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POTENTIOMETRIC MICROSENSORS FOR CYANOBACTERIAL
BIOFILMS MONITORING IN HYPOGEAN MONUMENTS Calvo
Quintana J1., Piermarini S.1, Albertano P.2,
Palleschi G .1, Moscone D. 1 1Dept. of Chemical
Sciences and Tecnologies 2Dept. of
Biology University of RomeTor Vergata, Italy
Introduction
Recently the need of non-invasive methods for the
conservation of hypogea and caves has encouraged
studies on the physico-chemical methods aimed at
controlling the development of phototrophic
microrganisms. Cyanobacteria are the major
organisms responsible for biofilm formation on
any hard surface exposed to the light in Roman
Catacombs (Fig.1). The strategies for the
adhesion to the stone seemed to be generally
based on the production and secretion at the cell
surface of mucilaginous compounds (exopolymeric
substances). One species, Scytonema julianum, is
of particular interest because of its ability to
precipitate carbonate crystals from surfaces on
its polysaccharide sheath (Fig.2).
The growth of these microbial films, as a result
of their photosynthetic and respiratory activity,
can induce more or less pronounced variation of
the chemical parameters that characterise the
microhabitat, and possibly cause deteriogenic
effects on the colonised substrate. The use of
microelectrodes in biofilm studies has greatly
increased our knowledge of the importance of pH
and diffusion gradients in controlling the
distribution of different microbial population in
this complex communities.
Microsensor construction
Applications on cyanobacterial biofilm
Potentiometric microsensors have been constructed
to study the concentration and variation of pH,
potassium and calcium in cyanobacterial biofilm
during dark-light periods. They have been
assembled pulling glass capillaries to obtain
tips with a diameter of 10-20 mm, silanising them
and filling the tips with liquid membranes
specific for each ions. To obtain more rugged
sensors, additional layers were added to the
specific liquid membrane, consisting in PVC,
cellulose acetate and bovine serum albumin plus
glutaraldehyde. The sensors so assembled showed
many advantages ?increased life time
?reproducibility of measured values of potential
?dry storage ?more stable and less noisy
signal ?short response time ?strong membrane
keeping ?high resistance to the biofilm damaging
effect ?possibility to perform measurements on
every kind of sample
The measurements were carried out both on
cyanobacterial strains cultured in laboratory and
on natural biofilm collected from Roman
Catacombs. They were submitted at light cycles of
increasing irradiance, and the pH, K and
Ca variations were continuously recorded
during the whole experiment.
pH measurements have been carried out at
incresing irradiance in parallel with
measurements of oxygen on three strains of
Scytonema (Fig.4a), isolated from Roman
Catacombs, and on natural biofilm (Fig.4b). The
results showed that in the transition from dark
to 1100 mmol photons m-2 s-1, values of H
increased according to the irradiance, starting
from values slightly below the neutrality.
Within the concentration range tested, the
microelectrodes showed a linear slope of 57 mV
(pH and potassium microelectrodes) and of 28 mV
(calcium microelectrode) for a tenfold variation
of concentration. Moreover, they presented a good
reproducibility of the values of potential
between different days. Each electrode has been
continuously used for more then fifteen days
without loss sensitivity.
Concerning K and Ca variations, different
behaviour were showed by natural biofilms, which
resulted no sensitive to the light variations.
However, the potassium and calcium values
measured in biofilm (Tab.1) resulted much higher
(20-50 times) in comparison with the
concentrations measured in the strains (data not
shown).
Conclusion
Potentiometric microsensors were constructed and
usefully employed to asses and quantify the
mobilisation of these ions by cyanobacterial
biofilm in the substrate and can be further
applied for the evaluation of the
biotransformation processes of lithic substrata
caused by the growth of biofilm-forming
cyanobacteria in Roman Catacombs.
Fig.3. a) Scheme of a potentiometric electrode.
Calibration curves of (b) pH microsensor in the
pH range 4 - 10 and (c) in standard solutions
10-1 - 10-5 M KCl (?) and CaCl2 (?), respectively
for potassium and calcium microelectrodes.
Acknowledgement This work was supported by the
financial contribution of the National Research
Council of Italy, C.N.R. - Progetto Finalizzato
Beni Culturali, grant 99 .03688.PF.36. and by EU
Energy Programme, Environment and Sustainable
Development in the frame of CATS contract n0
EVK4-2000-00028.