Fottea 11(1): 187-201, 2011 | DOI: 10.5507/fot.2011.018

The biodiversity of subaerophytic phototrophic biofilms from Maltese hypogea.

Gabrielle Zammit*,1, Daniela Billi2, Elliot Shubert3, Jan Ka¹tovský4, Patrizia Albertano2
1 Department of Physiology & Biochemistry, Faculty of Medicine & Surgery, University of Malta, Msida, Malta
2 Department of Biology, Faculty of Science, University of Rome 'Tor Vergata', Rome, Italy
3 Department of Botany, Natural History Museum, London, UK
4 Department of Botany, Faculty of Science, University of South Bohemia, Èeské Budìjovice, Czech Republic

The present study focuses on a description of the biodiversity of subaerial phototrophic biofilms occurring on archaeological surfaces in Maltese hypogean environments, namely St Paul's, St Agatha's and Abbatija tad-Dejr Catacombs, all situated in Rabat and the ancient Ħal Saflieni Hypogeum at Paola, Malta. Direct observation of the biofilms, carried out using light (LM), epifluorescent and confocal laser scanning microscopy (CLSM), allowed the description of the major cyanobacterial and microalgal taxa, and also the associated heterotrophic microorganisms, mainly actinobacteria. Some biofilm microorganisms were able to grow in culture and this allowed the isolation of cyanobacterial, microalgal and chemoorganotrophic bacterial strains. Thylakoid arrangement and cell division were examined using transmission electron microscopy (TEM). The cytomorphology of isolated microorganisms was described. The undisputed phototrophic protagonists in these subaerial biofilms of Maltese hypogean environments were the non-heterocytous (Oscillatorialean) cyanobacteria. In order to increase the limited data available for Leptolyngbya spp. from aerophytic epilithic biofilms in catacombs, the 16S rRNA genes of isolated Leptolyngbya strains were sequenced and compared with those obtained for related strains. Phylogenetic trees of cyanobacterial 16S rRNA sequences were constructed using parsimony and Bayesian analyses. Microorganisms forming biofilms in Maltese hypogea were found to be similar, both cytomorphologically and genetically, to those colonising lithic surfaces of caves and catacombs in other Mediterranean countries.

Keywords: catacombs, cyanobacteria, cyanoprokaryotes, hypogea, Leptolyngbya, microalgae, Oscillatoriales, phototrophic biofilms, Stigonematales, subaerophytic

Published: March 1, 2011Show citation

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Zammit, G., Billi, D., Shubert, E., Ka¹tovský, J., & Albertano, P. (2011). The biodiversity of subaerophytic phototrophic biofilms from Maltese hypogea. Fottea11(1), 187-201. doi: 10.5507/fot.2011.018
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References

  1. Aboal, M., Asencio, A. D. & López-Jiménez, E. (2003): Morphological, ultrastructural and ecological study of Asterocapsa divina Komárek (Chroococcaceae, Cyanobacteria) from a cave of Southeastern Spain. - Algological Studies 109: 57-65. Go to original source...
  2. Allen, M. M. (1968) Simple conditions for growth of unicellular blue-green algae on plates. - Journal of Phycology 4: 1-3. Go to original source...
  3. Asencio, A. D. & Aboal, M. (2000): A contribution to knowledge of chasmoendolithic algae in cave- like environments. - Algological Studies 98: 133-151. Go to original source...
  4. Albertano, P. & Bellezza, S (2001): Cytochemistry of cyanobacterial exopolymers in biofilms from Roman hypogea. - Nova Hedwigia 123: 501- 518.
  5. Albertano, P. & Kovacik, L. (1994): Is the genus Leptolyngbya an homogeneous taxon? - Algological Studies 75: 37-51. Go to original source...
  6. Albertano, P. & Urzì, C. (1999): Structural interactions among epilithic cyanobacteria and heterotrophic microorganisms in Roman hypogea. - Microbial Ecology 38: 244-252. Go to original source...
  7. Albertano, P., Luongo, L. & Grilli Caiola, M. (1989): Ultrastructural investigations on algae deteriorating Roman frescoes. - In: Baer, N., Sabbioni, C. & Sors, A.I. (eds): Science, Technology and European Cultural Heritage. - pp. 501-504, Butterworth-Heinemann - Oxford. Go to original source...
  8. Albertano, P., Kováèik, L., Marvan, P. & Grilli Caiola, M. (1995): A terrestrial epilithic diatom from Roman Catacombs. - In: Marino, D. &. Montresor, M. (eds): Proceedings of the Thirteenth Inter. Diatom Symp. - pp. 11-21, Biopress Limited - Bristol.
  9. Albertano, P., Barsanti, L., Passarelli, V. & Gualtieri, P. (2000): A complex photoreceptive structure in the cyanobacterium Leptolyngbya sp. - Micron 31: 27-34. Go to original source...
  10. Altschul, S.F., Gish, W., Miller, W., Myers, E.W. & Lipman D.J. (1990): Basic local alignment search tool. - Journal of Molecular Biology 215: 403-410. Go to original source...
  11. Anagnostidis, K. & Komárek, J. (1985a): Modern approach to the classification system of Cyanophytes 1 - Introduction. - Algological Studies 38-39: 291-302.
  12. Anagnostidis, K. & Komárek, J. (1985b): Modern approach to the classification system of Cyanophytes 3 - Oscillatoriales. - Algological Studies 50-53: 327-472.
  13. Berard, A., Dorigo, U., Humbert, J.F. & Martin- Laurent, F. (2005): Microalgae community structure analysis based on 18S rDNA amplification from DNA extracted directly from soil as a potential soil bioindicator. - Agronomie 25: 1-7. Go to original source...
  14. Billi, D. & Potts, M. (2002): Mini-review Life and death of dried prokaryotes. - Research in Microbiology 153: 7-12. Go to original source...
  15. Bischoff, H.W. & Bold, H.C. (1963): Phycological Studies IV. Some algae from Enchanted Rock and related algal species. - University of Texas Publications 6318: 1-95.
  16. Boison, G., Mergel, A., Jolkver, H. & Bothe, H. (2004): Bacterial Life and Dinitrogen Fixation at a Gypsum Rock. - Applied and Environmental Microbiology 70: 7070-7077. Go to original source...
  17. Boyer, S.L., Flechtner, V.R. & Johansen, J.R. (2001): Is the 16S-23S rRNA Internal Transcribed Spacer region a good tool for use in molecular systematics and population genetics? A case study in cyanobacteria. - Molecular Biology and Evolution 18: 1057-69. Go to original source...
  18. Bruno, L., Billi, D. & Albertano, P. (2006): Genetic Characterization of Epilithic Cyanobacteria and Their Associated Bacteria. - Geomicrobiology Journal, 23:293-299. Go to original source...
  19. Bruno, L., Billi, D., Bellezza S. & Albertano, P. (2009): Cytomorphological and Genetic Characterization of Troglobitic Leptolyngbya Strains Isolated from Roman Hypogea. - Applied and Environmental Microbiology 75: 608-617. Go to original source...
  20. Diez, B., Pedros-Alio, C., Marsh, T.L. & Massana, R. (2001): Application of Denaturing Gradient Gel Electrophoresis (DGGE) To Study the Diversity of Marine Picoeukaryotic Assemblages and Comparison of DGGE with Other Molecular Techniques. - Environmental Microbiology 67: 2942-2951. Go to original source...
  21. Dodds, W.K., Gudder, D.A. & Mollenhauer, D. (1995): The ecology of Nostoc. - Journal of Phycology 31: 2 -18. Go to original source...
  22. Ettl, H. (1978) Xantophyceae 1. Teil. - In: Ettl, H., Gerloff, J. & Heynig, H. (eds): Süßwasserflora von Mitteleuropa, Vol. 3. - 530 pp., Gustav Fischer Verlag, Jena.
  23. Ettl, H. (1983): Chlorophyta I (Phytomonadina). - In: Ettl, H., Gerloff, J., Heynig, H. & Mollenhauer, D. (eds): Süßwasserflora von Mitteleuropa, Vol. 9. - 807 pp., Gustav Fischer Verlag, Jena.
  24. Ettl, H. & Gärtner, G. (1988): Chlorophyta II (Tetrasporales, Chlorococcales, Gloeodendrales). - In: Ettl, H., Gerloff, J. & Heynig, H. (eds): Süßwasserflora von Mitteleuropa, Vol. 10. - 436 pp., Gustav Fischer Verlag, Jena.
  25. Ettl, H. & Gartner, G. (1995): Syllabus der Boden-, Luft- und Flechtenalgen. - 721 pp., G. Fischer Verl., Stuttgart.
  26. Evans, R.D. & Johansen, J.R. (1999): Microbiotic crusts and ecosystem processes. - Critical Reviews in Plant Sciences 18:183-225. Go to original source...
  27. Friedl, T. & O'Kelly, C.J. (2002): Phylogenetic relationship of green algae assigned to the genus Planophila (Chlorophyta): evidence from 18S rDNA sequence data and ultrastructure. - European Journal of Phycology 37: 373-84. Go to original source...
  28. Friedl, T., Kostikov, I., Müller, J., Beck, A., Zufall-Roth, E. & Hoffmann, L. (2007): An investigation of Pseudococcomyxa (Chlorophyta, Trebouxiophyceae): morphology, rDNA sequencing and AFLP fingerprinting to define species. - Proc. Abstr. Int. Conf. Taxonomy and Biology of Green Algae V., Smolenice.
  29. Garcia-Pichel, F., Nübel U. & Muyzer, G. (1998): The phylogeny of unicellular, extremely halotolerant cyanobacteria. - Archives of Microbiology 169: 469-482. Go to original source...
  30. Geitler, L. (1932): Cyanophyceae. - In: Rabenhorst's Kryptogamenflora 14/2. - 1196 pp., Akad. Verlagsges, Leipzig.
  31. Gugger, M.F. & Hoffmann, L. (2004) Polyphyly of true branching cyanobacteria (Stigonematales). - International Journal of Systematic and Evolutionary Microbiology 54: 349-357. Go to original source...
  32. Hall, T.A. (1999): BioEdit: a user-friendly biological sequence alignment editor and analysis program for Windows 95/98/NT. - Nucleic Acids Symposium Series 41: 95-98.
  33. Hasegawa, M., Kishino, H., & Yano, T. (1985): Dating of the human-ape splitting by a molecular clock of mitochondrial DNA. - Journal of Molecular Evolution 22:160-174. Go to original source...
  34. Helm, R.F., Huang, Z., Edwards, D., Leeson, H., Peery, W. & Potts, M. (2000): Structural Characterization of the Released Polysaccharide of Desiccation-Tolerant Nostoc commune DRH-1. - Journal of Bacteriology 182: 974- 982. Go to original source...
  35. Hernández-Mariné, M., Asencio-Martínez, A., Canals, A., Ariño, X., Aboal, M. & Hoffmann, L. (1999): Discovery of populations of the lime incrusting genus Loriella (Stigonematales) in Spanish caves. - Algological Studies 94: 121- 138. Go to original source...
  36. Huss, V.A.R., Frank, C., Hartmann, E.C., Hirmer, M., Kloboucek, A., Seidel, B.M., Wenzeler, P. & Kessler, E. (1999): Biochemical taxonomy and molecular phylogeny of the genus Chlorella sensu lato (Chlorophyta). - Journal of Phycology 35: 587-598. Go to original source...
  37. Ionescu, D., Oren, A., Hindiyeh, Y. & Malkawi, H.I. (2007): The thermophilic cyanobacteria of the Zerka Ma'in thermal springs in Jordan. - In: Seckbach, J. (ed.): Algae and cyanobacteria in extreme environments. - pp. 413-424, Springer, Dordrecht, The Netherlands. Go to original source...
  38. John, D.M. (1988): Algal growths on buildings: a general review and methods of treatment. - Biodeterioration Abstracts 2, 81-102.
  39. John, D.M., Whitton, B.A. & Brook, A.J. (2002): The Freshwater Algal Flora of the British Isles. - 702 pp., Cambridge University Press, UK.
  40. King, A.D., Hocking A.D. & Pitt, J.I. (1979): Dichloran-rose bengal medium for enumeration and isolation of molds from foods. - Applied and Evnironmental Microbiology 37: 959- 964. Go to original source...
  41. Komárek, J. (2002): Problems in cyanobacterial taxonomy; implication for most common toxin producing species. - In: Melchiorre, S., Viaggiu, E. & Bruno, M. (eds): Rapporti Istituto Superiore di Sanitá. - pp. 6-43, Roma.
  42. Komárek, J. (2003): Planktic oscillatorialean cyanoprokaryotes (short review according to combined phenotype and molecular aspects). - Hydrobiologia 502: 367-382. Go to original source...
  43. Komárek J. (2007): Phenotype diversity of the cyanobacterial genus Leptolyngbya in the maritime Antarctic. - Polish polar Research 28: 211-231.
  44. Komárek, J. & Fott, B. (1983): Chlorococcales. - In: Huber-Pestalozzi, G. (ed.): Das Phytoplankton des Süßwassers, Vol. 7. - 1043 pp., Schweizerbart, Stuttgart.
  45. Komárek, J. & Anagnostidis, K. (1998): Cyanoprokaryota 1 (Chroococcales). - In: Ettl H., Gärtner, G. & Heynig, H. & Mollenhauer D. (eds): Süßwasserflora von Mitteleuropa, 19/1.- 548 pp., Gustav Fischer, Jena.
  46. Komárek, J. & Anagnostidis, K. (2005): Cyanoprokaryota 2 (Oscillatoriales). - In: Büdel, B., Krienitz, L., Gärtner, G. & Schagerl, M. (eds): Süßwasserflora von Mitteleuropa, 19/2. - 759 pp., Elsevier/ Spektrum, Heidelberg.
  47. Kostikov, I., Darienko, T., Luke¹ová, A. & Hoffmann, L. (2002): Revision of the classification system of Radiococcaceae Fott ex Komárek (except the subfamily Dictyochlorelloideae) (Chlorophyta). - Algological Studies 104: 23-58. Go to original source...
  48. Krammer, K. & Lange-Bertalot, H. (1986) Bacillariophyceae 1. Teil. - In: Ettl, H., Gerl off, J., Heynig, H. & Mollenhauer, D. (eds): Süßwasserflora von Mitteleuropa, 2/1. - 875 pp., Gustav Fischer Verlag, Jena.
  49. Krammer, K. & Lange-Bertalot, H. (1988): Bacillariophyceae 2. Teil. - In: Ettl, H., Gerloff, J., Heynig, H. & Mollenhauer, D. (eds): Süßwasserflora von Mitteleuropa, 2/2. - 596 pp., Gustav Fischer Verlag, Jena.
  50. Krammer, K. & Lange-Bertalot, H. (1991a): Bacillariophyceae 3. Teil. - In: Ettl, H., Gerloff, J., Heynig, H. & Mollenhauer, D. (eds): Süßwasserflora von Mitteleuropa, 2/3. - 576 pp., Gustav Fischer Verlag, Stuttgart.
  51. Krammer, K. & Lange-Bertalot, H. (1991b): Bacillariophyceae 4. Teil. - In: Ettl, H., Gärtner G., Gerloff, J., Heynig, H. & Mollenhauer, D. (eds): Süßwasserflora von Mitteleuropa, 2/4. - 436 pp., Gustav Fischer Verlag, Stuttgart.
  52. Krienitz, L., Hegewald, E., Hepperle, D. & Wolf, A. (2003): The systematics of coccoid green algae: 18S rRNA gene sequence data versus morphology. - Biologia 58: 437-446.
  53. Lamprinou, V. Pantazidou, A., Papadogiannaki G., Radea, C. & Economou-Amill, A. (2009) Cyanobacteria and associated invertebrates in Leontari Cave, Attica (Greece). - Fottea 9: 155-164. Go to original source...
  54. Lerch, K & Ettlinger, L. (1972): Purification and characterization of a tyrosinase from Streptomyces glaucescens. - European Journal of Biochemistry 31: 427-437. Go to original source...
  55. Lewis, L.A. & McCourt, R.M. (2004): Green algae and the origin of land plants. - American Journal of Botany 91: 1535-1556. Go to original source...
  56. Lopez-Bautista, J., Rindi, F. & Casamatta, D. (2007): The systematics of subaerial algae. - In: Seckbach J. (ed.): Algae and Cyanobacteria in Extreme Environments. - pp. 599-617, Springer, The Netherlands. Go to original source...
  57. Meeks, J.C., Elhai, J., Thiel, T., Potts, M., Larimer, F., Lamerdin, J., Predki, P. & Atlas, R. (2001): An overview of the genome of Nostoc punctiforme, a multicellular, symbiotic cyanobacterium. - Photosynthesis Research 70: 85-106. Go to original source...
  58. Migula, W. (1907): Algen. - In: Kryptogamen-Flora von Deutschland, Deutsch-Österreich und der Schweiz 2, Teil 1, Zezschwitz Verlag, Gera.
  59. Neustupa, J. & Nemjová, K. (2008): Quantitative assessment of morphological variation of six Pseudococcomyxa strains by geometric morphometric descriptors. - Fottea 8: 111-116. Go to original source...
  60. Paerl, H.W. (1992): Epi- and endobiotic interactions of cyanobacteria. In: Reisser, W. (ed) Algae and Symbioses: Plants, Animals, Fungi, Viruses, Interactions Explored. - pp. 537-565, Biopress, Bristol.
  61. Pointing, S.B., Warren-Rhodes, K.A., Lacap, D.C., Rhodes, K.L. & McKay, C.P. (2007): Hypolithic community shifts occur as a result of liquid water availability along environmental gradients in China's hot and cold hyperarid deserts. - Environmental Microbiology 9: 414-424. Go to original source...
  62. Prescott, G.W., Croasdale, H.T. & Vinyard, W.C. (1972): A synopsis of North American desmids. Part 1, Mesotaeniaceae: Saccodermae. - In: Rogerson, C.T. (ed.): North American Flora, II/6. - 84 pp., The New York Botanical Garden.
  63. Printz, H. (1939): Vorarbeiten zu einer Monographie der Trentepohliaceen. - Nytt. Mag. Naturvbidensk. 80: 137-210.
  64. Printz, H. (1964): Die Chaetophoralen der Binnengewässer. - Hydrobiologia 24: 1-376. Go to original source...
  65. Reynolds, C.S. (1963): The use of lead citrate at high pH as an electron-opaque stain in electron microscopy. - Journal of Cellular Biology 17: 208-212 Go to original source...
  66. Rindi, F., Sherwood, A.R. & Guiry, M.D (2002): Diversity, Life History and ecology of Trentepohlia and Printzina (Trentepohliales, Chlorophyta) in urban habitats in western Ireland. - Journal of Phycology 38: 39- 54. Go to original source...
  67. Rindi, F., Sherwood, A.R. & Guiry, M.D. (2005): Taxonomy and distribution of Trentepohlia and Printzina (Trentepohliales, Chlorophyta) in the Hawaiian Islands. - Phycologia 44: 270- 284. Go to original source...
  68. Rodríguez, F., Oliver, J.L., Marín, A., Medina, J.R. (1990): The general stochastic model of nucleotide substitution.- Journal of Theoretical Biology 142: 485-501. Go to original source...
  69. Roeselers, G., Norris, T.B., Castenholz, R.W., Rysgaard, S., Glud, R.N., Kuhl, M. & Muyzer, G. (2007): Diversity of phototrophic bacteria in microbial mats from Arctic hot springs (Greenland). - Environmental Microbiology 9:26-38. Go to original source...
  70. Roland, J.C., Lembi, C.A. & Morre, D.G. (1972): Phosphotungstic acid chromic as a selective electron dense stain for plasma membrane of plant cells. - Stain Technology 45: 195-200. Go to original source...
  71. Roldan, M. & Hernandez-Marine, M. (2009): Exploring the secrets of the three-dimensional architecture of phototrophic biofilms in caves. - International Journal of Speleology 38: 41-53. Go to original source...
  72. Ronquist, F. & Huelsenbeck J.P. (2003) MrBayes 3: Bayesian phylogenetic inference under mixed models. - Bioinformatics 19: 1572-1574. Go to original source...
  73. Saarela, M., Alakomi, H.M., Suihko, M.L., Maunuksela, L., Raaska, L. & Mattila- Sandholm T. (2004): Heterotrophic microorganisms in air and biofilm samples from Roman catacombs, with special emphasis on actinobacteria and fungi. - International Biodeterioration and Biodegradation 54: 27 - 37. Go to original source...
  74. Stomp, M., Huisman, J., de Jongh, F., Veraart, A.J., Gerla, D. & Rijkeboer, M. (2004): Adaptive divergence in pigment composition promotes phytoplankton biodiversity. - Nature 432: 104- 107. Go to original source...
  75. Swofford, D.L. (2002): PAUP*. Phylogenetic Analysis Using Parsimony (and other methods). Version 4. Sinauer Associates, Sunderland, Massachusetts.
  76. Taton, A., Grubisic, S. & Wilmotte A. (2002): Cyanobacterial diversity in Antarctic microbial mats.- In: Solheim, B. (ed), Cyanobacteria and Nitrogen Fixation in Extreme Environments. - p. 22, Eur. Sci. Found. CYANOFIX, Longyearbyen.
  77. Thiéry J.P. (1967): Mise en évidence des polysaccharides sur coupes fines en microscopie électronique. - Journal of Microscopy 6: 987-1018.
  78. Urzi, C., & De Leo, F. (2001): Sampling with adhesive tape strips: an easy and rapid method to monitor microbial colonization on monument surfaces. - Journal of Microbiological Methods 44: 1-11. Go to original source...
  79. Uyenco, F.R. (1965): Studies on some lichenized Trentepohlia associated in lichen thalli with Coenogonium. - Trans. Am. Microsc. Soc. 84:1-14. Go to original source...
  80. Watanabe, M.M. (1999): Network approach to make biodiversity information accesible worldwide. - In: Species 2000, Abstracts of the International Workshop for Studies on Biodiversity. - p. 13, Tsukuba.
  81. Wehr, J.D. & Sheath, R.G. (eds) (2003): Freshwater Algae of North America: Classification and Ecology. - 918 pp., Academic Press, New York.
  82. Wilmotte, A. & Golubiæ, S. (1991): Morphological and genetic criteria in the taxonomy of Cyanophyta/ Cyanobacteria. - Algological Studies 64: 1-24.
  83. Zammit, G., De Leo, F., Albertano, P. & Urzì, C. (2008a): A preliminary comparative study of microbial communities colonizing ochre- decorated chambers at the Ħal Saflieni Hypogeum at Paola, Malta. - In: Lukaszewicz, J.W. & Niemcewicz P. (eds): Prodeedings of the 11th International Congress on Stone Deterioration. - pp. 555-562, Nicolaus Copernicus University Press, Torun, Poland.
  84. Zammit, G., Psaila, R. & Albertano, P. (2008b): An investigation into biodeterioration caused by microbial communities colonising artworks in Maltese Palaeo-Christian Catacombs. - In: Notea A. & Shoef Y. (eds): Art 2008 Conference Proceedings, -9 th International Conference on Non-destructive testing, microanalysis and preservation in the conservation of cultural and environmental heritage. - pp. 1-10, ISAS International Seminars Ltd., Jerusalem, Israel.
  85. Zammit, G., De Leo, F., Urzì, C., & Albertano, P. (2009): A non-invasive approach to the polyphasic study of biodeteriogenic biofilms at St Agatha Crypt and Catacombs at Rabat, Malta. - In: Meli, G. (ed.): Science and Cultural Heritage in the Mediterranean Area- Prodeedings of the third International Conference on Materials and the Signs of History. - pp. 323-327, Department of Environmental and Cultural Heritage, Palermo, Italy.
  86. Zammit, G., Ka¹tovský, J. & Albertano, P. (2010): A first cytomorphological and molecular characterisation of a new Stigonematalean cyanobacterial morphotype isolated from Maltese catacombs. - Algological Studies 135:1-14. Go to original source...