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DETERIOGENIC CYANOBACTERIA ON HISTORIC

BUILDINGS DETECTED BY CULTURE AND

MOLECULAR TECHNIQUES

Cezar A. Crispim 1, Peter M. Gaylarde 1, Christine C. Gaylarde 1,

Brett A. Neilan 2

1MIRCEN/Soils Dept., Federal University of Rio Grande do Sul, Porto Alegre,

Brazil, e-mail [email protected]; 2University of New South Wales, Sydney,

Australia.

Keywords: Cyanobacteria, PCR, dendrogram, biodeterioration, historic buildings

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Abstract

There are few modern analyses of the cyanobacterial communities in biofilms on

external surfaces of buildings. As the classification of cyanobacteria is rapidly

changing, we aimed to identify them on historic buildings in Brazil using both

traditional and molecular techniques. In mature biofilms, cyanobacteria of sub-sections I

and II were generally the major biomass; occasionally filamentous genera

(Scytonemataceae, Microchaetaceae and Rivularaceae) were dominant. Using culture

techniques, mainly filamentous organisms of sub-sections III and IV were isolated. PCR

products from morphologically identified organisms using cyanobacteria-specific 16S

rDNA primers were sequenced. Homologies with deposited sequences were generally

low. Phylogenetic analysis showed that the positions of many of the isolates in the

dendrogram were deeply-rooted. The results show that cyanobacteria on external walls

of historic buildings in Southern Brazil are considerably different from the majority of

those whose DNA sequences that have been deposited in data banks, which are mainly

from aquatic cyanobacteria.



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Introduction

Microbial biofilms on the external walls of buildings cause aesthetic deterioration and

degradation of the structure through production of acidic/alkaline conditions, retention

of humidity and differential heat absorption by coloured surface deposits [17]. When the

buildings are of architectural or historical importance, the resultant losses are not merely

economic, but involve the cultural heritage of a people.

Microrganisms found on external walls are algae, fungi, bacteria and actinomycetes,

myxomycetes and protozoa [5]. Of these, cyanobacteria and fungi usually consitute the

major biomass and can cause degradation of stone by the production of aggressive acid

or alkaline metabolites and surfactants, as well as by physical penetration of the cells

into the substrate [8].

Previous work has indicated that cyanobacteria constitute the major biomass on external

surfaces of ancient stone structures [7, 16]. Cyanobacteria are Gram-negative

photosynthetic prokaryotes that occur in both filamentous and coccoid forms. Some are

capable of fixing nitrogen. As a group, they are particularly resistant to desiccation and

high levels of UV-light [2, 3], giving them a distinct advantage over many other

organisms on exposed surfaces. The resistance to UV is generally associated with the

production of protective pigments, which adds to the deteriorative characteristics of the

biofilm. Cyanobacteria can also be found growing endolithically in stone buildings (See

Gaylarde & Gaylarde, these proceedings) and this leads to degradation of the structure

from within.

We report here the results of investigations on the presence of cyanobacteria, as well as

other photosynthetic microorganisms, on historic buildings in Brazil and try to relate

their presence to the biodeterioration process.



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Materials and Methods

Samples were taken from the external surfaces of the buildings using the non-

destructive adhesive tape sampling method of Gaylarde & Gaylarde [4]. Where obvious

degradation was present, small samples of the material could also be collected for

detection of endoliths. Samples were placed on plates of Modified Knops Medium

(MKM, [4]). They were examined after a few hours of rehydration on this medium,

using a binocular microscope and the lower power objectives of an optical microscope.

This allowed the identification of the major biomass in the biofilms. Plates were then

incubated at 25C with constant illumination for up to 8 weeks. Cyanobacteria were

isolated by micromanipulation and repeated subculture on both solid and liquid MKM.

Cyanobacteria in rehydrated biofilms and in culture were identified by traditional

morphological methods [1, 12]. Cyanobacteria were also identified by molecular

techniques [6]. Single colonies from solid medium, or DNA extracted from isolates,

were subjected to 16S rDNA PCR using the universal forward primer, 27F1, and the

cyanobacteria-specific reverse primer, 408R, [14, 15] and the PCR products sequenced

by the University of New South Wales Genomic Analysis Facility. The sequences were

submitted to the BLAST facility (www.ncbi.nlm.nih.gov/BLAST) and nearest matches

conforming to the morphological appearance of the cells recorded. Dendrograms were

constructed for the isolate sequences, along with sequences from the BLAST databases,

using the CLUSTAL-X programme [10] and bootstrap with 1000 comparisons.



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Results and Discussion

The cyanobacteria detected in the biofilms are shown in Table 1. The major biomass on

the external surfaces was almost invariably cyanobacteria of subsections I and II, that is,

the coccoid and colonial types. Subsection I cells were also found growing

endolithically. This confirms previous reports [7 ,16]. Many of these genera have been

shown to be capable of boring into natural stone, the Pleurocapsa -group [13],

Synechocystis and Gloeocapsa , as have the filamentous genera, Stigonema , Schizothrix

[11], Scytonema [9] and Mastigocladus [1]. All these genera, apart from Stigonema ,

were detected in our samples, indicating the deteriorative nature of the biofilms.

The dendrogram constructed from the sequences is shown in Fig. 1. Twenty three PCR

products were sequenced and these are indicated in the Fig. 1 as numbers, rather than by

their presumptive identifications, to allow easy comparison with the sequences obtained

from the BLAST databases.

The dendrograms show that the cyanobacteria detected in this study, although they often

conform reasonably well in their morphology to the nearest neighbour group, are

frequently a considerable distance from it. For example, sequences 28 and 32,

morphologically identical and typical of Plectonema , have almost identical sequences to

one another, but are very distant from their nearest sister group, which contains

Leptolyngbya , Phormidium and Plectonema . This shows that morphologically diverse

organisms like these 3 genera may cluster in the dendrogram. On the other hand,

sequences 8 and 38, although both identified microscopically as Plectomena , were

morphologically distinct. The dendrogram placed them close together, with their nearest

neighbour being Phormidium murrayii .



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The results suggest that molecular techniques for the identification of cyanobacteria

require considerable development before they can be considered as a mature tool. In

particular, more sequences of non-aquatic organisms must be deposited. Although our

organisms broadly fit the dendrogram, a number of inconsistencies remain.

Acknowledgements

We wish to thank the Brazilian agency CNPq for funding for materials and a

postgraduate grant to CAC. BAN thanks the Australian Research Council for financial

support.

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Fig. 1 Dendrogram constructed with incomplete 16S rDNA cyanobacterial sequences

(positions 106 to 340 on the E. coli notation). Morphological identity and code number:

Chlorogloeopsis 48, 50; Chroococcidiopsis 11; Leptolyngbya 34, 40, 51, 52; Lyngbya

14; Nostoc 10, 13, 19, 49; Plectonema 07, 08, 28, 32, 38; Scytonema 16, 25, 26;

Scytonematopsis 44; Subsection II 46; Tolypothrix 23, 24Sequences nos. 11 and 40 and of Microcoleus sociatus (M. soc) are short.



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