BIODEGRADATION OF HIGH DENSITY POLYETHYLENE AND POLYURETHANE FILMS BY FUNGI

Abstract

Total 12 fungal isolates were isolated from buried polyethylene film samples collected from riverbed and dumpsites. The growth characteristics and colony morphology of all the isolates were studied using standard methods. To select the potential plastic degrading microorganism, all the twelve fungal isolates were screened for cell-surface hydrophobicity, percentage adherence to hydrocarbon, extracellular esterase enzyme production, biofilm formation and survival of fungal isolates on polyethylene surface and in planktonic phase. Out of 12 fungal isolates, strain F-8, F-9 and F-12 were found to be highly hydrophobic with high percentage of adherence and hydrolytic enzyme secretion compared to the other isolates, which enabled it to form a dense biofilm on the polyethylene surface with a large number of viable spores along with hyphae. Based on this data, these three isolates were further selected for biodegradation studies and identified. These isolates were deposited at Identification services, Indian Type Culture Collection (ITCC), Indian Agricultural Research Institute (IARI), New Delhi for identification. Isolate number F-8, F-9 and F-12 were identified as Aspergillus flavus (ITCC No. 6051), Aspergillus fumigatus (ITCC No. 6050) and Aspergillus niger (ITCC No. 6052) respectively. All the degradation studies were carried out in pure shake flask system for 30 days. To facilitate the degradation activity, pretreatment of polyethylene film was done by giving thermal treatment at 70°C for a period of 10 days in hot air oven followed by chemical treatment in freshly made disinfectant to check microbial contamination. Degradation of the polyethylene film was studied for physical or chemical changes using n weight loss measurement, reduction in tensile strength, surface changes using Scanning Electron Microscopy (SEM), changes in functional groups using Fourier Transform Infra red Spectroscopy (FTIR), morphological changes such as melting (Tm) and onset Temperature (T0) using Differential Scanning Calorimetery (DSC), structural changes like changes in crystallinity (%C^) using X-Ray diffraction (XRD), degradation products analysis by Gas Chromatography-Mass Spectrometry (GC-MS). A. flavus (ITCC No. 6051), A. fumigatus (ITCC No. 6050) and A. niger (ITCC No. 6052) showed 4.41, 3.45 and 1.16 %reduction in weight and 61.33, 60 and 58.77% reduction in tensile strength of the polyethylene film after incubation with the respective isolate for 30 days. These data suggest that these isolates could be potential polyethylene (HDPE) degrader. Degradation was further investigated by scanning electron microscopy (SEM). All the three isolates A. flavus (ITCC No. 6051), A. fumigatus (ITCC No. 6050) and A. niger (ITCC No. 6052) showed good colonization on the polyethylene surface, though alittle variation was observed in the extent of colonization. SEM studies showed clear degradation features such as shearing, tearing, hyphae penetration, formation of cavities and holes onthepolyethylene film surfaces. FTIR spectrum of heat treated polyethylene film showed atypical carbonyl peak at 1715 cm-1. Incubation of heat-treated polyethylene film with all the three isolates showed a significant reduction in carbonyl content. The reduction in carbonyl content was also estimated in terms of carbonyl index (CI). The changes in onset temperature (T0) and melting temperature (Tm) was also evaluated to determine the morphological changes induced by biological treatment. The significant changes were observed in Tra and T0 after treatment with fungal isolates compared to control. Samples incubated with in these three isolates showed a significant reduction in %Cxrd- Culture broth was analyzed for the presence of degradation products of HDPE by GC-MS and identification of compounds was done by comparison with NBS database and major products identified were toluene, 1,2-Benzene Dicarboxylic acid, Diisooctyl ester, Propanoic acid, Phenol, 4,6-Di(l,l-Dimethylethyl)-2-Methyl, Methyl carbamate, Phenol,2,6-Bis(l,l- Dimethylethyl)-4-Methyl-,Methylcarbamate, 2,6-Di-T-Butyl-4-Methylphenol acetate (ester), 1,3,5-cycloheptatriene. Polyurethane degradation ability of these isolates was also studied under similar conditions using polyurethane as carbon source. In contrast to polyethylene (HDPE), polyurethane (PU) is reported to be relatively susceptible to microbial attack. All the three isolates A. flavus (ITCC No. 6051), A. fumigatus (ITCC No. 6050) and A. niger (ITCC No. 6052) showed great potential for polyurethane degradation as revealed from weight loss, SEM, FTIR and DSC studies. The degradation of polyurethane was faster as compared to polyethylene as expected due to its susceptibility for microbial attack. The degradation of polyurethane by A. flavus (ITCC No. 6051), A. fumigatus (ITCC No. 6050) and A. niger (ITCC No. 6052) reported inthe present study isthe first direct report of degradation by these fungal isolates. Hydrolytic enzymes particularly Esterase, Lignin Peroxidase (LiP) and Manganese Peroxidase (MnP) producing abilities of A. flavus (ITCC No. 6051), A. fumigatus (ITCC No. 6050) and A. niger (ITCC No. 6052) were measured in 30 days old minimal media culture broths inoculated by these isolates containing polyethylene or polyurethane as carbon source. High esterase activity (136U/50ml), (152U/50ml) and (130 U/50ml) respectively were observed in the culture broth of A. flavus (ITCC No. IV 6051), A.fumigatus (ITCC No. 6050) andAniger (ITCC No. 6052) containing HDPE as carbon source. SDS-PAGE and in gel assay showed the secreted molecular weight in the range of 45-48 kDa. Asignificant MnP (8-22U/50ml) and LiP (6-9U/50ml) activities were also observed in the culture broths of these isolates, though it was much lower compared to esterase activity. The presence of significant activities of esterase, LiP and MnP suggest their possible role in plastic degradation. The biodegradation of high density polyethylene (HDPE) and polyurethane (PU) by A. flavus (ITCC No. 6051), A. fumigatus (ITCC No. 6050) and A. niger (ITCC No. 6052) in the present study is the first direct report.