Antifungal Activity of Polyoxometalate-Ionic Liquids on Historical Brick

Moulds inhabiting mineral-based materials may cause their biodeterioration, contributing to inestimable losses, especially in the case of cultural heritage objects and architectures. Fungi in mouldy buildings may also pose a threat to human health and constitute the main etiological factor in building related illnesses. In this context, research into novel compounds with antifungal activity is of high importance. The aim of this study was to evaluate the antifungal activity of polyoxometalate-ionic liquids (POM-ILs) and their use in the eradication of moulds from historical brick. In the disc diffusion assay, all the tested POM-ILs inhibited growth of a mixed culture of moulds including Engyodontium album, Cladosporium cladosporioides, Alternaria alternata and Aspergillus fumigatus. These were isolated from the surfaces of historical brick barracks at the Auschwitz II-Birkenau State Museum in Oświęcim, Poland. POM-IL coatings on historical brick samples, under model conditions, showed that two compounds demonstrated very high antifungal activity, completely limiting mould growth and development. The antifungal activity of the POM-ILs appeared to stem from their toxic effects on conidia, as evidenced by environmental scanning transmission electron microscopy observations. The results herein indicated that POM-ILs are promising disinfectant materials for use not only on historical objects, but probably also on other mineral-based materials.


Introduction
The susceptibility of brick, concrete, mortar, stone and other mineral-based building materials to colonisation by various organisms is defined as bioreceptivity. This concept was introduced by Guillette [1] and included chemical and physical parameters such as mineralogical composition, water availability, temperature, pH, surface porosity and roughness influence on biological succession [2,3]. From a biological point of view, mineral-based building materials represent an extreme microniche for microbial growth due to large variations in environmental parameters and higher desiccation conditions in vertical and subvertical surfaces [3]. Bacteria and fungi inhabit pits, cracks and fissures on the surface of mineral-based materials, and they often may interact with substrate causing their deterioration [4]. Microorganisms, especially fungi, as a result of metabolic activity, secrete different corrosive organic

Results and Discussion
A total of eight silicotungstate and phosphotungstate-based POM-ILs were synthesized based on previously reported experimental procedures. Please refer to Supplementary Materials for further details on synthesis and Tables S1-S4 for characterisation data. Table 1 provides a summary of the eight different POM-ILs employed in this study. Table 1. Summary of the POM-ILs.

POM-IL POM-IL Short Name
A disc diffusion method was used to assess the in vitro antifungal activity of eight different but closely related POM-ILs and their capacity to prevent or inhibit mould growth on historical brick samples. The antifungal activity was examined against a mixed culture of moulds consisting of Engyodontium album, Cladosporium cladosporioides, Alternaria alternata and Aspergillus fumigatus, that were isolated from the surfaces of historical brick barracks at the Auschwitz II-Birkenau State Museum in Oświęcim, Poland. The use of mixed culture of moulds is especially important since masonry building parts are usually inhabited not by one single strain but by a mixture of microorganisms [29]. Moreover, the environmental strains which were isolated from historical brick objects were adapted to nutrient-poor materials.
In the disc diffusion assay, all the tested POM-ILs inhibited mould growth, despite their poor aqueous solubility, although clear differences in diameters of inhibition growth zones were observed ( Figure 1). The highest antifungal activity was exhibited by [PW 12 3 . Other compounds acted only when the disc was in direct contact with the mould cultures, and then the growth inhibition zones equalled 6 mm. None of the anionic controls of POM-ILs showed activity against moulds, while cationic controls were highly active. The highest activity was found for [THepA] + and [THexA] + (Figure 1). These results indicate that diffusion-based antifungal activity of POM-ILs resulted mostly from activity of the cation species, which is consistent with previous findings of Kubo et al. [26], who explained that the antibacterial activity of these compounds arises primarily from the cationic action.
While the diffusion method is a standard method for evaluating antimicrobial compounds and materials, however, its principal limitation is that the assay requires the diffusion of compounds out of the discs and into the agar nutrient medium. In the case of hydrophobic compounds and water insoluble compounds, their diffusion and delivery to microbial cells in aqueous media may be hampered [30,31]. The POM-ILs in this report are characterised by high viscosity and water immiscibility [27].
Considering the water immiscibility and high viscosity of these POM-ILs, their antifungal activity was also determined on historical brick samples under model conditions with high mould content (>10 6 cfu/cm 2 ). Briefly, 19th century brick samples were inoculated with moulds and allowed to colonise the brick surface in a climate chamber (80% relative humidity and 28 • C) for three weeks. Afterwards, POM-ILs were spray-coated up to three times and the number of microorganisms after successive POM-IL applications was determined by the contact plate method whereby the antifungal activity of POM-ILs was evaluated according to a calibration scale (please refer to Materials and Methods figure).  4 , can be effectively used in disinfection of historical brick, although the following key issues should be considered. Considering the water immiscibility and high viscosity of these POM-ILs, their antifungal activity was also determined on historical brick samples under model conditions with high mould content (>10 6 cfu/cm 2 ). Briefly, 19th century brick samples were inoculated with moulds and allowed to colonise the brick surface in a climate chamber (80% relative humidity and 28 °C) for three weeks. Afterwards, POM-ILs were spray-coated up to three times and the number of microorganisms after successive POM-IL applications was determined by the contact plate method whereby the antifungal activity of POM-ILs was evaluated according to a calibration scale (please refer to Materials and Methods figure). In contrast to the disc diffusion studies, the historical brick studies demonstrated In this study, POM-ILs solutions were applied by spraying and this method was chosen to minimise the risk of mechanical removal of fungal mycelium from the surface of bricks. Consequently, this method can result in a heterogeneous coverage of the compounds on the brick surface. For this reason, if POM-ILs are to be used as protective antimicrobial coatings, it is preferable to use the brush-coating method to protect the surface of materials against the development of microorganisms, as has been reported for POM-IL coatings on natural stones [27]. Other important factors determining the activity of biocides are their concentration, the number of applications, type of material and, what is particularly important, the intensity of microbial growth already present on the material surface. Only in objects heavily contaminated by microorganisms and in favourable environmental conditions, macroscopic signs of microbial growth are visible and the number of moulds exceeds 10 6 cfu/cm 2 , as is seen in this study. In general, for historical objects, fungi were detected at a lower level of 10 1 -10 3 cfu/100 cm 2 [32], and on building materials characterised by spore presence and active mould growth, the fungal contamination is estimated at 10 4 -10 5 cfu/m 2 [33]. In this study, POM-ILs solutions were applied by spraying and this method was chosen to minimise the risk of mechanical removal of fungal mycelium from the surface of bricks. Consequently, this method can result in a heterogeneous coverage of the compounds on the brick surface. For this reason, if POM-ILs are to be used as protective antimicrobial coatings, it is preferable to use the brush-coating method to protect the surface of materials against the development of microorganisms, as has been reported for POM-IL coatings on natural stones [27].
Other important factors determining the activity of biocides are their concentration, the number of applications, type of material and, what is particularly important, the intensity of microbial growth already present on the material surface. Only in objects heavily contaminated by microorganisms and in favourable environmental conditions, macroscopic signs of microbial growth are visible and the number of moulds exceeds 10 6 cfu/cm 2 , as is seen in this study. In general, for historical objects, fungi were detected at a lower level of 10 1 -10 3 cfu/100 cm 2 [32], and on building materials characterised by spore presence and active mould growth, the fungal contamination is estimated at 10 4 -10 5 cfu/m 2 [33]. Therefore, the real-world use of POM-ILs as antifungal agents in situ is expected to be more effective than under the more extreme model conditions reported here.
In this context, the results obtained for the tested POM-ILs are very promising in the aspect of effective use on masonry surfaces of buildings, including historical objects. In addition to the potent antimicrobial activity of the quaternary ammonium cations that largely affected the conidia plasma membranes, we hypothesised that POM-IL coatings interfere with electron transfer and respiration processes and the overall applicability and potential as protective surface coatings are also closely related to the viscosity and hydrophobicity of the material. To further analyse the antifungal activity of the most active POM-ILs from this study, environmental scanning transmission electron microscopy (ESTEM) was used to study the effect of [SiW 11  After the first application, solutions not only covered the mycelium and conidia, but also penetrated deep into the spatial moulds structure. After three applications, POM-ILs homogeneously covered the surface of the material together with the developed mycelium ( Figure 3) (Figure 3 and Figure S1). In control images, conidia were collapsed as a result of moderate dehydration and a loss of turgor pressure under the ESTEM sample collection conditions. Such morphological changes are typical for conidia with intact membranes [34]. After the POM-ILs application, conidia were generally spherical, which is indicative of membrane damage and collapse. Similar results have been published previously for quaternary phosphonium ionic liquids, the mechanism of action of which are involved in damaging conidia plasma membrane [35].
In this context, the results obtained for the tested POM-ILs are very promising in the aspect of effective use on masonry surfaces of buildings, including historical objects. In addition to the potent antimicrobial activity of the quaternary ammonium cations that largely affected the conidia plasma membranes, we hypothesised that POM-IL coatings interfere with electron transfer and respiration processes and the overall applicability and potential as protective surface coatings are also closely related to the viscosity and hydrophobicity of the material. To further analyse the antifungal activity of the most active POM-ILs from this study, environmental scanning transmission electron microscopy (

ESTEM) was used to study the effect of [SiW11O39][THTDA]8 and [SiW12O40][THTDA]4 POM-ILs on fungal mycelium and conidia.
After the first application, solutions not only covered the mycelium and conidia, but also penetrated deep into the spatial moulds structure. After three applications, POM-ILs homogeneously covered the surface of the material together with the developed mycelium ( Figure 3) (Figure 3 and Figure S1). In control images, conidia were collapsed as a result of moderate dehydration and a loss of turgor pressure under the ESTEM sample collection conditions. Such morphological changes are typical for conidia with intact membranes [34]. After the POM-ILs application, conidia were generally spherical, which is indicative of membrane damage and collapse. Similar results have been published previously for quaternary phosphonium ionic liquids, the mechanism of action of which are involved in damaging conidia plasma membrane [35]. The results presented herein for POM-ILs are consistent with those reported for ionic liquids; this reinforces the theorem on the key role of cation species in antifungal activity. The antimicrobial mechanism of ionic liquids results from their amphiphilic character allows their insertion into the phospholipid bilayer of plasma membranes [36]. These interactions lead to plasma membrane damage, leakage of intracellular content, and, in consequence, to cell death [37]. In moulds, substantial damages to conidia and hyphae provoked by long alkyl chains were also documented The results presented herein for POM-ILs are consistent with those reported for ionic liquids; this reinforces the theorem on the key role of cation species in antifungal activity. The antimicrobial mechanism of ionic liquids results from their amphiphilic character allows their insertion into the phospholipid bilayer of plasma membranes [36]. These interactions lead to plasma membrane damage, leakage of intracellular content, and, in consequence, to cell death [37]. In moulds, substantial damages to conidia and hyphae provoked by long alkyl chains were also documented [38]. The severe ultrastructural changes in cells were associated with membrane destruction, distending of cell wall structure, formation of residual organelles in the form of membranous structures and multivesicular bodies. On the other hand, the high negative charge of polyoxometalates means that they do not readily penetrate through cytoplasmic membranes, but they can cause impairments of vital cell functions in bacteria by interaction with the proteins involved in signal-transducing systems [20]. Furthermore, POMs can interfere with bacterial electron transfer, thereby affecting respiration processes [39]. The combination of two active components in the POM-IL molecule is clearly an important antimicrobial activity enhancing property, however, the exact antifungal mode of action of POMs is still not understood and will require further detailed mechanistic experimentation.

Synthesis and Characterisation of POM-ILs
POM-ILs were synthesised according to previously reported synthetic procedures [26,27]. Briefly, a desired amount of POM was dissolved in 50 mL of pre-heated distilled water at 50 • C. The mixture was stirred until complete dissolution. In a beaker, the appropriate amount of the corresponding ionic liquid was dissolved in 80 mL of the solvent and stirred until complete dissolution. Then, the POM solution was slowly added to the IL solution and the mixture was stirred for 3 h at room temperature. After this time, the mixture was transferred to a separation funnel and once the two phases were clearly separated, the organic phase-including the POM-IL-was extracted into a round bottom flask. The organic solvent was removed in a rotatory evaporator, and the product was isolated in the form of powder or viscous liquid at room temperature.

Assessment of Antifungal Activity of POM-ILs by Disc Diffusion Method
In the assay, four mould strains in a mixed culture were used, i.e., Engydontium album LOCK 0590, Cladosporium cladosporioides LOCK 0592, Alternaria alternata LOCK 0594 and Aspergillus fumigatus LOCK 0596. All moulds were isolated from the surfaces of historical brick barracks at the Auschwitz II-Birkenau State Museum in Oświęcim, Poland. Antifungal activity was determined for POM-ILs solutions in dimethyl sulfoxide (DMSO) at a concentration of 5 mg/mL, by the disc diffusion method. Briefly, after spreading 100 µL of mixed culture of moulds in sterile saline (10 6 conidia/mL) on malt extract agar (MEA, Merck KGaA, Darmstadt, Germany), paper discs with 20 µL of POM-ILs were placed on the agar surface. DMSO (100%) served as a control. Plates were incubated for up to 5 days at 28 • C. The results were expressed as diameters of growth inhibition zones including the diameter of the disc (Ø 6 mm).

Evaluation of Antifungal Activity on Brick Samples
Antifungal activity of POM-ILs was assessed on historical brick under model conditions, as described previously [40]. One millilitre of mixed culture of moulds (10 6 conidia/mL) in the medium ((NH 4 ) 2 SO 4 0.075%, K 2 HPO 4 0.025%, MgSO 4 ·7H 2 O 0.125%, yeast extract 0.125%, glucose 0.5%, agar 0.1%, pH 6.0) was applied on the surface of brick. Brick samples (50 × 20 × 10 mm 3 ) came from the demolition of a 19th century residential building. The samples inoculated with moulds were incubated in a constant climate chamber with a relative humidity of 80% for 21 days at 28 • C. Afterwards, POM-ILs were applied up to three times at an interval of 24 h, using the spraying method. The number of microorganisms after successive applications of POM-ILs was determined by the contact plate method in MEA medium. Plates were incubated for 5 days at 28 • C. The antifungal activity of POM-ILs was evaluated according to a calibration scale (Figure 4).

Environmental Scanning Electron Microscopy (ESEM)
Data were collected on a Quanta FEG-250 (FEI Company, Hillsboro, OR, USA) field emission SEM for high-resolution imaging working in ESEM mode, using a gaseous secondary electron detector (GSED) detector under high relative humidity conditions.

Conclusions
The tested POM-ILs as part of this project showed high antifungal activity against a mixed culture of moulds that are well-adapted to the extreme conditions of mineral-based building materials. The inhibition of mould growth by POM-ILs occurs through toxic effects on conidia, and by coating the surface of the brick material, which may consequently limit the access to oxygen and nutrients. Although the antimicrobial activity of POM-ILs was mostly associated with cations, the combination of two components in the molecule gives them unique properties such as water immiscibility and creates the opportunity to design compounds with the desired characteristics for reducing microbially induced biodeterioration.
As a result of these promising features, POM-ILs could be used as antifungal agents in a variety of applications, not limited to historical brick, as described in this current study. Furthermore, the use of POM-ILs to disinfect or form protective coatings on other mineral-based building materials or historical objects, as well as in residential buildings can also be envisaged.  Table S1: Summary of the POM-ILs used in this work, their POM and ionic liquid precursors and the solvent used for their synthesis. Table S2: Thermogravimetric analysis of POM-ILs (MW = molecular weight). Table S3: Elemental analysis of POM-ILs. The values are given as mass percentages (%). Table S4: FT-IR analysis of POM-ILs (cm −1 ).

Conclusions
The tested POM-ILs as part of this project showed high antifungal activity against a mixed culture of moulds that are well-adapted to the extreme conditions of mineral-based building materials. The inhibition of mould growth by POM-ILs occurs through toxic effects on conidia, and by coating the surface of the brick material, which may consequently limit the access to oxygen and nutrients. Although the antimicrobial activity of POM-ILs was mostly associated with cations, the combination of two components in the molecule gives them unique properties such as water immiscibility and creates the opportunity to design compounds with the desired characteristics for reducing microbially induced biodeterioration.
As a result of these promising features, POM-ILs could be used as antifungal agents in a variety of applications, not limited to historical brick, as described in this current study. Furthermore, the use of POM-ILs to disinfect or form protective coatings on other mineral-based building materials or historical objects, as well as in residential buildings can also be envisaged.