International Biodeterioration & Biodegradation 94 (2014) 1e11
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International Biodeterioration & Biodegradation
journal homepage: www.elsevier .com/locate/ ibiod
Laponite micro-packs for the selective cleaning of multiple coherentdeposits on wall paintings: The case study of Casina Farnese on thePalatine Hill (Rome-Italy)
Matteo Mazzoni a, Chiara Alisi b, *, Flavia Tasso b, Adele Cecchini c, Paola Marconi b,Anna Rosa Sprocati b
a University of Rome, “Sapienza” Piazzale Aldo Moro 5, 00185 Rome, Italyb ENEA, Environmental Characterization, Prevention and Recovery Unit, Via Anguillarese 301, 00123 Rome, Italyc Free-lance Conservator, Via S. Croce in Gerusalemme 100, 00185 Rome, Italy
a r t i c l e i n f o
Article history:Received 11 April 2014Received in revised form9 June 2014Accepted 9 June 2014Available online
As a result of the research described in this article a safe, bio-based procedure has been established totreat hard-to-remove coherent deposits composed of gypsum, weddellite, calcium carbonate, apatite,nitrate, and aged proteinaceous matter from the wall paintings of the lower loggia of the Casina Farnese(Palatine Hill, Rome). Following a laboratory screening, three bacterial non-spore-forming strains wereselected from our laboratory collection to solubilise calcium sulphate and carbonate (Cellulosimicrobiumcellulans TBF11E), to degrade proteins (Stenotrophomonas maltophilia UI3E), to solubilise inorganic com-pounds and to degrade protein material (Pseudomonas koreensis UT30E). The living bacterial cells weresuspended in a Laponite gel, which is highly compatible with the survival of the cells and is also easilyapplied, and removed from, vertical walls. Compresses containing microorganisms (micro-packs) wereused in a series of biocleaning tests carried out in situ, from July 2012 to February 2013, in temperaturesranging from 6 �C to 37 �C. Each micro-pack contained a single bacterial strain (TBF11E, UI3E or UT30E).The micro-packs containing the different bacteria were applied, individually or in succession, dependingon the nature and layers of the deposits to be removed. Contact times of between 24 and 48 h wereestablished in accordance with the advice of the restorers. The strain TBF11E removed the inorganicdarker layer, UI3E dissolved the brownish layer (probably aged casein) and UT30E removed the mixeddeposits. No residues were left after the treatment and the restorers successfully completed therestoration.
During the period between the Venice Charter (1964) and themore recent documents issued by the European Parliament in 2001,the emphasis of European policy regarding the conservation of ourcultural heritage gradually changed and became geared toward amore sustainable approach. Principles of compatibility and re-treatability have come to replace those of reversibility andrepeatability, requiring changes in both the scientific and techno-logical research approach toward cultural heritage. Biorestorationis perfectly suited to this approach.
Microbial biotechnology, on which biorestoration is based,meets these requirements and offers an opportunity to overcome
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some of the problems posed by current methods, especially thoseproblems related to damage to the artworks and toxicity for boththe restorers and the environment. Some of the main advantagesoffered by bio-based technologies are selectivity, environmentalcompatibility, low cost of implementation, low aggression towardsartworks, protection of the health of the restorers and absence ofethical implications (Webster and May 2006).
Microbial biotechnology addresses the dual and opposing rmi-croorganisms play in the context of artistic heritage: on the one handcausing damage (biodeterioration), but on the other providing newsolutions for restoration (biorestoration). Indeed, biotechnologymakes a complete, culture-independent diagnosis of biodeteriora-tion possible, allowing selective control and early monitoring to becarried out; it also provides selected microbial strains and microbialproducts suitable to establishing sustainable restoration techniques.In fact, the same metabolic mechanisms by which naturally
Fig. 1. Loggia of the Casina Farnese on the Palatine Hill (Rome). Photo Franco Adamoand Adele Cecchini.
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occurring bacteria attack the materials of the artworks can beexploited for attacking organic and inorganic patinas and deposits,which are frequently hard to remove safely (biocleaning).
This research presents a case study of biocleaning, addressingthe removal of aged deposits from mural paintings.
Cleaning is one of the most common activities carried out byrestorers of artworks and many people identify cleaning with therestoration itself. Without entering into the passionate debate thathas developed on the subject, it is worth mentioning that, althoughcleaning is considered necessary in order to enable the imple-mentation of conservation measures, the need for cleaning shouldalso be carefully evaluated, because potentially risky procedures areinvolved (Torraca, 2009).
The currently available, well-tested methods are mainly chem-ical or physics-based and display many drawbacks. These aremainly related to toxicity for restorers and the risk of furtherdamage to the artwork. Nature also provides biological compoundswhich have been used for cleaning purposes in the past. Thesecompounds include wine, soaps and a variety of vegetables (i.e.potatoes, onions, garlic) as well as physiological fluids such as urine,blood, bile fluids and saliva (Mora et al., 1984). Nowadays innova-tive biological methods are based on the use of living microbes andtheir products. Biocleaning is still experimental, largely due to thesmall number of studies published so far.
Gauri et al. (1992) was one of the first researchers who realisedthe high potential of this process, exploiting a sulphate-reducingbacterium, Desulfovibrio desulfuricans, for the removal of blackcrusts from marble. Thereafter, biocleaning of highly polymerisedprotein material from frescoes (Ranalli et al., 2005), or from papermaterial (Barbabietola, 2012) and the removal of black crusts andsalts from stone (Ranalli et al., 1997; Troiano et al., 2013) representexamples of the positive contributions of microbial technologywith regard to restoration. Biocleaning has proved effective inremoving crusts and efflorescence from both stone surfaces andwall paintings, proving to be even more highly effective in com-parison to both the commonly used cleaning chemicals (Cappitelliet al., 2007) and the more recent laser cleaning technique(Gioventù et al., 2011).
The success and development of biorestoration techniques arerelated to their ability to extend the offer of suitable and eligibleproducts which operate selectively in function of the substrate tobe removed while respecting the nature of both the material andthe artistic content. Expanding the still scant knowledge of themicrobial world and its huge, and as yet, unexplored metabolicpotential, paves the way to creating and producing new ready-to-use materials for restorers, and also creates the possibility ofinnovation for manufacturers within the sector.
The case study presented here focused on the wall paintings inthe loggias of the Casina Farnese on the Palatine Hill in Rome andwas undertaken in collaboration with the Special Superintendencefor Archaeological Heritage of Rome. The research was driven bythe difficulties faced by restorers in cleaning the coherent depositsof inhomogeneous thickness covering some areas of the paintings.Deposits were variously composed of gypsum, calcium oxalatedihydrate (weddellite), calcium carbonate, apatite and a proteincompound, probably aged casein.
The main goal of this work was to develop a risk-free procedurefor applications on vertical walls and ceilings, which would be, atthe same time, economic and feasible. The objectives were i) toidentify bacterial strains capable of selectively degrading theorganic substances and/or solubilising the inorganic substratescomposing the deposits without altering the noble patina ordamaging the artworks and ii) to identify a support matrix, asappropriate as possible in consistency and compatibility with themicroorganisms.
1.1. Case study
The Casina Farnese is mentioned in various sources as theBelvedere or Casino della Veduta (view), or as Casino dei bagni di Livia(Livia's baths), and is a structure belonging to Nero's Domus tran-sitoria (Tomei and Rea, 2011). Situated at the top of the Palatine Hill,the monument consists of a small two-storey building. Each storeycomprises a rectangular room leading, through a door, to a loggia oftwo travertine arches with balustrades overlooking the Basilica ofSt. Peter. The walls and pillars of the loggias rest upon a structure ofRoman brickwork and clearly constitute a projection annexed to alower, pre-existing construction of the Middle Ages. Building star-ted in 1579, and the double order of loggias was completed by 1593.The Casina did not have a residential function: it was a point fromwhich to enjoy the view, intended for short stays, visits, appoint-ments, or secret talks. The loggias were decorated throughout, bothinternally as externally, as evidenced by the discovery of traces ofcolour on the short sides (Cecchini et al., 1990).
On the basis of correspondence between Cardinal Alessandroand Fulvio Orsini, the lower loggia's decoration was ascribed to acertain Silvio, a pupil of Taddeo Zuccari, and that of the upper loggiato Pasqualino Livio da Forlì, while some representations of poorerquality were ascribed to Giovanni Paolo da Pesaro, who was rec-ommended to the cardinal by the influential brother of the painter.
The decoration inside the loggias follows the architecturaldesign of the building (Fig. 1). In the slightly arched area in thecentre of the ceiling of each loggia, a moulding separates thedepiction of two episodes from the legend of Ercole and Caco,representing a political metaphor of contemporary events at thattime. In the upper loggia the giant Caco brings Hercules's oxen intohis cave, pulling them by the tail, while in the lower loggia Ercole iskilling Caco. The entrance and a large landscape, that is now theonly aspect for the wall paintings still visible in the lower loggia,occupy the long walls of the two loggias. These are the parts of thelower loggia that were involved in the biocleaning tests.
The state of conservation of the loggia and the paintings wasdiagnosed by Cecchini et al. (1990). Dust deposits, whose accu-mulation is due to the intrinsic porosity of the plaster, darkened allthe painted surfaces. The dust was held in place by a film of car-bonates formed on the surface due to the presence of moisture inthe masonry, infiltration and capillary rise, which had led to theformation of calcareous deposits on the lower parts of the walls.The blackening on the surfaces was accentuated where fixative
Table 1Laboratory screening for the selection of bacterial strains able to solubilise carbonates, phosphate and gypsum and to degrade proteins. The strains belong to the “ENEA-Lilith”collection.
Strain code GenBank accessionnumber
Origin CaCO3 sol CaSO4 sol PO4 sol Gelatine 1%on TSA
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agents had been previously applied. In addition, atmospheric par-ticles, besides causing aesthetic damage, had conveyed aggressivesubstances capable of chemically reacting with the substrate in thepresence of water. The presence of water had led to soluble salts ofendogenous or external origin, such as sulphates and nitrates,forming a whitish efflorescence. As well as the above, a patina of anorange colour covered some plastered and painted surfaces. Finally,damage of a biological nature was also detectable, though confinedto only the most exposed areas.
At the same time as this work was being carried out, archivalresearch was also conducted, which was mainly aimed at findinginformation concerning any previous restoration of the paintings ofthe loggias of the Casina Farnese. The only document foundreferred to a cleaning operation dating back to 1927, which did notdescribe the methods, nor specify the products used.
In March 2012, the Special Superintendence for ArchaeologicalHeritage of Rome undertook restoration of the paintings of theloggias of the Casina Farnese with funding provided by the WorldMonuments Fund Europe. The works were completed in May 2013.
2. Materials and methods
2.1. Sampling and characterisation of deposits
In the lower loggia three samples were taken from pointsconsidered representative of the most hard-to-remove deposits. Thethickness of the deposits was uneven, so small scales detached fromthe most superficial layer and some grains scratched from the layerin direct contact with the original painted surface were collected.
The samples were analysed, without any preparation, using theFourier transform infrared spectrometer (VERTEX 70 - Bruker Op-tics), recording the spectra in the range 4000 to 400 cm�1, with adiamond compression cell in attenuated total reflectance (ATR)with a resolution of 4 cm�1 and a scan speed of 36 min. After heattreatment, Sample 2 was analysed for a second time as a driedextract in order to confirm the results of the initial analysis.
2.2. Selection of microbial strains
On the basis of general metabolic information acquired in ourprevious researches, twenty original bacterial strains belonging to
the laboratory collection “ENEA-Lilith” were chosen to undergo ametabolic screening for the selection of the best strains able to reactwith the substrates detected by FTIR-ATR. The strains were native,and thus adapted to harsh environmental conditions (Table 1), andhad been previously identified by sequence analysis of the 16S r-RNA gene, deposited in the GenBank® database.
After revitalisation, the strains were grown on TSA medium(Tryptic Soy Agar; Liofilchem, Teramo, Italy) at 28 �C for 48 h beforeundergoing specific metabolic tests in relation to the substrates tobe removed.
To test the production of extracellular proteases, we referred toVermelho et al. (1996), with some modifications. Petri dishescontaining TSA medium supplemented with gelatine 1% (w/v), orR2A medium (Reasoner and Geldreich, 1985) supplemented withgelatine 0.4% (w/v) were prepared. After sowing a spot, the plateswere incubated at 28 �C and observed for a clarification haloappearance, according to the method.
To determine the ability of bacteria to solubilise the calciumcarbonate, a modified version of the method described by NorMaL9/88 (1988) was used to identify strains producing acidic metabo-lites. Petri dishes containing TSA were prepared and glazed on thesurface with a sterile suspension of CaCO3 at 1% w/v in distilledwater (pH 8.4). After sowing a spot, the plates were left at roomtemperature. The halo of clarification on calcium carbonate patinawas observed and measured after 3 and 7 days, recording the timeof formation of the halo as well.
A similar method was employed to determine the ability ofbacteria to solubilise the gypsum, glazing on the surface with asterile suspension of CaSO4 2 (H2O).
To select the strains capable of solubilising the phosphate, thePikovskaya's medium (PVK) was used. Bacterial strains streaked onthe agar plates were incubated for up to 14 days; phosphate sol-ubilisationwas evaluated by formation of halo around the bacterialcolonies (Pikovskaya, 1948).
2.3. Strain identification and characterisation for substrateutilisation patterns in Biolog™ microplates
The phylogenetic affiliation of the selected strains wasconfirmed by both a molecular method (r-RNA 16S genesequencing (Sprocati et al., 2013) and by metabolic
Fig. 2. Biocleaning restoration of the southeast wall (SEw) of the lower loggia. S-1andS-3 indicate samples for FTIR-ATR analysis; T-1 to T-5 indicate the “cleaning tests” forpreliminary trials; Bio-1, Bio-2 and Bio-5 indicate the areas to be treated with bio-cleaning applications. The bullets indicate monitoring points, sampled at different timeafter the treatment; Photo Franco Adamo and Adele Cecchini.
Fig. 3. Biocleaning restoration of the north east wall (NEw) of the lower loggia: S-2indicates samples for FTIR-ATR analysis; Bio-3 and Bio-4 indicate areas to be treatedwith bio-cleaning applications. The bullets points indicate monitoring points afterrestoration.
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characterisation, using GP2 or GN2 BIOLOG™ Microplates, ac-cording to BIOLOG™ protocols. The microplates contained a solecarbon source in each of 95 wells, belonging to the followingclasses: carbohydrates, glycosides, acids, amides, esters, aminoacids, nucleotides, amines and alcohol. The analysis of the colourdevelopment data provided qualitative and quantitative informa-tion on the fingerprint of individual strains, including the phylo-genetic identification and the kinetics of the oxidation ofsubstrates.
Table 2Experimental design: treatments applied to “cleaning tests” (T-1 to T-5) by combining bacbiocleaning procedure. PBS (Phosphate Buffered Saline), Na-Pyrophosphate (Na-PP), TSB
Cottonwool
Cellulosepulp
Laponite 5%(w/v)
UI3E in PBS T1.1UI3E in Na-PP T 2.4UI3E in Na-PPþ Bio-Z T 2.3Bio-Z T 1.2 T 1.3UT30E in Na-PP T3TBF11E in TBS T3Laponite (control) T 1.5
2.4. Selection of a matrix as support for the preparation of “micro-packs”
For the application on the walls, the methodology of the wetpack was preferred. The term “wet pack” indicates a matrix thatincorporates an adequate amount of water solution to keep thebacterial cells alive as long as possible; herein this paper it is spe-cifically called “micro-pack”. Therefore, it was necessary to identifysuitable matrices for the immobilisation of microbial cells, whichwould be capable of maintaining a microenvironment appropriateto their metabolic functions while allowing contact with the sur-face for as long as possible without damaging the painting.
Among the products available on the market and used as sup-port in the restoration, cottonwool, cellulose pulp and Laponite®RD(Rockwood Additives, CAS N. 53320-86-8) were chosen both asmeeting the requirements mentioned in the paragraph above andfor their compatibility with cell viability. All of these agents wereused as inert fillers in the preparation of the compresses.
Laponite was mixed in small amounts, stirring continuouslywith the help of a sterile metal rod to avoid the formation of lumpsor phase separation. As soon as the dispersion started to becomeviscous, we left the Laponite to stand so that it could reach theoptimal degree of swelling.
Laponite is a colloidal clay consisting of a mixture of silicates ofsodium, magnesium and lithium. In the art of restoration Laponiteis used to remove, or render less visible, any rust stains and ferrousencrustation on frescoes. It is sometimes used in combination withcitric acid and sodium citrate, but also often with water only, toavoid further damaging already very compromised materials.
2.5. Test for cell viability in Laponite
According to data in related literature, cottonwool and cellulosepulp matrices do not interfere with the cellular activity (Bosch-Roiget al., 2010; Lustrato et al., 2012). To verify whether Laponite wouldinhibit the growth of microorganisms, cell viability was tested forup to 144 h every 24 h, by serial dilution on a TSA medium, incu-bated at 28 �C for the time required by the individual strains.
2.6. Mapping of cleaning tests and biocleaning procedure
Biocleaning tests were carried out in selected areas located intwo different zones of the lower loggia (maps are shown in Figs. 2and 3): one area was up around the door located on the southeastwall (SEw) and another around the corner of the northeast wall(NEw). The sampling points for FTIR-ATR analysis of deposits (S-1 toS-3) and the “cleaning tests” (T-1 to T-5) are shown on the map.These cleaning tests represent small surface areas where pre-liminary assays were carried out in an attempt to find an appro-priate biocleaning procedure which could subsequently be testedon larger areas (Bio-1 to Bio-5). A few monitoring points, identifiedby bullets, were chosen to make sure that no treatment residues
terial strains with different matrices in different liquid media, to establish a selective(growth medium), Bio-Z (original microbial product belonging to SACs category).
Laponite 8%(w/v)
Laponite 9% (w/v) Laponite 10% (w/v)
T 1.4T 2.2 T 1.6eT 5T 2.1
T 1eT 3T 3T 4T 1.7eT 3 T 4eT 5
Fig. 4. Flow-chart of the biocleaning procedure.
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were left on the walls. Samples were collected with swabs imme-diately after the removal of themicro-packs and, then again, up to 4month after the biocleaning treatments at the end of the restorer'sintervention. A wide array of preliminary tests were performed oneach “cleaning test”:Table 2 shows a summary of the most signif-icant combinations evaluated to select the best biocleaning pro-cedure. Cotton wool, cellulose pulp and Laponite were tested asimmobilisation matrices. Laponite was tested at different concen-trations suspended in different solutions: water, phosphate buffersaline (PBS) and sodium pyrophosphate 0.05% w/v (Na-PP), ac-cording to the needs of the bacterial strain immobilised in it. Duringeach test an area was reserved as a control for the application of apack free of bacterial cells. A microbial product, the crude extract ofBio-Z (2 mg/mL) with emulsifier properties intended as a pre-treatment to facilitate the removal of the generic dirt trappedinto the porosity of the plaster, was also tested. Bio-Z is producedfrom the original strain Pedobacter sp. MCC-ZE, isolated frompolluted soil by the ENEA team (Sprocati et al., 2012; Grimaldi,2013).
According to restorers, the maximum safe contact time for theapplication of the micro-packs was established at 48 h, this beingthe longest exposure time compatible with the need to protect thepaintings from damage. Applications, in practice, ranged between24 and 48 h.
At the end of the trial of the “cleaning tests”, the work pro-gressed to larger areas (Bio-1 to Bio-5) with Laponite prepared atthe concentration of 9% w/v in a solution varying with the bacterialstrain used; the bacterial strains varied in turn according to thesubstrate to be removed. On the SWwall, the area Bio-1was treatedwith a uniquemicro-pack containing the strain UI3E in Na-PP 0.05%(w/v) for 48 h. This length of application is necessary to produce cellstarvation. Starvation is a physiological state obtained by keepingthe bacterial cells in a solution free of any carbon source in order toinduce the depletion of the intracellular reserves, therebyprompting a more aggressive attack on the organic deposit to beremoved (casein) when the micro-pack is applied. An identicalmicro-pack was applied on the area Bio-3 for a shorter contact timeof 24 h.
The area Bio-2 was treated for 24 hwith amicro-pack containingthe strain UT30E in a TSB medium diluted in distilled water 1:10 (v/v). This "light feeding" was necessary for the solubilisering strainsin order to support the production of the metabolites responsiblefor the solubilisation of the inorganic deposits. On the NE wall,areas Bio-4 and Bio-5 were treated applying two consecutivemicro-packs in succession, each containing different bacteria: a first mi-cro-pack containing the strain TBF11E in TSB and distilled water1:10 (v/v) was applied for 24 h to solubilise inorganic deposits, thena second micro-pack containing the strain UI3E in Na-PP 0.05% (w/v) was applied for 24 h to degrade the proteinaceous layerunderneath.
The flowchart in Fig. 4 summarises the steps of the procedure.
2.7. Monitoring of residual bacterial cells after treatment
To evaluate whether the removal of the pack was effective ineliminating any residual cells, samples were collected immediatelyafter washing the surfaces of the small testing area using a sterileswab with orthogonal movements from a sample area of10 � 10 cm. Swabs were immediately transported to the laboratoryin sterile Falcon tubes and processed for viability tests. Each swabwas suspended in saline and vortexed for 30 s to allow any cells stillpresent to move into suspension. The operation was repeated 3times and then 100 ml of suspension was plated in a Petri dishcontaining the solid medium TSA. The plates were incubated at28 �C for 48e72 h and then were left at room temperature and
monitored over several weeks for the formation of new colonies.Various samples were collected up to fourmonths after the removalof the lastmicro-pack with a view to monitoring for the presence ofthe microorganisms used in the biocleaning work over a period oftime. An adjacent, untreated surface was also sampled as a negativecontrol.
3. Results
3.1. Chemical composition of the deposits
Samples of the hard-to-remove deposits were analysed at thelaboratory of the CISTeC Sapienza-University of Rome and the re-sults are reported in Fig. 5.
The Samples 1 and 2 were primarily composed of gypsum,calcium oxalate dihydrate (weddellite) and calcium carbonate. Alsosome apatite and soluble nitrate salts were detected. In Sample 2the effect of absorption at 1682 cme1 attributable to the presence ofprotein phases was also observed. For a more thorough check, thesample was again analysed after heat treatment, revealing thepresence of the amide group, thus confirming the protein nature ofthe substance. Sample 3 consisted of gypsum with the presence ofsmall percentages of weddellite and calcium carbonate.
Fig. 5. FTIR-ATR spectra of three samples of deposits S-1, S-2, S-3 (performed by CISTeC, Sapienza e University of Rome).
Table 3Survival of bacterial cells in laponite gel, under starvation (UI3E) and in growthmedium TSB 1:10 (UT30E and TBF11E).
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3.2. Selection of bacterial strains for the development of micro-packs
The results of the microbial screening are shown in Table 1. Onlytwo strains, LAM33E and TBF11E, were able to solubilise the calciumcarbonate patina.
Calcium sulphate was solubilised by the strains UI24E andTBF11E, showing a clarification halo after 48 and 72 h of incubation,respectively.
However, the capacity to solubilise phosphate was more wide-spread amongst the strains. Nine strains, out of the twenty tested,produced a clarification halo between 48 and 96 h of incubation.
After the screening for proteolytic activity, the strains CONC11E,CONC18E, LAM21E showed a marked halo around colonies grownon a TSAmedium supplemented with gelatine 1% (w/v). The strainsUI3E and UT30E were the only strains also able to form the halo onR2A medium supplemented with gelatine 0.4% (w/v).
Merging these results, we selected three strains to be used inmicro-pack applications: Stenotrophomonas maltophilia UI3E for theremoval of organic deposits; Pseudomonas koreensis UT30E for theremoval of phosphate and organic deposits and Cellulosimicrobiumcellulans TBF11E for the removal of carbonate and gypsum deposits.
Biolog microplates confirmed the identity of the strains andprovided a metabolic fingerprint for each strain: UT30E was able tometabolise 51 out of the 95 substrates, UI3E metabolised 54 sub-strates, while TBF11E showed a high metabolic diversity, being ableto use 85 substrates as a sole carbon source.
3.3. Compatibility of Laponite with the viability of the bacterial cells
The survival tests (Table 3) proved that a suspension in Laponite,of up to 144 h, did not adversely affect the viability of the cell.Changes of the microbial load over time are more readily attrib-utable to the physiological conditions that strains are kept in:starvation for the strain UI3E, lightly decreasing the microbial load,and diluted growth medium for the strains TBF11E and UT30E,increasing the microbial load.
3.4. Biocleaning of painted walls
The areas selected for the biocleaning tests (Figs. 2 and 3) didnot exhibit any detachment of the pictorial film from its surface, norany chipping of the plaster supporting it. On the basis of theoutcome of the “cleaning tests”, Laponite was the only support thatallowed for the application of micro-packs to vertical surfaces and
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also on ceilings in such an easy and homogenous way, without anydripping of liquids. Moreover, when used at the concentration of 9%w/v in Na-PP, or in diluted TSB solution, Laponite showed optimalswelling, allowing for perfect adhesion to the vertical walls, thusprolonging the contact time of the packs. The addition of the bio-emulsifier Bio-Z did not have a significant influence as a pre-treatment, nor as adjuvant for bacteria. The strain TBF11E wasable to work on the grey deposits (gypsum and carbonates), UI3E onthe organic orange-brown layers (probably of protein origin), andUT30E on compounds based on phosphates and it was also able toremove the organic material.
Biocleaning using Laponite micro-packs was then carried out onlarger areas. A micro-pack containing UI3E was applied to theorange-brown layer covering the architrave over the door on the SEwall (Bio-1). After 48 h the deposit appeared almost completelyremoved, with the exception of a small portion of greater thicknessthan the rest (Fig. 6a and b). A micro-pack containing UT30E wasapplied on the area Bio-2 which was covered by a deposit of vari-able thickness and of a crusty and non-homogeneous colour (inweb version). When the micro-pack was removed after 24 h, evi-dence of a previous restoration was revealed: this was in the formof a white frame on a red (in web version) background creating ageometric design in imitation of the original and a filling made ofplaster to cover the recess of the door frame. An identical micro-pack was applied to the area Bio-3, which was covered by a mixeddeposit of variable thickness consisting of an orange-brown layer,directly apparent on the surface or covered by a grey layer. .A singlemicro-pack containing UI3E removed the orange-brown layer andthinned the greyish deposits after 24 h of contact.
On the NE wall, a thick and layered deposit, consisting of a darkgrey layer hiding an underlying orange-brown layer, covered thearea Bio-4 (Fig. 7). Two micro-packs were applied in succession,each being removed after 24 h: the first contained TBF11E, which iscapable of solubilising gypsum and carbonates, and the secondcontained UI3E, which is suited to degrading proteins. Afterremoving the first pack, the surface layer of the deposit waseffectively removed in both areas, revealing the layer underneath.After the removal of the second pack containing UI3E, the effect ofthe cleaning process appeared more homogeneous, with enhancedbrightness penetrating to the surface of a previous repainting of theplaster In the area Bio-5, showing the same deposits, the removalwas effective as well, although less uniform.
After the biocleaning, restorers were able to complete therestoration of the areas concerned using compresses of ammoniumcarbonate. This process was followed by the pictorial retouching.The final result obtained on the architrave (Bio-1) is shown inFig. 6c.
3.5. Monitoring after treatments
The results of viability tests conducted on samples collectedover a period of time, starting immediately after the biocleaningtreatments and continuing up to the conclusion of the restoration,indicated a rare presence of bacterial colonies, ranging from 0 to
Fig. 6. Biocleaning of the area B-1 using a single micro-pack containing S. maltophilia U
30 CFU per plate (corresponding to an area of 10 � 10 cm). Thedetectable morphotypes were different from the strains used in themicro-packs. They changed over time and were detected also onuntreated areas (control samples), thus representing environ-mental transitory populations unrelated to the biocleaningtreatment.
4. Discussion
Deposits that were more hard-to-remove covering the wallpaintings of the lower loggia of the Casina Farnese appeared mostlycompact and coherent, except for the areas close to the floor pavingwhich showed cracked or flaked surfaces. The presence of anorange-brown layer was not found throughout the loggia, but wasapparent only on the NE and SE walls, which had been affected inthe past by restoration, which had entailed the renovation of por-tions of the plaster. This past restoration work had resulted in anirretrievable loss of the pictorial decoration, which in some caseshad been restored in such away as to imitate the original paintings.
The spectroscopic analyses, carried out on three samples takenfrom the walls of the lower loggia, revealed a varied composition inthe layer covering the walls, where the main compounds weregypsum, calcium oxalate dihydrate (weddellite) and calciumcarbonate.
The presence of calcium carbonate can be attributed to thecarbonate nature of the plaster on which the paintings wereexecuted: the calcium carbonate could have been drawn up to thesurface or could have been fixed in the surface by moisture as aresult of wind erosion. Gypsummay have been deposited as a resultof the plaster sulphatation processes reacting with the pollutedatmosphere, which may have also led to the creation of the blackcrusts on the travertine areas. The gypsum may also have partiallycome frommortar and have been brought to the surface bywater asthe result of capillary infiltration of the walls. The presence ofcalcium oxalate on the exterior of stone monuments is the subjectof a discussion based on two opposite assumptions concerning itsorigins. Some authors argue that the production of oxalate canoccur following the microbial degradation of organic substancesused in the process of repainting and the subsequent secretion ofacids that react with the calcium carbonate of the support(Lazzarini and Salvadori, 1989), while others do not rule outconcomitant causes of abiotic origin (Martín-Gil et al., 1999; Cariatiet al., 2000; Pavía and Caro, 2006). The almost complete absence ofvisible microflora in the lower loggia would initially indicate thatcalcium oxalate is linked to an abiotic origin. However, as thespectroscopic analyses detected the presence of apatite and ageneric protein substance as well, we can advance the hypothesisthat the calcium oxalate is the product of the degradation of a su-perficially applied compound. Unfortunately, the archival researchcarried out did not provide documentation describing themethods,or the products used in past restoration.
In the case of paintings of the lower loggia, we can assume that asurface treatment was applied with a brush in order to consolidatethe most deteriorated portions of plaster, namely those on the NE
I3E. a) before biocleaning; b) after biocleaning and c) after the pictorial retouching.
Fig. 7. Biocleaning of the area Bio-4, using two micro-packs applied in succession. Left panel: a) dark and thick patina covering the painting; b) after the application of the firstmicro-pack containing C. cellulans TBF11E; c) after the application of the second micro-pack containing S. maltophilia UI3E. The gradual removal of the patina is shown in the rightpanel: plot profiles display a two-dimensional graph of the intensities of the pixels along the line selection corresponding to the vertical bar on the pictures (Grey value ¼ pixelintensity, Image J 1.47).
M. Mazzoni et al. / International Biodeterioration & Biodegradation 94 (2014) 1e118
and SE walls which had been affected by renovations in the pastand that this process had also been extended to the surroundingpainted surfaces. The consolidation could have been made withcasein (a phosphoprotein) or a derivative thereof, i.e. calciumcaseinate (a traditional material used in the restoration). Thiswould explain the presence of apatite, a calcium phosphate(Lazzarini and Salvadori, 1989; Martín-Gil et al., 1999; GarofanoMoreno, 2011).
The removal of these deposits has been deemed difficult byrestorers, as packs of cellulose pulp kneaded in a solution ofammonium carbonate have not produced the expected outcome.Therefore, based on some positive results already obtained by otherauthors on different substrates and conditions (Antonioli et al.,2005; Alfano et al., 2011) we experimented with a process of bio-logical cleaning.
The first phase of this work was carried out in the laboratory toidentify the most suitable microbial strains to use from ourcollection. Non-pathogenic bacteria and preferably non-spore-forming ones were selected in order not to leave dormant forms,capable of revitalise subsequently.
Numerous studies have investigated the ability of microorgan-isms to precipitate calcium carbonate, a process that has attractedthe interest of researchers as a possibility for use in applications ofbiorestoration, while studies aimed specifically at identifying mi-crobial species able to solubilise carbonates are still rare (Boquetet al., 1973; Friis et al., 2003). This reflects what some authorshave found in the environment, where biomineralisation is a verywidespread function among microorganisms, while solubilisation
is much rarer. In a previous work we found that 5% of microor-ganisms isolated from the Etruscan tomb of Mercareccia were ableto solubilise carbonate patinas, while about 65% showed the abilityto precipitate calcite crystals varying in size, colour and quantity(Barbabietola et al., 2012).
Among the 20 strains that underwent screening for carbonatesolubilisation, only two developed a clarification halo around thecolonies after 168 h of incubation: Pseudomonas fluorescens LAM33E
and C. cellulans TBF11E, which probably owe this capacity to theirextracellular metabolites (Davis et al., 2007).
The ability to solubilise calcium sulphate was expressed by onlytwo strains: Plantibacter sp. UI24E and TBF11E. In biocleaning litera-ture a similar case of aerobical removal of gypsum from art work hasnot yet been reported; rather, anaerobic sulphur bacteria have beenused for the removal of black crusts (Ranalli et al., 2000; Cappitelliet al., 2007; Gioventù et al., 2011). These black crusts, while pre-senting a different morphology, are similar in chemical compositionto the deposits on wall paintings, as they mainly consist of gypsum.
More prevalent instead, among the strains tested, was the abilityto mobilise phosphates. After all, the ability of bacteria, especiallysoil bacteria, to mobilise phosphate from the minerals present inthe soil is known, making it available for plants (Ivanova et al.,2006; Sprocati et al., 2013). The mechanism of solubilisation ofphosphates by bacteria is mainly made possible thanks to theproduction of organic and inorganic acids, the pH decrease and thecations complexation (Chen et al., 2006; Malboobi et al., 2009).
Following the screening, the strain C. cellulans TBF11E wasselected to remove both gypsum and calcium carbonate. Given the
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presence of a carbonatic plaster immediately underlying thesuperimposed substances, the strain TBF11E was used in preferenceto the other carbonate solubilisers, as it was only moderatelyaggressive, displaying a longer clarification time (168 h) and a smallhalo (<1 mm), thus safeguarding the plaster from a possibleaggressive attack. On the contrary, the strain UT30E was preferredas a phosphate solubiliser, being considered the most effective andthe fastest acting and forming the greatest clarification halo(3e4 mm) after only 48 h of incubation.
Proteolytic activity was tested on strains in the laboratorycollection previously characterised by the ability to effectivelydegrade organic substances, such as resins, wax and for attackingshellac (Barbabietola, 2012). Among the positive strains Pseudo-monas stutzeri CONC11E, Achromobacter xylosoxidans CONC18E,Acinetobacter calcoaceticus LAM21E S. maltophilia UI3E and P. kore-ensis UT30E, the strains UT30E and UI3E were preferred, as theywere able to hydrolyse gelatine on both of the culture media used.The proteolytic activity found in UI3E is compatible with another S.maltophilia strain, described as producer of an extracellular alkalineprotease (Miyaji et al., 2005).
After selecting the suitable strains, we proceeded to test thebiocleaning procedure in situ at the Casina Farnese using the strainUI3E for the proteinaceous layers, UT30E both for the phosphatesand proteinaceous matter and TBF11E for the gypsum andcarbonates.
First, it was decided to adopt the wet pack method traditionallyused for cleaning architectural and painted surfaces, as it is aconvenient way of increasing the contact with the reagent whilelimiting, as much as possible, the penetration of the reagent to thelower layers of the artwork. We therefore identified the mostsuitable support among three different matrices: cotton wool, cel-lulose pulp, and Laponite. Laponite, unlike the other two matrices,showed a good capacity to preserve the viability of the immobilisedbacteria throughout the application time, and could be applied onvertical surfaces without suffering any dripping due to leakage ofthe cell suspension. It was further noted that the PBS buffer, inwhich the bacterial cells were usually resuspended, hindered thefull swelling of Laponite, which did not reach the ideal viscosity. Byreplacing the PBS buffer with a solution of sodium pyrophosphate(0.05% w/v) the suspension jellified in a few minutes. These abovephenomena were unexpected, but are integral to the innovation,since they have brought about improvements in the method ofusing the Laponite, with respect to the current practices.
We must also specify that Laponite is not a source of nutritionfor these strains and does not negatively interfere with cellularmetabolism. As compared to other matrices used as delivery sys-tems for biocleaning, Laponite proved to be highly compatible withcell viability. Cappitelli et al. (2006) reported the viability ofDesulfovibrio vulgaris in three delivery systems, finding a reductionin the number of cells by two orders of magnitude in Carbogel, byfour orders in Hydrobiogel and by five orders in Sepiolite. InLaponite we measured a decrease of microbial load only for thestrain UI3E (one order after 24 h and three orders after 144 h)which, however, can be explained by the starvation condition. Forthe strains TBF11E and UT30E, in contrast, an increase of one orderof magnitude was noted. This is most probably due to the sus-pension in the diluted growth medium.
After applying the micro-pack on the surface, the gel wascovered by a film of PVC, principally to maintain the moisturecontent at a constant level, thus preventing the micro-pack fromdrying out.
The contact time was determined by the restorers according tothe thickness of the deposit to be removed and the results obtainedin the preliminary tests, but the decision had also to take into ac-count the weather conditions.
The micro-packs were left in situ for 24 or 48 h, but the contacttime does not appear to have affected the level of clean-up ach-ieved. Generally, a single micro-pack application was never suffi-cient, due to the thickness, the layers and the variable compositionof the deposit to be removed. During the preliminary tests in situ itwas observed that S. maltophilia UI3E had a greater affinity inremoving the orange-brown (protein) deposit, C. cellulans TBF11E
was the most effective as regards the inorganic grey deposit and P.koreensis UT30E had a good cleaning action on both the deposits.
The strains were tested in various combinations: singly, in aconsortium and in succession. The application of micro-packscontaining individual strains produced more effective results thanthose containing a microbial consortium. The application in suc-cession of micro-packs containing individual bacterial strainsappeared to be the best solution when deposits were layered. Anexample of the effectiveness of the treatment in succession isshown in Fig. 7, where the gradual removal of the dark and thickpatina is confirmed by the enhancement of brightness on the wall.After biocleaning, the most hard-to remove deposits had beenremoved from the areas most severely damaged and the restorershad achieved excellent results with regard to the restoration(Fig. 6).
The procedure of biocleaning established by the study proved tobe selective, as the microorganisms restricted their metabolic ac-tion to degrading and/or solubilising specific components of thedeposits. As a result, the cleaning was more gradual, controllableand able to safeguard the surface of the painting.
A further positive point demonstrated is that the Laponite mayhave facilitated the process of cleaning by softening the layer ofdeposit to be removed, as seen in Rozeik (2009), with a synergisticeffect.
Some authors argue that, for a cleaning operation using micro-organisms to be successful, the temperature of the surface areashould remain stable (Bosch-Roig et al., 2013). In this study, itwould have been difficult to thermally isolate the loggia, which isaffected not only by the seasonal climate, but also by daily varia-tions in temperature and, in addition, by the restorers' presence andthe incandescent lamps used during the working operations. As aresult of these uncontrollable circumstances, the micro-packs hadto be applied in temperatures ranging between 6 �C and 37 �C.Despite this, no significant differences were detected in the efficacyof the cleaning treatment.
The use of viable microbial cells represents then a greatadvantage, compared to the use of purified enzymes, which usuallyrequire rather more restrictive conditions (Wolbers, 2002; Ranalliet al., 2005). In fact, bacteria are capable of homeostasis, whichrefers to self-regulating processes that living organisms use tomaintain their internal stability. Bacteria can also self-regulate,adjusting to the ever-changing environmental conditions thatsurround them. Themain homeostatic processes that guarantee thesurvival of bacteria include iron and metal homeostasis, pH ho-meostasis and membrane lipid homeostasis (Hutkins and Nannen,1993; Andrews et al., 2003; Zhang and Rock, 2008; Krulwich et al.,2011).
To answer the question of the duration on surfaces of unwantedresidues attributable to the biological treatment, especially bacte-ria, we can say that the monitoring carried out immediately after,and up to four months after the removal of the micro-packs,confirmed that the microbial species used during biocleaning donot persist on the surface. However, it must be mentioned thatsome products used in the finishing process may have positivelyaffected this result.
The use of Laponite on textile (feathers), paper and parchmenthas already been reported (da Silveira, 1997; Totten, 2003) and hasdemonstrated some negative side effects, including that of gel
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residues altering the artefacts. The modification of the Laponitepreparation we proposed (concentration and solvent) provided amore resistant and malleable gel, which was easily removablewithout leaving either gel, or bacteria residues on the walls. Oncethe gel was removed, the deposit layers were reduced and softened,so that the residual deposits could be gently removed simply with asponge dampened with water and an appropriate brush.
Finally, the procedure does not damage the pictorial layer or theunderlying noble patina and does not use toxic products. Theprocedure is thus safe for the artwork, for the restorer's health andfor the environment. The procedure is under a patent claim (n.RM2013A000519). The bacterial strains are an integral part of thebiotechnological process and are deposited at the DSMZ microbialstrains collection with access numbers DSM 27225 (S. maltophiliaUI3E), DSM 27226 (P. koreensis UT30E) and DSM 27224 (C. cellulansTBF11E). The phylogenetic affiliation of the strain TBF11E is acceptedby DMSZ on a provisional basis as C. cellulans, since it could be anew genus; its entire genome is now undergoing sequencing.
5. Conclusion
Laponite micro-packs containing individual original bacterialstrains, non-pathogenic and non-spore-forming, were appliedsingly or in succession to degrade organic substrates such as casein(S. maltophilia UI3E), to solubilise gypsum and carbonates (C. cel-lulans TBF11E) and mobilise phosphates (P. koreensis UT30E). Thenovel combination of bacteria with Laponite is here proposed forthe first time as a delivery system for biocleaning, being highlycompatible with cell viability and allowing easy application andremoval of themicro-packs on vertical surfaces or ceilings, withoutany dripping.
The outcome of this study is a novel biocleaning procedure(patent claim N. RM2013A000519) for the selective removal ofcoherent deposits, even when layered on wall paintings. The pro-cedure is safe for the artwork, the restorer's health and the envi-ronment and has contributed to overcoming the difficultiesencountered by restorers in cleaning hard-to-remove aged depositson the wall paintings of the lower loggia of Casina Farnese on thePalatine Hill, in Rome. No operating limitations are required; theprocedure is easy, effective in aerobic conditions and has beenproven to work in a range of temperatures from 6 �C to 37 �C.
The use of several original strains with diversified metabolicfunctions, at times used in combination, represents an effort toextend the offer of safe and selective products to create a bio-restoration market.
Improvements such as shortening the contact time, realising aready-to-use kit for restorers and adjusting the procedure for othermaterials, can be envisaged in the perspective of implementingmarketable applications.
Acknowledgements
The financial support for this research was provided by ENEA.Authors thank Dr. Maria Laura Santarelli of CISTeC “Sapienza”-University of Rome, for supervising the FTIR-ATR analysis of de-posits, the World Monuments Fund Europe, the architect GiuseppeMorganti of the Special Superintendence for Archaeological Heri-tage of Rome, and the restorers Franco Adamo, Chiara SciosciaSantoro and Corinna Ranzi for their kind invitation to test innova-tive cleaning solutions.
Appendix A. Supplementary data
Supplementary data associated with this article can be found, inthe online version, at http://dx.doi.org/10.1016/j.ibiod.2014.06.004.
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