Author Correction: Unprecedented rains decimate surface microbial communities in the hyperarid core of the Atacama Desert.

A correction to this article has been published and is linked from the HTML and PDF versions of this paper. The error has not been fixed in the paper.

Unprecedented rains decimate surface microbial communities in the hyperarid core of the Atacama Desert.

The hyperarid core of the Atacama Desert, the driest and oldest desert on Earth, has experienced a number of highly unusual rain events over the past three years, resulting in the formation of previously unrecorded hypersaline lagoons, which have lasted several months. We have systematically analyzed the evolution of the lagoons to provide quantitative field constraints of large-scale impacts of the rains on the local microbial communities. Here we show that the sudden and massive input of water in regions that have remained hyperarid for millions of years is harmful for most of the surface soil microbial species, which are exquisitely adapted to survive with meager amounts of liquid water, and quickly perish from osmotic shock when water becomes suddenly abundant. We found that only a handful of bacteria, remarkably a newly identified species of Halomonas, remain metabolically active and are still able to reproduce in the lagoons, while no archaea or eukaryotes were identified. Our results show that the already low microbial biodiversity of extreme arid regions greatly diminishes when water is supplied quickly and in great volumes. We conclude placing our findings in the context of the astrobiological exploration of Mars, a hyperarid planet that experienced catastrophic floodings in ancient times.

Miniaturized Raman instrumentation detects carotenoids in Mars-analogue rocks from the Mojave and Atacama deserts.

This study is primarily focused on proving the potential of miniaturized Raman systems to detect any biomolecular and mineral signal in natural geobiological samples that are relevant for future application of the technique within astrobiologically aimed missions on Mars. A series of evaporites of varying composition and origin from two extremely dry deserts were studied, namely Atacama and Mojave. The samples represent both dry evaporitic deposits and recent evaporitic efflorescences from hypersaline brines. The samples comprise halite and different types of sulfates and carbonates. The samples were analysed in two different ways: (i) directly as untreated rocks and (ii) as homogenized powders. Two excitation wavelengths of miniaturized Raman spectrometers were compared: 532 and 785 nm. The potential to detect carotenoids as biomarkers on Mars compared with the potential detection of carbonaceous matter using miniaturized instrumentation is discussed.

Fluorescent fingerprints of endolithic phototrophic cyanobacteria living within halite rocks in the Atacama Desert.

Halite deposits from the hyperarid zone of the Atacama Desert reveal the presence of endolithic microbial colonization dominated by cyanobacteria associated with heterotrophic bacteria and archaea. Using the λ-scan confocal laser scanning microscopy (CLSM) option, this study examines the autofluorescence emission spectra produced by single cyanobacterial cells found inside halite rocks and by their photosynthetic pigments. Photosynthetic pigments could be identified according to the shapes of the emission spectra and wavelengths of fluorescence peaks. According to their fluorescence fingerprints, three groups of cyanobacterial cells were identified within this natural extreme microhabitat: (i) cells producing a single fluorescence peak corresponding to the emission range of phycobiliproteins and chlorophyll a, (ii) cells producing two fluorescence peaks within the red and green signal ranges, and (iii) cells emitting only low-intensity fluorescence within the nonspecific green fluorescence signal range. Photosynthetic pigment fingerprints emerged as indicators of the preservation state or viability of the cells. These observations were supported by a cell plasma membrane integrity test based on Sytox Green DNA staining and by transmission electron microscopy ultrastructural observations of cyanobacterial cells.

Towards a more realistic picture of in situ biocide actions: combining physiological and microscopy techniques.

In this study, we combined chlorophyll a fluorescence (ChlaF) measurements, using pulse-amplitude-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.

Whole lichen thalli survive exposure to space conditions: results of Lithopanspermia experiment with Aspicilia fruticulosa.

The Lithopanspermia space experiment was launched in 2007 with the European Biopan facility for a 10-day spaceflight on board a Russian Foton retrievable satellite. Lithopanspermia included for the first time the vagrant lichen species Aspicilia fruticulosa from Guadalajara steppic highlands (Central Spain), as well as other lichen species. During spaceflight, the samples were exposed to selected space conditions, that is, the space vacuum, cosmic radiation, and different spectral ranges of solar radiation (λ ≥ 110, ≥200, ≥290, or ≥400 nm, respectively). After retrieval, the algal and fungal metabolic integrity of the samples were evaluated in terms of chlorophyll a fluorescence, ultrastructure, and CO(2) exchange rates. Whereas the space vacuum and cosmic radiation did not impair the metabolic activity of the lichens, solar electromagnetic radiation, especially in the wavelength range between 100 and 200 nm, caused reduced chlorophyll a yield fluorescence; however, there was a complete recovery after 72 h of reactivation. All samples showed positive rates of net photosynthesis and dark respiration in the gas exchange experiment. Although the ultrastructure of all flight samples showed some probable stress-induced changes (such as the presence of electron-dense bodies in cytoplasmic vacuoles and between the chloroplast thylakoids in photobiont cells as well as in cytoplasmic vacuoles of the mycobiont cells), we concluded that A. fruticulosa was capable of repairing all space-induced damage. Due to size limitations within the Lithopanspermia hardware, the possibility for replication on the sun-exposed samples was limited, and these first results on the resistance of the lichen symbiosis A. fruticulosa to space conditions and, in particular, on the spectral effectiveness of solar extraterrestrial radiation must be considered preliminary. Further testing in space and under space-simulated conditions will be required. Results of this study indicate that the quest to discern the limits of lichen symbiosis resistance to extreme environmental conditions remains open.

Microbial colonization of Ca-sulfate crusts in the hyperarid core of the Atacama Desert: implications for the search for life on Mars.

The scarcity of liquid water in the hyperarid core of the Atacama Desert makes this region one of the most challenging environments for life on Earth. The low numbers of microbial cells in the soils suggest that within the Atacama Desert lies the dry limit for life on our planet. Here, we show that the Ca-sulfate crusts of this hyperarid core are the habitats of lithobiontic micro-organisms. This microporous, translucent substrate is colonized by epilithic lichens, as well as endolithic free-living algae, fungal hyphae, cyanobacteria and non photosynthetic bacteria. We also report a novel type of endolithic community, "hypoendoliths", colonizing the undermost layer of the crusts. The colonization of gypsum crusts within the hyperarid core appears to be controlled by the moisture regime. Our data shows that the threshold for colonization is crossed within the dry core, with abundant colonization in gypsum crusts at one study site, while crusts at a drier site are virtually devoid of life. We show that the cumulative time in 1 year of relative humidity (RH) above 60% is the best parameter to explain the difference in colonization between both sites. This is supported by controlled humidity experiments, where we show that colonies of endolithic cyanobacteria in the Ca-sulfate crust undergo imbibition process at RH >60%. Assuming that life once arose on Mars, it is conceivable that Martian micro-organisms sought refuge in similar isolated evaporite microenvironments during their last struggle for life as their planet turned arid.

Microbial colonization of halite from the hyper-arid Atacama Desert studied by Raman spectroscopy.

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 micro-organisms 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.

Laboratory-induced endolithic growth in calcarenites: biodeteriorating potential assessment.

This study is aimed to assess the formation of photosynthetic biofilms on and within different natural stone materials, and to analyse their biogeophysical and biogeochemical deterioration potential. This was performed by means of artificial colonisation under laboratory conditions during 3 months. Monitoring of microbial development was performed by image analysis and biofilm biomass estimation by chlorophyll extraction technique. Microscopy investigations were carried out to study relationships between microorganisms and the mineral substrata. The model applied in this work corroborated a successful survival strategy inside endolithic microhabitat, using natural phototrophic biofilm cultivation, composed by cyanobacteria and algae, which increased intrinsic porosity by active mineral dissolution. We observed the presence of mineral-like iron derivatives (e.g. maghemite) around the cells and intracellularly and the precipitation of hausmannite, suggesting manganese transformations related to the biomineralisation.

Advances in the ultrastructural study of the implant-bone interface by backscattered electron imaging.

The biocompatibility of titanium implants in bone depends on the response shown by cells in contact with the implant surface. Several developments have been targeted at achieving successful implant treatment. The aim of this study was to develop a novel preparation procedure to evaluate the bone cell response produced at the bone-implant interface using the technique scanning electron microscopy with backscattered electron imaging (SEM-BSE). Dental prostheses with an SLA-modified or TOP-modified surface were implanted in a toothless part of the mandibula in female pigs. The animals were sacrificed 12 weeks after surgery, at which time block specimens containing the implants were obtained. These specimens were then processed for SEM-BSE by optimizing a protocol involving chemical fixation and heavy metal staining. In addition, element distribution maps for the implant-bone tissue interface were obtained using a microanalytical system based on energy-dispersive X-ray spectrometry (EDS). This novel visualisation approach enabled a comprehensive study of the extracellular matrix and cell components of the host tissues neoformed around the implant. SEM-BSE images also provided ultrastructural details of the bone cells. This technique appears to be an effective and very promising tool for detailed studies on the implant-bone tissue interface and the host response to the bone incorporation process.

Lichens survive in space: results from the 2005 LICHENS experiment.

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 protection against solar radiation. Moreover, after extreme dehydration induced by high vacuum, the lichens proved to be able to recover, in full, their metabolic activity within 24 hours.

Endolithic growth of two Lecidea lichens in granite from continental Antarctica detected by molecular and microscopy techniques.

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

Viability of endolithic micro-organisms in rocks from the McMurdo Dry Valleys of Antarctica established by confocal and fluorescence microscopy.

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.

New approaches to the study of Antarctic lithobiontic microorganisms and their inorganic traces, and their application in the detection of life in Martian rocks.

Microbial life in the harsh conditions of Antarctica's cold desert may be considered an analogue of potential life on early Mars. In order to explore the development and survival of this epilithic and endolithic form of microbial life, our most sophisticated, state-of-the-art visualization technologies have to be used to their full potential. The study of any ecosystem requires a knowledge of its components and the processes that take place within it. If we are to understand the structure and function of each component of the microecosystems that inhabit lithic substrates, we need to be able to quantify and identify the microorganisms present in each lithobiontic ecological niche and to accurately characterize the mineralogical features of these hidden microhabitats. Once we have established the techniques that will allow us to observe and identify these microorganisms and mineral substrates in situ, and have confirmed the presence of water, the following questions can be addressed: How are the microorganisms organized in the fissures or cavities? Which microorganisms are present and how many are there? Additional questions that logically follow include: What are the existing water relationships in the microhabitat and what effects do the microorganisms have on the mineral composition? Mechanical and chemical changes in minerals and mineralization of microbial cells can give rise to physical and/or chemical traces (biomarkers) and to microbial fossil formation. In this report, we describe the detection of chains of magnetite within the Martian meteorite ALH84001, as an example of the potential use of SEM-BSE in the search for plausible traces of life on early Mars.

New approaches to the study of Antarctic lithobiontic microorganisms and their inorganic traces and their application in the detection of life in Martian rocks / Un nuevo enfoque al estudio de microorganismos litobiontes de la Antártida y sus indicios inorgánicos: aplicaciones en la detección de vida en rocas marcianas

Microbial life in the harsh conditions of Antarctica's cold desert may be considered an analogue of potential life on early Mars. In order to explore the development and survival of this epilithic and endolithic form of microbial life, our most sophisticated, state-of-the-art visualization technologies have to be used to their full potential. The study of any ecosystem requires a knowledge of its components and the processes that take place within it. If we are to understand the structure and function of each component of the microecosystems that inhabit lithic substrates, we need to be able to quantify and identify the microorganisms present in each lithobiontic ecological niche and to accurately characterize the mineralogical features of these hidden microhabitats. Once we have established the techniques that will allow us to observe and identify these microorganisms and mineral substrates in situ, and have confirmed the presence of water, the following questions can be addressed: How are the microorganisms organized in the fissures or cavities? Which microorganisms are present and how many are there? Additional questions that logically follow include: What are the existing water relationships in the microhabitat and what effects do the microorganisms have on the mineral composition? Mechanical and chemical changes in minerals and mineralization of microbial cells can give rise to physical and/or chemical traces (biomarkers) and to microbial fossil formation. In this report, we describe the detection of chains of magnetite within the Martian meteorite ALH84001, as an example of the potential use of SEM-BSE in the search for plausible traces of life on early Mars (AU)

La vida microbiana en las duras condiciones del frío desierto de la Antártida puede considerarse un análogo de la vida que posiblemente hubo en un principio en el planeta Marte. Para explorar el desarrollo y la supervivencia de esta forma de vida microbiana epilítica y endolítica, son necesarias las más sofisticadas tecnologías de visualización. El estudio de todo ecosistema requiere el conocimiento de sus componentes y de los procesos que tienen lugar en él. Para entender la estructura y función de cada componente de los microecosistemas de los sustratos líticos, necesitamos poder cuantificar e identificar los microorganismos presentes en cada uno de los nichos ecológicos que allí se encuentran y caracterizar adecuadamente los rasgos mineralógicos de estos microhábitats ocultos. Una vez se haya establecido las técnicas que nos permitirán observar e identificar estos microorganismos y sustratos minerales in situ, y que se haya confirmado la presencia de agua, pueden estudiarse las siguientes cuestiones: ¿cómo se organizan los microorganismos en las fisuras y cavidades?; ¿qué microorganismos están presentes y cuántos hay? Preguntas a las que, de manera lógica, les suceden: ¿qué relaciones se establecen con el agua presente en el microhábitat y qué efectos tienen los microoganismos sobre la composición mineral? Los cambios químicos y físicos en los minerales y la mineralización de las células microbianas pueden dar lugar a marcadores físicos y/o químicos (biomarcadores) y a la formación de fósiles microbianos. En este artículo, se describe la detección de cadenas de magnetita en el meteorito marciano ALH84001 como ejemplo del posible uso de la SEM-BSE en la búsqueda de rastros de vida en el Marte primitivo. (AU)

Microhabitats and chemical microenvironments under saxicolous lichens growing on granite.

Lasallia 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 accumulation of microorganisms at an interface forming a biofilm, which interacts with the lithic substrate both geophysically and geochemically. Because of their fruticose and foliose morphology, the saxicolous species examined here are mainly involved in geophysical processes, but in the proximity of their attachment structures, geochemical processes may also be observed. On the other hand, fungi, algae and cyanobacteria forming crustose lichens, as well as free-living lithobiontic microorganisms, are known to show combined geophysical and geochemical action, mainly on laminar minerals. The substrate zone where the saxicolous lichens are attached is most affected by weathering reactions and shows the highest co-occurrence of lithobiontic microorganisms. The physical and chemical properties of the substrate, along with lichen and microorganism activity, determine different microenvironments and microhabitats. The ecological functioning of these lithobiontic communities is not yet fully understood, and research efforts similar to the present are needed to confirm that their development is influenced by interrelations between different community members and the substrate, as suggested here.

Chains of magnetite crystals in the meteorite ALH84001: evidence of biological origin.

The presence of magnetite crystal chains, considered missing evidence for the biological origin of magnetite in ALH84001 [Thomas-Keprta, K. L., Bazylinski, D. A., Kirschvink, J. L., Clemett, S. J., McKay, D. S., Wentworth, S. J., Vali, H., Gibson, E. K., Jr., & Romanek, C. S. (2000) Geochim. Cosmochim. Acta 64, 4049-4081], is demonstrated by high-power stereo backscattered scanning electron microscopy. Five characteristics of such chains (uniform crystal size and shape within chains, gaps between crystals, orientation of elongated crystals along the chain axis, flexibility of chains, and a halo that is a possible remnant of a membrane around chains), observed or inferred to be present in magnetotactic bacteria but incompatible with a nonbiological origin, are shown to be present. Although it is unlikely that magnetotactic bacteria were ever alive in ALH84001, decomposed remains of such organisms could have been deposited in cracks in the rock while it was still on the surface on Mars.

Study of lichens with different state of hydration by the combination of low temperature scanning electron and confocal laser scanning microscopies.

The use of techniques such as low temperature scanning electron microscopy (LTSEM) and confocal laser scanning microscopy (CLSM) allows the study of lichen thalli in different states of hydration and also near the natural state. The spatial organization of desiccated thalli, with reduced, very compact algal layers, is different from that of hydrated ones. Sometimes, the observation with transmission electron microscopy (TEM) of photobiont pyrenoids from desiccated thalli reports pyrenoids with a central part of a weak stained matrix lacking pyrenoglobuli, named "empty zones". "Empty zones" are not distinguishable with LTSEM and do not present immunolabelling with rubisco antibody in TEM. These zones could be originated by an expansion process during rehydration produced in chemical fixation.

Specific inclusion bodies are associated with replication of lettuce infectious yellows virus RNAs in Nicotiana benthamiana protoplasts.

Nicotiana benthamiana mesophyll protoplasts, either mock-inoculated or inoculated using in vitro transcripts derived from lettuce infectious yellows virus (LIYV) RNA 1- and/or RNA 2-cloned cDNAs were analysed by transmission electron microscopy (TEM) and, in some cases, also by immunogold labelling. TEM revealed the main cytopathological effects of LIYV infections in N. benthamiana protoplasts infected with RNAs 1 and 2: (a) typical closterovirus-induced (beet yellows virus-type) accumulations of vesiculated cytoplasmic membranes as inclusion bodies, sometimes with associated virions; (b) scattered aggregations of virions within the cytoplasm; and (c) electron-dense plasmalemma deposits. These were not seen in mock-inoculated protoplasts. Protoplasts inoculated only with LIYV RNA 1 contained vesiculated cytoplasmic inclusion bodies, but not virions or plasmalemma deposits. Thus, infection by only LIYV RNA 1 is sufficient to induce characteristic closterovirus vesiculated cytoplasmic inclusion bodies. However, both LIYV RNAs 1 and 2 are needed for production of virions and plasmalemma deposits.

[New applications of submicroscopic techniques in the study of biodegradation caused by lichen thalli]. / Nuevas aplicaciones de las técnicas submicroscópicas en el estudio del biodeterioro producido por talos liquénicos.

The advances that have been made on the effect of lichens on rock substrate have been numerous. The first techniques used in order to know the type of biodeterioration produced by lichen thalli, were observations by light microscopy. The microchemical techniques that followed this, such as X-Ray Diffraction and Infrared Spectroscopy, provided knowledge of the mineral composition of the substrate which is in contact with the lichen thalli. This zone is known as interface. More recently, the Scanning Electron-Microscope has allowed observations of the relationship between the hyphae of the lower part of the thallus and the topography of the superficial part of the rock. In the current work, new applications of Scanning Electron Microscopy are presented. The backscattered electron image permits observation of the penetration of living thalli elements in the rock. With this technique, it is possible to follow the fissures underneath the lichen thalli and see at a cellular level the biological elements. This technique is an important step in understanding the biodeterioration produced by lichens. It allows clarification of such aspects as the ability of embedded minerals detached from the rock among the hyphae and also the possibility of investigating organic and inorganic compounds mixed with hyphae and algae. This technique, moreover, is promising for use in future investigations of biodeterioration where the cellular morphology of the different organisms are interesting, taking into account treatments with biocides.