Soil burial biodegradation studies of starch grafted polyethylene and identification of Rhizobium meliloti therefrom

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1 Vol. 5(6), pp , June 2013 DI: /JECE ISSN X 2013 Academic Journals Journal of Environmental Chemistry and Ecotoxicology Full Length Research Paper Soil burial biodegradation studies of starch grafted polyethylene and identification of Rhizobium meliloti therefrom Neena Gautam 1 * and Inderjeet Kaur 2 1 Department of Chemistry, Government Degree College, Dhalpur, Kullu , Himachal Pradesh, India. 2 Department of Chemistry, Himachal Pradesh University, Summer Hill, Shimla , Himachal Pradesh, India. Accepted 18 February, 2013 Present study report on biodegradation of starch grafted polyethylene (PE), successfully synthesized by graft copolymerization method using benzoyl peroxide as the radical initiator. Biodegradable behavior of the grafted PE was ascertained by soil burial test. Percent weight loss was measured as a function of number of days and observed that it increased with increasing number of days. Microanalysis of the soil containing samples was carried out which substantiated the degradation. The biochemical tests was performed on the microorganism isolated from the soil in microanalysis, identified the organism as Rhizobium meliloti which helped in biodegradation of PE-g-Starch samples. The degradation was further confirmed by carrying out the physical characterization of the original samples and the degraded samples by scanning electron microscope (SEM) and thermogravimetric analysis (TGA). Hydrolysis of grafted samples, taken out from the soil after a specified number of days also corroborated in the findings, revealing continuous loss of weight with increasing number of days. Key words: Polyethylene, starch, graft copolymer, biodegradation. INTRDUCTIN In this era of many astonishing industrial developments, probably no industry has undergone such a rapid growth and development as the plastic industry. However its lack of biodegradability has caused much concern because of its role in causing environmental pollution. Various attempts, have, therefore been made to develop biodegradable plastics. A vast number of biodegradable polymers have been synthesized recently and some microorganisms and enzymes capable of degrading them have been identified. Biodegradation is a process whereby bacteria, fungi, yeasts and their enzymes consume a substance as a food source so that its original form disappears. Degradation of starch-blended polyethylene (PE) in soil burial test have been studied and confirmed under various compost field environmentgreizerstein (1993). According to Bastioli (1995) starch promotes the biodegradability of a nonbiodegradable plastic and it can also be used together with fully biodegradable synthetic plastic producing biodegradable blends of low costs. Starch blended biodegradable PE films grafted with vinyl acetate showed better printability performance without affecting the biodegradation properties (Ghosh et al., 2002). Thakore et al. (1999) tested the biodegradability of low-density polyethylene (LDPE)/Starch and LDPE/ starch/ starch acetate blends and observed the biodegradability on the starch acetate content. They found that incorporation of starch acetate along with starch improved properties, particularly elongation at break and toughness. Krupp and Jewell (1992) studied biodegradability of plastic films in controlled biological environments. PE with 6% starch in PE matrix was reported to degrade in landfills. Maharana and Singh *Corresponding author. neeeenachem@yahoo.co.in. Tel:

2 148 J. Environ. Chem. Ecotoxicol. (2006) carried out graft copolymerization of LDPE onto starch with glucose-cerium (IV) redox initiator in aqueous sulfuric acid medium under nitrogen atmosphere and tested the biodegradability of starch grafted PE by applying soil burial test. LDPE films containing starch were tested for biodegradation by Dave et al. (1997). and it was found that films containing 30% starch showed maximum biodegradation leading to a loss of 6.3% in weight and 84.5% starch upon burial in a soil compost mixture for 48 weeks. Ratanakamnuan and Aht-ng (2006) exposed binary polymer films containing different percentages of corn starch and LDPE to soils over a period of 8 months and monitored for starch removal and chemical changes of the matrix using Fourier transform infrared spectroscopy (FTIR) spectroscopy. IR analysis did not show significant chemical changes in the PE matrix after 240 days. However, the matrix did show evidence of swelling, an increase in surface area, and removal of low molecular weight components. Griffin (1974) determined the rate and extent of deterioration of starch-plastic composites over a 2-year period for samples buried in a municipal solid waste landfill. The deterioration of the starch-plastic composites following exposure was determined by measuring changes in tensile properties, weight loss, and starch content of samples retrieved from the landfill. Elongation decreases of 92 and 44% were measured for starchplastic composite LDPE and linear low-density polyethylene (LLDPE) films, respectively, while elongation decreases of 54 and 21% were measured for their corresponding control films following two years of burial. Starch loss of 25% for LLDPE and 33% for LDPE starch-plastic composite films was measured following two years of landfill burial. Starch-plastic composites did not fragment or lose mass during the two year landfill burial. The limited degradation observed for the starchplastic composites was attributed to the ineffectiveness of the prooxidant additive to catalyze the thermal oxidation of the PE or polypropylene component of the starchplastic composite under the environmental conditions present within the landfill. Because of the biodegradable behaviour of starch, we grafted PE with starch and in the present manuscript we report on the biodegradation studies of starch grafted PE by soil burial test. Weight loss of the grafted PE kept for soil burial studies were measured and it was followed by hydrolysis to corroborate the degradation. Weight loss of the grafted PE and PE kept for soil burial studies were measured, microbial studies of the soil containing samples in NutrientAgar and CzapecDox and was followed by hydrolysis to corroborate the biodegradation. MATERIALS AND METHDS Commercial LDPE was obtained as beads from Thukral Trading Company, Delhi, India. PE was dissolved in p-xylene and was precipitated with the addition of methanol. PE was irradiated from source housed in Gamma Chamber-900 (BARC, Trombay, Mumbai, India) at a constant dose rate of 3.40 kgy/h. Starch and benzoyl peroxide (BP) (S.D. fine chemicals) were used as received. Co 60 Synthesis of graft copolymer Graft copolymerization has been achieved for the systems where monomer is polymerized in the presence of preformed polymer backbone using different kinds of initiating systems. However, in the present work graft copolymer of two preformed polymers that is, PE and starch has been synthesized. The activation of PE is initiated through gamma radiations which generate macro oxy radicals (I) through the peroxidation process. These peroxidic linkages, generate macro peroxy radicals upon heating where the formation of graft copolymer can take place through ether linkage (Baldev et al., 2004; Inderjeet et al., 2008). γ-rays CH 2CH2 + 2 CH 2 CH CH 2CH2 + CH 2 CH. C H C H 2 C H 2 CH 2. CH CH 2 CH CH 2 C H CH 2. (I) + H. Benzoyl peroxide (BP) is a known free radical initiator for carrying polymerization and graft copolymerization reactions. The generation of active sites on starch (II) can take place by the abstraction of hydrogen by the phenyl radical. C6 H5 H C. H5C C H. CC C C 2.. PH + C H P + C 6 5 6H6 (II) where ~~~ PH ~~~ represents starch. The two macro radicals (I) and (II) intercross link to give the graft copolymer. P. + CH 2 CH. CH 2 CH Graft copolymerization of starch onto PE was carried out as a function of different reaction variables such as concentration of BP, temperature, time of reaction and amount of water. ptimum conditions pertaining to maximum percentage of grafting (135%) was obtained at [BP] = mol/l at 60 C in 90 min using 200 mg of PE, 300 mg of starch and 40 ml water. The grafted PE containing both unreacted PE and starch is referred to as PE-g- Starch Composite while the graft from which the unreacted starch and PE are removed by washing respectively with water and xylene is referred to as PE-g-Starch True graft. P

3 Gautam and Kaur 149 Bio degradation studies Biodegradation behavior of PE, PE composite and true graft was studied by ascertaining the loss in weight during the soil burial test. Soil burial method Garden soil (1200 g) was taken in different pots. A weighed amount (1 g) of each of the samples that is, PE, PE-g-Starch (composite and true graft separately) wrapped in synthetic net were placed in the beaker such that the soil covered the polymer from all the sides. The pots were covered with the aluminum foil and kept at room temperature. The weight of all the samples, PE and the grafted PE, were taken at regular interval of time (10 days) to check for any weight loss. Percent wt. loss as a function of number of days was determined as (Kim et al., 2000): Total percent weight loss after 4 months was calculated as: % Wt.Loss = Initial wt. at the beginning _ Final wt. after ten days X 100 Initial wt. at the beginning % Wt.Loss = (after every 10 days) Microanalysis Initial wt. before ten days _ Final wt. after ten days X 100 Initial wt. before ten days In order to corroborate degradation, assay of the soil containing samples for degradation was studied in Nutrient Agar and Czepeck Dox media. The growth of microorganisms such as bacteria was checked at definite intervals of time. Samples of the soil (1%) containing PE, PE-g-Starch (composite and true graft) were separately mixed well with the 10 ml of saline solution. Six test tubes were taken for making serial dilutions. Each test tuber was marked with serial sequence from 10-1 to Now 1 ml of the prepared solution was added to 9 ml of double distilled water, which was marked as Now from this tube 1 ml of solution was added into its serial sequential tube marked as Proceed till tube six which is marked as The number of bacteria in soil was determined by a serial dilution method. The growth pattern was observed and the numbers of colony forming units (CFU) were counted. Some of the growth colonies were submitted in IMTECH (Institute of Microbial Technology), Chandigarh, India for their identification (Kim et al., 2000). Direct microbial studies Direct microbes study of PE and PE-g-Starch (both composite and true graft) with isolated culture/microbes was studied by using a 20 ml of medium which is prepared by adding 1.25 g peptone and 0.75 g beef extract in 250 ml of water. The reaction was carried out for 7 days in rbitek Shaker (RPM = 160) at 30 C. After completion of the reaction, the sample was collected by centrifuging it in Remi Cooling Compufuge CPR 23 by setting at 7000 rpm for 10 min and filtered it very carefully. Sample was washed with water and dried at 40 C until it gives constant reading. Dried sample was weighed and % weight loss was calculated as follows (Kim et al., 2000): W 0 -W d %Wt. Loss = 100 W 0 Where W o is the initial weight of sample and W d is the weight of dried sample after degradation. Hydrolysis studies For hydrolysis studies, definite weight (500 mg) of different samples of PE, PE-g-Starch (composite and true graft) separately wrapped in synthetic net was buried in soil. The samples were taken out after a definite interval of time (10 days), washed with water to remove the adhered soil, filtered, dried and calculated the % weight loss as (Kim et al., 2000): % Wt.Loss = Initial wt. at the beginning _ Final wt. (after every ten days interval) X100 Initial wt. at the beginning Dried sample was weighed and hydrolyzed with 20 ml of 6N HCl for 4 h. After the hydrolysis, the residue was dried and weighed. Growth of plants In order to find out whether or not the degradation products from the polymer samples are harmful to the growth of the plants, the growth of soybean plants and wheat plants was checked from the germination stage. The soybean seeds and wheat seeds were placed uniformly in different pots containing the samples for degradation along with a reference pot containing no grafted sample. The plants were allowed to grow in the open for 35 days. The length and height of the roots and shoot respectively were measured. For wheat plants average of six plants have been used for measuring the length and height of the roots and shoot respectively (Kim et al., 2000). RESULTS AND DISCUSSIN Biodegradation studies Soil burial studies Starch grafted PE has pendant chains of starch suspended from the PE backbone where the process of degradation begins because starch is well known for its biodegradable behavior. Biodegradation of grafted samples both composite and true graft buried in the soil was monitored as a function of number of days and the results are presented in Figure 1. Percent weight loss due to degradation was determined by subtracting the weight of the sample taken out on a particular day (that is, after every 10 days) from the initial weight that is, the weight of the sample at the start of the degradation study each time. It is observed from the figure that percent weight loss of both the samples increase continuously with increase in the number of days. Maximum % wt. loss 88 and 84% of composite and true graft sample respectively was observed after a period of four months indicating that the samples continuously degrade with increase in the length of time. In another set up, percent weight loss of the sample was measured after every ten days, but the loss in percent weight of the sample was taken as the weight of the sample on each tenth day minus the preceding weight of the sample prior to ten days and results are presented in Figure 2. It is observed from the figure that

4 150 J. Environ. Chem. Ecotoxicol Percent weight loss % Wt. loss C o m p o s it e o f P E - g - S t a r c h T r ue G r a f t o f P E - g - S t a r c h NNumber of o f days s Figure 1. Percent weight loss as a function of number of days in simple soil Percent weight loss % Wt. Loss C o m p o sit e o f P E - g - St a r c h T r ue G r a f t o f P E - g - St a r c h N Number r of o f days d a y s Figure 2. Percent weight loss as a function of number of days in soil. composite sample shows an initial decrease in % weight loss of 8.88% during first 20 days. It then increases up to 24.53% in the next 40 days. After a decrease in % weight loss on the next 10 days to 10%, increase in percent weight loss of 28% with a small decrease in weight loss on the 90th day is observed. In case of true graft, the sample shows 7.14% of percent weight loss in the first 30 days, which increases to 22.22% in the next 30 days. The percentage weight loss drops to 11.9% on the 70th day and increases to maximum 28% in 100th day beyond which it decreases. The decrease in the percent weight loss is due to the invasion of microorganisms into the substrate and absorption of the moisture by the samples. The microorganisms when feed upon the substrate increase the percent weight loss of the sample. However for PE percent weight loss shows 0% degradation in all respective degradation studies. Thermogravimetric analysis of degraded PE and PEg-starch (composite) sample Thermal gravimetric analysis of PE, PE-g-Starch (composite), degraded PE and degraded PE-g-starch (composite) are represented in Figures 3, 4, 5 and 6, respectively. The IDT, FDT and DT values at every 10%wt. loss are presented in Table 1. It is observed from the table that the IDT, FDT and DT values at every 10% wt. loss of degraded starch grafted PE and degraded PE are almost same. However, on comparison of the TG

5 Gautam and Kaur 151 Weight loss ( ) (%) Figure 3. Primary thermogram of PE. Temperature ( C) Weight loss ( ) (%) Temperature ( C) Figure 4. Primary thermogram of PE-g-starch. TG (%) Temp Cel Figure 5. Primary thermogram of degraded PE.

6 152 J. Environ. Chem. Ecotoxicol. T G (% ) Figure 6. Primary thermogram of degraded PE-g-starch. Table 1. Primary thermographs of PE, PE-g-Starch (composite) and degraded (PE and composite) sample. S/N Sample IDT (%) FDT (%) DT ( C) at every 10% weight loss Residue left (%) 10% 20% 30% 40% 50% 60% 70% 80% 90% 1. PE (10.81) (97.37) PE-g-starch(composite) (11.41) (93.26) PE-g-starch (degraded composite) PE(degraded) data of the grafted PE before and after degradation it is observed that DT values of the degraded sample are parallel to those of the DT values of PE and degraded PE. These observations indicate that the degradation begins at the grafted starch chains and approaches towards the PE chains. Scanning electron micrographs of degraded PE-g-starch (composite) sample Scanning electron micrograph of PE, PE-g-starch (composite) and degraded PE-g-starch (composite) at x4000 magnification and is represented in Figures 7, 8 and 9, respectively. Comparing the SEM of the degraded PE-g-starch (composite) with the SEM of PE-g-starch (composite) (Figure 8) it is observed that the grafted starch appearing as a thick homogeneous deposite on the PE surface has been reduced to scattered and thin deposites after degradation. These observations therefore symbolize of

7 Gautam and Kaur 153 Figure 7. Scanning electron micrograph of PE at x4000 magnification. Figure 8. Photograph of scanning electron micrographs of PE-g-starch (composite) at x4000 magnification. degradation process during soil burial studies of starch grafted PE. Micro analysis During micro analysis studies it was observed that pure starch completely degraded in 10 to 20 days time period. Soil containing PE and the soil sample without any sample show a very small growth of the bacteria, while composite material and the true graft of PE on the other hand show a rich growth of colonies of the bacteria. Growth of microorganisms w.r.t. the number of colonies as a function of number of days in Czapek Dox Media and Nutrient Agar media is presented in Tables 2 and 3, respectively. Careful perusal of the Table 2 reveals that in

8 154 J. Environ. Chem. Ecotoxicol. Figure 9. Photograph of scanning electron micrograph of degraded PE-g-starch (composite) at x4000 magnification. Table 2. Growth of microorganisms as a function of number of days in Czapek Dox medium. No. of days 10 Dilution PE Total number of colonies in (CFU) PE-g-starch (composite) PE-g-starch (true graft) case of PE-g-starch (composite), the growth (total count of all the colonies) of the microorganisms in Czapek Dox medium increases from 333 to maximum 518 in 10-2 dilution in 90 days and from 75 to 105 in 10-4 dilution in

9 Gautam and Kaur 155 Table 3. Growth of microorganisms as a number of days in Nutrient Agar medium. No. of days 10 Dilution Total number of colonies in (CFU) PE No. of Days Dilution days and minimum growth of 92 in 10-2 and 13 in 10-4 is observed in 20 and 90 days respectively. For true graft sample growth of the microorganisms increases continuously from 205 to maximum 305 in 10-2 dilution in 60 days and in 10-4 dilution growth of microorganisms decreases from 132 to 10 in 120 days and minimum growth of 70 in 10-2 dilution is observed in 120 days. However for PE maximum growth of colonies of 88 in 30 days and of 4 in 120 days is observed in 10-2 in 10-4 dilution respectively. When the microanalysis are made in Nutrient Agar (Table 3) the growth of colonies are less than that observed in Czapek Dox media. From the table it is observed that the growth of colonies of microorganisms for composite sample increases from 185 to maximum 305 in 10-2 dilution in 60 days and for 10-4 dilution maximum growth of 145 is observed in 10 days, which thereafter decreases to 10 in 60 days and increases to 40 in 120 days. For true graft sample maximum growth of colonies of microorganisms 530 and 380 is observed for10-2 in 10-4 dilution respectively in 10 days and 30 days. However for PE maximum growth of colonies of 32 in 30 days and of 4 in 120 days is observed in 10-2 in 10-4 dilution respectively. However, for PE maximum growth of colonies of 32 in 30 days and of 1 in 10 days is observed in 10-2 in 10-4 dilution respectively. n comparing the growth of and decrease in the number of colonies with the % weight loss as a function of number of days during soil burial studies, it is observed that the increase and decrease in percent weight loss go parallel to the growth and decrease in number of colonies with increasing number of days. These observations implies to the fact that the microorganisms attack the polymer, feed upon it leading to decrease and increase in the percent weight loss accordingly. The cultures were isolated for direct attack on the samples. Identification of one culture was done by performing the biochemical tests on the organism and identified as Rhizobium meliloti given Microbial Type Culture Collection (MTCC) number of The microbial degradation was further substantiated by the direct attack of the isolated microbe R. meliloti from microanalysis studies of soil on the grafted samples. It was observed that the rate of degradation by direct attack was faster than the degradation rate in soil burial studies. The study was carried out for one week. From results it is observed that PE shows no loss in weight when placed in the control sample containing no cells and when it is treated with cells of isolated cultures containing R. meliloti observed percent weight loss was 5%. PE-gstarch composite and true graft on the other hand when treated with simple medium without cells shows 25 and 20% weight loss. However, grafted PE on treatment with

10 156 J. Environ. Chem. Ecotoxicol. Table 4. Hydrolysis of PE-g-starch (composite) as a function of number of days of soil burial. No. of days Weight of sample (g) Weight of PE after hydrolysis (g) % weight loss Table 5. Hydrolysis of PE-g-Starch (true graft) as a function of number of days of soil burial. No. of days Weight of the sample (g) Weight of PE after hydrolysis (g) % weight loss Table 6. Growth measurements of soybean plants grown in soil containing PE and PE grafted samples. Variable Length of root (cm) Simple soil Length of shoot (cm) Urea enriched soil Length of root (cm) Length of shoot (cm) Reference plant without sample PE-g-starch (composite) PE-g-starch (true graft) PE cultures containing R. meliloti shows a total 85% wt. loss in case of composite and 75% wt. loss in case of true graft. The results thus indicate that the presence of microorganisms facilitates degradation. This further substantiates the observed degradation. Hydrolysis of the sample leaves behind PE, the amount of which decreases continuously with increasing number of days. Hydrolysis studies The results of hydrolysis of the PE-g-starch (composite) and PE-g-starch (true graft) placed for degradation in soil is presented in Tables 4 and 5, respectively. It is observed that percent weight loss increases with increasing number of days. Maximum percent weight loss for composite and true grafted sample is 86% (Table 4) and 84% (Table 5), respectively in 120 days. The percent weight loss is comparable to the percent weight loss presented as a function of number of days (Figure 1) where maximum percent weight loss for composite and true graft (88% and 84%) was observed. Effect of degradation of grafted samples on the growth of plants The length of roots and shoot of soybean and wheat plants was measured after 35 days and the results are presented in Tables 6 and 7, respectively. It is observed that the growth of the plants (from root to tip) is better in urea enriched soil. From Table 6 it is clear that the length of root of plants grown in simple soil containing composite and true graft sample is 5.3 and 5.4 cm, respectively which is higher by 0.1 and 0.2 cm from the reference plant with length of 5.2 cm in comparison to

11 Gautam and Kaur 157 Table 7. Growth measurements of wheat plants grown in soil containing PE and PE grafted samples. Variable Average length of root (cm) Simple soil Average length of shoot (cm) Average length of root (cm) Urea enriched soil Average length of shoot (cm) Reference plant without sample PE-g-starch (composite) PE-g-starch (true graft) PE urea enriched soil where the length of root of plant grown in soil containing composite sample is 5.7 cm (higher by 0.1 cm than the reference plant) and for true graft sample is 5.3 cm lower by 0.3 cm than the reference with 5.6 cm root length. It is interesting to note that for plant grown in soil containing PE, length of root measured is 5.1 and 5.5 cm for simple soil and urea enriched soil, respectively. The length of shoot of the plant grown in soil containing composite and true graft sample is 4.2 and 4.3 cm, which is higher by 0.1 and 0.2 cm, respectively than the reference plant with length of 4.1 cm. While the length of shoot for plants grown in soil containing PE is same as that of reference plant. In urea enriched soil the length of shoot of plant grown in soil containing composite and reference plant is of 4.9 cm and for true graft and PE is 4.8 cm, which is smaller than the length of shoot of reference plant. Table 7 shows growth measurements of wheat plant from where it is clear that for the plant grown in simple soil containing grafted samples and reference plant, the average length of root of is same, that is, 6.1 cm and of PE is 6.0 cm which is smaller by 0.1 cm than the reference plant. While for urea enriched soil average length of root of plant grown in soil containing composite (6.7 cm) and PE (6.7 cm) is higher by 0.1 cm than the reference plant with average root length of 6.6 cm. However, for the plant grown in soil containing true graft sample average root length is 6.6 cm, which is same as that of reference plant. Results for shoot measurements shows that average length of shoot of plant grown in soil containing true graft (6.7 cm) and PE (6.7 cm) is smaller by 0.1 cm and the plants grown in soil containing composite 6.9 cm is higher by 0.1 cm than the reference plant with average length of shoot of 6.8 cm in simple soil. However, in urea enriched soil plant grown in soil containing composite sample show increase of 0.2 cm in average length of shoot of 7.4 cm in comparison to the reference plant of average shoot length of 7.2 cm while plant grown in soil containing true graft and PE does not shows any change in average length of shoot from the reference plant. Thus, it is observed that the growth of the plants is much better in urea enriched soil. In case of soil containing the grafted samples the length of root and shoot show slight increase while in urea enriched soil, not much variation in length of root and shoot is observed indicating that the presence of the PE or the grafted PE samples in soil undergoing degradation process will not harm the growth of the plants. Conclusions Grafting of biodegradable polymer, starch, induces biodegradability into the otherwise stubborn PE. A contrast difference in the topological morphology and the thermal behavior between the original and the degraded samples further substantiate the degradability induced into the grafted PE. R. meliloti can be used for degradation of grafted PE in lab conditions also. The synthesis of starch grafted PE will be beneficial in making best use of PE without the risk of environmental pollution as clear from the consistent growth of plants. REFERENCES Baldev R, Udaya SK, Siddaramaiah (2004). 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