PHB Processability and Property Improvement with Linear-Chain Polyester Oligomers Used as Plasticizers

In 2021, global petroleum-based plastic production reached over 400 million metric tons (Mt), and the accumulation of these non-biodegradable plastics in the environment is a worldwide concern. Polyhydroxybutyrate (PHB) offers many advantages over traditional petroleum-based plastics, being biobased, completely biodegradable, and non-toxic. However, its production and use are still challenging due to its low deformation capacity and narrow processing window. In this work, two linear-chain polyester oligomers were used as plasticizers to improve the processability and properties of PHB. Thermal analyses, XRD, and polarized optical microscopy were performed to evaluate the plasticizing effect on the PHB and the reflection on the mechanical behavior. Both oligomers acted as PHB plasticizers, with a reduction in Tg and Tm as a function of the plasticizer concentration, which can make it easier to handle the material in thermal processing and reduce the probability of thermal degradation. Plasticizer 2 proved to be the most promising between the two with an optimized condition of 20%, in which there was a decrease in elastic modulus of up to 72% and an increase in the maximum elongation of 467%.


Introduction
In 2021, global plastic production exceeded 400 million metric tons (Mt), and around 85 percent of the total was petroleum-derived [1]. While petroleum-based plastics are essential to virtually all industrial segments, the accumulation of these non-biodegradable plastics in marine, freshwater, and terrestrial ecosystems is a global concern [2][3][4]. In this context, researchers have focused their attention on developing and producing biodegradable plastics.
Polyhydroxyalkanoates (PHAs) are biodegradable polymers that are partially crystalline, with a comparatively high melting temperature (T m ≈ 175 • C) and a high degree of crystallinity classically produced from different types of microorganisms, such as Alcaligenes, Azobacter, Bacillus, and Pseudomonas. These ferment organic matter and accumulate PHA in the presence of carbon and other nutrient-deficient conditions (typically through N or P limitation) [5][6][7][8]. Cyanobacteria have been reported to synthesize PHB from CO 2 as a sole carbon source [9,10]. Methanotrophic bacteria can yield PHB from CH 4 as a sole carbon and energy source [11,12]. Thereby, the latter two classes of microorganisms do not require feed or food sources such as glucose for PHB production and can offer a sustainable route for scale-up.
Polyhydroxybutyrate (PHB) is the most common form of PHA. It is a naturally occurring compound. PHB has thermoplastic properties and offers many advantages over and that both plasticizers have a critical concentration of 20 wt%; higher concentrations cause phase separation and consequently mechanical deterioration. Thus, at the limit of mixture miscibility, the addition of oligomer plasticizers provides effective plasticization with greater thermal stability than low-molecular-weight plasticizers.

Materials
The PHB in this study was obtained from the cyanobacterial strain Synechocystis sp. PCC 6714 feeding on CO 2 as a sole carbon source.
An axenic culture of wild-type strain Synechocystis sp. PCC 6714 was purchased from the Pasteur Culture Collection of Cyanobacteria (Pasteur Institute, Paris, France). The cells were grown in a modified BG-11 medium at pH 8.2. In order to induce nitrogen deficiency, cells were cultured in BG-11 medium without nitrate and ammonia. (NH 4 ) 5 [Fe(C 6 H 4 O 7 ) 2 ] and Co(NO 3 ) 2 ·6H 2 O were substituted with equimolar concentrations of FeC 6 H 5 O 7 and CoCl 2 ·6H 2 O with regard to iron and cobalt content. For phosphorus limitation, KH 2 PO 4 was replaced with an equimolar concentration of KCl for potassium content [10].
The highest average volumetric PHB production rate was obtained during two-step cultivation with a value of 14 mg L −1 d −1 , and the highest specific PHB production rate was determined during a one-step process with a value of 5.4 mg g −1 d −1 . The strain could produce up to 16% (DCW) PHB under nitrogen and phosphorous limitation [9].
The pilot-scale cultivations were performed in a 40 L glass reactor of tubular with a vertical design (airlift). The circulation was performed using sterile filtered air [31].
For PHB extraction, the biomass was lyophilized and suspended in chloroform at 30 mL g −1 of biomass. The suspension was put on a heating block and allowed to boil for one hour under continuous shaking at 300 rpm. The hot suspension was filtered through filter paper. The PHB was extracted using 10 times volume of ice-cold methanol. The polymer was separated using centrifugation at 30,000 rpm, and then was air-dried. The PHB was finally washed using cold acetone [31].
The GPC analysis showed that the molecular weight was (Mw = 1,051,900 g mol −1 ) and the number average of the molecular weight of the PHB was (Mn = 316,060 g mol −1 ). The polydispersity index (PDI-M w /M n ) of the cyanobacterial PHB was determined to be 3.328 [31].

X-ray Photoelectron Spectroscopy
The plasticizer surfaces were analyzed by X-ray photoelectron spectroscopy (XPS) using a K-alpha+ spectrometer (ThermoFisher Scientific, Waltham, MA, USA) with Alkα radiation (1486.6 eV) and a pass energy of 200 eV for the survey and 50 eV for the high-resolution spectra. A flood gun was used for static charge compensation. The X-ray beam size was 400 µm. The operation was carried out at a base pressure of 10 −7 Pa. The background was subtracted according to the Shirley model, and the peak fit was performed with a product of Gaussian and Lorentzian shapes. Atomic concentration was based on Scofield sensitivity factors [32].

Preparation of Plasticized PHB
The plasticized PHB samples were obtained via solution using chloroform as solvent, mixing PHB with different weight percentages (wt%) of either P1 or P2 (10, 20 and 30%). Each solution was prepared at a concentration of 0.2 g mL −1 and heated to 40 • C under continuous agitation for 2 h, including a solution of neat PHB. The solutions were then Polymers 2022, 14, 4197 4 of 16 poured into Petri dishes and kept in a fume hood until the complete evaporation of the solvent had taken effect. The plasticized PHB and neat PHB samples were obtained by pressing the remaining material in a hydraulic press with a pressure of 0.5 MPa, at a temperature of 180 • C, for 5 min. The resulting samples were identified as PHB/xxP1 or PHB/xxP2, where xx is the weight fraction of the incorporated plasticizer.

Thermal Characterization
Thermogravimetric analysis (TGA) was carried out using a TGA Q500 (TA instruments, Waltham, MA, USA), from room temperature to 600 • C, at a heating rate of 10 • C min −1 , in an inert nitrogen atmosphere. Differential scanning calorimetry (DSC) measurements were carried out on a DSC Q200 equipment (TA Instruments, Waltham, MA, USA). A first heating scan from room temperature to 200 • C (isothermal for 3 min) was used to erase the thermal history of the polymer, followed by cooling to −80 • C and reheating to 210 • C. All heating and cooling cycles were performed at a rate of 20 • C min −1 in a nitrogen atmosphere.
The crystallinity index (X DSC ) was estimated as shown in Equation (1) [33]: where ∆H m is the PHB melting enthalpy in the sample, ∆H 0 m is the melting enthalpy for 100% crystalline PHB (∆H 0 m = 146 J g −1 ), and w i is the weight fraction of PHB in the plasticized sample (w i = 0.9, 0.8 or 0.7).

Crystalline Morphology
Polarized optical microscopy (POM) was used to evaluate the effect of the plasticizer addition on the spherulitic morphology of PHB. In a heating module T95 HS (Linkam, Salfords, UK) coupled to an Axio Scope A1 optical microscope (Carl Zeiss, Oberkochen, Germany), samples in the order of 4 mg were heated to a temperature of 210 • C, kept in isotherm conditions for 3 min, and cooled at a rate of 15 • C min −1 to a temperature of 60 • C, where they were kept in an isotherm for 1 h.

X-ray Diffraction (XRD) Analysis
The crystalline structure of PHB was studied with Stadi P equipment (Stoe, Darmstadt, Germany), with Cu-K-α (λ = 0.1542 nm) in 2θ range 5 • -30 • with a scan rate of 1 • min −1 . Fityk software (version 1.3.1) was used to analyze the data. For comparison purposes, the crystallinity index was also calculated using the XRD analysis (X XRD ) from Equation (2): where A c is the sum of the areas under the crystalline peaks extracted from the XRD diffractogram, and A a is the area of the amorphous halo.

Mechanical Properties
Tensile strength (σ), elastic modulus (E), and the elongation at break (ε) were determined using an universal testing machine model 3369 (Instron, Norwood, MA, USA). The samples were stored in a desiccator for 15 days before performing the analyses and cut into rectangles of 6.0 × 0.8 mm. Tests were carried out in ambient conditions with a crosshead speed of 2 mm min −1 , using a gauge length of 10 mm.

Structure of the Polyesters
XPS analyzes were performed on the plasticizers to obtain information on the surface composition of the polyesters [34]. Figure 1 shows the XPS spectra of the plasticizers, P1 and P2, where the photoemissions of C(1s) and O(1s) are observed. The high-resolution resolved spectra of C(1s) and O(1s) are shown in Figures 2 and 3, respectively. The atomic concentrations of carbon and oxygen were determined from binding energy and based on sensitivity factors. Table 1 presents the data obtained in the spectra photoemission, binding energy, area, and the atomic O/C ratio.
into rectangles of 6.0 × 0.8 mm. Tests were carried out in ambient conditions with a crosshead speed of 2 mm min −1 , using a gauge length of 10 mm.

Structure of the Polyesters
XPS analyzes were performed on the plasticizers to obtain information on the surface composition of the polyesters [34]. Figure 1 shows the XPS spectra of the plasticizers, P1 and P2, where the photoemissions of C(1s) and O(1s) are observed. The high-resolution resolved spectra of C(1s) and O(1s) are shown in Figures 2 and 3, respectively. The atomic concentrations of carbon and oxygen were determined from binding energy and based on sensitivity factors. Table 1 presents the data obtained in the spectra photoemission, binding energy, area, and the atomic O/C ratio.   head speed of 2 mm min −1 , using a gauge length of 10 mm.

Structure of the Polyesters
XPS analyzes were performed on the plasticizers to obtain information on the surface composition of the polyesters [34]. Figure 1 shows the XPS spectra of the plasticizers, P1 and P2, where the photoemissions of C(1s) and O(1s) are observed. The high-resolution resolved spectra of C(1s) and O(1s) are shown in Figures 2 and 3, respectively. The atomic concentrations of carbon and oxygen were determined from binding energy and based on sensitivity factors. Table 1 presents the data obtained in the spectra photoemission, binding energy, area, and the atomic O/C ratio.   The high-resolution resolved spectrum of C1s from P1 ( Figure 2a) shows three photoemission peaks-284.78 eV, 286.35 eV, and 288.71 eV-which correspond to the C-C, C-O, and C=O bonds, respectively. Still, for sample P1, the resolved spectrum of O1s (Figure 3a) presents two peaks, 531.91 eV and 533.26 eV, which refer to C=O and C-O, respectively. The adjusted binding energy peaks corresponded to polyester and were previously observed in the literature [35]. The resolved spectra, C1s and O1s, of sample P2 are shown in Figures 2 and 3, respectively. In C1s, in addition to the peaks corresponding to C-C, C-O, and C=O, a fourth photoemission peak was observed at 290.22 eV. This fourth peak possibly corresponds to an end-of-chain bond HO-C=O, or even HO-C(O)-O, originating from the 2-ethyl-1-hexanol used as a terminating agent in obtaining P2, which due to its greater electronegativity has a greater distance eV to the C-C/C-H bond [36]. Likewise, the resolved spectrum of O1s from P2 has three peaks, the two corresponding to C=O and C-O and an additional peak at 535.21 eV.  The high-resolution resolved spectrum of C1s from P1 ( Figure 2a) shows three photoemission peaks-284.78 eV, 286.35 eV, and 288.71 eV-which correspond to the C-C, C-O, and C=O bonds, respectively. Still, for sample P1, the resolved spectrum of O1s ( Figure  3a) presents two peaks, 531.91 eV and 533.26 eV, which refer to C=O and C-O, respectively. The adjusted binding energy peaks corresponded to polyester and were previously observed in the literature [35]. The resolved spectra, C1s and O1s, of sample P2 are shown in Figures 2 and 3, respectively. In C1s, in addition to the peaks corresponding to C-C, C-O, and C=O, a fourth photoemission peak was observed at 290.22 eV. This fourth peak possibly corresponds to an end-of-chain bond HO-C=O, or even HO-C(O)-O, originating from the 2-ethyl-1-hexanol used as a terminating agent in obtaining P2, which due to its greater electronegativity has a greater distance eV to the C-C/C-H bond [36]. Likewise, the resolved spectrum of O1s from P2 has three peaks, the two corresponding to C=O and C-O and an additional peak at 535.21 eV.  The atomic O/C ratio of plasticizers has a slight difference-0.337 and 0.306-for P1 and P2, respectively. The main difference between them occurs in the proportion of each kind of binding energy. P1 has a ratio of approx. 52% C=O and 48% C-O, i.e., each bond involving O corresponds to an ester group. The ester group is formed in the stoichiometric reaction between dicarboxylic acid (adipic acid), diol (propylene glycol), and the carboxylic acid and hydroxyl groups present in lactic acid.
On the other hand, P2 has a higher proportion of C-O compared to C=O: approx. 69% and 31%, respectively. In this case, proportionally, there is an additional bond of the C-O type for each ester group. This occurs due to the reaction of adipic acid (dicarboxylic acid) with two diols, ethylene glycol, and 1,4-butanediol.

Thermal Properties
TGA and DSC analyses were carried out to evaluate the dependence of the thermal properties of PHB blends on the plasticizer used. Figure 4 shows the weight loss curves (Figure 4a,c) and their corresponding derivative curves (DTG Figure 4b,d) of neat PHB and plasticized PHB/P1 and PHB/P2. At the same time, the main thermal degradation parameters are presented in Table 2, in which it is possible to evaluate the effect of plasticizers on the thermal stability of PHB. reaction between dicarboxylic acid (adipic acid), diol (propylene glycol), and the carboxylic acid and hydroxyl groups present in lactic acid.
On the other hand, P2 has a higher proportion of C-O compared to C=O: approx. 69% and 31%, respectively. In this case, proportionally, there is an additional bond of the C-O type for each ester group. This occurs due to the reaction of adipic acid (dicarboxylic acid) with two diols, ethylene glycol, and 1,4-butanediol.

Thermal Properties
TGA and DSC analyses were carried out to evaluate the dependence of the thermal properties of PHB blends on the plasticizer used. Figure 4 shows the weight loss curves (Figure 4a,c) and their corresponding derivative curves (DTG Figure 4b,d) of neat PHB and plasticized PHB/P1 and PHB/P2. At the same time, the main thermal degradation parameters are presented in Table 2, in which it is possible to evaluate the effect of plasticizers on the thermal stability of PHB.    Neat PHB has a single weight loss event, with a temperature of the maximum degradation rate (T d1 ) at 288 • C, a value commonly observed in the literature [19,24] and which can be attributed to the random chain scission of PHB by intramolecular cis-elimination [18]. Using either P1 or P2 plasticizers did not significantly change either temperature corresponding to 5% weight loss (T 5% ≈ 270 • C) or the first DTG peak (T d1 ≈ 290 • C) in relation to PHB decomposition, respectively. The second weight loss event for plasticized PHB was around 360 • C for P1 and 390 • C for P2. The peak's intensity is proportional to each component's weight fraction and, therefore, can be associated with plasticizer degradation. Thus, the TGA results showed that the addition of plasticizers does not cause the degradation of PHB. Figure 5 shows DSC curves for the second reheating cycle and the cooling cycle for the PHB plasticized with P1 (Figure 5a,b) and P2 (Figure 5c,d) of the main thermal parameters and X DSC (calculated from Equation (1)) are summarized in Table 3.
relation to PHB decomposition, respectively. The second weight loss event for plasticized PHB was around 360 °C for P1 and 390 °C for P2. The peak's intensity is proportional to each component's weight fraction and, therefore, can be associated with plasticizer degradation. Thus, the TGA results showed that the addition of plasticizers does not cause the degradation of PHB. Figure 5 shows DSC curves for the second reheating cycle and the cooling cycle for the PHB plasticized with P1 (Figure 5a,b) and P2 (Figure 5c,d), respectively. The values of the main thermal parameters and XDSC (calculated from Equation (1)) are summarized in Table 3.  Table 3. Main parameters obtained from DSC analysis of PHB, PHB/P1, and PHB/P2. Tg is the glass transition temperature; Tc is the crystallization peak temperature; ΔHc is the enthalpy of crystallization; Tcc is the cold crystallization peak temperature; ΔHcc is the enthalpy of cold crystallization; Tm is the melting peak temperature; ΔHm is the enthalpy of melting; XDSC is the calculated crystallinity. The melting temperature peak of neat PHB was 175.24 °C. The addition of plasticizers resulted in a slight decrease in the Tm of the PHB blends, proportional to the concentration of each plasticizer added. The most significant reduction occurred for the addition of 30%  Table 3. Main parameters obtained from DSC analysis of PHB, PHB/P1, and PHB/P2. T g is the glass transition temperature; T c is the crystallization peak temperature; ∆H c is the enthalpy of crystallization; T cc is the cold crystallization peak temperature; ∆H cc is the enthalpy of cold crystallization; T m is the melting peak temperature; ∆H m is the enthalpy of melting; X DSC is the calculated crystallinity.

Material
T The melting temperature peak of neat PHB was 175.24 • C. The addition of plasticizers resulted in a slight decrease in the T m of the PHB blends, proportional to the concentration of each plasticizer added. The most significant reduction occurred for the addition of 30% of P1, resulting in T m = 169 • C; when 30% of P2 was added, T m was 171 • C. As a result of the T m decrease, the processing temperature window is increased, making the PHB blends easier to process. The enthalpy of melting (∆H m ) of PHB was also reduced with the addition of the plasticizers.
A similar behavior was observed using the low-molecular-weight plasticizer tributyl 2-acetyl citrate (TAC) in PHB. The TAC addition induced a systematic reduction in the melting temperature values of the PHB blend, with 5-13 • C, along with its increasing content in PHB. On the other hand, in the same work, the addition of poly(3-hydroxyoctanoate) (PHO), a biosynthesized homopolymer as a plasticizer, did not induce significant changes in T m due to the low influence of PHO on the mobility of PHB chains [28].
Thus, the thermal results observed in this work indicate that the plasticizers used favor the segmental movement of PHB due to the plasticizer-PHB compatibility.
As shown in Table 3, there was an increase in the degree of crystallinity in the plasticized PHB. This increase was more significant in samples with P1, showing a X DSC up to 8% higher than neat PHB, while samples with P2 showed X DSC up to 5% higher. The addition of plasticizers can cause either a decrease in crystallinity due to the dilution effect or its increase due to the incorporation of an additive into the amorphous phase of a semicrystalline polymer, decreasing the melting viscosity, which results in higher chain diffusion and a faster crystallization rate [19,37]. The crystallinity index values calculated for both plasticizers increased, indicating promising candidates to improve PHB processing, with a possible reduction in melt viscosity accelerating the crystallization rate.
PHB/P1 cooling curves (Figure 5b) exhibited a non-linear relationship with the plasticizer concentration. At lower concentrations, the addition of P1 causes a reduction in T c compared to that of the neat PHB. However, as the P1 content increases, so does T c , surpassing the PHB T c by 6 • C (PHB T c = 68 • C, PHB/30P1 T c = 72 • C). These results can be related to the interaction between PHB and P1. Both components demonstrate a complex influence on crystallization as this mixture has been reported as miscible in the molten state and partially miscible after crystallization [37]. On the other hand, adding P2 ( Figure 5d) decreased T c by up to 15 • C for PHB/10P2 followed by a slight increase for higher P2 concentrations, but this value was still approximately 9 • C lower than PHB T c . The reduction in T c from the addition of P2 indicates a pronounced plasticizing effect [24], in which the interaction with the polyester oligomer possibly hinders the crystallization of PHB.
In addition, a second exothermic peak is observed in the reheating curve (Figure 5a,c). In this case, these peaks are related to cold crystallization effects and occur at temperatures (T cc ) above the T g of PHB, which allows sufficient chain mobility for crystallization to occur [19]. In both plasticizers, there was a decrease in the T g of PHB, which favored the occurrence of the cold crystallization effect in the plasticized PHBs.
As both plasticizers resulted in a reduction in T g and T m , both are seen to be good choices to improve PHB processability, with special attention given to P2, which demonstrated greater interaction in the polymer matrix.
To estimate the T g of mixtures of polymers from data of the pure components and the miscibility of the mixtures, several approaches have been developed. They are commonly based on the additivity of basic thermophysical properties, and one of the most widely used equations to predict the T g of amorphous mixtures and random copolymers is said to be the Fox equation. The Fox equation was used to calculate the theoretical T g for each blend according to Equation (3): where w PHB is the mass fraction of PHB, and T g PHB and T g Plasticizer are the glass transition temperatures of PHB and the plasticizer used, respectively. The resulting model obtained using the Fox equation along with the practical T g results of the PHB blends is shown in Figure 6. The T g values of P1 (−34.2 • C) and of P2 (−64.9 • C) were obtained using DSC.
As shown by the experimental data, adding both plasticizers causes a significant decrease in T g , which is more pronounced for PHB/P2. This indicates that the plasticizer interaction occurs in the amorphous phase of PHB. This effect has already been observed in the work of Bibers et al. [38]. As shown by the experimental data, adding both plasticizers causes a significant decrease in Tg, which is more pronounced for PHB/P2. This indicates that the plasticizer interaction occurs in the amorphous phase of PHB. This effect has already been observed in the work of Bibers et al. [38].
The reduction in Tg, and effective plasticization, occurs from the choice of plasticizer with a balance of molecular weight, spatial structure, and the content of functional groups [25]. In this case, the plasticizers used have aliphatic chains as spacers, providing structural mobility and the ester group as a linking segment with the polymer [39]. The highest C-O/C=O ratio in P2 demonstrated greater effectiveness in plasticizing PHB. It is possible to observe that the Fox model describes the variation in Tg well for both plasticizers up to a concentration of 20%. Comparatively, the best fit occurs in samples with P2, demonstrating a more significant interaction with the PHB matrix, also associated with a more pronounced plasticizing effect (greater reduction in Tg) in the DSC results.
The Tg deviation between the data obtained experimentally and the theoretical model can be associated with the lack of miscibility in the polymer matrix [37]. Thus, these results indicate phase separation at high plasticizer concentrations (30%), as indicated by the more expressive mismatch of practical and theoretical Tg results for the plasticizer concentration of 30%.

Morphology
Polarized optical microscopy (POM) was used to evaluate the effect of the addition of plasticizers on the spherulitic morphology of PHB. The samples were heated to 210 °C and cooled to 60 °C (isothermal crystallization temperature) to promote the samples' crystallization. The POM images of the morphology obtained after crystallization are shown in Figure 7.
All samples showed large spherulites with the characteristic Maltese cross. Neat PHB under controlled conditions presents spherulites with sizes of 350-500 μm [40]. The presence of additives (nucleating agents) or plasticizers can change the size of the spherulites with variation in the nucleation rate [22,24,41]. The reduction in T g , and effective plasticization, occurs from the choice of plasticizer with a balance of molecular weight, spatial structure, and the content of functional groups [25]. In this case, the plasticizers used have aliphatic chains as spacers, providing structural mobility and the ester group as a linking segment with the polymer [39]. The highest C-O/C=O ratio in P2 demonstrated greater effectiveness in plasticizing PHB. It is possible to observe that the Fox model describes the variation in T g well for both plasticizers up to a concentration of 20%. Comparatively, the best fit occurs in samples with P2, demonstrating a more significant interaction with the PHB matrix, also associated with a more pronounced plasticizing effect (greater reduction in T g ) in the DSC results.
The T g deviation between the data obtained experimentally and the theoretical model can be associated with the lack of miscibility in the polymer matrix [37]. Thus, these results indicate phase separation at high plasticizer concentrations (30%), as indicated by the more expressive mismatch of practical and theoretical T g results for the plasticizer concentration of 30%.

Morphology
Polarized optical microscopy (POM) was used to evaluate the effect of the addition of plasticizers on the spherulitic morphology of PHB. The samples were heated to 210 • C and cooled to 60 • C (isothermal crystallization temperature) to promote the samples' crystallization. The POM images of the morphology obtained after crystallization are shown in Figure 7.
All samples showed large spherulites with the characteristic Maltese cross. Neat PHB under controlled conditions presents spherulites with sizes of 350-500 µm [40]. The presence of additives (nucleating agents) or plasticizers can change the size of the spherulites with variation in the nucleation rate [22,24,41].  The addition of plasticizers increased the size of the spherulites. This behavior proportionally to the plasticizer concentration, but its effect is more evident from P2, which has larger and more uniform spherulites. These results may be associate the more significant influence that P2 has on the PHB structure compared to P1 an verge with the results presented by DSC, especially for the variation in Tc. Thus, th tion of P2 caused a reduction in the nucleation rate and, consequently, promoted crease in the diameter of the spherulites. A similar phenomenon is observed in w Umemura et al. [22] with the addition of triethyl ci-treat in PHB. Furthermore, in th ples with 30 wt% of plasticizers, there was a higher incidence of dark spots throu spherulites (yellow arrows); this phenomenon is reported as amorphous phases phase separation [37].

Crystalline Structure
The analysis of the crystalline structure of neat PHB and its plasticized blen performed by XRD, and the resulting diffractograms, with the assigned planes, are The addition of plasticizers increased the size of the spherulites. This behavior occurs proportionally to the plasticizer concentration, but its effect is more evident from adding P2, which has larger and more uniform spherulites. These results may be associated with the more significant influence that P2 has on the PHB structure compared to P1 and converge with the results presented by DSC, especially for the variation in T c . Thus, the addition of P2 caused a reduction in the nucleation rate and, consequently, promoted an increase in the diameter of the spherulites. A similar phenomenon is observed in work by Umemura et al. [22] with the addition of triethyl ci-treat in PHB. Furthermore, in the samples with 30 wt% of plasticizers, there was a higher incidence of dark spots through the spherulites (yellow arrows); this phenomenon is reported as amorphous phases due to phase separation [37].

Crystalline Structure
The analysis of the crystalline structure of neat PHB and its plasticized blends was performed by XRD, and the resulting diffractograms, with the assigned planes, are shown in Figure 8. The PHB and PHB blend diffractograms exhibited similar profiles corresponding to the orthorhombic unit cell [28,42] normally obtained for neat PHB.
As seen in Figure 8, the crystalline peaks present in the spectra are not modified by the addition of plasticizers, which corroborates the crystalline results obtained using DSC analysis. All samples presented two strong crystalline peaks at 2θ ≈ 13.5 • assigned to the (020) plane and 2θ ≈ 17 • to the (110) plane of the orthorhombic unit cell, while also containing a less intense peak set to the (021) plane (2θ ≈ 20 • ), indicating that the samples have a small amount of orthorhombic β-form crystals with zigzag conformation [28].
presented in Table 4. The XXRD of PHB from XRD was 70.17%, whereas XDSC was 61.78%. While the addition of plasticizers to PHB slightly increased the XDSC value, neither P1 or P2 content significantly altered this value. The addition of plasticizers caused a decrease in the XXRD, and the increase in plasticizer content resulted in the reduction in the crystallinity index calculated. The difference between the crystallinities reported by DSC and XRD can be associated with the difference between the methods; XRD emphasizes surface crystallinity while DSC represents bulk behavior [28].

Mechanical Behavior
The effect of the plasticizers' concentration on the mechanical properties of PHB, i.e., elastic modulus (E), maximum tensile strength (σ), and elongation at break (ε), is shown in Figure 9. Neat PHB is a material with high rigidity due to its crystallinity [23]; it has a high elastic modulus and low elongation at break. Plasticizers with greater free volume than the polymer reduce the relative number of polymer-polymer contacts, providing the flexibility of the structure and thereby decreasing the rigidity of the three-dimensional structure, resulting in higher ε values [17,43].
As shown in Figure 9a, the addition of plasticizers caused a decrease in E. Initially, this behavior is more pronounced for P2; however, as plasticizer content increases, the E The X XRD (%) values obtained from the XRD spectra have the same order of magnitude as those obtained from the DSC, but with different absolute values and behavior, as presented in Table 4. The X XRD of PHB from XRD was 70.17%, whereas XDSC was 61.78%. While the addition of plasticizers to PHB slightly increased the X DSC value, neither P1 or P2 content significantly altered this value. The addition of plasticizers caused a decrease in the X XRD , and the increase in plasticizer content resulted in the reduction in the crystallinity index calculated. The difference between the crystallinities reported by DSC and XRD can be associated with the difference between the methods; XRD emphasizes surface crystallinity while DSC represents bulk behavior [28].

Mechanical Behavior
The effect of the plasticizers' concentration on the mechanical properties of PHB, i.e., elastic modulus (E), maximum tensile strength (σ), and elongation at break (ε), is shown in Figure 9. Neat PHB is a material with high rigidity due to its crystallinity [23]; it has a high elastic modulus and low elongation at break. Plasticizers with greater free volume than the polymer reduce the relative number of polymer-polymer contacts, providing the flexibility of the structure and thereby decreasing the rigidity of the three-dimensional structure, resulting in higher ε values [17,43].
As shown in Figure 9a, the addition of plasticizers caused a decrease in E. Initially, this behavior is more pronounced for P2; however, as plasticizer content increases, the E value for both starts to match, showing a decrease of 72% in E for blends with 30% of plasticizer when compared to neat PHB. However, the tensile strength (Figure 9b) only showed an increase for samples with 10% of P2 and no significant changes for PHB blends with 10% of P1. As the plasticizer concentration increases, the σ for both the PHB/P1 and PHB/P2 blends decreases.
The effects of plasticizer concentration on the elongation at break are seen in Figure 9c. The PHB ε value increases for all plasticizer concentrations studied in this work. For PHB blends with 10% plasticizer, for P2 there was an increase of 359% in the PHB elongation at break, while P1 showed an increase of 170%. At 20% of plasticizer, ε increases approximately 450% for both plasticizer-PHB formulations compared to neat PHB. At 30% plasticizer, there is a decrease in the maximum elongation compared to their value at 20%. PHB/P2 blends decreases.
The effects of plasticizer concentration on the elongation at break are seen in Figure  9c. The PHB ε value increases for all plasticizer concentrations studied in this work. For PHB blends with 10% plasticizer, for P2 there was an increase of 359% in the PHB elongation at break, while P1 showed an increase of 170%. At 20% of plasticizer, ε increases approximately 450% for both plasticizer-PHB formulations compared to neat PHB. At 30% plasticizer, there is a decrease in the maximum elongation compared to their value at 20%. This behavior is also observed in other plasticized PHB sys tributed to the high crystallinity of PHB, which hinders the di chains in the crystalline regions and causes a concentration satur system, reducing its mechanical properties. These results supp hypothesis generated from the Tg results for samples with the ad Thus, both oligomers P1 and P2 are effective when used wherein P2 indicates better mechanical properties than P1. D properties of the final products, the optimal concentration of pla with a critical concentration of 20%, as higher concentrations co ration and the consequent deterioration of mechanical propertie

Conclusions
Polymers are indispensable materials, and their production of steel. To a large extent, they are used in short-lived, singlepackaging, and classic fossil plastics have two main drawbacks: t and their longevity in the environment. "White littering" and m a huge area of concern because plastics cause harm to the envi biobased and/or biodegradable materials, can be part of the so transition of plastics. PHA materials can play a pivotal role here b This behavior is also observed in other plasticized PHB systems [24,28,44] and is attributed to the high crystallinity of PHB, which hinders the diffusion of the plasticizer chains in the crystalline regions and causes a concentration saturation of plasticizer in the system, reducing its mechanical properties. These results support the phase separation hypothesis generated from the T g results for samples with the addition of 30% plasticizer.
Thus, both oligomers P1 and P2 are effective when used as plasticizers for PHB, wherein P2 indicates better mechanical properties than P1. Depending on the desired properties of the final products, the optimal concentration of plasticizer could be selected with a critical concentration of 20%, as higher concentrations could present system saturation and the consequent deterioration of mechanical properties.

Conclusions
Polymers are indispensable materials, and their production, by volume, exceeds that of steel. To a large extent, they are used in short-lived, single-use applications such as packaging, and classic fossil plastics have two main drawbacks: their depletable feedstock and their longevity in the environment. "White littering" and microplastics have become a huge area of concern because plastics cause harm to the environment. Bioplastics, i.e., biobased and/or biodegradable materials, can be part of the solution towards a circular transition of plastics. PHA materials can play a pivotal role here because they are degradable in different environments, including challenging ones such as cold sea water. PHB is the simplest representative of PHA, and it resembles the commodity plastic PP (polypropylene) in most properties. However, PHB is stiff and brittle, with a small processing window which, coupled with higher material price, limits its application potential. What is needed is a more flexible PHB formulation. Work has been conducted on several copolymers and blends extensively. In this study, the authors have proposed a novel approach: they have developed and tested two linear-structured polyester oligomers as plasticizers for polyhydroxybutyrate (PHB) to positively alter its mechanical and thermal properties.
TGA demonstrated the excellent thermal stability of PHB-plasticizer mixtures, while DSC showed a reduction in T g by 16 and 19 • C, and T m by 5 and 4 • C for PHB/30P1 and PHB/30P2, respectively. The miscibility of the mixtures was qualitatively evaluated using T g calculated with the Fox equation, which showed good miscibility for up to 20% (by weight) plasticizer. The POM images revealed increased spherulite size using P2, and emphasized its good interaction with PHB.
The best plasticizing effect occurred with the addition of P2, which had the highest C-O/C=O ratio: 2.226 versus 0.923 for P1. PHB/P2 presented an increase in tenacity and demonstrated an optimized concentration of 20%. With a concentration of 30% in both plasticizers, there was an indication of phase separation which resulted in the deterioration of mechanical properties.
Therefore, the aliphatic polyesters used provide the effective plasticization of PHB with superior thermal stability compared to low-molecular-weight plasticizers. It is assumed that this work contributes to the advancement of PHA formulation development by offering a route to improved material properties through novel, biobased and biodegradable plasticizing agents.

Data Availability Statement:
The data presented in this study are available in this article.