One microbial bioassay for a method of cyanobacterial and
algal removal – microalgae causing biodeterioration
Bohuslav Uher
Department of Botany and Zoology, Faculty of Science, Masaryk University, Kotlářská 2, 611 37 Brno, Czech Republic; uherius@sci.muni.cz
Stone is the oldest entity of Earth, it creates ecosystem which includes also environmental
factors (light, nutritions, climate, pollution). The factors of weathering never affect separatly
but together in a lot of antagonistic or synergic interactions. The significant deterioration of
monuments has been begun since yaers 1870-1880, this phenomenon has not been possible to
stop since 1950 when the defects of buildings have gone over the level of carrying-capacity.
Intensity of colonisation of organisms, biotransport and biodeterioration are directly
proportionated to the poriness of stone, this phenomenon confirmed Vendellsaz et al. (1996)
and Laiglesia et al. (1994) in Spain. Limestone, dolomite, sandstone, bricks and cement
materials are the most colonized materials by microorganisms (Hueck van der Plas,
1968).Color changes of stone surface include biological aspects, the black color represents
colonisations of micromycetes or of cyanobacteria, the green, orange, brown or yellow
growths represent derivates of green chlorofyles of algae, red or pink biofilms represents the
chemoautotrofic bacteria (Urzi et al., 1992). Biofilms induce changes of mineral and
chemical composition of stone, microorganisms change its stability, permeability, density and
color (van der Molen et al., 1980). Usually, the changes of the surface are very good visible,
the presence of some species can be also endolitic, which are not visible by eye, there are
species, mainly cyanobacteria, specialised to the rifts of substrata or to cavernities and to
pores of material (Golubic et al., 1981; Asencio & Aboal, 2000).
Actual state of the protection of the monuments stones is different in Europe. Research of the
photosynthetic lithobionts on monuments is in exces of importance to understood the stucture
of the characteristic microbial associations considering the biodecay effects. It is one part of
the doctoral thesis “Cyanobacteria and algae as agents of biodeterioration of stone substrata”.
This part of thesis titled “El Estuco’s Experiments” was accomplished in Laboratory of
Algology (Department of Botany, Murcia University, Spain).
- to examine the taxonomic and autecological traits of
subaerial mikroalgae occuring in urban habitats in the
Region of Murcia
- to reassume in research of subaerial algae in apllied
way, i.e. to design a growth inhibition test on subaerial
microalgae and to test a microalgae causing actively
biodeterioration which potentially damage the building
stone. A type of growth inhibitor was used white powder
called in Spain “el estuco”.
Samples were taken from the 16 subaerial places, include monuments (12: Real Monasterio
de Encar-nacion de Monjas Clarisas, Castillo de los Vélez, Monasterio de San Francisco,
Iglesia del Santo Cristo, Iglesia del Salvador, Iglesia Nuestra Señora de la Asuncini, Colegiata
de San Patricio, Palacio de Guevara, Palacio Episcopal, Catedral de Murcia, Medina Siyasa,
Castillo de Monteagudo, see Fig. 1), calcareous rocks (3 – La Puerta) and a new building
(Faculdad de Biologia). The representative substrata hosting the algal biofilm were limestone,
sandstone and in one case concrete. The samples were samplied and collected during the
spring and summer seasons of years 1996 and 2003. The part of scrapied field material was
aseptically spread into test tubes and over surface of Petri dishes containing medium BG11
(Rippka et al. 1979), BG110 (Rippka et al. 1988) and BBM (Smith & Bold 1967), which was
liqiud or agarized. The test tubes and Petri dishes were incubated by constant condition t =
20ºC, humidity 64.2 %, irradiation 75 μE m2 s-1, light 3000 lx and in 8/16 dark-light regime in
laboratory of the S.A.C.E. (Servicio de Apoyo a las Ciencias Experimentales, Murcia
University). Observations of samples were done by stereomicroscope OLYMPUS SZH and
microscope OLYMPUS BH2. From the all cultivated samples were isolated 58 unialgal
strains, which are maintaing in LAUM collection (Laboratory of Algology, Murcia
University, Spain). Colected samles preserving dry and the photographic documentation of
species are stored in Laboratory of Algology, Murcia University (Spain). The powder mixture
(el estuco) is used traditionally for coating buildings in Mediterranean regions, mainly in
Spain. There are a lot of kind of procedures how to prepare coating for buildings, usually is
used mixture with water 1:1, 1:2 or 1:3 ratio. This coating is the natural powder, prepared
from calcareous material or rocks. The exact composition of this material was not known.
The powder diffraction analysis of this material was made in Laboratorio de Geologia (Dpto.
de Química Agrícola, Geología y Edafología, Facultad de Química) by María Teresa
Fernández Tapia. The analysis showed that it is mineral CaRuO4.2H2O - Calcium Ruthenium
Oxide Hydrate. For grow inhibition test it was finally used 4 successfully grown algal strains,
which were incubated in constant conditions in the laboratory of the S.A.C.E. (Servicio de
Apoyo a las Ciencias Experimentales, Murcia University). In general, the BBM medium
(Smith & Bold, 1966), either liquid or agarized was used. Four strains FAB1/02 (Diadesmis
cf. contenta), FAB1/03A (Xanthonema sp., Fig. 5), FAB2/01A (Klebsormidium flaccidum),
BES5/03A (Chlorella kessleri) are maintained in a LAUM culture collection (Laboratory of
Algology, Murcia University, Spain).
The qualitative composition of species is different in several places of Europe,
the results of them shown clearly that the habitats with a high water potencial,
ensures that the cells remain aquatic for short periods when the habitat is dry,
hosting mainly green algae. It is typical for temperate humid areas (Schlichting,
1975; Rifón-Lastra & Noguerol-Seoane, 2001; Godyová et al., 2003; Darienko
& Hoffmann, 2003; Rindi & Guiry, 2003), with very short exposition of extrem
osmotic stress on the cell. Others artificial stone habitats exposed to extrem
osmotic stress very long time such as 4 months and more, are hosting
predominantly by epi- or endolithic cyanobacteria, which are adapted to extrem
dessication and teperature conditions. Firstly it is clear, that the important
influence on the composition of subaerial phycoflora has mainly the moisture
as the most important environmental parameter in lithic environment, which
confirms also Grilli Ciaola et al. (1987), Potts (1994), Nienow (1996).
Secondarilly the type of lithic substrata has less or non-significant influence on
composition of lithic phycoflora in the case of our localities. The epi- and
(chasmo)endolithic algae from subaerial monument-like environment, show a
dominance of prokaryotes microorganisms, as occurs in hot regions, in caves,
and others extrem environments.
Specific laboratory tests of biodeterioration were investigated by laboratories at
the universities in Messina and Oldenburg (Anagnostidis et al., 1992). The
selection of the most suitable method depended on the type and the stage of
organisms which were deteriorating monuments. Sometimes a good effect was
obtained by the combination of many methods which were successfully applied
by Krumbein et al. (1992) on the 50 architectonic monuments. Dupuy et al.
(1976), Pietrini et al. (1985) and Tiano (1979) investigated the effect of
algicides and its reversibility effects. The biotest with antibiotics were
investigated by Lefévre et al. (1964) and the effects of UV-rays were
investigated by Molen et al. (1980). The new technique designed and
successfully used in laboratory required considerable modification when used
on others photosynthetic organisms. This methodology is a pioneer test for
culture-testing of algae required both technical and biological expertise,
furthermore one needed to be able to improvise. It was developed the
taxonomical and afterwards experimental program, including the compilation of
bibliographical data on some strains. In addition, this collection has active
interests in developing miniaturized algal bioassays, algal biosensors and
development of cultivation tests. This experiment has considerable expertise in
algal culture and the spectrum of cultivation units from colonies on agar plates.
In addition, work on subaerial algae caused biodeterioration has highlighted
their practical potencial, e.g. use in the new growth inhibition test.
Acknowledgements.
I am grateful to Prof. Dra. Marina Aboal Sanjurjo for her invitation to work in
her lab and for providing the research work at the University of Murcia in Spain
for the successful realization of experiments conducted these expertiments
confirmed the inhibition effect of “Estuco” material for the development and
growing of microalgae, obtained results that are able to be applied in the control
of biodeterioration and are currently in the acceptation process of patent of
invention. Special thanks are expressed to Lcd. Jose Pedro Marín Murcia, Dra.
Antonia Dolores Asencio Martinez, Juana María García Reverte, Lcda. Encarna
Zafra Pastor and Lcda. Elena Ruíz Valero for their cooperation in the lab
(Laboratorio de Algologia and S.A.C.E.) I further would like to thank Dra.
María Teresa Fernández Tapia (Laboratory of Geology) for her research
cooperation in the “Estuco” project.
The study was supported by the project PCT 95/25 of Comunidad Autónoma de
Murcia, the Erasmus/Socrates Grant for mobility No. 10/2003 and by the
Funding of Ministry of Education of Czech Republic project No.
MSM0021622416.
The 47 epilithic and chasmoendolithic algae were identified: 22 cyanophytes/ cyanobacteria
(48%), 4 heterokontophytes (9%) and 20 chlorophytes (43%). 47 species: Pseudocapsa
dubia, Cyanobacterium cedrorum, Synechocystis sp. 1, Synechocystis sp. 2, Aphanocapsa
muscicola, Chroococcidiopsis kashaii, Hyella balani, Pseudanabaena sp., Leptolyngbya sp.
1, Leptolyngbya sp. 2, Leptolyngbya sp. 3, Leptolyngbya nostocorum Leptolyngbya sp. 5,
Leptolyngbya sp. 6, Leptolyngbya sp. 7, Schizothrix friesii , Phormidium autumnale,
Microcoleus vaginatus, Scytonema julianum, Tolypothrix byssoidea, Calothrix fusca var.
crassa, Nostoc sphaericum (Fig. 4), Botrydiopsis sp., Heterothrix (Xanthonema) sp.,
Heteropedia cf. simplex, Hantzschia amphioxys, Diadesmis cf. contenta, Nautococcus
terrestris, Tetracystis sarcinalis, Trebouxia arboricola, Myrmecia cf. globosa, Apatococcus
lobatus, Chlorosarcinopsis sp. 1, Chlorosarcinopsis cf. Arenicola (Fig. 6), Ecdysichlamys
obliqua, Oocystis asymetrica, Muriella terrestris, Chlorella vulgaris, Chlorella miniata,
Chlorella kessleri, Scenedesmus obtusiusculus, Klebsormidium nitens, Klebsormidium
flaccidum, Klebsormidium crenulatum, Stichococcus allas, Stichococcus bacillaris,
Stichococcus minutus. The above data confirm that coccoid species outnumber filamentous
species (totally represent 55 % of all investigated algae). The inoculated algae have not grown
on the all agar plates contained “el estuco”powder this material Calcium Ruthenium Oxide
Hydrate totally inhibited and eradicated growths of algae in all case of used concentrations
(Fig. 3).
Fig. 1: Sampling site - Castillo de Monteagudo (author: Jose Pedro Marin Murcia)
Fig. 5: The subaerial heterokonthophyte alga Xanthonema)sp. (author: Bohuslav Uher)
Fig. 4: The subaerial cyanobacterium Nostoc sphaericum (author: Bohuslav Uher) Fig. 6: The subaerial green alga Clorosarcinopsis cf. arenicola (author: Bohuslav Uher)
Fig. 2: 1 – Preparing the basic concentration in ratio 1:2, agarised BBM medium :
sterile powder Calcium Ruthenium Oxide Hydrate; 2 – scheme of the new inhibition
test, Petri dishes with diameter 3.5 cm and 5 inoculation per agar plate, first row of
Petri dishes is powder negative, the next rows with powder in mixture with different
ratio. (author: Bohuslav Uher)
Fig. 3: The results of El estuco experiment (author: Bohuslav Uher)