Biodegradability of Epoxidized Soybean Oil Based ... - Springer Link

J Polym Environ (2014) 22:140–147 DOI 10.1007/s10924-013-0615-x

ORIGINAL PAPER

Biodegradability of Epoxidized Soybean Oil Based Thermosets in Compost Soil Environment W. S. Chow • S. G. Tan • Z. Ahmad K. H. Chia • N. S. Lau • K. Sudesh

•

Published online: 28 July 2013 Ó Springer Science+Business Media New York 2013

Abstract In this study, bio-thermoset from epoxidized soybean oil (ESO) was prepared in the presence of methylhexahydrophthalic anhydride curing agent and 2-ethyl-4methylimidazole catalyst. The crosslink densities of the synthesized ESO are ranged from 0.109 9 10-3 to 0.308 9 10-3 mol/cm3. The ESO bio-thermosets were exposed to the soilburial test for 8 months. Weight change and morphology of the degraded ESO specimens were assessed. It was found that the weight loss of ESO was governed by the materials compositions, crosslink density and the soil-burial exposure time. The 3 mm thickness ESO bio-thermosets with crosslink density of 0.109 9 10-3 mol/cm3 had fully biodegraded after soil-burial for 6 months. In addition, 16S rDNA sequencing was carried out to identify the soil microorganisms. It was suggested that Comamonas sp., Bacillus sp., Streptomyces sp. and Acinetobacter sp. are the possible soil microbes that degrade the ESO bio-thermosets in the compost soil environment. Keywords Bio-thermosets Biodegradation Soybean oil Soil microbes Introduction Biopolymers produced from sustainable and renewable natural resources have received considerable attention in

W. S. Chow (&) S. G. Tan Z. Ahmad School of Materials and Mineral Resources Engineering, Universiti Sains Malaysia, Engineering Campus, 14300 Nibong Tebal, Pulau Pinang, Malaysia e-mail: [email protected]; [email protected] K. H. Chia N. S. Lau K. Sudesh Ecobiomaterial Research Laboratory, School of Biological Sciences, Universiti Sains Malaysia, 11800 Penang, Malaysia

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recent years. Natural oils derived from both animal and plant sources are important renewable raw materials used in the polymer industries. This is attributed to the environmental friendliness and cost effectiveness of vegetable oils. Epoxidized soybean oils (ESO), as one of the sustainable biopolymer, has been investigated as starting materials to prepare thermosetting materials owing to their ready availability and versatility. ESO thermosetting material can be synthesized by thermal- and UV-curing processes. In the published literature, plenty of efforts have been used to modify the properties of ESO, e.g. adding fiber/filler, blending with other thermosets [1–17]. In recent years, there have also been a few studies carried out to investigate the biodegradability of epoxidized vegetable oil-based thermoset under the natural environment [18–21]. Uncross-linked ESO was found to be readily biodegradable when buried in soil [18]. This is due to the fact that the ester linkages in ESO can be easily attacked by lipase secreted by bacteria and the resulting products can be further mineralized into water and carbon dioxide by various microorganisms [19]. On the other hand, the biodegradability of crosslinked ESO bio-thermosets is greatly controlled by the crosslink densities, types of crosslink linkages and the nature of curing agents. It has been reported that highly-crosslinked ESO bio-thermosets with the non-degradable ether linkages and with the amine type linkages were hardly mineralized and biodegraded, while the ESO crosslinked with the hydrolysable ester linkages were more readily cleaved by esterases and biodegraded by microbial enzymes in soil [20, 21]. Although ESO bio-thermosets could be biodegraded under the natural environments and mineralized by the soil microorganisms, up-to-date, there is no work done to identify the soil fungus and bacteria which are capable of degrading the ESO bio-thermosets. In this study, we aimed

J Polym Environ (2014) 22:140–147

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to evaluate the biodegradability of ESO bio-thermosets in compost soil environments. This work reports on the weight changes and the morphological properties of ESO bio-thermosets after exposure to the compost soil environment for 8 months. In addition, we isolated and identified using molecular techniques the soil microbes which colonised the buried ESO bio-thermosets.

at 1 Hz and 0.05 mm, respectively. The crosslink density was calculated by using Eq. 1.

Experimental

Biodegradation Study of ESO Bio-thermosets

Materials

Soil Burial Test

Epoxidized soybean oil resin with 6.1 wt% of epoxy content and molecular weight of about 950 g/mol was supplied by Shangdong Longkou Longda Chemical Industry (China). Methylhexahydropthalic anhydride (MHHPA, CAPE Technology, Malaysia) and the 2-ethyl-4-methylimidazole (EMI, Sigma-Aldrich, Malaysia) were selected as the curing agent and the catalyst, respectively. Table 1 shows the material designations and compositions of the ESO bio-thermosets.

Soil burial test according to BS standard EN ISO 846 was conducted by burying the sample with the dimensions of 60 mm 9 20 mm 9 3 mm (length 9 width 9 thickness) completely in the compost soil for 240 days, under an average environmental temperature of 28.3 °C, 80 % relative humidity and 5.9 mm rainfall. The daily weathering report was provided by the Malaysian Meteorological Department (Malaysia). A total of 40 samples for each formulation were prepared and buried in vertical configuration with the sorted distance of 3 cm from each other at a depth of approximately 10 cm. Five samples were taken out at 60, 120, 180 and 240 days of exposure in the compost soil. The compost soil was made up of nutrient-rich humus compound, red burnt soil, charcoal and river sand. Soil analysis using the Elemental Analyzer (EA; model: Perkin Elmer 2400, USA) revealed that the compost soil consisted of 10.62 % carbon, 0.57 % hydrogen and 0.17 % nitrogen.

Preparation of ESO Bio-thermosets The MHHPA curing agent was first mixed with the EMI catalyst at a stoichiometric ratio of 1:1. Next, the ESO resin and MHHPA/EMI were then mixed at room temperature and stirred mechanically at 300 rpm for 5 min in order to prepare a homogeneous mixture. Finally, the ESO mixture was poured into a silicone mould and subjected to thermal curing process in an oven at 140 °C for 3 h.

0

vc ¼

E 3RT

ð1Þ

where vc is the crosslink density, E0 is the storage modulus of the sample in rubbery region at Tg ? 40 °C, R is the gas constant, and T is the absolute temperature.

Weight Loss Measurement

Crosslink Density Determination of ESO Bio-thermosets Crosslink density measurement on the ESO bio-thermosets was conducted using a dynamic mechanical analyzer (DMA SDTA861e, Mettler Toledo, USA). The ESO specimen with the dimension of 25 mm 9 10 mm 9 3 mm (length 9 width 9 thickness) was heated from 15 to 200 °C at a heating rate of 5 °C/min under nitrogen atmosphere. The frequency and the displacement were set Table 1 Materials designation and compositions of ESO biothermosets Material designation

ESO/MHHPA ratio

EMI loading (phr)

ES_0.5I

1.0

0.5

ES_1.0I

1.0

1.0

ES_1.5I

1.0

1.5

ES_2.0I

1.0

2.0

The weight loss assessment of the ESO samples was carried out at a regular time intervals (i.e., 60, 120, 180 and 240 days) of exposure in the compost soil. Samples were gently washed with distilled water after being removed from the soil and were dried to a constant weight at 80 °C in a vacuum oven. The percentage of weight loss was calculated using Eq. 2. Wloss ¼

Wo Wd 100 % Wo

ð2Þ

where Wloss is the percentage of weight loss; Wo and Wd is the dry weight of sample before and after the soil burial test, respectively. Morphological Characterization The surface morphologies of the ESO bio-thermosets were studied by means of field emission scanning electron microscopy (SEM; model: Supra 35VP, Zeiss, Germany).

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The ESO sample was gold coated prior to the FESEM examination. Isolation and Identification of Bacterial Strain Samples after soil burial test were washed and resuspended with sterile distilled water. Dilutions were made and spread

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on the nutrient agar plates. These plates were incubated at approximately 30 °C for 1–3 days to permit the growth of bacterial colonies. 16S rDNA fragment corresponding to Escherichia coli at the positions of 8 and 1541 was amplified with the primers BSF 8 (50 -AGAGTTTGATC CTGGCTCAG-30 ) and BSR 1,541 (50 -AAGGAGGTGAT CCAGCCGCA-30 ) by polymerase chain reactions (PCR). The 25 lL PCR amplification process consisted of 5 lL of 59 PCR buffer, 2 mM of MgCl2, 0.2 mM of deoxynucleoside triphosphate, 1 lL each of forward and reverse primers, and 1.25 U of Taq DNA polymerase (Promega, USA). The colony PCR was conducted using the MJ Mini Personal Thermal Cycler (Bio-Rad, USA) with 10-min initial denaturation at 94 °C, followed by denaturation at 94 °C for 30 s, annealing at 60 °C for 1 min and elongation at 72 °C for 2 min. Two microliter of the PCR product was electrophoresed through the 1.0 % agarose gel and stained with the ethidium bromide, followed by photographed under UV light. The amplified PCR product was purified using WizardÒ SV Gel and PCR Clean-up System (Promega USA), and the purified PCR product was then

Fig. 1 Weight loss of the ESO bio-thermosets as a function of soilburial time

Fig. 2 Photographs of soil-buried ESO bio-thermosets a ES_0.5I, b ES_1.0I, c ES_1.5I and d ES_2.0I

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Fig. 3 SEM micrographs taken from the surfaces of the ES_0.5I biothermosets after being soil-buried for a 2 months and b 4 months

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sent for sequencing at 1st BASE Laboratory (Malaysia). 16S rDNA sequences obtained were compared with the GenBank databases using the Basic Local Alignment Search Tool (BLAST, National Center for Biotechnology Information).

Results and Discussion Weight Changes and Physical Appearance Figure 1 shows the percentage of weight loss of the ESO bio-thermosets after being subjected to the soil burial test for 8 months. One can observe that the ESO bio-thermoset experienced a significant weight loss on exposure to the compost soil. The weight losses of samples were presumable due to the biodegradation of ESO bio-thermosets in soil. Also, it is believed that the weight loss of the ESO biothermosets in the compost soil can be attributed to the actions of soil microorganisms that are responsible to degrade the bio-thermosets. It has been reported that the ESO can be readily attacked and degraded by the lipase secreting bacteria found naturally in the soil [18, 19]. The

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microbial lipase enzymes produced by these soil microbes especially fungi and bacteria have been documented to be able to convert the triacylglycerols of soybean oil to intermediates with much lower molar mass, such as diacryglycerols, monoacrylglycerols, free fatty acids and glycerols. These intermediates would then be absorbed by the cells of lipase-producing bacteria and be utilized as a main carbon source [22]. The fungal and bacterial degradations may eventually result in weight loss of the ESO bio-thermosets as a function of degradation time during the soil burial test. Furthermore, it is also found in Fig. 1 that the weight loss curve of the ESO bio-thermosets follows a typical sigmoid shape. In the initial stage of biodegradation, it is determined that there is no weight change for ESO biothermosets. As the soil-burial exposure time increased, the ESO bio-thermosets experience a significant weight loss as a result of the action of the soil microorganisms to attack and disintegrate the ESO bio-thermoset into smaller fragments in the soil compost. Such weight loss behaviour as illustrated in Fig. 1 also suggests that the biodegradation process of the ESO bio-thermosets in the compost soil is autocatalytic in nature. It is known that the autocatalytic

Fig. 4 SEM micrographs taken from the surfaces of the ES_1.0I bio-thermosets after being soil-buried for a 2 months, b 4 months, c 6 months and d 8 months

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degradation reaction involves the cleavage of an ester linkage in the backbone of a polymer in the initial stage of reaction to yield the carboxyl end group, which can autocatalyze the hydrolysis of other ester bonds during the later stage of biodegradation reaction [23]. Accordingly, the extent of biodegradation is relatively slow during the initial stage of reaction, and then increases profoundly as the concentration of the autocatalytic species is sufficiently high to promote and accelerate the disintegration of a polymer as the biodegradation reaction proceeds. As can be seen from Fig. 1 the weight loss of an ESO bio-thermoset catalyzed with a lower EMI catalyst content is much higher than those with a higher EMI content. This indicates that the ESO bio-thermoset with lower crosslink density has the tendency to biodegrade more readily under the attack of soil microbes in the compost. It is noteworthy to mention that the crosslink density of the ESO bio-thermoset increased approximately 182.6 % when the EMI content is increased from 0.5 to 2.0 phr. Accordingly, the susceptibility of a polymer to biodegradation highly depends on the crosslink density and the amount of overall free volume in the polymer matrix [24]. This is due to the fact that highly crosslinked thermoset tends to act as a

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barrier to restrict the penetration of soil microbes into its network and thus limit the local colonization of the soil microorganisms in the free volume of ESO system. As a consequence, the formation of autocatalytic species (i.e., carboxyl end groups) becomes more difficult ascribed to the steric interference of the enzyme accessibility, resulting in lower level of biodegradability. Figure 2 displays the photographs of the ESO bio-thermosets after being soil buried for 0, 2, 4, 6 and 8 months. One may observe that the ESO bio-thermosets with 0.5 and 1.0 phr of EMI catalyst content (ES_0.5I and ES_1.0I) had disintegrated into a smaller piece by 4–6 months, while those catalyzed with higher EMI content (ES_1.5I and ES_2.0I) almost remained their geometries throughout the degradation study for 8 months. This observation again suggests that the ESO bio-thermosets with higher crosslink density are less susceptible to biodegradation due to their higher resistance towards soil microbial attack in soil. Morphological Properties Figures 3, 4, 5 and 6 show the SEM micrographs taken from the surfaces of the ESO bio-thermosets after being


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soil-buried for 2, 4, 6 and 8 months. From Figs. 3, 4, 5 and 6, one may observe that the surface morphology of the ESO became progressively rougher as the degradation reaction proceeded. The surface cracking also became more visible throughout the surfaces of the biodegraded ESO bio-thermosets. Apart from that, it was determined that the ESO bio-thermosets catalyzed with lower EMI catalyst content displayed more severe surface degradation as compared to those with higher EMI catalyst content (c.f. Figs. 3, 4, 5 and 6). This suggests that less tightly crosslinked ESO biothermoset may be more labile to biodegradation because of their poor chemical barrier to penetration of soil microbes. Moreover, Fig. 7 shows the SEM micrographs (taken at high magnification) of the bacterial colonies and the networks of fungal hyphae colonized on the surfaces of the ESO bio-thermosets. Shogren [20] also found that a similar fungal colonization on the surface of ESO/citric acidcoated paper after 6 weeks of burial in soil. Such colonization by the soil microbes is one of the basic requirements to meet in order for biodegradation to take place in polymer [25]. Considering these facts, in this study, the observations clearly revealed the signs of biodegradation of ESO bio-thermosets by the soil microbes under the

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compost soil environment. Additionally, we suggest that the degradation mechanisms of the ESO bio-thermosets in the compost soil involved a lipase-catalyzed reaction of water molecules and triglycerides that can liberate the bound fatty acids. The hydroxide nucleophile (from water molecule) attacked the ester bonds of the triglycerides and broke the bond of C=O and thus created an intermediate. The intermediate would then collapse and reform the C=O, and thus yielding carboxylic acid but resulting in the loss of alkoxide groups. In order to achieve a rapid equilibrium, the alkoxide group tends to function as a base to deprotonate the carboxylic acid, and lead to the formation of free fatty acids [26]. It is believed that these free fatty acids will be further broken down by cleavage into smaller units prior to cell metabolism. Accordingly, the breakdown of the complex triglycerides can be correlated to the weight loss of the ESO samples, as discussed earlier. Isolation and Identification of Bacterial Strains 16S rDNA sequencing was performed on the 16 isolates present in the biodegraded ESO bio-thermosets in order to determine the possible soil microbes and bacterial colonies


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Fig. 7 SEM micrographs of bacterial colonies and network of fungal hyphae present on the surface of ESO bio-thermosets

that were involved in the biodegradation. Figure 8 illustrates the agarose gel electrophoresis of the PCR products for the 16 isolates. As can be seen in Fig. 8 almost fully length of the 16S rDNA gene at about 1,500 bp of the 16 isolates was amplified. The obtained 16S rDNA sequences for 16 isolates were compared with the GenBank databases using the Basic Local Alignment Search Tool for the identification of the microbial populations that were present on the biodegraded ESO bio-thermosets. The predominant soil microbes identified from the soil-buried ESO biothermosets were Comamonas sp., Bacillus sp., Streptomyces sp. and Acinetobacter sp. In the published literatures, it has been reported that Comamonas sp., Bacillus sp. and Streptomyces sp. are few examples of lipase-producing soil microbes [27, 28]. These soil microbes have been known to be involved in secretion of the lipases which in turn hydrolyze the ester linkages of polymers in the presence of water molecules into smaller units allowing for the metabolisms by the cells of microorganisms [29, 30]. Specifically, the Comamonas sp., Bacillus sp., and Streptomyces sp. used the ester-based polymer as sole source of carbon and energy in order to support the microbial lipase production [31–33]. Gupta et al. [28] reported that lipases from Comamonas sp. show

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Fig. 8 Ethidium bromide-stained agarose gel electrophoresis of polymerase chain reaction (PCR) products for the 16 isolates. [Note: Lane M, 1,500 bp DNA ladder (Promega, USA), Lane 1–16, colony PCR products]

a preference for triglycerides of long-chain fatty acid moieties, while lipases from Bacillus sp. prefer small and medium-chain fatty acids. According to Xiang et al. [34], lipases from Streptomyces sp. shows specificity for unsaturated fatty acids, particularly the linoleate. On the other hands, Acinetobacter sp. is a gram negative soil microbe that was reported to be an efficient vegetable oil-degrading soil bacterium [35]. Recall that ESO thermosets consisting

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of ester linkages. Considering this fact, we suggest that Bacillus sp., Comamonas sp., Streptomyces sp. and Acinetobacter sp. are among the bacterial colonies responsible for biodegradation of the ESO bio-thermoset.

Conclusions Based on the study devoted to examine the biodegradability of ESO bio-thermosets, the following conclusions can be made. The soil-burial biodegradability of ESO bio-thermosets was studied by buried the ESO specimens in the compost soils for 8 months. Surprisingly, it was found that the ESO bio-thermosets (with 3 mm thickness) experienced a significant weight loss on exposure to the soil environment over a period of 8 months. This finding implied that the ESO bio-thermoset can be degraded under these environmental conditions. It was also determined that ESO biothermosets with lower crosslink density showed the tendency to biodegrade more readily in compost soil. From FESEM micrographs, the surface morphology of the ESO bio-thermosets gradually became rougher with noticeable holes and cracks. In addition, the network of fungal hyphae was found to colonize on the surfaces and interiors of the ESO bio-thermosets. From the 16S rDNA sequencing technique, it was found that Comamonas sp., Bacillus sp., Streptomyces sp. and Acinetobacter sp. are the potential soil microbes to biodegrade the ESO bio-thermosets. Acknowledgments This study was funded by the Universiti Sains Malaysia Incentive Grant (Grant Number: 8021013), Research University Postgraduate Research Grant Scheme (USM-RU-PRGS; Grant Number: 8045001), the Ministry of Higher Education, Exploratory Research Grant Scheme (MOHE, ERGS; Grant Number: 6730084) and the Ministry of Science, Technology and Innovation, National Science Foundation (MOSTI, NSF) fellowship.

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