2.2. Strain isolation and screening

Surface polluted sludge (1–10 cm) was collected from Rumaila oil field. MSM with 1% crude oil was used to feed the microorganisms with oil degradation abilities in soil samples. Five grams of soil samples were cultivated in 250‐ml Erlenmeyer flasks containing 100 ml MSM supplemented with 1% crude oil as a sole source of carbon and energy at 30°C with shaking at 130 r/min for 7 days. Primary inoculums (10 ml) were transferred in fresh MSM with 1% crude oil, incubated for 7 days. The enrichment cultivation was carried out under the same conditions, and repeated four times totally for 4 weeks. The microorganisms detected in the enrichment culture were transferred onto 15 g/L MSM agar plates (sprayed with 1% crude oil). Morphologically separated colonies were picked and purified on fresh potato dextrose agar (PDA) plates at least three times.

After that, a 5 mm mycelial disc from the margin of the actively growing cultures on PDA plate was transferred into 100 ml of potato dextrose broth (PDB) medium in a 250 ml Erlenmeyer flask and incubated at 30°C with shaking at 130 r/min for 3 days as primary inoculums. Then, 10 ml of primary inoculums were inoculated into fresh PDB medium and incubated at the same conditions. After 2 days, mycelial pellets were harvested by centrifugation at 7000 g for 5 min at 4°C and washed thrice with sterile MSM. Then, the mycelial pellets were resuspended in MSM, transferred to a sterile Waring blender cup, and homogenized.

For the growth of isolated strains, the optimum conditions were as follows: temperature of 30°C, pH 7, with 250 mg/L crude oil, 50 mg/L NAP, 20 mg/L PHE, and 20 mg/L PYR. To screen oil‐degraded strains, crude oil and PAHs (NAP, PHE, and PYR) were used as sole carbon sources.