Bioremediation & Biodegradation A Review on Biodegradation of Polythene: The Microbial Approach

The use of polythene is increasing day by day and its degradation is becoming a great challenge. Annually about 500 billion to 1 trillion polythene carry bags are being consumed around the globe. Polythene is durable and needs up to 1000 years for natural degradation in the environment. In the present review, an attempt has been made to pool all the available literature on the biodegradation of polythene under the following objectives: (1) to highlight the level of polythene pollution; (2) the cost effective methods; (3) the source of polythene degrading microbes; (4) to mechanism of polythene degradation; (5) for biodegradation of polythene; (6) discuss


Introduction
The contamination of soil due to dispersal of industrial and urban wastes generated by the human activities is of great environmental concern [1]. Various plants possess the capacity to convert the toxic compounds into non-toxic forms and the process is known as phytoremediation. The concept of cleaning contaminated environment using plants is about 300 years old [2]. One of the major environmental threat is the slow/least rate of degradation or nonbiodegradability of the organic materials under natural condition, e.g. plastics. The plastics of various forms such as nylon, polycarbonate, polyethylene-terephthalate, polyethylene, polypropylene, polystyrene, polytetraflouroethylene, polyurethane, polyvinyl chloride are being continuously used in our day-to-day life [3]. Among the synthetic plastics waste produced, polythene shares about 64% [4]. As per the reports the most commonly used non-degradable solid waste is polythene which is a linear hydrocarbon polymers consisting of long chains of the ethylene monomers (C 2 H 4 ). The general formula of polyethylene is C n H 2n , where 'n' is the number of carbon atoms [5]. Polythene is made from the cheap petrochemical stocks extracted from oil or gas through efficient catalytic polymerization of ethylene monomers [6]. Polythene finds a wide range of applications in human's daily use because of its easy processing for various products used for carrying food articles, for packaging textiles, for manufacturing laboratory instruments and automotive components [5]. Various polymers such as lignin and paraffins were reported to be degraded by various microorganisms [6,7]. Jen-hou and Schwartz [8] carried out the comparative degradation study of paraffins and polythene for the first time and recorded utilization of polythene in terms of the growth of various bacteria on these alkenes. They concluded that microbes can degrade only low molecular weight polythene (MW up to 4800). Nineteen years later, degradation of high density polythene (HDPE) film (Mw 93000) was performed and it was documented that the main degraded component contained in HDPE film is the short-chain oligomer [9]. There is no such structural similarity between polythene and lignin except to have carbon-carbon bonding which is being broken by these microbes and using the polymers as a carbon source.
In the literature, various reviews had been written on biodegradation of the plastic [10][11][12][13][14][15][16][17][18]. Only a few review [19,20] deals with polythene but a comprehensive review on the polythene is lacking, so we tried to highlight the glimpses of the polythene biodegradation. We also tried to discuss, how to encounter the polythene pollution in future.

Status of Polythene Pollution
The use of plastic, especially polythene is growing day by day. Every year 25 million tons of synthetic plastics are being accumulated in the sea coasts and terrestrial environment [4][5][6][7][8][9][10][11][12][13][14][15][16][17][18][19][20][21]. Polythene constitutes 64% of the total synthetic plastic as it is being used in huge quantity for the manufacture of bottles, carry bags, disposable articles, garbage containers, margarine tubs, milk jugs, and water pipes [4]. Similarly, in the marine environment alone, out of total marine waste, plastic shares about 60-80% by mass [10]. All the polythene waste along with other plastic wastes generated by the human activity finally enters into marine water through rivers, canals/channels and municipal drainages. Therefore, the beaches were reported to be the excellent depository sites for the polythene (plastic) wastes. At dumping sites, polythene waste degraded with both chemical and mechanical weathering but it takes long time for mineralization and may remain in the microscopic form for long time [22]. Annually 500 billion to 1 trillion polythene bags are being used routinely all over the world. Polythene is strong and highly durable and takes up to 1000 years for natural degradation in the environment. Furthermore, plastic degrades by sunlight into smaller toxic parts contaminating soil and water where they can be accidentally ingested by animals and thereby enter the food chain especially in the marine biota [23]. To the marine life polythene waste is recognized as a major threat. Sometimes, it could cause intestinal blockage in the fishes, birds and marine mammals [23][24][25]. As per report [26] due to plastic pollution in the marine environment minimum 267 species are being affected which includes all mammals, sea turtles (86%) and seabirds (44%). The death of terrestrial animals such as cow was reported due to consumption of polythene carry bags [27]. The polythene leads to blockage of their digestive tract. It is also found that the polythene remains undigested in the stomach of the animals, after the death of the animals the polythene is again being eaten by some other animal and the cycle continues [27]. The undigested polythene was found to be responsible for various problems in the animals such as (1) during the digestion the fermentation process and mixing of the other contents were hampered due to ingested polythene and leads to indigestion; (2) the ingested polythene blocks the opening between omasum and reticulum which leads to death of the animal if the polythene will not be removed, (3) impaction: due to accumulation of large quantity of polythene bags rumen becomes impact which leads to remenatony; (4) tympany: due to blockage of the reticulum and omasum with polythene, accumulation of gases takes place in rumen, which leads to death of the animal if not removed properly; (5) polybezoars: In the digestive track around the polythene deposition of salt takes place that leads to formation of stone like structure which hampers the food passages and leads to pain and inflammation of rumen; (10) immunosuppression: the accumulation of polythene in the stomach of the animals (cow) leads to increased sensitivity to infections such as haemorrhagic septicemia [27]. The widely used packaging plastic (mainly polythene) constitutes about 10% of the total municipal waste generated around the globe [28]. As per literature, every year hundred thousand tons of plastics have been degraded in the marine environment resulting death [29]. The use of polythene is increasing every day and its degradation is becoming a great challenge. In the year 2000 about 57 million tons of plastic waste was generated around the world annually [30]. Only a fraction of this polythene waste is recycled whereas most of the wastes enter into the landfills and take hundreds of years to degrade [28][29][30][31].

Cost Effective Methods of Polythene Degradation
The process which leads to any physical or chemical change in polymer properties as a result of environmental factors (such as light, heat and moisture etc.), chemical condition or biological activity is said to be polymer degradation [32]. Based on the factors responsible for the degradation of the polymers, three types of polymer degradation methods are cited in the literature such as photodegradation, thermooxidative degradation and biodegradation [13]. The biodegradation is a natural process of degrading materials through microbes such as bacteria, fungi and algae [29]. The biodegradation involves microbial agents and does not require heat. Organic material can be degraded in two ways either aerobically or anaerobically. In landfills and sediments, plastics are degraded anaerobically while in composite and soil, aerobic biodegradation takes place. Aerobic biodegradation leads to the production of water and CO 2 and anaerobic biodegradation results in the formation of water, CO 2 and methane as end products [33]. Generally, the conversion of the long chain polymer into CO 2 and water is complex process. In this process, various different types of microorganisms are needed, with one leads to breakdown of the polymer into smaller constituents, one utilizes the monomers and excrete simple waste compounds as by products and one uses the excreted waste. The efficiency of this method is moderate but is environment friendly. This method is cheap and widely accepted [13]. Depending upon the formulation of the biodegradable polythene carry bags, three types along with one standard polythene, were studied for their degradation potential in the marine water. It was reported that after 40 weeks of exposure period the surfaces of the biodegradable polythene carry bags degraded less than 2% whereas the degradation of standard polythene was negligible [34]. The major consequences in the bio-degradation of polythene are enlisted briefly in the Table 1.

Sources of The Polythene Degrading Microbes
Following sites (Table 1)

Mechanism of Polythene Biodegradation
The degradation of polythene begins with the attachment of microbes to its surface. Various bacteria (Streptomyces viridosporus T7A, Streptomyces badius 252, and Streptomyces setonii 75Vi2) and wood degrading fungi produced some extracellular enzymes which leads of degradation of polythene [35,36,7]. In wood degrading fungi, the extracellular enzymatic complex (ligninolytic system) contains peroxidases, laccases and oxidases which leads to the production of extracellular hydrogen peroxide [37]. Depending upon the type of the organism or strain and culture condition, the characteristics of this enzyme system varies [38]. For degradation of lignin, three enzymes such as lignin peroxidase (LiP), manganese peroxidase (MnP) and phenoloxidase containing copper also known as laccase [7,39]. Based on the capabilities of these lingolytic enzymes, they are being used in various industries such as agricultural, chemical, cosmetic, food, fuel, paper, textile, and more interesting point is that they are also reported to be involved in the degradation of xenobiotic compounds and dyes [39]. During lignin degradation, phenolic compounds are being oxidized in the presence of H 2 O 2 and manganese by manganese peroxidase (MnP). MnP oxidizes Mn-II to Mn-III and monomeric phenols [40], phenolic lignin dimmers [41] and synthetic lignin [42] are in turn oxidized by Mn-III via the formation of phenoxy radicals [36]. There is no such report in case of polythene degradation but a similar trend is predicted. The byproducts of the polythene varied depending upon the conditions of degradation. Under aerobic conditions, CO 2 , water and microbial biomass are the final degradation products whereas in case of anaerobic/ methanogenic condition CO 2 , water, methane and microbial biomass are the end products and under sulfidogenic condition H 2 S, CO 2 and H 2 O and microbial biomass are reported to be the end products [5].

Determination of Polythene Degradation
The level of polythene degradation can be determined by the various methods as well as analytical techniques and the detail is given in Table  1. At topographical level, the Scanning Electron Microscopy (SEM) are being used to see the level of scission and attachment of the microbes on the surface of the polythene before and after the microbial attack [43]. The microdestruction of the small samples is widely analyzed by an important tool such as Fourier Transform Infrared spectroscopy (FT-IR), and due to the recent up-gradation of this instrument the map of the identified compounds on the surface of the sample can be documented via collection of large number of FT-IR spectra [44]. To measure the physical changes of the polythene after the microbial attack various parameters are usually used to determine the weight loss, percentage of elongation and change in tensile strength ( Table  1). The products from polythene degradation are also characterized using various techniques such as Thin Layer Chromatography (TLC), High Performance Liquid Chromatography (HPLC) and Gas Chromatography-Mass Spectrometry (GC-MS) ( Table 1).

Maximum Biodegradation of Polythene both In Vitro and In Vivo
The maximium 61.0% (Microbacterium paraoxydans) and 50.5% (Pseudomonas aeruginosa) of polythene degradation in terms of Fourier Transform Infrared coupled Attenuated Total Reflectance (FTIR-ATR) was recorded [45] within two months. But in terms of weight loss was the degradation of polythene was recorded as 47.2% after 3 months of incubation with the A. oryzae [46] followed by 50% weight loss of the polythene discs using fungus, Phanerochaete chrysosporium after 8 month of regular shaking with pH= 4.00 at room temperature [47]. But due to biodegradation, weight loss of the polythene is not always reported. Some workers [48] reported gain in the polythene weight after cultivation of the microbes on the polythene, incubated at regular shaking for one month at 30 o C. Only three out of 10 microbes lead to weight loss. The maximum weight gain (2.02%) was reported with Streptomyces humidus. The possible reason for gaining of the polythene weight after the cultivation of the microbes on the strips is accumulation of cell mass on the polythene surface [48]. In case of in vivo study after 32 years of polythene dumping in the soil only partial degradation was reported [49].

Toxicity Level of the Biodegraded Polythene Products
To the best of our knowledge there is no report on this aspect except Aswale [47]. She tested the toxicity level of all the polythene biodegraded products on both the animal and plant systems. Among the plant systems, she tested the toxicity level of the degraded polythene products along with culture filtrate on the seed germination rate of the Arachis hypogaea (groundnut), Glycine max. (soybean), Sesamum laciniatum (oil seed, sesame), Helianthus annuus (sunflower) and Carthamus tinctorius (safflower). Moderate decrease in the germination of the seeds was recorded. For the animal system, she calculated the mortality rate of Chironomous larvae, and had not reported any significant difference in the mortality rates as compare to control.

Future Needs
The status of polythene pollution should be updated area wise. The awareness campaign of the polythene pollution should be promoted at mass level among the public. The idea of using starch based polythene or biodegradable polythene should be encouraged. The microbes responsible for the degradation of polythene should be isolated from all the sources, screened to know the efficient isolates. The efficient microbes are needed to characterize at molecular level. Some extracellular enzymes are responsible for the biodegradations of the polythene [56]. These enzymes needed to be characterized and the genes responsible for those enzymes should be worked out. Once the genes responsible for the degradation of polythene would be known, the genes would be used to enhance the polythene degrading capacity of the other easily available microbes. After field trials, the most efficient polythene degrading microbes should be multiplied at large scale to decompose the polythene at commercial level.

Conclusions
Based on the literature survey, it can be concluded that polythene is very useful in our day to day life to meet our desired needs. It can be used for wrapping the goods, food material, medicine, scientific instruments etc. Due to its good quality its use is increasing day by day and its degradation is becoming a great threat. Only in the marine biota annually almost one million marine animals are dying due to 33 their intestinal blockage. Various polythene degradation methods are available in the literature but the cheapest, eco-friendly and acceptable method is degradation using microbes. The microbes release the extracellular enzymes such as lignin peroxidase, manganese peroxidase to degrade the polythene but the detailed characterization of these enzymes in relation to polythene degradation is still needed to be carried out. It was also been known that microbes from various sources are responsible for the degradation of polythene. But efficient polythene degrading microbe is still needed to screen from all the sources. The characterization of efficient polythene degrading microbes at molecular level is still not available up to the mark, which can be multiplied at large scale to commercialize the polythene biodegradation.