This protocol is significant because it isolates fungal strains with high degradative potential directly from soil samples to be considered as candidates for bioremediation purposes. This protocol is a low-cost screening to find fungi with bioremediation potential. Moreover, it gives results that are easy to interpret.
Since this technique involves handling microorganisms, it is very important to work in sterility using a laminar flow cabinet or a Bunsen burner to avoid contamination. To begin, sample the soil of interest and sieve the soil using a two-millimeter mesh to remove any roots and plant debris. Under a laminar flow cabinet, add one gram of the sieved soil into a sterile 15-milliliter tube.
Then, add 10 milliliters of sterile deionized water to make a 10 to the minus one dilution. Shake the tube horizontally for 30 minutes. Before the suspension settles, take one milliliter of 10 to the minus one dilution with a sterile pipette and transfer it to a nine-milliliter of deionized water blank to make 10 to the minus two dilution.
Vortex the suspension thoroughly. Similarly, make a 10 to the minus three dilution from a 10 to the minus two dilution tube. For plating soil dilutions on selective media, collect 100 microliters of the 10 to the minus three dilution using a micropipette with a sterile point and transfer it onto a humic agar Petri dish.
Distribute the dilution evenly on the dish surface using a sterile, disposable, L-shaped cell spreader. Prepare four to five such dishes. Similarly, prepare lignocellulose dishes from a 10 to the minus three dilution tube.
Allow the dishes to air dry for 10 to 15 minutes before incubating at 25 degrees Celsius in the dark for 15 days. Check all the fungal colonies present for similarities. If necessary, prepare a slide to be observed under a light microscope.
Under a laminar flow cabinet or at a Bunsen burner, isolate each chosen fungal strain by gently removing a small part of the mycelium from the colony with a sterile inoculation needle. Transfer the isolated colony to a new malt extract agar, or MEA, dish. Transfer 20 milliliters of Bushnell Haas, or BH medium, into 50-milliliter glass vials.
Transfer seven milliliters of deionized water into 15-milliliter vials and add pieces of broken glass cover slips into each vial. Sterilize the vials by autoclaving at 121 degrees Celsius for 20 minutes. To test growth on petrolatum, add one milliliter of petrolatum to each 50-milliliter BH vial using a sterile pipette under the laminar flow cabinet.
Keep some vials with only BH as the negative control. Remove the mycelium on the surface of the medium from the MEA plate with the chosen fungal strain using a sterile needle and transfer it to a 15-milliliter glass vial with the broken cover slips. Agitate the vial on a vortex mixer for two minutes, allowing the broken cover slips to cut the cube of the fungal colony, making a suspension.
Inoculate each petrolatum vial with 200 microliters suspension. Make two replicates for each fungal strain. For the negative control, add 200 microliters of fungal suspension into the vial containing only BH medium.
Also, keep the vial with petrolatum without any fungal inoculation to check for contamination in the medium. Incubate the vials at 25 degrees Celsius in the dark and observe them after 15 and 30 days from the inoculum. To test growth on used engine oil, transfer 500 microliters of used engine oil onto the BHA Petri dish and distribute the oil evenly.
Then, inoculate the plate with fungal strain by collecting the mycelium from MEA plates. To test growth on plastics, transfer 200 microliters of fungal suspension into a 96-microwell plate. Make three replicates of the fungal strain-plastic combination.
Add 10 milligrams of a different plastic powder to each well with the fungal suspension. For the negative control, add 200 microliters of BH medium to the well with the fungal suspension. Moreover, for each plastic powder, fill a well with 200 microliters of BH solution and 10 milligrams of each plastic dust to check for contamination.
Incubate the plates at 25 degrees Celsius in the dark and observe them after 15 and 30 days. For qualitative laccases calorimetric test, prepare one liter of potato dextrose agar, or PDA, and autoclave for 20 minutes. After autoclaving, when the medium has cooled down but is still liquid, add 400 microliters of guaiacol under a laminar flow hood and plate it into Petri dishes.
Inoculate the PDA guaiacol plate with fungal strain by collecting the mycelium from the MEA plate. For negative controls, prepare PDA plates with each fungal strain and one plate with PDA and guaiacol. Incubate the plates at 25 degrees Celsius in the dark and observe them after seven days.
For the qualitative esterases test, prepare one liter of Tween 80 medium. Then, place the medium on a magnetic stirrer without heating with a stirring bar inside the medium. After 10 minutes, when everything is melted, transfer five milliliters of the medium into 20-milliliter sterile vials and autoclave.
Add 200 microliters of fungal suspension to the Tween 80 medium sterilized vial. For the negative control, add five milliliters of autoclaved BH medium into a 20-milliliter vial. Then, add 200 microliters of fungal suspension to the vial.
Incubate the vials at 25 degrees Celsius in the dark for 21 days and observe them every seven days. The percentage of fungal strains able to grow on petrolatum, used engine oil, and plastic powders as their sole carbon source with different degrees of success was evaluated. The ability of fungi to exploit petrolatum was assessed as the difference in colony growth between controlled BH and containing petrolatum.
Almost 75%of the tested strains were able to use petrolatum as their sole carbon source and 21%exhibited maximum growth. The growth for plates containing only BHA and in the presence of engine oil was also monitored. Almost 90%of the strains grew on used engine oil, with 39%showing maximum growth.
The formation of a red-brownish halo around the fungal colony indicated higher production of laccases and other ligninolytic enzymes. Approximately 60%of the fungi were able to produce laccases. Moreover, 17%of the strains produced an intense dark halo around the colony in the guaiacol medium.
Esterases activity was assessed by the formation of a white precipitate in Tween 80 medium. Almost 60%of strains showed esterases activity and 13%demonstrated higher precipitate formation. This protocol can be tailored to screen for fungi able to degrade any pollutant by adding the recalcitrant substance of interest to a minimal culture medium.
