Entry and dispersion of microorganisms inside Altamira Cave. New evidences from aerobiological and atmospheric gases surveys

Altamira Cave, a UNESCO World Heritage Site, houses one of the world's most prominent Palaeolithic rock art. The conservation of the rock art is threatened by microorganisms (bacteria and recently fungi). Previous studies revealed that the entry and dispersion of microorganisms and nutrients to the interior of the cave depends on the exchange rates between the cave atmosphere and the exterior through the entrance. Nevertheless, the data obtained in an aerobiological study combined with a multiparametric survey of atmospheric gases have shown that there is another possible entry and spread of microorganisms in the innermost area of the cave (the Well Hall), far from the single and most elevated entrance. Figure 1. (A) Altamira Cave map with location of the aerobiological and atmospheric gases sampling points inside the cavity. (B) Geographic location of the cave. Samples were obtained by filling Tedlar air sampling bags with capacity of 1 L. Air is introduced inside the bag by using a hand air pump. Special seal system ensures the sample during transport and temporal storage. Samples were analyzed afterwards in laboratory by using an optic spectrometer Picarro G2101-i. The device identifies and quantifies different compounds in the optical spectrum obtained. Isotopic relationship δCO2 is measured as a calculation from CO2 and CO2 concentrations analyzed by the spectral system. Precisions of 200 ppb, 10 ppb and 0.3% are guaranteed for CO2, CO2 and δCO2 respectively. Precision of 0.1 ppm has been obtained for CH4 compound in the concentration range studied (0-2 ppm). An extensive aerobiological sampling all over the cave was conducted in June 2009 to quantify the level of airborne bacteria and fungi. The methodology used was published elsewhere (Fernandez-Cortes et al. 2011). 3 RESULTS AND DISCUSSION A cave environment is characterized by high levels of CO2 with very low δC isotopic signal values and low levels of CH4 compared with the external atmosphere. Average values recorded in Altamira Cave and its surroundings in the samplings carried out in July 2012 are shown in Table 1. Table1. Environmental parameters in Altamira Cave Air CO2 (ppm) δC (CO2) ‰ CH4 (ppm) Cave 1257 -19.9 0.62 Top soil 2887 -20.5 1.38 Exterior 420 -8.5 1.70 Data analysis using Keeling’s Plot (Keeling 1958) indicated that the CO2 present in the ecosystem has a soil-derived origin. Figure 2 shows that inside the cave the lowest CO2 concentration correspond to the Entrance Hall due to the connection with the external atmosphere. This external influence is progressively reduced from the entrance inwards reaching the lower isotope signal values, maximum CO2 and minimum CH4 concentrations in the innermost areas (between the Great Hall and the Well Hall). However, in the last hall (Well Hall) is noticed a change of trend despite being the farthest hall from the only known accessible entrance. The slight increase of CH4 and a lower concentration of CO2 with heavier isotope values indicate the presence of an unknown connecting track to the outside near or in the Well Hall area. Regarding the level of airborne microorganisms (bacteria and fungi), a distinct behavior along the cave has been observed. The concentration of fungal spores outdoor the cave is higher than inside the cave. Throughout the cave, the concentration of fungal spores is higher in the halls and galleries close to the entrance (Kitchen Hall, Crossing, Polychrome Hall) and is reduced towards the interior (Walls Hall). The concentration of fungal spores reaches its minimum in the Grave Hall and Great Hall, ranging between 10 and 21 colony forming units (CFU)/m. However, the Well Hall reached 50 CFU/m. This can be attributed to a direct connection of the Well Hall with the exterior. Interestingly, a major abundance of Cladosporium spores was observed in Altamira, which is not common in other caves studied (FernandezCortes et al. 2011, Porca et al. 2011). In the halls near the entrance, considering Walls Hall, Crossing, and Polychrome Hall all together, Cladosporium attained 79.6%, followed by Epicoccum (18.0%), Penicillium (1.0%), and Aspergillus (0.5%). Because Cladosporium and Epicoccum are considered common outdoor fungi, it is suggested that the abundance of Cladosporium all over the cave can be a consequence of the existence of a connection with the exterior not only at the entrance but also at the end of the cave, in the Well Hall. In fact, 84% of the fungal spores present in the Well Hall belong to Cladosporium. Figure 2. In profile, spatial distribution of airborne bacteria and fungal spores in Altamira Cave (June 2009) and CO2, δCO2 and CH4 (July 2012) in the air of Altamira Cave. The airborne concentration of bacteria inside the cave is higher than that recorded outside and also much higher than of the fungi. This can be understood taking into account that the ceiling and walls of Altamira Cave (Kitchen Hall, Crossing, Polychrome Hall and Walls Hall) are widely colonized by bacteria (Cuezva et al. 2009, Saiz-Jimenez et al. 2011). Thereby, the higher concentrations of airborne bacteria were found in the Crossing and entrance to Polychrome Hall. Considering all samplings together, the most abundant bacteria in Altamira Cave air was Micrococcus luteus (83.2%), followed by different species of Pseudomonas (7.8%) and Bacillus (2.1%). In the halls near the entrance (Walls Hall, Crossing, and Polychrome Hall), M. luteus concentrations ranged between 77.3 and 88.9%, while in some other halls (Wells Hall, Grave Hall) was absent. Again the influence of a possible connection exterior-interior in the Well Hall is confirmed by the increasing number of bacteria in this hall (740 CFU/m, from which 89.2% belonged to Bacillus weihenstephanensis, a soil-dwelling bacterium) in comparison with the samplings obtained in the nearby halls (Great Hall: 360 CFU/m and Horse Tail Gallery: 280 CFU/m). The Grave Hall that is an inferior hall, isolated in some way from the rest of the cave, shows the lower bacteria concentration in the cave (150 CFU/m). The caves are considered oligotrophic environments, and areas with direct communication with the outside favor the entry of microorganisms and the development and dissemination of microbial colonizations. In Altamira Cave the area near the entrance has a higher level of airborne microorganisms in relation to nearby halls. However, an anomaly in the Well Hall in relation to nearby halls reveals a connection with the outer atmosphere at this site.