10.6092/issn.1973-9494/7123
Negli ultimi decenni è cresciuto il contributo che le diverse discipline scientifiche hanno dato al recupero e alla conservazione del patrimonio artistico per trovare soluzioni sempre più efficaci e rispettose del bene, dell’operatore e dell’ambiente.Un esempio è un brevetto sviluppato dall’Università degli studi di Milano che prevede l’utilizzo di alcuni specifici batteri come agenti di pulitura. Tale metodo, indicato col termine di bio-pulitura propone, tra gli altri, l’impiego del batterio solfato riduttore Desulfovibrio vulgaris per la rimozione di alterazioni solfatiche da superfici litoidi. Recentemente, il brevetto è stato acquisito dallo spin-off universitario Micro4yoU che ha avviato una serie di investimenti per sviluppare, a partire dal prototipo di laboratorio, un prodotto commerciale.Nel presente lavoro verranno illustrati: il meccanismo di desolfatazione ad opera di Desulfovibrio vulgaris, il processo di trasferimento tecnologico affrontato per ottenere un formulato microbico utilizzabile in cantiere e due casi studio applicativi.
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Daniele Daffonchio
Conservation Science in Cultural Heritage
artwork
Sulfate Reducing Bacteria as Bio-cleaning Agents: Development of New Methodologies and Study Cases
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https://conservation-science.unibo.it/
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Articles
2017-06-01
2017-07-04
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2017-09-04
109-119
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Daniele Daffonchio
2015
10.6092/issn.1973-9494/7123
109
Università degli Studi di Milano; King Abdullah Univ. of Science and Technology (KAUST)
1973-9494
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Sulfate Reducing Bacteria as Bio-cleaning Agents: Development of New Methodologies and Study Cases
https://conservation-science.unibo.it/article/download/7123/6833
15
S     ULFATE REDUCING BACTERIA AS BIO-CLEANING
      AGENTS: DEVELOPMENT OF NEW METHODOLOGIES
      AND STUDY CASES

    Annalisa Balloi*9
    Micro4yoU S.r.l., via Carlo Freguglia 2, 20122 Milan, Italy
    Emanuela Lombardi
    Micro4yoU S.r.l., via Carlo Freguglia 2, 20122 Milan, Italy
    Dipartimento di Scienze per gli Alimenti, la Nutrizione e l’Ambiente, Università
    degli Studi di Milano, via Celoria 2, Milan 20133, Italy
    Federica Troiano, Andrea Polo, Francesca Capitelli
    Dipartimento di Scienze per gli Alimenti, la Nutrizione e l’Ambiente, Università
    degli Studi di Milano, via Celoria 2, Milan 20133, Italy
    Davide Gulotta, Lucia Toniolo
    Dipartimento di Chimica, Materiali, e Ingegneria Chimica (CMIC) “G. Natta”, Po-
    litecnico di Milano, Via Mancinelli 7, 20131 Milan, Italy
    Anna Lucchini
    Anna Lucchini Restauri S.r.l., Via Cardinale Mezzofanti, 20133 Milan, Italy
    Daniele Daffonchio
    Dipartimento di Scienze per gli Alimenti, la Nutrizione e l’Ambiente, Università
    degli Studi di Milano, via Celoria 2, Milan 20133, Italy
    King Abdullah Univ. of Science and Technology (KAUST), Biological and Environ-
    mental Sciences and Engineering Division, Thuwal 23955-6900, Saudi Arabia




                                                                                                   CONSERVATION SCIENCE IN CULTURAL HERITAGE
    Keywords: sulfation; Desulfovibrio vulgaris; bio-cleaning; SRB; artwork


    1. Introduction

   Sulfation is a very common pathology in stone, especially in urban areas, due to air
pollution.
   Sulfur dioxide (SO2) is a pollutant which converts to sulfite ions (SO32-) in the pres-
ence of moisture and is then oxidized to sulfate ions (SO42-) by oxidizing agents such
as oxygen, ozone and hydrogen peroxide [1]. In these conditions, sulfates interact
with the calcium carbonate of the stone forming bi-hydrate calcium sulfate or gypsum
(CaSO4∙2H2O). Gypsum undergoes processes of dissolution and recrystallization on
areas exposed to rain, resulting in the erosion of the altered stone. Moreover, if the
surface is not exposed to rain, minerals and smog particles could become entrapped in
the porous surface of the gypsum, leading to the formation of black crusts [1].
   Traditional chemical and mechanical treatments are not selective towards the al-
teration, attacking not only the black crust but also the sound stone. An alternative
cleaning technology, employing sulphate-reducing bacteria (SRB), was first proposed


*   Corresponding author: annalisa.balloi@micro4you.eu

                                                                                             109
                                                                                                                                      by Atlas and colleagues (1988). They used a specific strain of Desulfovibrio for the re-
Balloi, Lombardi, Troiano, Polo, Capitelli, Gulotta, Toniolo, Lucchini, Daﬀonchio - SULFATE REDUCING BACTERIA AS BIO-CLEANING



                                                                                                                                      moval of sulfates from a stone cornice of the Museum of Natural History in Chicago by
                                                                                                                                      immerging the sample in a broth culture for 24 hours. The experiment was successful
                                                                                                                                      and the bacteria were able to dissociate the gypsum [2]. In fact, due to its metabolism,
                                                                                                                                      Desulfovibrio vulgaris reduces sulphate ions while calcium ions react with carbon diox-
                                                                                                                                      ide allowing the precipitation of calcite, according to the following reaction:

                                                                                                                                                   6 CaSO4 + 4 H2O + 6 CO2 → 6 CaCO3 + 4 H2S + 2 S + 11 O2

                                                                                                                                          From that first experiment, other tests were done until one research group at the
                                                                                                                                      University of Milan patented some microbial cells as cleaning agents, including Desul-
                                                                                                                                      fovibrio vulgaris.
                                                                                                                                          This innovative and environmental friendly cleaning technology has been applied
                                                                                                                                      successfully on important cultural monuments, including two artworks by the sculptor
                                                                                                                                      J. Eberle, used to decorate the Castle of Buon Consiglio of Trento [3]; G. Lazzerini’s
                                                                                                                                      sculpture “Allegoria della Morte” [4]; one tile from the Cathedral of Milan [5]; and one
                                                                                                                                      area of the base of the Rondanini Pietà [6].
                                                                                                                                          In light of interesting development opportunities and potential market externalities,
                                                                                                                                      the University spin-off Micro4yoU acquired the exclusive patent by initiating the project
                                                                                                                                      Micro4Art, aimed at implementing a line of biological products and diagnostic ser-
                                                                                                                                      vices, and training and assistance for restoration professionals. The first line product
                                                                                                                                      is Micro4Art-sulfates, an appropriate microbial formulation for treating stone surfaces
                                                                                                                                      and for effectively removing sulfate alterations [7].
                                                                                                                                          Initially, the product was a prototype, it was produced in laboratory and directly ap-
                                                                                                                                      plied, but with huge limitations such as: i) low productivity; ii) high production costs; iii)
                                                                                                                                      impossibility to store the product for long periods. For the removal of particularly thick and
                                                                                                                                      persistent black crusts, the application of the microbial formulation has to be carried out
                                                                                                                                      several times and though effective, affects the global cost of restoration. Lastly, although
                                                                                                                                      mural paintings suffer from this phenomenon of sulfation, no testing has yet been done.

                                                                                                                                         2. Optimization of the productive process and bacterial biomass production

                                                                                                                                          The research project, finalized to optimize the productive process of D. vulgaris and
                                                                                                                                      to develop a correct method for bacterial biomass long-time conservation, consisted
                                                                                                                                      of several steps.
                                                                                                                                          Initially, the previous laboratory protocol was reproduced in 3 L flasks in order to es-
                                                                                                                                      tablish the bacterial growth curve, verify its metabolic response at different growth con-
                                                                                                                                      ditions, and identify the parameters to act on in order to improve biomass production.
                                                                                                                                      By shifting the fermentation process from flasks to pilot plants (9 and 75 L fermenters),
                                                                                                                                      it was possible to control the main parameters, such as pH and H2S concentration.
                                                                                                                                      Indeed, an over-accumulation of hydrogen sulfide is toxic for bacteria and causes a
                                                                                                                                      drastic reduction in pH, leading to unfavourable growing conditions.
                                                                                                                                          In their entirety, these adjustments have increased the production of bacterial biomass
                                                                                                                                      (at least two orders of magnitude) and have reduced production times by about 40%.
                                                                                                                                          Regarding its conservation for long periods, the biomass was lyophilized testing dif-
                                                                                                                                      ferent cryoprotectants. The addition of lactose was the most effective in terms of post-
                                                                                                                                      rehydration cellular vitality among all the methods utilized, ensuring product stability for
                                                                                                                                      not less than 12 months [8].


                                                                                                                                110
  3. Study cases

  3.1 The statue of Neera

   The development of new application methods using the microbial Micro4Art-sul-
fates formulation, finalized to reduce the time and number of applications needed for
sulfate alteration removal, had been successfully applied on the funeral monument of
the writer Anna Zuccari, pen name Neera, built at the beginning of the 1900s and cur-
rently located in the Famedio of the Monumental Cemetery of Milan [7].

  3.1.1 Conservation state of the artwork

    Before restoration, the overall state of conservation of the artifact was fair. Moreo-
ver, before the statue was moved, it was subject to the constant mechanical, physical
and chemical actions of atmospheric agents, as well as the natural aging of the marble,
all of which contributed to deterioration. In particular, before starting restoration, two
different kinds of alteration were present: one typical of run-off zones, in which the
surface of the marble appeared compact but very rough and grainy, also characterized
by an extensive superficial and intergranular deposit involving widespread darkening
of the surface; the other, typical of those areas protected by run-off, was characterized
by black crusts with sulfates. The clear division of these areas gave rise to a marked
chromatic alteration of the artwork.

  3.1.2 Biocleaning assays

    The “Soprintendenza per i Beni Architettonici e Paesaggistici per le Provincie di
Milano, Bergamo, Como, Lecco, Lodi, Pavia, Sondrio e Varese” (arch. L. Corrieri,
official responsible for the procedure – reference number 6732) commissioned the




                                                                                                   CONSERVATION SCIENCE IN CULTURAL HERITAGE
University spin-off Micro4yoU to perform biocleaning assays on alteration by sulfides
on the statue. The method suggested by the spin-off included the use of Micro4Art-
sulfates bio-formulation based on sulfate-reducing bacteria.
    The Micro4Art bio-formulation was applied both alone or with a non-ionic surfac-
tant, commercially known as Tween20. Tween20 is commonly used in restoration and
cleaning procedures on stone surfaces with a concentration range between 3%-10%.
Being a very gentle aqueous detergent, Tween20 guarantees high compatibility with
microbial cells and hence it can be used with the Micro4Art bio-formulation. The treat-
ments on small tested areas of the statue were done as follows:
    Treatment 1: 7 applications of 22 h each with Bio-treatment (Micro4Art)
    Treatment 2: 3 applications of 22 h each with Surfactant (Tween20)
    Treatment 3: 1 application of 22 h with Surfactant + 2 applications of 22 h with
    Bio-treatment.

   Micro-fragments from different treated surfaces were sampled and analyzed. Un-
treated surface samples were also collected as control. The collected samples, type of
treatment and reaction times used for the complete removal of black crusts are listed
in Table 1.




                                                                                             111
                                                                                                                                                                      Table 1. Analyzed samples.
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                                                                                                                                                                                                Number of applications and
                                                                                                                                         Sample              Type of Treatment
                                                                                                                                                                                                     reaction times
                                                                                                                                                      Untreated area (solid black crust
                                                                                                                                        TZ CRc                                                                 -
                                                                                                                                                                  sample)
                                                                                                                                                    Solid black crust sample treated with
                                                                                                                                        TZ CHb                                                          3 appl. of 22 h
                                                                                                                                                                 Surfactant
                                                                                                                                                    Solid black crust sample treated with
                                                                                                                                        TZ BIOc                                                         7 appl. of 22 h
                                                                                                                                                                  Micro4Art
                                                                                                                                                    Solid black crust sample treated with   1 appl. of 2 h (Surfactant) + 2 appl. of
                                                                                                                                       TZ CH BIO
                                                                                                                                                           Surfactant+Micro4Art                         22 h (Micro4Art)



                                                                                                                                        3.1.3 Evaluation of biocleaning assays

                                                                                                                                         Biocleaning assays carried out on the funeral monument are globally effective in the
                                                                                                                                      removal of black crusts, while displaying some differences.
                                                                                                                                         The Micro4Art treatment used alone or with Tween20 was more selective for the
                                                                                                                                      gypsum component with respect to the treatment with the surfactant alone. Moreover,
                                                                                                                                      at microscopic level, the result obtained with the treatment using only bio-formulation
                                                                                                                                      and with the surfactant, a certain heterogeneity was observed; this effect was not vis-
                                                                                                                                      ible when the combined treatment was done. Another advantage of the combined ef-
                                                                                                                                      fect of surfactant + bio-formulation is the reduction in application time and duration,
                                                                                                                                      conferring considerable economic benefits, as well as respecting the artwork. Indeed,
                                                                                                                                      a long-term application of aqueous packs on stone substrates may have negative ef-
                                                                                                                                      fects on the artwork’s conservation.

                                                                                                                                        3.1.4 Restoration of the artwork

                                                                                                                                         In accordance with the results obtained from the biocleaning tests and with the So-
                                                                                                                                      printendenza, a proper restoration strategy was designed for the entire statue surface
                                                                                                                                      (Figure 1), and was performed by the restorer Dr. Eleonora Gioventù. The restoration
                                                                                                                                      procedure consisted of different steps, summarized as follows:
                                                                                                                                         •     removal of dust and micro-suction;
                                                                                                                                         •     stone surface cleaning and removal of black crusts using a Tween20 pack and fol-
                                                                                                                                               lowing application of Micro4Art-sulfates bio-formulation, provided by Micro4yoU;
                                                                                                                                         •     removal of base stucco and reintegration;
                                                                                                                                         •     surface and aesthetical trimmings.
                                                                                                                                         Sulfate removal from mural paintings of the Queen Theodolinda Chapel in Monza
                                                                                                                                      Cathedral
                                                                                                                                         The object of the experimentation was the pictorial cycle decorating the Queen The-
                                                                                                                                      odolinda Chapel situated in Monza Cathedral (Figure 2). The artwork was subjected
                                                                                                                                      to an intervention of mild conservation, involving the most prestigious Italian research
                                                                                                                                      Institutes1 and important sponsors. The experimentation carried out until now is not
                                                                                                                                      exhaustive and is still in progress and is intended to be a first innovative approach to
                                                                                                                                      the delicate issue of sulfate removal from mural paintings.



                                                                                                                                112
Figure 1. Upper body of the statue of Anna Zuccari, pen name Neera. On the left: before
restoration; on the right: after restoration.

   3.2.1 Artwork description and state of conservation

    The pictorial cycle “Delle Storie della Regina Teodolinda” painted by “La Bottega
degli Zavattari”, was started in the first half of 1400 and completed in 1446, as stated
in the contract. It is composed of 45 scenes which narrate episodes of life at court, with
a precious description of clothes and armours, presenting an extraordinary description
of courtly life in 15th century Milan.
    The artwork was almost entirely realized using colors diluted in organic media, some-




                                                                                                       CONSERVATION SCIENCE IN CULTURAL HERITAGE
times using milk of lime as a basis or, very seldom, the “buon fresco” technique. This
technical choice has allowed for a particular chromatic vivaciousness through consider-
able recourse to gilding carried out with the method of “pastiglia a rilievo” (“tablet re-
lief”) using gypsum and glue, whereas the dresses, damasks and armours are made of
golden or silver tin foil and veiled with copper resinate and red lacquer. At the same time,
the choice of the organic binders is in itself the cause of extreme weakness, exacerbated
by decay owing to ageing and manifold past restoration interventions.




Figure 2. Pictorial cycle decorating the Queen Theodolinda Chapel situated in Monza Cathedral.


                                                                                                 113
                                                                                                                                         The trial with sulfate-reducing bacteria was carried out on two scenes belonging
Balloi, Lombardi, Troiano, Polo, Capitelli, Gulotta, Toniolo, Lucchini, Daﬀonchio - SULFATE REDUCING BACTERIA AS BIO-CLEANING



                                                                                                                                      to the pictorial cycle of the Queen Theodolinda Chapel: Scene 16 (upper rectangle in
                                                                                                                                      Figure 2) and 33 (lower rectangle in Figure 2).
                                                                                                                                         Scene 16 belongs to the group describing the preliminaries to the wedding of
                                                                                                                                      Theodolinda and Autharis. It represents the episode in which Autharis is informed
                                                                                                                                      of Theodolinda’s arrival in Lombardy by the delegates (Figure 3a). This scene,
                                                                                                                                      which has never undergone cleaning, was characterized by exfoliation and blister-
                                                                                                                                      ing of the pictorial film, caused by previous sulfation, still ongoing at the moment
                                                                                                                                      of treatment.
                                                                                                                                         Scene 33 describes the apparition in Theodolinda’s dream of the Holy Spirit, in the
                                                                                                                                      form of a dove, suggesting the place where she should build her church (Figure 3b).
                                                                                                                                         In this scene the superficial salts appeared in a crystalline form, previously treated
                                                                                                                                      for sulfate removal by means of packs of sepiolite, deionized water and ion-exchange
                                                                                                                                      resins.




                                                                                                                                                                    Figure 3. Scene 16 (a) and 33 (b).

                                                                                                                                         3.2.2 Experimental setting and results

                                                                                                                                          For sulfation removal, Micro4Art-sulfates were used in a freeze-dried form; before
                                                                                                                                      application, it was rehydrated with deionized water to the concentration of 5´107 cells/
                                                                                                                                      ml. Two different thickeners were selected for the installation of the packs: Carboneu-
                                                                                                                                      tralgel and Arbocel BWW40 (Bresciani Srl).
                                                                                                                                          With respect to Scene 16 (Figure 4), initially, a test application was performed in or-
                                                                                                                                      der to check the compatibility of the pack and time contact with the surface. According
                                                                                                                                      to the results and due to the frailty of the pictorial film, successive tests were carried out
                                                                                                                                      using two Japanese paper sheets with a short contact time (approximately 2 hours), in
                                                                                                                                      order to avoid chromatic alteration and to enhance the result of the application.
                                                                                                                                          On the Lapis lazuli surface of Autharis’ tunic, a preliminary cleaning step was carried
                                                                                                                                      out using deionized water, then the left hem of the tunic was subjected to Micro4Art-
                                                                                                                                      sulfate treatment using Carboneutralgel as thickener. Two samples (sample A and B)
                                                                                                                                      were collected before and after biocleaning, respectively.
                                                                                                                                          In the following table (Table 2) the results of the chemical analyses carried out on
                                                                                                                                      sample A and B are reported.


                                                                                                                                114
        Table 2. Results of chemical analyses carried out on sample A and B of scene 16.
                                                        Chlorides       Nitrates      Sulfates
 Scene                      Sample
                                                        (mass %)       (mass %)      (mass %)
           A) on the Lapis lazuli of the left hem of
           Autharis’ tunic. Before biocleaning, after     0.08            0.41          3.20
           a soft water-cleaning.
   16      B) on the Lapis lazuli of the left hem
           of Autharis’ tunic. After biocleaning
                                                          0.01            0.12          0.75
           by means of Micro4art-sulfates in
           Carboneutralgel (Bresciani).

   In Scene 33 the superficial salts appeared in a crystalline form (Figure 3b). After
a mechanical dusting, the complete area was repeatedly rinsed using deionized wa-
ter across the Japanese paper. An extracting action through a pack of Sepiolite and
Arbocel BWW40 (2:1), applied by spatula on two Japanese paper sheets and left on
the painting until completely dry (about 15 hours), followed. Afterwards this area was
repeatedly rinsed. Once dry, observation of the surface showed a thinning of the saline
patina, but not its complete removal.
   After pre-consolidation treatment of the Lapis lazuli painting of the background, by
calcium-hydrate nanomolecules applied using a brush, two other neutral extracting
packs as mentioned above were carried out. The area was then treated by means of
anionic ion-exchange resins, applied over two Japanese paper sheets and left until
completely dry (about one hour) (sample 4, Figure 4). Sulfation removal gave good
results, as proved by the percent of sulfates, which was very high before treatment and
0.36% after treatment (sample 4, Table 2).




                                                                                                       CONSERVATION SCIENCE IN CULTURAL HERITAGE




        Figure 4. Scene 33 with the areas treated using different methodologies (in red).

   The area of vermilion, in the mantle of one of the women, indicated as sample 9,
was treated with Micro4Art-sulfates in Arbocel BWW40 (Figure 4). In this area too, the
pictorial film remained intact and the chemical analyses recorded the lowest sulfate
values: 0.19% (mass percentage).


                                                                                                 115
                                                                                                                                        In the following table (Table 3) the results of the chemical analyses carried out on
Balloi, Lombardi, Troiano, Polo, Capitelli, Gulotta, Toniolo, Lucchini, Daﬀonchio - SULFATE REDUCING BACTERIA AS BIO-CLEANING



                                                                                                                                      sample 4 and 9 are reported.

                                                                                                                                              Table 3. Results of chemical analyses carried out on sample 4 and 9 of scene 33.
                                                                                                                                                                                               Chlorides         Nitrates      Sulfates
                                                                                                                                       Scene                     Sample                        (mass %)         (mass %)      (mass %)
                                                                                                                                                 Before treatments*                                ND              ND              >2
                                                                                                                                                 4) on ground vegetation, above the tail
                                                                                                                                                 of the dress of Theodolinda in the act of
                                                                                                                                                 prayer. Treatment by means of packs of           0.20             2.06            0.36
                                                                                                                                         33      sepiolite, Arbocel, deionized water, ion-
                                                                                                                                                 exchange resins.
                                                                                                                                                 9) on the vermilion dress of the woman
                                                                                                                                                 holding a book. After treatment with Mi-
                                                                                                                                                                                                  0.20             1.33            0.19
                                                                                                                                                 cro4Art-sulfates supported on an Arbocel
                                                                                                                                                 BWW40 tablet.

                                                                                                                                         Due to their high solubility and deliquescence (property of some hygroscopic sub-
                                                                                                                                      stances which pass in solution through absorption of water vapor from the environ-
                                                                                                                                      ment, holding humidity), chlorides and nitrates constitute very hazardous salts for
                                                                                                                                      stone materials. For this reason, their acceptability limit is very low and near to the
                                                                                                                                      instrumental detectability limit. Instead, for sulfates, tolerability is higher.
                                                                                                                                         In the following table (Table 4) the tolerability of these salts is reported:

                                                                                                                                       Table 4. Acceptability limits for chlorides, nitrates and sulfates on stone materials constituting
                                                                                                                                                                                cultural heritage.
                                                                                                                                                                        Low                   Significant                   High
                                                                                                                                              Chlorides             0.01 - 0.09 %            0.10 - 0.99 %                  >1 %
                                                                                                                                              Nitrates              0.01 - 0.09 %            0.10 - 0.99 %                  >1 %
                                                                                                                                              Sulfates              0.10 - 0.99 %               1-2%                        >2 %

                                                                                                                                          Nevertheless, this work should be considered a “first step” in the employment of
                                                                                                                                      sulfate-reducing bacteria for the restoration of mural paintings, which constitute a very
                                                                                                                                      complex category of art works. More research must be conducted in order to verify
                                                                                                                                      the compatibility of the method with different kinds of materials, such as pigments
                                                                                                                                      containing metals (e.g. copper and lead), which could be susceptible to the hydrogen
                                                                                                                                      sulfide released by sulfate-reducing bacteria, or pigments with oily binders, based on
                                                                                                                                      malachite and copper resinate, which are very delicate. Moreover, another interesting
                                                                                                                                      issue to analyze is exposure time.
                                                                                                                                          According to the results obtained from the Ionic Chromatography analyses, an inter-
                                                                                                                                      esting consideration can be made on the reduction of nitrates after bacterial applica-
                                                                                                                                      tion. The mass % of nitrates has passed from 0.41 to 0.12 in Scene 16 and from 2.06 to
                                                                                                                                      1.33 in Scene 33. These data can be correlated to the nitrate-reducing ability of sulfate-
                                                                                                                                      reducing bacteria, already discussed in previous studies. In fact, D. vulgaris possesses
                                                                                                                                      the proteins FprAs which provide constitutive protection against nitric oxide exposure
                                                                                                                                      [10, 12]. Moreover, SRB of the genus Desulfovibrio are able to carry out dissimilatory
                                                                                                                                      nitrate reduction to ammonia thanks to the presence of NrfA, a gene encoding for a


                                                                                                                                116
nitrite reductase. NrfA plays an important role in those strains able to use nitrate and
nitrite as an electron acceptor when sulfate is not freely available and in those bacteria
capable of reducing nitrite but not nitrate [9, 11]. Further investigations and trials for the
removal of nitrates using sulfate-reducing bacteria could be interesting.
    On account of this, the effectiveness of Micro4Art-sulfates in the removal of nitrates
can be considered another interesting and promising result.


      Acknowledgements

   The authors are grateful to Alberto Gnesutta, descendant of the poetess Anna Zuc-
cari and patron of the restoration work, and the Soprintendenza per i Beni Architettonici
e Paesaggistici di Milano, Italy.
   For the work on the mural paintings of the Queen Theodolinda Chapel, the authors
thank Gian Carlo Lanterna of OPD and Claudio Seccaroni, researcher at ENEA.
   A special thank you to Bresciani s.r.l. for the financial support.


      Notes
1
      The restoration of the Queen Theodolinda Chapel in Monza Cathedral was promoted by Fon-
      dazione Gaiani and financed by Regione Lombardia, Fondazione Cariplo, the World Monu-
      ment Fund Europe.
      Scientific coordination was arranged by “Opificio delle Pietre Dure (OPD)” of Florence, under
      the supervision of the “Soprintendenza per i Beni Architettonici ed il Paesaggio” of Milan.
      Scientific investigations were carried out by OPD, ENEA UTTMAT-DIAG of Rome and IFAC-
      CNR of Florence.




                                                                                                            CONSERVATION SCIENCE IN CULTURAL HERITAGE
      References

[1]      Sorlini, C., Ranalli, G., Zanardini, E., (2010) Stone bioconservation. In: Mitchell
         R. and McNamara (eds.), Cultural Heritage Microbiology: Fundamental Studies
         in Conservation Science. Washington, DC: C.J. ASM Press.
[2]      Atlas, R.C., Rude, P.D., (1988) Complete oxidation of solid phase sulfides by
         manganese and bacteria in anoxic marine sediment. Geochim Cosmochim Acta,
         52, pp. 751-766.
[3]      Polo, A., Cappitelli, F., Brusetti, L., Villa, F., Giacomucci, L., Ranalli, G., Sorlini,
         C., (2010) Feasibility of removing surface deposits on stone using biological and
         chemical remediation methods, Microbial Ecology, 60(1), pp. 1-14.
[4]      Gioventù, E., Lorenzi, P., (2013) Bio-removal of black crust from marble surface:
         comparison with traditional methodologies and application on a sculpture from
         the Florence’s English Cemetery, Procedia Chemistry, 8, pp. 123-129.
[5]      Cappitelli, F., Toniolo, L., Sansonetti, A., Gulotta, D., Ranalli, G., Zanardini, E.,
         Sorlini C., (2007) Advantages of using microbial technology over traditional
         chemical technology in removal of black crusts from stone surfaces of historical
         monuments, Applied and Environmental Microbiology 73(17), pp. 5671-5675.
[6]      Cappitelli, F., Zanardini, E., Toniolo, L., Abbruscato, P., Ranalli, G., Sorlini, C.,
         (2005) Bioconservation of the marble base of the Pietà Rondanini by Michelan-


                                                                                                      117
                                                                                                                                           gelo Buonarroti, Geophysical Research Abstracts, vol. 7, 06675. SRef-ID: 1607-
Balloi, Lombardi, Troiano, Polo, Capitelli, Gulotta, Toniolo, Lucchini, Daﬀonchio - SULFATE REDUCING BACTERIA AS BIO-CLEANING



                                                                                                                                           7962/gra/EGU05-A-06675, European Geosciences Union.
                                                                                                                                      [7] Troiano, F., Gulotta, D., Balloi, A., Polo, A., Toniolo, L., Lombardi, E., Daffonchio,
                                                                                                                                           D., Sorlini, C., Cappitelli, C., (2013) Successful combination of chemical and
                                                                                                                                           biological treatments for the cleaning of stone artworks, International Biodeterio-
                                                                                                                                           ration & Biodegradation, 85, pp. 294-304.
                                                                                                                                      [8] Salimbeni, R., La scienza dedicata al restauro, Documento per l’Assessorato alle
                                                                                                                                           attività produttive Regione Toscana.
                                                                                                                                      [9] Ranalli, G., Alfano, G., Belli, C., Lustrato, G., Colombini, M.P., Bonaduce, I., Za-
                                                                                                                                           nardini, E., Abbruscato, P., Cappitelli, F., Sorlini, C., (2005) Biotechnology ap-
                                                                                                                                           plied to cultural heritage: biorestoration of frescoes using viable bacterial cells
                                                                                                                                           and enzymes, Journal of Applied Microbiology, 98(1), pp. 73-83.
                                                                                                                                      [10] Silaghi-Dumitrescu, R., Yong Ng, K., Viswanathan, R., Kurtz, D.M.Jr., (2005) A
                                                                                                                                           Flavo-Diiron Protein from Desulfovibrio vulgaris with Oxidase and Nitric Oxide
                                                                                                                                           Reductase Activities. Evidence for an in Vivo Nitric Oxide Scavenging Function,
                                                                                                                                           Biochemistry, 44(9), pp. 3572-3579.
                                                                                                                                      [11] Giacomucci, L., Purdy, K.J., Zanardini, E., Polo, A., Cappitelli, F., (2012) A New
                                                                                                                                           Non-Degenerate Primer Pair for the Specific Detection of the Nitrite Reductase
                                                                                                                                           Gene nrfA in the Genus Desulfovibrio, J Mol Microbiol Biotechnol, 22(6), pp. 345-
                                                                                                                                           351.


                                                                                                                                        Biographical notes


                                                                                                                                        Annalisa Balloi, CEO Micro4yoU
                                                                                                                                        Emanuela Lombardi, Laboratory technician Micro4yoU
                                                                                                                                        Federica Troiano, Post-doc fellow, University of Milan
                                                                                                                                        Davide Gulotta, Scientific Secretariat, Centro Beni Culturali, Politecnico di Milano
                                                                                                                                        Andrea Polo, Post-doc fellow, Bozen Free University
                                                                                                                                        Anna Lucchini, Anna Luchini Restauri S.r.l.
                                                                                                                                        Lucia Toniolo, President, Centro Beni Culturali, Politecnico di Milano
                                                                                                                                        Francesca Capitelli, Associate Professor, University of Milan
                                                                                                                                        Daniele Daffonchio, Associate Professor, University of Milan




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   Summary
   In the last decades, the contribution of different scientific disciplines in the field
of restoration and conservation of cultural heritage to finding alternative methods of
investigation that are even more effective and fully respect artworks, operators and
environment, has greatly increased.
   An example is the University of Milan patent that provides for the use of some
specialized bacteria as cleaning agents. This method, known as biocleaning, is based
on the use of Desulfovibrio vulgaris, a sulfate reducing bacterium, for the removal of
sulfate alterations from stone surfaces.
   Recently, the university spin-off Micro4yoU purchased the patent by initiating a se-
ries of investments aimed at enhancing the commercial product from prototype.
   The present work describes: the desulfation mechanism operated by Desulfovibrio
vulgaris and the technological shift necessary to obtain a biological formulation usable
in situ, with two practical case studies.


   Riassunto
   Negli ultimi decenni è cresciuto il contributo che le diverse discipline scientifiche
hanno dato al recupero e alla conservazione del patrimonio artistico per trovare solu-
zioni sempre più efficaci e rispettose del bene, dell’operatore e dell’ambiente.
   Un esempio è un brevetto sviluppato dall’Università degli studi di Milano che pre-
vede l’utilizzo di alcuni specifici batteri come agenti di pulitura. Tale metodo, indicato
col termine di bio-pulitura propone, tra gli altri, l’impiego del batterio solfato riduttore
Desulfovibrio vulgaris per la rimozione di alterazioni solfatiche da superfici litoidi.
   Recentemente, il brevetto è stato acquisito dallo spin-off universitario Micro4yoU
che ha avviato una serie di investimenti per sviluppare, a partire dal prototipo di labo-
ratorio, un prodotto commerciale.
   Nel presente lavoro verranno illustrati: il meccanismo di desolfatazione ad opera di




                                                                                                     CONSERVATION SCIENCE IN CULTURAL HERITAGE
Desulfovibrio vulgaris, il processo di trasferimento tecnologico affrontato per ottenere
un formulato microbico utilizzabile in cantiere e due casi studio applicativi.




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