Open access peer-reviewed chapter

Biotechnology and Cultural Heritage Conservation

By Franco Palla

Submitted: September 2nd 2019Reviewed: November 27th 2019Published: March 2nd 2020

DOI: 10.5772/intechopen.90669

Downloaded: 350


The deterioration of cultural asset is induced by biological, chemical, and physical factors, influenced by anthropogenic activity and environmental conditions. In this study, the contribution of biotechnology is emphasized to define the conservation strategy, for a marble Fountain (Two Dragons, XV century) located in Palermo city center, based on an integrated approach and eco-friendly procedures. Biotechnological protocols are preliminarily applied as an integrated approach, based on microscopy observation, in vitro culture and genomic DNA analysis to recognize and characterize microbial communities. Several biological systems have been identified: green algae (Chlorella) and cyanobacteria (Cyanobium, Oscillatoria); bacteria (Arthrobacter, Bacillus, Micrococcus, Paracoccus); fungi (Alternaria, Aspergillus, Penicillium, Phoma, Fusarium, Cladosporium). In order to address biological colonization, the commercial Tea Tree Oil (Melaleuca alternifolia) and laboratory-distilled (Calamintha nepeta and Allium sativum) EOs, have been assayed by in vitro Agar disc diffusion, Well-plates diffusion, and Micro-dilution methods; the result allows to define the most appropriate EOs concentration to use. In a green conservation prospective, this study highlighted that EOs can potentially replace the traditional biocides, but the activity must be preliminary evaluated by centring the choose specifically on each microbial taxon identified.


  • stonework deterioration
  • integrated approach
  • biocides
  • essential oil
  • green conservation.

1. Introduction

The biological colonization of stone artifact is basically related to the mineral components and bio-receptivity of the constitutive material, the presence of particulate on the surface, the environmental condition, and the availability of nutrients [1, 2, 3]. Generally, for outdoor Fountains, the biodeterioration is mainly induced by microalgae and cyanobacteria [4], but other biological agents such as bacteria, fungi, mosses, and lichens were frequently revealed [5, 6]. Moreover, the biological colonization is enhanced by the occurrence of water that cooperate in deterioration processes [7], acting mechanically and chemically, producing visible effects on stonework surface (cracking, detachment, crusts formation, and chromatic alterations) allowing to structural damage and loss of material [8, 9, 10].

Fungi (such as Alternaria, Cladosporium, Epicoccum, Aureobasidium, Phoma) have a significant biodeteriorative action and may penetrate into the stone surface, causing the bio-pitting; fungi colonies can be in close association with lichens [11, 12].

Autotrophic (photolithotrophs and chemolithotrophs) and heterotrophic bacteria have also been isolated from stonework and since many of these microorganisms contain pigments (β-carotene, α-bacterioruberin, and derivatives) and salinixanthin in their cell membranes, their proliferation can produce typical rosy stains on the stone surface [10, 13, 14].

Furthermore, the deterioration is also the direct result of atmospheric pollution due to soot, grease, dust, etc., implying the deposition of suspended particles on the stonework surface, enhancing the SO2 deposition, a very reactive compound with a significant corrosive effect on marble surface [15, 16]; especially for outdoor monument, anthropogenic factors must be also considered [17].

To control biodeteriogen growth of powerful biocides, as well as water-repellents, with a broad spectrum of action are usually utilized against green and brown algae, bacteria, yeasts, lichens, molds, and micro-fungi [18, 19, 20, 21].

In the last decades, integrated approaches (based on microscopy, in vitroculture and molecular biology analysis) have been applied to reveal and identify the greater number of microorganisms involved in the deterioration processes of cultural assets [22, 23, 24, 25, 26, 27, 28, 29, 30, 31].

In this case study, in order to define adequate conservative strategies, the identification and evaluation of biological colonization of the Two Dragons fountain (sculptured by Nunzio La Mattina, XV century) were carried out, providing needful information to choose the appropriate biocide both for active compound and concentration.

Recently, non-toxic natural compounds (essential oils, EOs), in order to replace the chemical compounds, have been utilized to control artworks biological colonization and to inhibit re-colonization events [32, 33, 34, 35, 36, 37].

The aim of this work has been the revealing of microbial communities on the stonework surface, evaluating the antimicrobial activity of traditional (Benzalkonium chloride) and green biocides (Melaleuca alternifolia– TTOil, Calamintha nepetaand Allium sativumEOs) vsthe identified microbial taxa [38, 39, 40, 41].

The results of in vitroassays and controlled step by step application on stonework samples, prompt us to hypothesize the EOs as valid alternative to traditional biocides, in respecting human health and environment, according to modern restoration procedures.


2. Material and methods

2.1 Sampling

Samples were collected from different Fountain areas, affected by chromatic alterations, deposits, exfoliations, incrustation, or biological patinas, by sterile swabs moistened with NaCl-Tween solution (0.9% Sodium Chloride, 0.02% Tween-80, Polyoxyethylene sorbitan monooleate) or sterile scalpel Figure 1.

Figure 1.

Stonework altered areas, sampling performed by sterile swab o scalpel: (A) dark-rust red area; (B) light – green calcareous deposit; (C) dark-green area.

2.2 In vitromicrobial culture

Nutritive media specific for bacteria or fungi colonies (Nutrient or Sabouraud agar, Difco) were inoculated by the swab collected samples, incubating at 30°C for 18–48 hours.

2.3 Morphological analysis

Morphological profiles of algae and bryophytes were revealed by stereomicroscope (Wild Heerbrugg) and digital microscope (DinoLite) observations. After Lugol’s iodine staining, the reproductive structures of isolated fungal colonies were also distinguished by Optical Microscope (Leica). Coccoid bacteria have also been noticed by Scanning Electronic Microscope (Leica Cambridge – Leo 400), after coating (Agar-Auto-Sputter – Coater B7341) by gold particles (13 nm).

2.4 Molecular biology investigation

Patina sample of approximately 200 mg, collected by sterile scalpel, undergone to three freezing (−80°C) and thawing (+ 55°C) cycles, in presence of 500 μl – 1X TE Buffer (10 mM Tris-HCl pH 8.0/1 mM EDTA), to achieve the lysis of microbial cells; genomic DNA was extracted by QI Amp DNA stool Kit(Qiagen), partially modified (+ Proteinase K (5 mg/ml) and incubation at 65°C for 4 hours). Instead, from in vitroisolated microbial colony, the Genomic DNA Purification Kit(Fermentas) has been appropriate.

Genomic DNAs were utilized as template molecules in Polymerase Chain Reaction (PCR), in order to amplify bacterial or fungal target sequences, specifically, the Internal Transcribed Sequences (ITS) 16-23S rRNA for bacterial and ITS 18-26S rRNA for fungal species [25, 26, 40]. Each PCR reaction solution consisted of: microbial Genomic DNA as template; 10 μM Primer Forward; 10 μM Primer Reverse; 3.0 mM dNTP mix; 1X Reaction Buffer including MgCl2; 0.5Us Taq DNA polymerase (Sigma).

PCR products were resolved by electrophoresis on 2.5% agarose gels (1X TAE – Tris-HCl/Acetate/EDTA, in 1X SYBER-safe DNA gel stain) and related aliquots were sequenced by Eurofins MWG-Operon sequencing service (Germany). Referring to genomic databases (EMBL-Germany, NIH-USA), the sequences were analyzed (percentage of similarity) by BLAST analyzer [42].

2.5 Commercial (CB) and natural (EOs) biocides

The antimicrobial activity of: (i) commercial EOs, Melaleuca alternifolia(Maiden and Betche) Cheel -Tea Tree Oil; (ii) laboratory distilled EOs (Calamintha nepeta(L.) Savi, Allium sativumL.; (iii) Benzalkonium chloridecommercial biocides (CB), was tested by outlined in vitroassays [36, 37, 38].

The microbial taxa were Bacillus subtilis, Micrococcus luteus, Penicillium chrysogenum, Aspergillusspp.

2.6 Antimicrobial activity assays

Three in vitromethods, Agar disc diffusion, Well-plates diffusion, and Micro-dilutionin micro-titer plates [38, 43, 44] were performed:

  • Agar disc diffusion: paper disc (4 mm in diameter) was placed onto the surface of Nutrient or Sabouraud agar (90 mm Petri dish), previously wetted with 10 μl of CBs (25, 50%) or EOs (12.5, 25, 50, 100%) The agar surface has been previously seeded by microbial cells (bacterial cells = 1 × 106 CFU/ml or fungal suspension = 1 × 104 conidia/ml) and incubated for 18–48 h at 30 ± 1°C. Confluent microbial growth was observed and the diameter (mm) of growth-inhibition-halo measured (> 6 mm = sensible; < 6 mm = resistant); CB was Benzalkonium chloride (25, 50%). Each test was performed in triplicate.

  • Well plate diffusion: the microbial inoculum was uniformly spread on Nutrient or Sabouraud agar surface, then holes of 4 mm in diameter were punched aseptically [38] and 10 μl aliquots (12.5, 25, 50, 100%) of each essential oil solutions loaded. After 18/48 h of incubation at 30 ± 1°C, the diameter (mm) of growth inhibition halos were measured. Each test was performed in triplicate.

  • Micro-dilution: was performed in 96-wells micro-titer, in order to define the Minimum Inhibitory Concentration (MIC) and Minimum Bactericidal Concentration (MBC), distinguishing between biocide or biostatic action [39]. In each well, 30 μl of plant extracts (6.25, 12.5, 25, 50, 100%)/ liquid nutritive medium and an equal volume of microbial suspension were added; to facilitate the dispersion of the oil in the medium solution, 1% of Tween 80 (not toxic for microbial cells) was added. Benzalkonium chloride (0.2%, vol/vol) was utilized as CB. Microbial growth, after 18 h of incubation at 30°C was evaluated by estimating the optical density at 500–600 nm. The MIC value was measured as the lowest concentration corresponding to any visible microbial growth, after incubation at 30°C. The MBC and MFC were determined as the lowest concentration of antimicrobial agent able to kill the 99.5% of the original inoculum, evaluating on antimicrobial-free sub-culture [45].

3. Results

Green algae as Chlorella(Figure 2) and cyanobacteria as Cyanobium and Oscillatoriagenera were revealed in fountain samples, classified as biodeteriogen and also as first pioneering of stone substrates colonization. Particularly, algae can induce carbonate precipitation on stone substrates and their metabolic processes also generate organic acids (aspartic, citric, glutamic, glycolic, oxalic, and uric) promoting the dissolution of same minerals [3, 46, 47]. Cyanobacteria, algae, and lichens contribute to the weathering of stone in humid as well as in semiarid and arid environments [48, 49, 50]. Furthermore, cell compounds such as chlorophyll, carotenoid, and melanin may generate chromatic alteration from yellow, orange, and red to brown [10, 13, 51].

Figure 2.

Chlorellagreen algae, optical microscope images; bar = 10 micromillimeters.

Bacterial and fungal diversity was also distinguished, bacteria or fungi genera mainly belonging to Arthrobacter, Bacillus, Micrococcusor Alternaria, Fusarium, Cladosporium, Penicillium, and Aspergillus, respectively (Figures 35). Moreover, bacteria of the Bacillusgenus are able to produce crystalline aggregates and precipitates (carbonate and phosphate), which can form insoluble complexes with pigments, producing different spots on stonework surface [52, 53]. Fungi, in relationship to their metabolic activities, are able to produce efflorescence and patina, breaking and cracking processes, contributing to chemical-physical alteration of the constitutive materials [54, 55]. Fungi also represents an important group of deteriogen systems for stonework exposed to the environment, due to the release of acids compounds during hyphae development or in the apical growth zones, able to penetrate inside the stone surface [56, 57].

Figure 3.

Morphological profile of pigmented bacterial cells isolated from the sampled areas on nutrient agar: (A)Microcossussp. colonies; (B) different bacterial colonies;Bacillussp. colonies agar; plates incubated at 30°C for 18 h.

Figure 4.

SEM micrograph of Coccoid bacterial cell; bar = 1 micromillimeter.

Figure 5.

Morphological profile ofAspergillussp. colony isolated on Sabouraud agar (A), related fungal spore and reproductive structure stained by Lugol’s iodine reactive (B–C); optical microscopy (40× magnification).

Finally, biological systems referable to Mosses[58] were revealed in a green patina, Figure 1C, with a detrimental action related to the keeping of moisture, the production of carbonic acid and, after their death, the indirect damages by enriching and increasing the humus content of stone surfaces, supporting the consequent growth of plant species [59].

In order to inhibit biological colonization, traditional (benzalkonium chloride) or green (Melaleuca alternifolia, Calamintha nepeta, and Allium sativumEOs) biocides have been tested.

In Figure 6, the inhibition activity of Melaleuca alternifolia(TTOil) vs. Bacillus subtilis(A) or Micrococcus luteus(B) has been evaluated by the Well plate diffusionmethod; the size of inhibition halos is related to the essential oil concentration.

Figure 6.

Well plate diffusionmethod. Antimicrobial activity ofMelaleuca alternifolia(TTOil)vs. Bacillus subtilis(A) orMicrococcus luteus(B). The inhibition halos show a different antimicrobial activity related to the EO concentration.

The antimicrobial activity has been also performed using the three EOs or CB at different concentration (12.5, 25.0, and 50.0%) vsmicrobial taxa identified in the stonework colonized areas; the results have been summarized in Table 1. Particularly, a relevant inhibition on bacterial growth was performed by M. alternifoliaand A. sativumEOs against B. subtilisand M. roseus, so strong that the halo inhibition was equal to the petri dish diameter.

Microbial taxaEssential oils (EOs)Classical biocide (CB)
(%)Melaleuca alternifoliaCalamintha nepetaAllium sativumBenzalkonium chloride
Micrococcus roseus50.00*8*9.0
Penicillium chrysogenum50.08.25.0104.0≥1

Table 1.

Well plates diffusionmethod: Measurement of microbial growth inhibition as halo diameter (mm): Diameter ≥ 9 mm. (sensible strain); 6–9 mm. (relative sensible strain); ≤ 6 mm (resistant strain).

Total inhibition of microbial growth.

Minimum Inhibitory Concentration (MIC) vsbacterial colonies has been evaluated by the Microdilution method. Particularly, biocidal activity vs M. luteusand B. subtilishas been showed by M. alternifoliaand C. nepetaEOs; while A. sativumEO showed both biocidal and biostatic activity vs M. luteusand biocidal activity against B. subtilis(Table 2); the MIC related to benzalkonium chloride was also performed.

EOs or CBMicrococcus luteus(%)Bacillus subtilis (%)
Tea tree oil0.60.6
Calamintha nepeta1.561.56
Allium sativum100100
Benzalkonium chloride0.00310.0031

Table 2.

Minimum inhibitory concentration (MIC) %, of EOs and CB vs. bacterial taxa.

4. Conclusions

The results showed that the fountains are differently colonized by several biological systems (Table 3).

A. Dark-rust redChlorellasp.
B. Light-green calcareous depositChlorellasp.Bacillussp. Arthrobactersp.
C. Dark-greenishChlorellasp.
Bacillus sp. Micrococcus sp.Alternariasp.
class Bryopsida

Table 3.

Microbial taxa colonizing stonework areas showed in Figure 1.

Particularly for the dark-greenish area, Mosses[58] were also revealed, enhancing the bio-detrimental action due to the keeping of moisture, the production of carbonic acid and, after their death, enriching and increasing the humus content helping a following growth of plants on the stonework surface.

The identified colonizers were utilized to test the antimicrobial activity of three EOs Melaleuca alternifolia, Calamintha nepeta, and Allium sativum,in order to test natural product as alternative biocide. In Figure 7, the growth inhibition activity, measured by both Agar discand Well platediffusion methods of the three EOs was performed in parallel to a commercial biocide benzalkonium chloride.

Figure 7.

Evaluation of the growth inhibition activity of the three EOs and the CB, against two identified bacterial and fungal taxa. Histograms represent the medium value obtained performing bothAgar discandWell plate diffusionmethods for each sample, in triplicate.

The results of this study confirm the need of a fuller identification of microbial colonizers in order to perform an adequate biocidal treatment, focalizing the attention on green alternatives.

The innocuousness of essential oils in respecting of human health and environment protection, prompt us to hypothesize the use of these plant products as natural biocides, although more studies on permanence and durability on artifacts surfaces are needed.

The antimicrobial efficiency of these and other vegetal biocompatible extracts is on-going in our laboratory in order to set up green strategiesto control the biodeteriogen growth and colonization on cultural assets.


Authors are indebted with prof. Maurizio Bruno, Laboratory of Chemistry of Natural Products, University of Palermo. Thank are also due to the Soprintendenza BB.CC.Palermo and the Salvare PalermoFoundation for their faithful collaboration. This study is part of the research project It@cha, PON “Ricerca e Competività 2007-2013”, PON 01_00625 and was partially financed by Salvare PalermoFoundation.


Conflict of interest

The authors declare no conflict of interest.

© 2020 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution 3.0 License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

How to cite and reference

Link to this chapter Copy to clipboard

Cite this chapter Copy to clipboard

Franco Palla (March 2nd 2020). Biotechnology and Cultural Heritage Conservation, Heritage, Daniela Turcanu-Carutiu, IntechOpen, DOI: 10.5772/intechopen.90669. Available from:

chapter statistics

350total chapter downloads

2Crossref citations

More statistics for editors and authors

Login to your personal dashboard for more detailed statistics on your publications.

Access personal reporting

Related Content

This Book

Next chapter

Automation of the Expertise of the Roman Mosaic Arts in Constanta: Analytical and Statistical Models for a Fuzzy Inference-Based System

By Silviu Ioniță and Daniela Ţurcanu-Caruțiu

Related Book

First chapter

Introductory Chapter: Environmental Characteristics of a Dobrudja Famous Archeological Monument

By Daniela Turcanu-Carutiu and Rodica-Mariana Ion

We are IntechOpen, the world's leading publisher of Open Access books. Built by scientists, for scientists. Our readership spans scientists, professors, researchers, librarians, and students, as well as business professionals. We share our knowledge and peer-reveiwed research papers with libraries, scientific and engineering societies, and also work with corporate R&D departments and government entities.

More About Us