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International Biodeterioration & Biodegradation 84 (2013) 307e313Contents lists availableInternational Biodeterioration & Biodegradation
journal homepage: www.elsevier .com/locate/ ibiodBiomimetic system for removal of fungal melanin staining on paper

Crtomir Tavzes a,e,*, Jernej Pal
ci
c b, Karin Fackler c, Franc Pohleven b, Robert J. Koestler d
aResearch Institute, Conservation Centre, Institute for the Protection of Cultural Heritage of Slovenia, Ljubljana, Slovenia
bDepartment of Wood Science and Technology, Biotechnical Faculty, University of Ljubljana, Slovenia
c Institute of Chemical Engineering, Vienna University of Technology, Austria
dMuseum Conservation Institute, Smithsonian Institution, Suitland, MD, USA
e Institute of Wood Science and Technology, and Sustainable Development, Celov
ska cesta 268, SI-1000 Ljubljana, Sloveniaa r t i c l e i n f o
Article history:
Received 31 January 2012
Received in revised form
22 June 2012
Accepted 14 July 2012
Available online 7 November 2012
Keywords:
Biomimetic system
Colorimetry
Copperepyridine complex
FTIR
Melanin degradation
UVeVIS* Corresponding author. Institute of Wood Sci
Sustainable Development, Celov
ska cesta 268, S
Tel.: þ386 51 369 311; fax: þ386 1 423 50 35.
E-mail addresses: crtomir.tavzes@iltra.si, ceromir@
0964-8305/$ e see front matter 
 2012 Elsevier Ltd.
http://dx.doi.org/10.1016/j.ibiod.2012.07.022a b s t r a c t
Fungal melanin staining is a problem on many cultural objects, ranging from the French Palaeolithic cave
at Lascaux to books and papers in museum collections. Melanin, because it is insoluble and resistant to
bleaching, may leave behind undesirable stains long after the fungal infestation has been controlled.
Research into removal of melanin stains from paper and other sensitive substrates using industrial
biomimetic oxidizing systems has shown considerable success. We studied relative concentration of the
bleaching reagents and the reaction kinetics both in liquid suspensions of melanin and on melanized
paper samples. Liquid suspension samples were tested for changes in their chemical composition
(appearance and relative representation of functional groups and chemical bonds) with FTIR spec-
trometry. Changes in color of melanized paper samples were investigated with a CIE L*a*b system, where
the effectiveness of the treatment (bleaching) was determined as a change in lightness (DL). Melanin was
oxidized in the liquid suspensions, and the intensity of modification depended on the procedure
employed. Bleaching of melanin with the biomimetic copperepyridine complex proved to be far superior
to the effect of white-rot fungal oxidizing enzymes, previously reported on by this group.

 2012 Elsevier Ltd. All rights reserved.1. Introduction
Melanin-producing fungi infestmany art objects, especially when
the artifacts are stored under conditions of high relative humidity.
They produce the black pigment melanin that causes undesirable
stains on or in the objects. Fungal melanins are dark-pigmented
(generally black) high-molecular-weight phenolic polymers found
in the cell walls of spores, sclerotia, mycelia, and fruiting bodies, or as
extracellularpolymers formed inthemediumaround fungal cells, and
they are synthesized by oxidative polymerization of phenolic and
indolic monomers (Bartnicki-Garcia and Reyes, 1964; Durrell, 1964;
Bell and Wheeler, 1986; Takano et al., 1997; Butler and Day, 1998b).
They are thought to have a number of natural precursors, including
1,8-dihydroxynaphthalene (DHN), g-glutaminyl-4-hydroxybenzene
(GBH), catechol, catecholamines, and tyrosine. The most extensively
analyzed and possibly the most prevalent fungal melanin is often
called “DHNmelanin”, after its immediate precursor monomer, DHNence and Technology, and
I-1000 Ljubljana, Slovenia.
yahoo.com (
C. Tavzes).
All rights reserved.(Bell and Wheeler, 1986). This precursor is produced from acetate
through several intermediates in the pentaketide pathway
(Tokousbalides and Sisler,1979; Siehr,1981; Fogarty and Tobin,1996).
These intermediates are secreted across the cell membrane into the
surrounding medium, with some remaining in the cell wall. Outside
thecell, theyoxidizeorareoxidativelypolymerizedbyphenoloxidase
(Carlile and Watkinson, 1994). Synthesis, chemical structure, func-
tionality, and distribution of various melanins in the fungal kingdom
were initially reviewed by Butler and Day (1998b), andmore recently
by Eisenman and Casadevall (2012). Issues regarding melanin struc-
ture (including proposed schemata) and chemical changes that occur
during its abiotic degradation have been proposed and discussed by
several researchers (Blois, 1978; Bell and Wheeler, 1986; Korytowski
and Sarna, 1990; Kaim and Schwederski, 1994; Jacobson, 2000).
Melanins were often considered as non-biodegradable poly-
mers and defined on the basis of their supposed inertness and
resistance to chemical attack (Prota, 1992). However, contradictory
reports suggested slow fungal melanin biodegradation (Luther and
Lipke, 1980; Liu et al., 1995; Rättö et al., 2001). Groundbreaking
work by Butler and Day (1998a) showed the ability of Phaner-
ochaete chrysosporium manganese peroxidase (MnP) system to
degrade melanin.

C. Tavzes et al. / International Biodeterioration & Biodegradation 84 (2013) 307e313308The biomimetic copperepyridine complex (Cuepy system) was
developed for bleaching of residual lignin in cellulose pulps
(Watanabe et al., 1997). In the presence of hydrogen peroxide,
highly oxidative reaction intermediates, putatively copper-oxo or
coppereperoxo complexes, mimic the reaction of lignin peroxidase
with hydrogen peroxide and lignin (Fackler et al., 2001).
Even if fungal infestation of paper is eradicated, melanin
remains attached (or entangled) to or under surfaces that were
previously overgrown by the mycelia. Melanin is an extremely
recalcitrant polymer and cannot be removed from the art by clas-
sical conservation techniques. In our previous study on enzymatic
degradation of melanin stains on paper, laccase and MnP were
tested for their ability to break down the pigmented fungal struc-
tures from the surface of the paper, with varying degrees of success
(Tavzes et al., 2009). The biomimetic Cuepyeperoxide system, with
its relatively small size, has a unique opportunity to deliver
oxidizing potential in close proximity to an aromatic polymer, i.e.,
melanin, even if this polymer is embeddedwithin cellulose fibers of
the paper and/or encapsulated in proteinaceous and carbohydrate
(chitin) hyphal cell walls.
This article reports on chemical changes induced in melanin as
a result of treatment with the Cuepy-peroxide system, and
bleaching of fungal melanin stains on paper brought about by
exposure to this system. The implications of these findings for
applied research in art conservation science are also discussed.
2. Materials and methods
Chemicals were purchased in p.a. grade (equivalent to reagent
grade) from Acros Organics, Fluka, and SigmaeAldrich, and used
without further purification. Potato dextrose agar (PDA) was
purchased from Difco, and malt extract broth (MEB) and soy
peptone from BectoneDickinson. Strains of Amorphotheca resinae
Parbery (syn. Cladosporium resinae Vries) and Sydowia polyspora
(Bref. & Tavel) E. Müll. (syn. Sclerophoma pithyophila (Corda) Höhn.)
were obtained from the fungal collection of the Department of
Wood Science and Technology, Biotechnical Faculty, University of
Ljubljana, Slovenia. In all experiments, water of MiliQ quality (miliQ
H2O, Millipore, USA) was used, with the exception of solid growth
medium preparation, where distilled water (dH2O) was used.
2.1. Preparation of experimental samples
2.1.1. Liquid DHN melanin suspensions
Themelanin-producing fungus S. polysporawas cultured on PDA
plates. The inoculum was transferred from the plates into 500-ml
Erlenmeyer flasks with 150 ml of nutrient medium (1 L of the
medium contained 30 g MEB, 5 g soy peptone, and 100 mM CuCl2
[final concentration]). The submerged culture was grown on a non-
rotary shaker at 100 shakes per minute for 8 days (28 C). After the
fermentation the extracellular DHNmelaninwas purified according
to a modified procedure described by Liu et al. (1995). The content
of the Erlenmeyer flasks was filtered through a Büchner funnel
(Whatman filter paper #4, F 125 mm). The filtrate was adjusted to
pH 3 with 6 M HCl and centrifuged for 1 h (8000 rpm, g ¼ 11500),
the supernatant was discarded, and the melanin was re-suspended
in miliQ H2O. The suspension was finally adjusted to pH 8
(10 M NaOH).
2.1.2. Melanized paper
Pieces of autoclaved filter paper (Whatman filter paper #2, F
55 mm) were put in Petri dishes, and each piece was inoculated
evenly with 1 ml of the suspension containing spores of A. resinae,
prepared by the procedure described above (Section 2.1.1). In
approximately two weeks, the paper was overgrown with themelanin-producing fungus (with its hyphae entangled in its
structure) and “blackened”. Blackened filter papers were dried in an
oven for 3 days at 60 C.2.2. Treatment procedures
2.2.1. Liquid DHN melanin suspensions
The samples were prepared with the aim of comparing the
influence on melanin decolorization of the biomimetic system and
its respective components, and different hydrogen peroxide
concentrations.
Each of the experimental samples (initial total volume 1 ml) con-
tained 300 ml of the extracellular melanin suspension (Section 2.1.1.).
The samples were supplemented by either water (700 ml miliQ H2O),
copper (10 ml 50mMCu(II)Cl2) and water (690 ml miliQ H2O), pyridine
and water (100 and 600 ml, respectively), or copper, pyridine and
water (10,100 and 590 ml, respectively)e the complete Cuepy system.
Hydrogen peroxide solutions (0.5, 1, or 3%) were continuously
administered to the samples with a Cole-Palmer 74900 syringe pump
(24 h; 4.17 ml h1). Residual peroxide in the reaction was measured
with QUANTOFIX Peroxide 100 (MachereyeNagel) test strips at each
measurement point.
Aside from the experimental samples, control (I) samples rep-
resented each combination of components, but instead of contin-
uously adding hydrogen peroxide, 100 ml of miliQ H2O was added
once every 24 h. Control (I) samples were used to eliminate the
influence of dilution on the results, due to the constant addition
of H2O2.
Control (II) samples were prepared representing the same
combinations of the system components, but without melanin.
They were used to determine the contribution of the system and its
components to absorbance in the UVeVIS range. The hydrogen
peroxide was added to these samples in the same manner as it was
to the experimental samples.
2.2.2. Melanized paper
Melanized and control filter papers were cut into squares
(10 10mm) andwere scanned prior to the decolorizationprocess, to
obtain the initial color value of the samples. They were put into
Eppendorf tubes (five samples each) containing1mlof thebiomimetic
systemor its respective components (eitherwater [1000mlmiliQH2O],
copper [10 ml 50mMCu(II)Cl2] andwater [990 ml miliQ H2O], pyridine
and water [100 and 900 ml, respectively], or copper, pyridine and
water [10,100 and 890 ml, respectively]). Hydrogen peroxide solutions
(3, 15, or 30%) were continuously administered to the treatment solu-
tions (24 h; 4.17 ml h1). Residual peroxide in the reaction was
measured with QUANTOFIX Peroxide 100 (MachereyeNagel) test
strips at each measurement point. After the treatment, samples were
removed fromtheEppendorf tubes, carefullywashed indistilledwater,
and oven-dried (3 days at 60 C).2.3. UVeVIS spectrometry of extracellular melanin
UVeVIS absorption measurements of experimental samples,
controls (I), and controls (II) were made with a Perkin Elmer -
Lambda 2 spectrophotometer (data interval 1 nm, measurement
speed 120 nm/min, measurement range 250e800 nm) and data
were gathered and processed with “Lambda 2” software (Perkin
Elmer, USA). Quartz spectroscopy cells (0.7 ml, Perkin Elmer) were
used for the measurements (reference and samples).
Measured absorbance of experimental samples was corrected
for the effects of dilution (due to the continuous addition of H2O2)
using the results of control (I), and the contribution of the system or
its components to the absorbance [control (II)] was subtracted.

C. Tavzes et al. / International Biodeterioration & Biodegradation 84 (2013) 307e313 3092.4. Chemical measurements
Liquid suspension samples were tested for any changes in their
chemical composition (appearance and relative representation of
functional groups and chemical bonds) with diffuse reflectance
FTIR spectroscopy (DRIFT). Before and after the treatment with the
biomimetic system or its components, 50 ml of the experimental
samples, controls (I), and controls (II) solutions were transferred
onto separate reflective-flake-covered abrasive pads (Perkin Elmer,
USA) and thoroughly dried with a fan at room temperature.
DRIFTmeasurements of the dried melaninwere performedwith
a Spectrum One spectrometer (Perkin Elmer, USA) using a TGS
detector at a spectral resolution of 4 cm1 and expressed as
apparent absorbance log10[1/R].
Each reflective-flake-covered abrasive pad was measured in 16
scans (400e4000 cm1) and an average spectrum was created
using Spectrum ONE software (www.PerkinElmer.com). These
average spectra were unit vector normalized using Opus software
(version 6.0, www.brukeroptics.com) in the spectral region
between 1850 and 750 cm1 and baseline-correction using
a rubber-band method was drawn in the respective region. The
amount of melanin modification was estimated by comparing the
relative intensity of relevant absorption bands in the fingerprint
region of the investigated spectrum. The assignment of bands
observed in the spectra to structural components (as determined
by other researchers) is provided in Table 1.2.5. Color changes in melanized paper samples
Changes in color of melanized paper samples were investigated
with a scanner (HP Scanjet G4050), using Corel Photo-Paint soft-
ware (Corel Corporation, version 8.232). Effectiveness of the
treatment (bleaching) was determined as a change in lightness (DL)
in the CIE L*a*b system (L* e lightness values from 0 (absolute
black) to 100 (absolute white); a* e a color value on red-green axis;
b* e a color value on yelloweblue axis).
Each melanized paper sample was measured for its color before
and after the treatment, and the difference in L values indicated the
effectiveness of the particular treatment. For better graphical
presentation, L (lightness) values were recalculated into “darkness”
values [darkness¼ 100 (L/99.6 100) %e the value 99.6 presents
maximal measured L value (non-melanized, control filter paper
samples)].Table 1
Infrared (FTIR) band identities of DHN melanin spectra. Assignment of bands
observed in the spectra to structural components.
Wavenumber
(cm1)
Assignment Sourcea
3440 OeH stretch 2,3
3300 NeH stretching 2
w2900 CeH stretching in methyl and methylene groups 2,3
1800e1740 C]O stretching in free carboxylic acids 4
1740e1730 C]O stretching in aliphatic aldehydes, ketones,
and carboxyls not conjugated with benzene ring
2,4
1670e1650 Amide I: C]O stretching in amides
Aldehydes, ketones, and carboxyls conjugated
with C]C or benzene ring, conjugated quinone
structures
2,4,5,6
1550 Amide II: CeN and NeH deformation in amides 2,4
1480e1350 CeH deformation vibrations of CH3 and CH2 2,4
1240 Phenolic CeO 1
1100e1050 Alcoholic CeO 1
a 1e Bilinska (1996); 2e Polanc and Stanovnik (1993); 3e Zink and Fengel (1990);
4e Gottwald and Wachter (1997); 5e Bode and Zeeck (2000); 6e Korytowski and
Sarna (1990).3. Results
3.1. Liquid DHN melanin suspensions
Melanin was oxidized in the liquid suspensions, and the inten-
sity of modification varied on the procedure employed. The most
pronounced changes in melanin were observed in the treatment
with the complete copperepyridine system with the continuous
administration of 3% hydrogen peroxide.
The first support for this statement is given in Fig. 1, where it is
shown that treatments with both pyridine-only and the complete
copperepyridine systemyielded a very good decrease of absorption
at 450 nm (UVeVIS measurements).
The DRIFT spectrum of isolatedmelanin from S. polyspora (Fig. 2)
shows a broad absorption band with a maximum between 3460
and 3300 cm1, where bands assigned to the stretching vibrations
of free OeH and NeH occur. Near 2920 cm1 a more differentiated
region of stretching vibrations of various CeH groups is situated. In
the fingerprint region, prominent bands are due to C]O stretching
(1738 cm1 due to carbonyls and 1600 cm1 due to amidese Amide
I and/or aromatic C]C double bonds). A weaker Amide II band
(shoulder) is found at 1550 cm1. An Amide III band, expected as
a very weak absorption between 1430 cm1 and 1395 cm1, was
not found, due to the stronger absorption of various CeH vibrations
at 1400 (methylene scissoring) and 1455 cm1 (aliphatic CeH
deformations). Phenolic groups (CeO) of DHN melanin absorb
near 1240 cm1. Finally a strong region of bands due to CeO
deformation vibrations of aliphatic alcohols was found between
1100 and 1050 cm1 (Table 1).
Figs. 2e5 show the chemical changes (measured by DRIFT) that
occurred in the melanin during the various treatments. In Fig. 2, the
relative decrease of the absorption bands, assigned toOeH,NeH, and
CeHbonds, can be observed in the overall mid-IR absorption spectra.
The higher the concentration of the continuously administeredH2O2,
the more pronounced differences in the spectra were observed.
Fig. 3 shows the same spectra, but only in the so-called fingerprint
region (1800e800 cm1). It can easily be discerned that the bands in
the melanin spectra, assigned to less-oxidized bonds (the region
between 1450 and 1350 cm1 e CeH; the region around 1050 cm1
e OeH of primary alcohols), become much less prominent after the
treatment with the higher (1% and even more 3%) concentration of
the H2O2 solution. Additionally, the band assigned to the aromatic
ring-conjugated carbonyls (1700 cm1) is replaced with that0%
10%
20%
30%
40%
50%
60%
70%
80%
90%
100%
Day 1 Day 2 Day 3 Day 4 Day 5
R
e
l
a
t
i
v
e
 a
b
s
o
r
b
a
n
c
e
 (
4
5
0
 n
m
)
A
B
C
D
Fig. 1. Relative absorbance (at 450 nm) of extracellular melanin after treatment with
(3% solution, 4.17 ml h1) H2O2 only (A), H2O2 and copper (B), H2O2 and pyridine (C),
and H2O2 and the complete copperepyridine system (D).
0.00
0.04
0.08
0.12
0.16
8001050130015501800
N
o
rm
a
lis
e
d 
lo
g 1
0
(1/
R)
Wavenumber [cm-1]
Day 1 Day 2 Day 3
Day 4 Day 5 Melanin Control
C=O
conj.
C=O
C-H
C-O
Fig. 4. FTIR (DRIFT) spectra of melanin (fingerprint region), treated with the complete
copperepyridine system and 3% H2O2 solution for different time periods (1e5 days).
0.00
0.04
0.08
0.12
0.16
800130018002300280033003800
N
o
rm
a
lis
e
d 
lo
g 1
0
(1/
R)
Wavenumber [cm-1]
A B C Melanin Control
C-H
O-H, N-H
IR-fingerprint
Fig. 2. FTIR (DRIFT) spectra of melanin treated for five days with the complete coppere
pyridine system and increasing concentrations of H2O2 (0.5% e A, 1% e B, and 3% e C).

C. Tavzes et al. / International Biodeterioration & Biodegradation 84 (2013) 307e313310assigned to non-conjugated carbonyls (1730e1770 cm1). All these
differences in the spectra indicate that a strong oxidation was taking
place on the melanin molecules during treatment with the Cuepy
biomimetic system and the continuously administered H2O2.
It is important to note that measurements of excess H2O2 (data
not shown) showed that the continuous administration of 3% H2O2
solution was optimal, as there was no excess H2O2 accumulation
measured, and the Cuepy systemwas fully functional. Overall, even
after five days of continuous administration of H2O2, the highest
concentration of residual H2O2 measured was 0.01%, with the
typical concentration being 0.003%.
Very similar differences in the treated melanin spectra were
observed if the variable in the treatment was not the concentration
of continuously administered H2O2, but the duration of addition of
3% H2O2 (Fig. 4). Again, it is clear that the prominence of the differ-
ences in the spectra is correlated to the amount of added H2O2.0.00
0.04
0.08
0.12
0.16
8001050130015501800
N
or
m
a
lis
e
d 
lo
g 1
0
(1/
R)
Wavenumber [cm-1]
A B C Melanin Control
C-H
C-O
conj.
C=O
C=O
Fig. 3. FTIR (DRIFT) spectra of melanin (fingerprint region), treated for five days with
the complete copperepyridine system and increasing concentrations of H2O2 (0.5% e
A, 1% e B, and 3% e C).Fig. 5 shows that when the melanin was treated with continu-
ously administered H2O2 and the full biomimetic system, and not
with the pyridine alone, the differences in the spectra were much
more pronounced. This shows that although the treatment with
continuously administered H2O2 and pyridine only does cause
substantial oxidation (and thus discoloration of the melanin), these
changes were much greater when the whole biomimetic system
was used (Fig. 5).3.2. Melanized paper
The superior efficacy of the complete biomimetic system can also
be seen in Fig. 6, where the treatment effect on lightening of the
melanized paper samples is shown. Compared to treatment in the
presence of only pyridine, bleaching of melanin staining appeared
after fewer additions of hydrogen peroxide (expressed in days of
continuous administration); this happened also when the treatment0.00
0.04
0.08
0.12
0.16
8001050130015501800
N
o
rm
a
lis
e
d 
lo
g 1
0
(1/
R)
Wavenumber [cm-1]
C D Melanin Control
C-O
C-H
conj.
C=O
C=O
Fig. 5. FTIR (DRIFT) spectra of melanin (fingerprint region), treated with 3% H2O2
solution and e pyridine only (C) or the complete copperepyridine system (D).
Fig. 6. Melanized paper samples, treated with continuously administered H2O2 (3 or 30%) alone (MEL), or in the presence of only copper (MEL þ Cu), only pyridine (MEL þ Py), or
the complete biomimetic system (MEL þ Cu þ Py); non-melanized paper samples were treated in the presence of the biomimetic system (Cu þ Py) as control.
0%
20%
40%
60%
80%
100%
Day 1 Day 2 Day 3 Day 4
pe
rc
en
ta
ge
  “
da
rk
ne
ss
”
 
v
al
ue
 
A
B
C
D
Fig. 7. Percentage decrease of the “darkness” value of melanized paper samples;
the samples were treated with continuously administered 3% H2O2 solution alone (A),
or in the presence of only copper (B), only pyridine (C), or the complete biomimetic
system (D).

C. Tavzes et al. / International Biodeterioration & Biodegradation 84 (2013) 307e313 311was with a lower concentration of H2O2, and was generally much
more pronounced. In other samples, the bleaching occurred only at
the higher concentration of the oxidant, was much less intense, and
took longer to appear (Fig. 6). The treatment did not have any
influence on the color of control, non-melanized paper samples.
Melanized paper samples were also measured for color in the
CIELAB system, before and after treatment. Lightening of the samples
(degradation of melanin staining) was expressed as the percentage
decrease of the “darkness” value (the higher the L value, the lighter
the sample, the lower the “darkness” value). In Figs. 7e9, it can be
seen that the lower concentration (3%) of continuously administered
H2O2 did not have the desired effect (thorough bleaching of the
melanized samples) (Fig. 7). The highest concentration (30%) ach-
ieved complete bleaching (Fig. 9), but so did the middle concentra-
tion (15%) of continuously administered H2O2 (Fig. 8). However, in
the latter case, the treatment was more targeted, as pyridine alone
achieved less bleaching than the complete biomimetic system.
Therefore, treatment with the middle concentration (15%) of
continuously administered H2O2 in the presence of the complete
biomimetic system was chosen as most appropriate for the removal
of melanin staining on paper substrates (Fig. 8).
As in the liquidmelaninsuspensions,measurementsofexcessH2O2
(data not shown) showed that the continuous administration of 15%
H2O2 was optimal, as there was no excess H2O2 accumulation and the
Cuepy system was fully functional. The highest concentration of
residual H2O2 measured in any of the treatment solutions was 0.01%.
4. Discussion
4.1. Liquid DHN melanin suspensions
The prominent decrease of UVeVIS absorbance of the melanin-
containing reaction solutions to just above 10% of the initial value
indicated that Cuepy system/H2O2 treatment caused strong
chemical change and/or degradation of the extracellular melanin
polymer.The DRIFT spectrum of S. polyspora melanin is comparable to IR
spectra of isolated microbial melanins before acid hydrolysis (Zink
and Fengel, 1990) and to A. resinae pigment (Tavzes et al., 2009).
However, it exhibits weaker IR absorptions at bands assigned to
amide-bound nitrogen, which is not incorporated in pure DHN
melanin, than the melanin of A. resinae. Nevertheless, this was
sufficient to reconfirm that fungi contain not only DHNmelanin but
also significant amounts of protein, or that proteins are associated
with fungal melanins.
The intensity of the chemical change in the melanin due to the
activity of the CuepyeH2O2 systemwasmuch higher in the present
study than previously reported by this group for enzymatic treat-
ment of this fungal pigment (Tavzes et al., 2009), although a very
high efficacy of HBT as laccase mediator (LMS) is known (Thurston,
1994; Yaropolov et al., 1994; Call and Mücke, 1997; Böhmer et al.,
0%
20%
40%
60%
80%
100%
Day 1 Day 2 Day 3 Day 4 Day 5
pe
rc
en
ta
ge
  “
da
rk
n
es
s”
 
v
al
u
e 
A
B
C
D1
D2
Fig. 8. Percentage decrease of the “darkness” value of melanized paper samples; the
samples were treated with continuously administered 15% H2O2 solution alone (A), or
in the presence of only copper (B), only pyridine (C), or the complete biomimetic
system (D1 and D2).

C. Tavzes et al. / International Biodeterioration & Biodegradation 84 (2013) 307e3133121998; Cantarella et al., 2003). This is true for laccase/HBT treat-
ments (which resulted in more prominent changes of melanin than
all other enzymatic treatments), as well as for different MnP
melanin treatments.
There are also qualitative differences among the three (laccase,
MnP, and CuepyeH2O2) melanin oxidizing systems. Treatment
with laccase/HBT caused a general absorbance increase of the C]O
attributed band, but the absorbancemaximumwas shifted to above
1755 cm1, indicating the increased presence of free carboxylic acid
groups. However, unlike in the laccase/HBT system, the band shift
to higher wavenumbers did not occur at either MnP (Tavzes et al.,
2009) or in the present experiment with the CuepyeH2O2
system, indicating less intensive formation of free carboxylic
acids. As a consequence, oxidation by these two systems resulted in
a reduction of bands attributed to aliphatic structures, and alcoholic
and phenolic groups. This similarity between the MnP and Cuepye
H2O2 system in melanin oxidation further indicates that highly
oxidative reaction intermediates of the reaction of the Cuepy
complex with H2O2 mimic the oxidative intermediates of fungal
peroxidases (Fackler et al., 2001).
4.2. Melanized paper
As with degradation of the extracellular melanin, the biomi-
metic system had a much greater effect on melanized paper0%
20%
40%
60%
80%
100%
Day 1 Day 2 Day 3 Day 4 Day 5
pe
rc
en
ta
ge
  “
da
rk
n
es
s”
 
v
al
u
e 
A
B
C
D1
D2
Fig. 9. Percentage decrease of the “darkness” value of melanized paper samples; the
samples were treated with continuously administered 30% H2O2 solution alone (A), or
in the presence of only copper (B), only pyridine (C), or the complete biomimetic
system (D1 and D2).bleaching than did isolated laccase (even with the mediator HBT).
Although laccase could change the appearance of melanized paper
in the desired direction via degradation and oxidation of DHN
melanin, colorimetric measurements with the CIELAB system
showed that this change was not higher than 10% of the L value
(Tavzes et al., 2009). Conversely, CuepyeH2O2 system treatment
easily achieved total lightening of the samples. Furthermore, even
separate components of the system accelerated H2O2 degradation
of the melanin, albeit not as successfully as in the treatment with
the complete system.
It is also very encouraging that a thorough visual inspection of
treated melanized paper samples did not reveal any significant
damage to the integrity of the paper as a consequence of the
treatment, regardless of the procedure employed. It was reported
previously that the oxidative reaction intermediates are highly
selective for aromatic structures, oxidizing aromatic lignin struc-
tures selectively and not affecting cellulose (Rahmawati et al.,
2005). The near-neutral conditions (pH ca. 8.0) and ambient
temperature used in this experiment also prevent swelling of the
cellulose and end-group oxidation [peeling reactions (Fackler et al.,
2001)]. However, even in conditions similar to those used in paper
pulp processing, the biomimetic copperepyridine complex did not
damage the cellulose molecules as significantly as other processes
(Watanabe et al., 1997).
5. Conclusions
The CuepyeH2O2 treatment procedure looks very promising
for the removal of fungal melanin staining on paper, but it needs
to be optimized and paper integrity, possible oxidative damage to
paper samples, and degree of polymerization of cellulose (and
other indicators of possible degradation) after the procedure
should be carefully evaluated. After scientifically verifying that this
melanin degradation procedure does not cause damage to the
paper and that it fulfils paper conservators’ requirements, the
procedure will be further developed. These procedures could be
readily tested also for applications on other materials such as
stone (no dissolution of stone expected due to near neutral pH of
the reaction solutions), wood, and textiles, all of which may be
negatively affected by classical conservation procedures for
melanin staining removal.
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