Microscopic and biomolecular complementary approaches to characterize bioweathering processes at petroglyph sites from the Negev Desert,Israel

Throughout the Negev Desert highlands, thousands of ancient petroglyphs sites are susceptible to deterioration processes that may result in the loss of this unique rock art. Therefore, the overarching goal of the current study was to characterize the composition, diversity and effects of microbial colonization of the rocks to find ways of protecting these unique treasures. The spatial organization of the microbial colonizers and their relationships with the lithic substrate were analysed using scanning electron microscopy. This approach revealed extensive epilithic and endolithic colonization and close microbial–mineral interactions. Shotgun sequencing analysis revealed various taxa from the archaea, bacteria and some eukaryotes. Metagenomic coding sequences (CDS) of these microbial lithobionts exhibited specific metabolic pathways involved in the rock elements' cycles and uptake processes. Thus, our results provide evidence for the potential participation of the microorganisms colonizing these rocks during different solubilization and mineralization processes. These damaging actions may contribute to the deterioration of this extraordinary rock art and thus threaten this valuable heritage. Shotgun metagenomic sequencing, in conjunction with the in situ scanning electron microscopy study, can thus be considered an effective strategy to understand the complexity of the weathering processes occurring at petroglyph sites and other cultural heritage assets.     

Life, decay and fossilisation of endolithic microorganisms from the Ross Desert, Antarctica

The adaptation and survival of the endolithic microorganisms that colonise the near-surface layer of porous sandstone rock in the Ross Desert (Antarctica) depend upon a precarious equilibrium of biological, geological and climatic factors. Any unfavourable change in external conditions can result in the death and disappearance of microscopic organisms, and this may be followed by trace microfossil formation. The sequence of events leading to the extinction of life in the Antarctic desert is considered to be a terrestrial analogue of the disappearance of possible life on early Mars. The present paper reviews the current state of knowledge on the endolithic microorganisms of the Ross Desert with particular reference to their decay and fossilisation processes. Ideas for in situ further research on this microbial ecosystem are also proposed, including several new microscopy techniques such as CLSM, LTSEM, SEM-BSE and EDS. Preliminary images are presented and it is proposed that, for the first time, such techniques will permit the in situ study of the ecology of Antarctic lithobiontic microorganisms and the identification and characterisation of fossilised traces of past life.     

Phylogenetic Composition of Rocky Mountain Endolithic Microbial Ecosystems

The endolithic environment, the pore space in rocks, is a ubiquitous microbial habitat. Photosynthesis-based endolithic communities inhabit the outer few millimeters to centimeters of rocks exposed to the surface. Such endolithic ecosystems have been proposed as simple, tractable models for understanding basic principles in microbial ecology. In order to test previously conceived hypotheses about endolithic ecosystems, we studied selected endolithic communities in the Rocky Mountain region of the United States with culture-independent molecular methods. Community compositions were determined by determining rRNA gene sequence contents, and communities were compared using statistical phylogenetic methods. The results indicate that endolithic ecosystems are seeded from a select, global metacommunity and form true ecological communities that are among the simplest microbial ecosystems known. Statistical analysis showed that biogeographical characteristics that control community composition, such as rock type, are more complex than predicted. Collectively, results of this study support the idea that patterns of microbial diversity found in endolithic communities are governed by principles similar to those observed in macroecological systems.     

Phylogenetic composition of Rocky Mountain endolithic microbial ecosystems

The endolithic environment, the pore space in rocks, is a ubiquitous microbial habitat. Photosynthesis-based endolithic communities inhabit the outer few millimeters to centimeters of rocks exposed to the surface. Such endolithic ecosystems have been proposed as simple, tractable models for understanding basic principles in microbial ecology. In order to test previously conceived hypotheses about endolithic ecosystems, we studied selected endolithic communities in the Rocky Mountain region of the United States with culture-independent molecular methods. Community compositions were determined by determining rRNA gene sequence contents, and communities were compared using statistical phylogenetic methods. The results indicate that endolithic ecosystems are seeded from a select, global metacommunity and form true ecological communities that are among the simplest microbial ecosystems known. Statistical analysis showed that biogeographical characteristics that control community composition, such as rock type, are more complex than predicted. Collectively, results of this study support the idea that patterns of microbial diversity found in endolithic communities are governed by principles similar to those observed in macroecological systems.     

Molecular characterization of endolithic cyanobacteria inhabiting exposed dolomite in central Switzerland

The phototrophic microbial community inhabiting exposed dolomite in the alpine Piora Valley (Switzerland) forms a distinct endolithic bilayer that features adjacent red dolomite (exterior) and green dolomite (interior) layers that are c. 0.5-1 mm below the rock surface. Characterization of the community, with an emphasis on cyanobacteria, was conducted with culture-dependent and -independent approaches. Direct microscopy of green dolomite revealed four distinct morphotypes consistent with Chlorophyta genera Chlorella and Stichococcus and the Cyanobacterial genera Nostoc and Calothrix, whereas only Stichococcus and Nostoc were observed in the red dolomite. Enrichment in BG-11 media resulted in the growth of Chlorella and Stichococcus. Denaturing gradient gel electrophoresis (DGGE) analysis of DNA extracted from the enrichment revealed two dominant phylotypes with sequence similarity to Chlorella osrokiniana chloroplast and the cyanobacteria genus Leptolyngbya. 16S rRNA gene-based DGGE analysis of DNA extracted directly from both layers indicated that although both layers harboured phylotypes most similar to the Cyanobacterial genera Nostoc, Chroococcidiopsis, and Microcoleus, and the Chlorophyte Stichococcus bacillaris, the two layers also harboured unique genera such as Scytonema, and Symploca (red, external layer of dolomite) and Chlorella (green, internal layer of dolomite). The unique community structure of each layer suggests a selection process directed by the pressures of the endolithic environment. We conclude that the overall composition of the phototrophic community closely resembles that of endolithic communities located in extreme habitats, suggesting that a cosmopolitan community inhabits this defined niche.     

Phototrophic Community in Gypsum Crust from the Atacama Desert Studied by Raman Spectroscopy and Microscopic Imaging

The hyperarid core of the Atacama Desert represents one of the driest places on Earth with an exceptional occurrence of microbial life coping with extreme environmental stress factors. The gypsum crusts have already been found to harbor diverse communities in this area. Here, we present a Raman spectroscopic study, complemented by correlative microscopic imaging using SEM-BSE and fluorescence microscopy, of the endolithic microbial communities inside the Ca-sulphate crusts dominated by phototrophic microorganisms. Differences of pigment composition within different zones follow the cyanobacterial and algal colonization and also reveal the degradation of phycobiliproteins within the decayed biomass of cyanobacteria. Carotenoids of at least three different types were recognized, differing in dependence on the particular phylum of phototrophic microorganisms. Moreover, calcium oxalate monohydrate – whewellite – was found to be associated with the algae and hyphal associations living in the lower regions of the crust. The 785 nm excitation wavelength employed here was found to be the correct source for studying pigment composition as well as for the detection of the oxalate. A comparison of these results with those using 514.5 nm laser excitation which is widely adopted for the detection of carotenoids due to the resonance Raman effect is made and discussed.     

Mapping the lithic colonization at the boundaries of life in Northern Victoria Land,Antarctica

The endolithic microbial communities of the Antarctic represent a borderline lifestyle in the most hostile ice-free areas of the continent. The extreme adaptation of microbes in these communities renders them very sensitive to environmental changes. To date, the actual distribution of these communities has never been investigated; yet, this information would define the geographic limits for life at present and supply a useful tool for monitoring any possible future variation related to climate change. In this study, most of the outcrops of Northern and of one site in Southern Victoria Land were recorded by altitudinal and sea distance gradients. The presence of endolithic life was determined by in situ observation, by microscopic observation of rock fragments in the laboratory, and, for doubtful samples, by culture experiment. Colonizers were present in more than 87 % of the visited sites. The presence of lithic life in Victoria Land appears to be wider than that reported 14 years earlier. The colonization trend follows climatic variation, with epiliths prevailing in coastal sites and decreasing towards the interior, while chasmoendoliths and cryptoendoliths increase and become predominant from the coast towards the inland sites. Typical cryptoendolithic colonization was exclusive on porous rocks as sandstone, chasmoendolithic colonization occurred even in less porous but translucent rocks as granite and quartz. Multivariate analysis of the combined results clearly indicates the pivotal role of the rock type in the colonization of endolithic micro-organisms; sandstone allows lithobionts to push themselves towards areas characterized by harsher conditions.     

Phylogenetic diversity of endolithic bacteria in Bole granite rock in Xinjiang

The endolithic environment is a ubiquitous microbial habitat for microorganisms, such as lichens, Cyanobacteria and fungi, and it provides mineral nutrients and growth surfaces. In extremely environments, such as hot and cold desert, endolithic communities are often the main form of life. More recently, endolithic microbial communities have been observed inhabiting a variety of rock types ranging from hard granite to porous rocks such as basalt, dolomite, limestone, sandstone and granites. Regardless of geographic location and rock type, each of these habitats is characterized by a subsurface microclimate that prevents endolithic microorganisms growth. Photosynthesis-based endolithic microbial communities commonly inhabit the outer millimeters to centimeters of rocks exposed to the surface. The ability to fix carbon dioxide and in some cases atmospheric dinitrogen, gives the Cyanobacteria a clear competitive advantage over heterotrophic bacteria, so it is been called the main primary producer. Light quality and intensity appear to be the main determinant of the maximum depth to which growth occurs in endolithic phototrophic communities. Valleys of Fantastic Rocks in Bole is close to Alashankou Port of Xinjiang which belongs to extreme continental climate. In order to investigate the structure, composition and diversity of endolithic bacterial community in exposed granitic porphyry in the Valleys of Fantastic Rocks, environmental DNA was directly extracted from granite rock, the 16S rRNA genes were amplified from the total DNA by PCR with bacterial-specific primers, and an endolithic bacterial clone library was constructed. Positive clones were randomly selected from the library and identified by Restriction Fragment Length Polymorphism (RFLP). The unique rRNA types clones were sequenced, analysised and then constructed phylogenetic tree. In total, 129 positive clones were screened and grouped into 46 operational taxonomic unites (OTUs). The clone coverage C value was 89.15%, indicating that most of the estimated endolithic bacterial diversity was sampled. BLAST analysis indicated that 46 OTUs were divided into seven phyla (Acidobacteria, Actinobacteria, Bacteroidetes, Chloroflexi, Cyanobacteria, Planctomycetes, Proteobacteria) and five unknown groups. Cyanobacteria (43%), especially the Gp I, form the functional basis for an endolithic bacteria community which contain a wide spectrum species of chemotrophic bacteria (33%) with mainly Actinobacteria, α-Proteobacteria, Acidobacteria. Additionally, most clones that derived from the endolithic bacteria clone library showed high similarity to the sequence deposited in GenBank database with 97%–99%. Besides, 35% of the clones showed less than 97% of sequence similarity, of which 12% sequences were affiliated to genus Rubrobacter. The results suggested that endolithic bacteria in Valleys of Fantastic Rocks in Xinjiang were highly diverse in species richness, and maybe have a diversity of potential novel species and lineages.     

Nd-YAG laser irradiation damages to Verrucaria nigrescens

Epilithic and endolithic microorganisms and lichens play an important role in stone biodeterioration. The structural and physiological damage caused by nanosecond pulsed laser of 1064 nm from Nd:YAG laser to Verrucaria nigrescens lichen as well as to endolithic algae and fungi were investigated in the present study. Ultrastructural laser effects on lichen and endolithic microorganisms were study without disturbing the relationship between lichen and lithic substrate by taking lichen-containing rock fragments and processing both together. SEM-BSE, LT-SEM and FM were used to determine cell integrity and ultrastructure, which reflect microorganism viability. Photobiont vitality was determined using a PAM chlorophyll fluorescence technique. The lichen thalli were completely removed by irradiation with 5 ns pulses at a fluence of 2.0 J/cm² with no stone damage as showed by micro-Raman spectroscopy. The fungal and algal endolithic cells located below were completely destroyed or presented a high plasmolysis degree resulting from heating their microenvironment. The lichen and endolithic mycobiont near the irradiated zone were also damaged. Algal photosynthetic damage prevents fungal survival and lichen viability. This is the first report of laser removal and inactivation of lichen and lithic microorganisms, and thus provide an environmentally friendly and efficient method to control stone biodeterioration.     

In vitro receptivity of carbonate rocks to endolithic lichen-forming aposymbionts

Sterile cultured isolates of lichen-forming aposymbionts have not yet been used to investigate lichen-rock interactions under controlled conditions. In this study mycobionts and photobiont of the endolithic lichens Bagliettoa baldensis and Bagliettoa marmorea were isolated and inoculated with coupons of one limestone and four marbles commonly employed in the Cultural Heritage framework. After one year of incubation, microscopic observations of polished cross-sections were performed to verify if the typical colonization patterns observed in the field may be reproduced in vitro and to evaluate the receptivity of the five lithotypes to endolithic lichens. The mycobionts of the two species developed both on the surface of and within all the lithotypes, showing different penetration pathways which depend on mineralogical and structural features and highlight different receptivity. By contrast, algae inoculated with the coupons did not penetrate them. Observations suggest that the hyphal penetration along intrinsic discontinuities of rocks is a relatively fast phenomenon when these organisms are generally considered as slow-growing. Samples from limestone outcrops and abandoned marble quarries, colonized by the same species or other representatives of Verrucariaceae, showed penetration pathways intriguingly similar to those reproduced in vitro and highlighted that lichen-driven erosion processes only increase the availability of hyphal passageways after a long-term colonization. These results show that in vitro incubation of sterile cultured lichen-forming ascomycetes with rock coupons is a practicable experimental system to investigate the lichen-rock interactions under controlled conditions and, together with analysis in situ, may support decisions on conservative treatments of historical and cultural significant stone substrata.     

Further Readings in Geomicrobiology

Evaporite accumulations produced by artesian waters in the arid zones of southern Tunisia led to the formation of subrounded, gypsiferous mounds consisting of irregular alternations of mineral precipitates and aeolian sand. The joint occurrence of gypsum crusts and plant colonization determined the stabilization of their top surface. Careful examination of the pigmented (green-brown) crusts revealed endolithic microbial communities just below the surface. In previous optical and scanning electron microscope studies cyanobacteria were the dominant component of these communities. Molecular diversity studies based on small subunit ribosomal RNA (SSU rRNA) gene analysis revealed that Flavobacteria, Actinobacteria, Deinococcales, Alpha- and Gamma- Proteobacteria are also important components of the microbial assemblage. Their pigment analyses, determined by high performance liquid chromatography (HPLC), detected the presence of carotenoids and chlorophyll (chl) a and b. Microbial communities that produce pigmentation and display an endolithic lifestyle typify the extreme environments as those found in arid/semiarid and hot desert regions.     

Diversity of endolithic fungal communities in dolomite and limestone rocks from Nanjiang Canyon in Guizhou karst area, China

The endolithic environment, the tiny pores and cracks in rocks, buffer microbial communities from a number of physical stresses, such as desiccation, rapid temperature variations, and UV radiation. Considerable knowledge has been acquired about the diversity of microorganisms in these ecosystems, but few culture-independent studies have been carried out on the diversity of fungi to date. Scanning electron microscopy of carbonate rock fragments has revealed that the rock samples contain certain kinds of filamentous fungi. We evaluated endolithic fungal communities from bare dolomite and limestone rocks collected from Nanjiang Canyon (a typical karst canyon in China) using culture-independent methods. Results showed that Ascomycota was absolutely dominant both in the dolomite and limestone fungal clone libraries. Basidiomycota and other eukaryotic groups (Bryophyta and Chlorophyta) were only detected occasionally or at low frequencies. The most common genus in the investigated carbonate rocks was Verrucaria. Some other lichen-forming fungi (e.g., Caloplaca, Exophiala, and Botryolepraria), Aspergillus, and Penicillium were also identified from the rock samples. The results provide a cross-section of the endolithic fungal communities in carbonate rocks and help us understand more about the role of microbes (fungi and other rock-inhabiting microorganisms) in rock weathering and pedogenesis.     

Fundamental drivers for endolithic microbial community assemblies in the hyperarid Atacama Desert

In hyperarid deserts, endolithic microbial communities colonize the rocks’ interior as a survival strategy. Yet, the composition of these communities and the drivers promoting their assembly are still poorly understood. We analysed the diversity and community composition of endoliths from four different lithic substrates – calcite, gypsum, ignimbrite and granite – collected in the hyperarid zone of the Atacama Desert, Chile. By combining microscopy, mineralogy, spectroscopy and high throughput sequencing, we found these communities to be highly specific to their lithic substrate, although they were all dominated by the same four main phyla, Cyanobacteria, Actinobacteria, Chloroflexi and Proteobacteria. Our finding indicates a fine scale diversification of the microbial reservoir driven by substrate properties. The data suggest that the overall rock chemistry and the light transmission properties of the substrates are not essential drivers of community structure and composition. Instead, we propose that the architecture of the rock, i.e., the space available for colonization and its physical structure, linked to water retention capabilities, is ultimately the driver of community diversity and composition at the dry limit of life.     

Geomicrobiology of the ocean crust: a role for chemoautotrophic Fe-bacteria

The delicate balance of the major global biogeochemical cycles greatly depends on the transformation of Earth materials at or near its surface. The formation and degradation of rocks, minerals, and organic matter are pivotal for the balance, maintenance, and future of many of these cycles. Microorganisms also play a crucial role, determining the transformation rates, pathways, and end products of these processes. While most of Earth's crust is oceanic rather than terrestrial, few studies have been conducted on ocean crust transformations, particularly those mediated by endolithic (rock-hosted) microbial communities. The biology and geochemistry of deep-sea and sub-seafloor environments are generally more complicated to study than in terrestrial or near-coastal regimes. As a result, fewer, and more targeted, studies usually homing in on specific sites, are most common. We are studying the role of endolithic microorganisms in weathering seafloor crustal materials, including basaltic glass and sulfide minerals, both in the vicinity of seafloor hydrothermal vents and off-axis at unsedimented (young) ridge flanks. We are using molecular phylogenetic surveys and laboratory culture studies to define the size, diversity, physiology, and distribution of microorganisms in the shallow ocean crust. Our data show that an unexpected diversity of microorganisms directly participate in rock weathering at the seafloor, and imply that endolithic microbial communities contribute to rock, mineral, and carbon transformations.     

甲硝唑与乳杆菌活菌制剂对细菌性阴道病患者阴道微生物群影响的对比性研究

目的对300例细菌性阴道病（Bacterial Vaginosis,BV）患者随机使用甲硝唑（100例）、乳杆菌活菌制剂定君生（100例）及甲硝唑联合定君生（100例）,观察乳杆菌在阴道内定植情况及用药前后阴道内微生物多样性的变化,为临床治疗细菌性阴道病提供实验依据。方法分别提取300例BV患者用药前和用药后的阴道微生物总基因组DNA,以德氏乳杆菌16SrDNA特异性引物进行PCR,检测患者用药后德氏乳杆菌在阴道内的定植情况。以细菌16SrRNAV3区通用引物进行PCR,扩增产物进行变性梯度凝胶电泳（DGGE）,对所得指纹图谱进行分析,研究用药对微生态变化的影响状况。结果甲硝唑治疗组用药前后均没有德氏乳杆菌检出;定君生治疗组用药后德氏乳杆菌检出率为54％;甲硝唑联合定君生治疗组用药后德氏乳杆菌检出率为76％。甲硝唑治疗组用药后微生物多样性显著降低,微生物群结构失衡;定君生治疗组用药后微生物多样性有所降低,微生物群结构无显著差异;甲硝唑联合定君生治疗组用药后微生物多样性降低,但较前两组有所增加,微生物群结构差异无统计学意义。结论甲硝唑显著降低BV患者阴道微生物多样性,引起微生态失衡;定君生对BV患者阴道微生态的影响明显小于甲硝唑;定君生的使用有利于甲硝唑f预后阴道微生物多样性的恢复和微生态平衡的重建。     

Monitoring of microbial diversity and activity during bioremediation of crude oil-contaminated soil with different treatments

The present study compared the microbial diversity and activity during the application of various bioremediation processes to crude oil-contaminated soil. Five different treatments, including natural attenuation (NA), biostimulation (BS), biosurfactant addition (BE), bioaugmentation (BA), and a combined treatment (CT) of biostimulation, biosurfactant addition, and bioaugmentation, were used to analyze the degradation rate and microbial communities. After 120 days, the level of remaining hydrocarbons after all the treatments was similar, however, the highest rate (k) of total petroleum hydrocarbon (TPH) degradatioN was observed with the CT treatment (P < 0.05). The total bacterial counts increased during the first 2 weeks with all the treatments, and then remained stable. The bacterial communities and alkane monooxygenase gene fragment, alkB, were compared by denaturing gradient gel electrophoresis (DGGE). The DGGE analyses of the BA and CT treatments, which included Nocardia sp. H17-1, revealed a simple dominant population structure, compared with the other treatments. The Shannon-Weaver diversity index (H') and Simpson dominance index (D), calculated from the DGGE profiles using 16S rDNA, showed considerable qualitative differences in the community structure before and after the bioremediation treatment as well as between treatment conditions.     

Diversity of phototrophic components in biofilms from Piperno historical stoneworks

We investigated phototrophic microorganisms dwelling on stone walls made of Piperno, a volcanic rock frequently used as construction material in historical buildings in Naples, Italy. Biofilms from three historical buildings in the center of the city and from a natural Piperno quarry located in a suburban area were examined. Light and electron microscopy, and molecular biology techniques allowed the identification of 17 species belonging to Cyanobacteria, Rhodophyta, Bacillariophyta, and Chlorophyta. Cyanobacteria were the dominant components in all the biofilms. No significant differences in microbial composition were observed for biofilms collected in autumn and spring, with minor exceptions for the quarry samples, where environmental conditions were relatively more stable than in the city. Results are discussed in comparison with information on microbial communities dwelling on other kinds of substrata commonly used in historical buildings in the Neapolitan area.     

The diversity and abundance of bacteria inhabiting seafloor lavas positively correlate with rock alteration

Young, basaltic ocean crust exposed near mid-ocean ridge spreading centers present a spatially extensive environment that may be exploited by epi- and endolithic microbes in the deep sea. Geochemical energy released during basalt alteration reactions can theoretically support chemosynthesis, contributing to a trophic base for the ocean crust biome. To examine associations between endolithic microorganisms and basalt alteration processes, we compare the phylogenetic diversity, abundance and community structure of bacteria existing in several young, seafloor lavas from the East Pacific Rise at ∼9°N that are variably affected by oxidative seawater alteration. The results of 16S rRNA gene analyses and real-time, quantitative polymerase chain reaction measurements show that the abundance of prokaryotic communities, dominated by the bacterial domain, positively correlates with the extent of rock alteration – the oldest, most altered basalt harbours the greatest microbial biomass. The bacterial community overlap, structure and species richness relative to alteration state is less explicit, but broadly corresponds to sample characteristics (type of alteration products and general alteration state). Phylogenetic analyses suggest that the basalt biome may contribute to the geochemical cycling of Fe, S, Mn, C and N in the deep sea.     

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.     

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. Electronic Publication