Biosynthesis and characterization of polyhydroxyalkanoates by Pseudomonas guezennei from alkanoates and glucose

International Journal of Biological Macromolecules 51 (2012) 1063–1069

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Biosynthesis and characterization of polyhydroxyalkanoates by Pseudomonas guezennei from alkanoates and glucose Christelle Simon-Colin a,∗ , Christelle Gouin a , Pierre Lemechko a,b , Sophie Schmitt a , Amandine Senant a , Nelly Kervarec c , Jean Guezennec a a

Institut Franc¸ais de Recherche pour l’Exploitation de la Mer, Centre de Brest, RBE/BRM/LBMM, B.P. 70, 29280 Plouzané, France Institut de Chimie et des Matériaux de Paris Est (ICMPE) UMR 7182, Université Paris Est, 2 à 8 rue Henri Dunant, 94320 Thiais, France c Service Commun de RMN-RPE, UBO, 6 Avenue Le Gorgeu, 29200 Brest, France b

a r t i c l e

i n f o

Article history: Received 24 May 2012 Received in revised form 5 August 2012 Accepted 19 August 2012 Available online 27 August 2012 Keywords: Polyhydroxyalkanoates mcl-PHAs PHOU Pseudomonas Double bond

a b s t r a c t The biosynthesis of medium chain length poly(3-hydroxyalkanoates) mcl PHAs by Pseudomonas guezennei using glucose, sodium octanoate, and 10-undecenoic acid as sole or mixed carbon sources was investigated. Chemical composition of polyesters was analyzed by GCMS and NMR. The copolyester produced by P. guezennei from glucose mainly consisted of 3-hydroxyoctanoate and 3-hydroxydecanoate, and the presence of 3-hydroxydodec-5-enoate was demonstrated. Using sodium octanoate as the sole nutrient, the microorganism produced a poly(3-hydroxyoctanoate) (PHO) polymer containing up to 94 mol% 3-hydroxyoctanoate. Biosynthesis of poly[(3-hydroxyoctanoate)-co-(3-hydroxyundecenoate)] (PHOU) copolymers bearing terminal reactive double bonds on its side chains with unsaturation degree ranging from 8.8% to 78.2% was obtained by tuning the ratio of sodium octanoate/10-undecenoic acid in the medium. Thermal analysis indicated semi-crystalline polymers with melting temperatures (Tm ) ranging from 46 to 55 ◦ C, fusion enthalpy (H) comprised between 3 and 35 J/g and glass transition temperature (Tg ) from −36 to −44 ◦ C, except for the highly amorphous 78.2% unsaturated PHOU with a low Tg (−50 ◦ C). Molecular weights determined by GPC ranged from 119 000 and 530 000 g/mol. The biosynthesis of natural polyesters with controlled ratio of vinyl-terminated side chains is of great interest for further chemical modifications. © 2012 Elsevier B.V. All rights reserved.

1. Introduction Poly-␤-hydroxyalkanoates (PHAs) are biopolymers stored in intracellular inclusion bodies by a wide variety of bacteria as an energy reserve, in response to excess carbon under nutrientlimited conditions [1,2]. The monomeric composition of PHAs can be related both to the nature of the carbon source supplied and to the bacteria. PHAs exhibit a great variety of properties and thus may have different applications. Regarding the length of their carbon chains, PHAs can be divided in three groups: short chain length PHAs (3–5 carbon atoms, scl-PHAs), medium chain length (6–15 carbon atoms, mcl-PHAs) and long chain length (more than 15 carbon atoms, lcl-PHAs). Due to structural differences, the physical properties of mcl-PHAs are quite different from scl-PHAs such as poly(3-hydroxybutyrate) (PHB) that is highly crystalline and stiff material whereas mcl-PHAs have elastomeric properties. Due to their biodegradability and thermoplastic properties, PHAs have attracted great scientific and technological interest and have been

∗ Corresponding author. Tel.: +33 2 98224528; fax: +33 2 98224757. E-mail address: [email protected] (C. Simon-Colin). 0141-8130/$ – see front matter © 2012 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.ijbiomac.2012.08.018

extensively studied in the last two decades [3–6]. They are regarded as promising substitutes for petrochemicals plastics, and thus for use in tackling the problem of plastic waste in the future. Moreover, PHAs can be produced from natural renewable carbon sources and represent a new way of utilizing waste from low cost carbon stocks [7–11]. Moreover, owing to their inherent biocompatibility, PHAs are considered as good candidates for biomedical applications [12] such as drug delivery [13] and tissue engineering [14,15]. However, these biopolymers in their native state are hydrophobic and their hydrolytic degradation is very slow in the tissue, which is not favorable for biological applications because polymers with enhanced hydrophilicity are usually more biocompatible in therapeutic and biomedical applications [16]. Consequently, PHAs need to be chemically modified to modulate their hydrophilic/ hydrophobic balance and make them suitable for medical applications. PHAs containing long chain saturated and unsaturated monomers have unique properties, and double bonds hold the possibility for further chemical modifications. New functional groups such as epoxide [17], carboxylic acid [18,19], chlorine [20], hydroxyl groups [16,21,22] and alkyne groups [23] have been introduced to produce new polymers having different physico-chemical

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properties. These functional groups can be used to further conjugate oligomers, bioactive compounds or targeting molecules. Moreover, while many different PHAs have been reported, relatively few have been produced in useful quantities and fewer still meet properties necessary to replace synthetic polymers. Therefore, modification of natural PHAs is often necessary to impart properties at least equivalent to their synthetic counterparts and make PHAs viable replacement candidates. In the present study we investigated the ability of the bacterium Pseudomonas guezennei, isolated from microbial mats on the atoll of Rangiroa in French Polynesia [24], to produce saturated and unsaturated mcl-PHAs from glucose, sodium octanoate and 10-undecenoic acid supplied in different concentrations. As polyesters with unsaturated side chains are reactive for functionalization, we focused on the tailor-made production of poly[(3-hydroxyoctanoate)-co-(3-hydroxyundecenoate)] PHOU with unsaturation rate ranging from 8.8 to 78.2% by adjusting the sodium octanoate/10-undecenoic acid ratio in the culture medium. GCMS, NMR, DSC and GPC analyses were used to study chemical, thermal and physical properties of these polymers. 2. Experimental 2.1. Biosynthesis of mcl-PHAs P. guezennei (strain CNCM-I-3358 in the Collection Nationale de Cultures de Microorganismes, Institut Pasteur, Paris, France) was cultivated in a two-steps batch cultivation process. In the first step, the cells were inoculated at 10% (v/v) with a suspension of cells in exponential phase and grown in 5 l fermenter (Infors, Massy, France) containing 3 l of rich marine broth medium (10 g peptone, 5 g yeast, 15 g sea salts/l distilled water). The temperature was maintained at 35 ◦ C and the pH was adjusted at 7.0 by automatic addition of 2 M NaOH. The air flow was fixed at 30 l/h and the agitation rate from 200 to 800 rpm to maintain the level of dissolved O2 at its maximum. After the cultivation for 8 h, cells were harvested by centrifugation (5000 × g, 20 min), and transferred into 5 l fermenter containing 3 l nitrogen-free medium (15 g/l sea salts) enriched with one of the following carbon source: 20 g/l of glucose as the sole carbon source, sodium octanoate (2 g/l, 3 g/l, 5 g/l) and/or 10-undecenoic acid (0.2 g/l, 0.5 g/l, 1 g/l, 2 g/l, 3 g/l). Culture was incubated at 35 ◦ C, 200–400 rpm, and dissolved O2 maintained around 25%. Following cultivation for 60 h, cells were harvested by centrifugation (10 000 × g for 15 min), washed-up three times with diluted sea-water, and the pellets lyophilized prior to PHAs extraction. Pellets of freeze-dried cells were ground with a mortar and pestle, the resulting powder was extracted with chloroform for 4 h at 50 ◦ C. The PHAs-containing chloroform phase was washed once with water to remove residual solid particles, and concentrated. The organic phase was evaporated to dryness, and purified PHAs were obtained by repeated precipitations in 10 volumes of cold methanol. 2.2. Gas chromatography mass spectroscopy Samples of polymer were subjected to methanolysis in the presence of MeOH/HCl 37% (17/2, v/v) at 100 ◦ C for 4 h in Pyrex test tubes (volume 10 ml) with screw Teflon-lined caps. After phase separation and two washes with 1 ml distilled water, the organic phase was dried with MgSO4 and evaporated under nitrogen. TMSi derivation of 3-hydroxyalkanoates methyl esters was accomplished by adding 100 ␮l pyridine and 100 ␮l sylon (BSTFA–TMCS, 99:1) to 1 ml methanolized sample. The reaction mixture was heated at 70 ◦ C for 45 min. The TMSi derivatives of methyl esters

were analyzed by GCMS allowing the determination of carbon chain length and number of double bonds within the 3-hydroxyalkanoic monomers. For localization of olefin groups on monounsaturated chains, methyl esters were acetylated in the presence of 100 ␮l acetyl chloride at 65 ◦ C as described by Johnson and Trinh [25]. The mixture was evaporated to dryness with nitrogen. Hexane (500 ␮l), dimethyldisulfide DMDS (100 ␮l) and iodine I2 (20 ␮l) were added, and the mixture heated at 50 ◦ C for 48 h as described elsewhere [26,27]. Samples were diluted with 200 ␮l of hexane, and the iodine in excess was reduced by addition of Na2 SO3 . After centrifugation, thiomethyl acetylated methyl esters derivatives were recovered from the supernatant, dried under nitrogen and dissolved in 500 ␮l dichloromethane prior to analysis by GCMS. The TMSi methyl esters derivatives and thiomethyl acetylated methyl esters derivatives were analyzed by GCMS using an Agilent 6890N chromatograph coupled to a quadrupole Agilent 5975 inert XL mass selective spectrometer, equipped with a HP-5-MS fused silica capillary column (30 m × 0.25 mm, 25 ␮m film thickness). A 1 ␮l sample was injected (split ratio 100:1) with helium as carrier gas and the temperature was programmed for the separation of peaks (60 ◦ C for one min, ramp of 4 ◦ C/min to 140 ◦ C, 15 ◦ C/min to 280 ◦ C and 5 min at 280 ◦ C). The ionizing energy for MS operation was 70 eV. 2.3. NMR spectroscopy NMR spectroscopy was performed at 25 ◦ C on samples of PHAs dissolved in deuterated chloroform on a BRUKER 400 DRX spectrometer (Bruker, Germany) equipped with a 5 mm triple resonances 1 H/{BB}/31 P operating at 400 MHz for 1 H and 100 MHz for 13 C. Chemical shifts are reported in ppm relative to signal of 3,3,3,4 tetramethyl silane. 2.4. Thermal analysis Differential scanning calorimetry (DSC) was performed with a TA Instrument 2920 Modulated DSC. Sample of about 10 mg was encapsulated between aluminum pan and lid, and heated from −80 ◦ C to 100 ◦ C at a heating rate of 10 ◦ C/min under 50 ml/min nitrogen purge. The melting temperature (Tm ) and enthalpy of fusion (H) were taken from the melting endotherm and glass transition temperature (Tg ) was taken as the midpoint of the heat capacity change. 2.5. Gel permeation chromatography Molecular weights were determined by gel permeation chromatography on a system consisting of a 2695 Alliance Module (Waters), three Ultrastyragel columns (HR2, HR4, HR6) placed in series, a 2414 differential refractometer (Waters) and a laser light scattering detector miniDawn Treos (Wyatt Technology Corporation). Tetrahydrofuran (THF) was used as a mobile phase at a flow rate of 1 ml/min. The samples were injected as solutions in THF, filtered through a 0.45 ␮M polytetrafluoroethylene filter and 100 ␮l of 5 mg/ml solutions were injected. The value of the specific refractive index dn/dc was determined as equal to 0.065 ± 0.002 ml/g for mcl-PHAs in THF at room temperature [28]. 3. Results and discussion GCMS analyses of TMSi methyl esters and thiomethyl acetylated methyl esters derivatives were used to determine the monomeric composition of PHAs obtained from P. guezennei. The mass spectra of TMSi derivatives of 3-hydroxyalkanoic acids exhibited characteristic fragments [(CH3 )3 SiO+ = CHCH2 CO2 CH3 ] at m/z 175 and

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Fig. 1. Mass spectra of the TMSi derivatives of 3-hydroxydecanoic acid (3HD) (a) and 3-hydroxydodecenoic acid (3HDD:1) (b) methyl esters. The characteristic peaks and molecular ion-related fragments were assigned as described in the text.

[RCH = O+ Si(CH3 )3 ] at m/z [M•+ −73] resulting from ␣-cleavage of the derivatized hydroxyl group. The TMSi ethers do not usually show a parent molecular ion but the molecular ion related fragment at m/z [M•+ −15] is quite prominent and can be used to determine the chain length of monomers (Fig. 1). The existence of a double bond was deduced from the molecular weight of the fragment at m/z [M•+ −15] which is two amu less than that of the corresponding saturated monomer (Fig. 1b), and from the shift of the base peak [29]. Others fragments of lower mass were also common for the 3-hydroxyl functional group and readily assignable: m/z 73 [(CH3 )3 Si+ ], m/z 89 [(CH3 )3 SiO+ ], m/z 131 (C5 H11 SiO2 )+ , m/z 133 (C5 H13 SiO2 )+ and m/z 159 (C6 H11 SiO3 )+ . GCMS analysis of thiomethyl acetylated methyl esters was carried out to determine the position of double bond in monounsaturated monomers. The mass spectra of thiomethyl acetylated methyl esters of 3-hydroxyalkenoic acids showed recognizable molecular ions at m/z [M•+ ] and key fragments [A+ ] and [B+ ] that

allow us to clearly identify the position of the double bond. Key fragments [A+ ] and [B+ ] derived from the cleavage of the carbon–carbon bond between the two methyl sulphide groups CH3 S (Figs. 2 and 3). Fragment [B+ ] is often detected in low intensity because it further decomposes and gives [B+ −HOAc] via loss of the O-acetyl group. Additional ions in a lower mass range were detected for [B+ −HOAc] after successive loss of the HSCH3 group and/or acetyl group, thus confirming the olefin group position. The mass spectra of TMSi methyl esters and thiomethyl acetylated methyl esters derivatives allowed a complete characterization of the monomeric composition and location of unsaturation of mcl-PHAs produced by P. guezennei cultivated on glucose, sodium octanoate plus/or 10-undecenoic acid. The composition of PHAs synthesized by P. guezennei from glucose was found to mainly consist of 3-hydroxydecanoate (3HD) and 3-hydroxyoctanoate (3HO), along with low fractions of 3-hydroxydodecanoate (3HDD), 3-hydroxydodec-5-enoate

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Fig. 2. Mass spectra of thiomethyl acetylated methyl esters derivative of 3-hydroxydodec-5-enoic acid (3HDD:15) ([M+ ] = 364) as a constituting monomer of mcl-PHAs produced by P. guezennei cultivated on glucose.

(3HDD:15) and 3-hydroxyhexanoate (3HHx), as identified by GCMS (Table 1). The 5 position of the double bond of the 3hydroxydodecenoate unit was deduced from the fragmentation pattern of thiomethyl acetylated methyl esters derivative showing parent ion [M•+ ] at m/z 364, fragment [A+ ] at m/z 145 and [B+ −HOAc] at m/z 159 (Fig. 2). DSC analysis showed glass transition temperature (Tg ) of PHAs produced by P. guezennei grown on glucose at −44 ◦ C and an obvious peak of melting temperature (Tm ) appeared at 55 ◦ C with associated enthalpy H = 35 J/g. The molecular weight (Mw )

and polydispersity index (Mw /Mn ) were 119 000 g/mol and 1.8 respectively. Using sodium octanoate as the sole carbon source, P. guezennei synthesized a mcl-PHAs mainly containing of 3-hydroxyoctanoate (3HO) accounting for up to 94 mol% whatever the substrate concentration, and lower amounts of 3-hydroxyhexanoate (3HHx) and 3-hydroxydecanoate (3HD) (Table 1). These poly(3hydroxyoctanoate) (PHO) copolymers were characterized by similar molecular weights ranging from 254 000 g/mol to 337 000 g/mol and a polydispersity index near 2. DSC experiments

Fig. 3. Mass spectra of thiomethyl acetylated methyl esters derivatives of 3-hydroxynon-8-enoic acid (3HN:18) ([M+ ] = 322) (a) and 3-hydroxyundec-10-enoic acid (3HUD:110) ([M+ ] = 350) (b) as constituting monomers of PHOU produced by P. guezennei from a mixture of sodium octanoate plus 10-undecenoic acid.

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Table 1 Monomeric composition of mcl-PHAs as determined by GCMS, produced by P. guezennei grown on glucose, sodium octanoate (Oct) and/or 10-undecenoic acid (Und) mixed with different ratios. PHAs monomeric composition mol% Carbon source

3HHx

Glucose 20 g/l Oct 5 g/l Oct 3 g/l Oct 2 g/l Und 3 g/l Oct 2 g/l + Und 2 g/l Oct 3 g/l + Und 0.2 g/l Oct 3 g/l + Und 0.5 g/l Oct 3 g/l + Und 1 g/l

1 3.5 4 3 1 1.6 3 1

3HHp:16

3HO

6 2.6 0.7 1.5 1.1

24 94 93 92 7 49.3 85.2 73 69

3HN:18

3HD

53 30 6.8 13.3 19

66 2.5 3 5 8 4.7 3 3.2 2.7

3HUD:110

3HDD 7

25 12.4 2.5 5.4 7.2

3HDD:15

%C C

2

1 45 0.2 0.2

0.1

2

84 45 10 20.6 27.3

Table 2 Unsaturation degree (%C C) as determined by 1 H NMR, Tm , Tg , H and molecular weight of mcl-PHAs obtained from P. guezennei grown on glucose, sodium octanoate (Oct) and/or various concentrations of 10-undecoic acid (Und). Carbon source

% C C 1 H NMR

Tm (◦ C)

Tg (◦ C)

H (J/g)

Mw (g/mol)

Mw /Mn

Glucose 20 g/l Oct 5 g/l Oct 3 g/l Oct 2 g/l Und 3 g/l Oct 2 g/l + Und 2 g/l Oct 3 g/l + Und 0.2 g/l Oct 3 g/l + Und 0.5 g/l Oct 3 g/l + Und 1 g/l

nd nd nd nd 78.2 42.3 8.8 19.7 26.3

55 48 50 52 nd 46 54 50 52

−44 −36 −37 −38 −50 −43 −39 −40 −41

35 19 21 20 nd 8 11 13 3

119 000 264 000 254 000 337 000 239 000 474 000 319 000 365 000 530 000

1.8 2.5 1.9 2 2.8 1.5 1.7 1.8 1.6

indicated an average Tg of −37 ◦ C and Tm of 50 ◦ C with a heat of fusion H of 20 J/g (Table 2). Conversely, the use of sodium octanoate in mixture with 10-undecenoic acid as carbon sources led to the formation of mcl-PHAs with more unsaturated monomers. Up to eight different TMSi methyl esters peaks were identified: 3-hydroxhexanoate (3HHx), 3-hydroxyheptenoate (3HHp:1), 3-hydroxyoctanoate (3HO), 3-hydroxynonenoate (3HN:1), 3hydroxydecanoate (3HD), 3-hydroxyundecenoate (3HUD:1), 3hydroxydodecanoate (3HDD) and 3-hydroxdodecenoate (3HDD:1) present in different proportions depending on the composition of the culture medium. Mcl-PHAs produced by P. guezennei from sodium octanoate plus 10-undecenoic acid mainly consisted of 3HO along with 3HN:1 and 3HUD:1, thus defining a poly[(3-hydroxyoctanoate)-co-(3-hydroxyundecenoate)] (PHOU) copolymer. The terminal position of double bond in monounsaturated monomers in PHOU was deduced from mass spectra of thiomethyl acetylated methyl esters showing high intensity fragment [A+ ] at m/z 61 (CH2 = + SCH3 ) together with the detection of [B+ −HOAc] at m/z 173, 201, 229 and 243 for 3HHp:16, 3HN:18, 3HUD:110 and 3HDD:111 respectively, characterizing the SH CH3 group in terminal position (Fig. 3, Table 3). The terminal position of double bond in PHOU was confirmed by NMR analysis. Signals at 4.95 (h) and 5.77 (g) ppm on the 1 H NMR spectra corresponded to the ethylenic protons of terminal vinylic group (CH CH2 ), whereas signal at 5.2 ppm (b,b ) was attributed to Table 3 GCMS fragmentation pattern of thiomethyl acetylated methyl esters derivatives of monounsaturated 3-hydroxyalkenoic acids issued from mcl-PHAs produced by P. guezennei. [M• + ] 3HHp:16 3HN:18 3HUD:110 3HDD:15 3HDD:111

294 322 350 364 364

[A+ ] 61 61 61 145 61

[B+ ]

[B+ −HOAc]

233 261 289 219 303

173 201 229 159 243

the methine protons (CH) of both saturated and unsaturated side chains, and signal at 0.9 ppm (e) was assigned to protons of the CH3 in terminal position of saturated side chains (Fig. 4). No other CH signal was detected on the proton spectra, thus confirming the absence of unsaturation in other location. The multiplet resonance at 2.47–2.60 (a,a ) ppm was assigned to the methylene protons (CH2 ) of second carbon atom. The methylene protons in fourth position yielded a signal at 1.59 (c,c ) ppm, while all other methylene hydrogens produced a signal at 1.28 (d,d ) ppm, with the exception of methylene group next to the terminal vinylic group that gave a signal at 2.04 (f,f ) ppm. An estimation of the unsaturation contents in PHOU could be made from the 1 H NMR spectra using the intensity ratio of the signal at 5.77 ppm (g) versus the signal at 5.2 ppm (b,b ). It was found to be 8.8, 19.7 and 26.3% for concentrations of 10-undecenoic acid ranging from 0.2, 0.5 and 1 g/l respectively plus 3 g/l sodium octanoate (Fig. 5a–c). In the presence of 2 g/l of both sodium octanoate and 10-undecenoic acid, the degree of unsaturation elevated to 42.3% (Fig. 5d). Those results as determinated by the analysis of 1 H NMR spectra are consistent with data obtained from GCMS analysis. Cells cultivated with 3 g/l of 10-undecenoic acid as the sole carbon source led to the production of a highly amorphous polymer with 78.2% terminal olefinic groups (Fig. 5e), including 53 mol% 3-hydroxynon-8-enoate (3HN:18), 25 mol% 3-hydroxyundec-10-enoate (3HUD: 10), 6 mol% 3-hydroxyhept6-enoate (3HHp:16) and less significant amounts of saturated monomers i.e. 7 mol% 3-hydroxyoctanoate (3HO), 8 mol% 3hydroxydecanoate (3HD) and 1 mol% 3-hydroxydodecanoate (3HDD) (Table 1). The high degree of side-chain unsaturation resulted in an amorphous polymer with a consistency of a viscous, sticky, unhandle gum at room temperature. Moreover, this PHOU sample became insoluble in chloroform after a few days, as the result of cross-linking reactions caused by the high concentration of unsaturated groups. Physical properties of all PHAs polymers produced by P. guezennei during this study are shown in Table 2. With mixed sodium octanoate and 10-undecenoic acid used as carbon substrates,

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Fig. 4. 1 H NMR spectrum in CDCL3 of PHOU containing 26.3% unsaturated units, produced by P. guezennei from 3 g/l sodium octanoate and 1 g/l 10-undecenoic acid. The percentage of olefin groups was calculated from the ratio of peak at 5.77 ppm (g) to peak at 5.2 ppm (b,b ).

molecular weights of PHAs ranged from 319 000 to 530 000 g/mol, with polydispersity values under 2, indicative of the uniform formation of PHAs. From DSC analysis, Tg of PHOU copolymers with 8.8, 19.7 and 26.3% unsaturated pendant groups were near −40 ◦ C and a fusion peak was observed between 50 ◦ C and 54 ◦ C (Table 2, Fig. 6). However, a decrease of the associated enthalpy was

evidenced and could be related to the presence of more unsaturated side chains in these polymers. This is confirmed by the calorimetric analysis of PHOU sample with 78.2% unsaturation degree composed by 25 mol% 3HUD:110 units together with 53 mol% 3HN:18, for which no peak of fusion was observed on the DSC curve, whereas a low glass transition temperature was observed at −50 ◦ C, thus

Fig. 5. 1 H NMR spectra in CDCL3 of PHOU copolymers produced by P. guezennei cultivated in the following conditions: Oct 3 g/l plus Und 0.2 g/l (a), Oct 3 g/l plus Und 0.5 g/l (b), Oct 3 g/l plus Und 1 g/l (c), Oct 2 g/l plus Und 2 g/l (d), and Und 3 g/l (e). The percentage of unsaturated units was determined from the ratio of the integration of peak at 5.77 (g) ppm to peak at 5.2 (b,b ) ppm.

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chemical modifications. These samples are now studied as precursors for the design of novel biopolyesters based on PHAs for biomedical applications. References

Fig. 6. DSC thermograms of mcl-PHAs produced by P. guezennei in the presence of glucose (a), Oct 3 g/l plus Und 0.2 g/l (b), Oct 2 g/l plus Und 2 g/l (c), Oct 5 g/l (d) and Und 3 g/l (e).

characterizing a highly amorphous elastomer. The average molecular weight for 78.2% unsaturated PHOU was 239 000 g/mol with a polydispersity index of 2.8. 4. Conclusions In this study, we demonstrated the ability of P. guezennei to synthesize different saturated and unsaturated mcl-PHAs from glucose, sodium octanoate and/or 10-undecenoic acid. More particularly, PHAs containing unsaturated monomers can be further modified by chemical reactions such as cross-linking, double bond hydratation and epoxidation to produce new polymers having different thermal and mechanical properties, and chemical modification of biosynthetic PHAs thus constitutes a promising approach to expend bacterial polyesters to be used in the medical and environmental areas. Compared to other aliphatic polyesters, poly[(3-hydroxyoctanoate)-co-(3hydroxyundecenoate)] PHOU has the major advantage to bear accessible terminal double bond on its side chains, that favors post-polymerisation modification. In this study, we succeeded to produce PHOU samples with different unsaturation degree by controlling the ratio of sodium octanoate to 10-undecenoic acid, thus allowing us to make mcl-PHAs with suitable properties for further

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