Keywords

2.1 Indoor Environments (Libraries, Museums, Storerooms, Hypogea, Churches)

Several aspects of indoor environments need to be defined. Their main function is to preserve the objects, made of different materials that make up a country’s historical and artistic cultural heritage, in addition to serving educational purposes and representing cultural and social identity. Indoor environments may either be constructed in situ to preserve artworks in their place of origin (hypogea) or custom-built using innovative techniques and with air-conditioned premises.

Alternatively, they may be realised in existing monumental buildings which constitute artworks themselves, but also house works of art. The type of building structure will determine the risk and potentially the type of deterioration and problems associated with managing the indoor environment and collections.

In turn, the location of indoor environments, in urban or rural centres, industrialised or green areas and coastal or mountain regions (Thomson 1986; Camuffo 2019) will determine their different external and internal microclimatic conditions, the aerosol composition and its biological impact. As indoor air is linked to the atmosphere surrounding the building (Brimblecombe 1990), also a wide number of visitors will have a negative effect on the indoor environment. Moreover, the shape of indoor spaces may have a further influence, as they can range from large, very high premises to small interconnecting areas arranged either on several floors or on a single level, attics or basements, with or without windows, varying exposure, adjoined or separate, with showcases, display cabinets or clima boxes representing micro-environments enclosed within the main exhibition macro-environments (Michalski 1994; De Guichen and Kabaoglu 1985; De Guichen 1980; Cassar 1995; IBC 2007; Lazaridis et al. 2015).

Indoor microclimatic conditions deeply depend on whether active heating/air-conditioning indoor and lighting systems are present or not in a given environment. Most indoor environments, especially those constructed in loco (hypogeum), or in historical buildings or premises mainly destined for other uses (churches), lack active microclimate control systems. Even when conditioning systems are present, they are often adjusted to the needs of human comfort on the part of museum staff or church worshippers rather than those of artefact conservation. In addition, unless these systems are regularly serviced, they can be a further source of potential chemical and, above all, biological pollutants (Sedlbauer 2002; Varas-Muriel et al. 2014).

The study and control of the microclimate (Cavallini et al. 1991; IBC 2007; Bernardi 2009; Camuffo 2019) is essential to curb “biological risk”, as microclimatic conditions have a major impact on spore germination. The main indoor microclimatic parameter is relative humidity. Very high relative humidity levels in the winter months, or in conditions of moist warm wind, may result in condensation followed by microbial colonisation on the internal or external surfaces of a building and even worse on the frescoes of the masonry walls (Camuffo 2007; Ranalli et al. 2018).

Under these conditions, churches are more at risk of biodeterioration than hypogea due to the different materials they contain ranging from stone architectural and decorative features to organic materials like wood used for floorings, ceilings and furnishings and objects linked to worship (Nugari 2003). The thermo-hygrometric excursions during daily liturgical celebrations typical of the mountain churches, have not to be overlooked (Camuffo et al. 1999). With the possible exception of storerooms, another problematic parameter in all these indoor environments is natural or artificial light. Light heats the illuminated surface generating hygrometric and mechanical stress and warms the circulating air mass triggering convection that not only enhances the inertial deposition of suspended particulate matter but also pigment discoloration and biodegradation (Fig. 2.1). Light is needed to display objects, but existing lighting systems are often outdated and obsolete. Nonetheless, a variety of lighting systems are currently available that ensure optimum object preservation, saving both energy and money, including through the use of sensors that trigger light only when visitors enter an environment (IBC 2007).

Fig. 2.1
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Hypogeum archaeological site, in addition to the glass ceiling (dark area at the top), allowing natural light to penetrate, two older-generation lighting systems are visible: fluorescent tubes lacking UV guards and halogen spotlights generating considerable heat and relative air movement. This lighting system combined with high relative humidity levels increases the risk of biodegradation. The site is currently under monitoring and closed to the public

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Biological contamination can also occur in objects newly acquired from uncontrolled indoor environments (donations from private collections or excavated materials in contact with the soil), but is also common after natural or man-made disastrous events like flooding. Visitors also act as carriers of outdoor spores transported inside the building through clothes, skin and hair, they contribute to increasing CO2 linked to breathing, temperature, water vapour and microorganisms through sweating, talking and sneezing (Table 2.1). Indoor exhibition/conservation environments such as museums and churches often contain different materials and multi-material artworks, which give rise to a number of conservation problems linked to the intrinsic features of the materials (Hueck 2001) and their microclimatic requirements for conservation and management. Related to this aspect are storerooms located in attics or basements, which are often poorly maintained, full of dust and insects, and have suboptimal ventilation and unstable microclimatic conditions due to their location and frequent lack of air-conditioning systems. These environments are often neglected in terms of conservation measures as they are deemed less important, despite housing large quantities of precious artefacts made of different materials for long periods of time. In addition, storerooms often house restored works of art that emit further pollutant gases generated by restoration treatments (Cappitelli et al. 2004).

Table 2.1 Average contribution ascribed to individual museum visitors
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Artworks dust protection has been recently achieved using the Compactus shelves, but these microenvironments can cause fungal colonisation, especially in historical libraries on leather or fabric books binding (Micheluz et al. 2015). A regular and adequate cleaning, is a highly recommended preventive conservation strategy in every environment (Getty Conservation Institute 1994); in fact, humidity associated with mild-moderate temperatures and accumulated dust may create a favourable habitat for a biodeterioration of many materials (Sterflinger and Pinzari 2012). In the already contaminated material, moisture is well preserved in the biofilm, so even conditions of RH > 50% are sufficient for the survival of some microorganisms (Camuffo 2019).

Finally, a complete analysis should not neglect the assessment of building vulnerability, considering the physical and managerial criticalities related to poor maintenance of roofs, windows and masonry (Bonazza et al. 2021; Cacciotti et al. 2021, Sesana et al. 2021), especially caused by the extreme hydrometeorological phenomena, due to climate change and closely connected to the microclimate and indoor microbial exposure. The use of water-sensitive building materials in areas with hot and humid climates can also lead to indoor mould growth.

Environmental monitoring continues to be the main tool not only for recording but above all for actively keeping under control the causes of material degradation. Based on a series of data recorded over a sufficiently long period of time, continuous monitoring will permit to correctly analyse indoor and outdoor environmental measurements based on specific targets. The importance of on-site monitoring is also reiterated in the Sendai Framework for Disaster Risk Reduction 2015–2030 (Sendai Framework - United Nations 2015)Footnote 1 Priority 4 (Enhancing disaster preparedness for effective response and to “Build Back Better” in recovery, rehabilitation and reconstruction) in the sectors of risk due to climate change which also includes Cultural Heritage (Bonazza et al. 2018). The knowledge of climate change impact on cultural heritage in Italy is principality based on the identification of the main climatic parameters for degradation of indoor and outdoor artworks. A specific threat to cultural heritage concerns changes in biodegradation processes due to projections of seasonal rainfall (National Plan for Adaptation to Climate Change—PNACCFootnote 2 which makes effective the National Strategy for Adaptation to Climate Change—SNACCFootnote 3).

2.2 Airborne Particles: Organic and Vegetable Dust and Biohazards

Bioaerosol is the scientific term used to define a suspension of aerosols or particulate matter of microbial, plant or animal origin and may consist of pathogenic or non-pathogenic, live or dead bacteria and fungi, algae, viruses, pollen, plant fibres, high molecular weight allergens, bacterial endotoxins, mycotoxins, peptidoglycans or glucans (Douwes et al. 2003) passively carried by air (Cox and Wathes 1995). For this reason, the term bioaerosol does not include insects (Mandrioli and Ariatti 2001).

Airborne biological material is composed of particles generated from natural sources by active or passive mechanisms and resuspended in the atmosphere as individual organisms or often aggregated with each other or with non-biological solid or liquid particles in suspension (Lighthart and Stetzenbach 1994). The biological aerosol includes many types of airborne particles varying widely in morphology and size that can be seen with a magnifying glass or microscope. Their aerodynamic diameter may range from molecular size to large and giant particle size (Jaenicke 2005; Hinds 1999; Pöschl 2005), e.g. viruses (1 nm–1 μm), bacteria (0.1–1 μm), fungal spores (0.5–50 μm), lichen propagules (10 μm–1 mm), bryophyte spores (1–100 μm), algal cells (1 μm–1 mm) and pollen grains (10–100 μm) (Després et al. 2012; Fuzzi et al. 2015). Airborne particles are associated with other biological materials such as protozoan cysts found in aggregates or incorporated in solid or liquid particles, pteridophyte spores, plant fragments and products from metabolic activities. Another term commonly used to describe airborne particles derived from biological organisms is primary biological aerosol particles (PBAPs) that differentiates biogenic particles from secondary organic aerosols formed by further physical processes and chemical reactions in the atmosphere such as photo-oxidation (Després et al. 2012). When microorganisms are incorporated, for instance, into fog droplets for many hours, the environmental conditions are favourable for rapid growth, giving rise to secondary biological aerosol particles, commonly called SBAPs (Fuzzi et al. 1997, 2015; Després et al. 2012).

Aerobiology is a relatively new scientific discipline specifically dealing with airborne particles, how they behave in the air once generated, how the environment influences their dispersion and deposition and the impact these particles have on other organisms or materials such as artworks. The presence of bioaerosol in the atmosphere is strictly correlated to an active source able to produce material through physiological processes generating microorganisms or physical processes resulting in disaggregation and fragmentation of organisms. Sources of bioaerosol emission can be natural, for example, bacteria found in the air often belong to groups commonly present in the ground, and in fresh and sea water, while bacteria, algae and fungi are released into the air by a bubble-bursting mechanism influenced by the wind. Forests and vegetation are sources of pollen, spores and fragments, while anthropogenic sources include farming and agricultural processing and industrial activities.

The particles produced are emitted into the atmosphere by both physiological (e.g. the catapult expulsion mechanism for the dispersal of Parietaria (officinalis, judaica, lusitanica, creatica) allergens and physical processes (e.g. fungal spores released by the action of wind and rain on vegetation) (Mandrioli 1985). The process of fungal spore release mainly depends on atmospheric agents, relative humidity, air temperature, dew point temperature and wind turbulence (Jones and Harrison 2004). Peak fungal spore concentration in temperate and Mediterranean climates coincides with summer and autumn when relative humidity is higher but is strongly influenced by the frequency of precipitations that attenuate relative humidity values. Seasonal variations in pollen production have a major impact on the ratio between indoor and outdoor spore concentrations. In spring and summer, the peak concentration of most fungal spores detected in indoor environments is similar to that found outside, whereas in winter the indoor concentration is higher (Mandrioli et al. 1998; Sabbioni et al. 2008), thereby confirming the trapping effect of buildings. Post-injection conditions in the atmosphere are due to the survival of microorganisms and are controlled by physical and chemical parameters. The dynamic nature of the atmosphere makes difficult to assess the individual importance of each parameter. The major limiting factors are temperature, relative humidity, ultraviolet radiation, oxygen, carbon monoxide, nitrogen dioxide and formaldehyde (Mandrioli 1998). Although bioaerosol is ubiquitous its concentration in the atmosphere varies with season and location, and it has been estimated to constitute up to 25% of total aerosol mass and is sometimes numerically close to 50% of all aerosol particles on a global basis (Jones and Harrison 2004; Jaenicke 2005; Walser et al. 2015).

Recent studies implementing molecular techniques demonstrated that the fungal spectrum suspended in the air is much richer than previously known (Fröhlich-Nowoisky et al. 2009; Després et al. 2012; Pashley et al. 2012).

The transport or dispersal of bioaerosol in the atmosphere is a physical process based on kinetic energy exchanged when gas particles in the atmosphere clash with motionless air-dispersed particles. Particles do not behave consistently so that each stage is random in both duration and direction. Due to unfavourable environmental conditions such as dehydration and UV radiation (Griffith and De Cosemo 1994), the atmosphere contains not only vegetative forms but many forms of resistance like bacterial and fungal spores. Small particles, ranging in size from 1.0 to 5.0 μm, remain suspended in the air for a longer time, whereas larger particles tend to settle more quickly on surfaces due to their larger mass. Bioaerosol can be transported in the atmosphere for long distances (Gregory 1973; Schlesinger et al. 2006) and a longer time due to its vicinity to particle sources and resuspension of deposited particles (Tampieri et al. 1977; Mandrioli et al. 1980, 1984; Rantio-Lehtimäiki 1994). Kellogg and Griffin (2006) identified the global transport of desert dust as the main mechanism responsible for the transport of aerosol microbiota: pollens, fungi and bacteria. The average residence time of biological particles in the atmosphere can range from less than a day to a few weeks, depending on their size and aerodynamic properties (De Nuntiis et al. 2003; Després et al. 2012).

Particle deposition is the aspect of most interest to cultural heritage and, in particular, the biodeterioration of artworks. However, bioaerosol in the atmosphere is only one of the potential risks arising when deposited material encounters favourable environmental conditions for the colonisation of artefacts. Particles are usually removed from the air by sedimentation and deposition on all surfaces, not only horizontal planes. Deposition occurs by gravitational settling, molecular diffusion and impact. A highly effective but discontinuous means of bioaerosol removal in outdoor environments is rainout and washout, which happens during precipitations when damp deposition captures the particulate in precipitations and deposits it on the ground. Precipitation is the most efficient removal mechanism for particles 0.1–10 μm in diameter. Computation of bioaerosol deposition velocity is a complex problem as particles are irregular in shape and their structural features hamper calculation of particle density, e.g. despite its size, the two air bladders of Pinus pollen grains make them particularly light, thereby increasing dispersal distance (Schwendemann et al. 2007). In addition, particles vary in relation to atmospheric humidity changes: small particles are dispersed among air molecules increasing in velocity, whereas large particles shift the surrounding air creating vortices and falling more slowly. Particle deposition is slowed down if the descending particle trajectory is close to a vertical surface, whereas the velocity changes when particles are clustered together. Particles are also affected by thermophoresis and diffusiophoresis, temperature and concentration gradients and electrostatic forces that not only induce particle accumulation with blackening of the surfaces involved but also biodeterioration when conditions are favourable. Particles settled by dry or wet deposition can be involved in resuspension mechanisms and hence return once again into the atmosphere. Once deposited, biological particles can interact with the substrate, be it the nasal mucosa, a leaf surface or a fifteenth-century fresco, giving rise to an allergic reaction in sensitive patients, a plant disease following fungal or bacterial colonisation or mechanical and aesthetic deterioration of a painted surface. Fungal attack of a fresco surface, due to its porosity, can lead to hypha penetration of the painted layer resulting in flaking and detachment of the fresco surface, coloured stains obscuring the painting and the production of acid metabolites or enzymes able to transform complex molecules into simple water-soluble molecules. Over time, this process will weaken the painted layer damaging the material and value of the artwork. Studies on biodeterioration are a constantly evolving field for scientific and technological research. They currently focus not only on bioaerosol sampling and identification methods but also on transport and deposition mechanisms and above all on the ecology of the species involved.

2.3 Impact of Colonised Aerosol on Artwork Surfaces and Potential Enemies of Human Health

Studies on the biodeterioration of cultural heritage are not confined to the microbiology of biodeteriogens and material degradation processes; they require a multidisciplinary approach to understand the chemical and biological relations between the air and the materials it surrounds. Research in this sector not only serves to identify potential risk factors for artwork preservation, devise specific preventive conservation protocols or to identify the most suitable treatments, but it also serves to safeguard the health of operators (conservationists, restorers) and visitors from the risk of exposure through inhalation and contact with contaminated surfaces or objects. The risk for human health stems not only from harmful microbial species or the products of their metabolic activity, e.g. allergens, present on artefacts or in the air, but also from the hazardous residues of biocide treatments used in the cultural heritage sector. Another aspect, neglected in the past, is that some components of the bioaerosol as well as some environmental factors may have synergistic effects; for instance, you may think of the hypothesis in the health sector that hypersensitivity pneumonitis can develop after infection with the influenza virus (Gudmundsson et al. 1999). Indeed research on SARS-CoV-2 virus and COVID-19 disease has been strongly oriented, as never before, to study environmental and biological synergies, having impacts that will also affect the field of cultural heritage protection. Bioaerosol research started thanks to the interest of the health sector (allergology) and agriculture (phytopathology). The cultural heritage sector is just one of many areas in which bioaerosol can cause damage to persons and/or objects, with major economic and other related consequences. The biodegradation of cultural heritage, therefore, has a primary cultural, scientific and economic impact. As previously mentioned, bioaerosol on artworks is only harmful for preservation in concomitance with other factors: microclimatic conditions, the nature of the object, its state of preservation and chemical and physical degradation processes already in place. Biological degradation is seldom caused by a single microorganism but is produced by complex communities, real ecosystems that develop on the artefact. Under favourable microclimatic conditions (RH, T and light), the bioaerosol deposited on the surface can grow and reproduce itself using the substrate as a nutrient (heterotrophic) or support (autotrophic), causing damage to the material component (Hueck 2001) of the cultural heritage whether it consists of traditional materials and/or modern materials like polymers. The substrate may be a statue, painting, old parchment, cave painting, glass window, liturgical vestment or fresco, made of a single organic or inorganic material or several different materials combined. The inorganic substrate can provide microorganisms with an exclusive supply of mineral salts and a limited amount of water depending on the material’s porosity. The microorganisms colonising these artworks are therefore photo- or chemoautotrophic, i.e. able to self-synthesise the molecules required for their development by photosynthetic reaction (photosynthesis) or chemical reactions (chemosynthesis). Heterotrophic species can only penetrate the same artwork after autotrophic organisms, whereas organic materials are colonised by heterotrophic bacteria able to utilise the nutrients available in the material itself. The organisms causing damage to works of art are called biodeteriogens, but do not correspond to all the bioaerosol deposits generally found on them. Some of these particles may be viable but not culturable as they form colonies on solid media under certain growth conditions (time, temperature and nutrients). Many bioaerosol particles cannot be cultured on conventional media, but their existence can be proved using other methods (Năşcuţiu 2010; Blais-Lecours et al. 2015). An indoor environment (museums, galleries, archives, churches and hypogea) can be particularly suitable for microbial growth as it protects the microorganisms themselves from extreme variations in outside temperature and UV rays that can damage the bioaerosol. Desiccation, radiation, oxygen, ozone and its reaction products together with various pollutants can operate cumulatively affecting the viability of microorganisms (Griffith and De Cosemo 1994). For this reason, biological spores survive better in air than vegetative cells, as the humidity in the air is an important potential source of microorganism stress. Fungi are among the most harmful organisms associated with the biodeterioration of organic and inorganic materials (Sterflinger 2010). Many organisms excrete waste metabolic products, including pigmented or acid compounds that may disfigure materials, altering their colours or causing mechanical damage. The presence of heavy metals in some pigments used in paintings on the other hand can increase the resistance to microbial deterioration (Ciferri 1999).

Aesthetic biodeterioration should not be underestimated as it severely alters the perception of beauty and the legibility of artworks although generally it is less aggressive towards the materials. For example, the fungi present on different types of materials can determine the formation of visible films, spots, exfoliation, disruption and pitting. Chemical processes lead to the transformation, alteration and decomposition of the substrate and are much more common than in the past. In addition, the pores and fractures caused by chemical and mechanical interactions can host further biodeteriogens (Urzì et al. 2000). A chemical action may be attributed both to assimilation processes (when the organisms use the material as nourishment by means of extracellular enzyme activity or ion exchange) and to the excretion of metabolic intermediates or substances having an inhibitory or waste function (such as acid, alkaline and complexing substances and pigments). In physical biodeterioration, the organism breaks or simply deforms the material with growth or movement. Although there are many studies in the literature on microbial contamination relating to works of art preserved in different structures such as museums, crypts, churches, hypogea, libraries and archives (Valentin 2003; Nugari et al. 2003; Gaüzère et al. 2014; Tarsitani et al. 2014; Kavklera et al. 2015; Ruga et al. 2015; Ranalli et al. 2018), there is still much to do on the definition of danger thresholds for biodeterioration processes.

2.4 Revealing and Identifying Microbial Particles and Products

No automatic instruments are currently available for the direct measurement of viable and non-viable microorganisms in the air or on surfaces (Caneva et al. 2020). Nor is there a universal bioaerosol sampler: available devices must provide a representative sample trying to minimise stress (e.g. dehydration) and damage to the biological activity of microorganisms. The main aim of sampling is to identify the biological particles (qualitative sampling) presents in the sampled location and measure the variation of their concentration in the atmosphere (quantitative sampling).

Before embarking on sampling, it is important to establish what is being sought and where, which is the best sampling point in relation to the environmental characteristics and the presumed degree of contamination so as to set appropriate sampling times. Alongside biological sampling, it is particularly important to undertake parallel sampling of the main physical and chemical environmental parameters. Last but not least, the most suitable analysis techniques must be chosen to identify and quantify the bioaerosol isolated, especially in the case of viable material. It should be emphasised again that the exact bioaerosol fraction cannot be known using only culture-dependent methods since microbes may be viable but non-cultivable, underestimating both microbial diversity and concentration (Blais-Lecours et al. 2015). Nevertheless, the combination of culture-dependent and molecular analysis should be promoted to increase the observed microbial diversity (Palla 2015; Saiz-Jimenez and Gonzalez 2007) and should be adopted also in the field of cultural heritage diagnostics.

2.4.1 Sampling by Passive or Active Methods

The simplest technique, and hence the most commonly used by non-experts, is gravitational deposition, exposing a horizontal surface on which particles settle by gravity and remain trapped by an adhesive placed on the sampling surface or directly on a semisolid culture. Sampling efficacy will depend on air conditions, wind direction and speed, as well as particle concentration and dimensions. The passive sampling allows a qualitative investigation, as the volume of sampled air and the efficiency of capture are not known. It is only suitable for undisturbed indoor environments and is also used for scientific tests in Italian heritage sites, such as the Sistine Chapel in Rome, the Palatina Library in Parma and in the crypt of St. Peter in Perugia (Montacutelli et al. 2000; Pasquarella et al. 2015; Ruga et al. 2015). Petri dishes measuring 90 cm in diameter, containing semisolid culture media (Sabouraud or Nutrient agar), are normally left open to the air for 1 h at 1 m from the floor and 1 m away from walls and then incubated at 30 °C for 16–72 h reaching bacterial or fungal colonies (Fig. 2.2). Culture plate sampling results first need to undergo culture analysis and are then expressed in colony-forming units per surface area (CFU/dm2). To estimate microbial air contamination, an index of microbial air (IMA) is used, based on the count of microbial fallout on Petri dishes, expressed in CFU/dm2/h or CFU/h (Pasquarella et al. 2000).

Fig. 2.2
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Passive sampling carried out in the same indoor environment using (a) Sabouraud medium (fungal colonies) and (b) Nutrient agar medium (bacterial colonies)

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A sterile glass slide treated with adhesive can be used to sample non-viable bioaerosol with subsequent direct observation of the particulate under an optical microscope, the results are expressed in this case in particles per surface area (particles/cm2). In addition to air sampling the affected surfaces of cultural objects can be sampled in a non-destructive and non-invasive way, using strips of adhesive tape (Fungi-Tape™) or sterile cotton swabs or nitrocellulose membrane filters (Sartorius AG, Göttingen, Germany), followed by observation under optical microscope or scanning electron microscope (SEM) after adequate sample preparation. The nitrocellulose membrane filters consisted of a 47 mm square disc pressed onto the sample surface for 30 s and then transferred to Petri dishes containing agar for bacteria or fungi isolation (Pitzurra et al. 1997; Pasquarella et al. 2015, Micheluz et al. 2015). To avoid material loss in very small sampling points it is possible to perform a slight aspiration, using a vacuum pump for a short time, in order to guarantee the sufficient quantity of material for subsequent analyses (Paiva de Carvalho et al. 2019).

The Andersen microbial air sampler is a cascade impactor commonly used in diagnostics in the cultural heritage sector as it samples viable bioaerosol (Andersen 1958). The device is particularly suited to indoor sampling as the aspiration tube cannot be oriented according to the wind direction. The sampler has six or three stages in which the particles are separated by size and collected on Petri dishes containing culture medium. Each stage contains plates with 400 precision-drilled holes of decreasing diameter. The slower air speed in the first stages allows the larger particles to be captured, while the smaller particles are accelerated thanks to the narrower diameter of the holes through which they must pass before being captured by the agar-treated surface. The Petri dishes can then be removed and incubated to allow the captured microbes to grow for subsequent quantification and identification. Airflow is sampled at 28.3 l per minute, wall deposition is negligible and the particle capture rate is close to 100%. Sampling time depends on bioaerosol concentration but is generally in the order of a few minutes. The only limitation of this type of sampler is the high number of dishes generated during each sampling.

The Surface Air System (SAS Super ISO, PBI International, Milan, Italy) is a much more manageable and practical portable single-stage impactor specifically designed for indoor environments of hospitals and pharmaceutical and food companies. At the international level, it is currently considered the reference instrument for microbiological air sampling with an environmental bioaerosol capture rate of 100%. The device aspirates the air at a constant flow for periods varying from a few seconds up to an hour depending on the microbial contamination. The SAS SUPER ISO 100 (180 l of air per minute) is commonly adopted in the cultural heritage sector using 55 mm diameter contact plates, but if high fungal contamination is expected, maxi 84 mm plates or 9 cm Petri dishes can be used with a special adaptor because moulds tend to spread and consequently make counting difficult after incubation. The number of colonies counted on the surface must first be corrected for the statistical possibility of multiple particles passing through the same hole, and then the CFU per cubic metre of air sampled can be calculated.

It must be mentioned that the choice of a specific microbiological growth medium, the incubation temperature and the cultivation time result to differences on the selection of the viable, cultivable, airborne microbial community. Media can be specially prepared in the laboratory or purchased ready-to-use to allow a comparison of results. This is a key feature in the cultural heritage sector where national technical standards provide indications, but standardisation is still a long way off. Tryptic soy agar (TSA) is the medium most commonly used for a total microbial count (incubation for 24–48 h a 32 °C), while Sabouraud dextrose agar (SDA) is used to count yeasts and moulds and usually includes an antibiotic (e.g. chloramphenicol) to avoid bacterial growth (incubation for 5 days at 25 °C).

Other SAS samplers are based on the capture of microorganisms by membrane filtration (SAS dust) or liquid filtration (SAS PCR) which allows identifying the material collected by means of a real-time PCR method. The air samplers that operate in impaction mode (AirPort MD8, Sartorius) collect airborne microorganisms by suctioning a defined air volume through a gelatine membrane filter (Fig. 2.3a) or a culture agar plate. Gelatine filters (Sartorius, AG Germany) allow the material collected to be transported to a culture medium for incubation as shown in Fig. 2.3b, c (Di Carlo et al. 2016).

Fig. 2.3
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Crypt environment. (a) Aerosol active sampling by portable sampler equipped with sterile disposable gelatine filter; (b) nutrient medium inoculated by Gelatine filter (completely water-soluble); (c) dissolving of gelatine filter during the contact with Nutrient Agar (the filter completely disappears in a few minutes)

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Aspiration samplers are frequently used for the non-biological fraction of atmospheric particulate adopting membrane filters with pores of a few micron for non-viable bioaerosol (pollen grains and spores) where microorganisms remain trapped on the surface and can be examined under the light microscope either directly or after diaphanization, depending on the filter matrix.

Hirst impact sampler is used to measure the concentration trends of bioaerosol components like pollen, fungal spores, algae and other particles morphologically recognisable under optical microscope (Hirst 1995). The collection surface can be a microscope slide (for daily sampling) or a transparent plastic tape (for 7-day monitoring) treated with the application of a silicon layer to retain the impacted particles. Sampling efficiency is around 95% for particles with a diameter larger than 20 μm and 50% for particles with diameters 2–5 μm. The 2 × 14 mm slit allows the sampled air to move over a surface at 2 mm per hour so that the time trend of particulate concentration can be measured by subsequent light microscopy Observation. The sampler’s suction rate is 10 l per minute, equivalent to the average human breathing rate (Mandrioli et al. 1998), and for this reason is principally used in the field of health, in centres belonging to aeroallergen monitoring networks worldwide, performing the continuous monitoring of pollens and fungal spores (EAN, RIMA, REA, RNSA, NAB, etc.)Footnote 4 by a procedure already standardised in Italy (UNI 11108:2004)Footnote 5 and in Europe (CEN EN 16868:2019).Footnote 6

The resulting data bank could be an interesting source of information on the daily concentrations of outdoor fungal spores in urban and rural environments for the cultural heritage sector. Three manufacturers (Lanzoni S.r.l., Bologna, Italy, Burkard Manufacturing Co. Ltd., Hertfordshire, England, Cavazza Sas, Bologna, Italy) currently adopt international recommendations to manufacture the commonly used outdoor samplers (VPPS 2000, volumetric spore trap and easy SPT100) and the portable version more often used indoors (VPPS 1000, indoor volumetric spore trap and easy SPT100 light).

2.4.2 Biochemical and Biomolecular Techniques

The analytical approach aimed at identifying the biological particles in the indoor air environment that can represent biodeterioration and health hazards includes a broad spectrum of methods. They are based on both conventional microbiological procedures and advanced techniques of molecular biology (Letch 2016; MacNeil et al. 1995). The identification of colonies isolated from air samples can be performed by observing the morphological features according to different manuals or identification keys. In many cases, however, it may be necessary to identify microbial consortia using specific staining methods (e.g. Lugol’s staining, Gram staining), biochemical tests (e.g. enzymatic assay, metabolite profiling, ATP bioluminescence assay) and molecular analysis (Di Carlo et al. 2016; Lavin et al. 2014; Šimonovičová et al. 2015; Sanmartín et al. 2016; Castillo et al. 2016).

The application of molecular methods has allowed cultivation-independent investigations of microbial communities in diverse environments. Since not every microorganism in a microbial community can be isolated or cultivated, extraction and sequencing of total microbial DNA are useful to identify those microorganisms defined “not culturable” (Puškárová et al. 2016). Culture-independent methods (CIMs) are based on genetic identification (qualitative analysis) of bacteria and fungi as well as DGGE profiling and PCR that have been developed to study microbial communities from various environments (Šimonovičová et al. 2015; Letch 2016). The strategy of PCR-mediated amplification of targeted sequences, followed by sequencing and comparative data analysis, has been used successfully on samples from air (Palla et al. 2014; Pasquarella et al. 2015). Similarly, non-PCR-based molecular techniques, such as microarray and fluorescence in situ hybridisation, have also been adopted (Su et al. 2012).

Molecular fingerprinting techniques (bacteria and fungi quantitative PCR, capillary electrophoresis single-strand conformation polymorphism fingerprinting) have been applied by many authors to analyse airborne bacteria and fungi in enclosed spaces, also in relation to bioaerosol in outdoor air, and the influence of microclimate parameters and total dust content on microbial contamination (Gaüzère et al. 2013; Skóra et al. 2015). Studies utilising culture-independent analyses of microbial communities in indoor environments give a complete overview, also based on their level of detail in documenting built environment data (Ramos and Stephens 2014).

Recent studies in DNA sequencing techniques have been carried out focusing on airborne and dust-borne microorganisms in selected museum, archive and library environments. An analytical approach using molecular fingerprinting has been applied to monitoring and characterising the airborne microbial diversity in the Louvre Museum over a long period of time (Gaüzère et al. 2014).

Microbiological contamination has been analysed in several Polish museums, libraries and archives by Skóra et al. (2015). The resulting nucleotide sequences of the identified microorganisms were analysed and compared to the sequences published in the National Center for Biotechnology Information (NCBI) database, using the BLASTN programme, confirming genetically the identified bacteria and yeasts that were previously macroscopically and microscopically characterised using Gram staining and catalase and oxidase tests. Combining cultivation-independent and cultivation-dependent studies, the fungal diversity in indoor environments was performed in order to shed light on the components of microbial consortia (Micheluz et al. 2015; Ortega-Morales et al. 2016). A widespread fungal infection was revealed in compactus shelves of Venetian library by Micheluz et al. (2015); particularly, xerophilic fungi were identified using a polyphasic approach based on morpho-physiological features and molecular studies. Molecular identification was performed by amplification and sequencing of internal transcribed spacers (ITS) of β-tubulin and actin genes. Moreover, airborne fungi possess great enzymatic potential to degrade materials, so their hydrolytic activity in attacking, proliferating and degrading these important artistic-historical items can be successfully detected using enzymatic assays and can be considered valuable data in completing the typical identification list of isolated strains. Recently, the biodegradative action of fungal microflora from mummified remains and fungal airborne communities was investigated using hydrolytic assays (Šimonovičová et al. 2015). Borrego et al. (2012) have determined indoor air quality in Argentine archives and the biodeterioration of documentary heritage using an analytical approach based on the qualitative determination of enzymatic fungal activity and acid production by fungi.