org/10.1039/a905407c

22. Dobircau, L.; Delpouve, N.; Herbinet, R.; Domenek, S.; Le Pluart, L.; Delbreilh, L.; Ducruet, V.; Dargent, E. Molecular mobility and physical ageing of plasticized poly(lactide). *Polym. Eng. Sci.* **2015**, *Volume 55, Issue 4*, pp. 858-865. https://doi.org/10.1002/pen.23952

23. White, J.L.; Kim, E.K. (Eds.) "Twin Screw Extrusion: Technology and Principles", Hanser Publishers, New York, **1991**

24. Carter, J.G.; Clark, G.R. Classification and phylogenetic significance of molluscan shell microstructure. In: Molluscs, Notes for a Short Course. University of Tennessee, Department of Geological Sciences, Studies in Geology Publishers, Volume 13; Bottjer, D.J.; Hickman, C.S.; Ward, P.D.; Broadhead, T.W. (Eds.) **1985**, pp. 50-71

25. Chateigner, D.; Hedegaard, C.; Wenk, H.R. Mollusc shell microstructures and crystallographic textures. *J. Struct. Geol.* **2000**, *Volume 22, Issue 11*, pp. 1723-1735

26. Chateigner, D. (Ed.), "Combined Analysis", Wiley-ISTE Publishers, **2010**.

27. Rietveld, H.M. A profile refinement method for nuclear and magnetic structures. *J. Appl. Cryst.* **1969**, *Volume 2*, pp. 65-71. https://doi.org/10.1107/S0021889869006558

28. Lutterotti, L.; Bortolotti, M. Object oriented programming and fast computation techniques in Maud, a program for powder diffraction analysis written in java. *IUCr: Com. Powd. Diff. Newsletter* **2003**, *Volume 1*, pp. 43-50

29. Ouhenia, S.; Chateigner, D.; Belkhir, M.A.; Guilmeau, E.; Krauss, C. Synthesis of Calcium Carbonate Polymorphs in the Presence of Polyacrylic Acid. *J. Cryst. Growth.* **2008**, *Volume 310, Issue 11*, pp. 2832-2841

30. Grazulis, S.; Chateigner, D.; Downs, R.T.; Yokochi, A.F.T.; Quiros, M.; Lutterotti, L.; Manakova, E.; Butkus, J.; Moeck, P.; Le Bail, A. Crystallography Open Database- an open- Access collection of crystal structures. *J. Appl. Cryst.* **2009**, *Volume 42, Issue 4*, pp. 726-729

31. Caspi, E.N.; Pokroy, B.; Lee, P.L.; Quintana, J.P.; Zolotoyabko, E. On the structure of aragonite. *Acta Cryst.* **B 2005**, *Volume 61*, pp. 129-132

32. Popa N.C. The (hkl) dependence of diffraction-line broadening caused by strain and size for all Laue groups in Rietveld refinement, *J. Appl. Cryst.* 1998, *Volume 31*, 176-180

33. Esteban-Delgado, F.J.; Harper, E.M.; Checa, A.G.; Rodriguez-Navarro, A.B. Origin and expansion of foliated microstructure in pteriomorph bivalves. *Biol. Bull.* **2008**, *Volume 214, Issue 2*, pp. 153-165. https://doi.org/10.2307/25066672

34. MacDonald, J.; Freer, A.; Cusack, M. Alignment of Crystallographic c-Axis throughout the Four Distinct Microstructural Layers of the Oyster *Crassostrea gigas*. *Cryst. Growth Des.* **2010**, *Volume 10, Issue 3*, pp. 1243-1246. https://doi.org/10.1021/cg901263p

35. Checa, G.; Esteban-Delgado, F.J.; Ramírez-Rico, J. Crystallographic reorganization of the calcitic prismatic layers of oysters *J. Struct. Biol.* **2009**, *Volume 167, Issue 3*, 261-270

36. Taylor, P.D.; Weedon, M.J. Skeletal ultrastructure and phylogeny of cyclostome bryozoans. *Zool. J. Linnean Soc.* **2000**, *Volume 128, Issue 4*, pp. 337-399. https://doi.org/10.1111/j.1096-3642.2000.tb01521.x

37. England, J.; Cusack, M.; Dalbeck, P.; Perez-Huerta, A. Comparison of the Crystallographic Structure of Semi Nacre and Nacre by Electron Backscatter Diffraction. *Cryst. Growth Des.* **2007**, *Volume 7, Issue 2*, pp. 307-310. https://doi.org/10.1021/cg060374p

38. Chateigner, D.; Morales, M.; Harper, E.M. QTA of prismatic calcite layers of some bivalves, a link to trichite ancestral. *Mater. Sci. Forum* **2002**, *Volume 408-412*, 1687-1692

39. Misra, R.D.K; Nerikar, P.; Bertrand, K.; Murphy, D. Some aspects of surface deformation and fracture of 5-
20% calcium carbonate-reinforced polyethylene composites. *Mat. Sci. Eng. A* **2004**, *Volume 384*, *Issue 1-2*, 284-298

40. Shi, X.; Rosa, R.; Lazzeri, A. On the coating of precipitated calcium carbonate with stearic acid in aqueous medium. *Langmuir* **2010**, *Volume 26*, *Issue 11*, pp. 8474-8482. https://doi.org/10.1021/la904914h

41. Kiss, A.; Fekete, E.; Pukanszky, B. Aggregation of CaCO₃ particles in PP composites: effect of surface coating. *Compos. Sci. Technol.* **2007**, *Volume 67*, *Issue 7-8*, pp. 1574-1583

42. Sahebian, S.; Zebarjad, S.M.; Sajjadi, S.A.; Sherafat, Z.; Lazzeri, A. Effect of both uncoated and coated calcium carbonate on fracture toughness of HDPE/CaCO₃ nanocomposites. *J. Appl. Polym. Sci.* **2007**, *Volume 104*, *Issue 6*, pp. 3688-3694. https://doi.org/10.1002/app.25644

43. Kutz, M. (Ed.) "Handbook of environmental degradation of Materials", **2012** William Andrew Publisher, UK
<figure><img src="image_8.png" /></figure>

20% calcium carbonate-reinforced polyethylene composites. *Mat. Sci. Eng. A* **2004**, *Volume 384*, *Issue 1-2*, 284-298

40. Shi, X.; Rosa, R.; Lazzeri, A. On the coating of precipitated calcium carbonate with stearic acid in aqueous medium. *Langmuir* **2010**, *Volume 26*, *Issue 11*, pp. 8474-8482. https://doi.org/10.1021/la904914h

41. Kiss, A.; Fekete, E.; Pukanszky, B. Aggregation of CaCO₃ particles in PP composites: effect of surface coating. *Compos. Sci. Technol.* **2007**, *Volume 67*, *Issue 7-8*, pp. 1574-1583

42. Sahebian, S.; Zebarjad, S.M.; Sajjadi, S.A.; Sherafat, Z.; Lazzeri, A. Effect of both uncoated and coated calcium carbonate on fracture toughness of HDPE/CaCO₃ nanocomposites. *J. Appl. Polym. Sci.* **2007**, *Volume 104*, *Issue 6*, pp. 3688-3694. https://doi.org/10.1002/app.25644

43. Kutz, M. (Ed.) "Handbook of environmental degradation of Materials", **2012** William Andrew Publisher, UK
Supplementary Materials

<figure><img src="image_8.png" /><figcaption>**Supplementary Figure 1.** XRD pattern and corresponding Combined Analysis fit of the ground *Pecten maximus* layers of Figure 1. CoFe peaks come from the sample holder. Goodness of Fit is 1.68. Residual aragonite is seen as very small peaks. Inset is the refined anisotropic mean shape of crystallites.</figcaption></figure>

<figure><img src="image_9.png" /><figcaption>**Supplementary Figure 2.** XRD pattern and corresponding Combined Analysis fit of the ground *Crepidula fornicata* layers of Figure 3. Goodness of Fit is 1.33. Insets are the refined anisotropic mean shape of crystallites and SEM image of the powder.</figcaption></figure>
Supplementary Materials

<figure><img src="image_9.png" /><figcaption>**Supplementary Figure 1.** XRD pattern and corresponding Combined Analysis fit of the ground *Pecten maximus* layers of Figure 1. CoFe peaks come from the sample holder. Goodness of Fit is 1.68. Residual aragonite is seen as very small peaks. Inset is the refined anisotropic mean shape of crystallites.</figcaption></figure>

<figure><img src="image_10.png" /><figcaption>**Supplementary Figure 2.** XRD pattern and corresponding Combined Analysis fit of the ground *Crepidula fornicata* layers of Figure 3. Goodness of Fit is 1.33. Insets are the refined anisotropic mean shape of crystallites and SEM image of the powder.</figcaption></figure>
<figure><img src="image_10.png" /><figcaption>**Supplementary Figure 3.** XRD pattern and corresponding Combined Analysis fit of the ground *Crassostrea gigas* layers of Figure 5. Goodness of Fit is 1.66. Insets are the refined anisotropic mean shape of crystallites and SEM image of the powders. Residual quartz comes from incorporated sand in the outer part of the shell</figcaption></figure>

<figure><img src="image_11.png" /></figure>
<figure><img src="image_11.png" /><figcaption>**Supplementary Figure 3.** XRD pattern and corresponding Combined Analysis fit of the ground *Crassostrea gigas* layers of Figure 5. Goodness of Fit is 1.66. Insets are the refined anisotropic mean shape of crystallites and SEM image of the powders. Residual quartz comes from incorporated sand in the outer part of the shell</figcaption></figure>

<figure><img src="image_12.png" /></figure>
<figure><img src="image_12.png" /><figcaption>**Supplementary Figure 4.** a) Synthesized aragonite powder and b) XRD pattern and fit of Commercial calcite as received (GoF = 2.2) c) XRD pattern and fit of the ground commercial calcite (GoF = 1.4). Insets in b) and c) are zooms of the calcite diffraction line broadening increase due to grinding in the 2θ = 56°-58.5° range, with their respective mean crystallite shapes</figcaption></figure>

<figure><img src="image_13.png" /><figcaption>**Supplementary Figure 5.** SEM images of commercial calcite samples a) before and b) after grinding</figcaption></figure>

<figure><img src="image_14.png" /><figcaption>**Supplementary Figure 6.** SEM pictures of SA coated a) C. Fornicata b) P. Maximus and c) C. Gigas powders</figcaption></figure>
<figure><img src="image_13.png" /><figcaption>**Supplementary Figure 4.** a) Synthesized aragonite powder and b) XRD pattern and fit of Commercial calcite as received (GoF = 2.2) c) XRD pattern and fit of the ground commercial calcite (GoF = 1.4). Insets in b) and c) are zooms of the calcite diffraction line broadening increase due to grinding in the 2θ = 56°-58.5° range, with their respective mean crystallite shapes</figcaption></figure>

<figure><img src="image_14.png" /><figcaption>**Supplementary Figure 5.** SEM images of commercial calcite samples a) before and b) after grinding</figcaption></figure>

<figure><img src="image_15.png" /><figcaption>**Supplementary Figure 6.** SEM pictures of SA coated a) C. Fornicata b) P. Maximus and c) C. Gigas powders</figcaption></figure>