William C. Ghiorse

Microbial Ecology of the Terrestrial Subsurface

We have presented a current view of the microbial ecology of the terrestrial subsurface by considering primarily the ecology of shallow aquifer sediments. The properties of the aquifer sediments and groundwater determine their ability to support microbial life and control the abundance and activities of microorganisms. Pore size, nutrient limitations, availability of electron acceptors, and large surface area for attachment all may have major effects on microbial abundance and activities in aquifer material. Microorganisms are the predominant forms of life in the subsurface. They will be found wherever enough space, nutrients, and water are available for them to live. Environmental factors such as pH, temperature, hydrostatic pressure, and dissolved salts also may influence subsurface microbial populations, but these factors do not exhibit great extremes in shallow water table aquifers, and thus only in very deep formations might they limit diversity or preclude the existence of microorganisms. Although the presence and activity of microorganisms in most subsurface environments are predictable, only recently have subsurface microbial populations in shallow subsurface zones been characterized. Aseptic sampling methods have been employed and microbiological and biochemical methods have been adapted to determine the types, abundance, and metabolic activities of microorganisms in subsurface material. Bacteria dominate, but eukaryotic microorganisms also are present. Vertical profile studies of a shallow aquifer in Oklahoma showed that active microbial biomass declined with depth to the unsaturated zone, but was variable in saturated sediments. Such a distribution of active biomass may be common in shallow aquifers. Studies on the lateral distribution of microorganisms in shallow and deep aquifers suggest that microorganisms are transported or migrate over fairly long distances in aquifer sediments. Surficial aquifers may be colonized by vertical or lateral transport and migration of surface microorganisms from recharge areas, but microorganisms could also have colonized when sediments were originally deposited. The biological and physical mechanisms controlling the migration of microorganisms in aquifers are not well understood. The function of shallow aquifers was considered with regard to nutritional ecology. Most pristine aquifers are oligotrophic. Heterotrophic life in these unique ecosystems is supported by secondary organic compounds that filter down from the soil above. The quantity and quality of organic nutrients depend on the age of water and rate of recharge of the aquifer.(ABSTRACT TRUNCATED AT 400 WORDS)

Distribution of aerobic bacteria, protozoa, algae, and fungi in deep subsurface sediments

Abstract The distribution of microorganisms in deep subsurface profiles was determined at three sites at the Savannah River Plant, Aiken, South Carolina. Acridine orange direct counts (AODC) of bacteria were highest in surface soil samples and declined to the 106 to 107 per gram range in the subsurface, but then did not decline further with depth. In the subsurface, AODC values varied from layer to layer, the highest being found in samples from sandy aquifer formations and the lowest in clayey interbed layers. Sandy aquifer sediments also contained the highest numbers of viable bacteria as determined by aerobic spread plate counts (CFU) on a dilute heterotrophic medium. In some of these samples bacterial CFU values approached 100% of the AODC values. Viable protozoa (amoebae and flagellates, but no ciliates) were found in samples with high bacterial CFU values. A variety of green algae, phytoflagellates, diatoms, and a few cyanobacteria were found at low population densities in samples from two of the thr...

In situ biodegradation: microbiological patterns in a contaminated aquifer

Conventional approaches for proving in situ biodegradation of organic pollutants in aquifers have severe limitations. In the approach described here, patterns in a comprehensive set of microbiological activity and distribution data were analyzed. Measurements were performed on sediment samples gathered at consistent depths in aquifer boreholes spanning a gradient of contaminant concentrations at a buried coal tar site. Microbial adaptation to polyaromatic hydrocarbons (PAHs) was demonstrated by mineralization of naphthalene and phenanthrene in samples from PAH-contaminated, but not adjacent pristine, zones. Furthermore, contaminant-stimulated in situ bacterial growth was indicated because enhanced numbers of protozoa and their bacterial prey were found exclusively in contaminated subsurface samples. The data suggest that many convergent lines of logically linked indirect evidence can effectively document in situ biodegradation of aquifer contaminants.

Distribution and Activity of Microorganisms in Subsurface Sediments of a Pristine Study Site in Oklahoma

Distribution and activity of microorganisms in surface soil and subsurface sediments were studied in depth profiles of six different microbial biomass and activity indicators (total direct counts, number of cells capable of electron transport system activity, viable cell plate counts, most Probable numbers of protozoa, and 4-hydroxybenzoate-degrading microorganisms, and ATP content). The profiles showed the same general trends on two different dates (January and June 1985). Seasonal variations were noted, but they were not extreme. Biomass and activity values declined sharply with depth in the unsaturated zone, reaching minima in a clay confining layer in the interface zone between 3 and 4 m. Contiguous 10-cm samples from the interface zone showed significant textural and microbiological variability. Higher and more stable biomass and activity values were detected in the saturated zone, the highest being a very permeable gravelly loamy sand layer at approximately 7.5 m. In this layer, viable counts were nearly equal to total counts and they approached the viable counts in surface soil. Surface-type protozoa and cyanobacteria also were detected in this layer, suggesting that it was connected hydrologically to a nearby river. Lowest values were detected in an underlying bedrock clay layer at 8 m, which, despite its impermeability and low viable counts, did contain measurable total counts, 4-hydroxybenzoate-degrading microorganisms, and ATP. Correlations were noted between sediment texture and microbial activity (i.e., sandy texture=high activity, clayey texture=low activity), but other hydrogeological and geochemical factors probably also influenced microbial distribution and activity in the profile.

A quantitative analysis of DNA extraction and purification from compost

We quantified both DNA and humic acid concentrations during the extraction and purification of DNA from compost. The DNA extraction method consisted of bead-beating with SDS for cell lysis, poly(ethylene glycol)-8000 precipitation for preliminary DNA purification, and chromatography on a 10-ml Sephadex G-200 column for final DNA purification. Direct microscopic observation of pre- and post-lysis samples revealed that 95.3+/-2.3% of native cells was lysed. Sixty-three percent of the original DNA was lost during purification, resulting in a final DNA yield of 18.2+/-3.8 microg DNA/g of wet compost. The humic acid content was reduced by 97% during the purification steps resulting in a final humic acid concentration of 27+/-4.7 ng humic acid/microl. The purified DNA fragments were up to 14 kbp in size and were sufficiently free of contaminants to allow both restriction enzyme digestion by four different enzymes and PCR amplification of 16S rDNA.

Mobilization of adsorbed cadmium and lead in aquifer material by bacterial extracellular polymers

The mobility of cationic trace metals, such as Pb and Cd, in porous media can be severely limited by their adsorption at the solid/solution interface. The transport of metals can be enhanced by complexation with a ligand of “carrier” that (i) is soluble in water and does not strongly sorb to surfaces, (ii) has a high metal binding affinity and (iii) is not readily altered in soil by chemical or biological reactions. Extracellular polymers of bacterial origin are plausible carriers for metals in soil or aquifer systems. Bacterial extracellular polymers occur naturally in groundwaters and some have well established metal binding properties. In this study, extracellular polymers from 13 bacterial strains, including five subsurface isolates, were screened for their ability to mobilize Pb and Cd adsorbed to an aquifer sand. Batch adsorption isotherms were employed to screen polymers for their effect on metal phase distribution. All of the extracellular polymers tested reduced the linear distribution coeffients of Cd and Pb. Reductions in metal adsorption by over 90% were achieved at an extracellular polymer concentration of 10.6 mg l−1 The sorption isotherm of a selected extracellular polymer indicated that it had a low affinity for the sand sorbent and suggested that the polymer would be mobile in the porous sand medium. The distribution coefficient of the polymer for the sand was not effected by the presence Cd at low concentrations. Independently determined distribution constants for Cd and extracellular polymer with the sand and the binding constant for Cd to polymer yielded reasonable estimates of the observed distribution of Cd in the presence of the extracellular polymer. Column experiments performed with Cd in the presence and absence of the selected extracellular polymer confirmed that application of polymer solutions can enhance metal mobility in porous media.

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, O2, 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.

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

Abstract 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/m 2 , 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 × 10 7 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 expopolymer 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 Γ max = 50 μ mol 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.

Preferential Flow and Transport of Cryptosporidium parvum Oocysts through the Vadose Zone: Experiments and Modeling

in the form of 4- to 6-m-long ovoid-shaped oocysts, with a double wall that is resistant to most oxidation As a result of Cryptosporidium parvum in drinking water, several processes such as ozonation and chlorination (Current, outbreaks of cryptosporidiosis have occurred in the last 10 yr. Al1986; Atwill et al., 1997). though it is generally believed that movement of pathogens through the soil is minimal, recent research has shown that appreciable num- During the past two decades, the presence of C. parbers of C. parvum oocysts may be transported via preferential or vum in surface- and groundwaters in the United States fingered flow to groundwater. The objective of the present research and Great Britain (Galbraith et al., 1987; Rose et al., was to further investigate and model the transport of oocysts through 1991; Craun et al., 1998) has been associated with several preferential flow paths in the vadose zone under a “worst-case” sce- major outbreaks of cryptosporidiosis (Hayes et al., 1989; nario. This was studied by adding calves feces containing C. parvum MacKenzie et al., 1994). Among the different pathways oocysts with a Cl tracer to undisturbed silt loam columns and disfor the transport of oocysts to drinking water sources, turbed sand columns during a simulated steady-state rain. The sand columns exhibited preferential flow in the form of fingers whereas downward percolation is usually considered to be insigmacropore flow occurred in the undisturbed cores. In the columns nificant, because soils are generally assumed to be an with fingered flow, oocysts and Cl were transported rapidly with the effective filter for a wide range of pathogens. Studies same velocity through the columns. Although only 14 to 86% of the of packed columns with saturated flow by Brush et al. amount applied, the number of oocysts transported across the columns (1999) and Harter et al. (2000) and undisturbed columns was several orders of magnitude above an infective dose. The macwith unsaturated flow (Mawdsley et al., 1996), however, ropore columns had only a very limited breakthrough of oocysts, showed that C. parvum oocysts could be transported which appeared several pore volumes after the Cl broke through initially. A simulation model for the transport of oocysts via preferen- rapidly downward through the soil. Although transport tial flow was developed on the basis of an existing preferential flow of C. parvum oocysts in saturated flow has been studied model for nonadsorbing solutes, with addition of a first-order sink experimentally and described mathematically (Brush et term for adsorbance of the C. parvum to the air–water–solid (AWS) al., 1999; Harter et al., 2000), detailed observations of interfaces, and with velocity and dispersivity parameters derived from the transport and persistence of C. parvum oocysts in Cl transport. The breakthrough of C. parvum oocysts could be de- unsaturated soils with preferential flow are still lacking, scribed realistically for the sand columns. However, the model could particularly in the presence of preferential flow pronot describe oocyst transport in the columns with macropores. cesses.

Determination of spatial distributions of zinc and active biomass in microbial biofilms by two-photon laser scanning microscopy.

ABSTRACT The spatial distributions of zinc, a representative transition metal, and active biomass in bacterial biofilms were determined using two-photon laser scanning microscopy (2P-LSM). Application of 2P-LSM permits analysis of thicker biofilms than are amenable to observation with confocal laser scanning microscopy and also provides selective excitation of a smaller focal volume with greater depth localization. Thin Escherichia coli PHL628 biofilms were grown in a minimal mineral salts medium using pyruvate as the carbon and energy source under batch conditions, and thick biofilms were grown in Luria-Bertani medium using a continuous-flow drip system. The biofilms were visualized by 2P-LSM and shown to have heterogeneous structures with dispersed dense cell clusters, rough surfaces, and void spaces. Contrary to homogeneous biofilm model predictions that active biomass would be located predominantly in the outer regions of the biofilm and inactive or dead biomass (biomass debris) in the inner regions, significant active biomass fractions were observed at all depths in biofilms (up to 350 μm) using live/dead fluorescent stains. The active fractions were dependent on biofilm thickness and are attributed to the heterogeneous characteristics of biofilm structures. A zinc-binding fluorochrome (8-hydroxy-5-dimethylsulfoamidoquinoline) was synthesized and used to visualize the spatial location of added Zn within biofilms. Zn was distributed evenly in a thin (12 μm) biofilm but was located only at the surface of thick biofilms, penetrating less than 20 μm after 1 h of exposure. The relatively slow movement of Zn into deeper biofilm layers provides direct evidence in support of the concept that thick biofilms may confer resistance to toxic metal species by binding metals at the biofilm-bulk liquid interface, thereby retarding metal diffusion into the biofilm (G. M. Teitzel and M. R. Park, Appl. Environ. Microbiol. 69:2313-2320, 2003).