# Designing poly(3-hydroxybutyrate-co-3-hydroxyvalerate) P(3HB-co-3HV) films with tailored mechanical properties

Estelle Doineau, Coline Perdrier, Fanny Allayaud, Elise Blanchet, Laurence Preziosi-Belloy, Estelle Grousseau, Nathalie Gontard, Helene Angellier-Coussy

► To cite this version:

Estelle Doineau, Coline Perdrier, Fanny Allayaud, Elise Blanchet, Laurence Preziosi-Belloy, et al. Designing poly(3-hydroxybutyrate-co-3-hydroxyvalerate) P(3HB-co-3HV) films with tailored mechanical properties. Materials Today Communications, 2023, 36, pp.106848. 10.1016/j.mtcomm.2023.106848. hal-04248218

HAL Id: hal-04248218

https://hal.science/hal-04248218v1

Submitted on 18 Oct 2023

HAL is a multi-disciplinary open access
archive for the deposit and dissemination of sci-
entific research documents, whether they are pub-
lished or not. The documents may come from
teaching and research institutions in France or
abroad, or from public or private research centers.

L'archive ouverte pluridisciplinaire HAL, est
destinée au dépôt et à la diffusion de documents
scientifiques de niveau recherche, publiés ou non,
émanant des établissements d'enseignement et de
recherche français ou étrangers, des laboratoires
publics ou privés.

<figure><img src="image_1.png" /></figure>

Distributed under a Creative Commons CC BY 4.0 - Attribution - International License
# Designing poly(3-hydroxybutyrate-co-3-hydroxyvalerate) P(3HB-co-3HV) films with tailored mechanical properties

Estelle Doineau<sup>a,1</sup>, Coline Perdrier<sup>a,1</sup>, Fanny Allayaud<sup>b</sup>, Elise Blanchet<sup>b</sup>, Laurence Preziosi-belloy<sup>a</sup>, Estelle Grousseau<sup>a</sup>, Nathalie Gontard<sup>a</sup>, Hélène Angellier-Coussy<sup>a,*</sup>

## ARTICLE INFO

Keywords:

Polyhydroxyalkanoates

Crystallization behavior

Differential scanning calorimetry

Processing temperature

Tensile tests

High-pressure homogenization

## ABSTRACT

Increasing the 3HV molar fraction up to 45 mol% in the poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (P(3HB-co-3HV)) monomeric composition is assumed to improve the ductility of P(3HB-co-3HV)-based materials. The objective of the paper was to assess how the polymer recovery treatment and the melt processing temperature could counterbalance this effect. For that purpose, copolymers with different 3HV contents (18, 28 and 40 mol%) and recovered both using a solvent free high-pressure homogenization (HPH) based treatment and a chloroform-based treatment were used to produce non-formulated and self-supported films by thermopressing. The improvement of ductility with increased 3HV content was confirmed whatever the recovery treatment. Lower elongation at break values were obtained for HHP-based extracted copolymers, due to the presence of impurities, including non-PHAs cell mass, that favored thermal degradation during the film shaping process. An in-depth study by differential scanning calorimetry (DSC) analysis of the melt crystallization behavior, coupled to observations in polarized optical microscopy (POM), allowed to predict the optimal processing temperature, i. e. the temperature allowing the formation of microstructures in favor of improved ductility. Tensile properties were discussed in relation to the thermal properties and crystalline structure of copolymers evaluated by wide-angle and small-angle X-ray scattering analyses (WAXS/SAXS).

## 1. Introduction

The problems of recyclability of petroleum-based plastics currently on the market, as well as their persistence in the environment and the danger they constitute to human health [1], are driving the scientific community to develop more environmentally friendly polymers, particularly in the field of packaging which constitutes around 40% of the global plastic consumption [2]. In this way, the microbial polyesters polyhydroxyalkanoates (PHAs) appear as polymers of the future [3,4]. Indeed, PHAs are biodegradable under natural conditions and biobased, as they are synthesized by a biological process using microorganisms fed by organic biomass and possibly agro-residues [5]. Among PHAs, the semi-crystalline and thermoplastic copolymer poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (P(3HB-co-3HV)) is promising due to its better processability compared to the poly(3-hydroxybutyrate) P(3HB), and the possibility to tailor its intrinsic properties depending on the molar content of 3HV units. The 3HB/3HV monomeric ratio depends on the

kind of strains, the carbon sources, the nutrient limitation and its intensity used to promote the production of the copolymer as well as the feeding strategy [6,7].

The glass transition temperature of the polymer is known to linearly decrease from 6 °C to – 13 °C by increasing the 3HV content from 0 to 100 mol%, while the degree of crystallinity decreases from 60% to 47% (from WAXD patterns) by increasing 3HV content up to 45 mol% [8]. Both modifications may induce in turn a significant improvement of the material ductility [9]. Elongation at break values can reach between 700% and 800% for a 3HV content of 17–29 mol% [5,10,11]). Such conclusions have been drawn on films most often obtained by solvent casting, i.e. dissolution of the polymer in chloroform, then spreading and evaporation [12,13], which is a process very useful at lab scale to investigate the properties of materials when only a small amount of polymer is available. When targeting packaging applications at industrial scale, solvent free processes such as thermo-mechanical processes (e.g. extrusion, compression molding or injection molding) must be

<footnote><sup>a</sup> JRU IATE 1208, INRAE, Montpellier SupAgro, University of Montpellier, CEDEX 02, 34060 Montpellier, France</footnote>

<footnote><sup>b</sup> CRITT Bio-industries, INSA Toulouse, 31400 Toulouse, France</footnote>

<footnote>* Corresponding author.</footnote>

<footnote>E-mail address: helene.coussy@umontpellier.fr (H. Angellier-Coussy).</footnote>

<footnote n="1">¹ first co-authors</footnote>
favored. Worth to note that much lower values of elongation at break are obtained when melt processing are used, with values reaching only 10–30% at similar 3HV content [14,15]. Holmes explains that solution-cast films would provide higher ductility due to low crystallization temperature compared to melt-processed films, inducing a favored nucleation density and thus a smaller crystal size [10], and/or plasticizing effect of residual solvent. However, most of studies dealing with the impact of 3HV content on the mechanical properties of films prepared by melt processing focused on P(3HB-co-3HV)s produced and extracted in different ways. Some studies use commercial grades as comparative data although production and recovery processes are partially unknown, with possible formulation, that could confuse the comparison with other studies [16–18]. The only authors who produced their own P(3HB-co-3HV)s in comparable conditions, used different films processing with solvent casting for 3HV mole fraction lower than 20%, and compression molding for higher 3HV mole fraction, which could hide or distort possible opposite effects [15]. Furthermore, due to intrinsic differences in thermal stability and melt flow of P(3HB-co-3HV) with different 3HV content, optimization of melt processing conditions is also particularly important for controlling the final properties of the materials [19]. An inappropriate processing temperature can annihilate the positive effect of increased 3HV content on the mechanical properties of P(3HB-co-3HV) films [18].

The recovery step is also a key factor in the whole PHAs production chain. Eco-friendly PHAs extraction routes have been recently investigated to substitute polluting, toxic and non-recyclable halogenated solvents such as chloroform, considered today as the benchmark for PHAs recovery but hardly to up-scale [20–22]. They are based on the use of “green” solvents, or mechanical, enzymatic, chemical or surfactant based treatments. Among non-PHAs cell mass (NPCM) mechanical disruption methods, the high-pressure homogenization (HPH) appears very promising for high biomass concentrations [23], due to its industrial scalability allowing PHAs recovery in large quantities [24,25]. It has been reported that conditions of recovery can strongly impact the overall purity of the polymer with the presence of residual biomass mostly constituted of DNA, proteins, lipids and/or inorganic residues [20,26]. Depending on their nature, the presence of NCPM is known to induce degradation reactions during P(3HB-co-3HV) thermomechanical processing, by favoring chain scission via β-elimination reactions [27, 28]. This leads therefore to a decrease of polymer molecular weight and thermal degradation temperature, as well as a modification of the semi-crystalline structure, and the melting and crystallization behavior [22,28,29]. This will in turn have an impact on the processability of P (3HB-co-3HV) and final performances of P(3HB-co-3HV)-based materials [18,30].

In this context and based on the research hypothesis that increasing the 3HV content allows to improve the ductility of P(3HB-co-3HV) materials, the scientific question we proposed to answer is: How both the recovery treatment and the melt processing temperature can counterbalance the effect of the P(3HB-co-3HV)'s monomeric composition on the mechanical properties of P(3HB-co-3HV) films and thus be controlled to design materials with improved ductility?

For this purpose and to allow reliable comparisons between samples, a panel of three P(3HB-co-3HV)s with distinct 3HV contents (18, 28 and 40 mol%) were produced from propionic acid under phosphorus depletion conditions, using Cupriavidus necator as pure strain [31]. All P (3HB-co-3HV)s were recovered via a chloroform-based treatment and characterized in Perdrier et al. (2023) [31] in terms of molecular structure by SEC-MALS (molecular weight, polydispersity index), as well as melting temperatures by differential scanning calorimetry (DSC). P (3HB-co-3HV)s with 18 and 28 mol% 3HV were also recovered using an alternative solvent free high-pressure homogenization (HPH)-based treatment and further characterized and compared to chloroform recovered references. In this study, additional investigations about crystalline structure, assessed by both wide-angle and small-angle X-ray scattering analysis, and melt crystallization behavior, characterized by

DSC and polarized optical microscopy (POM), were carried out. Moreover, an in-depth study of the thermal behavior of copolymers was performed by DSC. Cycles of heating-cooling-heating were recorded by imposing different values of pre-heating temperatures in order to estimate the optimum processing temperature based on the crystallization behavior of copolymers. Self-supported and non-formulated (i.e. without any additive) films were prepared by compression molding. Visual aspect and mechanical properties were discussed in relation to the structure of the polymers.

## 2. Materials and methods

### 2.1. Materials

Three grades of poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (P (3HB-co-3HV)) with different 3-hydroxyvalerate (3HV) molar contents, i.e. 18%, 28% and 40%, were biosynthesized by specific propionic acid feeding of Cupriavidus necator bacteria via phosphorus depletion. Growing and synthesis conditions are described in Perdrier et al., 2023 [31]. At the end of the cultivation, biomass was washed twice by resuspension in a volume of deionized water equivalent to the sample volume followed by centrifugation (16 000 rcf, 10 min, 4 °C).

All P(3HB-co-3HV) copolymers were recovered by a solvent reference method with chloroform [31]. For that purpose, the washed biomass was then stored at – 80 °C and lyophilized under vacuum (Christ LDC-1 Alpha 2–4, –85 °C, 3bar). Then, the lyophilized biomass was refluxed (4 h, 65 °C) with chloroform (analytical grade, Carlo Erba). After filtration under vacuum on PVDF membranes with a 0.45 µm pore size (Merck Durapore), the retentate was rinsed with a small amount of chloroform, and the filtrate containing solubilized polymers was recovered was concentrated approximately 5-fold by rotary evaporation (at 30 °C under vacuum). P(3HB-co-3HV) was then precipitated by dropwise addition into a large amount of ice-cold absolute ethanol (chloroform/ethanol ratio: 1:10 v/v) under continuous magnetic stirring, recovered by vacuum filtration over sintered glass (porosity of 10–16 µm) and then washed with ethanol. The polymer was finally dried at 30 °C under vacuum for 24 h. They are noted chloro-18%, chloro-28% and chloro-40% in the following paragraphs.

An alternative recovery method based on a high-pressure homogenization (HPH) treatment was developed by the CRITT Bio-Industries (Toulouse, France) and used for P(3HB-co-3HV) with 18 and 28 mol% 3HV content, noted HPH-18% and HPH-28% in the following paragraphs. In that case, the washed and centrifugated biomass was further stored at – 80 °C. To recover intracellular polymer, the frozen and wet biomass was thawed at room temperature and the cells were washed with deionized water by centrifugation (6000 g, 20 min, 20 °C) to remove supernatant. The washed cells were then suspended in deionized water (dry cell concentration of about 40 g.L⁻¹) and diluted in carbonate buffer (50 mM, Na₂CO₃ + NaHCO₃) at pH 10 to reach final dry cell weight cell of 20 g.L⁻¹. The resulting cell suspension was passed through a two-stage HPH (PandaPlus 2000, GEA Niro Soavi) fitted with a ceramic cell disruption valve at 900 bar. Cell lysis was achieved after 2 passes at room temperature. The disrupted cells were then post-treated adding 0.2% (w:v) SDS to the suspension, to promote solubilisation of the non-PHA cell mass (NPCM). The resulting suspension was adjusted to pH 10 using sodium hydroxide and stirred 1 h at 37 °C and the polymers were recovered by centrifugation. Depending on the amount of material to process, batch centrifugation (7800 rcf, 20 min, 20 °C) or semi-continuous centrifugation (16000 rcf, 70 min, 25 L.h⁻¹, 20 °C) was performed for HHP-18% and HHP-28%. The polymers were then washed with deionised water (12000 g, 20 min, 20 °C) and further dried at 30 °C for 3 days. HHP-18% was manually milled with a mortar-pestle. HHP-28% consistency did not allow to proceed in the same way. Thus, the latter was knife grinded (IKA M20) during 10 s using nitrogen liquid cooling circuit at 6 °C. Polymer molecular weight was checked and was shown not affected by knife milling compared to manual milling.
Moreover, in order to investigate the impact and possible P(3HB-co-HV) degradation of milling step, three different particle sizes were obtained by sieving (Retsch sieves 1 mm and 0.5 mm), i.e. 85.8 wt% of < 0.5 mm, 9.5 wt% of 0.5–1 mm and 4.7 wt% of > 1 mm, and compared to an unground sample. Heterogeneities of films with orange spots and zones were obvious for unground and > 1 mm samples, contrary to < 0.5 mm and 0.5–1 mm giving homogeneous films (see Fig. S1 in supplementary data). It was then decided to keep and mix the first two fractions, i.e. < 0.5 mm and 0.5–1 mm, based on visual aspect but also on additional mechanical tensile tests and thermal analysis, not presented in this article.

All recovered P(3HB-co-3HV)s were finally stored in a desiccator at 23 °C before further characterization.

## 2.2. Production of P(3HB-co-3HV)-based films

Recovered polymers were dried at 30 °C under vacuum during 24 h and then shaped in films by compression molding using a heated hydraulic press (20 T, Pinette Emidecau Industries). 2.2 g of polymers were heated between two Teflon coated plates in a 100 µm thick mold to obtain films of 12 × 12 cm² following 5 processing steps: contact with plates at heating temperature ($T_{heat}$) for 5 min, then pressing at $T_{heat}$ for 30 s under 50 bar, followed by pressing at $T_{heat}$ for 30 s under 100 bar, then pressing at $T_{heat}$ for 60 s under 150 bar and finally cooling during 10 min under a tray filled with cold water. Produced films were then stabilized at 25 °C and 50% HR for a minimum of 14 days before further characterization.

## 2.3. Polymer purity (acid butanolysis)

3HB and 3HV monomers in the recovered P(3HB-co-3HV)s were quantified by an acid butanolysis method directly on polymers adapted from Werker et al., 2008 and Jan et al., 1995 [32,33] followed by gas chromatography (GC-FID), and modified in Perdrier et al., 2023 [31]. Purity was calculated from quantified monomer masses (respectively $m_{3HB}$ and $m_{3HV}$) and polymer mass ($m_{P(3HB-co-3HV)}$) (20 mg) on which the butanolysis was carried out, as follows: $\frac{m_{3HB} + m_{3HV}}{m_{P(3HB-co-3HV)}}$, then expressed as a percentage. Standard deviation of purity measurement was determined to be 0.5% for 9 repetitions.

## 2.4. Elementary analysis

The residual nitrogen content of recovered polymers was quantified by elementary analysis using Vario Micro Cube and a thermal conductivity detection. These analyses were performed by the Laboratoire de Mesures Physiques, analytical facilities of Montpellier University from washed and freeze-dried biomass. Weight percentages of nitrogen N were obtained after a catalytic combustion of recovered polymers in a tin capsule at 1150 °C followed by a flue gas reduction on copper at 850 °C.

## 2.5. Size Exclusion Chromatography (SEC)

The weight-average molecular mass ($\bar{M}_w$), as well as the polydispersity index (PDI) were determined by the Toulouse White Biotechnology (TWB) institute using size exclusion chromatography associated with multi-angle light scattering (SEC-MALS) and refractive index detectors (both from Wyatt Technology Corp) [31].

## 2.6. Wide-angle X-ray scattering (WAXS) and small-angle X-ray scattering (SAXS)

Wide-angle X-ray scattering (WAXS) and small angle X-ray scattering (SAXS) experiments were performed with an in-house setup of the Laboratoire Charles Coulomb at the University of Montpellier (France).

A high brightness low power X-ray tube, coupled with aspheric multi-layer optic (GeniX³D from Xenocs) was employed. It delivers an ultralow divergent beam (0.5 mrad, $\lambda$ = 0.15418 nm). Scatterless slits were used to give a clean 0.7 mm beam diameter with a flux of 35 Mphotons.s⁻¹ at the sample. We worked in a transmission configuration and scattered intensity was measured by a 2D “Pilatus” 300 K pixel detector by Dectris (490 *600 pixels) with pixel size of 172 × 172 µm², at a distance from the sample of 1.9 m and 0.2 m for SAXS and WAXS configuration, respectively. All intensities were corrected by samples transmission and the empty cell contribution was subtracted.

The crystallinity degree was assessed from the WAXS spectra by the ratio between the crystalline diffraction areas and the total area of the diffraction profile. The crystalline diffraction areas were obtained by deconvoluting the diffraction profile using the Fityk application, according to the PseudoVoigt mode. The P(3HB-co-3HV) crystallization occurs according to an orthorhombic crystalline lattice. The lattice parameters (a, b, c) of the P(3HV-co-3HB) crystalline lattice were calculated from the (020), (110) and (121) diffraction peaks using (i) the relation between the d-spacing ($d_{hkl}$, distance between adjacent (hkl) lattice planes), the Miller indexes (hkl) and a, b, and c for an orthorhombic lattice: $d_{hkl} = 1/\sqrt{h^2/a^2 + k^2/b^2 + l^2/c^2}$.

The d-spacing values were calculated from the Bragg's law: $n\lambda = 2 \times d_{hkl} \times \sin\theta_{hnhkl}$.

The crystal size (cs, in nm) was determined by the Sherrer equation from the (020) reflection, which was the more intense peak (at ca. 2θ = 13.2°), using a Warren correction correction [34,35]: $cs_{020} = K \cdot \lambda / (\beta - \beta_0) \cdot \cos\theta$. 2θ is the reflection angle (rad), K the Sherrer constant (taken as equal to 0.9 [36]), $\lambda$ the wavelength (nm), $\beta$ the peak width of half intensity (rad) being the experimental $\beta$ corrected by $\beta_0$ = 0.0026 rad for instrumental line broadening.

The scattering vector $q$ used in SAXS patterns relates to the scattering angle (2θ) via the relation $q = (4\pi/\lambda) \cdot \sin\theta$. The long period ($L_p$, in nm), corresponding to the average distance between crystalline domains, was estimated from the SAXS profile as $L_p = 2\pi/q_{max}$, where $q_{max}$ is the position of the first maximum value of the curve $I(q) = I(q) \cdot q^2$ (Lorentz-corrected scattering curve).

## 2.7. Differential Scanning Calorimetry (DSC)

Differential Scanning Calorimetry (DSC) analysis was carried out using a Q200 equipment (TA Instruments, New Castle, DE, USA) with nitrogen as purge gas (50 mL.min⁻¹). Samples of 5–10 mg were placed in hermetic aluminum pans (TA Instruments, New Castle, DE, USA). For both P(3HB-co-3HV) powders and films, the melting behavior was studied by applying a heating ramp of 10 °C.min⁻¹ from – 30 °C up to 200 °C. For P(3HB-co-3HV) powders, an in-depth study of their thermal behavior was carried out by applying a heating-cooling-heating cycle: a first heating ramp from – 30 °C up to a pre-heating temperature $T_{ph}$, depending on the experiment followed by an isotherm at $T_{ph}$ during 5 min, then a cooling ramp down to – 30 °C during 3 min, and finally, a second heating ramp up to 200 °C, with heating/cooling rates of 10 °C.min⁻¹. Experiments were done at least in duplicate. The melting temperatures $T_{m,1}$ and $T_{m,2}$, glass transition temperatures $T_{g,1}$ (mobile amorphous fraction) and $T_{g,2}$ (rigid amorphous fraction), the melt crystallization temperature $T_c$ and the occurrence or not of cold-crystallization were determined from the obtained DSC curves.

## 2.8. Polarized optical microscopy (POM)

POM observations were performed on a Laborlux 11 POL S (Leitz, Germany) microscope in the transmission mode, equipped with a monochroic Leica DFC420 camera (resolution 1728 × 1296 pixels) and piloted by the Archimed® software (Microvision Instruments, France). Each sample was placed between two glass slides, then heated onto a LTS420 hot stage (Linkam, UK) up to pre-heating temperature $T_{ph}$ at 10 °C.
min⁻¹ and annealed for 3 min before being cooled at 80 °C (at 20 °C. min⁻¹) and stabilized 60 min before observation.

## 2.9. Tensile properties

Tensile tests were carried out at room temperature using a INSTRON 68SC-5 machine, equipped with a 50 N force sensor at a speed of 10 mm. min⁻¹, on samples cut in dumbbell shape with a width of 4 mm, an effective length (L₀) of 22 mm and a distance between jaws of 45 mm. At least six samples were done for each sample. The thickness of each specimen was measured using a Digimatic micrometer (Mitutoyo, precision 0.001 mm) and was the average of three measurement points.

## 2.10. Thermogravimetric analysis (TGA)

TGA was carried out using a Mettler TGA2 device (Schwerzebbach, Switzerland) equipped with a XP5U balance. Samples were heated from 25 °C to 600 °C at 10 °C.min⁻¹ under nitrogen flow of 50 mL.min⁻¹. Results were analyzed using STARE software. The maximum degradation temperature (Tₐ₈₉) corresponded to the temperature at which the degradation rate was maximum. Temperature range ΔT was calculated by the formula ΔT = Tₐ₉₅₄ - Tₐ₈₉₄. The onset and offset degradation temperatures (Tₐ₈₉₄ and Tₐ₉₅₄, respectively) were measured respectively when the first derivative of the weight loss became higher than 0.1%.°C⁻¹ and lower than 0.1%.°C⁻¹. Analyses were done at least in triplicate.

# 3. Results and discussion

## 3.1. Impact of 3HV content and recovery conditions on the intrinsic properties of P(3HB-co-3HV) copolymers

### 3.1.1. Purity and molecular weight

The purity of P(3HB-co-3HV) copolymers recovered using the chloroform-based treatment was very high for the three grades going from 102% to 104% (Table 1). The purity of copolymers obtained by the alternative HPH-based recovery process was however reduced to 95% and 98% for HPH-18% and HPH-28% respectively, due to the presence of residual biomass, as revealed by the nitrogen content measured by elementary analysis on copolymers. This could be explained by the reduced ability of the HPH-based process to well separate biomass debris mostly constituted of proteins and DNA [20,26] from P(3HB-co-3HV) polymer produced by the cell. Those impurities may favor the occurrence of degradation reactions during thermo-mechanical processing [30] and/or impact the solid-state morphology of polymers [37], which in turn will impact the functional properties of P(3HB-co-3HV)-based films.

$M̄_w$ values were around 1000 kDa for all copolymers, with no significant impact of the 3HV molar fraction or the recovery treatment (Table 1). The fact that the molecular weight was similar for all the copolymers was a first very positive point for our study since it allowed comparing structural characteristics without a possible competitive

effect of difference in molecular weight. Values of around 1000 kDa were a second positive point in view of targeted increase in ductility. Indeed, with the objective to use P(3HB-co-3HV) for thermoplastic applications, it has been shown that the PHA molecular weight should be higher than 600 kDa to get materials with good mechanical properties [5]. Additionally, given that thermo-mechanical processes used to produce films, e.g. compression molding, are known to thermally degrade P (3HB-co-3HV), the molecular weight of the raw polymers has to be as high as possible to compensate this most likely negative effect.

### 3.1.2. Micro-crystalline structure

The wide angle X-ray scattering patterns showed that all P(3HB-co-3HV)s considered in the study crystallized in the P(3HB) crystalline lattice (Fig. 1). The three lattice parameters a, b and c undergo a moderate but gradual increase by increasing the 3HV molar fraction (Table 2), showing an expansion of crystals in all directions that was ascribed to the inclusion of 3HV units within the P(3HB) crystals. Lattice parameters were in the same range as previously published results [35], the only difference being a slight increase, rather than constancy, of the c parameter. The overall degree of crystallinity was also significantly impacted by increasing the 3HV molar fraction with a decrease from 59% to 47% for a 3HV content increasing from 18 to 40 mol%, in agreement with previous results [8,35]. The crystal size slightly increased with decreasing the degree of crystallinity, i.e. with increasing HV content, which was in agreement with the decrease in the width of half intensity (WHI) of the (020) reflection peak. This result is however in contradiction with some previous published data [34].

SAXS data allowed to provide information on the lamellar structure of polymers, i.e. the internal structure of polymer spherulites. All P(3HB-co-3HV) copolymers recovered with chloroform presented a crystalline arrangement in smaller angles, as evidenced by a distinct interference peak on Lorentz-corrected SAXS curves located from about q = 0.6 to q = 1 nm⁻¹ depending on the 3HV molar fraction (Fig. 1). This single interference peak is due to the periodical stacks of alternate lamellar crystals and amorphous layers. The long period (Lp), representing the average distance between the two centers of gravity in two adjacent crystals, was estimated from the interference peak. Larger Lp values were estimated with increasing 3HV content (Table 2), corroborating the conclusion that the 3HV units are incorporated into the 3HB crystal lattice and suggesting the dilation of the amorphous lamella, in accordance with the decreased of the crystallinity degree, which is consistent with previous work [35,38].

When a high-pressure homogenizer HPH-based treatment was used to recover P(3HB-co-3HV) instead of a chloroform-based treatment, a similar effect of the increased 3HV content on the crystalline structure of the polymers was noticed, that is a decrease of the overall crystallinity degree and an increase of the lattice parameters with increasing 3HV content. However, significant differences were reported between the two recovery methods. A slightly higher degree of crystallinity was obtained for polymers extracted using a HPH-based method (Table 2). This devitrification phenomenon could be ascribed to a possible nucleating effect of impurities present in the samples. HPH extraction also seems to lead to less expanded crystals than in the case of polymers

**Table 1**

Purity (obtained by acid butanolysis), nitrogen content (obtained by elementary analysis), molecular weight $M̄_w$ and polydispersity index (PDI) (measured by SEC-MALS) of P(3HB-co-3HV) copolymers.

<table><thead><tr><th>Recovery method</th><th>3HV molar fraction (%)</th><th>Codification</th><th>Purity<sup>a</sup> (%)</th><th>Nitrogen content (%)</th><th>M̄<sub>w</sub> (kDa)</th><th>PDI (M̄<sub>w</sub> / M̄<sub>n</sub>)</th></tr></thead><tbody><tr><td rowspan="3">Chloroform</td><td>18</td><td>chloro-18%</td><td>103</td><td>&lt; detection limit</td><td>901 ± 0<sup>b</sup></td><td>1.42 ± 0.00 *</td></tr><tr><td>28</td><td>chloro-28%</td><td>102</td><td>&lt; detection limit</td><td>1112 ± 27<sup>b</sup></td><td>1.61 ± 0.02 *</td></tr><tr><td>40</td><td>chloro-40%</td><td>104</td><td>n.d.</td><td>1055 ± 54<sup>b</sup></td><td>1.59 ± 0.01 *</td></tr><tr><td rowspan="2">HPH</td><td>18</td><td>HPH-18%</td><td>95</td><td>0.381 ± 0.055</td><td>935 ± 25</td><td>1.48 ± 0.11</td></tr><tr><td>28</td><td>HPH-28%</td><td>98</td><td>0.366 ± 0.006</td><td>1075 ± 14</td><td>1.73 ± 0.02</td></tr></tbody></table>

<footnote>a standard deviation value of 0.5%</footnote>

<footnote>b data already published in Perdrier et al., 2023 [31]</footnote>
<figure><img src="image_2.png" /><figcaption>**Fig. 1.** (A) WAXS patterns and (B) Lorentz-corrected SAXS curves of P(3HB-co-3HV) copolymers.</figcaption></figure>

extracted by chloroform, as highlighted by lower values of lattice parameters, meaning that a part of 3HV units are excluded from the crystal. The width of half intensity of the (020) reflection peak was more narrow in the case of the HPH-based treatment, indicating the presence of crystals of higher quality, in accordance with the increased crystal size values (Table 2), with no significant effect of the 3HV content. The

long period was not impacted by the recovery method, even if we have to note that the interference peak was almost not visible on SAXS patterns of HPH-recovered polymers.

### 3.1.3. Melting behavior

The effect of both the 3HV molar fraction and the recovery method (chloroform-based vs. HPH-based) on the melting behavior of P(3HB-co-3HV) copolymers has been assessed by differential scanning calorimetry (DSC). Thermograms corresponding to a heating ramp up to 200 °C are shown in Fig. 2 and data are detailed in Table 3.

Glass transition temperatures $T_{g,1}$ and $T_{g,2}$, which were respectively attributed to the glass transition temperatures of the mobile amorphous fraction (MAF) and the rigid amorphous fraction (RAF) [39,40], linearly decreased by increasing the 3HV molar fraction from 18% to 40%, indicating an increase of the segmental mobility in the non-crystalline phase. Moreover, associated heat capacities $\Delta C_{p,1}$ and $\Delta C_{p,2}$ also increased by increasing 3HV content, highlighting greater chain mobility and suggesting increased ductility of resulting P(3HB-co-3HV) materials. Two successive endothermic peaks were then observed (Fig. 2). The main endothermic peak, centered around $T_{m,1}$, ranged from 76° to 133°C depending on the 3HV content and corresponded to the melting of the random copolymer P(3HB-co-3HV). The second one, corresponding to $T_{m,2}$ = 160 °C in the case of copolymers extracted by chloroform, was of lesser enthalpy and remained constant whatever the 3HV content. This second melting peak was attributed to the melting of the granule core constituted of P(3HB) [31]. Heating the polymer at temperatures allowing the melting of the entire crystalline phase will allow heterogeneous crystallization, while partially melting these crystals will favor self-nucleation and thus faster crystallization. So, fully or partially melting those P(3HB) core crystals when using thermal processes is a key point in the subsequent crystallization behavior that could be beneficial depending on targeted properties. Finally, overall melting

<figure><img src="image_3.png" /><figcaption>**Fig. 2.** DSC thermograms registered during the first heating ramp from –30–200 °C for P(3HB-co-3HV) copolymers.</figcaption></figure>

**Table 2**

d-spacings of the (020), (110) and (121) crystalline planes (measured by WAXS analysis), lattice parameters a, b and c (estimated from d-spacing values), width of half intensity (WHI) of the (020) reflection peak, crystal size determined from the (020) reflection peak, the degree of crystallinity of the P(3HB-co-3HV) copolymers, and long period Lp (estimated from SAXS patterns).

<table><thead><tr><th>Copolymer</th><th>d<sub>020</sub><br>(Å)</th><th>d<sub>110</sub><br>(Å)</th><th>d<sub>121</sub><br>(Å)</th><th>a (nm)</th><th>b (nm)</th><th>c (nm)</th><th>Crystal size<br>(nm)</th><th>WHI (°)</th><th>Crystallinity degree<br>(%)</th><th>Lp (nm)</th></tr></thead><tbody><tr><td>chloro-18%</td><td>6.61</td><td>5.28</td><td>3.52</td><td>0.577</td><td>1.323</td><td>0.602</td><td>21.7</td><td>0.517</td><td>59 ± 1</td><td>6.5</td></tr><tr><td>chloro-28%</td><td>6.68</td><td>5.37</td><td>3.56</td><td>0.587</td><td>1.337</td><td>0.604</td><td>30.5</td><td>0.370</td><td>53 ± 1</td><td>7.1</td></tr><tr><td>chloro-40%</td><td>6.69</td><td>5.40</td><td>3.57</td><td>0.590</td><td>1.339</td><td>0.606</td><td>36.2</td><td>0.419</td><td>47 ± 1</td><td>9.2</td></tr><tr><td>HPH-18%</td><td>6.56</td><td>5.26</td><td>3.50</td><td>0.574</td><td>1.313</td><td>0.597</td><td>29.6</td><td>0.353</td><td>63 ± 1</td><td>6.5</td></tr><tr><td>HPH-28%</td><td>6.60</td><td>5.32</td><td>3.53</td><td>0.582</td><td>1.321</td><td>0.598</td><td>39.7</td><td>0.350</td><td>55 ± 1</td><td>7.2</td></tr></tbody></table>
<caption>**Table 3**</caption>

Glass transition and melting parameters obtained by DSC from the first heating ramp. $T_{g,1}$ and $T_{g,2}$: glass transition temperature of mobile and rigid phase respectively; $\Delta C_{p,1}$ and $\Delta C_{p,2}$: heating capacity of both glass transition phases; $T_{m,1}$: first melting temperature peak; $T_{m,2}$: second melting temperature peak.

<table><thead><tr><th>Copolymer</th><th><i>T</i><sub>g,1</sub><br>(°C)</th><th>Δ<i>C</i><sub>p,1</sub><br>(J.g<sup>-1</sup>.°C<sup>-1</sup>)</th><th><i>T</i><sub>g,2</sub><br>°C</th><th>Δ<i>C</i><sub>p,2</sub><br>(J.g<sup>-1</sup>.°C<sup>-1</sup>)</th><th><i>T</i><sub>m,1</sub><br>(°C)</th><th><i>T</i><sub>m,2</sub><br>(°C)</th></tr></thead><tbody><tr><td>Chloro-18%</td><td>4.7<br>± 0.7</td><td>0.11<br>± 0.03</td><td>61.3<br>± 0.3</td><td>0.17<br>± 0.02</td><td>133.0<br>± 1.1*</td><td>159.5<br>± 0.1*</td></tr><tr><td>Chloro-28%</td><td>-1.4<br>± 0.5</td><td>0.20<br>± 0.01</td><td>48.9<br>± 1.0</td><td>0.37<br>± 0.01</td><td>101.6<br>± 0.1*</td><td>160.1<br>± 0.5*</td></tr><tr><td>Chloro-40%</td><td>-6.4<br>± 0.5</td><td>0.25<br>± 0.01</td><td>-</td><td>-</td><td>76.0<br>± 0.4*</td><td>162.3<br>± 0.3*</td></tr><tr><td>HPH-18%</td><td>1.0<br>± 0.2</td><td>0.14<br>± 0.02</td><td>64.5<br>± 0.1</td><td>0.16<br>± 0.01</td><td>120.8<br>± 0.0</td><td>174.4<br>± 0.0</td></tr><tr><td>HPH-28%</td><td>-4.4<br>± 1.4</td><td>0.13<br>± 0.02</td><td>52.8<br>± 0.4</td><td>0.43<br>± 0.07</td><td>94.5<br>± 0.5</td><td>173.5<br>± 1.2</td></tr></tbody></table>

enthalpy $\Delta H_{m,tot}$ decreased by increasing 3HV content, in accordance with the decrease of the overall degree of crystallinity.

Similar results were obtained with the alternative HPH-based recovery treatment. However, for a given 3HV content, significant differences between the two recovery treatments were noticed. First, lower values of $T_{g,1}$ and $T_{m,1}$ were recorded for the HPH-recovered P(3HB-co-3HV), suggesting a plasticizing effect of the HPH-based treatment (Table 3). In view of predicting the mechanical properties of films, this effect in favor of an improved ductility may compete with the previously mentioned increase of crystal size and degree of crystallinity (Table 2). On the contrary, $T_{m,2}$ was increased, with values for HPH-18% and HPH-28% around 174 °C against 160 °C for P(3HB-co-3HV) recovered with chloroform. This result revealed that the P(3HB) core displayed a different melting behavior depending on the type of extraction. It was assumed that the interaction between the P(3HB) granule core and the P (3HB-co-3HV) chains was stronger in the case of successive solubilization in chloroform and reprecipitation (i.e. favored co-crystallization, as highlighted by higher values of lattice parameters), thus inducing a melting peak closer to the copolymer one. In the case of the HPH-based recovery method, the melting temperature of the P(3HB) core approached the value of a pure P(3HB), i.e. around 180 °C [10]. Finally, the overall melting enthalpy values of copolymers were difficult to determine because of either two not well-separated melting peaks (e.g. for chloro-18%) and/or a possible overlap of the glass transition of the RAF and the first melting peak (e.g. for chloro-28%) (see Fig. 2). The overall melting enthalpy is therefore not reported in the Table 3.

## 3.2. Estimating the thermal processability of P(3HB-co-3HV) through DSC analysis

An improvement of ductility of P(3HB-co-3HV)-based materials is known to be favored by the formation of small and homogeneous spherulites during the melt crystallization [10]. If spherulites are too large, i.e. higher than around 20 µm in diameter, cracks may occur within the spherulite in the radial or tangential direction [41]. It was also shown that the absence of secondary cold crystallization was also in favor of improved ductility [18]. Indeed, the free volume size of cold-crystallized polymers is known to be reduced compared to melt-crystallized polymers, resulting in more rigid materials. Based on the knowledge of intrinsic properties and thermal behavior of produced copolymers, the objective of this study was thus to find the optimum processing temperature $T_{opt}$ that should be applied for each grade of P (3HB-co-3HV) to obtain a microstructure in favor of improved ductility, depending possibly on the 3HV molar fraction and/or the recovery method.

For this purpose, the melting and non-isothermal crystallization behavior of copolymers was investigated through an in-depth DSC

analysis carried out by applying heating-cooling-heating cycles coupled to observations by polarized optical microscopy (POM). Based on previous results obtained from the first heating ramp up to 200 °C, a range of pre-heating temperatures ($T_{ph}$) values was selected for each P(3HB-co-3HV) copolymer with the objective to voluntary favor heterogeneous nucleation by erasing the thermal history of the copolymer (complete melting of the crystals) or on the contrary favor self-nucleation by partially melting the crystals. Each range of selected $T_{ph}$ corresponded to temperatures higher than the first melting temperature $T_{m,1}$, going from 150 °C to 200 °C for chloro-18%, chloro-28%, HPH-18% and HPH-28%. For chloro-40%, given that it was characterized by a melting temperature $T_{m,1}$ of 76.0 °C, the pre-heating temperature range started at a lower temperature, i.e. 90 °C (Table 3).

Whatever the 3HV molar fraction and the recovery method, it was observed that increasing the $T_{ph}$ resulted in a decrease of the melt crystallization temperature ($T_c$) until the complete disappearance of melt crystallization for pre-heating temperatures of 200 °C (Figs. 3B and 3K) and 180 °C (Figs. 3E and 3N) for 3HV molar contents of 18% and 28%, respectively. Worth to note that in the case of 40 mol% of 3HV content, the melt crystallization enthalpy was too low and the peak too broad to be able to measure a melt crystallization temperature, whatever the pre-heating temperature (Fig. 3H). The disappearance of melt crystallization with increasing pre-heating temperature was previously explained by the difficulty for P(3HB-co-3HV) to reach a new ordered system if all the nuclei are eliminated, especially due to the drop of viscosity that hinders heterogeneous nucleation [42,43]. For 3HV contents of 18 and 28 mol%, the disappearance of melt crystallization gave rise to the occurrence of cold crystallization during the second heating ramp, as observed by the presence of exothermal peaks (Figs. 3C, 3F, 3L, 3O), what we targeted to avoid to favor the production of materials with improved ductility. The shape of the melt crystallization peak as well as the melt crystallization enthalpy were also key indicators to be discussed. Increasing $T_{ph}$ up to a given value, i.e. 170 and 180 °C for copolymers recovered using chloroform and HPH respectively (independently of the 3HV content), led to both an increase of melt crystallization enthalpy and the occurrence of more narrow crystallization peaks (Fig. 3B, E, K, N), indicating respectively an increase of the crystallinity degree and a reduction in crystals size polydispersity. Passing these values of $T_{ph}$, the melt crystallization peak intensity decreased again until the previously described disappearance of melt crystallization. These critical $T_{ph}$ may correspond to $T_{opt}$.

In this way, by coupling DSC analysis with POM observations, $T_{opt}$ values have been estimated for the four processable copolymers under study, based on a case-by-case analysis. It was deduced that $T_{opt}$ was around 170 °C for chloro-18%, since it resulted in the finest and most intense melt crystallization peak at $T_c = 88$ °C (Fig. 3B), with the absence of cold crystallization during the second heating ramp (Fig. 3C). This was corroborated by POM images showing the evolution of the size and shape of spherulites with increasing $T_{ph}$ (Fig. 4). For $T_{ph} = 170$ °C, spherulites were uniform and displayed a very little diameter of a few micrometers (Fig. 4A). By increasing the pre-heating temperature, the spherulites diameter increased up to about 200 µm at $T_{ph} = 190$ °C. At $T_{ph} = 190$ °C, spherulites were characterized by a wring-banded structure, regularly concentric, with visible Maltese cross (Fig. 4E).

In the case of chloro-28%, the determination of the optimum $T_{ph}$ was not so obvious since at $T_{ph} = 170$ °C a more intense and narrow melt crystallization peak was obtained, but together with a little cold crystallization phenomenon, while at $T_{ph} = 160$ °C a less intense and broader melt crystallization peak was observed, but no secondary cold crystallization (Fig. 3F). The optimum pre-heating was thus deduced to be in between these two temperatures.

As mentioned above, for chloro-40%, no or very little crystallization from the molten state was noticed for the entire $T_{ph}$ temperature window tested (Fig. 3H). The second heating ramp in Fig. 3I showed less and less re-melting of the crystals without however showing a significant

<footnote>* data already published in Perdrier et al., 2023 [31]</footnote>
Chloroform recovery – P(3HB-co-3HV) 18 mol%, 28 mol% and 40 mol% of 3HV

<figure><img src="image_4.png" /></figure>

<figure><img src="image_5.png" /></figure>

<figure><img src="image_6.png" /></figure>

<figure><img src="image_7.png" /></figure>

<figure><img src="image_8.png" /></figure>

<figure><img src="image_9.png" /></figure>

<figure><img src="image_10.png" /></figure>

<figure><img src="image_11.png" /></figure>

<figure><img src="image_12.png" /></figure>

High-pressure homogenizer (HPH) recovery – P(3HB-co-3HV) 18 mol% and 28 mol% of 3HV

<figure><img src="image_13.png" /></figure>

<figure><img src="image_14.png" /></figure>

<figure><img src="image_15.png" /></figure>

<figure><img src="image_16.png" /></figure>

<figure><img src="image_17.png" /></figure>

<figure><img src="image_18.png" /></figure>

Fig. 3. DSC thermograms of the first heating ramp, the cooling step and the second heating ramp of respectively (A, B and C) chloro-18%, (D, E and F) chloro-28%, (G, H and I) chloro-40%, (J, K and L) HPH-18% and (M, N and O) HPH-28% P(3HB-co-3HV). The curves corresponding to pre-heating temperatures *T*<sub>ph</sub> defined as optimum are shown in thicker lines. This *T*<sub>opt</sub> is also indicated in red in the first heating ramp cycle.
<figure><img src="image_19.png" /><figcaption>Fig. 4. POM images of P(3HB-co-3HV) chloro-18% (A, C and E), or HPH-18% (B, D and F), after heating to various T<sub>ph</sub> of 170 °C, 180 °C, 190 °C or 200 °C at 10 °C·min<sup>-1</sup>, and quick cooling at 25 °C·min<sup>-1</sup> until 80 °C and 1 h stabilization before observation (x40).</figcaption></figure>

peak of cold crystallization. The great difficulty of P(3HB-co-3HV) with 40 mol% 3HV to crystallize could be ascribed to its low viscosity inducing significant rubbery and elastomeric properties [44,45]. Such a behavior hinders the production of non-formulated and self-supported films, requiring the addition of nucleating agents to promote crystallization. This has not been tested in the present study.

Using the alternative HPH recovery method, an optimum T<sub>ph</sub> around 170-180 °C and 160-170 °C was determined for copolymers with 18 and 28 mol% 3HV content, respectively. A small shift was therefore observed towards higher pre-heating temperatures for 18 mol% of 3HV as compared to the chloroform reference extraction. This was ascribed to the fact that P(3HB) core crystals melted at higher temperatures compared to chloroform-recovered polymers. Therefore, the HPH-recovered polymers may need to be heated a little more to allow an equivalent proportion of melted P(3HB) core crystals, and thus achieve a similar crystallization. Moreover, for a same T<sub>ph</sub>, relatively smaller spherulites were observed when P(3HB-co-3HV) was recovered by HPH instead of chloroform (Fig. 4). Finally, at T<sub>ph</sub> = 200 °C, the sporadic heterogeneous crystallization induced by the complete melting of crystals led to the formation of spherulites with diameters larger than 200 µm with the presence of non-crystallized zones (evidencing the zones where cold crystallization will further take place) (Fig. 4F).

To conclude, increasing the 3HV molar fraction from 18% to 28% led to a slight decrease of the optimum pre-heating temperature for a same recovery method, i.e. T<sub>ph</sub> = 170 °C for chloro-18% against 160-170 °C

for chloro-28% and T<sub>ph</sub> = 170-180 °C for HPH-18% against 160-170 °C for HPH-28%. The higher processing temperature that seems to be required for HPH-18% was ascribed to the presence of a more thermally-resistant P(3HB) core. On the other hand, a high molar content of 3HV such as 40% makes the production of self-supported films impossible due to the absence of crystallization from the melt, whatever the pre-heating temperature. Worth to note that the shearing forces applied during the compression molding process were not taken into account in this approach.

### 3.3. *Validating the optimum processing temperature in view of enhancing the mechanical performance of non-formulated P(3HB-co-3HV) films*

The hypotheses established in the previous section are here refined through the preparation of non-formulated, i.e. without additives such as nucleating agents, and self-supported films using a compression molding process and by applying processing temperatures within the above recommended ranges. Films were mechanically characterized through uniaxial tensile tests (Fig. 5). Given that the objective is to improve the ductility of P(3HB-co-3HV)-based materials, the optimum processing temperature (T<sub>opt</sub>) was considered when P(3HB-co-3HV)-based films displayed the highest elongation at break (e<sub>b</sub>) values.

Due to the small quantity of copolymers recovered by chloroform, only two processing temperatures were tested for this recovery process, i.e. 165 °C and 170 °C for chloro-18%, and 160 °C and 165 °C for
<figure><img src="image_20.png" /><figcaption>Fig. 5. (A and B) Elongation at break, (C and D) tensile strength, (E and F) Young's modulus of respectively P(3HB-co-3HV) 18 mol% and 28 mol% extracted by chloroform or high-pressure homogenization HPH. Processing temperature is indicated in grey. Optimum processing temperatures $T_{opt}$ are indicated in black and bold.</figcaption></figure>

chloro-28%. For chloro-18%, higher values of elongation at break, but also tensile strength and Young's modulus were obtained for $T_{ph}$ = 170 °C, which was thus considered as the optimum processing temperature for this grade of P(3HB-co-3HV) copolymer (Fig. 5A, 5C and 5E). For chloro-28%, the determination of $T_{opt}$ was not so obvious, as already highlighted from DSC analysis, with contrary effects on elongation at break, tensile strength and Young's modulus. Considering as main criterion the increase in elongation at break, $T_{opt}$ was assumed to

be 160 °C (Figs. 5B, 5D and 5F). This result was in correlation with the DSC study and confirmed that the compression molding temperature should be adequately chosen to obtain the tailored mechanical properties.

The HPH-based alternative recovery method was very interesting as it delivered much higher quantities of polymer, allowing to test three or even four processing temperatures (Fig. 5). It was shown that $T_{opt}$ was not strongly impacted by the recovery method, with $T_{opt}$ = 170 °C for
chloro-18% against 175 °C for HPH-18%, and 160 °C for both recovery types at 28 mol% of 3HV, which was in line with DSC results. It was observed that for a processing temperature of 185 °C, corresponding to an increase of only 10 °C compared to $T_{opt}$, mechanical properties of HPH-18% were significantly reduced and particularly in the fracture domain, with a 25% decrease in tensile strength and a 31% decrease in elongation at break. This strong decrease in ductility is mainly due to the formation of bigger spherulites during cooling by increasing processing temperature above $T_{opt}$, as previously observed on POM images (Fig. 4), creating more defects within P(3HB-co-3HV) films and thus preferential crack paths. Such a loss of mechanical properties was also ascribed to the thermal degradation of HPH-18%, as shown by the decrease of the molecular weight from $\bar{M}_w$ = 220 ± 7 kDa at 175 °C to $\bar{M}_w$ = 136 ± 4 kDa at 185 °C. Thermal degradation occurs mainly through intra-molecular cis-elimination, which induces random chain scission and thus, a drastic reduction of the polymer chain length [46,47].

It is worth noting that the optimum processing temperatures estimated in the present study for P(3HB-co-3HV)s with a 3HV molar fraction of 18 mol%, i.e. 170 °C for chloro-18% and 175 °C for HPH-18%, were much higher than the value of 145 °C reported by Bossu et al. for copolymer displaying similar 3HV molar fraction and average molecular weight [18]. This was ascribed to presence of P(3HB) core granules melting around respectively 160 °C and 174 °C for chloro-18% and HPH-18%.

### 3.4. Impact of 3HV content and recovery conditions on the mechanical behavior of P(3HB-co-3HV) films produced in optimum conditions

Typical uniaxial tensile curves of films produced using the previously deduced optimum processing temperature are presented in Fig. 6. The hypothesis that the ductility of self-supported and non-formulated P (3HB-co-3HV) films is increased by increasing the 3HV molar fraction was here validated for melt processed films, whatever the recovery process, with $\epsilon_b$ = 62% for chloro-28% and $\epsilon_b$ = 26% for HPH-28% against $\epsilon_b$ = 14% for chloro-18% and $\epsilon_b$ = 8% for HPH-18%. It has to be noticed that standard deviations values are very high for chloro-28% with few samples showing very high deformations, against the majority of samples displaying premature cracks driving to faster ruptures. Samples displaying high elongation at break values, i.e. higher than 20–25%, showed necking phenomenon on dumbbells with the presence of a yield point, involving an alignment of the copolymer chains, visible

<figure><img src="image_21.png" /><figcaption>Fig. 6. Typical uniaxial tensile curves of all P(3HB-co-3HV) films at 18 mol% and 28 mol% of 3HV and recovered by chloroform or high-pressure homogenizer HPH method.</figcaption></figure>

by a whitening of the necking zone. The increase in ductility together with the 3HV content from 18 to 28 mol% was accompanied by a strong reduction of both the stiffness, as revealed by a 46% decrease in the Young's modulus, and the resistance, as revealed by a 42% decrease of the tensile strength (Table 4). This was likely related to the significant decrease of the degree of crystallinity from 62% to 46%, since no significant modification of the glass transition temperature was observed (Table 5). Worth to note that the occurrence of a broad single interference peak on SAXS patterns (Fig. 7B) is characteristic of spatial correlations of crystalline domains of fairly well defined size, but not ordered. The fact this interference peak was more intense and less wide than for unprocessed copolymers (Fig. 1B) would indicate that crystalline domains were more homogeneous in size and more ordered after the thermopressing step, but still scattered randomly in the film.

The overall mechanical behavior was significantly impacted by the recovery method (Fig. 6). The mechanical properties were globally poorer for HPH-recovered copolymer based films compared to chloroform ones, in spite of lower values of the degree of crystallinity for HPH-18%, higher Lp values (Table 5) and less intense scattering peaks on WAXS patterns (Fig. 7A). Elongation at break values were 42% and 58% lower for HPH-18% and HPH-28% compared to chloro-18% and chloro-28%, respectively (Table 4). The tensile strength was also decreased for HPH-18% compared to chloro-18%, but similar mechanical resistance was noticed for chloro-28% and HPH-28%. Conversely, Young's modulus was similar for chloro-18% and HPH-18% while a slight decrease was noticed for HPH-28% compared to chloro-28% (Table 4). The poorer mechanical properties of HPH-recovered P(3HB-co-3HV) based films could be ascribed to the favored degradation of the polymer during the thermal process, as shown by the decrease of molecular weight from around 1000 kDa for raw powders down to 220 kDa for processed HPH-18% (instead of 502 kDa for processed chloro-18%) and (Table 5) and the yellow/orange color of films produced from HPH-recovered copolymers (Fig. 8). Such a degradation was ascribed to the lowest purity of HPH-recovered polymers (Table 1), with the previously pointed out presence of NPCM.

From a visual aspect, films prepared with HPH-28% were however more homogeneous with no orange spots and zones (Fig. 8). This could be due to the higher purity, i.e. 98% for HPH-28% against 95% for HPH-18%, and also to the way the recovered polymers were milled. Indeed, HPH-18% was manually ground using a mortar and a pestle while HPH-28% was milled using a knife miller under liquid nitrogen due to the difficulty to grind it manually. HPH-28% powder was then sieved and the powder fraction with the biggest particle size (> 1 mm) was excluded from the final P(3HB-co-HV) powder for the preparation of films (see Materials and Methods part). The heterogeneities observed in HPH-18% films were assumed to cause premature defects in the material, resulting in lower tensile strength values.

### 4. Conclusions

The hypothesis that increasing 3HV content in P(3HB-co-3HV) polymers increased the ductility of materials was validated for the two recovery methods considered in the study, i.e. chloroform and HPH-based treatments. Crystallization from the melt was not possible for P (3HB-co-3HV) with the highest 3HV content of 40 mol%, making the

<table><caption>Table 4
Tensile properties of P(3HB-co-3HV) films processed at optimum temperatures.</caption><thead><tr><th>Copolymer</th><th><i>T</i><sub>opt</sub><br>(°C)</th><th>Young's<br>modulus<br>(MPa)</th><th>Tensile<br>strength<br>(MPa)</th><th>Elongation at<br>break (%)</th></tr></thead><tbody><tr><td>Chloro-<br>18%</td><td>170</td><td>693 ± 47</td><td>24.9 ± 1.7</td><td>14 ± 2</td></tr><tr><td>Chloro-<br>28%</td><td>160</td><td>371 ± 7</td><td>14.5 ± 0.5</td><td>62 ± 55</td></tr><tr><td>HPH-18%</td><td>175</td><td>696 ± 33</td><td>18.5 ± 1.3</td><td>8 ± 1</td></tr><tr><td>HPH-28%</td><td>160</td><td>334 ± 6</td><td>14.7 ± 0.4</td><td>26 ± 4</td></tr></tbody></table>
<table><caption>**Table 5**
Structural (crystallinity degree, $\bar{M}w$, PDI) and thermal properties of P(3HB-co-3HV) films processed at optimum temperatures.</caption><thead><tr><th>Copolymer</th><th>T<sub>opt</sub><br>(°C)</th><th>Crystallinity degree<br>(%)</th><th>Lp<br>(nm)</th><th>M̄w<br>(kDa)</th><th>PDI<br>(M̄w/M̄n)</th><th>Tdeg<br>(°C)</th><th>T<sub>m,1</sub><br>(°C)</th><th>T<sub>m,2</sub><br>(°C)</th><th>T<sub>r,1</sub><br>(°C)</th></tr></thead><tbody><tr><td>Chloro-18%</td><td>170</td><td>62 ± 1</td><td>7.40</td><td>502 ± 10</td><td>1.97 ± 0.18</td><td>253 ± 1</td><td>128.6 ± 3.6</td><td>166.5 ± 0.8</td><td>-3.5 ± 0.1</td></tr><tr><td>Chloro-28%</td><td>160</td><td>46 ± 1</td><td>8.38</td><td>-</td><td>-</td><td>279 ± 2</td><td>104.8 ± 1.5</td><td>166.2 ± 0.3</td><td>-3.2 ± 0.1</td></tr><tr><td>HPH-18%</td><td>175</td><td>55 ± 1</td><td>7.24</td><td>220 ± 7</td><td>1.74 ± 0.10</td><td>262 ± 4</td><td>128.9 ± 0.3</td><td>162.4 ± 0.3</td><td>-0.7 ± 1.3</td></tr><tr><td>HPH-28%</td><td>160</td><td>52 ± 1</td><td>8.17</td><td>-</td><td>-</td><td>291 ± 0</td><td>102.2 ± 0.4</td><td>173.4 ± 0.1</td><td>-4.5 ± 0.9</td></tr></tbody></table>

<figure><img src="image_22.png" /><figcaption>**Fig. 7.** (A) WAXS and (B) SAXS patterns of P(3HB-co-3HV)-based films thermopressed at the optimum processing temperature ($T_{opt}$).</figcaption></figure>

preparation of self-supported and non-formulated films impossible. Based on the assumption that film ductility was favored by the creation of small and homogeneous spherulites during the melt crystallization, with the least possible cold crystallization, a case-by-case in-depth study of the impact of the melt processing temperature on the melt crystallization behavior of raw powders coupling DSC analysis and POM observations was carried out to determine a range of optimum processing temperatures allowing to design this type of microstructure. This

original approach was validated by tensile tests performed on self-supported and non-formulated films. By playing on the 3HV content through the culture strategy, but also the recovery process as well as the processing temperature, an elongation at break of 62% was obtained for films processed at 160 °C from chloroform-extracted P(3HB-co-3HV) with a 3HV molar fraction of 28 mol%. Therefore, in order to design P (3HB-co-3HV)-based materials with tailored mechanical properties, the key point seems to be to work on a case-by-case basis to thermally process the copolymer.

Worth to note that a satisfactory increase in ductility was nevertheless obtained for the alternative HPH-based recovery method, even if mechanical properties were still poorer compared to the chloroform route, due to the presence of impurities favoring thermal degradation during the melt process and also creating preferential pathway for the fracture. During the chloroform-based extraction process, some of the polymers with smaller molecular weights and most other impurities are dissolved in chloroform and removed, favoring the formation of larger spherical crystals of P(3HB-co-3HV). While the use of the high-pressure homogenization method causes thermal degradation of the chain due to the presence of residual impurities, the smaller molecular weight could beneficial to enhance the crystallization ability of the material, preventing the loss of mechanical properties caused by thermal degradation and physical defects, whether a certain amount of impurities will promote the heterogeneous nucleation of the polymer, tending to form smaller spherical crystals to enhance the ductility of the material.

As a perspective to this work, it would be interesting to characterize the residual impurities present in the final HPH-recovered P(3HB-co-3HV) powders and to elucidate their role on the overall performance of P (3HB-co-3HV)-based materials in order to consider strategies to remove them depending on the targeted application. Finally, increasing the 3HV content up to 28 mol% is a very encouraging strategy, especially as the decrease of P(3HB-co-3HV) crystallinity and glass transition temperature may offer opportunities for further investigations in the formulation, for example by the addition of nucleating agents or blending with other polymers.

## Funding

This work was performed in the framework of the LOOP4PACK project https://projetloop4pack.fr/en/welcome/, which is supported by the French National Research Agency (ANR). [Grant Agreement N#ANR-19-CE43-0006].

## CRediT authorship contribution statement

**Estelle Doineau:** Conceptualization, Investigation, Formal analysis, Visualization, Writing – original draft, Writing – review & editing.

**Coline Perdrier:** Conceptualization, Investigation, Formal analysis, Visualization, Writing – original draft, Writing – review & editing.

**Fanny Allayaud:** Methodology, Investigation. **Elise Blanchet:** Supervision, Project administration. **Laurence Preziosi-Belloy:** Conceptualization, Supervision, Writing – review & editing, Project administration, Resources. **Estelle Grousseau:** Conceptualization, Supervision, Writing – review & editing, Project administration, Funding acquisition, Resources.

**Nathalie Gontard:** Conceptualization, Funding acquisition, Resources. **Hélène Angellier-Coussy:** Conceptualization, Methodology,
<figure><img src="image_23.png" /><figcaption>**Fig. 8.** Pictures of P(3HB-co-3HV)-based films with different 3HV mole contents (18 and 28 mol%) and extraction types (chloroform and/or high-pressure homogenization HPH). Processing temperature is specified in the top left corner.</figcaption></figure>

Investigation, Formal analysis, Visualization, Supervision, Writing – original draft, Writing – review & editing, Project administration, Funding acquisition, Resources.

## Declaration of Competing Interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

## Data Availability

Data of Figures and Tables are available in the following repository: https://doi.org/10.57745/0YNWP6. Raw data will be made available on request.

## Acknowledgements

This work was performed in the framework of the LOOP4PACK project https://projet-loop4pack.fr/en/welcome/, which is supported by the French National Research Agency (ANR) [Grant Agreement N#ANR-19-CE43-0006]. The entire co-authors would like to thank Alexis Bessière for the technical support, Philippe Dieudonné-George from University of Montpellier for WAXS/SAXS experiments and Nicolas Le Moigne from IMT Mines Alès for the formation on the use of the polarized optical microscope.

## Declaration of competing interest

None.

## Appendix A. Supporting information

Supplementary data associated with this article can be found in the online version at doi:10.1016/j.mtcomm.2023.106848.

## References

[1] N. Gontard, G. David, A. Guilbert, J. Sohn, Recognizing the long-term impacts of plastic particles for preventing distortion in decision-making, Nat. Sustain 5 (2022) 472–478, https://doi.org/10.1038/s41893-022-00863-2

[2] V. Guillard, S. Gaucel, C. Fornaciari, H. Angellier-Coussy, P. Buche, N. Gontard, The next generation of sustainable food packaging to preserve our environment in a circular economy context, Front. Nutr. 5 (2018), https://doi.org/10.3389/ fnut.2018.00121.

[3] M. Koller, Poly(hydroxyalkanoates) for food packaging: application and attempts towards implementation, Appl. Food Biotechnol. 1 (2014) 3–15, https://doi.org/ 10.22037/afb.v1i1.7127.

[4] G. Keskin, G. Kızıl, M. Bechelany, C. Pochat-Bohatier, M. Öner, Potential of polyhydroxyalkanoate (PHA) polymers family as substitutes of petroleum based polymers for packaging applications and solutions brought by their composites to form barrier materials, Pure Appl. Chem. 89 (2017) 1841–1848, https://doi.org/ 10.1515/pac-2017-0401.

[5] B. Laycock, P. Halley, S. Pratt, A. Werker, P. Lant, The chemomechanical properties of microbial polyhydroxyalkanoates, Prog. Polym. Sci. 39 (2014) 397–442, https:// doi.org/10.1016/j.progpolymsci.2013.06.008.

[6] G. Policastro, A. Panico, M. Fabbricino, Improving biological production of poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV) co-polymer: a critical review, Rev. Environ. Sci. Biotechnol. 20 (2021) 479–513, https://doi.org/10.1007/s11157- 021-09575-z.

[7] Ö. Kökpınar, M. Altun, Evaluation of different nutrient limitation strategies for the efficient production of poly(hydroxybutyrate-co-hydroxyvalerate) from waste frying oil and propionic acid in high cell density fermentations of *Cupriavidus necator* H16, Prep. Biochem. Biotechnol. (2022) 1–10, https://doi.org/10.1080/ 10826068.2022.2114009.

[8] Y. Wang, S. Yamada, N. Asakawa, T. Yamane, N. Yoshie, Y. Inoue, Comonomer Compositional Distribution and Thermal and Morphological Characteristics of Bacterial Poly(3-hydroxybutyrate-co-3-hydroxyvalerate)s with High 3-Hydroxyvalerate Content, Biomacromolecules 2 (2001) 1315–1323, https://doi.org/ 10.1021/bm010128o.
[9] L.S. Serafim, P.C. Lemos, C. Torres, M.A.M. Reis, A.M. Ramos, The influence of process parameters on the characteristics of polyhydroxyalkanoates produced by mixed cultures, Macromol. Biosci. 8 (2008) 355–366, https://doi.org/10.1002/mabi.200700200.

[10] P.A. Holmes, Biologically produced (R)-3-Hydroxy- alkanoate polymers and copolymers, in: D.C. Bassett (Ed.), Developments in Crystalline Polymers, Springer Netherlands, Dordrecht, 1988, pp. 1–65, https://doi.org/10.1007/978-94-009-1341-7-1.

[11] P.J. Barham, Physical properties of poly(hydroxybutyrate) and poly (Hydroxybutyrate-Co-hydroxyvalerate), in: E.A. Dawes (Ed.), Novel Biodegradable Microbial Polymers, Springer Netherlands, Dordrecht, 1990, pp. 81–96, https://doi.org/10.1007/978-94-009-2129-0-8.

[12] L. Savenkova, Z. Gercberga, I. Bibers, M. Kalnin, Effect of 3-hydroxy valerate content on some physical and mechanical properties of polyhydroxyalkanoates produced by *Azotobacter chroococcum*, Process Biochem. 36 (2000) 445–450, https://doi.org/10.1016/S0032-9592(00)00235-1.

[13] E.N. Pederson, C.W.J. McChalicher, F. Srienc, Bacterial synthesis of PHA block copolymers, Biomacromolecules 7 (2006) 1904–1911, https://doi.org/10.1021/bm0510101.

[14] C.W.J. McChalicher, F. Srienc, Investigating the structure–property relationship of bacterial PHA block copolymers, J. Biotechnol. 132 (2007) 296–302, https://doi.org/10.1007/jbiotec.2007.04.014.

[15] I.N. Gogotov, V.A. Gerasin, Ya.V. Knyazev, E.M. Antipov, S. Kh. Barazov, Composite biodegradable materials based on polyhydroxyalkanoate, Appl. Biochem Microbiol 46 (2010) 607–613, https://doi.org/10.1134/S0003683810060086.

[16] S.J. Modi, Assessing the Feasibility of Poly-(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV) and Poly-(lactic acid) for Potential Food Packaging Applications, The Ohio State University, 2010. https://etd.ohiolink.edu/apexpro/d/rws_olink/r/1501/10?clear=10&p10_accession_num=osu1268921056) (accessed June 15, 2021).

[17] M.C. Righetti, P. Cinelli, N. Mallegni, C.A. Massa, S. Bronco, A. Stäbler, A. Lazzeri, Thermal, mechanical, and rheological properties of biocomposites made of Poly (lactic acid) and potato pulp powder, Int. J. Mol. Sci. 20 (2019) 675, https://doi.org/10.3390/ijms20030675.

[18] J. Bossu, N. Le Moigne, P. Dieudonné-George, L. Dumazert, V. Guillard, H. Angellier-Coussy, Impact of the processing temperature on the crystallization behavior and mechanical properties of poly[R-3-hydroxybutyrate-co-(R-3-hydroxyvalerate)], Polymer 229 (2021), 123987, https://doi.org/10.1016/j.polymer.2021.123987.

[19] L.-J. Vandi, C.M. Chan, A. Werker, D. Richardson, B. Laycock, S. Pratt, Extrusion of wood fibre reinforced poly(hydroxybutyrate-co-hydroxyvalerate) (PHBV) biocomposites: Statistical analysis of the effect of processing conditions on mechanical performance, Polym. Degrad. Stab. 159 (2019) 1–14, https://doi.org/10.1016/j.polymdegradstab.2018.10.015.

[20] M. Koller, H. Niebelschütz, G. Braunegg, Strategies for recovery and purification of poly(R)-3-hydroxyalkanoates (PHA) biopolyesters from surrounding biomass, Eng. Life Sci. 13 (2013) 549–562, https://doi.org/10.1002/elsc.201300021.

[21] C. Kourmentza, J. Plácido, N. Venetsaneas, A. Burniol-Figols, C. Varrone, H. N. Gavala, M.A.M. Reis, Recent advances and challenges towards sustainable polyhydroxyalkanoate (PHA) production, Bioengineering 4 (2017), https://doi.org/10.3390/bioengineering4020055.

[22] M. Koller, Established and advanced approaches for recovery of microbial polyhydroxyalkanoate (PHA) biopolyesters from surrounding microbial biomass, Eur. J. 4 (2020) 113–126, https://doi.org/10.2478/ebtj-2020-0013.

[23] I.M. Tamer, M. Moo-Young, Y. Chisti, Disruption of *Alcaligenes latus* for recovery of Poly(β-hydroxybutyric acid): comparison of high-pressure homogenization, bead milling, and chemically induced lysis, Ind. Eng. Chem. Res. 37 (1998) 1807–1814, https://doi.org/10.1021/ie9707432.

[24] Y. Ling, H.H. Wong, C.J. Thomas, D.R.G. Williams, A.P.J. Middelberg, Pilot-scale extraction of PHB from recombinant E. coli by homogenization and centrifugation, Bioseparation 7 (1998) 9–15, https://doi.org/10.1023/A:1007900416356.

[25] M.S. Ghatnekar, J.S. Pai, M. Ganesh, Production and recovery of poly-3-hydroxybutyrate from Methylobacterium sp V49, J. Chem. Technol. Biotechnol. 77 (2002) 444–448, https://doi.org/10.1002/jctb.570.

[26] M. López-Abelairas, M. García-Torreiro, T. Lú-Chau, J.M. Lema, A. Steinbüchel, Comparison of several methods for the separation of poly(3-hydroxybutyrate) from *Cupriavidus necator* H16 cultures, Biochem. Eng. J. 93 (2015) 250–259, https://doi.org/10.1016/j.bej.2014.10.018.

[27] F.-D. Kopinke, M. Remmler, K. Mackenzie, Thermal decomposition of biodegradable polymers—I: Poly(β-hydroxybutyric acid), Polym. Degrad. Stab. 52 (1996) 25–38, https://doi.org/10.1016/0141-3910(95)00221-9.

[28] A. Burniol-Figols, I.V. Skiadas, A.E. Daugaard, H.N. Gavala, Polyhydroxyalkanoate (PHA) purification through dilute aqueous ammonia digestion at elevated temperatures, J. Chem. Technol. Biotechnol. 95 (2020) 1519–1532, https://doi.org/10.1002/jctb.6345.

[29] Y. Jiang, G. Mikova, R. Kleerebezem, L.A. van der Wielen, M.C. Cuellar, Feasibility study of an alkaline-based chemical treatment for the purification of poly(hydroxybutyrate) produced by a mixed enriched culture, AMB Expr. 5 (2015) 5, https://doi.org/10.1186/s13568-015-0096-5.

[30] L. Hilliou, P.F. Teixeira, D. Machado, J.A. Covas, C.S.S. Oliveira, A.F. Duque, M.A. M. Reis, Effects of fermentation residues on the melt processability and thermomechanical degradation of PHBV produced from cheese whey using mixed microbial cultures, Polym. Degrad. Stab. 128 (2016) 269–277, https://doi.org/10.1016/j.polymdegradstab.2016.03.031.

[31] C. Perdrier, E. Doineau, L. Leroyer, M. Subileau, H. Angellier-Coussy, L. Preziosi-Belloy, E. Grousseau, Impact of overflow vs. limitation of propionic acid on poly(3-hydroxybutyrate-co-3-hydroxyvalerate) biosynthesis, Process Biochem. 128 (2023) 147–157, https://doi.org/10.3390/polym13142257.

[32] S. Jan, C. Roblot, G. Goethals, J. Courtois, B. Courtois, J.E. Nava Saucedo, J.-P. Séguin, J.-N. Barbotin, Study of parameters affecting poly(3-hydroxybutyrate) quantification by gas chromatography, Anal. Biochem. 225 (1995) 258–263.

[33] A. Werker, P. Lind, S. Bengtsson, F. Nordström, Chlorinated-solvent-free gas chromatographic analysis of biomass containing polyhydroxyalkanoates, Water Res. 42 (2008) 2517–2526, https://doi.org/10.1016/j.watres.2008.02.011.

[34] B.E. Warren, J. Appl. Phys. 12 (1945) 875.

[35] M. Scandola, G. Ceccorulli, M. Pizzoli, M. Gazzano, Study of the crystal phase and crystallization rate of bacterial poly(3-hydroxybutyrate-co-3-hydroxyvalerate), Macromolecules 25 (1992) 1405–1410, https://doi.org/10.1021/ma00031a008.

[36] E. Ten, L. Jiang, M.P. Wolcott, Crystallization kinetics of poly(3-hydroxybutyrate-co-3-hydroxyvalerate)/cellulose nanowhiskers composites, Carbohydr. Polym. 90 (2012) 541–550, https://doi.org/10.1016/j.carpol.2012.05.076.

[37] J. Bossu, H. Angellier-Coussy, C. Totee, M. Matos, M. Reis, V. Guillard, Effect of the molecular structure of Poly(3-hydroxybutyrate- co-3-hydroxyvalerate) (P(3HB-3HV)) produced from mixed bacterial cultures on its crystallization and mechanical properties, Biomacromolecules 21 (2020) 4709–4723, https://doi.org/10.1021/acs.biomac.0c00826.

[38] Y. Xie, I. Noda, Y.A. Akpalu, Influence of cooling rate on the thermal behavior and solid-state morphologies of polyhydroxyalkanoates, J. Appl. Polym. Sci. 109 (2008) 2259–2268, https://doi.org/10.1002/app.28278.

[39] M.L. Di Lorenzo, M.C. Righetti, Crystallization-induced formation of rigid amorphous fraction, Polym. Cryst. 1 (2018) 1–14, https://doi.org/10.1002/pcr2.10023.

[40] Y. Chen, G. Yang, Q. Chen, Solid-state NMR study on the structure and mobility of the noncrystalline region of poly(3-hydroxybutyrate) and poly(3-hydroxybutyrate-co-3-hydroxyvalerate), Polymer 43 (2002) 2095–2099, https://doi.org/10.1016/S0032-3861(01)00792-3.

[41] P.J. Barham, A. Keller, The relationship between microstructure and mode of fracture in polyhydroxybutyrate, J. Polym. Sci. Part B: Polym. Phys. 24 (1986) 69–77, https://doi.org/10.1002/polb.1986.180240108.

[42] A. El-Hadi, R. Schnabel, E. Straube, G. Müller, M. Riemtschneider, Effect of melt processing on crystallization behavior and rheology of Poly(3-hydroxybutyrate) (PHB) and its blends, Macromol. Mater. Eng. 287 (2002) 363–372, https://doi.org/10.1002/1439-2054(20020501)287:5<363:AID-MAME363>3.0.CO;2-D.

[43] M. Di Lorenzo, P. Sajkiewicz, A. Gradys, P.L.P.- E-Polymers, U. 2009, Optimization of melting conditions for the analysis of crystallization kinetics of poly (3-hydroxybutyrate), E-Polym. 9 (9) (2009).

[44] H.J. Choi, S.H. Park, J.S. Yoon, H.S. Lee, S.J. Choi, Rheological study on biodegradable Poly(3-Hydroxybutyrate) and Its copolymer, J. Macromol. Sci., Part A 32 (1995) 843–852, https://doi.org/10.1080/10601329508018975.

[45] M. Koller, The Handbook of Polyhydroxyalkanoates: Postsynthetic Treatment, Processing and Application, CRC Press, 2020.

[46] E. Leroy, I. Petit, J.L. Audic, G. Colomines, R. Deterre, Rheological characterization of a thermally unstable bioplastic in injection molding conditions, Polym. Degrad. Stab. 97 (2012) 1915–1921, https://doi.org/10.1016/j.polymdegradstab.2012.03.021.

[47] C. Bonnenfant, N. Gontard, C. Aouf, Biobased and biodegradable polymers in a circular economy context: understanding quercetin and gallic acid impacts on PHBV thermal properties, Polym. Degrad. Stab. 201 (2022), 109975, https://doi.org/10.1016/j.polymdegradstab.2022.109975.