Long-amplicon MinION-based sequencing study in a salt-contaminated twelfth century granite-built chapel

Vol.:(0123456789) 1 3 https://doi.org/10.1007/s00253-022-11961-8 ENVIRONMENTAL BIOTECHNOLOGY Long‑amplicon MinION‑based sequencing study inasalt‑contaminated twelfth century granite‑built chapel JelenaPavlović1· PilarBosch‑Roig2· MagdalenaRusková1· MatejPlaný1· DomenicoPangallo1,3· PatriciaSanmartín4,5 Received: 19 March 2022 / Revised: 14 April 2022 / Accepted: 2 May 2022 © The Author(s) 2022 Abstract The irregular damp dark staining on the stonework of a salt-contaminated twelfth century granite-built chapel is thought to be related to a non-homogeneous distribution of salts and microbial communities. To enhance understanding of the role of microorganisms in the presence of salt and damp stains, we determined the salt content and identified the microbial ecosystem in several paving slabs and inner wall slabs (untreated and previously bio-desalinated) and in the exterior surrounding soil. Soluble salt analysis and culture-dependent approaches combined with archaeal and bacterial 16S rRNA and fungal ITS fragment as well as with the functional genes nirK, dsr, and soxB long-amplicon MinION-based sequencing were performed. State-of-the-art technology was used for microbial identification, providing information about the microbial diversity and phylogenetic groups present and enabling us to gain some insight into the biological cycles occurring in the community key genes involved in the different geomicrobiological cycles. A well-defined relationship between microbial data and soluble salts was identified, suggesting that poorly soluble salts (CaSO4) could fill the pores in the stone and lead to condensation and dissolution of highly soluble salts (Ca(NO3)2 and Mg(NO3)2) in the thin layer of water formed on the stonework. By contrast, no direct relationship between the damp staining and the salt content or related microbiota was established. Further analysis regarding organic matter and recalcitrant elements in the stonework should be carried out. Key points • Poorly (CaSO4) and highly (Ca(NO3)2, Mg(NO3)2) soluble salts were detected • Halophilic and mineral weathering microorganisms reveal ecological impacts of salts • Microbial communities involved in nitrate and sulfate cycles were detected Keywords Long amplicons· MinION sequencing· Salt contamination· Stone· Batrachochytrium· Bio-desalination Introduction Stone monuments provide habitats for multispecies microbial communities, including bacteria, fungi, lichens, algae, and archaea (Hoppert etal. 2004; Gadd 2017). The presence of microbes on heritage stonework is generally considered negative owing to the physical and/or chemical damage that they cause via biodeterioration mechanisms. However, the presence of these communities can also have negligible effects, such as surface deposition with no substrate interaction and only an esthetic impact (Sanmartín etal. 2020), and even positive effects, such as bioprotection, biomineralization, and bio-desalination phenomena (Kembel etal. 2014; Pinna 2014; Gadd 2017; Schröer etal. 2021; OrtegaMorales and Gaylarde 2021; Bosch-Roig etal. 2021). As Jelena Pavlović and Pilar Bosch-Roig contributed equally. * Patricia Sanmartín patricia.sanmar[email protected] 1 Institute ofMolecular Biology, Slovak Academy ofSciences, Dúbravská cesta 21, 84551Bratislava, Slovakia 2 Instituto Universitario de Restauración del Patrimonio, Universitat Politècnica de València, 46022Valencia, Spain 3 Caravella, s.r.o., Tupolevova 2, 85101Bratislava, Slovakia 4 Departamento de Edafoloxía e Química Agrícola, Facultade de Farmacia, Universidade de Santiago de Compostela, 15782SantiagodeCompostela, Spain 5 CRETUS, Universidade de Santiago de Compostela, SantiagodeCompostela, Spain / Published online: 21 May 2022 Applied Microbiology and Biotechnology (2022) 106:4297–4314 1 3 geomicrobial agents on the built environment, stone colonizers are involved in elemental cycling, rock transformation, soil formation, organic matter decomposition, and cycling of elements, among other processes (Barton and Northup 2007; Gadd 2017) . The biological population associated with stone-built structures is determined by a combination of factors such as environmental conditions (e.g., humidity, temperature, light, and pollution), architectural design, the chemical and mineralogical composition, and petrophysical properties of the stone (e.g., roughness and pore structure) and anthropogenic influences (e.g., human occupancy and restoration processes) (Gorbushina and Broughton 2009; Gadd 2017). The presence of salts also affects stone-associated microbial communities, which are often rich in highly specialized microorganisms such as halophilic microorganisms (Schabereiter-Gurtner etal. 2004). Indeed, desalination treatments may affect the endogenous microbial communities (Caneva etal. 2005). Salt contamination is widespread and very difficult to eradicate from stone monuments. Salts can cause both esthetic and physical damage, and salt contamination is one of the most important factors involved in the deterioration of stone monuments, leading to important cultural and economic losses (Freedland 1999; Germinario and Oguchi 2021). The behavior of salt on stone-built cultural heritage surfaces is a complex phenomenon and involves multiple variables, such as salt type, salt combinations and quantity (supersaturation), substrate properties (porous size), and environmental factors (temperature and humidity). In addition, there are multiple sources of soluble salts in stone material (Freedland 1999; Barton and Northup 2007), such as the building material itself (e.g., manufacturing process), water (e.g., seawater), atmospheric pollution (e.g., acid rain), modern interventions/treatments (e.g., restoration products), and microorganisms (e.g. ammonia-oxidizing bacteria). Furthermore, salt contamination, often related to the presence of water, is an important factor influencing microbial growth (Hoppertet al. 2004; Dedesko and Siegel 2015). The present study concerns the salt-contaminated twelfth century granite-built Cristo Chapel of the Santa María de Conxo Monastery in Santiago de Compostela (NW Spain). The stonework is affected by irregular, damp-to-touch dark staining that may be caused by a non-homogeneous distribution of salts and biological factors. To enhance understanding of the role of microorganisms in the presence of salts and in the staining, stonework (including chapel and church paving slabs and the slabs in the inner wall of the chapel) and the soil surrounding the chapel were analyzed to determine the salt content and identify the microbes present. Different strategies were used to identify culture-dependent and culture-independent microorganisms (and thus identify nonculturable microorganisms) (Schröer etal. 2020; Elert etal. 2021). High-throughput sequencing, which has revolutionized the analysis of microbiota on cultural heritage objects (Marvasi etal. 2019), was also used. Among the sequencing approaches, the third-generation MinION sequencing platform (Oxford Nanopore Technologies, ONT, Oxford, UK), also used in this work, is an affordable device that can be used to identify the microorganisms present in samples, therefore enabling detailed study of microbial communities (Bosch-Roig and Sanmartín 2021; Pavlović etal. 2021). One of the advantages of the MinION sequencing approach is the ability of the technique to generate and analyze long reads (including long amplicons), which can enhance identification of the microbiota of interest (Pavlović etal. 2021). A high presence of salt related to the microbial communities on the pavement causing the damp dark staining is the hypothetical premise of the present study. To address it, analysis of soluble salts was coupled with a culture-dependent analysis and a long-amplicon MinION-based sequencing strategy focusing on archaeal and bacterial 16S rRNA, fungal ITS fragment, and the nirK (nitrite reductase), dsr (dissimilatory sulfite reductase), and soxB (sulfite oxidase) functional genes. Materials andmethods Site description The Santa María de Conxo Monastery, located in Santiago de Compostela (UNESCO World Heritage City since 1985, capital of Galicia, NW Spain) is a baroque complex of seventeenth century buildings, constructed around a Romanesque twelfth century chapel called the Cristo Chapel. The monastery complex includes a direct connection between the chapel, the church, and the cemetery. The walls and the paving of the Cristo Chapel and the church consist of granite slabs. The paving slabs of the Cristo Chapel (occupying an area of 233 m2) are strongly affected by salt contamination and irregular damp dark staining. In order to eliminate or at least reduce the dark staining, different treatments were applied between 2013 and 2020, including architectural interventions and bio-desalination treatments by the addition of live Pseudomonas stutzeri denitrifying bacteria (García Morales etal. 2016; Bosch-Roig etal. 2019, 2021). Although the treatments/interventions reduced the damp dark staining, it is still present. Sampling The sampling methodology was designed to evaluate both the salt content and the microbiome in order to study their relationship and their potential involvement in causing the damp staining. Sampling was carried out on January 4298 Applied Microbiology and Biotechnology (2022) 106:4297–4314 1 3 21, 2020, i.e., one to three months after a bio-desalination treatment. Seven sampling points were selected, including damp to touch dark patches and dry darkened areas, biodesalinated and untreated areas, and four different locations (chapel paving, church paving, chapel inner wall, and the soil surrounding the chapel). Of these, five samples (IC3, IC4, IC6, IC7, IC9) were taken from inside and two samples (ICS1, ICS2) from the soil outside the building (Fig.1, Table1). Samples IC3 and IC4 were taken respectively from two damp dark patches on chapel paving slabs, with different degrees of discoloration relative to the rest of the chapel paving. Thus, IC3 was from a darker, more heterogenous slab with a southern orientation, and IC4 was from a lighter, more homogeneous slab, with a northern orientation. Both areas had been treated by bio-desalination with the bacterium P. stutzeri. This treatment involves the addition of live P. stutzeri directly to the stone surface, followed by the application of ground 2% agar gel (as a delivery system and source of humidity) and of an electric heating mat (26 ± 4°C for 48h) (as described in Bosch-Roig etal. 2019, 2021). After the treatment, the thermal and delivery systems are removed, and the surface is cleaned with distilled water and a sponge. Likewise, the exogenous introduction of P. stutzeri and its permanence is checked with CFU (colony-forming unit) content, not obtaining significant differences with respect to the beginning (before bio-desalination) (BoschRoig etal. 2021). Sample IC6 was taken from a damp dark patch of chapel paving slab that had been partially treated (unfinished treatment) by bio-desalination, in which the P. stutzeri had been added, but the agar gel and heat had not been applied and the final cleaning step with water had not been carried out. This sample was taken to evaluate the potential change in microbial diversity on the stone slab during the bio-desalination treatment. Sample IC7 was taken from a slab from the inner wall of the chapel, not affected by damp staining and not treated by bio-desalination. Sample IC9 was taken from an untreated church paving slab affected by damp dark staining, for purposes of comparison. Finally, two samples of soil surrounding the chapel were collected: from an area adjoining the cemetery (ICS1) and from an area not adjoining the cemetery (ICS2). For each sampling point inside the building, a granite slab was selected and the entire surface was scratched with a sterile scalpel to yield approximately 0.3–0.8g of surface deposit, which was placed in sterile 50-mL plastic tubes. For each sampling point outside the building, composite soil samples were prepared by combining four subsamples from the same area. The soil subsamples were collected within an area of ~ 1m diameter, to a depth of 50cm, with an ethanolsterilized soil probe or hand auger. The subsamples were Fig. 1 Plan of the Santa María de Conxo Monastery (provided by Alicia Noia and Lourdes Pérez from the Consorcio de Santiago) showing the seven sampling points in red: IC3, IC4, IC6, IC7, IC9, ICS1, and ICS2 4299Applied Microbiology and Biotechnology (2022) 106:4297–4314 1 3 mixed together in a sterile 500-mL glass container, which was vigorously shaken to produce a homogenous sample. In both cases, the samples were transported to the laboratory, where each of the seven samples was divided into four subsamples for soluble salt analysis, DNA-based analysis in the MiniON device, and culture-dependent techniques. One subsample was reserved for possible further analyses. Material characterization: soluble salt content andsoil texture The soluble salts were extracted from all samples by a simple shaking-assisted method with water, for 24h (100mg of sample in 100mL of MilliQ water), following an optimized methodology based on UNE-EN 16, 455:2016. The ionic composition was determined in an ion chromatographer (Metrohm 930 Compact IC Flex, Riverview, FL, USA). The results are expressed as milligram of ion (anion or cation) per gram of sample. The texture of soils was determined according to a standard procedure, in which the soil samples were air-dried and sieved through a 2-mm-mesh-size sieve, and any plant roots were removed. Particle size distribution was determined by wet sieving and the pipette method (Gee and Bauder 1986) after the removal of organic matter and iron oxides. Isolation ofmicroorganisms About 20mg of each sample from the monastery (IC3, IC4, IC6, IC7, IC9) and 50mg of each sample from the surrounding soil (ICS1, ICS2) were placed in plastic tubes with 2mL of physiological solution and stirred briefly. The seven suspensions were serially diluted to 1 × 10−8, and 100 µL of each dilution was then plated in 9 different agar media including general isolation media and specific isolation media (according to the salt concentration present on the pavement) without addition of any colorant, to isolate the microbes present on the slabs. For the isolation of fungi, 3 different specific agar media were used: malt extract agar (MEA; Himedia, Mumbai, India); MEA with 3% NaCl and 2% Mg2SO4; and dichloran-glycerol (DG-18; Merck, Darmstadt, Germany). For the isolation of bacteria, 6 different Table 1 Visual description and details of the seven sampling points Code Description. OrientationPicture IC3 Heterogenous dampto touch dark staining and darker*,biodesalinated slab from the chapel paving. South IC4 Homogeneous damp to touch dark staining and lighter*, biodesalinated slab from the chapel paving. North IC6 Heterogenous damp to touch dark staining and darker*, partiallybiodesalinated chapel paving slab. North IC7 No damp dark staining, untreated slab from theinner wall of the chapel. South IC9 Homogeneous damp dark staining and darker*, untreatedchurch paving slab. South-west ICS1 Soil surrounding the chapel and adjoiningthe cemetery. North ICS2 Soil surrounding the chapel and not adjoinng the cemetery. East * Relative to the chapel paving slabs 4300 Applied Microbiology and Biotechnology (2022) 106:4297–4314 1 3 specific agar media were used: Reasoner’s 2A (R2A; Himedia); R2A with 3% NaCl and 2% Mg2SO4; ammonia oxidation agar (AOA medium containing (NH4)2SO4, 0.5g; KH2PO4, 0.2g; MgSO4.7H20, 0.2g; CaCI2.2H20, 0.02g; distilled water, 1l, with pH of adjusted to 8.2 with 0.1N NaOH, Sarathchandra 1979); denitrification screening agar (DSA, including KNO3 1g; CaCl2 0.2g; KH2PO4 1g; FeCl3·6H2O 0.5g; MgSO4·7H2O 1g; sodium succinate 8.6g; agarose 21g; distilled water 1l, the final pH of the medium was adjusted to 7.0 ± 0.2, Shao etal. 2016), Thiobacillus agar (TBA, for the isolation of sulfate-oxidizing bacteria, containing (NH4)2SO4 0.4g; MgSO4 7H2O 0.5g; CaCl2 0.25g; KH2PO4 4g; FeSO4 0.01g; Na2S2O3 5g; agar 12.5g; distilled water 1l, the final pH of the medium was adjusted to 4.0–4.5; Starosvetsky etal. 2013); and actinomycete isolation agar (AIA; Himedia). All plates were incubated at room temperature (24–26°C) for 3weeks. After the selection of pure colonies, based on their macro-morphology and color, the fungi were maintained on MEA and the bacteria were maintained on R2A plates. Specific plate assays The isolated bacteria and fungi were tested for different activities/capacities using specific agar plate assays. For screening microorganisms involved in nitrate and sulfate cycles, the following media were used: AOA with phenol red (phenol red 7.5mg/L; Merck, Darmstadt, Germany) for detecting ammonium-oxidizing bacteria; DSA with bromothymol blue (BTB, 1mL/L; Merck, Darmstadt, Germany) for screening aerobic denitrifiers; TBA with bromocresol green (BCG, 2mL/L; Merck, Darmstadt, Germany) for detecting sulfate-oxidizing bacteria. All plates were incubated at room temperature and the color change in agar media was monitored over a period of 1–3weeks. The screening assay is based on the color change of the pH indicator. Screening for the ability to solubilize and precipitate CaCO3 was conducted using CaCO3 glucose agar (glucose, 10g; CaCO3, 5g; agar, 15g, distilled water, 1L, Albertano and Urzì, 1999) and B4 medium (calcium acetate, 2.5g; yeast extract, 4g; glucose, 10g; agar, 15g; distilled water, 1L, the final pH of the medium was adjusted to 8.0, Boquet etal. 1973) respectively. On CaCO3 glucose agar, a clear zone was observed around positive strains, whereas on B4, the positive strains produced crystals. All assays were performed in triplicate 60-mm Petri plates. Identification ofthemicroorganisms isolated Pure bacterial strains were collected from plates, and the DNA was extracted with the DNasy Blood & Tissue Kit (Qiagen, Hilden, Germany) according to the manufacturer’s protocol. The fungal strains were inoculated in Malt Extract Broth (MEB. HiMedia, Maharashtra, India) at 26°C until growth. The fungal pellets were then separated from the broth by filtration through sterile filter paper. The DNA of the fungal pellets was extracted with the DNeasy Plant Mini Kit (Qiagen, Hilden, Germany) according to the manufacturer’s protocol. For identification of the isolated bacteria by sequencing, the 16S rRNA gene was amplified using the primers 27F (5′–AGA GTT TGA TCC TGG CTC AG-3′) and 685R (5′-TCT ACG CAT TTC ACC GCT AC-3′) according to Lane (1991). The fungal isolates were identified by PCR amplification and by Sanger sequencing of the ITS fragment with the primers ITS1 (5′-TCC GTA GGT GAA CCT GCG G-3′) and ITS4 (5′-TCC TCC GCT TAT TGA TAT GC-3′) according to White etal. (1990). Twentyfive microliters of PCR mixture contained 50pmol of each primer, 200µmol/L of dNTP (Life Technologies, Gaithersburg, MD, USA), 1.5 U HotStar Taq plus DNA polymerase (Qiagen), 1 × PCR buffer and 3 µL of the extracted bacterial or fungal DNA. The PCR program consisted of initial denaturation at 94°C for 5min, followed by 30 cycles (denaturation at 94°C for 30s, annealing at 54°C for 45s, extension at 72°C for 1min) and a final polymerization step at 72°C for 8min. PCR products from bacterial and fungal isolates were purified using ExoSAP-IT (Affymetrix, Cleveland, OH, USA) and sequenced at a commercial facility (Eurofins Genomics, Ebersberg, Germany). The sequences obtained were directly compared with sequences in GenBank by using the BLAST program (http:// blast. ncbi. nlm. nih. gov/ Blast. cgi) and were subsequently deposited in GenBank under the accession numbers OL423372–OL423396 (bacterial isolates) and OL439055-OL439059 (fungal isolates). MinION sequencing DNA extraction andPCR amplification For MinION sequencing, the total DNA of the seven samples, i.e., five samples from the monastery (IC3, IC4, IC6, IC7, IC9) and two samples from the surrounding soil (ICS1, ICS2) was extracted from the rest of suspensions (whose quantities of material have been described in the “Isolation of microorganisms” section) using the DNeasy PowerSoil Pro extraction kit (Qiagen) following the protocol provided by the producer. Prior to sequencing, total DNA was amplified by specific PCR reactions targeting generic genes for fungal ITS (the same primers and PCR program as in the previous section), for bacterial 16S rRNA (27F: 5′–AGA GTT TGA TCC TGG CTC AG-3′/1492R: 5′–AGA GTT TGA TCC TGG CTC AG-3′) and for archaeal 16S rRNA (Arc344F-mod: 5′-ACG GGG YGC ASS AGK CGV GA-3′/Arch958R-mod: 5′-YCC GGC GTT GAV TCC AAT T-3′) according to Kraková etal. (2016). The presence of microorganisms involved in nitrate and sulfate cycles was 4301Applied Microbiology and Biotechnology (2022) 106:4297–4314 1 3 established by identifying the functional genes encoding the enzyme nitrite reductase (nirK, denitrifying bacteria; nirKC1F: 5′-ATG GCG CCA TCA TGG TNY TNC C-3′/ nirKC1R: 5′-TCG AAG GCC TCG ATN ARR TTR TG-3′, according to Wei etal. 2015), the enzyme dissimilatory sulfite reductase (dsr, sulfate-reducing bacteria; DSR1Fdeg: 5′-ACS CAY TGG AAR CAC G-3′/DSR4Rdeg: 5′-GTG TAR CAG TTD CCR CA-3′, according to Wagner etal. 1998), the Sox enzyme system for sulfur oxidation (soxB, sulfur-oxidizing bacteria; soxB432F: 5′-GAY GGN GGN GAY ACN TGG-3′/soxB1446B: 5′-CAT GTC NCC NCC RTG YTG-3′, according to Petri etal. 2001), and the enzyme ammonia monooxygenase (amoA, ammonia-oxidizing bacteria; amoA-1F: 5′-GGG GTT TCT ACT GGT GGT-3′/amoA2R: 5′-CCC CTC KGS AAA GCC TTC TTC-3′, according to Rotthauwe etal. 1997). The PCR programs used were the same as those applied in the above cited studies (inside the brackets following the primer sequences). Library preparation andsequencing The sequencing library was prepared according to instructions provided in the protocol for Rapid PCR Barcoding Kit (SQK-RPB004. Oxford Nanopore Technologies, ONT, Oxford, UK), downloaded from the ONT website. The concentration of purified amplicons was determined with a DeNovix QFX Fluorometer (DeNovix Inc., Wilmington, DE, USA) and the DeNovix dsDNA Broad Range Kit (DeNovix). All amplicons were subsequently diluted to appropriate input concentrations ranging between 0.5 and 1.5ng/µL. Three µL of 1–5ng template DNA of each sample were used, following slight modifications of the protocol instructions for all the amplicons, except the bacterial 16S rRNA and dsr gene in the purification steps performed with AMPure XP beads (Beckman Coulter, Pasadena, CA, USA). For the ITS, archaeal 16S rRNA, nirK, and soxB amplicons, the previously optimized volume of AMPure beads (60 μL) was used, rather than the recommended 30 µL, to prevent the amplicons from being washed out because of their different, relatively short lengths. Twelve barcoded libraries were pooled in desired ratios to a total molar concentration ranging between 50 and 100 fmoles in 10 µL and ligated with RAP adapters included in the SQK-RPB004 kit (ONT). The prepared libraries were used for loading into the MinION flow cell FLO-MIN 106D R9 Version (ONT). The sequencing was performed in two separate runs (12 libraries for each run). The library features (correlation of samples and amplicons sequenced in each run) are provided in Supplementary material TablesS1 and S2. Sequencing data were split by barcodes with EPI2ME Desktop Agent (ONT). Taxonomic classification and quantitative analysis of the reads derived from bacterial 16S rRNA amplicons were performed using the EPI2ME 16S workflow in EPI2ME Desktop Agent. For the fungal ITS, archaeal 16S rRNA, and other specific markers, the “What’s in my pot” tool (EPI2ME WIMP workflow; ONT) was used. The minimal quality score in both cases was set by default to 7. The percentage of appropriate taxa on the genus taxonomic level was calculated from the total number of classified reads and the number of reads identified as given taxa. The percentage of given taxa was graphically visualized using bar plots. The sequences obtained by the metagenomic analysis are registered and publicly available as BioProject PRJNA767009. Results Salt content The soluble ion contents of each of the seven samples are shown in Table2. The salt content was higher in the samples from inside the building than in the samples from outside of the building, where the total ion content did not exceed 0.198mg/g soil. This finding can be explained by the coarse texture of the soils (Supplementary material TableS3). The particle size distribution was very similar in both soils, with a major sandy fraction of 72.4% and 69.4%, followed by silt content of 20.7% and 23.0%, and little clay, only 6.9% and 7.5%. In these coarse-textured soils, the loss of mobile particles (such as cations and anions) by leaching is frequent and is greatly favored by the climate of Santiago de Compostela, characterized by high rainfall throughout the year (MartínezCortizas and Pérez-Alberti 1999). Table 2 Concentration of soluble ions (mg) per gram rock or soil material from the seven sampling points Sample Na+NH4+K+Ca2+ Mg2+ Cl−NO3−SO42− PO43− Total IC3 1.702 0.002 0.177 2.610 0.393 1.250 9.464 1.139 0.083 16.820 IC4 1.952 0.005 0.422 34.177 0.369 1.778 13.869 54.635 0.041 107.248 IC6 0.011 0.450 17.834 0.129 20.671 0.538 23.788 0.973 20.671 85.062 IC7 1.628 0.013 0.961 17.044 0.108 2.026 16.883 19.271 0.024 57.958 IC9 0.563 0.001 0.656 3.060 0.060 0.616 5.743 3.260 0.013 13.972 ICS1 0.017 0.004 0.010 0.029 0.002 0.020 0.012 0.010 0.006 0.110 ICS2 0.013 0.003 0.018 0.122 0.002 0.015 0.008 0.015 0.002 0.198 4302 Applied Microbiology and Biotechnology (2022) 106:4297–4314 1 3 The building samples with the highest total salt contents were IC4 (107.248mg/g rock) and IC6 (85.062mg/g rock), followed by IC7 (57.958mg/g rock), and the samples with the lowest total salt contents were IC3 (16.820mg/g rock) and IC9 (13.972mg/g rock). The most abundant anions were nitrate (NO3−), with up to 23.788mg/g of rock in sample IC6, and sulfate (SO42−), with up to 54.635mg/g rock in sample IC4. Phosphate (PO43−) was not common, except in sample IC6, with 20.671mg/g rock. The most abundant cations were calcium (Ca2+), with up to 34.177mg/g rock in sample IC4, magnesium (Mg2+), with up to 20.671mg/g rock in sample IC6, and potassium (K+), with up to 17.834mg/g rock in sample IC6. Thus, the main salts present are expected to be calcium nitrate, potassium nitrate (niter), sodium nitrate (nitratine), calcium sulfate (gypsum), magnesium nitrate, and to a lesser extent sodium chloride (halite) and potassium chloride. Sample IC6 also contained phosphates, probably magnesium phosphate in the form of struvite ((NH4)MgPO4·6H2O), a typical by-product of bacterial activity (Rivadeneyra etal. 1992), and potassium and ammonium phosphates. Culture‑dependent analysis andproperties ofisolates A concentration in the range 101–103CFU/mL was evidenced on the agar media used in the culture-dependent analysis. The highest values of CFU/mL (103) were recorded in sample IC3, and the constituent microorganisms also grew in all nine agar media, while the lowest values of CFU/mL (101) were recorded in sample IC7. Clear differences between the microbial communities associated with the stonework of the building and the surrounding soil -and also between the samples of these materialswere observed (Fig.2). The R2A agar and R2A agar supplemented with NaCl (3%) and MgSO4 (2%) were the most effective media for isolating bacteria from all samples, except sample IC6. The few fungi isolated were mainly cultivated on MEA with NaCl (3%) and MgSO4 (2%). A total of 43 morphologically different colonies of microorganisms were isolated from the five sampling points in the monastery: i.e., 13 from IC3, 10 from IC9, 7 from IC4 and from IC7, and 6 from IC6 (Table3). Identification of the isolates revealed the presence of members of the genus Pseudomonas in all samples. The genus Flavobacterium was also isolated from samples IC3, IC7, and IC9. Members of the class Actinomycetes appeared in each sample and were particularly diverse in sample IC3. Representative members of the classes Bacilli and Alphaproteobacteria were also isolated. Fungi belonging to the genera Purpureocillium (IC6 and IC9), Penicillium (IC9), and Aspergillus (IC3) were only isolated from samples IC3, IC6, and IC9. The denitrification capacity of the isolates and their ability to solubilize and precipitate CaCO3 and to oxidize ammonium are indicated in Table3. Denitrification capacity was only observed in one fungal isolate (strain F_IC6_A1 Purpureocillium sp.). All activities tested, except sulfate oxidation activity, were demonstrated by the bacterial isolates although some differences were observed, i.e., the different bacteria displayed different activities/capacities. Fig. 2 Microorganisms isolated from all samples (monastery and surrounding soil) using the nine different agar media, described in the “Isolation of microorganisms” section. AIA: Actinomycete Isolation Agar, DG18: Dichloran-Glycerol (fungi), AOA: Ammonia Oxidation Agar, TBA: Thiobacillus agar, DSA: Denitrification Screening Agar, R2A: Reasoner’s 2A agar, MEA: Malt Extract Agar (fungi) 0% 10% 20% 30% 40% 50% 60% 70% 80% 90% 100% IC6 IC4 IC9 IC3 IC7ICS1ICS2 AIA DG18 AOA TBA DSA R2A 3% NaCl and 2% MgSO4 R2A MEA 3% NaCl and 2% MgSO4 MEA 4303Applied Microbiology and Biotechnology (2022) 106:4297–4314 1 3 Table 3 Identification and capacities/activities of the microorganisms isolated from the five sampling points in the monastery Sample Isolates Morphology Identification CaCO3 solubilization CaCO3 precipitation Ammonium oxidation Denitrification IC6 B_IC6_A Milky, circular MH127765 Pseudomonas vancouverensis 99.84% + - + + + + B_IC6_D Pink, raised, oval LK020736 Methylobacterium populi 100% - - - + B_IC6_H1 Dark yellow, irregular margins MF948937 Pseudomonas sp. 100% - - + + + + B_IC6_L Pale yellow, circular LT718481 Pseudomonas sp. 99.51% + - + + + + B_IC6_N White with yellow margin LR596321 Streptomyces sp. 100% - - - + F_IC6_A1 White, hairy, raised MT606206 Purpureocillium sp. 100% - - - + IC4 B_IC4_E Orange, circular colony MK241856 Bacillus sp. 99.84% + + - - B_IC4_J White, filamentous MT586023 Bacillus mycoides 100% - - + + B_IC4_N Bright yellow, circular colony MG705620 Corynebacterium sp. 100% - + + + + B_IC4_O Large light orange colonies, slime CP054880 Pseudomonas sp. 100% - - + + + + B_IC4_P Milky, slime MH337920 Rhizobium sp. 100% - - + + + + B_IC4_T Pinkish, circular KY992915 Paenibacillus sp. 100% - - - + B_IC4_W Pale cream color, regular margins AY167846 Janibacter limosus 100% + - - + IC9 B_IC9_B Pink, circular MN098864 Sporosarcina aquimarina 100% - + - - B_IC9_C Yellow, circular, raised MK670519 Flavobacterium sp. 99.17% - - - - B_IC9_G White, small colonies MH699287 Streptomyces sp. 100% - - + + + B_IC9_H Milk, opaque, circular MK371085 Pseudomonas sp. 99.84% - - + + + + B_IC9_I Yellow, slime MN098866 Paenarthrobacter nicotinovorans 100% - - + + + + B_IC9_N White with yellow margin LR596321 Streptomyces sp. 100% - - - + B_IC9_S White, slime MH549189 Flavobacterium resistens 100% - - - + + B_IC9_U Yellow-white, lobate margin MT102121 Pseudomonas putida 100% + - + + F_IC9_M White, hairy, raised MT529633 Purpureocillium lilacinum 100% - - - - F_IC9_R White, irregular MK450691 Penicillium decumbens 100% - - - - IC3 B_IC3_C Yellow, circular, raised MK670519 Flavobacterium sp. 99.17% - - - - B_IC3_D Pink, raised, oval LK020736 Methylobacterium populi 100% - - - + B_IC3_E Orange, circular colony MK241856 Bacillus sp. 99.84% + + - - B_IC3_H Milk, opaque, circular MK371085 Pseudomonas sp. 99.84% - - + + + + B_IC3_H1 Dark yellow, irregular margins MF948937 Pseudomonas sp. 100% - - + + + + B_IC3_I Yellow, slime MN098866 Paenarthrobacter nicotinovorans 100% - - + + + + B_IC3_L White, irregular margins KJ816785 Brevibacterium linens 100% + + + - + B_IC3_O Big light orange colonies, slime CP054880 Pseudomonas sp. 100% - - + + + + B_IC3_S White, slime MH549189 Flavobacterium resistens 100% - - - + + B_IC3_S1 Pale yellow, irregular margins JQ291594 Microbacterium pumilum 100% + + - + + B_IC3_V Glossy white, irregularly shape MN758847 Rothia endophytica 100% - + - + F_IC3_A3 Green–brown, hairy KT832076 Aspergillus medius 97.56% - - - - F_IC3_A4 Green–brown, hairy MT582752 Aspergillus pseudoglaucus 100% - - - - 4304 Applied Microbiology and Biotechnology (2022) 106:4297–4314 1 3 Several Pseudomonas and Actinomycetes (Corynebacterium sp., Brevibacterium linens, Microbacterium pumilum) isolates exhibited CaCO3 solubilization and precipitation, ammonium oxidation and denitrification capacities. Isolates belonging to the genus Pseudomonas and the classes Actinomycetes, Bacilli, and Alphaproteobacteria exhibited ammonium oxidation and denitrification capacities. Considering all bacterial isolates, 84% were capable of denitrification, 55% showed a positive reaction in the ammonia oxidation activity test, 26% solubilized CaCO3 and 18% formed CaCO3 crystals in B4 medium. Although several bacteria were isolated in Thiobacillus agar (medium specific for the isolation of sulfate-oxidizing bacteria), all of them displayed a negative reaction when bromocresol green was added as an indicator of sulfate-oxidizing activity. MinION sequencing analysis All seven samples produced positive PCR results when the bacterial 16S rRNA and the fungal ITS fragments were targeted. The archaeal 16S rRNA was successfully amplified from samples IC9 and IC7. No archaeal DNA amplification or poor amplification was obtained in both samples of surrounding soil (ICS1 and ICS2). Amplification of the nirK gene (nitrite reductase/denitrifying bacteria) produced amplicons from samples IC6, IC7, and IC9. The dsr gene (dissimilatory sulfite reductase/sulfate-reducing bacteria) was detected in samples IC7 and IC9. The soxB gene (Sox enzyme system for sulfur oxidation/sulfur-oxidizing bacteria) was detected in samples IC6, IC7, and IC9 (Supplementary material TablesS1 and S2). The amoA gene (ammonia monooxygenase/ammonia-oxidizing bacteria) only produced results with the soil samples ICS1 and ICS2, and therefore these amplicons were not included in the MinION analysis. For characterization of the taxa, different minimum thresholds of relative abundance were established for amplicons for purposes of visualization. The reason for this was the different numbers of reads obtained for the different amplicon groups and the limited number of pre-defined reference sequences in the reference database utilized in EPI2ME Desktop Agent (ONT). After sequencing each run for 48h, a total of 3,537,353 reads (first sequencing run, Supplementary TableS1) and 9,480,778 reads (second sequencing run, Supplementary TableS2) were obtained, with a total yield of 3.8 and 12.2 Gbases, respectively. The average quality scores were 10.15 and 8.78. The average sequence lengths were 984 and 1063 bases. Bacterial 16S rRNA sequencing evaluation The results of the bacterial community analysis, performed by using long amplicons (about 1400bp) that encoded the 16S rRNA gene, of the five samples from the monastery building (IC6, IC4, IC9, IC3, IC7) and the two samples from the surrounding soil (ICS1, ICS2) are summarized in Figs.3A and B, respectively. Pseudomonas spp. predominated in the bacterial communities from the interior environment especially in samples IC3, IC4, and IC6 (53.80%, 12.11%, and 96.83%, respectively, Fig.3A). The second most abundant genus was Aliifodinibius, which dominated in sample IC9 (56.81%). This genus also occurred in sample IC4, but to a lesser extent (2.33%). The bacterial consortia in sample IC9 also included bacteria identified as Salinisphaera (24.42%). Halomonas, the 4th most abundant taxa detected, was mainly present in samples IC3 and IC4 (3.42% and 17.03%, respectively). The composition of the bacterial consortia in samples IC3 and IC4 were qualitatively similar. Other genera present in IC3 and IC4 included Cellvibrio (15.32% and 2.71%, respectively), Paenibacillus (2.96% and 12.43%), Bacillus (3.37% and 0.54%), Brevundimonas (1.55% and 1.79%), Stenotrophomonas (0.81% and 1.42%), Sphingobacterium (0.64% and 0.69%), and Raoultella (0.79% and 0.26%). The presence of these taxa was negligible in the other samples. Arthrobacter (5.92%) and Pseudonocardia (3.88%) were also detected in IC4. -: no reaction displayed; + : positive reaction; + + : extensive and/or rapid positive reaction Table 3 (continued) Sample Isolates Morphology Identification CaCO3 solubilization CaCO3 precipitation Ammonium oxidation Denitrification IC7 B_IC7_A Milky, circular MH127765 Pseudomonas vancouverensis 99.84% + - + + + + B_IC7_A2 White-orange, flat AY996839 Nocardia sp. 100% + - + - B_IC7_C Yellow, circular, raised MK670519 Flavobacterium sp. 99.17% - - - - B_IC7_F Pink, slime MT065733 Ensifer adhaerens 100% - - + + + B_IC7_H Milky, opaque, circular MK371085 Pseudomonas sp. 99.84% - - + + + + B_IC7_K Orange, slime MH698769 Arthrobacter sp. 100% - - + + + B_IC7_P Milky, slime MH337920 Rhizobium sp. 100% - - + + + + 4305Applied Microbiology and Biotechnology (2022) 106:4297–4314 1 3 Bartossek R, Nicol GW, Lanzen A, Klenk HP, Schleper C (2010) Homologues of nitrite reductases in ammonia−oxidizing archaea: diversity and genomic context. 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