European Polymer Journal 39 (2003) 1385–1394 www.elsevier.com/locate/europolj
Medium-chain-length polyhydroxyalkanoic acids (PHAmcl ) produced by Pseudomonas putida IPT 046 from renewable sources Ruben J. S anchez a,*, Jan Schripsema a, Luziana F. da Silva b, rio C. Gomez Marilda K. Taciro b, Jose G.C. Pradella b, J. Grego a
b
b
Universidade Estadual do Norte Fluminense, Centro de Ci^ encias e Tecnologia, Avenida Alberto Lamego, 2000-Horto, 28015-620, Campos dos Goytacaze, RJ, Brazil Instituto de Pesquisa Tecnol ogica-Divis~ ao de Quımica––Agrupamento de Biotecnologia, Avenida Prof. Almeida Prado, 532 Pr edio 31-Butant~ a, 0550890, S~ ao Paulo, SP, Brazil Received 13 October 2002; received in revised form 22 January 2003; accepted 23 January 2003
Abstract Medium-chain-length polyhydroxyalkanoates are produced by Pseudomonas putida strain IPT046 growing on carbohydrates. Analysis by gas chromatography and nuclear magnetic resonance of the elastomeric material revealed that PHAmcl is composed from essentially hydroxydecanoate (60–70%) and hydroxyoctanoate (20–25%) sequence units with a non-terminal double bond in about 6% of the side chains. The average molecular weight of PHAmcl is 223 kDalton and the X-ray diffractogram showed that 24% of the solid phase is crystalline. This biodegradable polyester presents a relative low glass transition temperature ()39.7 °C) and melting point (56 °C), is thermically stable (234 °C) and displays appropriate thermomechanical properties for potential use as packaging film. Ó 2003 Elsevier Science Ltd. All rights reserved. Keywords: Elastomers; Medium-chain-length polyhydroxyalkanoates; Biodegradable polyester
1. Introduction Polyhydroxyalkanoic acids (PHAs) are produced by a wide number of bacteria as carbon and energy reserve granules [1,2]. PHAs are produced in large amounts under carbon source excess and limitation of at least one nutrient essential for cell multiplication [3]. These polymers have attracted great scientific and technological interest due to its thermoplastic properties [4]. Poly-3hydroxybutyric acid (P3HB) is a well-known member of this family of polymers and was discovered in the 20s [5]. P3HB and copolymers containing 3-hydroxyvaleric acid
*
Corresponding author. Tel.: +55-22-27261525; fax: +55-2227261533. E-mail address:
[email protected] (R.J. Sanchez).
units (P3HB-co-3HV) were produced and commercialised under the trade name Biopol [6,7]. In general, PHASCL are plastics with limited stiffness and brightness, as a consequence of crystallinity. All PHASCL are highly crystalline materials and therefore possess a high modulus. The cracks grow in the amorphous region among lamelar crystals by a process of secondary crystallisation at room temperature [8]. On the other hand, the related copolymers (P3HB-co-3HV) show a lower crystallinity and melting point compared to P3HB due to the 3HV contents and in this material a discrete decrease in stiffness and an increase in toughness was observed [9]. As a consequence of their intrinsic rigidity these materials are not suitable for all potential applications of biodegradable polymers particularly as films to medical and special devices.
0014-3057/03/$ - see front matter Ó 2003 Elsevier Science Ltd. All rights reserved. doi:10.1016/S0014-3057(03)00019-3
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In the 1980s, it was found that Pseudomonas strains accumulate PHA containing medium-chain-length monomers (PHAmcl –C6 –C12 ) from alkanes, alkenes, alkanols and carboxylic acids showing a composition clearly related to the carbon source supplied [10–14]. Pseudomonas strains are also able to accumulate PHAmcl from carbohydrates and other unrelated carbon sources [15,16]. Presently, more than 100 different monomers have been identified as constituents of PHA produced by bacteria [17,18]. Recently, a bacterial strain (Pseudomonas putida IPT 046) able to accumulate large amounts of PHAmcl from carbohydrates [19] and plant oils [20] was isolated from soil. P. putida IPT 046 accumulated higher amounts of PHAmcl from glucose and fructose compared to P. putida KT2440, reaching 60% and 50% of the cell dry weight, respectively. P. putida KT2440 was used as a reference since it and other related strains have been used in studies aiming at the establishment of industrial processes [21,22] and its complete genome sequence is in the final editing (www.qiagen.com/sequencing/psputida.html). In this work, P. putida IPT-046 was cultivated in a bioreactor using carbohydrates as sole renewable carbon source. The PHAmcl -046 was extracted, purified and characterized, and the perspective for film application was analysed.
magnetic detector MAGNOS 6G, an infrared CO2 analyser URAS 10P and an inlet gas mass flowmeter. The exit gas flow was then calculated with the hypothesis of nitrogen molar flow conservation.
2. Experimental procedures
2.2.4. Ammonium concentration Ammonium concentration was measured utilising a specific Orion electrode, mod. 95-12.
2.1. Biosynthesis of PHAmcl 2.1.1. Bacterial strain and inoculum preparation P. putida IPT 046 was isolated from soil [19] and was cultivated in nutrient broth (NB––5 g/l of bacteriological peptone and 3 g/l of meat extract) for 15 h, followed by 15 h cultivation in mineral salts medium [22] for inoculum preparation. The cultivation was made in a rotatory shaker New Brunswick Sci. Mod G-25 at 150 rpm and 30 °C. 2.1.2. Bioreactor The experiment was conducted in a New Brunswick bioreactor (10 l of volume), 30 °C, pH 7.0 and dissolved oxygen maintained above 10% of saturation, utilising a modified mineral culture medium [23], containing glucose (10 g/l) and fructose (10 g/l). The experiment was performed in batch mode to promote balanced cell growth. After nitrogen exhaustion, carbon source (glucose and fructose) was offered in a fed-batch mode (0.23 g of sugar/g of cells h) leading to polymer accumulation. At the end of cultivation, the temperature was elevated to 85 °C and cooled down to room temperature to perform cell inactivation. The respiration rate was determined by gas mass balance using an oxygen para-
2.2. Analytical methods 2.2.1. Biomass concentration Cell dry weight was determined by membrane filtration (0.45 lm, Millipore) and dried at 105 °C to constant weight. The residual biomass concentration (Xr) was obtained subtracting the PHA biomass content [24]. 2.2.2. PHA amount and composition Polymer was estimated by gas chromatographic method [25]. The Riis and Mai [26] method was used which utilizes a sample propanolysis instead of a methanolysis. The propyl esters were assayed with a Hewlett Packard HP 6890 Series gas chromatograph equipped with a HP-5 capillary column as described by Silva et al. [27]. 2.2.3. Carbohydrates concentration Glucose and fructose concentrations were determined by liquid chromatography in a Waters 510 HPLC equipped with a Shodex SC 1011 column as described by Gomez et al. [28].
2.2.5. PHAmcl -046 extraction The culture was harvest centrifuged at 6000 rpm, 20 min, 4 °C in a Sorval RC5B.centrifuge. The cell harvested (pellet) was then freeze-dried and submitted to polymer extraction. Lyophilized cells were suspended in chloroform and extracted for 6 h in a Soxhlet extractor. The chloroform solution was concentrated by evaporation and the polymer was precipitated on 96% ethanol (1:10 v/v chloroform–ethanol), it was washed with ethanol and dried in vacuum oven at 30 °C. 2.3. Structural characterization 2.3.1. Average molecular weight The measurements were performed with polymer solution in chloroform (1% w/v) after filtration through 0.45 lm Sartorious membranes. The average molecular weights were determined using a Lachrom Merck–Hitachi gel permeation chromatography system with refractive index detector and PS4000, PS400, PS40 and PS4 Licrogel columns placed in series with exclusion limits 106 , 105 , 104 , and 103 Dalton. Chloroform was used as eluent at a flow rate of
R.J. Sanchez et al. / European Polymer Journal 39 (2003) 1385–1394
1.0 ml/min and injection volumes of 20 ll were used. Polystyrene Merck standards with narrow polydispersity were used for the calibration curve. 2.3.2. Nuclear magnetic resonance Samples of PHAmcl were dissolved in deuterated chloroform. NMR spectra were obtained on a JEOL Eclipse þ 400 spectrometer operating at 400 MHz for 1 H and 100 MHz for 13 C. Chemical shifts are given in ppm relative to the signal of TMS.
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Thermal stability of PHAmcl -046 was examined using TG system SDT 2960––TA instruments with balance sensitivity of 0.1 lg. About 11 mg of sample was transferred to platinum sample pans and were heated at 10 °C/min under dynamic nitrogen atmosphere (10 ml/ min) from room temperature.
3. Results and discussion 3.1. PHAmcl -046 production by P. putida IPT 046
100.0
16
80.0
12
60.0
8
40.0
4
20.0
0
0
5
10
15
20
25
0.0 30
Time (h)
Fig. 1. Typical biomass ( ), residual biomass () and polymer PHA () evolution during an experiment to produce PHAmcl 046.
60.0
3.00
50.0
2.50
40.0
2.00
30.0
1.50
20.0
1.00
10.0
0.50
0.0
0
5
10
15
20
N (g/L)
The thermal properties of the PHAmcl -046 were determined on TA Instruments Systems employing differential scanning calorimetry (DSC) thermogravimetry (TG) and dynamical mechanical (DMA) techniques. Differential scanning calorimetric data of polymers were recorded on a TA Instruments DSC-2910 instrument calibrated with indium. Samples weighing between 8 and 10 mg were encapsulated in hermetic aluminium pans and heated from )100 to 90 °C at heating rate of 10°/min. The second thermogram was recorded after quenching to )100 °C with liquid nitrogen accessory of calorimeter system using the same heating rate and temperature range as in the first scan. Dynamic measurements were made in the extension mode employing a clamp film with a film dimension (12.4 4.8 0.4) mm. The frequency of oscillation force was kept at 1 Hz above the temperature range from )150 to +45 °C with heating rate of 1 and 3 °C/min. Films to DMA measurements were prepared from 5% w/v chloroform solution of PHAmcl . Films were cast in glass petri dishes and the solvent evaporated in a nitrogen atmosphere. The films were vacuum dried at 25 °C, for 7 h after them its were aged to 3 weeks before measurements.
Biomass (g/L)
2.4. Thermal analysis
20
PHA (%)
2.3.4. X-ray diffraction A Seifert-FPM model URD65 X-ray generator with a Ni filter to provide a CuKa radiation (k ¼ 0:1542 nm) was used. Every scan was recorded in the range of 2h ¼ 10–40° at a scan speed of 3°/min at the same acquisition time. Crystallinity percentages were calculated by integration of semi-crystalline and amorphous polymer area in the diffractogram and additionally, from mathematical model functions (Lorentzian and Gauss functions). All the difractograms were normalized and both methods employed gave a good correlation to Xc (% of crystallinity).
Figs. 1–4 describe a typical fermentation run for PHAmcl -046 production by P. putida IPT 046. Cells grow exponentially up to 10 h at lmax equal 0.46 h1 with virtually no PHA accumulation. With nitrogen exhaustion and carbohydrate feeding, PHA biosynthesis started and continued until the end of the experiment. A level of 14 g/l of biomass with approximately 40% of PHA content was reached (Figs. 1 and 2). During PHA accumulation, virtually no cell growth was observed. It should be mentioned however that these results were obtained in non-optimised experiments.
Carbon source (g/L)
2.3.3. Fourier transform infrared spectroscopy Infrared spectras were recorded on FTIR-8300 Shimadzu, between 400 and 4500 cm1 and 45 scans recorded. The polymer sample were casted on KBr pellets.
0.00 30
25
Time (h)
Fig. 2. Consumed carbon source Ccons [g/L] ( ), residual carbon source Cres [g/L] () and ammonium N [g/L] () concentration along the assay.
R.J. Sanchez et al. / European Polymer Journal 39 (2003) 1385–1394
100
25
80
20
60
15
40
10
20
5
0
0
5
10
15
20
25
A fed-batch strategy is currently being developed in order to have high biomass with high PHAmcl cell content from sugar cane carbohydrates [32].
Qo 2 (mmol/gh)
OUR (mmol/Lh), CER (mmol/Lh)
1388
0 30
Time (h)
Fig. 3. Oxygen uptake rate OUR [mmol/L.h] ( ), specific oxygen uptake rate Qo2 [mmol/g.h] () and CO2 evolution rate CER [mmol/L.h] () along the fermentation assay.
Oxygen uptake and CO2 evolution rates increased and went up to 80 mmol/l h at the end of exponential growth. During the accumulation phase they went down again due to cell growth cessation (Fig. 3). Maximum specific oxygen uptake rate (Qo2 max ) was 12 mmol/g of residual biomass per hour and it occurred during exponential growth phase. The carbohydrate cell yield during exponential growth, YX=C and carbohydrate PHA yield during accumulation phase (YPHA=C ) calculated as shown (Fig. 4(a) and (b)) were respectively 0.35 (r2 ¼ 0:996) and 0.2 (r2 ¼ 0:95). The PHAmcl -046 productivity was about 0.23 g/l h and specific polymer production rate per unit of biomass was calculated to be 0.023 g/g h with a polymer content of 35–40% of biomass. Similar results on polymer content and high cell concentration fed-batch strategies have been reported in the literature using P. putida and oleic acid as substrate [22,29–31]. The reported PHAmcl productivity was up to 1.8 g/l h and the specific polymer production rate per unit of biomass calculated from the authors data [22,31] were between 0.007 and 0.025 g/g h comparable to the value obtained in this study.
3.2. Structural characterization The medium side chains polyester (PHAmcl -046) was characterized with respect to average molecular weight, monomer composition, representative structure, crystallinity and thermal properties. The yield of the extraction of the polyester, calculated on the basis of the cellular dry weight was 30% and the average molecular weight reported in Table 1. The average molecular weight was one order higher than the medium side chain polyester produced by P. olevorans growing on natural oils [33,34] as carbon source. This difference should introduce a positive effect on the rheological behaviour. FTIR spectrum (Fig. 5) shows the typical structure of a polyester. An intense absorption bands at 1730, 1740 cm1 characteristic of valence carbonyl vibration (C@O groups) and intense 2955, 2925, 2856 cm1 large bands to aliphatic C–H. Medium signals could be seen at 1000– 1500 cm1 range due to the bendings of CH2 and CH3 , –C–O– and –C–C– groups. Between 1280 and 1050 cm1 signals due to valence antisymmetric and symmetric vibration of C–O–C. At 723 cm1 the transmission signal could associated to extend contribution of CH2 at side chain. Gas chromatographic analysis of PHAmcl revealed peaks which are in agreement with the following typical composition: 60–70 mol% of 3 hydroxydecanoic acid
Table 1 Average molecular weight of PHAmcl -046 biosynthesized by P. putida IPT046 Average molecular weight
M/Dalton
Mn Mw Mv M w /M n
(8.8 0.5) 104 (2.23 0.14) 105 (2.05 0.13) 105 2.53 0.05
4.0
12.0
(B) 3.0
8.0
PHA (g)
Biomass (g)
(A)
4.0 0.0 0.0
10.0
20.0
30.0
C consumed (g)
40.0
2.0 1.0 0.0 10.0
15.0
20.0
25.0
30.0
C consumed (g)
Fig. 4. Relationship between biomass (A) and PHA (B) and consumed carbon source for YX=C and YPHA=C calculation.
R.J. Sanchez et al. / European Polymer Journal 39 (2003) 1385–1394
1389
180
Trasmitance (%)
160 140 120 100 80 60 40 20 0 4000
3500
3000
2500
2000
1500
1000
500
cm -1 Fig. 5. FTIR spectrum of PHAmcl -046 biosynthesized by P. putida IPT 046.
(3HD), 20–25 mol% of 3-hydroxyoctanoic acid (3HO), 1–5 mol% of 3-hydroxyhexanoic acid (3HHx), 1–5 mol% of 3-hydroxydodecanoic acid (3HDD) and 4–5 mol% of 3 hydroxy-5-dodecanoic acid (3HDDD5 ). The presence of monomers differing by two carbon atoms as well as the presence of constituents with more than 10 carbon atoms containing unsaturation clearly reflects the use of the fatty acid biosynthesis pathway as supplier of intermediates for PHA biosynthesis [35]. The precursors for fatty acid biosynthesis are derived from the acetyl-CoA pool [36], which are generated from several substrates including carbohydrates. Acetyl-CoA is first converted to alonyl-CoA which is used to elongate the fatty acid carbon chain in two atoms. Thus, intermediates containing 4, 6, 8, 10, etc. carbon atoms are generated progressively. A key enzyme (b-hydroxydecanoyl-ACP dehydrase) allow the divergence between saturated and unsaturated fatty acids [36] resulting in unsaturated constituents with 12 or more carbon atoms. The PHAmcl -046 is characterized by an heterogeneous sequence from 8, 10 and 12 carbon atoms. The PHAmcl -046 1 H-NMR spectrum (Fig. 6) shows the signals of the methine protons at 5.20 ppm. The methylene protons of C-2 show a set of double doublets at 2.58 and 2.51 ppm. The signal of the terminal methyl group appears at 0.89 ppm. The methylene protons of C-4 give a signal at 1.59 ppm, while all other methylene hydrogens of the saturated side chains give a signal at 1.28. A weak signal at 5.52 ppm showed the presence of a small percentage of side chains containing the –CH@CH– sequences. At 2.01 ppm a small signal related to methylenes linked to double bonds (C5 –C6 ) is observed. In the 13 C-NMR spectrum of PHAmcl (Fig. 7), the signals from the major units can be distinguished. The signal assignment is shown in Table 2. From these sig-
nals it is apparent that the main structural units are derived from 3 hydroxydecanoic acid and 3HO, which are present in a molar ratio of about 2.2–1.0, respectively. One other relevant feature derived from the NMR analysis was the fact that in the units with unsaturation in the side chain the double bond was not located at the end of the chain, as has been reported for PHAmcl from P. olevorans [33,37]. Major signals of the olefenic carbons were observed at 122.8 and 133.7 ppm, indicating as more probable a localization of the double bond between C5 and C6 in the side chains of 3HDD units (Scheme 1). Minor olefinic signals were observed at 128.6 and 130.5 ppm, which could correspond to the localization of the double bonds between C7 and C8 in the side chains of 3-HDD units. The different olefinic position in the side chains should result in differences in the reactivity and properties of the polymers. An estimation of the unsaturation contents could be made from the 13 C-NMR spectrum using the intensity ratio of the signals of the methine groups (ICH@ ) at 133.7 and 122.8 ppm for 3HDDD5 , and 130.6 and 128.7 ppm for 3HDDD7 in relation to the methylene groups (ICH2 ) of 3HD between 10 and 40 ppm. 3HDDD5 would be present in a quantity of 5–6% of the 3HD content and 3HDDD7 in a quantity of 0.8%. The quantity of 3HHx was estimated from the intensity of the signal at 18.2 ppm as 7% of the 3HD content. The quantitative data from the NMR analysis correspond very well to those from the GC analysis. The polymer with average molecular weight Mw ¼ 223:000 Dalton, could be represented as a chain sequence with about 1600 units of which about 80 units could be unsaturated side chain units. The molecular weight of the polymer produced from carbohydrates was
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R.J. Sanchez et al. / European Polymer Journal 39 (2003) 1385–1394
Fig. 6. 1 H-NMR spectrum of PHAmcl -046 in CDCl3 (P. putida IPT 046).
Fig. 7.
13
C-NMR spectrum of PHAmcl -046 in CDCl3 .
R.J. Sanchez et al. / European Polymer Journal 39 (2003) 1385–1394
2 3 4
O
O
O
O
2500
O
O
O
2000
5
5
6
6 7
(b)
7 8
CH3
I / cps
O 1
1391
1500
(a)
1000
CH3 500
CH3 CH3
0
Scheme 1. Representative sequences structure of PHAmcl -046.
10
15
20
25
30
35
40
2θ
d (ppm)
Assignment
169.37 133.72 130.56 128.66 122.84 33.79 33.73 31.76 31.50 29.33 29.16 25.05 24.69 22.61 22.47 18.19 13.95
C-1 C-6 C-8 C-7 C-5 C-4 C-4 C-8 C-6 C-7 C-6 C-5 C-5 C-9 C-7 C-5 C-8
(3HD, 3HO) (3HDDDs) (3HDDD7 ) (3HDDD7 ) (3HDDDs) (3HD) (3HO) (3HD) (3HO) (3HD) (3HD) (3HD) (3HO) (3HD) (3HO) (3HHx) (3HO)
higher than the molecular weight reported for polymer obtained from oleic acid and fatty acid mixture and similar to PHAmcl from octanoic acid [31]. The crystallinity degree of PHAmcl -046 was determined from X-ray diffraction patterns (Fig. 8) of cast polymer films. The DRX (a) correspond to cast film without ageing and DRX (b) aged for 3 weeks at room temperature, time where it attained the maximum of crystallinity. The PHAmcl achieve 24% crystallization. Its relative low crystallinity is probably a consequence of large and irregular pendant side groups which increase the difficulty of close packing in a regular three-dimensional fashion to form a crystalline array. Axial geometry in a chain is the major factor in determining the ability of a chain to form crystallites. The crystalline contribution probably due to isotactic or syndiotactic structure sequences [38].
Fig. 8. X-ray diffractogram of cast films to amorphous polymer (a) and semicrystalline polymer (b).
3.3. Thermal properties The calorimetric analysis (DSC) of PHAmcl -046 shown from the first scan heating thermogram (Fig. 9) an endothermic transition at 56 °C due to melting point with associated enthalpy DHm ¼ 22 J/g. The melting point and percentage of crystalline phase of this PHAmcl 046, obtained from unrelated carbon sources, were similar to PHAmcl obtained from octanoic acid [31]. The length of side chains increase the entropy contribution to crystallite formation and it has a strong influence to crystallite formation and melting temperature associated to crystalline phase. An decrease in the lattice energy of crystallite could be consequence to polymer losing of hydrogen-bonding capability. The random inclusion of 8, 10 and 12 carbon atoms sequences in the chain
2 0
Heat Flow (W/g)
Table 2 13 C-NMR chemical shifts of PHAmcl -046
-2 -4
°
-48.15 C
°
-39.77 C First Scan
°
-42.96 C (I)
-6 Second Scan -39.36°C
22.51 J/g
-8 -10 56.46oC
-12 -80
-60
-40
-20
0
20
40
60
Temperature°(C) Fig. 9. DSC thermogram of PHAmcl -046.
80
R.J. Sanchez et al. / European Polymer Journal 39 (2003) 1385–1394
disturbs both the symmetry and the regular spacing of the hydrogen-bonding sites resulting in a drop of melting point relative to polyhydroxyalkanoates with short side chains. Its thermal behaviour could not be detected to medium side chains polymer from Natural oils to P. olevorans [35] identify as highly amorphous elastomers. The Tg of this medium-chain-length polymer is low as consequence to internal plasticizer effect of the side chains. The glass transition (Tg ) observed from the first scans, )39.7 °C (Fig. 9) was light lower that Tg obtained in second scan ()42.9 °C), probably due to the existence of crystallite anchoring points before first scan. These points were disintegrate as the temperature approaches the melting temperature and the mobility of macromolecules increase as reflected in the Tg from second scan. The DMA data (Fig. 10) gave a more exact detection of glass transition that DSC, based on Cp change. The dynamic storage modulus (E0 ) show the relative mechanical changes at lowest temperature ()39.9 °C) and the loss modulus (E00 ) ()39.4) reflected closely the physical properties change. It reflected molecular process agrees with the idea of Tg as temperature at the onset of segmental motion. The tan d gave change at highest temperature accord to midpoint between the glass and rubbery states Tg ()30.8 °C) to this relaxation process in the viscoelastic spectrum. The mechanical response of PHAmcl -046 films is influenced by crystallite disperse in the matrix. The major effect is to act as a crosslink in the polymer matrix. This makes the elastomer behave as crosslinked network but the crystallite anchoring points at not extense (24%) and it are thermically liable. The PHAmcl -046 elastomer did not show secondary transition up to )150 °C which is associated to aliphatic side mobility as some crankshaft motion or more complex twisting motion. This thermomechanical behaviour most be consequence to relative side chains lower re-
0.3
1500
o
-39.47 C
0.2
1000 -39.95 oC (I)
500
450
300
150
0.1
0
0 -120
-90
-60 -30 0 Temperature°(C)
30
Fig. 10. DMA thermogram of PHAmcl -046 films.
Loss Modulus (MPa)
Storage Modulus (MPa)
2000
0.4
Tan Delta -.-.-
-30.80oC
2500
6
12 o
281 C
10 o
234.92 C
4
8
Weight (mg)
1392
6 98.07 % 2 (10.13 mg)
4 2
0
0 50
100
150
200
250
300
350
Temperatureo(C) Fig. 11. TG-DTG thermogram of PHAmcl -046.
striction which it additional difference between PHAmcl reported from P. olevorans by Ashby and co. [33] where could be appreciate this secondary transition at )80 °C. The thermal degradation of PHAmcl -046 samples (Fig. 11) takes place within a narrow temperature range from 234 °C with maximum degradation rate at 281 °C. Its temperature is in off higher than to melting temperature of crystalline phase. This behaviour gives more processability than PHASCL from to melting process. The reaction pathway of thermal degradation to PHAmcl -046 could be follow similar route of that for polyhydrobutyrate PHB, which is known to involve a random, chain scission reaction of ester groups to form olefinic and carboxylic acid groups out the impact to low unsaturated and heterogeneous contribution in the polymer structure. The thermal stability of biodegradable elastomer produced from renewable sources associated to thermomechanical behaviour give a particularly feature to potential use as film. Furthermore recently research of permeability properties employed carbon dioxide and oxygen as probe molecule [39] confirm these sentences.
4. Conclusions Biodegradable elastomers PHAmcl -046 was obtained by modify strains P. putida-IPT046 from no-relation renewable carbon source which produce 14 g/l of biomass with almost 40% of PHA content. The structural composition was characterized by 3-hydroxydecanoate and 3-hydroxyoctanoate sequence contribution and 6% of olefinic non-terminal sequence in these side chains. This elastomers are semi-crystalline materials with 24% of crystallinity, and a relative low melting point and glass transition temperature. A high thermal stability
R.J. Sanchez et al. / European Polymer Journal 39 (2003) 1385–1394
(234 °C) and appropriate thermomechanical film properties characterize this elastomers and give good prospects for film application.
Acknowledgement The authors thank CNPq to the financial support from PADCT III-02 SBIO.01/97.02/01-25.
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