Marine microbe-mediated biodegradation of low- and high-density polyethylenes

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International Biodeterioration & Biodegradation 61 (2008) 203–213 www.elsevier.com/locate/ibiod

Marine microbe-mediated biodegradation of low- and high-density polyethylenes M. Sudhakara, Mukesh Doblea,, P. Sriyutha Murthyb, R. Venkatesanb a

Department of Biotechnology, Indian Institute of Technology Madras, Chennai 600 036, India b OSTI, National Institute of Ocean Technology, Chennai 601 302, India Received 29 June 2007; received in revised form 23 July 2007; accepted 23 July 2007 Available online 19 September 2007

Abstract Unpretreated and thermally pretreated low- and high-density polyethylenes (LDPE and HDPE) and unpretreated starch-blended LDPE were subjected to in vitro biodegradation. In this study two marine micro-organisms were selected, specifically Bacillus sphericus GC subgroup IV and Bacillus cereus subgroup A, for a duration of 1 year, at pH 7.5 and temperature 30 1C with the polymer as the sole carbon source. FTIR spectrum showed that initially carbonyl index increased, probably due to oxidation by dissolved oxygen (abiotic factor). Prolonged exposure to organisms led to decrease in carbonyl index due to biodegradation (biotic) through Norrish-type mechanism or through the formation of ester. The weight loss of the thermally treated LDPE and HDPE samples were about 19% and 9% respectively, and unpretreated samples were 10% and 3.5% respectively with B. sphericus in 1 year. Weight loss of unpretreated starch-blended LDPE was 25% with B. cereus. Tensile strength of thermally pretreated LDPE and HDPE and unpretreated starchblended LDPE decreased by 27%, 14.8% and 30.5%, respectively, with B. sphericus and the corresponding decrease in crystallinity was 8%, 2.2% and 8.5%, respectively. Decrease in contact angle indicated that the surfaces turned more hydrophilic after exposure. Surface morphological changes of the biological-treated samples were observed by atomic force microscopy. r 2007 Elsevier Ltd. All rights reserved. Keywords: LDPE; HDPE; Starch-blended LDPE; Biodegradation; Marine bacteria

1. Introduction Synthetic plastics accumulate at a rate of 25 million tons per year in the terrestrial and marine coastal environment. Polyethylene represents 64% of the synthetic plastics produced and they are mainly used for manufacturing plastic bags, bottles, disposable containers, which are discarded within a short time (Byuntae et al., 1991). The degradation of polymers involves several physical and chemical processes accompanied by small structural changes which lead nevertheless to significant deterioration of the quality of the material (Brown et al., 1974; Doi et al., 1992). Degradation is an irreversible change, resembling the phenomenon of metal corrosion. Polymeric materials are not easily biodegraded. Efforts have been directed to develop mild physicochemical procedures which includes Corresponding author. Tel.: +91 44 22574107; fax: +91 44 22574102.

E-mail address: [email protected] (M. Doble). 0964-8305/$ - see front matter r 2007 Elsevier Ltd. All rights reserved. doi:10.1016/j.ibiod.2007.07.011

thermal and radiation pretreatments (Albertsson et al., 1987) to enhance the biodegradation process. The degradation of plastics in nature is a very slow process which is first initiated by environmental factors followed by wild micro-organisms. The environmental factors include temperature, humidity, pH and UV. Biodegradation is the ability of micro-organism to influence abiotic degradation through physical, chemical or enzymatic action (Albertsson et al., 1987; Lee et al., 1991; Erlandsson et al., 1997; Chiellini et al., 2003). Interplay between biodegradation and different factors in the biotic and abiotic environments is very important. The micro-organism reported for the biodegradation of the polyethylene include fungi (Aspergillus niger, Aspergillus flavus, Aspergillus oryzae, Chaetomium globusum, Penicillium funiculosum, Pullularia pullulan), bacteria (Pseudomonas aeruginosa, Bacillus cereus, Coryneformes bacterium, Bacillus sp., Mycobacterium, Nocardia, Corynebacterium, Candida and Pseudomonas) and Actinomycetales

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(Streptomycetaceae). Their activity on the polymer was studied by growth tests on solid agar medium for a definite period of time. The changes in molecular weight, structure, crystallinity, density, weight loss, mechanical, optical or dielectric properties, etc., were also measured (Weiland et al., 1995; Volke et al., 2001; Yamada et al., 2001; Orhan and Buyukgungor, 2000; Saha et al., 2003; Pinchuk et al., 2004). The rate of biodegradation of polyethylene, even after prolonged exposure (10–32 years) to microbial consortia of soil, was found to be very low, thus accounting for carbon mineralization of less than 1% (Albertsson and Karlsson, 1990; Ohtake et al., 1998). More recently it has been demonstrated in soil burial tests that the use of suitable additives in polyethylene films induced substantial oxidation with consequent fragmentation, drop in molecular weight, increase in wettability, ultimately followed by high mineralization (60–70%) and fixation of about 8–10% of carbon into cell biomass (Chiellini et al., 2003; Jakubowicz, 2003). The low rate of biodegradation of plastics is usually due to lack of water solubility and due to the size of the polymer molecules which prevents it to get transported directly into the cells (Gilan et al., 2004; Sivan et al., 2006). The two major problems with polyethylene are its high hydrophobicity (due to the presence of only –CH2 groups) and its high molecular weight (more than 30 kDa). The biotic mechanism reported for the degradation of high molecular weight polymers are due to the extra cellular enzymes produced by micro-organisms which degrade the main polymeric chain and result in intermediates of lower molecular weight with modified mechanical properties, making it more accessible for the microbial assimilation (Palmisano and Pettigrew, 1992). Thermal or radiation treatments on polyethylene reduce the polymeric chain size and form oxidized groups such as carboxyl, carbonyl and hydroxyl. These treatments modify the properties (crystallinity level, morphological changes) of the original polymer and facilitate the polymer biodegradation (Lee et al., 1991). In the present work, unpretreated and thermally pretreated LDPE and HDPE and unpretreated starch-blended LDPE were incubated with marine microorganisms namely Bacillus sphericus GC subgroup IV (Alt) and B. cereus subgroup A (BF20) for a period of 1 year under in vitro conditions using sea water medium with polymer as the sole carbon source. The degradation was analyzed through physicochemical and mechanical changes that took place in the polymers. 2. Materials and methods 2.1. Polyethylene In the present investigation pure LDPE and starch-blended (with 12–15% of starch) LDPE films of size 80  25 mm with thickness 0.125 mm and HDPE films of size 80  25 mm with thickness 0.0981 were kindly provided by Excelcier Pvt. Ltd., Guindy, Chennai 600 025, India. Thermal treatment of LDPE and HDPE involved treating them at 801C for 10 days in a hot air oven (Sigma, USA).

2.2. Marine bacterial systems The marine bacterial culture of B. sphericus GC subgroup IV (Alt) and B. cereus subgroup A (BF20) was isolated from the shallow waters of the Indian Ocean by National Institute of Ocean Technology (NIOT), Chennai, India. The same sea water was used as a medium for all the bacterial cultures. These marine organisms were cultured on Zobell marine nutrient agar (Himedia Labs, Mumbai 86, India) and maintained in slants at 30 1C.

2.3. Chemical disinfection of films The disinfection procedure adopted consisted of washing the unpretreated and thermally pretreated films into a fresh solution containing 7 ml Tween 80 (Himedia Labs), 10 ml bleach and 983 ml of sterile water and stirred for 30–60 min. The films were removed and placed into a covered beaker filled with sterile water, stirred for 60 min at room temperature and aseptically transferred into ethanol (Hong Yang chemicals, China) solution 70% (vol/vol) and left for 30 min. Each film was placed into a preweighed sterile petridish and incubated at 45 1C for 24 h to dry and allowed to equilibrate at room temperature and weighed by using five-digit balance (Model CP64, Satorious, Germany).

2.4. Culture conditions Preweighted disinfected unpretreated or thermally pretreated films (80  25 mm) were aseptically added to 100 ml aliquots of filtered (0.22 mm Millipore filter paper) and autoclaved seawater medium. Log phase seed culture (100 ml) was added to these aliquots and they were kept under shaking at 180 rpm at 30 1C. The numbers of films taken were such that samples could be withdrawn after 1, 3, 6, 8 and 12 months. Also, all the samples were prepared and run simultaneously in triplicate. Sea water containing polymer without the microorganism was maintained as positive controls (Doi et al., 1992). Streak culture (surface plating) method was used for identifying any contamination in the medium or any possible changes to the morphology of the organisms. The growth of the bacterial culture containing polymer was monitored at an absorbance of 600 nm (UV–vis spectrophotometer, Jasco, Japan, Model V550) as well as by counting the colony forming units (CFU). Fresh filtered sea water was added after 6 months to make up for the evaporation.

2.5. Colony forming unit Marine bacterial culture growths for the first 6 months were monitored every 15 days by plating method. In this method 20 ml of bacterial culture was serially diluted and inoculated on the surface of Zobell marine agar solid media in sterile petridish and incubated for 24 h. Viable colony counts were obtained by appropriately diluting the sample (Moat and Foster, 1995). Colonies were counted by using digital colony counter (Scigenics, Chennai, India).

2.6. Viability of the bacterial biofilm Viability of the marine bacterial biofilm was determined by the LIVE/ DEAD BacLight Bacterial Viability Kit (Molecular Probes, USA), according to the manufacturer’s instructions. At prescheduled times, polyethylene samples containing the biofilm were removed from the medium, washed in sterile water and stained according to manufacturer’s instructions and viewed under an epifluorescent microscope equipped with a fluorescein isothiocyanate (FITC) filter (Axioplan 2E microscope coupled with a camera). The images were analyzed with Visiolab 1000 software (Biocom, Les Ulis, France) (Chavant et al., 2002).

2.7. Determination of enzymatic assay Veratryl alcohol and 2,4-dichlorophenol peroxidase (Sigma, USA) activity were determined for the bacterial culture concentrate after

ARTICLE IN PRESS M. Sudhakar et al. / International Biodeterioration & Biodegradation 61 (2008) 203–213 filtration and sterilization using the method suggested by Ramachandra et al. (1987, 1988). To measure the enzyme activity, increase in the absorbance at 470 and 510 nm were monitored at 37 1C on a UV–vis spectrophotometer, Jasco, Japan, Model V-550. Laccase (Sigma, USA) activity was measured by monitoring the oxidation of 2, 2-azinobis (ABTS) at 415 nm. Peroxidase activity was determined by subtracting laccase activity from the oxidation of ABTS at 415 nm.

2.8. Spectroscopy

2.12. Surface changes The films were washed in 70% ethanol to remove cell mass from the residual film as much as possible and then dried at 45 1C for 24 h. These films were used to evaluate the surface topography and bio deterioration by a multimore atomic force microscopy (AFM) with a Nanoscope IV digital (Veeco technologies, USA) with an ADCS controller.

2.13. Weight loss

Fourier transform infrared spectroscopy (FTIR) analysis is a useful tool to determine the formation of new or disappearance of functional groups. So degradation products, chemical moieties incorporated into the polymer molecules such as branches, co-monomers, unsaturation and presence of additives such as antioxidants can be determined by this technique. A Jasco N4200 spectrometer at a resolution of 2 cm1, in the frequency range of 4000–400 cm1, calibrated with polystyrene standards was used for the current study. Relative absorbance intensities of the ester carbonyl band at 1740 cm1, keto carbonyl band at 1715 cm1, terminal double bond (vinyl) band at 1650 cm1 and internal double bond at 908 cm1 to that of the methylene band at 1465 cm1 were evaluated using the following formulae (Albertsson et al., 1987): Keto carbonyl bond index ¼

I 1715 , I 1465

Ester carbonyl bond index ¼

I 1740 , I 1465

Vinyl bond index ¼

205

I 1640 , I 1465

Internal double bond index ¼

I 908 . I 1465

The percentage crystallinity of the polymer was measured based on the method suggested by Zerbi et al. (1989).   1  ðI a =1:233I b Þ 100, % crystallinity ¼ 100  1 þ ðI a =I b Þ where Ia and Ib are the absorbance values determined from the bands at 1474 and 1464 cm1 or from the bands at 730 and 720 cm1, respectively.

2.9. Thermal analysis Thermal analysis of polyolefin generally involves heating or cooling a sample at a controlled rate while monitoring some of its physical characteristics. Changes in heat capacity, glass transition temperature, melting temperature of the polymer samples are measured by Differential Scanning Calorimeter (Model 204, Netzsch, Germany).

A simple and quick way to measure the biodegradation of polymers is by determining the weight loss. Microorganisms that grow within the polymer lead to an increase in weight due to accumulation, whereas a loss of polymer integrity leads to weight loss. Weight loss is proportional to the surface area since biodegradation usually is initiated at the surface of the polymer. This method cannot be used on polymers that absorb water. Multiple samples were weighed with an accurate five-digit balance and average values are reported here.

3. Results and discussion Pure culture colonies (CFU) were quantified every 15 days for a period of first 6 months (Figs. 1 and 2). Optimal density (OD) results also showed a similar pattern to CFU data. Initial growth of organism could be due to carbon, nitrogen and some inorganic salts present in the sea water (Moat and Foster, 1995). For unpretreated LDPE, the growth had reached the stationary phase in the first 50 days, whereas it took almost 90 days for the thermally pretreated LDPE exposed to strain Alt (Fig. 1). Thermally pretreated LDPE exposed to BF20 exhibited the highest live colonies (8  104 CFU/ml in 180 days). Thermally pretreated HDPE also had more CFU than the corresponding unpretreated polymer. Since biodegradation and utilization of polyethylene is a slow process, it requires the biofilm to be active over a long period of time. The number of live organisms on the LDPE and HDPE surfaces with both the organisms after 1-year treatment was determined with BacLight Bacterial Viability Kit and is shown in Figs. 3 and 4, respectively. More live organisms (green spots) are seen on thermally treated LDPE and HDPE than on unpretreated films. Dead organisms appear as red in color. Also, more live

2.10. Contact angle

2.11. Mechanical properties

Unpretreated LDPE and BF 20

Thermalpretreated LDPE and BF20

Starch blend LDPE and Alt

Thermal pretreated LDPE and Alt Starch blend LDPE and BF 20

1.0E+05 8.0E+04 CFU/ ml

Contact angle is an indication of the hydrophobicity or wettability of the surface, higher the contact angle higher is the hydrophobicity. Samples supported on glass slide were analyzed using a Camtel (Royston, UK) Goniometer model FT200. The wetting liquid was Millipore grade distilled water. Calculations were averaged from five measurements carried out at appropriate times.

Unpretreated LDPE and Alt

6.0E+04 4.0E+04 2.0E+04 0.0E+00

The tensile properties of the samples were measured as per the ASTM D882 procedure on Instron machine (no. 4301, from Germany) at 30 1C, 50% humidity and cross-head speed of 25 mm/min. Relative elongation and relative tensile strength of the samples treated with marine bacteria were compared with untreated control samples.

0

20

40

60

80 100 Days

120

140

160

180

Fig. 1. CFU count for various LDPE samples treated with two marine organisms.

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organisms are seen on BF20 than on Alt-treated films, which was also confirmed from the above-mentioned colony count. In the biodegradation of polyethylene, an initial abiotic step involves oxidation of the polymer chain due to the dissolved oxygen or that which is present in the ambient leading to the formation of carbonyl groups. These eventually form carboxylic groups, which subsequently undergo b-oxidation (Albertsson et al., 1987) and are totally degraded via the citric acid cycle resulting in the formation of CO2 and H2O. b-Oxidation and the citric acid cycle are catalyzed by micro-organisms. Monitoring the formation or disappearance of acids (1715 cm1), ketones (1740 cm1) and double bonds (1640 and 915 cm1) using FTIR is necessary to elucidate the mechanism of the Unpretreated HDPE and Alt

Unpretreated HDPE and BF 20

Thermal pretreated HDPE and Alt

Thermal pretreated HDPE and BF20

8.0E+04 CFU/ml

6.0E+04 4.0E+04 2.0E+04 0.0E+00 0

20

40

60

80 100 Days

120

140

160

180

Fig. 2. CFU count for various HDPE samples treated with two marine organisms.

biodegradation process. Figs. 5–9 show the various FTIR indices for unpretreated and thermally pretreated LDPE and HDPE, and starch-blended LDPE as a function of time exposed to B. sphericus (Alt). Initially carbonyl index increases, probably due to oxidation by the dissolved oxygen (abiotic factor). Prolonged exposure to organism leads to decrease in carbonyl index probably due to biodegradation (biotic) through Norrish-type mechanism (Fig. 10) or through the formation of ester (Fig. 11). The rate of decrease in carbonyl index in HDPE is much less than the rate of decrease in LDPE indicating lower biodegradation. This abiotic carbonyl formation and reduced biotic degradation also leads to higher carbonyl index build up in HDPE. Starch-blended LDPE shows lower carbonyl index, and it also undergoes highest biodegradation. FTIR as a tool for differentiating between abiotic and biotic degradation of LDPE has also been reported by Albertsson et al. (1987). They have observed that samples stored in air increased their carbonyl index with time, but all samples in contact with soil showed a decrease of carbonyl index with time. Others have also observed a continuous increase in the amount of carbonyl compounds with exposure in an abiotic environment as against a decrease in the biotically aged samples (Chiellini et al., 2003; Gilan et al., 2004; Hadad et al., 2005). Dolezel (1967) also observed that the amount of carbonyl groups decreased with prolonged exposure to a biotic environment. Albertson et al. (1995) and Weiland et al. (1995) observed a reduction in the carbonyl group after 150 days of incubation with a mixed fungal culture. Norrish type II

Fig. 3. Live and dead cells attached on LDPE surfaces after 1 year (A) unpretreated and exposed to B. sphericus (Alt), (B) thermally pretreated and exposed to B. sphericus (Alt), (C) unpretreated and exposed to B. cereus (BF20) and (D) thermally pretreated and exposed to B. cereus (BF20).

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Fig. 4. Live and dead cells attached on HDPE surfaces after 1 year (A) unpretreated and exposed to B. sphericus (Alt), (B) thermally pretreated and exposed to B. sphericus (Alt), (C) unpretreated and exposed to B. cereus (BF20) and (D) thermally pretreated and exposed to B. cereus (BF20).

0.15

Absorbance

Absorbance

0.15

0.1

0.1

0.05

0.05 0 1

0 1

3

6 Months

8

12

Fig. 5. FTIR of unpretreated LDPE exposed to B. sphericus (Alt) for 1 year ( —ester carbonyl; —keto carbonyl; —interior double bond; —terminal double bond).

reaction leads to the formation of double bonds in the polymer chain (Fig. 10). Ester and keto carbonyls have also been reported as major products formed during abiotic oxidation of polymer under thermal oxidation or in the presence of enzymes such as oxidoreductase (Karlsson and Albertsson, 1998). A negligible increase in carbonyl index was observed for all samples during the thermal pretreatment at 70 1C for 10 days similar to the observations made by Khabbaz et al. (1998, 1999). These authors observed increase in carbonyl index after treatment at 100 1C for 10 days (Khabbaz et al.,

3

6 Months

8

12

Fig. 6. FTIR of thermally pretreated LDPE exposed to B. sphericus (Alt) for 1 year ( —ester carbonyl; —keto carbonyl; —interior double bond; —terminal double bond).

1999). The carbonyl and double bond indices for the thermally treated PE reached a higher value than the unpretreated samples when exposed to microbes. This could be due to the formation of loose PE chain fragments during the heat treatment and when later exposed to microbes underwent higher oxidative products leading to the formation of carbonyl and double bond. In the present study, fraction of internal double bond (–CHQCH–) was higher than that of the terminal/vinyl double bond (–CHQCH2). Similar observations were made with BF20-treated samples and hence are not shown here. Tables 1–4 list the changes in contact angle, percentage crystallinity, glass transition temperature (Tg), melting temperature (Tm), tensile strength, percentage elongation

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0.2

Absorbance

0.15

0.1

0.05

0 1

3

6 Months

8

12

Fig. 7. FTIR of unpretreated HDPE exposed to B. sphericus (Alt) for 1 year ( —ester carbonyl; —keto carbonyl; —interior double bond; —terminal double bond).

0.2

Absorbance

0.16 0.12 0.08 0.04 0 1

3

6 Months

8

12

Fig. 8. FTIR of thermally pretreated HDPE exposed to B. sphericus (Alt) for 1 year ( —ester carbonyl; —keto carbonyl; —interior double bond; —terminal double bond).

0.08

Absorbance

0.06 0.04 0.02 0 1

3

6 Months

8

12

Fig. 9. FTIR of unpretreated starch-blended LDPE exposed to B. sphericus (Alt) for 1 year ( —ester carbonyl; —keto carbonyl; — interior double bond; —terminal double bond).

and breaking load for unpretreated and thermally treated LDPE and HDPE as well as starch-blended LDPE over a 1-year period with both the organisms. Tensile strength of thermally pretreated LDPE and HDPE and untreated starch-blended LDPE decreased by 27%, 14.8% and 30.5%, respectively, with B. sphericus and the corresponding decrease in crystallinity was 8%, 2.2% and 8.5%, respectively. The decrease in these parameters was less with

B. cereus. Decrease in crystallinity was also seen in the form of decrease in Tg. The temperature decreased by 7, 6.2 and 9 1C, for thermally treated LDPE and HDPE and untreated starch-blended LDPE, respectively with B. sphericus. Decrease in Tg was less with B. cereus treated samples. Decrease in contact angle in all cases indicated that the surfaces turned more hydrophilic after exposure due to the attachment of the micro-organisms on the polymer surface followed by surface degradation. Initial increase in crystallinity is probably due to the fact that the micro-organisms preferably degraded first the amorphous regions or disrupt the crystalline order (Khabbaz et al., 1999). The overall decrease in crystallinity may be due to biodegradation and biodeterioration. The annealing effect at the oxidation temperature may be contributing to the increase in crystallinity and Tg observed with the thermally pretreated control samples when compared unpretreated samples. Dolezel (1967) has shown that the tensile strength of polyethylene changes in a biotic environment. Previous reports of the degradation of paraffin and polyethylene (Karlsson and Albertsson, 1998) on agar and solution by two species of fungi indicated enhanced rate of degradation in solution compared to solid substrate, which is due to the greater contact between the polymer and the micro-organism in the solution phase. Another factor to be considered is the better buffering effect of solution. Albertsson and co workers (1987) reported a decrease (2%) in the crystalline fraction of thermally oxidized LDPE (100 1C) prior to 10 months biological treatment using Arthrobacter paraffinneus. However, in the current work, large decrease in crystallinity of thermally pretreated LDPE reacted with B. sphericus was observed probably due to a prolonged pretreatment (10 days). Weiland et al. (1995) report that the crystalline lamellae have a very low permeability to oxygen, and are insensitive to thermal oxidation below the melting point, thus oxidation being mainly restricted to the amorphous inter-lamellar phase. Karlsson and Albertsson (1998) studied the degradation of 14C-labelled LDPE in soil over a 15-year period. They found that the total weight loss during degradation was 16% and in addition to H2O and CO2, shorter hydrocarbons, alcohols, organic acids, ketones, aldehydes, etc., are also formed (Carlsson and Wiles, 1969a, b; Albertsson and Karlsson, 1990). In this current study, a maximum weight loss of 25% was observed with untreated starchblended low-density polyethylene (LDPE) reacted with B. cereus. Weight loss of 19% with thermally pretreated LDPE, 10% with unpretreated LDPE, 9% with thermally pretreated high-density polyethylene (HDPE) and 3.5% with unpretreated HDPE reacted with B. sphericus were observed here (Figs. 12–15). These results could be due to the fact that Bacillus species utilize carbon source more compared to that of Brevundimonas sp. and Curtobacterium sp. reported by others. Previous study reports that Bacillus sp. is capable of producing extra cellular enzyme (oxidoreductase) in the nutrient starvation condition to acclimatize itself (Seneviratane et al., 2006).

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O H2C-CH2 O

C

O H2 C

CH2

Uv

H2C-CH2

H 2C

C

CH2

OH

H

HC

CH CH2

209

H2 C

C

CH2

CH

CH2

O C

CH3

Fig. 10. Norrich type I and type II biotic reactions (Albertsson et al., 1987).

O

C H2

H2 C

C

O CH2

C C H2 CH2

CH

CH2

O CH2

CH

CH2

O

C

CH2

O IR 1740 cm-1 Fig. 11. Mechanism of abiotic conversion of carbonyl to ester (Albertsson et al., 1987).

The presence of extracellular enzymes such as oxidoreductase could be one of the key factors for the biodegradation of LDPE and HDPE observed here. Surface morphological changes after biological treatment are observed in AFM pictures (Fig. 16). The figure compares the AFM of control with thermally pretreated polymer samples after exposure to both the marine organisms for 1 year. One-year-old samples display

considerable (more than a factor of 50) increase in the surface roughness when compared to the control samples, probably due to surface degradation and deterioration. 4. Conclusion The biodegradation of LDPE and HDPE by two marine microbes, namely, B. sphericus and B. cereus, are reported

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Table 1 Observed changes in physical and mechanical properties of low-density polyethylene treated with B. sphericus (Alt) for 1 year Month

Contact angle

% Crystallinity

Tg (1C)

Tm (1C)

Tensile strength (N/ mm2) MPa

Elongation (%)

Breaking load (N)

Untreated LDPE

Control 3 6 12

90.070.3 8774.0 8673.4 8670.1

75 77 76 73

112.12 113.13 111.12 109.14

124.57 124.12 125.46 124.70

13.71 13.11 13.00 13.18

163.64 165.12 164.90 168.13

48 46 45 44

Thermally pretreated LDPE

Control 3 6 12

8871.2 8770.5 85.172.5 8571

76 78 74 70

112.11 107.13 106.18 95.10

124.21 124.98 125.10 123.12

13.98 13.20 11.25 10.16

163.43 164.14 166.90 169.18

48 44 40 40

Untreated starchblended LDPE

Control 3 6 12

8070.9 81.272.3 79.872.5 65.071.4

65 66 63 60

100.34 100.23 93.13 90.98

120.13 122.13 123.98 119.0

11.20 11.00 10.78 7.78

175.11 175.11 177.78 180.21

34 34 32 22

Table 2 Observed changes in physical and mechanical properties of low-density polyethylene treated with B. cereus (BF20) for 1 year Month

Contact angle

% Crystallinity

Tg (1C)

Tm (1C)

Tensile strength (N/ mm2) MPa

Elongation (%)

Breaking load (N)

Untreated LDPE

Control 3 6 12

90.070.3 8871.0 8772.4 8770.1

75 76 76 73

112.12 113.76 112.76 110.09

124.57 124.98 125.65 124.94

13.71 12.86 14.95 13.13

163.64 164.12 164.90 166.13

48 44 45 43

Thermally pretreated LDPE

Control 3 6 12

8871.2 8870.5 86.170.5 8671

76 78 75 72

112.11 113.53 110.09 110.05

124.21 126.78 128.95 125.93

13.98 14.22 11.67 12.16

163.43 166.34 170.19 168.90

48 43 43 40

Untreated starch blend LDPE

Control 3 6 12

8070.9 8071.0 78.072.0 63.0471.3

65 68 63 58

100.34 100.10 96.10 93.19

120.13 125.09 123.10 122.10

11.20 11.13 10.12 9.54

175.11 175.00 179.12 186.13

34 33 30 27

Table 3 Observed changes in physical and mechanical properties of high-density polyethylene treated with B. sphericus (Alt) for 1 year Month

Contact angle

% Crystallinity

Tg (1C)

Tm (1C)

Tensile strength (N/ mm2) MPa

Elongation (%)

Breaking load (N)

Untreated HDPE

Control 3 6 12

73.073.5 73.072.9 73.070.5 71.971.3

85 86 87 86

113.12 113.61 112.87 110.14

128.65 128.69 129.38 128.10

18.90 18.12 18.78 17.06

201.12 203.00 198.12 219.10

56 54 54 53

Thermally pretreated HDPE

Control 3 6 12

73.072.0 73.171.5 71.72.7 71.370.1

86 87 86 84

115.37 112.10 109.90 109.10

132.09 134.15 129.09 128.13

18.90 18.00 17.34 16.10

201.12 203.90 222.79 222.13

56 53 53 54

here under in vitro conditions in the sea water medium. Thermal pretreatment seems to play a vital role in enhancing the rate of biodegradation. Blend with natural

polymer (starch) exhibits a higher weight loss when compared to unblended polymers. Although starchblended LDPE exhibits higher biodegradation, if one

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Table 4 Observed changes in physical and mechanical properties of high-density polyethylene treated with B. cereus (BF20) for 1 year Contact angle

% Crystallinity

Tg (1C)

Tm (1C)

Tensile strength (N/ mm2) MPa

Elongation (%)

Breaking load (N)

Untreated HDPE

Control 3 6 12

73.073.5 73.071.0 73.071.0 72.171.0

85 85 87 85

113.12 113.90 112.10 110.32

128.65 128.94 129.03 129.30

18.90 18.90 18.45 18.16

201.12 200.90 207.12 209.10

56 56 56 54

Thermally pretreated HDPE

Control 3 6 12

73.072.0 73.271.0 73.171.7 72.370.2

86 85 86 84

115.37 113.90 112.09 110.04

132.09 132.10 134.90 129.45

18.90 17.05 16.94 16.00

201.12 213.10 232.19 232.73

56 55 50 49

25

10

20

8

% Weight loss

% Weight loss

Month

15 10 5

6 4 2 0

0 1

3

6

8

1

12

2

4

5

Months

Months Fig. 12. Percentage weight loss of LDPE exposed to B. sphericus (Alt) ( —unpretreated; —thermally pretreated; —unpretreated starch blended).

Fig. 14. Percentage weight loss of HDPE exposed to B. sphericus (Alt) ( —unpretreated; —thermally pretreated).

8

30 % Weight loss

25 % Weight loss

3

20 15 10 5

6 4 2 0 1

0 1

3

6

8

12

Months Fig. 13. Percentage weight loss of LDPE exposed to B. cereus (BF20) ( —unpretreated; —thermally pretreated; —unpretreated starch blended).

eliminates the 12–15% of starch present in the polymer blend (which would have degraded first), then the amount of polymer that has degraded matches with the extent of degradation of unpretreated LDPE. LDPE degrades faster than HDPE, possibly due to the fact that the latter polymer has closely packed chains than the former, leading to easy approachability of the organisms. The rate of biodegradation is highest with starch-blended LDPE followed by thermally pretreated

3

6 Months

8

12

Fig. 15. Percentage weight loss of HDPE exposed to B. cereus (BF20) ( —unpretreated; —thermally pretreated).

LDPE and HDPE. Abiotic and biotic degradation show synergistic relationship during the process. The formation and disappearance of ester, keto, double bond and vinyl groups are clearly seen from the FTIR spectra, which help in the elucidation of the biodegradation mechanism. Initially abiotic oxidation increases and the subsequent biotic process decreases carbonyl group. Increase in wettability of the polymer, decrease in tensile strength and percentage crystallinity are seen after biological degradation.

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Fig. 16. Atomic force microscopy images of polymers (A) and (B) HDPE and LDPE—control; (C) and (D) thermally pretreated HDPE and LDPE exposed to B. sphericus (Alt), respectively; and (E) and (F) thermally pretreated HDPE and LDPE, respectively, exposed to B. cereus (BF20).

Acknowledgment We extend our sincere gratitude to Naval Research Board, N. Delhi, for providing us the funding to carry out this research work. We are thankful to Dr. Abhijit Deshpande, Department of Chemical Engineering, IIT Madras, and Regional sophisticated instrument facility

(RSIC) IIT Madras for permitting us to measure Tensile strength, DSC and contact angle. We thank the Director of NIOT for allowing us to use the available facilities. First author thanks Mr. Sangram and Ms. Mamoni of Department of Biomaterials, University of Pisa, Italy and Ms. Sanghamitra of Scuola Normale Superiore, Pisa, Italy, for helping with the manuscript preparations.

ARTICLE IN PRESS M. Sudhakar et al. / International Biodeterioration & Biodegradation 61 (2008) 203–213

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