Biotechnology and Cultural Heritage Conservation

*Franco Palla*

### **Abstract**

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.

**Keywords:** 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 [1–3]. 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 [8–10].

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 [18–21].

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 cultural assets [22–31].

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 [32–37].

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

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 restoration procedures.
