PHBV copolymer blends

Accepted Manuscript The morphology of crystallisation of PHBV/PHBV copolymer blends Alexandra Langford, Clement Matthew Chan, Steven Pratt, Christopher J. Garvey, Bronwyn Laycock PII: DOI: Reference:

S0014-3057(18)31994-3 https://doi.org/10.1016/j.eurpolymj.2018.12.022 EPJ 8754

To appear in:

European Polymer Journal

Received Date: Revised Date: Accepted Date:

12 October 2018 11 December 2018 14 December 2018

Please cite this article as: Langford, A., Matthew Chan, C., Pratt, S., Garvey, C.J., Laycock, B., The morphology of crystallisation of PHBV/PHBV copolymer blends, European Polymer Journal (2018), doi: https://doi.org/ 10.1016/j.eurpolymj.2018.12.022

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The morphology of crystallisation of PHBV/PHBV copolymer blends Alexandra Langford a, Clement Matthew Chana, Steven Pratt a, Christopher J. Garvey b, Bronwyn Laycock a,1 a

School of Chemical Engineering, The University of Queensland, St Lucia, QLD 4072,

Australia. b

ANSTO, Locked Bag 2001, Kirrawee DC NSW 2232, Australia.

Abstract The crystallisation of a range of as-produced poly(3-hydroxybutyrate-co-3-hydroxyvalerate) copolymer blends from a mixed culture polyhydroxyalkanoate production process, with low (15%), high (82%) and intermediate (62% and 50%) average 3-hydroxyvalerate (3HV) contents, was studied at different temperatures using polarised optical microscopy, differential scanning calorimetry and X-ray crystallography. The low-3HV content material had narrow compositional distribution and crystallised in the typical highly regular banded spherulite morphology, while the other materials displayed varying degrees of interpenetrating crystallisation of separate crystal phases comprising either the 3HV or 3hydroxybutyrate (3HB) crystal lattice structure. Because of the differing and competing crystallisation kinetics of these phases coupled with diffusion effects, there was a very strong influence of crystallisation temperature on the resulting morphology. Thus manipulation of the final material properties of such copolymeric materials is dependent on understanding these effects and controlling their processing and crystallisation temperatures and conditions. Keywords

1

Corresponding author telephone: P +61 (0)7 3346 3188; Email: [email protected]

Polyhydroxyalkanoates, mixed culture, solvent fractionation, crystallisation, microstructure

1. Introduction Polyhydroxyalkanoates (PHAs) are a family of microbially synthesised and biocompatible polyesters [1]. They can be processed using conventional equipment and are fully biodegradable under ambient conditions in soil and aquatic environments [2]. As a result they have attracted significant commercial attention [3, 4]. Poly(3-hydroxybutyrate) (PHB) is the most common homopolymer of the PHAs. But it typically crystallises into large, dense, radially orientated lamellar spherulites, which make the material exceptionally crystalline and too brittle for many commercial applications without the use of additives and additional processing [1]. This can be addressed through copolymerization or blending. Copolymers of PHB have been developed by incorporating other monomers such as 3-hydroxyvalyrate (3HV) [5], 3-hydroxyhexanoate (3HHx) [6, 7], 3hydroxypropionate (3HP) [6], and 4-hydroxybutyrate (4HB) [6, 8, 9], though only 3HV can be co-crystallised within the 3-hydroxybutyrate (3HB) crystal lattice [1, 9-12]. In such 3HB lattice crystallisation, the bulkier 3HV units lead to a gradual expansion of the a-axis in the unit cell [13], reducing packing density within the unit cell due to internal stresses, and in turn reducing packing density of the lamellae in the crystals. This inclusion of foreign monomer units into the unit cell is responsible for the improved mechanical properties of these copolymeric materials, as the perfect crystalline structure of PHB crystals is disturbed, leading to larger amorphous regions with better flexibility. By contrast, when 3HB units are incorporated into the 3HV lattice, a decrease in the length of the b- and c-axes occurs, leading to a more compact unit cell [1, 14]. In some crystals, this leads to a characteristic ‘eye-like’ region in the crystal (Figure 1), thought to be due to shift of direction of lamellar twisting [14]. As a result, PHBV copolymers exhibit a minimum crystallinity of 45% at 47-52% 3HV

content (for carefully fractionated materials of narrow compositional distribution) [15, 16], a pseudoeutectic which also represents the transition from the 3HB to the 3HV lattice crystals. Pure PHB and PHV homopolymers have higher crystallinities of 60-68% [1, 17] and ~64%, respectively [15], although there can be very significant variability depending on production method, processing history, degree of secondary crystallisation, etc. [18].

Figure 1 (ref: [14]) (single-column fitting)

PHB and low 3HV content PHBVs are miscible, provided that the 3HV content of the PHBV is less than ~15 mol% [19]. However, blends of the PHBV copolymers are immiscible if the compositional distribution difference is larger than 15 mol% [19]. As a result, PHBV blends containing both higher- and lower-HV content copolymers represent a system of polymers which may exhibit “interpenetrating crystallisation” where the spherulites of the component with slower crystal growth rate intrude in the spherulites of the one with faster growth [20]. While Jungnickel et al. [20, 21] and Yoshi and Inoue et al. [19, 22] have developed useful frameworks for studying the crystallisation of blends, there have to our knowledge been no reports on the crystallisation of blends of PHBV with very wide compositional distributions (i.e. greater than 50% differences in 3HV contents). The

crystallisation

behaviour

of

the

fractionated

poly(3-hydroxybutyrate-co-3-

hydroxyvalerate) (PHBV) copolymeric polyhydroxyalkanoates with narrow compositional distributions are well studied. In carefully fractionated materials, these copolymers are understood to undergo isodimorphic crystallisation at a range of temperatures, producing crystals of regular ring-banded spherulitic morphology, with increasing rejection of 3HV from the 3HB crystal and increases in band spacing at higher temperatures (lower degrees of undercooling) when the 3HV content is less than 50%, and a transition to the 3HV crystal

lattice unit above 50% 3HV content [15, 16, 23, 24]. It is also known that the rate of crystallisation from the melt decreases with an increase in 3HV content up to the pseudoeutectic, then increases again [1, 25]. In reality, however, much of the PHBV material as-produced comprises blends of copolymers with different degrees of compositional distribution, particularly when using a mixed culture production process [26], but even when using pure culture production [25, 26]. The direct use of the as-produced PHBV blends without further fractionation is attractive as it offers cost-advantages. This work builds on the studies of Inoue and co-workers [19, 22] and Organ and co-workers [27-29] who studied PHB blends with PHBV copolymers with 3HV contents of up to 27%, and Jungnickel and co-workers who extended the understanding of the behaviour of crystalline-crystalline blends [20, 21]. It further develops the previous studies of PHBV blends by using as-produced materials with 3HV contents up to 82% and a broad compositional distribution. The fractionated components of one of these materials, with narrow compositional distributions, are also included for comparison. The crystallisation properties of these materials were studied at a range of temperatures using polarized optical microscopy (POM), with the material properties being further characterized using DSC and WAXS to understand the behaviour of these PHBV blends in the melt and after crystallisation. SAXS is also used to probe the lamellar structure and the so-called linear crystallinity of the crystallised polymer [30].

2. Experimental 2.1. Materials HPLC grade chloroform and laboratory grade hexane were obtained from Sigma-Aldrich and used as received (99.9% and >99 % purity respectively). Fermented whey permeate waste

that was used as a carbon source was obtained from a cheese production site near Lund, Sweden. Acetic and propionic acids, which were used as carbon sources were obtained from Merck and used as-is. 2.2. Samples Samples used in this study were produced in an activated sludge-based Aerobic Dynamic Feeding (ADF) pilot plant operated by AnoxKaldnes, Sweden, as described in full in previous reports [31, 32]. In summary, samples were produced using 100 L of enriched biomass in a 150 L accumulation reactor, in accordance with International patent WO2001/070544 A3 [33]. The accumulation process was controlled according to the methods described in US Patent US20130029388A1, the equipment details provided therein [34]. Fermented whey permeate waste at 50 g/L COD concentration was used as a carbon substrate in the enrichment reactor while acetate and propionate, applied as acids, were used as carbon sources during PHA accumulation. The proportion of acetate and propionate varied, as did the feeding strategy, with acetate and propionate being combined together to produce random copolymers or fed in an alternating fashion to produce blocky copolymers. The properties of the samples used are shown in Table 1.

Table 1 (ref: [35])

2.3. Fractionation Samples were fractionated on the basis of 3HV content by solvent-nonsolvent fractionation using chloroform and hexane at ambient temperature using the procedure described in full in Laycock et al. [35]. 2.4. Differential Scanning Calorimetry (DSC)

Thermal properties of the polymers were investigated using DSC (TA DSC-Q2000). The apparatus was calibrated using the onset of melting of indium (429.6 K) and the heat of fusion of indium (28.45 J g-1). All runs were performed on 2.0 – 4.0 mg samples in a nitrogen atmosphere. No thermal pretreatment was applied in this case. The data obtained were used to calculate the glass transition temperature (Tg), crystallisation temperature (Tc), cold crystallisation temperature (Tcc), melting point (Tm), and fusion enthalpies (ΔHf). Samples were heated at 10°C min−1 to 185°C, kept isothermal for 0.1 min then cooled at 10°C min−1 to −70°C and kept isothermal for 5 min. After this, the sample was once again heated at 10°C min−1 to 185°C, followed by rapid cooling at 100°C min−1 to −70°C before being kept isothermal for 3 min. In a final heating ramp, the sample was heated at 10°C min−1 to 200°C. The Tm and enthalpy of fusion, ∆Hm, were determined from the first heating ramp. The Tc and ∆Hc were determined from the cooling cycle following the first heating ramp. The Tg and Tcc were determined from the final heating cycle. 2.5. Polarised Optical Microscopy (POM) The spherulitic morphology and crystal growth of the as-produced PHBV copolymers were investigated using an OXJP304 Polarising microscope equipped with a 3.2 Mpixel ODCM310 Digital camera with an OZ9-TCS temperature controlled microscope stage and separate hot plate. Samples were dissolved in chloroform to a concentration of 10 wt%, then filtered through a 0.45 μm PTFE filter on to a clean glass cover slip. Samples were allowed to dry at room temperature for a minimum of 10 minutes then sealed after film formation under another glass coverslip before testing. At the start of each test, slides were melted on a hot plate at 190°C for 60 seconds, then transferred directly to a hot melt stage which was set at a pre-set temperature where they were crystallised isothermally for times ranging from 1 hour to 10 days, depending on the crystallisation kinetics. Test results were collected at intervals of 2 –

50,000 seconds, again depending on the crystallisation kinetics of the samples. The spherulitic growth rate (G) was calculated from the change of radius (R) with time (t) and nucleation rates were calculated on an area basis. The latter had inherently high variability due to heterogeneous nucleation at the film boundary, variability in film thickness, the presence of environmental impurities in the melt, and nucleation during cooling from the melt temperature to the isothermal crystallisation temperature. 2.6. Wide Angle and Small Angle X-ray Scattering X-ray scattering measurements were undertaken at the Australian Synchrotron’s SAX/WAXS beamline (Clayton, Victoria, Australia) [36]. Wide angle X-ray scattering (WAXS) was recorded on a Pilatus 200K detector (Dectris, Baden, Switzerland) positioned to cover a solid angle and effective range of scattering vectors, 0.83 < q < 3.76 Å-1, where q = 4.sin(/, 2 is scattering angle and  is the wavelength of incident X-rays). SAXS measurements were made on a Pilatus 1M (Dectris, Baden, Switzerland) 1683 mm from the sample to cover 0.15 < q, 0.71 Å-1. Both measurements were made simultaneously using X-rays of wavelength 0.729 Å (17 keV). The two dimensional scattering patterns were converted to the radially averaged background subtracted scattering curves with the NIKA [37] macros for IgorPro (Wavemetrics, Oswego, USA) using sample and background transmission, X-ray wavelength, measurement geometry; the isotropic intensity distribution of counts on the detector; and signal from the sample holder. From the one-dimensional scattering curves (I(q)) of the major 3HB and 3HV units the main crystal peaks were assigned at 0.96, 1.55 and 1.20 Å-1 for 3HB lattice crystals and 0.92 and 1.27 Å-1 for 3HV lattice crystals [38, 39]. Each peak was fitted a Gaussian function using the software Peakfit v4.12 using the literature values of the peaks for the initial values in the fitting. There is a broad underlying intensity contribution from the amorphous halo, scattering from the disordered material [40], which could not be accurately modelled in terms of its

amplitude but this had little effect on fitting relatively sharp crystalline peaks. The peak area at each of these locations was calculated from the peak deconvolution and the ratio of 3HB peak area to 3HV peak area was used as an estimate of the proportion of material crystallising in the 3HB and 3HV lattice in a method previously described by Garvey et al. [41]. SAXS data was analysed using a cosine transformation [30, 42] which yields the linear correlation function (LCF) of a disordered lamellar structure (Figure 2). Prior to transformation the background corrected SAXS data was first smoothed using a spline function and then extrapolated to zero and  angle for transformation into the LCF and subsequent extraction of the important structural parameters the long period (LP), and linear crystallinity (LC) using the Windows program SAXDAT [43]. Structural parameters (Figure 2) were then extracted from the LCF curves by SAXSDAT.

Figure 2 (single-column fitting)

2.7. Gas Chromatography (GC) PHA monomeric composition (3HB and 3HV) was determined using the gas chromatography method described in Arcos-Hernandez et al. using a Perkin-Elmer gas chromatograph (GC) [44]. Calibration was based on reference standards of a biologically sourced PHBV copolymer (30 mol% 3HV) (Sigma Chemicals, USA). The calibration standards were prepared using the same method. 2.8. Gel Permeation Chromatography (GPC) GPC analyses were performed using a Waters 1515 HPLC solvent delivery system combined with a Wisp 717 auto-injector, a column set consisting of a Waters Styragel guard column (20 µm, 4.6 x 30 mm) and a set of linear columns in series (Waters Styragel HR5 (5.5 µm,

7.8 x 300 mm), Waters Styragel HR1 (5 µm, 7.8 x 300 mm) and Waters Styragel HR4 (4.5 µm, 4.6 x 300 mm)) and kept at 35°C with a refractometer/UV-Vis detector set at 37°C. HPLC grade chloroform was utilized as the continuous phase at a flow rate of 1 mL/min. The polymer molecular weight was calibrated with reference to polystyrene standards.

3. Results and Discussion 3.1.1. Thermal properties of the as-produced polymer Figure 3a, b and c shows the DSC thermograms of the first heating scan (melt transition), first cooling scan (crystallisation transition), and final heating scan after quench cooling (glass and melt transitions), respectively, of the as-produced PHBV materials. The asdetermined thermal properties are presented in Table 2. As can be seen from Figure 3a, sample A1 (15% 3HV random copolymer) only exhibits endotherms in the high temperature range (at 152 and 166°C), consistent with high-3HB copolymer co-crystallisation into the PHB crystal lattice. Sample A2 (82% 3HV random copolymer) has both multiple melt endotherms in the low temperature region (<110°C), plus a very minor high-melting peak at 173°C, indicating crystallisation associated with both high 3HV copolymer content crystallisation in the PHV crystal lattice and high 3HB copolymer (high temperature) crystallisation in the PHB crystal lattice, although the proportion associated with the latter component is small. Samples A3 and A4 (64% 3HV “random” copolymer and 50% 3HV “blocky” copolymer, respectively) both show four melting points, associated with both PHV and PHB lattice crystallisations. When comparing the cooling thermograms of all samples in Figure 3b, one can see that only sample A1 showed a relatively sharp crystallisation peak at 86oC with the exotherm of crystallisation being equivalent to the original endotherm of melting, which indicates relatively rapid and complete crystallisation. By contrast, Sample A4 has a broad

crystallisation peak at a lower temperature (61oC), or, in other words, crystallises at a higher degree of undercooling (lower temperature) and is slower to crystallise compared with A1. Samples A2 and A3 both fail to crystallise over the time range of this test, indicating slower crystallisation dynamics. The glass transitions of all samples were measured during the final heating scan following rapid quench cooling (Figure 3c). Sample A1 had a single glass transition at 1.5°C, a value that is consistent with literature expectations for this high 3HB content PHBV [1], with either a single copolymer being present (rather than a blend) or immiscibility in the melt if there is any other component in the mix, given that no other glass transition was observed. Sample A2 also has a single Tg at -13.2°C, which is again consistent with literature expectations for this 3HV content and indicates miscibility of the low melting components, with the high 3HB component being such a small fraction that its glass transition is not evident. Samples A3 and A4 (64% 3HV “random” copolymer and 50% 3HV “blocky” copolymer, respectively) both showed two glass transition temperatures, indicating immiscibility of the components of the blends. In both cases, the Tg values obtained were consistent with low- and high-3HV PHBV copolymers, although somewhat depressed relative to expectation [1].

Figure 3 (1.5-column fitting)

Table 2 (ref: [35])

3.1.2. Crystal morphology The crystallisation of polymers is possible at temperatures between the glass transition temperature and the melt temperature, and has a maximum value within this range. PHB has a much higher melt temperature (at 179°C) than PHV (at 107°C for a solvent cast film) [45],

while PHBV copolymers with intermediate 3HV content (> 35%) generally have melt temperatures below 110°C [1]. It has been reported that the spherulitic growth rate at crystallisation temperatures ranging from 60 oC to 90oC decreases with increasing 3HV content up to a 3HV content of 27 mol% (Figure 4) [22]. In other words, a lower temperature is needed for higher 3HV content materials to achieve a similar growth rate to that of PHB. For example, PHB has a maximum crystal growth rate of 3-4 μm/s at approximately 90°C [46]. High 3HV content PHBV (>83% 3HV) by contrast reaches growth rates of the same order of magnitude at an optimum crystallisation temperature of 60°C [24]. However, PHBV copolymers at compositions approaching the pseudo-eutectic are up to four orders of magnitude slower to crystallise than the pure polymers [47]. As a result, crystallisation of the fastest crystallising component drives the crystallisation process. The nucleation rate is also important in determining crystalline morphology. Mid-range 3HV content PHBV materials (of between 25 – 75% 3HV content) have lower nucleation rates than PHB or PHV due to the reduced packing affinity which restricts crystallisation [22, 29, 48].

Figure 4 (ref: [22]) (single-column fitting)

Understanding this context, the performance of the as-produced PHA materials was examined. Sample A1 (15% 3HV Random copolymer) Sample A1 (15% 3HV Random copolymer) exhibited growth of a single, highly regular crystal type at all temperatures. As can be seen in Figure 5, dense, uniform 3HB spherulites were formed at all temperatures, with 3HV repeat units presumably incorporated into the unit cells and any residual non-blended copolymer presumably being included in the intraspherulitic domains between or within the lamella stacks. If so, these were not evident in

the DSC or other analytical techniques. No 3HV-type crystal growth was observed. This implies that the compositional distribution of this PHBV copolymer is narrow. A reduction in the melt temperatures (152oC, 166oC) (Table 2) compared with pure PHB, which melts at 172oC, is consistent with the expected isodimorphism of this copolymer at this 3HV content, and aligns with observations from POM. High 3HB component polymer crystal growth rates are likely to be much higher than the growth rates of any high 3HV copolymer crystals present due to the much larger proportion of high 3HB copolymers in the melt.

Figure 5 (1.5-column fitting)

The morphology shown in this case is that of non-banded spherulites at lower temperatures, with some irregular banding at 78oC. Crystal uniformity was found to be lower at higher temperatures, likely due to an increase in the rate of diffusion. The growth of spherulites is an intricate process and the final morphology depends on many factors, with the possibility of banded or non-banded spherulites being formed. The banded spherulites are more commonly formed in thinner films and at intermediate crystallisation temperatures [49]. Overall, the polymer crystallisation process is decided by the competition between the crystallisation rate (νc) and the diffusion rate (νd) of melt molecules (Figure 6). If νd > νc, then non-banded spherulites are formed.

Figure 6 (ref: [49]) (single-column fitting)

Sample A2 (82% 3HV random copolymer) Sample A2 (82% 3HV random copolymer) also exhibited growth of a single crystal type (presumed to comprise 3HV unit cells) at temperatures of 36°C and under (Figure 8). As

discussed above, any 3HB units in the PHBV copolymer chains should be included (cocrystallised) in the 3HV unit cell. This is evidenced by the presence of lamellar twisting (Figure 8) which does not occur in 3HB lattice crystals. The temperature was too low for significant growth of the high-3HB copolymer component observed in the DSC. However, at the slightly higher temperature of 43°C, two crystal types were observed: the 3HB lattice crystals which incorporated only a small fraction of polymers into its structure, as well as the 3HV lattice crystal, which initially grew within the faster crystallising crystals then overtook them (Figure 8). The boundaries between the 3HV spherulites can be clearly observed following full crystallization. This suggests that crystallization of separate crystal phases occurs at around 43°C, a temperature at which both low and high 3HV content crystals grow at an observable rate under the conditions used herein. In the case of crystalline/crystalline polymer blends, there are a range of interactions that can take place, balancing phase separation and crystallisation, these being (i) simultaneous phase separation

and

crystallisation,

(ii)

phase-separation

induced

crystallisation,

(iii)

crystallisation-induced phase separation and (iv) crystallisation in the separated phases [50]. The other consideration in this is when the rate of crystal growth is faster than the rate of phase separation, there are again differing morphologies that can occur (Type I to IV, Figure 7), depending on whether the differing copolymer chains can exhibit complete cocrystallisation (I), partial cocrystallisation with enrichment of one copolymer as amorphous material in the interlamellar regions (II), partial cocrystallisation with enrichment of one copolymer as crystalline material in the interlamellar regions (III), and complete rejection of one copolymer from the primary lamellae and crystallisation as separate microcrystals in the interlamellar regions (IV).

Figure 7 (ref: [19]) (single-column fitting)

For Sample A2, there is likely a combination of these factors at play. It appears that the 3HB crystal lattice spherulites had favourable nucleation kinetics but were present at low concentrations in the melt. Hence, the overall growth rate of the high-3HB PHBV copolymer was low and these crystals were overtaken by the faster crystallising space filling 3HV crystal lattice spherulites, which crystallised through the high 3HB crystal and filled the space, inhibiting the high 3HB component spherulite from growing further except through the much slower interpenetrating crystallisation. In this case the high 3HB and the high 3HV copolymeric materials phase separated into different crystals due to the wide compositional difference between them.

Figure 8 (1.5-column fitting)

Sample A3 (62% 3HV Random Copolymer) Sample A3 underwent clear morphology changes when the isothermal crystallisation temperature rose above 43oC. Figure 9 shows the crystal structure captured by POM at temperatures ranging from 24oC to 78oC. At temperatures below 43oC, a combination of dendrite and compact globular crystal morphologies were observed. Most crystals began growing in a compact globular morphology. These spherulites grew slowly, rejecting high 3HB crystals into the melt at the crystal growth front, leading to the initial formation of dense, perfect spherulites. This process caused the melt composition to change over time, lowering the proportion of high 3HV material in the melt (and thus the rate of diffusion, vd) and leading to dendrite morphology then being observed, with the high 3HB phase being an unfavourable material included in intra-spherulitic regions (Figure 9b). Some crystals also grew separately in dendritic morphologies, which may be due to local variations in material

composition occurring due to solvent casting of the material onto the slides, or it could represent nucleation of a 3HB lattice crystal under less favourable nucleation kinetics (Figure 9a). At higher isothermal crystallisation temperatures, phase separation in the melt became more pronounced. The transition from a crystal-melt separation dominated morphology to a meltmelt separation morphology occurred at 43oC. As can be seen in Figure 9c, interpenetrating crystallisation was observed. This indicates the immiscibility in the melt and separation of high 3HV and higher 3HB crystals due to the wide compositional distribution of the PHBV mix. Phase separation occurred both in the melt and at the crystal growth front, with the high 3HB fractions crystallising quickly. The high-3HV copolymer phase then nucleated both heterogeneously on the high-3HB crystal and within it, growing both outside and within the high 3HB component amorphous domains in a structure that appeared to be interpenetrating crystallisation, leading to interspherulitic spherulites with interlocking crystalline structure. The dominance of the fast crystallising high-3HB copolymer phase grew with increasing temperatures. This pattern of interpenetrating crystallisation over time and the resulting morphologies are shown in Figure 10.

Figure 9 (1.5-column fitting)

Figure 10 (2-column fitting)

Sample A4 (50% 3HV Block Copolymer) Sample A4 behaved very similarly to Sample A3. No structural difference between the crystals for random and “block” copolymers was observed. The crystal morphology changed from compact globular morphology to show some extent of interpenetrating crystallisation

when the isothermal crystallisation temperature increased, but at a lower transition temperature compared to A3, at 29oC. Figure 11 shows the crystal structure captured by POM at temperatures ranging from 24oC to 78oC. At temperatures higher than 29oC, the higher 3HV fractions and the higher 3HB fractions separated into two crystal structures due to immiscibility. The faster-growing 3HB-rich crystals dominated and the slower-growing 3HV-rich crystals grew within the 3HB-rich crystals, demonstrating interpenetrating crystallisation.

Figure 11 (1.5-column fitting)

Interspherulitic spherulites in both Samples A3 and A4 developed interlocking morphology as the PHBV (high 3HV) crystallised within the existing PHBV (high 3HB matrix). The high 3HB component of the melt crystallised quickly into a porous, continuous matrix partially obscured by high 3HV material in the melt (Figure 12a-b). This high 3HV material crystallised subsequently, nucleating at several points within the high 3HB matrix (Figure 12c-d) and then growing into a continuous structure as is commonly seen with amorphouscrystalline blends of other polymers, but rarely observed in PHA blends.

Figure 12 (1.5-column fitting)

3.1.3. Kinetics of crystallisation The growth rates, which were calculated from the change in radius of the crystals over time, of each crystal type in each material are plotted in Figure 13, with the exception of the high 3HB component crystal growth in sample A2, which could not be measured due to its low

nucleation rate and very slow growth. The growth rates plotted at each temperature are averages of all crystals observed under POM. As can be seen in Figure 13, the crystal growth rates increased with temperature at lower isothermal crystallisation temperatures and then decreased or plateaued at higher temperature, in the typical fashion. The temperature at which the highest growth rate was achieved depended on the average copolymer content and the compositional distribution of the samples. It could be interpreted that the crystallisation of PHA could be slowed down by cooling. Overall, the crystallisation rate and the morphology can both be controlled by temperature but it will be material dependent. Two types of crystal growths were observed from the samples with a wide distribution of composition (samples A3 and A4). In both of these, high 3HB crystals had a faster growth rate than 3HV crystals at all tested temperatures and the difference was more pronounced at higher temperature (Figure 13). It should be noted that the observed crystal growth rates are confounded for A3 and A4 in particular by crystallisation in a constrained environment with shifting diffusion rates and nucleation and growth rates with temperature leading to changes in morphology and thus are not reflective of the isolated crystals. Interpenetrating crystallisation was observed when the difference in growth rate was significant, at >40oC for sample A3 and >30oC for sample A4.

Figure 13 (2-column fitting)

3.1.4. Lamellar Morphology, Degree of Crystallinity and Quantitative Phase Analysis To test the impact of temperature on the long-term phase behaviour, all samples were allowed to crystallise for 3 days at 30°C and 70°C, then conditioned at room temperature and ambient pressure for at least 6 months to allow for secondary crystallisation, before being

characterised using WAXS and SAXS analysis. Figure 14 shows the 1D WAXS profiles of all samples, with the peaks corresponding to both PHB and PHV type lattices being annotated. In Figure 15, we examine in more detail the region of the 1-D WAXS pattern corresponding to the 110 reflection for PHB and 020 reflection for PHV at 30°C. While only sample A2 shows no discernible crystallisation of the PHB lattice, there is a clear shift in position of the PHV 020 peak between samples A2 and A3 (0.92 Å) and sample A4 (0.89 Å). This is clearly a distortion of the PHV lattice but it is difficult to quantify due to the limited q of this instrumental set-up. Thus the initial literature based fitting based parameters were allowed to relax to reflect the uncertainty in the q-scale and its resolution and we confine our discussions to qualitative aspects.

Figure 14 (1.5-column fitting)

Figure 15 (single-column fitting)

The percent of 3HB lattice and the overall degree of crystallinity were calculated from the 1D WAXS profiles using software Peakfit as described in section 2.6. The data is presented in Table 3. The percent of 3HB lattice present followed the same rough order as the average 3HB content determined using GC. However, there were very distinct differences. Sample A1 appears to have crystallised predominantly (>95%) in the 3HB crystal lattice, despite having 15% 3HV present, meaning that some of the 3HV present must be in the 3HB lattice or in the amorphous regions between lamellae (Type 1 or 2 crystals as per Yoshie and Inoue (Figure 7)) [19]. By contrast, sample A2 was almost completely present in the 3HV crystal lattice form, while samples A3 and A4 both contained a higher proportion of 3HV crystal lattice than would be expected from the bulk composition, indicating exclusion of high 3HB

content copolymers into amorphous regions. Using a higher isothermal crystallisation temperature resulted in a slightly higher percent of 3HB lattice in all samples. This supports the observation that 3HB crystals have a higher growth rate or, in other words, need less “under-cooling” energy to grow, than 3HV crystals at the same isothermal crystallisation temperature. Higher isothermal crystallisation temperatures led to higher crystallinity which was preserved in the long term. This is likely due to a higher diffusive rate, leading to a greater degree of separation between high 3HB and high 3HV components, while at 30°C a greater degree of co-crystallisation occurs.

Table 3

Figure 16 shows the radially averaged SAXS curves for the materials A1, A2, A3 and A4 at 30oC. All curves are typical of semicrystalline polymers where the poorly defined lamellae give rise to a broad diffraction feature with no visible higher order Bragg features [30]. The position of this peak is indicative of the lamellar spacing, LP (Figure 2).

Figure 16 (single-column fitting)

We have used the cosine transform implemented in the program SAXSDAT [43] to extract further structural information. A summary of the results extracted this transformation are given in Table 4. Sample A2, with the most clearly defined lamellar peak, is the most crystalline. Sample A1 has an intermediate linear crystallinity. Samples A3 and A4 both have a similar lower linear crystallinity. The values of crystallinity are quite different, and lower, from those determined from the deconvolution of WAXS patterns (Table 3) but maintain the same order in terms of crystallinity. This difference, between the linear crystallinity and the

bulk crystallinity determined from WAXS measurements, is usually observed and rationalized in terms of a crystalline phase formed outside the lamellar structure [51]. Because of the low resolution nature of SAXS measurements it is not possible to provide chemical localization of this structure. There is a clear difference between the lamellar spacing/long period between the 4 materials in the order A1
Table 4

3.1.5. Fractionation To verify the interpretation that the observed interpenetrating crystallisation in PHBV is due to the wide compositional distribution within the PHBV copolymer blend, solvent/nonsolvent fractionation was performed on Sample A3 to quantify the compositional distribution of this as-produced PHBV. Sample A3 was fractionated into three components: fraction B1, B2 and B3. The characteristics of the fractions are summarised in Table 5. Fraction B1 (11% by mass) has 26 mol% of 3HV in the copolymer, Fraction B2 (17% by mass) has 50 mol% 3HV and Fraction B3 (72% by mass) has 80% 3HV. The DSC melt thermograms showing the melt transitions for the fractions are shown in Figure 17. Their corresponding thermal properties are presented in Table 6. As can be seen in Figure 17, more distinct and narrower melting peaks are observed from the fractions when compared to the as-produced Sample A3. This indicates that as-fractionated PHBV fractions consist of narrow chemical compositional distributions. It is obvious that Fraction B1 has a higher melting point than B2 and B3, and B3 has a slightly higher melting point than B2, which aligns with the determined 3HV content such that PHBV with 3HV content of between 40 – 60 mol% has a lower melting point than those below 40 mol% and above 60 mol% [52].

The crystallisation morphology of the as-fractionated PHBV was imaged using POM and is presented in Figure 18. POM of Fraction B1 showed that it grew dense, uniform 3HB-rich spherulites at all temperatures as expected (Figure 18a). Two crystal morphologies were observed from POM of Fraction 2. The first had a low proportion in the melt and favourable nucleation kinetics. It nucleated first and then grew in a small, non-space filling area. A second crystal type then nucleated and grew through the first type in a dendritic crystal structure, including all material and stopping growth of the initial type. This pattern was observed only at temperatures higher than 29oC (Figure 18b). At 36oC (Figure 18c), the 3HB crystal type was larger due to the slower crystallisation of the 3HV component. At 24oC (image not shown), only the dendritic 3HV lattice crystal (with characteristic lamellar twisting region) was observed. Fraction 3 grew in dense, uniform spherulites at all temperatures (24 – 42oC) (e.g. Figure 18d). One instance of a second morphology was observed at 36oC (Figure 18e) – a small dendritic, non-space filling structure likely to represent a very small proportion of high 3HB fractions. All three fractions showed minimal crystal growth from the minor component, indicating narrow compositional distribution, which is expected from fractionated PHBV. The 3HV content of the fractions ranged from 26 to 80 mol% (>15 mol%) with the majority (72% by mass) at 80 mol% 3HV. The fractionation results support the observations from the POM of the as-produced sample. It should be noted that there was some molecular weight loss on fractionation.

Table 5

Figure 17 (single-column fitting)

Table 6

Figure 18 (1.5-column fitting)

Conclusion The crystallisation morphology and growth kinetics of as-produced mixed culture PHBV blends produced using different combinations of feeding sequences were characterised. POM images indicate that the crystal morphology of the samples was governed by the compositional distribution of the blend and the temperature of crystallisation. PHBV with a narrow, low 3HV content composition (Sample A1) showed only single-phase 3HB crystals, where the 3HV monomer units predominantly co-crystallised within the 3HB lattice. When the compositional distribution of the blend was broad (Samples A2, A3 and A4), which is commonly observed in as-produced mixed culture PHBV, interpenetrating crystallisation was observed at crystallisation temperatures higher than 43oC, 43oC and 29oC for A2, A3 and A4, respectively, with the slow-growing high 3HV crystals intruding into the high 3HB crystals and growing as islands within the 3HB spherulites. At isothermal crystallisation temperatures lower than the transition point (43oC, 43oC and 29oC for A2, A3 and A4, respectively), PHBV blends exhibited a range of morphologies, forming either in single phase 3HB or 3HV crystal lattices or in both simultaneously or consecutively, depending on the 3HV content of the blend components, the nucleation kinetics and the similarity of growth rates of the different phases, with a potential effect of local compositional variations on the slide due to solvent casting adding additional complexity. This indicates that different materials could be developed by manipulating the crystal growth conditions, such as through changing crystallisation temperature and compositional distributions as well as through initial processing methods applied. Alternatively, the crystallisation rates could be manipulated through the addition of nucleating agents, as is common. Overall, understanding and

regulating the phase-separation kinetics, diffusion kinetics, crystallisation kinetics and nucleation rate and the resultant morphological structure from the combination of these is essential for optimising the properties of the blends. This further confirms that optimising the crystallisation temperature is key to the preparation of high performing materials. Future work will be directed to investigating the effect of crystallisation behaviours on PHBV material properties, leading to an analysis of the structure-property relationships of PHBV/PHBV blends.

Acknowledgement This work is supported by the Australian Research Council for funding through ARC linkage grant LP0990917. The ARC had no role in the study design, collection, analysis, or interpretation of the data. The authors also thank A. Werker, L. Karabegovic, P. Johansson, and P. Magnusson from AnoxKaldnes for their valuable assistance with pilot plant operation.

Data availability The raw/processed data required to reproduce these findings cannot be shared at this time as the data also forms part of an ongoing study. The data will be made available on request.

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Figure Captions

Figure 1: Spherulitic morphology of PHV homopolymer isothermally crystallised at (a) 88 oC and (b) 70oC. The white arrow indicates the characteristic ‘eye-like’ region. [14] © ACS Publications. Reproduced by permission from ACS Publications.

Figure 2: Cartoon representation of model used for the analysis of SAXS curves by transformation into linear correlation function. It assumes on these length scales (10’s of Å) that the semicrystalline polymer may be represented by a linear stack of alternating crystalline and amorphous regions with electron density ρc and ρa, respectively. The linear crystallinity is the proportion of crystalline material in the semicrystalline lamellae (Lc/LP).

Figure 3: DSC thermograms showing (a) melt transitions during first heating scan, (b) crystallisation transition during first cooling scan, and (c) glass, cold crystallisation and melt transitions during final (third) heating scan following rapid quenching for as-produced PHBV copolymers.

Figure 4: Spherulitic growth rates of PHB and PHBV copolymers of varying compositions as a function of temperature. [22]. ○ = PHB; ▲= PHBV (7% 3HV content); □ = PHBV (11% 3HV content); ● = PHBV (16% 3HV content); ∆ = PHBV (20% 3HV content); ■ = PHBV (27% 3HV content) © Elsevier. Reproduced by permission from Elsevier.

Figure 5: Spherulitic morphologies of Sample A1 at (a) 36oC for 800 s; (b) 43oC for 650 s; (c) 49oC for 2000 s; (d) 58oC for 900 s; (e) 68oC for 900 s; (f) 78oC for 7000 s (Tc: Isothermal crystallisation temperature; tc: time allowed for crystallisation). Sample A1 exhibited growth of a single, highly regular crystal type (PHB lattice) at all temperatures.

Figure 6: The transition of morphology with change of the relationship between crystallisation rate νc, and diffusion rate νd with crystallisation temperature. [49] © Springer. Reproduced by permission from Springer.

Figure 7: The crystallisation process in PHA – PHA blends with differing degrees of miscibility. [19] © Wiley. Reproduced by permission from Wiley.

Figure 8: Spherulitic morphologies of Sample A2 at (a) 29oC for 3000 s; (b) 36oC for 6000 s; (c) 43oC for 13,000 s; (d) 43oC for 30,000 s (Tc: Isothermal crystallisation temperature; tc: time allowed for crystallisation). Sample A2 exhibited growth of a single, highly regular crystal type (3HV lattice) at temperatures below 36 oC. It exhibited interpenetrating crystallisation (slow-growing copolymer crystals growing within the fast-growing copolymer crystals) at 43oC.

Figure 9: Spherulitic morphologies of Sample A3 at (a) 23oC for 11 hrs; (b) 36oC for 11 hrs; (c) 43oC for 6 hrs; (d) 78oC for 7 hrs (Tc: Isothermal crystallisation temperature; tc: time allowed for crystallisation). Sample A3 exhibited growth of a single, highly regular crystal type (3HV lattice) at temperatures below 43oC. It exhibited interpenetrating crystallisation (where the slow-growing 3HV lattice phase intrudes into the fast-growing 3HB lattice phase and forms an island within it) at 43oC.

Figure 10: Time series of crystallisation of Sample A3 at 43oC from 2 mins to 340 mins.

Figure 11: Spherulitic morphologies of Sample A4 at (a) 24oC for 10 hrs; (b) 29oC for 6 hrs; (c) 33oC for 8 hrs; (d) 40oC for 6 hrs; (e) 43oC for 6 hrs; (f) 78oC for 7 hrs (Tc: Isothermal crystallisation temperature; tc: time allowed for crystallisation). Sample A4 exhibited growth of a single, highly regular crystal type (presumably 3HV lattice) at temperatures below 29 oC. It exhibited interpenetrating crystallisation at 29oC and above.

Figure 12: Comparison of the spherulitic morphologies between Sample A3 and Sample A4 at 78oC (Figure 12 (a) and (b) respectively), and 43oC (Figure 12 (c) and (d) respectively), (Tc: Isothermal crystallisation temperature; tc: time allowed for crystallisation.

Figure 13: Average growth rates of each crystal type of all samples at isothermal crystallisation temperatures ranged from 20oC to 78oC.

Figure 14: 1D WAXS patterns of all PHBV samples (dashed and solid lines denote samples which were isothermally crystallised at 70oC and 30oC respectively)

Figure 15: Magnification of low-q region of 1-D WAXS pattern.

Figure 16: Radially averaged SAXS curves from 4 samples.

Figure 17: DSC thermograms showing melt transitions during the first heating scan for asproduced Sample A3 (62% 3HV random) and its three fractions (B1, B2 and B3)

Figure 18: Spherulitic morphologies of the 3 fractions from solvent/nonsolvent fractionation of Sample A3: (a) Fraction B1 at 36oC for 1 hr; (b) Fraction B2 at 29oC for 18 hrs; (c)

Fraction B2 at 36oC for 35 hrs; (d) Fraction B3 at 24oC for 9 hrs; (e) Fraction B3 at 36oC for 32 hrs (Tc: Isothermal crystallisation temperature; tc: time allowed for crystallisation) Tables Table 1 Properties of the as-produced polymers. Data also reported in Laycock et al. [35] Exp

Feeding Sequence

PHA Type

HV

(Mn)

(Mw)

(Relative percent

Anticipated

content

g mol-1

g mol-1

(mol%)

x10-5

x10-5

15

3.5

82

as gCOD) A1

HAc:HPr

Random

combined 75:25

Copolymer

PDI

D

R

6.3

1.8

6.8

0.72

2.6

5.0

1.9

2.2

0.91

62

2.6

4.6

1.9

2.3

0.95

50

2.7

4.5

1.63

4.5

0.63

(8h) A2

HAc:HPr

Random

combined 33:67

Copolymer

(8h) A3

HAc:HPr

Random

combined 50:50

Copolymer

(11.95h) A4

HAc (1h)

(A-B)n

alternating HPr

repeating

(0.5h) – (total 8h)

multiblocks (including diand tri-blocks)

Table 2 Thermal properties of the as-produced PHBV. Data also reported in Laycock et al. [35] Sample

3HV

Tm

ΔHm

Tm

ΔHm

ΔHm

Tc

ΔHc

Tg

Tcc

ΔHcc

Code

content

(under

(under

(over

(over

(total)

(˚C)

(J g- 1)

(˚C)

(˚C)

(J g-1)

(mol%)

110˚C)

110˚C)

110˚C)

110˚C)

(J g-1)

(˚C)

(J g-1)

(˚C)

(J g-1)

-

-

152.1/

54.4

54.4

86.0

55.2

1.5

56.6

31.2

0.2

57.9

-

-

-

-

-

-

-

75.8

12.7

A1

15

165.5 A2

82

92.7

57.7

173.4

13. 2 A3

62

80.6/

33.3

94.9

143.1/

2.5

35.8

-

-

156.6

12. 8/0. 1

A4

50

84.6/ 94.7

38.3

141.7/ 160.1

16.2

54.5

61.3

141

16.3 /1.0

Table 3 The percent of 3HB lattice and the overall degree of crystallinity values of each samples calculated from the X-ray diffraction patterns Percent of 3HB

Percent of 3HB lattice (%)

Overall degree of crystallinity (%)

by GC (mol%) Isothermal

Isothermal

Isothermal

Isothermal

crystallisation at

crystallisation at

crystallisation at

crystallisation at

30oC

70oC

30oC

70oC

A1

85

95.7

97.0

54

58

A2

18

1.7

2.3

60

66

A3

38

18.3

23.6

48

53

A4

50

36.7

37.4

49

50

Table 4 Parameters extracted from the cosine transformation of SAXS data (see Figure 2) Sample

LP (Å)

LC (%)

Lc (Å)

A1

54.8

40

21.4

A2

56.9

54

24.7

A3

58.6

32

19.5

A4

60.9

31

18.8

Table 5 Properties of fractions of solvent/nonsolvent fractionated Sample A3 % mass

3HV content by GC

Mn

MW

fraction

(mol%)

(kDa)

(kDa)

As-produced

100

62

260

460

1.9

B1

11

26

150

340

2.3

B2

17

50

150

310

2.0

B3

72

80

130

270

2.1

Fraction

PDI

Table 6 Thermal properties of the as-produced Sample A3 (62% 3HV random) and its three fractions (B1, B2 and B3) Sample

3HV

Tm

ΔHm

Tm

ΔHm

ΔHm

Tc

ΔHc

Tg

Code

content

(under

(under

(over

(over

(total)

(˚C)

(J g- 1)

(˚C)

(mol%)

110˚C)

110˚C)

110˚C)

110˚C)

(J g-1)

(˚C)

(J g-1)

(˚C)

(J g-1)

80.6/

33.3

143.1/

2.5

-

-

- 12.8/

A3

62

94.9 Fraction

26

-

35.8

156.6 -

130.0/

0.1 61.0

61.0

84.1

56.7

-2.3

138.6/

B1

154.0 Fraction

50

B3

58.7

-

-

58.7

-

-

-9.8

77.7

-

-

77.7

-

-

-12.3

96.7

B2 Fraction

85.6/

80

94.9

Graphical abstract

Highlights 

Crystal morphology of PHBV depends on the composition distribution of copolymers



Crystal structure was studied using optical microscopy, DSC and X-ray crystallography



Narrowly distributed copolymers crystallised

in regular

banded spherulite

morphology 

Copolymer blends displayed interpenetrating crystallisation of separate crystal phases



Optimizing the crystallisation temperature is key to high performing materials