681

POLYCYCLIC AROMATIC HYDROCARBON-DEGRADING BACTERIA IN BUILDING STONES

ORTEGA-CALVO, J. J. and SAIZ-JIMENEZ, C.

lnstiluto de Recursos Naturates y Agrobiologia, C.S.l.C., Apartado 1052, 41080 Sevilla, Spain

SUMMARY Two European cathedrals (Seville, Spain, and Mechelen, Belgium) were sampled for investigating the

presence of polycyclic aromatic hydrocarbon-degrading bacteria in their stones. These two cities are

representatives of polluted environments, either by the effect of city traffic (Seville) or nearby industries (Mechelen). The presence of such bacteria was evidenced by the conversion to 14C02 of freshly-added [9-

14C]phenanthrene and by the isolation of bacteria capable of using this compound as the sole source of carbon

and energy. The mineralization of aromatic hydrocarbons demonstrated that an active microflora transforming

deposited airborne pollutants on to building stones is active in different climates and environments.

1. INTRODUCTION

Anthropogenic activities, particularly the burning of fossil fuels, are significant sources of polycyclic aromatic

hydrocarbons (PAHs) in the environment. Monuments, buildings, statues, located in urban environments act as

repositories of organic and inorganic pollutants, which accumulate at the surfaces. They are natural and

passive samplers, constantly present in polluted areas which have been used for pollution studies (1).

Generally, black and white zones can be observed on the exposed surfaces of any building. Surfaces directly

exposed to rainfall show a white color since the deterioration products formed on the stone surface are

continuously washed out. Black patches, found in zones protected from direct rainfall and from surface runs,

are covered by an irregular, dendrite-like, dirty, grey-to-black hard crust composed of crystals of gypsum mixed

with aerosols, spores, pollen, dust and every class of particulate matter which are entrapped in the mineral

matrix. This crust, known as black crust or sulfated crust, is originated by wet and dry deposition processes in

which sulfuric acid, a sulfur dioxide oxidation product, attacks carbonate rocks, resulting in gypsum formation

(2, 3).

Seville and Mechelen are cities located in very different climatic regions. Seville is one of the southern cities

with highest average temperatures in Europe. The climate of Mechelen in nothem Belgium is much colder.

Also the sources of pollution are different. The city of Mechelen is located on the industrial axis between the

major cities of Antwerp and Brussels, with refineries, electrical power plants and non-ferrous industries make

this area the most important emitter of industrial S02 and NOx in Belgium. The pollution levels of Seville are

due to the dense traffic, areas of car parking, and narrow sheltered streets, a typical configuration of historic

centers in old Andalusian cities (4). The organic composition of weathered stones in polluted environments has been studied by Saiz-Jimenez (1,

3). Alkanes, fatty acids and PAHs were the main components. In general, these compounds originated from

combustion of petroleum derivatives (gasoline and diesel fuel). Air pollution represents an organic matter input on the bare stone, usually depleted of organic compounds in the quarry, which obviously enriches the original

substratum making it colonizable by microorganisms. Consequently, the deposition of anthropogenic

compounds on to stones may affect the colonization and growth pattern of microorganisms in building stones

located in polluted environments when compared with the growth of microorganisms in the same stone in

quarries. From previous studies it was suggested that air pollution modifies the chemical composition of

building stones in urban environments, resulting in the selection of microorganisms with specific nutrient

requirements or with a defined metabolic capability (5). In this paper, the presence of an active phenanthrene-degrading microflora in the walls of two European

cathedrals is reported.

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2. MATERIAL AND METHODS

Samples of weathered stones were removed aseptically from the different pilasters supporting the fence defending the Prince's Gate, cathedral of Seville, Spain. Calcarenite from a quarry historically documented to be the same as that employed for the construction was used as control. Also, pieces of limestones and black crusts, were sampled from the cathedral of Mechelen, Belgium. Organic carbon content of black crust samples ranged between 3.5 and 4.8 %. A more detailed study of the two cathedrals has been reported elsewhere (4). Phenanthrene was used because it is representative of the PAHs present in many building stones and urban environments (4, 5). To measure mineralization, duplicate portions of ground samples were placed in 250-mL Er1enmeyer flasks, and distilled water was added to bring the moisture level to 20 % (w/w). Twenty-gram portions were used for the Seville samples, while 2 g were used for the Mechelen samples. In the latter case the samples were placed in 20-ml glass vials that were introduced upright into the flasks. The samples were amended with 0.66 mg of [9-14CJ phenanthrene (Sigma, Germany) dissolved in 250 ml of dichloromethane, the latter then being allowed to evaporate. The flasks, maintained at laboratory temperature (20-22°C), were closed with teflon-lined stoppers, which prevented drying out of the samples during the measurements. 14C02 production was measured as radioactivity appearing in an alkali trap, with a Beckman model lS5000TD liquid scintillation counter. Sterile controls were run with autoclaved samples. Maximum rates of mineralization were calculated as previously described (6). Viable counts were determined as colony-forming units (CFU) per gram of sample by plating serial dilutions of original samples on plates containing 0.3 % (wt/vol) of Trypticase-soy broth, 1.5 % agar, and an inorganic salts solution (6). Bacteria capable of using phenanthrene as a sole carbon and energy source were isolated from stone samples through enrichment procedure. Characterization of the isolates was based on standard microbiological methods (l). Experiments with stone sample suspensions were performed in 250-ml Er1enmeyer flasks containing 20 g of ground, autoclaved samples, 40 ml of sterile inorganic salts solution, and 250 ml of dichloromethane with [9-14C] phenanthrene and sufficient unlabeled phenanthrene to give a final concentration of 0.1 mg/ml. The flasks were maintained for 12 hon a rotary shaker operating at 100 rpm for equilibration. Then, an inoculum of phenanthrene-preconditioned bacteria was added to the flasks, shaken at 100 rpm on a rotary shaker at 30°C, and the 14C02 production measured. The results are given, unless otherwise stated, as mean of duplicate measurements ± standard deviation. Statistical analyses were performed at P = 0.05 with t-test for comparisons between two means and with F-test for multiple comparisons.

3. RESULTS

Table 1 shows the sites, description of sampling, microbial numbers and extents of phenanthrene mineralization. Microbial numbers revealed that most samples had a significant heterotrophic microbial population, while quarry stones had no detectable heterotrophs. Phenanthrene was readily mineralized to C02 by the natural microbiota in samples from the cathedrals (Figure 1). Mineralization in samples S1 and S2 from the cathedral of Seville started rapidly with no apparent lag phase and reached a final extent of 35.0 and 26.9 % of substrate mineralized after 100 days (Table 1). An acclimation period of 1 O days and a statistically lower rate and extent of mineralization characterized the mineralization in the sample S3, where only 15.3 % of the compound was converted to co2 in 100 days. Mineralization in samples from other pilasters also occurred after an acclimation period but differed in the rates and extents. In these samples the percentage of phenanthrene mineralized to C02 in 1 oo days ranged from 18.0 to 28.1 %. Sample 01, from a quarry, showed a reduced activity, close to background levels. Mineralization of phenanthrene in samples from the cathedral of Mechelen also occurred (Figure 1). Mineralization in samples M2 and M7, was reduced when compared to other samples, as only 3.2 and 0.7 % of phenanthrene, respectively, was mineralized after 100 days.

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TABLE 1

Description, microbial numbers and mineralization of phenanthrene in samples from monumentsa

Site and Crust CFU/g % mineralized sample location Code formationb (x105)c in 100 days

Seville Cathedral

Pilaster 1 S1 8.2S ± 1.76A 3S.O ± 2.1A

S2 7.00 ± 1.06A 26.9 ± 2.38

S3 + 2.80 ± 0.268 1S.3 ± 1.7C

Pilaster 2 S4 + 1.26 ± 0.04 21.4 ± 2.3

Pilaster 3 SS + 0.68 ± 0.17 18.0±1.4

Pilaster4 S6 + 2.28 ± 0.02 28.1±1.6

Quarry Q1 Not detectedd 1.1±0.0

Mechelen Cathedral

East M1 1.40 ± 0.23A 23.3 ± 1.4A

M2 + 0.09 ± 0.008 3.2 ± 0.28

North M3 2.54 ± 0.31A 37.6 ± 9.7A

M4 + O.S8± 0.148 28.9± 2.7A

North east MS 1.46 ± 0.48Ae 24.S1

M6 + 0.19±0.0288 19.S1

South M7 + Not detectedd o_i

aFor each sample location, values in a column followed by the same capital letter are not significantly different (P = O.OS)

b - and + indicate crust-free- and black crust samples, respectively

cCFU/g presented are for total heterotrophic bacteria

dDetection limit was 1, 000 CFU/g 9Significantly different at P = 0.10

Values from non-duplicate measurement

Bacteria able to grow with phenanthrene as the sole source of carbon were isolated from some stone samples.

From sample S4 two Gram-negative rods identified as Pseudomonas sp. 1 and 2, and two Gram-positive rods,

a Bacillus sp. and a (tentatively identified) Nocardia sp., were obtained. From sample M3 a Gram-negative rod was identified as Pseudomonas sp. 3.

Bacillus sp., Pseudomonas sp. 1, and Pseudomonas sp. 2, which showed the fastest growth, were tested for

mineralization of phenanthrene in laboratory cultures. These bacteria mineralized phenanthrene in liquid

culture, at an initial concentration of 0.1 mg/ml, both with and without stone samples (Figure 2). The influence

of stone samples on maximum rates of mineralization was different depending on the isolate. Whereas Bacillus sp. mineralized the substrate at a maximum rate irrespective of whether the medium contained stone

or not, the presence of stone induced different maximum rates of mineralization in the two Pseudomonas sp.

Interestingly, the higher rate of mineralization for each Pseudomonas sp. strain occurred concomitantly with

the presence of the type of stone from which it was isolated.

4. DISCUSSION

Previous studies have shown that PAHs are widely distributed in the stones of the cathedrals of Seville and Mechelen (4). The compounds deposited on to the two cathedrals have a petrogenic origin, as denoted by the

carbon preference index of hydrocarbons and other characteristic molecular markers of petroleum (8).

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Petroleum residues are a major and usually predominant component of the lipids extractable from aerosols in

urban environments and PAHs have been identified, among other sources, in smoke particles and diesel

engine soot (9). It is known that organic compounds adsorbed on to the particulate phase of diesel exhaust can

be accounted for by PAHs (10). Exposed building stones act as a non-selective surface, passively entrapping all deposited airborne particulate

matter and organic compounds, and therefore, building stones represent a peculiar environment subjected to

continuous deposition and washing off of airborne pollutants. The stones contain mineral salts, organic

compounds, and the pores and crevices are good niches for the colonization and growth of microorganisms.

Phenanthrene-degrading bacteria have been used previously as indicators in the evaluation of microbial

activity in PAH-polluted environments. For instance, Bogardt and Hemmingsen (11) detected and enumerated

the phenanthrene-degrading bacteria in petroleum-contaminated sites; and phenanthrene-utilizing and

phenanthrene-cometabolizing microorganisms have been evidenced in estuarine sediments (12).

Building stones also contain bacteria able to utilize PAHs. This kind of microflora is not only restricted to fuel­

contaminated soils or sediments, PAH-contaminated wastes, etc. but can also be found in urban

environments, where the bacteria have adapted to specific site conditions which include high PAH

concentrations for long periods. In fact, the Seville public bus service started to operate in the ear1y 60's, and

some of the stops were located just in front of the sampling zone, at about 20 m from it (1). The city of

Mechelen is located in a very polluted industrial area. High concentrations of PAHs were found on the

limestone of this cathedral (4).

Black crusts contain compounds such as phenols, benzoic acids, lead, etc. (4) which can inhibit bacterial

growth. This could explain a significantly reduced bacterial population in some areas with black crusts with

respect to adjacent crust-free zones. However, these compounds did not suppress PAH mineralization. On the

contrary, a clear1y stimulatory effect in 14C-phenanthrene mineralization was found in cultures of Pseudomonas sp. 2, with black crust suspension, which indicates the presence in the crust of nutrient elements required for an

adequate growth and to the adaptation of the bacteria to the polluted environment from which they were isolated.

The data herein reported indicate that microbial degradation of phenanthrene is common in crust-free and

black crust stones from European cathedrals. Although the experiments performed in this work required the

removal of the samples from the walls, the rapid and significant phenanthrene mineralization observed

strongly suggests that microbial transformation reactions also occur in situ. Therefore, biological activity plays a

role in the fate of organic compounds deposited on building stones located in urban (polluted) environments.

Mineralization of organic compounds is characteristic of growth-linked biodegradation and part of the

phenanthrene is converted to cell components and degradation products that could remain in the stone. It is

also possible that the particular conditions prevailing in the stone niches promote the selection of

microorganisms able to transform, either by growth-linked reactions or by cometabolism, other anthropogenic compounds that have been so far considered as recalcitrant.

ACKNOWLEDGEMENTS

The samples from the cathedral of Mechelen were kindly provided by Prof. R. van Grieken, Antwerp, Belgium.

The identification of the bacterial strains was carried out with the help provided by Prof. A. Ramos, Granada, Spain.

REFERENCES 1.Saiz-Jimenez, C. 1993. Deposition of airborne organic pollutants on historic buildings. Atmos. Environ.

27B, 77-85.

2.Saiz-Jimenez, C. and Garcia del Cura, M.A. 1991. Sulfated crusts; a microscopic, inorganic and organic analysis. In: N.S. Baer, C. Sabbioni, and A.I. Sors (eds.), Science, Technology and European Cultural Heritage. Butterworth-Heinemann, Oxford, pp. 527-530.

3.Saiz-Jimenez, C. 1991. Characterization of organic compounds in weathered stones. In: Baer, N.S.,

Sabbioni, C. and Sors, A.I. (eds.), Science, Technology and European Cultural Heritage. Butterworth­Heinemann, Oxford, pp. 523-526.

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4.Fobe, B., Veugels, G., Roekens, E., van Grieken, R., Hennosin, B., Ortega-Calvo, J.J., Sanchez del Junco, A. and Saiz-Jimenez, c. 1995. Organic and inorganic components in limestone weathering crusts from cathedrals in Southern and Western Europe. Environ. Sci. Technol. 29, 1691-1701.

5.Saiz-Jimenez, C. 1995. Deposition of anthropogenic compounds on monuments and their effect on airborne microorganisms. Aerobiologia 11, 161-175.

6.0rtega-Calvo, J.J., I. Binnan and M. Alexander. 1995. Effect of varying the rate of partitioning of phenanthrene in nonaqueous-phase liquids on biodegradation in soil slurries. Environ. Sci. Technol. 29, 2222-2225.

7.Holt, J.G., Krieg, N.R., Sneath, P.H.A., Staley, J.T., Williams, S.T. 1994. Bergey's Manual of Detenninative Bacteriology. Williams and Wilkins, Baltimore.

8.Simoneit, B.R.T 1986. Characterization of organic constituents in aerosols in relation to their origin and transport: a review. Int. J. Environ. Anal. Chem. 23, 207-237.

9.Yu, M.-L and Hites, R.A. 1981. Identification of organic compounds on diesel Chem. 53, 951-954.

engine soot. Anal.

10.Bayona, J.M., Markides, K.E. and Lee, M.L. 1988. Characterization of polar polycyclic aromatic compounds in a heavy-duty diesel exhaust particulate by capillary column gas chromatography and high-resolution mass spectrometry. Environ. Sci. Technol. 22, 1440-1447.

11.Bogardt, A.H. and Hemmingsen, B.B. 1992. Enumeration of phenanthrene- degrading bacteria by an over1ayer technique and its use in evaluation of petroleum-contaminated sites. Appl. Environ. Microbial. 58, 2579-2582.

12.Cemiglia, C.E. 1993. Biodegradation of polycyclic aromatic hydrocarbons. Curr. Op. Biotechnol. 4, 331-338.

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