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Appl Biochem Biotechnol DOI 10.

1007/s12010-012-9688-6

Environmental Degradation of Microbial Polyhydroxyalkanoates and Oil Palm-Based Composites


Y. S. Salim & A. Sharon & S. Vigneswari & M. N. Mohamad Ibrahim & A. A. Amirul
Received: 9 May 2011 / Accepted: 10 April 2012 # Springer Science+Business Media, LLC 2012

Abstract This paper investigates the degradation of polyhydroxyalkanoates and its biofiber composites in both soil and lake environment. Time-dependent changes in the weight loss of films were monitored. The rate of degradation of poly(3-hydroxybutyrate) [P(3HB)], poly (3-hydroxybutyrate-co-4-hydroxybutyrate) [P(3HB-co-23 mol% 4HB)] and poly(3-hydroxybutyrate-co-3-hydroxyvalerate-co-4-hydroxybutyrate) [P(3HB-co-9 mol% 3HV-co-19 mol% 4HB)] were investigated. The rate of degradation in the lake is higher compared to that in the soil. The highest rate of degradation in lake environment (15.6 % w/w week1) was observed with P(3HB-co-3HV-co-4HB) terpolymer. Additionally, the rate of degradation of poly(3hydroxybutyrate-co-3-hydroxyvalerate) [P(3HB-co-38 mol% 3HV)] was compared to PHBV biofiber composites containing compatibilizers and empty fruit bunch (EFB). Here, composites with 30 % EFB displayed the highest rate of degradation both in the lake (25.6 % w/w week1) and soil (15.6 % w/w week1) environment. Keywords Polyhydroxyalkanoates (PHAs) . Biodegradation . Biocomposites . Environment

Introduction Synthetic plastic products in modern society have been widely used. It is famous for its versatility, imperviousness to water, as well as resistance to degrade. Unfortunately, these features, which were thought to be the forte of synthetic plastic, are detrimental to the environment due to their nonbiodegradability [15]. The increasing environmental problems

Y. S. Salim and A. Sharon contributed equally to this work.

A. Sharon : S. Vigneswari : A. A. Amirul (*) School of Biological Sciences, Universiti Sains Malaysia, 11800 Minden, Penang, Malaysia e-mail: amirul@usm.my Y. S. Salim : M. N. Mohamad Ibrahim School of Chemical Sciences, Universiti Sains Malaysia, 11800 Minden, Penang, Malaysia Y. S. Salim Department of Chemistry, Universiti Malaya, 50603 Serdang, Selangor, Malaysia S. Vigneswari : A. A. Amirul Malaysian Institute of Pharmaceuticals and Nutraceuticals, MOSTI, 11900 Minden, Penang, Malaysia

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associated with discarded plastics have brought many research studies directed towards the development of suitable eco-friendly materials that can replace the commodity plastics. One of the biotechnologically important eco-friendly materials is polyhydroxyalkanoate (PHA), which is synthesized by a wide variety of microorganisms as carbon and energy storage under nutrient-limiting conditions in the presence of excess carbon source [8]. Having several desired characteristics such as biodegradability and biocompatibility (with living cells), these polymers are often used for biomedical and pharmaceutical applications, including drug delivery systems, implant materials, and nontoxic surgical sutures [23]. Poly(3-hydroxybutyrate) [P(3HB)], a homo-biopolymer, exists naturally in many organisms [10]. Several related copolymers associated to P(3HB) such as poly(3-hydroxybutyrate-co3-hydroxyvalerate) [also termed as P(3HB-co-3HV) or PHBV] and poly(3-hydroxybutyrateco-3-hydroxyhexanoate) [termed as P(3HB-co-3HHx) or PHBHHx] are also available in the market. Although some of these biopolymers are commercially available, the production cost is the main cause of the slow-pace growth of their global large-scale production. Several attempts are still underway to improve the bacterial strain through the genetic engineering, sourcing of cheap and renewable carbon energy, and high recovery efficiency of PHAs [20]. Incorporation of natural fibers to form biocomposites is an alternative way to reduce the overall production cost, and it is believed that biocomposites provide an alternative positive solution to all the issues mentioned [13]. Biocomposites are made from natural or hybrid fibers and biodegradable polymer [12]. Many have reported the use of kenaf, jute, bamboo, and empty fruit bunch (EFB) fibers as biocomposites. Empty fruit bunch fibers are lignocellulosic waste produced from palm oil mills. These authors have successfully revealed the properties of the resulting composites through several analyses involving thermal analyses and chromatography analyses [3, 5, 14]. Yet, it is essentially important to explore the biodegradability of PHA-based composites as a continuation study from previous work [18]. In this paper, we study the degradation of biodegradable PHA-based composites, namely P (3HB-co-38 mol% 3HV)/EFB composites, in natural environment, followed by a comparison studies between the degradation behaviors of these composites and various PHAs, such as P (3HB), P(3HB-co-23 mol% 4HB), P(3HB-co-38 mol% 3HV), and P(3HB-co-9 mol% 3HV-co19 mol% 4HB). The degradation process was studied by monitoring the time-dependent changes in weight loss of the samples over a period of 8 weeks. As the degradation of PHA and biocomposites was hypothesized to be caused mainly by microbial activity, isolation of microorganisms capable of degrading PHA and biocomposites was also carried out.

Materials and Methods Bacterial Strains and Culture Media Cupriavidus sp. USMAA2-4 was locally isolated from a soil sample collected in Sg. Pinang, western Penisular Malaysia [2]. The growth medium employed in this study was nutrient broth, which consists of 5 g/l peptone (Oxoid, England), 2 g/l yeast extract (Oxoid, England), 1 g/l meat extract (Oxoid, England), and 5 g/l NaCl (R&M Chem, UK). Preculture preparation has been described elsewhere [2]. Microbial PHAs of various monomer compositions (Table 1) were prepared by inoculating Cupriavidus sp. USMAA2-4 in 2-l fermenter (Biostat B; B Braun Biotech Inc., USA) containing mineral salt medium [3.7 g/l KH2PO4 (R&M Chem), 5.8 g/l K2HPO4 (R&M Chem), 1.1 g/l (NH4)2SO4 (R&M Chem), 10 ml/l 1.0 M MgSO4 7H2O (R&M Chem), and 1 ml/l trace elements]

Appl Biochem Biotechnol Table 1 Microbial PHAs of various monomer type and compositions Sample no. 1 2 3 4 5 6 7 PHA samples P(3HB) P(3HB-co-4HB) P(3HB-co-3HV) P(3HB-co-3HV-co-4HB) PHBV55/CA5/EFB40 PHBV60/CA5/EFB35 PHBV65/CA5/EFB30 Carbon substrate Oleic acid Oleic acid and -butyrolactone Oleic acid and 1-pentanol Oleic acid, 1-pentanol and -butyrolactone

for 72 h at 30 C. The trace elements were prepared by mixing the following chemicals in 1 l of HCl [0.1 M] to 2.78 g FeSO4 7H2O, 1.98 g MnCl2 4H2O, 2.81 g CoSO4 7H2O, 1.67 g calcium chloride (CaCl2) 2H2O, 0.17 g CuCl2 2H2O, and 0.29 g ZnSO4 7H2O. Sample 1, i.e., P(3HB) homopolymer, was produced from oleic acid. Samples 2 and 3, i.e., P(3HB-co-38 mol%-3HV) and of P(3HB-co-23 mol%-4HB) copolymers, respectively, were produced from mixture of oleic acid and 1-pentanol, and mixture of oleic acid and -butyrolactone. Sample 4, i.e., P(3HB-co-9 mol%-3HV-co-19 mol%-4HB) terpolymer, was prepared from mixture of oleic acid, -butyrolactone, and 1-pentanol. Samples 57, i.e., P(3HB-co-38 mol%-3HV)/EFB blends of different ratios, were prepared as previously described [18]. The composites were coded as PHBV65/CA5/EFB30, PHBV60/CA5/EFB35, and PHBV55/CA5/EFB40.

Preparation of PHAs Films Prior to the preparation of films, lyophilized cells obtained from the fermentation were extracted with chloroform (ratio to methanol 1:200) and further purified in ice-cold methanol. Analyses of monomer compositions and molecular weights were done using gas chromatography and gel permeation chromatography respectively [17]. PHA films of samples 14 were prepared by solvent casting technique. Approximately 1 g of polymer was dissolved in 20 ml of chloroform and poured into an 11-cm diameter petri dish. The petri dish was left overnight at room temperature to evaporate the chloroform. Complete evaporation resulted in the formation of PHA films. Samples 57 were prepared by hot compression molding at 165 C for 5 min. The difference between two preparations (solvent casting technique and hot compression molding) is due to the solubility of the samples in the solvent. Samples 14, which are neat PHAs, can be dissolved in chloroform and form a smooth film. They were prepared by solution casting method to avoid any thermal degradation that may occur during melt press. On the other hand, samples 57 are made up of the mixture of PHA and undissolved constituents such as lignocellulosic short fibers. Thus, they were prepared by melt press. All degradation samples were cut into smaller pieces measuring 1.51.5 cm (Fig. 1). The thickness of all the films was 0.060.10 mm.

Experimental Design and Sampling Apparatus Polymer degradation studies were carried out in natural ecosystem of Harapan Lake and Science Garden of Universiti Sains Malaysia, Penang, Malaysia. Harapan Lake is

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Immersion Test Method

Soil Burial Test Method

Fig. 1 Schematics preparation of polymer films

a highly eutrophic aquatic ecosystem, whereas the soil of the Science Garden of Universiti Sains Malaysia is anoxic. The average temperature of these two places was 30 C. All the samples with respective compositions were weighed and placed into a nylon mesh pocket measuring 22 cm. The pockets were sewn using different color threads to distinguish one sample to another. Two types of test methods were employed: immersion test method to study the PHAs biodegradation in the lake and soil burial test method to study the PHAs biodegradation in the soil. In the immersion method, after inserting all the samples into respective pockets, they were attached to strings measuring 20 cm in length. The strings were tied to a wire inside a polyvinylchloride (PVC) core measuring 1560 cm. The PVC contained several holes which were randomly drilled to ensure unrestricted water exchange throughout the 8 weeks of the study period (Fig. 1). The samples were immersed at approximately 15 cm below the surface of the lake water [19]. As for the soil burial method, a dimension of 10.3 m flower bed was made, and it was divided into eight portions. In each portion, approximately 710-cm-deep holes were dug and the samples were buried in the respective holes. All the degradation experiments were performed in triplicates. The samples were retrieved after the stipulated time, and the soil particles rinsed with distilled water prior to oven drying at 80 C.

Isolation of PHA-Degrading Microorganisms The microorganisms capable of degrading the samples were isolated using P(3HB) liquid medium. The medium consisted of two solutions, where the first solution is made up of 2.56 g/l K2HPO4, 2.08 g/l KH2PO4, 0.5 g/l MgSO4 7H2O, 1.0 g/l NH4Cl,

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and 0.25 % P(3HB). The second solution was made up of 1.0 g/l of ammonium ferric citrate and 0.1 g/l of CaCl2. Approximately 0.5 % (v/v) of solution 2 was transferred aseptically into solution 1. P(3HB) agar medium was prepared by adding 15 % (w/v) of agar powder. One milliliter of lake water sample was transferred into the P(3HB) liquid medium. As for the soil sample, 1 g of soil was transferred into 10 ml of distilled water. After thorough mixing, 1 ml of the mixture was transferred into the P(3HB) liquid medium. Both cultures (obtained from lake water and soil samples) and control were incubated for 72 h until there is a clearing of the medium color. Once a clear zone was observed, the culture was streaked onto P(3HB) agar medium to obtain a single colony. The single colony was transferred onto a new P(3HB) agar medium prior to 16s rRNA analysis.

Analyses PHA content and polymer composition of the lyophilized cells were determined using gas chromatography (GC-2014, Shimadzu, Kyoto, Japan) based on a standard method [4]. Molecular weight of the extracted and purified copolymers were analyzed using gel permeation chromatography (GPC; Waters 600E GPC system and Waters 410 refractive index detector with a PL gel Mixed C column; Polymer Laboratories, Ltd., UK). Chloroform was used as the eluent at a flow rate of 1.0 ml/min. The sample concentrations and injection volumes were 2 mg/ml and 20 l, respectively, at 35 C. Polystyrene standards with low polydispersity were used to construct a calibration curve. Number average molecular weight (M n), weight average molecular weight (Mw), and polydispersity index (Mw/Mn) were calculated using Clarity chromatography software (Version 2.4). The surface morphologies of the copolymers films were observed under a scanning electron microscope (Leo Supra 50 VP Field Mission SEM; Carl-Ziess SMT, Oberkochen, Germany) as previously mentioned [6].

Results and Discussion Degradation of a series of PHA samples and their composites in lake water and soil was carried out. The weight loss percentage was given in Eq. 1 as a function of final weight over initial weight. Also, degradation rate was calculated as a function of weight changes over the time period of degradation, which is presented in Eq. 2.  % weight loss Initial weight Residual weight Initial weight of film  100% 1

AA0 kt

A is the balance weight (percent, w/w), A0 refers to initial weight (percent, w/w), k refers to constant degradation rate (percent, w/w each week), and t represents time taken to degrade (in weeks). The obtained k value is then used to estimate the time for PHA samples to degrade partially and completely. t1=2 ln 2=k 3

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Degradation Pattern of Various PHA Samples Biodegradation rates of PHA and biocomposites films depend on few factors, notably those related to the environment such as pH, availability of water, nutrient supply, temperature and microbial activity [7, 16]. Besides the environment influencing factors, degradation rate is also affected by the physical properties of PHA and its composite materials such as crystallinity, blending composition, surface area and presence of additives [7]. Figure 2 shows time dependence of weight loss (in percent) of various PHA samples in the soil and lake. Overall, the degradation rate of neat PHAs (Fig. 2b) was similar in the soil to that of PHBV/EFB composites (Fig. 3b), which were 14.9, 15.0, and 15.6 % w/w week1, respectively. The degradation of PHAs in the soil has similar pattern, with P(3HB) showing the lowest degradation rate, while terpolymers P(3HB-co-9 mol%-3HV-co-19 mol%-4HB) have

Weight Loss (%)

Fig. 2 Degradation of various PHA samples in the a lake and the b soil for a period of 8 weeks. P(3HB) (empty circles), P(3HB-co-4HB) (filled square), P(3HB-co-3HVco-4HB) (multiplication sign)

100 90 80 70 60 50 40 30 20 10 0 0 2 4 Time (week) 6 8

30

B
25 Weight Loss (%)

20

15

10

0 0 2 4 Time (week) 6 8

Appl Biochem Biotechnol Fig. 3 Degradation of PHBV biocomposites in the a lake and the b soil for a period of 8 weeks. Neat P(3HB-co-3HV) (empty circles), 30 % EFB fibers content (filled square), 35 % EFB fibers content (multiplication sign), 40 % EFB fibers (filled circles) content in PHBV/EFB composites, respectively
100 90 80 Weight Loss (%) 70 60 50 40 30 20 10 0 0 2 4 Time (week) 6 8

20 18 16 Weight Loss (%) 14 12 10 8 6 4 2 0 0 2 4 Time (week) 6 8

the highest rate of degradation. Meanwhile, all PHAs exhibited similar pattern of degradation in the lake (Fig. 2a). This is due to the fact that PHA undergoes hydrolysis in the presence of water. Since polymers are subjected to hydrolysis during degradation, water contributes to the ease of hydrolytic activity by the PHA depolymerases secreted by microorganisms. In the lake, where water is abundant and is constantly flowing, collision between water and polymers per unit time increases. This increases the rate of degradation. A similar behavior was observed by Yoshie et al. [25] in their study of P (3HB-co-3HV). Hydrolysis of PHA samples also occurred in the soil, but in limited condition. The movement of water in soil plays an important role in the degradation of PHA in the soil [11]. Therefore, the rate of degradation in the soil is hypothesized to be lower compared to lake.

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According to Sudesh et al. [21] and Abe et al. [1], copolymers have larger amorphous region than homopolymers. Since the amorphous region of P(3HB-co-3HV), P(3HB-co4HB) and P(3HB-co-3HV-co-4HB) films is larger than P(3HB) films, P(3HB) films have the lowest rate of degradation and percentage of weight loss (Fig. 2b). It is wellknown that the existence of longer side carbon chain in copolymers made the second monomers, such as 3HHx, 3HV, or 4HB, unable to co-crystallize in P(3HB) crystal lattice [26]. These characteristics enabled the films to have higher rate of degradation compared to P(3HB) films. Another possible reason for high degradation rate of both copolymers was due to the existence of steric hindrance, caused by the ethyl side chain of the 3HV monomer. The hindrance also exists between adjacent carbonyl oxygen atoms of 4HB monomer, and this requires the P(4HB) lattice to be deformed to accommodate the 3HB monomers [9]. All PHA samples are to be partially degraded within 2125 weeks in soil, and 34 weeks in lake. Figure 3 shows the time dependence of weight loss (in percent) of PHBV-based composites in the soil and lake. To elucidate the degradation pattern of PHBV/EFB composites in the lake, it is crucial to understand the definition of biocomposites. Biocomposites consist of biodegradable polymers as the matrix material and biodegradable fibers as the filler. The fillers serve as reinforcement to improve the stiffness of the composites. Salim and co-workers [18] reported the improvement of toughness when fillers such as EFB short fibers were incorporated. As the percentage of EFB fibers increases, the biocomposites become stiffer and more stable, hence decreasing the rate of degradation. This hypothesis was consolidated by the degradation pattern of PHBV/ EFB composites with 35 and 40 % EFB fiber content. In the lake, the calculated degradation rate (based on Eq. 2) of both PHBV/EFB composites with 35 and 40 % EFB fibers content showed similar trend, which was 10.1 and 9.0 % w/w week1, respectively (Fig. 3a). In contrast, PHBV/EFB composites with 30 % EFB fibers degraded at a faster rate (k025.6 % w/w week1) after 6 weeks. This could be due to the difficulty in collecting the composite fragment after degradation (shown later in Fig. 5). In addition, Teramoto et al. [22] reported an acceleration of weight loss in PHBV composites when untreated abaca fibers were used. However, in our study, it showed preferential degradation towards neat PHBV. This could be due to the higher content of PHBV that triggered the hydrolysis and biodegradation by the microorganisms present in the lake. As shown in Fig. 3b, PHBV/EFB composites demonstrated higher degradation rate in the soil compared to neat PHA. PHBV/EFB composites were degraded at a rate of 4.3 4.7 w/w week1, while the degradation rate of PHA films varied from 2.3 to 2.6 % w/w week1 with not much variation among samples. During the degradation period, the color of the biocomposites changed. The initial biocomposites films were brown in color, and the color faded gradually over the experiment period resulting in a very light shade of brown. Degradation of the lignin portion of the EFB fibers is suggested to be responsible for the distinctive color change. Biofibers serve as reinforcement of the biocomposites. So, as the percentage of the biofiber increases in the biocomposite, the biocomposite becomes stiffer and more stable, hence decreasing the rate of degradation. All biocomposites are to be partially degraded within 2124 weeks in soil, and 6 11 weeks in lake. Physical changes of the films after degradation over the period of experiment were observed, and illustrated in Figs. 4 and 5. The original shape of PHA was retained (Fig. 5a), while PHBV/EFB composites cracked after 8 weeks of degradation in the soil

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P(3HB)

P(3HB-co-4HB)

P(3HB-co-3HV-co-4HB)

4 Weeks

P(3HB-co-3HV)

55%HV/5%CA/40%EFB

60%HV/5%CA/35%EFB

65%HV/5%CA/30%EFB

4 Weeks

Fig. 4 Degradation in the lake illustrated by physical changes in the a PHA films, and b PHBV/EFB fiber composites over the duration of the experiment

(Fig. 5b). This made the recovery of the degraded fragments difficult after 60 days. Similar observations were also reported in another study [22]. Due to the failure in determining the molecular weight of biocomposites, discussion on changes in monomer compositions (Tables 2 and 3) and molecular weight (Table 4) are limited to PHA samples. From the results, no significant changes were observed in the P(3HB) monomeric units before and after degradation. Nevertheless, significant changes (p<0.05) were observed in copolymers having 3HV fraction, and terpolymers having 3HV and 4HB fractions. It was observed in copolymers and terpolymers that the fractions of monomer such as 4HB and 3HV were significantly degraded, and 3HB monomers fraction increased with time. Furthermore, there were slight reductions in

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P(3HB)

P(3HB-co-4HB)

P(3HB-co-3HV-co-4HB)

P(3HB-co-3HV)

55%HV/5%CA/40%EFB

60%HV/5%CA/35%EFB

65%HV/5%CA/30%EFB

Fig. 5 Degradation in the soil illustrated by physical changes in the a PHA films and in the b P(3HB-co3HV) with PHBV biofiber composites over the duration of the experiment Table 2 Changes in monomer compositions of various PHA samples in the lake Films Monomer units (%mol) Initial 3HB P(3HB) P(3HB-co-4HB) P(3HB-co-3HV-co-4HB) 100 c 77 a 72 a 3HV 0a 0a 9b 4HB 0a 23 e 19 d 3HB 100 c 84 b 76 a Final 3HV 0a 0a 16 c 4HB 0a 16 c 8b

Analyzed using GC with three replicates. Means in the same row with different letters are significantly different (p<0.05)

Appl Biochem Biotechnol Table 3 Changes in monomer compositions of various PHA samples in the soil Films Monomer units (%mol) Initial 3HB P(3HB) P(3HB-co-4HB) P(3HB-co-3HV-co-4HB) 100 e 77 b 72 a 3HV 0a 0a 9b 4HB 0a 23 e 19 d 3HB 100 e 87 d 80 c Final 3HV 0a 0a 15 c 4HB 0a 13 c 5b

Analyzed using GC with three replicates. Means in the same row with different letters are significantly different (p<0.05)

molecular weight of all PHA samples in the lake, while no changes were observed in P(3HB) samples in the soil. This may be attributed to high crystallinity of P(3HB) samples.

Isolation of Microorganisms from the Site of Degradation Isolation and identification of bacteria were done to observe the different types of PHA degraders present at the site of research. Bacteria were isolated from soil on the surface of the degraded PHA films and from water sample obtained at the site where the films were submerged. PHA degraders were identified by observing the formation of clear zones surrounding the bacteria colonies grown on P(3HB) agar. Formation of the clear zones indicates that the bacteria are capable of excreting extracellular PHA depolymerases. Positive isolates were then subjected to biochemical characterization as well as 16S rRNA cloning and DNA sequencing. In general, the isolates were found belong to two main genera, Acidovorax and Ralstonia with a confidence level of 100 % (Fig. 6). Bacteria from these genera are well-known for PHA degradation [24]. Results here indicate the predominance of these bacteria/groups at the research site and that they could have mainly contributed to the degradation of the PHA films.
Table 4 Changes of molecular weight of various PHA samples in the lake and soil Condition Lake Initial Final Soil Initial Final Molecular weight Mn (103 g/mol) PDI Mn (103 g/mol) PDI Mn (103 g/mol) PDI Mn (103 g/mol) PDI P(3HB) 180 g 2.4 x 90 d 2.4 x 180 g 2.4 x 180 g 1.7 v P(3HB-co-4HB) 170 f 2.0 w 70 c 2.4 x 170 f 2.0 w 140 e 2.5 x P(3HB-co-3HV-co-4HB) 40 a 4.5 z 40 a 3.2 y 40 a 4.5 z 60 b 3.2 y

Analyzed using GPC with three replicates. Means in the same column with different letters are significantly different (p<0.05)

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HQ704414 Ralstonia sp. DPS1

HQ704415 Acidovorax sp. DPS2

Fig. 6 Neighbor-joining phylogenetic tree of bacteria isolated at the place of degradation both in soil and lake; Ralstonia sp. DPS1 and Acidovorax sp. DPS2 based on 16S rRNA sequence comparisons. Accession numbers are given

Conclusions Degradation rates of several types of PHA and its composites were studied in natural ecosystem. Biodegradation was evaluated by the weight loss. It was found that degradation rates of neat PHA were higher in lake compared to the composites. All samples undergo microbial degradation through hydrolysis and enzymatic pathways. The order of weight loss percentage among the PHA films in the lake can be given in the following order of PHBV65/ CA5/EFB30 > P(3HB-co-38 mol%-3HV) > P(3HB-co-9 mol%-3HV-co-19 mol%-4HB) > P (3HB-co-23 mol%-4HB) > P(3HB) > PHBV60/CA5/EFB35 > PHBV55/CA5/EFB40. In the soil, however, the degradation rate of composites was higher than neat PHA. The order of weight loss percentage among the PHA films is P(3HB-co-38 mol%-3HV) > PHBV65/CA5/ EFB30 > PHBV60/CA5/EFB35 > PHBV55/CA5/EFB40 > P(3HB-co-9 mol%-3HV-co19 mol%-4HB) > P(3HB-co-23 mol%-4HB) > P(3HB). Since both the components of

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biocomposites and PHA are discretely biodegradable, the composite as the integral part is also biodegradable. On the basis of this study, we concluded that neat PHA and its composites can be degraded upon disposal of end usage.
Acknowledgments The authors wish to thank the Ministry of Science, Technology and Innovation (MOSTI), Malaysia and Universiti Sains Malaysia for the research grant to this work. Y. S. Salim acknowledges Ms. Hanisah Kamilah for her guidance in statistical analyses.

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