Natural Additives for Poly (Hydroxybutyrate–CO

Natural Additives for Poly (Hydroxybutyrate – CO - Hydroxyvalerate) – PHBV: Effect on Mechanical Properties and Biodegradation

Daiane Gomes Brunela, Wagner Maurício Pachekoskib*, Carla Dalmolinc, José Augusto Marcondes Agnellia

aDepartamento de Engenharia de Materiais, Universidade Federal de São Carlos – UFSCar, Rod. Washington Luis, Km 235, CEP 13565-905, São Carlos, SP, Brasil bUniversidade Federal de Santa Catarina – UFSC, Campus Joinville, Rua Presidente Prudente de Moraes, 406, CEP 89218-000, Joinville, SC, Brasil cDepartamento de Química, Centro de Ciências Tecnológicas – CCT, Universidade do Estado de Santa Catarina – UDESC, Rua Paulo Malschitzki, s/n, Campus Universitário Prof. Avelino Marcante, CEP 89219-710, Joinville, SC, Brasil

Received: August 28, 2013; Revised: August 21, 2014

In this work, the improvement of mechanical properties in biodegradable materials was obtained through the incorporation of natural and also biodegradable plasticizers and nucleation agents into the PHBV copolymer. PHBV production with different quantities of additives was obtained by extrusion followed by injection. The additives in the copolymer were efficient, resulting in an adequate processing due to the presence of nucleate and an improvement of the mechanical properties of the resulting material provided by the action of the plasticizer. The formulation with the minimum amount of additive content, 5% epoxidized cottonseed oil and 0.1% Licowax, was the most effective showing 35% reduction in the elastic modulus, and 18% in the PHBV crystallinity; 58% increase in impact resistance and 46% increase in elongation. Furthermore, it is important to emphasize that the natural additives were very efficient for biodegradation, showing a mass loss higher of pure PHBV.

Keywords: poly(hydroxybutyrate-co-hydroxyvalerate), PHBV, biodegradable polymers, additives, mechanical properties

1. Introduction Poly (3-hydroxybutyrate) – PHB – is a well-known with glucose, as a carbon source. The amount of propionic biologically derived and biodegradable polymer1. Since it acid that is found in the nourishment of the bacteria is can be produced from renewable resources, it has received responsible for the concentration of hydroxyvalerate increasing attention due to the potential applications such (HV) in the copolymer. As HV content increases, Tg, Tm as in environment-friendly products, tissue engineering, and crystallinity decrease, improving the processing and and control release devices2,3. Nowadays, bacterial toughness in PHB5. fermentation is the main source for PHB production. The By being thermoplastic, of renewable sources, process basically consists of two stages: a fermentative biodegradable, compostable and biocompatible, PHB and stage, in which the microorganisms are fed in reactors PHBV are of great interest in the production of fast usage containing butyric acid or fructose, where they metabolize products, such as disposable materials, packages, medical the sugar available and accumulate the PHB in the inner artifacts for human or veterinary, automobile industry cell as a power supply source; and the extraction stage, products, among others. To be suitable for these industrial where the polymer accumulated in the microorganism applications, however, PHB and PHBV should be processed inner cell is removed and purified with adequate solvents in large scale, mostly by melt processing techniques such 4 until obtaining the final product, that is solid and dry . as extrusion and/or injection. In this case, the polymeric However, the commercialization of these materials did not chains are submitted not only to high temperatures, but result in a major replacement of the conventional plastics also to shearing tension, which may lead to a scission on because of the higher costs of PHB, its brittleness, and a the polymeric chain, causing reduction in the molar mass narrow process window due to the lack of thermal stability. and characterizing a further degradation6. Incorporation The PHB copolymer, i.e. poly (3-hydroxybutyrate-co- of additives is another resource to modify some polymer 3-hydroxyvalerate) – PHBV – has been developed in an properties in order to achieve better processing or to adjust effort to improve its properties for industrial application. It their mechanical and thermal behavior. However, when is produced by a fermentative process similar to the PHB dealing with biodegradable polymers, it is preferable that process, only differing in the use of propionic acid, together these additives are biodegradable as well. Indeed, some *e-mail: [email protected] authors have reported the use of soybean oil7, β-carotene8 1146 Brunel et al. Materials Research

and low molecular weight additives9 as plasticizing agents thread extruder. Next, the pelletized formulations were dried for PHB and PHBV in order to improve their mechanical in an oven at 60°C for 24h. The injection of impact and properties for industrial applications. Other approaches also tension specimen according to ASTM D-638[12] and D-256 include the use of nucleating agents and compatibilizers to standards13 was carried out in a 270V 300-120 Arburg All accelerate the crystallization process and refine morphology Rounder injector, with 12 cm3/s flow and 20 cm/s injection and thermal stabilizers, also known as antioxidants, which speed. can prevent various effects such as oxidation, chain scission An Instron 5569 Universal Test Machine, in ASTM and uncontrolled recombination that may occur during the D-638 standard12, with a 10 mm clutch gap, 5 mm/min process10. speed and 50 kN load cell was used to measure mechanical Among various natural biodegradable additives, an properties (Young’s modulus, stress and elongation at epoxidized cottonseed oil plasticizer and a nucleate based on break). The notched Izod impact test was carried out in fatty acids were efficient in the improvement of processing a 65451000 code CEAST Impact Machine, with a 2 J by extrusion and injection. Futhermore, the mechanical pendulum, under controlled temperature, according to properties and biodegradation increased when they were ASTM D-256 standard13. All procedures were done in 11 mixed together in a PHBV formulation . However, triplicate, three days after processing. optimized results still can be obtained through the study of The thermo gravimetric (TG) and the derived thermo the influence of both plasticizer and nucleating agent when gravimetric (DTG) curves were obtained in a TA Instrument their contents inside the formulation are changed. Therefore, TGA2950 at a 20°C.min–1 heating rate between room different PHBV formulations with an epoxidized cottonseed temperature (23°C) and 600°C, under N2 atmosphere oil as the plasticizer and a nucleate based on fatty acids (50 mL/min) in an alumina sample rest. A TA Instruments were processed by both extrusion and injection to result in DSC Q100 calorimeter was used for the DSC characterization materials with adequate mechanical properties for industrial ranging from –50 to 200°C at both a heating and cooling rate usage and composition with approximately 100% weight in of 20°C.min–1, under N atmosphere. The PHBV crystallinity biodegradable materials. To evaluate the better composition 2 was calculated by dividing the heat of fusion of each sample and the effect of additives in PHBV properties, specimens (ΔH ) and the heat of fusion of the hypothetically 100% with different formulations were tested by mechanical, m crystalline PHB, determined as 146 J/g[14]. thermal, microscopy and biodegradation analysis. The influence of nucleating agent contents in the 2. Experimental spherulites growth rate in relation to temperature was studied by optical microscopy with polarized light, PHBV – ( of 650,000 g/mol; 3.5% HV, 1.22 g/cm3) was using a heating plate. The optical microscope used had manufactured by biological fermentation from renewable a DMRXP Leica polarized light and a KAPPA webcam sugarcane carbohydrate at PHB Industrial S/A. P902 coupled to a computer with software for capturing images. (Logos Química Ltda), an epoxidized cottonseed oil, was To get the experiments into controlled temperatures, a chosen as the plasticizing agent, and the nucleating agent THMS 600 Linkan heating plate was used, monitored was the fatty acid based compound Licowax (Clariant). by a TMS92 Linkan temperature controller. The samples Contents of plasticizer (P) and nucleate (N) are listed in were heated at 50°C.min–1 up to 190°C and were kept at Table 1 for all formulations in this study. To guarantee this temperature for 3 minutes to guarantee the complete the homogeneity between both powder PHBV and liquid fusion of the spherulites, destroying the previous thermal plasticizer, the different blends were mixed into a Henschel history, but paying attention not to initiate a possible thermal blender for 10 minutes, with 450 rpm rotation. These degradation process. Subsequently, the samples were cooled compounds were dried in an air circulation oven (Soc. Fabbe down at 100°C.min–1, up to the isothermal crystallization 170) at 60°C for 24h. The different amounts of nucleating temperature (60°C, 70°C, 80°C, and 90°C), and were kept agents were manually blended. The pure copolymer and the there for 20 minutes. different formulations described in Table 1 were processed To evaluate the biodegradation of the processed in a DC-R 30:40 IF Imacom co-rotational double screw copolymer, the Sturm methodology was used, which is considered the most trustable for the evaluation of polymer biodegradability in active microbial medium15,16. This Table 1. Nucleating agent and plasticizer contents used in all methodology consists of embedding the test specimen formulations. in an activated organic compound and evaluating its Formulation Plasticizer (% mass) Nucleate (% mass) biodegradation through the mass loss and the modification PHBV 0 0 in its visual aspect. In this study, the test was carried out 5P01N 5 0.1 in a Compound Organic Fertilizer (40% minimum organic 5P03N 5 0.3 matter, 45% maximum humidity, pH 6 and 18/1 maximum 5P05N 5 0.5 C/N ratio), supplied by PROVASO, under room temperature and controlled humidity, according to ASTM D-6003[17] 7P01N 7 0.1 and ASTM G-160 standards18. Three distinct systems 7P03N 7 0.3 were prepared for withdrawing after 60, 120 and 180 days 7P05N 7 0.5 of test. Each sample system was formed by 5 tension 10P01N 10 0.1 specimens for each compound. Besides the mass loss after 10P03N 10 0.3 the biodegradable tests, samples were analyzed by Scanning 10P05N 10 0.5 Electron Microscopy (SEM) (Stereoscan 440), and the Natural Additives for Poly (Hydroxybutyrate – CO - Hydroxyvalerate) – PHBV: 2014; 17(5) Effect on Mechanical Properties and Biodegradation 1147

modification of the mechanical properties was evaluated filament became rigid, showing good stability during by mechanical tests. extrusion. The temperature profile was similar for the pure copolymer and also for the additive formulations; however, 3. Results and Discussion less darkening in the compounds with additives (Figure 1) was observed, suggesting a lower thermal degradation. In During the extrusion, pure PHBV had high cast viscosity the injection mold, where the same conditions were used and slow extruded crystallization, characteristics that in the processing of all formulations, it was observed that resulted in higher pelletizing difficulties. The formulations the higher the percentage of nucleate in the formulation, the with additives were easier to processing, because the joint more pressure in injection was necessary. The formulations action between the plasticizer and the nucleate resulted in a with 0.5% nucleate, by crystallizing faster than the others, more stable extrusion. The larger the amount of nucleating did not show homogeneity in the filling of the mold cavities, agent content in the formulation, the faster the extruded with constant failure in the injected specimen.

Figure 1. Aspect of the pellets produced by extrusion of a) pure PHBV and b) PHBV with additives.

Figure 2. Stress-strain curves with a variation of the plasticizer content and nucleating contents equal to a) 0.1%; b) 0.3%; and c) 0.5%. 1148 Brunel et al. Materials Research

3.1. Mechanical tests were observed for the additive formulations when compared to the pure PHBV; however, the increase of the percentage of Figures 2a-c shows the PHBV copolymer stress-strain plasticizer and nucleate did not have a significant influence curves compared to the additive formulations with 0.1%, in this variation. 0.3% and 0.5% nucleate contents and varied plasticizer The values of the thermal properties (T – glass contents. All the additive formulations showed lower stress g transition; T – cold crystallization; T – melting point; in the rupture, lower elastic modulus and higher deformation cc m if compared to the formulation without additives, indicating and crystalline degree) obtained by DSC during the second heating run are found in Table 3. It was observed that T the effectiveness of the plasticizer in reducing rigidity and g fragility of the copolymer, according to the values presented was reduced with the increase of the plasticizer content in Table 2. Comparing the results of additive formulations, and, in general, the increase of the amount of nucleate it is observed that maintaining the concentration of the in the formulation restricted this effect. In general, the plasticizer constant, the increase in the nucleate content additive formulations differed from the pure PHBV with the displacement of T , T and T down to lower temperatures. causes a reduction of strain in the rupture. Moreover, g cc m this effect is minimized when the nucleate and plasticizer In the formulations with 0.1% nucleate, it can be noted contents increase. It is verified that the formulation with that with the increase in the plasticizer content, there was a minimum amount of nucleate and plasticizer (5P01N) a gradual reduction of the melting, crystal and vitreous showed a reduction of PHBV’s stiffness together with a transition temperatures. In compounds with 0.3% nucleate, higher tensile strength, implying an improvement of its the increase of plasticizer from 5% to 7% significantly mechanical properties. reduced the temperatures; however, the increase from 7% According to Table 2, the formulation with pure to 10% shows a similarity of curves, which means that the copolymer had the lowest impact resistance of all tested addition of more plasticizer did not reduce the temperature formulations. These results also showed that the PHBV any more. This behavior suggests a possible exudation of impact resistance rose up to 60% due to the presence of the plasticizer. The formulations with plasticizer and 0.5% additives; however, it was observed that increasing the nucleate had analogous behavior. quantity of the nucleating agent and/or the plasticizer content It can be observed an important reduction in the Tcc of caused a reduction of the measured impact resistance. The the additive formulations when compared with pure PHBV same performance was observed in the stress and strain in (from 68°C to 36-40°C). This reduction causes a delay on rupture, where the most efficient formulation was that with the melting stiffness and, consequently, increases the time the lower concentration of plasticizer and nucleates, 5P01N. needed for the cooling and molding releasing steps. This effect explains the difficulty found during the processing 3.2. Thermal analysis for the injection of the compositions with high additive Pure PHBV and other additive formulations were contents. Also, the presence of additives in the formulation submitted to DSC thermal analysis with the purpose of made the PHBV crystallization more difficult, reducing its verifying the alterations in transition temperatures caused crystallinity. The increase in the nucleating content did not by the different tested additive contents. The DSC curves cause an increase in crystallinity. referent to the second heating run for pure PHBV compared PHBV based materials were examined by TGA with the curves for formulations with 0.1%, 0.3%, or 0.5% combined to its first derivative (DTGA) to access their of nucleate and varied plasticizer contents are presented in thermal degradation data in order to verify the efficacy of Figures 3a-c. It can be observed that all formulations showed the addition of plasticizer in copolymer thermal stability. two melting temperatures. The first and lower temperature, The onset decomposition temperature (Td) was defined as characterized by a small peak in the DSC curve, corresponds that corresponding to 2% weight loss due to degradation; to the melting of the crystalline poly (hydroxyvalerate), and peak decomposition temperature (Tp) was obtained from while the second temperature corresponds to the melting of the maximum amount of DTGA. Table 4 summarizes the 19,20 the poly(hydroxybutyrate) . Lower melting temperatures events observed, initial decomposition temperature (Ti), Td,

Table 2. Mechanical properties of the formulations mentioned in Table 1.

Formulation Stress in the rupture Strain in the rupture Elasticity module Notched impact (MPa) (%) (GPa) resistance (J/m) PHBV 31.7 ± 0.5 4.6 ± 0.4 2.0 ± 0.04 25.6 ± 0.7 5P01N 27.3 ± 0.1 8.5 ± 0.4 1.3 ± 0.01 61.3 ± 2.1 5P03N 25.7 ± 1.8 6.6 ± 1.9 1.4 ± 0.05 56.3 ± 3.1 5P05N 25.8 ± 0.4 6.0 ± 0.3 1.5 ± 0.04 51.7 ± 2.7 7P01N 24.9 ± 0.1 7.1 ± 0.4 1.3 ± 0.03 56.5 ± 2.0 7P03N 24.3 ± 0.2 6.3 ± 0.3 1.4 ± 0.02 45.8 ± 1.3 7P05N 24.4 ± 0.2 5.9 ± 0.5 1.5 ± 0.08 36.5 ± 1.7 10P01N 22.6 ± 0.2 6.6 ± 0.3 1.3 ± 0.04 42.3 ± 0.9 10P03N 23.0 ± 0.2 6.3 ± 0.2 1.3 ± 0.04 36.0 ± 3.5 10P05N 24.0 ± 10.1 5.8 ± 0.4 1.3 ± 0.03 32.3 ± 1.8 Natural Additives for Poly (Hydroxybutyrate – CO - Hydroxyvalerate) – PHBV: 2014; 17(5) Effect on Mechanical Properties and Biodegradation 1149

Figure 3. DSC curves for the PHBV second heating with a variation in the plasticizer content and in the nucleate contents that are equal to a) 0.1%; b) 0.3%; and c) 0.5%.

Table 3. Thermal properties obtained by DSC from different formulations of pure PHBV and PHBV with additives.

Formulation Tg (ºC) Tm1 (ºC) Tm2 (ºC) Tcc (ºC) *Crystalline Degree PHBV 2.6 155 169 68 46.3 5P01N –7.8 152 165 40 38.1 5P03N –6.4 154 167 43 36.8 5P05N –8.4 153 165 42 39.3 7P01N –9.3 150 164 38 44.7 7P03N –9.1 150 164 37 35.8 7P05N –8.6 152 165 38 37.3 10P01N –10.1 150 163 37 37.1 10P03N –8.9 150 164 36 37.6 10P05N –8.6 151 165 36 36.1 *Calculus of crystalline degree considering, exclusively, the copolymer mass.

Tp, the organic material content, determined as the mass loss decomposition range is also narrow, the copolymer has more from 25°C up to 600°C, and stable residue content at 600°C. thermal stability, characterized by the major temperatures For the pure copolymer sample, there was a standard presented. The additive formulations showed a minor mass mass loss that occurs in a single stage and in a narrow loss corresponding to the additive decomposition, which temperature range, initially with 263°C and ending with a occurs before the PHBV continuous mass loss. The 5P01N final decomposition temperature of approximately 315°C. formulation shows a 5% mass loss, from 179°C up to If compared to the main range of PHB decomposition (from approximately 280°C, related to the amount of plasticizer in 220°C to 250°C)[10], it can be noted that although the PHBV the formulation. The 7P01N formulation lost the equivalent 1150 Brunel et al. Materials Research

Table 4. Thermal events observed during the thermogravimetric analysis of pure PHBV and the formulations with 0.1% nucleate with different plasticizer contents.

Formulation Ti (°C) Td (°C) Tp (°C) Organic material Residues (%) %) PHBV 263 284 303 99.6 0.4 5P01N 179 285 306 99.5 0.5 7P01N 164 261 275 98.5 1.5 10P01N 166 270 281 98.8 1.2 of 5% additives, while for the 10P01N sample, this loss was Figure 5 presents the rate of spherulite formation for around 7%. This reduction in the amount of additives in each isothermal crystallization temperature (60°C, 70°C, each formulation indicates the exudation (or migration to 80°C and 90°C) studied, obtained through the measurement the surface) of the plasticizer after the process. It is probable of spherulite radius according to time. Immediately, it is that the addition of the PHBV with the P-902 plasticizer verified that, for all formulations, the maximum increase is viable up to 6% additive, which would explain why the rate occurred at 80ºC, as already reported in other works21. It incorporation of more plasticizer to PHBV did not improve is also noted that the formulations with additives presented the mechanical properties of the formulations. almost the same increase on the spherulite growth rate, The temperature where the decomposition rate is at superior than pure PHBV. The faster spherulite growth maximum stage varied with the formulations: the PHBV rate indicated that the additives had an effect on the PHBV copolymer had a 303°C decomposition peak temperature; crystallization kinetics. However, from these results, it the 7P01N and 10P01N formulations were less stable, can be attested that the formulations with Licowax were showing the lowest temperatures, 275°C and 281°C, not so efficient in relation to the refining of the crystalline respectively. It was observed that the 5P01N blend was structure. The reduction of stable nuclei in the formulations the formulation with the greatest thermal stability, because with additives shows that this additive did not perform as the greatest decomposition rate occurred in the highest a classical nucleating agent, a characteristic previously 11 temperature (306°C), in which the effective mass loss starts observed through DSC thermal analysis . Nevertheless, at approximately 30°C, being above the other formulations. the efficient crystallization provoked by the presence of Licowax is noticed in the performance presented by the 3.3. Polarized Light Optical Microscopy (PLOM) formulations containing this additive in relation to the mechanical properties. The effect of additives to the nucleating agent in the crystalline morphology of pure PHBV and PHBV 3.4. Biodegradability evaluation compositions with 5% of plasticizer was studied through Test specimens of pure PHBV and PHBV with Polarized Light Optical Microscopy (PLOM). Figure 4 additives were exposed to biodegradation during 60, presents the PLOM images for isothermal crystallization 120 and 180 days of organic compound, according to at 60ºC. The first image corresponds to the isotherm initial the Sturm methodology15,16. Five test specimens for time, while the second shows the morphology after 20 each formulation were weighed before being submitted minutes of crystallization. In the images of the first column, to testing for subsequent calculation of their mass loss. small spherulites that grew from stable nuclei (non-visible) These results are presented in Figure 6. In general, it can be seen. At this temperature, the effect of the nucleating was observed that the longer the test specimen exposure agent with additives in the formulations is expected and time in organic compound, the greater was the mass loss desired, i.e., it has a large amount of stable nuclei, resulting presented by them. The additive formulation had a greater in smaller sizes of spherulites and more even distribution mass loss than the pure copolymer, indicating that the when compared to the pure copolymer. The formulation chosen additives (P902 and Licowax) did not affect the with the highest concentration of nucleate showed a more polymer biodegradation, inclusively, accelerating the refined structure, suggesting that under these conditions PHBV microbiological degradation. Given this finding, the 5P05N formulation would have the best mechanical it is possible to affirm that the additives used in this work properties. On the other hand, at higher temperatures can accelerate PHBV biodegradation, probably due to (80°C and 90°C), the formulations with additives had the reduction of its crystallinity. Considering pure PHB a lower amount of stable nuclei if compared to the pure biodegradation studies16,21, it showed approximately formulation, indicating that under these temperatures no 5% mass loss after 180 days, a value very close to the nucleating effect of Licowax occurred. At the same time, result reached in this work for PHBV without additives. there was an effect of the plasticizer in restraining the Calculating the mass variation for each formulation in each formation of these nuclei. Comparing the images based removal (mf - mi) by the number of test days, it was possible on the crystallization temperature for each formulation, to come to an average biodegradation rate of 3.4 mg/day it is clear that the number of visible stable nuclei was and 8.7 mg/day for the pure PHBV and the PHBV with reduced with the increase in the temperature, suggesting additive, respectively. that under the PHBV melting temperature, Licowax also The presence of additives in PHBV resulted in an reached fusion. increase of approximately 5% in the result of the mass Natural Additives for Poly (Hydroxybutyrate – CO - Hydroxyvalerate) – PHBV: 2014; 17(5) Effect on Mechanical Properties and Biodegradation 1151

loss after biodegradation tests. The visual aspect of these results are shown in Table 5. There was a gradual decrease biodegradable samples, presented in Figure 7, shows more in the mechanical properties with the increasing time of evident alterations in the surface of the copolymer with biodegradation. As we know, the microbiological attack additive. occurs, initially, in the polymer amorphous phase with In order to verify changes in the mechanical properties the production of small “blanks” that contribute to the of the biodegraded specimen tests, stress in the rupture breaking of the material with little or no deformation22,23. It

Figure 4. PLOM images (100x) in the initial (left) and final (right) times for each formulation, in the isothermal crystallization temperature of: a) 60°C; b) 80°C; and c) 90°C. 1152 Brunel et al. Materials Research

Figure 4. Continued... Natural Additives for Poly (Hydroxybutyrate – CO - Hydroxyvalerate) – PHBV: 2014; 17(5) Effect on Mechanical Properties and Biodegradation 1153

Figure 4. Continued... 1154 Brunel et al. Materials Research

Figure 5. Rate of spherulites formation for each isothermal Figure 6. Test specimen mass loss percentage submitted to crystallization temperature (60°C, 70°C, 80°C and 90°C). biodegradation tests by 60, 120 and 180 days.

Figure 7. Visual aspect of pure PHBV and additive PHBV samples after biodegradation tests. Natural Additives for Poly (Hydroxybutyrate – CO - Hydroxyvalerate) – PHBV: 2014; 17(5) Effect on Mechanical Properties and Biodegradation 1155

Table 5. Stress in the rupture (MPa) of pure and additive PHBV during the different times of biodegradation.

Days of Biodegradation in organic compound Formulation 0 60 120 180 PHBV 36.0 ± 0.3 34.7 ± 1.0 31.8 ± 1.3 29.6 ± 1.0 5P01N 27.3 ± 0.4 23.9 ± 0.7 20.8 ± 1.5 18.6 ± 1.5

Figure 8. Photomicrographs (1000x resolution) of pure PHBV (left) and additive PHBV (right) before and after 180 days of biodegradation test.

was observed that the formulation with additives suffered plasticizer action. The increase in the plasticizer percentage greater damage in the tensile mechanical properties than was not proportional to the increase of the properties due PHBV, which makes sense, since this formulation shows to its migration to the surface. The formulation with the greater mass loss than the pure copolymer. Figure 8 presents minimum amount of additive content, 5% P-902 and 0.1% the photomicrographs of pure PHBV and additive PHBV Licowax, was the most effective in adding additives, with before and after biodegradation tests, where the structural the best results: 35% reduction in the elastic constant, and changes that resulted from the attack of microorganisms 18% in the PHBV crystalline degree; and also 58% increase are evident. in impact resistance and 46% increase in elongation. Furthermore, it is important to emphasize that the use of 4. Conclusions lower additive content has an effect on the lower cost in the final product. In general, the addition of PHBV copolymer with the plasticizer P-902 and the nucleating Licowax with different contents resulted in an improvement in the properties of the Acknowledgements pure copolymer, characterized by the reduction of rigidity. The authors gratefully acknowledge PHB Industrial S/A, Nevertheless, the increase in the amount of additives in the and PHBV supply; and also the Brazilian research funding formulations did not make them more efficient. The increase agencies CAPES and CNPq for the financial support and in the nucleating content served as an inhibitor of the the scholarships. 1156 Brunel et al. Materials Research

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