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Phosphonium-based ionic liquids as antifungal agents for conservation of heritage sandstone

With a view to preventing fungal deterioration of historical stone artworks, we report the use of phosphonium-based ionic liquids (ILs) as potent antifungal agents against dematiaceous fungi commonly found on heritage stones. Three ILs: tributyldodecylphosphonium polyoxometalate [P44412][POM], tributyltetradecylphosphonium polyoxometalate [P44414][POM], and trihexyltetradecylphosphonium polyoxometalate [P66614][POM] were prepared and their thermal stabilities and in vitro antifungal activities were evaluated. From the ramped temperature thermogravimetric analysis and antifungal experiments it can be clearly observed that the alkyl chain length of the tetraalkylphosponium cation has a significant influence on the thermal and antifungal properties. The thermal stability and antifungal activity decreased as the number of carbon atoms of the alkyl substituents increased and, thus, followed the order [P44412][POM] > [P44414][POM] > [P66614][POM]. In addition, inoculation of four fungal species on IL-coated sandstone surfaces showed significant inhibition of fungal growth, endowing the materials with potential applications in heritage sandstone conservation.


Introduction
The essential roles of fungal communities in the process of heritage stone deterioration are well known. 1-3 Different fungal species are mainly associated with the active biodeterioration process, thereby resulting in aesthetic and structural impairment of cultural heritage. 4,5 One of the most evident signs of aesthetic appearance issues is the colonization of melaninproducing dematiaceous fungi. [6][7][8] These dematiaceous fungi are not only able to survive under adverse environmental conditions, 9,10 but also may serve as potential human and plant pathogens. 11 Moreover, melanized fungal cells may resist killing by antibiotics or other anti-microbial treatments. 12,13 Some commercially available chemical products have been widely used to inhibit or eliminate fungal growth. [14][15][16] These methods do exhibit signicant disadvantages, 17,18 however, like their harmfulness to humans and the environment, their limited long-term efficiency, and their unwanted negative effects on stone monuments. A large number of novel biocides based on nanomaterials and natural compounds appear very promising to prevent the biodeterioration of historical stone materials. 19,20 Nevertheless, further studies are really needed to evaluate their potentially hazardous effects, antifungal efficiency, and durability. 21,22 Ionic liquids (ILs) as an innovative class of nonmolecular, tuneable, and remarkable chemical compounds, have applied in many areas of modern science. 23 In the eld of cultural heritage conservation, ILs have been successfully used for the removal of corrosion crusts and varnish from cultural heritage surfaces. 24,25 Moreover, ILs are also being considered as antibacterial and antifungal agents, 26 and have been widely exploited for preserving wood, 27,28 paper, 29,30 stone, 31,32 and brick 33 heritages against biodeterioration. The organic cations, typically imidazolium, alkylammonium, and alkylphosphonium, contribute to their superior antimicrobial activities. 34 Meanwhile, the biocidal activity is greatly affected by the alkyl chain length and positive charge density of cations, in which the higher charge density or moderate side chain length results in improved antimicrobial activity. 35,36 The anions, including simple halides, and organic and inorganic species, usually are employed to tune the physicochemical properties and play a signicant role in the design of multifunctional ILs. 37 Polyoxometalates (POMs) are a family of inorganic metal oxygen anion clusters and have received signicant interest because of their redox and topological properties. 38 Bijelic et al. focused on the antibacterial activity of POMs and underlined that POMs can also directly interact with proteins and enzymes, leading to serious damage within the bacterial cell. 39 Herrmann et al. designed a polyoxometalate supported ionic liquid phase (POM-SILP) composite for the removal of multiple contaminants from water, and results indicate that the multi-functional composite can effectively remove toxic heavy metals and organic pollutants. 40 Regarding the application of POMs-based ILs for the protection of cultural artifacts, to our knowledge, limited work has been done. 33 Misra et al. reported a multifunctional POM-ILs transparent coating for natural stones, and results show that the coating protects the stone surfaces from acid corrosion and bacterial colonization. 41 Considering the structural stability of POM anion and high positive charge of tetraalkylphosphonium cation, we designed to synthesize three ILs, namely [ (Fig. 1), aiming to prevent fungal contamination of stone surfaces. Phosphonium-based POM-ILs may possess higher thermal stability in comparison with its ammonium analogue. 42,43 Moreover, improved antimicrobial activity could also be achieved since the phosphorus atom has larger atomic radius and lower electronegativity, preferential binding to the negatively charged cell membrane and killing them. [44][45][46] 2. Materials and methods

Synthesis and characterization of ILs
Three phosphonium-based ILs were synthesized according to procedures reported previously. 41,47 Briey, 5 g of K 8 [a-SiW 11 O 39 ]$13H 2 O was dissolved in 100 mL of water and then the mixture was stirred at 50 C until completely dissolution. Subsequently, 1.55 mmol of the corresponding tetraalkylphosponium salt (dissolved in 100 mL of toluene) was added to the rst solution. The resulting biphasic solution was stirred vigorously for 25 min. Aer this time, the layers were separated, and the aqueous phase was extracted with toluene. The combined organic layers were evaporated under reduced pressure and the resulting light-yellow highly viscous liquid was washed once with toluene and twice with chloroform, and then dried under vacuum at room temperature for 96 h.
The elemental composition of K 8 [a-SiW 11 O 39 ]$13H 2 O was determined using inductively coupled plasma atomic emission spectrometry (ICP-AES, Thermo iCAP 6300). The elemental composition (C and H) of the three ILs was assayed on an Elementar Vario Micro Cube instrument. Fourier transform infrared (FTIR) spectra of the prepared ILs were recorded on a Thermo Scientic Nicolet iS10 spectrometer. Thermal gravimetric analysis (TGA) was performed on a Netzsch STA409PC instrument under nitrogen conditions, with a heating rate of 10 C min À1 in the temperature range of 25-600 C. Proton nuclear magnetic resonance ( 1 H NMR) spectra were recorded on a Bruker AVANCE III 500 MHz spectrometer. 1

In vitro assessment of antifungal activities of ILs
A disk diffusion assay was performed to determine the antifungal efficacy of different concentrations of ILs. Briey, 150 mL each of the conidial and mycelial suspensions was spread on potato dextrose agar (PDA) plates using a sterile glass spreader. Seven cylindrical wells with diameters of 7.5 mm were radially punched in the agar plate with a stainless-steel cylinder. Each well was lled with 100 mL of the as-prepared ILs dissolved in DMSO to obtain different concentrations 0.05, 0.1, 0.5, 1, 5, and 10 mg mL À1 . The well without ILs served as a negative control and uconazole served as a positive control. The plates were incubated at 30 C for 48 h and the inhibition diameter was calculated to evaluate the antifungal activities of the corresponding ILs. All assays were performed in triplicate.
A colony biolm formation test was performed on agar plates supplemented with different concentrations of ILs. Briey, all the synthesized ILs were dissolved in DMSO and mixed with a sterile molten PDA medium to achieve nal concentrations of 0.05, 0.1, 0.5, 1, 5, and 10 mg mL À1 . The PDA medium with a nal concentration of 1% DMSO (v/v) was used as a control. The mycelial disks (5 mm in diameter) of isolated fungi were inoculated on PDA plates and then incubated at 30 C for 3 d. All experiments were repeated three times. The percentage reductions in the biolm were calculated according to the following formula: where C and T are the diameters of the fungal biolms of the control and treatment samples, respectively. XTT-reduction assay was used to determine the in vitro activities of the ILs against fungal biolms (ESI Methods 1.1 †). 48 All experiments were performed in triplicate.

Propidium iodide uptake assay and scanning electron microscope observations
The fungal membrane permeabilization and cell viability were studied using a propidium iodide (PI) uptake assay. Briey, fungi were inoculated into 50 mL of potato dextrose broth and grown at 30 C for 4 d with vigorous shaking (160 rpm). Subsequently, the culture was washed with phosphate-buffered saline (PBS) three times and transferred to a 24-well plate wherein each well contained a mixture of RPMI 1640 medium and ILs. Final concentrations of 0.1 and 1 mg mL À1 were set up for each IL, and the well containing 1% DMSO served as a control. The cell suspensions were then incubated at 30 C for 12 h with continuous shaking (150 rpm). Aer incubation, the cell suspensions were centrifuged and washed twice in PBS. Staining was detected using a uorescence microscope (Olympus CX41) aer fungal cells were stained with PI for 30 min at room temperature.
A uorescence-based test was also conducted to determine the fungal cell viability on simulated sandstone samples (diameter, 20 mm; height, 8 mm) coated with three ILs. Sandstone samples were prepared according to procedures reported previously. 49  , respectively (10 mg mL À1 of IL dissolved in acetone, $3.18 mg cm À2 per sample), and an additional four samples were le uncoated. All samples were placed in an oven at 55 C for 2 h to allow the evaporation of the solvent, thereby yielding a thin IL layer. The fungal inoculum was deposited on sandstone surfaces and incubated in an incubator with a constant climate of 30 C and 80% relative humidity for 10 d. All samples were directly stained with PI and the generated biolm was visualized on a uorescence microscope. Additionally, the fungal morphology was observed using scanning electron microscope (SEM).
The color parameters of CIE L*a*b* were determined using a colorimeter Konica Minolta CR-10. The simulated sandstone samples were measured before and aer treatment with the ILs. The total colorimetric change was determined by DE, which was calculated by following equation: where, DL*, Da*, and Db* are the luminosity change, red-green parameter, and blue-yellow parameter, respectively.  Fig. 2a, together with the spectrum of the POMs. A decrease in the absorption peak intensity at 3415 and 1632 cm À1 is observed in the three ILs, suggesting that little water is present in the ILs. Three bands are observed at 2956, 2923, and 2853 cm À1 , which are attributed to the presence of -CH 3 and -CH 2 groups. Meanwhile, three ILs present the new characteristic band at 1463 cm À1 which are assigned to P-CH 2 vibration. 50,51 The structure of the ILs is also conrmed by 1 H NMR spectroscopy. As shown in Fig. 2b, the NMR signals correspond to the tetraalkylphosponium groups. Therefore, it is concluded that the three ILs are synthesized successfully. TGA of the three ILs was performed under an N 2 atmosphere, from room temperature to 600 C. The TGA spectra of the ILs and its derivative curves are shown in Fig. 2c and  [POM] occur at 350.5 C (T onset ; T max ¼ 391 C), 335.8 C (T onset ; T max ¼ 382.6 C), and 308.3 C (T onset ; T max ¼ 381.2 C), respectively, these onset temperatures consistent with literature reports indicating thermal stabilities of many phosphonium-based ILs exceeding 300 C. 43 Apparently, increasing the alkyl chain length decreases the decomposition temperature and thermal stability, and the same trend is observed in a series of ILs, 52,53 which may be attributed to the increased stability of the carbocation and/or carbon radicals when the alkyl chain increases. 54,55 Moreover, it was clear that phosphonium-based POM-ILs are found to have a higher thermal stability than its ammonium analogue. 42  ramped temperature TGA method exhibits a wide working temperature range of three ILs, time consuming isothermal TGA analyses will be necessary to obtain more profound and accurate information on the thermal stability properties. 57

The antifungal activities of [P 44412 ][POM], [P 44414 ][POM], and [P 66614 ][POM]
were determined by a disk diffusion method in terms of the zone of inhibition of fungal growth (Fig. 3a). Fig. 3b-e show the antifungal activities of different concentrations (0.05, 0.1, 0.5, 1, 5, and 10 mg mL À1 ) of ILs and uconazole against Curvularia sp. F3, A. aculeatinus F6, Aporospora sp. F7, and Alternaria sp. F8. Remarkably, A. aculeatinus F6, Aporospora sp. F7, and Alternaria sp. F8 are especially sensitive to the most common antifungal uconazole and the diameter of the inhibition zone as high as 27.5 AE 2.3 mm is observed (Fig. 3c) does not exhibit any evident inhibition zone against the four fungal isolates, which is likely due to its longer alkyl chains compared to those of the other two ILs. Specically, there are no signicant differences in the reduction rates of the control and treatment (0.05 and 0.1 mg mL À1 ) groups, suggesting that the four fungi are less susceptible to lower doses of ILs. Fig. 4 shows the inhibition ratios of the three ILs at different concentrations (0.05, 0.1, 0.5, 1, 5, and 10 mg mL À1 ). For Curvularia sp. F3 (Fig. 4a) shows similar antifungal activities when the concentrations of the ILs are lower than 1 mg mL À1 , even though the alkyl groups of the two ILs are different (Fig. 4c). In the assays of the three ILs against Alternaria sp. F8 (Fig. 4d) (Fig. 5a) (Fig. 5b). Aer treatment with [P 44412 ] [POM] and [P 44414 ][POM] at concentrations of 0.05 mg mL À1 , the metabolic activity of Aporospora sp. F7 is reduced by 56% and 55.2%, respectively, and thus, these ILs exhibit a higher antifungal activity than that of [P 66614 ][POM] (26%) (Fig. 5c). To achieve 50% growth inhibition of Alternaria sp. F8 with the control group, a concentration of 0. groups, the required concentration is higher than 0.5 mg mL À1 (Fig. 5d).
As shown in Fig. 3 does not exhibit any evident inhibition zone, even at a high concentration (5 and 10 mg mL À1 ). In the colony biolm formation and XTT-reduction assays, [P 66614 ][POM] is found to have high antifungal activity, especially when the [P 66614 ][POM] concentration is higher than 0.5 mg mL À1 . The fact may be related to the strong electrostatic interaction between phosphonium cation and bulky POM anion limits the diffusion of antimicrobial cation species into the agar medium and subsequent delivery to microbial cells. 58 This conclusion is consistent with previous ndings of Rajkowska et al., 33 who assayed the antifungal activities of ammonium-based POM-ILs and each component alone and revealed that diffusion-based antifungal activities of POM-ILs arise primarily from the cation species. Additionally, it is clear that cationic biocides kill bacteria by binding to negatively charged cell surface and disrupting the cell membranes, in which different charge distributions and alkyl chain length play a key role in the antibacterial efficacy. 46,59 In this study, three phosphonium-based ILs with different long alkyl chains (C 12 and C 14 ) exhibit antifungal activity, which is consistent with previous reports indicating that phosphonium compounds containing a long alkyl chain (C 8 -C 18 ) usually exert antimicrobial capabilities. 45,60,61 Regarding the [P 44412 ][POM] with a shorter alkyl chain, a high level of antifungal activity is observed, which may be affected by many potential important factors, such as hydrophobic-hydrophilic balance, 62,63 adsorption, and critical micelle concentration. 64 Because phosphonium-based cations can adsorb negatively charged cell walls and membranes via electrostatic interactions and disrupt the cytoplasmic membrane via the hydrophobic alkyl chains, 44,46 thereby leading to cell death, PI uorescent dye was used to evaluate the antifungal efficiencies of the three ILs. As shown in Fig. 6 (Fig. S1 †).
To determine the antifungal effectiveness of the ILs that are applied to the simulated sandstone, the inhibition of the growth of Curvularia sp. F3, A. aculeatinus F6, Aporospora sp. F7, and Alternaria sp. F8 is studied using a uorescence-based test. Fig. 7 shows the optical and uorescence microscopy images of  Fungi incubated over the [P 44412 ][POM]-coated sandstone were further examined by SEM and the typical images are displayed in Fig. 8. In control images, fungal spores and mycelium exhibit a normal shape with intact membranes/walls. In contrast, aer exposure to [P 44412 ][POM], signicant cellular morphologic changes (smooth surfaces become rough and irregular) are observed, which are indicative of irreversible membranes damage. 65 Additionally, the color variation is characterized before and aer the ILs application by spectrophotometric technique. The changes in the colorimetric values (i.e., DE) of the sandstone samples coated with 10 (Fig. S2 †), showing no signicant color changes and thus their potential as antifungal agents for heritage sandstones.

Conclusions
In summary, three phosphonium-based ILs, [ , conrming that the optimized chain length of alkyl substitution on tetraalkylphosponium cation enhances the antifungal activity. More importantly, the application of ILs-based coatings on sandstone sample exhibits strong antifungal activity and no signicant color changes, which makes it a promising candidate for the treatment of invasive fungi colonizing heritage sandstone. These results are preliminary and future studies will further investigate the antifungal performances of ILs under in situ conditions.

Conflicts of interest
There are no conicts to declare.