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Contract n° |
EVK4-CT-2000-00028 |
Reporting period: |
1/1/2001- 31/12/2003 |
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Title |
CATS - Cyanobacteria Attack Rocks: control and preventive strategies to avoid
damage caused by cyanobacteria and associated microorganisms in Roman
hypogean monuments |
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Objectives: The overall objective of CATS was to achieve a
better understanding of biotransformation and biodecay processes of lithic substrata caused by the growth of
biofilm-forming cyanobacteria in hypogean monuments, and to evaluate the
applicability of a two-phase (physical plus biotechnological) strategy to
decrease and inhibit the growth of phototrophic and heterotrophic
microorganisms that cause severe damage mostly to calcareous rock surfaces in
Roman hypogea. The specific
objectives of the project were: - to characterise the
geological, geochemical, hydrochemical and physical environment of rocks
unaffected or colonised by cyanobacterial communities inside Roman hypogea,
and to evaluate possible preferences of cyanobacteria and associated
microorganisms for specific
lithologies; - to describe the
architecture and functioning of biofilms built by cyanobacteria and
associated microorganisms on different types of lithic surfaces; - to ascertain the most
critical physical, chemical and biological factors that control colonisation
of rock surfaces; - to assess and quantify
the damage caused by cyanobacterial biofilms to different types of surface; - to develop new physical
methods to control and prevent biofilm growth using wavelengths in the
visible part of the light
spectrum that are, at best,
poorly used by photosynthesis; - to identify siderophores
and cell-to-cell signalling biomolecules and experimentally to test their
potential to interfere with biofilm development; - to develop an innovative
monitoring method using a multiparametric microsensor device for the
measurement of biogeochemical parameters on endangered rock surfaces; - to test the response and
expectation of citizens to the innovative strategies proposed. Scientific achievements: During the three years research, sampling
campaigns have been organised in two Roman hypogea (Catacombs of Domitilla
and St. Callistus in Rome, Italy) and in a pre-historical cave (Cave of Bats
in Zuheros, Spain). These campaigns led to the collection of physical,
chemical, mineralogical and biological data on rock surfaces and biofilms there
developing. The geological setting of the sampling sites has been studied and
the structure of both unaffected and colonised rock
surfaces characterised. The catacomb host rock was soft and highly
porous (around 40%), consisting on volcanic glasses and alumino-silicate
minerals like feldspars, plagioclase (orthoclase-Ba) as well as the products
of its diagenetical alteration: zeolites (analcime), carbonates (calcite),
gypsum and clays (biotite, illite). Substrata deterioration was due both to
inorganic and organic processes. Destructive and constructive processes have
been observed in particular at substratum/biofilm interface. Microbial
colonisation of the rocks was characterised by a biological succession. In
well-illuminated areas and mainly close to the lamps, a great development of
green cyanobacterial biofilms was observed. These areas daily suffered strong
environmental changes involving: (1) high increase of the air and rock
temperature induced by the lit lamp; (2) evaporation of available water and
decrease of air relative humidity, that raises the saturation water vapour
pressure in the air; (3) total air humidity and CO2 air
concentration increase induced by visitors; (4) rapid decrease of temperature
when turning off the lamp, that causes fast condensation of acid CO2-enriched
water on substrata and biofilms; (5) micro-corrosion of calcareous substrata
(stuccoes) dissolving soluble minerals (i.e. calcite) and increasing
roughness. Condensation and microdissolution processes on rocks and artificial
building materials favour the accessibility of microorganisms to nutrients
and the spreading of microbial
communities. A preference of microorganisms
for the substrata richest in calcium carbonate, i.e. frescoes and stuccoes,
has been assessed. In addition, light intensity gradients have been measured
at all sampling sites and differences in the biofilm ability to absorb and
reflect light of different wavelength have been detected in Domitilla and St.
Callistus using non-destructive spectroradiometric methods. These results
indicated that the wavelengths required by
cyanobacteria to perform photosynthesis span over the all visible spectrum
with the exception of reduced absorbance in narrow bands of the blue and
green region.
The biofilm architecture was studied using novel CLSM methods appositely
developed, and different biofilm typologies were described on the basis of
phototrophic species composition and distribution within the microbial
community. An intimate relationships between phototrophs and
heterotrophs within the biofilms in catacombs was observed. Biodiversity has
been determined both in natural samples and in cultures using a combination
of microscopy and PCR-based techniques. The taxonomic position of phototrophs
and associated heterotrophic bacteria has been defined using phenotypic and
genotypic methods. A total of about 50 strains of phototrophic and more than
1000 of heterotrophic microorganisms has been isolated and is conserved in
participant culture collections. Microsensors have been constructed and
experimentally applied for oxygen, pH, calcium, potassium, ammonium, nitrate
and phosphate measurements. The respiratory and photosynthetic capability of
biofilms has been assessed at increasing irradiances showing low values of
photosynthesis maxima yet high efficiencies, as expected for these
sciaphilous phototrophic communities. During the illumination time, the
increase of pH was shown to almost parallel the increase of oxygen evolution,
while calcium and potassium concentrations did not vary and showed a
light-independent response. Although the high variability found between
biofilms, potassium and calcium mean values measured in natural biofilms
resulted 20-50 times higher when compared with monospecific biofilms
cultured in laboratory, while the average ammonium concentration was below
the millimolar range, nitrate mean concentration was 4-20 millimolar and the
amount of phosphate was 50-360
mg phosphorous/g dry weigth.
These data have been used to design and construct the multiparametric
“Portable Sensor Monitor” that was applied to measure threshold values of
ionic species within cyanobacterial biofilms and uncolonised calcareous
slides previously placed at the sampling sites and removed at intervals,
after in situ development of
biofilms. Once transferred to standard laboratory conditions, slides were
exposed to a range of different wavelengths provided by monochromatic lamps
to assess the effect of light quality on biofilm viability. A new non
destructive methods based on the use of a portable spectrorameter was
developed to detect phototrophic colonisation and growth and to characterise
lamp emission. The results achieved were used to set up the experimental
installation of blue and green monochromatic lamps in situ. The
response of citizen to the blue lighting system installed for some months
inside the Ocean’s cubiculum at the Catacombs of St. Callistus was collected.
A total of 1500 questionnaires was statistically evaluated and these data
revealed a positive answer of respondents to the possible application of
innovative strategies to limit the development of biofilms in archaeological
areas. Socio-economic relevance and policy implications: The problem of conservation, restoration and exploitation
of Roman hypogea is part of the more general need to safeguard of the
Cultural Heritage of Europe. This heritage has a significant influence on the
economy of nations rich in archaeological remains, including most of the
Mediterranean countries, and influences two main socio-economic factors: the
significant amount of human and financial resources needed to preserve
important archaeological sites and the improvement of both tourism and the
quality of life through a sustainable management of the artistic patrimony of
Europe. The results achieved by the CATS project can, therefore, have an
economic relevance for both European and Mediterranean countries,
particularly concerned with safeguarding of monuments. In this sense, CATS
developed tools and methodologies to provide conservation and restoration
managers with a better understanding of the development of
cyanobacteria-dominated biofilms and therefore an ability to predict the
development of biofilm-induced damage. In addition, new strategies became
available for the monitoring and control of rock biodecay. In fact, through
CATS, we obtained, for the first time, data that provide evidence (a) of the
type of damage produced on rock surfaces through biological activity in Roman
hypogea and (b) on the identity of the major factors that control the
development and growth of biofilms, their biodiversity and function. Protection and management
of the artistic legacy found in hypogean monuments has been addressed by a
complete, and complex multidisciplinary scientific study. CATS approached the
problem by including geological, geochemical, hydrochemical, microclimatic,
environmental, microbiological, ecophysiological, biochemical and genetic
studies. The combination of all these data has been used to construct models
of the complexity of the hypogean environment, of deterioration processes, of
biofilm architecture and of the biological activity occurring on lithic
faces. In addition, the research has determined that changing the wavelengths
used for illumination cause a decrease in the growth of unwanted
cyanobacteria and that active biomolecules are potentially useful to prevent
and control the development of cyanobacteria-dominated biofilms. In this
context, new microscopy methods have been developed for the study of biofilm
structure, function and diversity that can be applied to other microbial
communities. Furthermore, the development of non-destructive and safety
methods for monitoring of cyanobacterial biofilms has been accomplished
through the development of a “Portable Sensor Monitor” holding together
different microsensors for the measurement of threshold values of
chemical species on endangered
stones, and the establishment of a promising new methodology based on the use
of a portable spectroradiometer to detect phototrophic biofilm development
and growth. CATS also focused on other
aspects of biodeterioration in Roman hypogea. The influence of visitors was
continuously monitored at one site in St. Callistus catacomb and it has been
shown to be relevant in changing microclimatic conditions that influence
water condensation and, in turn, biological colonisation. The
characterisation of lithic faces resulted in the understanding of Roman
procedures for plaster preparation and allowed to recommend similar old
material for restoration intervention in catacombs. The use of a biological
anti-cyanobacterial strategy was never attempted before, and the possibility
to apply new alternative biomolecules that interfere with biofilm formation
will represent a future development. Microorganisms, abundantly detected in
air samples, can attach to exposed surfaces and there establish microbial
communities thanks to the favourable environmental conditions. The study of
exopolymeric substances is, therefore, extremely promising because of the
role of these biopolymers in biofilm adhesion and cohesion, and stone decay
and could lead to future “bio-cleaning” procedures. Microorganisms causing
decay of lithic surfaces in Roman hypogea were poorly known. Most of them
have been identified using new PCR-based molecular tools, isolated and
characterised in culture. Therefore, CATS addressed also the issue of
preservation of environmental biodiversity in Roman hypogea. A further output
from this project is the large number of clonal isolates of heterotrophic
bacteria. There is therefore the potential for future exploitation of the
organisms and challenge for global market of such genetic resources. The
incorporation of the two-phase methodology, that was pursued by the CATS project,
within under- and postgraduate courses in restoration, environmental and
biological sciences will endeavour to improve knowledge and understanding of
the problems and implication connected to the managing of archaeological
sites and conservation. While sensor technology is inherently perceived as
simplicistic in nature, biotechnology is still an obscure matter for most
people. However, the application of the two-phase strategy to the safeguard
of cultural heritage can provide opportunities to increase the knowledge base
and understanding of the workforce. The more techno- and biotechnologically
advanced the workforce the greater the understanding of the safety issues and
willingness to overcome conservation and environmental problems. Conclusions: CATS consortium is being, therefore, proposing
methods that are not harmful to the environment and contributing to increase
knowledge of (new) organisms. Moreover, the feed-back of the public (the
final end-users) towards the new lighting technique in hypogean
archaeological areas has been evaluated for the first time and the positive
feeling expressed by the catacomb visitors has hopefully initiate a new means
of interaction with citizens. Therefore, most of the CATS results provide
possible solution for the conservation of Roman hypogea. In addition,
methodologies developed in the frame of CATS can be adapted to apply to any
other archaeological or historical site at which biological decay of rock
surfaces is occurring. This might generate new employment for highly skilled
technical staff members of SMEs active in the cultural heritage domain of
conservation and promote a sustainable use of cultural resources on the basis
of which touristic activities, new infrastructures and job opportunities
could develop. Expert input from end-users
in different EU Member States can be now expected for a commercial
development in the market of sustainable technologies for the safeguarding of
cultural heritage. As a commitment for improving the social and economical
cohesion, links between the CATS consortium and other SMEs and public
authorities will be developed during the next years. In medium and long term,
results achieved within the CATS project will be relevant to the EU policy on
the conservation of the European cultural heritage. Keywords: phototrophic biofilms,
cyanobacteria, hypogea, lighting systems, microsensors, quorum sensing
molecules, siderophores, environmental biotechnologies, microorganism
monitoring, stone conservation. |
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