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Synthesis, photocatalytic, and antifungal properties of MgO, ZnO and Zn/ Mg oxide nanoparticles for the protection of calcareous stone heritage Aránzazu Sierra Fernández, Susana Del Carmen De la Rosa García, Luz Stella GomezVillalba, Sergio Gomez-Cornelio, María Eugenia Rabanal, Rafael Fort, and Patricia Quintana ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.7b06130 • Publication Date (Web): 05 Jul 2017 Downloaded from http://pubs.acs.org on July 6, 2017
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Synthesis, photocatalytic, and antifungal properties of MgO, ZnO and Zn/Mg oxide nanoparticles for the protection of calcareous stone heritage A. Sierra-Fernandez1, 2*, S.C. De la Rosa-García3*, L.S. Gomez-Villalba1, S. Gómez-Cornelio3, M.E. Rabanal2, 4 , R. Fort1, P. Quintana5 1
Instituto de Geociencias (CSIC, UCM), C/ José Antonio Novais 12, CP 28040, Madrid, Spain. Carlos III University of Madrid, Department of Materials Science and Engineering and Chemical Engineering, Avda. Universidad 30, 28911 Leganés, Madrid, Spain. 3 Laboratorio de Microbiología Aplicada, División de Ciencias Biológicas, Universidad Juárez Autónoma de Tabasco (UJAT), Tabasco, México. 4 Instituto Tecnológico de Química y Materiales “Álvaro Alonso Barba” (IAAB), Avda. Universidad 30, 28911 Leganés, Madrid, España 5 Departamento de Física Aplicada, CINVESTAV-IPN, A.P.73, Cordemex, Mérida, Yucatán, México. 2
* Corresponding author. Email address:
[email protected] (A. Sierra-Fernandez),
[email protected] (S.C. De la Rosa-García) Abstract More recently, the biological colonization of stone heritage and consequently its biodeterioration has become the focus of numerous studies. Among all microorganisms, fungi are considered to be one of the most important colonizers and biodegraders on stone materials. This is why the development of new antifungal materials requires immediate action. ZnMgO nanoparticles (NPs) have several exciting applications in different areas, highlighting as an efficient antimicrobial agent for medical application. In this research, the application of Zn-doped MgO (Mg1−xZnxO, x=0.096) NPs obtained by sol-gel method as antifungal coatings on dolomitic and calcitic stones has been explored as a means to develop effective protective coatings for stone heritage. Moreover, the photocatalytic and antifungal activity of Mg1−xZnxO NPs were comparatively studied with single ZnO and MgO NPs. Thus, compared to the MgO and ZnO nanomaterials, the Mg1−xZnxO NPs exhibited an enhanced photocatalytic activity. After UV irradiation for 60 min, 87% methylene blue was degraded over Zn-doped MgO NPs, whereas only 58% and 38% of MB was degraded over ZnO and MgO NPs, respectively. These nanoparticles also displayed a better antifungal activity than that of single pure MgO or ZnO NPs, inhibiting the growth of fungi Aspergillus niger, Penicillium oxalicum, Paraconiothyrium sp., and Pestalotiopsis maculans, which are especially active in the bioweathering of stone. The improved photocatalytic and antifungal properties detected in the Mg1−xZnxO NPs was attributed to the formation of crystal defects by the incorporation of Zn into MgO. The application of the MgO and Zn-doped MgO NPs as protective coatings on calcareous stones showed important antifungal properties, inhibiting successfully the epilithic and endolithic colonization of A. niger and P. oxalicum in both lithotypes, and indicating a greater antifungal effectiveness on Zndoped MgO NPs. The use of Zn-doped MgO NPs may thus represent a highly efficient antifungal protection for calcareous stone heritage. Keywords: Stone biodeterioration; Sol-gel method; MgO nanoparticles; ZnO nanoparticles; Mg1−xZnxO nanoparticles; Antifungal coatings; Photocatalytic property; Photoluminescence 1 ACS Paragon Plus Environment
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1. Introduction The effects of microclimate variations and biological decay on the historic monuments are of great importance in the field of stone conservation. One of the main degradation processes of stone heritage is produced by the growth of a wide variety of microorganisms, among which fungi represent one of the most important for the generation of undesirable changes in the properties of the stone material (biodeterioration) and for the biotic erosion and decay of stone substrates and minerals (bioweathering). 1-2 At the same time, bioweathering of stone is an important field in geomicrobiology. 3 The main types of building materials affected by fungal growth are limestone, 4 dolostone, 5-6 sandstone, 7 marble, 8 granite 9 as well as mortars and stuccos, which can also be extremely susceptible to bioweathering. 10-11 Thus, filamentous microorganisms may grow on the rock surfaces and building stone (epilithic), in pores, cracks and fissures of the stone sub-surface (endoliths), as well as inside cavities and among crystal grains (cryptoendoliths), 1 leading directly and indirectly to bioweathering. This growth and activity of the microorganisms on monuments or stone surface involves important physical and biochemical alterations: penetration by filamentous microorganisms, bioweathering through excretion of H+, CO2 or organic and inorganic acids, among other, staining or color alteration, surface alterations (pitting, etching, stratification and complete dissolution), and transformation of crystal into small size. 1 Besides, microbial exopolymers and organics acids are also involved in biocorrosion by metal complexation as well as acid effects 12 and the growth of black fungi on white stone surfaces causes a selective absorption of solar radiation. This leads to a local extension of crystals and thus discontinuities in the stone matrix which also causes crystal decohesion. 13 In this context, nanostructured semiconductors might be an effective tool for controlling the bioweathering because present great advantages (high surface to volume ratio and small particle size). 14-15 Among these nanomaterials, titanium dioxide has attracted special attention in the development of antibacterial and antifungal coatings. 16-17 However, the presence of light is generally required for achieving good performance, which limits its potential applications. 18 Thus, magnesium oxide is particularly interesting as a low cost and environmental friendly material, with interesting antimicrobial activity without the presence of light. 19-20 As well as MgO NPs, nanostructured zinc oxide (ZnO) has shown to be bio-safe and bio-compatible and possesses antibacterial and antifungal activity due to their surface-chemical activity. 21-22 In addition, ZnO NPs are regarded as an excellent photocatalyst for the degradation of organic pollutants due to their high photosensitivity. 23 The present research aims to combine the strong antimicrobial activity of ZnO with the safe-to use antimicrobial effectiveness and good compatibility of MgO with stone materials in order to develop antifungal coatings highly potential for stone conservation. The idea to join both types of metal oxides is particularly interesting because the presence of both lattices could develop point defects into the crystals, modifying the photocatalytic and antifungal properties. This is why, MgO, ZnO and Zn-doped MgO (Mg1−xZnxO, x=0.096) NPs obtained by sol-gel method were studied. This synthesis is an effective way to obtain nanoparticles with controlled-morphology, narrow particle size distribution and high phase compositional homogeneity. 24 The photocatalytic properties of MgO, ZnO and Mg1−xZnxO NPs 2 ACS Paragon Plus Environment
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were further evaluated in terms of methylene blue (MB) solution degradation under UV-light irradiation. The antifungal activity of single and mixed-metal oxides NPs were assessed against Aspergillus niger, Penicilium oxalicum, Paraconiothyrium sp., and Pestalotiopsis maculans. All these fungus were isolated from stones with a black biogenic surface, and are of substantial concern because they have shown to be potentially active in solubilising calcium carbonate plates and limestone through the production of oxalic acid. 25 Moreover, the application of these NPs as antifungal coatings has been studied both on glass slides and two types of calcareous stones, widely used in the cultural heritage of Spain and Mexico. The microorganisms-stone interfaces have been also investigated in all stone samples, with a particular focus on the spread and the depth of the hyphal penetration component. The aim was to examine the influence of the type of nanoparticles-based treatment and the petrophysical properties of the stone over the susceptibility of stone substrates for fungal colonization. 2. Experimental Section 2. 1. Synthesis and characterization of metal oxide nanoparticles MgO, ZnO and Zn-doped MgO (Mg1−xZnxO, x=0.096) NPs were prepared by sol-gel method. All the chemical reagents used in this work, magnesium ethoxide (Mg(OC2H5)2), zinc acetate dihydrate (Zn(CH3COO)2 2H2O ) and sodium hydroxide (NaOH), were purchased from Sigma-Aldrich and used as received, without further purification. In a typical procedure, MgO was synthesized from denatured ethanol mixing magnesium ethoxide (Mg(OC2H5)2, 0.5M, (Aldrich, 98%) with NaOH 0.2M, (Aldrich, 98%). ZnO was obtained by adding zinc acetate dihydrate (Zn(CH3COO)2 2H2O at a concentration of 0.62M, (Aldrich, 98%) into denatured ethanol. Then, sodium hydroxide (NaOH) at a concentration of 0.3M (Aldrich, 98%) was gradually added. In both synthesis procedures, the two solutions were vigorously stirred at room temperature for 24 h. Mg1−xZnxO NPs (x=0.096) were synthesized from denatured ethanol using Mg(OC2H5)2 (0.5M) and Zn(CH3COO)2·2H2O (0.62M). The two solutions were mixed and kept at room temperature with vigorous stirring. Subsequently, NaOH (2M) water-based solution was added to the solution. The reaction mixture was stirred for 24 h at room temperature. The obtained samples were centrifuged and washed using distilled water and ethanol and then, were dried in an inert gas atmosphere at 70 °C. Finally, the samples were annealed in a programmable muffle furnace at 650 °C at a rate of 2 °C/min for 3 h. For the application on glass and stone substrates, the nanoparticles were dispersed in ethanol by vigorous stirring followed by ultrasound treatment for 15 min. The concentration of the nanoparticles in the dispersion was 2.5 gL-1. The phase purity and crystallographic structures of the oxide nanoparticles were studied by XRay Diffraction (XRD). Measurements were carried out on a Siemens D-5000 diffractometer (Cu Kα radiation, λ=1.5418 Å). Rietveld refinements were determined for unit cell parameters calculation and phase quantification using the Fullprof program. 26 The crystallite size of the nanoparticles was determined by the Scherrer´s formula, D=Kλ/βcosθ, where λ is the X-ray wavelength used (λ=1.54 Å), K is the particle shape factor which is a constant taken as 0.9, β the full width at half maximum height (FWHM), and θ is the diffraction angle (in rad) used in calculus. 27 The most intensive reflection peaks of the samples were used in the line broadening analysis. The morphological microstructural and chemical analysis of the different samples were conducted by Field emission scanning electron microscopy (FESEM) (JEOL JSM-7600F) and Transmission 3 ACS Paragon Plus Environment
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Electron Microscopy-Selected Area Electron Diffraction (TEM-SAED), equipped with Energy Dispersive X ray spectrometer (EDS) (JEOL JEM 2100 at 200 kV). The distribution and average size of the nanostructures obtained were determined from TEM images using the Digital Micrograph™ (DM, Gatan Inc.) software. The photocatalytic efficiency was evaluated by degradation of methylene blue solution under UV-light irradiation. Typically, 0.003 g of synthesized nanoparticles was dispersed in 50 mL (1.88 × 10−5 M) MB aqueous solution with continuous stirring, and was enclosed in order to avoid effects of spurious external light. Prior to irradiation, the suspensions were magnetically stirred in dark for 2 h to reach the adsorption-desorption equilibrium. Then, the photocatalytic degradation of synthesized samples was carried out in continuous stirring under UV-light irradiation (250 nm) from a 20W halogen lamp (Pen-Ray PS1 (model UVP-11Sc-1) with a typical intensity of 4.1 mW.cm2. The solutions, collected at definite intervals, were centrifuged in order to remove the catalysts; their absorbance was analyzed on a UV-Vis spectrophotometer (Agilent, 8453 diode array). Room temperature photoluminescence (PL) spectra were recorded by fluorescence photoluminescence spectrophotometry (Edinburg Instruments Co., 235 nm xenon lamp). The PL emission spectra were recorded in the wavelength range from 375 to 600 nm using an excitation wavelength λexc = 320 nm. The measurement of the PL excitation spectra were performed from 250 to 420 nm for the emission wavelength λem = 450 nm. In all measurements, the scan speed was 100 nm/min with a slit size of 10 nm. For sample preparation, the obtained samples were made as thin layer on the ultrasonically cleaned (in ethanol) glass slides and loaded in the sample holder. 2.2. Fungal suspension preparation Fungal inoculums were prepared by growing Aspergillus niger (R3T2M774) and Penicillium oxalicum (R3T3M877), Paraconiothyrium sp. (R1T2C113), and Pestalotiopsis maculans (R3T2C756) on Potato Dextrose Agar (PDA, Difco) plates at 28 °C for 4-15 days. All fungi were isolated from limestones with a black biogenic surface by washing and filtration of particles technique (25). This technique reduces the isolation of propagules from spores, favoring only the isolation of fungi attached to rock particles and with metabolism active in the substrate. These fungi are particularly interesting because they have shown to be potentially active in solubilising calcium carbonate plates and limestone because they secrete enzymes and organic acids during their metabolic processes (data not shown). Fungal growth from plates was flooded using a sterile saline solution (0.85%) with 0.025% of Tween 20 (Sigma, Aldrich), and stirred gently with a sterile swab. The spore concentrations in the stock suspensions were determined using a Neubauer chamber and were adjusted to 1 × 106 conidia/mL. 2.3. In vitro evaluation of antifungal activity of metal oxide nanoparticles The antifungal activity of the different synthesized nanoparticles was studied using the agar well diffusion assay for A. niger and P. oxalicum. Briefly, 3.5 mL of each conidial suspensions were mixed with 31.5 mL molten PDA and poured slowly on the square Petri dish with 12 stainless steel cylinder, when the agar layer solidified, the cylinders were retired carefully. Each well was filled with 100 µL of the asprepared samples dissolved in dimethylsulphoxide (DMSO) to get different concentrations 10, 5 and 2.5 mg/mL. 100 µL of DMSO was utilized as negative control. The Petri dishes were incubated at 5 °C for 4 h
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to permit good diffusion and then incubated at 28 °C for 48-72 h. The antifungal activities were measured as the diameter (mm) of clear zone for growth inhibition. The assays were carried in triplicate and the mean inhibition zone ± S.D. were calculated for each fungal species. The results of these analyses served as guidance for the design of the experiments on dolostone and limestone samples. Moreover, the minimum inhibitory concentrations (MICs) were determined by microdilution method in culture broth, according to the National Committee for Clinical Laboratory Standard recommendations using 96-well microtiter plates. The MIC value is defined as the lowest NPs concentration that resulted in a 100% reduction in the visible growth compared with that of NPs free growth control well. In order to test MICs with a higher number of fungi, two species of the Coelomycetos group were incorporated (Paraconiothyrium sp., and P. maculans), which have a different sporulation system than those tested on agar plates. The inoculum of each fungus (A. niger, P. oxalicum, Paraconiothyrium sp., and P. maculans) was prepared, and the suspension was adjusted to 5 × 105 conidia/mL. All metal oxide nanoparticles were dissolved in DMSO and diluted in microtiter plates by cultural medium (Potato Dextrose Broth, Difco) in a geometrical progression from 2 a 2048 times. Thus, the obtained concentrations of NPs were from 5 mg/mL-0.003 mg/mL (5, 2.5, 1.25, 0.65, 0.31, 0.15, 0.07, 0.03, 0.015, 0.007, 0.003, and 0.0015 mg/mL). After NPs were diluted, a standard amount of the test fungal was inoculated onto microtiter plate so that the inoculum density in the wells was equal 2.5 × 105 conidia/mL. The microtiter plates were incubated at 28˚C for 48 h. DMSO was utilized as negative control. The MIC values were determined visually and by optical microscopy (OM) as the lowest concentration of NPs where no visible fungal growth was observed in the wells of the microtiter plates. 2.4. Determination of antifungal activity of the nanoparticles as coatings Based on the results of the in vitro evaluation, the antifungal activity of ZnO NPs was not tested as coating. Thus, the antifungal activity of the MgO and Mg1−xZnxO NPs was studied using glass microscope slides, dolostone and limestone coupons of 2 cm x 2 cm x 1 mm size as substrates. The experimental setup carried out is schematically shown in Figure 1. The selected lithotypes were Laspra dolostone (Asturias, Spain) and conchuela limestone (Yucatán, México), differing in porosity and mineralogical composition. In particular, Laspra dolostone is composed mainly of dolomite (90 wt%) and characterized by its high open porosity (around 37%), while Conchuela limestone is mainly composed of calcite (around 80 wt%) and exhibits a total open porosity of about 27%. Both types of stones were selected considering their regional significance and frequency of usage in the stone heritage objects of Spain and Mexico, respectively. The different stone samples were completely dried in an oven at 60 ˚C, until the dry weight was achieved. Firstly the antifungal activity of the metal oxide NPs was studied on grease free glass slides. Glass slides were cleaned with ethanol and coated on one side using drop-wise addition of 1 mL of nanoparticle suspensions and allowed to dry overnight at room temperature (23 ± 5 ˚C). After allowing the covered slides to dry completely, 20 µL of fungal inoculum (1 × 105 conidia/ml) was applied to an area of 1 cm2. All the slides were prepared in triplicate and fungal growth was compared with control glass slides without coatings. Fungal growth was inspected daily by visual counting of colonies using a stereoscopic and optic microscope (Carl Zeiss).
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Dolostone and limestone samples were freshly cut with a diamond disk saw, without polishing the obtained stone faces. Subsequently, the samples were treated drop-by-drop with 2 mL of ethanol dispersions of MgO and Mg1−xZnxO nanoparticles (2.5 gL-1) on the dry and clean top surfaces of each stone. After that, the coupons were left to dry in air at 23 ± 5 ˚C and 55 ± 5% relative humidity for 2 days. Then, a sufficient volume to homogeneously cover the stone surface of 1 mL of the fungal spore suspension was applied using a micropipette. Control samples were not subjected to any treatment. All stone samples were assayed in tetraplicate. Antifungal activity of the treatments on the stone surfaces and the cross sections were inspected by a stereoscopic microscope (Nikon), and an Environmental Backscattered Electron Scanning Microscopy (ESEM-BSE) (JEOL JSM 6400). The use of ESEM-BSE is reported as one of the most effective methods for determining the success of a treatment due to the fact that the lithotypes can be studied without disturbing the microhabitats. 28 In addition, the epilithic colonization for each sample was quantitatively estimated by means of image analysis software (Image J, W. Rasband, National Institutes of Health, Bethesda, MD, USA. v. 1.5b, http://rsbweb.nih.gov/ij/) using the K-means-like method and the same procedure was applied to all images following the process described by. 29 Furthermore, to ensure that the treatments had no negative effects on the physical characteristics of the lithotypes, Spectrophotometry was performed to study the color changes induced by the treatments. The measurements were evaluated by means a portable spectrophotometer (MINOLTA CM-700d), before and after the treatment and the inoculation. Conditions used were Standard illuminant D65 and observer angle 10˚. The CieLab Color Space (L*, a* and b*) were chosen for the study, i.e., lightness (L*), which ranges from 0 for black to 100 for white, and the chromatic coordinates a* and b*. Coordinate a* ranges in value from +60 (magenta) to −60 (green) and b* from +60 (yellow) to −60 (blue). The global color change in the different stone samples was determined according to the formula ΔE = (ΔL∗2 + Δa∗2 + Δb∗2)1/2, using the non-colonized dolostone and limestone color as the reference value. For each treatment condition, the average for 3 measurements was considered. The porosity is a key factor in determining the primary bioreceptivity of stone types and consequently for weathering processes related to fungal growth. 5, 30 This is why porosimetric analyses were performed on untreated and treated stone samples with Mercury Intrusion Porosimetry (MIP). These measurements included total porosity (P), macro- and micro-porosity, which have a pore diameter of > 5µm and < 5 µm, respectively 31 and Pore Size Distribution (PSD). Readings were taken at pore diameter range of 0.005
