trans -1,2-Dichloroethene as the Major Product of Tetrachloroethene (PCE) Degradation. B.M. GRIFFIN, M.E. DOLLHOPF, J.M. TIEDJE, and F.E. LOEFFLER†.

NSF Ctr. for Microbial Ecology, Michigan State Univ., East Lansing, MI 48824, and † School of Civil and Environmental Engineering, Georgia Institute of Technology, Atlanta, GA 30332-0512.

Tetrachloroethene (PCE) is an abundant subsurface pollutant. Due to the toxicity of chlorinated ethenes, the complete dechlorination of PCE to ethene is desired during natural attenuation and in engineered bioremediation. Dichloroethenes (DCE's), however, often accumulate under field conditions and in laboratory microcosms. It is our current understanding that the cis$ form is the most common DCE isomer and is more readily reduced to vinyl chloride than the trans form. Hence, biodegradation research is focused on the fate of cis -DCE. In this study, we describe the microbial production of trans -DCE as the primary end product of PCE degradation. We obtained five enrichment cultures that reduced PCE and trichloroethene (TCE) primarily to trans -DCE. In all these cultures the cis and trans isomers of DCE were formed in a similar ratio of 1:3 (±0.5), and no further dechlorination was observed. 2-Bromoethanesulfonate (BES) inhibited PCE dechlorination beyond TCE in all cultures. However, after methanogens had been diluted out and BES was omitted from the medium, trans -DCE accumulated again as the major end product. In all cultures PCE was dechlorinated to TCE before further dechlorination to trans -DCE and cis -DCE occurred. A sporeforming desulfitobacterium, strain Viet 1, was isolated from one of the enrichments. Strain Viet 1 coupled the reduction of PCE to TCE to the oxidation of hydrogen, formate, lactate, and pyruvate as electron donors. Strain Viet 1 could not dechlorinate TCE. In another enrichment culture, pasteurization experiments indicated the presence of a sporeforming population capable of stoichiometrically reducing PCE to cis -DCE. Like strain Viet I, this interesting PCE to cis -DCE dechlorinating sporeformer did not produce trans -DCE. We are currently characterizing the physiologies and nutritional requirements of the different dechlorinating populations in the enrichment cultures.

Isolation and Characterization of a Novel Sulfate-Reducing Bacterium which is able to Utilize Chlorobenzenes, Phenol, and p-Cresol

HARD, B.C. and BABEL, W. UFZ Ctr. for Environmental Research, Department of Environmental Microbiology, Leipzig, Germany

Subsurface samples were taken from a pristine site near Birsay, 200 km southeast of Saskatoon, Canada. A strain of sulfate-reducing bacteria was isolated from core samples at 2.5 m depth using a modification of Postgate's medium with pyruvate as sole source of carbon and energy at a cultivation temperature of 4 °C. The strain grew in liquid culture only, agar plate cultivation was not possible. The strain, designated UFZ B 454, tested Gram-positive and had very large, up to 10 µm long chain forming cells. Spore formation was only observed at temperatures above 20 °C. 16 sRNA comparison revealed a 97% similarity with the type strain Desulfotomaculum orientis. However, fatty acid analyses and physiological characterization of UFZ B 454 and two Desulfotomaculum orientis strains, including the type strain showed significant differences between the strains. It seems therefore likely that a novel strain has been isolated. UFZ B 454 was found to be psychrotolerant, growing at temperatures between 1 and 30°C. The substrates which serve as sole source of carbon and energy include a number of xenobiotics which are not found at the sample site and which the strains has never been in contact with prior to cultivation in the laboratory. Such substrates are phenol, p-cresol, monochloro-, 1,2- dichloro- and 1,4- dichlorobenzene which the strain UFZ B 454 utilizes to reduce sulfate and to produce biomass. The possible role of this strain in low temperature bioremediation processes is discussed.

Anaerobic Microcosm Studies in Support of a Field Demonstration of Enhanced TCE Transformation at IRP Site 24, Point Mugu Naval Weapons Station, CA

M. T. KEELING and L. SEMPRINI. Oregon State Univ., Corvallis, OR

Laboratory scale microcosm studies were performed using groundwater and aquifer solids from IRP Site 24 at the Point Mugu Naval Air Weapons Station, CA to assess the feasibility of enhanced anaerobic in-situ transformation of trichloroethylene (TCE). Long-term (302 days) TCE anaerobic studies compared lactate, benzoate and methanol as potential anaerobic substrates. Site groundwater containing 1300 mg/L of sulfate and 2.3 mg/L of TCE was added to the microcosms. The substrates were added at 1.5 times the stoichiometric electron equivalent of sulfate. Nutrient addition and bioagumentation were also studied. Both benzoate and lactate stimulated systems achieved complete sulfate-reduction and prolonged dechlorination of TCE to VC and ethylene. Dechlorination was observed after 15 to 20 days of incubation in the lactate fed microcosms after sulfate concentrations were reduced to 300 mg/L, while benzoate fed microcosms required 120 to 120 days and complete sulfate reduction. Acetate and propionate were produced in both the lactate and benzoate fed microcosms, and acetate persisted until rapid methanogenesis occurred after 150 days of incubation. Methanogenesis did not accelerate the rates of dechlorination in either system. After 302 days of incubation TCE was transformed to VC (85 to 93%) and ethylene (7 to 15%). Re-additions of TCE to both systems resulted in rapid transformation to VC, while VC transformation to ethylene was very slow, and rates appeared to increase when TCE was added. Direct hydrogen addition at 10-3 and 10-4 atmospheres had no effect on the transformation of VC. Rapid methanol utilization resulted in nearly stoichiometric conversion to methane and carbon dioxide without significant sulfate-reduction or dechlorination. Bioagumentation with a TCE dechlorination culture from a previously benzoate amended Point Mugu microcosm effectively decreased lag-times and increased overall dechlorination. Nutrient addition enhanced and inhibited dechlorination in the lactate and benzoate fed microcosms, respectively. Based on the results of these tests, a pilot-scale field demonstration is currently being conducted where lactate is being added to the subsurface at IRP Site 24.

Biotransformation Of Tetrachloroethene To Ethene During Anaerobic Degradation Of Toluene

SEWELL, G.W. and SHEN, H. US-EPA and Dynamac Corporation, Ada, OK

Reductive biotransformation of tetrachloroethene (PCE) to ethene was achieved during anaerobic degradation of toluene in a subsurface derived enrichment culture. Ethene was detected as a dominant daughter product of PCE dechlorination over a wide range of PCE and toluene concentrations, with negligible accumulation of other less-chlorinated ethenes. PCE dechlorination can be described by a Monod-like equation, and followed a zero order kinetic at high levels of PCE. In addition to toluene, benzoate and lactate were also used as sole electron donors for reductive dechlorination of PCE in the culture. In terms of dechlorination rate, lactate was the best electron donor followed by benzoate and then toluene. The kinetic constants were unique to each electron donor. The dechlorination rate was found to correlate with the level of hydrogen produced during fermentation of the three organic compounds, suggesting that hydrogen serves as the direct electron donor for PCE dechlorination. Nitrate and sulfate were observed to be preferred electron acceptors for the culture, and their presence completely blocked electron transport to PCE. However, exposure to nitrate and sulfate did not destroy the dechlorination capability of the culture, since PCE dechlorination was immediately re-established after depletion of nitrate and sulfate. This anaerobic system simulates the natural attenuation of chlorinated solvents and fuel hydrocarbons at contaminated sites. The implications of the findings to the implementation of monitored natural attenuations will be discussed.

Degradation Of Both Enantiomers Of The Chiral Herbicide Mecoprop By A Groundwater Bacterium

JOHANNESEN, H.¹*, KOHLER, H.-P. E.² HANSEN, R. R¹. AND AAMAND, J.¹ 1.Geological Survey of Denmark and Greenland (GEUS), Copenhagen, Denmark, 2. Swiss Federal Institute for Environmental Science and Technology (EAWAG), Duebendorf, Switzerland.

Burkholderia cepacia PM, a bacterial strain isolated from an aerobic aquifer, was able to grow on both enantiomers of the chiral herbicide mecoprop ((RS)-2-(4-Chloro-2-Methylphenoxy)Propionic Acid) as the sole source of carbon and energy. When cells grew on the racemic mecoprop as the substrate, the S enantiomer disappeared from the medium before the R enantiomer did. When racemic mecoprop, the pure R, or the pure S enantiomer of mecoprop was the substrate for growth, disappearance of all the substrate from the medium occured in the order racemic, R- and S-mecoprop. Resting cells of strain PM which had grown on pure S-mecoprop or on pure R-mecoprop took up both enantiomers with slightly different rates. This indicates that the enzyme- and/or transport systems for degradation and uptake of mecoprop by strain PM do not distinguish between the two enantiomers. Another possibility is two enantio-specific systems, which are either both induced by each of the enantiomers or constitutively expressed. Cells of strain PM which adhered to sand surfaces in columns were also able to degrade both enantiomers of racemic mecoprop which was eluted through the columns. Of the racemic mecoprop eluted through the sand columns, 80-90% of the total degraded mecoprop was the S enantiomer, and 10-20% was the R enantiomer of mecoprop.

Ecophysiological Parameters Of Importance For Microbial Degradation of MCPP in an Aerobic Aquifer ALBRECHTSEN, H.-J., CHRISTIANSEN, T.G., SCHOUW, N.L. AND RÜGGE, R.

Department of Environmental Science and Engineering, Groundwater Research Centre, Technical Univ. of Denmark.

Distribution of Microbial Communities in Sediments that Contain Methane Hydrates. F.S. COLWELL, M.E. DELWICHE, D.B. BLACKWELDER, R.M. LEHMAN, Y. FUJITA, M.S. WILSON, T. UCHIDA

Idaho Natl. Engineering and Environmental Laboratory, Humboldt State Univ., Japanese Petroleum Exploration Research Center.

Methanogens are believed to be responsible for producing the large quantities of methane that are trapped in gas hydrate deposits in deep sediments around the world. This research focuses on the distribution of microbial communities in subsurface environments where methane hydrates are present. Abiotic factors that may control microbial distribution include high hydrostatic pressures, the proximity of methane hydrates, high concentrations of dissolved or free phase methane, total organic carbon, and sediment texture. We enumerated total cells using acridine orange direct counts (AODC) and culturable methanogens by most probable number (MPN) in gas hydrate-bearing sediment cores from the Nankai Trough off the coast of Japan and from a borehole on the Mackenzie River delta, Canada. AODC on Nankai Trough sediment cores obtained from 0.24 to 250 m below the seafloor indicated cell numbers as high as 1x10⁷ near the surface decreasing to less than 1x10⁵ cells/g at the greatest depth. AODC on Mackenzie River delta samples obtained from 912 to 950 m below land surface had similar values of 1.1x10⁵ to 2.8x10⁶ cells/g with sandy sediments exhibiting lower AODC values than silty sediments. Methanogen MPNs from Nankai Trough samples indicated 10 to 100 cells/g and did not decrease significantly with depth. In Mackenzie Delta samples, methanogen numbers constituted as much as 1% of the cells that were seen in AODCs with the highest numbers estimated at greater than 10³ cells/g in sandy strata that were rich in methane hydrate. These data confirm the widespread distribution of methanogens and other microorganisms in deep sediments which contain methane hydrates, including the unique observation of cells in subpermafrost zones. Understanding the distribution and constraints on these microbial communities is important considering the potentially significant economic and environmental impacts associated with methane hydrates.

Oceanic basalt, an important deep biosphere.

T. TORSVIK, H. FURNES, I.H. THORSETH, O. TUMYR, R. B. PEDERSEN AND K. MUEHLENBACHS. T.Torsvik; Dept. of Microbiology, Univ. of Bergen, Jahnebk. 5, N-5007 Bergen, Norway. H. Furnes, I.H.Thorseth, O.Tumyr, R.B.Pedersen; Geological Inst., Univ. of Bergen, Allegt.4, N- 5007 Bergen, Norway. K.Muehlenbachs; Dept. of Geology, Univ. of Alberta, Alberta, Canada

Evidence for microbial activity has been found in basalt from the mid Atlantic Ridge, the Costa Rica Rift and the Lau basin, in recent to 38 Ma old samples and at in situ temperature ranging from 0 to 100 °C. At the Costa Rica Rift the depth of active biodegradation appeared to reach 380 m into the volcanic basement. Microbes were found in the glassy part of the basalt, predominately at the glass-alteration interface. The evidence for the presence of microbes include morphological evidence and detection of DNA and ribosomal RNA. Scanning electron microscopic analysis of samples from the Costa Rica Rift showed that stalkforming bacteria were common. Analysis of ribosomal RNA in samples from the Costa Rica Rift showed the presence of both Bacteria and Archaeae. Geochemical evidence showed that microbial activity resulted in pitting of the glass. In the process C, N, P, and K accumulated at the alteration front. Where measured the N/C ratios were comparable to those of nitrogen-starved bacteria. Stable isotopic analysis of disseminated carbonates showed that microbes predominately obtained energy from oxidation of organic material to CO2, resulting in a negative ^13? signature. In some samples from the Mid Atlantic Ridge positive 13C signatures indicated methanogenic activity.

Microbes in Igneous Rocks of the Ocean Crust

M. R. FISK, I. H. THORSETH, S. J. GIOVANNONI, H. POINAR. Oregon State Univ., Corvallis, Oregon 97331; University of Bergen, 5007 Bergen, Norway; Oregon State Univ., Corvallis, Oregon, 97331; Max Planck Inst. for Evolutionary Anthropology, Leipzig, D-04103 Germany

About 70 percent of the Earth is covered by oceans, and 70 percent of the oceans are floored by igneous crust that formed at ocean ridges. The upper several hundred meters of this igneous crust erupted as molten lava into cold sea water to form a jumble of glass-covered basalts. Chemical reactions involving sea water and basalt glass and minerals produce clay and other secondary minerals. Several lines of evidence support the hypothesis that microbes are associated with the production of secondary minerals from glass in basalts in the oceans. First, the alteration front between glass and secondary minerals is enriched in carbon, phosphorus, nitrogen, and nucleic acids. Second, examination of rocks by electron microscopy identifies cells at the interface of glass and secondary minerals. The location of organic matter at the boundary between the fresh basalt and secondary minerals suggests that some of these microbes may promote the alteration of the basalts. Observations of hundreds of surface and subsurface oceanic basalts indicates that microbes are widespread in the ocean crust. Nucleic acids from cleaned samples show that a variety of microorganisms are present in the rocks, some of which appear to be closely aligned with barophilic Shewanella sp., an iron-oxidizing bacterium, and a proteobacterium from the deep sea. The release of some elements from fresh basalt and the sequestering of others in secondary minerals suggest the possibility that the geochemical cycles of some elements my be controlled by these microbes. Carbon and metabolic energy may be derived from the rocks themselves, making the subsurface microbial communities independent of surface processes.

Thermodynamic and Experimental Constraints on Energy Sources for Microbial Metabolism in Mid-Ocean Ridge Hydrothermal Systems. J L HOUGHTON, W E SEYFRIED III, P J NOVAK

Dept. of Geology, Univ. of Minnesota, Minneapolis, MN; Dept. of Civil and Engineering, Univ. of Minnesota, MN

Evidence suggests the presence of bacteria in subseafloor hydrothermal systems, perhaps utilizing the minerals present as growth substrates. The nature of such autotrophic metabolism with regard to redox is ultimately determined by the availability of dissolved H₂ and O₂ in the system. Each of these is derived from the endmember fluids (oxygenated seawater and reduced vent fluid) present during hydrothermal mixing in the subsurface. Experiments conducted with dissolved H₂ and O₂ in de-ionized water confirm that at relatively high temperatures (>325?C), the Knallgas reaction (H_2(aq) + 1/2 O_2(aq) = H₂O) proceeds spontaneoulsy. At temperatures between 250?C and 325?C, however, the kinetics of this reaction are sufficiently slow to allow instantaneous formation of metastable hydrogen peroxide. At lower temperatures (* 100?C), both H_2(aq) and O_2(aq) coexist, thereby making possible the simultaneous microbial use of oxidative and reductive metabolic pathways. A series of thermodynamic calculations were performed, simulating hydrothermal plume and subsurface mixing environments in contrasting sediment-hosted and basalt-hosted systems. Model results indicate that there is sufficient energy available at all temperatures where life is sustainable for a variety of metabolic reactions, including methanogenesis, methanotrophy, and reactions linked to Fe and S oxidation and reduction. Open system models that allow mineral precipitation and separation from the system quantitatively remove iron, also removing the potential for microbial iron reduction or oxidation. Owing to the relatively high Fe and low dissolved C in non-sedimented hydrothermal systems, the models predict less diversity in potential metabolic pathways relative to the sedimented systems. Consideration of the chemistry in natural aqueous environments may be key to understanding and predicting the full range of biodiversity at different vent systems. Further experiments will examine the relative kinetics of other redox reactions in the Fe, S, and C cycles to better constrain the chemical environment available to the subsurface biosphere in marine hydrothermal systems.

Anaerobic Microbial Activities in Deep Gulf Coastal Sediments. Lee R. Krumholz¹ Sonal Patel¹, Ralph S.Tanner¹, Dwayne A. Elias¹ and David B. Carson². ¹University of Oklahoma, Norman, OK and ²Monsanto Corporation, St. Louis MO.

Biogeochemical processes within the deep sediments below the gulf coast of the South-eastern United States have not been previously studied in detail. These sediments are saline, often rich in organic materials with an increase in temperature with depth. Our interests lay in a determination of the type of microbial activities and the level of these activities occurring within these sediments. Sidewall cores were obtained during a deep subsurface drilling operation in the southern part of Louisiana. At the deepest sampling sites, in-situ temperature was 52^oC. Core incubations with silver foils carried out in the presence of ³⁵S-sulfate demonstrated that sulfate reduction was occurring in the sediment cores. Activity within sandy sediments was detectable whereas activity within clay layers was low or undetectable. Serum bottle incubations carried out in the presence of sediments slurried with a mineral solution showed that Fe(III) nitrilotriacetate was reduced under those conditions. Sulfate reduction also occurred at rates as high as 20 nmol/gm sediment/day in unamended bottles. The addition of electron donor (formate) appeared to enhance the activity of microorganisms living in clay rich samples and not in other sediments. In the clays, both Fe(III)-and sulfate-reduction rates were greatly increased with the addition of formate. Neither nitrate reduction nor methanogenesis were detectable in any of these sediments. Enumerations carried out with sandy sediments demonstrated the presence of anaerobic heterotrophic bacteria (at 10⁴ cells/gm) and sulfate reducing bacteria at 20 cells/gm. In clay rich sediments heterotrophs were present at 10³ cells/gm and sulfate reducers were not detectable. Methanogen concentration, determined by measurement of coenzyme-M in sediments was estimated at approximately 100 cells/gm. These experiments have demonstrated the presence of an active anaerobic population of microorganisms living in the deep subsurface of the Louisiana coast area.

Three Types of Interaction of Carbon and Sulfur Cylcles in Subseafloor Biosphere. IVANOV, M.V and LEIN A.Y. Institute of Microbiology, Moscow, Russia, and Shirsov Institute of Oceanology, Moscow, Russia

Use of radiolabeled sulfur and carbon compounds and investigation of the dynamics of the chemical composition of pore waters showed that microbial processes of the sulfur and carbon cycles in the subseafloor biosphere can be traced to a depth of several meters from the sediment surface. In most shelf and continental slope sediments, it is the organic matter produced by phytoplankton that serves as the main energy source for the heterotrophic microbial community. Anaerobic degradation of this organic matter yields diagenetic carbonates, methane, and reduced sulphur compounds (as a result of the activity of sulfate-reducing bacteria). In deep-water valleys of mid-oceanic ridges, reduced gases of thermal abiogenic origin (H₂S, CH₄, H₂, CO, NH₃) are the main energy source. Oxidation of these gases by chemolithoautotrophic microorganisms yields organic matter, which is utilized by heterotrophic organisms, particularly by sulfate-reducing bacteria, which produce secondary biogenic hydrogen sulfide. The third type of interaction of the microorganisms, particularly by sulfate-reducing bacteria, which produce secondary biogenic hydrogen sulfide. The third type of interaction of the microorganismsof the sulfur and carbon cycles occurs at the outlets of cold methane seeps. In this case, methanotrophic bacteria are the main providers of organic matter for sulfate-reducing bacteria.

Geomicrobiological investigations of secondary mineral deposits in the subsurface environment of Lechuguilla Cave, Carlsbad Caverns National Park, New Mexico NORTHUP, D.E.; BEAN, L.E.; SPILDE, M.N.; BOSTON, P.J.; BARNS, S.M.; CONNOLLY, C.A.; SKUPSKI, M.P.; NATVIG, D.O. and C.N. DAHM. Univ. of New Mexico, Albuquerque, NM

Limestone wall rock of Lechuguilla Cave and Spider Cave, both in Carlsbad Caverns National Park, shows extensive corrosion that has resulted in large expanses of deposits known as ``corrosion residues'' (CRs). These CRs may be colored black, gray, pink, orange, red, or ocher and are distributed throughout both caves. EDS analysis of these CRs and underlying wall rock reveals the presence of aluminum, silicon, manganese and iron oxides, phosphorus, vanadium, sulfur, and rare earth elements. Geologists have hypothesized that Lechuguilla's extensive CRs are the long-term result of upwelling corrosive air, but the widely distributed presence of microorganisms has led us to investigate the possibility of microbial involvement in the dissolution of the wall rock. With scanning electron microscopy, we have noted pits on the surface of the wall rock underlying CRs that contain a mesh of filaments, each approximately 200-400 nm in diameter. We believe these filaments to be bacterial. The presence of Hyphomicrobium sp. cells (a known iron-oxidizer) with attachment structures and nearby crater-like structures of approximately the same size as the bacterial cells on the surface of the calcite has been noted in CRs. In order to more fully characterize the microbial community associated with corrosion residues (CRs), we are utilizing molecular phylogenetic techniques. Results from the phylogenetic analysis of the small-subunit ribosomal RNA (rRNA) gene from clones showed that the nearest relatives of several of the clones are Crenarchaeota and actinomycetes. Most of the sequences are very dissimilar to any other known 16S rDNA sequences. Identification of novel organisms within this low-nutrient environment may give us insight into the unusual microbial communities which inhabit these immense underground systems.

Chemolithotrophic Based Ecosystem in the Crystal Beach Spring Cave System GARMAN, K.M.(1), PAUL, J.(1), HARWOOD, V.J.(2) AND ROBBINS, L.(3) Univ. South Florida Department Of Marine Sciences (1), Department Of Biology (2), Depapartment Of Geology (3)

Microbial Productivity in Sulfidic Groundwater Systems PORTER M.L. Univ. of Cincinnati, Cincinnati, OH

Since the discovery of the deep-sea hydrothermal vent systems in the 1970s, numerous ecosystems utilizing chemoautotrophy as an energy source have been documented. However, the contribution of chemoautotrophy to ecosystem energy budgets is still poorly understood. Therefore the rates of production of microbial communities from sulfidic groundwater were examined using an ecosystems approach. Using caves as access points to groundwater systems, microbial mat communities from four sites were used for determining productivity: the Frasassi Cave system, Italy; Movile Cave, Romania; Cesspool Cave, West Virginia; and Lower Kane Cave, Wyoming. Microbial production was measured in each community using parallel time-course radiotracer studies. Autotrophic productivity was measured using [¹⁴C]bicarbonate incorporation while heterotrophic productivity was measured using [¹⁴C]leucine. Results indicate that chemoautotrophic productivity (approximately 390 ng C/L/hr) in these communities is similar to published values for open ocean and mesotrophic lake systems. Heterotrophic productivity peaked at rates of 2.1 ug C/L/hr. Continuing studies will determine the incorporation of both autotrophic and heterotrophic production into different cellular components.

The Witwatersrand Deep Microbiology Project: A Window Into The Extreme Environment Of Deep Subsurface Microbial Communities. MOSER D1, ONSTOTT T1, PFIFFNER S2, WHITE DC2, PEACOCK A2, PHELPS T3, DEFLAUN M4, HOEK J5, GHIORSE WC6, COLWELL F7, KIEFT T8, REYSENBACH A-L9, FREDRICKSON JK10, SOUTHAM G11, KOTELNIKOVA S12, SLATER G13, OMAR G5, PRATT L14, BOONE D9, PEDERSEN K12, and SHERW. Princeton Univ., NJ1, Univ. Tenn., TN2, ORNL, TN3, Envirogen Inc., NJ4, Univ. Penn., PA5, Cornell Univ., NY6, INEEL, ID7, New Mexico Tech, NM8, Portland State Univ., OR9, PNNL, WA10, Northern Arizona Univ., AZ11, Univ. Göteborg, Sweden12, Univ. of Toronto, Canada

A major challenge to studying microbial life at great depths in the Earth’s crust is the limited access to this environment. The ultradeep (>3 km) Au mines of the Witwatersrand in South Africa, offer an excellent opportunity to probe microbial diversity and biogeochemical processes in the continental lithosphere. To this end, we recently collected rock, water and biofilm samples from several of these mines. The rock sampling focused on an actively-mined, Au, UO₂ and sulfide rich organic layer located 3.2 km beneath the surface (kmbls.), where in situ rock temperatures were 45°C. Samples were collected using aseptic techniques to assess microbial mining contamination. Rock samples were processed anaerobically on-site. The groundwater samples were collected from boreholes and dripping fissures at 0.9 to 3.3 kmbls. Biofilms were collected from a dolomite chamber at 1.0 kmbls. and from beneath a weeping borehole and dripping fracture at 3.2 kmbls. A diverse array of microbiological media were inoculated to culture aerobic and anaerobic, mesophilic and thermophilic and saline tolerant bacteria capable of utilizing organic and inorganic compounds as energy sources. Nucleic acid, lipid, and microscopy samples were also collected for direct determination of microbial community structure. Groundwater and rock strata were sampled for geochemical, mineralogical, and physical analyses to determine biogeochemical processes. Rock samples for fission track apatite analyses were collected to determine the origin of the subsurface bacteria. We thank Turgis Technology (PTY) Ltd., East Driefontein Mine, and the Dept. of Microbiology, Univ. of Witwatersrand for their support and the National Geographic Society, and the NSF LExEn and DOE NABIR programs for sponsoring this research.

The Witwatersrand Deep Microbiology Project: Groundwater geochemistry and potential biogeochemical processes. ONSTOTT TC¹, MOSER DP¹, SHERWOOD-LOLLAR B², SLATER G², DEFLAUN MF³, HOEK J⁴ and PRATT LM⁵, Princeton Univ.¹, Univ. of Toronto², Envirogen Inc.³, Univ. Penn.⁴, Indiana Univ.⁵

The Witwatersrand Deep Microbiology Project: Methane and Hydrogen Dependent Metal Reduction.

KOTELNIKOVA S1, PEDERSEN K1, MOSER D2, and TC ONSTOTT2 Gothenburgs Univ., Department of Cell and Molecular Biology, Microbiology, Box 462, 40530 Gothenburg, Sweden; Princeton Univ., Department of Geosciences, Princeton, N.J.

The Witwatersrand mining levels at Western Deep Levels Inc. are the deepest in the world. The most productive "reef" is a thin organic-rich layer, called the Carbon Leader, which also contains high uranium concentrations. The gold mines of South Africa provide a unique "window" into the deep, continental biosphere. During the Witwatersrand Deep Microbiology Project we have collected biofilms from a dolomite chamber at 1000 meters, from beneath a weeping borehole and dripping fracture at 3100 meters, as well as Carbon Leader rock and groundwater samples. These samples were inoculated into a variety of microbiological media of different pH to culture aerobic and anaerobic, mesophilic and thermophilic bacteria capable of utilising methane and hydrogen as energy sources. Methane and hydrogen occur frequently in the mines. Our results of most probable number, radiotracer experiments and enrichment cultures showed that methane and hydrogen were consumed by microbes both in the presence and in the absence of oxygen. At anaerobic conditions methane and hydrogen were respired with ferric iron and manganese (Mn³⁺, Mn⁴⁺). We assume the presence of syntrophic consortia of hydrogen-producing methanotrophs and anaerobic organisms respiring sulphate, ferric iron and/or manganese thereby able to oxidise methane under anoxic conditions. Methane and hydrogen may contribute to the organic carbon production of the ultradeep system, constituting the energy base of subterranean microbial ecosystems. An understanding of the energy giving mechanisms under the Earth´s surface, in deep hot methane and hydrogen rich-environments, may be a key for approaching studies of metabolisms of extraterrestrial life.

Occurrence of Viruses in Groundwater: A National Study. Morteza Abbaszadegan, Charles P. Gerba, Mark LeChevallier. American Water Works, Belleville, IL : Univ. of AZ, Arizona : American Water Works, Voorhees, NJ

The enteroviruses can cause a variety of illness ranging from gastroenteritis to myocarditis and aseptic meningitis. The overall aim of this project was to investigate the occurrence of viruses such as: enteroviruses, hepatitis A virus and rotavirs in 500 groundwater sources. A comprehensive research plan was developed to evaluate the occurrence of enteroviruses, hepatitis A virus, norwalk virus and rotavirus in groundwater samples. The samples were collected from different geographical locations, with a variety of physical and chemical characteristics and different geological settings. The minimum sample volume was 1,500 liters, collected using a 1MDS cartridge filter. Five hundred and thirty-nine samples were collected for this study. Each sample was assayed for: virus infectivity using cell culture assay, the presence of virus nucleic acid using RT-PCR, bacteriophage using three different bacteria hosts, total coliforms, enterococci, total organic carbon, various minerals and metals. Additionally, the participating utilities completed a detailed questionnaire for each well, providing information on the geology, construction, location, and physical characteristics. The five hundred and thirty-nine samples represent 448 wells (25 wells were sampled more than once),from 117 different utilities or state agencies in 35 states (Figure 4.1). Sites were selected from a pool of 750 wells which were volunteered by utilities. Some of the samples were initially collected to evaluate the applicability of an RT-PCR method for the detection of pathogenic viruses in groundwater. Twenty-two samples (4.1% of 529), representing 21 different sites (4.8% of 442), tested positive for viral infectivity by exhibiting cytopathic effect (CPE) in Buffalo green monkey (BGM) kidney cells. The sample concentrates were also assayed for viral RNA by the RT-PCR method. Four different pairs of primers, specific for enterovirus, rotavirus, hepatitis A virus, or Norwalk virus were used. Each sample was also assayed twice with each primer pair - once after having been seeded with a positive control virus, and once unseeded. The seeded reactions were performed to determine whether the sample would inhibit the RT-PCR reaction. The RT-PCR analyses of the samples resulted for enterovirses15.2%, rotavirus, 13.8%, Hepatitis A virus, 6.9% and Norwalk viruses 0.9%. The samples were also assayed for bacteriophage, using three different hosts. Twenty one percent were positive for one or more phage. Ten percent were positive for total coliform. Results of the study will be incorporated in a database on virus occurrence and virus levels in public groundwater systems. The data will be analyzed in a manner to permit an assessment of the applicability of the Ground Water Rule.

Bacteriophage Migration Patterns in High Velocity Peri-Glacial Aquifers K. KENNEDY(1) & I. MUELLER(1) (1)Hydrogeology Ctr.-CHYN, Univ. Neuchatel, Emil Argand 11, CH-2007, Neuchatel,CH.

Documenting colloid transport rates in well developed water supply aquifers has been done with 'instantaneous' injections under natural gradient field conditions at three Swiss sites. Marine (H4, H40) and non-marine (Psf2, MS2) bacteriophages and at one site, microspheres, were variously compared with the solute tracer,uranine. The trials were done in three relatively prolific gravel-sand aquifers over distances from about 10m to 65m. Hydraulic gradients varied from 0.001 to 0.004. Velocity based on first arrival time varied from about 20 to 400 m/day. Arrival appeared similar for the colloidal particles and solute regardless of location in the aquifer heterogeneity. Velocity based on tracer 'peaks' was 15 to 160 m/d and, except in one extremely fast flowing setting, colloids peaked consistently two to three times earlier than uranine. Subsurface heterogeneity precludes comparing migration rates to individual wells without a more dense observation network. Colloid breakthroughs were consistent at each point. Their behavior was distinctive from, and predictably small in relative recovery compared to, uranine. In these and related clean sand tank experiments, phage H40 had higher relative breakthrough and total recovery than any other phage used, about 15 to 55 percent more than MS2. H40 may become a preferred biocolloid surrogate for viral field studies and ground water protection programs requiring tracer experiments. Bacteriophages, under these relatively fast flow conditions: 1) can likely be used to typify expected colloidal behavior to distances of many hundreds of meters given longer injection periods, 2) illustrate particle mass transport phenomena and characteristics not obtainable or predictable using solutes, 3) have patterns that do not appear to upscale well from the larger behavioral differences observed in laboratory column data.

The Use of a Mass Balance Approach to Characterize Virus Attachment During Transport In A Coarse Grained Aquifer WOESSNER, W. W.1, BALL, P. N.2, DEBORDE, D.C.2, TROY, T. L. 1 1 Dept. of Geology, 2 Division of Biological Sciences, Univ. Montana, Missoula, MT.

The coliphages MS2, PhiX 174 and PRD1, attenuated polio virus type-1 (CHAT strain), and bromide were seeded into the capture zone of a well pumping at a steady rate of 408 L/min. The site is composed of floodplain sediments that are dominated by gravel and cobbles with minor sand and contain a rapid flowing (29 m/d), cold (5 to 10 C), neutral (pH= 7.2), unconfined groundwater system. Viruses and bromide were injected into the aquifer as a slug 21.5 m from the pumping well. Normalized with respect to bromide mass (78% collected over 47 hr), 37 % of PRD1, 20% of MS2, 4.6% Phix174 and 0.15% of polio virus were recovered. The percentage of virus recovery appears correlated with virus pI. Relative Breakthrough analyses at the pumping well over estimated the PRD1, PhiX174 and poliovirus mass collected and underestimated the MS2 mass observed. Analyses of MS2 and bromide data show the majority of attachment occurred within the first few meters and hours of transport. Over 70% of the attachment process occurred within 0.84 m of transport and 80 to 90% by 7.5 m of transport. In this aquifer system, aqueous virus transport is modified by attachment rapidly and within a few meters of the injection point. Total attachment was greatest for attenuated polio virus and least for PRD1.

Use of Batch Adsorption Isotherm Data to Predict Virus Transport. F. GHODRATI, M.V. YATES, Y. JIN, AND S.R. YATES Univ. of California, Riverside, CA; Univ. of Delaware, Newark, DE; USDA/ARS/USSL, Riverside, CA

The ability to accurately assess the potential for virus contamination of ground water is of increasing importance, especially in view of the forthcoming Ground Water Rule. This Rule is being promulgated by the U.S. Environmental Protection Agency to protect potable ground water supplies from fecal contamination. Determination of the potential for virus transport is based, in part, on the extent of adsorption to the porous medium, which is usually measured in a laboratory batch study. The objective of this study was to examine the predictive capabilities of batch adsorption data for virus transport in a flowing system. Batch adsorption isotherms were conducted using poliovirus 1 and MS2 bacteriophages in Ottawa sand. Saturated column studies were conducted using the same viruses and sand. Little or no adsorption of either virus was measured in the batch studies (poliovirus: K = 0.000044; MS2: K~0). In column studies, no adsorption of MS2 was seen: the effluent concentration reached the influent concentration (C/Co = 1) after approximately 1 pore volume. However, in the poliovirus studies, the effluent concentration peaked at less than 0.01% of the influent concentration (C/Co = 0.0001). This suggested that "irreversible" adsorption or inactivation of the virus occurred in the column. Elution of the column using 1.5% beef extract, pH 9.5, resulted in the recovery of approximately 40% of the applied viruses. This analysis was conducted using cell culture, so only infective viruses were enumerated. Studies are in progress to determine whether inactivated viruses were also recovered. Our results suggest that traditional batch adsorption isotherm studies may not provide accurate information on the potential for all viruses to be transported through the subsurface.

Transport and fate of wastewater microorganisms in soil infiltration systems: Intermediate-scale 3-D lysimeters and multicomponent tracer studies S. VAN CUYK, A. LOGAN, S. MASSON, R. SIEGRIST, L. FIGUEROA, R. HARVEY, D. METGE

S. Van Cuyk, A. Logan, S. Masson, R. Siegrist, L. Figueroa, Colorado School Of Mines; R. Harvey, D. Metge, United States Geological Survey

Understanding the transport and fate of bacteria and virus during wastewater infiltration through the unsaturated subsurface is critical to effective performance of soil-based treatment systems to ensure the protection of drinking water. Experiments were initiated at CSM using intermediate-scale 3-D tank lysimeters to study purification processes in soil-based wastewater systems having 60- or 90-cm depth to ground water. Over the past year, four lysimeters have been intermittently dosed with real wastewater effluent at an effective loading rate of 5 cm/day. Applied wastewater and percolate were sampled weekly and analyzed for a suite of parameters. Purification with respect to removal of indigenous fecal and total coliforms and specific pathogens was monitored as a function of time and depth. In addition, multicomponent tracer studies were conducted during system startup (wk 8) and after early maturation (wk 45). In each case, the wastewater was amended for three days with KBr solute, MS-2 and PRD-1 bacteriophages, and an INA pseudomonas bacteria. Temporally and spatially intensive sampling of the lysimeters was conducted to define the breakthrough and elution behavior during each operational period. Lysimeter coring and analysis of soil solids for microbial levels and key environmental parameters occurred at week 48. Purification exhibited temporal and spatial effects that were strongly correlated with hydraulic retention times and effective contact area between the media and the percolating effluent. Episodic events relating to release of immobilized microorganisms into the pore water were also observed over the year-long study. Probabilistic multimedia risk modeling is now being applied to assess the human health risks that might occur due to common soil-based wastewater systems and environmental conditions.

Adsorption of Bacteriophages to Light-Weight Aggregate. T.Z. HARRIS, G.P. BREIDENBACH, and G.W. SEWELL ManTech Environmental Research Services Corp., Ada, OK, U.S. Environmental Protection Agency, Robert S. Kerr Environmental Research Ctr., Ada, OK. A series of batch adsorption and column experiments were conducted to find a suitable viral sorbent for use in a passive, diffusion, and multi-layer sampler. Batch adsorption studies were conducted with the following materials: activistic carbon, iron oxide, magnetic iron oxide, silica beads, and several light-weight aggregates (LWA). Three bacteriophages, MS-2, T-2 and PhiX-174 were used in these adsorption studies. Virus adsorption isotherms of the tested materials were obtained. The adsorbed bacteriophage recovery rates were investigated. LWA was found to have a high virus adsorption capacity and a high recovery rate. Column experiments were conducted using ground water, bacteriophage, LWA and sand columns under saturated condition. A simplified form of the governing equations for one-dimension colloid transport in porous medium was used to described the virus adsorption in the LWA and sand columns.

Comparison of Microbial Numbers in Deep Marine and Artic Methane Hydrate Bearing Sediments

D.B. BLACKWELDER¹, M.E. DELWICHE,¹ M.WILSON², AND F.S. COLWELL¹. (1) Idaho Natl. Environmental and Engineering Lab,Idaho Falls Id. (2) Humbolt State Univ., Arcata CA

Few studies have looked at the microbial communities associated with methane hydrate deposits even though much of the methane in these deposits is believed to be biogenic in origin. Cell numbers are important indicators of the biomass of bacteria in the subsurface and accurate measurements of microorganisms are essential to understanding the microbial ecology of methane hydrates. Deep core samples associated with methane hydrate deposits were obtained from the Nankai Trough located off the east coast of Japan and from the Mackenzie River Delta near the Beaufort Sea. The ocean sediments consisted of clay, to clayey silt, to siltstone, and occasional ash layers in the upper boreholes with more sandy sediments lower in the boreholes. The arctic borehole contained sands, sandy silts, and sandy gravels in the upper borehole with dolomite-cemented sand, clayey silt, silty sand, and low-rank coal lower in the borehole. Direct counts of microbes were performed using acridine orange and a confocal microscope equipped with a laser imaging system. Ocean sediment counts produced 1.8X107 cells/gm at 0.24 mbsf (meters below sea floor) and 2.6X105 cells/gm at 247 mbsf Delta sediment counts showed 1.1X105 cells/gm at 912 meters and 2.8X106 cells/gm at 951 meters. Generally, ocean sediment cell counts decreased with depth while the Arctic cell counts increased with depth.

Bacterial Biomass and Bioderadation Potential related to heterogeneous sandy Subsurface Soils. JACOBSEN, O.S. and VINTHER, F.P. Geological Survey of Denmark and Greenland, Copenhagen, Denmark and Danish Institute of Agriculural Sciences, Tjele, Denmark

In sandy soils the vertical flow of dissolved and particulate organic matter were formerly believed to take place as a piston flow. However, results from some recent projects seem to confirm that water and substrate flow in sandy profiles is quite heterogeneous. Transport experiments and microbial activity experiments are carried out in the laboratory as well as in the field. The field site is located in central Jylland, Denmark on a site where a well-drained podzolic soil has developed on glacial outwash sand sediment. Determination of the microbial biomass and the diversity of substrate utilisation patterns of the microbial communities revealed different microbial activity at different locations in the soil profile. Further, the biodegradation potential of selected pesticides in different compartments of the soil profile varied and an inverse relationship between sorptive capacity and microbial degradation of pesticides was observed. The results of the direct measurements in the compartments confirmed the ’flow channels’ as preferential pathways for transportation of solutes in the sandy soils. The higher numbers of bacterial cells in the preferential flow path environments may be a combined effect of a more optimal substrate supply and transportation of bacterial cells through the macropore channels.

Chemical and Physical Controls on Shifts of Microbial Populations in an Aquifer Contaminated with Crude Oil. B.A. BEKINS, I.M. COZZARELLI, E.M. GODSY, E. WARREN, M.E.TUCCILLO. Authors 1-4 at U.S. Geological Survey; 5 at U.S. EPA

The problem of delineating zones of different dominant terminal electron processes in a plume of organic contaminants has been noted by many authors. We provide insight into this problem with a combined data set that includes microbial populations, grain size, pore water chemistry, and sediment iron content. Cores containing both sediment and pore water were collected with a freezing drive shoe from an aquifer, near Bemidji, MN, contaminated with hydrocarbons from a crude oil spill. Pore water was drained at 15 cm intervals and analyzed for hydrocarbons, DOC, methane, and major ions. Sediment samples were collected at 20-70 cm intervals and analyzed for populations of methanogens, fermenters, sulfate-reducers, iron-reducers, and aerobes, using the most probable number method. Sediment grain-size distributions and extractable Fe (III) and Fe (II) were also determined at each of these locations. Data from three vertical profiles through the anaerobic portion of the plume show similar patterns in the microbial populations. Within each profile, numbers of iron-reducers vary from lows of 10² -10⁴ /g sediment to highs of 10⁵ -10⁶ /g. Areas that are evolving from iron-reducing conditions to sulfate-reducing or methanogenic conditions are clearly indicated by lower numbers of iron-reducers and the presence of culturable methanogens and sufate reducers (10¹ -10² /g). These conditions are found in areas of high contaminant flux either in the vicinity of the non-aqueous oil or where higher concentrations in the dissolved plume are associated with local increases in aquifer permeability. The methanogenic niche spans a vertical distance of 0.5-1 m or about half of the dissolved plume at two locations under the oil body and 1 m or one-fourth of the plume at a site 30 m downgradient from the non-aqueous oil. In all locations where methanogens are found, culturable iron reducers are also present. Moreover, in these areas, significant extractable Fe (III) (>10 umol/g) is still present, suggesting that the remaining iron on the sediments may be less energetically favorable for microbial reduction. In three locations, low numbers of iron reducers (10² /g) correspond to very high concentrations of extractable Fe (II) (30-60 umol/g) and moderate to high Fe(III) (10-30 umol/g), indicating Fe (III) utilization may be inhibited by a surface coating of an Fe(II) phase or a mixed Fe(II)/Fe(III) phase.

Spatial Variability Of Redox Processes, Microbial Activity And Degradation of Herbicides In Eight Shallow Danish Aquifers PEDERSEN, P. G., ALBRECHTSEN, H.-J., MOSBÆK, H.

Department of Environmental Science and Technology, Groundwater Research Center, Technical University of Denmark, Lyngby, Denmark.

Adjacent groundwater and sediment samples were collected from eight aquifers to investigate spatial variability in redox processes, microbial activity and potential herbicide degradation. Samples from 2-4 different depths at each aquifer were characterized with respect to geochemistry and water chemistry, and redox processes were investigated by measuring redox process endproducts or intermediates (e.g. Fe(II) or N₂O). The microbial activity was evaluated by measuring the uptake and mineralization of ¹⁴C-benzoic acid, and the herbicide degradation potential was assessed by amending the samples with different herbicides and analyzing subsamples using HPLC. Briefly the redox investigations showed that six samples were aerobic, three were denitrifying, three were manganese reducing, seven were iron reducing, ten were sulfate reducing, and one methane producing, and often more than one redox process occured simultanously in the same sample. The redox processes corresponded well with the geochemistry and water chemistry of the samples. ¹⁴CO₂ -production was found in all but four of the samples showing microbial activity in most samples. In aerobic samples the benzoic acid was degraded within a few hours or half a day, in anaerobic samples the degradation took up to 3-4 weeks. MCPA, 2,4-D and 2,4,5-T were primarily degraded aerobically. DNOC was degraded in all anaerobic samples. Atrazine was only degraded anaerobically, primarily in samples with sulfate reduction, but no correlation was found between atrazine degradation and microbial activity. The microbial behaviour varied substantially in the investigated aquifers, and the investigation may contribute to answers to how redox conditions or microbial activity governs herbicide degradation, thereby providing a tool for assessing the fate of herbicides in groundwater.

Potential Effects of the Spatial Distribution of Bacteria on Subsurface Bioremediation MURRAY, C. J., STREILE, G. P., BROCKMAN, F. J., SCHEIBE, T.D., and CHILAKAPATI, A. Pacific Northwest National Laboratory, Richland, WA

Previous studies at two field sites have quantified the spatial heterogeneity in the distribution of microbial activity in subsurface sediments, and its relationship to the geological and geochemical heterogeneity. One site consists of horizontally-bedded lacustrine sand and silt beds. The silt beds have low permeability but contain the vast majority of active microorganisms in the section, while the interbedded high-permeability sand layers are nearly barren. The second field site contains interfingering horizontally-bedded and trough cross-bedded sand deposited in the nearshore marine environment. The structure of the marine sediments is more irregular than that of the lacustrine sediments, but the contrasts between the microbiological and geological properties of the horizontally- and cross-bedded sand beds are not as strong. Geostatistical methods were used to generate numerical realizations of the distribution of microbiological, geological, and geochemical properties at the two sites, based on the results of the field studies. The numerical realizations of the site properties were input to a reactive flow and transport simulator to determine the potential effects of the combined microbiological and geological heterogeneity on aerobic degradation of trichloroethylene (TCE) and phenol at the two sites. The degradation results for the heterogeneous, spatially correlated microbiologic and geologic distributions were compared with the amount of degradation occurring when the microbiological distributions were assumed to be either randomly distributed or homogeneous. Results from modeling of the lacustrine site indicate that assuming homogeneous distribution of biodegradation potential would result in a 21% underestimation of the mass of TCE and phenol degraded, but that it was not necessary to fully characterize the spatial distribution of the bacteria. The preliminary results suggest that detailed and expensive characterization of microbiological spatial heterogeneity may not be necessary for successful predictive modeling of sitewide bioremediation at the lacustrine site, and that the less intensive sampling required to simply determine the univariate histogram of biodegradation potential at the site would be sufficient. However, full spatial characterization would still be necessary to predict the bioremediation occurring at particular spatial locations within the site.

The Effect of Various Geochemical Conditions on the Inactivation of Bacteriophage in Microcosms G.P. BREIDENBACH*# AND G.W. SEWELL@ ManTech Environmental Research Services Corp., Ada, OK, US EPA, Ada OK

The effect of various geochemical conditions on bacteriophage inactivation was investigated. It was found that anaerobic incubation of MS-2 and Phi-X 174 bacteriophage produced a greater inactivation than aerobic incubation. Nitrate reducing conditions did not produce an increased rate of inactivation of any of the test bacteriophage, but potassium nitrate at a concentration of 200 mg nitrate-nitrogen L-1 in saturated soil microcosms or soil free suspensions inactivated MS-2 bacteriophage. Sulfate reducing conditions was found to inactivated all three bacteriophage by the tenth day in microcosms. These results suggests that various geochemical conditions which may exist in an aquifer could contribute to the natural attenuation of contaminating enteric viruses.

Role Of Anionic Surfactants In The Transport And Inactivation Of The Bacteriophage Prd-1 In Aquifer Sediments: Results Of Field And Laboratory Experiments David W. Metge , Theresa Navigato, Joseph N. Ryan , and Ronald W. Harvey. Metge and Harvey -U.S. Geological Survey, WRD, Boulder, CO. ; Ryan and Navigato -Univ. of Colorado, Boulder, CO

Laboratory experiments were conducted with dual radioisotope-labeled bacteriophage PRD-1 to investigate factors influencing viral inactivation within sandy aquifer sediments. PRD-1, containing ³²P-labeled nucleic acids and ³⁵S-labeled proteins, was used as a model virus to assess the effects of different anionic surfactants (commonly found in treated sewage) upon viral inactivation. Radiolabeled virus (~ 10⁷ PFUs per g sediment) were introduced to 20 mL static mini columns filled with aquifer sediments and (or) unamended and surfactant-amended groundwater. At 1-day intervals, one pore volume was collected. Linear regression analyses were performed to determine inactivation rates for virus in the presence and absence of surfactants. In the presence of groundwater alone, the rate of PRD-1 inactivation dropped by less than one log unit over a 7-day period. Inactivation rates were about 20 to 30 times higher for virus in the presence of sediments than in the presence of groundwater alone. When sediments were subjected to modest levels (~25 mg/L) of linear alkylbenzene sulfonate (LAS) or sodium dodecyl benzene sulfonate (SDBS) surfactants, the rate of viral inactivation was about 15% less than the rate in sediments alone. This suggests that surfactants serve to protect the virus against inactivation. In PRD-1 injection and recovery experiments in which sediments within a Cape Cod aquifer were conditioned apriori with SDBS, relative breakthroughs of infective virus at 1 m downgradient from point of injection were 1-2 orders of magnitude greater than was observed in a previous field experiment involving LAS surfactants. Also, retardation and apparent dispersion were significantly reduced. In contrast to results from earlier experiments at the same site, labeled infective viruses in the SDBS experiment were detected several meters downgradient. These findings may have significant implications for setback distances of water supply wells from potential sources of domestic sewage.

Interactive Effect of Water Content and Ionic Strength on Virus Transport through Sand Columns

Y. JIN, Y. CHU, AND M. V. YATES. Univ. of Delaware, Newark, DE; Univ. of Delaware, DE; Univ. of California, Riverside, CA

Concern over virus contamination of groundwater resources has increased in recent years. To accurately assess the vulnerability of groundwater to viral contamination, better understanding of virus behavior in the subsurface is needed. Many factors contribute to virus sorption and inactivation processes and their impact on virus transport through porous media. Among these factors, the role that the vadose zone plays in microbial retention and transport has received increasing attention in recent years. The presence of an air-water interface (AWI) has been associated with the observed increase in sorption/inactivation of microorganisms in unsaturated systems. Therefore, the vadose zone is generally perceived as a barrier for microbial transport. However, most of these studies were conducted under conditions that promoted attachment of microorganisms on the AWI. In this study, column experiments were conducted using Ottawa sand and two bacteriophages (MS2 and phi X174) in three buffer solutions of various ionic strength and composition. The effect of water content on the retention and transport behavior of the two phages was examined and quantified. We found that the removal (sorption and/or inactivation) of both viruses increased as water content decreased in all buffers and the extent of the water content effect varied depending on the ionic strength of the buffer solutions and the type of virus used. The importance of properly relating laboratory experimental results to natural subsurface conditions (e.g., ionic strength of soil extracts and groundwater samples) in evaluating the role of the vadose zone in the elimination, retardation and migration of viruses will be discussed.

Attenuation of PRD-1 Land MS-2 During Recharge at a Constructed Research Basin in Los Angeles County ¹ANDERS, ROBERT*; ²YANKO, W.A; ¹SCHROEDER, R.A; ²JACKSON, J.L. ¹US Geological Survey, San Diego, CA; ²County Sanitation Districts of Los Angeles County, Whittier, CA

Augmentation of ground-water supplies through artificial recharge with treated wastewater (recycled water) is increasing, especially in the arid Southwest. Where this practice exists, it is important to ensure the absence of human viruses from ground water at its point of withdrawal for potable use. Analysis of the water supply and bench-scale studies of virus removal and inactivation in a laboratory setting can help provide such ensurance, but field-scale experiments in the area of recharge also are needed for a complete understanding of virus transport and attenuation. Results from experiments in the field can provide the site-specific information needed to quantify the effects of distance and time on viral attenuation during subsurface transport. We have recently begun such experiments at a small research basin constructed adjacent to a large recharge facility (2-km² spreading grounds) located at the Whittier Narrows in Los Angeles County. Nearly 0.2 km³ of water, about one-third of which is recycled water, is diverted annually to these spreading grounds where it infiltrates and recharges the aquifer. The research basin has an infiltration area of about 400 m² and can be supplied with recycled water at a rate of 750 L/min. The research basin was filled to a depth of about 1 m with recycled water, then sprayed over the water?s surface with concentrated stock solutions of bacteriophage and KBr to yield initial concentrations of about 10⁴ to 10⁵ PFU/ml and 100 mg/L, respectively, in the basin. In the first of two experiments, only PRD-1 was used and no further additions took place after the initial seeding. For the second experiment, two bacteriophages, PRD-1 and MS-2, were used and both bacteriophage and KBr concentrations were maintained for an additional 8 hours through continuous metering from the concentrated stock as recycled water was added to the research basin. The continuous addition during the second experiment allowed for monitoring the attenuation of bacteriophage to greater depths and distances. Ground-water samples withdrawn and analyzed from wells beneath and outside the research basin show that: (1) breakthrough of bacteriophage and Br was coincident, (2) no dilution occurred at depths less than 3 m, (3) bacteriophage was attenuated by removal and(or) inactivation, and (4) attenuation of PRD-1 was similar in both experiments and was greater than attenuation of MS-2. These results indicate that further experiments may permit reliable projections of the traveltime and distance over which the several-log removal desired by regulators is accomplished.

Subsurface Removal of Salmonella by On-Site Alternative Wastewater Treatment Systems

Role of Preferential Flow for the Subsurface Transport of Cryptosporidium parvum Oocysts through the Vadose Zone DARNAULT, C.J.G¹; GARNIER, P. ²; STEENHUIS, T.S. ¹; PARLANGE, J. Y. ¹; BAVEYE, P.C. ¹; JENKINS, M. ³; GHIORSE, W.C^{3 1}Depart. of Agricul. and Biol. Engineer., Cornell Univ; ²INRA Laon, France; ³Section of Microbiol, Div. of Biol. Sci., Cornell University

The objective of this study is to conduct a risk-cost analysis of the regulatory setback distance between municipal wells and virus sources to groundwater. The proposed groundwater disinfection rule of the 1986 Safe Drinking Water Act (SDWA) recommended a maximum virus concentration of 1 X 10^-7 virus particles/L at the well. One option for a municipal water system is to demonstrate that a sufficient setback distance exists between the well and a virus source (e.g., septic tank and sewer line) to allow natural disinfection. The Nebraska proposed regulatory set-back distance of 160m provides a sufficient travel time to the well by allowing natural disinfection through viral die-off. Groundwater transport modeling was used to calculate needed setback distances to Nebraska’s 2,000 municipal wells based on viral die-off times. Conservative unsaturated and saturated zone properties were assumed for the wells. Factors evaluated in the modeling included the depth of the viral source; depth to water; advection, dispersion and adsorption of the viral particles; and other standard groundwater transport parameters. The frequency of the modeled setback distance that exceeded the SDWA requirement was the risk of viral contamination at the well. The relative costs of different setback distances were computed by multiplying the capture zone area by an average urban land price. Based on risk-cost trade-off, the costs were evaluated against risks to determine the "optimum" setback distance. The optimum setback distance was 122m for virus source depths of 1.2 to 3.7 m.

Development of New Methods for Monitoring Bacterial Transport in Subsurface Environments

FULLER, M.E., DONG, H., ONSTOTT, T.C., and DEFLAUN, M.F. Envirogen, Inc., Lawrenceville, NJ; Princeton Univ., Princeton, NJ; Princeton Univ., Princeton, NJ; Envirogen, Inc., Lawrenceville, NJ

There is a need for new methods to track injected cells during in situ subsurface bacterial transport research. Labeling cells with fluorescent stains like DAPI and acridine orange has been done previously, but resulted in undesirable changes in the viability and transport properties of the cells. This research focuses on evaluating alternative fluorescent stains for labeling bacteria for transport experiments. Of the seven green fluorescent stains screened, only CFDA/SE (5-(and-6-)-carboxyfluorescein diacetate, succinimidyl ester) fulfilled the criteria that a stain must: 1) exhibit a high efficiency and uniformity of staining, 2) cause no decrease in organism culturability, and; 3) cause no significant change in cell adhesion to subsurface sediment. Greater than 95% of the CFDA/SE-stained cells of a indigenous groundwater isolate, Comamonas sp. DA001, remained fluorescent during 28 days of incubation in artificial groundwater at 15°C. The survival and culturability of stained DA001 cells was not significantly different from unstained DA001 cells in groundwater and sediment microcosms, indicating that the fitness of the cells was not adversely affected by the presence of the CFDA/SE stain inside the cell. The bright, yellow-green cells were easily distinguished from autofluorescing sediment particles during epifluorescence microscopy, and stained cells could be visualized in thin sections of sediment which had been dried and epoxied. The detection limit for CFDA/SE-stained DA001 in groundwater was on the order of 500-1000 cells/ml using a fluorescent microplate reader and an HPLC fluorescence detector attached to a low pressure flow cell and autosampler. Screening of red fluorescent stains, as well as evaluating the stained bacteria during laboratory-scale bacterial transport experiments, is currently underway. These methods will allow the movement of bacteria introduced into subsurface environments to be accurately and easily assessed.

A Mathematical Basis for Active Bacterial Adhesion Kinetics Modeling T. R. Ginn, Univ. California, Davis, California

The fate of microorganisms undergoing subsurface transport depends on the time of exposure of the microbes to other materials present in the system. In particular, the rates of bacterial partitioning between the solid and aqueous phases in the subsurface can appear dynamic, where physiological changes that occur in the microbe over time incur significant changes in the partitioning kinetics. One instance of dynamic partitioning occurs when the propensity for a microorganism to become irreversibly attached to a solid phase depends on the residence-time of the microorganism on or near the mineral surface, during an individual attachment event. Residence-time is defined here as the amount of time a microorganism is reversibly associated with a surface through a specific interaction, such as electrostatic or van der Waals forces. Irreversible attachment is usually associated with active adhesion processes on the part of the microbe. Conventional descriptions of partitioning kinetics at the bulk phase scale are incapable of capturing this behavior because such models cannot track the distribution of biomass over residence-time. A new theoretical approach is introduced which provides such a basis, in such a way that residence-time may be treated as an additional independent variable that is utilized in the differential equations expressing the attachment/detachment kinetics. This is accomplished by extending the usual mathematical distribution of microbial density in balance equations over space and time, to balance equations over space, time, and residence-time. The approach is summarized, and an application to published data of McCaulou et al. (1995) is described in detail.

Role of growth conditions upon the subsurface transport behavior of a groundwater nanoflagellate

MAYBERRY, N.A.1, HARVEY, R.W.2, METGE, D.W.2, and KINNER, N.E.3. 1Wright Pierce, Topsham, ME; 2U.S. Geological Survey, Boulder, CO; 3Univ. New Hampshire, Durham, NH

A low-nutrient, slightly acidic, porous-media growth procedure was used to grow Spumella guttula, a groundwater nanoflagellate, for an injection and recovery transport experiment involving an organically contaminated aquifer, Cape Cod, Massachusetts. The new growth procedure mimiced conditions within the aquifer and maintained the nanoflagellates' small (2-3 microns) size. This allowed assessment of its potential for advective transport through the aquifer sediments. Results from the in-situ transport experiment suggest a high transport potential, which was about two orders of magnitude greater than was observed in an earlier experiment using the same nanoflagellate grown in conventional liquid-broth media. Calculated collision efficiencies were on the order of 10 ^-2. The high transport potential of this nanoflagellate was due, in part, to a combination of its 2-3 micron size and relatively low specific gravity (~1.02 g/cm ³), as determined by density gradient centrifugation and its observed sedimentation rate relative to that of bromide in the aquifer. In spite of its larger size, transport potential of porous-media grown S. guttula was comparable to that for many of the uncultured, unattached groundwater that serve as its prey. However, the attachment behavior of S. guttula to aquifer sediments in static minicolumns was very different than that of the groundwater bacteria, judging from the pH-dependency of sorption in static minicolumns containing aquifer sediments. This suggests that the mechanisms by which S. guttula attach to surfaces may be very different from the ones used by bacteria. The high potential of S. guttula for advective mobility in the aquifer sediments has important ecological implications for the nanoflagellates within the contaminated aquifer sediments.

Biocolloid And Chemical Mass Transport In a Well Defined Heterogeneous Porous Media Tank Model

K. KENNEDY (1) & P. ACKERER (2). (1) Hydrogeology Ctr.-CHYN, Univ. Neuchatel, CH (2) Fluid Mechanics Inst., Univ. Louis Pasteur, Strasbourg, FR

Our research in this laboratory environment focussed on distinguishing flow path and mass transport rates of biocolloids and dissolved solutes in heterogeneous porous media. The clear objective is to develop better predictive tools for particulate, particulate-assisted and dissolved constituent contaminant migration/reaction processes. This began with seven tracer test campaigns repeated in a physical tank model with a well-defined physical heterogeneity (MARCEAUs). Six different biocolloids (marine-H4, H6, H40, non-marine-T7, Psf2, MS2 bacteriophages) were variably co-mingled with both salt and uranine to begin the experiments with a uniform input signal. The 1m x 1m x 6m tank with three different-permeability sands arranged discretely in 1080 'boxes' (10cmx10cmx40cm) has scale and pore-size conditions similar to some field sites currently used for viral migration studies. The three sand materials are washed and therefore the environment does not containing noticeable silt or clay size fractions. MARCEAU's advantage was a controllable gradient, temperature and chemistry. Salt, a 'conventional' tracer, was monitored on-line and continuously at 360 nodes and gave a fundamental solute migration setting with which to compare the bacteriophage and uranine responses. The phages and uranine were measured at six 'wells' in three of the tank's distinct transport fields. Results showed consistent salt and uranine transport throughout the tank with uranine and phage breakthrough patterns changing in response to ionic strength variations. Double peaks were clear evidence of multiple route contributions in the longer path, lower permeability areas. Biocolloid behavior differed from the solutes. Marine phages were consistent and throughout with similar breakthrough time to salt in the higher permeability regions. The subdued second response of the bacteriophages suggested more mass attachment and selectivity in pathway routing. Results have provided the starting point for currently ongoing transport modeling and biocolloid migration process evaluation.

The Determination of Aqueous Concentration Profiles within Intact Sediment Cores During Transport Experiments. Mailloux, B. J.1, Onstott, T. C.1, Moline, G.2, Dong, H.1, DeFlaun, M.3, Fuller, M.3, Gillespie, K.3, and Streger, S.3. 1 Dept. of Geosciences, Princeton Univ., Princeton, NJ 08544 2 Environmental Sciences, Division, Oak Ridge National Laboratory, OakRidge, TN 3 Envirogen Inc., Lawrenceville, NJ 08648

A multiport permeameter was utilized to assess the rate of and the factors controlling sorption and desorption during bacterial transport experiments conducted using intact sediment cores (7.3 x 50 cm) from Oyster, Virginia. The multiport permeameter consists of a needle inserted into the shelby tube and attached to either a manometer or a sampling port. The unimpeded hydraulic connection to the core was obtained using either pneumatic inflation or 16 gauge syringe needles with nine side holes (<0.5 mm diameter) drilled within the first 2 cm of the needle. Six needles were inserted halfway into the core at 7 cm intervals. The relatively small diameter needles did not disturb the intact sediments. The needles are initially attached to manometers to determine the permeability of the sediments. The needles are subsequently utilized to extract aqueous samples during the injection of a small pulse (<1 pore volume) of non-reactive tracers or bacteria. The aqueous samples are used to produce concentration profiles at discrete time intervals along the length of the core. A small sample volume is extracted through the sampling ports at a rate that does not reverse the direction of flow nor alter the observed break through curve. Bromide and tritium were injected simultaneously to determine if bromide reacted with the iron rich sediments. The collection of multiple samples from the side ports throughout the bacteria transport experiment showed the change in the aqueous bacteria concentration through time. A high resolution cat-scan of one core shows the distribution of porosity and grain size in the core. The permeability, grain size, porosity, side-port concentrations, breakthrough curve, and final sorbed profile were used to assess the factors controlling transport.

Health Effects posed by Giardia in Support of the Interim Enhanced Surface Water Rule. LATISHA S. PARKER EPA, Washington D.C.

Giardia is currently regulated under the Surface Water Treatment Rule (SWTR). Although the SWTR does not establish a Maximum Contaminate Level (MCL) for Giardia, it specifies treatment requirements to achieve at least 99.9% removal and/or inactivation of Giardia cysts. This regulation requires that all drinking water systems using surface water or ground water under the influence of surface water must disinfect and filter the water. Recently, the Interim Enhanced Surface Water Treatment Rule (IESWTR) was passed. This rule will serve to improve control mechanisms of microbial pathogens in drinking water and address risk trade-offs of disinfection byproducts. In addition, the IESWTR will require States and water systems to work together to ensure that there are no significant increases in microbial risk when public water systems act to implement new requirements to control disinfection byproducts under the Stage 1 Disinfectants and Disinfection Byproducts Rule (DBPR). In the United States, Giardia is the most commonly identified protozoan pathogen in waterborne disease outbreaks since 1971. Since 1965, 133 waterborne outbreaks and almost 28,000 cases of giardiasis have been reported in the United States (56%), primarily from unfiltered surface water systems. In support of the IESWTR the Environmental Protection Agency has written a Human Health Criteria Document which covers all aspects of Giardia such as; general properties of the organism, health effects, and treatment techniques. The opinions in this abstract are those of the author and do not necessarily reflect those of the EPA.

Enhanced colloidal tracer particle enumeration and identification methodologies as applied to subsurface contaminant migration and transport behavior research. Pierre Rossi(1) and Keith Kennedy(2)

1.Univ. of Neuchatel, Microbiology Laboratory (LAMUN), Neuchatel, Switzerland 2.Univ. of Neuchatel, Hydrogeology Department (CHYN), Switzerland

We have used bacteriophage and latex microsphere colloidal particles parallel to conventional solute tracers for hydrogeological and contaminant transport characterization in variable porous media and fractured subsurface environments in Switzerland. These colloids can represent models emulating bacterial and viral migration and facilitated transport. For phages, we reduced the analysis time required, increased the precision and eliminated the non-discriminating nature associated with earlier methods. Our first attempts using bacteriophages as subsurface tracers showed we needed increased analytical reliability and reproducibility with reduced cost/time. We did this by adapting the classical double-layer technique in Petri dishes to each system of bacteriophage/host bacteria (B/HB). Introducing a contact period between the phage and the host bacteria before plating and carefully selecting buffers and growth media allowed both increased numbers of phages detected (>250%) and reduced analysis variability (<5%). An important step was using efficient marine B/HB systems. High salt concentration in the growth medium eliminated background and interference signal noise given by allochtonous bacteria from freshwater subsurface systems. For microspheres, we cooperate with our ETH (Zurich) colleagues who have developed a field durable, high sensitivity (1 particle/ml), on-line particle counting system. This laser-based equipment identifies fluorescent colloids with virus to bacteria size diameters. The real-time colloid identification, along side the solute data, dramatically reduces uncertainty in, and optimizes, sampling programs yielding better migration test performance without sacrificing accuracy. High-accuracy data now available for both phages and 'inactive' microspheres under field trial conditions may help us better evaluate the relevance of behavioral discrepancies frequently observed among colloid species in smaller scale laboratory column and batch tests.

Free-living Bacteria Degrade Sorbed 2,4-Dichlorophenol

CARLSSON C M; BENGTSSON, G. University of Lund, Department of Ecology, Ecologybuilding, Lund, S-223 62 Sweden

The influences of sorption of bacteria as well as 2,4-dichlorophenol (2,4-DCP) on the microbial degradation of 2,4-DCP was studied in a continuos flow system. The experiments were performed on columns using sterilized aquifer sand as the solid support and filter sterilized ground water as the mobile phase. The soil-water system could be characterized as an extreme oligotrophic system with a TOC content of about 0.2 %, and a subsequent slow turnover of carbon and nutrients. Sterile ground water, spiked with 14C-labelled 2,4-dichlorophenol was pumped through the columns along with a tritiated 2,4-DCP degrading Pseudomonas putida strain EST4021. The evolved 14CO2 was measured and the breakthrough curves for 2,4-DCP were compared and evaluated for experiments without and with adsorbed bacteria, and with free living bacteria in order to clarify the partitioning of the degradation between the aqueous phase and the solid phase. The result showed that 2,4-DCP was mainly degraded in solution by free living bacteria. Sorbed 2,4-DCP was also degraded by free-living bacteria, but to a lesser extent than 2,4-DCP in solution. Very little or no degradation was associated with adsorbed bacteria. The results are consistent with an earlier batch study on microbial degradation of aniline in a corresponding oligotrophic environment which showed that degradation occurred in both phases and that the degradation rate per cell was about ten times higher in the aqueous phase than in the solid phase.

Remediation of AMD Sites by Inducing Iron Respiration MARCHAND, E. AND SILVERSTEIN, J.

Department of Civil, Environmental, and Architectural Engineering, University of Colorado at Boulder

Formation of acid mine drainage (AMD) is a serious environmental problem that is catalyzed at low values of pH by iron-oxidizing autotrophic bacteria such as Thiobacillus ferrooxidans. The high concentration of dissolved metals in drainage water seriously impacts receiving waters and can be problematic to treat, particularly in the subsurface environment. An attractive alternative is to induce conditions within the AMD environment that will prevent acid generation in situ before the drainage water enters the aquatic environment. Bench-scale experiments have been performed in which competition for oxygen has been induced between T. ferrooxidans and native non-iron-oxidizing heterotrophic bacteria in a limited oxygen environment, by supplementing with glucose. Iron oxidation was inhibited (55% lower concentration of oxidized iron) compared to a control without the heterotrophic bacteria. Additionally, once oxygen became limiting in the mixed culture systems, glucose was consumed while ferric iron was reduced during iron respiration. It was also found that when the acidophilic heterotrophs reduced iron under acidic conditions (pH_initial = 2.5), the pH increased significantly (pH > 4.0). These results suggest that biologically mediated acidification of drainage water in the subsurface environment can be reversed by the addition of a degradable, soluble organic carbon substrate such as glucose. Additionally, the increase in pH following iron reduction could act as a mechanism to remove metals via precipitation before they can enter the environment.

In-Situ Bioremediation of Chloroethene-contaminated Groundwater Using Halorespiring Bacteria - A Pilot Evaluation. FATHEPURE, B.Z. ¹, LOFFLER, F.E. ², TIEDJE, J.M. ³ and ADRIAENS, P. ¹ 1The University of Michigan,Ann Arbor, MI; 4Georgia Institute of Technology, Atlanta, GA; 3Michigan State University,East Lansing, MI

The Bachman aquifer (Oscoda, Michigan) is primarily contaminated with tetrachloroethene (PCE) and trichloroethene and has shown evidence of PCE dechlorination in the field since cis-DCE was detected, primarily in the deep aquifer. The goal of this study was to evaluate the spatial distribution of halorespiring bacteria within the aquifer, and the potential for enhanced dechlorination rates through bioaugmentation. Aquifer materials were collected at different depths along a transect perpendicular to groundwater flow. Batch microcosms were established with chlorinated ethenes as electron acceptors. PCE was stoichiometrically transformed to ethene in bottles with deep aquifer material and amended with formate, lactate, pyruvate, glucose, or whey. In contrast, cis-DCE or VC was detected in microcosms with shallow zone aquifer material. Halorespiration activity was confirmed by (i) developing dechlorinating enrichment cultures that exhibited high fe values for PCE dechlorinations (0.65 to 0.7), and (ii) based on hydrogen thresholds near 1 nM. The potential for bioaugmentation by chlororespiring bacteria was evaluated in continuously-fed columns established with shallow aquifer material. A pure chlororespirer, Desulfuromonas sp. strain BB1 that couples oxidation of acetate or lactate to PCE to cis-DCE dechlorination was injected into the column. Gene probe analysis showed that strain BB1 colonized the Bachman aquifer solids and also rapidly converted PCE to cis-DCE. More than 70% of the influent PCE was dechlorinated in the first 1 to 2-inches of the column at a rate of approximately 6 umol /L/hr (the pseudo first-order rate constant was 0.18 hr -1). To achieve complete transformation of PCE, the column was reinoculated with mixed chlororespirers that dechlorinated cis-DCE or VC to ethene. After about 2 months, chloroethenes were removed to non-detectable levels.

Field Studies of Aerobic Bioremediation of Groundwater Contaminated by Sanitary Landfills.

T. C. Hazen, Lawrence Berkeley National Laboratory

Leachate from sanitary landfills at many sites has contaminated groundwater with chlorinated solvents, usually trichloroethylene (TCE), vinyl chloride (VC), and chlorobenzene (CB). In situ aerobic bioremediation was selected from over 50 remediation strategies to be the most cost effective while minimizing environmental impact. Treatability tests and optimization tests were used to establish the operating parameters required for aerobic biodegradation of the contaminants of concern. These processes have even been demonstrated to encourage reductive dechlorination of PCE in soil pore spaces under bulk aerobic conditions. Since direct air injection into the subsurface will also increase metal precipitation and decrease metal solubility by increasing the pH and redox potential, biosparging may provide the best conditions for treating mixed waste in situ and for controlling metal mobility at heavy metal/radionuclide contaminated sites. Our own field studies indicate that air injection can significantly modify pH, redox potential, and conductivity of the adjacent groundwater. Our understanding of how these anaerobic/aerobic biodegradation processes are changed by biosparging and how they effect metal solubility are very poorly understood and discussed in this paper. Bioremediation strategies (both natural and accelerated) that both contain the metals, eliminate the organic contaminants and stabilize the waste site are extremely advantageous since they will decrease or eliminate the environmental and health risks of these sites. These points are demonstrated through field studies using air injection and leachate recirculation at several landfills in South Carolina and Georgia.

Batch studies of microbial processes and metal removal in material proposed for a permeable sub-surface reactive barrier, Sweden. T. A. MORALES, R.HERBERT and R.HALLBERG. Stockholm University, Department of Geology and Geochemistry

One method for the remediation of metal-contaminated ground water is the installation of sub-surface treatment walls consisting of geochemically reactive material. Such a reactive geobarrier is planned to be installed at the Kristineberg mine site where elevated levels of heavy metals, sulphate and acidity are found in the groundwater. Anaerobic batch studies have been performed to evaluate the rates of bacterial sulphate reduction and metal removal in different types of barrier material (bark shavings, saw dust and leaf compost). Fe (II/III), Zn, Pb, Cu, sulphide, sulphate, phosphate, alkalinity, pH and TOC have been analysed in the effluents. Initial batch studies, with the compost leaf material, showed a sulphate removal of 450 mg/l over a period of six weeks (1000 mg/l to 550 mg/l). High iron and zinc removal were also observed (40 to 80 %) followed by a rise in both pH and alkalinity within six weeks. Batches with high initial sulphate and metal content were most effective in terms of metal removal. Sulphide production is observed suggesting that bacterial sulphate reduction is an important process governing sulphate removal and that growth of sulphate reducing bacteria (SRB) is promoted despite initially acidic conditions. Determinations of the existence of SRB such as <i>Desulfovibrio desulfuricans</i>, were performed using viable count and MPN method, and are used for the evaluation of batch performance. Variations in material composition and different bacterial activities in the various batch studies account for the different removal rates of sulphate in the batches. In general, a positive correlation was found between bacteria and sulphide concentrations.

Overproduction of Bacterial Alkaline Phosphatase as a Mechanism for Metal Sequestration.

L.G. POWERS, P.A. SOBECKY, AND A.V. PALUMBO Georgia Inst. Technology, Atlanta, GA and Environmental Sciences Division, Oak Ridge Natl. Laboratory,Oak Ridge, TN

Traditional approaches to the remediation of metals and radionuclides utilize dissimilatory reduction processes. However, in oxygenated environments such as the vadose zone dissimilatory reduction often proves to be problematic. Thus, new technologies are needed to broaden the scope of available bioremediation approaches. We plan to immobilize contaminants in aerobic environments by coupling the introduction of bacteria genetically modified to constitutively overproduce the enzyme alkaline phosphatase. We have previously introduced plasmid pJH123 containing a pglA-phoA hybrid gene encoding a fusion protein into marine bacteria. The presence of the plasmid in the marine bacterial host elevated extracytoplasmic alkaline phosphatase production >40-fold and did not adversely effect growth and survival of the bacterium. We have introduced plasmid pJH123 into a number of pseudomonads via electroporation chosen for their potential in field-scale delivery systems. Preliminary data indicate maintenance and stability of the plasmid in several of the pseudomonads. Confirmation of the overproduction of alkaline phosphatase and characterization of the effects of growth on the host pseudomonads are underway. Ultimately, this information will assist in developing new strategies to remediate contaminants in aerobic systems utilizing natural and genetically modified bacterial variants.

Copper Bioavailability And Its Effects On Methanotrophic Activity J. D. SEMRAU, J. MORTON, S. LONTOH, J. I. HAN AND K. F. HAYES Univ. of Michigan, Ann Arbor, Michigan

Many chlorinated hydrocarbons can be co-oxidized by the methane monooxygenase (MMO) of methanotrophs. Due to their ubiquity and ability to degrade priority pollutants, methanotrophs have been extensively studied for in situ bioremediation. The utility of methanotrophs, however, is complicated by the fact that two forms of the MMO exist with very different substrate ranges and transformation kinetics. A cytoplasm-associated or soluble methane monooxygenase (sMMO) is expressed at low copper:biomass ratios while a membrane-associated or particulate methane monooxygenase (pMMO) is expressed at high copper:biomass ratios. The sMMO has been extensively characterized while relatively little is known about the pMMO. To enhance the use of methanotrophs for bioremediation, it is necessary to know more about what compounds can be degraded by the pMMO as well as what forms of copper are actually bioavailable and can cause changes in microbial activity. Data presented here shows that the pMMO can degrade a variety of halogenated hydrocarbons and that the kinetics of pMMO-mediated pollutant degradation depend strongly on the availability of copper and reducing equivalents as well the presence of competing substrates. We have also correlated copper speciation as determined by atomic adsorption spectroscopy and ion-selective electrode measurements with microbial activity, MMO expression, and cell-associated copper to determine copper bioavailability. The assessment of copper bioavailability and its resulting effect on methanotrophic activity will help develop better strategies for the effective use of methanotrophs for bioremediation.

Coupled Chemical Reaction and Biodegradation of Metal-Ethylenediaminetetraacetic acid (EDTA) Complexes WILLETT, A.I. AND RITTMANN, B.E.

Northwestern Univ., Evanston, IL

The subsurface co-disposal of the synthetic chelating agent ethylenediaminetetraacetic acid (EDTA) with radioactive and heavy metal waste can increase the dissolution, dispersion, and subsurface transport of radionuclides and heavy metals. Biodegradation of EDTA at contaminated sites would enhance adsorption and immobilization of radionuclides and heavy metals. In addition to the effects of total substrate and biomass concentrations, the rate and extent of EDTA biodegradation are affected by the EDTA aqueous speciation. The goal of this research is to quantify the effect of aqueous speciation on the biodegradation of EDTA using a combination of computer modeling and laboratory experimentation. The computer model used in this study is CCBATCH, a biogeochemical model developed at Northwestern University that couples equilibrium EDTA speciation reactions with kinetically-controlled EDTA biodegradation reactions. Detailed modeling with CCBATCH shows that experimentally-observed EDTA biodegradation trends are correlated with the concentration of a single complexed form of EDTA. Specifically, CCBATCH modeling trials show that the lack of EDTA biodegradation by the EDTA-degrading bacterium BNC1 when EDTA and Fe(III) are present in equal amounts is due to a high concentration of Fe(III)-EDTA complexes, which are resistant to biodegradation, and a low concentration of the Ca(II)-EDTA complex. Increasing the pH of the EDTA and Fe(III) solution from pH 7 to pH 9, increases the biodegradation rate. At pH 9, CCBATCH output shows that the hydroxide ligand competes with EDTA for Fe(III). The result is a decrease the concentration of Fe(III)-EDTA complexes and an increase in the concentration of Ca(II)-EDTA, which results in an increase in the EDTA biodegradation rate.

Anaerobic Degradation of Naphthalene by a Pure Culture of a Novel Type of Marine Sulfate-Reducing Bacterium GALUSHKO A.¹, MINZ D.², SCHINK B.¹, WIDDEL F.^{3 1}Fakultät für Biologie, Universität Konstanz, Konstanz, Germany. ²Volcani Research Center, Soil Microbiology, Israel. ³Max-Planck-Institut für marine Mikrobiologie, Bremen, Germany

Incubation of marine sediment in anoxic, sulfate-rich medium in the presence of naphthalene resulted in a sulfide-producing enrichment culture. Pure cultures of sulfate-reducing bacteria with short, oval cells (1.3 by 1.3 to 1.9 µm) were isolated that grew with naphthalene as the only organic carbon source and electron donor for sulfate reduction to sulfide. One strain, NaphS2, was characterized. It affiliated with completely oxidizing sulfate-reducing bacteria of the delta subclass of the Proteobacteria, as revealed by 16S rRNA sequence analysis. 2-Naphthoate, benzoate, pyruvate and acetate were utilized as growth substrates in addition to naphthalene. Quantification of substrate consumption, sulfide formation and formed cell mass revealed that naphthalene was completely oxidized with sulfate as the electron acceptor.

Natural Attenuation of Trichloroethylene in a Surficial Aquifer/Wetland Discharge Area. J.L. BANKSTON, T.M. TWESME, and D.F. DWYER

Univ. of MN, Minneapolis, MN

Groundwater and wetland ecosystems were studied to determine the fate of trichloroethylene (TCE) contaminated groundwater discharging from a sandy surficial aquifer into a freshwater wetland. Based on laboratory and field studies, the aquifer and wetland ecosystems functioned as a three-stage TCE remediation system: 1) The indigenous bacteria in the anaerobic aquifer dehalogenated TCE to the primary dehalogenation product, cis-1,2-dichloroethene; 2) At the groundwater discharge area, TCE and its anaerobic degradation products were mineralized by aerobic bacteria in the soil and also by aerobic bacteria in the rhizosphere of Populus deltoides (cottonwood tree); 3) Within the wetland, the remaining TCE and anaerobic degradation products were mineralized by methanotrophic bacteria within the rhizosphere of Typha latifolia (broad-leaved cattail). Half-lives of TCE in the three zones were determined experimentally using microcosms that simulated aquifer and wetland conditions. Aquifer microcosms contained: aquifer sediment, indigenous bacteria, groundwater, and TCE (300 mg/kg); wetland microcosms contained: soil, wetland water, indigenous bacteria, cattails or cottonwoods, and radio-labeled 14C-trichloroethylene (35 mg/kg, 1.27 mCi). Pseudo-first-order rate constants were calculated for the degradation of TCE in the wetland and aquifer microcosms and then used in an advection/dispersion mass transport model to simulate the fate of TCE in an anaerobic aquifer and a freshwater wetland.

Anaerobic microbial degradation of chloroethenes in the subsurface: recent findings. P.M. BRADLEY, F.H. CHAPELLE U.S. Geological Survey, Columbia, South Carolina

Over the last two decades a number of pathways have been described for microbial degradation of chloroethenes in the subsurface. Under aerobic conditions, the more common chloroethenes, tetrachloroethene and trichloroethene, are recalcitrant except in the presence of organic co- substrates which support co-metabolic oxidation to CO2. However, under anaerobic conditions these highly chlorinated chloroethenes are readily transformed via reductive dechlorination to the less chlorinated intermediates, dichloroethene (DCE) and vinyl chloride (VC). Although widely observed to occur in subsurface environments, reductive dechlorination is rarely complete, generally results in accumulation of toxic compounds (DCE and VC), and, therefore, cannot be considered bioremediation. Recent studies conducted in this lab indicate that indigenous microorganisms can catalyze a net oxidation of DCE or VC to CO2 via a number of anaerobic terminal electron accepting processes. These processes include chloroethene oxidation under Mn(IV)-reduction, Fe(III)-reduction, SO4-reduction and net oxidation of VC coupled to humic acids reduction. Recent observations also indicate that a net fractionation of VC to equal molar amounts CO2 and CH4 can be catalyzed under acetotrophic methanogenic conditions. These recent results indicate that the microbial ecology of chloroethene contaminants in the subsurface is diverse and an important determinant of the fate of these compounds in the environment.

Cave Microbes; Microbial Mats Lining Hydrogen Sulfide Springs P. BOSTON, L. KLEINA, D. SOROKA, K. LAVOIE, D. NORTHUP, M. SPILDE

Complex Systems Research, Inc., Boulder, CO and UNM; Caves of Tabasco Project, NSS, Huntsville, AL; Caves of Tabasco Project, NSS, Huntsville, AL; SUNY-Plattsburgh, Plattsburgh, NY; Univ. of New Mexico, Albuquerque, NM; Univ. of New Mexico, Albuquerque, NM

We have discovered dense microbial mats lining springs in a sulfide-dominated cave (Cueva de Villa Luz) in Tabasco, Mexico. These mats range from 1 to several centimeters in thickness. Light microscopy has revealed organism types including gram negative rods, cocci, and many different filamentous forms. Scanning electron microscopy with accompanying EDS analysis of mat segments shows the presence of elemental sulfur and gypsum crystals. Transmission electron micrography shows the presence of some distinctive morphological organism types. Patches of the mats exhibit auto-fluorescence macroscopically and pink and green areas when illuminated in visible light. Of course, they grow entirely in the dark in the cave environment. The springs give forth hydrogen sulfide, carbon monoxide and possibly other reduced gases. Studies of microbial isolates in culture have shown that sulfate-reducing bacteria are present, as are several strains that can metabolize thiosulfate and elemental sulfur. The mats occur in clear water springs within the cave. Our initial investigations have uncovered no mats in the cloudy water springs. The spring within which the original discovery was made has coarse sand-grain sized sediments which are continuously roiling from the outflow of waters. Presumably, finer sediments have been washed away. The mats were recently discovered during our January 1999 expedition by D. Soroka. We will present our preliminary characterization of these mats, their constituent organisms, and basic chemistry of the waters and sediments.

Metabolic Diversity in Ultra-Deep Groundwater

DEFLAUN, M.F., KOTELNIKOVA, S., BALKWILL, D., STREGER, S., ONSTOTT, T.C., and MOSER, D.

Envirogen, Inc., Lawrenceville, NJ, Univ. Goeteborg, Sweden, Florida State Univ., Tallahassee, FL, Princeton Univ., Princeton, NJ.

Ultra-deep groundwater was collected from fissures in the Boonton Shales at a depth of 3.1 km bgs in the East Driefontein Mine. East Driefontein is one of the Witwatersrand gold mines located in the Transvaal region of South Africa near Johannesburg. In situ rock temperatures at this depth range from 40 to 45° C and the pH of this fissure water sample was 8.0. Microbial cell counts in the groundwater were 5.36 x 105 cells per ml. This water was inoculated into a variety of both solid and liquid media under aerobic and anaerobic conditions. Mesophilic microorganisms respiring oxygen or iron at the expense of hydrogen or methane were successfully enriched. The groundwater on GelRite gellan gum plates containing 1 mg/ml phenanthrene yielded a fast growing organism when incubated aerobically at 60° C. Further investigation indicated that this isolate (GE-7) grew on both GelRite and biotechnology grade agarose (Agarose IV, Amresco) with no additional carbon source, but did not grow on silica gel plates. Growth was observed in high liquid media (LB and R2A) with glucose and sodium acetate as sole carbon sources, but not with sodium lactate, naphthalene, phenanthrene, phenol, BTEX or #2 fuel oil. No growth has been observed at either 30 or 37° C. The GE-7 isolate is a long (4 mm) gram positive rod currently being identified by 16srRNA sequence analysis.

Survey of Presumptive Iron-oxidizing Bacteria in a Deep South African Gold Mine. GHIORSE, W. C., EAGLESHAM, B. S., and GAREN, R. Cornell University, Ithaca, NY

As part of the Witwatersrand Deep Microbiology Project, samples were obtained in November 1998 from the East Driefonein mine shaft No. 5 levels 46 and 48 (= 3,100 mbs). Level 46 samples were rusty-colored microbial mats fed by 37°C-salty water seeping from two exploratory boreholes in an access tunnel wall. Spot tests indicated abundant iron oxides, but no manganese oxides in the mat material. In one borehole the mat formed a crust that overlayed inky-black iron sulfide-containing water. Water chemistry data supported field observations suggesting that sulfate and iron reduction was occurring in anaerobic zones upstream of the iron oxide mats. Confocal laser scanning microscopy showed bacterial assemblages and protozoan cysts within a porous iron-oxide-polymer matrix. Transmission electron microscopy showed intact rod-shaped bacteria with electron-dense oxide particles attached to their cell walls, and mineralized walls. Enrichment cultures were set up in agarose-stabilized, iron-oxygen gradient tubes (Emerson and Moyer, 1997, Appl. Environ. Microbiol. 63:4784) and incubated at room temperature (22-25), 37 and 60°C. Presumptive iron-oxidizing bacteria grew in all tubes incubated at room temperature and at 37°C. No growth was observed in tubes incubated at 60°C. Level 48 samples consisted of freshly mined, carbon leader and adjacent quarzite country rock. Samples were pulverized and slurried under anaerobic conditions. Presumptive iron-oxidizers grew at room temperature, but not at 60°C. Tracer analyses indicated that these samples had been contaminated by mine service water. These observations suggest that mesophilic iron-oxidizing bacteria inhabit the borehole iron mats. Similar bacteria may also exist in the service water.

The Witwatersrand Deep Microbiology Project: Microbial Community Assessment By Phospholipid Fatty Acid Analysis. PFIFFNER SM1, PEACOCK A1, MACNAUGHTON S1, WHITE DC1,2, PHELPS TJ2, MOSER D3, and ONSTOTT TC3. 1The Univ. of Tennessee, Knoxville, TN, 2 Oak Ridge Natl. Laboratory, Oak Ridge, TN, 3 Princeton Univ., Princeton, NJ.

As part of an interdisciplinary research project funded by the NSF LExEn program, research at the gold mines of South Africa has provided a unique opportunity to study microbial communities at depths ranging from 2000 to 3500 meters below land surface. Highly organic carbon leader, quartz rock, weeping borehole biofilms from mineshaft walls, mining water, borehole water, and filtered air samples were collected and analyzed for phospholipid fatty acids (PLFA; off-site) and for microbial enrichments (incubation initiated on-site). Results of the PLFA analyses showed biomass estimates ranging from 10 to 200 pmol/g in carbon leader and quartz samples to greater than 1200 pmol/g in weeping borehole biofilms. Diverse community profiles were observed in the weeping borehole water and the carbon leader, with biomarker lipids indicating the presence of thermophiles (cyclic fatty acids), sulfate reducers (i17:1w7c, 10Me16) and iron reducers (dioic fatty acids). Confirmatory data obtained from microbial enrichment assays indicated the presence of sulfur and iron reducing bacteria. Also addressed, was the potential contamination of rock and water samples by mining activity. Epoxide fatty acids were detected in the service water, indicating exposure of bacteria to high chlorine concentrations. These epoxides are currently being investigated as potential tracers for service water contamination of recovered material. Analyses for other membrane lipid components, the community diversity of the cultivated microbial enrichments, and development of better sampling strategies are ongoing. The gold mines have proven to be a worthy site for investigating microbial ecology of extreme environments.

Mineral precipitates within Sulphur Spring, Florida

M. GARMAN, L. ROBBINS, V. HARWOOD

Dept. of Marine Science, Univ. of South Florida, Tampa, FL; Dept. of Geology, University of South Florida, Tampa, FL., Dept. of Biology, Univ. of South Florida, Tampa, FL

Carbonate caves receiving reduced water from deep aquifers can provide one model of a contemporary terrestrial analogue for chemosynthetically driven extraterrestrial life. Furthermore, these unique systems can provide information on the formation and co-existence of biotically (microbially) and abiotic derived mineral precipitations. Sulphur Spring in Tampa, Florida, is a freshwater spring fed by an extensive, submerged cave system that has been developed in the carbonate rock. The cave system also receives reduced, saline groundwater from deep in the aquifer through many small vents. While the water temperature remains fairly constant (26 -27 C), the pH ranges between 8.5-10.30. This cave system is particularly interesting because it is microbially active. Throughout the cave the limestone walls are covered by a dark biofilm that includes sheathed filamentous iron bacteria, fungi, small rods, cocci, as well as a variety of minerals. In proximity to the saline groundwater vents, the biofilm is white and is tentatively identified as sulfur oxidizing bacteria. In one side room, the Crystal Room, the saline groundwater is concentrated. The walls of the upper part of the Crystal Room are covered by aragonite needles that have apparently formed underwater. The lower part of the Room is coated with a black precipitate. Preliminary analyses have identified sulfur reducing bacterial communities in the lower water column and sediments. Iron-related bacteria have been identified throughout the water column. The current investigations are focusing on microbial interactions involved in the precipitation of minerals, including the aragonite needles in the Crystal Room.

Interpreting high resolution profiles of bacterial and viral abundance in Holocene sediments – ODP Leg 169S JUNIPER, SK, RIGAULT-RICCIARDI, M., BIRD, D., PRAIRIE, Y. MARTINEU, P. Université du Québec à Montréal, Montréal, Québec, Canada

Little is known of long term processes affecting microbial abundance in buried marine sediments. In collaboration with geochemists and sedimentologists involved in ODP Leg169S we undertook a study of bacterial and viral abundance throughout the entire Holocene sediment section in Saanich Inlet, British Columbia, Canada. Sediments were sampled at 1.5m intervals from the sediment surface down into Pleistocene sequences at depths of >100m. Preparations of formalin-fixed sediment were stained with Yo-Pro and bacteria and viruses were enumerated under epifluorescence microscopy. Viral presence was confirmed by TEM. Limited measurements of ATP, pore water H2, CH4 and CO, and 3H-leucine assimilation were used as indices of microbial metabolic activity. Bacterial and viral abundances were high in these organic rich sediments (>109/g), relative to oceanic areas and were highly correlated. The upper Holocene section was characterized by a downcore increase in microbial abundance that was correlated negatively with sediment organic matter content. The interpretation of this and other significant trends will be discussed in relation to the Holocene history of organic matter sedimentation and diagenetic processes.

Metabolic Formation of Filamentous Sulfur by Hydrogen Sulfide Oxidizing Bacteria From the Shallow Subsurface of Deep Sea Hydrothermal Vents. WIRSEN, C. O. and TAYLOR, C. D., Biology Department, Woods Hole Oceanographic Institution, Woods Hole, MA

Investigation of Intrinsic Bioremediation at NAB Little Creek, Site 12: I. Hydrogeochemical Assessment WIDDOWSON, M.A.(1), NOVAK, J.T.(1), BERRY, D.F.(2), MACEWEN, S.J.(3), DRONFIELD, D.G.(3), ERRETT, A.H.(3), LADE, N.A.(1). Department of Civil and Environmental Engineering, Virginia Tech, Blacksburg, Virginia 24061,(1); Department of Crop and Soil Environmental Sciences, Virginia Tech, Blacksburg, Virginia 24061,(2) and CH2M Hill Federal Group, Ltd., Herndon, Virginia

Hydrogeochemical, hydrologic, and microbiological data have been collected and analyzed to assess the rate and extent reductive dechlorination of tetrachloroethene (PCE) and chlorinated ethene daughter products in a contaminated shallow aquifer located at Site 12 of the Naval Amphibious Base (NAB) Little Creek, Virginia Beach, Virginia. Multilevel groundwater sampling devices (MLS) were installed in 8 locations at Site 12 to delineate the vertical and horizontal extent of contamination and delineate microbially-active zones of variable electron acceptor processes. Each MLS consisted of 6-8 sample ports comprised of 0.25-inch diameter tubing installed at 2.5-ft intervals. Groundwater concentrations of chlorinated ethene contaminants, H2 (electron donor), electron acceptor (O2, NO3, SO4), reduced Fe and Mn, and biodegradation end products (CH4, ethene, ethane, Cl-, CO2) varied considerably with depth. Concentrations of PCE varied from not-detected (ND) to 23,000 ppb. Concentrations of microbially-mediated daughter products varied from ND to compound-specific peaks in parts per billion: trichloroethene (1,300), cis- and trans- 1,2-dichloroethene (532 and 18, respectively), vinyl chloride (105), and ethene, (47). Total organic carbon was present at measurable levels in the plume, as were elevated levels of Mn2+, Fe2+, CH4, ethene, ethane, and CO2. Relative to data from the shallow depths, deeper groundwater samples were characterized by elevated levels of CH4 and Cl-, depleted SO4, and products of reductive dechlorination. Results of this study demonstrate the value of the MLS as both an instrument for effective regulatory-driven monitoring programs and a research tool to delineate plumes and assess reductive dechlorination at chlorinated solvent-contaminated sites.

Investigation of Intrinsic Bioremediation at NAB Little Creek, Site 12: II. Microbiological Assessment

BERRY, D.F.,(1) HIGGINS, M.J.,(2) RECTANUS, H.,(3) WIDDOWSON, M.A.,(3) NOVAK, J.T.,(3)

Department of Crop and Soil Environmental Sciences, Virginia Tech, Blacksburg, VA 24061,(1) Department of Civil and Environmental Engineering, Bucknell Univ., Lewisburg, PA, 17837,(2) and Department of Civil and Environmental Engineering, Virginia Tech, Blacksburg, VA

Decisions regarding the efficacy of intrinsic bioremediation should be site- specific and based on various lines of evidence. We have combined hydrological, hydrogeochemical, and microbiological methods to assess natural attenuation of tetrachloroethene (PCE) and chlorinated daughter products in a contaminated shallow aquifer located at Site 12 of the Naval Amphibious Base (NAB) Little Creek, Virginia Beach, Virginia. To document the degradative capabilities of naturally occurring microorganisms and to determine rates of reductive dechlorination for the purpose of model development, anoxic soil slurry microcosms were established with subsurface soil from heavily contaminated, slightly contaminated or uncontaminated locations at Site 12. Concentration data from eight multilevel groundwater sampling devices provided information on locating microbially-active zones and uncontaminated areas in the aquifer. These zones were targeted for aquifer sediment sampling to construct microcosms. Results of groundwater sample analysis revealed the presence of microbially mediated (PCE) breakdown products including ethene, vinyl chloride, cis- and trans- 1,2-dichloroethene, and trichloroethene. In the microcosm studies we observed complete transformation of PCE to cis-DCE within the first 90 days (ranging from 0.8 to 0.3 mmol/L/d for carbon supplemented vs unsupplemented microcosms) for the heavily contaminated location (shallow zone). Microcosms exhibiting rapid PCE transformation also produced much larger quantities of CH4. Results to-date, indicate that biodegradation is a significant contributing factor in the natural attenuation of chlorinated ethenes at Site 12. Biodegradation kinetic parameters derived from microcosm results will serve as input to a fate and transport site model developed using SEAM3D.

Field and Laboratory Evidence for the Intrinsic Bioremediation of Gas Condensate Hydrocarbons

L.M. GIEG, R.V. KOLHATKAR, M.J. MCINERNEY, R.S. TANNER, S.H. HARRIS, K.L. SUBLETTE, J.M. SUFLITA. Univ. Of Oklahoma, Norman, Ok; Univ. Of Tulsa, Tulsa, Ok; Amoco Research Ctr., Naperville, Il

Gas condensate hydrocarbons, including BTEX, have contaminated a shallow aquifer underlying a natural gas production site in Colorado. In order to investigate the prospect for the intrinsic bioremediation of these hydrocarbons, geochemical monitoring was conducted at this site for over four years. Correlating with an overall decrease in sediment-associated BTEX concentrations, oxygen and sulfate concentrations were decreased, and Fe(II), sulfide, and methane were elevated in the hydrocarbon-impacted groundwater relative to upgradient, uncontaminated groundwater. Measurements of dissolved hydrogen levels in the contaminated groundwater indicated that sulfate reduction and methanogenesis were the predominant biological processes in-situ, which correlated with higher numbers of sulfate-reducing bacteria and methanogens in the contaminated sediments. Using GC-MS, several putative bacterial metabolites were detected in organic extracts of contaminated groundwater, including methylbenzylsuccinic acid. This compound is a signature addition metabolite in anaerobic xylene decay, suggesting that anaerobic biodegradation was occurring in-situ. In laboratory incubations, microorganisms from contaminated sediments biodegraded all six BTEX hydrocarbons using sulfate as an electron acceptor whereas only toluene was utilized under methanogenic conditions. Benzylsuccinic acid was detected in toluene-amended incubations, as were methylbenzylsuccinic and toluic acids in xylene-amended incubations. In ethylbenzene-amended incubations, a metabolite eluting with the same GC retention time and mass spectral profile as 3-phenyl-1,2-butanedicarboxylic acid was detected, suggesting an analogous addition mechanism of decay for this hydrocarbon. Collectively, these field and laboratory results argue that intrinsic bioremediation, including anaerobic biodegradation, is occurring in this hydrocarbon-impacted aquifer.

The Potential For Biostabilization Of Coal Tar In The Aqueous Environment. MORRISON, K.A. (1), RAMASWAMI, A. (2) 1. Department of Environmental Sciences, Univ. of Colorado, Denver 2. Department of Civil Engineering, Univ. of Colorado, Denver

Coal tar is a multicomponent dense non-aqueous phase liquid (DNAPL) typically associated with subsurface contamination at former manufactured gas plant facilities. Coal tar is composed of several hundreds of polycyclic aromatic hydrocarbons (PAH) compounds. Because of the complex and often unpredictable migration of DNAPLs in heterogeneous subsurface systems, it is often technologically impossible to achieve complete removal or destruction of all of the DNAPL mass present at a site, particularly when large DNAPL pools are entrapped within fine-grained media. In situ biodegradation surrounding the DNAPL source may stabilize the DNAPL source region and offer a cost-effective risk management strategy for DNAPL contaminated sites. The goal of this project is to develop rapid-screening tests to quantitatively evaluate the potential for near-source biostabilization of multicomponent coal tar in batch biometer reactors under aerobic conditions. Biostabilization refers to microbial activity in the vicinity of the DNAPL source area that can result in: (1) consumption of aqueous phase pollutants released from the DNAPL source, resulting in plume prevention, (2) consumption of the more soluble components of the DNAPL over the long-term, leaving a more stable and less soluble post-degradation DNAPL residue, and (3) lowered aggregate toxicity of the aqueous plume due to a combination of the above two processes. DNAPL biostabilization is assessed by monitoring four parameters in the biometer batch reactors: (1) microbial activity determined by cell counts, oxygen consumption and CO2 evolution, (2) aqueous phase contaminant concentrations surrounding a pool of DNAPL coal tar, (3) aggregate toxicity of the aqueous phase, (4) composition of the DNAPL residue. The four parameters are assessed in well-mixed biometers representative of above-ground slurry systems with no mass transfer constrains, as well as in quiescent, unmixed systems representative of the subsurface. Preliminary results from these experiments indicate lower levels of microbial activity, and lower aqueous phase contaminant concentration and aggregate toxicity in unmixed quiescent systems with low mass transfer rates and reduced bioavailability versus the mixed system. These results suggest that low mass transfer rates and reduced bioavailability in quiescent systems, combined with low-level microbial activity surrounding coal tar pools can facilitate toxicity reduction and risk management at subsurface sites contaminated with DNAPL coal tar. Acknowledgment: This project is funded by the USEPA through the EPA/NSF/DOE/ONR Program on Bioremediation, 1997.

Fate of Linear Alkybenzenesulfonate in the Subsurface Environment. BARBER, L.B., FIELD, J.A., AND KRUEGER, C.J.

Linear alkylbenzenesulfonate (LAS) compounds are extensively used biodegradable anionic surfactants that are discharged directly into the aquatic environment through common waste disposal practices. Although LAS compounds are rapidly biodegraded in aerobic surface waters, degradation rates in anaerobic groundwater can be very slow. Extensive studies of the

fate and transport of LAS and related compounds have been conducted in a wastewater-contaminated aquifer on Cape Cod, Massachusetts. A plume of contaminated groundwater that is over 5 kilometers miles long has resulted from 60 years of wastewater disposal into rapid infiltration beds. Surfactant products detected in the plume include LAS, sulphophenylcarboxylate (SPC) metabolites of LAS, dialkyltetralinsulfonates (DATS) alicyclic reaction byproducts, and branched-chained

alkylbenzenesulfonate (ABS). In situ tracer experiments indicate that LAS compounds introduced in the suboxic (0.1-1.0 mg/L O2) and anoxic zones (<0.1 mg/L O2) of the contaminant plume were only partially degraded and that degradation was limited by availability of oxygen. LAS compounds introduced in the oxic (>1.0 mg/L O2) groundwater overlying the plume did not degrade due to the lack of an acclimated microbial population. The AS compounds were more strongly sorbed in the oxic uncontaminated zone than in the contaminated zone, possibly because competition from other ions was lower in the oxic zone. Degradation of LAS compounds was isomer specific, with longer chain and external isomers being preferentially removed. The SPC metabolites

were stable under the low oxygen conditions in the plume. The DATS compounds also were resistant to biodegradation.

13C/12C Isotope Fractionation of Aromatic Hydrocarbons During Microbial Degradation

MECKENSTOCK R, MORASCH B, WARTHMANN R, SCHINK B, ANNWEILER E, MICHAELIS W, and RICHNOW HH. Univ. Konstanz, Konstanz, Germany

Bacterial Iron Mineral Respiration: A Natural Strategy for Enhanced Bioavailability. FRANK CACCAVO, JR. Univ. of New Hampshire, Durham, New Hampshire

Dissimilatory iron-reducing bacteria (DIRB) use ferric iron as a terminal electron acceptor for anaerobic respiration and growth. This metabolism greatly influences the geochemistry of anaerobic soils, sediments and subsurface aquifers. It may also provide a mechanism for both intrinsic and engineered bioremediation of metal-, hydrocarbon- and halogenated solvent-contaminated subsurface environments. Although ferric iron is abundant in many non-sulfidogenic anaerobic environments, it is essentially insoluble at circumneutral pH. DIRB are thus faced with the paradox of using an abundant, energetically favorable electron acceptor that is essentially insoluble in the environments in which they live. The rate and extent of dissimilatory Fe(III) reduction, and thus the potential for bioremediaton by DIRB, are limited by the bioavailability of Fe(III) in natural environments. A fundamental understanding of the molecular-level interactions between Fe(III)-reducing bacteria and insoluble Fe(III) minerals is thus requisite to the effective application of this metabolism in bioremediation. Our recent work has led to a model of the molecular interactions between DIRB and insoluble ferric iron minerals. In this model, the hydrophobic nature of the DIRB cell surface mediates the initial reversible contact between the cell and Fe(III) minerals. Specific adhesion proteins on the cell surface then promote cell adhesion to the iron mineral particle. Once the DIRB cell attaches, an iron-chelating protein solubilizes ferric ions from the Fe(III) mineral, allowing the efficient transfer of reducing power from the outer membrane-bound respiratory system. This research suggests that DIRB have evolved specific mechanisms to overcome the complex chemical nature of iron and exploit Fe(III) as a potential substrate for anaerobic respiration and growth. This information provides insight into how DIRB compete in subsurface environments, and can ultimately be used to facilitate the application of this metabolism in bioremediation.

TEAP Distribution and Geochemical Modeling of Redox-active Constituents in the Magothy Aquifer, Long Island, New York. *BROWN, C.J.*1, SCHOONEN, M.A.A.2, COATES, J.D.3

1 Water Resources Division, U.S. Geological Survey, Coram, NY, 2 Department of Geosciences, Stony Brook Univ., Stony Brook, NY, 3 Department of Microbiology, Southern Illinois Univ., IL

Fe(III) reduction in the Magothy aquifer of Long Island, N.Y., results in high dissolved-iron concentrations that degrade water quality. Analytical results of aquifer geochemistry, terminal electron-accepting process (TEAP) assays, and geochemical modeling were used to constrain iron-related geochemical processes and redox zonation along a flow path. The observed increase in dissolved inorganic carbon is consistent with lignite oxidation coupled to the reduction of O₂ and sulfate in the aerobic zone, and to the reduction of sulfate in the anaerobic zone; estimated rates of CO₂ production through reduction of Fe(III) were relatively minor by comparison. The overall increase in sulfate concentrations along the flow path, together with the results of mass-balance calculations, and variations in delta S-34 values along the flow path, indicate that sulfate loss through microbial reduction is exceeded by sulfate gain through diffusion from sediments and through the oxidation of FeS₂. Geochemical and microbial data on cores indicate that Fe(III) oxyhydroxide coatings on sediment grains in local, organic carbon- and sulfate-rich zones have been depleted by microbial reduction and resulted in localized sulfate-reducing zones in which the formation of iron disulfides decreases dissolved iron concentrations. These localized zones of sulfate reduction, which are important for assessing zones of low dissolved iron for water-supply development, could be overlooked by aquifer studies that rely only on ground-water data from well-water samples for geochemical modeling.

Microbial Iron Reduction: Indirect Mechanism for Chromate Stabilization B. WIELINGA, M.M. MIZUBA, S.E. FENDORF Univ. of Idaho, Moscow, Idaho; Univ. of Idaho, Moscow, Idaho; Stanford Univ., Palo Alto, California

Ferric iron is ubiquitous in nature and in many suboxic soil, sediment and subsurface environments it is likely to be the most abundant terminal electron acceptor for microbial respiration. Chromate is known to be rapidly reduced by aqueous Fe(II). In this study, the microbially mediated, indirect reduction of chromate by a dissimilatory iron reducing bacterium Shewanella alga, strain BrY was studied. A continuous stream of chromate was introduced to flow-cells containing BrY and a common short-range order iron hydroxide (ferrihydrite). Chromate was reduced at a rate of 195 ug Cr per mg ferrihydrite per hour over a period of 110 h. In such systems, Fe(II) produced as a result of microbial respiration serves as a catalyst (or an electron shuttle), reducing Cr(VI) and thus being reoxidized. This cycling of iron suggests that a limited quantity of iron can potentially cycle indefinitely serving to stabilize chromate in situ. Furthermore, a magnetic chromium/iron solid-phase product (a chromite-like phase) of very limited solubility resulted from these reactions.

Trace Metal Immobilization Via Microbial Reduction of Iron Oxide Minerals. D.C. COOPER, F.W. PICARDAL, J. RIVERA. Indiana Univ., Bloomington, IN

Metal contaminants in iron-oxide-dominated soils and sediments may be immobilized by sorption to the oxide surface. It has been hypothesized that microbial reduction of iron oxide minerals may release sorbed metal contaminants as a result of (i) dissolution of the mineral matrix, or (ii) competition for adsorption sites by biogenic Fe(II). Long term laboratory batch experiments were performed with the iron-reducing bacterium, Shewanella putrefaciens 200, in an artificial groundwater medium containing 10 mM lactate and either 50 mM synthetic lepidocrocite or goethite. Microbial lepidocrocite reduction resulted in formation of magnetite, as shown by x-ray diffraction analysis. Magnetite was not observed during microbial reduction of goethite and the ferrous minerals formed during goethite reduction have not yet been identified. When Zn (0.30 mM) was adsorbed onto the surface of the goethite or lepidocrocite prior to inoculation with S. putrefaciens, microbial Fe(III) reduction sequestered the ‘weakly bound' Zn (soluble in cold, 0.5 N HCl) into a ‘strongly bound' phase (soluble in 6 N HCl, but insoluble in 0.5 N HCl). The observed sequestration of adsorbed Zn into the new ‘strongly bound' phase coincided with the formation of magnetite and various other ferrous minerals. Microbial iron reduction, therefore, may immobilize sorbed zinc by incorporating it into a new mineral phase that is resistant to release by ion exchange. These experiments have implications for understanding metal speciation at sites containing sediments dominated by iron oxide minerals.

Dynamics of Nitrate and Nitrite Inhibition of Fe(II) Production via Goethite Reduction by Shewanella putrefaciens 200. COOPER D.C., PICARDAL F., LLOYD R., RIVERA J. Indiana Univ., Bloomington, IN 47405, USA, Indiana Univ., Bloomington, IN 47405, USA, Indiana Univ., Bloomington, IN 47405, USA, Indiana Univ., Bloomington, IN 47405, USA,

Previous research has shown that nitrate and nitrite can inhibit microbial iron reduction, and that a similar inhibition of the reduction of natural ferric oxyhydroxides can prevent the biogeochemical changes that result from Fe(II) production. Long term batch experiments using S. Putrefaciens 200 in an artificial groundwater medium containing 20 mM lactate, 50 mM goethite, and approximately 2.0 x 10⁶ cells/mL indicate that nitrate inhibits the production of Fe(II) via goethite reduction, and that nitrite production is primarily responsible for this inhibition. Additionally, the presence of goethite decreased the nitrite reduction rate 20-fold, increased the degree of N₂O production 100-fold, and may have inhibited nitrate reduction. Subsequent investigation into the causes of this "mutual inhibition" of Fe(III) and nitrite reduction indicate that extensive surface-catalyzed chemical nitrite reduction, Fe(II) reoxidation, and subsequent ferric oxyhydroxide precipitation may occur in combined systems. When goethite was reduced in the absence of nitrate (conditions as described above), the initial Fe(II) production rate ranged from 0.08 - 0.2 mmol/L hr. When the reduced goethite was chemically oxidized by 2 mM nitrite (1-2% of total Fe present as Fe(II) ), reoxidation rates typically ranged from 0.04 - 0.5 mmol/L hr and the observed 4 to 1 molar ratio of iron oxidized to nitrite reduced was indicative of N₂O production. Experiments to utilize ?N¹⁵ ₂O to distinguish between the chemical and biological reduction of nitrite are currently underway. The potential for this geochemical inhibition of a microbiological process has important implications toward the global iron and nitrogen cycles, and also towards the impact of microbial iron reduction on the mobility of trace metals in systems where re-adsorption of the metal onto iron oxide surfaces (following reductive dissolution of the original iron oxide) is strongly affected by site limitation or the presence of chelating agents.

The Role of Heavy Metal Tolerance in the Bacterial Reduction of Copper-Contaminated Hydrous Ferric Oxide P. J. S. COLBERG (1) and J. T. MARKWIESE (2) ; (1) Univ of Wyoming, Laramie, WY and (2) Neptune and Co., Los Alamos, NM

Fe(III) oxide is an important heavy-metal sink. Bacteria are responsible for much of the Fe(III) reduced in nonsulfidogenic aquatic environments, yet factors governing the bacterial reduction of heavy-metal contaminated iron oxide are largely unknown. In this study with a bacterial co-culture enriched from metal-contaminated sediments, we demonstrate that copper toxicity impedes anaerobic carbon oxidation and bacterial reduction of hydrous ferric oxide (HFO). In the enrichment culture, a Clostridium sp. fermented lactate to propionate and acetate, and Fe(III) reducers coupled acetate oxidation to HFO reduction. Increasing the amount of Cu in the culture medium significantly extended the time before Fe(III) reduction occurred and decreased the reduction rate, but generally did not affect the extent of HFO reduction. The Clostridium had a higher Cu-complexation capacity than the Fe(III) reducer, Shewanella alga BrY. Iron reduction was inhibited until almost all of the dissolved Cu was removed from solution and occurred two to seven times faster if the co-culture was fed lactate instead of acetate. Our findings suggest that fermentative bacteria may play a role in ameliorating heavy metal toxicity to iron-reducing bacteria, thereby enhancing the potential for metal release in sediments by facilitating the reductive dissolution of heavy metal-contaminated HFO.

Nitrate Stimulates Microbial Mediated Disappearance of Monochlorobenzene. LORBEER, H., VOGT, C., SEIFERT, K., WUENSCHE, L., and BABEL, W. UFZ Ctr. for Environmental Research, Department of Environmental Microbiology, Leipzig, Germany

Monochlorobenzene (MCB) is known to be a final product of reductive dechlorination of higher chlorinated chlorobenzenes and to be persistent under methanogenic conditions. The autochthonous bacteriocenoses of an anoxic quaternary aquifer contaminated with this chemical was able to mineralize MCB under aerobic conditions completely within a few days. Without supply of air the autochthonous bacteria were not able to metabolize MCB under in situ conditions. In model experiments in the laboratory as well as in semi-technical scale using an on-site reactor continuously flowed by ground water which contained about 25 mg MCB per liter it was obvious that addition of nitrate accelerates the disappearance of MCB and that extra nitrate is reduced. In order to investigate the fate of MCB, a method was used which is based on ¹⁴C-labelled MCB both in the presence of oxygen or nitrate. In contrast to aerobic cultures the formation of ¹⁴CO₂ was very weak in denitrifying cultures within 28 days of incubation, although about 50 % of the MCB disappeared during this period.

Feasibility of Residual Bioremediation Strategy for a Chlorinated Solvent Contaminated Aquifer Following Solvent Extraction Remediation Biotreatment R.R. HELTON 1, S.J. FLYNN 1, F.E. LOEFFLER 1, G.W. SEWELL 2, J.M. TIEDJE 1 1 NSF Ctr for Microbial Ecology, MSU, E. Lansing, MI , 2 US EPA-NRMRL/SPRD, Ada, OK

An aquifer in Jacksonville, Florida, contaminated with chlorinated solvents, including chlorinated ethenes, was treated with a solvent extraction remediation biotreatment (SERB). Greater than 90% of the chlorinated compounds were extracted using this technique, however a mixture of residual chlorinated solvents and ethanol remained. In this study we have established techniques to monitor microbial community structure and chloroethene dechlorination activity for feasibility testing of residual remediation. Samples from injection and monitoring wells taken before the SERB were chosen for laboratory studies. The potential and extent of PCE dechlorination was assessed in microcosms amended with nine different electron donors. PCE dechlorination to TCE and cis DCE was detected in both the absence and the presence of several electron donors. DTAF staining and computer image analysis of the indigenous microorganisms revealed low cell count estimations of 7.5 E9/g soil for the injection well and 6.8 E9/g soil for the monitoring well. Terminal restriction fragment length polymorphism is being performed to further assess the microbial community structure. These techniques will be used to examine changes in community structure and dechlorination activity in samples following the SERB.

Monitoring Of Enhanced In-situ Bioremediation Of Trichloroethylene Using Stable Carbon Isotopes

1-D.L. SONG,2-L. ALVAREZ-COHEN,3-M.E. CONRAD,4-K. SORENSON. 1-D.L. SONG, Univ. of California at Berkeley, Berkeley, CA; 2-L. ALVAREZ-COHEN, Univ. of California at Berkeley, Berkeley, CA; 3-M.E. CONRAD, Lawrence Berkeley Natl. Laboratory, Berkeley, CA; 4-K. SORENSON, Idaho Natl. Engineering Laboratory, Idaho Falls

Groundwater and soil gas samples from monitoring wells at the Test Area North Site (TAN) , Idaho National Engineering and Environmental Laboratory, are currently being analyzed for stable carbon isotope ratios (delta ¹³C) of organic compounds and inorganic carbon. The study is a component of an extensive monitoring program to evaluate the effectiveness of enhancing in-situ bioremediation of trichloroethylene with the injection of lactate as a substrate for anaerobic reductive dechlorination. The site geology consists of a fractured basalt aquifer. Contaminants, primarily trichloroethylene (TCE), related volatile organics, and radionuclides such as strontium, were released into the groundwater from an injection well formerly used to dispose of the laboratory’s liquid wastes. Stable carbon isotopic composition of TCE and anaerobic degradation products (cis-DCE, VC), organic acids (lactate, acetate, proprionate, butyrate), dissolved inorganic carbon, carbon dioxide, and methane are being analyzed for possible fractionation during biological remediation. Samples taken from ten monitoring wells prior to lactate injection and then monthly thereafter will be analyzed using a combination of purge-and-trap extraction, continuous flow-GC-combustion isotope ratio mass spectrometry. Preliminary results indicate that the delta ¹³C values of trichloroethylene from before and after lactate ranged from –24 per mil to –26 per mil. The delta ¹³C of injected lactate was –11per mil while the degradation products acetate and proprionate detected at two wells during the first round of monthly samples yielded delta ¹³C of –8 per mil and –12 per mil, respectively. Dissolved inorganic carbon delta ¹³C values at the site range from –10 per mil to –11 per mil. Carbon and hydrogen isotope ratios (delta ¹³C and dD) of dissolved methane will be quantified in order to determine mechanisms of methanogenesis. The test study will continue until November 1999 and groundwater will be sampled monthly for the duration of the project.

Triethylphosphate Utilizing Bacteria and Bioimmobilization. A. V. PALUMBO, R. A. HURT, L. A. FAGEN, AND S. P. SCARBOROUGH. Oak Ridge National Laboratory, Oak Ridge, TN

Field Evaluation of the Solvent Extraction Residual Biotreatment Technology.

MRAVIK, S.C., SEWELL, G.W., and WOOD, A.L. USEPA/NRMRL/SPRD

Aerobic Cometabolic TCE Transformation in Propane-fed Aquifer Micrococsms. M.E. DOLAN, L. SEMPRINI. Oregon State Univ.

Batch microcosms were used to determine the potential for aerobic cometabolic chlorinated aliphatic hydrocarbon (CAH) degradation using propane as the primary substrate. The microcosms were constructed using aquifer material and/or groundwater aseptically obtained from two CAH-contaminated sites at McClellan Air Force Base, CA. CAH contamination included trichloroethylene (TCE) at 350 to 1100 ug/L, cis-dichloroethylene (c-DCE) at about 50 ug/L, 1,1-dichloroethane (1,1-DCA) at 10 ug/L, 1,1-dichloroethylene (1,1-DCE) at 6 ug/L, and chloroform (CF) at 2 ug/L. Each microcosm was prepared with approximately 200 ml of site groundwater and 100 ml headspace. Some microcosms also contained 5, 10, or 70 g of aquifer material from the respective sites. Mixed results were obtained from the microcosms without aquifer solids. Of the 32 microcosms prepared only 19 exhibited propane utilization. TCE transformation was observed in only two of the microcosms. Successful enrichment of propane-utilizers occurred in all 20 of the microcosms containing aquifer material. Lag times for the onset of propane utilization ranged from 15 days for microcosms containing the most aquifer material (with little CAH contamination) to approximately 50 days for those with less aquifer solids and higher CAH concentrations. Rapid CAH transformation following propane utilization was found in many of the microcosms with up to 140 ug TCE transformed from a single addition of 8 mg propane, resulting in TCE concentrations below 5 ug/L. Rapid TCE transformation occurred only after the successful transformation of the other CAHs present. In many microcosms, a transient build-up of propylene was observed during propane degradation. In microcosms fed commercial grade propane containing 18% propylene, propane degradation began only after propylene transformation was complete. Propane degradation stopped due to nitrogen limitations, but resumed upon the addition of nitrate or ammonia gas. Nitrous oxide did not cause the resumption of propane degradation within the 100-day test. Dinitrogen trioxide is currently being tested as an alternate gaseous nitrogen source.

Dissolution of Fe Oxides by an Aerobic Microorganism. HERSMAN, L.E., FORSYTHE, J.H., MAURICE, P.A., AND G. SPOSITO Life Sciences Division, Los Alamos Natl Laboratory, Los Alamos, NM; Dept. of Geology, Kent State Univ., Kent, OH; and, Environmental Geochemistry Group, Univ. Calif., Berkeley, CA

To sustain minimal growth, nearly all microorganisms have an absolute requirement for iron (Fe) at mM concentrations. However, in aerobic environments at circum neutral pH Fe is subject extreme insolubility that limits its aqueous concentration to less than 10-17 M (in the absence of organic complexing ligands). Thus, microorganisms must overcome a concentration disparity of greater than 10 orders of magnitude between Fe's aqueous (inorganic) availability and their physiological requirement. We have investigated Fe acquisition by a strict aerobe, Pseudomonas mendocina var., that unlike other commonly studied microorganisms (e.g., Shewanella putrifacians, Geobacter sulfurreducans, etc.) cannot reduce Fe as a terminal electron acceptor. In addition to producing siderophores, this organism also produces at least 2 reductants (membrane associated and extracellular) in response to either Fe depravation, Fe supplied as FeEDTA, or Fe in the form of goethite, hematite, or ferrihydrite. Surprisingly, its growth on goethite nearly equals its growth on FeEDTA, exceeds its growth on the less crystalline ferrihydrite, and increases with increasing Al substitution in the goethite lattice - all counterintuitive to current paradigms. Also puzzling is our observation that maximum siderophore production appears to follow, and not precede, maximum growth on hematite. Siderophore (desferrioxamine B) attachment to the mineral surface is hindered by its positively-charged amino group (the goethite surface charge is positive at pH <8). Replacement of this group by a neutral acetyl group leads to sorption that is comparable (when suitably normalized for the number of hydroxamate groups) to that of acetohydroxamic acid at the same pH. Finally, atomic force microscopy (AFM) reveals that for hematite the microorganism attaches to only a small percentage of the mineral surface, implying surface attachment selectivity by P. mendocina. AFM coupled with epifluorescence microscopy reveals the presence of abundant exopolysaccharides - potentially significant in the dissolution process.

Dissolution of Potassium-Feldspar by Heterotrophic Bacteria M^cELDOWNEY, S¹, VALSAMI-JONES, E²., GAZE, W.² and M^cLEAN, J.² 1 School of Biosciences, University of Westminster, London W1M 8JS

2 Department of Mineralogy, The Natural History Museum, Cromwell Road, London SW7 5BD.

Mineral dissolution is normally described in purely physical and chemical terms. There are sites, however, where evidence suggests a biological contribution. A relatively high proportion of sub-surface bacterial communities associated with minerals is likely to be heterotrophic bacteria. Heterotrophs are potentially able to interact with minerals in a variety of ways including: through exoproduct formation e.g. organic acids, chelating agents, and exopolymers; through cell surface or internal accumulation of metals; and through attachment to the mineral surfaces. The aim of this study was to address the hypothesis that heterotrophic bacteria can enhance the dissolution of potassium-feldspar. Potassium-feldspar is of particular interest since it is abundant in rocks and is common in soils. A variety of heterotrophic bacteria were isolated from soils formed by weathering of feldspar rich rock and outcropping weathered crystals at Shap Granite (UK). Isolations selected for aerobic and facultative anaerobic bacteria including denitrifying bacteria capable of growing on glucose as a sole carbon and energy source, isolates able to grow under low iron conditions or low potassium conditions. Twenty-seven of these isolates were screened for their ability to enhance potassium-feldspar dissolution. The majority were able to enhance dissolution. The extent of dissolution varied with organism and growth condition, and was time dependent. Dissolution of K-feldspar by Serratia marcescens, and the release of Al and Si from the mineral was initiated during the early stationary phase of growth in batch culture e.g. approximately 16 hrs in C/N sufficient medium. Longer term studies indicated continued dissolution of K-feldspar. The dissolution was not related to changes in pH. The evidence suggests that heterotrophic bacteria may contribute significantly to the dissolution of minerals, influencing the circulation of base-cations on the earth’s surface.

Nutrient-Driven Colonization and Weathering of Silicates ROGERS, J.R., BENNETT, P.C., HIEBERT, F.K., and CHOI, W.J. The Univ. of Texas at Austin, Austin, TX, 78712

The dynamics of microbial attachment to mineral surfaces is a complex combination of surface charge, solution and mineral composition, and the types of organisms present in the ground water. Another potential influence is the presence of limiting inorganic nutrients in the mineralogy of the aquifer, or conversely the presence of toxic metals. In this study we examined microbial colonization and weathering of silicate minerals using in situ field microcosms and controlled laboratory microcosms, as a function of mineral composition. We found that in environments where P is scarce, feldspars that contain micro-inclusions of P-minerals such as apatite are preferentially colonized over similar feldspars without P. Further, we found that colonization of mineral surfaces directly correlates to the degree of mineral weathering and weathering is only found near attached organisms. This suggests that colonizing organisms produce a reactive microenvironment at the mineral surface altering the mineral-water equilibria at that surface. Degradation of substrate in laboratory microcosms containing P-silicates was substantially faster than in those containing non P-silicates, suggesting that P released from silicates influences metabolism of the microbial community. We propose that colonization behavior can be influenced by the availability of P, and that native subsurface microorganisms preferentially colonize and weather silicates, which contain apatite. The implications of this interaction is that nutrient-bearing silicates will be colonized, and destroyed, while non P bearing silicates are uncolonized and unweathered. The dissolution of P-bearing silicates releases P to the colonizing organisms, offering them a competitive advantage over planktonic organisms or those colonizing non P-bearing silicates.

Modern calcite precipitation in a fractured aquifer waste plume: reconciling laboratory and natural microbial CO2 mineralization TOBIN, K.J.1, COLWELL, F.S., ONSTOTT, T.C., FREDRICKSON, J., and SMITH, R. 1 Univ. of Illinois at Chicago, Department of Earth and Environmental Sciences

Minor modern calcite cementation is confined to the distal tips of the latest generation of calcite that is in contact with a contaminated aquifer at the Test Area North (TAN), INEEL. Modern calcite is geochemically distinct from ancient calcite. Specifically, modern calcite has elevated Mn values when compared with more ancient cement. It is difficult to envision anoxic conditions within the aquifer before the time of waste disposal at TAN that would be required to form Mn-rich calcite. Away from the plume the aquifer is open with respect to diffusion from the atmosphere and has low dissolved organic carbon. Consequently, it is conceivable that the distal tips of the latest generation of calcite were formed in association with anaerobic microbial activity that developed within the plume. Calcite is thermodynamically favored to be precipitated from the modern TAN aquifer based on speciation modeling with PHREEQE. Calculated saturation indexes for calcite in the TAN area range from 0.1 to 0.7. A value of 20 µg/L/yr of calcite precipitation has been predicted based on NETPATH modeling. Assuming a nominal 10% porosity and a maximum duration for contamination of 47 years (from 1952 to 1999) then 0.04 µg of calcite should have formed in one gram of rock over this period. The observed abundance of modern calcite (0.6 to 37.9 µg of calcite per g of total sample) are broadly consistent with results indicated from NETPATH modeling particularly when considering that modern calcite was present in only 3 out of the 30 samples from TAN 37. Bulk aerobic and anaerobic heteorotrophic microbial respiration (measured using 14C-labeled acetate) from fresh TAN basalt chips range from 56 to 243 nmoles of carbon dioxide/g of rock/yr. If this rate has been constant over the last 47 years and if all of this respired carbon dioxide has been incorporated into calcite then 263 to 1142 µg of calcite would potentially have been formed per g of sample, which is significantly higher than what is observed. Significantly, our results are consistent with previous studies of unfractured sandy lithologies in which laboratory microbial activity measurements tend to overestimate natural respiration rates by a factor of 1000 to 1000000.

Moderately Thermophilic, Calcium-Precipitating Bacteria Found In Calcite Deposits At Yucca Mountain. T.A. ELSE¹, C. PANTLE², P.S. AMY¹, Y. DUBLYANSKY³, G. SOUTHAM⁴, and R. MIELKE⁴. University of Nevada, Las Vegas, NV¹, Community College of Southern Nevada, Las Vegas, NV², Institute of Mineralogy and Petrography, Russian Academy of Sciences, Novosibirsk, Russia³, and Northern Arizona University, Flagstaff, AZ⁴.

Recent findings indicate that 160,000 years ago, thermal waters rose inside Yucca Mountain, the proposed site for a high-level nuclear waste repository. Fluid inclusion data provide evidence for thermal deposition of calcite within fractures of the tunnel walls. Evidence for microbially-influenced calcite precipitation in these deposits is indicated by the presence of moderately-thermophilic, calcium-precipitating bacteria. Calcite and volcanic tuff specimens were collected and aseptically prepared to prevent contamination from outside sources. The calcite and volcanic tuff were separately crushed and slurries were prepared using 1:10 dilutions of calcite or tuff in 0.1% sodium pyrophosphate. The slurries were plated onto R2A and B-4 media and incubated at 23^o C, 41^oC, and 60^oC for two weeks. Growth on B-4 medium was observed along with the formation of calcite crystals. Preliminary data show growth of calcium-precipitating bacteria at 23^oC, 41^oC, and 60^oC obtained from calcite deposits at two different locations in Yucca Mountain. Volcanic tuff specimens from the same two locations, and an independent site, yielded calcium-precipitating bacteria that grew at 23^oC and 41^oC, but showed no growth at 60^oC. Initial TEM analysis reveals one of the isolates to be large, spore-forming bacilli. SEM analysis shows inverted cone-shaped crystals in the center of the bacterial colonies and EDS analysis verified these crystals as calcite. Microbial identification by 16s rDNA sequencing is underway. These findings may have an impact on the suitability of Yucca Mountain for storing high-level nuclear waste.

Bacterially-Induced Carbonate Precipitation in Anoxic Environments WARTHMANN R., VASCONCELOS C., VAN LITH Y. and McKENZIE J. A. Geological Inst., Laboratory of Geomicrobiology, ETH-Zurich, Switzerland

Recent studies of modern anoxic environments provide valuable insights into natural microbial processes related to carbonate mineral precipitation. One example is the intensive microbially mediated precipitation of carbonates with variable crystal shapes such as, dumbbells, star-shapes, and oval crystals, observed in Lagoa Vermelha, a hypersaline lagoon located ~100 km east of Rio de Janeiro, Brazil. In order to study mechanisms of biomineralization taking place at the sediment surface and in the subsurface, strains of marine sulfate-reducing bacteria were isolated from the Lagoa Vermelha sediment. Growth experiments were carried out with bacterial pure cultures under different environmental conditions. Our experimental results show that the studied strains precipitate carbonate minerals when they are incubated in a synthetic liquid medium with formiate as a carbon source. Dissimilation of formiate causes a slight alcalinization of the environmental water, which most probably promotes mineral precipitation. The minerals formed during an incubation time of 30 days were identified by XRD as calcite and Mg-calcite with up to 20 mol% Mg. The apparent precipitation rate was ~153 mg /l month. The precipitates (length 50-100 ?m) have remarkable dumbbell and spherical crystal morphologies, which are also observed in the lagoon sediment. Further investigations will elucidate whether the mineral shape and composition are actively influenced by the bacteria, that are sometimes directly attached to the mineral surface. These preliminary results show that sulfate-reducing bacteria may influence the carbonate system, which suggests that anaerobic microorganisms may have played a key role in Precambrian carbonate sedimentation.

Degradation Of Herbicides In Ground Water At Different Redox Conditions LARSEN, L.; JØRGENSEN, C.; AAMAND, J. Larsen and Aamand: GEUS, Geological Survey of Denmark and Greenland, Department of Geochemistry, Copenhagen, Denmark. Jørgensen: VKI, Water Quality Inst., Hørsholm, Denmark

Herbicides are found increasingly often in ground water. The only way to reach complete degradation (mineralization) of the compounds to CO₂ and H₂O is by microbial activity. Important factors determining the microbial ability to degrade herbicides are the interaction of bacteria and redox condition present in the aquifer. This study describes the potential for mineralization of herbicides in ground water in two aquifers and a freshwater wetland area. The herbicides studied were mecoprop, isoproturon, atrazine, and metsulfuron-methyl. Sediment and ground water were sampled at sites representing different redox conditions. The sites were characterized as regards water chemistry and rates of reduction of terminal electron acceptors. Slurries of sediment, ground water, and 25 microg/l [ring-U-¹⁴C]herbicide were incubated at 10?C in the presence of either O₂, NO₃, SO₄ + CO₂, or CO₂ as terminal electron acceptors corresponding to the conditions prevailing at the sampling site. Atrazine was not mineralized under any of the conditions applied. Mecoprop, metsulfuron-methyl, and isoproturon were mineralized under aerobic conditions, 33%, 14-20%, and 14%, respectively, after 360 days. In NO₃ amended slurries no mineralization was observed. Surprisingly, mecoprop was mineralized under anaerobic conditions, 8-12% was recovered as ¹⁴CO₂ in sulfate reducing and methanogenic sediments, whereas neither metsulfuron-methyl nor isoproturon were mineralized. The results indicate that, whereas some herbicides are completely degraded aerobically, mineralization of most herbicides is absent or extremely slow under anaerobic conditions.

Mineralisation of 2,4-D, Mecoprop, and 2,4,5-T In An Aerobic Sandy Aquifer As Effect Of Substrate Concentration And Addition Of Organic And Inorganic Nutrients. de Lipthay, J.R., and Aamand, J.

Geological Survey of Denmark and Greenland (GEUS), Department of Geochemistry, Copenhagen, Denmark.

Phenoxyalkanoic acid herbicides have been used extensively throughout the world, and due to their relatively high solubility they may be transported with infiltrating rainwater to the groundwater. This study examines the mineralisation (as measured by 14CO2) of the three phenoxyalkanoic acid herbicides 2,4-D, Mecoprop (MCPP), and 2,4,5-T in sediment from an aerobic sandy aquifer at low temperature (10&#61616;C). The effect of herbicide concentration as well as addition of different sources of organic and inorganic nutrients on mineralisation were investigated. Generally, the most rapid mineralisation was observed for 2,4-D giving rise to sigmoidal kinetics for a wide range of substrate concentrations (0.0005 – 10 ppm) and to linear mineralisation kinetics for concentrations below. By increasing the concentration of 2,4-D the maximum level of percentage evolved 14CO2 increased as well. For MCPP the pattern of mineralisation was similar although the threshold level for linear mineralisation kinetics was higher. For 2,4,5-T only little mineralisation took place. Addition of organic and inorganic nutrients showed no effect on mineralisation of 2,4,5-T. However, significant effects resulting in a decreased lagphase were imposed on mineralisation of 2,4-D and MCPP when high concentrations of herbicide (10 ppm) were used indicating either a toxic effect or a nutrient limitation. When small concentrations (0.001 ppm) of 2,4-D and MCPP were used no significant effects were observed. By the end of mineralisation all experiments were examined for 14C in different fractions, and effects on the microbial biomass was studied.

Exploring the Dependence of PKO1 Degradative Activity on Substrate Exposure History in Porous Media. L. M. ABRIOLA 1, J. PARK 1, Y.-M. CHEN 1, and J. J. KUKOR 2. 1. Univ. of Michigan, Ann Arbor, MI 2. Rutgers Univ., New Brunswick, NJ

Potential biodegradative capacity, as assessed by treatability studies performed in laboratory batch assay systems, is often unrealized in contaminated subsurface environments. Reduced degradation may be attributed to many factors related to the coupling of physical, chemical and biological processes. This study presents the results of batch and column experiments designed to explore the effects of substrate concentration history (level and contact time) on enzymatic regulation and biotransformation rates in the subsurface. To investigate enzymatic regulation, an indigenous toluene oxidizer, Ralstonia pickettii PKO1, was chosen as a well-characterized strain, with an inducible enzyme (toluene 3-monooxygenase). Aerobic toluene degradation was investigated in a PKO1 inoculated sand column. The column experiment was conducted under constant flow conditions at 25 ± 0.5 oC by perturbing influent toluene concentrations from 0 ppm to 2 ppm. Influent and effluent toluene concentrations were monitored over time, and the spatial distributions of living cells and specific toluene activity were quantified at the start and end of the experiment. A Michaelis-Menten based numerical model was then used to predict system behavior. Simulations employed independently measured parameters, including microbial kinetic parameters for fully induced suspended cells harvested from an exponential growth phase, initial biomass, retardation factor, and dispersivity. Comparison of experimental observations and model predictions provided evidence of de-activation of toluene degradative capacity before the initiation of the column experiment when toluene was absent. Column observations were also consistent with subsequent enzymatic induction following a lag period. Comparison of model predictions and the observed effluent toluene breakthrough curves, following perturbations in influent toluene concentration levels, further suggested that the observed substrate concentration-dependent regulation was hysteretic as well as reversible. The presence of hysteresis was independently confirmed through a series of batch experiments, conducted with suspended cells which had been starved for different periods. Batch experimental results revealed that specific degradative activity was inversely correlated to the length of the starvation period. In these experiments, a fixed lag time was not observed. Instead, Vmax or maximum specific degradation rate was found to be a function of time, with its rate of change reduced as the length of the starvation period increased. The observed hysteresis in enzymatic regulation may be attributed to changes in physiological state which occur as a consequence of substrate concentration history. These observations may have significant implications for intrinsic bioremediation rates under the low concentrations typical of many contaminant plumes.

Operational Regimes for Surfactant-Enhanced PAH Biodegradation. BROWN, D.G., STENCEL, J.R., AND JAFFÉ, P.R. Princeton University, Princeton, NJ

Patchy Distribution of Microbial Activity in Low Recharge Vadose Zone Sediments F. J. BROCKMAN J. STULTS, S. LI, C. SPADONI, S. PFIFFNER, C. J. MURRAY , E. MURPHY all authors Pacific Northwest Natl. Lab. EXCEPT Pfiffner is Univ. Tennessee

At a site with approximately 15 micrometers of average annual recharge, aerobic microbial activity was not detected in 30% of replicate 10 g samples (n=200) after a 244 day incubation with a substrate mixture in the absence or presence of inorganic nutrients. In separate experiments, microbial activity was not detected in 0.1 g or 1 g samples but was encountered in 37% of the 10 g samples and in 75% of the 100 g samples. These results indicate that viable microorganisms exist in "hotspots" separated by extensive regions of excluding conditions. At a second site with approximately 2 cm of average annual recharge, 8 cores were sampled from the B horizons of ancient buried soils in a 100-foot thick loess deposit composed of the same parent material. From each core, 0.1 g samples (n=50) from a 17-cm "transect" were analyzed by the highly sensitive tritiated acetate incorporation into membrane lipids assay. The frequency of positive samples ranged from 100% for 60 year old pore water to 6% for 900 year old pore water. The frequency of positive samples was strongly correlated to both pore water age and pore water organic carbon concentration. The assay was able to detect incorporation equivalent to the de novo synthesis of tens of cells per gram sediment. Phospholipid fatty acid analysis performed on adjacent 100 g samples showed a spatially averaged density of approximately log4 viable cells/g in all cores, suggesting that the distribution of microorganisms capable of growth is much more discontinuous, with extensive volumes lacking detectable growth, in the cores with older pore water ages and lower porewater organic carbon.

Physiological and Kinetic Characterization of Soil Microbial Fractions in Response to Nutrient Fluctuations in the Subsurface A. N. RIHANA, and P. ADRIAENS The Univ. of Michigan, Ann Arbor, Michigan

Variably-attached microbial fractions were derived from unsaturated soils ranging in carbon content using a serial elution technique, and were physiologically and kinetically characterized to (i) understand their adaptive response to substrate concentrations and nutrient fluctuations in natural systems, and (ii) to develop cause-effect relationships between cell attachment and biokinetic endpoints as a function of soil matrix and acclimation substrate and concentration. The results, using principal component analysis, from Biolog and FAME characterization of fractions derived from a sandy soil adapted to various substrates showed that the ecological indications for loosely attached fractions diverge as a function of adaptation substrate, whereas indicators for tightly-attached fractions cluster independent of the adaptation substrate. For high organic carbon soils, this trend becomes less distinct and the attachment factor does no longer differentiate the ecological characteristics of the microbial communities. This work is presently being complemented using denaturing gradient gel electrophoresis (DGGE) profile analysis. Biokinetic characterization using toluene indicates that substrate affinity constant, K_s, and maintenance energy requirement, q_m, decrease with increasing strength of attachment, resulting in a low threshold concentration, C_t. Even though adapted to high toluene concentration, the tightly-attached populations exhibit a greater adaptability to substrate fluxes than the loosely-attached populations. The convergence of biokinetic and physiological endpoints may indicate a distribution of microbial activity and community composition as a function of the occupied niche. This distributed activity concept may have implications for the prediction of achievable biodegradation endpoints in soils, especially in low residual concentrations systems.

Bacterial Biomass, Activity and community structure related to macropore channels in Danish subsurface soils. VINTHER¹, F. P., ELSGAARD¹, L. and JACOBSEN², O. S. ¹ Danish Institute of Agricultural Sciences, Dept. of Crop Physiology and Soil Science, Research Centre Foulum, DK-8830 Tjele, Denmark. ² Geological Survey of Denmark and Greenland, Dept. of Geochemistry, Thoravej 8, DK-2400 Copenhagen NV, Denmark.

In clay and loamy soils the vertical flow of dissolved and particulate organic matter primarily takes place through macropore channels, thereby supplying the attached microorganisms with nutrients and substrates for their growth. Consequently the walls of the macropore channels may have a higher microbial biomass, a higher biodegradation potential, and a different microbial community structure, than the soil matrix not affected by macropores. This was tested by determining the size of the microbial biomass, the degradation of selected pesticides, and the carbon source utilization patterns by microbial communities in soil adjacent to macropore channels (macropore soil) and in soil not affected by macropores (matrix soil) from agricultural and forest sites in Denmark. It was shown that macropore soil had the largest bacterial biomass and that the microbial communities in macropore soil were different from those in the matrix soil. However, the pesticide degradation potential in the two types of soil did not seem to be different. These results of direct measurements in macropore soil and matrix soil agreed with the role of macropore channels as preferential pathways for transportation of solutes. The higher numbers of bacterial cells in the macropore environments may be a combined effect of a more optimal substrate supply and transportation of bacterial cells through the macropore channels.

Effects of Water Content and Temperature on Subsurface Production, Transformation, Transport and Flux of Nitric Oxide to the Troposphere

J. JEFFREY PEIRCE. Department of Civil and Environmental Engineering, Duke Univ., Durham. NC

Characterization of the Subsurface Microorganisms in the Vicinity of the Liquid Radioactive Waste Repository. NAZINA T.N.1, DAVIDOV A.S.2, KOSAREVA I.M.2 1-Institute of Microbiology, Russian Academy of Sciences, Moscow, Russia; 2 -Institute of Physical Chemistry, Russian Academy of Sciences,Moscow, Russia

It is now generally acknowledged that microorganisms presented within repositories of liquid radioactive waste (LRW). Structure of the bacterial cenosis, hysicochemical and geochemical characteristics of the strata waters from control wells located in the vicinity of the low level radioactive waste repository were investigated. In subsurface samples presented aerobic saprotrophic bacteria (102 -104 cells/ml) and anaerobic bacteria -fermenters (105 cells/ml), sulfate reducers (102 cells/ml), nitrate reducers (103 -106 cells/ml) and methanogens (single cells/ml). The aim of further investigation was to find microorganisms useful for cleaning LRW from two main environment contaminants, that are – nitrate and acetic acid. Although aerobic bacteria that are able to mineralize acetate are ubiquitous in the environment, an anaerobic treatment under denitrifying condition can be advantageous due to the fact that the supply of the subsurface with sufficient oxygen is problematic. Very promising is microbiological treatment combining utilization of acetate with nitrate as electron acceptor. Many denitrifying bacterial strains were isolated from the indigenous community of the stratal water in the vicinity of repositories of LRW. Strains were selected by a protocol that enriched for phenotypes important for radioactive waste cleaning. The resulting organisms were resistant to high levels of radioactivity, and high content of acetic acid and nitrate. Nitrogen and carbon dioxide were the main products of denitrification. Phenotypic and phylogenetic analyses of pure cultures of denitrifying bacteria were also performed. The results of this study show that main components of radioactive waste – acetate and nitrate – can be degraded in subsurface by a consortium of different radiation-resistant bacterial strains, clearly indicating that bioremediation of nuclear waste-contaminated environments is feasible.

Evidence for and significance of Microbial Fe(III) Reduction in Mine Tailings-Enriched Anoxic Lake Sediments CUMMINGS D. ¹; CACCAVO, F. JR²; MARCH, A³; SPRINGS, S. ⁴; BOSTICK, B. ⁵; FENDORF, S. ⁵; ROSENZWEIG, F. ^{3 1}Dept of Microbiol. Univ. of ID, Moscow, ID; ²Dept. of Microbiol. Univ. of New Hampshire, Durham, NH; ³Dept. of Biol. Sci. , Univ. of Idaho, Moscow, ID; Lehrstuhl fur Mikrobiol, Technische Univer. Muchen, Munich, Germany; ⁵ Dept. of Geol., Stanford Univ, CA

Previous work in our lab has demonstrated that sediments of Lake Coeur d’Alene (CDA), ID consist of ca. 10% iron, are highly reduced, and support abundant microbial populations in spite of severe metal contamination from upriver mining activities. Depth profiles of redox-active elements such as iron and arsenic suggest that diagenesis has occurred, resulting in upward migration of these elements. Herein we present evidence that dissimilatory Fe(III)-reducing bacteria (DIRB) are capable of mobilizing As(V) from crystalline iron-arsenate minerals, as well as that adsorbed onto Lake CDA sediments. We also present geochemical and microbiological evidence indicating that microbial iron reduction occurs within this environment. In cell suspension experiments, the DIRB Shewanella alga mobilized As(V) bound to Fe(III) in the mineral scorodite (FeAsO_4· 2 H₂O) and to ferric oxides in whole Lake CDA sediments, coupled to the reduction of Fe(III). The oxidation state of the arsenic was not changed. Controls receiving heat-killed cells or live cells with no electron donor did not reduce Fe(III) or mobilize As(V), indicating that the mechanism of arsenic mobility was respiratory reduction of Fe(III) to which As(V) was bound. Geochemical evidence consistent with biotic iron reduction in Lake CDA sediments includes the following observations: (1) dissolved Fe(II) in pore waters reached concentrations as high as 41 mg L^-1; (2) the fraction of weak-acid extractable Fe(II) increased disproportionately with depth compared to total iron; (3) magnetite, a common reduction product of amorphous ferric hydroxides, was abundant, ranging from 8.5 to 15.5 g kg^-1 dry sediment. Microbiological evidence indicating the potential for biotic iron reduction includes the following: (1) most probable number (MPN) estimates of cultivable DIRB ranged between 10⁶ per g in the upper 0-15 cm of the sediments and 10⁴ per g at 15-30 cm; (2) purified bacterial isolates from Fe(III)-reducing enrichments were all capable of respiratory Fe(III) reduction. Altogether, our results indicate that DIRB have influenced the patterns of iron speciation in this environment, and suggest that these organisms may play a critical role in the post-depositional mobilization of arsenic.

Micheal Addition of Hydrogen Sulfide onto Juglone, A Naturally-Occuring Quinone. PERLINGER, J.A*; KALLURI, V Michigan Technological University, Department of Civil & Environmental Eng.

Under sulfate-reducing conditions, humic substances in subsurface environments can react with reduced inorganic sulfur forms through additions reactions. The additions under ambient conditions, of hydrogen sulfide onto, unsaturated carbonyl compounds that are found in the humics has been demonstrated in earlier studies in the laboratory. We have studied the Micheal addition of hydrogen sulfide onto 1,4-hydroxynaphthoquinone or juglone, a quinone found in many plants that facilitate electron transport and photosynthesis. In parallel experiments, we examined the reactivity of jugions with respect to transformations of polyhalogenated alkanes, which are found at numerous ground water contaminated sites. Electrochemically-reduced juglone was unreactive with respect to reductive dehalogenation of hexachloroethane. However, aqueous solutions containing juglone and hydrogen sulfide were reactive in reductive dehalogenation of the polyhalogenated alkanes including hexachloroethane. The increased reactivity in the latter system appears to be caused by the mercaptojuglone hydroquinone addition product, which is expected to have a lower reduction potential and higher nucleophilicity than the juglone hydroquinone. Products of the Micheal addition reaction were separated and characterized using ¹H and ¹³ C nuclear magnetic resonance spectroscopy, by X-ray chromatography-fluorescence detection. The structure of the product(s) observed and the influence of pH on the yield of the addition reaction will be presented. This study demonstrates the need to study the interplay between the biogeochemistry of a given subsurface environment and transformation of organic xenobiotic compounds in disturbed subsurface environments.

New Substances Notification Regulations - Biotechnology Products. D. H. CARON

Environment Canada, Québec, Canada

As of September 1, 1997, certain new biotechnology products, namely organisms, microorganisms and products of microorganisms (biochemicals and biopolymers), have been regulated under the New Substances Notification Regulations (NSNR) of the Canadian Environmental Protection Act (CEPA). Products for uses regulated under other federal Acts are exempted. Biotechnology products subject to the CEPA regulations must be notified to Environment Canada by the importer or manufacturer. The information provided is assessed by both Environment Canada and Health Canada to determine whether the product will or may have adverse effects on the environment or human health. This poster will describe what products are subject to the NSNR, the notification process and the risk assessment process.

Use of Microbially-Induced Calcite in Cementation of Geological Formations. Sookie S. Bang, V. Ramakrishnan, and Patrick R. Zimmerman. South Dakota School of Mines and Technology, Rapid City, SD

A novel technique of the selective microbiological cementation process has been developed to consolidate structural formations. In principle, a mineral precipitation of CaCO₃ resulting from metabolic activities of a soil microorganism, Bacillus pasteurii, is environmentally benign and persistent in the natural environment. Microbial urease enzyme catalyzes urea to produce CO₂ and ammonia and increases pH in surroundings to induce CaCO₃ deposition mainly as a form of calcite. As a microbiological sealant, calcite exhibits its potential to selectively cement highly permeable rock formations and even to fix cracks and fissures in subsurface areas of natural structures. Kinetics of microbial precipitation is compared with chemical precipitation of calcite. Direct participation of microorganisms in selective consolidation of porous media is examined by Scanning Electron Microscopy. Cementation profiles of CaCO₃ is quantified by X-ray diffraction analysis. The surface and subsurface remediation of microfissures in granite and concrete has shown a significant increase in the compressive strength of the material. Currently, attempts are being made to employ an immobilization technique that encapsulates microorganisms in polyurethane matrix to concentrate cells and to maintain active metabolic activities. The microbial precipitation of CaCO₃ also occurs naturally in soils and aquatic ecosystems. This process is likely to be accelerated in alkaline irrigated farmlands and in agriculturally influenced prairie potholes of the Northern Great Plains. The potential importance of this process with respect to the global carbon cycle and the potential for enhancing microbially-produced CaCO₃ formation as a strategy for atmospheric CO₂ sequestration are discussed.

Sources And Quality Of Dissolved Organic Carbon In The Shingobee River, Minnesota: Implications For Microbial Utilization. DUFF, J.H., AIKEN, G.R., JACKMAN, A.P., TRISKA, F.J. U.S. Geological Survey, Menlo Park, CA, U.S. Geological Survey, Boulder, CO, Univ. of California, Davis, CA, U.S. Geological Survey, Menlo Park, CA

DOC is an important source of organic energy in headwater streams. Primarily of terrestrial origin, it enters the channel in both surface runoff and groundwater. The aromatic content of DOC, influenced by terrestrial, aquatic, and subsurface processes, influences microbial utilization. The Shingobee River is situated on recently glaciated sediments draining forested and pastured uplands. It connects lowland aquatic features including lakes, wetlands, groundwater seeps, and beaver ponds, providing a complex array of DOC sources. DOC decreases from about 6 to 4.5 mg C L-1 over a 1400-m reach because of dilution by low DOC groundwater (2 mg C L-1) and in situ processing. Up to 88% of the DOC mass exported from the reach (2010 g C h-1) is supplied by the upstream fluvial system compared to 8% by groundwater discharge. An additional 4% is released from the streambed to the overlying water following leaching and microbial processing of organic matter. DOC mass flux from the streambed to the overlying water is 140 mg C m-2 h-1. The Specific UV Absorption coefficient of stream water, an indication of the aromatic content of DOC, does not change along the reach, and is significantly higher than groundwater (0.023 vs. 0.01 L (mg C-1) cm-1). CFC dating indicates that most groundwater is older than 45 years and, as an energy source, is highly recalcitrant. We conclude that the DOC released from the streambed is probably the most labile and thus functional energy source for microorganisms in the channel.

Patterns Of Dissolved Oxygen Concentration And Nitrification Rates Among Different Substrates In Prairie Streams. MELODY J. KEMP & WALTER K. DODDS. Division Of Biology, Kansas State Univ., Manhattan, KS

Preliminary data was taken to determine the role of shallow subsurface micro-sites in prairie stream nitrogen cycling. Substrates sampled included diatom mats, leaf and wood packs, filamentous algae, and fine benthic organic matter (FBOM). Diurnal spot measurements of dissolved O2 were taken at Konza Prairie Research Natural Area (KPRNA) using microelectrodes. These data were compared to nitrification rates of different substrata. Nitrification rates were determined by taking core samples in the field and using the N-serve method in the lab. Significant differences in O2 concentrations were found among the different substrata. Anoxic zones were found in deep pool sediment and litter packs. Filamentous green algae, leaves and wood had O2 concentrations near saturation and intermediate rates of nitrification. Diatom mats had the highest concentration of O2 and the highest rate of nitrification. Whole stream nitrification rates were estimated by using two methods. The first method used laboratory-derived rates that were weighted by the occurrence of each type of substrate. The second method determined nitrification rates by using the measured rate of 15NO3- production following 35 days of a whole-stream 15NH4+ release. These methods yielded nitrification rates of 0.64 and 0.65 mg N d-1 m-2 respectively. The oxic groundwater had only a moderate input of ammonium to the stream, suggesting that shallow subsurface ammonium generation is the primary source of energy for nitrifying bacteria in this system.

Evidence For Anaerobic Mineralization Of Lignite

KUEVER, J., DETMERS, J., SCHULTE, U., WEBER, A., STRAUSS, H. Max-Planck-Institute for Marine Microbiology, Celsiusstr. 1, D-28359 Bremen, Germany, Institute for Geology, Ruhr-University Bochum, Universitätsstr. 150, D-44801 Bochum, Germany

Lignite is generally considered to be recalcitrant to anaerobic microbial mineralization. Chemical, isotopic and microbiological results characterize a multiple aquifer system consisting of Tertiary marine sands and three intercalated lignite seams. These data provide strong evidence for the anaerobic mineralization of bituminous coal under in-situ conditions. Depth profiles of dissolved species across the aquifer system display a downward increase in inorganic carbon (DIC) paralleled by a decrease in sulfate and an increase in hydrogen sulfide. In addition, DIC is characterized by negative ?¹³C values around -17 ‰, indicating mineralization of organic matter as its source. Strongly positive ?³⁴ S values up to +50 ‰ for dissolved sulfate suggest sulfate reduction under closed system conditions. Dissolved hydrogen sulfide shows sulfur isotope values between -28 and +10 ‰. Both, hydrochemical and isotopic data are, thus, consistent with the recent activity of sulfate reducing bacteria. Quantification of bacterial abundance through standard microbiological techniques (MPN counts) display a maximum (1520 cells/gram sediment dry weight) for sulfate reducers and fermenting microorganisms at the transition from aquifer sand to lignite seam. Increasing rates of sulfate reduction in close proximity to the lignite with a maximum turnover of 4.16 mM SO₄^2- /l-1 * y-1 yields additional support. In summary, chemical, isotopic and microbiological results provide clear evidence for current bacterial sulfate reduction. The observation of maximum sulfate turnover in close proximity to the coal seam points strongly to the anaerobic mineralization of lignite.

The Modelling Of Geochemical Activity Of Methanotrophs ALEXANDER A. OBORIN, LJUDMILA M. RUBINSHTIEN, IGOR A. SELEZNEV, VADIM T. KHMURCHIK, GEORG G. ZERNIN A.A.Oborin, L.M.Rubinshtien, G.G.Zernin :Lab.of geological microbiology, Inst. of Ecology and Genetics of Microorganisms, Ural Branch of the Russian Academy of Sciences (IEGM UB RAS), Perm, Russia; I. A.Seleznev, V.T.Khmurchik : Lab. of technogenic ecosystem

The modelling of geochemical activity of aboriginal and pure cultures of methanotrophs under the conditions of regular supply of gas-air mixture with 5-10% of gaseous hydrocarbons was conducted. Variations of Eh and pH, rock magnetic sensibility, formation of carbonates, montmorillonite by feldspar grains and quartz grain corrosion were observed in test samples as compared to the control. Hydrocarbon gases in the absence of organic substance in rock were nearly completely oxidized to CO2. Carbonate enrichment in isotope-light carbon was detected which supported formation of secondary carbonates at the sacrifice of hydrocarbon microbiological oxidation. By the results of radioautography in experiments with «labelled» methane it was proved that methanotrophy appears to be the main coupling factor of methane in soil. Methane sorption was not observed in the control sample neither in pelitic nor in humus fractions of soil. The rate of methane oxidation by one cell was 0.002 ng C-CH4/day. An essential increase in ferruginization in the form of microaggregates of iron hydroxides of reddish-brown color which cloth the fragments of effusive rocks, biotite, hydromicas was observed. The microaggregates often displayed divergent, more rarely worm-like, arched or wedge-shaped structure of biogenic type.

Chemical and Mineralogical Characterization of Iron Carbonate Formed by Psychrophilic, Mesophilic, and Thermophilic Bacteria

Y. ROH, R. D. STAPLETON, C. -L. ZHANG, A. V. PALUMBO, J. -Z. ZHOU, T. J. PHELPS

Y. ROH, R. D. STAPLETON, A. V. PALUMBO, J. -Z. ZHOU, T. J. PHELPS, Oak Ridge Natl. Lab., C. -L. ZHANG, Univ. of Missouri-Columbia

Understanding biogenic Fe-rich carbonate formation by iron reducing bacteria may complement carbon management strategies in marine or terrestrial subsurface environments. The objective of this study is to identify environmental factors that control mineral formation by iron reducing bacteria. Psychrophilic (from Pacific Ocean sediments and Alaskan tundra), mesophilic (from lake and estuary sediments), and thermophilic (from deep subsurface) iron reducing bacteria were used to examine iron mineral formation in the presence of N₂, H₂ /CO₂ and N₂ /CO₂ headspace. These iron reducing bacteria exhibited diverse mineral precipitation capabilities including the formation of magnetite, maghemite, siderite, calcite, and sergeevite over wide ranges of temperatures (4 - 70 ^oC) and salinities (0.1 - 3%). The partial pressure of CO₂ and ionic species composition exhibited profound influences on the type of minerals in these anaerobic cultures. Minerals precipitated under a nitrogen atmosphere were predominantly magnetite. In the presence of 20% headspace CO₂, a mixture of magnetite, and iron rich carbonates such as siderite were formed. Results of this study also showed that the chemical milieu and species of bacteria affected the composition and morphology of iron minerals formed by bacterial iron reduction. The formation of iron carbonate and calcium carbonate by iron reducing bacteria may play a role in carbon management in subsurface and ocean sediments.

An Ecosystem Model For Predicting The Methane Flux Into The Archean Atmosphere J. S. SIEFERT and J. F. Kasting. Rice University, Houston, TX and Penn State University, Statge College, PA

Sulfate-Reduction in High-Temperature Oil-Field White Tiger of Vietnam ROZANOVA E.P.* ¹; BORZENKOV I.A¹; BELYAEV S.S. ¹; TARASOV A.L. ¹; SUNTSOVA L.A. ¹; IVANOV M.V¹; TIEN, H.D²; DONG T.L. ^{2 1}Moscow Institute of Microbiology Russian Acad. Sci., Russia; ²Vung Tau City, NIPI-Sea-oil-gas, Vietnam

Deposits of Miocene, Oligocene and Base content high-paraffinic Petroleum. Collectors are flooded with sea water with sulfate. Mineralization of stratal waters is 2-7 g per liter. Temperature of the strata ranges up to 150 degrees C, some areas are cooled to 50-90 degrees. Thermophilic SRB oxidizing water-soluble organic matter locate in water of oil-separation tanks, inner tube space, near-bottom zones of injection wells, and coproduced water. Oil-oxidizing thermophilic SRB mainly were distributed in oil phase. They were isolated in pure cultures and were shown to be able to destroy C₁₂ - C₁₆ n-alkanes. Distribution of SRB correlates with more heavy soluble S/SO₄^2- isotopic composition of coproduced and tanks waters (* ³⁴S from 19.9 to 29.9°/4 ). As compared with the same pumping and stratal waters ((* ³⁴S 19.6 and 15.2°/4 ) . The sulfur of sulfide (* ³⁴S from -0.2 to 11.2°/4 ) is more light than S/SO₄^2-. It was concluded that in some ecotops of White Tiger deposits the process of biogenic sulfate reduction have been started. This process is partly connected with oxidation of light paraffins. Concentration of sulfide in stratal gas increases from 3 to 130 ppm. Concentration of sulfate in sea water from oil deposits is less than in origin sea waters. Sulfate-reduction intensity measured in some samples of stratal water has values 0.02-19.94 mkg S per liter per day (radioisotopic method).

Characterization of cold-climate crude oil-degrading microbial communities S. Rowsell

The rate at which environmental contaminants are biodegraded is often assumed to follow the Arrhenius equation, where the rate will is halved with every 10E C decrease in temperature. This conventional reasoning implies that the activities of the microorganisms involved in bioremediation become relatively insignificant at cold temperatures. However, laboratory studies involving the bioventing of crude oil-contaminated soil columns have indicated the evolution of carbon dioxide at low temperatures, implying microbial activity. These preliminary studies have demonstrated that significant biodegradation continues to occur at temperatures as low as 5E C and that microorganisms likely play a major role in the mineralization of crude oil in cold environments. It is hypothesized that there are certain populations of microbes responsible for this elevated activity at low temperatures. These populations may be the same as those occurring at ambient temperatures or they may be populations unique to cold temperatures. It is also hypothesized that some of these microorganisms are capable of degrading crude oil at elevated rates, compared to other microorganisms. The purpose of this study is to characterize the microbial communities responsible for the degradation of weathered crude oil during bioventing applications in cold climates. Characterization of these communities will occur through the isolation and identification of pure bacterial cultures using the Biolog system, and the development of a rapid bioassay to measure the biodegradation capabilities of the oil degraders.

Thermophilic and Hyperthermophilic Bacteria and Archaea from Deep Subsurface Oil Reservoir of Western Siberia. E.A. BONCH-OSMOLOVSKAYA, N.A. CHERNYH, M.L. MIROSHNICHENKO, A.I. SLOBODKIN, T.G. SOKOLOVA, T.N. NAZINA, H. HIPPE, C. JEANTHON E.A. BONCH-OSMOLOVSKAYA, N.A. CHERNYH, M.L. MIROSHNICHENKO, A.I. SLOBODKIN, T.G. SOKOLOVA, T.N. NAZINA, - Institute of Microbiology, Russian Academy of Sciences, Moscow, Russia, H. HIPPE - DSMZ, Braunschweig, Germany, C. JEANTHON - Station Biologique, CN

Samples from 23 oil wells of Samotlor oil reservoir (1800-2500 m depth, temperature 60-84 oC, pH 6.8-7.0, salinity 1.8-3.2 %) were used for the inoculation of anaerobically prepared media with H2, acetate, peptone, sucrose or starch as energy sources and, if any, electron acceptors: sulfate, sulfur, thiosulfate, Fe(III). After the inoculation at in situ temperatures microbial growth of diverse thermophilic procaryotes was observed. Rod-shaped methanogens developed in most of samples at 60 or 70 oC. Organotrophic anaerobes appeared to be the most widely distributed group, developing in the temperature range from 60 to 85 oC. 10 strains have been isolated and partial or complete 16S rRNA sequences obtained. Isolates growing at 60-70 oC were found to belong to the genera Thermoanaerobacter, Thermosipho; and Thermotoga. Hyperthermophilic organotrophs were represented by bacteria (Thermotoga maritima) and three archaeal isolates, belonging to a new species Thermococcus sibiricus. Extremely broad temperature, pH and salinity ranges of growth were the most important phenotypic feature of this species. Lithotrophic (using molecular hydrogen) and organotrophic (growing on peptone) Fe(III)-reducing thermophiles were found in most samples studied. The ability to grow on molecular hydrogen via iron reduction was also found in all organotrophic strains isolated from Samotlor oil reservoir in the course of this study. Thus, finding of diverse thermophilic microbial community inhabiting Siberian oil reservoir and adapted to the in situ temperature and salinity supports the existance of deep subsurface biosphere.

Stimulation of Native Crude Oil Oxidizing Bacteriocenoses by Rhodococcus Biosurfactant Complexes IVSHINA I.B.1, KUYUKINA M.S.1, PHILP J.C.2 , CHRISTOFI N.2 1Institute of Ecology and Genetics of Microorganisms, Ural Branch of Russian Academy of Sciences, Perm, Russia2Napier University, Edinburgh, Scotland, UK

Ecological monitoring and the evaluation of populations of soil hydrocarbon oxidizing bacteriocenoses demonstrated a community of certain Rhodococcus species in areas of hydrocarbon accumulation from natural or anthropogenic sources. Two species, R. ruber and R. rhodochrous, were found to predominate (90-100%) in subsurface bacterial populations of oil and gas deposits. Representatives of R. erythropolis, R. opacus and R.

A New Petrotoga Strain Isolated from Oil Reservoirs under Microaerophilic Conditions

COMBET-BLANC Y.1, SELLIN S.1, PATEL B.K.C.2, MAGOT M.3, OLLIVIER B.1 IRD, Marseille, France1, Griffith Univ., Australia2, Sanofi Recherche, Labège, France3

From the point of view of biotechnology, phylogeny, and ecology, oil fields, because of their physico-chemical conditions, are an extraordinary subterrestrial ecosystem from which methanogens, sulfate reducers, and fermentative bacteria have been isolated. Amongst fermentative bacteria belonging to the five genera of the Thermotogales (Thermotoga, Thermosipho, Fervidobacterium, Geotoga, and Petrotoga), only Thermotoga, Geotoga, and Petrotoga strains have been so far isolated from oil field reservoirs. All Petrotoga and Geotoga species currently described do not share the extreme thermophilic nature of the Thermotoga and are considered rather as moderate to strict thermophiles with a range of temperature for growth between 30 and 65°C. The presence of Petrotoga species has been established in oil field reservoirs, very distant geographically, thus suggesting their ubiquity in these ecosystems. Recently, we isolated a new strain of the genus Petrotoga (strain LOMA 37) from a French oil well. The sequence analysis of the 16S rRNA gene showed a close phylogenetic relationship with Petrotoga miotherma (97% of similarity). The result of DNA/DNA hybridization between Petrotoga miotherma and strain LOMA 37 indicated 69% homology of the DNA. Despite Petrotoga species were described as anaerobic microorganisms, strain LOMA 37 was isolated under microaerophilic conditions. In contrast to Petrotoga miotherma, strain LOMA 37 exhibited a low fermentative activity on sugars. However, luxuriant growth was obtained in presence of yeast extract with hydrogen as the electron donnor and elemental sulfur as the electron acceptor, indicating that ATP was generated from hydrogen oxidation. Strain LOMA 37 can be considered aerotolerant to microaerophilic since the presence of oxygen, at a low partial pressure, stimulated its growth in a medium containing glucose as the energy source.

Acetogenic Bacteria From Stratal Waters Of Oil Deposits Olga V. Boiko, Sergey A.Ilarionov, Alexander A.Oborin O.Boiko: Lab. of geological microbiology, Institute of Ecology and Genetics of Microorganisms, Ural Branch of the Russian Academy of Sciences, Perm, Russia; S.Ilarionov: Lab. of technogenic ecosystems, IEGM UB RAS, Perm, Russia; A.Oborin: Lab

Microbiological processes in stratal waters of oil wells with various degree of mineralization were investigated. For the objects of investigation, the stratal waters of three oil wells in Churovsky, Moskudinsky and Kokuisky deposits of Perm Pre-Urals were chosen. The depth of stratum bedding comprised 1850,1600, and 1292, respectively. Stratal waters of oil wells are characterized by high degree of mineralization (over 200 g/l), and high sulphate content (over 440 mg/l). We did not manage to obtain extreme halophilous forms of acetogenes, but as the microbiological assay has shown, in samples of stratal water mesophillous halotolerant acetogenic microflora is presented that is able to synthesize acetic acid from the mixture of hydrogen and carbon dioxide. Of particular interest is the fact that both rod-shaped and coccal forms of acetogenic bacteria are detected in obtained enrichment cultures. Earlier investigations (Davidova-Charakchjan et al., 1992; Kotelnikova, Pedersen, 1997, 1998) supported the occurrence of acetogenic microflora in the underground ecosystems. However, solely rod-shaped forms were so far isolated from similar ecosystems. Of 40 present known forms of acetogenes, only two representatives have coccal form: Peptostreptococcus productus and Syntrophococcus sucromutans, isolated from the sludge of stratal waters and scar liquid, that are characterized by high content of organic substance. Apparently, the coccal form of acetogenic microorganisms being detected by us represents a new non-described species, which systematic fitness will be determined in further investigations

References:

Davidova-Charakchjan L.A. et al (1992) Microbiology 61 (2): 306-315.

Kotelnikova S., Pedersen K. (1997) FEMS Microbiol. Rev. 20 (3-4): 339-349.

Kotelnikova S., Pedersen K. (1998) FEMS Microbiol. Ecol. 26(2): 121-134.

Diversity and Activity of Microorganisms in Stratal Water of Daqing Oil Field and Their Biotechnological Potential NAZINA T.N.1, XUE YAN-FEN2 , WANG XIUYUAN2, NOVIKOVA E.V.1, GRIGORIYAN A.A.1, BELYAEV S.S.1 1 ã Institute of Microbiology, Russian Academy of Sciences, , Moscow, Russia

2 -Institute of Microbiology, Chinese Academy of Sciences, Beijing, 100080, China.

To-date, little information exists on microbiology and biogeochemistry of Chinese subsurface ecosystems. We investigated the number of microorganisms and rates of sulfate-reduction and methanogenesis in stratal water of the largest in China Daqing oil field. Physiological groups like aerobic saprotrophic and hydrocarbon-oxidizing, anaerobic fermentative, acetogenic, sulfate-reducing and methanogenic bacteria were determined with MPN-counts. Aerobic bacteria inhabited mostly near-bottom zone of injection wells, where their number reached 104-105 of cells/ml. In stratal water from oil-producing wells the number of aerobic and anaerobic bacteria didn't exceed 100 and 1000 of cells/ml respectively; rates of sulfate reduction varied from 0 to 3.6 More than 20 strains of aerobic bacteria have been isolated from Daqing oil field. To identify bacteria we tested their physiological properties, G+C content, partial 16S rRNA gene nucleotide base sequences. Many of the Gram-positive isolates fall into the genera Bacillus, Rhodococcus, Kocuria, Dietzia. The most detected Gram- negative genera are Pseudomonas and Acinetobacter. The isolates are also characterized by their capability to produce exopolysaccharides and surfactants in pure and mixed culture (artificial biocenoses). High biotechnological potential of stratal microorganisms was revealed. This research was performed in the framework of Russian-Chinese project and supported by the Ministry of Science and Technology of Russia and Ministry of Science of China.

Microbiological and Geochemical Investigation of Subsurface Ecosystem Subjected to Strong Technological Impact NAZINA T.N.1, IVANOVA A.E.1, IBATULLIN R.R.2, BELYAEV S.S.1, IVANOV M.V.1 1 – Institute of Microbiology, Russian Academy of Sciences, Moscow, Russia; 2 – Tatarstan Research Institute for Petroleum Industry, Bugulma, Russia

This work devoted to monitor of ecosystem of oil field subjected to technological impact. The use of the microbial enhancement of oil recovery (MEOR) method based on the introduction of molasses and fermentative bacteria resulted in a change in the structure of the bacterial cenosis, physicochemical and geochemical characteristics in the carbonate collector (oil bed 302) of the Romashkinskoe oil field. The cell number and activity of fermentative and methanogenic bacteria considerably increased in oil field. Due to the high initial content of sulfates and sulfate-reducing bacteria, sulfate reduction remained the dominant terminal process in the stratum, although, in certain periods of the technological treatment, its rate fell to zero values. The molasses injected was mostly consumed by the introduced and indigenous microflora; the metabolites formed – volatile fatty acids, lower alcohols, carbon dioxide, molecular hydrogen, surfactants – caused changes in the stratal water, gas, carbonate matrix, and oil. In the periods of the largest technological impact, variations in the isotopic composition of carbon compounds in the stratum were revealed. Isotopically light carbonates appeared in the stratum that formed from molasses, having a d13C value of –27.9…-28.50/00. The d13C of dissolved carbonates value decreased from –12.2 to –20 and even –28.30/00. The interaction of microbial metabolites (CO2, volatile fatty acids) with the carbonate matrix (having a d13C value of +0.4 to –4.00/00) resulted in its dissolution and release of isotopically heavy carbonates. Consumption of isotopically light carbon dioxide be hemolithotrophic methanogens led to a decrease in the d13C/CH4 value from –42.3 to –54.80/00. Oil bed 302 functioned as an integral ecosystem. After the cessation of molasses injection, the rate of biogenic processes decreased, confirming the possibility of their purposeful regulation.

Biosorption Of Metallic Cations By Bacterial Biomass. P. GINISTY- C.SAHUT- CEA ST Paul lez Durance FRANCE. French Atomic Energy Commission –Nuclear research Center of Cadarache - Department of Nuclear Waste Management - St Paul lez Durance - FRANCE

Sulfur-turf microbial mats in Japanese hot springs

K.KATO(1), T.KOBAYASHI(1), A.KATO(1), Y.MAKI(2), H.YAMAMOTO(3), A.HIRAISHI(4) AND H.CHIURA(5) (1)Shinshu Univ., Matsumoto, 390, Japan, (2)Iwate Univ., Morioka, 020,Japan, (3)St.Marianna Univ.Sch. of Med.,Kawasaki,216,Japan, (4)Toyohashi Univ. of Technology, Toyohashi, 441, Japan, (5)Intrl.Christian Univ.,Mitaka,181,Japan.

Sulfur-turf microbial mats, which are macroscopic white filamentous or bundles consisting of large sausage-shaped bacteria and elemental sulfur particles, occur in sulfide-containing hot springs in Japan. Since first reported by Miyoshi in 1887, this kind of microbial mat has been recorded for several geographically remote hot springs in Japan. The sulfur-turf mats generally develop within a temperature range of 50 to 75 C, within a pH range of 6 to 9, and at sulfide-oxygen interfaces in geothermal springs. However, no thermophiles from sulfur-turf mats have yet been isolated as cultivable strains. 16SrRNA sequencing suggested that the sausage-shaped large bacteria formed a major cluster with members of the Aquifex-Hydrogenobacter complex at the order or subclass level. We discuss similarity of the common clones isolated from different geothermal springs, their possible growth rate and physiological characteristics, and environmental control of their distribution in a given thermal microbial mat community.

Phylogenetic Characterization of Bacteria in Atlantic Coastal Plain Sediments at a Site in Virginia

CL BUEKER¹, GR DRAKE¹, MF DEFLAUN², S STREGER², and DL BALKWILL¹ (1) Florida State University and (2) Envirogen, Inc.

Samples of saturated and unsaturated Atlantic coastal plain sediments (mostly sands) were obtained from a study site near Oyster, Virginia. Sample depths ranged from 5 to 20 meters below land surface. Aerobic and facultatively anaerobic chemoheterotrophic bacteria were isolated from these samples by plating on R2A and dilute peptone-tryptone-yeast extract-glucose (1% PTYG) media. Representative isolates were then characterized by phylogenetic analysis of their 16S rRNA gene sequences, to obtain information on subsurface microbial community structure. The culturable heterotrophic communities in the sediments were quite diverse, representing six major taxonomic-phylogenetic groups: the high- and low-G+C Gram positive bacteria; the alpha-, beta-, and gamma-subdivisions of the Proteobacteria, and the Flexibacter-Cytophaga-Bacteroides group. Within these major groups, isolates were placed in at least 26 distinct genera. The most frequently detected Gram-positive genera were: Arthrobacter, Aureobacterium, and bacillus. The most frequently detected Gram-negative genera were: Agrobacterium, Burkholderia, Caulobacter, Pseudomonas, and variovorax. Two groups of isolates with distinctive physiological characteristics were also characterized phylogenetically: (i) strains that can degrade citrate under anaerobic conditions in the presence of nitrate and (ii) strains that can reduce Fe (III). All but four of the citrate degraders were strains of pseudomonas belonging to at least four species (and, possibly, two or three novel species). The remaining citrate-degrading isolates were found to be strains of Arthrobacter, Azospirillum, Chromobacterium, and ralstonia. The iron-reducing isolates included strains of bacillus (several isolates), Paenibacillus, Klebsiella, Enterococcus, and enterobacter.

Widespread Antibiotic Resistance among Bacteria Isolated from Subsurface Environments

GR DRAKE¹, MF DEFLAUN², S. STREGER², SB LEVY³, CL BUEKER¹, and DL BALKWILL¹

(1)Florida State Univ, (2)Envirogen, Inc., and (3)Tufts Univ.

More than 1,800 strains of bacteria isolated from two shallow (< 30 m below land surface) and three deep (> 200 m) terrestrial subsurface environments were tested for resistance to eight antibiotics: ampicillin (50 ?g/ml), erythromycin (30 ?g/ml), gentamicin (30 ?g/ml), kanamycin (50 ?g/ml), nalidixic acid (50 ?g/ml), rifampicin (50 ?g/ml), streptomycin (50 ?g/ml), and tetracycline (12.5 ?g/ml). Resistance was common among the isolates from all environments tested, with 85% of the total being resistant to one or more drug and 17% resistant to four or more. Multiple resistance was detected somewhat more frequently among isolates from shallow environments (38% resistant to three or more drugs; 5% resistant to six or more) than among those from deeper environments (25% resistant to three or more drugs; <1% resistant to six or more). Approximately 2% of the strains from shallow environments (but none of those from deeper environments) were resistant to all eight antibiotics tested. Resistance to tetracycline, ampicillin, and nalidixic acid was observed most frequently (59%, 50%, and 38% of the strains, respectively); resistance to the other drugs was less common (8-18% of the isolates). Isolates from shallow and deep environments displayed somewhat different resistance patterns. A larger proportion of the shallow isolates were resistant to ampicillin (52% versus 32% of the deep isolates) and erythromycin (21% versus 7%), whereas a larger proportion of the deep isolates were resistant to nalidixic acid (52% versus 36% of the shallow isolates). Phylogenetic analyses of 16S rRNA gene sequences for selected isolates placed antibiotic-resistant strains into six major phylogenetic groups: the high- and low-G+C Gram positive bacteria, the alpha-, beta-, and gamma-subdivisions of the Proteobacteria, and the Flexibacter-Cytophaga-Bacteroides group. The most frequently detected genera were Agrobacterium, Arthrobacter, Bacillus, Burkholderia, Caulobacter, and pseudomonas. These findings demonstrate an unexpectedly high frequency of antibiotic resistance among subsurface bacteria. These organisms may serve as reservoirs of known as well as yet unknown drug resistance genes.

Use of 16S rRNA-Targeted Oligonucleotide Probes and FAME Analysis for Community Structure Identification. N. L. DURAN and G. W. SEWELL

U.S. Environmental Protection Agency, Ada, OK

Interest of subsurface microbial communities has increased due to their recognized roles in geochemical processes as well as in biodegradation of groundwater contaminants. Thus, understanding of the microbial diversity and community structure composition of the subsurface will not only provide valuable scientific information regarding biogeochemical process but also information of practical use for in situ bioremediation technologies. The present study involves the use of 16S rRNA-targeted oligonucleotide probes and FAME analysis in the characterization of aquifer enrichment cultures and other subsurface samples. Previous studies established that the microbial composition of the original ground water samples were physiologically and phylogenetically very diverse with reductive dehalogenation activity. Probes S-F-Dsv-0687-a-A-16 and S-*-Dsb-0804-a-A-18 were used to identify the presence of type I and type II sulfate-reducing bacteria (SRB), respectively. However, since probe S-*-Dsb-0804-a-A-18 cross hybridized to RNA of Desulfovibrio gigas and homology of probe S-F-Dsv 0687-a-A-16 to iron-reducing bacteria has been recently reported, we tested other probes with higher specificity. These probes included DSV 407 and DSV 698 for the identification of members of the Desulfovibrionaceae family and DSB 985 for the Desulfobacteriaceae family. Hybridization of probe S-D-Arch-0915-a-A-20 to RNA samples suggested the presence of Archeae while probe S-G-Dsbm-0221-a-A-20 demonstrated the absence of the Desulfobacterium genera from the community. Additionally, FAME pattern analysis by the MIDI microbial identification system were performed and comparisons to the hybridization studies will also be presented.

Assessing the Prevalence of pDTG1-like Naphthalene Catabolic Plasmids in the Groundwater Microbial Community at a Coal Tar- Contaminated Site

A. M. Hohnstock and E. L. Madsen, Cornell Univ. Ithaca NY

Horizontal gene transfer may play a significant role in the adaptation of bacterial communities to environmental pollutants. Plasmids responsible for horizontal transfer of naphthalene catabolic genes between bacteria at a coal tar-contaminated site were found to be homologous to pDTG1 from P. putida NCIB 9816-4, which was isolated decades ago in Bangor Wales (Stuart-Keil et al. Appl. Environ. Microbiol. 64: 3633-3640, 1998). While the previous study focused on the bacterial community associated with contaminated surface sediments, the objective of the present study was to examine the presence of these plasmids in the contaminated groundwater bacterial community. Filter matings between the bacterial community native to contaminated groundwater and five different cured recipient bacteria (rifampicin-resistant and unable to grow on naphthalene) were performed. Groundwater bacteria (10⁷; approximately 10⁵ naphthalene degrading potential donors) were collected on site and mixed with 10⁹ cells each recipient. The matings proceeded overnight on a rich media plate. After plating on selective media, isolating retrieved plasmids, and characterizing them, we found that the filter matings retrieved two distinct but similar naphthalene catabolic plasmids closely related to pDTG1 (as determined by RFLP analysis and Southern hybridization to labeled pDTG1). These two plasmid types were also the only types previously recovered from the contaminated surface sediment. In order to document the distribution of these plasmids in the groundwater community, fluorescent in situ hybridization (FISH) techniques using a probe specific for the pDTG1-like plasmids are currently under development. FISH techniques have the potential to address plasmid ecology by assessing the prevalence of these mobile genetic elements in cells that comprise the groundwater microbial community.

Terminal Restriction Fragment Length Polymorphism (T-RFLP) Analysis to Characterize the Differences in Microbial Diversity between Groundwater and Sediment Communities Undergoing Bioremediation. K. D. LINNING¹ *, F. GEBHARD², M. J. DYBAS¹, T. L. MARSH¹, AND J.M. TIEDJE¹ Michigan State Univ., Ctr. for Microbial Ecology, East Lansing, MI 48824 and Federal Inst. of Biology, Biochemistry Division, Braunschweig, Germany 38104

Engineered bioremediation technologies are being conducted in Schoolcraft, Michigan at an aquifer contaminated with carbon tetrachloride (CT) from 30 to 80 feet below the ground surface. Bioremediation of the site requires the introduction of the denitrifying microorganism Pseudomonas stutzeri strain KC (strain KC) into the aquifer environment. Strain KC is the only known isolate capable of transforming CT to nonvolatile end products without the production of chloroform (CF). The requirements for rapid transformation of CT by strain KC include an iron limiting environment (established by raising the pH to approximately 8.2), the presence of an appropriate electron donor (carbon source) and an electron acceptor (nitrate), and a small factor secreted by strain KC. Environmental adjustments in recent field applications included raising the pH to approximately 8.2 and supplementing with acetate. A new bioremediation technology to be implemented at the site involves the use of Crisco? as a slow release carbon source. Continuous-flow microcosms simulating Schoolcraft site conditions are used to evaluate the efficiency of remediation and changes in the microbial community in response to acetate or Crisco? as a carbon source. Previous studies using Terminal Restriction Fragment Length Polymorphism (T-RFLP) analysis of 16S ribosomal DNA indicate gradual changes in the structure of the microbial community over the course of the bioremediation process when acetate is employed as the carbon source. The differences between both the cultivable versus total microbial communities sediment and groundwater are identified with T-RFLP. Evaluating the changes to microbial communities under field simulated conditions will aid in determining the optimal protocol for remediation.

The Influence of Chemical Environment and Spatial Separation in the Distribution of Microbial Communities in Anaerobic and Aerobic Zones of a Shallow Coastal Plain Aquifer

R. B. FRANKLIN, D. R. TAYLOR, AND A. L. MILLS

Univ. of Virginia, Charlottesville, VA 22903

Several ground water wells, penetrating a shallow coastal plain aquifer on the Eastern Shore of Virginia, were sampled, and overall genetic similarity between the communities of unattached microorganisms was estimated. Randomly amplified polymorphic DNA (RAPD) fingerprinting was applied to determine the genetic similarity of whole-community DNA extracts, and the results indicate that chemically distinct zones of the aquifer support different microbial consortia. The wells are located in a single 1.7 ha field that overlies distinct regions of aerobic and anaerobic ground water. Determination of the water table surface gradients suggests that the aerobic portion of the field is the water source for the anaerobe region. Several wells in each region were sampled in August 1997 and principal component and cluster analyses showed a clear separation of the microbial communities in the two zones of the aquifer. Within these zones, however, no relationship was observed between sampling distance and community genetic relatedness; the communities in a pair of nearby wells (i.e., 10 m) were as similar as a pair of distant communities (e.g., 140 m). To compliment the spatial analysis, two additional sets of samples were taken (June 1997 and January, 1998). Using a subset of the original wells, this analysis revealed the same general trend at each time, a clear separation between communities in the anaerobic and aerobic regions of the field. However, the specific relationships between wells within each zone did change with time and the magnitude and direction of these changes corresponded to concurrent changes in the groundwater chemistry at each well.

Horizontal Gene Transfer In Subsurface Microbial Community Under Heavy Metal Stress ZELENNIKOVA O. A., BILLMAYER J. S., PANCIERA M. A., and SMETS, B. F.

1,4: Microbiology Program, Dept. of Molecular and Cell Biology; 2,3,4:Environmental Engineering Program, Dept. of Civil and Environmental Engineering, Univ. of Connecticut

Horizontal gene transfer may be determinant in adaptation of microbial communities to changing environments. Our research aims to quantify and compare migration kinetics of genetic elements in subsurface microbial communities under different levels of heavy metal stress. To that effect, a set of detailed microcosm experiments is being conducted wherein the horizontal mobility of several type genetic elements is being monitored as a function of degree of heavy metal stress. The microcosms contain subsurface soils derived from a historically contaminated site. We are examining the extent to which soil microbial communities acquire a non-conjugal IncQ plasmid carrying czc marker genes via retrotransfer. The rates of conjugal transfer to the indigenous soil microbial community are being measured using the IncP TOL plasmid. Studies to date have revealed an abundance of soil isolates cross-resistant to Cd, Ni and Zn. Preliminary mating experiments indicated high incidence of horizontal transfer of heavy metal resistant genes in the isolates. Mating experiments are being conducted to distinguish self-transmissible and mobilizable gene transfer. Genetic fingerprinting of microbial communities in the soils is via amplification of 16S rDNA genes and computation of pair-wise similarity indices of resulting ARDRA patterns. Preliminary experiments showed substantial variability of ARDRA profiles among communities derived from different sampling locations at the test set. In the microcosms, the fate and migration of introduced plasmids is determined by whole community DNA hybridization with probes for all relevant genetic fragments (czcD, IncP, IncQ, xylE). We anticipate that our studies will provide quantitative insight in the significance of horizontal plasmid transfer as short-term adaptive response to heavy metal stress in subsurface microbial communities.

Abundance of subsurface microbial communities in the Tono area, central Japan MURAKAMI, T. IWATSUKI and T. NAGANUMA. Hiroshima Univ., Higashi-hiroshima, Japan; Tono Geoscience Ctr, Toki, Japan; Hiroshima Univ., Higashi-hiroshima, Japan

Abundances of total and selected microbial populations in the core and water samples from granite and sedimentary rocks up to 840 m deep were determined. Subsurface interstitial waters were collected from the geologically and geochemically well-characterized boreholes in the Tono area, central Japan, using a multiple piezometer-sampler system. The Tono area provides a good contrast of granite-dominated and sediment-dominated geology, and may serve as a good field for subsurface microbiolgy. We report the result of our 1-year pilot study in the Tono area. Maximum abundance (total counts) of 10^5 to 10^6 cells/ml was observed in the granite and sedimentary rock waters, respectively. Higher numbers (10^7 to 10^8 cells/g dry rock) were estimated for the core samples from other Tono boreholes by the phospholipid fatty acid analysis. By culturing the subsurface waters, iron-oxidizing/reducing bacteria were detected at the depths having specific pH-Eh regimes corresponding to the ferrous/ferrric or pyrite/ferrous interface conditions. Also, sulfate-reducing bacteria were detected at the depths having the Eh of -100 to -200 mV, which is generally regarded as optimum for sulfate-reduction. It is implied that these bacteria are involved in geochemical processes such as precipitation, dissolution and cycling of sulfur, iron and related elements.

Eukaryotic Microbes In The Granitic Äspö Groundwater, Sweden EKENDAHL, S.¹, THOMSSON, E.¹, AND PEDERSEN, K¹. ¹ Göteborg University, CMB – Microbiology, Box 462, SE – 405 30 Göteborg, Sweden.

Evidence Of A Deep Hydrogen-Driven Biosphere In Hard Rock Aquifers HAVEMAN, S. A., and PEDERSEN, K. Inst. for Cell and Molecular Biology, Microbiology, Goteborg Univ., Box 462, SE-40530 Goteborg, Sweden

Deep groundwater is thousands of years old and depleted in anaerobically biodegradable organic carbon. However, deep shield groundwaters can contain up to mM concentrations of hydrogen. Therefore, it has been hypothesised that subsurface microbial populations are supported by hydrogen-driven autotrophic primary production. Groundwater samples were collected from Fennoscandian Shield aquifers at 5 sites from depths of 60 to 950 m. Total cell numbers were on the order of 10⁴ to 10⁶ cells/ml. Culturable anaerobic microbes were enumerated by the most probable number (MPN) method. Media were prepared for each sample either by using groundwater as a base or by designing synthetic media based on groundwater chemistry. The MPN method resulted in culturing an average of 5% of the total cells, with up to 35% cultured. The predominant populations at all sites and depths were heterotrophs: iron-reducing bacteria (IRB), sulfate-reducing bacteria (SRB), and heterotrophic acetogens. Populations of IRB and SRB correlate with iron- and sulfur-containing fracture minerals at each site. Autotrophic acetogens growing with hydrogen and carbon dioxide were cultured at low levels from 4 of the sites from depths of 119 to 866 m. Autotrophic methanogens were also cultured from 2 samples from one site. One of these samples contained only culturable methanogens, but none of the other groups. Autotrophic capacity is evident at a wide range of sites and depths in Fennoscandian Shield groundwaters. The level of in situ activity of these populations, and their potential to support heterotrophic populations remains to be investigated.

Microbiological And Geochemical Description Of The Russian Superdeep Boreholes Core Samples

SERGEY A. ILARIONOV, ALEXANDER A. OBORIN, LEV A. PEVZNER. S. A. Ilarionov - Lab.of technogenic ecosystems, Institute of Ecology and Genetics of Microorganisms UB RAS,Perm, Russia; A. A. Oborin - Lab. of geological microbiology IEGM UB RAS, Perm, Russia; L. A. Pevzner- The Academy of Natural Sciences, The Verkhn

Microbiological and geochemical investigation of Vorotilovskaya, Kolvinskaya, Tyumen and Ural superdeep boreholes core samples has been carried out for same years. The core samples were taken out and transported with a special apparatus PPU-2 giving a possibility to take out, transport and keep core samples in sterile anaerobic conditions. Most studied core samples was taken from Ural superdeep borehole. Free and adsorbed gases were present in almost all samples: H2, CO2, CO, N2, CH4, C2H6, C3H8. Active fluid- and gas-dynamics were characteristic for most samples. The maximum depth where microbial activities were recorded was 5111 m in the Ural superdeep borehole and 6840 m in the Tyumen one. The depth of 6840 m can be supposed at present the lower bound of biosphere. The number of cells in the samples, as a rule, wasn’t more than 100 cells in 1 cm3 of the core sample. Microbiological analyses were carried out for presence of following functional groups of microorganisms: methanogens, sulfate and sulfur reducers, nitrate reducers, FeIII-reducers, hydrocarbon oxidizers, hydrogen users and acetogens. Practically no sample was sterile. Bacterial producing methane and sulfate reducing were recorded in most samples. Hydrocarbon oxidizers and nitrate reducers were not detected. The investigation was carried out at 30, 65 and 80o C. The best grown of the microorganisms was observed at 65o C. Therefore, most microorganisms detected belong to anaerobic thermophilic chemolithotrophs. Thus, this data show that in studied depths the microbiological processes bounded with organic and inorganic matter transformation are carrying constantly.

Comparison of culture- and non-culture- based methods for measuring abundance of methanotrophic bacteria in a deep basalt aquifer. M.S. WILSON*, R.M. LEHMANÝ, and F.S. COLWELL. *Humboldt State Univ., Arcata CA; ÝIdaho Natl Engineering and Environmental Laboratory, Idaho Falls ID

We are attempting to correlate the presence and abundance of methanotrophic bacteria with physical, hydrological, and geochemical properties of groundwater from the Snake River Plain Aquifer in southeastern Idaho, USA. The aquifer consists of a series of ydrogeologically complex saturated basalt flows and interbedded poorly consolidated sedimentary and pyroclastic deposits. Sampling wells were located in a pristine area and within a contaminant plume which resulted from the injection of significant quantities of chlorinated solvents, radionuclides, and sewage. The number of methanotrophs was expected to be low, and if present, to represent a small fraction of the total bacterial community. Replicates of serial dilutions of the extracted DNA were PCR-amplified in a semi-nested two-step PCR Most Probable Number Assay (MPN) using primers targeted at conserved signature regions of Type I and Type II methanotrophic 16S rDNA sequences. Culture-based MPN analyses were carried out in mineral salts medium supplemented with methane and scored for growth by turbidity, depletion of methane, and production of carbon dioxide. Using both approaches, methanotrophic bacteria were shown to be present in water from all three monitoring wells examined. Although methanotrophs are typically difficult to culture, results using the independent culture- and non-culture-based approaches were in close agreement. These values may indicate the actual numbers of methanotrophs present, or the independent methods might each have separate but equivalent biases. The PCR method suffered from difficulties associated with low biomass and low concentrations of target DNA sequences.

Diversity of TCE-degrading bacteria from a contaminated basalt aquifer as determined by enrichment culturing and PCR-DGGE

MOBARRY, BK, GRIFFITHS, EC, ELY, RL, and CRAWFORD, RL. Univ. of Idaho, Moscow, Idaho

Roles of Unique Martian Subsurface Environments in Microbial Energy Metabolism. B. C. CLARK

Lockheed Martin Astronautics, Denver, CO

Environmental factors at the surface-atmosphere interface on Mars appear to be quite inhospitable even to microbial extremophiles. The subsurface may, however, provide refuge from sterilizing UV light, extreme desiccation, low water activity, and pervasive highly-oxidizing free radicals and compounds produced photochemically from atmospheric gases. The only missing protective factor is a temperature regime conducive to the stability of liquid water. Overlying layers can provide needed thermal insulation to assure a biotic zone, once a thermal source is present. Sources of subsurface thermal energy include: magmatic intrusions, the lower layers of volcanic extrusions, buried heat associated with impact craters, and the natural geothermal gradient which slowly dissipates accretion energy and the heat from decay of buried radionuclides. A liquid-water zone will span various depths, depending on the characteristics of the heat source. This poses, however, an energetics dilemma for metabolism. Although photosynthesis cannot be direct, it is shown that many constituents of the martian atmosphere are out of thermodynamic equilibrium, allowing an

Accessing the Subsurface of Mars to Search for Life

R. L. Mancinelli and G. Briggs. NASA Ames Research Center, Moffett Field, CA

Data from the Viking Mission suggests that there is no extant life on the Martian surface. Research conducted on Earth has revealed that life can exist deep beneath the surface of a planet. Current data from Mars missions suggesting the presence of liquid water early in Mars’ history and mathematical modeling of the fate of water on Mars suggest that liquid water may exist deep beneath the surface of Mars. The existence of liquid water beneath the Martian surface leads to the hypothesis that life may exist deep beneath the Martian surface. Acquisition and analyses of Martian subsurface samples will shed light on the possibility of extant or extinct life on Mars, the processes leading to the origin of life, and the size of the Solar System's habitable zone. One requirement for sample acquisition compatible with planetary protection guidelines and with biological and chemical characterization include is that the sample be in the form of a core that can be sub-sampled rather than small cuttings or chips. For a mission to Mars the power, mass and volume of the drilling apparatus must be kept to a minimum. One attractive technique is an electrically heated probe that penetrates and cores by melting through rock. This approach eliminates the need for casing material, drilling fluids, and handling potentially contaminated debris from the borehole. The probe is attached to an umbilical cable to provide power and to allow periodic retrieval of the probe.

How bacteria fossilise: looking for subsurface fossil bacteria in extraterrestrial environments

F. WESTALL . NASA-Johnson Space Ctr, Houston TX

An understanding of the mechanisms of the fossilisation of bacteria is important to the search for fossil life on a planet such as Mars. Microorganisms can be preserved in the rock record in four ways: (1) by compression or as impressions in very fine-grained sediments such as shales, (2) by impregnation with silica (permineralisation), (3) by mineral replacement, and (4) as empty moulds in a mineral deposit (Westall, in press). Permineralisation and mineral replacement take place as follows (Westall et al., 1995): One of the components of the bacterial cell walls, peptidoglycan, contains a large number of functional groups, such as carboxyls, hydroxyls and phosphoryls, which are implicated in the chelation of mineral and metal ions. After chelation, the nucleating phase then dehydrates and polymerises in the shape of the microorganism, forming crusts and casts. There are more available functional groups in dead bacteria and the walls of Gram positive bacteria than in Gram negative bacteria. Gram positive bacteria are, therefore, more readily fossilised than Gram negative bacteria.

Evidence for Mineralized Bacteria in the Martian Meteorite Nakhla. D.S. MCKAY1, S.J. WENTWORTH2, F. WESTALL3, K.L. THOMAS-KEPRTA2, AND E.K. GIBSON, JR. 1. NASA Johnson Space Center, Houston, TX; 2Lockheed Martin, Houston, TX; 3NRC, NASA Johnson Space Center, Houston, TX. Nakhla, an igneous Martian meteorite which fell in Egypt in 1911, provides

and excellent opportunity to search for possible biogenic material because it is known that secondary minerals present in this meteorite are the result of low-temperature, aqueous weathering processes on Mars. Using optical microscopy and field emission scanning electron microscopy (FE-SEM) we find scattered occurrences of round to ovoid objects in chips of Nakhla, individual objects range from 0.2 - 1.5 micrometers in diameter. Some of them are found on surfaces formed by shock fracturing that occurred on Mars, but some are embedded within fine-grained Martian clay veins. We suggest that the round and ovoid features in Nakhla are possible mineralized bacteria for the following reasons: (1) uniform size: most are 0.5 -1.5 micrometers in diameter; (2) attachment: many are connected together in configurations identical to those of dividing bacteria; (3) surface texture: much like those of some mineralized terrestrial bacteria; (4) appendages: filamentous

material attached to some individuals have a similar appearance to appendages commonly found on terrestrial bacteria; (5) possible mineralized biofilms: webby or lacy material associated with the ovoids is much like mineralized terrestrial biofilm; (6) colonies: ovoids and spheres are found in colonies or patches, which are distributed sporadically in the rock. Most chip surfaces lack those features. Supposing that these features are indeed fossilized bacterial remains, then their planet of origin still has to be determined. Considering Nakhla's terrestrial history, it is hard to imagine that terrestrial bacterial would have had either the environment of the time that would be sufficient to invade, colonize, and fossilize.

Bacterial Mineralization Patterns in the Columbia River Basaltic Aquifers: Implications for Possible Life in Martian Meteorite AL84001. S.J. WENTWORTH1, K. THOMAS-KEPRTA1, D.S. MCKAY2, T.O. STEVENS3, A.E. TAUNTON4, C.C.ALLEN1, A. COLEMAN5, AND E.K. GIBSON, JR.2 1. Lockheed Martin, Houston, Tx; 2nasa/Johnson Space Center, Houston, Tx; 3batelle, Pacific Northwest Laboratory, Richland, Wa; 4univ. Of Wisc., Dept. Of Geology And Geophysics, Madison, Wi; 5. Brown Univ., Providence, R.I.

We examined organisms in experimental microcosms using scanning and transmission electron microscopy. These microcosms simulated natural growth conditions in the deep subsurface of the Columbia River Basalt (CRB). Four types of microorganisms were recognized on the basis of size, morphology, and chemical composition. The average size for these cells is about 1.2 micrometers in diameter, well within the range of terrestrial bacteria. Some of the organisms mineralized rapidly, whereas others show no evidence of mineralization. Many mineralized cells are hollow and do not contain evidence of microstructure. Filaments, either attached or no longer attached to organisms, are common. Filaments range in size from 0.02-0.2 micrometers wide and up to six micrometers in length. Filaments are generally longer and more narrow than the cells themselves, and some are also mineralized. The mineralized filaments are composed mainly of Fe and other minor elements including P. The presence of P indicates that these filaments are most likely biogenic appendages (e.g. prosthecae). Features strikingly similar in size and morphology to unattached, mineralized filaments from the CRB are recognized in Martian meteorite ALH84001. This work does not prove the biogenicity of the ALH84001 features. However, on the basis of the criteria of size morphology, an interpretation of the Martian features as mineralized, unattached cellular filaments appears reasonable.

Assessing Earth-based Contamination with Lipid Biomarkers in Martian Extraterrestrial Life Detection. WHITAKER, K. W.1, KERN, R. G.2, CHUNG, S.2, BURKHALTER, R. S.3, MACNAUGHTON, S. J.3, AND WHITE, D. C.2,3,4 Microbial Insights, Inc., Knoxville TN, 2- Jet Propulsion Laboratory , Pasadena, CA, 3-Univ. Tennessee, Knoxville TN, 4- Oak Ridge Natl. Laboratory, Oak Ridge, TN.

Detecting evidence of life on Mars in the 2008 sample-return mission will involve ultrasensitive detection of fossil biomarkers. The mission could be seriously compromised with forward contamination of the spacecraft and, particularly the sample cache, by terrestrial biomarkers. A rapid comprehensive and ultrasensitive detection method involves the assay of polar lipids by extraction/fractionation of the lipids, high performance liquid chromatography, electrospray ionization, and mass spectrometry. Lipid biomarker analysis provides the viable biomass and community composition, status at femtomolar sensitivity without isolation and culture of microbes. Aluminum coupons contaminated with microbes, spores, and human desquamated cells were subjected to cleaning/sterilizing procedures and the residual contamination determined. Witness plates from spacecraft assembly facilities were likewise analyzed for contaminant composition and viability. Quantitative detection of cell lysis is ascertained from the Polarlipid fatty acid to diglyceride ratio. Polar lipid fatty acid distribution reflects the contaminant composition.

Longitudinal and Vertical Variations in the Microbial Ecology of a Fractured Basalt Aquifer with Respect to a Contaminant Plume R.M. LEHMAN, F.S. COLWELL, R. SMITH, M. DELWICHE, S.P. O'CONNELL, J.K. FREDRICKSON, F. BROCKMAN, A.L. REYSENBACH, T. KIEFT, T.J. PHELPS, D.B. RINGELBERG, D.C. WHITE. INEEL, Idaho Falls, ID; INEEL, Idaho Falls, ID; INEEL, Idaho Falls, ID; INEEL, Idaho Falls, ID; INEEL, Idaho Falls, ID; PNNL, Richland, WA; PNNL, Richland, WA; Oregon Graduate Institute, Beaverton, OR; New Mexico Tech, Socorro, NM; ORNL, Knoxville, TN; CE

The microbial ecology of a basalt aquifer was characterized along two transects with respect to a contaminant plume - longitudinally along the plume axis and vertically within each corehole. The plume containing sewage, chlorinated hydrocarbons and radionuclides is the result of an injection well used for historical waste disposal at a Department of Energy facility in southeast Idaho. The aquifer consists of multiple basalt flows where dense fracture networks conduct the groundwater. Core was collected for microbiological analyses from the saturated zone in three coreholes from ca. 67 m below land surface (BLS) to 134 m BLS. One corehole (TAN-37) was located ca. 30 m from the injection well and is influenced heavily by the waste; the second hole (TAN-33) was located more distally (300 m) where the major contaminant is thought to be dissolved trichloroethylene (TCE) (ca. 500 ppb); and, the third hole (TAN-48) was located further yet (ca. 1 km) from the source with lower concentrations of TCE (< 200 ppb). Microbiological analyses conducted on pared core samples include biomass and community structure by phospholipid fatty acid (PLFA) analyses; cultural enumerations of methanotrophs, propanotrophs, phenol-oxidizers, ammonia oxidizers, iron reducers, sulfate reducers, methanogens; community-level physiological profiles (CLPP); 16s rRNA sequencing of PCR products and acetate mineralization. Data from intentionally introduced tracers (microspheres and perfluorocarbons) and indigenous microbial tracers indicated that several orders of magnitude reduction in potential drilling contaminants was achieved by paring of the cores. Microbial biomass in the basalt from the distal two coreholes (TAN-33 & 48) was at the limit of detection for most assays; minimal acetate mineralization was detected for selected samples. On the other hand, basalt from TAN-37, proximal to the injection well, showed measurable biomass by PLFA (ca. 3 pmol/g), much higher levels of acetate mineralization and positive enrichments for nearly all physiological types in all samples. In TAN-37, considerable vertical variability existed in the microbiology of the cores and correlations between vertical distributions of hydraulic conductivity and geochemical characteristics were performed. Vertically-averaged data from the longitudinal transect indicate that bacteria associated with basalt may be present in low numbers in pristine areas of the aquifer while a substantial stimulation of similar types of organisms may result from organic contamination. The variability in microbiological parameters along a vertical axis within a single corehole may rival that seen across a longitudinal gradient although the predictability of this vertical variation is more elusive.

A Facultative Anaerobic Co-Culture Capable of Using a Variety of Electron Donors and Acceptors Including Trichloroethylene. GRIFFITHS E.C., MOBARRY B.K., ELY R.L., CRAWFORD R.L. Univ. of Idaho, Moscow, ID, USA

Groundwater at Test Area North (TAN), located at the Idaho National Engineering and Environmental Laboratory (INEEL), is contaminated with perchloroethylene (PCE), trichloroethylene (TCE), dichloroethylene (DCE), radionuclides and sewage sludge. A TCE contaminant plume is located in a fractured basalt aquifer at a depth of 60-120 meters and is 3,000 meters long and 300 meters wide. An aseptically cored well drilled in the aerobic portion of the contaminant plume was used to obtain aquifer material, which was used to enrich for anaerobic bacteria capable of utilizing TCE as a terminal electron acceptor. We obtained a stable, facultative, anaerobic co-culture from the enrichments that is capable of degrading 1-2 mg/l of a 10 mg/l aqueous solution of TCE in about two days. The co-culture was enriched using lactate as the carbon source/electron donor and TCE as the electron acceptor. The co-culture is also capable of utilizing a variety of other electron donors including acetate, propionate, and hydrogen and other electron acceptors including oxygen, nitrate, sulfate, bicarbonate and a combination of ferric iron and humics. Two organisms comprising the co-culture were identified by polymerase chain reaction/denaturing gradient gel electrophoresis (PCR/DGGE). DGGE indicated that one of the organisms is present in relatively higher numbers than the other. Results of growth experiments will be presented for each of the carbon source/electron donor and electron acceptor combinations used.

Biostimulation of Reductive Dechlorination of TCE in a Fractured Basalt Aquifer. SORENSON, K. S., JR.; L. N. PETERSON; AND R. L. ELY Sorenson and Peterson: Idaho Natl. Engineering and Environmental Laboratory, Idaho Falls, ID; Ely: Univ. of Idaho, Moscow, ID

Test Area North (TAN) at the Idaho National Engineering and Environmental Laboratory is the site of a nearly 2-mile long trichloroethene (TCE) plume resulting from the injection of liquid waste directly into the fractured basalt of the Snake River Plain Aquifer during the 1960s. Historical ground water monitoring data suggest that co-disposed organic materials have provided an electron source to drive limited intrinsic reductive dechlorination in the vicinity of the former injection well. The potential for complete dechlorination of TCE by indigenous microorganisms at TAN was demonstrated through fed-batch bioreactor studies using basalt core collected from the plume as the inoculum. Rapid TCE disappearance accompanied by ethene and ethane generation was observed in bioreactors fed lactate. Accumulation of ethene and ethane was not observed in the controls. Based on the preliminary field and laboratory data, a 1-year field evaluation of enhanced reductive dechlorination through electron donor (lactate) addition was initiated in the fall of 1998. The field evaluation was designed as a 150-m long treatment cell between the former injection well and a downgradient extraction well. Following a baseline tracer test, sodium lactate addition began in the former injection well. Lactate and its breakdown products as well as chloroethenes, reaction products, electron acceptors, biological nutrients, tritium (a co-disposed compound serving as a conservative tracer), and redox conditions were sampled biweekly throughout the treatment cell. In situ instruments were also used successfully to monitor electron donor transport through the treatment cell almost continuously in selected observation wells. Less than two weeks after initiating lactate injection, dissolved oxygen, redox potential, and inorganic electron acceptor concentrations were observed to decrease significantly within 40 m of the injection well. In February 1999, after just 5 weeks of lactate addition, significant conversion of TCE to cis-1,2-dichloroethene was observed in the nearest observation well. Monitoring of the treatment cell is ongoing.

Vertical Variations in Composition for TCE Contaminated Groundwater at the Test Area North (TAN), Idaho National Engineering and Engineering Laboratory, Idaho. R.W. SMITH, F.S. COLWELL, R.M. LEHMAN, T.L. MCLING, J.P. MCKINLEY

Idaho National Engineering and Environmental Laboratory, Idaho Falls, ID, Pacific Northwest National Laboratory, Richland, WA

Understanding the vertical variations in microbial activity within a plume and its relationship to the distribution of contaminants is key to accessing natural attenuation or designing in situ bioremediations strategies. Because subsurface microbial processes often influence the chemical composition of groundwater, vertical composition variations can serve as indicators of zones with differing microbial activities. A 21-m vertical profile (40 cm resolution) of groundwater composition has been developed for the TAN 33 well located in the trichloroethene (TCE) plume at the Test Area North (TAN) of the Idaho National Engineering and Environmental Laboratory (INEEL), Idaho. Groundwater composition exhibits subtle but distinct variations that are correlated with geologic features (interflow zones, fractured flow-interiors, and unfractured flow-interiors) of the aquifer. Most distinct are variations in dissolved inorganic carbon (DIC) that may reflect differences in microbial activity between highly permeable interflow zones and less permeable flow-interiors.

Microbial Adhesion to Fractured Chalk from a Desert Aquitard and its Effect on the Fate of Organic Contaminants SAADI I NEJIDAT A RONEN Z

Blaustein Inst. For Desert Res. Ben-Gurion Univ. of the Negev, Sede Boker Campus 84990 ISRAEL 4990 ISRAEL

Where groundwater flows via preferential pathways such as fractures in low permeable rock matrix, contaminants migration is relatively fast with a potential that small volume of effluent might affect a large saturated area. In this study, the activity of bacteria adhered to chalk was investigated. Crushed samples of fractured chalk from a depth of 85 m. and which was transmitted and exposed to industrial effluents for more than 20 years from the were used. Organic effluents have percolated through the fractured chalk and contaminated a shallow groundwater aquitard. The compound 2,4,6-tribromophenol was selected as a representative contaminant. For the study a strain of 2,4,6-tribromophenol degrading bacteria designated as a Psedumonase TBPZ was used. The bacteria adhered rapidly to the crushed gray chalk and more than 96% of the cells adhered within 15 min. After 72h the bacteria were strongly attached to the chalk and no significant detachment was observed. The degradation of 2,4,6-tribromophenol by attached bacteria was comparable to the activity of a suspended culture. The information gathered in this study should be helpful in remediation of the contaminated fractured aquitered.

Critical Biogeochemical Parameters for Bioremediation of Solvents in Fractured Rock Aquifers. T. C. Hazen. Lawrence Berkeley National Laboratory, Berkeley, CA

In-Situ Determination of Microbial Biomass Using Single-Well Push-Pull Tests in Contaminated Aquifers

J.A. Field, K.M. Radakovich, J.D. Istok. Oregon State Univ. Corvallis, OR

A soil enzyme assay for glucosidase activity was modified for use in determining the in-situ microbial biomass of contaminated aquifers. Laboratory experiments were conducted to determine the relation between the hydrolysis rate of p-nitrophenyl-&#61538;-D-glucopyranoside (PNG) to p-nitrophenol (PNP) and microbial biomass. A range in biomass was obtained by filtering 0.5 to 3.0 L groundwater from a single groundwater well through 0.2 &#61549;m filters and placing the filters into solutions containing 40 mg/L PNG. The PNG hydrolysis rates were linear over 8 hr and linearly correlated with biomass. Rates of PNG hydrolysis also were determined for sediment cores collected in the vicinity of the groundwater well. In the field, the rate of PNG hydrolysis under in-situ aquifer conditions was determined using a single-well "push-pull" test. A test solution containing 40 mg/L PNG and 100 mg/L bromide was injected into a single well and then the test solution/groundwater mixture was extracted from the same well. The concentration of PNP was measured with time and a zero-order reaction rate was calculated from the field breakthrough curve. The rate determined from the field test was then used to determine the in-situ biomass using the correlation between PNG hydrolysis rate and biomass previously obtained for the groundwater collected from the test well. Assay reproducibility was determined from replicate push-pull experiments conducted in the same well. Spatial variability in PNG activity assay was determined by performing assays on groundwater collected from several wells located at a uranium-, BTEX-, and TCE-contaminated field sites.

Synchrotron FTIR Spectromicroscopy - A Chemical/Biological Probe for Monitoring Biogeochemical Processes on Geological Material Surfaces HOLMAN, H.-Y. N.; PERRY, D.L.; MARTIN,M.C.; McKINNEY W.R.; HUNTER-CEVERA, J.C. E.O.Laawrence Berkeley National Laboratory

Synchrotron Radiation-based (SR) Fourier transform infrared (FTIR) spectromicroscopy in the mid-IR region is a powerful chemical/biological probe to provide insights into the localization and progress of biogeochemical processes on surfaces of geologic materials. This hypothesis was tested by validating and demonstrating the use of SR FTIR to study the mechanisms of Cr(VI) reduction on basalt mineral and basalt rock surfaces. Time-resolved SR FTIR spectra indicated that microbial reduction of Cr(VI) to Cr(III) proceeded in at least a two-step reaction with Cr(V) compounds as intermediate products. The reduction reaction significantly increased during the concomitant biodegradation of a volatile compound (toluene). Analyses of spatially resolved SR FTIR spectra showed that maximum reduction of Cr(VI) to Cr(III) occurred on surfaces that were densely populated by microorganisms. The reduced Cr(III) state was confirmed by x-ray absorption fine structure (XAFS) spectroscopy. Both the time- and space-resolved SR FTIR spectra showed that Cr(VI) abiotic reduction was insignificant when compared to the microbial-mineral system. With this experimental effort, the usefulness of SR FTIR as a promising tool to complement existing techniques for studying the Cr(VI) reduction by specific clusters (microcolonies) of microorganisms on a mineral surface, and the potential of this technique in the study of other geomicrobiological phenomena have been demonstrated.

Biofilm:Porewater Ratios Of Microbial Numbers, Biomass And Activity Provide Useful Information On Nutrient Conditions Of Groundwater Ecosystems

GRIEBLER, C. & MINDL, B. Inst. of Limnology, Austrian Academy of Sciences, Gaisberg 116, A-5310 Mondsee, Austria

Biofilm:Porewater ratios of bacterial cell numbers and biomass seem to reflect the nutrient conditions of groundwater environments. Although the mentioned ratios increase with decreasing nutrient availability and vice versa, we found that these ratios provide useful information about short term changes of the nutrient conditions in the direct past. Experiments were carried out in groundwater model systems (flow-through and batch systems, supplied with natural groundwater), which were amended periodically (11 to 14 days periods) with an easy biodegradable organic carbon source (BDOC, i.e. leucine). Bacterial cell numbers, biomass and several activity parameters were followed for the biofilm and the planktonic porewater community. For the planktonic community the experiments showed significant differences between the flow-through model system, where the produced biomass was steadily transported out, and the batch model system with an increasing cell density, biomass and a higher proportion of active cells. On contrary the biofilm community of the flow-through system exhibited similar pattern as the batch system biofilm concerning cell density, biomass, bacterial secondary production and the percentage of active cells. The Biofilm:Porewater ratios for some tested microbial variables shifted up with the amendment of BDOC in the flow-through system, which were used to simulate natural waterflow conditions. Subsequently a remarkable high Biofilm:Porewater ratio indicates changed nutrient conditions in the direct past.

A Highly Sensitive Method for Tracking of Stable-Labeled Microbes for Bioaugmentation BURKHALTER RS1, FULLER ME2 and WHITE DC1,3, 1The University of Tennessee, Center for Environmental Biotechnology, Knoxville TN 2 Envirogen Inc., Lawrenceville, NJ 3 Oak Ridge National Laboratory, Environmental Sciences DivisionOak Ridge, TN

Augmentation with specific microbes could increase bioremediation effectiveness. Comamonas species with excellent transport properties was grown for several cycles on per 13C-labeled acetate as carbon source,

membrane components were extracted, the lipids fractionated, and phospholipids further purified by reversed-phase high performance liquid chromatography and identified online by electrospray ionization mass

spectrometry (HPLC/ESI/MS). Addition of basic modifiers resulted in the formation of negative ions of phosphatidylglycerol (PG) and phosphatidylethanolamine (PE) in the ratio of 1.6:1. Elevated cone voltages resulted in negative ion currents from fatty acyl constituents and the polar head group. Major fatty acyl constituents were per 13C-labeled 16:0, 16:1w7, Cyclopropyl 17:0, and 18:1w7. The major fatty acyl constituent, per 13C-18:1w7, provided a "window", devoid of possible interference with ion currents attributable to other fatty acids in their naturally occurring isotopic distributions. Initial work has established the detection limit of

less than 1000 cells based on the selected ion monitoring of the per 13C-labeled 18:1w7 acylated PG & PE "signatures." Current efforts are focused

on determining the stability of the 13C-label in the bacteria over time in subsurface microcosms. Improvements in 13C enrichment or miniaturization of

the HPLC system should result in even greater sensitivity. An HPLC/ESI/MS method also exists for the monitoring of fatty acyl constituents in diglycerides that will provide valuable information regarding the viability of strains injected during bioaugmentation activities.

Development and Use of a High-Efficiency RNA Isolation Protocol for Subsurface Sand

X. YIN* and F. J. BROCKMAN. Pacific Northwest Natl. Lab., Richland, WA

Increasing emphasis has been placed on the analysis of RNA from environmental samples to determine microbial community structure and activity. Due to small amounts of biomass present, it is difficult to isolate RNA efficiently from terrestrial subsurface sediments. We have developed a high-efficiency RNA isolation protocol for a vadose zone sand sediment. This simple and direct extraction protocol is a modification of the acid buffered, hot phenol extraction method of Lin and Stahl (Appl. Environ. Microbiol. 61:1348-1351, 1995). We evaluated properties of the lysis buffer (pH, SDS concentration, temperature, and different organic mixtures and reagents for deactivation of RNases), RNase deactivation before versus after physical lysis, and the method of physical lysis (freeze, bead-beat, boil, microwave, and combinations) and its duration. The modified protocol was used to detect rRNA in the equivalent of 0.2 to 3 mg of subsurface sand (ca. log6 to log7 indigenous cells/g) by hybridization to a non-radioactive universal probe. By using bacteria, eucarya, archaea, and reverse universal probes, the data indicated that the nucleic acids are predominantly RNA and that most of the RNA is bacterial. The modified protocol and a suite of group-specific phylogenetic probes were used to investigate the impact of different levels and combinations of chromate, nitrate, and supplemental carbon on vadose zone microbial community structure and activity.

Substrate Specific-Direct Viable Counts (SS-DVC) Identify Naphthalene Degrading Bacteria Within Microbial Communities C. BAKERMANS AND E. L. MADSEN Cornell Univ.

A microscopy-based procedure has been developed to identify cells in a naturally occurring groundwater microbial community capable of growth on naphthalene. Substrate specific-direct viable counts (SS-DVC) use naphthalene as the only added growth substrate; while typical DVC methods use a complex carbon source such as yeast extract. Substrate responsive cells are elongated because they grow while antibiotics inhibit their division. Pure cultures of Pseudomonas putida NCIB 9816-4 were used to develop the following method. Cultures were grown in 5% PTYG at 30 ?C, 250 rpm for 2 days. Cells are harvested by centrifugation, washed 3 times in PBS, resuspended in PBS, and starved by further incubation at 30 ?C, 250 rpm for 3 hours. Cells were concentrated on Isopore polycarbonate membrane filters and placed on plates of Stanier's Minimal Basal Salts medium containing antibiotics (20 mg/l nalidixic acid, 10 mg/l piromidic acid, 10 mg/l pipemidic acid, and 10mg/l cephalexin). Samples were exposed to vapors of naphthalene as appropriate. Following overnight incubation at 30 ?C, samples were fixed by immersion in 2% formaldehyde. Samples were stained with acridine orange and visualized by epifluorescent microscopy. After incubation with naphthalene, 44.0% of P. putida 9816-4 were elongated (substrate responsive); while only 0.7% of unamended P. putida 9816-4 were elongated. When SS-DVC was used on groundwater samples from a coal tar-contaminated site, naphthalene responsive cells were easily distinguished from unresponsive cells and debris. The concentration of naphthalene responsive cells was 347 ± 140 cells/ml (4.1% of the total cell count) relative to negative controls (P value less than 0.05). By using SS-DVC in combination with nucleic acid-based techniques, relationships between phenotypes and genotypes can be explored for individual members of naturally occurring microbial communities.

Characterization of the Bacterial Communities from Pristine and Contaminated Aquifer Sediment Using T-RFLP RILEY, M. S.1,2, MARSH, T. L.1,3

1Ctr. for Microbial Ecology, 2Department of Crop and Soil Science, and 3Department of Microbiology, Michigan State Univ., East Lansing, MI 48824

We have characterized the bacterial diversity in two different aquifer sediments using both culture dependent and independent approaches. High resolution comparative community analysis was performed with terminal restriction fragment length polymorphism (T-RFLP) of 16s rDNA. This approach detects restriction fragment length polymorphisms differing by as little as a single base pair. Sediments from a pristine aquifer on the Delmarva Peninsula (Oyster, Virginia) and from an aquifer contaminated with trichloroethylene (Oscoda, Michigan) were taken at several depths, chilled, and transported to the laboratory. The sediments were used to inoculate an MPN dilution series that was cultivated at 10°C. Community DNA was harvested directly from the aquifer sediment as well as from the MPN dilution series, PCR amplified, and profiled with T-RFLP. Up to 24 unique bacterial ribotypes (based on terminal fragment size) were identified in community DNA directly extracted from sediments and up to twelve different ribotypes were detected in the cultivable communities, suggesting that representatives of roughly 50% of the detected ribotypes could not be cultivated under the selected conditions. T-RFLP was used to evaluate the effects of TCE contamination on aquifer sediment communities as well as to assess community heterogeneity as a function of depth and geochemistry. In addition, bacterial diversity was characterized by sequence analysis of cloned PCR-amplified 16s rDNAs from representative samples.

Groundwater Microbial Diversity of a Porous Aquifer Revealed by Temporal temperature gradient ectrophoresis (TTGE) and Canonical Correspondence Analysis (CCA) Pierre Rossi (1), Michel Aragno (1), Ronald Kozel (2) 1. Univ. of Neuchatel, Microbiology Laboratory (LAMUN), Neuchatel, Switzerland 2.Univ. of Neuchatel, Hydrogeology Department (CHYN), Switzerland

The studied quaternary Seeland aquifer is located in the alpine foreland basin of NW Switzerland. Its total extent is about 50 km2 with a mean thickness of 25 m of sand and gravel. These sediments show an average hydraulic conductivity of 3*10-3 m/s with a high local heterogeneity (1*10-2 to 1*10-5 m/s). The groundwater chemistry is strongly influenced by 3 main factors: important river infiltration, intense agricultural land use and leachates from waste deposits of a sugar refinery. The dense observation network and the intense groundwater monitoring since the 1950th allows to delimit hydrochemical groundwater zones with dilution along the rivers, growing nitrate concentrations in groundwater flow direction and a large plume of reducing conditions downstream the waste deposits. The goal of this work is to assess the microbial diversity in relation to the different hydrochemical conditions, particularly under waste influence. 60 water samples were collected in Fall '98, covering an important part of the aquifer. Major hydrochemical parameters were analyzed in-situ and in the lab with a special attention to redox parameters, nitrogen compounds and pesticide residues (triazine compounds and metabolites).Total bacterial counts (DAPI counts) and DNA extraction were obtained from 1L water samples which were collected in a sterile way using teflon samplers. All samples were filtered through 0.2mm polycarbonate filters for DNA extraction. DNA was processed further to obtain PCR fragments of the 16S rDNA gene from the domain Bacteria. Cloning procedure and T-RFLP patterns were carried out to obtain a representation of the microbial diversity. These data were then pooled with physico-chemical data and analyzed using a canonical correspondence analysis (CCA). Analysis are still ongoing and results showing the relationship between microbial diversity and groundwater chemistry will be presented.

Assesment of Microbial Community Structure in Contaminated and Pristine Areas of Two Aquifers by Denaturing Gradient Gel Electrophoresis of 16S rDNA Sequences HARRINGTON, C. P., RICHARD, D. E., DWYER, D. F. Univ. of Minnesota, Minneapolis, MN

Environmental contaminants can produce changes in microbial community structure. These changes may include an increase in contaminant degraders and/or a decrease in overall community diversity and health. Applying biological remediation technologies to contaminated aquifers requires an understanding of these changes. Denaturing gradient gel electrophoresis (DGGE) can be used to assess contaminant caused changes amongst dominant members of the microbial community. DGGE is a rapid, culture-independent method that can separate 16S rDNA sequences into unique bands that represent individual microbial species. Using DGGE we are characterizing the structure of microbial communities within two contaminated aquifers and comparing them to the structure of their respective pristine surroundings. One aquifer is contaminated with polycyclic aromatic hydrocarbons (PAHs), and the other with trichloroethylene. First, bacterial community DNA is extracted directly from aquifer sediments. Variable regions of bacterial 16S rDNA are then amplified by the polymerase chain reaction and separated using DGGE. Also, using standard culture methods we have isolated bacterial strains that have the ability to degrade PAHs. Nucleic acid probing of DGGE separated bands for strains isolated on PAHs can elucidate how contamination effects the density of known PAH degraders. The number of bands in DGGE gels can be used to assess the effect of contamination on microbial diversity. DGGE in combination with nucleic acid probing provides more rapid and complete insight into microbial community structure than culture based methods. This information can then be used to assess the feasibility of using bioremediation to remediate contaminated aquifers.

Microbial Community Shift in TNT-contaminated Groundwater Monitored by Genetic Fingerprints and Principal Component Analysis (PCA)

P. WIKSTROM, L. HAGGLUND, M. FORSMAN

P. WIKSTROM and M. FORSMAN Department of Microbiology, Defence Research Establishment, Umea, Sweden. L. HAGGLUND Department of Chemistry, Defence Research Establishment, Umea, Sweden.

Diversity of Groundwater Protists in Relation to Environmental Pollution. NOVARINO, G.(1), WARREN, A.(1) & KINNER, N. E. (2)

1: Department of Zoology, Natural History Museum, London SW7 5BD, UK 2: Environmental Research Group, Department of Civil Engineering,

University of New Hampshire, Durham, NH, USA

The relationships between microbial diversity and environmental pollution have received scant attention. Even in those instances where these relationships have been investigated, results are conflicting and overall

this topic is poorly understood. The diversity (species richness) of protists from an organically contaminated aquifer on Cape Cod, Mass., has been studied for a number of years in laboratory cultures using a range of microscopical techniques. The observations suggest that diversity is higher in the contaminant plume than

in pristine, uncontaminated sites outside the plume. Despite the inherent limitations of the culture method, the observations raise interesting questions regarding the origin, mode of life, and long-term survival of groundwater protistan populations.

Response of Bacteria and Protists in a Pristine Aquifer to an Organic Perturbation N.E. KINNER, R.W.HARVEY, L.E.HINES, D. METGE, G. NOVARINO Univ. of New Hampshire, Durham, NH; U.S. Geological Survey, Boulder, CO; Weston and Sampson Engineers, Peabody, MA; U.S. Geological Survey, Boulder, CO; Natural History Museum, London, UK

Though numerous laboratory experiments have been conducted to study bacterial biodegradation of organic contaminants in aquifers, little data is available on how protists in the pristine subsurface respond to addition of contaminants. Our field injection studies involved addition of an organic carbon source (acetate) into a pristine sandy aquifer located on Cape Cod (MA, USA). The injectate solution contained 10 mg/L acetate and nutrients in ratios similar to those at the head of a nearby contaminant plume. Acetate was chosen as the organic carbon source because the state regulatory agency would permit its injection into the aquifer because it is highly biodegradable. The field injection was performed using a 61-cm injection well and two multi-level sampling wells 0.5 and 1.0 m downgradient. Every 3 days, 100 L of pristine groundwater were pumped out of a well 5-m upgradient and filtered to remove microorganisms. Nutrients, acetate and a chloride tracer were added prior to injection. Water samples were collected in the downgradient wells every 1 to 2 days for 16 days. Acetate was degraded at the 0.5 m downgradient well to <1 mg C/L with corresponding increases in the bacterial population (10³ /mL to 10⁶ /mL). The protistan population then increased in response to the change in the bacterial population. The data indicated that there is a fairly classical predatory:prey relationship between protists and unattached bacteria in the aquifer. As in other environments, flagellate grazing on bacterial populations likely impacts the rate of bacterial biodegradation of organic contaminants.

Ecology of bacterial and protozoan communities in an aquifer contaminated with monoaromatic hydrocarbons. B. ZARDA, R.G. MATTISON, A. HESS, D. HAHN, P. HOEHENER, AND J. ZEYER

Department of Soil Biology, Inst. of Terrestrial Ecology, Swiss Federal Inst.of Technology (ETH Zuerich),Grabenstr.3, CH-8952 Schlieren, Switzerland Zürich), Grabenstr. 3, CH-8952 Schlieren, Switzerland Zürich)

Bacterial and protozoan communities were examined in three sediment cores (A,B and C) from an aquifer at an abandoned refinery near Huenxe, Germany. Cores were removed along a transect through a contaminant plume containing a mixture of hydrocarbons dominated by xylenes (50-70%). These hydrocarbons could not be detected in the unsaturated zone in any core but were present in the saturated zones of core C (up to 42600 ?mol kg ^-1) of core material [dry wt.]) and in cores A and B (up to 190 ?mol kg ^-1). The number of DAPI-stained bacteria was found to increase from the less contaminated cores A and B (ca. 0.2 x 10⁸ cells g ^-1) of core material [dry wt.]) to the highly contaminated core C (2.4 x 10⁸ cells g ^-1). Proteobacteria of the ?-subdivision (which includes many sulfate-reducing bacteria) were the most common group detected by in situ hybridization (7-15% of DAPI-stained bacteria). The total numbers of protozoa and bacteria determined by in situ hybridization occurred in a ratio of approx. 1: 10³, respectively, which was independent of depth or core examined. Most-probable-number (MPN) analysis combined with a classification of the culturable protozoa revealed nanoflagellates as the major component of the protozoan community. Ciliates were confined to the surface layer (less than 1 m depth) in each core. Naked amoebae became increasingly more encysted with depth, except in core C where vegetative trophozoites were also present in the saturated zone. The co-occurrence of bacteria and protozoa in association with high concentrations of hydrocarbons suggests the involvement of trophic interactions in the process of biodegradation.

Colloid Transport of Cryptosporidium parvum in Sandy Porous Media. T. Harter, S. Wagner, E. R. Atwill Univ. of California, Davis, CA

In situ Assessment of the Transport and Microbial Consumption of Oxygen in Ground Water, Cape Cod, Massachusetts T. YOSHINARI*, R. L. SMITH**, J. K. BOHLKE***, K. M. REVESZ***, P. B. HATZINGER*, C. T. PENARRIETA**, AND D. A. REPERT**

*New York State Dept. Health, Albany, New York, **U.S. Geological Survey, Boulder, Colorado, ***U.S. Geological Survey, Reston, Virginia

Oxygen is a key ground-water constituent, controlling both the geochemistry and microbiology of an aquifer. Accordingly, aerobic respiration, the microbial metabolic process that consumes oxygen, is fundamentally important to the overall functioning of the aquifer. However, despite its significance, few studies have directly examined this process in the subsurface. This study has used several different approaches to investigate oxygen consumption on several different scales in parts of a large (> 5 kilometers) plume of dilute sewage contamination in a sand and gravel aquifer on Cape Cod, Mass. First, oxygen concentration profiles and stable isotope ratios were used to infer the net effect of aerobic respiration on the aquifer scale. Second, natural gradient tracer tests were used at an intermediate scale to measure in situ rates of aerobic respiration within different contours of the ground-water oxygen gradient. Third, two different types of laboratory incubations using aquifer core material, potential electron transport activity (ETS) and oxygen uptake activity, were used for small-scale examination of the process. The latter methods yield estimates of rates and kinetic parameters, which can be compared with the tracer test and isotope results. The sum of these approaches views the aquifer within the context of a subsurface ecosystem, integrating the combined effects of the hydrology, geochemistry and microbiology on the process of oxygen consumption.

Thermal Subsurface Factors influecne on Allochthonic Bacilli Survival Ways. N.V. SHEKHOVTSOVA, T.A. RODIONOVA, D. J. BRUMA, E.S. LITVINENKO Department of Microbiology, Yaroslavl State University, pr. Matrosova, 9, Yaroslavl, 150014, Russia

The growth and survival of slime producing bacteria capable to jam into underground concrete filters are studied under model circumstances. Pure culture of Bacillus sp. Isolated by glasses of colonization at 60⁰C from boring solution and thermal stratum water mixture out of Medyaginskaja borehole at the depth 2086-2095 m. Batch culture was grown during 20 days into mineral medium with 1 g/1glucose by varying of temperature (28, 40, 60⁰C) and solids nature (sand, Fe(III)-oxide). Bacteria growth continued 8-14 days in different experiments up which the starvation state had set in. It was revealed that under examinated conditions the spores formation was not enough to keep microbial population viability at high level. Spore number declined during starvation parallel with vegetative cells amount. It's rate was equal both in homogeneous and heterogeneous media at 28 ^oC and in a whole became less in the presence of solids at 40 and 60⁰C. The sand stimulated spores formation most evidently at 40 ⁰C . In this one spores number three times increased during starvation. In all cases solids intensified the cell division and at least didn't accelerate their death. So Fe(III)-oxide prolongated bacilli growth owing to immobilized cells activity (89%) until the end of observations at 60⁰C and inhibited three times specific death rate to 0.04 days^-1 at 40 ⁰C. Since the slime production was correlated with vegetation cells amount, it should be concluded that nutrient rich subsurface is favorable habitat for these usually soil bacilli.

Comparison of Allochtonic and Autochtonic Bacteria Isolated from Deep Aquifer of the Vorotilov Research Borehole N.V. VERHOVTSEVA, G.V. KONDAKOVA, O.M. RUTKOVSKAYA Department of Biology, Yaroslavl State University

The object of the research is the subsurface waters (SW) of the Vorotilov research deep borehole. The results of SW microbiological research from five depths: 1550m(t⁰ of the layer is 28⁰C), 2500 and 3800 m (60⁰C), 4500 (80⁰C) are presented in the work. Cultivable subterranean bacterial populations of carbon cycle were found at all depths investigated. They appear in number from 10⁰ to 10⁶ Bacterial/ml SW for such aerobic groops as saprotrophic, methylotrophs and hydrogen and hydrocarbon oxidizing bacteria, and also anaerobic groops such as SRB and methanogens. Saprotrophic bacteria and heterotrophic methanogens (HM) were groops dominant. Comparing these numbers on general selective media (with salt SW concentrations at depths investigated approximately 80- 130 g/l) confirms autochtonic nature of all groups investigated besides SRB. Disappearance of SRB on high SW mineralisation selected media was noted. It was also found the count data for methanogens (AM and HM) on SW selected media at depths 3800-4500 m more than they appear on general media. So autochtonic activity of autotrophic and heterotrophic carbon cycles bacteria was found for the Vorotilov SW populations. All cultivable autochtonic bacteria demands the use of SW selected media and SW temperature.

Bacterial growth and protistan grazing in a nitrate-contaminated, coastal sandy aquifer. KEUN-HYUNG CHOI, J. PAUL RICHARDSON, FRED C.DOBBS. Department of Ocean, Earth & Atmospheric Sciences Old Dominion Univ. Norfolk, VA

A series of laboratory incubation experiments was performed to investigate the effects of nutrients and temperature on bacterial growth in a nitrate-contaminated, coastal sandy aquifer, together with an experiment to assess protistan predation on unattached bacteria. The study area (DOE NABIR site, South Oyster, VA) is characterized by high concentrations of organic carbon and nitrate, but low microbial biomass. Growth of bacteria, as measured by incorporation of tritiated thymidine was little affected by a 5^oC increase relative to in situ temperatures. The addition of phosphate significantly increased thymidine uptake rates, but the addition of glucose had little effect, indicating the bacterial community in this environment is P-limited. Approximately 71%and 56% of exogenous thymidine and leucine, respectively, were incorporated into protein as determined by acid hydrolyses, indicating appreciable nonspecific incorporation of both precursors. Protists’ uptake and clearance of bacteria were 2.69 nl protist ^-1 h ^-1 (FLB cell volume ca. 0.23 micro m³) and 30.9 cells protist ^-1 h ^-1, respectively. These rates would remove 0.5 to 2.2 % of bacterial standing stock daily at the study site. The implications of these results will be discussed with respect to transport of bacteria through the aquifer

Bacterial Signalling Molecules As Regulators In Microbial Biofilms BACHOFEN R.*, SCHENK A., STETTLER R. Institute for Plant Biology, University of Zürich, Zollikerstr. 107, CH 8008 Zürich, Switzerland

Most natural or artificial surfaces are covered with microbial biofilms, e.g. skin, plant leaves and roots, rocks and concrete. As a consequence of the bacterial metabolism deterioration of the surface material starts. It is of great importance to know more on:

• the structural and organismic composition of the biofilms,
• the regulation of microbial growth and metabolism in biofilms,

Recently a variety of bacterial signalling compounds have been described. In Gram-negatives, N-acylated homoserine lactones act as autoinducers for many physiological inductions, mostly under conditions of high cell density (quorum sensing). Towards the end of the exponential growth phase they induce the transcription of organismic specific genes, such as bioluminescence in Vibrio fischeri or the synthesis of exoenzymes in Pseudomonas aeruginosa. High concentrations of microorganisms similar to stationary phase conditions are found in nature, specifically in microbial biofilms, bacterial mats and algal blooms. Screening for the presence of AHL in various biofilm materials, e.g. from various natural surfaces shows so far that homoserine lactones are detected in many systems where the biofilm is visible, but also on surfaces from leaves and roots of plants where only an invisible film is present. Homoserine lactone(s) are detected in such samples by using a bioassay with mutants in which the synthesis of the signal compound is deleted. Biofilms often form on surfaces under nutrient limited conditions. Such cells excrete extracellular polymers (EPS) which serve as a "glue" in biofilms. It has been recently shown that the signal molecules effect both biofilm growth and EPS formation, as well as detachment of the cells from the surface and induction of specific hydrolytic enzymes. AHLs must therefore play a crucial role in the regulation of biofilms.

Development Of A New Technique (ISSEM) For The Study Of Groundwater Biofilms Under In Situ Conditions MINDL, B., SLEZAK, D. & GRIEBLER, C. Inst. of Limnology, Austrian Academy of Sciences, Gaisberg 116, A-5310 Mondsee, Austria, birgit mindl@oeaw.ac.at

Studies about the development of biofilms in groundwater systems are scarce, and furthermore in most cases biofilm development were followed on artificial substrates (e.g. glass beads), exposed to water of groundwater observations wells or were placed in the hyporheic zones of streams. Recent investigations, including personal experiences, documented the tremendous differences between "well" water and "real" groundwater. Beside these, the ratio of microbial parameters (bacterial numbers, biomass, ...) between the porewater and the sediment surfaces (biofilm) may vary by a factor of 1:101 to 1:104. Therefore the investigation of the easy extractable interstitial water (free groundwater) alone is not anymore sufficient, and biofilms have to be considered more seriously. In order to avoid this discrepancy, a new technique was developed, which allows the exposition of sediments to real groundwater under In Situ conditions. The ISSEM (In Situ Sediment Expositions Microcosm) consists of a stainless metal cage (varying mesh size), which contains the sediment, exposed for microbial colonization. The cage is connected by a tube to an electric pump, which manages a moderate but permanent water flow from the aquifer through the exposed substrate up the observation well. The ISSEM may be introduced to any depth of multi level wells. To avoid the already discussed contamination with well water, the ISSEM has two rubber packing rings, which are inflated after introduction, in order to separate the microcosm from well water above and below. Well water which is enclosed with the exposed material, is immediately flushed out of the system through the exchange with real groundwater. The ISSEM technique allows regular subsampling of the exposed material and can further be easily combined with datalogger for automated recordings of several abiotic variables. First results concerning the development of biofilms on sterile sediments exposed to pristine and contaminated groundwater are presented.

Multiplication Of Heterotrophs In The Absence Of Organic Substratum Souses And Sporadic Variations Of Background Noise Of Spontaneous Mutagenic Into Local Areas E.coli Population. V. A.GUSEV. Sobolev Institute for Mathematics Siberian Division of Russian Academy of Sciences, Russia.

Cells of some heterotrophs after a certain preparation gain the capacity to multiply in media containing no organic substratum [1]. The final concentration of the cells is achieved for 1-3 days as a rule and thereafter quasistationary variations of viable cell number near stationary concentration K that equal to 2-5 million cells per ml are observed. Repeated re-seeding of populations grown to stationary state in new portions of water has shown that the concentration K is achieved in every re-seeding. Results of these experiments exclude a possibility of increase of population in indicated conditions through the use of cell biomass or cryptic substrate of cells. Presence of glucose in the water leaves population growth up to cell concentration K unaffected. However, unbalance of the media of molecular growth factors leads then to paradoxical situation: at small dozes of glucose less than 0.004 % in pure water population dynamics manifests itself in variation a viable E.coli cell number near stationary state K and a physiological dozes (0.2-0.4%) a peculiar shock and kill it [2]. Only dissolved atmospheric components could be the sources of nitrogen and carbon under conditions these experiments. Equilibrium concentration of carbon oxide dissolved in 1 ml of water is enough for biomass synthesis of one million cells per ml (nitrogen, hydrogen and oxygen present in large quantities). All other elements, including phosphorus, sulphur, sodium, potassium and other microelements can enter to the water by glass leaching. Thus, the material balance between the media and cells is not broken in process of the cell multiplication into distilled water. However, direct utilization of the carbon oxide by E.coli cells is impossible. We should notice that experimental results on E.coli cell multiplication and other heterotrophs under free substratum conditions need a critical analysis of traditional ideas about intra-cell metabolism of carbon substratum. The experimental results of investigation of the spontaneous mutations of E.coli cell genomes are presented. The areas of heightened number of mutant cells arise into population volume. Mutagenic process doesn’t stop throughout the all time of observation from a few hours to a few days. The number of mutant cells determined from the availability of resistance to antibiotics doesn’t arise though. Therefor variations of mutagenic background only are observed but the tendency for a increase of the background is lacking. 1.Gusev V.A., Bagayev S.N., Orlov V.A., Panov S.V. // 11th InternationalConference on the Origin of Life. Orleans, France, July 7-12, 1996. Book of Abstracts. P.107.2.Gusev, V.A., Orlov V.A., Panov S.V. // Biophysics (Rus), v. 43, N 4, p.p.746-750, 1998.

A Chromium VI Reductase Activity from Bacillus thuringiensis. N. NURHIDAYAT AND G.L. MARCHIN Division of Biology, Kansas State Univ., Manhattan, KS

A chromium VI resistant bacterial strain identified as Bacillus thuringiensis was isolated from the soil of an historic mining area in Southeastern Kansas. The chromium resistant isolate contains soluble NADH2 dependent chromium VI reductase activity. Cells of the isolate accumulate chromium containing, electron dense granules identified as an insoluble chromium III compound using energy dispersive X-ray spectrometry. The bacterial isolate does not grow anaerobically in the presence of chromium VI; thus, the function of the reductase activity appears to be detoxification. A 4.8 kb HindIII fragment from a 9.2 kb plasmid in the Bacillus strain was found to simultaneously confer Cr VI resistance and the reductase activity when cloned into Escherichia coli hosts. Subcloning from the Hind III fragment of a 1.2 kb Sac I fragment also yielded chromium resistant recombinants. Sequencing this 1.2 kb Sac I fragment has been completed. The termini of the sequence correctly identify the Sac I insertion site. Five open reading frames can be identified in the sequence. The largest, containing 803 nucleotides with the capacity to code for a protein composed of 269 amino acids, has a nucleotide sequence identical to that of the nucleotide sequence specifying the amino terminus of the transposase gene of the transposon Tn10. Curiously, the amino acid sequence derived from our cloned gene has a strong functional relationship to cytochrome P450 a molecule with the requisite enzymatic properties for chromium detoxification.

Life In Frozen Soil NÜSSLEIN, K., J. MCGRATH, AND J. M. TIEDJE Ctr. for Microbial Ecology, Michigan State Univ., East Lansing, Michigan

Recently, viable microorganisms have been recovered from Siberian Arctic permafrost soil samples after survival for 2-3 million years at low temperatures (-10 to -12°C) and low water activity. We investigated isolated permafrost microbes and permafrost soils themselves to see if they are metabolically active in situ as well as to examine any cold induced changes in cell composition. We assessed the use of the dye CTC (5-cyano-2,3-ditolyl tetrazolium chloride) as an appropriate test to determine metabolic activity. The red fluorescent precipitate of CTC-formazan formed during bacterial respiration allowed us to directly distinguish metabolically active bacteria from nonrespiring bacteria and abiotic material. The results established that the detection of metabolic activity using CTC reduction is possible in all of the conditions of interest to subsurface studies. These include: both gram-positive and gram-negative permafrost strains grown at both low (-4°C) and high temperatures, with and without high salt concentrations, in cells grown in liquid or on solid medium, and in assays done at low (-4°C) or high temperatures. Activity was also measured as CTC reduction in permafrost slurries held at -4°C. Control experiments show that only intact, living, and respiring cells exhibit CTC reducing activity. We will use laser confocal microscopy to determine metabolic activity in situ in permafrost soil samples in their original state. In addition, cold induced molecular adaptations determined for permafrost isolates, such as compatible solute formation and changes in fatty acid composition, revealed physiology adaptive for this extreme environment. The conditions for the prokaryotic community in permafrost soils resemble somewhat those proposed for extraterrestrial soils, namely severe cold, low water activity and age.

Shallow Aquifers Do Not Reflect The General View That Groundwater Habitats Are Stable Ecosystems

SLEZAK, D., GRIEBLER, C. & MINDL,B. Inst. of Limnology, Austrian Academy of Sciences, Gaisberg 116, A-5310 Mondsee, Austria

Groundwater systems are generally considered as stable ecosystems with little variations in abiotic and biotic parameters. In a recent study, a shallow aquifer (mean groundwater table at 3.3 m, sampling depth at 7 m below soil horizont) was monitored over a hydrological season and the bacterial density and activity were determined together with physico-chemical parameters. The rational was to investigate whether the system is characterized by a strong hydrological dynamic and how hydrology influences the physico-chemical factors and further the microbial community. In total, changes in the groundwater level and the temperature were moderate (almost 1 m and 1°C, respectively), but after periods of precipitation the groundwater level and the temperature changed rapidly down to 7 m within hours, indicating dramatic fluctuations in the environmental conditions for the microbial community on a short term base. Some of the chemical parameters, which were rather stable under general conditions showed a 2-3 fold increase or decrease after strong precipitation events (for instance dissolved organic carbon or sulfate). We further determined the bacterial density and the activity via the incorporation of radiolabeled leucine and thymidine and the dimethylsulfoxide (DMSO) reduction rate as a respiratory activity parameter. Bacterial production and respiratory activity varied considerably with no clear relation to seasonal changes. DNA synthesis, biomass production and respiraory activity showed individual pattern and did not always covary. Nevertheless some peaks in the bacterial activity coincidenced with rapid changes in the hydrological conditions and the chemical parameters. This study proves that shallow groundwater systems are characterized by a pronounced hydrological dynamic, which determines the physico-chemical conditions and has therefore – to some extend – a strong influence on the bacterial community and its activity.

Unusual Restriction Enzyme Profile of Bacillus spp. Isolated from Lake Baikal Water and Sediment Samples. V. REPIN1, TOROK, T.*2, M. I. KUZMIN3, and J. C. HUNTER-CEVERA2 1State Research Center of Virology and Biotechnology, VECTOR, Koltsovo, Novosibirsk region, Russia, 2Lawrence Berkeley National Laboratory, Berkeley California, and 3Institute of Geochemistry of the Russian Academy of Sciences, Irkutsk, Russia

Earlier work in our laboratory yielded unusual restriction enzyme profile information for many Bacillus spp. Basic and applied research importance of this finding justifies the use of the information in characterizing Bacillus spp. biodiversity. As part of an ongoing collaborative project, a number of Bacillus spp. was isolated from Lake Baikal water samples collected at depths down to 90 m, from hot springs surrounding the lake, and from sub-bottom sediment core samples obtained during the 1998 drilling of the international Baikal Drilling Project (BDP). (The latter project has been investigating the paleoclimatic history and tectonic evolution of the Lake Baikal sedimentary basin.) Following preliminary characterization of the strains, their biodiversity is being assessed based on the unusual restriction enzyme profile. Since this work is in progress, inter- and intraspecies diversity and ecological conclusions for the sampling sites will be discussed at the meeting.

Microbial Diversity in Lake Baikal Water and Sediment Samples as Determined by an Extensive Isolation Program TOROK, T.*1, V. REPIN2, V. GELETIJ3, and J. C. HUNTER-CEVERA1 1Lawrence Berkeley National Laboratory, Berkeley California, 2State Research Center of Virology and Biotechnology, VECTOR, Koltsovo, Novosibirsk region, Russia, and 3Institute of Geochemistry of the Russian Academy of Sciences, Irkutsk, Russia

Lake Baikal in Russia, the world's oldest and deepest continental lake lies in south central Siberia. It contains one-fifth of all the fresh water on Earth. Lake Baikal was formed when water from a number of rivers in the region flowed into crevasses that appeared in the Earth’s crust over 30 million years ago. Sampling this pristine ecological niche provides a unique opportunity in the search for microorganisms. In an ongoing collaborative project, over 200 strains were isolated from Lake Baikal water samples collected at depths down to 90 m, from hot springs surrounding the lake, and from sediment core samples obtained during the 1998 drilling of the international Baikal Drilling Project (BDP). The extensive isolation program used a selection of microbe-specific low-nutrient content media and incubation conditions. The isolated strains were preliminarily identified based on fatty acid methyl ester (FAME) analysis data. Some 20% of the strains seem to be novel microorganisms. Strains were preserved immediately after isolation and are maintained at –86ºC. Further screening will investigate the unique capabilities and/or novel biologically active compound production of the isolates.

Hydrogen Consumption by Hydrogen Oxidising Bacteria in Nuclear Environment.. M.F.LIBERT Inst. French Atomic Energy Commission –Nuclear research Center of Cadarache - Department of Nuclear Waste Management - St Paul lez Durance - FRANCE

To characterise the long term behaviour of nuclear waste in a deep repository, the effect of several parameters need to be studied. These parameters concern chemical effects, radiolytic effects, mechanical properties, water composition, and microbiological activity. To evaluate microbial activity in such environment, we focuses our work on an inventory of key nutrients (C, H, O, N, P, S) and energy sources required for bacterial growth. In this way, the production of hydrogen in the environment of nuclear waste leading to the development of hydrogen oxydising bacteria, needs to be evaluated. In deep repository, several sources of hydrogen are investigated such as :

-Radiolysis of water.

-Corrosion of metal containers.

-Radiolysis of embedding matrices such as bitumen.

In each case, kinetic values of hydrogen production are given and bacterial activity is measured. Bacteria are using hydrogen as the sole energy source with oxygen or nitrates as electron acceptors :

:

Enzyme Activity-Dependent Probes for Trichloroethylene (TCE) Degradative Pathways in Bacteria. A.R.MILLER(1,2), M.E.KAUFFMAN(1,2), W.K.KEENER(2), F.F.ROBERTO(2), M.E.WATWOOD(1), P.K.LINK(1); (1)Idaho State University, Pocatello, ID, (2) Idaho Natl. Engineering and Environmental Laboratory, Idaho Falls, ID

Environmental bacteria can be screened for enzymes capable of degrading trichloroethylene (TCE) by exposing them to specific chemical substrates, which form colored and/or fluorescent products when biotransformed by the enzymes of interest. We have identified suitable probe substrates for two TCE-degrading enzymes, toluene ortho-monooxygenase (TOM) and methane monooxygenase (MMO).The soluble form of the methane monooxygenase enzyme, sMMO, has been shown to cometabolically oxidize several classes of industrially and environmentally important substrates, including aliphatic and alicyclic hydrocarbons and chlorinated compounds. Only some methanotrophic species exhibit sMMO activity, and the enzyme is inhibited by certain growth conditions, such as elevated copper levels. Currently, sMMO activity can be estimated by gas chromatography, which requires technical expertise and expensive equipment, or by the naphthalene oxidation technique, but this assay is only linear in a narrow range and relies on an extremely unstable colored product. Therefore, a fluorescent probe that provides a specific, sensitive, and quantitative estimate of sMMO enzyme activity is desirable for numerous environmental and industrial applications. We have screened numerous compounds for fluorescent products due to sMMO enzymatic conversion. One substrate that appears to be oxidized by sMMO to a fluorescent product is coumarin. The production of the fluorescent oxidation product, 7-hydroxycoumarin, appears to be directly dependent on sMMO activity. Currently, the limits of linearity, inhibition, and rates of the reaction are being determined. To enhance the quantitative function of the probe, fluorescence is being correlated with methanotrophic cell numbers and sMMO activity (naphthalene oxidation assay). Burkholderia cepacia G4 catabolizes toluene via the TOM (toluene ortho-monooxygenase) pathway and is also capable of cometabolizing TCE. We have identified 3-hydroxyphenylacetylene (3-HPA) as a potential probe substrate, which undergoes conversion by TOM to yield a fluorescent product. Toluene and phenol have been shown to induce TOM activity in Burkholderia cepacia G4. Initial experiments have consisted of growing Burkholderia cepacia G4 cells in the presence of toluene or phenol, trapping them on black polycarbonate filters, exposing the trapped cells to 3-HPA, and examining them for fluorescence under an epifluorescent microscope. Control experiments consist of growing G4 cells on succinate, which does not induce TOM activity, exposing them to 3-HPA in the same manner, and examining the cells for fluorescence. G4 mutants, blocked at various steps of the TOM pathway, as well as bacteria with other toluene catabolic pathways, have also been analyzed for comparison.

Improvement of Organic Solvent Tolerance of Escherichia coli by Overexpression of Stress-Responsive Genes via Activity of the AcrAB-Tolc Efflux Pump R. AONO Department of Bioengineering, Faculty of Bioscience and Biotechnology, Tokyo Institute of Technology, Nagatsuta 4259, Midori-ku, Yokohama 226-8501, Japan

Hydrophobic organic solvents can be toxic to microorganisms. Bacteria have some defensive systems for organic solvents, and these systems are enable bacteria to grow in the presence of such solvents. Magnitude of the defence ability by the system is likely strain-specific because each bacterium shows the intrinsic level of organic solvent tolerance. The organic solvent tolerance level is determined genetically in Escherichia coli. We previously constructed mutants from E. coli, of which organic solvent tolerance levels were improved. The mutants were defective in marR gene encoding a repressor for mar operon responsible for multiple antibiotic resistance of E. coli. We show that overexpression of marA, rob or soxS gene improves organic solvent tolerance of E. coli. Gene soxS is responsible for superoxide resistance. MarA, Rob and SoxS are transcriptional activators belonging to AraC subfamily, and activate expression of sox-mar regulon genes responsible for various environmental stress factors. The regulon includes acrAB and tolC coding for AcrAB-TolC efflux pump. Mutant that is defective in acrAB or tolC shows extremely low level of organic solvent tolerance. The pump contributes to maintaining the intrinsic tolerance of E. coli.

Use Of Short-term, Single-well Injection Experiments To Study In Situ Nitrite Production In A Sand and Gravel Aquifer. BAUMGARTNER, LK, SMITH, RL, and *MILLER, DN U. S. Geological Survey, Boulder, CO, *U. S. Department of Agriculture, Clay Center, NE

Subsurface microbial processes such as nitrification and denitrification are usually very slow and are estimated using long-term incubation assays at the field site or in the laboratory. However, the greater sensitivity afforded by new analytical techniques, such as chemiluminescence analysis of nitrite, enables in situ studies of nitrite turnover in groundwater in much shorter time periods. We utilized this technique to study nitrite production and consumption with short-term, single-well injection experiments in a nitrogen-contaminated sand and gravel aquifer on Cape Cod, Massachusetts. Separate experiments were conducted in two zones, (i) a microaerobic, nitrifying zone where oxygen and ammonium coexist, and (ii) an anoxic, denitrifying zone of high ammonium concentrations. Groundwater from these zones, having natural concentrations of nitrite (20 nM) and controlled concentrations of oxygen (15-76 ?M) and ammonium (10-169 ?M), was amended with bromide tracer (1.6-2.4 mM). The amended groundwater was injected into selected vertical horizons of the aquifer via multi level sampler. Samples were taken from the injection well for up to 96 hours after injection, initially at one-hour intervals and increasing to three-hour intervals. Rapid nitrite increases were observed in nitrification zone experiments, with peak nitrite concentrations (up to 680 nM) occurring well after the bromide cloud had moved past the injection port, suggesting that the increase is probably due to nitrification. Added ammonium (40 ?M or twenty-four fold above background) in the nitrifying zone caused a six-fold increase in nitrite concentration, demonstrating that in situ nitrification was ammonium limited. Concomitant nitrite decreases were evident in experiments in the denitrification zone. Overall, short-term, single-well injection experiments proved to be an acceptable alternative to long-term tracer tests for studying nitrogen fluxes in situ in groundwater.

Use of Species-Specific DNA Amplification for Identification of Rhodococcus Species and Their Detection in Environmental Samples. KUYUKINA M.S.1, BELL K.S.2, IVSHINA I.B.1, PHILP J.C.3, CHRISTOFI N.3 1Institute of Ecology and Genetics of Microorganisms (IEGM), Ural Branch, Russian Academy of Sciences, Perm, Russia, 2Scottish Crop Research Institute, Dundee, Scotland, UK, 3Napier University, Edinburgh, Scotland, UK

Molecular techniques based on species-specific amplification of 16S rRNA gene using polymerase chain reaction (PCR) have been developed for the specific ex-/in-situ identification of Rhodococcus species isolated from a wide range of environments including shallow and deep subsurface geological material containing hydrocarbons. The Rhodococcus strains used were obtained from the Regional Specialised Alkanotrophic Micro-organism Collection IEGM, Perm, Russia; the National Collection of Industrial and Marine Bacteria (NCIMB), Aberdeen, UK; the Collection of the Institute of Microbiology, Belorus Academy of Sciences (INMIB), Minsk, Belorus and isolates from various environments. Primers were designed for 5 Rhodococcus species (R. erythropolis, R. globerulus, R. opacus, R. rhodochrous and R. ruber). PCR was carried out on DNA extracted both from pure bacterial cultures and directly from environmental (e.g. soil) samples. A high percentage similarity in the identification of rhodococci using PCR analysis, and, classical phenotypical (92.5%) and immunochemical (98.7%) methods was demonstrated. The PCR techniques developed were successfully applied to the identification of 28 bacterial isolates and to culture collection Rhodococcus sp. The strains submitted as Rhodococcus sp. IEGM 743 (NCIMB 9741) and IEGM 744 (NCIMB 9784) were identified as R. ruber, and, Rhodococcus sp. IEGM 745 (NCIMB 13261) as R. erythropolis. Culture collection strains listed as R. erythropolis IEGM 246 (INMIB DSS-7), IEGM 247 (INMIB DSS-31), IEGM 248 (INMIB 29F), IEGM 249 (INMIB DSS-29), IEGM 261 (INMIB 7-F), IEGM 262 (INMIB 14-F), IEGM 263 (INMIB 70-F), IEGM 264 (INMIB DSS-14) have now been changed to R. opacus. R. rhodochrous IEGM 656 (NCIMB 11273), IEGM 657 (NCIMB 11277), IEGM 759 (NCIMB 13259) were changed to R. ruber. The PCR based techniques were successfully used to detect the species R. erythropolis in natural and spiked soil.

Reporter Construct For Use With Dissimiulatory Sulfur Reducing Bacteria. S. TECHKANJANARUK, G. GEESEY. Montana State Univ., Bozeman, MT

Evaluating the activities of a specific bacterial population within a community of microorganisms growing on mineral surfaces offers a window to micro-scale processes that are important to bioremediation. We have recently modified the broad host range plasmid pDSK519 of the IncQ group and successfully transferred it into the sulfur reducing bacterium Desulfovibrio desulfuricans G20. The plasmid contains a constitutively expressed green fluorescent protein (gfp) gene under the control of the kanamycin promoter, Pnpt2, which causes the plasmid-containing cells to autofluoresce. Individual cells containing this plasmid can be distinguished from other non-plasmid-containing cells and surrounding sulfide deposits when viewed by epifluorescence microscopy. A derivative of this plasmid is presently being constructed in which gfp is under the control of a dissimilatory sulfite reductase (dsr) gene, allowing the evaluation of its expression among individual members of a population under different environmental conditions. These plasmids should facilitate the establishment of micro-scale relationships between biological sulfur reduction and other biogeochemical processes on mineral surfaces.

Functional Analysis of Two Chromosomal Virulence Genes, chvA and acvB, of Agrobacterium tumefaciens Using Avirulent Mutants with A Transposon 5 Insertion in Respective Gene

Parimal MAJUMDER, Kenichiro TAKAGI, Hidenari SHIOIRI, Masayuki NOZUE AND Mineo KOJIMA

Shunshu Univ., Faculty of Text. Sci. & Tech., Dept. of Applied Biol., Tokida 3-15-1, ueda, Nagano 386-8567, Japan

A mutant (M3 strain) of Agrobacterium tumefaciens was isolated by transposon (Tn5) mutagenesis. The M3 strain was avirulent on all host plants tested; Daucus carota, Solanum tuberosum and Kalanchoe pinnata. Avirulent phenotype of M3 strain was shown to be due to an insertion of Tn5 in chvA chromosomal virulence gene. Using this M3 strain (chvA::Tn5) and two other strains, B119 strain (acvB::Tn5) and A208 strain (parent, virulent), the functions of chvA and acvB chromosomal virulence genes of A. tumefaciens were analyzed. When M3, B119 and A208 strains were co-cultured with tobacco BY2 suspension cultured cells in the presence and absence of acetosyringone (As), efficient T-DNA transfer from Agrobacterium cell to nucleus of tobacco cell occurred only with A208 strain in the presence of As. However, T-strand (an intermediate form of T-DNA for transfer) was produced normally in all three strains in the presence of As. Analyses with tricine-SDS-polyacrylamide gel electrophoresis and electron microscopy demonstrated that pili, which are assumed to mediate T-DNA transfer to host cell, were not produced on cell surface of M3 strain in both media with and without As. On the other hand, perfect pili were produced on the cell surface of A208 strain in the presence of As. On the cell surface of B119 strain, imperfect pili which were thinner than ones of A208 strain were produced in the presence of As. Taken together these results, chvA and acvB likely play roles in virulence through influence on formation of pili through which T-strands are transferred to host cells.

Are VBNC Organisms a Significant Reservoir of Reactive Biomass In The Rock. SP O'CONNELL, RM LEHMAN, ME DOWNING, DB RINGELBERG AND FS COLWELL. Idaho Natl. Engineering and Environmental Laboratory.

Basalt cores were collected from two locations in the Snake River Plain Aquifer to evaluate the potential for intrinsic remediation of a trichloroethylene (TCE) plume. Dilution series of crushed basalt were plated on R2A solid media to estimate numbers of aerobic heterotrophic bacteria and also added to liquid minimal salts media (MSM; with no dissolved organic carbon) for long-term enrichment. In core samples from the upgradient site nearest the source of TCE and other organic contaminants, immediate plating onto R2A showed high levels of CFU (ca. 10^6) and good morphological diversity (ca. 6 morphotypes). Core samples from the downgradient location produced no growth upon direct plating onto R2A; however, after prolonged enrichment in MSM with no soluble carbon, subsequent plating showed a relatively high (4 to 6 morphotypes) diversity of culturable morphotypes. Most-Probable-Number estimates of these organisms in the original core material range up to 10^5 cells/g. Total biomass in the upgradient cores ranged up to 20 pmol/g PLFA which could be largely accounted for by the number of culturable organisms while biomass in the downgradient cores was below the limit of detection (1 pmol/g). Isolations were performed on solid media from samples plated before and after MSM enrichment for one upgradient core and two downgradient cores. Fatty acid methyl ester (FAME) profiles for these isolates were produced using the Microbial Identification System (MIS) and comparisons were made between the groups of isolates. While several of the isolates recovered From the MSM enrichment of the upgradient core were identical to those recovered by immediate plating, there were two new isolates that were not previously seen. In the downgradient cores, the appearance of 4 to 6 organisms following MSM enrichment can be compared to 0 isolates seen after immediate plating. In general, the isolates recovered after MSM enrichment are dissimilar to organisms seen in other samples (e.g., groundwater) from this site and were unknown to the MIS database. While it has been demonstrated that low-nutrient media is useful to maximize recovery of aerobic heterotrophs from oligotrophic subsurface environments, we found that pre-enriching with MSM supported the recovery on solid media of an even greater diversity of isolates. This work demonstrates that in addition to the use of multiple solid media or low-carbon media, other

Trichloroethylene Biodegradation in Large Undistributed Columns of Fractured Weathered Shale in East Tennessee M. LENCZEWSKI, L. D. MCKAY, A. LAYTON Univ. of Tennessee, Knoxville, TN 37996

A plume of trichloroethylene (TCE) contaminated groundwater was detected at the Oak Ridge Reservation (ORR) in eastern Tennessee adjacent to shallow waste trenches in a highly weathered and fractured shale. Monitoring wells at the site indicate a downgradient decline in concentration of TCE and the appearance of its daughter products (dichloroethylene, vinyl chloride, ethylene, and methane). This suggests that anaerobic and possibly aerobic biodegradation of TCE is occurring and is the first documented case of natural attenuation of TCE in fractured shale. A research program is currently underway to investigate factors influencing biodegradation of TCE in these materials. The study is being performed using large undisturbed column of fractured and weathered shale from a nearby uncontaminated site. The experiment involves slowly pumping groundwater containing dissolved phase TCE (100ppb) through the undisturbed soil column for one year. This is intended to simulate condition downgradient from the waste trenches or from a TCE spill. The influent and effluent is being monitored for TCE, daughter products, and microorganisms. Conventional geochemical and microbial techniques, as well as molecular techniques are being used to investigate TCE biodegradation. The microbial community structure at the contaminated site was examined using 16S rDNA analysis; this data will be used for comparison to the charges that occur in the column and assess the shifts in microbial community structure with the exposure to TCE. This research will help develop a better understanding of the potential for TCE biodegradation in these widespread soils.

Microbial Respiration of Oxygen in Granitic Ground Water. KOTELNIKOVA S and K, PEDERSEN

Gothenburgs Univ., Department of Cell and Molecular Biology, Microbiology, Box 462, 40530 Gothenburg, Sweden

Safety assessment of high level radiation nuclear waste disposal considers the maintenance of redox stability of the engineered barriers. Rates of microbial oxygen consumption followed with carbon dioxide production were measured and could be inhibited in Äspö Hard Rock Laboratory ground water. Oxygen uptake rates were reproducible in different ground water and with time. Oxygen consumption exponentially depended on time. The ground water and granite surfaces contain abundant and diverse microbial populations which include not only anaerobic organisms as could be expected in the reduced environment, but also facultative aerobic and microaerophilic organisms. The microbial mineralisation of carbon sources in the presence of oxygen and respiration in the ground water were measured in vitro at conditions close to the granite surroundings. Substrates such as methane, hydrogen, acetate and formate were actively metabolised in the presence of oxygen. The ground water contains the energy giving gases hydrogen and methane. The structure of the mixed population depends on the energy sources available. We showed that hydrogen and methane-utilising biota is widely distributed and responsible for oxygen consumption in oligotrophic Äspö ground waters. Methane oxidation is one of the dominant processes in microbial oxygen consumption. Methanotrophs react up to 80% of the available methane The results on microbial community structure, activity and respiration was used for modelling of oxygen uptake in situ.

Transport of Viral and Bacteria Sized Colloids in Granite Fracture Zones. K. KENNEDY(1), S. NIEHREN(2), P. MARSCHALL(3), W. KINZELBACH(2) & T. FIERTZ (4). (1)Hydrogeology Ctr. CHYN/Univ.Neuchatel, Neuchatel,CH (2)ETH-Inst.Hydromechanik & Wasserwirtschaft,Zurich,CH (3)NAGRA-Natl. Coop. Storage of Radioactive Waste, Wettingen,CH (4)Solexperts SA.,Zurich,CH

Subsurface colloid migration through fracture porosity can differ considerably from granular porous media yet is particularly critical to hazardous waste site-selection and wellhead protection programs. Colloids intensively attach to mineral surfaces resulting in smaller concentration along advective flow paths. Different colloids are being used to assist characterize shear-zone transport and retardation mechanisms and effective porosity quantification at a high altitude Swiss underground research site. Two colloids (bacteriophage H40, carboxylate latex microspheres) and a solute (uranine) were injected into a 40 cm long core at 18 ml/hr for 36 hours followed by formation water for 8.5 days. On-line uranine and colloid particle (microsphere) data, updated every minute, guided sampling. Both tracer types eluted together 1.8 hours after injection. The solute 'plateau' began at 20 hours and continued during tracer injection. In contrast, both colloids 'peaked' and began decreasing 14 hours prior to ending injection. Recovery and C/C0 maximums were 90 and 82 percent for uranine and 15 to 20 and 25 to 45 percent for the phages and microspheres, respectively. Bacteriophage and microsphere relative breakthrough was 17 and 22 percent. In the field, a 30 day pilot transport test was completed in 1999. Napthionate and dissolved helium migrate over a distance of 1.2m under injection/extraction 'dipole' conditions with a flow rate of 1 ml/min. Tracers arrived at 12 hours, had double peaks at 20 and 30 hours with 30 and 40 percent total recovery at 5 and 19 days, respectively. The core particle and field pilot tests were done to provide planning input to this year's bacteriophage and solute migration tests over greater fracture lengths. Biocolloid responses from 1999 field tests will be presented and compared to results from testing to date.

MICROANALYSIS OF PYROXENE, FELDSPAR, AND SILICA GLASS IN ALH84001; M.S. Bell^1,2, K.L.Thomas-Keprta², and S. J. Wentworth²; and D.S. McKay³, ¹University of Houston, Houston, TX 77058, ²Lockheed Martin, 2400 Nasa Rd. 1, Houston, Tx, 77058, ³NASA/Johnson Space Center, Houston, TX 77058.

Fine-grained magnetite crystals, in the Fe-rich rims of carbonate globules of martian meteorite ALH84001, have been proposed as fossil remains of primitive Martian organisms (McKay et al., 1996). We report observations on the size and shape distributions and chemical compositions of magnetites from ALH84001 and compare them to biogenic and inorganic magnetite crystals of terrestrial origin. Magnetite is found in a wide variety of terrestrial rock types and in meteorites, but none has been reported with the specific and unique characteristics of magnetites produced by various kinds of magnetotactic bacteria. We suggest that magnetite in ALH84001 carbonate globules might be explained by low temperature, inorganic and biogenic processes. A significant fraction of magnetite has a pure chemical composition, unique morphology, and length-to-width ratio that are indistinguishable from a variety of terrestrial biogenic magnetite but distinct from all known inorganic forms of magnetite. Unless an inorganic analog for these crystals is found, the presence of elongated prismatic magnetite crystals associated with the carbonate globules in Martian meteorite ALH84001 must be considered as strong evidence for primitive life on early Mars. Although high temperature mechanisms have been suggested for the formation of whisker-shaped magnetite in ALH84001 (Bradley et al., 1998), we suggest that all ALH84001 magnetite likely formed in the presence of low temperature fluids.

1. McKay, D.S., E.K. Gibson, Jr., K.L. Thomas-Keprta, H. Vali, C.S. Romanek, S.J. Clemett, X.D.F. Chillier, C.R. Maechling and R.N. Zare (1996) Science 273, 924-930
2. Bradley, J.P., H.Y. McSween, and R.P. Harvey (1998) Meteoritics and Planetary Science 33, 765-773.

Antarctic Subsurface Permafrost - Model for Methodology Development in Astrobiology

E.Vorobyova, V.Soina, A.Mamukelashvili, D.Zvyagintsev, D.Gilichinski, L.Polanskaya

Soil Biol. Dpt.,Moscow StateUniv.,Inst.Fundament.Biol.Problems Rus.Acad.Sci.

Tracking Subsurface Bacterial Transport: Comparing Multiple Detection Strategies. W.E. HOLBEN, W.P. KOVACIK, AND T.C. GSELL The Univ. of Montana, Missoula, MT

Numerous studies have been performed which required the ability to monitor the transport of microorganisms through subsurface media under conditions of saturated flow. These have typically employed stained bacterial cells, radioactively labeled cells, plating approaches, or DNA-based detection strategies. However, it is not always feasible to employ radioactive labels or organisms that are genetically engineered or otherwise modified to facilitate monitoring. For example, in situ experiments being performed in a shallow East Coast aquifer require organisms used to be indigenous to the aquifer, and not genetically modified, radioactively labeled, or resistant to clinically important antibiotics. To underpin these experiments, we have developed a novel monitoring technology based on altering the stable isotopic signature of bacterial cells to facilitate their specific and sensitive detection in environmental samples. Advantages of this approach are its high sensitivity, quantitative capabilities and ability to monitor an unmodified organism in the presence of the native community including "self". Potential concerns include label turnover and that it is largely untried under field conditions. To assess the feasibility and utility of this approach we conducted a series of transport experiments with intact and repacked cores containing sediments from the field transport site and employing multiple bacterial detection strategies simultaneously. These experiments compared detection based on direct microscopic enumeration, plating on culture media, quantitative PCR and radioactive and stable isotopic labeling. The results suggest that both microscopic counting and plating techniques suffer from high non-specific background and typically result in underestimation of the number of organisms of interest present. However, microscopic enumeration has value in reliably and directly detecting peak concentrations of transported cells when above background community numbers. The PCR-based detection strategy holds promise for rapid and quantitative monitoring of

specific populations but is hampered in that it also detects the indigenous "self" that is present in the native community when non-engineered organisms are used. The isotopic tracking techniques appeared to be superior in their detection capability, specificity and sensitivity since they specifically detect added labeled organisms in the presence of unlabeled "self". Our data further indicate that cells that are isotopically enriched using acetate as substrate retain label very well and that the sediments effectively absorb lysed cell parts (i.e. only intact bacteria are transported). Thus, isotopic label recovered in aqueous phase samples appears to truly represent added and still intact bacteria. Additionally, stable isotopic labeling can be employed where use of radioactivity is not feasible.

Mechanisms of Bacterial Retention in Subsurface Porous Media Revealed by Phosphor and Electron Imaging DONG, H1, ONSTOTT, T.C.1, DEFLAUN, M.F.2, FULLER, M. E.2, GILLESPIE, K.M.2, STREGER, S. H.2, and MAILLOUX, B. J.1

1: Princeton Univ., Princeton, NJ; 2: Envirogen Inc., Lawrenceville, NJ

Bacterial transport experiments were conducted using intact sediment cores (7 x 50 cm) collected from sites on the Delmarva Peninsula near Oyster, VA. The sediments consisted of quartz and feldspar sand with minor amounts of Fe and Al hydroxides. Two indigenous groundwater strains, Erwinia herbicola OYS2-A and Comamonas sp. DA001, which had relatively low adhesion, were labeled with either ¹⁴C or ₃₅S. OYS2-A is a motile, gram-negative rod (1.6 x 0.5 micron) with a strongly hydrophilic membrane, a neutral charge and percent adhesion to site sediment of 54%. DA001 is a nonmotile, gram-negative rod (1.2 x 0.6 micron) with a similar hydrophilicity, a neutral charge and percent adhesion of 33%. A finite pulse of bacteria was injected into the cores. After breakthrough was observed, the core was split, and one half was analyzed using scintillation counting to determine the distribution of radiolabeled bacteria retained in the sediment. The other half was dried, epoxied and thin sectioned. The distribution of radiolabeled bacteria in the thin sections was mapped using phosphor imaging. Physical/chemical heterogeneities of the core were characterized by scanning electron microscope. The two strains exhibited markedly different retention behaviors. In the case of OYS2-A, the highest retention (up to 99%) of the injected bacteria was observed when the cells moved through a fine-grained core (mean grain size 150 micron), and more than 70% of the injected bacteria was retained within 12 cm from the influent end. The cells exhibited no preferential attachment as a function of certain grain size or mineralogy. In the case of DA001, the degree of retention was considerably less even within cores of similar lithology. The sediment retained ~30% of the injected bacteria, and bacterial retention was much greater in the fine-grained cores than that in the coarse-grained cores. However, the total amount of retention was similar for both types of cores. Bacterial retention was inversely correlated with grain size. In cores with alternating zones of fine- and coarse-grained minerals, the fine-grained zones retained 4-5 times more bacteria than the coarse-grained zones. Where the grain size was homogeneous, the degree of bacterial retention was positively correlated with the areal abundance of the Fe and Al hydroxides. These results strongly demonstrate that bacterial transport is controlled by both bacterial (e.g., % adhesion) and sedimentary properties. Among the sediment properties, grain size is the primary controlling factor, and mineralogy the secondary.

Vibration-Accelerated Microbial Transport In Subsurface Media PHELPS TJ1, KINSALL BL1, DOLL WE1, KISNER R1, and PFIFFNER SM2

1 Oak Ridge Natl. Laboratory, Oak Ridge, TN, 2 The Univ. of Tennessee, Knoxville, TN

Low transport rates of water, microorganisms, and substrates through sediment matrices can pose severe limitations on the applicability of bioaugmentation/ biostimulation for in situ remediation. Preferential flow, low matrix diffusion rates, and adsorption/filtration can all inhibit the transport and activities of degrading microbes. This research investigates the use of vibrational-based energy (sonication) to increase solute diffusion and microbial transport through sediments. Investigations focus on evaluating the applicability of vibration-induced transport through detailed hypothesis testing in laboratory experiments to be complemented by field-scale verification. We hypothesize that sonication will result in increased flow and microbial transport through sediment matrices. Alterations in preferential flowpaths, pore-size distributions, and sorption capacities between microbes/sediment particles could hypothetically increase spatial distributions of active microbial populations. Our results indicate that sonic energies may well be effective for increased bacterial transport. Vibrational energies do indeed increase dispersion, presumably by mixing and opening pore spaces. Results indicate accelerated flow and colloidal transport in controlled laboratory experiments. Comparisons with tracer regimens and non-vibrated columns revealed stark differences in the flow, distribution, and dispersion of fluorescent microspheres. Current experiments are focusing on quantifying vibrational energies; power and frequency (watts and hertz) within field applicable ranges. Current results suggest frequencies between 40-200 Hz with power levels of several Kw/m³) of sediment may be best suited for microbial transport. In addition, impacts of vibration energies on adsorption/desorption mechanisms are being examined by comparing elution profiles and adsorption profiles between vibrating and non-vibrating columns treated with differing ionic strength waters.

Effect of Pulse Length, Grain Size, and Surface Coatings on Bacterial Retention Capacity of Sand

BOLSTER, C. H., MILLS, A. L., HORNBERGER, G. M., HERMAN, J. S. Univ. Virginia, Charlottesville, VA

Miscible displacement experiments were performed on short sand columns (~5 cm) to determine how bacterial deposition is affected by previously deposited bacteria. A 10-pore-volume pulse of radiolabeled cell suspension was introduced into the columns followed by a 2-pore-volume flush of artificial groundwater (AGW) for comparison with deposition in 1-pore-volume pulse injections of bacteria. Effluents were collected with a fraction collector. Following the flush with AGW, the columns were dissected along the axis of flow. At approximately 0.5-cm intervals, sediment was removed for the enumeration of sediment-associated bacteria. The effects of sand grain size and sand grain coatings were evaluated. Treatments included columns packed with clean Unimin sand and Fe-coated Unimin sand, and comparisons were also made between columns packed with either sand ranging from 0.4 to 0.5 mm or 0.85 to 1.0 mm. A simple first-order model (classic filtration theory) could not adequately describe the average spatial distribution of bacteria with depth within the columns. Rather, a second-order model was required to describe the observed decreases of deposited bacteria with depth. Parameter values obtained from the effluent data were compared to parameter values obtained from the deposited bacteria concentrations. Distributions of bacteria in the long- vs. short-pulse columns were similar except that high concentrations of bacteria penetrated deeper into the columns with longer injection pulses. Results demonstrate that both sand grain size and sand grain surface coatings affect the total amount of bacteria deposited and the bacterial retention capacity of the sediments.

Bacteria Transport in Sand Column Experiments: Effect of Media Preparation BROWN, D.G., STENCEL, J.R., AND JAFFÉ, P.R. Princeton University, Princeton, NJ

Prediction of bacterial transport through a porous media requires knowledge of the filtration parameters for the system of interest. Unfortunately, bacteria filtration parameters can vary widely from one experiment to the next. Given the importance of estimating bacteria transport through porous media, it is important to understand the degree of reliability within which the filtration parameters can be determined. This reliability is an issue that has not been addressed much in the literature. When sand is used as the porous media, mechanical sieving is frequently employed to fraction the sand into tight size ranges. We found that there are inherent problems associated with using mechanically sieved sand for bacterial transport experiments. First, we found that mechanical sieving does not provide the tight size fractioning required for laboratory experiments. Second, we found that mechanical sieving with standard brass sieves results in extensive zinc and copper contamination of the sand grains. The amount of metal contamination showed very strong correlation to the sieving time and occurred with new and old sieves. These two artifacts of mechanical sieving had a significant effect on the bacteria filtration parameters. To address the inadequacies of mechanical sieving, we developed a sand preparation procedure that combines a form of wet-sieving of the sand to obtain a tight size distribution along with a slightly acidic column flush to remove any residual metal contamination. Bacteria filtration experiments, which were not reproducible utilizing mechanical sieving, were reproducible using the new sand preparation procedure. Thus, this method provides a consistent sand grain surface from one sand batch to the next, and facilitates determination of filtration parameters with a high reliability.

Long Term Low Concentration Transport of Microspheres and Bacteria ZHANG P., JOHNSON W. P. Univ. of Utah, Salt Lake City, UT 84112

Simulating Bioremediation of Uranium Contaminated Aquifers; an Evaluation of Model Robustness

P.R. JAFFE, S. WANG, H.A. RABITZ

all at Princeton Univ., Princeton NJ

Mobility of trace metals and radioisotopes in groundwater can be controlled to a significant extend via in-situ manipulation of several biogeochemical reactions that directly or indirectly alter their solubility. For this purpose, specified redox profiles need to be established in the groundwater. These redox profiles develop, in response to biostimulation schemes, from the availability and transport of different electron acceptors, and their utilization by different bacteria during the degradation of an organic substrate. The objective of this research is to assess modeling techniques to simulate the fate of trace metals and radionuclides in the subsurface, in response to different biostimulation schemes. A time-dependent one-dimensional reactive transport model has been developed. The model consists of a set of coupled, steady state mass balance equations, accounting for advection, diffusion, dispersion, and a kinetic formulation of the transformations affecting an organic substrate, electron acceptors, corresponding reduced species, and contaminant metals of interest. This set of equations is solved numerically, using a finite element scheme. The redox conditions of the domain are characterized by estimating the pE, based on the concentrations of the dominant terminal electron acceptor and its corresponding reduced specie. This pE and the concentrations of relevant species are passed to a modified version of MINTEQA2, which calculates the speciation and solubilities of the species of interest. Kinetics of abiotic reactions are described as being proportional to the difference between the actual and equilibrium concentration. Simulations are performed to illustrate the effect of biostimulation on the transport of uranium in the subsurface.

Removal of Nitrate and Uranium from Groundwater and Soil using Indigenous Bacteria using indigenous bacteria H. E. NUTTALL*, A. ABDELOUAS*, W. LUTZE* & B. A. STRIETELMEIER**

*Ctr. for Radioactive Waste Management, The Univ. of New Mexico, Albuquerque NM 87131 The University of New Mexico, Albuquerque NM 87131 ; ** Los Alamos Natl. Laboratory, Environmental Science and Waste Technology CST-7, Los Alamos, NM 87545

Given the large number of sites, e.g., uranium mill tailings dumps, where soils and groundwaters are polluted by nitrate, uranium, and other contaminants, there is demand for the development of innovative remediation technologies. We are studying in situ remediation with indigenous bacteria. This method relies on ubiquity of certain bacteria in soils and groundwater, capable of mediating reactions at depth that lead to elimination or precipitation of contaminants. In this paper, we report on removal of nitrate and on sequential removal of nitrate and uranium from groundwaters and soils. Nitrate alone is frequently encountered in groundwaters and in soils in the vadoze zone at dairies. We conducted experiments with groundwater and host rock from a dairy and from mill tailings sites in the U.S. and Germany. Water was amended with nutrients (acetate, ethanol) to stimulate growth of indigenous bacteria (information on strains will be given) and reduction of nitrate and U(VI). Nitrate was quantitatively reduced to nitrogen and uranium was precipitated as uraninite (UO2) on the sandstone. Stability of uraninite against reoxidation is provided by iron sulfide that precipitates upon microbially mediated reduction of Fe(III) from the rock and sulfate. The final concentration of uranium was on the order of 1µg/L. The protection of uraninite by iron sulfide is strong enough to limit the U-concentration at this level. Field experiments were conducted very successfully to clean up groundwater contaminated with nitrate. The process and its results will be presented along with the results from laboratory experiments with uranium.

Microbiological and Biogeochemical Characteristics of Subsurface Sediments at Uranium Mill Tailings Sites at Gunnison, Colorado and Shiprock, New Mexico: Implications for In Situ Remediation .

PHILIP E. LONG¹, DWAYNE ELIAS³, KEVIN FINNERAN⁴, JAMES K. FREDRICKSON¹, CRAIG GOODKNIGHT⁶, JONATHAN ISTOK², LEE R. KRUMHOLZ³, DEREK R. LOVLEY⁴, JAMES P. MCKINLEY¹, SARAH MACNAUGHTON⁵, STAN MORRISON⁶, JOHN STEPHEN⁵, JOSEPH M. SUFLITA³, AND D. C. WHITE^{5 1}Pacific Northwest National Laboratory, Richland, WA; ²Oregon State University, Corvallis, OR; ³University of Oklahoma, Norman, OK; ⁴University of Massachusetts, Amherst, MA; ⁵Univ. of Tennessee, Knoxville, TN; ⁶MACTEC ERS, Grand Junction, CO.

Uranium Mill Tailings Remedial Action (UMTRA) Sites provide an opportunity to study the microbiology and biogeochemistry of field sites contaminated with metals and radionuclides. Subsurface sediments at UMTRA sites have been in contact with contaminants for 30 to 50 years, a time period comparable to that for contaminants at DOE weapons complex sites. Contaminants include a wide range of metals dominated by U. Other anthropogenic solutes include nitrate, ammonium, and sulfate. Given these characteristics, the DOE’s NABIR Program is collaborating with the UMTRA Program to study selected UMTRA sites. The near-term objectives of this research are to 1) determine the dominant electron accepting processes at sites with long-term metal contamination and 2) define the biogeochemical transformations that may be important to either natural or accelerated bioremediation. Exploratory sampling has been completed at Shiprock, NM (alluvial sediments and shale) and Gunnison, CO (groundwater). Preliminary results for Gunnison indicate 1) low to moderate microbial activity (based on p-nitrophenol production rates); 2) enrichment of viable sulfate-reducing, nitrate-reducing, and Fe(III)–reducing bacteria. A peak in sulfide, acetate, and p-nitrophenol activity in a single sample suggests that microbial activity is relatively heterogeneous at this site. Preliminary results for the Shiprock site include 1) phospholipid fatty acids ranging from 50 to 200 picog/g sediment indicating the presence of a diverse, active microbial community, including SRB’s and actinomycetes; 2) viable anaerobic bacteria including NRB, SRB and methanogens in flood plain sediments; 3) low levels of Fe-oxidizing bacteria that use nitrate as an electron acceptor under anaerobic conditions; 4) flood plain sediments also exhibit U-reducing bacteria but none in shale samples. These results suggest the importance of biogeochemical processes in the natural attenuation of U in alluvial sediments, particularly at the Shiprock UMTRA site and that indigenous microorganisms are present at both sites that could contribute to in situ stabilization of U via reduction to insoluble U(IV) species .

Bacterial Diversity and Activity in Uranium Waste Piles SELENSKA-POBELL S., FLEMMING K., KUTSCHKE S., PANAK P., SATSCHANSKA G.

Inst. of Radiochemistry, Forschungszentrum Rorf, D-01314 Dresden, Germany

Bacterial diversity in subsurface soil samples drawn from different depths of several uranium waste piles was studied using the 16S rDNA retrieval. Extremely high diversity was found in all samples investigated. In particular, the presence of several dominant 16S rDNA groups related to the genera Thiobacillus, Bacillus, Pseudomonas, Desulfovibrio was demonstrated. One of these 16S rDNA groups was affiliated to the species T. ferrooxidans. Interestingly, this group includes three 16S rDNA types which possess slightly different sequences. Strains of T. ferrooxidans corresponding to two of the mentioned three 16S rDNA types were recovered from two soil samples polluted in different extend with heavy metals. The two groups of isolates have different genomic organization. In addition, the members of the group recovered from the more polluted sample are tolerant to higher concentrations of uranyl ions which are lethal for the isolates of the second group. The expression of at least three genes of the U-tolerant strains is influenced by the presence of uranyl ions in the nutrient medium. The capability of the U-mine isolates to interact with U(VI) was studied, and it was found that they accumulate significantly higher amounts of U(VI) in comparison to several reference T. ferrooxidans strains recovered from other environments. Using time-resolved laser fluorescence spectroscopy it was shown that the complexes build by one of the U-waste isolates with U(VI) are much stronger than those build by the reference strains. This is the first report which demonstrates a microdiversity in closely related natural isolates of T. ferrooxidans. We suggest that the microdiversity observed reflects the genetic adaptation of the strains studied to the different heavy metal concentrations in their natural environments.

Bacillus subtilis As A Nucleation Template For Ferric Oxyhydroxide Formation: Implications For Uranium Removal From Groundwaters

LEES, K.S. ¹, ABBOTT, A. ¹, KONHAUSER, K. ²

Westlakes Research Inst., Moor Row, Cumbria, England., Univ. of Leeds, Leeds, England.

The potential of microorganisms to accelerate the formation of inorganic compounds such as amorphous ferric oxyhydroxides, is being increasingly recognised. However, little work has been carried out on the utilisation of these biogenically formed precipitates for the removal of trace elements, such as uranium. In a subsurface environment where ferric oxyhydroxides and microbial cells frequently constitute important components of the solid phase matrix this may have implications in actinide transport and attenuation. Uranium, used in this study as a model contaminant, exhibits a strong affinity for biosorption. Preliminary ICP-MS and TEM/EDS data indicates substantial metal removal by Bacillus subtilis 168 cells when initial concentrations of iron and uranium were 1.8M and 3.4mM respectively. Uranium removal appears to occur via three mechanisms: direct sorption to untreated cells; adsorption to a pre-formed, biogenically nucleated, iron phase; co-precipitation with a biogenically nucleated iron phase. Further studies will consider uranium binding to biogenically precipitated ferric oxides at lower initial concentrations, representative of natural groundwater systems. The effect of environmental parameters upon uranium removal, including pH, complexing agents and competing ions, will be investigated. The efficiency of uranium removal to biogenically formed ferric oxyhydroxides will be compared to an abiotic equivalent, such as smectitic clay particles.

Toxicity of uranyl to Pseudomonas fluorescens -- implications for uranyl mobility in the subsurface

R. BENCHEIKH-LATMANI, J.O. LECKIE

Department of Civil and Environmental Engineering, Stanford Univ., Stanford, CA

Transport and Attenuation of Carboxylate-Modified-Latex Microspheres in Fractured Bedrock M W BECKER Department of Geology, State Univ. of New York at Buffalo, Buffalo, New York

There is a need for field data concerning the transport of microbes in ground water, for purposes such as understanding pathogen distribution in drinking water and the prediction of in-situ bioremediation efforts. Tracer tests involving latex microspheres as microbial analogs have been used successfully to investigate microbe transport in porous geologic media at the field scale. Little information is available, however, concerning the transport of colloid-sized particles in fractured geologic media, particularly in crystalline bedrock. Data will be presented from field colloid transport experiments performed at two fractured bedrock sites. The tests were conducted in a fractured granite in northern California and a fractured granite/schist in central New Hampshire, using a similar two-well injection/withdrawal design and fluorescent carboxylate-modified-latex (CML) microspheres (0.19 - 0.98 mm diameter). The results indicate that the first-arrival and mean transport time of the microspheres was shorter than simultaneously injected solute tracers. Only a fraction of the injected microsphere mass was recovered, and the apparent filtration increased with increasing microspheres size and decreasing transport velocity. These trends suggest, according to filtration theory, that microsphere immobilization is not dominated by inertial or Brownian contact with collectors. The dominant filtration mechanism may have been gravity settling, possibly combined with particle aggregation. It may be, therefore, that conceptual models of filtration that have been successful for predicting colloid transport in porous media, may not be directly transferable to fractured geologic media.

Predicting Bacterial Transport in Heterogeneous Soil

SEILIUTE L. and BENGTSSON G., Univ. of Lund, Lund, Sweden

Heterogeneity of the soil is a major challenge in predicting bacterial transport through the soil, resulting in large errors in predicting movement rates in the subsurface. The proper way of dealing with the spatial heterogeneity in nature may be a stochastic approach. The large variability in bacterial sorption coefficients may then be analyzed from relationship between the spatial differences in the soil properties and hydraulic conductivity. If there is a correlation between particle size, flow rate and sorption, it would not be necessary to perform laborious sorption/transport experiments with soil samples from the boreholes but rather to measure the particle size distribution of the samples and hydrodynamic conductivity to be able to predict the transport of bacteria in the field. Bacterial sorption to different size fractions of sand was examined in water-saturated sand columns at three different flow rates: 0.1, 0.2 and 1ml/min. Groundwater with ³H-labelled bacteria was pumped through columns filled with sand particles sieved to different size fractions and mixtures thereof. All other conditions were kept constant. The ³H activity in the effluent solution was measured by liquid scintillation. An advection-dispersion-sorption model and two-site model were used to calculate the bacterial sorption coefficient K_b. Relationship between hydraulic conductivity, particle size distribution and bacterial sorption to the soil have been investigated. The results support the idea that bacterial sorption increases with decreasing particle size of soil fractions and with increasing hydrodynamic conductivity. The relationships were used to predict sorption in a heterogeneous soil from hydraulic conductivity and particle size distribution.

Modeling Bacterial Transport based on Laboratory Experiments using Intact Cores TD SCHEIBE, TR GINN, ME FULLER, TC ONSTOTT, MF DEFLAUN

Pacific Northwest Natl. Laboratory Richland WA 99352, Univ. of California Davis CA 95616, Envirogen Inc. Lawrenceville NJ 08648, Princeton Univ. Princeton NJ

Intact sediment cores were collected horizontally from an excavation near Oyster, Virginia, USA and subjected to a series of laboratory transport experiments using inert and bacterial tracers. Experimental data includes observations of effluent concentrations over time and the two-dimensional spatial distribution of attached bacteria in a longitudinal cross-section of the core sediment at the end of the transport experiment. Numerical models of solute and bacterial transport were applied to the experimental results to estimate transport parameters at the core scale. These parameters are being used to predict and evaluate field observations of bacterial transport using a high-resolution three-dimensional field-scale numerical model. Because the scale of the intact core experiments and that of grid elements of the field-scale model are comparable, the model parameters derived from the core experiments are hypothesized to be useful for field-scale modeling. Several alternative model approaches have been developed and tested using the experimental data. Fate and transport processes simulated are advection and dispersion, bacterial attachment and detachment, and size exclusion. Bacterial attachment is simulated using a linear kinetic model representative of filtration theory. Detachment is also simulated using a kinetic model, but linear and non-linear forms are considered, including time-dependent detachment, heterogeneous microbial properties, and heterogeneous attachment sites. Effects of a number of variables have been explored using the numerical models, including the type of bacteria (two different organisms), variations in geological materials (sediments from three different facies classes), and effects of in-situ conditions (sediments from saturated and unsaturated zones of the aquifer).

Influence of rotational shear on the random motility of Escherichia coli at laminar flowrates. H. L. REDDY, R. M. FORD SymBiotech, Inc., Wallingford, CT; Univ. of Virginia, Charlottesville, VA

Modelling of bacterial transport in the subsurface must account for the influence of fluid flow. In addition to contributions of fluid flow to advective and dispersive movement of microorganisms, fluid flow can also influence the diffusive characteristics of bacteria. Bacteria will rotate in shear flow, and this rotation may alter the observed diffusion of non-spherical bacteria. If these non-spherical bacteria are also motile, their behavior will be altered even further. In this work, the transport of E. coli in laminar flow was examined to determine how the diffusion-like behavior (random motility) of the motile, wild type organism is altered by rotational shear. A mathematical model was developed to predict bacterial transport in a channel; such a channel is analogous to fractures or to long pores in the subsurface, where a laminar flow profile would become fully developed. The model incorporates either isotropic or anisotropic (appropriate for axisymmetric particles) diffusion, and the resulting average diffusion coefficients and bacterial profiles in the channel are compared. At low shear rates, the predicted concentration profiles for the two models are not significantly different. Bacterial diffusion in areas of high shear, similar to what would be observed near the walls in a fracture or pore, is predicted over a range of shear rates.

Chemotaxis of Pseudomonas putida G7 to naphthalene in porous media R.B. MARX AND M. D. AITKEN

Univ. of North Carolina, Chapel Hill, NC

The capillary assay was modified to study chemotaxis in porous media by introducing a packed bed of glass beads into both the culture chamber and a 10-microliter capillary tube. Using this modified method, chemotaxis of Pseudomonas putida G7 to naphthalene was demonstrated for a range of grain sizes. Compared to a free-liquid system, the accumulation of cells in capillaries of the porous-media system was lower. This first example of chemotaxis to a groundwater pollutant in porous media suggests that chemotaxis may lead to enhanced microbial degradation in the subsurface environment.

Transport of Motile Bacteria through a Model Porous Network R. M. Ford, K. C. Chen, P. T. Cummings and L. M. Lanning Univ. of Virginia, Charlottesville, VA 22903; Univ. of Virginia, Charlottesville, VA 22903 (Current: NYU Medical Ctr., New York, NY 10016; Univ. of Tennessee, Knoxville, TN 37996 and Oak Ridge Natl. Laboratory, Oak Ridge, TN 37831; Univ. of Virginia, Charlottesville

Many bacterial species which inhabit the subsurface environment are able to swim independently of the surrounding groundwater movement. In a bulk liquid environment the trajectories of motile bacteria trace out a three-dimensional random walk characterized by runs of about a second in duration interrupted by a brief period during which the bacterium reorients itself before running in a new direction. Our efforts are directed toward quantifying the impact of bacterial motility and chemotaxis on the transport of bacteria in the subsurface by appropriately modifying the macroscopic transport parameters which are used for nonmotile colloids in advection-dispersion equations. We consider a simple model of porous media consisting of interconnected capillary tubes (or pores) with diameters comparable to the size of a bacterial run length, so that bacterial interactions with the pore walls are significant. Observations reported in the literature suggest that the bacterial flux increases as the tube diameter decreases from 50 microns to 10 microns due to guidance from the pore walls. However, a further decrease in pore diameter to 6 microns results in a shift from a diffusive mechanism for transport to wavelike motion which no longer allows a chemotactic response. We have developed a mathematical description of bacterial transport in capillaries that is qualitatively consistent with these observations. The next step is to apply this model to bacterial migration in experimental systems with well-defined patterns of pore networks (micromodels) to evaluate the predictions of our mathematical modeling efforts on a laboratory scale.

Immobilization of Aqueous Strontium During Carbonate Mineral Formation Coupled to Microbial Fe(III) Oxide Reduction E.E. RODEN, M.R. LEONARDO, V.K. KEITH, F.G. FERRIS Univ. of Alabama, Tuscaloosa, AL; Univ. of Alabama, Tuscaloosa, AL; Univ. Alabama, Tuscaloosa; Univ. of Toronto, Toronto, Ontario, Canada

Environmental Actinide Mobility: Actinide Interactions With Capsules And Siderophores Of Aerobic Soil Microbes. VANDERBERG, L.A.; NEU, M.P.; FAIRLEE, J.R.; RUGGIERO, C.E.; HE, L.M. Chemical Science & Technology Division, Los Alamos National Laboratory, Los Alamos NM 87545 USA.

In order to apply bioremediation to actinide and heavy metals contaminated environments, the entire range of subsurface interactions must be understood. Aerobic microbes are dominant in some subsurface saturated and unsaturated zones and siderophores and capsules of these organisms are likely significantly affect the mobility of actinide contaminants. This study focuses on the interactions of environmentally relevant forms of U and Pu with the polyglutamate capsule (PGA) of Bacillus lichenformis, the desferrioximine siderophore (DFO) of Streptomyces pilosus, and an uncharacterized siderophore (RRS) from Rhodococcus rhodochrous OFS. PGA surface charge and conformation vary with ionic strength and ion type. The change in conformational structure for the capsule may influence the exposure of surface functional groups, the sites responsible for metal complexation. U(VI)-capsule complexes were investigated using ATR-FTIR and SAXS. DFO forms inner-sphere complexes with UO22+, PuO22+, and Pu4+, but not with the lesser charged PuO2+. Pu(VI) cleaves the DFO molecule and is rapidly and irreversibly reduced to Pu(V). RRS is a catecholate siderophore and GC-MS indicates that dihydroxybenzoic acid is the aromatic nucleus. Amino acid analysis suggests that a glycine moiety is part of the RRS. Optimal RRS production occurs at 0.5-2.0mM iron with C13 - C16 n-alkanes as sole carbon and energy source. Further characterization of RRS is ongoing. The results of our continuing research program indicate that these biomolecules do indeed impact actinide contaminant behavior.

Modeling the Sorption of Uranium to Gram-Negative Bacteria L. L. LANDKAMER, B. D. HONEYMAN AND L. FIGUEROA Colorado School of Mines, Golden CO. (all authors)

The transport of metal ions through saturated soil or rock is often regulated by sorption to surfaces. This study focuses on modeling the sorption of uranium to the surface of Desulfovibrio vulgaris, a gram-negative sulfate reducing soil bacterium. Gram-negative bacteria differ from gram-positive bacteria in that along with sorption of metal ions to the outer membrane, ions can diffuse through porins into the periplasmic space. In this study, the time dependent behavior of acid/base titrations of live bacteria was analyzed to estimate the rate of diffusion of ions into the cell and the quantity of proton-reactive functional groups present. Finally, uranium sorption data as a function of pH, and sorption kinetic data were generated while attempting to inhibit bacterial metabolism, which is known to enzymatically reduce uranium from U(VI) to U(IV), without affecting the uranium solution chemistry or bacterial surface.

Most often, modeling of metal sorption by surfaces adopts an electrical double layer to account for the electrostatics of metal ions sorbing to charged, non-porous planer surfaces. Contrastingly, bacterial surfaces are a porous, three-dimensional matrix of moiety-bearing polysaccharides and proteins infused with inert electrolyte ions. In this work, the Donnan model was used to simulate the electrostatics of this surface in a physically realistic manner. Using a combined model including diffusion, solution and surface complexation along with Donnan electrostatics, the system sorptive characteristics were successfully modeled. Uranium sorption increased with increasing pH until a maximum near pH 6, and then decreased as non-sorbing uranium-carbonato species start to dominate the solution chemistry.

Characterization of the Subsurface Microorganisms in the Vicinity of the Liquid Radioactive Waste Repository NAZINA T.N.1, DAVIDOV A.S.2, KOSAREVA I.M.2 1-Institute of Microbiology, Russian Academy of Sciences, Moscow, Russia; 2 -Institute of Physical Chemistry, Russian Academy of Sciences,Moscow, Russia

It is now generally acknowledged that microorganisms presented within repositories of liquid radioactive waste (LRW). Structure of the bacterial cenosis, physicochemical and geochemical characteristics of the strata waters from control wells located in the vicinity of the low level radioactive waste repository were investigated. In subsurface samples presented aerobic saprotrophic bacteria (102 -104cells/ml) and anaerobic bacteria -fermenters (105 cells/ml), sulfate reducers (102 cells/ml), nitrate reducers (103 -106 cells/ml) and methanogens (single cells/ml). The aim of further investigation was to find microorganisms useful for cleaning LRW from two main environment contaminants, that are – nitrate and acetic acid. Although aerobic bacteria that are able to mineralize acetate are ubiquitous in the environment, an anaerobic treatment under denitrifying condition can be advantageous due to the fact that the supply of the subsurface with sufficient oxygen is problematic. Very promising is microbiological treatment combining utilization of acetate with nitrate as electron acceptor. Many denitrifying bacterial strains were isolated from the indigenous community of the stratal water in the vicinity of repositories of LRW. Strains were selected by a protocol that enriched for phenotypes important for radioactive waste cleaning. The resulting organisms were resistant to high levels of radioactivity, and high content of acetic acid and nitrate. Nitrogen and carbon dioxide were the main products of enitrification. Phenotypic and phylogenetic analyses of pure cultures of denitrifying bacteria were also performed. The results of this study show that main components of radioactive waste – acetate and nitrate – can be degraded in subsurface by a consortium of different radiation-resistant bacterial strains, clearly indicating that bioremediation of nuclear waste-contaminated environments is feasible.

Desorption of 239Pu by Pseudomonas Mendocina HERSMAN, L.E, J.H. FORSYTHE AND E.A STREITELMEIER Environ Molecular Biology Grp.

We investigated the desorption of plutonium (Pu) from Fe(III)(hydr)oxides by the common soil microorganism, Pseudomonas mendocina. To acid washed, 125 mL Teflon flasks, we added aseptically 10 mL Fe free succinate medium, and 0.0417 g of synthetic goethite to give 29 m2/L surface area. Additionally, 10 mL medium was added to a second set of flasks that were used as goethite negative controls. Stock solution (2.2 x 10-4 M)of 239Pu(IV) was added to sterile succinate medium, resterilized by filtration, and 10 mL was transferred to each of the flasks from above, yielding a final concentration of 1.1 x 10-6 M in 20 mL succinate medium per flask, pH = 7.2. The flasks were incubated in the absence of light at 18° C, 50 rpm, for four days. Then, the entire contents of the sterile goethite flasks were transferred aseptically and individually into acid washed, sterile 125 mL Teflon flasks. These fresh flasks containing Pu sorbed to goethite were inoculated with an early log growth phase culture of P. mendocina (100 mL, 0.2 absorbance at 600 nm wave number), incubated as described previously, and alpha counts and optical densities were recorded. After 4 d of incubation the microorganisms grew to approximately .552 absorbance units, and removed 19.2% of the sorbed Pu. This experiment was performed in triplicate, and repeated. The results of the two replications are consistent with one another. The fact that these cells, growing aerobically, removed nearly 20% of the sorbed Pu is significant by itself. However when one considers that the microorganisms did not achieve maximaum optical density, then the results assume even greater significance. Pu desorption from Fe(III)(hydr)oxides, promoted by P. mendocina, may be common to other aerobic species as well. Thus, these results reach out far beyond an examination of esoteric or exotic phenomenon, but rather it has relevance to common ecologically based principals - principals that appear to effect significantly the desorption of radionuclides and metals, and thus can be utilized for bioremediation.

Factors Affecting Reduction of Hexavalent Chromium by Aquifer Materials. T.L. MARSH AND M.J. MCINERNEY Univ. of Oklahoma

Anaerobic sediments from an aquifer contaminated with landfill leachate were used to study the potential for microbial reduction of Cr(VI) to Cr(III). The lightly colored, sandy sediments slowly reduced Cr(VI) in viable, but not heat-killed, microcosms indicating a biological process. Microcosms containing sandy sediments and mineral medium were amended with various electron donors to determine those most important for biological Cr(VI) reduction. More than 500 µM Cr(VI) was reduced when formate, lactate, hydrogen, and glucose, but not benzoate or acetate, were added as electron donors. Chromate reduction in microcosms was not inhibited by the addition of sulfate, selenate, or Fe(III). However, the presence of nitrate partially inhibited Cr(VI) reduction. Nearly complete inhibition of Cr(VI) reduction was observed when microcosms were shaken in the presence of oxygen. The addition of molybdate to the microcosms did not affect Cr(VI) reduction in sandy sediments until very high concentrations (40 times the Cr(VI) concentration) were used. The addition of bromoethanesulfonic acid (BESA) in amounts less than, or slightly greater than, the Cr(VI) concentration partially inhibited Cr(VI) reduction in the microcosms. An anaerobic Cr(VI)-utilizing enrichment was obtained that was dependent upon hydrogen for both growth and Cr(VI) reduction. Growth and Cr(VI) reduction by the enrichment were completely inhibited by the addition of formaldehyde, partially inhibited by molybdate, and completely unaffected by BESA. No methane was produced by the enrichment, which reduced about 750 µM Cr(VI) in less than six days. These studies showed that Cr(VI) reduction in sandy aquifer sediments is a biologically mediated, anaerobic process that is inhibited by oxygen and nitrate. In addition, electron donors which resulted in increased available hydrogen showed a greater extent of Cr(VI) reduction.

Purification, Physiology, And Properties Of Chromate Reductase In Pseudomonas Putida. C-H. PARK, M. KEYHAN, AND A.C. MATIN*. Stanford University School of Medicine, Stanford, California