International Biodeterioration & Biodegradation 54 (2004) 121–124www.elsevier.com/locate/ibiod

Bio�lms on church walls in Porto Alegre, RS, Brazil,with special attention to cyanobacteria

C*ezar Augusto Crispima, Christine C. Gaylardeb;∗, Peter M. Gaylardea

aSoils Department, MIRCEN, Federal University of Rio Grande do Sul (UFRGS), Cx. P. 776, Porto Alegre-RS 91501-970, BrazilbBiophysics Department, UFRGS, Av. Bento Goncalves 9500, Porto Alegre-RS 91501-970, Brazil

Abstract

The populations of phototrophs in bio�lms on the external walls of six churches in Porto Alegre were identi�ed. Cyanobacterial isolateswere identi�ed morphologically, according to Bergey, and PCR products were obtained for sequencing using the universal forward primer,27F1, and the cyanobacteria-speci�c reverse primer, 408R. The sequences were submitted to the BLAST search analysis.

Eleven cyanobacterial taxa were found and among algae three genera predominated. Three of the cyanobacterial taxa were coccoid groupsand eight were �lamentous. Two orders (Pleurocapsales and Chroococcales) and two �lamentous genera (Lyngbya and Scytonema)were present on �ve of the six buildings. Cyanobacterial genera with reported stone-boring activity which were detected on the churcheswere Synechocystis, Gloeocapsa, Scytonema and Mastigocladus, as were members of the Order Pleurocapsales. The best-maintainedchurch had the lowest cyanobacterial biodiversity; in damp areas of this church many algae were present in bio�lms and none of thecyanobacterial groups mentioned above were detected.? 2004 Published by Elsevier Ltd.

Keywords: Bio�lms; Biodeterioration; Cyanobacteria; Monuments; PCR; Weathering

1. Introduction

Bio�lms on the external walls of buildings lead toaesthetic deterioration, production of acidic/alkaline condi-tions, retention of moisture and altered temperatures causedby di;erential heat absorption by coloured surface deposits(Gri<n et al., 1991; Warscheid et al., 1991; GarciaValleset al., 1997). The �nal result is, in most cases, biodegra-dation of structural materials. When the buildings are ofarchitectural or historical importance, the resultant lossesare more than economic.The most frequent microrganisms present on external

walls are cyanobacteria, algae, fungi, bacteria and acti-nomycetes, myxomycetes and protozoa (Gaylarde andGaylarde, 2000). Of these, cyanobacteria and fungi usuallyconstitute most biomass. These organisms can cause degra-dation of stone by the production of aggressive acid oralkaline metabolites and surfactants, as well as by physicalpenetration of their cells into the substrate (Gaylarde andMorton, 1999).

∗ Corresponding author.E-mail address: cgaylarde@yahoo.com (C.C. Gaylarde).

Previous work has indicated that cyanobacteria constitutethe main portion of the biomass on ancient stone struc-tures (Ortega-Morales et al., 2000; Gaylarde et al., 2001).Cyanobacteria are Gram-negative photosynthetic prokary-otes that occur in both �lamentous and coccoid forms. Someare capable of �xing nitrogen. As a group, they are partic-ularly resistant to desiccation and high levels of UV-light(Garcia-Pichel et al., 1992; Chazal and Smith, 1994), giv-ing them a distinct advantage over many other organismson exposed surfaces. The resistance to UV is generally as-sociated with the production of protective pigments, addingto the deteriorative characteristics of the bio�lm.We report here the results of investigations on the presence

of cyanobacteria, and other photosynthetic microorganisms,on churches in the southern Brazilian city of Porto Alegreand try to relate their presence to the biodeterioration process.

2. Materials and methods

2.1. Sampling sites

Six churches were chosen: Sao Jos*e, Sao Judas, SantoAntonio, Nossa Senhora dos Navegantes, Nossa Senhora

0964-8305/$ - see front matter ? 2004 Published by Elsevier Ltd.doi:10.1016/j.ibiod.2004.03.001

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Table 1Cyanobacterial taxa and algal abundance on six churches in Porto Alegre

Order/Genus Church

Sao Sao Santo Sao Judas Navegantes DoresJos*e Geraldo Antonio

Calothrix + +Chroococcales: + + + + +

Synechocystis + + + +Gloeocapsa + + + +

Leptolyngbya + + + + +Lyngbya + + + + +Nostoc + + + +Oscillatoria + + +Plectonema + + + +Pleurocapsales + + + + +Scytonema + + + + +Mastigocladus +Algae + +=− +++ ++ +++ +++

das Dores, Sao Geraldo. These are all situated within themunicipality of Porto Alegre, a city located in the south ofBrazil. It has a sub-tropical climate, with high temperaturesin the summer months and high humidity throughout theyear.The churches are in various stages of deterioration. Sao

Judas is the only church without a rendering over the brickstructure and shows considerable degradation, with crum-bling mortar colonized by mosses and higher plants, deteri-orating bricks and, in a few areas, loss of whole bricks, pro-viding nesting sites for pigeons. The cement renderings ofSao Jos*e and Santo Antonio are in an advanced state of de-terioration, with blown areas, while the rest of the churchesare relatively well conserved, but show various degrees ofbio�lm formation.

2.2. Sampling and culture techniques

Samples were taken from the surfaces of the buildingsusing a non-destructive adhesive tape sampling methodof Gaylarde and Gaylarde (1998). Tape samples taken tothe laboratory were placed on plates of modi�ed Knop’smedium (MKM, Gaylarde and Gaylarde, 1998). The sam-ples were examined after a few hours of rehydration on thismedium, using a binocular microscope and the lower powerobjectives of an optical microscope. This allowed the iden-ti�cation of the major biomass in the bio�lms. Plates werethen incubated at 28◦C with constant illumination for up to8 weeks. Cyanobacteria were isolated by micromanipulationand repeated subculture on both solid and liquid MKM.

2.3. Identi<cation

Microorganisms in rehydrated bio�lms and in cul-ture were identi�ed by traditional morphological methods(Prescott, 1964; Holt et al., 1994). Cyanobacteria were also

identi�ed by molecular techniques (Gaylarde et al., 2004).Single colonies from solid medium, or DNA extracted fromisolates, were subjected to the PCR using the universal for-ward primer, 27F1, and the cyanobacteria-speci�c reverseprimer, 408R (Neilan et al., 1997, 2002), and the PCR prod-ucts were sequenced. The sequences were submitted to theBLAST facility (www.ncbi.nlm.nih.gov/BLAST) and near-est matches conforming to the morphological appearanceof the cells recorded.

3. Results

Microorganisms seen on direct examination of rehydratedbio�lms were cyanobacteria, algae and �lamentous fungi.Yeasts, slime moulds, protozoa and other bacteria, includ-ing actinomycetes, were also observed during incubation.Bio�lms on Sao Judas and N. Sra. das Dores were less richthan on other churches, with no protozoa and fewer �la-mentous fungi present. The principal genera of green algaedetected were Chlorella, Klebsormidium and Trentepohlia.Thirteen di;erent algal taxa were identi�ed, the majoritybeing coccoid morphotypes. Diatoms were present only onSanto Antonio and N. Sra. dos Navegantes, and the formeralso showed a high number of slime moulds.The degree of algal colonization and the cyanobacterial

taxa identi�ed on the churches are shown in Table 1. Ofthe 11 cyanobacterial taxa detected, three were coccoid andeight �lamentous. However, the major biomass in “mature”bio�lms, i.e. those with high biodiversity, was of the coccoidorder Pleurocapsales.

4. Discussion

The number of cyanobacterial types detected (Table 1)emphasizes the low biodiversity in bio�lms on N. Sra. das

C.A. Crispim et al. / International Biodeterioration & Biodegradation 54 (2004) 121–124 123

Fig. 1. Nossa Senhora das Dores, showing dark bio�lms on water drainagesites.

Dores, which was also one of the visually cleaner build-ings. Of the �ve commonly detected cyanobacteria in thissurvey, only two, Lyngbya and Leptolyngbya, were foundon this church. Samples from this building were taken fromdiscoloured areas, close to leaking down pipes and corners(Fig. 1) and the greater moisture content is probably thereason for the presence of large numbers of algae (Gillattand Tracey, 1987; Crispim et al., 2002) in what is an appar-ently “young” bio�lm. These eukaryotic phototrophs werethe major biomass on this church. Santo Antonio was one ofthe other buildings showing large numbers of algae, but inthis case, together with many other microorganisms. This isan unpainted cement-rendered structure on which the typicalorange subaerial bio�lms (thalli) of the alga Trentepohlia,a member of the Chlorophyta, occur at the base (Fig. 2).Most of the cyanobacteria were identi�ed by traditional

techniques. Only in few cases did the sequences submittedto the BLAST facility show close similarity (¿ 95% se-quence match) to organisms with sequences deposited in thedatabases. This is undoubtedly the consequence of the factthat almost all cyanobacterial sequences deposited to dateare from aquatic or polar environments. The cyanobacteriain the particular environment of the church walls, subjectedto repeated desiccation and high UV levels, are apparentlydi;erent from those which have been investigated in othermilieux and other parts of the world.The presence of the Pleurocapsales in all the long-

established bio�lms on the churches con�rms previous

Fig. 2. Santo Antonio, showing dark bio�lms. The pediment showedtypical orange growth of Trentepohlia at the base.

suggestions of this group as indicators of mature bio�lmson buildings (Gaylarde and Gaylarde, 1999; Shirakawaet al., 2002). Members of the colonial, baeocyte-formingcyanobacterial order Pleurocapsales detected in cul-tures from walls are at present di<cult to classify, sincemany of the isolates do not conform to the genera listedin standard modern works. The most frequent identi�-able organisms on walls belong to the genera Xenococ-cus, Stanieria, Chroococcidiopsis, Myxosarcina and thePleurocapsa-group (our unpublished data). In the presentinvestigation, Chroococcidiopsis, the Pleurocapsa-groupand an unknown member of the Pleurocapsales were de-tected. The Pleurocapsales and the Chroococcales werethe major components of biomass on the most deterioratedchurches, i.e. Sao Judas and Santo Antonio. Members ofthe Pleurocapsa group have previously been indicated asstone-boring genera, which release a carbonate-digestingliquid from apical cells (Mao-Che et al., 1996). Syne-chocystis and Gloeocapsa (Order Chroococcales) havealso been stated to be capable of stone-boring activity,along with the �lamentous genera, Stigonema, Schizothrix(Ho;man, 1989), Scytonema (Golubic et al., 2000) andMastigocladus (Boone et al., 2001). Most of these generawere found on our churches.Although the number of samples is small, the results con-

�rm our earlier impressions from hundreds of samples fromboth modern and historic buildings, viz. for biodeterioration,the coccoid cyanobacterial species are the most importantbio�lm members. We have seen these morphotypes grow-ing within stone, as epiliths (Ortega-Morales et al., 2000),

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as well as on surfaces. In bio�lms examined in situ, �lamen-tous cyanobacteria are rarely the major biomass (Crispimet al., 2002; Ortega-Morales et al., 2000), although they maybe the principal isolates obtained by standard culture meth-ods, as in the present case.In summary, this study demonstrates that known stone-

degrading cyanobacteria are present on the external wallsof churches in Porto Alegre. These organisms are the ma-jor microbial biomass in bio�lms on churches with obviousdeterioration.

Acknowledgements

We wish to thank the Brazilian agency CNPq for fundingfor materials and CAPES for a post-graduate grant to CAC.

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