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THE GENERAL LIBRARIES

THE UNIVERSITY OF TEXAS AT AUSTIN

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THE UNIVERSITY OF TEXAS AT AUSTIN THE GENERAL LIBRARIES

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Franz Kunkel Hiebert

1994

MICROBIAL DIAGENESIS IN TERRESTRIAL AQUIFER CONDITIONS: LABORATORY AND FIELD STUDIES

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MICROBIAL DIAGNESIS IN TERRESTRIAL AQUIFER CONDITIONS: LABORATORY AND FIELD STUDIES

by

FRANZ KUNKEL HIEBERT A. 8., M.A.

DISSERTATION

Presented to the Faculty of the Graduate School of The University of Texas at Austin in Partial Fulfillment of the Requirements for the Degree of

DOCTOR OF PHILOSOPHY

THE UNIVERSITY OF TEXAS AT AUSTIN

May, 1994

Dedication

This dissertation is dedicated to my parents

Talmage Gordon Hiebert

Laura Franz Hiebert

Acknowledgements

Thanks to Julie, Samuel, Simon, and Jacob Hiebert for their enduring patience and support. Special thanks to my advisors, Philip C. Bennett and Robert L. Folk for their guidance, support, and constant and unrepentant enthusiasm for science. Dennis Brown and his students at the Institute for Cell Research gave expert guidance and help with SEM. E. Wheeler fabricated the field columns and many SEM stubs. Prof. Larry Lake (Dept, of Petroleum Engineering) helped with grant writing and provided lab space for bioreactor experiments. Jeff Horowitz drafted the figure of bacteria on quartz from drawings by Hiebert and Bennett. I received graduate support funding from grants from the USDOE (to Robert Folk) and NSF (to Philip Bennett). Special thanks to the Geology Foundation for financial support to present research at professional meetings and for occasional life-support loans.

MICROBIAL DIAGENESIS IN TERRESTRIAL AQUIFER CONDITIONS: LABORATORY AND FIELD STUDIES

Publication No.

Franz Kunkel Hiebert, Ph.D. The University of Texas at Austin, 1994

Supervisors: Philip C. Bennett and Robert L. Folk

The results of laboratory and field experiments demonstrated that bacteria indigenous to a shallow oil-contaminated aquifer caused minerals to weather rapidly in microreaction zones created in the near vicinity of attached bacteria. Bacteria readily colonized clean mineral surfaces in both laboratory and field microcosms. Nannobodies (0.1 pm diameter spheres) coated all minerals collected from organic rich and bacterially active waters. Nannobodies may be metabolizing bacteria, starved or shrunken bacteria, clots of congealed amorphous organic matter, and/or inorganic calcite crystal nuclei.

Feldspars, quartz, and, in some cases, calcite dissolved in the immediate vicinity of attached bacteria, even though the saturation indices for these minerals in the bulk ground water indicated that little to no dissolution should occur. Metabolic production of CO2 and HCO3 resulting from acetate methanogenesis and the oxidation of aromatic hydrocarbons coupled with iron reduction provided a supply of reactants to the aquifer-scale bulk ground water for calcite precipitation. Dolomite, with no evidence of direct bacterial involvement, dissolved as expected from results of geochemical modeling and observations of increased dissolved Mg concentrations in the anaerobic zone. Calcite, in contrast, precipitated in a wide variety of spiky morphologies to an absolute uniform elevation above the underlying crystal surface The uniform ultimate elevation of calcite precipitate suggests a physical control on reactant supply.

Bacteria colonizing quartz and microcline were in close proximity to etch pits, exhibiting a spatial correlation between bacteria location and etch pits. The etch pits, however, were not localized at the actual contact surface of the microbe, and the euhedral shape and extent of etching suggest the mineral surface was in contact with an aqueous weathering fluid. Localized mineral etching is proposed to have occurred in a reaction zone at the bacteria/mineral interface where high concentrations of organic acids, formed by bacteria during metabolism of hydrocarbon, selectively mobilized silica from the mineral surface.

Table of Contents

List of Figures xii

List of Tables xvii

Introduction 1

Microbes in the Subsurface 4

Extent and Limits 5

Controls on Growth 7

Bacterial Morphology and Taphonomy 11

Microbial Diagenesis ...14

Carbonates 15

Dissolution: Occurrence and Mechanisms 15

Precipitation: Occurrence and Mechanisms 16

Silicates 20

Dissolution: Occurrence and Mechanisms 20

Precipitation: Occurrence and Mechanisms 22

Bacterial Use of Hydrocarbons 22

Alkanes 24

Metabolism and Byproducts 24

Aromatics 29

Metabolism and Byproducts 30

Purpose of the Study 31

Approach 32

Laboratory Experiments 33

Field Experiments: Background 33

Regional Geology 38

Study Site 39

Hydrogeology 40

Mineralogy 41

Microbiology 42

Organic Geochemistry 42

Ground Water Chemistry 45

Methods and Materials 51

Bacterial Culturing, Isolation, and Identification 52

Bacterial Morphology and Taphonomy 53

Batch Reactors 57

In-situ Microcosms 59

Etching of Calcite Collected From In-Situ Microcosms 67

Field Columns 68

Mineral Surface Characterization 68

Scanning Electron Microscopy 68

Sample Preparation 71

Image Collection 73

ED AX 74

Acid Leach 75

Geochemistry 76

Results 77

Bacterial Culturing, Isolation, and Identification 77

Bacterial Morphology and Taphonomy 80

Batch Reactors 87

Bacterial Colonization and Growth 88

Cell Morphology 88

Colony Morphology 90

Patterns of Colonization 90

Field Microcosms 90

Ground Water Chemistry 91

Silicates 97

Colonization 97

Surface Coatings 107

Dissolution 109

Precipitation 115

Carbonates 118

Colonization 118

Dissolution 119

Precipitation 123

Field Column Experiments ; 143

Discussion 145

Section 1 145

Bacterial Culturing, Isolation, and Identification 145

Bacterial Morphology and Taphonomy 146

Bacterial Colonization and Growth 151

Section 2 154

Effect on the Bemidji Aquifer of Microbial Activity 154

Bemidji Aquifer Diagenesis 159

Silicate Dissolution 159

Silicate Precipitation 165

Carbonate Dissolution 168

Carbonate Precipitation 170

Summary 180

Conclusions 182

Bibliography 185

Vita 205

List of Figures

Figure 1. Research site and well locations 36

Figure 2. Cross section of aquifer flow path in the field study area 37

Figure 3. Aromatic hydrocarbons and their oxidized intermediate forms identified from water collected from beneath and down-gradient of the oil body 46

Figure 4. Cross section through the center of the oil body at the study site 47

Figure 5. Generalized bacterial growth curve showing the phase of mixed culture growth estimated to correlate with samples of calcite crystals collected at 2 hours, 20 days, 30 days and 18 months. The growth curve is a semi-log plot with the y-axis on a log scale and the x-axis on an arithmetic scale 56

Figure 6. Schematic diagram of microcosm design and position in water wells 60

Figure 7. Electron micrographs of quartz grains prepared for microcosm experiments 63

Figure 8. Electron micrographs of microcline grains prepared for microcosm experiments 64

Figure 9. Electron micrographs of calcite grains prepared for microcosm experiments 66

Figure 10. Schematic design of the field flow-through column 69

Figure 11. Cell and colony morphology 79

Figure 12. Calcite crystal collected from the growth experiment after two hours incubation 81

Figure 13. Calcite crystal collected from the growth experiment after two hours incubation contrasting smooth and cracked surface film 82

Figure 14. Calcite crystal collected from the growth experiment after 20 days incubation 84

Figure 15. Calcite crystal collected from the growth experiment after 18 months incubation 86

Figure 16. Bacteria strains are not identifiable by morphology after incubation in batch reactor experiments 89

Figure 17. X-ray diffraction analysis of precipitate formed in ground water samples collected from wells 015 and 017 in October, 1992 94

Figure 18. Precipitate formed in ground water samples collected during the recovery of microcosms from wells 015 and 017 95

Figure 19. Electron micrographs of authigenic calcite from precipitate formed in ground water samples 96

Figure 20. Electron micrographs of the surface coating of authigenic calcite from precipitate formed in ground water samples 98

Figure 21. Electron micrographs of authigenic calcite and Fe (?) precipitate formed in ground water samples 99

Figure 22. Electron micrograph of vegetative bacteria from precipitate formed in ground water samples 100

Figure 23. Electron micrographs of growth phase bacteria attached to surfaces of prepared minerals 102

Figure 24. Electron micrographs of bacterial attachment mechanisms observed on microcosm mineral surfaces 103

Figure 25. Electron micrographs of various colonization morphologies observed on surfaces of the microcosm minerals 105

Figure 26. Electron micrographs of prepared minerals recovered from field columns 106

Figure 27. Electron micrographs of various minerals recovered after 14 months in-situ below the oil layer (zone 3) at the Bemidji, MN research site 108

Figure 28. Stereo pair image of bacteria colonizing the surface of quartz overgrowths on a sand grain from the batch reactor experiments. .110

Figure 29. Electron micrographs of quartz crystals recovered from anaerobic zone microcosms after 14 months 112

Figure 30. High resolution scanning electron micrographs of "cleaned"quartz grains 114

Figure 31. Electron micrographs of surface of microcline crystal fragment recovered from in-situ microcosm after 14 months 116

Figure 32. Electron micrographs of authigenic clay minerals on the surface of microcline grains 117

Figure 33. Electron micrographs of calcite crystals recovered from in-situ microcosm after 14 months 120

Figure 34. Freshly fractured crystal of Iceland spar calcite etched for 120 seconds in 10% HCI 121

Figure 35. Dolomite recovered after 14 months from Zone 3 in the Bemidji aquifer 124

Figure 36. Morphologies of precipitate covering the surface of calcite crystals recovered from Bemidji microcosm experiments 125

Figure 37. Transition areas or borders between spike morphologies on precipitate-covered calcite 127

Figure 38. Three dimensional cross-section of a single layer of surface precipitate 128

Figure 39. High resolution SEM imaging of calcite spikes and flat area between spikes 130

Figure 40. Atomic force microscopy image of the surface of a precipitate- covered calcite crystal collected from well 018 at the Bemidji research site 132

Figure 41. Overview of a precipitate-covered calcite grain which has been incised by a scratch to reveal the unaltered interior calcite 133

Figure 42. Diagrammatic map of the surface of a calcite grain recovered from the Bemidji microcosms which has been etched in HCI and incised with a scratch 135

Figure 43. Unetched column-type precipitate 136

Figure 44. Lightly etched column-type spikes from the ten second-etched zone 138

Figure 45. Medium etched column-type spike from the 30 second etched zone 139

Figure 46. Cross section of the unaltered mother grain calcite and surface precipitation spikes that have been etched almost to their bases. ...140

Figure 47. Surface features of precipitate-coated calcite recovered from the microcosm experiment in well 018 at the Bemidji study site which has been bleached or heated to remove organic matter from the surface 142

Figure 48. Conceptual model of bacterially generated zone at the surface of a quartz crystal fragment 163

Figure 49. Congruent dissolution of microcline, local transport of dissolved constituents, and local precipitation of clay minerals 167

Figure 50. Bacterial etching of calcite and availability of dissolved constituents for precipitation 171

Figure 51. Unetched surface precipitate on calcite crystals which show a sequence of precipitate growth 174

Figure 52. Calcite precipitation catalyzed by bacterial nucleation 176

Figure 53. Supply of reactants for calcite precipitation 179

List of Tables

Table 1. Mineralogy of 2-4 phi size fraction aquifer sediments in the study area 41

Table 2. Physical and chemical characteristics of the oil spilled at Bemidji 43

Table 3. Chemical analysis of ground water samples collected June, 1987 49

Table 4. Nutrient-enriched seawater formula 54

Table 5. Etching of unaltered Iceland spar calcite in HCI 75

Table 6. Key to results of biochemical testing 78

Table 7. Water well chemistry on October 10, 1992, Bemidji, MN research site. Microcosms were recovered from well 017 and 015 after 14 months of in-situ reaction with anaerobic ground water and indigenous bacteria 91

Table 8. Results of analysis of calcite precipitate from microcosm experiment in well 018 at the Bemidji study site. Values for magnesium, calcium, and strontium are reported in pg/ml 143

Introduction

This dissertation focuses on an infinitesimal target in order to address a big question: do bacteria which occur naturally in the terrestrial subsurface effect mineral diagenesis?

Until recently, bacteria were thought to be absent from the subsurface greater than 10m deep (Ghiorse and Wilson, 1988). In fact, the ground water saturated zone is an excellent environment for bacterial life (e.g. Balkwill 1989; Fredrickson et al., 1989). The small size and ability to adapt to extreme environmental conditions allow bacteria to flourish in many subsurface environments. At low temperatures (<BO°C), interconnected porosity, abundant surface area, and the presence of dissolved organic matter in interstitial pore water allow bacteria to grow in and move through a sedimentary rock environment. Recent drilling and sampling for indigenous bacteria has extended the depth to which bacteria are known to live in the subsurface to 1160 m (Fliermans et al., 1994).

The study of subsurface microbiology has exploded during the past eight years. Microbiologists, microbial ecologists, hydrogeologists and geochemists have turned their attention to the saturated zone as an environment that includes microorganisms as well as rocks, water, and dissolved organic and inorganic solutes (DOE, 1992). For example, of 432 papers produced from 1985 - 1992 as the result of U. S. Department of Energy funding of deep subsurface microbiology research, 260 papers address hydrologic and contaminant transport issues, 78 address microbial ecology of terrestrial subsurface environments, 51 focus on microbial transformation of some type of contaminant, and 24 are taxonomic descriptions of novel or unexpected subsurface microorganisms. However, only 19 papers could be considered to address issues of mineral alteration and of these 19, only four actually investigate questions of the role of bacteria in the transformation of minerals or metal oxides in aquifers. Francis and Dodge (1988, 1992) investigated the role of a species of the strictly anaerobic bacteria Clostridium on the dissolution of metal oxides, Fredrickson et al. (1989) studied chemolithotrophic and heterotrophic bacteria and pore water chemistry, and Kelly and Herman (1992) ran theoretical models of ground water chemistry that included the effects of benzene metabolism and concluded that both calcite and iron oxyhydroxides dissolve as a result of bacterial metabolism.

The geochemical effect of bacterial metabolism within rock pores is a component of early and shallow-burial diagenesis that has been largely unrecognized and under-appreciated by most geologists. Bacteria are the major recyclers of naturally occurring organic matter, gaining energy and carbon for growth from the metabolic transformation of complex organic molecules ultimately to CO2 and H2O. Organic matter, inorganic nutrients and minerals from the environment surrounding bacteria are actively transported into the cell via enzymatically catalyzed reactions, and metabolic by-products are discarded from the cell back to the environment (e.g. Brock and Madigan, 1988).

In the saturated-zone bacterial activity occurs in the pore space microenvironment. The cell spends energy to grow, and in the process, concentrates or dilutes biochemicals and minerals at non-equilibrium concentrations for its own uses. Many of these biochemicals are reactive with dissolved and solid phase minerals. Bacterial cells also have certain characteristics, such as electrical surface charge and the capability to concentrate ions at high concentrations on their surfaces, that increase their probability of effecting a rock-water interaction. Thus, bacteria have the potential to directly effect mineral dissolution and precipitation.

Whereever organic matter is found in sufficient concentrations and for extended periods of time, some type of bacteria is likely to exist that has a capability to use it as an energy source. Crude oil and various fractions of crude oil are commonly found in deep subsurface environments as natural deposits and increasingly in shallow aquifers as contaminants. Many bacteria use hydrocarbon as a source of carbon for energy and growth in both aerobic and anaerobic conditions. The intermediate by-products of hydrocarbon metabolism, alcohols, aldehydes and especially organic acids, build up in the extracellular microenvironment of the surrounding pore water, are highly soluble in water, transport with water flow and in many cases are highly reactive with both dissolved and solid phase minerals and rocks.

Physically, bacteria bodies may be catalysts for mineralization or dissolution effects by acting as nucleation sites or as "lightning rods" for the concentration of cations (e.g., Morita, 1980; del Moral et al., 1987; Folk, 1993). At the scale of individual depositional units within an aquifer, extensive or prolonged bacterial metabolism may substantially alter pH and redox conditions and fundamentally alter large areas of an aquifer's chemistry and therefore its diagenetic processes.

Given these observations, it is reasonable to assume that direct evidence of bacterially mediated diagenesis would be abundant in the geological literature. It is not. The purpose of this study is to investigate if naturally occurring bacteria that exist in the terrestrial subsurface effect mineral diagenesis.

Microbes in the Subsurface

The utilization and cycling of organic matter by microorganisms in the shallow subsurface (to 50 cm depth) by microorganisms has been recognized as fundamental to organic-enhanced mineral weathering in unsaturated soil systems (e.g. Huang and Schnitzer, 1986; , Berthelin, 1988; Sposito, 1989; Folk, 1993). Recent investigations of ground water-saturated sedimentary rocks, however, demonstrate that viable and biochemically active heterotrophic bacteria and fungus in densities up to 10$ cells per gram of sediment are present to a depth of 1,600 m (Fliermans et al., 1994). Indigenous bacteria have been recognized from oil reservoirs and associated brines since the 1920's (Bastin, 1926; Davis 1967). Russian workers in the 1950’s and 60's examined in detail bacteria from oil reservoirs (e.g. Kuznetsov, 1950; Ashirov and Sazonova, 1962). Further, these bacteria occur as both planktonic and sessile forms, with mineral surfaces of pores providing the surface for bacterial attachment. Since viable bacteria have been shown to exist to a depth of at least 1,600 m, it is reasonable to investigate what role, if any, they may have in in-situ mineral transformations.

Extent and Limits

The extent of bacterial colonization of the terrestrial subsurface is poorly understood. Recent work, however, has shown that bacteria live and are metabolically active in deeper and more varied aquifer conditions than previously expected (Chapelle, 1993). Local flow systems, with rapid recharge from infiltration, often rapid ground water flow, and discharge to local topographic lows, represent an excellent environment for bacteria. McNabb and Dunlap (1975) reported dense and diverse populations of heterotrophic bacteria in shallow aquifers contaminated by hydrocarbons. Wilson et al. (1983) found bacteria in concentrations up to 10$ cells/gm of sediment in an investigation of the distribution and diversity of bacteria indigenous to a shallow aquifer. Ghiorse and Wilson (1988) reviewed microbial ecology of the terrestrial subsurface in a paper that sparked increased interest in the subject from both pure and applied scientists. Since the early 1980's many workers from hydrogeology, microbiology, and civil engineering disciplines have studied the microbiology of shallow contaminated aquifers in search of new tools in the battle against contaminated ground water. The technique of "bioremediation", the use of naturally occurring microorganisms to transform organic pollutants into non-hazardous materials, has become a leading technique for the clean-up of hydrocarbon-contaminated soil and ground water. The importance and popularity of the subject is reflected in the flood of journal articles published and a number of recent volumes dedicated to understanding the state of the art of bioremediation (e.g. Riser-Roberts, 1992; Chapelle, 1993; National Research Council, 1993).

Russian workers recognized that bacteria were indigenous to deeper aquifers which were part of intermediate flow systems in the 1950's (e.g. Kuznetsov, 1950). Chapelle and Knobel (1985) reported that indigenous bacteria produced CO2 in a regional aquifer in Maryland. Sinclair and Ghiorse (1989) reported high densities, from to 10$ cells/gram of sediment, and high diversity of both bacteria and eucaryotes from the Pee Dee, Black Creek and Middendorf formations of S. Carolina which range from 150 -300 m in depth. Chapelle et al. (1987, 1988), Chapelle and McMahon (1991) and Chapelle and Lovley (1992) reported on bacterial metabolism and CO2 production in deep coastal plain aquifers along the southern east coast of the United States. An important observation from each of these studies was that the density of bacteria increased with increasing porosity and permeability. Where as fine grained sediments did contain bacteria, the density per gram of sediment was lower by up to several orders of magnitude than in coarser-grained sediments.

The study of bacteria in deep, regional flow systems has been carried out largely by petroleum microbiologists (e.g. Kuznetsov et al., 1963; Davis, 1967). Although many workers were convinced that indigenous bacteria were responsible for a wide range of geochemical activity, they could not prove conclusively that bacteria were active in-situ. Collection of bacteria from oil wells is accomplished by taking produced water at the well-head, a process that allows "contamination" by surface bacteria and bacteria that live in the upper pipes of the wells. The possibility that the bacteria reported to be collected from deep reservoirs actually came from surface facilities was impossible to disprove, subsequently much of the early published work on deep subsurface bacteria was not taken seriously by most geologists. Recent deep drilling and recovery of core material with aseptic techniques for the purpose of establishing the presence of bacteria, however, has resulted in the confirmation of active bacteria from the surface to a depth of 3800 ft (Chapelle, 1993; Fliermans et al., 1994).

Controls on Growth

The microbial ecology of indigenous bacteria in hydrocarbon-rich aquifer environments has been studied intensively (e.g. McNabb and Dunlap, 1975; Freeze and Cherry, 1979; Mathess, 1982; Bouwer and McCarty, 1984; Balkwill and Ghiorse, 1985; Ghiorse and Wilson, 1988; Phelps et al., 1988; Lovley et al., 1989; MacQuarrie and Sudicky 1990 a and b; Sinclair and Ghiorse, 1991; Baedecker and Cozzarelli, 1991; Chang et al., 1991; Bennett et al., 1993; Baedecker et al., 1993; Eganhouse et al., 1993; and National Research Council; 1993). Bacteria survive in a wide range of environmental conditions, but the conditions of optimal metabolic activity are constrained to a much narrower range of conditions.

Environmental conditions control microbial abundance and activity in terrestrial aquifers. For heterotrophic bacteria (those that use organic carbon for energy), the type, distribution and concentration of organic matter in the aquifer material and interstitial pore waters limits the extent of their growth. In most subsurface waters the concentration of natural organic carbon is low, often less than lmg DOC/liter and aquifer solids contain only trace amounts of water- soluble OM, but waters associated with oil reservoirs, peat, and coal deposits may contain 2-10 mg/1 DOC or more (Ghiorse and Wilson, 1988).

The type of organic matter found in aquifers depends on the source material of the geologic formation, water recharge rates and biological activity in the recharge zones. Reactive organic matter may be degraded by aerobic soil microorganisms as it moves with infiltrating precipitation towards ground water. Organic matter commonly found in shallow terrestrial aquifers includes humic substances, naphthenic acids and phenolic compounds derived from degrading plants.

Bacteria require more than carbon for growth. Nitrogen is required for synthesis of amino acids, phosphorus for synthesis of ATP (energy) NADPH (reducing power) and potassium for enzyme production. Trace amounts of metals are also required for metabolism and growth. Lack of any of the required inorganic nutrients will limit or stop growth. In pristine aquifers, nitrogen and phosphorus are usually not found in levels high enough to support intense and sustained microbial growth (Ghiorse and Wilson, 1988; Chapelle, 1993). Oxidation of organic nitrogen from decomposing plants and animals occurs only in aerobic conditions and therefore mostly in the upper soil zone. However, phosphate can be readily mobilized from mineral phases by microbes and nitrogen can be recycled to the extent that concentrations of N and P and trace minerals occur in amounts sufficient to support at least a vestigial population of indigenous microorganisms (Ghiorse and Wilson, 1988).

Many shallow aquifers contain significant amounts of dissolved oxygen which is essential for aerobic metabolism. Aerobic bacteria use molecular oxygen as a terminal electron acceptor during the metabolism (oxidation) of organic matter. Aerobes are the dominant type of bacteria in the soil and shallow ground water environment in which trace amounts of oxygen are present. Anaerobic bacteria use electron acceptors such as sulfate and nitrate in the oxidation of organic matter. Manganese oxides and iron oxyhydroxides which may act as electron acceptors in bacterial respiration are abundant in many subsurface zones. Microbial oxidation of these solid phase minerals may account for the oxidation of organic matter and mobilization of Fe 2+ and Mn 2+ in some anaerobic ground waters (Matthes, 1982; Lovley and Phillips, 1987). The concentration of oxygen, Fe 3+ , nitrate, and sulfate for use as a terminal electron acceptor in bacterial metabolism controls what type of bacteria will be dominant in a subsurface environment.

Solute concentration of ground water alters the availability of water and osmotic pressure of bacterial cells. Solutes such a salt and sugar have an affinity for water and the water associated with such solutes becomes unavailable to microorganisms (Brock, 1988). In hypertonic solutions, microorganisms may shrink and become desiccated and in hypotonic solutions the cell may burst.

Most bacteria have a wide pH range in which they can survive and much narrower range in which they grow optimally. Most ground water environments have pH values between 5 and 9, a range in which most bacteria grow well (Baas- Becking, 1965). Microbial growth rates are greatly influenced by pH values because of their effect on proteins and enzymes. At extreme ends of the pH spectrum the charge interactions of the amino acid R-groups cause enzymes to be inactive. Enzyme functions are essential to bacterial metabolism, growth and reproduction. If enzymes are inhibited by extreme pH conditions, bacteria stop growing and may stop functioning completely.

In ground water environments, temperature is one of the most important environmental factors controlling what type bacteria survive, bacterial growth rates, and rates of chemical transformations related to microbial activity. Most bacteria have a range of temperature tolerance of between 30 and 40°C. The temperature of ground water in the temperate region of North America favors psychrophilic bacteria, defined as those bacteria that grow at an optimum temperature between 0 and 20°C (Brock and Madigan, 1988). Shallow ground water temperatures typically show little annual variation around a mean temperature, and this relatively constant temperature favors sustained bacterial activity.

Increasing temperature causes bacterial metabolic functions to increase in rate to a point and then suddenly stop functioning. Loss of function occurs at the temperature at which proteins and protein-related cell components lose their structure. Bacteria vary in their capability to resist heat damage and some thermophyllic bacteria thrive at temperatures greater than the boiling point of water. Heat sterilization of surfaces is routinely carried out in hospital and laboratory autoclaves at 121 °C for 15 minutes at 15 lb/in 2 pressure. Autoclaving of thermophiles requires longer times and higher heats to achieve sterilization. Temperature in the subsurface saturated zone generally increases with depth. The depth limit for the existence of deep subsurface bacteria may ultimately be determined to be a function of depth-related upper temperature limits on protein function of the indigenous organisms.

Bacterial morphology and taphonomy

The shape of a bacterial cell is a characteristic of limited value in the determination of species since the size and shape of bacteria depend largely on environmental conditions and can change with changing conditions. Bacterial identification is based largely on elucidation of biochemical transformations that the cell carries out and on biochemical and genetic characteristics.

The recognition of cells by morphological characteristics as a method to identify that bacteria are present, however, is a useful survey technique. Examination of geological samples for analysis of composition, texture, and relationship of mineral dissolution and precipitation events is usually carried out by microscopy, and therefore the recognition of the morphology of bacterial cells is an important element of reconnaissance petrography or electron microscopy. Bacteria occur in a narrow range of sizes and only in a few basic shapes. The four major shapes of bacteria are rods, spheres, spiral rods, and curved or v-shaped cells. Most vegetative, or growing, bacteria range in size from 0.5 to 2.opm in length and about o.spm in diameter (Brock and Madigan, 1988).

Bacteria smaller than o.4pm diameter have been recognized by workers studying non-optimal growth environments. In the soil, the diameter of indigenous bacteria ranges from less than o.4pm to >o.Bpm, in some cases with a preponderance of the cells in the <o.4pm category. Casida (1977) found that the common soil microorganism Agomyces ramosus, which is known from liquid culture growth experiments to be usually greater than o.spm diameter, was shown to maintain active metabolism as shrunken cells ranging from 0.2 to o.3pm in diameter. The small size was induced by nitrogen starvation and growth on a solid surface, both common conditions in subsurface environments. Bakken and Olsen (1987) separated soil bacteria by filtering into <o.4pm, 0.4 to o.6pm, and >o.6pm diameter size fractions. 82% of the total soil microbiota fell in the size range of < o.4pm diameter, 15% in the mid-size range and 3% in the >o.6pm diameter cells. 10% of the smallest cells increased in size during growth experiments, suggesting that the majority of these bacterial cells were not small forms of ordinary-sized bacteria, but rather fully functioning "dwarf" cells. However, the viability (those capable of growth on the test media) was only 0.2% for the smallest cells, 10% for mid-range cells, and 30-40% for largest cells. Thus, if one assumes that dwarf and normal cells have equal capability to grow on the test media, the results suggest that while large numbers of < o.4pm diameter cells exist, larger cells may be responsible for most of the bacterial metabolic activity in soil.

Novitsky and Morita (1977), and Torella and Morita (1981) described minute forms of bacteria which range between 0.1 and o.spm in diameter. These "ultramicrobacteria" were reported to form as the result of the bacteria's survival response to "stress" such as starvation or other harsh environmental conditions. They reported that as stress increased, full-sized bacteria stopped growing and reduced to 0.1 to o.4pm diameter spheres.

Folk (1993) reviewed the occurrence of fossilized "nannobacteria" in carbonate sediments and rocks. He described abundant 0.1 pm spherical bodies which he interpreted to be bacteria (nannobacteria) entombed in a wide diversity of carbonate rocks. The nannobacteria were revealed by etching the carbonate rock with HCI and studying the specimens with high magnification SEM. Folk proposed that the nannobacteria play an active role in the precipitation of carbonate materials. Pentecost and Folk (1992) reported preliminary results that nannobacteria collected from travertine were cultured and reproduced multiple o.lmm diameter spheres. However, these results have not yet been published in detail or confirmed by other workers.

For the purpose of describing SEM images in the results and discussion section, the following terms will be used in this dissertation:

Bacteria - cells in the 0.5 to 2.opm diameter size range

Nannobodies - 0.05 to o.4pm diameter spherical and eliptical particles that morphologically resemble nannobacteria or stressed bacteria

Microcurdle- Bumps or hemispherical particles < 0.05 pm in diameter.

The term nannobodies is defined heein in order to describe round particles in the size range of 0.05 to o.4pm in diameter without conveying an interpretation of origin. The term "microcurdle" is synonymous with the grain-coating surface texture described by Westall and Rince (1994) as a "retracted granulated" surface.

Microbial Diagenesis

Many geologic investigations of ancient rocks suggest that bacteria have played an important role in rock formation and alteration. For example, Kuznetsov et al., (1963), Ehrlich (1981), Krumbein (1983), and Chilingarian and Wolf (1988) have reviewed in broad terms bacterial roles in a variety of diagenetic processes in both silicate and carbonate rocks. Fossil bacteria are well known in the rock record, preserved as fossils and by evidence of their geochemical activity such as framboidal pyrite, manganese nodules, and carbonate precipitates such as stromatolites, carbonate concretions, and travertine deposits (e.g. Folk and Chafetz, 1980; Folk, Chafetz and Tiezzi, 1985; Folk, 1990). The occurrence of fossil bacteria establishes that bacteria were present in ancient depositional and diagenetic environments. However, the presence of fossil bacteria does not establish what role, if any, the bacteria played in the alteration, either precipitation or dissolution, of their entombing rocks. Gathering direct evidence of the mechanism of bacterial alteration of minerals and rocks requires experiments with, or observations of the effect of, live bacteria.

As a chemical system, living bacteria are not in equilibrium with their external aqueous environment. The cell is an open system in which energy is taken from the surroundings and used to grow and to maintain the cell structure (Brock and Madigan, 1988). Chemical gradients of inorganic and organic compounds are maintained within and around the cell that may be, but are not necessarily, in equilibrium with the chemistry of the surrounding fluid (Atlas, 1984; Gibson 1984). Bacteria can be considered to be chemical machines which convert large molecules into smaller ones and build small molecules into large complex molecules all the while catalyzing oxidation-reduction reactions by means of their enzymes.

It is the capability of bacteria to act as chemical machines that make them especially interesting in the study of diagenesis. Depending on the geochemical environment, byproducts of microbial metabolism may build up to measurable quantities in the surrounding extra-cellular environment and directly effect mineral dissolution and precipitation.

Carbonates

Dissolution: Occurrence and Mechanisms

Bacterial dissolution of carbonates has been reported by a number of dissolution mechanisms including organic and amino acids leaching and direct cell-substrate dissolution (Arnold et al., 1988). Strasser, Burgstaller, and Schinner (1990) reported that copper and antimony metals were released from dolomite by the metabolic action of a strain of Pseudomonas during an eight day incubation in the presence of starch and glutamine. They concluded that the metals were complexed by amino acids instead of carboxylic acids. In marine sediments near saturation with respect to aragonite (the principal component), Walter and Burton (1990) reported dissolved carbonate released to the water column from the oxic upper meter of sediment. They attributed the dissolution to acid generation by aerobic respiration and oxidation of dissolved sulfide in burrow and sediment pores, but did not explicitly identify the role of heterotrophic bacteria in the process. Mass losses from the carbonate sediment were on the order of 500p.m01/cm 2 /year.

Precipitation: Occurrence and Mechanisms

The role of bacteria in the precipitation of calcium carbonate has been reported from many field studies of ancient rock. Ashirov and Sazonova (1962) studied a calcite layer beneath the Pokrovka oil reservoir in the Volga-Ural Basin and determined that it was formed as the result of the reduction of sulfate by sulfate reducing bacteria. Folk and Chafetz (1980, 1983) and Chafetz and Folk (1984) reported that bacteria formed low magnesium, microporous calcite travertines and pisoliths in a variety of natural spring environments, and described five morphologies of bacterially precipitated calcite aggregates. Chafetz and Meredith (1983) reported a bacterial role in the formation of travertine pisoliths in Idaho hot springs. Folk, Chafetz, and Tiezzi (1985) further described the role of sulfur bacteria in the deposition of Italian travertines.

Tiezzi and Folk (1988) described calcite cements from within stromatactis-type cavities of the Mississippian Lodgepole Formation in Montana as cloudy with bacterial bodies. They attributed the precipitation of these calcite cements to the biochemical activity of live bacteria in the original reef. Folk (1986, 1987) proposed that gray colored carbonate nodules in the Triassic black Portoro Limestone of Italy are the result of local early bacterial diagenesis. Chafetz (1986) identified high magnesian calcite peloids with fossilized clumps of bacteria at their center. He proposed that the nuclei of many marine peloids originated as a fine-grained precipitate of high magnesian calcite within and around active clumps of bacteria and that the vital activity of the bacteria influenced the precipitation of the calcite (p. 816). Hiebert (1988) described fossil bacteria trapped in calcite cements of the Posidonienschiefer Shale and proposed that when the bacteria were alive, they performed a catalytic role in the precipitation of the entombing cements. Folk (1993, and in press) has illuminated the virtually übiquitous occurrence of ultra-microbacteria or nannobacteria found within carbonate rocks, and has proposed their catalytic involvement in calcite precipitation.

The ability of a variety of bacteria to initiate carbonate precipitation has been studied in varying degrees of detail in the laboratory. Drew (1911) was an early worker who noted that denitrifying marine bacteria could precipitate carbonate in tropical and temperate sea water. Zobell (1946) reviewed processes of bacterial participation in carbonate precipitation and suggested that 1) bacterial production of ammonia during metabolism caused a pH rise, 2) that sulfate reduction was necessary, and 3) that only certain bacteria were capable of initiating precipitation events. LaLou (1957) experimentally produced a carbonate film on the surface of a mud-enriched sea water aquarium and attributed its formation to bacterially mediated changes in pH, CO2 and H2S. Berner (1969, 1971) systematically investigated carbonate during bacterial degradation of marine organisms. He found that the type of organic matter available to bacteria determined pore water conditions. He concluded that in restricted pore water conditions, the kinetics of base versus acid production during metabolism determined whether carbonate precipitated or dissolved.

In an open sediment-seawater system, bicarbonate will diffuse away from local concentrations into the open seawater reducing local bicarbonate alkalinity below the equilibrium carbonate ion product and thereby keeping carbonate in solution (Berner, 1970). However, if the microbial degradation of organic matter is occurring in a closed pore-water system, diffusion is restricted and the products of both reactions will build up high local concentrations in the sediment. The production and retention of ammonia in the sediment will tend to raise pH, thus favoring carbonate precipitation. Berner (1969) and Morita (1980) experimentally determined that during anaerobic microbial decay of marine organic matter in restricted conditions, pH of the sample waters rose above 8.5 and remained alkaline for the duration of experiments ( 8.5 months and 24 days, respectively). In restricted environments, bicarbonate concentration may exceed the equilibrium ion product for calcium carbonate and precipitation of calcite cement may begin.

The production of organic acids as a by-product of microbial degradation of organic matter in addition to the CO2aq formed during metabolism may have the effect of acidifying the sediment pore waters and inhibiting carbonate precipitation. Berner (1970) points out, however, that the rate of base production (ammonia and bicarbonate) versus the rate of acid production in the local restricted environment is the factor which ultimately controls whether calcite precipitates or stays in solution. Under sediment conditions where abundant nitrogen-rich organic matter is available, such as the deposition of a fresh fish carcass or a sudden release of reactive organic matter from sediment stirring, the production of ammonia may outstrip acid production and interstitial calcite cements may precipitate.

Besides the chemical role of providing bicarbonate and raising pH, bacteria may also contribute to carbonate precipitation by providing a discrete point of Ca ++ concentration and therefore a nucleation site for the growth of calcium carbonate crystals. Greenfield (1963) showed that marine bacteria concentrated Ca ++ on their cell walls. Morita (1980) also described bacteria that loosely bound Ca ++ with cell wall proteins. If free Ca ++ ions are concentrated on a bacterial cell wall in the presence of high concentrations of bicarbonate, calcium carbonate may form.

Morita (1980) described a microbial mechanism that produces calcium carbonate crystals on the exterior surface of bacterial cell walls. As the bacteria internally degrades organic matter, CO2 is produced as a by-product and diffuses through the cell wall, out into the surrounding fluids. The CO2 is available to combine with a base such as NH3 a q to form bicarbonate and ammonium. As a final result, bicarbonate is locally available to combine with Ca++ ions bound to the cell wall and precipitate calcium carbonate. Once a single crystal of calcium carbonate is formed, it may act as a nucleation site allowing the rapid abiotic precipitation of a generation of cement. Krumbein (1974) observed the precipitation of aragonite on the surface of marine heterotrophic bacteria in the presence of high concentrations of various types of organic matter. Morita (1980) and del Moral (1987) also found that marine and halophilic bacteria produced calcium carbonate crystals during laboratory experiments.

Oppenheimer (1961) noted that spherical crystals of aragonite formed in the presence of marine bacteria which were indigenous to the carbonate muds of the Bahamas. McCallum and Guhathakurta (1970) described bacterial bodies forming the nucleus of aragonite needles precipitated from nutrient-enriched sea water solutions. Krumbein (1974) found that marine heterotrophic bacteria which precipitate aragonite, Mg-calcite and monohydro-calcite under both aerobic and anaerobic conditions were first coated, and then encapsulated with carbonate. Krumbein noted that the actual cells were destroyed by later crystal growth. Morita (1980) and del Moral et al., (1987) also reported bacteria incorporated in carbonate precipitated from nutrient enriched sea water in laboratory experiments. Buczynski and Chafetz (1991) defined unique crystal morphologies of calcite crystals that were precipitated around the bodies of bacteria, both in the laboratory and in the field. Chafetz and Buczynski (1992) demonstrated that live bacteria from a cyanobacterial mat directly precipitated calcite, but killed bacteria did not.

Silicates

Dissolution: Occurrence and Mechanisms

The alteration of silicate minerals by microorganisms has been reported via several mechanisms. Eno and Reuzer (1955) experimented with Aspergillus niger (a fungus) and found that potassium was released from biotite, muscovite and microcline because of metabolically increased acidity of the soil environment. Webley, Duff, and Mitchel (1960) and Duff et al. (1963) made studies of silica degrading soil bacteria. They correlated silicate degradation with bacterial production of organic acids, especially 2-ketogluconic acid. Savotsin (1972) studied non-heterotrophic silicate degrading bacteria from tropical soils and reported that 26% of total potassium was released from orthoclase in 40 days of growth, an order of magnitude faster rate than weathering with carbonic acid alone.

In laboratory experiments with three strains of carbohydrate-utilizing bacteria, Rades-Rohkohl et al. (1978) showed bacterially colonized synthetic quartz crystals covered with shallow pits complete with bacterial bodies within each, but felt chemical data did not support an interpretation that bacteria dissolved quartz in their experiments. They reported total dissolved SiO2 in solution decreased for six days, then increased to saturation in six days but did not result in statistically significant changes. Eckhardt (1979) found that organic acids produced by heterotrophic yeasts released K, Mg, Fe, Al and Si from biotite over a 12 week growth experiment.

Ehrlich (1981) reviewed silicate dissolution by glucose-utilizing heterotrophic soil bacteria. He reported that the weathering and dissolution of silicate minerals was caused by chelation of cations by organic acids and bases and did not comment on any precipitation effects. Krumbein and Jens (1981) reviewed indirect physical evidence of bacterial dissolution of silicates, and bacterial precipitation of silicates, but not mechanisms of dissolution. They proposed that carboxylic acids are important in silicate weathering as chelating agents and concluded that microcrystalline quartz can be dissolved under natural conditions by a variety of microorganisms. However, they left the question of direct or indirect bacterial dissolution unresolved.

Bennett and Siegal (1987) reported dissolution of quartz at near-neutral pH by organic acid complexation in a shallow, crude-oil contaminated sand aquifer in Bemidji, MN. They proposed that the source of organic acids was the microbial metabolism of crude oil. Bennett et al. (1988) suggested that biodegradation of the organic component of a silica complex could release the silica and create supersaturated conditions. This would favor quartz overgrowths, precipitation of cherts and the replacement of carbonate cements by silica during diagenesis.

Precipitation: Occurrence and Mechanisms

Ferris, Fyfe, and Beveridge (1988) proposed a geochemical mechanism for the fossilization of individual bacteria based on experiments with Bacillus subtilis grown in a solution saturated with iron and colloidal silica. Weaver (1989) described microbial alteration of silicates in clay and shales.

Bacterial Use of Hydrocarbons

Hydrocarbon is a naturally produced and occurring organic substance widely distributed in sedimentary basins. Many aerobic and anaerobic bacteria are capable of using hydrocarbons as a carbon source for growth and energy (e.g. Atlas, 1984; Gibson, 1984; and Lovley et al., 1989). Metabolism of hydrocarbons often results in the production of a variety of metastable intermediate byproducts.

This investigation will focus on bacteria that live on hydrocarbons and the resultant geochemical reactions of the bacterial metabolism of hydrocarbons in crude oil. Crude oil is a commonly distributed organic substance in sedimentary basins. In addition, hydrocarbons have entered the shallow subsurface environment as pollutants as the result of accidental spills and normal operational leakage in the global petroleum business.

Soil and aquatic bacteria use petroleum hydrocarbons as a source of carbon for energy and growth., with paraffins most susceptible followed by isoparaffins and aromatics (Perry, 1984). Diverse bacteria have evolved with the ability to utilize hydrocarbon as a sole energy and carbon source using various metabolic pathways. The role of bacteria in hydrocarbon degradation has been studied for many years (e.g., Atlas, 1984). Pre-1940 work was primarily in Russia and Germany, and represented some of the earliest studies of oil-bacteria interactions. During the 19405, interest shifted to the role of microorganisms in petroleum formation and preservation in reservoirs. The decade of the 1950-60 s was dominated by advances in the understanding of metabolic pathways, cataloging taxa, and in pioneering studies in microbial ecology. During the 19705, major marine oil spills stimulated interest in the fate of petroleum in the environment and the role of natural bacteria in hydrocarbon pollution remediation. The 1980's have been a period of rapid advances in the knowledge of the genetics and molecular biology of bacterial hydrocarbon degradation coupled with a renewed interest in the microbial ecology of pollution-stressed environments.

Certain species of bacteria, actinomycetes, yeasts, fungi, protozoans, and microalgae are all reported to utilize hydrocarbons as food sources (Floodgate, 1984). Even mammals can transform polyaromatic hydrocarbons in liver tissue; unfortunately the byproducts are carcinogenic (Cerniglia, 1984). This review will focus on bacterial degradation of the three main fractions of crude oil: straightchain alkanes, cyclic alkanes, and aromatics.

Crude oil is a complex mixture of tens of thousands of compounds that differ markedly in volatility, solubility, and susceptibility to biodegradation (Cooney, 1984). The bulk of petroleum components can be grouped into three broad categories of hydrocarbon type. The straight chain and branched alkanes range from 27-72% of a crude petroleum, cyclic alkanes may constitute from 22- 67%, and aromatics 6-14%. Other organic compounds in crude oil are characterized by the inclusion of nitrogen, sulfur, or oxygen in their molecular structures and make up only a few percent of any crude oil. These compounds will not be discussed here.

Alkanes

Many types of bacteria, molds and yeasts have evolved that can utilize alkanes as a sole carbon and energy source. While a great diversity of genera are capable of degrading alkanes, strains of the genus Pseudomonas, Acinetobacter, and Bacillus are the most common and vigorous alkane utilizers (Chapelle, 1993).

Metabolism and Byproducts

In the most general terms, alkanes are degraded by bacteria via a sequence of oxidations to the corresponding alcohol, aldehyde and monobasic fatty acid. The oxidation state of the oxidized carbon falls from -3 in the terminal alkyl group to + 3 in the fatty acid, as the organism derives energy from the reaction. The fatty acids are further degraded to acetyl-CoA and ATP via the beta-oxidation pathway. Acetyl-CoA is then available as fuel for the Krebs-cycle. Each oxidation step is catalyzed by highly specific enzymes.

The utilization of alkanes as a sole source of carbon for bacteria is considered by most microbiologists to be strictly an aerobic process (Atlas, 1984). Molecular oxygen is required for the first, and rate limiting, step of the oxidation process. Three mechanisms have been proposed to describe the primary oxidation of an alkane to a corresponding alcohol, they are: 1) hydroxylation, 2) dehydrogenation, and 3) hydroperoxidation.

Hydroxylation occurs at the C-l position, monoterminal methyl group. The first reaction requires an alkane, 02, a monooxygenase enzyme and the coenzyme NADH. The alkane and the oxygen are available to the cell from the external environment and, if hydrocarbon metabolism is internal, must be transported across the cell membrane. One of the oxygen atoms from O 2 bonds with the methyl group to form an alcohol with the same carbon chain number, the other oxygen is reduced to water (Singer and Finnerty, 1984). This primary alcohol is oxidized to the corresponding aldehyde by an alcohol dehydrogenase plus coenzyme NAD, and the aldehyde oxidized to a fatty acid in the presence of an aldehyde dehydrogenase plus NAD. Methyl group oxidation of alkanes yields fatty acids of identical carbon number and these fatty acids are assimilated directly into cellular lipids and/or are exported from the cell and accumulate in the surrounding aqueous environment. These extracellular organic acids are water soluble, mobile with water flow and provide both oxidizable substrates for other bacteria and are geochemically reactive compounds. Fatty acids derived from alkane that stay within the cell and are used for food are oxidized by an inducible beta-oxidation system to the level of acetate for even-chain alkanes and to propionate for odd-chain alkanes (Singer and Finnerty, 1984). Acetate is subsequently oxidized in the Krebs cycle. Final end-products are new cells, CO2 and H2O.

The accumulation of fatty acids in the extracellular aqueous environment may act as a stimulus to further alkane use. If the residual fatty acid (now a water-soluble polar molecule) emulsifies alkane, alkane surface area and solubility increases. Thus more alkane is available from the bulk phase to microbial degradation mechanisms. Increased surface area or solubility allows the more rapid oxidation, or metabolism, of alkane.

Dehydrogenation of alkanes to alkenes has been proposed by several authors (e.g., Gallo et al., 1973 a). This mechanism would result in the transformation of alkane to alcohol without the interaction of molecular oxygen, instead relying on the breakup of water molecules to supply the oxygen to form the hydroxyl group at the terminal methyl group. This mechanism has not been substantiated by enzymological studies (Singer and Finnerty, 1984).

Hydroperoxidation is another alternative mechanism for the first step of the alkane oxidation, in which a dioxygenase incorporates both oxygen atoms from O 2 into the terminal methyl group to form an n-alkyl hydroperoxide, which in turn is reduced to an alcohol:

R-CH2-CH3 + 02 — > R-CH2-CH2-OOH + NAD(P)H + H+ -—>

R-CH2-OH + NAD(P)+ + H2O.

The final result of this pathways is a fatty acid, which is available for betaoxidation.

There is no net difference between the hydroxylation, hydrogenation, and hydroperoxidation mechanism in terms of the production of final metabolic products, but the pathways are considerably different. This is significant as a measure of the diversity of mechanisms that have evolved to utilize alkanes as an energy source.

Although most published degradation mechanisms of alkane indicate that O 2 is essential to the oxidation process, several researchers have reported evidence of anaerobic degradation of alkanes. The initial hydroxylation of alkanes is the major barrier to anaerobic degradation. Once the alcohol is formed, further oxidation is not restricted, provided that there is an oxidation-reduction balance (Britton, 1984). Traxler and Bernard (1969) reported that a strain of Pseudomonas metabolized alkanes to fatty acids in strict anaerobic conditions. Sulfate-reducing bacteria have been noted as possible hydrocarbon degraders, in which methane acted as the electron donor, sulfate as the electron acceptor and acetate as carbon source. Nitrate has also been suggested as a final electron acceptor in place of oxygen (Parekh, Traxler, and Sobek, 1977).

Most research on metabolic pathways of alkane degradation is based upon laboratory studies of pure cultures. Several reports of anaerobic degradation of alkanes have been reported from field studies in which degradation would be the result of the activity of a mixed population of symbiotic bacteria. In these circumstances microbial utilization of organic matter other than alkane could serve as energy source and allow cometabolism of alkanes.

Less attention has been focused on branched-chain compounds than on straight chain alkanes. In general, dibasic acids are produced as by-products of branched alkane degradation. Secondary alkanes are easily degraded but tertiary alkanes are significantly more recalcitrant to metabolism. McKenna (1972) reported that secondary alkanes were readily utilized by diverse bacteria as carbon sources, but that tertiary branched alkanes were not. Pirnik et al., (1974) suggested that branched alkane metabolism is repressed by products of n-alkane oxidation.

Cyclic-alkanes are produced in nature by plants and microorganisms and have been preserved in crude oil and other geologically stored hydrocarbons. Cyclic-alkanes form a part of carotenoids, plant terpenoids and are incorporated into the lipids of many bacteria. Cyclic alkanes are the most recalcitrant fraction of petroleum to bacterial degradation, yet microbes are known that can utilize these compounds.

The number of types of bacteria capable of oxidizing cyclo-alkanes is far fewer than those known to utilize straight-chain alkanes. Most attempts to isolate pure strains of cyclo-alkane metabolizing bacteria have met with failure. However, cyclo-alkane metabolism has been documented in field studies and in laboratory studies with natural mixed communities of microbes. The difficulty of isolating pure cultures of cyclo-alkane utilizing bacteria coupled with the observations of cyclo-alkane utilization under natural conditions has led investigators to propose that such compounds may be degraded in the environment by mixed microbial cultures in a system of cooxidation and commensalism (Perry, 1984). Cooxidation is the incidental degradation of a recalcitrant organic compound by bacteria that utilize a second more readily utilizable carbon compound in the environment for their energy source.

The basic pathway of microbial degradation of cyclohexane is similar to the hydroxylation sequence of straight chain alkane oxidation. The first step is the hydroxylation of the cyclohexane to an alcohol, and then future transformation to the corresponding aldehyde. The ring is cleaved by the insertion of an oxygen forming a dicarboxylic acid as the final oxidation product. As with alkanes, a fatty acid is available for beta-oxidation which supplies acetyl CoA to the Krebs cycle.

Aromatics

The bacterial degradation of the aromatic fraction of petroleum has been studied in great detail (e.g., Gibson, 1977; Cerniglia, 1984; Lovley et al., 1989; Cozzarelli et al., 1989; Eganhouse et al., 1993). Many types of bacteria have been identified as capable of utilizing aromatic hydrocarbons as a carbon and energy source.

Metabolism and Byproducts

The metabolism of aromatic hydrocarbons by bacteria involves the action of either oxygenase or mixed-function oxygenase enzymes. Bacteria initially oxidize aromatic hydrocarbons by incorporating two atoms from molecular oxygen onto the ring structure (Gibson, 1977). This reaction is catalyzed by a complex enzyme system that consists of three interacting enzymes. Dihydrodiols are oxidized to catechols. Degradation of catechol is catalyzed by a dioxygenase and at this point the aromatic ring is broken. Aromatic compounds known to be degraded by bacteria include: benzene, toluene, naphthalene, phenanthracene, anthracene, benzo (a) pyrene, and benz (a) anthracene.

Benzene is an important aromatic component of petroleum and of interest in itself since it is often found contaminating natural waters. Bacteria oxidize benzene in the presence of molecular oxygen via a multicomponent enzyme system to dihydroxy dihydro benzene. This molecule is further transformed to catechol. Catechol formed from benzene can be broken down in a variety of ways, leading ultimately to either acetyl-CoA or tricarboxcylic acid intermediates. Catechol is oxidized via the ortho ring-cleavage pathway which involves cleavage of the bond between carbon atoms of the two hydroxyl groups of catechol to yield mechanic acid, or via the meta ring-cleavage pathway which involves cleavage of the bond between a carbon atom with a hydroxyl group ( Cerniglia, 1984). If the benzene ring is substituted with methyl groups or other constituents, these substitutions may be attacked before the ring (Brock and Madigan, 1988).

Microbial oxidation of toluene was demonstrated to occur in anaerobic sediments by Lovley and Phillips (1988) and Lovley et al., (1989). 25 ml of sediments from the anaerobic zone of a shallow oil-contaminated aquifer were transferred into serum bottles filled with anaerobic ground water from the same location. The bottles were sealed without a headspace. Toluene was added to provide an initial concentration of 600pM. After 45 days of incubation, less than 2% of the toluene remained. In sediments in which the microorganisms had been killed with heat (121 °C for 1 h) before incubation, there was no loss of toluene and no Fe(III) reduction. A pure strain of bacteria, GS-15, was isolated that reduced Fe(III) oxyhydroxide to Fe(II) while oxidizing toluene.

Purpose of the Study

The purpose of this study was to investigate the hypothesis that bacterial activity, i.e., attachment, colonization, metabolism and growth, causes diagenetic changes in minerals and rocks in terrestrial aquifer conditions. Research questions include: Do bacteria colonize mineral surfaces in optimal laboratory conditions and in natural aquifer conditions? If so, are there colonization patterns, and what if any effect does colonization have on the mineral itself? Can cell morphology be used to identify types of bacteria in natural mixed cultures from laboratory or field samples? Can the full size range of bacteria, from < 0.4p,m to > I.spm diameter cells, be identified on the basis of morphology by means of scanning electron microscopy? Most importantly, do bacteria dissolve, precipitate, or otherwise alter minerals they colonize? Is it possible to elucidate the mechanisms by which bacteria alter rocks and at what scale are the reactions occurring?

Research tasks included the design of laboratory and field experiments which isolated the effects of bacteria on mineral surfaces and the adaptation of analytical methods to study and quantify these effects. In order to focus the scope of this study of bacterial diagenesis, the environment of investigation was constrained to terrestrial aquifer conditions with mixed hydrocarbons as the main source of organic matter.

Approach

Research methods in this investigation include descriptive natural history, experimental microbiology, geology and geochemistry in the field and in the laboratory. The field of geomicrobiology, especially in the terrestrial subsurface environment, is in its infancy, and the natural history of the subject, i.e., description of the phenomena of bacteria on minerals, trapped within minerals, and the interaction of bacteria, water and rock in aquifer environments is sparse. Description of basic bacteria-rock interaction is an essential research element in the development of the field. Microbiology is essentially an experimental science and the experimental approach was integrated with geological and geochemical research in this study. Experiments were designed to test hypotheses developed from direct observation. Observations of bacterial interactions with minerals were tested with laboratory and field experiments.

Laboratory Experiments

Laboratory experiments in batch reactors that simulated aquifer conditions were performed to develop basic data on bacteria/mineral interactions, to guide the design of field experiments, and to inform the interpretation of results from microscopy and chemical analyses of the microbial alteration of mineral surfaces. The hypothesis that individual strains of bacteria could be isolated, characterized by morphology and then recognized by morphology in mixed communities under growth conditions was tested. A series of experiments were carried out to document the response of the mixed culture to environmental and nutritional stress and to elucidate the origin of nannobodies. Calcite collected from a microbially active aquifer was sequentially etched in hydrochloric acid and studied with SEM to determine the internal structure of the surface precipitate. Similar calcite was etched with nitric acid and the eluent analyzed to determine the stoichiometry of the surface precipitate. Scanning electron microscopy was an essential technique in the analysis of laboratory experiments.

Field Experiments: Background

The influence of indigenous surface-adhering bacteria on diagenesis of minerals in an hydrocarbon-rich shallow aquifer was investigated in situ using controlled microcosm and flow-through column experiments. The goals of the microcosm experiments were to capture bacterially colonized geologic materials for analysis and to document the influence of surface-adhering microbes on mineral alteration under actual aquifer conditions.

A microcosm ("little world") is an experimental system that models a complex natural environment with both biologic and physical components (Ehrlich, 1981). The design of microcosms may be tailored to the purpose of studying a specific question, and have been designed to model aquifer systems and to study the effects of changing environmental conditions on bacterial communities (Wilson and Noonan, 1984). Another use of microcosms is to define the processes that effect the transport and fate of specific environmental pollutants in subsurface conditions (Bouwer and McCarty, 1984). While microcosms have been utilized for studies of bacteria and the fate of organic compounds, there has been little published research to date on the diagenetic effect that various bacteria might have on the rock substrates included in microcosm experiments. This work is the first to use in-situ microcosms for the purpose of studying bacterially caused diagenetic effects on the rock component of the saturated zone

A microbially active, petroleum-rich, shallow unconfined aquifer was selected as the site for field studies. This pristine aquifer was contaminated in 1979 when a petroleum transport pipeline burst spilling crude oil onto the ground surface. 400,000 liters of oil infiltrated the soil overlying the aquifer and eventually pooled on the water table 3-5 m below the surface (Bennett et al., 1993). Although the accident polluted a formerly pristine area with xenobiotic organic matter, it also created the conditions for a controlled study of the fate, mobilization and transport of crude oil in a natural sedimentary environment (Hult, 1991).

The ground water chemistry at this site has been monitored annually since 1983 as part of a research effort sponsored by the U.S. Geological Survey (Mallard and Aronson, 1991). This site was chosen for the in-situ microcosm experiments because it represented on a small scale a common geologic condition in sedimentary basins. In addition, the high concentration of labile organic matter dissolved in the ground water as a result of the recent release of petroleum hydrocarbons offered the opportunity to observe the effects on aquifer mineralogy and ground water chemistry of accelerated microbial activity. The aquifer was well characterized, with over one hundred wells installed for sampling in order to characterize the background chemistry of the aquifer and to study the fate of the oil in the aquifer (Fig. 1). Beneath the main plume of oil floating on the ground water, a flow cell was selected for this study that included a variety of chemical and environmental conditions that affect bacterial growth and diagenetic reactions (Fig. 2). In addition to USGS work, a research group from the University of Texas at Austin (UT) has studied the hydrogeologic, chemical, and geomicrobiological characteristics of this aquifer for the past seven years (Bennett and Siegel, 1987; White, 1991; Belir, 1992; Hiebert and Bennett, 1992; Bennett et al., 1993).



Figure 1. Research site, well locations and contours of surface elevation at Bemidji, MN. Inset shows detail of the flow cell chosen as the location of the experimental microcosms for this study. Microcosms were placed below the oil lens in wells 015, 017, and 018. (Modified from Belir, 1992).



Figure 2. Cross section of aquifer flow path from upgradient of the oil spill, through the floating oil body to below the contaminant plume. Microcosms (M) were located in wells 018, 017 and 015 as indicated. An additional microcosm was located 100 m upgradient of the oil body in an uncontaminated portion of the aquifer. Crosses indicate the location of well screens.

Regional Geology

The study site occurs within the Bagley outwash plain (Wright, 1972 b) and is bordered by the Bigstone Moraine to the north, and the Itasca Moraine to the south and the west (Hobbs and Goebel, 1982). On a regional scale, the aquifer would be considered of homogeneous glacial outwash composition. At a local level, and certainly at a microbial scale, the aquifer is heterogeneous.

The depositional architecture of the sediments that compose the aquifer is complex. Franzi (1987, p. C-7) described the aquifer material in detail.

Glacial deposits are composed of a complex assemblage of four lithofacies; a basal till, two units of ice-contact stratified drift, and outwash sands and gravels. Erosional contacts and intercalation of lacustrine sediments indicate separate phases of late glacial sedimentation with each lithofacies. A basal till unit underlies the entire sequence at depths of 23- 31m and is known to be at least 1-6 min thickness. The stratified moraine drift is a unit consisting of poorly sorted medium to coarse grained sand and gravely sand of up to 10 m in thickness. Individual beds, within the stratified drift, extend no more than 10 to 25 m laterally and a few centimeters to meters in thickness. Discontinuous lenses of till and lacustrine silt and clay form semi-confining layers that occur occasionally throughout the stratified drift. Lacustrine sediments consisting of silt and very fine sand form bodies of 1 m thick patches are believed to extend laterally more than 50m.

The base of the unconfined aquifer slopes to the east at a gradient of 0.0019 (10 ft/mile). Saturated thickness ranges from 0 to 38 m. Generally, the regional hydraulic gradient is 0.001 (5.3 ft/mile) (Stark et al., 1991) and flow is to the northeast.

Meteorological conditions in the Bemidji area are typical for northern Minnesota. The air temperature from 1941-1970 ranged between average annual monthly extremes of -23°C in January and 27°C in July (NOAA, 1982; Kuhenast, 1972). Annual precipitation averages 56.5 cm (NOAA, 1982; Kuhenast, 1972; NOAA 1988-90), and ground water recharge occurs primarily during spring snow melt.

Study Site

The study site is located approximately 29 km northwest of Bemidji, MN, in an undeveloped forest area and represents a small portion of the regional glacial aquifer. The forest is within the drainage that sources the headwaters of the Mississippi River. The area selected for characterization by the USGS encompasses 28 hectares (Belir, 1992). The focus of USGS and UT studies at this site is a shallow unconfined aquifer with a locally extensive low permeability layer at 23-31 m below the land surface. The aquifer is composed of discontinuous low permeability layers which cause flow heterogeneities within the sediments. As a result of both deposition of sediments and formation of kettles, the aquifer has large hydraulic discontinuities in the overall flow field (Franzi, 1987). Permeabilities of adjacent beds may differ by more than three orders of magnitude. Thick accumulations of outwash and small bodies of finegrained sediment, form layers of varying permeability that affect the rate of ground water flow and the shape and advancement of a plume of dissolved hydrocarbons derived from crude oil.

The surface topography at the site is characterized by rolling sandy hills with wetlands and shallow small lakes. Topographic relief is 11.5 m, ranging from 422.5 to 434.0 m, mean sea level (Belir, 1992). Dense second-growth mixed deciduous and conifer forest covers most of the region, but the surface area directly above the study portion of the aquifer is a large stony meadow with clumps of small pine trees. The meadow was created as a pipeline right-of-way and expanded during the surface remediation efforts immediately following the pipeline break that introduced oil into the aquifer.

Hydrogeology

The site is located on a local flow system that discharges into a lake 300 m down gradient from the initial break in the pipeline. The regional hydraulic gradient is towards N57°E, but can range in direction from N49°E to SBl° (White 1991). The average local horizontal hydraulic gradient is 0.0027 (14.3 ft. mile) and ranges from 0.002 to 0.003 (10.6 to 15.8 ft/mile). In the northeast section of the site the water table contours parallel the shoreline of lake 1386 indicating flow towards and into the lake. Lake levels fluctuate less than 0.3 meters/yr and the lake freezes during the winter months thus making fishing a waste of time (Miller 1988).

White (1991) documented two predominant hydraulic conductivities in the study area. North of the study site, the average hydraulic conductivity is 1.5X10" 3 m/s, while to the south, hydraulic conductivities averaged 3.1X10'4 m/s.

Mineralogy

The glacial outwash sands and gravels that make up the aquifer lithology consist of approximately 57% quartz, 29% feldspars, 3-5% calcite, 1-2% dolomite, 2% hornblende, less than 1% clay minerals and less than 0.2 % organic carbon (Table 1). Clay minerals were identified as kaolinite, smectite, and chlorite (Bennett et al., 1993).

Cozzarelli and Baedecker (1989) examined aquifer carbonates by SEM and EDAX. Calcite and dolomite were found but siderite was not. However, grain coatings with more iron than calcium were present on some calcite grains. Published SEM images were not at a high enough magnification to resolve bacteria or nannobodies, if they were present.

Surface etching of native silicate sand grains collected from the contaminated zone was previously noted, and both quartz and feldspar reported to be rapidly weathered (Bennett and Siegel, 1987; Bennett, 1991).

Zone 3 3 4 4 4 5 Average Well# 534 533 532 531 530 527 Depth (m) 9.58 9.76 9.86 10.4 10.5 11 Quartz % 61 54 56 60 55 55 57 K-Spar % 11 13 11 13 12 8 11 Plagioclase % 18 17 18 18 19 20 18 Calcite % 3.2 5.8 4 4.3 5.6 3.3 4 Dolomite % 0.92 1.1 0.82 0.52 0.98 1.2 1 Hornblende % 2.4 1.7 2.1 1.4 3.3 2.1 2 Other % 3.7 9.4 8.1 2.4 3.7 10.4 6

Table 1. Mineralogy of 2-4 phi size fraction aquifer sediments in the study area.

Microbiology

Nine genera of common soil and ground water bacteria were identified by culturing from samples collected from the sediment and ground water of the Bemidji site and include Acinetobacter, Aeromonas, Vibrio, Pseudomonas, Mycobacterium, Micrococcus, and Achromobacter, and Alcaligenes (Chang et al., 1991). Most of the bacteria in the genera identified are rod-shaped during loggrowth phase, with the exception of Mycobacterium and Micrococcus which are usually coccoid. Strains from each genera collected from the Bemidji site were determined by Chang et al. to be capable of using hydrocarbon as a source of energy

Organic Geochemistry

A one-time pulse of crude oil has supplied the study area with a massive amount of labile organic matter. Crude oil released during the 1979 oil spill pooled in low areas of the site and gradually seeped downward through the surface sediments. Up to Im of sediment at the water table is saturated with crude oil and defines a distinct floating oil pool on the ground water. Directly under the oil, the ground water has been depressed by almost 0.5 meters and the depressed oil lens behaves as a non-penetrable barrier to flowing ground water.

A plume of reactive materials which consists of dissolved organic components of oil and metabolic products of biodegradation such as methane, CO2, and organic acids, inorganic compounds, and ions mobilized from sediments has migrated down gradient with local ground water flow (Baedecker et al., 1989; Cozzarelli et al., 1989; Cozzarelli et al., 1990). Hult (1984) suggested that the oil pool is being depleted of volatiles and becoming more dense and viscous. Landon and Hult (1991) calculated yearly oil-mass rate-loss of 0-1.25 %, averaging 0.5% within the oil pool ( Belir, 1992).

The crude oil at the water table is a paraffinic oil composed primarily of hydrocarbons in the C-l to C-35 range with trace amounts of sulfur, oxygen and nitrogen (Table 2). Crude oil has migrated 30 m down gradient over the past 14 years. The aromatic hydrocarbons, benzene, and alkybenzenes (C-6 to C-10) are soluble in water and have migrated in the ground water up to 200 m down gradient of the oil body. Along a 100 m flowpath down gradient from the oil the concentrations of aromatic and aliphatic hydrocarbons decreased by 40 to 99 percent (M. J. Baedecker, pers. com., 1993).

Aromatic hydrocarbons have leached from the crude oil into the ground water. Concentrations of benzene and alkylbenzenes have been measured at high concentrations in the anaerobic ground water and in diminishing concentrations down gradient in the more oxidized portions of the flowpath (Lovley et al., 1989). The disappearance of different isomers of the same alkylbenezene compound at different rates and the generation of isotopically light methane in the anaerobic plume indicates that microbial metabolism of the aromatics is occurring (Baedecker, pers. com. 1993).

Chang et al. (1991) carried out 6-month static incubations of Bemidji aquifer sediment, aquifer water, and added crude oil to study the effect of indigenous microorganisms on oil biodegradation kinetics. For the temperature range closer to actual aquifer temperatures (7-12°C) the maximum biodegradation rate of oil by the mixed population of microbes in the incubation vessels ranged from 6.98 - 12.57 micrograms per milliliter per hour. Chang et al. (1991) also reported that the maximum rate that oil could dissolve into the water varied from .006 - .009 mg/l/hr.

The rate of oil dissolution into the ground water, and therefore the amount of dissolved hydrocarbon available for microbial metabolism, as presented by Chang et al. (1991) may be substantially underestimated. Chang et al. (1991) simplified the equation describing the total loss of oil by omitting the rate constant for the disappearance of the oil, Ks. Since Ks should increase with time as a results of the solvent effect on oil of the accumulation of organic acids, the subsequent compounding increase in solubility of oil in water was omitted. Therefore Chang's estimate of oil dissolution rate underestimated. While oil dissolution rate in water did not vary significantly within the temperature range of the aquifer, biodegradation rates and the rate constant Kt did vary significantly. This suggests that the intermediate byproducts of oil biodegradation increased the solubility of hydrocarbons in the crude oil and thus the bioavailibility of the oil to microbes. Partitioning of the aromatic fraction of hydrocarbon in the water could have been substantially higher than the 0.006 to 0.009 mg/l/hr calculated by Chang et al. (1991).

Additional evidence for the degradation of aromatic hydrocarbons was the identification of organic acids (Fig. 3) in anoxic ground water that were structurally related to the hydrocarbons and are probably intermediates in the degradation process (Cozzarelli et al., 1989; Eganhouse et al., 1993). These acids were found only in the anoxic water zone.

Specific Gravity 0.859 Fe 4-9 ppm Viscosity 14.6 Mn <1 ppb Sulfur 0.56% Si <5 ppb Oxygen 10.50% Ni 4.3 ppm Nitrogen 0.27% Al <10 ppb Hydrogen 10.50% V 3.2 ppm Carbon 78.20% (from Bennett et al., 1993)

Table 2. Physical and chemical characteristics of the oil spilled at Bemidji.

Ground Water Chemistry

The chemical composition and physical characteristics of the native ground water evolve as it flows horizontally down-gradient through and beneath the surface area affected by the spilled oil. Baedecker et al., (1989) and Bennett et al., (1993) recognized five geochemical zones within the study area characterized by similarities in physico-chemical properties of the ground water (Fig. 4) Ground water data were collected from over 100 wells scattered across the entire 28 hectare research site with most of the wells concentrated in the area below and surrounding the pool of crude oil that floats on top of the ground water . Each geochemical zone is bounded on the top by the water table surface and extends downward and down gradient. The microcosms described in this study were placed beneath the floating pool of oil in the area characterized as zone 3. Zone 3 represents an inner core of anaerobic geochemical conditions surrounded by transitional disaerobic zone which separates the anoxic core from the aerobic conditions of zones 1 and 5.

Under the floating pool of oil, oxygen has been consumed, and the ground water is near neutral pH and is highly reducing. In this zone, silica is mobilized, and the waters approach equilibrium with amorphous silica. Water flowing under the oil pool is locally reducing, slightly acidic, with no detectable oxygen, and contains a high concentration of dissolved organic carbon. The carbon is a combination of unaltered volatile petroleum components and partially degraded petroleum in the form of simple and complex organic acids (Hult, 1984; Bennett and Siegal, 1987; Cozzarelli, Eganhouse, and Baedecker, 1991).

The aqueous chemistry of the principle flow path through the study area is summarized in Table 3. While the values reported here represent the chemistry at a single point in time, there was little variation over the previous 4 years, thus values in Table 3 represent a characteristic geochemistry.

Dissolved silica increases along the flow-path from a background concentration of 0.2 mmol/1 to a maximum of about 1 mmol/1 under the center of the oil pool. The microcosms were located in water with about 0.7 mmol/1 Si, and where the change in dissolved silica with distance along the flow path is greatest. Dissolved total aluminum is very low, increasing from a background concentration of less than 1 |Limol/l to about 3 pmol/1. Whereas the increase in aluminum corresponds to the increase in silicon, the nearly 500:1 ratio of silica to aluminum in a water where feldspar is dissolving suggests that aluminum is essentially conserved in a solid phase. Calculations using chemical speciation and equilibrium models show that the contaminated ground water is supersaturated with respect to quartz and approaches equilibrium with amorphous silica (Bennett et al. 1993). The waters are supersaturated with respect to gibbsite, smectite, and kaolinite, but under-saturated with respect to the primary silicate phases such as anorthite, microcline, and albite, within the uncertainty of the available thermodynamic database (Hiebert and Bennett, 1992).

Five years after the spill had released hydrocarbons into the aquifer, Fe 2+ concentrations were measured to be high in the anaerobic zone but non-detectable in the pristine control areas. Fe 3+ was measured to be up to four times less in the anaerobic area than in the pristine background control areas (Lovley et. al. 1989).

This background data base provided an unusually detailed geological, chemical, and microbiological context in which to interpret experimental results and the direct observations of in-situ mineral weathering.



Figure 3. Aromatic hydrocarbons and their oxidized intermediate forms identified from water collected from beneath and down-gradient of the oil body at the Bemidji research site by Cozzarelli, Eaganhouse and Baedecker (1991). The parent products are slightly soluble in water and thus are available for microbial use. Both aerobic and anaerobic metabolic pathways may generate the oxidized intermediates listed in the right-hand column. The oxidized compounds are polar, water soluble, and the COOH moiety may act to chelate metal ions in solution or from mineral surfaces (Bennett and Siegel, 1989; Bennett, 1991).



Figure 4. Native ground water flows from the background area (zone 1) under the area contaminated with sprayed oil (zone 2), where oxidation of oil in the unsaturated zone and reaction with minerals increases the concentrations of inorganic solutes. This water flows under the floating oil body (zone 3) , where mass transfer of organic compounds to the aqueous phase occurs, resulting in extremely high concentrations of organic carbon. Surrounding this highly contaminated "flow tube" is a less contaminated transitional zone (zone 4). Water from zone 3 exits from under the oil body, producing a plume of dissolved organic and inorganic solutes that extends several hundred meters down-gradient. This plume passes thorough the oxygen limited transition zone (zone 4) where mixing, dilution and biogeochemical reactions reduce the concentration of dissolved solutes. Beneath the flow tube that is directly affected by the free crude oil, and down gradient of zone 4, a contaminated water mixes with native ground water (zone 5).

Well 1 D T SC pH C 0, Aik Ca Mp Na K Si Fe Mn SO4 Cl ZONE 1 310wt -199 80 255 7.66 0.23 0.28 3.41 1.27 0.58 0.084 0.021 0.29 0001 0 0002 0 03 0.022 707-1.5 -69 8.0 340 7.60 0.10 024 3.95 1.32 0.54 0.071 0.024 0.34 0.001 0.0004 — 707-2.5 -69 8.0 312 7.6 0.11 0.28 3.56 1.19 0.49 0.080 0.021 0.33 0 001 0.0002 — ZONED 709wt -139 8.5 298 7.72 0.28 0.28 3.97 1.47 0.62 0.082 0.028 0.32 0.002 0.0001 — 603wt -117 8.5 630 7.25 1.33 0.24 7.22 3.02 1.10 0.083 0.024 0.30 0.002 0.0009 0.11 0.023 708wt -96 10.0 510 7.13 0.71 0.22 6.92 2 87 0.87 0.075 0.035 0.32 0.001 0.0006 0.04 0014 707wt -69 9.0 620 7.11 1.09 0.10 8.08 2.99 1.28 0.099 0.033 0.37 0.009 0.0050 0 08 0.033 707-1 -69 9.0 312 7.15 0.13 0.23 3.74 1.34 0.56 0.072 0.025 0.31 0.001 0.0005 0.02 0.013 523wt -68 13.0 1200 6.45 2.59 0.22 14.2 5.39 2.39 0.121 0.052 0.38 0.004 0.145 0.25 0.032 ZONE HI 421-1.8 0 10.0 830 6.78 4.17 <0 01 12.1 3.31 1.77 0.135 0.169 1.02 1.11 0.0164 <0 01 0.010 522wt 28 11.0 780 6.93 4.02 <0.01 11.2 3.32 1.88 0.137 0.033 0.81 0.843 0.0473 0.02 0.031 533wt 38 90 745 6.95 3.59 <0.01 11.7 3.79 1.85 0.149 0.041 0.62 0.093 0.123 <001 0012 533-1.5 38 9.0 540 6.93 1.52 <0.01 8 12 3.04 1.37 0.091 0.035 0.52 0.096 0.127 0.01 0.019 532wt 49 9.5 725 692 2.78 <0.01 10.7 3.47 1 77 0.131 0.031 0.60 0.075 0 .117 <0 01 0.011 532-2 49 9.0 690 6.95 1.53 <0.01 10 3 3.32 1.67 0.118 0.036 0.60 0 036 0.146 <0 01 0.010 518wt 59 9.0 675 6.88 3.14 <0.01 10.7 3.84 1.72 0.149 0.046 0 47 0.016 0.127 <0.01 0.020 531wt 70 10.0 680 6.93 0.57 <001 10.1 3.62 1.77 0.150 0.036 045 0.091 0.146 0 01 0.018 ZONE rv 604-1.6 -34 10 0 503 7.27 0.89 0.03 6.77 2.54 1.05 0.094 0.031 0.33 0.003 0.014 0.03 0.015 533-3 38 9.0 400 7.40 0.32 Oil 5.94 2.09 0.92 0.091 0.027 0.32 .001 0.0005 0.03 0.010 532-2.6 49 9.0 490 6.99 1.10 0.07 7.89 2.97 1.32 0.097 0.032 0.40 0.003 0.0519 0.02 0 014 513wt 80 9.0 600 6.96 1.33 0.04 8.92 3.34 1.48 0.127 0.033 0.36 0.002 0.131 0.01 0.012 530wt 94 8.5 515 6.91 1.10 0.02 8.00 2.99 1.30 0.121 0.027 0.34 0.003 0.0856 0.01 0.011 530-1.7 94 9.0 610 7.01 1.44 0.01 9.03 3.42 1.52 0.137 0.034 0.37 0.007 0.124 0.03 0.012 530-3 94 8.0 375 7.05 0.48 0.02 6.06 2.27 1.03 0.098 0.027 0.35 0016 0.0293 0.02 0.011 510wt 107 8.0 500 7.09 0.45 tr 8 16 2.97 1.28 0.095 0.032 0.37 0.001 0.0561 0.01 — ZONE V 515wt 139 8.0 500 7.09 0.73 0.03 8.28 3.09 1.40 0.101 0.034 0.38 0.002 0.0001 0.03 0.023 527wt 180 7.5 360 7.36 0.18 0.11 5.32 2.02 0.91 0.097 0.029 0.30 0.002 0.0001 0.03 0.010 529wt 185 7.5 410 7.25 0.13 — 4.97 1.83 0.85 — 0.025 0.30 0.001 0.0001 —

Table 3. Chemical analysis of ground water samples collected June, 1987. All concentrations are reported in mmol/1. Well numbers correlate to those presented in Figure 1 and 2. The desnignation "wt" means water table well, numbers following the designation "wt" indicates the depth below water table of the well screen. "D" = distance from oil body center, negative distances are up-gradient, positive are down gradient. "C" = total dissolved organic carbon, equal to the sum of the non-volatile and volatile fractions. Specific conductance in microsiemans at 25°C. From Bennett et. al., 1993.

Methods and Materials

Methods from geology, geochemistry, and microbiology were combined in an interdisciplinary approach to study the effect of bacteria on the alteration of minerals and to document the effect of diagenetic conditions on bacterial morphology. Methods included experimental microbiology, geochemistry, microscopy, and field work. Batch reactor experiments that simulated aquifer conditions were performed to develop basic data on bacteria/mineral interactions and to guide field experiments and observations. In particular, the hypothesis that individual strains of bacteria could be isolated, characterized by morphology and then recognized by morphology in mixed communities under growth conditions was tested. A series of experiments were carried out to document the response of the mixed culture to environmental and nutritional stress and to elucidate the origin of nannobodies. Scanning electron microscopy was an essential technique in the analysis of laboratory experiments.

The influence of indigenous surface-adhering bacteria on diagenesis of minerals in a hydrocarbon-rich shallow aquifer was investigated in-situ using controlled microcosm and flow-through column experiments. Geochemical measurements of the ground water in the study area were collected. Surface features of minerals from the experiments were studied with scanning electron microscopy and atomic force microscopy. Calcite collected from the aquifer was sequentially etched in hydrochloric acid and studied with SEM to determine the internal structure of the surface precipitate. Calcite from the same field experiment was etched with nitric acid and the eluent analyzed to determine the stoichiometry of the surface precipitate.

Samples of geological materials were prepared using microbiological techniques in order to preserve evidence of bacteria and their effects. Basic methods of bacteria culture, isolation and identification, and direct observation by light and electron microscopy were used to collect data on bacterial morphology, surface colonization, and direct evidence of bacteria-mineral alteration. Video tape was used to collect mass amounts of SEM images for further study. Laboratory experiments focused on the investigation of the bacteria/mineral contact. Field geochemical measurements and data review was on an aquifer scale and focused on determining bulk geochemical changes within zones of the aquifer. Extensive experimentation with mechanical and chemical sample preparation and alteration of various minerals was carried out to establish biologic and non-biologic controls. Experiments that simulated saturated zone conditions were undertaken in the laboratory using batch reactors. Novel in-situ microcosms were developed for the study of microbial diagenesis in the field. In-field flowthrough columns were prepared and studied to supplement the data from the insitu microcosms. Geochemical background data were analyzed and new data collected at the scale of geochemical zones within the field study aquifer.

Bacterial Culturing, Isolation, and Identification

A mixed culture of naturally occurring bacteria that use hydrocarbon for food was grown in simple laboratory batch reactors. The culture, courtesy of Alpha Environmental, Inc., Austin, Texas, was a mixture of unidentified strains collected from hydrocarbon-contaminated soil and oilfield core samples. The mixed culture was maintained under aerobic conditions in a media of nutrientenriched seawater with crude oil as the sole source of carbon. Inoculation of the batch reactors was made with culture in the log growth phase.

Isolation of strains from the batch reactor was by standard enrichment techniques using tripticase soy broth agar coated with hydrocarbon evaporated from crude oil (Brock and Madigan, 1988; Pope, 1989). Isolates were incubated at 30°C for three to nine days. Colony morphologies were described and documented by binocular microscope photography. Four of the bacterial isolates from the mixed community were tested with a series of biochemical evaluations.

Bacterial Morphology and Taphonomy

Following Folk's 1993 publication on the presence of nannobacteria in carbonate precipitates, the SEM work supporting this dissertation was reevaluated for evidence of nannobacteria. The presence of 0.1 - 0.4 ptm diameter spherical particles associated with organic-rich fluids and bacteria-rich rocks is virtually übiquitous. The particles are not artifacts of gold coating or SEM examination. However, on the basis of morphology alone, it is unclear whether these particles are bacteria, congealed organic matter, inorganic or mineral material, or even effects of sample preparation. Laboratory experiments with live mixed cultures of hydrocarbon utilizing bacteria were carried out with the goal of investigating the origin of nannobodies.

One gram each of freshly crushed o.smm diameter calcite crystals was added to 40 ml of growth media inoculated with a mixed culture of bacteria in the log phase of growth. The growth media was nutrient-enriched filtered seawater. Seawater from Port Aransas Pass was collected from the University of Texas Marine Station Seawater settling tanks, and filtered through a 25 micron polypropylene filter, steam autoclaved, and enriched with inorganic nutrients (Table 4). Five drops of light "sweet" crude oil similar to that spilled at Bemidji was used as the source of organic carbon for all the bacterial growth experiments.

Pure crystals of calcite were separately crushed in a tungsten shatterbox (SPEX Industries model 8500-115), dry sieved to collect the o.smm size fraction, and briefly rinsed in distilled water. Crystal surfaces were cleaned of rock dust by brief low-power ultrasonification. Following inoculation, the mineral sediment was hand mixed by vigorous shaking so that each mineral grain was completely wetted by the bacteria-rich fluid. The vials were agitated occasionally to mix the sediment and supernatant fluid.

The inoculation of the calcite sediment was made under optimal growth conditions for the mixed culture. Immediately following inoculation, certain environmental parameters that effect bacterial growth were changed in order to stress the aerobic bacteria in the mixed culture. The growth media was not replenished with nutrients or carbon, and the vials were kept tightly capped to prevent oxygen from being replaced as it was used during bacterial metabolism. The vials were stored at @25°C in the dark. At intervals of 2 hours, 20 days, 30 days, and 18 months, aliquots of each mineral type were collected, spread on a gold-coated aluminum stub, air dried, gold-coated and studied with the SEM.

The sampling schedule was determined in order to collect bacteria-coated calcite crystals from the vials at different phases in the culture's growth cycle. Samples were collected at the two hour interval to represent the culture in optimal growth conditions with no limiting factors. It was assumed that under the stressed conditions imposed after initial inoculation, the culture would begin to enter the stationary phase in which growth is limited by some environmental or nutrient deficiency condition. The samples collected between 20 and 30 days probably represent the state of the culture during late exponential phase growth and the onset of stationary phase. Samples collected after 18 months represent the state of the culture under extreme limiting conditions or in death phase (Fig. 5).

Additional experiments to determine the fate of the bacteria bodies as a result of heating and chemical stress were carried out on calcite crystals from the same experiment. A sample of the calcite crystal sediment was collected at the 30 day interval, and split into four aliquots. One was treated with 5 ml of chlorine bleach for 30 minutes and rinsed briefly with distilled water, one was heated to 500°C for 6 hours, one was treated with gluteraldehyde for 30 minutes, rinsed with distilled water and air dried, and one was simply air dried as a control. Each sample was spread on a gold-coated aluminum stub, air dried, gold-coated and studied with the SEM.



Figure 5. Generalized bacterial growth curve showing the phase of mixed culture growth estimated to correlate with samples of calcite crystals collected at 2 hours, 20 days, 30 days and 18 months. The growth curve is a semi-log plot with the yaxis on a log scale and the x-axis on an arithmetic scale.

Component Filtered seawater 1 liter Ammonium Sulfate 1.0 gm/1 Potassium Sulfate 0.1 gm/1 Trace elements (Fe, Mg, Zn, Mo) 0.025 ml/1

Table 4. Nutrient-enriched seawater formula.

Batch Reactors

Experiments to determine if bacteria would colonize mineral surfaces in water-saturated conditions and to develop bacteria-mineral materials with which to practice imaging the cell-mineral contact were undertaken in laboratory batch reactors.

The previously described culture was grown in a laboratory batch reactor with a sediment of pure minerals and bits of lithified sandstone. Quartz, calcite and sandstone were selected and prepared as the geologic components of bacterial growth experiments. Rounded quartz sand from a high energy shallow marine environment (Mobile Bay, Alabama, courtesy of R.L. Folk) was wet sieved, and the 1-2 phi fraction (0.25-0.5 mm) saved for further preparation. The quartz was cleaned by continuous washing with distilled water, surface detritus was dislodged by 5 minutes of sonic dissagregation, and washed again with distilled water until no visible debris was contained in the rinse water. The sand was then steam autoclaved and stored in sealed sterile glass flasks until used in experiments. Calcite crystals with fresh smooth surfaces were prepared by crushing a large single crystal of Iceland spar and cleaned in the same manner as the quartz grains.

Sandstone was selected from core drilled from the Cretaceous Shannon Formation, Bar Margin facies, Natrona County, Wyoming. The rock was broken into pieces approximately 2 cm in diameter, steam autoclaved at 121 °C for 30 minutes at 15 pressure, allowed to dry in a covered beaker, and stored in sealed sterile bottles until used in experiments.

The cleaned mineral grains and rocks were characterized in the SEM as controls for comparison with bacterially altered material. The sandstone chips were removed from the reactor fluid, and immediately prepared for SEM viewing by the critical point drying method. Once dried, but prior to gold coating, the sandstone chips were fractured apart with a razor blade and hammer. The interior surfaces of the fractured chips were oriented upwards on aluminum stubs and sputter gold coated for 40 seconds at 40 milliamperes.

The reactors were inoculated with the mixed culture and incubated at room temperature. Mineral grains and sandstone were examined for evidence of bacterial colonization and diagenesis periodically throughout the experiments. Microbially colonized mineral surfaces were prepared for SEM study.

Reactor vessels were constructed of 500 ml sterile beakers that contained components of mineral or rock, fluid, organic matter, and bacteria as previously described. The beakers were covered, kept at room temperature and atmospheric pressure. The bacterial inoculum was adjusted so that the initial concentration of bacteria in the microcosm fluid was IXlO$ cells/ml. This population density is equivalent to values reported for indigenous subsurface bacteria in deep aquifers (Balkwill, 1989; Phelps et al., 1988, 1989). The experiments were observed over a three to nine month period.

The batch reactors and a control were maintained as semi-open systems in which the organic matter and media fluids were partially replaced on a daily basis. Every 24 hours, 250 ml of fluid was removed from the microcosm and replaced with fresh water plus 2.5 ml crude oil. In this system, nutrients were replaced, and by-products and cells removed. Ehrlich (1981) considered a semi-open batch reactor system to be most representative of subsurface conditions.

In-situ Microcosms

Microcosm chambers were constructed of 15 or 60 ml polypropylene (Nalgene) bottles (Fig. 6). Approximately fifty 5.0 mm holes were bored in each bottle to allow for free flow of ground water and planktonic bacteria through the holes. The diameter of the bottles was chosen to allow a 1.0 cm tolerance between the well wall and the microcosm wall. At the time of microcosm emplacement and recovery, ground water samples were collected from the contaminated and uncontaminated regions. At the end of 14 months, the microcosms were recovered, the contents preserved and studied for evidence of bacterial activity and chemical alteration..

Upon recovery the sampled microcosm-materials were split into archive and study portions and treated with a 2% gluteraldehyde solution to fix biological tissue. Mineral grains were prepared for SEM inspection by the critical point drying method or air drying, and sputter coated with gold film (e.g., Costerton, 1980).

Aliquots of quartz, feldspars, and calcite, were prepared, mixed together in porous polyethylene cylinders (microcosms) and submerged in an oilcontaminated aquifer for 10-14 months. There was no direct contact between the prepared minerals inside the microcosms chamber and aquifer sediment. The microcosm minerals were protected from being coated by the floating layer of oil on the ground water by the outer chamber, and by establishing a water wet surface on each mineral fragment prior to emplacement. The contents of each microcosm were dipped in freshly collected ground water from the well in which the microcosm was to be emplaced. The water-wet surface helped repel nondissolved phase hydrocarbons, which would not be representative of the native aquifer grains.

The wells used for this study were open to ground water (screened) just below the floating pool of oil. The microcosms were suspended by nylon line in the screened flow-through portion of the well approximately 10 cm below the oil layer. Following installation of the microcosms, the well bore was sealed and left undisturbed for the duration of the experiment.

Pure crystals of microcline (Wards # 46E5125, Keystone, South Dakota), Iceland spar calcite, quartz (Wards # 46E6605, Red Horse Lake, Lyndhurst, Ontario, Canada) and dolomite (Wards # 46E2705, Franklin, New Jersey) were separately crushed in a tungsten shatterbox (SPEX Industries model 8500-115), dry sieved to collect the 5-10 mm size fraction, and rinsed in distilled water. Crystal surfaces were cleaned of rock dust by brief low-power ultrasonification. 50 ml of crystal sediment was added to 100 ml distilled water in an acid-rinsed beaker, sonicated for 10-30 seconds and the supernatant decanted. This process was repeated until no visible suspended particles washed out with rinse water.

Samples of each prepared mineral type were reserved as reference and controls and characterized by SEM and EDAX analysis. Not all phases of the preparation of the mineral materials for these experiments were carried out under strictly sterile conditions. However, standard techniques for the aseptic handling of microbial cultures and of sterile materials were used and every effort was made to minimize the possibility of contamination of the mineral specimens by opportunistic microorganisms.

Examination of the reference mineral grains by SEM showed that the surfaces are clean and without evidence of chemical or mechanical weathering. The successful preparation and characterization of the mineral surfaces prior to use in reaction experiments represents an important difference between this study and most before and after-type experiments. There is no question that each mineral surface was as flat and debris-free as the mineral surface would allow. This clean starting point allowed definitive observations on surface alteration to be made following recovery of the samples from both laboratory and field experiments. Quartz grains are angular and irregular, and the surface consists of flat micro step-fractures (Fig. 7). Grains produced from larger crystals by crushing were largely equidimensional with concoidal fracture patterns. The quartz surface appears glass-smooth at magnifications less than 1000 X. Many of the quartz grains exposed broken isolated vacuoles or lines of vacuoles. These vacuoles appear to be former fluid inclusions and are easily distinguished from evidence of chemical etching by their large size, sharp-edged morphology and great depth. At high magnifications, the surface of the quartz grains appears flat with sheet-like 0.2 to o.s|im overlapping steps. No roughness, nannobodies, bacteria, or particles of micro-debris are evident.

Microcline surfaces were smooth and irregular, and cleavage can be distinguished (Fig. 8). Microcline, a low temperature potassium feldspar is the most stable of potassium feldspars at earth surface environmental conditions. At a magnification of 5000 X, the surface of the prepared microcline appears as highly ordered sheets of sub-micron thickness cut by cleavage or parting planes at 2-3 micron intervals. The straight lines and smooth surfaces on the crystal result from mechanical fracturing of the mother grain and provides a "clean slate" upon which to document any experimental chemical weathering effects. Some vacuoles are present and exhibit the same morphology as in quartz grains. The vacuoles were mostly isolated and did not form contiguous areas of pitting. The vacuoles are located most commonly along cleavage or parting planes. The larger size and more variable vacuole morphology could lead an interpreter to mistake a virgin vacuole for chemically produced etching. Careful documentation of preexperiment surface features that might be confused with the effects of chemical etching was important in guiding the interpretation of post-experiment mineral surfaces.

Calcite fragments used in the microcosm experiments were prepared in a similar manner as the previously described silicates, except for much shorter water rinses. The clean rhombohedral crystal fragments had flat surfaces with regular 0.1- 0.5 pm high steps where cleavage planes had been fractured during crushing (Fig. 9). Each calcite crystal surface had a very uniform microstepped surface, clean and flat steps and uniform step height of between 0.5 and I.opm. Damage to the stepped surface was common and is the result of the crushing process. Prepared calcite crystals showed substantially more surface abrasion at high magnifications than did the harder silicate minerals. These flat, clean surfaces represented well documented pre-reaction starting points for comparison with post-experiment surface alteration.



Figure 6. Schematic diagram of microcosm design and position in approximately to scale. Ground water and planktonic bacteria pass from the aquifer, through the well screen, through the holes in the microcosm chamber and contact the prepared mineral grains within the chamber. The objective of the design was to capture indigenous aquifer bacteria on the mineral grains.



Figure 7. Electron micrographs of quartz grains prepared for microcosm experiments. A. Quartz prepared by crushing, sieving, and cleaning fragments of large single crystals. Scale bar = 100p.m. B. Conchoidal patterns caused by mechanical fracturing of larger grains are common surface features. Isolated vacuoles on the grain surface represent former fluid inclusions and do not represent effects of chemical etching of the fractured surface. Scale bar = 50p.m. C. A typical grain surface of quartz prepared for microcosm experiments. Flat, regular microstepped surfaces were clear of debris and exhibited no evidence of chemical weathering. Scale bar = Ipm. Note: All of the micrographs presented in this dissertation have been reduced to 77% of the original. The scale bars are correct, but magnifications printed on the micrographs should be scaled down accordingly.



Figure 8. Electron micrographs of microcline grains prepared for microcosm experiments. Scale bars = sp.m. A. The prepared microcline surface is clear of debris and shows cleavage or parting planes with 2-3 pm thick sheets. The straight lines and smooth surfaces on the crystal result from mechanical fracturing of the mother grain and provides a "clean slate" upon which to document any experimental chemical weathering effects. B. Large irregular vacuoles were common on the surface of most prepared microcline grains. The vacuoles were mostly isolated and did not form contiguous areas of pitting. Careful documentation of pre-experiment surface features that might be confused with effects of chemical etching was important in guiding the interpretation of post-experiment mineral surfaces.



Figure 9. Electron micrographs of calcite grains prepared for microcosm experiments. Scale bars are included in each separate micrograph. A. Large crystals of Iceland spar calcite crushed into very uniform rhombic microcrystals. The edges and some surfaces showed much greater mechanical damage resulting from the shatter-box crushing. Note rounded corners and gouged surfaces on the two crystals in the center and upper right. B. Characteristic view of the prepared calcite surface at high magnification. Note the very uniform microstepped surface, clean and flat steps and uniform step height of between 0.5 and 1.0p.m. Damage to the stepped surface (in upper right hand corner) was common and is the result of the crushing process. Prepared calcite crystals showed substantially more surface abrasion at high magnifications than did the harder silicate minerals.

Etching of Calcite Collected From In-Situ Microcosms

Mineral fragments recovered from the field microcosm experiment were preserved in 2.0% gluteraldehyde. Some calcite crystals were selected for detailed examination of surface precipitation features and in order to investigate if bacteria were trapped within the surface precipitation. The crystals were handled only with plastic tweezers in order to avoid inadvertent chipping, contaminating the surface with trace amounts of metal, and to avoid transfer of opportunistic bacteria to the sample. A small portion of the bottom of each crystal was slightly chipped with a sterile steel needle to expose the interior of the crystal. Individual crystals were fixed in a plastic vice and slowly lowered into 0.5% HCI. The bottom of the crystal was etched for 60 seconds, then dipped deeper into the acid. The second zone was etched for 20 seconds, a third zone for 10 seconds and the top of the crystal was not etched at all. This process created a pattern of gradually increasing etching on the crystal surface, thus allowing comparisons of etch effects to be made without changing samples.

A scratch was inscribed in the surface of the crystal across the different etch zones with a sterile steel needle. The scratch was deep enough to reveal the pristine interior of the calcite crystal and expose a cross section of the altered surface and non-altered interior. The crystal was gold coated and examined in the SEM.

Field Columns

Flow-through columns filled with quartz, microcline, and calcite prepared as previously described and clean Bemidji sand were constructed for field experiments at the Bemidji study site. In the field, ground water from well 018 and 017 (zone 3. anaerobic conditions) was pumped directly from the level at which the microcosms were suspended through the columns for up to three days (Fig. 10). The columns were returned to the lab intact, disassembled and the sediments preserved for SEM inspection.

Mineral Surface Characterization

Scanning Electron Microscopy

Scanning Electron Microscopy (SEM) has largely been ignored or dismissed as an appropriate tool for the study of subsurface microbial ecology for reasons of scale, i.e., geochemical methods provide better information about overall microbiological activity in aquifers (Ghiorse and Wilson. 1988; Chapelle. 1992). SEM documentation in publications of deep subsurface microbiology is virtually absent and the lack of such data helps explain the lack of recognition of the role of bacteria in mineral alteration.

SEN! is well suited to the study of bacteria/mineral interactions with appropriate depth of field and resolution to image micron and sub-micron size bacteria on rugged micro-topographic surfaces (Weise and Rheinheimer. 1978). Bacteria are generally around 1.0 urn diameter and smaller, and millions may coat the surface of a single sand grain.

The observation of a single bacteria on a mineral surface at 10,000 X magnification by electron microscopy can reveal data on bacteria morphology, attachment mechanism, and direct evidence of mineral alteration at the cell/mineral contact. Only 90 of mineral surface area is covered in such a micrograph which is hardly representative of an aquifer stratum, or even the single grain's entire surface. However, this is exactly the scale at which direct microbe-mineral interactions occur.

SEM and coordinated EDAX analysis represent petrographic control on geochemical measurements. Bulk geochemical measurements of pore water composition can leave multiple reaction pathways as reasonable options to the end product. Direct observations of mineral dissolution and precipitation help constrain interpretation of geochemical data.

The effect of bacteria on mineral surfaces was studied by SEM and ED AX techniques. For general reconnaissance review, samples were fixed with gluteraldehyde and air dried. For studies of bacterial colonization, surface coatings, and detailed documentation of bacteria-mineral contact, samples were prepared by critical point drying method. In order to determine if bacteria were trapped within the calcite precipitate, samples were etched with dilute HCI. Some samples were etched in entirety, some were partially etched, some were etched in a pattern of gradually decreasing etch time to show a range of etching effects.

SEM observations were made with a Philips Model 515 (Cell Research Institute, UT) and a JEOL JSM T33OA Scanning Microscope. Microscope settings, magnification, and scale bar are recorded on each micrograph presented in the figures. In general, highest resolution of bacteria-mineral contact was observed with accelerating voltage of 30kv, close working distance (9-12 mm and small spot size. Data were collected on video tape and Polaroid film.



Figure 10. Schematic design of the field flow-through column. Columns were composed of polycarbonate resin with plastic caps. The dimensions are 3.5 cm interior diameter by 50 cm in length.

Sample Preparation

Petrographers have studied mineral surfaces and pore spaces of sedimentary rocks with scanning electron microscopes for years, yet rarely has notice of bacteria been reported in the geologic literature (some exceptions include: May and Perkins 1979; Maurin, et al., 1981; Danielli and Edington 1983; Chafetz and Folk 1984; Barker, 1985; Folk, Chafetz, and Tiezzi, 1985; Hiebert, 1988; Weaver, 1989; Buczynski and Chafetz, 1991; Chafetz and Buczynski, 1992; Hiebert and Bennett 1992; and Folk, 1993). Of course, bacteria are not present in all samples. If bacteria are present, however, it is likely that most petrographers missed their occurrence for several reasons. In most cases typical geological sample preparation techniques destroy the delicate structure of bacterial tissue by agitation, heat, dessication, and chemical additives.

Drying is a highly destructive process to cell wall morphology. The cell wall is composed of lipoprotein membranes and the presence of water is essential for its structural integrity. As a liquid/gas interface moves through drying tissue, relatively large surface tension forces can cause severe distortions. Distortion can be reduced or eliminated by the addition of chemicals to the biological sample that causes the cell wall to solidify in-place. Fixation by gluteraldehyde causes proteins in the bacterial cell wall to cross-link, thus stabilizing and inhibiting tissue decay (e.g. Hayat, 1981).

Once fixed, the surface morphology of cells can be preserved with the critical-point drying (CPD) technique (e.g. Costerton 1980; and Newbury, 1981). At any given temperature and at equilibrium in a closed vessel, the space above a fluid will be saturated by molecules of the fluid, according to the partial pressure of the liquid. If the temperature is increased, more molecules will be driven from the liquid phase into the vapor phase resulting in an increase in the density of the vapor phase and a decrease in density of the liquid phase. As the temperature is raised to the critical point, the density of the two phases will be equal, and the phase boundary between liquid and vapor will disappear. At temperatures and pressures above the critical point only the vapor phase will exist. This vapor can be removed to leave a dried, yet morphologically intact cell specimen suitable for further SEM preparation (Coleman, 1975).

Two methods of sample preparation were studied and adapted that preserved bacterial tissue on the mineral and rock surfaces of interest: 1) fixation and air drying, and 2) fixation and Critical Point Drying (CPD).

1. Fixation/Air-Dry Method.

Mineral grains with attached bacteria were collected from growth experiments and soaked in a 2% gluteraldehyde solution for 10-60 minutes to fix the bacterial cell walls. Following fixation the sample was air dried, stub mounted, gold coated, and stored in a desiccator.

2. Fixation/Critical Point Drying Method.

Mineral grains with attached bacteria were fixed with gluteraldehyde, and dehydrated in serial baths of 25, 50, 75 90, 95, and 100% ethyl alcohol (e.g., Postek et al., 1980). The alcohol was replaced with amyl acetate in serial baths of 25, 50, 75, and 100% acetate/alcohol solution. Samples were stored in amyl acetate until critical point drying. Following CPD, each sample was stub mounted, gold coated, and stored in a desiccator.

Image Collection

In addition to collecting data in a series of isolated micrographs, data was also collected on video tape. One of the most important benefits of collecting SEM data on tape was the number of images that could be saved. The tape data allowed long segments of multi-hour SEM sessions to be recorded for detailed later review and at a reasonable cost (@ $4/tape as opposed to @s2/micrograph). Another advantage to video data collection was that the sense of scale of the images was greatly enhanced. The operator could "zoom in" on a point of interest starting from an overview at low (50 -100 X) magnification and bring the viewer to high magnification in a zoom mode, thus never losing the point of interest while demonstrating visually the enormous difference from the scale of the whole grain to a spot on the grain surface at the bacteria scale. Tape 1 is a short compilation of data compiled from hours of tape collected during the analysis of the batch reactor experiments. Video recording equipment was available on the Philips SEM at the Institute for Cell Research.

EDAX

The ED AX beam on the JEOL JSM T33OA Scanning Microscope used for these analysis does not detect elements with an atomic weight less than 18.9, (fluorine) and therefore does not detect the important compositional elements for organisms: carbon, nitrogen and oxygen. For example, a sample of bacteria mounted on a glass cover slip analyzed only as Si and Fe. The glass cover slip is the source of the silica and Fe is measured as background contamination from the steel in the SEM column. The ED AX was essential in determining the elemental composition of sub-micron sized spots on the surface of the mineral grains studied. Since pure minerals were used in each experiment, any elements not found in the original grains were considered to have accumulated on the surface as the result of reactions in the experiments or during sample preparation. Data collected with the EDAX must be interpreted with caution, however, since the volume of the area analyzed by the beam extends into the sample surface up to several microns deep, deeper than the thickness of the surface coatings observed to have formed during reaction experiments. The high intensity of the focused electron beam during EDAX analysis also was useful in blasting holes in organic coatings and bacteria bodies. The EDAX beam will create a bright spot on the surface of an organic material in less than five seconds. Production of the same intensity of burn mark on a mineral surface requires an EDAX beam duration of greater than 30 seconds.

Acid Leach

A series of etching experiments were carried out in order to characterize the effects of non-biologic etching as a comparison to the crystals recovered from the Bemidji microcosms. Calcite crystals between 250 and 700 pm in diameter were prepared from the same material used to produce the microcosm crystals by crushing large clear crystals of Iceland spar, rinsing and ultrasonically cleaning the surfaces to remove clinging microdebris and allowed to air dry. Calcite crystals were placed in four separate clean beakers and etched with four different concentrations of HCI for a given amount of time (Table 5). At the end of the etching period, the acid was diluted to extinction by adding a steady stream of distilled water until the pH returned to near neutral. The calcite was air dried, and prepared for SEM.

Calcite crystals collected from the microcosms experiment in well 018 at the Bemidji study site and un-reacted fresh calcite crystals for controls were etched in 0.2 to 0.3% nitric acid for 0.5, 1.0 and 2.0 seconds. The extent of etching was calculated to remove as much of the surface coating precipitate as possible and as little of the underlying crystal as possible. P. C. Bennett performed the analysis using a Waters lon Chromatograph. An ion chromatograph was chosen instead of an ICP for analysis since the sample size was so small. Each sample was only 2.0 ml total fluid.

Sample 1 Sample 2 Sample 3 Sample 4 HC1, % G 0.1 1.0 10.0 Time. Seconds 60 10 60 120

Table 5. Etching of unaltered Iceland spar calcite in HCI.

Geochemistry

Geochemical data was collected from a field site in Bemidji, MN, where in-situ microcosms experiments were located. The geochemistry of the study-site aquifer has been monitored for the past 10 years by well water analysis from approximately 100 wells by the USGS and by researchers from the University of Texas at Austin. Well construction and standard geochemical sampling, sample handling, and analysis methods were followed and are described in detail in Bennett et al., 1993, and Baedecker et al., 1993. Geochemical measurements made in the field during microcosm placement and recovery and during field column experiments were consistent with techniques used to collect the past ten years' worth of data.

The pH, temperature and redox potential for each well sampled were determined in the field using a flow cell connected directly to the discharge of a submersible or peristaltic pump. Prior to collecting water samples for analysis of inorganic and organic constants, the wells were purged by pumping 2-3 well volumes of water until pH and temperature readings stabilized. Water samples were filtered to o.2gm (Gelman Super 200). Waters for cation analysis were also acidified using ultra-pure HNO3. Nitrate, dissolved oxygen and iron were measured by colorimetry with Chemetrix Chemistries in the field using a Spectronics 20 (Bausch and Lomb).

Results

Bacterial Culturing, Isolation, and Identification

Thirteen pure strains of bacteria were isolated from fluid and sediment of the batch reactor experiments. Four strains, which propagated colonies over several plate transfers, were characterized by biochemical tests (Table 6). Biochemical differentiation tests measure whether the subject bacteria has the ability to produce a specific enzyme that catalyzes a reaction with a known chemical in the test media.

Colony and cell morphology were characterized with transmitted light microscopy and SEM (Fig. 11). Strains were labeled 1,2, 3, and 4. Strains 2,3, and 4 were rod-shaped, with slight differences in overall morphology. Strain 1 was most column-like, uniformly 1.0 pm in length and 0.2 to 0.4 pm in width. The surface was covered by well defined bumps. Strain 1 was identified as a member of the genus Bacillus. Strain 2 was a shorter and fatter rod, with a rough surface. Strain 3 was a long narrow rod, almost 2.0 pms in length and 0.5 pms wide with slightly tapering ends and was identified as a member of the genus Pseudomonas. The surface was rougher than strain 1 or 2. Strain 4 appeared to have two distinct morphologies, a long and narrow twisted-rod form and a short, fat-rod form. Isolates 2 and 4 were not identified to the genus level. As isolates each strain had distinct morphologies and could be distinguished by observation.



Figure 11. Colony morphology (left) and cell morphology (right) of four strains of bacteria isolated from the mixed culture. Strains are labeled 1-4 and correspond to strains identified in Table 6.

Bacteria Strain 12 3 4 Gram Reaction - Motility - - - - Succinate - + - - Mannitol - + - - Lactose - + - - Glucose - + - - Indole . _ _ _ Gelatin Liquifaction _ _ _ _ Oxidase + + + - Catalase + + + + Gram Stain: A differential staining method that distinguishes between Gram Positive or Gram Negative cell wall. Motility: A test of the ability of the subject bacteria to move through a semi-soft gel medium. A positive result indicates presence of flagella, but a negative result does not always indicate lack of flagella. Carbohydrate Fermentation: Acid and or gas is produced if fermentative growth occurs on sugar or sugar alcohols. Positive results indicate that the subject sugar- utilizing bacteria are facultative anaerobes. Indole Test. Detects the presence of the intracellular enzyme complex tryptophanase which degrades the amino acid tryptophane. Gelatin Liquefaction Test. Determines the presence of the extra-cellular enzyme- complex gelatinase which breaks large proteins into smaller pieces. Catalase. This test determines the presence or absence of the enzyme catalase which decomposes hydrogen peroxide. Oxidase. This test determines the presence or absence of the enzyme oxidase.

Table 6. Results of biochemical testing. Key: Biochemical differentiation tests measure whether the subject bacteria has the ability (+) or not (-) to produce a specific enzyme that catalyzes a reaction with a known chemical in the test media. .

Bacterial Morphology and Taphonomy

Calcite crystals retrieved from the experiment described on p. 70-74 after 2 hours were coated with a uniform and smooth film of evaporated organic-rich growth media. Few bacteria were observed on top of the film. Beneath the surface-coating film, rod-shaped and coccoid bacteria ranging from 0.5 to 2.o|im in length were evident as single isolated cells and in clumps of cells (Fig. 12). The coating appeared to be less than o.spm thick since bacteria that are o.spm in diameter protrude through the film and are easily identified. There were so many cells (approximately 3 cells every no pattern of preferential location was evident. All calcite surfaces were colonized with approximately equal density of bacteria.

Completely spherical nannobodies were rare on the surface of the evaporated coating of the grains but they did occur. A more common morphology of the nannobodies was as slightly raised bumps or hemispheres protruding from the surface-coating film (Figure 13A).

In some areas the morphology of the film changed during examination with the SEM. As the electron beam of the SEM focused on small areas of the film, the film shriveled and cracked open. The morphology of the cracks is similar to the familiar large-scale mud cracks on desiccated mud and evaporite surfaces and suggests that the film on the calcite grains was drying out and cracking as a result of heat generated by the electron beam (Fig. 13B). At high magnification, the surface topography of the shriveled organic film showed a surface texture similar to the microcurdled texture described on the surface of grains recovered from batch reactor and Bemidji microcosm experiments.

At the edges of the cracks distinct o.lpm diameter nannobodies were apparent in a range of morphologies (Fig. 13C). Spherical nannobodies were exposed at the very edge of the coating film. Some nannobodies are only partially exposed and appear as hemispheres and some as rounded bumps. Most nannobodies are smooth surfaced at magnifications of 75K and are surrounded by surface film that is characterized by the microcurdled surface.

Samples of the calcite crystals collected after 20 days of incubation showed a reduced or thinner coating on the surface. Bacteria were generally more pronounced as discrete cells on the crystal surface and were not buried as deeply in the coating film (Fig. 14A). Bacteria were forming colonies on the calcite at the time of sampling as evidenced by the presence of attached dividing cells on most surfaces. No obvious change in bacterial colonization density was observed on the mineral surfaces. The density of nannobodies on the surface coating was not substantially different from the 2 hour samples but the nannobodies were more pronounced on the surface, sticking out further from the coating film.

The EDAX beam (spot size of approximately 0.05 pm diameter) was focused for 5 seconds at a time on different places of the crystal surface and burned identical bright spots on the surface of bacteria, nannobodies, and the surface-coating film (Fig 14B). On uncoated calcite surfaces, the effect of a 5 second beam application produced a virtually unnoticeable burn spot. The coating film is much more susceptible to damage from the beam than the underlying uncoated calcite.

Calcite collected after 18 months incubation revealed a much different surface morphology than observed on the samples collected after 2 hours and after 20 days. Most of the calcite surface was either clear of the contiguous graincoating film or revealed only very thin and/or ragged patches of coating (Fig. 15 A). ED AX beam application on the surface of the calcite did not produce the distinct bright spot as similar applications produced on the 2 day and 20 day grain-coating film and on bacteria bodies. In contrast, in some areas densely colonized by bacteria, the microcurdled surface that characterized the batch reactor and Bemidji microcosms grain surfaces was distinct on the grain surfaces.

Bacteria and nannobodies were bimodally distributed on the 18 month calcite grain surfaces. Many bacteria appeared to be identical in surface morphology to those observed in the 2 and 20 day samples. The cell walls of most bacteria were smooth. Some cells were preserved in a growth phase, either doubling or in dense colonies of dividing cells. Many bacteria, however, showed signs of decay. Large (>l.sgm in length) rod-shaped bacteria showed lengthwise cracks from end to end with the edges of the cracked cell wall turned inwards.

In the vicinity of decayed bacteria, swarms of 0.1 to o.s|im nannobodies were common (Fig. 15B). Some of the nannobodies were spherical in shape. However, rod-shaped nannobodies were also observed. The rod-shape nannobodies ranged between 0.1 and 0.5p,m in length and had the smooth and uniformly rounded ends characteristic of rod-shaped bacteria. These rod-shaped nannobodies were associated with an underlying microcurdled surface texture.

Calcite grains recovered from the experiment after 30 days and bleached to remove organic matter showed virtually no evidence of bacteria >o.6pm in diameter. Several cells were observed with smooth, well rounded cell morphologies, but in general, the bulk of the bacterial biomass was removed. The surface of the calcite appeared smooth and flat in some areas, but in most areas the microcurdled surface texture was übiquitous. Abundant and densely packed 0.05 to o.lpm diameter nannobodies were associated with the microcurdled surface, especially evident at the edges of cracks in the residual surface film.

Calcite grains recovered from the experiment after 30 days and heated to 500°C for 2 to 6 hours revealed a remarkably similar surface texture to the bleached grains. In some areas flat mineral surfaces were observed, but in general, the grains were coated with unorganized, amorphous debris. Beneath and surrounding the debris, the surface was characterized by a patchy microcurdled surface as the dominant surface texture. Few bacteria >0.6 pm in diameter were observed, most appeared to have been destroyed by the heat. 0.05 to o.lpm diameter spherical and hemispherical nannobodies were abundant over the surface of the calcite and in the surface debris. Heating the grains for 2 to 6 hours at 500°C had little to no effect on the number, density of packing, and morphology of nannobodies on the surface of the calcite and within the surface-coating film.



Figure 12. Calcite crystal collected from the growth experiment after two hours incubation. A. Overview of calcite grains coated with organic-rich surface film. Note the soft rounded appearance of the crystal edges and the surface coating film stretched between grains. B. Flat surface of calcite grain with coccoid and rod-shaped bacteria on the mineral surface partially covered by the coating-film. Note the desiccation cracks in the film which occurred while the electron beam was focused on this spot. Cracks cut across cells as well.



Figure 13. Calcite crystal collected from the growth experiment after two hours incubation contrasting smooth and cracked surface film. A. Overview of the surface coating film showing a contact between smooth and dried and cracked area. The cracking occurred while the electron beam was focused on this field of view. B. Detail of cracked surface showing development of microcurdled surface and nannobodies at the edge of cracks. C. Detail of the edges of cracked surface film. Note bumpy surface showing distinct rounded shapes ranging from 0.01 to 0.05 pm in diameter. Note also 0.1 pm diameter nannobodies in the film matrix and on the surface of the film.



Figure 14. Calcite crystal collected from the growth experiment after 20 days incubation. A. Note the bimodal distribution of normal sized cells and nannobodies. The bacteria are more pronounced as discrete cells and the surface coating film is thinner than in the two day sample. B. Three blast points produced by the EDAX beam focused on the surface for 5 seconds. The effect is the same on bacteria, nannobody and on the surface film.



Figure 15. Calcite crystal collected from the growth experiment after 18 months incubation. A. Calcite surface showing sparse colonization by bacteria and patches of smooth un-coated mineral surface. In many areas the formerly übquitous surface coating was patchy or gone. B. Near decaying bacteria, 0.1 to o.spm diameter rod-shaped nannobodies occurred in swarms. This is the first evidence of rod-shaped nannobodies.

Batch Reactors

Within three days of inoculation, bacteria had colonized some surfaces of unconsolidated quartz sand and penetrated to the interior of 2 cm sandstone chips and colonized internal pore surfaces. Bacteria that appeared in the center of the sandstone chip moved from the reactor fluid, through the pores in the rock to the location where they were observed in three days or less. The distance to the center was approximately 1.0 cm from any direction.

Bacterial Colonization and Growth

Cell Morphology

Mineral surfaces colonized by the mixed culture from which the isolates were recovered were studied for colonization patterns and to determine if the isolates could be identified from the mixed communities on the basis of cell morphology.

After growth of the mixed culture for three or more days, individual strains could not be unambiguously identified on the basis of cell morphology on the surface of minerals (Fig. 16). New morphologies of rod-shaped bacteria were evident. Rod-shaped bacteria appeared in morphologies intermediate between the three distinct morphologies of Strains 2,3, and 4. Only rarely were recognizable cells unambiguously identified. Coccoid bacteria were as common as rods on surfaces.

Most bacteria showed no sign of polymer adhesion, flagella, or attachment filament and may have adhered directly or by cryptic filaments at the bacteria/mineral contact (Marshall 1976, Rades-Rohkohl 1978, and Marshall 1980). Some bacteria did attach with visible filaments, for example, an elongate rod attached to a feldspar surface within a sandstone pore by some type of polymer (Figure 16).



Figure 16. Bacteria strains are not identifiable by morphology after incubation in batch reactor experiments.

Colony Morphology

Bacteria were most commonly observed colonizing rock and mineral surfaces as single isolated cells. Microcolonies, (clusters of 10-100 bacteria) were also commonly observed. On sand grains microcolonies were observed in cracks and in depressions of the surface. Within the pores of sandstone, microcolonies occurred randomly, even on the sharp terminations of quartz overgrowths (Figure 16).

Patterns of Colonization

On quartz sand grains, bacteria were observed to be concentrated in cracks and crevices, while sparse colonization was observed on exposed smooth surfaces. Bacterial colonization of internal pore surfaces of sandstone chips were distributed across smooth surfaces and within cracks more evenly. A variety of bacterial types were observed to colonize smooth quartz overgrowth surfaces and clay surfaces with nearly equal density .

Field Microcosms

Upon recovery from the aquifer after 14 months, previously clean mineral surfaces were coated with an amorphous sub-pm thick organo-iron precipitate and colonized by indigenous bacteria. Beneath the surface coating, it was evident that both the silicates and carbonates had been substantially altered. Calcite crystals were less densely colonized by vegetative bacteria than the silicates. Dolomite was observed to have dissolved, however no evidence of colonization by vegetative bacteria was found. Most calcite crystals showed surface-wide precipitation features of sub-pm spikes which coalesced into 0.5 pm thick sheets.

Ground Water Chemistry

Field analysis of ground water from both wells in which the microcosms were placed and surrounding wells was carried out upon recovery of the microcosms (Table 7). The chemistry of the ground water is roughly consistent by geochemical zone with data taken for the past four years. B

The pH of water in wells where oil is not present ranges from 0.5 to 1.0 pH units higher than in the oiled wells where the microcosms were placed. Positive Eh and dissolved oxygen values of greater than 2.0 ppm in non-oiled wells confirmed oxidizing conditions. In contrast, the oiled wells had negative Eh readings and dissolved oxygen values of substantially less than 1.0 ppm, the lower limit for aerobic bacterial activity. Total iron concentrations were non-detectable in the non-oiled wells, but in the oiled wells Fe was measured at 0.6 ppm and greater. Interestingly, temperature was slightly (1-2°C) elevated in the oiled wells. Increased temperatures of 1-2 degrees above background temperatures have been noted in microbially active ground water during bioremediation of hydrocarbon pollution activities (Chris Hollingsworth, Biotech Remediation, pers. com. 1993).

Suspended particles and precipitates were observed in ground water from which the microcosms were collected. A rusty-red precipitate formed immediately upon contact with air and sank to the bottom of the sample jars. The precipitate consisted of quartz and calcite silt, authigenic calcite, amorphous iron, minor amounts of kaolinite or chlorite and goethite, and live bacteria. In the light microscope, the mineral silt was large enough to show quartz and calcite birefringence in polarized light. At 600-1000 X in normal transmitted light, swarms of motile and sessile bacteria were observed mixed throughout the precipitate. Observation of the bacteria was greatly enhanced by standard microbiological staining techniques.

X-ray analysis of the precipitate confirmed the presence of calcite, quartz, kaolinite or chlorite, and the iron hydroxide goethite. Goethite is a common weathering product of iron-bearing minerals formed under oxidizing conditions. It is known to occur as a direct precipitate from iron-rich ground and bog water (Deer, Howie and Zussman, 1966). A large background peak in the diffraction pattern revealed that the bulk of the sample consisted of amorphous non- crystalline material (Fig. 17). The large amount of amorphous material in the precipitate is consistent with the observed high levels of dissolved and reactive mineral and organic compounds present in the anaerobic ground water in which the microcosms were placed.

SEM observations and EDAX analyses confirmed that most of the precipitate consisted of microspherical orbs with a dominant Fe composition, authigenic carbonate, and a wide variety of bacteria (Fig. 18). Large rod-shaped cells (> 1.0 Jim in length) were identified as vegetative bacteria. Nannobodies (@ o.2s|im in diameter) are probably a mixture of bacterial cells and inorganic precipitates. EDAX analysis resulted in major Fe and minor Si and Ca peaks, consistent with X-ray analysis. Dividing cells indicate active growth in the ground water. Microcurdle or bumps on the surface of cells may be indicative of mineralization of a cell surface (amorphous Fe precipitation) or alternatively represent normal cell surface morphology (Atlas, 1984, p. 59).

Visually striking elements of the precipitate were euhedral authigenic calcite crystals that ranged in size from less than 4.o|im to greater than 200p.m in length (Fig. 19) The surface of larger calcite crystals showed a similar sub-|im stepped surface architecture as the clean prepared calcite grains produced for microcosm experiments. The surface, however, was heavily coated with a mixture of large and small bacteria, and bacteria-like framboidal particles. Many larger bacteria did show distinctive bacteria morphology and were identified unambiguously as bacteria (Fig. 20). The surface of the authigenic calcite also exhibited a bumpy sub-jim coating on rounded faces of the crystals (Fig. 21). A high density of bacteria was present in the ground water sampled from the microcosm wells. A similar density of cells was never observed on samples of microcosm minerals. The large number of bacteria in the precipitate from the microcosm wells demonstrates that the ground-water beneath the oil body is bacteria-rich, and a likely location for observing intense biochemical activity.

At high magnifications (20,000 to 50,000 X), the surfaces of some large bacteria were noted to be covered with bumpy micro-coating (Fig. 22). Every surface in the frame of the micrograph is uniformly coated including the 0.25 pm diameter spheres in the upper right. It is unlikely that the coating represents bacterial budding since the surface effect is uniform across various cell morphologies at the same instant in time. The large bacteria may be coated with minerals or the cell walls may have mineralized. No morphological differences in large bacteria were noted between samples prepared by fixing with gluteraldehyde and those that were simply air dried prior to gold coating for SEM.



Figure 17. X-ray diffraction results of precipitate formed in ground water samples collected during the recovery of microcosms from wells 015 and 017 in October, 1992. Identical precipitate formed on mineral surfaces at the inlet and first four centimeters of field columns. Goethite, an iron hydroxide, kaolinite, chlorite, calcite and quartz peaks are identified. The large "hump" in the center of the diffraction pattern indicates that a large amount of the sample consists of amorphous, non-crystalline material. The large amount of amorphous material in the precipitate is consistent with the observed high levels of dissolved and reactive mineral and organic compounds present in the anaerobic ground. water in which the microcosms were placed. X-ray diffraction by F.L. Lynch.



Figure 18. Precipitate formed in ground water samples collected during the recovery of microcosms from wells 015 and 017. A. Overview of precipitate: mixed cells, nannobodies, and spherical mineral precipitates. Large rod-shaped cells (> 1.0 pm in length) are identified as vegetative bacteria. Smaller orbs (@ 0.25 pm in diameter) are probably a mixture of bacterial cells and inorganic precipitates. EDAX analysis results in major Fe and minor Si and Ca peaks, consistent with X-ray analysis. B. Dividing cells (D) indicate active growth in the ground water. Note 0.1 pm nannobodies. C. Two distinct morphologies of 0.25 pm cells: smooth (S) and bumpy (B).



Figure 19. Electron micrographs of authigenic calcite from precipitate formed in ground water samples. Magnification and scale bars are included in each separate micrograph. A. Euhedral calcite crystal coated with amorphous Fe and cells. B. Stepped crystal face exhibits similar surface morphology as the fractured Iceland spar calcite used for microcosm work. C. 0.25p.m and 1.0p.m cells colonizing the surface of calcite. At magnifications > 10,000 X, the surface of authigenic calcite appears rough in contrast to the surfaces of prepared Iceland spar.

Well# Zone PH Eh Cond Temp Fe DO 707b 1- aerobic 7.81 -45 490 8.5 75 >2 709 1 - aerobic 7.05 75 480 7.5 nd >2 603 2- oxidizing 7.23 180 750 8.9 nd >2 708 2- oxidizing 6.85 185 900 9.1 nd >2 707a 2- oxidizing 7.09 150 930 8.8 nd nd 523 2-oxidizing 6.98 nd 1320 10.2 nd 2.15 017 3- anaerobic 6.48 -60 1900 10.2 0.6 78 015 3- anaerobic 6.62 -55 1030 9.3 >high 27ppb 604b 4- mixing 7.18 15 830 9.3 82 42.5

Table 7. Water well chemistry on October 10, 1992, Bemidji, MN research site. Microcosms were recovered from well 017 and 015 after 14 months of in-situ reaction with anaerobic ground water and indigenous bacteria.

Silicates

Colonization

Scanning electron microscope examination of the surfaces of minerals recovered from the microcosms showed evidence of colonization by bacteria in a vegetative (growing) state. A wide variety of morphological types of bacteria were documented. The bacteria ranged in size from as small as 0.25 pm in length and .05pm in diameter (not including flagella) to over 1.5 pm in length and o.Bpm in diameter. The most common morphology was medium-sized rods, which averaged I.opm by 0.5 pm. Pseudomonas-like bacteria, rods 1-1.5 pm in length and 0.5 pm in diameter were common as isolated cells and as microcolonies (Fig. 23). Larger-diameter rods with a characteristically bumpy surface were also observed (Fig. 23). The bumpy surface illustrates the rigid structure of the peptidoglycan layer of the cell wall. Short peptide chains cross-link repeating units of N-acetylglucosamine and N- acetylmuramic acids to form a porous but rigid cell wall, which maintains the cell's shape. If these peptide links are disrupted (for example by drying or chemical alteration) the cell is unprotected from osmotic shock and may burst. Critical point drying permanently cross-links the peptidoglycan layer and preserves cell morphology during sample preparation. An alternative explanation of surface bumps is that they represent spore formation or surface colonization by nannobacteria (Folk, 1993). Some cells were preserved as they were in the process of dividing, thus indicating that the attached bacteria were actively growing during the microcosm emplacement experiment..

In a few instances, the attachment mechanism of bacteria was observed. A mucus-like film extended from the cell wall to the mineral surface (Fig. 24). The development of a sticky membrane for attachment, glycocalyx, is common in many heterotrophic bacteria. The web-like strands surrounding each cell are an extra-cellular glycocalyx, or slime layer. The glycocalyx is a mechanism for aquatic bacteria to bind to surfaces and may also assist the cell in binding nutrients required for growth. Most of the cells observed on the microcosm mineral surfaces did not exhibit such pronounced glycocalyx development. Whereas most bacteria observed appeared to be attached, some bacteria capable of free motility as evidenced by the presence of long (2.0 pm) flagella were also observed (Fig. 248). Bacteria that colonized the minerals in the microcosms must have been passively transported by water flow or actively moved across the "water gap" between the aquifer sediment and the microcosm surfaces. The flagella of the smaller rod indicates the capability of active motility.

Several colonization morphologies were documented, primarily individual cells and small patches (Fig. 25). Isolated bacteria were the most commonly observed. The 0.25 pm and smaller spherical nannobodies are abundant and appear similar in overall texture to nannobodies from acid-etched aragonite needles and oolites described by Folk (1993). Microcolonies (2-100 cells clumped together) were also common. Unexpectedly, no large-scale biofilms of full-sized bacteria were observed. However, some mineral surfaces were coated with o.lpm diameter nannobodies.

Distinct boundaries between lightly coated and more heavily coated regions were clearly revealed and at high magnification (50,000 X a sharp "front" of surface coating composed of o.lpm nannobodies was observed (Fig. 26). Although ground water was pumped directly from the well into the columns, it is likely that inflow and top centimeters of each column represented oxidizing conditions. Coating of the prepared grains occurred quickly. The grains in the columns were partially coated in just three days. A sharp linear edge was observed at the leading edge of the microcurdle texture and suggests a chemical or physical control on distribution. Whereas some bacterial colonization is known to form straight-edged colonies, the most common colony edge morphologies are lobate, undulate, curled, filamentous and erose (spiky) (Atlas, 1993). In addition, the small size of the individual particles, which are uniformly about 0.05p,m, are approximately the size of most viral capsids, the protein sheath that protects the simple genetic material of a complete virus, and would generally be considered too small to represent a complete cell capable of growth and reproduction. If these bumps do represent growing cells, they are smaller than any complete bacterial cells recognized by microbiologists (Casida, 1977; Bakken and Olsen, 1987; Brock and Madigan, 1988).



Figure 20. Electron micrographs of the surface coating of authigenic calcite from precipitate formed in ground water samples. A and B. Overview of various bacterial morphologies found on authigenic calcites. C. Bacteria with bimodal distribution of long rods and 0.1 pm -0.25 pm nannobodies. Note smaller hemisphere-shaped bumps on the surface of rodshaped bacteria. Folk (1993) has identified similar bumps on bacteria from travertine-forming springs as nannobacteria and/or vegetative bacteria budding structures.



Figure 21. Electron micrographs of authigenic calcite and Ferich precipitate formed in ground water samples. A. Authigenic calcite and framboidal Fe-rich precipitate from well 017. The calcite crystal faces are flat with no coating of cells or Fe. B. The backside of the crystal does show rounded bumps which could be interpreted as an organic coating or alternatively as the exposed and rounded edges of stepped crystal sheets as seen in previous micrographs of prepared and authigenic calcite. C. Detail of B.



Figure 22. Electron micrograph of vegetative bacteria from precipitate formed in ground water samples. Magnification and scale bars are included in the micrograph. A rod-shaped bacteria, caught in the early stages of cell division, is coated with euhedral to semi-euhedral Fe-rich coating. EDAX analysis indicates that the coating is predominantly Fe with a minor Si component. B. Surface features of authigenic calcite from the ground water precipitate. This is a typical view of calcite showing the micro-curdled surface texture of 0.02-o.o4|im diameter bumps.



Figure 23. Electron micrographs of vegetative (growth phase) bacteria attached to surfaces of prepared minerals recovered after 14 months of submersion in the anaerobic ground water of the Bemidji, MN, study area. A. The most common morphology of attached vegetative bacteria observed during this study was pseudomonad-like rods. Note that the large bacteria in the center of the micrograph is resting on a surface completely coated with micro-curdled texture and spherical, and roughly 0.1 |im diameter nanno-bodies. B. High magnification detail of surface morphology of a short-rod bacteria. The spot in the center of the bacteria is the result of "shooting" the cell for 30 seconds with the electron beam focused for EDAX analysis. "Shooting" bacteria with the EDAX beam most often deflated or destroyed non-fixed cells. This cell did not pop, suggesting that the surface had hardened or mineralized.



Figure 24. Electron micrographs of bacterial attachment mechanisms observed on microcosm mineral surfaces. A. A micro-colony of attached rod-shaped bacteria. Most of the cells observed on the microcosm mineral surfaces did not exhibit such pronounced glycocalyx development. B. Two rod shaped bacteria of vastly different sizes illustrating attachment and motility. The central 1.0p.m long bacteria is in the advanced stages of dividing and just above the cell a flat sheet of glycocalyx (G) is spread across the mineral surface. Abutting the right-hand side of the dividing bacteria note a o.3|im long rodshaped bacteria (B) which exhibits a long flagella (F).



Figure 25. Electron micrographs of various colonization morphologies observed on surfaces of the microcosm minerals. A. The most common observation of vegetative bacteria on mineral surfaces was as single cells. The surface texture of the underlying mineral (quartz) is completely micro-curdled. B. Microcolony of mixed spherical and rod-shaped bacteria attached to a quartz grain surface collected from the field flowthrough column experiments. Mixed in with the cells are framboidal particles of ambiguous composition (Fe, organic, or microbiologic?). Note unambiguous rod-shaped bacteria with nannobody or budding structure at lower right.



Figure 26. Electron micrographs of prepared minerals recovered from field columns through which ground water from anaerobic microcosms wells had flowed for three days. A. Surface of a quartz grain that shows a boundary between the clean prepared surface and a portion of the surface coated with Fe-rich precipitate. B. High magnification of a boundary between smooth and curdled surface on a quartz grain. The sharp linear edge of the curdling suggests a chemical or physical control, not biolgocial, on the texture formation. Straight boundaries are rarely observed in bacterial colonization processes.

Surface Coatings

Virtually all of the prepared mineral surfaces were dramatically changed during their residence in the aquifer. The most obvious and übiquitous alteration was the coating of mineral surfaces, regardless of mineral type, by a sub-|im thick layer of hemispherical bumps and micro-spherical orbs ranging from very uniform arrangements to mixed jumbles, the previously defined microcurdled surface texture (Fig. 27). The microcurdled texture is not an effect of gold coating of specimens. Many non-bumpy surfaces of minerals prepared in an identical manner have been observed at the same high magnifications. Folk (1978) has described a similar surface texture, characterized as a "turtle-skin" coating on quartz grains collected in the Simpson Desert of Australia. The micro-curdled surface texture on the microcosm grains is similar to turtle-skin but the features on the microcosms grains occur on a smaller size-scale. EDAX analysis indicated an overall elemental composition of Si and Fe. ED AX analysis revealed presence of K, Na, Al, Si and a large Fe peak. The size of the bumps that comprise the microcurdled surface range from 0.02-0.25ym in diameter, with clumps of 0.02ym bumps joined together to form larger diameter spheres.

ED AX analysis of the elemental composition of the coatings showed that iron (Fe) was übiquitous and in high relative concentrations to all other elements. The surface of coated quartz crystals showed only Si and Fe. Analysis of coated feldspar grains showed expected composition of K, Al, and P plus a large Fe peak. Calcite grains showed Ca, Fe and a small Si peak, which suggested that the coating had a minor Si component in the overall composition.



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Dissolution

Possible dissolution effects on quartz were noted on sand grains recovered from the batch reactor experiments. On smooth quartz overgrowth surfaces, several types of bacteria (spheres, curved rods, and some rods) were observed to be attached in "micro-divots" and runnels which ranged from 0.1- 0.5 yms deep, and from between 1-3 yins in length and width (Figures 28). The margins of the divots are not sharply defined, rather they gently grade from the deepest directly beneath the bacteria outwards into the plane of the mineral surface. The bacterial divots were most commonly observed on the quartz overgrowths of the Shannon sandstone samples. Not all types of bacteria were observed in divots. There appeared to be a correlation between the occurrence of micro-divots and attached bacterial bodies.

The divots are not apparent in top-down views of bacteria on mineral surfaces because of the lack of divot edges. The divots are visible only in side views (profiles) at an angle that allows observation of the bacteria/mineral surface contact. These divots were not noticed for a long time since most SEM imaging at high magnifications (10K-50KX) was from a top-down perspective. The divots are not an effect of the SEM. Stereo pair micrographs of bacteria in divots confirm that a real depression is present (Figure 28). In addition, bacteria not in divots were documented attached next to bacteria in divots, thus substantiating that the divot is not an artifact.

Slight but distinct etching was noted on the surface of quartz grains recovered from the Bemidji microcosms. The etching occurred in the vicinity of attached bacterial cells (Fig. 29). Distinct triangular etch pits formed on a formerly smooth quartz surface and near various types of attached bacteria. The triangular morphology of the pits is characteristic of chemical etching. The chemistry of the bulk pore water, however, indicated that dissolution of quartz should not have occurred. Etching of quartz occurred in water many times supersaturated with respect to quartz (Baedecker et al., 1993; Bennett, 1993). Triangular etch pits are grouped together in discrete areas, while other areas of the quartz surface are apparently unetched. The triangular shape of the etch pits is diagnostic of crystallographic control of quartz dissolution (Bennett, 1991). The occurrence of these pits suggest that a volume of weathering fluid more reactive than ground water persisted at the mineral interface long enough to initiate etching. Etch pits in the shape of bacterial cells were not noted in this study, in contrast to the results of the batch reactor study and a recent report of bacterial weathering of basaltic glass (Thorseth, Furnes and Heidel, 1992).

In order to observe diagenetic effects beneath the micro-coating observed on many grains, some grains were "cleaned" by ultrasonically dislodging the surface coating and rinsing with distilled water. Most surface coating and debris was removed by this procedure, but at high magnifications (40,000 +) the quartz surface revealed 0.05 pm bumps that formed a micro-curdled surface (Fig. 30). The micro-curdled surface texture was not observed on un-reacted control grains. Extremely fine etching effects were observed on cleaned quartz surfaces studied with high-resolution high magnification SEM. Open-ended and very regular triangular shaped etch pits ranged from o.spm to I.opm in diameter. Microrunnels (0.02 pm X l.Op) traversed I.opm steps in the quartz surface between triangular etch pits and appeared to be etching effects as well (Fig. 30). Microrunnels connect etch pits which occur on different stepped surfaces. These may be the initiation of new etch pits or simply be an artifact of the crystal edge structure of the stepped surface. At 40,000 X the quartz surface appeared cracked and curdled. Individual bumps that compose the surface are still too small to be resolved, but resemble a shrunken version of the microcurdle surface previously described.

Microcline grains straight from the microcosms, without surface cleaning, revealed a surface coating similar to that on quartz: loose mineral debris, microcurdled texture, single bacteria, and isolated microcolonies. In contrast to the light weathering of quartz, some microcline surfaces were intensely etched and weathered (Fig. 31). Deep linear chemical etching of the microcline surface was observed near colonies of attached bacteria. Dendritic fibrils were scattered between the etch pits. The etch pits are prismatic in shape and orient along crystal planes, suggesting that the shape was controlled by crystallographic constraints such as cleavage or twinning planes. Diverse attached bacteria were present on the surface of microcline grains.

Microcline grains were ultrasonically cleaned of surface coating, bacteria and debris to reveal the morphology and extent of etching. The cleaning procedure involved low power ultrasonification and short immersion times to avoid any possibility of creating a surface artifact on the microcline grains. The etch pits observed are similar to those produced in laboratory studies of chemical dissolution of feldspars (Berner and Holdren, 1979; Knauss and Wolery, 1986). However, the volume of etching is unexpected given the bulk geochemistry of the surrounding pore water and suggests that a fluid more highly reactive than ground water was present at the immediate mineral surface.



Figure 28. Stereo pair image of bacteria colonizing the surface of quartz overgrowths on a sand grain from the batch reactor experiments. Bacteria were often found in slight depressions in the mineral surface. Note abundant nannobodies



Figure 29. Electron micrographs of quartz crystals recovered from anaerobic zone microcosms after 14 months. A. Quartz surface showing microcurdled coating, attached vegetative bacteria and ragged-edged triangular etch pits near the bacteria. B. Detail of micrograph A. Note the 0.3p.m diameter etch pits (P) immediately next to the bacteria on the left. C. Distinct triangular etch pits (P) on a formerly smooth quartz surface and near various types of attached bacteria.



Figure 30. High resolution scanning electron micrographs of quartz grains that have been "cleaned" of the microcurdled coating, attached debris and bacterial cells. A. Quartz surface showing the regular stepped surface characteristic of the prepared surface prior to submersion in microcosm experiments. Isolated pockets of regular triangular etch pits were apparent and confirmed observations of etching documented in the "uncleaned" microcosm samples. Scale bar = 1 pm. B. Detail of micrograph A. Micro-runnels connect etch pits which occur on different stepped surfaces. Scale bar = o.lpm. C. High magnification view of the surface within an etch pit. The entire surface appears as a miniature version of the micro-curdled texture of uncleaned microcosm mineral surfaces. Scale bar = o.lpm.

Precipitation

Some microcline grains had patches of surface covered with a honey-comb shaped matrix of precipitate (Fig. 32). In some cases, feldspar grains etched on one end had a build-up of this precipitate on the other end of the same grain. High magnification inspection of the precipitate revealed that each component "blade" of the honeycomb matrix was coated with, or composed of, spherical or rounded particles. The particles range in size from 0.02 to 0.05 pm in diameter and appeared to agglomerate in linear ribbons which protruded above the underlying grain uniformly at a height of between 0.1 to 0.5 pm.

EDAX analysis of the clay-coated surface showed the same elemental constituents as the underlying microcline, but these results are ambiguous since the area of analysis of the electron beam could not resolve just the surface coating and included elements in the underlying grain. Decomposition of microcline by normal weathering processes produce kaolinite, halloysite, smectite, sericite, quartz, and gibbsite. Halloysite, a hydrated form of kaolinite is reported to have a fibrous, tubular or scroll-like morphology when examined with the SEM (Deer, Howie and Zussman, 1966). The EDAX and SEM data suggest that the precipitate on the microcline surface may be halloysite, but this has not been confirmed by other analytical methods. Cozzarelli, et. al. (1993) reported the occurrence of small amounts of kaolinite and smectite from the anaerobic sediments in this study area. The morphology, texture, and composition of the precipitate is similar to smectite which occurs in the Frio Formation of the Texas Gulf Coast (F. Leo Lynch, pers. com. 1994) The bladed morphology of the clay has also been recognized as an artifact of dehydration during sample preparation, but the morphology is typical of smectite (E. F. Mcßride pers. com. 1994).



Figure 3 1. Electron micrographs of surface of microcline crystal fragment recovered from in-situ microcosm after 14 months. The etch pits are prismatic in shape and orient along crystal planes, suggesting that the shape of etch pits was controlled by crystallographic constraints such as cleavage or twinning planes. B. At least four morphologies (1-4) of attached bacteria are present in the surface area of this micrograph, only 300 gm 2 , which indicates the wide diversity of bacteria that colonized feldspar surfaces. C. Surface of microcline grain as in A and B, which was ultrasonically cleaned of surface coating, bacteria and debris to reveal the morphology and extent of etching.



Figure 32. Electron micrographs of authigenic clay minerals on the surface of microcline grains. A. Many feldspar grains were coated in patchily distributed areas with authigenic clays. On some microcline grains, clays patches were found immediately adjacent to areas of microbial attachment and etching. The clays in this micrograph are configured in a honeycomb fence pattern with a uniform fence height of 0.25j1m. B. and C. High magnification views of A. Note übiquitous micro-curdled texture on mineral surface. Also note that at highest magnification, the clay fence contains nannobodies.

Carbonates

Colonization

The SEM analysis of several batch reactor calcite experiments indicated fewer vegetative bacteria attached to the calcite surface than to silicate surfaces. SEM analysis of the calcite crystals recovered from the Bemidji field microcosm experiments also showed sparser colonization of calcite than of silicates by vegetative bacteria. The 0.5 pm short rod-shaped bacteria were noted in abundance in isolated patches on some flat calcite surfaces (Fig. 33).

Dissolution

In the immediate vicinity of bacterial colonization, deep (1.0 to > 3.0 p.) etching of the calcite surface was observed. Etching was evident in two morphologies of pits. Sub-pm diameter pits appear as irregular spots randomly distributed in the vicinity of cells or cell-debris grain coatings. The pits are estimated to be less than 0.1 pm in depth. In contrast, much larger pits, I.opm to >6.opm in length, have distinct prismatic edges and depths of 2.opm and deeper. Pits intermediate between the suggested end members show combinations of irregular and prismatic edges and highly variable shapes. The larger the pit, the more uniform and prismatic the edges.

Results from the control etching experiments helped interpret the etching observed from field microcosms. Samples 1 and 2, with 0.0% and 0.1% acid showed no effects of etching. Sample 3 was very lightly etched and sample 4 was deeply etched. Well defined parallel linear patterns of dissolution occurred along crystallographically defined planes. The distance between the linear features is 3.opm. Step heights from one sheet to the next measured o.spm in height. Along the edge of the step which exposed the o.spm face to the dissolving fluid, pits with prismatic leading edges formed at roughly a 45° angle to the linear etching. The edges of the etch pits are euhedral and smooth, even at magnifications of up to 35,000 X. (Fig. 34). The etched calcite revealed no nannobodies or curdled surface texture, as was commonly observed in the calcite materials collected from laboratory and field experiments. The etched crystal face also revealed long tabular steps with rounded ends. Similar rounded features have been described by Berner and Morse (1974) as the result of diffusion-limited dissolution, which is expected considering the strength of the acid used for etching.

Along entire surfaces of some crystals, fields of uniform points of calcite protrude from a flat background face (Fig. 34 C). The points have uniform terminations and range from o.2pm to 3.opm in height. The points are arranged in linear patterns and in isolated clumps. The orientation of the etch features reflects the crystallographic structure of calcite. At magnifications above 10,000 X, the points emerge from a flat base, like pyramids in the Sahara. Close examination of the flat area between exposed points shows the tips of points similar to the larger ones, but just barely emergent.

Neither bacteria nor nannobodies were observed on or in any of the etched spar, nor was any micro-curdled surface texture observed, both of which are übiquitous features on the calcite recovered from the microcosms. The deep, but highly localized, etching associated with concentrations of attached bacteria on the calcite recovered from the microcosms is consistent in size and general shape, but not of exact morphology, with etch pits produced in the HCI etching experiment.

Dolomite crystals recovered from the Bemidji microcosms showed deep etching especially along crystal edges (Figure 35). Few bacteria were observed colonizing the dolomite crystals. The microcosms were placed in ground water that has been shown to be undersaturated with respect to dolomite ( Baedecker et al., 1993, Bennett et. al, 1993). Evidence of dissolution of dolomite was expected and is consistent with the chemistry of the ground water.



Figure 33. Electron micrographs of calcite crystals recovered from in-situ microcosm after 14 months. A. At low magnifications, calcite grains appear unaffected by the 14 month reaction period in anaerobic ground water. Sharp fractured crystal edges and smooth flat surfaces appear as in the prepared materials prior to the microcosms experiments. B. Colonization of calcite by vegetative bacteria was sparse compared with silicate minerals. Where bacteria colonization did occur however, deep chemical etching occurred in the immediate vicinity of the attached cells. This image represents a rare occurrence of attached bacteria and a variety of similarly shaped but widely varying sized etch pits.



Figure 34. Freshly fractured crystal of Iceland spar calcite ( from the same stock of calcite used in all studies) etched for 120 seconds in 10% HO. The entire surface was etched in distinct linear patterns along crystallographic axes and along cleavage planes. A) Etching along steps in the crystal face caused by fracturing of the crystal prior to etching. Step height is approximately 0.5 pm. B) Detail of etch pit termination. Note the smooth surface and euhedral shape of the etch pit terminations. The etched calcite revealed no nannobodies or curdled surface texture, as was commonly observed in the calcite materials collected from laboratory and field experiments. C) Effect of heavy etching on the fractured edge of a calcite crystal. Note the parallel edges and stacked appearanc of the etch features. The etching has "sharpened" the edges of the fractured steps and the angle of the electron beam has highlighted the edges of the etched steps.

Precipitation

A übiquitous thin layer of uniform-height calcite precipitate covered the surface of all calcite crystals recovered from field microcosm experiments in Bemidji. The precipitate is composed of a carpet of morphologically diverse calcite spikes, elongated parallel to the c-axis of the mother crystal. The density of spikes across the surface of the underlying crystal varies. In some small areas, the original flat crystal surface is exposed. Most of the underlying crystal surface is covered by a mass of spikes which forms a continuous flat-topped surface, approximately 0.5 to 2.opm above the flat face of the underlying crystal.

Three basic morphologies of spikes were observed (Fig. 36). Sharp-tipped pyramidal bladed spikes stand on a rhombic base and extend a uniform 0.5 pm out from the underlying crystal surface. Spikes with a rounded base, which is one to two times thicker than the tip, occur over most of the calcite that was studied. The smallest of these spikes, 0.05 to o.lopm in diameter, appear as irregular hemispheres against a flat crystal surface. Larger spikes, 0.10 to o.spm in height, occur with both rounded and sharp euhedral points. Column-type spikes with a uniform diameter from base to tip range from 0.1 to 0.2 pms in diameter. As best could be determined, all spikes regardless of morphology are oriented parallel to the c-axis of the underlying crystal.

Individual spikes form straight rows across the surface of the underlying crystal at equal 1.0 to 2.0 pm intervals. Some lines of spikes intersect but most spikes occurred in parallel lines. Some sets of spikes formed "corrals" which were empty on the inside or filled with spikes to the point of forming isolated plateaus o.spm in height. The sharp pyramidal spikes in straight lines and the corrals of sharp spikes represent the most visually beautiful mineral precipitation encountered in this study.

Near depressions in the calcite surface caused by the original mineral preparation process, areas of rounded spikes grade into areas of sharper spikes (Fig 37). While the spike morphology gradually changed, the ultimate height of the precipitate stayed exactly the same, regardless of the total size of the spike.

The total height of the precipitate from the surface of the underlying grain is often obscured by flat layers of an amorphous, non-crystalline material spread between the tips of the spikes (Fig. 38). A calcite crystal was fractured revealing edges and cross-sections of the surface precipitate. In cross-section, column spike crystals proved to be the surface expression of 2.0 pm long crystals. The base of the crystal was sub-pm in diameter and gradually expanded to o.spm diameter at the top. The single crystal bifurcated at the surface into eight to ten rounded terminations. The crystal tips gathered to form a new surface with a bumpy texture. Distinct wispy layers of amorphous material occurred throughout the surface precipitate at several levels perpendicular to the long axis of the crystals. This layer appears flat and continuously horizontally bisecting the crystal. This layer may represent the flat "floor" that appears beneath and next to areas of bumpy surface texture. In cross-section, these layers appear as wispy bands, less than 0.1 pms in thickness that are continuously connected between individual spikes of precipitate. From a map-view perspective, the layer between the spikes appears to be the flat underlying surface of the mineral. In some cases, however, spikes that appear to be 0.3 pms in height, actually are the tips of 2.0 pm spikes with a layer of amorphous material spread between the tips at the 0.3 pm height. The total height of the surface precipitate on most of the calcite grains from the Bemidji microcosms was approximately 2.0 pms.

High resolution SEM of the calcite precipitate and the flat layer between spike tips revealed details of the surface features of the spikes and inter-spike layers (Fig. 39). A field of o.spm high spikes showed flattened tops and evidence that narrow spikes coalesced as they increased in width. The surface of each spike which appeared smooth even at 30,000 X magnifications appeared bumpy at 80,000 X. The surface did not appear as an agglomeration of microspheres, but did appear very similar to the previously described curdled bumpy texture common on calcite as well as quartz surfaces.

Examination of the flat areas between spikes at 100,000 X magnification revealed a generally curdled, microgranular surface with distinct rounded spherules on top of the surface. The spherules ranged from approximately 0.02 to 0.07 pm in diameter. These particles appear to be almost identical in size and shape to calcite nucleation points described by Dove and Hochella (1993) in calcite growth experiments observed with Scanning Force Microscopy.

The precipitate-coated surface of one of the calcite grains described above was studied with atomic force microscopy (Nannoscope 111, Digital Instruments, Santa Barbara, California) in order to evaluate the surface morphology with a different line of evidence and at higher magnification and resolution. The instrument is in the Chemistry Department, University of Texas at Austin. A plan view image of an area of individual spikes and coalesced plateaus was prepared from a sequence of linear scans across the surface of the crystal. The basic morphology of the surface as described by SEM was confirmed, however, the profile of the transition from flat mineral surface to spike base and up to the spike tip was clarified and the angles quantified (Fig. 40). This technique offers great potential for future work, since the atomic force microscope can study wet surfaces. Samples could be collected from an aqueous experiment or aquifer environment and studied without drying thus avoiding the potential for creating surface artifacts that may be associated with the drying process. In the analysis of the Bemidji calcite, however, no major differences in surface interpretation were warranted as a result of data gathered by AFM.

A precipitate-covered calcite grain was incised by a scratch to reveal unaltered interior calcite and the precipitate-crystal surface contact (Fig. 41). A distinct contact between the surface of the mother grain and the beginning of the precipitate is clearly revealed in cross-section. In areas of the calcite where the precipitation has been removed by scraping, the surface of the calcite grain appears smooth and unaltered. Spikes of precipitate in linear and box patterns grow perpendicularly to the surface and parallel to each other to a uniform height. The precipitate has a total height of approximately 2.0 gm. The calcite underlying the precipitation has a flat smooth surface identical with the condition of the prepared calcite grains prior to exposure to aquifer conditions in the Bemidji microcosms.

Calcite crystals that were uniformly covered with the column type spikes were etched to reveal the interior morphology of the spikes (Fig. 42). The crystal was lowered into 0.5% HCI in stages in order to create a gradual sequence of increasing etch effects on the surface. The bottom of the crystal was etched for the longest time, the two middle zones for intermediate times and the top zone not etched at all. In the zone that was etched the longest, the surface precipitation was completely dissolved. The two zones of intermediate etching revealed spikes that were partially etched, and the unetched zone remained unchanged. Following the etching, a deep scratch was incised on the surface across the etch zones with a sterile steel needle. The scratch revealed the unaltered interior calcite and created a continuous cross-section through out the various etch zones that exposed the precipitate-mother grain contact.

The tips of unetched spikes were rounded and in some cases revealed bead-like terminations (Fig. 43). The columns range from between 0.1 to 0.5 pm in base diameter, and narrow to 0.1 to 0.2 pm diameter at the tip. The tip is rounded, and in many cases is capped with a 0.05 to o.lpm bead. The bead composition is the same as the column as analyzed by ED AX. The column of the spike has a micro-curdled texture. Some of the columns have a uniform 0.1 pm diameter and others taper to a rounded point from 0.3 to 0.5 pm diameter bases. Light etching removed the tips of the spikes and partially dissolved columns of precipitate. The geometric pattern of the column arrangement on the calcite grain surface is much more evident when the coalesced tips have been dissolved (Fig. 44A). The spikes are composed of rounded 0.1 to 0.2 pm particles with a smooth matrix filling the space between the particles (Fig. 44 B). The morphology of the particles ranges from irregular euhedral shapes to rounded spherical beads. Each spike is composed of agglutinated sub-pm particles and what appears to be matrix cement. At high magnification, irregular and spherical-shaped particles slightly smaller than, or the same diameter as the column appear stacked to make the column.

Medium etching dissolved more of the spike precipitate away, revealing the lower portions and the base of the spikes. The bases of the spikes were geometrically arranged and connected (Fig. 45). Nannobodies were less evident than in the lightly etched material. Grains that were etched all the way to the original crystal surface revealed flat unaltered calcite identical with the premicrocosm condition.

In cross-sectional areas which showed the base crystal-precipitate contact, linear crystallographic features, such as cleavage planes, were observed to intersect exactly with linear voids in the surface precipitation (Fig. 46). Cleavage planes in the underlying crystal propagated as voids into the surface precipitate. The general occurrence of linear rows of spikes, euhedral boxes of spikes and fused spikes in linear patterns suggests a crystallographic control on the nucleation sites of individual spikes.

Experiments were carried out to determine the effect of removing organic matter from the surface of calcite recovered from the Bemidji microcosm experiments. A single crystal from the well 018 microcosm which was coated with the column-type surface precipitate was split in two parts. One half was treated with 5 ml of chlorine bleach for 30 minutes, rinsed briefly with distilled water and air dried. The other half was heated to 500°C for 2 hours, and cooled to room temperature. Each sample was mounted on an aluminum stub, gold-coated and studied with the SEM.

The surface of the bleached crystal revealed a übiquitous microcurdled texture with abundant spherical and hemispherical nannobodies (Fig. 47A). The bumps that constitute the microcurdle texture were more pronounced as discreet points following the bleaching, suggesting that material between the bumps had been removed or that the bumps had accreted slightly.

The heated crystal showed a smooth undulating surface. Cracks in the smooth surface revealed precipitation spikes beneath the surface. The heat sealed or smoothed over the tips of the calcite precipitate but left them revealed beneath the cracked surface. Calcite is resistant to heat alteration up to 700°C. The surface coating layer on the grain congealed or burned onto the top of the calcite precipitate spikes. Within cracks, individual nannobodies were abundant. Heating accentuated the linear bead-like appearance of the calcite spikes (Fig. 478).

Samples of precipitate-coated calcite collected from the microcosm in well 018 were etched in nitric acid in order to dissolve the surface precipitation for analysis of the stoichiometry of the precipitate (Table 8). The dissolved precipitate was pure calcite with no trace of magnesium or strontium. No iron was detected in the calcite.



Figure 35. Dolomite recovered after 14 months from Zone 3 in the Bemidji aquifer. A) Overview of dolomite crystals showing uniform dissolution features along crystal edges and in discrete central areas of each crystal. B) Detail of A showing deep dissolution along the upper edge and in the center of the crystal. C) Detail of B. Depth of etching extends from the surface to 5- 10 pm depth. Few bacteria were observed in the immediate vicinity of dolomite dissolution.



Figure 36. Morphologies of precipitate covering the surface of calcite crystals recovered from Bemidji microcosm experiments.



Figure 37. Some areas on the mother crystal show transitions or borders between spike morphologies. A. Rounded spikes on the left of this image grade into sharper, bladed spikes of the same size. A sharp boundary separates the smaller spikes from multimicron-diameter euhedral spikes. Note that while the large spikes originate in a hole, well below the level of the flat surface of the mother crystal, the maximum height of the small and large spikes is exactly the same. B. Detail of the termination of the spike shown on the right-center of A. C. Detail of the rounded spikes on the left of A.



Figure 38. Three dimensional cross-section of a single layer of surface precipitate. A calcite crystal from the Bemidji microcosms was fractured to produce edges and cross-sections of the surface precipitate. Note the column spike-type terminations as the surface expression of the 2.0 pm long crystal. The base of the crystal is sub-micron in diameter and gradually expands to o.spm diameter at the top. The single crystal bifurcates at the surface into eight to ten rounded terminations. The crystals appear to bundle together to form a new surface with a bumpy texture. Note the distinct wispy layers that occur at several levels perpendicular to the long axis of the crystals. This feature may represent a layer of organic matter through which the crystals grew. This layer may represent the flat "floor" that appears beneath and next to areas of bumpy surface texture.



Figure 39. High resolution SEM imaging of calcite spikes and flat area between spikes, a) Overview of area of spikes that are intermediate in morphology between rounded and pyramidal forms. Each spike stands on a flat base, or is protruding though a thin wispy layer of amorphous material. At a uniform height, the spikes develop a flat-top termination. In areas where the spikes are close together, they coalesce into larger spikes and into linear features. Magnification = 30,000 X, Scale bar = Ip.m. B) Detail of A. The spikes have a curdled surface texture. The terminations of each spike is flat. Note the flat surface from which the spikes emanate. Magnification = 80,000 X, scale bar = o.spm. C) Detail of the surface between the spikes. At high magnification the "flat" surface between spikes has a microcurdled texture. Individual semi-spherically- shaped nannobodies rest on the surface or are imbedded in the surface material. Magnification = 100,000 X, scale bar = o.spm.



Figure 40. Atomic force microscopy image of the surface of a precipitate-covered calcite crystal collected from well 018 at the Bemidji research site.



Figure 41. A) Overview of a precipitate-covered calcite grain which has been incised by a scratch to reveal the unaltered interior calcite. The light colored flat-looking surface is the precipitate covered surface. The laterally-oriented dark band is the scratch made with a sterile steel needle. The extreme right side of the crystal has the surface chipped away and also appears dark compared to the spiky surface. The scratch provided a cross-section through the surface into the unaltered interior of the mother crystal. B) Detail of A showing the contact between surface precipitate and underlying flat crystal surface. The precipitate has a total height of approximately 2.0 |im. The calcite underlying the precipitation has a flat smooth surface and appears to be identical with the condition of the prepared calcite grains prior to exposure to aquifer conditions in the Bemidji microcosms. C) Detail of the lower surface of B, showing a cross-section of the precipitate height.



Figure 42. Diagrammatic map of the surface of a calcite grain recovered from the Bemidji microcosms which has been etched in HCI and incised with a scratch. The crystal was lowered into 0.5% HCI in stages in order to create a gradual sequence of increasing etch effects on the surface. The bottom of the crystal was etched for the longest time, the two middle zones for intermediate times and the top zone not etched at all. In the zone that was etched the longest, the surface precipitation was completely dissolved. The two zones of intermediate etching revealed spikes that were partially etched, and the unetched zone remained unchanged. Following the etching, a deep scratch was incised on the surface across the etch zones with a sterile steel needle. The scratch revealed the unaltered interior calcite and created a continuous cross section through out the various etch zones that exposed the precipitate-mother grain contact.



Figure 43. Unetched column-type precipitate. The location of the image is indicated on a reduced-size map of the entire crystal. The columns range from between 0.1 to 0.5 pm in base diameter, and narrow to 0.1 to 0.2 pm diameter at the tip. The tip is rounded, and in many cases is capped with a 0.05 to o.lpm bead. The bead composition is the same as the column as analyzed by EDAX. The bead may be a nannobacteria or alternatively an inorganic form of calcite.



Figure 44. A) Lightly etched column-type spikes from the ten second-etched zone (see map). The image illustrates the columntype spike halfway dissolved, revealing a horizontal cross-section through the center of the spike. Gross patterns of the spikes’ base arrangement on the crystal surface are still evident. Note the overall linear arrangement of the etched spikes and euhedral corners of clumps of spikes. B) The interior morphology of the column-type spikes is revealed by the partial etching. Each spike is composed of agglutinated sub-micron particles and what appears to be matrix cement. At high magnification, irregular and spherical-shaped particles slightly smaller than, or the same diameter of, the column appear stacked to make the column.



Figure 45. Medium-etched column-type spike from the 30 second etched zone (see map). These spikes are more dissolved than those in the ten-second zone, but have not completely dissolved away. The connected bases of each column-type crystal protrude approximately 0.5 pm above the surface of the mother grain. The overall linear pattern of spike base on mother crystal is still evident. The occurrence of discrete particles within the column base is less evident than in the ten-second section, however, faint outlines of 0.2 to 0.5 pm spheres are evident in the central and lower left portions of this image.



Figure 46. A) Cross-section of the unaltered mother grain calcite and surface precipitation spikes that have been etched almost to their bases (see map for image location). Cleavage planes in the mother crystal propagate as voids into the surface precipitate. The general occurrence of linear rows of spikes, euhedral boxes of spikes and fused spikes in long narrow “fences” suggests a crystallographic control on the nucleation sites of individual spikes. B) Detail of A. Note the clear extension of the cleavage plane as a void in the surface precipitate. Also note the abundance of 0.5 to 1.0 pm spherical and rod-shaped bodies exposed within the calcite of the spike base.



Figure 47. Surface features of precipitate-coated calcite recovered from the microcosm experiment in well 018 at the Bemidji study site which has been bleached or heated to remove organic matter from the surface. A) Microcurdle texture and nannobodies were not removed a a result of bleach treatment. B). The heat sealed or smoothed over the tips of the calcite precipitate but left them revealed beneath the cracked surface. Cracks in the smooth surface revealed precipitation spikes beneath the surface. The surface coating layer on the grain congealed or burned onto the top of the calcite precipitate spikes. Within cracks, individual nannobodies were abundant. Heating accentuated the linear beadlike appearance of the calcite spikes

Sample Etch Time, Secs. Magnesium Calcium Strontium Nitric acid blank 0 1.0284 0 018 calcite 0.5 0 69.9511 0 Fresh calcite 0.5 0 55.1199 0 018 calcite 1.0 0 65.9419 0 Fresh calcite 1.0 0 62.4014 0 018 calcite 2.0 0 100.3682 0 Fresh calcite 2.0 0 83.9247 0

Table 8. Results of analysis of calcite precipitate from microcosm experiment in well 018 at the Bemidji study site. Values for magnesium, calcium, and strontium are reported in pg/ml.

Field Column Experiments

In field experiments at the Bemidji study site, ground water was pumped for three days directly from the level at which the microcosms were suspended through columns of quartz sediment prepared as previously described. The columns were originally designed to collect a data set of geochemical measurements comparable to the chemistry of water collected from the wells. However, mechnical problems with the columns and pumps due to cold weather largely scuttled the intent. The columns were useful as filters for capturing bacteria, suspended solids, and precipitates from the anaerobic ground water as it came in contact with the cleaned and well characterized mineral sediment filling the column. A red precipitate formed at the inlet of the columns which was identical in composition and morphology to that which formed in the anaerobic ground water from zone 3 when it was exposed to oxic surface conditions. Quartz sediment recovered from these columns and examined by SEM showed that the extent of surface coating by the organo-iron precipitate was intermediate between the clean pre-test prepared surface and the completely coated surfaces of the minerals recovered from the wells after 14 months, thus providing a rough guide as to how fast the surface coatings developed on the in-situ microcosm material.

Discussion

The discussion is organized in two major subject areas of the dissertation results: 1) progress in improving methods for studying bacteria in geologic materials and in interpretation of bacteria-mineral interaction data, and 2) evidence of bacterially mediated diagenesis in aquifer conditions.

The environmental conditions in laboratory experiments were optimized for vigorous bacterial growth and the field conditions at the Bemidji aquifer also represent good, if not optimal conditions for bacterial growth. Both environments were good candidates for the observation of bacteria - mineral interaction. Not all geologic environments are as "supercharged" with labile organic matter, or have as abundant a supply of dissolved oxygen and alternative electron acceptors. The good conditions for growth in lab and field experiments allowed observation over the course of months of bacteria-mineral interactions that, under less favorable conditions, might occur over years, decades, or centuries

Section 1.

Bacterial Culturing, Isolation, and Identification

The technique of using SEM to identify specific types of bacteria on mineral surfaces after incubation in batch reactor experiments or collection from the field did not work. It was impossible to identify various strains of bacteria on the basis of morphology once they had been released into a rock-water environment and started growing. The size, shape, and surface features of the various pure strains changed enough while growing under aquifer conditions and in batch reactor experiments that they could not be unambiguously identified or even matched to their original "mug shots". This was a disappointing, but not unexpected, finding. In fact it confirms the dogma that microscopy alone is insufficient for bacterial identification (e.g.. Brock and Madigan, 1988; Chapelle, 1993). Bacteria are protean, and known to change morphology with changing environmental conditions such as concentration of organic matter, salinity, and temperature. While it has been proposed that different types of bacteria colonize different types of minerals (Mills, Herman, and Hornberger, 1993) this question cannot be resolved by microscopy alone. Future work combining biochemical methods to determine the presence of strain-specific genetic material with direct microscopy may prove to be successful in addressing this question.

Bacterial Morphology and Taphonomy

There is no doubt that the coccoid, rod-shaped, and spiral-shaped particles with diameters of between 0.5 and I.opm observed in laboratory and field experiments on the surface of minerals are bacteria. The repeated experiments in which bacteria of known size and shape were allowed to colonize mineral surfaces and then characterized by SEM confirm what bacteria on rocks look like. Alongside these "normal" bacteria, virtually übiquitous nannobodies were observed on mineral and organic surfaces. The nannobodies were observed as spheres and hemispheres, and in one instance as rods, and ranged in size from 0.05 to 0.5 pm in diameter. The most common nannobody size and shape observed in this work was o.lpm diameter hemispheres. Although nannobodies can be seen in the background of many high magnification, high resolution SEM micrographs of bacteria in organic-rich environments, they have only recently been recognized and described as cells of suspected organic origin (Folk, 1992; 1993 a; 1993 b; 1993 c; 1993 d). Little is known about nannobodies, including their origin. What is the origin of nannobodies? Are all nannobodies bacteria? If they are bacteria, are they actively metabolizing cells, or rather are they stressed inactive cells? Are nannobodies inorganic material or congealed organic matter? Are they artifacts of sample preparation, for example, does simple drying cause nannobodies to form? While it is beyond the scope of this dissertation to address these questions in general, experiments were carried out to investigate the origin of the nannobodies observed in the mixed culture used in lab experiments and on the surface of mineral grains recovered from the Bemidji aquifer experiments.

The evidence that these particles are bacteria is entirely morphological. Clusters of nannobodies appear similar to common colony morphologies of coccoid bacteria. Areas of high concentration of nannobodies observed in this study are similar to closely packed colonies of normal bacteria that form on surfaces (e.g., Fig. 16, 17). Yet nannobodies also are commonly observed spread evenly and equi-distantly across flat mineral surfaces (Fig. 208, 238, 24). The particles are identical in shape with larger coccoid bacteria and are the same size and shape as starved cells described by Novitsky and Morita (1977) and Morita (1980), but smaller than starved cells (0.25 by o.spm) described by Lapin-Scott et al., (1988). Nannobodies fall into the same <o.4pm diameter size range as the voluminous soil bacteria described by Bakken and Olsen (1987).

Some nannobodies may be congealed non-living organic matter or organic matter that dried into round forms during sample preparation. Nannobodies were only observed on mineral surfaces that were exposed to organic-rich fluids. No nannobodies were found on minerals prepared by crushing and surface cleaning with distilled water (Fig. 7,8, 9). Clean Iceland spar calcite deeply etched with HCI revealed only smooth surfaces (Fig. 34). Calcite recovered from the Bemidji microcosms that was scratched to reveal the underlying grain surface not exposed to the aquifer water also did not show any evidence of nannobodies (Fig. 41).

Other workers have noted the occurrence of nannobody-like particles associated with organic-rich fluids and sediments. Westall and Rince (1994) documented the development of individual and hemispherical masses of exopolymeric substances generally <loonm in diameter at the edges of biofilm layers. The hemispheres occur as curved chains or connected beads along the rounded edges of discrete sections of biofilm. They noted that when such edges were viewed end-on, they looked like segmented filaments similar in appearance to filaments of bacteria bodies. Transmission electron microscope observations of biofilm cross-sections, however, confirmed that the origin of these features were organic matter-coated clays and thickened organic coatings, not bacterial bodies. The cross-section images from TEM allow the identification of cell wall and internal cell features as distinct from amorphous organic coatings and organic matter-clay clots.

Accumulations of masses of exopolymeric material also created hemispherical bumps, lumpy aggregates, and isolated spherical balls on the surface of the biofilm. The balls and hemispheres were observed to range from 0.1 to 0.25 pm in diameter, similar in morphology and size range to nannobacteria as described by Folk (1993) and nannobodies described in this dissertation. It should be noted, however, that while Westall and Rince did experimentally determine the origin of a granulated surface as the sequential development of an organic coating on previously smooth diatom frustules, they only observed the occurrence of the spherical exopolymeric aggregates on and in the biofilm and did not prove their origin.

The material of bacteria cell walls can be destroyed by heat and oxidizing compounds such as chlorine bleach. If nannobodies are metabolizing, living bacteria, it is reasonable to expect that they would behave in a manner similar to normal-sized bacteria when attacked by high heat or bleach. Mineral grains coated with both live bacteria and nannobodies from the mixed culture used in dissertation experiments were heated to 500°C for 2 to 6 hours or soaked in bleach for 30 minutes and observed with SEM. Most normal bacteria were destroyed, yet abundant nannobodies were observed, seemingly undisturbed. Nannobodies on the surface of calcite grains collected from the Bemidji microcosm experiments which were heated or bleached also survived intact (Fig. 47). It may be that nannobacteria are resistant to high heat and oxidation by bleach, but normal bacteria were shown to have been destroyed under such conditions.

Some nannobodies could also be inorganic. Calcite precipitation under abiotic experimental conditions has been shown to begin by the formation of rounded, 100 -200 nm diameter nuclei, which gradually and uniformly grow larger until they coalesce into a sheet structure (Dove and Hochella, 1993). Dove and Hochella precipitated calcite in a closed cell containing a solution of 0.2 M calcium chloride and 2 M ammonium chloride. Carbonate ion was supplied from solid ammonium carbonate suspended above the growth solution. Nuclei grown in phosphate-free solutions were euhedral in shape. Nuclei grown in the presence of phosphate, however, were distinctly rounded. The authors postulate that phosphate "poisons" or "roughens" the nuclei edges and produced the rounded effect. In experiments under marine and lacustrine conditions, Berner et. al, (1978) and Reynolds (1978) reported that low concentrations of aromatic carboxylic acids, tannic acids and polyphenolic acids had the same effect on inhibiting calcite precipitation as did orthophosphate, thus suggests that low concentrations of dissolved organic matter may have a similar effect on the rounding of calcite nuclei morphology. All of the nannobodies observed on calcite surfaces in this dissertation work were associated with fluids rich in aromatic hydrocarbons and their derivative organic acids.

Nannobodies are most likely not one type of object, living or non-living. Bacteria are known to occur in the 0.1 to 0.25 diameter size range. Organic matter makes balls under certain conditions. Calcite forms rounded nuclei in the presence of phosphate. The resolution of the question of the origin of nannobodies will require taxonomic patience, and interdisciplinary biogeochemical research methods.

Bacterial Colonization and Growth

The large diversity and number of attached bacteria that were observed on samples collected from the zone 3 in-situ microcosms indicates that the aquifer was densely populated by a variety of bacteria. Bacteria readily colonized mineral surfaces under organic-rich aquifer conditions in the laboratory and in the field. The hydrocarbon-utilizing bacteria colonized mineral and rock surfaces as individual cells and as microcolonies, but true "biofilms" were observed only in the 2 hour and 20 hour samples collected from laboratory growth experiments. Biofilms are defined as a slime or mucus of organic macromolecules such as proteins or polysaccharides mixed with a layer of microbial cells which are absorbed or attached to particle surfaces (Westall and Rince, 1994). Much of the literature that describes growth of bacteria in experimental porous media reports that adhering bacteria eventually form a continuous biofilm over the surface they colonize (e.g.., Marshall, 1976, 1980; Characklis and Cooksey 1983; Costerton et al., 1985) and several numerical models of subsurface bacterial degradation of organic pollutants assume the development of a biofilm as the basis for estimating contaminant degradation kinetics (Moltz et al., 1986; MacQuarrie et al., 1990 a, 1990 b). Given the high concentration of bacteria known to inhabit the zone 3 area of the field site, it is surprising that biofilms did not form or formed sparingly over the fourteen month in-situ experiment.

The lack of evidence of biofilm development at Bemidji may represent an artifact of sample preparation. Although some areas of dense bacterial colonization were observed it is estimated that some of the attached biomass may have dislodged during the critical point drying, even though efforts were made to minimize sample disruption. Once cell materials have been cross-linked by gluteraldehyde fixation, cell adhesion to surfaces may be reduced and many cells may be dislodged. All samples of microcosm material were fixed with 2% gluteraldehyde in the field or immediately upon return to the laboratory, so no comparison study of non-fixed cell adhesion was possible. In any case, where we see individual cells and small colonies established on mineral grain surfaces, larger colonies of bacteria may have occurred in the undisturbed sediments.

Another possibility to explain the lack of a well developed biofilm on mineral surfaces is that hydrocarbon bacteria may not produce excessive extracellular slime or glycocalyx. Most experimental work on biofilm development is carried out with carbohydrate-metabolizing bacteria (e.g.. Lapin- Scott et al., 1988; Hoyle et al., 1993). Simple carbohydrates (sugars) are constructed into polysaccharides within the cell and some of the polysaccharide is used in the formation of an external glycocalyx or slime layer which forms the bulk of a biofilm. Hydrocarbon-utilizing bacteria may not produce the copious extracellular polysaccharide slime that is common among sugar-eating bacteria. Intermediate metabolic byproducts of hydrocarbon degradation include alcohols, aldehydes and organic acids (see Introduction section). These byproducts are polar and are readily soluble in water, thus may not be as likely to stick to the external surface of cells.

Dissolved organic carbon, including aromatic hydrocarbons and organic acids, does however sorb to mineral surfaces (Freeze and Cherry, 1979). The pore water in the zone 3 area of the Bemidji aquifer has high concentrations of total dissolved organic carbon (TDOC), in the range of 2.1 to 4.1 mM (Baedecker et al. 1993). At least some of this TDOC will partition to the surface of the minerals and form an organic layer. The übiquitous microcurdle texture observed by SEM on the surface of all grains collected from the Bemidji microcosms and from batch reactor experiments in which crude oil was used as a carbon source is interpreted as a sorbed organic layer.

In experiments in which a biofilm was grown on the surface of diatom frustules and carefully preserved by fixation, dehydration and critical point drying, Westall and Rince (1994) documented the sequential development and morphology of an organic coating of the diatom surface. In an early stage of biofilm development, they noted that formerly smooth diatom surfaces were uniformly coated with an organic film ranging from 10 to 200 nm in thickness. The surface of the organic layer was described as a retracted granular texture. The granules ranged from 10-100 nm in diameter and formed a contiguous layer of bumps across the diatom surface. This texture is identical to the microcurdled texture described in this dissertation (e.g. Fig. 228, 238, 26C, and 358) which occurred as a übiquitous feature of quartz, microcline, and calcite grain surfaces that were exposed to organic-rich water, both in the lab and in the subsurface at Bemidji.

Westall and Rince (1994) determined that the granular texture was not an artifact of sputter coating with metal, but did suggest that the bumpy or granular texture was the result of dehydration of the organic film during sample preservation. The results of my experiments in which the surface coating on calcite grains dried while in the SEM column and showed a transformation of surface morphology from smooth to microcurdled and cracked (Fig. 12, 13, 14) confirms the dehydration hypothesis of the origin of the retracted granular surface texture.

The documentation of a thin organic coating on crystal surfaces is significant in the interpretation of mineral precipitation, especially calcite, and will be discussed further in the carbonate precipitation section.

Section 2.

Effect on the Bemidji Aquifer of Microbial Activity

The dissolved hydrocarbons present at the Bemidji research site are known to be degradable by aerobic heterotrophs and anaerobic or facultative microorganisms. The concentration of total dissolved organic carbon (C = sum of volatile and non-volatile fractions) in the ground water varies substantially between geochemical zones. In Zones 1 and 2, C ranges between 0.10 and 2.59 mg/1, high enough to support a reproducing population of bacteria. Zones 1 and 2 are up gradient of the oil source and the C in the water probably represents natural humic compounds derived from decaying plants. Supply of this type of carbon to the aquifer is controlled by seasonal recharge to ground water by infiltration of surface waters. Zone 3, the area immediately below the floating crude oil which acts as a long term source for dissolved phase hydrocarbons has four times as much C as the background. Total concentration of hydrocarbon in the ground water is limited only by the rate of diffusion of water soluble components from the oil phase to the aqueous phase. The distribution of dissolved hydrocarbon in the plume down-gradient of the oil source is apparently uniform in the immediate microcosm study area (zone 3), but on a microbe scale, may fluctuate substantially as a result of micro-flow paths, permeability and porosity variations. No data exists on C distribution on a microbial scale from the study site.

Three categories of biogeochemical reactions are occurring at the Bemidji site and correspond to the distinct geochemical zones that have developed since the pulse of crude oil. Aerobic hydrocarbon metabolism occurs in the unsaturated zone and in the oxygenated ground water at the up-gradient edge of the oil-water contact, where molecular oxygen serves as the terminal electron acceptor, i.e.:

C 6 H 6 + 7.5O 2 -> 6C02 + 3H 2 O

(1).

In anaerobic waters below and down-gradient of the oil body, oxidation continues via the reduction of Fe 3+ to Fe 2+ , i.e.:

C 6 H 6 + 30Fe(OH)3 -> 6HCO3- + 30Fe 2+ +54 OH’ +lB H 2 O

(2)

(e.g. Lovley et al., 1989; Chapelle, 1992; Bennett et al., 1993). In addition, methane is produced in the most anoxic regions, probably by acetate methanogenesis:

CH3COOH -> CH4 +CO2

(3) (Baedecker et al., 1993).

Both of the oxidation processes produce polar organic compounds as metabolic intermediates, such as organic acids (Cozzarelli et al., 1990), which react with the mineral solid phase, producing a plume of organic and inorganic solutes that has migrated down-gradient with local ground-water flow. Polar by-products of hydrocarbon metabolism are also readily used as sources of carbon and energy by a wide variety of bacteria

Nitrogen is most likely a limiting factor to microbial growth in the study area. The bulk oil contains 0.27% nitrogen but may not be available for bacterial use. Nitrogen in the bulk oil is unavailable for metabolism by bacteria that live in the aqueous phase since they require a water-oil contact during hydrocarbon metabolism. However, a robust population of hydrocarbon degrading bacteria are present and active. Recycling of the nitrogen in the microbial biomass is a mechanism for maintaining a supply of nitrogen for microbial growth in this aquifer (Brock and Madigan, 1988), but no research has directly addressed

questions of nitrogen supply or cycling at this site. Elements required in trace amounts of microbial metabolism such as Ca, Mg, Na, K, Si, Fe, and Mn are present in amounts sufficient to support robust microbial growth in each of the five geochemical zones at Bemidji.

The optimum temperature for the growth of the eight genera of bacteria identified by Chang et al.( 1991) is in the range of 15-25°C (Brock and Madigan, 1992), substantially higher than the recorded temperature range of the aquifer, 8.0 to 13.0 °C. The low temperature may have the effect of reducing the metabolic rate of the bacteria below expected under optimal conditions and thus the growth rate or doubling time. Under optimal conditions doubling times for bacteria in the genera identified at Bemidji can be as fast as 20 minutes. The constant low temperature may also have the long term (years) effect of selecting for psychrophyllic bacteria that preferentially grow in the range of 8.0 °-13.0 ° C.

The circum-neutral pH conditions in the study site ground water are well within the range of normal microbial enzyme activity and represent close to optimal conditions for growth ( Baas-Becking, 1965). The availability of water, necessary for microbial metabolism (most microorganisms require a water activity above 0.9 for active metabolism (Atlas, 1984) and total surface area of sediment for bacteria to colonize are not growth limiting factors at the Bemidji.

An indicator of ionic strength, specific conductance, changes substantially from zone 1 and 2, where the average value is about 400 microsiemans, and zone 3 where the average value is around 700 microsiemans. This change occurs over a space of only 60-90 meters and may inhibit bacteria growing in areas of low

ionic strength from flourishing in zone 3. Data taken during this study did not address this question.

No xenobiotic anti-microbial materials are known to have been added to the study site aquifer. An analysis of the presence of the wide range of possible natural growth inhibitors at the study site is beyond the scope of this work, but the presence of a robust mixed community of indigenous microorganisms at the study site suggests that no wide spectrum inhibitor is present, at least in concentrations that greatly influence the microbiology of the aquifer.

The geochemical zones are largely determined by the amount of oxygen and alternative electron acceptor dissolved in the ground water. The mineralogy, background water composition, supply of soil organic matter and original microbial populations were most likely very homogeneous across the study site prior to the oil spill. Following the introduction of high amounts of labile dissolved organic carbon, aerobic bacteria grew rapidly until the dissolved oxygen in the ground water was used up. Diffusion of oxygen from the unsaturated zone into ground water was either prevented by a layer of oil or did not match the oxygen utilization rate of the microorganisms.

The concentration of dissolved organic matter and the resulting gradationally decreasing oxygen concentration in the ground water exhibit the strongest controls over which type of microbes can grow and what metabolic mechanisms are utilized during growth. As oxygen is used during aerobic metabolisms and is not replaced by diffusion (blocked by the overlying oil layer) microbes adapted to use alternative electron acceptors become the dominant micro-fauna. In discrete areas of the aquifer where molecular oxygen is no longer available, organisms capable of using Fe^ + and CO2 as terminal electron acceptors flourished. The biochemistry of these reactions and the extra-cellular build-up of biochemicals in the vicinity of bacteria directly affect the chemistry of the ground water and mineral surfaces.

Bemidji Aquifer Diagenesis

Silicate Dissolution

Silicate solubility and aqueous dissolution kinetics are controlled by many variables, including crystal lattice defects, solution pH, temperature, ionic strength, and the presence or absence of inorganic and organic ligands (Huang and Keller, 1970; Schott, Berner, and Sjoberg, 1981; Chau and Wollast, 1985; Holdren and Speyer, 1985; Knauss and Wolery, 1986; Mast and Drever, 1987). Geochemical reaction models have been constructed based upon principals of equilibrium geochemistry and are used to predict or model geochemical reactions along fluid flow paths in aquifers (e.g. Plummer et al., 1991). In some organicrich aqueous environments, however, both quartz and feldspar dissolve faster than predicted by estimates based on the bulk ground water geochemical environment (Bennett and Siegel, 1987; Bennett, 1991; Bennett et. al., 1991).

Feldspars dissolve in most aqueous systems by proton or ligand-promoted hydrolysis (Bennett and Siegel, 1987). In the pH range of most natural waters, feldspar dissolution rate is extremely slow, with the slowest rate at about pH 6.2. In the absence of complexing ligands, silica release rate from microcline at pH 7 and 25° C, for example, is approximately 10-15.5 moles SiO2 cm-^s-l (Bennett, Hill, and Glaser, 1991) Quartz dissolution rate at near-neutral pH is directly influenced by the concentration of mono- and divalent cations (Stumm and Furrer, 1987). In dilute natural waters the 25° C dissolution rate is predicted to be about 10-16.5 moles SiO2 cm-2 s-k

Organic substances, especially organic acids, influence the dissolution of both feldspars and quartz. Organic acids accelerate the dissolution of feldspar at mildly acidic pH via surface complexation of metals, accelerating the breakdown of framework metal-oxygen bonds. Organic acids also complex aluminum in solution, resulting in an increase in feldspar solubility (Bennett, Hill and Glaser, 1991; Bennett, 1991). In contrast, organic acids accelerate quartz dissolution at near-neutral pH, but only with a few multi-functional organic acids at concentrations well in excess of that found in ground water. At neutral pH, organic acid anions complex silica on the quartz surface (and possibly in solution) resulting in a surface ligand exchange reaction similar to the feldspar system.

Quartz and feldspar solubilities are controlled by conventional inorganic equilibrium in ground water at near-neutral pH except in the presence of organic acid anions in excess of 50 milliequivalents per liter. With high concentrations of chelating organic acids at neutral pH, however, silica will be mobilized preferentially over aluminum, and the rate of both quartz and feldspar dissolution will increase.

Whereas the zone 3 waters are undersaturated with respect to the introduced aluminosilicate phases, feldspar dissolution rate should be at a minimum at the prevailing ground water pH (e.g., Huang and Keller, 1970). Over a period of 14 months at 10° C, only a few molecular layers might be expected to dissolve in the ground water, in contrast to the deep weathering actually documented. Further, examination of native feldspar grains collected from uncontaminated regions of the aquifer has yielded little evidence of chemical weathering (Bennett and Siegel, 1987).

Ground water in the anaerobic zone 3 was calculated to be supersaturated with respect to quartz using the geochemical model NETPATH by Baedecker et al., (1993). The saturation index (Log ion activity product/equilibrium constant) for quartz in the immediate vicinity of the in-situ microcosms was 1.274, which indictes that quartz should not be dissolving. Yet a major finding in this dissertation research was that quartz was directly observed to have dissolved during the 14 month period in the aquifer. This finding is a significant anomaly and merits close attention. The bulk geochemical characteristics of the ground water which were measured and used for calculating the saturation index did not accurately predict the fate of quartz and under estimates the dissolution of microcline.

One approach to resolve the anomaly is to assume that the measurements of dissolved silica in the zone 3 area were too high and recalculate the saturation index. However, high values for dissolved silica have been consistently recorded in this zone for many years. Different researchers have sampled the same water, analyzed the samples in different labs and arrived at very similar values (e.g., Baedecker et al, 1993; and Bennett et al., 1993). The chemical data are consistent and the analytical methods rigorous. The dissolution of quartz in 14 months was observed directly by SEM. Distinct triangular etch pits were present on the surface of quartz grains that, prior to immersion in the aquifer for 14 months, had pristine flat surfaces. Microcline weathered faster than expected, showing distinct evidence of surface etching to several microns in depth. The data appears to be good and therefore, in this case, the theoretical paradigm used to interpret the data should be re-evaluated to see if it requires modification (Kuhn, 1970).

The saturation index of quartz was calculated using NETPATH, which is a geochemical reaction model that assumes rock-water interactions are controlled by equations of equilibrium geochemistry. While the model does take into consideration the oxidation state and isotopic composition of carbon in mass balance equations, it does not consider the effect of microorganisms that use energy to stay out of equilibrium with their aqueous surroundings. If we add in the missing element of the effects of bacteria adhered directly to the mineral surface, an alternative hypothesis to explain the rapid and localized dissolution of quartz and microcline presents itself.

A hypothesis to explain the rapid surface etching of microcline, and to a lessor but perhaps even more significant degree quartz, is that surface-adhering bacteria create a chemical reaction zone in the immediate vicinity of the cell/mineral interface (Fig. 48). Organic acids, produced within the cell and released extracellularly, are concentrated at the cell/solution/mineral interface. Organic acids are known to chelate Si and in some cases Al in aqueous conditions, but must occur at concentrations of over 1200 ppm carbon (as citrate) in the bulk porewater to have a chelating effect (Bennett, 1991). This concentration is far in excess of that measured in bulk samples of ground water from zone 3 and from natural waters in general (Cozzarelli et al., 1990). In the near vicinity of carbon-utilizing microbes, however, it is possible that bacteria cause a high concentration of organic acids to occur immediately near their cell wall.

During metabolism, hydrocarbon substrate is consumed by heterotrophic bacteria while enzymes and metabolic byproducts such as organic acids are exported from the cell interior to the nearby exterior fluid environment thereby producing a non-equilibrium chemical gradient between the cell surface and the surrounding bulk fluid. High concentrations of these byproducts may be present in the near vicinity of the cell body thus creating a localized chemical reaction zone. Within this microenvironment, defined by the organic acid concentration gradient, complex organic acids bind silica at the mineral surface, thus causing dissolution despite the supersaturated status of the bulk pore water. Si, and possibly Al, thus chelated and in solution, is available for transport away from the dissolution site along the ground water flow path.

Other workers, including Bennett and Siegal (1987), Chafetz et al. (1991), and Folk (1993) have suggested that in aqueous conditions bacterial microreaction zones effect mineral alteration without regard to the bulk geochemical conditions of the surrounding aqueous environment. However, data presented in this dissertation is the first direct evidence of bacterial dissolution of a silicate mineral surface which is well constrained by geochemical data in a subsurface aquifer environment.



Figure 48. Conceptual model of a bacterially generated zone at the surface of a quartz crystal fragment. Surface adhering bacteria create a reaction zone in their immediate vicinity by producing and releasing organic acids during metabolism. The organic acids released by the cell create a gradient between the cell surface and the surrounding pore water. In this microenvironment, complex organic acids chelate SiO2 from the mineral surface and cause dissolution despite the quartz-supersaturated bulk pore water.

Silicate Precipitation

The silicates studied in this research largely showed evidence of dissolution near the attachment points of colonizing bacteria. However, on some microcline grains, a honey-comb textured precipitate was identified. The precipitate did not occur directly adjacent to dissolution pits but did occur on the face of the same grain. The dissolved feldspar could represent the source of material for the formation of the precipitate. Constituents of silicate minerals such as Si, Al, Fe and K complexed by bacterially produced organic acids are polar compounds that react with water. The water soluble complex may be transported down-gradient from the dissolution site by local ground water flow. It is unknown how stable these complexes may be and how long the complex would last before breaking apart and freeing the metal ion for further chemical reaction or precipitation. The clays growing at the end of the dissolving feldspars may represent the reprecipitation of clay from metal ions solubilized by bacterially produced organic acids and transported down-gradient.

Petrographic observations of the dissolving feldspar indicate that dissolution is congruent: all the components of the feldspar are going into solution and none are remaining behind as "clay halos". Closely associated with the dissolution features are attached bacteria. The bacteria produce organic acids during metabolism of hydrocarbon, and as previously postulated, create a microreaction zone in the immediate vicinity of the bacteria-mineral contact. Bacterial metabolism of ample hydrocarbon in the aquifer represents a source of acid to drive a feldspar weathering reaction that consumes acid:

KAISi3OB + 4H+ + 4H20 -> K+ + Al 3 + + 3H4SiO4.

(4)

The honeycomb clay precipitate occurs separated by 10 to 100's of microns from the dissolution features. Is the silica and aluminum forming these precipitating clays coming from the dissolution of the same grain? Ground water measurements show that aluminum in solution increases slightly, but not in an amount that could account for the volume of feldspar observed to be dissolved, thus indicating that aluminum is behaving conservatively and is not likely to be transported a long distance from the dissolution site. One hypothesis that relates the observed petrography with geochemical measurements and the presence of bacteria on the feldspar grains is that Si, Al, and K have been complexed by bacterially produced organic acids, and in the complexed form are mobile for a short distance only (Fig. 49). What would cause the organic-metal complex to break and free the dissolved constiuents to re-precipitate? No evidence was discovered to support the fomation of a hypothesis, but it is known that bacteria commonly use organic acids as a carbon source. Bacterial use of the acids themselves for metabolism would represent a mechanism for breaking the complex, but this problem has not been studied yet.

These observations just scratch the surface of the research possibilities in investigating bacteria/clay and organic matter/clay interactions in organic rich aqueous environments. Future work needs to focus on identifying organic/metal- ion ligand molecules and determining the duration of the ligand attachment before the ion breaks away from the organic and is available for other chemical reactions.



Figure 49. Congruent dissolution of microcline, local transport of dissolved constituents, and local precipitation of clay minerals. Highly localized acid production is caused by metabolizing, adhering bacteria which complex Si and Al with organic acids. The complex is transported locally 10 to 1000's of microns where, outside the influence of the bacterial micro-reaction zone, the cations re-precipitate as clay.

Carbonate Dissolution

Geochemical modeling by Baedecker et al. (1993) and Bennett et al. (1993) indicated that ground water in zone 3 was very near equilibrium with respect to calcite (-0.020 to - 0.040) and undersaturated with respect to dolomite. Under such conditions, both precipitation and dissolution of calcite could be expected in close proximity to each other. Only small perturbations in carbonate equilibria cause calcite to dissolve or to precipitate. Dolomite was predicted to dissolve and was observed to dissolve. In the case of calcite and dolomite, the bulk geochemistry of the aquifer in zone 3 did predict the behavior of the mineral phase, and no alternative hypothesis to the control of aquifer rock-water interactions by inorganic chemical equilibria is required to explain the behavior of these minerals in the aquifer.

However, it should be noted that it is the activity of bacterial oxidation of hydrocarbon that created the geochemical conditions in the zone 3 of the Bemidji aquifer. Oil is mostly unreactive in water. Most fractions have low to no solubility. In an aseptic environment, a simple plume of dissolved aromatic hydrocarbons would be expected to develop in response to chemical diffusion and down-gradient transport of the solute with ground water. In the microbially active Bemidji aquifer, however, distinct hydrochemical zones developed, loaded with reactive organic matter and bacteria. The biochemical reactions of hydrocarbon metabolism modifies the geochemistry of the ground water and thus affects mineral alteration.

For carbonate minerals in the Bemidji aquifer, the major effect of bacteria on diagenesis is on a local aquifer scale and not a microbe scale. The infusion of reactive organic matter into the aquifer is the direct result of microbial metabolism of non-polar, non-reactive hydrocarbons into polar, highly reactive intermediate compounds such as alcohols, aldehydes, and especially organic acids. The aquifer is probably supporting log phase growth of bacteria that can use hydrocarbon as a food source with a variety of terminal electron acceptors. The geochemical zonation of the aquifer, now 14 years after the spill, largely reflects the net effect of chemical reactions of specialized microbial metabolism. Shifts in the hydrogeochemical conditions occur rapidly (within ten years) in response to changes in the dominant microbial processes and the resultant biogeochemical reactions.

The two types of dissolution pits observed on the surface of calcite grains recovered from the field microcosms may represent a sequence in a single dissolution mechanism, which has been documented in various stages of development. Once tiny pits have been established on the calcite surface, the pits may enlarge along the lines of the calcite crystallographic lattice. The random orientation of the tiny pits is suggestive of microbial colonization which could provide a focus for the start of a dissolution event. Once the dissolution is started, both microbially assisted dissolution and, in the appropriate geochemical environment, inorganic calcite dissolution could proceed thus providing dissolved constituents for calcite precipitation down gradient (Fig. 50).

Maclnnis and Brantley (1992) used concentrated formic acid (concentration not reported) to etch calcite and document the morphology of the resultant pits. They identified two major types of pits: " Gl" , large 0.5 to s.opm deep pointed pits, and "G 2 smaller pointed, flat, and terraced pits. G 2 pits occurred beside and within Gl pits. Etching longer than several hours caused sharp pyramid and truncated-top pyramid-shaped points to appear against a background of terraced flat ledges, which they interpreted as the stepped edges of larger pits. The pits observed in few instances of etched calcite are analogous to the pits described by Maclnnis and Brantley (1992).

Carbonate Precipitation

The spikes observed on the microcosm calcite surfaces are interpreted to be precipitation instead of dissolution features for several reasons. In comparison with the HCI etched spar samples, which revealed extensive areas of 90°angle terminated protrusions from the crystal surface, the spikes observed on the microcosm calcites are longer and narrower, with point termination angles in the range of 15 to 30°. The spikes are arranged in discrete narrow lines and in geometrically defined shapes rather than in the surface-wide pattern of the HCI etched surface. In most areas studied, the spikes merge together and tips coalesce, in comparison with the isolated points observed in the HCI etched samples. Folk, Chafetz and Tiezzi (1985) described spiky calcite as the result of both precipitation and dissolution mechanisms. They proposed that spikes were the effect of mineral dissolution if they meet at their bases with sharp angles and at various depths. The same interpretation was confirmed by Maclnnis and Brantley (1992). It appeared that as spikes either widened at their bases or new spikes grew in-between existing ones, they coalesce into continuous lines or fill the space between spikes until each individual spike was obscured.

The question of whether the spikes have sharp or rounded tips is an important one in investigating the mechanism of crystal growth. Sharp euhedral terminations would suggest an inorganic, or at least non-biologic, crystal growth mechanism. Round-tipped spikes indicate that some influence, other that simply supply of reactants has affected crystal growth. As previously discussed, zone 3 is rich in dissolved organic carbon as aromatic hydrocarbons and as polar daughter products of hydrocarbon oxidation. The übiquitous coating of organic matter on minerals placed in zone 3, as evidenced by the microcurdled surface, suggests that normal calcite crystal growth may be inhibited by the organic coating as described by Berner et al. (1978) for aragonite crystals. However, it is obvious that, although the spike morphology is rounded and not euhedral, massive amounts of calcite did precipitate, despite the high concentrations of dissolved organic matter in the surrounding fluid and coating the mineral surfaces.

High magnification examination of many samples revealed a diversity of tip-types. Most spikes are uniformly o.s|am in height, but occur with ball-shaped tips, sharp spear-like tips and flattened tops. Some sharp spikes occur with o.3pm rounded balls on their tips. Regardless of tip-type, however, the height of each spike was remarkably uniform. Spikes of calcite from travertine hot springs (Folk et al., 1985) that are morphologically very similar to these, have a much larger size variation, ranging from 1.0 to 10.0 pm in height.

A sequence of precipitation growth is proposed in which the various morphologies of calcite spikes have a common growth pattern, ultimately resulting in the formation of a new flat layer of calcite cement on the surface of the mother grain (Fig. 51). Nucleation of individual calcite spikes represents the first step. On the surface of calcite that revealed the flat underlying crystal surface, particles o.olpm diameter and less were observed occurring in scattered and linear patterns. From this nucleation point, a rounded spike grew upward and in some cases widened upwards from the mother grain base. At the height of 0.5 to 2.0 pm in height, upward growth ceased and lateral growth continued until the base of spikes grew together, fusing into linear ridges and box-like corrals. The linear features gradually fuse into sheets of uniform height and with a flat surface.

This sequence is similar to that described by Dove and Hochella (1993). They precipitated calcite under near equilibrium aqueous conditions in the viewing chamber of a scanning force microscope, which resolves 10 nm objects. Precipitation began with the formation of surface nuclei. Calcite nuclei are euhedral 10-80 nm diameter bumps on the smooth calcite background. Calcite nuclei in solution with 10 pmol phosphate appear as rounded bumps of the same size (Dove and Hochella, 1993, Fig. 2). These nuclei "roughen" the formerly smooth calcite surface. With continued precipitation, the nuclei coalesce to form a new smoother surface that grows by the spreading of monolayer steps. At these saturation states, a mechanism resembling spiral growth appears to become the dominant reaction process only after longer reaction times. The rows and rhombs apparently coalesce into sheets of smooth calcite, thus adding a new layer to the original calcite grain.

Whereas Dove and Hochella (1993) resolve the nucleation points with greater accuracy than is possible with the SEM, they are not able to document the latestages of precipitate growth. Considering the similarities in chemical conditions between the Dove and Hochella (1993) experiments and the zone 3 water in Bemidji, a comparison between the experiments seems valid. The results of the Bemidji microcosms experiment represent the first field data to validate the laboratory observations of the nucleation and growth of calcite precipitate on a calcite surface under natural conditions.

An alternative hypothesis of spike nucleation and growth is that nannobacteria directly cause the precipitation of calcite on or near their cell wall (Folk, in press). Bacteria can play both an indirect chemical role and a direct catalytic role in the local precipitation of calcium carbonate. Bacteria that bind Ca2+ ions to their cell walls act as nucleation sites for further precipitation of calcium carbonate. Bacteria that produce CO2 and bind Ca2+ to their cell walls may actively produce a crystal of calcium carbonate around themselves or between themselves and the mineral they sit on (Fig. 52). Evidence supporting this theory includes the nannobodies revealed as the calcite spikes were gradually etched and the bead-like tips on many spikes. These nannobodies are similar in size and shape to nannobacteria, are found in clusters similar to colony morphologies of coccoid bacteria, and analyze as calcite by EDAX. Bleach treatment and heating to 500°C did not remove or alter the nannobodies on the surface of the spike precipitate, suggesting that the bodies are not now live bacteria. If the nannobodies within the etched calcite are bacteria, they are now presumably calcitized.

It is also possible that rounded bumps are the normal nucleation shape for abiotic calcite crystal growth when the surrounding fluids contain low concentrations of "poisoning" reactants such as phosphate or aromatic organic molecules. In a study of calcite formation in spelean environments, Gonzalez, Carpenter and Lohmann (1992) propose that inorganic calcite crystal form is determined by the number of nucleation points and degree of supersaturation of the surrounding fluids with respect to calcite. For saturation levels of up to six times, they propose that one or two simple forms of crystal form would be present consisting of shallow to moderately steep rhombohedrons. Although the authors present SEM photos of each of the described crystal forms, none of the photos are presented at a scale or magnification appropriate for investigating the precipitation mechanisms of crystal formation, or for checking to see if any microorganisms were involved at the mineral/fluid surface.

In a cave environment, rate of fluid flow affects the supply of reactants, and supply of nucleation points. With increased flow, more nucleation particles are available, and more reactants per unit of time are supplied to the growing crystal face. In non-flow environments, reactants are available at the growing crystal face via diffusion or convection.

The pores within the Bemidji sediments could be considered micro-caves for the sake of comparison of the calcite precipitated on the microcosms seed crystals to the results of Gonzalez, Carpenter and Lohmann (1992). The pore fluids of the Bemidji water are only slightly supersaturated with respect to calcite (Bennett et al., 1993). Fluid flow rates in the area of the microcosms would supply an ample supply of reactants to the crystal surface. Examination of the highest magnification SEM in the paper revealed fine overlapping sheets, which appear similar in morphology and scale to the sheet structures of coalesced crystal points on the surface of the Bemidji calcites (Fig. 53). It is difficult to imagine a cave environment devoid of organic material and bacterial activity, therefore it would be interesting to examine the finer surface features of the cave calcite crystals at magnifications that could resolve the surface features.

Berner et al., 1978 reported the inhibition of aragonite precipitation in highly supersaturated marine waters by the presence of molecular layers of aromatic carboxylic acids produced by bacterial metabolism on the surface of carbonate particles. Similar organic acids are present in the pores of the Bemidji site and these compounds may be "poisoning" the crystal growth at the 0.5 p, height. The aromatic organic acids which Berner et al., reported in the marine sediments are "humic" type acids and are considered substantially recalcitrant to rapid microbial decay. The organic acids at Bemidji are more "bioavailable" and are assumed to be rapidly further metabolized to CO2 and H2O. At Bemidji, the organic acids are found at a much lower concentration down-gradient of the microcosm location, indicating their breakdown, or at least removal from the ground water.



Figure 50. Bacterial etching of calcite and dissolved reactants available for re-precipitation.



Figure 51. Unetched surface precipitate on calcite crystals which snow a sequence of precipitate growth. The sequence is proposed as follows: (1) nucleation, shown here as particles from o.olpm to o.lpm in diameter (2) individual spike growth upwards from the base, (3) termination of upwards growth at the uniform height of 0.5 pm, (4) lateral growth or thickening, (5) fusion of individual spikes into linear ridges and rhombohedral shapes, fusion of ridges and shapes into a new uniform flat surface covering the flat surface of the mother crystal. Note the wide diversity of spike size and shape in the 0. 1 to o.spm range, and the remarkable uniformity spike height and shape at the height of o.spm. Figures A and B illustrate spikes coalescing into linear ridges and sharp crystal terminations. Figure C shows the sequence of ridge and box formation and how the spikes fill in spaces on the mother crystal surface between the linear and box features.



Figure 52. Hypothetical mechanism of the catalytic effect of nannobacteria on the precipitation of column-like calcite spikes. Negative charge on the cell wall of each bacterial body attracts Ca 2+ , concentrating the cations in the near vicinity of the cell. Once nucleation of a calcite crystal has occurred, the crystal will grow abiotically or with the aid of another nannobacteria on the end of the calcite stalk. (After Folk, in press).



Figure 53. Ca 2+ and HCO3" released from calcite which dissolved in the aerobic zone 2, upgradient from the anaerobic zone 3, are supplied by fluid flow past the growing precipitate crystals. The uniform ultimate altitude of o.s|im. suggests a physical control on the supply of reactants.

Summary

Fourteen years after the spill of crude oil, the anaerobic zone of the contaminant plume is populated with diverse and active bacteria. The microbial metabolism of hydrocarbons has mobilized sparingly soluble metals and metalloids from aquifer minerals to the point of accelerating natural mineral diagenetic processes. On a localized aquifer scale, a highly reactive ground water is produced as a result of microbial metabolism which reacts with the bulk aquifer material according to inorganic equilibrium chemical reactions between mineral and water phases. On a microenvironment scale however, individual bacteria attached to mineral surfaces are metabolizing hydrocarbons and producing polar organic compounds as metabolic byproducts. These byproducts build up on the mineral surface immediately adjacent to the microbe, producing water enriched in dissolved inorganic species. This fluid is then subject to rapid changes in composition during diffusion out of the surface micro-reaction zone and flow through aquifer-scale geochemical zones.

Feldspars, quartz, and, in some cases, calcite dissolve in the immediate vicinity of attached bacteria, even though the saturation indices for these minerals in the bulk ground water indicate that little to no dissolution should occur.

Dolomite, with no evidence of direct bacterial involvement, is dissolving as expected from results of geochemical modeling and observations of increased dissolved Mg concentrations in the anaerobic zone. Calcite, in contrast, precipitates in the anaerobic Zone 3 waters. Metabolic production of CO2 and HCO3 resulting from acetate methanogenesis and the oxidation of aromatic hydrocarbons coupled with iron reduction provides a supply of reactants to the aquifer-scale bulk ground water for calcite precipitation.

Bacteria colonizing quartz and microcline are observed in close proximity to etch pits, exhibiting a spatial correlation between bacteria location and etch pits. The etch pits, however, are not localized at the actual contact surface of the microbe, and the triangular shape and extent of etching suggest the mineral surface is in contact with an aqueous weathering fluid. Localized mineral etching is proposed to have occurred in a reaction zone at the bacteria/mineral interface where high concentrations of organic acids, formed by bacteria during metabolism of hydrocarbon, selectively mobilized silica from the mineral surface.

Conclusions

Bacteria readily colonized mineral surfaces under organic-rich aquifer conditions. Surface adhering bacteria that metabolize hydrocarbons and produce organic acids have been shown to affect mineral diagenesis on an aquifer-wide scale and within microreaction zones only microns in diameter.

Investigation of bacterial colonization patterns revealed that individual strains of bacteria could not be unambiguously identified by morphology in the SEM, but relative densities of bacteria colonizing various minerals were easily quantified.

Fixing and air drying geological samples is an acceptable preparation technique for reconnaissance viewing. Geologists who study diagenetic reactions should routinely prepare and check their samples for evidence of bacterial colonization and alteration of minerals. For detailed study of bacteria-mineral surface interactions, sample preparation by critical point drying is essential. Nannobodies are übiquitous in samples associated with high organic matter content and bacterial activity. Rounded particles of 0.1 pm diameter may be metabolizing bacteria, starved or shrunken bacteria, clots of congealed amorphous organic matter, or even inorganic calcite crystal nuclei. Biochemical methods which can determine if nannobodies contain unique genetic material or culturing experiments in which o.lpm diameter bacteria are shown to reproduce themselves are required to demonstrate that specific nannobodies are bacteria.

The surface of quartz and microcline dissolved in highly localized areas that were colonized by bacteria. Microenvironments created by attached bacteria caused silicates to dissolve, probably by complexation with organic acids produced during hydrocarbon metabolism. Congruent dissolution of K-feldspar and precipitation of illite/smectite in the immediate vicinity indicated that a weathering process occurred in which acid was consumed. Local transport of Al and Si was indicated by the precipitation of clay near the dissolution site.

Modeling of the expected geochemical changes in carbonate minerals from measurement of the bulk ground water correctly predicted carbonate diagenesis. Calcite dissolved and precipitated in water that was near-equilibrium with respect to calcite. Dolomite dissolved in water that was undersaturated with respect to dolomite. In near-equilibrium conditions, calcite precipitated by a process in which linearly arranged particles grow into larger spikes, merged with surrounding spikes and finally formed a flat uniform height sheet of calcite precipitate over the surface of the original calcite grain.

Microbial metabolism of hydrocarbons in the subsurface resulted in enhanced weathering of both quartz and aluminosilicates. The probable mechanism is the production of complex organic acids which complex silica and/or aluminum at the silicate surface, resulting in accelerated mineral dissolution. The concentration of the microbially produced chelating agents is high in the vicinity of the microbe, resulting in enhanced chemical weathering. Further away from the microbe/mineral interface, the concentration of organic chelators returns to equilibrium concentration by process of diffusion. The chemistry of the microreaction zone in the immediate vicinity of an attached bacteria cannot be characterized by bulk geochemical analyses.

This research extends our knowledge of the role of microorganisms in the weathering of silicate minerals in terrestrial aquifer conditions. The activity of indigenous microorganisms represents a commonly overlooked factor in modeling subsurface geochemistry and in interpreting diagenetic mechanisms. Mechanisms of bacterial mineral alteration documented in the shallow aquifer conditions at Bemidji, MN can be applied in the interpretation of basin-wide diagenetic reactions in the deeper sub-surface as long as the presence of bacteria can be documented. The rapid rates of dissolution of silicates and precipitation of calcite at Bemidji have important implications for models of global biogeochemical cycles involving silicate and carbonate minerals near the earth surface.

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