Benzanthracene

A modified protocol of El Hanafy et al. (2015) was used to screen for fungal utilization of hydrocarbons. Briefly, Bushnell‐Hass agar comprising of K₂HPO₄ (1 g/L), MgSO₄ (0.2 g/L), KH₂PO₄ (1 g/L), CaCl₂ (0.02 g/L), NH₄NO₃ (1 g/L), FeCl₂ (0.05 g/L), and Agar (1.25% (w/v)) was prepared in 1 L of distilled water. The agar was poured into 90 mm diameter petri plates which were then overlaid with 100 μl of a 1:1 (v/v) mixture of 0.2 mg/ml naphthalene solution prepared by dissolving 10 mg of naphthalene in 50 ml of methanol and 1 mg/ml benzanthracene prepared by dissolving 10 mg of benzanthracene (Sigma, Pretoria, RSA) in 10 ml of methanol. The polycyclic aromatic hydrocarbon (PAHs) solution was evenly spread over the agar surface using sterile disposable spreaders and the plates left in the lamina flow for the solvent to evaporate, leaving behind a visible thin white layer of PAHs on the surface of the agar. The plates were then spot inoculated with square fungal plugs measuring approximately 1 cm by 1 cm and incubated at 27°C for 1 week. Fungal plugs which grew beyond their original size (>1 cm in diameter) after incubation were deemed positive for hydrocarbon utilization.