International Biodeterioration & Biodegradation 46 (2000) 299–303www.elsevier.com/locate/ibiod

Changes in the bio�lmmicro ora of limestone causedby atmospheric pollutantsRalph Mitchella ;∗, Ji-Dong Gub

aDivision of Engineering and Applied Sciences, Harvard University, 29 Oxford Street, Cambridge, MA 02138, USAbLaboratory of Environmental Toxicology, Department of Ecology & Biodiversity, The Swire Institute of Marine Science,

The University of Hong Kong, Pokfulam Road, Hong Kong SAR, People’s Republic of China

Accepted 23 October 2000

Abstract

Historic limestone materials in urban environments are continually exposed to air pollutants, including sulfur compounds and hydro-carbons. We investigated the e�ects of air pollution on the bio�lm micro ora of historic limestone gravestones located at two locationsMassachusetts, USA. Our data showed that the culturable populations of chemolithotrophic and heterotrophic bacteria, and fungi weresuppressed in the polluted habitat comparing with the unpolluted location. The diversity of the micro ora was also reduced in the sur-face bio�lms on gravestones in the city contaminated by air pollution. However, both the sulfur-oxidizing and hydrocarbon-utilizingmicro ora were enriched in the bio�lms exposed to air pollution. In a laboratory study, low concentrations of the polluting chemicalsstimulated growth of these bacteria, and resulted in rapid acid production. Scanning electron microscopy demonstrated that the bio�lmsof both the sulfur-oxidizing bacteria and the hydrocarbon-degrading micro ora penetrated into the limestone. The enrichment of sulfur-and hydrocarbon-utilizing bacteria in the bio�lms may contribute to dissolution of the stone. However, further research is required todetermine the e�ects of speci�c metabolites of these microorganisms on stone deterioration. c© 2001 Published by Elsevier Science Ltd.

Keywords: Stone deterioration; Bio�lms; Pollutants; Air pollution; Sulfur; Hydrocarbons; Historic stone; Limestone

1. Introduction

Deposition of sulfur dioxide on historic buildings andmonuments is well documented in both Europe and theUnited States (Yerrapragada et al., 1994). The resultingacidity has been shown to cause severe decay. For example,Gauri and Holdren (1981) observed that increased levels ofSO2 in the atmosphere were responsible for deterioration ofmarble monuments in both Athens and Chicago.Urban air pollutants are also rich in both aliphatic and

aromatic hydrocarbons. Saiz-Jimenez demonstrated that theblack crusts coating buildings in European cities where theair pollution is high are rich in both aliphatic and polycyclicaromatic hydrocarbons (Saiz-Jimenez, 1993). These chemi-cals were found to be trapped in the mineral matrices of thebuildings.Microorganisms have been implicated in the attack of

both natural limestone materials and concrete by sulfur

∗ Corresponding author. Tel.: +1-617-495-2846; fax: +1-617-495-1471.E-mail addresses: mitchell@deas.harvard.edu (R. Mitchell), jdgu@

hkucc.hku.hk (J.-D. Gu).

compounds (Gu et al., 2000a). The chemolithotrophicthiobacilli have been shown to cause severe damage to con-crete sewer pipes exposed to volatile sulfur compounds (Guet al., 2000b; Sand and Bock, 1991). We have observedthat, in addition to Thiobacillus spp., the fungus Fusariumsp. also plays an important role in concrete deterioration(Gu et al., 1998).There is extensive evidence for the involvement of a

hydrocarbon utilizing micro ora in the biodeteriorationof historic buildings (Siaz-Jimenez, 1997). Chemoorgano-trophic microorganisms isolated from rocks have beenshown to utilize hydrocarbons as sole carbon sources and toproduce signi�cant quantities of organic acids (Warscheidet al., 1991). The aim of our research was to determine thee�ect of atmospheric pollutants on the bio�lm micro ora oflimestone gravestones. We have compared the bio�lm mi-cro ora on limestone gravestones in two locations. One setof gravestones is in a highly polluted urban environment,while the other is in a less polluted rural location. In a labo-ratory study, we have determined the capacity of the bio�lmcommunity, isolated from limestone gravestones in thepolluted habitat, to produce acidity and penetrate limestone.

0964-8305/01/$ - see front matter c© 2001 Published by Elsevier Science Ltd.PII: S 0964 -8305(00)00105 -0

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2. Materials and methods

2.1. Sampling sites and methods

Gravestones were selected in two cemeteries in the North-east of the United States. The polluted location was HarvardSquare, Cambridge, MA. The cemetery is located close tothe urban center where there is heavy continuous tra�c. Thegravestones in this cemetery date from the 17th century. Forour study, we selected limestone gravestones dating from themid-19th century. For our less polluted location we chose acemetery in Lexington, MA. The cemetery, approximately15 km from Cambridge, is in an area with minimal exposureto urban pollution. We sampled from limestone gravestonesdating from the mid-19th century.In all cases we prepared cotton swabs by dipping them

in 10ml of sterile distilled water containing one drop ofnon-toxic surfactant Triton X-100 (Sigma Chemical Co.,St. Louis, MO, USA). The gravestones were swabbed withthe damp cotton swabs over one square centimeter areas.Ten swabs were pooled for each sample. We analyzed threesamples for each location. After bringing them into the lab-oratory, the swabs were homogenized in 10ml of steriledistilled water before microbial analysis.

2.2. Enumeration of bio�lm microorganisms

All microorganisms were enumerated by plate counts fol-lowing one week of incubation at 30◦C. Heterotrophic bac-teria were enumerated on nutrient agar (Difco Lab., Detroit,MI, USA). Chemolithotrophic bacteria were enumerated onthe following medium: (g l−1) NH4Cl, 1.0 g; MgSO4, 0.5 g;K2HPO4, 0.5 g; KH2PO4, 0.5 g; Fe(SO4)3, 0.5 g; Na2S2O3,1.0 g; agar 10.0 g. For growth of thiobacilli the mediumused consisted of (g l−1): Na2S2O3, 5H2O, 10.0 g; NH4Cl,1.0 g; MgCl2, 0.5 g; K2HPO4, 0.6 g; KH2PO4, 0.4 g; FeCl3,0.02 g; chlorophenol red, 0.08 g; agar 10.0 g (Atlas, 1993).Fungi were enumerated following growth on malt extractagar medium (Difco Lab, Detroit, MI, USA). Penicillin G,97; 5000 U l−1 and bacitracin 6500 U l−1 were added to thefungal medium to inhibit bacterial growth.

2.3. Identi�cation of microorganisms

Samples from the gravestones were inoculated onto nu-trient agar plates for identi�cation of heterotrophic bacteriaand to the fungal growth medium described above for iden-ti�cation of fungi. Following 1 week of incubation at 30◦C,distinctive individual colonies were subcultured and puri�edby streaking on fresh agar plates. Characterization of thebacteria was achieved using the Biolog identi�cation system(Biologic Inc., Hayward, CA, USA). Fungi were identi�edmicroscopically as described elsewhere (Gu et al., 1998).

2.4. Scanning electron microscopy

In order to determine the penetration of bio�lms of hydro-carbon degrading bacteria into the limestone, we carried outa scanning electron microscope study.We inoculated 102=mlof hydrocarbon degrading bacteria to samples of limestoneto which 10 ppm of kerosene had been added. Parallel sam-ples of limestone were treated with 102=ml sulfur-oxidizingbacteria and treated with 10 ppm of thiosulfate. The sam-ples were incubated at 30◦C and 80% relative humidity for30 days at which time they were prepared for electron mi-croscopy. A diamond cutter was used to prepare surfacelayers of the limestone for examination. These pieces were�xed for 12 h in 3% gluteraldehyde–0.2M sodium cacody-late, previously �ltered through a 0.2-�m-pore-size poly-carbonate membrane �lter (Gelman Science, Ann Arbor,MI, USA). After washing in cacodylate and dehydration ina series of increasing ethanol concentrations the sampleswere critical point dried in liquid carbon dioxide (SamdriPV T-3b, Tousimis Research Co., Rockville, MD, USA).Immediately after drying, the specimens were coated withgold–palladium and viewed under an AMR 1000 scanningelectron microscope.

3. Results and discussion

3.1. Changes in the total bio�lm community

We analyzed the di�erences in the population size offungi, and heterotrophic and chemolithotrophic bacteria inbio�lms on the limestone at two locations, one less pollutedin Lexington and one polluted in Cambridge. The fungalpopulation was suppressed to a minor degree on the stone inthe polluted city (Fig. 1). However, the bacterial populationsin the bio�lms were dramatically di�erent between these twosites. Both the heterotrophic and chemolithotrophic bacte-ria on the limestone in the less polluted area were orders ofmagnitude higher than on the stone in the cemetery in thepolluted city. All three groups of microorganisms, fungi, het-erotrophic and chemolithotrophic bacteria were suppressed

Fig. 1. Comparison of the bio�lm populations of heterotrophic andchemolithotrophic bacteria and fungi on limestone gravestones in a loca-tion exposed to atmospheric pollution in Cambridge and a less-pollutedarea in Lexington, MA, USA.

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in the polluted location, presumably by the atmospheric pol-lutants emitted by the motor vehicles.When we compared the predominant populations of mi-

croorganisms in the bio�lms at the two locations, we foundthat there were twice as many di�erent genera of both bac-teria and fungi present in the bio�lms on gravestones in theless polluted city. Xanthomonas, Vibrio and Bacillus werevery common in bio�lms from both locations. A numberof species of Pseudomonas spp., found in bio�lms in theless polluted area, were absent from bio�lms in the pollutedcity. Among the fungal genera, Penicillium, Cladosporium,Fusarium, andAureobasidiumwere predominant in both lo-cations.Epicoccum andAlternaria species, common in Lex-ington bio�lms, were rarely found in Cambridge bio�lms.Our data showed that the presence of atmospheric pollu-

tants inhibited both the size and diversity of the bio�lm mi-crobial community. These observations provide new insightsinto the e�ects of stress on microbial community ecology.It is unusual for pollution stress to suppress microbial ac-tivity. Typically, one population becomes dominant, at theexpense of others (Saylor et al., 1982). However, the totalsize of the community usually remains stable. In our study,the data suggest that the air pollutants suppress the totalcommunity of microorganisms in these bio�lms.

3.2. E�ects on the sulfur and hydrocarbon utilizingmicro ora

The concentrations of atmospheric pollutants in urban en-vironments in the United States have increased dramaticallyduring the past quarter century. Sulfur dioxide is a majorpollutant in most cities. Concentrations range from 20 to200 ppb depositing on surfaces in urban environments, com-pared to less than 10 ppb in rural areas for a duration of1 h (Seinfeld, 1986). Similarly, organic pollutants, particu-larly hydrocarbons, are present in high concentrations in theurban atmosphere. Hydrocarbons typically deposit at ratesof 500–1500 ppb for 1-h duration in major United Statescities. This compares with rates of less than 100 ppb in lesspolluted habitats (Seinfeld, 1986). These pollutants depositand accumulate on limestone materials, providing potentialnutrients for a bio�lm community on the surface.As a means of determining the e�ect of sulfur pollu-

tion on limestone bio�lm, we measured the percentage ofsulfur-utilizing bacteria in the chemolithotrophic commu-nity. Fig. 2 shows that only 20% of the chemolithotrophicbacteria in the bio�lms on limestone in Lexington, theless-polluted location, were capable of utilizing sulfur com-pounds. Apparently 80% of the bio�lm bacteria utilizeother inorganics in the stone as sole energy sources. In con-trast, we found that 50% of the bio�lm chemolithotrophiccommunity on the limestone in Cambridge, the locationcontaminated by air pollution, used sulfur compounds astheir sole energy source. The data indicate that, despite thesuppression of the total population of chemolithotrophic

Fig. 2. Comparison of the percentage of chemolithotrophic bacteria capa-ble of utilizing sulfur compounds in limestone bio�lms from a pollutedand less polluted location.

bacteria by air pollutants, there is an enrichment of sulfuroxidizers. Presumably their selective enrichment resultsfrom the accumulation of sulfur compounds on the stone.Similarly, the e�ect of air pollution on the hydrocarbon-

utilizing bacteria in the bio�lms was evident by comparinggrowth on a complete medium, and a minimal medium con-taining hydrocarbons as the sole carbon source. While therewas an enrichment of hydrocarbon-utilizing bacteria in thebio�lms on limestone in Cambridge relative to those in lesspolluted Lexington, the di�erences were not great. Almost90% of the heterotrophic bacteria in the bio�lms from bothlocations were capable of using kerosene as a sole carbonsource. The results are not surprising, since we would expectthat even in the absence of contamination of the stone byhydrocarbons, a proportion of the indigenous heterotrophicbacterial population would be capable of utilizing hydro-carbons at relatively low concentrations of the chemicals.However, we found no evidence of an enrichment of fungiin the bio�lms exposed to air pollutants.It is probable that, in Massachusetts, where there is fre-

quent precipitation throughout the year, the concentrationof sulfur compounds and hydrocarbons depositing and thenremaining on the limestone in the cemeteries remains quitelow. We studied the e�ect of sulfur and hydrocarbon con-centrations on the bio�lm bacterial population from the pol-luted city to estimate the ability of the micro ora to utilizelow concentrations of the pollutants. In the current study,we found no evidence that the bio�lms in the presence ofsulfur compounds contained fungi. We did not observe, inour scanning electron microscopic study, that the thiobacillipenetrating the limestone was associated with fungal hy-phae. In contrast to sewage pipes which are exposed to highconcentration of organic matter (Sand and Bock, 1991), thelimestone in the current investigation was not heavily con-taminated with organic chemicals likely to be utilized byfungi.Both aliphatic and polycyclic aromatic hydrocarbons are

found in the black crusts of monuments (Saiz-Jimenez,1997). He suggested that exposed building materials actas non-selective surfaces, passively entrapping depositedairborne particulate matter and organic compounds. Anumber of investigators have suggested that fungi play an

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Fig. 3. Acid production by sulfur-utilizing bacteria and mixed populationsof hydrocarbon-utilizing bacteria isolated from limestone gravestones inCambridge, MA, USA.

important role in the degradation of hydrocarbons (Saiz-Jimenez, 1993; De la Torre et al., 1993). However, noevidence was obtained in the current investigation linkingbio�lm development on limestone in the presence of hydro-carbons with a fungal population. Neither our comparisonof microbial populations in polluted and less polluted lo-cations nor our investigation of the speci�c hydrocarbonutilizing microbial population yielded evidence of stimula-tion of a fungal community in the bio�lms. It is probablethat the porosity of the limestone favored the preferentialdevelopment of a community of hydrocarbon-degradingbacteria.

3.3. Acid production and stone penetration

As part of our study of the e�ects of pollutants on thebio�lm sulfur-utilizing population, we measured the produc-tion of acidity by the micro ora growing at di�erent con-centrations of sulfur. At all concentrations, down to 50 ppb,acidity began to develop after ten days of incubation (Fig.3). After 15 days the pH had declined to 6. Even at lowconcentrations of sulfur, the bio�lm micro ora is capable ofproducing acid. We also tested the ability of hydrocarbondegrading bacteria from the bio�lms on stone in the pollutedlocation to produce acid from low concentrations of hydro-carbons. We inoculated microorganisms from gravestonesin Cambridge to a minimal liquid medium containing hy-drocarbons as the sole carbon source at concentrations be-tween 1 and 10 ppm. During incubation, we determined thesize of the bacterial populations at the di�erent hydrocarbonconcentrations. Our data showed that there were more than106 hydrocarbon-degrading bacteria per cm2 of gravestoneat all concentrations tested.When the acidity of the culture aliquots was determined, it

was found that substantial quantities of acid were generatedby hydrocarbon degraders in the bio�lm. In all cases the pHhad declined from 7.0 to 6.1 in 15 days (Fig. 3). Since theacids produced by these bacteria are organic, it is surprisingto �nd such a rapid production of acidity. Presumably therewas su�cient production of acidic metabolites to overcomethe bu�ering capacity of the growth medium.

Fig. 4. Scanning electron micrographs of bio�lms of sulfur-oxidizingbacteria on limestone: (a) surface growth of the bio�lm (magni�cation5000×); (b) the bio�lm growing into the pores of the stone (magni�cation10,000×).

The presence of large populations of sulfur and hydro-carbon degrading bacteria in bio�lms on limestone grave-stones in polluted locations suggested that these bacteria arecapable of penetrating the limestone, and accelerating dete-rioration. When sulfur-utilizing bacteria were inoculated tolimestone and incubated at 25◦C and 80% relative humidityfor 30 days, a thick bio�lm was observed on the surface oflimestone (Fig. 4a). We observed that the bio�lm had pen-etrated into the pores of the limestone (Fig. 4b). It is prob-able that growth was controlled by the depth of penetrationand availability of the pollutant in the pores of the lime-stone. Similar results were observed in a scanning electronmicroscope study of bio�lm development of hydrocarbondegrading bacteria on limestone. However, it is not knownif the penetration of the bacteria into the interior of the stonea�ects deterioration.Further research is needed to determine if su�cient acid is

produced by the bacteria to cause dissolution. It is possiblethat other bacterial metabolites may stimulate penetrationand accelerate stone dissolution. Warscheid et al. (1991)suggested that extracellular polymers, produced by bacte-ria on stone in polluted environments, act as surfactants,

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permitting increased capillary action of water into the stone.Surfactants would also facilitate the penetration of bacteria.The deposition of acid precipitates on limestone is well

documented. Gauri and his colleagues have demonstratedthe presence of sulfates on gravestones that had been in-stalled less than 5 years earlier (Gauri and Holdren, 1981).The potential for thiobacilli to produce corrosive sulfuricacid in limestone bio�lms has been demonstrated by Sandand Bock (1991). They observed severe corrosion of con-crete sewage pipes exposed to sulfur compounds originat-ing in the sewage. They found that the degree of corrosionwas proportional to the size of the population of thiobacilli.Recently, we observed that a fungus growing in the con-crete bio�lm facilitates the attack by thiobacilli (Gu et al.1998). The fungus, identi�ed as a Fusarium species, signif-icantly increased both calcium release and weight loss fromthe limestone. However, in the current study no evidence offungal involvement was detected.Despite the suppression of the total community of

microorganisms by atmospheric pollutants, we observedthat a speci�c population of bacteria capable of utilizingsulfur compounds was stimulated. The presence of a largepopulation of these bacteria in limestone bio�lms in thepolluted location is a strong indication of the involvementof metabolic products of the sulfur bacteria in deteriorationof historic limestone in habitats contaminated by atmo-spheric sulfur pollution. Hydrocarbon-degrading bacteriahave also been observed to excrete su�cient quantities oforganic acids to damage stone materials in historic buildings(Warscheid et al., 1991).Our data show that atmospheric pollutants cause a sup-

pression and change in the bio�lm micro ora on grave-stones. The dominant micro ora stimulated by the pollutantspenetrates into the stone. It is not known if these changesin the microbial community structure accelerate stone dete-rioration. We are currently investigating the e�ects of thebio�lm bacteria stimulated by atmospheric pollutants onlimestone dissolution.

Acknowledgements

This work was supported in part by Grant NumberMT2210-7-NC-033 from the US Department of the Interior,National Park Service, Center for Technology & Training.

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