Influence of Nucleating Agents on the Crystallization, Thermal, and Mechanical Properties of Poly(3-hydroxybutyrate-co-3-hydroxyhexanoate) (P3HBHHx)

Featured Application

The Findings of this study can be applied to improve the injection molding process of P3HBHHx by accelerating its crystallization using nucleating agents. This enables shorter cycle times during manufacturing, making P3HBHHx more suitable for industrial-scale applications as a sustainable alternative to conventional plastic.

Abstract

This study investigates the impact of various nucleating agents on the crystallization behavior, thermal stability, and mechanical properties of poly(3-hydroxybutyrate-co-3-hydroxyhexanoate) (P3HBHHx) with 6 mol% 3-hydroxyhexanoate (3HHx) units. Nucleating agents, including boron nitride (BN), poly(3-hydroxybutyrate) (PHB), talc, ultrafine cellulose (UFC), and an organic potassium salt (LAK), were incorporated to enhance the crystallization performance. Differential scanning calorimetry (DSC) revealed that BN and PHB significantly increased the crystallization temperature and reduced the crystallization time by half, with BN exhibiting the highest nucleation efficiency. Isothermal kinetics modeled using the Avrami and Lauritzen–Hoffman theories confirmed faster crystallization and reduced nucleation barriers in nucleated samples. Polarized optical microscopy (POM) revealed that the nucleating agents altered the spherulite morphology and increased the growth rates. Under fast cooling, only BN induced crystallization, confirming its superior nucleation activity. Thermogravimetric analysis (TGA) indicated minimal changes in thermal stability, while mechanical testing showed a slight reduction in stiffness without compromising the tensile strength. Overall, BN emerged as the most effective nucleating agent for enhancing the P3HBHHx crystallization kinetics, providing a promising strategy for improving processing efficiency and reducing the cycle times in industrial applications.

1. Introduction

Polyhydroxyalkanoates (PHAs) are biodegradable polyesters produced via microbial fermentation using renewable carbon sources, such as sugars, fatty acids, and agricultural by-products. Their biosynthesis is regulated by a sequence of enzymatic reactions, with PHA synthases playing a key role in catalyzing the polymerization of hydroxyalkanoate monomers [1,2]. By carefully controlling the carbon source, fermentation conditions, and microbial strain, it is possible to produce various types of PHAs with a wide range of monomer compositions and material properties [3,4,5,6,7,8,9].

Among the different PHAs, poly(3-hydroxybutyrate-co-3-hydroxyhexanoate) (P3HBHHx) stands out due to its superior flexibility and mechanical performance compared to poly(3-hydroxybutyrate) (PHB). The incorporation of 3-hydroxyhexanoate (3HHx) comonomer units increases the elasticity and toughness of the polymer, making P3HBHHx particularly suitable for applications in packaging, agriculture, and biomedicine [10,11,12,13].

Despite these promising properties, a key limitation of P3HBHHx for industrial use is its slow crystallization rate, especially in copolymers with a high 3HHx content [14]. This slow crystallization behavior can hinder manufacturing processes, such as injection molding and extrusion, leading to longer cycle times, incomplete solidification, and product defects. One effective strategy to accelerate crystallization is the addition of nucleating agents, which facilitate crystal formation and enhance the crystallization rate [15,16,17]. These additives have been widely applied to biodegradable polymers, including polylactic acid (PLA) and other PHAs [15,18,19,20,21].

In this study, a range of nucleating agents, including boron nitride (BN), poly(3-hydroxybutyrate) (PHB), talc, organic potassium salts (LAK), and ultrafine cellulose (UFC), were incorporated into P3HBHHx to evaluate their effects on crystallization behavior, thermal stability, and mechanical performance. These nucleating agents were selected based on their distinct chemical properties, morphology, and compatibility with biopolymers. Inorganic fillers, such as BN and talc, are known for their high thermal conductivity and surface activity, which can promote heterogeneous nucleation [22]. Organic nucleating agents, like LAK and PHB, offer potential benefits in terms of biocompatibility and chemical affinity with the P3HBHHx matrix [23,24]. UFC, on the other hand, represents a natural fiber-based additive with a high surface area and the potential to act as a reinforcement agent [25,26].

Both non-isothermal and isothermal crystallization methods were employed to comprehensively characterize the crystallization behavior. The non-isothermal approach simulates industrial cooling conditions, while isothermal crystallization enables kinetic modeling using the Avrami and Lauritzen−Hoffman theories. This dual approach provides valuable insights into nucleation efficiency, growth mechanisms, and structure-property relationships under both practical and theoretical conditions.

In addition to the crystallization behavior, thermal stability was examined using thermogravimetric analysis (TGA), and the molecular degradation was evaluated via gel permeation chromatography (GPC). The mechanical properties were assessed using tensile testing. Polarized optical microscopy (POM) was used to study spherulitic morphology, crystal growth rates, and nucleation density. Self-nucleation experiments were conducted to quantify the nucleation efficiency and provide deeper insight into the effectiveness of each additive.

By systematically comparing the crystallization behavior and material performance of P3HBHHx with various nucleating agents, this study aims to identify the most suitable additive for improving the processability and expanding the industrial applicability of P3HBHHx-based material.

2. Materials and Methods

2.1. Materials

Poly(3-hydroxybutyrate-co-3-hydroxyhexanoate) (P3HBHHx, 6 mol% 3HHx, grade BP330_05, Helian Polymers, Venlo, The Netherlands) was used as the base polymer in this study. The nucleating agents included boron nitride (LL-SP 010, HeBoFill GmbH, Heilbronn, Germany), organic potassium salt (LAK-301, Takemoto Oil & Fat Co., Ltd., Nagoya, Japan), poly(3-hydroxybutyrate) (PHB, grade PB3000G, PhaBuilder, Beijing, China), ultrafine cellulose (UFC 100, Arbocel, J. Rettenmaier & Söhne, Rosenberg, Germany), and talc (Intalc 10CG, euroMinerals GmbH, Sankt Georgen im Schwarzwald, Germany). All nucleating agents were used as received without further purification.

2.2. Sample Preparation

P3HBHHx samples containing each nucleating agent at concentrations of 0.5 wt% and 2 wt% were prepared by melt blending. Blending was performed using a Process 11 twin-screw extruder (Thermo Scientific, Waltham, MA, USA) with a length-to-diameter (L/D) ratio of 40:1. The barrel temperature profile was set between 145 °C and 160 °C. Following extrusion, the materials were pelletized and subsequently dried in an oven at 60 °C overnight.

The injection molding of the dried pellets was performed using a MiniJet Pro injection molding machine (Thermo Scientific, Waltham, MA, USA) at an injection temperature of 160 °C and a mold temperature of 50 °C, producing standardized test specimens suitable for thermal and mechanical analyses.

Neat P3HBHHx (without any additives) was used as the control sample. The detailed compositions of all the prepared samples are summarized in

Table 1. P3HBHHx samples prepared with various nucleating agents at concentrations of 0.5 wt% and 2 wt%.

Sample Code	Nucleating Agent	Concentration (wt%)
P3HBHHx	None	0
PHH_05BN/PHH_2BN	Boron nitride	0.5/2
PHH_05PHB/PHH_2PHB	P3HB	0.5/2
PHH_05Talc/PHH_2Talc	Talc	0.5/2
PHH_05LAK/PHH_2LAK	Organic potassium salt	0.5/2
PHH_05UFC/PHH_2UFC	Ultrafine cellulose	0.5/2

.

2.3. Sample Characterization

Fourier-transform infrared (FTIR) analysis was performed using a Jasco FTIR 4700 spectrometer (JASCO Corporation, Tokyo, Japan) equipped with a Specac MKII Golden Gate single-reflection diamond ATR system (Specac Ltd., Orpington, UK). Samples were scanned over a wavenumber range of 4000–650 cm⁻¹, and the resulting spectra were compared to identify any changes in functional groups or molecular interactions.

Gel permeation chromatography (GPC) was used to assess whether the addition of nucleating agents led to any significant degradation of the polymer chains. The analysis was performed using a Shimadzu LC-8A liquid chromatographic pump controlled by LC Solution software, version 5.114 (Shimadzu Corporation, Tokyo, Japan). For each sample, 10 mg was dissolved in 1 mL of 1,1,1,3,3,3-hexafluoroisopropanol (HFIP) containing 0.05 M sodium trifluoroacetate (CF₃COONa), which was also the eluent for the column. The flow rate was set at 1 mL/min, and the injected volume was 20 µL with a sample concentration of 10 mg/mL. A PL-HFIP-gel column (Agilent Technologies Deutschland GmbH, Böblingen, Germany) and detection was achieved with a Shimadzu RID-20A refractive index detector (Shimadzu Corporation, Tokyo, Japan). The molecular weights (M_w and M_n) and polydispersity index (PDI) of each sample were determined using polymethyl methacrylate standards.

Thermogravimetric analysis (TGA) was performed using a Q50 thermogravimetric analyzer (TA Instruments, New Castle, DE, USA) to investigate the thermal stability of the P3HBHHx samples in the presence of the nucleating agents. The samples were heated from room temperature to 550 °C at a constant heating rate of 10 °C/min under a nitrogen atmosphere (flow rate: 50 mL/min) to ensure an inert environment and minimize oxidative degradation. This heating rate is commonly used in TGA protocols to evaluate the thermal stability of biodegradable polymers and aligns with the recommendations of ISO 11358-1:2022 [27,28].

The degradation behavior was assessed by analyzing the temperatures at which specific percentages of mass loss occurred: T_5% (5% mass loss), T_50% (50% mass loss), and T_90% (90% mass loss) temperatures. These parameters provide insights into the onset, midpoint, and extent of thermal degradation. In particular, T_5% is widely used to indicate the initial degradation temperature, which is critical for assessing the thermal resistance of a material during processing. Additionally, the temperature at the maximum rate of mass loss (T_max) was determined from the derivative thermogravimetric (DTG) curve, which represents the point of the highest decomposition rate. These parameters together offer a comprehensive understanding of how different nucleating agents influence the thermal stability of P3HBHHx.

Differential scanning calorimetry (DSC) analysis was performed using a Q100 series DSC instrument (TA Instruments, New Castle, DE, USA) equipped with a refrigerated cooling system capable of operating within a temperature range of −90 to 550 °C. Each experiment was performed under a flow of dry nitrogen (flow rate: 50 mL/min) using approximately 4 mg of sample. The instrument was calibrated for both temperature and heat of fusion using an indium standard to ensure measurement accuracy and reproducibility.

To evaluate the effect of various nucleating agents on the crystallization behavior of P3HBHHx, a three-step DSC thermal cycle was performed. First, the samples were heated to 200 °C to erase any previous thermal history and ensure a fully amorphous state. This temperature is well above the melting point of P3HBHHx and enables the complete melting of the crystalline phase. The samples were then cooled to −50 °C to monitor the crystallization during cooling. This low cooling limit ensured the complete detection of crystallization, even in slowly crystallizing samples. Finally, the sample was reheated at 200 °C to evaluate its melting behavior and any cold crystallization events.

This thermal protocol is commonly used for the characterization of PHA copolymers [29,30,31,32]. The specific heating and cooling rates applied in each step are indicated within the corresponding experimental sections, depending on whether the test was performed under isothermal or non-isothermal conditions.

The spherulitic growth rate was measured using a polarized light microscope (Axioskop 40 Pol, Carl Zeiss Microscopy GmbH, Oberkochen, Germany) equipped with a temperature-controlled hot stage (THMS 600) connected to an LNP 94 liquid nitrogen cooling system (Linkam Scientific Instruments, Surrey, UK). Spherulites were grown from homogeneous thin films prepared by melt pressing, with the film thickness controlled to approximately 10 µm. To remove any prior thermal history, the samples were first heated to 100 °C and held for 5 min. They were then cooled to the desired isothermal crystallization temperature and held to allow measurable spherulitic growth. The radius of the growing spherulites was recorded over time using micrographs captured at regular intervals with a Zeiss Axioscam MRC5 digital camera (Carl Zeiss Microscopy GmbH, Oberkochen, Germany). These measurements were used to calculate the spherulitic growth rates.

The mechanical properties of the P3HBHHx samples containing nucleating agents were evaluated by tensile testing using an Instron Universal Testing Machine (Model 34SC-5 Single Column, Instron, Norwood, MA, USA), following with the ASTM D527 standard. Test specimens measured 75 mm × 5 mm × 2 mm. The crosshead speed was set to 20 mm/min, and the load cell capacity was 100 kN. The mechanical parameters, including tensile strength, Young’s modulus, and elongation at break, were determined using the Instron Bluehill software, version 4.47 (Instron, Norwood, MA, USA). For each sample, at least five specimens were tested to ensure reliable and reproducible results.

2.4. Non-Isothermal Crystallization

The non-isothermal crystallization behavior was studied using a three-step protocol. First, the samples were heated from room temperature to 200 °C at a rate of 10 °C/min to eliminate any previous thermal history. This was followed by a cooling run from 200 °C to −50 °C at the same rate. Finally, a second heating run was performed from −50 °C to 200 °C at the same rate. From the second heating cycle, the melting temperature (T_m) and apparent enthalpy of fusion (ΔH_f) were determined.

2.5. Isothermal Crystallization

The isothermal crystallization process was also performed using a three-step protocol. First, the sample was rapidly heated to 200 °C at a rate of 50 °C/min to eliminate any thermal history. Next, it was cooled at the maximum rate (i.e., 100 °C/min) allowed by the apparatus to the predetermined isothermal crystallization temperature and held isothermally to allow crystallization. Finally, the sample was reheated at 200 °C at a rate of 10 °C/min to evaluate the thermal properties of the isothermally crystallized material.

2.6. Self-Nucleation Experiments

Self-nucleation experiments were designed to quantitatively evaluate the nucleation efficiency of the selected additives on the crystallization behavior of P3HBHHx. This procedure was based on the methodology developed by Wittmann et al. [33,34]. The protocol consisted of the following steps: First, the samples were heated to 200 °C at a rate of 50 °C/min to erase their thermal history. Subsequently, they were cooled to 60 °C at 5 °C/min to establish a standard thermal history, followed by isothermal crystallization at 60 °C for 10 min to ensure complete crystallization. Once crystallized, the samples were reheated at 10 °C/min to a defined self-nucleation temperature (T_s) within the partial melting zone and maintained at this temperature for 5 min to induce self-nucleated crystalline seeds. Finally, the samples were cooled to 30 °C at 5 °C/min, and the resulting crystallization peaks were recorded during the cooling run.

The nucleation efficiency (NE) of each nucleating agent was calculated using Equation (1):

$$\% NE={\displaystyle \frac{T_{\mathrm{c}}-T_{\mathrm{c}}^{\mathrm{m}\mathrm{i}\mathrm{n}}}{T_{\mathrm{c}}^{\mathrm{m}\mathrm{a}\mathrm{x}}-T_{\mathrm{c}}^{\mathrm{m}\mathrm{i}\mathrm{n}}}}\times 100$$

(1)

where $T_{\mathrm{c}}^{\mathrm{m}\mathrm{i}\mathrm{n}}$ is the minimum crystallization temperature corresponding to the fully molten state of the polymer (i.e., no residual crystals remain), and $T_{\mathrm{c}}^{\mathrm{m}\mathrm{a}\mathrm{x}}$ is the maximum crystallization temperature obtained when the polymer was partially melted at a carefully selected T_s. Both $T_{\mathrm{c}}^{\mathrm{m}\mathrm{i}\mathrm{n}}$ and $T_{\mathrm{c}}^{\mathrm{m}\mathrm{a}\mathrm{x}}$ were determined using neat P3HBHHx (without nucleating agents), while T_c refers to the crystallization peak temperature of each nucleating agent loaded P3HBHHx during the DSC cooling run.

3. Results and Discussion

3.1. Non-Isothermal Crystallization Behavior of P3HBHHx—Effect of Nucleating Agents

3.1.1. Thermal Behavior of Neat P3HBHHx

Figure 1 shows the DSC thermograms of neat P3HBHHx during non-isothermal crystallization, revealing two distinct crystallization events. During the cooling run at 10 °C/min (see inset of Figure 1), a weak and broad crystallization peak appeared at 36.4 °C, with a relatively low enthalpy of 8.9 J/g. This indicates that only a small fraction of the polymer crystallized during cooling. The broad shape and low intensity of the peak suggest non-uniform and kinetically limited crystallization, likely due to the random incorporation of 3HHx comonomer units, which disrupts chain regularity and hinders nucleation and crystal growth.

Upon reheating, a second crystallization peak appears at 44.7 °C, with a significantly higher enthalpy of 44.6 J/g. This cold crystallization event accounts for the majority of the crystalline fraction and suggests that more stable and ordered crystalline domains preferentially form from the amorphous solid state rather than directly from the melt. It is noticeable that the cold crystallization peak exhibits a long tail extending beyond 100 °C, highlighting the difficulty in achieving complete crystallization due to the presence of 3HHx units.

A double melting peak was observed in the reheating curve. This behavior is typical of semi-crystalline polymers and is commonly attributed to the coexistence of multiple crystalline phases. The lower melting peak at 130 °C corresponds to less ordered, imperfect crystals formed during the initial crystallization, while the higher melting peak at 150 °C is associated with more stable lamellae formed during the heating process.

These results highlight a significant challenge in processing neat P3HBHHx for industrial applications, especially its slow and incomplete crystallization behavior. In manufacturing processes such as injection molding and extrusion, where rapid cooling is essential, a limited crystallization rate can lead to incomplete solidification, uneven mechanical properties, and defects such as warping. DSC analysis clearly demonstrates the need for nucleating agents to overcome these limitations. By increasing the crystallization rate, these additives are expected to promote faster crystal growth, improve the uniformity of crystalline regions, and reduce processing times. As a result, the overall suitability of P3HBHHx for industrial use can be significantly improved.

3.1.2. Effect of Nucleating Agents on Non-Isothermal Crystallization of P3HBHHx

The non-isothermal crystallization behavior of P3HBHHx containing 0.5 wt% nucleating agents was investigated. The results are summarized in

Table 2. DSC summarized the data of P3HBHHx with 0.5 wt% nucleating agents obtained from non-isothermal processes.

Samples	Tc (°C)	ΔHc (J/g)	Tg (°C)	Tcc (°C)	ΔHcc (°C)	Tm1 (°C)	Tm2 (°C)	ΔHf (J/g)
P3HBHHx	36.4	8.9	1.4	44.7	44.6	130.0	150.2	55.6
PHH_05LAK	45.6	18.3	1.4	45.5	38.4	129.8	149.5	58.0
PHH_05UFC	58.9	48.5	4.8	-	-	132.1	150.2	56.5
PHH_05Talc	66.5	46.3	3.7	45.3	0.8	133.2	149.9	49.7
PHH_05PHB	81.9	59.5	4.4	-	-	138.1	152.7	60.0
PHH_05BN	84.1	56.5	3.3	-	-	136.6	150.7	56.0

and illustrated in Figure 2a,b. The incorporation of nucleating agents led to a significant increase in the crystallization temperature (T_c) compared to that of neat P3HBHHx.

The most pronounced improvements were observed for boron nitride (PHH_05BN) and PHB (PHH_05PHB), which raised T_c from 36.4 °C (neat P3HBHHx) to 84.1 °C and 81.9 °C, respectively. This substantial increase indicates that both additives effectively promote crystal formation during cooling. In addition, the crystallization enthalpy increased markedly from 8.9 J/g in neat P3HBHHx to 59.5 J/g in the PHB-loaded sample. The absence of cold crystallization peaks during the second heating run further confirms that crystallization was completed during the cooling phase, leaving no residual amorphous regions to crystallize upon reheating. These findings suggest that both BN and PHB are highly effective in enhancing nucleation and accelerating the crystallization kinetics.

The superior performance of BN and PHB can be attributed to their nucleation efficiency and compatibility with the P3HBHHx matrix. BN possesses a highly crystalline and thermally conductive structure that promotes heterogeneous nucleation. Its high surface area and heat dissipation capacity may lower the nucleation barrier, thereby enabling faster crystal formation [22]. In contrast, PHB is chemically similar to P3HBHHx and may facilitate epitaxial crystallization due to its structural compatibility, providing an efficient interface for chain alignment and crystal growth [35].

Talc and UFC also improved the crystallization behavior of P3HBHHx, increasing T_c to 66.5 °C and 58.9 °C, respectively. However, the presence of a cold crystallization peak in the PHH_05Talc sample, along with its relatively low crystallization enthalpy (46.3 J/g), indicates incomplete crystallization. This suggests that while talc provides nucleation sites, its effectiveness may be limited by agglomeration or less favorable surface interactions. Although offering additional surface area, it contains hydroxyl groups that may interfere with the crystallization process, resulting in moderate performance [36,37].

The addition of organic potassium salt (LAK) resulted in only a slight increase in T_c to 45.6 °C. The limited shift in the crystallization temperature, along with the presence of cold crystallization during reheating, indicates that LAK is less effective in promoting crystallization from the melt. A substantial portion of the polymer remained amorphous after cooling, further confirming its low nucleation efficiency.

The addition of nucleating agents had little impact on the melting temperatures (T_m1 and T_m2) of P3HBHHx, except in the case of PHB, where a slight increase was observed, as highlighted by the dashed ellipses in Figure 2b. For PHH_05PHB, the first melting peak increased to 138.1 °C, and the second peak increased to 152.7 °C. This shift is likely due to the development of thicker lamellae during the crystallization process (note the difference of more than 40 °C between the crystallization temperatures of the neat polymer and PHB-loaded sample). Similar but less pronounced effects were observed for boron nitride, where the first melting peak increased to 136.6 °C while the second melting peak remained unchanged.

These results confirm that the addition of nucleating agents can significantly alter the crystallization behavior of neat P3HBHHx by enhancing the crystallization kinetics and increasing the degree of crystallinity. Among the tested additives, boron nitride and PHB exhibited the highest efficiencies, resulting in faster and more complete crystallization during cooling. Talc and ultrafine cellulose provided moderate improvements, while LAK had a minimal impact.

3.1.3. Effect of Increased Nucleating Agent Concentration on the Crystallization Behavior of P3HBHHx

To evaluate whether 0.5 wt% of the nucleating agents was sufficient to enhance the crystallization behavior of P3HBHHx, a higher concentration of 2 wt% was also tested. As shown in Figure S1, increasing the concentrations of BN and PHB to 2 wt% resulted in only minor improvements. The T_c increased by approximately 3 to 5 °C, while ΔH_c increased by 2 to 6 J/g (Table S1). These slight increases suggest that the nucleation efficiency of BN and PHB is already near saturation at 0.5 wt%, indicating that higher concentrations are unnecessary.

In contrast, talc exhibited a more pronounced concentration-dependent effect on the crystallization behavior of P3HBHHx. Figure 3 compares the samples with talc loadings ranging from 0.5 wt% to 20 wt%. At 2 wt%, the T_c increased significantly from 66.5 to 72.6 °C, and the ΔH_c increased from 46.3 to 57.1 J/g. Additionally, the cold crystallization peak observed at 0.5 wt% disappeared at 2 wt%, as shown in the inset of Figure 3. These results suggest that talc is considerably more effective as a nucleating agent when present at higher concentrations. This improvement may be attributed to the increased number of active nucleation sites provided by the higher talc content, enhancing the probability of crystallite formation during cooling.

In contrast, BN and PHB appear to reach their optimal nucleation performance at 0.5 wt%, as only marginal improvements were observed when their concentration increased to 2 wt%. Therefore, while BN and PHB demonstrate early saturation in nucleating efficiency, talc exhibits a more pronounced concentration-dependent effect. This is likely due to its initial moderate nucleation activity, which becomes significantly more effective as the concentration increases.

Higher talc concentrations, including 10 wt% and 20 wt%, were also evaluated and resulted in further improvements in crystallization behavior. However, at these elevated loadings, the large quantity of talc not only affects crystallization but also has a considerable influence on other properties such as mechanical performance, processability, and potentially biodegradability. Therefore, 2 wt% talc was selected for further investigation in the following studies.

For ultrafine cellulose (UFC), increasing the concentration to 2 wt% resulted in a more defined and sharper crystallization peak, indicating improved nucleation uniformity and more consistent nucleation site formation. However, ΔH_c remained largely unchanged compared to the 0.5 wt% sample, suggesting that UFC primarily enhances crystalline uniformity rather than improving crystallization efficiency.

Lastly, increasing the concentration of LAK to 2 wt% showed no improvement in either T_c or ΔH_c (Table 2 and Table S1, Figure S1). These results indicate that the organic potassium salt-based nucleating agent lacks sufficient nucleating activity to influence the crystallization behavior of P3HBHHx, even at higher concentrations.

3.1.4. Nucleation Efficiency

As shown in Figure 4, self-nucleation experiments revealed that fully crystallized P3HBHHx exhibits distinct crystallization behavior when reheated to different self-nucleation temperatures (T_s). At T_s = 145 °C, no crystallization occurred during the subsequent cooling phase. Since melting did not occur at this temperature, the original crystalline structures remained intact, and recrystallization was not initiated.

When T_s increased to 150 °C, partial melting occurred, leaving some crystalline domains intact. These residual structures acted as nucleation sites, resulting in rapid crystallization during cooling. Under these conditions, the crystallization temperature was recorded as $T_{\mathrm{c}}^{\mathrm{m}\mathrm{a}\mathrm{x}}$ = 127.7 °C for neat P3HBHHx.

As T_s increased further, the crystallization temperature progressively decreased due to the melting of additional crystalline domains. At T_s = 170 °C, all nuclei completely melted, leaving no residual sites for nucleation. In this case, the crystallization temperature was identified as $T_{\mathrm{c}}^{\mathrm{m}\mathrm{i}\mathrm{n}}$ = 54.5 °C. It is important to note that in a previous non-isothermal crystallization study, the crystallization temperature of neat P3HBHHx was reported as 36.4 °C under conditions where the material had also been fully melted. This difference can be attributed to the effect of the cooling rate on the crystallization behavior. At slower rates, such as 5 °C/min, the polymer chains have more time to align and form crystalline regions, resulting in a higher T_c. In contrast, faster cooling rates, such as 10 °C/min, reduce the time available for nucleation and crystal growth, leading to a lower T_c.

The nucleation efficiency (% NE) of each nucleating agent was quantified using Equation (1). The results are summarized in

Table 3. Crystallization temperature of P3HBHHx with different nucleating agents at a cooling rate of 5 °C/min and corresponding nucleation efficiency calculated from Equation (1).

	Tc (°C) a	NE (%) b
P3HBHHx	54.5	-
PHH_05LAK	57.0	3.4
PHH_05UFC	68.3	18.9
PHH_05Talc	76.9	30.7
PHH_2Talc	83.9	40.2
PHH_05PHB	85.8	42.8
PHH_05BN	88.3	46.2

^a Determined from a cooling run at 5 °C/min. ^b Calculated using Equation (1).

and Figure 5. The NE results revealed significant differences in the performance of the tested nucleating agents, highlighting their varying abilities to enhance the crystallization behavior of P3HBHHx.

PHH_05BN and PHH_05PHB exhibited the highest % NE values of 46.2% and 42.8%, respectively. These results demonstrate their strong ability to promote nucleation and significantly increase the number of effective crystallization sites during cooling. Their high efficiency suggests that BN and PHB facilitate early-stage crystal formation, allowing P3HBHHx crystallization to occur more rapidly and uniformly. The effectiveness of BN can be attributed to its high surface area, thermal conductivity, and stable crystalline structure, which favor heterogeneous nucleation. PHB, which is structurally similar to P3HBHHx, allows epitaxial crystallization due to molecular compatibility, leading to efficient chain alignment and crystal growth.

Talc also demonstrated strong nucleation effects, particularly at a concentration of 2 wt%, where it reached an % NE value of 40.2%, which is comparable to those of BN and PHB. This enhanced crystallization behavior is particularly beneficial in industrial processes, such as injection molding, where reduced cycle times are essential.

In contrast, PHH_05UFC and PHH_05LAK exhibited considerably lower NE values of 3.4% and 18.9%, respectively, indicating limited nucleating activity. These low values suggest that both ultrafine cellulose and the organic potassium salt provide insufficient nucleation sites to significantly influence the crystallization of P3HBHHx. The poor performance of UFC may be related to its abundant hydroxyl group, which can form hydrogen bonds with the polymer matrix and interfere with local chain mobility, as well as its tendency to agglomerate, reducing the effective number of nucleating surfaces. Similarly, LAK may suffer from poor dispersion in the hydrophobic P3HBHHx matrix and lack chemical affinity with the polymer. These characteristics limit their ability to trigger early or uniform nucleation, resulting in a minimal impact compared to more efficient nucleating agents such as BN, PHB, and talc.

3.2. Structural Comparison of P3HBHHx with Different Nucleating Agents

3.2.1. FTIR Analysis

Fourier-transform infrared spectroscopy (FTIR) was used to assess whether the incorporation of nucleating agents induced any structural changes in P3HBHHx. As shown in Figure 6, the characteristic absorption bands of P3HBHHx were preserved in all samples, regardless of the nucleating agent. These include the carbonyl stretching vibration (C=O) at approximately 1720 cm⁻¹ and the C-O stretching and bending vibrations in the range of 1300–1050 cm⁻¹, which are associated with the ester groups in the polymer backbone [38]. The absence of new peaks, significant shifts, or changes in the fingerprint region further confirms that the molecular structure of P3HBHHx remained unaffected by the presence of the nucleating agents.

3.2.2. Degradation of Nucleated Samples During Processing

The potential degradation of P3HBHHx during the extrusion process in the presence of nucleating agents was evaluated by analyzing the molecular weights of the samples using gel permeation chromatography (GPC). As shown in Figure 7 and summarized in Table S2, both the weight-average (M_w) and number-average (M_n) molecular weights of most nucleated samples remained consistent with those of the neat P3HBHHx copolymer (M_w = 245,280 g/mol; M_n = 122,440 g/mol). This consistency indicates that the incorporation of nucleating agents did not lead to polymer degradation during extrusion.

An exception was observed in the sample containing ultrafine cellulose (PHH_05UFC), which exhibited a significant increase in M_w to 300,520 g/mol, approximately 50,000 g/mol higher than that of the neat polymer. A slight increase in M_n was also observed (M_n = 124,650 g/mol). This increase was not attributed to chemical modification but was likely due to physical interactions between the hydroxyl groups on the cellulose surface and the polymer matrix, which may have promoted chain entanglement during processing [39]. This phenomenon appears to be specific to PHH_05UFC, as it was not observed in samples containing other nucleating agents that lack surface functional groups capable of inducing such interactions.

Overall, these results demonstrate that most nucleating agents, when used at low concentrations, do not cause degradation of P3HBHHx during extrusion. This thermal and molecular stability is critical for industrial applications, as it ensures that the crystallization performance can be enhanced without compromising the polymer’s structural integrity. As a result, the mechanical properties of the material are likely to be preserved, rendering these formulations suitable for a wide range of practical applications.

3.2.3. Influence of Nucleating Agents on the Thermal Stability of P3HBHHx

Figure 8 presents the results of TGA, showing the remaining mass percentage of each sample as a function of temperature, along with the corresponding derivative thermogravimetric (DTG) curves. The temperatures at which 5%, 50%, and 90% mass loss occurred (T_5%, T_50%, and T_90%), the temperature at the maximum rate of mass loss (T_max), and the residual mass at 450 °C are summarized in

Table 4. Thermogravimetric parameters of P3HBHHx and the nucleated samples.

	Tmax (°C)	T5% (°C)	T50% (°C)	T90% (°C)	Residue at 450 °C (wt%)
P3HBHHx	275.7	246.9	269.1	276.7	0
PHH_05LAK	273.5	246.8	267.3	275.1	0.57
PHH_05UFC	261.1	236.3	255.4	263.6	0.50
PHH_05PHB	273.6	247.1	267.8	275.3	0.52
PHH_05BN	258.3	236.1	255.2	263.8	0.58
PHH_05Talc	271.4	246.7	266.9	274.6	0.53
PHH_2Talc	279.5	247.9	264.3	281.7	1.76

.

According to the results, neat P3HBHHx exhibited T_5% of 246.9 °C. Samples containing PHB (PHH_05PHB), LAK (PHH_05LAK), and 0.5 wt% talc (PHH_05Talc) showed nearly identical T_5% values around 246 °C, indicating that these nucleating agents did not significantly affect the initial thermal resistance of the material. A slight increase to 247.9 °C was observed for PHH_2Talc, suggesting a modest improvement in thermal stability at a higher talc content.

In contrast, PHH_05BN and PHH_05UFC exhibited significantly lower T_5% values of 236.1 °C and 236.3 °C, respectively, indicating a reduction in thermal stability. A similar trend was observed for T_50% and T_90%, reinforcing the observation that these two additives accelerated degradation. This behavior is likely associated with the specific interactions between the nucleating agents and the polymer matrix. Boron nitride, known for its high thermal conductivity, may enhance heat transfer within the material, leading to localized heating and an earlier onset of decomposition [40]. Similarly, ultrafine cellulose introduces numerous hydroxyl groups that can act as additional sites for thermal degradation. These groups are prone to hydrolysis, potentially accelerating the degradation process by initiating the earlier thermal breakdown of the polymer chains [36].

T_max trends were largely consistent with the T_5% results, with PHH_05BN and PHH_05UFC exhibiting the lowest T_max values (258.3 °C and 261.1 °C, respectively), indicating that these samples underwent the most rapid decomposition during thermal breakdown. In contrast, PHH_2Talc exhibited the highest T_max of 279.5 °C. This behavior is consistent with the enhanced crystallinity and thermal resistance observed for this sample. This improvement is likely related to the increased crystallinity observed with 2 wt% talc (Figure 3), as crystalline regions are inherently more thermally stable than amorphous ones. These ordered domains require more energy to be disrupted, which contributes to the elevated degradation temperature of the PHH_2Talc sample.

3.2.4. Mechanical Testing

Mechanical tests were conducted to assess the effects of the nucleating agents on the mechanical properties of P3HBHHx. As shown in

Table 5. Mechanical properties of P3HBHHx and the corresponding nucleating agents.

Sample	Young’s Modulus (MPa)	Tensile Strength (MPa)	Elongation at Break (%)
P3HBHHx	2264 ± 138	33.7 ± 0.4	4.0 ± 0.2
PHH_05LAK	2260 ± 213	30.8 ± 0.2	3.8 ± 0.1
PHH_05UFC	2309 ± 42	34.1 ± 0.4	3.9 ± 0.1
PHH_05PHB	1866 ± 119	32.4 ± 0.3	4.8 ± 0.2
PHH_05BN	2060 ± 19	34.5 ± 0.4	4.5 ± 0.2
PHH_05Talc	1906 ± 43	30.0 ± 0.4	3.6 ± 0.1
PHH_2Talc	1943 ± 46	31.2 ± 0.6	3.9 ± 0.2

and Figure 9, most nucleated samples maintained mechanical performance comparable to that of the neat polymer, with variations in Young’s modulus, tensile strength, and elongation at break, generally within 10%. All specimens were injected into a mold set at 50 °C, allowing consistent crystallization across the samples. However, as discussed later, the heterogeneous nucleation introduced by the additives and homogeneous nucleation in the neat polymer result in different spherulite sizes due to their distinct induction times.

Specifically, samples with enhanced crystallization and higher nucleation efficiency, such as PHH_05PHB, PHH_05BN, and PHH_05Talc or PHH_2Talc, exhibited a more pronounced decrease in modulus, from 2264 MPa in the neat sample to values between 1866 and 2060 MPa, representing a reduction of up to 17%. These samples also exhibited a slight increase in the elongation at break. This behavior is attributed to changes in crystalline morphology, particularly the formation of larger spherulites through heterogeneous nucleation, which results in reduced stiffness.

Although an inverse relationship between the modulus and elongation is often expected in polymer systems, such a trend was not clearly observed here. This is primarily because the absolute differences in the elongation at break across the samples are relatively small, typically within ±0.5%, as indicated in Table 5. Given this narrow range, it is difficult to establish a correlation between stiffness and ductility.

To further explore the influence of processing conditions, additional tests were conducted on PHH_05BN at different mold temperatures (50 °C, 60 °C, 70 °C, and 80 °C). As shown in Figure 10, increasing the mold temperature led to a further decrease in stiffness, as evidenced by the lower modulus and higher elongation at break. This trend is consistent with the expected formation of larger spherulites in heterogeneously nucleated samples under slow cooling conditions.

These findings highlight the importance of considering the interplay between nucleating agents and processing parameters when optimizing the mechanical properties of P3HBHHx. While efficient nucleators improve crystallization kinetics and potentially reduce cycle time, their influence on crystalline morphology can affect stiffness. Therefore, achieving a balance between enhanced processability and acceptable mechanical performance is essential for industrial applications.

3.3. Isothermal Crystallization and Kinetic Analysis

3.3.1. Crystallization of Neat and Nucleated P3HBHHx at Different Isothermal Temperatures

The non-isothermal DSC results identified boron nitride (BN) as the most effective nucleating agent for P3HBHHx, increasing the crystallization temperature (T_c) from 36.4 °C in neat P3HBHHx to 84.1 °C. PHB and talc (2 wt%) also showed significant effects, increasing the T_c to 81.9 °C and 72.6 °C, respectively. To further investigate the crystallization behavior and kinetics, isothermal crystallization experiments were conducted at various temperatures for these samples.

Figure 11 shows the DSC exothermic peaks corresponding to the isothermal crystallization of the neat and nucleated P3HBHHx samples. When the isothermal temperature closely approaches the crystallization temperature identified in the non-isothermal analysis, the crystallization peaks are sharp and well-defined, indicating rapid nucleation and crystal growth. In contrast, at higher isothermal temperatures, the peaks become broader and appear after longer induction times, reflecting a reduced thermodynamic driving force for crystallization and slower kinetics.

It is important to note that for a reliable kinetic analysis, the selected isothermal temperature must enable full crystallization during the isothermal hold. For example, although the crystallization of neat P3HBHHx (Figure 11a) at 50 °C occurs rapidly with a sharp and defined peak, the crystallization enthalpy determined was only 41.5 J/g (

Table 6. Summarized data of the isothermal crystallization process for P3HBHHx and various nucleating agents at different temperatures obtained from DSC.

Sample	Isothermal Temperature (°C)	Enthalpy of Crystallization (J/g)	Half-Time of Crystallization τ(1/2) (s)	Crystallization Rate k × 103 (s−1)
P3HBHHx	50	41.5	77	13.06
	70	55.1	159	6.27
	80	60.3	340	2.94
	85	60.1	726	1.38
	95	60.8	1404	0.71
PHH_05BN	95	48.1	68	14.67
	100	64.3	99	10.05
	105	66.5	173	5.77
	110	66.7	306	3.27
PHH_05PHB	85	46.9	104	9.66
	90	59.4	165	6.06
	95	60.2	193	5.18
	100	60.5	242	4.13
PHH_2Talc	85	44.5	84	11.84
	90	55.6	106	9.47
	95	58.0	133	7.53
	100	58.4	244	4.09
	105	60.3	434	2.31

), significantly lower than the 60.3 J/g measured at higher temperatures (80 °C and above). This suggests that a substantial portion of the material had already crystallized during the rapid cooling step prior to reaching the isothermal temperature. Consequently, this condition does not accurately reflect isothermal crystallization and is unsuitable for kinetic modeling.

The same criterion applies to the nucleated samples. For example, PHH_05BN required isothermal temperatures of 100 °C or higher to avoid partial crystallization during cooling. At this temperature, PHH_05BN exhibited significantly higher enthalpy values (64.3 J/g) than neat P3HBHHx (Table 6), which can be attributed to the high nucleation site density introduced by boron nitride, promoting the formation of well-ordered crystalline structures.

Both PHH_05PHB and PHH_2Talc exhibited enhanced crystallization behavior, achieving complete crystallization at isothermal temperatures slightly lower than those of PHH_05BN. PHH_05PHB reached a stabilized enthalpy of approximately 60 J/g at 90 °C, while PHH_2Talc achieved a similar value at 95 °C. These results are comparable to the enthalpy observed for neat P3HBHHx but with significantly shorter crystallization times, confirming the effective nucleating capabilities of PHB and talc.

3.3.2. Evolution of the Relative Crystallinity over Time

To gain deeper insight into the crystallization kinetics of neat P3HBHHx and its nucleated variants, the evolution of relative crystallinity over time was analyzed at various isothermal temperatures, as shown in Figure 12. The half-crystallization time (τ_1/2), defined as the time required to reach 50% relative crystallinity, was extracted from each curve, and the crystallization rate (k) was calculated as the inverse. The corresponding values are listed in Table 6.

Only data obtained under isothermal conditions, where full crystallization was achieved, were considered valid for the kinetic analysis. For neat P3HBHHx (Figure 12a), valid data began at 80 °C, where τ_1/2 was 340 s, corresponding to a crystallization rate of 2.94 × 10⁻³ s⁻¹. As the isothermal temperature increased, the crystallization process became significantly slower, with τ_1/2 exceeding 1400 s at 95 °C.

The incorporation of BN (Figure 12b) significantly reduced τ_1/2 to 99 s at 100 °C (k = 10.05 × 10⁻³ s⁻¹), indicating a high nucleation efficiency and rapid crystal growth. The crystallization curves were sharper and more symmetrical than those of the neat polymer, reflecting more uniform nucleation and a faster crystallization process.

Similarly, PHB (PHH_05PHB, Figure 12c) and talc (PHH_2Talc, Figure 12d) improved the crystallization kinetics. At 90 °C, PHH_05PHB had a τ_1/2 of 165 s (k = 6.06 × 10⁻³ s⁻¹), while PHH_2Talc crystallized even faster, with a τ_1/2 of 133 s (k = 7.53 × 10⁻³ s⁻¹). The shapes of the curves for both samples remained relatively sharp, especially at intermediate temperatures (85–95 °C), indicating effective nucleation. However, the deduced rate constants were slightly lower than those of PHH_05BN, suggesting that while PHB and talc efficiently promote nucleation, they provide fewer active nucleation sites than boron nitride.

3.3.3. Avrami Modeling of Crystallization Kinetics

The Avrami equation was applied to model the isothermal crystallization kinetics of neat P3HBHHx and selected nucleated formulations. This approach provides quantitative insights into the nucleation mechanism and crystal growth dimensionality by evaluating the evolution of relative crystallinity over time. The relative crystallinity, χ(t − t₀), was calculated from the isothermal DSC exotherms using Equation (2), as follows:

$$\chi \left(t-t_0\right)={\int }_{t_0}^t\left(dH∕dt\right)dt /{\int }_{t_0}^{\infty }\left(dH∕dt\right)dt$$

(2)

where dH/dt is the heat flow rate, and t₀ is the induction time, defined as the time at which crystallization begins. This approach normalizes the area under the exothermic peak to the total crystallization enthalpy, thereby enabling the determination of the crystallization degree as a function of time.

When plotted, the resulting curves exhibited a characteristic sigmoidal shape, as shown in Figure 12, which is typical of semi-crystalline polymers and reflects the distinct stages of nucleation and crystal growth [41,42]. These curves were fitted to the Avrami equation as follows:

$$1-\chi \left(t-t_0\right)=\exp \left[{-Z\left(t-t_0\right)}^n\right]$$

(3)

where Z is the temperature-dependent crystallization rate constant, and n is the Avrami exponent, which provides insight into the nucleation mechanism and dimensionality of crystal growth.

To facilitate comparisons across systems, the normalized crystallization rate constant k was calculated as

$$k=Z^{{\displaystyle \frac{1}{n}}}$$

(4)

This normalization ensures that k has consistent units of s⁻¹, allowing for direct comparisons across different nucleating agents and temperatures.

The crystallization half-time (τ_1/2) was calculated as follows:

$${\tau }_{\left(1/2\right)}={\left({\displaystyle \frac{\ln 2}{Z}}\right)}^{1/n}$$

(5)

Both Avrami exponent n and the rate constant Z were determined by plotting $log\left[-ln\left(1-\chi \left(t-t_0\right)\right)\right]$ against $log\left(t-t_0\right)$, which yielded linear fits, as illustrated in Figure 13. In this representation, the x-axis corresponds to the logarithm of the time elapsed since the onset of crystallization, while the y-axis represents a transformed expression of relative crystallinity. The kinetic parameters deduced from the Avrami analysis, including τ_1/2 and k, are listed in

Table 7. Isothermal crystallization kinetic parameters deduced from the Avrami equation for P3HBHHx and the corresponding nucleating agents.

Sample	Tc (°C)	n	Z × 106 (s−n)	k × 103 (s−1) Avrami	τ(1/2) (s) Avrami
P3HBHHx	70	2.10	16.11	5.26	160
	80	2.34	0.890	2.63	326
	85	2.54	0.046	1.29	670
	95	2.75	0.002	0.64	1366
PHH_05BN	100	2.39	11.98	8.76	98
	105	2.20	8.38	4.98	170
	110	2.39	0.87	2.88	298
PHH_05PHB	90	2.29	5.76	5.15	165
	95	2.10	11.23	4.43	189
	100	2.12	5.79	3.43	246
PHH_2Talc	95	2.32	8.42	6.45	132
	100	2.68	0.27	3.54	246
	105	2.88	0.02	2.04	431

.

3.3.4. Analysis of Avrami Plots

The linearity of the Avrami plots for neat P3HBHHx and the nucleated samples across all tested isothermal temperatures confirms that the crystallization behavior follows the Avrami model with a good approximation (Table S3). The slope of each plot corresponds to the Avrami exponent (n), which provides insights into the nucleation mechanism and the dimensionality of crystal growth.

For neat P3HBHHx (Figure 13a), n ranged from 2.10 at 70 °C to 2.75 at 95 °C (Table 7), suggesting three-dimensional spherulitic growth with some degree of spatial confinement. These values are consistent with a combination of instantaneous and homogeneous nucleation, which is typically observed in systems with limited nucleation sites. The increase in n with temperature suggests reduced spatial confinement, as fewer active nuclei are formed at higher temperatures, allowing larger spherulites to grow before impingement.

In the case of PHH_05BN (Figure 13b), n values remained relatively stable between 2.20 and 2.39 (Table 7) across the tested range (100–110 °C), suggesting a combination of instantaneous homogeneous nucleation and additional heterogeneous nucleation induced by the additive. The steeper slopes of these plots compared to those of the neat polymer reflect a higher rate constant (Z), confirming enhanced crystallization kinetics.

PHB (PHH_05PHB, Figure 13c) and talc (PHH_2Talc, Figure 13d) also improved the crystallization behavior of P3HBHHx. The Avrami exponent (n) ranged from 2.10 to 2.29 for PHH_05PHB and 2.32 to 2.88 for PHH_2talc (Table 7), indicating that the overall crystallization mechanism remained consistent with that of neat polymer. In both cases, the crystallization rate constant was significantly higher than that of neat P3HBHHx, reflecting an improved nucleation efficiency.

The crystallization rate constants followed the trend BN > PHB ≈ Talc > neat P3HBHHx. This observation is consistent with previous results from half-time and relative crystallinity analyses, reinforcing the conclusion that BN is the most effective nucleating agent for enhancing the crystallization of P3HBHHx.

Figure 14 illustrates the validation of the Avrami model by comparing the experimental crystallization rates and half-crystallization times obtained from DSC measurements with the theoretical values predicted by the Avrami equation. The strong correlation between the experimental and calculated data confirms that the crystallization behavior of both neat and nucleated P3HBHHx closely follows the Avrami model, with minimal deviations.

3.4. Spherulitic Morphology and Crystal Growth of P3HBHHx Samples

3.4.1. Spherulitic Morphology

The spherulitic morphologies of neat P3HBHHx and the nucleated samples were examined at various isothermal temperatures using polarized optical microscopy (POM). Representative micrographs are shown in Figure 15, highlighting the significant differences in morphology and nucleation density among the samples.

For neat P3HBHHx (Figure 15a), no spherulites were observed at temperatures above 65 °C, even after prolonged observation (e.g., approximately 30 min at 70 °C). The absence of crystalline structures demonstrates the difficulty in forming homogeneous primary nuclei, which require significant supercooling to initiate. At 65 °C, large spherulites with extensive radial growth appeared, although the density of the primary nuclei remained low. These spherulites exhibited coarse textures with irregular Maltese-cross patterns, indicative of non-uniform lamellar packing, likely caused by variations in lamellar orientation and thickness [43].

At 60 °C, the spherulites began to display regular bands, indicating a transition in the crystallization mechanism. This banded structure is associated with the periodic twisting of lamellae during radial growth [44], and the resulting birefringence contrast between edge-on and flat-on lamellae leads to the observed alternating bright and dark rings. Surface stresses have been proposed to drive this twisting behavior [45,46,47]. At 50 °C, the morphology remained banded, but the spherulite size decreased, and the nucleation density increased markedly, resulting in a dense arrangement of small, banded spherulites.

For the nucleated samples (Figure 15b–d), spherulites were observed even at elevated temperatures, such as 100 °C, indicating that heterogeneous crystallization was effectively promoted by the incorporated nucleating agents. At this temperature, the morphology was characterized by coarse textures and irregular Maltese-cross patterns, similar to those of neat P3HBHHx at 65 °C. As the isothermal temperature decreased, the nucleation density increased, leading to the formation of finer and more densely packed spherulites with banded structures. This morphology results from the combined effects of heterogeneous nucleation induced by the additives and homogeneous nucleation inherent to the neat polymer at high degrees of supercooling.

Interestingly, the nucleation density in the nucleated samples was lower than that in the neat polymer, resulting in the formation of larger spherulites. Polarized optical micrographs revealed the coexistence of two spherulitic populations: larger spherulites attributed to rapid heterogeneous nucleation and smaller spherulites associated with slower homogeneous nucleation. In particular, the PHH_05BN samples continued to exhibit irregular Maltese-cross patterns even at lower temperatures, suggesting a distinct crystallization mechanism that may be influenced by specific interactions between boron nitride and the P3HBHHx matrix.

3.4.2. Nucleation Density

Figure 16 presents the nucleation densities of neat P3HBHHx and the nucleated samples at low (50 °C) and high (100 °C) isothermal crystallization temperatures. At 100 °C, spherulites were observed only in the nucleated samples, each exhibiting a nucleation density of approximately 20 nuclei/mm². This density was similar for all these samples since similar concentrations of the nucleating agent were incorporated (0.5 or 2 wt%), which remained active even at elevated temperatures.

At 50 °C, the nucleation density increased significantly in all samples due to the combined effects of heterogeneous and homogeneous nucleation. For example, PHH_05BN exhibited a sharp increase in nucleation density from approximately 20 to 850 nuclei/mm². This increase is attributed to the earlier activation of heterogeneous nucleation sites introduced by boron nitride, followed by homogeneous nucleation as supercooling progressed. In contrast, PHH_2Talc exhibited a lower nucleation density under the same conditions, suggesting that the homogeneous nucleation process was slightly hindered by the presence of the added inorganic particles.

Interestingly, at 50 °C, neat P3HBHHx exhibited a high nucleation density of approximately 1440 nuclei/mm². This high value is attributed entirely to homogeneous nucleation, which becomes increasingly favorable at low temperatures due to higher degrees of supercooling. However, in the nucleated samples, the early formation of heterogeneously nucleated spherulites likely reduced the available material/space for homogeneous nucleation, resulting in overall lower densities.

Above 65 °C, no nuclei were detected in neat P3HBHHx, confirming that homogeneous nucleation becomes ineffective due to the increased energy barrier for primary nucleation at higher temperatures. In contrast, the nucleated samples still displayed measurable nucleation densities at 100 °C, demonstrating that the additives significantly lowered the energy barrier required for nucleation. This ability to promote nucleation at elevated temperatures confirms that the nucleating agents provide energetically favorable sites for the formation of stable nuclei.

3.4.3. Crystal Growth Rates Determined from POM

The crystal growth rates (G) of neat P3HBHHx and the nucleated samples at different crystallization temperatures were determined using polarized optical microscopy (POM) by measuring the increase in the spherulite radius over time. The slopes obtained from the linear correlations of the radius versus crystallization time (Figure 17) were used to calculate the growth rates, which are summarized in

Table 8. Summarized data of the growth rate of P3HBHHx and nucleated samples at different temperatures, determined from the slope of the linear correlation of the crystal radium increase with time.

Sample	P3HBHHx	G (µm/s)	Induction Time (s)
P3HBHHx	50	0.23	1
	60	0.57	45
	65	0.49	50
PHH_05BN	80	0.71	1
	90	0.65	71
	100	0.48	150
PHH_05PHB	80	0.76	1
	90	0.73	22
	100	0.50	80
PHH_2Talc	80	0.87	1
	90	0.79	75
	100	0.43	100

.

For neat P3HBHHx, the maximum growth rate was observed at 60 °C, with a G value of 0.57 µm/s, suggesting that this temperature offers an optimal balance between the polymer chain mobility and the efficiency of secondary nucleation. In contrast, the nucleated samples exhibited maximum growth rates at higher temperatures, around 80 °C. This shift indicates that the presence of nucleating agents enhances the nucleation efficiency, allowing effective crystal growth to occur at elevated temperatures compared to the neat polymer.

Among the tested formulations, PHH_2Talc exhibited the highest crystal growth rate, reaching 0.87 µm/s at 80 °C. Interestingly, this result differs from the trends observed in the DSC-based crystallization data, where PHH_05BN exhibited the fastest overall crystallization rate. This discrepancy demonstrates the different aspects of crystallization captured by the POM and DSC techniques. In DSC, the measured crystallization rate reflects a combination of primary nucleation (formation of stable nuclei) and crystal growth from existing nuclei. In contrast, POM focuses solely on crystal growth at each temperature, which is directly related to secondary nucleation mechanisms rather than initial nucleation events.

The superior growth rate of PHH_2Talc can be attributed to several factors, including the following: Although talc is a less potent nucleator than BN or PHB, its moderate nucleation density at 2 wt% (Figure 16) allows more space for each spherulite to grow, reducing early impingement and enabling faster radial expansion. Furthermore, the well-dispersed lamellar structure of talc may facilitate lateral chain alignment, thereby supporting faster crystal growth once nucleation has occurred. Therefore, although BN excels at initiating nucleation, talc is more effective at promoting rapid crystal expansion under isothermal conditions.

These findings suggest that while boron nitride excels at initiating crystallization (primary nucleation), talc is more effective at promoting crystal growth (secondary nucleation) once the nuclei are present. As a result, talc exhibited a higher growth rate in POM, but boron nitride remained superior in accelerating the overall crystallization process, as captured by DSC.

3.4.4. Determination of Equilibrium Melting Temperature ($T_{\mathrm{m}}^{°}$)

Figure 18 presents the equilibrium melting temperatures ($T_{\mathrm{m}}^{°}$) of neat P3HBHHx and its nucleated samples, determined from the first melting peak and using the Hoffman–Weeks method. This approach involves plotting the melting temperature (T_m) as a function of the corresponding crystallization temperature (T_c) and extrapolating the linear fit to the point where T_m equals T_c. The intercept represents the equilibrium melting temperature ($T_{\mathrm{m}}^{°}$), which defines the temperature at which the crystalline and melt phases can coexist.

For neat P3HBHHx, the extrapolation process may introduce intrinsic error due to the significant difference between the highest accessible melting temperatures and the actual $T_{\mathrm{m}}^{°}$. This discrepancy can lead to an underestimation of $T_{\mathrm{m}}^{°}$ for neat P3HBHHx (143 °C), as the limited range of higher temperature data may bias the linear fit.

In contrast, the presence of nucleating agents enables crystallization at higher temperatures, making it possible to obtain data at higher temperatures (i.e., closer to the real equilibrium temperature) and, therefore, decreasing the inherent error associated with the extrapolation.

3.4.5. Kinetic Analysis Based on the Lauritzen−Hoffman Model

The crystal growth rate (G) of the neat P3HBHHx and nucleated samples were simulated using the Lauritzen−Hoffman (LH) theory, which describes polymer crystallization kinetics by considering secondary nucleation and molecular transport as rate-determining steps [48,49,50]. This model provides insights into the activation energy and nucleation barrier that control the crystallization process.

The Lauritzen−Hoffman equation expresses the growth rate G as follows:

$$G=G_0\times \exp \left[-{\displaystyle \frac{U^{*}}{R\left(T_{\mathrm{c}}-T_{\mathrm{\infty }}\right)}}\right]\times \exp \left[-{\displaystyle \frac{K_{\mathrm{g}}}{T_{\mathrm{c}}\left(\Delta T\right)f}}\right]$$

(6)

where G₀ is the pre-exponential factor related to molecular mobility; U* is the activation energy for segmental motion above the glass transition temperature; R is the universal gas constant; T_c is the crystallization temperature; $T_{\infty }$ is the reference temperature, typically approximated as T_g − 30 K; K_g is the nucleation constant, which accounts for the energy barrier of secondary nucleation; $\Delta T=T_{\mathrm{m}}^{°}-T_{\mathrm{c}}$ is the supercooling (the difference between the equilibrium melting temperature $T_{\mathrm{m}}^{°}$ and the crystallization temperature T_c); f is a correction factor for temperature dependence, given by:

$$f={\displaystyle \frac{2T_{\mathrm{c}}}{T_{\mathrm{m}}^{°}+T_{\mathrm{c}}}}$$

(7)

To determine K_g and validate the applicability of the LH model, the experimental spherulitic growth rates obtained from POM observations were analyzed. The data were plotted according to the LH equation as follows:

$$\ln G+{\displaystyle \frac{U^{*}}{R\left(T_{\mathrm{c}}-T_{\mathrm{\infty }}\right)}} \mathrm{versus} {\displaystyle \frac{1}{T_{\mathrm{c}}\left(\Delta T\right)f}}$$

(8)

In such calculations, it is common to adopt the “universal” values reported by Suzuki and Kovacs [51], namely U* = 1500 cal/mol and $T_{\infty }$ = T_g − 30 K. At low supercooling conditions (i.e., when $\Delta T$ is small), crystallization kinetics are primarily governed by the nucleation term. As a result, the overall growth rate becomes relatively insensitive to variations in the U* and $T_{\infty }$, allowing for reliable fitting of the Lauritzen−Hoffman (LH) model using these standard values. The linearized form of the LH equation follows the relation y = mx + b, where y = $\ln G+{\displaystyle \frac{U^{*}}{R\left(T_{\mathrm{c}}-T_{\infty }\right)}}$ and x = ${\displaystyle \frac{1}{T_{\mathrm{c}}\left(\Delta T\right)f}}$. The slope m corresponds to the negative secondary nucleation constant −K_g, and b represents the intercept associated with the pre-exponential growth terms. The results are presented in

Table 9. Lauritzen−Hoffman parameters obtained from POM analyses for P3HBHHx and nucleated samples.

Sample	Linear Plot	R2	Kg 10−5 (K2)
P3HBHHx	y = −4.097x + 22.295	0.9946	4.10
PHH_05BN	y = −1.707x + 13.463	0.9924	1.71
PHH_05PHB	y = −1.734x + 13.644	0.9957	1.73
PHH_2Talc	y = −2.107x + 15.293	0.9996	2.11

.

As shown in Figure 19, the LH plots for all samples exhibited strong linearity, with correlation coefficients (R²) exceeding 0.99. This high degree of linear correlation supports the applicability of the LH model in describing the crystallization kinetics of both neat and nucleated P3HBHHx.

Two distinct slopes can be observed in Figure 19: one corresponding to neat P3HBHHx and the other to the nucleated samples. This distinction suggests that crystallization proceeded in different kinetic regimes: regime III for the neat polymer and regime II for the nucleated samples due to the different temperature ranges used in each case. In regime III, which is typical of lower crystallization temperatures, crystal growth is governed by isolated nucleation events that result in higher K_g values. Conversely, in regime II, which occurs at higher crystallization temperatures, crystal growth is characterized by prolific multiple nucleations with a niche separation similar to the stem width, leading to lower K_g values.

For neat P3HBHHx, the highest K_g value was obtained (4.10 × 10⁵ K²), consistent with its crystallization occurring at lower temperatures within regime III. In contrast, the nucleated samples crystallized at higher temperatures, consistent with regime II, and exhibited lower K_g values, averaging around 2.0 × 10⁵ K². These findings are in agreement with the Lauritzen−Hoffman theory, which predicts a K_g ratio close to 2 between regimes III and II.

Figure 20 illustrates the simulated dependence of the crystal growth rate (G) on the crystallization temperature for neat P3HBHHx and its nucleated samples based on Equation (6) and the corresponding parameters derived from the LH model. The simulated curves exhibit a bell-shaped profile typical of polymer crystallization, reflecting the competing effects of chain mobility and nucleation barriers. The maximum growth rate for neat P3HBHHx was predicted to be near 65 °C, whereas, for the nucleated samples, the optimal crystallization temperature shifted to approximately 80–85 °C. This shift confirms that nucleation agents enhance crystallization kinetics at higher temperatures, taking advantage of the decrease in the energy barrier for secondary nucleation. Note that there is also a slight shift between the predicted and experimental temperatures for the maximum growth rate of the neat polymer. This feature is a consequence of employing standard U* and T_∞ values without performing additional refinement.

3.5. Fast Cooling Behavior of P3HBHHx and Nucleated Samples

Figure 21 shows the DSC thermograms of the P3HBHHx samples with different nucleating agents during fast cooling (100 °C/min) and subsequent reheating (10 °C/min). This experiment was designed to evaluate the influence of rapid cooling on the crystallization behavior of P3HBHHx by comparing the crystallization temperatures and enthalpies of the samples.

During the fast-cooling step, only the sample containing boron nitride (PHH_05BN) exhibited a crystallization exotherm, with a peak at 57.4 °C and an associated enthalpy of 16.8 J/g. This result confirms that BN substantially enhances nucleation efficiency, even at high cooling rates. However, the relatively low enthalpy suggests that while nucleation is initiated, crystal growth is limited by the insufficient time available during rapid cooling, resulting in partial crystallization.

In contrast, neat P3HBHHx, PHH_05PHB, and PHH_2Talc did not exhibit any exothermic peaks during cooling, indicating that crystallization was not triggered under these conditions. This suggests that while PHB and talc are effective at promoting crystallization under moderate cooling rates, they are not sufficiently active to induce nucleation during fast cooling at 100 °C/min.

Upon reheating (Figure 21b), all the samples exhibited a cold crystallization peak, confirming that most of the crystallization occurred during this second step. The cold crystallization temperatures (T_cc) ranged from 42.6 °C to 44.4 °C, with PHH_05BN showing the lowest T_cc. The cold crystallization enthalpy (ΔH_cc) ranged from 22.7 to 37.6 J/g, reflecting the extent of crystallization that occurred during reheating. Surprisingly, the total cold crystallization enthalpy was approximately 20 J/g lower than the melting enthalpy (approximately 53 J/g for all samples; see the dashed zone in Figure 21b). This discrepancy suggests that full crystallization was only achieved during the high-temperature ramp (see the dashed line in Figure 21), likely due to kinetic limitations, restricted chain mobility, or the inability of certain amorphous domains to reorganize into crystalline structures.

Despite the differences in crystallization behavior, all samples, including the neat polymer, exhibited a melting peak around 150 °C with similar melting enthalpies (~53 J/g). This consistent melting temperature confirms that the addition of nucleating agents does not alter the fundamental crystalline structure of P3HBHHx, reinforcing that their primary role is to influence the crystallization kinetics.

4. Conclusions

This study systematically evaluated the effects of various nucleating agents on the crystallization behavior, thermal stability, and mechanical properties of P3HBHHx. BN and PHB were identified as the most effective nucleating agents, significantly enhancing the crystallization kinetics by increasing the crystallization temperature, reducing the crystallization half-time, and eliminating cold crystallization upon reheating. Talc showed moderate nucleation efficiency at 0.5 wt%, but its effectiveness increased markedly at 2 wt%, becoming comparable to that of BN and PHB. In contrast, UFC and LAK had a limited influence on nucleation. Avrami and Lauritzen−Hoffman analysis confirmed that BN, PHB, and talc accelerated crystallization and shifted the crystal growth regime from III (in neat P3HBHHx) to II. Under fast cooling, only BN enabled crystallization during quenching, highlighting its unique performance under industrial conditions. TGA results indicated that most additives had minimal impact on thermal stability, with talc improving degradation resistance at higher loadings and BN and UFC slightly reducing the onset degradation temperature. Mechanical testing showed that the nucleating agents preserved the tensile strength and elongation at break, with BN, PHB, and talc slightly reducing the modulus due to the formation of larger spherulites. Overall, BN emerged as the most effective additive for promoting nucleation at elevated temperatures, while talc exhibited a strong concentration-dependent effect on crystal growth. These findings provide valuable insights for improving the processability of P3HBHHx in industrial settings. Future work could explore the combined use of nucleating agents or their impact on biodegradation behavior to further enhance the performance of P3HBHHx-based materials.