Molecular identification of cyanobacteria associated with stromatolites from distinct geographical locations

Modern stromatolites represent a significant resource for studying microbial ecology and evolution. A preliminary investigation was undertaken employing specific genetic probes to characterize the cyanobacteria responsible for stromatolite construction in a range of environments, including microbial mats found in Australia not previously examined with molecular methods. Isolates of cyanobacteria were collected from stromatolites in thermal springs, hypersaline lakes, and oceanic fringes on two continents. A polymerase chain reaction specific for DNA of cyanobacterial 16S rRNA was developed, the resulting products of the DNA amplification reaction were sequenced, and the data were used to infer relatedness between the isolates studied and other members of the cyanobacterial radiation. Complete sequence was generated for the region from position 27 to 408 for 13 strains of cyanobacteria associated with stromatolites. All stromatolite-derived sequences were most closely related to cyanobacteria, as indicated by local sequence alignment. It was possible to correlate genetic identity with morphological nomenclatures and to expand the phylogeny of benthic cyanobacteria. These inferences were also expanded to temporal variation in the dominant resident cyanobacterial species based on sampling of surface and core sinter laminations. Under the methods employed, only one cyanobacterial strain was detected in each sample, suggesting the possible dominance of a specific clonal population of cyanobacteria at any one time in the biota of the samples tested. The data indicate that internal core samples of a stromatolite at least 10 years old can be successfully analyzed by DNA-based methods to identify preserved cyanobacteria.     

Calcification and silicification: fossilization potential of cyanobacteria from stromatolites of Niuafo'ou's Caldera Lakes (Tonga) and implications for the early fossil record

Calcification and silicification processes of cyanobacterial mats that form stromatolites in two caldera lakes of Niuafo'ou Island (Vai Lahi and Vai Si'i) were evaluated, and their importance as analogues for interpreting the early fossil record are discussed. It has been shown that the potential for morphological preservation of Niuafo'ou cyanobacteria is highly dependent on the timing and type of mineral phase involved in the fossilization process. Four main modes of mineralization of cyanobacteria organic parts have been recognized: (i) primary early postmortem calcification by aragonite nanograins that transform quickly into larger needle-like crystals and almost totally destroy the cellular structures, (ii) primary early postmortem silicification of almost intact cyanobacterial cells that leave a record of spectacularly well-preserved cellular structures, (iii) replacement by silica of primary aragonite that has already recrystallized and obliterated the cellular structures, (iv) occasional replacement of primary aragonite precipitated in the mucopolysaccharide sheaths and extracellular polymeric substances by Al-Mg-Fe silicates. These observations suggest that the extremely scarce earliest fossil record may, in part, be the result of (a) secondary replacement by silica of primary carbonate minerals (aragonite, calcite, siderite), which, due to recrystallization, had already annihilated the cellular morphology of the mineralized microbiota or (b) relatively late primary silicification of already highly degraded and no longer morphologically identifiable microbial remains.     

Comparative Metagenomics of Two Microbial Mats at Cuatro Ciénegas Basin II: Community Structure and Composition in Oligotrophic Environments

Abstract Microbial mats are self-sustained, functionally complex ecosystems that make good models for the understanding of past and present microbial ecosystems as well as putative extraterrestrial ecosystems. Ecological theory suggests that the composition of these communities might be affected by nutrient availability and disturbance frequency. We characterized two microbial mats from two contrasting environments in the oligotrophic Cuatro Ciénegas Basin: a permanent green pool and a red desiccation pond. We analyzed their taxonomic structure and composition by means of 16S rRNA clone libraries and metagenomics and inferred their metabolic role by the analysis of functional traits in the most abundant organisms. Both mats showed a high diversity with metabolically diverse members and strongly differed in structure and composition. The green mat had a higher species richness and evenness than the red mat, which was dominated by a lineage of Pseudomonas. Autotrophs were abundant in the green mat, and heterotrophs were abundant in the red mat. When comparing with other mats and stromatolites, we found that taxonomic composition was not shared at species level but at order level, which suggests environmental filtering for phylogenetically conserved functional traits with random selection of particular organisms. The highest diversity and composition similarity was observed among systems from stable environments, which suggests that disturbance regimes might affect diversity more strongly than nutrient availability, since oligotrophy does not appear to prevent the establishment of complex and diverse microbial mat communities. These results are discussed in light of the search for extraterrestrial life. Key Words: Cuatro Ciénegas-Metagenomics-Microbial mats-Oligotrophic-Phosphorus limitation-Stromatolites. Astrobiology 12, 659-673.     

Comparative metagenomics of two microbial mats at cuatro ciénegas basin I: ancient lessons on how to cope with an environment under severe nutrient stress

Abstract The Cuatro Ciénegas Basin (CCB) is an oasis in the desert of Mexico characterized by low phosphorus availability and by its great diversity of microbial mats. We compared the metagenomes of two aquatic microbial mats from the CCB with different nutrient limitations. We observed that the red mat was P-limited and dominated by Pseudomonas, while the green mat was N-limited and had higher species richness, with Proteobacteria and Cyanobacteria as the most abundant phyla. From their gene content, we deduced that both mats were very metabolically diverse despite their use of different strategies to cope with their respective environments. The red mat was found to be mostly heterotrophic, while the green mat was more autotrophic. The red mat had a higher number of transporters in general, including transporters of cellobiose and osmoprotectants. We suggest that generalists with plastic genomes dominate the red mat, while specialists with minimal genomes dominate the green mat. Nutrient limitation was a common scenario on the early planet; despite this, biogeochemical cycles were performed, and as a result the planet changed. The metagenomes of microbial mats from the CCB show the different strategies a community can use to cope with oligotrophy and persist. Key Words: Microbial mats-Metagenomics-Metabolism. Astrobiology 12, 648-658.     

Following the kinetics: iron-oxidizing microbial mats in cold icelandic volcanic habitats and their rock-associated carbonaceous signature

Icelandic streams with mean annual temperatures of less than 5 °C, which receive the cationic products of basaltic rock weathering, were found to host mats of iron-cycling microorganisms. We investigated two representative sites. Iron-oxidizing Gallionella and iron-reducing Geobacter species were present. The mats host a high bacterial diversity as determined by culture-independent methods. β-Proteobacteria, Actinobacteria, α-Proteobacteria, and Bacteroidetes were abundant microbial taxa. The mat contained a high number of phototroph sequences. The carbon compounds in the mat displayed broad G and D bands with Raman spectroscopy. This signature becomes incorporated into the weathered oxidized surface layer of the basaltic rocks and was observed on rocks that no longer host mats. The presence of iron-oxidizing taxa in the stream microbial mats, and the lack of them in previously studied volcanic rocks in Iceland that have intermittently been exposed to surface water flows, can be explained by the kinetic limitations to the extraction of reduced iron from rocks. This type of ecosystem illustrates key factors that control the distribution of chemolithotrophs in cold volcanic environments. The data show that one promising sample type for which the hypothesis of the existence of past life on Mars can be tested is the surface of volcanic rocks that, previously, were situated within channels carved by flowing water. Our results also show that the carbonaceous signatures of life, if life had occurred, could be found in or on these rocks.     

Long-term manipulations of intact microbial mat communities in a greenhouse collaboratory: simulating earth's present and past field environments

Photosynthetic microbial mat communities were obtained from marine hypersaline saltern ponds, maintained in a greenhouse facility, and examined for the effects of salinity variations. Because these microbial mats are considered to be useful analogs of ancient marine communities, they offer insights about evolutionary events during the >3 billion year time interval wherein mats co-evolved with Earth's lithosphere and atmosphere. Although photosynthetic mats can be highly dynamic and exhibit extremely high activity, the mats in the present study have been maintained for >1 year with relatively minor changes. The major groups of microorganisms, as assayed using microscopic, genetic, and biomarker methodologies, are essentially the same as those in the original field samples. Field and greenhouse mats were similar with respect to rates of exchange of oxygen and dissolved inorganic carbon across the mat-water interface, both during the day and at night. Field and greenhouse mats exhibited similar rates of efflux of methane and hydrogen. Manipulations of salinity in the water overlying the mats produced changes in the community that strongly resemble those observed in the field. A collaboratory testbed and an array of automated features are being developed to support remote scientific experimentation with the assistance of intelligent software agents. This facility will permit teams of investigators the opportunity to explore ancient environmental conditions that are rare or absent today but that might have influenced the early evolution of these photosynthetic ecosystems.     

Degradation of cyanobacterial biosignatures by ionizing radiation

Primitive photosynthetic microorganisms, either dormant or dead, may remain today on the martian surface, akin to terrestrial cyanobacteria surviving endolithically in martian analog sites on Earth such as the Antarctic Dry Valleys and the Atacama Desert. Potential markers of martian photoautotrophs include the red edge of chlorophyll reflectance spectra or fluorescence emission from systems of light-harvesting pigments. Such biosignatures, however, would be modified and degraded by long-term exposure to ionizing radiation from the unshielded cosmic ray flux onto the martian surface. In this initial study into this issue, three analytical techniques--absorbance, reflectance, and fluorescence spectroscopy--were employed to determine the progression of the radiolytic destruction of cyanobacteria. The pattern of signal loss for chlorophyll reflection and fluorescence from several biomolecules is characterized and quantified after increasing exposures to ionizing gamma radiation. This allows estimation of the degradation rates of cyanobacterial biosignatures on the martian surface and the identification of promising detectable fluorescent break-down products.     

Putative fossilized fungi from the lithified volcaniclastic apron of Gran Canaria, Spain

We report the discovery of fossilized filamentous structures in samples of the lithified, volcaniclastic apron of Gran Canaria, which were obtained during Leg 157 of the Ocean Drilling Program (ODP). These filamentous structures are 2-15 μm in diameter and several hundred micrometers in length and are composed of Si, Al, Fe, Ca, Mg, Na, Ti, and C. Chitin was detected in the filamentous structures by staining with wheat germ agglutinin dye conjugated with fluorescein isothiocyanate (WGA-FITC), which suggests that they are fossilized fungal hyphae. The further elucidation of typical filamentous fungal morphological features, such as septa, hyphal bridges, and anastomosis and their respective sizes, support this interpretation. Characteristic structures that we interpreted as fossilized spores were also observed in association with the putative hyphae. The fungal hyphae were found in pyroxene phenocrysts and in siderite pseudomorphs of a basalt breccia. The fungal colonization of the basalt clasts occurred after the brecciation but prior to the final emplacement and lithification of the sediment at ～16-14 Ma. The siderite appears to have been partially dissolved by the presence of fungal hyphae, and the fungi preferentially colonized Fe-rich carbonates over Fe-poor carbonates (aragonite). Our findings indicate that fungi may be an important geobiological agent in subseafloor environments and an important component of the deep subseafloor biosphere, and that hydrothermal environments associated with volcanism can support a diverse ecosystem, including eukaryotes.     

Biomarker and stable isotope characterization of coastal pond-derived organic matter, McMurdo Dry Valleys, Antarctica

Small coastal ponds that contain photosynthetic microbial mat communities represent an extreme environment where a potentially significant source of labile organic carbon can be found within the McMurdo Dry Valleys, Antarctica. To distinguish coastal pond-derived organic matter from other sources of organic matter in the Dry Valleys, bulk organic carbon, nitrogen, and sulfur isotope signatures and phospholipid fatty acid (PLFA) profiles of benthic microbial mats located at two sites--Hjorth Hill coast and Garwood Valley--were investigated. The average isotope values at Hjorth Hill coast and Garwood Valley are, respectively, -10.9 per thousand and -10.2 per thousand for delta(13) C, 3.7 per thousand and -1.3 per thousand for delta(15)N, and 8.1 per thousand and 16.7 per thousand for delta(34)S. Microbial mats from all ponds are dominated by monounsaturated PLFAs (indicative of Gram-negative bacteria) and polyunsaturated PLFAs (indicative of microeukaryotes). Biomarkers specific to aerobic prokaryotes, eukaryotes, and photoautotrophic microeukaryotes, as well as sulfur-reducing bacteria, are present in all samples. Benthic mats at Garwood Valley are thicker and more laminated, have a higher biomass, and have a greater carbon and nitrogen content, which suggests greater productivity than mats at Hjorth Hill coast. Greater productivity is supported, as well, by higher dissolved oxygen contents likely derived from heightened photosynthetic productivity. More productivity at Garwood Valley likely results from a larger influx of terrestrial surface waters together with a concomitant nutrient loading.     

Biogenicity of morphologically diverse carbonaceous microstructures from the ca. 3400 Ma Strelley pool formation, in the Pilbara Craton, Western Australia

Morphologically diverse structures that may constitute organic microfossils are reported from three remote and widely separated localities assigned to the ca. 3400?Ma Strelley Pool Formation in the Pilbara Craton, Western Australia. These localities include the Panorama, Warralong, and Goldsworthy greenstone belts. From the Panorama greenstone belt, large (> 40?μm) lenticular to spindle-like structures, spheroidal structures, and mat-forming thread-like structures are found. Similar assemblages of carbonaceous structures have been identified from the Warralong and Goldsworthy greenstone belts, though these assemblages lack the thread-like structures but contain film-like structures. All structures are syngenetic with their host sedimentary black chert, which is associated with stromatolites and evaporites. The host chert is considered to have been deposited in a shallow water environment. Rigorous assessment of biogenicity (considering composition, size range, abundance, taphonomic features, and spatial distributions) suggests that cluster-forming small (<15 μm) spheroids, lenticular to spindle-like structures, and film-like structures with small spheroids are probable microfossils. Thread-like structures are more likely fossilized fibrils of biofilm, rather than microfossils. The biogenicity of solitary large (>15?μm) spheroids and simple film-like structures is less certain. Although further investigations are required to confirm the biogenicity of carbonaceous structures from the Strelley Pool Formation, this study presents evidence for the existence of morphologically complex and large microfossils at 3400?Ma in the Pilbara Craton, which can be correlated to the contemporaneous, possible microfossils reported from South Africa. Although there is still much to be learned, they should provide us with new insights into the early evolution of life and shallow water ecosystems.     

Hypersaline microbial systems of sabkhas: examples of life's survival in "extreme" conditions

Life and living systems need several important factors to establish themselves and to have a continued tradition. In this article the nature of the borderline situation for microbial life under heavy salt stress is analyzed and discussed using the example of biofilms and microbial mats of sabkha systems of the Red Sea. Important factors ruling such environments are described, and include the following: (1) Microbial life is better suited for survival in extremely changing and only sporadically water-supplied environments than are larger organisms (including humans). (2) Microbial life shows extremely poikilophilic adaptation patterns to conditions that deviate significantly from conditions normal for life processes on Earth today. (3) Microbial life adapts itself to such extremely changing and only ephemerally supportive conditions by the capacity of extreme changes (a) in morphology (pleomorphy), (b) in metabolic patterns (poikilotrophy), (c) in survival strategies (poikilophily), and (d) by trapping and enclosing all necessary sources of energy matter in an inwardly oriented diffusive cycle. All this is achieved without any serious attempt at escaping from the extreme and extremely changing conditions. Furthermore, these salt swamp systems are geophysiological generators of energy and material reservoirs recycled over a geological time scale. Neither energy nor material is wasted for propagation by spore formation. This capacity is summarized as poikilophilic and poikilotroph behavior of biofilm or microbial mat communities in salt and irradiationstressed environmental conditions of the sabkha or salt desert type. We use mainly cyanobacteria as an example, although other bacteria and even eukaryotic fungi may exhibit the same potential of living and surviving under conditions usually not suitable for life on Earth. It may, however, be postulated that such poikilophilic organisms are the true candidates for life support and survival under conditions never recorded on Planet Earth. Mars and some planets of other suns may be good candidates to search for life under conditions normally not thought to be favorable for the maintenance of life.     

Using a mini-Raman spectrometer to monitor the adaptive strategies of extremophile colonizers in arid deserts: relationships between signal strength, adaptive strategies, solar radiation, and humidity

Abstract The survival strategies of one cyanobacteria colony and three terricolous lichen species from the hot subdesert of Tabernas, Spain, were studied along with topographical attributes of the area to investigate whether the protective strategies adopted by these pioneer soil colonizers are related to the environmental stressors under which they survive. A handheld Raman spectrometer was used for biomolecular characterization, while the microclimatic and topographic parameters were estimated with a Geographic Information System (GIS). We found that the survival strategies adopted by those organisms are based on different combinations of protective biomolecules, each with diverse ecophysiological functions, such as UV-radiation screening, free-energy quenching, antioxidants, and the production of different types and amounts of calcium oxalates. Our results show that the cyanobacteria community and each lichen species preferentially colonized a particular microhabitat with specific moisture and incident solar radiation levels and exhibited different adaptive mechanisms. In recent years, a number of studies have provided consistent results that suggest a link between the strategies adopted by those extremophile organisms and the microclimatic environmental parameters. To date, however, far too little attention has been paid to results from Raman analyses on dry specimens. Therefore, the results of the present study, produced with the use of our miniaturized instrument, will be of interest to future studies in astrobiology, especially due to the likely use of Raman spectroscopy at the surface of Mars. Key Words: Hot desert-Raman spectroscopy-Topography-Terricolous lichens-Cyanobacteria-Planetary exploration. Astrobiology 12, 743-753.     

Lava cave microbial communities within mats and secondary mineral deposits: implications for life detection on other planets

Lava caves contain a wealth of yellow, white, pink, tan, and gold-colored microbial mats; but in addition to these clearly biological mats, there are many secondary mineral deposits that are nonbiological in appearance. Secondary mineral deposits examined include an amorphous copper-silicate deposit (Hawai'i) that is blue-green in color and contains reticulated and fuzzy filament morphologies. In the Azores, lava tubes contain iron-oxide formations, a soft ooze-like coating, and pink hexagons on basaltic glass, while gold-colored deposits are found in lava caves in New Mexico and Hawai'i. A combination of scanning electron microscopy (SEM) and molecular techniques was used to analyze these communities. Molecular analyses of the microbial mats and secondary mineral deposits revealed a community that contains 14 phyla of bacteria across three locations: the Azores, New Mexico, and Hawai'i. Similarities exist between bacterial phyla found in microbial mats and secondary minerals, but marked differences also occur, such as the lack of Actinobacteria in two-thirds of the secondary mineral deposits. The discovery that such deposits contain abundant life can help guide our detection of life on extraterrestrial bodies.     

Lossless compression of stromatolite images: a biogenicity index?

It has been underappreciated that inorganic processes can produce stromatolites (laminated macroscopic constructions commonly attreibuted to microbiological activity), thus calling into question the long-standing use of stromatolites as de facto evidence for ancient life. Using lossless compression on unmagnified reflectance red-green-blue (RGB) images of matched stromatolite-sediment matrix pairs as a complexity metric, the compressibility index (delta(c), the log ratio of the ratio of the compressibility of the matrix versus the target) of a putative abiotic test stromatolite is significantly less than the delta(c) of a putative biotic test stromatolite. There is a clear separation in delta(c) between the different stromatolites discernible at the outcrop scale. In terms of absolute compressibility, the sediment matrix between the stromatolite columns was low in both cases, the putative abiotic stromatolite was similar to the intracolumnar sediment, and the putative biotic stromatolite was much greater (again discernible at the outcrop scale). We propose tht this metric would be useful for evaluating the biogenicity of images obtained by the camera systems available on every Mars surface probe launched to date including Viking, Pathfinder, Beagle, and the two Mars Exploration Rovers.     

浅谈温度变化对光学镜面的影响

环境温度变化将导致光学镜内的温度分布发生变化,文章分析了光学镜内不同的温度分布对其面形造成的危害,以及对光学系统成像质量的影响,对空间光学系统光学镜温度场设计具有一定参考价值.     

Control of Lunar and Martian dust--experimental insights from artificial and natural cyanobacterial and algal crusts in the desert of Inner Mongolia, China

Studies on the colonization of environmentally extreme ground surfaces were conducted in a Mars-like desert area of Inner Mongolia, People's Republic of China, with microalgae and cyanobacteria. We collected and mass-cultured cyanobacterial strains from these regions and investigated their ability to form desert crusts artificially. These crusts had the capacity to resist sand wind erosion after just 15 days of growth. Similar to the surface of some Chinese deserts, the surface of Mars is characterized by a layer of fine dust, which will challenge future human exploration activities, particularly in confined spaces that will include greenhouses and habitats. We discuss the use of such crusts for the local control of desert sands in enclosed spaces on Mars. These experiments suggest innovative new directions in the applied use of microbe-mineral interactions to advance the human exploration and settlement of space.     

Cyanobacterial emergence at 2.8 gya and greenhouse feedbacks

Apparent cyanobacterial emergence at about 2.8 Gya coincides with the negative excursion in the organic carbon isotope record, which is the first strong evidence for the presence of atmospheric methane. The existence of weathering feedbacks in the carbonate-silicate cycle suggests that atmospheric and oceanic CO2 concentrations would have been high prior to the presence of a methane greenhouse (and thus the ocean would have had high bicarbonate concentrations). With the onset of a methane greenhouse, carbon dioxide concentrations would decrease. Bicarbonate has been proposed as the preferred reductant that preceded water for oxygenic photosynthesis in a bacterial photosynthetic precursor to cyanobacteria; with the drop of carbon dioxide level, Archean cyanobacteria emerged using water as a reductant instead of bicarbonate (Dismukes et al., 2001). Our thermodynamic calculations, with regard to this scenario, give at least a tenfold drop in aqueous CO2 levels with the onset of a methane-dominated greenhouse, assuming surface temperatures of about 60 degrees C and a drop in the level of atmospheric carbon dioxide from about 1 to 0.1 bars. The buildup of atmospheric methane could have been triggered by the boost in oceanic organic productivity that arose from the emergence of pre-cyanobacterial oxygenic phototrophy at about 2.8-3.0 Gya; high temperatures may have precluded an earlier emergence. A greenhouse transition timescale on the order of 50-100 million years is consistent with results from modeling the carbonate-silicate cycle. This is an alternative hypothesis to proposals of a tectonic driver for this apparent greenhouse transition.     

Endolithic cyanobacteria in halite rocks from the hyperarid core of the Atacama Desert

In the driest parts of the Atacama Desert there are no visible life forms on soil or rock surfaces. The soil in this region contains only minute traces of bacteria distributed in patches, and conditions are too dry for cyanobacteria that live under translucent stones. Here we show that halite evaporite rocks from the driest part of the Atacama Desert are colonized by cyanobacteria. This colonization takes place just a few millimeters beneath the rock surface, occupying spaces among salt crystals. Our work reveals that these communities are composed of extremely resistant Chroococcidiopsis morphospecies of cyanobacteria and associated heterotrophic bacteria. This newly discovered endolithic environment is an extremely dry and, at the same time, saline microbial habitat. Photosynthetic microorganisms within dry evaporite rocks could be an important and previously unrecognized target for the search for life within our Solar System.     

Life support system with autonomous control employing plant photosynthesis

This research was aimed at obtaining a closed control system. This was achieved by placing all the technological processes providing for human vital activities within the hermetically sealed space, and by transferring the entire control and guidance of these processes to people inhabiting the system. In contrast to existing biological life support systems, man has been included not only as a participant of metabolism, but as an operator who is the central figure in collecting information, making decisions and controlling all technological processes. To tackle this problem, the "BIOS-3" experimental complex was created for performing long-term experiments using different structures of biological life-support system. The experiment lasted six months and consisted of three stages. During the first stage the system was comprised of two equivalent phytotrons with the culture of wheat and an assortment of vegetable plants, and the living compartment. At the second stage, one of the phytotrons was removed while a compartment of chlorella cultivators was introduced. The third stage differed from the second, the former using wheat phytotron and the latter employing phytotron with an assortment of vegetable cultures. Three men inhabited the system simultaneously. The experiment demonstrated that a biological life support system controlled autonomously from the inside is feasible within a small confined space. However, immunological and microbiological research shows, that the medium created by the system is not fully adequate for man. In conclusion, some prospects have been outlined for further studies of biological life support systems.     

Microbial influences on local carbon isotopic ratios and their preservation in carbonate

Analysis of the carbon isotopic composition of carbonates is a valuable tool for studying microbial processes and looking for evidence of life. Microbial fixation of CO2 and conversion of organic carbon to CO2 can produce measurable delta 13C shifts in a microbial mat environment. Diffusion modeling demonstrates that substantial isotopic shifts can develop within the mat and in the diffusion boundary layer in the fluid when CO2 fixation is rapid and prolonged for several hours. Carbonates that precipitate during rapid CO2 fixation can preserve these microbially produced isotopic shifts. However, continued precipitation during intervals when respiration dominates or after the cessation of active microbial growth commonly dilutes autotrophic isotopic signatures. Thus, preserved isotopic signatures rarely reflect the magnitude of isotopic shifts within the mat. Interpretation of observed isotopic shifts in microbial mat carbonate depends on fully characterizing ambient delta 13C and eliminating other origins for isotopic shifts. The carbon isotopic composition of reservoirs can vary substantially, both on Earth and on other planets. Characterizing the reservoir composition and any changes through time is critical to evaluating microbially induced shifts. In addition, careful evaluation of non-microbial causes for shifts in isotopic composition is essential for a reliable interpretation. Complicating processes include recrystallization, calcite precipitation over extended periods of time, variable precipitation rates and water chemistry, and mixing of carbonates having different isotopic signatures.