2.1.1. Soil sample collection

Soil samples were collected from the different dumping sites of Hardwar district. The chosen dumping sites have been used to dump the plastic for a long time. Plastic degrading organisms were expected to be present at these sites. The samples were collected in sampling sterile polythene bags. These samples were then carefully transported to the laboratory and stored at a temperature of 4°C.

2.1.2. Test sample preparation

The commercially used plastic water bottles (HDPE) were selected to be the test samples for the study as they are dumped large in numbers. The water bottles were purchased from the local market and cut into square pieces of 2 cm length. These HDPE strips were washed manually with absolute ethanol using cotton wool and rinsed severally with distilled water to remove dirt and other organic particles adhering to the surface and then allowed to dry in air for 30 min. The stripes were then dipped into the solvent (xylene) which is heated at a temperature of 140°C and stirred continuously. The resulted mushy mixture was crushed and filtered with the help of a white cloth and then rinsed severally with acetone to remove the solvent. The acetone got evaporated and the leftover was the powdered HDPE. The HDPE powder was stored in closed containers at room temperature [4-5].

2.1.3. Isolation and enrichment

From the soil samples (Section 2.1.1.), fungal colonies were isolated on Czapek-Dox Agar (CDA) using pour plate method and serial dilution method. The plates obtained were incubated at 28°C for 3–4 days. Carefully chosen fungi from CDA plates were subcultured for storage and further characterization.

2.1.4. Screening of fungus

Polyethylene powder was contained in a minimal salt medium and acts as a sole carbon source. Isolated fungus was inoculated on minimal salt medium at 28°C for 4 weeks, thereafter; the colony diameter was measured [5-6].

2.1.5. Identification of selected fungi

Selected fungi were identified by colony morphology and staining. Morph taxonomical identification was performed at NFCCI, Agharkar Research Institute, Pune, India.

2.2.1. Treatment of HDPE

The following three pretreatment strategies were employed for the present study.

2.2.2. Biodegradation study using shake flask method

The pre-weighted treated as well as untreated HDPE strips were aseptically shifted into the conical flask containing 150 ml of minimal salt medium and then inoculated with selected fungus. Control was maintained with HDPE strips in the sterile medium.

Experiments were performed with three sets of flasks and with three isolates, as shown in Table 1. The three sets of flasks were maintained at a temperature of 28°C and shaked at 120 rpm. After every 30 days of incubation, the plastic discs were collected, washed thoroughly using distilled water, dried in hot air oven at 50°C overnight, and then weighed for residual polyethylene (Swomya et al., 2012). The percentage degradation of polyethylene was calculated by the following formula:

Table 1: Percentage degradation of treated and non-treated HDPE by dry weight of polyethylene

Percentage degradation = (W₀ − W_t)/W₀ ×100 (1)

Where

W₀ = Initial weight of polyethylene

W_t = Final weight of residual polyethylene

2.2.3. Field emission scanning electron microscopic (FE-SEM)

The pieces of HDPE (after incubation with fungal cultures) were taken out from the culture and repeatedly rinsed with distilled water. The fungal mycelium was removed carefully and the polymer film was dried at room temperature. FESEM was carried out to study the surface morphology and structural changes in HDPE film before and after biotic exposure, following the biodegradation in shake flask method. The images of treated as well as untreated test samples were captured and compared. FESEM provides evidence of physical deterioration of the polymer surface caused by the microorganisms.

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