Journal of Water Process Engineering 37 (2020) 101357

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Journal of Water Process Engineering

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Coupling Laccase production from Trametes pubescence with heavy metal T

removal for Economic Waste Water Treatment
Naeimeh Enayatizamira,b, Jing Liua, Li Wanga, Xiuying Lina, Pengcheng Fua,*
a
State Key Laboratory of Marine Resource Utilization in South China Sea, Hainan University, 570228, China
b
Soil Science and Engineering Department, Faculty of Agriculture, Shahid Chamran University of Ahvaz, Ahvaz, Iran

ARTICLE INFO ABSTRACT

Keywords: The objective of this work is to evaluate simultaneously the impact of nickel and lead on the fungus Trametes
Biosorption pubescence growth and laccase production, and their removal efficiency by T. pubescence. Here laccase activity
Fungus and production were investigated in both in vitro and in vivo fungal growth conditions at the presence of either Pb
Heavy metal or Ni, with the results showing inhibitory effects of Pb and Ni on T. pubescence growth and laccase activity. In
Laccase
particular, T. pubescence was seen to be able to withstand 1000 mg L−1 of Pb and Ni, while the fungus was
Trametes pubescence
capable of removing 99 % of Pb and 8.6 % of Ni, respectively. Most importantly, it was found that although only
live cells will secrete laccase, both live and dead fungal biomass can accumulate heavy metals in the aqueous
solutions. The laccase production can thus be coupled with heavy metal removal for potential waste water
treatment.

1. Introduction oxygen oxidoreductase, EC 1.10.3.2) is a group of copper containing

Heavy metals have been considered as hazardous elements which
may accumulate in plants to inhibit their growth and yield for the soil
ecosystem [1], or harm fishes and other aquatic organisms in water
through inducing reactive oxygen species [2]. The main sources of
heavy metals pollution are such as mining extraction operations, metal
processing in refineries, coal burning in power plants, petroleum
combustion, plastics, microelectronics and textile industries [3–8].
Metal ions such as copper, iron and nickel can act as co-factors to
benefit cellular growth with even trace level, they would become toxic
in excess amount to majority of the living systems [9]. Other metals
such as aluminium (Al), antinomy (Sb), arsenic (As), beryllium (Be),
bismuth (Bi), cadmium (Cd), gallium (Ga), gold (Au), indium (In), lead
(Pb), lithium (Li), and mercury (Hg) have no biological functions [10].
It needs to be noted that the non-degradable nature of heavy metals has
caused them to remain and accumulate in the environment and threat
seriously the viability of eco-systems [4]. Microbial approaches to di­
minish the toxicity of some heavy metal ions or transform them to less
harmful reduced states have been reported in the literature [11–17].
Some fungi are found to be effective in degrading compounds with
lignin structure. Among them, Trametes is mostly reported to be an
efficient degrader [18–20]. Among the species of this genus, Trametes
pubescence is selected to perform the present work, since it is able to
produce considerable amounts of laccase [21]. Laccase (benzenediol:
oxidase which can degrade a wide range of aromatic and non-aromatic
compounds [22–27] which is found in many plants, fungi and bacteria
[28,29].
So far there are only few reports to investigate the production of
laccase of fungi in the presence of heavy metals for the degradation of
lignocellulosic materials [30,31] and some recalcitrant compounds
such as Azo dyes [21] in the wastewater environments. It was found
that the dead fungal biomass can accumulate heavy metals in the lit­
erature [32–34]. Furthermore, it was seen that the heavy metal ions
entrapped by dead cells were bound to functional groups of cell wall
first [35], then transformed into less toxic elements by live cells
through different biosorption mechanisms such as binding with protein
and efflux pumping [36–38].
This study aimed to determine the effect of two heavy metals, Pb
and Ni, on the laccase activity in Trametes pubescence and at the same
time the heavy metal removal by the fungus which, to our knowledge,
has not been reported to date. In particular, the mechanisms of Pb and
Ni removal from the aqueous solutions using viable biomass of T.
pubescence cells were ascertained through Scanning Electron
Microscopy, SEM; Transmission Electron Microscopy, TEM; and Energy
Dispersive X-ray, Fourier Transform Infrared, FTIR assay. It was found
that although only live cells will secrete laccase, both live and dead
fungal biomass can accumulate heavy metals. The laccase production
can thus be coupled with heavy metal removal for potential waste water

⁎
Corresponding author.
E-mail address: pcfu@hainanu.edu.cn (P. Fu).

https://doi.org/10.1016/j.jwpe.2020.101357
Received 3 September 2019; Received in revised form 30 March 2020; Accepted 10 May 2020
Available online 28 June 2020
2214-7144/ © 2020 The Author(s). Published by Elsevier Ltd. This is an open access article under the CC BY license
(http://creativecommons.org/licenses/by/4.0/).
N. Enayatizamir, et al.
treatment.
2. Material and methods
2.1. Fungal strain
Trametes pubescens MB 89 (CBS 696.94; Centraalbureau voor
Schimmelcultures, Utrecht, The Netherlands) was obtained from Dr.
Susana Rodriguez couto (The Technology Centre Ceit-IK4 (Donostia-
San Sebastian, Spain).
2.2. Pb and Ni effect on fungal growth on plates
Effect of heavy metals (Pb and Ni) on the T. pubescence radial
growth was monitored on PDA medium (1/4-Strenghth PDA) con­
taining different concentrations of Pb and Ni (0, 10, 25, 50. 100, 200
ppm) using Pb (NO3)2 and Ni (Cl) 2. 6H2O, respectively. A plug of
fungal inoculum was placed on medium and incubated at 30 °C for 6
days. Radial growth of fungus was measured daily and the inhibition
percentage of fungal growth was evaluated. The half maximal effective
concentration (EC50) of Ni and Pb was determined using Origin 9.1
software.
2.3. Laccase production
The fungus was grown in an Erlenmeyer flask containing 100 ml of
culture medium according to Enayatzamir et al. [21]. The Erlenmeyer
flasks containing T. pubescence were statically incubated at 30 °C and
supplemented with 0.5 mM Cu2+ to stimulate laccase production on
third day of cultivation. Laccase activity assay was conducted using
reaction mixture consisted of 1.15 ml diluted crude enzyme extract
(50−100 μl) in succinate buffer (pH 4.5) and 0.35 mL ABTS reagent in
the cuvette. The absorbance increase was measured at 436 nm for 2 min
via spectrophotometer at 436 nm using according to the method de­
scribed by Niku-Paavola et al. [39]. One activity unit (U) was defined as
the amount of enzyme that oxidized 1 μmol of ABTS per min. The ac­
tivity was expressed in U/L.
2.4. In vitro stability of Laccase activity in the presence of Ni and Pb
1150 ml mixtures of the diluted crude enzyme extract from Trametes
pubescens in succinate buffer (25 mM, pH 4.5) and Ni or Pb solutions
(10, 25, 50, 100, 200, 400, 600, 800 and 1000 ppm) were kept at 25 °C
for 8 and 24 h, then the laccase activity was determined via spectro­
photometer by adding 350 μl substrate (0.5 mM ABTS) and reading the
absorbance at 436 nm for 2 min. The experiment was run using a
control in which the inhibitor (Pb or Ni) was replaced with the succi­
nate buffer.
2.5. In vivo laccase production in the presence of Ni and Pb
Trametes pubescens was grown in Erlenmeyer flask containing 100
ml of above mentioned culture medium with reduced amount of glu­
cose and peptone to 5 g and phosphate-citrate buffer to 1 mM and in­
cubated at 30 °C under stationary condition, then the flasks were
transferred into shaking incubator on 6th day and shaked at 140 rpm. It
was supplemented with different concentration of Pb and Ni (0, 10, 25,
50. 100, 200 ppm) on 8th day of cultivation. It was then observed that
T. pubescence started to produce laccase. Afterwards the activity of
laccase was measured every day for 5 days.
2.6. Biosorption of Pb and Ni by T. pubescence in aqueous solutions
Biosorption experiment was carried out in 250 mL Erlenmeyer
flasks, as mentioned above, with 1000 mg L−1 of Pb (II) and Ni (II)
addition on 8th day of cultivation. The flasks were agitated at a
2
Journal of Water Process Engineering 37 (2020) 101357
constant speed of 140 rpm in a shaking incubator. The pH of the so­
lutions was adjusted with 1 mM of phosphate citrate buffer at 4.5. The
samples were taken once a day for 5 days, and filtered through 0.2 μm
Millipore membrane filters to measure laccase activity, and Pb and Ni
concentration by atomic absorption spectrometer (TAS-990 Super AFG,
Beijing Purkinje General Instrument Co., Ltd., Beijing, China). At the
end of experiment, the T. pubescence biomass was separated and washed
two time with sterile ultra-pure water to release the adsorbed metals
from the surface [40] (Albert et al., 2018). Afterwards the collected
biomass was dried at 60 °C and digested by aqua regia to extract metals
from the fungus [40]. The liquid and digested samples were analyzed
by atomic absorption spectrometer. All experiments were repeated
three times and the average results were presented. A control was
prepared in the same condition in a medium containing heavy metals
and without fungus to evaluate the effect of medium on metals. The
metal removal and bioaccumulation were calculated using the fol­
lowing equation:
(C0 Cf )100
R=
C0 (1)
(C0 Cf ) V
R=
M (2)
Where R: removal; q: bioaccumulation; C0: initial metal concentration
(mg L−1); Cf: final metal concentration (mg L−1); M: dry weight of
fungal cells (g); V: volume of solution (L).
The adsorption percentage was calculated by dividing the amount of
measured metals in washing solution on initial concentration of metals.
The absorption ratio was determined from the ratio metals amounts
inside the fungus on initial concentration of metals.
2.7. Ion exchange examination
To determine the mechanisms of metal ions absorption by T. pub­
escence the amount of K in withdraw sample at different days was
measured by Flame photometer (410 Falme photometer, Sherwood
Scientific Ltd, Uk).
2.8. Scanning electron microscopy (SEM) with energy dispersive X-ray
analysis (EDX)
The filtered mycelium samples were treated both with a 1000 ppm
of metal solution and without metal. They were washed twice with
ultra-pure water and then prepared for analysis by drying under la­
minar flow at room temperature. A thin film of carbon, approximately
30−50 nm has been applied as coating material for SEM (Hitachi S-
4800 Hitachi, Ltd, Chiyoda-ku, Japan). Energy-dispersive X-rays ana­
lysis of samples was conducted with the coated samples on Au, using an
EDAX EDS Silicon Drift 2017 (FEI ESEM Quanta200, FEI, Holand).
Point and region analyses were performed at 25 kV. The EDX spectra
with Pb peaks (0−14 keV) were recorded.
2.9. Transmission electron microscopy (TEM)
Transmission electron microscopy imaging of live treated mycelium
with Pb and also control mycelium without any Pb was performed via
transmission electron microscopy (TEM; JEM-2100, JEOL, Tokyo,
Japan) with an operating voltage of 30kv.
2.10. X-ray diffraction (XRD) and atomic force microscopy(AFM)
The dried mycelium with and without metal ions was powdered and
used to measure X-ray diffraction of samples via a Bruker operated at a
voltage of 40 kV and a current of 40 mA with Cu-Ka radiation over the
range of 10° -100° (2teta) with a step length of 0.05° (2teta). Pb loaded
and free metal mycelium surface topography was measured using AFM
N. Enayatizamir, et al.
(DME 95−50E, Denmark) in contact mode using Silicon tips (Nano
World Pointprobe) with force constant of 0.07−0.4 N m−1.
2.11. Fourier transform infrared spectroscopy (FTIR)
The metal biosorption by T. pubescence cells was investigated via
FTIR analysis and functional groups responsible for metal binding on
the cell surface were characterized. The air dried biomass with and
without metals treatment were mixed with KBr powder. They were then
scanned via FTIR spectrophotometer (Perkin Elmer Frontier, Perkin
Elmer Inc., USA) within the Ir region of 500−4000 cm−1.
2.12. Viability examination of fungus cells in the presence of metals
Viability of T. pubescence inside the flasks with 1000 ppm of Pb and
Ni was evaluated by subculture of three plugs (4 mm diameter) of
fungus from each flask containing fresh medium in the absence of the
above mentioned metals. Laccase activity of each flask was measured
for 5 days from 8th day of cultivation.
2.13. Statistical analysis
All the experiments were conducted at three replications and the
means of replications are presented along with standard deviation
calculated using SPSS (version 16).
3. Results and discussion
3.1. Metals effect on fungal growth in agar plates
Radius growth diameter of T. pubescence in solid medium supple­
mented with different concentration of Pb and Ni has been presented in
Fig. 1. Radius growth of Trametes pubescence inhibition on PDA medium (1/4-
Strenghth PDA) containing different concentrations of Pb and Ni (0, 10, 25, 50.
100, 200 ppm) (A) and Half maximal effective concentration (EC50) of Pb and
Ni (B) against T. pubescence.
3
Journal of Water Process Engineering 37 (2020) 101357
Fig. 1. The EC50 and EC80 of Pb and Ni (Fig. 1) showed different effect
of metals on the T. pubescence growth. The growth was decreased with
the increase in both Pb and Ni concentrations, the figure shows that T.
pubescence was more tolerant against Pb than Ni. In comparison, it has
been reported in the literature that growth of the fungus Absidia cy­
lindrospora was viable in the medium containing lead at 10, 50 and 100
ppm while the growth was inhibited at 1000 ppm [40]. In another
study, IC50 of 50 mg L−1 for Pb has been recorded for Absidia cylin­
drospora [40]. Joshi et al. [41], reported growth inhibition of different
fungi isolates such as Phanerochaete chrysosporium, Aspergillus awamori,
Aspergillus flavus, Trichoderma viride in the presence of Pb and Ni with
different concentration (25, 50, 100 and 400 ppm). They have shown
the Pb and Ni tolerance at the concentration of 25 ppm only. Our result
indicated by EC50 of 73.4 for Pb that is higher than that in the men­
tioned above literature and the fungus was able to tolerate Pb better
than Ni (EC50 of 9.9) in solid culture.
3.2. Effects of Pb and Ni on T. pubescence laccase activity stability and
production
The results obtained by different concentration of Pb and Ni on the
laccase activity in Figs. 2 showed the stability of laccase activity in the
presence of heavy metals, although the laccase activities for the growth
with Pb or Ni were slightly lower comparing to the control. It can be
seen in Fig. 2 that when Pb concentration was 100 ppm, the laccase
stability was decreased while it kept increasing at 200, 400 and 600
ppm of Pb due to precipitation of Pb. Further increase in Pb con­
centration would lead to constant decrease of the laccase activity. The
negative effect of Pb is mainly due to the inactivation of enzyme by the
metal.
The laccase production in the presence of Ni was different, 10 and
25 ppm of Ni caused to increase of the activity, while it would decrease
by the further increase in the concentration of Ni. The impact of Pb and
Ni on laccase production in flasks can be seen in Fig. 3. It shows that
comparing to the control, the laccase production was decreased in the
presence of Pb, while it was increased in the presence of Ni up to 50
ppm after 144 h of exposure to the heavy metal. In the literature, in­
hibition effects of Cd2+ and Mn2+ and Pb2+ on activity of laccase from
Trametes versicolor, Daedalea quercina and Pleurotus ostreatus have been
reported, respectively [29,42,43]. In general, our results are in agree­
ment with their findings. It is found that the induction of the laccase
activity only occurred at low concentration of Ni. When the Ni con­
centration was increased to higher concentration, the laccase produc­
tion would be declined. Similarly, the induction of laccase activity of
micromycetes exposed to 0.005 M Ni has been recorded after 21 days of
cultivation that increased 1.3–3.3 times in comparison with the control
[44]. Whereas concentration of 0.01 M of NiCl2 depressed the laccase
activity in all micromycetes, Galactomyces geotrichum achieved 1.5
times increase of the laccase activity on the 18th day of cultivation in
comparison with the control. In summary, production of laccase was
found to be related to the presence of metal ions in fungal cell cultures
[44,45].
3.3. Pb and Ni removal from the aqueous solutions
Our previous experimental data [21] indicates that there were two
cultivation phases of T. pubescence growth in the liquid cultures: Phase
1. fungal growth for about 8 days (at day 3 0.5 mM Cu2+ was added as
an inducer for later laccase production) and Phase 2. T. pubescence cell
growth into stationary phase, laccase production was started. We thus
introduced Pb and Ni into liquid culture of T. pubescence after 8 days of
fungal growth.
Fig. 4 is the figure for the biosorption and removal of Pb and Ni at
1000 mg L−1 by T. pubescence. On exposure to Ni, the highest bio­
sorption rate (8.6 %) by T. pubescence was obtained, while a maximum
Pb removal and biosorption was 97.3 % and 99.56 % after 72 h,
N. Enayatizamir, et al.
Fig. 2. In-vitro measurement of the laccase activity stability after 6 and 24 h exposure at different concentration of Ni (A) and Pb (B) (in cuvette).
respectively. In the literature, about 66.3 % maximum Pb removal by
Pleurotus ostreatus HAU-2 in liquid culture contain 1500 mg L−1 of Pb
has been reported after 10 days exposure to Pb [30]. Additionally, a
maximal Pb removal rate and uptake of 71.04 % and 76.06 mg g−1 by
A. lentulus FJ172995 was observed at 1000 mg L−1 of Pb after 5 days
exposure to Pb. The Pb uptake capacity of fungus increased by initial
concentration of P b [46]. In another report, the Pb biosorption capacity
for A. flavus was 20.75–93.65 mg/g with initial concentration
200−1400 ppm [47].
It can be observed that our T. pubescence strain was more effective
for Pb removal at the high concentration such as 1000 ppm after 3 days
exposure to the heavy metal. One of promising strategy in treatment of
contaminated the waste water containing heavy metals is the use of
filamentous fungi to remove those toxic metals [46]. Extracellular ad­
sorption and intracellular bioaccumulation are mainly mechanisms of
heavy metal removal by fungi [48]. Metal ions transported through the
cell wall is facilitated via different mechanisms such as ion-exchange,
hydrolytic adsorption and surface precipitation [30], while the metal
ions taken into the cells are reduced by intracellular metal-binding and
sequestration sites. The maximum Pb bioaccumulation by our fungus
has reached 367 mg g−1, which was higher than those recorded by
other fungi, such as 165 mg g−1 for P. ostreatus HAU [30]. T. pubescence
biomass was measured in the presence of Pb and Ni (Fig. 5).
The results showed the decrease in biomass exposed to Ni compared
to control and increase of that in the presence of Pb. The increased
biomass exposed to Pb was due to absorbed Pb weight. After 4 days of
Pb exposure, the biomass of T. pubescence was 50 % higher than that for
the control (without Pb). On the other hand, the biomass of T.
4
Journal of Water Process Engineering 37 (2020) 101357
pubescence grown with Ni reached 34 % of that for the control only at
day 4. Similar results were observed by Moore et al. [49], who had
reported that Ni (II) caused a significant inhibitory effect on the fungal
biomass production.
An interesting difference was observed between the fungal tolerance
in agar and liquid medium containing Pb and specially Ni. Our toler­
ance test in agar medium (Fig. 1), shows that Ni was toxic metal to
fungi. However, in liquid media, they could tolerate both Pb and Ni
elements even at 1000 mg L−1. This difference might be due to the
varying bioavailability of metals in agar and liquid medium. We have
found that T. pubescence was not to remove Ni from the medium, while
there are some reports in the literature that other fungi may be able to
remove Ni [50]. For this research, the authors have isolated A. niger
strain tolerant to cadmium, chromium, cobalt, copper and nickel. The
strain was able to remove nickel from the solution at maximum level of
98 % after 100 h of culturing with the metal (6.5 mM), while it failed to
remove other metals. In another study, 98 % removal rate of nickel by
Aspergillus lentulus FJ172995 at 140 mg L−1 with the Ni concentration
of 11.05 mg g−1 in medium has been reported [46].
The viability of fungi exposed to heavy metals was determined by
cultivation of three plugs of T. pubescence in fresh medium. The laccase
activity of each treatment showed the ability of T. pubescence to grow
and produce laccase, although laccase production was lower in flasks
containing Pb, compared to T. pubescence exposed to Ni (Fig. 6). These
tests show also the possibility of reusing fungi to remove Pb from the
environment. Up to now, most of the researches applied non-viable
microorganisms for removal of heavy metals [51,52]. Preparation of
these biosorbents needs to harvest and dry collected biomass which
N. Enayatizamir, et al.
Fig. 3. In vivo measurement of Trametes pubescence laccase production after
several days’ exposure at different concentration of Ni (A) and Pb (B) (in flasks).
Fig. 4. Trametes pubescence Pb (A) and Ni (B) biosorption and removal from the
solution (initial concentration :1000 ppm).
disables continuous operation of the systems [53,54]. While in living
system, there is no need to separate biomass. Self-replenishment and
continuous metabolic absorption would thus give benefits for the use of
living microorganisms to treat contaminated environments [55].
5
Journal of Water Process Engineering 37 (2020) 101357
Fig. 5. Dry biomass of Trametes pubescence after 24, 48 and 72 h exposure to
1000 ppm Ni and Pb in flask cntaining liquid medium compared to control flask
without heavy metal addition.
Fig. 6. Viabilty test of Ni and Pb (1000 ppm) exposured Trametes pubescence
after changing the medium with new one without heavy metals and measuring
laccase production in new culture medium compared with control (cultured
fungus without heavy metals).
3.4. K content of liquid culture
Heavy metals exchanged with K, Na, Ca and Mg is one of the bio­
sorption mechanisms of fungi [56–58]. In our work, potassium content
of liquid culture showed the difference between control and Pb treated
medium. K concentration of medium in control was 239.04 mg L−1 and
increased significantly (P < 0.05) to 268.82 mg L−1 after 48 h of
treatment with Pb.
3.5. FTIR analysis and XRD pattern
Shifts in the cellular functional group of fungal biomass exposed to
Pb and Ni were observed in FTIR spectra. Table 1 displays the present
peaks for the samples of mycelium after 72 h in contact with Pb and Ni,
in comparison with the control in free metal medium. It is seen that
changes in vibrational frequencies using FTIR analysis on the metal-
treated T. pubescence revealed the involvement of the fungus in binding
of Pb. In particular, the shift in the region of 3500−3300 cm−1 in­
dicates that the NHe or carboxyl group stretching vibration [59] hap­
pened to the Pb and Ni treated fungal culture. Additionally, for the Ni
treated T. pubescence, a distinct change in the FTIR spectra in the region
of 3000 to 1600 cm−1 was seen, which was related to the carboxyl
group (C]O of COOH) in appearance of a band of 2091 cm−1. A new
peak at 957 cm−1 corresponding to phosphate (PO4) appeared in the
spectrum for the T. pubescence growth with Ni. Appearance of the peak
at 927 cm−1 correspond to phosphate group were detected for Zn
treated Penicillium simplicissimum [60].
Furthermore, the peaks at 1077 cm−1 (control) corresponding to the
groups of amide band [61] or phosphate groups [58] was changed to
1079.5 cm−1 for the T. pubescence growth with Ni loaded. The complete
masking of peaks related to the carboxylic (1314.00 cm−1) and phos­
phate group (1155.2 and 1077 cm−1) was observed in the presence of
N. Enayatizamir, et al. Journal of Water Process Engineering 37 (2020) 101357

Table 1
The FTIR bands frequencies (cm−1) and assignments for T. pubescence with and without Pb and Ni in broth medium.
Untreated fungus Pb treated Ni treated Suggested assignment

3331.4 3402.1 3410.1 OeH and NeH stretching vibrations

2926.9 2926.6 2928.9 eCH asymmetric stretching
– – 2091.4 C]O of COOH stretching
1655.1 1654.8 1645.5 C]O stretching and NeH deformation (amide I region)
1545.4 1547.3 – NeH deformation (amide II region)
– 1574.3 – eNH bending in amide II and CeN stretching in –COeNHe
1408 1402.8 CeN stretching in aliphatic amine
– 1385.1 1385.6 amide III group
1314.7 – 1319.7 Ce0eC of carbohydrate of polysaccharide
1241.6 1251.5 1244 COOH stretching
1155.2 – 1155.2 Phosphate group
1077.3 – 1079.5 Phosphate group
1043.3 1043.7 CeN stretching vibration of aliphatic amine
– – 957.7 Phosphate (PO4)
622.1 617.7 – Aromatic CeH or CeS stretching
– 571.6 – Nitro compounds and disulfide groups
530.4 540.6 – Nitro compounds and disulfide groups
– 519.5 – Nitro compounds and disulfide groups

Pb (II). In the literature, disappearance of carboxylic group in the FTIR
spectra of Bacillus sp. in contact with Pb (II) was reported previously
[62]. In that research, peaks at 1385, 571.6, 540.6 and 519.6 cm−1 for
amide III, Nitro compounds and disulfide groups were observed in the
presence of Pb (II). It was explained that the peak at 2926 cm−1 region
in the spectrum of metal-free mycelia was related to the alkyl groups,
whereas in Ni loaded samples, the peak was slightly shifted to 2928
cm−1. Moreover, broad peak of 3402 cm-1 appeared in the spectrum of
mycelium in contact with Pb, in comparison to the control which in­
dicated stretching vibration of OH or NH group.
Compared to the control, shift appeared in the spectrum of Pb and
Ni loaded mycelium in the region 1655−1545 cm−1 represented the
NHe amide I and amide II shift. Peaks at 519- 571 cm−1 of the Pb
loaded fungus presented nitro compounds and disulfide group’s role at
metal compelxation. These FTIR results revealed a possible involvement
of negatively charged functional groups of fungus mycelium in metal
interaction for Pb removal from the medium [33,63].
The XRD pattern of Pb treated samples and control shown in Figure
S1 revealed the difference between the two types of samples, which
exhibited the main peak at 29.96° for the Pb loaded mycelium.
3.6. SEM- EDX, TEM and AFM analysis
The SEM images of fungal biomass (control and Pb treated) showed
a visible difference in surface morphology of mycelia pellets exposed to
Pb. A clear, cylindrical mycelium without any particles (Figure S2A)
was observed in the control while the rough and ruptured mycelium
was observed in the surface of the T. pubescence cells grown with Pb.
Some particles were observed on the mycelia surface (Figure S2B). The
mycelia surface changes were also reported in the study with Aspergillus
terreus [64] and Trametes versicolor [65] grown with heavy metals. It
must be noted that the hyphae were denser and packed in the presence
of Pb (II) which might be the fungi’s strategy to prevent the toxicity of
Pb [9].
Our EDX results indicate that the elemental composition of myce­
lium which mainly was carbon, oxygen, phosphorous and lead (Figure
S3). The peak for the T. pubescence growth in the presence of lead
confirmed that Pb was bound to mycelium surface. Binding metal ions
in cell wall or extracellular matrix through the adsorption or pre­
cipitation has been reported as mechanisms of white rot fungi to pre­
vent cellular damage by heavy metals [66]. Our results are in agree­
ment with the previous studies.
The Pb (31.1 wt %) and P (1.8 wt %) elements was respectively
detected in EDX analysis of mycelia surface that suggested precipitation
of Pb [60]. Since no phosphorus was in the culture medium, we con­
cluded that the low peak of phosphorus in analysis was most probably
originated from T. pubescence cells. The T.pubescence hyphae grown at
free Pb medium indicated well- defined structure (Figure S4A) via TEM
image, whereas visible dark spots was indicated inside the Pb loaded
sample (Figure S4B) which was might be due to Pb bioaccumulation.
Dark area was also reported in cell membrane and cytoplasm of Pb
treated Beauveria bassiana [67,68].
A visible change in morphology of fungal cells was also observed in
presence of Pb through AFM image, compared to the control. The AFM
analysis of T. pubescence cells grown with Pb (II) clearly indicated the
increase in roughness of the fungal surface, in comparison to the con­
trol. The height distribution of mycelia surface showed the difference
between mycelia exposed to pb and control (Figure S5). The increase in
height confirmed the precipitation of Pb on the surface of mycelium.
4. Conclusions
This work examined the impact of Ni and Pb on laccase stability of
T. pubescence and the removal of two heavy metals from aqueous so­
lutions. Analysis of the laccase production and activity stability in­
dicated varying responses of T. pubescence against these metals. FTIR
results demonstrated the involvement of intracellular functional groups
in removal of heavy metals.
Fungi are beneficial microorganisms that exist widely in soil and
water ecosystem. The fungus Trametes pubescens from the Trametes
genus is known to be abundant with laccase. Our study indicates that
the fungal Trametes pubescens is effective in the heavy metal removal
from the water environment, while their laccase production is not in­
hibited. The approach developed in this work may be further extended
into the waste water treatment with other heavy metals.
Declaration of Competing Interest
We declare that we have no financial and personal relationships
with other people or organizations that can inappropriately influence
our work, there is no professional or other personal interest of any
nature or kind in any product, service and/or company that could be
construed as influencing the position presented in, or the review of the
manuscript entitled “Coupling Laccase production from Trametes
pubescence with heavy metal removal for Economic Waste Water
Treatment”.

6
N. Enayatizamir, et al.
Acknowledgements
All the authors are thankful to the financial support by the Research
Start-Up Funds from Hainan University in China (KYQD_ZR2017212).
NE is grateful for the support by Shahid Chamran University of Ahvaz,
Iran.
Appendix A. Supplementary data
Supplementary material related to this article can be found, in the
online version, at doi:https://doi.org/10.1016/j.jwpe.2020.101357.
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