Polythene waste alone shares 64% of the total plastic waste produced annually across the globe12,16. Among all the methods available to deal with plastic waste disposal, bioremediation technology succeeded with wide range of acceptance throughout the globe3,12. In literature, polythene deteriorating fungi were reported from various sources viz. marine water, plastic dumping sites and mangrove rhizosphere soil3,12,16. In the current investigation, we selected only those sites, for collecting of the rhizosphere soil samples to isolate polythene degrading fungi which represent all these sources. There are two reports4,54 from East Coast of India and one report4 from South East Coast of India and each report exhibits either utilization of mangrove rhizosphere soil or marine water for isolation of polythene degrading fungi. To the best of our knowledge, from the West coast of India, we have reported for the first time, polythene degrading fungi from all the available polythene degrading sources (dumping site, mangrove rhizosphere, marine water). Polythene degradation using fungal isolates had been assessed by determining the changes in some of the key characteristics of the polythene before and after the treatment of the fungal isolates viz. reduction in weight, reduction in tensile strength, reduction in percent elongation, reduction in viscosity, reduction in crystallinity, formation of cracks/scars/holes on the surface of the polythene, changes in the carbonyl index, estimation of CO 2 released etc3,12. The rate of polythene degradation is highly influenced with various other factors viz. incubation time, pH, temperature, treatment of the polythene with some acids e.g. nitric acid (to remove the plasticizers) etc3,12. The incubation time period (period from the date of treatment to harvesting of the polythene strips for assessing the level of polythene deterioration) of the fungal isolates used to determine the rate of polythene degradation reports to be varied from 10 days to 32 years.

Previously after 30 days of incubation maximum 28.80 ± 2.40 percent weight loss (%WL) of the polythene was recorded with Aspergillus glaucus4, 23.11% WL (pre-treated UV and nitric acid) with Aspergillus niger55, 12.25%WL with Aspergillus niger56, 11.11% WL (LDPE) with Aspergillus japonicas and 5.8% with Aspergillus niger57. Even after increasing the incubation duration to 60 days, only 28–40% WL the polythene with Aspergillus niger was recorded but our results are more promising and efficient comparatively, we have reported ≈50% WL of the pretreated polythene strips with Aspergillus terreus MANGF1/WL at pH 9.5 during the same incubation period (60 days). Various workers used 3 months incubation time to determine the level of polythene degradation using fungi and recorded maximum 58.45% WL in pretreated polythene (2 days old chemically treated polythene followed by UV irradiation for 50 minutes before treatment) with Aspergillus oryzae, whereas in case of untreated polythene, only 6.3%WL with the same fungi during the same test period was recorded58 followed by 5.95% WL (with Aspergillus niger)59, 1.2% WL (with Curvularia lunata), 0.8% WL (with Alternaria alternate), 7.7% WL (Penicellium simplicissimum) and 0.7% WL (with Fusarium sp.) but the consortia of all these fungi (Curvularia lunata + Alternaria alternate + Penicellium simplicissimum + Fusarium sp.) results in 27% WL during the same test period60. After 6 months of incubation, only 26.17% WL (with Aspergillus niger)61 and 20.63%WL (with Aspergillus flavus)62 of polythene was documented. Aswale63 reported maximum 50%WL (with Phanerochaete chrysososporium) of the polythene at pH 4 after 8 months of incubation period. Abdullahi et al.64 recorded polythene degradation after 9 months of test period in terms of percent weight loss in two different types of degradation sets (polythene seeding in cow dung mixed fadama soil (PECDS) and poultry dropping mixed fadama soil (PEPDS) and reported 18.1% WL in the set of PECDS whereas only 6.0% WL with set PEPDS. Finally they concluded Aspergillus niger, A. fumigatues, and A. flavus mixed with PECDS and PEPDS leads to highest weight loss of the polythene compared to fadama soil mixed with inorganic fertilizer (NPK) and control. The possible reason for weight reduction in polythene in all the studies is due to breakdown of carbon backbone (enzymatic degradation)65 and utilizing the resulting monomers and oligomers as a carbon source by the fungi61,66. Otake et al.67 buried different kinds of plastic (including polythene) in the soil and assessed the level of degradation after 32 years and reported only whitening patches on the surface of the polythene due to the microbes (both fungi and bacteria) and recorded no evidence of degradation of other types of plastics. Among the various reports68,69,70,71,72 published during 2017–2018 on fungal based plastic degradation, Penicillium sp. was recorded as the most efficient fungi with percent weight reduction 43.4%70 in just 30 days.

Besides reduction in weight due to the degradation potential of the fungi, reduction of the tensile strength is also one of the widely studied parameter by different research groups around the globe. In the current investigation, we reported highest percent reduction in TS (94.44 ± 2.40%) with A. sydowii strain PNPF15/TS at pH 3.5 after 60 days of continuous shaking at ambient temperature. Our results are in agreement with the previous studies, previously, after 10 days of incubation maximum reduction (60%) in tensile strength (TS) of the heat treated polythene was reported with Mucor rouxii [NRRL 1835]73. After three months of testing period maximum reduction in TS (63%) of the polythene was reported with A. oryzae58 followed by 51% reduction in PE (Mangnease sterate treated LDPE exposed to UV irradiation) with the same fungi (A. oryzae)74. Vijaya and Reddy52 followed the ASTM standard and assessed the degradation (by compositing) of polythene (HDPE) along with municipality solid waste and recorded highest 20% reduction in tensile of HDPE after 1 year of testing duration. Vijaya and Reddy52 studied correlation coefficient among WL and TS and suggested strong correlation coefficient; if one factor is affected by microbial attack other factor also gets affected at the same time.

The degradation of the polythene was further authenticated using SEM and FTIR analysis. The SEM analysis revealed the degradation level on the surface of the polythene in the form craks/scions/holes (Fig. 2). Our observations are similar with the previous reports. Due to use of SEM analysis, structural changes and erosions on the surface of the polythene in the form of porosity, cavities, holes/scions/cracks were reported with fungal consortia60, Mucor circinilloides and Aspergillus flavus45, Aspergillus and Penicillium75, Chaetomium globosum76, Aspergillus niger and Aspergillus japonicas57,77. After SEM analysis, the level of polythene degradation was further authenticated by FTIR analysis. In the present investigation, the FTIR data confirmed the level of structural changes in the polythene. Abiotically treated sample (HNO 3 treatment, 20 min UV treatment) shows generation of carbonyl peak, carboxylic acid and its derivatives. We observed the peak of carboxylic acid in the range of 1633.73–1812.08 cm−1 and reported the reduction of this peak up to 1629.53 cm−1 and no peak was observed at 1812.08 cm−1 on the PE strips degraded by A. terreus strain MANGF1/WL. A. sydowii strain PNPF15/TS based degraded polythene strip depicts the reduction of peak from 1633.73–1812.08 cm−1 to 1628.60 cm−1 and similarly no peak was recorded at 1812.08. Similarly in past, Konduri et al.74 also reported carboxylic acid peak in the range of 1630–1840 cm−1.

Chatterjee et al.78 reported the formation of C-H stress group at peak 2915 and we also reported similar peak at 2912.10 cm−1 in abiotic treated PE (control) and documented the reduction in the peak with both the fungal strains (A. terreus strain MANGF1/WL and A. sydowii strain PNPF15/TS) to 2912.09 cm−1 and 2912.92 cm−1 respectively. Chatterjee et al.78 reported occurrence of CH 2 peak at 718, same functional group was observed in our study in the control PE strip at peak 721.52 cm−1 and compared to control we reported reduction in the peak with A. terreus strain MANGF1/WL (720.88 cm−1) and A. sydowii strain PNPF15/TS (721.05 cm−1). Balasubramanian et al.79 reported Keto carbonyl band at 1715, we reported the similar peak (1716.66 cm−1) in the control PE strips, but due to action of both the fungal strains this Keto carbonyl peak was not recorded on the degraded PE strips. Konduri et al.74 reported the peak of C=O stretching in between 1710–1740 cm−1, we got nearly same but smallar peak in between 1716–1766 cm−1 in the control PE strips and similar to Keto carbonyl band, C=O stretching was also not observed in degraded PE strips with both the fungal strains. In case of untreated PE strips (control), carboxylic group peak was not recorded, whereas CH 2 was recorded at peak 721.28 cm−1 and reduction in CH 2 peak was reported in the untreated PE strips degraded by both the fungi (718.45 cm−1 by A. terreus strain MANGF1/WL and 720.51 cm−1 by A. sydowii strain PNPF15/TS). CH stress peak was observed in the control PE (untreated) strips at 2913.03 cm−1 and only A. sydowii strain PNPF15/TS was reported to lead reduction of CH peak to 2912.02. In agreement with the Konduri et al.74 we also recorded C=C stretching at two peaks 1739.35 cm−1 and 1792.57 cm-1 only in case of PE strips degraded by A. terreus strain MANGF1/WL. Microbes are also reported to be responsible for decreasing the carbonyl index58 which in turn depicts the level of degradation. Manzur et al.80 reported maximum (40%) reduction in Carbonyl Index (CI) after 3 months of incubation. Yamada et al.81 studied the effect of Penicillium simplicissimum (soil fungi) on the degradation of low density polythene and after 3 months of incubation in liquid culture, Penicillium simplicissimum was reported to utilize polythene as a carbon source before irradiating with UV and nitric acid treatment. They also suggested that time required for degradation of polythene is depend on the time needed for the growth phase pure culture and they also reported that degradation is directly proportional to the addition of functional groups. Konduri et al.58,74 observed reduction in carbonyl group after three and six months of incubation with the fungi A. oryzae and A. flavus. Similarly in the current investigation there was a change in carbonyl groups, carboxylic groups after incubation with A. terreus strain MANGF/WL and A. sydowii strain PNPF15/TS for 60 days of continuous shaking at ambient temperature.

The previous literature depicts that polythene deteriorating fungi were mostly characterized based on morphological keys82,83,84. There are only few reports of identification of polythene degrading fungi at biochemical level82,85 and at molecular level5,58,69. As per the literature the traditional methods of fungal identification are time consuming, labour extensive and needs the utility of wide range of culture media with experienced personnel to characterize commonly occurring fungal strain variants86,87,88. The traditional methods are usually based on morphological keys and biochemical tests such as the identification of yeast based on biochemical test such as carbohydrate assimilation and fermentation tests which are unmanageable in non-specialized laboratory of microbiology89. There are various kits available in the market which leads to the rapid identification of the fungi but these kits are also not reliable and may takes few weeks to get the final results90,91. So, it is needed to have fast and accurate method of fungi identification. In the present scenario, the strategy utilized to identify many important fungi is the combined usage of morphological keys and biochemical tests with molecular diagnostics92. Presently, molecular tools are employed to aid the traditional method of fungi identification at greater pace93,94,95. Analysis of the variation in the internal transcribed spacer (ITS) regions of the rDNA is widely used for accurate identification of fungi96. Identification species and strain are more accurate based on variation in the ITSl/ITS2 domains than the 18S region (small subunit), the 5.8S region and the 28S region (large subunit)96,97. As per reports98,99 sequence based method is most rapid and authentic. Furthermore, molecular tools are the authentic and more reliable than the morphological and biochemical analysis and are considered as gold standard for the identification of any micro-organism. In the current investigation, based on morphological and molecular level (ITS gene sequence variation analysis), Aspergillus, Penicillium and Meyerozyma were reported as three main polythene degrading fungal genera. In case of genus Aspergillus, only four species in agreement with the previous reports such as Aspergillus awamori100; A. niger52,83,101, A. terreus57,102 and A. versicolor102 were recorded with polythene degradation potential. Besides the above species of the Aspergillus 7 more species such as A. candidus52, A. cremeus52, A. glaucus4,101, A. japonicus57, A. nidulans82,62, A. flavus52,82, A. oryzae103, A. ornatus52 were reported to have polythene degradation potential. The fungus, Aspergillus sydowii from the genus Aspergillus was reported for the first time with the polythene degradation potential, however, it was reported to degrade PVC plastic5. In genus Penicillium only Penicillium chrysogenum was recorded in the current investigation to degrade polythene. Sowmya et al.60 studied the degradation of rubber due to Penicillium chrysogenum, however, in literature Penicillium simlicimmum81, Penicilliumsp.82, P. pinophilum83, P. frequentans82, P. funiculosum104, also reported to have polythene degradation capacity. In the literature there is no report of Meyerozyma guilliermondii with polythene deteriorating potential, instead gasoline was reported to be degraded with same fungi105. Further, the polythene degradation-products (PE-DP) produced with the elite polythene degrading fungi (Aspergillus terreus strain MANGF1/WL and Aspergillus sydowii strain PNPF15/TS) were subjected to Gas Chromatography and Mass Spectra analysis followed by followed by their deleterious potential effect on Sorghum seeds and tiger shark fish were assessed, fungi based by-products of the polythene were found least toxic to both the plants and animal system106.