Análisis de comunidades microbianas presentes en la cueva de Doña Trinidad (Ardales, Málaga) utilizando cultivos y métodos moleculares basados en ADN y ARN

Abstract

Microorganisms constitute an extremely heterogeneous group of organisms on our planet and microbial communities generally show an extraordinary potential of metabolic capacities (Brock and Madigan, 2000). However, only a small number of microorganisms are known and have been properly described, since only an extremely low portion of microorganisms can be cultivated through traditional microbiological techniques (Curtis et al., 2002). Thanks to the development of new molecular methods it is now possible to identify previously undetected microorganisms considered uncultivable in the laboratory. Historical Cultural Heritage sites often offer an adequate scenario for the growth of microorganisms, which can thus cause damage, sometimes irreversible, to the artistic representations. In particular, underground environments such as caves and catacombs, in which the temperature is relatively constant throughout the year and the humidity generally high, offer a favourable habitat for the growth of numerous microorganisms (Agarossi, 1992). The present study is part of a multidisciplinary project focused on the conservation of the works of art of Ardales’s Cave and represents the first microbiological study in this cave. The Cave of Doña Trinidad is located in the town of Ardales, Malaga (Spain). The cave has a length of 1.5 km with several halls and galleries. The interior contains labyrinths of columns, underground lakes, and beautiful formations of stalactites and stalagmites. The cave contains paintings and engravings dating back to about 20,000 years, belonging to the Upper Paleolithic. The samples analyzed were collected from the cave’s soil and from the main artistic representations (engraving of a fish, serpents, red pigments and black tracks from the cave). In particular, the microbial composition of white colonizations present in the cave was studied. In recent years, those colonizations have spread abundantly throughout the cave. Results showed a dominance of Proteobacteria in the studied samples except in white colonizations. Among the metabolically active communities of the cave, Gammaproteobacteria and Alphaproteobacteria were the most abundant in soil samples, engravings, red pigments and black tracks. Firmicutes, Bacteroidetes, Deinococci, Chloroflexi, Planctomycetes and Nitrospirae were present at lower proportions (as revealed by DNA-based molecular methods) while Acidobacteria and Sulphate reducing bacteria showed high diversity. The analysis of the bacterial communities of the white colonizations showed significant differences with respect to the other studied samples. The Actinobacteria phylum and in particular the genus Pseudonocardia, was the most metabolically active component of these colonizations as a result of DNA and RNA libraries. Molecular fingerprints of the metabolically active microbial communities also showed a major, almost exclusive, band corresponding to sequences belonging to the genus Pseudonocardia showing that Pseudonocardia played a key role in white colonizations’ expansion in Ardales’s Cave (Stomeo et. al., 2007). Considering these results and the important role played by Pseudonocardia in the cave, we tried to cultivate this bacterium in the laboratory. We obtained three strains of this genus and they were used to carry out enzymatic and metabolic studies. To investigate

the metabolism of Pseudonocardia and the best conditions for its growth we carried out colonization’s experiments (in the laboratory) with the isolated strains using cave’s soil as substrate, with and without the addition of different nutrients (carbon, nitrogen and phosphate), at 16 ºC (cave’s temperature) and at 28ºC. The results indicated that Pseudonocardia grows better in the presence of glucose and phosphate with a significant statistical value (P<0.05) with respect to others nutrients showing that this bacterium’s proliferation is maintained by the cave’s temperature and its development is favoured by organic nutrients and phosphate’s sources which might being limiting Pseudonocardia’s growth in situ. Rarefaction curves of bacterial phyla detected in this study through molecular methods indicated that, even if a higher number of clones were analyzed it would not have been possible to detect additional bacterial phyla; the OTUs curves suggested the presence of an elevated bacterial diversity in the cave under study. In the case of isolated strains, OTUs rarefaction curves indicated that, culture techniques only allowed the detection of a reduced fraction of microorganisms in the cave because an asymptote was reached well below the curve obtained by culture-independent methods based on DNA, cloning and sequencing. With regard to the fungal diversity in Ardales’ Cave, the results indicated the presence of Ascomycota and Basidiomycota. The most frequent orders in the cave were Hypocreales and Eurotiales (Ascomycota) and Tremellales among Basidiomycota. Fusarium (Hypocreales) was the unique fungal genus detected through DNA libraries. This suggested its high presence in the cave and that its spread by spores’ production could seriously deteriorate Ardales’ Cave paintings and engravings. As for Pseudonocardia, this fungus was isolated in the laboratory and colonization assays were performed. The results obtained indicated that this fungus’s growth is favored at 28 °C and in the presence of nitrogen sources (p <0.05). Rarefaction curves showed that during this study, it was possible to detect an extremely significant portion of fungal species present in the cave of Doña Trinidad.

Results obtained during this study have led to the following general conclusions:

- Proteobacteria represented the most characteristic phylum in the analyzed samples with the exception of white colonizations. Gammaproteobacteria and Alphaproteobacteria represented the main classes showing metabolic activity in the studied samples.

- Other bacterial phyla revealed during this study are: Acidobacteria, Sulphate reducing Bacteria, Chloroflexi, Planctomycetes, Deinococci, Nitrospirae and Bacteroidetes. The role these phyla play in the cave is unknown, since their metabolism and physiology are not yet well understood.

- White colonizations present in the cave were composed mainly by Actinobacteria, in particular by the genus Pseudonocardia.

- The presence of fungal species was detected in the Cave of Doña Trinidad. Among these, the orders Hypocreales, Eurotiales (Ascomycota) and Tremellales (Basidiomycota). Fusarium (Hypocreales) was the most abundant fungal genus detected in the cave during this study.

- Molecular methods based on DNA were useful to identify the bacterial and fungal members of the microbial communities in the studied cave. Molecular techniques based on RNA allowed the identification of those bacteria metabolically active in the analyzed

communities.

- The combination of standard culture techniques and molecular methods is essential for fungal classification.

- Through this study it was possible to determine the majority of bacterial and fungal components of the microbial communities of Doña Trinidad Cave. Among these, Pseudonocardia (Bacteria) and Fusarium (Fungi) represented the major risks for the cave’s conservation.

- The cultivation of bacteria and fungi allowed the study of the strains’ capability to grow under different conditions.

- In order to reduce the proliferation of Pseudonocardia throughout the cave, it would be necessary to limit the amount of organic material and phosphate in the cave.

- With regard to Fusarium, it would be necessary to maintain stable the temperature in the cave and limit nitrogen’s sources, in order to reduce the possibility of proliferation of this fungal species.