Soil Sample Collection
Soil samples were collected from three different crude oil reservoirs at Al Faisaliyyah, Al Sina’iyah, and Ghubairah, located in Riyadh, Saudi Arabia. Briefly, 400 g of soil samples were collected at 0-10 cm depth, under aseptic conditions. Samples were sieved by 2.5 mm pore size sieves, mixed with Tween 80, homogenized, and stored at 4ºC until use .
Sources of Different Hydrocarbons
Different samples of crude oil, kerosene, diesel, and used oil were collected from the tankers of Saudi Aramco Company (Dammam, Saudi Arabia). The oil samples were sterilized by Millex® Syringe Filters (Merck Millipore co., Burlington, MA, United States) and stored at 4°C for further usage.
Isolation and identification of fungal species
The fungal species in the soil contaminated by crude oil were identified using the dilution method. Briefly, 10% of each soil sample was dissolved in distilled water and vortexed thoroughly. Then, 0.2 mL of each sample was cultured on a sterile PDA plate incubated at 28°C for three days until the growth of different fungal colonies. Carefully, each colony was isolated, re-cultured on new PDA McCartney bottles of PDA slant, and incubated at 28°C for three days. The morphology of pure cultures was tested and identified under a light microscope as described before .
The incidence of each strain was calculated as follows:
Hydrocarbon Tolerance Test
The growth rate of isolated strains was tested in a liquid medium of MSM mixed with 1% of either crude oil, used oil, diesel, kerosene, or mixed oil. Furthermore, a control sample of MSM liquid medium without any of the oils tested, and all culture media were autoclaved at 121°C for 30 min. After cooling, 1 ml of each isolate was inoculated with one of the above mixtures and incubated at 25°C on an orbital shaker. The growth rate was measured every three days for a month for each treatment versus the control. All experiments were performed in triplicates.
Scanning Electron Microscopy (SEM)
The morphology of different strains of the isolated fungi was tested by SEM, as previously described , with some modifications. Briefly, 1 ml of each growing strain, in the liquid media, was centrifuged at the maximum speed (14000 rpm) for 1 min, followed by fixation with 2.5% glutaraldehyde, and overnight incubation at 5°C. Later, the sample was pelleted, washed with distilled water, then dehydrated with different ascending concentrations of ethanol (30, 50, 70, 90, 100 (v/v)) for 15 mins at room temperature. Finally, samples were examined in the Prince Naif Research Centre (King Saud University, Riyadh, Saudi Arabia) by the JEOL JEM-2100 microscope (JEOL, Peabody, MA, United States), according to the manufacturer instructions.
Crude Oil Degradation Assay
A modified version of the DCPIP assay  was employed to assess the oil-degrading ability of the fungal isolates. For each strain, 100 ml of the autoclaved MSM was mixed with 1% (V/V) of one of the hydrocarbons (crude oil, used oil, diesel, kerosene, or mixed oil), 0.1% (v/v) of Tween 80, and 0.6 mg/mL of the redox indicator (DCPIP). Then, 1-2 ml of different fungi growing in liquid media (24-48 h) add to the Crude Oil Degradation media, prepared previously, and incubated for two weeks in a shaking incubator at 25°C. A non-inoculated Crude Oil Degradation media was used as the negative control. Afterward, the colorimetric analysis for the change in DCPIP color was estimated, spectrophotometrically, at 420 nm. All experiments were performed in triplicates.
Preparation of Cell-Free Supernatant (CFS)
To prepare the Cell-Free Supernatant (CFS), all isolates were grown in MSM broth medium with 1% of either crude oil, used oil, diesel, kerosene, or mixed oil for 30 days in a shaking incubator at 25°C. After incubation, the cells were removed by centrifugation at 10000 rpm for 30 min at 4°C. The supernatant (CFS) was collected and filter-sterilized with a 0.45 μm pore size sterile membrane. CFS was screened for the production of different biosurfactants. All the experiments were carried out in triplicates, and the average values were calculated.
The Drop-Collapse assay was performed as previously described , with some modifications. 100 µl of crude oil was applied on glass slides, then 10 µl of each CFS was added to the center of the slide surface, and incubated for a minute at room temperature. The slides were imaged by a light microscope using the 10X objective lenses. The spreading on the soil surface was scored by either « + » to indicate the level of positive spreading, biosurfactant production, or « - » for negative spreading. Biosurfactant production was considered positive at the drop diameter ≥ 0.5 mm, compared to the negative control (treated with distilled water).
Oil Spreading Assay
An amount of 20 ml of water was added to the Petri plate and mixed with 20 µl of crude oil or mixed oil, which created a thin layer on the water surface. Then, 10 µl of CFS was delivered onto the surface of the oil, and the clear zone surrounding the CFS drop was observed. The results were compared to the negative control (without CFS) and positive control of Sodium Dodecyl Sulfate (SDS) . The assay was performed in triplicates.
Emulsification Activity Assay
The emulsification activity of each isolate was assessed by mixing equal volumes of MSM broth medium of each isolate with different oils in separate tubes. The samples were homogenized by vortex at high speed for two minutes at room temperature (25°C) and allowed to settle for 24 hrs. The tests were performed in duplicate. Then, the emulsification index was calculated as follows :
Recovery of Biosurfactants
The recovery of biosurfactants from CFS was tested through different assays:
a) Acid Precipitation Assay
3 ml of each CFS was adjusted by 6N HCl to pH 2 and incubated for 24 h at 4°C. Later, equal volumes of chloroform/methanol mixture (2:1 v/v) were added to each tube, vortexed, and incubated overnight at room temperature. Afterward, the samples were centrifuged for 30 min at 10000 rpm (4°C), the precipitate (Light brown colored paste) was air-dried in a fume hood, and weighed .
b) Solvent Extraction Assay
The CFS containing biosurfactant was treated with a mixture of extraction solvents (equal volumes of methanol, chloroform, and acetone). Then, the new mixture was incubated in a shaking incubator at 200 rpm, 30°C for 5 h. The precipitate was separated into two layers, in which the lower layer (White) was isolated, dried, weighed, and stored .
c) Ammonium Sulfate Precipitation Assay
The CFS containing biosurfactant was precipitated with 40% (w/v) ammonium sulfate and incubated overnight at 4°C. The samples were centrifuged at 10,000 rpm for 30 min (4°C). The precipitate was collected and extracted with an amount of acetone equal to the volume of the supernatant. After centrifugation, the precipitate (Creamy-white) was isolated, air-dried in a fume hood, and weighed .
d) Zinc Sulfate Precipitation Method
Similarly, 40% (w/v) zinc sulfate was mixed with the CFS containing biosurfactant. Then, the mixture was incubated at 4°C, overnight. The precipitate (Light Brown) was collected by centrifugation at 10000 rpm for 30 min (4°C), air-dried in a fume hood, and weighed .
All experiments were performed in triplicate, and the results were expressed as the mean values ± standard deviation (SD). One-way ANOVA and Dunnett's tests were used to estimate the significance levels at P<0.05. Statistical analyses were performed using the SPSS statistical package (version16) (IBM, Armonk, NY, United States).