Biocide Treatments on Stone Materials from Pompeii: Microbial Selection, Efficacy and Emerging Risks

Abstract

At the archeological site of Pompeii, the deterioration of exposed structures is frequently associated with the combined action of microbial colonization and soluble salts, both recognized as major agents of decay affecting ancient surfaces. Although biocides are commonly applied during cleaning procedures to reduce microbial biomass, their incorporation into restoration-oriented formulations for the protection of porous stone substrates requires careful assessment of efficacy, microbiological risks, and sustainability. This study evaluated the performance of 2,4,5,6-tetrachloroisophthalonitrile (chlorothalonil) and iodopropynyl butylcarbamate (IPBC) as candidate active ingredients for conservation applications in activated new mortars. Yellow tuff, gray tuff, and brick samples collected from different sectors of Pompeii were investigated through culture-based analyses, ATP quantification, and metabolic profiling. Biocidal treatments were subsequently tested under laboratory conditions. The investigated substrates exhibited variable microbial counts and metabolic activity, generally reflecting different degrees of deterioration. Chlorothalonil showed negligible inhibitory effects, whereas IPBC reduced fungal growth in a dose-dependent manner. However, the highest IPBC concentration induced a red chromatic alteration associated with the selection of a bacterial strain preliminarily identified as Micrococcus roseus. Phenotype microarray analyses revealed broad chemical tolerance. Overall, biocidal treatments may alter microbial communities, favor tolerant microorganisms, and produce undesirable aesthetic effects. Finally, the study also assessed the environmental impact associated with laboratory and field activities, highlighting potential mitigation strategies to support more sustainable conservation research practices.

1. Introduction

The archeological site of Pompeii, recognized by UNESCO as a World Heritage Site, covers approximately 66 hectares, of which about 50 are currently excavated. It represents a remarkably well-preserved set of public and private buildings, monuments, sculptures, paintings, and mosaics. The ashes and lapilli that buried the city following the eruption of Vesuvius in AD 79 played a key role in its extraordinary preservation, offering a highly detailed picture of Roman urban organization and daily life [1,2,3,4]. The origins of the settlement are generally attributed to an Etruscan foundation, with early fortifications in local gray tuff, later replaced in the 5th century BC by a Sarno limestone wall [5,6].

The conservation of Pompeii, however, presents significant challenges due to the site’s vast extent, the variety of construction materials, exposure to atmospheric agents, and constant anthropogenic and tourist pressure. Since the mid-20th century, systematic conservation interventions and excavation campaigns have been carried out, with the aim of stabilizing and protecting exposed structures [7,8,9]. In this context, preventive conservation has assumed an increasingly central role, as it allows early identification of risk factors and reduces the need for invasive interventions, in accordance with current international guidelines [10,11,12].

Biodeterioration represents one of the main threats to cultural heritage, as microorganisms colonize surfaces and interact with substrates through chemical and physical processes that may compromise their stability. Their development is influenced by environmental factors such as humidity, temperature, light exposure, and climatic variations [13,14]. The intrinsic properties of materials, such as porosity, surface roughness, and mineralogical composition, also play a crucial role in either promoting or limiting microbial colonization [15].

A well-documented case of biodeterioration in Pompeii is the necropolis of Porta Nocera, where decorative surfaces that appeared intact at the time of excavation deteriorated drastically within a few decades [16]. Damage to plaster and wall paintings often results from the interaction between microbial activity and soluble salts, which are considered among the most destructive agents affecting ancient surfaces [17,18,19,20].

In recent years, advances in conservation science have introduced integrated three-dimensional survey techniques, such as orthophotos, digital elevation models, and multispectral imaging, to support preventive conservation in large archeological sites [21]. At the same time, more traditional conservation management approaches have also proven effective: in the case of the House of Ariadne in Pompeii, for example, the replacement of transparent coverings with opaque materials contributed to reducing thermal fluctuations and improving the microclimatic stability of wall paintings [22,23].

Biocides such as benzalkonium chloride (BAC) and isothiazolinone-based formulations are primarily used during cleaning procedures to reduce microbial biomass, whereas long-term prevention of recolonization relies on antimicrobial or protective coatings [24,25]. When applied as stand-alone treatments, biocides may leave exposed surfaces susceptible to rapid recolonization, particularly in outdoor environments, unless integrated within a broader conservation strategy. Therefore, conventional biocides may pose risks to human health and the environment, cause aesthetic or chromatic alterations to stone substrates, and promote the selection of resistant microorganisms or shifts in microbial community structure [26,27]. These concerns have stimulated the development of safer and more environmentally sustainable formulations [28,29]. Consequently, increasing attention has been directed toward multifunctional restoration materials capable of combining structural stabilization with long-term protection against biological recolonization. Among these, salt-free hydraulic lime mortars can act as carriers for hydrophobic agents and biocides, while microencapsulation strategies enhance the safe and controlled delivery of active compounds [30].

Chlorothalonil and Iodopropynyl butylcarbamate (IPBC) were selected as candidate biocidal agents because of their broad-spectrum antimicrobial activity and their documented use in protective formulations for highly porous and bioreceptive stone materials, such as tuff [30,31,32]. Both compounds show good stability and limited reactivity with mineral substrates, supporting their potential incorporation into restoration mortars [31]. Their use in protective systems may help prevent the establishment of pioneer microorganisms responsible for the onset of biodeterioration. Furthermore, encapsulation-based systems may reduce operator exposure and dermal absorption risks while ensuring a more gradual release of biocidal agents, as previously demonstrated for compounds such as diuron and terbutryn [30].

Nevertheless, stone materials may act as selective niches for microorganisms adapted to chemical treatments. From this perspective, the issue has a dual significance: on one hand, the vulnerability of the artifact, which is unique and irreplaceable; on the other, the possible selection of more tolerant microbial communities, which are more difficult to control and potentially capable of causing damage in treated surfaces [33,34].

In parallel with the evaluation of treatment effectiveness and microbiological risks, increasing attention is being devoted to the environmental sustainability of research activities themselves. Scientific studies traditionally focus on objectives, materials and methods, results, and their interpretation, considering existing literature, whereas the direct and indirect environmental impacts associated with conducting the research are rarely quantified or reported. In this context, the present study also aims to assess the greenhouse gas (GHG) emissions generated by both laboratory and field activities, with the objective of identifying potential mitigation strategies and promoting more sustainable research practices [35].

On this basis, the present study aims to: (i) evaluate the performance of two biocidal active ingredients embedded in restoration-oriented formulations, combining mechanical stabilization with protective action against biodeteriogenic microorganisms; (ii) analyze the microbiological risks associated with their use, and (iii) assess the sustainability of the proposed approach by quantifying greenhouse gas emissions associated with laboratory and field activities and identifying mitigation measures.

2. Materials and Methods

2.1. Experimental Design

The experimental design illustrated in Figure 1 was developed in collaboration with the Archaeological Superintendency of Pompeii.

The research activities were mainly structured into the following phases: (i) selection and collection of stone materials from different areas of the archeological site of Pompeii; (ii) execution of microbiological analyses aimed at the quantification and characterization of bacterial, fungal and algae communities present in the samples; (iii) evaluation of the inhibitory effectiveness of the biocides chlorothalonil (2,4,5,6-tetrachloroisophthalonitrile) and IPBC (iodopropynyl butylcarbamate); (iv) data processing and interpretation and preliminary assessment of the environmental impact of the entire study in terms of CO₂ equivalent emissions.

2.2. Sampling and Material Collection

Representative tough samples were collected from 20 areas of the archeological site of Pompeii by extracting stone fragments. The samples were sampled using sterile materials as described in previous work [36]. Quantities from 1.0 g to 50.0 g were sampled from each sampling site by scalpel. Before microbiological analysis, all samples were aseptically powdered in a sterile mortar. The samples exhibited different types of alterations attributable to physico-chemical causes (e.g., lacunae, alveolization, and cracking) and biological causes (e.g., patinas and chromatic alterations).

For the subsequent microbiological analyses, samples were collected in sterile containers and stored at 4 °C to limit secondary contamination and changes in the original microbiota.

For each sampling point, descriptive records and photographic documentation of the conservation state of the surfaces were also performed, as shown in Figure 2.

2.3. Microbial Counts and Culture Media

Samples of powdered samples from Pompeii sites were diluted in physiological solution (9.0 g L⁻¹) NaCl by the standard dilution technique, inoculated in the cultural media under appropriate conditions according to Zanardini et al., 1997 [37]. All determinations were performed in triplicate.

The total aerobic viable microbial load was determined by inoculating Petri dishes containing Plate Count Agar (PCA) (Oxoid, UK) and incubating them at 37 °C for 72 h.

For the quantification of the fungal component and subsequent isolation procedures, PDA (Potato Dextrose Agar—BD Difco™, Milan, Italy) and WL (BD Difco™, Milan, Italy) agar media were used. Plates were incubated at 28 °C for 48 h. Bacterial and fungal counts were expressed as colony-forming units per gram of sample (CFU × g⁻¹).

The presence or absence of algae was evaluated by light microscopy (Leica DMI 3000BI, Milan, Italy) on fresh samples.

2.4. Isolation and Characterization of Microbial Community

The diagnosis of alterations observed in the samples was carried out using traditional microbiological methods, including culture-based, morphological, and biochemical approaches to investigate the presence of microorganisms potentially associated with the observed alterations.

2.4.1. Phenotypic Characterization

Fungal and bacterial isolates were grown on PDA (BD Difco™, Milan, Italy) and PCA (BD Difco™, Milan, Italy), respectively. Developed colonies were purified by streaking on the same growth medium and subsequently characterized based on macroscopic and microscopic colony morphology (size, margin, color, elevation, and consistency). Microscopic observations were performed using an optical microscope (Leica DMI 3000BI, Milan, Italy). The red-pigmented bacterial isolate recovered from IPBC-treated stone specimens was further characterized by colony morphology, microscopic examination, Gram staining, KOH test, catalase activity, and physiological and biochemical assays according to previously described methodologies [38,39].

2.4.2. Analysis of Metabolic Activity and Microbial Viability

Microbial isolates were cultured in LB liquid medium (Luria–Bertani). Aliquots of 100 µL of cultures at the exponential phase, with OD₆₀₀ values between 0.7 and 0.8, were subjected to biochemical-enzymatic assays (ATP assay) and bioelectrical analyses (impedance variation measurements, expressed as Detection Time, DT, in hours), following the protocols described by Ranalli et al. [40] and Liberatore et al. [41], respectively.

Multiple enzymatic activities of microbial cultures were also determined using the API ZYM system (BioMérieux Italia, Bagno a Ripoli, Italy). The semi-quantitative evaluation of 19 hydrolytic enzymatic activities was carried out according to Viti et al. [42] and Ranalli et al. [43]. In brief, using a sterile Pasteur pipette, each gallery was inoculated with two drops of 10⁻¹ or 10⁻² suspensions of 20.0 g of soil in 180 mL of sterile saline solution (0.9% NaCl, w/v). After incubation at 28 °C for 4 h, the galleries were activated by adding one drop of Reagent ZYM A and Reagent ZYM B (bio-Merieux Italia, Bagno a Ripoli, Italy) and after 5 min at room temperature, the results were recorded referring to a colorimetric standard table. All determinations were performed in triplicate. Experimental reproducibility exceeded 95%.

2.5. Phenotype Microarray (PM) Technology

The metabolic capacity and chemical tolerance of the isolated bacterial strain were investigated using Biolog Phenotype Microarray (PM) system (Biolog, Hayward, CA, USA). According to a previous study [44], this technology was applied to further explore the metabolic capabilities of the isolated strain. The strain was tested on three PM panels (PM12, PM16, and PM17), specifically designed for chemical sensitivity assays. In these panels, each toxic compound (heavy metal, antibiotic, biocide, antimetabolitie, etc.) is present in four adjacent wells at four different doses (from the lowest to the highest).

The strain was grown on BUG agar (Biolog) and incubated at 30 °C for 24–48 h. Colonies were then collected with a sterile swab and suspended in 15 mL of 0.8% NaCl solution. Cell density was adjusted to 79% transmittance using a Biolog turbidimeter.

Subsequently, the inoculum for PM12, PM16, and PM17 was prepared by diluting the suspension 15-fold in IF-10b. Dye Mix A (1×) was added, and the mixture was dispensed into PM plates (100 µL per well). Incubation was carried out at 30 °C using an Omnilog reader (Biolog), with automatic readings every 15 min to detect color changes associated with tetrazolium reduction.

Measurements were recorded for 96 h and analyzed using Omnilog-PM software (version 2.3.01), which generated kinetic color development curves. Two independent experiments were conducted for each panel. For the analysis of kinetic curves, the IC₅₀ parameter was used. In this context, IC₅₀ is expressed in well units and is defined as the well or fraction of a well at which the area of the kinetic curve is half of its maximal value over a concentration series. IC₅₀ ranges from 0.6 (metabolic activity is totally inhibited at all four concentrations tested) to 4.4 (metabolic activity is not affected at all four concentrations tested) [44].

2.6. Microbial Inocula on Stone Materials

Different fungi from the previously isolated ones were used as inocula. Additionally, a reference microbial inoculum previously used in other studies [32], consisting of Alternaria infectoria strain NIS4, was included. Experiments were conducted in controlled microenvironments at 28 °C using the substrates described above. Control microorganisms included fungal spore suspensions of Penicillium citrinum, Penicillium funiculosum, Aspergillus flavus, Aspergillus niger, and Cladosporium herbarum.

2.7. Use of Biocides on Stone Materials

Two biocides were applied to multiple specimens (in triplicate; dimensions 30 mm × 30 mm × 25 mm) of yellow tuff, gray tuff, and brick. Specimens were pretreated in an oven at 50 °C for 12 h. The biocides used in this study in their pure form were: (i) chlorothalonil (Sipcam, Lodi, Italy), an organochlorine fungicide (Figure 3a) [45]; and (ii) IPBC (Sipcam, Lodi, Italy), a common anti-mold agent (Figure 3b) [46].

Experiments were conducted on the three stone substrates using increasing concentrations of both biocides, with untreated materials serving as controls. A final volume of 1.0 mL was applied per specimen, resulting in the following surface doses:

• chlorothalonil: 7.5 g × m⁻², 20.0 g × m⁻² and 40.0 g × m⁻²;
• IPBC: 1.0 g × m⁻², 2.5 g × m⁻² and 5.0 g × m⁻².

Inhibitory effectiveness was evaluated on inoculated specimens using a grid with 3 mm × 3 mm calibrated units applied to the surface.

2.8. Environmental Impact

For a preliminary sustainability assessment, the main inputs associated with laboratory activities were considered, including energy consumption, reagents, and consumables.

We proposed a survey sheet (

Table 1. Main indicators suggested calculating an environmental footprint originated by a research activity, impacts and green mitigation acts.

Type Consumptions	Evaluation of CO2 Eq. Emission
	Parameter	Unit	Amount	K Conversion to CO2	CO2 Yield (kg)

) useful for the inventory of each identified methodology, the different parameters and translated into CO₂ eq. emissions, useful for estimating the social costs and the mitigation of impacts in terms of tree planting, as adopted in previous studies [35,47].

3. Results and Discussion

3.1. Preliminary Microbiological Characterization of Stone Materials

Samples of stone materials (yellow tuff, gray tuff, and brick) were collected under sterile conditions and subjected to microbiological analysis as described in the Materials and Methods section. The materials investigated were sampled from different sectors of the archeological site of Pompeii, as reported in

Table 2. Assessment of decay phenomena and conservation condition of Pompeian masonry structures.

Samples	Location	Surface Powdering/Disintegration	Mortar Joint Erosion	Lacunae	Structural Stability	Conservation Condition
3	Northern Sector	Severe	Severe	Diffuse	Fair	Poor
4	Eastern Sector	Severe	Severe	Diffuse	Poor	Poor
7	Northern Sector	Moderate	Severe	Localized	Fair	Fair
9	Western Sector	Severe	Severe	Diffuse	Poor	Very poor
10	Domus A	Moderate	Moderate	Localized	Good	Fair
12	Thermae	Moderate	Severe	Localized	Fair	Fair
13	Insula Occidentalis	Moderate	Moderate	Localized	Fair	Fair
16-16bis	Domus C	Severe	Severe	Extensive	Poor	Very poor
20	Thermae	Severe	Severe	Extensive	Poor	Very poor
1a	Insula Occidentalis	Severe	Severe	Diffuse	Poor	Poor
2a	Amphitheater	Severe	Moderate	Diffuse	Fair	Poor

. The selected masonry structures exhibited variable lithological compositions but shared common signs of advanced deterioration and weathering.

Visual assessment of the sampled structures revealed widespread degradation phenomena affecting both stone materials and mortar joints (Table 2). In particular, the investigated masonry surfaces showed: (i) diffuse surface powdering and disintegration of stone substrates and mortars, resulting in loss of cohesion and pronounced surface irregularity; (ii) severe erosion of mortar joints accompanied by partial washout of the binding matrix; (iii) lacunae and interstitial voids between masonry elements; (iv) chromatic alterations associated with weathering processes; (v) cracking and fissuring related to structural settlement; and (vi) irregular and degraded upper margins, indicating a progressive loss of the original wall-top configuration. Although the masonry texture remains generally recognizable, the observed decay patterns highlight an advanced deterioration of binding materials and significant erosive processes, suggesting the need for continuous conservation monitoring and targeted consolidation interventions.

To obtain a preliminary evaluation of the biological colonization affecting these substrates, bacterial and fungal loads, algal presence, adenosine triphosphate (ATP) content, and impedometric measurements were determined (

Table 3. Microbiological data: samples collected from the archeological site of Pompeii.

Samples	Bacteria (log CFU × g−1)	Fungi (log MFU × g−1)	Algae	ATP (pg × g−1)	DT (h)
3	5.0	4.0	-	High	3
4	5.1	4.0	-	High	3
7	1.9	2.2	+	Low	6
9	2.0	4.9	+	Very high	2
10	1.5	2.0	-	Low	6
12	1.7	2.3	-	Low	5
13	1.5	2.0	+	Low	6
16-16bis	6.3	4.9	+	Very high	2
20	6.2	2.7	+	Very high	2
1a	4.0	4.1	+	High	3
2a	4.0	4.1	+	High	3

Legend: CFU = Colony Forming Unit × g⁻¹ of sample; MFU = Mycelium Forming Unit × g⁻¹ of sample. Algae: + = at least in 2 optical fields of 5; - = not detected on slide. ATP: Very high >1000 pg × g⁻¹; High 100–1000 pg × g⁻¹; Low <100 pg × g⁻¹. impedometric measurements DT (h): DT < 3 ore—high microbial activity, high presence of biodeteriogens; DT > 4 ore—low microbial activity, low presence of biodeteriogens.

). These parameters provide complementary information on both the abundance and metabolic activity of microbial communities potentially involved in biodeterioration processes.

The microbiological characterization of stones highlights a marked variability among the collected samples, both in terms of total microbial count and metabolic activity (Table 3).

Bacterial counts ranged from 1.5 to 6.3 log CFU g⁻¹, whereas fungal abundance varied between 2.0 and 4.9 log MFU g⁻¹. The highest bacterial concentrations were detected in samples 16-16bis and 20 (6.3 and 6.2 log CFU g⁻¹, respectively), while samples 10, 12, and 13 exhibited the lowest bacterial loads. Fungal colonization was particularly pronounced in samples 9 and 16-16bis, both reaching 4.9 log MFU g⁻¹, suggesting favorable conditions for fungal establishment on these substrates.

Algal cells were detected in seven out of the eleven samples analyzed, including both moderately and severely deteriorated substrates. Their occurrence suggests the presence of microenvironmental conditions favorable to phototrophic colonization, including enhanced moisture retention, increased surface porosity, and prolonged exposure to light. As primary colonizers, algae may contribute to the establishment of more complex biofilms, thereby facilitating the subsequent development of heterotrophic microbial communities [48].

When microbiological data were compared with the conservation assessment of the sampled masonry structures (Table 2 and Table 3), a general relationship emerged between the severity of material decay and the level of biological colonization. Samples collected from structures classified as having poor or very poor conservation conditions, such as 9, 16-16bis, and 20, exhibited the highest ATP levels and the lowest impedometric detection times (DT = 2 h), indicating intense microbial metabolic activity. These samples were also characterized by severe surface disintegration, extensive mortar joint erosion, and widespread lacunae. Conversely, samples obtained from structures in fair conservation conditions (e.g., 7, 10, 12, and 13) generally showed lower microbial loads, low ATP content, and higher DT values, consistent with reduced biodeteriogenic activity.

Notably, ATP content and impedometric measurements appeared to provide a more reliable indication of the metabolic state of the colonizing communities and their biodeteriorative potential than microbial counts alone [49]. For instance, sample 9 showed relatively low bacterial abundance (2.0 log CFU g⁻¹) but very high ATP levels and the shortest DT value, suggesting the presence of a highly active microbial community. This apparent discrepancy may be explained by the substantial fungal colonization detected in the same sample (4.9 log MFU g⁻¹), indicating that fungal biomass likely contributed significantly to the overall metabolic activity. Similar observations have been reported in biodeteriorated stone substrates, where fungal communities often play a major role in colonization processes and deterioration mechanisms [50]. Therefore, ATP and impedometric analyses represent valuable complementary tools for assessing the actual biological impact on stone materials and for identifying substrates at greater risk of biodeteriorative damage.

Although the limited number of samples precludes robust statistical analyses, the observed trend suggests that advanced physical deterioration may promote microbial colonization by increasing surface roughness, porosity, and water retention capacity. The presence of cracks, eroded mortar joints, and interstitial voids likely creates favorable microenvironments for the establishment and persistence of biodeteriogenic communities, thereby contributing to the progression of stone decay [48,50].

The results of qualitative assessments of environmental contamination, obtained through microbial growth tests in PVC containers performed in triplicate, are shown in Figure 4.

Observation of the stone materials revealed abundant and heterogeneous microbial growth, characterized by a highly diverse microbial community. In the brick sample (Figure 4a), fungal growth with cotton-like morphology, white or greenish-gray in color, attributable to fast-growing filamentous molds, was observed, along with rough or cerebriform structures referring to different microbial populations. In the yellow tuff sample (Figure 4b), widespread growth was observed, including filamentous white fungal development, mucous bacterial growth, and pigmented green and yellowish formations, likely of fungal origin.

The morphological diversity of the observed growth represents a qualitative indicator of the complexity of the microbial communities involved in biodeterioration processes affecting the examined archeological materials [51,52,53].

3.2. Biocide Tests

In general, microbial growth on stone materials in open environments is controlled through the application of biocidal substances [54].

The results obtained using IPBC at concentrations of 1.0, 2.5, and 5.0 g × m⁻², using A. infectoria NIS4 [38] as the control microorganism, are shown in Figure 5. After 96 h of incubation at 28 °C, the three untreated control replicates (a) showed complete fungal growth (100% surface coverage). In tests with increasing doses of IPBC (b, c, d), a dose-dependent inhibition of fungal growth was observed on the yellow tuff surface, with inhibition levels ranging approximately from 10 to 30%, 30–50%, and 50–100%, respectively. Simultaneously, the appearance of additional microbial populations on the treated surfaces was observed.

Under the adopted experimental conditions, the fungal inoculum of A. infectoria showed an evident growth inhibition starting from the third day. However, increasing doses of IPBC induced the formation of a clear red chromatic alteration (Figure 5).

Chlorothalonil resistance experiments showed negligible (<10%) inhibitory effectiveness against the control microorganism.

Similar results were obtained with the other control microorganisms (P. citrinum, P. funiculosum, A. flavus, A. niger, and C. herbarum), without any evidence of red chromatic alterations.

The results obtained on yellow tuff samples indicate that IPBC, while exerting inhibitory effects on fungal growth, may be associated with significant side effects, including microbial selection and the appearance of secondary chromatic alterations, as reported in the literature [26,55,56,57,58].

Although reduced dosages and controlled application techniques may help to limit such effects, the treatment efficacy may be temporary, and the possible migration of compounds into the environment highlights the need for prudent use and periodic monitoring of treated surfaces [55].

Our data are consistent in showing limited effectiveness or the generation of undesirable outcomes [59]. Studies conducted on archeological stone materials from Pompeii suggested a difference in effectiveness between IPBC and chlorothalonil, likely related to factors such as solubility, adsorption onto porous matrices, and diffusion within pores. In this context, chlorothalonil, despite its intrinsic chemical activity, may exhibit a low bioavailability due to its limited water solubility and a strong adsorption to substrate minerals, whereas IPBC, characterized by higher solubility and mobility, appears more effective in exerting direct inhibitory action [26,60,61].

3.3. Preliminary Studies on Red Chromatic Alteration

The red chromatic alteration observed in Figure 5 at the highest IPBC concentration (5.0 g × m⁻²) was further investigated. To determine whether this alteration was biological in nature, samples were collected using sterile loops and inoculated onto general media (PDA for fungi and PCA for bacteria). After incubation at 37 °C for 72 h, the PCA plates showed red-pigmented bacterial colonies (Figure 6). The strain was subsequently characterized through morphological, biochemical, and physiological tests as shown in

Table 4. Morphological and biochemical characteristics of the pigmented bacterial strain.

Colonies Characteristics	Data
Colony in PCA	Spherical
Colony color	Red
Temperature range (°C)	5–37
Temperature optimum (°C)	25–35
Gram reaction	+
Morphology cells	Tetrads
Motility	-
NaCl range (% w/v)	0–5
KOH test	-
NaCl optimum (% w/v)	0–2.0
Catalase test	+
Oxidase test	+
Coagulase test	-
Glucose fermentation	+
Aerobic	+
API 20NE activity of Aesculin	+
API 50CH acid production on
Glycerol	+
n-Xylose	+
n-Glucose	+
Inositol	+

Legend. Reactions: - negative; + positive.

and

Table 5. Physiological and biochemical characteristics of the pigmented bacterial strain.

API ZYM Substrate	Activity
alkaline phosphatase	++++
esterase (C4)	+
esterase–lipase (C8)	+
lipase (C14)	-
leucine arylamidase	+++
valine arylamidase	++
cistine arylamidase	+++
trypsin	-
α-chymotripsin	-
acid phosphatase	++
phosphoamidase	++++
α -galactosidase	-
β-galactosidase	-
β -glucuronidase	-
α -glucosidase	+
β -glucosidase	+
N-acetil- β -glucosamidase	+++
α -mannosidase	-
α -fucosidase	-

Legend.: Nanomoles of products of enzymatic reactions during incubation time of API ZYM strip test: - < 5 mmol; + 10 mmol; ++ 10–20 mmol; +++ 20–30 mmol; ++++ >40 mmol of substrate hydrolyzed.

.

Based on the results obtained, the microbial origin of the chromatic alteration observed on the yellow tuff sample was strongly supported. A red-pigmented bacterial isolate noted on the tuff sample tested in the laboratory was assigned to the genus Micrococcus and preliminarily identified as Micrococcus roseus, designated as strain 2356 [39]. The occurrence of this pigmented isolate in correspondence with the red spot suggests its possible involvement in the observed chromatic alteration. This interpretation is consistent with literature reports describing M. roseus pigments as intracellular carotenoids linked to growth phase and metabolism [62,63].

However, since neither molecular identification nor chemical characterization of the pigment was performed, the causal role of M. roseus in the discoloration process cannot be conclusively demonstrated and should be regarded as a preliminary hypothesis requiring further investigation. A possible explanation for the inhibition of fungal growth and the subsequent appearance of red pigmented bacterial growth includes: (i) the composition and dynamics of the native microbial community; (ii) the chemical nature and mechanism of action of the biocide; (iii) the metabolic activity of microorganisms selected under high biocide concentrations.

Biocidal treatments are known to alter microbial community structure, promoting the selection of more tolerant taxa. Exposure to biocides may also induce adaptive responses, including pigment production and increased chemical tolerance [57,58,64].

Pigmentation became clearly visible after 72–96 h of incubation at 28 °C. Although the pigment produced by M. roseus 2356 has not yet been chemically characterized, macroscopic observations suggest it is an intracellular pigment associated with bacterial growth and biomass accumulation. Future studies based on molecular identification of the isolate and chemical characterization of the pigment will be necessary to confirm the direct involvement of M. roseus 2356 in the formation of the red chromatic alteration.

3.4. M. roseus Phenotype Microarray

To further investigate the metabolic capabilities of M. roseus. Phenotype Microarray (PM) technology was employed. The isolate was tested on PM12, PM16, and PM17 panels, designed for sensitivity assays toward antibiotics, bacteriostatic agents, and enzymatic inhibitors. The strain was incubated in the Omnilog system, and colorimetric data were collected to generate kinetic curves (Figure 7) and calculate the IC₅₀ parameter (Figure 8).

Based on IC₅₀ values, chemical sensitivity data were summarized in

Table 6. Sensitivity of M. roseus to compounds in PM12, PM16, and PM17.

Chemical Sensitivity	Samples	%
High resistance (IC50: 3.0–4.4)	53	73.61
Intermediate resistance (IC50: 2.1–2.99)	6	8.33
Low resistance (IC50: 1.0–2.09)	5	6.94
No resistance (IC50: 0.6–0.9)	8	11.11
Total compounds	72	100.00

.

The analyses revealed a strong prevalence of high tolerance: 73.61% of compounds showed high resistance (IC₅₀: 3.0–4.4); 8.33% intermediate resistance; 6.94% low resistance; 11.11% no resistance.

These results indicate a remarkable ability of M. roseus to tolerate a broad spectrum of chemical compounds. Under the experimental conditions, IPBC has inhibited fungal growth but promoted the appearance of a chemically tolerant bacterial population. This suggests that its application on large archeological surfaces should be carefully evaluated due to potential selective effects on resident microbial communities. Our findings highlight that the use of biocides use can cause a strong selective pressure, favoring microorganisms with multi-compound tolerance, with implications for conservation practices [65,66,67].

The evidence obtained suggests that biocide applications are not microbiologically neutral but can reshape microbial communities. Repeated exposure may lead to co-selection and cross-resistance phenomena [68,69,70].

Broad-spectrum biocides such as IPBC may reduce microbial biodiversity and favor opportunistic microorganisms, potentially affecting material durability and microbial balance [71]. Furthermore, the presence of tolerant strains in accessible environments may contribute to antimicrobial resistance spread [72]. Thus, critical evaluation and sustainable strategies are required in conservation protocols [71].

The data from the phenotype microarray analyses confirm a marked prevalence of high-tolerance phenotypes, indicating that M. roseus 2356 strain can survive in the presence of a broad spectrum of chemical compounds. This pattern is consistent with the hypothesis of a selective pressure exerted by the biocide, which may favor microorganisms endowed with nonspecific tolerance mechanisms [73,74]. The wide tolerance spectrum observed also suggests possible cross-resistance or co-selection phenomena, previously reported following exposure to biocides and other antimicrobial agents [70]. Such phenomena may include the activation of efflux pumps, changes in membrane permeability, and phenotypic adaptations capable of conferring multiple tolerances to chemically diverse compounds [62,65]. From an applied perspective, these features represent a critical issue, as they may reduce the long-term effectiveness of treatments and promote the persistence of strains that are difficult to eradicate and capable of producing chromatic alterations [70,74]. At the same time, the metabolic capabilities of M. roseus 2356 strain could be explored for bioremediation applications, although further genetic and metabolic studies are required.

3.5. Environmental Impact of the Study

In applied research, sustainable restoration and conservation practices are gaining increasing attention, driven by sustainable research practices, enhancing operator safety through reduced use of hazardous substances, and ensuring greater respect for cultural heritage. In addition to microbiological effects, use of biocides, water, reagents, electricity and fuel consumption results in environmental impacts measurable as CO₂ equivalent emissions. In this study, a preliminary assessment estimated total emissions of approximately 1200 kg CO₂ eq., corresponding to a social cost of € 148.00 [35,47].

As a mitigation strategy, it was assumed that 100 kg CO₂ eq. could be offset over 10 years through tree planting [35]. Based on this, the planting of Quercus robur and Quercus cerris, mycorrhized with Tuber aestivum, was proposed. These species were selected for their adaptability and ecological compatibility. Planting was carried out at the Green Park—Unimol Campus.

4. Conclusions

At the archeological site of Pompeii, material decay and biodeteriogenic processes were investigated. The altered substrates hosted heterogeneous microbial communities, which were characterized through culture-dependent analyses, ATP quantification, and metabolic profiling.

The experimental results led to the following main findings:

(i)

IPBC exhibited a clear dose-dependent antifungal activity, whereas chlorothalonil showed negligible inhibitory effects under the tested conditions, likely due to reduced bioavailability within porous stone matrices;

(ii)

IPBC treatment promoted the emergence of a red-pigmented bacterial population, preliminarily assigned to the species M. roseus, which was associated with chromatic alteration of the substrate. Phenotype Microarray analyses revealed a broad tolerance of this isolate to numerous chemical compounds, suggesting potential co-selection and cross-resistance mechanisms. Although IPBC effectively reduced fungal growth, it also favored the emergence of a highly tolerant bacterial population and induced undesirable chromatic changes;

(iii)

The estimated environmental impact of the experimental activities (approximately 1200 kg CO₂ eq.) underscores the importance of integrating sustainability criteria into conservation research and practice.

Overall, these findings reveal important limitations of biocide-based treatments for porous archeological stone materials. While IPBC demonstrated antifungal efficacy, it also promoted secondary effects that may compromise long-term conservation outcomes. In contrast, chlorothalonil exhibited negligible inhibitory activity under the experimental conditions adopted in this study.

Future conservation strategies should move beyond a predominantly biocidal approach and instead emphasize integrated preventive conservation. Priority should be given to reducing substrate bioreceptivity through moisture control, improved surface stability, and the use of compatible natural restoration materials, such as plant essential oils. In this context, multifunctional mortars, controlled-release systems, and protective coatings may represent promising alternatives for limiting microbial recolonization while minimizing environmental impacts and reducing the risk of selecting resistant microbial communities.