Marine Antifouling for Underwater Archaeological Sites : TiO 2 and Ag-Doped TiO 2

1 Università della Calabria, Dipartimento di Biologia, Ecologia e Scienze della Terra (DiBEST), Via Pietro Bucci, 87036 Arcavacata di Rende, Italy 2 Syremont S.p.A., Via Pietro Bucci, 87036 Arcavacata di Rende, Italy 3 Università di Messina, Dipartimento di Scienze Biologiche e Ambientali, Viale F. Stagno d’Alcontres 31, 98166 Messina, Italy 4Università della Calabria, Dipartimento di Fisica, Via Pietro Bucci, 87036 Arcavacata di Rende, Italy


Introduction
Biofouling plays a major role in degradation of submerged stone [1,2].This process develops within a few hours on material surfaces exposed to natural marine environments.At long exposure times, marine fouling occurs in the form of a complex community of plants, animals, and microorganisms that considerably alter and influence the immediate surroundings of the stone surface.Biofouling and biofilm formation are the result of an accumulation process, which is not necessarily uniform in time and space.On an active rough marble surface, the gelatinous structure of the biofilm (formed by extracellular polymeric materials "EPS, " bacterial cells, and water) is mixed with alteration products found on the stones that are formed within the same time scale [3].The role played by microorganisms, both endolithic and epilithic species, in the alteration of submerged stone materials is well known.This leads to the depletion and destruction of the substrate, by means of secretion of acids, surface deposits, and development of vegetative structures within the colonized stone.Research in this work has been focused on the experimentation of some innovative antifouling coatings suitable for stone materials.The antifouling coating technology has a long history, being studied since the 15th century [4].Toxic antifoulants have been widely employed in the past as a method for controlling the fouling, but biocides such as lead, arsenic, mercury, and their organic derivatives have been banned due to the environmental risks associated with their use.A revolutionary self-polishing copolymer technique, using a similar heavy metal toxic action to deter marine organisms, consisted in the use of tributyltin-(TBT-) based antifoulant [5].However, this latter caused shellfish International Journal of Photoenergy deformities and bioaccumulation of tin in some ducks, seals, and fish [6], resulting in legislations that culminated in the global ban of tributyltin [7,8].In the last decade, the scientific research has focused attention on alternative products, which have "acceptable" environmental impacts [9].Nanometric oxides, such as TiO 2 and ZnO, are promising antimicrobial agents suitable for stone protection [10,11] that act thanks to their photocatalytic effect [12][13][14]; in particular the anatase crystalline phase shows higher efficiency with respect to rutile [15].
The effectiveness of titania against marine antifouling has been studied only on glassy substrate [16].Several papers have described the doping of titania with several elements [17][18][19][20][21] in order to enhance the bio-and photoactivity [22].
There are numerous studies on the properties and applications of Ag-TiO 2 systems used as photocatalysts, optical applications, and antibacterial treatment [23,24].
Noble metals deposited or doped with TiO 2 have high Schottky barriers among the metals and thus act as electron traps, facilitating electron-hole separation and promoting the interfacial electron transfer process [25,26].Silver can trap the excited electrons from titanium dioxide and leave the holes for the degradation reaction of organic species.It also results in the extension of their wavelength response towards the visible region [27].In addition, silver nanoparticles possess the ability to absorb visible light due to localized surface plasmon resonance (LSPR) [28].These properties have led to tremendous range of applications of Ag-TiO 2 nanoparticles, for instance, antibacterial textiles, engineering materials, medical devices, food preparation surfaces, air conditioning filters, and coated sanitary wares.
In this work, we have synthesized titania and silver-doped titania and characterized them by means of XRD diffraction, reflectance spectroscopy, and XPS measurements.We have assessed the antifouling properties of these materials by performing microbiological tests.Finally we have dispersed such materials in a binder and applied them on marble specimens, and we have monitored the coating behavior in simulated marine environment.

Materials and Methods
Undoped TiO 2 was prepared using Ti(OBu) 4 as precursor.5 mL of Ti(OBu) 4 were dissolved in 20 mL ethanol under ultrasonic stirring (20 minutes) and then 0.5 mL of HNO 3 solution (VHNO 3 : VH 2 O = 1 : 1), and 1 mL of H 2 O were added dropwise to the above solution.The mixture was sonicated at room temperature for 30 min to obtain a white colloidal dispersion.The dispersion was dried at 100 ∘ C for 24 h and calcined at different temperatures for 6 h.The Ag-doped TiO 2 photocatalysts were prepared by the same procedure adopted for the pure TiO 2 except that an aqueous AgNO 3 solution was used instead of H 2 O, and the Ag/TiO 2 ratio was 5% wt.
The X-ray diffraction (XRD) patterns of TiO 2 and doped TiO 2 were recorded on a D8 Advance Bruker X-ray diffractometer using Cu K radiation as the X-ray source.The diffractograms were recorded in the 2 range of 10-80 ∘ .
Measuring conditions were set at 40 kV voltage, 30 mA current, 0.02 ∘ 2 step size, and 3.0 sec step time.
XPS measurements were conducted in a UHV chamber equipped for standard surface analysis with a base pressure in the range of low 10 −9 torr.Nonmonochromatic Mg-K Xray (h = 1253.64eV) was used as excitation source.The XPS spectra were calibrated with the C1s peak of a pure carbon sample (binding energy 284.6 eV).
Photoluminescence (PL) and optical absorbance measurements were taken with an Olympus microscope (Horiba-Jobyn Yvon) mounting objectives of 10x, 50x, and 100x magnification.The microscope is equipped with a laser source at 378 nm (12 mW of power) for PL, a white lamp for absorbance measurements, and a Triax 320 (Horiba-Jobyn-Yvon) spectrometer working in the 200-1500 nm range.
We measured directly the reflected spectrum (  ()) and obtained, by simply assuming the transmittance to be null for bulk samples, the reflectance (()) and the absorbance (()) as a function of wavelength by the following relation: where   () is the source spectrum.Marble probes (20 × 10 × 2 mm) were treated with a dispersion of TiO 2 (or Ag-TiO 2 ) dispersed in a binder (5% wt of acetone solution of Paraloid B72).As reference, untreated samples and samples treated with binder only have been also analysed.
Chromatic variations were assessed through colorimetric tests using a CM-2600d Konica Minolta spectrophotometer.Chromatic values were expressed in the CIE  *  *  * space, where  * is the lightness/darkness coordinate,  * the red/green coordinate (+ * indicating red and − * green), and  * the yellow/blue coordinate (+ * indicating yellow and − * blue).The colour modification on the surface (Δ) was calculated using the following relation [29]: where Δ * , Δ * , and Δ * represent the difference between the value of a specific chromatic coordinate in altered and fresh samples.TiO 2 , Ag, and Ag-doped TiO 2 were suspended in ultrapure water and sonicated for 10 min at 20% of power (Bandeling electronic UV2070) and then sterilized in an autoclave.
To determine the efficiency of TiO distilled water and then suspended to reach a final concentration of 1 × 10 6 cell/mL.They were inoculated in plates containing 0.1% and 0.01% (final concentration) of each product in double.Plates were incubated for 20 h under UV (UV 70030005/3V 25W Black) and solar lamp (Fluoradym R63 40 W).
The survival percentage was calculated after spreading on the surface of agarized medium (TSA, Oxoid) 100 L of each suspension and respective decimal dilution.After incubation at 28 ∘ C per 24-48 h, the percentage of survival was calculated as colony forming units per mL (cfu/mL).
Another test was carried out with marble slabs (treated and untreated) in simulated natural marine habitat.In this case, a simulation of submerged archeological artifacts was created in a container filled with marine water and a natural colonization of enriched marine bacteria was let to start and left for 24 h and for 72 h under natural daylight.Marble slabs were immerged in the marine water with an angle of 45 ∘ degrees in order to avoid bacterial passive sedimentation on  the marble surface.Both upper part and lower part of marble slabs were analyzed.After the incubation time, each marble slab was cut in two parts, one set fixed in Glutaraldehyde phosphate, dehydrated with a crescent series of alcohols (30-50-70-80-90-absolute), chrome metallized, and observed by SEM microscopy.For this purpose, an FEI Quanta 200F (Philips) scanning electronic microscope (SEM) has been used.All measurements were carried out with an acceleration voltage of 20 kV and under low vacuum conditions (10 −5 mbar pressure).

Results and Discussion
In Figure 1, it has been shown the XRD pattern of titania and Ag-doped titania.It is worth to note that an annealing temperature of 550 ∘ C leads to the formation of anatase crystalline phase, while the presence of silver affects the crystallinity of the product, since a calcination temperature of 550 ∘ C leads to the formation of a mixture of anatase and rutile, while at 400 ∘ C only anatase is detected.
Information on the bonding nature and superficial composition are obtained with XPS analysis (Figure 2).Survey spectra indicate the presence of titanium, oxygen and silver, with traces of carbon and nitrogen probably adsorbed from atmosphere.
The main XPS lines for each elements are indicated in Table 1.Ti 2p and O 1s lines are characteristic of TiO 2 in all International Journal of Photoenergy The optical absorbance, showed in Figure 3, changes strongly in visible region for doped samples.The absorbance of Ag-doped sample is about four time larger than pure TiO 2 .On the contrary, only little changes are visible in UV region.
The absorption of TiO 2 composite moved to longer wavelength in comparison with pure TiO 2 suggesting that the band gap was decreased by doping with Ag [31].
Synthetized powders were dispersed in different proportions (powder/polymer ratios: 1/1, 1/10, and 1/100) in Paraloid B72 solution in order to assess their suitability in terms of colour variations once applied to stony surface.Colorimetric measurements have been performed to assess chromatic variations induced by treatments (Figure 4); results are reported in Table 2.
Ag-doped titania provides strong colorimetric variations with respect to pure titania.The best compromise between aesthetic issue and product amount is represented by the 1/10 ratio, so this has been chosen as mixture to be applied on stone samples.
Results obtained from biological experiments showed that both bacterial strains were sensitive to the powders tested at a concentration of 0.1% and 0.01%.In particular, with TiO 2 tested at concentration of 0.1%, the percentage of survival cells was 55% and 19%, respectively, for S. maltophilia and for Micrococcus sp.(Figure 5), while the lower concentration of TiO 2 (0.01%) was more effective in determining the complete killing of bacteria tested.The doped TiO 2 with Ag determined a complete killing of bacteria at both concentrations; Ag alone was tested at the same concentration present in the doped TiO 2 gave comparable results.Exposure of untreated and treated marble slabs showed a different response to the treatments after 24 h and 72 h of spontaneous colonization by microorganisms.
In fact, while the microbial colonization was irrelevant after 24 hours, after 72 h significant differences among the marble slabs were noticed (Figure 6).Untreated marble showed a higher production of EPS on the surface, while on the marble treated with the binder alone the surface was covered by occasional or no patches of EPS.Marbles treated with TiO 2 or Ag-TiO 2 or Ag alone showed no EPS production and no microbial colonization.
Our results showed that treatments with titanium oxides and their Ag derivate were very effective to control bacteria population, the process of adhesion, and consequent extracellular polymeric substances (EPS) formation.The results obtained were also comparable with the action of Ag alone.
However, due to the different sets of the experiments used, we can draw some conclusions.
In fact, in the first set of experiments carried out to test the survival of bacterial cells we could state that the photocatalytic effects of the tested products under laboratory  conditions were effective reducing dramatically the number of viable cells.However, this method suffers from limitation due to the fact that in field experiments the application of products on the treated surfaces is necessary through the use of a binder and thus testing products alone is not comparable to the experiments in field conditions.The other set of experiments, carried out with untreated or treated submerged marble slabs, showed that the products tested were effective against the first steps of colonization that is the main cause of the progression of biofouling of submerged surfaces.In fact, our experiments were successful because no EPS or bacteria colonization was observed after 24 h and 72 hrs of immersion of marble slabs treated with the products.On the contrary, control surfaces (untreated marble) showed the presence of EPS produced by the bacteria underneath (Figure 6).

Conclusions
This work has dealt with the synthesis, structural characterization, and biological assessment of pure and Ag-doped titania in order to explore the possibility to use them as antifouling agent suitable for the protection of submerged archeological stone artefacts.
A treatment, with a mixture of Ag-TiO 2 /binder ratio of 1/10, represents the limit that should not be exceeded to avoid aesthetical issues on the stone surface.
As far as the antimicrobial tests were concerned, the goal of this study was to test a reliable standard method for testing the antimicrobial efficacy of TiO 2 photocatalytic products that could work on submerged objects.To achieve this objective, we used two methods that gave us promising results.The first method of determining the survival curve of tested bacteria gave us information on the sensitivity of bacterial strains.In this condition, bacterial sensitivity could be tested at different concentrations.The second method seems to be more promising but still some adjustments especially regarding the proper time of exposure are needed.
Although these results have a preliminary nature, it can be stated that a decreasing of biofouling activity has been observed on treated stony surfaces.

2 Figure 5 :
Figure 5: Histogram showing the percentage of survival cells in water of two bacterial strains tested against products under UV and solar lamp for 20 h.Controls were carried out with only bacteria.

Figure 6 :
Figure 6: SEM pictures of the untreated and treated marble slabs before and after 72 hours of immersion in sea water simulation.

Table 1 :
XPS main line position and element amount (%) obtained from XPS data.