Isolation and Screening from Soil Biodiversity for Fungi Involved in the Degradation of Recalcitrant Materials
Summary
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.
Abstract
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.
Introduction
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, 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.
Protocol
Representative Results
Discussion
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…
Declarações
The authors have nothing to disclose.
Acknowledgements
We acknowledge Scuola di Alta Formazione Dottorale (SAFD) of the University of Pavia and Professor Solveig Tosi for providing the opportunity for this work.
Materials
| 96 microwell plate | Greiner bio-one | 650185 | |
| Agar | VWR | 84609.05 | |
| Bushnell-Haas Broth | Fluka | B5051 | |
| CaCl2 | Sigma-Aldrich | C5670 | |
| Chloroamphenicol | Eurobio | GABCRL006Z | |
| Chlortetracycline | Sigma-Aldrich | Y0001451 | |
| CoCl2·6H2O | Sigma-Aldrich | C8661 | |
| CuCl2·2H2O | Sigma-Aldrich | C3279 | |
| Ethanol | VWR Chemicals | 20821.296 | |
| FeCl3·6H2O | Sigma-Aldrich | 236489 | |
| Filter 0.2 µm | Whatman | 10462200 | |
| gallic acid | Sigma-Aldrich | G7384 | |
| Glass cover slips | Biosigma | VBS634 | |
| Glass vials 15 mL | SciLabware | P35467 | |
| guaiacol | Sigma-Aldrich | G5502 | |
| High-density polyethylene (HDPE) | Sigma-Aldrich | 434272 | |
| Humic acids | Aldrich Chemistry | 53680 | |
| K2HPO4 | Sigma-Aldrich | P8281 | |
| KH2PO4 | Sigma-Aldrich | P5655 | |
| Lignocellulose | / | / | Sterilized bioethanol production waste |
| L-shaped cell spreader | Laboindustria S.p.a | 21133 | |
| magnetic stirrer | A.C.E.F | 8235 | |
| Malt Extract Broth | Sigma-Aldrich | 70146 | |
| MgSO4·7H2O | Sigma-Aldrich | M2643 | |
| Micropipette 1000 μL | Gilson | FA10006M | |
| Micropipette 200 μL | Gilson | FA10005M | |
| MnCl2·4H2O | Sigma-Aldrich | M5005 | |
| Na2MoO4·2H2O | Sigma-Aldrich | M1651 | |
| NaCl | Sigma-Aldrich | S5886 | |
| Neomycin | Sigma-Aldrich | N0401000 | |
| Penicillin | Sigma-Aldrich | 1504489 | |
| peptone | Sigma-Aldrich | 83059 | |
| Polyethylene terephthalate (PET) | Goodfellow | ES306031 | |
| Petri dishes | Laboindustria S.p.a | 21050 | |
| Petrolatum (Paraffin liquid) | A.C.E.F | 009661 | |
| Potato Dextrose Broth | Sigma-Aldrich | P6685 | |
| Polystyrene (PS) | Sigma-Aldrich | 331651 | |
| Polyurethane (PUR) | Sigma-Aldrich | GF20677923 | |
| Polyvinyl chloride (PVC) | Sigma-Aldrich | 81388 | |
| Sterile falcon tube | Greiner bio-one | 227 261 | |
| Sterile glass vials 20 mL | Sigma-Aldrich | SU860051 | |
| Sterile point 1000 μL | Gilson | F172511 | |
| Sterile point 200 μL | Gilson | F172311 | |
| Sterile polyethylene bags | WHIRL-PAK | B01018 | |
| sterile syringe | Rays | 5523CM25 | |
| Streptomycin | Sigma-Aldrich | S-6501 | |
| Tween 80 | Sigma-Aldrich | P1754 | |
| Used engine oil | / | / | complex mixture of hydrocarbons, engine additives, and metals, provided by an Italian private company |
| Vials 50 mL | Sigma-Aldrich | 33108-U | |
| ZnCl2 | Sigma-Aldrich | Z0152 |
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