Influence of atmospheric pollutants on the biodeterioration of stone E. Zanardini a, *, P. Abbruscato a , N. Ghedini b,c , M. Realini d , C. Sorlini a a Department of Food Science and Microbiology, Section of MAAE, Agricultural Faculty, University of Milan, Via Celoria 2, 20133 Milan, Italy b Department of Pharmaceutical Sciences, Pharmacy Faculty, University of Bologna, Via Belmeloro 6, 40126 Bologna, Italy c Institute ISAO-CNR, Via Gobetti 101, 40129 Bologna, Italy d Centre CNR ‘‘Gino Bozza’’, Piazza L. da Vinci 32, 20133 Milan, Italy Received 28 September 1999; accepted 5 April 2000 Abstract Studies have been carried out on specimens of Saltrio stone, Carrara and Candoglia marbles, exposed for some years to the polluted atmosphere of Milan. The test specimens, which were directly exposed to the washing away, showed alterations in the form of grey-black stains which were dierent in dimensions and quantity according to the stone material, while those sheltered showed only a uniform grey deposit of particulate matter. The decay on the exposed specimens has been tested by gravimetric analyses and scanning electron microscopy (SEM) observations. Microbiological investigations evidenced that microorganisms, either chemoautotrophic or photosynthetic, were not present in any sample. On the washed out test specimens, the presence of heterotrophic bacteria was considerable, while the sheltered specimens showed negligible values. Chemical analysis, carried out through gas chromatography–mass spectrometry (GC/MS), of the particulate deposited on the test specimens, evidenced the presence of a wide range of aromatic and aliphatic hydrocarbons and fatty acids. Cultural tests of mixed cultures and isolated strains were carried out by using a mineral medium added with 14 organic compounds, typical representatives of atmospheric pollution, as a sole source of carbon and energy. The results evidenced that the mixed cultures in particular, as well as some of the isolated strains, are capable of using many of the tested molecules (fatty acid, aliphatic compounds, p-xylene). 7 2000 Elsevier Science Ltd. All rights reserved. 1. Introduction The studies carried out in the last decades on out- door artistic stoneworks particularly in urban areas evidenced diused and rapid phenomena of corrosion. It is well known that the atmospheric pollution is the cause of the accelerated deterioration of the exposed stonework, since the atmospheric pollutants interact with the carbonatic matrices, damaging them, some- times seriously and irreversibly (Torraca, 1976; Del Monte et al., 1981; Amoroso and Fassina, 1983; Brim- blecombe, 1988; Fassina, 1988; Lorusso et al., 1992; Realini et al. 1995). Following the chemical deterioration comes micro- biological deterioration, caused by microorganisms growing on the material surfaces. Until a short time ago, biodeteriorative colonisers of the stone materials were considered to be autotrophic microorganisms, capable of surviving on inorganic substrates, because of their capacity to utilise CO 2 , and it was considered that the heterotrophics could colonise the stone substrates only later on, in accord- ance to a real ecological succession, utilising the or- ganic substances released by the cellular lysis of the autotrophics (Sorlini, 1984; Tiano, 1986; Giacobini et al., 1987; Grin et al., 1991). Saiz-Jimenez and subsequently other authors recently demonstrated that numerous heterotrophic microorganisms present on the stonework can utilise, for their growth, the airborne organic compounds settled on the stone surface. They mainly arise from International Biodeterioration & Biodegradation 45 (2000) 35–42 0964-8305/00/$ - see front matter 7 2000 Elsevier Science Ltd. All rights reserved. PII: S0964-8305(00)00043-3 www.elsevier.com/locate/ibiod * Corresponding author.
Transcript
In¯uence of atmospheric pollutants on the biodeteriorationof stone
E. Zanardinia,*, P. Abbruscatoa, N. Ghedinib,c, M. Realinid, C. Sorlinia
aDepartment of Food Science and Microbiology, Section of MAAE, Agricultural Faculty, University of Milan, Via Celoria 2, 20133 Milan, ItalybDepartment of Pharmaceutical Sciences, Pharmacy Faculty, University of Bologna, Via Belmeloro 6, 40126 Bologna, Italy
cInstitute ISAO-CNR, Via Gobetti 101, 40129 Bologna, ItalydCentre CNR ``Gino Bozza'', Piazza L. da Vinci 32, 20133 Milan, Italy
Received 28 September 1999; accepted 5 April 2000
Abstract
Studies have been carried out on specimens of Saltrio stone, Carrara and Candoglia marbles, exposed for some years to the
polluted atmosphere of Milan. The test specimens, which were directly exposed to the washing away, showed alterations in theform of grey-black stains which were di�erent in dimensions and quantity according to the stone material, while those shelteredshowed only a uniform grey deposit of particulate matter. The decay on the exposed specimens has been tested by gravimetric
analyses and scanning electron microscopy (SEM) observations. Microbiological investigations evidenced that microorganisms,either chemoautotrophic or photosynthetic, were not present in any sample. On the washed out test specimens, the presence ofheterotrophic bacteria was considerable, while the sheltered specimens showed negligible values. Chemical analysis, carried out
through gas chromatography±mass spectrometry (GC/MS), of the particulate deposited on the test specimens, evidenced thepresence of a wide range of aromatic and aliphatic hydrocarbons and fatty acids. Cultural tests of mixed cultures and isolatedstrains were carried out by using a mineral medium added with 14 organic compounds, typical representatives of atmosphericpollution, as a sole source of carbon and energy. The results evidenced that the mixed cultures in particular, as well as some of
the isolated strains, are capable of using many of the tested molecules (fatty acid, aliphatic compounds, p-xylene). 7 2000Elsevier Science Ltd. All rights reserved.
1. Introduction
The studies carried out in the last decades on out-door artistic stoneworks particularly in urban areasevidenced di�used and rapid phenomena of corrosion.It is well known that the atmospheric pollution is thecause of the accelerated deterioration of the exposedstonework, since the atmospheric pollutants interactwith the carbonatic matrices, damaging them, some-times seriously and irreversibly (Torraca, 1976; DelMonte et al., 1981; Amoroso and Fassina, 1983; Brim-blecombe, 1988; Fassina, 1988; Lorusso et al., 1992;Realini et al. 1995).
Following the chemical deterioration comes micro-
biological deterioration, caused by microorganismsgrowing on the material surfaces.
Until a short time ago, biodeteriorative colonisers ofthe stone materials were considered to be autotrophicmicroorganisms, capable of surviving on inorganicsubstrates, because of their capacity to utilise CO2,and it was considered that the heterotrophics couldcolonise the stone substrates only later on, in accord-ance to a real ecological succession, utilising the or-ganic substances released by the cellular lysis of theautotrophics (Sorlini, 1984; Tiano, 1986; Giacobini etal., 1987; Gri�n et al., 1991).
Saiz-Jimenez and subsequently other authorsrecently demonstrated that numerous heterotrophicmicroorganisms present on the stonework can utilise,for their growth, the airborne organic compoundssettled on the stone surface. They mainly arise from
International Biodeterioration & Biodegradation 45 (2000) 35±42
0964-8305/00/$ - see front matter 7 2000 Elsevier Science Ltd. All rights reserved.
PII: S0964-8305(00 )00043 -3
www.elsevier.com/locate/ibiod
* Corresponding author.
the incomplete combustion of fossil fuels (Saiz-Jime-nez, 1995, 1997; Krumbein et al, 1996; Wolf andKrumbein, 1996). This evidence induces the belief thatheterotrophic microorganisms can act as ®rst coloni-sers in the areas with a high level of organic pollu-tants.
The objective of this work is to investigate the in¯u-ence of the atmospheric pollutants on the biodeteriora-tion of the stone surface. To reach this objective, astudy about the deterioration undergone by some testspecimens of Saltrio stone, Carrara and Candogliamarbles (these stones have been largely employed inmonuments and statues since ancient times), exposedsince 1989 to the polluted atmosphere of Milan, hasbeen carried out; in particular, the alteration under-gone and the growth capacities of the coloniser micro-¯ora were analysed.
2. Materials and methods
2.1. Samples
Sixty specimens of each lithotype (50� 50� 20 mm)were exposed on the roof of the Polytechnic of Milan:they were placed on a Plexiglas support, with an incli-nation of 458 toward the North. Ninety specimenshave been sheltered from meteoric precipitation by ashed, whilst the others were directly exposed to therain-washing away. The specimen surfaces were onlycut and they were left unpolished in order to facilitatethe start of the deterioration mechanisms.
Every 6 months, six specimens of each lithotypewere transferred, in sterile conditions, into the labora-tory and analysed.
2.2. Microscopical observations
The morphology of the unsheltered stained surfaceswas observed by Wild Makroskop M420 Heerbruggstereomicroscope, provided with Olympus OM1 cam-era and Leica Wild MPS 52 exposure meter.
SEM observations of the unstained surface were car-ried out on gold coated samples, and in order to esti-mate the presence of bacteria and fungi on thedecayed surface, samples were ®xed overnight in 2%glutaraldehyde in 0.01 M phosphate bu�er, dehydratedin serial higher concentrations of ethanol (50%, 60%,70%, 80%, 90% and absolute), dried at critical pointand gold coated. Observations were carried out in aJeol JSM 35 C microscope.
2.3. Weight measurements
Before the exposure, each specimen was dried at608C until it reached a constant weight (di�erence
between two subsequent weighings <0.5 mg). Thesame procedure was adopted when specimens wereweighed after the exposure.
2.4. Cultural media and microbiological analyses
Sampling was carried out by scraping the super®ciallayer of every sample using a sterile scalpel, to about 1mm depth. On sheltered specimens it was carried outrandomly, both deeply (as far as about 3 mm) andsuper®cially (1 mm); on unsheltered specimens thesamples were taken from the areas characterised bythe presence of stains and, as a control, from the areaswithout any type of alteration.
Samples were suspended in physiological solution (9g/l NaCl) and, after the preparation of the serial di-lutions, they were directly inoculated or transferred in:Plate Count Agar (PCA) for the count of aerobic het-erotrophic bacteria and Malt Agar for the moulds andyeasts, incubation at 288C for 5/6 days; Chu mediumfor Cyanobacteria and Detmer medium for Chloro-phyta, incubation at 258C for 30 days in the light; inStanier medium for nitrifying bacteria and Pochon andTardieux media for sulphur-oxidizing bacteria, incu-bation at 288C for 30 days (Normal recommendations9/88, 1990).
2.5. Strain identi®cation
For the identi®cation of some isolated strains, afterprevious procedures (Gram staining, oxidase, catalase,etc.), a biochemical characterisation using API Staphand API 50 CH was carried out. Other strains wereidenti®ed by the Biolog's automated Micro Station,after the inoculation in the GP Microplate panel.
2.6. Chemical analysis
The atmospheric particulate settled on the plexiglasssupports was collected and analysed by gas chroma-tography±mass spectroscopy (GC/MS). Samples weresubmitted to an extraction by ultrasonic agitation andthe extracts were reduced to about 250 ml using a rota-vapor (below 408C) under high-purity N2 stream evap-oration. The organic acids and the hydroxylatedaromatic compounds were converted into the respect-ive methyl-ester through reaction with freshly pro-duced diazomethane.
Identi®cation of organics was carried out in a GC/MS device. The instrument used was a HP 5890 GC,interfaced to a HP 5971 selective mass detector, oper-ating with an ionisation energy of 70 eV, a m/z massrange of 50±500 and a cycle of 2 s. The separation ofthe components was reached through the use of a silic-eous capillary column (30 m � 0.25 mm), coated withHP-5 phenyl±methyl±silicone. The temperature pro-
E. Zanardini et al. / International Biodeterioration & Biodegradation 45 (2000) 35±4236
gramming consisted of the following steps: (a) injectionat 658C, (b) isothermal hold at 658C for 10 min, (c)temperature ramp of 108C/min and (d) an isothermalhold at 2758C for another 49 min. For solvent extrac-tion, the sample was ground and extracted using ®rstlyn-hexane and then methylene chloride/methanol 1:1mixture. Inorganic materials were eliminated by atreatment with bi-distilled cold water.
Main organic molecules were identi®ed by computermatches to a standard reference mass fragmentogramsin the NBS75K.L library.
2.7. Cultural tests
For the cultural tests on the organic pollutants, themixed cultures and the isolated strains were inoculatedin M9 mineral medium (Kunz and Chapman, 1981),added with a sole source of carbon and energy atdi�erent concentrations (250 and 500 mg/l). The com-pounds used are summarised in Table 1.
Microbial growth was estimated until the 21st dayof incubation by the turbidity of the cultural broth, byspectrophotometric determinations at 540 nm usingBioscreen (Labsystems, Helsinki, Finland), an auto-matic turbidimetric analyser, and by spectrophoto-metric analyses (Beckman DU640).
Moreover, to verify the p-xylene removal, a spectro-photometric scanning of the cultures at the beginning�T0� and at the end of the incubation �T21� was carriedout in a range of wavelengths between 200 and 600nm.
3. Results
3.1. Morphological observations
Direct observation of the alterations allowed topoint out the di�erent distribution and the extensionof the stains over the lithotypes. In fact, Saltrio stoneshows very evident dark stains (Fig. 1), bigger andmore numerous than those on the two marbles. Onmarbles the stains are similar to black speckles, moreor less di�used.
Stereomicroscopic observations showed that, inde-pendently from the extension and the number, thestains on all lithotypes are formed by black fungal ®la-ments (Fig. 2), closely connected to the stone surface,which seems to be very corroded.
SEM observations evidenced traces of chemical cor-rosion, which appear di�erently because of the di�er-ent granulometry of the lithotypes.
On Carrara marble and Saltrio stone many micro-fractures forming a grid were observed, while on Can-doglia marble, microfractures are preferentiallyarranged along the twin-planes of the calcite crystals(Figs. 3 and 4).
Moreover on stained zones fungal hyphae andspores have been observed (Fig. 5), while their pre-sence was very scarce in unstained areas.
On the sheltered specimens also, after the removalof the surface deposition, no evidence of stains wasobserved.
3.2. Weight measurement
The amount of particulate matter found on the shel-tered surface is quite similar on all the specimens. Thelowest values of particulate were always found onCandoglia marble; this fact probably depends on thedi�erent grain size of the lithotypes which directly in-
Fig. 1. Grey stains on Saltrio stone specimens.
Fig. 2. Stereomicroscopic observation of a grey stain on Saltrio
stone. Black fungal ®laments are well evident on the corroded sur-
¯uences the surface decay as previously reported, pro-ducing, on Carrara marble and Saltrio stone, a morerough surface which can more easily retain the particu-late.
The di�erent decay of the lithotypes is more evidenton the unsheltered specimens. In this case we havequanti®ed the material loss due to the combined actionof pollution and rain, which produced decohesion ofcrystals and their removal (Fig. 6). Carrara marble isone of the more decayed lithotypes, and this is evidentfor all the exposure period. After 30 months of ex-posure, the di�erence between Carrara and Candogliais very considerable; Saltrio stone shows intermediatevalues, even if it seems to be more similar to Carraramarble, reaching a signi®cant amount of lost material.
3.3. Microbiological analyses
Microbiological analyses were carried out with the
aim of verifying if there was a microbial colonisation,both on the sheltered specimens and on those washedaway.
In both cases autotrophic microorganisms, such asnitrifying and sulphur-oxidizing bacteria and photo-synthetic microorganisms, were not present.
As far as heterotrophic microorganisms are con-cerned, on the sheltered specimens, their countsresulted low, in the order of <10 CFU/g, both in thesuper®cial and in the deeper sampling.
Instead, on the unsheltered specimens (Fig. 7), theheterotrophic bacterial and fungi count in the stainsresulted 105±106 CFU/g.
In the unstained areas, the heterotrophic bacterialcount resulted 104 CFU/g for all lithotypes, while themoulds, which were in a negligible quantity (<10CFU/g) on Carrara and Candoglia marbles, wereunexpectedly high (104 CFU/g) on Saltrio stone. Thelatter value correlated well with both the considerabledimensions of the stains on this lithotype, and the greycolour of the surface, which does not permit an easydistinction of the limit between stained and unstainedareas.
Moreover, a wide phenotypic biodiversity of bac-terial colonies grown from the unstained areas wasobserved, while this fact is not so evident for thestained areas.
The high bacterial counts and low biodiversity isusually related to the phenomena of selective pressure;it is possible that the species able to degrade the or-ganic pollutants prevail on the others and grow inmore considerable quantities.
Subsequently, numerous bacterial strains were iso-lated and some of them were identi®ed as Bacillus sub-tilis, B. licheniformis, B. coagulans, Staphylococcuslentus, Micrococcus roseus, Promicromonospora entero-phila. The fungi found in every lithotype belonged
Fig. 3. Chemical corrosion of unsheltered Saltrio stone specimen
after 30 months of exposure (SEM).
Fig. 4. Chemical corrosion of unsheltered Candoglia marble speci-
men after 30 months of exposure. The decay morphology is very
di�erent compared to Saltrio stone (SEM).
Fig. 5. SEM observation of a grey stain on Saltrio stone. Fungal
hyphae are closely connected to the corroded crystals.
E. Zanardini et al. / International Biodeterioration & Biodegradation 45 (2000) 35±4238
only to two species Moniliella sp. (prevalent in the twomarbles) and Fusarium sp.
3.4. Chemical analysis
GC/MS analysis of the atmospheric particulate pre-sent on the test specimen supports were conductedwith the aim of identifying the atmospheric pollutantsdeposited on the samples. The resulting Total Ion Cur-rent (TIC) is reported in Fig. 8.
The main organic compounds found are: fatty acids,dibenzofurane and complex aromatic and aliphatichydrocarbons.
Subsequently, according to these results and the lit-
erature data (Saiz-Jimenez, 1991, 1993), 14 compoundsusually found in the polluted atmosphere of the urbanareas were chosen: aliphatic hydrocarbons (cyclopen-tane, dodecane and hexadecane), aromatic hydrocar-bons (benzene, toluene, xylenes, naphthalene,anthracene and phenanthrene), fatty acids (palmiticand caprylic acids) and dibenzofuran.
3.5. Cultural tests
The heterotrophic mixed culture is able to degrade alarge part of the tested compounds, whilst the singlestrains reveal more specialisation, growing on a morelimited number of compounds (Table 2).
Fig. 6. Trend of the weight loss caused by decay on the unsheltered specimens.
Fig. 7. Microbial growth in stained and unstained areas on the unsheltered specimens.
E. Zanardini et al. / International Biodeterioration & Biodegradation 45 (2000) 35±42 39
The substrates used by the larger number of isolatedstrains were aliphatic, like hexadecane, dodecane andfatty acids; the aromatic hydrocarbons were rarelyused, except p-xylene, that was degraded by nearly allthe strains. This fact is demonstrated by the spectro-photometric scanning of the cultures, grown in the pre-sence of p-xylene, which showed the completedisappearance of the compound peak after 21 days ofincubation.
Comparing the degradative abilities, no signi®cantdi�erences were evidenced among the microorganisms,except for mixed culture from Saltrio stone and the rRstrain isolated from it, which showed much wider abil-ities.
The growth curve of the mixed culture from Saltrio
stone in the presence of palmitic acid is reported inFig. 9.
4. Discussion
The absence of grey stains and the low bacterial andfungal counts on the sheltered specimens, suggest thatthere is not a biodeteriorative process on the surface;this probably depends on the very low water contentof the substrate, which represents a limiting factor forthe microbial growth.
By contrast, on the unsheltered specimens, high bac-terial and fungal counts were measured connected withthe stains which are however very di�erent in numberand extension on the lithotypes.
Fig. 8. Total Ion Current (TIC) from GC/MS data for a sample of particulate matter. Organic compounds, with relative number, are listed
below.
E. Zanardini et al. / International Biodeterioration & Biodegradation 45 (2000) 35±4240
The environmental interaction (rain and pollution)has caused a di�erent decay of the surfaces, inducingsigni®cant di�erences of the bioreceptivity of thestones.
This situation results in a major presence of biologi-cal colonisation (grey stains) on the Saltrio stone, evenif the morphology and microbial count of a singlestained area are similar in all the lithotypes.
Table 2
Results of growth tests in M9 mineral medium added with organic compounda
a + = abundant; +/ÿ = medium; ÿ = absence.
Fig. 9. Growth trend of the mixed culture from Saltrio stone on M9 mineral medium added with palmitic acid (250 mg/l).
E. Zanardini et al. / International Biodeterioration & Biodegradation 45 (2000) 35±42 41
Frequently black fungi (Dematiaceae ), black yeastsand actimomycetes have been identi®ed as responsiblefor the colour change on the stone surface, even in avery extreme environment, such as high temperature,drought, hypersaline habitat, with direct exposure tosunlight. Studies regarding their morphological, eco-logical and molecular characterisation have been car-ried out by many authors (UrzõÁ et al., 1995; Eppard etal., 1996; Ster¯inger et al., 1997; Wollenzien et al.,1995, 1997).
The observed alterations on our specimens arecaused by heterotrophic micro¯ora, autotrophic micro-organisms have not been detected. The fungal strainMoniliella, which is able to produce a non di�usibleblack pigment, seems to be the main agent responsiblefor the chromatic aspect of the stains.
The growth tests on the di�erent organic com-pounds showed that the mixed cultures, in particularthe ones obtained from Saltrio stone, have a higherdegradative ability as compared to the pure microbialstrains, which are specialised for a limited number ofcompounds.
It is correct to hypothesise that mutualist and com-mensalist microbial associations can develop on thespecimen surfaces. This particular situation, togetherwith co-metabolic processes, give rise to the mixed cul-tures with a wider degradative ability.
Our conclusions are in agreement with Saiz-Jime-nez's, which hypothesise that heterotrophic micro¯oracould often be the ®rst colonisers of stone materialsexposed to the atmospheric pollution, because of theirability to grow utilising organic pollutants.
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