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NASA NTRS2002

Hydrogen in rocks: an energy source for deep microbial communities

To survive in deep subsurface environments, lithotrophic microbial communities require a sustainable energy source such as hydrogen. Though H2 can be produced when water reacts with fresh mineral surfaces and oxidizes ferrous iron, this reaction is unreliable since it depends upon the exposure of fresh rock surfaces via the episodic opening of cracks and fissures. A more reliable and potentially more voluminous H2 source exists in nominally anhydrous minerals of igneous and metamorphic rocks. Our experimental results indicate that H2 molecules can be derived from small amounts of H2O dissolved in minerals in the form of hydroxyl, OH- or O3Si-OH, whenever such minerals crystallized in an H2O-laden environment. Two types of experiments were conducted. Single crystal fracture experiments indicated that hydroxyl pairs undergo an in situ redox conversion to H2 molecules plus peroxy links, O3Si/OO\SiO3. While the peroxy links become part of the mineral structure, the H2 molecules diffused out of the freshly fractured mineral surfaces. If such a mechanism occurred in natural settings, the entire rock column would become a volume source of H2. Crushing experiments to facilitate the outdiffusion of H2 were conducted with common crustal igneous rocks such as granite, andesite, and labradorite. At least 70 nmol of H2/g diffused out of coarsely crushed andesite, equivalent at standard pressure and temperature to 5,000 cm3 of H2/m3 of rock. In the water-saturated, biologically relevant upper portion of the rock column, the diffusion of H2 out of the minerals will be buffered by H2 saturation of the intergranular water film.

Hydrogen/analysis↗

NASA NTRS2002

Parallel characterization of anaerobic toluene- and ethylbenzene-degrading microbial consortia by PCR-denaturing gradient gel electrophoresis, RNA-DNA membrane hybridization, and DNA microarray technology

A mesophilic toluene-degrading consortium (TDC) and an ethylbenzene-degrading consortium (EDC) were established under sulfate-reducing conditions. These consortia were first characterized by denaturing gradient gel electrophoresis (DGGE) fingerprinting of PCR-amplified 16S rRNA gene fragments, followed by sequencing. The sequences of the major bands (T-1 and E-2) belonging to TDC and EDC, respectively, were affiliated with the family Desulfobacteriaceae. Another major band from EDC (E-1) was related to an uncultured non-sulfate-reducing soil bacterium. Oligonucleotide probes specific for the 16S rRNAs of target organisms corresponding to T-1, E-1, and E-2 were designed, and hybridization conditions were optimized for two analytical formats, membrane and DNA microarray hybridization. Both formats were used to characterize the TDC and EDC, and the results of both were consistent with DGGE analysis. In order to assess the utility of the microarray format for analysis of environmental samples, oil-contaminated sediments from the coast of Kuwait were analyzed. The DNA microarray successfully detected bacterial nucleic acids from these samples, but probes targeting specific groups of sulfate-reducing bacteria did not give positive signals. The results of this study demonstrate the limitations and the potential utility of DNA microarrays for microbial community analysis.

Non-NASA Center↗

NASA NTRS2001

A microwave-powered sterilizable interface for aseptic access to bioreactors that are vulnerable to microbial contamination

Novel methods and apparatus that employ the rapid heating characteristics of microwave irradiation to facilitate the aseptic transfer of nutrients, products, and other materials between microbially sensitive systems and the external environment are described. The microwave-sterilizable access port (MSAP) consists of a 600-W magnetron emitting at a frequency of 2.45 GHz, a sterilization chamber with inlet and outlet flow lines, and a specimen transfer interface. Energy is routed to the sterilization chamber via a coaxial transmission line where small quantities of water couple strongly with the incident radiation to produce a superheated vapor phase. The efficiency of energy transfer is enhanced through the use of microwave susceptors within the sterilization chamber. Mating surfaces are thermally sterilized through direct contact with the hot gas. Efficacy has been demonstrated using the thermophile Bacillus stearothermophilus.

Validation Studies↗

NASA NTRS1998

Acronema sippewissettensis Gen. Nov. Sp. Nov., microbial mat bicosoecid (Bicosoecales = Bicosoecida)

A heterotrophic mastigote from the flat laminated Microcoleus-dominated intertidal microbial mat at the Sippewissett salt marsh, Cape Cod, Massachusetts, was isolated into monoprotist culture in the same anoxic medium that led to spirochete and other anaerobic bacterial enrichments. The protist grew vigorously and was transferred indefinitely in oxic marine medium. Videomicroscopy as well as scanning and transmission electron microscopy were used to document its features. The swimming and perching behavior, nutritional mode (bactivory) and morphology including ultra-structure identify it as an aloricate bicosoecid. The presence of heteromorphic acronematic undulipodia, bilateral bipartite tubular mastigonemes, absence of a cytostome, absence of extrusomes, and presence of "Dauerstadien" (duration stages) distinguish this from other Cafeteriaceae bicosoecids. Cell division involves a closed intranuclear spindle. The unspecialized bicosoecid morphology and behavior juxtaposed with oomycete-like vesicles and mastigonemes suggest that this protist may be an extant descendant of a common ancestor of bicosoecids and other stramenopiles (e.g. labyrinthulids, thraustochytrids and oomycetes). A new genus and species, Acronema sippewissettensis, are proposed.

Non-NASA Center↗

NASA NTRS1986

Organically preserved microbial endoliths from the late Proterozoic of East Greenland

Diverse microorganisms ranging from cyanobacteria to eukaryotic algae and fungi live endolithically within ooids, hardgrounds and invertebrate shells on the present-day sea floor. These organisms are involved in the mechanical destruction of carbonates, and are useful ecological indicators of water depth and pollution. The Phanerozoic history of microbial endoliths has been elucidated through the study of microborings (the trace fossils of endolithic microorganisms) and rare cellularly preserved individuals, but nothing was known of the possible Precambrian evolution of comparable microorganisms until Campbell documented the occurrence of microborings in late Proterozoic ooids from central East Greenland. We now report the discovery of large populations of organically preserved endolithic microorganisms in silicified pisolites from 700-800-Myr-old Limestone-Dolomite Series of East Greenland. This fossil assemblage is significant for three reasons: (1) It confirms the prediction that oolites, pisolites and hardgrounds--the substrates for pre-Phanerozoic endoliths--provide a hitherto poorly explored but rewarding set of environments into which the search for early microfossils must be broadened; (2) the assemblage is diverse, containing about 12 taxa of morphologically distinct and previously unknown endolithic cyanobacteria, plus associated epilithic and interstitial populations; and (3) at least six of the fossil populations are indistinguishable in morphology, pattern of development, reproductive biology and inferred ecology from distinctive cyanobacterial species that bore ooids today in the Bahama Banks.

Non-NASA Center↗

NASA NTRS1995

On the use of antibiotics to reduce rhizoplane microbial populations in root physiology and ecology investigations

No straightforward method exists for separating the proportion of ion exchange and respiration due to rhizoplane microbial organisms from that of root ion exchange and respiration. We examined several antibiotics that might be used for the temporary elimination of rhizoplane bacteria from hydroponically grown wheat roots (Triticum aestivum cv. Veery 10). Each antibiotic was tested for herbicidal activity and plate counts were used to enumerate bacteria and evaluate antibiotic kinetics. Only lactam antibiotics (penicillins and cephalosporins) did not reduce wheat growth rates. Aminoglycosides, the pyrimidine trimethoprim, colistin and rifampicin reduced growth rates substantially. Antibiotics acted slowly, with maximum reductions in rhizoplane bacteria occurring after more than 48 h of exposure. Combinations of nonphytotoxic antibiotics reduced platable rhizoplane bacteria by as much as 98%; however, this was generally a reduction from about 10(9) to 10(6) colony forming units per gram of dry root mass, so that many viable bacteria remained on root surfaces. We present evidence which suggests that insufficient bacterial biomass exists on root surfaces of nonstressed plants grown under well-aerated conditions to quantitatively interfere with root nitrogen absorption measurements.

NASA Discipline Life Support Systems↗

NASA NTRS1993

Did surface temperatures constrain microbial evolution?

The proposition that glaciation may not have occurred before the Cenozoic--albeit not yet a consensus position--nevertheless raises for reconsideration the surface temperature history of the earth. Glacial episodes, from the Huronian (2.3 billion years ago; BYA) through the late Paleozoic (320 to 250 million years ago; MYA) have been critical constraints on estimation of the upper bounds of temperature (Crowley 1983, Kasting and Toon 1989). Once removed, few if any constraints on the upper temperature limit other than life remain. Walker (1982) recognized that life provides an upper limit to temperature in the Precambrian. We propose a more radical concept: the upper temperature limit for viable growth of a given microbial group corresponds to the actual surface temperature at the time of the group's first appearance. In particular, we propose here that two major evolutionary developments--the emergence of cyanobacteria and aerobic eukaryotes--can be used to determine surface temperature in the Precambrian, and that only subsequent cooling mediated by higher plants and then angiosperms permitted what may possibly be the earth's first glaciation in the late Cenozoic.

NASA Program Exobiology↗

NASA NTRS1995

Diverse Thermus species inhabit a single hot spring microbial mat

Through an effort to characterize aerobic chemoorganotrophic bacteria in the Octopus Spring cyano-bacterial mat community, we cultivated four Thermus isolates with unique 16S rRNA sequences. Isolates clustered within existing Thermus clades, including those containing Thermus ruber, Thermus aquaticus, and a subgroup closely related to T. aquaticus. One Octopus Spring isolate is nearly identical (99.9% similar) to isolates from Iceland, and two others are closely related to a T. ruber isolated from Russia. Octopus Spring isolates similar to T. aquaticus and T. ruber exhibited optimal growth rates at high (65-70 degrees C) and low (50 degrees C) temperatures, respectively, with the most abundant species best adapted to the temperature of the habitat (50-55 degrees C). Our results display a diversity of Thermus genotypes defined by 16S rRNA within one hot spring microbial community. We suggest that specialization to temperature and perhaps other local environmental features controls the abundance of Thermus populations.

Non-NASA Center↗

NASA NTRS1993

Microscale characterization of dissolved organic matter production and uptake in marine microbial mat communities

Intertidal marine microbial mats exhibited biologically mediated uptake of low molecular weight dissolved organic matter (DOM), including D-glucose, acetate, and an L-amino acid mixture at trace concentrations. Uptake of all compounds occurred in darkness, but was frequently enhanced under natural illumination. The photosystem 2 inhibitor, 3-(3,4-dichlorophenyl)-1,1-dimethyl urea (DCMU) generally failed to inhibit light-stimulated DOM uptake. Occasionally, light plus DCMU-amended treatments led to uptake rates higher than light-incubated samples, possibly due to phototrophic bacteria present in subsurface anoxic layers. Uptake was similar with either 3H- or 14C-labeled substrates, indicating that recycling of labeled CO2 via photosynthetic fixation was not interfering with measurements of light-stimulated DOM uptake. Microautoradiographs showed a variety of pigmented and nonpigmented bacteria and, to a lesser extent, cyanobacteria and eucaryotic microalgae involved in light-mediated DOM uptake. Light-stimulated DOM uptake was often observed in bacteria associated with sheaths and mucilage surrounding filamentous cyanobacteria, revealing a close association of organisms taking up DOM with photoautotrophic members of the mat community. The capacity for dark- and light-mediated heterotrophy, coupled to efficient retention of fixed carbon in the mat community, may help optimize net production and accretion of mats, even in oligotrophic waters.

NASA Discipline Exobiology↗

NASA NTRS1989

Distribution of inorganic species in two Antarctic cryptoendolithic microbial communities

Chemical differences were noted between two Antarctic cryptoendolithic (hidden within rock) microenvironments colonized by different microbial communities. Microenvironments dominated by cyanobacteria (BPC) had a higher pH (pH 7-8) than those dominated by lichen (LTL) (pH 4.5-5.5). In order to understand the interactions between the microbiota and the inorganic environment, the inorganic environment was characterized. Water-soluble, carbonate-bound, metal-oxide, organically bound, and residual inorganic species were sequentially extracted from rock samples by chemical means. Each fraction was then quantified using inductively coupled plasma atomic emission spectrometry. BPC contained much more water-soluble and carbonate-bound Ca and Mg than LTL. Metal-oxide species of Al, Fe, and Mn were more abundant in LTL than BPC. Metal oxides appeared to be mobilized (in the order Mn > Fe > Al) from the LTL lichen zone but remained immobile in BPC sandstone. The distribution of K and P bound to metal oxide reflected the distribution of iron oxide in LTL, an indication of the importance of iron in controlling the availability of nutrients in this ecosystem. Metal oxides in turn were likely controlled or influenced by organic matter associated with the lichen community. Despite overall depletion of Fe, Al, and K in the lichen zone, SEM X-ray analysis showed that they were enriched in fungal hyphae. Water-soluble P was present despite the presence of metal oxides, which sequester phosphate. This has biological relevance since P is an essential nutrient.

NASA Program Exobiology↗

NASA NTRS1987

The cryptoendolithic microbial environment in the Ross Desert of Antarctica: satellite-transmitted continuous nanoclimate data, 1984 to 1986

A satellite mediated station for monitoring nanoclimate (climate in the millimeter range) data, suitable for use in polar regions is described. The station, located in the Ross desert of Antarctica, has been in operation for more than 3 years, measuring rock temperatures, air temperature, light, snow, wind, and moisture. The data indicate that biological activity in the cryptoendolithic microbial ecosystem is limited to the period from mid November to mid February. The total number of hours of biological activity, based on assumptions of the minimum light, temperature and moisture requirements of the community, is less than 1000 h/year. The time above 0 degrees C, representing more nearly optimal conditions, is between 50 and 550 h/year, depending on the orientation of the surface.

Non-NASA Center↗

NASA NTRS1990

Microbial mats and the early evolution of life

Microbial mats have descended from perhaps the oldest and most widespread biological communities known. Mats harbor microbes that are crucial for studies of bacterial phylogeny and physiology. They illustrate how several oxygen-sensitive biochemical processes have adapted to oxygen, and they show how life adapted to dry land long before the rise of plants. The search for the earliest grazing protists and metazoa in stromatolites is aided by observations of mats: in them, organic compounds characteristic of ancient photosynthetic protists can be identified. Recent mat studies suggest that the 13C/12C increase observed over geological time in stromatolitic organic matter was driven at least in part by a long-term decline in atmospheric carbon dioxide levels.

NASA Center ARC↗

NASA NTRS1990

The diffusive boundary layer of sediments: oxygen microgradients over a microbial mat

Oxygen microelectrodes were used to analyze the distribution of the diffusive boundary layer (DBL) at the sediment-water interface in relation to surface topography and flow velocity. The sediment, collected from saline ponds, was covered by a microbial mat that had high oxygen consumption rate and well-defined surface structure. Diffusion through the DBL constituted an important rate limitation to the oxygen uptake of the sediment. The mean effective DBL thickness decreased from 0.59 to 0.16 mm as the flow velocity of the overlying water was increased from 0.3 to 7.7 cm s-1 (measured 1 cm above the mat). The oxygen uptake rate concurrently increased from 3.9 to 9.4 nmol cm-2 min-1. The effects of surface roughness and topography on the thickness and distribution of the DBL were studied by three-dimensional mapping of the sediment-water interface and the upper DBL boundary at 0.1-mm spatial resolution. The DBL boundary followed mat structures that had characteristic dimensions > 1/2 DBL thickness but the DBL had a dampened relief relative to the mat. The effective surface area of the sediment-water interface and of the upper DBL boundary were 31 and 14% larger, respectively, than a flat plane. Surface topography thereby increased the oxygen flux across the sediment-water interface by 49% relative to a one-dimensional diffusion flux calculated from the vertical oxygen microgradients.

NASA Center ARC↗

NASA NTRS1990

Mobilifilum chasei: morphology and ecology of a spirochete from an intertidal stratified microbial mat community

Spirochetes were found in the lower anoxiphototrophic layer of a stratified microbial mat (North Pond, Laguna Figueroa, Baja California, Mexico). Ultra-structural analysis of thin sections of field samples revealed spirochetes approximately 0.25 micrometer in diameter with 10 or more periplasmic flagella, leading to the interpretation that these spirochetes bear 10 flagellar insertions on each end. Morphometric study showed these free-living spirochetes greatly resemble certain symbiotic ones, i.e., Borrelia and certain termite spirochetes, the transverse sections of which are presented here. The ultrastructure of this spirochete also resembles Hollandina and Diplocalyx (spirochetes symbiotic in arthropods) more than it does Spirochaeta, the well known genus of mud-dwelling spirochetes. The new spirochete was detected in mat material collected both in 1985 and in 1987. Unique morphology (i.e., conspicuous outer coat of inner membrane, large number of periplasmic flagella) and ecology prompt us to name a new free-living spirochete.

Non-NASA Center↗

NASA NTRS1988

The cryptoendolithic microbial environment in the Ross Desert of Antarctica: mathematical models of the thermal regime

Microbial activity in the Antarctic cryptoendolithic habitat is regulated primarily by temperature. Previous field studies have provided some information on the thermal regime in this habitat, but this type of information is limited by the remoteness of the site and the harsh climatic conditions. Therefore, a mathematical model of the endolithic thermal regime was constructed to augment the field data. This model enabled the parameters affecting the horizontal and altitudinal distribution of the community to be examined. The model predicts that colonization should be possible on surfaces with zenith angle less than 15 degrees. At greater zenith angles, colonization should be restricted to surfaces with azimuth angles less than 135 degrees or greater than 225 degrees. The upper elevational limit of the community should be less than 2,500 m. The thermal regime probably does not influence the zonation of the community within a rock.

Non-NASA Center↗

NASA NTRS1994

Distribution of cultivated and uncultivated cyanobacteria and Chloroflexus-like bacteria in hot spring microbial mats

Oligodeoxynucleotide hybridization probes were developed to complement specific regions of the small subunit (SSU) rRNA sequences of cultivated and uncultivated cyanobacteria and Chloroflexus-like bacteria, which inhabit hot spring microbial mats. The probes were used to investigate the natural distribution of SSU rRNAs from these species in mats of Yellowstone hot springs of different temperatures and pHs as well as changes in SSU rRNA distribution resulting from 1-week in situ shifts in temperature, pH, and light intensity. Synechococcus lividus Y-7c-s SSU rRNA was detected only in the mat of a slightly acid spring, from which it may have been initially isolated, or when samples from a more alkaline spring were incubated in the more acid spring. Chloroflexus aurantiacus Y-400-fl SSU rRNA was detected only in a high-temperature mat sample from the alkaline Octopus Spring or when lower-temperature samples from this mat were incubated at the high-temperature site. SSU rRNAs of uncultivated species were more widely distributed. Temperature distributions and responses to in situ temperature shifts suggested that some of the uncultivated cyanobacteria might be adapted to high-, moderate-, and low-temperature ranges whereas an uncultivated Chloroflexus-like bacterium appears to have broad temperature tolerance. SSU rRNAs of all uncultivated species inhabiting a 48 to 51 degrees C Octopus Spring mat site were most abundant in the upper 1 mm and were not detected below a 2.5-to 3.5-mm depth, a finding consistent with their possible phototrophic nature. However, the effects of light intensity reduction on these SSU rRNAs were variable, indicating the difficulty of demonstrating a phototrophic phenotype in light reduction experiments.

Non-NASA Center↗

NASA NTRS1986

Model system for studies of microbial dynamics at exuding surfaces such as the rhizosphere

An autoclavable all-glass system for studying microbial dynamics at permeable surfaces is described. Standard hydrophobic or hydrophilic membranes (46-mm diameter) of various pore sizes were supported on a glass frit through which nutrient solutions were pumped by a peristaltic pump. The pump provided a precisely controlled flow at speeds of 0.5 to 500 ml of defined or natural cell exudates per h, which passed through the membrane into a receiving vessel. The construction allowed a choice of membranes, which could be modified. The system was tested with a bacterium, isolated from rape plant roots (Brassica napus L.), that was inoculated on a hydrophilic membrane filter and allowed to develop into a biofilm. A defined medium with a composition resembling that of natural rape root exudate was pumped through the membrane at 0.5 ml/h. Scanning electron microscopic examinations indicated that the inoculum formed microcolonies embedded in exopolymers evenly distributed over the membrane surface. The lipid composition and content of poly-beta-hydroxybutyrate in free-living and adhered cells were determined by gas chromatography. The bacterial consumption of amino acids in the exudate was also studied.

NASA Discipline Number 61-10↗

NASA NTRS1985

The cryptoendolithic microbial environment in the Antarctic cold desert: temperature variations in nature

In the Antarctic cold desert, cryptoendolithic microorganisms live under the surface of porous sandstone rocks. During the austral summer, the environment of the near-surface rock layers colonized by organisms is characterized by two kinds of temperature oscillations, both occurring across the freezing point. Low-frequency (diurnal) and large-amplitude (up to about 20 degrees C) oscillations on the sunlit surface of rocks result in a daily freeze-thaw cycle. This is a result of the diurnal changes in the sun altitude and angle with respect to the rock surface. The biological effect of this oscillation is the regulation of the onset and cessation of metabolic activity. The high-frequency (few minutes) oscillations occur only under certain weather conditions (sunny days with light winds) and are superimposed on the low-frequency oscillations. They are caused by the cooling effect of wind gusts on rock surfaces that are much warmer than ambient air temperatures. High-frequency oscillations result in a rapid freeze-thaw cycle on the surface, which, however, does not reach the microbial zone. These high-frequency freeze-thaw oscillations are probably the cause of the abiotic nature of the rock surface. Both oscillations seem to have an effect on rock weathering.

Cold Climate↗