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PHBV) blends
Polymer Degradation and Stability 94 (2009) 575–583
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Polymer Degradation and Stability journal homepage: www.elsevier.com/locate/polydegstab
Thermal properties and degradability of poly(propylene carbonate)/poly (b-hydroxybutyrate-co-b-hydroxyvalerate) (PPC/PHBV) blends Jian Tao a, b, Cunjiang Song a, b, Mingfeng Cao b, Dan Hu b, Li Liu b, Na Liu b, Shufang Wang a, * a
Key Laboratory of Bioactive Materials for Ministry of Education, NanKai University, Tianjin 300071, China Department of Microbiology, Key Laboratory of Molecular Microbiology and Technology for Ministry of Education, Life Science College, NanKai University, Tianjin 300071, China b
a r t i c l e i n f o
a b s t r a c t
Article history: Received 4 August 2008 Received in revised form 9 January 2009 Accepted 12 January 2009 Available online 3 February 2009
Poly(propylene carbonate)/poly(b-hydroxybutyrate-co-b-hydroxyvalerate) (PPC/PHBV) blends were prepared via the solution casting method at different proportions. Their thermal characteristics were studied by means of differential scanning calorimetry (DSC) and thermogravimetry (TG). The degradability of the blends was investigated in soil suspension cultivation and in vitro degradation testing. The changes of structure and molecular weight for blends were also studied by 1H nuclear magnetic resonance spectroscopy (1H NMR), scanning electron microscopy (SEM) and gel permeation chromatography (GPC) before and after degradation. Although the PPC/PHBV blends were immiscible, the addition of PHBV could improve the thermal stability of PPC. PHBV was degraded mainly by the action of microbial enzymes in the soil suspension, which biodegraded it more rapidly than PPC in a natural environment. PPC was degraded mainly by chemical hydrolysis and random hydrolytic scission of chains in the PBS solution in vitro, and degradation of PPC was more rapid than that of PHBV in a simulated physiological environment. Ó 2009 Elsevier Ltd. All rights reserved.
Keywords: PPC/PHBV blends Thermal properties Biodegradability Degradation in vitro
1. Introduction It is well known that the white pollution caused by petroleumbased materials has become an international environmental problem, and the shortage of petroleum resources has driven efforts to produce biodegradable or bio-based materials. Additionally, global warming, known as the greenhouse effect, is caused mostly by the massive release of carbon dioxide (CO2) into the atmosphere [1]. Thus, the incorporation of CO2 into materials as a means of sequestration has attracted scientific and practical interests recently as a means to reduce greenhouse gas pollution and overcome shortages in classical fuel supplies. Polypropylene carbonate (PPC) was first synthesized by Inoue et al. via the copolymerization of CO2 and propylene oxide (PO) with the molecular structure shown in Fig. 1a [2]. PPC is a biodegradable aliphatic polycarbonate that can be degraded to H2O and CO2, and its synthesis can fix and recycle CO2 in the environment. This polymer has good properties such as compatibility, impact resistance, translucence, innocuousness etc. However, its thermal stability, viscosity and biodegradability still need to be improved.
* Corresponding author. Tel./fax: þ86 22 23503866. E-mail address: [email protected] (S. Wang). 0141-3910/$ – see front matter Ó 2009 Elsevier Ltd. All rights reserved. doi:10.1016/j.polymdegradstab.2009.01.017
Therefore, many approaches to improve these properties have been considered [3–8]. PHBV is a member of the polyhydroxyalkanoate (PHA)-family. PHAs are biodegradable polyesters that are synthesized and accumulated intra-cellularly as a carbon or energy storage material during unbalanced growth by a large variety of bacteria. Currently, more than 80 hydroxyalkanoates have been detected as constituents of PHAs, and more than 300 different microorganisms are known to synthesize and accumulate PHAs intra-cellularly [9]. Poly(b-hydroxybutyrate-co-b-hydroxyvalerate) (PHBV) is an optically active thermoplastic aliphatic polyester with high stereoregularity. Its structure is shown in Fig. 1b [10]. It can be produced by bacterial fermentation from renewable natural materials. It possesses many desirable properties such as biodegradability, biocompatibility, piezoelectricity, etc., and has been used as a biomedical and packing material. However, its high cost, poor processability, and especially low impact resistance at room temperature, resulting from its very high crystallinity, hinder its commercial application on a large scale [11–13]. Many methods have been developed to improve the properties of biodegradable polymers, such as random and block copolymerization, which improve both the biodegradation rate and the mechanical properties of the final products. Physical blending is another effective and simple way to prepare biodegradable
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a
O
CH3
C O
CH
CH2
O n
80
m
)
CH3 CH2 O O CH CH2 C
CH3 O O CH CH2 C
Mass (
b
a : PPC/PHBV=100/0 b : PPC/PHBV=80/20 c : PPC/PHBV=60/40 d : PPC/PHBV=40/60 e : PPC/PHBV=20/80 f : PPC/PHBV=0/100
100
n
a
60
b 40
f
e d
Fig. 1. (a) Structure of PPC. (b) Structure of PHBV.
20
composites with different morphologies and physical characteristics. Studies of PPC blending with other materials such as starch, wood flour, poly(3-caprolactone) (PCL), poly(lactic acid) (PLA), PHBV etc. were reported previously [3–6]. Peng et al. reported the miscibility and crystallization behaviour of PHBV/PPC blends [7], Li et al. researched the crystallinity and morphology of PPC/PHBV blends [8,10]. Wang et al. reported the effect of compatibiliser poly(vinyl acetate) (PVAc) on the thermal behaviour and mechanical properties of PHB/PPC blends [14]. Up to now, there have been few reports on the degradation of PPC/PHBV blends. In this study, the thermal properties of PPC/PHBV blends were studied using differential scanning calorimetry (DSC) and thermogravimetric analysis (TGA), and the degradability was investigated in soil suspension and by in vitro degradation testing. Scanning electron microscopy (SEM), gel permeation chromatography (GPC) and nuclear magnetic resonance (NMR) were also used to investigate the changes of PPC/PHBV blends before and after degradation. 2. Materials and methods 2.1. Materials PPC was obtained from the Inner Mongolia Meng Xi High-Tech Group, China. The weight-average molecular weight (Mw) was 1.5 105. PHBV with 5 mol% HV was obtained from Zeneka Ltd. Co., Japan. The weight-average molecular weight (Mw) was 1.0 105. 2.2. Preparation of the blends
0 0
100
200
300
400
500
T (°C) Fig. 3. TGA thermal diagrams of PPC/PHBV blends.
cast from chloroform. The samples were dried in vacuo at room temperature to a constant weight [15]. 2.3. Analysis of the properties 2.3.1. Differential scanning calorimetry (DSC) A Netzsch DSC 204 differential scanning calorimeter was used to study the glass-transition temperature (Tg) and the melting and crystallization behaviours of the blends. About 20 mg of each sample was heated from 100 to 200 C at a rate of 10 C/min. The midpoints of the transitions in the heating scan were taken as the values of Tg, and the sites of the endothermic peaks on the thermograms were taken as the melting temperature (Tm). 2.3.2. Thermogravimetry analysis (TGA) TGA of the blends was performed with a Netzsch TG 209 thermal analyzer. About 11 mg of each sample was heated from room temperature to 600 C at a heating rate of 10 C/min. The peak temperatures of the derivative thermogravimetry (DTG) curves were taken as the maximum mass-loss rate temperature (Tp). 2.4. Degradation of PPC/PHBV blends
PPC/PHBV blends with weight ratios of 100/0, 80/20, 60/40, 40/ 60, 20/80 and 0/100 were prepared by solution blending and then
2.4.1. Biodegradation in soil suspension The soil sample was collected from topsoil (0w10 cm in depth) in a Dengdian farm field (Xiqing District, Tianjin, China) that had no
a: PPC/PHBV=100/0 b: PPC/PHBV=80/20 c: PPC/PHBV=60/40 d: PPC/PHBV=40/60 e: PPC/PHBV=20/80 f: PPC/PHBV=0/100
0
f e d
/min)
-10
DTG (
DSC(mW/mg)
c
d
-20
e a
-30
c
c b a
-40
b
a : PPC/PHBV=100/0 b : PPC/PHBV=80/20 c : PPC/PHBV=60/40 d : PPC/PHBV=40/60 e : PPC/PHBV=20/80 f : PPC/PHBV=0/100
f -50 -50
0
50
100
150
T (°C) Fig. 2. DSC thermal diagrams of PPC/PHBV blends.
200
0
100
200
300
400
T (°C) Fig. 4. DTG thermal diagrams of PPC/PHBV blends.
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J. Tao et al. / Polymer Degradation and Stability 94 (2009) 575–583 Table 1 Thermal decomposition of PPC/PHBV blends.
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6.9
Tp
Tm
T0
T0 Tm
100/0 80/20 60/40 40/60 20/80 0/100
247 267 270 274 286 286
– 162 165 162 164 163
187 190 192 196 198 199
– 28 27 33 34 36
6.8
pH
Compositions (PPC/PHBV)
6.7
PPC/PHBV = 100/0 PPC/PHBV = 80/20 PPC/PHBV = 60/40 PPC/PHBV = 40/60 PPC/PHBV = 20/80 PPC/PHBV = 0/100
6.6
history of exposure to PPC or PHBV, and measured using standard methods [16]. Some of the soil properties were as follows: pH(H2O), 8.10; pH(KCl), 7.65; H2O, 15.41%; C, 2.14%; H, 0.28%; N, 0.19%. The numbers of total microorganisms and PHA-degraders in the soil were estimated by the MPN method and film-MPN method [17] to be 9.2 107/g wet-soil, and 1.1 105/g wet-soil, respectively. A 500 mL conical flask containing 190 mL mineral salt solution was sterilized. The mineral salt solution contained (/L): KH2PO4, 2.2695 g; Na2HPO4$12H2O, 5.9707 g; NH4Cl, 1 g; MgSO4$7H2O, 0.5 g; CaCl2$2H2O, 0.005 g; ferric ammonium citrate, 0.05 g; yeast extract, 0.05 g; pH, 6.8, and 0.3 g of each PPC/PHBV blend (100/0, 80/20, 60/40, 40/60, 20/80, 0/100) was dried to constant weight and added into the conical flask. The original soil–mineral salt solution was prepared as previously described [18]. A sample of 100 g soil was added into 1000 mL of the sterilized mineral salt solution. Soil was dispersed with a magnetic stirrer for 30 min at 4 C. Then 10 mL of the liquid suspension of the original soil–mineral solution was added into each conical flask described above. All conical flasks were incubated in a reciprocal shaker (120 rpm) at 28–30 C under aerobic conditions in the dark. Each treatment was performed in triplicate. During the biodegradation, the number of PHBV degraders in the soil suspension was estimated via the film-MPN method. The pH of the soil suspension was measured periodically. After biodegradation, the PPC/PHBV blends were removed and washed with 75% ethanol and distilled water to stop any further microbial reaction and dried to constant weight under vacuum. The weights of the blends were then determined. 2.4.2. Degradation in vitro Each of PPC/PHBV blends (100/0, 80/20, 60/40, 40/60, 20/80, 0/100) was submerged in 30 mL of 0.1 M phosphate-buffered saline (PBS) solution in a 50 mL test tube at physiological temperature
6.5
6.4 0
5
10
15
20
(37 2 C). The ionic concentration was adjusted to the physiological range as described in the ASTM Designation: F 1635-04a [19]. Sodium azide (0.1% (w/w)), penicillin (100 U/mL) and streptomycin (100 mg/mL) were added to the solution as the antimicrobials to prevent bacterial growth. The blends were immersed in the physiological solution for the specified period of time, and removed at regular intervals (two weeks, three weeks or four weeks), washed with 75% ethanol and distilled water, and dried to constant weight under vacuum. The weight of the remaining material was determined, and the molecular weights of the blends were measured by GPC. 2.4.3. Scanning electron microscopy (SEM) The surfaces of the PPC/PHBV blends before and after biodegradation in soil suspension were studied with a HITACHI S-3500N scanning electron microscope. The surfaces were vacuum-coated with gold for SEM. 2.4.4. Nuclear magnetic resonance (NMR) Proton (1H) nuclear magnetic resonance spectra (1H NMR) were recorded on a Varian UNITY-plus Spectrometer at 400 MHz for the blends before and after biodegradation. About 20 mg of the blends
60
40
PPC/PHBV = 100/0 PPC/PHBV = 80/20 PPC/PHBV = 60/40 PPC/PHBV = 40/60 PPC/PHBV = 20/80 PPC/PHBV = 0/100
20
log (the number of PHBV-degraders)
Weight remaining ( )
80
PPC/PHBV = 100/0 PPC/PHBV = 60/40 PPC/PHBV = 20/80 PPC/PHBV = 0/100
11 10 9 8 7 6 5 4 0
0 0
5
10
15
20
25
30
30
Fig. 6. Changes of pH in the soil suspension during biodegradation.
12 100
25
Time (days)
5
10
15
20
25
30
Time (days)
Time (days) Fig. 5. Weight changes of PPC/PHBV blends in soil suspension.
Fig. 7. Changes in the number of PHBV degrading microbes in the soil suspension during biodegradation.
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J. Tao et al. / Polymer Degradation and Stability 94 (2009) 575–583
Fig. 8. SEM micrographs of PPC/PHBV blends before biodegradation (1) and after 9 days (2) at magnification of 2000, (a) 100/0, (b) 80/20, (c) 60/40, (d) 40/60.
were dissolved in 1 mL of CDCl3. 1H NMR peak areas were determined by spectrometric integration and are reported as relative intensities representing a given number of hydrogens. The compositions of the blends were determined before and after biodegradation.
510 system with an HP Mix ED-D column. Chloroform was used as the eluent at 40 C, and the flow rate was 1.0 mL/min.
2.4.5. Gel permeation chromatography (GPC) The number average molecular weight (Mn), weight-average molecular weight (Mw) and polydispersity (Mw/Mn) were determined by gel permeation chromatography (GPC) using a Waters
3.1. Thermal characterization and miscibility of the blends
3. Results and discussion
It is well known that the thermal characterization of polymer blends is a feasible method for the determination of their miscibility.
J. Tao et al. / Polymer Degradation and Stability 94 (2009) 575–583
579
Fig. 9. 1H NMR spectra of pure PPC and PHBV.
The miscibility between any two polymers in the amorphous state was detected by the shift of Tg. So, the Tg behaviours of PPC/PHBV blends were measured to evaluate their miscibility. Miscibility and phase behaviour of the different blend systems were investigated by DSC. Fig. 2 shows the Tg of PPC/PHBV blends as a function of composition. Two Tgs were observed in the blends: one was about 28 C, close to the Tg of PPC, and the other was about 6 C, which is the Tg of PHBV. These transitions were nearly unchanged with the variation of PHBV content. The Tgs of PPC and PHBV were independent of the ratio of the components in the blends. The Tm could also be used to examine the miscibility of the system. As shown in the curves, there was no Tm for PPC because it was an amorphous polymer [6]. Meanwhile, PHBV was a crystalline polymer, and the Tm of pure PHBV was about 163 C. Furthermore, the Tm value was unchanged with increasing concentrations of PPC
in the PPC/PHBV blends. These observations were an indication of the immiscibility of the PPC/PHBV blends, which has been reported previously [7,8]. 3.2. Thermal stability TGA was performed for the blends, and the weight loss due to the volatilization of the degradation products was monitored as a function of temperature as shown in Figs. 3 and 4. The TGA curves showed that the blends displayed obvious thermal weight losses. PPC showed low thermal stability. Peng et al. reported that PPC was easily decomposed to cyclic carbonate at a temperature of only about 180 C, and underwent a reduction in molecular weight in which ‘‘back-biting’’ reactions of the free hydroxyl groups were said to play a significant part [7].
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J. Tao et al. / Polymer Degradation and Stability 94 (2009) 575–583
The DTG curves for PPC/PHBV blends in Figs. 3 and 4 show the temperatures of decomposition for PPC and PHBV in the blends. The temperature of decomposition increased with increasing PHBV content. The thermal properties of the blends are shown in Table 1. The thermal decomposition temperature (T0) of PPC was 187 C, the T0 of PHBV was 199 C, and that of blends rose gradually with increasing PHBV content. The Tp of the blends behaved in a similar fashion. The results indicated that PHBV could improve the thermal stability of PPC efficiently. 3.3. The biodegradability of PPC/PHBV blends in soil suspension 3.3.1. Weight changes during biodegradation As shown in Fig. 5, the biodegradation rate of pure PPC was low, and the weight loss was only 3.3% after 29 days in soil suspension, while pure PHBV was completely biodegraded after 12 days. In fact, the higher the concentration of PHBV in the blends, the more quickly the blends were biodegraded. PPC/PHBV blend (20/80) was completely biodegraded after 14 days. PPC/PHBV blend (40/60) was completely biodegraded after 24 days. The results showed that the biodegradability of PHBV was higher than that of PPC in the soil suspension. In the polymer blends, higher concentrations of PHBV led to more rapid biodegradation. This may be due to a larger number of PHBV degraders in the soil than PPC degraders. PPC is an aliphatic polycarbonate that was synthesized artificially by chemical methods. Because there is no PPC in the natural environment, the number of PPC degraders in the soil is very low. Obviously, PHBV was synthesized by bacteria and could also be utilized by bacteria easily. On the other hand, when PPC was blended with the more rapidly degraded PHBV, PPC in the blends would be biodegraded faster than pure PPC. The degradation enzymes excreted by the microorganisms first degraded the PHBV, and then diffused to the interfaces of the blend films. More surface area of PPC was exposed to the environment, and the great amount of –COOH end groups and organic acids produced previously catalyzed the later biodegradation of PPC and PHBV. The enzymatic hydrolysis and chemical scission occurred at the interfaces as well at the film surface. The results mentioned above revealed that hydrolysis of PPC in the blend films was accelerated by the presence of PHBV. 3.3.2. The change of pH during biodegradation Fig. 6 shows the pH changes of soil suspension during biodegradation. With the biodegradation of PPC/PHBV blends, the pH of soil suspension decreased gradually. This might be because of organic acids produced by PHBV degraders. The pure PHBV was metabolized by the microorganisms most easily, so the pH changed most dramatically. The more easily the blends could be used, the faster the microorganisms grew, and the faster the pH would be changed. After the pH reached its minimum, it rose slightly due to consumption of organic acids by microorganisms in the soil suspension during the process of biodegradation. As there was no carbon source other than the PPC/PHBV blends in the soil suspension and as the PPC/PHBV was biodegraded to completion, the microorganisms in the soil suspension began to consume the organic acids as a carbon source. 3.3.3. The number of PHBV degraders during biodegradation Fig. 7 shows the changes in the number of PHBV degraders during biodegradation in the soil suspension. The initial number of PHBV degraders in the soil was 1.1 105/g wet-soil, and the number of PHBV degraders increased dramatically during the degradation of PHBV, then decreased again after PHBV degradation was complete. The greater the concentration of PHBV in the blends, the faster the number of PHBV degraders would increase. This implied
Fig. 10. 1H NMR spectrum of PPC/PHBV blend (60/40) before degradation.
that the PHBV in the blends was utilized as a carbon and energy source for bacterial growth. 3.3.4. SEM analysis The surface morphology of PPC/PHBV blend films (100/0, 80/20, 60/40, 40/60) before and after biodegradation in soil suspension was studied via SEM (Fig. 8). Prior to biodegradation, all blends showed a relatively compact and smooth surface. The transmittance and thickness of blend films were reduced after nine days of biodegradation. Some of the blend films (20/80, 0/100) could not be studied by SEM after nine days of biodegradation. Blend films failed to maintain their integrity when the content of PHBV was over 80% in the PPC/PHBV blend; the surfaces of the blends were eroded by the action of microorganisms, which made the blend films change their shape and the thickness. Therefore, after nine days of biodegradation, the blend films were too fragmented to perform SEM analysis. It is well known that the enzymatic hydrolysis of biodegradable polyesters such as PCL, PHB, and PLA proceeds mainly via a surface erosion mechanism [20]. The high density of pore formation on the surface of the blend films accelerated the enzymatic hydrolysis inside the blend films as well as at the film surface. The depth and extent of holes depended upon the composition of the blends. These holes inside the film matrix were due to easier penetration of water into amorphous parts of the film, and hydrolysis occurred at these locations [21]. There were many deep holes and cracks over surface of some PPC/PHBV blend films (60/40, 40/60) after biodegradation for nine days. This demonstrates that PPC/PHBV blend films with higher PHBV content were eroded more severely by microbes in the soil suspension.
Table 2 –CH2 mole fraction of PPC and PHBV in PPC/PHBV blend (60/40) during biodegradation. Time (d)
Ratio of –CH2 integral area between PPC and PHBV
Ratio of mole fraction between PPC and PHBV in blends
0 9 19 29
10.00/6.79 10.02/2.61 10.00/0.54 10.00/0.43
59.56/40.44 79.33/20.67 94.88/5.12 95.88/4.12
J. Tao et al. / Polymer Degradation and Stability 94 (2009) 575–583
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3.3.5. NMR analysis The 1H NMR spectra of the PPC/PHBV blends (100/0, 0/100 and 60/40) are shown in Figs. 9 and 10, respectively. One PPC/PHBV blend (60/40) was taken as an example to investigate the changes in composition during the process of biodegradation. For PPC, the peaks at 1.25–1.4 (–CH3), 4.1–4.3 (–CH2) and 4.95–5.05 (–CH) were attributed to methyl, methylene and methine protons. For PHBV, the peaks were at 0.85–0.95 (–CH3 of HV), 1.25–1.4 (–CH3 of HB), 2.45–2.65 (–CH2 of HB and HV) and 5.2–5.3 (–CH of HB and HV). In order to study the composition changes of the PPC/PHBV blends before and after biodegradation, the peaks at 2.45–2.65 (–CH2 of PHBV) and 4.1–4.3 (–CH2 of PPC) of the 1H NMR spectra were selected for investigation. The actual mole fractions of PPC and PHBV could be calculated by integrating the areas of –CH2 peaks on the graph [22]. The composition changes in each blend were investigated by calculating both mole fractions in the blends. In Fig. 10, the integrated areas of 1H NMR spectra of the methylenes of the PPC/PHBV blend (60/40) before biodegradation were 10.00 (integral area of –CH2 for PPC) and 6.79 (integral area of –CH2 for PHBV), respectively. The mole fraction of PPC was 10.00/ (10.00 þ 6.79) ¼ 59.56%; the mole fraction of PHBV was 6.79/ (10.00 þ 6.79) ¼ 40.44%. Therefore, the actual initial mole fraction of the PPC/PHBV blend (60/40) was 59.56/40.44. Table 2 shows the integral area and mole fraction of each component during biodegradation. All integral values are taken from the data in Figs. 10 and 11. The mole fraction of PPC/PHBV was 79.33/20.67 at the ninth day of biodegradation. After 19 and 29 days, the mole fractions of PPC/PHBV changed to 94.88/5.12 and 95.88/4.12, respectively. We observed that the mole fraction of PHBV in the PPC/PHBV blend gradually decreased during biodegradation, while the mole fraction of PPC in the PPC/PHBV blend increased. PPC was biodegraded slowly in the soil suspension because PPC-degrading microorganisms in the soil were rare. These data demonstrated that the weight loss was mainly due to degradation of PHBV.
3.4. Degradation in vitro Fig. 12 shows the remaining weight of PPC/PHBV blends (100/0, 80/20, 60/40, 40/60, 20/80 and 0/100) degraded in vitro for more than 200 days. The chemical decomposition rate for PPC/PHBV (0/100) was slowest in all blends. Meanwhile, higher concentrations of PPC in the blends resulted in more rapid degradation in vitro. In fact, PPC/PHBV blends immersed in PBS solution could be hydrolyzed, with hydrolysis first appearing on the surface, followed
100
Weight Remaining (
)
98 96 94 92 90 PPC/PHBV=100/0 PPC/PHBV=80/20 PPC/PHBV=60/40 PPC/PHBV=40/60 PPC/PHBV=20/80 PPC/PHBV=0/100
88 86 84 0
50
100
150
200
Time (days) Fig. 11. 1H NMR spectrum of PPC/PHBV blend (60/40) after degradation in soil suspension.
Fig. 12. Weight changes of PPC/PHBV blends during in vitro degradation.
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Table 3 Molecular weights and distributions of PPC and PHBV before and after degradation. Before degradation
PPC PHBV
After degradation for 140 days in vitro
After biodegradation for 24 days (PPC) and 4 days (PHBV) in soil suspension
Mn
Mw
Polydispersity
Mn
Mw
Polydispersity
Mn
Mw
Polydispersity
49,900 38,800
143,500 95,300
2.9 2.4
24,200 38,300
71,500 78,100
3.0 2.0
49,500 24,000
15,0200 65,700
3.0 2.7
by swelling of the materials after absorption of water leading to an increase in the bulk of the material. This expansion led to the presentation of interior surface area, and allowed the PBS solution to penetrate into the material for further degradation. As the chemical bonds in the main chain broke down to form large molecular fragments, and these fragments were further hydrolyzed to oligomers and monomers that then dissolved in the PBS solution. This process led to a decrease of molecular weight or weight loss in the PPC/PHBV blends. It was reported that the hydrolysis rate of some aliphatic polyesters was affected by their degree of crystallinity [21,23]. Since PPC was fully amorphous and PHBV had a relatively high degree of crystallinity as shown in Fig. 2, water penetration in the PPC/PHBV blend film was very difficult and the chemical bonds in PHBV appeared to be difficult to hydrolyze. Therefore, the different degradation rates of PPC and PHBV in vitro might be attributed to their degrees of crystallinity. The changes of molecular weight of PPC and PHBV described below supported the above explanations. 3.5. The comparison between biodegradation in soil suspension and in vitro Table 3 shows the molecular weight changes of PPC and PHBV before and after degradation in soil suspension and in vitro. These results indicate that the Mn of PPC was reduced from 49,900 to 24,200 after 140 in PBS solution, while the PHBV was largely intact under the same conditions (from 38,800 to 38,300). The polydispersity of PPC changed from 2.9 to 3.0, and that of PHBV changed from 2.4 to 2.0. This suggested that the degradation of PPC was mainly through chemical hydrolysis and random hydrolytic scission of polymer chains in the PBS solution. PHBV was hardly degraded in vitro, and only small molecular fragments dissolved in the PBS, but the main chain could not be broken down. In contrast, PHBV was easily biodegraded in the soil suspension, and the Mn changed from 38,800 to 24,000 after only four days, while the polydispersity changed from 2.4 to 2.7. The weight loss of PPC/PHBV blends in soil suspension was caused mainly by degradation of the PHBV component. Enzymatic degradation involved hydrolysis of ester bonds and the chemical scission of macromolecular chains into small fragments or oligomers. These low molecular weight fragments are soluble in water, and for this reason the films start to fragment on the surface [21]. However, because of a lack of PPC degraders in the soil suspension, PPC could not be degraded. Even after extension of the degradation time to 24 days, the Mn of PPC changed from 49,900 to 49,500 and the polydispersity of PPC changed from 2.9 to 3.0. There was nearly no change in the Mn for PPC, but the polydispersity widened a little. This may have been due to chemical hydrolysis in the soil suspension. 4. Conclusions PPC/PHBV blends formed immiscible mixtures. However, the addition of PHBV improved the thermal stability of PPC. PHBV was biodegraded more rapidly than PPC in the natural environment due to the action of microorganisms, and there were more
microorganisms capable of degrading PHBV than PPC. PPC was degraded more rapidly than PHBV in PBS in a simulated physiological environment in vitro, because degradation of the blends was mainly through chemical hydrolysis and random hydrolytic scission of chains. PPC was fully amorphous, allowing greater solvent access, while PHBV had a relative high degree of crystallinity. In order to make PPC/PHBV show a better application potential, a third component (such as PLA or OMMT) can be added into the binary blends, and this will be the focus of future work. Acknowledgements This study was supported by Chinese Natural Science Foundation (No. 20374032), and Key Project, Tianjin, China (No. 06YFGZSH06800). References [1] Li XH, Meng YZ, Chen GQ, Zhu Q, Tjong SC. Thermal properties and rheological behaviour of biodegradable poly(propylene carbonate) derived from carbon dioxide and propylene oxide. J Appl Polym Sci 2004;94(2):711–6. [2] Inoue S, Koinuma H, Tsuruta TJ. Copolymerization of carbon dioxide and epoxide. J Polym Sci Part B Polym Lett 1969;7:287–92. [3] Lu XL, Du FG, Ge XC, Xiao M, Meng YZ. Biodegradability and thermal stability of poly(propylene carbonate)/starch composites. J Biomed Mater Res A 2006;77(4):653–8. [4] Ge XC, Zhu Q, Meng YZ. Fabrication and characterization of biodegradable poly(propylene carbonate)/wood flour composites. J Appl Polym Sci 2006;99:782–7. [5] Hirotsu Toshihiro, Ketelaars AAJ, Nakayama Kazuo. Biodegradation of poly(3-caprolactone)–polycarbonate blend sheets. Polym Degrad Stab 2000;68: 311–6. [6] Ma XF, Yu JG, Wang N. Compatibility characterization of poly(lactic acid)/ poly(propylene carbonate) blends. J Polym Sci Polym Phys 2006;44:94–101. [7] Peng SW, An YX, Chen C, Fei B, Zhuang YG, Dong LS. Miscibility and crystallization behavior of poly(3-hydroxybutyrate-co-3-hydroxyvalerate)/poly(propylene carbonate) blends. J Appl Polym Sci 2003;90:4054–60. [8] Li J, Lai MF, Liu JJ. Effect of poly(propylene carbonate) on the crystallization and melting behaviour of poly(b-hydroxybutyrate-co-b-hydroxyvalerate). J Appl Polym Sci 2004;92:2514–21. [9] Song CJ, Zhao LX, Shin Ono, Shimasaki C, Inoue M. Production of poly(3hydroxybutyrate-co-3-hydroxyvalerate) from cottonseed oil and valeric acid in batch culture of Ralstonia sp. strain JC-64. Appl Biochem Biotech 2001;94:169–78. [10] Li J, Lai MF, Liu JJ. Control and development of crystallinity and morphology in carbonate) poly(b-hydroxybutyrate-co-b-hydroxyvalerate)/poly(propylene blends. J Appl Polym Sci 2005;98:1427–36. [11] Brandl H, Bachofen R, Mayer J, Wintermantel E. Degradation and applications of polyhydroxyalkanoates. Can J Microbiol 1995;41(1):143–53. [12] Delafield FP, Doudoroff M, Palleroni NJ, Lusty CJ, Contopoulos R. Decomposition of poly-b-hydroxybutyrate by Pseudomonas. J Bacteriol 1965 Nov: 1455–66. [13] Hankermeyer CR, Tjeerdema RS. Polyhydroxybutyrate: plastic made and degraded by microorganisms. Rev Environ Contam Toxicol 1999;159:1–24. [14] Wang XY, Peng SW, Dong LS. Effect of poly(vinyl acetate) (PVAc) on thermal behavior and mechanical properties of poly(3-hydroxybutyrate)/poly(propylene carbonate) (PHB/PPC) blends. Colloid Polym Sci 2005;284:167–74. [15] Caballero KP, Karel SF, Register RA. Biosynthesis and characterization of hydroxybutyrate–hydroxycaproate copolymers. Int J Biol Macromol 1995;17(2):86–92. [16] Ishikuri S, editor. New compilation soil microbiology experimental method. Japan: YOKENDO; 1992 [chapter 2, in Japanese]. [17] Song CJ, Uchida U, Ono S, Shimasaki C, Inoue M. Estimation of number of polyhydroxyalkanoate (PHA)-degraders in soil and isolation of degraders based on the method of most probable number (MPN) using PHA-film. Biosci Biotechnol Biochem 2001;65(5):1214–7. [18] Wang SF, Song CJ, Chen GX, Guo TY, Liu J, Zhang BH, et al. Characteristics and biodegradation properties of poly(3-hydroxybutyrate-co-3-hydroxyvalerate)/
J. Tao et al. / Polymer Degradation and Stability 94 (2009) 575–583 organophilic montmorillonite (PHBV/OMMT) nanocomposite. Polym Degrad Stab 2005;87(1):69–76. [19] ASTM Designation: F 1635-04a. Standard test method for in vitro degradation testing of hydrolytically degradable polymer resins and fabricated forms for surgical implants. [20] Tsuji H, Miyauchi S. Poly(L-lactide) VI effects of crystallinity on enzymatic hydrolysis of poly(L-lactide) without free amorphous region. Polym Degrad Stab 2001;71:415–24.
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[21] Seretoudi G, Bikiaris D, Panayiotou C. Synthesis, characterization and biodegradability of poly(ethylene succinate)/poly(3-caprolactone) block copolymers. Polymer 2002;43:5405–15. [22] Sheth M, Kumar RA, Dave V, Gross RA, McCarthy SP. Biodegradable polymer blends of poly(lactic acid) and poly(ethylene glycol). J Appl Polym Sci 1997;66:1495–505. [23] Gan Z, Liang Q, Zhang J, Jing X. Enzymatic degradation of poly(3-caprolactone) film in phosphate buffer solution containing lipases. Polym Degrad Stab 1997;56:209–13.
Contents lists available at ScienceDirect
Polymer Degradation and Stability journal homepage: www.elsevier.com/locate/polydegstab
Thermal properties and degradability of poly(propylene carbonate)/poly (b-hydroxybutyrate-co-b-hydroxyvalerate) (PPC/PHBV) blends Jian Tao a, b, Cunjiang Song a, b, Mingfeng Cao b, Dan Hu b, Li Liu b, Na Liu b, Shufang Wang a, * a
Key Laboratory of Bioactive Materials for Ministry of Education, NanKai University, Tianjin 300071, China Department of Microbiology, Key Laboratory of Molecular Microbiology and Technology for Ministry of Education, Life Science College, NanKai University, Tianjin 300071, China b
a r t i c l e i n f o
a b s t r a c t
Article history: Received 4 August 2008 Received in revised form 9 January 2009 Accepted 12 January 2009 Available online 3 February 2009
Poly(propylene carbonate)/poly(b-hydroxybutyrate-co-b-hydroxyvalerate) (PPC/PHBV) blends were prepared via the solution casting method at different proportions. Their thermal characteristics were studied by means of differential scanning calorimetry (DSC) and thermogravimetry (TG). The degradability of the blends was investigated in soil suspension cultivation and in vitro degradation testing. The changes of structure and molecular weight for blends were also studied by 1H nuclear magnetic resonance spectroscopy (1H NMR), scanning electron microscopy (SEM) and gel permeation chromatography (GPC) before and after degradation. Although the PPC/PHBV blends were immiscible, the addition of PHBV could improve the thermal stability of PPC. PHBV was degraded mainly by the action of microbial enzymes in the soil suspension, which biodegraded it more rapidly than PPC in a natural environment. PPC was degraded mainly by chemical hydrolysis and random hydrolytic scission of chains in the PBS solution in vitro, and degradation of PPC was more rapid than that of PHBV in a simulated physiological environment. Ó 2009 Elsevier Ltd. All rights reserved.
Keywords: PPC/PHBV blends Thermal properties Biodegradability Degradation in vitro
1. Introduction It is well known that the white pollution caused by petroleumbased materials has become an international environmental problem, and the shortage of petroleum resources has driven efforts to produce biodegradable or bio-based materials. Additionally, global warming, known as the greenhouse effect, is caused mostly by the massive release of carbon dioxide (CO2) into the atmosphere [1]. Thus, the incorporation of CO2 into materials as a means of sequestration has attracted scientific and practical interests recently as a means to reduce greenhouse gas pollution and overcome shortages in classical fuel supplies. Polypropylene carbonate (PPC) was first synthesized by Inoue et al. via the copolymerization of CO2 and propylene oxide (PO) with the molecular structure shown in Fig. 1a [2]. PPC is a biodegradable aliphatic polycarbonate that can be degraded to H2O and CO2, and its synthesis can fix and recycle CO2 in the environment. This polymer has good properties such as compatibility, impact resistance, translucence, innocuousness etc. However, its thermal stability, viscosity and biodegradability still need to be improved.
* Corresponding author. Tel./fax: þ86 22 23503866. E-mail address: [email protected] (S. Wang). 0141-3910/$ – see front matter Ó 2009 Elsevier Ltd. All rights reserved. doi:10.1016/j.polymdegradstab.2009.01.017
Therefore, many approaches to improve these properties have been considered [3–8]. PHBV is a member of the polyhydroxyalkanoate (PHA)-family. PHAs are biodegradable polyesters that are synthesized and accumulated intra-cellularly as a carbon or energy storage material during unbalanced growth by a large variety of bacteria. Currently, more than 80 hydroxyalkanoates have been detected as constituents of PHAs, and more than 300 different microorganisms are known to synthesize and accumulate PHAs intra-cellularly [9]. Poly(b-hydroxybutyrate-co-b-hydroxyvalerate) (PHBV) is an optically active thermoplastic aliphatic polyester with high stereoregularity. Its structure is shown in Fig. 1b [10]. It can be produced by bacterial fermentation from renewable natural materials. It possesses many desirable properties such as biodegradability, biocompatibility, piezoelectricity, etc., and has been used as a biomedical and packing material. However, its high cost, poor processability, and especially low impact resistance at room temperature, resulting from its very high crystallinity, hinder its commercial application on a large scale [11–13]. Many methods have been developed to improve the properties of biodegradable polymers, such as random and block copolymerization, which improve both the biodegradation rate and the mechanical properties of the final products. Physical blending is another effective and simple way to prepare biodegradable
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a
O
CH3
C O
CH
CH2
O n
80
m
)
CH3 CH2 O O CH CH2 C
CH3 O O CH CH2 C
Mass (
b
a : PPC/PHBV=100/0 b : PPC/PHBV=80/20 c : PPC/PHBV=60/40 d : PPC/PHBV=40/60 e : PPC/PHBV=20/80 f : PPC/PHBV=0/100
100
n
a
60
b 40
f
e d
Fig. 1. (a) Structure of PPC. (b) Structure of PHBV.
20
composites with different morphologies and physical characteristics. Studies of PPC blending with other materials such as starch, wood flour, poly(3-caprolactone) (PCL), poly(lactic acid) (PLA), PHBV etc. were reported previously [3–6]. Peng et al. reported the miscibility and crystallization behaviour of PHBV/PPC blends [7], Li et al. researched the crystallinity and morphology of PPC/PHBV blends [8,10]. Wang et al. reported the effect of compatibiliser poly(vinyl acetate) (PVAc) on the thermal behaviour and mechanical properties of PHB/PPC blends [14]. Up to now, there have been few reports on the degradation of PPC/PHBV blends. In this study, the thermal properties of PPC/PHBV blends were studied using differential scanning calorimetry (DSC) and thermogravimetric analysis (TGA), and the degradability was investigated in soil suspension and by in vitro degradation testing. Scanning electron microscopy (SEM), gel permeation chromatography (GPC) and nuclear magnetic resonance (NMR) were also used to investigate the changes of PPC/PHBV blends before and after degradation. 2. Materials and methods 2.1. Materials PPC was obtained from the Inner Mongolia Meng Xi High-Tech Group, China. The weight-average molecular weight (Mw) was 1.5 105. PHBV with 5 mol% HV was obtained from Zeneka Ltd. Co., Japan. The weight-average molecular weight (Mw) was 1.0 105. 2.2. Preparation of the blends
0 0
100
200
300
400
500
T (°C) Fig. 3. TGA thermal diagrams of PPC/PHBV blends.
cast from chloroform. The samples were dried in vacuo at room temperature to a constant weight [15]. 2.3. Analysis of the properties 2.3.1. Differential scanning calorimetry (DSC) A Netzsch DSC 204 differential scanning calorimeter was used to study the glass-transition temperature (Tg) and the melting and crystallization behaviours of the blends. About 20 mg of each sample was heated from 100 to 200 C at a rate of 10 C/min. The midpoints of the transitions in the heating scan were taken as the values of Tg, and the sites of the endothermic peaks on the thermograms were taken as the melting temperature (Tm). 2.3.2. Thermogravimetry analysis (TGA) TGA of the blends was performed with a Netzsch TG 209 thermal analyzer. About 11 mg of each sample was heated from room temperature to 600 C at a heating rate of 10 C/min. The peak temperatures of the derivative thermogravimetry (DTG) curves were taken as the maximum mass-loss rate temperature (Tp). 2.4. Degradation of PPC/PHBV blends
PPC/PHBV blends with weight ratios of 100/0, 80/20, 60/40, 40/ 60, 20/80 and 0/100 were prepared by solution blending and then
2.4.1. Biodegradation in soil suspension The soil sample was collected from topsoil (0w10 cm in depth) in a Dengdian farm field (Xiqing District, Tianjin, China) that had no
a: PPC/PHBV=100/0 b: PPC/PHBV=80/20 c: PPC/PHBV=60/40 d: PPC/PHBV=40/60 e: PPC/PHBV=20/80 f: PPC/PHBV=0/100
0
f e d
/min)
-10
DTG (
DSC(mW/mg)
c
d
-20
e a
-30
c
c b a
-40
b
a : PPC/PHBV=100/0 b : PPC/PHBV=80/20 c : PPC/PHBV=60/40 d : PPC/PHBV=40/60 e : PPC/PHBV=20/80 f : PPC/PHBV=0/100
f -50 -50
0
50
100
150
T (°C) Fig. 2. DSC thermal diagrams of PPC/PHBV blends.
200
0
100
200
300
400
T (°C) Fig. 4. DTG thermal diagrams of PPC/PHBV blends.
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J. Tao et al. / Polymer Degradation and Stability 94 (2009) 575–583 Table 1 Thermal decomposition of PPC/PHBV blends.
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6.9
Tp
Tm
T0
T0 Tm
100/0 80/20 60/40 40/60 20/80 0/100
247 267 270 274 286 286
– 162 165 162 164 163
187 190 192 196 198 199
– 28 27 33 34 36
6.8
pH
Compositions (PPC/PHBV)
6.7
PPC/PHBV = 100/0 PPC/PHBV = 80/20 PPC/PHBV = 60/40 PPC/PHBV = 40/60 PPC/PHBV = 20/80 PPC/PHBV = 0/100
6.6
history of exposure to PPC or PHBV, and measured using standard methods [16]. Some of the soil properties were as follows: pH(H2O), 8.10; pH(KCl), 7.65; H2O, 15.41%; C, 2.14%; H, 0.28%; N, 0.19%. The numbers of total microorganisms and PHA-degraders in the soil were estimated by the MPN method and film-MPN method [17] to be 9.2 107/g wet-soil, and 1.1 105/g wet-soil, respectively. A 500 mL conical flask containing 190 mL mineral salt solution was sterilized. The mineral salt solution contained (/L): KH2PO4, 2.2695 g; Na2HPO4$12H2O, 5.9707 g; NH4Cl, 1 g; MgSO4$7H2O, 0.5 g; CaCl2$2H2O, 0.005 g; ferric ammonium citrate, 0.05 g; yeast extract, 0.05 g; pH, 6.8, and 0.3 g of each PPC/PHBV blend (100/0, 80/20, 60/40, 40/60, 20/80, 0/100) was dried to constant weight and added into the conical flask. The original soil–mineral salt solution was prepared as previously described [18]. A sample of 100 g soil was added into 1000 mL of the sterilized mineral salt solution. Soil was dispersed with a magnetic stirrer for 30 min at 4 C. Then 10 mL of the liquid suspension of the original soil–mineral solution was added into each conical flask described above. All conical flasks were incubated in a reciprocal shaker (120 rpm) at 28–30 C under aerobic conditions in the dark. Each treatment was performed in triplicate. During the biodegradation, the number of PHBV degraders in the soil suspension was estimated via the film-MPN method. The pH of the soil suspension was measured periodically. After biodegradation, the PPC/PHBV blends were removed and washed with 75% ethanol and distilled water to stop any further microbial reaction and dried to constant weight under vacuum. The weights of the blends were then determined. 2.4.2. Degradation in vitro Each of PPC/PHBV blends (100/0, 80/20, 60/40, 40/60, 20/80, 0/100) was submerged in 30 mL of 0.1 M phosphate-buffered saline (PBS) solution in a 50 mL test tube at physiological temperature
6.5
6.4 0
5
10
15
20
(37 2 C). The ionic concentration was adjusted to the physiological range as described in the ASTM Designation: F 1635-04a [19]. Sodium azide (0.1% (w/w)), penicillin (100 U/mL) and streptomycin (100 mg/mL) were added to the solution as the antimicrobials to prevent bacterial growth. The blends were immersed in the physiological solution for the specified period of time, and removed at regular intervals (two weeks, three weeks or four weeks), washed with 75% ethanol and distilled water, and dried to constant weight under vacuum. The weight of the remaining material was determined, and the molecular weights of the blends were measured by GPC. 2.4.3. Scanning electron microscopy (SEM) The surfaces of the PPC/PHBV blends before and after biodegradation in soil suspension were studied with a HITACHI S-3500N scanning electron microscope. The surfaces were vacuum-coated with gold for SEM. 2.4.4. Nuclear magnetic resonance (NMR) Proton (1H) nuclear magnetic resonance spectra (1H NMR) were recorded on a Varian UNITY-plus Spectrometer at 400 MHz for the blends before and after biodegradation. About 20 mg of the blends
60
40
PPC/PHBV = 100/0 PPC/PHBV = 80/20 PPC/PHBV = 60/40 PPC/PHBV = 40/60 PPC/PHBV = 20/80 PPC/PHBV = 0/100
20
log (the number of PHBV-degraders)
Weight remaining ( )
80
PPC/PHBV = 100/0 PPC/PHBV = 60/40 PPC/PHBV = 20/80 PPC/PHBV = 0/100
11 10 9 8 7 6 5 4 0
0 0
5
10
15
20
25
30
30
Fig. 6. Changes of pH in the soil suspension during biodegradation.
12 100
25
Time (days)
5
10
15
20
25
30
Time (days)
Time (days) Fig. 5. Weight changes of PPC/PHBV blends in soil suspension.
Fig. 7. Changes in the number of PHBV degrading microbes in the soil suspension during biodegradation.
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Fig. 8. SEM micrographs of PPC/PHBV blends before biodegradation (1) and after 9 days (2) at magnification of 2000, (a) 100/0, (b) 80/20, (c) 60/40, (d) 40/60.
were dissolved in 1 mL of CDCl3. 1H NMR peak areas were determined by spectrometric integration and are reported as relative intensities representing a given number of hydrogens. The compositions of the blends were determined before and after biodegradation.
510 system with an HP Mix ED-D column. Chloroform was used as the eluent at 40 C, and the flow rate was 1.0 mL/min.
2.4.5. Gel permeation chromatography (GPC) The number average molecular weight (Mn), weight-average molecular weight (Mw) and polydispersity (Mw/Mn) were determined by gel permeation chromatography (GPC) using a Waters
3.1. Thermal characterization and miscibility of the blends
3. Results and discussion
It is well known that the thermal characterization of polymer blends is a feasible method for the determination of their miscibility.
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579
Fig. 9. 1H NMR spectra of pure PPC and PHBV.
The miscibility between any two polymers in the amorphous state was detected by the shift of Tg. So, the Tg behaviours of PPC/PHBV blends were measured to evaluate their miscibility. Miscibility and phase behaviour of the different blend systems were investigated by DSC. Fig. 2 shows the Tg of PPC/PHBV blends as a function of composition. Two Tgs were observed in the blends: one was about 28 C, close to the Tg of PPC, and the other was about 6 C, which is the Tg of PHBV. These transitions were nearly unchanged with the variation of PHBV content. The Tgs of PPC and PHBV were independent of the ratio of the components in the blends. The Tm could also be used to examine the miscibility of the system. As shown in the curves, there was no Tm for PPC because it was an amorphous polymer [6]. Meanwhile, PHBV was a crystalline polymer, and the Tm of pure PHBV was about 163 C. Furthermore, the Tm value was unchanged with increasing concentrations of PPC
in the PPC/PHBV blends. These observations were an indication of the immiscibility of the PPC/PHBV blends, which has been reported previously [7,8]. 3.2. Thermal stability TGA was performed for the blends, and the weight loss due to the volatilization of the degradation products was monitored as a function of temperature as shown in Figs. 3 and 4. The TGA curves showed that the blends displayed obvious thermal weight losses. PPC showed low thermal stability. Peng et al. reported that PPC was easily decomposed to cyclic carbonate at a temperature of only about 180 C, and underwent a reduction in molecular weight in which ‘‘back-biting’’ reactions of the free hydroxyl groups were said to play a significant part [7].
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The DTG curves for PPC/PHBV blends in Figs. 3 and 4 show the temperatures of decomposition for PPC and PHBV in the blends. The temperature of decomposition increased with increasing PHBV content. The thermal properties of the blends are shown in Table 1. The thermal decomposition temperature (T0) of PPC was 187 C, the T0 of PHBV was 199 C, and that of blends rose gradually with increasing PHBV content. The Tp of the blends behaved in a similar fashion. The results indicated that PHBV could improve the thermal stability of PPC efficiently. 3.3. The biodegradability of PPC/PHBV blends in soil suspension 3.3.1. Weight changes during biodegradation As shown in Fig. 5, the biodegradation rate of pure PPC was low, and the weight loss was only 3.3% after 29 days in soil suspension, while pure PHBV was completely biodegraded after 12 days. In fact, the higher the concentration of PHBV in the blends, the more quickly the blends were biodegraded. PPC/PHBV blend (20/80) was completely biodegraded after 14 days. PPC/PHBV blend (40/60) was completely biodegraded after 24 days. The results showed that the biodegradability of PHBV was higher than that of PPC in the soil suspension. In the polymer blends, higher concentrations of PHBV led to more rapid biodegradation. This may be due to a larger number of PHBV degraders in the soil than PPC degraders. PPC is an aliphatic polycarbonate that was synthesized artificially by chemical methods. Because there is no PPC in the natural environment, the number of PPC degraders in the soil is very low. Obviously, PHBV was synthesized by bacteria and could also be utilized by bacteria easily. On the other hand, when PPC was blended with the more rapidly degraded PHBV, PPC in the blends would be biodegraded faster than pure PPC. The degradation enzymes excreted by the microorganisms first degraded the PHBV, and then diffused to the interfaces of the blend films. More surface area of PPC was exposed to the environment, and the great amount of –COOH end groups and organic acids produced previously catalyzed the later biodegradation of PPC and PHBV. The enzymatic hydrolysis and chemical scission occurred at the interfaces as well at the film surface. The results mentioned above revealed that hydrolysis of PPC in the blend films was accelerated by the presence of PHBV. 3.3.2. The change of pH during biodegradation Fig. 6 shows the pH changes of soil suspension during biodegradation. With the biodegradation of PPC/PHBV blends, the pH of soil suspension decreased gradually. This might be because of organic acids produced by PHBV degraders. The pure PHBV was metabolized by the microorganisms most easily, so the pH changed most dramatically. The more easily the blends could be used, the faster the microorganisms grew, and the faster the pH would be changed. After the pH reached its minimum, it rose slightly due to consumption of organic acids by microorganisms in the soil suspension during the process of biodegradation. As there was no carbon source other than the PPC/PHBV blends in the soil suspension and as the PPC/PHBV was biodegraded to completion, the microorganisms in the soil suspension began to consume the organic acids as a carbon source. 3.3.3. The number of PHBV degraders during biodegradation Fig. 7 shows the changes in the number of PHBV degraders during biodegradation in the soil suspension. The initial number of PHBV degraders in the soil was 1.1 105/g wet-soil, and the number of PHBV degraders increased dramatically during the degradation of PHBV, then decreased again after PHBV degradation was complete. The greater the concentration of PHBV in the blends, the faster the number of PHBV degraders would increase. This implied
Fig. 10. 1H NMR spectrum of PPC/PHBV blend (60/40) before degradation.
that the PHBV in the blends was utilized as a carbon and energy source for bacterial growth. 3.3.4. SEM analysis The surface morphology of PPC/PHBV blend films (100/0, 80/20, 60/40, 40/60) before and after biodegradation in soil suspension was studied via SEM (Fig. 8). Prior to biodegradation, all blends showed a relatively compact and smooth surface. The transmittance and thickness of blend films were reduced after nine days of biodegradation. Some of the blend films (20/80, 0/100) could not be studied by SEM after nine days of biodegradation. Blend films failed to maintain their integrity when the content of PHBV was over 80% in the PPC/PHBV blend; the surfaces of the blends were eroded by the action of microorganisms, which made the blend films change their shape and the thickness. Therefore, after nine days of biodegradation, the blend films were too fragmented to perform SEM analysis. It is well known that the enzymatic hydrolysis of biodegradable polyesters such as PCL, PHB, and PLA proceeds mainly via a surface erosion mechanism [20]. The high density of pore formation on the surface of the blend films accelerated the enzymatic hydrolysis inside the blend films as well as at the film surface. The depth and extent of holes depended upon the composition of the blends. These holes inside the film matrix were due to easier penetration of water into amorphous parts of the film, and hydrolysis occurred at these locations [21]. There were many deep holes and cracks over surface of some PPC/PHBV blend films (60/40, 40/60) after biodegradation for nine days. This demonstrates that PPC/PHBV blend films with higher PHBV content were eroded more severely by microbes in the soil suspension.
Table 2 –CH2 mole fraction of PPC and PHBV in PPC/PHBV blend (60/40) during biodegradation. Time (d)
Ratio of –CH2 integral area between PPC and PHBV
Ratio of mole fraction between PPC and PHBV in blends
0 9 19 29
10.00/6.79 10.02/2.61 10.00/0.54 10.00/0.43
59.56/40.44 79.33/20.67 94.88/5.12 95.88/4.12
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3.3.5. NMR analysis The 1H NMR spectra of the PPC/PHBV blends (100/0, 0/100 and 60/40) are shown in Figs. 9 and 10, respectively. One PPC/PHBV blend (60/40) was taken as an example to investigate the changes in composition during the process of biodegradation. For PPC, the peaks at 1.25–1.4 (–CH3), 4.1–4.3 (–CH2) and 4.95–5.05 (–CH) were attributed to methyl, methylene and methine protons. For PHBV, the peaks were at 0.85–0.95 (–CH3 of HV), 1.25–1.4 (–CH3 of HB), 2.45–2.65 (–CH2 of HB and HV) and 5.2–5.3 (–CH of HB and HV). In order to study the composition changes of the PPC/PHBV blends before and after biodegradation, the peaks at 2.45–2.65 (–CH2 of PHBV) and 4.1–4.3 (–CH2 of PPC) of the 1H NMR spectra were selected for investigation. The actual mole fractions of PPC and PHBV could be calculated by integrating the areas of –CH2 peaks on the graph [22]. The composition changes in each blend were investigated by calculating both mole fractions in the blends. In Fig. 10, the integrated areas of 1H NMR spectra of the methylenes of the PPC/PHBV blend (60/40) before biodegradation were 10.00 (integral area of –CH2 for PPC) and 6.79 (integral area of –CH2 for PHBV), respectively. The mole fraction of PPC was 10.00/ (10.00 þ 6.79) ¼ 59.56%; the mole fraction of PHBV was 6.79/ (10.00 þ 6.79) ¼ 40.44%. Therefore, the actual initial mole fraction of the PPC/PHBV blend (60/40) was 59.56/40.44. Table 2 shows the integral area and mole fraction of each component during biodegradation. All integral values are taken from the data in Figs. 10 and 11. The mole fraction of PPC/PHBV was 79.33/20.67 at the ninth day of biodegradation. After 19 and 29 days, the mole fractions of PPC/PHBV changed to 94.88/5.12 and 95.88/4.12, respectively. We observed that the mole fraction of PHBV in the PPC/PHBV blend gradually decreased during biodegradation, while the mole fraction of PPC in the PPC/PHBV blend increased. PPC was biodegraded slowly in the soil suspension because PPC-degrading microorganisms in the soil were rare. These data demonstrated that the weight loss was mainly due to degradation of PHBV.
3.4. Degradation in vitro Fig. 12 shows the remaining weight of PPC/PHBV blends (100/0, 80/20, 60/40, 40/60, 20/80 and 0/100) degraded in vitro for more than 200 days. The chemical decomposition rate for PPC/PHBV (0/100) was slowest in all blends. Meanwhile, higher concentrations of PPC in the blends resulted in more rapid degradation in vitro. In fact, PPC/PHBV blends immersed in PBS solution could be hydrolyzed, with hydrolysis first appearing on the surface, followed
100
Weight Remaining (
)
98 96 94 92 90 PPC/PHBV=100/0 PPC/PHBV=80/20 PPC/PHBV=60/40 PPC/PHBV=40/60 PPC/PHBV=20/80 PPC/PHBV=0/100
88 86 84 0
50
100
150
200
Time (days) Fig. 11. 1H NMR spectrum of PPC/PHBV blend (60/40) after degradation in soil suspension.
Fig. 12. Weight changes of PPC/PHBV blends during in vitro degradation.
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Table 3 Molecular weights and distributions of PPC and PHBV before and after degradation. Before degradation
PPC PHBV
After degradation for 140 days in vitro
After biodegradation for 24 days (PPC) and 4 days (PHBV) in soil suspension
Mn
Mw
Polydispersity
Mn
Mw
Polydispersity
Mn
Mw
Polydispersity
49,900 38,800
143,500 95,300
2.9 2.4
24,200 38,300
71,500 78,100
3.0 2.0
49,500 24,000
15,0200 65,700
3.0 2.7
by swelling of the materials after absorption of water leading to an increase in the bulk of the material. This expansion led to the presentation of interior surface area, and allowed the PBS solution to penetrate into the material for further degradation. As the chemical bonds in the main chain broke down to form large molecular fragments, and these fragments were further hydrolyzed to oligomers and monomers that then dissolved in the PBS solution. This process led to a decrease of molecular weight or weight loss in the PPC/PHBV blends. It was reported that the hydrolysis rate of some aliphatic polyesters was affected by their degree of crystallinity [21,23]. Since PPC was fully amorphous and PHBV had a relatively high degree of crystallinity as shown in Fig. 2, water penetration in the PPC/PHBV blend film was very difficult and the chemical bonds in PHBV appeared to be difficult to hydrolyze. Therefore, the different degradation rates of PPC and PHBV in vitro might be attributed to their degrees of crystallinity. The changes of molecular weight of PPC and PHBV described below supported the above explanations. 3.5. The comparison between biodegradation in soil suspension and in vitro Table 3 shows the molecular weight changes of PPC and PHBV before and after degradation in soil suspension and in vitro. These results indicate that the Mn of PPC was reduced from 49,900 to 24,200 after 140 in PBS solution, while the PHBV was largely intact under the same conditions (from 38,800 to 38,300). The polydispersity of PPC changed from 2.9 to 3.0, and that of PHBV changed from 2.4 to 2.0. This suggested that the degradation of PPC was mainly through chemical hydrolysis and random hydrolytic scission of polymer chains in the PBS solution. PHBV was hardly degraded in vitro, and only small molecular fragments dissolved in the PBS, but the main chain could not be broken down. In contrast, PHBV was easily biodegraded in the soil suspension, and the Mn changed from 38,800 to 24,000 after only four days, while the polydispersity changed from 2.4 to 2.7. The weight loss of PPC/PHBV blends in soil suspension was caused mainly by degradation of the PHBV component. Enzymatic degradation involved hydrolysis of ester bonds and the chemical scission of macromolecular chains into small fragments or oligomers. These low molecular weight fragments are soluble in water, and for this reason the films start to fragment on the surface [21]. However, because of a lack of PPC degraders in the soil suspension, PPC could not be degraded. Even after extension of the degradation time to 24 days, the Mn of PPC changed from 49,900 to 49,500 and the polydispersity of PPC changed from 2.9 to 3.0. There was nearly no change in the Mn for PPC, but the polydispersity widened a little. This may have been due to chemical hydrolysis in the soil suspension. 4. Conclusions PPC/PHBV blends formed immiscible mixtures. However, the addition of PHBV improved the thermal stability of PPC. PHBV was biodegraded more rapidly than PPC in the natural environment due to the action of microorganisms, and there were more
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