Evaluation of the efficiency of water-repellent and biocide compounds against microbial colonization of mortars

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International Biodeterioration & Biodegradation 60 (2007) 25–34 www.elsevier.com/locate/ibiod

Evaluation of the efficiency of water-repellent and biocide compounds against microbial colonization of mortars$ Clara Urzı` , Filomena De Leo Department of Microbiological, Genetic and Molecular Sciences, University of Messina, Salita Sperone 31, 98166 Messina, Italy Received 12 July 2006; received in revised form 30 October 2006; accepted 6 November 2006 Available online 3 January 2007

Abstract Mortars are building material with a high primary bioreceptivity and thus, they are easily colonized by different microorganisms. To reduce this problem, especially if it concerns restoration and conservation of works of art, water repellents and biocides can be applied alone or in combination. This paper describes experimental data carried out in laboratory conditions as well as outdoors with artificially infected mortars to reproduce natural colonization. The effects of three hydrophobic compounds (RHODORSIL RC80, HYDROPHASE SUPERFICI and HYDROPHASE MALTE) applied alone or in combination with the biocide ALGOPHASE and with the new water miscible formulation (ALGOPHASE PH025/d) were studied. Effectiveness was compared with the behavior of untreated mortars. In both types of experiments, it was clearly shown that water repellents alone do not stop microbial colonization, while water repellents plus biocides prevent microbial growth. In addition, it was shown under indoor and outdoor conditions that fungi are able to colonize untreated mortars as well as those treated only with hydrophobic compounds before phototrophic microorganisms. No significant differences were observed among the compounds tested in their efficacy in preventing colonization. r 2006 Elsevier Ltd. All rights reserved. Keywords: Biocides; Water repellents; Microbial colonization; Mortars; Bioreceptivity

1. Introduction Mortar is a mixture of sand with a powdered adhesive, such as cement, and water. Once applied as paste, it dries hard, forming pores of different size that create suitable microhabitats for many types of microorganisms. According to Guillitte (1995), mortars thus possess a high primary bioreceptivity. In fact, visible patterns of colonization (green, orange, black and grey patinas) can be formed by lichens, algae, cyanobacteria, fungi, actinomycetes and other bacteria of various phylogenetic affiliation; those microorganisms are present in a rather variable number of species and biocenoses ( ¼ a group of interacting (micro)$ Scientific relevance of paper: Increase understanding of the preventive effect of biocides and water repellent substances on microbial colonization of mortars. Corresponding author. Tel.: +39 90 6765196; fax: +39 90 392733. E-mail address: [email protected] (C. Urzı` ).

0964-8305/$ - see front matter r 2006 Elsevier Ltd. All rights reserved. doi:10.1016/j.ibiod.2006.11.003

organisms that live in a particular habitat and form an ecological community), and may cause the powdering of material (Urzı` and Realini, 1998). Water is one of the most important abiotic factors of decay of porous materials (Amoroso and Fassina, 1983; Warscheid and Braams, 2000). Once it penetrates into the pores by capillary force, water carries out its deteriorating effect through the chemical dissolution of the calciumcarbonate component of the stone, through physical phenomena such as freezing-and-thawing, salt crystallization and deposition, and through the growth of microorganisms due to deteriorative processes connected to their colonization. To prevent this multi-factorial damage, treatment of the surfaces with hydrophobic products is often suggested (Malagodi et al., 2000). Water repellents, when impregnated into the porous stones reduce the surface energy and the force of capillarity, slowing down the adsorption of water into the stone (Puterman, 2000).

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The main group of hydrophobic compounds in use are ‘‘silicones’’: alchyl-silanes or alchyl-alcoxi-siloxanes. Once applied on the stone surface, they form a hydro-repellent silicone film by hydrolysis and condensation reaction (polymerization). Hydro-repellency is developed generally within 24 h and can be effective for 5–10 y depending on the exposure, light radiation, etc. (Balzarotti-Ka¨mmlein et al., 1999). Biocide treatments are considered to be one of the practical approaches for conservation of artworks including stone materials. Their application aims to prevent and/ or control microbial growth (Koestler, 2000; Warscheid and Braams, 2000; Caneva et al., 2005). Two main categories of biocides are known: those applied before the treatments to eliminate microorganisms already present, and those that should have a preventive effect that slow down the re-colonization of restored surfaces. It has been proposed that the application of hydrophobic compounds and biocides of the second group is more effective against microbial colonization of surfaces (Commissione Normal, 1991, Malagodi et al., 2000; Urzı` et al., 2000a; Arin˜o et al., 2002; Caneva et al., 2005; Nugari and Salvadori, 2003). This application can be carried out in a single step: the water repellent and biocide are mixed together and applied on the surface, or in two steps, in which the biocide is applied before the treatment of water repellent or after it (Nugari and Salvadori, 2005). Nugari and Salvadori (2003) stressed that before any application on monument surfaces, products need to be shown to be completely harmless to the material treated and to be effective against the microorganisms (Koestler and Salvadori, 1996). To fulfill this requirement and to test the efficiency of mortar treatments, we have carried out two different types

of experiments on mortar probes; in the laboratory as well as under outdoor conditions. In the first case, the efficiency of treatments was tested against a massive colonization of different kinds of microorganisms (bacteria, fungi and a mixture containing cyanobacteria, algae, bacteria and fungi). In the second set of experiments, under outdoor conditions, we observed the natural settlement of airborne microbiota on untreated and treated mortars. 2. Material and methods 2.1. Mortars probes Freshly prepared mortar probes (size 1  1  0.5 cm) with different compositions were used for the experiments: lime+sand (L+ S) and Pozzolana+lime (P+L). The selection and preparation of these types of mortars was based on the compositions of masonry dating up to the mid19th century and was carried out as in the frame of EC project ENVK4 0707 Development of an Innovative Water-Repellent/Biocide Surface Treatment for Mortars: Assessment of their Performance by using Modern Analytical tools and Surface Analysis, acronym SILICON, by MT Blanco Varela of Instituto de Ciencias de la Construccio´n, CSIC, Madrid, Spain.

2.2. Chemical compounds Three water-repellent products were chosen: non-water miscible HYDROPHASE SUPERFICI (PHASE srl), water miscible HYDROPHASE MALTE PH 19503 (PHASE srl); non water miscible RHODORSIL RC80 (Rhone-Poulenc); two biocides produced by PHASE srl: ALGOPHASEs non water miscible and a new water miscible formulation ALGOPHASE PH025/d were also selected (Table 1). Application of products was carried out by complete immersion of probes in each solution as specified in Table 1, left for 3 s, and then they were removed and let to dry on paper cloth for 72 h at room temperature.

Table 1 Water repellents and biocides composition, type of solvent used and final concentration Mortar Ref.

Product and active ingredient

Composition

Concentration and solvent used

T0 T1 T2 T3

40% in isopropyl alcohol 70% in white spirit 50% of active ingredient in mineralized H2O As T1

as supplied as supplied 1:1.5 (v:v) in distilled H2O

T4

Untreated (control) HYDROPHASE SUPERFICI (alkyl alcoxy silane) RHODORSIL RC80 (polymethylsiloxane) HYDROPHASE MALTE PH91503 (alchyl tri-alcoxi silane) HYDROPHASE SUPERFICI+ALGOPHASE

T5

RHODORSIL RC80+ALGOPHASE

As T7 As T2

T6

HYDROPHASE MALTE PH91503 (alchyltrialcoxisilane) +

As T7 50% of active ingredient in mineralized H2O

ALGOPHASE PH025/d (2,3,5,6-tetrachloro-4methylsulfonyl-pyridine)

T7

ALGOPHASE (2,3,5,6-tetrachloro-4-methylsulfonylpyridine)

5% of active ingredient in suspending solution, anti-thicking agents (attagel 50-like), gelifiers, xantogum and polyvinylpirrolidone 30% of active ingredient in Nmethyl-2-pirrolidone

3.5% of ALGOPHASE directly in HYDROPHASE SUPERFICI 3.5% of ALGOPHASE directly in RHODORSIL RC80 3% of ALGOPHASE PH025/d directly in HYDROPHASE MALTE PH91503 already diluted in distilled H2O (1:1.5 v:v)

3,5% of ALGOPHASE diluted in isopropyl alcohol

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2.3. Indoor experiments 2.3.1. Microorganisms and media for the laboratory tests Three different microbial suspensions were prepared. (1) Fungal suspension: a mixture of two black fungal strains, Alternaria alternata MC 816 and Cladosporium sp. MC 781, isolated respectively from mortar surfaces of two monuments: Schloss Weissenstein, Pommesfelden, Germany and Mura Farnesiane Parma, Italy studied in the frame of SILICON project. The isolated strains were kept in the Department Collection. For the preparation of suspension, each fungal strain was cultivated in solid medium (Potato Dextrose Agar— PDA, Oxoid) for 7 d at 28 1C, then fungal colonies were scraped from the agar surface and conidia suspended in physiological solution (0.9% of NaCl in distilled water). The number of fungal units (conidia) was determined through a direct microscopic count in a counting chamber (Bu¨rker chamber, ProSciTech, Australia) and adjusted at a concentration of 1  106 cfu ml1. (2) Bacterial suspension: two bacterial strains: a red pigmented Micrococcus sp. strain and an unidentified Gram positive rod slime producer were used. The two strains were isolated from mortar surfaces of two monuments: Schloss Weissenstein, Pommesfelden, Germany and Mura Farnesiane, Parma, Italy and kept in the Bacterial Collection of the Department as strain BC623 and BC624, respectively. A mixed bacterial suspension was obtained from freshly prepared cultures of each strain grown in liquid medium BRII (Urzı` et al., 2000b) for 7 d at 28 1C, harvest by centrifugation at 10,000 rpm for 10 min and suspended in physiological solution. Density of suspension was compared to the 0.5 MacFarland standard (Biome`rieux Italia, Cat. n. 692801) and adjusted at a concentrations of 1  107 cells ml1. (3) Non axenic algal and cyanobacterial suspension: a non axenic fresh algal culture obtained after inoculation in liquid BG 11 medium (Commissione Normal, 1990) of a portion (100 mg) of biofilm sample taken from a mortar surface in the Templete de Mudejar in Guadalupe, Spain (Urzı` et al., 2000a) was used to inoculate the mortar probes. The culture grown after 15 d of incubation at room temperature and day light, contained filamentous cyanobacteria Nostoc sp., unicellular green algae Chlorella sp. and filamentous ones; also black fungi of the genera Humicola, Ulocladium, Phoma, coryneform bacteria and strains of the genus Micrococcus were present. The culture was concentrated by centrifugation at 10,000 rpm for 10 min and suspended in physiological solution. The density of suspension was adjusted comparing it to the standard MacFarland 3 (Biome`rieux Italia, Cat. n. 69280). The mortar probe surfaces were inoculated with 0.1 ml of each microbial suspension.

2.3.2. Susceptibility tests of treated and untreated mortars to microbial colonization Untreated and treated mortar probes were inoculated in duplicate with the fungal, bacterial and algal suspension separately and maintained in constant humid condition at room temperature and day light. The progression of microbial colonization was monitored through stereomicroscopic observations (Leica WILD M10) at intervals of one month. Photographic documentation was also carried out. Fifteen months after the inoculation, all the experiments were stopped and one replica of each mortar probe was utilized for light and epifluorescent microscopic analysis. For this purpose, samples were taken from each mortar surface with adhesive tape (Fungi Tape DID, Milan, Italy) as described in Section 2.4.1.

2.4. Outdoor experiments Untreated and treated mortar probes were exposed in outdoor conditions in the terrace of the Faculty of Science of the University of Messina, Italy.

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Eight replicas for each type of mortar were placed into the racks placed on the exposition table. The racks were oriented to North East and kept inclined at an angle of 451 degree. The progress of colonization of mortar surfaces was monitored every month under binocular microscope. After 15 months the exposure experiment was stopped and microscopic and cultural analyses were carried out on the specimens as described in Sections 2.4.1 and 2.4.2, respectively. 2.4.1. Microscopic observations Monitoring of mortar probes colonization was carried out by using the non destructive sampling method of adhesive tape strips as reported by Urzı` and De Leo (2001). This technique was chosen in order to simulate a sampling method used on valuable surface. In fact, this method produces a mirror image of the sampled surface, which can be observed under a microscope and used to carry out cultural and molecular analysis. Adhesive tape strips (Fungi Tape DID, Milan, Italy) were gently applied on the top surface of each mortar probe and small pieces (about 5  5 mm) were observed directly under light and epifluorescent microscopy (Leica DMR/HCS microscope equipped with a 50 W mercury lamp HBO) with or without the addition of the fluorescent dye Acridine Orange (AO). 2.4.2. Cultural analysis Microbiological analyses were carried out on each type of mortar sample according to the Normal Committee recommendations (Commissione Normal, 1990). Samples were powdered in a mortar and suspended (1:10) in a physiological solution with addition of 0.001% Tween 80 and continuously stirred for 1 h, in order to have a better separation and distribution of microorganisms living in/on the rock material. For the growth of phototrophic microorganisms, detected as presence/ absence, 1 ml of suspension was inoculated in triplicate into liquid medium BG11 (Commissione Normal, 1990). Incubation was carried out in the light and at room temperature for 75 d. For the enumeration of cultivable chemo-organotrophic microorganisms, 1 ml of suspension and following decimal dilutions were inoculated in duplicate in Petri dishes with the addition of solid medium BRII (Urzı` et al., 2000b) for bacteria and DRBC medium (King et al., 1979; Urzı` et al., 1992) for fungi. Incubation was carried out at 28 1C for one month. Counts of viable microorganisms were determined as cfu g1 sample. Cyanobacteria were assigned to morphological groups according to Rippka et al. (1979). Identification of fungi was carried out according to Barnett and Hunter (1972), Fassatiova` (1986) de Hoog (1987) and Ellis (1971, 1976).

3. Results 3.1. Indoor experiments 3.1.1. Mortars inoculated with bacterial suspension Bacterial colonization was scarce and non uniform on the mortar surfaces. Microbiological analysis carried out 15 months after inoculation revealed the presence only of the slime-producing Gram-positive rods (Coryneform strain) in both types (L+S and P+L) of the untreated mortars (T0) and in mortar samples treated with the hydrorepellent (T1, T2, T3) or with biocide alone (T6). Only T4 and T5 mortar probes (both types, L+S and P+L) treated both with water-repellent products plus biocide (HYDROPHASE plus ALGOPHASEs and RHODORSIL RC80 plus ALGOPHASEs, respectively), showed the total absence of bacterial colonization.

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Fig. 1. (a)–(c) L+S and (d)–(f) P+L mortars infected with mixed fungal suspension observed under binocular microscope (magnification 80x). (a), and (d): Mortar probe surface immediately after the inoculation; (b),and (d): mortars treated with hydrophobic compounds alone (T2 and T1 respectively) after 15 months of incubation; (c), and (f): mortar samples treated with hydrophobic compounds plus biocide (T5 and T6, respectively) after 15 months of incubation.

Fig. 2. L+S mortar probes infected with a mixed algal suspension observed under binocular microscope. (a)–(c): Untreated mortar probes: (a) immediately after the inoculation, magnification 8x; (b) after 15 months from the inoculation, magnification 8x; (c) Blow up of (b) showing the evident algal and fungal colonization, magnification 80x. (d)–(f): Mortar probes treated with hydrophobic compounds (T2): (d) immediately after the inoculation, magnification 8x; (e) after 15 months from the inoculation, magnification 8x; (f) blow up of (e) showing the algal and fungal colonization on mortar probe, magnification 80x. (g)–(i): Mortar probe treated with biocide plus hydrophobic compound (T5): (g) immediately after inoculation, magnification 8x; (h) after 15 months of incubation, magnification 8x; (i) blow up of (h) showing the pink spots on the surface, magnification 80x.

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3.1.2. Mortars inoculated with fungi On both untreated mortar probes (L+S and P+L) an increment of fungal colonization was clearly observed 3 months after inoculation. Fungal growth appeared as black hyphae and/or as chains of black conidia. A similar pattern of growth was observed on the mortars treated with hydrophobic compounds alone (T1, T2, T3). Contrary to this, the mortars treated with biocides alone or in combination with hydrophobic compounds did not show fungal growth; fungal units were observed as resting conidia. This situation was observed for the whole length of the incubation time (15 months) (Fig. 1). 3.1.3. Mortars inoculated with algae and cyanobacteria Changes in the size and aspect of the phototrophic-based patina were observed only after six months of incubation on the surface of both types (L+S and P+L) of untreated mortar samples (T0) as well as on mortars treated with hydrophobic compounds alone (T1, T2, T3). Besides the algal patina, the presence of fungal growth as black masses

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and/or as black hyphae was also observed in all the samples. This fact was particularly evident in the T3 mortar samples (L+S and P+L) treated with HYDROPHASE MALTE and in the T1 (L+S) mortar samples treated with HYDROPHASE SUPERFICI. On the contrary, the disappearance of the green patina on mortar surfaces treated with biocide alone (T7) or in combination with hydrophobic compounds (T4, T5, T6), was observed as early as three months after the inoculation. Remains of dead algal cells were evidenced as pink-orange colored spots or patinas. Microbiological analysis confirmed what was observed under the stereomicroscope. Direct observations of adhesive tape strips under microscopy showed the presence of phototrophic microorganisms (mostly filamentous Nostoclike cyanobacteria) on the untreated mortar samples (T0) and on those treated with hydrophobic compounds alone (T1, T2, T3). Absence of algal colonization was confirmed on the mortar probes treated with biocides alone (T7) or in combination with hydrophobic compounds (T4, T5, T6).

Fig. 3. P+L mortar probes infected with a mixed algal suspension observed under binocular microscope. (a)–(c): Untreated mortar probe: (a) immediately after the inoculation magnification 8x; (b) after 15 months from the inoculation, magnification 8x; (c) blow up of (b) showing the evident algal and fungal colonization, magnification 80x. (d)–(f): Mortar probe treated with hydrophobic compounds (T2): (d) immediately after the inoculation, magnification 8x; (e) after 15 months from the inoculation, magnification 8x; (f) blow up of (e) showing the algal and fungal colonization on mortar probe, magnification 80x. (g)–(i), Mortar probe treated with biocide plus hydrophobic compound: (g) immediately after the inoculation, magnification 8x; (h) after 15 months of incubation, magnification 8x; (i) blow up of (h) showing the pink spots on the surface, magnification 80  .

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Figs. 2 and 3 show the results obtained by inoculating the algal suspension onto untreated and treated mortar surfaces. 3.2. Outdoor experiments 3.2.1. Microscopic observations Direct examination of samples taken from mortar surfaces by adhesive tape strips showed the presence of black fungal clusters or chains of meristematic cells in both types (L+S and P+L) of untreated mortar probes and treated with water repellent products alone (Fig. 4(a)–(c)). In some cases (T0 and T1), a close adherence of microorganisms to the stone material was observed. 3.2.2. Cultural analysis Results of cultural analysis showed the occasional presence of chemo-organotrophic bacteria on untreated mortars and on those treated with water repellent alone. Fungi were found in all untreated and treated mortar probes (Fig. 5), while algae were present in all L+S mortars and found only on P+L mortar treated with HYDROPHASE MALTE PH91503 with and without the addition of biocide (T3, T6) (Table 2). L+S mortars: in almost all treated mortar probes the number of fungi was lower (ranging from 9.7  102 to 1.8  104 cfu g1) than in the untreated mortar probes (2.4  104 cfu g1). Only sample T2 (treated with RHODORSIL RC80 showed an higher number of fungi (4.5  104 cfu g1). The treatment of mortars with the biocide alone or in combination with hydrophobic compounds reduced drastically the number of fungi (range of reduction of 78–98%). This fact was particularly evident in the mortar probes treated with HYDROPHASE SUPERFICI+ALGOPHASEs (T4) and RHODORSIL RC80+ALGOPHASEs (T5) (Fig. 5). Algae were detected in the untreated sample (T0) and in all L+S mortar probes treated with hydrophobic compounds alone, except on the sample treated with HYDROPHASE SUPERFICI (T1). The biocide alone or in association with hydrophobic compounds did not allow the algal colonization except for the sample T5 (RHODORSIL RC 80 plus ALGOPHASEs) (Table 2). P+L mortars: The treatment with hydrophobic compounds alone was not sufficient to prevent the fungal colonization. In fact, the number of fungi recovered was in all cases even higher than the untreated mortar probe (ranging from 3.6  104 to 2.2  104 cfu g1). The association of the hydrophobic compounds with biocides or the biocide alone decreased dramatically the number of fungi (reduction rate between 78% and 99%). This was particularly evident in the sample treated with HYDROPHASE SUPERFICI +ALGOPHASEs (T4) (1.8  102 cfu g1), that showed a reduction of 99.5% in the number of fungi with respect to the sample treated with HYDROPHASE SUPERFICI (T1) (3.6  104 cfu g1) (Table 2 and Fig. 5). In P+L mortar probes, algae were not detected, except in

Fig. 4. Light microscopy observations of adhesive tape samples taken from mortar surface exposed in outdoor condition after 15 months. (a) Untreated mortar surface (T0). Several fungal conidia (arrows) are observed on the surface. (b) and (c) Mortars treated with hydrophobic compounds (T1) and (T3) alone. The presence of fungal conidia is still observed even if scarce. Bar is 10 mm.

the case of the sample treated with HYDROPHASE MALTE (T3) and in scarce amounts on mortar treated with HYDROPHASE MALTE+PH025/d (T6).

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Fig. 5. Enumeration of fungi (cfu g1) colonizing mortar probes after 15 months of outdoor exposure. It is evident how the combined treatments of water repellent plus biocide (samples T4 and T5) reduce dramatically the colonization by fungi.

Table 2 Results of cultural analysis carried out after 15 months of outdoor exposure L+S

A/Ca

F (cfu1 g)

B (cfu1 g)

P+L

A/C1

F (cfu1 g)

B (cfu1 g)

T0 T1 T2 T3 T4 T5 T6 T7

++ + ++ ++ +/ +/ +/ +/

2.4  104 1.0  104 4.5  104 1.7  104 1.3  103 9.7  102 1.5  104 5.5  103

1.7  103 * * — — — — —

T0 T1 T2 T3 T4 T5 T6 T7

— — — ++ — — +/ —

1.8  104 3.6  104 2.2  104 2.8  104 1.8  102 4.9  103 3.8  103 2.7  103

* * * * — — — —

(B ¼ bacteria, F ¼ fungi, A/C ¼ algae and cyanobacteria) a Phototrophic microorganisms were found only after 75 d of incubation; ++ ¼ abundant growth;+ ¼ visible growth; +/ scarce growth; * ¼ occasional growth.

Concerning the fungal isolates, they were mostly fast growing airborne dematiaceous species. Isolates of the species A. alternata were predominant in all treated and untreated mortar probes (both L+S and P+L), except in the L+S mortar probe treated with HYDROPHASE SUPERFICI+ALGOPHASEs (T4), in which non pigmented yeasts of the genus Trichosporon were found to be the most representative mycoflora. Strains of the genera Cladosporium, Ulocladium, and Phoma were isolated with a minor frequency in all type of mortars and treatments (Table 3). Of the algae, only unicellular green algae (Chlorella like) were those observed on the mortar surfaces. 4. Discussion and conclusions Our experiments demonstrated that untreated mortars of both types considered in this study (L+S and P+L)

possessed a high primary bioreceptivity and were suitable surface for microbial colonization by heterotrophic and phototrophic microorganisms. In particular, the two types of mortars used showed a slightly different porosity (higher for L+S than P+L) and this fact in turn determined a higher rate of deterioration processes and higher colonization (Saiz-Jimenez, 2000). It was also shown that the application of the hydrophobic compounds alone was not sufficient to prevent biofilm growth on the surface. On the contrary, it seems evident that the combination of waterrepellent compounds and biocides applied in a single step create unfavorable conditions for microbial growth for up 15 months of incubation under laboratory conditions. Similar results arose from the experiments carried outdoors. In this case, as well, the spontaneous microbial colonization, comparable to that occurring on monument surfaces, showed that the combined action of the water repellent plus biocide has a protective effect for the mortars

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Table 3 Fungal strains isolated from the mortars exposed outdoors and their percentage of isolation L+S

Fungi

P+L

Fungi

T0

Alternaria alternata (95%) Cladosporium sp. (5%) Alternaria alternata (85%) Ulocladium sp. (10%) Cladosporium sp. (5%) Alternaria alternata (85%) Cladosporium sp. (15%) Alternaria alternata (80%) Cladosporium sp. (20%) Phoma sp. (+) Yeast (90%) Alternaria alternata (10%)

T0

Alternaria spp. (95%) White mould (5%) Alternaria alternata (90%) Cladosporium spp. (10%) Black Yeast (+) Alternaria alternata (90%) Cladosporium spp. (10%) Alternaria alternata (50%) Black yeasts (50%) Phoma sp. (+) Cladosporium sp. (100%) Black Yeast (+) Aspergillus sp. (+) Alternaria alternata (70%) Cladosporium sp. (+) Ulocladium sp. (30%) Ulocladium sp. (60%) Alternaria alternata (40%)

T1

T2 T3

T4

T1

T2 T3

T4

T5

Alternaria alternata (60%) Cladosporium sp. (40%)

T5

T6

Alternaria alternata (85%) Ulocladium sp. (5%) Cladosporium sp. (5%) Phoma sp. (5%) Alternaria alternata (85%) Cladosporium spp. (7%) Phoma sp. (8%)

T6

T7

T7

Alternaria alternata (60%) Ulocladium sp. (30%) Phoma sp. (10%)

(+): Occasional presence.

especially against algae and bacteria. No significant differences on the microbial colonization were noticed among the type of hydrophobic compounds tested (HYDROPHASE SUPERFICI, RHODORSIL RC80, and HYDROPHASE MALTE). However, the results obtained from outdoor exposure showed that fungi, and in particular black fungi (dematiaceous fungi), were the main colonizers of both the untreated mortars and/or those treated with water repellent products alone. This fact can be explained by capacity of fungi to grow at lower water availability than algae, cyanobacteria and bacteria, as already observed by Mansch et al. (1999) on monument surfaces. In addition, ubiquitous dematiaceous fungi such as Alternaria, Cladosporium, and Ulocladium possess a very high metabolic versatility that together with their morphological characteristics allow them to survive in dry and oligotrophic conditions. These results are in agreement with recent observations demonstrating that melanized fungi are the most common species isolated from freshly exposed stone surface (Sterflinger, 1999; Urzı` et al., 2000c; De Leo and Urzı` , 2003). Furthermore, fungi are able to utilize monomers of hydrophobic compounds as their sole carbon source and to grow on polymers of different nature (Leznicka, 1992; Koestler, 2000, Urzı` et al., 2001b). Under natural conditions, nutrient availability, even if small, enhances the chance of fungal units (single cells, conidia, or hyphae fragments) surviving and starting the colonization (Urzı` et al., 2001a). As the fungi form explorative hyphae to seek

nutrients on the surface, or mycelium during the growth, they can break the hydrophobic layer, provoking the loss of water repellency of the layer; thus by reaching the rock underneath they can exert their mechanical and enzymatic action directly against the material. Thus only the association of water repellent compounds and biocides can prevent and slow down the colonization of clean or newly exposed surfaces. The application of water repellent plus biocide in the same solution seems to be very effective as suggested also by other studies (Balzarotti-Kammlein et al., 1999; Urzı` et al., 2000a; Arin˜o et al., 2002). Inhibition of microbial growth by biocide treatment can persist for a period of 5–10 y except under continuous conditions of rising damp; under these conditions massive phototrophic-based microbial community were observed. Concerning the possible interference of the biocide on the water repellent, if they are applied in two steps, it was reported (Malagodi et al., 2000) that the application of biocide before the treatment with water repellent can modify the characteristic of this latter, reducing the water repellency properties. Contrary to this, no effect was noticed when the biocide was applied after the hydrophobic treatment. The good performance of single-step application of biocide and water repellent can be explained by the random distribution of biocide compound below, between and above the hydrorepellent film. In this manner, the biocide can exert its action against the remains of old colonization below, and against new colonizers on the surface.

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