Biotechnology: Innovative Contributions to the Conservation and Restoration of Cultural Heritage

As exhaustively showed during the conference "Molecular Biology and Cultural Heritage", held in Seville in 2003, molecular biology represents an important source of insight for the development of innovative protocols for the detection and characterization of microbial consortia colonizing historic-artistic manufacts. In particular, fungi and bacteria (frequently associated with green algae, cyanobacteria, lichens, etc), wide-spread in biosphere environments, are the main biological systems related to deterioration of cultural asset. Moreover in the aerosol of indoor environments, whre the manufacts are exposed or stored, complex microbiai communities may release some molecules with human (visitors, professionals) health damaging properties, that may persist during the time. In order to identify the biological systems on artworks surface andJor dispersed in the aerosol, an integrated morphological-molecular protocol based on microscopy (OM, SEM, CLSM), in vitro culture (colonies isolation) and amplification of DNA target sequences (PCR, sequencing, sequence-comparison) have been applied. Non invasive samplings were performed by sterile swab, adhesive tape or Nylon H+ membrane (Amersham) on work of art surfaces, while aerosol sampling by portable AirPort MD8 sampler (Sartorius), equipped with disposable gelatine filters. Recently, in our laboratory, new bioactive molecules (BMs) extracted from marine invertebrates organisms have been characterized and tested in order to remove protein layers (bio-c1eaning by Proteolytic-peptides = BMP) or to control the bacteria and/or fungi colonization onto artifacts (Antimicrobial-peptide = BMA). The action of c1eaning represents one of the most important step in restoration projects, such as take out organi c layers (animai glue or other protein mixtures, frequent1y deteriored) stratified onto the manufact surface. Cleaning must be selectively performed, making distinction between different areas, removing the deposits without acting direct1y to originaI materials ofthe manufact. Particularly interesting is that the Proteolytic-peptides (BMP) start to act from 4°C up to 37°C. We tested the BMP molecules on different substrates and temperatures, between 19-26°C. The related commerciaI enzymes, actually used in bio-c1eaning procedures, work at specific range of temperature ~ 37°C; temperature value represents a limit in the use of these enzymes, since the heating of manufact surface is usually not available. The opportunity to apply BMP molecule on several substrates, both organic and inorganic (canvas, wooden, ceroplastics, mosaic, frescos), without heating (surface or enzyme solution), improve the efficiency of bio-c1eaning protocols, according to the conservative-restoration procedure. Concerning BMA peptides, their Antimicrobial (biostatic-biocide) activity was in laboratory preliminary assessed; particular1y against Bacillus / Micrococcus and Aspergillus / Penicillium colonies. We focalized the attention on the painting lining process, usually performed by new canvas (natural or synthetic) layers glued by rabbit- skin or others animai adhesives to the verso of degraded paintings; generally, this procedure is performed by using a heat source (iron). This heating-treatment and the presence of organic compounds (glue) can induce microbial colonization. Since these methodologies are totally safe for the operators and environment, are low time-consuming, they can be considered as a sustainable alternative to the traditional procedures

Blue-Biotechnology e la pulitura biologica di manufatti storico-artistici

The search for novel cleaning strategies in restoration procedures is growing fast. One innovation/innovative breakthrough in cleaning procedures is represented by the use of enzymes. This is due to the fact that their action is highly selective and they effectively remove undesired layers without adversely affecting the constituent or surrounding materials of the artifact undergoing treatment. In this study we present the results of tests performed by a new set of enzymatic proteins, esterases, extracted from marine organisms, for the bio-removal of Paraloid B72.La ricerca di nuove strategie di pulitura è in costante crescita, trovando applicazione in molte procedure di restauro. Possiamo affermare che il ricorso a proteine enzimatiche, utilizzate in questi decenni, rappresenta un’innovazione nel campo della pulitura, poiché gli enzimi agiscono selettivamente sugli strati da rimuovere, senza incidere negativamente sui materiali costitutivi del manufatto. In questo studio si presentano i risultati preliminari della bio-rimozione del Paraloid B72 ricorrendo all’uso di un nuovo set di proteine enzimatiche, esterasi, estratte da organismi marini

Biotechnology and Cultural Heritage Conservation

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

Chromatic alteration on marble surfaces analysed by molecular biology tools

The patina represents a superficial natural alteration of the constituting matter of the work of art. It emerges from the natural and usual stabilization process that the materials of the surface undergo because of the interaction with outdoor agents characterizing the surrounding environment. Besides, it is not linked to an obvious phenomenon of degradation that can be noticed through the change in the original colour of the matter. This is what we intend when we talk about biological patina usually generated by macro and/or micro-organic colonization (fungi, bacteria, alga) which contributes to surface bio-deterioration and thus lead to the formation of orange, red or even brown and dark pigmented areas. The presence of chromatic alterations (rose-coloured areas), as a consequence of bacterial colonization, was most particularly pointed out in different sites, such as in the marble slabs on the facades of both the Cathedral of Siena (Duomo di Siena) and the Certosa of Pavia. The present study shows an example of chromatic alteration of the surface of marble works due to bacterial colonization

What does happen when an insect pest follows its host plant and viceversa?

Invasive species are usually well known but only make up a small percentage of the European’s alien plants and insects. The ways these exotic species interact with our natural and cultural heritage are very complex and even some species considered invasive had and could have positive aspects on some European landscape and culture. A close look at the complicated relationship between non-native and native plants and insects in the Mediterranean areas reveals some unexpected twists in the story. Here are some cases of when aliens can threaten our cultural heritage or being part of i

Multiple approach to identify bacteria into archaeological waterlogged wood

This study was carried out in collaboration with Soprintendenza del Mare (SM) that started, since 2004, to plan and realize underwater archaeological parks, such as in the Sicilian islands of Pantelleria (Gadir), Levanzo (Cala Minnola), Ustica (Falconiera), Panarea (Basiluzzo) and Filicudi (Capo Graziano). In situ conservation, as well as virtual exhibitions of the same topics, can contribute to ensure the protection and best fruition of underwater cultural heritage. The focus of this study was the identification of bacterial colonies in waterlogged wood samples from the rostrum of a excellent workmanship, that is very likely one of the wrecks attributed to Sextus Pompey fleet (36 BC) and discovered in Acqualadroni, Messina, Sicily, Italy (2008). Samples were analyzed by light and Scanning Electron Microscopy (SEM), in vitro culture and molecular technique (DNA base techniques). The results, focused on bacterial consortia, allowed us to reveal the presence of Pseudomonas sp., Sphingomonas sp., Xanthomonas sp. besides Marinobacter sp. and Desulforudis audaxviator. A prompt and accurate characterization of bacterial colonization represents one of the preliminary step in preservation/restoration projects, especially for waterlogged wood since the metabolic activity of specific bacteria induce and accelerate the deterioration processes. Although it is reported in a case study, this multiple approach is useful for reveal and identify bacterial colonizing both organic and inorganic artifacts.This study was carried out in collaboration with Soprintendenza del Mare (SM) that started, since 2004, to plan and realize underwater archaeological parks, such as in the Sicilian islands of Pantelleria (Gadir), Levanzo (Cala Minnola), Ustica (Falconiera), Panarea (Basiluzzo) and Filicudi (Capo Graziano). In situ conservation, as well as virtual exhibitions of the same topics, can contribute to ensure the protection and best fruition of underwater cultural heritage. The focus of this study was the identification of bacterial colonies in waterlogged wood samples from the rostrum of a excellent workmanship, that is very likely one of the wrecks attributed to Sextus Pompey fleet (36 BC) and discovered in Acqualadroni, Messina, Sicily, Italy (2008). Samples were analyzed by light and Scanning Electron Microscopy (SEM), in vitro culture and molecular technique (DNA base techniques). The results, focused on bacterial consortia, allowed us to reveal the presence of Pseudomonas sp., Sphingomonas sp., Xanthomonas sp. besides Marinobacter sp. and Desulforudis audaxviator. A prompt and accurate characterization of bacterial colonization represents one of the preliminary step in preservation/restoration projects, especially for waterlogged wood since the metabolic activity of specific bacteria induce and accelerate the deterioration processes. Although it is reported in a case study, this multiple approach is useful for reveal and identify bacterial colonizing both organic and inorganic artifacts

Biotechnology a Source of Knowledge in Agreement with Green Strategies for the Conservation of Cultural Assets

In this study, conservative strategies are applied in order to limit any irreversible damage and to characterize and quantify microbial colonization that may induce the deterioration of constitutive heritage materials; subsequently, suitable antimicrobial interventions are performed based on the use of biotechnology. This paper presents a summary of the basic and applied biotechnology research developed in the Laboratory of Biology and Biotechnology for Cultural Heritage (LaBBCH, University of Palermo, Italy). It shows that biotechnology has an evidently important role in the preservation and restoration of cultural assets, also taking into consideration that “Prevention is better than cure”, a milestone in the field of human health that can be shifted and applied to the “cure and preservation” of cultural assets. Moreover, biotechnological tools offer great potential for application, thanks to the increasing interaction between the worlds of art and science, thus opening the way for advanced innovation in the conservation and restoration field of art works

DNA analysis as tool for identification of bacteria in archaeological waterlogged wood

Abstract In this work molecular techniques were applied in order to integrete the results obtained by Optical (OM) and Scanning Electron (SEM) Microscopy, to understanding and assessing the changes in the anatomical structure of archaeological waterlogged wood (Pinus sp.) induced by bacteria colonization. Observation of wooden thin sections revealed by OM showed the presence of black and dark -brown areas (must probably due to sulphur compounds) and mineral concretions. The SEM micrographs revealed a specific cell wall alteration attributable to bacterial activity and abundant pyrite framboids (as single structure or clustered). The presence of sulfur compounds in archaeological waterlogged wood, indicate both long-term burial in anoxic environment and colonization by sulfate-reducing bacteria. Molecular biology investigation was performed through ad hoc protocols by direct DNA extraction from wood samples and in vitro amplification of bacteria DNA target sequence (16S, ITS regions-r RNA). The results reveal and identify bacterial genus as Pseudomonas, Cellulomonas, Xanthomonas and Bacillus that, as reported in the related scientific literature, are the most common cellulosolytic and ligninolytic bacteria. Moreover were also revealed the presence of Marinobacter sp. and Desulforudis audaxviator, respectively iron- oxidizing and sulfate- reducing bacteria. The investigation protocol set up in this work can be applied to a range of wooden artifacts of archaeological findings for both identification of bacteria colonization shed some light on the degradation phenomena, indispensable for correct conservation and restoration strategies

Molecole bioreattive: un contributo innovativo delle biotecnologie per il restauro dei Beni Culturali

SummaryOne of the first and most important steps during the conservative restoration of an artifact, is the action of cleaning, which enables the removal of organic layers such as animal glue or other protein mixtures that cover the original surface. Cleaning must be performed carefully and selectively, making a distinction between the different areas, so as not to cause irreversible damage to the surface of the object, and remove the deposits without acting directly on the original surface materials. In this study hydrolases isolated from marine organisms, whose peculiarity is to be active in a range of temperature between 4° C and 37° C, are proposed as an alternative to classic cleaning methods. RiassuntoLa pulitura, azione che permette la rimozione di strati soprammessi dalla superficie di un manufatto, è una delle prime e più importanti fasi attuate in interventi di restauro conservativo. Questa deve essere eseguita in modo da non causare danni irreversibili, quindi effettuata in modo selettivo, differenziando l’azione tra le diverse zone e rimuovendo in successione gli strati di deposito, senza interagire in modo diretto con i materiali originali della superficie. In questo studio sono state utilizzate nuove idrolasi isolate da organismi marini, la cui attività proteasica è stata saggiata in laboratorio, presentano la rilevante peculiarità è di essere attivi in un range di temperature compreso tra 4°C e 37°C e sono proposte come valida alternativa ai classici metodi di pulitura.RésuméLe nettoyage est l’action qui permet d’enlever les couches accumulées sur la surface d’un ouvrage, et c’est l’une des premières phases les plus importantes mises en œuvre dans les interventions de restauration-conservation. Le nettoyage doit faire en sorte de ne pas provoquer de dommages irréversibles : il est donc effectué de manière sélective en différenciant l’action suivant les zones, et en enlevant en succession les couches de dépôt sans interagir directement avec les matériaux originaux de la surface. Dans cette étude, on a utilisé de nouvelles hydrolases isolées d’organismes marins, dont l’activité protéasique a été testée en laboratoire. Elles présentent la particularité importante d’être actives dans un intervalle de températures compris entre4°Cet37°C. Elles sont proposées comme une alternative valable aux méthodes classiques de nettoyage. ZusammenfassungDie Säuberung, d.h. das Abtragen von abgelagerten Schichten auf einem Gegenstand, ist eine der ersten und wichtigsten Phasen bei konservativen Restaurierungsmaßnahmen. Sie muss so erfolgen, dass keine nicht wieder gutzumachenden Schäden entstehen, d.h. sie muss selektiv vorgenommen werden, durch unterschiedliche Bearbeitung der verschiedenen Zonen und aufeinanderfolgendes Abtragen der abgelagerten Schichten ohne direkt auf die originalen Materialien der Oberfläche einzuwirken. In dieser Studie wurden neue, von Meeresorganismen isolierte Hydrolasen eingesetzt, deren eiweißspaltende Wirkung im Labor getestet wurde. Diese haben die relevante Eigenschaft, dass sie in einem Temperaturbereich zwischen 4 und 37°C aktiv sind und eine gute Alternative zur den herkömmlichen Säuberungsmethoden bieten. ResumenLa limpieza, acción que permite remover capas superpuestas de la superficie de una obra, es una de las primeras y más importantes fases llevadas a cabo en los trabajos de restauración conservativa. Dicha fase ha de ser realizada sin provocar daños irreversibles, por lo tanto es efectuada de forma selectiva, diferenciando el tipo de acción en las distintas zonas y quitando sucesivamente las capas de depósito, sin interaccionar directamente con los materiales originales de la superficie. En este estudio se han utilizado nuevas hidrolasas aisladas de organismos marinos, cuya actividad proteasa ha sido probada en laboratorio, con la importante peculiaridad de que permanecen activas en una franja de temperatura situada entre 4°C y 37°C y se proponen como una alternativa apropiada ante métodos clásicos de limpieza. РезюмеОчистка, действие, которое позволяет удалить накопленные слои с поверхности артефакта, является одним из первых и наиболее важных шагов, применяемых при консервативной реставрации. Она должна производиться при недопущении необратимых повреждений, следовательно,  ее проведение должно быть выборочным, при дифференциации вмешательств в различных зонах и, удаляя в последовательности слои накопления, не соприкасаясь напрямую с оригинальными материалами поверхности. В этом исследовании были использованы новые гидролазы, выделенные из морских организмов, протеазная активность которых была проанализирована в лаборатории, и которые имеют важную особенность, состоящую в том, что они могут быть активными в диапазоне температур от 4 ° С до 37 ° С и могут являться эффективной альтернативой традиционным методам очистки. 摘要清洁，即去除物品表面层层覆盖物的操作，是分阶段修复措施中最首先和最重要的一步。这必须在不会造成不可弥补伤害的前提下进行，所以应该用选择性的清洁，在不同区域要用不同的操作方式并且相继除去覆盖层，切忌直接与表面原始材料相互影响。这项研究中从海洋生物内提取了新的水解酶，其蛋白酶活性已通过实验室检测，它表现出的相关特性是在4°C ~37°C的温度范围内活跃并被提议为很好的替代传统清洁的新方法。SummaryOne of the first and most important steps during the conservative restoration of an artifact, is the action of cleaning, which enables the removal of organic layers such as animal glue or other protein mixtures that cover the original surface. Cleaning must be performed carefully and selectively, making a distinction between the different areas, so as not to cause irreversible damage to the surface of the object, and remove the deposits without acting directly on the original surface materials. In this study hydrolases isolated from marine organisms, whose peculiarity is to be active in a range of temperature between 4° C and 37° C, are proposed as an alternative to classic cleaning methods. RiassuntoLa pulitura, azione che permette la rimozione di strati soprammessi dalla superficie di un manufatto, è una delle prime e più importanti fasi attuate in interventi di restauro conservativo. Questa deve essere eseguita in modo da non causare danni irreversibili, quindi effettuata in modo selettivo, differenziando l’azione tra le diverse zone e rimuovendo in successione gli strati di deposito, senza interagire in modo diretto con i materiali originali della superficie. In questo studio sono state utilizzate nuove idrolasi isolate da organismi marini, la cui attività proteasica è stata saggiata in laboratorio, presentano la rilevante peculiarità è di essere attivi in un range di temperature compreso tra 4°C e 37°C e sono proposte come valida alternativa ai classici metodi di pulitura.RésuméLe nettoyage est l’action qui permet d’enlever les couches accumulées sur la surface d’un ouvrage, et c’est l’une des premières phases les plus importantes mises en œuvre dans les interventions de restauration-conservation. Le nettoyage doit faire en sorte de ne pas provoquer de dommages irréversibles : il est donc effectué de manière sélective en différenciant l’action suivant les zones, et en enlevant en succession les couches de dépôt sans interagir directement avec les matériaux originaux de la surface. Dans cette étude, on a utilisé de nouvelles hydrolases isolées d’organismes marins, dont l’activité protéasique a été testée en laboratoire. Elles présentent la particularité importante d’être actives dans un intervalle de températures compris entre4°Cet37°C. Elles sont proposées comme une alternative valable aux méthodes classiques de nettoyage. ZusammenfassungDie Säuberung, d.h. das Abtragen von abgelagerten Schichten auf einem Gegenstand, ist eine der ersten und wichtigsten Phasen bei konservativen Restaurierungsmaßnahmen. Sie muss so erfolgen, dass keine nicht wieder gutzumachenden Schäden entstehen, d.h. sie muss selektiv vorgenommen werden, durch unterschiedliche Bearbeitung der verschiedenen Zonen und aufeinanderfolgendes Abtragen der abgelagerten Schichten ohne direkt auf die originalen Materialien der Oberfläche einzuwirken. In dieser Studie wurden neue, von Meeresorganismen isolierte Hydrolasen eingesetzt, deren eiweißspaltende Wirkung im Labor getestet wurde. Diese haben die relevante Eigenschaft, dass sie in einem Temperaturbereich zwischen 4 und 37°C aktiv sind und eine gute Alternative zur den herkömmlichen Säuberungsmethoden bieten. ResumenLa limpieza, acción que permite remover capas superpuestas de la superficie de una obra, es una de las primeras y más importantes fases llevadas a cabo en los trabajos de restauración conservativa. Dicha fase ha de ser realizada sin provocar daños irreversibles, por lo tanto es efectuada de forma selectiva, diferenciando el tipo de acción en las distintas zonas y quitando sucesivamente las capas de depósito, sin interaccionar directamente con los materiales originales de la superficie. En este estudio se han utilizado nuevas hidrolasas aisladas de organismos marinos, cuya actividad proteasa ha sido probada en laboratorio, con la importante peculiaridad de que permanecen activas en una franja de temperatura situada entre 4°C y 37°C y se proponen como una alternativa apropiada ante métodos clásicos de limpieza. РезюмеОчистка, действие, которое позволяет удалить накопленные слои с поверхности артефакта, является одним из первых и наиболее важных шагов, применяемых при консервативной реставрации. Она должна производиться при недопущении необратимых повреждений, следовательно,  ее проведение должно быть выборочным, при дифференциации вмешательств в различных зонах и, удаляя в последовательности слои накопления, не соприкасаясь напрямую с оригинальными материалами поверхности. В этом исследовании были использованы новые гидролазы, выделенные из морских организмов, протеазная активность которых была проанализирована в лаборатории, и которые имеют важную особенность, состоящую в том, что они могут быть активными в диапазоне температур от 4 ° С до 37 ° С и могут являться эффективной альтернативой традиционным методам очистки. 摘要清洁，即去除物品表面层层覆盖物的操作，是分阶段修复措施中最首先和最重要的一步。这必须在不会造成不可弥补伤害的前提下进行，所以应该用选择性的清洁，在不同区域要用不同的操作方式并且相继除去覆盖层，切忌直接与表面原始材料相互影响。这项研究中从海洋生物内提取了新的水解酶，其蛋白酶活性已通过实验室检测，它表现出的相关特性是在4°C ~37°C的温度范围内活跃并被提议为很好的替代传统清洁的新方法