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Brett A. Neilan 2
1
MIRCEN/Soils Dept., Federal University of Rio Grande do Sul, Porto Alegre,
Australia.
Abstract
techniques, mainly filamentous organisms of sub-sections III and IV were isolated. PCR
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
Introduction
Microbial biofilms on the external walls of buildings cause aesthetic deterioration and
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
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
Previous work has indicated that cyanobacteria constitute the major biomass on external
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
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.
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
(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
isolated by micromanipulation and repeated subculture on both solid and liquid MKM.
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
constructed for the isolate sequences, along with sequences from the BLAST databases,
using the CLUSTAL-X programme [10] and bootstrap with 1000 comparisons.
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
[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
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,
one another, but are very distant from their nearest sister group, which contains
organisms like these 3 genera may cluster in the dendrogram. On the other hand,
morphologically distinct. The dendrogram placed them close together, with their nearest
The results suggest that molecular techniques for the identification of cyanobacteria
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.
References
8. Gaylarde C.C., Morton L.H.G. (1999) Deteriogenic biofilms on buildings and their
control: a Review. Biofouling 14: 59-74.
9. Golubic, S., Seong-Joo, L., Browne, K.M. (2000) Cyanobacteria: architects of
sedimentary structures. In: Riding, R., Awrami, S.M. (eds.), Microbial Sediments,
Springer-Verlag, Berlin, pp. 57-67.
10. Higgins, D.G., Bleasby, A.J. and Fuchs, R. (1992) CLUSTAL V: improved
software for multiple sequence alignment. Com. Appl. Biosc. 8: 189-191.
11. Hoffman, L. (1989) Algae of terrestrial habitats. Botanical Reviews 55: 77-105.
12. Holt, J.G., Krieg, N.R., Sneath, P.H., Staley, J.T., Williams, S.T. (1994)
Bergey’s Manual of Determinative Bacteriology, William & Wilkins, Baltimore.
13. Mao-Che, L., Le-Campion-Alsumard, T., Boury-Esnault, N., Payri, C.,
Golubic, S.,Bezac, C. (1996) Biodegradation of shells of the black pearl oyster,
Pinctada margaritifera var. cumingii, by microborers and sponges of French
Polynesia. Marine Biology 126: 509-519.
14. Neilan, B.A., Jacobs, D., Del Dot, T., Blackall, L.L., Hawkins, P.R., Cox, P.T.,
Goodman, A.E. (1997) rRNA sequences and evolutionary relationships among toxic
and nontoxic cyanobacteria of the genus Microcystis. International Journal of
Systematic Bacteriology 47: 693-697.
15. Neilan, B.A., Burns, B.P., Relman D., Lowe, D.R. (2002) Molecular identification
of cyanobacteria associated with stromatolites from distinct geographical locations.
Astrobiology 2: 271-280.
16. Ortega-Morales O., Guezennec J., Hernandez-Duque G., Gaylarde C.C.,
Gaylarde P.M. (2000) Phototrophic biofilms on ancient Mayan buildings in
Yucatan, Mexico. Current Microbiology 40: 81-85.
17. Warscheid, T., Oelting, M., Krumbein, W.E. (1991) Physico-chemical aspects of
deterioration process on rocks with special regard to organic pollution. International
Biodeterioration 28: 37-48.