1. Introduction
Mycoremediation as a form of bioremediation may be an effective, eco-friendly technique for decontamination of polluted environmental matrices because of its simplicity and highly efficient implementation process [
1,
2,
3,
4,
5,
6,
7]. It is also one of the least costly forms of remediation, and both micromycetes and macromycetes may be used [
8,
9]. Fungi-mediated remediation as a cost-effective method may use mycelium to effectively secrete extracellular enzymes, finally transforming organic pollutants into non-toxic compounds (bioaugmentation) or accumulating toxic elements [
10,
11,
12].
There are numerous literature data about biodegradation, bioconversion, or biosorption for the degradation of common pollutants using different mushroom species [
13,
14]. Biodegradation of polycyclic aromatic hydrocarbons by
Phanerochaete chrysosporium,
Trametes versicolor,
Pleurotus ostreatus, and
P. eryngii; pesticides and herbicides by
Botryosphaeria laricina,
Aspergillus glaucus,
T. pavonia,
Penicillium spiculisporus, and
P. verruculosum; antibiotics and pharmaceuticals by
Leptospaherulina sp,
Irpex lacteus,
Lentinula edodes,
Mucor hiemalis, and
Phanerochaete chrysosporium were reviewed by Akhtar and Mannan [
9]. Effective biosorption of heavy metals (aluminium (Al), chromium (Cr), cobalt (Co), copper (Cu), lead (Pb), iron (Fe), nickel (Ni), manganese (Mn), and zinc (Zn)) with the use of spent
Agaricus bisporus from production has also been described in the literature [
4,
15,
16]. In terms of heavy metals, mycoremediation by easily cultivable, fast-growing, and highly accumulating white rot fungi
P. sajor-caju,
A. bitorquis, and
Ganoderma lucidum with a potential for Cr, Cu, and Pb remediation was described by Hanif and Bhatti [
17]. This method may also use macromycetes to accumulate toxic elements in their biomass [
18]. An example of such a species is
Pleurotus spp., which is known to effectively accumulate selected trace elements in whole fruit bodies [
19]. This method limits the initial toxicity of elements after their accumulation without the risk of the production of toxic metabolites, which are usually present in bioremediation with microbes [
20].
It should be remembered that despite many advantages, biological methods of environmental remediation also have limiting factors. In the review of Boopathy [
20], various factors that limit the use of bioremediation technologies were summarized. Some information can be found in the literature on the critical aspects and limitations of the use of mycoremediation. They should not be forgotten and strategies to overcome them are necessary [
9]. One of the examples of limiting factors is the reduced bioavailability of pollutants. Puglisi et al. [
21] observed that some fungi overcome this limitation by the production of unique proteins (hydrophobins), due to their ability to dissolve hydrophobic molecules into aqueous media. For an effective process, optimal conditions should be present (temperature: 10–28 °C, pH: about 6.5, humidity: 86–90%, and CO
2 level: 15–20%) [
22,
23]. In the case of the in situ process, maintaining the above-mentioned conditions can be difficult, which is another limitation. Rubichaud et al. [
24] confirmed that cold environmental conditions can impede the activity of various fungal enzymes necessary to degrade toxic pollutants. Therefore, the selection of suitable macrofungi for the particular substrate is essential. A high rate of element accumulation combined with a more frequent harvest cycle is a clear argument for using this method in practice [
25].
A separate issue is the efficiency of metal accumulation by fruit bodies related to their concentration in naturally and artificially polluted soil [
26,
27]. Sithole et al. [
28] studied accumulation in mushrooms growing around three mining areas and reported that heavy metal contents can be significantly different. This confirms that both element concentration and soil chemistry influence the bioavailability of metals and their contents in fruit bodies. It seems that the enrichment of samples may be diverse, which is finally related to the potential toxicity of mushrooms and differing contents of essential inorganic elements [
29].
Among the many industrial activities with negative environmental effects, the production of hazardous wastes poses serious environmental and social problems around the world. One group of such wastes is metal processing tailings. Flotation is a common mineral processing method used to upgrade copper sulfide ores where copper mineral particles are concentrated in froth, and associated gangue minerals are separated as tailings [
30,
31,
32,
33]. According to Zhai et al. [
34], 60 million tons of Cu slag are generated annually worldwide during flotation and cause irreversible water and soil pollution. Finding an environmentally friendly remediation technology is crucial. There is potential for transforming tailing wastes into valuable products due to their considerable concentrations of many critical metals/metalloids. The recovery of elements and the use of the mineral residues in high- and low-value products can be very profitable from an industrial point of view (for producers of these pollutants and companies experienced in these methods). The amounts of generated wastes are so significant that a combination of several different approaches (reduce, reprocess, upcycle, downcycle) is needed [
35]. One of the stages may be effective mycoremediation.
Agaricus bisporus is the most important commercially cultivated mushroom, contributing approximately 40–45% to world mushroom production [
36,
37]. Since it is so commonly cultivated, it would seem to be ideal for mycoremediation purposes. There are more and more reports of such use in the literature. Kryczyk et al. [
38] presented in vitro cultures of
A. bisporus demonstrating their remediation capacity for Cd and Pb from a supplemented medium. Kumar et al. [
39] described an integrated approach for sustainable management of industrial wastewater and agricultural residues in
A. bisporus production while minimizing the risks associated with their disposal. Ugya and Imam [
40] described the effectiveness of
A. bisporus in the remediation of refinery wastewater. The species showed high reduction efficiency for sulphate, phosphate, nitrate, alkalinity, electrical conductivity (EC), biological and chemical oxygen demands (BOD and COD), and heavy metals (Ag, Hg, Mn, Pb, and Zn).
In view of the above, the aim of this study was to evaluate the mycoremediation ability of A. bisporus. The content of 51 elements in the mushroom fruit bodies growing on compost mixed with highly polluted flotation tailings in different quantities (1, 5, 10, 15, and 20% addition) enriched with flotation tailing was determined. The biomass of the collected fruit bodies was also assessed to estimate how polluted materials affect mushroom development. An experiment was performed to show the mineral composition of fruit bodies after the mycoremediation process.
2. Materials and Methods
2.1. Experimental Design
The substrate used for the experimental cultivation came from the commercial production of compost for mushroom growing (WRONA Company, Pszczyna, Poland). The compost was based on wheat straw, chicken manure, and gypsum and was prepared using a conventional method characteristic for phase II compost (fermentation and pasteurization). The compost was mixed with flotation tailings in quantities of 0 (control), 1 (FT
1), 5 (FT
5), 10 (FT
10), 15 (FT
15,), and 20% (FT
20) by weight of the compost. Granulation [%] of flotation tailings was 11, 88, and 1 for clay, silt and sand, respectively. The pH of this component was 7.19, with an EC of 6.98 dS m
−1. The characteristics of element concentration in flotation tailings are described in
Table 1.
The compost was inoculated with commercial
A. bisporus mycelium in 5% of the compost weight. The strain EuroMycel 58 was used. The mixtures were placed in plastic containers (15 × 18 × 14 cm) at 1 kg per container for each particular experimental system (
Figure 1). The compost layer thickness was 8 cm. Eight containers for each treatment were prepared. Incubation was carried out in a growing chamber at a temperature of 24–25 °C and 85–90% of humidity. Once the compost was completely overgrown with mycelium, a 3.5 cm layer of casing soil was applied to the substrate surface. The moisture content of the casing soil was 75%. The casing soil came from the Wokas Company (Łosice, Poland) and was prepared based on sphagnum peat moss with chalk (pH 6.7). Incubation was continued until the mycelium overgrew the casing soil. When it appeared on the casing soil surface, the air temperature was lowered to 17–18 °C. The casing soil was watered to maintain constant moisture content. The growing chamber was aerated to keep CO
2 concentration below 1500 ppm. The fruit bodies were collected fully developed but still completely closed. Individuals from the control and treatment FT
1 were harvested simultaneously, whereas from the other experimental systems (FT
5, FT
10, FT
15, and FT
20) 2, 3, 4, and 5 days later, respectively. The delay in the harvesting of fruiting bodies was due to the fact that increasing the amount of sludge (waste) addition delayed their setting.
Just two flushes of fruit bodies were produced and harvested. The interval between consecutive harvests was 8 days in each case. The yield included whole fruit bodies collected from 1 container, and the determined yield was a mean value calculated based on 8 containers belonging to the same treatment. None of the fruit bodies showed any signs of distortion or discoloration (
Figure 1). The experiment was performed in May 2020.
2.2. Analytical Procedure
All collected fruit bodies were carefully washed with distilled water from a Milli-Q Academic System (non-TOC) (Merck Millipore, Darmstadt, Germany) to remove substrate particles, and subsequently dried with paper towels and weighed. The mushrooms were then dried at 40 ± 1 °C to a constant weight in an electric oven (SLW 53 STD, Pol-Eko, Poland) and ground in a laboratory Cutting Boll Mill PM 200 (Retsch, Haan, Germany).
An accurately weighed 0.200–0.500 (±0.001) g of a sample was digested with 10 mL of concentrated nitric acid (HNO3; 65%; Sigma-Aldrich, Darmstadt, Germany) in closed Teflon containers in a microwave digestion system (Mars 6 Xpress, CEM, Matthews, NC, USA). Finally, the samples were filtered (Qualitative Filter Papers Whatman) and diluted to a volume of 15.0 mL with demineralized water (Direct-Q system, Millipore, USA). The inductively coupled plasma mass spectrometry system PlasmaQuant MS Q (Analytik Jena, Germany) was used to determine the following conditions: plasma gas flow 9.0 L min−1, nebulizer gas flow 1.05 L min−1, auxiliary gas flow 1.5 L min−1, radio frequency (RF) power 1.35 kW. The interferences were reduced using the integrated collision reaction cell (iCRC) working sequentially in three modes: with helium (He) as the collision gas, hydrogen (H) as the reaction gas, and without gas addition. The uncertainty for the analytical procedure, including sample preparation, was at the level of 20%. The detection limits were determined at the level of 0.001–0.010 mg kg−1 dry weight (DW) for all elements determined (3 times the standard deviation of the blank analysis (n = 10)). The accuracy was checked by analysis of the reference materials CRM 2709—soil; CRM S-1—loess soil; CRM 667—estuarine sediments; CRM 405—estuarine sediments; CRM NCSDC (73349)—bush branches and leaves, and the recovery (80–120%) was acceptable for most of the elements determined. For uncertified elements, recovery was defined using the standard addition method.
All the determined major and trace elements were divided into 6 groups, according to Kalač (2019):
- (a)
Major essential elements (MEEs): calcium (Ca), potassium (K), magnesium (Mg) and sodium (Na);
- (b)
Essential trace elements (ETEs): boron (B), cobalt (Co), copper (Cu), chromium (Cr), iron (Fe), manganese (Mn), nickel (Ni), selenium (Se) and zinc (Zn);
- (c)
Trace elements with detrimental health effects (TEWDHE): silver (Ag), arsenic (As), barium (Ba), cadmium (Cd), lead (Pb) and thallium (Tl);
- (d)
Rare earth elements (REEs): cerium (Ce), dysprosium (Dy), erbium (Er), europium (Eu), gadolinium (Gd), holmium (Ho), lanthanum (La), lutetium (Lu), neodymium (Nd), praseodymium (Pr), scandium (Sc), samarium (Sm), terbium (Tb), thulium (Tm), yttrium (Y) and ytterbium (Yb);
- (e)
Platinum group elements (PGEs): iridium (Ir), palladium (Pd), platinum (Pt), rhodium (Rh), ruthenium (Ru);
- (f)
Nutritionally non-essential elements (NNEs): aluminium (Al), gold (Au), bismuth (Bi), gallium (Ga), germanium (Ge), indium (In), lithium (Li), rhenium (Re), antimony (Sb), strontium (Sr), and tellurium (Te).
2.3. Statistical Analysis and Calculation
All statistical analyses were performed using the Agricole package (R). The analyses were performed in accordance with the procedure implemented in the R 3.6.1 environment [
41]. To compare the content of determined elements in compost with different proportions of flotation tailings, a one-way ANOVA with Tukey’s HSD (statistically significant difference) post hoc: test was used. The same analysis was performed to compare the content of elements in fruit bodies growing in particular experimental treatments, separately for both flushes. This analysis used the Stat and Agricole package. A heatmap with a cluster analysis (implemented in the package Heatmaply) was performed to visualize multidimensional data separately for particular groups of elements and all elements jointly. An empirical normalization transformation brings data to the 0 to 1 scale and it allows comparison of variables of different scales, but it also keeps the shape of the distribution. A dendrogram was computed and reordered based on row and columns means. Additionally, to compare Ca, K, Mg, and Na contents together in compost with flotation tailings and fruit bodies from both flushes produced from particular experimental treatments, the rank-sum test was performed [
42].
To estimate the efficiency of the element accumulation by mushrooms growing under particular treatments, the bioaccumulation factor (BAF) was calculated as a ratio of metal content in the whole fruit body dry matter to its concentration in substrate dry matter.
4. Discussion
Recently, there have been more and more reports on the possibility of using
A. bisporus in remediation. In our experiment, five different quantities of flotation tailings were used to study the mycoremediation potential of this species. In the first flushes, a reduction in the yield was confirmed with the addition of flotation tailings. The literature confirms that supplementation of the substrate with metals/metalloids may reduce the growth dynamics of this species. Rzymski et al. [
43] showed supplementation with Cu, Se, and Zn resulted in the biomass of fruiting bodies decreasing significantly at higher element addition (0.8 mM). Also, the addition of higher Hg concentrations (0.4 and 0.5 mM) to the growing medium reduced the growth of
A. bisporus biomass [
44].
The substrate in our experiment was richer in MEEs than mixtures of substrate and flotation tailings. The same tendency was confirmed in fruit bodies growing in experimental systems with their use. A different trend was observed concerning other groups of elements. The control substrate (compost) contained a significantly lower content of other elements than the substrate with flotation tailings. Because of the ability of
A. bisporus to accumulate selected elements, the composition of waste affects the composition of its fruit (different amounts of waste addition resulting in different levels of elements in the substrate). Differences in the accumulation of heavy metals (Cd, Cr, and Pb) in
A. bisporus fruiting bodies depending on the concentration of these elements in the growing medium were confirmed by Zhou et al. [
45]. Nagy et al. [
46] also confirmed that the maximum removal efficiencies from monocomponent aqueous solutions by
A. bisporus for Cd and Zn took place at the highest concentrations of the substrate. The ability of
A. bisporus to effectively accumulate selected elements shown in our study, is also well demonstrated by the results of our previous studies on supplementation during the cultivation of these mushrooms. Effective uptake of Cu, Se, and Zn from the enriched medium was confirmed by Rzymski et al. [
43]. Supplementation with 0.6 mmol L
−1 of Cu, Se, and Zn resulted in an over 3-fold, 2.5-fold, and 10-fold increase in their concentrations in fruiting bodies, respectively, whereas Rzymski et al. [
44] demonstrated that Hg uptake increased in a concentration-dependent manner and exceeded 116 mg kg
−1 in
A. bisporus caps after 0.5 mM was added to the substrate.
BAF values may measure the mycoremediation efficiency of different mushroom species growing in polluted substrates and provide direct information about the potential of particular mushroom species to accumulate elements. This is crucial because this process has numerous dependent factors (environmental and genetic) [
47], and is also the case for mushroom cultivation using different substrates as previously described [
48]. In this study, effective accumulation was observed for selected elements only, which reflected the chosen additions of flotation tailings added to the compost. It suggests that
A. bisporus application can be limited and/or used for the accumulation of selective elements only [
13,
49].
In general, mushroom fruit bodies collected after the mycoremediation process are waste that can be a substrate for the recovery of elements in the case of very high contents of especially precious elements [
50]. The risk of contaminated food is genuine [
51]. The problem of assessing the quality of fruit bodies (especially for significant and toxic trace elements) that could be consumed has been closely associated with a lack of appropriate legal regulations for many years [
52,
53]. However, even in countries where legislation exists, the regulations are limited to specific and usually toxic trace elements only [
54,
55]. The analysis and the further possibility of using the “product” from the mycoremediation of post-industrial wastes seems to be unlikely. Simultaneously, the risk of consuming contaminated fruit bodies is high, as may be shown in the study of Pająk et al. [
56], who collected 10 mushroom species from polluted forest ecosystems.
5. Conclusions
The possibility of using mushroom fruiting bodies to decontaminate contaminated substrates effectively is an essential aspect of bioremediation due to their ability to accumulate elements effectively. The A. bisporus strain tested in this study effectively accumulated selected elements (Ag, Au, B, Cu, Ga, Ge, In, Ir, K, Mg, Pd, Pt, Re, Rh, Ru, Sb, Se, and Te), as evidenced by values of BAF > 1. Although this efficiency was not spectacular, to be able to recover elements in pure form, fundamentally enriched the fruiting bodies. The results of these studies indicate the potential for using A. bisporus fruiting bodies after the mycoremediation process in industry, although this must be preceded by larger-scale tests. This application seems to be the most favorable for media contaminated with selected elements, the absorption of which by fruiting bodies is the most efficient. Our study confirmed that the key to mycoremediation is determining the right fungal species to target a specific pollutant. However, due to the particularly effective accumulation of As and Cd, the post-flotation sediment subjected to this process should contain the lowest possible concentrations of both of these elements. Further research is necessary to determine the long-term potency of such a method.