Simultaneous Heavy Metal-Polycyclic Aromatic Hydrocarbon Removal by Native Tunisian Fungal Species

Citation: Hkiri, N.; Olicón-Hernández, D.R.; Pozo, C.; Chouchani, C.; Asses, N.; Aranda, E. Simultaneous Heavy Metal-Polycyclic Aromatic Hydrocarbon Removal by Native Tunisian Fungal Species. J. Fungi 2023,9, 299. https://doi.org/ 10.3390/jof9030299 Academic Editor: Birgitte Andersen Received: 28 January 2023 Revised: 16 February 2023 Accepted: 21 February 2023 Published: 24 February 2023 Copyright: © 2023 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/). Fungi Journal of Article Simultaneous Heavy Metal-Polycyclic Aromatic Hydrocarbon Removal by Native Tunisian Fungal Species Neila Hkiri 1,2,*, Dario R. Olicón-Hernández 3, Clementina Pozo 1,4, Chedly Chouchani 2, Nedra Asses 5 and Elisabet Aranda 1,4,* 1Institute of Water Research, University of Granada, 18071 Granada, Spain 2 Laboratory of Environmental Sciences and Technologies, Higher Institute of Sciences and Technologies of the Environment, University of Carthage, Tunis 1000, Tunisia 3 Laboratorio de Bioquímica y Biotecnología de Hongos, Departamento de Microbiología, Escuela Nacional de Ciencias Biológicas, Instituto Politécnico Nacional, Ciudad de México 07738, Mexico 4Department of Microbiology, University of Granada, 18071 Granada, Spain 5Laboratory of Microbial Ecology and Technology, National Institute of Applied Science and Technology, University of Carthage, Tunis 1000, Tunisia *Correspondence: [email protected] (N.H.); [email protected] (E.A.) Abstract: Multi-contamination by organic pollutants and toxic metals is common in anthropogenic and industrial environments. In this study, the five fungal strains Chaetomium jodhpurense (MH667651.1), Chaetomium maderasense (MH665977.1), Paraconiothyrium variabile (MH667653.1), Emmia lacerata, and Phoma betae (MH667655.1), previously isolated in Tunisia, were investigated for the simultaneous removal and detoxification of phenanthrene (PHE) and benzo[a]anthracene (BAA), as well as heavy metals (HMs) (Cu, Zn, Pb and Ag) in Kirk’s media. The removal was analysed using HPLC, ultra-high performance liquid chromatography (UHPLC) coupled to a QToF mass spectrometer, transmission electron microscopy, and toxicology was assessed using phytotoxicity (Lepidium sativum seeds) and Microtox ® (Allivibrio fisherii) assays. The PHE and BAA degradation rates, in free HMs cultures, reached 78.8% and 70.7%, respectively. However, the addition of HMs considerably affected the BAA degradation rate. The highest degradation rates were associated with the significant production of manganese-peroxidase, lignin peroxidase, and unspecific peroxygenase. The Zn and Cu removal efficacy was considerably higher with live cells than dead cells. Transmission electron microscopy confirmed the involvement of both bioaccumulation and biosorption processes in fungal HM removal. The environmental toxicological assays proved that simultaneous PAH and HM removal was accompanied by detoxification. The metabolites produced during co-treatment were not toxic for plant tissues, and the acute toxicity was reduced. The obtained results indicate that the tested fungi can be applied in the remediation of sites simultaneously contaminated with PAHs and HMs. Keywords: polycyclic aromatic hydrocarbons; heavy metals; ascomycetes fungi; mycoremediation; phenanthrene; extracellular enzymes; microtoxicity; phytotoxicity; transmission electron microscopy 1. Introduction Environmental quality and human health are increasingly being affected by various pollutants of anthropogenic origin. Polycyclic aromatic hydrocarbons (PAHs) and heavy metals (HMs) are some of the most important environmental contaminants (https://www. epa.gov/ accessed on 21 January 2023) and characterised by a high persistence, recalcitrance, abundance, and toxicity [1,2]. The PAHs are mainly released by industrial operations and during the incomplete combustion of organic materials such as coal, fuel, and wood. Exposure to PAHs is associated with various serious diseases due to their teratogenic, carcinogenic, and mutagenic properties. The HMs are widespread in the environment and are mainly derived from industrial wastewater (tanning industry, sewage sludge usage, mining activities, fertilisers, among others). Some HMs, such as Cu (II), Zn (II), and J. Fungi 2023,9, 299. https://doi.org/10.3390/jof9030299 https://www.mdpi.com/journal/jof J. Fungi 2023,9, 299 2 of 18 Fe (III), are essential for the biological activities of plants and microorganisms. However, at high concentrations in soils, HMs can be toxic, threatening human and environmental health. The prevalence of typical potentially toxic metals such as Pb, Zn, Cu, Cd, and Ag in paddy soils and in different organs of rice plants in a typical industrial zone in China were investigated [ 3 ]. The study demonstrated that the rice containing some heavy metals might cause serious non-carcinogenic and carcinogenic health risks for residents, especially for aged persons. Frequently, PAHs and HMs co-occur, mainly in industrial areas, when different pollutants sources converge in combination with phenomena such as transportation from air and waters from different emission points. Both pollutant types tend to be accumulated in organic matter, making soil a major pollutant reservoir [4]. Among the technologies for the removal of hazardous pollutants such as PAHs and HMs, bioremediation represents an eco-friendly approach in comparison with physical and chemical methods [ 5 , 6 ]. In particular, the use of fungi has several advantages over the use of bacteria since fungi can produce a wide range of unspecific degrading enzymes, possess a large surface-to-cell ratio, and are able to biomineralize or change the valence of HMs [ 7 ]. In addition, the interactions between HMs and both live and dead cells lead to a better biological elimination of metals via mechanisms such as cellular surface adsorption, bioleaching, intracellular bioaccumulation, biomineralization, and biotransformation [ 8 ]. In the last decades, bioremediation technology studies have focused on the removal of individual PAHs or HMs. For instance, Aspergillus flavus and Aspergillus fumigatus strains were found to play an important role in bioremediation of 16 PAHs compounds with a degradation rate of 82.7% and 68.9% of the total PAHs after 15 days of incubation [ 9 ]. Podoscypha elegance strain was also able to degrade 99% of phenanthrene and 98.9% of pyrene in-vitro conditions [ 10 ]. In addition, fungal HMs removal is well-documented. For example, the strain Penicilium simplicissium showed a great ability to remove Cd, Cr, Cu, Pb, and Zn involving both bioaccumulation and biosorption mechanisms [ 11 ]. The removal of Cu, Cr, Cd, and Zn by Trichoderma brevicompactum was investigated, which demonstrated a high removal rate for both individual and multi-metal mixture [12]. However, although pollutants rarely occur individually, the bioremediation of sites with various pollutants has rarely been reported [ 13 – 15 ]. The coexistence of both pollutant types can negatively affect bioremediation processes since microbes are unable to efficiently bioremediate a co-contaminated environment. As HMs are potent inhibitors of various microbial activities, they can disrupt their regulation and expression by competing for metal binding sites or with enzymes, i.e., oxygenases [ 16 ], thus inhibiting the enzyme functions or proteins. Intermediates of PAHs, such as salicylic acid, can also impact microbial cell metabolism, and induce reactive oxygen species (ROS) production, thus affecting the adsorption capacity of HMs [17,18]. The present study investigated the simultaneous PAH biotransformation and HM removal by five selected fungi isolated from polluted Tunisian salt water [ 19 ]. Two PAHs, namely phenanthrene (PHE) and benz[a]anthracene (BAA), were used in this work. Four HMs, namely Cu(II), Zn(II), Pb(II), and Ag(II), were tested in mixtures with the PAHs for fungal removal. This study represents a step forward to develop biological processes designed to treat pollutants in complex mixtures. 2. Materials and Methods 2.1. Chemicals and Strains The compounds 1-amonibenzothiazole (ABT, 97% purity), 2,2 0 - azinobis (3-ethylbenzo thiazoline-6-sulfonic acid) (ABTS, 98% purity), phenanthrene (PHE, 98% purity), and benz[a]anthracene (BAA, 98% purity) were purchased from Sigma (St. Louis, MO, USA). Copper sulphate and zinc, lead, and silver nitrate metal salts were acquired from Merck (Madrid, Spain). All solvents used were of HPLC grade: acetonitrile (PanReac, AppliChem, Barcelona, Spain), HPLC water (PanReac, AppliChem, Barcelona, Spain), and phosphoric acid (Fisher Chemical, Madrid, Spain). All other chemicals and reagents were of analytical grade or higher purity. J. Fungi 2023,9, 299 3 of 18 The used fungi were Chaetomium jodhpurense (MH667651.1), Chaetomium maderasense (MH665977.1), Paraconiothyrium variabile (MH667653.1), Emmia lacerata, and Phoma betae (MH667655.1), previously isolated from a salt environment in Tunisia and molecularly identified [ 19 ]. The fungal genera Chaetomium,Paraconiothyrium,Phoma, belonging to Ascomycota, were known by their large application in bioremediation due to their great ability to produce extracellular enzymes [ 20 – 22 ]. The strain Emmia lacerate is a Basidiomycota belonging to polypore species. As a white rot fungus it can produce a large wide of extracellular enzymes [23]. 2.2. Culture Conditions Mycelium from 5-day-old cultures of the five strains on PDA (potato dextrose agar, BD DIFCO, NJ, USA) petri dish medium was added to 80 mL of sterile distilled water and aseptically homogenised with an ultra-turrax (IKA, Germany) for 10 s. Five hundred µ L of the suspension used as pre-inoculum was added into individual flasks containing 25 mL of Kirk’s medium prepared with half-strength artificial seawater [ 24 ]. The composition of the artificial seawater was as follows: 29.8 g/L NaCl, 0.73 g/L KCl, 10.7 g/L MgCl 2• 6H 2 O, 5.4 g/L MgSO 4• 7H 2 O, and 1.1 g/L CaCl 2• 2H 2 O. The culture was incubated at 28 ◦ C under agitation at 120 rpm. After 2 days of growth, half of the flaks were heat-inactivated (autoclaved for 20 min at 121 ◦C) and used as biotic control. All experiments were performed in triplicate. 2.2.1. Phenanthrene and Benz[a]anthracene Biodegradation Experiments The PHAs (PHE and BAA) were prepared individually in a stock solution (5 mM dissolved in acetonitrile) and added into Kirk’s medium after 2 days of fungal growth to reach final concentrations of 100 µ M for PHE and 50 µ M for BAA. The experiment was maintained for 18 days at 28 ◦ C under agitation at 120 rpm. Each treatment was performed with three replicates. Flasks were sacrificed at regular intervals of 9 days. 2.2.2. Bioremediation of Combined Pollutants The influences of HMs on PAH biodegradation and the bioaccumulation properties of the fungi were investigated in a pollutant cocktail of 20 mg/L total concentration, with the addition of a multi-metal mixture [5 mg/L Cu(II), Zn(II), Pb(II), and Ag(II) each] in combination with the PAHs, as previously described in Section 2.2.1. The metal concentration was selected according to a previous experiment to study the minimum inhibitory concentration (MIC) with the selected fungi. A metal stock solution of 1000 mg/L was prepared by dissolving their respective salts CuSO 4• 7H 2 O, ZnNO 3 , Pb(NO 3 ) 2 , and Ag(NO 3 ) in distilled water, followed by sterilisation by filtration using a syringe filter with a pore size of 0.22 µ m. The sterilised metal stock solutions were added separately to the PAH medium flasks to obtain final total metal concentrations of 20 mg/L. Additionally, to investigate the involvement of CYP450 in the bioremediation processes, the CYP450 inhibitor 1-aminobenzotriazole (ABT) was used at a final concentration of 1 mM in additional flasks, and ABT was added together with the pollutants. 2.2.3. Sampling After each treatment, half of the content of each flask was centrifuged and stored at − 20 ◦ C for enzyme activities, biomass, pH, glucose content, HMs content, Microtox ® Bioassay, and phytotoxicity experiments, as described below. Biomass was calculated gravimetrically by filtrating and drying in an oven for 72 h until constant weight. The pH was measured using a reactive strip (Panreac Quimica, Barcelona, Spain). The remaining content was treated with ethanol (1.6:1 v:v) to stop the incubation and to obtain the PAHs. After sonication for 15 min, the samples were centrifuged (12,000 × g), disposed in HPLC glass vials, and analysed by chromatography (HPLC and UHPLC-QTOF). J. Fungi 2023,9, 299 4 of 18 2.3. Enzymatic Activity Samples of each treatment were filtered and centrifuged at 10,000 × gfor 20 min, and the supernatant was used for enzymatic analyses. Subsequently, Mn peroxidase (MnP), unspecific peroxygenase (UPO), lignin peroxidase (LiP), and laccase (Lac), were analysed on a UV-visible spectrophotometer (Shimadzu UV-1800 UV). Manganese peroxidase (MnP) activity (EC 1.11.1.13) was monitored at 270 nm through the oxidation of 0.5 mM MnSO 4 in sodium malonate buffer (50 mM, pH 4.5) with 0.5 mM H 2 O 2 [ 25 ]. Unspecific peroxygenase (UPO) activity (EC 1.11.2.1) was measured using 5 mM veratryl alcohol as substrate in potassium phosphate buffer (50 mM, pH 7.0) in the presence of 1 mM H 2 O 2 [ 26 ]. Lignin peroxidase (LiP) activity (EC 1.11.1.14) was determined by measuring the rate of oxidation of veratryl alcohol toveratryl aldehyde at 310 nm, as described by Tien and Kirk [ 27 ]. The mixture reaction contained 2 mM veratryl alcohol and 0.4 mM H 2 O 2 in sodium tartrate buffer (50 mM, pH 3). Laccase activity (EC 1.10.3.2) was measured using 2,2-azino-bisethylbenthiazolina (ABTS) according to Novotny et al. (1999) [ 28 ]. The rate of ABTS oxidation (5 mM) in sodium acetate buffer (0.1 M, pH 4.5), was determined at 420 nm. One unit of enzyme was defined as the amount of enzyme necessary to release 1 µ mol of product per minute under the assay conditions and expressed as UI. 2.4. Chemical Analyses 2.4.1. Estimation of Glucose Content Glucose was analysed according to Miller (1959) [ 29 ], using DNS (dinitrosalicyclic acid reagent). The supernatant of each treatment was centrifuged and used in a proportion of 1:1 (sample: DNS reagent). The mixture was boiled at 90 ◦ C for 10 min in a water bath, and after cooling, 1 mL of distilled water was added. The obtained solution was read at a wavelength of 540 nm using a spectrophotometer (Shimadzu UV-1800 UV). The total glucose content was estimated using a standard curve of glucose, and the results are expressed as mg/L. 2.4.2. Phenanthrene and Benzo[a]anthracene Quantification and Metabolite Identification The removal of PHE and BAA was analysed using an Agilent 1200 HPLC (Agilent, Technologies, Palo Alto, CA, USA), coupled to a DAD detector. The compounds were separated using an RP-C18 Synergy Fusion column (80 Å; 4 µ m, 4.6 × 150 mm; Phenomenex ® , Madrid, Spain). Separation was performed according to the methodology described in Aranda et al. (2009) [ 30 ], using water + 1% phosphoric acid (A) and acetonitrile (B) as eluent buffers in isocratic mode, and a flow rate of 1 mL/min. Quantification was performed based on a calibration curve with the pure standards. The metabolites produced were analysed using an ultra-high performance liquid chromatography (UHPLC), Acquity I-Class System (Waters, Milford, MA, USA), and a Synapt G2S QToF mass spectrometer (Waters, Milford, MA, USA) coupled to a CORTECSUHPLC ® HILIC ® C18 1.6 µ m column (2.1 × 50 mm; Waters, Milford, MA, USA), connected to a PDA (photodiode array). The flow rate was 0.350 mL/min, using a gradient flow of water, 0.1% NH 3 (A), and acetonitrile, 0.1% NH3 (B) (5 min A 80%: B 20%; 10 s A 30%: B 70%; 1.40 min A 0%: B 100%; 1.10 min A 80%: B 20%). The data were analysed using the MassLynx software (version 4.1, Waters, Milford, MA, USA). Metabolites were analysed on positive and negative mode. 2.4.3. Heavy Metal Analysis The removal of Cu, Zn, Pb, and Ag from the culture medium was evaluated by inductively coupled plasma-optical emission spectrometry (ICP-OES) (Perkin-Elmer Optima 8300) at the Center of Scientific Instrumentation of the University of Granada (Granada, Spain). At the end of the incubation time, 10 mL of the samples from HM treatment cultures (biotic control and fungal treatments) were taken and centrifuged at 12,000 × g. The supernatant was filtered through 0.22- µ m Millipore filters and injected into the ICP- J. Fungi 2023,9, 299 5 of 18 OES. Standards with different concentrations were prepared with pure water (Millipore). Recovery value for all metals was 99%. 2.4.4. Transmission Electron Microscopy Coupled with Energy-Dispersive X-ray Spectroscopy (TEM EDX) Fungal pellets were harvested and washed with phosphate saline buffer (100 mM, pH 6.8). The obtained fungal cells were fixed using 2.5% v/v glutaraldehyde overnight at 4 ◦ C, and 2% osmium tetroxide was used as a secondary fixative for 2 h. The fixed cells were washed with 0.1 M phosphate buffer. Subsequently, fungal cells were dehydrated in a series of ethanol, cut into ultra thin sections using an ultramicrotome, and fitted on TEM grids for examination [ 11 ]. Samples were visualised using an HR-TEM TALOS F200X (Thermo Fisher Scientific) equipped with a detector of a high-angle annular dark-field (HAADF), Cannon type: Schottky-type field emission (FEG), using the Microanalysis system EDX type (using X-ray energy dispersion) at the Center of Scientific Instrumentation of the University of Granada (Granada, Spain). 2.5. Toxicity Bioassays 2.5.1. Phytotoxicity Test The phytotoxicity test was performed according to the method of Zucconi et al. (1981) [ 31 ], using cress seeds (Lepidium sativum) to evaluate the toxicity of the culture medium after treatment with the selected fungi. The L. sativum seeds were soaked in tap water for 1 h; subsequently, 20 seeds per replicate were positioned at equal distances in glass Petri dishes with Whatman paper N º 1, and 2 mL of each sample was added at different concentrations (100%, 40%, 20% and 10%), followed by incubation at 28 ◦ C in the dark. Distilled water was used as control, and the abiotic control was prepared using non-inoculated media amended with PHE and BAA, and HMs. After 48 h, the germination index (GI%) was calculated from the number of germinated seeds and the root length, according to the following formula: GI% = (G ×L)/G0×L0)×100 •G and G0: number of germinated seeds in the samples and the control; •L and L0: root length values in the samples and the control. 2.5.2. Microtox®Test The Microtox ® bioassay was employed to evaluate acute toxicity (as EC 50 ) of the supernatant after fungal treatment of PAHs and HMs, abiotic, and biotic samples at time 0 and at the end of each experiment. For this, the Microtox ® M500 toxicity analyser (Instrumentación Analítica S.A. Madrid, Spain) was employed. Toxicity is expressed as EC 50 (%), the concentration of the sample that causes a 50% of bioluminescence reduction in the bacterium Aliivibrio fischeri after 5 and 15 min of exposure to the sample [ 32 , 33 ]. The analyses were performed in triplicate. 3. Results 3.1. Removal of Phenanthrene and Benz[a]anthracene The removal of PHE and BAA was studied in salt Kirk media every 9 days for 18 days. The biodegradation ability of the fungi varied for the different PAHs (Figure 1). For PHE, E. lacerate showed the highest biodegradation rate (78.8%), followed by P. variabile (74.1%), C. jodhpurense (69.3%), C. maderasense (48.4%), and P. betae (46.3%). However, for BAA, P. variabile exhibited the largest removal rate (70.7%) (Figure 1), followed by P. betae (58.7%), E. acerate (57.8%), C. maderasense (28.8%), and C. jodhpurense (26.9%). J. Fungi 2023,9, 299 6 of 18 J. Fungi 2023, 9, x FOR PEER REVIEW 6 of 20 3. Results 3.1. Removal of Phenanthrene and Benz[a]anthracene The removal of PHE and BAA was studied in salt Kirk media every 9 days for 18 days. The biodegradation ability of the fungi varied for the different PAHs (Figure 1). For PHE, E. lacerate showed the highest biodegradation rate (78.8%), followed by P. variabile (74.1%), C. jodhpurense (69.3%), C. maderasense (48.4%), and P. betae (46.3%). However, for BAA, P. variabile exhibited the largest removal rate (70.7%) (Figure 1), followed by P. betae (58.7%), E. 6acerate (57.8%), C. maderasense (28.8%), and C. jodhpurense (26.9%). Figure 1. Residual phenanthrene and benzo[a]anthracene (μM) in the culture medium, fungal biomass (g/L) (red color), pH variation, and glucose concentration (g/L) (blue color). (a) C. jodh- Figure 1. Residual phenanthrene and benzo[a]anthracene ( µ M) in the culture medium, fungal biomass (g/L) (red color), pH variation, and glucose concentration (g/L) (blue color). ( a )C. jodhpurense, ( b )C. maderasense, ( c )P. variabile, ( d )E. lacerata, and ( e )P. betae. Error bars indicate the average of tree replicates (n = 3). The influence of the presence of the mixture of HMs (Cu, Zn, Pb, Ag) on PHE and BAA removal was evaluated under the same experimental conditions. The PHE degradation rate varied from 41.1% to 78.0%, and for BAA, the degradation rate ranged between 25.6% and 49.9%. J. Fungi 2023,9, 299 7 of 18 The degradation rate for BAA coexisting with the mixture of HMs was considerably reduced compared to that in free HM cultures, except for C. maderasense, in which the rate of BAA degradation in free HM media increased from 33.7% to 49% in the presence of HMs. The highest degradation inhibition of BAA in the presence of HMs was observed with P. betae,E. lacerata, and P. variabile, with a degradation rate of approximately 30%. However, the ability of the tested strains to biodegrade PHE coexisting with HMs increased from 48.4% to 51.7% in C. maderasense, and from 69.3% to 78.0% in C. jodhpurense. A moderate decline in the PHE biodegradation rate by approximately 3% was observed in E. lacerata, and 5% in P. betae and P. variabile was detected in the presence of HMs. The role of the cytochrome P450 enzymatic system (CYP) in the degradation of the cocontaminated media was evaluated by the addition of the inhibitor 1-aminobenzotriazole. A lower degradation activity was observed in cultures containing CYP inhibitor with only the P. betae strain, in which the degradation rate decreased by 12.8% and 14.6% for PHE and BAA, respectively. This indicates that P. betae is involved in both extra- and intracellular enzymatic PAH removal. For the other strains (C. jodhpurense,C. maderasense,P. variabile and E. lacerata), inhibition of the CYP did not significantly influence the biodegradation rate of PAHs (Figure 1), indicating that PHE and BAA could be transformed mainly via extracellular pathways. Regarding the pH profile, glucose use, and biomass production by fungi in the absence and presence of HMs in the growth medium, a shift of pH from 5 to 7 and an increase in biomass accumulation during the first 9 days with or without HMs were detected. A rapid decrease in the glucose concentration was also observed in the first 9 days, followed by a moderate and stable decline in the remaining 9 days of incubation. The biomass produced by the fungi was increased considerably. The presence of HMs did not affect fungal growth throughout the experiment. However, in the last 9 days, biomass accumulation in P. betae and C. jodhpurense increased from 8 g/L in the PAH treatment to 10 g/L in the presence of HMs. 3.2. Enzyme Production during Degradation Extracellular ligninolytic enzyme production was also monitored after 9 and 18 days of incubation in Kirk media supplemented with the PAHs, and in the presence or absence of HMs (Table 1). Table 1. Enzymatic activities. MnP—manganese peroxidase; UPO—unspecific peroxygenase; LiP—lignin peroxidase; Lac—Laccase, expressed as (UI) during PAH treatment after 9 and 18 days of cultivation in the absence and presence of HMs. ±Value indicates the average of tree replicates (n = 3). MnP UPO LiP Lac T9 T18 T9 T18 T9 T18 T9 T18 C. jodhpurense PAH 87.6 ±7.4 16.0 ±1.4 n.d. 41.5 ±6.4 32.4 ±10.3 25.7 ±1.8 n.d. 8.8 ±2.5 PAH + HM + ABT 8±2n.d. n.d. 8.98 ±1.94 16.6 ±3.9 43.2 ±9.2 n.d. 8.4 ±0.9 PAH + HM n.d. n.d. n.d. 12.2 ±2.8 23.9 ±3.8 99.4 ±3.0 n.d. 8.0±1.3 C. maderasense PAH 274.0 ± 12.3 9.1 ±4.5 15.2 ±1.4 17.8 ±2.3 n.d. n.d. 43.7 ±5.1 225.1 ± 13.8 PAH + HM + ABT 259 ±12 9.0 ±1.7 26.0 ±0.5 n.d. 10.2 ±2.3 n.d. n.d. n.d. PAH + HM 120.6 ±5.7 3.6 ±0.7 175.7 ± 36.2 n.d. 10.9 ±2.4 n.d. n.d. 122.1 ±3.5 PAH 14.9 ±0.9 85 ±14 55.5 ±5.6 37.9 ±6.1 309.1 ± 41.2 n.d. n.d. n.d. P. variabile PAH + HM + ABT 88.7 ±3.9 66.1 ±6.2 77.2 ±4.8 n.d. 111 ±31 n.d. n.d. n.d. PAH + HM 51 ±11 7.4 ±2.8 200.4 ± 37.6 181.4 ± 17.0 329.4 ± 15.9 n.d. n.d. n.d. E. lacerata PAH 25.0 ±3.7 n.d. n.d. 41.8 ±12.1 62.2 ±4.6 42.6 ±2.7 29.3 ±1.7 n.d. PAH + HM + ABT n.d. 66.5 ±26.1 n.d. 105.7 ±7.3 66.2 ±5.7 46.6 ±5.5 n.d. n.d. PAH + HM n.d. 15.6 ±2.3 n.d. 71.4 ±13.8 183.9 ± 25.4 62.9 ±6.7 n.d. 0 P. betae PAH 4.2 ±0.9 25.4±8.4 21.6 ±2.4 65.0 ±11.4 19.5 ±2.0 69.4 ±4.7 47.3 ±3.9 63.5 ±6.5 PAH + HM + ABT 24.7 ±3.2 25.9 ±3.4 22.4 ±2.4 10.5 ±1.3 9.1 ±2.3 77.4 ±10.5 65.6 ±11.8 39.1 ±0.9 PAH + HM 18.5 ±2.7 n.d. 27.3 ±3.5 45.5 ±10.2 32.8 ±6.9 76.0 ±11.9 687.5 ± 28.3 523.4 ± 21.5 n.d. = not detected. J. Fungi 2023,9, 299 8 of 18 The production of MnP, LiP, UPO, and laccase enzymes was detected in most of the treatment cultures of the five tested fungi (Table 1). Maximum MnP production was observed in C. maderasense, which reached 274.0 ±12.3 UI in the PAH treatment. The presence of HMs with PAHs negatively affected enzyme activity; MnP was not detected in C. jodhpurense, and its level decreased considerably in C. maderasense (from 274.0 ± 12.3 UI to 120.6 ± 5.7 UI). The highest level of production was found after 9 days of incubation. Among the peroxidase enzymes, UPO had the highest levels. The fungus P. variabile produced the maximum amounts of 329.4 ± 15.9 UI in the presence of HMs, and of 309.4 ±41.2 UI in PAH media. Co-exposure to HMs and PAHs enhanced UPO activity three-fold in C. jodhpurense and four-fold in E. lacerate; LiP activity was lower than MnP and UPOs. The highest value was detected in P. variabile (200.4 ± 37.6 UI). In treatments amended with HMs, LiP activity increased in C. maderasense (from 15.2 ± 1.4 UI to 175.7 ±36.2 UI), P. variabile (from 55.4 ± 5.6 UI to 200.4 ± 37.6 UI) and E. lacerata (from 41.8 ±12.1 UI to 71.4 ± 13.8 UI). In C. jodhpurense and P. betae, LiP activity decreased from 41.55 ±6.4 UI to 12.2 ±2.8 UI and from 65.0 ±11.4 UI to 45.5 ±10.2 UI, respectively. Only C. maderasense and P. betae produced considerable levels of Lac in both PAH treatment and PAHs with HMs. Interestingly, the addition of HMs to PAHs in P. betae increased Lac production 14- and 8-fold, respectively, after 9 and 18 days of cultivation. 3.3. Metabolic Products after PHE and BAA Biodegradation The intermediate metabolites of PHE and BAA after biodegradation by the strains detected by LC-MS are shown in Figure 2. No significant differences were observed in the metabolism of the PAHs among the strains or between the presence of ABT and HMs. The analysis revealed the presence of 1-phenanthrol (P1), phenanthrene 9,10-dihydrodiol (P2), anthracene (B1), anthrone (B2), anthraquinone (B3), phthalic anhydride (B4), and phthalic acid (B5), being the main metabolites produced under the tested conditions, although we also found other unspecific metabolites (Appendix ATable A1). Phthalic anhydride and phthalic acid could be part of both BAA and PHE degradation metabolic pathways as a consequence of ring cleavage reactions under in vivo conditions after the first oxidation steps. The hydroxylated metabolites confirm the action of peroxidases in PAH biotransformation. J. Fungi 2023, 9, x FOR PEER REVIEW 9 of 20 Figure 2. Proposed pathways for the biotransformation of phenanthrene and benz[a]anthracene by the selected fungi. The same metabolites were found with the five tested strains for all experimental variants. 3.4. Heavy Metal Removal 3.4.1. Residual Heavy Metal Content Figure 3 shows the residual metal (Cu, Zn, Pb and Ag) concentrations in the culture media. After 18 days of cultivation, both live and inactivated mycelia showed high removal efficiencies for Ag and Pb. The highest amounts of Ag(II) and Pb(II) absorbed by inactivate mycelium were 4 and 4.4 mg/L, respectively. These amounts were increased to reach 4.9 mg/L with live cells at the end of the experiment. However, only live cells removed large amounts of Zn(II) and Cu(II), with a maximum of 4.7 mg/L for Zn(II) and 3.9 mg/L for Cu(II), contrary to inactivated mycelia, which did not exceed 0.357 mg/L for Cu and 0.801 mg/L for Zn. At the end of the experiment, after 18 days of incubation, the total amounts of metals removal by live cells were 4.9 mg/L for Ag(II) and Pb(II), 4.7 mg/L for Zn(II), and 3.9 mg/L for Cu(II). Figure 2. Proposed pathways for the biotransformation of phenanthrene and benz[a]anthracene by the selected fungi. The same metabolites were found with the five tested strains for all experimental variants. J. Fungi 2023,9, 299 9 of 18 3.4. Heavy Metal Removal 3.4.1. Residual Heavy Metal Content Figure 3shows the residual metal (Cu, Zn, Pb and Ag) concentrations in the culture media. After 18 days of cultivation, both live and inactivated mycelia showed high removal efficiencies for Ag and Pb. The highest amounts of Ag(II) and Pb(II) absorbed by inactivate mycelium were 4 and 4.4 mg/L, respectively. These amounts were increased to reach 4.9 mg/L with live cells at the end of the experiment. However, only live cells removed large amounts of Zn(II) and Cu(II), with a maximum of 4.7 mg/L for Zn(II) and 3.9 mg/L for Cu(II), contrary to inactivated mycelia, which did not exceed 0.357 mg/L for Cu and 0.801 mg/L for Zn. At the end of the experiment, after 18 days of incubation, the total amounts of metals removal by live cells were 4.9 mg/L for Ag(II) and Pb(II), 4.7 mg/L for Zn(II), and 3.9 mg/L for Cu(II). J. Fungi 2023, 9, x FOR PEER REVIEW 10 of 20 Figure 3. Residual concentrations of Cu, Zn, Pb, and Ag, expressed in mg/L in the culture medium of inactivate mycelium (IM), and for each fungus in the presence of PAH (MXT) and ABT for (a) C. jodhpurense, (b) C. maderasense, (c) P. variabile, (d) E. lacerata, and (e) P. betae after18 days of incubation. Error bars indicate the average of tree replicates (n = 3). 3.4.2. Transmission Electron Microscopy Coupled with EDX Analysis Figure 4 shows the transmission electron micrographs of the five fungi. The TEM images clearly show the morphological structures of the fungi after exposure to HMs. The EDX spectrum of strain C. maderasense shows only the presence of Cu and Zn inside the cell, reflected by the presence of some dark areas (Figure 4a). Ultrastructure analysis of C. jodhpurense, with the presence of an intact and regular cell membrane and a well-organised cytoplasm distribution (Figure 4b), and EDX analysis revealed no interesting findings in relation to the tested metals. The dark electron granules (Figure 4c) found in P. variable in the cell wall/cell membrane and in the cytoplasm were identified by EDX as Cu. Dark, and dense spots were observed in various areas in the cytoplasm of E. lacerate, and EDX analysis confirmed the prevalence of the four metals (Cu, Zn, Pb, and Ag) (Figure 4d), indicating bioaccumulation. Strain P. betae accumulated Cu, Zn, and Ag throughout the cell wall/cell membrane, and the cytoplasm showed visible dark spots inside the fungal cell. The cell wall/cell membrane was surrounded by electron-dense granules identified by the EDX spectrum as Pb (Figure 4e). The obtained results indicate that this tolerance towards Cu, Zn, Pb, and Ag could be associated with both biosorption and bioaccumulation mechanisms. Figure 3. Residual concentrations of Cu, Zn, Pb, and Ag, expressed in mg/L in the culture medium of inactivate mycelium (IM), and for each fungus in the presence of PAH (MXT) and ABT for ( a )C. jodhpurense, ( b )C. maderasense, ( c )P. variabile, ( d )E. lacerata, and ( e )P. betae after18 days of incubation. Error bars indicate the average of tree replicates (n = 3). 3.4.2. Transmission Electron Microscopy Coupled with EDX Analysis Figure 4shows the transmission electron micrographs of the five fungi. The TEM images clearly show the morphological structures of the fungi after exposure to HMs. The EDX spectrum of strain C. maderasense shows only the presence of Cu and Zn inside the cell, reflected by the presence of some dark areas (Figure 4a). Ultrastructure analysis J. Fungi 2023,9, 299 16 of 18 Table A1. Cont. Metabolite Elemental Composition Chemical Structure Molecular Weigh Error (ppm) Retention Time (min) Benz[α]anthracene Original molecule C18H12 J. Fungi 2023, 9, x FOR PEER REVIEW 17 of 20 D.R.O.-H.,C.C., N.A., C.P. and E.A.; supervision, C.C., N.A. and E.A.; project administration, C.C., N.A. and E.A.; funding acquisition, C.C., N.A. and E.A. All authors have read and agreed to the published version of the manuscript. Funding: This research was funded by Ministry of Higher Education and Scientific Research in Tunisia, grant for N.H.; FEDER/Junta de Andalucía-Consejería de TransformaciónEconómica, Industria, Conocimiento y Universidades (B-RNM-204-UGR20) and Secretaria de Investigación y posgrado SIP of IPN (Project number 20230427). Institutional Review Board Statement: Not applicable Informed Consent Statement: Not applicable Data Availability Statement: Not applicable Acknowledgments: Neila Hkiri gratefully acknowledges the scholarship from the Ministry of Higher Education and Scientific Research in Tunisia. E.A. would like to thank the FEDER/Junta de Andalucía-Consejería de Transformación Económica, Industria, Conocimiento y Universidades(B-RNM-204-UGR20) for providing funding. Olicón-Hernández would like to thank the Secretaría de Investigación y posgrado SIP of IPN (Project number 20230427) as well as CONACyT. Authors acknowledge Gabriela Angeles de Paz and Tatiana Robledo-Mahón for her support and the Center of Scientific Instrumentation of the University of Granada (Granada, Spain) for the analytical support. Conflicts of Interest: The authors declare no conflict of interest. Appendix A Table A1. Phenanthrene and benz(α)anthracene metabolites after fungal biodegradation. Metabolite Elemental Composition Chemical Structure Molecular Weigh Error (ppm) Retention Time (min) Phenanthrene Original molecule C 14 H 10 179.0861 1.7 5.30 P1 C 14 H 10 O OH 195.0810 −10.3 0.70 P2 C 14 H 12 O 2 OH H H OH 213.0916 −6.1 0.52 Benz[α]anthracene Original molecule C 18 H 12 229.1017 −4.8 5.90 B1 C 14 H 10 179.0861 1.7 5.30 229.1017 −4.8 5.90 B1 C14H10 J. Fungi 2023, 9, x FOR PEER REVIEW 17 of 20 D.R.O.-H.,C.C., N.A., C.P. and E.A.; supervision, C.C., N.A. and E.A.; project administration, C.C., N.A. and E.A.; funding acquisition, C.C., N.A. and E.A. All authors have read and agreed to the published version of the manuscript. Funding: This research was funded by Ministry of Higher Education and Scientific Research in Tunisia, grant for N.H.; FEDER/Junta de Andalucía-Consejería de TransformaciónEconómica, Industria, Conocimiento y Universidades (B-RNM-204-UGR20) and Secretaria de Investigación y posgrado SIP of IPN (Project number 20230427). Institutional Review Board Statement: Not applicable Informed Consent Statement: Not applicable Data Availability Statement: Not applicable Acknowledgments: Neila Hkiri gratefully acknowledges the scholarship from the Ministry of Higher Education and Scientific Research in Tunisia. E.A. would like to thank the FEDER/Junta de Andalucía-Consejería de Transformación Económica, Industria, Conocimiento y Universidades(B-RNM-204-UGR20) for providing funding. Olicón-Hernández would like to thank the Secretaría de Investigación y posgrado SIP of IPN (Project number 20230427) as well as CONACyT. Authors acknowledge Gabriela Angeles de Paz and Tatiana Robledo-Mahón for her support and the Center of Scientific Instrumentation of the University of Granada (Granada, Spain) for the analytical support. Conflicts of Interest: The authors declare no conflict of interest. Appendix A Table A1. Phenanthrene and benz(α)anthracene metabolites after fungal biodegradation. Metabolite Elemental Composition Chemical Structure Molecular Weigh Error (ppm) Retention Time (min) Phenanthrene Original molecule C 14 H 10 179.0861 1.7 5.30 P1 C 14 H 10 O OH 195.0810 −10.3 0.70 P2 C 14 H 12 O 2 OH H H OH 213.0916 −6.1 0.52 Benz[α]anthracene Original molecule C 18 H 12 229.1017 −4.8 5.90 B1 C 14 H 10 179.0861 1.7 5.30 179.0861 1.7 5.30 B2 C14H10O J. Fungi 2023, 9, x FOR PEER REVIEW 18 of 20 B2 C14H10O O 195.0810 −1.5 5.72 B3 C14H8O2 209.0603 2.4 6.84 B4 C8H4O3 O O O 149.0239 −9.4 6.31 B5 C8H6O4 O OHO OH 167.0344 −3.6 6.27 References 1. Arul, M.N.; Alemu, A.K.; Goswami, L.;Pakshirajan, K.; Pugazhenthi, G. Waste litchi peels for Cr (VI) removal from synthetic wastewater in batch and continuous systems: Sorbent characterization, regeneration and reuse study. J. Environ. Eng. 2016, 142, C4016001. https://doi.org/10.1061/(ASCE)EE.1943-7870.0001099. 2. Titaley, I.A.;Chlebowski, A.; Truong, L.; Tanguay, R.L.; Massey Simonich, S.L. Identification and toxicological evaluation of unsubstituted PAHs and novel PAH derivatives in pavement sealcoat products. Environ. Sci. Tech. Let. 2016, 3, 234–242. https://doi.org/10.1021/acs.estlett.6b00116. 3. Wang, J.; Deng, P.; Wei, X.; Zhang, X.; Liu, J.; Huang, Y.; She, J.; Liu, Y.; Wan, Y.; Hu, H.; et al. Hidden risks from potentially toxic metal (loid) s in paddy soils-rice and source apportionment using lead isotopes: A case study from China. Sci. Total Environ. 2023, 856, 158883.https://doi.org/10.1016/j.scitotenv.2022.158883. 4. Ali, M.; Song, X.; Ding, D.; Wang, Q.; Zhang, Z.; Tang, Z. Bioremediation of PAHs and heavy metals co-contaminated soils: Challenges and enhancement strategies. Environ. Pollut. 2021, 295, 118686. https://doi.org/10.1016/j.envpol.2021.118686. 5. Kadri, T.;Rouissi, T.;Brar, S.K.;Cledon, M.; Sarma, S.; Verma, M. Biodegradation of polycyclic aromatic hydrocarbons (PAHs) by fungal enzymes: A review. J. Environ. Sci.2017, 51, 52–74. https://doi.org/10.1016/j.jes.2016.08.023. 6. Janicki, T.;Długoński, J.;Krupiński, M. Detoxification and simultaneous removal of phenolic xenobiotics and heavy metals with endocrine-disrupting activity by the non-ligninolytic fungus Umbelopsisisabellina. J. Hazard. Mater. 2018, 360, 661–669. https://doi.org/10.1016/j.jhazmat.2018.08.047. 7. Deshmukh, R.; Khardenavis, A.A.; Purohit, H.J. Diverse metabolic capacities of fungi for bioremediation. Indian J. Microbiol.2016, 56, 247–264. https://doi.org/10.1007/s12088-016-0584-6. 8. Priyadarshini, E.; Priyadarshini, S.S.; Cousins, B.G.; Pradhan, N. Metal-Fungus interaction: Review on cellular processes underlying heavy metal detoxification and synthesis of metal nanoparticles. Chemosphere 2021, 274, 129976. https://doi.org/10.1016/j.chemosphere.2021.129976. 9. Al-Dossary, M.A.; Abood, S.A.; Al-Saad, H.T. Factors affecting polycyclic aromatic hydrocarbon biodegradation by Aspergillus flavus. Remediation 2020, 30, 17–25. https://doi.org/10.1002/rem.21658. 10. Agrawal, N.; Barapatre, A.; Shahi, M.P.; Shahi, S.K. Biodegradation pathway of polycyclic aromatic hydrocarbons by ligninolytic fungus Podoscyphaelegans strain FTG4 and phytotoxicity evaluation of their metabolites. Environ. Process. 2021, 8, 1307–1335. https://doi.org/10.1007/s40710-021-00525-z. 11. Chen, S.H.;Cheow, Y.L.; Ng, S.L.; Ting, A.S.Y. Mechanisms for metal removal established via electron microscopy and spectroscopy: A case study on metal tolerant fungi Penicilliumsimplicissimum. J. Hazard. Mater. 2019, 362, 394–402.https://doi.org/10.1016/j.jhazmat.2018.08.077. 12. Zhang, D.; Yin, C.; Abbas, N.; Mao, Z.; Zhang, Y. Multiple heavy metal tolerance and removal by an earthworm gut fungus TrichodermabrevicompactumQYCD-6. Sci Rep. 2020, 10, 6940. https://doi.org/10.1038/s41598-020-63813-y. 13. Zhang, W.; Zhuang, L.; Yuan, Y.; Tong, L.; Tsang, D.C. Enhancement of phenanthrene adsorption on a clayey soil and clay minerals by coexisting lead or cadmium. Chemosphere 2011, 83, 302–310. https://doi.org/10.1016/j.chemosphere.2010.12.056. O O 195.0810 −1.5 5.72 B3 C14H8O2 J. Fungi 2023, 9, x FOR PEER REVIEW 18 of 20 B2 C14H10O O 195.0810 −1.5 5.72 B3 C14H8O2 209.0603 2.4 6.84 B4 C8H4O3 O O O 149.0239 −9.4 6.31 B5 C8H6O4 O OHO OH 167.0344 −3.6 6.27 References 1. Arul, M.N.; Alemu, A.K.; Goswami, L.;Pakshirajan, K.; Pugazhenthi, G. Waste litchi peels for Cr (VI) removal from synthetic wastewater in batch and continuous systems: Sorbent characterization, regeneration and reuse study. J. Environ. Eng. 2016, 142, C4016001. https://doi.org/10.1061/(ASCE)EE.1943-7870.0001099. 2. Titaley, I.A.;Chlebowski, A.; Truong, L.; Tanguay, R.L.; Massey Simonich, S.L. Identification and toxicological evaluation of unsubstituted PAHs and novel PAH derivatives in pavement sealcoat products. Environ. Sci. Tech. Let. 2016, 3, 234–242. https://doi.org/10.1021/acs.estlett.6b00116. 3. Wang, J.; Deng, P.; Wei, X.; Zhang, X.; Liu, J.; Huang, Y.; She, J.; Liu, Y.; Wan, Y.; Hu, H.; et al. Hidden risks from potentially toxic metal (loid) s in paddy soils-rice and source apportionment using lead isotopes: A case study from China. Sci. Total Environ. 2023, 856, 158883.https://doi.org/10.1016/j.scitotenv.2022.158883. 4. Ali, M.; Song, X.; Ding, D.; Wang, Q.; Zhang, Z.; Tang, Z. Bioremediation of PAHs and heavy metals co-contaminated soils: Challenges and enhancement strategies. Environ. Pollut. 2021, 295, 118686. https://doi.org/10.1016/j.envpol.2021.118686. 5. Kadri, T.;Rouissi, T.;Brar, S.K.;Cledon, M.; Sarma, S.; Verma, M. Biodegradation of polycyclic aromatic hydrocarbons (PAHs) by fungal enzymes: A review. J. Environ. Sci.2017, 51, 52–74. https://doi.org/10.1016/j.jes.2016.08.023. 6. Janicki, T.;Długoński, J.;Krupiński, M. Detoxification and simultaneous removal of phenolic xenobiotics and heavy metals with endocrine-disrupting activity by the non-ligninolytic fungus Umbelopsisisabellina. J. Hazard. Mater. 2018, 360, 661–669. https://doi.org/10.1016/j.jhazmat.2018.08.047. 7. Deshmukh, R.; Khardenavis, A.A.; Purohit, H.J. Diverse metabolic capacities of fungi for bioremediation. Indian J. Microbiol.2016, 56, 247–264. https://doi.org/10.1007/s12088-016-0584-6. 8. Priyadarshini, E.; Priyadarshini, S.S.; Cousins, B.G.; Pradhan, N. Metal-Fungus interaction: Review on cellular processes underlying heavy metal detoxification and synthesis of metal nanoparticles. Chemosphere 2021, 274, 129976. https://doi.org/10.1016/j.chemosphere.2021.129976. 9. Al-Dossary, M.A.; Abood, S.A.; Al-Saad, H.T. Factors affecting polycyclic aromatic hydrocarbon biodegradation by Aspergillus flavus. Remediation 2020, 30, 17–25. https://doi.org/10.1002/rem.21658. 10. Agrawal, N.; Barapatre, A.; Shahi, M.P.; Shahi, S.K. Biodegradation pathway of polycyclic aromatic hydrocarbons by ligninolytic fungus Podoscyphaelegans strain FTG4 and phytotoxicity evaluation of their metabolites. Environ. Process. 2021, 8, 1307–1335. https://doi.org/10.1007/s40710-021-00525-z. 11. Chen, S.H.;Cheow, Y.L.; Ng, S.L.; Ting, A.S.Y. Mechanisms for metal removal established via electron microscopy and spectroscopy: A case study on metal tolerant fungi Penicilliumsimplicissimum. J. Hazard. Mater. 2019, 362, 394–402.https://doi.org/10.1016/j.jhazmat.2018.08.077. 12. Zhang, D.; Yin, C.; Abbas, N.; Mao, Z.; Zhang, Y. Multiple heavy metal tolerance and removal by an earthworm gut fungus TrichodermabrevicompactumQYCD-6. Sci Rep. 2020, 10, 6940. https://doi.org/10.1038/s41598-020-63813-y. 13. Zhang, W.; Zhuang, L.; Yuan, Y.; Tong, L.; Tsang, D.C. Enhancement of phenanthrene adsorption on a clayey soil and clay minerals by coexisting lead or cadmium. Chemosphere 2011, 83, 302–310. https://doi.org/10.1016/j.chemosphere.2010.12.056. O O 209.0603 2.4 6.84 B4 C8H4O3 J. Fungi 2023, 9, x FOR PEER REVIEW 18 of 20 B2 C14H10O O 195.0810 −1.5 5.72 B3 C14H8O2 209.0603 2.4 6.84 B4 C8H4O3 O O O 149.0239 −9.4 6.31 B5 C8H6O4 O OHO OH 167.0344 −3.6 6.27 References 1. Arul, M.N.; Alemu, A.K.; Goswami, L.;Pakshirajan, K.; Pugazhenthi, G. Waste litchi peels for Cr (VI) removal from synthetic wastewater in batch and continuous systems: Sorbent characterization, regeneration and reuse study. J. Environ. Eng. 2016, 142, C4016001. https://doi.org/10.1061/(ASCE)EE.1943-7870.0001099. 2. Titaley, I.A.;Chlebowski, A.; Truong, L.; Tanguay, R.L.; Massey Simonich, S.L. Identification and toxicological evaluation of unsubstituted PAHs and novel PAH derivatives in pavement sealcoat products. Environ. Sci. Tech. Let. 2016, 3, 234–242. https://doi.org/10.1021/acs.estlett.6b00116. 3. Wang, J.; Deng, P.; Wei, X.; Zhang, X.; Liu, J.; Huang, Y.; She, J.; Liu, Y.; Wan, Y.; Hu, H.; et al. Hidden risks from potentially toxic metal (loid) s in paddy soils-rice and source apportionment using lead isotopes: A case study from China. Sci. Total Environ. 2023, 856, 158883.https://doi.org/10.1016/j.scitotenv.2022.158883. 4. Ali, M.; Song, X.; Ding, D.; Wang, Q.; Zhang, Z.; Tang, Z. Bioremediation of PAHs and heavy metals co-contaminated soils: Challenges and enhancement strategies. Environ. Pollut. 2021, 295, 118686. https://doi.org/10.1016/j.envpol.2021.118686. 5. Kadri, T.;Rouissi, T.;Brar, S.K.;Cledon, M.; Sarma, S.; Verma, M. Biodegradation of polycyclic aromatic hydrocarbons (PAHs) by fungal enzymes: A review. J. Environ. Sci.2017, 51, 52–74. https://doi.org/10.1016/j.jes.2016.08.023. 6. Janicki, T.;Długoński, J.;Krupiński, M. Detoxification and simultaneous removal of phenolic xenobiotics and heavy metals with endocrine-disrupting activity by the non-ligninolytic fungus Umbelopsisisabellina. J. Hazard. Mater. 2018, 360, 661–669. https://doi.org/10.1016/j.jhazmat.2018.08.047. 7. Deshmukh, R.; Khardenavis, A.A.; Purohit, H.J. Diverse metabolic capacities of fungi for bioremediation. Indian J. Microbiol.2016, 56, 247–264. https://doi.org/10.1007/s12088-016-0584-6. 8. Priyadarshini, E.; Priyadarshini, S.S.; Cousins, B.G.; Pradhan, N. Metal-Fungus interaction: Review on cellular processes underlying heavy metal detoxification and synthesis of metal nanoparticles. Chemosphere 2021, 274, 129976. https://doi.org/10.1016/j.chemosphere.2021.129976. 9. Al-Dossary, M.A.; Abood, S.A.; Al-Saad, H.T. Factors affecting polycyclic aromatic hydrocarbon biodegradation by Aspergillus flavus. Remediation 2020, 30, 17–25. https://doi.org/10.1002/rem.21658. 10. Agrawal, N.; Barapatre, A.; Shahi, M.P.; Shahi, S.K. Biodegradation pathway of polycyclic aromatic hydrocarbons by ligninolytic fungus Podoscyphaelegans strain FTG4 and phytotoxicity evaluation of their metabolites. Environ. Process. 2021, 8, 1307–1335. https://doi.org/10.1007/s40710-021-00525-z. 11. Chen, S.H.;Cheow, Y.L.; Ng, S.L.; Ting, A.S.Y. Mechanisms for metal removal established via electron microscopy and spectroscopy: A case study on metal tolerant fungi Penicilliumsimplicissimum. J. Hazard. Mater. 2019, 362, 394–402.https://doi.org/10.1016/j.jhazmat.2018.08.077. 12. Zhang, D.; Yin, C.; Abbas, N.; Mao, Z.; Zhang, Y. Multiple heavy metal tolerance and removal by an earthworm gut fungus TrichodermabrevicompactumQYCD-6. Sci Rep. 2020, 10, 6940. https://doi.org/10.1038/s41598-020-63813-y. 13. Zhang, W.; Zhuang, L.; Yuan, Y.; Tong, L.; Tsang, D.C. Enhancement of phenanthrene adsorption on a clayey soil and clay minerals by coexisting lead or cadmium. Chemosphere 2011, 83, 302–310. https://doi.org/10.1016/j.chemosphere.2010.12.056. O O 149.0239 −9.4 6.31 B5 C8H6O4 J. Fungi 2023, 9, x FOR PEER REVIEW 18 of 20 B2 C14H10O O 195.0810 −1.5 5.72 B3 C14H8O2 209.0603 2.4 6.84 B4 C8H4O3 O O O 149.0239 −9.4 6.31 B5 C8H6O4 O OHO OH 167.0344 −3.6 6.27 References 1. Arul, M.N.; Alemu, A.K.; Goswami, L.;Pakshirajan, K.; Pugazhenthi, G. Waste litchi peels for Cr (VI) removal from synthetic wastewater in batch and continuous systems: Sorbent characterization, regeneration and reuse study. J. Environ. Eng. 2016, 142, C4016001. https://doi.org/10.1061/(ASCE)EE.1943-7870.0001099. 2. Titaley, I.A.;Chlebowski, A.; Truong, L.; Tanguay, R.L.; Massey Simonich, S.L. Identification and toxicological evaluation of unsubstituted PAHs and novel PAH derivatives in pavement sealcoat products. Environ. Sci. Tech. Let. 2016, 3, 234–242. https://doi.org/10.1021/acs.estlett.6b00116. 3. Wang, J.; Deng, P.; Wei, X.; Zhang, X.; Liu, J.; Huang, Y.; She, J.; Liu, Y.; Wan, Y.; Hu, H.; et al. Hidden risks from potentially toxic metal (loid) s in paddy soils-rice and source apportionment using lead isotopes: A case study from China. Sci. Total Environ. 2023, 856, 158883.https://doi.org/10.1016/j.scitotenv.2022.158883. 4. Ali, M.; Song, X.; Ding, D.; Wang, Q.; Zhang, Z.; Tang, Z. Bioremediation of PAHs and heavy metals co-contaminated soils: Challenges and enhancement strategies. Environ. Pollut. 2021, 295, 118686. https://doi.org/10.1016/j.envpol.2021.118686. 5. Kadri, T.;Rouissi, T.;Brar, S.K.;Cledon, M.; Sarma, S.; Verma, M. Biodegradation of polycyclic aromatic hydrocarbons (PAHs) by fungal enzymes: A review. J. Environ. Sci.2017, 51, 52–74. https://doi.org/10.1016/j.jes.2016.08.023. 6. Janicki, T.;Długoński, J.;Krupiński, M. Detoxification and simultaneous removal of phenolic xenobiotics and heavy metals with endocrine-disrupting activity by the non-ligninolytic fungus Umbelopsisisabellina. J. Hazard. Mater. 2018, 360, 661–669. https://doi.org/10.1016/j.jhazmat.2018.08.047. 7. Deshmukh, R.; Khardenavis, A.A.; Purohit, H.J. Diverse metabolic capacities of fungi for bioremediation. Indian J. Microbiol.2016, 56, 247–264. https://doi.org/10.1007/s12088-016-0584-6. 8. Priyadarshini, E.; Priyadarshini, S.S.; Cousins, B.G.; Pradhan, N. Metal-Fungus interaction: Review on cellular processes underlying heavy metal detoxification and synthesis of metal nanoparticles. Chemosphere 2021, 274, 129976. https://doi.org/10.1016/j.chemosphere.2021.129976. 9. Al-Dossary, M.A.; Abood, S.A.; Al-Saad, H.T. Factors affecting polycyclic aromatic hydrocarbon biodegradation by Aspergillus flavus. Remediation 2020, 30, 17–25. https://doi.org/10.1002/rem.21658. 10. Agrawal, N.; Barapatre, A.; Shahi, M.P.; Shahi, S.K. Biodegradation pathway of polycyclic aromatic hydrocarbons by ligninolytic fungus Podoscyphaelegans strain FTG4 and phytotoxicity evaluation of their metabolites. Environ. Process. 2021, 8, 1307–1335. https://doi.org/10.1007/s40710-021-00525-z. 11. Chen, S.H.;Cheow, Y.L.; Ng, S.L.; Ting, A.S.Y. Mechanisms for metal removal established via electron microscopy and spectroscopy: A case study on metal tolerant fungi Penicilliumsimplicissimum. J. Hazard. Mater. 2019, 362, 394–402.https://doi.org/10.1016/j.jhazmat.2018.08.077. 12. Zhang, D.; Yin, C.; Abbas, N.; Mao, Z.; Zhang, Y. Multiple heavy metal tolerance and removal by an earthworm gut fungus TrichodermabrevicompactumQYCD-6. Sci Rep. 2020, 10, 6940. https://doi.org/10.1038/s41598-020-63813-y. 13. Zhang, W.; Zhuang, L.; Yuan, Y.; Tong, L.; Tsang, D.C. Enhancement of phenanthrene adsorption on a clayey soil and clay minerals by coexisting lead or cadmium. Chemosphere 2011, 83, 302–310. https://doi.org/10.1016/j.chemosphere.2010.12.056. O O 167.0344 −3.6 6.27 References 1. Arul, M.N.; Alemu, A.K.; Goswami, L.; Pakshirajan, K.; Pugazhenthi, G. 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