Abstract
The diversity and load of heterotrophic fungi associated with the polythene degradation in polythene polluted sites around Chennai, Tamil Nadu were studied. The fungi were isolated and identified by plating and staining techniques. Isolated fungal strains were identified as Aspergillus niger, A. japonicus, A. terreus, A. flavus and Mucor sp. Predominant fungal strains such as A. niger and A. japonicus were selected for polythene degradation under laboratory conditions. Their effectiveness on the degradation of commercial polythene carry bags of low density polyethylene (LDPE) was studied over a period of 4 weeks in shaker culture. Biodegradation was measured in terms of mean weight loss, which was nearly 6 to 11% after a period of 4 weeks. Further, Scanning electron microscopic (SEM) study confirmed the degradation by revealing the presence of porosity and fragility of the polythene surface degraded by fungal. A. japonicus showed as much as 11.11% degradation potential when compared to A. niger (5.80%) for the same period of 4 weeks.
Introduction
Plastic is the most versatile synthetic ‘manmade’ substance created out of the fossil fuel resources that enabled most of the industrial and technological revolutions of the 19th and 20th centuries. During the past 25 years, plastic materials have gained widespread use as they have been increasingly used in food, clothing, shelter, transportation, construction, medical and leather industries. Plastics are composed of petroleum based materials called resins (e.g. polythene and polypropylene) which are resistant to biodegradation. Due to this resistance, plastics that are disposed off in landfills remain in their original form in perpetuity. Plastics offer a number of advantages over alternative materials– they are light weight, low cost, extremely durable and relatively unbreakable. Production of plastics has grown significantly in the last 3 decades averaging 10% of annual growth rate. A general estimate of worldwide plastic waste generation is annually about 57 million tons (Shristi Kumar et al., 2007). However, plastic materials have several disadvantages, the most important one being that they do not break down in the environment. Due to their buoyancy, long term persistence and ubiquity in the marine environment, plastic waste poses a variety of hazards to marine life (Spear et al., 1995). In the recent years, public concern has been increasing over the environmental deterioration associated with the disposal of conventional plastics. Discarded plastics, besides being highly visible are a rapidly increasing percentage of solid waste in landfills, resistant to biodegradation leading to pollution, harmful to the natural environment. Most of the biodegradation studies on plastics are being carried out using microorganisms. Most of the organic wastes undergo microbial degradation and contribute to the biological productivity either directly or indirectly. Since microorganisms are capable of degrading most of the organic and inorganic materials, there is a growing interest in the microbial degradation of plastic and polythene waste material. Kambe et al. (1999) have isolated and characterized a bacterium from soil which utilizes polyester polyurethane as a sole carbon and nitrogen source. Two strains with good polyurethane degrading activity were isolated and identified as Comamonas acidovorans. Oda et al. (1998) have studied polycaprolactone depolymerase produced by the bacterium Alcaligenes faecalis and isolated several bacteria capable of degrading polycaprolactone (PCL) from soil and activated sludge. Webb et al. (2000) have studied the fungal colonization and biodeterioration of plasticized polyvinyl chloride in in-situ and ex-situ conditions and suggested that microbial succession may occur during the long periods of exposure in in-situ conditions. They also identified Aureobasidium pullulans which was the principal colonizing fungus and a group of yeast and yeast-like fungi, including Rhodotorula aurantica and Kluyveromyces spp. Incidence of marine and mangrove bacteria accumulating polyhydroxy-alkanoates on the mid-west coast of India has been reported by Rawte et al. (2002). Microbial degradation of PCL -poly vinyl butyral (PUB) blends has been studied by Rohindra et al. (2003). Kathiresan (2003) has revealed that the high diversity of micro organisms in mangrove soil is capable of degrading plastics, although at a slower rate. Low–density polyethylene (LDPE) accounts for 60% of the total plastic production and the most commonly found solid waste is the non-degradable polythene carry bags. The indiscriminate use of polythene shopping bags by the public is increasingly becoming an environmental problem in India. Most of the municipal and garbage sites are littered with large quantity of this highly recalcitrant waste materials. Against these backdrops, this study was aimed to estimate the diversity of heterotrophic fungi with special emphasis on determining their ability to degrade the polythene waste.
Materials and methods
Estimation of heterotrophic fungi in the polythene samples
The polythene bags were collected in a sterile plastic box from the soils of polluted areas of Chennai, Tamil Nadu. The samples were serially diluted and pour plated in sterile Potato Dextrose Agar to estimate and isolate heterotrophic fungi respectively. The plates were incubated at 37°C for 48 hrs. After incubation, plates with 30-300 colonies were selected for counting and the total plate count for fungi was expressed as number of colony forming units per gram (CFU/gm) of soil.
Characterization of the heterotrophic fungi
After counting and estimation of total colonies, morphologically different colonies were picked up using sterile needle and forceps and aseptically transferred to sterile potato Dextrose Agar slants for further characterization. Fungi were chosen for characterization and identified by macroscopic and microscopic observations (microscopic/staining techniques).
Screening and identification of polyethylene degrading fungi
Agar amended with substrates such as 1% starch, 1% gelatin, 1% tween-80 served as the suitable medium for the action of enzymes present in culture extracts of fungi. Agar amended with each substrates were separately sterilized at 15 lbs for 15 min. A volume of 5-20 mL of sterilized substrate was poured into sterile Petri-dish. The plates were surface dried overnight and the wells were cut aseptically to load the culture filtrates of isolated fungi. The plates loaded with culture filtrates were incubated at 37°C for 3 - 4 hrs. After incubation, the opacity was observed around the well surface which indicated the positive result for the respective substrates.Further quantitative assay was also performed.
Assessment of Microbial degradation of plastics
The pre-weighed LDPE strips (W1) of 1 cm diameter were aseptically transferred to the conical flask containing 50 mL of Rose Bengal broth medium, and separately inoculated with the selected fungal strains. Control was also maintained with LDPE strip in the fungus free medium. Four flasks were maintained for each treatment and incubated in a shaker for 2 and 4 weeks. After one month of shaking, the polythene strips were collected and washed thoroughly using distilled water, shade-dried and weighed for final weight (W2). The weight loss of the polythene strip was calculated by deducting W2 from W1. Further, the surface of degraded LDPE was analyzed by Scanning Electron Microscope (SEM), as outlined by Webb et al. (2000).
Results and Discussion
Fungi collected from the soil sample of the polluted sites were identified as Aspergillus niger, A. japonicus, A. terreus, A. flavus and Mucor sp. Among the five, Aspergillus niger and A. japonicas, the dominant fungi, were selected for further studies (Figs.1a, b). A. niger showed degradation of LDPE up to 5.80% in one month, while A. japonicus showed more capability to degrade the LDPE up to 11.11% under laboratory conditions (Fig. 2). SEM study reveals that the control polythene strips displayed a normal surface topography but the polythene strips treated with A. niger and A. japonicus showed an appreciable surface corrosion, folding and cracks.
a. Aspergillus niger; b. Aspergillus japonicus
Figs. 1 a,b. Photomicrographs of isolated fungal cultures
This may be due to the fungal extracellular metabolites and fungal enzymes (Figs. 3 & 4). Both control and experimental polythene strips were heated upto 200°C at the heating rate of 10°C/min. The melting point was found to have reduced in the experimental polythene strips to 151.98ºC when compared with the control (162.20ºC) polythene strips (Fig. 5); suggesting the degradation of polythene strips by the fungi.
Fig. 2. Plastic degradation by the fungal strains
Fig. 3. SEM of polythene film treated with A. japonicus
Fig. 4. SEM of polythene film treated with A. niger
Surface topography of LDPE films treated with experimental fungal strains showing the degradability after 2 and 4 weeks. Note the porosity and fragility of polyethylene membrane (for details refer to text) Microorganisms play a significant role in biological decomposition of materials, including synthetic polymers in natural environments.
Fig. 5. Melting point of polythene strips
High-density and low-density polyethylenes are the most commonly used synthetic plastics and they are slow in degradability in natural environments, causing serious environmental problems. In this regard, there is a growing interest in non-degradable synthetic polymer biodegradation using effective microorganisms (Lee et al., 1991; Gu, 2003). There is no report on polythene degradation by Aspergillus japonicus so far. This is the first experimental report of LDPE degradation under laboratory conditions by showing effective ability of A. japonicus. The potency of degradation by A. japonicus is two times higher than that of A. niger, i.e. A. japonicus degraded 11.11% while A. niger degraded 5.8% per month.
The polythene bags in the soil of polluted sites have been degraded by the presence of fungi; other than abiotic factors such as moisture, heat, temperature etc. of the soil (Anonymous, 1999). The mechanism of degradation by fungi is not exactly known. The surface of plastic material has turned from smooth to rough with cracking and the molecular weight reduction, increase in carbonyl double bond groups, erosion on the surface of polyethylene is due to the microorganisms. In the process of depolymerization, at least two categories of enzymes are actively involved in biological degradation of polymers: extracellular and intracellular depolymerases (Gu et al., 2000). During degradation, exo-enzymes from microorganisms break down complex polymers yielding smaller molecules of short chains, e.g. oligomers, dimers and monomers, that are smaller enough to pass the semi-permeable outer membranes of the microbes, and then to be utilized as carbon and energy sources (Frazer, 1994; Hamilton et al., 1995). Hence, further study on microbial enzymes or organic acids in degradation of the polythene and plastics will pave way for finding technology for degrading these environmentally hazardous plastic materials.
Conclusion
Fungal strains Aspergillus niger and A. japonicus were selected for polythene degradation under laboratory conditions. Their effectiveness on the degradation of commercial polythene carry bags of low density polyethylene was studied over a period of 4 weeks. Biodegradation was measured in terms of mean weight loss, which was nearly 6 to 12% after a period of 4 weeks. Further, SEM study confirmed the degradation of plastic by the presence of porosity and fragility of the fungal degraded polythene surface. Efficiency of polyethelene degradation was high with A. japonicus s compared to A. niger for a period 4 weeks.
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