AEROBIOLOGÍAMecanismos de dispersión de los microorganismos en cuevas turísticas.

Resum

It is well known that caves are ecosystems with low nutrient content, stable environmental conditions, low temperatures, high humidity, and are colonized by a variety of microorganisms (Cunningham et al., 1995; Engel et al., 2004; Barton and Northup, 2007). The biotic communities dwelling in caves are not very diverse, contain simple structures and are sensitive to changes, such as the introduction of organic matter from the outside (Chelius et al., 2009). Spain has more than thirty thousand known caves. Many of them were declared as natural monuments and a few of them were declared World Heritage by UNESCO (www.cuevasturisticas.es). Caves are extremely susceptible to energetic disturbances induced by anthropogenic activity. Some of them include geological, biological and/or cultural features that should be preserved. These changes, and the contributions of organic matter from the outside, can cause a progressive alteration on the microenvironment that result in irreversible damages to a medium-term (Sanchez-Moral et al., 1999; Sanchez-Moral et al., 2000). Each visitor produces several changes in the microclimate of the cave due to the human metabolism. Aerobiology is defined as the study of the sources, dispersion, and effects of airborne biological materials, such as pollen, spores and microorganisms. Aerobiology in caves is still in its infancy. Few works have been published using different methods. At present, no information has been accomplished on the concentration of fungi in different caves, as well as on its limits of acceptance. These measurements permit to characterize the atmosphere of subterranean environments, particularly those containing rock-art paintings, in order to preserve these assets. AIMS This PhD thesis arises from the growing national and international interest on the aerobiology of caves as one of the most complex areas of microbiology. This study aimed to characterize microbial diversity (bacteria and fungi) in the air of several caves using the air samplers DUO SAS SUPER 360 and Coriolis ¿. The caves studied were Castañar de Ibor (Extremadura, Spain), Ardales (Andalucía, Spain), Altamira (Cantabria, Spain) and Lascaux (France). MATERIAL AND METHODS The cave of Castañar de Ibor (Extremadura, Spain) was opened to public visits in 2003. Since then a strict control of visits was designed. Visits were cancelled (on September, 2008) due to an accidental organic matter input (vomit of a visitor) and a subsequent fungal outbreak. Since 2009 the cave is only visited by scientists and staff people. The cave of Ardales (Andalucía, Spain) was opened to visitors in 1852, but the visits were discontinued in 1896. Actually, this cave is characterized by a very moderate to low visitors impact (1.000 visitors/ year). The cave of Altamira (Cantabria, Spain) was opened to public in 1917. Massive visits during the 60s and 70s (175,000 visitors) strongly altered the microclimatic conditions resulting in an increasing deterioration of the impressive Paleolithic paintings. The cave was closed to the public in 1977, but reopened in 1982. In 2002 the cave was closed again and nowadays the cave is still close to visitors. The cave of Lascaux (France) is a paradigm of the effects of massive visits on a rock-art cave. Briefly, after its discovery in 1940, the cave was seriously disturbed by several destructive interventions. In 1963, the cave was closed due to algal growth on the walls. In 2001, the ceiling, walls and sediments were colonized by the fungus Fusarium solani (Bastian et al., 2009, 2010). Later, black fungal strains appeared on the walls. Biocide treatments, including quaternary ammonium derivates, were applied during several years until January 2008. The cave is now closed to visits and periodical controls are being carried out (Bastian et al., 2009, 2010). In all the caves several sampling campaigns were conducted. Different samplings points were selected (visited and non visited areas), in order to cover as much as possible all cave areas. For the identification of microorganisms (bacteria and fungi), classical and molecular biology techniques were performed. The samples were taken using the DUO SAS SUPER 360 and Coriolis ¿. Subsequently, isolation and identification of the microorganisms were accomplished. CONCLUSIONS To the light of the results of this study, the following general conclusions can be reached: ¿ Fungal spores concentrations in the air are related to the number of visits. This relationship is not so clear concerning the air concentrations of bacteria. ¿ A classification has been proposed to describe the atmosphere cleanness of a touristic cave. We propose five categories ranging from no danger to potential danger, related to fungal air concentration: - category 1: a cave with no fungal problem (< 50 CFU/m3); - category 2: cave with alarm signal and recommends a series of periodic inspections and studies to disclose the problem (50-150 CFU/m3); - category 3: a cave threatened by fungi and this case involves a series of different measures such as control visits, drafts, closing doors, etc. (150-500 CFU/m3); - category 4: cave greatly affected by fungi as a result of visits, spillage, etc. (500-1.000 CFU/m3); - category 5: a cave in danger (>1.000 CFU/m3). The closing of the cave is recommended while studies to determine the source of microbial air contamination and conservation strategies are performed. This classification should be considered as a working hypothesis and must be validated carrying out further samplings in other caves with different visitation regime. For comparison purposes the methodology and conditions applied in this work should be followed for the study of other caves. We attempted to know the problems caused by the touristic use of caves and their damage in Paleolithic art. Finally, this work proposes a series of measures to preserve the caves, rock-art paintings and manage a sustainable tourism.