The electrolyte generated by dissolving superalloy waste from an enterprise in Shenyang was used as a heavy metal stock solution. The electrolyte, which is a type of strongly acidic wastewater (pH = 2.6), mainly contained five heavy metal elements (Co, Cr, Mo, Re, and Ni) at concentrations of 10317, 4537, 35, 2557, and 39088 mg/L, respectively.

All five fungal strains were isolated from soil in the Yangjiazhangzi Mine in Huludao City (120°36′E, 40°53′N). Potato Dextrose Agar medium (200 g potato, 20 g glucose, 15 g agar, and 1000 mL water), Autotrophic Inorganic Salt medium [3.0 g (NH₄)₂SO₄, 0.1 g KCl, 0.5 g K₂HPO₄, 0.5 g MgSO₄⋅7H₂O, 0.02 g NaNO₃, 0.01 g CaCl₂, 0.5 g FeSO4⋅7H₂O, 15 g agar, and 1000 mL water], and Heterotrophic Inorganic Salt medium [3.0 g (NH₄)₂SO₄, 2.0 g Na₂CO₃, 0.1 g KCl, 0.5 g K₂HPO₄, 0.5 g MgSO₄⋅7H₂O, 0.01 g Ca (NO₃)₂, 0.5 g FeSO₄⋅7H₂O, 15 g agar, and 1000 mL water] were used for screening, with 10% electrolyte added as the screening condition. In a 10% electrolyte solution, the concentrations of each heavy metal, namely Co, Cr, Mo, Re, and Ni, were 1031, 453, 3.5, 255, and 3908 mg/L, respectively. These three culture media can comprehensively cover the growth requirements of different types of microorganisms, facilitating the effective and comprehensive screening of target microorganisms that can tolerate electrolytes (Talukdar et al., 2020; Henagamage et al., 2022).

In the end, five fungi grew successfully in the culture medium containing the electrolyte, indicating that they were resistant to the heavy metals in the electrolyte. Fungal DNA was extracted using urea extraction (Sun et al., 2000). PCR amplification was performed using the universal primers ITS4 and ITS5. A 1.0% (w/v) agarose gel was prepared and the PCR products were verified by gel electrophoresis using the DNA Marker from BM2000 as a control. After successful amplification, the PCR products were sent to Tsingke Biotechnology Co., Ltd. for sequencing, and the obtained ITS sequences were compared using BLAST in the NCBI database to identify the fungal strains.

The wet bodies of the five selected fungal strains were collected separately, and the fungal spores on the solid medium were inoculated into the PDA liquid medium with glass beads using an inoculation loop and incubated in a shaker (BSD-YX2200, BOXUN, China) at 28°C and 150 rpm. After 36 h of incubation, the spores were reintroduced into the PDA liquid medium at 10% inoculum and incubated at 28°C and 150 rpm. After 48 h of incubation, the fungal bodies were collected by centrifugation (5804R; Eppendorf, Germany) at 10000 rpm for 10 min and rinsed twice with sterile water. We then added 1.5 g of the wet bodies of the five selected fungal strains to 150 mL of the 10% electrolyte solution and placed in shaker (1 h, 28°C, 150 rpm) to conduct an adsorption reaction. After the adsorption reaction was complete, the concentrations of the five heavy metals in the electrolyte (Co, Cr, Mo, Re, and Ni) remaining in the supernatant of each sample were quantified after centrifugation (10000 rpm, 10 min). We found that all five heavy metals remained in the solution. Their concentrations were then quantified by ICP-AES (PlasmaQuant PQ9000, Analytik Jena AG, Germany).

The adsorption rate η and adsorption capacity q were used to evaluate the adsorption ability of the five fungal strains for heavy metals (Equations 1 and 2). The strain with the highest adsorption capacity was selected for further analysis:

In section “2.3 Screening of strains with high adsorption capacity for heavy metals in a superalloy electrolyte,” we selected the fungus with the best adsorption capacity of the five heavy metals in the electrolyte (Co, Cr, Mo, Re, Ni) as the object for further research. The initial dosage was set at 1.5 g, and the initial conditions for the biosorption process in the shaker were 25°C and 150 rpm. In 150 mL of the 10% electrolyte solution, we studied the effects of the selected fungus’ biosorption of the five heavy metals (Co, Cr, Mo, Re, and Ni) at different pH values, initial concentration, adsorption times, adsorption dosages, and adsorption temperatures: (1) To study the effect of pH (3, 4, 5, 6, 7). (2) To investigate the effect of initial electrolyte concentration, the electrolyte solution was diluted 0, 5, 10, 20, and 50-fold respectively. The pH was adjusted to the optimized conditions based on (1) above. (3) To study the effect of adsorption time (0.1, 0.5, 1, 2, 4, 8, 12, 16, 20, and 24 h), the pH and initial electrolyte concentration was adjusted to the optimized conditions based on (1) and (2) above. (4) To investigate the effect of adsorbent dosage (1.5, 2.0 2.5, 3.0, 3.5, 4.0, and 4.5 g), the pH and adsorption time were adjusted to the optimized conditions based on (1), (2), and (3) above. (5) To determine the effect of adsorption temperature (15, 20, 25, 30, 35°C), the pH, initial concentration, adsorption time, and adsorption dosage were adjusted to the optimal conditions based on the above tests, The calculation method for the adsorption effect of all samples was similar to that described in section “2.3 Screening of strains with high adsorption capacity for heavy metals in a superalloy electrolyte.”

During the biosorption of heavy metal ions, biosorption enters a state of equilibrium, which can be regarded as the equilibrium state of the metal adsorption on the adsorbent and the residual metal ions in the solution. Langmuir and Freundlich adsorption isotherms have often been used in related studies (Huang et al., 2020). We diluted the original electrolyte 2, 4, 6, 8, 10, 15, 20, 30, and 40 times. We used the fungus selected in section “2.3 Screening of strains with high adsorption capacity for heavy metals in a superalloy electrolyte” in 150 mL electrolyte solution under its optimal adsorption conditions, determined in section “2.4 Effect of different factors on the adsorption effect of five heavy metals.” The concentrations of heavy metal ions (Co, Cr, Mo, Re, and Ni) adsorbed onto the fungal cells at different electrolyte concentrations were determined. Langmuir and Freundlich equations were applied according to the approach of Tran and Chao (2018).

Kinetics studies were conducted using the selected fungus (in section “2.3 Screening of strains with high adsorption capacity for heavy metals in a superalloy electrolyte”) in 150 mL electrolyte under its optimal adsorption conditions (see section “2.4 Effect of different factors on the adsorption effect of five heavy metals”). The concentrations of heavy metal ions (Co, Cr, Mo, Re, and Ni) adsorbed onto fungal cells at 1, 5, 10, 20, 30, 60, 120, 240, 360, 480, 600, and 720 min were determined. Quasi-first-order and quasi-second-order kinetics models were applied according to Dai et al. (2020) and Rohit et al. (2021).

The wet fungal cells selected in section “2.4 Effect of different factors on the adsorption effect of five heavy metals” with the best adsorption before and after the adsorption test were used for the drying treatment. The wet cells before and after adsorption with the KBr powder were mixed, ground evenly in an agate mortar, pressed into thin slices, and scanned using a Nicolet iS 10 infrared spectrometer.

Five fungal strains were obtained through screening and named F-1, F-2, F-3, F-4, and F-5. The morphology of the fungi grown on PDA solid medium for 10 days is shown in Figure 1. The diameter of the F-1 colony was approximately 55 mm; it was dark green, the edge was black, the back was dark green, and the mycelia were tightly felt with concentric rings. The diameter of the F-2 colony was approximately 80 mm; it was flat, fluffy gray-green, and the back was light yellow. The diameter of the F-3 colony was approximately 70 mm; it was rose-gray, fluffy, and raised, and the back was light yellow. The diameter of the F-4 colony was approximately 60 mm; it was white, thick, hairy, raised with a dense structure, and the back was golden-yellow. The diameter of the F-5 colony was approximately 85 mm; it was dark green flocculent, the color darkened after maturity, and the back was dark green.

The internal transcribed spacer (ITS) sequences obtained from the sequencing of the five fungal strains were compared by BLAST using the GenBank database. The results of the strain sequence comparison and identification are listed in Table 1. Based on morphological characteristics and sequence homology comparison, the five fungal strains, F-1, F-2, F-3, F-4, and F-5, were tentatively identified as Cladosporium subuliforme, Penicillium solitum, Paecilomyces lilacinus, Simplicillium cylindrosporum, and Aspergillus fumigatus, respectively.

Table 2 shows that among the five fungi, the highest dry matter density was recorded for Penicillium solitum (23.76 g/L), followed by Simplicillium cylindrosporum and Paecilomyces lilacinus at 23.12 and 22.05 g/L, respectively. The maximum biomass was observed for Simplicillium cylindrosporum (1.29 g/L), followed by Penicillium solitum and Paecilomyces lilacinus at 0.61 and 0.49 g/L, respectively.

Paecilomyces lilacinus (F-3) had the best adsorption effect on Co, Cr, Mo, and Re, with adsorption rates of 16.42, 28.77, 24.77, and 22.13%, respectively (Figure 2). However, its adsorption rate of Ni was poor.

Heavy metal adsorption capacity was calculated using moisture content, adsorption efficiency, and pre- and post-adsorption concentrations of heavy metals across the five fungal strains. Overall, Paecilomyces lilacinus had the highest adsorption capacities for Co, Cr, Mo, and Re, with 764.06, 0.33, 0.26, and 249.36 mg/g of dry matter, respectively. Therefore, P. lilacinus was selected as the biological adsorbent for the subsequent adsorption experiments.

Many studies have indicated that P. lilacinus possesses broad-spectrum resistance to heavy metals, indicating that it is an excellent microbial resource for remediating heavy metal pollution (Zeng et al., 2010) found that P. lilacinus showed resistance to Cd, Zn, Mn, Cu, Pb, and Co, and that the Cd biosorption capacity of the fungus reached 24.23 mg/g during growth. A novel microbial nanocomposite, Paecilomyces lilacinus—silica nanoparticle-calcium-alginate beads, has shown excellent removal efficiency of Pb (II) in aqueous solution (85.54%) at an initial concentration of 200 mg/L (Ruan et al., 2020). Paecilomyces lilacinus demonstrates notable efficacy in the adsorption of Cd (Chen et al., 2023b) and may be used as an inexpensive, environmentally friendly, and effective bioremediation agent to remove Cd²⁺ from wastewater (Xia et al., 2015). In this study, the electrolyte containing a high concentration of heavy metals was a complex system. Nonetheless, P. lilacinus exhibited tolerance to the electrolyte and demonstrated a high adsorption capacity for Co, Re, and Ni, reaching 764, 358, and 315 mg/g of dry matter (Table 3), respectively, indicating that it has considerable potential as a bioremediation strain for superalloy electrolytes.

In Figure 3A, At a pH range of 3–7, the adsorption rate of the five heavy metals by Paecilomyces lilacinus showed a trend of increasing and then decreasing. The adsorption rates of Co, Cr, and Ni by Paecilomyces lilacinus increased rapidly with increasing pH at pH 3–5, and reached a maximum of 18.58%, 28.87%, and 11.35% at pH 5, respectively, decreasing gradually at pH – 5. The adsorption rates of Mo and Re increased rapidly at pH 3–4 and reached a maximum of 24.50 and 20.99% at a pH of 4, respectively, whereas the adsorption rate at a pH of 5 was similar to that at a pH of 4 decreasing gradually at pH > 5. Therefore, the optimal adsorption pH for heavy metals in the superalloy electrolyte is 5. In general, at pH < 7, cobalt is mainly present as Co²⁺ (Hayrettin et al., 2008). Hexavalent chromium exists mainly as HCrO^4–, Cr₂O7^2–, and CrO₄^2– in acidic aqueous solutions (Tandon et al., 1984) and is always negatively charged (Castro-Rodriguez et al., 2015). The two main ionic forms of molybdenum are molybdate (MoO₄^2–) and tetrathiomolybdate (MoS₄^2–) (Xu et al., 2006). Rhenium is stable in solution as ReO₄^– (Srivastava et al., 2015). Under acidic conditions, the concentration of H₃O⁺ in the solution increases, and the surfaces of suspended microorganisms can be covered by H₃O⁺. Due to electrostatic repulsion, it is difficult for metal ions to bind to adsorption sites on the microbial surface (Mengmei and Shuliang, 2018). The repulsive force may lead to a reduced adsorption of Co and Ni on Paecilomyces lilacinus, but the increased electrostatic adsorption may enhance the adsorption effect of Mo and Re. The poor adsorption effect of Cr at pH = 4 may be due to the competitive adsorption between Mo, Re, and Cr. Competitive adsorption commonly occurs in multi-metal coexistence systems for the variations in the metal characteristics, electronegativity, the resultant affinity for sorption sites and the hydrated radius among metals (Lee and Chang, 2011; Park et al., 2016; Huang et al., 2018). The initial solution pH affects the degree of protonation of functional groups on the adsorbent surface. At low pH values, the protonation of the surface functional groups positively charges the surface of the adsorbent. With increasing pH, the number of negatively charged functional groups on the surface of the adsorbent gradually increases (Wen et al., 2024). Therefore, the adsorption capacity of Co and Ni gradually increased, but this was not conducive to the adsorption of Cr, Mo, and Re, so their adsorption rates decreased. When the pH continued to increase, metallic Co and Ni formed hydroxide precipitates, which were not conducive to adsorption by P. lilacinus.

In Figure 3B, the adsorption capacities for the five heavy metals (Co, Cr, Mo, Re, and Ni) increased with increasing initial concentration of the electrolyte. After reaching a certain value, the adsorption capacity decreased. The optimal adsorption performance was achieved when the electrolyte was diluted by a factor of 5. Generally, as the initial concentration of heavy metals in a solution increases, the adsorption capacity of the adsorbent increases until it reaches the maximum point of adsorption site saturation (Ahsaine et al., 2017). Beyond this point, further increases in the metal concentration may not result in an increased adsorption capacity and may even cause a decrease. The reasons for this phenomenon may include site saturation or electrostatic repulsion between metal ions (Ong et al., 2018) and the toxicity of high metal concentrations to microorganisms (Karthik et al., 2017).

In Figure 3C, Removal of the five heavy metals by P. lilacinus first increased rapidly and then slowly, reaching an adsorption dynamic equilibrium state. During the first 30 min of the reaction, P. lilacinus had a large number of binding sites on the surface, and the adsorption rate increased significantly, after which the rate slowed down. The adsorption rate of P. lilacinus to Cr peaked at 32.66% when the adsorption time was 30 min. In contrast, the adsorption rates of Co (16.82%) and Mo (25.04%) were the highest at an adsorption time of 1 h, whereas the adsorption rates of Re (20.03%) and Ni (10.68%) were highest when the adsorption time was 2 h. The reaction time after 2 h was considered the dynamic adsorption equilibrium time. Thereafter, P. lilacinus could no longer provide sufficient adsorption sites. Consequently, the adsorption reached saturation and the removal rate fluctuated and decreased to a certain extent. Therefore, based on the adsorption status of the five heavy metals, the ideal adsorption time of P. lilacinus for heavy metals in the electrolyte is 1 h.

In Figure 3D, When the dosage of P. lilacinus was 1.5 g, the adsorption rates of Co, Cr, Mo, Re, and Ni were 12.42, 29.45, 24.05, 17.94, and 10.11%, respectively. With an increase in the dosage, the adsorption rate of P. lilacinus for various metals increased linearly. The adsorption rates of Co, Cr, Mo, Re, and Ni were 40.16, 62.38, 44.91, 46.89, and 31.76%, respectively, when the dosage of P. lilacinus was 4 g. The removal rate did not increase significantly as the fungal dosage continued to increase. With an increased dosage of P. lilacinus, more surface area was available for adsorption owing to the increase in active sites on the adsorbent (Goh et al., 2019), resulting in the adsorption rate continuing to increase. However, beyond a certain range, metal adsorption reaches a saturation state (Usman et al., 2016), which may leads to a decline in the adsorption capacity for solute availability, electrostatic interaction, andinterference between the binding site (Wu et al., 2019). This phenomenon can also be explained by the “mass effect,” whereby the aggregation of abundant biosorbents reduces the effective surface area, resulting in insufficient functional sites on the cell surface for bonding metal ions and a decrease in biosorption capacity (Xia et al., 2015). Therefore, from the perspective of the comprehensive removal rate and treatment cost, the optimal dosage of P. lilacinus for treating 150 mL of electrolyte was 4 g, i.e., 26.67 g/L.

In Figure 3E, The adsorption temperature increased continuously in the range of 15–35°C, and the adsorption of the five heavy metals by P. lilacinus first increased slowly and then decreased slowly. When the adsorption temperature was 25°C, the adsorption rate of P. lilacinus to Co (38.58%) and Re (45.36%) peaked. When the adsorption temperature was 30°C, the adsorption rates of Cr (64.41%), Mo (47.87%), and Ni (25.38%) were at their highest. Above 30°C, the adsorption rate decreased, possibly because the increased temperature may have affected the metabolic activity of P. lilacinus. Microbial metabolic activity increases with increasing temperature up to an optimum level. However, excessive heat can lead to enzyme denaturation, diminished microbial activity, and potentially cause organismal inactivity or death (Thomson et al., 2017). Furthermore, temperatures exceeding the optimum temperature may induce changes in the ribosomal conformation, leading to decreased protein synthesis (Sanchez Sanchez de Groot and Ventura, 2006). It has also been reported that at high temperatures, the rate of interaction between biomass and heavy metal ions decreases. With increasing temperature, the boundary layer diminishes, resulting in a decreased removal rate of heavy metal ions (Sharma et al., 2023). In general, the best adsorption temperature range of P. lilacinus for heavy metals in electrolytes is 25–30°C. This may be due to the maximum activity of microbial enzymes under optimal mesophilic conditions (Jacob et al., 2018).

Based on the above conclusion, under the conditions of adsorption pH = 5, adsorption time 1 h, dosage 26.67 g/L, and adsorption temperature 25–30°C, the adsorption rates of P. lilacinus for Co, Cr, Mo, Re, Ni in the electrolyte were 37.09, 64.41, 47.87, 41.59, and 25.38%, respectively. Various studies have consistently observed that the optimal conditions for fungal adsorption of heavy metals, such as Co (II) (El-Bondkly and El-Gendy, 2022), Ni (II) (Sharma et al., 2021), and Cr (II) (Boujelben et al., 2022), commonly involve a pH around 5.0. Additionally, the temperature range of 25–30°C to be favorable for the maximum uptake of these heavy metals by different fungal strains (Bourzama et al., 2020; Nguyen Van et al., 2021; Kerga et al., 2023). These conclusions are consistent with the findings of the present study, indicating that these conditions could be considered optimal for the fungal adsorption of heavy metals.

The FTIR spectra of P. lilacinus before and after biosorption are shown in Figure 6. The main functional groups related to the strong peaks observed have been identified in previous studies (Zhang et al., 2011; Chen and Wang, 2016; Chen et al., 2020) and are listed in Table 6.

As shown in Table 6, P. lilacinus contains–OH,–NH,–CH,–C = O, C-N, PO₄^3–, and other active groups. Figures 7A, B shows that the broad stretching bands at 3408.48 and 531.99 cm^–1 strongly shifted to 3425.04 and 519.52 cm^–1 after heavy metals loaded onto P. lilacinus. The other three peaks at 2924.99, 1641.00 and 1075.29 cm^–1 are observed to become sharper after heavy-metal loading. These changes in the absorbance peaks correspond to hydroxyl (-OH), amino (-NH₂), amide (-CONH₂), carbonyl (-C = O), and phosphate (PO₄^3–) groups, which could be active functional sites (Xia et al., 2015). The bands at 1546.53 and 1240.24 cm^–1 disappeared and the peaks at 1744.44 cm^–1 only appeared in the spectra of metals-loaded biomass. These results indicate the presence of interactions between Co, Cr, Mo, Re, Ni, carboxyl (-COOH), and amide groups. Thus, the FTIR spectral results suggest that the five aforementioned groups on the surface of P. lilacinus were significant functional sites for binding heavy metal ions.

Yildirim et al. (2020) utilized Pleurotus ostreatus powder for Ni (II) adsorption and observed a redshift in the absorption peak of -OH (-NH) from 3275 to 3369 cm^–1, consistent with our findings. Ou employed Paecilomyces lilacinus for Ag (I) adsorption and detected redshifts in the -OH peak from 3297 to 3304 cm^–1, blueshifts in the -CH peak from 2928 to 2926 cm^–1 and in the -C = O peak from 1653 to 1652 cm^–1 (Ou et al., 2011). These alterations in functional groups align with our study’s findings. It was confirmed that functional groups such as -OH, -NH₂, -COOH, and PO₄^3– are chelated to Cr by fungi (Vale et al., 2016).