south oyster natural and accelerated bioremediation ... · south oyster science plan u.s....

South OysterScience Plan

U.S. Department of EnergyOffice of Energy ResearchOffice of Biological and Environmental ResearchEnvironmental Sciences Division

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Field Experimentationin Bacterial Transport

Bacterial TransportElement

October 28, 1998Updated: October 29, 1999

Glossary

CPT Cone penetrometer test

DAPI A chemical stain used in counting bacteria. Its full name is4',6-diamidino-2-phenylindole.

DIRB Dissimilatory iron-reducing bacteria

DOC Dissolved organic carbon

DOE U.S. Department of Energy

EMSL Environmental and Molecular Sciences Laboratory

GC-CRIMS Gas chromatography-chemical reaction interface massspectrometry

HPLC-ESI High Performance Liquid Chromatography-ElectrosprayIonization.

INEEL Idaho National Engineering and EnvironmentalLaboratory

IR Infrared

IRB Iron-reducing bacteria

ISE Intermediate-scale experiment

LBL Lawrence Berkeley Laboratory

MIRP Microbial FE reduction potential

MLS Multi-level sampler

NABIR Natural and Accelerated Bioremediation Research

NC Narrow Channel Focus Area

ORNL Oak Ridge National Laboratory

PCR Polymerase chain reaction

PCR-DGGE Denaturing gradient gel electrophoresis

PNNL Pacific Northwest National Laboratory

SMCC Subsurface microbial culture collection

SOFA South Oyster Focus Area

TNC The Nature Conservancy

TOC Total organic carbon

VaDEQ Virginia Department of Environmental Quality

Contents

Introduction .............................................................................. 1

Integrated Approach ............................................................... 5

Hypotheses to be Tested ......................................................... 7

Collaborating Team Members ............................................... 15

Research Plan............................................................................ 17

1.0 Site Design and Development .................................. 19

1.1 Site Selection .............................................................. 19

1.2 Detailed Site Characterization ................................ 20

1.3 Flow Cell Parameter Integration ............................ 23

1.4 Excavation Site .......................................................... 26

1.5 Microbial Analyses of Site Materials ...................... 28

1.6 Flow Cell Design ....................................................... 29

1.7 Design, Construction, and Installation ofMulti-Level Samplers ............................................... 32

1.8 Scaling-Up From Laboratory to Field .................... 32

2.0 Tracking Bacteria in Porous Media .......................... 35

2.1 Detection Strategies, New MethodsDevelopment, and Bacterial Survival .................... 35

2.2 Development of Fermentation, Storage,and Transportation Protocols .................................. 36

3.0 Processes Controlling BacterialTransport in Porous Media ........................................ 37

3.1 Intact Core Bacterial Transport Studies ................. 37

3.2 Modeling Bacterial Transport in Intact Cores ....... 38

3.3 Conservative/Reactive Tracer TransportExperiments in Aerobic/Suboxic Flow Fields ...... 39

3.4 Field Injection Experiments Planned for theNarrow Channel Aerobic Flow Cell ....................... 40

3.5 Integration of Field and Laboratory Data into aHigh-Resolution 3D Numerical Simulation ofField Data ................................................................... 43

4.0 Environmental Factors ControllingBacterial Transport ....................................................... 45

4.1 Screening for IRB/DIRB .......................................... 45

4.2 Characterization of Facultative IRB ....................... 45

4.3 Survival of Facultative IRB in SouthOyster Sediment Microcosms ................................. 46

4.4 Bacterial Transport Under Suboxic Conditions .... 46

4.5 Evaluation of Cell Surface Characteristics ............ 47

4.6 Role of Humics in Bacterial Transport ................... 48

4.7 Intact Core Studies with Facultative IRB .............. 48

4.8 Field Injection Experiments Planned for SouthOyster Suboxic Flow Cell ........................................ 49

4.9 Intermediate Flow Cell Experiments ..................... 51

5.0 Technology Transfer Opportunities ......................... 55

5.1 Seek Opportunities for Research Transfer............. 55

5.2 Design and Test Methods for InformationTransfer ....................................................................... 57

5.3 Public/Community Outreach Program ................ 57

References ................................................................................. 59

Appendix A ............................................................................... A.1

1

Introduction

B acterial transport is of increasinginterest to those involved in reme-diating subsurface environments

because adding and dispersing bacteria thatcan degrade or transform contaminants (bio-augmentation) is an attractive and viableremedial option. Treatment regimes for con-taminated soils and aquifer sediments havemost often involved separating the contami-nant from the solid, followed by treatmentor disposal.

Soils and near-surface sandy aquifers com-monly contain iron (Fe), manganese (Mn),and aluminum (Al) oxyhydroxides. Toxicmetals and radionuclides can stronglyadsorb onto the surfaces of these oxyhy-droxides, and their desorption kinetics intothe groundwater can be extremely slow. Sim-ply pumping the groundwater to the surfacefor treatment, therefore, is not always aneffective remediation strategy for aquiferscontaminated with metals or radionuclides.

Bacterial interactions with metals are variedand complex. These biogeochemical reactionscan result in the physical removal of metalsor radionuclides by mobilization, or the pre-cipitation of metals or radionuclides toimmobilize them. Bioaugmentation—theaddition of microorganisms—is being consid-ered (Caccavo et al. 1996; Gorby et al. 1994;Phillips et al. 1999) as a means to modify thebehavior of metals and radionuclides concen-trated in the oxyhydroxides of sandy aquifers.

Bioaugmentation promises to be very usefulwhere natural attenuation or biostimulationare inappropriate or do not work. For example,biostimulation can be ineffective where: 1) thenecessary nutrients/inducers to promotethe desired microbial activity are absent

and cannot be added to the environment, or2) organisms with the appropriate activityare absent from the indigenous population.In these cases, bioaugmentation may be aneffective biological treatment strategy andperhaps the only feasible option. There arealso cases where the natural microbial popu-lation may be insufficient to achieve reme-diation with biostimulation; in this case,bioaugmentation is essential to increase therate and thereby shorten the time frame andcosts for full-scale remediation.

There are two distinct approaches for usingbioaugmentation to remediate contaminatedareas (Unterman et al. 1999):

• Growth Strategy

In the first bioaugmentation approach,microorganisms that are selected for long-term survival and the ability to occupy aselective niche within the contaminatedenvironment are introduced; electrondonors, nutrients, electron acceptors, orselective co-substrates may then be addedto aid survival. Thus, the goal of thisapproach is to achieve prolonged survivaland growth of the introduced organismsand the concomitant treatment of the tar-get contaminants.

This approach is most effective in subsur-face environments that contain a suitablegrowth substrate or where a selectivegrowth substrate can be added to promotethe survival of the microbial amendments.This approach has been most effectivewhen the microorganisms are immobi-lized in the subsurface and form a per-meable barrier or “biocurtain” thatintercepts the migrating contaminant

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plume. To predict the effectiveness of thisapproach, the size of the treatment zoneand the in situ transport behavior andphysiological characteristics of the intro-duced strain must be known.

Although microbial barriers represent veryeffective containment strategies, they arestill a variation of subsurface pump andtreat technology, and therefore, are con-strained by the same problems associatedwith pump and treat remediation pro-grams. Specifically, pump and treat meth-ods may require a decade or more tocomplete clean up, and are controlled bythe rate of the flow of the contaminatedgroundwater passing through the per-meable barrier. Because contaminantssorb strongly to aquifer sediments, reme-diation by pump and treat is limited bydesorption kinetics, which are typicallyvery slow.

• Biocatalyst Strategy

In the second bioaugmentation approach,large numbers of bacteria are introducedinto a contaminated environment as bio-catalysts that will degrade, mobilize, orimmobilize a significant amount of thetarget contaminant before becominginactive or perishing. The long-term sur-vival, growth, and establishment of thebiocatalyst is not a primary goal of thistreatment approach. Additional injectionsof bacteria (biocatalyst) can be added asneeded to further the remediation process.

Applications of this approach are con-trolled by the cost of culturing sufficientmasses of organisms and the extent towhich organisms are distributed in situthroughout the area of contamination. Apivotal requirement for successful sub-surface remediation using this secondapproach is reliable delivery and dis-persion of the injected microorganismsthroughout the contaminated area.Development of this bioaugmentation

technology, however, is currently limitedby a paucity of data on field-scale migra-tion of bacteria through porous sedimen-tary aquifers (DeFlaun et al. 1997; Dybaset al. 1998; Steffan et al. 1999).

The transport of bacteria in groundwater isinversely related to the extent of their attach-ment to sediment grain surfaces. The extentof bacterial attachment to grain surfaces isrelated to the surface chemistries of the bac-teria and sediment (Van Loosdrecht et al.1989; Van Loosdrecht et al. 1990; Costonet al. 1995; Neu 1996; Johnson and Logan1996; Jucker et al. 1998), and to the physicalproperties of the sediment, as described byfiltration theory (Rajagopalan and Tien 1976;Logan et al. 1995).

Hence, the extent of bacterial transport varieswith changes in both the physical and chemi-cal characteristics of the aquifer, i.e., physicaland chemical heterogeneity in groundwateraquifers, as well as with variations in thephenotype of the bacterial strain (DeFlaunet al. 1999).

Whereas the effects of physical heterogeneitieson bacterial (colloidal) transport has been asubject of significant investigation over thepast decade (Bales et al. 1989; Buddemierand Hunt 1988; Champ and Schroeter 1988;Toran and Palumbo 1992; Harvey et al. 1993;McKay et al. 1993; Vilks and Bachinski 1996),the significance of chemical heterogeneity inbacterial transport has received less attention.

The best known, and arguably the mostcommon transport-relevant, chemical het-erogeneities in sedimentary materials are Feand Al oxyhydroxides. At pH values lessthan 8, these oxides may display positivelycharged surfaces that may serve as points ofbacterial attachment within a sedimentarymatrix dominated by negatively-chargedsilicate minerals.

The extent of bacterial transport in a numberof sedimentary materials has been shown to

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be inversely related to the amount of Fe andAl oxyhydroxides present in the sediment(Johnson and Logan 1996; Knapp et al. 1998).Obfuscating the effects of Fe and Al oxyhy-droxide are variations in groundwater pHand ionic strength, both of which serve toalter the extent of bacterial attachment tosediment with a given oxyhydroxide content(Fontes et al. 1991; Li and Logan 1999; Scholland Harvey 1992; Ryan et al. 1999).

Additionally, the presence of dissolved naturalorganic matter (such as humics) in ground-water may further complicate the effect ofoxyhydroxide on bacterial transport due totheir ability to mask the positive surfacecharge of the oxyhydroxide (Tipping andCooke 1982; Davis 1982; Thurman 1985;Johnson and Logan 1996). Another potentialeffect of the natural organic matter in ground-water is that its transport may enhance thetransport of organic contaminants, metals, andradionuclides and perhaps even injected bac-teria (McCarthy et al. 1996).

Suboxic conditions may prevail in mixedmetal-organic contaminated sediments.Within this setting, Fe oxyhydroxide fre-quently becomes a major electron acceptor insuboxic microsites, promoting the growthof iron-reducing bacteria (IRB). The abilityof IRB to transform, mobilize, or immobi-lize toxic metals and radionuclides makesthem good candidates for bioaugmentation(Caccavo et al. 1996). Unfortunately, modelsfor suboxic bioaugmentation are inadequategiven the absence of either column-scale orfield-scale bacterial transport experimentsunder suboxic conditions (Ginn 1995).

In many cases, the best source of microor-ganisms for bioaugmentation is the contami-nated zone itself. The environment alreadymay have selected for bacteria with thedesired metabolic and enzymatic capabilities,but their activity may be limited by nutrientavailability. A potentially successful bioaug-mentation strategy is to isolate and culturebacteria from the contaminated zone and

select strains that 1) possess low adhesion forthe aquifer minerals, 2) tolerate starvation,3) can be readily grown to high densities, and4) possess the appropriate enzymatic capa-bilities to remediate the contaminant.

The most important advantage to isolatingand the reinjecting members of the in situ bac-terial populations is that indigenous strainsare “pre-adapted” to in situ biogeochemi-cal conditions. Another advantage of thisapproach is that use of natural microbiotamay be more attractive to stakeholders andlocal government agencies, because such bac-teria already exist in the environment andare not genetically altered.

To obtain high-quality data on the transportof injected, indigenous bacterial strains, how-ever, reliable and sensitive detection methodsspecific to the injected bacteria are required(Errampalli et al. 1999). Selective plate counts,fluorescent cell stains, and whole-cell label-ing with the stable isotope of carbon (13C)(DeFlaun et al. 1997) have all been tested tomonitor microbial transport in groundwater;however, each of these techniques has poten-tial limitations.

The detection limit for plate counts may behigher than is needed to document the pres-ence of the injected target organism in a down-gradient sampling point, and it may not beable to enumerate those target organismsthat are still able to carry out the function forwhich they were injected but are no longerable to form colonies on solid media (Oliver1993). Fluorescent cell stains allow all theinjected cells to be detected regardless oftheir culturability, but most stains are knownto affect the activity, viability, or adhesiveproperties of the cells (Parolin 1990). Recentwork with viable stains shows promise fortracking bacteria without affecting theirphysiology (Fuller et al. 1999; Fuller et al. inprep). Injection of cells with high amountsof 13C incorporation, with subsequent detec-tion of the stable isotope enrichment of theparticulate carbon downgradient, is not

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dependent on cell culturability and is notexpected to alter cell activity, viability, oradhesive properties. However, these bulkdeterminations of 13C enrichment are notable to unequivocally document that the 13Crepresents live target cells, since the 13C inthe injected organisms may have been trans-ferred or incorporated into other microbes astarget organisms died and lysed or becameprey for protozoa. Compound-specific 13Canalyses when applied to microbial lipidsshould be able to ascertain the relativeimportance of these processes

These new bacterial tracking methodologies,as well as refinement of traditional ones, meritfurther investigation, particularly in light ofrecent scientific and technological advances.

The feasibility of remediating metal andradionuclide contamination in either aerobicor suboxic porous aquifers using bioaugmen-tation strategies is theoretically conceivable,

but it cannot be rigorously evaluated becauseso few field experiments have been per-formed. The purpose of this field researchproject is to 1) develop new insights intothe basic processes that control bacterialtransport in aquifers, and 2) extend thisexperience to metal-contaminated DOEsites. A pristine site was sought for initialfield experiments to elucidate the basicmechanisms controlling field-scale trans-port before transitioning to contaminatedsites where this basic understanding couldbe applied. To meet these needs, the OysterScientific Team has selected an appropriatefield site, are developing procedures for select-ing bacteria strains from in situ communities,developing new procedures for tracking bac-teria, testing scale-up approaches, and gen-erally designing strategies to facilitate thetransport of bacteria within heterogeneousFe and Al oxyhydroxide-bearing subsurfacesystems where so much toxic metal andradionuclide contamination resides.

5

Integrated Approach

Numerous published studies of bench-scale, aerobic bacterial transportexperiments in columns packed with

glass beads and quartzsand are the basis for fil-tration theory (Martinet al. 1996; Johnson et al.1995) and the realizationof the importance of vari-ous bacterial desorptionkinetic parameters.Applying these theoriesto field-scale bacterialtransport experimentsshows promise (Harveyand Garabedian 1991;Bales et al. 1997), but theveracity of these theories is compromised bythe physical and geochemical heterogeneityof the field environment (Harvey et al. 1993;DeFlaun et al. 1997).

Field transport experiments are essential toconfirm whether an understanding of theprocesses believed to be responsible foradhesion and detachment of viable bacteriafrom the aquifer solids can be used to reli-ably predict the rate and extent of bacterialmigration at a well-characterized field sitewith an accurate monitoring technique. Fieldtransport experiments also provide a test-bedfor future studies at NABIR Field ResearchCenters and ultimate transfer to a variety ofindustrial and federal sites.

To perform bacterial transport experimentsunder field conditions, the following majorquestions must be addressed:

■ What level of field characterization isrequired to develop an accurate bacterialtransport model?

Heterogeneity in grain size, porosity,hydraulic conductivity, and mineralcomposition all affect the migration ofbacteria. To address this question, thesubsurface environment must be charac-terized using: 1) surface geophysicaltechniques; 2) coring and logging; 3) sub-surface geophysical tomography;4) examination of nearby stratigraphi-cally equivalent strata; and 5) conserva-tive, reactive, and particle tracers. Thesuite of observations and the spatialresolution required for adequate charac-terization have yet to be determined, butan overall approach recently has beendeveloped (DeFlaun et al. 1997).

■ How can results from laboratory trans-port experiments be used and scaled-upto the field?

Laboratory transport experiments arecurrently being performed under condi-tions simulating the natural ground-water environment using intact cores.These studies document the effect ofbacterial adhesion on the transport dis-tance, bacterial concentrations, and flowrates that maximize transport, the stabil-ity of the labels used to track the micro-organisms, and the effects of sedimentaryfacies on transport. These data are crucialto flow-cell design and to the bacterialgrowth, labeling, and injection protocols.

Intact core studies also provide a way totest methods for facilitating bacterial trans-port in advance of field trials. Examinationof the cores following a bacterial trans-port experiment enables documentationof the bacterial attachment/detachmentprocess at the millimeter scale.

Field experi-

ments are

essential to

confirm that

process-level

understanding

can be used

to reliably pre-

dict bacterial

transport.

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Although the effects of physical andchemical heterogeneity on bacterial trans-port can be tested by intact core experi-ments, heterogeneities are difficult toquantify because of the challenge ofcharacterizing the core in three dimen-sions. A useful approach to quantifyingattachment/detachment phenomena inheterogeneous media is the use of inter-mediate (meter-scale) laboratory flow cellsin which defined heterogeneities can bebuilt into the porous media, and theirrole in controlling bacterial transportcan be assessed (Murphy et al. 1997).

Finally, parametric models of laboratory-scale bacterial transport studies using

intact cores and meter-scale flow cells havebeen developed that will yield bacterialattachment/detachment parameters forsub-facies-scale physical and chemical het-erogeneity (Ginn 1995). These parameteri-zations can be combined with facies-scalecharacterization of physical heterogeneityto develop quantitative models for field-scale bacterial transport. A comparisonof the success of this scaling-up approachin capturing field-scale heterogeneityand reproducing the observed bacterialbreakthrough profiles, as opposed toalternative scaling-up strategies, is oneof this program’s goals.

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Hypotheses to be Tested

T his document outlines the majorresearch tasks that, in total, comprisethe South Oyster Science Plan. The

plan is a tool that seeks to 1) promote collabo-rations among various investigators andinstitutions, and 2) assist scientists outside ofthe program to better understand the researchin progress.

The principal goal of the experiments out-lined herein is to increase our scientific under-standing of the role of microbial adhesionand biogenic Fe(III) reduction on field-scalebacterial transport. The field experiments willresult in protocols that will generally lead toa new understanding of bacterial transportin sediments containing Fe, Mn, and Aloxyhydroxides, the sites of metal and radio-nuclide contamination, in circum- to sub-neutral pH groundwater.

A specific focus of the planned research is todevelop field strategies for bioaugmentationwhereby the preferential adsorption of facul-tative IRB to Fe oxyhydroxides and the totalmicrobial Fe(III) reduction rate are increased.The plan relies on a series of integrated fieldand laboratory experiments, each of which isdesigned to test one of the following coreconcepts:

• Physical heterogeneity controls bacte-rial transport—Wild-type and adhesion-selected strains of bacteria typically yielda range of collision efficiencies and chargedensities (Glynn et al. 1998; Baygents et al.1998). Theoretically, hydrophilic bacterialstrains with low or neutral surface chargeshould be insensitive to the variable min-eral surface charges encountered in achemically heterogeneous aquifer. Bac-terial adhesion to aquifer sediment is

controlled primarily by physical hetero-geneity, and specifically, by grain and porethroat size distributions. In sedimentswith variable grain size, e.g., silt to sand,the bacterial strains will move fasterthrough the coarse-grained layers thana conservative solute tracer. The scale atwhich this is observed is on the order ofmeters and controlled by the spatial scaleof the sedimentary layering and on theinter-layer variability of grain size. Thisphenomenon, often accredited to either“pore exclusion” or “size exclusion,” hasbeen documented in the laboratory, butnot observed in the field. The mecha-nisms governing this behavior are poorlyunderstood.

• Chemical heterogeneity controls bacterialtransport—For quartz-bearing sedimentscontaining Fe, Mn, and Al oxyhydroxidecement and circum- to sub-neutral pHgroundwater, the quartz has a negativesurface charge, whereas the oxyhydrox-ide cement has a positive surface charge.Bacterial strains that are hydrophobic orthat bear high negative surface chargewill be repelled by quartz grains lackingoxyhydroxide coatings and attracted tosurfaces dominated by oxyhydroxidephases.

If the oxyhydroxides are uniformly dis-tributed as grain coatings throughout theaquifer sediment, then these negativelycharged bacterial strains will not migrateas far as neutrally charged bacteria. If theconcentration, mineralogy, and grain sizeof the oxyhydroxides are heterogeneouslydistributed in layers with uniform ground-water chemistry, however, then the nega-

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tively charged bacteria are likely to movefaster than the conservative solute tracerthrough the layers containing littleoxyhydroxide.

The concentrations of negatively chargedbacteria entrained in the groundwaterwill diminish rapidly, however, as moreof the population adheres to the layersrich in oxyhydroxide phases. Bacterialadhesion in aquifer sediment, therefore,is controlled by a combination of in situchemical and physical heterogeneity.

• Microbial Fe(III) reduction will indirectlyenhance IRB transport by several mecha-nisms—Caccavo et al. (1997) has proposedthat IRBs are hydrophobic and adherereversibly to hydrophobic Fe, Mn, and Aloxyhydroxide minerals. Membrane pro-teins then cause strong bacterial adhesionto Fe oxyhydroxide mineral surfaces.

The high affinity of IRB for oxyhydro-xides improves their chances of adheringto near-field contaminated micrositesbut impairs their ability to move throughthe sediment to far-field contaminatedoxyhydroxide loci. In those cases wherehigh collision efficiency limits bacterialpenetration of a formation, bacterialtransport can be significantly improvedif the desorption rate of the bacteria isincreased.

Enhancement of the Fe(III) reduction rateof the IRB may increase the desorptionrate through several mechanisms. Fe(III)reduction will reduce the bioaccessibleFe(III) surface area and should encour-age the IRB to desorb. Increases in theadsorbed, microbially produced Fe(II)on the Fe(III) surfaces may also reduceIRB adhesion.

One approach to enhancing Fe(III) reduc-tion is to provide a humic acid analogue,e.g., anthroquinone disulfonate, whichacts as an electron shuttle from the Fe(III)mineral surface to the membrane-bound

respiratory system (Fredrickson et al.1999). Naturally occurring humic acidsmay also increase Fe(III) reduction activ-ity and thereby increase IRB desorptionrates and transport. Elevated Fe(III)reduction rates could locally increase thepH, which in turn, may promote desorp-tion of IRB by reducing the positivesurface charge of the oxyhydroxide min-erals. Finally, if the Fe(III) reduction activ-ity is high enough to induce significantIRB growth, the desorption of daughterIRB cells will increase net transport rates.Under growth conditions, however, theeffects of bacterial predation by protistsmust be considered (Kinner et al. 1997).

The results of prior lab and field bacterialtransport studies provide a conceptual frame-work for the planned series of aerobic andanaerobic injection experiments.

The pH of the groundwater where publishedfield-scale bacterial transport experimentshave been conducted to date, e.g., Cape Codand Borden sites, ranges from 5 to 6.5. In thisrange, the surface charges of Fe, Al oxides,and oxyhydroxides are positive as opposedto the negative surface charges of bacteria,quartz, and feldspar (Sverjensky and Sahai1996). These minerals may act as stronglyadsorbing and perhaps irreversible sites forbacterial adhesion, and ample laboratoryevidence indicates that bacterial retention incolumns packed with by Fe(III)-coated quartzis higher than columns packed with justquartz (Mills et al. 1994; Johnson et al. 1996;Johnson and Logan 1996; Knapp et al. 1998).

Variations in the amount of bacterial break-through detected in the multi-level sampler(MLS) arrays at either the Borden or CapeCod site could be mitigated by variations ofFe, Mn, Al oxyhydroxide coatings of thesediments, but extensive characterization ofthese chemical parameters has not been per-formed for these sites. In preliminary intactcore experiments, the spatial distribution ofbacteria remaining in the sediment appears

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to correlate with the distribution of Fe, Mn,Al oxyhydroxide minerals (Dong et al. 1999).But grain size also controls bacterial adhe-sion, and if grain size and oxyhydroxideconcentrations are correlated in the aquifersediment, then deconvolution of the trans-port controlling mechanisms will requiresophisticated experimental approaches.

Absorption/desorption experiments per-formed on Cape Cod sand (Scholl and Harvey1992) that contain Fe(III) oxyhydroxideminerals indicated that significant desorp-tion of bacteria from these sand-sized mate-rials occurred when the pH was increasedfrom 5 (typical for uncontaminated Cape Codgroundwater) to 8. This is attributed to thereversal of the Fe(III) oxyhydroxides surfacecharge from positive to negative as the pHwas increased from 5 to 8.

Scholl and Harvey (1992) also performed afield bacterial injection experiment withDAPI-stained cells at Cape Cod and then fol-lowed the bacterial injection experiment withan injection of groundwater adjusted to pH 8using a phosphate buffer. In this case, littledesorption was reported, which they attrib-uted to the neutralization of the pH 8 water asit moved away from the tracer injection well.

Bales et al. (1997) appear to have been moresuccessful at introducing a high pH solutionsubsequent to an injection of viruses andmicrospheres at the Borden site. As the pHincreased from 7.2 to 8.4, a pulse of bacte-riophage, which had been deposited onthe sediment by the first injection, traveledthrough the MLS array. Differences betweenfield and laboratory results were attributedto the uncharacterized chemical heterogene-ity in the field.

Ryan et al. (1999) followed a bacteriophageand Si-colloid injection into the Cape Codaquifer with a high-pH (8-10) solution.Release and breakthrough of adsorbed bac-teriophage and Si-colloids were correlatedwith pH breakthrough.

Quantifying the influence of chemical het-erogeneity on field-scale bacterial transport,therefore, requires multiple experimentswhere either the pH of the groundwater isadjusted to weaken mineral surface chargedifferences, or the surface charge of the bac-terial strain (or fluorescent microspheres) isvaried, or some competitive anion is addedto saturate the positively charged sites. Withrespect to remediation of toxic metals andradionuclides adsorbed to Fe, Mn, and Aloxyhydroxides, a bacterial strain that pref-erentially adsorbs to the oxyhydroxides,i.e., a negatively charged bacterium, ismost desirous.

Capillary electrophoresis can be used toidentify strains with a specific charge (Glynnet al. 1998). Adsorption/desorption or adhe-sion experiments performed as a functionof pH and synthetic Fe, Al oxyhydroxideconcentrations can be used to evaluate thedominance of electrostatic interactions ver-sus hydrophobicity effects.

Laboratory experiments performed byJohnson et al. (1996) suggest that dissolvedorganic carbon (DOC) will reduce the bacterialadsorption on quartz surfaces and increasebacterial adsorption on Fe(III) oxide-coatedquartz. Scholl and Harvey (1992) reportedreduced bacterial adsorption on Cape Codsediments with groundwater containing4 ppm dissolved organic carbon versus arti-ficial groundwater containing <0.4 ppm dis-solved organic carbon. This relationshipswitched, however, at pH >6.5.

Dissolved organic carbon is frequently char-acterized by negatively charged moieties,which, if present in groundwater over a longperiod of time, will preferentially coat the Fe,Mn, and Al oxyhydroxides, thereby maskingthe differential surface charges. In repackedcore experiments, Johnson and Logan (1996)showed that humic coatings on Fe(III) oxideswith sedimentary humics reduces bacterialadhesion to Fe(III) oxides by 60%.

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If laboratory experiments do suggest acorrelation between bacterial absorption/desorption rates, and either high pH or theconcentration of Fe, Mn, and Al oxyhydrox-ide phases, then the surface charge of the sitesediments should be measured as a functionof pH by using the “streaming potential”(Ryan et al. 1999). Such measurements canbe used to determine if charges are maskedcompared to artificially coated sand grains.If they are, then the addition of high pHwater to intact sediment cores should resultin the release of adsorbed dissolved organiccarbon or colloids from the sediment, therebyenhancing the charge differential betweenquartz and Fe, Mn, and Al oxyhydroxidephases.

Provided that intact core experiments withOyster sediments look promising, then afield-scale bacterial injection might then befollowed by a high pH injection. MeasuringpH breakthrough at the MLS’s is straightfor-ward. The challenge will be to see if the num-ber of injected bacteria that adsorbed to thesediment and released during the high pHinjection can be correlated with the chemicalheterogeneity of the sediment as determinedfrom core analyses. The total organic carbon(TOC) content of the water also should bemonitored during the high pH injection, inthe event that organic matter adsorbed to thesediment is partially desorbed.

Another approach for enhancing the surfacecharge differences between the quartz andthe Fe, Al oxyhydroxide phases is to decreasethe ionic strength. Camesano and Logan(1998), Johnson et al. (1996), and Hornbergeret al. (1992), have all reported enhanced trans-port of bacteria through quartz sands bylowering the ionic strength of the water. Lowionic strength groundwater would producea thick, repulsive double layer on the surfaceof quartz grains, leading to lower collisionefficiencies (Spielman and Friedlander 1974).

The same may be true for Fe and Al oxyhy-droxides at pH 8, but, at a pH of 5, the elec-trostatically repulsive barriers of quartz willbe increased relative to the electrostatic at-traction of the Fe, Mn, and Al oxyhydrox-ides. Preferential attraction of negativelycharged bacteria to the Fe and Al oxyhy-droxides should be increased. Such labora-tory or field experiments, however, have notbeen reported.

Combined with pH changes, ionic strengthvariations could prove to be a powerful toolfor manipulating electrostatic processes andfacilitating the attachment of bacteria tometal-contaminated sediments. Such experi-ments could be designed to investigate thebacterial adsorption process by injecting thebacteria with low ionic strength artificialgroundwater. The bacterial desorption pro-cess could be investigated by injecting bacte-ria with natural groundwater, followed byan injection of low ionic strength water. Itmay also be possible to lower the surfacecharge differential by injecting the bacteriawith artificial groundwater with the samesalinity as the natural groundwater, but withgreater proportion of divalent ions.

Such field experiments should be precededby measurements of bacterial surface chargeas a function of ionic strength and by bacterialtransport experiments using Oyster intactcores and low ionic strength artificial ground-water. If these experiments produce promis-ing results, then a field injection of bacteriain the presence of varying ionic strengthnatural groundwater might be useful. Meas-uring conductivity breakthrough at the MLS’sis straightforward. The challenge wouldbe to determine if the number of bacteriaadsorbed or desorbed during the low ionicstrength injection can be correlated with thechemical heterogeneity of the sediment.TOC and colloids in the groundwater should

11

be monitored during the low ionic strengthinjection, in the event that they are partiallydesorbed during the injection.

Groundwater temperature is another possibleparameter that affects bacterial transport, butpublished reports are inconclusive. Labora-tory experiments indicate that the higher thetemperature, the greater the adhesion of bac-teria to sediment (Hendricks et al. 1979;Fletcher 1977). Bellamy et al. (1985) observed100 times greater adsorption of bacteria tosand at 17°C versus 2°C.

For bacteria that are motile at high tempera-ture, but not at low temperature, the oppo-site behavior was observed (McCaulou et al.1995). Strain A0500, a motile subsurface strain,exhibited a lower collision efficiency whenpassed through a repacked sand column at18°C, where it was motile, versus 4°C, whereit was nonmotile. Strain A0500 also exhibitedfaster detachment rates compared to nonmo-tile bacteria. Enhanced column transport ofmotile bacteria over nonmotile bacteria underlow flow velocities (0.6 m/day) has also beenreported by Camesano and Logan (1998).

Low temperature injections could enhancebacterial transport for nonmotile species rela-tive to ambient temperature injections andthe electrostatic potential difference betweenpositively and negatively charged mineralssurfaces could also increase. The metabolicactivity and growth of the adsorbed bacteriawill also be affected by changes in ground-water temperature. Although no papersreport varying the groundwater temperatureduring field-scale bacterial transport studies,Davis et al. (1985) did report injecting hotwater and monitoring the temperature break-through in a sandy aquifer using thermistors.

The effect of Fe(III) reduction upon bacterialtransport requires performing experimentsunder anaerobic conditions. To date, almostall laboratory experiments on bacterial trans-port and all field bacterial transport experi-ments have been conducted under aerobicconditions. An anaerobic field injection ofxenobiotic compounds has been recentlyreported by Rügge et al. (1999), but bacteriawere neither injected nor was the transportof indigenous bacteria investigated. Bioaug-mentation has been performed in the field atcontaminated sites that are suboxic (Steffanet al. 1999), but the injected microorganismswere aerobic, and the injection medium wasoxygenated. Although parts of the Cape Codaquifer are anaerobic due to organic contami-nation, the mechanisms mitigating bacterialtransport in that portion of the aquifer havenot yet been delineated.

To determine whether long-term Fe(III) reduc-tion leads to enhanced bacterial transport,field experiments must be conducted in anundisturbed, uncontaminated aquifer wherenatural Fe(III) reduction has been in progresssince the deposition of the sediments.

By altering the laboratory flow system toprevent leakage to air and by co-injectingdeoxygenated artificial groundwater withthe bacteria, the same experiments can berepeated on intact cores obtained from thesuboxic portion of the field site. These coresmust be collected and stored so as to bestmaintain original physical/chemical condi-tions before incorporation into the laboratoryflow system. Numerical models of theseintact core experiments will be used to definebacterial adhesion and desorption param-eters for scaling up for the suboxic fieldbacterial transport study.(1)

(1) An Intact Core Workshop was held in April 1999 to review and improve procedures for conducting intact coreexperiments. Representatives from ORNL, PNNL, Princeton University, LBNL, INEEL, and Envirogen, Inc.attended the workshop.

12

To test the stated hypotheses in the field,however, flow cells(2) to support this researchmust be located at a site where aerobic andsuboxic groundwater exist in proximitywithin approximately the same hydrogeo-logical unit. The pH of the site groundwaterneeds to be neutral to sub-neutral to test theeffects of oxyhydroxide surface charge onbacterial transport. The salinity of the sitegroundwater needs to be <0.1 wt% to avoid

Figure 1. Location of Oyster Field Site on the Delmarva Peninsula. Alternatecandidate sites included the Cape Charles Air Station and Townsend. The AbbottPit site is a satellite site that provides ready access to sediments for laboratorymicrobial adhesion and transport studies.

strong bacterial adhesionto the mineral grainsby suppression of theelectrical double layer.Finally, the presence ofrelatively high, aqueousFe(II) in the suboxicportion of the aquiferis important as it indi-cates that microbial Fereduction is probablytaking place.

A field site south ofOyster, Virginia (Fig-ure 1), has been identi-fied that meets all theserequirements. At thislocation, the dissolvedoxygen in the ground-water of the surficial,unconfined aquiferdecreases from 6 mg/Lto <1 mg/L as it flowsfrom a local topographichigh toward the tidalmarsh. This trend towardhypoxia is observed innumerous locals in theDelmarva Peninsulaand is related to theupwelling of suboxicwater from deeperconfined aquifers (Fig-ures 2a,b; Speiran 1996).

IRB have been enriched from indigenouspopulations at this site. Once adhesion-deficient, indigenous facultative IRB strainshave been selected, they will be labeled andinjected with conservative tracers into boththe aerobic and suboxic flow fields.

Satellite field sites, established on theDelmarva Peninsula are also being usedthroughout this project as sources of both

(2) The “flow cells” being constructed are forced gradient cells with a series of injection and extraction wellsdesigned to create desired groundwater flow.

13

Figure 2a. Cross-Section of Delmarva Peninsula IllustratingHydrostratigraphy and Regional Groundwater Streamlines.

E

Water Table

Stream

Surface

Atl

anti

c O

cean

Freshwaterand Saltwater

Wetlands

W

Ch

esap

eake

Bay Land

Upper Yorktown Eastover Aquifer

Middle Yorktown Eastover Aquifer

Lower Yorktown Eastover Aquifer

Surficial Aquifer

Figure 2b. Cross-Section of Townsend Site Illustrating the Decrease in Dissolved O2

in Proximity to the Margin of the Delmarva Peninsula.

W E

Feet

30

20

10

S.L.

-10

-20

-30

-40

-50

Agricultural Fields

Surficial Aquifer

Confining Unit

Water Table Woodland

Wetland Wetland

Leaky Confining Unit

Confined Aquifer

bulk sediments and for comparative pur-poses. Sites such as Abbott’s Pit (Figure 1)have sediments that are sandy, but differfrom South Oyster in the degree of hetero-geneity, distribution of grain sizes, andmetal oxyhydroxide content. Sediments fromthese sources will be used to both verifyresults obtained with South Oyster sedi-ments as well as to determine differences inresults obtained in sediments with differingphysical/chemical heterogeneity.

A number of researchers from all the DOENABIR scientific program elements will usematerials from the Abbott’s Pit, South Oys-ter, and other established satellite sites onthe Delmarva Peninsula (Figure 1). Use ofthese common reference materials providesunique opportunities for collaboration amongresearchers from many scientific disciplinesand elements in the NABIRprogram. This sedimentcollection and distributionis administered by Dr. GaryJacobs (Oak Ridge NationalLaboratory).

Plan Reviews

Visiting scientists will con-duct periodic reviews toevaluate the status of theresearch and the science plan.Annual “stocktaking meet-ings” are also held annuallyto review progress and topromote collaborations.

15

Collaborating Team

Members

uccessful field experiments in bacte-rial transport require seamless inte-gration of the results from bench-scale

core studies with the characterization andsample acquisition activities in the field.Timely feedback fromthe investigators respon-sible for modeling bacte-rial transport is pivotalto the design and execu-tion of the laboratoryand field experiments.Drs. T.C. Onstott, M.F.DeFlaun, and Mr. Tim Griffin share overallresponsibility for coordinating laboratoryresearch, theoretical modeling, and field re-search activities among the PrincipalInvestigators (PI’s) on this project.

The following summarizes research activitiesassociated with the South Oyster bacterialtransport project and lists co-PIs and col-laborators responsible for or participatingin each activity.

■ Enrichment and selection of IRB fromthe site—Drs. D. Balkwill (Florida StateUniversity(3)) and J. Fredrickson (PacificNorthwest National Laboratory).

■ Intact core bacterial transport studies—Dr. M. DeFlaun (Envirogen, Inc.) andDr. W. Holben (University of Montana).Dr. W. Johnson (University of Utah).

■ Understanding the effects of predationby protozoa on bacterial transport—Dr. F. Dobbs (Old Dominion University).

■ Adhesion studies of obligate anaerobicand facultative bacteria—Dr. M. Fletcher(University of South Carolina).

■ Theoretical analysis of intact coreexperiments—Dr. T. Ginn (Universityof California at Davis).

■ Coring, logging, excavation, and logisti-cal support at the field site—Mr. T. Griffinand Dr. B. Hallett (Golder Associates).

■ Installation of flow cells—Drs. DeFlaun,Onstott (Princeton University), Holben,T. Phelps (Oak Ridge National Labora-tory), and Mr. Griffin.

■ Methods for tracking bacteria in thefield—Drs. Holben, Johnson, D. White(University of Tennessee), and M. Fuller(Envirogen, Inc.).

■ Photographic infrared imaging of theSouth Oyster excavations and cores—Dr. P. Long (Pacific Northwest NationalLaboratory) and J. Wilson (New MexicoInstitute of Mining and Technology).(4)

■ Intermediate flow cell experimentsexamining heterogeneity and growthof IRB on scaling bacterial transport—Dr. E. Murphy (Pacific NorthwestNational Laboratory/EMSL).(4,5)

■ Geophysical characterization of the site—Drs. E. Majer and S. Hubbard (LawrenceBerkeley Laboratory).

SSuccessful field

experiments

require seam-

less integration

of results.

(3) SMCC (Subsurface Microbial Culture Collection) supports this research by archiving and characterizaing theOyster culture collection.

(4) Environmental Molecular Science Laboratory at PNNL.(5) Related projects in other NABIR program elements at the South Oyster Site.

16

■ Alternative methods to enhance transport;genetic engineering to reduce bacterial cellsize—Dr. A. Matin (Stanford University).(6)

■ Analyses of the effects of colloids on bac-terial transport—Drs. Phelps, Onstott,and DeFlaun.

■ Geostatistical analysis of geological, geo-physical, and microbiological data—Drs.C. Murray (Pacific Northwest NationalLaboratory) and D. Swift (Old DominionUniversity), and M. McInerney (Univer-sity of Oklahoma).(6)

■ Geochemical analyses of groundwaterand sediments—Dr. Onstott.

■ Development of advanced bacterialdeployment strategies in the field—Dr. Phelps.(6)

■ Nitrate/Fe geochemistry—Dr. E. Roden(University of Alabama).(6)

■ Three-dimensional finite difference mod-eling of conservative tracer and bacte-rial transport field data—Dr. T. Scheibe(Pacific Northwest National Laboratory)and Mr. T. Griffin.

■ In situ assessment of effective reactivesurface area of chemically heterogeneous

porous media using reactive tracers—Dr. R. Smith (Idaho National Engineer-ing and Environmental Laboratory).(6)

■ Physical characterization of sediments,stratigraphy and structure—Dr. Swift.

■ Microbiological site characterization—Drs. White, Balkwill, and T. Marsh(Michigan State University).(6)

■ Repacked core studies to assess effectsof microbial reduction of Fe oxyhy-droxides on bacterial transport—Drs. J.Fredrickson and J. Zachara (PacificNorthwest National Laboratory/EMSL).

■ Spatial heterogeneity of microbial Fereduction potential—Drs. C. Murray,E. Roden, S. Hubbard, E. Majer,Y. Gorby, and F. Brockman (PacificNorthwest National Laboratory).(6)

■ Ferrographic method for tracking bacte-ria—Dr. W. Johnson (University of Utah).

■ Role of humics in enhancing bacterialtransport—Dr. J. McCarthy (Oak RidgeNational Laboratory) and M. Fuller.

(6) Related projects in other NABIR program elements at the South Oyster Site.

17

Research Plan

19

Expected Results

■ Selection and characterization of a fieldresearch site.

■ Design and installation of flow cells fordetection of heterogeneity effects on bac-terial transport.

1.1 Site Selection

Task Leader and Collaborators

Griffin, DeFlaun, Onstott, and Swift

Goal

■ To select a site that meets the criteria setforth in the proposed research.

Approach

The challenges of site selection includeaccessibility and the presence of geochemical,geological, microbiological, and hydrologicalproperties that are adequate to test the statedresearch hypotheses. In this case, site-selectionefforts focused on the Delmarva Peninsula.

In the past, the DOE Subsurface Science Pro-gram, under direction of Dr. Frank J. Wobber,conducted bacterial transport research at asite owned by The Nature Conservancy (TNC)in Oyster, Virginia. In the interest of buildingon the experience developed at that site, siteslocated in similar geological formations on theDelmarva Peninsula were screened. Selectedsites had to meet the following criteria:

■ Areas of oxic and suboxic groundwaterof sufficient extent (100 m x 100 m) to con-struct two bacterial transport flow cells.

1.0 Site Design and

Development

■ Circumneutral pH, low salinity, andelevated solid Fe(III) or dissolved Fe(II).

■ Close proximity to a site of exposedsediments for collecting intact coresfor aerobic and suboxic experimentsand to develop stratigraphic/structuralinformation for incorporation into 3Dmodels of the field flow cells.

■ Groundwater not contaminated byorganic pollutants.

■ Groundwater or sediments containingindigenous facultative or obligate IRB.

■ Satellite sites (e.g., Abbott’s Pit) availableto support comparative research.

Site selection began with a thorough analysisof existing information on the local andregional hydrology and geology. A modelof deep upwelling groundwater as the sourceof suboxic groundwater along the flanks ofthe Delmarva Peninsula (Figures 2a,b; Speiran1996) was used as a guide. The literature sur-vey was followed by visual inspection for thepresence of outcrops or pit exposures anddiscussion with local agencies regardinginstitutional ownership and access. Morethan 20 potential sites were investigated inNorthampton and Accomack counties. Basedon visual inspection, the number of candi-date sites was narrowed to three located atthe Cape Charles Air Base, Townsend, andSouth Oyster (Figure 1). The Townsend andSouth Oyster Sites are owned by TNC, andthe Cape Charles Air Base is occupied andoperated by the U.S. Fish and Wildlife Service.

In January 1998, a field investigation wascompleted at these three sites to verify

20

whether they met the stated criteria. Holeswere advanced by direct-push cone penetrom-eter test (CPT) technology, using to the degreepossible “real-time” probe data for site char-acterization. CPT holes are small diameter(~2 in.), require no circulation media, andare plugged immediately after the test iscompleted.

A cluster of CPT holes was made at each loca-tion to acquire all the data desired. Field con-firmation of suboxic groundwater oxygenlevels of <2 mg/L determined whether addi-tional CPTs were conducted at that location.If suboxic conditions were encountered, asecond CPT cluster was installed 50 to 100 maway, either on or perpendicular to deposi-tional strike (depending on physical siteconstraints), to help determine the lateralcontinuity of the suboxic groundwater. Thefollowing parameters were evaluated insamples from each location:

Groundwater

■ Dissolved oxygen, pH, salinity, Fe(II),and total Fe and were measured in thefield (Griffin).

■ Volatile organic carbon samples werecollected and measured in the lab tocheck for potential anthropogeniccontamination (DeFlaun).

■ Suboxic groundwater samples (sealed inserum bottles) were collected and shippedovernight to a microbiology laboratory toenrich for IRB (Fredrickson and Balkwill).

Sediment

■ Sediment samples sealed in tubes werecollected for stratigraphic and grain sizeanalyses (Swift).

■ Sediment samples sealed in tubes(suboxic locations only) were collectedand shipped overnight to a microbiologylaboratory to enrich for IRB (Fredricksonand Balkwill).

■ Sediment samples sealed in tubes(suboxic locations only) were shippedovernight to a geochemical lab foranalysis of total Al, Fe, and Fe(III) insediment (Onstott).

A brief sampling plan was prepared to out-line the sample procedures and protocols tobe used in the field. This plan was madeavailable to personnel at the National Wild-life Refuge, located at the old Cape CharlesAir Station Site, and TNC on request, andwas circulated to all PI’s for input before thefield work.

Finally, all field-related activities wererecorded in detail in field logbooks, includ-ing CPT cluster configurations, samples col-lected (with ID numbers), and results of fieldanalyses.

A brief report was prepared that includedresults of all field measurements and labo-ratory analyses of groundwater and sedimentsamples. It was circulated to PI’s at a meet-ing held in Salt Lake City, January 24-25,1998. The report identified accessible suboxicgroundwater at South Oyster and CapeCharles Air Base, but not at the Townsend Site(Figures 2a,b). Based on the data obtained,the consensus among the PI’s was that theSouth Oyster Site would be the primary siteand Cape Charles Air Base the back-up site.The South Oyster Site is located in a largeopen field just south of the small village ofOyster, Virginia (Figure 3).

1.2 Detailed Site Characterization


Griffin, Onstott, Swift, Majer, Balkwill, andScheibe

Goals

■ To determine in situ geological heteroge-neity across the site; to assess hydro-geological units.

21

■ To identify any hidden, man-made struc-tures in the subsurface.

■ To locate any saline groundwaterintrusions.

■ To establish direction of groundwater flow.

■ To locate the boundary between oxic andsuboxic groundwater.

Approach

For purposes of the detailed site character-ization, the site was partitioned into threefocus areas: 1) South Oyster Focus Area in thenortheastern portion of the field near the

town limits, 2) NarrowChannel Focus Areaon the southern flankof the field adjacentto Narrow ChannelBranch, and 3) thenorth-south trendingWheatfield Focus Arealocated between thefirst two focus areas.

Geologically, all threefocus areas are locatedin the WachapreagueFormation, which over-laps from the east ontothe flank of the Mapps-burg Scarp. The SouthOyster Focus Area issituated farther downthe flank of the scarp,while the Narrow Chan-nel Focus Area (SOFA)to the south is well upthe scarp flank. TheWheatfield Focus Areais oriented north-north-east along the scarpflank, parallel to depo-sition strike. Based onresults from the initialfield reconnaissance, the

detailed site characterization efforts werefocused on the South Oyster Focus Area,where suboxic groundwater conditions hadbeen identified, and on the Narrow Chan-nel Focus Area, where aerobic groundwaterhad been identified.

To address the first three goals, a non-intrusivesurface geophysical survey was performedwith ground-penetrating radar at the site inMarch 1998. The purpose of the survey was toidentify any hidden subsurface structures thatwould interfere with the natural groundwaterflow gradient or chemistry and correlate thestratigraphy between the respective flow cellsand any potential excavation point(s) (Majer).

Figure 3. South Oyster Field Site and Locations of Suboxic and Aerobic Flow Cells

22

To address the other goals, a limited seriesof 23 CPT investigations was performedbetween April 6 and 18, 1998, along the geo-physical survey lines. The purpose of theinvestigations was to establish the boundarybetween the suboxic and aerobic water, deter-mine the hydrological gradient, and establisha stratigraphic and geophysical correlation.Measurements of depth to water were taken,and a geographic survey was performed toaccurately establish surface topography andwater table elevations. Sediment and ground-water samples were collected and processedanaerobically in the field. These holes wereplugged immediately after coring and testswere completed. Results of the field workwere compiled as a report to the PI’s andposted on the Princeton University web sitein June 1998 (Griffin).

Based on the results of this more detailedsite characterization, the suboxic flow cell islocated in proximity to sample station SO-3in the South Oyster Focus Area (Figure 4)while the aerobic flow cell is located nearNC-4 in the Narrow Channel Focus Area(Figure 5). Major cation and anion concen-trations from representative groundwatersamples from these two locations are similar,but the redox-sensitive species are distinct(Table 1). The size of the flow cells is approxi-mately 20 m x 30 m with the long dimensionoriented parallel to the groundwater flowdirection (east-southeast for both flow cells).The CPT logs and ground-penetrating radarindicate that the sandier portion of the aqui-fer located between 6 m and 9 m will be thetarget zone for the injections and monitor-ing wells.

Figure 4. South Oyster Focus Area Showing Approximate Location of Suboxic Flow Cell(dissolved O

2 in mg/L)

23

The Nature Conservancy, which owns theSouth Oyster site, has a number of require-ments that must be fulfilled to obtain andmaintain a research permit at this site. Theserequirements include: 1) working with countyand state officials and the local Oyster com-munity to educate, inform, and gain publicacceptance of the field research to be con-ducted there; 2) fostering and fulfillingcollaborations with other researchers involvedat TNC sites; 3) working with TNC to designa field site that will be both physically attrac-tive and educational; 4) keeping the countyOffice of Planning and Zoning informed ofour progress; 5) providing results of ourresearch to TNC; and 6) prior permission fromthe Virginia Department of EnvironmentalQuality to conduct injection experiments.

The Nature Conservancy also requires asingle contact person for each project, whichis Dr. DeFlaun’s responsibility. Dr. DeFlaun,with the assistance of Drs. Swift and Onstott,have met with a number of county officialsas well as with the Water Quality Consor-tium of Northampton County to informthem of our plans for the site and to elicittheir comments. The research application to

TNC for a research permit was approved byTNC in September 1988.

A variance to the groundwater quality stan-dards from the Virginia Department of Envi-ronmental Quality and a variance for injectioninto a drinking water supply from the U.S.Environmental Protection Agency (EPA)Region III were approved in August 1998.The NEPA Categorical (CX) Exclusion wasapproved by the Chicago Operations Officein September 1998. Although these experi-ments have no impact on groundwater qual-ity in the area, a variance must be grantednonetheless to conduct injection experiments.

1.3 Flow Cell Parameter

Integration


Onstott, Swift, Griffin, Long, and Majer

Goals

■ To provide a detailed 3D-depiction of thepermeability structure of the flow cells.

Figure 5. Narrow Channel Focus Area Showing Approximate Location of Aerobic Flow Cell (dissolved O2 in mg/L)

24

■ To document the physical, chemical, andmineralogical variations in the flow-celltarget horizons.

■ To calibrate geophysical signals that willbe used to map physical and chemicalvariations in 3D.

■ To obtain more refined geological andgeophysical surveys of the site to

constrain the exact location and orienta-tion for the forced gradient flow cells andMLS’s.

■ To provide a detailed 3D picture of thepermeability structure of the flow cells toconstrain the vertical distribution of sam-plers in the MLS’s.

Table 1. Groundwater and Sediment Properties for Narrow Channel Aerobic (NC) andSouth Oyster Suboxic (SOFA) Flow Cells. Concentration are in parts per billion unlessotherwise specified.

Groundwater Parameters

NC(6-7 m-bgs)

Aerobic Flow Cell

SO(6-7 m-bgs)

Suboxic Flow Cell

Dissolved O2 (mg/L) 3.5-5.5 0.4-1.0

Eh (mv) 431 to 437 -25 to 69

pH 5.6-6.1 5.6-5.9

Total organic carbon 1000 10,000

Total inorganic carbon 40,000-55,000 65,000-80,000

Cl 19,400-33,210 45,975-85,300

NO3

25,180-43,110 5330-15,870

SO4

22,560-64,100 52,905-69,450

Na 13,300-17,300 13,300-46,500

Mg 3090-6240 3600-10,800

Ca 20,300-29,200 23,400-25,000

Filtered Fe total 100 430-1980

Filtered Fe(II) 0 330-1960

Unfiltered Fe total 20 1440-3720

Sediment Parameters

Total Fe (ppm) 1892-8847 3731-22,413

Fe(II)/Fe Total 0.02-0.13 0.09-0.73

HCI-Amine extractable Fe 24-442 14-1626

HCI-Amine extractable Mn 3-12 1-78

HCI extractable Al 48-1072 18-653

Porosity (%) 20-45 15-36

Hydraulic conduct. (10-5 m/s) 5-28 0.7-4.1

Mean grain size (µ) 151-423 89-304

25

Approach

At the flow cell sites, characterization is basedon intact vertical cores, cross borehole radar,and seismic tomography. The accuracy ofthese geophysical approaches is optimizedwhen calibrated against the chemical andphysical characteristics of the core material.

From the tomographic data and grain sizeanalyses of the cores, the sedimentary bedstructure and permeability structure of theflow cell can be estimated. The modeledstructure can be refined, however, from obser-vations of bed geometry and permeabilityvariation at the excavation site. This structuralbasis is pivotal to the development of a newfluid flow/transport modeling approach.

The following analyses on the vertical coresand horizontal cores from the excavationwill be required to achieve the stated goals:

■ Permeability and seismic velocity meas-urements (Figure 6; Swift and Majer).

■ Grain size and porosity determinationsfor initial conditioning of permeabilityvariation in hydrological models (Swift).

■ Mineralogical characterization and poresize variation by scanning electron micros-copy (Onstott).

■ Fe, Mn, and Al oxyhydroxide concentra-tions (Onstott).

Figure 6. Cross Borehole Radar and Seismic Tomographic Profiles Along the Axis of the Narrow Channel Flow Cell(from Borehole B2 to NC-S24 in Figure 12). High velocities and attenuation correlate with high hydraulicconductivity.

26

■ Measurement of bed geometry and ori-entation and grain size variation withinthe bedded structures at the excavationsite (Swift).

■ Infrared, air permeability, and multi-spectral measurements on vertical coresto establish joint relationship betweenphysical/geochemical heterogeneities(Long, Wilson, and Onstott).

1.4 Excavation Site


Griffin, DeFlaun, Onstott, Swift, Majer,McInerny, Long, and Wilson

Goals

■ To determine the 3D spatial geometry ofsedimentary beds that correlate with thosethat constitute the flow-cell target zone.

■ To compare physical, chemical, and min-eralogical heterogeneities and bed geo-metry to geophysical tomography data.

■ To collect intact cores for bacterial trans-port experiments, permeability measure-ments, and microbial analyses.

Approach

A limited excavation was made in the NarrowChannel Focus Area where the water tabledips down in elevation close to a streamincision. This permits access to subsurfaceformations that are present below the watertable in the flow cell. A plan for stabilization/reclamation was drafted in cooperation withTNC to minimize visual and environmentalimpact. An excavation took place in August1998 along the east-west scarp on the northside of Narrow Channel Branch (Figure 7)and was approximately 20 m long x 1 m deep.This task involved the following activities:

■ Draft of field sampling plan circulatedto the PI’s. Annual, DOE-sponsored

meetings (including external reviewers)with PI’s were also used to formulate afield sampling plan. A formal draft wascirculated to all PI’s before the date ofexcavation (Griffin).

■ Detailed ground-penetrating radar andcross borehole tomography were used tocharacterize the subsurface at the excava-tion site prior to excavating (Majer andGriffin).

Figure 7. Photographic IR Imaging of the Excavationin the Narrow Channel Focus Area in August 1998.Subsequent to opening and smoothing the face ofthe excavation, an IR camera was used to obtainhigh-resolution images in conjunction with air mini-permeameter measurements. Research by Dr. Phil Long(in collaboration with Griffin and Swift) has shownthat IR response can be correlated with permeability,potentially obviating the need for exhaustive small-scale sampling. His work at the South Oyster Site willfurther verify this technique as a means of obtainingquantitative representations of the heterogeneousphysical and geochemical properties of the subsurface.

27

■ After excavating down to the water table,mapping and sediment facies descrip-tions, infrared (IR) digital photography,and mini-air permeability measure-ments were made across the smoothedsurface of the excavation (Figures 7 and8; Swift, Long, and Wilson).

■ Samples were collected from the exca-vated face for falling head permeability,permeability anisotropy, grain size,porosity, microbial activity, microbialbiomass, geochemistry, and mineralogy.Oriented intact cores were collected forbacterial transport and for core manipu-lation experiments (Figure 9). Bulk sedi-ment samples were collected for microbialreduction experiments and microbialadhesion assays. All sampling points onthe excavation face were surveyed andanalyzed to provide an accurate 2D rep-resentation of all data (DeFlaun, Swift,Onstott, McInerney, and Griffin).

■ Oriented intact cores (Figure 10) werecollected from below the water table bytrenching down with the back hoe andquickly extracting core before the trenchcollapsed (DeFlaun, Onstott, and Griffin).

■ Finally, the excavation site was buriedand reclaimed (Griffin).

Excavation was done in collaboration withanother NABIR project (“Heterogeneity ofSedimentary Aquifers: Role in MicrobialDynamics Assessed by Radar Imaging andAcoustic and Radar Tomography”). Princi-pal investigators on this project are Drs. Swiftand Majer, who will characterize the pit by

Figure 8. Collection of Air Mini-Permeametry Dataat the Excavation Site in the Narrow Channel FocusArea in August 1998. Mini-air permeametry is atechnique developed by Dr. John Wilson at the NewMexico Institute of Mining and Technology to meas-ure the permeability of geological formations in thefield.

Figure 9. End View of Three Intact Cores Collectedin the Vadose Zone During Excavation of the Nar-row Channel Focus Area in August 1998. Vadosezone intact cores were collected from each of threevisually distinct facies in the vadose zone duringexcavation at Narrow Channel. Samples collectedduring the excavation will be used to characterizeeach of these facies. The blue foam end pieces wereplaced into the shelby tube to keep the sediments fromslumping as the cores were being driven into the faceof the excavation. Each core was driven into the facein the orientation of the local groundwater flow.

28

geological and geophysical techniques. Dr.McInerney will conduct the microbial char-acterization, and Dr. Murray will integrateall these characterization activities with geo-statistical techniques. They will share thefield work, samples, and data with the inves-tigators in this project.

1.5 Microbial Analyses of Site

Materials


Balkwill, DeFlaun, Holben, Fredrickson,White, Dobbs, and Marsh

Goals

■ To quantify and characterize the indig-enous microbial communities in bothgroundwater and sediment samples.

■ To identify candidate strains for injection.

■ To provide a baseline for assessing theimpact of transport experiments on theindigenous microbial community.

Approach

The methods outlined below are designedto determine the size and composition of themicrobial community and microbial distri-butions (heterogeneity). Culturable hetero-trophs and IRB, and (depending on theneeds/requirements of TNC) non-culturablemicroorganisms will be characterized usingthe following approaches:

■ Phospholipid fatty acid analyses of sedi-ment and water samples to determinebiomass and general community struc-ture. PCR-DGGE (Denaturing GradientGel Electrophoresis) of PCR-amplifiedeubacterial rDNA to provide a diversityfingerprint will be performed on thesame samples as the phospholipid fattyacids (White).

■ Viable counts (plate counts) on severalmedia will be used to enumerate cultur-able aerobes and facultative heterotrophsand to provide a source of isolates forscreening (Balkwill).

■ Enrichment of sediment and groundwa-ter samples for IRB, including facultativeIRB reducers, will be conducted. Isolationof cultures from positive enrichments andphysiological characterization to assessutilization of electron donors/acceptors(Balkwill and Fredrickson).

■ Colony morphology and diversity analy-ses (done on plates used for viable counts)will be undertaken to estimate the numberof distinct culturable types in each sampleand to facilitate comparison of samplesfrom different depths, boreholes, andtime points e.g., before and after trans-port experiments (Balkwill and DeFlaun).

■ Representative colony types will beisolated and deposited in the DOESubsurface Microbial Culture Collection(Balkwill).

Figure 10. Collection of Horizontal Intact Cores fromBelow the Water Table (saturated zone) During theExcavation in the Narrow Channel Focus Area inAugust 1998. The excavator was used to create asmooth surface below the water table and aluminumshelby tubes were driven into the face of the exca-vation in the orientation of local groundwater flow.These intact cores will be used for transport studiesthat will be used to design the field bacterial transportexperiments.

29

■ Analysis of 16S ribosomal RNA sequencesfor selected isolates will be done toprovide genus-level identification ofnumerically predominant culturableforms, if required by TNC (Balkwill andHolben).

■ Isolates will be screened for phenotypesof interest for injection. Low adhesion tosite sediments, non-pathogenicity, andsensitivity to clinical antibiotics will be theselection criteria (Table 2; DeFlaun andBalkwill).

■ Restriction Fragment Length Profile(RFLP) analyses of water samples willbe performed to detect the presence ofpreviously characterized Oyster isolatesat the new site (Holben and Balkwill).

■ Characterization of nonculturable micro-bial communities will be undertakenby direct extraction of DNA, followedby PCR amplification, PCR-DGGE ofeubacterial rDNA to provide a diversityfingerprint on the same samples as the

phospholipid fatty acids, cloning, andsequencing of 16S rRNA genes (Balkwilland White).

■ Direct counts of protozoans in ground-water and sediments will be made (Dobbs).

■ Microbial community phylogeneticanalysis by terminal restriction fragmentlength polymorphisms of PCR-amplified16S rRNA genes (Marsh).

1.6 Flow Cell Design


Scheibe, Onstott, DeFlaun, Ginn, Griffin,Majer, Murray, and Hallett

Goal

■ To design flow cells based on integrationof the site characterization data to provideuseful information on bacterial transport.

Table 2. Strain Characteristics

Strain IDAntibioticResistance HIC/ESIC

Electrophoretic

(10-8 m2V-1s-1)

ZetaPotential Size

Lowest %Adhesion

Recent %Adhesion

PL2W31 Arthrobactersp.

Nal 50 Weakly hydrophilic/

high negative

-1.82 ± 0.04 0.9 x 0.9 µm 55% 99%

Mach 1 Arthrobactersp.

Nal 50 Strongly hydrophilic/

negative

-1.16 ± 0.02 1.5 x 0.5 µm 20% 20%

OYS50 Erwiniaherbicola

Sensitive Weakly hydrophilic/

weak negative

-0.45 ± 0.05 0-2 1.8 x 0.5 µm 64% 64%

OYS2-A Erwiniaherbicola

Sensitive Strongly hydrophilic/

neutral

-0.37 ± 0.04 0-2 1.6 x 0.5 µm 14% 54%

DA001 Comamonassp.

Rif 50 Strongly hydrophilic/

neutral

-0.45 ± 0.01 0-2 1.2 x 0.6 µm 0% 11%

DA001-R Comamonassp.

Rif 150 Strongly hydrophilic 17% 35%

B2-4 Comamonassp.

Rif 50 Very weak/negative -1.60 ± 0.08 0-2 95% 95%

FER-02 Paenibacilluspolymyxa

Sensitive Very weak

hydrophilic/neutral

93% 99%

(a) Electrophoretic mobility was measured in Narrow Channel artificial groundwater of ionic strength 0.0034 M.

30

Approach

The South Oyster Site will include bothsuboxic and aerobic flow cells. The flowcells will be circumscribed by nine injection/extraction wells in a 20-m x 30-m grid (Fig-ure 11). Six to eight additional wells willbe installed in the center of the flow cells forborehole tomography, along with a series ofapproximately 25 MLS’s. In addition, fourmonitoring wells will be positioned around

the MLS’s to monitor any release of bacteria ortracer beyond the sampling array (Figure 12).Such a release would require remedial actiondesigned to confine the bacterial or tracerplume to within the flow cell.

A baseline groundwater characterization,monitoring, and contingency plan will be pre-pared and implemented to ensure that pre-existing groundwater quality at the site ismaintained in the vicinity of the flow cells.

Figure 11. Flow Cell Configuration for Narrow Channel and South Oyster Focus

31

Figure 12. Multi-level Sample Array for Narrow Channel Flow Cell. Groundwatermonitoring system consists of tranduces placed in NC-B1, M2, and M4 and a probefor monitoring dissolved O

2, pH, conductivity, and temperature in M3.

This task involves the following steps:

■ Install the injection/extraction wells andtomography boreholes and collect coresfrom boreholes and wells (Griffin).

■ Collect groundwater for geochemical andmicrobial analyses (Onstott, DeFlaun, andGriffin).

■ Collect bulk groundwater for laboratorybacterial transport experiments (DeFlaunand Holben).

■ Perform down-hole seismic and radartomography on between six to eighttomography holes (Majer).

■ Perform pump/slug tests and analyzefield data to determine vertical varia-tion in hydraulic conductivity using aflow meter; perform water level meas-urements over a period of days to testfor tidal influences (Griffin and Hallett).

■ Perform geological description and IRimaging of split cores on site (Swift andLong).

32

■ Collect samples from split cores for micro-bial and geochemical analyses and forfalling head permeability measurements(Swift, DeFlaun, Holben, White, Balkwill,and Onstott).

■ Archive remaining cores, and performgrain size analyses (Swift).

■ Correlate geophysical tomography datawith grain size analyses, falling headpermeability, flow meter measurements,and geochemical analyses (Figure 6;Majer, Swift, Onstott, and Griffin).

■ Develop a preliminary 3D model of theflow cell using geophysical, grain size,pump test, flow meter, and permeabilitydata (Scheibe, Murray, and Griffin).

■ Determine the optimum horizontal dis-tribution of MLS’s within the flow cellsbased on this 3D model and on bacterialtransport intact core experiments (Scheibeand Ginn).

1.7 Design, Construction,

and Installation of Multi-Level

Samplers


Griffin, Onstott, Phelps, and Scheibe

Goal

■ To design, install, and field test semi-automated sample collectors for aerobicand suboxic flow cells.

Approach

Multi-level samplers were designed based ondata obtained in previous CPT campaignsthat allowed us to obtain samples for grainsize analysis and estimate the conductivityof the aquifer with depth. The MLS’s willhave 10-cm-long samplers with a 0.01-in. slot

size. These samples will be distributed acrossan interval from 6 to 9 mbls. The MLS’s will beconnected to the surface by a 3/8-in. OD rein-forced PVC tubing attached to a central PVCrod for stability. Peristaltic pumps will be usedto obtain samples from various depths.

The flow cell for the Narrow Channel FocusArea was installed during Fall 1998, andthe flow cell for the South Oyster Focus Areawas installed during Spring 1999. Theseactivities entailed the following:

■ Research, design, and construction ofsamplers and shipment to site (Phelpsand Griffin).

■ Installation of samplers into cased wellwith sampler depths established usingthe geophysical data (Griffin, Hallett,and Phelps).

■ Accurately surveying and measuringelevation of MLS’s and depths to sam-plers (Griffin and Hallett).

1.8 Scaling-Up From

Laboratory to Field

Task Leader and Collaborator

Scheibe and Long

Goal

■ To develop and use parametric modelsgenerated at the core and field scales todetermine whether an understanding ofheterogeneity at the core scale can beused to predict field-scale transport.

Parametric models of laboratory-scale bacte-rial transport studies will be developed usingintact cores that yield cellular attachment/detachment parameters for sub-facies-scalephysical and chemical heterogeneity. Theseparameterizations can be combined withfacies-scale characterization of physical het-erogeneity to develop quantitative models

33

for field-scale bacterial transport. Establish-ing the relationship between model accuracy/precision and the level of characterizationdetail will be one aim of the field experiment.

Approach

The approach will be to assess the researchvalue of intact core experiments according tothe following hypotheses:

■ The use of intact core experiments to testbacterial transport enables the design offield-scale bacterial transport experiments.

■ Intact core experiments test the veracityof bacterial tracking methodologies.

■ Intact core experiments provide a testbed for developing deployment method-ologies to facilitate transport.

Intact core experiments relating the amountof bacterial breakthrough to the concentra-tion and duration of inoculation and velocityof the groundwater will lay the groundwork

for a successful field experiment. New deploy-ment strategies, such as development ofthe vibrational approach of Dr. Phelps orthe co-injection of humics, may improvethe dispersion of bacteria.

Research using intact cores will test whethercentimeter-sized physical and chemicalheterogeneity control bacterial transport atthe meter scale. The diameter of these cores(7.3 cm) captures considerable variation in thesedimentary fabric across a distance of 50 cm.Because this scale is directly comparable tothe field experiment, the core experimentsprovide a direct test of scaling up. Largerdiameter cores can be collected if necessary.

Cores from the intact core experiments will beincluded in the IR and multispectral imag-ing experiments of Long/Scheibe/Wilson toestablish the relationship between physicaland geochemical heterogeneity and to includethis information in appropriate scale-upmodels (Long, Onstott, and Wilson).

35

Expected Result

■ Development of new field methods fortracking indigenous subsurface organismsthat are candidates for in situ bioreme-diation by means of bioaugmentation.

2.1 Detection Strategies, New

Methods Development, and

Bacterial Survival


Holben, Onstott, White, Johnson, and Fuller

Goal

■ To develop and implement appropriate,sensitive, and reliable methods to trackinjected bacteria in field and laboratorytransport experiments. These methodswill be used to determine transport aswell as survival of injected strains.

Approach

■ Further develop the 13C stable isotopetracking method of Holben et al. (inpreparation) to determine stability oflabel in the injected cells and for stream-lining the method for quick and efficientanalysis of a large number of samples(Holben).

■ Establish dual stable-isotope labelingcapabilities for co-injection and unam-biguous detection and monitoring ofdifferent strains. This incorporates statis-tical tools to determine precision and

2.0 Tracking Bacteria in

Porous Media

detection limits for labeled strains innatural groundwater (Holben, Onstott,White, and Fuller).

■ Assess the suitability of specific isotopesand growth substrates for isotopic enrich-ment of bacteria to maximize specificactivity (hence sensitivity) and mini-mize label turnover, exchange, or othercompromising label factors (Fuller).

■ Develop and optimize isotopic enrich-ment strategies for the candidate strains(DeFlaun, Holben, and Fuller).

■ Develop a more specific labeling strategyfor the injected bacteria that uses gaschromatography-chemical reaction inter-face mass spectrometry (GC-CRIMS),gas-chromatography-isotope ratiomass spectrometry (GC-IRMS), andhigh-performance liquid chromatogra-phy electrospray ionization massspectrometry (HPLC-ESI-MS).

By this method, 13C-labeled and 15N-labeled cellular components, such asfatty acids, proteins, or nucleic cells areextracted, purified, and separated via GCand converted in the chemical reactioninterface to oxidized products that thenare measured in the mass spectrometer.

By comparing the isotopic enrichment ofspecific cellular components of the targetcells before injection with those of down-gradient samples, the presence and quan-tity of the target cells in the post-injectionsamples can be determined. The majoradvantage of this approach is that the

36

13C-labeled and 15N-labeled cellular com-ponents establish a “signature” for thetarget cells. Variations in this signatureindicate changes in the target cells them-selves, cell death, or incorporationof labeled cellular materials by othermicrobes, all of which would allow foradjustments to be made in the calculatednumber of injected microorganisms(Onstott, White, and Fuller).

■ Assess the use of newly developed fluo-rescent dyes for tracking organisms in thesubsurface. These dyes may allow cells tobe stained without loss of activity, viability,or changes in transport properties. Somenewer dyes specifically stain cell mem-branes while others cross the membraneand covalently bond to intracellular pro-teins. In either case, some dyes have beenshown to be retained in cells for up to3 to 4 weeks, without loss of cell viabilityor alterations in cell function or adhesion.

These stains will be evaluated for theirapplicability for use in the bacterial trans-port studies. Their longevity in cells,retention of cell viability, and retentionof adhesion/transport properties will allbe tested in the laboratory before beingtransitioned to the field. Adhesion prop-erties will be tested in the sand columnadhesion assay, and transport propertieswill be tested in intact cores. Equally

important is their lack of toxicity and suit-ability for field use (Fuller).

■ Explore and develop alternative molecu-lar detection strategies, such as quantita-tive PCR, that may provide additionaland independent monitoring capabilitiesin support of field and laboratory trans-port experiments (Holben).

2.2 Development of

Fermentation, Storage, and

Transportation Protocols


DeFlaun, Holben, and Griffin

Goal

■ To develop protocols for growth, labeling,storage, and transportation for organismsselected for injection that will result in via-ble, labeled cells on arrival at the field site.

Approach

The bacterial strain selected for injection willbe grown in a manner that maximizes theamount of the label to be used in trackingthe organism in the field. Protocols will thenbe developed to deliver the maximum num-ber of viable organisms to the site.

37

Expected Results■ Quantification of the relative impacts of

physical and chemical heterogeneity onmicrobial transport.

■ Evaluation of the incremental value ofcharacterization data in terms of the pre-dictive ability of the resulting model.

■ Preliminary understanding of the poten-tial impacts of grazing by protozoa.

■ Determination of how core-scale bacterialtransport experiments can be scaled-upto field-scale transport experiments.

■ Quantification of the effect of aerobicbacterial adhesion on transport throughaquifer sediments.

3.1 Intact Core BacterialTransport Studies

Task Leader and CollaboratorsDeFlaun, Onstott, Ginn, Scheibe, Holben,Long, and Dobbs

Goals■ To determine kinetic adsorption/

desorption parameters for bacterialstrains in intact cores from the SouthOyster excavation to constrain the hori-zontal spacing of the MLS’s for the newflow cells.

3.0 Processes ControllingBacterial Transport inPorous Media

■ To determine the effect of grain size,mineralogy, texture, and porosity on bac-terial transport.

■ To quantify protozoan bactivory of injectedbacteria in South Oyster intact cores.

ApproachThe activities for intact core experimentsare as follow:

■ Radiolabeled bacteria are introducedfrom the bottom of the core, with waterflow in the upward direction. A pressuretransducer measures the pressure differ-ence between the influent and effluentends. A conservative tracer (chloride orBr) is injected to assess water flow dynam-ics. The cores are run in an environmen-tal chamber at 15°C, corresponding to theaverage groundwater temperature of theSouth Oyster Site. Water flow rates varyfrom 0.5 m to 2 m/day, bracketing therange of the in situ groundwater veloci-ties measured at the South Oyster Siteduring the forced gradient transportexperiments (Figure 13). Bacterial break-through is monitored by measuring bothplate counts and the radiolabel in theeffluent fractions, which are collected at20-min intervals throughout the experi-ment (DeFlaun, Onstott, and Holben).

■ For protozoan bactivory studies, bothbacteria and cultured protozoan grazerswill be injected simultaneously. A time

38

course of population dynamics for bothpredator and prey will be determined.Bacteria will not be radiolabeled in thesestudies. Protozoan population dynamicswill be assessed by direct epifluorescencetechniques. Bactivory will be directlyassessed by enumerating fluorescentbacteria in protozoan vacuoles (Dobbs).

■ At the completion of the experiments,the cores are split and the distribution

of attached bacteria measured on the 2Dface with phosphorimage screens andin 3D with liquid scintillation countingof the subsampled core (Onstott andDeFlaun).

■ For selected cores, IR and multispectralimaging will be performed on the non-epoxied half of the core (Long, DeFlaun,and Onstott).

3.2 Modeling BacterialTransport in Intact Cores

Task Leader and CollaboratorsScheibe, Ginn, and Onstott

Goal■ To derive kinetics of bacterial adsorption/

desorption for incorporation into a pre-dictive field-scale transport model.

Approach■ Data from breakthrough curves and the

cells retained by the intact cores will besimulated by a 1D advection/dispersionmodel (Onstott, Ginn, and Scheibe).

■ The kinetic adsorption/desorption termsderived from multiple core experimentswill be used to develop distribution func-tions of these parameters for each faciesand/or empirical functions relating theseparameters to physical, chemical, andmineralogical properties (Onstott, Ginn,Scheibe, Swift, and Holben).

■ The kinetic adsorption/desorption termsfor intact core experiments will be com-pared with kinetic parameters derivedfrom intermediate-scale experiments con-taining representations of the facies chemi-cal and physical heterogeneities (Murphy,Ginn, and Scheibe).

Figure 13. Bacterial Transport Experiments arePerformed Using Intact Cores Collected in ShelbyTubes from the Excavation and Groundwater Col-lected from the Flow Cell Site. Experiments are run ina cold room maintained at 15°C, the ambient ground-water temperature at the site. Groundwater and radio-labeled bacteria enter the core from the bottom andmove upward. Effluent is collected with fraction col-lectors and the radioactivity measured with a liquidscintillation counter. At the end of the experiment, thecore is split, the sediment subsampled and thin sec-tioned to determine distribution of adsorbed bacteria.

39

■ These kinetic parameter distributions orempirical functions will be used to con-dition the kinetic parameters for the 3Dmodel by stochastic analyses (see Task 3.5;Scheibe and Majer).

■ Inject conservative tracer and reactivetracers into select intact cores and moni-tor breakthrough curves. Split core,subsample, and determine concentrationof adsorbed reactive tracer. Determinedistribution of reactive tracer with respectto the Fe, Mn, and Al oxyhydroxides(Smith and Onstott).

■ Develop 1D advective/dispersion modelfor reactive tracers. Explore 2D and 3Dmodels if reactive tracer concentrationsin core are heterogenous. Use these dataand models to select an appropriate reac-tive tracer for field injection that will beaccepted by TNC and regulatory agen-cies (Smith).

3.3 Conservative/ReactiveTracer Transport Experimentsin Aerobic/Suboxic Flow Fields

Task Leader and CollaboratorsScheibe, Onstott, and Smith

Goals■ To assess the effects of physical/chemical

heterogeneity on the movement of waterand reactive constituents within theflow field.

■ To establish the relationships betweenheterogeneity in permeability and hetero-geneity in biogeochemical reactivity.

Approach■ Inject a conservative tracer (e.g., Br-)

at different depths, and monitor tracerbreakthrough in multilevel samplersto track groundwater flow across faciesboundaries (Griffin and Scheibe).

■ Model conservative tracer experimentsconditioned by measured permeabilityin intact cores, and the orientation andsize of sedimentological structures (Fig-ure 14). Measurements obtained byconducting intermediate-scale (tensof centimeters) permeability measure-ments (slug tests) at different intervalsin the injection borehole are used toobtain conditioning data for the physi-cal model (Griffin Scheibe, Swift, andMajer).

■ Conduct pump tests and inject reactivetracers (e.g., F-, Sr2+ or others) simulta-neously with a conservative tracer atdifferent depths and monitor tracer break-through in multilevel samplers to trackreactive transport across facies bound-aries. Model predictions will be com-pared to field observations to evaluatethe predictability of field-scale advectiontransport (Griffin, Smith, and Scheibe).

■ Model reactive tracer experiments condi-tioned by the measured spatial distribu-tions of Fe and Al oxyhydroxides andthe results of the conservative tracerexperiments to establish the integratedbiogeochemical reactivity along flowpaths. Based on the results of the tracerexperiments, select vertical intervals andtimes for sampling during the bacterialinjection experiment (see Task 3.4; Smith,Scheibe, and Onstott).

40

3.4 Field Injection ExperimentsPlanned for the NarrowChannel Aerobic Flow Cell

Task Leader and CollaboratorsDeFlaun, Griffin, Onstott, Swift, Majer,Holben, Balkwill, Dobbs, Ginn, Scheibe,Johnson, and White

Goals• To determine the effect of grain size and

hydraulic conductivity distribution andthe spatial distribution and type (compo-sition and crystallinity) of Fe, Mn, and Aloxyhydroxide phases on bacterial adhe-sion and transport.

• To understand the role of microbial adhe-sion to Fe, Mn, and Al oxyhydroxide sur-faces on bacterial transport.

Figure 14. Comparison of Br Breakthrough at One of the MLS’sDuring a Tracer Injection Experiment at Narrow Channel. Theminimum, average, and maximum curves represent the range ofpredicted Br breakthroughs from a 3D stochastic model of theNarrow Channel flow cell.

• To understand the influenceof Fe(II) adsorption to Fe,Mn, and Al oxyhydroxideand cell surfaces on micro-bial adhesion.

• To develop rapid and sensi-tive methods for quantifyingbacteria introduced to thesubsurface in groundwaterand sediment samples.

• To develop efficient scaling-up methodologies thatyield accurate predictionswhile economizing on datarequirements.

• To assess the importance ofbacterial predation on thereduction of microbial popu-lations introduced as part ofbioaugmentation strategies.

The Narrow Channel Focus Area offers sig-nificant opportunities for addressing theseobjectives, because 1) it contains sedimentsthat have a range of Fe(III), Al and to a lesserextent Mn oxyhydroxide contents, grain sizeand hydraulic conductivity distributions;2) the groundwater is aerobic and has apH 5.5 to 6, low DOC, and low ionic strength;and 3) anaerobic groundwater with elevatedDOC and Fe(II) is available nearby at theSouth Oyster Focus Area (see Table 1 in Sec-tion 1.2).

ApproachA series of field injection experiments havebeen planned that collectively will contrib-ute to the scientific goals identified in thistask (see Table A.1).

41

• The first experiment at the Narrow Chan-nel Focus Area will involve injection ofan adhesion-deficient, aerobic, hydro-philic, bacterium with neutral surfacecharge (Comamonas DA001 in Table 2).Multiple bacterial tracking tools(7) willbe used to quantify the transport of themicroorganism in the field (see Section 2).The resiliency, the detection limit, therapidity of analyses, and the economicsof implementation for each trackingtechnique will be compared in this experi-ment to determine which subset of tech-niques will be used in future fieldinjections. The variations in the grainsize and hydraulic conductivity distribu-tion at this site will provide insight intothe influence of these properties on bac-terial transport relative to that of a con-servative solute tracer, Br.

This experiment will also be the firstevaluation of model predictions of fieldtransport based on results of core-scalebacterial transport experiments comple-mented by field imaging of physicalheterogeneity using radar and seismictomography. The number and type ofindigenous protozoa will be determinedbefore, during and after the injection toascertain the potential impact of bactivoryon the breakthrough profiles of the intro-duced microorganism. If protozoanpredators are present, a subsequent fieldexperiment (see below) will be designedto ascertain the rates at which the protistcommunities respond to the injectedmicroorganisms.

• The second injection at the NarrowChannel Focus Area will include thesimultaneous injection of strain DA001and a negatively charged, facultativeIRB, that preferentially adheres to Fe-oxyhydroxides (see Table 2), into theaerobic flow cell. Multiple tracking tools

will be applied to delineate the transportbehavior of the two injected bacterialstrains.

The specific methods for labeling andindependently tracking each bacterialstrain will be selected on the basis oftheir performance during the first fieldinjection experiment. This experiment isalso designed to determine the relativeimportance of the spatial distributionand type (composition and crystallinity)of Fe, Mn, and Al oxyhydroxide phasesversus the sediment grain size andhydraulic conductivity distribution onbacterial transport.

Depending on the results of intact coreexperiments with the facultative IRB,additional measures may be explored tofacilitate the transport of the IRB. Thesemeasures include, for example, increas-ing the pH of the injectate water to 8 byequilibrating groundwater with air,reducing the ionic strength by dilutionwith distilled water, or co-injection withcolloids filtered and concentrated fromthe groundwater. The type and numberof indigenous protozoan communitieswill be monitored before, during, andafter the injection to further assess thepotential for bactivory.

• The third injection experiment at theNarrow Channel Focus Area will bedesigned to evaluate coupled bioaug-mentation and biostimulation approachesfor potential bioremediation strategies.Specifically, a facultative IRB enrichedfrom SOFA will be injected into the aero-bic flow cell. The oxidation potential of aportion of the flow cell will then be pro-gressively lowered by injecting a narrowplume of suboxic groundwater from theSouth Oyster Focus Area.

(7) The tracking tools will include IRMS, GC-IRMS, and HPLC-ESI-MS analyses of 13C labelled bacterial cells, platereader or cytometer counts of CFDA-stained cells, quantitative PCR, plate counts and ferrographic separa-tion immunoassay.

42

By reducing the injection rate relative tothe extraction rate, the dispersion of theplume will be less than that of the bacte-rial injection. A portion of the flow cell,therefore, will remain aerobic as a control.The transport of the facultative IRB, theconcentration of DOC, Fe(II), pH and tracemetals will be monitored as the oxidationpotential declines. The high concentra-tion of DOC (i.e., carboxylic acid portion)will act as an additional electron donorpromoting activity and growth of theindigenous bacterial populations. Growthof the IRB may enhance its transport.

These experiments will also determineif the DOC (i.e., humic acids portion)adsorbed to the positively charged Feand Al oxyhydroxides enhances the des-orption and transport of the IRB. Theconcentration of Fe(II) will be monitoredduring these manipulations as a proxyfor IRB activity. Select trace metals will bemonitored to determine if they are beingimmobilized as precipitates by the bio-genically produced Fe(II). Since growthof the indigenous bacterial populationsmay stimulate growth of the protozoancommunity feeding upon them, preda-tion rates may increase for the injectedIRB as well. Consequently, the numbersof protozoa will be monitored to deter-mine if their increase corresponds to anydecreased in injected IRB.

• The fourth injection experiment at theNarrow Channel Focus Area will entailcoinjection of the facultative IRB andanionic complexes. If the results ofprevious injections indicate that theadsorption of the facultative IRB to Fe-oxyhydroxides strongly inhibits thebacterium’s transport, then alternativemeans may be required to reduce theadsorption of the IRB.

Strongly binding anionic complexescompete with negatively charged bacte-ria for the positively charged sites on thesurfaces of the Fe and Al oxyhydroxides.

Injection of such a complex prior to andduring the injection of the facultative IRBstrain into the aerobic flow cell shouldreduce the adsorption of IRB.

Hypothetically, the anionic complexshould have little effect on the transportof neutrally charged DA001. Coinjectionof DA001 and the facultative IRB withthe anionic complex should enhance thetransport of the IRB relative to that ofDA001 and Br. Na-pyrophosphate mayrepresent one candidate complex, butbecause is solublizes Fe(III) phases, itsimpact on sediment mineralogy has tobe evaluated.

Any anionic complex will be tested inintact core experiments prior to propos-ing their use at the Oyster site to theVaDEQ. The bacterial injection will befollowed by injection of suboxic SouthOyster groundwater to lower the oxida-tion potential into the Fe(III) reductionregime. The total IRB activity will bedetermined to assess the net affect ofadding the anionic complexes to the sys-tem. As before, a portion of the flow cellwill be kept aerobic as a control.

• The fifth injection experiment at the Nar-row Channel Focus Area will use theobservations on protozoan populationsduring the previous injections and asequential injection strategy for DA001that will test models of bactivory. Thisexperiment will monitor how rapidlythe indigenous protozoan populationresponds to the introduction of DA001and how quickly the population dimin-ishes after the departure of DA001.

The rise and decline of the protozoanpopulation during the subsequent injec-tion will be compared to the first injectionto determine the optimal frequency ofbacterial injections. These results shouldalso assess the impact of the protozoanbactivory on the cost versus potentialbenefit arising from multiple injections.

43

• The sixth injection experiment at the Nar-row Channel Focus Area will examine theimportance of bacterial size in enhancingbacterial transport. The higher velocityof DA001 with respect to Br observed inthe intact core experiments is hypoth-esized to be caused by the preferentialexclusion of DA001 from fine-grainedzones within the sedimentary fabric.As this exclusion is a size-dependent phe-nomena, decreasing the size of DA001should reduce its average velocity. Col-loid filtration theory also predicts that theamount of adsorption is size dependent.DA001 will be injected simultaneouslywith a dwarf strain of DA001 into theaerobic flow cell to monitor their relativevelocities and amounts of adsorption.

3.5 Integration of Field andLaboratory Data into a High-Resolution 3D NumericalSimulation of Field Data

Task Leader and CollaboratorsScheibe, DeFlaun, Onstott, Ginn, Holben,Majer, Swift, and Murray

Goals■ To construct a high-resolution 3D sto-

chastic numerical model, representingbacterial transport in the experimentalflow cells, based on integrated laboratory-and field-scale data.

■ To use the model system in conjunctionwith the field experimental observationsto evaluate the effects of field-scale physi-cal and chemical heterogeneity and inparticular the existence of preferentialflow paths on field-scale bacterialtransport.

■ To test the predictability of field-scalebacterial transport based on laboratory-scale parameterization of attachment/

detachment models combined withfacies-scale characterization of physicalheterogeneity (scaling-up).

■ To provide a quantitative framework tosupport interpretation of field experimentsand for testing detailed hypotheses.

ApproachA 3D numerical framework representing non-reactive (Br) tracer transport and simulationtools has been developed. This frameworkhas been adapted to the South Oyster Site con-ditions and configuration. Laboratory resultsfrom intact core experiments have been usedto condition biogeochemical parameteriza-tion of the aquifer model and to expand itspredictive capabilities to bacterial (reactive)and non-reactive tracer transport.

Specific tasks are as follow:

■ Use the model framework to assist inexperimental design of the MLS array(Scheibe, Majer, and Deflaun).

■ Following emplacement of the flow cellsand geological and geophysical charac-terization, update the model to reflectsite-specific conditions (Scheibe, Swift,Majer, and Murray).

■ Use the updated model to predict Brbreakthrough at sampler locations andcompare to field observations to validatemodel performance (Scheibe, DeFlaun,and Onstott).

■ Develop a method based on intact coreexperimental results for assigning bacte-rial transport properties to grid cells ofthe numerical model (scaling-up;DeFlaun, Holben, Scheibe, and Onstott).

■ Use the numerical model to predict bac-terial breakthrough at sampler locationsand evaluate experimental hypothesesby comparison with field observations(DeFlaun, Holben, Scheibe, and Onstott).

45

Expected Results■ Development of new concepts for facilitat-

ing bacterial transport in the subsurface.

■ Quantification of the effect of bacterialadhesion on transport through aquifersediments.

■ Determination of the influence of reduc-tive dissolution of Fe oxyhydroxides byiron-reducing bacteria (IRB) on bacterialtransport in the field.

■ Determination of the effect of adsorbedmetals on bacterial adhesion and transport.

4.1 Screening for IRB/DIRB

Task Leader and CollaboratorsBalkwill, Fredrickson, and White

Goal■ Enrichment, isolation, and characteriza-

tion of facultative IRB or dissimilatoryIRB (DIRB) that can be injected into boththe aerobic and suboxic flow cells.

Approach■ Isolate (by enrichment culture) DIRBs for

use in field experiments (Fredrickson).

■ Screen existing Oyster culture collectionand new isolates (from Task 1.5) for IRBusing a soft agar indicator medium(Balkwill).

4.0 Environmental FactorsControlling BacterialTransport

■ Screen candidate IRB for the abilityto reduce South Oyster sediment Feand Mn oxyhydroxides (Fredricksonand Zachara).

■ Screen existing Oyster collection andnew isolates for presence of Shewanellastrains, using a genus-specific 16S rRNA-based gene probe (Balkwill).

■ Identify IRB strains that meet TNCrequirements for antibiotic resistance byscreening candidate strains for resistanceto eight different antibiotics: ampicillin,nalidixic acid, rifampicin, streptomycin,and kanamycin (50 mg/mL); gentamicinand erythromycin (30 mg/mL); and tet-racycline (12.5 mg/mL). Those resistantto the clinical antibiotics ampicillin,streptomycin, gentamicin, erythromycin,and tetracycline will be eliminated fromfurther consideration (Balkwill).

■ Develop alternative methods to enhancetransport including genetic engineeringto reduce cell size.(8)

4.2 Characterization ofFacultative IRB

Task Leader and CollaboratorsDeFlaun, Balkwill, Fredrickson, Zachara,and Fletcher

(8) This task is associated with the South Oyster Site but is not an integral part of the Bacterial Transport project.

46

Goal■ To identify and select strains with accept-

able antibiotic resistance profiles that havereduced adhesion for facilitated transport.

ApproachAdhesion screening and selection for adhe-sion variants are performed in a sand columnassay (DeFlaun et al. 1990). Percent adhe-sion as determined in this assay is generallyhigh for soil isolates (>90%). This assay selectsfor strains with <50% adhesion to an Ottawasand standard. Candidates from this screen-ing are tested further in the same assay withSouth Oyster Site sediment and selection fora less adhesive variant is performed. Thisselection involves use of the same sand col-umn assay, except that the variants areselected by repeated passage of the effluentfrom the column over subsequent columns.This enriches for cells that pass through thecolumn i.e., the non-adhesive variants. Thestability of the non-adhesive phenotype istested by growing strains of interest for morethan 100 generations in a non-selectivemedium and retesting for percent adhesion.

Sand column assays run under both aerobicand suboxic conditions will be used to deter-mine percent adhesion values for thesestrains. Percent adhesion will be comparedin sediments with and without ferrous iron.Adsorption of Fe to cells will be tested as ameans of facilitating transport. Other meansof facilitating transport, (i.e., humics, col-loids) will also be tested.

4.3 Survival of Facultative IRBin South Oyster SedimentMicrocosms

Task Leader and CollaboratorsBalkwill, Fredrickson, Holben, Matin, andWhite

Goals■ To determine the longevity of candidate

IRB at various population densities insite sediment and groundwater.

■ To determine the limit of detection byseveral methods (viable plate counts,stable isotopes, PCR).

ApproachMicrocosm survival studies will be conductedunder both aerobic and suboxic conditionsand will include studies with DIRB strains.Studies will assess the survival of strains thatare candidates for injection at high concen-trations under in situ conditions in the pres-ence of the indigenous microbial community.These microcosms will also test bacterialtracking methods. PCR-DGGE of eubacterialrDNA will provide a diversity fingerprint onthe same samples as the phospholipid fattyacid analysis.

4.4 Bacterial Transport UnderSuboxic Conditions

Task Leader and CollaboratorsDeFlaun, Onstott, Holben, Ginn, Scheibe,Zachara, and Fredrickson

Goals■ To relate mineralogical and chemical prop-

erties of South Oyster sediments (hetero-geneity) to transport of IRB.

■ To obtain retardation coefficients of IRBin aerobic and microbially reduced SouthOyster sediments.

ApproachThe primary purpose of these experiments isto obtain adsorption/desorption kineticparameters for candidate bacteria being

47

transported through aerobic South Oystersediment and sediment in which the Fe, Mnoxyhydroxide phases have been partially orquantitatively reduced.

Experiments will be conducted with faculta-tive IRB under both aerobic and suboxic con-ditions. The Fe, Mn oxyhydroxide phases inSouth Oyster sediments will be character-ized before and after biogenic reduction. Thebiogenically reduced sediments will then beused to assess the impact of Fe(III) reductionon bacterial transport in repacked sedimentcolumns.

Future studies will include evaluating theimpact of biogenic reduction in the presenceof natural humic acids on the transport ofbacteria in repacked and intact South Oystersediment cores. Humic acids can facilitatethe reduction of Fe, Mn oxyhydroxides byfunctioning as electron shuttles between thebacterial and oxide surfaces and by com-plexing Fe(II), preventing its absorption tooxyhydroxides and bacterial cells.

Experimental systems and methods include:

■ Kontes Chromaflex Chromatographycolumns (4.8 cm x 15 cm) are filled with410 gm of air-dried, pre-homogenizedOyster sediment. The columns are runin an upflow manner with groundwaterpumped through with a peristaltic pump.Flow rates analogous to those in theintact cores and in previous field experi-ments will be used (0.5 m to 2.0 m/day).These experiments will be performed ataverage South Oyster groundwater tem-perature (15°C).

■ Groundwater will be pumped throughthe columns for at least 24 h before pro-ceeding with conservative tracer tests.The conservative tracer will be used todetermine the exact groundwater flowrate through the column.

■ Radiolabeled bacteria will be injectedinto the influent flow and collected at theeffluent in a fraction collector. The num-ber of cells in the effluent will be deter-mined by scintillation counting.

4.5 Evaluation of Cell SurfaceCharacteristics

Task Leader and CollaboratorsFletcher, DeFlaun, Onstott, and Holben

Goal■ To determine physiological conditions

that modify surface properties related tobacterial adhesiveness.

Approach■ Relative adhesiveness of test strains will

be established by simple in vitro adhe-sion assays (DeFlaun).

■ Relative surface hydrophobicity andsurface charge of test strains will be deter-mined by hydrophobic interactionchromatography and electrostatic inter-action chromatography, respectively.Any relationships between adhesive-ness and surface property evaluationswill be identified (DeFlaun).

■ Changes in adhesiveness and relativesurface properties that alter relevantphysiological responses will be assessed.These include differences in strains thata) have been equilibrated to aerobic orsuboxic conditions or b) have been pre-pared for stable isotope incorporationand according to the injection protocol(and are therefore adapted to low nutri-ent conditions) and then are subjected tohigher nutrient levels, stimulating meta-bolic activity (DeFlaun, Holben, andFletcher).

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■ Compare surface charge of candidateaerobic and facultative IRB strains to thesurface charge of fine-grained diageneticFe, Mn, and Al oxyhydroxides that arepresent in the South Oyster sediments.This will be performed with Zetameterstreaming potential measurements andcapillary electrophoresis using SouthOyster groundwater. The data willbe used to calculate the double-layerthickness for reversible absorption tooxyhydroxides versus quartz (Onstott,DeFlaun, and Fletcher).

4.6 Role of Humics in BacterialTransport

Task Leader and CollaboratorMcCarthy and Fuller

Goals■ Determine if humics alter the retention

of bacteria to mineral surfaces througheffects on surface potentials or blockingof sorption sites.

■ Evaluate the redox behavior of humicsand their potential effects on bacterialtransport through humic-enhancedreduction of mineral oxides.

Approach■ Humics in groundwater at South Oyster

will be characterized with respect totheir chemical composition to evaluatedifferences in organic matter between theaerobic and microaerophilic conditions.

■ The composition of the South Oysterhumics will be compared with that ofthe NABIR reference humics to facilitatetransfer of information from investiga-tors working with the reference humicsto observations at South Oyster.

■ The transport and retention of humics inSouth Oyster sediments will be meas-ured in batch and column experiments toevaluate the extent of humic interactionswith sediments and the potential of usinghumics to facilitate bacterial transport.Measurements of streaming potential orelectrophoretic mobility will be used todetermine the effect of humics on surfacecharge of the sediments.

■ Effects of humics on bacterial transportwill be evaluated first by screening humic-coated sediments in repacked columnadhesion assays and then by core-scaleexperiments similar to those described inTask 3.1. Results will relate the extent ofhumic retention and transport to themobility of bacteria.

■ The redox behavior of humics will beevaluated in batch potentiostat experi-ments to evaluate the reduction capacityof humics under different Eh potentials.These data will provide an initial indica-tion of humic-bacteria-iron oxide interac-tions under varying redox conditionsduring experiments with IRBs.

4.7 Intact Core Studies withFacultative IRB

Task Leader and CollaboratorsDeFlaun, Onstott, Holben, Zachara, Ginn,Scheibe, and Fredrickson

Goal■ To compare transport of the same strain

of facultative IRB in intact cores collectedfrom the aerobic and suboxic flow cellsunder controlled laboratory conditions.

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Approach■ Intact cores will be collected in both the

aerobic and suboxic sites at South Oysterduring construction of the flow cells.Care will be taken to preserve the redoxconditions in the cores during collectionand transport to the laboratory (DeFlaun,Onstott, Holben, and Griffin).

■ A strain of facultative IRB will be usedin transport experiments in these coresto compare transport in the aerobicand suboxic sediments with the samemicroorganism. These experiments willbe performed under conditions that mostclosely mimic field conditions (DeFlaun,Onstott, and Holben).

■ Intact core experiments will be performedas described in Task 3.1. Groundwaterfrom the two flow cells will be used inthese experiments. The intact core fromthe suboxic flow cell will be kept at lowredox values throughout the experiments(DeFlaun, Onstott, and Holben).

4.8 Field Injection ExperimentsPlanned for the South OysterSuboxic Flow Cell

Task Leader and CollaboratorsDeFlaun, Griffin, Onstott, Swift, Majer,Holben, Balkwill, Dobbs, Ginn, Scheibe,Johnson, and White

Goals• To determine the effect of grain size and

hydraulic conductivity distribution andthe amount and type of Fe, Mn, and Aloxyhydroxide on bacterial transport.

• To understand the role of IRB adhesionto Fe, Mn, and Al oxyhydroxide surfaceson IRB transport and activity.

• To determine the effect of IRB activity andbacterially produced Fe(II) on the mobili-zation and precipitation of trace metals.

• To determine the effect of bacterially pro-duced Fe(II) on IRB transport.

• To adapt and refine the bacterial trackingtools for suboxic environments.

• To assess the importance of bactivory insuboxic environments and designingeffective bioaugmentation strategiesthat minimize the detrimental impactof bactivory.

The South Oyster Focus Area (SOFA) offers anexcellent opportunity for fulfilling these objec-tives, because 1) it contains Fe-oxyhydroxidecoated sediments of varying Fe content, grainsize and hydraulic conductivity distribution;2) the Fe(II)/Fe(III) of the sediments is vari-able within the flow cell; 3) the groundwaterhas varying, but low O2 concentrations andoxidation potentials; 4) the groundwater hasa subneutral pH, high DOC, and low ionicstrength (see Table 1 in Section 1.2); and 5) theconcentrations of certain trace metals, Ni, Co,and Zn, are correlated with Fe(II), whereasthose of other trace metals, e.g., Sn, areinversely correlated with Fe(II).

ApproachA series of field injection experiments havebeen identified that collectively will contrib-ute to these scientific goals (see Table A.1).

• The first field injection at SOFA willemploy an adhesion deficient, facultativeIRB that will be co-injected with Br intothe suboxic flow cell under suboxic con-ditions. This bacterial injection experi-ment will provide the first field test ofmultiple bacterial tracking tools undersuboxic conditions. It will also be the firstfield injection of the IRB and will seek toverify numerical model predictions based

50

upon core-scale, suboxic bacterial trans-port experiments in conjunction withfield-scale physical heterogeneity deter-mined by radar and seismic tomography.Intact core experiments performed usingSOFA sediments and the candidateIRB may predict that detectable break-through of the IRB at the MLS’s of theflow cell cannot be achieved withoutemploying additional measures. Thesemeasures include increasing pH to 8 byequilibrating groundwater with air,reducing the ionic strength by dilutionwith distilled water, or co-injection withcolloids filtered and concentrated fromthe groundwater. The intact core experi-ments will be used to evaluate which ofthese measures will be implemented inthe field. The number and type of indig-enous protozoa will be measured before,during, and after the injection to assess,for the first time, the field rate of bactivoryin suboxic conditions.

• During the second field injection at SOFAthe facultative IRB will be co-injectedwith DA001. As DA001 is most sensitiveto physical heterogeneity (i.e., grain sizeand hydraulic conductivity distribution),this injection is designed to determinethe relative importance of spatial distri-bution and type (composition and crys-tallinity) of Fe, Mn, and Al oxyhydroxidephases versus the sediment grain sizeand hydraulic conductivity distributionon IRB transport.

This experiment will also be compared tothe results of a similar experiment per-formed at the Narrow Channel flow cellwhere the chemical and physical hetero-geneity of the Narrow Channel sedimentsdiffer somewhat from those of SOFA (seeTable 1). The number and type of indig-enous protozoa will be quantified before,during and after the injection to furtherconstrain the field rate of bactivory.

• The third experiment at SOFA will bedesigned to simulate potential bioreme-diation approaches that combine bioaug-mentation with biostimulation strategies.The facultative IRB strain used in the pre-vious field experiments will be injectedinto the suboxic flow cell. The more oxicportions of the SOFA flow cell will betargeted with an injection of groundwa-ter with the highest DOC from the flowcell to lower the oxidation potential ofthe targeted zone.

The portions of the flow cell untouchedby the low O2, low Eh plume will bemonitored as controls. The transport ofthe facultative IRB, the concentration ofDOC, Fe(II), pH and trace metals will bemonitored as the oxidation potentialdeclines. These experiments will deter-mine if the DOC enhances IRB activity.The concentration of Fe(II) will be moni-tored during these manipulations as aproxy for IRB activity. The high concen-tration of DOC (i.e., carboxylic acid por-tion) will also promote growth of theindigenous bacterial populations. Growthof the IRB may enhance its transport.

Select trace metals will be monitoredto determine if they are being immobi-lized as precipitates by the biogenicallyproduced Fe(II). These experiments willalso determine if the DOC (i.e., humicacids portion) adsorbed to the posi-tively charged Fe and Al oxyhydroxidesenhances the desorption and transportof the IRB. Since growth of the indig-enous bacterial populations may stimu-late growth of the protozoan communityfeeding upon them, predation rates mayincrease for the injected IRB as well. Con-sequently, the number and type of proto-zoa will be monitored to determine if theirincrease corresponds to any decrease inthe injected IRB.

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• The fourth experiment at SOFA is designedto investigate the effect of enhanced IRBtransport on total IRB activity. If theresults of previous injections indicatethat the adsorption of the facultative IRBto Fe, Mn and Al oxyhydroxides undersuboxic conditions strongly inhibits thebroad dissemination of the IRB through-out the flow cell, then alternative meansmay be required to reduce the adsorptionof the IRB. Strongly binding anionic com-plexes compete with negatively chargedbacteria for the positively charged siteson the surfaces of the Fe, Mn, and Aloxyhydroxides. Injection of such a com-plex (see Section 3.4) before and duringthe injection of the facultative IRB straininto the aerobic flow cell should reducethe adsorption of IRB. Hypothetically,the anionic complex will have little effecton the transport of neutrally chargedDA001. Coinjection of DA001 and thefacultative IRB with the anionic complexshould enhance the transport of the IRBrelative to that of DA001 and Br. Theeffect of the anionic complex on net IRBactivity (Fe[III] reduction) will also bemonitored.

• The fifth injection experiment at SOFAwill use the observations on protozoanpopulations during the previous injec-tions, the experiments performed atNarrow Channel and a sequential injec-tion strategy for the IRB to determinewhether the conceptual models of bacti-vory in oxic settings apply to suboxicenvironments. This experiment willdetermine how rapidly the indigenousprotozoan population responds to theintroduction of the IRB and how quicklythe population diminishes after thedeparture of the IRB.

The rise and decline of the protozoanpopulation during the subsequent injec-tion will be compared to that of the first

injection. A frequency for bacterial injec-tions will be derived which minimizesthe impact of bactivory and enhancesIRB total activity. The rates of bactivoryderived from this experiment will beused to assess the impact of protozoanbactivory on the cost versus potentialbenefit arising from multiple injections.

• The sixth injection experiment at SOFAwill examine how the relative size of IRBto the pore size affects IRB transport andactivity. Petrographic observations haverevealed that the Fe, Mn, and Al oxyhy-droxides are concentrated in the finergrained portions of the sediment. If thesefine-grained layers are physically lessaccessible to the normal-size, facultativeIRB than to a dwarf strain of IRB, and thenatural humic acids are not acting aselectron shuttles, then the total IRB activ-ity may be greater following the injectionof dwarf cell IRB. To determine whethersize is important, the facultative IRB andthe dwarf strain will be coinjected andthe relative rates of migration monitored.IRB activity will be evaluated by meas-urements of Fe(II) in the groundwaterand related to the distribution of the fac-ultative IRB and dwarf IRB adsorbed tothe sediment.

4.9 Intermediate Flow CellExperiments

Task Leader and CollaboratorsMurphy, Ginn, Scheibe, and Zachara

Goals■ To determine the effect of geochemical

heterogeneity on bacterial transport.

■ Intermediate-scale experiments will beused to test specific theories for scaling

52

up process-based modules. The stochasticconvective reaction model works wellwith nonlinear reactions that are charac-teristic of biological systems. The ISEprovides a complete data set to rigor-ously test this theory before applying itto a less-controlled field environment.

ApproachIRB cause dynamic concentrations of aqueousFe(II). This bioreaction may produce tran-sient transport events to occur in the IRBpopulation through adsorption of the Fe(II)with IRB extracellular polysaccharides ormembrane components (e.g., polar lipids)and/or reaction of Fe(II) with mineralogicalcomponents of the solid-phase. These proc-esses may facilitate or retard IRB transportin porous media in the short term. In thelong term, IRB activity should decrease theFe(III) mineral content and enhance IRBtransport. To test these hypotheses in anintermediate-scale experiment, informationis needed to characterize the reactions ofFe(II) with IRB and mineralogical surfaces:

■ How does Fe(II) reaction with IRB changethe surface charge properties of the cells?One experimental approach may be tomonitor electrophoretic mobility of IRBwith increasing concentrations of Fe(II).

■ What are the transport properties(attachment/detachment kinetics) of IRBin selected mineralogies when surfacesites on the IRB are saturated with Fe(II)compared to IRB where Fe(II) is absent?

■ What are the transport properties(attachment/detachment kinetics) of IRBwhen the Al and Fe(III) oxyhydroxidecontain adsorbed Fe(II)?

Pacific Northwest National Laboratory hasestablished an intermediate-scale flow cellfacility for collaborative research at EMSL.

This facility is being used to perform experi-ments in support of this and other NABIRprojects.

■ Prior to the design of the intermediate-scale flow cell experiment for this project,a column packed with a homogeneousmineralogy of quartz, Al oxyhydroxide,or Fe(III) oxyhydroxide-coated sandwill be used to monitor the breakthroughof an IRB pulse under different Fe(II)treatments.

■ An IRB strain either from the Oyster siteor an existing strain of IRB (e.g., Shewanellaputrifaciens CN32) will be chosen forthese column experiments and theexperiments completed.

■ The effect of Fe(II) on electrophoreticmobility of the IRB will be determined toexamine the mechanisms responsible forthe behavior observed in the columns.

■ Kinetic parameters from these columnexperiments will then be used to designthe intermediate-scale flow cell andrefine the experimental hypothesis.

■ Intermediate-scale flow cell transportexperiments will also be used to test theeffect of spatial heterogeneity on bacte-rial transport and the veracity of theintact core experiments.

Affiliated NABIR Task: SpatialHeterogeneity of Microbial FeReduction Potential

Task Leader and CollaboratorsMurray, Roden, Swift, Majer, Hubbard,Gorby, and Brockman

53

Goal■ Develop the ability to predict microbial

Fe reduction potential (MIRP) at unsam-pled locations using geostatistical meth-ods that integrate the spatial model forMIRP and geological/geophysical infor-mation on the distribution of sedimen-tary facies.

Approach■ Identify a batch measurement that can

be used as a proxy to characterize thespatial distribution of in situ MIRP.

■ Employ geostatistical methods to charac-terize the spatial heterogeneity of MIRPand relate it to the spatial distribution ofenvironmental properties.

■ Use a combination of batch and intactcore microcosm incubations to developan inexpensive measure to assess thepotential for MIRP in sediments. Theincubations would be performed usinga medium consisting of electron donors,inorganic nutrients, vitamins, and traceminerals designed to selectively promotemicrobial Fe reduction. The batch meas-urements will be performed on samplesfrom vertical boreholes at the Oyster orAbbott Site.

Geostatistical techniques will then be usedto characterize the spatial heterogeneityof the MIRP and its relationship to sedi-mentary facies. Assuming that a relation-ship exists, geological and geophysicaldata on facies distributions and the spa-tial heterogeneity models will be usedto predict the distribution of MIRP atunsampled locations.

Affiliated NABIR Task:Vibration-Accelerated Transportof Microbes in Subsurface Media

Task LeaderPhelps

GoalDevelop and demonstrate a conceptual frame-work for vibration-facilitated microbial trans-port through subsurface porous media byexamining the effects of vibrational energieson particulate and microbial transport usingintact core columns and by field testinghypotheses for accelerated transport inporous media.

Approach■ Examine effects of vibration energies on

particulate and microbial transport usingintact sediment cores recovered from theEastern Shore.

■ Determine processes and variables con-trolling vibration-facilitated microbialtransport in laboratory-based columnstudies.

■ Identify the impacts of frequency, power,sediment structure, and vibration durationon transport processes using intact cores.

■ Using similar sites, organisms, and pro-cedures to those used by the South OysterField Experimentation Program, con-duct field applicability test of vibration-accelerated microbial transport.

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5.0 Technology Transfer

Opportunities

Goal

■ To promote transfer of new concepts,information, and research tools to DOEgroups responsible for site remediationand to industry.

Approach

It is anticipated that new, field-relevantinformation, research methods, and noveltechnologies will emerge from research atthe South Oyster Site. Experiments at thesite are being conducted under aerobic andsuboxic conditions that are relevant to theenvironments commonly associated withcontaminated groundwater. Spinoffs fromthese experiments will benefit remedialaction programs at contaminated sites underDOE stewardship.

Intrinsic bioremediation offers a viable optionat a number of DOE sites, but in situ micro-bial populations at some locations are limitedin numbers, especially in thick vadose zonesand deep aquifers in the western UnitedStates. Under these conditions, bioaugmen-tation will be needed to accelerate in situbioremediation activity by increasing micro-bial numbers.

Even at sites where microbial numbers aregenerally elevated, it is known that micro-bial spatial distribution (microbial heteroge-neity) in the subsurface is controlled bynatural hydrogeologic variations such thatinjection of bacteria may be needed to ensurethat isolated zones of contamination, includ-ing poorly permeable regions and pockets of“dead end” pore space can be treated. Givennational concern regarding the release of

genetically engineered bacteria to the environ-ment, the reintroduction of strains obtainedfrom indigenous microbial communitiesmay be an economically and environmen-tally viable approach. This conceptualapproach underpins the field experimentsbeing conducted at the South Oyster Site.

5.1 Seek Opportunities for

Research Transfer

The most significant opportunities for tech-nology transfer are likely to emerge follow-ing analysis of results from field campaigns.As research proceeds, and especially duringthe conduct of annual investigator “stock-taking meetings,” progress will be assessedwith the goal of identifying research resultsthat may contribute to ongoing and antici-pated DOE remediation efforts.

Idaho National Engineering and Environ-mental Laboratory-Test Area North (INEEL-TAN) represents a potential opportunity fortransfer of research results (Smith and Colwell1998). At INEEL-TAN, intrinsic bioremedia-tion is targeted for a zone of sewage-metal-TCE contamination, with the goal of reducingcleanup costs in a fractured bedrock aquifer.The Record of Decision permits the use ofnew and emerging research approaches atthe site. INEEL and University of Idaho sci-entists are seeking to exploit in situ microbialcommunities that may prove to be insufficientin distribution, numbers, and/or activity toremediate the contaminant plume. Anotheropportunity exists at the Hanford Site,where injection of indigenous, iron-reducingmicroorganisms and a simple electron donormight serve as a cost-effective, less disruptive,

56

and safer alternative to chemical injection,which has been shown to modify redox con-ditions and attenuate the movement of chro-mium in an initially aerobic aquifer. Theapplicability of this microbially basedapproach is being explored as a means ofattentuating chromium, uranium, and tech-netium mobility at the Hanford 100-H Areawhere these elements are impinging onsalmon redds in the Columbia River.

Opportunities for research transfer also existat other sites at which intrinsic bioremedia-tion is planned in zones with complex geologyand where a need may exist to supplementindigenous populations with bacterialamendments.

5.1.1 Facilitate General Site

Remediation Problem Solving

Field-tested methods and technologies withbroad applications to remediation across theDOE site complex potentially include thefollowing:

■ Field protocols for core and groundwatersampling and analysis under aerobic andsuboxic conditions, which also retain themicrobiological integrity of samples.

■ Core experimentation and modelingapproaches for scaling mechanistic infor-mation, such as microbial attachment/detachment, to the field.

■ A suite of bacterial markers to tag andtrack bacteria, and for examination ofin situ microbial ecology, such as changesin community structure and dynamicsthat result from manipulation.

■ Field-tested, low-cost multi-level sam-plers for mid- to long-term groundwatermonitoring.

■ Three-dimensional models for design andanalysis of field injection experiments.

5.1.2 Complete Transfers Relevant

to Bioaugmentation at DOE Sites

At DOE sites where bioaugmentation isanticipated, examples of research transfer fromfield research experiments at South Oystermay include any or all of the following:

■ Innovative protocols for identifyingadhesion-resistant strains from amongin situ populations at laboratory (core)and field scales.

■ High throughput methods for screeningand culturing indigenous bacterialvariants for bioaugmentation via aqui-fer injection.

■ Combined (tracer, bacterial marker)approaches for tracking cells in the sub-surface and monitoring locations andrates of movement within contaminatedzones.

■ Predictive, field-scale bacterial transportmodels that address natural physical andgeochemical heterogeneity and are appli-cable across a range of sedimentologicaland geochemical regimes.

The South Oyster Site also provides an oppor-tunity to study the microbial ecology of anatural redox gradient in which N and Fecycling occurs under suboxic conditions. Asa result, this site could provide field valida-tion of laboratory studies and source materi-als for the “Biogeochemical DynamicsElement” and other elements of the NABIRprogram.

5.1.2 Complete Transfers Relevant

to In Situ Remediation Technologies

Remedial action programs at DOE and othersites include establishing, maintaining, and/or enhancing the longevity of in-ground,permeable biotic or abiotic (e.g., zero-valence

57

Fe) barriers to contaminant migration.Spinoffs may lead to new or improved bar-rier technology that may be useful at DOEsites. Opportunities include the following:

■ Field-tested methods for conductingforced-gradient injections under aerobicand suboxic groundwater conditions.

■ New discrete-interval tracer injectiontechnologies to predict bacterial, andpossibly chemical, dispersion withinaquifers and for monitoring effective-ness of installed permeable barriers.

■ New evaluation methods that predictthe adhesion of metal-reducing bacte-ria to Fe mineral surfaces—informationuseful to predict the potential for trans-port of these organisms through Feoxide-containing sediments, the micro-bial reductive dissolution of Fe oxide-contaminant coprecipitates, and themicrobial reduction of structural Fe forcontaminant attenuation.

5.2 Design and Test Methods

for Information Transfer

A future mechanism of technology transferis likely to be through the NABIR Field

Research Centers at DOE sites where bio-augmentation may be a necessary part ofin situ remediation. As the research proceedsat the South Oyster site, results will also betransferred as informational briefs to all DOEsites. Other transfer vehicles that have beenshown to be suitable include onsite work-shops and training of DOE site personnel atthe South Oyster Site itself.

5.3 Public/Community

Outreach Program

Insights into public concerns about bioreme-diation may be gained as a result of interac-tions with local environmental groups, stateand county officials, other local organiza-tions, and the citizens of coastal Virginia.These insights may range from determiningthe public acceptance of in situ bioremedia-tion as a cleanup tool to improved methodsfor effective communication of field researchactivities.

This experience will be useful to thoseinvolved in public outreach at DOE sites.Although no formal program of public par-ticipation or outreach is planned, any experi-ence that is generated at the South Oystersite will be transferred to NABIR’s BASICProgram.

59

Bales, R.C., C.P. Gerba, G.H. Grondin, andS.L. Jensen. 1989. Bacteriophage Transport inSandy Soil and Fractured Tuff. Appl. Environ.Microb. 55:2061-2067.

Bales, R.C., Jim Li Shimin, T.C. Yeh, M.E.Lenczewski, and C.P. Gerba. 1997. Bacte-riophage and Microsphere Transport inSaturated Porous Media: Forced-GradientExperiment at Borden, Ontario. Water Res.Res. 33:639-648,

Baygeants, J.C., J.R. Glynn, Jr., O. Albinger,B.K. Biesemeyer, K.L. Ogden, and R.G.Arnold. 1998. Variation of Surface ChargeDensity in Monoclonal Bacterial Populations:Implications for Transport Through PorousMedia. Environ. Sci. Technol. 32:1596-1603.

Bellamy, W.D., D.W. Hendricks, and G.S.Logsdon. 1985. Slow Sand Filtration: Influ-ences of Selected Process Variables. J. Am.Water Well Assoc. 12:62-66.

Buddemier, R.W., and J.R. Hunt. 1988. Trans-port of Colloidal Contaminants in Ground-water: Radionuclide Migration at the NevadaTest Site. Appl. Geochem. 3:535-548.

Caccavo, Jr., F., N.B. Ramsing, and J.W.Costerton. 1996. Morphological and MetabolicResponses to Starvation by the DissimilatoryMetal-Reducing Bacterium Shewanella algaBrY. Appl. Environ. Micro. 62:4678-4682.

Caccavo, Jr., F., P.C. Schamberger, K. Keiding,and P.H. Nielsen. 1997. Role of Hydropho-bicity in Adhesion of the DissimilatoryFe(III)-Reducing Bacterium Shewanella Algato Amorphous Fe(III) Oxide. App. Environ.Microbio. 63:3837-3843.

References

Camesano, T.A., and B.E. Logan. 1998. Influ-ence of Fluid Velocity and Cell Concentra-tion the Transport of Motile and NonmotileBacteria in Porous Media. Environ. Sci.Technol. 32:1699-1708.

Champ, D.R., and J. Shroeter. 1988. BacterialTransport in Fractured Rock—A Field ScaleTracer Test at the Chalk River Nuclear Labo-ratories. Wat. Sci. Tech. 20:81-87.

Coston, J.A., C.C. Fuller, and J.A. Davis.1995. Geochim. Cosmochim. Acta 59:3535-3547.

Davis, J.A. 1982. Adsorption of DissolvedNatural Organic Matter at the Oxide/WaterInterface. Geochim. Cosmochim. Acta 46:2381-2393.

Davis, S.N., D.J. Campbell, H.W. Bentley,and T.J. Flynn. 1985. Ground Water Tracers,Chap. 4. National Water Well Association 61-66.

DeFlaun, M.F., C.J. Murray, W. Holben,T. Scheibe, A. Mills, T. Ginn, T. Griffin,E. Majer, and J.L. Wilson. 1997. PreliminaryObservations on Bacterial Transport in aCoastal Plain Aquifer. FEMS Microbiol. Revs.20:473-487.

DeFlaun, M.F., S.R. Oppenheimer, S. Streger,C.W. Condee, and M. Fletcher. 1999. Alter-ations in Adhesion, Transport, and MembraneCharacteristics in an Adhesion-DeficientPseudomonad. Appl. Environ. Microbiol.65:759-765.

60

DeFlaun, M.F., A.S. Tanzer, A.L. McAteer,B. Marshall, and S.B. Levy. 1990. Develop-ment of an Adhesion Assay and Character-ization of an Adhesion-Deficient Mutant ofPseudomonas fluorescens. Appl. Environs.Microbiol. 56:112-119.

DeFlaun, M.F., C.J. Murray, W. Holben,T. Scheibe, A. Mills, T. Ginn, T. Griffin,D. Majer, and J.L. Wilson. 1997. PreliminaryObservations on Bacterial Transport in aCoastal Plain Aquifer. FEMS MicrobiologyReviews 20:473-487.

Dong, H., T.C. Onstott, M.F. DeFlaun,M. Fuller, K. Gillespie, and J.K. Fredrickson.1999. Development of Radiographic andMicroscopic Techniques for the Character-ization of Bacterial Transport in Intact Sedi-ment Cores from Oyster, Virginia. J. Microbiol.Methods 37:139-154.

Dybas, M.J., M. Barcelona, S. Bezborodnikov,S. Davies, L. Forney, H. Heuer, O. Kawka,T. Mayotte, L. Sepulveda-Torres, K. Smalla,M. Sneathen, J. Tiedje, T. Voice, D.C. Wiggert,M.E. Witt, and C.S. Criddle. 1998. Pilot-ScaleEvaluation of Bioaugmentation for In-SituRemediation of a Carbon Tetrachloride-Contaminated Aquifer. Environ. Sci. Technol.32:3598-3611.

Fletcher, M. 1977. The Effects of CultureConcentration and Age, Time, and Tempera-ture on Bacteria Attachment to Polystyrene.Can. J. Microbiol. 23:1-6.

Fontes, D.E., A.L. Mills, G.M. Hornberger,and J.S. Herman. 1991. Physical and Chemi-cal Factors Influencing Transport of Microor-ganisms Through Porous Media. Appl.Environ. Microbiol. 57:2473-2481.

Fuller, M.E., M.F. DeFlaun, and T.C. Onstott.1999. Development and Evaluation of Fluo-rescent Labeling Techniques for TrackingBacteria Injected into Subsurface Environ-ments. Abstracts of the 99th General Meeting

of the American Society for Microbiology,May 30-June 3, 1999. Chicago, Illinois.

Fredrickson, J.K., J.M. Zachara, D.W. Kennedy,H. Dong, T.C. Onstott, N.W. Hinman, andLi Shu-mei. 1999. Biogenic Iron MineralizationAccompanying the Dissimilatory Reductionof Hydrous Ferric Oxide by a GroundwaterBacterium. Geochim. Cosmochim. Acta62:3239-3257.

Fuller, M.E., K.M. Gillespie, H. Dong, B.S.Parsons, T.C. Onstott, and M.F. DeFlaun.Examining Bacterial Transport in Intact Coresfrom Oyster, Virginia: Effect of Facies Typeon Bacterial Breakthrough and Retention.Microbial Ecology (Submitted 1999).

Fuller, M.E., S.H. Streger, R. Rothmel, H. Dong,and M.F. DeFlaun. Staining and DetectionProtocols for Bacterial Cells Labeled withViable Fluorescent Stains. J. Bacterio. (Inpreparation).

Ginn, T.R. 1995. A Brief Review of BacterialTransport in Natural Porous Media. PNL-10876.Pacific Northwest Laboratory, Richland,Washington.

Glynn, Jr., J.R., B.M. Belongia, R.G. Arnold,K.L. Ogden, and J.C. Baygents. 1998. Capil-lary Electrophoresis Measurements of Elec-trophoretic Mobility for Colloidal Particlesof Biological Interest. App. Environ. Microbio.64:2572-2577.

Gorby, Y.A., J.E. Amonette, and J.S. Fruchter.1994. Remediation of Contaminated Subsur-face Materials by a Metal-Reducing Bacterium,In: G.W. Gee and N.R. Wing eds. In-SituRemediation: Scientific Basis for Current andFuture Technologies, Battelle Press, Columbus.

Harvey, R.W., and S.P. Garabedian. 1991. Useof Colloid Filtration Theory in ModelingMovement of Bacteria Through a Contami-nated Sandy Aquifer. Environ. Sci. Technol.25:178-185.

61

Harvey, R.W., N.E. Kinner, D. MacDonald,D.W. Metge, and A. Bunn. 1993. Role ofPhysical Heterogeneity in the Interpreta-tion of Small-Scale Laboratory and FieldObservations of Bacteria, Microbial-SizedMicrosphere, and Bromide Transport ThroughAquifer Sediments. Water Res. Res. 29:2713-2721.

Harvey, R.W., L.H., George, R.L. Smith, andR.D. LeBlanc. 1989. Transport of Microspheresand Indigenous Bacteria Through a SandyAquifer: Results of Natural and Forced Gra-dient Tracer Experiments. Environ. Sci.Technol. 23:51-56.

Hendricks, D.W., F.J. Post, and D.R. Khairmar.1979. Adsorption of Bacteria on Soils. WateAir Soil Pollut. 12:219-232.

Johnson, W.P., K.A. Blue, B.E. Logan, andR.G. Arnold. 1995. Modeling Bacterial Detach-ment During Transport Through PorousMedia as a Residence-Time-Dependent Pro-cess. Water Res. Res. 31:2649-2658.

Johnson, W.P., and B.E. Logan. 1996. EnhancedTransport of Bacteria in Porous Media bySediment-Phase and Aqueous-Phase NaturalOrganic Matter. Water Res. Res. 30:923-931.

Johnson, W.P., J.J. Martin, M.J. Gross, andB.E. Logan. 1996. Facilitation of BacterialTransport Through Porous Media by Changesin Solution and Surface Properties. Colloidsand Surfaces A: Physicochem. Engineer.Asps. 107:263-271.

Jucker, B.A., A.J.B. Zehnder, and H. Harms.1998. Quantification of Polymer Interactionsin Bacterial Adhesion. Environ. Sci. Technol.32:2909-2915.

Knapp, E.P., J.S. Herman, G.M. Hornberger,and A.L. Mills. 1998. The Effect of Distribu-tion of Iron-Oxyhydroxide Grain Coatingson the Transport of Bacterial Cells in PorousMedia. Environ. Geo. 33:243-248.

Kinner, N.E., R.W. Harvey, andM. Kazmierkiewica-Tabaka. 1997. Effect ofFlagellates on Free-Living Bacterial Abun-dance in an Organically Contaminated Aqui-fer. FEMS Microbiology Reviews 20:249-259.

Knapp, E.P., J.S. Herman, G.M. Hornberger,and A.L. Mills. 1998. The Effect of Distribu-tion of Iron-Oxyhydroxide Grain Coatingson the Transport of Bacterial Cells in PorousMedia. Environ. Geol. 33:243-248.

Li, Q., and B.E. Logan. 1999. EnhancingBacterial Transport for Bioaugmentation ofAquifers Using Low Ionic Strength Solutionsand Surfactants. Wat. Res. 33:1090-1100.

Logan, B.E., D.J. Jewett, R.G. Arnold, E.J.Bouwer, C.R. O’Melia. 1995. J. Environ. Eng.121:869-873.

Martin, J.J., B.E. Logan, W.P. Johnson, D.G.Jewett, and R.G. Arnold. 1996. Scaling Bacte-rial Filtration Rates in Different Sized PorousMedia. J. Environ. Eng. May 1996:407-415.

McCarthy, J.F., B. Gu, L. Liang, J. Mas-Pla,T.M. Williams, and T.C.J. Yeh. 1996. FieldTracer Tests on the Mobility of NaturalOrganic Matter in a Sandy Aquifer. WaterRes. Res. 32:1223-1238.

McKay, L.D., J.A. Cherry, R.C. Bales, M.T.Yahya, and C.P Gerba. 1993. A Field Exampleof Bacteriophage as Tracers of Fracture Flow.Environ. Sci. Technol. 27:1075-1079.

Mills, A.L., J.S. Herman, G.M. Hornberger,and T.H. DeJesus. 1994. Effect of SolutionIonic Strength and Iron Coatings on MineralGrains on the Sorption of Bacteria Cells toQuartz Sand. App. and Environ. Microbiol.60:3300-3306.

Murphy, E.M., T.R. Ginn, A. Chilakapati,C. Thomas Resch, J.L. Phillips, T.W. Wietsma,and C.M. Spadoni. 1997. The Influence ofPhysical Heterogeneity on Microbial Degra-dation and Distribution in Porous Media.Water Res. Res. 33:1087-1103.

62

Neu, T.R. 1996. Significance of Bacterial Sur-face Active Compounds in Interaction ofBacteria with Interfaces. Microb. Rev. 60:151.

Oliver, J. 1993. Formation of Viable but Non-culturable Cells, In: Starvation in Bacteria(S. Kjelleberg, Ed.), p. 239. Plenum Press,New York, New York.

Parolin, C., A. Montecucco, G. Ciarrocchi,G. Pedrali-Noy, S. Valisena, M. Palumbo, andG. Palu. 1990. The Effect of the Minor GrooveBinding Adent DAPI (2-Aminodiphenyl-Indole) on DNA-Directed Enzymes: AnAttempt to Explain Inhibition of PlasmidExpression in Escherichia Coli. FEMS Micro-biology Letters 68:341.

Phillips, P., J. Bender, and C.F. Thornton.1999. Field-Scale Evaluation of MicrobialMats for Metal Removal in a Funnel-and-Gate Configuration. In: A. Leeson and B.C.Alleman eds. Bioremediation of Metals andInorganic Compounds, Battelle Press, Colum-bus, Ohio.

Rajagopalan, R., and Tien C. 1976. TrajectoryAnalysis of Deep Bed Filtration With theSphere-in-Cell Porous Media Model. AIChEJ. 22:523-533.

Rügge, K., P.L. Bjerg, J.K. Pedersen, H.Mosbaek, and T.H. Christensen. 1999. AnAnaerobic Field Injection Experiment in aLandfill Leachate Plume, Grindsted, Den-mark. 1. Experimental Setup, Tracer Move-ment, and Fate of Aromatic and ChlorinatedCompounds. Water Res. Res. 35:1231-1246.

Ryan, J.N., M. Elimelech, R.A. Ard, R.W.Harvey, and P.R. Johnson. 1999. Bacterioph-age PRD1 and Silica Colloid Transport andRecovery in an Iron Oxide-Coated SandAquifer. Environ. Sci. Technol. 33:63-73.

Scholl, M.A., and R.W. Harvey. 1992. Labora-tory Investigations on the Role of SedimentSurface and Groundwater Chemistry in

Transport of Bacteria Through a Contami-nated Sandy Aquifer. Environ. Sci. Technol.26:1410-1417.

Smith, R.W., and F.S. Colwell. l998. Test AreaNorth (TAN), Idaho National Engineering Labora-tory (INEEL) Microbial Ecology and Biogeo-chemistry Well Field. INEEL Paper, 12 pp.

Speiran, G.K. 1996. Geohydrology and Geo-chemistry Near Coastal Ground-Water-DischargeAreas of the Eastern Shore, Virginia. USGSWater-Supply Paper 2479, 73 pp.

Spielman, L.A., and S.K.J. Friedlander. 1974.Colloid Interface Sci. 46:22-31.

Steffan, R.J., K.L. Sperry, M.T. Walsh, S.Vainberg, and C.W. Condee. 1999. Field-Scale Evaluation of In Situ Bioaugmentationfor Remediation of Chlorinated Solvents inGroundwater. Environ. Sci. Technol. 33:2771-2781.

Sverjensky, D.A., and N. Sahai. 1996. Theo-retical Prediction of Single-Site Surface-Protonation Equilibrium Constants for Oxidesand Silicates in Water. Geochim. Cosmochim.Acta 6:3773-3797.

Thurman, E.M. 1985. Organic Geochemistryof Natural Waters. Martinhus Nijhoff/Junk,Dordrecht, The Netherlands.

Tipping, E., and D. Cooke. 1982. The Effectsof Adsorbed Humic Substances on the Sur-face Charge of Goethite in Freshwaters.Geochim. Cosmochim. Acta 46:75-80.

Toran, L., and A.V. Palumbo. 1992. ColloidTransport Through Fractured LaboratorySand Columns. J. Cont. Hyd. 9:289-303.

Unterman, R., M.F. DeFlaun, and R.J. Steffan.1999. Advanced In Situ Bioremediation—AHierarchy of Technology Choices. In: Bio-technology, Wiley-VCH, Germany. In Press.

63

van Loosdrecht, M.C.M., J. Lyklema,W. Norde, and A.J.B. Zehnder. 1989. Bacte-rial Adhesion: A Physicochemical Approach.Microb. Ecol. 17:1-15.

van Loosdrecht, M.C.M., J. Lyklema,W. Norde, and A.J.B. Zehnder. 1990. Hydro-phobic and Electrostatic Parameters in Bac-terial Adhesion. Aquat. Sci. 52:103-114.

Vilks, P., and D.B. Bachinski. 1996. Colloidand Suspended Particle Migration Experi-ments in a Granite Fracture. J. of Contam.Hyd. 21:269-279.

Appendix A

Table A.1. Calendar of Field Experiments at Oyster Site

A.1

Dates Location Experiment

1999 Narrow Channel First field injection of DA001.

2000 South Oyster First field injection of facultative IRB.

2000 Narrow Channel Dual injection of DA001 and facultative IRB.

2001 South Oyster Dual injection of DA001 and facultative IRB.

2001 Narrow Channel Co-injection of facultative IRB and high DOC SOFA water.

2002 South Oyster Co-injection of facultative IRB and high DOC SOFA water.

2002 Narrow Channel Co-injection of facultative IRB, anionic complexes and high

DOC SOFA water.

2003 South Oyster Co-injection of facultative IRB, anionic complexes and high

DOC SOFA water.

2003 Narrow Channel Sequential injections of DA001 to test bactivory rates.

2004 South Oyster Sequential injections of the facultative IRB to test bactivory

rates.

2004 Narrow Channel Dual injection of DA001 and a dwarf cell.

2005 South Oyster Dual injection of facultative IRB and a dwarf cell

south oyster natural and accelerated bioremediation ... · south oyster science plan u.s....

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