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International Biodeterioration & Biodegradation 89 (2014) 115e125

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International Biodeterioration & Biodegradation

journal homepage: www.elsevier .com/locate/ ibiod

Algal growth inhibition on cement mortar: Efficiency of waterrepellent and photocatalytic treatments under UV/VIS illumination

Thomas Martinez a, Alexandra Bertron a,*, Gilles Escadeillas a, Erick Ringot a,b

aUniversité de Toulouse, UPS, INSA, LMDC (Laboratoire Matériaux et Durabilité des Constructions), 135 avenue de Rangueil, 31 077 Toulouse Cedex 04,Franceb LRVision SARL, Zi de Vic, 13 rue du Développement, 31320 Castanet-Tolosan, France

a r t i c l e i n f o

Article history:Received 23 September 2013Received in revised form29 January 2014Accepted 29 January 2014Available online 20 February 2014

Keywords:PhotocatalysisTiO2

CoatingWater repellentAlgaeUV illuminationBuilding materialsBioreceptivity

* Corresponding author. Tel.: þ 33 5 61 55 99 31.E-mail addresses: bertron@insa-toulouse.fr, alexan

(A. Bertron).

http://dx.doi.org/10.1016/j.ibiod.2014.01.0180964-8305/� 2014 Elsevier Ltd. All rights reserved.

a b s t r a c t

Building materials are regularly affected by the growth of microalgae. The consequences are mainlyaesthetic but the colonization can cause biodeterioration of the material in the most extreme cases. Thisstudy investigates two building material treatments that can potentially inhibit or slow down suchgrowth: photocatalytic coatings and water repellent treatments. The efficiency of these treatments interms of biological growth inhibitionwas tested on the algae species Graesiella emersonii. Algal growth onbuilding materials was investigated using two accelerated tests simulating different types of humidifi-cation (water capillary ascent and water run-off) under different lighting conditions. Mortars treated withphotocatalytic coating or with water repellent were studied. The algal growth on the mortar surface wasevaluated using image analysis (area covered and intensity of fouling). No slow down of the biologicalgrowth kinetics could be attributed to photocatalytic substrates. However, for mortars impregnated with awater-repellent preparation, algal growth slowed significantly under water run-off and even stoppedunder water capillary ascent.

� 2014 Elsevier Ltd. All rights reserved.

1. Introduction

Colonization of walls by microorganisms can be observed onlyseveral months after a building is built (Wee and Lee, 1980). Theaesthetics of the facade is then altered by the appearance of green,red or black streaks. Microscopic observations have shown thatthese unsightly stains are mainly related to the development ofmicroalgae on the surface (Gaylarde and Gaylarde 2005; Duboscet al., 2001). Fungi, lichens and mosses can also be found onthese surfaces in very advanced cases of colonization (Escadeillaset al., 2007). These microorganisms may extend to the surface ofbuilding materials under certain conditions of humidity (waterflow and/or high relative humidity), temperature and sunshine. Themain material-related physical factors responsible for the bio-receptivity of a surface are: porosity, roughness and mineralcomposition (Guillitte and Dreesen, 1995; Escadeillas et al., 2007;Tran et al., 2012, 2013, 2014; Giovannacci et al., 2013). Significantroughness increases adherence, retention and therefore growth of

dra.bertron@insa-toulouse.fr

microorganisms. High porosity promotes the retention of waterafter any humidification. Finally, the mineral composition of thesurface material plays a major role in bioreceptivity since it caneither favour the colonization, for example by providing a source ofnutriments (calcite and silica from the cementitious substrate(Ribas Silva, 2000)), or prevent colonization, for example because ofits high pH value (pH of cementitious materials before theircarbonation inhibits algal growth (Dubosc et al., 2001; Tran et al.,2012)) or because of the presence of metal ions (Wessel, 2011).

Architectural measures can be implemented, at the buildingdesign step, to avoid the further development of biological growth.They may consist of the installation of components to preventwater run-off on walls and thus prevent colonization, or the use ofconstructionmaterials of low bioreceptivity (e.g. a smoothmaterial,low roughness, low porosity)(Dubosc et al., 2001; Tran et al., 2012,2013, 2014; Giovannacci et al., 2013).Wessel et al. (2011) also reportthe results of installing zinc strips on the ridge of the StanfordMausoleum roof. Rainwater in contact with the metal strips ischarged with metal ions and flows over the roof, inhibiting thegrowth of microorganisms.

Existing walls can be treated by chemical methods. Biocidalproducts are usually applied to remove microorganisms that have

T. Martinez et al. / International Biodeterioration & Biodegradation 89 (2014) 115e125116

grown on concrete walls. Depending on the type of product, acurative or preventive effect can be obtained (Shirakawa et al.,2002; Urzi and De Leo, 2007). The utilization of water repellentcompounds has been suggested as a preventive method and hasproved to be effective under laboratory conditions (De Muyncket al., 2009; Zhang et al., 2013). The presence of water repellentsreduces the availability of water and therefore the bioreceptivity ofthe materials. Moreover, water repellents and biocidal compoundscan be combined to improve treatment effectiveness (Urzi and DeLeo, 2007; Moreau et al., 2008; De Muynck et al., 2009).

Another potentially effective method is to apply a photocatalytictreatment to materials subject to biological growth. The process isbased on the irradiation of semiconductor materials, here TiO2particles, with high energy photons (hn) that raise electrons e�from the valence band (vb) to the conduction band (cb), thusleaving electron holes hþ (reaction 1). The pairs of mobile chargesproduced can reach the surface of the semiconductor particle andinitiate a reduction-oxidation process. Moreover, through reactionswith the adsorbed oxygen and water from the surrounding air,reactive oxygen species such as HO� and O2�

� are created (reactions2, 3) and act as strong oxidants with the potential to decompose ormineralize a wide range of compounds (Hoffmann et al., 1995;Blake, 2001).

TiO2!hnTiO2 þ hþvb þ e�cb (1)

H2Oads þ hþ/Hþ þ HO$ (2)

ðO2Þads þ e�/O$�2 (3)

The efficiency of the photocatalysis process has been widelyreported for the decomposition of gaseous and aqueous pollutants.Regarding microorganisms, TiO2 photocatalysts have been found tokill bacteria, viruses and algae under UV illumination (Huang et al.,2000; Sunada et al., 2003; Peller et al., 2007). On the basis of studieson the photokilling of Escherichia coli (Migula, 1895) (Castellani andChalmers, 1919) cells in suspension pipetted onto TiO2 coated glass,Sunada et al. (2003) proposed a three-stage mechanism for thedegradation of microorganisms on illuminated TiO2: (1) creation ofdefects in the outer membrane by the reactive oxygen speciesgenerated by photocatalysis (OH�, H2O2, O2�

�); (2) destruction ofthe cytoplasmic membrane causing death of the cell; (3) decom-position of the dead cell.

Additionally, surfaces containing TiO2 as a photocatalyst canhave superhydrophilic properties under UV illumination (Yu et al.,2003; Diamanti et al., 2008). This phenomenon reduces the con-tact angle of a drop of water to near zero. When the surface istilted, the water film thus formed falls by gravity (easy rinsing)removing the accumulated dirt and the products of the photo-catalytic reaction. The photoinduced superhydrophilicity of TiO2combined with the degradation of organic pollutants by photo-catalysis can therefore impart self-cleaning properties to thesurface.

However, despite these biocidal and self-cleaning properties,few studies concern the application of photocatalysis as a pre-ventive treatment against biofouling of construction materials.Gladis and Schumann (2011a) studied the development ofChlorella, Stichococcus and Coccomyxa on photocatalytic glassplates. The growth of algae in an atmosphere of saturated hu-midity under different types of illumination (visible light alone,mix of visible light and UV-A) was not impacted by the phe-nomenon of photocatalysis although the oxidative nature of thematerial had been validated by the degradation of methylene

blue dye. Moreover, measurements of the cells’ permeability oncoated and uncoated materials showed no significant differences.However, in another work (Gladis et al., 2009), the same authorsobserved an inhibition of growth of the algae Stichococcus byphotocatalysis using a filter on which a suspension of ZnO hadbeen dried. Growth inhibition was obtained by Ramirez and DeBelie (2009) and Graziani et al. (2013) respectively on aeratedconcrete and brick specimens coated with TiO2 exposed to cyclicwater run-off but the nano-coating was unable to stop algalgrowth. However, in a similar experiment, no visible algal growthwas observed on a cement paste prepared with commercial TiO2-bearing cement, whereas laboratory-made photocatalytic cement(containing 5 and 10% TiO2) did not appear to be efficient to avoidalgal growth (Maury-Ramirez et al., 2013). Outdoor weatheringover several years showed that photocatalytic coating on rooftiles was not effective against phototrophic growth (Gladis andSchumann, 2011b).

Materials with photocatalytic properties have been proved toreduce air and water pollution under UV irradiation (Ollis, 2000).Studies on their potential to reduce biological growth on buildingmaterials are becoming more common but have not reached aconsensus. Further research on this topic is needed.Water repellenttreatments are commonly used to increase the durability of con-crete structures by reducing the penetration of water into theconcrete and, for this reason, this type of treatment could be usedfor the protection of surfaces against algal growth.

In this study, prevention of Graesiella emersonii (Nozaki et al.,1995) (or Chlorella emersonii, Shihira and Krauss, 1965) growthon mortar by application of photocatalytic treatment or waterrepellents was investigated under different UV exposures. G.emersonii is widely used as a model in antifouling tests andecotoxicology (Anton et al., 1993; Nyström and Blanck, 1998;Escadeillas et al., 2007). Two laboratory test set-ups(Escadeillas et al., 2007) were adapted to study the efficiency ofthe photocatalysis process and different conditions of illumina-tion were applied. These experimental arrangements were cho-sen to simulate different growth conditions (humidification bywater capillarity ascension or by water run-off). The develop-ment of biological stain was evaluated by two non-destructivemethods using image analysis. The first method was developedin the framework of this study and measured the intensity offouling at the pixel level. The second one, developed byEscadeillas et al. (2009), evaluates the percentage of sample areacovered by algae.

2. Materials and methods

2.1. Preparation of mortar substrates

CEM I cement mortars with water to cement ratios of 0.7 wereused as the substrate (treated or control specimens) for the algalgrowth. The formulation and manufacturing of the mortars wasadapted from the NF EN 196-1 standard. The mortars were cast in5*5*0.5 cm3 PVC moulds sprayed with demoulding oil and sub-jected to about ten shocks (by gravity) to help the mortar fill themould properly. The moulds were then put in a storage room (100%relative humidity and temperature 21 �C) for 24 h. Afterdemoulding, the mortar specimens were stored in a room with aregulated atmosphere (20 �C, 50% RH) for 15 days. The mortarspecimens were then kept in an accelerated carbonation chamber(50% air, 50% CO2, RH in the 60e70% range) until completecarbonation was obtained (around 1 month). This carbonationperiod was necessary to reduce the pH of the mortar substratebefore algal inoculation (the initial pH of cementitious materials,around 13, inhibits algal growth).

T. Martinez et al. / International Biodeterioration & Biodegradation 89 (2014) 115e125 117

2.2. Application of water repellent and photocatalytic treatment

Two types of treatment were studied:

- Photocatalytic glazes were formulated using silicate/acrylic-based binders. Water was used as the solvent to limit the useof hazardous chemical products. Thickeners and wetting agentswere also incorporated to ensure good wettability and unifor-mity of aspect of the coating. The UV-activatable TiO2 was acommercial slurry solution from Millennium Chemicals (S5-300B). The treatment did not alter the aesthetic aspect of thesurface and contained 2% wt TiO2

- Awater repellent impregnationwas formulated using silane andfluorinated compounds

- As control specimens, mortar samples free of any coating, werealso tested.

Water repellent and photocatalytic treatment were applied tomortar after the carbonation period. The photocatalytic coatingswere applied to the cast plane surface of the mortars using a brush.The amount of coating deposited was about 80 g m�2 (determinedby weighing). For the water repellent treatment, the product wasapplied until all open superficial pores were saturated.

Before the introduction of the photocatalytic samples into thealgae test set-up, the photocatalytic activity of the samples wasassessed using a NOx degradation experiment (Martinez et al.,2010, 2011). Degradation rates of 39% were obtained under UVillumination (6 W m�2, 310e400 nm).

Changes of the surface properties of the mortar after applicationof the water repellent compounds were verified by visual obser-vation. Fig. 1 shows the increased contact angle of drops of waterinduced by the presence of the water repellent on the surface of themortar. The surface energy and the force of capillarity were reducedafter impregnation of the porous material by the water repellentcompound; the surface modification was expected to prevent orreduce algal growth on the surface because of the limited avail-ability of water.

2.3. Accelerated testing of biological stain growth

2.3.1. Choice of algal species and preparation of algaeThe information available on the distribution and ecology of

microorganisms on Façades in urban habitats is quite limited.Cyanobacteria, are most frequently present as major biomass inLatin America, followed by fungi, whereas in Europe algae are most

Fig. 1. Increase of the contact angle of water drops on mortar surface. (a) referencemortar (b) mortar treated with a water repellent compound.

common, followed by cyanobacteria. Algae are more frequent thanother groups on all substrates in Europe (Gaylarde and Gaylarde,2005). Barberousse et al. (2006b) carried out an investigation onthe diversity of algae and cyanobacteria on building façades inFrance (71 samples were collected all over France). Green algaewere the most frequent microorganisms, Chlorella genius beingamong the most frequent microorganisms encountered, afterKlebsormidium flaccidum (the most frequently recorded species inurban environments in Europe (Rindi, 2007)), Trebouxia and Sti-chococcus bacillaris. The choice of the algal species for the labora-tory tests was made considering representativeness, growthrapidity and ease of liquid culture. The results of our previous work(Escadeillas et al., 2007) led us to choose an algal species of theGraesiella genus: G. emersonii ((Nozaki et al., 1995), initial taxon-omy: C. emersonii (Shihira and Krauss, 1965)) provided by theCulture Centre of Algae and Protozoa (U.K.) reference CCAP 211/8G).

The algae were cultivated in BG11 medium under artificial light(1600 lux, OSRAM L18/765). The growth rate was monitored usingspectrophotometric and dry mass measurement so that the algaecould be inoculated into the experimental device during an expo-nential growth phase (Prescott et al., 2003).

2.3.2. Test by water run-off2.3.2.1. Principle. In order to accelerate algal growth on mortarspecimens, the water run-off dynamic test equipment developedby Escadeillas et al. (2007, 2009) was used and adapted to thisstudy. The aim of this test is to simulate the water run-off thatfrequently occurs on some parts of constructions.

The device (Fig. 2) consisted of a transparent polycarbonatechamber (1*0.5*0.5m3) divided into 4 alveoli, each containing a 45�

tilted support for the mortar samples under test. This chamber,fitted with a lid, was stored in an air-conditioned room (21 �C)where no outside light could penetrate.

Since the photocatalytic process is activated by the UV part ofthe solar spectrum, and the growth of algae and cyanobacteria isessentially dependent on visible light (red and blue in particular),the chamber was fitted with a “visible-spectrum” fluorescent tube(ARCADIA Natural Sunlight�) and a UV-A tube (Sylvania�). Thesetubes illuminated the mortar samples and the culture mediumcontaining algae to ensure their development (Fig. 2).

Pumps and watering ramps allowed algal growth medium(BG11) to flow over the sample surface. The imposed photoperiodwas 12 h day and 12 h night. The run-off was set at 1.5 h per day. Itwas ensured by immersed pumps (300 l/h aquarium-type pump)connected to spraying ramps. The liquid from the spraying rampsfirst fell on PVC samples situated above the mortars under test andhaving the same size, in order to favour homogeneous flow on themortar. 10 l of algal growth medium was placed in each of thealveoli. A circuit of immersed, closed pumps ensured constantmovement and mixing of the medium. The samples were inocu-lated through the water run-off.

2.3.2.2. Light conditions. UV light intensity in the chamber had tobe representative of the exposure of a facade in a real situation.High UV intensity could lead to the efficiency of photocatalysisactivity being overestimated and could alter microorganisms byphotochemical effects (molecular, cell or physiological damage(Karsten et al., 2006)). The intensity of UV-A light from the sun is inthe 6e30 W m�2 range depending on the sunlight conditions(Fujishima and Zhang, 2006; Blöß and Elfenthal, 2007; Martinezet al., 2011).

Irradiance was measured at the mortar surface in the 310e400 nm (UV light) and 400e800 nm (visible light) ranges using aradiometer (Gigahertz-Optik X11) at the different zones inside thechamber.

Fig. 2. a) schematic diagram and b) top view of the run-off test (adapted from Escadeillas et al. (2007)).

T. Martinez et al. / International Biodeterioration & Biodegradation 89 (2014) 115e125118

Because of the difference of light emission between the centreand the sides of the tubes, different illumination conditions wereavailable in the test set-up. The irradiance measurements indicatedfour zones of different UV light conditions: 12, 7.5, 3, and 1.5Wm�2

for zones 1 to 4 respectively (Fig. 3). For visible light, measurementsin the 400e800 nm range showed two zones with different in-tensities: one at the centre of the fluorescent tube with 5.5 W m�2

and the other at the ends with 3.0 W m�2 (Fig. 3).

2.3.3. Test by water capillary ascent2.3.3.1. Principle. The aim of this test was to simulate the algalgrowth at walls bases, where water supplies essentially occur bycapillary ascent (Escadeillas et al., 2007). Fig. 4 show a schematicdiagram of the experimental device. The principle of the test was toplace mortar samples on vermiculite humidified with a BG11nutrient medium. Before inoculation, the samples were placed onthe bed of vermiculite for 48 h to allow capillary ascent to takeplace. The samples were then removed from the test set-up and leftin the air for 2 h so that the surfacewas dry, thus allowing the liquidcontaining the algae to be more rapidly absorbed and the initial

Fig. 3. Distribution of UV-A (310e400 nm) and vi

distribution of algae on the sample to be facilitated. The inoculationwas performed on the cast surface of the sample where the coatinghad been applied. The liquid containing the algae was spreadevenly using a pipette tip.

The samples were then deposited in the box containingvermiculite and culture medium. The box was fitted with a boro-silicate glass lid transmitting the UVevisible radiation produced byfluorescent tubes (Philips� and FSA�). The lid enabled the samplesto be kept in a highly humid environment. The day/night cycles of18/6 h were selected according to Escadeillas et al. (2007).

2.3.3.2. Lighting conditions. Photocatalytic samples were illumi-nated with two UV-A tubes (Philips�) and two tubes emitting in thevisible (FSL�). Radiometric measurements showed 6 � 0.5 W m�2

in the 310e400 nm range and 6.5 � 0.2 Wm�2 in the 400e800 nmrange.

Samples treated with water repellent compounds were illumi-nated with fluorescent tubes emitting in the visible (FSL�).Radiometric measurement resulted in 5.0 � 0.2 W m�2 (400e800 nm).

sible light (400e800 nm) in the run-off test.

Fig. 4. Schematic diagram of the test set-up for capillary ascent.

T. Martinez et al. / International Biodeterioration & Biodegradation 89 (2014) 115e125 119

2.4. Quantification of growth using image analysis

2.4.1. Image acquisitionDuring colonization by algae, the surfaces of the mortars were

photographed using a scanner (EPSON Perfection 2580) at a reso-lution of 600 dpi. This device is suitable for image analysis becauseit ensures reproducible conditions of light and image size. Beforeeach acquisition, samples were stored for 3 h in the atmosphere ofthe room in order to avoid any influence of moisture on the greylevel (Escadeillas et al., 2009).

2.4.2. Intensity of foulingThe colour of each pixel of the photograph was characterized by

3 coordinates in the RGB colourimetric space (R: red, G: green, B:blue). The first step of the analysis was to convert the image intogrey levels using equation (4):

Grey ¼ 0:299Red� 0:587Green� 0:114 Blue (4)

The coefficients in Equation (4) (the sum of which is 1) accountfor the way the human eye perceives red, green and blue colours(recommendation 601 of the International Commission on Illumi-nation, CIE). New pixels were characterized by one component, thevalue of which indicated the grey level over 256 values fromwhite(255) to black (0).

When the image was converted into grey levels, the zones withthe most intensive colonization were characterized by a grey levelclose to black (Fig. 5).

To evaluate the intensity of colonization, pixels were sorted intoeight classes according to grey level and then counted. This methodgave the area occupied by algae at various intensities of coloniza-tion and thus the total colonized area of each sample.

Fig. 6 shows the distribution of pixels in the eight classes of a5*5 cm2 mortar specimen during the progression of colonization ofthe sample. The image of the non-colonized specimen is mainlycomposed of Class 5 and Class 4 pixels. The progression of coloni-zationwas accompanied by a darkening of the image and thus by anincrease in the number of pixels in classes 0e4. When colonizationbecame total and intense, the image tended to be composedexclusively of pixels of class 0.

Fig. 5. Conversion of the image of a colonized mortar specimen into 8 grey levels.

The colonization index I (%) was determined as follows:

Ið%Þ ¼ 100� 100� 800� Ct800� Ct¼0

With:

Ct ¼8� C0t þ 7� C1

t þ 6� C2t þ 5� C3

t þ 4� C4t þ 3

� C5t þ 2� C6

t þ 1� C7t

where Ct is the intensity of the colonization level after t days of testand Cn

t is the percentage of pixels in class n after t days of test.The results of discrimination of colonization intensity levels

were similar in significance to those obtained using reflectancemeasurements with a spectrophotometer (Escadeillas et al., 2007,2009; De Muynck et al., 2009). The present method allowed theheterogeneity of the intensity of development to be better repre-sented than the reflectance measurements since the analysis wasperformed at pixel scale whereas a spectrophotometer measureson an area between 50 and 490 mm2 depending on the charac-teristics of the device (one average reflectance value is obtained permeasuring area).

2.4.3. Colonized areaThe “K-Means-like” method was used for the quantification of

the area colonized by algae (Escadeillas et al., 2009). This algorithmperforms an automatic classification that partitions the image inthe parameter space (RGB) into a given number of classes. In thecase of colonized surfaces, the K-means-like method allowedgreenish pixels representing algae to be grouped separately fromthe grey ones of non-colonized zones.

Quantification was performed with a separation into threeclasses. After identifying the classes belonging to the colonized areaby comparison with the original image, the area colonized wasdetermined by calculating the percentage of pixels belonging to thecolonized classes.

Fig. 7 shows each step of the quantification performed by thesoftware process: (a) image acquisition of the sample, (b) gatheringof the pixels of the image in three classes by the “K-Means-like”method, comparison with the original image shows that themethod can isolate the colonized areas in two classes, (c) calcula-tion of the area colonized.

3. Results and discussion

3.1. Influence of UV irradiance

Image acquisitions of the reference mortar during the waterrun-off experiment and in the different zones of illumination arepresented in Fig. 8 and the results of the image analysis process forthe three types of samples (reference, photocatalytic and waterrepellent treatment) are presented in Figs. 9 and 10.

The kinetics of the colonization index and the colonized areaprogression seemed to be sigmoidal. A latency period was firstobserved where no sign of colonization was detected on the mor-tars. This latency period lasted between 50 and 100 days dependingon the lighting conditions. The latency period matched the initialgrowth of the algae in the culture medium circulating in thepumping system and the attachment phase of the algae to themortar substrate. Then the first algal stains were detected throughimage analysis (first at the edge of the samples) and the evolutionwas very rapid. The surfaces of the mortar specimens then becamecompletely covered in less than 160 days in the most rapid case.This kinetic behaviour was also observed by Tran et al. (2013)

Fig. 6. Evolution of the area occupied by the 8 pixels in classes based on the colonization status of the sample.

T. Martinez et al. / International Biodeterioration & Biodegradation 89 (2014) 115e125120

during the fouling of Portland cement mortars by the green algae K.flaccidum (Kützing) (Silva et al., 1972). The area colonized by algaedetermined by image analysis versus time followed a sigmoidal-type curve.

UV intensity had a great impact on the development of algalcolonization on mortars whatever the treatment applied, the lowerlighting conditions leading to the most intense colonization. Colo-nization on control samples placed under low UV intensities (1.5e3 W m�2) were detectable earlier than on the samples placed inareas of high UV intensities (7.5e12 W m�2). Moreover, between100 and 169 days, the intensity of fouling under high UV radiation(between 7.5 and 12 W m�2), was half that observed at lower in-tensity (1.5e3 W m�2) (Fig. 9).

In a study by Karsten et al. (2006), the effect of 8Wm�2 artificialUV irradiation showed an inhibitory effect on the photosyntheticperformance of Chlorella luteoviridis Chodat (Conrad et Kufferath,1912). However, after 8 days, the photosynthetic performancewas the same as that observed in the absence of UV irradiation.Moreover, algal growth was not impacted by the UV irradiation.This last observation confirms that made by Gladis and Schumann(2011a) with a UV-A irradiation of 4e7 W m�2.

During exposure of the specimens to UV-A, Karsten et al. (2006)were able to measure an increase in intracellular concentrations ofamino acids such as mycosporin (MAA), a compound whichstrongly absorbs ultraviolet radiation and acts as a sunscreen. TheMAA production and lower photosynthetic performance observed

Fig. 7. Image analysis process using K-M method (a) image obtained by the scanner (b

by Karsten et al. (2006) show that the presence of UV light has aninfluence on the metabolism of algae. In the water run-off test, themetabolism of the algae could have had difficulty in adapting dueto the sudden change of environment (changes in light conditions:UV light was less intense in the culture medium contained in thebottom of the device than at the surface of the mortar specimens,apparition of dryewet cycles and accessibility of nutriment afterthe attachment of the cells to the mortar surface). Therefore, theresults obtained in the present study could be explained by aperiod of acclimation of the algae depending on the level of UVradiation.

Fig. 11 shows images of the three types of samples acquiredunder VIS and UV/VIS light during the water capillary test. Unlikethe results obtained by water run-off, the edges of the samples arenot the first zones colonized. The growth of algae on the surface ofthe samples is relatively homogeneous in both lighting conditions.These observations can be explained by the method of inoculationand by the humidification phenomenon of capillary ascent. Inho-mogeneous zones, distinguishable at the surface of few samples,may have been caused by a slightly improper horizontal placementof the samples after inoculation, which could create an accumula-tion of algae on an edge.

Visual observations provided by Fig. 11 show that the presenceof UV radiation causes the darkening of algae during growth ascompared to the brightest green colour of the algae obtained underthe visible fluorescent tube. Because of this difference of colour,

) gathering of the pixels into three classes (c) determination of the colonized area.

Fig. 8. Macroscopic aspect of reference mortar samples (5*5 cm2) exposed to water run-off testfor different times of exposure and different UV illumination.

T. Martinez et al. / International Biodeterioration & Biodegradation 89 (2014) 115e125 121

clearly visible from the beginning of the test, that can affect theconversion to grayscale for the same state of fouling, determinationof the colonization index was not performed to quantify the in-fluence of UV light on the development.

3.2. Evaluation of the anti-fouling activity of the photocatalytictreatment

During thewater run-off test, image analysis (Figs. 9 and 10) andvisual observations (Fig. 12) did not enable the samples treatedwith the photocatalytic coating to be distinguished from the un-treated samples. The growth of algae was not reduced or sloweddown by the presence of a photocatalyst in any lighting condition,even the most favourable to photocatalytic reaction kinetics (7.5e12 W m�2).

On the simulation by capillary ascent, the photographs providedin Fig. 11 does not exhibit differences in algal fouling that can beattributed to a photocatalytic process. Moreover, the rate of foulingon reference and photocatalytically treated mortar measured bythe two image analysis methods are not significantly different(Fig. 13).

Under the experimental conditions used in the two testset-ups, the photocatalytic surfaces investigated did not reduce

Fig. 9. Evolution of the colonization index of the samples exposed

algal fouling by C. emersonii, although these surfaces have beenshown to be photoactive under similar light conditions anddegrade nitrogen oxides (Martinez et al., 2011). Moreover, thesame tests performed on mortars loaded with photocatalyticparticles (5% TiO2 P25 relative to the cement weight) led to similaroutcomes (results not shown). Several hypotheses may explainthese results:

- The test acceleration factor of the water run off simulation mayhave been too high and therefore inappropriate for the identi-fication of the anti-algal properties of the photocatalytic surface.

- Algae may be resistant to photocatalysis. Studies on thedestruction of microorganisms mainly focus on bacteria forwhich photokilling is regularly obtained in presence of a pho-tocatalyst and begins with the degradation of cell walls (Sunadaet al., 2003). The photocatalysis process may be less effective onalgae because of their generally more complex and thicker cellwalls (Hoiczyk and Hansel, 2000; Barsanti and Gualtieri, 2006).Moreover, the attachment of aero-terrestrial algae to a surface isprovided by extracellular polysaccharides which remain incontact with the cell walls and the substrate (Barberousse et al.,2006a). These extracellular polysaccharides can act as a barrierbetween the photocatalytic surface and the algal cell wall. Algal

to water run-off test(a) UV 7.5e12 W m�2 (b) 1.5e3 W m�2.

Fig. 11. Macroscopic aspect of samples exposed to water capillary ascent test for different times of exposure (0, 35 and 74 days).

Fig. 10. Evolution of the colonized area of the samples exposed to water run-off test (a) UV 7.5e12 W m�2 (b) 1.5e3 W m�2.

T. Martinez et al. / International Biodeterioration & Biodegradation 89 (2014) 115e125122

Fig. 12. Macroscopic aspect of mortar samples exposed to run-off test for different times of exposure (84, 131 and 158 days). Comparison of the algal development between aspecimen coated with UV-TiO2 glaze, a specimen coated with a water repellent and a control specimen. The specimens presented were kept in zone 2 of the run-off test set-up (UV-A: 7.5 W m�2, VIS: 3.0 W m�2).

T. Martinez et al. / International Biodeterioration & Biodegradation 89 (2014) 115e125 123

cells in general have evolved defences with antioxidant enzy-matic and non-enzymatic mechanisms to protect the cellsagainst the danger posed by the presence of reactive oxygenspecies (Mallick and Mohn, 2000). Therefore, aero-terrestrialalgae may not be altered by the oxidative process ofphotocatalysis.

- Dead algae may create a barrier effect. This biofilm could reducethe contact between the new algae deposited and the photo-catalytic surface. Moreover, the presence of algae minimizes the

Fig. 13. (a) Evolution of the colonization index and (b) evolution of the colonized area of the rascent test (UV/VIS illumination).

amount of UV light that reaches the photocatalytic surface andthe diffusion of the reactive oxygen species on the algaedeposited by the water run-off.

3.3. Evaluation of the anti-fouling effect of the water-repellenttreatment

On the water run-off simulation, it can be observed that waterrepellent treatment is able to notably slow the progression of

eference mortar and of the photocatalytically treated mortar exposed to water capillary

T. Martinez et al. / International Biodeterioration & Biodegradation 89 (2014) 115e125124

colonization compared to that obtained with the control specimensand photocatalytic samples (Figs. 9, Fig. 10, Fig. 12).

Image acquisitions (Fig. 11) show that, in contrast to referencemortars, mortars treated by water repellent supported no devel-opment of algae during the test by capillary ascent. In addition,visual observation revealed that: (1) the surfaces of samples treatedbywater repellent remained dry, whichwas certainly the reason forthe absence of algal fouling, (2) few algal cells deposited during theinoculation were visible to the naked eye but they changed colourduring the test - from a greenish to a yellowish tint, reflecting astress condition that can lead to a reduction of the pigment contentand/or even a loss of cell viability.

The application of the water repellent reduced the availabilityof water on the surface of the specimen, and water supply is amajor parameter of algal growth, together with light, nutrientsupplies and temperature (Escadeillas et al., 2007). Therefore, thedifference between the two simulation results can be explained bythe mode of humidification and inoculation. The application of thewater repellent prevented capillary ascent reaching the surface ofthe sample, which prevented the growth of the inoculum depos-ited on the sample surface. In the water run-off test, the samplesurface was directly wetted by the watering system and inocula-tion occurred constantly during the water run-off period. Theapplication of a water repellent caused the surface to remain dryfor longer than the untreated surface. Therefore, the growth ofmicroorganisms on the water repellent treated surface was notcompletely prevented under water run-off. This observation underlaboratory conditions supports findings in real situations (Nugariand Pietrini, 1997; Urzi and De Leo, 2007; Moreau et al., 2008),where algae were detected in biological analyses performed onbuilding material treated with hydrophobic compounds (differentchemical composition from the water repellent used in this study)and exposed outdoors. The results obtained in the present studyclearly show that the bioreceptivity of mortar decreases after theapplication of a water repellent. Further tests should be carried outunder natural weathering to confirm the effectiveness and thedurability of this protection against microbial colonization.

4. Conclusion

The performance of photocatalytic and water repellent treat-ments in inhibiting algal growth (G. emersonii) were assessed withtwo different accelerated algae growth tests (water run-off andcapillary ascent).

� Increasing UV intensity (1.5e12 W m�2) caused an inhibition offouling during the water run-off test and a darkening of thefouling during the test by capillary ascent.

� The development of biofouling was not limited by the photo-catalytic treatment in the various test set-ups and lightingconditions used in this study.

� Treating the mortar with water repellent compounds decreasedthe rate of fouling obtained in the water run-off test. Moreover,no colonization was observed during the 74-day test when itwas performed in the capillary ascent set-up. These results arecertainly related to the reduced availability of water at the sur-face, caused by the water repellent treatment.

All the tests performed and the results reported in the literatureshow that it is necessary to multiply the types of biological growthsimulation to characterize the influence of surface treatments onbioreceptivity. It would be interesting to investigate the perfor-mances of such coatings against algal colonization using realcommon algal biofilms common biofilm (Klebsormidium, Sticho-coccus Apatococcus, Coccomyxa, Chloroidium or Trentepohlia).

Moreover, results in this study obtained with the water-repellent-treated mortars suggest formulating a coating combining hydro-phobic and photocatalytic properties to reduce the level of airpollution and to limit the development of microorganisms onwalls.Recent findings in the literature showed that it should be possible(Yoshida et al., 2006).

Acknowledgements

The authors would like to thank LRVision for its financial andscientific support and ANRT and Région Midi Pyrénées (France) fortheir financial support.

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