Radiation-induced degradation of poly(3-hydroxybutyrate) and the copolymer poly(3-hydroxybutyrate-co-3-hydroxyvalerate)

PolymerDegradationand Stability45 (1994)11-17

1994 ElsevierScienceLimited Printed in Northern Ireland. All rights reserved 0141-3910/94/$07.00

ELSEVIER

Radiation-induced degradation of poly(3hydroxybutyrate) and the copolymer poly(3hydroxybutyrate-co-3-hydroxyvalerate) Hiroshi Mitomo Department of Biological and Chemical Engineering, Faculty of Engineering, Gunma University, Tenjin-cho I-5-I, Kiryu, Gunma 376, Japan

Yuhei Watanabe, Isao Ishigaki Japan Atomic Energy Research Institute, Takasaki Radiation Chemistry Research Establishment, Watanuki, Takasaki-shi, Gunma 370-12, Japan

&

Temmi Saito Department of Biological Sciences, Faculty of Science, Kanagawa University, Hiratsuka, Kanagawa 259-12, Japan (Received 14 December 1993; accepted 22 December 1993)

Poly(3-hydroxybutyrate) {P(3HB)} and the copolymer poly(3-hydroxybutyrate-co-3-hydroxyvalerate) {P(3HB-co-3HV)} were irradiated with y-rays at 25°C in air and in vacuum. Melting points (T.,) and glass-transition temperatures (Ts) were measured by differential scanning calorimetry. Numberaverage molecular weights (M.) were analyzed by gel permeation chromatography. No significant differences were observed between T,. values of P(3HB) and P(3HB-co-3HV) irradiated in air and in vacuum, which decreased almost linearly with increasing irradiation dose. The Mn values of both samples decreased sharply with increasing dose, reflecting typical random chain scission. The decrease in M. of the sample irradiated in vacuum was smaller than that irradiated in air with the same dose, implying the occurrence of crosslinking. The Ts values for both polymers irradiated in vacuum remained almost unchanged over a wide dose range, while those irradiated in air decreased as the irradiation dose increased. Both the Tm and T8 of samples irradiated in air were inversely proportional to M.. Biodegradability was clearly promoted with decreasing molecular weight.

amine treatment caused selective hydrolysis in the amorphous regions. 8 A number of studies have been made of the radiation-induced degradation of common polymers such as polyethylene 9 and polypropylene, t° where it was found that the resistance to radiation-induced oxidative degradation increased with increasing amorphous fraction and a greater amount of crosslinking occurred in lower density than in higher density material. Recently, an NMR study of the structural changes in butyl

INTRODUCTION Poly(3-hydroxybutyrate) {P(3HB)} and the copolymer poly(3-hydroxybutyrate-co-3-hydroxyvalerate) {P(3HB-co-3HV)} are biodegradable polyesters produced by many types of bacteria.t.2 Degradation processes in these polymers have been reported in a number of papers; enzymatic degradation occurred by a depolymerization process at the chain ends, 3'4 thermal degradation by random scission of polymer chains, ~-7 and 11

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Hiroshi Mitomo et al.

rubber after y-irradiation has been reported, where the yields of main-chain scission and crosslinking were estimated. 11 In the present study, P(3HB) and P(3HB-co3HV) were irradiated with 6°Co y-rays at 25°C in air and in vacuum. Number-average molecular weight, melting point and glass-transition temperature were measured as a function of radiation dose, and the mechanism of radiationinduced degradation is discussed. The relationships of melting points and glass-transition temperatures to molecular weight were investigated. The biodegradability of these irradiated samples was studied using an enzyme. The regularity in the P(3HB) crystal lattice and the relative distortion or irregularity in the P(HBHV) crystal lattice were indirectly confirmed by the degradation behavior.

EXPERIMENTAL Materials P(3HB) and P(3HB-co-3HV) containing 20 mol% 3HV (hereinafter abbreviated as 20M sample) were purchased from Aldrich Chemical Co. Both polymers were isolated from the bacterium Alcaligenes eutrophus.

Irradiation P(3HB) and the 20 M sample were sealed in glass ampoules in an atmosphere of dried air or in vacuum (evacuated to 10-3 Pa), then irradiated at 25°C with ~°Co y-rays at a dose rate of 10 kGy/h for various periods of time. Irradiated samples were kept in the sealed ampoules at 25°C for at least a week before making analytical measurement.

Analytical procedures The melting points Tm and glass-transition temperatures Tg of each irradiated sample (3 mg) were measured in the temperature range -100 to 200°C in a Perkin-Elmer Model DSC-7 differential scanning calorimeter (DSC) at a heating rate of 10°C/min under a nitrogen or helium flow of 30 ml/min. The temperature and calorimetric scales were calibrated with highpurity standards, benzoic acid and indium (Tin,

121 and 156.6°C, heat of fusion, 142 and 28.4 J/g, respectively). Tg was taken as the mid-point of the heat capacity change. Gel permeation chromatography (GPC) was carried out with an HLC-802A (Tosoh Co.) instrument equipped with a series of four TSK gel columns and an RI-8 differential refractometer at 38°C. The eluent was chloroform with a flow rate of lml/min, and the polymer concentration was c. l%(w/v). The count of 6000 corresponded to an elution volume of 34 ml. The number-average molecular weight /f/. was calibrated using six typical radiation-degraded P(3HB) and 20 M samples with various M, values (from 4.29 x 103 t o 5.64 x 105) evaluated by GPC and a low-angle laser light scattering system.

Enzymatic degradation Enzymatic degradation of irradiated samples was studied by monitoring the weight loss of film samples (initial weight, 10-15mg; dimensions, 10 mm x 10 mm and 0-1 mm thick) at 37°C in a 0.1M phosphate buffer solution (pH 7.4) of extracellular PHB depolymerase purified from Alcaligenes faecalis T1.12 Film samples were placed in a glass tube with 1.0 ml of the buffer containing 8~g of PHB depolymerase. The solution was incubated at 37 + 0.1°C with shaking. The film samples were removed, washed with warm water and dried to constant weight in vacuo.

RESULTS AND DISCUSSION Melting points and molecular weights of irradiated samples Typical DSC heating curves of P(3HB) and the copolymer 20 M irradiated in air with various irradiation doses are shown in Fig. 1. The peak temperature for P(3HB) decreased sharply and the peak decreased in area and became broader as the irradiation dose increased, except for the sample irradiated with 50 kGy, which showed a slight increase (c. 5%) in peak area. The samples irradiated above 50kGy showed a doublet melting peak composed of a main peak and a small peak appearing at temperatures 10-20°C lower than the main one, which implies that further degradation proceeded heterogeneously into crystalline regions. The peak temperature

Radiation-induced degradation of poly(3-hydroxybutyrate)

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100

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Temperature

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I 140

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I 160

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1 180

(°C)

Fig. 1. DSC heating curves of P(3HB) and 20 M samples irradiated in air with various doses. Doses (kGy): (a) 0, (b) 50, (c) 500, (d) 1000, (e) 3000 for P(3HB); and (f) 0, (g) 500, (h) 1000, (i) 2000 for 20 M.

and the peak area for the 20M sample also decreased as the radiation dose increased, except for the sample irradiated with 50kGy, which showed a slight increase (c. 3%). The melting peak area of the 20 M sample was far smaller than that of P(3HB), reflecting its low crystallinity and slow crystallization rate. 13 The curves for a second DSC run for the samples heated once to 190°C and cooled to room temperature were essentially similar to those of DSC first run. The Tm values of P(3HB) and 20 M samples irradiated in air and in vacuum are plotted against the radiation dose in Fig. 2 (the Tm at lower temperature is omitted). In air, Tm of P(3HB) decreased sharply up to 200kGy and then decreased gradually with almost the same gentle gradient as that of 20 M. It is considered that the decrease in Tm is caused mainly by the introduction of irregularities, such as the partial destructuon of crystalline regions by chain scission. The Tm values of P(3HB) and 20M irradiated in vacuum are very close to that irradiated in air up to 1 MGy. However, Tm of those samples irradiated in vacuum above 1 MGy decreases slower than the latter, implying that

I

40

0

1000 Irradiation

. 300

dose (kGy)

Fig. 2. Plots of melting points (a) and enthaipies of fusion (b) of the irradiated samples against the radiation dose. P(3HB) irradiated in air (O) and in vacuum (0); 20M sample in air (A) and in vacuum (A).

the degradation proceeds more slowly than in air because of the introduction of crosslinking. In Fig. 2, the enthalpies of fusion (AHm) of both samples irradiated in dried air and in vacuum were plotted against the irradiation dose. The crystallinity of P(3HB), calculated from AHm (=86J/g) using the heat of fusion of PHB (146J/g), 14 is 59%, which increased to 62% (at 100 kGy) then decreased sharply again to 30 and 39% respectively after irradiation in air and in vacuum up to 3MGy. The initial increase in crystallinity is considered to be caused by random chain scission in amorphous regions and removal of them by partial volatilization. The AHm of 20M showed a similar variation, though the value is approximtely half that of P(3HB). Figure 3 shows GPC chromatograms of P(3HB) irradiated in air with various doses. The number-average molecular weight /f/, values of P(3HB) and 20M samples are 2.46 x 105 and 3.85 x 105, respectively. The AS/,values decreased significantly at first, then more slowly as the irradiation dose increased. In the lower dose

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Hiroshi Mitomo et al. ~.0 10 ~

Irradiation dose (kGy)

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200 2

1000

d

10

0

3000

4000

5000

6000

7000

8000

1000 Irradiation

2000 dose (kGy)

0 3000

Fig. 4. Plots of ~/. and degree of main-chain scission of irradiated P(3HB) and 20M samples against irradiation dose. Samples and symbols are as in Fig. 2.

Count Fig. 3. GPC chromatograms of the irradiated P(3HB) samples. Values of /1~/, (xl0 -4) and h~/w//~/, are: (a) 23.5, 1.77; (b) 12.9, 1.71; (c) 3.15, 1.70; (d) 0.52, 1.43; (e) 0.10, 1.06, respectively.

range (0-100 kGy), main-chain scission occurred mainly at the folded chain regions of the crystal surface. This chain scission occurred preferentially within the less stable regions, such as the crystal surface where the residual strain had been introduced as a result of forcible chain folding. At higher doses, above 100 kGy, chain Scission began to occur even in the crystalline regions, and Mw/M. (polydispersity) became gradually close to 1 (monodisperse), as shown in the caption to Fig. 3. The decrease in /~/. values of 20 M was very similar to that of P(3HB). The /fit. values of both samples irradiated in vacuum decreased more slowly than those irradiated in air as the irradiation dose increased. The /17/, values P(3HB) and 20M samples irradiated in air and in vacuum are plotted against the irradiation dose in Fig. 4. The M, of each sample decreased sharply at first, then more slowly along almost parallel curves with increasing irradiation dose. The hT/. value of P(3HB) irradiated in air is less than half that irradiated in vacuum over the whole dose range. This implies that degradation in air proceeds with random

scission of polymer chains, while that in vacuum proceeds with the introduction of crosslinking in addition to random chain scission. The/k/, values of P(3HB) and 20 M irradiated in air are very close to each other, while the A~/. of 20M irradiated in vacuum decreased slightly faster than that of P(3HB). It can be considered that reactions, such as mutual recombination or crosslinking, became more difficult because the molecular chain packing of 20 M sample is looser than that of P(3HB).t5 The degree of main-chain scission (a~= 1/(/f/.-1//(/.)0) shows linear dependence on the irradiation dose as shown in Fig. 4, where M, and (M,)o are the molecular weights of irradiated and unirradiated samples, respectively. Ttius, the G values for main-chain scission were evaluated as 3.0 (3.1 #mol of main-chain bonds were cleaved per 10 kGy of irradiation) for P(3HB) and 2-6 for 20 M samples irradiated in air. In contrast, those irradiated in vacuum do not show a linear change with increasing dose. Nevertheless, the G values were roughly estimated as 0-8 and 0-9 for P(3HB) and 20 M samples from the initial slopes up to 1000 kGy. Those of polypropylene irradiated in air and in vacuum have been reported to be 3.1 and 0.64, respectively, 1° which are very close to the present authors' results.

Radiation-induced degradation of poly O-hydroxybutyrate)

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Glass-translation temperatures of irradiated samples

Relationship of melting point and glasstransition temperature to molecular weight

Figure 5 shows glass-transition temperatures Tg of P(3HB) and 20M samples plotted against the irradiation dose. Since the sample having high crystallinity, which was aged at room temperature for a long time, hardly showed a glass transition in the DSC curve, every sample was melted at 190°C and quenched in liquid nitrogen prior to the DSC measurement. As decomposed and shortened molecular chains show high molecular mobility Tg for samples irradiated in air decreased almost linearly with increasing irradiation dose. On the other hand Tg values of both samples irradiated in vacuum showed S-shaped curves, i.e. decreasing sharply at first, then hardly decreasing in the dose range 0.5-2 MGy, followed by a gentle decrease at the higher radiation dose. The reason why the Tg values of both samples irradiated in vacuum remained almost constant irrespective of decrease in 2f/, is due to low molecular mobility by the introduction of crosslinking between polymer chains.

Figure 6 shows plots of Tm and T~ of P(3HB) and 20M irradiated in air and in vacuum against 1/37/.. At doses above 200kGy, Tm and Tg of P(3HB) and 20M irradiated in air decrease almost linearly, and the following equations of straight lines are obtained: for P(3HB): Tm: 1 6 2 - (5.45 × 104)//~n

(1)

Tg = 0.6 - (2.38

(2)

× 104)/Mn

for 20 M: Tm= 111 - (6.30 × 104)/M,

(3)

Tg = - 3 . 2 - (2.35 × 104)//~n

(4)

The melting points of n-paraffins have been expressed in terms of the carbon number x as follows: t6

Tm= T°m(X+ b)/(x + a) = 414.1(x - 1-5)/(x + 5.0)

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1 / ~ . (×104) Fig. 5. Plots of Tg of irradiated P(3HB) and 20 M samples against the irradiation dose. Samples and symbols are as in Fig. 2.

Fig. 6. Plots of T,, and T~ of irradiated P(3HB) and 20 M samples against 1/hT/o.Samples and symbolare as in Fig. 2.

lOO,

Hiroshi Mitomo et al.

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where T ° is the equilibrium melting point (K) and a and b are constant. If x becomes far larger than a, eqn (5) is close to Tin°(1+ b/x), which is similar to eqns (1) and (3). For both samples, the linear relationships are observed over a range of AS'/n f r o m 10 3 to 10 4, implying that a is far smaller than b in this case. Equation (3) shows a slightly steeper slope than eqn (1), which suggests that main-chain scission in crystalline regions of 20 M is more effective because of the presence of local irregularities compared with P(3HB). The decrease in Tg is also linear, as Fox and Flory proposed for polystyrene considering the free volume: 17

s ~ S S

60 m

20

0

Tg = T:(1 - c/1(4,) = 100 - (1-7 x 105)/.(/, (6) where c is a constant and T~ is the glasstransition temperature when M, is very large or infinity. Equation (6) is equivalent to eqns (2) and (4). From eqns (1) and (2), Tm ° and T~ are obtained as intercept values on the vertical axis, i.e. 162 and 0-6°C, while those of the unirradiated P(3HB) are 177 and 4°C, respectively. From eqns (3) and (4), T ° and Tg are 111 and -3.2°C, while those of the unirradiated 20 M are 118 and - I ° C , respectively. The discrepancy of T ° and Tg of the latter from those of the unirradiated sample is half of the former. The reason why the discrepancy for the 20 M sample is smaller than that of P(3HB) is considered to be that selective degradation in amorphous regions becomes indistinguishable because of the presence of slight irregularities in the crystalline regions by inclusion of the 3HV component. Tm and Tg of samples irradiated in vacuum did not show any linear relationship with 1/A7/,, as shown in Fig. 6. It is considered that this deviation from linearity is due to the occurrence of crosslinking during irradiation. It is to be supposed that new chemical residues such as - - O H or - - C O O H originating from hydroxybutyric acid residues are introduced at the polymer chain ends as a result of oxidative degradation in air, while residues such as ---CH--CH---CH3 or crosslinking are preferentially formed in vacuum as reported elsewhere. 1°'1~ An N M R study of these irradiated polymers will be published in a subsequent paper. Figure 7 shows weight loss curves for the enzymatic degradation of irradiated samples. o

_ _ .-i[

200

500

Irradiation dose

1000 (kGy)

Fig. 7. Plots of weight loss of the irradiated samples by biodegradation against the irradiation dose. P(3HB) irradiated in air (O) and vacuum (0); 20M in air (A) and vacuum (&). Solid and broken lines show the treatment time of 8 and 16 h, respectively. Weight loss values for P(3HB) were 15 and 29% after treatment for 8 and 16 h, whereas those for 20 M were slightly smaller, namely 11 and 21%, respectively. The irradiated P(3HB) films show a faster degradation rate than 20 M irradiated with the same dose. Both P(3HB) and 20M films irradiated with doses higher than 120 and 210kGy were completely degraded after treatment for 16 h, while both irradiated in vacuum showed a weight loss of c. 50% under the same conditions. This implies that biodegradation is clearly promoted with decreasing M,. The effect of crosslinking on biodegradability is quite large, e.g. the weight loss of P(3HB) irradiated in vacuum with 1000 kGy (39%) was approximately half of that irradiated in air with 500kGy (79.5%). Nevertheless the A7/. value of the former (1.8 × 104) was comparable with that of the latter (1.1 x 104). Moreover, the weight loss of P(3HB) irradiated in vacuum almost leveled off at a dose of 1000 kGy, due to the presence of crosslinking, while that irradiated in air continued to increase. The same tendency was observed for the 20 M sample. The rate of enzymatic degradation increased in the order: (the original s a m p l e ) < (that irradiated in v a c u u m ) < (that irradiated in air). Comparing weight loss curves of both samples irradiated in air with M, curves in Fig. 4, it can be said that the weight loss is roughly inversely proportional to logarithmic value of M,.

Radiation-induced degradation of poly(3-hydroxybutyrate)

CONCLUSIONS At lower irradiation doses ( 0 - 1 0 0 k G y ) , chain scission occurs mainly within the folded chains or a m o r p h o u s regions at the crystal surface, while r a n d o m chain scission p r o c e e d s farther into crystalline regions at higher doses. Experimentally, Tm and Tg were inversely proportional t o / f / , at doses a b o v e 500 k G y in air. U n d e r irradiation in v a c u u m , molecular chain scission mainly occurs a c c o m p a n i e d by crosslinking. These samples no longer show the linear relationship m e n t i o n e d above. The rate of biodegradation of the irradiated samples increases with increasing irradiation dose or decreasing Mn values. The rate of those irradiated in v a c u u m was considerably slower than those irradiated in air because of the introduction of crosslinking.

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Microbiol. Biotechnol., 28 (1988) 330. 3. Doi, Y., Kanesawa, Y., Kunioka, M. & Saito, T., Macromolecules, 23 (1990) 26. 4. Kumagai, Y. & Doi, Y., Polym. Deg. Stab., 37 (1992) 253. 5. Grassie, N., Murray, E. J. & Holmes, P. A., Polym. Deg. Stab., 6 (1984) 47; Polym. Deg. Stab., 6 (1984) 95; Polym. Deg. Stab., 6 (1984) 127. 6. Kunioka, M. & Doi, Y., Macromolecules, 23 (1990) 1933. 7. Mitomo, H. & Ota, E., Sen-i Gakkaishi, 47 (1991) 89. 8. Mitomo, H., Sen-i Gakkaishi, 48 (1992) 595. 9. Geetha, R., Torikai, A., Yoshida, S., Nagaya, S., Shirakawa, H. & Fueki, K., Polym. Deg. Stab., 23 (1988) 91. 10. Kagiya, T., Nishimoto, S., Watanabe, Y. & Kato, M., Polym. Deg. Stab., 12 (1985) 261. 11. Hill, D. J. T., O'Donnell, J. H., Senake Perera, M. C. & Pomery, P. J., Radiat. Phys. Chem., 40(2) (1992) 127. 12. Shirakura, Y. et al., Biochim. Biophys. Acta, 880 (1986) 46. 13. Mitomo, H., Barham, P. J. & Keller, A., Polymer J., 19 (1987) 1241. 14. Barham, P. J., Keller, A., Otun, E. L. & Holmes, P. A., J. Mater. Sci., 19 (1984) 2781. 15. Scandola, M., Ceccorulli, G., Pizzoli, M. & Gazzano, M., Macromolecules, 25 (1992) 1405. 16. Broadhurst, M. G., J. Chem. Phys., ~ (1962) 2578. 17. Fox, T. G. & Flory, P. J., J. Appl. Phys., 21 (1950) 581.