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.

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.

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.

Biodegradation of chlorinated aliphatics and aromatic compounds in total-recycle expanded-bed biofilm reactors.

Ground-water contamination by chlorinated aliphatic compounds is a major cause for concern because of their toxicity. This study examined the biodegradation of trichloroethylene and aromatic compounds by microbial consortia enriched from contaminated subsurface sediments. The consortia were capable of utilizing methane and propane as sources of carbon and energy. Two continuously recycled expanded-bed bioreactors were inoculated with (1) the subsurface consortium, and (2) P. fluorescence, P. putida (strains pRB1401 and pWWO), and M. trichosporium OB3b. An uninoculated reactor containing 0.2% sodium azide and 0.5% formalin served as the control. Methane (5% v/v) and propane (3% v/v) were maintained by batch feeding through the course of the experiment. Greater than 97% degradation of trichloroethylene was observed over a period of 12 d. More than 99% of benzene, toluene, and xylene were degraded within the first 7 d. Dissolved oxygen levels were measured and found to be in the range 4.9-6.5 mg/L throughout the experiments.

Biodegradation of chlorinated aliphatic hydrocarbon mixtures in a single-pass packed-bed reactor.

Aliphatic chlorinated compounds, such as trichloroethylene (TCE) and tetrachloroethylene (PCE), are major contaminants of ground water. A single-pass packed-bed bioreactor was utilized to study the biodegradation of organic waste mixtures consisting of PCE, TCE, and other short-chain chlorinated organics. The bioreactor consisted of two 1960-mL glass columns joined in a series. One column was packed with sand containing a microbial consortia enriched from a contaminated site. The other column provided a reservoir for oxygen and a carbon source of methane/propane that was recirculated through the reactor. Sampling was accomplished by both direct headspace and liquid effluent concentration analyses. The reactor was operated in a single-pass mode. Greater than 99% degradation of trichloroethylene, approaching drinking water standards, was observed when the bioreactor residence time ranged from 1.9 to 3.2 d. Typically, when the reactor was pulse-fed with methane, propane, and air, 1 mol of TCE was degraded/110 mol of substrate utilized. Perturbation studies were performed to characterize reactor behavior. The system's degradation behavior was affected by providing different carbon sources, a pulse feeding regime, supplementing microbial biomass, and by altering flow rates.

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.

Development of a differential volume reactor system for soil biodegradation studies.

A bench scale experimental system was developed for the analysis of polycyclic aromatic hydrocarbon (PAH) degradation by mixed microbial cultures in PAH contaminated Manufactured Gas Plant (MGP) soils and on sand. The reactor system was chosen in order to provide a fundamental protocol capable for evaluating the performance of specific mixed microbial cultures on specific soil systems by elucidating the important system variables and their interactions. The reactor design and peripherals are described. A plug flow differential volume reactor (DVR) was used in order to remove performance effects related to reactor type, as opposed to system structure. This reactor system could be well represented mathematically. Methods were developed for on-line quantitative determination of PAH liquid phase concentrations. The mathematical models and experimental data are presented for the biodegradation of naphthalene on artificial and MGP soils.

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.

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.

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.

Influence of pH on Terminal Carbon Metabolism in Anoxic Sediments from a Mildly Acidic Lake.

The carbon and electron flow pathways and the bacterial populations responsible for transformation of H(2)-CO(2), formate, methanol, methylamine, acetate, glycine, ethanol, and lactate were examined in sediments collected from Knaack Lake, Wis. The sediments were 60% organic matter (pH 6.2) and did not display detectable sulfate-reducing activity, but they contained the following average concentration (in micromoles per liter of sediment) of metabolites and end products: sulfide, 10; methane, 1,540; CO(2), 3,950; formate, 25; acetate, 157; ethanol, 174; and lactate, 138. Methane was produced predominately from acetate, and only 4% of the total CH(4) was derived from CO(2). Methanogenesis was limited by low environmental temperature and sulfide levels and more importantly by low pH. Increasing in vitro pH to neutral values enhanced total methane production rates and the percentage of CO(2) transformed to methane but did not alter the amount of CO(2) produced from [2-C]acetate ( approximately 24%). Analysis of both carbon transformation parameters with C-labeled tracers and bacterial trophic group enumerations indicated that methanogenesis from acetate and both heterolactic- and acetic acid-producing fermentations were important to the anaerobic digestion process.

Effect of fall turnover on terminal carbon metabolism in lake mendota sediments.

The carbon and electron flow pathways and the bacterial populations responsible for the transformation of H(2)-CO(2), formate, methanol, methylamine, acetate, ethanol, and lactate were examined in eutrophic sediments collected during summer stratification and fall turnover. The rate of methane formation averaged 1,130 mumol of CH(4) per liter of sediment per day during late-summer stratification versus 433 mumol of CH(4) per liter of sediment per day during the early portion of fall turnover, whereas the rate of sulfate reduction was 280 mumol of sulfate per liter of sediment per day versus 1,840 mumol of sulfate per liter of sediment per day during the same time periods, respectively. The sulfate-reducing population remained constant while the methanogenic population decreased by one to two orders of magnitude during turnover. The acetate concentration increased from 32 to 81 mumol per liter of sediment while the acetate transformation rate constant decreased from 3.22 to 0.70 per h, respectively, during stratification versus turnover. Acetate accounted for nearly 100% of total sedimentary methanogenesis during turnover versus 70% during stratification. The fraction of CO(2) produced from all C-labeled substrates examined was 10 to 40% higher during fall turnover than during stratification. The addition of sulfate, thiosulfate, or sulfur to stratified sediments mimicked fall turnover in that more CO(2) and CH(4) were produced. The addition of Desulfovibrio vulgaris to sulfate-amended sediments greatly enhanced the amount of CO(2) produced from either [C]methanol or [2-C]acetate, suggesting that H(2) consumption by sulfate reducers can alter methanol or acetate transformation by sedimentary methanogens. These data imply that turnover dynamically altered carbon transformation in eutrophic sediments such that sulfate reduction dominated over methanogenesis principally as a consequence of altering hydrogen metabolism.

Gas metabolism evidence in support of the juxtaposition of hydrogen-producing and methanogenic bacteria in sewage sludge and lake sediments.

We developed new techniques to measure dissolved H(2) and H(2) consumption kinetics in anoxic ecosystems that were not dependent on headspace measurements or gas transfer-limited experimentation. These H(2) metabolism parameters were then compared with measured methane production rates, and estimates of H(2) production and interspecies H(2) transfer were made. The H(2) pool sizes were 205 and 31 nM in sewage sludge from an anaerobic digestor and in sediments (24 m) from Lake Mendota, respectively. The H(2) turnover rate constants, as determined by using in situ pool sizes and temperatures, were 103 and 31 h for sludge and sediment, respectively. The observed H(2) turnover rate accounted for only 5 to 6% of the expected H(2)-CO(2)-dependent methanogenesis in these ecosystems. Our results are in general agreement with the results reported previously and are used to support the conclusion that most of the H(2)-dependent methanogenesis in these ecosystems occurs as a consequence of direct interspecies H(2) transfer between juxtapositioned microbial associations within flocs or consortia.

Sulfate-Dependent Interspecies H(2) Transfer between Methanosarcina barkeri and Desulfovibrio vulgaris during Coculture Metabolism of Acetate or Methanol.

We compared the metabolism of methanol and acetate when Methanosarcina barkeri was grown in the presence and absence of Desulfovibrio vulgaris. The sulfate reducer was not able to utilize methanol or acetate as the electron donor for energy metabolism in pure culture, but was able to grow in coculture. Pure cultures of M. barkeri produced up to 10 mumol of H(2) per liter in the culture headspace during growth on acetate or methanol. In coculture with D. vulgaris, the gaseous H(2) concentration was

Survival of subsurface microorganisms exposed to UV radiation and hydrogen peroxide.

Aerobic and microaerophilic subsurface bacteria were screened for resistance to UV light. Contrary to the hypothesis that subsurface bacteria should be sensitive to UV light, the organisms studied exhibited resistance levels as efficient as those of surface bacteria. A total of 31% of the aerobic subsurface isolates were UV resistant, compared with 26% of the surface soil bacteria that were tested. Several aerobic, gram-positive, pigmented, subsurface isolates exhibited greater resistance to UV light than all of the reference bacterial strains tested except Deinococcus radiodurans. None of the microaerophilic, gram-negative, nonpigmented, subsurface isolates were UV resistant; however, these isolates exhibited levels of sensitivity similar to those of the gram-negative reference bacteria Escherichia coli B and Pseudomonas fluorescens. Photoreactivation activity was detected in three subsurface isolates, and strain UV3 exhibited a more efficient mechanism than E. coli B. The peroxide resistance of four subsurface isolates was also examined. The aerobic subsurface bacteria resistant to UV light tolerated higher levels of H2O2 than the microaerophilic organisms. The conservation of DNA repair pathways in subsurface microorganisms may be important in maintaining DNA integrity and in protecting the organisms against chemical insults, such as oxygen radicals, during periods of slow growth.