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Article

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

1
Department of Sciences, University Roma Tre, Viale Marconi 446, 00146 Rome, Italy
2
Department of Economics, Engineering, Society and Business Organization (DEIM), University of Tuscia, Largo dell’Università snc, 01100 Viterbo, Italy
3
National Research Center (CNR), Institute of Heritage Science, SP35d, 9, 00010 Montelibretti, Italy
4
Soprintendenza Speciale di Roma Archeologia Belle Arti Paesaggio, Ministero della Cultura, Piazza dei Cinquecento, 67, 00185 Rome, Italy
5
National Biodiversity Future Center (NBFC), Università di Palermo, Piazza Marina 61, 90133 Palermo, Italy
*
Author to whom correspondence should be addressed.
These authors contributed equally to this work.
Microorganisms 2023, 11(7), 1770; https://doi.org/10.3390/microorganisms11071770
Submission received: 5 June 2023 / Revised: 2 July 2023 / Accepted: 3 July 2023 / Published: 6 July 2023

Abstract

:
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.

Graphical Abstract

1. 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 for sudden changes in underground microbial communities [2,7,8]. Allochthonous carbon energy sources can be tied to outdoor airborne particles, arthropods, roots, and materials used in wall paintings [2,9,10,11,12] or frequently to mass tourism [13,14,15]. Indeed, human presence can affect microclimatic conditions since it increases the indoor carbon dioxide, temperature, and humidity content [16,17,18]; otherwise, the visitors’ shoes and clothes are vehicles for organic and inorganic outer particles [1,5,19,20].
These conservation problems of hypogeal sites are widely known, and general suggestions for their maintenance is often reported. It has been observed that along the visitor trails the growth is higher than others [13,16,19,21]. The most common actions aimed to limit the access/flow of visitors and their permanence in the site, and, in extreme cases, provide the use of protecting clothing to avoid the input of outdoor contaminants and the changing of environmental condition [2,22,23,24]. Many attempts have been performed to maintain under control the lampenflora growth, using, for instance, the installation of monochromatic lights with limited wavelength emission [25,26] or tunable low-energy-consuming LED lamps with the emission spectra lying out of the absorption maxima of chlorophyll a [27,28,29,30]; moreover, this indirect control measure may fail in the long term [7].
The biodeterioration problems of Roman hypogea have been extensively studied for over 40 years. Different species of terrestrial epilithic cyanobacteria were detected as dominant phototrophic microorganisms [31,32,33] followed by bacteria and, in a lesser extent, by green algae and fungi [2,34,35,36]; mosses and roots of higher plants were also present [22,23,37,38,39]. In Roman hypogea, different biofilms whose composition changes according to microclimatic conditions and lamps are evident [33,35,40,41,41,42,43,44,45,46,47]. In fact, when light is available, a massive growth of cyanobacterial-dominated microbiota has been reported [25]. Otherwise, in the darkness, white biofilms of different consistency and origin were found [16,25,47,48]. In all cases, a high diversity of heterotrophic microflora was recorded, and several new bacterial species were described [49,50,51,52,53,54], confirming that the unexplored and/or extreme niches are reservoirs for unknown entities [5,55,56,57,58].
In cultural heritage conservation, the importance of long-term studies lies in measuring and documenting nature’s response to the changes imposed by treatments as previously done, for example, for the Matera’s paintings in a rocky habitat [59,60] and, meanwhile, allowing preventive actions when and if the first signs of a dangerous situation are recognized. In this light, the Roman Houses of the Caelian hill represent an interesting case since broad conservative interventions to reduce biodeterioration, and physical–chemical damages were carried out in the first years of such millennium before opening to visitors [22,23]. Hence, using the past data in this paper, we aim to: (a) analyze the current conservative conditions after about two decades from the first interventions; (b) compare the results of the previous monitoring work with the present state of conservation and biodeterioration of the site; and (c) assess the detrimental potential of the occurring species. This diachronic study will allow to establish who (referred to the microbial components), why, and what has changed. It will also allow for the identification of the potentially deteriogenic species and the improvement of the management strategies for the protection of the site.

2. Materials and Methods

2.1. Study Site, Previous Intervention, and Analyses

Roman houses, object of this study, are located between the Coliseum and the Circus Maximus in the heart of ancient Rome (41°53′11.04″ N 12°29′31.2″ E). In the first century B.C., high insula buildings flanked the Clivus Scauri, the western main route of the Caelian hill (Collis Caelius). Here, arcades, shops, and entrance doors of apartments on the upper floor made this street very busy and full of activities, which continued until the third century [61]. Many building transformations occurred to meet the needs of the population and owners during the time until the second half of the fourth century when it became a place of worship. The most 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.
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 thermal plant, BAL) in the deepest floor, never treated or restored and excluded from the visit path (Figure 1). The paintings decorating the CG and NP were prepared using the fresco technique with a whitish and blue dominant background colour respectively.

2.2. Present Investigations: Sampling, Environmental Measurements and Biodeteriogens Identification

2.2.1. Field Analysis: Microclimatic Measurements and Sampling

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.
During sampling, the temperature (°C) and relative humidity (RH%) measurements were made using portable thermo-hygrometer Extech MO290 (Extech Instruments, Nashua, NH, USA).

2.2.2. 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 DifcoTM 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/cm2 [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 micro-algae 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 MgSO4·7H2O (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.
Molecular identification of bacteria was performed using 16S as molecular marker and 27F/1492R as primer set (5′-AGAGTTTGATCMTGGCTCAG-3′ and 5′-GGYTACCTTGTTACGACTT-3′ respectively). Specific primer pair Cya106f (5′-CGGACGGGTGAGTAACGCGTGA-3′) and Cya781r (5′-GACTACWGGGGTATCTAATCCCWTT-3′; [64] for cyanobacteria were also used. The nuclear internal transcribed spacer (ITS) flanked by ITS4/ITS5 primers (5′-TCCTCCGCTTATTGATATGC-3′/5′-GGAAGTAAAAGTCGTAACAAGG-3′; [65]) was used for fungi and root sample identification. While primer sets targeted to chloroplasts such as ChloroF/ChloroR (5′-TGGCCTATCTTGTTGGTCTGT-3′ and 5′-GAATCAACCTGACAAGGCAAC-3′ respectively; [66]) and rbcLa/rbcLr590 (5′-ATGTCACCACAAACAGAGACTAAAGC-3′ and 5′-AGTCCACCGCGTAGACATTCAT-3′; [67]) were used for the molecular identification of green algae and roots respectively.
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].

2.3. 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/cm2 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.

2.4. 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 CaCO3 agar medium containing yeast extract 5 g, glucose 50 g, CaCO3 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.

3. Results

3.1. 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).

3.2. 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 light-green 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 3B) while Scytonema sp. was rarely found in fresh microscope preparation only. Wall B showed the prevalence of Chlorella vulgaris while molecular analysis evidenced the presence of Koliella sp. (93.04%, OQ540756) from this sampling site. All BAL sampling sites showed a varied presence of primary producers represented by Albertania sp. (A. skiophila 98.68%, OQ534285) and Coccomyxa sp. (97.21%, OQ540754) in Site P14, and Leptolyngbiaceae sp. (A. alaskaensis KL12 93.18%, OQ534284) in Site P15. The sample extracted from the vault (G) revealed the presence of Desmococcus vulgaris (Figure 3D). While Sample By evidenced the huge presence of young leafy gametophytes and mosses rhyzoids (Figure 3H), Stichococcus sp. along with Chlorococcum vulgaris were found in Site Bx.
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.

3.3. 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).
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).

3.4. 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.

4. 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 importance 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.

5. 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.

Author Contributions

Conceptualization, G.C., D.I.; methodology, D.I., F.B., formal analysis, D.I., F.B.; investigation, D.I., F.B.; writing—original draft preparation, D.I.; writing—review and editing, D.I., F.B., G.C., S.M.; supervision, G.C.; project administration, G.C., F.B.; funding acquisition, G.C. All authors have read and agreed to the published version of the manuscript.

Funding

This research was funded by a grant from the Italian Ministry Foreign Affairs and International Cooperation Program “Research of conservation environment and eco-friendly damage control of cultural heritage Korea and Italy”, grant number PGR01059.

Data Availability Statement

All data are available in this article and Supplementary Material.

Acknowledgments

The authors wish to thank the Soprintendenza Speciale di Roma Archeologia Belle Arti Paesaggio-Ministero della Cultura for supporting the planning of the Italian-Korean project such as for the useful conservative and historical information of the archaeological site and sampling. The authors also thank Maria Pia Nugari and the Istituto Centrale per il Restauro (ICR), Rome, for sharing their internal reports.

Conflicts of Interest

The authors declare no conflict of interest.

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Figure 1. Roman houses of the Caelian Hill. (A) Aerial view, Northern side, (B) Aerial view, Southern side; (C) Site map, in red, are the four rooms under study and after the pictures of them as they were during the early 2000s: (D) Chamber of Geniuses (CG), (E) Nymphaeum of Proserpine (NP), (F) The Winery, (W), and (G) The “Balneum” the white arrows indicate the A and B walls where sampling was performed (BAL). Within yellow are the sampling points used by Bartolini and colleagues [22,23].
Figure 1. Roman houses of the Caelian Hill. (A) Aerial view, Northern side, (B) Aerial view, Southern side; (C) Site map, in red, are the four rooms under study and after the pictures of them as they were during the early 2000s: (D) Chamber of Geniuses (CG), (E) Nymphaeum of Proserpine (NP), (F) The Winery, (W), and (G) The “Balneum” the white arrows indicate the A and B walls where sampling was performed (BAL). Within yellow are the sampling points used by Bartolini and colleagues [22,23].
Microorganisms 11 01770 g001
Figure 2. Deterioration patterns recorded and additional sampling points. (A,D) GC, niche—additional sampling point; (B,E) NP, lower wall–additional sample; (C) Light green opaque patina at the NP side chamber; (F) Lampenflora example along the visit path; (G,H) Winery deterioration pattern recorded on the A wall; the white arrow indicates a exfoliating flake from the wall (K,L) Winery-deterioration pattern recorded on the B wall characterized by a brown patina in the lower shaded part (K) and a green one close to the light (L); (I,J,M,N) Balneum: (I,J) Illuminated wall, By sampling site, and magnification of the yellow–green discoloration pattern; (M) Greenish colonized lower wall from where Bx sample was extracted, (L) Whitish patina on the non-irradiated lobby wall. Bar is equal to 10 cm.
Figure 2. Deterioration patterns recorded and additional sampling points. (A,D) GC, niche—additional sampling point; (B,E) NP, lower wall–additional sample; (C) Light green opaque patina at the NP side chamber; (F) Lampenflora example along the visit path; (G,H) Winery deterioration pattern recorded on the A wall; the white arrow indicates a exfoliating flake from the wall (K,L) Winery-deterioration pattern recorded on the B wall characterized by a brown patina in the lower shaded part (K) and a green one close to the light (L); (I,J,M,N) Balneum: (I,J) Illuminated wall, By sampling site, and magnification of the yellow–green discoloration pattern; (M) Greenish colonized lower wall from where Bx sample was extracted, (L) Whitish patina on the non-irradiated lobby wall. Bar is equal to 10 cm.
Microorganisms 11 01770 g002
Figure 3. Phototrophs optical microscope images. (A) NP, Scytonema sp. from sample Px; (B) W, wall A—Pseudostichococcus monallardoides; (C) W, wall B—Chlorella vulgaris; (D) BAL, Desmococcus vulgaris from site G; (E) Albertania sp.; (F) BAL, Phormidium laminosum from site B15; (G) BAL, Chlorococcum vulgaris from site Bx; (H) BAL, young moss gamethophytes, Site By. Scale bar 15 μm in (AG), 50 µm in (H).
Figure 3. Phototrophs optical microscope images. (A) NP, Scytonema sp. from sample Px; (B) W, wall A—Pseudostichococcus monallardoides; (C) W, wall B—Chlorella vulgaris; (D) BAL, Desmococcus vulgaris from site G; (E) Albertania sp.; (F) BAL, Phormidium laminosum from site B15; (G) BAL, Chlorococcum vulgaris from site Bx; (H) BAL, young moss gamethophytes, Site By. Scale bar 15 μm in (AG), 50 µm in (H).
Microorganisms 11 01770 g003
Figure 4. Bacterial isolates detrimental potential. (A) Acid production leading to carbonates dissolution; (BL) Carbonates precipitation differing in shape and colour. Magnifications from B4 plates of the strains: (B) Lysinibacillus sp. B35; (C) B106; (D) Amycolatopsis sp. B79; (E) Alcaligenaceae sp. B71; (F) Bacillaceae sp. B65; (G) Bacillaceae sp B66; (H) Stenotrophomonas cfr. maltophilia B105; (I) Microbacterium sp. B76b; (J) Lysinibacillus sp. B70; (K) Agromyces sp. B58; (L) Peribacillus sp. B61. Magnifications were 4× except (D), whicht was 2×.
Figure 4. Bacterial isolates detrimental potential. (A) Acid production leading to carbonates dissolution; (BL) Carbonates precipitation differing in shape and colour. Magnifications from B4 plates of the strains: (B) Lysinibacillus sp. B35; (C) B106; (D) Amycolatopsis sp. B79; (E) Alcaligenaceae sp. B71; (F) Bacillaceae sp. B65; (G) Bacillaceae sp B66; (H) Stenotrophomonas cfr. maltophilia B105; (I) Microbacterium sp. B76b; (J) Lysinibacillus sp. B70; (K) Agromyces sp. B58; (L) Peribacillus sp. B61. Magnifications were 4× except (D), whicht was 2×.
Microorganisms 11 01770 g004
Table 1. Identification and detrimental potential of the bacterial isolates.
Table 1. Identification and detrimental potential of the bacterial isolates.
Sampling PointIsolateClosest BLASTn Match% IdentityAccession NCaCO3B4
G1B59Streptomyces pluricolorescens NBCR12808 99.62OQ533438
B82Peribacillus simplex NBRC 15720 = DSM 1321
Peribacillus frigoritolerans DSM 8801
Peribacillus castriliensis N3
99.10OQ606394++
G2B103Phyllobacterium zundukense Tri-48
Phyllobacterium loti S658
Phyllobacterium trifolii PETP02
99.39OQ533475++++
G3B58Agromyces humi ANK07398.50OQ533437+++
B105[Pseudomonas] hibiscola ATCC 19867
Stenotrophomonas maltophilia ATCC 19867
99.47OQ533476+++
B107Staphylococcus capitis DSM 20329 97.53OQ533477+
B108Staphylococcus capitis subsp. capitis DSM 20326 Staphylococcus capitis JCM 242099.30OQ533478+
G4B53Cellulomonas cellasea DSM 2011899.62OQ533432++
G5B56Alcaligenes faecalis subsp. phenolicus DSM 16503
Alcaligenes javaensis JG3
99.59OQ533435+++
B57Phyllobacterium endophyticum PEPVI591.52OQ533436++
B61Peribacillus castrilensis N3
Peribacillus frigoritolerans DSM 8801
97.15OQ533440+++
B62Rhodococcus opacus DSM 4320/
Rhodococcus wratislavensis DSM 44107
98.65OQ533441+
B63Peribacillus frigotolerans DSMZ 8801100OQ533442++
B64Psychrobacillus lasiicapitis NEAU-3TGS1798.55OQ533443++
B65Psychrobacillus lasiicapitis NEAU-3TGS17 92.58OQ533444++
B66Peribacillus frigoritolerans DSM 8801
Peribacillus castrilensis N3
96.69OQ533445++
B76aMicrobacterium foliorum DSM 1296698.23OQ533454++
B76bMicrococcus luteus NCTC 2665
Micrococcus yunnanensis YIM 65004
95.11OQ606393++
GxB80Peribacillus frigoritolerans DSM 8801
Peribacillus castrilensis N3
98.42OQ533458+
P6B79Amycolatopsis jiguanensis CFHS0158098.96OQ533457++
B83Paenibacillus mobilis S8/
Paenibacillus tundrae A10b
98.81OQ533460+++
B112Agromyces terreus DS-1099.11OQ533479
P7B37Lysinibacillus fusiformis NBRC 1571799.04OQ512987++
B44Lysinibacillus fusiformis NBRC 1571799.00OQ533428+++
B89Promicromonospora xylanilytica YIM61515 99.27OQ533466++
B97Micrococcus endophyticus YIM 5623897.37 OQ533472+
P8B60Advenella kashmirensis WT001 98.43OQ533439+++/−
B67Phyllobacterium zundukense Tri-48
Phyllobacterium sophorae CCBAU 03422
Phyllobacterium loti S658
96.81OQ533446+
B71Advenella kashmirensis WT001 95.01OQ533449++++
B81Peribacillus simplex NBRC 15720 = DSM 1321
Peribacillus frigoritolerans DSM 8801
98.78OQ533459++
B85Phyllobacterium zundukense Tri-48
Phyllobacterium loti S658
Phyllobacterium trifolii PETP02
98.64OQ533462+++
B92Microlunatus parietis CCM 7636 99.27OQ533468
B102Microlunatus nigridraconis CPCC 203993/
Microlunatus parietis CCM 7636
99.31OQ533474+
P9B13Mesorhizobium atlanticum CNPSo 3140
Mesorhizobium comanense 3P27G6
Mesorhizobium plurifarium NBRC 102498
96.81OQ512977
B15Delftia acidovorans JCM 583399.45OQ512978+
P10B04Achromobacter pestifer LMG 3431
Achromobacter piechaudii CCUG 724
96.39OQ512006++
B06Achromobacter xylosoxidans FDAARGOS_78999.37OQ512007+++
B07Bacillus thuringiensis ATCC 10792 99.25OQ512008+
B08Brevundimonas diminuta b7198.52OQ512009++
B09Nocardia ninae DSM 44978 94.95OQ512010+++
B10Achromobacter insolitus NCTC13520
Achromobacter spanius DSM 23806
Achromobacter deleyi LMG 3458
99.40OQ512011+
B11Delftia acidovorans JCM 583399.09OQ512975++
B12 bStenotrophomonas geniculata ATCC 19374 = JCM 1332499.40OQ512976++
B41Bacillus altitudinis 41KF2b
Bacillus aerophilus 28K
Bacillus australimaris MCCC 1A05787
Bacillus aerius 24K
98.78OQ533427++
B54Peribacillus castrilensis N3
Peribacillus frigoritolerans DSM 8801
99.23OQ533433++
B55Phyllobacterium zundukense Tri-48
Phyllobacterium trifolii PETP02
99.45OQ533434+++
B70Lysinibacillus fusiformis NBRC 1571799.05OQ533448+++
B74Peribacillus frigoritolerans DSM 880198.13OQ533452++
B84Phyllobacterium zundukense TRi-48
Phyllobacterium loti S658
Phyllobacterium trifolii PETP02
Phyllobacterium bourgognense STM 201
99.13OQ533461++++
B86Phyllobacterium endophyticum PEPV15
Phyllobacterium zundukense Tri-48
Phyllobacterium loti S658
98.05OQ533463++++
PXB50Peribacillus frigoritolerans DSM 8801
Peribacillus castrilensis N3
99.17OQ533430+
B72Peribacillus frigoritolerans DSM 8801
Peribacillus castrilensis N4
99.43OQ533450++
B75Peribacillus simplex NBRC 15720 = DSM 1321
Peribacillus frigoritolerans DSM 8801
Peribacillus castrilensis N5
99.27OQ533453++
B77Peribacillus castrilensis N3/
Peribacillus frigoritolerans DSM 8801
99.63OQ533455+
B78Inquilinus ginsengisoli Gsoil 080 97.81OQ533456+++
P11B25Myroides odoratus FDAARGOS_113196.76OQ512979+
B26Providencia rettgeri FDAARGOS 145098.20OQ512980++
B27Providencia rettgeri FDAARGOS 145098.45OQ512982++/−
B28Providencia rettgeri FDAARGOS 145098.42OQ512983++
B30Myroides odoratus DSM 2801 NBRC 14945 98.51OQ512984+
B35Lysinibacillus fusiformis NBRC 1571798.63OQ512985++
B36Lysinibacillus fusiformis NBRC 1571799.87OQ512986++
B38Lysinibacillus fusiformis NBRC 1571799.91OQ607474++
B20, B40Peribacillus castrilensis N3
Peribacillus frigoritolerans DSM 8801
99.37OQ512988++
B87Inquilinus ginsengisoli Gsoil 08098.91OQ533464++
B88Promicromonospora kermanensis UTMC 53399.29OQ533465++
B90Inquilinus ginsegisoli GSOIL 080 98.98OQ533467+
B94Microbacterium shaanxiense CCNWSP60
Microbacterium arthrosphaerae CCM 7681
Microbacterium murale 01-Gi-001
99.33OQ533470++
B95Leucobacter aerolatus CCM 7705 97.37OQ533471+++
B96Serratia liquefaciens ATCC 2759298.61OQ606395
B125Achromobacter insolitus NCTC13520
Achromobacter spanius DSM 23806
99.48OQ533487+++
W-11/bB116Microlunatus parietis 12-Be-011 98.66OQ533482++
B123Paenibacillus lautus NBRC 1538096.69OQ533486+++
W-AB150Micromonospora cremea CR3098.93OQ533480++
B151Micromonospora coriariae DSM 44875
Micromonospora cremea CR30
98.81OQ533481++
W-BB121Nocardioides panzhihuensis KLBMP 105098.28OQ533485++
B153Leucobacter salsicius M1-8
Leucobacter exalbidus K-540B
99.14OQ533489++
BALNB48Peribacillus castrilensis N3
Peribacillus frigoritolerans DSM 8801
98.53OQ533429++
B52Peribacillus frigoritolerans DSM 8801
Peribacillus castrilensis N4
99.72OQ533431+++
B69Lysinibacillus cavernae SYSU K30005/
Lysinibacillus fusiformis NBRC 15717/
Lysinibacillus pakistanensis NCCP-54
97.18OQ533447++
B73Acinetobacter iwoffii FDAARGOS 139399.47OQ533451+
B93Prolinoborus fasciculus CIP 10357998.30OQ533469+/−
B98Advenella mimigardefordensis DPN7/
Advenella kashmirensis subsp. methylica PK1
97.99OQ533473++
B119Metabacillus sediminilitoris DSL-17 99.52OQ533483++
B120Nocardioides panzhihuaensis KLBMP105098.72OQ533484+/−+
B-14B128Leucobacter aerolatus CCM 7705/
Leucobacter salsicius M1-8
98.74OQ533488++
Negative result is indicated by −, +/− indicates a weak response, while +, ++, and +++ a positive increasing response.
Table 2. Fungal strains identification.
Table 2. Fungal strains identification.
Sampling PointIsolateClosest BLASTn Match% IdentityAccession N
G1F13Penicillium polonicum KMM471999.02OQ512946
G2F29Aspergillus pseudoglaucus CBS 126221
Aspergillus ruber CBS 126220
Aspergillus glaucus CBS 126.55
99.81
99.81
99.81
OQ512947
F06Penicillium oxalicum DUCC574497.82OQ512948
G3F19Cladosporium neolangeronii CPC 22267
Cladosporium psychrotolerans DTO:305-G3
Cladosporium langeronii CPC 22326
100
100
100
OQ512949
G4F23Penicillium chrysogenum NBPen2012A0299.33OQ512950
F14Chrysosporium undulatum CBS 964.9799.84OQ512951
F22Torula hollandica CBS 220.6999.18OQ512952
G5F28Pseudogymnoascus pannorum CBS 12691391.60OQ512953
GXF38 Cladosporium halotolerans CBS 127370
Cladosporium parahalotolerans CPC 22373
99.43
99.43
OQ512954
F39Penicillium chrysogenum SCSGAF007099.34OQ512955
F31[Coniosporium] MA 4640 97.18OQ512956
P6F08Penicillium brevicompactum CBS 287.53100 OQ512957
P7F32Penicillium dipodomyus CBS 110412
Penicillium flavigenum CBS 419.89
Penicillium lanosocoeruleum CBS 334.48
97.90
97.90
97.90
OQ512958
P8F17Cladosporium halotolerans CBS 11406599.84OQ512959
F04Cladosporium parahalotolerans CPC 22373
Cladosporium halotolerans CBS 127370
99.81
99.81
OQ512960
P9F01Malassezia restricta CBS 799198.42OQ512961
F02Penicillium brevicompactum CBS 287.53
Penicillium kongii AS3.15329
100
100
OQ512962
F09aCyphellophora olivacea CCFEE 991699.33OQ512963
P10F18Cladosporium endophyticum MFLUCC 17-059998.46OQ512964
P11F05Cladosporium herbarum CBS 128234
Cladosporium macrocarpum CBS 12778
Cladosporium variabile CBS 121635
Cladosporium ossifragi CBS 842.91
Cladosporium allicinum CBS 188.54
99.61
99.61
99.61
99.61
99.61
OQ512965
PxF11Penicillium rubens CBS 132206
Penicillium chrysogenum CBS 127368
99.82
99.82
OQ512966
W11F34Penicillium rubens CBS 132206
Penicillium chrysogenum NEF9
97.03
97.03
OQ512967
CV- AF36Penicillium chrysogenum CBS 127368 Penicillium rubens CBS 205.5799.09
99.09
OQ512968
CV- BF37Penicillium rubens CBS 132206
Penicillium oxalicum DUCC 5744
97.44
97.44
OQ512969
BAL14F40Cladosporium subuliforme CBS 126500
C. verrucocladosporioides CBS 126363
Cladosporium xylophilum CBS 125997
99.81
99.81
99.81
OQ512970
BAL15F41Penicillium goetzi CBS 285.73
Penicillium rubens CBS 129667
99.07
99.07
OQ512971
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/cm2: +/− 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.
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/cm2: +/− 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.
RoomSite20022003July 2019 Nov 2019
PhototFungiBacteria PhototFungiBacteriaPhotot.FungiBacteria Photot.FungiBacteria
CGG1////++++++
G2////++++++
G3/++/+++/++/++
G4/+++/++++/++/++++
G5+++++++++++++++++
NPP6//+++/++/++
P7++++++++
P8//+/++++/++
P9/+/++/++++/++
P10+++++++++++++/−++
W11/++/++/+/+
11/b/++/++/+/+
A////++++++++++
B////++++++++++
BAL14/+++++/++++++++++++++
15/+++++/++++++++++++++
G+++//+++//++++
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.
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.
RoomSampleDeter. Pattern2002 2003 Deter. Pattern2019
NPP10GBPNavicula sp.+
Px / / LGPScytonema sp. +++
WAGP GP(*) Pseudostichococcus cfr. Monallardoides
Scytonema sp.
+++
+/−
BYP GPChlorella vulgaris+++
BAL14 / / FGP(*) Albertania cfr. skiophila
(*) Coccomyxa sp.
++
+
15 / / FGP(*) Albertania sp.
Phormidium laminosum
++
+++
GDGPPlectonema gracillium
Desmococcus sp.
Navicula gallica
Moss protonemata
+++
++
+
++
Plectonema gracillium
Desmococcus sp.
Navicula gallica
Chlorococcum sp.
Stichococcus bacillaris
Moss protonemata
+++
++
+
+
+
++
Desmococcus vulgaris
Fern’ spores
+++
++
By / / GOPMoss gametophyte +++
Bx / / DGPChlorococcum vulgaris
Stichococcus sp.
+++
+
Quantitative data are based on microscope observations or by the number of extracts from which have been sequenced the species recorded. GBP, grey–brown patina; GP, green patina; YP, yellow patina; DGP, dark-green patina; LGP, light-green patina; FGP, fair-green patina; GOP, green-ochre patina; DGP, dark-green patina.
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Isola, D.; Bartoli, F.; Morretta, S.; Caneva, G. The Roman Houses of the Caelian Hill (Rome, Italy): Multitemporal Evaluation of Biodeterioration Patterns. Microorganisms 2023, 11, 1770. https://doi.org/10.3390/microorganisms11071770

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Isola D, Bartoli F, Morretta S, Caneva G. The Roman Houses of the Caelian Hill (Rome, Italy): Multitemporal Evaluation of Biodeterioration Patterns. Microorganisms. 2023; 11(7):1770. https://doi.org/10.3390/microorganisms11071770

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Isola, Daniela, Flavia Bartoli, Simona Morretta, and Giulia Caneva. 2023. "The Roman Houses of the Caelian Hill (Rome, Italy): Multitemporal Evaluation of Biodeterioration Patterns" Microorganisms 11, no. 7: 1770. https://doi.org/10.3390/microorganisms11071770

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