Bioresource Technology Reports 22 (2023) 101427

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Bioresource Technology Reports

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Biodecolorization of anthraquinone and azo dyes by dark septate

endophytic fungi
Irma Melati a, b, Gayuh Rahayu a, *, Surono c, d, Hefni Effendi e, Cynthia Henny b,
Dede Heri Yuli Yanto d, f
a
Microbiology Study Program, Department of Biology, Faculty of Mathematics and Natural Sciences, Bogor Agricultural University, Jl. Raya Dramaga, Bogor 16680,
Indonesia
b
Research Center for Limnology and Water Resources, National Research and Innovation Agency (BRIN), Jl. Raya Bogor Km. 46 Cibinong, Bogor 16911, Indonesia
c
Indonesian Soil Research Institute, Indonesian Agency for Agricultural Research and Development, Jl. Tentara Pelajar No. 12, Ciwaringin, Bogor 16114, Indonesia
d
Research Center for Applied Microbiology, National Research and Innovation Agency (BRIN), Jl. Raya Bogor Km. 46 Cibinong, Bogor 16911, Indonesia
e
Department of Fishery Resource Management, Faculty of Fisheries and Marine Science, Bogor Agricultural University, Jl. Raya Dramaga, Bogor 16680, Indonesia
f
Research Collaboration Center for Marine Biomaterials, Jatinangor 45360, Indonesia

A R T I C L E I N F O A B S T R A C T

Keywords: Mycoremediation has been proposed as a useful approach for treating dye wastewater. In the present study, six
Anthraquinone dyes dark septate endophytic (DSE) strains (KSP, CPP, PP, DD, K.III.3.4, and TKC) were evaluated for their ability to
Azo dyes decolorize anthraquinone (RBBR) and azo (AB 113) dyes. The decolorization ability was evaluated in solid and
Decolorization
liquid media in conjunction with ligninolytic enzyme production and fungal biomass dry weight. The results
Dark septate endophytic strains
Ligninolytic enzyme
showed that all of the tested DSE fungi successfully decolorized 89 % of the RBBR and 97 % of the AB 113 dyes.
Three selected DSE strains (KSP, PP, and CPP) demonstrated high decolorization of RBBR (74 %–99 %) and AB
113 (79 %–99 %) dyes at pH 3–11 while producing ligninolytic enzymes. The decolorization occurred through
biosorption, biodegradation, and simultaneous detoxification of the dyes. These results suggest that the selected
DSE fungi may have the potential to bioremediate dye wastewater, which is usually under alkaline pH.

1. Introduction waste that pollutes the surroundings (Li et al., 2017).

Synthetic dyes are fabricated from organic molecules. Over 10,000
classes of synthetic dyes have been utilized, with an annual production
of >7 × 105 tons (Vikrant et al., 2018). Synthetic dyes have been cate­
gorized into 20–30 groups based on chemical structure, and the
anthraquinone (15 %) and the azo groups (60 %–70 %), are the major
dyes (Routoula and Patwardhan, 2020). Remazol Brilliant Blue R
(RBBR), an anthraquinone dye, is mainly used in textile manufacturing
and produces poisonous and hardly-degradable organic toxic waste
(Hadibarata et al., 2012). The azo dye Acid Blue 113 (AB 113) is present
in approximately 60 % of the total marketplace and is used in the textile
manufacturing (Asghar et al., 2019). Synthetic dyes are progressively
utilized in many industries, but primarily textiles because they are easy
to use, cost-effective, and have reasonably high balance facing light,
temperature, cleaner, and microbial attack. In addition, synthetic dyes
have more color variations than natural dyes (Couto, 2009). Unfortu­
nately, synthetic dyes have a low affinity, so almost 15 % of their use is
Dye molecules have enormous molecular substances and are difficult
to degrade. Additionally, dyes are poisonous, carcinogenic, and
dangerous to the environment and human health (Al-Tohamy et al.,
2022; Chung, 2016). Synthetic dyes decrease light penetration and
affect the growth of aquatic plants and algae when released into the
water. Dyes taken up by fish and other organisms may be converted into
toxic intermediates, which may be harmful to the health of fish (Elgar­
ahy et al., 2021). Furthermore, cramps, fever, and hypertension can
develop when fish or aquatic biota containing dyes are consumed by
humans. Dyes have been linked to everything from dermatitis to central
nervous system disorder and can irritate the skin and eyes when
consumed or inhaled, particularly if they are exposed as dust (Al-Toh­
amy et al., 2022; Chung, 2016; Sharma et al., 2022). Dyes are used
extensively, which makes it possible to identify them in the environment
and determine how they accumulate physiologically in the food webs of
freshwater flora and fauna, such as fish and algae. Humans contain
dangerous amounts of organic chemicals that are 1000 times higher than

* Corresponding author.
E-mail addresses: gayuhra@apps.ipb.ac.id (G. Rahayu), suro004@brin.go.id (Surono), dede.heri.yuli.yanto@brin.go.id (D.H.Y. Yanto).

https://doi.org/10.1016/j.biteb.2023.101427
Received 4 September 2022; Received in revised form 31 March 2023; Accepted 2 April 2023
Available online 7 April 2023
2589-014X/© 2023 Elsevier Ltd. All rights reserved.
I. Melati et al.
their initial concentrations in water (Al-Tohamy et al., 2022; Bera and
Tank, 2021).
Most physicochemical approaches that have been implemented
conventionally for dye remediation have proven effective. However,
these physicochemical approaches are less environmentally safe and are
costly due to the formation of sediment and poisonous metabolites and
intensive strength necessities. These arguments have driven many re­
searchers to search for and develop green technologies. One of the
technologies is a fungal-based approach (mycoremediation). Fungi are
effective biological agents for degrading and mineralizing the complex
structure of dyes because they have vital extracellular ligninolytic en­
zymes, extensive metabolic capabilities, and robust morphologies
(Rahimnejad et al., 2015). Several fungi decolorize synthetic dyes in
either live or inactive forms (mycelial state only) (Melati et al., 2022).
The mycelial structure of the fungus plays a vital role as a biosorbent
that removes dyes (Hao et al., 2000). Using fungi as dye decolorization
agents have been extensively reviewed (Vikrant et al., 2018). White-rot
fungi (WRF) have been widely investigated for the decolorization of
synthetic dyes because of their potential capability in the excretion of
the ligninolytic extracellular enzymes, such as lignin peroxidase, man­
ganese peroxidase (MnP), and laccase (Alam et al., 2023, 2021; Anita
et al., 2019; Ardiati et al., 2019; Yanto et al., 2022). However, some
studies have reported that the ideal conditions for dye decolorization by
WRF fungi are acidic to neutral rather than alkaline (Sudiana et al.,
2018).
In contrast, dye wastewater, particularly in the textile industry,
consists of many complex compounds and varies in pH (usually alkaline)
and salinity (Yaseen and Scholz, 2019). Therefore, it is crucial to identify
other sources of fungi as alternative dye decolorization agents in
extreme conditions, such as textile wastewater. Dark septate endophytic
(DSE) fungi could potentially fulfil these requirements because they can
adapt to a polluted environment and remediate pollutants individually
and in symbiosis with plants in a phytoremediation system (Likar and
Regvar, 2013).
DSE fungi have received attention due to their ability to colonize
plants that grow in stressful conditions, such as high CO2, nutritional
deficiency, drought, high salt, and pest stress (including plant patho­
gens) (Li et al., 2019). DSE fungi are tolerant in the range of pH 2–10
(Spagnoletti et al., 2017). A wide variety of DSE fungi have been found
in the roots of various plant species that grow in heavy metal-
contaminated soil (Berthelot et al., 2016). In addition, DSE fungi have
extracellular enzymes that degrade dyes, such as laccase, peroxidase,
and tyrosinase (Neoh et al., 2015). These observations have led to the
use of DSE fungi for decolorizing and degrading synthetic dyes. How­
ever, no scientific studies on the decolorization or degradation of azo
and anthraquinone synthetic dyes by DSE fungi have been reported.
Therefore, the objective of this study was to investigate the ability of
DSE fungi to decolorize azo and anthraquinone synthetic dyes.
2. Materials and methods
2.1. Fungal cultures
The DSE, i.e. Cladosporium tenuissimum KSP (GenBank accession
number: MH810309.1), Curvularia lunata PP (Genbank accession num­
ber: MN213745.1), Curvularia caricae-papaya TKC (GenBank accession
number: NR147458.1), and three unknown fungal isolates (CPP, K.
III.3.4, and DD) obtained from the Culture Collection of Biology Labo­
ratory of the Indonesian Soil Research Institute, Ministry of Agriculture,
Indonesia were used in this study. Stock cultures were maintained on
corn meal malt yeast agar (CMMYA), and working cultures were pre­
pared in potato dextrose agar (PDA) (HiMedia, Mumbai, India).
2
Bioresource Technology Reports 22 (2023) 101427
2.2. Qualitative screening of ligninolytic enzyme activity using the plate
test method
The DSE fungi were prescreened for ligninolytic enzyme activity
using the Bavendamm method (Sepwin and Munir, 2019). All isolates
were developed on PDA media supplemented with 0.1 % tannic acid
(Merck, Darmstadt, Germany). A brown-colored complex emerged on
the top of the PDA, indicating that the tested DSE fungus had phenol
oxidase activity. Moreover, the presence of a brown oxidation zone
around the colony was used to calculate the enzyme potency index using
the following formula (Fijai et al., 2019):
Area of the color zone (cm2 )
Potency index (PI) = (1)
Area of colony (cm2 )
DSE strains with phenol oxidase activity were investigated for lac­
case activity by testing on PDA media supplemented with 0.01 %
guaiacol (pH 5.5). The presence of laccase was indicated by the
appearance of a brownish-red color around the colony.
2.3. Decolorization of the dyes on solid media
After 14 days of growth on CMMYA, a mycelial plug (∅ 5 mm) of the
DSE isolate was transferred to 50 % corn meal agar (CMA) (HiMedia)
supplemented with either 50 mg/L Remazol Brilliant Blue R (RBBR) or
50 mg/L Acid Blue 113 (AB 113) (Sigma-Aldrich, Berlin, Germany) dye.
The cultures were incubated at room temperature for 14 d. The exper­
iment was performed in triplicate, and 50 % CMA without RBBR or AB
113 was the control. The decolorized zones surrounding the growing
colony were used as the base for the decolorization index (DI) calcula­
tion as follows:
decolorization diameter
Decolorization index (DI) = (2)
colony diameter
Furthermore, the diameter of the colony was used to determine the
growth rate (GR) and tolerance rate (TR) of fungi according to Eqs. (3)
and (4) (González-Abradelo et al., 2019; Melati et al., 2021),
respectively:
colony diameter final − colony diameter initial (cm)
GR (cm/day) = (3)
Incubation time (day)
GRT
TR (%) = × 100 (4)
GRC
GRT is the fungal growth rate in media containing RBBR or AB 113
individually, and GRC is the fungal growth rate of the control.
2.4. Decolorization of RBBR and AB 113 in liquid media
Three mycelial plugs (∅ 5 mm) were taken from 14-day-old colonies
of each isolate on CMMYA. They were inoculated into 50 mL potato
dextrose broth (PDB) (HiMedia) media supplemented with either the
RBBR (50 mg/L) or AB 113 (50 mg/L) dye. The cultures were incubated
at room temperature for 2 weeks. Fungal-free medium was used as the
control. At the end of the incubation period, the cultures were centri­
fuged at 5000 rpm for 10 min to separate the biomass. The supernatants
were collected, and absorbance was analyzed at a maximum wavelength
for RBBR (592.5 nm) and AB 113 (546.0 nm), respectively, using a
visible spectrophotometer (Shimadzu UV-1800, Tokyo, Japan) (Melati
et al., 2021). The decolorization percentage was calculated using the
following formula (5).
I. Melati et al.
Control absorbance − Treatment absorbance
Decolorization percentage (%) =
Control absorbance
The biomass pellet was washed in distilled water, stored in an oven at
80 ◦ C overnight, and weighed. The DSE strains with high decolorization
capacity were studied further to determine the effect of pH on dye
decolorization, ligninolytic enzyme activity, and biosorption capacity in
addition to kinetic and isothermal studies.
2.5. Biosorption test
The dye adsorbed in the mycelia after the decolorization experiment
was measured. The dye-adsorbed mycelia were separated from the
liquid medium using Whatman filter paper no. 41 and were transferred
into an Erlenmeyer flask. Then, 10 mL of 96 % ethanol and 10 mL of
fresh liquid medium were added. The Erlenmeyer flasks were agitated
on a shaker at 160 rpm for 20 min and the suspensions were centrifuged
at 5000 rpm for 10 min. The dye-contained supernatant was taken and
the concentration of dyes (expressed as the concentration of dyes
adsorbed in the mycelia) was analyzed using a standard calibration
curve based on the absorbance measurements using a visible spectro­
photometer (Shimadzu UV-1800, Tokyo, Japan). As a control, 10 mL of
the initial concentration of dyes (50 mg/L) without the addition of
mycelia was extracted as the same method above. The dye biosorption
rate was estimated using the formula:
Concentration of dye adsorbed in mycelia
Biosorption rate (%) = × 100
Concentration of dye in the control
The amount of dye absorbed (uptake capacity) by the various DSE
strains was determined using the following equation:
(Co − Ct)V
qt =
W
where qt is uptake capacity by the DSE fungi at time t (mg/g), Co is the
initial concentration of dye in the liquid medium (mg/L), Ct is the
concentration of dyes at time t (mg/L), V is the volume of liquid medium
(L), and W is the mass of the DSE fungus (g).
2.6. Visible spectral analysis
A visible spectral study was performed to determine the incidence of
dye biodegradation using a vis spectrophotometer (Shimadzu UV-1800,
Japan) at wavelengths from 400 to 800 nm (Ting et al., 2016). The
change in the peak of the untreated dye sample compared to the treated
dye indicated biodegradation.
2.7. Microbial toxicity test
The microbial toxicity test was conducted following Yanto et al.
(2021) with modifications. The toxicity of the synthetic dyes was tested
on Gram-negative (E. coli) and Gram-positive (Bacillus subtilis). The
bacterial inoculum was cultured in 20 mL of nutrient broth and incu­
bated on a shaker at 100 rpm at 30 ◦ C for 24 h on tryptic soy agar (TSA)
(Merck) plates. Fifty microliters of microbial inoculum was spread over
the TSA using a bacterial spreader. Before and after treatment with the
DSE fungi, the dye solutions were autoclaved at 121 ◦ C for 15 min.
Sterile filter paper discs (Ǿ 5 mm) were soaked in each dye solution for
10 min and a single paper disc was placed in the center of a TSA plate
containing either the B. subtilis or the E. coli culture. Sterile filter paper
discs containing chloramphenicol (4 mg/mL) were used as the positive
control. The cultures were incubated at 30 ◦ C for 1 d. Toxicity was
Bioresource Technology Reports 22 (2023) 101427
× 100 (5)
indicated by bacterial growth toward the filter paper disc.
2.8. The effect of pH on dye decolorization, biosorption capacity, and
ligninolytic enzyme activity
Three selected strains (KSP, PP, and CPP) from the screening ex­
periments were studied for the effect of pH on decolorization, bio­
sorption capacity, and ligninolytic enzyme activity toward the RBBR and
AB 113 dyes. The experiment was conducted in liquid culture at various
pH values (pH 3, 5, 7, 9, and 11). Ligninolytic enzyme activity, i.e., MnP
and laccase were measured quantitatively by spectrophotometry at 420
nm (laccase) and 470 nm (MnP). Laccase activity was analyzed based on
the oxidation of ABTS (2,2′ -azino-bis (3-ethylbenzthiazoline-6-sulfonic
acid). MnP activity was determined using 2,6-dimethoxyphenol as the
substrate (Anita et al., 2019). One unit of enzyme activity (U) repre­
sented the amount of enzyme required to metabolize 1 μmol of ABTS per
min.
2.9. Kinetic and isotherm study
2.9.1. Kinetic models
Experiments were performed on combinations of a single strain of
selected DSE fungi with one type of dye to collect data on the kinetic
assessment of RBBR and AB 113 decolorization. Three mycelial plugs (Ǿ
5 mm) from the selected DSE strain were added to 50 mL of PDB con­
(6)
taining either RBBR (50 mg/L) or AB 113 (50 mg/L). The concentration
of RBBR and AB 113 dye in the liquid medium was evaluated at 2, 5, 7, 9,
11, and 14 d of incubation. The kinetics of RBBR and AB 113 decolor­
ization caused by the selected DSE fungus was described using pseudo-
(7) first-order and pseudo-second-order adsorption models. Data were
collected as dye concentrations at a particular time to determine the
kinetic reaction order of the dyes. The kinetic formula used for studies of
the first (ln C vs. t) and second orders kinetics (1/C vs. t) is shown in Eqs.
(8) and (9), respectively:
Ct = C0 exp ( − k1 t) (8)
1 1
= + k2 t (9)
Ct C0
where Ct is the capacity for dye biosorption at a time (mg/g), C0 is the
amount of equilibrium (mg/g), k1 is the pseudo-first-order equilibrium
constant (min− 1), and k2 is the pseudo-second-order equilibrium con­
stant of the kinetic model (g/mg.min).
2.9.2. Isothermal models
Three mycelial plugs (Ǿ 5 mm) of the selected DSE strains were
added to 50 mL of PDB followed by either the RBBR or AB 113 dye to
final concentrations of 100, 150, 200, and 250 mg/L, respectively. The
Langmuir and Freundlich isotherm models (Parimelazhagan et al.,
2022) were applied to characterize the decolorization of the RBBR and
AB 113 dyes caused by the DSE fungi. The Langmuir model is expressed
as:
Ce 1 Ce
= + (10)
qe KL qm qm
where qe is the amount of dye adsorbed per unit mass of fungi (mg/g), Ce
is the concentration of dye at equilibrium (mg/L), qm is the maximum
adsorption capacity of the fungi (mg/g) and KL is the Langmuir
adsorption constant (L/mg).
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I. Melati et al. Bioresource Technology Reports 22 (2023) 101427

A B C

D E F

Fig. 1. Qualitative screening of the DSE fungi: (A) KSP; (B) CPP; (C) PP; (D) TKC; (E) K.III.3.4; (F) DD, on media containing 0.1 % tannic acid.

The Freundlich isotherm model was calculated as:
1
logqe = logKf + logCe
n
where qe is the amount of dye adsorbed per unit mass of fungi (mg/g), Kf
is the Freundlich isotherm constant (L/mg), and n is the heterogeneity
factor.
3. Results and discussion
3.1. Qualitative screening of ligninolytic enzyme activity using the plate
test method
All DSE fungal strains had different levels of phenol oxidase activity
(Fig. 1), as depicted by the various diameters of the coloration zones and
potency indices (Table 1). Of all of the DSE fungi tested, KSP, PP, and
CPP demonstrated the highest activity (2.4, 2.3, and 1.9, respectively).
The Bavendamm test based on the oxidative polymerization of
phenolic acids to brownish products is an accepted evaluation in which
Table 1
Qualitative screening of lignin degradation and laccase activities using the plate
test method.
the release of phenol oxidases (Gramss et al., 1998) indicates lig­
ninolytic activity (Sepwin and Munir, 2019). All DSE strains had lig­
(11) ninolytic activity, but only four DSEs, such as KSP, PP, CPP, and K.
III.3.4, were grouped into the higher-ligninolytic strains (P > 1) based
on the potency index value.
The phenol oxidase activity responses ranged from no color to weak,
and medium reddish-brown discoloration, which was visually observed
after 7 to 14 d of incubation. The reddish-brown discoloration indicated
laccase activity. Of the 6 DSE strains tested, only three (CPP, DD, and K.
III.3.4) exhibited laccase activity after 7 d of incubation.
The phenol oxidase enzymes are comprised of laccases and tyrosi­
nases. Not all DSE fungi with phenol oxidase activity possess laccase and
tyrosinase activities. Gramss et al. (1998) reported that some ectomy­
corrhizal fungi, such as Boletus luridus and Lactarius necator, produce
positive Bavendamm test results but are negative for laccase activity.
Thus, we suggest that the positive Bavendamm test for KSP, PP, and TKC
was due to tyrosinase activity. Tyrosinase plays a critical role in the
formation of the melanin (Halaouli et al., 2006). DSE fungi contain the
melanin pigment; thus, these DSE fungi may also have tyrosinase ac­
tivity. Several DSE fungi have laccase, peroxidase, and tyrosinase (Neoh
et al., 2015).
3.2. Decolorization of RBBR and AB 113 in solid media

DSE Colony area Color zone area PI Laccase

fungi (cm2) (cm2) activity
The DSE fungi tested were capable of decolorizing the RBBR and AB
113 synthetic dyes in solid and liquid media. The results showed that all
KSP 2.5 ± 0.1 6.2 ± 0.1 2.4 ± −
DSE fungi decolorized RBBR and AB 113 in solid media with DIs of
0.1
CPP 6.2 ± 0.1 11.9 ± 0.2 1.9 ± ++ 1.00–1.43 and 1.01–1.96 for RBBR and AB 113 (Table 2), respectively.
0.0 Strain KSP had the highest DI for both synthetic dyes, although it was
PP 6.2 ± 0.1 13.8 ± 0.2 2.3 ± − not significantly different from CPP (P = 0.05). Interestingly, the DI
0.0 value after the 7-d incubation was higher for RBBR or AB113 than that
DD 0.8 ± 0.1 0.8 ± 0.1 1.0 ±
after the 14-d incubation for all DSE fungi.
+
0.0
K.III.3.4 2.3 ± 0.1 3.5 ± 0.2 1.5 ± ++ The growth of the DSE fungi in PDA with the synthetic dyes was not
0.0 significantly different (P < 0.05) from the control, indicating that all of
TKC 4.5 ± 0.4 4.5 ± 0.4 1.0 ± − the DSE fungi used RBBR and AB 113 as carbon sources in solid media. In
0.0
addition, the GR of most of the DSE fungi was higher on media with
− No color, + weak, ++ medium. RBBR than that of the AB 113 synthetic dye. All of these data

4
I. Melati et al. Bioresource Technology Reports 22 (2023) 101427

Table 2 the fungi toward AB 113 (which is an azo dye) was higher than that of
Decolorization index (DI), growth rate (GR), and tolerance rate (TR) of the DSE RBBR. We assumed that AB 113 was more easily decolorized by the DSE
fungi on solid media consisting of the RBBR and AB 113 synthetic dyes after 7 fungi than RBBR. These results are in line with some previous studies.
and 14 d of incubation. Toker et al. (2021) reported that marine-derived fungi are more efficient
Dyes Isolate Incubation DI GR (cm/day) TR (%) in decolorizing azo dyes (methyl orange) than anthraquinone dyes
time (d)
Control Treatment (RBBR). Similarly, Khudhair et al. (2015) showed the decolorization rate
of RB 5 (an azo dye) by Rhizoctonia zeae SOL3, Candida sp. S1, and
RBBR KSP 7 1.43 ± 0.23 ± 0.25 ± 110.42
0.06a 0.04a 0.04a
Meyerzoma sp. S7 was higher than that of RBBR (anthraquinone dyes). In
14 1.34 ± 0.23 ± 0.25 ± 110.53 addition, it was more stable than RB5 due to the fusion of the aromatic
0.11ab 0.02a 0.04a structure of RBBR. Anthraquinone dyes are more resistant to degrada­
CPP 7 1.21 ± 0.31 ± 0.40 ± 127.27 tion, particularly compared to azo dyes. The percentage of RBBR
0.00bc 0.04b 0.00b
decolorization produced by KSP in this study was the same as that re­
14 1.19 ± 0.42 ± 0.44 ± 106.86
0.01 cd 0.02b 0.00b ported by Syafiuddin and Fulazzaky (2020), who used WRF to decol­
TKC 7 1.16 ± 0.83 ± 0.90 ± 108.57 orize RBBR dyes (88.8 %). The ability of KSP to decolorize AB 113 was
0.06 cd 0.04c 0.16c higher than that reported by Jasińska et al. (2019), who used WRF to
14 1.02 ± 0.59 ± 0.59 ± 101.21 decolorize AB 113, with a resulting decolorization percentage of 66.91
0.03e 0.02c 0.02c
DD 7 1.33 ± 0.25 ± 0.26 ± 101.89
%. This result indicates that the decolorization ability of DSE fungi is not
0.05ab 0.00d 0.02d less than WRF and has the potential as a candidate biodecolorization
14 1.09 ± 0.26 ± 0.27 ± 102.75 agent.
0.02 cd 0.00d 0.00d
PP 7 1.07 ± 1.00 ± 1.00 ± 100.00
3.4. Biosorption test
0.00ef 0.00e 0.00e
14 1.00 ± 0.61 ± 0.60 ± 99.61
0.01e 0.00f 0.00f Three modes occur during the myco-decolorization process,
K. 7 1.12 ± 0.33 ± 0.36 ± 119.05 including biosorption involving components in the cell walls, enzymatic
III.3.4 0.05 cd 0.04 g 0.00 g degradation, or their combination. In this study, different fungal strains
14 1.06 ± 0.44 ± 0.45 ± 101.61
0.01ef 0.02 h 0.01 h
had different dye adsorption capabilities (Table 3). In general, all of the
AB KSP 7 1.96 ± 0.23 ± 0.17 ± 72.920 tested DSE fungi, except DD adsorbed the RBBR synthetic dyes (>50 %)
II3 0.09a 0.04ab 0.01b at 14-d incubation. In contrast, all DSE fungi, except DD and K.III.3.4,
14 1.71 ± 0.23 ± 0.24 ± 107.368 adsorbed low levels of the AB 113 dye (<50 %). RBBR was more readily
0.26a 0.02ab 0.01a
adsorbed by the DSE mycelia than AB 113. In addition, more synthetic
CPP 7 1.75 ± 0.31 ± 0.31 ± 98.485
0.07a 0.04c 0.02c dye was adsorbed as the incubation time was extended. However, the
14 1.19 ± 0.42 ± 0.44 ± 106.857 uptake capacity (qt) for all DSE fungi at 7 d was higher than at 14 d.
0.01b 0.02d 0.00d Fungal biosorption is a standard mode to transform a synthetic dye.
TKC 7 1.11 ± 0.73 ± 0.75 ± 102.941 The presence of phosphate and carboxyl functional groups in glucuronic
0.03c 0.01e 0.03e
14 1.03 ± 0.58 ± 0.59 ± 101.235
acid and amino groups in the chitosan of the fungi cell wall makes the
0.04c 0.03f 0.02f cell wall positively and negatively charged. Thus, the dye binds to
DD 7 1.18 ± 0.25 ± 0.01 ± 3.50 functional groups by covalent or ionic exchange bonds or van der Waals
0.05b 0.03 g 0.00 h forces for removal during the textile wastewater treatment (Alexander
14 1.04 ± 0.36 ± 0.37 ± 101.987
and Thatheyus, 2021).
0.01c 0.03i 0.01i
PP 7 1.06 ± 0.76 ± 0.76 ± 100.000 Different biomass weights were observed among the DSE fungi
0.01c 0.03j 0.02j during the incubation. KSP had the highest biomass (0.49 g/50 mL) in
14 1.01 ± 0.60 ± 0.60 ± 99.605 RBBR-containing media, followed by CPP, DD, PP, TKC, and K.III of
0.01c 0.00 k 0.01 k approximately 0.43, 0.32, 0.26, 0.26, and 0.17 g/50 mL, respectively,
K. 7 1.4 ± 0.33 ± 0.44 ± 130.714
III.3.4 0.12b 0.04 l 0.07 lm
over the 14 d incubation. Similarly, KSP had the highest biomass (0.56
14 1.04 ± 0.44 ± 0.49 ± 110.215 g/50 mL) in AB 113-containing media.
0.01c 0.02 m 0.00 m

The values are the average of three experiments ± standard error of the mean
(SEM). The values were significantly different P < 0.05 by one-way ANOVA with
the Tukey comparison test.
demonstrate that these fungi have a high tolerance to the tested dyes and
that the dye concentration (50 mg/L) had no toxic effect (Table 2) (TR >
90 %). Similar results were reported for WRF and brown-rot fungi
(Vasdev, 2011). Some other synthetic dyes may have an inhibitory effect
on fungi (Rueda-Villabona et al., 2021).
3.3. Decolorization of RBBR and AB 113 in liquid media
All of the tested DSE fungi were highly efficient at decolorizing both
synthetic dyes (>50 %) after 14 d of incubation in liquid media. Sig­
nificant differences in decolorization were observed after the 14 d of
treatment (decolorization ranges: 49 %–89 % and 69 %–97 % for RBBR
and AB 113, respectively) (Fig. 2). The KSP strain had the greatest
decolorization ability, while DD strain had the lowest ability to decol­
orize the synthetic dyes. Interestingly, the decolorization ability of all of
3.5. Visible spectral analysis
The untreated RBBR and AB113 synthetic dyes exhibited peaks at
400–800 nm. The peaks decreased after treating all of the DSE fungi with
the RBBR and AB 113 for 14 d (Fig. 3). The spectral peaks decreased
during RBBR decolorization, whereas no peaks were detected in the AB
113 absorption experiment (Fig. 3), suggesting complete degradation of
the AB 113 dye. The decrease and the absence of peaks may indicate the
degradation of the dye. As Kalpana et al. (2012) and Ting et al. (2016)
reported, the decrease or absence of peaks suggests the dislocation of the
dye chromophore by the DSE fungi. All of the tested DSE fungi degraded
both RBBR and AB 113. However, most of the DSE fungi studied were
more effective at degrading AB 113 than RBBR, as shown by the percent
decolorization of AB 113 (Fig. 2).
3.6. Microbial toxicity test
The E. coli and B. subtilis colonies were suppressed by the untreated
RBBR dye solution with inhibition zones of 7.00 and 8.00 mm, respec­
tively. AB 113 repressed the growth of E. coli and B. subtilis with

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I. Melati et al. Bioresource Technology Reports 22 (2023) 101427

(A) 100
90

80

70

Decolorization (%)
60

50
7
40 14
30

20

10

0
KSP PP CPP TKC K.III.3.4 DD
DSE strains

(B) 100
90
80
70
Decolorization (%)

60
50
7
40
14
30
20
10
0
KSP PP CPP TKC K.III.3.4 DD
DSE Strains

Fig. 2. Decolorization of RBBR (A) and AB113 (B) dyes by DSE fungi within 7 and 14 d.

inhibition zones of 9 and 10 mm, respectively (Table 4). Based on this
result, we assumed that AB 113 was more toxic than RBBR. However,
after the DSE fungal treatment, no inhibition of the E. coli and B. subtilis
colonies was detected. These results indicate that the decolorization by
the DSE fungi also detoxified the RBBR and AB113 dyes. This agrees
with the findings of Guo et al. (2020), and Yanto et al. (2021).
3.7. The effect of pH on dye decolorization, ligninolytic enzyme activity,
and biosorption capacity
The effects of pH (3–11) on the decolorization efficiency and bio­
sorption of the RBBR and AB 113 dyes are presented in Fig. 4. The three
selected DSE fungi (KSP, PP, and CPP) decolorized 74 %–99 % of the
RBBR and 79 %–99 % of the AB113 dyes, respectively, at pHs of 3–11.
The decolorization ability of the KSP and CPP strains was slightly
affected at pH 11. This result agrees with Spagnoletti et al. (2017) who
reported that DSE fungi are pH tolerant in the range of 2–10. In contrast,
other studies have reported that the decolorization ability of some WRF
decreases under alkaline conditions (Sudiana et al., 2018) This finding
indicates that DSE fungi may be a good alternative for wastewater
bioremediation under alkaline conditions.
In contrast, the biosorption capacity of the fungal strains to the RBBR
and AB 113 dyes decreased with increasing pH (Fig. 4). The maximum
biosorption of the RBBR and AB 113 dyes by the DSE fungi occurred at
pH 3. This result agrees with other studies reporting that maximum
adsorption occurs at an acidic pH (Dhaif-Allah et al., 2020; Pourali et al.,
2021). The competitive adsorption of H+ and dye ions with adsorbates
has been used to explain the adsorption of different anionic and cationic
species on adsorbents. A higher pH surface adsorbs cations due to the
deposition of ions. In contrast, a lower pH surface favorably adsorbs
anions due to the presence of H + ions. A similar outcome was observed
in the present investigation. RBBR and AB 113 are anionic dyes with
negatively charged chromophore groups. These anionic dyes dissolve
and discharge colored dye ions into the solution. More protons are
available to protonate the amino groups of the chitosan molecules on the
fungal cell walls to generate positively charged NH+ 3 groups when the pH
of the solution drops. Dye adsorption increases due to increased elec­
trostatic attraction between the anionic groups of the dye and the pro­
tonated amino groups (NH+ 3 ) of chitosan. The increase in the distribution
of negative charges on the fungal biomass surface under alkaline con­
ditions would cause the electrostatic attraction between the adsorbent
and dye molecules, which would decrease biosorption capacity. The
competition for adsorption sites between the chromophores and the ions
increases at higher pHs as the number of OH ions increases, which de­
creases the sorption (Chen and Chen, 2009; Saraf and Vaidya, 2015).
The effects of pH (3–11) on laccase and MnP activities produced
during the decolorization of the RBBR and AB 113 dyes are displayed in
Fig. 5. All three selected DSE fungi secreted laccase and MnP under an

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I. Melati et al. Bioresource Technology Reports 22 (2023) 101427

Table 3
Biomass dry weight, biosorption rate and uptake capacity of DSE fungi during the decolorization process.
Dyes Isolate Incubation time (d) Biomass dry weight (g) Biosorption rate Uptake capacity

Control Treatment (%) (mg/g)

RBBR KSP 7 0.03 ± 0.01a 0.03 ± 0.01a 49.06 ± 3.51 66.78 ± 2.75
14 0.47 ± 0.05b 0.49 ± 0.07b 81.23 ± 1.74 3.94 ± 0.28
CPP 7 0.05 ± 0.01c 0.05 ± 0.00c 39.27 ± 0.61 22.63 ± 0.06
14 0.35 ± 0.06d 0.43 ± 0.06d 56.52 ± 6.41 3.80 ± 0.03
TKC 7 0.03 ± 0.01e 0.05 ± 0.02e 60.29 ± 0.48 29.52 ± 2.05
14 0.23 ± 0.01f 0.26 ± 0.06f 64.54 ± 3.42 5.82 ± 0.14
DD 7 0.02 ± 0.01 g 0.04 ± 0.03 g 43.76 ± 1.39 8.67 ± 0.23
14 0.31 ± 0.10 h 0.32 ± 0.20 h 44.81 ± 0.50 2.86 ± 0.26
PP 7 0.02 ± 0.01i 0.05 ± 0.04i 76.77 ± 0.51 28.57 ± 0.45
14 0.26 ± 0.02j 0.26 ± 0.04j 77.86 ± 0.01 5.86 ± 0.13
K.III.3.4 7 0.14 ± 0.04 k 0.09 ± 0.05 k 62.29 ± 1.41 10.43 ± 1.05
14 1.31 ± 0.01 l 0.17 ± 0.03 k 68.95 ± 3.06 8.71 ± 0.22
AB II3 KSP 7 0.02 ± 0.00a 0.37 ± 0.05b 24.43 ± 3.16 8.00 ± 0.19
14 0.47 ± 0.05b 0.56 ± 0.01b 55.14 ± 1.54 6.72 ± 1.20
CPP 7 0.05 ± 0.01c 0.10 ± 0.00c 20.24 ± 0.42 21.15 ± 0.09
14 0.35 ± 0.06d 0.23 ± 0.04e 48.07 ± 1.05 18.82 ± 0.79
TKC 7 0.02 ± 0.01f 0.15 ± 0.03 g 29.22 ± 0.77 18.90 ± 0.04
14 0.23 ± 0.01 g 0.23 ± 0.02 g 63.61 ± 4.80 16.92 ± 1.44
DD 7 0.02 ± 0.01 h 0.07 ± 0.01 h 45.38 ± 0.27 23.82 ± 2.26
14 0.48 ± 0.13i 0.22 ± 0.14 h 97.70 ± 3.91 10.70 ± 0.03
PP 7 0.02 ± 0.01j 0.23 ± 0.06 k 41.90 ± 0.27 12.08 ± 1.02
14 0.26 ± 0.02 k 0.27 ± 0.06 k 44.55 ± 3.78 11.31 ± 0.08
K.III.3.4 7 0.12 ± 0.02 l 0.09 ± 0.05 l 85.37 ± 3.90 32.42 ± 1.06
14 1.31 ± 0.01 m 0.17 ± 0.03 l 58.55 ± 3.48 24.64 ± 0.49

The values are the average of three experiments ± standard error of the mean (SEM). The values were significantly different P < 0.05 by one-way ANOVA with the
Tukey comparison test.

0.7 Table 4
Untreated RBBR dye
A KSP Microbial toxicity test of RBBR and AB 113 before and after treatment.
0.6 PP
CPP Dyes Inhibition zone (mm)
DD
K.III.3.4 E. coli B. subtilis
0.5 TKC
Untreated RBBR 7 8
Treated RBBR by
Absorbance

0.4
KSP 0 0
CPP 0 0
0.3
PP 0 0
TKC 0 0
0.2 DD 0 0
K.III.3.4 0 0
0.1 Untreated AB 113 9 10
Treated AB 113 by
0 KSP 0 0
400 500 600 700 800 CPP 0 0
Wavelength (nm) PP 0 0
TKC 0 0
DD 0 0
0.7 K.III.3.4 0 0
Untreated AB 113 dye
B KSP Control (chloramphenicol 0.4 %) 23 25
0.6 PP
CPP
DD
0.5 K.III.3.4 alkaline pH, suggesting that the selected DSE fungi may be used in
TKC
textile processes that require an alkaline pH. The limitation of applying
laccase in the industry is that it requires an acidic pH to function
Absorbance

0.4
properly, so high-activity laccase under alkaline conditions is urgently
0.3 needed (Novoa et al., 2019). In contrast, a previous study showed that
most fungal laccases have an optimal pH range of 3–5.5 when used with
0.2 phenolic substrates, and they lose their activity very quickly at pH 7 (Yin
et al., 2019).
0.1 The quantitative laccase activity in the liquid media differed slightly
from the qualitative laccase activity results, in which the KSP and PP
0 strains lacked laccase activity in agar media as described in Table 1. This
could be because the substrates used in the tests were different; the
400 500 600 700 800
Wavelength (nm)
quantitative test used the ABTS substrate, while the previous test
Fig. 3. The visible spectral analysis of RBBR (A) and AB 113 (B) solution within employed guaiacol. According to Li et al. (2008), substrates’ efficiency
the 14-d incubation in the presence of various DSE fungi. significantly affects enzyme test sensitivity. In addition, some research
has revealed that laccase activity is higher with the ABTS substrate than
with the guaiacol (Li et al., 2008).

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I. Melati et al. Bioresource Technology Reports 22 (2023) 101427

(A) (B)

100 100

Decolorization (%)
Decolorization (%)
80 80

60 60

40 40

20 20
0 0
KSP PP CPP KSP PP CPP
(A) DSE strain DSE strain
(B)

100 100
80 80
Biosorption (%)

Biosorption (%)
60 60

40 40

20 20

0 0
KSP PP CPP KSP PP CPP
DSE Strain DSE Strain

Fig. 4. Effect of pH on the decolorization efficiency and biosorption of RBBR (A) and AB 113 (B) dyes by different DSE fungi ( 3, 5, 7, 9, and 11).

20 20
(A) (B)
15 15
Laccase (U/L)

Laccase (U/L)

10 10

5 5

0 0
KSP PP CPP KSP PP CPP
DSE strain DSE strain

20 20
(A) (B)
15 15
MnP (U/L)

MnP (U/L)

10 10

5 5

0 0
KSP PP CPP KSP PP CPP
DSE strain DSE strain

Fig. 5. Effect of pH on laccase and MnP activities of DSE fungi in the presence RBBR (A) and AB 113 (B) dyes at different pH ( 3, 5, 7, 9, and 11).

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I. Melati et al. Bioresource Technology Reports 22 (2023) 101427

5 5
KSP KSP
4 4 PP
PP
CPP
3 CPP 3

Ln C
Ln C
2 2
1 1
0 0
0 5 t (d) 10 15 0 5 10 15
t (d)
(A) (B)
0.3 0.3
KSP
KSP 940
PP PP
0.2 CPP 0.2 CPP
1/C

1/C
0.1 0.1
941
0 0
0 5 10 15 0 5 10 15
t (d) t (d)

(C) (D)

Fig. 6. Pseudo-first-order kinetic linear model for RBBR (A) and AB 113 (B), and Pseudo-second-order kinetic linear model for RBBR (C) and AB 113 (D) dyes.

depicted by a pseudo-second-order kinetic model rather than a pseudo-

Table 5
first-order model. This result agrees with other studies reporting that the
The value of the parameters in the kinetic models.
pseudo-second-order model better explains the adsorption kinetics of
Dyes DSE Fungi Decolorization kinetic models RBBR and AB 113 rather than a first-order model (Anita et al., 2019;
Pseudo-first-order Pseudo-second-order Pourali et al., 2021).
2
k1 R k2 R2
3.8.2. Isothermal models
RBBR KSP 0.14 0.86 0.01 0.96
PP 0.14 0.69 0.01 0.88
The Freundlich and Langmuir adsorption equations were used to
CPP 0.14 0.93 0.01 0.98 explain the biosorption of the dyes by the DSE fungi. Based on the
AB 113 KSP 0.13 0.58 0.01 0.70 isotherm data and correlation coefficients (Table 6), the biosorption of
PP 0.13 0.57 0.01 0.69 the RBBR dyes by the DSE fungi fit the Langmuir model better, while AB
CPP 0.12 0.74 0.01 0.90
113 dye biosorption followed the Freundlich model, under the dye
concentration ranges studied. These results suggest that biosorption of
3.8. Kinetic and isotherm study the RBBR dye was by chemisorption, in which a single layer of adsorbed
material (dye) is on top of a uniform adsorbent surface (the cell wall of
3.8.1. Adsorption kinetic models the DSE) at a constant temperature. In contrast, biosorption of AB 113
The experimental data were analyzed using pseudo-first-order and was physical adsorption, in which adsorption takes place in several
pseudo-second-order kinetic formulas to understand the adsorption ki­ layers with a weak connection. The Freundlich model assumes that the
netics. Both adsorption kinetics fit a linear regression model (Fig. 6), in adsorption sites are heterogeneous.
which the adsorption capacity depended on the time of contact with the Numerous factors and an unusual adsorption isotherm pattern,
biosorption material. The correlation coefficients (R2) of the pseudo- including the type of adsorbent, the substance adsorbed, the surface
second-order equation for RBBR and AB 113 of the selected DSE area, the concentration of the substance adsorbed, and the temperature
strains were higher than those of the pseudo-first-order equation affect the adsorption process (Dallel et al., 2018; Yildirim et al., 2020).
(Table 5), indicating that the biosorption of RBBR and AB 113 was In the presence of such factors, any adsorbent that adsorbs one material
would not have the same adsorption pattern for other materials.
This is the first report on the capability of DSE fungi to decolorize
Table 6 RBBR (anthraquinone) and AB 13 (azo) dyes at different pHs. Thus, this
The value of the parameters in the isotherm models. study contributes significantly to knowledge about DSE fungi, particu­
larly their capability of degrading pollutants under alkaline conditions
Dyes DSE Adsorption isotherm models
strains in conjunction with their ligninolytic enzyme potential. DSE fungi have
Langmuir Freundlich
become promising candidates for green textile wastewater remediation
qe (mg/ KL (L/ R 2
n Kf (L/ R2 technology.
g) mg) mg)

RBBR KSP 14.53 7.02 0.98 9.24 9.16 0.19 4. Conclusions

PP 37.31 0.59 0.97 2.65 14.56 0.67
CPP 26.11 0.02 0.93 2.68 2.94 0.88
All of the DSE strains (KSP, CPP, PP, DD, TKC, and K.III.3.4) had
AB KSP 73.53 0.04 0.87 7.49 2.32 0.98
113 PP 28.09 0.05 0.96 0.58 1.85 0.99 phenol oxidase activity, and the ability to decolorize RBBR and AB113
CPP 96.15 0.04 0.86 1.46 5.98 0.95 on solid and liquid media. Three isolates, such as KSP, PP, and CPP,

9
I. Melati et al.
displayed high performance among the DSE fungi. The selected DSE
decolorized 74–99 % of the anthraquinone (RBBR) and 79 %–99 % of
the azo (AB 113) dyes at pH 3–11 using ligninolytic enzymes (laccase
and MnP). This study shows that the DSE fungi decolorized the dyes
through an adsorption and degradation mechanism, and simultaneously
detoxified the dyes. Based on the kinetic data, a pseudo-second-order
(R2: 0.69–0.98) equation was the best model for the biosorption of
both dyes. The Langmuir model (R2: 0.93–0.98) was the best-fit model
for RBBR dye biosorption and the Freundlich model (R2: 0.95–0.99) was
the best-fit model for AB 1113 biosorption based on the isothermic data.
These newly isolated DSE fungi are potential candidates for green
wastewater bioremediation, particularly under alkaline conditions.
CRediT authorship contribution statement
All authors are the main contributors to the research. Irma Melati:
methodology, formal analysis, investigation, writing of the original
draft. Gayuh Rahayu: supervision, conceptualization, writing, review­
ing, and editing. Surono: supervision, funding acquisition, project
administration, writing, reviewing, and editing. Hefni Effendi: super­
vision, conceptualization, writing, reviewing, and editing. Cynthia
Henny: supervision, funding acquisition, project administration,
writing, reviewing, and editing. Dede Heri Yuli Yanto: supervision,
funding acquisition, project administration, data analysis, writing,
reviewing, and editing.
Declaration of competing interest
The authors declare that they have no known competing financial
interests or personal relationships that could have appeared to influence
the work reported in this paper.
Data availability
No data was used for the research described in the article.
Acknowledgments
This research was supported by the Saintek Grant of the Ministry of
Research and Technology of Indonesia. We collaborated with the
Indonesian Soil Research Institute, the Ministry of Agriculture, Research
Center for Limnology and Research Center for Applied Microbiology,
National Research and Innovation Agency (BRIN), Indonesia and Inte­
grated Laboratory of Bioproducts (iLaB), National Research and Inno­
vation Agency (BRIN).
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