ORIGINAL PAPER

Gellan gum hybrid hydrogels for the cleaning of paperartworks contaminated with Aspergillus versicolor

Giovanni De Filpo . Anna Maria Palermo .

Riccardo Tolmino . Patrizia Formoso .

Fiore Pasquale Nicoletta

Received: 2 March 2016 / Accepted: 27 July 2016 / Published online: 1 August 2016

� Springer Science+Business Media Dordrecht 2016

Abstract The degradation of archive materials is

related to irreversible phenomena induced by light,

temperature, humidity, air pollution, micro-organ-

isms, and use. Among biological factors, fungi can

induce harmful effects in paper artworks. Further

forms of damage (e.g. artwork swelling, fibre lifting

and sheet delamination) can be caused by water

immersion, which is one of the most commonly used

methods for cleaning paper. To avoid damage it is

necessary to control the amount and absorption rate of

water by paper. Recently, gellan gum hydrogels have

been proposed as effective tools to allow contaminant

removal from paper supports, owing to the controlled

water release and adhesive properties of gellan gum.

In this study hybrid hydrogels were fabricated by

doping gellan gum either with calcium compounds

(calcium sulphate, hydroxide, chloride, and acetate) or

titanium dioxide nanoparticles in order to evaluate

their ability in cleaning different types of paper

samples affected by spots originating from Aspergillus

versicolor. The best decolourization results were

obtained by calcium acetate/gellan gum hydrogels

and titanium dioxide nanoparticle/gellan gum hydro-

gels, while no synergistic effect was found in paper

samples treated with calcium acetate/titanium dioxide/

gellan gum hydrogels. Hybrid hydrogels were tested

on a case-study book.

Keywords Hydrogels � Gellan gum � Aspergillusversicolor � Fungi � Photo-catalysis � Titanium dioxide

Introduction

Paper deterioration

Paper undergoes changes over time, due to modifica-

tions in the molecular structure of its components

(fragmentation and weakening of the polymer chains)

by chemical, physical and biological agents. Ageing is

a natural phenomenon and depends on several factors

including the raw materials (e.g. cellulose, hemicel-

lulose, lignin), the use of additives (e.g. alum, rosin,

dyes, fillers, heavy metals), and manufacture and

storage conditions (delignification, bleaching, pres-

ence of microorganisms, insects, rodents and pollu-

tants, and unsuitable humidity, temperature or light

intensity) (Zervos 2007; Zervos and Alexopoulou

2015).

G. De Filpo (&)Dipartimento di Chimica e Tecnologie Chimiche,

Università della Calabria, 87036 Rende, CS, Italy

e-mail: defilpo@unical.it

A. M. Palermo � R. TolminoDipartimento di Biologia, Ecologia, e Scienze della Terra,

Università della Calabria, 87036 Rende, CS, Italy

P. Formoso � F. P. NicolettaDipartimento di Farmacia e Scienze della Salute e della

Nutrizione, Università della Calabria, 87036 Rende, CS,

Italy

123

Cellulose (2016) 23:3265–3279

DOI 10.1007/s10570-016-1021-z

http://crossmark.crossref.org/dialog/?doi=10.1007/s10570-016-1021-z&domain=pdfhttp://crossmark.crossref.org/dialog/?doi=10.1007/s10570-016-1021-z&domain=pdf

Biodeterioration is ‘‘any unwanted change in the

properties of a material caused by the activity of vital

organisms’’ (Hueck 1968). In the case of paper

documents, this change may include both irreversible

transformations of the substrate and aesthetic damage,

which can prevent the correct reading/observation of

the document/artwork. The biodeterioration of paper

is caused by the mechanical and chemical action of

biodeteriogens, e.g. microalgae, bacteria, fungi,

insects.

The mechanical action of some fungi can cause

either micro- or macro-damage, while their metabolic

processes may result in the appearance of very

differently coloured spots/stains, the felting of sup-

ports and an odour change (pungent smell) (Gu-

tarowska et al. 2012). The temperature and relative

humidity affect the metabolism of micro-organisms by

influencing the kinetic rates of chemical and enzy-

matic reactions. High values of temperature and

relative air humidity (20 �C\T\ 45 �C and RHlarger than 60 %) favour the development of fungi.

These microorganisms can cause severe damage to

paper documents as they are able to produce enzymes

capable of hydrolysing a wide variety of natural

polymers, including cellulose (Silva et al. 2006).

The biocidal activity of titanium dioxide

The first research on the photocatalytic destruction of

fungi, specifically the yeast Saccharomyces cerevisiae

Meyen, was carried out by Matsunaga et al. (1985).

The antifungal activity of titanium dioxide has been

examined intensively on other fungal species, namely

Candida albicans (C.P. Robin) Berkhout, Penicillium

expansum Link, Diaporthe niger N.F. Sommer &

Beraha, Aspergillus niger Tiegh, Fusarium solani

(Mart.) Sacc., F. anthophilum (A. Braun) Wollenw, F.

equiseti (Corda) Sacc., F. oxysporum Schltdl., F.

verticillioides (Sacc.) Nirenberg and Penicillium

chrysogenum Thom (Chen et al. 2009; Sichel et al.

2007).

The physical principle of this method is based on

the ability of some semiconductors to accelerate the

radical oxidation of organic substances in the presence

of light (photocatalysis) (De Filpo et al. 2010). The

photocatalytic processes in which a semiconductor

catalyst, e.g. titanium dioxide (TiO2), is involved

when, in contact with water and oxygen, it is irradiated

by photons of suitable energy (i.e. larger than its

bandgap), can be summarized according to the

following reactions:

TiO2 þ hm ! e�cb þ hþvbO2 þ e�cb ! O��2H2Oþ hþvb ! OH� þ Hþ

O��2 þ H2O ! H2O2 ! 2OH�

OH� þ OC ! OCoxOCþ e�cb ! OCred

Here a titanium dioxide molecule absorbs a photon of

energy hm, creating a free electron and electron holepair (ecb

- ? hmb? ) (Hoffmann et al. 1995). Both electron

and hole move to the semiconductor surface and

produce reactive oxygen species, such as O2-� and OH�,

which can oxidize organic compounds (OH� ? -

OC ? OCox), whereas the electrons can reduce them(OC ? ecb

- ? OCred). It is known that some organiccontaminants can be removed if suitably oxidized or

reduced (De Filpo et al. 2015).

Titanium dioxide nanoparticles are considered an

ideal photocatalyst, as they are readily available,

inexpensive, and have a large surface area; however,

they present some difficulties for eventual recovery.

For this reason, titanium dioxide nanoparticles are

generally immobilized on substrates, such as polymer

matrices, glasses, steels, and hydrogels (Nicoletta

et al. 2012).

The TiO2-generated hydroxyl radicals (OH�) and

superoxide anions (O2-�) can inactivate bacteria,

viruses, fungi, and algae by either the photochemical

oxidation of intracellular coenzyme A (Matsunaga

et al. 1988) or damage to the cell membrane (Sunada

et al. 1998) and its polyunsaturated phospholipids

(Gutteridge 1987; Maness et al. 1999).

More recently, it has been found that titanium

dioxide nanoparticles prevent the biodeterioration of

mortars in cultural heritage buildings (Fonseca et al.

2010), inhibit the A. niger colonization of limestone

and Carrara marble (La Russa et al. 2012), and show

interesting antifungal and biocidal properties on wood

(De Filpo et al. 2013) and parchment (De Filpo et al.

2015).

In addition, a cellulosic nanocomposite of TiO2 was

used as a protective and consolidative coating on the

surface of paper fibres. This layered nanocomposite

could protect works of art on paper from the damaging

3266 Cellulose (2016) 23:3265–3279

123

effects of UV light, air pollutants, mould and bacteria.

The reversibility of the consolidation treatment was

tested on blank sheets of paper by washing the

coatings with ethanol (Afsharpour et al. 2011).

Standard methods for cleaning and disinfecting

paper

Fungi play a considerable role in the deterioration of

cultural heritage, as their enzymatic activity induces

fast decay in the raw materials (e.g. paper, leather,

parchment) of historical art objects. In paper conser-

vation, fungi represent an important problem due to

their ability to excrete cellulases, i.e. enzymes that

catalyse the decomposition of cellulose and of some

related polysaccharides into monosaccharides (Ster-

flinger 2010). Consequently, fungal colonization can

result in serious damage to paper documents, such as

weakening, brittleness, discolouration (due to weak

acids produced by fungi) and foxing (due to the

accumulation of reddish and/or brownish stains) (Arai

2000; Michaelsen et al. 2006).

The best way to prevent fungal contamination is to

keep the rooms clean and maintain appropriate

conditions of temperature and humidity (below

55 %) (Valentin 2007; Sterflinger 2010). When con-

tamination is already present, disinfection by physical

or chemical methods is necessary to fight the infection.

Different cleaning methods are available for paper

depending on the particular stains. The mechanical

removal of dust particles and dirt spots (by soft

brushes and cotton pads if they are located on the

support surface, or by scalpels, soft erasers and

brushes with different degrees of hardness if they are

located at depth) is a dry cleaning method, which

avoids possible swelling and bending due to the

absorption of water.

Laser cleaning technology has some advantages

over conventional cleaning methods, being a contact-

less and chemical-free technique (Friberg et al. 1997).

Stains and spots from foxing are generally removed

by washing the paper with demineralized water or with

detergent solutions, or by decolourization using solu-

tions of hydrogen peroxide, acetic acid, oxalic acid,

ammonia, potassium perborate, or sodium and cal-

cium hypochlorite, according to the stain type.

Chemical reactions involving the formation of acids

are the most harmful processes of biodeterioration for

paper, as they can seriously damage the structure of

cellulose (Arai 2000). The optimal pH of paper is

around 7.5, while at a pH below five paper deteriorates

in a short time (Tang 1981). In these cases aqueous de-

acidification is carried out by washing the paper with

basic solutions.

All the methods described in the previous para-

graph are wet treatments and are not always suit-

able for paper artworks. Therefore, in recent years,

researchers have looked for new cleaning and disin-

fection methods in order to achieve a more selective

and less invasive recovery of paper artworks and

books.

In addition to the mechanical removal of biodete-

riogens from paper artefacts, the most commonly used

methods for paper disinfection include chemical and

physical methods. As recently reviewed (Sequeira

et al. 2012), chemical methods can be grouped into

treatments using alcohols, alkylating agents, azole

antifungals, essential oils, phenol derivatives, photo-

catalysts, quaternary ammonium compounds, salts,

and esters of acids. Fumigants, such as ethylene oxide,

have been widely used for the inactivation of

microorganisms in cultural heritage preservation

(Mendes et al. 2007). Sterilization of paper with

ethylene oxide is an effective, but not long-lasting

treatment. In fact, subsequent contamination is possi-

ble as ethylene oxide does not remain as a preventive

biocide on materials (Michaelsen et al. 2006). Never-

theless, being an alkylating agent, ethylene oxide has

been banned in many countries for its carcinogenic

potential (NTP 2014).

The vapours of essential oils can exhibit antifungal

activity against the moulds commonly found on

library and archival materials. It has been shown that

the activity of linalool is fungistatic rather than

fungicidal. Linalool vapours do not affect the bright-

ness of paper or the degree of polymerization of

cellulose, but do reduce the pH of paper (Rakotoni-

rainy and Lavédrine 2005).

Physical methods include dehydration, gamma

irradiation, high-frequency current, low-oxygen envi-

ronments, ultraviolet radiation and temperature

extremes. Alternative techniques are based on silver

nanoparticles (Shirakawa et al. 2013). Gamma radia-

tion at doses ranging between 3 and 15 kGy is highly

effective in the decontamination of fungi, without

causing significant damage to supports such as cellu-

lose depolymerization, increased yellowing, or gen-

eral embrittlement (Adamo and Magaudda 2003).

Cellulose (2016) 23:3265–3279 3267

123

Freeze-drying of paper is a conservation method in

which water is frozen and then removed by sublima-

tion, thus killing conidia and stopping the growth of

fungi and bacteria (Florian 2002). Nevertheless, these

techniques can only be considered decontamination

treatments.

More recently, gellan gum rigid hydrogels have

been investigated as cleaning tools for works of art on

paper (Iannuccelli and Sotgiu 2010). The basic

principle of paper-cleaning by gellan gum hydrogels

is the controlled release of water from gel to paper and

the ability of gellan gum to absorb the surface deposits

and contaminants responsible for the acidic degrada-

tion of paper. The cleaning by gellan gum hydrogels is

based on the trapping and removal of the contaminat-

ing organic material (including hyphae and spores)

by the gel’s three-dimensional network. Gellan

gum is a water-soluble and high-molecular-weight

heteropolysaccharide. It is used as a gelling agent in

biomedical, pharmacological and food applications.

Gellan gum hydrogels are able to retain and control-

lably release large amounts of water. They are stable in

a wide pH range and can be prepared with different

degrees of viscosity (soft and rigid gels, according to

use) by a simple change of gellan gum concentration.

In addition, gellan gum hydrogels can be used in the

restoration and conservation of paper and parchment

artefacts. In fact, since 2003 ICRCPAL, the Italian

Institute for the Restoration and Conservation of

Papers and Books in Rome, has developed wet

cleaning treatments for works of art on paper based

on the use of rigid gellan gels. This technique is a valid

alternative to conventional aqueous solutions, which

can cause irreversible changes in the substrates, such

as discolouration, deformation and fragility. In fact,

the gradual and controlled release of water molecules

from the gel to the paper is less invasive than

immersion or buffing methods, and allows the removal

of the degrading substances according to a process of

concentration gradient, without causing any morpho-

logical changes to the paper. In addition, gellan gum

hydrogels are characterized by easy application and an

effective cleaning action, which guarantees the struc-

tural and aesthetic preservation of paper supports with

no residue on the samples during the treatment and no

rinsing after application (Iannuccelli and Sotgiu

2010).

More recently, the use of gellan gum/titanium

dioxide nanoparticle hybrid hydrogels proved an

effective method to both clean and disinfect parch-

ment contaminated by P. chrysogenum and Cladospo-

rium cladosporioides (Fresen.) G.A. de Vries (De

Filpo et al. 2015).

In the present study, the cleaning properties of pure

gellan gum, gellan gum/de-acidifying substances and

gellan gum/titanium dioxide nanoparticle hybrid

hydrogels on paper samples contaminated by Asper-

gillus versicolor (Vuillemin) Tiraboschi were inves-

tigated. It was expected that the cleaning action of

hydrogels combined with either the de-acidifying

properties of calcium compounds or the photo-

catalytic properties of titanium dioxide nanoparticles

could efficiently clean paper samples containing spots

caused by fungal contamination. In addition, photo-

catalytic disinfection and protection against fungal re-

colonization were expected in samples treated with

gellan gum/titanium dioxide nanoparticle hybrid

hydrogels. In order to explore possible synergistic

effects, paper samples were treated with gellan gum

hybrid hydrogels containing both de-acidifying com-

pounds and titanium dioxide nanoparticles. The hybrid

hydrogels that gave the best results were tested on a

case-study book.

Materials and methods

Paper characterization

The following three types of paper were used:

A. MUNKEN PRINT, which is a wood-free What-

man paper from sustainable forests with low ash

content (Munken Print by Artic Paper, Munkedal,

Sweden).

B. RECYCLED PAPER, which is a high quality

recycled paper, certified FSC, with 60 % recycled

fibres and 40 % recycled post-consumer fibres

(Freelife Cento 70x100 LL80 by Cartiere Fedri-

goni, Verona, Italy).

C. ARCOSET EDITION 1.3, which is made with

eco-friendly paper bleached without the use of

chlorine (Arcoset 70x100 LL80 by Cartiere

Fedrigoni, Verona, Italy).

The sample size was 2 9 2.5 cm2. In addition, the

third page of a textbook (printed in 1953 and

deteriorated by foxing from A. versicolor) was taken

3268 Cellulose (2016) 23:3265–3279

123

into account as a case study. A. versicolor (sequence

JN997427, GenBank) was determined as the culprit of

foxing by DNA sequence analysis (Jurjevic et al.

2012).

The pH of paper aqueous extract was determined

according to the procedure reported by ASTMMethod

D-778 and confirmed by a portable pH meter with a

flat-tip probe designed to optimize surface contact

with leather and paper (HI99171, Hanna Instruments,

Inc., Woonsocket, RI, USA). Briefly, 5 g of paper (cut

into small pieces of about 1 cm2) was boiled in 100 ml

of distilled water for 1 h. Then, the dispersion was

cooled to room temperature (T = 20 �C) and the pHwas measured.

The paper composition was characterized by

Herzberg’s reagent (Houck 2009). Fibres treated with

Herzberg’s reagent are differently coloured according

to their nature. In particular, a purple-red colour

indicates the presence of flax and/or bleached hemp,

while a blue colour is due to pulp flocks, and a yellow

colour reveals the presence of pulp, jute, hemp, and

generally lignin-rich fibres.

Fungal inoculation

The biodeteriogen was A. versicolor from the stock

culture collection of the Laboratory of Plant Biosys-

tematics at the University of Calabria. Although there

are many species frequently found on paper, such as A.

niger, Chaetomium globosum Kunze, C. cladospori-

oides and P. chrysogenum, A. versicolorwas chosen as

it was found to have contaminated the pages of the

case-study book.

The fungal species was grown in solid culture

medium (Malt Extract Agar) in a climatic chamber at

25 �C for 7 days. Once the microorganisms haddeveloped, the conidia were collected and stored in a

saline solution of 0.05 % NaCl. The conidial concen-

tration was 11.3 9 104 conidia/ml as evaluated by a

Thoma counting chamber. Paper samples were placed

in Petri dishes with no addition of any nutritional

substance, in order to ensure microbial growth at the

expense of the paper. Inoculation was performed by

micro-deposition of 50 ll drops of conidia solutionover the sample surface. All samples in Petri dishes

were covered by plastic lids and kept at 25 �C in athermostatic cell with a relative humidity of 80 %.

Two weeks after inoculation, optical microscope

observations (LaborLux 12 POL, Leica, Wetzlar,

Germany) showed the presence of both hyphae and

conidiophores in the specimens.

Then, the paper samples were subjected to various

cleaning test procedures as listed in Table 1, observed

by stereo and optical microscopy, and placed in a

climatic chamber with controlled temperature and

humidity (T = 25 �C and RH = 80 %) for a further15 days to allow possible fungal re-growth. The

biostatic/biocidal action was judged based on the

observations after cleaning and incubation in the

climatic chamber for 15 days. Similar tests were

performed under the harsher conditions of T = 30 �Cand RH = 80 %.

The pictures displayed in the following figures rep-

resent illustrative examples of the images taken with

the optical microscope.

Hydrogel preparation

Hydrogels were prepared from pure gellan gum

solutions (Gelrite�, Sigma-Aldrich, Milan, Italy) and

gellan gum solutions doped with either calcium

compounds (calcium sulphate, hydroxide, chloride,

and acetate, Sigma-Aldrich, Milan, Italy) or titanium

dioxide nanoparticles (P25, Evonik, Essen, Germany)

in order to evaluate their ability in cleaning different

types of paper samples containing spots. Hydrogels

were labelled Gel n, with n ranging from 1 to 7 (see

Table 1), and their thickness was about 5 mm.

Specifically, Gel 1 was simply a pure gellan gum

hydrogel (without any added compound), Gels 2–5

were gellan gum hydrogels doped with calcium

compounds (calcium sulphate, hydroxide, chloride,

and acetate, respectively), Gel 6 was a pure gellan gum

hydrogel (Gel 1) doped with titanium dioxide

nanoparticles and Gel 7 was a calcium acetate-loaded

gellan gum (Gel 5) doped with TiO2 nanoparticles.

Obviously, gellan gum hydrogels must be solid

enough to ensure a controlled water release and easy

manipulation, with no residue on the samples during

the treatments. In previous investigations on parch-

ment (De Filpo et al. 2015), the ideal weight concen-

tration of gellan gum to be used in the solutions was

found to be 3 wt.%. Lower concentrations of gellan

gum gave soft hydrogels with consequent manipula-

tion difficulties and residues on samples, while higher

concentrations of gellan gum resulted in too-rigid

hydrogels with poor cleaning properties. The contact

time between samples and hydrogels was no longer

Cellulose (2016) 23:3265–3279 3269

123

than 1 h in the present study, in order to avoid

excessive water release that could damage the paper.

Gel 1 was prepared in order to compare the cleaning

action of pure gellan gum hydrogels with those of

gellan gum/de-acidifying compounds and gellan gum/

titanium dioxide nanoparticle hybrid hydrogels.

Hydrogels doped with different calcium salts were

prepared using the salt solutions reported in the

literature (Placido 2012). Gellan gum/TiO2 nanopar-

ticle hybrid hydrogels were prepared by placing TiO2nanoparticles in their powder form on the upper face of

the pure gellan gum hydrogels before their complete

gelling (16 mg of TiO2 per g of hydrogel, see Fig. 1).

In this way, hybrid hydrogels with a high loading of

nanoparticles were obtained.

Even if the best cleaning results with gellan gum

gels doped with calcium compounds were obtained by

Gel 3, its strong base characteristics (pH 11.4) did not

suggest the use as a deacidificant tool. So, Gel 7 was

prepared by addition of TiO2 to Gel 5, which is

characterized by a slightly alkaline pH (7.4).

All hybrid hydrogels were stable against 10

swelling–deswelling cycles, and the reproducibility

in their preparation was confirmed by the relatively

constant value of their equilibrium swelling ratio.

In tests involving TiO2 nanoparticles, gels were

UV-irradiated for 5 min (k = 380 nm, power = 10mW/cm2, HPK 125, Philips, Amsterdam, Nether-

lands) in order to activate titanium dioxide. Paper

samples, previously inoculated and incubated with A.

versicolor, were covered with activated gels for 1 h.

Then, hydrogels were gently removed and samples

observed under the optical microscope. The gellan

gum hydrogels were almost transparent to radiation in

the Vis and near-UV regions, and the penetration

depth of the activating radiation was estimated to be

about 1 cm.

Table 1 Chemical composition of cleaning systems and their average cleaning action and biocidal/biostatic activity (±SD)

Gel id Concentration of de-acidifying compound TiO2 Cleaning action (%) Biostatic/biocidal activity (%)

Gel 1 0 0 64 0

Gel 2 [CaSO4] = 40 mg L-1 0 56 0

Gel 3 [Ca(OH)2] = 40 mg L-1 0 85 0

Gel 4 [Ca(Cl)2] = 40 mg L-1 0 52 0

Gel 5 [Ca(CH3COO)2] = 400 mg L-1 0 72 0

Gel 6 0 16 mg/g 90 100

Gel 7 [Ca(CH3COO)2] = 400 mg L-1 16 mg/g 93 100

The concentration of gellan gum water solution was 3 wt.%. Gel 1 was simply a pure gellan gum hydrogel (without any added

compound), Gels 2–5 were gellan gum hydrogels doped with calcium compounds (calcium sulphate, hydroxide, chloride, and acetate,

respectively), Gel 6 was a pure gellan gum hydrogel (Gel 1) doped with titanium dioxide nanoparticles and Gel 7 was a calcium

acetate-loaded gellan gum (Gel 5) doped with TiO2 nanoparticles

(a) (b)

(c)

Fig. 1 a Top view of a gellan gum hydrogel (Gel 1); b top viewand c cross section of a gellan gum hydrogel loaded withtitanium dioxide nanoparticles (16 mg of TiO2/g of hydrogel,

Gel 6). The upper side (that rich in TiO2 nanoparticles) of Gel 6

was placed on the contaminated paper samples. Typical

hydrogel dimensions: length 5 cm, width 5 cm, and height

0.5 cm. The bar size is: a 0.03 cm, b 0.03 cm and c 0.1 cm,respectively

3270 Cellulose (2016) 23:3265–3279

123

Evaluation of cleaning action and biostatic/

biocidal activity

The cleaning action and biostatic/biocidal activity

were evaluated using image treatment software

(Motic Images Plus 2.0). By using the Auto

Segment and Auto Calculation commands it was

possible to separate object regions from the back-

ground using a threshold value, in order to obtain

detailed data from the segmented images, such as

the total area and percentage of total area of the

respective objects. All experiments were performed

at least in triplicate. All statistical analyses were

performed with one-way analysis of variance

(ANOVA) using the Bonferroni post-test (Instat

software, version 3.36 GraphPAD Software Inc., San

Diego, CA, USA) to determine significant differ-

ences in the experimental data. P\ 0.05 wasconsidered statistically significant.

Results

The pH of paper depends on the substances used in its

manufacture and the presence of acid groups as a result

of hydrolysis and oxidation reactions. The experimen-

tal results showed that all the investigated types of

paper had similar pH values. Specifically, the pH

values were

pH paper Að Þ ¼ 8:21� 0:01;

pH paper Bð Þ ¼ 8:55� 0:01;

pH paper Cð Þ ¼ 8:08� 0:01;

where the slightly higher value of paper B can be

explained by its origin as recycled paper (Fedrigoni

2012).

The observation of different paper samples, treated

with Herzberg’s reagent, by polarizing optical micro-

scopy allows their fibre content to be identified

(Fig. 2). The results showed the presence of:

1. Yellow fibres from pulp, jute, hemp, raw, or

generally lignin-rich fibres in paper A samples

(Fig. 2a).

2. Purple-red fibres from flax or bleached hemp, blue

fibres from untreated pulp and yellow fibres from

pulp, jute, hemp, raw, or generally lignin-rich

fibres in paper B samples (Fig. 2b).

3. Only blue fibres from untreated/bleached pulp in

paper C samples (Fig. 2c).

4. Purple-red fibres from flax or bleached hemp in

the book case-study (Fig. 2d).

(a) (b)

(c) (d)

Fig. 2 Fibres fromdifferent paper samples,

treated with Herzberg’s

reagent, imaged by

polarizing optical

microscopy. a Wood-freepaper A, b recycled paper B,c eco-friendly paper C, andd book case-study. The barsize is 30 lm

Cellulose (2016) 23:3265–3279 3271

123

Fungal colonization of the paper samples began about

2 weeks after inoculation. In the case of paper A, made

with wood-free paper and characterized by a heavy

and rough texture, fungi spread both on paper surfaces

and edges, where several hyphae and conidiophores

were evident under the microscope. Therefore, this

type of paper presented an excellent substrate for

fungal growth and consequent fast deterioration.

Paper B is made with recycled paper and charac-

terized by the lightest, smoothest and whitest texture,

while paper C is obtained from eco-friendly cellulose,

i.e. bleached without the use of chlorine, and charac-

terized by a rather yellowish, rough, and wavy aspect.

In both cases paper contamination remained localized

around the initial infection points due to their partic-

ular composition (recycled and eco-friendly paper,

respectively).

Cleaning treatments were carried out by placing the

gellan gum hydrogels listed in Table 1 on inoculated

papers A, B and C. In all tests, the gellan gum

hydrogels left no residues in the paper. Only in the

case of hydrogels containing titanium dioxide (Gels 6

and 7) were some aggregated nanoparticles found by

optical microscope observations after treatment, but

they could be easily removed with a soft brush.

Cleaning action by gellan gum hydrogels (Gel 1)

In this test, paper samples infected with A. versicolor

were covered with pure gellan gum hydrogels (Gel 1)

for 1 h. Gel 1 was able to promote the removal of

particulate matter present on the paper supports as

evaluated by optical microscope observations. Never-

theless, samples placed in the climatic chamber for a

further 15 days showed fungal re-growth.

Cleaning and decolouring action by gellan

gum/calcium compound hybrid hydrogels (Gels

2–5)

In this test, paper samples infected with A. versicolor

were covered with gellan gum/de-acidifying com-

pound hybrid hydrogels (Gels 2–5, prepared with

calcium sulphate, hydroxide, chloride, and acetate,

respectively) for 1 h. Gels 2–5 promoted the removal

of particulate matter on the paper supports due to the

cleaning action of the gellan gum gel, and decoloured

the spots differently according to the calcium

compound used, as shown by the pictures in

Fig. 3a2–d2. Figure 3 shows that all the gels caused

an evident decolouration of the spots present at the

beginning of the experiments (Fig. 3a1–d1), with

better results for Gel 5 (Fig. 3d1, d2), containing

calcium acetate. All treatments were unable to prevent

fungal re-growth on the paper samples after a further

15 days in the climatic chamber (Fig. 3a3–d3). The

results show slight differences in the extent of fungal

re-growth depending on the type of paper (A, B and

C), as shown in Fig. 3d1–f1, d2–f2, and d3–f3.

Cleaning, decolouring action and biostatic/

biocidal activity of gellan gum/TiO2 nanoparticle

hybrid hydrogels (Gel 6)

As can be seen in Fig. 4, the paper samples were

cleaned in an excellent manner by Gel 6, and no fungal

re-growth was observed after a further 15 days in the

climatic chamber (Fig. 4).

Cleaning, decolouring action and biostatic/

biocidal activity of gellan gum/calcium acetate/

TiO2 nanoparticle hybrid hydrogels (Gel 7)

Before applying the gels to the cleaning of a book (the

case study), the cleaning tests were repeated using

calcium acetate-loaded gellan gum/TiO2 nanoparticle

composites in order to evaluate the presence of

synergistic effects. After inoculation with A. versi-

color, paper samples were covered with activated Gel

7 for 1 h. Then, the hydrogels were gently removed

and the samples observed under the optical micro-

scope. As can be seen in Fig. 5, the paper samples

were cleaned in an excellent manner by Gel 7, and no

fungal re-growth was observed after a further 15 days

in the climatic chamber.

Cleaning, decolouring action and biostatic/

biocidal activity of gellan gum hydrogel and gellan

gum/calcium acetate/TiO2 nanoparticle hybrid

hydrogel (Gel 1 and Gel 7) on the case-study book

On the basis of the results of the previous tests (see

section ‘‘Cleaning, decolouring action and biostatic/

biocidal activity of gellan gum/calcium acetate/TiO2nanoparticle hybrid hydrogels (Gel 7)’’), the gellan

3272 Cellulose (2016) 23:3265–3279

123

gum/calcium acetate/TiO2 nanoparticle hybrid hydro-

gel (Gel 7) was tested on the third cover page of the

case-study book, which was characterized by evident

foxing. The page of the case-study book was covered

with Gel 1 (pure gellan gum hydrogel blank) and Gel 7

(activated as previously described) for 1 h. Then, the

hydrogels were gently removed and the page was

observed with a Wood’s lamp. As can be seen from

Fig. 6c, the area treated with Gel 7 shows a better

cleaning than the area treated with Gel 1, as almost all

spots (present on the right of Fig. 6a) are removed,

while several spots are still present after treatment

with Gel 1 (bright spots on the left of Fig. 6c). These

spots are also responsible for an apparent higher

fluorescence arising from the left side of Fig. 6c.

These observations were confirmed by the image

treatment analysis described in section ‘‘Evaluation of

cleaning action and biostatic/biocidal activity’’, which

Gel 1 on paper A

Gel 2 on paper A

Gel 3 on paper A

Gel 4 on paper A

Gel 5 on paper A

Gel 5 on paper B

Gel 5 on paper C

(a1) (a2) (a3)

(b1) (b2) (b3)

(c1) (c2) (c3)

(d1) (d2) (d3)

(e1) (e2) (e3)

(f1) (f2) (f3)

(g1) (g2) (g3)

Fig. 3 a–e Cleaning anddecolouring action by pure

gellan gum hydrogel (Gel 1)

and by gellan gum/de-

acidifying compound hybrid

hydrogels (Gels 2–5) on

wood-free paper A. The first

picture of each row (a1–e1)shows the initial

contaminated condition,

while the second picture

(a2–e2) was taken after thetreatment. The third picture

(a3–e3) shows papersamples after a further

15 days in a climatic

chamber. e–g Cleaning anddecolouring action by gellan

gum/calcium acetate hybrid

hydrogels (Gel 5) on wood-

free paper A, recycled paper

B, and eco-friendly paper C.

The first picture of each row

e1–g1 shows the initialcontaminated condition,

while the second picture e2–g2 was taken after thetreatment. The third picture

e3–g3 shows paper samplesafter a further 15 days in a

climatic chamber. The bar

size in a1 is common to allpictures and 0.2 cm long

Cellulose (2016) 23:3265–3279 3273

123

resulted in an average cleaning action of 92 % for the

area treated with Gel 7 and of 70 % for the area treated

with Gel 1. Nevertheless, Fig. 6c shows the presence

of some ink bleeding around the red letters, may be

due to the particular composition of this ink. So, a

detailed investigation should be generally made on the

Gel 6 on paper A

Gel 6 on paper B

Gel 6 on paper C

(a1) (a2) (a3)

(b1) (b2) (b3)

(c1) (c2) (c3)

Fig. 4 a–c Cleaning, decolouring action, and biostatic/biocidalactivity by gellan gum/TiO2 nanoparticle hybrid hydrogels (Gel

6) on wood-free paper A, recycled paper B, and eco-friendly

paper C. The first picture of each row a1–c1 shows the initial

contaminated condition, while the second picture a2–c2 wastaken after the treatment. The third picture a3–c3 shows papersamples after a further 15 days in a climatic chamber. The bar

size in a1 is common to all pictures and 0.2 cm long

Gel 7 on paper A

Gel 7 on paper B

Gel 7 on paper C

(a1) (a2) (a3)

(b1) (b2) (b3)

(c1) (c2) (c3)

Fig. 5 a–c Cleaning,decolouring action, and

biostatic/biocidal activity by

gellan gum/calcium acetate/

TiO2 nanoparticle hybrid

hydrogels (Gel 7) on wood-

free paper A, recycled paper

B, and eco-friendly paper C.

The first picture of each row

a1–c1 shows the initialcontaminated condition,

while the second picture a2–c2 was taken after thetreatment. The third picture

a3–c3 shows paper samplesafter a further 15 days in a

climatic chamber. No

synergistic effect is evident

from the simultaneous

activity of calcium acetate

and titanium dioxide

nanoparticles. The bar size

in a1 is common to allpictures and 0.2 cm long

3274 Cellulose (2016) 23:3265–3279

123

particular ink composition present in real artefacts in

order to find the optimal number and time of gel

applications.

Discussion

Figure 7 shows optical microscope images of a pure

gellan gum hydrogel (Gel 1) after the cleaning of a

paper sample inoculated with A. versicolor. It is

possible to observe on the hydrogel surface the

presence of both hyaline hyphae and spores (Fig. 7a,

b, respectively), confirming that the hydration and

swelling of samples due to the hydrogels decreases the

adhesion of organic contaminants to the paper

substrates.

The gellan gum concentration of 3 wt.% used in the

hydrogels’ preparation allows their easy manipulation,

and no residue of gellan gum was present on the paper

samples when observed under the optical microscope.

All paper samples benefitted from evident cleaning by

the treatment with pure gellan gum hydrogels (Gel 1),

but not from biostatic/biocidal activity, as after

15 days there was evident fungal re-growth.

The addition of de-acidifying compounds to the

hydrogel formulation (Gels 2–5) results in a decolour-

ing action on the paper samples in addition to the

previously reported cleaning action by pure gellan

gum hydrogels. Decolourization is most effective with

gellan gum/calcium acetate hybrid hydrogels (Gel 5,

see Fig. 3d2), and no particular dependence is

observed on the type of paper sample, as shown in

Fig. 3d2, e2, and f2. Nevertheless, none of the calcium

compounds ensured protection against fungal re-

growth.

The addition of TiO2 nanoparticles into the Gel 1

formulation provides effective cleaning action,

decolourization of the paper support and finally a

biocidal (no fungal re-growth) or, at least, biostatic

activity (no spore development) 15 days after the

treatment. Samples were clearly decolourized and

almost all stains disappeared. In fact, the application of

gellan gum/TiO2 nanoparticle composite hydrogels on

all three types of paper cleaned the dark spots caused

by fungal growth, with the best results obtained on

eco-friendly paper samples.

As reported by Iannuccelli and Sotgiu (2010), the

cleaning mechanism of gels during the treatment can

be summarized as follows:

1. Spontaneous spread of water molecules from the

gel to the paper.

2. Solubilisation of degradation by-products of

cellulose.

Fig. 6 Cleaning, decolouring action, and biostatic/biocidalactivity by gellan gum/calcium acetate/TiO2 nanoparticle

hybrid hydrogels (Gel 7) on the case-study book. a initialcontaminated condition under a Wood’s lamp, b during thetreatment, and c after a further 15 days in a climatic chamberunder a Wood’s lamp. A pure gellan gum hydrogel (Gel 1) was

placed on the left as blank. Gel diameter is 5 cm

Cellulose (2016) 23:3265–3279 3275

123

3. Diffusion of concentrated solution of by-products

from the paper to the gel according to the gradient

of concentration.

4. Effective removal of both surface particulate

matter and a part of the substances that are present

within the paper.

So, a partial penetration to the back of the paper and

some migration of by-products to the sides cannot be

excluded as seen for some red inks in Fig. 6.

Obviously, the penetration and migration of by-

products within the paper artefact is dependent from

the application time and percentage of water present in

the gels, on the degree of porosity and wettability of

the paper, and from the preservation state of the

artefact.

The biostatic/biocidal activity of the hydrogels, due

to the photo-catalytic properties of the incorporated

titanium dioxide nanoparticles, was confirmed by the

absence of fungal re-growth after a further 15 days in

the climatic chamber under the harsher conditions of

T = 30 �C and RH = 80 %.Nevertheless, some TiO2 nanoparticles were found

on paper samples after treatment with gellan gum

hydrogels incorporating titanium dioxide. This appar-

ent drawback could undoubtedly prolong the biocidal

activity of the treatment.

As a consequence of the tests carried out on the

three types of paper, it is possible to confirm the

effectiveness of gellan gum hybrid hydrogels in the

cleaning and decolourization of infected papers. In

particular, hydrogels loaded with either calcium

acetate or titanium dioxide nanoparticles gave the

best results.

Similar results were obtained when the different

types of paper were cleaned by gellan gum/calcium

acetate/TiO2 nanoparticle hybrid composites (Gel 7):

spots were cleaned to a greater extent on the eco-

friendly cellulose paper but Gel 7 did not show any

additional cleaning effect compared with Gel 5 and

Gel 6. Evidently, Gel 7 has similar biostatic/biocidal

properties to Gel 6.

For the tests on the deteriorated book (case study),

Gel 1 and Gel 7 (i.e. the pure gellan gum and gellan

gum/calcium acetate/TiO2 hybrid hydrogels) were

applied simultaneously. Pictures taken after UV

irradiation demonstrate good results with no detri-

mental effect on the inks. The cleaning action and

biostatic/biocidal activity shown by the different

tested hydrogels are summarized in Table 1. To test

the activity of titanium dioxide nanoparticles alone,

paper samples were covered for 1 h with activated

TiO2 nanoparticles after inoculation with A. versi-

color. Then, the titanium dioxide powder was gently

removed from the samples using a soft brush and the

samples were observed under the optical microscope.

No difference in spots was evident between contam-

inated and treated samples, possibly due to the absence

of a wet hydrogel environment, which strongly limits

the decolouring action of TiO2 nanoparticles. Never-

theless, the biostatic/biocidal activity of treatment

with TiO2 nanoparticles was demonstrated by keeping

the sample in the climatic chamber for 15 days, after

which time no fungal re-growth was evident. It is well

known that titanium dioxide under UV irradiation can

cause the oxidation and, consequently, possible

degradation of cellulose and hemicellulose. However,

this possible drawback was mostly avoided in our

(a) (b)

Fig. 7 Optical microscope picture of a pure gellan gumhydrogel (Gel 1) surface after cleaning of a paper sample

inoculated with A. versicolor. It is possible to observe on the

hydrogel the presence of: a hyaline hyphae and b spores of A.versicolor. The bar size is 30 lm

3276 Cellulose (2016) 23:3265–3279

123

investigations, as the activation of TiO2 nanoparticles

in Gels 6 and 7 was performed before their placement

on the paper samples. In order to verify whether the

proposed treatments could change the physical–chem-

ical properties of the tested papers, the water activity,

pH, tensile strength and colour values were checked

2 days after the application of the gellan gum hybrid

hydrogels. No significant difference in these proper-

ties was found in the tested papers. In fact, the values

of the above mentioned physical–chemical properties

changed by\5 % from the initial ones. In particular,water activity values changed by an average 3.3 %

(3.0 % for paper A, 3.7 % for paper B and 3.2 for

paper C, respectively), pH values changed by an

average 1.3 % (1.2 % for paper A, 1.8 % for paper B

and 1.0 for paper C, respectively), paper tensile

strength values changed by an average 2.7 % (2.2 %

for paper A, 3.3 % for paper B and 2.6 for paper C,

respectively) and, finally, whiteness values changed

by an average 4.5 % (4.2 % for paper A, 4.8 % for

paper B and 4.6 % for paper C, respectively).

In this work, the ability of gellan gum hydrogels in

cleaning paper, as well as the capacity of de-acidifying

calcium compounds and titanium dioxide in decolour-

ing paper, were investigated. In particular, the results

showed that the treatment with pure gellan gum

hydrogels ensures the cleaning of paper supports due

to the ‘‘gelled water content’’ of these materials, while

gellan gum/calcium compound hybrid hydrogels

provide both cleaning and decolouring action. An

additional biostatic/biocidal activity was found only in

hybrid hydrogels incorporating titanium dioxide

nanoparticles. Although these results were achieved

using a single fungal species, and further work is

needed to test other species both alone and in

combination in order to simulate real environmental

conditions, hybrid gellan gum hydrogels incorporating

titanium dioxide nanoparticles can be considered an

effective tool to improve the conservation, protection

and usability of library and archive materials. The use

of these hybrid hydrogels is easy and inexpensive, and

allows the single-step cleaning and decolouring of

paper artworks contaminated by A. versicolor.

Conclusions

Paper is the raw material for most of the cultural heritage

preserved in libraries and archives. Since it essentially

consists of cellulose fibres, paper can undergo physical,

chemical and biological processes of degradation. In

particular, biological attacks due to fungi can cause

evident aesthetic damage through the appearance of spots.

Several techniques for cleaning paper have been

proposed and, in this study, the application of rigid

gellan gum hydrogels in combination with either de-

acidifying compounds or titanium dioxide nanoparti-

cles was investigated for the cleaning and decolouring

of paper supports infected by A. versicolor. In

particular, the best results in decolouring spots were

obtained with gellan gum hydrogels enriched with

either calcium acetate or titanium dioxide nanoparti-

cles, although only the photo-catalytic activity of

titanium dioxide was able to inhibit the re-growth of A.

versicolor. No synergistic effect was found in gellan

gum hydrogels incorporating both calcium acetate and

titanium dioxide. Experiments performed on a page of

an old book confirmed that hybrid hydrogels are

respectful of the inks used for book printing. Further

work is in progress in order to test other fungal species

and investigate the long-term effects of the proposed

treatments on the chemical and physical properties of

paper before their applications in real artefacts. In fact,

it is important to emphasize that TiO2 acts through the

generation of reactive species like hydroxyl radicals

and superoxide ions, which would most likely also

have detrimental effects on the paper fibres.

Acknowledgments MIUR, the Italian Ministry forUniversity, is acknowledged for financial supports (Grants

PRIN and EX 60 %).

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Gellan gum hybrid hydrogels for the cleaning of paper artworks contaminated with Aspergillus versicolorAbstractIntroductionPaper deteriorationThe biocidal activity of titanium dioxideStandard methods for cleaning and disinfecting paper

Materials and methodsPaper characterizationFungal inoculationHydrogel preparationEvaluation of cleaning action and biostatic/biocidal activity

ResultsCleaning action by gellan gum hydrogels (Gel 1)Cleaning and decolouring action by gellan gum/calcium compound hybrid hydrogels (Gels 2--5)Cleaning, decolouring action and biostatic/biocidal activity of gellan gum/TiO2 nanoparticle hybrid hydrogels (Gel 6)Cleaning, decolouring action and biostatic/biocidal activity of gellan gum/calcium acetate/TiO2 nanoparticle hybrid hydrogels (Gel 7)Cleaning, decolouring action and biostatic/biocidal activity of gellan gum hydrogel and gellan gum/calcium acetate/TiO2 nanoparticle hybrid hydrogel (Gel 1 and Gel 7) on the case-study book

DiscussionConclusionsAcknowledgmentsReferences