ENVIRONMENTAL BIOTECHNOLOGY

Laboratory study of fungal bioreceptivity of different fractionsof composite flooring tiles showing efflorescence

Segula Masaphy & Ido Lavi & Stephan Sultz &

Limor Zabari

Received: 22 January 2014 /Revised: 14 February 2014 /Accepted: 17 February 2014# Springer-Verlag Berlin Heidelberg 2014

Abstract Fungi can grow in extreme habitats, such as naturalstone and mineral building materials, sometimes causing de-terioration. Efflorescence—concentrated salt deposits—re-sults from water movement through building material; it candamage masonry materials and other bricks. Fungal isolateKUR1, capable of growth on, and dissolution of stone chipscomposing terrazzo-type floor tiles, was isolated from suchtiles showing fiber-like crystalline efflorescence. The isolate’sribosomal DNA sequences were 100 % identical to those ofNigrospora sphaerica. The ability of KUR1 to colonize anddegrade the different stone chips composing the tiles wasstudied in axenic culture experiments. When exposed to eachof the different mineral chip types composed of dolomite,calcite, or calcite–apatite mineral in low-nutrition medium,the fungus showed selective nutrient consumption, and differ-ent growth and stone mineral dissolution rates. Micromorpho-logical examination of the fungus-colonized chips by electronmicroscopy showed the production of a fungal biofilm withthin films around the hyphae on the surface of the examinedchips and disintegration of the calcite–apatite fraction. Morethan 70 % dissolution of the introduced powdered (<1 mmparticle size) mineral was obtained within 10 days of incuba-tion for the soft calcite–apatite fraction.

Keywords Biodegradation . Bioreceptivity . Efflorescence .

Floor tile .Nigrospora . XRD

Introduction

Fungi play an important role in the biogeochemical transfor-mation of rocks and minerals, contributing to either theirdeterioration or their formation (Gadd 2007; Masaphy et al.2009). Fungi can grow in extreme habitats, even those withlow organic nutrient content or low water activity, such asnatural stone and mineral building materials. Their involve-ment in the weathering of these materials has been reported inconjunction with physical and chemical processes (Hirschet al. 1995; Hoffland et al. 2004; Gadd 2007; Warscheid andBraams 2009; Sterflinger 2010; Sterflinger and Piñar 2013).Fungi colonizing building materials can cause varying levelsof damage: aesthetic, such as black spot, direct or indirecthealth risks via toxic products transferred by breathing orhuman contact (Singh et al. 2010), and damage to buildingconstructions, leading to deterioration via cracks and dissolu-tion of the mineral material, similar to their activity in theweathering of natural stone (Palmer et al. 1991; Gaylarde et al.2003). Fungal attack on building and man-made mineralmaterials has been studied mainly in terms of its effects oncultural heritage (Principi et al. 2007; Sterflinger 2010;Sterflinger and Piñar 2013; Pinzari et al. 2012). Increasingour understanding of their role in weathering processes willcontribute to the use of biotechnological approaches to con-serve stone material (Fernandes 2006) and to exploiting thefungi’s abilities to interact with minerals for different biotech-nological processes such as mineral dissolution and metalrecovery (Mapelli et al. 2012).

Salt efflorescence occurs in natural and constructed humidenvironments. This phenomenon involves salt accumulationin zones of highest evaporation, usually at the edges of mate-rials, due to water movement through the pore system(Zehnder and Arnold 1989). The salt concretions may resem-ble white powder or a fiber-like crystalline structure, depend-ing mainly on the type of salt formed and the environmental

S. Masaphy : I. Lavi : L. ZabariApplied Mycology and Microbiology Lab, MIGAL, P.O. Box 831,Kiryat Shmona 11016, Israel

S. Masaphy (*) : I. LaviTel Hai College, Upper Galilee 12210, Israele-mail: segula@migal.org.il

S. SultzInstitute of Soil Science, Leibniz University Hannover,D-30419 Hannover, Germany

Appl Microbiol BiotechnolDOI 10.1007/s00253-014-5628-4

conditions. The phenomenon is common in humid environ-ments, when excessively wet walls during the autumn–winterperiod begin to dry out via evaporation, and the salts accumu-late on the surface of the material when critical concentrationsin the pore solution, due to the water evaporation, are reached(Zehnder and Arnold 1989). Salt efflorescence is found inmasonry, on bricks and mortar, walls, floors, and ceilings(Zehnder and Arnold 1989; Singh et al. 2010).

Efflorescence can cause heavy losses to cultural heritage,for example, when murals are attacked. Moreover, it maycause deterioration of building materials due to physical ef-fects—volumetric expansion of the growing crystals and lossof rock mass (Zehnder and Arnold 1989). Efflorescence isconsidered to result from physicochemical processes; to thebest of our knowledge, microbiological contributions to thisprocess, such as fungal activity, have never been considered.

In the winter and early spring of 2007, we observed efflo-rescence events in a residential home in Northern Israel,showing massive fiber-like crystal blooming on indoor floortiles. The salt efflorescence was accompanied by breaking ofthe mineral floor tiles as a result of disconnection of the stonechips in the tiles, resulting in salt deposits on the uncoveredchip-less floor material as well. This caused damage to thetiles, and raised health concerns due to possible inhalation ofthe crystalline salts by the residents. This phenomenon wasnoted sporadically in other houses on different settlements inthe area in subsequent winters.

To increase our understanding of the mechanisms contrib-uting to this destructive phenomenon, we examined the pos-sible involvement of fungi isolated from the efflorescence siteon the mineral flooring material near the broken tile. Wedetermined the susceptibility of the different tile mineral frac-tions to fungal attack in axenic culture in the laboratory byscanning electron microscopy (SEM) observation of hyphalmicromorphology on the surface of the incubated stone spec-imens, carbon and nitrogen source consumption by the cul-tured fungi, and dissolution rates of the different stone chipcomponents of the tiles as characterized by X-ray diffraction(XRD).

Materials and methods

Flooring material sample collection

Samples of the filamentous crystals, and stone material 1–2 mm below the site of salt-crystal accumulation, were col-lected aseptically from seven locations on the house floor,each sample from a separate tile showing efflorescence. Thesesamples were pooled as one master sample and brought to thelaboratory for fungal isolation and for determination of thecrystals’ chemical composition. Additional original unusedfloor tiles which had been stored in the house, without

efflorescence, were collected and brought to the lab to exam-ine their potential deterioration by fungi in laboratory exper-iments. Different stone chips were separated out from thecomposite tile by their color and hardness and were poundedto <3-mm particle size with a hammer. The mineral composi-tion of four of these: light red stone (fraction A), hard dark redstone (fraction B), hard white stone (fractionC), and soft whitestone (fraction D), were characterized by XRD and werefurther tested for their bioreceptivity potential to a fungalisolate. FractionD and the concrete layer under the floor werealso examined for their soluble elemental contents.

Fungal isolation and identification

A Nigrospora sp. was isolated by sampling the lower part ofthe filamentous crystals and the surface below the efflores-cence site, pulverizing them, and spreading them over rosebengal chloramphenicol (RBC) agar medium (Himedia,Mumbai, India). The isolated fungal colonies were furthercultured on potato dextrose agar (PDA; BD-Difco, New Jer-sey, USA) supplemented with 1 g l−1 CaCO3. The fungalisolate was identified by molecular analysis, and was used infurther degradation studies. This isolate was deposited in theCBS culture collection, with designation no. DTO 115-H4.

Molecular identification of the fungal isolate

Genomic DNAwas extracted from dry pulverized myceliumby phenol/chloroform procedure (Henrion et al. 1994), and theinternal transcribed spacer (ITS) region of the rRNA geneswas amplified. PCR primer pair ITS1-5′-TCC GTA GGTGAA CCT GCG G-3′ and ITS4-5′-TCC TCC GCT TATTGA TAT GC-3′ was used to amplify the correspondinggenes. PCR was conducted with Taq DNA polymerase(Sigma-Aldrich, St Louis, USA), using a MJMini™ PersonalThermal Cycler (Bio-Rad, Singapore) under the followingconditions: initial denaturation at 94 °C for 4 min, followedby 35 cycles of 94 °C for 1 min, 54 °C for 1 min and 72 °C for1 min. Amplicons were purified with a PCR purification kit(AccuPrep- K-3034- Bioneer, Daejeon, Korea). Amplificationproducts were sequenced by HyLabs (Rehovot, Israel). Thesequence was submitted to GenBank (accession no.KF574432).

Sequences were assembled and edited by SeqMan programand aligned, and a phylogenetic tree was constructed using theprogram Lasergene MegAlign (DNASTAR, Madison, WA,USA). The phylogenetic tree was based on multiple sequencealignments and cluster analyses constructed by MEGA 4program (Tamura et al. 2007). A dendrogram (comparingthe ITS regions) was created along with 2,000 bootstrap repeattest of phylogeny using the neighbor-joining algorithm(Saitou and Nei 1987). Other ITS sequences were retrievedfrom GenBank [National Center for Biotechnology

Appl Microbiol Biotechnol

Information (NCBI); http://www.ncbi.nlm.nih.gov/] and usedfor comparison.

Fungal degradation of the tile’s different mineral chips:in-vitro examination

Fungal inoculum and culture conditions

Nigrospora sp. was cultured in potato dextrose broth (PDB;BD, Difco, Franklin Lakes, NJ, USA; 12 g l−1) at 25 °C. After3 days of incubation, the myceliumwas collected by filtration,washed twice with buffered water, and homogenized. Thehomogenized mycelium was further inoculated (at 1:100) inlow-nutrient medium consisting of buffered water(42.5 mg l−1 KH2PO4 and 405 mg l−1 MgCl2⋅6H2O, broughtto pH 7 with 0.1 N NaOH). For the biodegradation test,ground mineral chip components were added, or not, to themedium before sterilization. The flasks were incubated for10 days at 25 °C with shaking (50 rpm).

Bioreceptivity and biodegradation tests

To determine the tile material’s ability to support fungalgrowth and provide nutritional requirements, a mixture (5 g)of the four different types of ground mineral fractions (equalweights of each; <3 mm particle size) was added to a 250-mlflask containing 50 ml buffered water which was also supple-mented with 0.1 g l−1 (NH4)2HPO4 as the nitrogen source or1 g l−1 glucose as the carbon source, separately or together.Medium without the addition of either carbon or nitrogensource was used as a control. Nutrient consumption rates weremonitored every 2 days during the incubation period bymeasuring ammonium nitrogen (N-NH4) and glucose concen-tration in the medium. Medium without fungal inoculum wasused as an additional control. To determine the bioreceptivityand support of fungal growth of each of the four differentstone mineral fractions composing the tile, the tile fractiontypes were added separately to the medium supplementedwith 0.1 g l−1 (NH4)2HPO4 and 1 g l−1 glucose. For eachstone type, 0.75 g of powdered fraction (<1 mm) and 4.25 g oflarger fraction (1–3 mm) were combined and added to eachErlenmeyer. Nutrient consumption was determined by mea-suring the N-NH4 and glucose remaining after 5 days ofincubation.

To determine fungal growth and activity in the presence ofthe tile material as influenced the presence of nitrogen orcarbon, another similar experiment was conducted by cultur-ing the fungus in the presence of the different nutrients andground stone mixture concentrations delineated above, butusing 3 ml medium in a glass test tube (25 mm×135 mm)incubated in a shaker (50 rpm, 25 °C). After 6 days ofincubation, fungal activity was determined by the MTT assay

[reduction of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetra-zolium bromide] (Freimoser et al. 1999).

To determine the biodissolution of the stone material, stoneparticles of fraction D [0.75 g of powdered fraction (<1 mm)and 4.25 g of larger fraction (1–3 mm)] were added to the50 ml medium supplemented with 0.1 g l−1 (NH4)2HPO4 and1 g l−1 glucose in 250-ml Erlenmeyer flasks. Medium con-taining stone particles without fungal inoculum was used as acontrol. After 10 days of incubation, the percent dissolutionand disappearance of powdered fraction D was determined.The Erlenmeyer content was filtered through Whatman filterpaper no. 1 (Whatman International, Maidstone, UK) andashed in a 500 °C oven for 6 h. The fine fraction, <1 mm,was then collected by separation through a 1-mm mesh netand weighed to quantify the fraction remaining after incuba-tion with the fungus. Treatment without fungal inoculationwas used as a control. Similar Whatman filter papers withoutfiltrate were ashed and quantified and subtracted from allvalues. The pH level in the culture medium was recorded bypH electrode before inoculation and at the end of the experi-ment, after 10 days of incubation.

Analytical methods

Mineral composition

The mineral composition of the different composite tile stonechips was determined by XRD analysis (Amir Sandler, TheGeological Survey of Israel, Jerusalem, Israel). The elementalcomposition (anions and cations) of the salt deposits at theefflorescent site, the soluble elements of fraction D from thetile, and the concrete layer at the bottom of the building, underthe floor, were determined by inductively coupled plasmaoptical mass spectrometry (ICP-MS; Agilent Technologies,Bellevue, WA, USA( for chemical elemental analysis andion chromatography (IC) for anion determination (IC-PRO,Metrohm, Herisau, Switzerland) after extraction in double-distilled water.

Determination of fungal growth and nutrient concentration

The amount of glucose remaining in the medium was deter-mined by the colorimetric 3,5-dinitrosalicylic acid method(Miller 1959). The amount of N-NH4 remaining in the medi-um and of N-NH4 in the tile stone material after distilleddeionized water extraction (1:10 w/v) was determined by thephenol–hypochlorite reaction method (Weatherburn 1967).

SEM examination of fungal colonization of each type oftile stone was performed with an environmental scanningelectron microscope (Quanta 200, FEI, Hillsboro, OR, USA)after gold-coating the samples. Control stone particles, incu-bated without fungal inoculum, were also examined.

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Fungal mycelial growth and activity were determined byMTT assay according to Freimoser et al. (1999) with somemodifications. After 6 days of culture incubation in test tubes,the medium was removed from the pelleted stone and myce-lium, and 1 ml of 60 mM phosphate buffer, pH 7.0, wasadded. After further incubation for 1 h under the same condi-tions, 20 μl of a fresh filter-sterilized solution of MTT (stocksolution 5 mg ml−1) was added. The culture was incubated for2 h in the dark in a shaker (50 rpm at 25 °C). The liquidfraction was then removed from the pellets and was replacedwith 1 ml of acidified 1-propanol (5 % of 1MHCl). The tubeswere incubated for a further 30 min at room temperature. Thesuspension was centrifuged after transferring to 1.5-mlEppendorf tubes (19,083×g, 2 min) to remove stone particles,lysed cells, and debris. The purple color produced in thesupernatant was measured with a Jasco (UV–VIS 7800, To-kyo, Japan) spectrophotometer at 560 nm, with 690 nm as areference read-out. A blank with acidified isopropanol alonewas measured and subtracted from all values.

All treatments were carried out in three replicates. Allresults were expressed as mean and standard error. Eachexperiment was carried out at least twice.

Results

Characterization of the efflorescence site

The deteriorated flooring material was found in a residentialbuilding built on concrete pillars. The terrazzo-type floor tileswere made of a composite material—a combination of differ-ent colored marble and quartz chips bonded by a cementitiousfraction. The mineral composition of four of these chips wasas follows: light red stone (fraction A), hard dark red stone(fraction B), hard white stone (fractionC), and soft white stone(fractionD). The efflorescence at the site was characterized byfilamentous fiber-like crystals accumulated on the surface ofthe tiles all around the room (Fig. 1). The salt deposits wereaccumulated mainly, but not only, on the white cementitiousfraction that bonded the different stone types in the terrazzo-type floor tiles. Holes were also found in the tile surface,where the stone chips had been disconnected from the com-posite tile and removed (Fig. 1). The phenomenon appeared indifferent rooms of the house during the winter and earlyspring.

Chemical analysis of the salt deposits by ICP-MS and ICshowed that they were composed of Na2SO4. Sulfate andsodium were found to be the dominant soluble anions andcations, respectively, in the tile and in the concrete in the samehouse (Table 1). In the analyzed concrete and in the soft whitestone chips (Table 2; fraction D) from the tile, other elements,such as Mg, K, Ca, and Fe, were also found. The anion

composition consisted mainly of sulfate ions, but chloride,nitrite, and nitrates were also found in measurable quantities.

XRD analysis of the four types of stone chips (fractions A–D) showed that the more resistant hard stones containedquartz. Some stones consisted primarily of dolomite[CaMg(CO3)2], others of calcite [a stable polymorph of calci-um carbonate (CaCO3)]. The soft white fraction contained nomeasurable quartz, but rather carbonate–apatite with highphosphate content (Table 2).

Fungal identification

The fungal isolate produced black colonies on PDA. Geneticanalysis confirmed that the strain belongs to the genusNigrospora. Comparison of the 493-bp ITS region ITS1-5.8-ITS2 of the nuclear ribosomal RNA (nrRNA) sequence of thisisolate with other Nigrospora species sequences retrievedfrom GenBank indicated 100 % similarity of the ITS regionsequence toNigrospora sphaerica (HQ608063) and 99.8% toN. sphaerica (GQ258792), and it was termed NigrosporaKUR1 (Fig. 2).

Fungal growth and deterioration of the studied tile stone chips

A ground particle mixture of the four stone fractions compos-ing the tile supported fungal mycelial growth in a laboratoryexperiment, mainly in media supplemented with both N-NH4

and glucose (Fig. 3a, b and c). Poor growth was observed inmedium with the addition of N-NH4 alone, but a pronouncedgrowth was observed with the addition of only glucose(Fig. 3b), and significantly more mycelial growth was obtain-ed with added glucose and N-NH4 (Fig. 3c). Consumption ofthe added glucose and N-NH4 by the growing mycelium wasenhanced by the addition of tile material to the medium(Fig. 4). Glucose also disappeared in the treatment withoutaddition of external N-NH4 (Fig. 4b), after a delay of 2 dayscompared to the treatment with addition of N-NH4. No reduc-tion in N-NH4 or glucose concentrations was recorded in the

Fig. 1 Efflorescence on floor tiles. Filamentous fiber-like crystals anddegradation of the different stone chips of the composite tile

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non-inoculated treatment containing only tile materials. Fun-gal activity, measured by MTT assay, was also higher in theculture supplemented with the combination of glucose and N-NH4 than in the treatments with glucose or N-NH4 alone.Nevertheless, fungal activity in the presence of added glucosemeasured at day 6 of incubation was somewhat higher thanthat with added N-NH4, both showing higher purple colorproduction than in the treatment with no addition of anynutrient source (Fig. 5).

When the different stone types composing the tile wereadded separately to the medium inoculated and supplementedwith glucose and N-NH4, nutrient consumption was related tothe type of the stone component (Fig. 6). The decrease inglucose and N-NH4 concentrations during incubation wasmost rapid in the presence of stone fraction D (soft whitefraction; Table 2(. In the presence of fractions A and C,nutrient reduction was also recorded, but to a lesser extent.No significant reduction of either nutrient in the culture me-dium containing fraction B was recorded compared to thecontrol culture with no added tile material.

Micromorphological investigations by SEM showed dif-ferential fungal growth and hyphal attachment to the differentstone types (Fig. 7). In general, the mineral surfaces showedmarked surface roughness which might facilitate hyphal col-onization. Small-sized silt particles also seemed to be attachedto the surface. Broad polysaccharide films covered the hyphaeand the mineral phase. Fraction D stone was markedlydisintegrated after fungal growth (Fig. 7d-1 and d-2 comparedwith 7d-3). Fungal mycelium was clearly observed in and onthe surface of the stone particles and powdered particles of thisfraction (Fig. 7d-1 and d-2). Fractions A and C showed hyphalgrowth on the surface of the stone, but its disintegration wasnot recorded. On stone fraction B, only poor hyphal growthwas recorded.

In the presence of fractionD, we observed rapid disappear-ance of the powdered fraction suspended in the mediumsupplemented with both carbon and nitrogen sources, as wellas enhanced glucose and nitrogen disappearance. Fraction Dwas further examined to determine its dissolution rate byassessing the amount of insoluble stone particles remainingin the culture after incubation with the fungi in the mediumsupplemented with glucose and N-NH4. However, over 70 %of the fine stone particles (<1 mm size) from fraction Ddisappeared within 10 days of incubation with the funguscompared to the control treatment without fungal inoculum(Fig. 8). The culture medium was alkaline in the presence offraction D particles, reaching a pH of 8.6±0.3, and at the endof this experiment on day 10, the pH declined to 6.4±0.5.

Discussion

We studied the possible contribution of fungi isolated fromflooring tile showing efflorescence salt deposits to the tile’sdeterioration. Although the efflorescence phenomenon—pro-duction of salt deposits on a mineral matrix—is considered tobe only a result of physicochemical processes, based on theresults of the accelerated bioreceptivity found here in the labexperiments, we suggest that fungi might contribute to, andenhance, the deterioration of the flooring tile material in theefflorescence process.

The efflorescence studied here was accompanied by dete-rioration of the flooring tiles. The salt crystals were found tobe composed of Na2SO4. Sodium sulfate and its hydrate(Na2SO4⋅10H2O) are among the most common salt depositsin urban efflorescence events on stone and concrete (WinklerandWilhelm 1970). Not surprisingly, sulfate and sodiumwerethe dominant soluble anions and cations, respectively, in the

Table 1 Elemental and anionionic composition of the soluble fraction of concrete under the floor and fraction D (soft white stone chips) of the tile asdetermined by IC and ICP-MS

Element (microgram per gram stone) Na Mg K Ca Fe Cl NO2 NO3 SO4 NH4

Concrete 682.9 3.2 445.5 12.3 0.0 20.6 0.8 94.5 623.1 <1

Fraction D of the tile 75.0 18.7 30.7 24.8 0.1 10.3 2.3 5.8 84.3 2.8

Ammonia in the soluble fraction of the minerals was determined separately by Weatherburn (1967) method

Table 2 Mineralogical composi-tion of the different stones makingup the terrazzo-type floor tilesshowing the efflorescence phe-nomenon, as determined by XRDanalysis

Composite fraction Stone description Mineralogy

A Light red stone Major: dolomite; Minor: calcite, quartz

B Dark red stone Major: dolomite; Minor: calcite, quartz

C Hard white stone Major: calcite; Minor/trace: quartz

D Soft white stone Major: calcite (CaCO3); Minor: carbonate–apatiteand phosphate mineral

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concrete in the same house and in the soft white stone (fractionD) from the tile (Table 1), suggesting migration of the saltfrom the concrete under the tiles and from the tile itself uponto the tile surface during efflorescence formation. Breakageof the tile surface and disconnection of the stone chips duringthe process suggested that aside from the migration of solublesalts from the under-the-floor layers, the tile surface itself issubjected to deterioration and dissolution of its mineral mate-rial. As analyzed by XRD, the stone chip composition of theterrazzo-type tile was heterogeneous including resistant hardstones containing quartz, and others consisting primarily ofdolomite and calcite. A fraction of carbonate–apatite with highphosphate content was also included in the tile composition(fraction D, Table 2), and this fraction might be more suscep-tible to biodeterioration. Welch et al. (2002) showed thatwhereas apatite readily undergoes chemical dissolution, it ismore susceptible to biological-originated dissolution by mi-croorganisms and their metabolites, especially organic acids.Moreover, ectomycorrhizal fungi have also been shown tofacilitate P uptake from apatite by Pinus sylvestris seedlings(Wallander 2000).

N. sphaerica is a common indoor airborne fungus(McGinnis 2007). Aside from Nigrospora isolate KUR1, onlya few other fungi were isolated from the same microsite,beneath the salt deposit: Cladosporium sp., Alternaria sp.,and Penicillium sp. These genera have been reported to beubiquitously present in living environments and are frequentlyisolated from hypersaline environments (Cantrell et al. 2006);moreover, different species of Cladosporium, Alternaria,

Nigrospora, and Aspergillus, among others, are regularly iso-lated from deteriorated stones and building materials(Shirakawa et al. 2002).

To study the possible contribution of the fungi to thedeterioration and efflorescence processes, experiments werecarried out under the accelerated conditions of axenic culturerather than in situ. Axenic culture, with added stone materials,was chosen to enable separating the damage produced by

Fig. 2 Unrooted dendrogram showing the relationships betweenNigrospora isolate KUR1 and different Nigrospora sequences retrievedfrom GenBank. The dendrogram was created along with 2,000bootstrapping repeats of phylogeny using the neighbor-joining algorithm

Fig. 3 Fungal growth in the presence of stone particles from the flooring tile with nutrient added: a no inoculation, b glucose (1 g/1), c both glucose (1 g/1) and (NH4)2HPO4 (0.1 g/1)

A

/L)

mg

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(m

niu

mo

Am

B

L)

(g/L

ose

u

coG

l

40

35

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5

040 2 6 8 10

Time (days)

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0 8.8

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Fig. 4 Residual nutrient (glucose or N-NH4) during incubation of fungiin the presence of a mixture of ground tile components and added glucoseor N-NH4. a Reduction of N-NH4 in the culture with added glucose(1 g l−1) and (NH4)2HPO4 (0.1 g l−1), with (○) and without (♦) groundtile material. b Reduction of glucose in the culture containing addedglucose (1 g l−1) and: (▲) ground tile material; (○) (NH4)2HPO4

(0.1 g l−1) and tile material; and (♦) (NH4)2HPO4 (0.1 g l−1) without tilematerial. Bars represent standard error; n=3

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microorganisms from that caused by physical and chemicalagents at the natural site (Sbaraglia et al. 2003). This approachhas been used to study the biodegradation of other materialsincluding ivory (Pinzari et al. 2012).

The Nigrospora sp. grew better in the culture with addedtile particles, as shown by measuring nutrient consumptionand fungal viability and activity (MTTassay). The MTTassayhas been described as a fast and reliable method for colori-metric determination of viable fungal cell densities (Freimoseret al. 1999) including various filamentous fungi and yeast.The enhanced consumption of carbon and nitrogen sources inthe presence of the tile material suggested that the mineral

material supplies essential microelements and macroelementssuch as P, S, Ca, K, among others, to the growing mycelium(Table 1; Mg and P were also supplied by the buffering saltsadded to the culture medium). Moreover, aside from theessential elements supplied by the stone chips of the tile, thismaterial also contained a soluble nitrogen source as NO2,NO3, and NH3, which can be exploited by the fungi at thenatural site. The presence of endogenous nitrogen in the tilematerial might explain the fungal growth and glucose con-sumption in the medium that was not supplemented with anexternal nitrogen source (Fig. 4b). Moreover, nitrogen sourceswere also found in the soluble fraction from the concretebelow the floor, and these may have migrated onto the tilesurface to be consumed by the fungi.

Addition of glucose to the in vitro system accelerated thedegradation process, showing that the tile material itself is apoor carbon source for fungal growth. In natural stone, blackfungi, colonizing mineral materials, are adapted to thisnutrient-poor environment. For example, slow-growing,extremotolerant black meristematic fungi form facultativelichen-like associations with algae or cyanobacteria on thestone (Gostinčar et al. 2012). Other nutrient sources in thenatural environment include atmospheric pollution, especiallyin urban environments, containing NO2, SO2, benzene, CO,and other elements (Moroni and Pitzurra 2008), and in indoorenvironments, resins and wall paint as carbon sources(Cappitelli et al. 2007). An active family’s indoor residentialenvironment contains many sources of volatile or nonvolatilecarbon compounds that may also be present on or under-the-floor surface, providing nutrients for the fungi.

The different types of stone chips composing the tile werefound to have different abilities to support fungal growth, aswell as differential resistance to fungal dissolution activity.Fraction D (the calcite carbonate–apatite and phosphate miner-al) was the most vulnerable fraction to fungal activity, andunderwent dissolution in the presence of the growing myceli-um. The calcite–quartz type (Fraction C) was also affected, butto a lesser extent, while the dolomite–quartz stone chips (frac-tions A and B) were less supportive. The mycelium produced abiofilm and hyphae were attached to all stone types, but a moreextended and thicker exopolysaccharide matrix embedded thefungal mycelium on fractions C and D (the calcite stones),whereas fractions A and B (the dolomite stones) supportedfungal growth less well, i.e., were less bioreceptive to the fungi.The mineral composition of fraction D contributed to the bio-logical dissolution of this fraction. These results support previ-ous ones showing that apatite undergoes higher biological-originated dissolution rates (Welch et al. 2002).

Building materials containing different minerals have beenreported to undergo weathering and dissolution by fungi, suchas those including asbestos (Daghino et al. 2010), cementi-tious materials (Wiktor et al. 2009), limestone (Diakumakuet al. 1995; Li et al. 2009), and dolomite and calcite (De La

Fig. 5 Comparison between MTT assay results of isolate KUR1 grownwith various medium compositions in the presence of a mixture of groundtile components (<3 mm) after 6 days of culture incubation at 25 °C. Eachdata point represents the mean and standard error of three independentreplicates

0

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Treatment

Nu

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Fig. 6 Nutrients [glucose (dark gray) and N-NH4 (light gray)] remainingin the medium in the presence the different stone chip types, fractions A–D, after 120-h incubation at 25 °C with fungal inoculum. All media weresupplemented with 1 g l−1 glucose and 0.1 g l−1 ammonium salt. Control:treatments without stone fractions added to the medium. The residual N-NH4

+ concentrations are presented as values×0.03. Bars represent stan-dard error; n=3

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Torre et al. 1993). Dissolution of mineral material by fungi issuggested to be governed by enzymatic activity or by acidsproduced by the fungi and released into the medium (Palmeret al. 1991; De La Torre et al. 1993; Gómez-Alarcón et al.1994; Gadd 1999). Different organic acids are produced byfungi at the site of the deterioration. Studying the biochemicalweathering mechanisms used by fungi, De La Torre et al.(1993) reported that Penicillium frequentans produces oxalic,

citric, and gluconic acids on sandstone, granite, and limestone.Those authors suggested that in addition to causing extensivedeterioration of clay silicates, micas, and feldspars from sand-stone and granite, the organic acids also degrade calcite anddolomite from limestone, as a result of high cation release andthe formation of organic salts such as calcium, magnesiumand ferric oxalates, and calcium citrates. They also suggestedthat micronutrient uptake and trivalent cation chelation (Fe3+

and A13+) are ecologically adaptative mechanisms stemmingfrom fungal growth on stone monuments.

Gaylarde et al. (2003) summarized the different mechanismsby which microorganisms might alter mineral materials. Theseinclude biochemical effects such as hydrolytic enzyme produc-tion, filamentous growth, acid production, mobilization of ions,and chelation of constituent ions, along with physical effectssuch as discoloration and retention of water (Shirakawa et al.1999). In the present study, we also found a reduction in pHfrom 10.5 to 6 in the culture containing fractionD over 10 daysof incubation (data not shown). This suggests the release oforganic acids by the fungi, whichmay govern the dissolution ofthe minerals in the soft calcite–apatite fraction.

Based on the results of this work, it is suggested that areduction in the pH of the tile material due to fungal activityoccurs in situ, enhancing dissolution of the tile matrix. Thisdissolution might increase the concentration of soluble ele-ments that may then move with the water to the tile surfaceand concentrate there. A similar phenomenon resulting fromphysicochemical processes was observed by Wiktor et al.

A B C

D-1 D-2 D-3

Fig. 7 Backscattered electron images of the fungus-colonized tile stonecomponents (fractions a–d-1), showing different arrangements of hyphaeand polysaccharide film on the surfaces of the rock fragments. d-2 shows

colonized fraction D stone at higher magnification; d-3 shows fraction Dstone before inoculation. Bar=100 μm. Arrows indicate fungal mycelium

0

250

500

750

1000

Not inoculated Inoculated

Treatment

Po

wd

ered

fra

ctio

n (

mg

)

Fig. 8 Effect of fungal inoculation on the powdered fraction (<1 mm)remaining in 50 ml medium after 10 days of incubation; 5 g of the groundstone particles (<3 mm) were added, containing 0.75 g powdered fractionper 100 ml medium. Bars represent standard error; n=3

Appl Microbiol Biotechnol

(2009), who examined fungal biodeterioration of a cementi-tious matrix. By reducing the matrix’s alkalinity to acceleratefungal growth in a weathering laboratory test, they noticedthat the matrix surface becomes progressively covered with acalcium carbonate layer as weathering increases. While theresults of the present work indicate the fungi’s ability to causedeterioration of the flooring mineral tiles and contribute to thedissolution of certain mineral fractions of the tile in a labora-tory experiment, more studies need to be carried out to eluci-date the fungi’s actual activity in situ.

Acknowledgments We wish to thank Amir Sandler at The GeologicalSurvey of Israel, Jerusalem, for the XRD analysis of the stone and JanDijksterhuis from CBS for assisting in the fungal identification.

References

Cantrell SA, Casillas-Martínez L, Molina M (2006) Characterization offungi from hypersaline environments of solar salterns using mor-phological and molecular techniques. Mycol Res 110:962–970

Cappitelli F, Principi P, Pedrazzani R, Toniolo L, Sorlini C (2007) Bacterialand fungal deterioration of the Milan Cathedral marble treated withprotective synthetic resins. Sci Total Environ 385:172–181

Daghino S, Martino E, Perotto S (2010) Fungal weathering and implica-tions in the solubilization of metals from soil and from asbestosfiber. In: Mendez-Vilas A (ed) Current research, technology andeducation topics in applied microbiology and microbial biotechnol-ogy, vol 1, Formatex. Badajoz, Spain, pp 329–338

De La Torre MA, Gomez-Alarcon G, Vizcaino C, Garcia MT (1993)Biochemical mechanisms of stone alteration carried out by filamen-tous fungi living in monuments. Biogeochem 19:129–147

Diakumaku E, Gorbushina AA, Krumbein WE, Panina L, SoukharjevskS (1995) Black fungi in marble and limestones—an aestheticalchemical and physical problem for the conservation of monuments.Sci Total Environ 167:295–304

Fernandes P (2006) Applied microbiology and biotechnology in theconservation of stone cultural heritage materials. Appl MicrobiolBiotechnol 73:291–296

Freimoser FM, Jakob CA, Aebi M, Tuor U (1999) The MTT [3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide] assay is afast and reliable method for colorimetric determination of fungal celldensities. Appl Environ Microbiol 65:3727–3729

Gadd GM (1999) Fungal production of citric and oxalic acid: importancein metal speciation physiology and biogeochemical processes. AdvMicrob Physiol 41:47–92

Gadd GM (2007) Geomycology: biogeochemical transformations ofrocks, minerals, metals and radionuclides by fungi, bioweatheringand bioremediation. Mycol Res 111:3–49

Gaylarde C, SilvaMR,Warscheid T (2003)Microbial impact on buildingmaterials: an overview. Mater Struct 36:342–352

Gómez-Alarcón G, Muñoz ML, Flores M (1994) Excretion of organicacids by fungal strains isolated from decayed sandstone. IntBiodeterior Biodegrad 34:169–180

Gostinčar C, Muggia L, Grube M (2012) Polyextremotolerant blackfungi: oligotrophism adaptive potential and a link to lichen symbi-oses. Front Microbiol 3:390

Henrion B, Chevalier G, Martin F (1994) Typing truffle species by PCRamplification of the ribosomal DNA spacers. Mycol Res 98:37–43

Hirsch P, Eckhardt FEW, Palmer RJ (1995) Fungi active in weathering ofrock and stone monuments. Can J Bot 73:1384–1390

Hoffland E, Kuyper TW, Wallander H, Plassard C, Gorbushina AA,Haselwandter K, Holmström S, Landeweert R, Lundström US,Rosling A, Sen R, Smits MM, van Hees PAW, van Breemen N(2004) The role of fungi in weathering. Front Ecol Environ 2:258–264

Li W, Zhou PP, Jia LP, Yu LJ, Li XL, Zhu M (2009) Limestone dissolu-tion induced by fungal mycelia acidic materials and carbonicanhydrase from fungi. Mycopathologia 167:37–46

Mapelli F, Marasco R, Balloi A, Rolli E, Cappitelli F, Daffonchio D,Borin S (2012) Mineral–microbe interactions: biotechnological po-tential of bioweathering. J Biotechnol 157:473–481

Masaphy S, Zabari L, Pastrana J, Dultz S (2009) Role of fungal myceliumin the formation of carbonate concretions in growing media—aninvestigation by SEM and synchrotron-based X-ray tomographicmicroscopy. Geophys J Roy Astron Soc 26:442–450

McGinnis MR (2007) Indoor mould development and dispersal. MedMycol 45:1–9

Miller GL (1959) Use of dinitrosalicylic acid reagent for determination ofreducing sugar. Anal Chem 31:426–428

Moroni B, Pitzurra L (2008) Biodegradation of atmospheric pollutants byfungi: a crucial point in the corrosion of carbonate building stone. IntBiodeterior Biodegrad 62:391–396

Palmer RJ, Siebert J, Hirsch P (1991) Biomass and organic acids insandstone of a weathering building: production by bacterial andfungal isolates. Microb Ecol 21:253–266

Pinzari F, Tate J, Bicchieri M, Rhee YJ, Gadd GM (2012) Biodegradationof ivory (natural apatite): possible involvement of fungal activity inbiodeterioration of the Lewis Chessmen. Environ Microbiol 15:1050–1062

Principi P, Borin S, Sorlini C (2007) Synthetic consolidants attacked bymelanin-producing fungi: case study of the biodeterioration ofMilan (Italy) cathedral marble treated with acrylics. Appl EnvironMicrobiol 73:271–277

Saitou N, Nei M (1987) The neighbor-joining method: a new method forreconstructing phylogenetic trees. Mol Biol Evol 4:406–425

Sbaraglia G, Pitzurra L, Moroni B, Nocentini A, Vitali M, Poli G,MilianiC, Bistoni F (2003) Fungal colonization on stoneworks, interactionfungi-powdered stone samples. Ann Chim 93:889–896

Shirakawa MA, Tapper R, Cincotto MA, Beech I, Gambale W (1999)Fungal growth measurement by ESEM in four different mortars andproposal of a biodeterioration mechanism. Proc 21st Int ConfCement Microscopy, 25–29 April, Las Vegas, NV, InternationalCement Microscopy Association (ICMA), Dallas, Texas, USA, pp383–393

Shirakawa M, Gaylarde CC, Gaylarde PM, Vanderley J, Gambale W(2002) Fungal colonization and succession on newly painted build-ings and the effect of biocide. FEMS Microbiol Ecol 39:165–173

Singh J,Wah C, Yu F, Kim JT (2010) Building pathology investigation ofsick buildings—toxic moulds. Indoor Built Environ 19:40–47

Sterflinger K (2010) Fungi: their role in deterioration of cultural heritage.Fungal Biol Rev 24:47–55

Sterflinger K, Piñar G (2013) Microbial deterioration of cultural heritageand works of art—tilting at windmills? App Microbiol Biotechnol97:9637–9646

Tamura K, Dudley J, Nei M, Kumar S (2007) MEGA4: molecularevolutionary genetics analysis (MEGA) software version 4.0. MolBiol Evol 24:1596–1599

Wallander H (2000) Uptake of P from apatite by Pinus sylvestris seed-lings colonized by different ectomycorrhizal fungi. Plant Soil 218:249–256

Warscheid T, Braams J (2009) Biodeterioration of stone: a review. IntBiodeterior Biodegrad 46:343–368

Weatherburn MW (1967) Phenol-hypochlorite reaction for determinationof ammonia. Anal Chem 39:971–974

Welch SA, TauntonAE, Banfield JF (2002) Effect of microorganisms andmicrobial metabolites on apatite dissolution. Geophys J Roy AstronSoc 19:343–367

Appl Microbiol Biotechnol

Wiktor V, De Leo F, Urzì C, Guyonnet R, Grosseau P, Garcia-Diaz E(2009) Accelerated laboratory test to study fungal biodeteri-oration of cementitious matrix. Int Biodeterior Biodegrad 63:1061–1065

Winkler EM, Wilhelm EJ (1970) Salt burst by hydration pressures in archi-tectural stone in urban atmosphere. Geol Soc Am Bull 81:567–572

Zehnder K, Arnold A (1989) Crystal growth in salt efflorescence. J CrystGrowth 97:513–521

Appl Microbiol Biotechnol