Biosynthesis and Characteristics of Aromatic Polyhydroxyalkanoates

Polyhydroxyalkanoates (PHAs) are polyesters synthesized by bacteria as a carbon and energy storage material. PHAs are characterized by thermoplasticity, biodegradability, and biocompatibility, and thus have attracted considerable attention for use in medical, agricultural, and marine applications. The properties of PHAs depend on the monomer composition and many types of PHA monomers have been reported. This review focuses on biosynthesized PHAs bearing aromatic groups as side chains. Aromatic PHAs show characteristics different from those of aliphatic PHAs. This review summarizes the types of aromatic PHAs and their characteristics, including their thermal and mechanical properties and degradation behavior. Furthermore, the effect of the introduction of an aromatic monomer on the glass transition temperature (Tg) of PHAs is discussed. The introduction of aromatic monomers into PHA chains is a promising method for improving the properties of PHAs, as the characteristics of aromatic PHAs differ from those of aliphatic PHAs.


Introduction
Polyhydroxyalkanoates (PHAs) are microbial polyesters produced by numerous bacteria, including Ralstonia eutropha and Pseudomonas putida. PHAs are accumulated as an intercellular energy and carbon storage material under nutrient-limited conditions in the presence of excess carbon sources [1,2]. PHAs exhibit valuable characteristics, such as biodegradability, biocompatibility, and thermoplasticity, and therefore can be used for medical, agricultural, and marine applications [3,4].
PHAs are partially crystalline polymers. Therefore, their thermal properties are generally expressed in terms of the glass transition temperature (T g ) of the amorphous phase and the melting temperature (T m ) of the crystalline phase [5]. The most common type of PHA, poly (3-hydroxybutyrate) [P(3HB)], has a T m of 177 • C and T g of 4 • C [6]. The thermal properties of P(3HB) are similar to those of polypropylene; however, P(3HB) is a highly crystalline material with poor elasticity. Additionally, P(3HB) shows secondary crystallization at room temperature, which means that its physical properties change depending on the aging time [7,8]. These characteristics limit the practical uses of P(3HB).
The physical and mechanical properties of PHAs are dependent on the types of monomers and monomeric composition. To date, about 150 different hydroxyalkanoic acids have been reported as constituent monomers of biosynthesized PHAs [9,10]. Based on monomer structure, PHAs are divided into three groups; short-chain-length PHAs (scl-PHAs) comprising 3-5 carbon atoms like P(3HB), medium-chain-length PHAs (mcl-PHAs) comprising 6-14 carbon atoms, and long-chain-length PHAs (lcl-PHAs) comprising more than 14 carbon atoms [11]. These PHAs show different thermal and physical properties. Generally, mcl-PHAs show lower T m and T g and more flexibility compared with scl-PHAs. Alteration of the monomer types and/or the composition of PHAs could lead to desirable polymer properties. For example, P(3HB-5 mol % 3-hydroxyhexanoate) [P(3HB-5 mol % 3HHx)] has a T m of 138-147 • C and T g of 0 • C [12], whereas medium-chain-length PHAs (mcl-PHAs) with C6-C12 are elastomers with a T g between −53 and −28 • C and T m between 45 and 69 • C [13].
In 1990, Fritzsche et al. [14] reported the production of an aromatic PHA, P(3-hydroxy-5phenylvalerate) [P(3H5PhV)], from 5-phenylvaleric acid (5PhV) by Pseudomonas oleovorans. That was the first report of a biosynthesized PHA bearing an aromatic group as a side chain [14]. Recently, wide varieties of aromatic monomers have been introduced into biosynthesized PHA chains. These aromatic PHAs are attractive, not only in terms of novelty, but also for their possible functionality conferred by the benzene ring.
The purpose of this review is to summarize the types of biosynthesized PHAs bearing aromatic groups as side chains and their properties. Specifically, the effect of aromatic monomers on the T g of PHAs is demonstrably different from that of other mcl-monomers, and is thus described in detail. Incorporation of aromatic monomers into PHAs is one possible method of improving the properties of PHAs by conferring superior physical properties, surpassing those of aliphatic PHAs. Additionally, the aromatic side groups do not compromise the important characteristics of PHAs, including their biodegradability, biocompatibility, and thermoplasticity. Thus, it is proposed that the introduction of aromatic monomers into PHA chains is a promising method for producing PHAs with improved material properties.

Biosynthesized PHAs Bearing Aromatic Groups as Side Chains
The production of a PHA containing an aromatic monomer was reported for the first time by Fritzsche and coworkers [14]. Thereafter, various aromatic monomers have been introduced into PHA chains through biosynthesis. Figure 1 shows the aromatic monomers incorporated into biosynthesized PHAs, and Table 1 summarizes the cultivation conditions used for the production of PHAs containing these monomers (the details are presented below).
Incorporation of most of the monomers shown in Figure 1 into PHA chains was achieved using Pseudomonas strains. Although there are some reports of the production of aromatic PHAs using recombinant Escherichia coli and R. eutropha [15][16][17][18], these cases also required PHA synthase from Pseudomonas. To the best of our knowledge, only PHA synthase from Pseudomonas can polymerize aromatic monomers.
Generally, to generate aromatic PHAs, defined compounds containing aromatic groups are added to the medium as a precursor (i.e., 5PhV or 5-phenyl-2,4-pentadienoic acid is used as a precursor to produce PHAs containing 3H5PhV [14,15,19,20]). However, there are a few reports on the production of aromatic PHAs without any supplementation with these defined aromatic compounds [16,18]. Therefore, aromatic PHAs could be divided into two groups, products synthesized from corresponding aromatic compounds through chemo-bioprocesses and products synthesized through complete biosynthesis from biomass. It was also reported that Pseudomonas strains fed with aromatic compounds produced PHAs composed of aliphatic monomers only [14,[21][22][23].

Figure 1.
Structures of aromatic monomer units introduced into biosynthetic polyhydroxyalkanoates (PHAs). A monomer containing a nitrophenoxy group was also reported, but the detailed structure, including the carbon number, was not presented [24].

Aromatic PHAs Produced from Corresponding Aromatic Compounds
As mentioned above, most biosynthesized aromatic PHAs have been produced from defined compounds with chemical structures similar to those of the incorporated aromatic monomers. Using various aromatic compounds as a precursor, many types of aromatic monomers containing phenyl, phenoxy, methylphenyl, methylphenoxy, nitrophenyl, nitrophenoxy, cyanophenoxy, fluorophenoxy, thiophenoxy, or benzoyl groups have been incorporated into PHA chains. The details are described below.

Copolymer Composed of Monomers Bearing Phenyl Groups with Different Carbon Numbers
P. putida U produced random copolymers P(3H5PhV-3H7PhHp) and P(3H6PhHx-3H8PhO) from 7-phenylheptanoic acid (7PhHp) and 8-phenyloctanoic acid (8PhO), respectively [25,26]. It was reported that P. putida U produced the P(3H5PhV) homopolymer from 7PhHp [25]. These two different results might be derived from the method used for polymer analysis. This strain also produced a terpolymer containing 3H6PhHx, 3H8PhO, and 3H10PhD monomer units from 10-phenyldecanoic acid (10PhD) [25]. Monomers with different carbon numbers may be generated from the precursors through the β-oxidation cycle of the host strains, where the cycle is involved in fatty acid degradation. Similar results are documented in other reports [19,40]. Production of a terpolymer and tetrapolymer has also been reported [40].

Copolymer Containing Aliphatic and Aromatic Monomers
Copolymers composed of aliphatic and aromatic monomers have been reported. P. oleovorans cultured with 5PhV and n-alkanoic acid (octanoic acid (OA) or nonanoic acid (NA)) produced PHAs containing both 3H5PhV and aliphatic monomers [27]. The produced polymer was not a random copolymer, but comprised a mixture of two PHAs, and thus showed two different T g s (see Section 3). Similar results have been documented in other reports [27,41]. Transmission electron microscopy (TEM) observation demonstrated that these two polymers were formed in the same granule [41]. Therefore, there might be some distinguishing factor in the metabolism of the two substrates which results in the production of two separate polymers with different physical properties [41]. On the other hand, P. citronellolis and P. putida GPo1 produced a PHA random copolymer composed of aromatic and aliphatic monomers from a mixture of aromatic and aliphatic compounds [28,29].
Recombinant E. coli expressing mutated PhaC1 Ps synthesized PHA copolymer containing 2H3PhP (phenyllactate) from amino acids such as phenylalanine and sugars [16]. Surprisingly, as described in Section 2.2, the same PHA copolymer was produced from sugars as the sole carbon source without amino acid supplementation [16,18].

PHAs Containing Phenoxy Group
Incorporation of aromatic monomers containing the phenoxy group (C4-C9) was also reported [30]. P. putida BM01 produced a PHA composed of 3-hydroxy-5-phenoxyvalerate (3H5PxV) as the major component and 3-hydroxy-7-phenoxyheptanoate (3H7PxHp) as the minor component from 11-phenoxyundecanoic acid. Interestingly, 3-hydroxy-9-phenoxynonanoate (3H9PxN) was incorporated only when OA was added to the medium in conjunction with the aromatic compound. The production of PHAs containing the phenoxy group was also observed when P. oleovorans and P. putida were fed with ω-phenoxyalkanoic acids [31,44,45]. Similar to PHAs containing phenyl groups, these PHAs are highly or totally amorphous (see Section 3).

PHAs Containing Methylphenyl Group
To produce a crystalline aromatic PHA, the synthesis of PHAs containing the methylphenyl group was attempted based on the results of previous studies [46][47][48]. These studies reported that the polymer formed by the cationic polymerization of para-methyl-α-methylstyrene showed a high degree of crystallinity, whereas the polymer obtained from α-methylstyrene did not crystallize. As described in Section 3, the obtained methylphenyl-containing PHA was crystalline.
However, no aromatic monomer was detected in the polymers isolated from the cells grown with 8-(ortho-methylphenoxy)octanoic acid. In other studies, the polymer produced from 8-(orthomethylphenoxy)octanoic acid by P. putida KCTC2407 also did not contain any corresponding aromatic units [45]. These results indicate that the biosynthesis of PHAs bearing methylphenoxy substituents is highly dependent on the position of the methyl substitutent. Additionally, the position of methyl substitution also affects the crystallinity of the PHA (see Section 3).

PHAs Containing Nitrophenyl or Nitrophenoxy Group
The production of aromatic PHAs bearing mononitrated or dinitrated groups has been reported [34]. In that study, to obtain a new bacterial PHA, a modified version of 5PhV containing the nitro group was fed to P. oleovorans. The bacteria eventually produced yellow PHAs with 1.2-6.9% repeating units containing para-nitro and/or ortho,para-nitrophenyl rings from 5-(ortho,para-dinitrophenyl)valeric acid and NA. However, the content of the dinitrated monomer unit decreased depending on the cultivation time. The bacteria initially synthesized a polymer containing orthoand para-substituted aromatic groups, and then generated the polymer containing aromatic groups with only para-substitution. In fact, the final polymer showed two different T g s (see Section 3), indicating that the sample comprised a mixture of two PHAs [34]. Kim et al. reported the production of a PHA containing the nitrophenoxy monomer from 6-(para-nitrophenoxy)hexanoic acid and OA (the detailed monomer structure, including the carbon number, was not reported) [24]. Both studies showed that the extent of the incorporation of these monomers into the PHA was significantly lower than that of other aromatic groups, and -feeding of NA or OA along with the aromatic compounds was inevitably required.

PHAs Containing Cyanophenoxy Group
An aromatic monomer containing hyperpolarizable cyanophenoxy side groups was also reported. P. putida KT2440 grown on OA and 6-(para-cyanophenoxy)hexanoic acid produced a PHA containing a monomer bearing the cyanophenoxy group, 3-hydroxy-6-(para-cyanophenoxy)hexanoate (3H6pCPxHx) [35]. Thermal analysis revealed that the produced polymer was not simply a random copolymer, but was a heterogeneous polymer consisting of various chains and/or chain segments with different compositions of 3H6pCPxHx units. The generated polymer showed weak second harmonic generation (SHG) signals [35]. The incorporation of cyanophenoxy groups, including 3-hydroxy-5-(para-cyanophenoxy)valerate (3H5pCPxV), was also reported by Kim et al. [24]. Similar to PHAs containing nitrophenyl or nitrophenoxy groups, co-feeding of OA was required to incorporate this monomer into the PHA chains.

Aromatic PHAs Produced through Complete Biosynthesis
As described above, 2H3PhP-containing PHA was synthesized from phenylalanine and sugars [16]. Because phenylalanine could be produced from biomass in bacterial cells, it can be viewed that this PHA could be biosynthesized from biomass through complete biosynthesis. Similarly, it could be concluded that 3H3PhP-containing PHA could also be produced through complete biosynthesis because it was produced from sugars and cinnamic acid [15], which could be synthesized in bacterial cells. In this system, cinnamic acid is thought to be enantioselectively hydrated after ligation with CoA, and polymerized into the PHA chain. In fact, it was reported that 2H3PhP-containing PHA was produced from sugars as the sole carbon source [16,18]. These are rare reports showing the production of aromatic PHAs without any supplementation of corresponding aromatic compounds. Additionally, it is also suggested that 3H4PhB-or 2-hydroxy-4-phenylbutyrate (2H4PhB)-containing PHAs may be produced by complete biosynthesis, because homophenylalanine, which has a carbon skeleton identical to that of 3H4PhB, was reported to be biosynthesized from phenylalanine [51].

Physical and Chemical Properties of Aromatic PHAs
Aromatic PHAs show various characteristics depending on the types of aromatic monomers incorporated. Particularly, the thermal properties of aromatic PHAs have been extensively studied, demonstrating structure-specific behavior. In this section, the physical and chemical properties of aromatic PHAs are summarized.
In the case of PHAs bearing the nitrophenyl group, even with the introduction of a small amount of nitrophenyl units (1.2-6.9%), the physical properties became very different from that of mcl-PHA [34]. The polymer appeared yellow and had an elastic texture, whereas mcl-PHA produced from 3HN is whitish and sticky.

Mechanical Properties
The mechanical properties of P(3-hydroxydodecanoate-3H5PhV) [P(3HDD-3H5PhV)] with various 3H5PhV contents is summarized in Table 2 [52]. Introduction of the 3H5PhV unit into P(3HDD) resulted in a decrease in the yield strength, maximum tension strength, and elongation at break. Interestingly, P(3HDD-18.70 mol % 3H5PhV) showed a higher elongation at break than P(3HDD). On the other hand, the Young's modulus became higher than that of P(3HDD), except for P(3HDD-31.97 mol % 3H5PhV). These results indicate a non-linear relationship between the 3H5PhV content and the mechanical properties.

Surface Properties
The surface properties of P(3H5opFPxV) with two fluorine atoms were evaluated [36]. The surface contact angle of this polymer was 104 • , whereas that of the PHAs with a phenoxy or alkyl group (C3 and C5) in the side chain was approximately 50 • . In general, a surface contact angle of over 100 • is sufficient to allow utilization of the polymer as a non-wetting material. Therefore, this difluorinated PHA possessed water-shedding properties.

Degradability
The degradability of aromatic PHAs has also been studied. Degradability is one important property for the use of PHAs as biodegradable materials. For medical applications such as drug delivery systems, the stability of PHAs at physiological pH and the safety of the material released through hydrolysis should be evaluated by evaluating the chemical degradation. For agricultural and marine applications, degradation by microorganisms should also be studied.

Chemical Degradation
The chemical degradation of the P(3H6PhHx) homopolymer was analyzed, as reported in the literature [40]. This polymer is quite stable around pH 7.0. Therefore, it could be used as a drug vehicle [54] to achieve slow release of the active product. Additionally, its hydrolytic products, which can be β-oxidized in vivo to phenylbutyric acid, phenylacetic acid, or trans-cinnamic acid, may exert important pharmaceutical effects, thereby improving or broadening the clinical effects of the encapsulated drug. Olivera et al. synthesized polymeric microspheres of P(3H6PhHx) at atmospheric pressure by a solvent evaporation method, and demonstrated its potential use as a drug vehicle [40].

Biological Degradation
Although aromatic PHAs are unusual in nature, some reports claim that they can be degraded by microorganisms such as Pseudomonas strains [27,35,56]. However, aromatic PHAs are degraded more slowly than PHAs having no aromatic repeating units. Interestingly, the rate of degradation of P(3H5PhV) became much greater when aliphatic PHA produced from 3HN was also present in the same cell, whereas the rate of degradation of aliphatic PHAs was not significantly affected by the presence of P(3H5PhV) [57]. Aliphatic PHA and P(3H5PhV) were degraded by the same depolymerase, and the enzyme worked more efficiently in the presence of aliphatic PHAs [57].

Solubility and Solvent Fractionation
Generally, bacterial PHA copolymers exhibit a broad comonomer compositional distribution, which may arise from changes in the bacterial metabolism during PHA biosynthesis. In addition, as shown in Section 2, biosynthesized aromatic PHAs are sometimes produced as a mixture of two different PHAs, but not simply as a copolymer. In some studies, these aromatic polymers were separated by solvent fractionation [17,27].
As reported [17], P(3HB-3H3PhP) and P(3HB-3H4PhB) were fractionated into several fractions with a narrow comonomer compositional distribution by using a chloroform/n-hexane mixture. The content of aromatic units in the fractionated samples increased as the concentration of n-hexane increased. In contrast, the molecular weights of the fractionated samples decreased as the concentration of n-hexane increased. This means that the samples containing less of the aromatic monomer with a high PHA molecular weight are difficult to dissolve in the chloroform/n-hexane mixture compared with the samples containing more of the aromatic monomer with a low PHA molecular weight. On the other hand, as reported [45], the sample with a larger content of aromatic monomers was isolated as an insoluble fraction. This means that the sample with a higher aromatic monomer content showed lower solubility in n-hexane than the sample containing less of the aromatic monomer. Similarly, an aromatic PHA was obtained as a precipitated fraction from solvent fractionation of a blend of aliphatic and aromatic PHAs by using a methanol/chloroform mixture [30]. The resulting precipitated sample had the same monomer composition as that of the aromatic PHA before blending, and no aliphatic monomers. From these results, it was suggested that solvent fractionation depended mainly the PHA molecular weight, which varied with the ratio of aromatic monomers.

Thermal Properties
PHAs are partially crystalline polymers. Therefore, their thermal properties are normally expressed in terms of the T g of the amorphous phase and the T m of the crystalline phase [5]. The thermal properties of the aromatic PHAs determined in various studies are summarized in Table 3. The properties differ significantly from that of mcl-PHAs, which are elastomers with T g s between −53 and −28 • C and a T m between 45 and 69 • C [13], where the values vary according to the types of aromatic monomers.

Thermal Properties of PHAs Containing Phenyl Groups
Homopolymer P(3H5PhV), the first biosynthesized PHA bearing an aromatic group, has a T g of 13 • C [14]. This is higher than that of PHAs with n-alkyl pendant groups (mcl-PHA), which have T g s in the range of −53 to −28 • C [13]. The increases of T g resulted from the introduction of aromatic monomers have been also reported in other aromatic monomers as described below. This behavior is different from mcl-PHAs. On the other hand, the T m of P(3H5PhV) ranged from 54 to 69 • C, which is similar to those of the n-alkyl-substituted PHAs. As for the value of T m , P(3H5PhV) is most similar to mcl-PHAs. However, the endothermic peak for the melting transition in the differential scanning calorimetry (DSC) thermogram was very small. This observation indicates that this polymer has a very low degree of crystallinity and is thus highly amorphous [20,27,52]. This is unusual because the homopolymer has a highly ordered isotactic structure, which generally provides an ordered packing structure in the solid state [21]. P(3H5PhV) showed another unusual feature whereby recrystallization occurred rapidly when the polymer was cooled rapidly from the melt, unlike the case of n-alkyl-substituted PHAs [14].

Copolymers Composed of Monomers Bearing Phenyl Groups with Different Carbon Numbers
The P(3H4PhB-95 mol % 3H6PhHx) copolymer has a T g of 10 • C, which is higher than that of mcl-PHA, but lower than that of P(3H5PhV) [19]. Similar results were observed in another study [15]. The lower-temperature T g may reflect decreased intermolecular interaction between the backbone chains, thereby resulting in increased backbone chain mobility, caused by lengthening of the side-chains by one methylene unit relative to that of 3H5PhV [58]. This sample did not exhibit any endothermic melting peak and was totally amorphous [15,19]. This suggests that the phenyl group as the side chain impedes the formation of a crystalline domain, probably due to steric hindrance [19].
Similarly, other PHAs composed of only monomers bearing phenyl groups produced from phenylalkanoic acids showed the same characteristics, being totally amorphous and with a higher T g than that of mcl-PHA [26,40]. In the literature [26], the T g values followed the order: P(3H5PhV-38 mol % 3H6PhHx-50 mol % 3H7PhHp) > P(3H5PhV-77 mol% 3H7PhHp) > P(3H6PhHx-73 mol % 3H8PhO). This is attributed to the increasing flexibility of the PHA chains due to the introduction of structural irregularities and alkyl side chains longer than three methylene units.

Copolymers Containing Aliphatic and Aromatic Monomers
PHAs consisting of 3HA and 45 mol % 3H5PhV as aliphatic and aromatic monomers, exhibited only one T g at −20 • C [28]. On the other hand, another PHA consisting of 3HA and 40.6 mol % 3H5PhV showed two T g s at −31 and 5 • C [27]. Because these values are close to the T g of the polymer produced from NA alone and P(3H5PhV), this sample is deduced to be a mixture of two PHAs. As described in Section 2, some biosynthesized aromatic PHAs are produced as a mixture of two polymers. In that case, the sample has two T g s.
The effects of the aromatic monomer ratio were also investigated [52]. With an increase in the 3H5PhV ratio in the P(3HDD-3H5PhV) copolymer from 0 to 100 mol %, the T g increased from −49.3 to 5.90 • C and the T m decreased from 82.4 to 50.40 • C. Therefore, the thermal properties of the P(3HA-3HPhA) copolyester can be easily modified by changing the monomer content, which leads thermal properties intermediate between those of the two homopolymers.
The thermal properties of PHAs composed of 3HB and phenylalkanoic monomers were also determined [15,17]. The T m of P(3HB-3H3PhP) with 4.1 mol % and 8.9 mol % 3H3PhP was similar to that of P(3HB) with ∆H m = 7.1 − 27.8 J·g −1 , which is an index of the degree of crystallinity, suggesting that these samples still contained the P(3HB) crystal phase. However, these values are lower than those of other copolymers, such as P(3HB-3HA) with 6 mol % 3HA (C6-C8) with a T m of 126 • C and ∆H m of 31 J·g −1 [59], and P(3HB-5 mol % 3HHx) with a T m of 138 • C and ∆H m of 45 J·g −1 [12]. This suggests that the phenyl side group of the 3H3PhP monomer strongly inhibited the crystallization of P(3HB). In fact, P(3HB-3H3PhP) with 15-21 mol % 3H3PhP did not show any melting behavior [17], indicating the lack of a crystal phase. The T g s of P(3HB-3H3PhP) and P(3HB-3H4PhB) were in the range of 9-20 • C and 7-10 • C, respectively, where these values are substantially higher than that of P(3HB) (5 • C) [17]. This is unusual behavior compared with that of aliphatic comonomers such as 3HV, 3HHx, and 3-hydroxy-4-methylvalerate (3H4MV). Although 3H4MV has a bulky iso-propyl structure and does not co-crystallize with 3HB [60], the T g decreased as the 3H4MV content increased. The rigidity of the aromatic rings in the 3H3PhP and 3H4PhB monomers may account for these differences. The T m of P(3HB-8 mol % 3H4PhB) is similar to that of low-density polyethylene, one of conventional plastics. The T g of this PHAs (10-15 • C) is higher than that of low-density polyethylene (−30 • C), but it should be increased more to avoid secondary crystallization.

Thermal Properties of PHAs Containing Phenoxy Groups
PHAs containing phenoxy groups are highly or totally amorphous with T g s higher than 10 • C [30,31,38,44]. The PHA composed of 3H5PxV and 3H7PxHp had a T m of 70 • C with small ∆H m (2.9 J·g −1 ), indicating that it is highly amorphous [30]. In another study, PHAs composed of 3-hydroxy-ω-phenoxyalkanoic acid (C4-C8) did not show any crystalline melting endotherm, indicating that these polymers were totally amorphous [31].
3.6.3. Thermal Properties of PHAs Containing Methylphenyl Groups P(3H5pMPhV) containing the methylphenyl group as a side chain is a crystalline polymer with a T g of 18 • C and T m of 95 • C with a defined endothermic peak [21], whereas P(3H5PhV) with no methyl substituent on the phenyl group is highly amorphous. It was suggested that the presence of the methyl group at the para-position of the phenyl ring allows the polymer chains to form an ordered structure. In fact, P(3HA-15 mol % 3H5pMPhV) also showed a T m of 60 • C and T g of 14 • C. However, the ∆H m for this polymer is less than 1 J·g −1 [32], indicating that this sample is partially crystalline but highly amorphous. On the other hand, P(3H5PhV-64 mol % 3H5pMPhV), a PHA composed of monomers bearing phenyl-and methylphenyl-groups, is none crystalline [21], indicating that the 3H5PhV unit inhibits formation of the 3H5pMPhV crystalline structure.

Thermal Properties of PHAs Containing Methylphenoxy Groups
P(3H4pMPhB-71.5 mol % 3H6pMPhHx), a PHA consisting of monomers containing the para-methylphenoxy group, is crystalline [33]. Precipitated samples of this polymer exhibit the typical spherulitic structure under a polarizing microscope. On the other hand, the ∆H m of P(3H4mMPhB-70 mol % 3H6mMPhHx) is 0.2 J·g −1 , indicating low crystallinity. These results indicate that the position of methyl substitution has a profound effect on the crystallinity of the polymers. P(3H5PhV-6.7 mol % 3H4pMPxB-28.3 mol % 3H6pMPxHx), a PHA consisting of monomers containing methylphenoxyand phenyl-groups, is amorphous [33]. This indicates that the 3H5PhV unit inhibited the formation of a crystalline structure. This behavior is similar to that of aromatic PHAs bearing methylphenyl groups, as mentioned above.

Thermal Properties of PHAs Containing Nitrophenyl Groups
From DSC analysis, P(3HA-6.9 mol % 3H5opNPhV), a PHA produced from 5-(ortho,paradinitrophenyl)valeric acid and NA, showed one T m and two T g peaks [34]. The existence of two T g s indicates that the produced PHA is a mixture of two polymers [34]. The T m of 56.42 • C and the T g of −35.95 • C were assigned to the PHA produced from NA only, and the T g of 28.74 • C is attributed to the newly synthesized polymer containing nitrophenyl rings. The lack of a defined T m for this newly formed polymer indicates that the PHA containing the nitrophenyl group lacks crystalline domains and is thus amorphous.

Thermal Properties of PHAs Containing Cyanophenoxy Groups
Incorporation of the 3H6pCPxHx unit into P(3HA) led to a slight decrease in the ∆H m and T m [35]. This indicates that the 3H6pCPxHx unit was coexistent with other monomer units in the same polymer chain, and disrupted the crystalline organization of the n-alkyl side groups, resulting in depressed T m values. P(3HA-19.6 mol% 3H6pCPxHx) showed a new T g at −21 • C that was not observed for the samples with 0-6.8 mol % of the 3H6pCPxHx unit. This indicates that the product with 19.6 mol % 3H6pCPxHx is not a random copolymer, but is heterogeneous, having chains and/or chain segments enriched with the 3H6pCPxHx repeating units. Additionally, these segments would have unique crystal structures as the original melting peak of the sample occurred at >64 • C, where no melting peak was observed for P(3HA).

Thermal Properties of PHAs Containing Fluorophenoxy Groups
Generally, fluoropolymers exhibit good thermal resistance. PHAs with a fluorinated side group also exhibit the expected behavior, with T m s of 52 • C (monofluorinated P(3H5pFPxV-8.7 mol % 3H7pFPxHp)) and 102 • C (difluorinated P(3H5opFPxV)) [36]. The wide angle X-ray scattering (WAXS) diffraction patterns indicated that fluorophenoxy PHA was crystalline, whereas phenoxy PHA without the fluorine atom was highly amorphous [36]. Incorporation of fluorine atoms into the side group clearly has a significant effect.
3.6.8. Thermal Properties of PHAs Containing Thiophenoxy Groups P(3H5TPxV-3H7TPxHp), a PHA consisting of thiophenoxy monomer units, has a T g of 4 • C with no clear melting peaks [37], indicating that the polymer is amorphous, as supported by the X-ray diffraction pattern.

Thermal Properties of PHAs Containing Benzoyl Groups
The PHA containing 79.8% 3H5BzV and 3HA (as determined from the GC-MS peak area) is relatively hard at room temperature, which indicates that this polymer has characteristic thermal properties [38]. From DSC analysis, it was determined that the T g was 36 • C and the T m was 150 • C.
These values are higher than those of other aromatic PHAs. Generally, a high molecular weight can lead to high T g and/or T m values, but mcl-PHA (number-average molecular weight M n = 170,000, weight-average molecular weight M w = 350,000) and PHB (M n = 1,800,000, M w = 2,600,000), which have relatively high molecular weights, do not exhibit such high T g or T m values. Therefore, it was suggested that the incorporation of 79.8% of the 3H5BzV unit resulted in these high values. However, the thermal properties of other PHAs bearing benzoyl groups were similar to those of the other aromatic PHAs [38]. Thus, it could be concluded that the length of the side chain containing the benzoyl group and the number of monomeric units also influenced the thermal properties.

Effect of Incorporation of Aromatic Monomer on T g of PHAs
As described in Section 3, the incorporation of aromatic monomers into PHA chains results in a distinct change in the thermal behavior. With an increase in the number of phenyl side groups introduced into P(3HB), the T m and ∆H m decreased, whereas the T g increased. This trend in the T g is much different from the cases of aliphatic monomers. The relationship between the aromatic monomer content and the T g has been studied detail [15,17].
The T g of a copolymer (T gr ) can be calculated by applying the Fox equation, using the T g of the constituent polymers (T g1 and T g2 ) [61]: 1/T gr = W 1 /T g1 + W 2 /T g2 (1) where the T g values of the homopolymers are given in Kelvin, and W 1 and W 2 are the weight fractions of the respective polymers in the copolymer. In the literature [15], from three experimental data sets (P(3HB) (T g = 280.9 K, W 3H3PhP = 0), P(3HB-4.1 mol% 3H3PhP) (T g = 283.7 K, W 3H3PhP = 0.068), and P(3HB-8.9 mol% 3H3PhP) (T g = 287.6 K, W 3H3PhP = 0.143)), the T g of the P(3H3PhP) homopolymer was estimated to be 62 • C (335 K). This estimated value is much higher than ambient temperature and is very close to that of poly(lactide) (T g = 60 • C) [62]. Additionally, it was revealed that the estimated T g for P(3HB-3H3PhP) with 33 mol% 3H3PhP (W 3H3PhP = 0.45) is 30 • C, which is the same as the culture temperature for PHA production. PHAs with T g s exceeding ambient temperature will be produced as rigid amorphous polymers. If PHA synthases do not prefer to polymerize rigid amorphous PHA, the production of P(3HB-3H3PhP) with 3H3PhP fractions of over 33 mol% may be a challenging task [15].
The correlation between the average number of methylene units in the aromatic side chain and the T g are shown in Figure 2, based on the literature data [15]. This figure shows the experimentally determined T g s and those predicted from the Fox equation. With increasing alkyl chain length of the aromatic side group on the monomers bearing the phenyl group, the T g of the PHAs tends to decrease. This may be the result of an increase in the free volume. The rigidity of the side group further hampers the motion of the polymer backbone. Similar trends have been reported for mcl-PHAs [13], poly(alkyl methacrylate)s [63], and poly(alkyl itaconate)s [63]. In fact, the predicted T g values from another study using the Fox equation were 60 • C for P(3H3PhP) and 27 • C for P(3H4PhB) [17], which are in good agreement with the foregoing data. Compared with 3H4PhB, the phenyl side group of 3H3PhP is smaller, but the rigidness of the side group is higher. Thus, 3H3PhP is more effective for increasing the T g than 3H4PhB. These results suggest that the T g of aromatic PHAs bearing phenyl groups depends not only on the size of the side group, but also on its rigidness.
As shown in Figure 2, most of the T g values of aromatic PHAs bearing phenoxy, methylphenyl, methylphenoxy, and nitrophenyl groups followed trends similar to those of PHAs bearing the phenyl group.

Conclusions and Future Perspectives
The types of biosynthesized aromatic PHAs and their chemical and physical properties are summarized in this review. Various monomers containing phenyl, phenoxy, methylphenyl, methylphenoxy, nitrophenyl, nitrophenoxy, cyanophenoxy, fluorophenoxy, thiophenoxy, and benzoyl groups have been introduced into biosynthesized PHA chains. The chemical and physical properties of aromatic PHAs are different from those of aliphatic PHAs, and vary depending on the types of aromatic monomers. The Tg increases as the aromatic monomer content increases, which differs from the behavior of aliphatic monomers. Even with the introduction of a small amount of aromatic monomers, crystallization of P(3HB) is strongly inhibited. This behavior is attributed to the rigidity of the aromatic rings in the side groups. Additionally, the degradation rate of aromatic PHAs is slower than that of mcl-PHAs. This suggests that aromatic PHAs could be used as a drug vehicle which achieve slow release of the encapsulated drug.
The introduction of aromatic monomers broadens the range of applications by altering the properties of PHAs. Predicting the composition of PHAs that would provide the desired Tg using the Fox equation is a useful tool for controlling the properties of PHAs. The composition of PHAs can be controlled by changing the ratio of the carbon sources and by modification of the metabolic pathway, including the β-oxidation cycle of the microorganisms involved in PHA production [29,39].
For practical use of aromatic PHAs, certain issues must be addressed, such as the heterogeneity of the produced polymers, increasing the molecular weight of the PHAs, and reduction of the production cost. Under some conditions, aromatic PHAs are produced as heterogeneous polymers. Because the polymer homogeneity affects the material properties, the cultivation conditions should be optimized to produce homogeneous polymers rather than blend polymers. Additionally, increasing the molecular weight of PHAs is effective for improving the properties [65]. If the Average number (n) of methylene units in aromatic side chain  Figure 2. Correlation between T g and average number of methylene units in aromatic side chain. Red circles: T g of aromatic PHAs containing phenyl group; blue triangles: T g of aromatic PHAs containing phenoxy, methylphenyl, methylphenoxy, or nitrophenyl group; green circles: T g predicted from Fox equation. Values are from References [9,14,15,19,26,30,33,38,40,42,44,52,64].

Conclusions and Future Perspectives
The types of biosynthesized aromatic PHAs and their chemical and physical properties are summarized in this review. Various monomers containing phenyl, phenoxy, methylphenyl, methylphenoxy, nitrophenyl, nitrophenoxy, cyanophenoxy, fluorophenoxy, thiophenoxy, and benzoyl groups have been introduced into biosynthesized PHA chains. The chemical and physical properties of aromatic PHAs are different from those of aliphatic PHAs, and vary depending on the types of aromatic monomers. The T g increases as the aromatic monomer content increases, which differs from the behavior of aliphatic monomers. Even with the introduction of a small amount of aromatic monomers, crystallization of P(3HB) is strongly inhibited. This behavior is attributed to the rigidity of the aromatic rings in the side groups. Additionally, the degradation rate of aromatic PHAs is slower than that of mcl-PHAs. This suggests that aromatic PHAs could be used as a drug vehicle which achieve slow release of the encapsulated drug.
The introduction of aromatic monomers broadens the range of applications by altering the properties of PHAs. Predicting the composition of PHAs that would provide the desired T g using the Fox equation is a useful tool for controlling the properties of PHAs. The composition of PHAs can be controlled by changing the ratio of the carbon sources and by modification of the metabolic pathway, including the β-oxidation cycle of the microorganisms involved in PHA production [29,39].
For practical use of aromatic PHAs, certain issues must be addressed, such as the heterogeneity of the produced polymers, increasing the molecular weight of the PHAs, and reduction of the production cost. Under some conditions, aromatic PHAs are produced as heterogeneous polymers. Because the polymer homogeneity affects the material properties, the cultivation conditions should be optimized to produce homogeneous polymers rather than blend polymers. Additionally, increasing the molecular weight of PHAs is effective for improving the properties [65]. If the interaction between the aromatic rings can be strengthened by increasing the molecular weight, the physical and mechanical properties can be improved. As the types of PHA synthases affect the molecular weight of PHAs [66], the search for new PHA synthases (other than PHA synthases from Pseudomonas) that can polymerize aromatic monomers is a worthwhile undertaking. Reduction of the cost of the carbon sources and improving the productivity are critical approaches to reducing the production cost. The production of aromatic PHAs from biomass such as sugars without any precursor supplementation, which is generally expensive, would contribute to this end. However, the production of aromatic PHAs from sugars was just reported [16,18], so it may require additional research to achieve it. Utilization of biowastes as carbon source and co-production strategies would be beneficial methods for cost reduction [67].
All aromatic PHAs described above have aromatic rings in their side chains. The production of PHAs having aromatic rings in their backbone has not been reported to date, and achieving this structure would be a challenging task because there is no PHA synthase capable of polymerizing aromatic rings in the PHA backbone thus far. If PHAs having aromatic rings in the backbone can be produced, they may show good thermostability, similar to polyethylene terephthalate.
The incorporation of aromatic monomers is a promising method for improving the properties of PHAs and conferring physical properties superior to those of aliphatic PHAs. Further, this approach is not expected to compromise the important characteristics of PHAs, including their biocompatibility, biodegradability, and thermoplasticity. Thus, incorporation of aromatic monomers into the PHA chain is proposed as a promising method of improving the material properties of PHAs.

Conflicts of Interest:
The authors declare no conflict of interest.