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 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.
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.
1. Selection of fungal strains able to degrade recalcitrant materials from soil
2. Growth tests on recalcitrant substances
3. Qualitative enzymatic tests
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 selective tests isolated the strains able to degrade humic acids and lignocellulose, the growth tests screened the isolated fungi for those able to use a specific pollutant as their sole carbon source, and the qualitative enzymatic tests described their metabolic activities.
The growth tests were focused on the fungal ability to degrade hydrocarbons (petrolatum and used engine oil) and plastics (PET, PVC, HDPE, PS, PUR). Each of these substances was used in a test as the sole carbon source for the selected fungal strains. The fungal ability to exploit these substances was evaluated as the difference in colony growth between the samples with the added substance and the control samples with only minimal medium (BH or BHA, depending on the test). A qualitative scale was used to describe the results (Figure 1), ranging from the absence of growth difference with the control (0) to low (+), medium (++), and high (+++) levels of difference in growth.
Figure 1: Rate of growth success of fungal strains in the growth tests. The percentage of fungal strains able to grow on petrolatum, used engine oil, and plastic powders (polyethylene terephthalate, PET; polyvinyl chloride, PVC; high-density polyethylene, HDPE; polystyrene, PS; polyurethane, PUR) as their sole carbon source with different degrees of success (no growth, 0; low growth, +; medium growth, ++; high growth, +++). Please click here to view a larger version of this figure.
The petrolatum and used engine oil tests evaluated the hydrocarbon biodegradation potential of the selected fungal strains. Petrolatum, a mixture of long-chain alkanes (>C25), was used as the sole carbon source. The fungal ability to exploit this substance was evaluated as the difference in colony growth between vials with BH + petrolatum and control vials with only BH (Figure 2). Almost 75% of the tested fungal strains were able to use petrolatum as their sole carbon source, and 21% of the strains exhibited maximum growth (+++, Figure 1).
Figure 2: Pictures of the qualitative scale of growth on petrolatum as the sole carbon source. The qualitative scale is based on the growth difference between the treated samples and BH control. It ranges from the absence of growth difference (0), to low (+), medium (++), and high (+++) levels of growth difference. Please click here to view a larger version of this figure.
Used engine oil is a complex mixture of hydrocarbons, engine additives, and metals. It was used in the growth tests to evaluate the fungal ability to utilize a complex and toxic hydrocarbon blend as a carbon source. Like the other growth tests, growth was evaluated by comparing the fungal growth in plates with the engine oil with growth in the control plates containing just BHA (Figure 3). The growth results confirmed the efficacy of the selective media (Section 1 of protocol). Indeed, almost 90% of the isolated fungal strains were able to grow on used engine oil, with 39% showing maximum growth (+++, Figure 1).
Figure 3: Pictures of the qualitative scale of growth on used engine oil as the sole carbon source. The qualitative scale is based on the growth difference between the treated samples and BHA control. It ranges from absence of growth difference (0), to low (+), medium (++), and high (+++) levels of growth difference. Please click here to view a larger version of this figure.
Growth tests on plastic powders were used to select the fungal strains that could use specific plastic polymers as their primary carbon source; hence, these strains have a high potential for plastic bioremediation. The growth in multiwell plates was observed by stereomicroscope and evaluated using a qualitative scale, ranging from the absence of growth (0) to high production of hyphae (+++). PUR was the plastic powder that had the highest percentage of fungal strains showing growth (92%), with 31% of strains showing maximum growth (+++). Approximately 70% of the strains isolated from humic acid and lignocellulose media grew on PS powder, while almost 60%-65% of them were able to use HDPE, PVC, and PET as their sole carbon source. Therefore, a large percentage of fungi were able to grow on the different plastic powders in multiwell plates, but a very low number of them showed high colonization of plastics. Indeed, only 10% of the strains were able to thrive on PET and HDPE, and 13% were able to thrive on PS. The only plastic where high fungal growth was regularly observed was PUR, on which approximately 30% of fungal strains managed to grow very abundantly.
Qualitative enzymatic tests were performed to detect fungal strains that can produce enzymes involved in the biodegradation processes of recalcitrant substances. Peroxidases and laccases are ligninolytic enzymes whose production is indicated by a brownish halo on the underside of the medium in gallic acid and guaiacol tests (Step 3.1. and Step 3.2. of the protocol). In both tests, the increase in the intensity of the color indicated a higher production of these enzymes (Figure 4).
Figure 4: Pictures of the qualitative scale of qualitative ligninolytic enzymes. The qualitative scale is based on the production of a red/brownish halo around the colony. 0 = no activity, + = some activity, ++ = high activity, +++ very high activity. Please click here to view a larger version of this figure.
Approximately 60% of the fungi tested were able to produce ligninolytic enzymes, indicating the success of the selective media. Moreover, 12% of the isolated fungal strains produced an intense dark halo around the colony (+++) in gallic acid medium, with 17% doing so in guaiacol medium (laccases), indicating high fungal secretion of these enzymes (Figure 5).
Figure 5: Rate of enzyme activity by fungal strains during qualitative enzymatic tests. The percentage of fungal strains able to produce ligninolytic enzymes, laccases, proteases, and esterases with different levels of success (0 = no activity, + = activity, ++ = high activity, +++ very high activity) during their corresponding qualitative enzymatic tests. Please click here to view a larger version of this figure.
Another screening test used to analyze fungal activity was the proteases test, which was based on the difference in fungal growth between YES medium vials and control vials with BH. However, approximately 70% of the fungal strains in the YES medium showed similar growth to controls, or even decreased growth. No fungal strain reached the maximum level of difference (+++). These results suggest that this test may be unsuitable for screening for protease activity.
Finally, esterases activity was evaluated by the formation of a white precipitate in Tween 80 medium (Figure 5). Almost 60% of the selected fungal strains showed esterase activity, and 13% displayed a high formation of the precipitate (+++), suggesting that the strains in the 13% should be selected to scrutinize their biodegradation potential (Figure 5).
Regarding all the growth and enzymatic tests, the most successful strains (i.e., those that obtained +++ in at least one test) were the most interesting. These fungal strains were able to efficiently use recalcitrant substances as their sole carbon source, or they produced a high level of enzymes involved in the biodegradation of recalcitrant substances. Indeed, 39 of the 115 fungal strains tested were able to thrive (+++) on hydrocarbon sources (petrolatum or used engine oil), and 58 had high growth on at least one plastic polymer. Only 32 showed very high enzymatic activity (+++) in at least one of the enzymatic tests performed. These results show the high efficacy of all the reported screening tests, apart from the proteases test, which did not select any high-performing strains.
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 peroxidases31. These enzymes can degrade lignocellulose and humic acids, but also many different xenobiotics, including hydrocarbons, aromatic compounds, and chlorinated organic compounds32.
The described methods produce results that are easy to interpret and are low-cost to carry out. These characteristics allow them to be used by non-experts. However, certain aspects, such as sterility and morphological identification, require special attention. For example, the 30 min agitation of the soil suspension (Step 1.3.4.) is necessary so that the fungal spores and propagules are freed from the soil microstructure and can be suspended in the solution. In this way, by plating dilutions of this soil suspension, it is possible to isolate the maximum number of fungal strains. Macro and microscopic morphological identification of fungal strains are important in order to differentiate between strains and avoid isolating the same fungal strain multiple times. Moreover, sterility and avoidance of microbial contaminations are crucial because the presence of other active fungal strains could misrepresent the activity of the studied colonies.
After the selective tests, an array of growth tests were performed on different recalcitrant substances, such as petrolatum, used engine oil, and different plastic polymers. This stage can be customized according to the recalcitrant substances of interest. The important step is to add the selected substance to BH or BHA as the sole carbon source and to check the fungal growth against a control without the added recalcitrant substance.
The aim of the growth tests on plastics and hydrocarbons was to assess whether the fungus could use these substances as its sole carbon source by qualitatively observing its growth on the material. Unfortunately, due to the extreme recalcitrance of these materials, it is very difficult to quantify the actual decrease in the weight of the substrate and the increase in the weight of the fungal biomass, especially after a relatively short amount of time. If a fungal strain performs very well in these screening growth tests, further studies should be undertaken to quantify the degradation of the recalcitrant substance.
The used enzymatic tests (Section 3 of the protocol) were chosen because they proved to be efficient and fast to perform, but they produce only qualitative results. They highlight the degradation potential of a fungal strain, but they cannot precisely quantify it. If a strain of particular interest is isolated, further tests, such as spectrophotometric quantitative enzyme essays, could be carried out to describe the metabolic activity more precisely. The only enzymatic test that showed suboptimal efficacy was the qualitative proteases test, with a low number of strains able to grow on the YES medium. Indeed, the high percentage of strains able to grow on PUR suggests that the fungi produced proteases involved in breaking the amide and urethane bonds, with esterase targeting the ester bonds40. The almost complete absence of protease-producing fungi suggests there may have been a problem in the method used. Other tests could be adopted for protease activity, such as casein41, skimmed milk42, or milk powder43 agar plate tests. The gallic acid test proved to be very useful for detecting the production of ligninolytic enzymes, but, unfortunately, it was not very specific and could not distinguish the type of ligninolytic enzyme produced (either peroxidases or laccases).
The results of this work suggest that the use of lignocellulose or humic acids as selective media allows fungal strains with bioremediation potential to be isolated from the rich soil biodiversity. Indeed, more than 70% of the fungal strains isolated by the selective methods were able to grow on petrolatum or used engine oil, and 60% were able to grow on different plastic polymers as the sole carbon source. Moreover, the qualitative enzymatic tests described the metabolic activity of these fungi linked to biodegradation processes. The selection of fungal strains showing +++ activity in one or more tests is recommended because it increases the chances of finding a fungal strain that is very active in bioremediation. Tests using gas mass-chromatography (GC-MS) on the recalcitrant substances after prolonged contact with the fungal strains selected with our screening tests have shown actual biodegradation of the materials9,44. The use of these simple screening tests in different ecosystems will allow the investigation of fungal biodiversity in different soils. Searching for fungi that can degrade recalcitrant materials will increase the number of identified strains that have the potential for bioremediation and help tackle the growing problem of environmental pollution.
The authors have nothing to disclose.
We acknowledge Scuola di Alta Formazione Dottorale (SAFD) of the University of Pavia and Professor Solveig Tosi for providing the opportunity for this work.
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 |