We acknowledge Scuola di Alta Formazione Dottorale (SAFD) of the University of Pavia and Professor Solveig Tosi for providing the opportunity for this work.

1. Selection of fungal strains able to degrade recalcitrant materials from soil
<ol>
	<li>Preparation of antibiotic solution.
	<ol>
		<li>Put penicillin (50 mg/L), streptomycin (40 mg/L), chlortetracycline (40 mg/L), neomycin (100 mg/L), and chloramphenicol (100 mg/L) into 250 mL of deionized sterile water.</li>
		<li>Before adding chloramphenicol to the antibiotic solution, dissolve it into 3 mL of &#8805;99% ethanol.</li>
		<li>Place the antibiotic solution on a magnetic stirrer (no heat) with a magn...

The authors have nothing to disclose.

The rich biodiversity of soil is an abundant source of fungi that possess numerous metabolic abilities, some of which could be potential candidates for bioremediation. Selective media tests (Section 1 of the protocol) are easy-to-perform and effective methods for isolating fungi able to grow on natural complex polymers as their sole carbon source. Fungi can produce extracellular, non-specific hydrolases and oxidoreductases30 such as the ligninolytic enzymes laccases and peroxidases<sup class='xref...

Soil is a fundamental component of life on Earth and is the basis of many ecosystems. The minerals, organic matter, and microorganisms in the soil can be considered as one system, with close associations and interactions occurring between them. The interactions of these compounds have an important impact on terrestrial processes, environmental quality, and ecosystem health1. Soil pollution poses serious environmental problems worldwide. The indiscriminate, long-term, and excessive application of recalcitrant and toxic substances, such as pesticides, petroleum products, plastics, and other chemicals, has serious effects on soil ecology and, as a result, can alter soil microbiota. Microbial communities in soils are composed of a wide range of organisms in different physiological states, with the majority being bacteria and fungi. Many of the contaminants in soils have medium- to long-term stability, and their persistence can lead to the development of adaptive mechanisms that allow the microorganisms to utilize recalcitrant substances as nutrients2,3. These microorganisms can, therefore, be considered for bioremediation techniques.
Bioremediation tries to mitigate the effects of pollution by using microorganisms and their enzymes for the degradation or transformation of waste into less toxic or non-toxic compounds. Various species of archaea, bacteria, algae, and fungi possess this bioremediation ability4. As a result of their particular biodegradative actions, fungi are especially promising organisms for bioremediation. They can attack different substrates using their hyphal network, enabling them to penetrate the soil matrix more efficiently than other microorganisms. Additionally, they can reach inaccessible interstices where contaminants are difficult to remove5, and they can also survive low moisture levels6. Moreover, fungi synthesize different cassettes of unspecific enzymes, usually to degrade natural recalcitrant substances such as cellulose, lignin, and humic acids. Those that lack the target substrate can be involved in the degradation of a wide range of recalcitrant pollutants, such as hydrocarbons, plastics, and pesticides7,8,9,10. Therefore, although many fungal species have already been reported as bioremediation agents, there is increasing interest in exploring species that have not yet been studied to select candidates for the bioremediation of recalcitrant contaminating substances. The species already known to have bioremediation properties belong to the phyla Ascomycota11,12,13, Basidiomycota14,15,&#160;and Mucoromycota. For example, the genera Penicillium and Aspergillus are well known to be involved in the degradation of aliphatic hydrocarbons13, different plastic polymers16,17,18, heavy metals19, and dyes20. Similarly, studies carried out on basidiomycetes fungi, such as Phanerochaete chrysosporium and Trametes versicolor, have revealed their involvement in the oxidation of recalcitrant materials such as aromatic hydrocarbons13 and plastics21. Another example of fungi involved in the biodegradation processes are the zygomycetes Rhizopus spp., Mucor spp., and Cunninghamella spp.22,23. In particular, Cunninghamella is able to oxidase aromatic hydrocarbons and is considered a model organism for studying the detoxification of products from a wide range of xenobiotics13.
There are several fungal enzymes involved in the major degradative processes of recalcitrant materials24,25, such as esterase, laccase, peroxidase, and protease. Laccases are copper-containing oxidases produced in the cell and subsequently secreted, that allow the oxidation of a variety of phenolic and aromatic compounds. They can degrade ortho and para diphenols, the amino group-containing phenols, lignin, and the aryl group-containing diamines26. Peroxidases use hydrogen peroxide as a mediator to degrade lignin and other aromatic compounds. There are many different peroxidases, but the ones with the greatest potential to degrade toxic substances are lignin peroxidase and manganese peroxidase27.
Esterases and proteases belong to the group of extra- or ecto-cellular enzymes, which act outside their cells of origin but are still bound to them. These enzymes can catalyze the hydrolysis of large recalcitrant molecules into smaller ones. Due to their low substrate specificity, these enzymes can play a key role in the bioremediation of various pollutants, such as textile dyes, effluents released from the pulp and paper industries and leather tanning, petroleum products, plastics, and pesticides28,29,30.
A number of screening methods to select for bioremediative fungal strains have already been published. For example, straw-based agar medium has been used to screen for white-rot fungi with high potential in the polycyclic aromatic hydrocarbons (PAH) degradation31; and small pieces of rotting wood have been placed onto malt extract agar (MEA) to isolate wood-rotting fungi32. However, most of the methods that have already been proposed select very specific fungi for their activity of interest. This research proposes a wider approach for selecting soil fungi with a broader range of action. The method relies on the initial plating of serial dilutions of soil samples onto a medium amended with humic acids or lignocellulose mixed with antibiotics to select fungi with the ability to degrade these natural recalcitrant substances. Humic acids and lignocellulose, in fact, are substances that are extremely resistant to biodegradation since they have very complex molecular structures, and this allows them to be excellent indicators of the degradative ability of the tested fungi33,34. Subsequently, the fungi selected in the first tests are screened to identify those with the potential to degrade specific pollutants such as petrolatum, used engine oil, and plastics. Finally, qualitative enzymatic tests are performed to detect fungal strains able to produce enzymes involved in the biodegradation processes of recalcitrant substances. For this purpose, protease and esterase tests are conducted, while gallic acid and guaiacol are used as indicators of laccase and other ligninolytic enzyme production35,36. These substrates are used because a strong correlation has been found between the ability of fungi to oxidize them to their brown-colored form and the possession of ligninolytic ability37,38,39.
Through these protocols, it is possible to isolate fungal strains with high degradative potential and a broad spectrum of action directly from soil samples. The isolation of these fungal strains could help find new candidates for bioremediation purposes.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>96 microwell plate</td>
			<td>Greiner bio-one</td>
			<td>650185</td>
			<td></td>
		</tr>
		<tr>
			<td>Agar</td>
			<td>VWR</td>
			<td>84609.05</td>
			<td></td>
		</tr>
		<tr>
			<td>Bushnell-Haas Broth</td>
			<td>Fluka</td>
			<td>B5051</td>
			<td></td>
		</tr>
		<tr>
			<td>CaCl2</td>
			<td>Sigma-Aldrich</td>
			<td>C5670</td>
			<td></td>
		</tr>
		<tr>
			<td>Chloroamphenicol</td>
			<td>Eurobio</td>
			<td>GABCRL006Z</td>
			<td></td>
		</tr>
		<tr>
			<td>Chlortetracycline</td>
			<td>Sigma-Aldrich</td>
			<td>Y0001451</td>
			<td></td>
		</tr>
		<tr>
			<td>CoCl2&#183;6H2O</td>
			<td>Sigma-Aldrich</td>
			<td>C8661</td>
			<td></td>
		</tr>
		<tr>
			<td>CuCl2&#183;2H2O</td>
			<td>Sigma-Aldrich</td>
			<td>C3279</td>
			<td></td>
		</tr>
		<tr>
			<td>Ethanol</td>
			<td>VWR Chemicals</td>
			<td>20821.296</td>
			<td></td>
		</tr>
		<tr>
			<td>FeCl3&#183;6H2O</td>
			<td>Sigma-Aldrich</td>
			<td>236489</td>
			<td></td>
		</tr>
		<tr>
			<td>Filter 0.2 &#181;m</td>
			<td>Whatman</td>
			<td>10462200</td>
			<td></td>
		</tr>
		<tr>
			<td>gallic acid</td>
			<td>Sigma-Aldrich</td>
			<td>G7384</td>
			<td></td>
		</tr>
		<tr>
			<td>Glass cover slips</td>
			<td>Biosigma</td>
			<td>VBS634</td>
			<td></td>
		</tr>
		<tr>
			<td>Glass vials 15 mL</td>
			<td>SciLabware</td>
			<td>P35467</td>
			<td></td>
		</tr>
		<tr>
			<td>guaiacol</td>
			<td>Sigma-Aldrich</td>
			<td>G5502</td>
			<td></td>
		</tr>
		<tr>
			<td>High-density polyethylene (HDPE)</td>
			<td>Sigma-Aldrich</td>
			<td>434272</td>
			<td></td>
		</tr>
		<tr>
			<td>Humic acids</td>
			<td>Aldrich Chemistry</td>
			<td>53680</td>
			<td></td>
		</tr>
		<tr>
			<td>K2HPO4</td>
			<td>Sigma-Aldrich</td>
			<td>P8281</td>
			<td></td>
		</tr>
		<tr>
			<td>KH2PO4</td>
			<td>Sigma-Aldrich</td>
			<td>P5655</td>
			<td></td>
		</tr>
		<tr>
			<td>Lignocellulose</td>
			<td>/</td>
			<td>/</td>
			<td>Sterilized bioethanol production waste</td>
		</tr>
		<tr>
			<td>L-shaped cell spreader</td>
			<td>Laboindustria S.p.a</td>
			<td>21133</td>
			<td></td>
		</tr>
		<tr>
			<td>magnetic stirrer</td>
			<td>A.C.E.F</td>
			<td>8235</td>
			<td></td>
		</tr>
		<tr>
			<td>Malt Extract Broth</td>
			<td>Sigma-Aldrich</td>
			<td>70146</td>
			<td></td>
		</tr>
		<tr>
			<td>MgSO4&#183;7H2O</td>
			<td>Sigma-Aldrich</td>
			<td>M2643</td>
			<td></td>
		</tr>
		<tr>
			<td>Micropipette 1000 &#956;L</td>
			<td>Gilson</td>
			<td>FA10006M</td>
			<td></td>
		</tr>
		<tr>
			<td>Micropipette 200&#160; &#956;L</td>
			<td>Gilson</td>
			<td>FA10005M</td>
			<td></td>
		</tr>
		<tr>
			<td>MnCl2&#183;4H2O</td>
			<td>Sigma-Aldrich</td>
			<td>M5005</td>
			<td></td>
		</tr>
		<tr>
			<td>Na2MoO4&#183;2H2O</td>
			<td>Sigma-Aldrich</td>
			<td>M1651</td>
			<td></td>
		</tr>
		<tr>
			<td>NaCl</td>
			<td>Sigma-Aldrich</td>
			<td>S5886</td>
			<td></td>
		</tr>
		<tr>
			<td>Neomycin</td>
			<td>Sigma-Aldrich</td>
			<td>N0401000</td>
			<td></td>
		</tr>
		<tr>
			<td>Penicillin</td>
			<td>Sigma-Aldrich</td>
			<td>1504489</td>
			<td></td>
		</tr>
		<tr>
			<td>peptone</td>
			<td>Sigma-Aldrich</td>
			<td>83059</td>
			<td></td>
		</tr>
		<tr>
			<td>Polyethylene terephthalate (PET)</td>
			<td>Goodfellow</td>
			<td>ES306031</td>
			<td></td>
		</tr>
		<tr>
			<td>Petri dishes</td>
			<td>Laboindustria S.p.a</td>
			<td>21050</td>
			<td></td>
		</tr>
		<tr>
			<td>Petrolatum (Paraffin liquid)</td>
			<td>A.C.E.F</td>
			<td>009661</td>
			<td></td>
		</tr>
		<tr>
			<td>Potato Dextrose Broth</td>
			<td>Sigma-Aldrich</td>
			<td>P6685</td>
			<td></td>
		</tr>
		<tr>
			<td>Polystyrene (PS)</td>
			<td>Sigma-Aldrich</td>
			<td>331651</td>
			<td></td>
		</tr>
		<tr>
			<td>Polyurethane (PUR)</td>
			<td>Sigma-Aldrich</td>
			<td>GF20677923</td>
			<td></td>
		</tr>
		<tr>
			<td>Polyvinyl chloride (PVC)</td>
			<td>Sigma-Aldrich</td>
			<td>81388</td>
			<td></td>
		</tr>
		<tr>
			<td>Sterile falcon tube</td>
			<td>Greiner bio-one</td>
			<td>227 261</td>
			<td></td>
		</tr>
		<tr>
			<td>Sterile glass vials 20 mL</td>
			<td>Sigma-Aldrich</td>
			<td>SU860051</td>
			<td></td>
		</tr>
		<tr>
			<td>Sterile point 1000&#160; &#956;L</td>
			<td>Gilson</td>
			<td>F172511</td>
			<td></td>
		</tr>
		<tr>
			<td>Sterile point 200&#160; &#956;L</td>
			<td>Gilson</td>
			<td>F172311</td>
			<td></td>
		</tr>
		<tr>
			<td>Sterile polyethylene bags</td>
			<td>WHIRL-PAK</td>
			<td>B01018</td>
			<td></td>
		</tr>
		<tr>
			<td>sterile syringe</td>
			<td>Rays</td>
			<td>5523CM25</td>
			<td></td>
		</tr>
		<tr>
			<td>Streptomycin</td>
			<td>Sigma-Aldrich</td>
			<td>S-6501</td>
			<td></td>
		</tr>
		<tr>
			<td>Tween 80</td>
			<td>Sigma-Aldrich</td>
			<td>P1754</td>
			<td></td>
		</tr>
		<tr>
			<td>Used engine oil</td>
			<td>/</td>
			<td>/</td>
			<td>complex mixture of hydrocarbons, engine additives, and metals, provided by an Italian private company</td>
		</tr>
		<tr>
			<td>Vials 50 mL</td>
			<td>Sigma-Aldrich</td>
			<td>33108-U</td>
			<td></td>
		</tr>
		<tr>
			<td>ZnCl2</td>
			<td>Sigma-Aldrich</td>
			<td>Z0152</td>
			<td></td>
		</tr>
	</tbody>
</table>

isolation and screening from soil biodiversity for fungi involved in the degradation of recalcitrant materials

Environmental pollution is an increasing problem, and identifying fungi involved in the bioremediation process is an essential task. Soil hosts an incredible diversity of microbial life and can be a good source of these bioremediative fungi. This work aims to search for soil fungi with bioremediation potential by using different screening tests. Mineral culture media supplemented with recalcitrant substances as the sole carbon source were used as growth tests. First, soil dilutions were plated on Petri dishes with mineral medium amended with humic acids or lignocellulose. The growing fungal colonies were isolated and tested on different substrates, such as complex mixtures of hydrocarbons (petrolatum and used motor oil) and powders of different plastic polymers (PET, PP, PS, PUR, PVC). Qualitative enzymatic tests were associated with the growth tests to investigate the production of esterases, laccases, peroxidases, and proteases. These enzymes are involved in the main degradation processes of recalcitrant material, and their constitutive secretion by the examined fungal strains could have the potential to be exploited for bioremediation. More than 100 strains were isolated and tested, and several isolates with good bioremediation potential were found. In conclusion, the described screening tests are an easy and low-cost method to identify fungal strains with bioremediation potential from the soil. In addition, it is possible to tailor the screening tests for different pollutants, according to requirements, by adding other recalcitrant substances to minimal culture media.

The selective media methods (Section 1 of the protocol) allowed the rich biodiversity of soil to be screened and the fungi with high bioremediation potential to be selected. With the humic acid and lignocellulose media, more than 100 fungal strains were isolated. These fungi produced enzymes involved in the biodegradation of natural recalcitrant materials, which have a chemical structure resembling many pollutants. However, the fungal strains isolated with the selective media needed further screening. Specifically, the s...

Watch this Scientific Journal Video about Isolation and Screening from Soil Biodiversity for Fungi Involved in the Degradation of Recalcitrant Materials at JoVE.com

Isolation and Screening from Soil Biodiversity for Fungi Involved in the Degradation of Recalcitrant Materials

Here, we present a protocol for screening soil biodiversity to look for fungal strains involved in the degradation of recalcitrant materials. First, fungal strains able to grow on humic acids or lignocellulose are isolated. Their activity is then tested both in enzymatic assays and on pollutants such as hydrocarbons and plastics.

Environmental pollution is an increasing problem, and identifying fungi involved in the bioremediation process is an essential task. Soil hosts an ...

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.

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Biology

4.4K vistas.  University of Pavia. Este protocolo es significativo porque aísla cepas fúngicas con alto potencial degradativo directamente de muestras de suelo para ser consideradas como candidatas con fines de biorremediación. Este protocolo es un cribado de bajo coste para encontrar hongos con potencial de biorremediación. Además, da resultados que son fáciles de interpretar.Dado que esta técnica implica el manejo de microorganismos, es muy importante trabajar en esterilidad utilizando un gabinete de flujo lami...