**3. Results**

#### **3.1 Macroscopical and microscopical analysis**

Relevant areas for analysis were identified as affected areas of the mosaic (**Figure 4a** and **b**) and adjacent parts, such as the eastern wall, built of brick and with obvious biological contamination and degradation (**Figure 4c** and **d**). The autotrophic biofilm was noticed at this level, arranged in successive layers, parts with intense processes of fungal contamination, in the upper area, as well as mineral deposits.

Microalgae are commonly found on calcareous substrates and can induce degradation by release of acids, accumulation of ions and alteration by pigmentation, fragmentation of the substrate and the formation of crusty, dark or patinatedlooking surfaces [23].

Microscopic analysis shows lichen-like structures with an upper cortex formed exclusively by tightly woven and welded hyphae, as well as a gonidial layer with algal symbiosis indicating chlorophyll activity (red coloration) (**Figure 5a**). The middle layer (medullary) consists exclusively of mycelial hyphae, loosely braided (green coloration), (**Figure 5a**).

The biological activity by substrate attachment is very evident (**Figure 5**), as well as the formation of mobile structures, detached from the substrate or colony,

#### **Figure 4.**

*The affected areas of the mosaic by chromatic alteration and destructions (a, b), the biodegradation of wall surfaces (c, d).*

of the goniocyst type, which include photobiont cells surrounded by hyphae arranged in a single layer (**Figure 5c**).

In the S 8 sample (**Figure 6**) intense biological activities were identified, including various colonies of microalgae (**Figure 6d**) as well as fungal activity (**Figure 6a**–**c**).

The massive microorganisms colonization, highlighted in the samples, explains the various depigmentation, or changes in shades, or the biogenic pigmentation that was identified on the analyzed surface. Bacteria induce changes by discoloration as well as by dissolving various materials generating biodegradation of the substrate through specific metabolism [24].

The crushing of the substrate is noticeable in many samples and can be generated, for the most part, by the impact produced by the bacterial metabolism combined with the chemoheterotrophic metabolism of the fungi [25].

Fungi (hyphae or resistance morphotypes) induce degradation on the substrate by dissolution or the formation of pigmented or discolored areas [26].

The analyzed samples were noted free of hyphae and completely attached to the substrate by obvious interactions. The samples show emission in green by staining with AO which suggests active metabolism.

#### **3.2 Microbiota isolated on nutritive medium**

The isolated microorganisms from the museum space, respectively mosaic surfaces, included several forms of which 15 are bacteria (*Bacillus ssp. Aeromonas salmonicida,* 

#### **Figure 5.**

*The microscopical examination of biological samples a) autotrophic forms, in epifluorescence with acridine orange staining, b) biological associations in light microscopy images, c) goniocyst, d) fungal contamination, optical microscopy, bar = 100 μm (x400 magnification).*

*The Impact Study of the Exchanges between the Microorganism Communities on the Surfaces… DOI: http://dx.doi.org/10.5772/intechopen.99057*

**Figure 6.** *The microscopic details of fungal morphotypes (a, b, c) and microalgae (d).*

*Kocuria rosea, Clostridium histolyticum, Corynebacterium urealyticum, Acinetobacter lwoffii, Corynebacterium pseudodiphtericum, Bacillus licheniformis, Bacillus clausii, Staphylococcus epidermis, Micrococcus luteus, Corynebacterium ssp., Pseudomonas* ssp.) and 11 fungi (*Aspergillus flavus, Aspergillus niger, Rizopus sp., Penicillium ssp, Candida sp., Alternaria* sp.) and five unidentified species.

Quantitative evaluation of microorganisms highlighted the microorganism load of surfaces near the eastern wall (S 5, S 7) of the analyzed space (**Table 1**) where the number of bacterial colonies was 1921.8 CFU/cm2 and 2328.1 CFU/cm<sup>2</sup> , respectively. The fungal colonies being also remarkable in the S 5 sample, the data indicating 3146.8 CFU/cm<sup>2</sup> (**Table 1**). The low CFU/cm2 density values were recorded in the central area of the space (S 1, S 3).

The air is not a favorable environment for the development of microorganisms, but in the air, there are microorganisms that come from the substratum, humans contact and another different sources and these organisms could be transmitted through air currents.

The air microbiota is influenced by a series of factors: the degree of air ventilation, temperature, relative humidity, human presence and agglomeration, etc.

From the total samples analyzed by the isolated colonies, one can notice the density of fungi with over 4000 CFU/m3 in the area destined for the passage of tourists (**Table 2**), passage with stairs (A 1) as well as near the brick wall (A 5). These data confirm the possibility of favorable factors with risk of passage to the mosaic, in the portions with cement and brick substrate where humidity and porosity are the main factors that maintain the development of fungi.

A large part of the microorganisms with pathogenicity for humans can be transmitted by air inside the exhibition hall, but at the same time they can represent


**Table 1.**

*The microbial density (CFU/cm2 ) on mosaic surfaces.*


**Table 2.**

*The airborn microbial density (CFU/cm3 ), the index of microbial air contamination (IMA).*

degradation factors for the exhibited museum objects, mosaic and other vestiges of the museum complex **(Figure 7**).

The samples highlight the microbiological load in the air column, the isolation from repeated samples suggesting an amplified activity, especially of the species isolated on Columbia blood environment that show hemolytic activity.

Many species are found in the native, resident microbiota of the colon. Among the isolated species, *Bacillus cereus*, *Corynebacterium urealyticum*, they attracted attention on surface of mosaic samples.

In terms of habitat and pathological implications, resistance by the formation of spores at these bacteria can maked occasionally be isolated from environmental sites, which are only sources of transmission. The ease of airborne transmission of endospores is very obvious.

The most important colonies, present in all the analyzed points, are from the group of gram-positive bacilli, the genus *Bacillus* being highlighted in all the samples (*B. pumilus, B. cereus*) and *B. clausii* bacteria only in airborne (**Figure 8**).

The genus *Bacillus* includes aerobic or facultative anaerobic forms with a diversity of physiological characteristics: mesophilic, thermophilic, psychrophilic, alkalophilic, acidophilic, halotolerant or halophilic species. These characteristics also explain the presence of this genus on analyzed surfaces, with the highest frequency and diversity (**Figure 8**), being very tolerant.

*Bacillus cereus* grows in poor soils and frequently contaminates food. The implications in the current infectious pathology of the identified bacilli, which aerobically endospore, are the followings: wound infections, lung infections, urinary tract infections, food poisoning, rectal fistulas.

Another genus identified is *Clostridium*. Bacteria gathered from this genus are sporulated anaerobic bacteria, widespread in the soil and present in the colon of humans and animals. The pathogenic potential of anaerobes is manifested only in conditions that favor their access to the internal medium and allow them to grow. Regarding their sensitivity to antimicrobial agents, it is important to mention that *Clostridium* sp. and gram-positive non-sporulated anaerobes have retained their natural sensitivity to antibiotics, but infections with anaerobic bacteria are often mixed, including different morphotypes.

*The Impact Study of the Exchanges between the Microorganism Communities on the Surfaces… DOI: http://dx.doi.org/10.5772/intechopen.99057*

**Figure 7.** *The common and specific microbiota for surfaces and airborne samples diagram.*

The study of Perez et al. 2021, highlighted the fact that in the biodegradation process there are successive sequences of biochemical reactions that favor the growth of different microbial communities. The first stages of this reaction complex are hydrolysis and bacterial acidogenesis in which class *Clostridia* are involved followed by class *Bacilli*, which explains the large number of colonies in

#### **Figure 9.**

*Fungal frequency (%) isolated from all samples (ned 1-ned 5 unidentified species).*

these categories isolated in samples taken and analyzed from the surfaces of Roman mosaic pieces named *tesserae* [27].

Another bacterial category that attracts attention, isolated from the analyzed surfaces (S1) is *Staphylococcus epidermidis* and *Micrococcus luteus*. The species is ubiquitous in skin biotopes [28]. The species *Staphylococcus epidermidis senso stricto* is a species of major medical interest, being characterized by high pathogenic potential. The presence in the samples was repeated and confirmed in the laboratory by morphological analysis of the colonies, microscopy and biochemistry.

*Kocuria* ssp. appears in the analyzed samples and be noticeable it stands out by the fact that the genus includes commensal forms in humans, but also in animal species. *Kocuria* spp. is also present in the environment but may have pathogenic potential in people with compromised immune systems [29].

Another species identified is *Corynebacterium urealyticum* which has the human skin as its habitat. Coryneform bacilli are distinguished by the fact that they can occasionally move from one ecological niche to another and can accidentally cause diseases (urinary tract infections, skin infections, endocarditis [30].

From the category of fungi from Ascomycota, the most dominant presence of the forms of *Aspergillus* as well as of the type of lower fungi *Rhizopus* sp. can be noticed (**Figure 9**). Also, several genera grew on nutritive media *Penicillium* ssp., *Candida* sp., *Alternaria* sp., and other five colonies were unidentified.

The genus *Aspergillus* comprises over 461 species, mostly found in saprobiosis in the environment and are rarely parasitic [31].

*Aspergillus flavus* is known for its ability to generate mycotoxins (aflatoxins) when grown on food substrate. As a pathogenicity, in immunodeficient subjects, it produces pulmonary aspergillosis and chronic sinusitis.

*Aspergillus niger* is a cosmopolite micromycete, frequently isolated from soil, but also from many organic substrates. It develops a series of mycotoxins such as aflatoxins, aspergillin, aspergin. It is frequently involved in the appearance of aspergillomas and in immunocompromised subjects causes skin, lung and systemic infections.

The genus *Rhizopus* develops rapidly in culture media but also in natural environments an invasive mycelial apparatus, with a fluffy appearance at maturity colored in black, due to sporangia. Characteristic of the genus are the rhizoids with *The Impact Study of the Exchanges between the Microorganism Communities on the Surfaces… DOI: http://dx.doi.org/10.5772/intechopen.99057*

which it attaches to the substrate. *Rhizopus* species are generally non-pathogenic but can cause zygomycosis in debilitated patients manifested by processes of tissue necrosis [31].

By analyzing the microbiota in the air of the exhibition hall one can predict the degree of danger of a future infection of the mosaic carpet, the exhibits and which are the groups and species of microorganisms that represent the main threat to an infection of heritage objects. Last but not least, through this analysis it is possible to establish the risk of infecting the people present inside the museum complex, both employed staff and visiting public.

## **4. Conclusions**

The results showed that the Roman Mosaic are significatively affected by multiple biological systems with biodeterioration high potential (algae, lichens, bacteria and fungi).

The species thus isolated and identified were analyzed in correlation with the implications in biodegradation, they affect the components of archeological interest, as well as in terms of pathogenic risk, taking into account the sources of anthropogenic contamination of mosaic components.

The presence, in this exhibition space, of several pathogenic genera (bacteria and fungi), confirmed as having an important pathogenic potential, draws attention to the management of cleaning and preparation of the space before and after the passage of visitors.

#### **Acknowledgements**

This work was supported by a grant of the Romanian Ministery of Research and Innovation, CCCDI – UEFISCDI, project number PN-III-P1-1.2-PCCDI-2017- 0476/51-PCCDI/2018, within PNCDI III, ACRONIM: ARHEOCONS.

### **Conflict of interest**

The authors declare no conflict of interest.

*Heritage - New Paradigm*
