Degradation of endocrine-disrupting chemicals in wastewater by new thermophilic fungal isolates and their laccases

Abstract

Thermophilic fungi are known to develop different metabolic and catabolic activities that enable them to function at elevated temperatures. Screening heat-resistant fungi, as promising resources for enzymatic activities, are still recommended. A total of eleven wood-decay thermophilic fungal strains were isolated from decaying organic materials (DOM) collected from arid areas of Khulais (Saudi Arabia). Six of these isolates are laccase-producing thermophilic strains growing at 50 °C. Among Laccase positive (Lac+) isolates, Chaetomium brasiliense (G3), Canariomyces notabilis (KW1), and Paecilomyces formosus (KW3) were exploited to treat single selected endocrine-disrupting chemicals (EDCs) that belonged to different classes (synthetic steroid hormone: 17α-ethinyl estradiol (EE2), and alkylphenols:4-tert-butylphenol (4-t-BP)). Chaetomium sp. was selected due to its potentialities against target EDCs, and then, their laccases were extracted and exploited for the biocatalytic degradation of treated municipal sewage wastewaters (TMWW) mixed with 4-t-BP and EE2. The results show that within 2 h of catalyzing at 50 °C, laccase could degrade 60 ± 4.8% of 4-t-BP; however, it oxidized EE2 less efficiently, reaching 35 ± 4.1%. The influence of some redox mediators on the laccase oxidation system was investigated. The 1-hydroxybenzotriazole (HBT) and syringaldehyde led to the highest transformation rates of EE2 (approximately 80 ± 2.4%). Near-total removal (90 ± 7.2%) of 4-t-BP was achieved with TEMPO in 2 h. With the metabolites identified through gas chromatography-tandem mass spectrometry (GC–MS), metabolic pathways of degradation were suggested. The results highlight the potential of Chaetomium sp. strains in the conversion of micropollutants.

Supplementary Information

The online version contains supplementary material available at 10.1007/s13205-022-03439-1.

Introduction

Endocrine-disrupting chemicals (EDCs) as an emerging group of trace organic pollutants are becoming an environmental issue as they persistently pollute surface water worldwide, affecting biological functions and harming wildlife and human beings at extremely low doses (Teles et al. 2004; Křesinová et al. 2018). Endocrine disruptors (EDs) are anthropogenic compounds that can mimic hormones, thus interfering with organisms’ endogenous hormonal systems by disrupting physiological processes such as synthesis, metabolism, and the excretion of hormones in the body (Lange et al. 2002; Whitman 2017).

Toxicology studies, conducted both in vitro and in vivo in animal systems, have demonstrated that the effects associated with endocrine disruptive activity in humans include changes in neurological and immunological functions, female reproductive disorders, and reduced fertility (Esplugas et al. 2007; Sarangapani et al. 2017). The health effects of exposure to EDCs have been investigated elsewhere and include thyroid dysfunction, obesity, diabetes, metabolic disorders, and cancers (Kumar et al. 2020).

The wide range of EDCs is currently grouped into two major categories. The first group includes natural hormones found in humans and animals, such as oestrogens (estrone (E1), 17β-estradiol (E2), and estriol (E3)), progesterone, and testosterone. Phytoestrogens, such as isoflavonoids and coumestrol, are also naturally present in some plants (Vandenberg et al. 2009).

The second group includes man-made or synthetically produced hormones comprising oral contraceptives, such as ethinylestradiol (EE2), and animal feed additives. Moreover, a variety of industrially synthetized chemicals are also EDCs including polychlorobiphenyls (PCBs), phthalates, organochlorine pesticides (OCPs), plasticizers, and pharmaceutical compounds that are released from many sources into the environment (Combalbert and Hernandez-Raquet 2010; De Toni et al. 2017; Chen et al. 2018). This broad class of chemicals is characterized by their resistance to further degradation, bioaccumulation in biota, and potential to harm humans and wildlife (Barbosa et al. 2016).

Among the various EDCs from the environment, oestrogen and alkylphenols have received great attention due to their bioaccumulation and toxicity in the local environment.

Oestrogen is known for its highly oestrogenic activity in wastewater plants (WWP) at low concentrations (ng L⁻¹), including natural steroidal oestrogens such as 17β-estradiol (E2) and the synthetic contraceptive 17α-ethinyl estradiol (EE2) (Chen et al. 2018). EE2 is one of the most widely used medications for livestock and in aquaculture, as well as for humans.

Additionally, alkylphenol such as 4-tert-butyl-phenol (4-t-BP) is commonly used to produce phenolic, polycarbonate, and epoxy resins for industrial purposes. Toxicology studies, conducted both in vitro and in vivo in animal models, have demonstrated the hazardous effects of these chemicals on human welfare.

Estrogenic active compounds may enter the environment through several channels, including industrial activities and the disposal of trade wastes (Papaevangelou et al. 2016). These compounds with endocrine-disrupting potency are typically recalcitrant in the environment and must be removed from contaminated sites.

One approach to address the challenge of removing EDCs from the environment is reducing the production and usage of chemicals with endocrine-disrupting activity. Controlling the potential sources of EDCs is necessary to preserve our environment. Further, conventional wastewater treatment plant (WWTP) techniques have been extensively studied for ways to eliminate endocrine disruptors, including precipitation, flocculation, coagulation (Huang et al. 2021a, b), and photochemical oxidation (Wang et al. 2020), and adsorption (Wang et al. 2021). These traditional WWTP processes present certain limitations and disadvantages and most are ineffective to remove EDCs as persistent micropollutants (Syafrudin et al. 2021; Gao et al. 2020; Siegrist et al. 2005).

Therefore, as a viable alternative, biodegradation processes have received increasing interest; these involve using microorganisms or their enzymes to clean up or bioconvert EDCs. (Gao et al. 2020). Those processes are environmentally friendly, economical, effective, and offer a broad range of activity (Zhang et al. 2016).

According to the literature, the biodegradation of EDCs by ligninolytic fungi and their lignin-modifying enzymes (LMEs), i.e. laccase (Lac), lignin-dependent peroxidase (LiP), and manganese-dependent peroxidase (MnP), has attracted significant attention. Among the ligninolytic enzymes, laccases were applied as biocatalysts to transform a wide range of phenolic and nonphenolic substrates (Cajthaml 2015; Daâssi et al. 2016a; Taboada-Puig et al. 2017; Gao et al. 2020).

Most industrial processes occur at high temperatures that emphasize the need for thermostable commercial enzymes. Thermophilic and thermotolerant fungi are characterized by their potential to produce heat-tolerant enzymes that typically have higher thermostability, resistance to denaturing agents, and tolerance of pH variation.

This study aimed to (a) isolate thermophilic fungal strains from decaying wood collected from the desert of Khulais in Saudi Arabia (b) treat EE2 and 4-t-BP supplemented to effluent from WWTP with Chaetomium sp strain and their laccases in the presence of synthetic and natural mediators (d) identify the degradation products of the target EDCs and propose possible metabolic pathways.

Materials and methods

Sampling

Samples of the municipal sewage wastewater MWW were provided by a WWTP located in Jeddah city in Saudi Arabia. The plant was constructed for a maximum inflow of 40 m³ of wastewater per h and an average inflow of 200 m³ per day. The plant is composed of three treatment phases: primary and secondary treatments, followed by ozonation as tertiary treatment, and, finally, a sand filtration step.

A composite sample represents the mixed samples from the effluent (downstream from the secondary treatment) at WWTP during the period of February 2020. The treated samples (TMWW) were sampled in a dry, sterile Cap-Bottle Adaptor, 1 L bottle, PFE Teflon, which was kept on ice during transportation and then stored in the refrigerator (4 °C). The MWW was subsequently filtered through a glass microfiber filter (Whatman 827-055 934-AH, 1.5 μm pore size, Ø 90 mm) (S1 Table).

Chemicals

The EDCs used in this study were 17α-ethinyl estradiol (EE2) and 4-tert-butylphenol (99%) (4-t-BP) (Table 1). Dichloromethane (Cas. N. 75-09-2) and dimethyl sulfoxide (DMSO) (Cas. N. 67-68-5) were used to dissolve the EDCs.

Table 1.

The endocrine-disrupting chemicals used in the current study

The laccase substrates were 2,6-dimethoxyphenol (2,6-DMP) and 2-2′-azinobis (3 ethylbenzthiazoline-6-sulfonic acid) (ABTS). The mediators used were 1-hydroxy benzotriazole (HBT), p-coumaric acid (PCA), syringaldehyde (SYR), and 2,2,6,6-tetramethyl piperidine-1-yloxy (TEMPO). All chemicals were obtained from Sigma-Aldrich Chemical Company (St. Louis, MO, USA).

Isolation and screening of thermophilic laccase-producing fungal strains

Decaying organic materials (DOM) were collected from arid zones in the region of Khulais in Saudi Arabia. To isolate thermophilic fungal strains, DOM were air-dried at 50 °C for 2 to 5 days to enhance the thermotolerant pullulation. Fungal strains were isolated on an antibiotic-supplemented malt extract agar medium (MEA) (30 g L⁻¹, pH 5.5, ampicillin, and streptomycin at 0.01%) (Daâssi et al. 2016a). Plates were incubated at 50 °C for 2 to 7 days, and then, pure fungal strains were obtained by sub-culturing inoculum from the young mycelium developing on the initial medium. The purity of the isolated fungi was confirmed with microscopic examination of the culture at 40 × magnification using a light microscope.

To select laccase-producing fungi, all the fungal isolates were grown on a selective solid MEA medium supplemented with 150 µM copper sulphate (as a Lac inducer) and 5 mM of 2,6-DMP or 1 mM ABTS (Lac substrates) and then incubated at 50 °C for several days. Fungal strains secreting laccases were selected by a visual colour change in the MEA plates after incubation, due to the oxidation of screening substrates (Kiiskinen et al. 2004; Daâssi et al. 2016a).

The laccase-positive strains (Lac (+)) were transferred into 250-mL flasks of malt extract broth (MEB) with 150 µM CuSO4 as an inducer for further characterization.

The thermotolerant and Lac (+) fungal isolates were identified by their colonial and morphological characteristics (Cooney & Emerson 1964).

Optical microscopy

A VHX-5000 optical digital microscope was used to take microscopic pictures of the suspended mycelia prepared from 7-day-old MEA fungal culture plates.

Identification and phylogenetic tree of fungal thermophilic isolates

The selected thermophilic fungal strains were molecularly identified with ITS1/ITS2. After sequencing, ITS sequences were used for homology analysis using blastn as a default parameter for nucleotide sequence homology from the basic local alignment search tool (BLAST). All of the ITS sequences of the fungal isolates have been deposited in the GenBank database under accession numbers MZ841818, MW699894, OK668265, MZ817961, MZ817962, MZ817959.

The neighbour-joining method was used to calculate the evolutionary distances of the isolates (Tamura et al. 2004). The phylogenetic tree was constructed in the MEGA11 program (Tamura et al. 2021) with a total of 1,484 positions in the final dataset.

Comparison of thermophilic laccase-producing fungi performances towards single EDCs

To assess the capacity of fungal treatment to EDCs removal, liquid cultures using malt extract broth (MEB) were conducted. Precultures of the selected high laccase-producing fungal species previously identified from the thermophilic isolates (Chaetomium sp G3, Canariomyces notabilis KW1, Paecilomyces formosus KW3), were prepared in 250-mL cotton-plugged Erlenmeyer flasks containing 100 mL of MEB inoculating from four mycelial plugs (diameter, 3 mm), taken from a 5-day old fungal culture plate.

After 3 days of growth at 30 °C under shaking (150 rpm), fungal mycelia were homogenized using sterilized beaded glasses, and 2% (v/v) aliquot of mycelial suspensions were introduced in 250-mL flasks containing 150 mL of culture broth supplemented with 0.15 mM of CuSO4 as laccase-inducer and single EDCs (200 μM). The concentration of the target EDCs was selected based on preliminary studies, and on the scientific literature.

The Erlenmeyer flasks were incubated in the dark for 12 days under shaking (30 °C; 150 rpm). Aliquots of 3 mL were daily withdrawn and samples were tested for laccase activities and remaining EDCs concentrations over 12 days.

Culture controls were carried out as follows: Flasks not supplemented with EDCs and other flasks inoculated with heat-killed cells by autoclaving (at 121 °C for 20 min) for adsorption estimation. All experiments were performed in triplicate.

At the end of cultivation time, control flasks (without EDCs) cultures were filtered and centrifuged at 7000 rpm for 20 min at 4 °C. The supernatant was lyophilized to be further characterized and exploited in later enzymatic treatment against the selected EDCs in TMWW.

Assay of laccase activity

Laccase activity was determined spectrophotometrically based on the oxidation of 2,6-dimethoxyphenol (2,6-DMP) or 2-2′-azinobis (3-ethylbenzthiazoline-6-sulfonic acid) (ABTS) (Rodríguez et al. 2008). Oxidation of 2 mM ABTS as a nonphenolic substrate by laccase results in the production of a green–blue coloured radical cation (ABTS+·) measurable at 420 nm (ɛ = 36 × 10³ M^–1 cm^–1). However, the oxidation of 2,6-DMP as a phenolic substrate by laccase forms red-brown coloured quinones measurable at 469 nm (ε = 27,500 M⁻¹ cm⁻¹).

The reaction mixture usually consists of 500 µL of acetate buffer (100 mm, pH 5.0) with 500 µL of ABTS (0.4 mM) or 2,6-DMP (5 mM) as substrate and 500 µL of the fungal extracellular medium containing the laccase activity to be measured.

The enzymatic assays were conducted at room temperature. One unit of laccase activity is defined as the formation of 1 µmol of product per min. All assays were performed in duplicate with a Shimadzu UV–Vis 2600-spectrophotometer.

Chaetomium sp. laccase metabolic EDC-degrading capacity

Stock solutions of the studied EDCs were freshly prepared by dissolving them in dichloromethane with 1% DMSO and then diluting them with the TMWW for a later experiment. The sample solutions were filtered with 0.22 µm PTFE syringe filters to obtain final concentrations of 200 µM EE2 and 4-t- BP in the reaction mixtures (TMWW + EE2; TMWW + 4-t-BP). All experiments in EDCs’ degradation were performed using 100 ml disposable flasks in 20 ml final reaction volumes.

The reaction mixture, containing 50 mM acetate buffer at a pH of 5.0, 0.2 mM of the redox mediator, EDCs (200 µM for each of EE2 and 4-t-BP), and Chaetomium sp laccase (1.5 U mL⁻¹), was incubated in the dark at 50 °C for 2 h. Our laboratory had previously optimized these reactions.

Reactions were performed both in the presence and absence of laccase mediators. Synthetic redox mediators (1-hydroxy benzotriazole (HBT), p-coumaric acid (PCA), and natural (syringaldehyde (SYR)) were investigated for their ability to improve the degradation rates of laccase oxidation of EDCs.

A reaction mixture without an enzyme was prepared under the same conditions to detect possible degradation not due to enzyme activity. Controls contained heat-killed enzymes, whereas blanks used all components of the reaction mixture except for the EDCs. All experiments were performed in duplicate.

Sample solutions were filtered with 0.22 µm PTFE syringe filters and the concentrations of EDCs in the supernatant were determined with a Gaz chromatograph 1200 Series (Agilent Technologies, USA).

All experiments were performed in triplicate, and the average values and standard deviations are reported. The removal efficiency was calculated as:

$$\text{Removal efficiency}\left(\%\right)=\left(C_0-C_{\text{t}}\right)\ast 100/C_0$$ 1

where C₀ is the initial concentration of EE2 (mg L⁻¹) and C_t is the residual concentration of EDCs (mg L⁻¹) at a given time.

Controls and test samples were extracted with dichloromethane using a separating funnel. The organic part from the separating funnel was collected very carefully and treated with anhydrous sodium sulphate to eliminate water from the organic solution. The organic part was completely evaporated using a rotary evaporator (Concentrator 5301Eppendorf).

Gas chromatography–tandem mass spectrometry (GC–MS)

Controls and test samples were extracted with dichloromethane using a separating funnel. The organic part was completely evaporated using IKA rotary evaporator.

Before GC–MS analysis, the concentrated extracted samples were trimethylsilylated (TMS), as Daâssi et al. suggested (2016b).

After derivatization, the degradation products resulting from the laccase oxidation of the EE2 and 4-t-BP were identified with GC–MS equipment (GC-2010 Plus coupled to a GCMS-QP 2010 Plus mass spectrometer (Shimadzu)).

The GC column was (Rtx 5 ms). The injector and detector were programmed at 305 °C for 1 μL volume, splitless per 1 min. Helium at 100 kPa was the carrier gas. Temperature programming analysis was started at 120 °C per min, increasing at a rate of 10 °C per min, and finished at 300 °C per 6 min (30 min total). The MS analysis occurred in selected ion monitoring (SIM) mode with electron impact (EI) ionization for quantitation. EI experiments were performed at 70 eV as electron energy, 200 °C, and 45–800 m/z.

The residual DDT after enzymatic degradation and the metabolites extracted from the controls were identified and compared with the WILEY mass spectra database.

Results and discussion

Isolation and screening of thermophilic and laccase-producing fungi

Wood-rotting fungi were isolated from wood debris collected from soil desert areas of Khulais (Jeddah City, Saudi Arabia) and screened for newly isolated thermophilic and laccase-producing strains. A total of 20 fungal strains were isolated to obtain pure strains using the MEA plate-agar method. Among the isolates, 11 are thermophilic strains, growing at 50 °C.

The thermophilic strains included five with positive oxidative activities on 2,6-DMP and seven on MEA plates with ABTS (Table 2).

Table 2.

Screening of thermophilic laccase-producing fungal isolates on MEA supplemented with 2,6-DMP (5 mM) and ABTS (1 mM) after 5 days of cultivation at 50 °C

Isolates ID	ABTS		2,6-DMP		Growth at 50 °C
	Colony diameter [mm]	Oxidation	Colony diameter [mm]	Oxidation
G3	25	+++	35	+++	**
KW1	45	++	40	++	**
KW2	22	−	35	−	**
KW3	22	+	30	++	**
KW4	40	−	20	−	*
KW5	60	−	45	−	**
KW6	22	−	27	−	**
KBR3	20	−	45	±	**
KB14	45	+	25	+	**
KB17	22	−	50	+	***

Activity:+++high;++medium;+low; ± ambiguous; −none

Growth: ***Excellent growth (colony diameter > 40 mm); **good growth (colony diameter 21–40 mm); *low growth (colony diameter < 21)

Table 2 illustrates the qualitative test to check for laccase activity on agar plates containing phenolic as well as nonphenolic substrates.

The data presented in Table 2 demonstrate that among the tested thermophilic fungal isolates, the strains designated G3, KW1, KW3, and KB14 exhibited laccase activity, which was detected by an orange halo in plates containing 2,6-DMP, while ABTS oxidation was indicated by a green halo. The strains KBR3 and KB17 were able to oxidase the phenolic compound (2,6-DMP) but not the nonphenolic substrate (ABTS).

According to Daâssi et al. (2016a), screening for laccase producers on an MEA medium containing coloured indicator compounds can rapidly and qualitatively detect laccase activity; however, liquid cultivation is still essential to measure the quantity of laccase activity. Thus, the positive isolates showing high colour intensity in the plated agar assay were screened for the production of laccase on semi-solid-state cultures with 150 µM CuSO4 at 30 °C to quantify and characterize the laccase activity.

In agreement with our study, Saroj et al. (2018) isolated fifteen thermophilic fungi from soil that produced extracellular lignocellulolytic enzymes. According to Ben Younes et al. (2011), 37 thermophilic fungi were isolated from different Tunisian biotopes. Interestingly, enzymes from extremophilic sources, especially laccases, have enormous utility in several biotechnological processes. Mtibaà et al. (2017) described the production and purification of a thermostable laccase from Chaetomium sp. that was newly isolated from arid soil.

The morphological aspect and the purity of the six isolates were proven with microscopic observation (Fig. 1A–F). Then, liquid cultures of the pure isolates on MEB were conducted for molecular analysis, relying on primers coordinated with the DNA sequences of the ITS region. Based on the combination of micro-morphological observation and the ITS identification.

Fig. 1.

Microscopic image projection system (MIPS) photograph showing morphological characteristics of thermophilic fungi: (A) Paecilomyces formosus (OK668265), (B) Aspergillus fumigatiaffinis (MZ817962), (C) Neurospora sp. (MZ817961), (D) Chaetomium sp. (MZ841818), (E) Canariomyces notabilis (MW699894), and (F) Acrophialophora sp. (MZ817959)

Identification and phylogenetic analysis

Using Blastn to align findings with the National Center for Biotechnology Information (NCBI) databases, the GenBank database was searched for the highest percentage of identity to the query ITS sequence of the isolated strain. The ITS regions of the fungal strains G3, KW1, KW3, KB14, KB17, and KBR3 were submitted to the GenBank database with accession numbers MZ841818, MW699894, OK668265, MZ817961, MZ817962, and MZ817959, respectively.

Table 3 illustrates the closely related species (homologies greater than 97%) obtained from databases (Rossello Mora and Amman 2001).

Table 3.

Molecular identification of thermophilic fungal isolates

Isolates ID	Max identity (%)	Strains of the close match (accession number)	Identification	GenBank accession number(s)
G3	99.79	Chaetomium brasiliense isolate UM 235 [JX966545.1] Ovatospora brasiliensis strain CBS [MH865522.1]	Chaetomium sp	MZ841818
KW1	99.78	Canariomyces notabilis CBS [NR_165232.1]	Canariomyces notabilis	MW699894
KW3	98.38	Paecilomyces formosus strain CCTU140 [MH758718.1] Byssochlamys spectabilis isolate (MK397308.1)	Paecilomyces formosus	OK668265
KB14	99.44	Sordaria sp. P44E2 [JN207345.1]	Neurospora sp.	MZ817961
KB17	99.71	Aspergillus sp. isolate SR40 [KX009134.1]	Aspergillus fumigatiaffinis	MZ817962
KBR3	97.65	Acrophialophora sp. SQU-QU15 [KU945957.1]	Acrophialophora sp	MZ817959

BLAST search revealed that the ITS of the isolate G3 matched 99.79% with the sequences of Chaetomium brasiliense isolate UM 235 (accession n° JX966545.1) and Ovatospora brasiliensis strain CBS (accession n° MH865522.1).

Based on the morphological aspects of the Chaetomium sp. given in Fig. 1D, especially the grey colour of the plate, ball-shaped asci, and the perithecia with dichotomously branched terminal hairs (Hibbett et al. 2016), we propose to affiliate our strain with Chaetomium brasiliense.

The closest match for ITS sequence of KW1 (MW699894) was Canariomyces notabilis (NR 165232.1) with 99.78% identity and Thielavia subthermophila (MG551575.1) with 99.53% similarity.

As presented in Fig. 1E, the micromorphological examinations of the strain KW1 led us to attribute the isolate to the genus Canariomyces notabilis (ascoma wall similar to those of some Microascaceae). The genus Thielavia is known morphologically to have nonostiolate ascomata with a thin peridium of textura epidermoidea and smooth, single-celled, pigmented ascospores with one germ pore (Wang et al. 2020).

The KW3 isolate exhibited homology with close strains including Paecilomyces sp. (MH758718.1), Byssochlamys sp. (MK397308.1) (98.38%). According to Samson et al. (2009), Paecilomyces and Byssochlamys are taxonomically related strains and are often illustrated in the literature as heat-resistant and mycotoxin-producing fungal strains.

In GenBank, Blastn comparison of the ITS sequence showed that the KB14 strain was Sordaria sp. (JN207345.1) with 99.44% similarity, while the closest match for the ITS region was Neurospora tetraspora (MH859472.1) with 99.06% identity. As shown in Fig. 1C, the microscopic examination of KB14 traits demonstrated septate hyphae and typical perithecium showing young asci. The phylogenetic tree (Fig. 2) suggests that Neurospora tetraspora is the closest strain to the isolate KB14 (Huang et al. 2021a, b).

Fig. 2.

Neighbour-joining phylogenetic tree constructed based on the ClustralW alignment of ITS sequences of the isolated thermophilic fungi, with homologue sequences obtained from the NCBI GenBank

Chaetomium brasiliense (G3), Canariomyces notabilis (KW1), Paecilomyces formosus (KW3), Neurospora sp. (KB14), Aspergillus fumigatiaffinis (KB17), and Acrophialophora sp. (KBR3) were identified as thermophilic fungal laccase-producing strains. All of the fungal isolates were known in the literature to be heat-resistant, laccase-producing strains. Similarly, Agrawal et al. (2021) reported the isolation and identification of the species Acrophialophora levis as a thermophilic fungus. In agreement with our study, the thermophilous Ascomycota are restricted to the orders Sordariales, Eurotiales, and Onygenales (Hutchinson et al. 2019). Accordingly, Korniłłowicz-Kowalska and Kitowski (2012) reported that Aspergillus fumigatus exceeded 50% of the total fungi growing at 45 °C. Additionally, the genus Fusarium sp. was isolated from soil and selected as a thermophilic strain in Saroj et al.’s (2018) study.

Our collection of thermophilic strains is supported by previous findings in the literature. For instance, Ahirwar et al. (2017) reported the isolation of 68 thermophilic and thermotolerant fungi from different self-heated habitats based on their ability to grow at 50 °C.

Comparison of thermophilic laccase-producing fungi performances towards single EDCs

Degradative potential of Chaetomium brasiliense (G3), Canariomyces notabilis (KW1), Paecilomyces formosus (KW3) was tested against selected EDCs. All fungal cells were able to grow in the MEB culture added with single EDCs, displaying various removal rates of EDCs.

The biodegradative potentials of the selected laccase-producing strains (Chaetomium brasiliense (G3), Canariomyces notabilis (KW1), Paecilomyces formosus (KW3)) were tested against the investigated EDCs during incubation periods (Fig. 3).

Fig. 3.

EDCs removal rates (%) by Chaetomium brasiliense (G3), Canariomyces notabilis (KW1) and Paecilomyces formosus (KW3) after 2, 4, 8 and 12 days of treatment in broth culture (MEB). Single EDCs were applied to the culture broth at 200 μM, respectively. Standard deviations from three replicates of each series of results were less than ± 5%

All fungal cells were able to grow in the MEB culture added with single EDCs, allowing various removal rates of EDCs.

As shown in Fig. 3, during cultivations with heat-killed fungal cells (Control flasks), there is an assimilation of the target EDCs into the fungal mycelium, greatly with the heat-killed G3’s biomass, being able to remove up to 55 ± 2.4% of the 4-t-BP within 8 days. Whereas, in the test flasks, 4-t-BP degradation reached around 72 ± 4.1% indicating that the biodegradation of the last compound by Chaetomium sp may be attributed to mycelium adsorption rather than extracellular enzymes in the media culture. Moreover, a limited removal rate of 4-t-BP has been observed with the heat-killed cells of (KW3) and (KW1), reaching around 20 ± 1.5% and 12.5 ± 0.8% within 12 days, respectively. As for EE2, the G3 fungal pellets achieved the highest extent of removal, compared to the other strains. In the same line with our study, Hwang et al. (2008) reported the adsorption of Phthalates by Pleurotus ostreatus mycelial pellets. Several other studies on the adsorption potential of fungal mycelial pellets for EDCs removal described the role of hydrophobic interactions between the compound and the mycelium surface in enhancing their degradation by mycelium-associated enzymes (Pezzella et al. 2017; Armenante et al. 2010).

As depicted in Fig. 3, the best rate of the EDCs removal was recorded with Chaetomium sp culture, removing 95 ± 11.5% of EE2 after 12 days, and 72 ± 7.5% of 4-t-BP within 8 days of cultivation. Additionally, Paecilomyces formosus W3 significantly removed 4-t-BP after 4 days achieving a removal comparable to that of Chaetomium sp which is around 69 ± 6.7%. These findings demonstrated that laccase-producing strains efficiently remove the target EDCs compounds that represent different classes of chemicals (steroidal oestrogens and alkylphenols).

Similarly, Taboada-Puig et al. (2017) illustrated the potential biodegradation of the EDCs by fungal culture and their oxidative enzymes, especially laccases. Also, Křesinová et al. (2018) reported that in the case of Pleurotus ostreatus (a well-known laccase-producing fungus), ligninolytic enzymes have been directly implicated in the biodegradation processes of EDCs.

Fungal treatments of EDCs have been well documented in the literature, whereas only few reports until now with the Chaetomium sp. as an ascomycete strain.

Further, the strain Chaetomium sp (number accession MZ841818) ant its laccase was selected due to its high ability against target EDCs to be used for the enzymatic treatment of EDCs in TMWW.

Biocatalytic degradation of 17α-ethinyl estradiol and 4-tert-butylphenol with Chaetomium sp. crude enzymes

Screening potential redox mediators for laccase-catalyzed system EDC degradation

The enzymatic degradation of 4-tert-butylphenol (4-t-BP) and 17α-ethinylestradiol (EE2) was tested in solution at a pH of 5.0 in the presence of laccases and laccase–mediator systems for 2 h. The influence of natural and synthetic redox mediators (SYR, PCA, TEMPO, and HBT) on the laccase oxidation system was investigated (Fig. 4).

Fig. 4.

Effect of laccase (red bars) and laccase–mediator systems oxidation (grey bars for laccase–HBT; orange bars for laccase–p-coumaric acid; blue bars for the laccase–syringaldehyde system) on the degradation yields of 17α-ethinyl estradiol (EE2) and 4-tert-butylphenol (4-t-BP) in the wastewater. Reaction conditions: EE2 (200 µM), 4-t- BP (200 µM), pH 5.0 (50 mM sodium acetate buffer), at 50 °C, 1.5 U mL⁻¹ laccase, and 0.2 mM mediator redox, with a reaction time of 2 h. All results are averages from duplicated experiments, and the standard deviation is less than 8%

As shown in Fig. 4, within 2 h of catalyzing at 50 °C, laccase could degrade 60 ± 4.8% of 4-t-BP; however, it oxidized EE2 less efficiently, reaching 35 ± 4.1%. This may be attributed to the higher redox potential of the tested compounds. Figure 4 reveals that the syringaldehyde and the HBT allowed the best degradation yields of EE2 at about 80 ± 2.4% and 77 ± 3.6%, respectively.

On the other hand, for 4-t-BP treatment, Lac–TEMPO and Lac–HBT systems afforded similar rates of about 90 ± 7.2% degradation. Thus, the transformation yield of the EDCs was both compound-structure and redox-mediator dependent (Lloret et al. 2013). Previous studies presented evidence for the enhancement of laccase oxidation potential in the degradation of EDCs through redox mediators (Guardado et al. 2019).

Additionally, the selected redox mediators differ in their laccase oxidation pathways, their modes of action, and specificity (Morozova et al. 2007; Ashe et al. 2016). For instance, TEMPO selectively acts on specific functional groups (i.e. alcohols) (de Nooy et al. 1996). The results show that HBT (N–OH compound) can mediate a range of laccase-catalyzed bio-transformations of different EDC structures (Xu et al. 2000). According to Guardiol et al. (2019), syringaldehyde was the best redox mediator to enhance the laccase oxidation of pharmaceutical products such as amoxicillin. The potential of the laccase–mediator system strongly depends on the stability and reactivity of the mediator radicals.

GC–MS chromatogram analysis and degradation pathways

Oxides before and after EE2 and 4-t-BP removal were investigated with GC–MS analysis (Fig. 5). Chromatograms showed an effective reduction in the intensity of 4-tert butylphenol peaks after enzymatic treatment (Fig. 5B, C) compared with the control (Fig. 5A). The presence of peaks in the enzymatic reaction (chromatograms b and c) indicated the breakdown of the EDCs’ initial structure. The GC profile of the reaction catalyzed by laccases alone is similar to the profile of the reaction catalyzed by the laccase–HBT system, except for the disappearance of peak 3. Thus, the degradation of 4-t-BP depends on the redox mediator. Longe et al. (2018) indicated that oxidation is mediator-dependent and provides new insights into the enzymatic mechanism.

Fig. 5.

GC–MS chromatograms of the EDCs in wastewater (A) 4-tert-butylphenol (a) and its remaining metabolites during laccase (b) and laccase-HBT system (c) degradation processes. (B) 17α-ethinyl estradiol (EE2) (d) and its laccase-catalyzed (e) and laccase–HBT-catalyzed (f) transformation products

As shown in Table 4, the chromatogram shows detected substances and retention times of 4-tert-butylphenol (11.01 min), 4-tert-butycatechol (22.5 min), 3,3-dimethyl-2-butanone (17.03 min), and pyruvic acid (8.7 min). The data presented in both Fig. 5A and Table 4 indicate that Chaetomium sp. laccase can oxidize 4-t-BP through a meta-cleavage pathway (Fig. 6A).

Table 4.

Metabolites detected during the oxidative degradation of 4-tert-butyl-phenol and 17α-Ethynylestradiol (EE2) by Chaetomium sp laccase

Peak	Name	Linear Formula	MIa	RTb (min)
4-tert-butylphenol (4-t-BP)
1	4-t-BP	(CH3)3CC6H4OH	191	11,01
2	3,3-dimethyl-2-butanone	C6H12O	57	17.03
3	Pyruvic acid	C3H4O3	43	8.7
4	4-tert-butylcatechol	C10H14O2	151	22.5
17α-Ethynylestradiol (EE2)
1	EE2	C20H24O2	213	25.191
2	4-Hydroxyestrone |	C18H22O3	287.16	24.09
3	Pyridinestrone acid	C18H21O3N	300.16	22.56
4	Eicosane = Hydrocarbon + Oxygen gas	C20H42	71	21.14
5	Resorcylic acid Benzoic acid, 2,4-bis[(trimethylsilyl)oxy]-, trimethylsilyl ester	C16H30O4Si3	355	13.81
6	Propanoic acid, 3,3'-thiobis-, didodecyl ester	C30H58O4S	178	26.16

^aRetention time

^bMass ion

Fig. 6.

Proposed pathway of (A) 4-tert-butylphenol (4-t-BP) and (B) 17α-ethinylestradiol (EE2) in the wastewater through degradation by Chaetomium sp. laccases based on the metabolites identified through GC–MS analysis

The proposed breakdown pathway of the tested alkylphenol (Fig. 6A) revealed that the structure of 4-t-BP was hydroxylated to a 4-tert-butyl catechol structure, followed by forming 3,3-dimethyl-2-butanone via a meta-cleavage pathway with the original n-butyl side chain. Afterwards, the 3,3-dimethyl-2-butanone was degraded to form pyruvic acid. This metabolic degradation pathway is similar to the pathway proposed by Chang et al. (2020), who demonstrated that 2-tert-butylhydroquinone was the main metabolite of 4-t-BP degradation by rhizosphere microorganisms. A similar trend in the degradation pathway was reported by Toyama et al. (2010) for a newly isolated Sphingobium fuliginis.

The profiles of the EE2 degradation products in the laccase-catalyzed reaction and laccase–HBT-catalyzed reaction were also examined by GC–MS (Fig. 5D–F). In this study, 0.2 mM of 1-hydroxybenzotriazole (HBT) was used as a mediator and improved EE2 elimination.

As presented in Fig. 5, the chromatograms show a decrease in the area of the EE2 peak after 2 h of enzymatic treatment (Fig. 5E, F) compared with the control (Fig. 5D). The profile resulting from the laccase–HBT catalyzed transformation seems to be different from the control as well as the laccase reaction. The differences in treatment efficiency are functions of the redox mediator.

Additionally, as depicted in Table 4 and GC–MS profiles (Fig. 5F), peak 6 appeared only in the reaction catalyzed by the laccase–HBT system and represents the major transformation product of EE2. Lloret et al. (2013) demonstrated the increased laccase substrate range with the use of redox mediators. Similarly, Suzuki et al. (2003) investigated the removal of the steroidal hormone EE2 with a laccase–HBT system prepared from Phanerochaete chrysosporium ME-446. Similarly, Křesinová et al. (2012) reported the efficiency of laccases isolated from Pleurotus ostreatus in the degradation of EE2 (about 90%) within 24 h.

Different catabolic products resulting from the enzymatic degradation of EE2 by Chaetomium sp. laccases were identified with GC–MS and are presented in Table 4.

The chromatogram shows detected substances and retention times of EE2 (25.194 min), 4-hydroxyestrone (24.09 min), pyridinestrone acid (22.56 min), eicosane (21.14 min), and resorcylic acid (13.81 min). In the laccase–mediator system reaction, the most abundant metabolite was 4-hydroxyestrone.

Through mass spectrometry (MS), the four main EE2-derived degradation metabolites were identified after laccase and laccase-HBT-catalyzed transformations (Table 4). The other metabolites, such as compound I (Estrone E2) and compound II (meta-cleavage product), may be involved in the degradation pathway (Fig. 6B).

As depicted in Fig. 6B, EE2 was first oxygenized to Estrone (E1) (not detected) that could be further hydrolysed into 4-hydroxyestrone, followed by forming pyridinestrone acid through a meta-cleavage pathway. Moreover, the resorcylic acid from the laccase-catalyzed reaction and propanoic acid, 3,3'-thiobis-, didodecyl ester were detected as intermediate metabolites of the TCA cycle. All of these metabolic products are well illustrated in the literature as EE2-derived degradation metabolites (Palma et al. 2021). For instance, Chen et al. (2018) reported an analogous aerobic oestrogen degradation pathway in activated sludge. Also, previous studies documented the involvement of ligninolytic and non-ligninolytic enzymatic machinery in the oxidation of EE2, such as hydrogenase, dehydrogenase, and cytochrome P-450 (Křesinová et al. 2012; Mtibaà et al. 2020).

Conclusion

These findings support the enzymatic oxidation potential of laccase–redox-mediator systems in the degradation of xenobiotic phenolic pollutants and are presented alongside proposed metabolic pathways.

Supplementary Information

Below is the link to the electronic supplementary material.

Author contributions

DD proposed the research topic, provided necessary tools for the experiments, conceived, planned, and conducted all experiments, collected the data, contributed to the analysis and interpretation of the results, and contributed substantially to the writing and revision of the manuscript. DD was the academic supervisor of the student SRA and the principal investigator. SRA provided some necessary tools for the experiments and performed the GC–MS analysis. All authors read and approved the final manuscript.

Funding

This project was funded by the Deanship of Scientific Research (DSR) of the University of Jeddah, Jeddah, Saudi Arabia, under Grant No. (UJ-20-109-DR). Therefore, the authors acknowledge and thank the DSR for its technical and financial support.

Availability of data and materials

Not applicable.

Declarations

Conflict of interest

All authors declare no conflict of interest.