International Journal of Speleology 43 (3) 295-303 Tampa, FL (USA) September 2014 Available online at scholarcommons.usf.edu/ijs/ & www.ijs.speleo.it International Journal of Speleology Off icial Journal of Union Internationale de Spéléologie Airborne microorganisms in Lascaux Cave (France) Pedro M. Martin-Sanchez1, Valme Jurado1, Estefania Porca1, Fabiola Bastian2, Delphine Lacanette3, Claude Alabouvette2, and Cesareo Saiz-Jimenez1* 1Instituto de Recursos Naturales y Agrobiología de Sevilla, IRNAS-CSIC, Apartado 1052, 41080 Sevilla, Spain 2UMR INRA-Université de Bourgogne, Microbiologie du Sol et de l’Environnement, BP 86510, 21065 Dijon Cedex, France 3Université de Bordeaux, I2M, UMR 5295, 16 Avenue Pey-Berland, 33600 Pessac, France Abstract: Lascaux Cave in France contains valuable Palaeolithic paintings. The importance of the paintings, one of the finest examples of European rock art paintings, was recognized shortly after their discovery in 1940. In the 60’s of the past century the cave received a huge number of visitors and suffered a microbial crisis due to the impact of massive tourism and the previous adaptation works carried out to facilitate visits. In 1963, the cave was closed due to the damage produced by visitors’ breath, lighting and algal growth on the paintings. In 2001, an outbreak of the fungus Fusarium solani covered the walls and sediments. Later, black stains, produced by the growth of the fungus Ochroconis lascauxensis, appeared on the walls. In 2006, the extensive black stains constituted the third major microbial crisis. In an attempt to know the dispersion of microorganisms inside the cave, aerobiological and microclimate studies were carried out in two different seasons, when a climate system for preventing condensation of water vapor on the walls was active (September 2010) or inactive (February 2010). The data showed that in September the convection currents created by the climate system evacuated the airborne microorganisms whereas in February they remained in suspension which explained the high concentrations of bacteria and fungi found in the air. This double aerobiological and microclimate study in Lascaux Cave can help to understand the dispersion of microorganisms and to adopt measures for a correct cave management. Keywords: aerobiology; bacteria; fungi; caves; Palaeolithic paintings; culture-dependent methods; climatic simulations Received 3 December 2013; Revised 10 April 2014; Accepted 24 May 2014 Citation: Martin-Sanchez P.M., Jurado V., Porca E., Bastian F., Lacanette D., Alabouvette C. and Saiz-Jimenez C., 2014. Airborne microorganisms in Lascaux Cave (France). International Journal of Speleology, 43 (3), 295-303. Tampa, FL (USA), ISSN 0392-6672 http://dx.doi.org/10.5038/1827-806X.43.3.6 INTRODUCTION 2007). In the last 50 years the cave suffered several microbiological crises (outbreak of microorganisms Lascaux Cave and other sites in the Vézère Valley, threatening the paintings) due to the invasion of the France, were included in the UNESCO World Heritage alga Bracteacoccus minor in 1963 (Lefèvre, 1974) List in 1979. This cave, discovered in 1940, contains and the fungus Fusarium solani in 2001 (Dupont et valuable Palaeolithic paintings. The cave was open al., 2007). At present, the paintings are threatened to the public in 1948 and attracted a large audience by black stains produced by the fungus Ochroconis which reached 100,000 visitors/year in 1962. This lascauxensis (Martin-Sanchez et al., 2012a) as seriously disturbed the cave microclimate and had a documented in the pictures published by Bastian et strong impact on the ecosystem (Bastian et al., 2010). al. (2010) and Jurado Lobo et al. (2010). Unfortunately, at that time, there was no scientific In the last few years several reports on the knowledge of cave conservation problems and the microbiology of this cave were published, which huge number of visitors resulted in fatal management contributed to understand the complex processes of errors that marked the future of the cave. microbial colonization and biodeterioration of the cave Currently, Lascaux Cave is an endangered cavity and its paintings (Bastian et al., 2009, 2010; Martin- that was studied from a microbiological point of view Sanchez et al., 2012 a,b; Saiz-Jimenez et al., 2012). since the 60’s of the past century, but unfortunately One of the aspects more neglected in cave research very few data were published before 2009 (e.g. is the study of cave aerobiology, although this issue Lefèvre & Laporte, 1969; Lefèvre, 1974; Dupont et al., is of great interest for a correct cave management. *[email protected] 296 Martin-Sanchez et al. A historical overview revealed that until the 1970’s, The system is also dedicated to recreate convection a monitoring of the microbiological contamination currents during these months. When the air located of Lascaux Cave was carried out by the Institute in the deeper parts of the cave is colder than the Pasteur (Paris, France). According to our knowledge, upper parts air, convection currents disappear. The data about the aerobiological studies performed artificial creation of a cold point in an area close to the on those dates were no published. The task of the upper parts (the machine room) helps the formation sanitary monitoring, including the evaluation of of convection currents. When the convection currents airborne bacteria, fungi and algae was then passed are present, airborne microorganism dispersion is to the Laboratoire de Recherche des Monuments higher than the case of stratified air. When there Historiques (LRMH, Champs-sur-Marne, France). is no convection, microorganisms are staying in For twenty years (from 1970 to 1990), a passive suspension, and are no more evacuated. method based on the gravity effect was used to The objectives of the present study were to report collect airborne microorganisms by LRMH. Open Petri on airborne bacteria and fungi in Lascaux Cave as dishes were exposed on the floor for five minutes for well as to know their dispersion mechanisms inside bacteria and fungi, or 24 hours for algae. About ten the cave, data which allow integrating the knowledge sampling points located inside the cave and in the acquired in the last years on the cave microbial entrance airlock chamber were checked using this ecology. To reach it, aerobiological and microclimate protocol twice a year (Orial et al., 2011). The resulting studies were carried out in two different seasons, counts using the passive method varied within very when a climate system for preventing condensation low ranges, from 0 to 15 colony-forming units (cfu) of water vapor on the walls was active (September per plate for both bacteria and fungi. The sampling 2010) or inactive (February 2010). The microbial method was changed in 1990 to an active method, communities were characterized by isolation and based on the suction and pumping of air samples molecular identification of cultivable strains from onto culture media. The results published by Orial air samples collected by Duo SAS Super 360. It was et al. (2011) were focused on total quantifications of expected that these data would contribute to the cultivable bacteria and fungi corresponding to the conservation of this fragile cave. active monitoring carried out until 2009. In summary, they remarked different periods in Lascaux Cave. MATERIAL AND METHODS From 1990 to 2000, the counts remained within low ranges (0-150 cfu/m3 for bacteria and 0-50 cfu/m3 for Sampling fungi). In December 2001, when the Fusarium solani Lascaux Cave is located in the basin of Vézère River, outbreak started, the fungal quantifications reached near the town of Montignac, in the Black Perigord in general low-medium levels (50-250 cfu/m3). From area, Dordogne, France (45º3’13.3’’N, 1º10’12’’E). 2002 to 2003, the bacterial and fungal counts varied The total length of the known subterranean system between very low and medium ranges (0-250 cfu/m3). In is less than 250 m, with an accessible volume of 2004, the levels of bacteria and fungi were particularly 3,300 ± 500 m3, as shown in Fig. 1a. The galleries of increased reaching a high level (250-500 cfu/m3) in Lascaux Cave are situated at shallow depth, between December, and even surpassing it in some halls. This 10 and 25 m below the current ground level, in increase was directly related to the frequent human the heterothermal zone of the soil. The average air activities performed in those dates. Since then, from temperature, which is relatively constant all year 2006 to 2009, the microbial contamination fell notably round, is 12.5ºC, and the average relative humidity and returned to lower levels. Very little information is 99% (Malaurent et al., 2011). was provided on the identification of microorganisms Two surveys were conducted in Lascaux Cave at detected in this study, only the most abundant genera: different dates, on 16th February and 21st September Fusarium, Cladosporium, Gliomastix, Penicillium and in 2010, in which were collected air samples from the Aspergillus for fungi, and Bacillus, Staphylococcus and same nine points shown in Fig. 1a. Eight of them Micrococcaceae family for bacteria (Orial et al., 2011). were located at different halls within the cave (A2- Few works about aerobiology of caves have been A9), and one additional sampling point was located published using different collection methods, mainly outside the cave as control (A1). passive samplings (Monte & Ferrari, 2000; Nugari A Duo SAS Super 360 air sampler (VWR-pbi, & Roccardi, 2001), a Hirst-type volumetric spore Milan, Italy), shown in Fig. 1b, was used to detect trap (Docampo et al., 2011), or a six-stage Anderson cultivable microorganisms in both surveys. Two FA-1 sampler (Wang et al., 2010). Recently, Porca replicates of air samples (100 L each one) were et al. (2011) used a Duo SAS Super 360 air sampler directly aspirated, using a 219-hole impactor, onto for evaluating the cultivable fungal communities the appropriate culture media contained in 90 mm of caves and proposed a biological index of fungal Petri dishes.