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 d