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Biotechnology applied to cultural heritage: Biorestoration of frescoes using viable bacterial cells and enzymes


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To set up and employ, for the biorestoration of cultural heritage (altered frescoes), an advanced and innovative biotechnology method based on the sequential use of whole viable bacterial cells and specific enzymes. The bioremediation intervention consisted of the direct application onto an artwork surface of whole bacterial cells of the Pseudomonas stutzeri A29 strain (bioaugmentation), followed by, in a final step, a purified Protease enzyme. The bioremediation was performed on a Spinello Aretino fresco that had become altered by the animal glue residues of past restoration. For the reader's interest the fresco is the 14th century Conversione di S. Efisio e battaglia (Conversion of S. Efisio and battle), size 3.5 x 7.8 m at the Pisa Camposanto Monumentale, Italy. An assessment was made of the final costs of the biological tests (whole bacterial cells, enzymes) so as to compare them with other intervention techniques. A successful innovative biological approach to recover valuable frescoes was set up, and the best conditions for treatment efficiency were identified. Furthermore the cost of the biological cleaning using viable bacterial cells and enzymes (P. stutzeri, Protease, Collagenase, 1 : 3 : 10, ratio respectively) was much lower than that of other conventional methods, making this biotechnology not only very interesting but also very competitive. New biotechnologies with an innovative, soft approach to the 'biocleaning' and 'biorestoration' of cultural heritage are in constant demand, and our results are clear evidence that such an approach has been achieved; the technique could be of significant importance towards developing other goals.
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Biotechnology applied to cultural heritage: biorestoration of
frescoes using viable bacterial cells and enzymes
G. Ranalli
, G. Alfano
, C. Belli
, G. Lustrato
, M.P. Colombini
, I. Bonaduce
, E. Zanardini
P. Abbruscato
, F. Cappitelli
and C. Sorlini
DISTAAM, Universita
`del Molise, Campobasso, Italy,
Dip. Chimica e Chimica Industriale, Universita
`di Pisa, Pisa, Italy, and
`di Milano, Milan, Italy
2003/1162: received 17 December 2003, revised 2 July 2004 and accepted 17 July 2004
P . A B B R U S C A T O , F . C A P P I T E L L I A N D C . S O R L I N I . 2004.
Aims: To set up and employ, for the biorestoration of cultural heritage (altered frescoes), an advanced and
innovative biotechnology method based on the sequential use of whole viable bacterial cells and specific enzymes.
Methods and Results: The bioremediation intervention consisted of the direct application onto an artwork
surface of whole bacterial cells of the Pseudomonas stutzeri A29 strain (bioaugmentation), followed by, in a final step,
a purified Protease enzyme. The bioremediation was performed on a Spinello Aretino fresco that had become altered
by the animal glue residues of past restoration. For the reader’s interest the fresco is the 14th century Conversione di
S. Efisio e battaglia (Conversion of S. Efisio and battle), size 3Æ5·7Æ8 m at the Pisa Camposanto Monumentale,
Italy. An assessment was made of the final costs of the biological tests (whole bacterial cells, enzymes) so as to
compare them with other intervention techniques.
Conclusions: A successful innovative biological approach to recover valuable frescoes was set up, and the best
conditions for treatment efficiency were identified. Furthermore the cost of the biological cleaning using viable
bacterial cells and enzymes (P. stutzeri,Protease,Collagenase, 1 : 3 : 10, ratio respectively) was much lower than that
of other conventional methods, making this biotechnology not only very interesting but also very competitive.
Significance and Impact of the Study: New biotechnologies with an innovative, soft approach to the
biocleaningand biorestorationof cultural heritage are in constant demand, and our results are clear evidence that
such an approach has been achieved; the technique could be of significant importance towards developing other
Keywords: bacteria, biocleaning, biorestoration, enzymes, frescoes, gas chromatography coupled with mass
spectrometry (GC-MS) and pyrolysis/GC-MS analyses.
Outdoor artwork, especially where lithoid materials, stones,
frescoes and paint are involved, is very susceptible to
deterioration that is brought about by changes mainly caused
by ageing and, in recent decades, to pollution. In fact, in
urban areas, damage because of the increased pollution of
today is manifested on monument surfaces by alterations
such as black crusts, nitration, sulphation and the deposition
of dust and residual hydro-carbons (Saiz-Jimenez 1995).
In addition to air pollutants, the surfaces of man-made
artistic stonework can also be altered by organic matter that
has been applied, but then not completely removed, during
restoration; in many cases this can pose serious danger even
to the preservation of the artwork itself (Ranalli et al. 1996,
2000). In fact such leftovercompounds often act as a good
growth substrate for micro-organisms and mycetes that
Correspondence to: Giancarlo Ranalli, DISTAAM, University of Molise, Via De
Sanctis 46, 86100 Campobasso, Italy (e-mail:
ª2004 The Society for Applied Microbiology
Journal of Applied Microbiology 2005, 98, 73–83 doi:10.1111/j.1365-2672.2004.02429.x
destroy the surface, and allow hyphae penetration to some
Moreover, the process of detaching frescoes from walls
prior to restoration calls for notable quantities of organic
compounds (such as glue and casein) that become distributed
on both the painted surface and at the back of the fresco.
This was the condition of the Pisa frescoes (in the
Monumental Cemetery, Camposanto) that cover a surface
area of about 1500 m
; these frescoes date back to the XIV
century and are by famous painters such as Antonio
Bonaiuti, Antonio Veneziano, Benozzo Gozzoli, Taddeo
Gaddi, Francesco Traini, Bonamico Buffalmacco and Spi-
nello Aretino.
Most of the frescoes had been restored using traditional
chemical and physical techniques in which residual organic
substances and salts are removed by ammonium carbonate
solution and organic solvents. However even today’s
enzymes, surfactants and solubilizing agents have failed to
clear away past restoration material and restore some of the
frescoes (Makes 1988; Bellucci and Cremonesi 1994; Bonomi
1994; Wolbers 2000) that today exhibit a particular hydro-
phobic behaviour related to weathering and protein poly-
In order to choose suitable conservation practices, and
to resolve the above-mentioned problems, it is most
important to identify both the original organic materials
applied and those belonging to restoration work, and also
materials that have undergone transformation through
natural ageing and pollution. As artwork samples are
obviously unique and generally very small, and the
compounds to be determined are in low concentration,
it is fundamental to use sensitive and selective techniques
like gas chromatography coupled with mass spectrometry
(GC-MS) and pyrolysis/GC-MS (PY/GC-MS) (Colom-
bini et al. 1999, 2003).
Up until this realwork on the frescos we had only used
viable micro-organisms to degrade and remove substrate
supported organic matter under laboratory conditions
(Ranalli et al. 2003a). Thus we had to first identify the
unwanted organic matter on the frescos, and verify the
difficulty of removing such organic compounds by tradi-
tional methods. We then began experimenting the use of
viable bacterial cells to degrade the organic matter, i.e. using
them as biorestorationagents on the frescoes; as the organic
matter degraded the residues were rinsed offand removed
by enzymatic treatments.
The present research was aimed at dissolving the adhesive
(animal glue) between the painted fresco face and the
adhering gauze, and thus allow the gauze to be detached. We
took the following steps:
i the characterization of the organic adhesive matter on the
gauze used to detach the fresco from the wall: GC-MS
and PY/GC-MS analytical procedures identified the
natural organic compounds and their degradation prod-
ii the developing and improving of an innovative bioresto-
ration system, that we had already set up on a laboratory
scale, to remove the organic matter from the fresco
We also assessed the costs of the whole bacterial cell and
enzyme biotechnologies, and compared them with tradi-
tional methods.
Micro-organisms, media and cultural methods
We tested Pseudomonas cepacia strain DSMZ 7288
(Deutsche Sammlung von mikroorganismen und Zellk-
ulturen GmbH, Braunschweig, Germany), P. testosteroni
strain DSMZ 1455, P. fluorescens strain DSMZ 304,
P. flavescens strain DSMZ 12071, P. stutzeri strain A 29
(DISTAM-DISTAAM Strains Collections, University of
Milan–University of Molise, Italy), P. stutzeri strains
DSMZ 5190, and ATCC 23856 (American Type Culture
Collection, Rockville, MD, USA). To select the best
culture to apply to the altered fresco surfaces we assessed
the biodegrading activity of the culture by adding animal
glue at 0Æ5% and 1%, supplied as the complex organic
matter and energy sources in the mineral medium M9,
under aerobic conditions, at 10, 20, 28 and 37C (Ranalli
et al. 1996, 2003a). Microbial growth was monitored by
, and an assay was made of ATP content and the
viable cell count in plate count medium. The selected
strains were stored on standard plate count agar (Difco).
Preparation of the bacterial suspension for
Suspensions containing exponentially growing bacteria,
ca 10
CFU ml
, were obtained by inoculating 3 ml of
an overnight broth culture into 300 ml fresh broth
medium and incubating it in a shaker (200 rev min
for 24 h at 28C. The cells were centrifuged at 7000 gfor
10 min at 4C, washed twice with phosphate-buffered
saline, and re-suspended in sterile 0Æ8% (w/v) NaCl
solution, pH 7Æ2; the final cell concentration was above
cells ml
, corresponding to an O.D.
of ca 1Æ6;
the cells were used immediately or stored at 4C during
conservation and/or transport from the laboratory to the
field application.
A fully computerized laboratory batch fermenter (20 l
useable volume) (mod. Biostat C, B. Braun; Biotech
International, Helsungen, Germany) was adopted to obtain,
when needed, a relevant amount of viable bacterial cell
biomass. The best selected bacterium for the in situ
ª2004 The Society for Applied Microbiology, Journal of Applied Microbiology,98, 73–83, doi:10.1111/j.1365-2672.2004.02429.x
biorestoration was cultivated, at the same concentration as
the cultures, for 24 h on broth medium containing animal
glue as the complex organic matter. The cells were
recovered in vitroby centrifugation and re-suspended in
NaCl solution at a final abundance of 1–3 ·10
cells per ml.
Whole bacterial cell applications on a fresco
Viable cells of the selected strain were applied to the fresco
surface at ca 20C and 10C to evaluate the effect of
temperature on the removal of organic matter. The bacterial
culture was applied in three ways to test the application
effect: (i) spraying, (ii) brushing and (iii) a covering cotton
wool layer soaked with bacterial suspension.
In the first case a manually operated atomizer (useful
volume 0Æ5 l) was used to spray the fresco surface from a
distance of 25–30 cm, distributing the bacterial cells regu-
larly by consecutive left-right and top-bottom dispersions;
to favour distribution, large fresco areas were subdivided
into areas to form rectangular reticules (treated areas ca 200–
400 cm
For the brushing distribution we adopted a flat paintbrush
(8–12 cm wide) to deliver the cells manually, making gentle
brush strokes to deliver the suspension over all the surface in
a regular layer and taking great care to avoid detaching
altered pellicles.
In the third application type we employed the same
brushed-on cell distribution (as in application 2) and then
covered the fresco with a thin layer (0Æ5 cm depth, 10 cm
wide, 10–20 cm long) of hydrophilic sterile white cotton
wool wet with the same bacterial cell suspension.
Monitoring of viable microbial cell counts
Throughout the biorestoration of the fresco, we constantly
evaluated the total viable microbial cell numbers. For
application 3 this was carried out on small samples taken
from the cotton layer (ca 1Æ0 g), using the plate counts
technique in standard plate count agar (Difco), incubation
for 48 h at 28C expressed as CFU g
. In applications 1
and 2, when the microbial count was determined directly on
the fresco surface, we adopted the replica-plating technique;
the results are expressed as CFU per unit area. Sterile velvet
was pressed onto the cleaned surfaceof the fresco, and then
onto plate count agarized medium. Finally, in order to verify
microbial viability during bacterial cell application, ATP
assays (total and free) were carried out using the sterile swab
technique (Ranalli et al. 2003b).
Enzyme treatments
Enzyme treatments were performed after the bacterial cell
applications had removed as much glue as possible.
Five pure enzymes (Collagenase Type IA and Type V
from Clostridium histolyticum, 1–3 U mg
Protease Type XIV from Streptomyces griseus,4Umg
7Æ5–8Æ5; Protease Type XIX from Aspergillus sojae,
,pH8Æ4; Lipase Type VII from Candida
cylindracea, 400–900 U mg
,pH8Æ4–8Æ8; Sigma-Aldrich,
St Louis, MO, USA) were tested, both separately and
mixed, for the removal of the weathered glue.
Purified enzyme solutions were added delicately to the
altered fresco surface by three different techniques: (i) swab,
(ii) paper-disc and (iii) brush.
Enzymatic activity
The levels of enzymatic activity of the pure bacterial
cultures were determined using the API-ZYM
´rieux Italia, Rome, Italy). The specific galleries (nm
5, 11, 12) were inoculated with 65 llof10
suspension (O.D.
560 nm
of ca 1.1) of 1 ml of an overnight
broth culture (at 12th hour) to 9 ml of sterile solution (NaCl
). After incubation for 4 h at 20, 28 and 37C, the
galleries were activated by adding 30 ll of Reagent ZYM A
and Reagent ZYM B (bioMe
´rieux) and after 5 min we
assigned values ranging from 0 to 5 in relation to the colour
developed in each enzymatic reaction, using the colour chart
provided by the manufacturer (Principi et al. 2003). For the
purposes of this study, the results are reported as reactions
of low intensity (1), moderate intensity (2–3) and high
intensity (4–5) (Tiquia 2002).
The grade of enzymatic activity on native animal glue and
on animal glue cross-linked was determined using the Feller
test (Feller et al. 1985; Cremonesi 1999).
ATP determination
Total ATP assays to monitor both culture growth on the
laboratory scale, and the viability of the bacterial cell
suspensions during the in situ biorestoration processes,
were performed using a specific enzymatic kit (NRM/
Lumit-QM, code 9332-l; Lumac B.V., Landgraaf, the
Netherlands). A Biocounter 1500 P luminometer (Lumac
B.V.) equipped with a photomultiplier tube set at
7200 RLU with 200 pg ATP in 100 ll of Lumit buffer
and Lumit-QM reagent was used (Ranalli et al. 1998,
Microscope observations
Microbial growth and cell survival were determined by
optical microscope (Axiophot, Zeiss; LEO Elektronen-
mikroskopie GmbH, Oberkochen, Germany) and scanning
electron microscopy (SEM) observations throughout the
application of the biocleaning treatment to the altered
ª2004 The Society for Applied Microbiology, Journal of Applied Microbiology,98, 73–83, doi:10.1111/j.1365-2672.2004.02429.x
surface of the fresco. The samples for SEM observation
were treated overnight in a solution of 2% glutaraldehyde
(0Æ01 mol l
phosphate buffer), and then immersed in 1%
osmium tetroxide. A microscope operating at 10 kV was
used (Zeiss DSM 940A; LEO Elektronenmikroskopie
Chemical analyses
Reagents. All the solvents were Baker HPLC grade.
Hexadecane and tridecanoic acid were used as internal
standards (IS). Hexamethyldisilazane (HMDS) and
N,O,bis(trimethylsilyl)trifluoroacetamide (BSTFA) con-
taining 1% trimethylchlorosilane were purchased from
Sigma (Milan, Italy), N-tert-butyldimethylsilyl-N-methyl-
trifluoroacetamide (MTBSTFA) with 1% trimethylchlo-
rosilane was from Fluka (Milwaukee, WI, USA); all were
used without any further purification. Standard solutions of
amino acids in HCl 0Æ1
, norleucine (IS2) and hexadecane
(IS1) as injection internal standard were purchased from
Analytical procedure based on selective extraction and
hydrolysis assisted by microwave and GC-MS analy-
sis. The glue sample (50–100 lg) was extracted with
ammonia solution, subjected to microwave-assisted acidic
hydrolysis (microwave oven model MLS-1200 MEGA
Milestone; FKV, Sorisole, Italy), and the resultant hydro-
lysed solution derivatized with MTBSTFA and GC-MS
analysis (6890N GC System Gas Chromatograph coupled
with a 5973 Mass Selective Detector; Agilent Technologies,
Palo Alto, CA, USA) to determine the protein content. The
residual of the ammonia extraction was subjected to
saponification, extraction with n-hexane, acidification and
extraction with diethyl ether to obtain, respectively, the
neutral and acidic fractions, subsequently analysed by GC-
MS after derivatization with BSTFA to determine the
presence of natural resins, lipids and waxes. The detailed
procedure is reported in the literature (Colombini et al.
1999, 2003; Bonaduce and Colombini 2003).
Analytical procedure based on PY/GC-MS. A few lgof
samples together with 5 ll of a HMDS solution (20% in
acetone) were pyrolysed at 600C in a pyrolyser operating at
constant temperature mode Pyrojector II (SGE, Austin,
TX, USA). Experimental conditions are reported in detail
in previous work (Colombini et al. 2003).
Mass spectra assignment. Mass spectra assignment was
generally based on direct matching with spectra from the
Wiley 275 library: spectra were retained when the correla-
tion match index was >95%. We compared pure compounds
whenever possible.
Physical analyses. The thickness of the glue layers was
measured at different points of the fresco using a laser
sensor micrometer, Thrubeam LX2-V model (Keyence
Corp., Osaka, Japan).
Spinello Aretino fresco
During World War II the frescoes belonging to the complex
of the Monumental Cemetery of Pisa were ruined by a bomb
(1944) but, after detachment from the walls, were restored at
a later date. From among them we chose, for the present
study, the XIV century fresco, Conversione di S. Efisio e
battaglia, painted by Spinello Aretino; the surface area is ca
50 m
(26 m
treated) and the contained inorganic pigments
are known from relative documentation.
Unfortunately the restoration techniques and conservation
conditions were inappropriate, and ourfresco had soon
shown alteration phenomena such as swelling and detachment
of the painting pellicle. In the 1980s, the fresco was again
detached from the walls at Camposanto using tear off
techniques that involve, on site, the application of a wide gauz e
directly onto the fresco surface, using animal glue as the
consolidating agent; thus the gauze and fresco become one
layer that is then detached from the wall. The back of the
fresco is reinforced by a canvas cloth, applied using casein,
which is, in turn, supported on asbestos, again with casein.
One of the greatest difficulties during the restoration was
the detachment of gauze still adhering, from the previous
restoration, to the paint layer on the front surface; this was
despite soaking with solvents. Over the years the glue had
altered greatly, and had become very hard and resistant to
commonly used solvents. Our task was to dissolve the animal
glue on the paintwork itself to release the gauze residues
without removing the casein supporting the back of the fresco.
The conservation state was also worsened by the
contraction of the glue and the swelling of the casein at
the back; furthermore different synthetic organic substances
had been used in the past restoration and these had favoured
the polymerization of the compounds, making their removal
an onerous task.
Biological treatment
Selection of the strain for application in the bioresto-
ration. Table 1 shows the results of the laboratory scale trials
of bacterial culture growth rate at 28C on broth medium
containing animal glue as the complex organic matter.
There were marked growth rate differences among the
tested Pseudomonas species and the P. stutzeri strains;
P. stutzeri A 29 showed the highest cell density (1Æ8 O.D.
and >8Æ5 log CFU ml
), a relevant ATP content, higher
ª2004 The Society for Applied Microbiology, Journal of Applied Microbiology,98, 73–83, doi:10.1111/j.1365-2672.2004.02429.x
enzymatic protease activity and, from optical microscope
observations, the greatest number of bacterial cells per field.
The influence of temperature on the growth of the
selected bacterial strains was investigated by cultural,
enzymatic and microscopic tests, the response of P. stutzeri
strain A29 growing cells being noted at 10, 20, 28 and 37C.
Cell density increased slightly when the incubation tem-
perature was maintained under 10C for 36 h, and improved
a little at 20C (data not shown). However at constant
temperatures of 28C and 37C there was a very marked
increase in cell growth at 24 and 36 h, with abundant
biomass production. Moreover, notable increases in enzy-
matic activity were observed at the tested temperatures: in
fact, the response of the protease reaction in P. stutzeri strain
A29 on overnight broth culture at 12th hour, showed low
intensity (value of 1) at 10C, moderate intensity (value of
2–4) at 20C, and high intensity (value of 5) at both 28 and
37C (Table 1).
Figure 1 shows the SEM observations on the selected
P. stutzeri strain A29 cells.
Chemical analyses
The GC-MS and PY/GC-MS results from several fresco
samples (1–5 mg) evidenced both animal glue, used to glue the
gauze onto the front of the fresco, and casein which came from
the back of the painting. The PY/GC-MS identified, by
specific markers, the proteins indole for casein and pyrrole and
alkylpyrroles for the animal glue. The GC-MS amino acidic
profiles showed that all the proteins had undergone degrada-
tion. This was particularly evident in the samples taken from
the front of the fresco (Fig. 2) where there was a typical
collagen protein pattern; the samples from the back of the
fresco showed a high degree of degradation in the casein amino
acidic pattern, because of natural ageing and micro-organism
attack (Fig. 3 in GC-MS). In fact, this phosphoprotein had a
low content of amino acids like methionine, phenylalanine and
lysine. These results concord with those of samples from other
analysed frescoes (Colombini et al. 1995, Colombini et al.
1999). Moreover, as protein recovery was ca 20% w/w of the
sample weight, it would appear that cross-linking reactions
had occurred.
This hypothesis was also confirmed by the presence of
formalin, which had been added to the glue mixture to avoid
Table 1 Growth yields in minimal medium with added glue, assessed by O.D.
and CFU at 36th hour, at 28C; ATP content, protease activity at
10, 20, 28 and 37C and microscope observations at 12th hour
Bacterial culture
(36 h)
Log CFU ml
(36 h)
Total ATP
(RLU)* (12 h)
Protease activity
(trypsin)(C) Microscope
observation(12 h)
10 20 28 37
P. cepacia DSMZ 7288 1Æ5(0Æ15) <8 ++ 1334++
P. testosteroni DSMZ 1455 0Æ1(0Æ1) <3 +/)0000+/)
P. fluorescens DSMZ 304 1Æ45 (0Æ2) <8 ++ 0233++
P. flavescens DSMZ 12071 1Æ4(0Æ15) <8 ++ 0234++
P. stutzeri A29
1Æ8(0Æ2) >8Æ5 ++++ 2455+++
P. stutzeri DSMZ 5190 1Æ65 (0Æ15) 8 +++ 1344+++
P. stutzeri ATCC 23856 1Æ7(0Æ2) 8 +++ 1345+++
RLU, relative luminose unit.
*+/), very low; ++, low; +++, high; ++++, very high.
Overnight broth-culture at 12th hour: reaction of low intensity (value of 1: 5 nm of hydrolysed substrate); moderate intensity (value of 2–4: 10–
30 nm of hydrolysed substrate); high intensity (value of 5: 40 nm of hydrolysed substrate).
Presence of bacterial cells per field (+/), very rare; ++, low; +++, high; ++++, very high).
Fig. 1 Scanning electron microscopy of Pseudomonas stutzeri strain
A29 cells in 0Æ8% NaCl solution
ª2004 The Society for Applied Microbiology, Journal of Applied Microbiology,98, 73–83, doi:10.1111/j.1365-2672.2004.02429.x
fermentation in alkaline conditions; furthermore, formalin in
the presence of casein during drying gives rise to insoluble
compounds that are difficult to remove.
The relevant amounts of sulphates, phosphates and
oxalates were because of the eternit, that together with the
lime putty contributed to the sulphation of the frescoes, to
both the phospholipids of the casein and the final products
of the oxidative degradation of the animal glues and casein,
and to metabolic micro-organism degradation.
These salts were responsible for the whitish patina
observed on the surface of almost all the frescoes.
Our results have revealed that the painted surface of
Spinello Aretino’s fresco is embedded in a hard net of
polymerized and cross-linked proteins which, in turn, are
glued to the canvas.
Physical analyses
The thickness of the glue layer was demonstrated to vary
from 1 to 3 mm corresponding to ca 4mgcm
Biorestoration treatment with whole bacterial cells
The ex situ biorestoration treatments were performed by
distributing a suspension of viable precultured bacterial cells
of P. stutzeri strain A29 on the altered fresco.
Of the three application modes, the best results were
achieved by putting a cotton wool layer soaked with microbial
suspension onto the fresco surface. In fact, in terms of
humidity preservation, this technique resulted in constant,
and favourable, microenvironmental conditions between the
viable cells and the fresco surface throughout the biological
treatment (time of contact and adhesion). The spraying and
brushing cell applications produced unfavourable rapid and
excessive drying, with a consequent reduction in bacterial cell
viability and activity. Furthermore, in both cases it was also
necessary to apply further cell suspension aliquots from time
to time after the first application, placing an extra burden on
our resources with regard to microbial biomass availability,
and on the restorers from the point of view of labour. Thus, on
the basis of these findings, the bacterial culture mode of
50 000
100 000
150 000
200 000
250 000
300 000
350 000
400 000
450 000
500 000
550 000
600 000
650 000
700 000
750 000
Time (min)
12·00 14·00 16·00 18·00 20·00 22·00 24·00 26·00 28·00 30·00
IS ser
met hyp
Fig. 2 GC-MS chromatogram (acquired in
the SIM mode) of a sample taken from the
front of the fresco. Internal standards (IS),
alanine (ala), glycine (gly), valine (val), leucine
(leu), isoleucine (ile), methionine (met), serine
(ser), proline (pro), aspartic acid (asp), gluta-
mic acid (glu), lysine (lys), hydroxyproline
(hyp) and tyrosine (tyr)
12·00 14·00 16·00 18·00 20·00 22·00 24·00 26·00 28·00 30·00
Time (min)
phe asp glu
lysmet tyr
Fig. 3 GC-MS chromatogram (acquired in
the SIM mode) of a sample taken from the
back of the fresco. Internal standards (IS),
alanine (ala), glycine (gly), valine (val), leucine
(leu), isoleucine (ile), methionine (met), serine
(ser), proline (pro), aspartic acid (asp), gluta-
mic acid (glu), lysine (lys), hydroxyproline
(hyp) and tyrosine (tyr)
ª2004 The Society for Applied Microbiology, Journal of Applied Microbiology,98, 73–83, doi:10.1111/j.1365-2672.2004.02429.x
application adopted was the culture-soaked cotton wool layers
(40 cm wide, 6Æ0 m long); these were placed over the whole
fresco surface, guaranteeing homogeneous conditions and
saving time during application and recovery.
Different tests showed the marked influence of tempera-
ture on the biological treatment. An environmental tem-
perature below 10C resulted in no evident biological
effects, however at ca 20C the collagen began to dissolve
8–24 h after the daily cell suspension application; this did
not happen in the control areas.
At 28C the cellular activity was so intense, just 8–12 h
after applying the bacterial suspension (log 8 CFU g
means ca 100 million of viable cells per g), that the treated
areas were clean and freefrom the animal glue without any
structural damage.
Nevertheless the degree of glue removal depended on the
thickness of the glue layer at the start of the treatment, varying
between 80 and 100%; some areas were cleaned completely,
while others (where the glue was as thick as 3 mm) were left
with residues, but never greater than ca 0Æ5 mm.
When residues were still present after the treatment it was
found that prolonging the bacterial treatment gave the
possibility of obtaining complete removal, but there was
some risk to the fresco. In fact, for periods of longer than
15–17 h, the prolonged contact with the moist cotton led to
the swelling and detachment of paint fragments.
Figure 4 shows the removal of cotton layer enriched with
P. stutzeri strain A29 after 10-h biorestoration from ex situ
fresco altered by animal glue (Conversione di S. Efisio e
battaglia, XIV century) at Pisa Camposanto Monumentale
Biorestoration treatment with enzymes
After removing the gauze through bacterial activity, small
organic matter residues (the glues themselves) still remained
on the surface. These were treated by enzyme solutions.
The purified enzymes, Collagenase Type IA and Protease
Type XIX, used separately and in mixture (data not shown
here), showed the highest removal efficiency, both on native
animal glue and on animal glue cross-linked (fragment of
altered fresco), as reported in Table 2.
However, because of the high cost of the collagenase-
based enzyme, we adopted Protease Type XIX (1Æ0g/
100 ml
Tris buffer solution, pH 7Æ8–8Æ2, at 38C, for 10–
15 min).
During application a portable heating device was used to
maintain optimal temperatures for the fresh enzyme solu-
tions (38C). Throughout the fresco biorestoration process
and during the enzymatic treatment, the recorded indoor
environmental temperature showed an average of 28 ± 3C.
Of the three different enzyme application modes, swab,
paper-disc and brush, enzyme distribution was best by
brush; in fact its efficiency in removing residual organic
matter and animal glue was probably favoured by the gentle
pressure of the bristles of the brush and the repetitive and
gentle mechanical pressure of the manual application.
Monitoring of the biorestoration process
An important aspect of bioremediation is the process
monitoring. This was performed initially on the laboratory
scale using a microbiological approach. Instead the in situ
application called for more rapid, although not always
specific, techniques such as the determination of ATP
content by bioluminescence.
During the biorestoration process on a full scale fresco we
assessed how time and the environment affected the
Fig. 4 Removal of cotton layer enriched with Pseudomonas stutzeri
strain A29 after 10-h biorestoration from ex situ fresco altered by
animal glue (Conversione di S. Efisio e battaglia, XIV century) at Pisa
Camposanto Monumentale (Italy)
Table 2 Enzymatic activities on native animal glue and on animal glue
cross-linked (altered fresco)
Native animal glue
Animal glue
Collagenase Type IA 5* +++ 2* +
Collagenase Type V 4* +++ 1* +/)
Protease Type XIV 4++ 1+/)
Protease Type XIX 5+++ 2+
Lipase Type VII 1+/)0+/)
Assay of specific activity: *chymotrypsin; trypsin; lipase
+/), very low; +, low; ++, middle; +++, high.
Reaction of low intensity (value of 0–1: 0–5 nm of hydrolysed
substrate); moderate intensity (value of 2–4: 10–30 nm of hydrolysed
substrate); high intensity (value of 5: 40 nm of hydrolysed substrate).
ª2004 The Society for Applied Microbiology, Journal of Applied Microbiology,98, 73–83, doi:10.1111/j.1365-2672.2004.02429.x
dynamics of P. stutzeri A29 bacterial cell survival, measured
as log CFU ml
and ATP content and compared with a
control. The results from a repeated series of tests showed
that there were no significant statistical differences in the
first 12 h and 14 h of the biological process. However a
significant variation appeared in the parameters of the 16
and 24-h treatments using the P. stutzeri strain A29. The
recovered cells (fresco surface) showed a final mean value in
cell viability of log 4–log 5 CFU cm
, compared with
log 8 CFU cm
in the control set (initial fresco surface
enriched by viable bacterial cell suspensions). The total ATP
content showed little variation as a function of treatment
duration. In fact, at hours 6, 10 and 12, the ATP content was
comparable with the initial values, whereas at 16 h, it was
slightly lower than the control.
The pH values were continually monitored by a probe
located between the fresco surface, in direct contact with the
altered patina, and the cotton wool layer enriched with the
selected bacterial cell suspension. After 12 h of biorestoration,
only a slight reduction in pH value was noted (from 7Æ2to6Æ7).
At the end of the microbial cell cleaning of the fresco
surface, and after removing the remaining bacteria by a final
washing with distilled sterile water (three soft manual
sponge applications), microbiological analyses were made on
the fresco surface to check for the possible presence of
residual microbial cells. The lack of growth on the
inoculated plates of the replica-plating technique confirmed
that no microbial cells were detectable with the methods
used. The data were confirmed by the free and total ATP
content values.
Figures 5 and 6 show the effects of biocleaningand
biorestorationprocess obtained with P. stutzeri strain A29
cells and Protease enzyme on Conversione di S. Efisio e
battaglia, fresco (XIV century) at Pisa Camposanto Monu-
mentale, (Italy) (top and bottom, before and after treatment
Economic evaluation of the biorestoration
An analysis of the costs of the bioremediation process shows
that the use of bacterial cultures is more convenient
economically than the use of enzymes like Protease and
In fact, the total cost can be quantified as being ca 50 l
for bacterial culture, 150 l
for the Protease and 500 l
the Collagenase, using a litre of the bacterial or enzymatic
suspension for ca 2m
of fresco surface. Thus the cost ratio
was 1 : 3 : 10.
On comparing the costs of the bacterial biorestoration
process and the more frequently used chemico-physical
techniques, it is evident that the latter are less convenient;
this can be seen in terms of both the total time needed for
the restoration and the number and duration of the
application times (repetitive treatments every 6–8 h) as well
as in the greater costs involved for specialized personnel,
restorers and finally environmental safety.
The success of the viable bacterial cell application, compared
with that of enzymes is, presumably, attributable to the wide
versatility of the bacteria and their activity. Bacteria are
known to produce not only constitutive but also inducible
enzymes that attack and degrade different types of mole-
cules. The synthesis of inducible enzymes takes place only in
the presence of a substrate, creating a regulatory effect.
Thus the use of micro-organisms is more effective than just
the use of a single enzyme that attacks only specific linkages.
In fact, the alkaline properties of formalin, a common
component of the organic matter, are conducive to poly-
merization, protein cross-linking and reticulation during
drying, resulting in the formation of insoluble compounds.
Fig. 5 Effects of biocleaningand biorestorationprocess obtained
with Pseudomonas stutzeri strain A29 cells and Protease enzyme on
Conversione di S. Efisio e battaglia, fresco (XIV century) at Pisa
Camposanto Monumentale, (Italy) (top and bottom, before and after
treatment, respectively)
ª2004 The Society for Applied Microbiology, Journal of Applied Microbiology,98, 73–83, doi:10.1111/j.1365-2672.2004.02429.x
Thus in the case of frescoes where the glue and casein
compounds are protein weathered, and altered by the
presence of formalin, the effectiveness of the micro-organ-
isms is really important as the enzymes, which are highly
specific, may not be useful. Moreover, the constitutive and
induced enzymes of viable cells of versatile bacteria like
Pseudomonas are able to break up many organic compound
chemical linkages.
These heterotrophic bacteria were applied to the Spinello
Aretino fresco as it was known that Spinello used only
inorganic pigments; thus his frescoes would not be damaged
in any way during the short application time of the bacterial
Many factors such as temperature, relative humidity, pH,
carbon and energy sources, etc. can influence metabolic
activity. Thus, to optimize the metabolic efficiency we
expected on the fresco, we had to check these factors
accurately in the laboratory, our assumption being that
if biorestoration could not be achieved under optimal
conditions on the laboratory scale, it would be unlikely to
succeed in an uncontrolled environment.
Thus we had to verify whether our successful laboratory
results would be confirmed under real, on site, environ-
mental conditions, especially for outdoor artwork.
In artwork bio-remediation, bacterial treatment requires
the application, by spray, brush or compress, of selected
micro-organisms to the artwork surface. The way the
application is carried out depends on the type of alteration,
the artwork material, the location of the areas undergoing
treatment, and the metabolic activity of the selected
microflora (aerobic and anaerobic).
Indeed the effectiveness of the biological process is
defined by the time course of the treatment. Long treatment
times require a high biomass concentration and favourable
contact between the micro-organisms and the surface.
Consideration must also be given to an adequate cell-carrier
support; in the case of ourfresco the choice was cotton
wool. A previous laboratory study carried out on stone
artwork had shown the best support matrix for the bacteria
to be sepiolite (Ranalli et al. 2000).
All artwork biorestoration requires a final, accurate
cleaning phase. Indeed, at the end of specific biological
processes all residual microflora should be removed carefully
to avoid the continuation of undesired metabolic processes.
Thus an adequate strategy for artwork protection must be
planned; if the micro-organisms remain alive and active they
can cause material loss or damage, either directly or as a
consequence of their catabolic by-products.
The proposed biological technique, that constitutes an
accurate cleaning phase, was a fundamental mustfor the
side of the fresco called intelaggio(the painted side),
although long contact with a water solution could have
posed an indirect risk to the safety of the fresco. In this
phase it was very important to reduce to the minimum both
the volume of water addition and its contact time on the
fresco surface.
Possible damage because of prolonged bacterial treatment
has, till now, only been evidenced by Ranalli et al. (2000),
who discussed the removal of black crusts from stone. In this
case, sulphate-reducing bacteria reduced the sulphates and,
consequently, removed the crusts; however prolonged
contact between the bacteria and the stone caused the
precipitation of sulphide salts. Thus it is very important to
control the duration of the application.
Nevertheless, when artwork undergoing microbial reme-
diation has peculiarities that permit other forms of inter-
vention, adequate surfactant solutions and weak biocides can
be used.
In order to identify optimal conditions to develop a
bioremediation approach for the use of selected microflora
on artwork, it is most important to monitor the microbial
activity during, and at the end of, the biological process.
Fig. 6 Effects of biocleaning,biorestoration, process obtained with
Pseudomonas stutzeri strain A29 cells and Protease enzyme on
Conversione di S. Efisio e battaglia, fresco (XIV century) at Pisa
Camposanto Monumentale, (Italy) (top and bottom, before and after
treatment, respectively)
ª2004 The Society for Applied Microbiology, Journal of Applied Microbiology,98, 73–83, doi:10.1111/j.1365-2672.2004.02429.x
Monitoring techniques are routinely used to carry out
microbial counts based on culturing methods and direct
counts by microscope observation. In fact SEM and optical
microscope observations give important information on the
presence of microflora and their degree of adhesion to the
surface of the treated materials. Rapid analyses are funda-
mental to the monitoring of biorestoration processes, and
fast feedback in the monitoring of microbial activity can be
achieved through bioindicators like ATP content by low
light imaging and dehydrogenase activity (DHA) (Ranalli
et al. 2000, 2003a).
The greatest advantage of bacterial/enzymatic bioresto-
ration, compared with traditional methods (chemical,
physical and mechanical), is that this new method is not
destructive and removes only extraneous substances or
altered compounds from the fresco. Moreover, microbial
cultures (bacteria) have non-specific activity, while the use
of enzymes is highly specific and limited. Another advant-
age is the use of safe micro-organisms (not pathogenic
bacteria or yeasts, not spore-forming bacteria) for both the
operators and the environment. Finally, the evaluation of
costs and convenience has revealed the transferability of
this technological innovation to other fields outside cultural
Organic matter (i.e. collagen) on the surface of altered
frescoes can be removed, under appropriate conditions, by
treating the fresco surface with selected aerobic heterotro-
phic microbial cultures (bioaugmentation) and/or selected
pure enzymes. As a consequence of the bacterial activity,
carbon dioxide is released from the pollutants without the
use of toxic compounds, so the method must be considered
After optimizing the parameters for the biological process
on the laboratory scale, and subsequently checking the
results on real outdoor samples, we consider that the
application of the method in wider fields would require: (i)
an abundant activated biomass produced under environ-
mentally controlled conditions; (ii) a rapid and sensitive
method to monitor the biological activity and avoid undes-
irable effects; (iii) the correct and complete removal of
residual biological activity.
We are presently researching ways of applying, in situ,
biological methods to several works of art, and to reduce the
limitations and possible risks of such methods. Our results
have confirmed the potential of biorestoration processes as
soft innovative biotechnology. Such restoration, aimed at the
recuperation of degraded artwork, is nondestructive, and
uses micro-organisms and their metabolic activity in associ-
ation with purified enzymes.
The positive results we have achieved have led the
Technical Commission for Restoration (Pisa, Italy) to accept
and approve the use of a P. stutzeri cell culture suspension
and purified Protease enzyme solutions for biological
applications; thus the innovative biorestorationbioaug-
mentation process is already being used.
The biorestored fresco discussed here is an important case
study on the real, true to life scale where soft biotechno-
logies can play a positive role.
We foresee that appropriate micro-organisms will be used
for the remediation and recovery of artistic stonework
altered by atmospheric pollutants like nitrogen and sulphur
oxides, compounds that rapidly accelerate artwork deterior-
It has been shown that cultural heritage restoration now
has a new and innovative technology: biorestoration through
the use of viable cells of selected bacteria; such restoration
poses no risk at all to human health, or the environment, and
has shown great potential for removing undesirable com-
pounds from the surfaces of works of art.
The work was carried out with partial financial support from
the Opera Primaziale Pisana, Pisa, Ing. G. Bentivoglio. The
authors wish to thank Clara Baracchini, Soprintendenza
Beni Culturali ed Ambientali, Pisa, Italy; Restorer Sig.
Gianni Caponi of Conservazione and Restauro, Pisa;
Dr Lucia Maiuro, CSIM (for SEM observations). Finally,
the authors are grateful to Barbara Carey for editing the
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... ATP quantification, serving as a bioindicator of microbial activity, was conducted using a Hygiena SystemSURE Plus® luminometer [74][75][76]. ...
... During this experiment, AquaSnap® and UltraSnap® ATP devices were utilized. To differentiate free ATP from non-viable fragmented cells, it is necessary to employ two distinct devices [75,76,78]; in this particular case AquaSnap Total® and AquaSnap Free®. It is crucial to discriminate extracellular or free ATP in this context, so both devices had to be used in each sample. ...
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This research paper proposes Microbially Induced Calcium Carbonate Precipitation (MICP) as an innovative approach for palaeontological heritage conservation, specifically on deteriorated carbonate fossils. Due to its efficiency in bioconsolidation of carbonate ornamental rocks, Myxococcus xanthus inoculation on carbonate fossils was studied in this research.Treatment was tested on nine fossil samples from decontextualized fragments of Cheirogaster richardi specimens (Can Mata site, Hostalets de Pierola, Catalonia, Spain). The main objective was to evaluate whether treatment with Myxococcus xanthus improved fossil surface cohesion and hardness and mechanical strength without significant physicochemical and aesthetic changes to the surface. Chemical compatibility of the treatment, penetration capacity and absence of noticeable changes in substrate porosity were considered as important issues to be evaluated.Samples were analysed, before and after treatment, by scanning electron microscopy, weight control, spectrophotometry, X-ray diffraction analysis, water absorption analysis, pH and conductivity control, Vickers microindentation and tape test. Results show that hardness increases by a factor of almost two. Cohesion also increases and surface disaggregated particles are bonded together by a calcium carbonate micrometric layer with no noticeable changes in surface roughness. Colour and gloss variations are negligible, and pH, conductivity and weight hardly change. Slight changes in porosity were observed but without total pore clogging.To sum up, results indicate that Myxococcus xanthus biomineralisation is an effective consolidation treatment for carbonate fossils and highly compatible with carbonate substrates. Furthermore, bacterial precipitation of calcium carbonate is a safe and eco-friendly consolidation treatment.
... An eco-friendly cleaning approach for the removal of inorganic patinas and organic coatings is based on the use of bacteria and enzymes [16]. The use of bacteria, such as A29 strain of Pseudomonas Stutzeri, had already been successfully applied to degrade the organic matter of altered animal glue for bio-restoration and bio-cleaning by Ranalli et al., 2005 andLustrato et al. 2012, [16,17]. Lipases, on the other hand, are used in cleaning procedures to remove oil-based coatings or even acrylic varnishes [18][19][20][21]. ...
... An eco-friendly cleaning approach for the removal of inorganic patinas and organic coatings is based on the use of bacteria and enzymes [16]. The use of bacteria, such as A29 strain of Pseudomonas Stutzeri, had already been successfully applied to degrade the organic matter of altered animal glue for bio-restoration and bio-cleaning by Ranalli et al., 2005 andLustrato et al. 2012, [16,17]. Lipases, on the other hand, are used in cleaning procedures to remove oil-based coatings or even acrylic varnishes [18][19][20][21]. ...
... Por otro lado, trabajos desarrollados en el campo específico de la pintura mural han demostrado cómo ciertas soluciones bacterianas son capaces de metabolizar los sustratos tanto orgánicos como inorgánicos que por diversas razones afectan a las películas pictóricas murales (Ranalli et al 2005;Bosch et al., 2013). En este sentido, en el marco de la restauración del gran ciclo pictórico de la Iglesia de San Nicolás de Valencia, se ha aplicado con éxito esta novedosa tecnología. ...
... Esta zona fue sometida a un proceso de arranque y reubicación en 1919, proceso que si bien salvó la pintura de su desaparición, no evitó un enorme grado de deterioro. La presencia de restos de cola de arranque en la superficie pictórica que con el paso del tiempo endurecen y envejecen, hace que la limpieza de esta zona mediante técnicas tradicionales, no sea óptima, mientras que los sistemas de biolimpieza han demostrado ser capaces de eliminar este tipo de residuos (Ranalli et al., 2005;Bosch et al. 2013;Bosch et al., 2015) En lo que se refiere a infraestructura y métodos empleados se ha seleccionado la mejor de las instrumentaciones ideadas para tal fin, y se han aplicado con el rigor propio que se espera en toda intervención sobre el patrimonio. ...
... In this context, a multi-analytical approach, as demonstrated by Bersani et al. [24], Romani et al. [25], and Alberghina et al. [26], is extremely effective in providing information, from the macro to the micro domain [27], not only on pigments but also on specific binders (such as egg whites, oils, and resins) and alteration patinas [28]. The combination of this information can guide the restorer in the decision and implementation of specifically targeted conservation practices, useful for restoring the condition and readability of the artwork in case of ordinary and extraordinary maintenance [29][30][31], even with bioremediation techniques [32]. ...
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This study presents the petrographic and chemical characterization of the frescoes in the Church of San Panfilo in Tornimparte (AQ, Italy) by Saturnino Gatti, a prominent painter of the late 15th-early 16th century, known for his exquisite technique, composition, and use of color. The characterization of the frescoes is essential for understanding the materials and techniques used by Gatti, as well as for identifying the stratigraphy and painting phases. Eighteen samples were collected from the original paint layers, later additions (17th century), and restored surfaces, and analyzed by optical microscopy, cathodoluminescence microscopy, scanning electron microscopy (SEM-EDS), μ-Raman, and electron paramagnetic resonance (EPR). The analyses revealed a microstratigraphy often made of three main layers: (1) preparation, consisting of lime plaster and sand; (2) pigmented lime, applied by the fresco technique; and (3) additional pigmented layer on the surface. The most often recurring pigments are black, red, yellow (all generally linked with the fresco technique), and blue (applied "a secco"). The presence of two painting phases was also noted in one sample, probably resulting from a rethinking or restoration. These findings contribute to the understanding of the history and past restoration works of this cultural heritage site, providing important insights not only for conservators and restorers, but also for a broader understanding of Italian fresco painting and art history of the late 15th and early 16th centuries.
... The bio-cleaning methods are non-toxic, nonaggressive, non-invasive, and highly specific for removing surface contaminants, and thus represent a promising result in the field of bio-cleaning of murals [130]. It should be borne in mind that some of the exoenzymes could not be completely removed during the restorations and these compounds may cause aesthetic damage to the murals over time [131]. Currently, the exoenzymes that could be potentially used for mural cleaning are not produced at an industrial, and they are not commercially available. ...
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As non-renewable cultural heritages, murals have important implications in historical customs, religions, and philosophy as well as their aesthetic values. Recently, many murals are threatened by natural factors and human activities. During the past decades, there are increasing interest in the investigation of murals. Here we review the current status of murals and provide an up-to-date summary of achievements related to murals. The murals that draw the most attention are distributed in Mexico, Ireland, China, and Spain. The aesthetics, history, cultural, educational, and economic values of murals are comprehensively analyzed. The main research technologies used to detect the chemical compositions and physical structures of murals are also summarized. The restoration of murals includes several procedures such as stabilization, repair, surface cleaning, and pigment reconversion. Emerging technologies such as computer science benefit the research and conservation of murals. We also propose that tourism management and climate change should be incorporated into the conservation of murals in the future.
... Because biodeterioration can lead to the damage of historic documents, artwork, monuments, or buildings, its study is fundamental for the conservation of cultural heritage [1][2][3][4][5]. The prevention of biodeterioration and development of adequate conservation and restoration strategies cannot be an unscripted process; it is necessary to undertake diagnoses of these valuable pieces of our history and art, which include chemical characterization and the study of microbial diversity together with the physiological characteristics of biodeteriogens [1,6]. ...
Full-text available
We studied the physicochemical characteristics and mycobiota associated to five key historic documents from Costa Rica, including the Independence Act of Costa Rica from 1821. We used nondestructive techniques (i.e., ATR-FTIR and XRF) to determine paper and ink composition. Results show that some documents are composed of cotton-based paper, whereas others were made of wood cellulose with an increased lignin content. We also determined that the ink employed in some of the documents is ferrogallic. Cultivation and molecular techniques were used to characterize the fungi inhabiting the documents. In total, 22 fungal isolates were obtained: 15 from the wood-cellulose-based documents and seven from the other three cotton-based. We also tested the cellulolytic activity of the recovered fungi; 95% of the fungi presented cellulolytic activity correlated to their ability to cause deterioration of the paper. Results suggest that cotton-based paper is the most resistant to fungal colonization and that most of the isolates have cellulolytic activity. This work increases the knowledge of the fungal diversity that inhabits historic documents and its relationship with paper composition and provides valuable information to develop strategies to conserve and restore these invaluable documents.
Blends of solvents from non-renewable sources, often polluting and toxic to humans, are routinely used in the restoration of painted artifacts. Here we present the application of three different green solvents (and their mixtures) as a viable alternative to the standard triad of solvents (acetone, ethanol, and isooctane) used in the solubility test for cleaning polychromic artworks. Solketal (SOLK), γ-valerolactone (GVL), and 2-ethylhexyl pelargonate (ARGO) were selected among the solvents achievable from bio-based synthons such as glycerol, levulinic acid, and pelargonic acid, which are mainly produced from biomass and renewable feedstocks as exhausted vegetable oils, carbohydrates, and lignocellulose. Specifically, ARGO solvent was prepared by esterification reaction and characterized by nuclear magnetic resonance (NMR) and mass spectroscopy coupled to gas chromatography (GC-MS). Hansen solubility parameters for each solvent were determined by a group contribution method, thus enabling their placement in the Teas graph. Their penetration ability in wooden specimens was investigated by evaluating the volume retention of each solvent with different coated specimens. The solvent ability of the selected compounds was tested by visible and UV observations on specimens prepared with film-forming substances (Dammar, Mastic, Shellac, Paraloid® B72 and linseed oil) brushed onto glass plates. Our results pointed out the suitability of this solvent triad for application to panel painting surfaces. The effectiveness of mixtures made with the above green solvent was successfully tested to remove a terpenic varnish from a 16th century oil painting on a wooden panel.
Two different biocleaning techniques for stamp removal from different paper samples (handmade and machine-made) were investigated. Cellulose is the main component of handmade paper, while higher concentration of lignin is present in machine-made paper. Biocleaning methods included the direct application on paper surfaces of the extracellular enzymatic mixture (EEM) extracted from the yeast Sporidiobolus metaroseus and the recombinant protein CthediskatG of Chaetomium thermophilum var. dissitum. The produced microbial enzymes (EEM or CthediskatG) were also combined with agarose hydrogels. The effectiveness of the cleaning ability of the individual methods was determined using different spectrophotometer measurements based on colorimetric analysis and by Attenuated Total Reflectance Fourier Transform Infrared Spectroscopy (ATR-FTIR). Some tested samples were also subjected to microstructural and chemical analysis using Scanning Electron Microscope-Energy-Dispersive X-ray spectroscopy (SEM-EDX). The analysis showed that the EEM-based approaches were the most suitable, mainly they are less time-consuming and easy to produce, and moreover slight differences were displayed between EEM and CthediskatG during the removal of the stamp by hydrogel-enzyme approaches. Both EEM applications (direct and hydrogel) speed up the stamp removal process from real paper samples. However, for the complete elimination of the stamp smears a quick N,N-dimethylformamide post-treatment is advised too.
Magnetic audiovisual (AV) media makes up a significant part of cultural heritage. It contains elements such as sounds, images, and other data for future access. Mould growth can impact magnetic media deterioration. Over time, mould impedes element retrieval and can even cause its total loss, resulting in the loss of our cultural heritage. Therefore, proper maintenance of magnetic media is required to ensure data retention. This paper presents the findings of an international online survey to better understand current knowledge surrounding mould-affected magnetic media. Information on magnetic media is limited, resulting in the question: what are the current experiences and practices surrounding mould on magnetic media in the cultural heritage sector? The aim of the project was to seek audiovisual conservators and film practitioners’ opinions about magnetic media (full coat magnetic film, magnetic audio, and video tapes), conservation processes, and their experience with mould. An on-line survey using Qualtrics software was employed. The survey indicated that mould is present in magnetic media collections regardless of substrate but may be limited to less than a quarter of magnetic media with various dual mechanical–chemical treatments used to combat mould growth. Mould continues to impact element retrieval and thus mould treatment is an important factor in deterioration prevention to ensure data retention.
In this article bacterial carbonate mineralization treatments are proposed as a novel strategy for decayed fossils and palaeontological heritage conservation; specifically, by means of inoculation of Myxococcus xanthus , a bacterium of proven effectiveness in ornamental stone bioconsolidation. Bioconsolidation treatments can be very effective, stable, nontoxic, environmentally friendly, and chemically compatible with fossil heritage. The method reproduces what nature has been doing for millennia with fossils that have been permineralized by bacterial calcium carbonate precipitation. There is, however, some concern that bacterial inoculation could lead to the growth of undesirable microbiota, which could subsequently damage the fossil substrate. Because of this, the use of bacteria on heritage items must be meticulously monitored and analysis strategies should be carried out to detect bacteria viability during and after treatments. For this purpose, adenosine triphosphate assay is proposed in this article as a fast, affordable, portable, and easy‐to‐use system for conservators. as ATP assay results are relative and difficult to relate to colony forming unit, this study aims to improve their applicability by examining the correlation between ATP analysis and total viable bacteria count in the specific case of M. xanthus . This research provides reference and correlatable data to obtain an approximate estimation of M. xanthus viable bacterial colonies based on relative light unit data.
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A method to analyze the spatial distribution and activity of biodeteriogen agents in Cultural Heritage is reported. It employs a bioluminescent low-light imaging technique to determine the content of ATP, a potential bioindicator of microbes on monuments and other material of Cultural Heritage-related origin. The method offers reduced processing time (10 min compared with the 48 h needed for cultural microbiological analyses); spe-cific response and sensitivity, allowing the detection of active microbial contamination, an indicator of 'at biological risk' for materials under test (stone, natural fibre, paper, wood); spatial distribution and sample geometry of bioluminescence and quantification of the dif-ferent luminescence levels in relation to biodeteriogen activity; future in situ applicability. Apart from its advantage of allowing rapid diagnosis, the proposed method could be a use-ful addition to the traditional culture-based microbiological techniques used to study biode-teriogen agents in Cultural Heritage.
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The objectives of this work were to determine the extracellular enzyme profiles during composting, relate the activities of these enzymes to the changes in microbial population and compare the enzyme profiles between two manures. API ZYM assay was used to monitor the activities of 19 extracellular enzymes during poultry and pig manure composting. Results showed an overall increase in diversity and relative abundance of enzymes present. The relative abundance and activities of enzymes were higher in poultry manure than in pig manure. Among the 19 enzymes tested, esterase, valine amino-peptidase and alpha-galactosidase were the most abundant enzymes in poultry manure, whereas it was N-acetyl-beta-glucosaminidase for the pig manure. A number of these enzymes correlated with change in numbers of different microbial groups during composting. The composting process represented a combined activity of a wide succession of environments, as one enzyme/microbial group overlapped the other and each emerged gradually due to the continual change in temperature and progressive breakdown of complex compounds to simpler ones. The results presented here show the applicability of the API ZYM test not only in monitoring the quantitative and qualitative fluctuation of the available substrate during composting, but also in revealing differences in composts and compost maturity.
Among the different pathologies of stone materials, the presence of sulphates and nitrates is the most frequent. In addition, the presence of organic matter on artistic stoneworks can be attributed to inadequate past restorations, to the lysis of microbial cells of the primary surface colonisation, and to the presence of hydrocarbons originating from oil combustion. The latter appears to be a serious danger for the preservation of artworks themselves. Until today, chemical and physical techniques have been largely used to remove pollutants and residual substances from works of art by using surfactants and solubilizing agents. We developed a multiple bioremediation system for the biological removal of sulphates, nitrates and organic matter present on artistic stoneworks utilizing microbial cultures carefully selected and grown on a suitable support. The study consisted of: screening of microorganisms in order to select cultures with a high sulphate-reducing, denitrifying and biodegradative ability of organic matter; setting up of simulated laboratory tests with stone samples artificially enriched with nitrates, sulphates and organic matter; testing appropriate inert matrices on which to immobilize the selected bacterial strains; and testing sulphate, nitrate and organic matter removal from artificially enriched stones as well as from naturally degraded artworks. Sepiolite was used to develop bacterial biofilms with a high active biomass per cm3. However, in order to eliminate or reduce interferences and the release of undesirable ions and contaminant trace elements, Hydrobiogel97, a mixture of polyacrylamide at different molecular weights, appears to be a good carrier. As regards nitrate removal, Pseudomonas aeruginosa and P. stutzeri were selected for their high denitrifying activity. Treatment with colonised sepiolite of stone specimens artificially enriched with nitrates and of real stone samples showed that, after 30 h, a very high percentage (90% and 88%, respectively) of nitrate was removed. For sulphate removal, Desulfovibrio vulgaris and D. desulfuricans were selected and tested in liquid cultures, on stone specimens artificially enriched with sulphates, and on real marble samples. The highest removal efficiency (81%) was achieved on real marble samples after 36 h of treatment. Finally, for the removal of organic matter, bioremediation tests on ancient frescoes (XV century) located near Pisa, altered by the presence of undesirable residual collagen, were carried out before intervention with traditional restoration. The treatment with pure cultures of P. stutzeri, at a temperature of 17–22°C, showed the complete removal of collagen after 8 h. The results confirm the potentiality of bioremediation processes as soft innovative technology based on the use of microorganisms and their metabolic activity in the recovery of degraded artworks.
Two GC-MS procedures for the characterization of shellac, an animal resin widely used in the field of art, were developed. One procedure was based on pyrolysis assisted by the hexamethyldisilazane reaction on-line with GC-MS and the other on the saponification of the sample assisted by microwave followed by GC-MS analysis. The former is a rapid and valuable method for resin identification and the latter gives a more in depth understanding of its composition. Butolic, aleuritic and its derivative acids together with typical sesquiterpenoid compounds were the main molecules identified and used as markers for the molecular pattern recognition of the resin in fresh and old samples (19th century shellac from the Salvemini Collection and gilding samples from the 15th century frescoes of the Duomo of Monza).
Monuments and buildings act as repositories of airborne organic and inorganic pollutants, which accumulate at the surfaces in zones protected from direct rainwater. This enriches the substratum and anthropogenic compounds may influence to a great extent the colonization and growth pattern of microorganisms in polluted urban environments. This paper reviews recent and ongoing research on the deposition of pollutants on building stones and their possible utilization by microorganisms.
Two methods for suppressing the interference of inorganic pigments in the determination of amino acids in hydrolysates of wall painting samples by gas chromatography–mass spectrometry are described. One is based on the extraction of proteinaceous matter from the sample by a 2.5 M ammonia solution prior to the hydrolysis step, and the other on the elimination of inorganic ions from the hydrolysate by means of a cation-exchange resin. The proteinaceous binders present in the paint layer were identified using principal component analysis on the relative amino acid percentage. Some samples from “Giudizio Universale” in Florence Cathedral and from the Monumental Cemetery in Pisa (Italy), were analysed using both procedures. The presence of milk binder as the main organic component of the tempera was highlighted.
The feasibility of composting as disposal practice of husk mixed with olive mill wastewaters (OMW) was studied in the present research. The process was investigated with regards to some reliable and easy to be determined microbial activity parameters such as the ATP content, the activity of a pool of enzymes and the viable counts, keeping in mind the well known bias of this last technique. Two different composting technologies were compared: static pile and reactor by studying the cured composts obtained. Among the bioindicators tested, the composting process trend was described better by the ATP content and the activity of some enzymes. In fact, the ATP content showed an increase during the thermophilic phase in both the pile and the reactor, and decreased at the end of the process, it was also higher in the bioreactor-pile technology than in the pile. With regard to the enzymatic activity, with the reactor technology experiment, two peaks were evidenced in the thermophilic phase and during the curing phase in pile, and a drop during the transfer of material from the reactor to the pile. The quality of the cured product obtained using the reactor technology has been evaluated considering phytoxicity and hygienic features: the stabilised compost resulted to have neither phytoxical effect, nor faecal indicator contaminants.
The paper deals with the application of two analytical procedures, based respectively on on-line pyrolysis in the presence of hexamethyldisilazane followed by gas chromatography/mass spectrometric analysis (Py/GC/MS) and on selective extractions and microwave-assisted hydrolysis followed by GC/MS determination. These procedures give reliable data on the organic composition of micro-samples; moreover, when applied to the same micro-sample, they give comparable data on natural substances, and complementary information on synthetic compounds and degradation products. The two methods were used to characterise the organic matter present in several samples taken from scenes of the frescoes 'Giudizio Universale' and the 'Ascensione' by Bonamico Buffalmacco in the Monumental Cemetery of Pisa. The observed hydrophobicity of their surfaces, which hampers traditional means of restoration, is explained by the formation of a polymeric network due to reactions between a synthetic polymer used as adhesive, the proteinaceous material (animal glue and casein), and formaldehyde.