**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**.

#### **2.2** *In vitro* **microbial 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

**241**

**Figure 1.**

*Biotechnology and Cultural Heritage Conservation DOI: http://dx.doi.org/10.5772/intechopen.90669*

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

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 vitro* isolated microbial colony, the *Genomic DNA Purification Kit*

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

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).

coating (Agar-Auto-Sputter – Coater B7341) by gold particles (13 nm).

*Stonework altered areas, sampling performed by sterile swab o scalpel: (A) dark-rust red area;* 

**2.4 Molecular biology investigation**

*(B) light – green calcareous deposit; (C) dark-green area.*

(Fermentas) has been appropriate.

polymerase (Sigma).

*Biotechnology and Cultural Heritage Conservation DOI: http://dx.doi.org/10.5772/intechopen.90669*

#### **Figure 1.**

*Heritage*

the stone surface [10, 13, 14].

cultural assets [22–31].

restoration procedures.

**2.1 Sampling**

18–48 hours.

**2. Material and methods**

**2.2** *In vitro* **microbial culture**

**2.3 Morphological analysis**

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

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

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

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 coloni-

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 nepeta* and

The results of *in vitro* assays 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

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**.

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

Morphological profiles of algae and bryophytes were revealed by stereomicroscope (Wild Heerbrugg) and digital microscope (DinoLite) observations. After

In the last decades, integrated approaches (based on microscopy, *in vitro* culture and molecular biology analysis) have been applied to reveal and identify the greater number of microorganisms involved in the deterioration processes of

monument, anthropogenic factors must be also considered [17].

algae, bacteria, yeasts, lichens, molds, and micro-fungi [18–21].

zation and to inhibit re-colonization events [32–37].

*Allium sativum* EOs) *vs* the identified microbial taxa [38–41].

**240**

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

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 vitro* isolated 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].
