Stress response of methanogenic archaea from Siberian permafrost compared with methanogens from nonpermafrost habitats

We examined the survival potential of methanogenic archaea exposed to different environmental stress conditions such as low temperature (down to −78.5°C), high salinity (up to 6 M NaCl), starvation (up to 3 months), long-term freezing (up to 2 years), desiccation (up to 25 days) and oxygen exposure (up to 72 h). The experiments were conducted with methanogenic archaea from Siberian permafrost and were complemented by experiments on well-studied methanogens from nonpermafrost habitats. Our results indicate a high survival potential of a methanogenic archaeon from Siberian permafrost when exposed to the extreme conditions tested. In contrast, these stress conditions were lethal for methanogenic archaea isolated from nonpermafrost habitats. A better adaptation to stress was observed at a low temperature (4°C) compared with a higher one (28°C). Given the unique metabolism of methanogenic archaea in general and the long-term survival and high tolerance to extreme conditions of the methanogens investigated in this study, methanogenic archaea from permafrost should be considered as primary candidates for possible subsurface Martian life.     

Methanogenic communities in permafrost-affected soils of the Laptev Sea coast, Siberian Arctic, characterized by 16S rRNA gene fingerprints

Permafrost environments in the Arctic are characterized by extreme environmental conditions that demand a specific resistance from microorganisms to enable them to survive. In order to understand the carbon dynamics in the climate-sensitive Arctic permafrost environments, the activity and diversity of methanogenic communities were studied in three different permafrost soils of the Siberian Laptev Sea coast. The effect of temperature and the availability of methanogenic substrates on CH4 production was analysed. In addition, the diversity of methanogens was analysed by PCR with specific methanogenic primers and by denaturing gradient gel electrophoresis (DGGE) followed by sequencing of DGGE bands reamplified from the gel. Our results demonstrated methanogenesis with a distinct vertical profile in each investigated permafrost soil. The soils on Samoylov Island showed at least two optima of CH4 production activity, which indicated a shift in the methanogenic community from mesophilic to psychrotolerant methanogens with increasing soil depth. Furthermore, it was shown that CH4 production in permafrost soils is substrate-limited, although these soils are characterized by the accumulation of organic matter. Sequence analyses revealed a distinct diversity of methanogenic archaea affiliated to Methanomicrobiaceae, Methanosarcinaceae and Methanosaetaceae. However, a relationship between the activity and diversity of methanogens in permafrost soils could not be shown.     

Life on Mars

Abstract

There is evidence that at one time Mars had liquid water habitats on its surface. Studies of microbial communities in cold and dry environments on the Earth provide a basis for discussion of the possible nature of any life that may have existed on Mars during that time. Of particular relevance are the cyanobacterial communities found in hypolithic and endolithic habitats in deserts. Microbial mats found under ice-covered lakes provide an additional possible Martian system. Results obtained from these field studies can be used to guide the search for fossil evidence of life on Mars. It is possible that in the future life will be reintroduced on Mars in an effort to restore that planet to habitable conditions. In this case the organisms under study as exemplars of past life may provide the hardy stock of pioneering Martian organisms. These first organisms must be followed by plants. The feasibility of reviving Mars will depend on the ability of plants to grow in an abundance of CO₂ but at extremely low pressures, temperatures, O₂, and N₂ levels. On Mars, biology was, and is, destiny.     

Errata corrige

Abstract

Ecological research on extreme environments can be applied to exobiological problems such as the question of life on Mars. If life forms (fossil or extant) are found on Mars, their study will help to solve fundamental questions about the nature of life on Earth. Extreme environments that are beyond the range of adaptability of their inhabitants are defined as “absolute extreme.” Such environments can serve as terrestrial models for the last stages of life in the history of Mars, when the surface cooled down and atmosphere and water disappeared. The cryptoendolithic microbial community in porous rocks of the Ross Desert in Antarctica and the microbial mats at the bottom of frozen Antarctic lakes are such examples. The microbial communities of Siberian permafrost show that, in frozen but stable communities, long-term survival is possible. In the context of terraforming Mars, selected microorganisms isolated from absolute extreme environments are considered for use in creation of a biological carbon cycle.     

Methanogenic community composition and anaerobic carbon turnover in submarine permafrost sediments of the Siberian Laptev Sea

The Siberian Laptev Sea shelf contains submarine permafrost, which was formed by flooding of terrestrial permafrost with ocean water during the Holocene sea level rise. This flooding resulted in a warming of the permafrost to temperatures close below 0°C. The impact of these environmental changes on methanogenic communities and carbon dynamics in the permafrost was studied in a submarine permafrost core of the Siberian Laptev Sea shelf. Total organic carbon (TOC) content varied between 0.03% and 8.7% with highest values between 53 and 62 m depth below sea floor. In the same depth, maximum methane concentrations (284 nmol CH₄ g⁻¹) and lowest carbon isotope values of methane (−72.2‰ VPDB) were measured, latter indicating microbial formation of methane under in situ conditions. The archaeal community structure was assessed by a nested polymerase chain reaction (PCR) amplification for DGGE, followed by sequencing of reamplified bands. Submarine permafrost samples showed a different archaeal community than the nearby terrestrial permafrost. Samples with high methane concentrations were dominated by sequences affiliated rather to the methylotrophic genera Methanosarcina and Methanococcoides as well as to uncultured archaea. The presented results give the first insights into the archaeal community in submarine permafrost and the first evidence for their activity at in situ conditions.     

Methanogenic activity and biomass in Holocene permafrost deposits of the Lena Delta, Siberian Arctic and its implication for the global methane budget

Permafrost environments within the Siberian Arctic are natural sources of the climate relevant trace gas methane. In order to improve our understanding of the present and future carbon dynamics in high latitudes, we studied the methane concentration, the quantity and quality of organic matter, and the activity and biomass of the methanogenic community in permafrost deposits. For these investigations a permafrost core of Holocene age was drilled in the Lena Delta (72°22′N, 126°28′E). The organic carbon of the permafrost sediments varied between 0.6% and 4.9% and was characterized by an increasing humification index with permafrost depth. A high CH₄ concentration was found in the upper 4 m of the deposits, which correlates well with the methanogenic activity and archaeal biomass (expressed as PLEL concentration). Even the incubation of core material at −3 and −6°C with and without substrates showed a significant CH₄ production (range: 0.04–0.78 nmol CH₄ h⁻¹ g⁻¹). The results indicated that the methane in Holocene permafrost deposits of the Lena Delta originated from modern methanogenesis by cold‐adapted methanogenic archaea. Microbial generated methane in permafrost sediments is so far an underestimated factor for the future climate development.     

Mars Primordial Crust: Unique Sites for Investigating Proto-biologic Properties

The Martian meteorite collection suggests that intact outcrops or boulder-scale fragments of the 4.5 Ga Martian crust exist within tens of meters of the present day surface of Mars. Mars may be the only planet where such primordial crust samples, representing the first 100 Ma of a planet’s environment, are available. The primordial crust has been destroyed on Earth by plate tectonics and other geological phenomena and is buried on the Moon under hundreds or thousands of meters of megaregoltih. Early Mars appears to have been remarkably similar to early Earth, and samples of rock from the first few Ma or first 100 Ma may reveal “missing link” proto-biological forms that could shed light on the transition from abiotic organic chemistry to living cells. Such organic snapshots of nascent life are unlikely to be found on Earth. Presented at: National Workshop on Astrobiology: Search for Life in the Solar System, Capri, Italy, 26 to 28 October, 2005.     

Life on Mars: chemical arguments and clues from Martian meteorites

Primitive terrestrial life – defined as a chemical system able to transfer its molecular information via self-replication and to evolve – probably originated from the evolution of reduced organic molecules in liquid water. Several sources have been proposed for the prebiotic organic molecules: terrestrial primitive atmosphere (methane or carbon dioxide), deep-sea hydrothermal systems, and extraterrestrial meteoritic and cometary dust grains. The study of carbonaceous chondrites, which contain up to 5% by weight of organic matter, has allowed close examination of the delivery of extraterrestrial organic material. Eight proteinaceous amino acids have been identified in the Murchison meteorite among more than 70 amino acids. Engel reported that l-alanine was surprisingly more abundant than d-alanine in the Murchison meteorite. Cronin also found excesses of l-enantiomers for nonprotein amino acids. A large collection of micrometeorites has been recently extracted from Antarctic old blue ice. In the 50- to 100-μm size range, carbonaceous micrometeorites represent 80% of the samples and contain 2% of carbon, on average. They might have brought more carbon than that involved in the present surficial biomass. The early histories of Mars and Earth clearly show similarities. Liquid water was once stable on the surface of Mars, attesting the presence of an atmosphere capable of deccelerating C-rich micrometeorites. Therefore, primitive life may have developed on Mars as well and fossilized microorganisms may still be present in the near subsurface. The Viking missions to Mars in 1976 did not find evidence of either contemporary or past life, but the mass spectrometer on the lander aeroshell determined the atmospheric composition, which has allowed a family of meteorites to be identified as Martian. Although these samples are essentially volcanic in origin, it has been recognized that some of them contain carbonate inclusions and even veins that have a carbon isotopic composition indicative of an origin from Martian atmospheric carbon dioxide. The oxygen isotopic composition of these carbonate deposits allows calculation of the temperature regime existing during formation from a fluid that dissolved the carbon dioxide. As the composition of the fluid is unknown, only a temperature range can be estimated, but this falls between 0° and 90°C, which would seem entirely appropriate for life processes. It was such carbonate veins that were found to host putative microfossils. Irrespective of the existence of features that could be considered to be fossils, carbonate-rich portions of Martian meteorites tend to have material, at more than 1000 ppm, that combusts at a low temperature; i.e., it is an organic form of carbon. Unfortunately, this organic matter does not have a diagnostic isotopic signature so it cannot be unambiguously said to be indigenous to the samples. However, many circumstantial arguments can be made to the effect that it is cogenetic with the carbonate and hence Martian. If it could be proved that the organic matter was preterrestrial, then the isotopic fractionation between it and the carbon is in the right sense for a biological origin. Received: January 22, 1998 / Accepted: February 16, 1998     

Identification of Methanogenic archaea in the Hyporheic Sediment of Sitka Stream

Methanogenic archaea produce methane as a metabolic product under anoxic conditions and they play a crucial role in the global methane cycle. In this study molecular diversity of methanogenic archaea in the hyporheic sediment of the lowland stream Sitka (Olomouc, Czech Republic) was analyzed by PCR amplification, cloning and sequencing analysis of the methyl coenzyme M reductase alpha subunit (mcrA) gene. Sequencing analysis of 60 clones revealed 24 different mcrA phylotypes from hyporheic sedimentary layers to a depth of 50 cm. Phylotypes were affiliated with Methanomicrobiales, Methanosarcinales and Methanobacteriales orders. Only one phylotype remains unclassified. The majority of the phylotypes showed higher affiliation with uncultured methanogens than with known methanogenic species. The presence of relatively rich assemblage of methanogenic archaea confirmed that methanogens may be an important component of hyporheic microbial communities and may affect CH₄ cycling in rivers.     

Chemical and physical microenvironments at the Viking landing sites

Summary Physical and chemical considerations permit the division of the near-surface regolith on Mars into at least six zones of distinct microenvironments. The zones are euphotic, duricrust/peds, tempofrost, permafrost, endolithic, and interfacial/transitional. Microenvironments vary significantly in temperature extremes, mean temperature, salt content, relative pressure of water vapor, UV and visible light irradiance, and exposure to ionizing radiation events (100 Mrad) and oxidative molecular species. From what is known of the chemistry of the atmossphere and regolith fines (soil), limits upon the aqueous chemistry of soil pastesmay be estimated. Heat of wetting could reach 45 cal/g dry soil; initial pH is indeterminate between 1 and 10; ionic strength and salinity are predicted to be extremely high; freezing point depression is inadequate to provide quantities of liquid water except in special cases. The prospects for biotic survival are grim by terrestrial standards, but the extremes of biological resiliency are inaccessible to evaluation. Second-generation in situ experiments which will better define Martian microenvironments are clearly possible. Antarctic dry valleys are approximations to Martian conditions, but deviate significantly by at least half-a-dozen criteria.     

Using thermodynamics to obtain geochemical information from genomes

Thermodynamic characterization of the relative stabilities of chemical compounds is a pillar of conceptual models in various fields of geosciences. Analogous models applied to genomes can yield new information about the relationship between genomes and their geochemical environments. In this perspective article, we present a chemical and thermodynamic analysis of prokaryotic lineages that have been the target of previous phylogenomic studies of evolutionary adaptation to varying redox conditions. The thermodynamic model development begins by quantifying the effects of hydrogen activity (aH₂) and temperature on the relative stabilities of organic compounds with different carbon oxidation state. When applied to proteins instead of metabolites, the same techniques can be used to identify combinations of aH₂ and temperature at which reference proteomes for Class I or Class II methanogens are relatively stable. The calculated aH₂ values are compatible with reported measurements for habitats of methanogens ranging from highly reducing submarine hydrothermal systems to less reducing environments including methanogenic sediments. In contrast to the transition between the two classes of methanogenic archaea, that between basal and terrestrial groups of Thaumarchaeota (denoting the origin of ammonia-oxidizing archaea) occurs at a less-reducing redox boundary. These examples reveal the consequences of energy minimization driving evolution and show how geochemical calculations involving biomolecules can be used to quantify and better understand the coevolution of the geosphere and biosphere.     

Astrobiological significance of minerals on Mars surface environment

Despite the large amount of geomorphological, geodynamic and geophysical data obtained from Mars missions, much is still unknown about Martian mineralogy and paragenetic assemblages, which is fundamental to an understanding of its entire geological history. Minerals are not only indicators of the physical–chemical settings of the different environments and their later changes, but also they could (and do) play a crucial astrobiological role related with the possibility of existence of extinct or extant Martian life. This paper aims: (1) to present a synoptic review of the main water-related Martian minerals (mainly jarosite and other sulfates) discovered up to the present time; (2) to emphasize their significance as environmental geomarkers, on the basis of their geological settings and mineral parageneses on earth (in particular in the context of some selected terrestrial analogues), and (3) to show that their differential UV shielding properties, against the hostile environmental conditions of the Martian surface, are of a great importance for the search for extraterrestrial life.     

Colonization of rice roots with methanogenic archaea controls photosynthesis‐derived methane emission

The methane emitted from rice fields originates to a large part (up to 60%) from plant photosynthesis and is formed on the rice roots by methanogenic archaea. To investigate to which extent root colonization controls methane (CH₄) emission, we pulse‐labeled rice microcosms with ¹³CO₂ to determine the rates of ¹³CH₄ emission exclusively derived from photosynthates. We also measured emission of total CH₄ (¹²⁺¹³CH₄), which was largely produced in the soil. The total abundances of archaea and methanogens on the roots and in the soil were analysed by quantitative polymerase chain reaction of the archaeal 16S rRNA gene and the mcrA gene coding for a subunit of the methyl coenzyme M reductase respectively. The composition of archaeal and methanogenic communities was determined with terminal restriction fragment length polymorphism (T‐RFLP). During the vegetative growth stages, emission rates of ¹³CH₄ linearly increased with the abundance of methanogenic archaea on the roots and then decreased during the last plant growth stage. Rates of ¹³CH₄ emission and the abundance of methanogenic archaea were lower when the rice was grown in quartz‐vermiculite with only 10% rice soil. Rates of total CH₄ emission were not systematically related to the abundance of methanogenic archaea in soil plus roots. The composition of the archaeal communities was similar under all conditions; however, the analysis of mcrA genes indicated that the methanogens differed between the soil and root. Our results support the hypothesis that rates of photosynthesis‐driven CH₄ emission are limited by the abundance of methanogens on the roots.     

Extreme environments and exobiology

Ecological research on extreme environments can be applied to exobiological problems such as the question of life on Mars. If life forms (fossil or extant) are found on Mars, their study will help to solve fundamental questions about the nature of life on Earth. Extreme environments that are beyond the range of adaptability of their inhabitants are defined as "absolute extreme". Such environments can serve as terrestrial models for the last stages of life in the history of Mars, when the surface cooled down and atmosphere and water disappeared. The cryptoendolithic microbial community in porous rocks of the Ross Desert in Antarctica and the microbial mats at the bottom of frozen Antarctic lakes are such examples. The microbial communities of Siberian permafrost show that, in frozen but stable communities, long-term survival is possible. In the context of terraforming Mars, selected microorganisms isolated from absolute extreme environments are considered for use in creation of a biological carbon cycle.     

Community analysis of methanogenic archaea within a riparian flooding gradient

Anoxic soils in river floodplains (or riparian soils) are a source of methane emission. However, little is known about the ecology and community structure of archaeal methanogenic microbes, which are a crucial component of methane flux in those habitats. We studied the archaeal community in the vertical profile of four different sites along the River Waal in the Netherlands. These sites differ in their annual flooding regime ranging from never or seldom to permanently flooded. The archaeal community structure has been characterized by terminal restriction fragment length polymorphism (T-RFLP) and comparative sequence analysis of the archaeal SSU rRNA gene and the mcrA gene. The latter gene codes for the alpha-subunit of methyl-coenzyme M reductase. Additionally, the potential methanogenic activity was determined by incubation of soil slurries under anoxic conditions. The community composition differed only slightly with the depth of the soil (0-20 cm). However, the diversity of archaeal SSU rRNA genes increased with the frequency of flooding. Terminal restriction fragment length polymorphism analysis of mcrA gene amplicons confirmed the results concerning methanogenic archaea. In the never and rarely flooded soils, crenarchaeotal sequences were the dominant group. In the frequently and permanently flooded soils, Methanomicrobiaceae, Methanobacteriaceae, Methanosarcinaceae and the uncultured Rice Clusters IV and VI (Crenarchaeota) were detectable independently from duration of anoxic conditions. Methanosaetaceae, on the other hand, were only found in the permanently and frequently flooded soils under conditions where concentrations of acetate were < 30 microM. The results indicate that methanogens as well as other archaea occupy characteristic niches according to the flooding conditions in the field. Methanosaetaceae, in particular, seem to be adapted (or proliferate at) to low acetate concentrations.     

Mars ultraviolet simulation facility

Summary A facility was established for long-duration ultraviolet (UV) radiation exposure of natural and synthetic materials in order to test hypotheses concerning Martian soil chemistry observed by the Viking Mars landers. The system utilized a 2500 watt xenon lamp as the radiation source, with the beam passing through a heat-dissipating water filter before impinging upon an exposure chamber containing the samples to be irradiated. The chamber was designed to allow for continuous tumbling of the samples, maintenance of temperatures below 0° during exposure, and monitoring of beam intensity. The facility also provided for sample preparation under a variety of atmospheric conditions, in addition to the Mars nominal. As many as 33 sealed sample ampules have been irradiated in a single exposure. Over 100 samples have been irradiated for approximately 100 to 700 h. The facility has performed well in providing continuous UV irradiation of multiple samples for long periods of time under simulated Mars atmospheric and thermal conditions.     

Microbial life beneath a high arctic glacier

The debris-rich basal ice layers of a high Arctic glacier were shown to contain metabolically diverse microbes that could be cultured oligotrophically at low temperatures (0.3 to 4 degrees C). These organisms included aerobic chemoheterotrophs and anaerobic nitrate reducers, sulfate reducers, and methanogens. Colonies purified from subglacial samples at 4 degrees C appeared to be predominantly psychrophilic. Aerobic chemoheterotrophs were metabolically active in unfrozen basal sediments when they were cultured at 0.3 degrees C in the dark (to simulate nearly in situ conditions), producing (14)CO(2) from radiolabeled sodium acetate with minimal organic amendment (> or =38 microM C). In contrast, no activity was observed when samples were cultured at subfreezing temperatures (< or =-1.8 degrees C) for 66 days. Electron microscopy of thawed basal ice samples revealed various cell morphologies, including dividing cells. This suggests that the subglacial environment beneath a polythermal glacier provides a viable habitat for life and that microbes may be widespread where the basal ice is temperate and water is present at the base of the glacier and where organic carbon from glacially overridden soils is present. Our observations raise the possibility that in situ microbial production of CO(2) and CH(4) beneath ice masses (e.g., the Northern Hemisphere ice sheets) is an important factor in carbon cycling during glacial periods. Moreover, this terrestrial environment may provide a model for viable habitats for life on Mars, since similar conditions may exist or may have existed in the basal sediments beneath the Martian north polar ice cap.     

有氧产甲烷研究进展

甲烷是温室气体的一种,对全球气候变化和人们的生活都有着重要的影响。全球绝大部分甲烷来自于产甲烷古菌的代谢,因此主要发生在厌氧环境之中。然而一些最新的研究发现,自然界的有氧环境中同样存在着甲烷产生现象,而产甲烷微生物也不仅限于产甲烷古菌。本文从微生物的产甲烷作用出发,对有氧环境中的甲烷产生进行整体的归纳,总结已有的研究结果,为以后产甲烷方面的研究提供新的思路和方向。     

S.A.M., the Italian Martian Simulation Chamber

The Martian Environment Simulator (SAM “Simulatore di Ambiente Marziano”) is a interdisciplinary project of Astrobiology done at University of Padua. The research is aimed to the study of the survival of the microorganisms exposed to the “extreme” planetary environment. The facility has been designed in order to simulate Mars’ environmental conditions in terms of atmospheric pressure, temperature cycles and UV radiation dose. The bacterial cells, contained into dedicated capsules, will be exposed to thermal cycles simulating diurnal and seasonal Martian cycles. The metabolism of the different biological samples will be analysed at different phases of the experiment, to study their survival and eventual activity of protein synthesis (mortality, mutations and capability of DNA reparing). We describe the experimental facility and provide the perspectives of the biological experiments we will perform in order to provide hints on the possibility of life on Mars either autochthonous or imported from Earth. Presented at: National Workshop on Astrobiology: Search for Life in the Solar System, Capri, Italy, 26 to 28 October, 2005.     

Primary structure of selected archaeal mesophilic and extremely thermophilic outer surface layer proteins

The archaea are recognized as a separate third domain of life together with the bacteria and eucarya. The archaea include the methanogens, extreme halophiles, thermoplasmas, sulfate reducers and sulfur metabolizing thermophiles, which thrive in different habitats such as anaerobic niches, salt lakes, and marine hydrothermals systems and continental solfataras. Many of these habitats represent extreme environments in respect to temperature, osmotic pressure and pH-values and remind on the conditions of the early earth. The cell envelope structures were one of the first biochemical characteristics of archaea studied in detail. The most common archaeal cell envelope is composed of a single crystalline protein or glycoprotein surface layer (S-layer), which is associated with the outside of the cytoplasmic membrane. The S-layers are directly exposed to the extreme environment and can not be stabilized by cellular components. Therefore, from comparative studies of mesophilic and extremely thermophilic S-layer proteins hints can be obtained about the molecular mechanisms of protein stabilization at high temperatures. First crystallization experiments of surface layer proteins under microgravity conditions were successful. Here, we report on the biochemical features of selected mesophilic and extremely archaeal S-layer (glyco-) proteins.