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RESEARCH

Shakina et al, Microbial degradation of synthetic polyesters from renewable resources, Indian Journal of Science, 2012, 1(1), 21-28, www.discovery.org.in http://www.discovery.org.in/ijs.htm © 2012 discovery publication. All rights reserved

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Shakina J1, Sathiya Lekshmi K2, Allen Gnana Raj G3

1. Department of Chemistry, Sarah Tucker College, Tirunelveli, Tamilnadu, India, E-mail: [email protected] 2. Department of Chemistry, Sri Devi Kumari Women’s college, Kuzhithurai, Tamil Nadu, India, E-mail: [email protected] 3. Department of Chemistry, Scott Christian College, Nagercoil, Tamil Nadu, India, E-mail: [email protected]

Received 29 September; accepted 07 October; published online 01 November; printed 16 November 2012

ABSTRACT Environmental pollution by synthetic polymers such as waste polymers and water soluble synthetic polymers in waste water has been recognized as a major problem. In view of this, energetic, chemical and biological polymer-degrading techniques have been studied extensively during the last three decades. Biodegradation takes place through the action of enzymes and / or chemical deterioration associated with living organisms. To find out, the environmental resistance of the synthesized polyesters from corn oil, soil burial test and Sturm test, aerobic degradation test and bacterial adhesion test were carried out. The films were buried in the soil mixed with municipal sewage sludge for six months. Two fungal strains PV1 and PV2 and two bacterial strains S.aureus and E.Coli showed adherence on the surface of the vegetable oil based polyester film. The strain PV1 identified as Phanerochaete chrysosporium was more efficient than the other PV2 identified as candida albicans. The biodegradability of vegetable oil based polyesters was determined by Scanning electron microscopy (SEM) and visual changes in the polymer. The rate of degradation of polymers was mainly caused by bacteria, fungi and different soil conditions. Carbon dioxide evolution as a result of polymer biodegradation was determined by Sturm test. The CO2 produced after mineralization of polymer for 30 days was found to be greater for vinyl acetate polymers. It appeared that, for most of the samples that CO2 released increased near the beginning of the experiment then remained constant with time. This suggested that there may be a small fraction of the poly esters such as a lower molecular weight fraction that degrades quickly leaving the bulk recalcitrant higher molecular weight material. The presence of cross links or branches in the fatty acid portion of the triglyceride network probably inhibits binding and activity of lipases which cleave the glycerol ester bonds. More branched polyester cross links such as those formed by maleic anhydride are degraded more slowly probably as a result of steric interference of enzyme accessibility.

Key Words: Biodegradation, soil burial test, sturm test, polyesters.

1. INTRODUCTION Polymers that undergo a controlled biological degradation by micro-organisms have become of remarkable interest during the last years. Composting for instance could thereby be established as an alternative waste management system for parts of the plastic waste. Within this group of innovative polymers, polyesters play a predominant role, due to their potentially hydrolysable ester bonds [1]. While aromatic polyesters such as poly (ethylene terephthalate) exhibit excellent material properties but proved to be almost resistant to microbial attack, many aliphatic polyesters turned out to be biodegradable but lacking in properties important for applications. To combine good material properties with biodegradability, aliphatic-aromatic co polyesters have been developed as biodegradable polymers for many years. The biodegradability of polymers is influenced not only by the chemical structure of the polymers, especially the presence of functional groups and hydrophilicity-hydrophobicity balance, but also by the ordered structure such as crystallinity, orientation, and other morphological properties [2]. The degree of crystallinity was known to be one of the major rate-determining factors of the biodegradability of polymers in the condition that the biodegradation starts in the amorphous regions and then continues into the crystalline regions. In most cases, the primary biological attack is an enzymatically catalysed hydrolysis of ester, amide or urethane bonds in the polymers. This first step of depolymerization is a surface erosion process (enzymes can not penetrate into the polymer bulk) that leads to water-soluble intermediates, which then can be assimilated by microbial cells and thereby metabolized [3]. However, in many cases, the term "biodegradation" is also used if the primary degradation step is caused by a hydrolysis, which is not catalysed by enzymes, but the depolymerization intermediates are then finally metabolized by micro-organisms or reabsorbed by the body, in the case of medical applications. Since aromatic polyesters were found to be resistant to hydrolysis under mild conditions many attempts were made to increase their hydrolytic susceptibility by introducing aliphatic components into the aromatic polyester chains [4]. Tokiwa prepared co polyesters by a transesterification reaction between poly (butylenes terephthalate) or poly (ethylene isophthalate) and PCL and then studied their hydrolyzability by exposing them to Rhizopus delemar lipase [5]. Witt et al. have also studied co polyesters prepared by the poly condensation of aliphatic and aromatic monomers [6]. Reed and Gilding have investigated the biodegradability of PET copolymers consisting of 50-70 wt % PEG of molecular weight (Mw) 1500 as biodegradable elastomer [7]. Nagata et al. have studied the enzymatic degradation of PET copolymers with aliphatic dicarboxylic acids and/or PEG [8]. Early investigations on the biologically-induced degradation of aliphatic-aromatic co polyesters came to the conclusion, that a significant degradation can be observed only at relative low fractions of aromatic component [9]. However, only limited knowledge about the detailed mechanisms of the enzymatic attack on the different structures present in the co polyesters are available up to now. For the design of new and improved materials and the evaluation of the degradation behavior under other environmental conditions, work has to be done to elucidate the degradation mechanism of these interesting groups of biodegradable polymers. Polymerized vegetable oils such as oxidized linseed or tung oils have been used for centuries as protective coatings [10]. These oils harden on exposure to oxygen through the oxidation of double bonds to hydro peroxides followed by decomposition to free radicals and radical combination. Such ‘‘drying oils’’ as well as similar alkyd resins are still used today in paints, varnishes, inks and enamels. There has been renewed interest recently in developing new polymers from soybean and other plant oils as they offer a renewable feedstock vs. finite and unreliable petroleum sources [11]. Wool et al. [12] have developed a family of chemically modified oils which can be polymerized using free radical initiators into rigid composites, rubbers and adhesives. Larock et al. [13] have polymerized native vegetable oils with synthetic monomers such as divinylbenzene using a cationic initiator such as BF3. Petrovic et al. [14] have recently developed methods for preparing soy polyols from epoxidized soybean oil and for further reacting these with diisocyanates to form polyurethanes. Such materials have applications as rigid materials or as foams for insulation, carpet backing etc. Sperling et al. [15, 16] have prepared soft rubbers from epoxidized oils cured with diacids and used the interpenetrating

RESEARCH Indian Journal of Science, Volume 1, Number 1, November 2012

Microbial degradation of synthetic polyesters from renewable resources

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polymer networks to toughen polymers. Ro¨sch et al. [17] prepared soft to rigid thermosets from epoxidized oils cured with different anhydrides. Liu et al. [18] have prepared soft to rigid composites of polyamine-cured epoxidized soybean oil with fillers and fibers. Shogren [19] has prepared citric acid-cured epoxidized soybean oil resins for use as coatings and showed that that these disintegrate over time during soil burial. There has been very little work published on the biodegradability of polymerized vegetable oil-based polymers. There have been reports of microbial deterioration of oil-based paints and addition of fungicides to inhibit growth [20]. Erhan and Bagby [21] found that heat bodied (partially polymerized) soybean oil used in inks was nearly completely degraded to carbon dioxide and water. Many of the vegetable oil-based polymers described above are now used commercially or are in the process of commercialization so it would be desirable to understand the biodegradability of these systems. Such knowledge would be useful whether the polymer was intended to degrade safely in the environment or be durable and have long term resistance to degradation. In this study, we have prepared a variety of triglyceride corn oil-based polyesters and characterized the ability of soil microorganisms to mineralize these polymers into carbon dioxide and water through various biodegradation studies.

2. MATERIALS AND METHODS Refined corn oil, was purchased from the market were used as received. These vegetable oils are semidrying oil contains mainly oleic, linoleic and linolenic acid bearing one ,two and three double bonds on their chain respectively. Formic acid (98%, Merck), hydrogen peroxide (30%, Merck), Benzoyl peroxide (Aldrich) Dimethyl aniline (DMAN, Aldrich), Morpholine (MP,Aldrich) were used without further purification. The co monomers Vinyl acetate (VA), methyl methacrylate (MMA), N-vinyl pyrollidone (NVP) and styrene (STY) were purchased from Aldrich and purified by conventional drying and distillation procedure. Triethyleneglycoldimethacrylate (TEGMA) was used as cross linking agent.

2.1. Synthesis of Hydroxylated triglyceride corn oil (HTCO) Hydroxylation of corn oil was carried out using 30% hydrogen peroxide and formic acid 1000 ml of triglyceride oil was mixed with 1000 ml of 97% formic acid and 550 ml 30% (H2O2). Ice water was used externally to keep the temperature below 40oC. The reaction was vigorously stirred over night. The resulting emulsion was poured into a separatory funnel and extracted with ether. The aqueous layer was washed with water, then with dilute solution of Na2CO3 and finally with saturated sodium chloride solution. The resulting ether layer was dried over anhydrous sodium sulphate and the ether was removed by a rotatory evaporator. The resulting product was hydroxylated triglyceride corn oil resin (HTCO) with an average 8.9 hydroxyl groups per glyceride from the 1HNMR analysis.

2.2. Synthesis of macro monomer Oligomeric Poly (corn oil) fumarate resins (o-PCF) The hydroxylated triglyceride resin was then reacted with maleic anhydride to get oligomeric poly (corn oil) fumarate resins. 400g of hydroxylated corn oil and 144.4 g of MA was added when the mixture was warmed to 60oC under string. Morpholine was used as catalyst. The reaction mixture was refluxed for 5 hours at 70 - 80oC and then at 160oC ± 2oC for 30 minutes under stirring condition yield a brown transparent liquid resin oligomeric poly (corn oil) fumarate with a 10:1 molar ratio of MA to hydroxylated triglyceride of corn oil. The hydroxylated triglyceride corn oil resin (HTCO) and oligomeric poly (corn oil) fumarate resins (o-PCF) were analyzed by physicochemical methods like iodine value, saponification value, specific gravity and viscosity and spectral analysis by UV, IR and NMR analysis.

Figure 1 Reaction Scheme Synthesis of cross linked biopolyesters (CBP) based on Poly (corn oil) fumarate and vinyl monomers

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2.3. Synthesis of thermosetting polymeric materials from corn oil The macro monomer Oligomeric poly (corn oil) fumarate resins (o-PCF) was then copolymerized with, VA, MMA, NVP, and STY to produce triglyceride poly esters, poly (corn oil) fumarate –co-vinyl acetate(PCFVA), poly (corn oil )fumarate –co-methylmetacrylate(PCFMMA),poly(cornoil )fumarate - co-N-vinyl pyrollidone(PCFNVP), poly (corn oil )fumarate –co-styrene(PCFSTY). Benzoyl peroxide was used as the initiator and dimethyl aniline as the accelerator. To prevent oxygen free radical reaction the (o-PCF) resin was purged with nitrogen gas prior to curing and then casted on a clean silicone oil spreaded glass plate, cured for six hours at room temperature to yield different polymeric materials. All the cured poly triglyceride esters showed high toughness, elastomeric and good transparency.

2.4. Molecular Weight between cross links and crosslink density Molecular weights of linear polymers may be determined by appropriate physical measurements on very dilute solutions. Most commonly practiced methods are gel permeation chromatography (GPC) and intrinsic viscosity. Light scattering, ultracentrifugation and osmometry are also used in the

determination of molecular weight of polymers. However, all of these methods require the solubility of the polymer, and therefore cannot be used to determine the molecular weight of an insoluble, cross linked polymer. Crosslink density plays an important role in determining the properties of polyesters. The cross linked polymers only swell and do not dissolve in a non reactive solvent. The degree of swelling in a non reactive solvent determines the degree of cross linking and the molecular weight between cross links. The density of the polyesters was determined according to ASTM D 792. The crosslink density, γ of the polyesters was determined from the solubility parameter of the poly ester. The solubility parameter of the polyester was determined by conducting swelling experiments using small rectangular specimens in nine different solvents, having solubility parameters ranging from 7.2 J 1/2 cm -3/2 (n-hexane) to 16.5 J 1/2 cm -3/2 (glycerol). Solubility parameters of solvents used for the determination of solubility parameters of polyesters are given in table.

The solubility parameters of solvents were plotted against the swelling coefficient of the polyesters. The peak of the curve gives the solubility parameters of polyester. Among all solvents used, dimethyl acetamide (DMA) gave the maximum swelling; hence its solubility parameter was taken as being equal to the solubility parameter of polyesters. The polymer solvent interaction parameter χ is given by

Where, Vs is the molar volume of the solvent is the gas constant, T is the absolute temperature, δs are the solubility parameter of DMA, and δp is the

solubility parameter of polyester. If δs = δp , the polymer solvent interaction parameter becomes equal to the lattice constant. The crosslink density or effective number of moles of cross linked units per gram of polyester was determined using the modified Flory Rehner equation

Where, Vr = Volume fraction of the polyurethane in the swollen state,

(Vr = 1 / 1 +Q Where, Q is swelling coefficient) dr = Density of polyurethane, V0 = Molar volume of the solvent, χ = Polymer solvent interaction.

The swelling studies of the newly prepared polyurethanes have been carried out in solvents having different solubility parameters, methanol (13.5), ethanol (12.3), dimethyl acetamide (10.8), tetra hydro furan (9.3) and dimethyl formamide (12.1). All these polyesters undergo slight swelling in solvents methanol, ethanol, tetrahydrofuran, dimethyl formamide and benzene and undergo appreciable swelling in and dimethyl acetamide alone. Low swelling in solvents methanol, ethanol, tetrahydrofuran, dimethyl formamide and benzene reveal that present polyesters are also cross linked. Due to the higher degree of swelling (Swelling coefficient) only in diethyl acetamide, it is understood that the solubility parameter of the new poly ester is 10.8. For cross linked polymer, Bristo - Watson’s equation indicates that maximum swelling could occur when the solubility parameter of the solvent is almost equal to that of the polymer. The value of the swelling coefficient (degree of swelling) of the polyesters in dimethyl acetamide are characteristic of a cross linked system. Molecular weights of cross linked polymers are also characterized by the back bone chain length i.e., molecular weight between adjacent cross links (Mc). Each net work structure (a single particle) may be viewed as a single molecule. The molecular weight between cross links Mc indicates the degree of cross linking. The higher the value of Mc, the lower is the crosslink density. The effective crosslink density of polyesters is the total sum of chemical cross links. The physical cross links (hydrogen bonding) exerts its influence under solid state, but not in solvated and swelled state.

2.5. Biodegradation tests 2.5.1. Soil Burial Test The soil burial degradation test of polymer was carried as per ISO 846:1997.The replicate pieces of the poly triglyceride esters (5 cm x 3 cm) were buried in the garden soil at the depth of 30 cm from the ground surface for 3 months, inoculated with the sewage sludge having ability to adhere and degrade the polymer film. The test specimen was periodically removed from the soil and the specimen was then gently washed to remove attached soil and dust after being dried in vacuum oven. The extent of degradation was examined by weight loss and surface observation. Scanning Electron Microscope (SEM) was used for assessing surface damages of polymeric sheet subjected to soil burial test.

2.5.2. Aerobic test The aerobic bio degradability of polymeric resins in an aqueous medium was evaluated by determining the oxygen requirement in a closed resiprometry (Germany standard ISO 9408.1999).

2.5.3. Sturm Test Carbon dioxide evolution as a result of polymer biodegradation was determined by sturm test [22]. The pieces of polymer were added to culture bottles containing de mineralized water (285 ml) without any carbon source. Spore suspension of Phanerochaete chyrosporium PV1 (2.7 x 108 spores/ml) was used as inoculums 5 (v/v) in the test and control bottles (without plastic). Sterilized air was supplied to keep conditions aerobic and reaction bottles were stirred continuously by placing them on magnetic stirrer. After 30 days, gravimetric analysis of CO2 production was done by trapping the gas in adsorption bottle containing KOH (IM) of test and control were filtered, weighed and calculated for CO2 produced per liter.

2.6. Microbial studies Bacterial adhesion and antimicrobial activity were evaluated by agar diffusion method. For bacterial adhesion study samples of 1 cm square pieces were used and for antimicrobial activity spherical disc of 10 mm diameter were used. Both test samples were sterilized by autoclaving before performing the

Solubility parameters of solvents used for determination of solubility parameter of polyesters

Solvent Solubility parameter ( cal / cc ) 1/2 Hexane 7.3 Tetra hydro furan 9.1 Benzene 9.2 Acetone 9.9 Dimethyl acetamide 10.8 Dimethyl formamide 12.1 Ethanol 12.3 Ethylene glycol 14.6 Glycerol 16.5

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test and finished by agar diffusion method. The test was done in triplicates. Gentamycin (10 μ / disc) of positive control was used for antimicrobial activity testing. The microbial strains used for bacterial adhesion study were Escherichia coli ATCC 25922 and S.aureus ATCC 25923.

3. RESULTS AND DISCUSSION 3.1. Preparation of poly (corn oil fumarate) and crosslinked biopolyesters The Poly corn oil fumarate has been synthesized from the naturally available corn oil containing oleic acid and linoleic acid. Corn oil is a triglyceride of fatty acids that contain 34% monounsaturated fatty acid oleic acid (C18:1) and 52% omega-6 linoleic acid (C18:2).The hydroxylated oil was prepared by the reaction of the corn oil with #0% hydrogen peroxide and formic acid. The hydroxylated oil on further esterification with maleic anhydride at high temperature results Poly (corn oil fumarate) as per scheme given in Fig-1. The reaction between hydroxyl and anhydride group proceeds in two steps, in the first step, esterification of the anhydride occurs to form a free acid group, which is esterified in the second step. Maleic anhydride an unsaturated condensation monomer provides a site for crosslinking [23]. During high temperature polyesterification, maleate groups are converted intofumarates. Since fumarate double bonds are more reactive, the extend of maleate –fumarate isomerisation exerts a great influence on cross link density. Therefore the isomerisation was carefully controlled.In the unsaturated polyester chemistry, the number and position of double bonds is an important factor responsible for their properties [24]. The analysis of molecular weight of o-PCf resin reveals Mw-2175 and Mn-1786 with Polydispersity-1.118 which indicate the oligomeric nature of the resin. The biodegradable and crosslinked polyeasters were prepared by curing with the vinyl monomers, vinyl acetate (VA), methyl methacrylate (MMA), N-vinyl pyrollidone (NVP) and styrene (STY) as per scheme given in Fig-1.

3.1.1. Crosslink density of cross linked biopolyesters Crosslink density and voids plays an important role in determining the properties of cross linked polymers. Corn oil was used to prepare biostable polyester resins using condensation polymerization. Generally such condensation polymerization leads to generation of water and consequent formation of voids.In the present studies addition polymerization and crosslinking was adopted to generate void free thermosets. These crosslinked polymers only swell in a non reactive solvent and do not dissolve in a non reactive solvent. The degree of swelling in a non-reactive solvent determines the degree of cross linking and the molecular weight between the crosslinks. The swelling studies of the present polyesters, PCFVA, PCFMMA, PCFNVP, and PCFSTY have been carried out in solvents having different solubility parameters. All these polyesters undergo slight swelling in solvents methanol, ethanol, tetrahydrofuran, toluene, benzene and

Figure 2 AT-IR spectrum of cross linked biopolyesters (CBP) based on Poly (corn oil fumarate) biopolyester resin and vinyl acetate

Figure 3 AT-IR spectrum of cross linked biopolyesters (CBP) based on Poly (corn oil fumarate) biopolyester resin ad methyl methacrylate

Figure 4 AT-IR spectrum of cross linked biopolyesters (CBP) based on Poly (corn oil fumarate) biopolyester resin ad n-vinyl pyrollidone

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appreciable swelling in dimethyl formamide and dimethyl acetamide. The low swelling in solvents methanol, ethanol, tetrahydrofuran, toluene, and benzene reveal that the present polyester are also crosslinked. The value of the swelling coefficient (degree of swelling) of the polymer in Dimethyl acetamide is characteristic of the cross linked system [25]. The low molecular weight between crosslinks (Mc) is due to the multiple crosslinking of O-PCF resin with co monomer (VA, MMA, NVP, and STY) Table-1. In the present work, the rigid polyester PCFSTY was found to have higher crosslink density.

3.2. AT-IR spectral studies The cross linking was confirmed through the AT-IR spectral studies. The IR spectrum of the surface of the cured samples clearly indicates the significant disappearance of peak at 1,645 cm-1 and reduction of peak at 981.59 cm-1 for the unsaturated double bonds of poly (corn oil fumarate) biopolyester resin. This is due to the cross linking reaction of o-PCF with vinyl monomers. The strong peaks observed at 1,721 for the ester groups

indicate that the surface of the cured sample is comprised predominantly with ester linkages. The AT-IR spectrum of cross linked biopolyesters (CBP) based on Poly (corn oil fumarate) biopolyester resin and vinyl monomers are given in Figures 2-5.

3.3. Analysis of soil burial test Wider application of the polymers in packaging and agriculture has raised serious issue of plastic waste disposal and its pollution. To find out, the environmental resistance of the synthesized polyesters soil burial test was carried. The synthetic plastics normally resist the microbial degradation and persist in the environment for longer times. The objective of this study was the isolation of the microbial strain having ability to adhere and degrade the

vegetable oil based polymers. The films were buried in the soil mixed with municipal sewage sludge for six months. Two fungal strains PV1 and PV2 and two bacterial strains S.aureus and E.Coli showed adherence on the surface of the vegetable oil based polyester film. The strain PV1 identified as Phanerochaete chrysosporium was more efficient than the other PV2 identified as candida albicans. The biodegradability of vegetable oil based polyesters was determined by visual changes in the polymer. The polymer PCFVA shows highest weight loss and the sample PCFSTY shows the lowest weight loss in soil burial degradation test for corn oil based polyesters. Triglyceride networks containing hydrolysable ester bonds especially linear diesters are biodegraded fairly rapidly in soil. The extent of degradation was examined by the relative weight loss of all the four polymers are shown in the Table-2. Kimura et al in their study of degradation of plastics was mainly caused by bacteria, fungi and that different soil conditions affected the rate of degradation of plastics [26]. The SEM micrograph of polyesters before the soil burial degradation test and the SEM micrograph of polyesters after soil burial degradation test for 28 days were taken. The original polymer film exhibits a relatively smooth surface without any pinholes and cavities (Fig.6). However after 28 days in the soil Fig.7, large number of holes, cavities, and pin hole were observed in polymer film, indicated that the polymer surface was attacked by the microorganism under soil environment. Thus from the soil burial test the polymer synthesized is biodegradable and attacked by microorganisms.

3.4. Analysis by strum test The CO2 evolved as a result of break down of polyesters was determined gravimetrically by sturm test. It was found that the test sample (with polyester sheets), the total amount of CO2 produced was greater than in control (with no polyester sheets).The difference in cell number in reaction vessel of both test and control also showed that the bacterial consortia grew more in the case of test. It was much more active against the corn oil polyesters and utilized it carbon as energy source. Gravimetric analysis of CO2 produced in test and control was given in the Table 3. The CO2 produced after mineralization of polymer for 30 days was found to be greater for vinyl acetate PCFVA polymers. Moisture contents of the samples at the end of the experiment were typically 40%-45%. Theoretical yields of CO2 were calculated from the

carbon content of the samples. It appeared that, for most of the samples that CO2 released increased near the beginning of the experiment then remained constant with time. This suggested that there may be a small fraction of the poly esters such as a lower molecular weight fraction that degrades quickly leaving the bulk recalcitrant higher molecular weight material. There is also significant change in dry cell mass of Phanerochaete chyrosporium PV1 in test

Table 1 Crosslink properties of crosslinked biopolyesters of poly (corn oil) fumarate

Polyesters

Density ( g/cc )

Crosslink density ( x 10-3 )

Molecular weights between

crosslinks ( mol -1 )

PCFVA 1.120 2.714 460 PCFMMA 1.2120 5.078 200 PCFNVP 1.202 3.409 293 PCFSTY 1.360 6.024 166 Table 2 Weight loss in the soil burial degradation test

Polyesters

Cross link density ( x 10-3 )

Weight(g)

Initial After 30 days

Final after 60 days

PCFVA 2.714 1.214 ±1.23 1.132±0.13 0.893±0.32 PCFMMA 5.078 1.123 ±0.64 0.841±0.42 0.452±0.43 PCFNVP 3.409 1.534±0.43 1.434±0.43 1.234±0.67

PCFSTY 6.024 1.734±0.12 1.662±0.28 1.582±0.16

Table 3 Total viable count and gravimetric analysis of CO2 evolution during the breakdown of the poly esters by bacterial consortia as determined through sturm test

Polyester

Amount of CO2( g/l) Viable count Cross link

density ( x 10-3 )

Control

After 30

days

Before experiment

CFU/ml

After experiment

CFU/ml PCFVA 2.714 11.08 41.23±1.34 2.5 x109 19.5 x109 PCFMMA 5.078 11.08 25.12±2.11 2.2 x109 10 x109 CORVP 3.409 11.08 29.54±1.52 2.9 x109 13 x109 PCFSTY 6.024 11.08 21.22±1.47 2.3 x109 8.9 x109

Table 4 Crosslink density and aerobic test

Sample Code

Average BOD

Crosslink density ( x 10-3 )

COD Bio

degradation (%)

Degradation days

PCFVA 659 2.714 1.012 84±0.27 70 PCFMMA 492 5.078 0.741 61±0.09 70 PCFNVP 557 3.409 0.827 69±1.23 70 PCFSTY 390 6.024 1.634 41.2±1.02 70

Table 5 Viable Count / Sample for bio polyester

Sample

code

Number of Bacteria adhered / sample

E.Coli x 106 cfu S.aureus x 106cfu Pseudomonas

aeruginosa x109cfu

PCFVA 0.51 0.912 0.845 PCFMMA 2.63 2.52 0.687 PCFNVP 2.73 1.9 0.111 PCFSTY 2.22 2.0 0.432

Figure 5 AT-IR spectrum of cross linked biopolyesters (CBP) based on Poly (corn oil fumarate) biopolyester resin and styrene

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is higher than in control. The relatively rapid biodegradation of poly triglyceride esters suggests that there are likely parts of the network which contain chain ends having groups susceptible to biodegradation such as carboxylic acid or alcohols [27]. More branched polyester cross links such as those formed by maleic anhydride are degraded more slowly probably as a result of steric interference of enzyme accessibility. Different microbial populations and physical conditions are

present in different environments such as activated wastewater treatment sludge, composting centers, rivers, and oceans showed different rates of biodegradation for polymerized vegetable oils. Enhanced rates of biodegradation may be due to the presence of oxidized chain ends such as carboxylic acids within the network which may be more readily degraded by microbial enzymes [28]. There was also a significant change in the dry cell mass of Phanerochaete chyrosporium PV1 in test higher than in control.

3.5. Aerobic Test The aerobic bio degradability of polymeric resins in an aqueous medium was evaluated by determining the oxygen requirement in a closed resiprometry (Germany standard ISO 9408.1999), the degradation rate = BOD / COD x 100 was calculated from the BOD as a function of THOD of COD [28]. From the results in Table-4 and Fig.8 the degradation rate is faster for the polyester PCFVA than the other polymeric materials. The degradation rate of PCFSTY is less when compared to the other poly esters PCFMMA, PCFNVP due to the presence of high cross link density. The presence of cross-links or branches in the fatty acid portion of the triglyceride network probably inhibits binding and activity of lipases which cleave the glycerol ester bonds. Alternatively, some glycerol ester bonds might be cleaved but further metabolism of the fatty acids would be blocked by the branch points. Longer the sample is subjected to the aerobic test, the faster the degradation rate. When compared to soil burial test, aerobic degradation is much faster.

3.6. Bacterial adhesion Test The bio polyesters were incubated on mineral salt agar medium inoculated with isolated bacterial and fungi strain. Degradation of several of these polymers proceeds through adsorption of the micro organism to the polymer surface followed by hydrolytic cleavage. S.aureus species degrades polyesters [29]. The rate of adhesion of bacteria onto a solid surface may be influenced by the bacterial diffusion towards the surface and/or by the reaction of the bacteria with the surface. The transfer of bacteria to the surface is governed by the hydrodynamic conditions present. When bacteria approach the surface, long-range interactions, resulting from London dispersion forces and electrostatic forces occur. According to the D.L.V.O. theory, the potential energy between the bacteria and the surface depends mainly on the separation distance, the surface potentials and the composition of the liquid medium. For bacterial adhesion to occur the potential energy has to be negative [30]. The potential energy between bacteria and substrata with potentials of the same sign usually shows a maximum at a certain distance of separation, providing an energy barrier for adhesion. Bacteria may (reversibly) adhere at a distance from the surface due to the presence of a secondary shallow minimum in the potential energy. Bacteria which are able to overcome the energy barrier will reach the surface and may adhere if the potential energy due to short-range interactions (<4 A), resulting from chemical bonds, dipole interactions and/or hydrophobic interactions is sufficiently negative. Adhesion experiments with three bacterial strains differing in their surface characteristics showed that the numbers of adhering bacteria of all three strains were higher onto PCFVA (Fig.9) triglyceride polyester than onto the MMA and styrene copolymer. PCFNVP polyesters showed nil adherences of pseudomonas bacteria Table 5. Pseudomonas aeruginosa has received a great deal of interest because it is responsible for a variety of chronic bacterial infections [31]. Infections caused by opportunistic pathogens can migrate to locations within the body and can easily contaminate medical implants because of the fragments of bio films. Recently,

Table 6 Antimicrobial test

Polyesters Zone of inhibition (Gum concentration (40g/l) time 36h)

E.Coli S.typi S.aureus Pseudomonas aeruginosa

Candida albicans

PCFVA 12mm - 7mm 13mm 12mm PCFMMA 8mm 15mm - - - PCFNVP 12mm 13mm 9mm 17mm 13mm PCFSTY 9mm - 7mm 16mm - Gentamycin- 10 mcg 21mm 22mm 22(Flucanazole) 19mm 21mm

Figure 6 SEM photographs of polyesters before soil burial test a-PCFVA, b-PCFNVP, c-PCFMMA, d-PCFSTY

Figure 7 SEM images of degraded polyesters after 28 days of soil burial test A-PCFVA, B-PCFNVP, C-PCFMMA, D-PCFSTY

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researchers have focused on identifying the factors that initiate bacterial adhesion to tissues and biomedical implants, and increase antibiotic resistance [32].

3.7. Antimicrobial activity of triglyceride polyesters In a newly prepared corn oil based triglyceride poly ester resins has been studied against the bacterial species Escherichia coli, S. typi, (Gram Positive) and S.aureus, Pseudomonas aeruginosa (gram negative) and fungal species Candida albicans (ATCC 10261). The polymer resins showed potential antibacterial and anticandidal activity against microorganisms. The zone of inhibition is shown in the Table 6.

4. CONCLUSION Non-biodegradable, petroleum based polymeric materials have brought about environmental concerns the growing demands for such non renewable in destructive materials have increased our dependence on crude oil. Biodegradable polymeric material especially those prepared from readily available, renewable and inexpensive natural resources such as carbohydrates, starch, proteins have thus become increasingly important. The current interest in cheap, biodegradable polymeric materials have recently encouraged the developments of such materials from readily available, renewable inexpensive natural sources such as carbohydrates, starch, proteins have thus become increasingly important. The synthesis of polymers from renewable resources has attracted considerable attention from polymer scientists throughout the world because of their potential attributes as substitute petrochemical derivatives. Triglyceride oil is an abundant natural resource those have yet to be fully exploited as a source for polymers and composites. Natural oils are expected to be inexpensive renewable resources. Development of new polymeric materials from vegetable oil is highly desirable. The purpose of this work is to prepare high molecular weight polymers and it would be alternative petroleum based polymeric materials such as plastic and rubber materials. The present method of insitu hydroxylation and maleanation favours formation of biodegradable oligomeric polytriglyceride fumarate resin (O-PTF). Copolymerization of O-PTF resin with vinyl monomers without any solvent leads to a cross linked polymer with high molecular weight and good resistance to chemicals. The results showed a substantial improvement in polymer properties including increasing in use temperature and mechanical properties, as well as reduction in flammability, heat evolution and viscosity during processing. Soil and sewage sludge contain microorganisms (fungi) that are able to bring about some degradation of synthetic polymers. The fungal isolate Phanaerochaete chrsosporium PV1 showing adherence and growth on the surface of corn oil based polymers indicated their ability to utilize

polyesters as a source of nutrient. Production of carbon dioxide during the Sturm test indicated positive degradability test for the vegetable oil based polyesters. The cured polyester films are biodegradable and used for medical purposes as absorbable sutures and agricultural films. In bio-application their bio-compatibility and biodegradability play an important role.

ACKNOWLEDGEMENT One of the authors (JS) wishes to thank the management and Principal, Sarah Tucker College (autonomous),Tirunelveli for their support to undertake this research programme. The authors acknowledge the staff BMT wing of Sree Chitra Tirunal Institute for medical sciences and technology, Trivandrum for the assistance in the evaluation of polymer samples.

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Figure 8 Aerobic degradation of cross linked Biopolyesters of poly (Corn oil fumarate)

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