Microbial life in the deep terrestrial subsurface

The distribution and function of microorganisms is a vital issue in microbial ecology. The US Department of Energy`s Program, ``Microbiology of the Deep Subsurface,`` concentrates on establishing fundamental scientific information about organisms at depth, and the use of these organisms for remediation of contaminants in deep vadose zone and groundwater environments. This investigation effectively extends the Biosphere hundreds of meters into the Geosphere and has implications to a variety of subsurface activities

Isolation, Cultural Maintenance, and Taxonomy of a Sheath-Forming Strain of Leptothrix discophora and Characterization of Manganese-Oxidizing Activity Associated with the Sheath

Leptothrix discophora SP-6 was isolated from the outflow reservoir of an artificial iron seep. Its sheathforming phenotype was maintained by slow growth in a mineral salts-vitamin-pyruvate medium under minimal aeration at 20 to 25°C. A sheathless variant, SP-6(sl), was isolated from smooth colonies that appeared on spread plates after rapid growth of SP-6 in well-aerated cultures. SP-6 and SP-6(sl) are closely related but not identical to the previously studied sheathless strain SS-1 (ATCC 43182). Increasing Mn(2+) concentrations in the growth medium of SP-6 increased the phase density of the sheath, indicating increased Mn oxide deposition in the sheath. Electron microscopy of cultures grown without added Mn(2+) revealed that the sheath consisted of a well-defined inner layer, 30 to 100 nm thick, and a diffuse outer capsular layer of variable thickness. Mn oxides were identified in the sheath by their characteristic ultrastructure, electron density, and X-ray-dispersive energy spectra. In heavily encrusted sheaths, the Mn oxides were evenly distributed in both layers of the sheath. Sheathed cells retained more Mn-oxidizing activity than did sheathless cells after washing with distilled, deionized water; the sheath retained some of its activity after an EDTA-lysozyme-detergent treatment which removed the cells. An ultrafiltration-dialysis procedure significantly increased the recovery of activity from spent media of SP-6 over that reported previously for SS-1 (L.F. Adams and W.C. Ghiorse, J. Bacteriol. 169:1279-1285, 1987). A 108-kDa Mn-oxidizing protein was identified in concentrated spent media of SP-6 and SP-6(sl), and the activity of the concentrates showed stability in detergents comparable to that of SS-1 and patterns of heat inactivation and chemical inhibition similar to those of SS-1

Production of Biogenic Mn Oxides by \u3cem\u3eLeptothrix discophora\u3c/em\u3e SS-1 in a Chemically Defined Growth Medium and Evaluation of Their Pb Adsorption Characteristics

Biogenic Mn oxides were produced by the bacterium Leptothrix discophora SS-1 (= ATCC 3182) in a chemically defined mineral salts medium, and the Pb binding and specific surface area of these oxides were characterized. Growth of SS-1 in the defined medium with pyruvate as a carbon and energy source required the addition of vitamin B12. Complete oxidation of Mn(II) within 60 h required the addition of ≥0.1 μM FeSO4. Pb adsorption isotherms were determined for the biogenic Mn oxides (and associated cells with their extracellular polymer) and compared to the Pb adsorption isotherms of cells and exopolymer alone, as well as to abiotic Mn oxides. The Pb adsorption to cells and exopolymer with biogenic Mn oxides (0.8 mmol of Mn per g) at pH 6.0 and 25°C was 2 orders of magnitude greater than the Pb adsorption to cells and exopolymer alone (on a dry weight basis). The Pb adsorption to the biogenic Mn oxide was two to five times greater than the Pb adsorption to a chemically precipitated abiotic Mn oxide and several orders of magnitude greater than the Pb adsorption to two commercially available crystalline MnO2 minerals. The N2 Brunauer-Emmet-Teller specific surface areas of the biogenic Mn oxide and fresh Mn oxide precipitate (224 and 58 m2/g, respectively) were significantly greater than those of the commercial Mn oxide minerals (0.048 and 4.7 m2/g). The Pb adsorption capacity of the biogenic Mn oxide also exceeded that of a chemically precipitated colloidal hydrous Fe oxide under similar solution conditions. These results show that amorphous biogenic Mn oxides similar to those produced by SS-1 may play a significant role in the control of trace metal phase distribution in aquatic systems

Kinetics of Mn(II) oxidation by Leptothrix discophora SS1

The kinetics of Mn(II) oxidation by the bacterium Leptothrix discophora SS1 was investigated in this research. Cells were grown in a minimal mineral salts medium in which chemical speciation was well defined. Mn(II) oxidation was observed in a bioreactor under controlled conditions with pH, O\u3esub\u3e2, and temperature regulation. Mn(II) oxidation experiments were performed at cell concentrations between 24 mg/L and 35 mg/L, over a pH range from 6 to 8.5, between temperatures of 10°C and 40°C, over a dissolved oxygen range of 0 to 8.05 mg/L, and with L. discophora SS1 cells that were grown in the presence of Cu concentrations ranging from zero to 0.1 µM. Mn(II) oxidation rates were determined when the cultures grew to stationary phase and were found to be directly proportional to O2 and cell concentrations over the ranges investigated. The optimum pH for Mn(II) oxidation was approximately 7.5, and the optimum temperature was 30°C. A Cu level as low as 0.02 µM was found to inhibit the growth rate and yield of L. discophora SS1 observed in shake flasks, while Cu levels between 0.02 and 0.1 µM stimulated the Mn(II) oxidation rate observed in bioreactors. An overall rate law for Mn(II) oxidation by L. discophora as a function of pH, temperature, dissolved oxygen concentration (D.O.), and Cu concentration is proposed. At circumneutral pH, the rate of biologically mediated Mn(II) oxidation is likely to exceed homogeneous abiotic Mn(II) oxidation at relatively low (≈µg/L) concentrations of Mn oxidizing bacteria

Determination of Iron Colloid Size Distribution in the Presence of Suspended Cells: Application to Iron Deposition onto a Biofilm Surface

Transport and deposition of colloidal Fe, Mn and Al oxides play key roles in the cycling of toxic transition metals in aquatic environments because these colloids strongly bind transition metals. Further, attachment of biological cells and biofilm growth on surfaces can indirectly affect toxic metal distribution by influencing the deposition of colloidal oxides to surfaces. To elucidate the mechanisms governing these processes, deposition of colloidal oxides onto surfaces must be evaluated in the presence of suspended and adherent bacterial cells. Both particle size and concentration are expected to influence deposition. An experimental protocol was developed to determine the size distribution of iron colloids in mixtures with suspended cells. A Ti(III) reagent was used to reduce and dissolve colloidal Fe(III) from mixtures containing both suspended cells and Fe colloids. The size distribution of Fe(III) colloids in the original solution was then determined from the difference between size distributions before and after dissolution of Fe with Ti(III). The Ti(III) reagent dissolved over 95% of the Fe colloids without altering the size distribution of suspended bacterial cells, and the method accurately determined the size distribution of Fe colloids added to cell suspensions. The applicability of this protocol was tested by applying it to a study of the deposition of Fe(III) oxide particles onto glass surfaces with and without biofilms of the bacterium Burkholdaria cepacia 17616. Experimental results using a laboratory biofilm reactor indicated that the deposition rate of Fe(III) colloids was not significantly affected by the presence of B. cepacia biofilms or by the presence of previously deposited Fe. However, deposition of Fe to reactor surfaces other than the glass surfaces may have interfered with the analyses, and atomic absorption measurements showed a slight increase in Fe deposition onto glass surfaces with biofilms present. Fe deposition to the composite of all reactor surfaces increased with increasing colloidal particle size, indicating a dominance of interception and/or sedimentation in controlling Fe deposition on surfaces in the biofilm reactor

Lead Distribution in a Simulated Aquatic Environment: Effects of Bacterial Biofilms and Iron Oxide

Biofilms influence the transport and fate of heavy metals in aquatic environments both directly by adsorption and complexation reactions and indirectly via interactions with oxides of iron and manganese. These reactions were investigated by introducing lead into a continuous-flow biofilm reactor that was designed to simulate conditions in a flowing freshwater aquatic environment. The reactor provided controlled conditions, and use of a chemically-defined growth medium allowed calculation of lead speciation with a chemical equilibrium program (MINEQL). Pseudomonas cepacia was employed as a test cell strain because of its ability to grow and form biofilms in the defined medium. This bacterium affected lead distribution in the reactor by adsorbing lead both to adherent and suspended cells. When the aqueous bulk lead concentration was 1.4 ± 0.1 µM and biofilm coverage (measured as chemical oxygen demand, COD) was 50 mequiv COD/m2, lead adsorption was increased by about a factor of five relative to bare glass. Of the total lead in solution, only 1% was adsorbed to suspended cells (5 x 107 cells/ml). Lead adsorption to biofilms followed a Langmuir isotherm with a maximum adsorption (Γ max) of 56 µmol Pb/equiv COD and an adsorption equilibrium constant (K) of 0.64 liter/µmol Pb. Lead complexed with dissolved bacterial exopolymer was below detection limits. Pretreatment of glass slides with colloidal iron also significantly increased lead adsorption relative to bare glass. Lead adsorption to adsorbed iron fit a Langmuir isotherm with Γmax = 50 µmol Pb/mol Fe, and K = 1.3 liter/µmol Pb. Lead binding to glass coated with both cells and iron was additive, and could be predicted by summing adsorption predicted using isotherms for each constituent. The presence of iron surface coatings increased initial biofilm formation rates, but after reaching steady state conditions, biofilm coverage was similar for slides treated with iron and untreated slides. A concentration of 1 µM lead produced a transient reduction in suspended cell counts. Cell counts recovered to the original cell density over the course of five to ten reactor retention times. With iron present, the magnitude of the reduction in cell concentration in response to the addition of lead was greatly reduced, suggesting that toxic effects of lead may be reduced by iron