Increased diversification of polyhydroxyalkanoates by modification reactions for industrial and medical applications

MINI-REVIEW

Increased diversification of polyhydroxyalkanoates

by modification reactions for industrial

and medical applications

Baki Hazer&Alexander Steinbüchel

Received: 7 June 2006 / Revised: 16 October 2006 / Accepted: 18 October 2006 / Published online: 5 December 2006 # Springer-Verlag 2006

Abstract A wide range of diverse polyhydroxyalkanoates, PHAs, is currently available due to the low substrate specificity of PHA synthases and subsequent modifications by chemical reactions. These polymers are promising materials for a number of different applications due to their biocompatibility and biodegradability. This review summa-rizes the large variability of PHAs regarding chemical structure and material properties that can be currently produced. In the first part, in vivo and in vitro biosynthesis processes for production of a large variety of different PHAs will be summarized with regard to obtaining saturated and unsaturated copolyesters and side chain functionalized polyesters, including brominated, hydroxyl-ated, methyl-branched polyesters, and phenyl derivatives of polyesters. In the second part, established chemical mod-ifications of PHAs will be summarized as that by means of grafting reactions and graft/block copolymerizations, as well as by chlorination, cross-linking, epoxidation, hydrox-ylation, and carboxhydrox-ylation, reactions yield further function-alized PHAs.

Keywords Chemical modifications of PHAs . Copolyesters . Microbial polyesters .

Poly(3-hydroxyalkanoate)s . PHA . PHA constituents

Introduction

Bacterial polyesters—also referred to as microbial poly-esters and poly(3-hydroxyalkanoate)s, PHAs (Fig. 1)—are

stored as intracellular granules as a result of a metabolic stress upon imbalanced growth due to a limited supply of an essential nutrient and the presence of an excess of a carbon source (Lenz and Marchessault 2005; Lenz 1993; Doi 1990; Sudesh et al. 2000; Sudesh and Doi 2005; Steinbüchel and Füchtenbusch 1998; Steinbüchel and Valentin 1995; Steinbüchel1991).

Two types of PHAs according to the length of the side chain are distinguished. One type is consisting of short-chain-length hydroxyalkanoic acids, sclPHA, with an alkyl side chain having up to two carbon atoms that are produced by Ralstonia eutropha (Lenz and Marchessault 2005) and many other bacteria. The second type is consisting of medium chain length hydroxyalkanoic acids, mclPHA, with an alkyl side chain consisting of at least three carbon atoms that are produced by Pseudomonas oleovorans and other pseudomonads sensu strictu (Timm and Steinbüchel1990). Typical members of the sclPHAs are hydroxybuty-rate), PHB; hydroxyvalehydroxybuty-rate), PHV; and poly(3-hydroxybutyrate-co-3-hydroxyvalerate), PHBV copolymer. Typical members of mclPHAs are poly(3-hydroxyocta-noate), PHO, and poly(3-hydroxynonapoly(3-hydroxyocta-noate), PHN, that are mostly formed as copolymers of 3-hydroxyoctanoate or 3-hydroxynonanoate together with 3-hydroxyhexanoate, HHx; 3-hydroxyheptanoate and/or 3-hydroxydecanoate (Gross et al.1989).

Some sclPHAs may be too rigid and brittle and may lack the superior mechanical properties required for biomedical and packaging film applications. In contrast, mclPHAs may be elastomeric but have very low mechanical strength. Therefore, for packaging materials, biomedical applica-B. Hazer (*)

Department of Chemistry, Zonguldak Karaelmas University, Zonguldak 67100, Turkey

e-mail: bhazer2@yahoo.com A. Steinbüchel

Institut für Molekulare Mikrobiologie und Biotechnologie, Westfälische Wilhelms–Universität Münster,

Corrensstrasse 3, 48149 Münster, Germany e-mail: steinbu@uni-muenster.de

tions, tissue engineering, and other specific applications, the physical and mechanical properties of microbial poly-esters need to be diversified and improved (Grassie et al.

1984; Hrabak1992; Hazer2002,2003).

Owing to the low substrate specificity of PHA synthases that represent the key enzymes for PHA biosynthesis, the variability of bacterial PHAs that can be directly produced by fermentation is extraordinary large with about 150 different hydroxyalkanoic acids, and even mercaptoalka-noic acid, that are meanwhile known as constituents of these PHAs (Steinbüchel and Valentin1995). By choosing an appropriate production strain as well as suitable cultivation conditions and carbon sources, PHA with tailor-made compositions can be produced. The variability of available PHAs can even be extended if the biologically produced PHAs are subjected to chemical modification. This review aims at pointing to the large variability of PHAs regarding chemical structure and material properties that can be currently produced.

Variability of bacterial systems for PHAs

In the hot drawing process of PHB, one major problem is the rapid thermal degradation of the polymer near its melting temperature (Grassie et al.1984). The physical and thermal properties of PHB can be regulated by in vivo and in vitro copolymerization with other hydroxyalkanoic acids (Hrabak

1992). PHA accumulation as an energy reserve in a bacterium cell is called in vivo polymerization. In vitro polymerization systems in aqueous solutions have been established using purified PHA synthases from R. eutropha (Gerngross and Martin1995) and from Chromatium vinosum (Jossek et al.1998) for bioynthesis of PHAs. Both systems can be used to prepare new types of homopolymers, block copolymers, and random copolymers. Such studies indicate that the in vitro polymerization processes are chain growth polymerization reactions that can form living polymers (Kamachi et al.2001) and can provide greater insights into the control of substrate specificity and reaction kinetics within PHA accumulating cells (Zhang et al.2004).

sclPHA copolymers

PHBV copolymers are produced from Alcaligenes latus, Bacillus cereus, P. pseudoflava, P. cepacia, Micrococcus

halodenitrificans, and R. eutropha when cells are supplied with glucose (or sucrose in case of A. latus) and propionic acid or other propionogenic carbon sources under nitrogen-limited conditions (Ramsay et al.1990). The mixture of the substrates can also produce either PHB homopolymer or PHBV copolymer depending on their concentrations (Koçer et al. 2003). Broad chemical composition of the PHA copolymer and copolymers obtained by a pulse feeding substrate may affect the final material properties. For example, a PHBV copolyester with randomly distributed hydroxyvalerate units (12 mol% HV) showed a single melting peak, whereas samples with nonrandom composi-tion distribucomposi-tion showed multiple melting peaks in their thermograms (Zagar et al. 2006). Periodic substrate additions yield block copolymers and are, therefore, useful for controlling polymer synthesis with specific and desired properties (Pederson et al.2006). Drawing by the stretching gave highly ordered structures in PHBV high-strength fibers (Tanaka et al.2006).

Gene recombination

Many genes related to PHA biosynthesis from various bacteria have been cloned and were employed in metabolic engineering (Steinbüchel 2001). The obtained recombinant strains can exhibit more favorable features of PHA copoly-mer biosynthesis (Tsuge et al. 2004). Poly(3-hydroxybuty-rate-co-3-hydroxyhexanoate), PHBHx, copolymers with low 3HHx fraction are known as one of the most useful polymers among PHAs because they gave flexible materials, com-pared with PHB and polylactic acid (PLA), and have appropriate mechanical properties for use as films (Doi et al.1995).

A recombinant strain of R. eutropha equipped with ccrSc

and phaC-JAc can synthesize a PHBHx copolymer from

fructose consisting up to 33–49% of copolyester with 1.2– 1.6 mol% of 3HHx fraction (Fukui et al. 2002). Similarly, the recombinant strain of R. eutropha produces the copolymer with 3HHx unit up to 5.1 mol% (Tsuge et al.

2004).

Aeromonas hydrophila is able to synthesize PHBHx copolyesters containing about 15mol% 3HHx when grown in the presence of long chain fatty acids, such as dodecanoate, regardless of growth conditions (Lee et al.

2000a). When gluconate is used as a cosubstrate together

with dodecanoate, the recombinant strain produces PHBHx containing 3–12mol% of 3HHx, depending on the gluco-nate concentration in media (Qiu et al. 2004). The biosynthesis of PHBHx from unrelated carbon sources such as gluconate or glucose using recombinant strains of A. hydrophila and P. putida was also demonstrated (Qiu et al. 2005). The original copolymer is a mixture of copoly-mers with different comonomer unit compositions when the

O

[ OCHCH

C ]

R

Fig. 1 General chemical struc-ture of PHAs. In this strucstruc-ture, R is an alkyl side chain of natu-rally occurring PHAs depending on the substrates and the type of the bacteria

copolymer is fractionated using a solvent (chloroform) and a nonsolvent (n-heptane) at ambient temperature (Feng et al.2004).

A recombinant strain of P. putida accumulated P<3HB-co-3HHx-co-4HV) copolyester (Schmack et al. 1998). Copo-lyesters containing 4-hydroxybutyrate units P<3HB-co-4HB) can be produced by R. eutropha fromγ-butyrolactone alone or from mixtures ofγ-butyrolactone with fructose or butyric acid (Doi et al.1990). Hydrogenophaga pseudoflava is also able to synthesize P<3HB-co-4HB) having a high level of 4HB unit fromγ-butyrolactone (Choi et al.1999). Microbial synthesis of 3HB-based copolyesters containing 4HHx has also been reported (Steinbüchel and Valentin1995; Valentin et al.1994). The chemical structure of copolyester containing 3HB, 3HV, 3HHx, 3HO, and 4HV repeating unit is given in Fig.2.

PHA accumulating bacteria have also been shown to synthesize copolymers containing 3-mercaptopropionate, 3MP; 3-mercaptobutyrate, 3MB; 3-mercaptovalerate, 3MV, in addition to 3HB, representing the first examples of polythioesters, PTE, as a novel class of biopolymers (Lütke-Eversloh et al.2001a,b). PMP, PMB, and PMV homopol-ymers (see Fig.3) were produced employing a recombinant strain of Escherichia coli expressing a nonnatural PTE biosynthesis pathway (Lütke-Eversloh et al.2002).

Tmvalues are 170°C for PMP, 100°C for PMB, and 84°C

for PMV, whereas Tmvalues are 77°C for PHP, 175°C for

PHB, and 112°C for PHV, respectively (Kawada et al.2003; Lütke-Eversloh et al.2002).

One example of unsaturated sclPHAs is poly(3-hydroxy-4-pentenoic acid), PHPE. Burkholderia sp. grown on sucrose-containing mineral salt mediums with phosphate limitation accumulates a blend of two distinct homopoly-esters rather than a copolyester of 3HB and 3HPE (Valentin et al.1999). The fermentation product, a blend of PHB and about 10mol% PHPE, can be purified by removing the THF-soluble PHPE fractions. The PHPE homopolyester compared to PHB and PHV has very low crystallinity and a

lower melting temperature: Tg=−10.8°C and Tm=63°C.

Incorporation of 3HPE in a P<HB-co-HV-co-HPE) ter-polyester can be achieved when Rhodospirillum rubrum is grown on 4-pentenoic acid and on an equimolar mixture of 4-pentenoic acid and n-pentanoic acid (Ulmer et al.1994; Ballistreri et al.1995).

mclPHA copolymers

P. oleovorans can be grown on n-alkanoates and related carbon sources like alkanes to produce mclPHAs that are all copolymers (Gross et al. 1989). The carbon source, n-alkanoate, is converted by β-oxidation to a monomer unit that has two carbons less than the n-alkanoate.

Unsaturated polyesters like poly(3-hydroxy-10-unde-cenoate), PHU, and others are produced by various pseudomonads during cultivation on 10-undecenoic acid (Kim et al.1995), 1-alkenes (Lageveen et al.1988), edible oils (Ashby and Foglia1998; Ballistreri et al.2001), or oily acids (Eggink et al.1993; Hazer et al.1998). The chemostat (continuous process) culture seems to be a very suitable method to produce PHAs with an exact defined monomeric unit composition (Hartmann et al.2006).

PHAs containing arylalkyl substituent groups are obtained from P. oleovorans grown on 5-phenylvaleric acid (Fritzsche et al. 1990a), 7-phenylheptanoic acid, 9-phenyl-nonanoic acid, and 11-phenylundecanoic acid (Hazer et al.

1996). Similarly, a p-tolyl substituent of valeric acid as a

O O O O O

[ OCHCH

C ]

[OCHCH

C ]

[OCHCH

C ]

[ OCHCH

CH

C ]

[ OCHCH

C ]

CH

CH

CH

CH

CH

CH

CH

CH

CH

CH

CH

CH

3HV 3HO 3HB 4HV 3HHx

Fig. 2 Chemical structure of poly(3HV-co-3HO-co-3HB-co-4HV-co-3HHx)

O O O

[SCHCH

C] , [SCHCH

C] , [SCHCH

C]

CH

CH

CH

P3MP P3MB P3MV

Fig. 3 Chemical structure of poly(3-mercaptoalkanoate)s: P3MP, P3MB and P3MV

substrate was used for the production of poly(3-hydroxy-5-p-tolylvalerate) (Curley et al.1996). Tailored biosynthesis of aryl-olefinic mclPHA can be obtained from P. putida grown on the mixture of structurally related substrates (Hartmann et al.2004).

The nitro phenyl derivative of the polyester, poly(3-hydroxy-5-p-nitrophenylvalerate) (Arestogui et al.1999) is an amorphous polymer having a Tgof 28°C. PHAs bearing

phenoxy groups in the side chains were produced by P. oleovorans cultivated in the presence of ω-phenoxyalka-noates (Ritter and von Spee1994; Kim et al. 1996,1999,

2000).

Methyl-branched PHAs like poly(3-hydroxy-6-methyl-nonanoate), P(H6MN), were obtained from P. oleovorans after cultivation of the cells on methyl-branched alkanoic acids that were provided either as sole carbon source or as a mixture together with octanoic or nonanoic acid (Fritzsche et al.1990b;İbaoğlu et al. 2000). The crystalline structure of PH6MN is different from that of PHN (Hazer et al.

1994a), and this polymer also crystallizes much faster than

the others and has a Tmhigher than that of PHN (Tm for

PH6MN=65°C and Tm for PHN=58°C). The comparison

of the physical properties of some PHAs with polypropyl-ene was given in Table1.

P. cichorii produces PHAs containing the epoxidized side chain from C7 to C12 1-alkene (Imamura et al.2001).

PHAs containing brominated repeating units are pro-duced by P. oleovorans cultivated on mixtures of ω-bromoalkanoic acids and either nonanoic or octanoic acid (Kim et al. 1992). A copolyester of 3-hydroxy-6-chloro-hexanoate Cl-Hx), 3-hydroxy-8-chlorooctanoate (3H-Cl-O), 3HHx and 3HO units was synthesized in P. oleovorans when octane and 1-chlorooctane were used as carbon sources (Shah et al.2000).

Hydroxyl-terminated P(3HB-co-4HB) can be produced using glucose and sodium 4-hydroxybutyrate as carbon sources in the presence of ethylene glycol (Steinbüchel

2001).

Chemical modifications of PHAs

The chemical modification of PHAs involves grafting reactions and graft (b)/block (g) copolymerization, chlori-nation, cross-linking, epoxidation, and hydroxyl and car-boxylic acid functionalization of the PHAs.

Block/graft copolymers of PHAs

Insertions of an additional different polymer segment into an existing polymer backbone or at the side chain of an existing polymer yields block or graft copolymers,

respec-Table 1 The comparison of the physical properties of the poly(3-/4-hydroxyalkanoate)s with polypropylene (PP)

Polyester Tg(°C) Tm(°C) Crystallinity Elongation

at break (%)

Reference

P3HB 15 175 50–80 5 Doi1990

P3HV −15, 0 110, 112, 118 56 Choi et al.1999; Schmack

et al.1998

P3HB-co-20mol%3HV −1 145 50 Sudesh et al.2000

P3HB-co-10mol%3HHx −1 127 Doi et al.1995

P3HB-co-17mol%3HHx −2 120 850 Doi et al.1995

P3HB-co-47mol% 3HV-co-16mol% 4HV-co-15mol%3HHx-co-2mol%3HO

−15 118 1,000 Schmack et al.1998

P3MB 8 100 Kawada et al.2003

P4HB −40 53 1,000 Choi et al.1999

P(3HB-co-16%4HB) 43 444 Doi et al.1990

P3HPE −11 63 Ulmer et al.1994; Valentin

et al.1999

PHO −36 59, 61 Gross et al.1989; Hazer et al.

1994a,b

PHN −39, −29 48, 54, 58

P3H6MN Not

determined

65 Hazer et al.1994a,b

PH-p5TV 17 95 Curley et al.1996

PH6PHx 4 Not determined Hazer et al.1996

PH5PoxV 14 Not determined Ritter and von Spee1994

PH-p-nitroPV 28 Not determined Arestogui et al.1999

PH8-pMPoxO 14 97 Kim et al.1999

PP −15 176 50 400 Sudesh et al.2000

tively (Hazer 1989, 1996a, 1997; Nuyken and Weidner

1986). Such reactions are important to obtain new poly-meric materials. We have separated copolymerization reactions in two parts: (1) copolymerization based on esterification or urethane formation and (2) free radical graft copolymerization.

(1) Copolymerization based on esterification or urethane formation

PEO-b-PHB-b-PEO triblock copolymers: Amphiphilic block copolymers have attracted special attention in both fundamental and applied research because of their unique chain architecture and physical properties (Förster and Antonietti1998). Poly(ethylene oxide) (PEO) as a hydro-philic and biocompatible polyether is widely used in biomedical applications (Kukula et al.2002). An amphiphilic triblock copolymer (Li et al.2003,2005a) can be synthesized by coupling two chains of methoxy-PEO-monocarboxylic acid with a low-molecular-weight telechelic hydroxylated PHB (PHB-diol) chain in the presence of 1,3-N,N′-dicyclo-hexylcarbodiimid. The PHB-diol is obtained by the trans-esterification reactions of PHB with diethylene glycol (Andrade et al.2002; Hirt et al.1996).

PHB-B-PEO Diblock Copolymers: Aliphatic polyesters coupled with monomethoxy poly(ethyleneoxide), mPEO, are often used as drug delivery systems, and many applications in this field involve polylactides (PLA) coupled to mPEO because of the biodegradability and biocompatibility of PLA (Zhu et al. 1989). Similarly, because PHB is also a chiral aliphatic polyester, PHB-b-mPEO diblock copolymer can be synthesized. For this, PHB and poly(ethylene glycol) methyl ether are melted under vacuum at 190°C in the presence of bis(2-ethyl-hexanoate) tin catalyst (Ravenelle and Marchessault2003). PHA-g-chitosan and PHB-g-cellulose graft copolymers: The terminal carboxyl groups of PHAs are open to react with the chitosan amine functions and the cellulose hydroxyl functions (Yalpani et al.1991). For the synthesis of PHB-g-chitosan graft copolymers, chitosan solutions in dilute acetic acid are treated with different molar ratios of reduced-molecular-weight PHB. The partially polymerized PHB samples can be prepared either in situ or before use by dissolving the PHB in a mixture (1:50, v/v) of acetic acid– DMSO and stirring 16 h at ambient temperature (Yalpani et

al.1991). Although neither of the parent polymers is water soluble, the PHA-chitosan derivatives form opaque, viscous solutions in water. Upon the drying of such solutions, strong elastic films can be prepared. Tmof PHB shifts from

175 to about 150°C for PHB-g-chitosan. At the same time, the endotherm of chitosan also decreased from 116 to 105°C. Similarly, PHO is grafted onto the chitosan via condensation reactions between carboxylic acids and amine groups (Arslan et al. 2006). The plasticizer effect of PHO in PHO-g-chitosan lowered the Tmof the graft copolymer up

to 80°C depending on its PHO content. Similarly, PHB can be linked to a cellulose backbone via transesterification between cellulose acetate and hydrolyzed PHB in acetic acid–DMSO at room temperature (Yalpani et al.1991). The graft copolyester has a melt transition at 181°C.

PHB-b-PEO block copolyurethanes: PHB/PEO block copolyurethanes are prepared by a chain extension reaction between PHB-diol, PEO, and a diisocyanate (Li et al.

2005b) according to the reaction scheme shown in Fig.4.

Telechelic PHB-diol can be obtained by a transesterifi-cation reaction of PHB with diethylene glycol in the presence of p-toluene sulfonic acid as catalyst. The proper choice of the composition of the copolymers (input ratio of hard segment, PHB-diol, and soft segments, polycaprolac-tone-diol and polybutylene succinate-diol), as well as the type of macrodiols used, allows the production of a variety of different materials possessing a wide range of thermal and mechanical properties (Saad2001). PHB and PEO can also form miscible blends over the whole composition. The crystallinity of PEO segments has been greatly hindered by the presence of the PHB component. Thus, a combination of the linear, crystalline PHB with the flexible cross-linked PHB may produce a new type of hydrogel having desirable mechanical properties. The preparation of PHB/cross-linked PEO semi-interpenetrating networks (IPNs) can be per-formed under UV irradiation, owing to the photoactive nature of the terminal acrylate groups (Hao and Deng

2001). IPNs have tensile strength values varying from 2.5-8.5 MPa and have elongation at break values varying from 3.8 to 35.5%.

PHO-g-PEO graft copolymers: PHO and PHN are elastomeric mclPHAs that are better suited for biomedical applications. To make mclPHAs the material of choice in the biomedical field, the hydrophilicity of these polymers

PHB-diol + HO[CH2CH2O]zH + 2 OCNCH2CH2CH2CH2CH2CH2NCO

PEO Hexamethylene diisocyanate, HDI

O O O

HO [PHB-O]m[CNH(CH2)6NHC]pO [ POE-O]nCNH(CH2)6NCO

Poly (PHB-b-PEO) block co-polyurethane Fig. 4 Chemical synthesis of

poly(PHB-b-PEO) block co-polyurethane

needs to be tailored to suit specific applications. Mono-acrylate-PEO can be grafted onto PHO by UV irradiation in a chloroform solution containing benzoyl peroxide. UV irradiation treatment generates free radicals that can undergo cross-linking (Kim et al.2005). Water uptake of the PHO-g-PEO copolymer increases up to 30% in comparison with the water uptake of PHO that is only 2%. Additionally, PEO blocks also affect the protein adsorption. The amounts of protein adsorbed onto the PHO-g-PEO copolymers decrease as the degree of grafting of PEG onto PHO increases. This means that the adsorption of proteins is suppressed by the increased surface hydrophilicity.

PHO-b-PCL diblock copolymer: PHO oligomers can be obtained by methanolysis (acidic hydrolysis after esterifi-cation with methanol) with varying molecular weights from 800 to 20,000 Da (Timbart et al. 2004). The terminal hydroxyl groups of the PHO oligomers can be activated by the catalyst Et3Al to initiate the ring opening

polymeriza-tion of ε-caprolactone. The PHO segment is the soft segment, and the poly(ε-caprolactone) (PCL) segment is the hard segment in a PHO-b-PCL semicrystalline diblock copolymer. The copolymer has two glass transitions at−63 and −41°C corresponding to PCL and PHO, respectively, whereas only one Tmrelated to PCL segment at about 60°C

is observed. Table 2 lists PHA block/graft copolymers obtained by esterification or urethane formation.

(2) Free radical grafting reactions of PHAs

Macroazo initiators (MAI) based on PEO, PHB, PHBV, PHO, PHN, PHU, or PHA-soya graft copolymers of poly (methylmethacrylate) (PMMA), polystyrene (PS), polyiso-prene (PI), polyacrylamide (PAAm), and poly(acrylic acid) (PAA) are discussed in this section (Table 3). The vinyl polymers discussed in this part give PHAs some improved mechanical and film properties (PMMA, PS, PI) and hydrophilic properties (PAAm, PAA).

PEO-g-mclPHA with unsaturated side chains: MAI based on PEO (Hazer et al. 1994b; Hazer 1985, 1991; Walz et al. 1977; Laverty and Gardlund 1977) can be obtained from the reaction of PEO and 4,4′-azo-bis(4-cyano

pentanoyl chloride). Polyazo esters can also be obtained by the reaction of 2,2′-azo-bis(isobutyronitrile) and PEO via Pinner synthesis (Walz et al.1977). Polyazo esters produce free radicals at elevated temperatures, and PEO radicals attack the double bonds of unsaturated side chains of mclPHAs that were produced from anchovy (hamci) oily acids (Hazer et al. 1999). When the proportion of the unsaturated PHA side chains is higher than 5%, very large network structures occur during grafting reaction. The hydrophilicity coming from PEO segments can also be observed. By this, another PEO graft copolymer network of a PHA containing unsaturated side chains, PHU-g-PEO net-works, (Chung et al. 2003) can be prepared by irradiating homogeneous solutions of poly(3-hydroxyundecenoate) (PHU) and the monoacrylate of PEO with UV light. By this way, the surfaces and the bulk of the graft copolymer can be made more hydrophilic as the PEO grafting density in the polymer network increases. For a graft copolymer having 50:50% (w/w) of PHU/PEO, tensile strength is 219 kPa and elongation at break is 379%, whereas these values are 462 kPa and 621% for homo cross-linked PHU, respectively. These amphiphilic graft copolymers have the potential to be used as blood-contacting devices in a broad range of biomedical applications because of their excellent blood compatibilities (Chung et al.2003).

PHB-g-PI graft copolymers: PI is a tough polymer and also used in the field of medicine (Jiang and Hu2001). To improve the tenacity of PHB, isoprene is grafted onto PHB by directly irradiating PHB immersed in isoprene solution. Heptane is the most favorable solvent in this reaction. PHB-g-PI has much better ductility and tenacity than homo-PHB. The elongation at break of the graft copolymer reached values up to 17.2%, whereas that of the used homo-PHB was only 8.2% (Jiang and Hu2001).

PHBV-g-PAA graft copolymers: Graft copolymerization of acrylic acid (AAc) onto PHBV can be achieved by gamma-irradiation to induce surface hydrophilicity for biomedical applications (Grondahl et al.2005). The PHBV film samples are irradiated in AAc/methanol solution to prepare PAAc grafted onto PHBV films for tissue engi-neering applications.

PHA graft/block copolymers PEO-b-PHB-b-PEO triblock

copolymer

PHB-b-PEO diblock copolymer PHA-g-Chitosan graft copolymer PHA-g-Cellulose graft copolymer PHB-b-PEO block copolyurethane

PHO-g-PEO graft copolymer PHO-b-PCL diblock copolymer Table 2 List of PHA graft/

block copolymers obtained by esterification or urethane formation

Table 3 Free radical grafting reactions on PHAs Grafting reactions Product

1. PS (or PMMA) peroxides+PHN PS-g-PHN, PMMA-g-PHN 2. PHA-soya +MMA+Bz2O2 PMMA-g-PHA

3. PHA-soya +MAI PHA-g-PEO 4. PHA+vinyl monomer

(Irradiation)

PMMA-g-PHN, PHB-g-PI, PHBV-g-PAA, PHO-g-PAAm For abbreviations, see text.

PHO-g-PAAm graft copolymers: PAAm is highly hydro-philic and forms hydrogels that have good properties for use in biomedical materials that play important roles in cell adhesion, spreading, and growth (Kim et al.2002). For this, a PHO film is treated with plasma and then treated with an acryl amide solution to prepare films with surfaces that contained different amounts of amide groups.

PHN (or PHA-soya)-g-PMMA (or PS) graft copolymers: When a PHN solution of MMA is exposed to high energy irradiation, PHN-g-PMMA graft copolymers occur depend-ing on the location of the radical sites on the polymer chain (Eroğlu et al.1998). Double bonds of unsaturated microbial polyester participate in the free radical polymerization of methyl methacrylate initiated by benzoyl peroxide to obtain PHA-soya-g-PMMA graft copolymers (Ilter et al. 2001). Oligoperoxides are useful free radical initiators in vinyl polymerization to obtain active polymers having peroxide groups that can be used as initiator in the polymerization of another vinyl monomer to obtain block copolymers (Hazer and Baysal1986; Hazer and Kurt 1995). Chain extension reaction between a diacid chloride and a dihydroperoxide (or sodium peroxide) gives oligoperoxide containing 6 to 12 peroxide groups (Hazer 1987). The active polymer having undecomposed peroxide groups in the backbone is obtained when an oligoperoxide initiates a vinyl polymer-ization (Hazer 1995). Because of the undecomposed peroxide groups in the backbone, active polymers thermally produce polymer radicals at moderate temperatures of about 80–100°C. When a mixture of active PS (or active PMMA) and PHA-soya is thermally cured, PS radicals (or PMMA radicals) formed attack the double bonds of the PHA-soya, yielding a PS-g-PHA-soya (or PMMA-g-PHA-soya) graft copolymer (Cakmakli et al.2001). Thermal curing of PHN as a member of the saturated PHA yields PS (or PMMA)-g-PHN graft copolymers (Hazer1994). The elastomeric PS-g-PHN graft copolymers that were obtained by this method reached an elongation at a break value of 1,430% and a tensile strength of 4.84 N/mm2. In contrast, PMMA-g-PHN graft copolymers reached an elongation at a break value of only 23%, whereas the tensile strength value of 23 N/mm2 was much higher (Hazer1994,1996b). Table2summarizes the PS (or PMMA) grafting reactions on PHAs.

Functionalization of PHAs

PHA functionalization is considered as derivatization via epoxidation, cross-linking, carboxylation, chlorination, or attachment of vinyl groups.

PHB macromonomers The thermal degradation of PHB leads to PHB oligomers having terminal crotonate groups

with molecular weights of about 2,000 Da. PHB macro-monomers can be obtained by the esterification of the PHB oligomers at their carboxylic ends with hydroxyethyl methacrylate (Nguyen and Marchessault2004). Therefore, the products contain two terminal unsaturated groups.

Maleation of PHB-diol also gives PHB macromonomers (Deng and Hao 2001). PHB-diol oligomers can be produced by alkoholysis of microbial PHB with 1,6-hexanediol into prepolymer, then followed by selective maleation of the chain-end group. Thus, this macromer contains a double bond in addition to hydroxyl groups at the two respective chain ends (Deng and Hao2001).

Chlorination of PHAs Chlorination of unsaturated PHAs can be carried out by chlorine addition to the double bonds and by substitution reactions with the saturated hydrocarbons (Arkin et al. 2000). By this polymers with higher glass transition (Tg) varying from 2 to 50°C and melting transition (Tm)

varying from 62 to 125°C, the chlorinated PHAs were obtained. Hydrolysis of the PHAs during the chlorination process occurs. The chlorinated PHA derivatives can be converted to their corresponding quaternary ammonium salts, sodium sulfate salts, and phenyl derivatives. Friedel-Crafts reactions between benzene and chlorinated moieties can yield cross-linked polymers (Arkin and Hazer2002).

Cross-linking of PHAs Poly(3-hydroxy octanoate-co-3-hy-droxy undecenoate), PHOU, polymer films containing a peroxide and a multifunctional cross-linker obtained from solvent casting are cured thermally under vacuum to obtain cross-linked PHOU (Gagnon et al.1994a). Cross-linking of PHOU films by gamma-irradiation can be also carried out (Dufresne et al.2001).

A mixture of elemental sulfur or dipentamethylene thiuramhexasulfide as a vulcanizing agent, zinc dibutyl dithiocarbamate as an accelerator, and zinc oxide or stearic acid as an accelerator activator is kneaded into the softened PHOU sample and cured at 140°C (Gagnon et al.1994b). The reactive processing of PHBV with a free radical initiator, such as dicumyl peroxide leads to a branching of the polymeric structure of the material (D’Haene et al.

1999). An increase in the number of branches leads to an increased elasticity of the product.

Unsaturated mclPHAs produced by P. putida from linseed oil fatty acids (LOFA) and tall oil fatty acids (TOFA) can be used as a polymer binder (van der Walle et al. 1999). After TiO2pigment powder is added into the

PHA-TOFA or PHA-LOFA, the pigmented PHA film can be cured under either auto-oxidative or photo-oxidative conditions. PHA-soya produced by P. oleovorans from soybean fatty acids is cross-linked via free radical mecha-nism either thermally or under UV irradiation in the presence of benzoyl peroxide, benzophenon, and/or

ethyl-ene glycol dimethacrylate (Hazer et al.2001). PHA films become cross-linked at around 90% by this way. Chemical cross-linking can also be achieved by an electron-beam irradiation of the unsaturated side chains that were incorporated in this PHA by using 7-octenoic acid as carbon source for biosynthesis (Konig et al. 1994). Even after cross-linking, the material was still biodegradable (Konig et al.1994).

Epoxidation of unsaturated PHAs Epoxidation of the unsaturated side chains in PHOU by reacting the polymer with m-chloroperbenzoic acid (MCPBA) in homogeneous solution can be readily carried out (Park et al.1998a; Bear et al. 1997). The ester forming cross-linking reaction of epoxidized PHOU with succinic acid anhydride in the presence of a base catalyst such as 2-methyl 4-methylimi-dazol proceeds readily at relatively low temperatures (Park et al. 1998b). First, the epoxide group is opened by the attack of the tertiary amine (imidazole) to form an alkoxide ion that reacts with the anhydride to form a monoester carboxylate anion, and then this anion, in turn, reacts with another epoxide group to form the diester cross-link. PHAs obtained from edible oils such as linseed oil (PHA-L) are amorphous and take on the consistency of a viscose liquid at room temperature (Ashby et al.2000). To increase their application potential, the side chain olefinic groups of PHA-L can be partially converted to epoxy derivatives using MCPBA (Ashby et al.2000).

Hydroxylation of unsaturated PHAs PHAs containing pendant diol groups can be prepared by the chemical modification of unsaturated PHAs using KMnO4 in cold

alkaline solution (pH 8–9) at 20°C without a severe reduction in molecular weight (Lee et al. 2000b). The polymers that are 40–60% hydroxylated are completely soluble in polar solvents including an 80:20% acetone/ water mixture, methanol, and DMSO, indicating a consid-erably enhanced hydrophilicity of the modified PHAs. The hydroxylation of the double bonds of PHU can be carried out completely by hydroboration–oxidation reaction using 9-borobicyclononane (Eroğlu et al.2005).

Carboxylation of unsaturated PHAs The unsaturated groups of PHOU can be converted to carboxyl groups via oxidation with KMnO4in the presence of NaHCO3(Konig

et al. 1994). Unsaturated groups of PHOU can also be converted to carboxylic groups using a modified method in which the carboxylation is carried out with KMnO4as the

oxidation agent and a crown ether as the phase transfer and dissociating agent of KMnO4(Lee and Park2000; Kurth et

al.2002). With the use of osmium tetroxide and oxone, and potassium peroxomonosulfate (a mixture of 2KHSO5,

K2SO4, and KHSO4) to convert double bonds of PHOU

to carboxylic groups, the oxidation proceeds to completion with little backbone degradation (Stigers and Tew2003).

PHOU provides options for producing a variety of derivatives through reaction at the double bonds, for example, conversion of double bonds to thioethers via the free radical addition of 11-mercaptoundecanoic acid (Hany et al. 2004). The molecular weight remains essentially constant upon addition of 11-mercaptoundecanoic acid to the double bonds.

Sugar grafted PHAs Glycopolymers are emerging as a novel class of neoglycoconjugates useful for biological studies (Constantin et al.1999). For this purpose, the thiol sugar per-O-acetyl-1thio-β-maltose is added to the unsaturated side chains of PHU via anti-Markovnikov addition without any side reactions. This amphiphilic graft copolymer can also be prepared by the reaction between per-o-acetyl-thio- β-maltose and poly(11-bromo-3-hydroxyhexanoate) obtained from P. oleovorans grown on 11-bromo undec-anoic acid (Kim et al.1992).

Conclusions

Biosynthetic efforts have produced a variety of copolyes-ters, with the greatest majority containing nonpolar side chains. These polyesters are substantially more hydropho-bic than other polyesters such as poly(glycolic acid) (Stigers and Tew 2003). In contrast, amphiphilic PHA copolymers find interesting applications in drug delivery and tissue engineering (Li et al. 2003). Chemical modifi-cation reactions make it easier to insert hydrophilic seg-ments into the hydrophobic PHAs and to produce amphiphilic copolymers. The various naturally occuring microbial polyesters can be further diversified via both chemical modification reactions and the engineering of the biosynthetic pathways. One main purpose of the PHA modification reactions is to obtain more flexible or even rubber-like materials. The engineering of metabolic path-ways were successful for the biosynthesis of the elastomeric materials as represented by PHB-copolymers containing hydroxyvalerate, hydroxyhexanoate, and/or 4-hydroxybu-tyrate units. The chemical modification reactions of PHAs with synthetic commercial olefinic polymers gave elasto-plastic microbial polyester synthetic polymer–polymer composites for industrial applications that may be useful as environmentally friendly polymers. Another important aim of chemical modifications is to increase the hydro-philicity of PHAs for certain medical applications. For example, amphiphilic graft copolymers have the potential to be used as blood-contacting devices in a broad range of biomedical applications because of their excellent blood compatibilities. During biosynthetic production of PHAs,

the PHA synthases are highly selective to the substrate, and the bacteria produce only hydrophobic PHAs. Chemical modification of biosynthetic PHAs is also important in the materials research area to produce new polymer composites having different thermal and mechanical properties. Chem-ical modification is a promising approach to obtain new types of PHA-composite materials including a wide range of monomers for graft/block copolymerization with syn-thetic and other natural polymers that cannot be obtained by biotechnological processes.

Acknowledgment This work was financially supported by TUBITAK (Turkey) grant no. 104M128. Studies of A.S. were supported by the grant provided by the Deutsche Forschungsgemeinschaft in the past.

Glossary of PHAs

mclPHA medium chain length poly(3-hydroxyalkanoic acid) PHA polyhydroxyalkanoates PHB poly(3-hydroxybutyrate) PHBV poly(3-hydroxybutyrate-co-3-hydroxyvalerate) PHBHp poly(3-hydroxybutyrate-co-3-hydroxyheptanoate PHBHx poly(3-hydroxybutyrate-co-3-hydroxyhexanoate) PHD poly(3-hydroxydecanoate) PHN poly(3-hydroxynonanoate) PHO poly(3-hydroxyoctanoate)

PHOU poly(3-hydroxy octanoate-co-3-hydroxy undecenoate)

PHPE poly(3-hydroxy-4-pentenoic acid) PHU poly(3-hydroxy-10-undecenoate)

PHV poly(3-hydroxyvalerate)

PH5PoxV poly(3-hydroxy-5-phenoxy valerate) PH6PHx poly(3-hydroxy-6-phenyl hexanoate) PH8-pMPoxO poly(3-hydroxy-p-methylphenoxy octanoate) PH-p-nitroPV poly(3-hydroxy-p-nitrophenyl valerate) PH-p5TV poly(3-hydroxy-p-tolyl valerate) P(H6MN) poly(3-hydroxy-6-methylnonanoate) sclPHA short chain length poly(3-hydroxyalkanoic

acid)

3MB 3-mercaptobutyrate

3MP 3-mercaptopropionate

3MV 3-mercaptovalerate

References

Andrade AP, Witholt B, Hany R, Egli T, Li Z (2002) Preparation and characterization of enantiomerically pure telechelic diols from mcl-poly[(R)-3-hydroxyalkanoates]. Macromolecules 35:684–689

Arestogui SM, Aponte MA, Diaz E, Schröder E (1999) Bacterial polyesters produced by Pseudomonas oleovorans containing nitrophenyl groups. Macromolecules 32:2889–2895

Arkin AH, Hazer B (2002) Chemical modification of chlorinated microbial polyesters. Biomacromolecules 3:1327–1335 Arkin AH, Hazer B, Borcakli M (2000) Chlorination of

poly-3-hydroxy alkanoates containing unsaturated side chains. Macromolecules 33:3219–3223

Arslan H, Hazer B, Yoon SC (2006) Grafting of poly(3-hydroxyalka-noate) and linoleic acid onto chitosan. J Appl Polym Sci (in press)

Ashby RD, Foglia TA (1998) Poly(hydroxyalkanoate) biosynthesis from trigliceride substrates. Appl Microbiol Biotechnol 49:431– 437

Ashby RD, Foglia TA, Solaiman DKY, Liu CK, Nunez A, Eggink G (2000) Viscoelastic properties of linseed oil-based medium chain length poly(hydroxyalkanoate) films: effects of epoxidation and curing. Int J Biol Macromol 27:355–361

Ballistreri A, Montauda G, Impallomeni G, Lenz RW, Ulmer HW, Fuller RC (1995) Synthesis and characterization of polyesters produced by Rhodospirillum rubrum from pentenoic acid. Macromolecules 28:3664–3671

Ballistreri A, Giuffrida M, Guglielmino SPP, Carnazza S, Ferreri A, Impallomeni G (2001) Biosynthesis and structural characteriza-tion of medium-chain-length poly(3-hydroxyalkanoates) pro-duced by Pseudomonas aeruginosa from fatty acids. Int J Biol Macromol 29:107–114

Bear MM, Leboucher-Durand MA, Langlois V, Lenz RW, Goodwin S, Guerin P (1997) Bacterial poly-3-hydroxyalkenoates with epoxy groups in the side chains. React Funct Polym 34:65–77 Cakmakli B, Hazer B, Borcakli M (2001) Polystyreneperoxide and

poly(methyl methacrylate) peroxide for grafting on unsaturated bacterial polyesters. Macromol Biosci 1:348–354

Choi MH, Yoon SC, Lenz RW (1999) Production of poly(3-hydroxy-butyric acid-co-4-hydroxypoly(3-hydroxy-butyric acid) and poly(4-hydroxypoly(3-hydroxy-butyric acid) without subsequent degradation by Hydrogenophage pseudo-flava. Appl Environ Microbiol 65:1570–1577

Chung CW, Kim HW, Kim YB, Rhee YH (2003) Poly(ethylene glycol)-grafted poly(3-hydroxyundecenoate) networks for en-hanced blood compatibility. Int J Biol Macromol 32:17–22 Constantin M, Simionescu CI, Carpov A, Samain E, Driguez H (1999)

Chemical modification of poly(hydroxyalkanoates) copolymers bearing pendant sugars. Macromol Rapid Commun 20:91–94 Curley JM, Hazer B, Lenz RW, Fuller RC (1996) Production of poly

(3-hydroxyalkanoates) containing aromatic substituents by Pseu-domonas oleovorans. Macromolecules 29:1762–1766

Deng XM, Hao JY (2001) Synthesis and characterization of poly(3-hydroxybutyrate) macromer of bacterial origin. Eur Polym J 37:211–214

D’Haene P, Remsen EE, Asrar J (1999) Preparation and characteriza-tion of a branched bacterial polyester. Macromolecules 32:5229– 5235

Doi Y (1990) Microbial polyesters. VCH, New York

Doi Y, Segawa A, Kunioka M (1990) Biosynthesis and characteriza-tion of poly(3-hydroxybutyrate-co-4-hydroxybutyrate) in Alcali-genes eutrophus. Int J Biol Macromol 12:106–111

Doi Y, Kitamura S, Abe H (1995) Microbial synthesis and characterization of poly(3-hydroxybutyrate-co-3-hydroxyhexa-noate). Macromolecules 28:4822–4828

Dufresne A, Reche L, Marchessault RH, Lacroix M (2001) Gamma-ray crosslinking of poly(hydroxyoctanoate-co-undecenoate). Int J Biol Macromol 29:73–82

Eggink G, van der Wal H, Huijberts GNM, de Waard P (1993) Oleic acid as substrate for poly-3-hydroxyalkanoate formation in Alcaligenes eutrophus and Pseudomonas putida. Ind Crops Prod 1:157–163

Eroğlu MS, Çaykara T, Hazer B (1998) Gamma rays induced grafting of methyl methacrylate onto poly(β-hydroxynonanoate). Polym Bull 41:53–60

Eroğlu MS, Hazer B, Ozturk T, Caykara T (2005) Hydroxylation of pendant vinyl groups of poly(3-hydroxy undec-10-enoate) in high yield. J Appl Polym Sci 97:2132–2139

Feng L, Yoshie N, Asakawa N, Inoue Y (2004) Comonomer-unit compositions, physical properties and biodegradability of bacte-rial copolyhydroxyalkanoates. Macromol Biosci 4:186–198 Förster S, Antonietti M (1998) Amphiphilic block copolymers in

structure-controlled nanomaterial hybrids. Adv Mater 10:195– 217

Fritzsche K, Lenz RW, Fuller RC (1990a) An unusual bacterial polyester with a phenyl pendant group. Makromol Chem 191:1957–1965

Fritzsche K, Lenz RW, Fuller RC (1990b) Bacterial polyesters containing branched poly(β-hydroxyalkanoate) units. Int J Biol Macromol 12:92–101

Fukui T, Abe H, Doi Y (2002) Engineering of Ralstonia eutropha for production of poly(3-hydroxybutyrate-co-3-hydroxyhexanoate) from fructose and solid-state propserties of the copolymer. Biomacromolecules 3:618–624

Gagnon KD, Lenz RW, Farris RJ, Fuller RC (1994a) Chemical modification of bacterial elastomers: 1. Peroxide crosslinking. Polymer 35:4358–4367

Gagnon KD, Lenz RW, Farris RJ, Fuller RC (1994b) Chemical modification of bacterial elastomers: 2. Sulfur vulcanization. Polymer 35:4368–4375

Gerngross TU, Martin DP (1995) Enzyme-catalyzed synthesis of poly [(R)-(−)-3-hydroxybutyrate]: formation of macroscopic granules in vitro. Proc Natl Acad Sci USA 92:6279–6283

Grassie N, Murray EJ, Holmes PA (1984) The thermal degradation of poly(-(D)-β-hydroxybutyric acid): part 3—the reaction mecha-nism. Polym Degrad Stab 6:127–134

Grondahl L, Chandler-Temple A, Trau M (2005) Polymeric grafting of acrylic acid onto poly(3-hydroxybutyrate-co-valerate): surface func-tionalization for tissue engineering applications. Biomacromolecules 6:2197–2203

Gross RA, DeMello C, Lenz RW, Brandle H, Fuller RC (1989) Biosynthesis and characterization of poly(β-hydroxyalkanoates) produced by Pseudomonas oleovorans. Macromolecules 22:1106–1115

Hany R, Böhlen C, Geiger T, Hartmann R, Kawada J, Schimid M, Zinn M, Marchessault RH (2004) Chemical synthesis of crystalline comb polymers from olefinic medium-chain-length poly(3-hydroxyalkanoates). Macromolecules 37:385–389 Hao J, Deng X (2001) Semi-interpenetrating networks of bacterial

poly(3-hydroxybutyrate) with net-poly(ethylene glycol). Polymer 42:4091–4097

Hartmann R, Hany R, Geiger T, Egli T, Witholt B, Zinn M (2004) Tailored biosynthesis of olefinic medium-chain-length poly[(R)-3-hydroxyalkanoates] in Pseudomonas putida Gpo1 with im-proved thermal properties. Macromolecules 37:6780–6785 Hartmann R, Hany R, Pletscher E, Ritter A, Witholt B, Zinn M (2006)

Tailor-made olefinic medium-chain-length poly[(R)-3-hydroxyal-kanoates] by Pseudomonas putida Gpo1: batch versus chemostat production. Biotechnol Bioeng 93:737–746

Hazer B (1985) Multiblock copolymers by polymeric initiators via free radical mechanism. Angew Makromol Chem 129:31–41 Hazer B (1987) Polymerization of vinyl monomers by a new

oligoperoxide. Oligo (adipoyl 5-peroxy 2,5-dimethyl n hexyl) peroxide). J Polym Sci Polym Chem Ed 25:3349–3354 Hazer B (1989) Synthesis and characterization of block copolymers.

In: Cheremisinoff NP (ed) Handbook of polymer science and engineering, vol 1. Marcel Dekker, New York, pp 133–176

Hazer B (1991) Synthesis of PS-PEG and PMMA-PEG branched block copolymers by macroinimers. J Macromol Sci Pure Appl Chem A28:47–52

Hazer B (1994) Preparation of polystyrene-poly(β-hydroxynonanoate) graft copolymers. Polym Bull 33:431–438

Hazer B (1995) Grafting reactions onto polymer backbone with polymeric initiator. J Macromol Sci Pure Appl Chem A32 (5, 6):679–685

Hazer B (1996a) Macromonomeric initiators. In: Salamone JC (ed) Polymeric materials encyclopedia, vol 6. CRC Press, Boca Raton, pp 3911–3918

Hazer B (1996b) Poly(β-hydroxynonanoate) and polystyrene or poly (methylmethacrylate) graft copolymers : microstructure charac-teristics and mechanical and thermal behavior. Macromol Chem Phys 197:431–441

Hazer B (1997) Macrointermediates for block and graft copolymers. In: Cheremisinoff NP (ed) Handbook of engineering polymeric materials. Marcel Dekker, New York, pp 725–734

Hazer B (2002) Chemical modification of bacterial polyester. Curr Trends Polym Sci 7:131–138

Hazer B (2003) Chemical modification of synthetic and biosynthetic polyesters. In: Steinbüchel A (ed) Biopolymers, vol 10. Wiley– VCH, Weinheim, pp 181–208

Hazer B, Baysal BM (1986) Preparation of block copolymers using a new polymeric peroxycarbamate. Polymer 27:961–986 Hazer B, Kurt A (1995) Polymerization kinetics of styrene by

oligododecandioylperoxide, ODDP, and synthesis of poly(sty-rene-g-butadiene) graft copolymers. Eur Polym J 31:449–503 Hazer B, Lenz RW, Fuller RC (1994a) Biosynthesis of methyl

branched poly(β-hydroxy alkanoate)s with Pseudomonas oleo-vorans. Macromolecules 27:45–49

Hazer B, Erdem B, Lenz RW (1994b) Styrene polymerization with some new macro or macromer initiators having PEG units. J Polym Sci A Polym Chem 32:1739–1746

Hazer B, Lenz RW, Fuller RC (1996) Production of some new biopolyesters containing aromatic substituents by either Pseudomo-nas oleovorans or PseudomoPseudomo-nas putida. Polymer 37:5951–5957 Hazer B, Torul O, Borcakli M, Lenz RW, Fuller RC, Goodwin

SD (1998) Bacterial production of polyesters from free fatty acids obtained from natural oils by Pseudomonas oleovorans. J Environ Polym Degrad 6:109–113

Hazer B, Lenz RW, Çakmaklı B, Borcaklı M, Koçer H (1999) Preparation of poly(ethylene glycol) grafted poly(3-hydroxyalka-noate)s. Macromol Chem Phys 200:1903–1907

Hazer B, Demirel SI, Borcakli M, Eroğlu MS, Cakmak M, Erman B (2001) Free radical crosslinking of unsaturated bacterial polyester obtained from soybean oily acids. Polym Bull 46:389–394 Hirt TD, Neuenschwander P, Suter UW (1996) Telechelic diols from

poly[(R)-3-hydroxy-butyric acid] and poly[(R)-3-hydroxybutyric acid-co-[(R)-3-hydroxyvaleric acid]. Macromol Chem Phys 197:1609–1614

Hrabak O (1992) Industrial production of poly-β-hydroxybutyrate. FEMS Microbiol Rev 103:251–256

İbaoğlu K, Hazer B, Arkin AH, Lenz RW (2000) Production of poly-3-hydroxyalkanoates from methyl branched alkanoic acids by Pseudomonas oleovorans. Bulletin of the Chemists and Tech-nologists of Macedonia 19:41–48

Ilter S, Hazer B, Borcakli M, Atici M (2001) Graft copolymerization of methyl methacrylate onto bacterial polyester containing unsaturated side chains. Macromol Chem Phys 202:2281–2286 Imamura T, Kenmoku T, Honma T, Kobayashi S, Yano T (2001)

Direct biosynthesis of poly(3-hydroxyalkanoates) bearing epox-ide groups. Int J Biol Macromol 29:95–301

Jiang T, Hu P (2001) Radiation-induced graft polymerization of isoprene onto polyhydroxybutyrate. Polym J 33:647–653

Jossek R, Reichelt R, Steinbüchel A (1998) In vitro biosynthesis of poly(3-hydroxybutyric acid) by using purified poly(hydroxyalka-noic acid) synthase of Chromatium vinosum. Appl Microbiol Biotechnol 49:258–266

Kamachi M, Zhang S, Goodwin S, Lenz RW (2001) Enzymatic polymerization and characterization of new poly(3-hydroxyalka-noate)s by a bacterial polymerase. Macromolecules 34:6889–6894 Kawada J, Lütke-Eversloh T, Steinbüchel A, Marchessault RH (2003) Physical properties of microbial polythioesters: characterization of poly(3-mercaptoalkanoates) synthesized by engineered Escherichia coli. Biomacromolecules 4:1698–1702

Kim YB, Lenz RW, Fuller RC (1992) Poly(β-hydroxyalkanoate) copolymers containing brominated repeating units produced by Pseudomonas oleovorans. Macromolecules 25:1852–1857 Kim YB, Lenz RW, Fuller RC (1995) Poly-3-hydroxyalkanoates

containing unsaturated repeating units produced by Pseudomonas oleovorans. J Polym Sci A Polym Chem 33:1367–1374 Kim YB, Rhee YH, Han SH, Heo GS, Kim JS (1996)

Poly-3-hydroxyalkanoates produced from Pseudomonas oleovorans grown with ω-phenoxyalkanoates. Macromolecules 29:3432– 3435

Kim YB, Kim DY, Rhee YH (1999) PHAs produced by Pseudomonas putida and Pseudomonas oleovorans grown with n-alkanoic acids containing aromatic groups. Macromolecules 32:6058– 6064

Kim DY, Kim YB, Rhee YH (2000) Evaluation of various carbon substrates for the biosynthesis of polyhydroxyalkanoates bearing functional groups by Pseudomonas putida. Int J Biol Macromol 28:23–29

Kim HW, Chung CW, Kim SS, Kim YB, Rhee YH (2002) Preparation and cell compatibility of acrylamid-grafted poly(3-hydroxyocta-noate). Int J Biol Macromol 30:129–135

Kim HW, Chung CW, Rhee YH (2005) UV-induced graft copolymer-ization of monoacrylate-poly(ethylene glycol) onto poly(3-hydroxyoctanoate) to reduce protein adsorption and platelet adhesion. Int J Biol Macromol 35:47–53

Koçer H, Borcaklı M, Demirel S, Hazer B (2003) Production of bacterial polyesters from some various new substrates by Alcaligenes eutrophus and Pseudomonas oleovorans. Turk J Chem 27:365–373 Konig GJM, van Bilsen HMM, Lemstra PJ, Hazenberg W, Witholt B, Preusting H, van der Galien JG, Schirmer A, Jendrossek D (1994) A biodegradable rubber by crosslinking poly(hydroxyal-kanoate) from Pseudomonas oleovorans. Polymer 35:2090–2097 Kukula H, Schlaad H, Antonietti M, Förster S (2002) The formation of polymer vesicles or “peptosomes” by polybutadiene-block-poly(L-glutamate)s in dilute aqueous solution. J Am Chem Soc 124:1658–1663

Kurth N, Renard E, Brachet F, Robic D, Guerin Ph, Bourbouze R (2002) Poly(3-hydroxyoctanoate) containing pendant carboxylic groups for the preparation of nanoparticles aimed at drug transport and release. Polymer 43:1095–1101

Lageveen RG, Huisman GW, Preusting H, Ketelaar P, Eggink G, Witholt B (1988) Formation of polyesters by Pseudomonas oleovorans: effect of substrates on formation and composition of poly-(R)-hydroxyalkanoates and poly-(R)-3-hydroxyalkenoates. Appl Environ Microbiol 54:2924–2932

Laverty LJ, Gardlund ZG (1977) Poly(vinyl chloride)-poly(ethylene oxide) block copolymers: synthesis and characterization. J Polym Sci A Polym Chem 15:2001–2011

Lee MY, Park WH (2000) Preparation of bacterial copolyesters. Macromol Chem Phys 201:2771–2774

Lee SH, Oh DH, Ahn WS, Lee Y, Choi J, Lee SY (2000a) Production of poly(3-hydroxybutyrate-co-3-hydroxyhexanoate) by high-cell-density cultivation of Aeromonas hydrophila. Biotechnol Bioeng 67:240–244

Lee MY, Park WH, Lenz RW (2000b) Hydrophilic bacterial polyesters modified with pendant hydroxyl groups. Polymer 41:1703–1709 Lenz RW (1993) Biodegradable polymers. Adv Polym Sci 107:1–40 Lenz RW, Marchessault RH (2005) Bacterial polyesters: biosynthesis,

biodegradable plastics and biotechnology. Biomacromolecules 6:1–8

Li J, Li X, Ni X, Leong KW (2003) Synthesis and characterization of new biodegradable amphiphilic poly(ethylene oxide)-b-poly[(R)-3-hydroxy butyrate]-b-poly(ethylene oxide) triblock copolymers. Macromolecules 36:2661–2667

Li J, Ni X, Li X, Tan NK, Lim CT, Ramakrishna S, Leong KW (2005a) Micellization phenomena of biodegradable amphiphilic triblock copolymers consisting of poly(β-hydroxyalkanoic acid) and poly(ethylene oxide). Langmuir 21:8681–8685

Li X, Loh XJ, Wang K, He C, Li J (2005b) Poly(ester urethane)s consisting of poly[(R)-3-hydroxy butyrate] and poly(ethylene glycol) as candidate biomaterials: characterization and mechan-ical property study. Biomacromolecules 6:2740–2747

Lütke-Eversloh T, Bergander K, Luftmann H, Steinbüchel A (2001a) Biosynthesis of a new class of biopolymer: bacterial synthesis of a sulfur containing polymer with thioester linkages. Microbiology 147:11–19

Lütke-Eversloh T, Bergander K, Luftman H, Steinbüchel A (2001b) Biosynthesis of poly(3-hydroxybutyrate-co-3-mercaptobutyrate) as a sulfur analog to poly(3-hydroxybutyrate). Biomacromole-cules 2:1061–1065

Lütke-Eversloh T, Fischer A, Remminghorst U, Kawada J, Marchessault RH, Bögershausen A, Kalwei M, Eckert H, Reichelt R, Liu SJ, Steinbüchel A (2002) Biosynthesis of novel thermoplastic poly-thioesters by engineered Escherichia coli. Nature Mater 1:236– 240

Nguyen S, Marchessault RH (2004) Synthesis and properties of graft copolymers based on poly(3-hydroxybutyrate) macromonomers. Macromol Biosci 4:262–268

Nuyken O, Weidner R (1986) Graft and block copolymers via polymeric azo initiators. Adv Polym Sci 145:73–74

Park WH, Lenz RW, Goodwin S (1998a) Epoxidation of bacterial polyesters with unsaturated side chains. I. Production and epoxida-tion of polyesters from 10-undecenoic acid. Macromolecules 31:1480–1486

Pederson EN, McChalicher CWJ, Srienc F (2006) Bacterial synthesis of PHA block copolymers. Biomacromolecules 7:1904–1911 Park WH, Lenz RW, Goodwin S (1998b) Epoxidation of bacterial

polyesters with unsaturated side chains. III. Crosslinking of epoxidized polymers. J Polym Sci A Polym Chem 36:2389–2396 Qiu YZ, Ouyang SP, Shen Z, Wu Q, Chen GQ (2004) Metabolic engineering for the production of copolyesters consisting of 3-hydroxybutyrate and 3-hydroxyhexanoate by Aeromonas hydro-phila. Macromol Biosci 4:255–261

Qiu YZ, Han J, Guo JJ, Chen GQ (2005) Production of poly(3-hydroxybutyrate and 3-hydroxyhexanoate) by Aeromonas hydro-phila and Pseudomonas putida. Biotechnol Lett 27:1381–1386 Ramsay BA, Lomaliza K, Chavarie C, Dube B, Bataille B, Ramsay

JA (1990) Production of poly-( β-hydroxybutyric-co-β-hydroxy-valeric) acids. Appl Environ Microbiol 56:2093–2098

Ravenelle F, Marchessault R (2003) Self assembly of poly[(R)-3-hydroxybutyric acid)-block-Poly(ethylene glycol) diblock copolymers. Biomacromolecules 4:856–858

Ritter H, von Spee AG (1994) Bacterial production of polyesters bearing phenoxy groups in the side chains, 1 poly(3-hydroxy-5-phenoxy-pentanoate-co-3-hydroxy-9-phenoxy-nonanoate) from Pseudomo-nas oleovorans. Macromol Chem Phys 195:1665–1672

Saad GR (2001) Calorimetric and dielectric study of the segmented biodegradable poly(ester-urethane)s based on bacterial poly[(R)-3-hydroxy butyrate]. Macromol Biosci 1:387–396

Schmack G, Gorenflo V, Steinbüchel A (1998) Biotechnological production and characterization of polyesters containing 4-hydroxyvaleric acid and medium-chain-length hydroxyalkanoic acids. Macromolecules 31:644–649

Shah DT, Tran M, Berger PA, Aggarwal P, Asrar J, Madden LA, Anderson AJ (2000) Synthesis and properties of hydroxy-terminat-ed poly(hydroxyalkanoate)s. Macromolecules 33:2875–2880 Steinbüchel A (1991) Polyhydroxyalkanoic acids. In: Byrom D (ed)

Biomaterials. Macmillan, New York, pp 123–213

Steinbüchel A (2001) Perspectives for biotechnological production and utilization of biopolymers: metabolic engineering of poly-hydroxyalkanoate biosynthesis pathways as a successful exam-ple. Macromol Biosci 1:1–24

Steinbüchel A, Füchtenbusch B (1998) Bacterial and other biological systems for polyester production. TIBTECH 16:419–427 Steinbüchel A, Valentin HE (1995) Diversity of bacterial

polyhydroxy-alkanoic acids. FEMS Microbiol Lett 128:219–228

Stigers DJ, Tew GN (2003) Poly(3-hydroxyalkanoate)s functionalized with carboxylic acid groups in the side chain. Biomacromolecules 4:193–195

Sudesh K, Doi Y (2005) Polyhydroxyalkanoates. In: Bastioli C (ed) Handbook of biodegradable polymers. Rapra Technology, UK, pp 219–256

Sudesh K, Abe H, Doi Y (2000) Synthesis, structure and properties of polyhydroxyalkanoates: biological polyesters. Progr Polym Sci 25:1503–1555

Tanaka T, Fujita M, Takeuchi A, Suzuki Y, Uesugi K, Ito K, Fujisawa T, Doi Y, Iwata T (2006) Formation of highly ordered structure in poly[(R)-3-hydroxybutyrate-co-3-hydroxyvalerate] high-strength fibers. Macromolecules 39:2940–2946

Timbart L, Renard E, Langlois V, Guerin P (2004) Novel biodegrad-able copolyesters containing blocks of poly(3-hydroxyoctanoate) and poly(ɛ-caprolactone): synthesis and characterization. Macromol Biosci 4:1014–1020

Timm A, Steinbüchel A (1990) Formation of polyesters consisting of medium-chain-length 3-hydroxyalkanoic acids from gluconate by Pseudomonas aeruginosa and other fluorescent pseudomonads. Appl Environ Microbiol 56:3360–3367

Tsuge T, Saito Y, Kikkawa Y, Hiraishi T, Doi Y (2004) Biosynthesis and compositional regulation of poly(3-hydroxybutyrate-co-3-hydroxyhexanoate) in recombinant Ralstonia eutropha express-ing mutated polyhydroxyalkanoate synthase genes. Macromol Biosci 4:238–242

Ulmer HW, Gross RA, Posada M, Weisbach P, Fuller RC, Lenz RW (1994) Bacterial production of poly(β-hydroxyalkanoates) contain-ing repeatcontain-ing units by Rhodospirillum rubrum. Macromolecules 27:1675–1679

van der Walle GAM, Buisman GJH, Weusthuis RA, Eggink G (1999) Development of environmentally friendly coatings and paints using medium-chain-length poly(3-hydroxy-alkanoates) as the polymer binder. Int J Biol Macromol 25:123–128

Valentin HE, Lee EY, Choi CY, Steinbüchel A (1994) Identification of 4-hydroxyhexanoic acid as a new constituent of biosynthetic polyhydroxyalkanoic acids from bacteria. Appl Microbiol Biotechnol 40:710–716

Valentin HE, Berger PA, Gruys KJ, Rodrigues MFA, Steinbüchel A, Tran M, Asrar J (1999) Biosynthesis and characterization of poly (3-hydroxy-4-pentenoic acid). Macromolecules 32:7389–7395 Yalpani M, Marchessault RH, Morin FG, Monasterios CJ (1991)

Synthesis of poly(3-hydroxyalkanoate) (PHA) conjugates: PHA-carbohydrate and PHA-synthetic polymer conjugates. Macromolecules 24:6046–6049

Walz R, Bömer B, Heitz W (1977) Preparation and characterization of a branched bacterial polyester. Makromol Chem 178:2527–2534 Zagar E, Krzan A, Adamus G, Kowalczuk M (2006) Sequence distribution in microbial poly(3-hydroxybutyrate and 3-hydroxy-valerate) co-polyesters determined by NMR and MS. Biomacro-molecules 7:2210–2216

Zhang S, Kolvek S, Goodwin S, Lenz RW (2004) Poly(hydroxyalka-noic acid) biosynthesis in Ectothiorhodospira shaposhnikovii: characterization and reactivity of a type III PHA synthase. Biomacromolecules 5:40–48

Zhu KJ, Bihai S, Shilin Y (1989) Super microcapsules (SMC). I. Preparation and characterization of star polymethylene oxide (PEO)-polylactide (PLA) copolymers. J Polym Sci A Polym Chem 27:2151–2159