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In this study, we combined chlorophyll a fluorescence (ChlaF) measurements, using pulseamplitude-modulate (PAM) equipment, with scanning electron microscopy in backscattered electron mode (SEM-BSE) and transmission electron microscopy (TEM) images to evaluate the actions of Koretrel at lower concentrations on Verrucaria nigrescens colonising a dolostone. ChlaF measurements are good indicators of the damaging effects of biocides. However, these indicators only provide an incomplete view of the mechanism of biocides used to control biodeterioration agents. The death of the V. nigrescens photobiont at two biocide concentrations was revealed by PAM, SEM-BSE and TEM. Once Koretrel was applied, the Fv/Fm ratios markedly fell in the first few hours after the 1.5% treatment, and ratios for the 3% dilution remained close to zero throughout the study. The algal zone shows the plasmolysed appearance of the photobiont cells, and important aspects related to the action of the biocide on free and lichenised fungi were also detected using SEM-BSE. Many of the mycobiont cells had only their cell walls preserved; although, some fungal hyphae in lichen thalli and some microorganisms in endolithic clusters maintained lipid storage in their cytoplasm. These results indicated that the combination of physiological and microscopy techniques improves the assessment of biocide action in situ and this will help to optimize protocols in order to reduce the emission of these compounds to the environment.

This paper reports on the differential elimination of surface lichen thalli and their penetrating hyphae and epilithic and endolithic cyanobacteria by two biocide treatments applied on a small scale to the cloister of the Segovia cathedral. Both chemical treatments were followed by mechanical cleaning, and their effects were determined by scanning electron microscopy with backscattered electron imaging (SEM–BSE). Both treatments were effective at killing epilithic lichens and cyanobacteria, although they showed different effects on endolithic microbiota. The cleaning treatment involving Biotin T, dissolved in water, applied to areas extensively colonized by crustose lichens, resulted in a cleaner stone surface that was practically free of surface epilithic lichen remains, but the presence of living fungal hyphae was detected in fissures of the stone under these remnants in samples examined two months after treatment. The treatment using Biotin R, dissolved in White Spirit, left more superficial lichen remains but was better at eliminating mycobiont hyphae penetrating the stone. Endolithic cyanobacteria from zones showing green and black colourations were also more affected by Biotin R treatment. Our SEM–BSE observations highlight the need for a complete evaluation of treatment effects, including those produced on both epilithic lichen thalli and their penetrating components and on epilithic and endolithic microbiota. The causes and consequences of the different efficacy of treatments observed, as well as the potential risks of recolonization by viable cells left behind after treatment, are discussed.

This article reviews current knowledge on microbial communities inhabiting endolithic habitats in the arid and hyper-arid regions of our planet. In these extremely dry environments, the most common survival strategy is to colonize the interiors of rocks. This habitat provides thermal buffering, physical stability, and protection against incident UV radiation, excessive photosynthetically active radiation, and freeze-thaw events. Above all, through water retention in the rocks' network of pores and fissures, moisture is made available. Some authors have argued that dry environments pose the most extreme set of conditions faced by microorganisms. Microbial cells need to withstand the biochemical stresses created by the lack of water, along with temperature fluctuations and/or high salinity. In this review, we also address the variety of ways in which microorganisms deal with the lack of moisture in hyper-arid environments and point out the diversity of microorganisms that are able to cope with only the scarcest presence of water. Finally, we discuss the important clues to the history of life on Earth, and perhaps other places in our solar system, that have emerged from the study of extreme microbial ecosystems.

This study examines the microbial colonization of three fronts of an abandoned dolostone quarry (Redueña, Madrid, Spain) exposed to atmospheric conditions for different time periods since Roman times to the present. Through scanning electron microscopy in backscattered electron mode (SEM-BSE), endolithic colonization was predominantly detected in the most recently exposed front, while in the longer exposed quarry fronts, epilithic forms of growth were most often observed. These observations were confirmed by denaturing gradient gel electrophoresis (DGGE) analysis. Based on the distribution pattern of microbial colonization in the different quarry fronts, we then established a sequence of colonization events that took place over this long time frame. Bioalteration processes related to this sequential colonization were also identified. Characterizing these sequential processes can be useful for interpreting biodeterioration processes in historic dolostone monuments, especially those affecting constructions in the area of the Redueña stone quarry. In a second experimental stage, different biocide treatments were tested on this quarry rock to find the best way to avoid the microbial colonization effects identified. Through combined SEMBSE/ DGGE analysis, the efficacy of several biocides against the microorganisms inhabiting the dolostones was assessed after 4 and 16 months treatment. In general, all treatments were effective at reducing around 80% of the lichen cover, although effects on endolithic lithobiontic communities were dependant on how well the rock surface had been mechanically cleaned prior to treatment and gradually disappeared over time.

The ochreous speleothems examined here were obtained from a lava tube on Terceira Island (Azores) and show compact mineralized areas intermixed with zones in which bacterial structures are evident. Bacterial layers of filament-like structures are common throughout the deposits but differences in microstructure among the different speleothems were observed, reflecting a broad morphological range of deposits. The structures and minerals detected in the speleothems betray their biogenic origin. Gallionella and Leptothrix were the two most frequently observed morphotypes and probably the main contributors to speleothem formation. However, DGGE analysis indicated the presence of another bacterial population (with a predominance of proteobacteria) that could also contribute to iron hydroxide–oxide precipitation. The sheaths of Leptothrix cells and stalks of Gallionella cells were associated with large amounts of extrapolymeric substances (EPS), which play a role in biomineralization processes. Independently of the taxa present, mineral deposits were composed of poorly ordered Si-rich ferrihydrite, a typical mineral phase of biogenic Fe precipitates. In this microscopy study of interrelationships between mineral precipitates and associated microorganisms and their structures, we attribute bacteria to have an important role in constructing these speleothems. Through their metabolic activity, these bacteria cause the precipitation of ferrihydrite but their mineral structures could also act as nucleation points for passive mineral precipitation. Finally, the build-up of bacterial mineral structures and later cementation processes seems responsible for the formation of mineral layers that confer consistency to the speleothem. Our findings point to bacterial activity as the main factor determining speleothem structure and formation.

The hyper-arid core of the Atacama Desert (Chile) is the driest place on Earth and is considered a close analogue to the extremely arid conditions on the surface of Mars. Microbial life is very rare in soils of this hyper-arid region, and autotrophic microorganisms are virtually absent. Instead, photosynthetic micro-organisms have successfully colonized the interior of halite crusts, which are widespread in the Atacama Desert. These endoevaporitic colonies are an example of life that has adapted to the extreme dryness by colonizing the interior of rocks that provide enhanced moisture conditions. As such, these colonies represent a novel example of potential life on Mars. Here, we present non-destructive Raman spectroscopical identification of these colonies and their organic remnants. Spectral signatures revealed the presence of UV-protective biomolecules as well as light-harvesting pigments pointing to photosynthetic activity. Compounds of biogenic origin identified within these rocks differed depending on the origins of specimens from particular areas in the desert, with differing environmental conditions. Our results also demonstrate the capability of Raman spectroscopy to identify biomarkers within rocks that have a strong astrobiological potential.

Hygroscopic salts have been detected in soils in the northern latitudes of Mars, and widespread chloride-bearing evaporitic deposits have been detected in the southern highlands. The deliquescence of hygroscopic minerals such as chloride salts could provide a local and transient source of liquid water that would be available for microorganisms on the surface. This is known to occur in the Atacama Desert, where massive halite evaporites have become a habitat for photosynthetic and heterotrophic microorganisms that take advantage of the deliquescence of the salt at certain relative humidity (RH) levels. We modeled the climate conditions (RH and temperature) in a region on Mars with chloride-bearing evaporites, and modeled the evolution of the water activity (aw) of the deliquescence solutions of three possible chloride salts (sodium chloride, calcium chloride, and magnesium chloride) as a function of temperature. We also studied the water absorption properties of the same salts as a function of RH. Our climate model results show that the RH in the region with chloride-bearing deposits on Mars often reaches the deliquescence points of all three salts, and the temperature reaches levels above their eutectic points seasonally, in the course of a martian year. The aw of the deliquescence solutions increases with decreasing temperature due mainly to the precipitation of unstable phases, which removes ions from the solution. The deliquescence of sodium chloride results in transient solutions with aw compatible with growth of terrestrial microorganisms down to 252 K, whereas for calcium chloride and magnesium chloride it results in solutions with aw below the known limits for growth at all temperatures. However, taking the limits of aw used to define special regions on Mars, the deliquescence of calcium chloride deposits would allow for the propagation of terrestrial microorganisms at temperatures between 265 and 253 K, and for metabolic activity (no growth) at temperatures

In the space experiments Lithopanspermia, experimental support was provided to the likelihood of the lithopanspermia concept that considers a viable transport of microorganisms between the terrestrial planets by means of meteorites. The rock colonising lichens Rhizocarpon geographicum and Xanthoria elegans, the vagrant lichen Aspicilia fruticulosa, and endolithic and endoevaporitic communities of cyanobacteria and bacteria with their natural rock substrate were exposed to space for 10 days onboard the Biopan facility of the European Space Agency (ESA). Biopan was closed during launch and re-entry. In addition, in the Stone facility, one sample of R. geographicum on its natural granitic substrate was attached at the outer surface of the re-entry capsule close to the stagnation point, only protected by a thin cover of glass textolite. Post-flight analysis, which included determination of the photosynthetic activity, LIVE/DEAD staining, and germination capacity of the ascospores, demonstrated that all three lichen were quite resistant to outer space conditions, which include the full spectrum of solar extraterrestrial electromagnetic radiation or selected wavelength ranges. This high resistance of the lichens to space appears to be due to their symbiotic nature and protection by their upper pigmented layer, the cortex. In contrast, the rock- or halite-inhabiting bacteria were severely damaged by the same exposure. After atmospheric re-entry, the granite of the Stone sample was transformed into a glassy, nearly homogenous material, with several friction striae. None of the lichen cells survived this re-entry process. The data suggest that lichens are suitable candidates for testing the concept of lithopanspermia, because they are extremely resistant to the harsh environment of outer space. The more critical event is the atmospheric re-entry after being captured by a planet. Experiments simulating the re-entry process of a microbe-carrying meteoroid did not show any surv

Algae are primary producers in a wide variety of natural ecosystems, and these microorganisms have been used in bioremediation studies. Nevertheless, very little is known about the in vivo effect of heavy metals on individual living cells. In this paper, we have applied a method based on confocal laser scanning microscopy and lambda scan function (CLSM- scan) to determine the effect of lead (Pb), at different concentrations, on the DE2009 microalga. At the same time, we have optimized a method based on CLSM and image-analysis software (CLSM-IA) to determine in vivo biomass of this microorganism. The results obtained by lambda scan function indicated that the pigment peak decreases while the concentration of metal increases at pH 7. On the other hand at pH 4 there is no good correlation between the concentration of metal and the intensity of the emission of fluorescence of the pigment. Also, in some cases a displacement of the Chl a peak towards 680nm is produced. Total and individual biomass determined by CLSM-IA shows statistically significant differences between unpolluted and 10mM polluted cultures. Complementary studies using electron microscopy techniques coupled to energy dispersive X-ray microanalysis (EDX) demonstrate that the microalga can sequestrate Pb extra- and intracellularly.

In this study, the deterioration effects of lichens and other lithobionts in a temperate mesothermal climate were explored. We examined samples of dolostone and limestone rocks with visible signs of biodeterioration taken from the exterior wall surfaces of four Romanesque churches in Segovia (Spain): San Lorenzo, San Martín, San Millán and La Vera Cruz. Biofilms developing on the lithic substrate were analyzed by scanning electron microscopy. The most common lichen species found in the samples were recorded. Fungal cultures were then obtained from these carbonate rocks and characterized by sequencing Internal Transcribed Spacers (ITS). Through scanning electron microscopy in back-scattered electron mode, fungi (lichenized and non-lichenized) were observed as the most frequent microorganisms occurring at sites showing signs of biodeterioration. The colonization process was especially conditioned by the porosity characteristics of the stone used in these buildings. While in dolostones, microorganisms mainly occupied spaces comprising the rock's intercrystalline porosity, in bioclastic dolomitized limestones, fungal colonization seemed to be more associated with moldic porosity. Microbial biofilms make close contact with the substrate, and thus probably cause significant deterioration of the underlying materials. We describe the different processes of stone alteration induced by fungal colonization and discuss the implications of these processes for the design of treatments to prevent biodeterioration.

La colonización biológica induce en la piedra además de daños estéticos, una serie de procesos de alteración mecánica y química que generan biodeterioro del monumento. El grupo de investigación de Ecología Microbiana y Geomicrobiología del sustrato Lítico, del Instituto de Recursos Naturales del CSIC, cuenta con una estrategia de actuación para llevar a cabo el diagnóstico de los fenómenos del biodeterioro de piedra monumental en la que por combinación de técnicas de microscopía in situ y de biología molecular, es posible analizar los procesos de alteración producidos, los microorganismos implicados, y con ello, plantear y posteriormente evaluar tratamientos encaminados a eliminar o reducir los fenómenos de biodeterioro.

This experiment was aimed at establishing, for the first time, the survival capability of lichens exposed to space conditions. In particular, the damaging effect of various wavelengths of extraterrestrial solar UV radiation was studied. The lichens used were the bipolar species Rhizocarpon geographicum and Xanthoria elegans, which were collected above 2000 m in the mountains of central Spain and as endolithic communities inhabiting granites in the Antarctic Dry Valleys. Lichens were exposed to space in the BIOPAN-5 facility of the European Space Agency; BIOPAN-5 is located on the outer shell of the Earth-orbiting FOTON-M2 Russian satellite. The lichen samples were launched from Baikonur by a Soyuz rocket on May 31, 2005, and were returned to Earth after 16 days in space, at which time they were tested for survival. Chlorophyll fluorescence was used for the measurement of photosynthetic parameters. Scanning electron microscopy in back-scattered mode, low temperature scanning electron microscopy, and transmission electron microscopy were used to study the organization and composition of both symbionts. Confocal laser scanning microscopy, in combination with the use of specific fluorescent probes, allowed for the assessment of the physiological state of the cells. All exposed lichens, regardless of the optical filters used, showed nearly the same photosynthetic activity after the flight as measured before the flight. Likewise, the multimicroscopy approach revealed no detectable ultrastructural changes in most of the algal and fungal cells of the lichen thalli, though a greater proportion of cells in the flight samples had compromised membranes, as revealed by the LIVE/DEAD BacLight Bacterial Viability Kit. These findings indicate that most lichenized fungal and algal cells can survive in space after full exposure to massive UV and cosmic radiation, conditions proven to be lethal to bacteria and other microorganisms. The lichen upper cortex seems to provide adequate pro

Using electron microscopy techniques (SEM, LTSEM) coupled with analytical methods (XRD and EDS) the role of phosphorus has been assessed in the formation of freshwater calcite deposits (tufa) in a small pond of the Ruidera Lakes (Spain). Differences between the cell walls and sheaths of bacteria and eukaryotic algae as well as the existence of additional layers of extracellular polymeric substances (EPS) were features that lead to differences in the process of induced calcite biomineralization. Phosphorus has influence in the biomineralization of the EPS, sheaths and cell walls of cyanobacteria allowing for fossil preservation whereas does not participate in the calcite precipitation around algae and mosses. This variability may explain the different positive or negative roles played by natural or artificial inputs of phosphorus in hard water lakes and the different morphological features of calcite precipitates associated with eukaryotic and cyanobacteria picoplankton found in natural environments. The biomineralization observed is in agreement with the isotopic composition of the tufa layers that reflect the variations in environmental conditions around biological communities.

The present study was designed to gain insight into the protists and fungi that made up the microbiota in the past, fossilized in two different substrates: amber and sandstone. The amber, dated as Lower Cretaceous, was from Álava in northern Spain, while fossil- bearing sandstone rocks were collected from the Linnaeus terrace and Mount Fleming regions of Antarctica. When examining this type of inclusion in hard substrates, it first has to be established whether the microorganism is mummified or only partially mineralized. In the latter case, some of the organism’s autofluorescence may be preserved. In our amber samples, light microscopy revealed a very well preserved microcenosis in what must have been a semiaquatic habitat comprised of several types of protozoa including Amoeba, Paramecium and Astasia (Euglena), Amebas limax and the colpodid ciliate Pseudoplatyophrya nana, as well as an abundant fossilized mycelium. The SEM-BSE procedure provided us with ultrastructural details of the fungi and protozoa, especially amoebae and flagellates. In the sandstone samples from Linnaeus terrace, it was possible to identify presumptive diatoms. Ultrastructural details were well-preserved in a fossil Trebouxia-type microalga fromMount Flemming, including the inner chloroplast area normally occupied by the pyrenoid. This fossilized microalga was shown by energy dispersive spectroscopy (EDS) to contain high Si levels in the pyrenoid zone, and high Fe levels in the area corresponding to the chloroplast periphery, peripheric cytoplasm and cell wall. In sandstone from Linnaeus Terrace, fossilized algae showed no central core containing Si, and the entire cell appeared to be completely filled with Al, K and Fe, with Fe most intensely observedwithin the algal wall.The present observations suggest that awell-preserved cell ultrastructure is the best criterion of biogenicity.

In this study, the symbiont cells of several endolithic lichens colonizing granite in continental Antarctica and the relationships they have with the abiotic environment were analyzed in situ, in order to characterize the microecosystems integrating these lichens, from a microecological perspective. Mycobiont and photobiont cells, the majority classified as living by fluoresecent vitality testing, were observed distributed through the fissures of the granite. The fact that extracellular polymeric substances were commonly observed close to these cells and the features of these compounds, suggest a certain protective role for these substances against the harsh conditions of the environment. Different chemical, physical and biological relationships take place within the endolithic biofilms where the lichens are found, possibly affecting also the survival and distribution of these organisms. The alteration of bedrock minerals and synthesis of biominerals in the proximity of these lichens gives rise to different chemical microenvironments and suggests their participation in mineral nutrient cycling.

Through the combined use of molecular and microscopy techniques, the endolithiclichens Lecidea cancriformis and Lecidea sp. were identified, even in the absence offruiting bodies, and positioned under epilithic lichens. Cells of both algal and fungalsymbionts were observed in fissures and cracks of the lithic substrate with no clearheteromerous structure. At the ultrastructural level, the two lichens differed in termsof their algal–fungal relationships. Only one genotype of Trebouxia ITS sequence was identified from specimensof Lecidea sp., Umbilicaria aprina and Buellia frigida from the same zone,which could be mainly determined by low availability of alga in these extremeenvironments. These lichens showed features typical of both chasmoendolithic and euendolithicmicroorganisms. Signs of biogeophysical and biogeochemical action on thesubstrate were detected close to fungal cells. This action seemed to be mainlyconditioned by the local physico-chemical features of the substrate. Evidencefor the biomobilization of elements by these endolithic lichens was found. L. cancriformis was observed to accumulate substantial amounts of calcium-richbiominerals. The combined approach proposed is useful for mapping the distribution of endolithiclichens and analysing the processes that occur in their microscopic environment.

In situ microscopy consists of simultaneously applying several microscopy techniques without separating the biological component from its habitat. Over the past few years, this strategy has allowed characterization of the biofilms involved in biodeterioration processes affecting stone monuments and has revealed the biogeophysical and biogeochemical impact of the microbiota present. In addition, through in situ microscopy diagnosis, appropriate treatments can be designed to resolve the problems related to microbial colonization of stone monuments., La microscopía in situ consiste en aplicar simultáneamente varias técnicas de microscopía sin separación de los componentes biológicos de su hábitat en la roca. Durante los últimos años, esta estrategia ha permitido caracterizar las biopelículas implicados en los procesos del biodeterioro que afectan los monumentos de piedra y ha revelado el impacto biogeofísico y biogeoquímico de la microbiota presente. Además, el diagnóstico mediante microscopía in situ permite diseñar tratamientos apropiados para resolver los problemas relacionados con la colonización microbiana de los monumentos de piedra.

Microbial life in the hars conditions of Antarctica's cold desert may be considered an analogue of potential life on early Mars. The microbial ecology of these lithobiontic, extremophile microorganism communities could provide clues to the challenging question of how life (if ever oresent) became extinct on Mars. Application of the SEM-BSE plus EDS technique has allowed us to demonstrate the presence of microbial fossils and biomarkers within Antarctic sandstone rocks collected from Ross Desert for the first time. There is an obvious need for extensive further work on live, dead, mummified and mineralized lithobiontic Antarctic microorganisms. The in situ examination of the interior of Antarctic rocks might represent the best option available to improve our knowledge on these extreme cold and dry microbial habitats and it is foreseen that this type of work will have applications in future astrobiological investigations performed on geological material obtained from Mars.

In this microscopy study, we show that microorganisms comprising many endolithic communities of the McMurdo Dry Valleys of Antarctica appear in different physiological states. Live/dead microbial fluorescence stains were used to identify the state of microorganisms in the biofilms. The ultrastructures of these microorganisms were then characterized by transmission electron microscopy. Cyanobacteria were associated with heterotrophic bacterial cells, while fungal cells were free-living or formed partners with green alga as lichens. The extracellular polymeric substances, in which the endolithic microorganisms were embedded, formed an integral part of the biofilms observed. Extracellular polymeric substances probably play a significant role in nutrient interactions and protection of microorganisms from the environmental conditions outside the film. Living, moribund, dormant and dead microorganisms shared this microhabitat. The ecological impacts of the observed physiological dynamics are discussed.

The rocks of the McMurdo Dry Valleys desert in Antarctica harbour endolithic communities of micro-organisms such as lichens, fungi, cyanobacteria and bacteria. Establishing the physiological status and viability of these microbial colonies in their natural microhabitat has far-reaching implications for understanding the microbial ecology of the harsh environment of this polar desert. Here we describe the use of confocal microscopy and a specific fluorescent probe (FUN-1) to evaluate the metabolic activity of fungal cells. Application of confocal microscopy also served to identify living and dead bacteria or cyanobacteria using the fluorescent assay reagents Live/Dead SYTO 9 and propidium iodide or SYTOX Green, respectively. In addition, through the use of epifluorescence microscopy, live/dead bacteria and cyanobacteria could be detected by estimating fluorescence from their cell components provoked by simultaneously staining with nucleic acids stains such as DAPI and SYTOX Green.

This report describes the in situ evaluation of biodeterioration processes occurring in the stone (granodiorite and carbonate) of the Convento de Santa Cruz la Real, Segovia, Spain. Scanning electron microscopy with back-scattered electron imaging (SEM–BSE) and 11 X-ray microanalysis were used to examine stone samples from the convent. Free-living algae and fungi, lichen thalli, mosses, cyanobacteria and bacterial colonies were observed. Some of these organisms were located epilithically, while others were found to inhabit ssures 13 or cavities inside the lithic substrate, a few of which showed an euendolithic ecological niche. Biogeomechanical and biogeochemical processes associated with the presence and action of lithobiontic microorganisms were detected, as well as evidence for the biomobilization 15 of elements by epilithic and endolithic microorganisms. The relative contribution of each lithobiont to these biodeterioration processess were found to vary according to the type of stone and its position in the monument.

The three-dimensional structures of two types of cyanobacterium-dominated microbial mats from meltwater ponds on the McMurdo Ice Shelf were as determined by using a broad suite of complementary techniques, including optical and fluorescence microscopy, confocal scanning laser microscopy, scanning electron microscopy with back-scattered electron-imaging mode, low-temperature scanning electron microscopy, and microanalyitical X-ray energy dispersive spectroscopy. By using a combination of the different in situ microscopic techniques, the Antarctic microbial mats were found to be structures with vertical stratification of groups of cyanobacteria and mineral sediments, high contents of extracellular polymeric substances, and large void spaces occupied by water. In cyanobacterium-rich layers, heterocystous nostocalean and nonheterocystous oscillatorialean taxa were the most abundant taxa and appeared to be intermixed with fine-size deposits of epicellular silica and calcium carbonate. Most of the cyanobacterial filaments had similar orientations in zones without sediment particles, but thin filaments were tangled among thicker filaments. The combination of the microscopic techniques used showed the relative positions of biological and mineral entities within the microbial mats and enabled some speculation about their interactions.

Antarctic endolithic microecosystems harbour distinct biofilms. The lithic substrate and the microorganisms comprising these films are intimately linked, leading to complex mineral–microbe interactions. Hence, the microhabitats and microenvironments of these microecosystems are not only determined by the physicochemical features of the lithic substrate, but are also conditioned by the biological components of these biofilms. The Antarctic biofilms analysed in this study are characterized by the presence of extracellular polymer substances and acid microenvironments in the proximity of the cells; cyanobacteria appearing as key components. On ultrastructural analysis, these endolithic cyanobacteria showed differences in sheath organization, probably related to their spatial position in the lithic substrate. It is proposed that in this type of ecosystem, biofilm structure could favour the formation of microsites with specific physicochemical conditions appropriate for the survival of microbial communities in this extreme environment.

Transmission electrón microscopy. Ultrastructural study of lichen symbionts can provide valuable information about symbiotic performance complementary to that obtained using other techniques (Lallemant et al. 1986). Transmission Electron Microscopy (TEM) was first used to describe the cellular organelles of both symbionts (Jacobs and Ahmadjian 1969; Boissiére 1972; Galun et al. 1970,1974; Peveling 1973, 1974, 1976; Ascaso and Calvan 1975, 1976). Later, different aspects of lichen symbiosis were studied, for example cellular membranes and cell wall with the freeze-etching electrón microscopy technique (Ellis and Brown 1972; Peveling and Robenek 1980; Ascaso et al. 1985; Honegger 1986a; Rapsch et al. 1986). TEM has contributed to the understanding of different types of mycobiont-photobiont relationships in lichens, e.g. by observing the physical contacts between symbionts (for reviews see Honegger 1984, 1985, 1986b). The study of storage bodies in both symbionts provides indirect information on biotrophic relationships (Valladares and Ascaso 1994). Some authors have described the variability of lichen ultrastructure in relation to season or environment (Holopainen 1982; Scott and Larson 1986; Fiechter and Honegger 1988; Balaguer et al. 1999). In some investigations, TEM techniques have revealed structural changes due to different experimental conditions, ranging from desiccation to environmental pollution (Eversman and Sigal 1984, 1987; Ascaso et al. 1986, 1988; Brown et al. 1987, 1988; Balaguer et al. 1996, 1997; Tarhanen et al. 1997).

Much of the Antarctic continent's microbiota is restricted to endolithic microecosystems which harbour distinct microbial communities as biofilms. The lithic substrate and the microrganisms comprising these films are intimately linked, giving rise to complex mineral-microbe interactions. The Antarctic biofilms analysed in this study were characterised by the presence of extracellular polymer substances. Cyanobacteria appeared as key components of these biofilms in zones where there were no nearby lichen thalli.

Rimularia insularis forms an photosynthetic thallus on lichens of the Lecanora rupicola group. In the thalli of R. insularis, some hyphae of the host are detectable, and they are situated mainly in the basal part of the Rimularia's medulla. No Lecanora hyphae were present in the Rimularia's algal layer and they were indistinct in the upper parts of its medulla, from where we obtained clean ITS-sequences of Rimularia. Direct PCR from various sections of the algal layer detected only one photobiont genotype, although the algal cells could be assigned to two morphologically different types. Calcium deposits were found in the upper parts of the medulla, while abundant rock substrate fragments with different mineral compositions were present in the lower parts. Cavities and ®ssures in the rock below the infected thalli were ®lled by fungal cells, PCR analyses of such parts indicated that the Rimularia hyphae extend below the host, and into the rock.

The biodeterioration e!ects of microorganisms colonizing the cloister terrace wall of the Jeronimos Monastery (Lisbon) were evaluated using several microscopy techniques that allow the in situ examination of lithobiontic communities. The techniques applied were: scanning electron microscopy withback-scattered electron imaging (SEM-BSE), low temperature scanning electron microscopy (LTSEM), transmission electron microscopy (TEM) and an X-ray energy dispersive spectroscopy (EDS) microanalytical system. The stone was seen to be colonized by di!erent lichens and microorganisms and lichen thalli of Thyrea, Aspicilia, Verrucaria and Caloplaca were identi=ed. Cyanobacteria were frequently observed close-by, as single cells or colonies and heterotrophic bacteria were also found among these. The lithobiontic community showed biogeophysical and biogeochemical e!ects on the substrate. Cyanobacteria produced bowl- or pear-shaped cavities. Using SEM-BSE and TEM we were able to observe a mineral network structure adjacent to the cyanobacterial wall that might be related to calcium biomobilization processes. Neoformation of biogenic carbonate was detected in thalli of the lichen Thyrea. This information was complemented by observing details of the response of these biological components to the biocidal agents, ALGOPHASEJ, METATINJ and PREVENTOL R80J. After treatment, Thyrea remained on the stone, although ultrastructural alterations were observed in the photobiont. When the e!ects of the biocides on the ultrastructure of the cyanobacteria were analyzed, ALGOPHASEJ proved to be the least eDcient, while PREVENTOL R80J led to the complete disorganization of the prokaryotic cyanobacterial cell. These results point to the importance of evaluating biodeterioration processes and possible treatment measures without extracting the microorganisms from their microhabitat.

Some micro-organisms are able to withstand extreme environments and form communities within rocks. To characterise these endolithic microecosystems, several microscopy and microanalytical approaches need to be combined, including scanning electron microscopy with back scattered imaging(SEM-BSE), low temperature scanning electron microscopy (LTSEM), confocal scanning laser microscopy (CSLM), and the X-ray energy dispersive spectroscopy (EDS) microanalytical system. These techniques have alowed the simultaneous observation of these micro-organisms and their habitats. SEM-BSE and LTSEM serves to evaluate the biodiversity of the rock from a morphological and ultrastructural perspective LTSEM also permits water localisation in the cells and their microhabitats. Information on the spatial distribution of the micro-organisms inside the rock is provided by CSLM.

Lasalia hispanica, Parmelia omphalodes, and Cornicularia normoerica, saxicolous thalli growing on granite, show a close relationship with other lichens and microorganisms living in the lithic substrate beneath them. The lithobiontic community is an acumulation of microrganims at an interface forming,a biofilm, which interacts with the lithic substrate booth geophysically and geochemically. Because of their fruticose and foliose morphology, the saxicolous species examined here are mainly involved in geophysical processes.

Direct in situ microscopy visualization is strongly recommended in studies of saxicolous lichen ecology and endolithic microorganisms. With this in mind, lithobiontic microorganisms were detected, localizad and determined at the cytological level. A range of microscopy techniques and a number of procedures for preparing lichen colonizad rock samples were applied. Scanning Electron Microscopy operating in Back Scattered Electron mode (SEM-BSE), Confocal Láser Scanning Microscopy ,(CLSM) and occasionally Energy Dispersión Spectroscopy (EDS) were used to study the same sample zones. This observation strategy, called "Correlative Microscopy", allowed us to obtain important information on the physico-chemical state of the mineral substrate, deposition processes of calcium ' oxalate and the in situ ultrastructural determination of lithobiontic microorganisms. Moreover, the observation of the three dimensional organization of living endolithic cells was also possible. On occasions, when cytological discernment was not sufficient, the SEM-BSE signal was used to detect immunolabeled gold particles which had previously been linked to proteins in order

Processes involved in photosynthetic Co2 acquisition were characterised for the isolated lichen photobiont Trebouxia erici (Chlorophyta, Trebuxiophyceae) and compared with Coccomyxa (chlorophyta), a lichen photobiont without a photosynthetic Co2.contrating mechanism.