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FACTORS AFFECTING THE COLONIZATION AND DISTRIBUTION OF CYANOBACTERIA, ALGAE AND LICHENS IN ANCIENT MORTARS

ARINO, X. and SAIZ-JIMENEZ C ' .

lnstituto de Recursos Naturales y Agrobiologia, C.S.l.C., Apartado 1052, 41080 Sevilla, Spain

SUMMARY

The high level of colonization of ancient mortars by phototrophic communities is a consequence of the

susceptibility of this material to the action of organisms. Although physical and chemical characteristics of the

substratum play an important role in the selection of the colonizing organisms, microclimatic parameters -

mainly light intensity and relative humidity- must be taken into account to explain the composition, distribution and ecological strategies of phototrophic communities growing in mortars.

1. INTRODUCTION

Mortar is a mixture of sand grains joined together by a binder (lime, cement, etc.). It is of plastic consistency,

traditionally used as a building material. Ancient mortars were made mainly of lime and sand; on some

occasions other materials were added, such as clay or pozzolana, in order to improve its properties. Lime

mortars are characterized by high porosity with distribution of pore size in the range of mesopore, and they

possess low mechanical strength and a relatively high deformation capacity, together with high permeability to

water (1).

Mortars are used in construction to bind bricks or for coating stone surfaces, carved rocks, etc. Fresco, a

technique of wall painting on wet lime plaster, is based on carbonatation of the lime hydroxide of the rendering.

Deterioration of ancient mortars has been studied in recent years (1-3). In laboratory tests, mortar proved to be

a bioreceptive material, and this can also be observed in buildings and monuments. Mortars are easily colonized by biological communities, as their surfaces provide sites of attachment of airborne propagules and

accumulation of nutrients, which supply the mineral salts required for the growth of organisms. Moreover, the

pores facilitate the establishment and development of endolithic communities of cyanobacteria, algae and

lichens. After excavation, the mortars of several archaeological sites of southern Spain have been colonized by

phototrophic organisms, mainly cyanobacteria, algae and lichens. The aim of this paper is to investigate the

main factors promoting their colonization, distribution and adaptative strategies leading to biodeterioration

processes.

2. MATERIAL AND METHODS

The study was carried out at two archaeological sites of southern Spain. The Roman city of Baelo Claudia,

near present-day Tarifa, Cadiz, is representative of a small industrial city with an economy based on fishing,

especially tuna from the Straits of Gibraltar. The excavated city, constructed mainly in the first century A.O., is

protected by walls and comprises theatre, temples, forum, basilica, thermae, macellum, tabemae, necropolis,

aqueduct and a few factories. The forum and temples, after excavation in 1971, appeared relatively well

preserved, although in recent years a progressive deterioration has been observed in the mortars.

The Roman Necropolis of Carmona is located near Sevilla, and contains a high number of hypogean tombs, excavated in a calcarenite bedrock, with their walls coated with mortar. The tombs have particular

microclimates depending on their exposure and architecture. The tombs studied were the Elephant, T-2, T-

118, Amphitheatre, Mausoleum I and 11, Gar1ands, Quadrangular and Columbarium. Samples were obtained by scraping the wall surfaces. Where possible, small fragments of colonized mortars

were also removed with forceps. For cyanobacterial and algal identification, samples were placed in Petri

dishes with enrichment culture media. Measurements of temperature, relative humidity and radiation were taken at the surface of the mortars during

the spring -after the rainy period (May), and at the end of summer- after the dry period (September). The

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temperature was measured by means of thermistors placed on the stone surface. Relative humidity was

measured 1 cm above the surface with a hygrometer. Light irradiance was measured with a sensor placed on

the colonized surface. All probes were connected to a Squirrel Data Logger (Grant, Cambridge), and logged

every ten minutes, from 9:00 to 18:00. For scanning electron microscopy, specimens were fixed and dried at a critical point and gold coated (3).

Observations were carried out with a Hitachi S-2300 microscope.

3. RESULTS

Baelo Claudia Although the walls of the temples were composed of a homogeneous material, the spatial distribution of

organisms was different. In fact, the walls of the temples of Jupiter and Isis were colonized by

cyanobacteria/algae or by lichens, depending on their exposure to sunlight. Thus, the shaded and damp north­

facing walls of these temples were covered by an epilithic community of cyanobacteria and algae, forming a

greyish-brown layer on the mortar surface. At microscopical level, isolated cells and colonies were found

occupying the numerous cavities and fissures of the surface. From this microniche they may extend to

neighbouring areas. This community was made up of a similar number of taxa of cyanobacteria and algae

(Table 1), many of them typical of soils. There were few lichen taxa. Those that were present has a low

percentage of covering, located mainly near the run-off water tracks (Co//ema spp.) and macroscopic fissures

or holes (Lepraria Jesdainm). In constrast, a community dominated by lichens colonized the sunny walls of Isis and especially of the Forum

(Table 1), which has the longest period of sunlight. Lichens occupied large areas of the mortar surface,

although many of them were endolithic and only the reproductive bodies were visible. After the dissapearance

of the thalli, a high number of pits produced by the action of the lichen remained in the mortar. Cyanobacteria

and algae were also abundant, although not evident, as they were inside the mortar living in the pores of the

material, as cryptoendolithic communities. They comprised a high number of taxa of cyanobacteria and only a

few of algae. Many of them are typical of lithic environments, such as the cyanobacteria Borzia periklei, Cyanosarcina parthenonensis, Chroococcidiopsis sp., and Synechococcus e/ongatus. Other species typical of

soils were also found. A species of chlorophyta previously found only in aquatic and saline environments,

Ctenoc/adus circinnatus, was also identified.

TABLE 1

Number of taxa of cyanobacteria, algae and lichens in the walls of the Temples of Baelo Claudia.

Cyanobacteria

Algae

Lichen

Forum S

13

3 11

lsisE

12

4

7

Isis N

9

8 3

Jupiter N

9

9 6

The lichen colonization was studied along one of the walls of the Temple of Jupiter. A transect, from the

sheltered part to the exposed one, was studied and evidenced that slight differences in light intensity induced

the distribution of species and different ecological strategies of colonization, in spite of the fact that differences in temperature and relative humidity differences were almost inappreciable (Figure 1). On the sheltered part,

epilithic colonization was abundant. Lichen covering was approximately 50 % of the surface at the beginning of

the transect. Verrucaria viridula was the dominant species, and a non-corticate lichen, Lepraria /esdainni, was

also frequent in fissures and holes. On the opposite part, subjected to longer sunlight periods, epilithic growth

was very restricted, and although lichens were abundant, the majority of the thalli were deeply bedded in the

mortar. In this situation, the cover percentage reached almost 90 % of the surface, and the community was

dominated by Verrucaria hochstetteri. Other heliophilic species such as Ca/oplaca /actea, Sarcogyne regu/aris

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and Lecania turicensis were also frequent. The increasing number of lichen taxa is noticeable on the part of the

wall with higher light intensity (Figure 2).

Necropolis of Cannona The colonization of mortars by phototrophic c.ommunities was severely affected by the microclimatic

parameters prevailing, which were directly related to exposure. Two major types of environment were perfectly

delimited: the external and internal walls. Lichens were the main c.olonizers of the external walls, originating a community dominated by different taxa depending on the orientation. Some species, such as Veffucaria macrostoma, V. viridula or Calop/aca aurantia, were frequent and abundant on east- or south-facing walls,

enjoying long periods of sunlight. On the shaded walls, the community was morphologically distinct, mostly

composed of leprose lichens such as Lepraria niva/is, Leproptaca xantho/yta and Lepraria lesdainni, together

with some other squamulose (Toninia spp. and Squamarina spp.). This contrast in the type of thalli seemed to

be related with the exposure of the wall , as was observed in all the studied tombs (Table 2). A particular situation was found in some tombs where there was high relative humidity simultaneously with high

illumination: in this case a community of the epilithic alga Trentepohlia sp., and some lichens, such as Dirina massiliensis f. sorediata and Pseudosagedia linearis developed.

The entrances of the hypogean tombs, with high humidity and scarce sunlight, were profusely colonized by a

community mainly composed of two lichens, Lepraria /esdainni and Leprop/aca xantholyta, a moss, Fissidens bryoides

60

40

30 25

0 s 26 15

t-

22

3000 ~I/) 3000

E 2000 2000

0 E 1000 0 1000

9 DAYTIME

17 18 DAYTIME

A B

figure 1. Diurnal courses of microclimate the east wall of the Temple of Jupiter. Measurements correspond to

spring (A) and late summer (8). The continuous line corresponds to microclimatical data on the sheltered part

and the dotted line to the exposed part.

151

~ 12

u. 0 9

a: w CD :::? e ::> z

3

728

A B

0 1 2 3 4 5 8 7 8 9 10 11 12 13 1<4 15 18 17 18 19 20 21 22

RE LEVE

Figure 2. Number of lichen taxa at the points of the transect from the sheltered part to the exposed one.

TABLE2 Lichens of the Necroeolis of Carmona. Percentage according to the type of thallus in the walls of the tombs.

Sampling site Exposure1 Type ofthallus2 (%)

c s L

Mausoleum II , s +++ 88 12 0 Mausoleum II, 0 +++ 90 10 0 Elephant, S-E +++ 82 18 0 Columbarium, S +++ 79 16 5 Elephant S-W ++ 70 20 10

T-118 ++ 67 0 33 Columbarium, W-N + 57 14 29 Mausoleum I + 56 11 33 Columbarium, W ++ 55 30 15 Mausoleum II N + 50 20 30 Amphitheater + 50 0 50 Elephant E-N ++ 44 44 12 Gar1ands + 40 30 30 Columbarium N + 38 37 25 T-2 + 25 0 75 Amphitheater (inside) 0 0 100 T-2 (inside) 0 0 100 Elephant (inside) 0 0 100

1 Exposure: +++ = very exposed, long periods of direct sunlight; ++ = exposed, alternating periods of

sunlight with shadow; + = low exposed, shadow with short periods of direct sunlight; (-) = environments without direct sunlight. 2

Type of thallus: C = crustose; S = squamulose or microfruticulose; L = leprose.

and some cyanobacteria, especially Scytonema julianum. These species were distributed following the gradient

of light and humidity. Thus, Leproplaca xantholyta was usually growing on areas better illuminated than those

occupied by Lepraria lesdainni (but never in direct sunlight). In tombs with high relative humidity, Lepraria /esdainni and Fissidens bryoides were able to grow at very low light intensities, but inside the cavities, near the

limit of the photic zone, cyanobacteria (mainly Scytonema julianum, Gloeothece sp. and Geitleria calcarea) were the only phototrophic organisms able to develop stable communities in the mortar. They seemed to be

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perfectly adapted to low irradiance, as large patches of Geitleria ca/carea were observed growing at an average irradiance of 1-2 mmols/m·2.s·1.

4. BIODETERIORATION PROCESSES

The described communities have a strong influence on the alterations of the mortar. The surface colonized by

epilithic cyanobacteria and algae showed small cavities in which cells become installed. Mucilaginous

substances from the sheath contributed to the adherence of the cells to the substratum. Moreover, the

cryptoendolithic algal and cyanobacterial community developed profusely to depths of several millimetres,

often with evident processes of dissolution and reprecipitation of the carbonate matrix. Of particular interest

was the presence of the calcifying cyanobacteria Scytonema julianum and Geitleria ca/carea, two interesting

filamentous cyanobacteria able to precipitate calcium carbonate on their sheaths, forming calcified filaments,

and originating a grey layer of powdered appearance on the surface. On the walls of some tombs the formation

of a white, mould-like crust was observed. This is composed of an accumulation of dead and empty calcified

filaments. After death of the cyanobacteria, the calcified sheaths lose their original structure due to dissolution

and reprecipitation processes, originating compact amorphous calcified filaments in a biogenic calcite layer on the mortar surface.

The surfaces of the mortars colonized by lichens were also strongly altered, especially evident after elimination

of the thalli. The alteration affected both the carbonate matrix and the calcite crystals, often showing clear

pitting and etching. The intense activity of the hyphal network gave rise to considerable alterations in the first few millimetres of mortar below the thalli.

5. DISCUSSION

The high level of colonization by phototrophic communities on the mortar surfaces, in both Baelo Claudia and

Carmona, evidenced the high bioreceptivity of this material. Bioreceptivity is considered as the totality of the

material properties that contribute to the establishment, anchorage and development of fauna and/or flora (4).

Although physical and chemical characteristics of the substratum play an important role in the selection of the

colonizing organisms (5), microclimatic parameters, mainly light intensity and relative humidity, must be taken

into account to explain the composition, distribution and ecological strategies of phototrophic communities in

the mortars of the studied areas, and confirm the possible role of such communities as biomonitors of the

environment of the monument. Cyanobacteria and algae are affected by microclimate in similar terms,

although water availability seems to be the main factor. Epilithic growth takes place mainly on the walls with no

or scarce sunlight, but also on the exposed ones, on the water run-off tracks, where some cyanobacteria are

perfectly adapted to resist high irradiance and periodical wetting and drying. A high light intensity and dryness

of the mortar surface in open areas leads cryptoendolithic communities to develop inside, to protect

themselves from desiccation (Figure 3). Cells take advantage of the pore system of the substratum,

penetrating and establishing at a depth where the level of light is sufficient for photosynthesis. Cyanobacteria

are especially successful in this adaptation and, in the walls exposed to sunlight, they surpass the number of

algae. The resistance of cyanobacteria to extreme environments is recognized, where the usual presence of a

mucilaginous and pigmented external sheath can play an important role (6). Another extreme environment is

located at the end of the tombs, where there is very low light intensity. Here again the cyanobacteria are the

main colonizers, as lichens and mosses are unable to grow. The presence of calcifying cyanobacteria is

noticeable, especially Geitleria calcarea, a species known only in natural caves with a high environmental

stability. The deposition of calcite crystals on the sheath, a process induced by the organism itself, is a factor

which must be take into account in the alteration of mortars, as it represents a mobilization of calcium cations

from the substratum. The deposition of successive layers of dead filaments can originate an initial stage of

stromatolites, forming a biogenic calcite layer on the stone surface. In fact, a biodeterioration process of mural

paintings has been reported from a rock carved church colonized by Scytonemaju/ianum (!) .

...... r::Jj -N

~ 0 2500

E :1. ....._.,,

25

ENDOLITIDC LICHENS

CRYPTOENDOLITIDC

730

CY ANOBACTERIA AND ALGAE

CRUSTOSE AND SQUAMULOSE EPILITHIC LICHENS

Trentepoh/ia sp . AND RELATED LICHENS

EPILITHIC ALGAE AND CY ANO BACTERIA

LEPROSE LICHENS

CALCIFYING CY ANO BACTERIA

50 100

RELATIVE HUMIDITY (%)

Figure 3. Scheme of the distribution of lichens, algae and cyanobacteria in the studied areas.

Light intensity is a critical factor for understanding lichen distribution. Lichen resistance to desiccation makes

the colonization of mortar possible in dry environments with high exposure to sunlight, where cyanobacteria,

algae or mosses cannot grow. In such walls, many lichens seem to be well adapted to areas with high light

intensity, where they are more competitive and take advantage of several adaptative strategies, such as the

development of endolithic thalli and the presence of thick, strongly pigmented layers with dark (VeTTUcaria macrostoma) or bright colours (many Caloplaca species). This is reflected in the increasing percentage of

covering and number of lichen taxa identified in the part of the transect with higher exposure. In constrast,

lichens change their adaptative strategy when growing on damp and shaded walls, where they develop leprose

or microfruticulose thalli, such as in Lepraria /esdainii, because a cortical layer to protect them from desiccation is unnecessary, and would even be an obstacle to light.

Biological activity provokes and enhances the loss of binding properties, since organisms penetrate the cement

matrix due to chemical attack, leading to the decrease of cohesion. The excretion of organic acids and the

production of C02 as a result of cell respiration are important factors in the weathering processes, favouring

carbonate dissolution and originating extensive pitting, especially in the lichen/mortar interface, as found by

naked eye observation of the sun-exposed mortar supporting high lichen colonization. Cyanobacteria and

algae also have an important biodeteriorating role, due to the softness of the material, which is easy to

penetrate and consequently develops cryptoendolithic communities, which contribute to the disintegration of

the mortar. This facilitates the appearance of organisms such as bryophytes or vascular plants, with further structural damage.

ACKNOWLEDGEMENTS This is a contribution from the Cooperation Agreement between the Consejeria de Cultura, Junta de Andalucia, and the C.S.l.C. for the study of Andalusian Archaeological Sites. The facilities provided by Ms. Elisa Pinilla and the directors of the Sites are gratefully acknowledged.

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REFERENCES 1. Puertas, F., Blanco-Varela, M.T., Palomo, A., Ortega-Calvo, J.J., Arino, X. and Saiz-Jimenez, C. 1994.

Decay of Roman and repair mortars in mosaics from ltalica, Spain. Sci. Total Environ., 153, 123-131.

2. Saiz-Jimenez, C. and Arii'lo, X. 1995. Biological colonization and deterioration of mortars by phototrophic organisms. Mat. Constr., 45, 5-16.

3. Arino, X. and Saiz-Jimenez, C. 1996. Colonization and deterioration processes in Roman mortars by cyanobacteria, algae and lichens. Aerobiologia (in press).

4. Guillite, 0 . 1995. Bioreceptivity: a new concept fot building ecology studies. Sci. Total Environ., 167, 215-220.

5. Ortega-Calvo J.J., Arino X., Hernandez-Marine M. y Saiz-Jimenez C. 1995. Factors affecting the weathering and colonization of monuments by phototrophic microorganisms. Sci. Tot. Environ. 167:329-341 .

6. Whitton, B.A., 1992. Diversity, ecology and taxonomy of the cyanobacteria. In: Photosynthetic Prokaryotes, N.H. Mann and N.G. Carr (Eds.), Plenum Press, New York, pp. 1-51.

7. Pietrini A.M. y Ricci S., 1993. Occurrence of a calcareous blue-green alga, Scytonemaju/ianum (Kutz.) Meneghini, on the frescoes of a church carved from the rock in Matera, Italy. Cryptogam. Bot. 3:290-295.