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

Abstract

Unlabelled:
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.

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https://doi.org/10.1007/s13205-022-03439-1
ORIGINAL ARTICLE
Degradation ofendocrine‑disrupting chemicals inwastewater bynew
thermophilic fungal isolates andtheir laccases
DalelDaâssi1,2 · ShuruqRahimAlharbi1
Received: 23 May 2022 / Accepted: 14 December 2022
© King Abdulaziz City for Science and Technology 2022
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 formo-
sus (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-hydroxybenzotria-
zole (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.
Keywords Thermophilic· Chaetomium sp.· Laccase· Redox mediators· Metabolic pathway· 17α-ethinylestradiol· 4-tert-
butylphenol
Introduction
Endocrine-disrupting chemicals (EDCs) as an emerging
group of trace organic pollutants are becoming an envi-
ronmental issue as they persistently pollute surface water
worldwide, affecting biological functions and harming wild-
life 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 asso-
ciated with endocrine disruptive activity in humans include
changes in neurological and immunological functions,
female reproductive disorders, and reduced fertility (Esplu-
gas 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 (Van-
denberg etal. 2009).
* Dalel Daâssi
daleldaassi@yahoo.com; 04220334@uj.edu.sa
1 Department ofBiology, College ofSciences andArts,
Khulais, University ofJeddah, Jeddah, SaudiArabia
2 Laboratory ofEnvironmental Bioprocesses, Centre
ofBiotechnology ofSfax, P. B “1177”, 3018, Sfax, Tunisia

3 Biotech (2023) 13:26
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26 Page 2 of 15
The second group includes man-made or synthetically
produced hormones comprising oral contraceptives, such
as ethinylestradiol (EE2), and animal feed additives. More-
over, a variety of industrially synthetized chemicals are
also EDCs including polychlorobiphenyls (PCBs), phtha-
lates, 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 resist-
ance to further degradation, bioaccumulation in biota,
and potential to harm humans and wildlife (Barbosa etal.
2016).
Among the various EDCs from the environment, oestro-
gen 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, polycar-
bonate, and epoxy resins for industrial purposes. Toxicol-
ogy 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 environ-
ment through several channels, including industrial activi-
ties 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 precipita-
tion, flocculation, coagulation (Huang etal. 2021a, b), and
photochemical oxidation (Wang etal. 2020), and adsorp-
tion (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 pro-
cesses 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 trans-
form 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 typi-
cally 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 dur-
ing transportation and then stored in the refrigerator (4°C).
The MWW was subsequently filtered through a glass micro-
fiber 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.

3 Biotech (2023) 13:26
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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 ben-
zotriazole (HBT), p-coumaric acid (PCA), syringalde-
hyde (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, ampicil-
lin, and streptomycin at 0.01%) (Daâssi etal. 2016a). Plates
were incubated at 50°C for 2 to 7days, and then, pure fun-
gal 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 incu-
bated at 50°C for several days. Fungal strains secreting lac-
cases 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 iden-
tified 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 molecu-
larly identified with ITS1/ITS2. After sequencing, ITS
sequences were used for homology analysis using blastn
as a default parameter for nucleotide sequence homol-
ogy 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 num-
bers 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 posi-
tions 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
Table 1 The endocrine-
disrupting chemicals used in the
current study
Endocrine-
disrupting
chemicals
Reference
ion
Retention
time
(min)
StructureLinear FormulaCAS
Number
17α-
Ethynylestradiol
(≥98%)
(EE2)
213 25.191 C20H24O
25
7-63-6
4-tert-
Butylphenol
(99%)
(4-t-BP)
191 11.901 (CH3)3CC6H4OH98-54-4

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26 Page 4 of 15
thermophilic isolates (Chaetomium sp G3, Canariomyces
notabilis KW1, Paecilomyces formosus KW3), were pre-
pared in 250-mL cotton-plugged Erlenmeyer flasks con-
taining 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 treat-
ment 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 pro-
duction 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 tempera-
ture. 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 oxi-
dation 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 reac-
tion 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 super-
natant 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:
where C0 is the initial concentration of EE2 (mg L−1) and
Ct is the residual concentration of EDCs (mg L−1) at a given
time.
Controls and test samples were extracted with dichlo-
romethane 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 com-
pletely evaporated using a rotary evaporator (Concentrator
5301Eppendorf).
(1)
Removal efficiency
(%)=
(
C
0
−C
t)
∗100∕C
0

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Gas chromatography–tandem mass spectrometry
(GC–MS)
Controls and test samples were extracted with dichlorometh-
ane using a separating funnel. The organic part was com-
pletely evaporated using IKA rotary evaporator.
Before GC–MS analysis, the concentrated extracted sam-
ples were trimethylsilylated (TMS), as Daâssi etal. sug-
gested (2016b).
After derivatization, the degradation products resulting
from the laccase oxidation of the EE2 and 4-t-BP were iden-
tified 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 detec-
tor were programmed at 305°C for 1 μL volume, splitless
per 1min. Helium at 100kPa was the carrier gas. Tem-
perature 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 per-
formed 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 col-
lected 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 oxi-
dative activities on 2,6-DMP and seven on MEA plates with
ABTS (Table2).
Table2 illustrates the qualitative test to check for laccase
activity on agar plates containing phenolic as well as non-
phenolic substrates.
The data presented in Table2 demonstrate that among
the tested thermophilic fungal isolates, the strains desig-
nated 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 sub-
strate (ABTS).
According to Daâssi etal. (2016a), screening for laccase
producers on an MEA medium containing coloured indica-
tor compounds can rapidly and qualitatively detect laccase
activity; however, liquid cultivation is still essential to meas-
ure the quantity of laccase activity. Thus, the positive iso-
lates 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) iso-
lated 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 purifi-
cation 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.
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
Activity:+++high;++medium;+low; ± ambiguous; −none
Growth: ***Excellent growth (colony diameter > 40 mm); **good
growth (colony diameter 21–40 mm); *low growth (colony diam-
eter < 21)
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 + ***

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26 Page 6 of 15
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)
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

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Page 7 of 15 26
Identification andphylogenetic analysis
Using Blastn to align findings with the National Center for
Biotechnology Information (NCBI) databases, the Gen-
Bank 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).
BLAST search revealed that the ITS of the isolate G3
matched 99.79% with the sequences of Chaetomium brasil-
iense isolate UM 235 (accession n° JX966545.1) and Ovato-
spora 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 exam-
inations 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 phyloge-
netic tree (Fig.2) suggests that Neurospora tetraspora is the
closest strain to the isolate KB14 (Huang etal. 2021a, b).
Chaetomium brasiliense (G3), Canariomyces nota-
bilis (KW1), Paecilomyces formosus (KW3), Neuros-
pora 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-pro-
ducing 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 ther-
mophilic strain in Saroj etal.’s (2018) study.
Our collection of thermophilic strains is supported by pre-
vious findings in the literature. For instance, Ahirwar etal.
(2017) reported the isolation of 68 thermophilic and ther-
motolerant 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), Canari-
omyces notabilis (KW1), Paecilomyces formosus (KW3))
were tested against the investigated EDCs during incubation
periods (Fig.3).
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

3 Biotech (2023) 13:26
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26 Page 8 of 15
interactions between the compound and the mycelium sur-
face 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 (ste-
roidal 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 Pleu-
rotus 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 abil-
ity against target EDCs to be used for the enzymatic treat-
ment of EDCs in TMWW.
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

3 Biotech (2023) 13:26
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Page 9 of 15 26
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).
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 oxi-
dized EE2 less efficiently, reaching 35 ± 4.1%. This may be
attributed to the higher redox potential of the tested com-
pounds. 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 (Guar-
dado etal. 2019).
Additionally, the selected redox mediators differ in their
laccase oxidation pathways, their modes of action, and speci-
ficity (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 oxida-
tion of pharmaceutical products such as amoxicillin. The
Fig. 3 EDCs removal rates (%)
by Chaetomium brasiliense
(G3), Canariomyces notabi-
lis (KW1) and Paecilomyces
formosus (KW3) after 2, 4, 8
and 12days of treatment in
broth culture (MEB). Single
EDCs were applied to the cul-
ture broth at 200μM, respec-
tively. Standard deviations from
three replicates of each series of
results were less than ± 5%
EE2+G3
4-t-BP+G3
heat-killed cells G3+EE2
heat-killed cells G3+4-t-BP
EE2+W1
4-t-BP+W1
heat-killed cells W1+EE2
heat-killed cells W1+4-t-BP
EE2+W3
4-t-BP+W3
heat-killed cells W3+EE2
heat-killed cells W3+4-t-BP
2 d
4d
8d
12d
Fungal treatments
Removal efficiency of EDCs (%)
0
10
20
30
40
50
60
70
80
90
100
EE2 4-tert-But
% Removal
EDCs tested
lac system Lac/HBT system lac /PCA system lac/SYR system lac/TEMPO
Fig. 4 Effect of laccase (red bars) and laccase–mediator systems oxi-
dation (grey bars for laccase–HBT; orange bars for laccase–p-cou-
maric acid; blue bars for the laccase–syringaldehyde system) on the
degradation yields of 17α-ethinyl estradiol (EE2) and 4-tert-butylphe-
nol (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%

3 Biotech (2023) 13:26
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26 Page 10 of 15
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). Chroma-
tograms 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 (chromato-
grams 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 dis-
appearance of peak 3. Thus, the degradation of 4-t-BP
depends on the redox mediator. Longe etal. (2018) indi-
cated that oxidation is mediator-dependent and provides
new insights into the enzymatic mechanism.
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
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 pro-
cesses. (B) 17α-ethinyl estradiol
(EE2) (d) and its laccase-
catalyzed (e) and laccase–HBT-
catalyzed (f) transformation
products

3 Biotech (2023) 13:26
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Page 11 of 15 26
Chaetomium sp. laccase can oxidize 4-t-BP through a meta-
cleavage pathway (Fig.6A).
The proposed breakdown pathway of the tested alkyl-
phenol (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 pyru-
vic 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 micro-
organisms. 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 lac-
case-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 trans-
formation 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 transfor-
mation 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 identi-
fied 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 lac-
case–mediator system reaction, the most abundant metabo-
lite 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 hydro-
lysed into 4-hydroxyestrone, followed by forming pyri-
dinestrone acid through a meta-cleavage pathway. Moreo-
ver, 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
Table 4 Metabolites detected
during the oxidative degradation
of 4-tert-butyl-phenol and
17α-Ethynylestradiol (EE2) by
Chaetomium sp laccase
a Retention time
b Mass ion
Peak Name Linear Formula MIaRTb (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 C3H4O343 8.7
4 4-tert-butylcatechol C10H14O2151 22.5
17α-Ethynylestradiol (EE2)
1 EE2 C20H24O2213 25.191
2 4-Hydroxyestrone | C18H22O3287.16 24.09
3 Pyridinestrone acid C18H21O3N 300.16 22.56
4Eicosane = Hydrocarbon + Oxygen gas C20H42 71 21.14
5 Resorcylic acid Benzoic acid,
2,4-bis[(trimethylsilyl)oxy]-, trimethylsilyl
ester
C16H30O4Si3355 13.81
6 Propanoic acid, 3,3'-thiobis-, didodecyl ester C30H58O4S 178 26.16

3 Biotech (2023) 13:26
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26 Page 12 of 15
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

3 Biotech (2023) 13:26
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Page 13 of 15 26
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 The online version contains supplemen-
tary material available at https:// doi. org/ 10. 1007/ s13205- 022- 03439-1.
Acknowledgements All the authors acknowledge and thank the Chem-
ical Department of the College of Science and Arts of Khulais, for
allowing the use of spectrophotometer. Also, we thank the students
Lama Jamal Hussain Alssulime and Fatimah Qabil Abdulrahman
Almaghrabi for helping in wood and wastewaters sampling.
Author contributions DD proposed the research topic, provided neces-
sary tools for the experiments, conceived, planned, and conducted all
experiments, collected the data, contributed to the analysis and inter-
pretation 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 neces-
sary 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.
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