**3. Results**

Green algae as *Chlorella* (**Figure 2**) and cyanobacteria as *Cyanobium and Oscillatoria* genera were revealed in fountain samples, classified as biodeteriogen and also as first pioneering of stone substrates colonization. Particularly, algae can

**243**

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

and red to brown [10, 13, 51].

**Figure 2.**

growth of plant species [59].

biocides have been tested.

concentration.

to penetrate inside the stone surface [56, 57].

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 [48–50]. Furthermore, cell compounds such as chlorophyll, carotenoid, and melanin may generate chromatic alteration from yellow, orange,

Chlorella *green algae, optical microscope images; bar = 10 micromillimeters.*

Bacterial and fungal diversity was also distinguished, bacteria or fungi genera mainly belonging to *Arthrobacter, Bacillus*, *Micrococcus* or *Alternaria, Fusarium*, *Cladosporium*, *Penicillium*, and *Aspergillus*, respectively (**Figures 3**–**5**). Moreover, bacteria of the *Bacillus* genus 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

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

In order to inhibit biological colonization, traditional (benzalkonium chloride)

or green (*Melaleuca alternifolia*, *Calamintha nepeta*, and *Allium sativum* EOs)

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 diffusion* method; the size of inhibition halos is related to the essential oil *Biotechnology and Cultural Heritage Conservation DOI: http://dx.doi.org/10.5772/intechopen.90669*

#### **Figure 2.**

*Heritage*

*num*, *Aspergillus* spp.

**2.6 Antimicrobial activity assays**

ml or fungal suspension = 1 × 104

Referring to genomic databases (EMBL-Germany, NIH-USA), the sequences were

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 sativum* L.; (iii) B*enzalkonium chloride* commercial biocides

The microbial taxa were *Bacillus subtilis*, *Micrococcus luteus*, *Penicillium chrysoge-*

Three *in vitro* methods, *Agar disc diffusion*, *Well-plates diffusion*, and *Micro-*

• *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

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].

Green algae as *Chlorella* (**Figure 2**) and cyanobacteria as *Cyanobium and Oscillatoria* genera were revealed in fountain samples, classified as biodeteriogen and also as first pioneering of stone substrates colonization. Particularly, algae can

CFU/

conidia/ml) and incubated for 18–48 h at

analyzed (percentage of similarity) by BLAST analyzer [42].

**2.5 Commercial (CB) and natural (EOs) biocides**

(CB), was tested by outlined *in vitro* assays [36–38].

*dilution* in micro-titer plates [38, 43, 44] were performed:

**242**

**3. Results**

Chlorella *green algae, optical microscope images; bar = 10 micromillimeters.*

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 [48–50]. Furthermore, cell compounds such as chlorophyll, carotenoid, and melanin may generate chromatic alteration from yellow, orange, and red to brown [10, 13, 51].

Bacterial and fungal diversity was also distinguished, bacteria or fungi genera mainly belonging to *Arthrobacter, Bacillus*, *Micrococcus* or *Alternaria, Fusarium*, *Cladosporium*, *Penicillium*, and *Aspergillus*, respectively (**Figures 3**–**5**). Moreover, bacteria of the *Bacillus* genus 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].

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 sativum* EOs) 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 diffusion* method; the size of inhibition halos is related to the essential oil concentration.

#### **Figure 3.**

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

**245**

**Figure 5.**

**Figure 6.**

*concentration.*

was also performed.

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

The antimicrobial activity has been also performed using the three EOs or CB at different concentration (12.5, 25.0, and 50.0%) *vs* microbial 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. alternifolia* and *A. sativum* EOs against *B. subtilis* and *M. roseus*, so strong that the halo

Well plate diffusion *method. Antimicrobial activity of* Melaleuca alternifolia *(TTOil)* vs. Bacillus subtilis *(A) or* Micrococcus luteus *(B). The inhibition halos show a different antimicrobial activity related to the EO* 

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

Minimum Inhibitory Concentration (MIC) *vs* bacterial colonies has been evaluated by the *Microdilution method*. Particularly, biocidal activity *vs M. luteus* and *B. subtilis* has been showed by *M. alternifolia* and *C. nepeta* EOs; while *A. sativum* EO showed both biocidal and biostatic activity *vs M. luteus* and biocidal activity against *B. subtilis* (**Table 2**); the MIC related to benzalkonium chloride

inhibition was equal to the petri dish diameter.

**Figure 4.** *SEM micrograph of Coccoid bacterial cell; bar = 1 micromillimeter.*

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

*Heritage*

**244**

**Figure 4.**

**Figure 3.**

*for 18 h.*

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

*Morphological profile of pigmented bacterial cells isolated from the sampled areas on nutrient agar: (A)*  Microcossus *sp. colonies; (B) different bacterial colonies;* Bacillus *sp. colonies agar; plates incubated at 30°C*  *Morphological profile of* Aspergillus *sp. colony isolated on Sabouraud agar (A), related fungal spore and reproductive structure stained by Lugol's iodine reactive (B–C); optical microscopy (40× magnification).*

#### **Figure 6.**

Well plate diffusion *method. Antimicrobial activity of* Melaleuca alternifolia *(TTOil)* vs. Bacillus subtilis *(A) or* Micrococcus 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%) *vs* microbial 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. alternifolia* and *A. sativum* EOs against *B. subtilis* and *M. roseus*, so strong that the halo inhibition was equal to the petri dish diameter.

Minimum Inhibitory Concentration (MIC) *vs* bacterial colonies has been evaluated by the *Microdilution method*. Particularly, biocidal activity *vs M. luteus* and *B. subtilis* has been showed by *M. alternifolia* and *C. nepeta* EOs; while *A. sativum* EO showed both biocidal and biostatic activity *vs M. luteus* and biocidal activity against *B. subtilis* (**Table 2**); the MIC related to benzalkonium chloride was also performed.


#### **Table 1.**

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


#### **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**).

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 disc* and *Well plate* diffusion methods of the three EOs was performed in parallel to a commercial biocide benzalkonium chloride.

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

**247**

Foundation.

surfaces are needed.

diffusion *methods for each sample, in triplicate*.

**Figure 7.**

**Acknowledgements**

**Conflict of interest**

riogen growth and colonization on cultural assets.

The authors declare no conflict of interest.

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

*Cyanobium* sp.

*Oscillatoria* sp. *Cyanobium* sp.

*Microbial taxa colonizing stonework areas showed in* **Figure 1***.*

*Chlorella* sp. *Bacillus* sp.

A. Dark-rust red *Chlorella* sp.

C. Dark-greenish *Chlorella* sp.

B. Light-green calcareous deposit

**Table 3.**

natural biocides*,* although more studies on permanence and durability on artifacts

*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 both* Agar disc *and* Well plate

**Area Algae Bacteria Fungi Mosses**

*Arthrobacter* sp.

Bacillus sp. Micrococcus sp.

*Micrococcus* sp. *Aspergillus* sp.

*Cladosporium* sp. *Fusarium* sp.

*Alternaria* sp. *Aspergillus* sp. *Penicillium* sp

– –

–

*Bryophyta* class Bryopsida

The antimicrobial efficiency of these and other vegetal biocompatible extracts is on-going in our laboratory in order to set up *green strategies* to control the biodete-

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 Palermo* Foundation 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 Palermo*

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


#### **Table 3.**

*Heritage*

*Bacillus subtilis*

**246**

**4. Conclusions**

**Table 2.**

**Table 1.**

biological systems (**Table 3**).

*\*Total inhibition of microbial growth.*

tion on *green alternatives.*

The results showed that the fountains are differently colonized by several

helping a following growth of plants on the stonework surface.

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

performed in parallel to a commercial biocide benzalkonium chloride.

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

**EOs or CB** *Micrococcus luteus* **(%) Bacillus subtilis (%)**

**Microbial taxa Essential oils (EOs) Classical biocide (CB)**

*Calamintha nepeta*

50.0 \* 7.0 \* 9.2 25.0 8.4 6.5 9.2 7.0 12.5 5.0 3 5.5 4.0

25.0 8.0 6 9.0 7.0 12.5 2 2 4 4.0

25.0 6.5 3.8 7.0 3.0 12.5 5.0 2.5 4.2 ≥1

25.0 6.0 3.0 6.9 2.0 12.5 3.8 2.5 4.0 ≥1

*Allium sativum* *Benzalkonium chloride*

**(%)** *Melaleuca* 

*alternifolia*

*Micrococcus roseus* 50.00 \* 8 \* 9.0

*Penicillium chrysogenum* 50.0 8.2 5.0 10 4.0

*Aspergillus* spp. 50.0 6.8 5.0 10 3.0

Tea tree oil 0.6 0.6 *Calamintha nepeta* 1.56 1.56 *Allium sativum* 100 100 *Benzalkonium chloride* 0.0031 0.0031

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

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 disc* and *Well plate* diffusion methods of the three EOs was

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 atten-

The innocuousness of essential oils in respecting of human health and environment protection, prompt us to hypothesize the use of these plant products as *Microbial taxa colonizing stonework areas showed in* **Figure 1***.*

#### **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 both* Agar disc *and* Well plate diffusion *methods for each sample, in triplicate*.

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 strategies* to control the biodeteriogen growth and colonization on cultural assets.

#### **Acknowledgements**

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 Palermo* Foundation 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 Palermo* Foundation.

## **Conflict of interest**

The authors declare no conflict of interest.
