The Roman Houses of the Caelian Hill (Rome, Italy): Multitemporal Evaluation of Biodeterioration Patterns

Like other hypogeal environments, the Roman Houses of the Caelian Hill are prone to unwanted biological growth. Wide conservative interventions have been carried out at the beginning of this millenium to reduce biodeterioration and physical–chemical damages. Retracing the last monitoring work, we assessed the site’s current state of conservation and biodeterioration intending to check the previous treatments’ effectiveness and deepen the common knowledge of the subterranean biota and their possible biodeteriogenic effects. Starting from the past test areas and the previous identifications of the occurring biodeteriogens, we further isolated and identified the main eubacterial, fungal, and phototrophic settlers, focusing on some detrimental traits for wall paintings (i.e., acid production and carbonate precipitation). The achieved results proved the success of the performed interventions in reducing the wall’s water content. Otherwise, the new conditions raise, in the long term, new concerns about lampenflora, carbonate precipitations, and salt efflorescence. Here, the Caelian Houses’ new status is documented. The possible favouring conditions for the different groups of biodeteriogens, along with the taxonomical novelties, additional risks tied to the anthropization of the resident culturable microbial community, and the possible relation between the black fungus Cyphellophora olivacea and roots, are reported and discussed.


Introduction
Hypogeal sites are, in general, nutrient-poor ecosystems characterized by very high air relative humidity, sometimes close to saturation, high water content in the wall structures, constant relatively low temperatures, and poor ventilation, which make them particularly favourable for a wide number of organisms [1]. Moreover, underground environments are considered "extreme." As for Liebig's law of the minimum, in this environment, the main limiting factors are the light for photoautotrophs (for lichens also ventilation) and the availability of organic matter for heterotrophs; for this reason, common inhabitants of these sites are phototrophs adapted to the scarcity of light and oligotrophs adapted to long periods of starvation [2][3][4][5]. The environmental pressure expressed by limiting factors is so high that each small change determines rapid variation in the resident biotic communities, producing different visible biodeterioration patterns (BP) [6]. Changes in light, carbon dioxide, nutrient inputs, and temperature have been recognized as major driving factors striking example of this transformation is the series of bricked up windows that became part of the Basilica dei Santi Giovanni e Paolo (Basilica of Saints John and Paul), which is visible even from the outside ( Figure 1A,B). This inclusion in the new structure changed drastically the houses space ratios and the lines of floors overlapping and caching the pre-existing structures, transforming de facto houses into a false hypogeum. The insula was used until the 9th century, then abandoned and discovered at the end of the 19th century. In 1997, the Soprintendenza Speciale per i Beni Archeologici di Roma (Special Archeological Superintendence of Rome), with the collaboration with the Central Institute of Restauration (ICR), started the restoration of the Roman houses facing several problems of paintings conservation due mainly to water infiltration and hypogeal microclimatic conditions.  Bartolini and colleagues [22,23].
Before restoration and opening to visits (July 2002) the ICR performed a study along a two years' time lapse, to understand the biodeterioration dynamics, support a preventive measures plan, and schedule a prevention timetable [22,23]. With this purpose, a quali-quantitative investigation has been performed on surfaces' contamination by heterotrophic and autotrophic microorganisms. Four rooms along the visit path were used as a reference for the environmental conditions characterizing the site. In detail, the "Stanza dei Geni" (the Chamber of the Geniuses, CG, the household spirits protecting home) is close to the entrance, the "Ninfeo di Proserpina" (the Nymphaeum of Proserpine, NP) is a large room with the external wall partially buried and in contact with the external garden and by air with the space downstairs. More, the "cella vinaria" (the winery, W), a three-sides closed cell characterized by highly moist walls, and the "Balneum" (a private  Bartolini and colleagues [22,23]. Before restoration and opening to visits (July 2002) the ICR performed a study along a two years' time lapse, to understand the biodeterioration dynamics, support a preventive measures plan, and schedule a prevention timetable [22,23]. With this purpose, a qualiquantitative investigation has been performed on surfaces' contamination by heterotrophic and autotrophic microorganisms. Four rooms along the visit path were used as a reference for the environmental conditions characterizing the site. In detail, the "Stanza dei Geni" (the Chamber of the Geniuses, CG, the household spirits protecting home) is close to the entrance, the "Ninfeo di Proserpina" (the Nymphaeum of Proserpine, NP) is a large room with the external wall partially buried and in contact with the external garden and by air with the space downstairs. More, the "cella vinaria" (the winery, W), a three-sides closed cell characterized by highly moist walls, and the "Balneum" (a private thermal plant, BAL) in the deepest floor, never treated or restored and excluded from the visit path ( Figure 1). To obtain consistent and comparable data, we identified the previously analysed areas [22,23] and applied the same sampling techniques and types of qualitative-quantitative analyses. On 19 July and 15 November 2019 two sampling campaigns have been performed. The date choice was subject to sampling permissions, the absence of open visits to the public and the four-month gap to verify the presence of thermal inversion.
Using sterile swabs, 16 samples were taken from 9 cm per side squared areas defined by plastic sheets (one for each sampling point) as performed by Bartolini and colleagues in the previous studies ( [22,23]; Figure 1D-G). Additional samples were taken to investigate the areas showing signs of biological growth such as discolorations or areas at risk for biological growth (e.g., illuminated areas), adhesive tape sampling method was also used. Dried tiny roots were recorded in the NP below the fresco in the main wall (right side) and sterilely collected. Samples taken from green patinas were stored at 4 • C and processed within the following two days; the other samples instead were stored at −20 • C and processed immediately after.

Cultivation, Isolation, and Identification of the BP' Components
The samples were suspended in sterile saline (NaCl 0.9%), diluted scalarly and then plated in triplicate on Mycological agar (MYC, BD Difco TM Sparks, MD, USA) consisting of soy peptone 10 g/L, dextrose 10 g/L, agar 15 g/L and incubated at 28 • C following UNI NORMAL 9/88 protocol to count the total number of fungi and bacteria expressed as CFU/cm 2 [22,23,62].
Fresh material was observed in a Thoma chamber where frequency values were achieved for phototrophic taxa. An aliquot of 100 µL of sample suspension was inoculated into liquid BG11 freshwater medium (BG11, Sigma Aldrich, Darmstadt, Germany) and Bold Basal liquid Medium (BBM, Sigma Aldrich) developed for cyanobacteria and microalgae respectively. Cultures were maintained 6-8 weeks under cool-white fluorescent illumination (Osram Dulux L 36W/840 Lumilux, 2900 lumens, Osram GmbH, Munich, Germany), with a 12-h photoperiod, at 20 ± 2 • C [22,23,63]. Once plated onto a solid medium, was left to grow at the abovementioned conditions until colonies were detectable and picked up for molecular identification not performed in the previous survey.
To improve the isolation yields and knowledge on current microbial community, 100 µL of each serial dilution (from all the samples taken) were plated also on Luria-Bertani (LB) for bacteria, Starch Casein Agar (SCA) for actinomycetes, Trypticase Soy Agar (TSA) supplemented with NaCl (3% w/v) and MgSO4·7H 2 O (2% w/v) for salt tolerant bacteria, Dichloran Rose Bengal Chloramphenicol (DRBC, VWR International GmbH, Darmstadt, Germany) for fungi. Plates were incubated for two months at 20 ± 1 • C for bacteria and 15 ± 1 • C for fungi to be more consistent with site conditions and improve the detection of slow-growing microorganisms.
The isolates in pure cultures were transferred on tryptic soy agar (TSA) for bacteria and malt agar (MA, Malt extract 30 g/L, bacteriological agar 15 g/L) for fungi and selected for further processing based on major morphological colony features.
PCR reactions were performed in a total volume of 25 µL using BioMix (BioLine, Luckenwalde, Germany), 5 pmol of each primer and about 30 ng of template DNA were added. Amplifications were carried out using MyCycler™ Thermal Cycler (Bio-Rad Laboratories, Munich, Germany), the protocols used are listed at Table S1. Sequencing was performed by Macrogen (Madrid, Spain) and the electropherograms manually checked/assembled using Chromas Pro 1.41 (Technelysium, Southport, QLD, Australia). Similarity searches have been performed using the algorithm BLASTn limiting the search between the sequences coming from type strains for bacteria and excluding from the comparison "uncultured/environmental sample sequences" for fungi. The bacterial taxonomical ranking was fixed using NCBI taxonomy browser as reference. The obtained sequences were deposited in GenBank. Morphological identification of phototrophs was performed using the analytic keys of Guiry and Guiry [68].

Multitemporal Analysis
According to Bartolini and colleagues [22,23], the presence of phototrophic and heterotrophic microorganisms has been assessed, the latter discriminating between fungi and bacteria. To visually describe the variation that occurred over time in the communities analyzed, a quali-quantitative scale of frequencies has been set. In detail, CFU/cm 2 were reported as values (+) in order of magnitude with the power of ten (i.e., +, ++, and +++ were used instead of values 10, 100, and 1000 respectively); while +/− indicate positive values below 9, -not found; and / not investigated.
The availability of morphological identification of the phototrophic component [22,23] allowed the comparison at the species level. Otherwise, a comparison at the higher rank (i.e., as fungi and bacteria) was performed for the heterotrophic microorganisms.

Plate Assay for Bacterial Detrimental Potential
Fungal spreading is a rare event in hypogeal environments generally tied to sudden changes in carbon sources availability or the weakening of the resident bacterial community with antifungal activity due to biocide treatments [1,5,9,14]. Since no active fungal growth was recorded before the opening to visit [22,23] nor recently, we decided to focus on the heterotrophic bacterial component and its adverse potential. Using plate trials, we investigated the acid production and carbonate precipitation possibly leading respectively to substrate dissolution and pigments alteration [69], and interfere with the visual appreciation of the underlying artwork [70]. Acid production was assessed using CaCO 3 agar medium containing yeast extract 5 g, glucose 50 g, CaCO 3 5 g, agarose 15 g per liter, while precipitation test was carried out on B4 medium (yeast extract 1 g, glucose 1 g, calcium acetate monohydrate 5 g, agarose 15 g per liter of solution). Mineral phase precipitation was monitored twice a week under a stereomicroscope (Nikon SMZ80, Minato, Tokyo, Japan) and documented by Nikon Coolpix 500 camera.

Current Conservative Conditions
From a microclimatic point of view, the performed measurements evidenced as the rooms investigated are subject to a gradient of temperature and relative humidity (Table S2) and as the outdoor conditions significantly affect the Geniuses Chamber (CG). In contrast, stable conditions characterize the Balneum (BAL). Indeed, the variation recorded in CG during Summer and Autumn sampling sessions was about 4 • C and 8 RH%, while 0.2 • C and 1 RH% in BAL. Moreover, even in BAL, the RH levels were below 90%.
Comparing the presence of microclimatic conditions to the data extracted in 5 November 2002, we can note that temperatures are superimposable while major RH differences have been recorded up to 7% (Table S2).
The most common biodeterioration pattern resulted green biofilms close to lamps ( Figure 2F). Particularly evident in the walls of the winery ( Figure 2G,H,L), the green patinas seem to be here associated, in the closest part to the light, with mortar weakness since in wall A it exfoliates ( Figure 2G white arrow) while the wall B ( Figure 2L) when gently knocked it produced a dull sound.; more in the shaded part of the wall B a brown patina was also visible ( Figure 2K). Lampenflora has also been recorded in the side chamber of the NP where the bricks are covered by a powderly opaque light green patina ( Figure 2C, Px sampling point) and in the Balneum with two new sampling points namely By ( Figure 2I) and Bx ( Figure 2M). A white patina due to salt efflorescence, not recorded before ( Figure 1E), has been found in the lower wall in the NP ( Figure 2B,E).

Current Conservative Conditions
From a microclimatic point of view, the performed measurements evidenced as the rooms investigated are subject to a gradient of temperature and relative humidity (Table  S2) and as the outdoor conditions significantly affect the Geniuses Chamber (CG). In contrast, stable conditions characterize the Balneum (BAL). Indeed, the variation recorded in CG during Summer and Autumn sampling sessions was about 4 °C and 8 RH%, while 0.2 °C and 1 RH% in BAL. Moreover, even in BAL, the RH levels were below 90%.
Comparing the presence of microclimatic conditions to the data extracted in 5 November 2002, we can note that temperatures are superimposable while major RH differences have been recorded up to 7% (Table S2).
The most common biodeterioration pattern resulted green biofilms close to lamps ( Figure 2F). Particularly evident in the walls of the winery ( Figure 2G,H,L), the green patinas seem to be here associated, in the closest part to the light, with mortar weakness since in wall A it exfoliates ( Figure 2G white arrow) while the wall B ( Figure 2L) when gently knocked it produced a dull sound.; more in the shaded part of the wall B a brown patina was also visible ( Figure 2K). Lampenflora has also been recorded in the side chamber of the NP where the bricks are covered by a powderly opaque light green patina ( Figure 2C, Px sampling point) and in the Balneum with two new sampling points namely By ( Figure 2I) and Bx ( Figure 2M). A white patina due to salt efflorescence, not recorded before ( Figure 1E), has been found in the lower wall in the NP ( Figure 2B,E).

Identification of Biodeteriogens
The direct observation at microscope allowed for the identification of phototrophs in some cases improved by the molecular analysis. The NP side chamber (Px) showed a lightgreen opaque patina on the main wall composed exclusively by Scytonema sp. (Figure 3A). The Wall A of the winery is dominated by Pseudostichococcus cfr. monalloides (identity 98.32%; OQ540755; Figure

Identification of Biodeteriogens
The direct observation at microscope allowed for the identification of phototrophs in some cases improved by the molecular analysis. The NP side chamber (Px) showed a lightgreen opaque patina on the main wall composed exclusively by Scytonema sp. (Figure 3A). The Wall A of the winery is dominated by Pseudostichococcus cfr. monalloides (identity 98.32%; OQ540755; Figure   One hundred fifty-one bacterial isolates were achieved, while 87 were selected by colony morphology and further processed. The bacterial identification (Table 1) evidenced the genus Peribacillus as the most frequent, as it was recorded in three chambers (CG, NP, and BAL) and represented 17.44% of isolates. At a higher rank, the class Bacilli (36.78%), the order Bacillales (36.78%), and family Bacillaceae (32.18%) are the most frequent ( Figure  S1). One hundred fifty-one bacterial isolates were achieved, while 87 were selected by colony morphology and further processed. The bacterial identification (Table 1) evidenced the genus Peribacillus as the most frequent, as it was recorded in three chambers (CG, NP, and BAL) and represented 17.44% of isolates. At a higher rank, the class Bacilli (36.78%), the order Bacillales (36.78%), and family Bacillaceae (32.18%) are the most frequent ( Figure S1). Twenty-six fungal isolates were achieved (Table 2). They mainly belong to the genera Penicillium (42.85%), Cladosporium (23.8%), and Aspergillus (4.76%). The remaining strains (28.57%) are represented by strains of the genera Malassezia, Torula, Chrysosporium, and Cyphellophora (C. olivacea); the last two had 97.1% identity with Pseudogymnoascus pannorum and 96,45% with [Coniosporium] MA 4639. Centaurea, Echinops, and Saussurea resulted in the best matches for rbcL root sequencing (100%, OQ550265), while the ITS target led to the amplification of the fungus Cyphellophora olivacea (99.3%, OQ534303). Penicillium sp. was isolated from PX, CV-A, and CV-B (green patinas). Still, its finding should be considered "occasional" since only one colony per site has been found there, and direct microscope observation of green patinas never recorded the presence of hyphae.

The Multitemporal Evaluation Total Counts Comparison
Comparing the results of the previous monitoring work with the present and biodeterioration of the site, trough plate counts, we can observe that the number of heterotrophs decreased in the samples taken from Sites G5 and P10, which were previously affected by a brown and grey-brown patina respectively (Table 3). A decrease has also been recorded in the winery (Samples 11 and 11b) and Balneum (Sample 14). Otherwise, a marked increase on counts has been recorded especially in the Nymphaeum (NP) in Samples P7, P8, and P9. Meanwhile, G1 and G2, which were never investigated before for heterotrophs, revealed a high presence of fungi. An increased presence of phototrophs was found in the winery and in all sampled sites in the Balneum (Table 3). Table 3. Total counts of phototrophs and heterotrophs during the multitemporal investigations. To highlight differences occurred over time, the results have been reported as order of magnitude with power of 10 of CFU/cm 2 : +/− indicate positive values below 5; values of power of 10, 100, 1000, and 10,000 are reported as +, ++, +++ and ++++ respectively;); − not found; and / not investigated. Shaded columns are for new data.

Room
Regarding the phototrophic microorganisms, the comparison with the previous data showed their complete disappearance in P10 samples ( Table 4). The Winery's walls, characterized by an evident green patina, recorded Pseudostichococcus, Scytonema, and Chlorella species; otherwise, the Balneum recorded the disappearance of the main patina from Sample G extracted along the arch ( Figure 1G) and the appearance of new ones where even the presence of moss gametophytes have been recorded as in Sample By ( Figure 2I,L). Table 4. Phototrophs identification by morphological and molecular methods, the latter is indicated by asterisk (*). Negative finding has been recorded as -and +/− means not investigated, while +, ++, and +++ a positive increasing finding. Shades indicate new results and samples.

The Assessment of the Detrimental Potential of the Occurring Bacterial Species
The bacterial isolates frequently showed the ability to precipitate carbonates (84.7%) while the ability to dissolve them was recorded in 29.4%. A small portion of them (5.9%) demonstrate none of the deteriorative ability at the tested conditions, while 20% had both. In the tested strains, the carbonatogenic phenomenon varied by quantity (Table 1, indicated by +, ++, +++) and quality (Figure 4) The carbonatogenic phaenomenon varied by quantity and quality in the tested strains ( Figure 4, Table 1). Strains of the genera Achromobacter, Alcaligenes, Agromyces, Lysinibacillus, Nocardia, Peribacillus, and Stenotrophomonas were the most active precipitating carbonates. Furthermore, the colour of precipitates ranged from whitish to brown, and their shape was from spherical to crystalline.

The Assessment of the Detrimental Potential of the Occurring Bacterial Species
The bacterial isolates frequently showed the ability to precipitate carbonates (84.7%) while the ability to dissolve them was recorded in 29.4%. A small portion of them (5.9%) demonstrate none of the deteriorative ability at the tested conditions, while 20% had both. In the tested strains, the carbonatogenic phenomenon varied by quantity (Table 1, indicated by +, ++, +++) and quality (Figure 4) The carbonatogenic phaenomenon varied by quantity and quality in the tested strains ( Figure 4, Table 1). Strains of the genera Achromobacter, Alcaligenes, Agromyces, Lysinibacillus, Nocardia, Peribacillus, and Stenotrophomonas were the most active precipitating carbonates. Furthermore, the colour of precipitates ranged from whitish to brown, and their shape was from spherical to crystalline.

Discussion
Although the crucial importance of long-term surveys is widely recognised for the conservation of cultural heritage [71], only a few researches have been performed in this light. Changes in the conservative priority objectives for site conservation and diagnostic methods used are the primary constraints for this kind of investigation based mainly on data comparison and the ability in deciphering the future trends.

Discussion
Although the crucial importance of long-term surveys is widely recognised for the conservation of cultural heritage [71], only a few researches have been performed in this light. Changes in the conservative priority objectives for site conservation and diagnostic methods used are the primary constraints for this kind of investigation based mainly on data comparison and the ability in deciphering the future trends.
The biodeterioration patterns found in the Roman Houses are consistent with those reported in visited hypogeal sites worldwide [1,72,73] as well as the record of new taxa within the bacterial, fungal, and phototrophic settlers. Such data also confirms the impor-tance of the multi-temporal investigation for checking the effectiveness of the previous treatments [59,60]. The occurred changes appear to be a clear consequence of the direct and indirect control methods applied before opening to visit, but in the meanwhile, this study added new insights into the underground biota. The duration of application of biocides can have a maximum duration of 3-4 years in a favourable condition, such as humid places [74]. The interventions applied to reduce the walls dampness threatening the frescoes conservation are long lasting and effective [4] and it is confirmed here since the total counts notably decreased, and brown biofilm and diatoms disappeared from G5 and P10 in the CG and NP respectively. Diatoms also disappeared from BAL demonstrating as the applied interventions affected even the inners floors. Indeed, if we recently recorded an average RH of 88.1% before it was always above 90% (93% measured 5 November 2002) [22.23]. After that, following the basic Liebig's law the heterotrophic communities confidently redistributed themselves driven by water gradient and carbon sources availability. For this reason, even the most common distribution of bacteria and fungi on vertical surfaces (namely bacteria/down and fungi/up) deserves a reading up in the light of the species involved and their ecology. Bacteria largely prevailed in number and diversity, showing a dominant carbonates-precipitation trait. This feature, widely reported in the literature [75][76][77][78], could threaten wall painting conservation beyond the interest for biotechnological applications [47,79,80], and needs further knowledge improvements to understand their role in the underground environments better. Meanwhile, colourimetric measurements could be scheduled for the wall paintings to verify possible changes in lightness or colour. While RH environmental monitoring is required to reduce/avoid further saline efflorescence and ensure the best conditions for wall paintings conservation.
Other detrimental treats deserving attention could be indirectly ascertain by the taxonomical composition of bacterial community. The recorded bacterial community is composed for about one third (35.29%) by Bacilli, 27.06% by Actinomycetes and 35.30% by Alpha-, Beta-, and Gamma-proteobacteria. The recurrent presence of pathogenic or opportunistic species can be considered a mirror of the human impact on the environment [2,55,81], while microorganisms of anthropogenic origin a consequence of increased availability of organic matter introduced by visitors [13]. In this sense, the finding of species of the genera Inquilinus, Amycolatopsis, Nocardia, Nocardioides, Streptomyces, the family Micrococcaceae on the one hand [55] and Alcaligenes faecalis and Staphylococcus capitis, on the other seems to confirm this trend. Along with the human-impact on the microbial community there is another aspect deserving attention from a conservative point of view: the resistance to biocides and/or antimicrobials. Actinobacteria and Firmicutes (now Bacillota) are able of carrying and disseminating ARGs (Antibiotic resistant genes; [82]) and recently has been assessed that in stone monuments microbiotas Actinobacteria and Proteobacteria averagely accounted for 39.7% and 39.0% respectively of ACCs (Antimicrobial resistance Carrying Contigs; [83]). Moreover, some resident bacterial strains can have antifungal activities like species of Micromonospora, Rhodococcus Streptomyces, Bacillus, Pseudomonas, Stenotrophomonas [14]. So, in case of direct actions, is of utmost importance to carefully evaluate the biocide chemical features as well as the spectrum of efficacy on the resident community to avoid the selection on resistant bacteria or fungal spreading as reported for Lascaux cave [1,5,84].
This principle is valid also for fungi even if in the Roman Houses the situation recorded do not seem to raise concerns. The isolated strains, according to other subterranean cultural heritage sites, mainly belong to the cosmopolitan highly sporulating genera Penicillium, Cladosporium and, in lesser extent, Aspergillus and their presence is related to the air flow from the entrance and within the site [85][86][87]. More interest raises from the other isolates closest relative of the species Malassezia restricta (97.46% identity), a species associated to infections in humans [88], or the presence of "black fungi" relatives to [Coniosporium] MA 4639, previously isolated from marble monuments [89] and Cyphellophora olivacea a species previously found in subterranean environments and hydrocarbon contaminated sites [90,91]. While, the relative of [Coniosporium] MA 4639 was little represented in a site characterized by huge debris, Cyphellophora olivacea was dominant in both P9 samples taken at NP [92]. More interestingly this species was also amplified from the root sample taken there (NP). This fact and previous records of its siblings from roots [93] corroborates the hypothesis of endophytism (dark septate endophyte, DSE) and confirms the detrimental role played by roots in the conservation of hypogeal cultural heritage sites [5,11]. The root identification by rbcL sequencing should be confidently ascribed to Asteraceae family because the species recording the best match are not part of the Latium flora [94] and the well-known poor representation of the local flora in GenBank [11]. In any case, roots finding deserves attention and external perimeter walls inspected.
Aside from the widely reported aesthetical alteration, phototrophs have been associated with stone surface decay. They can cause chemical and physical damage to stone surfaces by producing chelating agents and acids [95]. Calcium carbonate precipitation has often been recorded in association with algal growth in caves and mural paintings threatened by calcite deposition [96]. Most phototrophs grow following the topology of the mineral surface layer, while others contribute to the formation of micro-fissures growing just below it or actively bore the mineral substrata [4,35,97]. This activity has been documented, for example, for Scytonema, where calcium carbonate deposition on cyanobacterial filaments has frequently been recorded at subterranean sites [2,33,35,36]. Anyway, the finding of new taxonomical entities within the genera Albertania, Coccomyxa and Koliella, confirms underground sites as reservoir for new species and underline the importance of this kind of investigations [86,90]. Algal and cyanobacterial components were differently space distributed. For example, even if cyanobacteria are the most adaptable phototrophs due to their tolerance to desiccation and low light intensity requirements [98], in habitats where water is available and, in general, characterized by less environmental stress like illuminated spots around lamps, they are quickly overgrown by fast-growing eukaryotic algae [99]. Indeed, algae prevailed in site characterized by wall dampness (close the ground) and directly illuminated as the two walls of the Winery, while cyanobacteria dominate aside lights far from ground (Px). In this light, is obvious that one of the first actions to be done concern the illuminating system with the use of new generation lights (e.g., LED) not closely facing walls and possibly timed. Furthermore, as the Balneum was (i) never treated before, (ii) is constantly illuminated even if out of the visit path, (iii) is in continuum with the upper floors and (iv) is subject to temperature inversion affecting the air flow, (v) showed a notable increase of the phototrophic patinas (e.g., young gametophytes where they are not) could serve as reservoir for phototrophs spreading.

Conclusions
Research strategies on biodeterioration are changing, and ecological studies applied to artwork conservation could represent a compelling tool to improve the intervention durability and apply the principle of "minimum intervention" best. Long-term studies also provide a model for preventive actions based on BPs as bioindicators of warning conditions (e.g., the carbonatogenic trait characterizing our bacterial isolates).
Environmental monitoring and understanding the ecological successions are essential in preventing site re-colonization through appropriate conservation plans. Still, the knowledge of the involved species and their main traits is superficial.
Supplementary Materials: The following supporting information can be downloaded at: https:// www.mdpi.com/article/10.3390/microorganisms11071770/s1. Figure S1: Bacterial isolates frequency at (A) class, (B) order and (C) family level; Table S1: PCR amplification programs applied for the targeted regions. The last line reports the cycling repeats; Table S2: Environmental conditions recorded during the two sampling sessions.