Introduction

Industrial development, overpopulation, and the development of human civilization have drawn attention toward the harmful environmental pollution concerns including soil, water and air contaminants. Out of these, heavy metal contaminants including Cr, Cd, Pb and Ni are released in the environment through industrial wastewaters that often increase toxicity level, thus, may cause various adverse effects to the human health and surroundings (Proshad et al. 2020). A variety of chemical, physical and biological systems for removal of these metal contaminants have already been studied but with limited efficiency. However, use of microbes as biosorbents can be an environmentally friendly and economic alternatives to remove of heavy metal contaminants (Tarekegn et al. 2020).

Various microorganisms such as yeast (Acosta et al. 2018), algae (Bangaraiah et al. 2021), bacteria (Mwandira et al. 2020) and fungi (Sharma et al. 2020) have been effectively applied to reduce the heavy metal contents from aqueous solutions. Recently, different versatile low-cost sorbents of microbial origin for removing metal ions have also been suggested and being investigated to improve the performance and applications. These include different biomaterial based biosorbents as efficient alternatives to synthetic materials for contaminants removal from wastewaters (Elgarahy et al. 2021a), applications of environmentally friendly nanomaterials in wastewater remediation (Elgarahy et al. 2021b), immobilizing microbial biomass on suitable carriers for heavy metals removal (Velkova et al. 2018), etc.

Among different microorganisms, fungi are environmentally friendly and can be grown easily without using the expensive growth medium. Accumulation of heavy metal ions by microbes might be manifested by several functional groups primarily including amine, carboxyl, hydroxyl, sulfhydryl, and phosphonate. It also involves some active components, e.g., chitin and cellulose derivatives, different polysaccharides, and some heterogeneous components inside (metallothionein and polyphosphate) and outside (extracellular polymeric substances-EPS) the cell (Yin et al. 2019). Apart from this, the fungal cell wall has been found to have high content of proteins and polysaccharides and is rich in numerous of functional groups (–OH, –NH, –COOH, etc.) that can bind with heavy metal ion (Lei et al. 2018; Li et al. 2019) for higher biosorption activity. Earlier, both dead and living fungal cells have been reported to be capable of adsorbing toxic metals (Wang and Chen 2009). The heavy metal ions might bind to cell wall of dead cells first (Shoaib et al. 2013) and then transform these metal ions into less toxic elements by live cells (Xu et al. 2015) through functional groups and different biosorption mechanisms (protein binding and efflux pumping), respectively.

Earlier, removal of heavy metal by various fungal species, e.g., Aspergillus niger (Okoya et al. 2020), Trichoderma brevicompactum (Zhang et al. 2020), Aspergillus ustus and Penicillium chrysogenum (Alothman et al. 2020) have been studied. Apart from these, an eco-friendly, cost-effective, and viable method is the use of white rot fungi, which may be grown on easily available plant materials (lignocellulosic biomass) (Sharma et al. 2020). Some white rot fungal species have also been used to decompose hazardous compounds (DNTs) (Kist et al. 2020), mineralize different organic compounds (bisphenol A, bisphenol S and nonylphenol) (Grelska et al. 2020) and decrease the adverse effects of various metals ions (Enayatizamir et al. 2020) present in the wastewater, which can be due to their unique ability to produce different lignocellulolytic enzymes, organic acids, and free radical scavenging capability (Rajhans et al. 2020). Moreover, preconditioning to a particular pollutant may not be required and could tolerate high concentrations of pollutants (Yang et al. 2017). Earlier, wood degrading fungi Phanerochaete chrysosporium, Phlebia brevispora and Phlebia floridensis have been efficiently used for bioremediation of chemically different textile dyes (Korcan et al. 2013). P. chrysosporium (Lu et al. 2020) and P. brevispora (Sharma et al., 2020) have also been used as biological biosorbents to remove heavy metals from aqueous solution. However, the metal removal efficacy of white rot fungus P. floridensis has not been demonstrated. Hence, during the present study, the potential of wood degrading, nonpathogenic, white rot fungi was evaluated, which could further be exploited for efficient wastewater treatment. Fast growth of these fungi on abundantly available lignocellulosic biomass and efficient degradation of pollutant makes these organisms suitable for this work. Thus, in the present study, P. chrysosporium, P. brevispora and P. floridensis have been explored for their heavy metal removal efficiency from synthetic metal solution and industrial wastewaters, which could provide positive insight in the development of basic concepts related to fungal accumulation of metals long with practical applications. Further, as compared to available chemical and physical methods, the proposed system seems to be eco-friendly, economic and self-sustainable.

Materials and methods

Microorganism and characterization of industrial wastewater (IWW)

White rot fungi P. brevispora (HHB-7024), P. chrysosporium (BKM-F-1767) and P. floridensis (HHB-7157) were obtained from the Center for Forest Mycology Research, USDA Forest Products Laboratory, Madison, Wisconsin. A medium containing peptone, yeast extract and dextrose was used to grow and maintain P. chrysosporium, P. brevispora and P. floridensis. For longer preservation, the cultures were maintained at 4 °C under mineral oil.

IWW was collected from local textile and printing industries and analyzed to detect the concentrations of different heavy metal ions present. For digestion of IWW, a conical flask containing 100 ml of IWW was kept for 2–3 h after addition of nitric acid and perchloric acid in the ratio 3:1. The digested sample was diluted with 50 ml of deionized water (Millipore water) through Whatman® filter paper No. 1 (pore size of ~ 11 μm). The aliquots obtained was kept at 4 °C for further analyses.

Effect of metals on fungal biomass

To evaluate the effect of metal solution of fungal growth, metal ion solution blend was prepared. Metal stock solution (1 M) containing Pb, Cd, Ni and Cr was prepared using analytical grade salts of lead acetate [Pb (CH3COO)2·3H2O], potassium dichromate (K2Cr2O7), cadmium carbonate (CdCO3) and nickel chloride (NiCl2·6H2O) in sterilized deionized water. The prepared synthetic metal ion blend was added to test tubes having 15 ml of 0.5% (w/v) malt extract at different concentrations, i.e., 5, 10 and 20 µmol/L.

Similarly, another set of tubes containing 15 ml industrial wastewater amended with 0.5% (w/v) malt extract was prepared, and then, all the tubes were sterilized in an autoclave. One mycelial disc of 4 days old culture of P. chrysosporium, P. brevispora or P. floridensis was inoculated aseptically into test tubes containing metal ions blend and IWW along with suitable controls. These tubes were incubated for 7 days at 30 °C along with suitable controls. The contents from each tube were aseptically filtered through Whatman® filter paper No. 1 and the residual fungal biomass was dried at 65–70 °C for 48 h and the filtrate was kept at 4 °C for further analyses. Dried fungal biomass was weighed using a digital weighing balance.

Atomic absorption spectroscopy (AAS)

Residual metal ion concentration from the filtered aliquots (synthetic metal ions blend and IWW samples) was analyzed through atomic absorption spectrophotometer (AA-7000F) using an air–acetylene flame. The results obtained were compared with known standard solution of each metal. The metal removal efficiency all the fungi viz. P. chrysosporium, P. brevispora and P. floridensis at different metal concentrations and IWW was obtained by using the following expression:

$$ RE \left( \% \right) = \frac{{C_{i} - C_{f} }}{{C_{i} }} \times 100 $$

where RE (%) represents metal removal efficiency; \({C}_{i}\) is initial and \({C}_{f}\) is final concentration of the metal.

Scanning electron microscopy (SEM)

Effect of metals and IWW contaminants on fungal mycelial surface morphology was observed under SEM at 20.0 kV and 1000 × magnification. Both untreated (control) and treated fungal biomass was mounted on a carbon tape followed by a gold coating under vacuum to enhance the electron conduction during the microscopic imaging. Further, the presence of metals on the mycelial surface was analyzed using and energy-dispersive X-ray (EDX).

Replication and statistical analyses

All the experiments were conducted as triplicate and repeated. The mean values along with standard deviation were represented and ANOVA was used to determine statistically significant difference.

Result and discussion

Effect of metals on fungal growth

Microorganisms can adapt and withstand toxic compounds by utilizing these components as a source of energy for their own growth. Various trace metal ions hold an important position in the nutrition of fungi because of their indispensability for their growth, however, a little higher concentration than the required dose may cause stress and thus become toxic. In the present study, Pb, Cd, Ni and Cr were targeted, which do not hold any known nutritional potential. Despite this, lower concentration of these metals demonstrated a favorable effect on the growth of P. chrysosporium, P. brevispora and P. floridensis. The fungal biomass increased gradually with an increase in the concentrations of metal ion blend (upto 20 μM/L). Maximum mycelial mass (0.65 g/L) was produced by P. floridensis followed by P. chrysosporium (0.46 g/L) and P. brevispora (0.34 g/L) under the stress of synthetic heavy metal blend, while minimum mycelial mass 0.18 g/L was produced by P. brevispora in the absence of metals (Fig. 1). However, P. brevispora has previously shown significant growth of biomass under the presence of individual metal ions (Cd, Ni and Pb) at concentrations ranging from 1 to 100 μM/L (Sharma et al. 2020). Earlier, colony diameter of P. chrysosporium increased with the increase in Pb (25−100 mg/L) and Cd (2–32 mg/L) ion concentration while showed significant growth under singe Cd ion stress and combined stress of Cd and Pb (Zhao et al. 2016). Nickel concentration of 400 mg/L, 500 mg/L, 1000 mg/L and 1500 mg/L were found to be a maximum tolerance level for the growth of P. chrysosporium, A. foetidus, A. niger and Penicillium simplicissimum, respectively (Falih et al. 1997; Anahid et al. 2011). It has been reported that Cr (VI) could be absorbed by microbes, especially fungi, on their cell wall (Gadd 1994). This uptake of chromium by fungal cell wall might result in reduction of Cr6+ to r3+ as it is less toxic and less harmful to the cell. Various fungal species have been reported to be resistant to Cr+6 concentration 100 mg/L (Smily et al. 2017). Earlier, Penicillium chrysogenum and Trichoderma viride have been reported as capable of tolerating 2 mM concentration of Cr+6. In the present study, P. floridensis, P. brevispora and P. chrysosporium showed significantly higher growth under IWW stress, when compared to heavy metal blend. This might be due to the availability of organic carbon and nitrogen in wastewater which could be utilized by fungi as energy sources for their growth (Verlicchi et al. 2010). Earlier, white rot fungus has been reported to transform micropollutants co-metabolically which could be the carbon source for enhanced growth (Wen et al. 2011). Simultaneously, presence of some essential heavy metals, e.g., Cu, Co, Mn, and Zn also improved the growth of white rot fungi. Enhancement in the growth of a white rot fungus, Pleurotus ostreatus was observed when 5 mM CuSO4 was amended in the growth medium (Bhattacharya et al. 2014) and 1–10 mM MnSO4 (Baldrian et al. 2005). It was observed that all the tested fungi P. chrysosporium, P. brevispora and P. floridensis showed growth and some level of tolerance toward the heavy metal stress and can be exploited for mycoremediation of heavy metals from the contaminated water.

Fig. 1
figure 1

Growth of Phlebia floridensis, Phlebia brevispora and Phanerochaete chrysosporium at different heavy metal concentrations after 7 days of incubation at 30 °C

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Metal removal efficiency of P. chrysosporium, P. brevispora and P. floridensis

IWW sample showed the presence of Pb+2 (0.057 µg/ml), Cd+2 (0.715 µg/ml) and Ni+2 (0.039 µg/ml) metal ions; however, the sample showed absence of chromium contamination. Heavy metal ion removal efficiency of P. chrysosporium, P. brevispora and P. floridensis at different concentration of metal ion blend and IWW sample ranged from 84.51–99.71%, 12.12–99.85%and 24.24–99.77%, respectively, (Table 1). Higher metal removal efficiency was observed with increasing concentration of Pb+2, Cd+2, Ni+2 and Cr+6 (metal ion blend) being maximum as 20.6 mg/L, 34.4 mg/L, 4.74 mg/L and 11.34 mg/L, respectively, suggesting prominent removal of different metal ions by tested white rot fungi. P. chrysosporium showed maximum removal of metals from the IWW (97.86%) followed by P. floridensis (72.79%) and P. brevispora (68.96%). This metal removal efficiency depends upon the high degree of interaction of metal ions with various functional groups including OH–, –PO3–, –SO2–, and –NH2, that are available on the fungal cell surface during bioaccumulation (Singha et al. 2020). All the fungal strains showed a significant removal of Cd+2 and Ni+2 from IWW with greater than 94.57% efficiency. Maximum removal of Pb+2 from the IWW sample was 98.10% by P. chrysosporium, followed by 12.12% and 24.24% by P. brevispora and P. floridensis, respectively. Earlier, P. chrysosporium has been reported to show the ability to absorb 18.1 mg/L and 4.53 mg/L of cadmium and 26.34 mg/L and 9.28 mg/L of chromium under individual and mixed ion stress, respectively (Rudakiya et al. 2018). Recently, biosorption efficiency of Cd+2 and Pb+2 by P. chrysosporium have been reported to increase with increase in bio-sorbent dosage and reach up to 98% and 96%, respectively (Lu et al. 2020). Also, P. chrysosporium accumulated 96.23% Cd+2 and 89.48% Ni+2 ions from 25 mg/L to 16 mg/L metal containing solutions, respectively (Noormohamadi et al. 2019). Recently, P. brevispora has been reported to remove 85.9%, 94.5% and 97.5% of Pb+2, 91.6%, 85.2%, 77.3% of Cd+2 and 62.9%, 66.7%, 72.7% of Ni+2 under individual ion stress at initial concentrations of 10 µM, 20 µM and 100 µM, respectively (Sharma et al. 2020). Similarly, the present results demonstrated that the white rot fungi P. chrysosporium, P. brevispora and P. floridensis could be efficiently used as biosorbents to remove metal contamination from aqueous solutions, especially, industrial wastewater.

Table 1 Removal efficiency of P. chrysosporium, P. brevispora and P. floridensis at different metal ion concentration and industrial wastewater after 7 days of incubation at 30 °C
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Effect of industrial wastewater on mycelial surface morphology

The control of P. chrysosporium, P. brevispora and P. floridensis showed the presence of intact long rod, cylindrical sheets or even ribbon shaped mycelial fibers which were extremely branched and tangled with no visible physical damages (Fig. 2 A, D, G, respectively). P. floridensis showed somewhat damaged mycelium with a few visual deformities; however, the hyphal shape was distinct and regular. On the other hand, distorted and shrunken mycelia were observed in P. brevispora and P. chrysosporium after metal ion blend and IWW treatment. This damage to the fungal mycelia could be due to uptake and accumulation of toxic metal ions which cause changes at physiological, morphological, cellular, and molecular levels. Irregular expansion of fungal mycelia was observed in P. chrysosporium (Fig. 2; I) and P. floridensis (Fig. 2; C), which might be due growth under heavy stress conditions resulting in increased area for the interaction of metal ions. Morphological changes in the fungal mycelia under heavy metal stress have been reported earlier (Liaquat et al. 2020), which might be due to the oxidation of protein and DNA molecules, changes in ultrastructure, or inhibition of antioxidant defense system in cell (Chen et al. 2014). However, the degree of damage might vary with different fungal strains depending on their ability to overcome stress conditions. SEM images of the present study also showed some changes in hyphal shape, which developed closely to form a thick mycelial mass in the present of different metals. Recently, dimorphism study on marine yeast Yarrowia lipolytica revealed changed in morphology as elongated, oval, or rounded in response to different heavy metals stress including Pb and Cd (Bankar et al. 2018).

Fig. 2
figure 2

Scanning electron microscopic images (1000 X) of fungus Phlebia floridensis (A, B, C), Phlebia brevispora (D, E, F) and Phanerochaete chrysosporium (G, H, I) being A, D and G, as control; B, E and H under metal ion stress and C, F and I under IWW stress after 7 days of incubation at 30 °C

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Trace amounts of Cd+2, Ni+2, Pb+2 and Cr+6 were observed to be adsorbed on the fungal mycelia after the treatment with IWW and metal ion blend (Fig. 3). Detectable peak of Cr+6 ion adsorbed by the cell wall of all three fungi has been observed in EDX spectra. However, no significant peaks for the metal ions (Cd+2, Ni+2 and Pb+2) were present showing an intracellular accumulation of these metals rather than binding on the cell surface. Similarly, a wood-rot fungus Schizophyllum commune demonstrated an intracellular accumulation of the metals including Cd in the hyphae. Laser scanning microscopy images suggested an intracellular localization primarily within vacuoles and vesicles. However, maximum amount of metal accumulation was observed in the apical cells along with the swelling of the hyphal tip (Traxler et al. 2021). This intracellular bioaccumulation might induce production reactive oxygen species (ROS) and thiol compounds (gluthathione-GSH and metallothionein-MT), which could result into increased membrane damage (oxidation of lipids, proteins, and DNA) and increased tolerance level (intracellular chelation of metal ions) and lower cytoplasmic toxicity (subcellular compartmentation), respectively (Sharma et al. 2020).

Fig. 3
figure 3

Metal content on fungal mycelium of Phlebia floridensis (A, B, C), Phlebia brevispora (D, E, F) and Phanerochaete chrysosporium (G, H, I), being A, D and G, as control; B, E and H under metal ion stress and C, F and I under IWW stress after 7 days of incubation at 30 °C as estimated by EDX analyses

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Conclusion

White rot fungi P. floridensis, P. brevispora and P. chrysosporium showed significant growth under synthetic metal ion stress and IWW. Deformation and irregular expansion of fungal mycelia confirmed the interaction of metal ions with surface morphology resulting in their adsorption on the mycelial surface and intracellular accumulation. However, this phenomenon was specific depending upon the type and concentrations of metal as well as fungal species. The studied fungal strains were also capable of efficiently removing the metal ions from synthetic blend and IWW. Overall, P. chrysosporium demonstrated the maximum removal of all the tested metals viz. Pb, Cd, Ni and Cr (upto 99%). On the other hand, P. floridensis and P. brevispora could not remove Pb efficiently under the experimental conditions. Thus, present study points toward effective utilization of white rot fungi in bioaccumulation of metal ions from aqueous solution and IWW.