International Journal of

Molecular Sciences

Article
Melanins from the Lichens Lobaria pulmonaria and Lobaria
retigera as Eco-Friendly Adsorbents of Synthetic Dyes
Anna Rassabina 1 , Venera Khabibrakhmanova 1 , Vasily Babaev 2 , Amina Daminova 1 and Farida Minibayeva 1, *

1 Kazan Institute of Biochemistry and Biophysics, FRC Kazan Scientific Center of RAS, 2/31, Lobachevsky Str.,
Kazan 420111, Russia
2 Arbuzov Institute of Organic and Physical Chemistry, FRC Kazan Scientific Center of RAS, 8,
Academician Arbuzov Str., Kazan 420088, Russia
* Correspondence: minibayeva@kibb.knc.ru or fminibayeva@gmail.com

Abstract: Synthetic dyes are widely used in the industry; they are chemically stable, difficult to
neutralize, and therefore they are a threat to the environment when released into wastewaters. The
dyes have a significant impact on plant performance by impairing photosynthesis, inhibiting growth,
and entering the food chain and may finally result in the toxicity, mutagenicity and carcinogenicity
of food products. Implementation of the dark piment melanin for the adsorption of the synthetic
dyes is a new ecologically friendly approach for bioremediation. The aim of the present work was
to study the physico-chemical characteristics of melanins from the lichens Lobaria pulmonaria and
Lobaria retigera, analyze their adsorption/desorption capacities towards synthetic dyes, and assess
the capacity of melanins to mitigate toxicity of the dyes for a common soil bacterium Bacillus subtilis.
Unique chelating properties of melanins determine the perspectives of the use of these high molecular
weight polymers for detoxification of xenobiotics.
Citation: Rassabina, A.;
Khabibrakhmanova, V.; Babaev, V.; Keywords: lichens; melanins; FT-IR; synthetic dyes; adsorbents
Daminova, A.; Minibayeva, F.
Melanins from the Lichens Lobaria
pulmonaria and Lobaria retigera as
Eco-Friendly Adsorbents of Synthetic 1. Introduction
Dyes. Int. J. Mol. Sci. 2022, 23, 15605.
Synthetic organic dyes are the largest group of all coloring substances. More than
https://doi.org/10.3390/
100,000 commercially available synthetic organic dyes are widely used in the textile, tanning,
ijms232415605
printing, paper and plastic industries, the pharmaceutical and cosmetics industries as well
Academic Editor: Gianfranco as in food processing [1]. These dyes pose a threat to the environment when released
Gilardi into wastewater. In particular, they can cause a significant negative impact on plant
Received: 9 November 2022
performance by impairing photosynthesis, inhibiting growth, and entering the food chain
Accepted: 5 December 2022
and may finally result in the toxicity, mutagenicity and carcinogenicity of food products.
Published: 9 December 2022
Synthetic dyes are chemically stable; therefore, they are difficult to neutralize. In this
regard, the use of various adsorbents to remove these dyes is an effective approach of
Publisher’s Note: MDPI stays neutral
remediation of wastewaters. The advantage of using biosorbents is due to their high
with regard to jurisdictional claims in
efficiency, ease of technical implementation, and the possibility of multiple use of sorbents
published maps and institutional affil-
in the adsorption/desorption cycles. To date, biosorbents obtained from natural sources are
iations.
of a particular interest. Unlike synthetic adsorbents, biosorbents practically do not cause
detrimental effects on the environment [2,3].
At present, melanins, the dark pigments synthesized in animals, plants, fungi, and
Copyright: © 2022 by the authors.
bacteria, are considered as perspective biosorbents. Melanins are high molecular weight
Licensee MDPI, Basel, Switzerland. hydrophobic polymers with intricate structural organization and chemical composition [4].
This article is an open access article Pigments can be synthesized via the acetate-malonate and shikimate pathways in the
distributed under the terms and reactions of oxidation and polymerization of phenolic and/or indolic compounds. As a
conditions of the Creative Commons result, different types of melanins such as eumelanin, allomelanin, pheomelanin, and others
Attribution (CC BY) license (https:// can be produced [5]. Melanins manifest high biological activities, including antioxidant,
creativecommons.org/licenses/by/ photo- and radioprotective activities, while they do not exhibit pronounced toxicity [6,7].
4.0/). Moreover, due to their high reactivity, melanins demonstrate pronounced adsorption

Int. J. Mol. Sci. 2022, 23, 15605. https://doi.org/10.3390/ijms232415605 https://www.mdpi.com/journal/ijms

Int. J. Mol. Sci. 2022, 23, 15605 2 of 14

capacity towards heavy metal ions and toxic elements. For example, melanin extracted from
micromycete Amorphotheca resinae can adsorb Pb, Cu, Cd, and Zn [8]. Melanin extracted
from marine bacterium Pseudomonas stutzeri and functionalized by Fe/Cu impregnation
can effectively bind As [9]. These micro-organisms with high content of melanin belong to
extremophilic organisms, which are able to degrade oil products and use them for their
own growth.
Lichens represent another example of organisms with high stress tolerance. They
dominate over large areas of terrestrial ecosystems, for example as components of deserts,
boreal, Arctic and Antarctic regions, therefore lichens can tolerate severe abiotic stresses.
Lichens are symbiotic photosynthesizing organisms whose thalli are formed by mycobiont
(fungi) and photobiont (algae and/or cyanobacteria) [10]. It seems likely that symbiotic
co-existence of myco- and photobionts results in the synthesis of numerous secondary
metabolites, which contribute to the high stress tolerance of lichens. Physico-chemical
properties and biological activity of the unique lichen substances, terpenes, sterols, and
fatty acids are well-studied [11–14]. However, only scarce information is available about
physico-chemical characteristics of lichen melanins, although the involvement of these
pigments in the tolerance of lichens to light stress has been documented [15]. To date, it
is still unknown whether chemical composition, structural organization, and biological
activity of lichen melanins differ from those of the melanins from other living organisms.
Earlier it was demonstrated that UV exposure of the green foliose lichen Lobaria pul-
monaria can induce melanization of the upper cortex of thalli [15]. Lichen Lobaria retigera
although being naturally dark-brown can also synthesize melanin in response to UV radia-
tion [16]. The aim of the present work was to study how physico-chemical characteristics
determine the capacity of the adsorption/desorption of synthetic dyes by melanins ex-
tracted from the lichens L. pulmonaria and L. retigera. Adsorption/desorption capacity of
lichen melanins was studied using the water-soluble synthetic dyes that differ in their
structure, charge, and size of molecule: thiazine dye Methylene Blue (MB), anthraquinone
dye Remazol Brilliant Blue R (RBBR), indigoid dye Indigo Carmine (IC). To mathematically
describe the adsorption equilibrium established during the interactions of dye molecules
with melanins, the nonlinear models of Langmuir and Freundlich were applied. Based on
the data on the desorption of the dyes bound to melanins, the optimal parameters for their
regeneration were established. To compare adsorption/desorption capacities of melanins
extracted from two lichens, the physico-chemical characteristics, such as content of the
elements and main functional groups, as well as the structural organization were analyzed.
Furthermore, the perspectives of application of melanins for remediation of wastewaters
was assessed based on their capacity to tightly bind adsorbed dyes and as a consequence to
reduce their biological toxicity towards a common soil bacterium Bacillus subtilis.

2. Results and Discussion

2.1. Physico-Chemical Characteristics of Melanins from Lichens L. pulmonaria and L. retigera
2.1.1. Melanin Yield and Qualitative Analysis
The melanins extracted from two lichens L. pulmonaria and L. retigera yielded in
average ca. 3% of the dry weight. Compared to other natural sources of melanins, the
amount of extracted pigments from lichens is higher than that from micro-organisms but
less compared to fungi [17].
Analysis of qualitative reactions of melanins demonstrated that in the presence of
H2 O2 melanins became discolored and in the presence of KMnO4 the color changed from
dark brown to green. The addition of 10% FeCl3 to melanin containing solution led to
precipitation of a sediment, which was further dissolved in the presence of excess FeCl3 .
The results of these qualitative reactions indicate the presence of phenolic fragments, typical
for melanins [18].
Int. J. Mol. Sci. 2022, 23, 15605 3 of 14

2.1.2. Elemental Analysis

Analysis of the elemental composition showed that lichen melanins differ in the
content of C, H, N and O from the standard, a commercial eumelanin from Sepia officinalis
(Table 1). Melanins from L. pulmonaria and L. retigera contained similar content of C and H,
48% and 6% H on average, respectively. However, the content of N significantly differed in
two melanins. In melanin from L. retigera the content of N was more than doubled compared
to that in melanin from L. pulmonaria. It can be suggested that this difference in the content
of N is due to the differences in the photobionts of two lichen species. L. pulmonaria has
two photobionts—non-N-fixing alga Symbiochloris and N-fixing cyanobacterium Nostoc,
while L. retigera has only cyanobacterial photobiont Nostoc with high N-fixing activity [19].

Table 1. The element content of C, H, N, O and atomic ratio in the melanins.

Atomic Ratio
Melanins C (%) H (%) N (%) O (%)
C/O C/H C/N
L. pulmonaria 49.62 ± 0.03 5.78 ± 0.10 4.44 ± 0.12 40.16 1.65 ± 0.12 0.72 ± 0.11 13.04 ± 0.01
L. retigera 47.38 ± 0.10 6.13 ± 0.11 9.47 ± 0.12 37.02 1.71 ± 0.14 0.64 ± 0.11 5.84 ± 0.11
S. officinalis 44.62 ± 0.20 3.39 ± 0.01 7.60 ± 0.05 44.39 1.34 ± 0.07 1.10 ± 0.14 6.85 ± 0.23

Ratio of C/O in the structure of lichen melanins was approximately 1.3 times higher
than that in the standard, indicating a higher content of oxygen-containing fragments. A
high degree of aromaticity (C/H) of lichen melanins, especially melanin from L. pulmonaria,
suggests that condensed systems occupy a large proportion in their structures (Table 1).
However, for commercial eumelanin from S. officinalis the degree of aromaticity was much
higher. The C/N ratio in melanin from the lichen L. retigera and from S. officinalis was similar,
and therefore, this pigment can be attributed to eumelanin. Melanin from L. pulmonaria
differed by a lower content of N, which is typical for allomelanins [20], suggesting a possible
mixed chemical nature of this melanin.

2.1.3. X-ray Fluorescence Analysis

X-ray fluorescence analysis revealed the presence of metals Fe, Al, Zn, Cu in lichen
melanins (Supplementary Table S1). Melanin from L. retigera contained Fe, Al and Cu 1.5,
1.6 and 1.3 times more than those in melanin from L. pulmonaria. Pigment from L. retigera
contained Zn 3.9 times less than in melanin from L. pulmonaria. It is known that in fungal
melanins Fe and Al are bound by hydroxyl, amine, imine, and acetate groups, while Zn and
Cu are bound by carboxyl groups [21]. It should be noted that Cu is an important cofactor
for enzymes involved in the synthesis of melanins, including tyrosinases and laccases [22].

2.1.4. FT-IR Spectroscopy

The complex chemical composition of melanins extracted from lichens was further
confirmed by FT-IR spectroscopy.
The broad absorption band at 3279–3283 cm−1 was attributed to the stretching vi-
brations of the –OH и-NH groups in structure of melanins (Figure 1). The absorption
peak at 2852–2853 cm−1 could be assigned to stretching vibration of aliphatic group. Ab-
sorption bands of the carboxyl groups at 1730 cm−1 and 1680 cm−1 were not observed,
probably because of their low content and/or overlapping with an intensive neighboring
absorption band in the region 1645–1630 cm−1 , which was attributed to the vibrations of
aromatic ring C=C. The band at 1500–1400 cm−1 was assigned to the bending vibrations
of –N–H and stretching vibration of –C–N indole fragment. The peak at 1250–1120 cm−1
was related to the C–O group. The intensive peak centered at 1034 cm−1 (for melanin from
L. pulmonaria) or 1047 cm−1 (for melanin from L. retigera) (Figure 1) could be an indication
of the C–H in-plane of aliphatic structure characteristic of melanin pigment, aromatic
CH groups, alkene CH substitution/conjugated systems, and/or symmetric contraction
vibration of C–O–C bond [23–25]. The presence of a simple ether bond (C–O–C) and peak
 stretching vibration of –C–N indole fragment. The peak at 1250–1120 cm−1 was related to
the C–O group. The intensive peak centered at 1034 cm−1 (for melanin from L. pulmonaria)
or 1047 cm−1 (for melanin from L. retigera) (Figure 1) could be an indication of the C–H in-
plane of aliphatic structure characteristic of melanin pigment, aromatic CH groups, alkene
Int. J. Mol. Sci. 2022, 23, 15605 CH substitution/conjugated systems, and/or symmetric contraction vibration of C–O–C 4 of 14
bond [23–25]. The presence of a simple ether bond (C–O–C) and peak at approximately
890 cm−1 and 900 cm−1 in the melanin from L. pulmonaria indicates the occurrence of poly-
saccharides
at with ß
approximately -type
890 cmbond
−1 and [26].
900Incm the
−1 FT-IR
in the spectra
melaninoffrom
melanins from lichens,
L. pulmonaria indicatesthe the
ab-
sorption bands were observed in areas characteristic of melanins from
occurrence of polysaccharides with ß-type bond [26]. In the FT-IR spectra of melanins various natural
sources
from [4,27–29].
lichens, However, bands
the absorption in comparison with melanin
were observed from S. officinalis,
in areas characteristic lichen mela-
of melanins from
nins lacked
various a pronounced
natural absorption
sources [4,27–29]. peak atin1356
However, cm because
−1
comparison of the presence
with melanin from S.of C=O in
officinalis,
aromatic
lichen conjugate
melanins lackedstructures, as well
a pronounced as an absorption
absorption peak at 1356peak −1 c.
cmat 1040 cm
because (Figure
of−1the presence 1).
Therefore,
of these results
C=O in aromatic suggeststructures,
conjugate that among asfunctional
well as angroups discovered
absorption peak atinc.lichen cm−1
1040 mela-
nins, hydroxyl
(Figure (–OH) these
1). Therefore, and carboxyl (–COOH)
results suggest thatgroups
among are the main
functional functional
groups groups,
discovered in
which can be involved in the adsorption of dyes.
lichen melanins, hydroxyl (–OH) and carboxyl (–COOH) groups are the main functional
groups, which can be involved in the adsorption of dyes.

FT-IR spectra
Figure 1. FT-IR spectraofofmelanins
melaninsextracted fromL.L.pulmonaria
extractedfrom pulmonaria(red
(red L. L.
line);
line); retigera
retigera (blue
(blue line);
line); S.
officinalis
S. (black
officinalis line).
(black line).

2.1.5. Chemical Analysis

2.1.5. Chemical Analysis of of Melanins
Melanins
Further differences between
Further differences between two two melanins
melanins were
were shown
shown in in the
the content
content of
of polysaccha-
polysaccha-
rides
rides (Table
(Table 2).
2). It
It is
is known
known thatthat melanin
melanin binds
binds strongly
strongly to
to the
the polysaccharides
polysaccharides ofof the
the cell
cell
tissue of the fungus [30]. It can be suggested that polysaccharides, which are structurally
tissue of the fungus [30]. It can be suggested that polysaccharides, which are structurally
linked
linked to
to melanins,
melanins, cancan increase
increase their
their sorption
sorption activity
activity towards
towards metals
metals and
and dyes.
dyes.

Table 2. The
Table 2. The content
content of
of polysaccharides
polysaccharides and
and –OH,
-OH, –COOH functional groups
-COOH functional groups in
in the
the melanins.
melanins.

Melanins
Melanins Content of Polysaccharides
Content (%)
of Polysaccharides (%)–OH (%)
–ОН (%) –COOH
–СООН(%)(%)
L.L.pulmonaria
pulmonaria 19.93 ±19.93
0.60 ± 0.60 14.54 ±14.54
1.85 ± 1.85 1.06 ± 0.01
1.06 ± 0.01
L.L.retigera
retigera 14.08 ± 0.19
14.08 ± 0.19 8.08 ± 1.28
8.08 ± 1.28 2.66 ± 0.75
2.66 ± 0.75
S. officinalis - 16.15 ± 0.90 2.19 ± 0.10
S. officinalis - 16.15 ± 0.90 2.19 ± 0.10

These data
dataare
areconsistent
consistentwith
with
thethe results
results of quantitative
of quantitative determination
determination of these
of these func-
functional groups by the titration in alkali solutions of melanins (Table 2). Indeed, the
tional groups by the titration in alkali solutions of melanins (Table 2). Indeed, the content
content of –OH groups was 1.8 times higher in melanin from L. pulmonaria, whereas the
content of –COOH groups was 2.7 times higher in melanin from L. retigera. Interestingly,
the content of –OH groups in the eumelanin from S. officinalis was similar to that in melanin
from L. pulmonaria, while the content of –COOH groups in the eumelanin from S. officinalis
was similar to that in melanin from L. retigera, indicating the differences in reactivity of
melanins including their chelating abilities.
 of –СООН groups was 2.7 times higher in melanin from L. retigera. Interestingly, the con-
tent of –ОН groups in the eumelanin from S. officinalis was similar to that in melanin from
L. pulmonaria, while the content of –СООН groups in the eumelanin from S. officinalis was
similar to that in melanin from L. retigera, indicating the differences in reactivity of mela-
nins including their chelating abilities.
Int. J. Mol. Sci. 2022, 23, 15605 5 of 14

2.1.6. Zeta (ζ) Potential of Melanins

Using dynamic light scattering (DLS) it was demonstrated that melanins possess neg-
2.1.6.zeta
ative Zeta(ζ)
(ζ)potential.
Potential In
of Melanins
melanin from L. pulmonaria the zeta (ζ) potential was −32.73 ±
Using
9.71 mV, dynamic
while light scattering
in melanin (DLS)it itwas
from L. retigera was−12.63
demonstrated
± 7.39 mV.thatFormelanins
melanin from possess
L.
negative zeta
pulmonaria (ζ) potential.
(ζ) potential was 2.6Intimes
melanin
higherfrom L. pulmonaria
compared themelanin
to that in zeta (ζ)from
potential was
L. retigera,
−32.73correlates
which ± 9.71 mV, while
with in melanin
a large numberfrom
of functional it was −in12.63
L. retigera groups ± 7.39 mV.
its structure For melanin
(Table 2). The
L. pulmonaria
(ζ) potential corresponds to the surface potential and therefore it may influence the from
from (ζ) potential was 2.6 times higher compared to that in melanin pro-
L. retigera,
cesses which correlates with
of adsorption/desorption a large
of dye ions.number of functional groups in its structure
(Table 2). The
Taken (ζ) potential
together, corresponds
data suggest to the surface
that melanins from two potential
lichenand therefore
species it may influ-
demonstrate dif-
ence the processes of adsorption/desorption of dye ions.
ferences in the degree of aromaticity, the ratio of C/N, metal chelating abilities, and the
number Taken
of –OHtogether, data suggest
and –COOH groups.that
Thesemelanins
differencesfrom two determine
might lichen species demonstrate
the discrepancies
differences in the degree of aromaticity, the ratio of C/N, metal chelating
in their physico-chemical properties, including the ability of adsorption/desorption abilities, and the
of
number of
synthetic dyes.–OH and –COOH groups. These differences might determine the discrepancies
in their physico-chemical properties, including the ability of adsorption/desorption of
synthetic
2.2. dyes.Studies
Adsorption
2.2.1. Chemical Studies
2.2. Adsorption Structure of Applied Synthetic Dyes
2.2.1.ToChemical
assess the adsorption
Structure capacity
of Applied of melanins
Synthetic Dyesfrom lichens L. pulmonaria and L. re-
tigera To
water-soluble synthetic dyes MB, RBBR
assess the adsorption capacity of melanins fromand IC, differing
lichens L. in pulmonaria
the charge andand L.molecu-
retigera
lar weight were chosen. The structure of synthetic dyes is shown
water-soluble synthetic dyes MB, RBBR and IC, differing in the charge and molecular in Figure 2. It is known
that thiazine
weight were dye MB The
chosen. is widely usedoffor
structure dyeing dyes
synthetic cotton, silk, and
is shown in wood
Figure[31].
2. It This is a cati-
is known that
onic dye and
thiazine dye therefore
MB is widelyit canused
participate in electrostatic
for dyeing cotton, silk,interactions,
and wood [31]. and at theis
This same time,
a cationic
itdye
is capable of hydrophobic
and therefore interactions.
it can participate RBBR dye belongs
in electrostatic to active
interactions, andanthraquinone
at the same time, dyesit
and is widely used in the textile and cotton industries [32]. It is an
is capable of hydrophobic interactions. RBBR dye belongs to active anthraquinone dyesanionic dye containing
SO
and3¯ is
groups.
widelyTheusedpoly-conjugate
in the textile and structure
cottonof the dye ensures
industries [32]. It isitsanaffinity
anionicwith non-polar
dye containing
SO3 ¯ groups.
compounds [33].
TheIndigoid dye IC isstructure
poly-conjugate used in the textile
of the dyeand food its
ensures industries [34]. The
affinity with pres-
non-polar
ence of two SO
compounds 3¯ groups
[33]. Indigoidprovides
dye IC anionic
is usedproperties to theand
in the textile dye,food
and industries
the presence of sub-
[34]. The
stituted
presenceheterocyclic ¯ groups provides
of two SO3fragments in the main structure
anionic ensures
properties toits
theparticipation
dye, and theinpresence
the inter-of
molecular
substituted interactions
heterocyclic [35–37].
fragments in the main structure ensures its participation in the
intermolecular interactions [35–37].

Chemicalstructure
Figure2.2.Chemical
Figure structureof
ofsynthetic
syntheticdyes
dyesMB,
MB,RBBR
RBBRand
andIС.
IC.

To reduce the toxicity of synthetic dyes in ecosystems [38,39], it is crucial to develop

an effective protocol of remediation of wastewaters, including adsorption of dyes. For this
purpose, adsorption of dyes by melanins from lichens represents a promising approach.

2.2.2. Evaluation of Adsorption Capacity of Lichen Melanins

The study of the adsorption capacity of lichen melanins towards synthetic dyes in-
cluded the study of adsorption under static conditions using model solutions of dyes
at a constant temperature. In the experiment, the concentration of dyes varied, while a
concentration of melanin in the solution remained constant. The adsorption capacity of
melanins from two lichens in relation to synthetic dyes was markedly different (Table 3).
Int. J. Mol. Sci. 2022, 23, 15605 6 of 14

Table 3. The adsorption capacity of melanins from L. pulmonaria and L. retigera.

Amount of Adsorbed Dye (µg)

Melanins Amount of Dye (µg) 1
MB RBBR IC
75 67.49 ± 1.00 51.57 ± 0.04 41.29 ± 2.30
L. pulmonaria 150 140.50 ± 2.26 64.46 ± 4.41 93.54 ± 1.89
300 260.40 ± 1.98 140.53 ± 0.42 197.19 ± 4.56
75 49.53 ± 0.34 38.40 ± 0.14 38.07 ± 1.10
L. retigera 150 70.15 ± 4.69 64.46 ± 1.05 50.39 ± 4.65
300 129.21 ± 3.50 82.96 ± 0.21 60.97 ± 1.97
1 Initial amount of dye in the reaction medium.

At the maximum dye content in the solution (300 µg), adsorption of MB, RBBR, and
IC by melanin from L. pulmonaria was 2, 1.7, and 3.2 times greater than that by melanin
from L. retigera. Probably, the higher adsorption capacity of melanin from L. pulmonaria was
due to the high content of –OH functional groups and polysaccharides (Table 2).
Langmuir and Freundlich isotherm models were used to describe the mechanism of
adsorption of synthetic dyes by lichen melanins. These models are widely adopted for
Int. J. Mol. Sci. 2022, 23, x FOR PEER investigating
REVIEW 7 of 14
the relationship between adsorption capacity of adsorbent and equilibrium
adsorbent concentration in an aqueous solution [40]. Adsorption isotherms are presented
in Figure 3.

Figure 3.
Figure 3. Langmuir
Langmuir and
and Freundlich
Freundlich isotherm
isotherm models
models of of adsorption
adsorption ofof synthetic
synthetic dyes
dyes MB,
MB, RBBR
RBBR and
and
IС by melanins from L. pulmonaria (red line); L. retigera (blue line) at 298.1 ◦°К. Solid line corresponds
IC by melanins from L. pulmonaria (red line); L. retigera (blue line) at 298.1 K. Solid line corresponds
to the
to the Langmuir
Langmuir isotherm
isothermmodel,
model,dotted
dottedline
linecorresponds
correspondsto tothe
theFreundlich
Freundlichisotherm
isothermmodel.
model.

The
The main
main parameters,
parameters, including
including the constants of the Langmuir and Freundlich equa-
tions,
tions, were
were calculated
calculated and
and presented
presented in
inSupplementary
Supplementary Table
TableS2.
S2. Values
Valuesqqeqeq using
using the
the
Langmuir and Freundlich models (Section 3.4.2) were consistent with the experimental
Langmuir and Freundlich models (Section 3.4.2) were consistent with the experimental
data
data (Figure
(Figure 3).
3). According to the
the Langmuir
Langmuir model,
model, the
the formation
formation of
of aa monomolecular
monomolecular
layer
layer on
on the
the surface
surface of
of the
the sorbent
sorbent does
does not
not involve
involve interactions
interactions between
between the
the adsorbate
adsorbate
particles [41,42]. According to the Freundlich model, adsorption occurs on an energeti-
cally heterogeneous surface [43]. According to the type of isotherm and the obtained val-
ues (R2), the adsorption of synthetic dyes MB and RBBR by lichen melanins could be cor-
rectly described by the Freundlich isotherm model (Figure 3; Supplementary Table S2),
Int. J. Mol. Sci. 2022, 23, 15605 7 of 14

particles [41,42]. According to the Freundlich model, adsorption occurs on an energetically

heterogeneous surface [43]. According to the type of isotherm and the obtained values
(R2 ), the adsorption of synthetic dyes MB and RBBR by lichen melanins could be correctly
described by the Freundlich isotherm model (Figure 3; Supplementary Table S2), due to the
presence of the heterogeneous adsorption centers in the melanin structure.
The maximum dye adsorption capacity (qmax ) was calculated according to the Lang-
muir model. For melanin from L. pulmonaria it was 47.63, 36.03, 92.96 mg/g, while for
melanin from L. retigera it was 34.97, 18.28, 14.72 mg/g for MB, RBBR and IC, respec-
tively (Supplementary Table S2). These data suggest that the melanin from L. pulmonaria
demonstrates higher dye adsorption capacity. Presumably, MB adsorption is caused by
electrostatic interactions between the -NH group dyes and negatively charged functional
groups of melanin. Total amount of negatively charged functional groups in the melanin
of L. pulmonaria was 5% more than that in the melanin of L. retigera (Table 3). Adsorption
of anionic dyes is associated with the presence of hydrophilic centers in the structure
of melanin [44]. RBBR contains both cationic and anionic functional centers, which can
interact with the aromatic skeleton of melanins via π–π or n–π interactions, and therefore
the mechanisms of adsorption of this dye by lichen melanins are more complex. A signifi-
cant limitation of the effective RBBR adsorption is high molecular weight and a complex
branched structure of this dye [45]. The highest adsorption capacity melanin from L. pul-
monaria demonstrates towards the anionic dye IC (92.96 vs. 14.72 mg/g for the melanin
from L. retigera) that can be explained by interactions of the dye with the aromatic skeleton
of melanins and a small size of its molecule. Moreover, the presence of polysaccharides in
the structure of melanins (Figure 1; Table 2) can facilitate the adsorption of dyes.
Therefore, the melanins from the lichens L. pulmonaria and L. retigera can adsorb
synthetic dyes. The efficiency of adsorption is determined by the chemical characteristics
of a dye and physico-chemical properties of lichen melanins.

2.2.3. Regeneration Studies

In biotechnological practice, an important property of adsorbents is the possibility for
their reuse in adsorption and desorption cycles [46]. The desorption of synthetic dyes was
studied under constant stirring and successive treatments with water, heating at 30 ◦ C, and
pH change (Table 4).

Table 4. Desorption of synthetic dyes adsorbed by melanins from L. pulmonaria and L. retigera.

Desorption (D) of the Adsorbed Dye (%) 1

Melanins Treatment
MB RBBR IC
H2 O 1.50 ± 0.12 9.14 ± 0.11 2.16 ± 0.21
H2 O, 30 ◦ C 0.16 ± 0.05 0.89 ± 0.06 0.60 ± 0.59
L. pulmonaria
H2 O, pH 3 1.91 ± 0.23 1.08 ± 0.80 0.89 ± 0.32
H2 O, pH 11 n.d. 2 n.d. n.d.
H2 O 6.48 ± 0.75 12.68 ± 1.10 16.06 ± 0.73
H2 O, 30 ◦ C 1.42 ± 0.21 3.00 ± 0.10 3.82 ± 0.11
L. retigera
H2 O, pH 3 61.83 ± 0.04 2.46 ± 0.05 3.89 ± 0.55
H2 O, pH 11 n.d. n.d. n.d.
1 On the amount of adsorbed dye. 2 Not determined.

It was found that the binding strength of dyes by melanins varies depending on the
chemical structure of a dye and exposure conditions. The RBBR, which is characterized by
a complex branched structure and a low total charge, when treated with water, was actively
released into the solution (9–13% on average) from both melanins. When treated with heat
and pH 3, desorption of RBBR is negligible—no more than 1% from L. pulmonaria melanin,
no more than 3% from L. retigera. These results indicate that RBBR is strongly bound by
melanins and cannot be easily desorbed.
Int. J. Mol. Sci. 2022, 23, 15605 8 of 14

Differences in the structural organization and reactivity of melanins depending on the

lichens from which they were extracted determined their different retention capacity with
respect to the anionic dye IC and the cationic dye MB. Following each treatment melanin
from L. retigera released more dye compared to melanin from L. pulmonaria (Table 4). This
may be due to the high content of negatively charged carboxyl groups in melanin from
L. retigera (2.6 times) (Table 2) and the lower content of polysaccharides. Melanin from L.
retigera demonstrated a 4.3 times higher desorption capacity for MB during incubation in
water compared to that for melanin from L. pulmonaria (Table 4). Gentle heating did not
facilitate release of the dye, while acidification of the incubation medium greatly (10 times)
increased the release of MB. This may be caused by the transfer to the main status (non-
dissociated) of negatively charged carboxyl groups of melanin from L. retigera and their
disability to interact with dye molecules.
Altogether, these data suggest that melanin from L. retigera has a higher capacity for
desorption of all synthetic dyes applied in this study. It is important to note that in the alkali
solution (pH 10) melanins were fully dissolved and dyes released (Table 4). This provides
a possible mechanism for regeneration of melanins including the alkali dissolvement of
melanin/dye complex followed by membrane filtration, removal of dyes and recovery of
purified melanins for further reuse.

2.2.4. Reducing the Biological Toxicity of Synthetic Dyes by Their Adsorption

Synthetic dyes, when released into natural ecosystems, have a pronounced toxic effect
on living organisms [47–50]. We analyzed how adsorption of dyes by melanins can mitigate
their biological toxicity using nonpathogenic soil bacteria B. subtilis. These micro-organisms
occupy an important niche in the microbial consortium of natural systems, and the impact
of dyes on their viability can shift the ecological balance [50,51]. In our preliminary
experiments, we tested the effects of lichen melanins on the growth of bacterium B. subtilis.
It was found that melanins on their own do not have a significant inhibitory or stimulating
effect on bacterial growth. Here, it was shown that synthetic dyes MB and RBBR cause
an inhibitory effect on the growth of B. subtilis (Table 5; Supplementary Figure S1). In
contrast to MB and RBBR, IC did not show a pronounced toxic effect on bacteria (data are
not present). In the presence of melanins extracted from lichens L. pulmonaria and L. retigera
the negative effect of synthetic dyes on bacterial flora was less (Supplementary Figure S1).
For example, the inhibitory effect of MB on bacterial growth was reduced by ca. 30% when
melanins containing the same amount of adsorbed dye were applied. When evaluating
the toxic effect of RBBR on B. subtilis, it was found that RBBR adsorbed by melanin from
L. pulmonaria does not display a bactericidal effect, while RBBR adsorbed by melanin from
L. retigera showed a weak inhibitory effect on the growth of B. subtilis.

Table 5. Effects of melanins on the bacterial growth retardation caused by MB and RBBR.

Content Zone of Growth Retardation (mm)

Dyes of Dye
MB RBBR
(µg/disk)
Dye without melanins 28.8 ± 1.8 10.9 ± 1.6
Dye adsorbed by melanin from L. pulmonaria 50 20.0 ± 0.8 0
Dye adsorbed by melanin from L. retigera 21.0 ± 0.8 8.3 ± 0.5

The results obtained in biological studies using B. subtilis demonstrated that effec-
tiveness of lichen melanins to reduce toxicity of synthetic dyes is determined by their
physico-chemical characteristics and adsorption properties. Indeed, melanin from L. pul-
monaria with higher adsorption capacity (Table 3) and lower desorption capacity (Table 4)
could more effectively reduce the toxicity of synthetic dyes (Table 5). Melanin L. retigera
was not able to effectively retain dyes and reduce the toxicity of synthetic dyes; however, it
showed higher desorption capacity (Table 4).
Int. J. Mol. Sci. 2022, 23, 15605 9 of 14

3. Materials and Methods

3.1. Chemicals/Reagents
Dyes MB, RBBR, IC; melanin from S. officinalis were analytical grade (Sigma-Aldrich,
St. Louis, MO, USA). H2 O2 , KMnO4 , FeCl3 , NaOH, LiOH, BaCl2 , (CH3COO)2 Ca, methyl
red, phenolphthalein and organic solvents were of reagent grade (Russia).

3.2. Extraction and Purification of Melanins from Lichens L. pulmonaria and L. retigera
Melanins were extracted from lichens L. pulmonaria and L. retigera. Lichen L. pulmonaria
was collected from the bark of oak trees at Langangen, South Norway (59.096◦ N; 9.792◦ E)
and L. retigera from the bush branches in Fort Nottingham in the province KwaZulu-Natal,
South Africa (29.414◦ S; 29.913◦ E). Dry lichen thalli were crushed to a powder with the
addition of liquid nitrogen, then powder (5 g) was incubated with 50 mL 2 M NaOH for
24 h, and extract centrifuged at 150 rpm (Hermle, Gosheim, Germany) for 10 min. The
supernatant was acidified by adding 2 M HCl, incubated for 12 h at room temperature,
and centrifuged at 150 rpm for 10 min. The precipitate containing melanin was rinsed
with distilled water, then successively purified with chloroform, ethyl acetate and acetone
(2 × 10 mL). The purified melanin was lyophilized. Yield of melanin from L. pulmonaria
was 40 ± 5 mg/g DW and melanin from L. retigera—42 ± 8 mg/g DW. Melanins extracted
from lichens were brown and dark brown powders. Melanin powders were sifted and
fractions no larger than 1 mm were stored at −20 ◦ C until use for analyses.

3.3. Physico-Chemical Characteristics of Melanins

3.3.1. Elemental Composition and Content of Polysaccharides
Solutions of melanins (0.4 mg/mL) were prepared in distilled water with the addition
of 10% NH4 OH to pH 7.0. Identification of melanins was carried out using qualitative
reactions with H2 O2 , KMnO4 and FeCl3 [4,51]. Elemental composition of melanins (C, N,
H) determined using an analyzer EuroEA 3028-HT-OM (Eurovector SpA, Pavia, Italy) [52],
the oxygen content was determined by calculation.
Content of ash including metals was assessed by X-ray fluorescence analysis (XRF)
using an energy-dispersive fluorescent X-ray spectrometer EDX-800HS2 (Shimadzu, Kyoto,
Japan). The measurements were carried out under the following conditions: maximum
input voltage 50 kV, maximum current 1 mA, helium atmosphere. XRF analysis of melanins
was processed using EDX software.
The total polysaccharides content in the extracted melanins was estimated by the
anthrone method [53] using glucose as a standard.

3.3.2. Functional Groups

The functional groups of melanins were analyzed using Fourier-transform infrared
(FT-IR) spectroscopy (IR-Affinty1, Shimadzu, Kyoto, Japan). The measurements were
carried out using following parameters: wavenumber range 4000–700 cm–1 , the spectral
resolution fixed to 4 cm−1 , the number of scans to 32. The FT-IR spectra of melanins were
processed using OriginPro 8 software.
Quantitative analysis of –OH and –COOH groups in melanins was carried out by
LiOH titration according to the protocol with modification [54]. The number of –OH groups
was calculated using the equation:

a0 − a × f × 1.7 × 1.25


[OH] = × 100% (1)
A
where a, a0 —volumes 0.1 M LiOH, used for titration in the working and blank experiments,
respectively (mL); f—factor 0.1 M LiOH; 1.7—weight of –OH groups equivalent to 1 mL
0.1 M LiOH (mg); 1.25—full volume conversion factor; A—weight of melanin sample (mg).
The number of –COOH groups was calculated using the equation:
Int. J. Mol. Sci. 2022, 23, 15605 10 of 14

a − a0 × f × 1.25 × 0.85


[COOH] = × 100% (2)
A
where a, a0 —volumes 0.05 M LiOH, used for titration in the working and blank experiments,
respectively (mL); f—factor 0.05 M LiOH; 0.85—weight of –COOH groups, equivalent to
1 mL 0.05 M LiOH (mg); 1.25—full volume conversion factor; A—weight of melanin
sample (mg).

3.3.3. Colloidal Properties

The zeta (ζ) potential determination of samples was carried out by a Malvern ZS
Nanosizer (Malvern Instruments Ltd., Malvern, UK) Dynamic Light Scattering (DLS).
Sample preparation, included the preparation of melanin solutions at a concentration
of 0.4 mg/mL, filtration through a membrane filter (45/13 Chromafill Xtra, 0.45 µm,
Düren, Germany).

3.4. Adsorption Properties of Melanins

3.4.1. Adsorption of Synthetic Dyes
Melanin powder (500 µg) was incubated with 3 mL of dyes in concentrations of 25, 50,
100 mg/L at 25 ◦ C with continuous stirring at 100 rpm for 30 min (BioSan, Riga, Latvia).
Solutions were then centrifuged at 240 rpm for 10 min (Eppendorf, Hamburg, Germany).
The absorbance of the supernatant was measured at dye absorption maxima (663 nm for
MB; 595 nm for RBBR; 612 nm for IC) using a UV-1900 spectrophotometer (Shimadzu,
Kyoto, Japan). Calculation of the amount of dye adsorbed by melanin was determined
using the experimentally obtained calibration curves of the dependence of the optical
density of dye solutions on their concentration (Supplementary Table S3). The amount of
dye adsorbed was calculated using the following equation:

V
q = (C0 − C1 ) (3)
W
where q—amount of dye adsorbed on melanin (mg/g); C0 —the initial concentration of the
dye in the solution (mg/L); C1 —dye concentration in solution after adsorption (mg/L);
V—volume of the solution (L); W—the weight of melanin taken for analysis (g).

3.4.2. Construction of Adsorption Isotherms

To determine the maximum of adsorption capacity of melanins, adsorption isotherm
according to the models of Langmuir and Freundlich were constructed. Using experimental
data (Section 3.4.1), calculations were carried out using the formulas:

qe = qmax KL Ce /(1 + KL Ce ) (4)

qe = KF C1/n
e (5)
where qmax —maximum adsorption capacity (mg/g); KL —Langmuir constant; Ce —initial
amount of dye (mg/L); KF —Freundlich constant; 1/n—the value of the adsorption index.

3.5. Desorption of Synthetic Dyes Adsorbed by Melanins

Based on the experiments described in Section 3.4.1, melanins (500 µg) after incubation
with 3 mL dye (50 mg/L) were sequentially washed according to the following scheme:
(1) incubation in distilled water at 100 rpm for 30 min at room temperature and centrifu-
gation at 240 rpm for 10 min three times, (2) resulting pellets were incubated in distilled
water at 100 rpm and a temperature of +30 ◦ C for 30 min and centrifuged, (3) resulting
pellets were incubated in water solutions with pH 3 and pH 11 at 100 rpm for 30 min and
centrifuged. Optical density of the resulting supernatants was measured as described in
Int. J. Mol. Sci. 2022, 23, 15605 11 of 14

Section 3.4.1. The calculation of the desorption of the dye adsorbed with melanin was
performed according to the equation:
qa
D = × 100% (6)
qd

where qa —amount of dye in solution (µg); qd —initial amount of dye in solution (µg).

3.6. Biotesting of the Toxicity of Synthetic Dyes Adsorbed by Melanins

Toxicity of synthetic dyes absorbed by melanins was tested using a common soil
bacterium Bacillus subtilis by the disk-diffusion method. The bacterial inoculum was grown
in Luria-Bertani medium (LB) [55] in a shaker-incubator ES-20/60 (BioSan, Riga, Latvia)
at 170 rpm, 37 ◦ C for 16 h, and after that 100 µL of bacterial culture was plated on 1.5%
agar gel in Petri dishes. The dye solution was applied to sterile paper discs (10–50 µg/disk)
and incubated at 37 ◦ C for 12 h. Melanin (150 µg) containing 50 µg of adsorbed dye was
dispersed in the calculated volume of distilled water; the resulting solution was applied to
a disk. Retardation zone of bacterial growth was measured (in mm). Toxicity of each dye
adsorbed by melanins was assessed using the same protocol.

3.7. Statistical Analysis

The experiments were carried out in three biological and at least three analytical
replicates. Calculations were made in Microsoft Excel 2016. The results of the experiments
were statistically processed and presented in the work at a confidence level p = 0.95, n = 3
(n is the number of experiments).

4. Conclusions
Various physico-chemical treatments, such as ion exchange, reverse osmosis, and
advanced oxidation processes, are used to remove toxic compounds from wastewater.
Adsorption is one of the most effective methods used in treatment [1]. The results obtained
in this study demonstrate the effectiveness of lichen melanins in absorbing and reducing
the toxicity of synthetic dyes. A comparative analysis of melanins extracted from the
lichens L. pulmonaria and L. retigera demonstrated differences in their chemical composition,
structure, and the content of functional groups. Melanin from the lichen L. pulmonaria has a
high degree of aromaticity, low content of N, high content of Zn, which correlates with high
content of hydroxyl groups. Melanin from the lichen L. retigera is characterized by high
content of N and based on the C/N ratio melanin from the lichen L. retigera can be attributed
to eumelanin. High content of Fe and Al correlates with a bigger amount of carboxyl groups.
Given their physico-chemical properties, melanin from L. pulmonaria is more effective in
terms of the adsorption capacity and retention ability of synthetic dyes compared to
melanin from L. retigera. Melanin from the lichen L. pulmonaria showed great heterogeneous
adsorption capacity for the cationic dye MB, anionic dye RBBR and monolayer adsorption
capacity for IC. The adsorption properties of melanin can be enhanced due to the presence
of polysaccharides in its composition. During the adsorption process, dyes easily interact
with functional groups of melanins, as well as with their aromatic polyconjugated structures
through hydrogen bonds and electrostatic and donor-acceptor interactions. As a result of
dye sorption, melanins can reduce their toxicity to living organisms, e.g., nonpathogenic
soil bacteria B. subtilis.
In the future, based on the fundamental knowledge of physico-chemical properties
of lichen melanins, melanin-like materials can be synthesized to develop new ecologically
friendly and highly efficient reusable remediation technologies. Application of lichen
melanin is a basis for a new ecologically friendly approach for remediation.

Supplementary Materials: The following supporting information can be downloaded at:

https://www.mdpi.com/article/10.3390/ijms232415605/s1.
Int. J. Mol. Sci. 2022, 23, 15605 12 of 14

Author Contributions: Conceptualization, V.K. and F.M.; methodology, A.R. and V.K.; investigation,
A.R., V.K., V.B. and A.D.; writing—original draft preparation, A.R. and V.K.; writing—review and
editing, F.M.; project administration, F.M.; formal analysis, A.R., A.D. and V.B.; funding acquisition,
F.M. All authors have read and agreed to the published version of the manuscript.
Funding: This work was partially performed within the framework of the state assignment of the
FRC KazSC RAS. Financial support was received from the Russian Science Foundation (grant No.
18-14-00198, experiments on adsorption/desorption capacity) and the Russian Foundation for Basic
Research (grant No. 20-34-90044, spectral analysis). Some measurements were carried out using
CSF-SAC FRC equipment of the KNC RAS (elemental analysis, XRF).
Institutional Review Board Statement: Not applicable.
Informed Consent Statement: Not applicable.
Data Availability Statement: Not applicable.
Acknowledgments: The authors are grateful to Knut Solhag (University of Life Sciences, Norway)
and Richard Beckett (University of KwaZulu-Natal, South Africa) for valuable discussions and their
help with collecting lichens.
Conflicts of Interest: The authors declare no conflict of interest. The funders had no role in the design
of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, or
in the decision to publish the results.

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