Mycoremediation of Tunisian tannery wastewater under non-sterile conditions using Trametes versicolor: live and dead biomasses

Abstract

Tannery wastewater contains high concentration of pollutants (nitrogen, organic matter, toxic metals, etc.). The present study investigated the efficiency of mycoremediation on the removal of organic and inorganic pollutants and the reduction of the toxicity from a tannery wastewater (TW) using live and dead biomasses of Trametes versicolor under non-sterile conditions. Batch experiments were carried out at the normal pH of the raw TW (8.25) and at pH = 5.5. Monitoring of chemical oxygen demand (COD) reduction, chromium removal (Cr), laccase activity, color and toxicity removals, and the fungal functional groups involved in the mycoremediation was conducted before and after the treatment. The results revealed that COD removal using live biomass was significantly higher (31.2% for pH = 8.25 and 45% for pH = 5.5) than the removal obtained using dead biomass (only 19%). Laccase activity reached a maximum of 7.975 U/L (pH = 8.25) and 20 U/L (pH = 5.5) after 5 days of treatment. Color and Cr removal reached a maximum of 66% and 80%, respectively, for live biomass at pH = 5.5. FTIR spectra revealed the involvement of hydroxyl amino, phospholipids, carboxylic, and carbonyl groups in the elimination of TW pollutants. The phytotoxicity test showed that the decrease in the toxicity levels was significantly higher for live biomass, reaching 44.1% at pH = 5.5 and did not exceed 33% for dead biomass. Overall, this study advocates the potential of TW mycoremediation under non-sterile conditions and the efficiency of live biomass compared to dead biomass in reducing the organic and inorganic pollutants and consequently the toxicity levels in the TW.

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Biomass Conversion and Biorefinery
https://doi.org/10.1007/s13399-022-02328-0
ORIGINAL ARTICLE
Mycoremediation ofTunisian tannery wastewater undernon‑sterile
conditions using Trametes versicolor: live anddead biomasses
RaouiaBoujelben1· MariemEllouze1· MariaJosenaTóran2· PaquiBlánquez2· SamiSayadi3
Received: 29 September 2021 / Revised: 24 December 2021 / Accepted: 10 January 2022
© The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature 2022
Abstract
Tannery wastewater contains high concentration of pollutants (nitrogen, organic matter, toxic metals, etc.). The present study
investigated the efficiency of mycoremediation on the removal of organic and inorganic pollutants and the reduction of the
toxicity from a tannery wastewater (TW) using live and dead biomasses of Trametes versicolor under non-sterile conditions.
Batch experiments were carried out at the normal pH of the raw TW (8.25) and at pH = 5.5. Monitoring of chemical oxygen
demand (COD) reduction, chromium removal (Cr), laccase activity, color and toxicity removals, and the fungal functional
groups involved in the mycoremediation was conducted before and after the treatment. The results revealed that COD
removal using live biomass was significantly higher (31.2% for pH = 8.25 and 45% for pH = 5.5) than the removal obtained
using dead biomass (only 19%). Laccase activity reached a maximum of 7.975 U/L (pH = 8.25) and 20 U/L (pH = 5.5) after
5days of treatment. Color and Cr removal reached a maximum of 66% and 80%, respectively, for live biomass at pH = 5.5.
FTIR spectra revealed the involvement of hydroxyl amino, phospholipids, carboxylic, and carbonyl groups in the elimina-
tion of TW pollutants. The phytotoxicity test showed that the decrease in the toxicity levels was significantly higher for live
biomass, reaching 44.1% at pH = 5.5 and did not exceed 33% for dead biomass. Overall, this study advocates the potential of
TW mycoremediation under non-sterile conditions and the efficiency of live biomass compared to dead biomass in reducing
the organic and inorganic pollutants and consequently the toxicity levels in the TW.
Keywords Trametes versicolor· Mycoremediation· Live and dead biomasses· Non-sterile conditions· Phytotoxicity
1 Introduction
Regardless of the importance of tannery to several econo-
mies, the concern about the environmental impacts of the
effluents generated by this industry has been increasing due
to the release of various recalcitrant pollutants [1]. During
the leather-making process, 25 to 80 m3/ton of raw hides
of turbid, colored, and smelly wastewater is produced [2].
These wastewaters typically contain high levels of organic
and inorganic pollutants such as nitrogen, ammonia (NH3),
sulfides, hydrogen sulfide (H2S), acids, alkalis, surfactants,
and chromium [3–7].
Due to the excessive quantity and the composition of
the effluents produced during the leather-making process,
research has never ceased attempting to find efficient tech-
nologies to treat tannery wastewater [1, 8, 9]. Several physi-
cal and chemical methods have been deployed for this pur-
pose [8, 10–12]. However, most of these methods require a
high operational cost and may generate toxic elements [13,
14]. To avoid these drawbacks, special attention has been
given to biological methods as an alternative or a comple-
ment to physical and chemical methods since they are cost-
effective and eco-friendly [6, 15–17].
During the last decade, mycoremediation, which
is based on the use of fungi to reclaim contaminated
Raouia Boujelben and Mariem Ellouze contributed equally to this
work.
* Mariem Ellouze
ellouzemariem41@gmail.com
* Sami Sayadi
sami.sayadi@gmail.com
1 Laboratory ofEnvironmental Bioprocesses, Centre
ofBiotechnology ofSfax, University ofSfax, PO Box1177,
3018Sfax, Tunisia
2 Departament d’Enginyeria Química, Biològica I Ambiental,
Escola d’Enginyeria, Universitat Autònoma de Barcelona,
08193 Bellaterra, Cerdanyola del Vallès, Barcelona, Spain
3 Center forSustainable Development, College ofArts
andSciences, Qatar University, 2713Doha, Qatar

Biomass Conversion and Biorefinery
1 3
environments, has been widely studied. Fungi play a key
role in bioremediation due to their robust morphology
and their ability to remove several recalcitrant via their
mycelia or their enzymes [18, 19]. Indeed, fungal myce-
lia exhibit the robustness of adapting to highly limiting
environmental conditions which makes them more use-
ful compared to other microorganisms [18]. Besides, it
is well known that fungal enzymes have the potential to
effectively transform hazardous substances and to reduce
the toxicity [13, 20, 21].
White rot fungi (WRF) are more often used to treat
complex effluents since they are able to cope and grow
under extreme conditions [19, 22]. Their non-specific
enzyme system is a powerful tool used to transform,
degrade, and mineralize a wide variety of recalcitrant pol-
lutants, such as polyphenols, dyes, aromatic compounds,
and even the insoluble chemicals [23–25]. Other mecha-
nisms such as biosorption and bioaccumulation could also
be involved in the mycoremediation [26–28]. Bioaccumu-
lation is based on the uptake of pollutants by living cells,
while biosorption is based on the passive uptake of pol-
lutants by non-living cells [26]. Both mechanisms depend
on the functional groups in the cell wall such as carboxyl,
amine, and phosphate [14]. One of the major advantages of
these mechanisms over conventional methods is the high
efficiency toward several pollutants even at low concentra-
tions [29].
Mycoremediation of leather dyes and tannery effluents is
scarcely reported [25, 30]. Moreover, few researches focus-
ing on the removal of polluting parameters (COD, BOD5,
TOC, chromium, etc.) and the reduction of toxicity after
remediation of real tannery effluents with WRF are reported
[6, 31–33]. In addition, the main problem encountered with
the application of WRF under non-sterile conditions is the
competition with indigenous microorganisms, mainly bacte-
ria [34–36]. Indeed, it has been largely demonstrated that the
presence of bacteria often compromises fungal activity [17,
35, 37]. Therefore, maintaining a stable fungal growth and
performance under non-sterile conditions is still challenging.
The objective of the present work is to study the poten-
tial capacity of the WRF Trametes versicolor in removing
the organic and inorganic pollutants as well as reducing the
toxicity of a tannery wastewater (TW) under non-sterile con-
ditions. To foster fungal growth over bacteria, batch tests in
flasks were performed using two pH values (8.25 and 5.5). In
order to investigate the biosorption and the bioaccumulation
potential of T. versicolor in TW’s treatment, live and dead
biomasses were used. The feasibility of the fungal treatment
under these conditions shall broaden the current knowledge
about mycoremediation of tannery effluents, without co-
substrate addition, representing the first step for the scale-
up of such processes to substitute or to complement current
technologies for recalcitrant compounds removal.
2 Materials andmethods
2.1 Tannery wastewater origin
Tannery wastewater (TW) was taken from a local tan-
nery in Sfax, Tunisia. Samples were collected from the
final drainage where effluents from all the leather-making
operations are collected together. Samples were stored at
4°C without any previous pre-treatment.
2.2 Fungal strain
Trametes versicolor (ATCC 42,530), provided by the
hosting Laboratory in Barcelona, was obtained from the
American Type Culture Collection and maintained in 2%
of malt extract agar (MEA) medium at 25°C (pH 4.5).
2.3 Media andculture conditions
2.3.1 Mycelial suspension
A mycelial suspension was obtained by inoculating four
plugs (diameter = 1cm) from the growing zone of the
fungus on MEA medium. After 5days of incubation in
an orbital shaker (135rpm) at 25°C, mycelial mass was
grounded using T25 basic ULTRA-TURRAX homogenizer
[21].
2.3.2 Pellet production
A volume of 1mL of the mycelial suspension was used for
pellet production, as reported by Blánquez etal. [21]. A
solution of 0.8% NaCl was used to stock the pellets until
use at 4°C where they remain active for up to 2months.
2.3.3 Preparation ofdead biomass
Dead fungal biomass was prepared by autoclaving fungal
pellets in Erlenmeyer flasks containing water. After auto-
claving, dead biomass was separated from water and used
for experiments.
2.4 Experimental procedures: batch‑mode
mycoremediation
Experiments were carried out in 250-mL flasks inoculated
with 6.4% (w/v) of fungal pellets [21]. The experiment
design is presented in Table1. Three sets of experiments
were conducted: C as a control where flasks contained
only TW; D: inoculated with dead fungal pellets; and L:
inoculated with live fungal pellets.

Biomass Conversion and Biorefinery
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In order to study the behavior of T. versicolor under
non-sterile conditions, two pH values were applied; in
experiment 1 (C1, D1, and L1), the initial pH of the TW
was maintained (pH = 8.25) and in experiment 2 (C2, D2
and L2), pH was adjusted at 5.5 with 1M HCl.
All flasks were incubated in orbital agitated conditions
(135rpm) at 25°C for 9days.
2.5 Analytical procedures
2.5.1 Physical andchemical characterization oftannery
wastewater
The characterization of the TW was carried out as described
by Boujelben etal. [6]. Several physical and chemical
parameters have been determined (Table2): pH, electri-
cal conductivity (EC), salinity, chemical oxygen demand
(COD), total and volatile solids (TS and VS), Kjeldhal nitro-
gen content (NTK), and chromium (Cr).
2.5.2 Chromium analysis anduptake capacity
Chromium concentration was determined by atomic absorp-
tion spectrometry (Hitachi Z 6100). Chromium removal was
calculated by measuring Cr concentration in the TW before
and after the fungal treatment using the following equation:
where C0: initial Cr concentration (mg/L); Cf: final Cr
concentration (mg/L).
2.5.3 Laccase activity
Laccase activity was determined by monitoring the absorb-
ance (420nm) changes related to the rate of oxidation of
1mM 2,2-azinobis (3-ethylthiazoline)-6-sulfonate (ABTS)
in 0.1M sodium tartrate buffer (pH 4.5) [38].
2.5.4 Color removal
The color removal of the TW after the fungal treatment (day
7) was analyzed by a UV-spectrophotometer (UV-1800, Shi-
madzu) and calculated as described by Zheng etal. [39].
2.5.5 Fourier transform infrared spectroscopy
Live and dead biomasses of T. versicolor before and after
the mycoremediation were lyophilized [40] then analyzed
by Fourier transform infrared spectroscopy (FTIR) to deter-
mine functional groups. FTIR spectra were obtained in the
range of 400–4000 cm−1 using Nicolet 380 FTIR spectrom-
eter. Interferograms were averaged for 32 scans at 4 cm−1
resolution.
Chromium r emoval
(%)=
C0− Cf
C0
×
100
Table 1 Experimental design for the mycoremediation of tannery
wastewater under non-sterile conditions
Experiment pH Trametes versicolor
8.25 5.5 Live biomass Dead
bio-
mass
Experiment 1 C1x – – –
D1x – – x
L1x – x –
Experiment 2 C2– x – –
D2– x – x
L2– x x –
Table 2 Characterization of tannery wastewater compared to Tunisian standards for discharges in public canalizations (NT 106.002). Values
given represent the mean of three replicates
Parameters (Unit) Raw Tannery waste-
water (TW)
L1L2D1D2NT 106.002
pH 8.25 ± 0.24 7.51 ± 0.055 7.8 ± 0.065 8.49 ± 0.23 7.14 ± 0.1 6.5–9
EC (mS/cm) 17.26 ± 0.91 14.9 ± 0.1 15.5 ± 0.5 14 ± 0.5 16.4 ± 0.5 -
Salt (g/L) 14.671 ± 0.17 12.66 ± 0.085 13.17 ± 0.425 11.9 ± 0.425 13.94 ± 0.425 -
COD (g/L) 4.35 ± 0.42 2.993 ± 0.0085 2.391 ± 0.25 3.524 ± 0.15 3.489 ± 0.25 1
TS (g/L) 20.04 ± 1.23 12.34 ± 0.515 9.78 ± 0.45 17.38 ± 0.586 11.21 ± 0.12 -
VS (g/L) 5.3 ± 1.05 3.867 ± 0.1485 3.12 ± 0.05 4.26 ± 0.1 3.64 ± 0.25 -
TKN (mg/L) 187.6 ± 13.47 165.14 ± 3 118 ± 2 181.16 ± 2.65 177.39 ± 3.5 100
Cr (mg/L) 4.2 ± 1.31 2.19 ± 1.02 0.83 ± 0.96 2.21 ± 1.24 1.06 ± 0.73 0.1

Biomass Conversion and Biorefinery
1 3
2.5.6 Phytotoxicity assays
Phytotoxicity assays were carried out using tomato seeds
(Lycopersicon esculentum). The choice of tomato seeds was
based on its availability, since it is a common agricultural
plant in Tunisia [41] and on the fact that it is among the spe-
cies that are commonly used for phytotoxicity test [42, 43].
The seed germination index (GI) was measured as described
by Zucconi [44]. GI is calculated by the root length and the
germination percentage of the seeds in the sample compared
to that in the control (tap water). Different concentrations
of raw and treated TW (10, 25, 50, 75, and 100%) were
tested. Triplicates were used and results are presented as
mean values of GI.
2.5.7 Fungal dry weight
After separating the fungal biomass from the liquid medium
by vacuum filtering the cultures through pre-weighed filters,
the dry weight of the biomass was determined as the con-
stant weight at 105°C after 24h.
2.6 Statistical analysis
Graphpad Prism software (version 6) was used for data
analyses.
3 Results anddiscussion
3.1 Tannery wastewater characterization
The average values of the different physical and chemical
parameters of the tannery wastewater (TW) are presented
in Table2. TW was brownish in color with a pungent odor.
The pH was alkaline around 8. The EC of the TW exceeded
17 mS/cm, displaying a high salt content equal to 14.67g/L.
Overall, the TW presented high values in most of the param-
eters surpassing the Tunisian standards of discharges “Tuni-
sian Standard” (NT. 106.002) [45] which indicates the high
pollution level of the effluent and further explains the need
for treatment. COD values reached 4.3g/L while the limita-
tion of NT 106.002 is 1g/L. Analyses also revealed high
chromium (Cr) value (4.2mg/L) exceeding the NT 106.002
(≤ 0.1ppm).
3.2 Effect ofnon‑sterile conditions onthegrowth
andperformance ofT. versicolor
In order to evaluate the bioremediation potential of dead
and live biomasses of T. versicolor for TW treatment, the
measurement of COD, Cr, laccase activity and FTIR analy-
ses were examined. The main challenge in this study was to
successfully reduce the pollution load and the toxicity level
of raw TW without losing the fungal biomass, taking into
account that the non-sterile condition affects the growth and
the metabolic capacity of fungi [34].
3.2.1 COD monitoring duringthetreatment withT.
versicolor
Monitoring of COD during fungal treatment showed dif-
ferent patterns depending on pH values and the state of the
fungal biomass (dead or live). As presented in Fig.1a, at
initial effluent pH, a slight decrease in COD during the first
48h was noticed in the control experiments (C) meaning that
the endogenous microorganisms were able to degrade a part
of the biodegradable organic fraction present in the TW, then
COD removal was maintained almost constant until the end.
This reveals that the autochthonous microorganisms are not
efficient in reducing the pollution load of the TW.
During the D experiments (dead biomass), at both pH
values, COD removal was around 19% after 7days of treat-
ment. In this case, the decrease of COD is related to the
adsorption of organic pollutants on the fungal cell surface. In
fact, it was reported that autoclaving (thermal pretreatment)
increases the surface area of fungal biomass due to the dena-
turation of the cell wall proteins and to plasmolysis, leaving
exposed multiple active sites and leading to the increase of
the adsorption rate [46–48]. Moreover, it is well known that
the exposure of some fungal strains to heat treatment alters
the physicochemical properties of their surfaces leading to
a better, an equivalent, or a less bioadsorptive capacity than
that of living cells depending on the pollutant and its interac-
tion with the fungal cell wall [49, 50].
When live biomass was used (L), there was 31.2 and 45%
reduction in COD at the initial pH value and at pH = 5.5,
respectively. These results revealed that the efficiency of the
fungal treatment was related to the decrease of pH, which is
associated with the onset of fungal metabolism [51]. In fact,
the pH value in the “L2” experiments is close to the optimum
for the WRF enzymes, which makes them more active and
effective [17]. Moreover, acidic conditions may be beneficial
to T. versicolor, not only by promoting its growth but also by
reducing the activity of other microorganisms [52, 53]. This
was in total agreement with previous studies which reported
the effect of pH on the efficiency of the fungal treatment [6,
35, 54].
Overall, when using live biomass, pollutant removal is
related to biosorption, bioaccumulation, or enzymatic degra-
dation [55]. However, when using dead biomass, the removal
is associated to biosorption of pollutants on the fungal cell
surface. Xin etal. demonstrated that the main mechanism for
the living pellets of Trichoderma sp. involved in the removal
of acid Brilliant Red B was the bioaccumulation, mainly
in the cell wall and the fungal cytoplasm. However, when

Biomass Conversion and Biorefinery
1 3
dead biomass was used, the monolayer adsorption onto the
surface of the pellets was the mechanism involved in the dye
removal [56].
As shown in Fig.1a, b, after 7days of fungal treatment,
a slight increase in COD values was noted during L1 and L2
experiments. This may be due to the release of metabolites
and enzymes by the fungi and to the depletion of nutrients
[17]. It was also reported that T. versicolor grows until the
ninth day, whereupon, it reached a stationary phase [57].
Therefore, a cellular lysis could occur and lead to an increase
in the COD.
3.2.2 Chromium uptake
While most of the researches reported Cr removal by fungal
strains from synthetic solutions, in this work, fungal chro-
mium uptake was studied in real tanning effluent. The per-
centage of Cr removal by live and dead biomasses of T. versi-
color, presented in Fig.2, showed that Cr uptake was clearly
affected by the pH since the removal percentage exceeded
80% and 74% at pH = 5.5 during L2 and D2 experiments,
respectively, while it was around 47% during L1 and D1
experiments at pH = 8.25. The higher Cr removal obtained
at pH = 5.5 can be explained by the interaction between the
negatively charged functional groups of the fungal cell wall
and the positively charged Cr ions [58]. Moreover, the dif-
ference in Cr uptake between live and dead biomasses rep-
resents the amount of Cr removed by metabolism-dependent
accumulation into T. versicolor. Indeed, the accumulation of
metals by live fungal biomass occurs in two phases with an
initial surface binding (biosorption) followed by a slower
phase of uptake, presumably an intracellular accumulation
[59]. The majority of intracellular metals are either fixed
in polyphosphate granules located in and near the vacuoles
or bound to metallothioneins, known as intracellular metal-
binding proteins [60].
Based on our results (Fig.2), Cr removal by T. ver-
sicolor is probably attributed to biosorption process. In
fact, the metabolism-independent adsorption of Cr by
dead biomass is usually associated to physical adsorption
Fig. 1 COD removal using live
(L) and dead (D) biomasses at
pH = 8.25 (a) and at pH = 5.5
(b) and compared to a control
(C)

Biomass Conversion and Biorefinery
1 3
or ion exchange at the cell surface [61, 62]. Ramrakhiani
etal. demonstrated that Cr biosorption onto dead fun-
gal biomass of Termitomyces clypeatus involved more
than one mechanism, including physical adsorption, ion
exchange, complexation, and electrostatic attraction.
The main functional groups involved in Cr biosorption
were found to be in the following order: carboxyl > phos-
phates > lipids > sulfhydryl > amines [62]. Additionally, it
has been demonstrated that amide and alkane groups are
also involved in Cr binding. All these chemical bonds are
responsible for providing the ligand atoms to form metal
ion complexes by attracting and retaining metals in the
biomass [27, 61, 62].
3.2.3 Laccase activity monitoring
The non-sterile conditions are not the only challenge in this
study. The alkaline pH of the raw TW is also an impor-
tant parameter that can affect the activity of the degrad-
ing enzymes and the fungal performance [63]. As shown
in Fig.3, during the non-inoculated experiments (C), no
laccase activity was detected, meaning that the endogenous
microorganisms of the raw TW did not produce laccases,
and consequently, any activity recorded in the inoculated
batches is accredited to T. versicolor. During the L experi-
ments, laccase activity was used as a possible indicator of
fungal activity.
Fig. 2 Chromium removal by
Trametes versicolor during the
mycoremediation
Fig. 3 Laccase profile during
the mycoremediation by Tram -
etes versicolor

Biomass Conversion and Biorefinery
1 3
According to the laccase profile presented in Fig.3, a
maximum peak on the 5th day was noted, then, it started
to decrease. When live biomass was used at the natural pH
of the TW (L1), the laccase activity reached a maximum of
7.975 U/L (day 5) and then reached a level of 1 U/L at the
end of the mycoremediation (day 9). During L2, when pH
was adjusted to 5.5, maximum of laccase activity was of 20
U/L at day 5 and decreased up to 2.16 U/L at the end of the
treatment (day 9). As shown in Fig.3, laccase activity was
higher in L2 experiments (pH = 5.5), which could be justi-
fied by the fact that acidic pH is more suitable for laccases
[63]. These results confirm previous studies showing that
an acidic pH is the optimal pH for T. versicolor enzyme
activity [64]. Moreover, previous studies revealed that in
order to improve laccase production by fungi when working
with real effluents, nutrients such as carbon and nitrogen are
needed [35, 64, 65]. In addition, these results indicate that
there is no direct connection between the laccase values and
the removal of organic pollutants from the TW and that the
mycelium is a sort of self-healing filter that targets specific
organic compounds and pollutants [66]. Indeed, Mir-Tutu-
saus etal. [67] demonstrated that laccase detection confirms
the fungus activity when treating effluents. However, a low
level of the enzyme does not mean that the fungus is inac-
tive. Similar results were recorded when working with real
wastewater [22, 54].
3.2.4 Color removal
The color removal percentage at the end of the fungal treat-
ment is given in Fig.4. The maximum removal was obtained
with live biomass of T. versicolor. The results revealed
that the color removal was higher when pH was adjusted
to 5.5 for both dead and live biomasses reaching 35 and
67%, respectively. Moreover, visual observations revealed
that the color intensity decreased by the end of the treat-
ment. It has been reported that color reduction is related to
the intracellular laccase activity [21, 68]. However, other
studies demonstrated the possibility and the advantages of
using dead fungal biomass for the removal of color from
wastewater [69, 70].
3.2.5 FTIR analysis ofthelyophilized mycelia ofT. versicolor
The FTIR analysis was performed to identify the major
functional groups on the cell surface of T. versicolor, and
consequently, the surface properties as well as the interac-
tion between the fungal cell wall and the pollutants of the
TW [71]. Figure5 displays the FTIR spectra of T. versicolor
biomass before and after the treatment. The comparison
between the spectra of the control (before treatment) and
T. versicolor grown on the TW at different conditions (L1,
L2, D1, D2) can deliberately reveal the functional groups
contributing to the mycoremediation.
The spectrum of the control biomass shows a prominent
broad peak at 3261.99 cm−1 that reveals the presence of
an -OH group, primary and secondary amine and amides,
and N–H stretching which represents the polysaccharide
chains [40]. A sharp peak at 2922.21 cm−1 is attributed to
C-H stretching which is assigned to CH2 lipid stretching
[72]. A strong peak at 1624.39 cm−1 is attributed to
protein amide I band mainly C = O stretching [73]. Sharp
peaks at 1402.77 cm−1 and at 1308.73 cm−1 are related
to CH2, CH3, or P = O of proteins, lipids, and phosphate
compounds; a sharp peak at 1032.47 cm−1 is attributed to
C–O–C stretching vibration in polysaccharides together
and to PO2 in the functional groups from nucleic acids
[74]. It was reported that the various functional groups
Fig. 4 Color removal efficien-
cies after the mycoremadiation
with dead and live biomasses of
Trametes versicolor

Biomass Conversion and Biorefinery
1 3
on the fungal cell surface such as lipids, proteins, and
carbohydrates made the fungal biomass as an efficient
biosrobent [75].
Figure5 and Table3 show FTIR spectra of T. versicolor
grown on the TW at different conditions. As compared to
control biomass, patterns of band intensities varied with the
pH and the state of the fungus (dead or live). In fact, the
disappearance and peak shifting indicate the involvement
of interaction of fungal functional groups with pollutants in
effluents [76]. It is known that the passive adsorption process
on cell surface was performed using inactivated (dead) fun-
gal biomass and the active adsorption process was performed
using live biomass [77]. According to Table3, the prominent
broad peak at 3261.99 cm−1 was shifted to 3328.86 cm−1
and the sharp peak at 2922.21 cm−1 disappeared only dur-
ing L2 experiments when live biomass was used and pH was
adjusted to 5.5. This could be attributed to the involvement
of -COOH and -OH groups, I and II amine and amides as
well as N–H and CH2 lipids in the bioaccumulation of TW
pollutants since these peaks were only shifted/disappeared
when using live biomass [76]. Moreover, there was a clear
disappearance of the sharp peak at 1402.77 cm−1, when
pH was adjusted to 5.5, corresponding to sulfonyl, sulfona-
mide, and phosphate groups in the fungal biomass, known
as metal-binding functional groups [62]. These chemical
bonds are responsible for providing the ligand atoms to form
metal ion complexes by attracting and retaining metals in
the biomass [78]. Furthermore, a complete disappearance
of the sharp peak at 1308.73 cm−1 in all the studied condi-
tions (D1, D2, L1, L2) compared to the control biomass was
noted. This means that proteins, lipids, and phosphate com-
pounds may be involved in the fungal treatment. Chaudhary
etal. demonstrated the involvement of phosphate groups
in the removal of chromium from a tannery effluent using
Fig. 5 FTIR spectra of Trametes versicolor biomass before (control) and after TW treatment
Table 3 Frequency (cm−1) and FTIR bands of Trametes versicolor
before and after treatment under the studied conditions
Before treatment After treatment
D1D2L1L2
- 616.47 613.97 617 –
- – – – 774.93
- 871.21 – 871 –
- – – – 886.86
- – – – 931.66
1032.47 – – – 1038.45
- – 1086.06 – –
- 1103.35 – 1104.81 –
- – 1192.97 – –
1308.73 – – – –
- – 1311.88 1318.64 1314.01
- – – – 1373.47
1402.77 1408.8 – 1410.7 –
- – 1423.39 – –
- 1539.63 – – –
- – 1619.58 – –
1624.39 – – 1629.44 1625.83
2922.21 2920.61 2923.52 2923.7 –
3261.99 3268.89 3269.17 3275.51 –
- – – – 3328.86

Biomass Conversion and Biorefinery
1 3
Aspergillus fumigates [79]. Contrastingly, new peaks were
detected within the region 700–400 cm−1 after the mycore-
mediation of the TW. These peaks are usually attributed to
the presence of nitro compounds and disulfide groups known
as metal-binding groups [62, 80].
Overall, results demonstrated that the functional groups
which may play a significant role as pollutant-binding sites
on the fungal cell wall in both dead and live T. versicolor
biomasses were predominantly amines, hydroxides, car-
boxyl groups, phospholipids, and proteins. Similar results
were reported by Chaudhary etal. [79] and Vajpai etal. [80]
showing that the main functional groups responsible for a
biosorption mechanism are the hydroxyl, carbonyl, carboxyl,
sulfonate, amide, phosphonate, and phosphodiester groups.
Other studies reported that the removal of some heavy
metals is due to the electrostatic interactions of negatively
charged functional groups (e.g., hydroxyl, carbonyl, and
phosphoryl groups) of the fungal cell wall with positively
charged metal ions present in the effluents [81]. Additionally,
FTIR analysis showed that both passive and active adsorp-
tion are involved in the fungal treatment and that pH plays an
important role regarding the efficiency of mycoremediation
since it influences the protonation of functional groups on
the surface of the adsorbent [82].
3.2.6 Phytotoxicity monitoring beforeandafterthe
mycoremediation
The seed germination test is a key parameter to check the
afterward effect of the treated wastewater by measuring ger-
mination index (GI %) [83]. The result of GI monitoring of
L. esculentum upon exposure to different concentrations of
TW is shown in Fig.6. At 10, 25, and 50% (v:v) of raw TW
concentrations, the GI were very low, around 18, 8, and 3%,
respectively, and a total inhibition of the germination was
observed at 75 and 100% of raw effluent. These results are
in line with our previously reported ones [6] showing that
raw tannery effluents are extremely toxic and completely
inhibit the growth of tomato seeds. This can be attributed to
the high salt concentration and the high organic and mineral
(mainly Cr) contents of the raw TW. Indeed, Chandra etal.
[84] indicated that a high salt level, toxic organic pollutants
and metals act as inhibitors for several phytohormones that
play a key role in seed germination and seedling growth. It
has also been reported that the inhibition of seed germina-
tion at a higher effluent concentration may be due to the high
solids content (TS) which increases the salinity and the con-
ductivity of the solute absorbed by seeds [85, 86]. Moreo-
ver, at higher effluent concentration, shoot and root lengths
are reduced due to cellular disruption by toxic pollutants
[83]. After mycoremediation, a clear decrease of phytotoxic-
ity occurred in the treated TW. At 75% of TW, the highest
GI percentage was reached when live biomass was used,
exceeding 49% at pH = 5.5, while it was around 34.4% for
dead biomass at the same pH. Additionally, results clearly
showed the effect of pH on the efficiency of the fungal treat-
ment since at pH = 5.5, GI was higher than at pH = 8.25 for
both live and dead biomasses.
3.3 Biomass change: live vs dead biomass ofT.
versicolor
Biosorption is a metabolically passive process that occurs
when proceeding with dead biomass whereas bioaccumu-
lation is a metabolically active process that occurs in the
presence of living cells [87]. As can be seen in Fig.7, the
biomass concentration increased in the L experiments after
7days of treatment. The increase was more important when
Fig. 6 Germination index (%) of
Lycopersicon esculentum grown
on TW before and after fungal
treatment

Biomass Conversion and Biorefinery
1 3
pH was adjusted to 5.5 (L2) reaching 48.6%, whereas it did
not exceed 18% at initial pH (Table4). The increase in the
biomass in L experiments means that T. versicolor was able
to grow on raw TW without adding any nutrients. During
D experiments, the increase in the biomass was around 4.6
and 10% at initial pH and pH = 5.5, respectively, which is
due to the adsorption of pollutants on the fungal cell wall.
After 7days, a decrease in the biomass was noted, which
can be attributed to the lysis of the mycelia caused by the
nutrients starvation. Indeed, several studies reported that
autolysis is usually induced by physical stress, carbon-star-
vation, or by limitations in other nutrients [88, 89]; there-
fore, a lack of nutrients can lead to a decrease in the biomass
concentration since the fungus needs nutrients for its mainte-
nance [35]. Moreover, the study described by Cruz-Morató
etal. [35] showed that a high pH may promote early lysis of
fungal strains. In addition, COD removal can be correlated
to bioaccumulation since higher reduction rates, exceed-
ing 45%, were achieved using live biomass whereas it was
around 19% when dead biomass was used at pH = 5.5.
Overall, the better performance of live biomass of T.
versicolor over the dead biomass can be explained by the
fact that the removal of organic (COD) and inorganic (Cr)
pollutants may be attributed to their interaction with the
surface functional groups, thus, their sequestration on the
fungal cell wall (biosorption) and a subsequent uptake into
the live cells (bioaccumulation), as opposed to only biosorp-
tion mechanism by dead cells. Indeed, the ability of live
cells to tolerate toxic pollutants, mainly metals, is attributed
to the detoxification mechanisms by cells that may include
intracellular binding with chelators, compartmentalization
within vacuoles, or enzyme-catalyzed transformation to less
toxic forms [90, 91].
4 Conclusion
In the present study, mycoremediation of TW using dead and
live biomasses of T. versicolor was investigated under non-
sterile conditions and without diluting the effluent or adding
any nutrients. The analyses of the conventional parameters
including COD, chromium, laccase activity, and color were
indicative of the efficiency of the fungal treatment. These
findings corroborate with the results of the phytotoxicity test
which showed a decrease in the toxicity of the treated TW
compared to the raw one. FTIR spectra of live and dead bio-
masses of T. versicolor revealed the involvement of hydroxyl
amino, phospholipids, carboxylic, and carbonyl groups in
the elimination of pollutants from the TW. Based on the
obtained results, the use of live biomass of T. versicolor is
promising and efficient for the bioremediation of the TW.
Additionally, pH is a key parameter affecting the perfor-
mance of the fungal treatment.
Overall, this paper highlighted that mycoremediation can
be a serious alternative for the removal of organic and inor-
ganic pollutants from a real tannery effluent even under non-
sterile conditions. However, further studies are necessary in
Fig. 7 Monitoring of dry weight
of Trametes versicolor during
the treatment of the TW
Table 4 Growth of Trametes versicolor (%) during the treatment of
the TW
L1L2D1D2
Biomass (g dw/L): day 1 1.911 1.911 1.911 1.911
Biomass (g dw/L): day 7 2.253 2.840 1.999 2.102
Fungal growth (%) 17.9 48.6 4.62 10.03

Biomass Conversion and Biorefinery
1 3
order to analyze other operational strategies and optimize the
process to extent and improve the treatment.
Acknowledgements Authors would like to thank Mr. Nidhal Baccar
for his technical assistance in the FTIR analyses and Prof. Mohamed
Ksibi for his assistance in the revision of the FTIR section. We thank
the members of the Laboratory of Environmental Bioprocesses, Centre
of Biotechnology of Sfax, Tunisia, and the members of the Depart-
ment of Chemical, Biological and Environmental Engineering, at
the Autònoma University of Barcelona, Spain. Authors would like to
thank Prof. Kamel Maaloul, English professor and scientific transla-
tion expert, for proofreading, correcting, and improving the English
of the manuscript.
Author contribution Raouia Boujelben performed most of the experi-
ments, data analysis, and drafted the manuscript; Mariem Ellouze
helped in the direction of the study and in the manuscript interpreta-
tion; Maria Josefina Tóran helped shape the research; Professor Paqui
Blánquez helped in the conceptualization of the research; and Professor
Sami Sayadi provided critical revision of the manuscript and directed
the study.
Funding The present research study was supported by a pre-doctoral
grant provided by the Tunisian Ministry of Higher Education, the Doc-
toral School of the Faculty of Sciences of Sfax, University of Sfax and
the Laboratory of Environmental Bioprocesses, Centre of Biotechnol-
ogy of Sfax, Tunisia. This work was also partially supported by the
BioremUAB group (project BECAS, CTM2016-75587-C2-1-R) in the
Department of Chemical, Biological and Environmental Engineering,
at the Autònoma University of Barcelona, Spain.
Data availability All data collected in this study are presented in the
current manuscript.
Declarations
Consent to participate This article does not contain any studies with
human participants or animals performed by any of the authors.
Consent for publication All authors have read the manuscript and have
approved the submitted version.
Conflict of interest The authors declare no competing interests.
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