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ANNUAL MICROBIOLOGICAL ANALYSIS OF ALTAMIRA CAVE (SANTILLANA DEL MAR), SPAIN.

ARROYO, I. and ARROYO, G.

Institute of Conseivation and Restoration of Cultural Property. Ministry of Culture. C/EI Greco n° 4, 28040 Madrid. Spain.

SUMMARY

A~a.mira Cave is located near Santillana del Mar (Cantabria, Spain). It is a cavity some 300 metrs long

ongmated by karstic activity on strata of lime rocks separated by layers of clay. The Polychromes Room was

discovered in 1879 by Sanz de Sautuola and his daughter. It is decorated with a herd of bison surrounded by

different animals, mainly horses and deer. The pigments are of mineral origin except for the black pigments, obtained from charcoal.

The cave acts a natural ecosystem defined by rocky limestone mass subject to hydrogeological processes.

The air and rock surface temperatures range from 14-17° C and the relative humidity fluctuates between 96 and 98%. The variations that take place in the ecosystem are related to external factors.

Samples were taken during the spring, summer, autumn and winter in order to check for possible seasonal

variations in the different groups of microorganisms researched. Two different techniques, MNP and plate

counts, were used to determine the number of microorganisms. The counts of most groups of microorganisms

was high in the four seasons studied, with variations due to the influence of certain factors. For example the

proximity of cattle, the vegetation cover, the abundants rain durring Autumn permitted the growth of most

groups of microorganisms. The analysis of variance shows a seasonal dependence in certains groups of

microorganisms. The influence of visitors have had less of an effect on the counts than seasonal changes.

1. INTRODUCTION

Altamira Cave is located near Santillana del Mar (Cantabria, Spain), a rural town that is given over to

agriculture, but is also of great artistic interest and a major tourist attraction. It is a cavity some 300 metres long

originated by karstic activity on strata of lime rocks separated by thin layers of clay.

The Cave was discovered in 1868, yet it was not until 1879 that Marcelino Sanz de Sautuola and his daughter

Maria came across the palaeolithic paintings and engravings. The paintings are to be seen throughout the

cave, although the most outstanding are in two perfectly defined areas (Fig. 1). The first, and best known, is

the Polychromes Room, which is decorated with a herd of bison (Fig. 2) surrounded by different animals,

mainly horses and deer. The pigments are of mineral origin except for the black pigment, obtained from

charcoal, which has made it possible to date the paintings to the Magdalenian period, by 14C (15 910 and 14

480 BP). The second area, which is narrow and hard to reach, is known as the Horse's Tail and contains

different engravings, as well as some black-coloured paintings that represent animal masks.

The Cave acts as a natural ecosystem defined by a rocky limestone mass subject to hydrogeological

processes. The cave is only connected to the exterior by the entrance, closed off by a metal door that permits

a certain degree of ventilation, as well as helping to stabilize the internal atmosphere, which is sensitive to any

type of disturbance. The air and rock surface temperatures range from 14 to 17°C and the relative humidity

fluctuates between 96 and 98%.

Therefore the changes that occur in the ecosystem are related to external factors. The climate in Santillana del

Mar is mild and humid, with a mean annual rainfall of 1,200 mm. The humidity of the soil causes filtrations of

water that give rise to several dropping points inside the cave. The outer surface is formed by grass vegetation

cover, and dwellings and cattle are to be found nearby. From the time that the paintings were discovered to the

present day, much action has been taken inside the Cave to ensure its stability and to make it suitable for

tourists to visit. For instance, between 1924 and 1935 the cracks in the Polychromes Room were filled with

hydraulic cement, pillars and close walls were installed and the floor was lowered. In 1941 new walls were built

and wooden struts were installed, remaining there until 1960. In the Sixties and Seventies, with the boom in

tourism in Spain, the cave became a tourist attraction and visitors were allowed in en masse. Later the number

was limited to 9,000 per year. All these events have contributed to the change of the physical, chemical and

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biological factors inside the Cave and, naturally, they have played a significant role in the rise in the degree of

contamination and in the conservation of the paintings. consequently a project for the global conservation of the Rupestrian Art of Altamira has been drawn up. One of the aims of this project is to design a model of systematic control of the environmental parameters. The model includes studies of the microbial contamination and the possible influence of microorganisms in the

deterioration of the paintings. The objective of this study has been to conduct a microbiological analysis of the Polychromes Room during the different seasons of the year in order to ascertain to what extent certain external factors affect the development of different microbial groups that may contribute towards the biodeterioration of the paintings. Likewise, we have endeavoured to observe the possible effect of the number of visitors on the paintings.

2. MATERIALS AND METHODS

- Sampling: Samples were taken in aseptic conditions at 12 different points of the Polychromes Room, corresponding to the floor, walls and slopes, throughout the year (spring, summer and autumn of 1995 and winter of 1996), placed in sterile bags and transported at ambient air temperature to the laboratory, where they underwent certain microbiological tests. In order to ascertain the degree of contamination of the paintings, contact plates containing Tryptone Soy Agar (TSA) and Sabourard Agar were applied to 12 points on the ceiling, near the paintings. The air contamination analysis was conducted at four points of the room by filtering 500 I of air by a Super Air System (SAS Super 90) in Petri dishes of 55 cm in diameter containing the above­mentioned media.

- Microbiological analysis: 1 O g of each sample were weighed and homogenized in flasks containing 90 ml of Salt Solution (0.9%), and then decimal dilutions were performed. The number of microorganisms was determined by the Most Probable Number (MPN) method and the plate count, using the media habitual in environmental microbiology and those recommended by the Normal, 9/88 (Manufacti Lapidei Regulations). Vegetative forms for sporulated and sulphite-reducing microorganisms were destroyed by heating at 85-900C for 10-15 min. A presence/absence test was used to determine the presence of ferroxidant bacteria.

- Microorganism groups analyzed: In accordance with the characteristics of the Cave as regards chemical composition, environmental conditions, external conditions and the objectives of the study, the following groups were studied:

1.- Microorganisms indicating the degree of contamination: Mesophilic aerobic viable bacteria Mesophilic sporulated bacteria Molds and Yeasts

2.- Environmental microorganisms: Pseudomonads Actinomycetes Cyanobacteria

3.- Faecal contaminants: Coliforms Enterococci Sulphite-reducing bacteria

4.- Nitrogen cycle microorganisms: Nz-free fixing bacteria Nitrous bacteria Nitrifying bacteria Denitrifying bacteria Ammonifying bacteria

5.- other microorganisms of interest: Elementary sulphur oxidisers Ferroxidants

Amylolytic bacteria Proteolytics

- Number of visitors per year: 1. - Spring Sample (April 1995) 2.- Summer Sample (July 1995) 3.- Autumn Sample (October 1995) 4.- Winter Sample (January 1996)

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1,925 visitors. 1,935 visitors. 2, 765 visitors. 2,340 visitors.

- Statistical Analysis: A one way analysis of variance (oneway anova) was carriet out in order to test intra­

season variation of microorganism counts. Also homocedasticity condition was analized first with a Bartlett-Box

test since anova requires homogenity in the variances of the groups considered (season). Table 1 shows

microorganism type, probability of F ratio and probability of Bartlett-Box. Given that ours is a balanced design,

global F is not very affected by a heterocedasticity condition (heterogenity of variances) and oneway anova was considered in every case.

3. RESULTS

The graphs (Fig. 3, 4, 5, 6, and 7) show the count measurements for each group of microorganisms each

season. There were seasonal variations in the degree of contamination of the Cave due to the influence of

certain factors. In summer there was an increase in the number of mesophilic aerobic viable bacteria and of

molds and yeasts (one logarithmic unit) (Fig. 3), whereas in autumn there was a considerable drop (at least

one logarithmic unit). In winter the number of microorganisms reached similar or higher levels than in spring.

The mesophilic sporulated bacteria (Fig. 3) dropped slightly over the period of time. The statistical analysis

shows a seasonal dependence for the molds and yeasts (P< 0.05). As regards the socalled environmental

organisms, the general trend has been an increase of one logarithmic unit in summer (Pseudomonadaceae

and Cyanobacteria) (Fig. 4). The Actinomycetes (Fig. 4) dropped slightly in autumn (by less than one

logarithmic unit). Table 1 shows seasonal dependence for Pseudomonads (P<0.05). The morphological and

biochemical tests carried out on the isolated Pseudomonadaceae confirmed the presence of numerous strains

beloging to fluorescent group. The faecal contaminants appeared in most of the samples. The number of

coliforms was greater in autumn, whereas the number of enterococci and sulphite-reducing spores was larger

in winter. The tests failed to confirm the presence of E. coli, but did reveal the presence of Enterobacteriaceae

of the Citrobacter, Enterobacter, Klebsiella and Serratia genus. The tests also conformed the presence of

Enterococcus faecalis . . The statistical analysis shows seasonal difference for enterococci (P<0.05). The

behaviour of the nitrogen cycle microorganisms was variable (Fig 6). The counts of N2-free fixing,

ammonifying and denitrifying bacteria were considerably large. The nitrous and nitrifying bacteria counts

revealed smaller numbers, but wich increased in winter by more than one logarithmic unit. The statistical

reveals a clearly seasonal variation except for Nr free fixing bacteria (Table 1). Very few sulphur oxidising

bacteria were found (Fig. 7). The number of proteolytic and Clmylolytic bacteria was high (Fig. 7). The first

present seasonal variation (P<0.05). Most of the samples shows presence of ferroxidants. The interpretation of the contact plates shows a high level of contamination, both by molds and yeasts and by viable bacteria (>10

4

and >105 / cm2. Numerous strains of the Bacillus genus, and some Actinomycetaceae were foun. Filtering of

the air above the plates produced similar results.

The results demonstrated the very limited influence of the number of visitors.

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4. DISCUSSION

Altamira cave has been described on many occasions as a stable ecosystem in which the environmental conditions vary very little, this paper demonstrates the influence that certain external factors have on the interior of the cave. It should be taken into account that rainfall during the spring and summer of 1995 was lower than normal, giving rise to a long, dry period that lasted until the start of autumn (October). Ou the contrary, the abundance of rainfall in autumn has resulted in a very humid winter. It thus follows that the microorganisms that indicate degree of contamination, and the environmental microorganisms, many of which require large amounts of water, decline in autumn, sometimes by as much as one logarithmic unit. This decline does not, of course affect the mesophilic sporulated bacteria so much because the spores are capable of withstanding extreme conditions of dryness. The faecal contamination indicators, in particular the coliforms group, which indicate recent contamination, increase during the summer. It is well-known that the number of Enterobacteriaceae is generally higher in summer, because the high temperatures favour their growth. At the same time, there is an increase in the contamination of water because cattle are released to graze. The fact that the tests failed to confirm the presence of E. coli could be due to the microorganisms !ability and to the time that elapsed between the collection of the samples and the start of the microbiological analysis. Nonetheless, the tests did reveal other Enterobacteriaceae that are important on account of their intense proteolytic activity. The filtered water which, in this case, affected the winter counts, the proximity of the cattle and the abundant vegetation cover, favour the growth of other groups of microorganisms in the Room, such as ammonifying, denitrifying and Nrfree fixing bacteria that pick up the nitrogen from the animal droppings and plant remains. The same can be said of the proteolytic and amylolytic bacteria, whose metabolic products (nitrogen-bearing compounds, carbohydrates) serve as a source of Carbon for the enhanced growth of other heterotrophic microorganisms such as molds and yeasts, which were abundant in all the seasons and capable of producing dark or ochre-coloured, earthy-looking spots, as well as earth falls. Moreover, as a result of their metabolism, they produce organic acids that attack the carbonate and thus form the corresponding salts. However, few nitric and nitrous microorganisms were found. The high number of actinomycetes found both in the counts and in the contact plates are a potential hazard for the paintings because they produce a greyish poY/der on the lime rock, use nitrites and nitrates, reduce the sulphate and attack the limestone and other minerals via their metabolic products such as sulphuric acid, nitric acid, etc. Cyanobacteria are an important group of microorganisms in the cave. They grew abundantly in all the seasons, but especially so in winter, after the rise in humidity as a result of the autumn rains. The fact that in summer and autumn they did not drop considerably is probably due to the fact that many strains were sheathed, allowing them to retain water. The higher number of visitors during the summer and autumn, and the installation of probes to measure the humidity and temperature, meant that the lighting was left on longer, and had a favourable influence on its growth. Generally speaking, it seems that the periods of greatest affluence of visitors have had less of an effect on the counts than seasonal changes. This might indicate that, despite the apparent stability of the Cave's ecosystem, to a large extent the cave is subject to external fluctuations, and this must be taken into account by the overall conservation project.

ACKNOWLEDGEMENTS

We are grateful to Dr. Eduardo Arroyo from Facultad de Medicina for assistance with the statiscal analysis.

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49-64.

Microorganism groups F Probability Bartlett-Box test

Mesophilic Aerobic Viable Bacteria 0.7454 0.000

Contamination indicator Microorganisms Mesophilic Aerobic Sporulated Bacteria 0.0550 0.000

Molds and Yeasts 0.0192 0.000

Pseudomonadaceae 0.0000 0.000

Environmental microorganisms Cyanobacteria 0.7250 0.000

Actinomycetes 0.1338 0000

Coliforms 0.3508 0.000

Faecal contaminants Enterococci 0.0070 0.010

Sulphite-Reducing Bacteria 0.6785 0.000

Ni-Free Fixing Bacteria 0.2913 0.982

Nitrous Bacteria 0.0000 0.000

Nitrogen cycle microorganisms Nitrifying Bacteria 0.0000 0.000

Oenitrifying Bacteria 0.0249 0.966

Ammonifying Bacteria 0.0013 0.813

Sulphur Oxidizing Bacteria 0.9113 0.440

Other Microorganisms of interest Amylolytic Bacteria 0.1561 0.373

Proteolytic Bacteria 0.0000 0.996

Table 1. - Analysis of vanance for each group of microorganisms eac h season.

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Fig.1. - Map of Altamira Cave. A: Entrance B: Polychromes Rooms

, .. -...

i - -~ -...:. -

. __ .. l . . . .·:

Fig.2. - A Bison of the Polychromes Room

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Log . No. ctu/g

~-------------------- -

7 / . .- ·

6

5

4

3

2

Autumn Winter

. I '1 M/Y 6 MASB AMAVB I Fig. 3.- Seasonal variation in the No. of contamination indicator microorganisms·

M/Y Molds and. Yeasts; MASB Mesophilic Aerobic Sporeforming Bacteri~· MAVB Mesoph1l1c Aerobic Viable Bacteria. '

Log . No. cfu/g

/

5 ·

5

4

3

/ /

2 .··~~~ 0-~-

Spring Summer Autumn Winter

/ ~ Pseud. & Cyan. A Actin. /

Fig. 4.- Seasonal variation in the No. of Environmental microorganisms; Pseud. Pseudomonadaceae; Cyan. Cyanobacteria; Actin. Actinomycetes

Log . MPN of Microorganisms/g; Log. No. Spores/g ____ ,, --· - ------------- ···-·-------- ---

5 /

4

3

2

o ~~~~~~~~~L{g,~~~~~~~~

Summer Autumn Winter Spring

/ ~ E .AsR b. c

Fig . 5.- Seasonal variation in the number of faecal contamination Indicators E faecal Enterococci (MPN); SR Sulphite-reducing Microorgan isms (spores) ;

C faecal Coliforms (MPN) .

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Log. MPN of Microorganisms/g - ------- ------------ --··--- -·,

I

!

Spring Summer Autumn Winter

[.& NO 6 NI & ON AAM ~ NFF I Fig . 6.- Seasonal variation in the counts of Microorganisms of Nitrogen Cycle;

NO Nitrous Bacteria; NI Nitrifying Bacteria; ON Denitrifying Bacteria; AM Ammonifying Bacteria; NFF N-Free Fixing Bacteria.

I nq . MPN nf l'vl ir :rr11 H qanisms/g

Sprinq Summer Autumn Winter

/ !\ SO A Prot. Ll1 Amyl. I I iq . 1 . ScuSOI 1al varialio11 of different interesting Microorganisms;

SO Sulphur-Oxidizing Bacteri a; Prot. Proteolyti c Bacteria; Amyl. Amilolytic Bacteria.