Does aspartic acid racemization constrain the depth limit of the subsurface biosphere?

Previous studies of the subsurface biosphere have deduced average cellular doubling times of hundreds to thousands of years based upon geochemical models. We have directly constrained the in situ average cellular protein turnover or doubling times for metabolically active micro-organisms based on cellular amino acid abundances, D/L values of cellular aspartic acid, and the in vivo aspartic acid racemization rate. Application of this method to planktonic microbial communities collected from deep fractures in South Africa yielded maximum cellular amino acid turnover times of ~89 years for 1 km depth and 27 °C and 1-2 years for 3 km depth and 54 °C. The latter turnover times are much shorter than previously estimated cellular turnover times based upon geochemical arguments. The aspartic acid racemization rate at higher temperatures yields cellular protein doubling times that are consistent with the survival times of hyperthermophilic strains and predicts that at temperatures of 85 °C, cells must replace proteins every couple of days to maintain enzymatic activity. Such a high maintenance requirement may be the principal limit on the abundance of living micro-organisms in the deep, hot subsurface biosphere, as well as a potential limit on their activity. The measurement of the D/L of aspartic acid in biological samples is a potentially powerful tool for deep, fractured continental and oceanic crustal settings where geochemical models of carbon turnover times are poorly constrained. Experimental observations on the racemization rates of aspartic acid in living thermophiles and hyperthermophiles could test this hypothesis. The development of corrections for cell wall peptides and spores will be required, however, to improve the accuracy of these estimates for environmental samples.

Fiber optic sensing technology for detecting gas hydrate formation and decomposition.

A fiber optic-based distributed sensing system (DSS) has been integrated with a large volume (72 l) pressure vessel providing high spatial resolution, time-resolved, 3D measurement of hybrid temperature-strain (TS) values within experimental sediment-gas hydrate systems. Areas of gas hydrate formation (exothermic) and decomposition (endothermic) can be characterized through this proxy by time series analysis of discrete data points collected along the length of optical fibers placed within a sediment system. Data are visualized as an animation of TS values along the length of each fiber over time. Experiments conducted in the Seafloor Process Simulator at Oak Ridge National Laboratory clearly indicate hydrate formation and dissociation events at expected pressure-temperature conditions given the thermodynamics of the CH(4)-H(2)O system. The high spatial resolution achieved with fiber optic technology makes the DSS a useful tool for visualizing time-resolved formation and dissociation of gas hydrates in large-scale sediment experiments.

Degeneration of biogenic superparamagnetic magnetite.

Magnetite crystals precipitated as a consequence of Fe(III) reduction by Shewanella algae BrY after 265 h incubation and 5-year anaerobic storage were investigated with transmission electron microscopy, Mössbauer spectroscopy and X-ray diffraction. The magnetite crystals were typically superparamagnetic with an approximate size of 13 nm. The lattice constants of the 265 h and 5-year crystals are 8.4164A and 8.3774A, respectively. The Mössbauer spectra indicated that the 265 h magnetite had excess Fe(II) in its crystal-chemistry (Fe(3+) (1.990)Fe(2+) (1.015)O(4)) but the 5-year magnetite was Fe(II)-deficient in stoichiometry (Fe(3+) (2.388)Fe(2+) (0.419)O(4)). Such crystal-chemical changes may be indicative of the degeneration of superparamagnetic magnetite through the aqueous oxidization of Fe(II) anaerobically, and the concomitant oxidation of the organic phases (fatty acid methyl esters) that were present during the initial formation of the magnetite. The observation of a corona structure on the aged magnetite corroborates the anaerobic oxidation of Fe(II) on the outer layers of magnetite crystals. These results suggest that there may be a possible link between the enzymatic activity of the bacteria and the stability of Fe(II)-excess magnetite, which may help explain why stable nano-magnetite grains are seldom preserved in natural environments.

Challenges for coring deep permafrost on Earth and Mars.

A scientific drilling expedition to the High Lake region of Nunavut, Canada, was recently completed with the goals of collecting samples and delineating gradients in salinity, gas composition, pH, pe, and microbial abundance in a 400 m thick permafrost zone and accessing the underlying pristine subpermafrost brine. With a triple-barrel wireline tool and the use of stringent quality assurance and quality control (QA/QC) protocols, 200 m of frozen, Archean, mafic volcanic rock was collected from the lower boundary that separates the permafrost layer and subpermafrost saline water. Hot water was used to remove cuttings and prevent the drill rods from freezing in place. No cryopegs were detected during penetration through the permafrost. Coring stopped at the 535 m depth, and the drill water was bailed from the hole while saline water replaced it. Within 24 hours, the borehole iced closed at 125 m depth due to vapor condensation from atmospheric moisture and, initially, warm water leaking through the casing, which blocked further access. Preliminary data suggest that the recovered cores contain viable anaerobic microorganisms that are not contaminants even though isotopic analyses of the saline borehole water suggests that it is a residue of the drilling brine used to remove the ice from the upper, older portion of the borehole. Any proposed coring mission to Mars that seeks to access subpermafrost brine will not only require borehole stability but also a means by which to generate substantial heating along the borehole string to prevent closure of the borehole from condensation of water vapor generated by drilling.

Estimates of biogenic methane production rates in deep marine sediments at Hydrate Ridge, Cascadia margin.

Methane hydrate found in marine sediments is thought to contain gigaton quantities of methane and is considered an important potential fuel source and climate-forcing agent. Much of the methane in hydrates is biogenic, so models that predict the presence and distribution of hydrates require accurate rates of in situ methanogenesis. We estimated the in situ methanogenesis rates in Hydrate Ridge (HR) sediments by coupling experimentally derived minimal rates of methanogenesis to methanogen biomass determinations for discrete locations in the sediment column. When starved in a biomass recycle reactor, Methanoculleus submarinus produced ca. 0.017 fmol methane/cell/day. Quantitative PCR (QPCR) directed at the methyl coenzyme M reductase subunit A gene (mcrA) indicated that 75% of the HR sediments analyzed contained <1,000 methanogens/g. The highest numbers of methanogens were found mostly from sediments <10 m below seafloor. By considering methanogenesis rates for starved methanogens (adjusted to account for in situ temperatures) and the numbers of methanogens at selected depths, we derived an upper estimate of <4.25 fmol methane produced/g sediment/day for the samples with fewer methanogens than the QPCR method could detect. The actual rates could vary depending on the real number of methanogens and various seafloor parameters that influence microbial activity. However, our calculated rate is lower than rates previously reported for such sediments and close to the rate derived using geochemical modeling of the sediments. These data will help to improve models that predict microbial gas generation in marine sediments and determine the potential influence of this source of methane on the global carbon cycle.

Micron-pore-sized metallic filter tube membranes for filtration of particulates and water purification.

Robust filtering techniques capable of efficiently removing particulates and biological agents from water or air suffer from plugging, poor rejuvenation, low permeance, and high backpressure. Operational characteristics of pressure-driven separations are in part controlled by the membrane pore size, charge of particulates, transmembrane pressure and the requirement for sufficient water flux to overcome fouling. With long term use filters decline in permeance due to filter-cake plugging of pores, fouling, or filter deterioration. Though metallic filter tube development at ORNL has focused almost exclusively on gas separations, a small study examined the applicability of these membranes for tangential filtering of aqueous suspensions of bacterial-sized particles. A mixture of fluorescent polystyrene microspheres ranging in size from 0.5 to 6 microm in diameter simulated microorganisms in filtration studies. Compared to a commercial filter, the ORNL 0.6 microm filter averaged approximately 10-fold greater filtration efficiency of the small particles, several-fold greater permeance after considerable use and it returned to approximately 85% of the initial flow upon backflushing versus 30% for the commercial filter. After filtering several liters of the particle-containing suspension, the ORNL composite filter still exhibited greater than 50% of its initial permeance while the commercial filter had decreased to less than 20%. When considering a greater filtration efficiency, greater permeance per unit mass, greater percentage of rejuvenation upon backflushing (up to 3-fold), and likely greater performance with extended use, the ORNL 0.6 microm filters can potentially outperform the commercial filter by factors of 100-1,000 fold.

Extracellular synthesis of magnetite and metal-substituted magnetite nanoparticles.

We have developed a novel microbial process that exploits the ability of Fe(III)-reducing microorganisms to produce copious amounts of extracellular magentites and metal-substituted magnetite nanoparticles. The Fe(III)-reducing bacteria (Theroanaerobacter ethanolicus and Shewanella sp.) have the ability to reduce Fe(III) and various metals in aqueous media and form various sized magnetite and metal-substituted magnetite nano-crystals. The Fe(III)-reducing bacteria formed metalsubstituted magnetites using iron oxide plus metals (e.g., Co, Cr, Mn, Ni) under conditions of relatively low temperature (<70 degrees C), ambient pressure, and pH values near neutral to slightly basic (pH = 6.5 to 9). Precise biological control over activation and regulation of the biosolid-state processes can produce magnetite particles of well-defined size (typically tens of nanometers) and crystallographic morphology, containing selected dopant metals into the magnetite (Fe(3-y)XyO4) structure (where X = Co, Cr, Mn, Ni). Magnetite yields of up to 20 g/L per day have been observed in 20-L vessels. Water-based ferrofluids were formed with the nanometer sized, magnetite, and metal-substituted biomagnetite particles.

Indigenous and contaminant microbes in ultradeep mines.

Rock, air and service water samples were collected for microbial analyses from 3.2 kilometres depth in a working Au mine in the Witwatersrand basin, South Africa. The approximately metre-wide mined zone was comprised of a carbonaceous, quartz, sulphide, uraninite and Au bearing layer, called the Carbon Leader, sandwiched by quartzite and conglomerate. The microbial community in the service water was dominated by mesophilic aerobic and anaerobic, alpha-, beta- and gamma-Proteobacteria with a total biomass concentration approximately 10(4) cells ml(-1), whereas, that of the mine air was dominated by members of the Chlorobi and Bacteroidetes groups and a fungal component. The microorganisms in the Carbon Leader were predominantly mesophilic, aerobic heterotrophic, nitrate reducing and methylotrophic, beta- and gamma-Proteobacteria that were more closely related to service water microorganisms than to air microbes. Rhodamine WT dye and fluorescent microspheres employed as contaminant tracers, however, indicated that service water contamination of most of the rock samples was < 0.01% during acquisition. The microbial contaminants most likely originated from the service water, infiltrated the low permeability rock through and accumulated within mining-induced fractures where they survived for several days before being mined. Combined PLFA and terminal restriction fragment length profile (T-RFLP) analyses suggest that the maximum concentration of indigenous microorganisms in the Carbon Leader was < 10(2) cells g(-1). PLFA, 35S autoradiography and enrichments suggest that the adjacent quartzite was less contaminated and contained approximately 10(3) cells gram(-1) of thermophilic, sulphate reducing bacteria, SRB, some of which are delta-Proteobacteria. Pore water and rock geochemical analyses suggest that these SRB's may have been sustained by sulphate diffusing from the adjacent U-rich, Carbon Leader where it was formed by radiolysis of sulphide.

Molecular characterization and diversity of thermophilic iron-reducing enrichment cultures from deep subsurface environments.

AIMS: The objectives of this work were to explore the diversity in Fe (III)-reducing enrichment cultures from the deep subsurface and to identify strains involved in metal reduction. METHODS AND RESULTS: Analyses of 16S ribosomal RNA (rRNA) of enrichments, supplemented with hydrogen, acetate or pyruvate as an electron donor, identified three dominant operational taxonomic units (OTUs). All cultures exhibited considerable diversity (36-24 OTUs), even after being transferred at least nine times. Two OTUs were present in all three cultures, constituting about 65% of the total clones examined. CONCLUSION: Dominant OTUs appeared to be most closely related to Thermoanaerobacter ethanolicus or T. kivui. One OTU, which is potentially responsible for autotrophic Fe (III) reduction, was only about 95% similar to T. ethanolicus and may represent a new species. SIGNIFICANCE AND IMPACT OF THE STUDY: An unexpectedly high diversity was found in these enrichments and this diversity may be a feature that can be exploited.

Low-substrate regulated microaerophilic behavior as a stress response of aquatic and soil bacteria.

Low-substrate regulated microaerophilic behavior (LSRMB) was observed in 10-54% of the bacteria isolated from several fresh-water lakes or ponds, subsurface soils, activated sludge, and Antarctic dry valley soils. Five Pseudomonas and two Bacillus type species showed LSRMB. A subsurface Pseudomonas jessenii strain was used as a model to show the metabolic interaction between substrate and oxygen concentrations, cell band movement, and the appearance of unique stress lipids and proteins. When the oxygen in the P. jessenii culture medium was increased from 11% to 100% saturation under atmospheric condition, the concentration of 17:0 cyclopropane fatty acid, a stress indicator, increased five-fold, and four unique proteins were also detected. This stress response occurred only in low-substrate media. It is our hypothesis that LSRMB is a common but under-appreciated trait of many aquatic and soil bacteria.

Determining chemotactic responses by two subsurface microaerophiles using a simplified capillary assay method.

A simplified capillary chemotaxis assay utilizing a hypodermic needle, syringe, and disposable pipette tip was developed to measure bacterial tactic responses. The method was applied to two strains of subsurface microaerophilic bacteria. This method was more convenient than the Adler method and required less practice. Isolate VT10 was a strain of Pseudomonas syringae, which was isolated from the shallow subsurface. It was chemotactically attracted toward dextrose, glycerol, and phenol, which could be used as sole carbon sources, and toward maltose, which could not be used. Isolate MR100 was phylogenetically related to Pseudomonas mendocina and was isolated from the deep subsurface. It showed no tactic response to these compounds, although, it could use dextrose, maltose, and glycerol as carbon sources. The chemotaxis results obtained by the new method were verified by using the swarm plate assay technique. The simplified technique may be useful for routine chemotactic testing.

What's up down there?

The development of careful quality assurance criteria assuring freedom from contamination in all aspects of sample recovery has opened the window to studies of a fascinating new microbial biome in the deep subsurface. Organisms have been recovered with unusual metabolic capabilities and a chemosynthetic lifestyle independent of the recent surface photosynthetically derived energy inputs. The properties of the subsurface microbiota are critical when assessing aspects such as the utility of burying radioactive waste, the remediation of mixtures of organics, metals, and nuclides, and the search for life in extreme environments on Earth as well as on Mars and other extraterrestrial sites. In addition this pioneering work provides a foundation for examining life processes in extreme environments, such as the environment beneath the ocean floor.

Spatial and temporal variations of microbial properties at different scales in shallow subsurface sediments.

Microbial abundance, activity, and community-level physiological profiles (CLPP) were examined at centimeter and meter scales in the subsurface environment at a site near Oyster, VA. At the centimeter scale, variations in aerobic culturable heterotrophs (ACH) and glucose mineralization rates (GMR) were highest in the water table zone, indicating that water availability has a major effect on variations in microbial abundance and activity. At the meter scale, ACH and microaerophiles decreased significantly with depth, whereas anaerobic GMR often increased with depth; this may indicate low redox potentials at depth caused by microbial consumption of oxygen. Data of CLPP indicated that the microbial community (MC) in the soybean field exhibited greater capability to utilize multiple carbon sources than MC in the corn field. This difference may reflect nutrient availability associated with different crops (soybean vs corn). By using a regression model, significant spatial and temporal variations were observed for ACH, microaerophiles, anaerobic GMR, and CLPP. Results of this study indicated that water and nutrient availability as well as land use could have a dominant effect on spatial and temporal variations in microbial properties in shallow subsurface environments.

Effects of nutrient dosing on subsurface methanotrophic populations and trichloroethylene degradation.

In in situ bioremediation demonstration at the Savannah River Site in Aiken, South Carolina, trichloroethyle degrading microorganisms were stimulated by delivering nutrients to the TCE-contaminated subsurface via horizontal injection wells. Microbial and chemical monitoring of groundwater from 12 vertical wells was used to examine the effects of methane and nutrient (nitrogen and phosphorus) dosing on the methanotrophic populations and on the potential of the subsurface microbial communities to degrade TCE. Densities of methanotrophs increased 3-5 orders of magnitude during the methane- and nutrient-injection phases; this increase coinclded with the higher methane levels observed in the monitoring wells. TCE degradation capacity, although not directly tied to methane concentration, responded to the methane injection, and responded more dramatically to the multiple-nutrient injection. tion. These results support the crucial role of methane, nitrogen, and phosphorus as amended nutrients in TCE bioremediation. The enhancing effects of nutrient dosing on microbial abundance and degradative potentials, coupled with increased chloride concentrations, provided multiple lines of evidence substantiating the effectiveness of this integrated in situ bioremediation process.

Effect of temperature and yeast extract on microbial respiration of sediments from a shallow coastal subsurface and vadose zone.

As a part of our study on microbial heterogeneity in subsurface environments, we have examined the microbial respiration of sediment samples obtained from a coastal site near Oyster, VA. The sediments at the site are unconsolidated, fine to coarse beach sand and gravel. A Columbus Instruments Micro-Oxymax Respirometer was used to measure the rate of carbon dioxide (CO2) production during the respiration of the sediment samples. The rate of respiration of the sediment samples ranged from 0.035-0.6 microL CO2/h/g of the sediment. The sediment samples showing maximum (0.6 microL CO2/h/g) and minimum (0.035 microL CO2/h/g) production of CO2 were selected to study the effect of micronutrient-yeast extract (0.5 and 1.0 micrograms/g of the sediment) and water (0.5 and 1.0 mL) on the rate of CO2 production. The rate of CO2 production increased with the addition of water, but increased approx 2 orders of magnitude (from 0.26 to an average of 23.5 microL CO2/h/g) when 1.0 g/g yeast extract was added to the sediment samples. In these coastal sediments, temperature, depth, and addition of water influenced microbial activity, but the addition of 1.0 microgram/g yeast extract as a micronutrient rapidly increased the rate of CO2 production 2 orders of magnitude.

Microbiological Comparisons within and across Contiguous Lacustrine, Paleosol, and Fluvial Subsurface Sediments.

Twenty-six subsurface samples were collected from a borehole at depths of 173.3 to 196.8 m in the saturated zone at the Hanford Site in south-central Washington State. The sampling was performed throughout strata that included fine-grained lacustrine (lake) sediments, a paleosol (buried soil) sequence, and coarse-grained fluvial (river) sediments. A subcoring method and tracers were used to minimize and quantify contamination to obtain samples that were representative of subsurface strata. Sediment samples were tested for total organic carbon, inorganic carbon, total microorganisms by direct microscopic counts, culturable aerobic heterotrophs by plate counts, culturable anaerobes by most-probable-number enumeration, basal respiration rates, and mineralization of (sup14)C-labeled glucose and acetate. Total direct microscopic counts of microorganisms were low, ranging from below detection to 1.9 x 10(sup5) cells g (dry weight)(sup-1). Culturable aerobes and anaerobes were below minimum levels of detection in most samples. Direct microscopic counts, basal respiration rates, and (sup14)C-glucose mineralization were all positively correlated with total organic carbon and were highest in the lacustrine sediments. In contrast to previous subsurface studies, these saturated-zone samples did not have higher microbial abundance and activities than unsaturated sediments sampled from the same borehole, the fine-textured lacustrine sediment had higher microbial numbers and activities than the coarse-textured fluvial sands, and the paleosol samples did not have higher biomass and activities relative to the other sediments. The results of this study expand the subsurface microbiology database to include information from an environment very different from those previously studied.

Aerobic mineralization of trichloroethylene, vinyl chloride, and aromatic compounds by rhodococcus species.

Two Rhodococcus strains which were isolated from a trichloroethylene (TCE)-degrading bacterial mixture and Rhodococcus rhodochrous ATCC 21197 mineralized vinyl chloride (VC) and TCE. Greater than 99.9% of a 1-mg/liter concentration of VC was degraded by cell suspensions. [1,2-C]VC was degraded by cell suspensions, with the production of greater than 66% CO(2) and 20% C-aqueous phase products and incorporation of 10% of the C into the biomass. Cultures that utilized propane as a substrate were able to mineralize greater than 28% of [1,2-C]TCE to CO(2), with approximately 40% appearing in C-aqueous phase products and another 10% of C incorporated into the biomass. VC degradation was oxygen dependent and occurred at a pH range of 5 to 10 and temperatures of 4 to 35 degrees C. Cell suspensions degraded up to 5 mg of TCE per liter and up to 40 mg of VC per liter. Propane competitively inhibited TCE degradation. Resting cell suspensions also degraded other chlorinated aliphatic hydrocarbons, such as chloroform, 1,1-dichloroethylene, and 1,1,1-trichloroethane. The isolates degraded a mixture of aromatic and chlorinated aliphatic solvents and utilized benzene, toluene, sodium benzoate, naphthalene, biphenyl, and n-alkanes ranging in size from propane to hexadecane as carbon and energy sources. The environmental isolates appeared more catabolically versatile than R. rhodochrous ATCC 21197. The data report that environmental isolates of Rhodococcus species and R. rhodochrous ATCC 21197 have the potential to degrade TCE and VC in addition to a variety of aromatic and chlorinated aliphatic compounds either individually or in mixtures.

Bioremediation of petroleum hydrocarbons in soil column lysimeters from Kwajalein island.

Soil column studies were used to evaluate petroleum hydrocarbon (PHC) remediation in soils from Kwajalein Atoll. Treatments included controls, and combinations of water, air, nutrients, and bioaugmentation with indigenous microbes (W, A, N, and M, respectively). Microbial colony forming units (CFU) decreased in the control columns and in treatments without air. Treatments including W+A+N and W+A+N+ exhibited increased CFU. One third of the PHC was removed by water and another third was removed by W+A+N and W+A+N+M treatments. Bioaugmentation with indigenous PHC degraders did not enhance bioremediation. Potential for bioremediation was demonstrated by air, water, and nutrient amendments.

Comparison between geochemical and biological estimates of subsurface microbial activities.

Geochemical and biological estimates of in situ microbial activities were compared from the aerobic and microaerophilic sediments of the Atlantic Coastal Plain. Radioisotope time-course experiments suggested oxidation rates greater than millimolar quantities per year for acetate and glucose. Geochemical analyses assessing oxygen consumption, soluble organic carbon utilization, sulfate reduction, and carbon dioxide production suggested organic oxidation rates of nano- to micromolar quantities per year. Radiotracer timecourse experiments appeared to overestimate rates of organic carbon oxidation, sulfate reduction, and biomass production by a factor of 10(3)-10(6) greater than estimates calculated from groundwater analyses. Based on the geochemical evidence, in situ microbial metabolism was estimated to be in the nano- to micromolar range per year, and the average doubling time for the microbial community was estimated to be centuries.

Factors influencing the abundance and metabolic capacities of microorganisms in Eastern Coastal Plain sediments.

The abundance and metabolic capacities of microorganisms residing in 49 sediment samples from 4 boreholes in Atlantic Coastal Plain sediments were examined. Radiolabeled time-course experiments assessing in situ mirobial capacities were initiated within 30 min of core recovery. Acetate (1-(14)C- and(3)H-) incorporation into lipids, microbial colony forming units, and nutrient limitations were examined in aliquots of subsurface sediments. Water-saturated sands exhibited activity and numbers of viable microorganisms that were orders of magnitude greater than those of the low permeability dense clays. Increased radioisotope utilization rates were observed after 6-24-h incubation times when sediments were amended with additional water and/or nutrients. Supplements of water, phosphate, nitrate, sulfate, glucose, or minerals resulted in the stimulation of microbial activities, as evidenced by the rate of acetate incorporation into microbial lipids. Additions of water or phosphate resulted in the greatest stimulation of microbial activities. Regardless of depth, sediments that contained >20% clay particles exhibited lower activities and biomass densities, and greater stimulation with abundant water supplementation than did sediments containing >66% sands and hydraulic conductivities > 200 µm sec.(-1).