Antibacterial Effect of Zinc Oxide-Based Nanomaterials on Environmental Biodeteriogens Affecting Historical Buildings

The colonization of microorganisms and their subsequent interaction with stone substrates under different environmental conditions encourage deterioration of materials by multiple mechanisms resulting in changes in the original color, appearance and durability. One of the emerging alternatives to remedy biodeterioration is nanotechnology, thanks to nanoparticle properties such as small size, no-toxicity, high photo-reactivity, and low impact on the environment. This study highlighted the effects of ZnO-based nanomaterials of two bacteria genera isolated from the Temple of Concordia (Agrigento’s Valley of the Temples in Sicily, Italy) that are involved in biodeterioration processes. The antimicrobial activities of ZnO-nanorods (Zn-NRs) and graphene nanoplatelets decorated with Zn-NRs (ZNGs) were evaluated against the Gram positive Arthrobacter aurescens and two isolates of the Gram negative Achromobacter spanius. ZNGs demonstrated high antibacterial and antibiofilm activities on several substrates such as stones with different porosity. In the case of ZNGs, a marked time- and dose-dependent bactericidal effect was highlighted against all bacterial species. Therefore, these nanomaterials represent a promising tool for developing biocompatible materials that can be exploited for the conservation of cultural heritage. These nanostructures can be successfully applied without releasing toxic compounds, thus spreading their usability.


Introduction
The biodeterioration process refers to any form of irreversible alteration and any undesirable change in the properties of materials, caused by the activity of living organisms. It is known that microbial activities are one of the main factors of the degradation of cultural heritage that, together with other causes such as climatic factors, contribute to the deterioration of inorganic substrates, leading to physical-chemical and aesthetic damages. Indeed, changes in temperature, humidity, rain, erosion,

Cell Viability Test
Viability in liquid assay was analyzed as described in [28]. Samples were incubated with minimal agitation at 30 • C, under diffuse visible light. After treatments, the aliquots of the different samples were diluted and then grown on agar-LB plates; after incubation at 30 • C overnight, the bacterial ability to form new colonies was measured by counting the number of colonies forming units (CFU).

Evaluation of Biofilm Formation
Biofilm formation was evaluated as previously described [28] with some differences: each well was filled with 200 µL of LB broth and 125 µg/mL of ZnO-NRs or ZNGs (in triplicate). Wells without nanoparticles were utilized as controls. Next, plates were incubated at 30 • C for 24 h under diffuse visible light and Crystal Violet assay was performed. The experiment was repeated three times with three replicates for each treatment.

LIVE/DEAD Assay
An amount of 1 × 10 7 cells/mL was inoculated in LB in the presence of a ZnO-NRs concentration of 125 µg/mL or a ZNGs concentration of 10 µg/mL. The suspension was incubated in a 35 mm Petri plate with a cover slide for 24 h at 30 • C under diffuse visible light. As control, strains grown in LB were inoculated in the absence of nanoparticles. After treatment, cultures were gently removed, washed twice with sterile water, and stained using a LIVE/DEAD BacLight-Bacterial Viability (Kit 7012, Invitrogen, Mount Waverley, Australia) for 15 min at room temperature in the dark. After washing with sterile deionized water, stained cells were observed under a Zeiss Axiovert 25 fluorescence microscope. Syto9 is able to stain live and dead cells, while propidium iodide is unable to penetrate the intact cell wall in viable cells, staining only dead cells.

Preparation of Stones
Suspensions of ZNGs to be used with the stones were produced at 250 µg/mL through bath sonication (at 37 kHz of working frequency) of 10 mg of ZNGs, prepared as described before, in 40 mL deionized water for 3 min. Noto stone, Carrara marble and yellow brick specimens (1 × 1 × 0.5 cm 3 ) Nanomaterials 2020, 10, 335 4 of 14 were then spray coated, through an airbrush, with the aqueous suspension of ZNGs. Full coverage of the specimens was ensured by repeating the spraying process four times. Each time 2 mL of suspension was sprayed and evaluated by FE-SEM analysis.

Characterization of Stone Materials
Porosity and density of stone specimens were characterized through mercury porosimetry. The water capillarity absorption experiments were performed following the UNI EN 15801:2010 [31]. Colorimetric measurements were performed on stone samples to verify color modification due to ZNGs exposition according to [32]. The ∆E value refers to the median values of the treated samples compared to the untreated ones. Furthermore, according to the literature, ∆E < 5 was considered as corresponding to a not-significant variation [33]. The measurements were made on untreated and treated samples, on three different zones on stone surfaces.

Antimicrobial Activity of Treated Stones
Treated surfaces of the three different specimens were submerged in a concentration of 1.0 × 10 7 cells/mL of bacterial cultures and then incubated at 30 • C for 24 h, under diffuse visible light. After that, the treated stones were dipped into 3 mL of sterile deionized water, shaken for 1 min and then, 1 mL was plated on LB plates. After incubation at 30 • C for 24 h the bacterial colonies were counted. Untreated stone samples were used as reference. The experiments were repeated three times with three replicates for each treatment.

FE-SEM Microscopy Imaging for Treated Stones
Biological samples were prepared according to the procedures described above. After 24 h of treatment, stones in the presence or not of bacteria were fixed with 2% glutaraldehyde in PBS for 1 h at RT in the dark. After 3 washes in PBS, samples were then dehydrated as described in [34]. The stone samples were successively utilized for the electron microscopy analysis.

Statistical Analysis
Experiments were performed at least in triplicate. Data are presented as mean ± SD, and Student's-test or one-way ANOVA analysis coupled with a Bonferroni post-test (GraphPad Prism 4.0 software) was used to determine the statistical significance between experimental groups. Statistical significance was defined as * p < 0.05, ** p < 0.01, and *** p < 0.001.

Bacteria Identification and ZnO-NRs Cell Viability Assay
In this study, antimicrobial and antibiofilm properties of ZnO-based nanomaterials were evaluated against two bacterial genera involved in biodeterioration processes. Especially, several bacterial colonies were isolated from the Temple of Concordia in Sicily and identified at the molecular level by the amplification of 16S rDNA. The comparison of the obtained sequences with those in the BLAST database allowed identification of the Gram-positive Arthrobacter aurescens, and two strains belonging to the Gram-negative Achromobacter spanius, which are involved in biodeterioration, as has been reported in many works [35][36][37]. The three different strains were confronted with the ZnO-NR-based nanomaterials at different concentrations.
Initially, bacterial survival was evaluated by CFU counting analysis after incubation with ZnO-NRs in liquid assay. After 2 h of treatment, a significant reduction in cell viability was observed at concentrations of 50 µg/mL and 100 µg/mL, with respect to untreated cells. In the case of A. spanius TC7, a notable effect was highlighted also at lower concentration of 10 µg/mL. These results were marked in 24 h-exposure, where a mortality rate of over 98% was observed already with 10 µg/mL for all the strains (Figure 1).

Evaluation of ZnO-NRs Antibiofilm Properties
Biodeterioration of monuments is mainly due to the ability of microorganisms to create biofilm: in fact, bacteria can stick to each other and to a surface, forming a group of cells producing an extracellular matrix composed of DNA, proteins and polysaccharides. In these biofilms, microorganisms resist adverse abiotic conditions [2]. Consequently, cultural heritage decay problems Figure 1. Effect of zinc oxide nanorods (ZnO-NRs) on different strains viability. Bacteria were treated or not (UT: untreated) with different concentrations of nanorods for 2 h (left column) or 24 h (right column) and bacterial survival was evaluated by CFU counting analysis. (a-c) indicate respectively A. aurescens TC4, A. spanius TC1, and A. spanius TC7. A one-way ANOVA analysis with the Bonferroni post-test was used to assess statistical significance (ns not significant; * p < 0.05 and *** p < 0.001 with respect to UT).

Evaluation of ZnO-NRs Antibiofilm Properties
Biodeterioration of monuments is mainly due to the ability of microorganisms to create biofilm: in fact, bacteria can stick to each other and to a surface, forming a group of cells producing an extracellular matrix composed of DNA, proteins and polysaccharides. In these biofilms, microorganisms resist adverse abiotic conditions [2]. Consequently, cultural heritage decay problems are related to the biofilm formation and its metabolic activity, such as production of organic and inorganic acids and bacteria exopolymers [38].
Biofilms, grown on stone monuments, increase rock deterioration through physicochemical processes, as has been observed in many stone structures of cultural heritage [39,40]. For this reason, antibiofilm effects of ZnO-NRs against the three isolated microorganisms were tested at a concentration of 125 µg/mL. After 24 h of treatment, the biofilm formation on glass slides was evaluated by fluorescence microscope utilizing LIVE/DEAD assay ( Figure 2). The Syto9 staining of A. aurescens TC4 and A. spanius TC7 strains showed a reduced number of treated cells on the surface (Figure 2a,c respectively). Instead, when the propidium iodide was used, no differences in the number of death cells were recorded comparing treated and untreated samples (data not shown).
are related to the biofilm formation and its metabolic activity, such as production of organic and inorganic acids and bacteria exopolymers [38].
Biofilms, grown on stone monuments, increase rock deterioration through physicochemical processes, as has been observed in many stone structures of cultural heritage [39,40]. For this reason, antibiofilm effects of ZnO-NRs against the three isolated microorganisms were tested at a concentration of 125 µg/mL. After 24 h of treatment, the biofilm formation on glass slides was evaluated by fluorescence microscope utilizing LIVE/DEAD assay ( Figure 2). The Syto9 staining of A. aurescens TC4 and A. spanius TC7 strains showed a reduced number of treated cells on the surface (Figure 2a,c respectively). Instead, when the propidium iodide was used, no differences in the number of death cells were recorded comparing treated and untreated samples (data not shown). These results suggest that the nanomaterial is able to inhibit the adhesion of these species, to the surface. Instead, in the case of A. spanius TC1 an increased number of treated cells stained with Syto9 was observed in comparison with the untreated counterpart, suggesting a stimulation of biofilm formation by the nanomaterial (Figure 2b). The biofilm on plastic surface was also quantified by the Crystal Violet method (Figure 3). In A. aurescens TC4 and A. spanius TC7 an inhibition of about 90% of biofilm was observed (Figure 3a,c). Conversely, ZnO-NRs seemed to stimulate almost 50% of These results suggest that the nanomaterial is able to inhibit the adhesion of these species, to the surface. Instead, in the case of A. spanius TC1 an increased number of treated cells stained with Syto9 was observed in comparison with the untreated counterpart, suggesting a stimulation of biofilm formation by the nanomaterial (Figure 2b). The biofilm on plastic surface was also quantified by the Crystal Violet method (Figure 3). In A. aurescens TC4 and A. spanius TC7 an inhibition of about 90% of biofilm was observed (Figure 3a,c). Conversely, ZnO-NRs seemed to stimulate almost 50% of biofilm forming capacity in A. spanius TC1 (Figure 3b). Those results demonstrated the effectiveness of ZnO-NRs in controlling biofilm growth of only two isolated microorganisms, involved in biodeterioration. Nanomaterials 2020, 10, x FOR PEER REVIEW 7 of 15 biofilm forming capacity in A. spanius TC1 (Figure 3b). Those results demonstrated the effectiveness of ZnO-NRs in controlling biofilm growth of only two isolated microorganisms, involved in biodeterioration. Surprisingly, A. spanius TC1 and A. spanius TC7 showed different behavior when treated with ZnO-NRs, highlighting the presence of intra-species differences between the two strains. Indeed, as reported in biodiversity studies of several bacterial species, intra-species analysis revealed extensive variation between isolates, both at the genotypic and phenotypic level [41,42]. Surprisingly, A. spanius TC1 and A. spanius TC7 showed different behavior when treated with ZnO-NRs, highlighting the presence of intra-species differences between the two strains. Indeed, as reported in biodiversity studies of several bacterial species, intra-species analysis revealed extensive variation between isolates, both at the genotypic and phenotypic level [41,42].

ZNGs Antimicrobial and Antibiofilm Activity
An innovative nanomaterial consisting of graphene nanoplatelets decorated by zinc oxide nanorods (ZNGs) was described in previous work for the antimicrobial and antibiofilm properties (Zanni et al., 2017). Therefore, experiments were performed with ZNGs nanomaterial: cell viability test was performed with ZNGs at different concentrations ( Figure S1). After just 2 h, ZNGs treatment (1 µg/mL) resulted in almost 50% antibacterial activity against A. aurescens TC4 and A. spanius TC7, and in 70% against A. spanius TC1 at a concentration of 10 µg/mL. These results show a time-and dose-dependent antimicrobial effect of ZNGs against the planktonic forms of two bacteria genera. After 24 h of treatment, an inhibition of bacteria growth of over 70% was observed with 1 µg/mL. Especially, when the LIVE/DEAD assay was performed with ZNGs, fluorescence images showed the ability of ZNGs to inhibit A. spanius TC1 biofilm already at a concentration of 10 µg/mL (Figure 4a). Similarly, A. aurescens TC4 and A. spanius TC7 biofilms were also inhibited ( Figure S2). The Crystal Violet assay on plastic surface underlined a high inhibitory activity of ZNGs also against A. spanius TC1 biofilm formation with a decrease of 50% compared to the control (Figure 4b). In the case of A. aurescens TC4 and A. spanius TC7 the data obtained with ZNGs treatment were comparable with that obtained with ZnO-NRs (data not shown).
An innovative nanomaterial consisting of graphene nanoplatelets decorated by zinc oxide nanorods (ZNGs) was described in previous work for the antimicrobial and antibiofilm properties (Zanni et al., 2017). Therefore, experiments were performed with ZNGs nanomaterial: cell viability test was performed with ZNGs at different concentrations ( Figure S1). After just 2 h, ZNGs treatment (1 µg/mL) resulted in almost 50% antibacterial activity against A. aurescens TC4 and A. spanius TC7, and in 70% against A. spanius TC1 at a concentration of 10 µg/mL. These results show a time-and dose-dependent antimicrobial effect of ZNGs against the planktonic forms of two bacteria genera. After 24 h of treatment, an inhibition of bacteria growth of over 70% was observed with 1 µg/mL. Especially, when the LIVE/DEAD assay was performed with ZNGs, fluorescence images showed the ability of ZNGs to inhibit A. spanius TC1 biofilm already at a concentration of 10 µg/mL (Figure 4a). Similarly, A. aurescens TC4 and A. spanius TC7 biofilms were also inhibited ( Figure S2). The Crystal Violet assay on plastic surface underlined a high inhibitory activity of ZNGs also against A. spanius TC1 biofilm formation with a decrease of 50% compared to the control (Figure 4b). In the case of A. aurescens TC4 and A. spanius TC7 the data obtained with ZNGs treatment were comparable with that obtained with ZnO-NRs (data not shown).  To assess statistical analysis a one-way ANOVA analysis with the Bonferroni post-test was used (*** p < 0.001 with respect to UT).
Results demonstrate that biofilm production was reduced by ZnO-based nanomaterial treatment, in agreement with the observation that bacteria in biofilms are more resistant to antibacterial agents than their planktonic form [43]. For the Gram-negative A. spanius TC1, ZNGs treatment was more efficient, even when lower concentrations were used. The effect of ZNGs on the biofilm of A. spanius TC1 can be related to a different cell wall composition with respect to A. spanius TC7, due to intra-species variation. Indeed, the Gram-negative Chlamydia psittaci isolated microorganisms showed variations in the putative outer membrane genes [44].

ZNGs Actions on Stones
A large percentage of the world's cultural heritage is made from stone, which is disappearing, because of the different deterioration phenomena, one of which is biodeterioration. The most common stones are calcareous stones, such as marble and limestone, and siliceous ones, such as sandstone, granite, and artificial stones. They are different in hardness, porosity, and alkalinity, and their properties affect their predisposition to biodeterioration. In this study, three materials with different structural features, as reported in Table 1, were used to test the antimicrobial properties of ZNGs: common yellow brick, Noto stone, and Carrara marble. The apical surfaces of the stone specimens were coated with 250 µg/mL ZNG-suspension, to cover the porous surfaces of the materials preventing the proliferation of the bacteria in depth. In order to evaluate if the porosity and pore size influenced the distribution and position of the nanostructures on the surface, an FE-SEM analysis of these materials was carried out. The micrographs of treated and untreated samples showed the different disposition of the nanostructures on surfaces with diverse pore size, highlighting the uniformity of ZNGs distribution on the less porous Carrara marble, unlike the other specimens having a higher porosity ( Figure 5). Furthermore, colorimetric analysis showed no significant variation between treated and untreated stone (Table S1). The antibacterial activities of the treated stones against the Gram-positive A. aurescens TC4 and A. spanius TC1, representative of Gram-negative isolated microorganisms, are reported in Figure 6. Treated Noto stones, typical diffused stones in historical buildings in Sicily and Malta, revealed a reduction of viability up to 60% for both bacterial strains (Figure 6a). At the same time, treated common yellow brick induced a strong reduction of A. aurescens TC4 and A. spanius TC1 cell viability of about 90% (Figure 6b). A reduction of about 70% in bacterial viability was obtained in the case of The antibacterial activities of the treated stones against the Gram-positive A. aurescens TC4 and A. spanius TC1, representative of Gram-negative isolated microorganisms, are reported in Figure 6. Treated Noto stones, typical diffused stones in historical buildings in Sicily and Malta, revealed a reduction of viability up to 60% for both bacterial strains (Figure 6a). At the same time, treated common yellow brick induced a strong reduction of A. aurescens TC4 and A. spanius TC1 cell viability of about 90% (Figure 6b). A reduction of about 70% in bacterial viability was obtained in the case of treated Carrara marble (Figure 6c). Similar results were also obtained when A. spanius TC7 strain was utilized (data not shown). Generally, these results indicated a correlation between the antibiofilm properties of ZNGs and nanostructure distribution, depending on the specimen's porosity. Indeed, the high porosity of Noto stone causes a reduced distribution of ZNGs on the surface, leading to a slight reduction of the antibiofilm effect. In Figure 7, it is possible to observe, as an example, the Noto stone covered by Generally, these results indicated a correlation between the antibiofilm properties of ZNGs and nanostructure distribution, depending on the specimen's porosity. Indeed, the high porosity of Noto stone causes a reduced distribution of ZNGs on the surface, leading to a slight reduction of the antibiofilm effect. In Figure 7, it is possible to observe, as an example, the Noto stone covered by ZNGs treated or not with A. aurescens TC4 cells (Panels a,b, respectively). The bacterial cell wall showed mechanical injuries caused by direct contact with the nanomaterial. In fact, ZnO-NRs function as nanoneedles that pierce the bacterial wall, while nanosheets of GNPs offer a large surface area for the oriented growth of the ZnO-NRs over the GNP surface. Consequently, this behavior already confirmed what has been observed in previous work [25], namely that ZnO-NRs deposited on the GNPs surface provoke mechanical aggression on the cell walls. Especially, the use of graphene nanoplatelets (GNPs) creates an advantage, forcing the growth of the ZnO-NRs in a specific orientation and extending the area of their action. Indeed, this shape contributes to increasing the adhesion of the nanostructures to the cell wall enhancing the penetration of the ZnO-NRs through the cell membrane, as highlighted in FE-SEM analysis performed in the previous work [25]. Therefore, this may cause greater ability to damage the bacterial surface, through mechanical damage produced by the ZnO-NRs [45,46]. Furthermore, the use of ZNGs could bypass the treatment of biodeterioration with traditional biocides, obtaining a safer solution than toxic chemical agents. Indeed, it has been demonstrated that ZnO-NRs and ZNGs lacked acute toxicity in vitro and in vivo: ZNO-based nanomaterials did not affect viability, fertility, and body length in different model organisms [26,28,47].
Generally, several studies suggested that ZnO and graphene-based nanomaterials represent good candidates to be used for restoration of cultural heritage, thanks to their many advantages, such as low cost and effectiveness in size dependency against a wide range of bacteria [48,49]. Commonly, many biocides used in cultural heritage cover a wide range of chemical classes, such as inorganic compounds (in e.g., Na and Ca hypochlorite) and organic ones, like the quaternary ammonium compounds [50,51]. Most of the commercial products based on these chemical toxic compounds have been forbidden due to their environmental and health hazards [52]. Indeed, traditional chemical biocides are usually aggressive towards the damaged substrates as well as to workers' health. The graphene-based nanomaterials, with their biocompatible properties, could represent an alternative to conventional treatments, to be applied as antimicrobial agents on stone monuments [53].

Conclusions
Our studies indicated that ZnO-based nanomaterials show antimicrobial and antibiofilm properties against different bacteria involved in stone biodeterioration. Indeed, results suggest that ZnO-NRs-decorated GNPs might be highly effective in preserving the surface of artwork in a costeffective way that enables control of bacteria growth and therefore biodeterioration processes also at very low concentrations. These nanomaterials could be used to fight biodeterioration, offering the advantageous possibility of removing biofilm and limiting the consequent physical and chemical damages. ZNGs could avoid the use of traditional bioremediation reagents (such as biocide products) Consequently, this behavior already confirmed what has been observed in previous work [25], namely that ZnO-NRs deposited on the GNPs surface provoke mechanical aggression on the cell walls. Especially, the use of graphene nanoplatelets (GNPs) creates an advantage, forcing the growth of the ZnO-NRs in a specific orientation and extending the area of their action. Indeed, this shape contributes to increasing the adhesion of the nanostructures to the cell wall enhancing the penetration of the ZnO-NRs through the cell membrane, as highlighted in FE-SEM analysis performed in the previous work [25]. Therefore, this may cause greater ability to damage the bacterial surface, through mechanical damage produced by the ZnO-NRs [45,46]. Furthermore, the use of ZNGs could bypass the treatment of biodeterioration with traditional biocides, obtaining a safer solution than toxic chemical agents. Indeed, it has been demonstrated that ZnO-NRs and ZNGs lacked acute toxicity in vitro and in vivo: ZNO-based nanomaterials did not affect viability, fertility, and body length in different model organisms [26,28,47].
Generally, several studies suggested that ZnO and graphene-based nanomaterials represent good candidates to be used for restoration of cultural heritage, thanks to their many advantages, such as low cost and effectiveness in size dependency against a wide range of bacteria [48,49]. Commonly, many biocides used in cultural heritage cover a wide range of chemical classes, such as inorganic compounds (in e.g., Na and Ca hypochlorite) and organic ones, like the quaternary ammonium compounds [50,51]. Most of the commercial products based on these chemical toxic compounds have been forbidden due to their environmental and health hazards [52]. Indeed, traditional chemical biocides are usually aggressive towards the damaged substrates as well as to workers' health. The graphene-based nanomaterials, with their biocompatible properties, could represent an alternative to conventional treatments, to be applied as antimicrobial agents on stone monuments [53].

Conclusions
Our studies indicated that ZnO-based nanomaterials show antimicrobial and antibiofilm properties against different bacteria involved in stone biodeterioration. Indeed, results suggest that ZnO-NRs-decorated GNPs might be highly effective in preserving the surface of artwork in a cost-effective way that enables control of bacteria growth and therefore biodeterioration processes also at very low concentrations. These nanomaterials could be used to fight biodeterioration, offering the advantageous possibility of removing biofilm and limiting the consequent physical and chemical damages. ZNGs could avoid the use of traditional bioremediation reagents (such as biocide products) which otherwise, would require excessive and unfavorable chemical conditions. Furthermore, commercial chemical treatments can be hazardous for workers, the environment, and in some case for the artwork itself.