Visualization of Paracoccus denitrificans on various types of stones used in European built heritage

Visualization of Paracoccus denitrificans on various types of stones used in European built heritage | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article Visualization of Paracoccus denitrificans on various types of stones used in European built heritage Selen Ezgi Çelik, Jafar Qajar, Laurenz Schröer, Veerle Cnudde This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-6836573/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted 7 You are reading this latest preprint version Abstract Microorganisms are increasingly recognized for their dual role in the deterioration and conservation of cultural heritage, with Paracoccus denitrificans emerging as a promising candidate for bio-based stone stabilization. This study investigates the biofilm formation of P. denitrificans on stone surfaces, with a focus on five sedimentary rocks -Euville, Savonnières, Bentheimer, Vosges, and Maastricht - selected for their varied porosity, composition, colour and importance for cultural heritage. The samples were inoculated under different nutrient-to-medium ratios to evaluate the impact of inoculation conditions on bacteria-stone interactions. A multi-scale imaging approach using SEM, µ-CT, CLSM, digital microscopy, and colour spectrophotometry provided complementary insights into bacterial distribution, EPS production, biofilm morphology, and mineral deposition. Depending on the stone type, P. denitrificans formed distinct biofilm architectures, including spider web-like networks, spherical aggregates, or uniform surface coatings. Moreover, clear evidence of bacterially induced mineral crystallization was observed. Results reveal that both stone type and medium composition significantly influence biofilm development and mineralization behavior. This integrative methodology demonstrates the potential of P. denitrificans in stone conservation and offers a novel framework for advancing bio-conservation strategies in cultural heritage science. Paracoccus denitrificans biofilm conservation MICP imaging techniques (SEM µ-CT CLSM) cultural heritage Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 1. Introduction The relationship between microorganisms and built cultural heritage has been extensively studied for many years, primarily focusing on their role in biodeterioration [ 1 – 6 ]. In literature, microorganisms such as bacteria, fungi, algae, and primitive plants have been highlighted for their predominantly detrimental effects on stone surfaces, leading to research aimed at developing anti-microbial treatments [ 7 – 12 ]. To this end, products such as biocides, protective coatings, and chemical inhibitors have been developed. However, in recent years, observations of unexpectedly well-preserved structures, particularly in rural areas, have revealed that certain microorganisms and primitive plants may contribute to the protection of these structures rather than their degradation [ 13 , 14 ]. This has led to a new research approach focusing on understanding how biological interactions enhance specific stone properties. To address this, research is being conducted on the structure of biological coverage and the dynamic interactions between the coverage and stone surfaces [ 15 ]. These studies aim to reveal the mutual effects of such interactions on stone preservation and alteration. Specifically, they have demonstrated that biological coverage can fill stone pores with biomass or microbial by-products, or form superficial mineral layers that enhance specific physical properties [ 15 ]. For instance, in areas covered by vegetation (“green walls”), layers composed of lichens may serve as thermal insulators, effectively reducing fluctuations in surface temperature Furthermore, lab experiments revealed that cyanobacterial biofilms affect the water-stone relationship by inducing e.g. near hydrophobic conditions or slightly reducing the drying rate, which could affect other forms of weathering [ 16 ]. On the other hand, more complex mechanisms have been observed, particularly involving certain bacteria capable of inducing microbially induced calcium carbonate precipitation (MICP), a process that promotes calcite deposition within the pores and on the surface of stones. This potentially influences the structural strength of the stone [ 17 , 18 ]. Given all the observations described above, the artificial cultivation of microorganisms on stone surfaces to achieve overall bio-protection has emerged as a viable approach [ 19 , 20 ] Despite its potential, the literature remains limited, with existing studies predominantly focusing on surface strengthening, porosity reduction, and thermal resistance improvements. In terms of microbial diversity, the selection of bacteria hinges primarily on their carbonate yield. Strains such as Bacillus cereus , Bacillus subtilis , Myxococcus xanthus and Sporosarcina pasteurii , which are commonly isolated from carbonate-rich environments like calcareous stones, sludge, and soil, have been extensively studied [ 20 – 26 ]. For example, B. cohnii and B. sphaericus achieve high precipitation rates via ureolysis (the breakdown of urea into ammonia and carbon dioxide). However, the ammonia byproducts of ureolytic pathways pose significant environmental risks. In contrast, denitrification-based MICP, as demonstrated by Diaphorobacter nitroreducens and Pseudomonas aeruginosa (yielding 14.1–18.9 g CaCO₃/g NO₃-N in two days) offers an environmentally friendly alternative [ 22 ]. Given the toxicity concerns associated with ammonia, denitrification-based strains represent a promising avenue for sustainable biomineralization [ 23 ]. Similarly, P. denitrificans , an Alphaproteobacterium, is distinguished by its significant role in the environmental nitrogen cycle [ 22 ]. These facultative anaerobes, characterized by their 0.8–1.2 µm size and coccoid to rod-shaped morphology, are highly adaptable to diverse environmental conditions. P. denitrificans produces CO₂ aerobically and N₂ anaerobically via denitrification. The bacterium's ability to induce MICP in the presence of calcium sources, leading to calcium carbonate formation (Fig. 1 ), and its potential for exopolysaccharide (EPS) synthesis under sufficient carbon availability, which could further stabilize stone surfaces, makes it a compelling candidate for stone conservation. Notably, among denitrifying bacteria, P. denitrificans emerges as a superior choice due to its high denitrification efficiency, environmental safety, and adaptability. Unlike Pseudomonas aeruginosa and Diaphorobacter nitroreducens , which may generate harmful intermediate byproducts like NO and N₂O, P. denitrificans efficiently converts nitrate (NO₃⁻) to nitrogen gas (N₂), minimizing toxic emissions [ 24 ]. Furthermore, it achieves high CaCO₃ precipitation (9.5 g/l in 48 hours) yields even under low-oxygen conditions, promoting stable and durable biofilm formation [ 25 ]. In contrast to ureolytic bacteria such as Sporosarcina pasteurii and Bacillus sphaericus , which produce toxic ammonia, P. denitrificans employs denitrification, offering a more sustainable approach. P. denitrificans influence stone resilience through two interrelated biochemical processes: extracellular polymeric substance (EPS) production and biologically induced mineralization via denitrification. Methanol (CH₄O), supplied as the carbon and energy source, is oxidized by P. denitrificans , while nitrate (NO₃⁻) serves as the terminal electron acceptor in denitrification. This metabolic activity results in the reduction of nitrate to nitrogen gas (N₂), accompanied by local pH elevation due to proton consumption. The increased alkalinity, in the presence of calcium ions (Ca²⁺), promotes the precipitation of calcium carbonate (CaCO₃), which enhances the consolidation of the stone surface. Concurrently, P. denitrificans produce EPS that facilitates bacterial adhesion, biofilm formation, and the filling of surface pores, thereby influencing the cohesion and stability of the substrate. The EPS matrix may also act as a nucleation platform for calcium carbonate crystals, effectively coupling microbial colonization with mineral precipitation. Notably, the extent and morphology of biofilm and mineralization are modulated by the physicochemical properties of the stone, such as porosity and composition. For instance, in highly porous stones like Bentheimer, enhanced EPS production may support ion retention and localized supersaturation, favoring calcite nucleation [ 27 ]. This integrated mechanism demonstrates how microbial metabolism dynamically interacts with material properties to achieve both structural stabilization and increased water resistance in stone conservation applications. One of the key challenges in understanding the power of bioprotection of stone, is the structural and chemical heterogeneity of stone, which can result in inconsistent bacterial colonization and biofilm formation. Moreover, liquid transport and nutrient availability can significantly influence bacterial activity and crystal formation efficiency. To address these challenges, P. denitrificans was inoculated on five structural and/or chemically different types of building stone commonly used in historic structures across the Netherlands, Belgium, Germany, and France. We optimized bacterial inoculation conditions to promote biofilm development and investigated the host structure-microorganism interaction, using advanced imaging techniques, including Scanning Electron Microscopy (SEM), micro-computed tomography (micro-CT), Confocal Scanning Laser Microscopy (CSLM), digital microscopy and spectrophotometry. Here, we specifically focus on the distribution of P. denitrificans and the resulting biofilm formation on the different stones, employing complementary imaging techniques to comprehensively characterize bacterial distribution and biofilm structure across scales ranging from micrometers to centimeters. This study provides new insights into the interaction of P. denitrificans on different types of sedimentary rocks by enhancing visualization methodologies. This is the first step for future research on the bio-protection potential of P. denitrificans in cultural heritage conservation. 2. Materials and Methods 2.1. Materials 2.1.1 Stones In this study, five natural stones were selected for their diverse mineralogical properties, including variations in chemical composition, colour, and porosity. The selection includes three limestones- Euville [ 27 ], Savonnières [ 28 ], and Maastricht [ 29 ] -and two sandstones -Bentheimer [ 30 ] and Vosges [31]. These stones originate from historically and geologically significant sites in Western Europe: Bentheimer from the Netherlands; Maastricht from the Belgium–Netherlands border; Euville and Savonnières from northeastern France; and Vosges from eastern France (Fig. 2 ). The materials were either sourced directly from quarries or obtained through specialized suppliers. The preliminary characterization data for the stone samples, including composition and porosity, were compiled from published literature and are summarized in Table 1 . The stones were cut to dimensions of 1x1x0.5 cm to meet the sampling criteria of different types of instruments. Before bacterial application, all samples were treated with a 15% isopropyl alcohol (IPA) solution for 15 minutes to eliminate any potential surface contaminants and then rinsed with autoclaved dionized water (DI). Table 1 Summary of the lithological properties of the selected stones, including a short description, main compositions, and average open porosity percentages. Name (Country) (Type) Short Description Main Composition Average Open Porosity Bentheimer (Germany) (Sandstone) Fine to medium-grained quartz arenite with fine and homogeneously spread pores; secondary pores due to feldspar dissolution. 93–99% quartz, 1–4% K-feldspar, 0–3% rock fragments 21–24% Vosges (France) (Sandstone) Fine-grained red to pink sandstone with a mix of macropores (> 20 µm) and micropores (< 1 µm). 65% quartz, 25% feldspar, 10% clay < 21% Euville (France) (Limestone) Grainstone with large crinoid fragments, syntaxial overgrowth of calcite, and fragments of echinoderms, brachiopods, corals, and pellets; granular cohesion due to syntaxial cement; heterogeneous porosity. 98% calcite 10–20% Savonnières (France) (Limestone) Oolitic limestone with hollow ooids, shell fragments, pellets, and traces of dolomite. 98–99% calcite 22–41% Maastricht (Netherlands) (Limestone) Well-sorted calcarenite with skeletal components of foraminifera, ostracods, sponges, bryozoans, and brachiopods, all cemented with calcite spar; loosely bonded grains with a grain-supported texture. 95–98% calcite, some glauconite, quartz, opaque minerals, and iron oxide < 53% 2.1.2. Bacterial Solution The P. denitrificans ATCC 19367™ was obtained from LGC Standards GmbH, Germany. The original vial was stored in frozen conditions at − 80˚C in 30% glycerol. The vial revived by spreading defrozen cells on Lysogeny Broth (LB) agar plates (containing 20g/L of LB and 15g/L agar) and incubating aerobically at 30˚C for 2–3 days. The temperature of 30˚C is close to its optimal growth conditions. However, as we want to mimic real conditions as close as possible, we lowered the working temperature to 25˚C. The routinary stock culture of P. denitrificans (used in all assays) was aerobically grown, and maintained in LB broth (containing 20g/L of LB broth mix), at 25˚C, under the shadow, and at 100 RPM shaking for 3 days. For bacterial growth validation; initially, a portion of P. denitrificans colonies grown on an agar plate were transferred into 100 mL of LB solution. The bacteria were cultured until an Optical Density (OD600) (Shimadzu UV-1800) value of 0.790 was achieved, and subsequent daily OD measurements were conducted to confirm the sustained viability of the culture. The raw genetic sequences were deposited in the database GenBank under the following accession number:Y16930 (Paracoccus denitrificans 16S rRNA gene, strain ATCC 19367). 2.1.3. Medium In this study, a modified M9 minimal medium, proposed in [ 23 ], was prepared to support bacterial activity while providing essential nutrients and maintaining a controlled chemical environment. The medium was composed of phosphate buffers, including 8.5 g/L Na₂HPO₄·7H₂O and 3 g/L KH₂PO₄, which stabilize pH and provide a phosphorus source; these components were sterilized separately to prevent precipitation [ 23 ]. The basal salt solution contained 0.5 g/L NaCl, 0.24 g/L MgSO₄, and 0.011 g/L CaCl₂, supplying essential ions for osmotic balance, enzymatic function, and biofilm formation. As the carbon source, 4 g/L methanol (CH₄O), a widely used electron donor, was included to support microbial growth and facilitate nitrate reduction. Additionally, 0.72 g/L potassium nitrate (KNO₃) served as the electron acceptor, enabling nitrate respiration under anoxic conditions. 2.2. Method 2.2.1 Experimental Setup In order to understand the effect of mixing ratio of P. denitrificans concentrations in LB and methanol media, the following mixtures were prepared: 100% P. denitrificans in LB medium (10:0), 50% P. denitrificans in LB and 50% methanol medium (5:5), and 10% P. denitrificans in LB and 90% methanol medium (1:9). 2.2.1.1 Glass Slides To investigate the interaction between P. denitrificans and the LB-methanol medium independently of stone surface properties, initial experiments were conducted. For this purpose, 10 µL of the prepared solutions were dispensed onto glass slides. 2.2.1.2 Stones Three replicates of each stone sample were inoculated into each mixture, resulting in a total of 15 test tubes. The stone samples were incubated at 25°C with shaking at 100 rpm for one week. To evaluate the influence of nutrient composition on bacterial colonization and mineralization, stone samples were inoculated with P. denitrificans under three different LB : methanol medium ratios: 10:0, 5:5, and 1:9 (v/v). For each condition, three replicate samples were prepared for five stone types (Euville, Bentheimer, Savonnières, Vosges, and Maastricht), labeled accordingly (e.g., E1–E9, B1–B9). Samples incubated in 10:0 LB : methanol were analyzed using digital microscopy, followed by rinsing with PBS and subjected to colour spectrometry analysis, SEM-EDS, and further digital imaging. Those treated with the 5:5 ratio were rinsed with PBS and stained with Nile Red for biofilm visualization, followed by confocal laser scanning microscopy and digital imaging. The 1:9 LB : methanol samples underwent PBS rinsing. For B9, specific staining (Isolugol) applied, and were analyzed via micro-computed tomography (µ-CT) to assess internal colonization and mineral infilling. 2.2.2 Visualization Methods 2.2.2.1 Digital Microscopy for Biofilm Observation Digital microscopy was employed as an initial, non-destructive method to assess biofilm formation and distribution on stone surfaces. Using a Keyence VHX-7000 digital microscope at 2000x magnification, high-resolution images were acquired to visualize the biofilm structures produced by P. denitrificans . This technique provided preliminary characterization of biofilm morphology and spatial distribution, establishing a basis for subsequent, higher-resolution analysis on glass slides and on stone surfaces. 2.2.2.2 SEM/EDS Analysis Zeiss EVO 15 environmental SEM (equipped with Peltier cooling, EDS, and automated mineralogy) was employed for surface topography and elemental composition analyses. Samples were sputter-coated with Pt to enhance conductivity. SEM/EDS is a fundamental tool for investigating biofilm-stone interactions and microbially-induced alterations, offering detailed information on surface morphology and elemental distribution. 2.2.2.3 Confocal Laser Scanning Microscopy (CLSM) The experimental workflow was designed to assess biofilm formation and surface colonization on both stone and glass substrates. For evaluation of biofilm on stone surfaces, after the incubation period was completed, the samples were stained with Nile Red solution [ 28 ] to visualize biofilm and lipid content. The staining procedure involved immersing the samples in the Nile Red solution and incubating them for 10 minutes in the dark at room temperature. Following incubation, samples were gently washed with PBS to remove excess dye. The confocal microscopy analysis was then conducted to determine confocal laser scanning microscopy (Nikon A1, Nikon Eclipse 90i microscope body). To conduct visualization tests on glass surfaces, 10 µL of the prepared microbial suspension was pipetted onto glass slides and they were left to dry under controlled conditions at 25°C for one month. The resulting formations were analyzed using both a digital microscope and CLSM to capture structural and morphological details. 2.2.2.4 Micro CT Based on observations from SEM and digital microscope images, the best-selected sample (Bentheimer 1:9) with dimensions of 1x1x0.5 cm was scanned using the CoreTOM micro-CT scanner (TESCAN XRE) at the Centre for X-ray Tomography (UGCT) at Ghent University. The sample was scanned twice—first in PBS and then after adding Lugol’s iodine, which enhances the visualization of biofilms by increasing contrast and hydration of the EPS matrix [ 29 ]. The scans were performed at a voxel size of 13.5 µm using 100 kV and 15 W. The exposure time was 1150 ms and 2142 projections were taken resulting in a scan time of about 45 minutes. A 1 mm Al filter was used to reduce beam hardening. The scans were reconstructed using Panthera 1.5.3. (TESCAN XRE) where beam hardening and ring filtering was applied. Acquisition and reconstruction parameters were maintained constant for both scans to enable direct comparison across different time points. All scans are available in Utrecht University’s YODA repository (see Data Availability section). Further volume analysis was conducted using Avizo 2023.2 software (Thermo Fisher Scientific) to assess structural characteristics and material integrity. 2.2.2.5 Colourimetric Evaluation In order to determine whether P. denitrificans application had any effect on the aesthetic properties of the stone and to assess potential colour changes, colourimetric values were measured using the CIELAB colour space, introduced in 1976 by the International Commission on Illumination (CIE) (UNI EN 15886, 2010). The colour difference (ΔE*) was calculated using the following equation: ΔE* = √(ΔL*² + Δa*² + Δb*²) where ΔL*, Δa*, and Δb* represent the differences in each chromatic coordinate between the measured samples and the reference. This parameter is significant for aesthetic considerations, as any treatment should not result in a ΔE* greater than 5 to preserve the original appearance of the surface. For colourimetric analyses, a Konica Minolta CM-600d Spectrophotometer was used. Measurements were conducted in SCI mode with an 8° standard observer. The instrument was calibrated using a white reference before each measurement session. Three measurements were taken per sample, and the average value was used for analysis. 3. Results and Discussion 3.1. Biofilm Formation on Glass Slides To rule out abiotic mineral precipitation, we conducted control experiments on glass slides without bacterial inoculation. These controls exhibited distinct crystal morphologies compared to bacterial samples, indicating that bacterial metabolic activity strongly influences mineral precipitation patterns. For all three composition ratios, the presence of bacteria clearly altered the crystallization patterns. In the 10:0 LB-methanol ratio, the broad outer crystallized ring observed in the absence of bacteria became significantly thinner when bacteria were present. Additionally, bacterial cells appeared to align with the crystalline structures formed by salt precipitation, indicating an adaptive interaction between bacteria and the crystal network. At 5:5 LB-methanol (Fig. 3 .d,e,f), a dense outer biofilm layer was prominent, while the inner regions displayed a mixed structure of crystalline formations and network-like bacterial organization. In contrast, at the highest methanol ratio (1:9 LB-methanol), an outer ring composed entirely of salt crystals was observed, with bacteria failing to reach the outermost layer. Instead, an extensive network structure spreading inward was evident. These findings highlight the independent crystallization behavior of the medium in the absence of stone surfaces, suggesting that variations in ionic composition due to stone dissolution could further influence bacterial organization. Additionally, they demonstrate how crystallization patterns on stone surfaces can vary significantly depending on the medium composition. While Fig. 3 presents control experiments without stone surfaces, supplementary data A include microscopic images of glass slides of solutions taken from inoculation of stones with bacteria. Differences on these images highlight the influence of stone chemical composition on bacterial behavior and biofilm formation. 3.2. Biofilm Formation on Stone 3.2.1. Visual Inspection This study investigated the ability of P. denitrificans to form biofilms on five different limestone types (Euville, Bentheimer, Maastricht, Vosges, and Savonnières), as well as the impact of varying volumetric LB : Methanol medium ratios (10:0, 5:5, and 1:9) on biofilm development. Microscopic analyses revealed distinct biofilm formation patterns across different stone types, with notable variations in extracellular polymeric substance (EPS) distribution, microbial aggregation, and mineral precipitation. Figure 6. CLSM microscopy images showing biofilm formation on five different stone types (Euville, Bentheimer, Maastricht, Vosges, and Savonnières) after bacterial treatment with varying LB : Methanol (ml:ml) ratios (10:0, 5:5, and 1:9). The yellow fluorescence highlights bacterial biomass distribution, with variations in coverage and structural organization observed across different stone types and medium compositions. 3.2.1.1. Euville limestone Digital microscopy revealed biofilm filling the pore spaces (Fig. 4 ), while SEM images (Fig. 5 ) showed a homogeneous biofilm layer covering the entire surface. Uniquely among the stone types tested, at a 1:9 LB : Methanol ratio, SEM images revealed bacteria embedded within a highly homogenous biofilm matrix, demonstrating a uniform distribution of both the EPS and the individual bacterial cells. Furthermore, Euville limestone showed evidence of crystal formation. Combined analysis of Fig. 4 and Fig. 6 suggests that these crystals appear to be generated, or 'hatched,' from within the EPS matrix produced by the bacteria. CLSM (Fig. 6) indicated that increasing methanol concentration promoted a more continuous and widespread biofilm, with enhanced EPS accumulation within the pore spaces, which could contribute to improved stone cohesion. This observation was corroborated by digital microscopy. At low methanol concentrations, there was no evidence of network formation. However, at a 1:9 LB : Methanol medium ratio, network formation was clearly visible under the digital microscope. 3.2.1.2. Bentheimer sandstone Digital microscopy highlights biofilm-mediated pore filling, while SEM images reveal a spider web-like biofilm network. This structure forms a continuous covering over the stone, suggesting a blanket-like effect that might impact stone permeability and mechanical stability (Fig. 5 f-g). Additionally, Bentheimer exhibits crystalline precipitates, potentially CaCO₃ formations, which are associated with bacterial-induced mineralization. These crystals appear to nucleate around bacterial clusters, and possibly reinforcing the structural integrity of the stone. Unlike the other stone types, Bentheimer stone exhibits clearly visible filaments even under CLSM (Fig. 6). This serves as additional evidence confirming that the dense structures observed in SEM are biofilm-derived. 3.2.1.3. Maastricht limestone Characterized by its significantly higher porosity compared to the other samples, it does not show distinct biofilm accumulation under digital microscopy. However, SEM images reveal the presence of "bioball" structures (Fig. 5 j-l), which consist of bacterial aggregates encased in an EPS matrix. These formations are widely dispersed across the stone surface and appear to bridge micro-pores (Fig. 5 ) suggesting a potential role in altering stone permeability. Additionally, in addition to the bioball-like biofilm formations, bacteria also tend to accumulate on the smoother surfaces of the stone (Fig. 5 l). CLSM data (Fig. 6) further confirm that not only spherical formations are present but also a surface-covering biomass layer, indicating more extensive biofilm development. 3.2.1.4. Vosges sandstone For this clay rich sandstone, primary observation under SEM is the formation of newly precipitated crystalline structures on the stone surface (Fig. 5 n). These precipitates result from bacterial activity and are distinctly different from the untreated control samples. Additionally, a weakly interconnected network of microbial EPS is present between clay particles (Fig. 5 o), albeit less developed compared to Bentheimer sandstone. There is also bacterial accumulation on the surface as seen on Fig. 5 p. Compared to other stones, Vosges exhibited lower overall bacterial coverage, with confocal microscopy detecting microbial presence primarily in specific surface regions rather than forming a continuous biofilm (Fig. 6). The low fluorescence signals further support that bacteria may have been more dispersed rather than forming structured layers. On the other hand, there is a trend of accumulation on the interface between pore and grains with higher medium : LB ratios for Vosges. 3.2.1.5. Savonnières limestone Unlike the other investigated stone types, no significant biofilm network was detected in SEM for Savonnières. However, bacteria accumulation covering the surface is visible in CLSM images (Fig. 6). Additionally, significant crystal formations within pores under digital microscopy were observed (Fig. 4 ). SEM imaging also confirms extensive mineral precipitation, with well-defined euhedral crystal morphologies, suggesting active biomineralization (Fig. 5 s-t). 3.2.2 Elemental Analysis of Biofilm and Mineralization on Stone Surfaces The EDS spectra provide elemental composition insights into microbial biofilm formation and mineral precipitation across different limestone types. Figures 7 provide the detection of the elemental compositions on treated stones. 3.2.2.1 Euville Limestone EDS spectra from different regions in Euville limestone indicate the presence of oxygen (O), calcium (Ca), and carbon (C) as primary elements, consistent with the composition of limestone. Figure 7 c, shows the stone surface without bacterial accumulation or EPS formation with no signal for nitrogen (N) or phosphorus (P) (which are indicative elements for biologic compounds). On the other hand, presence of P and N suggests microbial activity (Fig. 7 b, d). This circular shape and size (1 µm in diameter) is also a supportive evidence that these structures are P. denitrificans cells. and Fig. 7 a is likely associated with EPS production. Another important aspect of that in Fig. 7 e, crystal formation is visible, and Ca, C and O elements are present in the structure, probably indicating CaCO 3 precipitation. Notably, variations in elemental intensity across different spectra suggest heterogeneity in biofilm development and mineral deposition. 3.2.2.2 Bentheimer Sandstone The EDS spectrum from Bentheimer (f) has dominant peaks for Ca, O, Magnesium (Mg), and Silicone (Si), reflecting the sandstone matrix. However, the presence of sodium (Na) and chlorine (Cl) suggests possible salt precipitation or microbial metabolic byproducts or by the medium. The network-like biofilm observed in SEM images aligns with the EDS results, which suggest localized mineral deposition associated with bacterial structures. 3.2.2.3 Maastricht Limestone The unique bioball bridge formation in the Maastricht limestone is further evidenced by the EDS spectra (Figs. 7 g–j), which demonstrate various structural formations within a single image. Figure 7 g represents the stone matrix, while Fig. 7 h highlights EPS formation. Despite exhibiting similar spectral peaks to Fig. 7 i (which indicates a single bacterium), Fig. 7 h shows a lower nitrogen (N) peak, consistent with previous findings that EPS formations contain less organic content than bacterial cells (Supplementary Data B). In Fig. 7 j, salt crystals are present on the right side of the bridge, exhibiting higher sodium (Na) and chloride (Cl) peaks compared to Fig. 7 i. Although the crystal and bacterial cell appear morphologically similar, the higher Cl peak and lower N peak in Fig. 7 j confirm salt crystallization rather than bacterial biomass. Notably, in this case, salt crystallization acts as a supportive structural element for the EPS rather than contributing to deterioration of the cells or the structure, reinforcing the stability of the biofilm matrix. 3.2.2.4 Vosges Sandstone The EDS results for Vosges (Fig. 7 k–p) indicate significant variations in mineral composition across different regions. The SEM images show crystal-like formations, while there is almost no Si signals and more Ca signals in the crystals (Fig. 7 n) which could be a result of biofilm-induced mineralization and fully covering silicate surface. The detected phosphorus signals further support the hypothesis that bacterial activity is influencing local mineral deposition. 3.2.2.5 Savonnières Limestone EDS spectra from Savonnières (r–s) indicate a mixture of carbonate minerals and biofilm-related elements, with dominant peaks for Ca, O, Mg, and P. The SEM image suggests the presence of both homogenous biofilm structures and mineral deposits, aligning with the observed elemental composition. The relatively high phosphorus content in certain areas suggests microbial influence on mineral formation, possibly through biomineralization pathways (Supplementary Data B). 3.2.3 Micro-CT Analysis Figure 8 shows representative 2D slices (side and top views) extracted from the 3D images of untreated and Lugol-treated Sample B9. Upon visual inspection, three primary phases were initially identified: (1) a liquid phase occupying pore spaces and surrounding the sample, (2) a gas phase present both as residual gas within pores and as bubbles suspended in the liquid phase, and (3) a solid grain phase. However, a closer examination of the Lugol-treated sample (indicated by arrows in Fig. 8 (b)) revealed an additional minor phase covering parts of the sample surface. To enhance visibility and facilitate clear identification, a difference image was generated (an image created by subtracting 8a from 8b, to highlight changes or differences), as illustrated in Fig. 8 (c). In this image, the newly identified phase is highlighted in black (indicated by arrows) and clearly visible on the stone surface after Lugol treatment. We interpret this additional phase as biofilm. Apart from visually inspecting the images, the various phases present in the reconstructed 3D images were analyzed based on their gray-level values (Supplementary Data C). The histogram displays the intensity histogram of the sample image treated with Lugol. As shown, the histograms of gas, liquid, biofilm, and grain phases exhibit clear separation. Each phase exhibits a characteristic range of gray-scale values based on its X-ray attenuation [ 30 ]. Logically, the gas phase appears at the lowest gray-scale range, followed by the liquid phase, which is centered at slightly higher values. The biofilm phase overlaps with the upper range of the liquid phase and extends toward the grain phase, indicating its intermediate density. The grain phase is represented by the highest gray-scale values, reflecting its dense mineral composition. These distributions enable segmentation and phase identification in image-based analysis of pore-scale processes. 3.2.4 Colour Difference Table 2 shows the colour difference (ΔE) across different limestone types under varying LB: Methanol medium ratios (10:0, 5:5, 1:9). The measurements have been taken after rinsing with PBS. According to the results, Maastricht exhibited the highest ΔE values, particularly at 5:5, indicating significant colour variation due to bacterial activity. Bentheimer and Vosges showed a decreasing trend in ΔE as methanol concentration increased. Savonnières displayed a notable drop at 5:5, followed by an increase at 1:9, suggesting a non-linear relationship between biofilm formation and colour variation. Euville consistently exhibited the lowest ΔE values, indicating minimal colour shifts. Overall results show that a colour difference due to the bacterial treatment is under the observable limit (5) for all samples. Table 2 Colour difference (ΔE) values for five stone types (Euville, Bentheimer, Maastricht, Vosges, Savonnières) before and after bacterial treatment with different LB : Methanol (ml:ml) ratios (10:0, 5:5, and 1:9). Colour Difference (ΔE) LB: Methanol (ml:ml) Euville Bentheimer Maastricht Vosges Savonnières 10:0 0.981 2.837 3.110 2.981 3.234 5:5 0.305 1.691 3.761 3.649 1.012 1:9 1.339 1.232 2.513 2.736 2.652 4. Conclusion This study demonstrates that biofilm formation and biomineralization by P. denitrificans are strongly influenced by stone type, surface properties, and medium composition. In particular: Euville and Bentheimer supported extensive biofilm formation and uniform EPS layers, suggesting high potential for structural stabilization. Maastricht showed the formation of discrete bioball-like biofilm structures, which may offer localized improvements in pore sealing. Vosges exhibited lower bacterial coverage and more crystalline precipitates, indicating a more limited interaction. Savonnières had significant mineral precipitation but minimal biofilm network formation. To address potential interactions between stone chemistry and nutrient composition, we note that differences in calcium content, porosity, and mineralogy likely modulate bacterial colonization and biofilm formation. Our observations showed that calcium-rich stones, such as Euville and Savonnières, supported more uniform biofilm coverage and mineral precipitation, while quartz-rich sandstones like Bentheimer displayed more filamentous structures. This suggests that nutrient-stone interactions play a key role in shaping biofilm behavior. While visual observations of biofilm coverage and EPS accumulation suggest enhanced stone cohesion, we acknowledge that direct measurements of mechanical strength (e.g., compressive strength tests or ultrasonic pulse velocity) are necessary to quantitatively validate these findings. Such mechanical testing will be a focus of future research. These results emphasize the importance of tailoring bio-based conservation strategies to specific material characteristics. This work establishes a framework for targeted microbial treatments in stone conservation, guiding future efforts toward optimized and sustainable long-term protection. Declarations Acknowledgement This project was funded by the Dutch Research Council (NWO) through the BugControl project (project number VI.C.202.074) under the NWO Talent program, and by the EXCITE Network, part of the Horizon 2020 research and innovation program of the European Union under grant agreement no. 101005611. Furthermore, we are grateful for the Ghent University Special Research Fund (BOF-UGent) to support the Centre of Expertise UGCT (BOF.COR.2022.0008). Finally, we would also like to thank Alejandra Reyes-Amezaga, Dr. Carolin Rieg, Eric Hellebrand, Mahin Baghery, Leonard Bik, Nadia Breugelmans and Alexander Hoogenboom for their technical support. Data availability The micro-CT reconstructed data are available at the Yoda data repository of Utrecht University and accessible through https://doi.org/10.24416/UU02-SECJ1Q. CRediT SEÇ: Conceptualization, Data curation, Investigation, Methodology, Writing – original draft, Writing – review and editing, Visualization JQ: Data curation, Writing – review and editing, Visualization, Software LS: Data curation, Writing – review and editing VC: Writing – review and editing, Resources, Funding acquisition, Supervision, Project administration References Luo L, Gu J-D. Bridging the link between microbial biofilm and biodeterioration in cultural heritage research. Int Biodeterior Biodegrad. 2025;198:106001. Liu X, et al. Innovative approaches for the processes involved in microbial biodeterioration of cultural heritage materials. Curr Opin Biotechnol. 2022;75:102716. Branysova T, Demnerova K, Durovic M, Stiborova H. Microbial biodeterioration of cultural heritage and identification of the active agents over the last two decades. J Cult Herit. 2022;55:245–60. Beata G. The use of -omics tools for assessing biodeterioration of cultural heritage: A review. J Cult Herit. 2020;45:351–61. Pinheiro AC, et al. Limestone biodeterioration: A review on the Portuguese cultural heritage scenario. J Cult Herit. 2019;36:275–85. Vivar I, Borrego S, Ellis G, Moreno DA, García AM. Fungal biodeterioration of color cinematographic films of the cultural heritage of Cuba. Int Biodeterior Biodegrad. 2013;84:372–80. Zhu C, et al. Application and evaluation of a new blend of biocides for biological control on cultural heritages. Int Biodeterior Biodegrad. 2023;178:105569. Villar-dePablo M, et al. Innovative approaches to accurately assess the effectiveness of biocide-based treatments to fight biodeterioration of Cultural Heritage monuments. Sci Total Environ. 2023;897:165318. Fidanza MR, Caneva G. Natural biocides for the conservation of stone cultural heritage: A review. J Cult Herit. 2019;38:271–86. Becerra J, Mateo M, Ortiz P, Nicolás G, Zaderenko AP. Evaluation of the applicability of nano-biocide treatments on limestones used in cultural heritage. J Cult Herit. 2019;38:126–35. Stupar M, et al. Antifungal activity of selected essential oils and biocide benzalkonium chloride against the fungi isolated from cultural heritage objects. S Afr J Bot. 2014;93:118–24. Schröer L, Boon N, De Kock T, Cnudde V. The capabilities of bacteria and archaea to alter natural building stones – A review. Int Biodeterior Biodegrad. 2021;165:105329. Carter NEA, Viles HA. Bioprotection explored: the story of a little known earth surface process. Geomorphology. 2005;67:273–81. Elert K, et al. Degradation of ancient Maya carved tuff stone at Copan and its bacterial bioconservation. npj Mater Degrad. 2021;5:44. Viles HA, Wood C. Green walls? integrated laboratory and field testing of the effectiveness of soft wall capping in conserving ruins. Geol Soc Lond Spec Publ. 2007;271:309–22. Schröer L, et al. The effects of cyanobacterial biofilms on water transport and retention of natural building stones. Earth Surf Process Landf. 2022;47:1921–36. Ortega-Villamagua E, Gudiño-Gomezjurado M, Palma-Cando A. Microbiologically induced carbonate precipitation in the restoration and conservation of cultural heritage materials. Molecules. 2020;25:5499. Ortega-Morales BO, Gaylarde CC. Bioconservation of historic stone buildings—An updated review. Appl Sci. 2021;11:5695. De Muynck W, De Belie N, Verstraete W. Microbial carbonate precipitation in construction materials: A review. Ecol Eng. 2010;36:118–36. De Muynck W, et al. Influence of pore structure on the effectiveness of a biogenic carbonate surface treatment for limestone conservation. Appl Environ Microbiol. 2011;77:6808–20. Castanier S, Métayer-Levrel L, Orial G, Loubière G, J.-F., Perthuisot J-P. Bacterial carbonatogenesis and applications to preservation and restoration of historic property. In: Ciferri O, Tiano P, Mastromei G, editors. Microbes and Art: The Role of Microbial Communities in the Degradation and Protection of Cultural Heritage. Springer US; 2000. pp. 203–18. De Belie N. Application of bacteria in concrete: a critical evaluation of the current status. RILEM Tech Lett. 2016;1:56–61. Erşan YÇ, De Belie N, Boon N. Microbially induced CaCO₃ precipitation through denitrification: an optimization study in minimal nutrient environment. Biochem Eng J. 2015;101:108–18. Bergaust L, Mao Y, Bakken LR, Frostegård Å. Denitrification response patterns during the transition to anoxic respiration and posttranscriptional effects of suboptimal pH on nitrogen oxide reductase in Paracoccus denitrificans . Appl Environ Microbiol. 2010;76:6387–96. Trends. opportunities for greener and more efficient microbially induced calcite precipitation pathways: a strategic review. Geotech Res. 2024;11:161–85. Lin W, et al. Microbially induced desaturation and carbonate precipitation through denitrification: a review. Appl Sci. 2021;11:7842. Rodriguez-Navarro C, et al. Influence of substrate mineralogy on bacterial mineralization of calcium carbonate: implications for stone conservation. Appl Environ Microbiol. 2012;78:4017–29. Bordel S, van Spanning RJM, Santos-Beneit F. Imaging and modelling of poly(3-hydroxybutyrate) synthesis in Paracoccus denitrificans . AMB Express. 2021;11:113. Schröer L, et al. 3D visualization of cyanobacterial biofilms using micro-computed tomography with contrast-enhancing staining agents. Tomogr Mater Struct. 2024;4:100024. Cnudde V, Boone MN. High-resolution X-ray computed tomography in geosciences: a review of the current technology and applications. Earth-Sci Rev. 2013;123:1–17. Additional Declarations No competing interests reported. Supplementary Files VisualizationSupData.docx Cite Share Download PDF Status: Under Review Version 1 posted Editorial decision: Revision requested 11 Oct, 2025 Reviews received at journal 10 Jun, 2025 Reviewers agreed at journal 10 Jun, 2025 Reviewers invited by journal 10 Jun, 2025 Editor assigned by journal 09 Jun, 2025 Submission checks completed at journal 09 Jun, 2025 First submitted to journal 06 Jun, 2025 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-6836573","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":469704943,"identity":"b57e08b3-4747-4b27-8269-ec73be617ff3","order_by":0,"name":"Selen Ezgi Çelik","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAArUlEQVRIiWNgGAWjYPACCx4J9gYwg2gtEjwSPAfADOK1MEhIJBCpRb6B+QDDjwoJGcmZb8wkGHcQocXgAFsCY88ZCR5p6RygljPEaGHgMWBmbJPgkQNraSPKYfwfmBn/AbVIniFSC8MBHgZmxgagwyR4iNRicJjN4GDPMQkeyZ60YotEohzW3vzwwY8aG3uJ44c33vjYZkOEw5iBboNzEojQMApGwSgYBaOACAAAzO8oVgVGxOkAAAAASUVORK5CYII=","orcid":"","institution":"Utrecht University","correspondingAuthor":true,"prefix":"","firstName":"Selen","middleName":"Ezgi","lastName":"Çelik","suffix":""},{"id":469704944,"identity":"1fcd2fde-1f3c-4df9-83ef-14b6e5f42820","order_by":1,"name":"Jafar Qajar","email":"","orcid":"","institution":"Utrecht University","correspondingAuthor":false,"prefix":"","firstName":"Jafar","middleName":"","lastName":"Qajar","suffix":""},{"id":469704945,"identity":"7728557d-a25d-4509-b2ff-7d6ab2257c62","order_by":2,"name":"Laurenz Schröer","email":"","orcid":"","institution":"Ghent University","correspondingAuthor":false,"prefix":"","firstName":"Laurenz","middleName":"","lastName":"Schröer","suffix":""},{"id":469704946,"identity":"5e614bb2-8180-49a1-97d2-5a3995b990c1","order_by":3,"name":"Veerle Cnudde","email":"","orcid":"","institution":"Utrecht University","correspondingAuthor":false,"prefix":"","firstName":"Veerle","middleName":"","lastName":"Cnudde","suffix":""}],"badges":[],"createdAt":"2025-06-06 11:23:24","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-6836573/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-6836573/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":84664799,"identity":"8744b118-b4d8-4247-9953-8709d8b85da1","added_by":"auto","created_at":"2025-06-16 05:31:35","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":46728,"visible":true,"origin":"","legend":"\u003cp\u003eThe complete reactions of \u003cem\u003eP. denitrificans\u003c/em\u003e illustrating gas production, denitrification and calcite precipitation (modified from [26]).\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-6836573/v1/4e67ed921fb4cc61339a7216.png"},{"id":84664795,"identity":"c7c9ab0f-1fca-49e3-829c-473ea91d114c","added_by":"auto","created_at":"2025-06-16 05:31:35","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":155175,"visible":true,"origin":"","legend":"\u003cp\u003eGeographic locations of the selected limestone types (Bentheimer, Euville, Maastricht, Savonnières, and Vosges) used in this study. The map indicates the origin of each stone type, marked with color-coded points. The inset images show the prepared samples (1x1x0.5 cm) before experimentation, highlighting differences in color and texture among the stone types.\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-6836573/v1/7aaa8b581579601edeb1fcd5.png"},{"id":84664801,"identity":"93295712-13d5-42ad-89ae-c3ba93d64d86","added_by":"auto","created_at":"2025-06-16 05:31:36","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":373132,"visible":true,"origin":"","legend":"\u003cp\u003eDigital and confocal microscopy images of\u003cstrong\u003e droplets\u003c/strong\u003efrom different LB: methanol-M9 medium compositions (a,b,c: 10:0, d,e,f: 5:5 and g,h,i: 1:9), without (a, d, g) and with \u003cem\u003eP.\u003c/em\u003e \u003cem\u003edenitrificans \u003c/em\u003e(b–h). (c-i): Images of the same droplet as in respectively (b-h) but stained with Nile Red, to highlight bacteria regions in red.\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-6836573/v1/e1efd974fe206c0236b87103.png"},{"id":84666623,"identity":"f9a430a7-8013-4877-beae-9d943c6dd18d","added_by":"auto","created_at":"2025-06-16 05:47:36","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":522610,"visible":true,"origin":"","legend":"\u003cp\u003eMicroscopic images of five different limestone types (Euville, Bentheimer, Maastricht, Vosges, Savonnières) untreated and treated with \u003cem\u003eP. denitrificans\u003c/em\u003e in LB : methanol medium (10:0, 5:5 and 1:9).\u003c/p\u003e","description":"","filename":"4.png","url":"https://assets-eu.researchsquare.com/files/rs-6836573/v1/93e2eae6d161bbfb3f57710a.png"},{"id":84664818,"identity":"04f16b2f-7b7b-49cb-9f15-b7625e0b9e61","added_by":"auto","created_at":"2025-06-16 05:31:36","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":321626,"visible":true,"origin":"","legend":"\u003cp\u003eSEM images of five stone types (Euville, Bentheimer, Maastricht, Vosges, and Savonnières) without (left) and with treatment (\u003cem\u003eP. denitrificans\u003c/em\u003e, LB:Methanol 1:9) (right). Images show bacterial colonization, biofilm formation, and mineral precipitation.\u003c/p\u003e","description":"","filename":"5.png","url":"https://assets-eu.researchsquare.com/files/rs-6836573/v1/a68ee7c21bc556a16ed4a980.png"},{"id":84664800,"identity":"7a233b30-b5ce-499c-91fc-282ae6689683","added_by":"auto","created_at":"2025-06-16 05:31:35","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":546471,"visible":true,"origin":"","legend":"\u003cp\u003eCLSM microscopy images showing biofilm formation on five different stone types (Euville, Bentheimer, Maastricht, Vosges, and Savonnières) after bacterial treatment with varying LB : Methanol (ml:ml) ratios (10:0, 5:5, and 1:9). The yellow fluorescence highlights bacterial biomass distribution, with variations in coverage and structural organization observed across different stone types and medium compositions.\u003c/p\u003e","description":"","filename":"6.png","url":"https://assets-eu.researchsquare.com/files/rs-6836573/v1/4c000f3be3a72467818e7513.png"},{"id":84664805,"identity":"f4a22e50-e90e-4d81-8b46-6e88fdae754c","added_by":"auto","created_at":"2025-06-16 05:31:36","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":292404,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eSEM images indicating where EDS data of treated stones with 1:9 LB : methanol medium (ml:ml) ratio was determined. A table is give the most important elements detected on the spots indicated in each figure. (üü: Elements with higher signal in the spectra, ü: Elements with lower signal in the spectra, - : No signal)\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"7.png","url":"https://assets-eu.researchsquare.com/files/rs-6836573/v1/854e1ecb11deebc6add4f8f5.png"},{"id":84664832,"identity":"9a6ccb08-f4ba-42ed-83ee-4a5bcb9bd13d","added_by":"auto","created_at":"2025-06-16 05:31:37","extension":"png","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":282370,"visible":true,"origin":"","legend":"\u003cp\u003e(a,d) Side and top view slices of the µ-CT image of Sample B9 immersed in PBS, (b,e) the corresponding registered slices of the sample image when Lugol was added to the liquid phase, and (c,f) difference image between the untreated and treated sample with Lugol.\u003c/p\u003e","description":"","filename":"8.png","url":"https://assets-eu.researchsquare.com/files/rs-6836573/v1/19313141e72406bf4437e668.png"},{"id":84667311,"identity":"52fbd4f4-a630-4c7f-a1a3-e99ee659228e","added_by":"auto","created_at":"2025-06-16 05:55:37","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":3905414,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6836573/v1/bf0f121a-8320-4b7e-b330-ff33593b0536.pdf"},{"id":84664803,"identity":"35bbba46-322a-47fa-9068-3518133f3884","added_by":"auto","created_at":"2025-06-16 05:31:36","extension":"docx","order_by":0,"title":"","display":"","copyAsset":false,"role":"supplement","size":2123312,"visible":true,"origin":"","legend":"","description":"","filename":"VisualizationSupData.docx","url":"https://assets-eu.researchsquare.com/files/rs-6836573/v1/3a8e25bd58aa98ed68a9c618.docx"}],"financialInterests":"No competing interests reported.","formattedTitle":"Visualization of Paracoccus denitrificans on various types of stones used in European built heritage","fulltext":[{"header":"1. Introduction","content":"\u003cp\u003eThe relationship between microorganisms and built cultural heritage has been extensively studied for many years, primarily focusing on their role in biodeterioration [\u003cspan additionalcitationids=\"CR2 CR3 CR4 CR5\" citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]. In literature, microorganisms such as bacteria, fungi, algae, and primitive plants have been highlighted for their predominantly detrimental effects on stone surfaces, leading to research aimed at developing anti-microbial treatments [\u003cspan additionalcitationids=\"CR8 CR9 CR10 CR11\" citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]. To this end, products such as biocides, protective coatings, and chemical inhibitors have been developed.\u003c/p\u003e \u003cp\u003eHowever, in recent years, observations of unexpectedly well-preserved structures, particularly in rural areas, have revealed that certain microorganisms and primitive plants may contribute to the protection of these structures rather than their degradation [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e, \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]. This has led to a new research approach focusing on understanding how biological interactions enhance specific stone properties. To address this, research is being conducted on the structure of biological coverage and the dynamic interactions between the coverage and stone surfaces [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]. These studies aim to reveal the mutual effects of such interactions on stone preservation and alteration. Specifically, they have demonstrated that biological coverage can fill stone pores with biomass or microbial by-products, or form superficial mineral layers that enhance specific physical properties [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]. For instance, in areas covered by vegetation (\u0026ldquo;green walls\u0026rdquo;), layers composed of lichens may serve as thermal insulators, effectively reducing fluctuations in surface temperature Furthermore, lab experiments revealed that cyanobacterial biofilms affect the water-stone relationship by inducing e.g. near hydrophobic conditions or slightly reducing the drying rate, which could affect other forms of weathering [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]. On the other hand, more complex mechanisms have been observed, particularly involving certain bacteria capable of inducing microbially induced calcium carbonate precipitation (MICP), a process that promotes calcite deposition within the pores and on the surface of stones. This potentially influences the structural strength of the stone [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e, \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eGiven all the observations described above, the artificial cultivation of microorganisms on stone surfaces to achieve overall bio-protection has emerged as a viable approach [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e, \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e] Despite its potential, the literature remains limited, with existing studies predominantly focusing on surface strengthening, porosity reduction, and thermal resistance improvements. In terms of microbial diversity, the selection of bacteria hinges primarily on their carbonate yield. Strains such as \u003cem\u003eBacillus cereus\u003c/em\u003e, \u003cem\u003eBacillus subtilis\u003c/em\u003e, \u003cem\u003eMyxococcus xanthus\u003c/em\u003e and \u003cem\u003eSporosarcina pasteurii\u003c/em\u003e, which are commonly isolated from carbonate-rich environments like calcareous stones, sludge, and soil, have been extensively studied [\u003cspan additionalcitationids=\"CR21 CR22 CR23 CR24 CR25\" citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e]. For example, \u003cem\u003eB. cohnii\u003c/em\u003e and \u003cem\u003eB. sphaericus\u003c/em\u003e achieve high precipitation rates via ureolysis (the breakdown of urea into ammonia and carbon dioxide). However, the ammonia byproducts of ureolytic pathways pose significant environmental risks. In contrast, denitrification-based MICP, as demonstrated by \u003cem\u003eDiaphorobacter nitroreducens\u003c/em\u003e and \u003cem\u003ePseudomonas aeruginosa\u003c/em\u003e (yielding 14.1\u0026ndash;18.9 g CaCO₃/g NO₃-N in two days) offers an environmentally friendly alternative [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]. Given the toxicity concerns associated with ammonia, denitrification-based strains represent a promising avenue for sustainable biomineralization [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e]. Similarly, \u003cem\u003eP. denitrificans\u003c/em\u003e, an Alphaproteobacterium, is distinguished by its significant role in the environmental nitrogen cycle [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]. These facultative anaerobes, characterized by their 0.8\u0026ndash;1.2 \u0026micro;m size and coccoid to rod-shaped morphology, are highly adaptable to diverse environmental conditions. \u003cem\u003eP. denitrificans\u003c/em\u003e produces CO₂ aerobically and N₂ anaerobically via denitrification. The bacterium's ability to induce MICP in the presence of calcium sources, leading to calcium carbonate formation (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e), and its potential for exopolysaccharide (EPS) synthesis under sufficient carbon availability, which could further stabilize stone surfaces, makes it a compelling candidate for stone conservation. Notably, among denitrifying bacteria, \u003cem\u003eP. denitrificans\u003c/em\u003e emerges as a superior choice due to its high denitrification efficiency, environmental safety, and adaptability. Unlike \u003cem\u003ePseudomonas aeruginosa\u003c/em\u003e and \u003cem\u003eDiaphorobacter nitroreducens\u003c/em\u003e, which may generate harmful intermediate byproducts like NO and N₂O, \u003cem\u003eP. denitrificans\u003c/em\u003e efficiently converts nitrate (NO₃⁻) to nitrogen gas (N₂), minimizing toxic emissions [\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e]. Furthermore, it achieves high CaCO₃ precipitation (9.5 g/l in 48 hours) yields even under low-oxygen conditions, promoting stable and durable biofilm formation [\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e]. In contrast to ureolytic bacteria such as \u003cem\u003eSporosarcina pasteurii\u003c/em\u003e and \u003cem\u003eBacillus sphaericus\u003c/em\u003e, which produce toxic ammonia, \u003cem\u003eP. denitrificans\u003c/em\u003e employs denitrification, offering a more sustainable approach.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cem\u003eP. denitrificans\u003c/em\u003e influence stone resilience through two interrelated biochemical processes: extracellular polymeric substance (EPS) production and biologically induced mineralization via denitrification. Methanol (CH₄O), supplied as the carbon and energy source, is oxidized by \u003cem\u003eP. denitrificans\u003c/em\u003e, while nitrate (NO₃⁻) serves as the terminal electron acceptor in denitrification. This metabolic activity results in the reduction of nitrate to nitrogen gas (N₂), accompanied by local pH elevation due to proton consumption. The increased alkalinity, in the presence of calcium ions (Ca\u0026sup2;⁺), promotes the precipitation of calcium carbonate (CaCO₃), which enhances the consolidation of the stone surface.\u003c/p\u003e \u003cp\u003eConcurrently, \u003cem\u003eP. denitrificans\u003c/em\u003e produce EPS that facilitates bacterial adhesion, biofilm formation, and the filling of surface pores, thereby influencing the cohesion and stability of the substrate. The EPS matrix may also act as a nucleation platform for calcium carbonate crystals, effectively coupling microbial colonization with mineral precipitation. Notably, the extent and morphology of biofilm and mineralization are modulated by the physicochemical properties of the stone, such as porosity and composition. For instance, in highly porous stones like Bentheimer, enhanced EPS production may support ion retention and localized supersaturation, favoring calcite nucleation [\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e]. This integrated mechanism demonstrates how microbial metabolism dynamically interacts with material properties to achieve both structural stabilization and increased water resistance in stone conservation applications.\u003c/p\u003e \u003cp\u003eOne of the key challenges in understanding the power of bioprotection of stone, is the structural and chemical heterogeneity of stone, which can result in inconsistent bacterial colonization and biofilm formation. Moreover, liquid transport and nutrient availability can significantly influence bacterial activity and crystal formation efficiency. To address these challenges, \u003cem\u003eP. denitrificans\u003c/em\u003e was inoculated on five structural and/or chemically different types of building stone commonly used in historic structures across the Netherlands, Belgium, Germany, and France. We optimized bacterial inoculation conditions to promote biofilm development and investigated the host structure-microorganism interaction, using advanced imaging techniques, including Scanning Electron Microscopy (SEM), micro-computed tomography (micro-CT), Confocal Scanning Laser Microscopy (CSLM), digital microscopy and spectrophotometry. Here, we specifically focus on the distribution of \u003cem\u003eP. denitrificans\u003c/em\u003e and the resulting biofilm formation on the different stones, employing complementary imaging techniques to comprehensively characterize bacterial distribution and biofilm structure across scales ranging from micrometers to centimeters.\u003c/p\u003e \u003cp\u003eThis study provides new insights into the interaction of \u003cem\u003eP. denitrificans\u003c/em\u003e on different types of sedimentary rocks by enhancing visualization methodologies. This is the first step for future research on the bio-protection potential of \u003cem\u003eP. denitrificans\u003c/em\u003e in cultural heritage conservation.\u003c/p\u003e"},{"header":"2. Materials and Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003e2.1. Materials\u003c/h2\u003e \u003cdiv id=\"Sec4\" class=\"Section3\"\u003e \u003ch2\u003e2.1.1 Stones\u003c/h2\u003e \u003cp\u003eIn this study, five natural stones were selected for their diverse mineralogical properties, including variations in chemical composition, colour, and porosity. The selection includes three limestones- Euville [\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e], Savonni\u0026egrave;res [\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e], and Maastricht [\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e] -and two sandstones -Bentheimer [\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e] and Vosges [31]. These stones originate from historically and geologically significant sites in Western Europe: Bentheimer from the Netherlands; Maastricht from the Belgium\u0026ndash;Netherlands border; Euville and Savonni\u0026egrave;res from northeastern France; and Vosges from eastern France (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). The materials were either sourced directly from quarries or obtained through specialized suppliers.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eThe preliminary characterization data for the stone samples, including composition and porosity, were compiled from published literature and are summarized in Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e. The stones were cut to dimensions of 1x1x0.5 cm to meet the sampling criteria of different types of instruments. Before bacterial application, all samples were treated with a 15% isopropyl alcohol (IPA) solution for 15 minutes to eliminate any potential surface contaminants and then rinsed with autoclaved dionized water (DI).\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eSummary of the lithological properties of the selected stones, including a short description, main compositions, and average open porosity percentages.\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"4\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eName\u003c/p\u003e \u003cp\u003e(Country)\u003c/p\u003e \u003cp\u003e(Type)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eShort Description\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eMain Composition\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eAverage Open Porosity\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eBentheimer (Germany)\u003c/b\u003e\u003c/p\u003e \u003cp\u003e\u003cb\u003e(Sandstone)\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eFine to medium-grained quartz arenite with fine and homogeneously spread pores; secondary pores due to feldspar dissolution.\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e93\u0026ndash;99% quartz, \u003c/p\u003e \u003cp\u003e1\u0026ndash;4% K-feldspar, \u003c/p\u003e \u003cp\u003e0\u0026ndash;3% rock fragments\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e21\u0026ndash;24%\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eVosges\u003c/b\u003e\u003c/p\u003e \u003cp\u003e\u003cb\u003e(France)\u003c/b\u003e\u003c/p\u003e \u003cp\u003e\u003cb\u003e(Sandstone)\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eFine-grained red to pink sandstone with a mix of macropores (\u0026gt;\u0026thinsp;20 \u0026micro;m) and micropores (\u0026lt;\u0026thinsp;1 \u0026micro;m).\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e65% quartz, 25% feldspar, 10% clay\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u0026lt;\u0026thinsp;21%\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eEuville\u003c/b\u003e\u003c/p\u003e \u003cp\u003e\u003cb\u003e(France)\u003c/b\u003e\u003c/p\u003e \u003cp\u003e\u003cb\u003e(Limestone)\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eGrainstone with large crinoid fragments, syntaxial overgrowth of calcite, and fragments of echinoderms, brachiopods, corals, and pellets; granular cohesion due to syntaxial cement; heterogeneous porosity.\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e98% calcite\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e10\u0026ndash;20%\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eSavonni\u0026egrave;res (France)\u003c/b\u003e\u003c/p\u003e \u003cp\u003e\u003cb\u003e(Limestone)\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eOolitic limestone with hollow ooids, shell fragments, pellets, and traces of dolomite.\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e98\u0026ndash;99% calcite\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e22\u0026ndash;41%\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eMaastricht (Netherlands)\u003c/b\u003e\u003c/p\u003e \u003cp\u003e\u003cb\u003e(Limestone)\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eWell-sorted calcarenite with skeletal components of foraminifera, ostracods, sponges, bryozoans, and brachiopods, all cemented with calcite spar; loosely bonded grains with a grain-supported texture.\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e95\u0026ndash;98% calcite, some glauconite, quartz, opaque minerals, and iron oxide\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u0026lt;\u0026thinsp;53%\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section3\"\u003e \u003ch2\u003e2.1.2. Bacterial Solution\u003c/h2\u003e \u003cp\u003eThe \u003cem\u003eP. denitrificans\u003c/em\u003e ATCC 19367\u0026trade; was obtained from LGC Standards GmbH, Germany. The original vial was stored in frozen conditions at \u0026minus;\u0026thinsp;80˚C in 30% glycerol. The vial revived by spreading defrozen cells on Lysogeny Broth (LB) agar plates (containing 20g/L of LB and 15g/L agar) and incubating aerobically at 30˚C for 2\u0026ndash;3 days. The temperature of 30˚C is close to its optimal growth conditions. However, as we want to mimic real conditions as close as possible, we lowered the working temperature to 25˚C. The routinary stock culture of \u003cem\u003eP. denitrificans\u003c/em\u003e (used in all assays) was aerobically grown, and maintained in LB broth (containing 20g/L of LB broth mix), at 25˚C, under the shadow, and at 100 RPM shaking for 3 days.\u003c/p\u003e \u003cp\u003eFor bacterial growth validation; initially, a portion of \u003cem\u003eP. denitrificans\u003c/em\u003e colonies grown on an agar plate were transferred into 100 mL of LB solution. The bacteria were cultured until an Optical Density (OD600) (Shimadzu UV-1800) value of 0.790 was achieved, and subsequent daily OD measurements were conducted to confirm the sustained viability of the culture.\u003c/p\u003e \u003cp\u003eThe raw genetic sequences were deposited in the database GenBank under the following accession number:Y16930 (Paracoccus denitrificans 16S rRNA gene, strain ATCC 19367).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section3\"\u003e \u003ch2\u003e2.1.3. Medium\u003c/h2\u003e \u003cp\u003eIn this study, a modified M9 minimal medium, proposed in [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e], was prepared to support bacterial activity while providing essential nutrients and maintaining a controlled chemical environment. The medium was composed of phosphate buffers, including 8.5 g/L Na₂HPO₄\u0026middot;7H₂O and 3 g/L KH₂PO₄, which stabilize pH and provide a phosphorus source; these components were sterilized separately to prevent precipitation [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e]. The basal salt solution contained 0.5 g/L NaCl, 0.24 g/L MgSO₄, and 0.011 g/L CaCl₂, supplying essential ions for osmotic balance, enzymatic function, and biofilm formation. As the carbon source, 4 g/L methanol (CH₄O), a widely used electron donor, was included to support microbial growth and facilitate nitrate reduction. Additionally, 0.72 g/L potassium nitrate (KNO₃) served as the electron acceptor, enabling nitrate respiration under anoxic conditions.\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003e2.2. Method\u003c/h2\u003e \u003cdiv id=\"Sec8\" class=\"Section3\"\u003e \u003ch2\u003e2.2.1 Experimental Setup\u003c/h2\u003e \u003cp\u003eIn order to understand the effect of mixing ratio of \u003cem\u003eP. denitrificans\u003c/em\u003e concentrations in LB and methanol media, the following mixtures were prepared: 100% \u003cem\u003eP. denitrificans\u003c/em\u003e in LB medium (10:0), 50% \u003cem\u003eP. denitrificans\u003c/em\u003e in LB and 50% methanol medium (5:5), and 10% \u003cem\u003eP. denitrificans\u003c/em\u003e in LB and 90% methanol medium (1:9).\u003c/p\u003e \u003cdiv id=\"Sec9\" class=\"Section4\"\u003e \u003ch2\u003e2.2.1.1 Glass Slides\u003c/h2\u003e \u003cp\u003eTo investigate the interaction between \u003cem\u003eP. denitrificans\u003c/em\u003e and the LB-methanol medium independently of stone surface properties, initial experiments were conducted. For this purpose, 10 \u0026micro;L of the prepared solutions were dispensed onto glass slides.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec10\" class=\"Section4\"\u003e \u003ch2\u003e2.2.1.2 Stones\u003c/h2\u003e \u003cp\u003eThree replicates of each stone sample were inoculated into each mixture, resulting in a total of 15 test tubes. The stone samples were incubated at 25\u0026deg;C with shaking at 100 rpm for one week. To evaluate the influence of nutrient composition on bacterial colonization and mineralization, stone samples were inoculated with \u003cem\u003eP. denitrificans\u003c/em\u003e under three different LB : methanol medium ratios: 10:0, 5:5, and 1:9 (v/v). For each condition, three replicate samples were prepared for five stone types (Euville, Bentheimer, Savonni\u0026egrave;res, Vosges, and Maastricht), labeled accordingly (e.g., E1\u0026ndash;E9, B1\u0026ndash;B9). Samples incubated in 10:0 LB : methanol were analyzed using digital microscopy, followed by rinsing with PBS and subjected to colour spectrometry analysis, SEM-EDS, and further digital imaging. Those treated with the 5:5 ratio were rinsed with PBS and stained with Nile Red for biofilm visualization, followed by confocal laser scanning microscopy and digital imaging. The 1:9 LB : methanol samples underwent PBS rinsing. For B9, specific staining (Isolugol) applied, and were analyzed via micro-computed tomography (\u0026micro;-CT) to assess internal colonization and mineral infilling.\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv id=\"Sec11\" class=\"Section3\"\u003e \u003ch2\u003e2.2.2 Visualization Methods\u003c/h2\u003e \u003cdiv id=\"Sec12\" class=\"Section4\"\u003e \u003ch2\u003e2.2.2.1 Digital Microscopy for Biofilm Observation\u003c/h2\u003e \u003cp\u003eDigital microscopy was employed as an initial, non-destructive method to assess biofilm formation and distribution on stone surfaces. Using a Keyence VHX-7000 digital microscope at 2000x magnification, high-resolution images were acquired to visualize the biofilm structures produced by \u003cem\u003eP. denitrificans\u003c/em\u003e. This technique provided preliminary characterization of biofilm morphology and spatial distribution, establishing a basis for subsequent, higher-resolution analysis on glass slides and on stone surfaces.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section4\"\u003e \u003ch2\u003e2.2.2.2 SEM/EDS Analysis\u003c/h2\u003e \u003cp\u003eZeiss EVO 15 environmental SEM (equipped with Peltier cooling, EDS, and automated mineralogy) was employed for surface topography and elemental composition analyses. Samples were sputter-coated with Pt to enhance conductivity. SEM/EDS is a fundamental tool for investigating biofilm-stone interactions and microbially-induced alterations, offering detailed information on surface morphology and elemental distribution.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section4\"\u003e \u003ch2\u003e2.2.2.3 Confocal Laser Scanning Microscopy (CLSM)\u003c/h2\u003e \u003cp\u003eThe experimental workflow was designed to assess biofilm formation and surface colonization on both stone and glass substrates. For evaluation of biofilm on stone surfaces, after the incubation period was completed, the samples were stained with Nile Red solution [\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e] to visualize biofilm and lipid content. The staining procedure involved immersing the samples in the Nile Red solution and incubating them for 10 minutes in the dark at room temperature. Following incubation, samples were gently washed with PBS to remove excess dye. The confocal microscopy analysis was then conducted to determine confocal laser scanning microscopy (Nikon A1, Nikon Eclipse 90i microscope body).\u003c/p\u003e \u003cp\u003eTo conduct visualization tests on glass surfaces, 10 \u0026micro;L of the prepared microbial suspension was pipetted onto glass slides and they were left to dry under controlled conditions at 25\u0026deg;C for one month. The resulting formations were analyzed using both a digital microscope and CLSM to capture structural and morphological details.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section4\"\u003e \u003ch2\u003e2.2.2.4 Micro CT\u003c/h2\u003e \u003cp\u003eBased on observations from SEM and digital microscope images, the best-selected sample (Bentheimer 1:9) with dimensions of 1x1x0.5 cm was scanned using the CoreTOM micro-CT scanner (TESCAN XRE) at the Centre for X-ray Tomography (UGCT) at Ghent University. The sample was scanned twice\u0026mdash;first in PBS and then after adding Lugol\u0026rsquo;s iodine, which enhances the visualization of biofilms by increasing contrast and hydration of the EPS matrix [\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eThe scans were performed at a voxel size of 13.5 \u0026micro;m using 100 kV and 15 W. The exposure time was 1150 ms and 2142 projections were taken resulting in a scan time of about 45 minutes. A 1 mm Al filter was used to reduce beam hardening. The scans were reconstructed using Panthera 1.5.3. (TESCAN XRE) where beam hardening and ring filtering was applied. Acquisition and reconstruction parameters were maintained constant for both scans to enable direct comparison across different time points.\u003c/p\u003e \u003cp\u003eAll scans are available in Utrecht University\u0026rsquo;s YODA repository (see Data Availability section). Further volume analysis was conducted using Avizo 2023.2 software (Thermo Fisher Scientific) to assess structural characteristics and material integrity.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec16\" class=\"Section4\"\u003e \u003ch2\u003e2.2.2.5 Colourimetric Evaluation\u003c/h2\u003e \u003cp\u003eIn order to determine whether \u003cem\u003eP. denitrificans\u003c/em\u003e application had any effect on the aesthetic properties of the stone and to assess potential colour changes, colourimetric values were measured using the CIELAB colour space, introduced in 1976 by the International Commission on Illumination (CIE) (UNI EN 15886, 2010). The colour difference (ΔE*) was calculated using the following equation:\u003c/p\u003e \u003cp\u003eΔE* = \u0026radic;(ΔL*\u0026sup2; + Δa*\u0026sup2; + Δb*\u0026sup2;)\u003c/p\u003e \u003cp\u003ewhere ΔL*, Δa*, and Δb* represent the differences in each chromatic coordinate between the measured samples and the reference. This parameter is significant for aesthetic considerations, as any treatment should not result in a ΔE* greater than 5 to preserve the original appearance of the surface.\u003c/p\u003e \u003cp\u003eFor colourimetric analyses, a Konica Minolta CM-600d Spectrophotometer was used. Measurements were conducted in SCI mode with an 8\u0026deg; standard observer. The instrument was calibrated using a white reference before each measurement session. Three measurements were taken per sample, and the average value was used for analysis.\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003c/div\u003e"},{"header":"3. Results and Discussion","content":"\u003cdiv id=\"Sec18\" class=\"Section2\"\u003e \u003ch2\u003e3.1. Biofilm Formation on Glass Slides\u003c/h2\u003e \u003cp\u003eTo rule out abiotic mineral precipitation, we conducted control experiments on glass slides without bacterial inoculation. These controls exhibited distinct crystal morphologies compared to bacterial samples, indicating that bacterial metabolic activity strongly influences mineral precipitation patterns.\u003c/p\u003e \u003cp\u003eFor all three composition ratios, the presence of bacteria clearly altered the crystallization patterns. In the 10:0 LB-methanol ratio, the broad outer crystallized ring observed in the absence of bacteria became significantly thinner when bacteria were present. Additionally, bacterial cells appeared to align with the crystalline structures formed by salt precipitation, indicating an adaptive interaction between bacteria and the crystal network.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eAt 5:5 LB-methanol (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e.d,e,f), a dense outer biofilm layer was prominent, while the inner regions displayed a mixed structure of crystalline formations and network-like bacterial organization. In contrast, at the highest methanol ratio (1:9 LB-methanol), an outer ring composed entirely of salt crystals was observed, with bacteria failing to reach the outermost layer. Instead, an extensive network structure spreading inward was evident.\u003c/p\u003e \u003cp\u003eThese findings highlight the independent crystallization behavior of the medium in the absence of stone surfaces, suggesting that variations in ionic composition due to stone dissolution could further influence bacterial organization. Additionally, they demonstrate how crystallization patterns on stone surfaces can vary significantly depending on the medium composition.\u003c/p\u003e \u003cp\u003eWhile Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e presents control experiments without stone surfaces, supplementary data A include microscopic images of glass slides of solutions taken from inoculation of stones with bacteria. Differences on these images highlight the influence of stone chemical composition on bacterial behavior and biofilm formation.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec19\" class=\"Section2\"\u003e \u003ch2\u003e3.2. Biofilm Formation on Stone\u003c/h2\u003e \u003cdiv id=\"Sec20\" class=\"Section3\"\u003e \u003ch2\u003e3.2.1. Visual Inspection\u003c/h2\u003e \u003cp\u003eThis study investigated the ability of \u003cem\u003eP. denitrificans\u003c/em\u003e to form biofilms on five different limestone types (Euville, Bentheimer, Maastricht, Vosges, and Savonni\u0026egrave;res), as well as the impact of varying volumetric LB : Methanol medium ratios (10:0, 5:5, and 1:9) on biofilm development. Microscopic analyses revealed distinct biofilm formation patterns across different stone types, with notable variations in extracellular polymeric substance (EPS) distribution, microbial aggregation, and mineral precipitation.\u003c/p\u003e \u003cp\u003eFigure 6. CLSM microscopy images showing biofilm formation on five different stone types (Euville, Bentheimer, Maastricht, Vosges, and Savonni\u0026egrave;res) after bacterial treatment with varying LB : Methanol (ml:ml) ratios (10:0, 5:5, and 1:9). The yellow fluorescence highlights bacterial biomass distribution, with variations in coverage and structural organization observed across different stone types and medium compositions.\u003c/p\u003e \u003cdiv id=\"Sec21\" class=\"Section4\"\u003e \u003ch2\u003e3.2.1.1. Euville limestone\u003c/h2\u003e \u003cp\u003eDigital microscopy revealed biofilm filling the pore spaces (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e), while SEM images (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e) showed a homogeneous biofilm layer covering the entire surface. Uniquely among the stone types tested, at a 1:9 LB : Methanol ratio, SEM images revealed bacteria embedded within a highly homogenous biofilm matrix, demonstrating a uniform distribution of both the EPS and the individual bacterial cells.\u003c/p\u003e \u003cp\u003eFurthermore, Euville limestone showed evidence of crystal formation. Combined analysis of Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e and Fig.\u0026nbsp;6 suggests that these crystals appear to be generated, or 'hatched,' from within the EPS matrix produced by the bacteria.\u003c/p\u003e \u003cp\u003eCLSM (Fig.\u0026nbsp;6) indicated that increasing methanol concentration promoted a more continuous and widespread biofilm, with enhanced EPS accumulation within the pore spaces, which could contribute to improved stone cohesion. This observation was corroborated by digital microscopy. At low methanol concentrations, there was no evidence of network formation. However, at a 1:9 LB : Methanol medium ratio, network formation was clearly visible under the digital microscope.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec22\" class=\"Section4\"\u003e \u003ch2\u003e3.2.1.2. Bentheimer sandstone\u003c/h2\u003e \u003cp\u003eDigital microscopy highlights biofilm-mediated pore filling, while SEM images reveal a spider web-like biofilm network. This structure forms a continuous covering over the stone, suggesting a blanket-like effect that might impact stone permeability and mechanical stability (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ef-g). Additionally, Bentheimer exhibits crystalline precipitates, potentially CaCO₃ formations, which are associated with bacterial-induced mineralization. These crystals appear to nucleate around bacterial clusters, and possibly reinforcing the structural integrity of the stone.\u003c/p\u003e \u003cp\u003eUnlike the other stone types, Bentheimer stone exhibits clearly visible filaments even under CLSM (Fig.\u0026nbsp;6). This serves as additional evidence confirming that the dense structures observed in SEM are biofilm-derived.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec23\" class=\"Section4\"\u003e \u003ch2\u003e3.2.1.3. Maastricht limestone\u003c/h2\u003e \u003cp\u003eCharacterized by its significantly higher porosity compared to the other samples, it does not show distinct biofilm accumulation under digital microscopy. However, SEM images reveal the presence of \"bioball\" structures (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ej-l), which consist of bacterial aggregates encased in an EPS matrix. These formations are widely dispersed across the stone surface and appear to bridge micro-pores (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e) suggesting a potential role in altering stone permeability.\u003c/p\u003e \u003cp\u003eAdditionally, in addition to the bioball-like biofilm formations, bacteria also tend to accumulate on the smoother surfaces of the stone (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003el). CLSM data (Fig.\u0026nbsp;6) further confirm that not only spherical formations are present but also a surface-covering biomass layer, indicating more extensive biofilm development.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec24\" class=\"Section4\"\u003e \u003ch2\u003e3.2.1.4. Vosges sandstone\u003c/h2\u003e \u003cp\u003eFor this clay rich sandstone, primary observation under SEM is the formation of newly precipitated crystalline structures on the stone surface (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003en). These precipitates result from bacterial activity and are distinctly different from the untreated control samples. Additionally, a weakly interconnected network of microbial EPS is present between clay particles (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eo), albeit less developed compared to Bentheimer sandstone. There is also bacterial accumulation on the surface as seen on Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ep.\u003c/p\u003e \u003cp\u003eCompared to other stones, Vosges exhibited lower overall bacterial coverage, with confocal microscopy detecting microbial presence primarily in specific surface regions rather than forming a continuous biofilm (Fig.\u0026nbsp;6). The low fluorescence signals further support that bacteria may have been more dispersed rather than forming structured layers. On the other hand, there is a trend of accumulation on the interface between pore and grains with higher medium : LB ratios for Vosges.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec25\" class=\"Section4\"\u003e \u003ch2\u003e3.2.1.5. Savonni\u0026egrave;res limestone\u003c/h2\u003e \u003cp\u003eUnlike the other investigated stone types, no significant biofilm network was detected in SEM for Savonni\u0026egrave;res. However, bacteria accumulation covering the surface is visible in CLSM images (Fig.\u0026nbsp;6). Additionally, significant crystal formations within pores under digital microscopy were observed (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e). SEM imaging also confirms extensive mineral precipitation, with well-defined euhedral crystal morphologies, suggesting active biomineralization (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003es-t).\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv id=\"Sec26\" class=\"Section3\"\u003e \u003ch2\u003e3.2.2 Elemental Analysis of Biofilm and Mineralization on Stone Surfaces\u003c/h2\u003e \u003cp\u003eThe EDS spectra provide elemental composition insights into microbial biofilm formation and mineral precipitation across different limestone types. Figures\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e7\u003c/span\u003e provide the detection of the elemental compositions on treated stones.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cdiv id=\"Sec27\" class=\"Section4\"\u003e \u003ch2\u003e3.2.2.1 Euville Limestone\u003c/h2\u003e \u003cp\u003eEDS spectra from different regions in Euville limestone indicate the presence of oxygen (O), calcium (Ca), and carbon (C) as primary elements, consistent with the composition of limestone. Figure\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e7\u003c/span\u003ec, shows the stone surface without bacterial accumulation or EPS formation with no signal for nitrogen (N) or phosphorus (P) (which are indicative elements for biologic compounds). On the other hand, presence of P and N suggests microbial activity (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e7\u003c/span\u003eb, d). This circular shape and size (1 \u0026micro;m in diameter) is also a supportive evidence that these structures are \u003cem\u003eP. denitrificans\u003c/em\u003e cells. and Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e7\u003c/span\u003ea is likely associated with EPS production.\u003c/p\u003e \u003cp\u003eAnother important aspect of that in Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e7\u003c/span\u003ee, crystal formation is visible, and Ca, C and O elements are present in the structure, probably indicating CaCO\u003csub\u003e3\u003c/sub\u003e precipitation.\u003c/p\u003e \u003cp\u003eNotably, variations in elemental intensity across different spectra suggest heterogeneity in biofilm development and mineral deposition.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec28\" class=\"Section4\"\u003e \u003ch2\u003e3.2.2.2 Bentheimer Sandstone\u003c/h2\u003e \u003cp\u003eThe EDS spectrum from Bentheimer (f) has dominant peaks for Ca, O, Magnesium (Mg), and Silicone (Si), reflecting the sandstone matrix. However, the presence of sodium (Na) and chlorine (Cl) suggests possible salt precipitation or microbial metabolic byproducts or by the medium. The network-like biofilm observed in SEM images aligns with the EDS results, which suggest localized mineral deposition associated with bacterial structures.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec29\" class=\"Section4\"\u003e \u003ch2\u003e3.2.2.3 Maastricht Limestone\u003c/h2\u003e \u003cp\u003eThe unique \u003cem\u003ebioball\u003c/em\u003e bridge formation in the Maastricht limestone is further evidenced by the EDS spectra (Figs.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e7\u003c/span\u003eg\u0026ndash;j), which demonstrate various structural formations within a single image.\u003c/p\u003e \u003cp\u003eFigure \u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e7\u003c/span\u003eg represents the stone matrix, while Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e7\u003c/span\u003eh highlights EPS formation. Despite exhibiting similar spectral peaks to Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e7\u003c/span\u003ei (which indicates a single bacterium), Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e7\u003c/span\u003eh shows a lower nitrogen (N) peak, consistent with previous findings that EPS formations contain less organic content than bacterial cells (Supplementary Data B).\u003c/p\u003e \u003cp\u003eIn Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e7\u003c/span\u003ej, salt crystals are present on the right side of the bridge, exhibiting higher sodium (Na) and chloride (Cl) peaks compared to Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e7\u003c/span\u003ei. Although the crystal and bacterial cell appear morphologically similar, the higher Cl peak and lower N peak in Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e7\u003c/span\u003ej confirm salt crystallization rather than bacterial biomass. Notably, in this case, salt crystallization acts as a supportive structural element for the EPS rather than contributing to deterioration of the cells or the structure, reinforcing the stability of the biofilm matrix.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec30\" class=\"Section4\"\u003e \u003ch2\u003e3.2.2.4 Vosges Sandstone\u003c/h2\u003e \u003cp\u003eThe EDS results for Vosges (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e7\u003c/span\u003ek\u0026ndash;p) indicate significant variations in mineral composition across different regions. The SEM images show crystal-like formations, while there is almost no Si signals and more Ca signals in the crystals (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e7\u003c/span\u003en) which could be a result of biofilm-induced mineralization and fully covering silicate surface. The detected phosphorus signals further support the hypothesis that bacterial activity is influencing local mineral deposition.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec31\" class=\"Section4\"\u003e \u003ch2\u003e3.2.2.5 Savonni\u0026egrave;res Limestone\u003c/h2\u003e \u003cp\u003eEDS spectra from Savonni\u0026egrave;res (r\u0026ndash;s) indicate a mixture of carbonate minerals and biofilm-related elements, with dominant peaks for Ca, O, Mg, and P. The SEM image suggests the presence of both homogenous biofilm structures and mineral deposits, aligning with the observed elemental composition. The relatively high phosphorus content in certain areas suggests microbial influence on mineral formation, possibly through biomineralization pathways (Supplementary Data B).\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv id=\"Sec32\" class=\"Section3\"\u003e \u003ch2\u003e3.2.3 Micro-CT Analysis\u003c/h2\u003e \u003cp\u003eFigure \u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e8\u003c/span\u003e shows representative 2D slices (side and top views) extracted from the 3D images of untreated and Lugol-treated Sample B9. Upon visual inspection, three primary phases were initially identified: (1) a liquid phase occupying pore spaces and surrounding the sample, (2) a gas phase present both as residual gas within pores and as bubbles suspended in the liquid phase, and (3) a solid grain phase. However, a closer examination of the Lugol-treated sample (indicated by arrows in Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e8\u003c/span\u003e(b)) revealed an additional minor phase covering parts of the sample surface. To enhance visibility and facilitate clear identification, a difference image was generated (an image created by subtracting 8a from 8b, to highlight changes or differences), as illustrated in Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e8\u003c/span\u003e(c). In this image, the newly identified phase is highlighted in black (indicated by arrows) and clearly visible on the stone surface after Lugol treatment. We interpret this additional phase as biofilm.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eApart from visually inspecting the images, the various phases present in the reconstructed 3D images were analyzed based on their gray-level values (Supplementary Data C). The histogram displays the intensity histogram of the sample image treated with Lugol. As shown, the histograms of gas, liquid, biofilm, and grain phases exhibit clear separation. Each phase exhibits a characteristic range of gray-scale values based on its X-ray attenuation [\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e]. Logically, the gas phase appears at the lowest gray-scale range, followed by the liquid phase, which is centered at slightly higher values. The biofilm phase overlaps with the upper range of the liquid phase and extends toward the grain phase, indicating its intermediate density. The grain phase is represented by the highest gray-scale values, reflecting its dense mineral composition. These distributions enable segmentation and phase identification in image-based analysis of pore-scale processes.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec33\" class=\"Section3\"\u003e \u003ch2\u003e3.2.4 Colour Difference\u003c/h2\u003e \u003cp\u003eTable\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e shows the colour difference (ΔE) across different limestone types under varying LB: Methanol medium ratios (10:0, 5:5, 1:9). The measurements have been taken after rinsing with PBS.\u003c/p\u003e \u003cp\u003eAccording to the results, Maastricht exhibited the highest ΔE values, particularly at 5:5, indicating significant colour variation due to bacterial activity. Bentheimer and Vosges showed a decreasing trend in ΔE as methanol concentration increased. Savonni\u0026egrave;res displayed a notable drop at 5:5, followed by an increase at 1:9, suggesting a non-linear relationship between biofilm formation and colour variation. Euville consistently exhibited the lowest ΔE values, indicating minimal colour shifts.\u003c/p\u003e \u003cp\u003eOverall results show that a colour difference due to the bacterial treatment is under the observable limit (5) for all samples.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab2\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 2\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eColour difference (ΔE) values for five stone types (Euville, Bentheimer, Maastricht, Vosges, Savonni\u0026egrave;res) before and after bacterial treatment with different LB : Methanol (ml:ml) ratios (10:0, 5:5, and 1:9).\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"6\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colspan=\"6\" nameend=\"c6\" namest=\"c1\"\u003e \u003cp\u003eColour Difference (ΔE)\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eLB: Methanol (ml:ml)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eEuville\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eBentheimer\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eMaastricht\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003eVosges\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c6\"\u003e \u003cp\u003eSavonni\u0026egrave;res\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003e10:0\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e0.981\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e2.837\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e3.110\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e2.981\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e3.234\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003e5:5\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e0.305\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e1.691\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e3.761\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e3.649\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e1.012\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003e1:9\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e1.339\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e1.232\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e2.513\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e2.736\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e2.652\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003c/div\u003e \u003c/div\u003e"},{"header":"4. Conclusion","content":"\u003cp\u003eThis study demonstrates that biofilm formation and biomineralization by \u003cem\u003eP. denitrificans\u003c/em\u003e are strongly influenced by stone type, surface properties, and medium composition. In particular:\u003c/p\u003e \u003cp\u003e \u003cul\u003e \u003cli\u003e \u003cp\u003eEuville and Bentheimer supported extensive biofilm formation and uniform EPS layers, suggesting high potential for structural stabilization.\u003c/p\u003e \u003c/li\u003e \u003cli\u003e \u003cp\u003eMaastricht showed the formation of discrete bioball-like biofilm structures, which may offer localized improvements in pore sealing.\u003c/p\u003e \u003c/li\u003e \u003cli\u003e \u003cp\u003eVosges exhibited lower bacterial coverage and more crystalline precipitates, indicating a more limited interaction.\u003c/p\u003e \u003c/li\u003e \u003cli\u003e \u003cp\u003eSavonni\u0026egrave;res had significant mineral precipitation but minimal biofilm network formation.\u003c/p\u003e \u003c/li\u003e \u003c/ul\u003e \u003c/p\u003e \u003cp\u003eTo address potential interactions between stone chemistry and nutrient composition, we note that differences in calcium content, porosity, and mineralogy likely modulate bacterial colonization and biofilm formation. Our observations showed that calcium-rich stones, such as Euville and Savonni\u0026egrave;res, supported more uniform biofilm coverage and mineral precipitation, while quartz-rich sandstones like Bentheimer displayed more filamentous structures. This suggests that nutrient-stone interactions play a key role in shaping biofilm behavior.\u003c/p\u003e \u003cp\u003eWhile visual observations of biofilm coverage and EPS accumulation suggest enhanced stone cohesion, we acknowledge that direct measurements of mechanical strength (e.g., compressive strength tests or ultrasonic pulse velocity) are necessary to quantitatively validate these findings. Such mechanical testing will be a focus of future research.\u003c/p\u003e \u003cp\u003eThese results emphasize the importance of tailoring bio-based conservation strategies to specific material characteristics. This work establishes a framework for targeted microbial treatments in stone conservation, guiding future efforts toward optimized and sustainable long-term protection.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgement\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis project was funded by the Dutch Research Council (NWO) through the BugControl project (project number VI.C.202.074) under the NWO Talent program, and by the EXCITE Network, part of the Horizon 2020 research and innovation program of the European Union under grant agreement no. 101005611. Furthermore, we are grateful for the Ghent University Special Research Fund (BOF-UGent) to support the Centre of Expertise UGCT (BOF.COR.2022.0008). Finally, we would also like to thank Alejandra Reyes-Amezaga, Dr. Carolin Rieg, Eric Hellebrand, Mahin Baghery, Leonard Bik, Nadia Breugelmans and Alexander Hoogenboom for their technical support. \u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData availability\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe micro-CT reconstructed data are available at the Yoda data repository of Utrecht University and accessible through https://doi.org/10.24416/UU02-SECJ1Q.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCRediT\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eSE\u0026Ccedil;:\u003c/strong\u003e Conceptualization, Data curation, Investigation, Methodology, Writing \u0026ndash; original draft, Writing \u0026ndash; review and editing, Visualization\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eJQ:\u003c/strong\u003e Data curation, Writing \u0026ndash; review and editing, Visualization, Software\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eLS:\u003c/strong\u003e Data curation, Writing \u0026ndash; review and editing\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eVC:\u003c/strong\u003e Writing \u0026ndash; review and editing, Resources, Funding acquisition, Supervision, Project administration\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eLuo L, Gu J-D. 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Appl Environ Microbiol. 2010;76:6387\u0026ndash;96.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eTrends. opportunities for greener and more efficient microbially induced calcite precipitation pathways: a strategic review. Geotech Res. 2024;11:161\u0026ndash;85.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLin W, et al. Microbially induced desaturation and carbonate precipitation through denitrification: a review. Appl Sci. 2021;11:7842.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eRodriguez-Navarro C, et al. Influence of substrate mineralogy on bacterial mineralization of calcium carbonate: implications for stone conservation. Appl Environ Microbiol. 2012;78:4017\u0026ndash;29.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBordel S, van Spanning RJM, Santos-Beneit F. Imaging and modelling of poly(3-hydroxybutyrate) synthesis in \u003cem\u003eParacoccus denitrificans\u003c/em\u003e. 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Earth-Sci Rev. 2013;123:1\u0026ndash;17.\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"npj-heritage-science","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"hsci","sideBox":"Learn more about [Heritage Science](http://heritagesciencejournal.springeropen.com)","snPcode":"40494","submissionUrl":"https://submission.nature.com/new-submission/40494/3","title":"npj Heritage Science","twitterHandle":"@SpringerOpen","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"BMC/SO AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"Paracoccus denitrificans, biofilm, conservation, MICP, imaging techniques (SEM, µ-CT, CLSM), cultural heritage","lastPublishedDoi":"10.21203/rs.3.rs-6836573/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-6836573/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eMicroorganisms are increasingly recognized for their dual role in the deterioration and conservation of cultural heritage, with \u003cem\u003eParacoccus denitrificans\u003c/em\u003e emerging as a promising candidate for bio-based stone stabilization. This study investigates the biofilm formation of \u003cem\u003eP. denitrificans\u003c/em\u003e on stone surfaces, with a focus on five sedimentary rocks -Euville, Savonni\u0026egrave;res, Bentheimer, Vosges, and Maastricht - selected for their varied porosity, composition, colour and importance for cultural heritage. The samples were inoculated under different nutrient-to-medium ratios to evaluate the impact of inoculation conditions on bacteria-stone interactions. A multi-scale imaging approach using SEM, \u0026micro;-CT, CLSM, digital microscopy, and colour spectrophotometry provided complementary insights into bacterial distribution, EPS production, biofilm morphology, and mineral deposition. Depending on the stone type, \u003cem\u003eP. denitrificans\u003c/em\u003e formed distinct biofilm architectures, including spider web-like networks, spherical aggregates, or uniform surface coatings. Moreover, clear evidence of bacterially induced mineral crystallization was observed. Results reveal that both stone type and medium composition significantly influence biofilm development and mineralization behavior. This integrative methodology demonstrates the potential of \u003cem\u003eP. denitrificans\u003c/em\u003e in stone conservation and offers a novel framework for advancing bio-conservation strategies in cultural heritage science.\u003c/p\u003e","manuscriptTitle":"Visualization of Paracoccus denitrificans on various types of stones used in European built heritage","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-06-16 05:31:30","doi":"10.21203/rs.3.rs-6836573/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2025-10-11T20:36:45+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-06-11T00:54:09+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"296022373601464132453883726218450237739","date":"2025-06-10T23:05:20+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2025-06-10T16:59:49+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-06-09T11:29:30+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2025-06-09T11:27:05+00:00","index":"","fulltext":""},{"type":"submitted","content":"npj heritage science","date":"2025-06-06T11:12:09+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"npj-heritage-science","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"hsci","sideBox":"Learn more about [Heritage Science](http://heritagesciencejournal.springeropen.com)","snPcode":"40494","submissionUrl":"https://submission.nature.com/new-submission/40494/3","title":"npj Heritage Science","twitterHandle":"@SpringerOpen","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"BMC/SO AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"be941876-2049-4ff5-a152-e81130ffcbdc","owner":[],"postedDate":"June 16th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[],"tags":[],"updatedAt":"2026-05-15T09:53:16+00:00","versionOfRecord":[],"versionCreatedAt":"2025-06-16 05:31:30","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-6836573","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-6836573","identity":"rs-6836573","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}