Biodeteriogenic Potential of Bacteria and Fungi Isolated from Deteriorated Areas of Masjed-e Jāmé of Isfahan, UNESCO Cultural Heritage

Abstract The Masjed-e Jāme’ of Isfahan is one of the largest historic mosques in the Islamic world. It was listed as a World Heritage Site by UNESCO. Due to the arid climate of the region, the building is quite well preserved and presents only localized patterns of alteration. However, due to the risk of biodeteriogenic microorganisms which could be present on these valuable surfaces, this study aimed to isolate the microorganisms associated with the specific deteriorated areas of this monument and determine their deteriorative mechanism. Samples were taken from the deteriorated areas in order to isolate bacteria and fungi. These were tested for their potential to induce biodeterioration via CaCO3 dissolution, pH alteration, and pigment and mineral production. Results revealed that ubiquitous species such as Penicillium spp. and Bacillus spp. were the most abundant microorganisms isolated from the samples, some of which were able to release organic acids and induce CaCO3 dissolution. Very often, the isolated fungi showed a combined biodeteriogenic activity due to solubilization and precipitation of CaCO3. Rarely was CaCO3 solubilization combined with pigment release. These findings are a first step toward providing helpful information to assess the biodeteriogenic potential of colonizing microorganisms and planning a preventive method for the conservation of this monument which has never been studied in terms of biodeterioration risk.


Introduction
The Masjed-e J ame' of Isfahan is one of the oldest mosques in Iran and has undergone various architectural additions and renovations. It is also known as Masjed-e J ame' Atiq and Masjed-e J ame' Adineh or Friday mosque (Iranian government and ICHHTO 2011). The mosque is a unique cultural heritage in the city of Isfahan and has been continually developed from circa 771 until the end of the twentieth century (Seyedashrafi et al. 2017). It is a masterpiece of Islamic architecture in the world (Abdollahnejad et al. 2014;Assari and Mahesh 2011). This historic monument is situated in the historical city center of Isfahan and was listed by UNESCO in 2012 as a World Heritage Site. The foundations of the mosque belong to the eigth century (Bonner 2016). The mosque includes over a millennial of architectural experiences in the special style of Iranian mosques. Furthermore, many historical and architectural styles found in Iran and its neighboring countries can be identified in this building (Iranian government and ICHHTO 2011). This mosque has been a proper model for the design of other mosques in central Asia (WHC 1992(WHC -2015. Its special architectural forms, unique building materials, and antiquity are rather remarkable (Abdollahnejad et al. 2014).
Biodeterioration is a combination of physical, chemical and aesthetic damages in cultural heritage (Cappitelli et al. 2020). Fungi and bacteria have main role in the microbial deterioration of monument of cultural heritage due to secreting a huge amount of enzymes and acids, pigment production and biofilm formation (Corbu et al. 2021). The biodeterioration investigation of cultural heritage is a hot topic that is widely considered by the researchers, and protection of monument and material is one of the aims of such studies (De Leo et al. 2022). In some studies have been assessed the biodeteriogenic potential of microorganisms in various materials such as glazed tile, stone and mural painting (Coutinho et al. 2013;Ma et al. 2020;Rojas et al. 2012;Savkovi c et al. Ruibal).
It is well known that microorganisms can be present in most inorganic and organic built surfaces and their spread and biodeteriogenic effects are closely related to the climate (high humidity, rain, pollutants) or to the stress events that modify the normal equilibrium existing in surfaces and the growing microorganisms (Caneva et al. 1995; Stanaszek-Tomal 2020). Thus, to counteract these unwanted occurrences, it is necessary to monitor the presence of microorganisms that are associated with specific deterioration patterns. The objective of a preliminary screening was to isolate and identify microorganisms and demonstrate their bioderiorative potential in the decay process in order to select a more appropriate method to protect these monuments.
Due to the particular relevance of this monument, and being aware that the presence of microorganisms although confined to small areas could potentially cause an outbreak in favorable climatic conditions, this study mainly aimed to isolate the microorganisms colonizing some of the altered surfaces of Masjed-e J ame' of Isfahan and demonstrate their biodeteriorative potential using different methods. This is the first research aimed at studying some of the biodeteriorated surfaces of the Masjed-e J ame' of Isfahan.

Site description and sampling method
The mosque is located in the city of Isfahan (Figure 1(A)). The climate of Isfahan is moderate and dry with temperatures varying from 6 C to 10 C in the winter and up to 40 C in the summer. It presents an annual average precipitation of 121.1 mm (Abdollahnejad et al. 2014;Assari and Mahesh 2011). Masjed-e J ame' of Isfahan is located in north-east of Isfahan at 32 39 0 25.97204 00 longitudinal and 51 41 0 08.38904 00 latitudinal geographical coordinates. The building materials used in this monument consist mainly of bricks, stones, tiles, gypsum, mud mortar, and lime mortar (Iranian government and ICHHTO 2011). Today's mosque is a combination of different styles and its surroundings are now completely integrated with the structures located around the bazaar and the old town. Today, only a small exterior part is visible (Figure 1). The average of visitors per year (before the Covid-19 pandemy) was of about 66,000 domestic and foreign.
Ten samples were aseptically taken by using a sterile scalpel, in April and August 2019 from different areas in correspondence of noticeable deterioration patterns such as loose pieces, flacking minerals, black and white spot and salt efflorescence from gypsum, bricks and tile as shown in Figure 2.
Codes were the following: South and South-East areas of Masjed-e J am e named Nezam al-Molk (samples NM1 and

NM2), Southern
Ayv an (samples SI1 to SI4) and South-Eastern Shabestan (samples SSP1 to SSP3); while a sterile swab moistened with sterile Phosphate buffer solution (PBS) was used to sample the tile surfaces of Soffe's Saheb (sample SS1). The sampling sites are shown in the Figure 1(C). Further, several pieces of detached materials (samples NM3 and SSP4) were collected and placed in sterile bags. All samples were transfer to the laboratory for microscopic and cultural analysis; summary of samples is reported in Table 1.

SEM-EDS and XRD analyses
The samples were observed under stereomicroscope, and scanning electron microscope, which for scanning electron microscopic studies the following protocol was used. Samples were fixed in 4% glutaraldehyde solution, then washed with 0.1 M phosphate buffer, after that dehydrated through an ethanol series, 30% for 30 min, 50% for 30 min, 70% for 30 min, 80% for 30 min, 90% for 60 min, and 100% for 12 h (Mohammadi and Krumbein 2008), and finally dried using lyophilizator for 2 h. Next, the samples were mounted and coated with gold, and then visualized using Scanning Electron Microscope; FESEM (TESCAN MIRA-3, Czech Republic) with an accelerating voltage of 15 kV.
The XRD analysis was carried out to investigate gypsum components and its difference between the control with no visible deterioration pattern and sample containing microorganisms. For this step, sample was powdered using a mortar. Then, they were deposited onto a holder and flattened. Data collection was performed by an X-ray diffractometer (Philips PW1730 FEI) with diffraction patterns from 5 to 80 using CuK alpha rays and voltage of 40 KV (Ma et al. 2020). Phase identifications were performed using the standard database for X-ray powder diffraction pattern with Xpert High Score software.

Isolation and cultivation of microorganisms
In order to isolate fungi and bacteria, 100 mg of powdered samples were suspended in physiological saline (0.85% NaCl) supplemented with 0.001% (w/v) Tween 80. Then, the samples were vortexed to detach the cells from inorganic material. After that, 100 lL of suspensions were plated into the different media (Urz ı et al. 2001).
For the isolation of fungi, different media were used such as Potato Dextrose Agar (PDA, Biolife), Dichloran-Glycerol agar (DG-18, Biolife), Malt Extract Agar (MEA, Biolife) supplemented with 7.5% NaCl (Trovão et al. 2020) and Czapek agar (Ponizovskaya et al. 2019). Streptomycin at 0.5 g/L concentration was added to all media to inhibit the bacterial growth (Trovão et al. 2020). Plates were incubated at 28-30 C in dark condition for 30 days. Fungal subcultures have been set up on PDA medium. The isolated fungal strains were maintained on agar slants of PDA medium. The identification of fungi was done studying the macroscopic appearance of colonies grown on PDA medium and microscopic morphology of reproduction structures evidenced on slide cultures (Watanabe 2002). Sequence comparison analyses of ITS1, 5.8S, ITS2 rDNA of selected strains was also carried out to confirm or to obtain a more specific identification as specified in par. 2.4. The Bunt and Rovira medium (BRII) (Bunt and Rovira 1955) modified as described by Urz ı and coworkers (2001), Glycerol-nitrate-casein (GNC) (K€ uster and Williams 1964) and the Blue Green medium BG11 (Stanier et al. 1971) supplemented with cycloheximide were used to isolate chemoorganotrophic bacteria, actinobacteria and cyanobacteria. After the growth, colonies were picked up from the plates and subcultured onto Tryptic Soy Agar (TSA, Biolife) and on Nutrient agar, (NA, Merck) and were incubated at 28 C for 2-3 days for further purification. The isolated bacteria were preliminarily identified by their macroscopic and microscopic morphology, Gram staining, spore staining, catalase and oxidase tests (De . Then, selected strains were identified by molecular methods as specified in part 2.4. Samples taken from saline efflorescence were used to isolate halophile or halotolerant microorganisms. For this reason, the samples were plated in DSMZ medium 372 (yeast extract 5 g/L, casamino acids 5 g/L, sodium glutamate 1 g/L l, KCl 2 g/L, Na 3 Citrate 3 g/L, MgSO 4 .7H 2 O 5 20 g/L, NaCl 200 g/L, FeCl 2 .4H 2 O 36 g/L, MnCl 2 .4H 2 O 0.3 g/L, agar 20 g/L) and medium 1018 (yeast extract 5 g/L, MgCl 2 20 g/L, K 2 SO 4 0.5 g/L, CaCl 2 0.1 g/L, NaCl 150 g/L, agar 20 g/L) (Ettenauer et al. 2014).

Molecular identification of microorganisms
Seven bacterial strains that showed biodeteriorative capability were identified by partial sequencing of 16S rDNA. To this purpose DNA extraction was obtained using the boiling method as described by Junior (Junior et al. 2016). Briefly, the bacterial cells grown on NA medium were suspended in 200 mL microliters of TE buffer (10 mM Tris-HCl and 1 mM EDTA) with pH 8.0 and boiled for 15-20 min. Then, the microtubes were placed into ice for 15 min and then centrifuged for 5 min at 14,000 RPM. Finally, the supernatant that contains DNA was transferred to a sterile microtube.
Primers 27 F (5 0 AGAGTTTGATCCTGGCTCAG3 0 ) and 1492 R (5 0 CGGCTACCTTGTTACGACTT3 0 ) were used for amplification of 16S rDNA. The PCR amplification reaction included 12.5 mL master mix (2Â), 0.5 mL of each primer with 10 mM concentration and 0.5 mL DNA templates. The thermal cycling protocol included an initial denaturation step at 95 C for 2 min, followed by 35 cycles consisting of denaturation at 94 C for 1 min, annealing at 55 C, extension at 72 C for 90 s and a final extension at 72 C for 7 min. The PCR products about 1500 bp long were visualized on 1.5% agarose gel containing SYBR safe DNA stain.
Eleven fungal isolates showing deteriorating ability were identified by ITS1, 5.8S ITS2 rDNA sequencing analyses. The protocol proposed by Ruibal et al. (2009) was applied for DNA extraction and rDNA amplification. Fungal biomass grown on PDA was transferred to microtubes containing 500 mL of TES buffer (100 mM Tris with pH 8.0, 10 mM EDTA, 2% SDS), ground with a micro-pestle and then added 140 mL of NaCl (5 M) and 65 mL of 10% CTAB (w/v). After incubating the mixture at 65 C for 30 min, 700 mL of chloroform/isoamylalcohol (24:1) was added and placed on ice for 30 min. Next, the solution was centrifuged for 10 min at 10,000 RPM at 4 C. The supernatant was transferred to a sterile microtube and then the genomic DNA was precipitated using isopropanol. After washing the pellets with 70% ethanol, the recovered DNA was dried and dissolved in 60 mL of TE buffer.
The ITS of fungal rRNA genes was amplified with the following universal two primer pair, ITS1 (5 0 -TCCGTAGGTGAACCTGCGG-3 0 ) and ITS4 (5 0 -TCCTC CGCTTATTGATATGC-3 0 ). The PCR amplification reaction included 12.5 of mL master mix (2Â), 1 mL of each primer of ITS1 and ITS4 (10 mM) and 1 mL of DNA template. The thermal cycling protocol included an initial denaturation at 94 C for 4 min, followed by 30 cycles consisting of denaturation at 94 C for 1 min, annealing at 55 C, extension at 72 C for 1 min and a final extension at 72 C for 10 min. The amplified products were visualized on 1.5% agarose electrophoresis gels stained with SYBR safe DNA stain.
The PCR products were sequenced by a Niagen noor Biotechnology Company (Tehran, Iran) and homology of nucleotide sequences was assessed by comparison with the sequences in NCBI database and using BLASTn algorithm. Their accession numbers are shown in Table 3

Assessment of deteriogenic properties of isolates
In order to determine deteriorating ability of all the isolates, the following tests were carried out.

CaCO 3 dissolution
All isolated microorganisms were tested for CaCO 3 dissolution using CaCO 3 glucose agar plates comprising 5 g/L of CaCO 3 , 10 g/L of glucose and 15 g/L agar (Ponizovskaya et al. 2019). After 21 days, CaCO 3 dissolution was evaluated by measuring of the clear zone around the colonies.

Pigment production
This experiment was carried out following the protocol (Savkovi c et al. 2016) in a medium containing, 2 g/L of sodium nitrate; 1 g/L of dipotassium phosphate; 0.5 g/L of magnesium sulfate; 0.5 g/L of potassium chloride; 0.01 g/L of ferrous sulfate; 20 g/L of agar; 10 g/L of glucose with pH 5.5, and cultures were incubated at 25 C for 21 days. Color change was observed in the Petri dishes containing pigmentproducing fungi. For the isolated bacteria, pigment production was considered by formation of colored colonies in cultured media after their growth.
Acid and alkaline production Spore suspensions from each isolated strain was inoculated in a liquid medium as the same described above (section "Pigment production"), with pH adjusted to 7. The cultures were incubated at the same temperature for three days and then pH of the cultures was measured using a pH meter (Crison, Spain) (Savkovi c et al. 2016). For bacterial metabolites assessment, 1-2 colonies of isolates were suspended in BRII medium with pH 7.
Organic acid production Fungi and bacteria that showed highest reduction in pH of broth medium selected for analysis of organic acid production by using High-performance Liquid Chromatography (HPLC). The used HPLC system was Knauer, Smartline (Germany), HPLC columns Eurokat H (8 Â 250 mm, 10 mm), analytical columns, the mobile phase H 2 SO 4 (5 mM with pH 2.25) and 3% methanol. The temperature of the column was set at 25 C and the flow rate controlled to 0.6 ml/min. The wavelength 210 nm was used to detect of organic acids. Oxalic, malic, acetic, citric, succinic and fumaric acids were used as standards to compare with HPLC peaks (Inberg et al. 2020).

Microbial mineralization
The isolates were inoculated on B4 medium containing 4 g/L of yeast extract; 5 g/L of dextrose; 2.5 g/L of calcium acetate and 14 g/L of agar (Savkovi c et al. 2016). After a period of 21 days, the SEM-EDX and XRD analysis was used to determine the morphological characteristics and the type of elements and compositions of the crystals.

Results
Despite of the old age of the monument of Masjed-e J am e, because of arid climate, signs of deterioration are very limited in small parts of the building especially indoors. The different patterns of deterioration were observed most frequently as black spots, fragmentation, and biomineralization.

SEM and XRD analysis
Direct observation under SEM of samples taken in correspondence of alterations in the gypsum and brick substrates showed the constant presence of microorganisms on deteriorated surfaces (Figure 3). Microbes were often organized in biofilm (Figure 3(A,D)), and newly formed crystals were often observed in these samples (Figure 3(A-C)). Fungal conidia were found in close contact with minerals ( Figure 3(B-C)).
The XRD analysis showed that the main component of gypsum was CaSO 4 .2H 2 O in association with aragonite, calcite, and graphite. A decrease in the peak of CaSO 4 .2H 2 O covered by biofilm was seen when comparing to controls (Figure 4).

Preliminary characterization of isolates
A culture-dependent approach allowed to isolate 82 different microorganisms (23 fungal isolates and 59 bacterial isolates) in 10 deteriorated areas of Masjed-e J ame' of Isfahan from gypsum, tile, and brick samples, as shown in Table 2.
Fungal isolates were identified at genus level by using micro-and macro-morphological features. They belonged to Aspergillus sp., Penicillium sp., Cladosporium sp., Alternaria sp., Ulocladium sp., Parengyodontium sp.; others did not show any production of reproductive mycelium and were considered as Mycelia sterilia. Penicillium strains were isolated mainly from NM1 sample (17.39%). The most abundant isolated fungi (43.47%) were achieved from black spot and from gypsum samples (respectively sample SI1 and SI3).
Gram positive bacteria were 89.83% of total bacterial isolates and belonged to the genera of Bacillus, Kocuria, Citricoccus, Arthrobacter, Actinomycetes and Oceanobacillus. Furthermore, Bacillus sp. were isolated from brick (SSP1-3 sample with 23.72%) and gypsum (SI1-4 sample with 16.94%), and in NM2 sample (gypsum) was less (with 1.6%). Actinomycetes constituted the least percentage of bacterial isolates with 5.08%, and Gram negative bacteria were 10.16% of total bacterial isolates. No bacteria were isolated from sample NM1 and SS1.

Deteriogenic properties of isolates
The results of the assessment of potential biodeteriogenic activities of selected isolates are summarized in Table 3. CaCO 3 dissolution, seen as a transparent zone around the colonies, was well performed by Aspergillus and Penicillium strains isolated from black spots on the gypsum and on  the tiles. CaCO 3 dissolution was not observed for Cladosporium cladosporioides, Cladosporium limoniforme, Penicillium chrysogenum isolated from brick and Aspergillus flavus, Ulocladium sp. Parengyodontium album and Alternaria sp. isolated from gypsum without apparent presence of black spots. Among the bacterial isolates, only Bacillus sp. isolated from brick showed scarce abilities to solubilize CaCO 3 .
Pigment production was observed in fungal colonies isolated from black spot in gypsum, tile and flacking part and saline efflorescence of in brick. The produced colors by fungal colonies were: Aspergillus flavus brown color, Penicillium polonicum red-brown color and Penicillium chrysogenum yellow color.
þ represents one isolated microorganism. þþ represents two isolated microorganisms. þþþ represents three isolated microorganisms. þþþþ represents four isolated microorganisms. À represents no isolated microorganisms. from black spot in gypsum and from flacking part and saline efflorescence in brick that showed orange, red and yellow colonies, respectively. Regarding organic acid production as seen by the lowering of the pH of the tested medium, strains of Aspergillus niger isolated from tile and gypsum and Penicillium polonicum isolated from gypsum and Penicillium chrysogenum isolated from brick were able to reduce the pH of the medium significantly higher than other isolates. Other strains including Aspergillus flavus, Cladosporium limoniforme, Parengyodontium album and Naganishia diffluens were not displayed any significant reduction of pH. Among the bacterial isolates, only Bacillus cereus isolated from gypsum and Bacillus licheniformis isolated from brick were able to decrease the pH of medium to 3.84 and 4.14, respectively (Table 3).
HPLC was used to detect type of organic acid in the medium. Aspergillus niger and Penicillium polonicum isolated from tile and Penicillium chrysogenum and Bacillus licheniformis isolated from brick, as well as Bacillus cereus isolated from gypsum showed the highest reduction of pH.
The results of HPLC showed that these strains had the ability to produce oxalic, fumaric and acetic acids as well as succinic and malic acids. All above strains had the ability to produce oxalic acid and fumaric acid. Furthermore, the result showed that the oxalic acid produced by fungi was greater than bacteria, and the highest amount (10,347 ppm) was produced by Penicillium polonicum (supplemental Table S1).
Mineral precipitation by bacteria was only observed in Arthrobacter agilis isolated from gypsum. Also, 8 out of 23 fungal isolates, including Aspergillus niger, Aspergillus flavus Penicillium sp. and Alternaria sp. isolated from the gypsum and Aspergillus niger isolated from the tiles, were able to precipitate mineral phases in B4 medium. The SEM-EDS analysis revealed that minerals were close to the colonies with different sizes, shapes, and chemical compositions ( Figure 5). The EDS analysis showed presence of Ca (calcium), C (carbon) and O (oxygen) elements in all biominerals. Chemical composite of all biominerals identified by XRD analysis showed calcium carbonate or calcite (CaCO 3 ) in bacteria and calcium oxalate in form of weddellite Precipitation of weddellite by Aspergillus flavus and whewellite by Aspergillus niger and Penicillium sp. was identified in B4 medium. Penicillium polonicum and Alternaria sp. were able to precipitate both whewellite and weddellite. The EDS analysis and XRD pattern in Arthrobacter agilis confirmed presence of calcite (Figure 7).

SEM and XRD analysis
The SEM analysis revealed the presence of microorganisms and their biodeteriorative patterns in substrates. In SEM micrographs, the presence of biofilm and biominerals was seen in the gypsum and brick samples. The observation of close contact between the minerals and microbial cells can confirm the formation of biominerals by microorganisms. The results of XRD analysis showed that the contamination of samples with microbial biofilm was followed by a significant decrease in CaSO 4 .2H 2 O. These changes can be attributed to the growth and influence of microorganisms reported in other studies (Fiertak and Stanaszek-Tomal 2016). Also, the presence of CaO in the biofilm structures can be attributed to the biotransformation of calcite by microbial biofilm.

Microbial community
The microorganisms were isolated by culture-dependent methods using different media. The relationship between alteration types in the sampling site and the number and diversity of microorganisms was also investigated. As presented in the results, 23 fungal isolates and 59 bacterial isolates were identified based on classic methods. Penicillium sp. was the predominant genera in all three substrates of tiles, bricks, and gypsum. Moreover, the most diversity of fungi was obtained from the black spots on gypsum surfaces, which can be attributed to the biogenic origin of the black spots. Black spots on the gypsum and tiles including Penicillium spp., Aspergillus niger, Aspergillus flavus, Cladosporium spp., Alternaria sp., Arthrobacter sp., Bacillus spp., Kocuria spp., Citricoccus sp. and Actinomycetes.
Most of the microorganisms, including 28 bacteria and 13 fungi, were isolated from the gypsum. It seems that gypsum provides a favorable habitat with sufficient moisture. Furthermore, as reported by site office high humidity in the gypsum of the south Ivan due to the problems of water piping, can be a reason for high quantities of microorganisms isolated from gypsum, and has been provided suitable conditions for the rapid growth of fungi.
It is the same for more abundance of Bacillus genera isolated from the gypsum and bricks. However, bacteria were not isolated from tile samples. The glaze of tile surfaces with less roughness can be a reason for less microbial colonization (Gazulla et al. 2011). Also, the extreme temperatures needed to bake tiles can remove any biological structures inside the materials during its manufacturing. The surfaces of bricks used in the southeastern Shabestan were covered with saline efflorescence, providing the right ecological conditions for the growth of halophile/halotolerant microorganisms. This is the reason for the presence of Oceanobacillus sp. in this area. Oceanobacillus was reported as moderately halophilic bacterium (Amoozegar et al. 2016).
Although isolates were classified based on macroscopic and microscopic morphological characteristics, 11 fungal and 7 bacterial isolates were selected and identified using ITS region and 16S rRNA gene, respectively. Penicillium, Aspergillus, Alternaria, and Cladosporium genera were isolated from different substrata as in many other similar studies were reported (Aira et al. 2007;Inberg et al. 2020;Poyatos-Jim enez et al. 2021;Rojas et al. 2012).

Calcite dissolution
The assessment of fungal CaCO 3 dissolution is helpful to show the biodeteriorative impacts of isolates recovered from cultural heritage sites (Trovão and Portugal 2021). CaCO 3 dissolution has been reported by Penicillium sp., Aspergillus sp., Lecanicillium sp. Penicillium chrysogenum, Cladosporium sphaerospermum, Aspergillus elegans and Aspergillus niger in several studies (G amez-Espinosa et al. 2020;Inberg et al. 2020;Ponizovskaya et al. 2019;Unkovi c et al. 2018) as we isolated some of them in our study. Thus 33.3% of our fungal isolates, Aspergillus niger and Penicillium spp. dissolved CaCO 3 . Interactions between the microorganisms' metabolites and calcium can be the reason for calcium complex solubilization (Gu et al. 1998). In this study, all fungi that showed the ability to dissolve CaCO 3 were isolated from the black spots on tiles and gypsum, and biodeteriogentic role of them was confirmed. Cirigliano et al. (2018) isolated bacterial species from moonmilk deposits and tested them for dissolution or production of CaCO 3 . They demonstrated that 27 isolated strains revealed the ability of CaCO 3 dissolution (Cirigliano et al. 2018). Pangallo et al. investigated the deterioration potential of microflora isolated from fresco. They showed that Acinetobacter, Micrococcus, Enterobacter, Nocardioides and Pseudomonas can dissolve CaCO 3 (Pangallo et al. 2012). In this study, just an isolated bacterium from the brick showed a slight ability for CaCO 3 solubilization. Although few bacteria could produce acid, but was not able to solubilize calcite, which may be related to insufficient quantity of solubilizing acids production by these bacteria.

Pigment production
The colored biofilms can cause aesthetic damage in monuments of cultural heritage. The biofilm of fungi, algae, and bacteria have the ability to produce pigments (Tescari et al. 2018a). Discoloration is one of the most common demonstration of fungal contamination (Rojas et al. 2012). Some studies showed fungi can produce organic pigments by different colors, depending on the chemical compounds of the substrate (Borrego et al. 2010;G amez-Espinosa et al. 2020;Garg et al. 1995;Rojas et al. 2012;Unkovi c et al. 2018). Bacteria are also known to be agents of material discoloration. Tescari et al. showed that Arthrobacter agilis was the cause of rosy discoloration in Terme del Foro (Tescari et al. 2018b). Moreover, Rosado et al., stated that Rubrobacter sp., Arthrobacter sp., Roseomonas sp., and Marinobacter sp. were responsible for colorful biofilm (Rosado et al. 2020). In this study, pigments secretion was seen by Aspergillus flavus, Penicillium polonicum and Penicillium chrysogenum and colored colonies were observed by Arthrobacter sp, Kocuria sp., Citricoccus sp. These pigments could be an agent of black spot and aesthetical damage in surfaces and need proper removal techniques to maintain the aesthetics of surfaces.

Organic acid production
The most important impact of fungi on stone surfaces is the production of organic acid (Sazanova et al. 2014). Aspergillus and Penicillium species can produce oxalic acid (Del Monte et al. 1987) and play important roles in the biodeterioration of materials as well as reducing pH (Trovão and Portugal 2021). The production of organic acid by Penicillium aurantiogriseum, Penicillium chrysogenum, Penicillium commune, Actinomucor elegans, and Cladosporium sphaerospermum has been previously reported (Inberg et al. 2020). In this paper the isolated Aspergillus niger from tile represents the most reduction in pH to 3.58. This result is in agreement with studies by Unkovi c and G omez-Espinosa (G amez-Espinosa et al. 2020;Unkovi c et al. 2018). Organic acids produced by fungi and decrease the pH levels can be cause of more fungal growth compared to bacterial growth (Ma et al. 2020;Vasanthakumar et al. 2013). Another reason of not isolate bacteria from tile surfaces in our study was probably related to the presence of fungi producing acid in black spot on tile surfaces and making improper habitat for bacterial growth.
In the present study, Aspergillus niger and Penicillium polonicum isolated from tiles, Bacillus cereus from gypsum, and Penicillium chrysogenum and Bacillus licheniformis isolated from bricks displayed the highest reduction in pH of the liquid medium. It is the reason which was selected these strains to assess organic acids production. All these selected strains showed the ability to produce different acids. Acids can form complexes via dissolution of cations or can chelate metals (Savkovi c et al. 2016). As reported the substrate acidification can be increased fungal growth and chemical biodeterioration (Unkovi c et al. 2018).

Microbial mineralization
Fungi play an important role in the formation of biominerals such as oxalates (Burford et al. 2006;Ortega-Morales et al. 2016). Calcium oxalate is the most common oxalate produced on fungal biomass (Gadd et al. 2014;Unkovi c et al. 2017). The production of mycogenic minerals by Aspergillus, Penicillium, Alternaria, Cladosporium, Colletotrichum, Pestalotiopsis, and Trichoderma has been reported in several studies (Gadd et al. 2014;Inberg et al. 2020;Savkovi c et al. 2016;Trovão and Portugal 2021;Unkovi c et al. 2017). Oxalic acid secreted by fungi reacts with Ca 2þ or CaCO 3 to form biominerals. The observation of weddellite on the biodeteriorated substrates confirmed that the biological growth and secretion of metabolites led to mechanical and esthetic deterioration (Ma et al. 2020). In this study, all the microorganisms capable of biominerals were isolated from gypsum and tiles. The results of EDS and XRD analysis confirmed the presence of calcium oxalate in the form of weddellite and whewellite in Aspergillus and Penicillium cultures, as shown below: Ca 2þ þ 2CH 3 COO À þ 2H þ þ C 2 O 2À 4 þ nH 2 O ! CaC 2 O 4 Á nH 2 OðsÞ þ 2CH 4 ðgÞ þ 2CO 2 ðgÞ (Inberg et al. 2020;Savkovi c et al. 2016). The formation of oxalate minerals along with fungal growth and its metabolite production induce mechanical pressure and create patterns such as cracking and detachment in addition to asthetic damage (Ma et al. 2020). Pangallo et al. (2012) showed that Bacillus and Micrococcus strains isolated from fresco, and Pseudomonas, Staphylococcus, Psychrobacter, Curtobacterium fangii and Arthrobacter isolated from the air could produce minerals (Pangallo et al. 2012). In our study, Arthrobacter agilis precipitated calcite. The formation of calcite occurred in areas of Ca 2þ accumulation with dissolving CO 2 from the respiratory metabolism of microorganism as well as atmospheric CO 2 , as shown below: Ca 2þ þ H 2 O þ CO 2 ! CaCO 3 ðsÞ þ H 2 ðgÞ (Inberg et al. 2020). Some fungi isolated in this investigation such as Cladosporium cladosporioides did not show any deteriorative impacts in our experiments. Possibly, these fungi have other deteriorating impacts such as mechanical deterioration through the penetration of hyphae into the substrate or other chemical deteriorations which were not investigated in this study (Unkovi c et al. 2018).

Conclusions
The Masjed-e J ame' of Isfahan is located in a semi-arid area with low precipitation and moisture. It can be concluded that not only can fungi and bacteria be isolated from the biodeteriorated surfaces of Masjed-e J ame' of Isfahan, they also have a deteriorating potential especially in black spots via CaCO 3 dissolution, organic acid and pigment production, and mineral formation. Isolating microorganisms from this monument can help measure the efficiency of cleaning protocols with microbiocidal materials and polymers used in conservation and restoration, as well as testing the novel protectants and polymers.