Uranium Sequestration During 

Biostimulated Reduction and In 

Response to the Return of Oxic 

Conditions In Shallow Aquifers

Office of Nuclear Regulatory Research

NUREG/CR-7178
NRC Reference Material 

As of November 1999, you may electronically access 

NUREG-series publications and other NRC records at 

NRC’s Public Electronic Reading Room at 

http://www.nrc.gov/reading-rm.html. Publicly released 

records include, to name a few, NUREG-series 

publications; Federal Register notices; applicant, 

licensee, and vendor documents and correspondence; 

NRC correspondence and internal memoranda; bulletins 

and information notices; inspection and investigative 

reports; licensee event reports; and Commission papers 

and their attachments. 

NRC publications in the NUREG series, NRC 

regulations, and Title 10, “Energy,” in the Code of

Federal Regulations may also be purchased from one 

of these two sources. 

1. The Superintendent of Documents

 U.S. Government Printing Office

Mail Stop SSOP

Washington, DC 20402–0001

Internet: bookstore.gpo.gov

Telephone: 202-512-1800

Fax: 202-512-2250 

2. The National Technical Information Service 

Springfield, VA 22161–0002

www.ntis.gov

1–800–553–6847 or, locally, 703–605–6000 

A single copy of each NRC draft report for comment is 

available free, to the extent of supply, upon written 

request as follows:

Address: U.S. Nuclear Regulatory Commission

 Office of Administration 

Publications Branch

 Washington, DC 20555-0001

E-mail: DISTRIBUTION.RESOURCE@NRC.GOV

Facsimile: 301–415–2289 

Some publications in the NUREG series that are 

posted at NRC’s Web site address 

http://www.nrc.gov/reading-rm/doc-collections/nuregs 

are updated periodically and may differ from the last 

printed version. Although references to material found on 

a Web site bear the date the material was accessed, the 

material available on the date cited may subsequently be 

removed from the site.

Non-NRC Reference Material 

Documents available from public and special technical 

libraries include all open literature items, such as books, 

journal articles, transactions, Federal Register notices, 

Federal and State legislation, and congressional reports. 

Such documents as theses, dissertations, foreign reports 

and translations, and non-NRC conference proceedings 

may be purchased from their sponsoring organization. 

Copies of industry codes and standards used in a 

substantive manner in the NRC regulatory process are 

maintained at—

The NRC Technical Library

Two White Flint North

11545 Rockville Pike

Rockville, MD 20852–2738 

These standards are available in the library for reference 

use by the public. Codes and standards are usually 

copyrighted and may be purchased from the originating 

organization or, if they are American National Standards, 

from—

American National Standards Institute

11 West 42nd Street

New York, NY 10036–8002

www.ansi.org

212–642–4900 

AVAILABILITY OF REFERENCE MATERIALS

IN NRC PUBLICATIONS

Legally binding regulatory requirements are stated only 

in laws; NRC regulations; licenses, including technical 

specifications; or orders, not in NUREG-series 

publications. The views expressed in contractorprepared publications in this series are not necessarily 

those of the NRC.

The NUREG series comprises (1) technical and 

administrative reports and books prepared by the staff 

(NUREG–XXXX) or agency contractors (NUREG/CR–

XXXX), (2) proceedings of conferences (NUREG/CP–

XXXX), (3) reports resulting from international 

agreements (NUREG/IA–XXXX), (4) brochures 

(NUREG/BR–XXXX), and (5) compilations of legal 

decisions and orders of the Commission and Atomic and 

Safety Licensing Boards and of Directors’ decisions

under Section 2.206 of NRC’s regulations (NUREG–

0750).

DISCLAIMER: This report was prepared as an account 

of work sponsored by an agency of the U.S. 

Government. Neither the U.S. Government nor any 

agency thereof, nor any employee, makes any warranty, 

expressed or implied, or assumes any legal liability or 

responsibility for any third party’s use, or the results of 

such use, of any information, apparatus, product, or 

process disclosed in this publication, or represents that 

its use by such third party would not infringe privately 

owned rights.
Uranium Sequestration During 

Biostimulated Reduction and In 

Response to the Return of Oxic 

Conditions In Shallow Aquifers

Manuscript Completed: February 2014

Date Published: December 2014

Prepared by:

C. C. Fuller1, K. J. Johnson1*, K. C. Akstin1, 

D. M. Singer2, S. B. Yabusaki3, Y. Fang3, 

M. Fuhrmann4

1 U.S. Geological Survey, Menlo Park, CA

2 Kent State University, Kent OH

3 Pacific Northwest National Laboratory, Richland, WA

4 U.S. Nuclear Regulatory Commission, Rockville, MD

* current address: MWH Global, Inc., Steamboat Springs, CO

Mark Fuhrmann, NRC Project Manager

NRC Job Code: N6651

Office of Nuclear Regulatory Research

NUREG/CR-7178

iii

Abstract

A proposed approach for groundwater remediation of uranium contamination is to generate 

reducing conditions by stimulating the growth of microbial populations through injection of 

electron donor compounds into the subsurface. Sufficiently reducing conditions will result in 

reduction of soluble hexavalent uranium, U(VI), and precipitation of the less soluble +4 oxidation 

state uranium, U(IV). This process is termed biostimulated reduction. A key issue in the 

remediation of uranium (U) contamination in aquifers by biostimulated reduction is the long term 

stability of the sequestered uranium. Three flow-through column experiments using aquifer 

sediment were used to evaluate the remobilization of bioreduced U sequestered under 

conditions in which biostimulation extended well into sulfate reduction to enhance precipitation 

of reduced sulfur phases such as iron sulfides. One column received added ferrous iron, Fe(II), 

increasing production of iron sulfides, to test their effect on remobilization of the sequestered 

uranium, either by serving as a redox buffer by competing for dissolved oxygen, or by armoring 

the reduced uranium. During biostimulation of the ambient microbial population with acetate, 

dissolved uranium was lowered by a factor of 2.5 or more with continued removal for over 110 

days of biostimulation, well after the onset of sulfate reduction at ~30 days. Sequestered 

uranium was essentially all U(IV) resulting from the formation of nano-particulate uraninite that 

coated sediment grains to a thickness of a few 10’s of microns, sometimes in association with S 

and Fe. A multicomponent biogeochemical reactive transport model simulation of column 

effluents during biostimulation was generally able to describe the acetate oxidation, iron, sulfate, 

and uranium reduction for all three columns using parameters derived from simulations of field 

scale biostimulation experiments. 

Columns were eluted with artificial groundwater at equilibrium with atmospheric oxygen to 

simulate the upper limit of dissolved oxygen in recharge water. Overall about 9% of total 

uranium removed from solution during biostimulation was remobilized. Release of U during oxic 

elution was a continuous process over 140 days with dissolved uranium concentrations about 

0.2 and 0.8 mM for columns with and without ferrous iron addition, respectively. Uranium 

remaining on the sediment was in the reduced form. The prolonged period of biostimulation and 

concomitant sulfate reduction appears to limit the rate of U(IV) oxidative remobilization in 

contrast to a large release observed for columns in previous studies that did not undergo sulfate 

reduction. Although continued sulfate reduction may cause decreased permeability from 

precipitation of iron sulfide, the greater apparent stability of the sequestered U(IV) provided by 

the sustained biostimulation should be considered in design of field scale remediation efforts. 

v 

Foreword

The Nuclear Regulatory Commission has received license applications and inquiries about the 

acceptability of in situ bioremediation (ISB) techniques for the cleanup of uranium contaminated 

aquifers. This technology is based on the stimulation of indigenous bacteria that catalyze

reactions leading to the immobilization of uranium in the solid phase. Reduction of soluble 

hexavalent uranium, U(VI), to the much less soluble +4 oxidation state, U(IV), has been 

demonstrated to effectively remove uranium from solution. ISB has been considered for cleanup 

of shallow (less than 20 m depth) contaminated aquifers as well as the restoration of deep 

(greater than 150 m depth) in situ recovery sites from which uranium is commercially extracted. 

In this document, staff from the U.S. Geological Survey and Pacific Northwest National 

Laboratory report the results of experiments and modeling studies investigating the processes 

that impact the use of bioremediation as it is applied to a shallow site. In another report 

(NUREG/CR-7167), the potential for ISB of uranium in situ recovery sites was assessed using 

similar experimental and modeling techniques.

vii

Contents

Abstract ....................................................................................................................................iii

Foreword .................................................................................................................................. v

List of Figures ..........................................................................................................................ix

List of Tables..........................................................................................................................xiii

Executive Summary................................................................................................................xv

Acknowledgments ................................................................................................................xvii

1 Introduction .................................................................................................................... 1-1

1.1 Purpose and Scope.................................................................................................. 1-1

1.2 Bioremediation Processes ...................................................................................... 1-2

1.3 Forms of U(IV) Produced by Bioreduction ............................................................. 1-2

1.4 Stability of Bioreduced Uranium............................................................................. 1-3

1.5 Scope of Report ....................................................................................................... 1-4

2 Experimental Methods and Approach........................................................................... 2-1

2.1 Artificial Groundwater Design and Composition ................................................... 2-1

2.2 Column Apparatus and Operation .......................................................................... 2-3

2.2.1 Columns .............................................................................................................. 2-3

2.2.2 Plumbing and Pumps........................................................................................... 2-3

2.2.3 Gas Phase and Oxygen Control .......................................................................... 2-5

2.2.4 Column Packing................................................................................................... 2-5

2.2.5 Column Physical Parameters............................................................................... 2-6

2.3 Column Effluent ....................................................................................................... 2-8

2.3.1 Sampling.............................................................................................................. 2-8

2.3.2 Effluent Analyses ................................................................................................. 2-8

2.4 Solid Phase Sampling.............................................................................................. 2-9

2.4.1 Column Sectioning and Sediment Sample Preservation ...................................... 2-9

2.4.2 Total U Screening by Gamma Spectrometry...................................................... 2-10

2.4.3 Chemical Analyses ............................................................................................ 2-10

2.4.3.1 Extractions...................................................................................................... 2-10

2.4.3.2 Total carbonate, total sulfur and acid volatile sulfur ........................................ 2-11

2.4.4 Spectroscopic Measurements............................................................................ 2-13

2.4.4.1 Bulk XAS........................................................................................................ 2-13

2.4.4.2 Microfocused synchrotron XRF and XAS........................................................ 2-14

2.4.5 SEM-EDS Imaging............................................................................................. 2-15

2.4.6 Microbial Assay.................................................................................................. 2-15

2.4.6.1 Column sediments.......................................................................................... 2-15

2.4.6.2 Effluent filters.................................................................................................. 2-16

2.4.6.3 Measurement methods................................................................................... 2-16

3 Results ............................................................................................................................ 3-1

3.1 Column Effluent: Biostimulation............................................................................. 3-1

3.1.1 Column A Effluent History during Biostimulated Reduction .................................. 3-1

3.1.2 Column B Effluent History during Biostimulated Reduction .................................. 3-3

3.1.3 Column C Effluent History during Biostimulated Reduction.................................. 3-5

3.1.4 Integrated Column Effluent Concentrations during Biostimulation........................ 3-7

3.2 Oxic Elution............................................................................................................ 3-10

3.3 Solid Phase Analyses ............................................................................................ 3-13

3.3.1 Column Sediment Subsamples Collected .......................................................... 3-15
viii

3.3.2 Column A Sediment Chemistry after Biostimulated Reduction........................... 3-17

3.3.3 Column B Sediment Chemistry after Biostimulation ........................................... 3-29

3.3.4 Column C Sediment Chemistry after Biostimulation........................................... 3-32

3.3.5 Summary of Sediment U, Fe(II) and S during Biostimulation.............................. 3-35

3.3.6 Sediment Chemistry after Oxic Elution............................................................... 3-36

3.4 X-ray Absorption Spectroscopy and Imaging...................................................... 3-42

3.4.1 U Oxidation State............................................................................................... 3-42

3.4.2 Speciation of Sequestered Uranium................................................................... 3-45

3.4.3 Distribution of Uranium on Sediments................................................................ 3-49

3.4.4 SEM-EDS Imaging of Column Sediments.......................................................... 3-59

3.5 Microbial Analysis.................................................................................................. 3-63

3.5.1 TRFLP Fingerprinting......................................................................................... 3-63

3.5.2 qPCR................................................................................................................. 3-65

3.5.3 Clone Library Analyses ...................................................................................... 3-68

3.5.4 Summary of Microbial Analyses of the Column Experiments ............................. 3-72

4 Discussion of Column Experimental Results............................................................... 4-1

4.1 Biostimulated Reduction ......................................................................................... 4-1

4.1.1 Electron Balance during Biostimulation................................................................ 4-1

4.1.2 Biogeochemical Processes during Biostimulated Reduction ................................ 4-3

4.2 Remobilization of Uranium during Oxic Elution .................................................... 4-7

4.2.1 Effect of U(IV) Form and Morphology................................................................... 4-7

4.2.2 Competitive Oxidation Processes ........................................................................ 4-8

4.2.3 Observed Rate and Extent of U(IV) Release........................................................ 4-9

4.2.4 Processes Affecting Uranium Remobilization in Column Experiments ............... 4-11

5 Reactive Transport Modeling of the Rifle Sediment Columns during 

Biostimulation................................................................................................................. 5-1

5.1 Introduction.............................................................................................................. 5-1

5.1.1 Background ......................................................................................................... 5-1

5.1.2 History ................................................................................................................. 5-2

5.2 Model Components.................................................................................................. 5-2

5.2.1 HYDROGEOCHEM ............................................................................................. 5-2

5.2.2 Conceptualization of Processes........................................................................... 5-2

5.2.3 Model Description................................................................................................ 5-3

5.2.4 UCODE ............................................................................................................... 5-9

5.3 Approach for Modeling Biostimulation Stage of Column Experiments ............. 5-10

5.3.1 General Approach.............................................................................................. 5-10

5.3.2 Challenges......................................................................................................... 5-10

5.3.3 Parameter Estimation ........................................................................................ 5-11

5.3.4 Calibration ......................................................................................................... 5-12

5.3.5 Initial Simulations............................................................................................... 5-13

5.4 Model Simulation Results...................................................................................... 5-16

5.4.1 Column B Simulations........................................................................................ 5-16

5.4.2 Simulation of Column A Effluent using Column B Parameters ........................... 5-20

5.4.3 Simulation of Column C Effluent using Column B Parameters ........................... 5-25

5.5 Discussion of Model Simulations ......................................................................... 5-29

6 Summary......................................................................................................................... 6-1

7 References Cited ............................................................................................................ 7-1
ix

List of Figures

Figure 1. Column set up. ................................................................................................... 2-4

Figure 2. Effluent concentrations of (top) dissolved uranium, iron, sulfide and sulfate 

and (bottom) pH, carbonate alkalinity for column A during biostimulation and 

during oxic elution versus time from start of biostimulation................................. 3-2

Figure 3. Effluent concentrations of (top) dissolved uranium, iron, sulfide and sulfate 

and (bottom) pH, carbonate alkalinity for column B during biostimulation 

elution versus time from start of biostimulation................................................... 3-4

Figure 4. Effluent concentrations of (top) dissolved uranium, iron, sulfide and sulfate 

and (bottom) pH, carbonate alkalinity for column C during biostimulation and 

during oxic elution versus time from start of biostimulation................................. 3-6

Figure 5. Cumulative uranium loading on column sediments during biostimulated 

reduction, in micromoles of uranium per gram of column sediment 

versus time. ....................................................................................................... 3-9

Figure 6. Effluent dissolved uranium from column A and column C during oxic 

elution versus days following introduction of dissolved oxygen to 

column influent................................................................................................. 3-11

Figure 7. Change in sediment uranium concentration during oxic elution, plotted 

as (A) micromoles U per gram remaining on sediment versus days since 

the start of suboxic elution, and (B) cumulative mass of uranium release in 

micromoles versus time. .................................................................................. 3-12

Figure 8. Column A 26 days after the start of biostimulation (A), at the end of 

biostimulation (B), and after oxic elution (C)..................................................... 3-15

Figure 9. Column A sediments after oxic elution during subsampling depicting 

location of samples and variability in sediment appearance............................. 3-16

Figure 10. Column C at end of oxic elution, (Top) side view with degrees clockwise 

around column denoted (flow is from bottom to top); (bottom) surface of 

intervals view from inlet end prior to subsampling .....................................3-17

Figure 11. Total uranium concentration (mmol/g) of <2 mm sediment from column A 

after biostimulated reduction versus distance from inlet. .................................. 3-18

Figure 12. 0.5 N HCl extractable Fe(II) and total Fe (mmol/g) of <2 mm sediment from 

column A after biostimulated reduction versus distance from inlet. .................. 3-19

Figure 13. Total and acid volatile sulfur of <2 mm sediment from column A after 

biostimulated reduction versus distance from inlet. .......................................... 3-19

Figure 14. Total uranium concentration (mmol/g) of <2 mm sediment from column B 

after 221 days of biostimulated reduction stage prior to repacking versus 

distance from inlet. B. Total uranium in sediments at end of biostimulation 

experiment....................................................................................................... 3-30

Figure 15. A. 0.5 N HCl extractable Fe(II) (mmol/g) of <2 mm sediment from column B 

after 221 days of biostimulated reduction stage prior to repacking versus 

distance from inlet. B. Extractable Fe(II) in sediments at end of 

biostimulation experiment. ............................................................................... 3-31

Figure 16. A. Total and acid volatile sulfur of <2 mm sediment from column B after 

221 days of biostimulated reduction stage prior to repacking versus distance 

from inlet. B. Total and acid volatile sulfur in sediments at end of 

biostimulation experiment. ............................................................................... 3-32

Figure 17. Column C total uranium concentration (mmol/g) of the <2 mm sediment after 

biostimulated reduction versus distance from inlet. .......................................... 3-33
x 

Figure 18. Column C sediment 0.5N HCl extractable Fe(II) and total Fe concentration 

(m mol/g <2 mm fraction) after biostimulated reduction stage versus 

distance from inlet............................................................................................ 3-34

Figure 19. Column C sediment total and acid volatile sulfur following biostimulated 

reduction (mmol/g versus distance from column inlet)...................................... 3-35

Figure 20. Total uranium concentration in sediments of column A (mmol/g <2 mm 

fraction) after oxic elution versus position in column. ....................................... 3-37

Figure 21. 0.5N HCl extractable Fe(II) concentration in sediments of column A 

(mmol/g <2 mm fraction) after oxic elution versus position in column................ 3-37

Figure 22. Column A <2 mm sediment total and acid volatile sulfur following oxic elution 

(millimoles per gram versus distance from column inlet. .................................. 3-38

Figure 23. Column C sediment total uranium concentration (mmol/g <2 mm fraction) 

after oxic elution versus position in column. ..................................................... 3-39

Figure 24. Column C sediment 0.5N HCl extractable Fe(II) and total Fe concentration 

(mmol/g <2 mm fraction) after oxic elution versus position in column................ 3-40

Figure 25. Column C sediment total and acid volatile sulfur following oxic elution 

(mmol/g vs distance from column inlet)............................................................ 3-41

Figure 26. U-LII (A) and U-LIII (B) bulk XANES of column sediment samples................... 3-43

Figure 27. Bulk EXAFS (A) and (B) their Fourier transform of column sediment 

samples (lines) and best fit to spectra (dots).................................................... 3-47

Figure 28. mXRF maps of post-biostimulation column sediment thin section of 

column C sediment sample Cr-5.... ................................................................. 3-50

Figure 29. mXRF maps of post-biostimulation column sediment thin section of column 

B sediment sample Br-F4... ............................................................................. 3-51

Figure 30. mXRF maps of column C sediment sample Cr-5 grain 1................................... 3-53

Figure 31. Micro-XANES of points on column sediment grain coatings............................. 3-54

Figure 32. U-LIII mEXAFS of points on column sediment grain coatings. ........................... 3-55

Figure 33. mXRF maps of column B sediment sample F4 grain 1...................................... 3-57

Figure 34. mXRF maps of column A sediment sample Ax-23 grain 1. ............................... 3-58

Figure 35. SEM-BSE images of column C after biostimulation interval 5 (Cr-5) thin 

section grain 1. ................................................................................................ 3-60

Figure 36. SEM-BSE images of column C interval 5 (Cr-5), grain 2 after biostimulation.... 3-62

Figure 37. Cluster analysis for column sediments. See Table 14 for sample 

identification codes. ......................................................................................... 3-65

Figure 38. Abundances of sulfate reducing bacteria (SRB) and Geobacter in sediment 

of column A after biostimulated reduction and after oxic elution....................... 3-66

Figure 39. Comparison of abundances of sulfate reducing bacteria (SRB) and 

Geobacter in column B sediments....... ........................................................... 3-67

Figure 40. Comparison of sulfate reducing bacteria (SRB) and Geobacter abundance 

in column C sediment after biostimulated reduction. ........................................ 3-67

Figure 41. Geobacter abundances in columns A, B, C after biostimulated reduction, 

and after oxic elution (column A). Sample position in column from inlet 

to outlet is from left to right............................................................................... 3-68

Figure 42. Abundance of sulfate reducing bacteria in column sediments. Sample 

position in column from inlet to outlet is from left to right. ................................. 3-68

Figure 43. Comparison of effluent dissolved uranium (top), dissolved iron (middle), and 

dissolved sulfide (bottom) for columns A, B and C during biostimulation. The 

data are plotted for the duration of acetate addition to column A........................ 4-4

Figure 44. Total uranium on a whole column basis for oxic elution columns A and C, in 

micromoles, before and after oxic elution from (A) integrated effluent 

concentration profiles and (B) sediment U concentrations................................ 4-10
xi

Figure 45. Total sulfur in columns A and C sediments, in millimoles, before and 

after oxic elution............................................................................................. 4-122 

Figure 46. Model simulation column B effluent acetate. .................................................... 5-17

Figure 47. Model simulation column B effluent dissolved iron. .......................................... 5-18

Figure 48. Model simulation column B effluent dissolved uranium. ................................... 5-19

Figure 49. Model simulation column B effluent dissolved sulfate. ...................................... 5-20

Figure 50. Model simulation column A effluent acetate. .................................................... 5-21

Figure 51. Model simulation column A effluent iron........................................................... 5-22

Figure 52. Model simulation column A effluent uranium.................................................... 5-24

Figure 53. Model simulation column A effluent sulfate. ..................................................... 5-25

Figure 54. Model simulation column C effluent acetate..................................................... 5-26

Figure 55. Model simulation column C effluent iron........................................................... 5-27

Figure 56. Model simulation column C effluent uranium.................................................... 5-28

Figure 57. Model simulation column C effluent sulfate. ..................................................... 5-29

Figure 58. Time-dependent variation in adsorbed U(VI) (top) and U(IV) (bottom) for 

the grid cell closest to the column A effluent. ................................................... 5-30

xiii

List of Tables 

Table 1. Artificial groundwater composition...................................................................... 2-2

Table 2. Column physical parameters.............................................................................. 2-7

Table 3. Integrated loss and gain from column effluent concentrations during acetate 

biostimulation..................................................................................................... 3-8

Table 4. Grain size distribution of Rifle BH-02 sediment used in column experiments, 

as determined by sieve analysis ...................................................................... 3-14

Table 5. Mineral weight percent of Rifle BH-02 sediment used in column experiments 

(pre-column) as determined by quantitative X-ray diffraction............................ 3-14

Table 6. Sediment chemistry for column A after biostimulated reduction and after 

oxic elution...................................................................................................... 3-21

Table 7. Sediment chemistry for column B during and after biostimulated reduction...... 3-25

Table 8. Sediment chemistry for column C after biostimulated reduction and after 

oxic elution...................................................................................................... 3-27

Table 9. Fraction of U(IV) and U(VI) of column sediments determined by linear 

combination fits to U-LII and LIII bulk XANES spectra. ...................................... 3-44

Table 10. EXAFS fitting parameters for column sediments. ............................................. 3-48

Table 11. Fraction of U(IV) and U(VI) at thin section points column sediments 

determined by linear combination fits to U-LIII mXANES spectra....................... 3-56

Table 12. Atomic percent of elements detected in SEM-EDS points data from 

column C subsample Cr-5, thin section right panel, grain 1.............................. 3-61

Table 13. Atomic percent of elements detected in SEM-EDS points data from 

column C post reduction, interval Cr-5 grain 2. Location of point spectra 

are shown in Figure 36. .................................................................................. 3-63

Table 14. Column sediment microbial abundance data for sulfate reducing bacteria 

(SRB) and Geobacter (GEO)......... ................................................................. 3-64

Table 15. Geobacter clones from columns A outlet filter, Ar-f(out), and B effluent 

filter, Br-eff(1)......................................................................................... 3-69

Table 16. Sulfate reducing bacteria phylotypes, relative abundance, and closest 

BLAST Hits from column sediment samples recovered after biostimulated 

reduction and after oxic elution. ....................................................................... 3-71

Table 17. Summary of electron transfer during biostimulated reduction in 

columns A, B and C. .......................................................................................... 4-2

Table 18. Uranium species and their formation constants.................................................. 5-5

Table 19. Uranium surface complexation reactions and formation constants..................... 5-6

Table 20. Equilibrium Reactions ........................................................................................ 5-7

Table 21. Kinetic Reactions ............................................................................................... 5-8

Table 22. Rate Constants.................................................................................................. 5-8

Table 23. Hydrologic parameters for columns A, B, and C............................................... 5-12

Table 24. Calibrated model parameters for column B...................................................... 5-15

Table 25. Particle Size Distribution of Rifle Aquifer.......................................................... 5-16

Table 26. Simulated adsorbed U(VI), U(IV), FeS, elemental S, and phyllosilicate 

Fe(II) at the end of experiments in columns A, B, and C. ................................. 5-31

xv 

Executive Summary

Uranium extraction and processing has resulted in contamination of groundwater and sediments 

at many locations worldwide. The high mobility of U(VI) under oxidizing conditions compared to 

the sparingly soluble +4 oxidation state, U(IV), can lead to dispersal of uranium contamination in 

groundwater and aquifer sediments. Traditional remediation methods of extraction and/or pump 

and treat are expensive and time consuming. Because of the ability of metal reducing microbes 

to chemically reduce dissolved U(VI), producing lower solubility U(IV), in situ stimulation of 

indigenous microbial populations in aquifers by electron donor addition has led to the concept of 

biostimulated reduction as a remediation strategy to immobilize uranium contamination. This 

topic has been the focus of many field and laboratory investigations over the past 20 years. A 

key issue for reduction based bioremediation of U(VI) is the stability of the solid phase reaction 

products over time. Remobilization of U(IV) through oxidation following cessation of electron 

donor addition remains a critical component in understanding the long term effectiveness of 

biostimulated reduction.

The focus of this study is to evaluate remobilization of U after biostimulation in response to the 

return to oxic conditions through long term (>100 pore volumes) column experiments using 

sediments from the Old Rifle site, a shallow aquifer contaminated from uranium ore milling and 

remediated through the Uranium Mill Tailings Remedial Action (UMTRA) program. The primary 

goal of this study was to evaluate the remobilization of U sequestered during biostimulated 

reduction under conditions in which biostimulation and concomitant U reduction extended well 

into sulfate reduction to enhance precipitation of reduced sulfur phases such as iron sulfides. 

Ferrous iron was added to the influent of one column during biostimulation to enhance the 

formation of iron sulfide. The intent of producing these reduced phases was to test their ability to 

inhibit remobilization of the sequestered uranium, either serving as a redox buffer by competing 

for dissolved oxygen, or by armoring the reduced uranium by precipitating an iron sulfide 

coating on mineral grains. Chemical analysis, X-ray absorption spectroscopy, micro-focused Xray fluorescence mapping, and electron microscopy of sediments recovered from columns after 

biostimulation and after oxic elution were conducted to characterize the form of U on sediments 

and other solid phase constituents that may affect U remobilization, notably iron and sulfur. 

Reactive transport modeling of column effluent chemistry during biostimulation was conducted 

to understand uranium behavior in the context of 1) transient biostimulation with acetate 

electron donor, 2) functional indigenous microbial groups representing Fe(III)-reducing bacteria 

(FeRB) and sulfate-reducing bacteria (SRB) responding to the biostimulation, and 3) 

geochemical response to the biostimulation products.

During biostimulation of the ambient microbial population with acetate, the removal of dissolved 

U(VI) from the influent continued over 110 to 330 days of biostimulation, well after the onset of 

sulfate reduction at ~30 days. The concentration of dissolved uranium was lowered from the 

influent 10 mM to <4 mM. By day 60 of the reduction phase of the experiments, concentrations of 

uranium in the effluent of a column receiving Fe (II) in the influent, column C, were less than half 

of the other columns without Fe addition (A and B). The uranium sequestered during 

biostimulation was essentially all precipitated as U(IV) resulting in part from the formation of 

nano-particulate uraninite. The reduced U(IV) formed contiguous coatings on sediment grains 

with thicknesses of a few 10s of micrometers and was in association with S and Fe in some 

cases. Uranium spectra by Extended X-ray Absorption Fine Structure (EXAFS) of column 

sediments are consistent with U(IV) comprised of a mixture of both partially disordered 

nanoparticulate UO2 and non-uraninite U(IV) species associated with biomass. The non-
xvi

uraninite U(IV)-bearing phase may exist separately from the partially polymerized nano-UO2-like 

U(IV).

The multicomponent biogeochemical reactive transport model simulation of column effluents 

during biostimulation was generally able to describe the acetate oxidation, iron, sulfate, and 

uranium reduction for all three columns using parameters derived from simulations of field scale 

biostimulation experiments. Together, the effluent and solid phase measurements from columns 

during and after the biostimulation stage suggest that stimulation and growth of indigenous 

microbial population in the aquifer sediments from the Rifle site by addition of an electron donor 

and carbon source may be an effective means of removing dissolved uranium from 

contaminated groundwater, consistent with many field and laboratory studies.

Elution of bioreduced U(IV) associated with sediments from two columns (A and C) was 

conducted using artificial groundwater at equilibrium with atmospheric oxygen. This oxygen 

level was used to assess the upper limit of dissolved oxygen in recharge water. The difference 

between the two columns was the addition of 15 mM dissolved Fe(II) to column C during 

biostimulation to enhance formation of iron sulfides. Release of U during oxic elution was a 

continuous process over 120 to 140 days (300 to 350 PV). Remobilization of uranium was four 

times higher in column A compared to column C with effluent dissolved uranium concentrations 

approximately 0.8 and 0.2 mM for the column A and C, respectively. The effluent U 

concentration from column C decreased over time indicative of a decreasing re-oxidation rate. 

In contrast, uranium in the effluent from column A did not display this decreasing trend but was 

instead somewhat constant over 140 days. This suggests that the addition of Fe (II) to the 

influent and the subsequent precipitation of iron sulfide minerals may decrease the release of 

uranium. Overall, less than 9% of total uranium sequestered during biostimulation was 

remobilized. The limited oxidative remobilization of U(IV) in these column experiments is 

consistent with the decreased U remobilization where sulfate reduction occurred in contrast to 

the large release of U from columns reported in the literature which did not undergo sulfate 

reduction. Although continued sulfate reduction may cause a decrease in permeability from 

precipitation of iron sulfide, the greater apparent stability of the sequestered U(IV) provided by

the sustained biostimulation could be considered in design of field scale remediation efforts. 

However, even with the greater uranium stability caused by iron sulfide precipitation the effluent 

uranium concentration of approximately 0.2 μM did exceed EPA’s uranium MCL of 30 μg/L 

(0.126 μM). Advancement of biostimulated reduction strategies for remediation of groundwater 

uranium contamination could include additional field scale testing of the stability of bioreduced 

uranium to remobilization following the end of biostimulation.
xvii

Acknowledgments

We thank the following USGS scientists for their efforts on this study: Alex Blum for XRD 

analysis; Shelley Hoeft for assistance in HPLC measurements and guidance in microbial 

protocols; Steve Silva for total and isotopic composition of sulfur in sediments; Julie Kirshtein for 

microbial assay and providing a detailed report on the findings; and Larry Miller for many 

discussions on microbial and biogeochemical processes. Sam Webb and Ben Kocar of the 

Stanford Synchrotron Radiation Lightsource (SSRL) are acknowledged for assistance in microfocused X-ray imaging. John Bargar of SSRL is thanked for providing uranium reference spectra 

and guidance in XAS data collection. Ken Williams of the Lawrence Berkeley National 

Laboratory is acknowledged for providing aquifer sediments from the Old Rifle site. 

Portions of this research were conducted at the Stanford Synchrotron Radiation Lightsource, a 

national user facility operated by Stanford University on behalf of the US Department of Energy, 

Office of Basic Energy Sciences. The SSRL Structural Molecular Biology Program is supported 

by the Department of Energy, Office of Biological and Environmental Research, and by the 

National Institutes of Health, National Center for Research Resources, Biomedical Technology 

Program. 

Massively parallel processing simulations were performed on the Chinook supercomputer at the 

Environmental Molecular Sciences Laboratory (EMSL), a national scientific user facility 

sponsored by the Department of Energy's Office of Biological and Environmental Research and 

located at Pacific Northwest National Laboratory.

We thank D.L. Stoliker, USGS, and K. Czyscinski, US EPA, for helpful reviews that improved 

this report. We also thank Earl Greene (USGS) and Thomas Nicholson (NRC) for their help 

with this work.

Any use of trade, product, or firm names is for descriptive purposes, only, and does not imply 

endorsement by the U.S. Government. 

1-1 

1 Introduction 

1.1 Purpose and Scope 

Uranium extraction and processing has resulted in contamination of groundwater and sediments 

at many locations worldwide. The high mobility of U(VI) under oxidizing conditions compared to 

the sparingly soluble +4 oxidation state, U(IV), can lead to dispersal of uranium contamination in 

groundwater and aquifer sediments. Development of effective strategies for remediation of 

uranium contamination in groundwater has focused largely on using in situ treatment processes 

because of higher costs associated with traditional pump and treat methods. Surface 

complexation of U(VI) on sediment surfaces maintains dissolved uranium concentrations. This 

greatly increases the time for flushing U contamination from an aquifer and limits the success of 

pump and treat or natural attenuation strategies. Because of the ability of metal reducing 

microbes to reduce dissolved U(VI) (Lovley et al., 1991), in situ stimulation of indigenous 

microbial populations in aquifers by electron donor addition has led to the concept of 

biostimulated reduction as a remediation strategy to immobilize uranium contamination (Lovley 

and Phillips, 1992). This topic has been the focus of many field and laboratory investigations 

over the past 20 years. 

A key issue for reduction based bioremediation of U(VI) is the stability of the solid phase 

reaction products over time (Yabusaki et al., 2010). Remobilization of U(IV) through oxidation 

following cessation of electron donor addition remains a critical component in understanding 

the long term effectiveness of biostimulated reduction. Previous laboratory studies have shown 

that U(IV) produced during biostimulation is more stable and less prone to oxidative 

remobilization when biostimulation proceeds well into sulfate reduction compared to 

experimental systems with limited sulfate reduction (Komlos et al., 2008b; Moon et al., 2009). 

Enhanced stability of U(IV) has been attributed to iron sulfides produced during biostimulation 

(Abdelouas et al., 1999). Evaluating the long term stability of bioreduced uranium in field scale 

biostimulation experiments has not been adequately addressed, in part because of the long time 

scales required. Laboratory studies allow evaluating stability over many pore volumes in a much 

shorter time scale than can be studied practically in field-scale studies and thus may help 

predict long term stability in a remediated aquifer. 

The focus of this study is to evaluate remobilization of U(IV) after biostimulation in response to 

the return to oxic conditions through long term (> 100 pore volumes) column experiments using 

sediments from the Old Rifle site, a shallow aquifer contaminated from uranium ore milling and 

remediated through the UMTRA program. The site has been described in detail elsewhere (eg 

Anderson et al, 2003; Yabusaki et al, 2009). In these experiments, biostimulation was allowed to 

proceed for several months after the onset of sulfate reduction. Ferrous iron was added to the 

influent of one column during biostimulation to enhance the formation of iron sulfide to test its 

effect on oxidative remobilization of reduced uranium. Chemical analysis, X-ray spectroscopy, 

micro-focused X-ray fluorescence mapping, and electron microscopy of sediments recovered 

from columns after biostimulation and after oxic elution were conducted to characterize the form 

of U on sediments and other solid phase constituents that may affect U remobilization, notably 

iron and sulfur. 
1-2 

1.2 Bioremediation Processes 

The high mobility of U(VI) under oxidizing conditions compared to the sparingly soluble +4 

oxidation state, U(IV), has led to the concept of producing reducing conditions to immobilize U in 

situ as a remediation strategy. The demonstrated ability of microbes to either reduce U(VI) 

either directly through enzymatic processes (Lovley et al., 1991) or indirectly through reaction 

byproducts of iron and sulfate reduction is the basis for in situ bioreduction of U(VI) and 

precipitation of U(IV) through stimulation of indigenous microbial populations through electron 

donor amendment as a remediation strategy. Numerous studies have evaluated the 

effectiveness of biostimulated reduction based immobilization of aqueous U(VI) in laboratory 

(Komlos et al., 2008b; Moon et al., 2007; Sharp et al., 2011) and in field tests (Anderson et al., 

2003; Wu et al., 2006; Williams et al., 2011). The biostimulation approach is based on microbial 

mediated reduction and precipitation that enhances U(VI) immobilization over attenuation 

processes occurring under ambient groundwater conditions, such as reduction by pre-existing 

mineral phases. Considerable effort has been focused on identifying specific microbial 

populations responsible for U reduction. For example, a number of studies have identified the 

importance of Geobacteraceae during biostimulation for enhancing U immobilization through 

growth and concurrent reduction of both Fe(III) and U(VI), for example (Lovley et al., 1991; 

Gorby and Lovley, 1992; Lovley and Phillips, 1992). The importance of the iron reducing 

bacteria Geobacter has been demonstrated in field biostimulation tests using acetate as an 

electron donor (Vrionis et al., 2005; Williams et al., 2011). The ability of sulfate reducing bacteria 

(SRB) to mediate reduction of uranium has also been observed. Sulfate reducing bacteria have 

been shown to reduce uranium directly through enzymatic mechanisms (Lovley et al., 1993), 

and have been shown to be important in field scale biostimulation experiments using ethanol as 

an electron donor (Cardenas et al., 2008; Luo et al., 2007a). In contrast, no direct evidence has 

been found for U(VI) reduction by sulfate reducing bacteria (SRB) during biostimulation with 

acetate as an electron donor in field scale tests (Williams et al., 2011). 

In addition to direct enzymatic reduction by microbes, U(VI) reduction may also occur by abiotic 

reaction processes. Reduction of U(VI) by dissolved sulfide is thermodynamically favored but 

may be limited by the presence of elevated dissolved carbonate complexation of dissolved U(VI) 

(Sani et al., 2005). Fe(II) can serve as an electron donor for abiotic U(VI) reduction (Campbell et 

al., 2013), but is kinetically slow under pH and dissolved carbonate levels typical of many 

aquifers (Liger et al., 1999). U(VI) reduction by Fe(II) sorbed to sediment surfaces has been 

observed and increases at higher sorbed Fe(II) concentrations (Fox et al., 2013). Uranium 

reduction has been observed to occur by reaction with solid phases produced during microbial 

mediated reduction processes, such as iron sulfides (Veeramani et al., 2013; Hyun et al., 2012; 

Hua and Deng, 2008) and carbonate green rust, a mixed oxidation state iron mineral 

(O'Loughlin et al., 2010). Although abiotic uranium reduction by the iron sulfide mackinawite has 

been demonstrated during field biostimulation experiments (Bargar et al., 2013), the 

quantitative impact of abiotic uranium reduction has not been demonstrated in field studies. 

1.3 Forms of U(IV) Produced by Bioreduction 

Reduction of U(VI) and precipitation of U(IV) commonly forms uraninite (UO2). The uraninite 

formed as the result of direct or indirect microbial processes has been shown to be 

nanoparticluate in size (Schofield et al., 2008; Singer et al., 2009; O'Loughlin et al., 2010). This 

nanoparticulate biogenic uraninite has been found to be on the order of 3 nm in diameter and 

tends to form aggregates (Burgos et al., 2008; Schofield et al., 2008). Because of the small 
1-3 

particle size, Schofield et al. (2008) estimated that about 50% of the U is near the surface 

resulting in a lattice contraction compared to larger abiotic particles of bulk UO2. Although the 

surface is distorted, the particle core remained highly ordered consistent with stoichiometric UO2

(Burgos et al., 2008; Schofield et al., 2008; Singer et al., 2009). The net effect, however, is that 

strain on the interior is negligible and likely does not enhance the solubility or stability (Schofield

et al., 2008). Nanoparticulate UO2 has been shown to form aggregates, the size of which is, in 

some cases, due to the rate of U reduction (Burgos et al., 2008; Senko et al., 2002). 

In addition, laboratory and field studies have shown that bioreduced U(IV) also forms complexes 

with microbial biomass binding to phosphate or carbon sites on cell biomass (Bernier-Latmani et 

al., 2010; Sharp et al., 2011; Boyanov et al., 2011; Alessi et al., 2012; Bargar et al., 2013). 

These non-uraninite biogenic U(IV) species have been termed monomeric or molecular U(IV) 

and more recently have been referred to as “non-uraninite U(IV) species associated with 

biomass” or NUSAB (Stoliker et al., 2013). NUSAB U(IV) may be more labile than uraninite and 

may have a higher susceptibility to oxidation than the biogenic nanoparticulate UO2, but further 

characterization of its stability is needed. Thus, it is important to determine the forms of U(IV) 

produced during biostimulated reduction as part of evaluating the long term effectiveness of in 

situ bioremediation.

1.4 Stability of Bioreduced Uranium 

A key issue for the success of in situ bioremediation of U(VI) is the stability of the solid phase 

reaction products over time. Remobilization of U through oxidation following cessation of 

electron donor addition remains a critical component in understanding the long term 

effectiveness of biostimulated reduction. Determining the stability of the bioreduced uranium 

that was formed during biostimulated reduction is needed to evaluate if the biostimulated 

reduction process would be an effective tool for long term remediation of U-contaminated 

shallow aquifers. Specifically, evaluation of oxidative remobilization in response to changes in 

groundwater chemistry after biostimulation has ceased, such as the return to oxic conditions, is 

required. Oxidative remobilization in shallow aquifers may also occur in response to recharge 

and flooding events, which increase groundwater dissolved oxygen concentrations (Yabusaki et 

al., 2010). 

The stability of bioreduced U(IV) following biostimulation is dependent both on the form of the 

bioreduced U(IV) and on the rate and extent of oxidation of the biogenic U(IV). Oxidation of 

U(IV) occurs primarily through abiotic processes by reaction with dissolved oxygen, nitrate, and 

Fe(III) solid phases. In contrast, biotic oxidation is limited particularly at the circum neutral pH of 

many aquifers (Campbell et al., 2013). Similar rates of oxidation are reported for biogenic UO2

and synthetic abiotic UO2 on a surface area basis (Ulrich et al., 2009). The susceptibility of 

nanoparticulate biogenic UO2 to oxidation is thought to be controlled in part by the extent of 

aggregate formation that may offset the high surface area of nanoparticles (Burgos et al., 2008; 

Senko et al., 2007). Formation of strong complexes of U(VI) with dissolved carbonate present in 

most groundwater systems enhances U(VI) solubility, minimizing accumulation of oxidation 

products on UO2 surfaces such that the oxidation reaction is rate limiting (Campbell et al., 

2013). Oxidation of biogenic UO2 in the field under ambient geochemical and flow conditions 

was observed to be several orders of magnitude slower than laboratory rates (Campbell et al., 

2011). 
1-4 

Hydrologic, chemical and microbiological conditions expected at shallow, alluvial sediment sites 

will influence the stability of bio-reduced U(IV) and how it responds to the long-term reintroduction of oxidants to the system. The response of bio-reduced U(IV) during a return to 

oxidizing conditions has been the focus of several recent and ongoing research efforts (Moon et 

al., 2007; Moon et al., 2009; Komlos et al., 2008b; Senko et al., 2002; Zhong et al., 2005). It has 

been shown that Fe, sulfate, and electron donor concentrations during the bio-reduction phase 

can largely influence both the bioreduction of U(VI) and the oxidative remobilization of U(IV) 

(Komlos et al., 2008b; Moon et al., 2007; Wan et al., 2005; Zhong et al., 2005). In column 

experiments, Komlos et al. (2008b) found that U(IV) re-oxidation readily occurs upon 

introduction of solutions containing atmospheric oxygen concentrations. However, Zhong et al.

(2005) found that the addition of Fe(II) to an abiotic system containing biogenic U(IV) resulted in 

both a decrease in the extent of U(IV) re-oxidation and increased sorption of U(VI) to Fe(III) 

oxide phases formed during the re-oxidation process, thus retarding the mobility of uranium. 

Conversely, under certain conditions oxidation of U(IV) may occur by reaction with Fe(III) oxides 

produced from oxidation of Fe(II) phases produced during biostimulation following the reaction 

pathway described by Ginder-Vogel et al. (2010). (Abdelouas et al., 1999) and (Moon et al., 

2009)) found that iron sulfides produced during biostimulation decreased the rate and extent of 

U(IV) re-oxidation upon introduction of oxidants to column influents (e.g., dissolved O2, nitrate). 

However, the distribution of these secondary reduced Fe phases within the column sediments in 

relation to the distribution of reduced uranium was not determined. As a result, it is unclear if the 

decrease in U(IV) re-oxidation was the result of competition for oxidant (redox “buffering”), or 

armoring of biogenic U(VI) by iron sulfide precipitates. In addition, the presence of FeS along 

flow paths may limit transport of remobilized U(VI) through abiotic reduction (Hyun et al., 2012; 

Gallegos et al., 2013). Consumption of oxygen by biomass degradation also may affect the rate 

of uranium oxidation (Komlos et al., 2008b). 

1.5 Scope of Report 

The intent of this report is to present the results of laboratory column experiments designed to 

investigate biostimulation and subsequent oxic elution of shallow aquifer sediments from the Old 

Rifle site in Colorado. The objectives of the study are to evaluate:

• the forms of reduced uranium precipitated during biostimulation 

• biogeochemical processes occurring during biostimulation

• remobilization of bioreduced uranium upon return to oxic conditions following 

biostimulation

• the effect of ferrous iron added to the influent on uranium removal and subsequent 

remobilization.

In addition, the reactive transport model of Fang et al. (2009) developed for field scale 

biostimulation experiments is tested for its ability to simulate column effluent in response to 

biogeochemical processes likely occurring within the column during biostimulation. 

Chapter 2 of the report provides a detailed description of the methods used for the column 

experiments and for characterization of sediments recovered from the columns.

Chapter 3 presents the results of the column experiments in the order of 1) effluent chemistry

during biostimulation, 2) effluent chemistry during oxic elution, 3) solid phase analyses of 
1-5 

sediments after biostimulation reduction and after oxic elution; 4) X-ray spectroscopic and 

electron microscopy of column sediments, and 5) characterization of microbes involved in 

biogeochemical processes occurring in the columns.

Chapter 4 uses the findings of the various components of the results to discuss 1) the 

biogeochemical processes occurring during biostimulation resulting in uranium reduction and 2) 

the rates and extent of U remobilization observed in the two columns.

Chapter 5 describes reactive transport model simulation during biostimulation of the Rifle 

sediment column experiments. The approach couples process models to describe the interplay 

between the saturated flow, microbiology, and geochemistry during acetate biostimulation. The 

model is based on the reactive transport model developed to simulation field scale 

biostimulation experiments at the Old Rifle uranium mill site (Yabusaki et al., 2007; Fang et al., 

2009). The modeling is intended to provide insight on the processes affecting uranium in the 

context of 1) transient biostimulation with acetate electron donors, 2) Fe(III)-reducing bacteria 

and sulfate-reducing bacteria responding to the biostimulation, and 3) geochemical response to 

the biostimulation products.

Chapter 6 summarizes the key findings of this study.

The laboratory column experiments and solid phase analysis were conducted at the U.S. 

Geological Survey. X-ray absorption spectroscopy and microfocused X-ray fluorescence 

measurements were collected at the Stanford Synchrotron Radiation Lightsource. Reactive 

transport modeling was conducted at Pacific Northwest Environmental Laboratory by Steve 

Yabusaki, who authored Chapter 5. 

2-1 

2 Experimental Methods and Approach

2.1 Artificial Groundwater Design and Composition 

Artificial groundwater (AGW) used for column experiments was developed to simulate the 

average major ion chemistry of groundwater sampled at the Old Rifle site. AGW composition is 

shown in Table 1. The pCO2 was fixed at 3.5% based on groundwater chemistry with dissolved 

calcium concentration iteratively adjusted in PHREEQC simulations to yield the desired pH of 

7.0 for the column influent. The resulting calculated alkalinity of 6.6E-3 M was about a factor of 

2 lower than groundwater from the field site (1.1E-2 to 1.3E-2 M). Both calcite and gypsum, 

phases present in the aquifer material, are slightly undersaturated in the AGW with log 

Saturation Index (SI) of -0.21 and -1.07, respectively. For the biostimulation stage of the column 

experiments, the AGW was amended with dissolved phosphate, ammonia, trace elements, and 

vitamins, as shown in Table 1. Dissolved uranium as U(VI) was added from a 1 mM stock 

solution prepared from dissolution of reagent grade UO3 with sulfuric acid.

AGW for columns was prepared in 3.5 L batches in tared 5-L glass reservoirs (Kontes model 

KC14395 5000) fitted with 3-hole screw caps (Kontes 953930) that were threaded for gas inlet 

and outlet, and liquid outlet tubing fittings. Calcium sulfate was dissolved in 3 L deionized water,

with appropriate volumes of concentrated salt solutions then added to yield desired final 

concentrations of all major ion components except for NaHCO3 and U(VI). The reservoir was 

then sterilized by autoclave, along with inlet and gas purging tubing assemblies. After 

autoclaving, the reservoir was sealed until cooled. The remaining AGW components and 

amendments were then added through 0.22 mm filters to sterilize them. Reservoir weight was 

recorded after each addition, and after autoclaving. For the biostimulation stage, an electron 

donor was flowed continuously through the columns to stimulate the indigenous microbes to 

produce reducing conditions to immobilize dissolved uranium. Sodium acetate solution was 

added from a 1 M stock solution prepared from sodium acetate. This stock solution was filter 

sterilized and stored at 4° C in an autoclaved serum bottle flushed with nitrogen. Influent acetate 

was 10 mM for all of column A biostimulation and for the first 70 days of biostimulation of 

column B. Influent acetate was 8 mM for the remainder of column B and for all of the 

biostimulation period of column C. After addition of all components, the AGW reservoirs were 

flushed with 3.5% pCO2 balance N2 gas mixture for one hour at ~1 L/min through a gas 

dispersion stone. The inlet tubing apparatus was installed, and the reservoir was flushed for 

another 30 minutes before attaching to a column. A continuous flow of the gas mixture was 

bubbled through the reservoir at ~50 mL/min and vented through an airlock for the duration of 

the experiment. 

Ferrous iron was added to the influent stream of column C throughout the biostimulation phase 

and to column B after 211 days of biostimulation. Iron addition was intended to produce 

additional iron sulfide to assess its effect on oxic remobilization of U that was reduced and 

precipitated during biostimulation. Iron was introduced to the influent from a second reservoir 

containing 150 mM ferrous iron from FeCl2 adjusted to pH 4 with 4N HCl. This reservoir was 

prepared as outlined above by autoclaving the deionized water, purging with a CO2/N2 gas 

mixture, and then adding a 4 mM ferrous chloride solution that was sterilized by passing it 

through a 0.22 mm filter. Anoxic ferrous iron was mixed into the influent stream from the AGW 

reservoir through a three-way T-fitting about 40 cm before the column inlet to attain a 15 mM 
2-2 

ferrous iron concentration in the influent. The pH of the mixed influents was 7.0. Travel time for 

the Fe(II)-AGW mixture between the mixing T-fitting and the column inlet was about 4.5 

minutes.

Table 1. Artificial groundwater composition

Constituent

with 

acetate 

moles/L

without 

acetate

Ca 2.40E-03 2.40E-03

Na 1.84E-02 1.04E-02

Mg 4.00E-03 4.00E-03

K 4.00E-04 4.00E-04

U 1.00E-05 1.00E-05

NH4 1.40E-04 0

Cl 3.00E-03 3.00E-03

SO4 6.40E-03 6.40E-03

Total CO3 7.79E-03 7.79E-03

acetate 8.00E-03 0

PO4 7.14E-07 0

pH 7.0 7.0

alkalinity 6.56E-03 6.56E-03

Trace elements, vitamins (biostimulation only)

CoCl2•6H2O 8.0E-07

MnCl2•4H2O 5.1E-07

ZnCl2 5.1E-07

H3BO3 9.7E-08

Na2MoO4•2H2O 1.6E-07

NiCl2•6H2O 1.0E-07

CuCl2•2H2O 1.2E-08

CoCl2•6H2O 8.0E-07

p-amino-benzoic acid 1.2E-08

biotin 2.7E-09

folic acid 1.5E-09

pyridoxine•HCl 1.6E-08

thiamine•HCl 5.0E-09

riboflavin 4.4E-09

nicotinic acid 1.4E-08

pantothenic acid 3.5E-09

thioctic acid 8.1E-09

vitamin B12 2.0E-11

The AGW used for oxic elution of columns was prepared using the same procedure and recipe 

as AGW used for the biostimulation stage except no uranium, acetate, vitamins or nutrients 

were added. The suboxic AGW reservoir was purged with the desired gas mixture (see 3.2.3).
2-3 

2.2 Column Apparatus and Operation 

Three columns (A, B, and C) of identical dimensions were packed for this study. Two columns 

(B and C) had ferrous iron added during the biostimulation. All columns were subsampled at the 

end of the biostimulation stage and the recovered sediments used for geochemical and 

microbial analyses. After subsampling, the remaining sediment was removed, homogenized, 

and repacked into a new column for oxic elution. Remobilization of sequestered uranium (reoxidation of U(IV) and desorption or dissolution of U(VI)) was tested in response to oxic 

conditions to simulate conditions in an aquifer following cessation of electron donor addition. 

This is termed the oxidation stage. A pulse of bromide tracer was passed through column C at 

the end of biostimulation, and through columns A and C at the end of oxic elution to determine 

column hydrodynamic parameters, and changes resulting from biostimulation and oxic elution. 

Because of clogging, column B was not used for oxic elution or bromide tracer tests. 

2.2.1 Columns

Columns were Soil Moisture Equipment, Inc., 1400 Tempe pressure cells modified by using 

acrylic tubes of inner dimensions of 5.1 cm ID by 6 cm length. Flow dispersing end caps with 

glass fiber filters were used for both the column inlet and outlet. The column apparatus was 

sterilized prior to packing with sediment.

2.2.2 Plumbing and Pumps

Column inlet and outlet tubing was 1/16’’ OD polypropylene with 1⁄4”-20 flangeless fittings to 

connect to 4-way valves at about 12” from column inlet and outlet fittings. A pressure gauge was 

installed at a three-way (T-) fitting upstream of the inlet 4-way valve and downstream of the 

pump. All tubing from the reservoir cap to effluent collection outlet was encased within 1⁄4” ID 

1/8” wall Tygon R3607 tubing to jacket the influent and effluent tubing. The gas mixture was 

continuously flowed thought the jacketing to minimize gas exchange and oxygen contamination. 

Tygon tubing was attached to nylon T-connectors at each fitting and at the column inlet and 

outlet with a length of Tygon connecting adjacent T- connectors to provide a continuous purge 

of the entire length of influent and effluent tubing. The jacketing purge gas was flowed at 50 

mL/min in the same direction as the AGW flow starting at the feed line from the reservoir to the 

column effluent outlet, bypassing the peristaltic pump, the pressure gauge, valves and the 

columns (Figure 1).
2-4 

Figure 1. Column set up illustrating A) column, B) effluent tubing with gas purged jacketing to minimize 

oxygen invasion and effluent collection bottle, C) influent reservoir with gas mixture purge and 

outlet lines, D) influent lines with gas jacketing , E) Effluent filter for microbial assay, housed in 

gas jacketed housing. Photographs by C. Fuller.

The influent AGW was pumped from the reservoir through the columns using an ISMATEC 8-

roller multi-channel pump fitted with 1.54 mm ID Tygon R3606 double stop tubing for pumping 

the AGW influent and with 0.44 mm ID tubing for the ferrous iron influent. Pump tubing was

attached to jacketed reservoir and inlet tubing with nylon hose barbs. All tubing and fittings were 

autoclaved before use. Influent flow rates were 0.050 mL/min for the AGW and 0.005 mL/min for 

ferrous iron.

A B C

D E
2-5 

2.2.3 Gas Phase and Oxygen Control

Commercially-prepared, high-purity gas mixtures of 3.5% CO2, 96.5% N2 were used for the 

biostimulated reduction stage of the columns. An oxygen scrubber was fitted in line to remove 

residual oxygen in the commercial mixture. Reservoirs were continuously purged with the gas 

mixture at about 50 mL/min, after the initial flush, and vented through airlocks. Jacketed tubing 

(described above) also was continuously purged with this gas mixture. For the oxidation stage,

a 3.5% CO2, 20% O2, 76.5% N2 premixed gas was used. Because of gas exchange across 

pump tubing walls resulting from pressure from pump rollers, introduction of oxygen to the 

influent stream may have occurred during biostimulation. However, aerophyllic bacteria in the 

sediment likely consumed this oxygen at or near the upstream end of the column. 

2.2.4 Column Packing

Sediments from the Old Rifle IFRC site were used for column biostimulation experiments. The 

sediment was provided by K. Williams (Lawrence Berkeley National Laboratory) that was 

collected from the aquifer from site BH-02. The site was located within the contamination zone 

from the Old Rifle uranium mill but not in areas of the aquifer where in situ biostimulation 

experiments have been conducted. The sediment was stored in a sealed jar until use, and was 

used as is. The sediment was described as a <2 mm grain size based on visual observation 

when collected. Instead, it is a silty sand which contains a significant fraction of coarser grains 

of up to ~8 mm diameter grains. No attempt was made to remove coarser material prior to the 

experimental study. Columns were packed with wet aquifer sediment in an anaerobic glove 

chamber. Starting and ending weights of the sediment container were recorded during column 

packing. Splits of the sediment were subsampled into tared glass vials to determine water 

content upon drying. Each column with its inlet and outlet fittings and tubing including 4-way 

valves at each end was weighed empty. Dead volumes of tubing, end caps and fittings were 

determined by weighing the column assembly full of deionized water and subtracting the empty

weight. 

An empty column cylinder with the inlet end cap, filter and fittings attached was mounted 

vertically with the inlet end down. Columns were oriented with outlet on top for upwards flow to 

minimize preferential flow paths. AGW was added through the inlet valve with a syringe until it 

was a few millimeters above the inlet bed support frit. The sediment mixture was added with a 

spatula to the column in increments of about 0.5 cm. A rubber policeman was used to move any 

sediment that adhered to column walls to the sediment bed. The column was tapped and 

sediment was allowed to settle for about 5 minutes before adding the next increment. AGW was 

added as needed to maintain a lens of water (1-2 mm) above the sediment. This process was 

repeated until the sediment was at the top of the acrylic cylinder. The sediment in the packed 

column was allowed to settle overnight. On the following day, any overlying water (~5 mL) was 

removed from the top of the sediment bed with an automatic pipette and retained in a tared vial 

to determine the mass of water and sediment removed. A second filter was placed on top of 

sediment and the outlet end cap and fittings were installed. Additional AGW was pushed 

through the column with a syringe until it flowed out of the outlet tubing. Inlet and outlet valves 

were then closed and the column was removed from the anaerobic chamber and weighed. 

Columns are mounted vertically with the outlet end up, and the influent and effluent plumbing 

attached to the appropriate 4-way valves. Flow of the gas mixture through the inlet and outlet 

tubing jackets was initiated. After one hour, flow of influent AGW containing dissolved U(VI), 

electron donor and other amendments, including ferrous iron for column C, was started.
2-6 

Columns A and C were repacked after subsampling in the anaerobic chamber at the end of the 

biostimulation stage (see below). The remaining sediment in the column was removed and 

homogenized. A new column was repacked with this homogenized sediment following the 

method described above, with the exception that a ~0.5 cm layer of autoclaved Ottawa standard 

sand was added first to account for the volume of sediment removed for subsampling. The 

repacked columns were then used for oxidation stage of the experiment. Because of high back 

pressure due to clogging, column B was subsampled and repacked 10 days after addition of 

ferrous iron to the influent commenced. The biostimulation was continued using this repacked 

column. The remaining composited sediment not used for repacking was retained for solid 

phase analysis and stored as described below.

2.2.5 Column Physical Parameters

Measured dimensions and weights of the columns are shown in Table 2. Column pore volume 

and porosity were determined from total sediment weight used in packing the columns, total 

weight of column minus empty weight, and the dimensional volume minus volume of bed 

support fittings, inlet and outlet tubing. Pore volume and total dry sediment mass were similar in 

the three columns, which were intended to be identical.
2-7 

Table 2. Column physical parameters

Value Parameter

Biostimulation stage

Column A Column B Column C

5.13 5.13 5.13 Column diameter, cm

6 6 6 Column length, cm

267.3 262.296 258.97 Total sediment weight, wet, in column (g)

0.14 0.12 0.14 Fraction water in sediment

226.4 231.8 220.5 Dry sediment in column (g)

37.4 37.5 37.5 Water mass in column: ignores dead volume of inlet/outlet tubing (g)

4.0 2 1.20 Tubing dead volume (cm3)

33.4 35.5 37.5 Pore volume (cm3)

124.0 124.03 124.03 Total column volume (cm3)

0.27 0.29 0.30 Porosity

2.50 2.62 2.55 Sediment density, rho (g/cm3)

2 2 2 Flow rate pore volume/day

Oxic Elution (A and C); B repacking during biostimulated reduction

Column A Column B Column C

5.13 5.13 5.13 Column diameter (cm)

6 6 6 Column length (cm)

249.9 268.82 261.1 Total weight aquifer sediment and Ottawa sand, wet, in column (g)

0.13 0.12 0.14 Fraction water in sediment

177.4 183.6 168.0 Dry aquifer sediment in column (g)

40 45 48 Dry Ottawa sand (g)

1.1 1.3 1.4 Thickness Ottawa sand (cm)

23.5 26.3 28.1 Volume of Ottawa sand zone (cm3)

9.2 10.3 11.0 Pore volume Ottawa sand, porosity 0.39

32.5 31.2 34.2 Water mass in column: ignores dead volume of inlet/outlet tubing (g)

4.0 3 4.0 Tubing dead volume (cm3)

28.5 28.2 30.2 Pore volume (cm3)

124.0 124.0 124.0 Total column volume (cm3)

100.6 97.7 95.9 Geometric volume of sediment zone, 4.9 cm

0.28 0.29 0.31 Porosity of sediment zone

37.7 38.45 41.2 Total pore volume, sediment and Ottawa sand (cm3)

0.30 0.31 0.33 Overall porosity, sediment and Ottawa sand zone

2.46 2.64 2.56 Sediment density, rho (g/cm3)

2.4 2.46 2.46 Flow rate in sediment, pore volume/day
2-8 

2.3 Column Effluent

2.3.1 Sampling

Samples of column effluent were collected continuously throughout the duration of the 

biostimulation stage of the column experiments into tared autoclaved serum bottles that were 

flushed with the 3.5% CO2 balance N2 gas mixture. The outlet tubing was fitted with a sterile 

0.22-μm pore size 25-mm diameter sterile cartridge filter with an attached hypodermic needle 

that was inserted into a 1-cm thick butyl rubber serum bottle stopper. A second needle 

connected to an airlock was inserted that allowed the headspace of the bottle to vent during 

sample collection. This collection method was intended to maintain anaerobic conditions in the 

effluent sample bottle during collection and subsequent storage. Sample bottles were changed 

every 24 to 60 hours. Filters were replaced as needed with about three dead volumes allowed 

to flow through the filter and needle before being attached to the collection bottle. Sample 

volume was determined by the weight of effluent collected, and the flow rate from volume 

divided by the duration of sample collection. Typically, every other sample was acidified to 1% 

(v/v) with nitric acid for dissolved U and dissolved cation analyses. Samples were acidified at 

least 24 hours prior to subsampling and analysis, and may have been stored at room 

temperature for up to four weeks before subsampling. The other samples were stored under 

refrigeration and were not acidified. These unacidified samples were used for analysis of 

dissolved sulfate, headspace and total dissolved carbonate, and acetate. During the first 48 

days of column A, subsamples for sulfate, acetate, and sulfide were taken for every tenth 

sample, the sample was then acidified for dissolved U and sulfate analyses. Headspace and 

total CO2 measurements did not commence until 54 days (117 pore volumes) after the start of 

column A. Separate samples for dissolved sulfide analysis were collected periodically into 5-mL 

glass vacuvials previously flushed with the gas mixture and containing 1 mL 10% (wt/v) zinc 

acetate to preserve sulfide by precipitating as zinc sulfide. Sulfide samples were collected on a 

one to two week frequency during the biostimulation stage of columns A, B and C. 

Influent reservoirs were sampled through the 4-way valve between the pump and the column 

when each new reservoir was installed.

2.3.2 Effluent Analyses

Dissolved U in effluent and batch samples was measured using a kinetic phosphorescence 

analyzer (KPA, Chemchek Instruments Model KPA1), following pretreatment of an aliquot of the 

acidified sample. The pretreatment consisted of drying on a hotplate in a glass scintillation vial, 

reconstituting in 1 mL of concentrated nitric acid, adding 7-10 drops of 30% H2O2, and heating 

to dryness. The dried sample was reconstituted in 0.1N HNO3 and sonicated prior to KPA 

analysis. The pretreatment removes constituents (e.g. chloride, acetate) that quench uranium 

phosphorescence. KPA measures only U(VI), but in the unlikely event that any dissolved or 

colloidal U(IV)was present in the effluent samples it was likely oxidized to U(VI) during the 

pretreatment process and also measured as [U]. The KPA detection limit was 1E-9M with a 

precision of ±3%. 
2-9 

Dissolved cation concentrations (Fe, Ca, K, Mg, Mn, Na, etc) in effluent samples were 

measured by inductively coupled plasma optical emission spectrometry (ICP-OES) using 

Thermo Scientific iCAP6000. The ICP-OES also measures total dissolved sulfur which includes 

both sulfate and sulfide species. ICP-OES analyses required 10-fold sample dilution because of 

high dissolved Ca and Na. The detection limit for minor elements was higher as a result and 

was 3 mM for iron with a precision of ±3%. Dissolved sulfide concentrations were measured in 

preserved samples by the colorimetric method described by (Cline, 1969) using a Milton Rov 

Spectronic 21D spectrophotometer. Dissolved sulfide detection limit was 0.2 mM with a ±4% 

precision. Dissolved sulfate in the un-acidified samples was measured using a Dionex DX500

ion chromatograph, which had a detection limit of 0.05 mM and precision of ±2%. pH and 

alkalinity were calculated from the pCO2 and total dissolved carbonate measured in the unacidified samples. This method was used because acetate and sulfide contribute to acid

neutralization during Gran titration. Headspace CO2 of un-acidified samples was measured on a 

SRI 310C TCD detector gas chromatograph prior to other subsampling from these bottles. 

Subsequently a 5-mL aliquot of the effluent sample was transferred by syringe to a N2 flushed 

serum bottle and acidified. The CO2 evolved from dissolved carbonate was measured by GC. 

Alkalinity and pH were measured in influent AGW (without acetate) samples collected by the 

same method but directly from the pump outlet. These samples gave comparable values to pH 

measured by electrode and to alkalinity determined by Gran titration on separate aliquots of this 

AGW. Acetate in the un-acidified samples was measured using a Shimadzu SIL-10AF HPLC 

with a UV detector. The acetate analysis had a detection limit of 0.5 mM and precision of ±4%.

2.4 Solid Phase Sampling 

2.4.1 Column Sectioning and Sediment Sample Preservation

Sediment samples were recovered from columns A, B and C at the end of the reduction stage 

and from columns A and C at the end of the oxidation stage for chemical analyses and microbial 

assay. Columns were dismantled and the sediment subsampled in the anaerobic glove 

chamber. Column outlet caps were removed. A cut-off 10 mL syringe barrel was used to core 

column sediment vertically from outlet to inlet at the end of the biostimulation stage (A and C), 

and after the clogging event of column B. The syringe barrel was pushed downwards to the inlet 

end while the plunger was held at the sediment surface. The sediment was extruded out of the 

syringe barrel column into ~1-cm sections with the length of the remaining sediment measured 

before and after each subsection was extruded. Each subsection was weighed and 

homogenized by mixing with a sterilized Teflon spatula. Each column subsection was about 4 to 

5 grams of dry sediment. A split of sediment was taken to determine water content upon drying. 

A second split of about 1 g was transferred to a whirlpak bag for microbial assay. The remaining 

sediment was split between a 2-mL micro-centrifuge tube for gamma spectrometry, and a glass 

scintillation vial. These containers were double bagged in Mylar pouches containing oxygen 

scrubbers. The bags were heat sealed and stored at -80° C until analyzed. Column B was 

subsampled by removing five intervals with a spatula each about 1 cm thick starting from the 

outlet. The bottom interval subsampling was stopped when the Ottawa sand layer was reached. 

The Ottawa sand was retained as a subsample. Column C at the end of the oxidation stage was 

subsectioned by this method but with the addition of two smaller subsections that appeared 

more oxidized. Column A at the end of the oxidation stage was subsampled in more detail. 

Three syringe barrels were inserted for cores. Before removing the cores, sediments 

surrounding the syringe barrels were carefully removed using a spatula, with about 3 to 5 

samples collected at about 1-cm depth increments while attempting to discriminate between 
2-10

oxidized and reduced appearing sediments (see section 3.3.1). The syringe barrels were 

removed and subsampled as described above. A total of 26 subsamples were collected from 

column A after oxic elution, of which 23 were comprised of aquifer sediment. These samples 

ranged from 1.2 to 23.1 g of wet sediment.

2.4.2 Total U Screening by Gamma Spectrometry

Total uranium content of the column subsections and the pre-column sediment was determined 

by gamma spectrometry following the method outlined in (Fuller et al., 1999) The 63 KeV

gamma emission of the 238U daughter 234Th was measured on a high resolution gamma 

spectrometer. This method assumes that the 234Th daughter is in secular equilibrium. Because 

the column C samples were measured immediately at the end of the biostimulation reduction, 

they were likely were not in secular equilibrium, the total uranium of these samples measured by 

gamma spectrometry is considered a lower limit. These total uranium values were used 

primarily to determine which samples to use for X-ray adsorption spectroscopic measurements 

and as a guide for dilutions for chemical extraction analyses. Total uranium concentrations 

determined by hot nitric acid extraction were used for mass balance calculations and for 

comparison to loading calculated by integrating the difference between influent and effluent 

dissolved U.

2.4.3 Chemical Analyses

Two to five gram splits of recovered sediment from the columns were dried at room temperature 

in the anaerobic chamber for chemical analyses. The coarse fraction of sediments (>~2 mm) 

was removed from the dried sediment by hand in the anaerobic chamber. The small sample 

size was insufficient for separation by dry sieving. The coarse fraction was retained for analysis. 

The fine fraction of the dried sediment was homogenized and a 1-gram split was ground with an 

agate mortar and pestle for nitric acid extraction and total S measurement. The unground 

remainder was used for partial chemical extraction of ferric and ferrous iron, acid volatile sulfide, 

hot nitric acid dissolution, and total carbonate analysis. A split of the original BH-02 sediment, 

sieved to <2 mm, was also analyzed by the techniques outlined below to compare with this 

sediment following biostimulation reduction and suboxic elution. This material is referred to as 

the pre-column sediment.

2.4.3.1 Extractions

Hot nitric acid and peroxide extraction was used to determine total uranium and other 

constituents such as calcium and iron. This method is based on EPA method 3050 and 

dissolves most sedimentary phases except for silicates. Briefly, 0.1 gram of the dried, ground 

sediment was placed in a tared glass vial, 3 ml of concentrated HNO3 was added and a glass 

reflux bulb placed on top of the vial. The sample was refluxed for 24 hours at 90°C. After 

cooling, 0.5 mL of 30% H2O2 was added and allowed to stand for 30 minutes. The reflux bulb 

was removed and the sample evaporated to dryness at 90°C. The dry weight was recorded and 

10 mL of 0.1N HNO3 added to reconstitute the sample. The sample was sonicated for 30 

minutes and allowed to settle overnight. An aliquot of supernatant was removed with a syringe, 

passed through a 0.22 μm filter, and diluted appropriately for analysis by KPA for U and ICP. 

The U concentration determined using this hot nitric acid extraction and the gamma 

spectrometry total U measurement generally agreed within 10%. Extraction of replicate sample 

splits was not conducted.
2-11

Extractable ferrous iron (Fe(II)) and reducible iron were determined by separate 0.5 N HCl and 

0.5 N HCl/0.25M hydroxylamine hydrochloride (HH) extractions of dried, unground <2 mm 

sediments. The difference between these two extractions has been defined operationally as the 

non-crystalline ferric iron oxides available for microbial reduction (Lovley and Phillips, 1987). 

The extractions were conducted in the anaerobic chamber on anaerobically preserved 

sediments. Briefly, 0.5 g of sediment was transferred to a tared polycarbonate Oak Ridge 

centrifuge tube and 30 mL of either 0.5 N HCl or 0.5N HCl/0.25 M hydroxylamine hydrochloride 

was added. Because of limited sample, duplicates were extracted on about every fifth sample, 

or a minimum of two per column. Tubes were mixed on an end over end shaker (12 RPM), and 

3 mL of extraction solution recovered by filtering through 0.22 μm syringe filter. Extractions with 

0.5 N HCl were sampled at 1 and 24 hours. HH extractions were sampled after 1 hour and 24 

hours. Tubes were weighed after each subsample to account for the volume removed. Fe(II) 

concentrations in the HCl extraction solutions were determined by the ferrozine colorimetric 

method (Stookey, 1970) using a Milton Roy Spectronic 21D spectrophotometer. The total iron 

concentration in the 0.5 N HCl extraction solutions was determined by adding hydroxylamine 

hydrochloride to a separate aliquot of the filtered extraction solution to reduced dissolved Fe(III), 

and analyzed by the ferrozine method. Total Fe extracted by HH was determined directly by 

ferrozine since extraction reduces Fe(III). Extractable iron measurements of replicates agreed to 

within 10%.

2.4.3.2 Total carbonate, total sulfur and acid volatile sulfur

Total carbonate content of the pre-column sediments was determined by measuring the CO2 

evolved upon acidification. Briefly, 0.5 to 1.0 g splits of dried sediment were placed into 120 mL

serum bottles which were sealed and flushed with N2. Five mL of 2N HNO3 were injected into 

the bottle after removing an equal volume of headspace. After mixing on an orbital shaker, a 60-

mL syringe fitted with a 3-way valve and a needle was inserted into the stopper. The excess gas 

pressure was allowed to displace the plunger upwards and the volume of displacement 

recorded. After closing the valve and removing the syringe from the bottle, the syringe was 

connected to the injection loop of the TCD detector GC to measure CO2 content of the gas 

phase. The percent carbonate in the solid was calculated by multiplying the gas phase CO2

concentration by the sum of the bottle volume and syringe displacement and dividing by the 

initial sample weight. Post-biostimulation total carbonate was not determined on column 

sediments since the estimated mass of calcium carbonate precipitated based on the measured 

change in dissolved calcium would not be distinguishable above the carbonate of the precolumn sediments.

Total sulfur content and S isotope ratio of the dried, ground column sediment and pre-column 

material were measured using a Carlo Erba elemental analyzer (EA) coupled to a Micromass 

Isoprime mass spectrometer. This method provides a measure of all forms of S present in the 

sample. An increase in the % total S would reflect reduction of sulfate in the AGW during 

biostimulation and precipitation of reduced sulfur species, such as FeS. The S isotopic ratio, 

expressed as δ34S, can be used as an indicator of fractionation of S isotopes during microbial 

reduction of S (Habicht and Canfield, 1997), resulting in a lighter isotopic ratio (lower δ34S) of 

the reduced S. This fractionation results from the kinetic isotope effect in which the reaction 

rates for heavier isotopes (e.g 34S) are slower resulting in a depletion of the heavier isotope in 

the product relative to the reactants. Briefly, 3 mg of the homogenized ground <2 mm sediment 

sample was weighed on a microbalance into tin capsules with approximately 2 mg of V2O5

added. The samples are combusted at 10000C. The EA traps water and separates the resulting 

CO2 and N2 from the SO2 which is measured using an IR detector and then sent to the mass 
2-12

spectrometer. The N2 and CO2 are vented. The samples are run in batches of 30 at 10 sample 

intervals. Each group of 10 samples was surrounded by four reference standards (NBS SRM 

bovine liver) run in a range of sizes to capture the range of S in the samples. A blank is included 

in the run. The standards were calibrated against Canyon Diablo Troilite (CDT) for δ34S. The 34S 

isotope composition is expressed as a difference in the ratio relative to the CDT in parts per 

thousand or per mil (‰), termed δ34S.The δ34S and %S values were corrected for instrumental 

drift. δ34S values were also corrected for oxygen contribution. One standard deviation of the 

standards throughout a run was ± 0.5 per mil (‰) or better for δ34S, and 0.01 %S. Duplicates 

measured every fifth sample agreed within 0.05% S and within 0.5 per mil δ34S.

Acid volatile sulfur (AVS) content was measured on sediments recovered from the columns, and 

the pre-column sediment using a modified version of the diffusion method of (Hsieh and Yang, 

1989). AVS is a measure of sulfide (S-2) species in the sample such as FeS that are liberated 

upon acidification forming H2S gas. The method used does not reduce more oxidized forms of S 

such as elemental sulfur or S-1 in FeS2. In the anaerobic chamber, dried column < 2 mm 

sediment samples (0.3 g) are placed in 100 mL serum bottles along with 1 mL of 1 M ascorbic 

acid to eliminate oxidation of AVS by ferric minerals (Hsieh et al., 2002). A 10 x 75 mm test tube 

containing 3 mL of alkaline zinc acetate (3% w/v zinc acetate in 2N NaOH) is placed in the 

serum bottle, with its’ opening placed upwards and resting on the shoulder of the bottle. The 

alkaline zinc acetate solution traps the H2S gas evolved on acidification forming a zinc sulfide 

precipitate. A 1-cm magnetic spin bar is placed in the bottom of the bottle. The serum bottles 

are then sealed and 10 mL of 6N HCl is injected through the serum bottle stopper with a syringe 

directly onto the sediment sample carefully avoiding contact with the zinc acetate solution. The 

bottles are removed from the anaerobic chamber and placed on a stir plate with gentle stirring. 

After 30 hours, the zinc acetate tubes are recovered. After weighing each tube, an additional 0.5 

mL of 2N NaOH is added, and the tubes are stoppered, then sonicated for 30 minutes to 

disperse the ZnS precipitate. Weights are recorded at each step to determine actual volumes. 

The resulting suspension of zinc sulfide is subsampled and sulfide is determined colorimetrically 

(see section 2.2.2). The method was tested using a reference synthetic solid (70% FeS; 30%

FeS2). The measured AVS within 24 hours was equivalent to the S in the FeS component. No 

additional S was recovered after longer equilibration times consistent with previous studies that

showed that acid without added reductants only volatilizes the S-2, and not higher oxidation 

states such as S- in FeS2, or elemental S. Replicates of this reference sample agreed to within ± 

5%, and duplicates of column samples agreed within ±10%. The detection limit for AVS by this 

method is estimated at 0.1 mmole per gram. The effect of CO2 evolved from the carbonates 

present in the ISR samples during acidification on AVS recovery by alkaline zinc acetate was 

found to be negligible. Other treatments such as acidic Cr(II) to reduce higher oxidation states of 

S were not attempted. Instead, the difference between total sulfur increase (column minus precolumn total S) and the AVS is used as a measure of higher oxidation states of S precipitated 

during the biostimulated reduction stage of the column experiments. The coarse fraction (>2 

mm) from selected column sediment subsamples also was analyzed for AVS. The entire >2 mm 

column subsample was used. Following recovery of AVS in the zinc acetate tube, a measured 

mass of the 6N HCl was recovered for analysis of U and cations. Because chloride quenches U 

phosphorescence in KPA analysis, the 6N HCl leachate required multiple drying steps with nitric 

acid to remove chloride by volatilization. No attempt was made to evaluate the effectiveness of 

the cold 6N HCl extraction relative to the hot concentrated HNO3 digest used for the ground, <2 

mm sediments.
2-13

2.4.4 Spectroscopic Measurements

Sediments recovered from sub-sectioning columns A, B and C were used for X-ray absorption 

spectroscopic (XAS) measurement to determine oxidation state and the local molecular 

structure of sequestered uranium. Column intervals for XAS measurement were selected based 

on the total U content and included samples from both the biostimulation and oxidation stages 

of the column experiments. The pre-column material was not measured because its total U was 

too low for collection of usable XAS spectra.

2.4.4.1 Bulk XAS

X-ray absorption near edge spectroscopy (XANES) and Extended X-ray Absorption Fine 

Structure (EXAFS) measurements were made at the Stanford Synchrotron Light Source (SSRL) 

beam lines (BL) 4-1 and 11-2 on anaerobically stored sediments recovered from the columns. 

Analysis of XANES spectra is used to provide a quantitative measure of the different oxidation 

states of an element in a sample. EXAFS spectra are used to derive the local atomic structure 

surrounding the element of interest in the sample by fitting the spectra based on spectra and 

structure of reference or model compounds spectra to determine the molecular scale speciation. 

Bulk XAS measurements provide information on the entire mass of the element of interest in the 

portion of sample illuminated by the X-ray beam. Beam size was typically 1 mm high by 8 mm 

wide with the sample placed at a 45° angle to the incoming X-ray beam, which resulted in an 

illumination area of 1 x 11.3 mm for the 1.2 mm thick sample.

Un-dried samples were ground with agate mortar and pestle in the anaerobic chamber, with 

AGW added to facilitate grinding. The larger grains were removed prior to grinding. The 

resulting paste of ground, wet sediment was packed into 1/32” thick polycarbonate holders with 

10 mil Kapton tape windows. Uranium LII and LIII edge fluorescence spectra were collected in an 

anaerobic stage to eliminate exposure to oxygen during sample collection. 

For XANES, a minimum of three replicate scans of fluorescence spectra of each sample was 

collected across either the U-LII or U-LIII edge using either a 30-element (BL 11-2) or 13-element 

(BL 4-1) germanium array detector. Internal calibration of the monochromator was made using 

either a Y or Mo foil placed on a second ion chamber “down-stream” of the sample stage, with 

foil edges collected at the start of each scan. The sample spectra were deadtime corrected and 

adjusted for drift of the monochromator prior to averaging. The averaged spectra were 

background subtracted and normalized using SIXPACK software (Webb, 2004). The resulting 

corrected spectra were fit to one or more reference U(IV) and U(VI) model spectra using the 

least squares fitting module in SIXPACK. Spectra of crystalline uraninite, andersonite, 

phosphuranylite and U(VI) sorbed to ferrihyrdrite provided by John Bargar, SSRL, were used for 

model compounds (personal communication). The model spectra were collected at other beam 

time sessions but also calibrated with internal reference foil for either the U-LII or U-LIII edge. 

The least squares linear combination fitting (LC) procedure yields fractional components of 

U(IV) and U(VI) in the sample. The best fit was based on the lowest residual chi squared value. 

That is, the components that yielded the minimum difference between the sample spectra and 

the fit of the components.

Bulk U EXAFS spectra were collected on a limited number of samples because of availability of 

beam time. The low uranium concentration of the column samples limited the usable data 

collection range to about k of 10 at best. Up to 12 replicate scans were collected. Spectra were 

deadtime corrected and averaged as described above. Shell by shell fits of column sediment U 
2-14

EXAFS spectra were performed to determine the speciation of sequestered uranium both at the 

end of biostimulated reduction and after oxic elution. Phase-shift and backscattering amplitude 

functions for quantitative EXAFS fitting were generated using FEFF 7.0 from the crystal 

structures of uraninite, liebigite, and autinite (Ankudinov and Rehr, 1997; Locock and Burns, 

2003; Mereiter, 1982; Wyckoff, 1963). The phase shift and backscattering functions from 

models were used for shell by shell fits of sample EXAFS spectra. The spectra were fit over the 

k-range 3-9.5 Å-1. 

2.4.4.2 Microfocused synchrotron XRF and XAS

Thin sections of column sediment were prepared for imaging by microfocused synchrotron X-ray 

fluorescence (XRF) and for discrete point XANES at the SSRL BL 2-3 and BL 10-2 X-ray 

microprobes, which provide spatial resolution of 2 and 30 mm, respectively. The microfocused 

beam XRF (mXRF) provides elemental distributions or maps of elements in a thin section. The 

mapping can be conducted at several energies across the critical X-ray absorption edge of an 

element (e.g. U) of interest to provide maps of oxidation state for the area of interest (Mayhew

et al., 2011). 

Petrographic thin sections of sediment recovered from columns were prepared by embedding 

the sediment in epoxy resin, then cut and polished to 30-mm thickness, and mounted on a 

quartz slide. Briefly, about 2 to 3 grams of the anaerobically stored sediment was dried by 

spreading into a thin layer on a 6-inch diameter paper filter in the anaerobic chamber. After 

drying, the sediment was transferred to a 10 mL polypropylene beaker with clumps gently 

broken up, as needed, if formed during drying. This made about a 0.5 cm thick layer in the 

beaker. Epotek 301 2-FL resin (Epoxy Technologies, Inc) that had been outgassed in the 

anaerobic chamber was mixed and poured onto the dried sediment covering the sediment with 

at least another 0.5 cm layer. The beakers were then placed in the anaerobic chamber airlock 

which was then evacuated to -10” Hg to remove any gas phase entrained within the dried 

sediment. The resin cured at room temperature over the next three days with additionally 

vacuum applied to maintain the desired pressure. After hardening, the resin pucks are removed 

from the beakers, labeled, and sealed in Mylar bags with oxygen scrubbers for shipment for thin 

section fabrication by Spectrum Petrographics (Vancouver, WA). Fabrication of thin sections 

entailed vertical slicing of the pucks and recast the slices into larger blocks using the same room 

temperature curing resin. Slices were cut from the block, mounted and polished using low 

oxygen and heat methods. Each thin section consisted of two slices or panels from the epoxy 

cast. The completed thin sections were transported in Mylar bags and stored in the anaerobic 

chamber until measured on the SSRL X-ray microprobes.

Thin sections were imaged using a flatbed scanner with the scanned image enlarged to provide 

a location map. The entire area of each thin section was mapped at SSRL BL10-2 using a 20-

μm nominal beam size focused through a capillary tube using 30 mm steps and 50 msec dwell 

time. Fluorescence data were collected at each pixel as the sample was stepped under the 

beam at energies of 17,100 and 17,200 eV, below and above the U LIII edge. A difference map 

(above minus below the edge) was then constructed using the Microprobe Analysis Tool Kit 

(http://home.comcast.net/~sam_webb/smak.html) to remove the contribution of Rb fluorescence 

to the U fluorescence window. Single and multi-element XRF maps depicting relative 

concentration were then constructed for each thin section to depict the distribution of U, Fe, Ca 

and other elements of interest. These maps also are used to locate areas for more detailed 

mapping at BL 2-3.
2-15

Selected areas of thin sections with higher U concentration were imaged using the SSRL BL2-3 

X-ray microprobe using a 1 mm focused monochromatic X-ray beam with fluorescence data 

collected at 50 to 200 msec dwell time as the sample was translated across the beam. The 

effective pixel size was 2 x 2 mm in most cases, and 5 x 5 mm for larger grains. The U enriched 

areas of the thin sections mapped with the X-ray microprobe were either individual grains or 

coatings on grain exteriors. Maps were collected at multiple energies across the U-LIII

absorption edge at 17,170, 17,175, 17,178, and 17,190 eV. The resulting maps were processed 

using a least squares fitting routine in the SMAK software to calculate the fraction of U(IV) and 

U(VI) at each pixel based on the contribution of each component to the normalized fluorescence 

yield at these energies in XANES spectra of model U(IV) and U(VI) compounds. This XANES 

mapping routine is further described in previous studies (Mayhew et al., 2011; Sharp et al., 

2011). U-LIII XANES and EXAFS spectra were then collected at specific points using a 1 mm 

beam spot size on thin sections that had sufficient U to provide usable data. These points were 

chosen based on apparent distribution of U(IV) and U(VI) from the XANES maps, and locations 

were optimized for maximum signal.

2.4.5 SEM-EDS Imaging

Carbon-coated epoxy-impregnated thin sections were analyzed by SEM-EDS using a JEOL 

5600 full field emission scanning electron microscope operated at 15 keV. Selected thin 

sections were imaged using scanning electron microscopy both in secondary electron (SE) and 

back scatter (BSE) modes. Because of the presence of the epoxy impregnation, the vacuum in 

the electron microscope is unlikely to have induced morphological changes in the samples. 

Elemental compositions of the grain coatings and adjacent areas of U bearing grains were 

measured using electron stimulated energy dispersive spectroscopy (EDS) using X-ray 

fluorescence.

2.4.6 Microbial Assay

Microbial characterization of sediments and effluent filters were conducted by the USGS 

microbiology laboratory in Reston, VA. The general approach taken was to extract DNA, and 

perform bacterial Terminal Restriction Fragment Length Polymorphism (TRFLP) fingerprinting 

and quantitative Polymerase Chain reaction (qPCR) analyses of Geobacter, a and sulfate 

reducing bacteria (SRB) on all samples. In addition selected samples were further characterized 

by cloning Geobacter 16S rRNA gene, and/or dsrB gene coding for the dissimilatory sulfite 

reductase involved in sulfate reduction. The intent was to 1) gain an understanding of microbial 

dynamics in these experiments as biostimulation of U(VI) reduction occurred, as well as during 

potential remobilization of uranium, 2) to assess where in the column the microbial abundances 

were highest and relate that to chemistry and solid phase analyses, and 3) determine potentially 

important Geobacter sp. and sulfate reducing organisms involved in these processes in the 

column experiments.

2.4.6.1 Column sediments

Subsamples of sediments recovered from columns A, B and C at the end of the biostimulation 

stage, and from column A after the aerobic elution were processed for microbial 

characterization. Upon recovery from the column, about one gram of wet sediment was 

transferred to a whirl pak bag using a sterilized spatula. The sediment adhering to the filters on 
2-16

the inlet (columns A, B,C) and outlet (column C) bed support also were assayed. The filters 

were folded into quarters and placed in a whirl pak bag. All samples were sealed in Mylar bags 

and frozen at -80°C. Samples were shipped on dry ice to the microbiology lab.

2.4.6.2 Effluent filters

Millipore Sterivex 0.2 μm filter capsules were installed in the column effluent line within a gas 

purged holder to limit exposure to oxygen (Figure 1E). The effluent was collected as described 

above, downstream of the Sterivex filter. Following the collection period, the filter capsules ports 

were sealed and the cartridge was stored at -80°C. Filter capsules and sediment samples were 

shipped on dry ice to the microbiology lab. Effluent filters for microbial assay were collected 

during the biostimulation stage: one for column B during sulfate reduction (days 113 to 121); two 

from column C during iron reduction (days 7 to 14) and at the onset of sulfate reduction (days 

29 to 34). In addition, an effluent filter was collected from column A after cessation of acetate 

and U inflow but under anoxic conditions (days 179 to 188).

2.4.6.3 Measurement methods

DNA Extraction. The 0.5-1 g frozen sediment samples from the column experiments were 

thawed and then extracted using the MoBio ultraclean soil DNA megaprep kit according to 

manufacturer’s instructions (MoBio, Inc., Carlsbad, CA). The solid from the flat end-cap filters 

was scraped then extracted by the same method. Sterivex capsule effluent filters were thawed 

and extracted as previously described using the Qiagen (Gentra) puregene kit with slight 

modifications. (Qiagen, Inc., Valenica, CA) (Ward et al., 2007). Briefly, 0.9 mL of lysis buffer with 

4.5 μL proteinase K was added to the filters and incubated with gentle rotation for 10 minutes at 

80°C. Volumes of solutions used in subsequent protein precipitation and DNA precipitation 

steps were scaled up accordingly (3X) to reflect the 3X volume increase in lysis step compared 

to manufacturer’s instructions (0.9 mL instead of 0.3 mL).

Bacterial 16S rRNA Gene PCR and TRFLP Fingerprinting. The polymerase chain reaction 

(PCR) technique was used to amplify bacterial DNA from samples, targeting the evolutionally 

conserved ribosomal RNA gene, 16S rRNA. This technique allows for the amplification of a 

specific segment of DNA of interest from the bulk DNA. Enough of the amplification product 

from PCR (amplicon) is produced such that it can then be detected and characterized further 

(cloning and sequencing or fingerprinting depending on the question being asked). PCR was 

performed as previously described using the 16S rRNA gene primers 46f-FAM and 519r (Jones

et al., 2006; Lane, 1991; Brunk et al., 1996). A DNA fingerprinting method, the terminal 

restriction fragment length polymorphism (TRFLP) technique was also used to look at bacterial 

community differences between samples. This technique involves the enzymatic digestion of the 

PCR product, which has a fluorescent tag. The enzyme has a specific DNA recognition 

sequence and only cuts the DNA where that sequence exists. Therefore, depending on the PCR 

product’s DNA sequences, enzyme digested products of varying size will be produced. The 

fragments from the end which contains the fluorescent tag can then be electrophoresed (size 

separated) and the fluorescence detected, generating a fragment size profile which is unique to 

that population of organisms with those sequences. This fingerprint can be compared to other 

samples to evaluate similarities and differences between the sample communities or within a 

community over time. TRFLP was performed as previously described (Jones et al., 2006). 

Briefly, PCR amplicons were digested with Mn(II), precipitated, and electrophoresed on an ABI 

310 genetic analyzer to generate fingerprint profiles. Fingerprint data were binned using the 
2-17

software R, and imported into the SAS based JMP8 statistical package (SAS, Cary, NC) for 

clustering analysis.

Real time quantitative PCR (qPCR) for Geobacter and SRB. qPCR was performed on all 

samples for Geobacter using the 16S rRNA gene primers Geo494f and Geo825r (Anderson et 

al., 1998; Holmes et al., 2007), and for SRB using the functional gene dsrB primers drp-2060f 

and dsr4r (Wagner et al., 1998; Geets et al., 2006). The procedure including cycling 

temperatures and times are as previously published (Wilson et al., 2010). 

Clone Library Construction and Sequencing. Geobacter and SRB clone libraries were 

constructed for selected samples. PCR was performed using the Geobacter specific and SRB 

specific primer sets above. PCR conditions; Geobacter 30 cycles of 94°C (30 s), 53° C (30 s), 

and 72° C (30s) followed by a 7 minute 72°C extension incubation. SRB PCR was also 30 cycle 

and conditions were identical except the annealing temperature was 56°C rather than 53°C. 

Amplicons were purified by wizard prep (Promega, Madison, WI.), and cloned into TA vector 

according to manufacturer’s instructions (Invitrogen, Carlsbad, CA). Single clones were picked 

and analyzed for insertion by PCR with M13f and M13r primers. PCR products were sequenced 

by single pass PCR sequencing (Beckman Coulter Genomics, Danvers, MA.).

Sequencing Analysis. Plasmid vectors were trimmed using the AWK program “vbgone” 

(Varnum Engineering, Seattle, WA). Sequences were oriented and aligned in Macvector 12 

using ClustalW (MacVector, Cary, NC). Phylogenetic trees were built in MacVector12 using the 

Neighbor Joining method and TamuraNei distance with bootstrapping (1000 replicates) or 

BESTTREE. Representative sequences from each phylotype were checked for highest similarity 

to sequences in Genbank database using the Basic local alignment tool (Blast).

3-1 

3 Results 

3.1 Column Effluent: Biostimulation 

3.1.1 Column A Effluent History during Biostimulated Reduction

Flow of anoxic AGW containing acetate and dissolved U through column A was initiated on July 

7, 2009, at a flow rate of 0.05 mL/min. Using the sediment pore volume of 33.4 cm3 (PV) 

calculated from the column weights (Table 2), this flow rate is equal to 2.16 pore volumes per 

day. Effluent acetate concentration equaled the influent (10 mM) over the first 17 days. A 

decrease in effluent acetate was measured starting at day 24 and continued to decrease 

through day 70, and subsequently changed little, averaging 1.5 mM (Figure 2) through day 111 

when acetate was removed from the influent. Thus, acetate is not fully consumed but was 

utilized at a constant rate in the later stage of biostimulation. 

Effluent dissolved iron increased from the onset of flow of acetate electron donor, reaching a 

maximum concentration by 15 days. Subsequently, effluent iron decreased to < 3 mM by day 25. 

Dissolved sulfide in the effluent was first measurable at day 32. Sulfide increased over the next 

20 days and was then relatively constant throughout the remainder of acetate addition, 

averaging 2.2 mM between days 43 and 111. Effluent sulfate did not change measurably over

the first 17 days, but began decreasing at day 24. By day 36 sulfate was below 3 mM and 

subsequently averaged 1.2 mM between days 43 and 111. The net loss of sulfate, the

difference between influent and effluent sulfate (6.8 mM), during this period is about 3 times 

greater than the effluent sulfide concentration indicating precipitation and retention of reduced 

sulfur within the column. 

Effluent carbonate alkalinity and pH measurements did not commence until day 53. At this time, 

effluent pH was 7.8, 0.8 pH units above the influent. Effluent pH decreased to about 7.7 by the 

end of the acetate amendment. Subsequently, effluent pH decreased to that of the influent. 

Alkalinity was also elevated between day 53 and 111, averaging 20.1±1.2 meq/L compared to 

influent alkalinity of 8 meq/L. Effluent alkalinity decreased to the influent level when acetate 

amendment was stopped. 

Because of the low solubility of reduced U(IV), dissolved U, [U], is assumed to be all in the +6 

oxidation state as U(VI). Dissolved U(VI) is dominated (>90%) by calcium uranyl carbonato 

complexes at the pH and composition of the influent and effluent. Effluent [U] increased over the 

first 3 days of the experiment to about the influent concentration. The lower effluent [U] during 

this initial period is consistent with U(VI) sorbing to aquifer sediments to attain surface 

complexation equilibrium. Effluent [U] began to decrease starting at day 10, and continued to 

decrease somewhat linearly throughout the biostimulation period. Effluent [U] was 3 mM at the 

end of biostimulation at day 111. The observed [U] decrease starting at day 10 slightly lagged 

the apparent onset of iron reduction and likely reflects microbial uranium reduction. Effluent [U] 

increased slightly from day 22 to day 30, after which it decreased through day 56 when another 

increase occurred. The first increase in [U] coincided with the onset of both decrease in effluent 

sulfate and increase in sulfide.
3-2 

Figure 2. Effluent concentrations of (top) dissolved uranium, iron, sulfide and sulfate and (bottom) pH, 

carbonate alkalinity for column A during biostimulation and during oxic elution versus time from 

start of biostimulation. Iron concentrations below the 3mM method detection limit are plotted as 

the measured value.

Overall, the column A effluent profile displays the typical sequence of microbially mediated 

redox processes with iron reduction evidenced by the increase in effluent dissolved iron 

followed by the onset of sulfate reduction and the concomitant decrease in effluent Fe. It is 

unclear from effluent concentrations whether the resulting drop in effluent iron occurred because 

the pool of reducible iron was depleted or because sulfide production limited iron solubility.

5

6

7

8

0

5

10

15

20

25

0 50 100 150 200 250 300 350

pH Alkalinity

Acetate

Effluent pH

Carbonate Alkalinity (meq/L); Acetate (mM)

Time (Days)

Acetate

Off

Uranium

Off

Oxygen

On

0

2

4

6

8

10

12

0

10

20

30

40

50

60

70

0 50 100 150 200 250 300 350

U biostimulation 

U oxic elution

Sulfide

Sulfate

Fe biostimulation

Fe oxic elution

U(VI) (

mM); Sulfide, Sulfate (mM)

 Dissolved Fe (

mM)

Time (Days)

U(VI) Influent

Column A Effluent Concentration

Acetate

Off

Uranium

Off

Oxygen

On

DL
3-3 

Following cessation of acetate input at day 111, microbial activity decreased rapidly as indicated 

by the decrease in effluent sulfide to below detection, and the return of sulfate, pH and alkalinity 

to the influent levels. Effluent [U] immediately decreased to about 1 mM when acetate input 

stopped, then increased slowly approaching 5 mM [U] over the next 36 days, at which point U 

was removed from the influent. These data indicate sequestration of [U] continued after the 

acetate amendment was stopped. However, the rate of U uptake decreased during this period

of anoxic influent containing U but not acetate. The possible processes that resulted in the 

continued U uptake after biostimulation ended are discussed below. Effluent dissolved Fe 

increased about 10 days after acetate inflow was stopped and was at 25 mM when the anoxic 

stage of the experiment was ended at day 194.

At day 148, anoxic AGW without U or acetate was passed through column A until day 194. 

During this time effluent [U] decreased from 5 to <2 uM over the first 10 days and was relatively 

constant averaging1.6 uM between day 158 and 194. At this point sediments from column A 

were subsampled and the remaining sediment homogenized and repacked into a new column 

for oxic elution with AGW. 

3.1.2 Column B Effluent History during Biostimulated Reduction

Flow of acetate and uranium amended AGW through column B was started on Sept. 11, 2009. 

The same influent reservoir used for column A also was used to supply column B during its first 

44 days of column B. The flow rate of 0.05 mL/min is equivalent to 2.02 pore volumes per day 

using the calculated pore volume of 35.5 cm3 (Table 2). The general trends in effluent 

concentrations for column B prior to inclusion of 15 mM ferrous iron in the influent are similar to 

those in column A. The influent acetate was 10 mM over the first 70 days, then was lowered to 

the intended 8 mM influent concentration for the remainder of the biostimulation experiment. 

The decrease in effluent acetate was first observed at day 22 and continued to decrease 

through day 70 (Figure 3). Subsequently effluent acetate was relatively constant averaging 

2.0±.05 mM through day 221. 

Unlike column A, dissolved iron was not detected in the effluent until 5 days after the start of the 

acetate amendment of column B. Effluent iron then increased reaching a maximum 

concentration (~70 mM) at day 19. Subsequently, effluent [Fe] decreased to < 3 mM by day 28, 

and was low, averaging 2.5 mM, throughout the remainder of the biostimulation period prior to 

addition of ferrous iron to the influent. As observed in column A, the decrease in [Fe] from the 

maximum coincided with the onset of sulfate reduction and measurable effluent sulfide. Effluent 

sulfate began to decrease at day 22 from the influent concentration of 6.4 mM, and was 2 mM 

by day 70, after which it was relatively constant averaging 1.4±0.5 mM. Sulfide was measurable 

in effluent starting at day 25, and increased through day 70. Subsequently, effluent sulfide was 

somewhat constant, but varying irregularly, averaging 5.4±1.2 mM, through day 221. Although 

the effluent sulfate during this steady state period was comparable to column A, a factor of two 

higher steady state effluent sulfide was observed in column B compared to column A.
3-4 

Figure 3. Effluent concentrations of (top) dissolved uranium, iron, sulfide and sulfate and (bottom) pH, 

carbonate alkalinity for column B during biostimulation elution versus time from start of 

biostimulation. Iron concentrations below the 3mM method detection limit are plotted as the 

measured value.

Effluent alkalinity increased from the influent level about 20% during the first 10 days followed 

by a further increase to about 20 meq/L by day 45. Alkalinity was relatively constant and 

averaged 19.7±1.1 meq/L from day 45 to day 221. Concurrently, effluent pH increased to 7.6 by 

day 38, 0.6 pH units above the influent. Effluent pH was relatively constant and averaged 

7.65±0.04 from day 45 through day 221.

Effluent [U] increased to the influent concentration (10 uM) over the first three days of the 

experiment. [U] then decreased and was 6 mM by day 16. Effluent [U] then increased to about 8 

5

6

7

8

0

5

10

15

20

25

0 50 100 150 200 250 300 350

Alkalinity

Acetate

pH

Effluent pH

Carbonate Alkalinity (meq/L); Acetate (mM)

Time (Days)

Start

Influent

Iron

Column

Repacked

0

2

4

6

8

10

12

0

10

20

30

40

50

60

70

0 50 100 150 200 250 300 350

U

S

Sulfate

Fe

U(VI) (

mM); Sulfide, Sulfate (mM)

 Dissolved Fe (

mM)

Time (Days)

Column B Effluent Concentration

Start

Influent

Iron Column

Repacked

DL

U(VI) Influent
3-5 

mM by day 32. This increase coincided with the increases in pH and alkalinity during the onset of 

sulfate reduction. Subsequently, effluent [U] decreased slowly to 4 mM by day 211, although 

somewhat irregularly. By comparison, column A effluent [U] had decreased to 3 mM after 110 

days.

Addition of 15 mM ferrous iron to the influent flow started on day 211. Within 10 days of the start 

of iron addition, the back pressure in column B had increased to above 30 PSI the limit for the 

apparatus. To resolve the back pressure, the column was subsampled in the anaerobic 

chamber, and the remaining sediment homogenized and repacked into a new column. The 

biostimulation experiment was then continued with 15 mM ferrous iron added to the acetate and 

U influent stream. Effluent [U], acetate and sulfate all increased to about half the influent 

concentration (Figure 3). During this period [U] steadily decreased while acetate and sulfate at 

these levels until 40 days later (day 260). Both subsequently decreased to <1 mM for the 

duration of the experiment. Effluent [Fe] increased for a short period of time, then was very low 

(<3 mM) for the remainder of the experiment. Effluent sulfide was not sampled until 50 days after 

repacking of column B; from this point it was about 4 mM for the remainder of the biostimulation 

experiment. Effluent [U] decreased from day 221 to about 1 mM by day 240, remained constant 

through day 270.

The column B experiment suffered from two inadvertent errors in influent preparation. First, the 

ferrous iron reservoir was replenished at day 267 but with a factor of ten lower concentration. 

This error was not corrected until day 328. Second, the AGW reservoir that also contains U and 

acetate was prepared with a much lower [U]. This lower [U] influent was used from day 273 to 

day 296, at which point it was restored to the intended 10 mM. This period coincided with near 

zero effluent [U]. Three days after the influent [Fe] was restored to the intended concentration 

on day 328, the column back pressure exceeded its limit again. The experiment was terminated 

and column sediments sampled in the anaerobic chamber for analysis. Column B thus 

underwent biostimulation with acetate for 211 days with 10 mM [U] added in the influent. The 

influent included 15 mM ferrous iron for 40 days during which time the column was repacked. 

The influent then had 1.5 mM ferrous iron for 61 days. Acetate and [U] input continued 

throughout this time but U input was much lower.

3.1.3 Column C Effluent History during Biostimulated Reduction

Flow through the third column, C, was started on March 5, 2010. This column was intended to 

be identical to columns A and B and thus its pore volume and flow rate were similar to the other 

columns (Table 2). Ferrous iron (15 mM) was added to the influent stream from the start of 

biostimulation of column C with the intent of producing additional iron sulfides to test their effect 

on U sequestration during biostimulation and on U remobilization during oxic elution. 

The effluent profile for column C is similar to both columns A and B with respect to the timing of 

the onset of both iron and sulfate reduction. Acetate was near constant for the first 20 days 

during which time effluent [Fe] increased to 70 mM (Figure 4). Subsequently, both effluent 

acetate and [Fe] decreased. Effluent [Fe] decreased to <3 mM by day 40 at which time 

measurable sulfide was observed in the effluent. Effluent sulfate decreased from the influent 

concentration between days 24 and 31, and was <2 mM by day 60. Sulfate remained at about 2 

mM though day 130. Sulfide increased to 3 mM by day 40. No effluent sulfide samples were 

collected between day 61 and day 132. Sulfide varied between 4 and 5 mM from day 132 to day 

186.
3-6 

Figure 4. Effluent concentrations of (top) dissolved uranium, iron, sulfide and sulfate and (bottom) pH, 

carbonate alkalinity for column C during biostimulation and during oxic elution versus time from 

start of biostimulation. Iron concentrations below the 3mM method detection limit are plotted as 

the measured value.

Column C effluent pH and alkalinity began increasing on day 38 concurrent with the increase in 

sulfate reduction. pH varied little from this point through day 186 averaging 7.57±0.04. Effluent 

alkalinity also was elevated during this period, varied little and averaged 17.7±1.5 meq/L.

Effluent [U] increased through the first four days to 8 mM, 2 mM less than the influent 

concentration. Effluent [U] then decreased to 3 mM by day 28 followed by an increase to 4 mM 

during the onset of sulfate reduction and higher pH and alkalinity at about day 40. After 10 days, 

[U] decreased to 2 mM over the next 10 days, and subsequently varied little through day 132. At 

0

2

4

6

8

10

12

0

10

20

30

40

50

60

70

0 50 100 150 200 250 300 350

U biostimulation

U oxic elution

Sulfide

Sulfate

Fe biostimulation

U(VI) (

mM); Sulfide, Sulfate (mM)

 Dissolved Fe (

mM)

Time (Days)

U(VI) Influent

Column C Effluent Concentration

Oxygen

On

Uranium, 

Acetate

Off

DL

5

6

7

8

0

5

10

15

20

25

0 50 100 150 200 250 300 350

Alkalinity

Acetate

pH

Effluent pH

Carbonate Alkalinity (meq/L); Acetate (mM)

Time (Days)

Uranium, 

Acetate

Off

Oxygen

On
3-7 

this point [U] decreased to near zero at day 136 through day 159 as a result of the improper 

reservoir preparation described for column B. The biostimulation stage continued using the 

correct influent [U] from day 159 through day 186. As described for column B, column C also 

received the lower influent [Fe] starting at day132 through to the end of the biostimulation stage 

of the experiment at day 186. 

3.1.4 Integrated Column Effluent Concentrations during Biostimulation

The overall characteristics of effluent profiles in all three columns indicate that the sequence of 

biogeochemical processes that occurred was the same and had similar timing. A more detailed 

comparison among columns is provided by estimating the extent of reactions from the effluent 

profiles of each column during biostimulation. The effluent water chemistry data were integrated 

to estimate total loading of uranium, reduction and dissolution of iron, loss of sulfate, production 

of sulfide, and production of total dissolved carbonate. Column B effluent changes were 

integrated to the start of ferrous iron addition. For U, sulfate, and acetate loss (or consumption) 

was calculated for each sample by subtracting the measured effluent concentration from the 

average measured concentrations in the influent reservoir. The change in concentration was 

then multiplied by the volume, in liters, of effluent sample collected. For total dissolved 

carbonate (TCO2), the production during each sample collection period is the difference 

between the measured concentration and the average influent concentration measured in 

reservoirs multiplied by the volume of effluent collected for each sample. The change in 

concentration of samples not analyzed was set to the average of adjacent measured samples 

and then multiplied by its respective sample volume. The resulting change in each constituent in 

units of millimoles (micromoles for U) in each sample was then summed over the duration of the 

biostimulation. The integrated Fe release from column C is calculated both by assuming the Fe 

added to the influent is transported conservatively through the column, and by assuming the 

influent Fe was all retained in the column. The later provides an upper limit of total iron released 

from sediments and transported out of column C. The results of the total mass of production or 

loss of these constituents from the effluent integrations are summarized in Table 3. The 

uncertainty in these integrated changes is difficult to assess but likely is greater for constituents 

that were sampled or analyzed infrequently and at irregular intervals such as acetate, sulfide 

and sulfate.

The uptake of U during biostimulation was divided by the total dry mass of sediment in the 

column and plotted as micrograms U per gram solid versus time to illustrate the cumulative 

loading of U onto the sediments (Figure 5). Normalizing U uptake by total dry weight assumes 

uniform distribution of U uptake on the entire sediment mass in the column. The normalized 

cumulative U uptake in the three columns was similar over the first 20 days. Subsequently the 

rate of uptake in column A was greater than column B through 110 days despite the same 

influent amendments and nearly identical columns. Uranium uptake continued in column A after 

the acetate amendment was ended at 110 days, and occurred at a slightly greater rate. About 

one third (20.6 umol) of total U uptake in column A occurred after acetate amendment was 

halted (Table 3). Uranium input to column A ended at day 150, after which a decrease in 

calculated U loading was observed and equaled about 8% of the total U uptake. The total U 

uptake at the end of the biostimulation stage was about 62, 74, and 106 μmol in columns A, B 

and C, respectively, which equals retention of 52, 48 and 76% of the total U added to columns 

A, B and C, respectively (Table 3). The rate of U uptake for column C was substantially greater 

than column A or B after the first 20 days. The presence of 15 μM ferrous iron in the column C 

influent was the only difference in the three experiments.
3-8 

Table 3. Integrated loss and gain from column effluent concentrations during acetate biostimulation. 

Units are millimoles except for uranium, which is in micromoles.

Constituent A effluent A sediment B effluentd B sediment C effluent C sediment

Total pore volumes 315 415 401

U input 118.3 154.8 137.0

U uptake 61.6a 48.8 73.8 62.6 106 47

U (mmol/g)e 0.27 0.32 0.51 0.16

% U retained 52 48 76

U released (oxic) 10.6 3.4 2.5 2.3

Fe reduced 0.084 9.1 0.110 9.5 0.081b

0.110c 6.4

Fe released (oxic) 0.025 n.a. n.m.

Acetate consumed 46 85 71

Total CO2 produced n.m. 160 120

Sulfate reduced 33.2 64 78

Sulfide produced 12.2 66 44

S retained 5.7 8.3 7.8

a. Total U uptake during biostimulation and after cessation of acetate. A total of 20.4 mmol U uptake 

occurred over 36 days (71 pore volumes) after acetate was removed from influent.

b. Integrated effluent iron corrected for the 15 mM influent Fe assuming that the influent Fe was transported 

through column without reactive loss.

c. Integrated effluent iron not corrected for 15 mM influent Fe. 

d. Values for column B are prior to start of ferrous iron addition to influent.

e. Uranium uptake normalized to total dry mass of sediment in column.

n.m. and n.a. stand for not measured and not applicable, respectively. 

Assuming effluent Fe is all Fe2+ released as a result of microbial iron reduction, the transport of 

iron out of the columns ranged from 0.8 to 0.11 mmoles, with greater amount for column B, 

which had a longer biostimulation period (Table 3). The peak of effluent Fe in the first 30 days 

accounted for 70 to 95% of the total effluent iron. This comparison for column C was made 

assuming the influent Fe was not transported out of the column. Effluent samples at or below 

the Fe detection limit (3 mM after dilution correction) were considered zero in these integrations.
3-9 

Figure 5. Cumulative uranium loading on column sediments during biostimulated reduction, in micromoles 

of uranium per gram of column sediment versus time.

Comparison of the integrated sulfate decrease with the integrated effluent sulfide transport out 

of the columns indicates the sulfate reduction was about equal (column B) or exceeded the 

integrated effluent sulfide by a factor of 2.7 and 1.7 for columns A and C, respectively (Table 3).

This comparison does not include solid phase sulfide, such as FeS precipitation. Inclusion of 

solid phase sulfide measured in the sediment subsamples at the end of biostimulation does not 

close this balance for columns A and C (Section 4.1.1). There may be a component of colloidal 

iron sulfide in the effluent since it can be measured by the same method used here. However, 

the effluent stream passed through a 0.22 micron filter before entering the sample vial or bottle, 

so it is assumed that sulfide measured in the samples represent dissolved (<0.22 um) sulfide. 

Uncertainty in the integrated sulfate mass because of sampling infrequency and poorly 

constrained influent sulfate concentration likely contributed to this imbalance in columns A and 

C. 

The integrated acetate consumption was greater as expected for the longer biostimulation 

period (Table 3). Oxidation of acetate to CO2 has a 1:2 reaction stoichiometry. The molar ratio of 

total CO2 produced to acetate consumed was 1.9 and 1.7 for columns B and C, respectively. 

Total CO2 was not measured in column A effluent during the first half of the biostimulation 

period. Ratios lower than 2 may be the result of other microbial pathways of acetate 

consumption such as fermentation. Alternatively, these ratios may be the result of measurement 

and integration uncertainty. Assuming sulfate reduction is the dominant electron pathway for 

acetate oxidation, acetate consumption and sulfate reduction should be equivalent. The ratio of 

acetate consumed to sulfate reduced was 1.4, 1.3 and 0.9 for columns A, B and C, respectively. 

The contributions of iron and uranium reduction to electron balance are small. The deviation of 

0.0

0.1

0.2

0.3

0.4

0.5

0 50 100 150 200 250 300

Column A

Column B

Column C

Uranium Uptake (

mmol/g sediment)

Time (Days)

Acetate

Off

Uranium

Off
3-10

up to 40% from the balance between total sulfate reduction and acetate consumption from the 

inferred reaction stoichiometry (1:1) is attributed to the uncertainties in the integrated values 

resulting from sampling infrequency and uncertainty of the influent concentrations, but may also 

result from other pathways of acetate consumption.

3.2 Oxic Elution 

The large extent of sequestration of dissolved uranium over the course of the biostimulation 

stage in all three columns suggests that stimulation of the ambient microbial population in the 

Old Rifle aquifer sediments may be an effective means of remediating dissolved uranium in 

groundwater at contaminated sites. The removal of dissolved uranium, which is in the +6 

oxidation state, likely occurred predominantly by reduction to U(IV) and precipitation, since the 

+4 oxidation state has much lower solubility than U(VI) for the groundwater chemistry of the Old 

Rifle site. Measurement of the U oxidation state and characterization of the form of the U 

removed during biostimulation is presented in section 3.4.1. Determining the stability of the 

bioreduced uranium in response to changes in groundwater chemistry after biostimulation has 

ceased, such as the return to oxic conditions, is needed to evaluate if the biostimulated 

reduction process would be an effective tool for long term remediation of U-contaminated 

shallow aquifers. Elution of columns A and C sediment was conducted using AGW at 

equilibrium with atmospheric oxygen. This oxygen level was used to assess the upper limit of 

dissolved oxygen in recharge water.

Prior to oxic elution, sediments from both columns were subsampled, and the remaining 

sediment homogenized and repacked into new columns (see section 2.2.4). This sediment is 

subsequently referred to as the composite sediment. A layer of sterilized Ottawa sand was used 

at the inlet end (bottom) of the columns to account for mass of sediment removed during 

subsampling. The new columns had a uniform distribution of sequestered uranium prior to the 

start of oxic elution. 

After repacking, anoxic AGW was flowed through column A for 1 day. Effluent [U] increased in 

the first 5 hours to 4.5 mM, well above the 1.2 uM at the end of the anoxic stage prior to 

repacking. Effluent [U] then decreased. The oxic gas mixture was introduced to the influent 

reservoir 24 hours after flow commenced through the repacked column. Effluent [U] decreased 

over the next 15 days to 1 mM, and subsequently varied little for the duration of the experiment 

averaging 0.85±0.11 mM (Figure 6). Effluent [Fe] decreased from 18 mM during the initial anoxic 

AGW flow through the repacked column then decreased further to <5 mM over the first 3 days of 

oxic AGW influent flow (Figure 2). Subsequently, [Fe] was at or near the 3 mM detection limit for 

the duration of the oxic elution stage. No measurable difference was observed between influent 

and effluent sulfate, pH, alkalinity or major cations throughout the oxic elution.
3-11

Figure 6. Effluent dissolved uranium from column A and column C during oxic elution versus days following

introduction of dissolved oxygen to column influent.

Column C effluent had a much lower [U] (0.1 mM) during the first 24 hours of flow with anoxic 

AGW after repacking. Upon introduction of oxygen to the influent reservoir, effluent [U] 

increased to ~0.4 mM (Figure 6). Over the next 30 days of oxic elution effluent [U] varied slightly 

averaging 0.34±.07 mM. Subsequently, effluent [U] decreased gradually to 0.22 mM by the end 

of the experiment 86 days later. Effluent [Fe] was equal to or less than 3 mM throughout the oxic 

elution. No measurable difference was observed between influent and effluent sulfate, pH, 

alkalinity or major cations throughout the oxic elution of column C.

Overall, remobilization of uranium from column C during oxic elution resulted in about a factor of 

4 lower integrated effluent dissolved [U] than was observed for column A. After 121 days of oxic 

elution, 2.5 mmol U, or about 3% of the total U uptake during biostimulation, was remobilized 

from column C. In contrast, 10.6 mmol, or 23% of the total U uptake, was released from column 

A over 161 days. Based on the integrated effluent, the total U uptake by column C (106 mmols) 

during biostimulated reduction was about a factor of two greater than for column A (62 mmols), 

in part because of the longer biostimulation period. In addition, the total U uptake in column C 

was substantially greater after 110 days of acetate and U(VI) inflow, 65 mmols, compared to 41 

mmols U in column A at 110 days when acetate input was halted in column A. An additional 20 

mmols of uranium uptake occurred in column A over the 36 days after the acetate amendment 

ended. 

The release of uranium from sediments over time from the two columns is shown in Figure 7. 

These plots are based on incremental loss described by the mass of U measured in each 

effluent sample subtracted from the remaining sediment U mass at the start of each sampling 
3-12

interval. The initial total sediment U concentration at the start of oxic elution was set at the 

measured total U in composite sediment used to repack the columns for oxic elution. In both 

columns the decrease in sediment loading occurred at near constant rates with the change in 

sediment U per gram versus time yielding release rates of 3.4E-9 and 1.2E-9 mol U/g 

sediment/day for columns A and C, respectively. 

Figure 7. Change in sediment uranium concentration during oxic elution, plotted as (A) micromoles U per 

gram remaining on sediment versus days since the start of suboxic elution, and (B) cumulative

mass of uranium release in micromoles versus time.

The cumulative release of U in micromoles versus time (Figure 7B) further illustrates the 

significantly greater remobilization rate of sequestered U from the column A sediments

compared to column C. In both columns the release of U is a continuous process at near 

constant rates that continues for more than 120 days or 300 pore volumes. The primary 

difference between column A and C was the addition of 15 mM ferrous iron throughout the 

biostimulation period of column C. The ferrous iron may have contributed both to the greater 

sequestration of U through abiotic reduction of U(VI) by the additional FeS produced in column 

0

2

4

6

8

10

12

0 50 100 150 200

Uranium Release (

mmoles)

Time (Days)

Column A

Column C

0.00

0.05

0.10

0.15

0.20

0.25

0 50 100 150 200

Sediment Uranium (

mmol/g)

Time (Days)

Column A

Column C

A

B
3-13

C, and to limiting remobilization of U during oxic elution likely because of competition for 

oxygen, or by armoring of sequestered U shielding it from the solution phase.

3.3 Solid Phase Analyses 

The BH-02 shallow aquifer sediment used for the column experiments consisted of a coarse 

sand with about a 20% coarser fraction (Table 4) despite being purported to be <2 mm. This 

sediment is referred to from here on as the pre-column sediment. This material also had a large 

component of silt and clay size sediment (<63 mm), 12% by weight. Grain size was determined 

by wet sieving to remove the fines (<63 mm), followed dry sieving of the remaining coarser 

material. The bulk sediment surface area determined by N2-BET was 3.4 m2

/g. The <63 mm 

fraction accounted for about 60% of the surface area. Quantitative XRD analysis and chemical 

extractions of the pre-column sediment was conducted on the <2 mm fraction. The non-clay 

fraction of pre-column sediment is dominated by quartz (>50% by weight) with lesser amounts 

of feldspars (Table 5). Iron oxides comprised about 6%, and calcite 1.6%. Gypsum was not 

detected (<0.5%). Clays comprised about 7% of the sediment with muscovite the most 

abundant.

Total carbonate by CO2 evolution on acidification was 1.6% as CO3, or 2.7 % as CaCO3, which 

is comparable to 1.6% calcite determined by XRD. The pre-column sediment total U was 1.7 

mg/g or 0.007 mmol/g indicative of uncontaminated sediment. Extractable Fe and total S content 

are presented in Table 6 and discussed in relation to measurements of column sediment after 

biostimulated reduction and after oxic elution. 
3-14

Table 4. Grain size distribution of Rifle BH-02 sediment used in column experiments, as determined by 

sieve analysis

Grain Size Weight % % finer Surface Area

m2/g Fraction of

surface areaa

>2 mm 18.5 81.5 n.m.

1 - 2 mm 9.7 71.9 2.25 0.06

0.5 - 1 mm 30.8 41.0 1.60 0.14

250 - 500 mm 22.0 19.0 1.87 0.12

125 - 500 mm 3.1 15.9 2.20 0.02

63 - 125 mm 4.3 11.6 2.52 0.03

<63 mm 11.6 0 18.3 0.62

Bulk sedimentb 3.43

a. calculated from weight % times surface area of fraction divide by bulk sediment surface area. 

b. calculated from sum of measured surface area of grains size fractions times fraction of total mass

Table 5. Mineral weight percent of Rifle BH-02 sediment used in column experiments (pre-column) as 

determined by quantitative X-ray diffraction.

Mineral Weight %

Non-Clays

Quartz 53.3

Kspar (ordered Microcline) 6.8

Kspar (orthoclase) 6.2

Plagioclase (albite, var. cleavelandite) 8.2

Plagioclase (oligoclase; NC) 3.2

Plagioclase (oligoclase; Norway) 1.5

Plagioclase (andesine) 5.2

Plagioclase (anorthite) 2.1

Calcite 1.6

Ankerite 0.7

Magnetite 0.6

Hematite (fine grind) 1.7

Ferrihydrite (Humbug Creek) 4.0

Total non-clays 95.0

Clays

Halloysite 1.1

Kaolinite 0.7

Biotite 0.0

Phlogopite 0.3

Muscovite 5.5

Total clays 7.5

Total 102.5
3-15

3.3.1 Column Sediment Subsamples Collected 

At the end of biostimulation, column sediments appeared uniformly black, consistent with iron 

sulfide precipitation, in contrast to light brown color prior to biostimulation (Figure 8). During oxic 

elution, sediments changed from the black to brown. 

Sediment samples recovered from the columns are listed in Tables 6, 7 and 8. Subsamples 

from columns A and C were collected after biostimulated reduction by coring the column down 

the center axis from the outlet end to inlet. Column B was also sampled by coring with a syringe 

barrel when it was disassembled to repack because of clogging shortly after the onset of ferrous 

iron input. Sediment surrounding the bottom of column B subcore near the inlet was collected. 

After removing the subcore, the remaining sediment was removed, subsampled, and then mixed 

to form the composite used for repacking for oxic elution. The excess composite was retained. 

Column B was sampled again at the end of biostimulation by removing the entire sediment in 

approximate 1 cm intervals until the Ottawa sand bed at the inlet end was reached. Column B 

sediment at the end of biostimulation appeared uniformly black in color. 

Figure 8. Column A 26 days after the start of biostimulation (A), at the end of biostimulation (B), and after 

oxic elution (C). Photographs by C. Fuller.

During oxic elution the sediments of column A changed from black to brown with the transition 

proceeding from the inlet to the outlet in an irregular fashion suggesting preferential flow (Figure 

8). Because of apparent channelized flow and distinct zones of oxidized and reduced sediment, 

twenty eight subsamples were recovered from column A after oxic elution. These included three 

syringe cores near the center of the column vertical axis starting from the outlet end (Figure 9). 

Cores were extruded in 5 intervals, each about 1 to 1.5 cm thick. The first ~1 cm of the 

subcores was in the Ottawa sand layer at the inlet end of the column. These sand intervals were 

not analyzed. Prior to removal of the cores, sediments surrounding the core barrels were 

removed in about 1-cm increments along the flow axis. These subsamples were chosen based 

on appearance to provide subsamples with differing apparent extent of oxidation (see Figure 9 

and Table 6). A total of 28 sediment samples were recovered from column A following oxic 

elution, of which 23 were comprised of the aquifer sediment, with the remainder mostly Ottawa 

sand. 

A B

C
3-16

Figure 9. Column A sediments after oxic elution during subsampling depicting location of samples and 

variability in sediment appearance. (A) Column outlet end prior to subsampling. (B) After removal 

of syringe core S1 and ~1 cm of sediment. (C) After top 2 cm removed; note clockwise rotation of 

~90 degrees. (D) After top 3 cm removed. Photographs by C. Fuller.

Column C after oxic elution was sampled using a spatula from the top downward in about 1 cm 

increments along the flow axis until the Ottawa sand layer was reached. The intervals sampled 

comprised the entire cross section of the column with the exception of the top interval. This 

interval was separated into two sections based on appearance. One portion, Cx-7, was brown 

suggesting more oxidation while the other, Cx-6, was black in color indicating it was still largely 

reducing (Figure 10). The brown portion spanned about 120 degrees of arc along the outside of 

the column and extended about 1.5 cm inwards in a wedge. The interval closest to the inlet also

was brown. The other intervals were largely black.
3-17

Figure 10. Column C at end of oxic elution, (Top) side view with degrees clockwise around column denoted 

(flow is from bottom to top); (bottom) surface of intervals view from inlet end prior to 

subsampling. Subsample IDs are shown in red. Photographs by C. Fuller.

3.3.2 Column A Sediment Chemistry after Biostimulated Reduction

Total U in the <2 mm fraction of the sediment at the end of the biostimulation stage of column A 

was much higher (0.30 mmol/g) in the first 1.5 cm interval of the syringe core compared to other 

subsamples (Figure 11). Sediment recovered from the inlet filter was almost a factor of two 

higher (0.54 mmol/g), suggesting most of the U sequestration occurred near the inlet end of the 

column. The sediment composited after the column was sub-cored, had a total U (0.26 mmol/g) 

almost as high as the core interval closet to the inlet (Figure 11; Table 6). The mass of U in 

each depth interval was calculated by multiplying the core interval sediment U concentration by 

the mass fraction of the column that depth interval represents times the total dry mass of the 

column. Summing total U of the intervals yields a whole column U content of 24 mmoles, which 

is more than a factor two lower than the whole column U mass of 57.7 mmoles based on the 

composite sediment U concentration. This difference indicates that after the first 1.5 cm of 

sediment, water largely by-passed the center of the column where the core was collected, and 

instead preferentially flowed around the outside of the center zone that was cored. The coarse

grains (>2 mm), which were also black, contained 4 to 8% of the total U of the unfractionated 

sediment but account for 18.5% of the sediment mass. Based on the <2 mm composite 

sediment U concentration and accounting for the distribution among coarse and fine grain 

sediments yields a total column U uptake of 48.8 mmoles. This is about 20% lower than U 

uptake of 61 mmoles estimated by integrating the column effluent [U] (Table 3). About ~4 mmol 

were released during the period of flow without U or acetate in the influent, which is about one 

third of the difference between the two measurements of total U.

0° 90° 90° 180°

180° 0°

270°

Cx6

Cx7

Cx2 Cx3
3-18

Figure 11. Total uranium concentration (mmol/g) of <2 mm sediment from column A after biostimulated 

reduction versus distance from inlet.

In column A, Fe(II) extracted by 0.5 N HCl for 1 hour increased during the biostimulation phase 

by 6 to 8-fold compared to the pre-column BH-02 sediment (Figure 12). This comparison is 

based on the <2 mm fraction. The extractable Fe(II) of coarser sediment was not measured but 

likely would not impact this trend. The composite sediment was higher than any of the 

subsamples but in most cases only by about 10%, which is the variability in extraction of 

replicate samples (Table 6). The whole column increase in Fe(II) was 6.6 mmoles using the 

mass weighted sum of the core intervals, or 9.1 mmoles using the composite Fe(II) 

concentration. Both are about two orders of magnitude greater than the integrated effluent iron 

indicating that a large majority of iron reduction resulted in iron sulfide precipitation or was 

Fe(III) in layer silicates reduced in place and not mobilized into solution. Reduction of Fe(III) in 

phyllosilicate minerals by dissimilatory microbial iron reduction can be significant in aquifer 

sediments (Wu et al., 2012; Lee et al., 2012; Komlos et al., 2007). However, ferrous iron 

produced by reduction of Fe(III) silicates may not be quantitatively extracted by 0.5 N HCl since 

silicate phases are not dissolved by HCl. Thus, total iron reduction may be under estimated by 

an unknown amount. Total Fe (Fe(II) plus Fe(III)) in the 0.5N HCl extracts equaled the Fe(II) in 

the column sediments, whereas the pre-column sediment was about 20% Fe(III) (Table 3). This 

finding indicates the easily extractable Fe(III)in the pre-column sediment likely was reduced 

during biostimulation.

By the end of the biostimulation of column A, total sulfur increased about 3-fold from the precolumn sediment total S (Figure 13, Table 6) and averaged 0.031±.0004 mmol/g compared to 

0.01 mmol/g for the pre-column. The increase in total S was slightly higher in the subsection 

closest to the inlet. Acid volatile sulfur (AVS) accounted for most of the increase in total S and 

was not detected in the pre-column sediment (< 0.001 mmol/g). AVS is a measure of sulfide 

(S-2) species in the sample such as FeS that are liberated upon acidification forming H2S gas. 

0.0

0.1

0.2

0.3

0.4

0.5

0.6

0 - 1.5 1.5 - 3 3 - 4.5 4.5 - 6 inlet filter composite

Uranium (

mmol/g)

Distance from inlet (cm)

Column A after Biostimulation
3-19

Figure 12. 0.5 N HCl extractable Fe(II) and total Fe (mmol/g) of <2 mm sediment from column A after 

biostimulated reduction versus distance from inlet.

Figure 13. Total and acid volatile sulfur of <2 mm sediment from column A after biostimulated reduction 

versus distance from inlet. Pre-column is for total sulfur only; acid volatile sulfur not detected.

The method used does not reduce more oxidized forms of S such as elemental sulfur (So

) or S-1

in FeS2. For example, the column A composite sediment total S increased by 0.025 mmol/g 

after biostimulation and had an AVS of 0.019 mmol/g, which accounts for about 75% of the total 

S increase. The remaining S likely is in the form of elemental S or other less reduced sulfur

species such as FeS2. On a whole column basis, total S increased by 5.7 mmoles of which 4.4 

0

10

20

30

40

50

60

0 - 1.5 1.5 - 3 3 - 4.5 4.5 - 6 inlet filter composite

Extractable Fe (

mmol/g)

Distance from inlet (cm)

Column A after Biostimulation

pre-column Fe(II) pre-column Fe(total) Fe(II) Fe(total)

0.00

0.01

0.02

0.03

0.04

0.05

0 - 1.5 1.5 - 3 3 - 4.5 4.5 - 6 inlet filter composite

Sulfur (mmol/g)

Distance from inlet (cm)

Column A after Biostimulation

Pre-column Total S Total S AVS
3-20

mmoles were AVS. By comparison, the integrated effluent sulfide was 12.2 mmoles. Combined 

these yield a total sulfate reduction of 17.9 mmoles, which is significantly lower than the loss of 

sulfate (33.2 mmoles) calculated by integrating the change in effluent sulfate. Total S in the >2 

mm sediment was not measured but AVS of the >2 mm accounted for about 20% of the 

unfractionated sediment AVS. Assuming a similar contribution of the >2 mm sediment to the 

total S would not increase the retained S sufficiently to close the difference between the two 

methods of estimating total S reduction during biostimulation. The increase in total S retained in 

the column (5.7 mmoles) is about 60% of the increase in extractable Fe(II), with AVS equal to 

about 50% of the increase in sediment Fe(II), suggesting about half of the ferrous iron increase 

is in the form of FeS. These processes are illustrated in the following reactions: 

FeS + H2S ↔ FeS2 + H2

Transformation of FeS to FeS2 by reaction with H2S is the primary pathway for pyrite formation 

under anaerobic conditions in groundwater systems (Maurer and Rittmann, 2004). 

Stable sulfur isotopes measured during the total S analyses ranged from -1.14 to -3.18 per mil 

δ34S, which represents depletion in the heavier isotope, 34S, of -0.74 to -2.78 per mil (‰) δ34S 

relative to the pre-column sediment δ34S of -0.40 ‰. The decrease in δ34S is indicative of 

fractionation of S isotopes with the lighter isotope, 32S, preferentially reduced and precipitated. 

The 34S depletion was greater for samples which had the greatest increase in total S. The δ34S 

of the S retained or precipitated in the column was calculated based on two component mass 

balance,

δ34Stot ∗ Stot = δ34Sppt ∗ Sppt + δ34Spre∗ Spre, 

where, δ34Stot, δ34Sppt, δ34Spre are the sulfur isotope ratios for the total S after biostimulation, the 

S precipitated in the column, and the S initially in the sediment prior to biostimulation, 

respectively, and Stot, Sppt, Spre are the concentration of S in the sediment after biostimulation, S 

precipitated during biostimulation, and the initial total S in the sediment.

The δ34S of the precipitated S calculated from this equation ranges from -1.59 to -4.45 per mil, 

which is a fractionation of about -1.4 to -4.3 per mil relative to the AGW sulfate δ34S of 0.01 ‰. 

By comparison, the δ34S of the total S of the column sediment would range from -0.10 to -0.15

per mil if no fractionation of the AGW sulfate S occurred during reduction. This was calculated 

using the above equation substituting the AGW sulfate δ34S (0.01 per mil) for δ34Sppt and solving 

for δ34Stot. Microbial sulfate reduction typically results in isotope fractionation on the order -20 or 

more ‰ δ34S (Habicht and Canfield, 1997), but the extent of fraction is a function of reduction 

rate and availability of electron donor and sulfate (Sim et al., 2011). In systems such as these 

column experiments where there is a continuous supply of both and neither is depleted or 

limiting, the fractionation factors are expected to be small. Thus, the observed S isotope 

fractionation, although low, is consistent with a microbial reduction process, which is expected in 

the biostimulation columns. 
3-21

Table 6. Sediment chemistry for column A after biostimulated reduction and after oxic elution. Table continues on the fo

Interval ID

Interval, 

cm from 

inlet Description

Fraction 

of 

columna

Fraction

>2 mm

U 

gammab 

mmol/g

U 

<2 mm 

mmol/g

Interval 

U 

<2 mm 

mmolc

U >2 

mm 

mmol/

BH-02 pre-column sediment 0.007

Column A Post Reduction total dry sediment weight: 226.4 g

Ar-1 0 - 1.5 syringe core interval 0.25 0.39 0.220 0.297 17.0 

Ar-2 1.5 - 3 syringe core interval 0.24 0.26 0.163 0.026 1.4 

Ar-3 3 - 4.5 syringe core interval 0.23 0.15 0.016 0.8 

Ar-4 4.5 - 6 syringe core interval 0.28 0.16 0.159 0.082 5.1 0.025 

Ar-5 inlet filter inlet filter scrape 0.31 0.341 0.540 0.045 

Ar-6 compositef remainder of column 1.00 0.21 0.254 0.258 0.087 

Total of core intervalsg 24.4 

Total from compositeh 57.7 

a. Fraction of whole column dry sediment mass contained in sample interval. 

b. Total uranium by gamma spectrometry. 

c. Total U, Fe, or S in an interval calculated from <2 mm fraction concentration times interval fraction of total dry sediment mass.

d. Total U concentration of sediment calculated from the U concentration of the <2 and >2mm fractions times their mass fraction.

e. Uranium of whole interval calculated from U concentration of the <2 mm and >2 mm fractions times interval fraction of total mass

mm to <2 mm U concentration of all column samples is used to estimate the U contribution of the >2 mm fraction in cases where it w

f. Sediment remaining in column after syringe cores used to re-pack column for oxic elution.

g. Total mass of U, Fe or S in column calculated as the sum of the concentration measured in each interval times the interval sedimen

h. Total mass of U, Fe or S in column calculated from the concentration of the composite times the total sediment dry mass.

i. Fe(II) concentration of <2 mm size fraction extracted using 0.5N HCl for 1 hour. 

j. One sigma standard deviation of the average of the analyses of replicate splits of sample.

k. Total Fe concentration of <2 mm size fraction extracted using 0.5N HCl for 1 hour.

l. AVS is acid volatile sulfur concentration of sediment.

m. Total AVS S concentration of sediment calculated from the AVS S concentration of the <2 and >2mm fractions times their mass fra
3-22

Table 6. continued.

Interval ID

Fe(II)

<2 mm

mmoles/g i stdevj

<2mm 

interval 

total 

Fe(II) 

mmolc

Total Fe

<2 mm

mmoles/g j stdev

Total S 

mmol/g stdevj

Interval 

total S 

mmol δ34S stdevj

AVS S

<2 mm

mmol/gl stdevj

A

mm

>2 

BH-02 7.4 0.3 9.7 0.1 0.010 0.000 -0.40 0.21 <0.002

Column A Post biostimulation

Ar-1 39.6 2.3 39.6 0.036 2.08 -2.55

Ar-2 29.7 1.6 29.7 0.025 1.37 -1.06

Ar-3 31.9 1.7 32.0 0.028 1.44 -1.14

Ar-4 43.2 2.7 43.4 0.030 1.90 -2.52 0.024 0.000 0.

Ar-5 41.0 41.7 0.033 -3.18 0.025 0.

Ar-6 47.7 3.8 47.5 4.7 0.035 0.000 -2.90 0.22 0.020 0.002 0.

Total of core intervalsg 8.3 5.65

Total from composite 10.8 7.97
3-23

Table 6. continued.

Interval, 

cm from 

inlet Description

Fraction 

of 

columna

Fraction

>2 mm

U 

gammab 

mmol/g

U 

<2 mm 

mmol/g

Interval 

U 

<2 mm 

mmolc

U 

>2 mm 

mmol/

Post biostimulation composite 0.21 60 0.258 0.087 

Column A: Post oxic elution total dry sediment weight: 177.4 g

Ax-26 1 - 3 around syringe cores S2 and S3 0.076 0.2 0.152 0.194 2.6 0.108 

Ax-24 1 - 3 1 - 3: around S1 towards S3 0.057 0.25 0.133 0.187 1.9 

Ax-25 1 - 3 1 - 3: around S1 towards S3 0.034 0.25 0.073 0.093 0.6 

Ax-12 1 - 2.3 S2 interval 0.025 0.17 0.085 0.232 1.0 0.113 

Ax-18 1 - 2 S3 interval, browner on bottom 0.020 0.24 0.158 0.234 0.8 0.103 

Ax-6 1 - 2.6 S1 interval 0.021 0.3 0.109 0.160 0.6 

Ax-23 3 - 4 mostly black, mottled brown 0.127 0.24 0.142 0.226 5.1 0.024 

Ax-22 3 - 4 around S1 and S3 0.066 0.24 0.072 0.109 1.3 0.233 

Ax-19 2 - 3.3 S3 interval, mostly black 0.025 0.22 0.153 0.276 1.2 

Ax-13 2.3 - 3.5 S2 interval 0.024 0.29 0.179 0.251 1.1 0.118 

Ax-7 2.6 - 3.8 S1 interval 0.017 0.3 0.128 0.192 0.6 

Ax-10 4 - 5 around S1 0.093 0.27 0.188 0.242 4.0 0.077 

Ax-16 4 - 5 between S2 and S3 0.079 0.22 0.215 0.285 4.0 0.123 

Ax-8 3.8 - 4.9 S1 interval 0.022 0.41 0.109 0.113 0.4 

Ax-20 3.3 - 4.7 S3 interval 0.021 0.14 0.197 0.277 1.1 

Ax-14 3.5 - 4.8 S2 interval 0.021 0.25 0.140 0.246 0.9 0.101 

Ax-1 5 - 6 black below surface 0.070 0.19 0.188 0.244 3.0 0.085 

Ax-2 5 - 6 top mottled orange; black below 0.079 0.24 0.195 0.366 5.1 0.117 

Ax-3 5 - 6 between S1, S2: 0.063 0.23 0.197 0.288 3.2 

Ax-4 5 - 6 between S1, S2: orange sediment 0.007 0.222 0.165 0.2 

Ax-9 4.9 - 6 S1 interval 0.011 0.260 0.238 0.5 

Ax-15 4.8 - 6 S2 interval 0.021 0.19 0.181 0.279 1.1 

Ax-21 4.7 - 6 S3 interval 0.022 0.38 0.254 0.457 1.8 

Total of intervalsg 16.8 
3-24

Table 6. continued.

Interval ID

Fe(II) 

<2 mm

mmoles/g i stdevj

<2mm 

interval 

total 

Fe(II) 

mmolc

Total Fe

<2 mm

mmoles/g j stdevj

Total S 

mmol/g stdevj

Interval 

total S 

mmol δ34S stdevj

AVS S

<2 mm

mmol/gl stdevj

A

mm

>2 

Composite 47.7 3.8 47.5 4.7 0.035 0.000 -2.90 0.22 0.020 0.002 0.

Column A: Post oxic elution

Ax-26 29.0 0.39 30.3 0.033 0.45 -2.33 0.010 0.

Ax-24 31.7 0.32 31.7 0.027 0.27 -2.98

Ax-25 24.3 0.15 25.7 0.021 0.13 -1.39

Ax-12 31.6 0.14 32.2 0.031 0.13 -2.70 0.012 0.

Ax-18 41.1 0.15 41.6 0.036 0.13 -3.17 0.014

Ax-6 24.4 0.09 26.0 0.022 0.08 -1.87

Ax-23 32.1 0.7 0.72 32.6 0.7 0.034 0.002 0.77 -3.00 0.05 0.010 0.001 0.

Ax-22 26.1 0.31 27.5 0.023 0.27 -2.17 0.010 0.

Ax-19 38.1 0.17 38.6 0.038 0.17 -3.95 0.

Ax-13 46.7 0.20 46.7 0.033 0.14 -2.57 0.020 0.

Ax-7 26.0 0.08 28.1 0.026 0.08 -2.46

Ax-10 34.9 1.5 0.57 35.6 1.0 0.032 0.001 0.53 -3.06 0.67 0.017 0.000 0.

Ax-16 39.4 0.55 39.7 0.040 0.004 0.56 -5.09 0.019 0.000 0.

Ax-8 18.3 0.07 21.8 0.020 0.08 -1.59

Ax-20 42.0 0.16 44.4 0.046 0.17 -4.25

Ax-14 39.0 0.15 39.4 0.041 0.15 -3.76 0.

Ax-1 32.7 0.3 0.40 33.1 0.1 0.030 0.37 -3.15 0.019 0.000 0.

Ax-2 33.8 1.7 0.47 33.9 1.4 0.027 0.38 -2.45 0.016 0.

Ax-3 32.2 0.36 30.8 0.030 0.33 -2.98

Ax-4 33.6 0.04 33.2 0.022 0.03 0.27

Ax-9 27.7 0.05 27.7 0.024 0.05 -2.60

Ax-15 39.8 0.15 39.6 0.038 0.14 -3.07

Ax-21 37.6 0.15 38.1 0.037 0.14 -4.95

Total of intervalsg 5.8 5.56
3-25

Table 7. Sediment chemistry for column B during and after biostimulated reduction. See Table 6 for footnote description

the following page.

Interval ID

Interval, 

cm from 

inlet Description

Fraction 

of 

columna

Fraction

>2 mm

U 

gammab 

mmol/g

U 

<2 mm 

mmol/g

Interval 

U 

<2 mm 

mmolc

U 

>2 m

mmo

Column B: during biostimulation

total dry sediment weight: 231.8 g

B1-5 0 - 0.2 ~1 -2 mm on core hole bottom 2.27 n.a.

B1-6 0 - 0.5 surrounding core hole ~ 5 mm 0.26 0.756 1.030

B1-1 0 - 1.5 syringe core interval 0.28 0.34 0.102 0.085 5.6 0.03

B1-2 1.5 - 3 syringe core interval 0.26 0.19 0.024 0.030 1.8 

B1-3 3 - 4.5 syringe core interval 0.22 0.22 0.019 0.024 1.2 0.00

B1-4 4.5 - 6 syringe core interval 0.24 0.21 0.043 0.072 4.1 0.02

B1-8 Compositef surrounding core 1 0.28 0.202 0.331 0.05

Total of core intervalsg 12.6 

Total from compositeh 76.8 

Column B: after biostimulation (end of experiment)

total dry sediment weight: 183.6 g

Br-9 0 - 2 Ottawa sand: not analyzed 0.204

B-F4 2 - 3 black sediment 0.172 0.19 0.350 0.353 11.2 0.12

Br-6 3 - 4 black sediment 0.208 0.25 0.298 0.510 19.5 

Br-4 4 - 5 black sediment 0.171 0.26 0.251 0.351 11.0 0.12

Br-1 5 - 6 black sediment 0.245 0.14 0.354 0.390 17.6 0.17

B-F4 fines fines separated from bulk 1.781

Total of intervalsg 59.2 
3-26

Table 7. continued.

Interval ID

Fe(II) 

<2 mm

mmoles/g i stdevj

<2mm 

interval 

total 

Fe(II) 

mmolc

Total Fe

<2 mm

mmoles/g j stdevj

Total S 

mmol/g stdevj

Interval 

total S 

mmol δ34S stdevj

AVS S

<2 mm

mmol/g l stdevj

A

>2 

mm

Column B: initial subsampling during biostimulation

B1-5 47.4 47.6

B1-6 52.4 52.7 0.052 -2.86

B1-1 42.3 2.78 42.5 0.037 2.44 -0.33 0.026 0.

B1-2 40.3 2.40 40.3 0.036 2.17 -0.46

B1-3 39.0 1.97 39.0 0.039 1.99 -0.49 0.028 0.

B1-4 50.1 2.81 50.3 0.046 0.001 2.60 -1.40 0.04 0.032 0.

B1-8 48.3 1.2 48.5 1.2 0.046 0.030 0.02 0.

Total of core intervalsg 10.0 9.2

Total from composite 11.2 10.7

Column B: after biostimulation (end of experiment)

Br-9

B-F4 51.9 1.1 1.64 52.1 0.0 0.056 1.78 -1.98 0.027 0.000 0.

Br-6 50.6 1.93 51.2 0.070 2.65 -2.42 0.025 0.

Br-4 49.9 1.56 49.7 0.052 1.64 -2.17 0.028

Br-1 40.5 1.87 41.0 0.066 0.001 2.97 -2.17 0.01 0.029 0.001 0.

B-F4 fines 142.6 7.0 143.9

Total of all intervalsg 9.0
3-27

Table 8. Sediment chemistry for column C after biostimulated reduction and after oxic elution. See table 6 for footnote d

continues on the following page.

Interval ID

Interval, cm 

from inlet Description

Fraction 

of 

columna

Fraction

>2 mm

U 

gammab 

mmol/g

U 

<2 mm 

mmol/g

Interval 

U 

<2 mm 

mmolc

U >

mm 

mmo

Column C: Post Biostimulation total dry sediment weight: 220.5 g

Cr-4 0 - 1.7 syringe core interval 0.291 0.19 0.147 0.421 27.0 0.14

Cr-3 1.7 - 3.3 syringe core interval 0.218 0.24 0.039 0.011 0.5 0.01

Cr-2 3.1 - 4.4 syringe core interval 0.218 0.18 0.021 0.007 0.3 0.00

Cr-1 4.4 - 6 syringe core interval 0.273 0.20 0.041 0.077 4.6 0.02

Cr-5 0 - 1 surrounding core 0.167 0.18 0.406 0.580 21.3 0.34

Cr-6 1 - 2 surrounding core 0.167 0.18 0.126 0.183 6.7 

Cr-7 2 - 5 surrounding core 0.500 0.25 0.143 15.8 0.00

Cr-8 compositef composite of C5, C6, C7 1.000 0.27 0.213 47.0 0.00

Total of core intervalsg 32.5 

Total of C5, C6, C7 43.8 

Total from compositeh 47.0 

Column C: Post oxic elution total dry sediment weight: 167.8 g

Cx-2 1.4 - 2.1 brown 0.086 0.32 0.054 0.046 0.7 0.01

Cx-3 2.1 - 2.9 black 0.214 0.23 0.148 0.190 6.8 0.06

Cx-4 2.9 - 3.8 black 0.259 0.27 0.171 0.194 8.4 0.05

Cx-5 3.8 - 4.8 black 0.194 0.20 0.187 0.207 6.7 0.03

Cx-6 4.8 - 6 black 0.197 0.26 0.194 0.176 5.8 0.08

Cx-7 5 - 6 oxidized wedge at outlet 0.051 0.28 0.060 0.045 0.4 0.02

Cx-6 fines fines separated from bulk 0.965

Total of intervalsg 28.9 
3-28

Table 8. continued.

Interval ID

Fe(II) 

<2 mm

mmoles/g i stdevj

<2mm 

interval 

total 

Fe(II) 

mmolc

Total Fe 

<2 mm

mmoles/g j stdevj

Total S 

mmol/g stdevj

Interval 

total S 

mmol δ34S stdevj

AVS S

<2 mm

mmol/gl stdevj

A

>2 

mm

Column C: Post reduction

Cr-4 50.8 3.26 51.1 0.039 2.52 -3.92 0.022 0.

Cr-3 34.8 1.67 35.6 0.034 1.66 -0.08 0.012 0.

Cr-2 38.8 1.87 39.0 0.030 1.43 -0.85 0.012 0.

Cr-1 30.8 1.85 31.0 0.037 2.25 -2.34 0.019 0.

Cr-5 36.3 1.33 36.3 0.037 1.35 -2.24 0.018 0.

Cr-6 31.4 1.15 31.8 0.037 1.35 -2.37 0.019

Cr-7 40.6 4.47 39.7 0.043 4.71 -2.36 0.025 0.000 0.

Cr-8 36.6 8.06 39.4 0.046 0.003 10.08 -1.45 0.59 0.018 0.001 0.

Total of core intervalsg 8.7 7.9

Total of C5, C6, C7 7.0 7.4

Total from compositeh 8.1 10.1

Column C: Post oxic elution 

Cx-2 13.3 0.19 20.1 0.021 0.30 -0.72 0.001 0.

Cx-3 43.4 1.56 43.0 0.045 1.63 -3.82 0.022 0.

Cx-4 47.6 2.9 2.07 47.2 2.6 0.041 0.002 1.79 -3.54 0.11 0.020 0.

Cx-5 43.5 3.3 1.41 44.0 3.3 0.039 1.25 -3.80 0.023 0.001 0.

Cx-6 50.3 1.67 48.1 0.038 1.26 -3.43 0.016 0.001 0.

Cx-7 16.3 0.14 29.0 0.021 0.18 -1.57 0.004 0.

Cx-6 fines 165.3 168.3

Total of intervalsg 7.0 6.4
3-29

3.3.3 Column B Sediment Chemistry after Biostimulation

Total U in column B sediments recovered after 221 days of biostimulation was 0.33±0.01 mmol/g 

in the <2 mm composite sediment (Figure 14A, Table 7). The syringe core intervals had much 

lower total U ranging from 0.02 to 0.09 mmol/g in the <2 mm sediment (Figure 14A). The 

sediment collected near the inlet (B1-5 and B1-6) had much higher U (Table 7). Uranium in the 

bulk sediment taken from within 0.2 cm of the inlet measured by gamma spectrometry was

about 2.3 μmol/g. The >2 mm fraction accounted for 6 to 16% of the total U in all grain sizes. 

The whole column unfractionated sediment U is 62.6 mmol based on the composite sediment, 

which is comparable to the U uptake calculated from the integrated effluent, 73.8 mmol, given 

the uncertainty in integrating effluent. 

Extractable Fe(II) increased during biostimulation by 5 fold or more compared to the pre-column 

sediment, and ranged from 39 to 52 mmoles/g. Negligible Fe(III) was extracted by the 0.5N HCl 

compared to 24% for the pre-column sediment (Table 7). Higher Fe(II) was measured both near 

the inlet and outlet (Figure 15A, Table 7). As measured for column A, the increase in extractable 

Fe(II) on a whole column basis was about two orders of magnitude greater than integrated 

effluent [Fe] transported out of the column (Table 3), consistent with retention of reduced iron 

either as sulfide or in another form such as reduction in place of Fe(III) silicates. 

A 4 to 5-fold increase in total S was measured in column B sediments after 221 days of 

biostimulation (Table 7). AVS accounted for about 90% of the increase in total S. Higher total S 

was present in sediments closer to the column inlet and outlet (Figure 16A). Based on the 

composite sediment samples, the whole column total S increased by 8.3 mmoles of which 6.9 

mmoles was AVS. Integrated effluent sulfide was 66 mmoles, yielding a combined measured 

total S reduction of 74 mmoles (Table 7). By comparison, integrated effluent sulfate loss was 64 

mmoles. The increase in total S retained in the column (8.3 mmoles) is about 90% of the 

increase in extractable Fe(II), with AVS equal to about 70% of the increase in sediment Fe(II). 

The δ34S of total S decreased relative to the pre-column sediment δ34S by -0.33 to -2.86 ‰ with 

samples containing higher total S having a more negative δ34S. Calculated 34S fractionation 

factors for sulfate reduction ranged of -0.2 to -3.4 ‰ based on the change in δ34S of solid phase 

total S.

After repacking column B and continued biostimulation and U(VI) input for 110 days, sediment U 

concentration increased from 0.33 mmol/g in the composite used to repack the column to 

between 0.35 and 0.51 mmol/g for sediments recovered at the end of the experiment (Table 7, 

Figure 14B). Extractable Fe(II) did not increase significantly during the biostimulation after 

repacking suggesting that pool of reducible iron may have already been reduced prior to 

repacking (Table 7, Figure 15B). The total of the influent ferrous iron added to column B would 

result in a sediment concentration of 0.3 mmol/g, which is a small fraction of the measured 

extractable Fe(II) (39 to 50 mmol/g, Table 7). Total S increased about 15% during the second 

part of the biostimulated reduction while AVS did not change measurably from the composite

used to repack the column (Table 7, Figure 16B). Total S δ34S ranged from -1.98 to -2.42, and 

yielded 34S fraction factors ranging from -2.3 to -2.7 ‰. 
3-30

Figure 14. Total uranium concentration (mmol/g) of <2 mm sediment from column B after 221 days of 

biostimulated reduction stage prior to repacking versus distance from inlet. B. Total uranium in 

sediments at end of biostimulation experiment.

0.0

0.2

0.4

0.6

0.8

1.0

1.2

0 - 0.5 0 - 1.5 1.5 - 3 3 - 4.5 4.5 - 6 Composite

Uranium (

mmol/g)

Distance from inlet (cm)

Column B during biostimulation

0.0

0.1

0.2

0.3

0.4

0.5

0.6

2 - 3 3 - 4 4 - 5 5 - 6

Uranium (

mmol/g)

Distance from inlet (cm)

Column B at end of biostimulation

A

B
3-31

Figure 15. A. 0.5 N HCl extractable Fe(II) (mmol/g) of <2 mm sediment from column B after 221 days of 

biostimulated reduction stage prior to repacking versus distance from inlet. B. Extractable Fe(II) in 

sediments at end of biostimulation experiment.

0

10

20

30

40

50

60

0 - 0.2 0 - 0.5 0 - 1.5 1.5 - 3 3 - 4.5 4.5 - 6 Composite

Extractable Fe (

mmol/g)

Distance from inlet (cm)

Column B during biostimulation

pre-column Fe(II) pre-column Fe(total) Fe(II) Fe(total)

0

10

20

30

40

50

60

2 - 3 3 - 4 4 - 5 5 - 6

Extractable Fe (

mmol/g)

Distance from inlet (cm)

Column B at end of biostimulation

pre-column Fe(II) pre-column Fe(total) Fe(II) Fe(total)

A

B
3-32

Figure 16. A. Total and acid volatile sulfur of <2 mm sediment from column B after 221 days of biostimulated 

reduction stage prior to repacking versus distance from inlet. B. Total and acid volatile sulfur in 

sediments at end of biostimulation experiment.

3.3.4 Column C Sediment Chemistry after Biostimulation 

Total U in the <2 mm sediments recovered from column C at the end of the biostimulated 

reduction stage (186 days) ranged from the pre-column concentration (0.007 mmol/g) in the 

intermediate depth syringe core samples to 0.58 mmol/g in the sediment collected in the first 1 

cm from the inlet (Table 8, Figure 17). The distribution of sequestered U within the column 

0.00

0.01

0.02

0.03

0.04

0.05

0.06

0 - 0.5 0 - 1.5 1.5 - 3 3 - 4.5 4.5 - 6 Composite

Sulfur (mmol/g)

Distance from inlet (cm)

Column B during biostimulation

Pre-column Total S Total S AVS

0.00

0.01

0.02

0.03

0.04

0.05

0.06

0.07

0.08

2 - 3 3 - 4 4 - 5 5 - 6

Sulfur (mmol/g)

Distance from inlet (cm)

Column B at end of biostimulation

Pre-column Total S Total S AVS

A

B
3-33

indicates uptake occurred primarily near the inlet, but based on the composite sediment U 

concentrations also occurred away from the central axis of the column (Figure 17). Thus, flow 

may be preferential along the outer part of the column. The composite of the column sediment 

had a total U of 0.21 mmol/g, which equals a column sediment U uptake of 47 mmoles (Table 8) 

or less than half of estimated U uptake from integrating the effluent (Table 3). This calculation 

assumes that the >2 mm size fraction had the same U concentration as the <2 mm sediment. 

Accounting for the U in the >2 mm fraction and its mass contribution lowers this estimate 

slightly. The much lower total U uptake determined from sediment measurements indicates 

either 1) that the composite sediment did not include all sediment from the column (for example, 

the sediment closest to the inlet), 2) some fraction of U sequestration occurred on biomass 

adhering to the inlet filters, or 3) that some of the fine grain sediment was not included either in 

the composite or in its analysis. The sediment recovered from within 2 mm of the inlet in column 

B was a factor of 10 higher than the composite for this column (Table 7). The fines separated 

from the bulk sediment were also elevated 5-fold compared to the U of the bulk <2 mm 

sediment (Table 7 and 8). Fine grain sediments adhered to containers used for storing and 

drying sediments. In addition, fines were inadvertently loss lost during removal of coarse grains. 

Lastly, coarse sediments were separated from dried samples because of insufficient mass for 

wet sieving. As a result, an unknown but likely significant amount of fine grained material may 

have adhered to the coarse sediment.

Figure 17. Column C total uranium concentration (mmol/g) of the <2 mm sediment after biostimulated 

reduction versus distance from inlet. 

As observed in the other columns, extractable Fe(II) increased 5 to 7 fold during the 

biostimulation stage, with a negligible amount of Fe(III) extracted. Extractable Fe(II) was 

elevated near the inlet by 20 to 40% compared to other samples (Figure 18). Unlike U, this 

distribution suggests iron reduction occurs throughout the column, although somewhat 

enhanced near the inlet. The whole column increase in Fe(II) was 6.4 mmoles, which is nearly 2 

orders of magnitude greater than the integrated effluent iron transported out of the column 

(Table 8). The Fe(II) added with the influent throughout the biostimulation was 0.14 mmoles, 

which is almost a factor two greater than iron exported from the column but small compared to 

the increase in sediment extractable Fe(II).

0.0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

Uranium (

mmol/g)

Distance from inlet (cm)

Column C after Biostimulation

 0-1.7 1.7-3.3 3.1-4.4 4.4-6 0-1 1-2 2-5 0-6

 Syringe Cores Composites
3-34

Figure 18. Column C sediment 0.5N HCl extractable Fe(II) and total Fe concentration (mmol/g <2 mm 

fraction) after biostimulated reduction stage versus distance from inlet. Extractable iron of 

sediment used to pack column shown for comparison and is denoted as pre-column.

Total S increased during biostimulation ranging from 3 to 4.5 times the pre-column S 

concentration (Table 8), with no obvious trend in position within the column (Figure 19). AVS 

accounted for 50 to 75% of the S increase. The total S increase on a whole column basis was 

7.8 mmoles, which combined with the integrated sulfide exported from the column yields a total 

reduced S increase of 52 mmoles. By comparison, the estimated sulfate reduction from 

integrating the decrease in effluent sulfate was 78 mmoles. The increase in total S retained in 

the column (7.8 mmoles) is about 120% of the increase in extractable Fe(II), with AVS equal to 

about 60% of the increase in sediment Fe(II). The δ34S of total S decreased relative to the precolumn sediment δ34S with samples with higher total S having a more negative δ34S, ranging 

from -0.95 to -3.92‰. Calculated 34S fractionation factors for sulfate reduction ranged of 0.2 to -

5.1‰ based on the change in δ34S of solid phase total S.
3-35

Figure 19. Column C sediment total and acid volatile sulfur following biostimulated reduction (mmol/g) 

versus distance from column inlet). Pre-column is for total sulfur only; acid volatile sulfur not 

detected.

3.3.5 Summary of Sediment U, Fe(II) and S during Biostimulation

Bioreduction resulted in precipitation and accumulation of U in each column, with between 62 

and 104 μmoles retained on the columns. In all three biostimulation columns, U uptake occurred 

primarily at or near the inlet end of the column. In contrast, iron reduction as evidenced from the 

increase in extractable Fe(II) was relatively uniform over the entire column. Increases in 

extractable Fe(II) were about two orders of magnitude greater than the integrated effluent 

dissolved iron exported from the columns. The Fe(II) added to column C was about 2% of the 

extracted Fe(II). These findings indicate most of the iron reduced was retained within the 

column either as sulfide or as Fe(II) produced by in-place reduction of phyllosilicate Fe(III)

(Komlos et al., 2007). Total S increased 5-fold or more in all three columns with fully reduced S, 

measured as AVS (e.g. S-2), contributing 50 to 75% of the total S increase. The total S increase 

ranged from 50 to 120% of increase in extractable Fe(II), with AVS equal to 50 to 70% of the 

sediment Fe(II) increase. Assuming the 0.5N HCl extraction recovers all iron reduced during 

biostimulation and that sulfur measured as AVS is all from FeS, between 30 and 50% of the 

reduced iron is in a form other than FeS. This likely includes other iron sulfides such as FeS2, or 

iron reduced in place in iron silicate minerals. Precipitation of siderite (FeCO3) is only favorable 

during the pulse of high effluent [Fe] prior to the onset of sulfate reduction, however, the kinetics 

of precipitation is slow (Jimenez-Lopez and Romanek, 2004). Small fractionation factors of -0.2 

to -5.1‰ δ34S were observed indicative of microbial sulfate reduction in systems where sulfate 

and electron donor are not limiting. 

0.00

0.01

0.02

0.03

0.04

0.05

0.06

Sulfur (mmol/g)

Distance from inlet (cm)

Column C after Biostimulation

pre-column Total S AVS

 0-1.7 1.7-3.3 3.1-4.4 4.4-6 0-1 1-2 2-5 0-6

 Syringe Cores Composites
3-36

3.3.6 Sediment Chemistry after Oxic Elution

The sediment U concentration in column A after oxic elution had an overall trend of increasing 

from the inlet to the outlet end although some samples within each depth interval had up to 60% 

lower U than the composite used to repack the column for oxic elution (Figure 20). The apparent 

greater oxidative mobilization of U from those subsamples is likely the result of preferential flow 

paths. In contrast, a few subsamples near the outlet had U concentrations up to 77% higher 

than the composite, which represents the sediment U concentration throughout the column at 

the start of oxic elution. This increase may be due to uptake of the U released from sediments 

closer to the inlet. Sediment U decreased on a whole column basis by 3.9 mmols during oxic 

elution. This U loss was calculated by summing the U mass in the <2 mm sediments for each of 

the 23 subsamples and subtracting from the total U of the composite used to repack column A. 

Accounting for both the sediment mass and the U concentration of the >2 mm fraction results in 

a lower total U loss of 3.4 mmols. By comparison, a U release of 10.6 mmols was calculated by 

integrating the effluent [U] during oxic elution. The summed sediment U mass assumes that the 

entire sediment was recovered. About 58 grams of dry material that included the 40 g Ottawa 

sand was not analyzed. This unanalyzed sediment would account for an additional 4.7 mmols if 

all its U was remobilized and transported out of the column, partially closing the difference 

between sediment and effluent estimates of the total U remobilized from the column during oxic 

elution. Because of the uncertainty in sediment recovery and mass contributions to the whole 

column sediment, it is assumed that the integrated dissolved U in the effluent during elution, 

10.6 mmols, is a better estimate of the overall U remobilization from column A. 

Extractable Fe(II) was up to 50% lower than the composite (Figure 21; Table 6). The trend of 

lower Fe(II) among column A subsamples was similar to the trend in U concentration in that 

sediment with lower U generally had lower extractable Fe(II). The decrease in Fe(II) from the 

whole column was 2.6 mmol based on the sum of Fe(II) concentration of each of the 23 

subsamples minus the initial Fe(II) of the composite sediment times the subsample mass 

fraction. The overall Fe(II) decrease represents about 30% of the Fe(II) increase during 

biostimulated reduction. The integrated effluent dissolved iron during oxic elution was 0.025 

mmol (<1% of the decrease in Fe(II)), suggesting that nearly all of the Fe(II) decrease was by 

oxidation to Fe(III) which is retained in the column. The presence of ferric oxides was apparent 

in the sediment during sectioning of column A (Figure 9). Total iron (Fe(II) + Fe(III)) extracted by 

the 0.5N HCl increased up to 10% in some of the column A post oxic elution sediments with no 

increase observed in others indicating small increase in Fe(III) defined by this extraction. Iron 

extracted by 0.25 M hydroxylamine HCl at 50° C for 30 minutes ranged from 82 to 105 mmol/g 

compared to 96 mmol/g for the composite used to repack column A after biostimulation. Since 

this extraction is effective for dissolution of amorphous Fe(III) oxides (Chao and Zhou, 1983) as 

well as some Fe(II) phases, the small change from the composite indicates that iron that was 

oxidized and retained in the column during oxic elution is more crystalline and may include reoxidation of iron in phyllosilicate phases. Komlos et al. (2007) observed that re-oxidation of 

bioreduced Fe(II) in phyllosilicates resulted in a large fraction of the Fe(III) retained in the 

silicate structure in the same form as the Fe(III) prior to bioreduction. 
3-37

Figure 20. Total uranium concentration in sediments of column A (mmol/g <2 mm fraction) after oxic elution 

versus position in column.

Figure 21. 0.5N HCl extractable Fe(II) concentration in sediments of column A (mmol/g <2 mm fraction) after 

oxic elution versus position in column. The composite sediment used to repack the column for 

oxic elution is plotted for comparison and noted as “pre-oxic”. 

0.0

0.1

0.2

0.3

0.4

0.5

0.6

1 -

3

3 -

4

4 -

5

5 -

6

Uranium (

mmol/g)

Distance from inlet (cm)

Column A after Oxic Elution

Pre-oxic Post-oxic elution

 1-3 3-4 4-5 5-6

Interval ID 26 24 25 12 18 6 23 22 19 13 7 10 16 8 20 12 1 2 3 4 9 15 21

0

10

20

30

40

50

60

1 -

3

3 -

4

4 -

5

5 -

6

Extractable Fe (

mmol/g)

Distance from inlet (cm)

Column A after Oxic Elution

Pre-oxic Fe(II) Pre-oxic Fe(total) Fe(II) Fe(total)

 1-3 3-4 4-5 5-6

Interval ID 26 24 25 12 18 6 23 22 19 13 7 10 16 8 20 12 1 2 3 4 9 15 21
3-38

Total S decreased during oxic elution by 0.7 mmol (Figure 22) with a greater loss from 

subsamples that had a greater decrease in Fe(II) and U. Loss of total S was calculated by 

summing the product of the total S in each interval times its mass, and subtracting from the total 

S at the start of oxic elution defined by the composite sediment total S times the mass of 

sediment used to repack column A. The whole column loss of total S would result in an effluent 

sulfate concentration increase of 0.06 mM if the loss was uniform over the duration of oxic 

elution. This level of increase in effluent sulfate was too low to be measurable. After oxic elution 

AVS ranged from 0.010 to 0.020 mmol/g in the <2 mm fraction and averaged 26% less than the 

composite sediment (0.020 mmol/g) used to repack the column for oxic elution (Figure 22, Table 

6). Because of insufficient sample to measure AVS on all sediment samples after oxic elution, 

the whole column mass and change in AVS could not be calculated. The ratio of AVS to total S 

corrected for pre-column sediment total S ranged from 43 to 96% and averaged 70%, compared 

to 79% for the composite. Thus, AVS decreased preferentially to total S during oxic elution. The 

oxidation of sulfide S to higher oxidation states and retention in the column cannot be 

determined from data collected. 

Figure 22. Column A <2 mm sediment total and acid volatile sulfur following oxic elution (millimoles per 

gram versus distance from column inlet. Composite is sediment used to pack column for oxic 

elution.

Total uranium remaining in column C sediment after oxic elution ranged from 0.08 to 0.21 

mmoles/g compared to 0.21 mmoles/g in the composite used to repack the column for elution 

(Figure 23; Table 8). The largest decreases in U were in the interval nearest the inlet (Cx-2), 

and in a small section of column near the outlet (Cx-7; Figures 9, 23). Both of these samples 

appeared oxidized based on the transition in color from black, characteristic of sulfide in 

reduced sediment, back to the brown hues of the pre-column sediments. The net loss of U from 

the column is estimated from the difference between the measured sediment U concentration 

before and after elution. At the start of oxic elution the column had a total U of 26.4 mmol based 

on the composite post biostimulation sediment times the dry mass of sediment used to repack 

column C. The sum of the column C post oxic elution intervals (concentration times mass) is 

24.1 mmols. The difference of 2.3 mmols is the U loss from the sediments. By comparison, 2.5 

mmols of U remobilization was determined by integrating the effluent [U] during oxic elution. 

0.00

0.01

0.02

0.03

0.04

0.05

1 -

3

3 -

4

4 -

5

5 -

6

Sulfur (mmol/g)

Distance from inlet (cm)

Column A after Oxic Elution

Composite total S Composite AVS

Total S AVS

 1-3 3-4 4-5 5-6

Interval ID 26 24 25 12 18 6 23 22 19 13 7 10 16 8 20 12 1 2 3 4 9 15 21
3-39

Note that the net loss is in good agreement for the sediment and effluent based estimates, 

despite a factor of three higher integrated effluent U loading compared to the loading based on 

the measured U concentration of the composite sediment used to re-pack column C for oxic 

elution. The possible reasons for this difference are described in section 3.3.4. 

Figure 23. Column C sediment total uranium concentration (mmol/g <2 mm fraction) after oxic elution versus 

position in column. Pre-oxic composite represents sediment from biostimulation used to pack 

column for oxic elution.

A 3-fold decrease in 0.5N HCl extractable Fe(II) compared to the composite sediment was 

measured in the sediments from near the inlet and outlet that appeared oxidized (Figure 24; 

Table 8). The total Fe measured in these two samples by this extraction indicates about a third 

to half of the remaining Fe(II) was transformed to HCl extractable Fe(III). Because effluent [Fe] 

was not detected in column C during oxic elution, the decrease in total iron in these two 

samples (0.4 mmol) likely was due to transformation to a form not extracted by 0.5N HCl. Using 

the iron detection limit of 3 mM, the upper limit of iron transported out of the column during oxic 

elution is 0.02 mmol. All the other column samples had higher Fe(II) than the composite 

sediment at the start of the oxic elution, with Fe(II) comprising all of the total HCl extractable Fe 

indicating no measurable oxidation occurred in these intervals. The total increase in Fe(II) of 

these intervals compared to the composite (1 mmol) is greater than the loss of iron from the 

inlet-end interval (0.3 mmol). The reason for the difference is unknown since there was no 

source of Fe to the column during oxic elution. The fines (<63 mm) account for 14.3% of the <2 

mm sediment mass but account for 60% of the extractable iron. To account for the apparent Fe 

increase in sample Cx-6, the composite would need to have a 35% lower mass fraction of the 

<63 mm sediments than this sample.

0.00

0.05

0.10

0.15

0.20

0.25

1.4 - 2.1 2.1 - 2.9 2.9 - 3.8 3.8 - 4.8 4.8 - 6 5 - 6

Uranium (

mmol/g)

Distance from inlet (cm)

Column C after Oxic Elution

Pre-oxic Composite Post oxic elution
3-40

Figure 24. Column C sediment 0.5N HCl extractable Fe(II) and total Fe concentration (mmol/g <2 mm 

fraction) after oxic elution versus position in column. Sediment iron prior to oxic elution plotted 

for comparison.

Similar to extractable iron, total S decreased by more than a factor of two in the two oxidized 

intervals of column C sediments. The other intervals were similar in total S to the composite with 

a small decrease closer to the outlet (Table 8; Figure 25). AVS in the oxidized interval nearest 

the inlet (Cx-2) decreased by 95% during oxic elution. By comparison total S decreased 45% 

indicating loss of AVS accounts for about 70% of decrease in the total S. The decrease in total 

S was also about 45% in the oxidized interval near the outlet (Cx-7), with an 80% decrease in 

AVS. The proportion of AVS to total S in the other intervals was similar to the composite 

material used to pack the column for oxic elution. Thus, the loss of both total S and AVS 

occurred primarily in the two intervals that appeared more oxidized.

0

10

20

30

40

50

60

1.4 - 2.1 2.1 - 2.9 2.9 - 3.8 3.8 - 4.8 4.8 - 6 5 - 6

Extractable Fe (

mmol/g)

Distance from inlet (cm)

Column C after Oxic Elution

Pre-oxic Fe(II) Pre-oxic Fe(total) Fe(II) Fe(total)
3-41

Figure 25. Column C sediment total and acid volatile sulfur following oxic elution (mmol/g vs distance from 

column inlet). Pre-oxic total and AVS represent sediment after biostimulation used to pack 

column for oxic elution.

0.00

0.01

0.02

0.03

0.04

0.05

0.06

1.4 - 2.1 2.1 - 2.9 2.9 - 3.8 3.8 - 4.8 4.8 - 6 5 - 6

Sulfur (mmol/g)

Distance from inlet (cm)

Column C after Oxic Elution

Pre-oxic Total Pre-oxic AVS Total S AVS
3-42

3.4 X-ray Absorption Spectroscopy and Imaging 

3.4.1 U Oxidation State

XANES spectra of uranium in column A, B and C sediments recovered at the end of the 

biostimulation period all are very similar to the XANES spectrum for U(IV) in the uraninite model 

compound (Figure 26). For example, both the energy of the absorption maxima and the shape 

of the near edge shoulder between 20960 and 20980 eV in U LII spectra (Figure 26A) are 

consistent with U(IV). In contrast, for U(VI) the white line is at a higher energy and the post edge 

shoulder is significantly more pronounced, as illustrated by a spectrum for andersonite U(VI) 

model compound in Figure 26. The sediments recovered from columns A and C after oxic 

elution also are similar to the U(IV) model spectrum with the exception of the column C sample 

(Cx-7) near the outlet. This sample spectrum appears intermediate to the U(IV) and U(VI) model 

compounds based on a higher white line energy and a more prevalent post-edge shoulder than 

the other samples.

The oxidation state of uranium in the column sediments was quantified by linear combination 

fitting of the background corrected and normalized XANES spectra. The best fits were obtained 

using a synthetic crystalline uraninite for the U(IV) component and andersonite (a uranyl 

carbonate mineral) for the U(VI) component. Best fits were determined by the smallest residual 

chi squared (χ2

) value of the difference between the sample spectra and the linear combination 

fit of the components in the least squares fitting routine of SIXPACK software. The sum of the 

fractions of each component in the fits may not equal exactly one owing to uncertainty in the fit. 

Fitting of U XANES spectra typically can distinguish components to 5% at best (Singer et al., 

2009), such that a component ≤ 5% cannot be detected. Uncertainty in the fit fraction of U(IV) 

and U(VI) is estimated at 0.05.

The U was essentially all in the +4 oxidation state (U(IV)) in the column A and B sediments 

recovered during or after the biostimulation period (Table 9). The U in column C sediments 

recovered after biostimulated reduction were about 90% U(IV). Following elution with oxic AGW, 

the fraction of U(IV) ranged from 0.88 to 1.0 in column A sediments and 0.82 to 1.0 in column C 

sediments (Table 9). Sediments from column C that appeared more oxidized based on change 

in color from black to brown (Cx-2, Cx-7) had a greater U(VI) component than the other 

samples, which retained the black color through to the end of oxic elution. Column A sediments 

did not have any obvious trend in U oxidation state with visual changes in color. Overall, the U 

retained by the sediments and not remobilized was largely still reduced indicating that U 

oxidized during oxic elution was primarily transported out of the columns. Thus, little U(VI) was 

retained by surface complexation to sediment surfaces such as iron oxides. The fractions of 

U(IV) and U(VI) from post biostimulation and post oxic elution shown in Table 9 support this 

conclusion.
3-43

Figure 26. U-LII (A) and U-LIII (B) bulk XANES of column sediment samples. Sample IDs correspond to those 

listed in Tables 6, 7, and 8.

A B

17140 17160 17180 17200 17220

eV

U(IV) Model

Uraninite

Cx-4

Andersonite

U(VI) Model

Ar-1

Ar-5

Ar-6

Br-F4

Cx-6

20920 20940 20960 20980 21000

eV

U(IV) Model

Uraninite

Andersonite

U(VI) Model

Ax-2

Ax-4

Ax-9

Ax-10

Ax-16

Ax-23

Ax-25

Ax-26

Ax-24

B1-5

Cx-6 fines

B1-6

B1-8

Br-F4 fines

Cr-5

Cr-6

Cx-2

Cx-7
3-44

Table 9. Fraction of U(IV) and U(VI) of column sediments determined by linear combination fits to U-LII

and LIII bulk XANES spectra.

Sample U mg/ga U(IV) U(VI)

Column A after biostimulated reduction

Ar-1 71 1.00 0.00

Ar-5 129 0.97 0.03

Ar-6 composite 62 0.99 0.01

Column average 0.99 0.01

Column A Post-oxic elution

Ax-24 44 0.88 0.12

Ax-25 17 0.88 0.12

Ax-26 36 0.95 0.05

Ax-23 34 0.88 0.12

Ax-10 45 0.94 0.06

Ax-16 51 0.92 0.08

Ax-2 46 1.00 0.00

Ax-4 53 0.99 0.01

Ax-9 62 0.93 0.07

Column average 0.93 0.07

Column C after biostimulated reduction

Cr-4 100 0.84 0.16

Cr-5 138 0.86 0.14

Cr-6 44 0.99 0.01

Column average 0.90 0.10

Column C Post-oxic elution

Cx-2 11 0.86 0.14

Cx-4 46 0.93 0.07

Cx-6 42 1.00 0.00

Cx-7 14 0.82 0.18

Cx-6 fines 230 0.94 0.06

Column average 0.91 0.09

Column B during reduction (before repack)

B1-5 540 0.98 0.02

B1-6 246 0.96 0.04

B1-8 79 1.00 0.00

Column average 0.98 0.02

Column B after biostimulated reduction

Br-F4 84 0.92 0.08

Br-F4 fines 424 0.99 0.01

Column average 0.95 0.05

a. U concentration determined by hot HNO3 extraction.
3-45

3.4.2 Speciation of Sequestered Uranium

Extended X-Ray Absorption Fine Structure (EXAFS) spectra can provide information on the 

local bonding environment of an element through analysis of the spectrum and by comparison 

to model compounds. Shell by shell fits of column sediment U EXAFS spectra (Figure 27) were 

performed to determine the speciation of sequestered uranium both at the end of biostimulated 

reduction and after oxic elution. Phase-shift and backscattering amplitude functions for 

quantitative EXAFS fitting were generated using FEFF 7.0 from the crystal structures of 

uraninite, liebigite, and autinite (Ankudinov and Rehr, 1997; Locock and Burns, 2003; Mereiter, 

1982; Wyckoff, 1963). The phase shift and backscattering functions from models were used for 

shell by shell fits of sample EXAFS spectra, with results shown in Table 10. The spectra were fit 

over the k-range 3-9.5 Å-1. 

Because linear combination fitting of the U LIII- and LII-edge XANES spectra indicated that the 

samples are essentially 100% U(IV) (with an analytical error of 5%), no contribution from uranyl 

was considered in fitting the EXAFS spectra (Table 10). Further, the first shell of oxygen atoms 

at 2.36 Å in all spectra is consistent with the U(IV)-O interatomic distance, and not U(VI). The 

EXAFS spectra fits of the first four atomic shells are in agreement with recent results of U 

speciation in reduced sediments from Rifle (Stoliker et al., 2013). The presence of a U-U pair 

correlation in the EXAFS spectra at 3.85 Å is consistent with the U-U interatomic distance in 

uraninite (O'Loughlin et al., 2010; Schofield et al., 2008; Singer et al., 2009). This suggests that 

at least some polymerized U(IV) is present. However, the fit-derived coordination number is low 

(~ 3-7) compared to bulk UO2 (12 U nearest neighbors) or nano-UO2 (8-12 nearest neighbors) 

(O'Loughlin et al., 2010; Schofield et al., 2008; Singer et al., 2009). As found by (Stoliker et al., 

2013), the low coordination number and relatively high Debye-Waller factor of the first U-U pair 

is consistent with a small, partially disordered nano-UO2-like cluster. However, in the current 

EXAFS analysis (excluding sample B1-5), a second uranium-uranium pair correlation was 

included at ~5.6 (± 0.3) Å, which is attributed to the second U-U interatomic distance in uraninite 

(5.45 Å). Excluding this pair-correlation resulted in an unaccounted contribution to the EXAFS 

spectra. This higher R shell has been previously included in the fits of EXAFS spectra of 

nanoparticulate UO2 (Schofield et al., 2008), and suggests that a higher degree of U(IV) 

polymerization is present in the current samples compared to sediments examined by Stoliker et 

al (2013). A multiple-scattering path (U-O-U-O) was included at 4.5 Å, and is consistent with 

previous fits of nano-UO2 and partially polymerized U(IV) EXAFS spectra (Stoliker et al., 2013; 

Schofield et al., 2008; Singer et al., 2009). The exclusion of this pair-correlation resulted in an 

unaccounted contribution to the EXAFS spectrum, and other single-scattering contributions at 

this distance could not reproduce this feature.

In addition to the nano-uraninite or partially polymerized uraninite, contributions from U(IV) in 

contact with organic matter were included in the fit. This contribution is similar to previously 

described “monomeric” U(IV) bound to organic matter (Bernier-Latmani et al., 2010; Sharp et 

al., 2011; Alessi et al., 2012; Bargar et al., 2013), and more recently referred to as “nonuraninite U(IV) species associated with biomass” or NUSAB (Stoliker et al., 2013). For two of 

the samples (Ar-5 and Cx-4) a shell fit in the EXAFS spectra was modeled as ~3 carbon atoms 

at ~2.9 Å, typical of the observed U-C interatomic distance for U bonded to carbonate of 2.94 Å 

and for U(IV)-organic ligand moieties (Bernhard et al., 2001; Kelly et al., 2007; Singer et al., 

2012). Given that reducing conditions are strongly biologically-mediated through continuous 

input of acetate that stimulates microbial activity, it is not surprising that U(IV) is associated with 

organic carbon. The organic matter may bind to the reduced U(IV) and inhibit further 

polymerization to nanoparticulate UO2, as suggested for monomeric U(IV) (Bernier-Latmani et 

al., 2010; Sharp et al., 2011; Alessi et al., 2012; Bargar et al., 2013). Three of the samples (Br- 
3-46

F4, B1-5, and Br-F4_fines) were fit with ~3 phosphorus atoms at ~3.15 Å, in slight contrast to 

previous work that used two separate U-P distances (Bernier-Latmani et al., 2010).Including two 

U-P distances in the EXAFS model resulted in poorer fits for this sample. Together, the U-P and 

U-U distance are consistent with previous work suggesting that partially polymerized U(IV) is 

bound to P in organic matter (Bernier-Latmani et al., 2010; Sharp et al., 2011; Alessi et al., 

2012; Bargar et al., 2013). The fit quality obtained using either the C or P shells was similar; the 

reduced chi squared values were within 10% for each sample and each of the fits matched the 

EXAFS spectra well visibly. The atomic identification and coordination environment in Table 10 

are for the fits which better modeled the second shell in the FT. Ultimately, it is not possible to 

distinguish between P and C coordination on the basis of EXAFS alone. Other backscattering 

atoms and combinations of atoms were attempted in the fit, including Al, Fe, S, Si, N, and Ca. 

Carbon or phosphorous (with only one shell, respectively) could fully account for this 

contribution to the EXAFS spectra. Fits with other backscatters in addition to, or replacing, C or 

P yielded fits with equivalent or worse reduced chi-squared values and were not statistically 

significant. Fits with equivalent reduced chi-squared values were with Si and S, and it is 

challenging to distinguish their x-ray scattering from C and P, respectively. High concentrations 

of aqueous Si during U(VI) reduction could result in the precipitation of coffinite (USiO4), 

however the second U-U pair correlation in coffinite (~6.2 Å) is longer than the fit values which 

are consistent with UO2. With respect to sulfur, a U(IV)-S mineral is unlikely, however, some 

U(IV) could be associated with S-bearing functional groups on organic matter.

Overall, the fitting results suggest a sediment containing only U(IV) as a possible mixture of both 

partially disordered nano-UO2 and NUSAB associated with organic matter. This U(IV)-bearing 

phase could exist separately from the partially polymerized nano-UO2-like U(IV) and/or the 

nano-UO2 could exist as aggregates with organic matter. Ultimately, the fits of the EXAFS 

spectra indicate that some organic matter is intimately associated with U(IV); as potential 

NUSAB species do not have a U-U pair and dilute the signal from uraninite-like species. We 

could not successfully ascertain if both species were present as independent phases.
3-47

Figure 27. Bulk EXAFS (A) and (B) their Fourier transform of column sediment samples (lines) and best fit to 

spectra (dots). Sample IDs correspond to those listed in Tables 6, 7, and 8.

4

8

12

16

20

0 1 2 3 4 5 6

R (Å)

Fourier Transform Magnitude

Nano UO2

B1-5

Cx-4

Br-F4

Br-F4 fines

Ar-5

0

5

10

15

20

25

30

3 4 5 6 7 8 9 10

k (A-1)

χ(k)•k3

A B
3-48

Table 10. EXAFS fitting parameters for column sediments. The estimated standard deviations are listed 

in parentheses, representing the errors in the last digit; values without reported errors were 

fixed during fitting. Scattering paths are derived from uraninite (U, O, and MS), and liebigite (C) 

calculated at the LII-edge.

Sample Z N a R (Å) b σ2 (Å2

) c ∆E

0 e red. χ2

 

f

Ar-5 O 8.2(5) 2.36(1) 0.011(2) 0.6(1) 18.9

C 3(1) 2.97(1) 0.007(2)

U 4(1) 3.82(2) 0.007(2)

MS d 8(2) 4.7(1) 0.005(1)

U 4(2) 5.6(1) 0.008(2)

Br-F4 O 7.9(4) 2.35(1) 0.012(2) -0.5(1) 33.0

P 3(1) 3.15(2) 0.010(5)

U 4(2) 3.81(2) 0.009(2)

MS 7(3) 4.6(1) 0.008(2)

U 5(2) 5.6(2) 0.007(3)

Cx-4 O 8.4(5) 2.34(1) 0.013(1) 1.1(2) 6.5

C 3(1) 2.93(4) 0.008(3)

U 4(1) 3.80(2) 0.009(2)

MS 12(3) 4.48(3) 0.008(4)

U 4(2) 5.6(3) 0.01(2)

B1-5 O 8.6(3) 2.35(3) 0.011(1) 0.2(3) 8.3

P 3(1) 3.16(2) 0.010(1)

U 7(1) 3.84(1) 0.015(1)

MS 18(7) 4.64(4) 0.017(3)

Br-F4 fines O 7.7(2) 2.34(1) 0.011(1) -1.8(6) 3.0

P 3(1) 3.14(2) 0.012(1)

U 3(1) 3.82(1) 0.009(2)

MS 6(3) 4.48(5) 0.010(1)

U 4(2) 5.64(3) 0.006(2)

a. CN is Coordination number

b. R is Interatomic distance

c. σ2 is theDebye-Waller factor

d. MS = multiple-scattering path: U–O–U–O 

e. ∆E

0 is the difference in threshold Fermi level between data and theory

f. Reduced χ2

, a goodness-of-fit parameter
3-49

3.4.3 Distribution of Uranium on Sediments

Micro-focused synchrotron X-ray fluorescence (mXRF) was used to map the distribution of 

uranium and other elements in thin sections of column sediments recovered after biostimulation 

and after oxic elution. Thin sections of column sediments were mapped above and below the U 

LIII edge at 30 mm spatial resolution (SSRL beam line 10-2 X-ray microprobe) to locate areas or 

grains which were enriched in uranium. Selected areas or grains with elevated U were 

examined at higher resolution (2 mm step size, SSRL beam line 2-3 X-ray microprobe) to obtain 

higher spatial resolution elemental maps. The distribution of U oxidation states was mapped at 

selected locations using multi-energy imaging across the U LIII edge, or XANES imaging. U LIII

edge XANES spectra were collected at specific points (mXANES) both to verify the XANES 

imaging approach and for comparison to bulk XANES measurements. The results of these 

measurements on three grains with U-rich coatings are described below as examples. Four 

other grains in sediments after biostimulated reduction and after oxic elution were also mapped 

by this technique and yielded comparable results.

Uranium in the pre-column sediment was not apparent in the 30-mm resolution XRF maps 

indicating that the background U is widely distributed throughout the sediment grains and not 

concentrated in grain coatings. In contrast, the 30-mm resolution mXRF maps of column 

sediment thin sections showed several grains with distinct areas of U. The U was present 

largely as discrete spots or as coatings on the exterior of grains. Examples are shown for 

column C composite sediment sample Cr-5 and column B interval Br-F4 along with optical 

scans of the thin sections (Figures 28 and 29). The range of sediment grain size is illustrated in 

the optical scans of the thin sections. Clusters of grains may, in part, be aggregates from drying 

or be natural aggregates of aquifer sediments. Some grains are rock fragments but can appear 

dominated by single minerals. Dark grains are typically iron bearing. The 30-mm resolution 

mXRF image of the Cr-5 thin section shows only one grain with a distinct U coating (Figure 28B, 

C). U in sample Br-F4 exists as coatings of varying intensity or concentration around several 

grains as well as smaller areas of one or two pixels (Figure 29). Some thin sections made from 

column intervals had no obvious areas of elevated U at the 30 mm map resolution. Fine grained 

sediments (<63 mm) are abundant (~12% by mass, Table 4) and contain 3 to 4 times higher U 

concentrations (Tables 7, 8). However, discrete particles of this size range are not evident at the 

30-mm resolution mXRF mapping resolution. SEM images of U-bearing grain coatings do not 

appear to be comprised of fine grained sediments adhering to larger grains (Section 3.4.4). 
3-50

Figure 28. mXRF maps of post-biostimulation column sediment thin section of column C sediment sample Cr5. (A) optical scan; (B) mXRF tricolor map of uranium (red), iron (green) and calcium (blue); and (C) 

mXRF map of uranium. The color intensity increases with relative concentration in the mXRF maps. 

Scale bars are in microns. Maximum counts in ( A) are 587, 3753, and 630 for U, Fe and Ca 

fluorescence, respectively.
3-51

Figure 29. mXRF maps of post-biostimulation column sediment thin section of column B sediment sample BrF4. (A) optical scan; (B) tricolor mXRF map of uranium (red), iron (green) and calcium (blue); and 

(C) mXRF map of uranium. The color intensity increases with relative concentration in the mXRF 

maps. Scale bars are in microns. Maximum counts in( A) are 1021, 3756, and 644 for U, Fe and Ca 

fluorescence, respectively.

The 2-mm resolution mXRF map of the U-coated grain in the column C thin section Cr-5 shows a 

near-contiguous coating of varying thickness ranging from 5 to 30 mm in thickness (Figure 30). 

This sediment sample was collected at the end of the biostimulation stage from near the inlet 

after removing the syringe core (Table 8). The underlying grain is a rock fragment with an iron 

rich zone. The multi-energy U XANES map, as well as point XANES spectra, shows that U is 

primarily U(IV). The U coatings overlay iron phases (Figure 30C) or possibly are intermixed with 

Fe. The coating also contains S intermixed with U. Locations of 1-mm (nominal beam size) point 

spectra for XANES and EXAFS are depicted in Figure 30. Spectra for points 2, 3 and 4 are 

shown in Figure 31 and the EXAFS spectrum of point 5 is shown in Figure 32. Linear 

combination fitting of spectra yields U(IV) fractions ranging from 0.61 to 0.78 and averaging 0.7 

(Table 11), which is measurably less reduced in comparison to the XANES of the bulk sample 

(0.86 U(IV)). The lower fraction of U(IV) in the grain coating is attributed to calibration 

uncertainty in the mXANES spectra and partial oxidation of U(IV) during thin section fabrication 

instead of U(VI) sequestration in the coating. Multi-energy U XANES mapping conversely shows
3-52

 U(IV) is dominant, comprising nearly all of the uranium in the coating with comparatively low 

and infrequent points of U(VI). The ratio of the sum of U(IV) in all pixels to the sum of the total U 

is 0.93, which is more in line with bulk XANES U(IV) fraction. Back scatter SEM imaging and 

EDS analysis of this grain are presented in section 3.4.4. 

A near continuous coating of U was also present surrounding a large (~1 mm) grain in the thin 

section of sediment sample B-F4 (Figure 33) recovered from the first 1 cm near the inlet of 

column B at the end of the experiment. This sample is from the composite of column B after 221 

days of biostimulation which was then packed into a new column and underwent 110 days of 

additional biostimulation with U(VI) inflow. The underlying grain has a mottled distribution of iron 

suggesting it is a rock fragment or an aggregate of smaller grains. The uranium coating ranged 

from <5 up to 30 mm thick and was dominated by U(IV) in the XANES map. The coating in part 

appears to have formed on Fe rich areas of the underlying grain and is largely comprised of U 

and S but little Fe, Ca or other elements evident within the coatings in the mXRF images (Figure 

33E). The co-association of S with U may reflect U reduction by sulfides. Lower atomic number 

elements, notably carbon, cannot be detected by the BL2-3 X-ray microprobe. U(IV) from linear 

combination (LC) fits of the mXANES at individual points ranged from 0.74 to 0.90 and averaged 

0.81±0.19 (Table 11; Figure 31). The individual points show a slightly lower U(IV) component 

(0.81) than the LC fit of bulk XANES of the column sediment sample (0.92). A U-LIII EXAFS 

spectrum was collected at one point (Figure 32).

A 200-mm diameter iron-bearing grain from column A subsample after oxic elution (Ax-23 grain 

1) also has a near continuous coating of U of varying concentration. The coating ranges from a 

few microns to more than 30 microns (Figure 34). U(VI) is more abundant in this grain coating 

than in grain coatings from samples recovered after biostimulation. Based on total counts of the 

multi energy U XANES map, U(IV) comprises 65% of the total U counts. The thickest coating 

area near the top of the grain as shown in the mXRF maps appears more concentrated in U(VI) 

near the outside of the coating evidenced by the transition from red to green in the tri-color 

image in Figure 34C. Linear combination fits of XANES spectra from 5 points in the coating 

have U(IV) fractions ranging from 0.62 to 0.71, averaging 0.68±0.05 (Table 11; Figure 31). As 

noted for the other thin sections, the U in the Ax-23 thin section grain coating is more oxidized 

than determined from LC fit of the bulk XANES of the column sediment subsample (0.88 U(IV)). 

A U-LIII EXAFS spectrum was collected at one point (Figure 32). Little Fe or S was apparent in 

the coating imaged by mXRF. 
3-53

Figure 30. mXRF maps of column C sediment sample Cr-5 grain 1. (A) mXRF tricolor map of U(IV) in red, iron 

in green and calcium in blue; (B) mXANES map of U(IV); (C) zoom of area near point 5 in A showing 

of U(IV) in red, iron in green and sulfur in blue; (D) zoom of area near points 1 and 4 in A showing 

of U(IV) in red, iron in green and calcium in blue; (E) same area as D but showing of U(IV) in red, 

iron in green and sulfur in blue. The color intensity increases with relative concentration. Scale 

bars are in microns. Numbered points are locations of mXANES spectra. Maximum counts are 

4018, 328, 4064, 828, and 30 for U(IV), U(VI), Fe, Ca and S fluorescence, respectively.

10 20 20

1

4

5

B

C

A

D E
3-54

Figure 31. Micro-XANES of points on column sediment grain coatings. Sample IDs correspond to those listed 

in Table 11 and locations of points are depicted in Figures 30, 33 and 34. 

17160 17180 17200 17220

eV

U(IV) Model

U(VI) Model 

Absorption (Normalized)

Cx-4 G1 pt 3

Cx-4 G1 pt 2

Cx-4 G1 pt 1

Cx-3 G1 pt 2

Cx-3 G1 pt 1

Br-1 G1 pt 1

17160 17180 17200 17220

eV

Absorption (Normalized)

Ax-23 G1 pt 2

Ax-23 G1 pt 3

Ax-23 G1 pt 5

Br-F4 G1 pt 5

Br-F4 G1 pt 3

Br-F4 G1 pt 1

Cr-5 G1 pt 4

Cr-5 G1 pt 3

Cr-5 G1 pt 2
3-55

Figure 32. U-LIII mEXAFS of points on column sediment grain coatings. Sample IDs correspond to points 

depicted in Figures 30, 33, and 34. 

0

5

10

15

20

25

2 3 4 5 6 7 8

k (A-1)

χ(k)•k3

0

2

4

6

8

10

0 1 2 3 4 5 6

R (Å)

Fourier Transform Magnitude

Nano UO2

Ax-23 G1 pt 5

Br-F4 G1 pt 5

Cr-5 G1 pt 5
3-56

Table 11. Fraction of U(IV) and U(VI) at thin section points column sediments determined by linear 

combination fits to U-LIII mXANES spectra. 

Sample U(IV) U(VI)

Ax_23 pt 1 * 0.65 0.37

Ax_23 pt 2 0.66 0.34

Ax_23 pt 3 * 0.76 0.25

Ax_23 pt 4 0.71 0.31

Ax_23 pt 5 * 0.62 0.34

Ax_23 pt 5 EXAFS 0.69 0.28

average 0.68 0.32

standard deviation 0.05 0.04

Br-F4 pt 1 * 0.78 0.22

Br-F4 pt 2 0.81 0.19

Br-F4 pt 3 * 0.76 0.24

Br-F4 pt 4 0.85 0.17

Br-F4 pt 5 * 0.74 0.22

Br-F4 pt 6 0.9 0.13

Br-F4 pt 5 EXAFS 0.81 0.19

average 0.81 0.19

standard deviation 0.05 0.04

Br-F1 pt 1 0.95 0.09

Br-F1 pt 2 1.01 0.04

Br-F1 pt 3 0.93 0.12

average 0.96 0.08

standard deviation 0.04 0.04

Cr-5 pt 1 0.78 0.23

Cr-5 pt 2 * 0.68 0.3

Cr-5 pt 3 * 0.61 0.37

Cr-5 pt 4 * 0.7 0.27

Cr-5pt 5 EXAFS 0.69 0.28

average 0.69 0.29

standard deviation 0.05 0.06

BL 2-3 May 2012 U L-III 

Cx-3 g1 pt1 * 0.91 0.08

Cx-3 g1 pt2 * 0.83 0.18

average 0.87 0.13

standard deviation 0.06 0.07

Cx-4 g1 sp1 * 0.8 0.2

Cx-4 g1 sp2 * 0.75 0.29

Cx-4 g1 sp3 * 0.89 0.11

average 0.81 0.20

standard deviation 0.07 0.09

* denotes samples with spectra shown in Figure 31.
3-57

Figure 33. mXRF maps of column B sediment sample F4 grain 1. (A) mXRF tricolor map of U(IV) in red, iron in 

green and calcium in blue; (B) mXANES map of U(IV); (C) zoom of area near points 2 and 3 in A 

showing of U(IV) in red, iron in green and sulfur in blue; (D) zoom of area near points 2 and 3 in A 

showing of U(IV) in red, iron in green and calcium in blue. The color intensity increases with 

relative concentration. Scale bars are in microns. Numbered points are locations of mXANES 

spectra. Maximum counts are 3776, 116, 8521, 1126, and 51 for U(IV), U(VI), Fe, Ca and S 

fluorescence, respectively.

40

2

3

200 200

4

3

2

1

5

6

4

3

2

1

5

6

A B

C D

2

3
3-58

Figure 34. mXRF maps of column A sediment sample Ax-23 grain 1. (A) mXRF tricolor map of uranium in red, 

iron in green and calcium in blue; (B) mXRF map of uranium; (C) mXRF tricolor map of U(IV) in red, 

U(VI) in green and iron in blue. The color intensity increases with relative concentration. Scale 

bars are in microns. Numbered points are locations of mXANES spectra. Maximum counts are in 

(A) 2847, 3412, and 573 for U, Fe and Ca, respectively, and in (C) 2116, 881, and 3114 for U(IV), 

U(VI), an d Fe fluorescence, respectively.

1

2

3

4

5

A B

C
3-59

Micro-focused U EXAFS spectra were collected at one point on each of the three grains 

described above (Table 11). The points with the highest U fluorescence count rate were chosen 

to provide best signal quality. The k-space k3

-weighted spectra of points in the U-rich grain 

coatings are similar to the nano-partculate uraninite reference spectrum (Figure 27) and to the 

bulk EXAFS of column sediments (Figure 32). In addition the Fourier transforms of the EXAFS 

also have peaks at distances similar to the U-O and U-U shells described for the bulk samples 

at 1.8 and 3.5Å, uncorrected for phase shift. Because of the significant fraction of U(VI) (0.19 to 

0.28) present at these thin section grain points, shell by shell fits of the EXAFS were not 

possible because of the inability to accurately account for the contributions from U(VI) masking 

longer distance shells. Linear combination fits were not attempted because no suitable single 

component models exist for non-polymerized NUSAB U(IV). Nonetheless, qualitatively these 

spectra are consistent with a large component of nano-particulate uraninite in the coatings.

3.4.4 SEM-EDS Imaging of Column Sediments

Imaging of thin section grains was also conducted using back-scatter electron scanning electron 

microscopy (BSE-SEM). Because of its high atomic number, uranium has a much higher yield of 

back scattered electrons than lighter elements allowing qualitative identification of U-rich areas. 

However, because U in the column sediment thin sections was located in a relatively small 

number of grains, BSE-SEM was limited to U-rich grains previously identified in the 30-mm 

resolution mXRF maps. Energy dispersive X-ray spectral (EDS) analysis of elemental 

composition of selected points was conducted using a nominal 1 mm diameter beam size. 

Results of measurements of grains from column C recovered at the end of biostimulated 

reduction are presented below as examples.

The BSE-SEM image (Figure 35) of the entire grain of the column C sediment (Cr-5 thin section 

right panel grain 1) described above (Figure 30) has a bright white zone surrounding the grain. 

EDS analysis confirms that these bright regions have abundant U. Higher resolution images of 

areas containing U show a coating 1 to 5 mm in thickness on the grain exterior that fills 

embayment and voids in the sediment grain surface. The U coating consists of fibrous particles 

clustered or aggregated to form the coating, with other particles without U intermixed into the 

coating (Figure 30B) or adhering to the coating surface (Figure 30A). Uranium also extends up 

to 10 mm into cracks or fissures near the surface (Figure 30 area A). The U appears to follow the 

morphology of the underlying grain in the iron rich area of this grain (Figure 30C). Area A is near 

XANES point 4 depicted in Figure 30, and area C is near EXAFS point 5. EDS measurements of 

elemental abundances of selected points depicted in sub areas A and C of this grain confirm 

that the brighter areas have elevated U (Table 12). For example point A1 is 14 atomic % U, 

while an entrained grain (A2) has no measurable U but contains Fe and S. The EDS of the 

interior of the large grain at point A3 is consistent with quartz. The gray material, point A4, 

outside of the bright coating has lower U but contains elements common to aluminosilicate 

minerals such as clays that appear to be adhering to the coating on the larger grain. Point C2 

within the grain in area C has detectable U, but elevated Fe and other elements indicating an 

iron silicate. Points C2 and C3 in the bright coating have high U, no Fe or S, and minor amounts 

of other elements. Both points have measurable P and point C2 has 22% C, which may be 

organic carbon since Ca and Mg, which are elements indicative of carbonate phases, are not 

present. EDS data was not collected in area B.
3-60

Figure 35. SEM-BSE images of column C after biostimulation interval 5 (Cr-5) thin section grain 1. Location of 

EDS spectra are shown in higher magnification images of areas A, B, and C outlined in red in the 

upper left image (1). mXRF images of this grain are shown in Figure 30.

A

C

B

100 mm

20 mm B

10 mm

C

+ C2

C1 +

+ C3

20 mm A

+

+

A2

A3

+

A1

+

A4

1
3-61

Table 12. Atomic percent of elements detected in SEM-EDS points data from column C subsample Cr-5, 

thin section right panel, grain 1. See Figure 35 for location of EDS points.

Point Ua Fe S C O Si Al Mg Ca K P 

A1 14.41 73.28 2.58 2.61 4.33 2.79

A2 8.22 1.11 62.59 10.96 8.02 9.11

A3 68.64 31.36

A4 2.3 2.04 66.50 16.34 8.13 1.42 1.65 1.61

C1 8.59 62.26 10.97 7.82 8.53 1.85

C2 9.32 21.9 61.68 1.95 2.9 2.25

C3 11.65 74.78 2.54 2.52 4.98 3.54

a. No value equals element below detection.

A small grain in column C thin section sample Cr-5 that had elevated U in the 30-mm resolution 

XRF map (not shown) was imaged using BSE-SEM with selected points measured by EDS 

(Figure 36; Table 13). The ~30 x 50 mm grain appears to be a cluster of smaller grains that 

includes bright U-rich areas near the exterior of the top and running vertically through the center 

of grain. The bright areas appear surrounded by a lighter gray material. Both zones have voids 

or dark areas throughout. Elevated U ranging from 4.3 to 7 atomic % was measured in the 

brightest areas (points A3, A8, B1, C4; Figure 36; Table 13). Points in the surrounding light gray 

areas had lower U (points A4, A6, A10, C2, C3 and C5). Points in darker areas or in obvious 

discrete grains had no measurable U (e.g B3, B4, C1). The light gray areas have high Fe and S. 

The high U points had lower, but measurable Fe and S. The S to Fe ratio in all the points in the 

bright and light area ranged from 0.6 to 1.2, consistent with the presence of iron sulfides. The U 

and the putative iron sulfides areas appear to be comprised of submicron particles or 

amorphous material. The high U areas appear to be forming on the iron sulfide material (e.g. 

area C). The nominal beam size of 1 mm diameter for EDS measurements limits resolving 

whether U is adjacent to or intermixed with iron sulfide areas. Combined, the BSE-SEM imaging 

and EDS measurements suggest that this particle is in part comprised of aluminosilicate 

material cemented by iron sulfides which have coatings or zones of U. These results are 

consistent with abiotic reduction of U by iron sulfides (Hyun et al., 2012; Gallegos et al., 2013). 

Points A3 and A4 have elevated carbon perhaps resulting from microbial biomass.
3-62

Figure 36. SEM-BSE images of column C interval 5 (Cr-5), grain 2 after biostimulation. Location of EDS 

spectra are shown in higher magnification images of areas outlined in red in the upper left image

(1). 

+

A10

+

A4

+

A6

+ A3

+

A5

+

A8

5 mm

+ B1

+ B2

+ B3

5 mm

+

B4

+

+

C2

+ C3

+

C4

C5 +

5 mm

+ C1

10 mm

B

A

C A

B

C

1
3-63

Table 13. Atomic percent of elements detected in SEM-EDS points data from column C post reduction, 

interval Cr-5 grain 2. Location of point spectra are shown in Figure 36.

Point Ua Fe S S/Fe C O Si Al Ti Mg Ca K P 

A3 7.02 1.29 0.89 0.7 21.6 56.9 3.84 1.52 2.54 2.68 1.72

A4 0.79 3.57 4.34 1.2 9.79 58.44 3.77 1.6 15 2.23 0.46

A5 15.7 17.35 1.1 46.68 10.46 3.49 6.32

A6 0.48 9.52 10.38 1.1 55.26 10.69 4.78 8.22 0.67

A8 4.32 2.48 1.27 0.5 63.97 14.78 7.88 3.27 2.03

A10 1.3 14.48 13.28 0.9 48.66 12.16 4.38 5.75

B1 6.13 1.31 0.94 0.7 56.88 27.9 1.19 1.52 2.55 1.57

B2 10.56 11.58 1.1 49.11 22.93 2.46 3.36

B3 66.58 33.42

B4 0.93 64.97 29.89 3.38 0.83

C1 0.92 0.85 0.9 63.11 18.68 14.92 1.51

C2 0.83 10.52 11.55 1.1 53.55 11.31 5.43 5.81 0.99

C3 1.11 2.4 0.86 0.4 66.06 15.48 6.94 5.43 0.59 1.13

C4 5.56 3.38 2.02 0.6 61.78 11.05 5.27 7.17 2.62 1.15

C5 1.61 3.03 63.83 15.09 7.68 4.58 1.43 1.7 1.06

a. No value equals element below detection.

3.5 Microbial Analysis 

Biomass was generally plentiful in the sediment recovered at the end of the biostimulation stage 

and in effluent filter samples collected during biostimulation. In addition, sediments from column 

A after oxic elution had sufficient biomass for microbial assay. All of these samples were 

successfully amplified for Terminal Restriction Fragment Length Polymorphism (TRFLP) 

quantitative Polymerase Chain reaction analyses (qPCR) of the iron reducing bacteria 

Geobacter and sulfate reducing bacteria (SRB). 

3.5.1 TRFLP Fingerprinting

Figure 37 shows the cluster analysis of the bacterial TRFLP fingerprints for Rifle material. 

Samples from the same column generally grouped together, with some exceptions. Column A 

samples, for example, recovered after oxic elution all group together. Column B forms 

subclusters which include samples from column C. Both of these columns received input of 

ferrous iron; column B after 210 days of acetate and U input while column C had continuous 

ferrous iron input throughout acetate biostimulation. In addition, the effluent filter (taken from 

column C during the iron reduction phase, Cr-eff(1), Figure 37 and Table 14), clusters with the 

inlet and outlet filters of column B, as well as the outlet filters for column C. In contrast, the 

effluent filter taken during sulfate reduction (Cr-eff(2)), clusters more with sediment sections 1-4 

of column C and some of the column C inlet filters.
3-64

Table 14. Column sediment microbial abundance data for sulfate reducing bacteria (SRB) and Geobacter 

(GEO).

Sample 

Name Description Sediment extracted (g) SRB/mL SRB/gram GEO/mL GEO/gram

Column A: after biostimulation

Ar-1 syringe core 0.72 2.50E+05 1.74E+07 5.49E+05 3.81E+07

Ar-2 syringe core 0.96 1.38E+05 7.16E+06 4.46E+05 2.33E+07

Ar-3 syringe core 0.91 7.94E+04 4.36E+06 2.78E+05 1.53E+07

Ar-4 syringe core 0.85 1.60E+05 9.43E+06 2.08E+05 1.23E+07

Ar-f(out) outlet end filter, scrape 0.57 2.15E+06 1.89E+08 7.25E+05 6.36E+07

Ar-f(out) extract of outlet filter after 

scrape 1.52E+06 5.62E+05

Ar-f(in) sed sediment from inlet end 

filter 0.74 7.45E+05 5.03E+07 4.83E+05 3.26E+07

Ar-f(in) inlet end filter, scrape 0.4 2.24E+05 2.80E+07 9.25E+04 1.16E+07

Ar-f(in) extract of inlet filter after 

scrape 5.74E+05 1.78E+05

Ar-eff-1 effluent filter day 179-186 0.69 8.03E+04 5.82E+06 1.94E+03 1.41E+05

Column A: after oxic elution

Ax-2 column outlet end 1.02 1.58E+04 7.75E+05 6.43E+04 3.15E+06

Ax-23 mid-column lengthwise 0.65 6.37E+03 4.90E+05 3.26E+04 2.51E+06

Ax-26 column inlet end 0.62 2.39E+04 1.93E+06 1.28E+05 1.03E+07

Ax-f(out) outlet end filter, scrapes 0.24 4.12E+03 8.58E+05 9.94E+03 2.07E+06

Column B: after biostimulation

Br-eff-1 effluent filter day 113-121 0.584 4.95E+05 4.24E+07 2.26E+04 1.93E+06

Br-F1 sediment, 5-6 cm 0.53 8.84E+03 8.34E+05 1.04E+05 9.81E+06

Br-F3 sediment, 3-4 cm 0.46 4.37E+03 4.75E+05 5.77E+04 6.27E+06

Br-F4 sediment, 2-3 cm 0.64 3.88E+03 3.03E+05 6.93E+04 5.41E+06

Br-F5 sand pack; 0-2 cm 0.63 2.14E+04 1.70E+06 5.34E+04 4.24E+06

Br-f(in) inlet end filter, scrapes 0.36 1.18E+04 1.64E+06 1.70E+03 2.36E+05

Br-f(out) outlet end filter, scrapes 0.49 5.00E+03 5.10E+05 1.21E+03 1.23E+05

Column C: after biostimulation

Cr-1 sediment, 4-5.5 cm 0.47 4.74E+02 5.04E+04 1.24E+05 1.32E+07

Cr-2 sediment, 2.8-4 cm 0.39 2.51E+02 3.22E+04 5.29E+04 6.78E+06

Cr-3 sediment, 1-2.8 cm 0.45 4.50E+02 5.00E+04 8.73E+04 9.70E+06

Cr-4 sediment, 0-1.6 cm 0.41 3.71E+03 4.52E+05 8.77E+04 1.07E+07

Cr-f(out) CN outlet end filter, scrapes 0.23 3.54E+04 7.70E+06 1.69E+05 3.67E+07

Cr-f(out) GF outlet end filter, scrapes 0.35 2.12E+03 3.03E+05 1.66E+03 2.37E+05

Cr-f(in) CN inlet end filter, scrapes 0.20 1.22E+05 3.05E+07 1.62E+05 4.05E+07

Cr-6 sediment, composite 0.97 - - 1.28E+04 6.60E+05

Cr-eff-1 effluent filter during iron 

reduction, day 7-14 0.52 3.81E+01 3.70E+03 6.61E+05 6.42E+07

Cr-eff-2 effluent filter during sulfate 

reduction, day 28-34 0.52 3.72E+04 3.61E+06 3.12E+05 3.03E+07

Samples in bold were characterized further with clone library analysis. Sample identification codes f(in), f(out), and 

eff refer to column inlet filter, outlet filter, and effluent filter, respectively; others as shown in Tables 6, 7, and 8. 

Volume filtered in liters is listed for effluent filters under sediment weight extracted, with SRB/L and GEO/L 

replacing per gram values for these samples.
3-65

Figure 37. Cluster analysis for column sediments. See Table 14 for sample identification codes.

3.5.2 qPCR 

Table 14 shows the abundance of dissimilatory sulfate reducing bacteria (SRB) and the iron 

reducing bacteria Geobacter 16sSrRNA gene expressed as copy number/g sediment or copy 

number/L filtered for effluent filters. The copy number is measured in the qPCR assay, and 

refers to the number of gene copies detected; this number relates to the cell number but is not 

equivalent to cell number, as copy number per cell depends on the gene and the organism. 

Typical SRB copies are 1-3 per cell, and Geobacter usually have 1-2 copies of 16SrRNA per 

cell. 

Geobacter was higher in abundance than SRB in most samples. Column A, which was sampled 

during the reduction stage and later after oxic elution, showed decreases in both Geobacter and 

SRB abundances after oxic elution (Table 14). Figure 38 compares sediment abundances of the 

two groups for different locations within column A. Geobacter was in greater abundance than 

SRB in sediment samples for both reduction and oxidation stages. During reduction, SRB 

abundance was in the 1E7 copy/g range (4E6 to 1.7E7) in column sediments, with the highest 

abundance detected in filter scrapes from the effluent end of the column >1E8/g. Geobacter

was in the 1 to 4E7 copy/g range in column sediments, with higher concentrations also 

observed in filters from the effluent end of the column. There were not large differences in 

Cr-comp

Cr-1

Cr-3

Cr-eff-2

Cr-f(in)-GF-a

Cr-f(in)-GF-b

Cr-4

Cr-2

Br-F3

Br-F4

Br-f(in)

Br-f(out)

Cr-f(out) CN

Cr-f(out) GF

Cr-eff-1

Ax-f(out)

Ax-2

Ax-26

Ax-23

Br-F1

Br-eff-1

Br-f(in)-b

Cr-f(in)-CN-a

Cr-f(in)-CN-b
3-66

abundance among sediment samples, but the inlet end did contain higher abundances of both 

SRB and Geobacter. The trend of higher abundance of both in sediments near the inlet 

corresponds to higher sediment uranium measured near the inlet of the column. After oxic 

elution without electron donor amendment, abundances of both were much reduced compared 

to the reduction phase. SRB abundances had decreased to the 5E5 to 2E6 copy/g range, and 

Geobacter decreased to 2E6 to 1E7 copy/g range, with higher abundances present at the inlet 

end (Table 14).

Figure 38. Abundances of sulfate reducing bacteria (SRB) and Geobacter in sediment of column A after 

biostimulated reduction and after oxic elution.

Microbial analysis of column B sediments was conducted on samples recovered at the end of 

the biostimulated reduction experiment. That is, sediments assayed were after 210 days of 

anaerobic artificial groundwater influent with U(VI) and acetate and an additional 120 day period 

of influent also containing ferrous iron. The range of SRB in column B sediment was 3E5 to 

1.7E6 copies/g, and Geobacter was 4E6 to 1E6 copies/g. These abundances are lower than in 

column A sediments recovered at the end of the biostimulation stage despite a much longer 

biostimulation period. Column B sediments show that both SRBs and Geobacter abundances 

are lower at the inlet and highest at the outlet (Figure 39). It is important to note that column B 

was homogenized after 220 days of reduction stage (see section 3.1.2.) and that the sediments 

assayed were sampled after an additional 120 days of continued biostimulation. In addition, the 

sand pack at the inlet end of the column had substantially higher abundances (more than a 

factor of two) than adjacent column sediments. It appears that microbial activity and biomass 

growth was enhanced in the sand pack where electron donor first entered the column. 

Interestingly, although the Geobacter abundances were higher in the column sediments than 

SRB, the inlet and outlet filters had higher abundances of SRB (Table 14).

0.E+00

1.E+07

2.E+07

3.E+07

4.E+07

5.E+07

R1 R2 R3 R4 V26 V23 V2

Counts/g

Column A Sediments

SRB

Geobacter

inlet

inlet

reduction phase oxidation phase
3-67

Figure 39. Comparison of abundances of sulfate reducing bacteria (SRB) and Geobacter in column B 

sediments. 

Column C had simultaneous addition of ferrous iron, U(VI), and acetate for the entire 185 days 

of the biostimulation stage of the experiment. Column C sediments (Figure 40) showed the 

largest difference in abundance between SRB and Geobacter in sediments compared to the 

other columns, with Geobacter abundances 2 orders of magnitude greater than SRB. SRB was 

in the range of 3E4 to 4.5E5 copies/g, while Geobacter was in the range of 6.8E6 to 1.3E7

copies/g. Column C sediment SRB were highest nearest the inlet, an order of magnitude higher 

than the other column sediments, while there was little trend in Geobacter abundance within the 

column. Column C Geobacter sediment abundance was similar to column B, but substantially 

lower than column A (Table 14). SRB abundance in column C was up to a factor of 50 lower 

than column A. A large change in SRB, a 3 orders of magnitude increase, is observed between 

the filter samples taken during the Fe reduction stage and then at the sulfate reduction stage. A 

decrease of 6.4E7 to 3.0E7copies/L Geobacter abundance was observed during this transition.

Figure 40. Comparison of sulfate reducing bacteria (SRB) and Geobacter abundance in column C sediment

after biostimulated reduction. 

Figure 41 shows abundances of Geobacter in sediment from all columns, including column A 

after 180 days of oxic elution. Figure 42 shows abundance of SRBs in the same samples. 

Abundance of Geobacter in column A during the biostimulation of column A is highest at the 

column inlet and decreases from there to the outlet. This is generally true for the SRBs as well. 

Column A sediments had a greater abundance of Geobacter by a factor of two or more than the 

0.0E+00

2.0E+06

4.0E+06

6.0E+06

8.0E+06

1.0E+07

1.2E+07

inlet middle outlet

Counts/g

Column B Sediment

Geobacter

0.E+00

2.E+05

4.E+05

6.E+05

8.E+05

1.E+06

inlet middle outlet

Counts/g

Column B Sediments

SRB

0.E+00

2.E+05

4.E+05

6.E+05

8.E+05

1.E+06

0-1.6 1.6-2.8 2.8-4.0 4.0-5.5

counts/g

Distance from inlet (cm)

Column C Sediment

SRB

0.0E+00

2.0E+06

4.0E+06

6.0E+06

8.0E+06

1.0E+07

1.2E+07

1.4E+07

0-1.6 1.6-2.8 2.8-4.0 4.0-5.5

counts/g

Distance from inlet (cm)

Column C Sediment

Geobacter
3-68

other columns. Abundances of SRB in sediments are up to a factor of 50 greater than in the 

other columns. Interestingly, column B and C had comparatively longer biostimulation periods, 

334 and 186 days, respectively, versus 112 days for column A. The greater abundances in 

column A of both types were contrary to an expected increase in biomass growth in the other 

columns because of the longer period of continual addition of electron donor (acetate) and 

micronutrients. The primary difference between column A and the others is the absence of

ferrous iron input to column A. It is unknown if the presence of dissolved iron limited growth of 

SRB. Additionally, column A had an extended period of 81 days anoxic influent in the absence 

of electron donor, which would be expected to result in limited increase in biomass. These 

results will be discussed further in relation to electron balance of likely microbial redox 

processes characterized by integrated effluent profiles and sediment chemical analyses.

Figure 41. Geobacter abundances in columns A, B, C after biostimulated reduction, and after oxic elution 

(column A). Sample position in column from inlet to outlet is from left to right.

Figure 42. Abundance of sulfate reducing bacteria in column sediments. Sample position in column from 

inlet to outlet is from left to right.

3.5.3 Clone Library Analyses 

Six samples were chosen for SRB cloning and sequencing, and 3 were chosen for Geobacter

sequencing based primarily on cell abundance. Column A outlet filter at the end of 

biostimulation and the B effluent filter sampled in February 2010 during the biostimulated 

reduction stage were chosen for both SRB and Geobacter clone library construction. In addition, 

sediment from the outlet end of column A recovered after oxic elution and the column B inlet 

0.E+00

1.E+07

2.E+07

3.E+07

4.E+07

5.E+07

Col A Reduced Col A oxidized Col B reduced Col C reduced

Copies/g

Column Sediment Geobacter Abundances

0.0E+00

2.0E+06

4.0E+06

6.0E+06

8.0E+06

1.0E+07

1.2E+07

1.4E+07

1.6E+07

1.8E+07

2.0E+07

Col A reduced Col A oxic Col B reduced Col C reduced

Copies/g

Column Sediment Sulfate Reducing Bacteria
3-69

filter after biostimulation were used for SRB clone library construction. From column C, the inlet 

end filter at the end of biostimulation and an effluent filter collected during sulfate reduction were 

chosen for SRB characterization by cloning. The column C effluent filter collected during the Fe 

reduction phase was chosen for Geobacter clone library analysis. These samples are denoted 

in Table 14. 

Geobacter sequences were not very diverse and were dominated by those previously described 

as subsurface clade 1 of Geobacteraceae, believed to be important in U(VI) reduction in 

subsurface environments (Table 15). There are also a very few in the D. acetoxidans clade 

(Holmes et al., 2007). The subsurface clade includes closely related sequences identified from 

water filtered from wells M16, M18, M21 at the Rifle site (Holmes et al., 2007), as well as some 

Geobacter sequences found at other subsurface sites. Previously, these authors found the 

same trend at the Rifle site in their study of subsurface clade 1 domination with some other 

minor sequences such as the D. acetoxidans clade. The proportions of each sequence were not 

the same in each column, however. Geobacter prevalent at well M21 dominated in column A, 

and a sequence from well M16 was also prevalent representing 22% of the clones. The 3rd 

sequence found in column A, which was most closely related to “Riflegeoclone 4” from a 

previous study, represented 13% of the sequences in column A. However, this was the 

dominant sequence, representing 71% of the sequences in column B (Table 15). In addition, a 

sequence in the D. acetoxidans clade as described by (Holmes et al., 2007) represents 14% of 

the sequences in column B. At the Rifle field site, M16 is a location closer to the acetate 

injection wells than M21. This suggests that M16 is more reducing than M18 which may be 

more reducing than M21. Thus the differences in sequences may represent different degrees of 

reducing conditions as a result of the concentration and input duration of acetate amendment. 

Table 15. Geobacter clones from columns A outlet filter, Ar-f(out), and B effluent filter, Br-eff(1).

Phylotype 

Number

Relative 

abundance 

(%) Col A

Relative 

abundance 

(%) Col B

Closest Genbank 

relative (Blast) Blast source description

Sequence 

similarity %

1 65 14 CP001661.1 Geobacter M21 from Rifle 100

2 22 0 EF668531.1 uncult Geo from M16 Rifle 100

3 13 71 EF668606.1 Riflegeoclone4 98

4 0 14 AJ271656.1 Pelobacter, Geothermobacter? 99

The column C effluent filter collected during the Fe(III) reduction phase, Cr-eff(2), was also 

mostly comprised of the subsurface clade 1 of Geobacteraceae. However, the M21 sequence 

was minor, representing 2% of the clone library. A sequence identical to a Rifle clone from M16 

(Genbank accession #EF668531) was the dominant Geobacter representing 66% of the clones 

(accession #CP001661) with 15% of the clones related to Geobacter M18 

(accession#CP002479), and 5% of the clones were most closely related to Pelobacter 

propionicus in the D. acetoxidans clade (accession # X70954). The rest of the clones, 

representing 12% of the sequences, were related to uncultured sequences from other 

environments, and do not appear to be part of the subsurface clade. 

Table 16 summarizes SRB sequences found in each sample, the closest phylogenetic relatives 

based on BLAST searches, and the relative abundance in the clone library. The SRB 

sequences in column samples have relatively little diversity. For example, in column B effluent 

filter, Br-eff(1), collected well after the onset of sulfate reduction (day 113 to 121), only three
3-70

phylotypes were defined by SRB sequences. The two dominant phylotypes, representing 66% 

and 32% of the sequences, respectively, were most closely related to uncultured dsrB sequence 

obtained from a different uranium mill tailings site (AY015603), and a metal precipitation in 

groundwater experiment (AY731421) (Table M-3). The phylogenetic affiliations are 

Desulfobulbaceae and Desulfobacteraceae. The column A outlet filter recovered at the end of 

biostimulated reduction was dominated by the same type of Desulfobulbaceae organism (71% 

most similar to AY015603 from a uranium mill tailings site), but overall was more diverse than 

the column B sample, with 8 phylotypes (Table 16). The next most abundant sequence, 

representing 13% of the sequences retrieved from column A, was closest to an uncultured 

organism associated with steel corrosion in a French seaport (FR689663), and is in the 

Syntrophobacteraceae. This sequence identity has been found in many other Rifle samples, 

though never dominant. There are also representatives that group with Desulfotomaculum

(Peptococcaceae), and Desulfobactereaceae. 

The other column samples were low in diversity, typically with one or two dominant SRB, and 

several minor phylotypes. The column B inlet filter sample, Br-f(in) recovered at the end of 

biostimulated reduction contained 5 phylotypes, with 1 comprising 73% of the sequences (Table 

16). This phylotype was most closely related to a SRB sequence retrieved from another uranium 

mill tailings site (AY015603), and is in the family Desulfobulbaceae. The next two dominant 

organisms corresponding to 10% of the sequences each are likely Desulfotomaculum, and 

Syntrophobacteraceae. This dominant sequence is the same as the effluent filter collected 

earlier from this column, but the Desulfotomaculum has decreased from 32% to 10% of the 

sequences, and the Syntrophobacteraceae have increased over time, from not detected on the 

effluent filter (day 113 to 121) to 10% of the population at the end of the biostimulation.

The SRB phyllotypes and abundances changed dramatically in column A as a result of oxic 

elution compared to during biostimulated reduction. Four phylotypes were measured after oxic 

elution, with the dominant sequence (73% of the clones) from a different organism, though still 

in the family Desulfobulbaceae (Table 16). Interestingly a group of Syntrophobacteraceae

represent approximately the same percentage of SRB sequences as determined in the column 

outlet filter sample, Ar-f(out), but are a different Syntrophobacteraceae than observed earlier. 

No members of Desulfotomaculum or the Desulobacteraceae are evident in the samples 

collected after oxic elution.
3-71

Table 16. Sulfate reducing bacteria phylotypes, relative abundance, and closest BLAST Hits from column 

sediment samples recovered after biostimulated reduction and after oxic elution.

Group 

#

a #b

rel

abundc

Closest 

Genbank 

relative 

(Blast)

% 

IDd Source Blast source Identification

Column A: Ar-f(out) outlet filter scrape

1 2 4.4 EF065046.1 81 landfill leachate Desulfotomaculum? 

(Peptococcaceae)

2 1 2.2 DQ250766.1 95 landfill leachate Syntrophobacteraceae

3 1 2.2 AM901668.1 96 freshwater grassland soil Desulfosarcina? 

(Desulfobacteraceae)

4 1 2.2 FR689665.1 81 steel corrosion Syntrophobacteraceae

5 6 13.3 FR689663.1 82 steel corrosion Syntrophobacteraceae

6 32 71.1 AY015603.1 95 uranium mill tailings Desulfobulbaceae

7 1 2.2 FR689616.1 85 steel corrosion Syntrophobacteraceae

8 1 2.2 CP001720.1 87 isolate Desulfotomaculum? 

(Peptococcaceae)

Column A: sediment interval Ax-2 

1 29 72.5 EF065041.1 87 landfill leachate Desulfobulbaceae

2 6 15 AM901632.1 89 freshwater grassland soil Syntrophobacteraceae

3 1 2.5 AM901621.1 90 freshwater grassland soil Desulfobulbaceae

4 4 10 AY015603.1 86 uranium mill tailings site Desulfobulbaceae

Column B: Br-eff(1), effluent filter (day 113 to 121)

1 1 10.0 FR689629.1 landfill leachate Desulfosarcina? 

(Desulfobacteraceae

2 25 72.5 AY015603.1 uranium mill tailings Desulfobulbus? 

3 12 5.0 AY731421.1 metal precipitation 

experiments, 

groundwater

Desulfobacteraceae

Column B: Br-f(in), column inlet filter

1 4 10.0 AF334595.1 90 Desulfomonile tiedjei 

(Syntrophaceae)

Syntrophaceae

2 29 72.5 AY015603.1 94 uranium mill tailings site Desulfobulbus?

3 2 5.0 AY929594.1 88 anaerobic sludge blanket Desulfotomaculum?

4 4 10.0 AM901632.1 89 freshwater grassland soil Desulfotomaculum?

5 1 2.5 AY929594.1 82 bioreacter granules, 

wastewater treatment

Column C: Cr-eff(2), column effluent filter during sulfate reduction (day 28 to 34)

1 13 37.1 AY015603.1 90 uranium mill tailings site Desulfobulbaceae

2 21 61.8 AY015603.1 94 uranium mill tailings site Desulfobulbaceae

Column C: Cr-f(in), column inlet filter

1 14 37.8 AY015603.1 94 uranium mill tailings site Desulfobulbaceae

2 1 2.7 AY015603.1 82 uranium mill tailings site Desulfobulbaceae

3 22 59.5 FR689629.1 82 steel corrosion in port Desulfobacteraceae 

a. Number of phylotypes

b. Number of clones

c. Relative abundance of clones

d. Sequence similarity
3-72

Two samples from column C were analyzed by clone library for SRB, and these samples were 

the least diverse for SRB of all of the samples examined. The effluent filter collected just after 

the onset of the sulfate reduction phase, Cr-eff(2), had two dominant groups, both in the 

Desulfobulbaceae and are most closely related to AY015603 from a uranium mill tailings site 

(Table 16). The column inlet filter, Cr-f(in), recovered at the end of biostimulated reduction also 

has lower diversity with two dominant groups observed (Table 16). One group is the same as 

the effluent filter sample and is most closely related to AY015603 from a uranium mill tailings 

site. It represents 39% of the clones. The other main phylotype represents 58% of the clones is 

dissimilar to most of in the database. It is closest to FR689629, which is only 82% similar (Table 

16) and is in the phylotype Desulfobacteraceae. 

3.5.4 Summary of Microbial Analyses of the Column Experiments

There was some structure in the columns in terms of abundances of Geobacter and SRB. In 

column A, which underwent a post reduction oxic elution, the abundances of SRB and 

Geobacter were highest in the outlet filter scrapes, but within the column itself, the highest 

abundances are towards the inlet end. Not surprisingly, the abundances of both Geobacter and 

SRB decreased between the biostimulated reduction phase and after oxic elution. 

The distribution of SRB in column B was unlike column A. This is likely because sediments were 

homogenized when the column was repacked at day 220. However, the Geobacter abundances 

were actually higher at the outlet end of the column in this experiment. Geobacter may be more 

motile than SRBs and accumulated at the filter. Interestingly, although the Geobacter

abundances were higher in the column sediments, the inlet and outlet filters had higher 

abundances of SRB.

Column C which had simultaneous addition of Fe(II), acetate and U(VI) throughout the 

biostimulated reduction stage of the experiment showed the largest difference in abundance 

between SRB and Geobacter compared to the other columns, with Geobacter abundances 2 

orders of magnitude greater than SRB. This addition of ferrous iron throughout the experiment 

may have favored Geobacter over SRB. The abundances of Geobacter were similar throughout 

the column, suggesting the whole column was conducive to Geobacter growth. A small increase 

in SRB abundance (<20%) was observed in sediments near the inlet. A similar increase in total 

and acid volatile S was also observed in column C sediments. A 3-order of magnitude increase 

in SRB abundance in response to the onset of sulfate reduction was observed in the effluent 

filter compared to the filter collected before sulfate reduction during the Fe(III) reduction phase

The Geobacteraceae structure in clone libraries at Rifle are similar to those previously reported 

in this type of environment, dominated by subsurface clade 1 (Holmes et al., 2007), and 

believed to be important in U(VI) reduction. Most of the sequences detected were either very

similar or identical to those detected in other U(VI) remediation experiments performed at the 

Rifle site (Vrionis et al., 2005). There were some differences in dominant Geobacter by column. 

For example a clone from Rifle site M16 was dominant in column C, whereas M21 was 

dominant in column A. The significance of these shifts is unknown, but likely reflects a 

differential response to the oxidation in column A for example, and to the continuous Fe addition 

in column C.

Sulfate reducing microbes were not very diverse in the column sediments. They were 

dominated by Desulfobulbaceae followed by Desulfobacteraceae, and to a lesser extent 

Syntrophobacteraceae , except in column C, where no Syntrophobacteraceae were detected. 

Generally only two to four phylotypes were observed, with the exception of the more diverse 

column A sample recovered at the end of biostimulated reduction, which had 8 phylotypes. In a 
3-73

recent study, researchers investigated dsrAB transcript abundance during a U remediation 

experiment at the Rifle site and found these same 3 families to be dominant (Miletto et al., 

2011). They also found that Desulfobacteraceae appeared to respond most significantly to the 

acetate amendment. A fourth family, Peptococcaceae, also was found in columns A and C, 

though they were of much lower abundance. This group, which contains Desulfotomaculum, has 

also previously been found at a uranium contaminated site and is possibly important in the 

remediation (Chang et al., 2005). 

The abundance and diversity of SRB was lowest in column C, relative to columns A and B. 

Perhaps the competition with Geobacter allowed only a few groups of SRB to persist in the 

column. In column A, SRB changed dramatically after oxic elution compared to during reduction. 

The dominant sequence, representing 73% of the clones, is from a different organism, though 

still in the family Desulfobulbaceae. No members of Desulfotomaculum or the 

Desulobacteraceae are evident in the post oxidation sample, suggesting that they were 

sensitive to the oxic elution. An increase in sulfate reducers was found at the end of a field-scale 

remediation experiment at Rifle when mobilization of uranium was observed (Anderson et al., 

2003). Perhaps the sulfate reducing organism which became dominant was less oxygen 

sensitive than the other SRB, and possibly able to live under oxic conditions, and therefore 

outcompeted the other SRB.

4-1

4 Discussion of Column Experimental Results 

4.1 Biostimulated Reduction

4.1.1 Electron Balance during Biostimulation 

The integrated column effluent concentrations for acetate, total dissolved CO2, sulfate, sulfide, 

iron, and uranium combined with “whole-column” changes in sediment content of iron, sulfur, 

and uranium (Table 3) provide insight on the relative importance of different biogeochemical 

processes occurring during the biostimulation period of the column experiments. These 

processes are incorporated into the reactive transport model simulation of biostimulated 

reduction presented in Chapter 5, specifically acetate oxidation and sulfate, iron and uranium 

reduction. 

Acetate consumption was small during the first 20 days of biostimulation, but then increased 

throughout the remainder of the biostimulation period with effluent concentrations in column A 

and C showing near complete consumption of acetate by the end of the biostimulation period. 

The increase in acetate consumption starting at 30 days coincided with the increase in sulfate 

reduction as evidenced by the increase in effluent sulfide and decrease in effluent sulfate at this 

time. Oxidation of acetate by microbial processes requires equivalent reduction of other 

constituents to balance electron transfer from these electron donors. Likely terminal electron 

accepting processes (TEAP) under anaerobic conditions in aquifer sediments are sulfate, iron, 

manganese, and uranium reduction. An electron balance for electron donors (acetate oxidation) 

and TEAPs was constructed using the integrated column effluent data and solid phase analyses 

(Table 17). The total acetate consumption in columns B and C (85 and 71 millimoles, 

respectively) was greater than in column A (46 millimoles) consistent with the longer period of 

acetate amendment (Table 3). Oxidation of acetate to CO2 produces 8 electrons. A net increase 

of 160 and 120 mmol total dissolved CO2 was estimated for columns B and C, respectively, by 

integrating effluent sample concentrations (Table 17). Total consumption of acetate was 85 and 

71 mmoles for columns B and C, respectively, which yields a ratio of acetate consumption to 

CO2 production of 0.53 and 0.59, which are slightly higher than 0.5 for the 1:2 reaction 

stoichiometry for acetate oxidation to CO2. The ratio of acetate consumption to CO2 production 

for column A cannot be determined because total CO2 was not measured in column A effluent 

samples during the first half of the biostimulation period.

Reduction of sulfate to sulfide (S-2) requires an 8 electron transfer, 1 electron for reduction of 

Fe(III) to Fe(II), 2 electrons for U(VI) reduction to U(IV), and 2 electrons for Mn(IV) reduction to 

Mn(II) (Table 17). The sulfate reduction estimated from integrated loss of dissolved sulfate 

accounted for 72 to 110% of the electron transfer from acetate consumption. Total sulfate 

reduction also was estimated from the sum of the integrated effluent dissolved sulfide and the 

increase in solid phase S, assuming that solid phase S increase was all initially from complete 

reduction to S-2. Subsequent partial oxidation of sulfide to S-1 forming FeS2 or to So during 

reduction of Fe(III) phases by sulfide is thus accounted for by using the increase in total S. Total 

sulfur reduction from this estimate accounted for 39 to 87% of electron transfer from acetate 

oxidation (Table 17). Both of these estimates are consistent with sulfate reduction as the 

dominant TEAP process for oxidation of acetate for columns B and C. The comparison for 

column A indicates that the sulfate reduction TEAP accounts for 39 to 72% of the electron 
4-2

transfer during acetate consumption. The discrepancy between the two estimates for column A 

likely is the result of error in the effluent dissolved sulfide since the solid phase measurements 

of S retained are in line with the other two columns given the duration of biostimulation of each.

Table 17. Summary of electron transfer during biostimulated reduction in columns A, B and C.

Constituent Total mmoles Electrons mmoles Electrons donated (%)

Column: A B C A B C A B C

Electron donors:

Acetate consumeda 46 85 71 368 680 568

C2H3O2

-

+ 2H2O→2HCO3

-

+ 5H+ + 8eElectron acceptors:

SO4

-2

+ 8e-→S

-2 b 33.2 64 78 -265.6 -512 -624 72.2 75.3 109.9

Total sulfidec 17.9 74.3 51.8 -143.2 -594.4 -414.4 38.9 87.4 73.0

Fe(III) + e-→Fe(II)d 9.2 9.6 6.5 -9.2 -9.6 -6.5 2.5 1.4 1.1

U(VI) + 2e-→U(IV)e 0.041 0.074 0.104 -0.082 -0.148 -0.208 0.02 0.02 0.04

Mn(IV) + 2e-→Mn(II) 0.059 0.045 0.048 -0.118 -0.09 -0.096 0.03 0.01 0.02

Total TEAP -275 -522 -631 74.7 76.7 111.1

Unaccounted e

- 93 158 -63

Effluent Dissolved CO2

f n.m. 160 120

HCO3

-

producedg 92 170 142

a. Acetate consumed is the integrated loss of acetate from difference between influent and effluent acetate over 

the duration of biostimulation.

b. Sulfate reduction determined by integrated effluent sulfate decrease over the duration of biostimulation. This 

measure of sulfate reduction is used in total TEAP estimate.

c. Total sulfide is the sum of the integrated effluent dissolved sulfide and the sum of the increase in total sulfur 

measured in each column sediment subsample times the samples mass fraction and the total sediment weight in 

the column.

d. Iron reduction is the sum of whole column increase in extractable ferrous iron and the integrated effluent 

dissolved iron.

e. Total uranium reduction is from the difference between integrated influent and effluent dissolved uranium 

during the period of acetate amendment.

f. Effluent Dissolved CO2 is the integrated increase in effluent total dissolved CO2. 

g. HCO3 produced is equal to two times acetate consumed. 

Ferric iron reduction estimated from the integrated effluent iron and the increase in 1-hour 0.5N 

HCl extractable Fe(II) in column sediment accounts for 1.1 to 2.5% of electron transfer (Table 

17), a small fraction of the electrons needed to balance acetate oxidation. Reduction of Fe(III) in 

phyllosilicate minerals by dissimilatory microbial iron reduction can be significant in aquifer 

sediments (Wu et al., 2012; Lee et al., 2012; Komlos et al., 2007). However, it is unknown if 

Fe(II) produced by reduction of Fe(III) silicates is quantitatively extracted by 0.5 N HCl since 

silicate phases are not readily dissolved by HCl. As such, these estimates of iron reduction are 

likely lower limits. Reduction of uranium and manganese combined account for 0.6% of the 

electron transfer, at most. Combined, these terminal electron accepting processes account for 

75, 77 and 110% of the electrons donated in column A, B and C, respectively (Table 17), with 

sulfate reduction the dominant TEAP. It is unknown if this disparity is the result of uncertainties 

in the estimated amounts of acetate consumption and reduction of S, Fe, U, and Mn, or if the 

unaccounted for electrons in columns A and B are the result of some other pathway of acetate 

consumption such as fermentation (Muyzer and Stams, 2008). Alternatively, the much higher 

microbial abundances in column A may account for part of the acetate balance assuming 

greater biomass growth. The biomass inferred from copy numbers in the qPCR analysis was 

greater for column A sediments than column B or C (Table 14).
4-3

4.1.2 Biogeochemical Processes during Biostimulated Reduction

The sequence of biogeochemical processes in the biostimulation stage of each of the three 

columns follows the expected sequence of iron reduction followed by sulfate reduction. The 

column effluent profiles overall are similar among the three biostimulation columns. The onset of 

iron reduction evidenced by the increase in effluent [Fe] occurred within 10 days of the start of 

the acetate inflow. Peak iron reduction based on maxima in effluent [Fe] occurred between 15 

and 25 days in the three columns with effluent [Fe] maxima occurring later in columns B and C 

(Figure 43). Effluent [Fe] decreased to near the detection limit by 25 to 30 days, concomitant 

with the onset of sulfate reduction, based on the increase of effluent dissolved sulfide to 

measurable levels. The onset of U reduction occurred concurrent to iron reduction consistent 

with other lab (Komlos et al., 2008a; Moon et al., 2007) and field biostimulation experiments 

(Anderson et al., 2003; Vrionis et al., 2005; Williams et al., 2011). The extent of U uptake 

increased overall throughout the duration of biostimulation (Figure 43) with the greatest uptake 

occurring during sulfate reduction. Uranium uptake was substantially greater in column C than in 

columns A and B throughout the biostimulation. The [U] effluent profiles for columns A and B 

show an increase in [U] starting at about day 25 concurrent with the onset of sulfate reduction. 

The effluent [U] increase likely is the result of the increase in bioreduction generating the higher 

pH and dissolved carbonate that increased U(VI) complexation in solution causing a decrease in

the rate and extent of uranium reduction (Brooks et al., 2003; Ulrich et al., 2011) as well as

enhancing U(VI) desorption. The increase in effluent [U] occurs later in column C, between day 

40 and 50 days (Figure 43). The onset of sulfate reduction and the resulting pH and alkalinity 

increase also occurred later in column C (Figure 3, 4, and 5). Subsequently, the rate of uranium 

reduction increases in all columns. The increased reduction is evident from the decrease in 

effluent [U] to 4 mM by 110 days in columns A and B, and to 2 mM by 70 days in column C. 

The enhanced U reduction in column C compared to columns A and B suggests either greater 

activity of metal reducing bacteria, or a greater component of indirect or abiotic reduction. The 

increased U removal is evident throughout the biostimulation period (Figure 43). Column C had 

continuous input of 15 mM Fe(II) throughout biostimulation, 1.5 times the influent [U]. Fox et al. 

(2013) showed that reduction of U(VI) can occur by electron transfer from Fe(II) sorbed to 

aquifer sediments at sorbed Fe(II) concentrations >20 mmol/g. This process may account for the 

difference in U reduction observed. However, the extractable Fe(II) concentrations in column C 

were similar to column A, so enhancement of U(VI) reduction by sorbed Fe(II) in column C is not 

expected. Alternatively, the sustained Fe(II) concentrations may result in ongoing formation of 

iron sulfides such as mackinawite during sulfate reduction providing additional sites for abiotic 

reduction of U(VI) (Veeramani et al., 2013; Hyun et al., 2012; Hua and Deng, 2008; Gallegos et 

al., 2013). 
4-4

Figure 43. Comparison of effluent dissolved uranium (top), dissolved iron (middle), and dissolved sulfide 

(bottom) for columns A, B and C during biostimulation. The data are plotted for the duration of 

acetate addition to column A.

Sulfate reduction and retained S was greater in column C than column A (Table 17) consistent 

with enhanced U reduction by this abiotic process. Lastly, the abundance of Geobacter relative 

0

2

4

6

8

10

0 20 40 60 80 100

Column A

Column B

Column C

Dissolved Sulfide (mM)

Time (Days)

0

10

20

30

40

50

60

70

80

0 20 40 60 80 100

Column A

Column B

Column C

Dissolved Fe (mM)

Time (Days)

0

2

4

6

8

10

0 20 40 60 80 100

Column A

Column B

Column C

Dissolved U(VI) (mM)

Time (Days)
4-5

to SRB was higher in column C (Figures 38, 40; Table 14). This observation suggests greater 

activity of metal reducing microbes in column C relative to sulfate reducers, perhaps resulting in 

the enhanced U(VI) reduction. However, the overall microbial abundances were substantially 

higher in the other columns. The processes contributing to the enhanced U(VI) uptake in column 

C cannot be constrained by the data collected. 

The observed increase in uranium uptake during sulfate reduction contrasts with other studies 

that propose U reduction is greatest during iron reduction with acetate oxidizing sulfate reducing 

bacteria not as effective in uranium reduction (Anderson et al., 2003; Yabusaki et al., 2010) and 

field observations that show the greatest U(VI) reduction correlated with Fe(III) reduction and 

Geobacter abundance (Vrionis et al., 2005). Additionally, field studies have observed that U(VI) 

removal is lower following the transition to sulfate reducing conditions, which has been 

attributed in part to less efficient uranium reduction by SRB relative to Geobacter (Anderson et 

al., 2003; Williams et al., 2011). The observed increase in uranium reduction as sulfate 

reduction progresses in all three column experiments would require that dissimilatory metal 

reducing bacteria, such as Geobacter, are active and undergoing growth concurrent with sulfate 

reduction if these microbes are dominating uranium reduction. In field biostimulation 

experiments, groundwater and sediment chemistry data indicate simultaneous occurrence of 

iron and sulfate reduction where the electron donor was not limiting (Vrionis et al., 2005). 

Williams et al. (2011) concluded that the availability of sufficient acetate to sustain metalreducing microbes during sulfate reduction resulted in the ability of these microbes to control 

uranium removal from groundwater at the Rifle site. Because neither acetate, sulfate or 

reducible iron are depleted it is unlikely that the electron donor availability is limiting microbial 

processes in these columns, and thus competition between SRB and metal reducing microbes 

is not sufficient to diminish uranium reduction by metal reducing microbes. Similarly, Komlos et 

al. (2008a) observed that the rate of U(VI) reduction was unaffected by the sulfate reduction in 

biostimulation column experiments with Rifle sediment at low and high sulfate concentrations. In 

addition, these authors concluded that sulfate reduction commenced prior to depletion of 

reducible iron in part because electron donor was not limiting. Uranium reduction also could be 

the result of other microbial processes. For example, sulfate reducing bacteria have been 

shown to reduce uranium directly through enzymatic mechanisms using lactate and ethanol as 

electron donors (Lovley et al., 1993; Luo et al., 2007a), both of which can produce acetate 

during consumption. 

In addition to direct enzymatic uranium reduction by microbes, U(VI) reduction has been shown 

to occur by abiotic reaction with products of other biotic reduction processes, such as iron 

sulfides (Veeramani et al., 2013; Hyun et al., 2012; Hua and Deng, 2008; Gallegos et al., 2013). 

Association of U(IV) with reduced iron and sulfur has been observed in sediments recovered 

from in-situ columns exposed to groundwater during field-scale acetate biostimulation 

experiments at the Rifle site (Bargar et al., 2013). These authors suggest a role of sulfides in 

ongoing sustained U(VI) reduction that has been observed in field biostimulation tests well after 

electron donor addition had been halted (Williams et al., 2011). However, the importance of 

abiotic, non-enzymatic uranium reduction has not been conclusively demonstrated at the field 

scale. In the present experiments, continued U uptake was observed well after cessation of 

acetate inflow to column A (Figure 3). Over a 35 day period (74 pore volumes) under zero 

oxygen conditions and 10 mM U(VI) influent, effluent [U] remained low initially decreasing to <2 

mM, then slowly rising (Figure 3). During this period of U(VI) input in the absence of acetate, a 

total uptake of 20.4 mmol was observed, which is about half of U uptake during acetate addition 

over 110 days (41.2 mmol). Analysis of XANES spectra of sediments recovered at the end of the 

anaerobic stage (after biostimulation, U inflow without acetate, and 45 days with no acetate or 

U(VI)), show that >97% of the U is U(IV) (Table 9). The U uptake after acetate cessation (20.4 

mmol) is about one third of U uptake in the entire experiment, and is well above the ~5% 
4-6

detection limit for U(VI). About 4.6 mmol of U was remobilized and transported out of the column 

over the 45 days of flow of AGW without acetate, U(VI), or dissolved oxygen (Figure 3). If this 

amount is assumed to be U(VI) that was sorbed (instead of reduced and precipitated as U(IV)) 

after acetate input ceased, it would equal to 22% of U uptake during this period. This 

percentage of U(VI) is readily detectable by XANES, but essentially only U(IV) was measured. 

This comparison indicates that significant U reduction continued after electron donor addition 

was halted. Since sulfate reduction decreased rapidly when acetate input ended as evidenced 

by sulfate returning to influent concentrations and effluent sulfide decreasing to below detection, 

SRB activity stopped, and likely other microbial activity decreased. We propose that the 

observed continued uranium uptake occurred largely by reduction by abiotic processes. The 

increase of Fe(II) and reduced S in sediments are consistent with U reduction by interactions 

with iron sulfides (Veeramani et al., 2013; Hyun et al., 2012; Hua and Deng, 2008; Gallegos et 

al., 2013). Additionally, the co-association of U(IV) with Fe and S in some grain coatings (e.g. 

Figures 30, 33 and 36) support this conclusion. Abiotic U(VI) reduction may also occur by 

electron transfer from other forms of Fe(II), such as sorbed to sediment surfaces (Fox et al., 

2013). A component of sorbed Fe(II) is possible since effluent [Fe] increased during the period 

after acetate input was halted when sulfate reduction ended (Figure 3). This effluent [Fe] 

increase may be in part from desorption of Fe(II) or dissolution of reduced iron phases, both of 

which would be more favorable in the absence of dissolved sulfide.

In contrast to the continuously decreasing [U] in the effluent throughout the acetate addition, 

effluent Fe decreased substantially after 15 to 20 days, and reached a low level by 30 days that 

persisted for the duration of the biostimulation. The comparison of integrated effluent [Fe] with 

the increase in extractable Fe(II) in sediments recovered from columns after biostimulation 

shows that a large majority of reduced iron remained in the column. The increase in effluent 

sulfide following the iron effluent peak limited ferrous iron solubility and thus decreased 

dissolved iron transport out of the columns. In addition, previous studies have observed that 

only a small amount of the Fe(II) produced by biogenic reduction of phyllosilicate iron is 

solubilized with most of Fe(II) remaining in the silicate layer structure (Komlos et al., 2007). 

Although its unknown if iron reduction continued throughout the biostimulation period, the 

availability of electron donor in excess of sulfate reduction throughout the biostimulation period 

and the abundance of metal reducing microbes measured in the sediments at the end of 

biostimulation suggest that iron reduction is likely occurring for the entire duration of electron 

donor addition. Poorly crystalline iron comprises a small fraction of total iron in Rifle sediments 

(~1 %) of total iron with phyllosilicate iron accounting for about 56% of the total iron with the 

remainder crystalline iron oxides (Komlos et al., 2008b). The abundance of reducible iron, 

including phyllosilicate iron, well exceeds the extent of iron reduction measured in these column 

experiments. Thus, iron reduction likely continued throughout the duration of the biostimulation 

either directly by dissimilatory metal reducing microbes or indirectly through reaction of Fe(III) 

with dissolved sulfide. The formation of elemental S by reaction of free sulfide with Fe(III) solids 

results in oxidation of S-2 and reduction of Fe(III) to Fe(II) (Li et al., 2009). This process is 

illustrated in the following reaction: 

Fe(OH)3(s) + 0.5 H2S ↔ Fe2+ +So + H2O + 2 OH-

. 

This iron reduction pathway may also occur for phyllosilicate iron. Reduction of both Fe(III) 

oxides or phyllosilicate iron by sulfide or direct microbial reduction of phyllosilicate iron would 

result in little transport of dissolved iron from the columns after the onset of sulfate reduction. 
4-7

4.2 Remobilization of Uranium during Oxic Elution 

Combined the effluent and solid phase measurements from columns during and after the 

biostimulation stage suggests that stimulation and growth of the ambient microbial population in 

the aquifer sediments from the Rifle site by addition of electron donor and carbon source may 

be an effective means of removing dissolved uranium from contaminated groundwater. This 

conclusion is consistent with a number of both field and laboratory studies at Rifle (Anderson et 

al., 2003; Williams et al., 2011; Yabusaki et al., 2010; Komlos et al., 2008b; Sharp et al., 2011) 

and at other sites (Gu et al., 2005; Wu et al., 2006). Because it well known that reduced 

uranium phases can undergo oxidative dissolution upon exposure to oxygen (Bi et al., 2013; 

Komlos et al., 2008b; Moon et al., 2007; Moon et al., 2009) many of these studies have stressed 

that the effectiveness of biostimulated reduction as a viable long-term remediation strategy 

requires evaluating the stability of the bioreduced uranium in response to changes in 

groundwater chemistry, such as presence of dissolved oxygen at suboxic levels. For example, 

Yabusaki et al. (2010) stated that “A key issue for uranium bioremediation is the stability of 

solid-phase reduction products, including U(IV).” The stability of bioreduced U(IV) is dependent 

on maintaining reducing conditions within the aquifer. The primary goal of this study was to 

evaluate the remobilization of U sequestered during biostimulated reduction under conditions in 

which biostimulation and concomitant U reduction extended well into sulfate reduction to 

enhance precipitation of reduced sulfur phases such as iron sulfides. The intent of producing 

these reduced phases was to test their effect on remobilization of the sequestered uranium, 

either serving as a redox buffer by competing for dissolved oxygen, or by armoring the reduced 

uranium. In particular, increased production of iron sulfide phases was attempted through 

addition of ferrous iron in the influent stream throughout biostimulation (column C).

The removal of dissolved U(VI) from the influent continued over the course of the 110 to 330 

day biostimulation stage well after the onset of sulfate reduction at ~30 days. The resulting 

sequestered U was essentially all U(IV) consistent with the formation of nanoparticulate 

uraninite that may be associated with organic microbial biomass, and may in part be sorbed to 

C or P in the organic matter (Section 3.4.2). The speciation of U(IV) associated with sediments 

after oxic elution was essentially the same as that measured in samples recovered after 

biostimulation and prior to oxic elution. The reduced U(IV) formed coatings on sediment grains 

with thicknesses of a few 10s of microns (Figures 33, 34, 38). The coatings of U are largely 

contiguous with the coating infilling voids and cracks in the grain surface suggesting the 

coatings are formed in place as opposed to fine particles adhering to grains during sample 

drying.

4.2.1 Effect of U(IV) Form and Morphology 

Nanoparticulate biogenic uraninite has been found to be on the order of 3 nm in diameter and 

tends to form aggregates (Burgos et al., 2008; Schofield et al., 2008). Because of the small 

particle size, Schofield et al. (2008) estimated that about 50% of the U is near the surface 

resulting in a lattice contraction compared with larger abiotic particles of bulk UO2. Although the 

surface is distorted, the particle core remained highly ordered consistent with stoichiometric UO2

(Burgos et al., 2008; Schofield et al., 2008; Singer et al., 2009). The net effect, however, is that 

strain on the interior is negligible and likely does not enhance the solubility or stability (Schofield

et al., 2008). Nanoparticulate UO2 has been shown to form aggregates with the size in part due 

to the rate of U reduction in some cases (Burgos et al., 2008; Senko et al., 2007). Although the 

~3 nm UO2 nanoparticles cannot be resolved by either mXRF mapping or SEM imaging, the U- 
4-8

bearing coatings are likely comprised of aggregates of the UO2 nanoparticles and perhaps may 

include U(IV) associated with the microbial biopolymers and microbes that attach to sediment 

surfaces (Bargar et al., 2013). As noted above, the coatings in part appear to be comprised of 

smaller particles (e.g. Figure 35B), but these particles are well above the particle size inferred 

from fitting of EXAFS spectra. The fits to EXAFS spectra do suggest that U(IV) in the sediment 

may be present as both partially disordered nanoparticles and as monomeric U in association 

with organic matter. However, XRF does not measure all elements, in particular C and therefore 

do not provide information on other components comprising the grain coatings, for example 

microbial biomass. Thus, the coating morphology and accessibility of U within the coatings 

cannot be further constrained by the present measurements, but may influence the susceptibility 

of U(IV) to oxidative remobilization. 

4.2.2 Competitive Oxidation Processes

Previous studies have reported oxidative remobilization of U(IV) under anaerobic conditions 

resulting from continued reduction of Fe and Mn oxides that serve as terminal electron 

acceptors for U oxidation coupled with increased dissolved carbonate shifting the redox state to 

more favorable conditions for U oxidation (Wan et al., 2005). Remobilization of U has been 

studied following biostimulated reduction under low sulfate conditions in laboratory column 

experiments with aquifer sediments from the Rifle site inoculated with Geobacter 

metallireducens. These experiments showed about 88% and 97% remobilization of U within 54 

days when 0.27 mM dissolved oxygen (saturation with respect to air) or 1.6 mM nitrate were 

added to the influent, respectively (Moon et al., 2007). After 200 days of biostimulated reduction 

in similar column experiments under low sulfate conditions, re-oxidation of U(IV) resulted in the 

increase of effluent [U] to over 150 mM within 10 days of exposure to dissolved oxygen at 

saturation with air, with 60% of U remobilized after 60 days and nearly all by 120 days (Komlos

et al., 2008b). The extent of U remobilization in these experiments suggested little adsorption of 

U(VI) by iron oxides or other surfaces on the column sediments and that oxidation of ferrous 

iron provided negligible buffering of U(IV) oxidation. Cell decay and ferrous iron oxidation 

accounted for 43 and 23% of oxygen consumption, respectively, with U accounting for 12% 

(Komlos et al., 2008b). In contrast, Sharp et al. (2011) observed little remobilization of 

biologically reduced U in columns with Rifle sediments under low sulfate conditions upon 

exposure to oxygen. Instead, the increase in the relative proportion of U(VI) in the middle and 

outflow end of the column was attributed to retention of U(VI) by sorption following re-oxidation 

of U(IV) near the inflow end of the column.

The presence of iron sulfides has been proposed to limit re-oxidation of U(IV) by serving as a 

redox buffer or armoring the U(IV) precipitate (Abdelouas et al., 1999). The timing and extent of 

U remobilization following prolonged biostimulated reduction of sulfate, iron and U in column 

experiments with inoculated Rifle sediments show significantly less U re-oxidation and a delay 

in release of U upon addition of oxygen or nitrate to column influent (Moon et al., 2009), 

compared to low sulfate experiments which had little or no iron sulfide precipitation (Moon et al., 

2007). The iron sulfides formed during biostimulation served to partially limit biogenic U(IV) 

oxidation by competing for dissolved oxygen. Less than 1% of U was remobilized by 0.27 mM 

dissolved oxygen and no oxidation at 0.03 mM dissolved oxygen was observed through 50 days 

of elution. Sulfide oxidation accounted for 85% of oxygen consumption. Mackinawite (FeS) has 

been shown to inhibit UO2 oxidation provided that it is it is in physical proximity to the U(IV) 

phases and is in sufficient quantity to effectively scavenge oxygen to act as a redox buffer (Bi et 

al., 2013). 
4-9

4.2.3 Observed Rate and Extent of U(IV) Release

In the present study, the time dependence of U release in the absence of dissolved oxygen and 

subsequently during oxic elution provides a measure of the stability of the bioreduced U. Prior to 

introduction of dissolved oxygen to the column influent stream, column C effluent [U] averaged 

<0.1mM over 18 pore volumes after U(VI) and acetate were removed from the influent during the 

bromide tracer pulse prior to repacking for oxic elution. In contrast, effluent [U] in column A 

averaged 0.4 mM over 45 days (96 pore volumes) of flow under anoxic AGW without acetate or 

U(VI). It is not known If the release of U during these periods is from U(VI) desorption, oxidation 

of U(IV), or mobilization of U(IV) without oxidation. Dissolution and mobilization of uranium from 

reduced sediments has been observed previously in the absence of oxygen and was attributed 

in part to complexation by natural humic substances (Luo and Gu, 2009). The very low effluent 

[U] prior to introduction of dissolved oxygen combined with the predominance of U(IV) (>90%) in 

the sediment following biostimulation are consistent with negligible release of sorbed U(VI) from 

sediments prior to the introduction of oxygen. 

After the first 15 days of oxic elution, effluent [U] for column A averaged 0.85±0.11 mM and was 

near constant over the duration of the 140 day (350 pore volume) elution (Figure 6). By 

comparison, column C effluent [U] averaged 0.34±0.07 mM over the first 30 days, then 

decreased slowly to 0.2 mM by the end of 120 day (300 pore volume) elution. Remobilization of 

uranium from column C during oxic elution resulted in about a factor of 3 lower integrated 

effluent dissolved [U] than was observed for column A (Table 3). The primary difference 

between column A and C was the inclusion of 15 mM ferrous iron to the influent of column C 

throughout biostimulated reduction.

The measured concentration in sediment samples was about a factor of 3 lower for the 

composite sediment from column C than the U concentration based on integrating the effluent 

profile (Figure 44). As noted in section 3.3.4, this disparity may be the result of incomplete 

sampling of sediment from column C at the end of biostimulation. Alternatively, U loss may have 

resulted upstream of the sediment in column C, perhaps from interaction of ferrous iron with 

U(VI) in the influent stream. The U mass released estimated by integrating effluent [U] during 

oxic elution, 2.5 mmol U, was comparable to the difference between measured sediment U 

before and after oxic elution, 2.3 mmol U (Figure 44). This comparison suggests the measured

sediment U in the composite used to repack column C for oxic elution better represents the 

initial condition. The similarity in the measured U of intermediate depth intervals after oxic 

elution with the U concentration of the column C composite used for repacking (Figure 23) also 

supports the use of the measured sediment concentration of the composite to represent the 

whole column U at the start of oxic elution. In contrast, the total U at the start of oxic elution in 

column A based on the integrated effluent total U uptake was about 10% higher than the 

composite sediment U content (Figure 44). However, the total U remobilized from column A 

during oxic elution based on the integrated effluent, 10.6 mmol U, was about a factor of 3 greater 

than the sum of the uranium concentration of all sediment samples times their mass 

contribution, 3.4 mmol U. This discrepancy likely is the result of a greater uncertainty in the 

measured sediment U concentrations when a large number of sediment samples are summed 

to estimate changes in total U within a column (23 subsamples for column A). The uncertainty is 

in part from analysis of small sample masses, loss of fine grain material, and the poorly 

constrained contribution of the >2 mm size fraction (see sections 3.2.4 and 4.3.4), which all may 

have caused an underestimate of total U release from column A. As a result, this discussion of 

oxidative remobilization relies on effluent dissolved uranium. The effluent profiles also provide 

information on the time dependence of the release.
4-10

Figure 44. Total uranium on a whole column basis for oxic elution columns A and C, in micromoles, before 

and after oxic elution from (A) integrated effluent concentration profiles and (B) sediment U 

concentrations.

Previous studies of oxidative remobilization of reduced U report dissolution rates of synthetic 

and biogenic UO2 in flow through or batch reactors in units of mol g-1 sec-1, where g is the mass 

of reduced (or total) uranium (in grams) (Bi et al., 2013; Sharp et al., 2011; Ulrich et al., 2009). 

Because the remobilization of U from both columns occurred at near constant rates, the linear 

slope of the change in sediment U per gram versus time (Figure 7A) yields release rates of 

3.4E-10 and 1.2E-10 mol U/g sediment/day for column A and C, respectively. Normalizing to the 

initial mass of reduced U at the start of oxic elution yields dissolution rates of 6.5 E-11 and 3.4E11 mol g-1 sec-1, respectively. The initial U mass is based on the measured U concentration of 

the composite sediment used to repack the columns and includes the fraction of U associated 

with the >2 mm particles. Because other processes, such as adsorption of U(VI) by sediment, 

can lower the effluent dissolved [U], calculated rates from column effluent data are considered 

net rates. The dominance of the solution speciation of dissolved U(VI) in the AGW as calcium 

carbonato complexes (>90%) likely limits adsorption or buildup of oxidized uranium on the 

sediments. Indeed, the U oxidation state from bulk XANES measurements of column sediments 

from intervals that appeared more oxidized also were dominated by U(IV) (Table 9). The 

calculated net release rates from the column effluent data are two orders of magnitude lower 

than the 1.5 to 4.7E-9 mol g-1 sec-1 rates reported for synthetic and biogenic UO2 from flow 

through reactors (Ulrich et al., 2009). The visual apparent channeling or preferential flow paths 

(Figures 8 and 9) combined with column samples which had little change in U concentration in 

response to oxic elution (Figures 20 and 23) suggest that release may not be not occurring 

uniformly from the whole column. Therefore, the actual mass of sediment U undergoing 

oxidation may be lower than the whole column U mass used to normalize the release rates. If 

so, the normalized rates would be higher if the actual mass of sediment U involved in the 

oxidative remobilization could be used instead of bulk sediment U concentrations. 

0

10

20

30

40

50

60

70

80

90

A C

Uranium (micromoles)

Column

Column Total U from Effluent

Pre-oxic

Post-oxic

0

10

20

30

40

50

60

70

80

90

A C

Uranium (micromoles)

Column

Column Total U - Sediment

Pre-oxic

Post-oxic

A B
4-11

4.2.4 Processes Affecting Uranium Remobilization in Column Experiments

A number of factors likely contributed to the observed lower rate and extent of U oxidative 

remobilization measured in column C compared to column A. The primary difference in the two 

column experiments was the 15 mM Fe(II) addition to the influent of column C throughout 

biostimulation. Differences in other constituents in the two columns may contribute to the 

observe difference in U remobilization. The mXRF maps and SEM-EDS measurements show coassociation of U with Fe and S in some grain coatings, but not in others. In addition, thicker 

coatings would be expected for column C because of the longer biostimulation period and 

apparent greater rate of U reduction. Because of the few grains with U coatings that were found 

in thin sections of column sediments and imaged, no trend in coating thickness or composition 

between the two columns could be discerned. Column A went through a longer period without 

electron donor addition under low or zero oxygen conditions than column C. Decrease of 

biomass during this period would limit biomass contribution to oxygen consumption during oxic 

elution. For example, Komlos et al. (2008b) estimated that biomass decay accounted for 43% of 

oxygen consumption. However, the abundances of Geobacter and SRB based on cell numbers 

were substantially higher in column A compared to C (e.g Figures 41 and 42) suggesting 

biomass decay does not contribute to the observed trend. Column C had about 35% greater 

retention of S during biostimulation than column A (Figure 45), with the fraction of S as AVS 

about the same in both columns. The decrease in total sulfur during oxic elution was about 50% 

greater for column C than column A suggesting a greater consumption of oxygen by reduced 

sulfur species in column C perhaps limiting remobilization of U(IV) by buffering oxidation as 

proposed by Abdelouas et al. (1999) and observed in batch experiments (Bi et al., 2013). 

Although there was little loss from sediment intervals near the middle of column C, U was 

remobilized primarily from intervals nearest the column inlet and outlet (Figure 23). These 

intervals comprise 33% of the sediment mass but accounted for 75% of the U loss. Similarly 

these intervals accounted for most of the total S decrease with 70% of the decrease from loss of 

AVS, presumably oxidation. The decrease in extractable Fe(II) was primarily from these 

intervals (Figure 24). They also had a substantial increase in Fe(III) extracted by 0.5N HCl 

relative to the sediment at the start of oxic elution. U decrease generally was greater at the inlet 

end of column A, but occurred in subsamples throughout the column. Decrease in Fe(II) and in 

total S also occurred to a greater extent in column A subsamples with greater U loss but not 

consistently for all such subsamples. These variations illustrate the heterogeneity of flow within 

the columns during oxic elution which was also evident in the appearance of sediments at the 

exterior of the columns during the experiments (Figure 8) and on subsampling after oxic elution 

(Figure 9 and 10). Although the relative amount of reduced sediment exposed to preferential 

flow cannot be constrained, the difference in the fraction of sediments exposed to oxygen 

between the two columns may be the cause in the observed difference in uranium 

remobilization. 
4-12

Figure 45. Total sulfur in columns A and C sediments, in millimoles, before and after oxic elution.

Overall, the release of U during oxic elution is a continuous process with dissolved uranium 

concentrations on the order of 0.8 and 0.2 mM for columns A and C, respectively (Figure 6). The 

effluent concentration from column C decreased over time indicative of a decreasing reoxidation rate. In contrast, column A effluent [U] does not display this decreasing trend but is 

instead somewhat constant over 140 days. Overall, only about 9% of total uranium sequestered 

during biostimulation was remobilized. The prolonged period of biostimulation and concomitant 

sulfate reduction appears to limit the rate of U(IV) oxidative remobilization. The limited oxidative 

remobilization of U(IV) in the present column experiments is consistent with the decreased U 

remobilization where sulfate reduction occurred (Moon et al., 2009) compared to large release 

from columns which had limited sulfate reduction (Moon et al., 2007). Although continued 

sulfate reduction may cause a decrease in permeability resulting from precipitation of iron 

sulfide, as observed in column B, and a decrease in U(VI) removal in field studies (Anderson et 

al., 2003; Williams et al., 2011), the greater apparent stability of the sequestered U(IV) provided 

by the sustained biostimulation should be considered in design of field scale remediation efforts.

0

2

4

6

8

A C

Total Sulfur (millioles)

Column Total S - Sediment

Pre-oxic

Post-oxic
5-1

5 Reactive Transport Modeling of the Rifle Sediment 

Columns during Biostimulation

5.1 Introduction 

5.1.1 Background

An important challenge identified in previous studies (Fang et al., 2009; Williams et al., 2011) is 

the impact of biologically-mediated reaction products (e.g., biomass, bicarbonate, Fe(II), U(IV), 

H2S) on the behavior of uranium (Englert et al., 2009; Li et al., 2010; Li et al., 2009). In 

particular, the sensitivity of uranium mobility to pH, Eh, alkalinity, calcium, and reactive surface 

area requires detailed biogeochemical process modeling to mechanistically simulate changes to 

the aqueous and solid phase chemistry induced by acetate biostimulation (Davis et al., 2006; 

Dong et al., 2005; Li et al., 2009; Liu et al., 2005; Luo et al., 2007b). Biologically-mediated 

oxidation of acetate produces a large amount of bicarbonate that directly affects the alkalinity, 

and indirectly affects the pH, calcium and reactive surface area through precipitation of 

carbonate minerals (Li et al., 2009). The dissolution of Fe(III) minerals and the precipitation of 

sulfides and elemental sulfur affects pH and reactive surface area (Poulton et al., 2004). Ca–

UO2–CO3 complexes (Dong and Brooks, 2006), which are predicted to be predominant under 

Rifle geochemical conditions, have been shown to affect U(VI) bioreduction (Brooks et al., 2003)

and adsorption (Zheng et al., 2003). Furthermore, the calcium concentrations controlling 

uranium speciation can be altered through cation exchange with the sodium cation present in 

the dissolved acetate and bromide salts that comprise the injectate. Thus, the inclusion of a 

reaction network that accounts for uranium, terminal electron-accepting processes (TEAPs), 

biomass, major ion chemistry (Ca, Mg, K, Na, carbonate, sulfate, Cl), reduction products [Fe(II), 

U(IV), H2S], aqueous complexation (e.g., Ca-UO2-CO3), surface complexation [Fe(II), U(VI)], 

and precipitation/dissolution (goethite, calcite, FeS, S, UO2) is critical to capturing important 

biogeochemical interactions during biostimulation.

We describe the modeling of uranium biogeochemistry during the biostimulation stage of a set 

of laboratory column experiments that were performed by the USGS described in the preceding

sections of this report. The experiments were motivated by work conducted at the Rifle 

Integrated Field Research Challenge (IFRC) site in western Colorado demonstrating that the 

stimulation of indigenous dissimilatory metal-reducing bacteria, via the addition of the electron 

donor acetate, can reduce soluble hexavalent uranium [U(VI)] to immobile solid-phase U(IV)

(Anderson et al., 2003; Vrionis et al., 2005; Williams et al., 2011). This biologically mediated 

reduction of U(VI) can decrease groundwater concentrations of U in the shallow unconfined 

aquifer below relevant standards.

We use the systematic and quantitative coupling of process models to describe the interplay 

between the saturated flow, microbiology, and geochemistry during acetate biostimulation of the 

Rifle sediment column experiments. Modeling of these experiments is used to understand and 

quantify uranium behavior in the context of 1) transient biostimulation with acetate electron 

donor, 2) functional microbial groups representing Fe(III)-reducing bacteria (FeRB, such as 

Geobacter) and sulfate-reducing bacteria (SRB) responding to the biostimulation, and 3) 

geochemical response to the biostimulation products. 

The numerical model incorporates advection, diffusion with Fick’s law modified for the inclusion 

of dispersion, and mixed kinetic and equilibrium reactions. An operator splitting solution method 

is used for flow, transport, and reactions, each of which employs implicit time-stepping 
5-2

schemes. HYDROGEOCHEM was used to resolve the detailed coupled processes and large 

number of reactive components.

5.1.2 History

Previous 1-D saturated flow and multicomponent biogeochemical reactive transport modeling 

studies were performed to simulate the 2002, 2003, and 2007 field biostimulation experiments 

at the Rifle IFRC site (Fang et al., 2009; Yabusaki et al., 2007). A comprehensive reaction 

network was developed from these studies that included biologically-mediated TEAPs for solid 

phase Fe(III), aqueous U(VI), and aqueous sulfate; aqueous and non-electrostatic surface 

complexation for Fe(II) and U(VI); calcite, siderite, FeS, S secondary minerals; sulfide promoted 

dissolution of Fe(III) minerals, cation exchange, protonation/deprotonation of Fe(III) surfaces, 

and major ion chemistry.

The integration of the abiotic chemistry with the TEAPs in the modeling is necessary to account 

for the impact of biostimulation products on uranium behavior. For example, the TEAP reaction 

products affect the system pH and alkalinity, which are controls on uranium mobility (Davis et 

al., 2004). In laboratory and field experiments, the bulk of the bicarbonate and reduced phases 

(e.g., Fe(II), sulfide) produced by the biologically-mediated reactions are not observed in 

solution. This underscores the importance of accurately representing the solid phase reactions 

(e.g., mineral reactions for carbonates, iron, and sulfur; iron and uranium surface complexation) 

that control the pH, Eh, alkalinity, and aqueous components of interest.

5.2 Model Components 

5.2.1 HYDROGEOCHEM

All simulations were run with the HYDROGEOCHEM (HGC) simulator (Yeh et al., 2004). It 

provides the framework for incorporating and coupling the flow and reactive transport process 

models in this investigation. In particular, the multicomponent biogeochemical solver technology

(Fang et al., 2006; Fang et al., 2003) addresses the complexity of mixed kinetic and equilibrium 

reactions that describe the aqueous and surface complexation, redox, mineral precipitation and 

dissolution, and microbial mediated transformations.

5.2.2 Conceptualization of Processes

The biologically-mediated transfer of electrons to solid phase Fe(III) by FeRB and to aqueous 

sulfate by SRB provides the energy for cell maintenance, activity, and growth. Introducing 

acetate electron donor in solutions passing through the sediment stimulates FeRB that catalyze 

the reduction of Fe(III) minerals but also opportunistically catalyze the reduction of the trace 

amounts of aqueous U(VI) that are present (Lovley et al., 1991). Reduced uranium [U(IV)] 

becomes part of the solid phase, most likely associated with surface coatings or overgrowths on 

existing grains. Increases in aqueous Fe(II) and decreases in aqueous U(VI) occur very soon 

after biostimulation is initiated. Conversely, the SRB are slow growing and initially low in 

abundance. Although the SRB actively respond to the acetate biostimulation, their initial impact 

on acetate consumption and sulfate reduction is negligible. With time, however, the SRB growth 

continues and sulfate reduction becomes the dominant process after about 30 to 40 days. At 

this point, the bulk of acetate is being consumed by the SRB. In the sulfate TEAP reaction, ~1 
5-3

mole of sulfate is reduced and ~2 moles of bicarbonate are produced for each mole of acetate 

consumed. The increase in bicarbonate concentrations thermodynamically favors the formation 

of aqueous U(VI)- CO3 complexes. The resulting desorption of U(VI) increases aqueous U(VI) 

concentrations although there is still a net reduction in aqueous U(VI) relative to the influent 

concentrations. In the presence of elevated alkalinity, Fe(II), and sulfide; and lowered redox 

potential, calcite and iron sulfide secondary mineral formation are thought to be the most 

important volumetrically (Li et al., 2009). Lesser amounts of elemental sulfur, from the sulfide 

promoted dissolution of Fe(III) minerals (Fang et al., 2009), and siderite should also form.

5.2.3 Model Description

The simulation of the 194-, 219-, and 185-day biostimulation periods of experimental 

observation for columns A, B, and C, respectively, used variable time stepping with a maximum 

time step of 30 min. The 1-D model domain used to represent the experimental field plot was 6 

cm long, comprised of 15 grid cells with uniform 0.4 cm spacing. The influx of acetate was 

based on the flow rate and influent concentrations.

The initial reactive transport modeling of biostimulated reduction used a reaction network 

developed for the 2008 Big Rusty field experiment at the Rifle IFRC site (Fang et al., 2009; 

Yabusaki et al., 2007). In this case, the equilibrium and kinetic reactions in this reaction network 

(Tables 18-22) addressed 2 distinct functional microorganisms (i.e., FeRB and SRB), 4 TEAPs 

(i.e., phyllosilicate Fe(III), poorly crystalline Fe(III), U(VI), and sulfate), 102 biogeochemical 

species (including surface complexes, exchanged cations), and 7 minerals (i.e., FeOOH, Fe3O4, 

FeS, S, calcite, siderite, uraninite).

Key assumptions included: 1) FeRB are solely responsible for bioreduction of aqueous U(VI) 

that occurs throughout the biostimulation, 2) SRB are present and active throughout the 

biostimulation, 3) abiotic uranium reduction is negligible, and 4) geochemistry, including uranium 

surface complexation, is fully operative during biostimulation (e.g., biomass does not 

significantly alter geochemical reactivity).

The aqueous initial and boundary conditions were acetate, U(VI), pH, bicarbonate, calcium, 

magnesium, sodium, potassium, chloride, Fe(II), and sulfate. The initial sorbed U(VI) and Fe(II) 

were assumed to be in equilibrium with their aqueous counterparts through the respective 

surface complexation models (Tables 18 and 19). U(VI) minerals were not included in the 

reaction network as they have not been identified in sediment analyses and are undersaturated 

with respect to the solution chemistry. Other nonaqueous initial conditions include FeRB and 

SRB biomass, goethite, phyllosilicate iron and surface complexation sites.

Both functional microbial groups, FeRB and SRB, are present and active during the initial period 

of biostimulation (Callister et al., 2010). The kinetics of the microbially mediated TEAP reactions 

is of the Monod type with thermodynamic control. The acetate consumption rate, , as 

described by Fang et al. (2009) is: 

( )r

N

eA s eAeA

eA

CCs

C

m eA C

bio

C Gf CK

C

CK

C R Bs eA

∆ 















+ 















+ −= ∑ , ,

m ,

where NeA = number of terminal electron acceptors

bio RC
5-4

sC = stoichiometric coefficient of acetate in the TEAP reaction

Cc = acetate concentration

B = biomass concentration of microorganism mediating the TEAP reaction

CeA = terminal electron acceptor concentration

μm,eA = acetate oxidation rate for the terminal electron acceptor

Ks,C = half-saturation coefficient for acetate

Ks,eA = half-saturation coefficient for the terminal electron acceptor

f(ΔGr) = 1 – exp[(ΔGr- ΔGmin)/RT] 

ΔGr = free energy change of the corresponding TEAP reaction

ΔGmin = minimum free energy change required to drive ATP Synthesis

R = gas constant

T = absolute temperature.

The half-saturation coefficient is the substrate concentration at which the reaction rate occurs at 

half of its maximum rate. The FeRB and SRB are assumed to be attached populations whose 

biomass is a control on the rates of the TEAP reactions they catalyze. The initial FeRB and SRB 

biomass amounts are initially low (Table 24). As the FeRB and SRB biomass grow, the 

respective TEAP reaction rates increase commensurately. A first-order decay model (dB/dt = -

kB) is used to describe the loss of biomass via decay. Over time (typically 20-30 days), the net 

growth of SRB biomass is sufficient for sulfate reduction to become the dominant TEAP reaction 

and principal consumer of acetate. A maximum rate is used to limit the biomass-dependent 

reduction rates.
5-5

Table 18. Uranium species and their formation constants

Reaction logK (I = 0) (a)

Aqueous Species 

UO2

2+ + H2O = UO2OH+ + H+ -5.25

UO2

2+ + 2H2O = UO2(OH)2(aq) + 2H+ -12.15

UO2

2+ + 3H2O = UO2(OH)3-

+ 3H+ -20.25

UO2

2+ + 4H2O = UO2(OH)42-

+ 4H+ -32.40

2UO2

2+ + H2O = (UO2)2OH3+ + H+ -2.70

2UO2

2+ + 2H2O = (UO2)2(OH)22+ + 2H+ -5.62

3UO2

2+ + 4H2O = (UO2)3(OH)42+ + 4H+ -11.90

3UO2

2+ + 5H2O = (UO2)3(OH)5+ + 5H+ -15.55

3UO2

2+ + 7H2O = (UO2)3(OH)7-

+ 7H+ -32.20

4UO2

2+ + 7H2O = (UO2)4(OH)7+ + 7H+ -21.90

UO2

2+ + CO32-

= UO2CO3(aq) 9.94

UO2

2+ + 2CO32-

= UO2(CO3)2

2- 16.61

UO2

2+ + 3CO32-

= UO2(CO3)3

4- 21.84

3UO2

2+ + 6CO32-

= (UO2)3(CO3)6

6- 54.00

2UO2

2+ + CO32-

+ 3H2O = (UO2)2CO3(OH)3

-

+ 3H+ -0.85

3UO2

2+ + CO3-- + 3H2O = (UO2)3CO3(OH)3+ + 3H+ 0.66

UO2

2+ + Cl-

= UO2Cl+ 0.17

Ca2+ + UO2

2+ + 3CO32-

= CaUO2(CO3)3

2- 25.40 (b)

2Ca2+ + UO2

2+ + 3CO32-

= Ca2UO2(CO3)3

0

(aq) 30.55 (b)

UO2

2+ + 2Cl-

= UO2Cl2(aq) -1.10

Mg2+ + UO2

2+ + 3CO32-

= MgUO2(CO3)3

2- 26.11 (c)

UO2

2+ + SO42-

= UO2(SO4)(aq) 3.15

UO2

2+ + 2SO42-

= UO2(SO4)2

2- 4.14 a Values from (Guillaumont et al., 2003), unless otherwise indicated. b (Bernhard et al., 2001). c (Dong and Brooks, 2006).
5-6

Table 19. Uranium surface complexation reactions and formation constants 

Reaction logK (estimated*)

Surface Species

SSOH + UO2

2+ = SSOUO2+ + H+ 12.28

SOH + UO2

2+ = SOUO2+ + H+ 6.95

WOH + UO2

2+ = WOUO2+ + H+ 2.74

SSOH + UO2

2+ + H2O = SSOUOOH + 2H+ 0.033

SOH + UO2

2+ + H2O = SOUOOH + 2H+ -2.12

WOH + UO2

2+ + H2O = WOUOOH + 2H+ -5.01

SSOH denoting very strong binding sites: 0.01% of total sites

SOH denoting strong binding sites: 0.1% of total sites

WOH denoting weak binding sites: 99.89% of total sites

*Values from (Fang et al., 2009)
5

-7

Table 20. Equilibrium Reactions

Reaction LogK

*

CH

3COO

-

+ H

+ = AcH 4.76

Ca2+ + H

2O = CaOH

+ + H+

-12.60

Ca2+ + CH

3COO

-

= CaCH

3COO

+ 1.18

Ca2+ + CO

3

2-

+ H

+ = CaHCO

3

+ 11.33

Ca2+ + CO

3

2-

= CaCO

3 3.15

Ca2+ + SO

4

2-

= CaSO

4 2.31

CO

3

2-

+ 2H

+ = H

2CO3 16.68

CO

3

2-

+ H

+ = HCO

3

- 10.33

Fe2+ + H

2O = FeOH

+ + H+

-9.50

CH

3COO

-

+ Fe2+ = FeCH

3COO

+ 1.82

Fe2+ + CO

3

2-

+ H

+ = FeHCO

3

+ 12.33

Fe2+ + CO

3

2-

= FeCO

3(aq) 5.50

Fe2+ + 2CO

3

2-

= Fe(CO

3

)

2

2- 7.10

Fe2+ + SO

4

2-

= FeSO

4 2.25

Fe2+ + Cl

-

= FeCl

+ 0.90

Fe2+ + 2HS

-

= Fe(HS)

2(aq) 8.95

Fe2+ + 3HS

-

= Fe(HS)

3

- 10.99

HS

-

+ H

+ = H

2S(aq) 6.99

HS

-

= H

+ + S2-

 -12.92

K

+ + SO

4

2-

= KSO

4

- 0.85

Mg2+ + H

2O = MgOH

+ + H+

-11.79

CH

3COO

-

+ Mg2+ = MgCH

3COO

+ 1.14

CO

3

2-

+ Mg2+ + H

+ = MgHCO

3

+ 11.40

CO

3

2-

+ Mg2+ = MgCO

3(aq) 2.98

Mg2+ + SO

4

2-

= MgSO

4(aq) 2.25

Na

+ + CH

3COO

-

= NaCH

3COO

-0.18

Na

+ + CO

3

2-

+ H

+ = NaHCO

3(aq) 10.08

CO

3

2-

+ Na

+ = NaCO

3

- 1.27

Na

+ + SO

4

2-

= NaSO

4

- 0.70

NH

4

+ = NH

3(aq) + H

+

-9.25

NH

4

+ + SO

4

2-

= NH

4SO4

- 1.11

SO

4

2-

+ H

+ = HSO

4

- 1.99

H

2O = OH

-

+ H

+

-14.00

>FeOH + H

+ = >FeOH

2

+

 7.47

>FeOH = >FeO

-

+ H

+

-9.51

>FeOH + Fe++ = >FeOFe

+ + H+

 -5.00

>FeOH + Fe++ + H

2O = >FeOFeOH + 2H

+

 -11.96

* Values from the EQ3/6 database.
5-8

Table 21. Kinetic Reactions

Calcite dissolution/precipitation

Ca2+ + HCO3

-

= Calcite(s) + H+

Siderite dissolution/precipitation

Fe2+ + HCO3

-

= Siderite(s) + H+

Iron sulfide dissolution/precipitation

Fe2+ + HS-= FeS(s) + H+

Silicate Iron(III) bioreduction

0.125 CH3COO-+ Fe(III)(ls) + 0.5 H2O = 0.25 HCO3

-

+ 0.9 Fe(II)(ls) + 0.1 Fe++ + 1.125H+

where (ls) refers to phyllosilicate Fe 

Goethite bioreduction

0.125 CH3COO− + FeOOH(s) + 1.875 H+ = Fe2+ + 1.5 H2O + 0.25 HCO3

−

Uranium bioreduction

0.125CH3COO-+ 0.3538H2O + 0.0113NH4

+ + 0.3875UO22+ = 0.0113BM_iron + 0.855H+ + 

0.1938HCO3

-

+ 0.3875UO2(s) 

where BM_iron is the FeRB biomass

Sulfate bioreduction

0.125CH3COO-+ 0.0057H+ + 0.0038NH4

+ + 0.1155SO42- = 0.0038BM_sulfate + 0.0114H2O + 

0.231HCO3

- + 0.1155HS-

 

where BM_sulfate is the SRB biomass

Sulfide-promoted goethite dissolution 

2FeOOH(s) + HS-+ 5H+ = 2Fe2+ + S0 + 4H2O

Table 22. Rate Constants

Parameter Value*

Precipitation rate for calcite 1.644×10-7 Md-1

Dissolution rate for calcite 1.37×10-6 d-1

Precipitation rate for siderite 1.37×10-6 Md-1

Dissolution rate for siderite 1.37×10-7 d-1

Precipitation rate for FeS(s) 5.11×10-6 Md-1

Dissolution rate for FeS(s) 6.85×10-6 d-1

Rate constant for phyllosilicate iron bioreduction 0.075 M-1d-1

Rate for Fe(III) bioreduction 0.016 d-1

Half-Saturation for acetate 5.0×10-4 M

Threshold based on silicate iron Fe(III) 0.14 mMg-1

Rate for sulfate bioreduction 1.22×10-3 Md-1

Half-Saturation for sulfate bioreduction 10-5 M 

Rate for U(VI) conversion by iron reducers 2.5×10-6 Md-1

Half-Saturation for U(VI) conversion by iron reducers 10-7 M

Half-Saturation for U(VI) conversion by sulfate reducers 10-7 M 

Dissolution rate of goethite by sulfide 20.8 M-1d-1

*Values from (Fang et al., 2009)
5-9

5.2.4 UCODE

We used UCODE_2005 version 1.021 (Poeter et al., 2008) with the HGC reactive transport 

simulator to calibrate model parameters for the simulation of the three Rifle IFRC sediment 

column experiments. The “black box” model application includes pre- and post-processing 

utilities that update the HGC input files using UCODE output and extract the HGC output to be 

used as UCODE input. 

UCODE is invoked by executing a ~.bat file (e.g., ucode_HGCflowchem.bat). The principal input 

file is a ~.in file (e.g., ucode_HGCflowchem.in). We have created a separate input file 

(HGCflowchemupdate.dat) for the black box application that contains only the parameters that 

UCODE is updating. A template file (HGCflowchemupdate.dat.tpl) is used to guide the formatted 

updates.

The black box model is executed by a Linux script (runHGCflowchem) which 

1) executes updateHGCflowcheminput, a utility program developed to take the changes 

UCODE has made to the HGCflowchemupdate.dat file and creates usgs_1d_fast.inp, 

the HGC input file

2) executes hgc_usgs.x, the HGC executable that creates the or1.bst binary output file 

3) executes tecFLOWCHEM, which takes the or1.bst binary output file and creates 

HGCflowchem.out, the formatted output of selected concentrations (Ac, Fe(II), U(VI), 

sulfate) expected by UCODE.

UCODE then reads the HGCflowchem.out file using the HGCflowchem.out.ins instruction file to 

extract the model output that will be compared with the observation data in the UCODE input 

file, ucode_HGCflowchem.in.
5-10

5.3 Approach for Modeling Biostimulation Stage of Column Experiments 

The goal is to interpret the observed behavior of the column experiments using the existing Rifle 

IFRC field-scale modeling reaction framework. The principal objective is to develop a single 

reactive transport modeling parameterization to simulate the bioreduction of sulfate, Fe(III), and 

U(VI) in the three acetate biostimulation column experiments using Rifle IFRC sediments.

5.3.1 General Approach

The starting point for the model development is the existing parameterization of the field-scale 

reactive transport model. The approach is based on the assumption that the modeled processes 

and reactions in the field-scale model are appropriate and sufficient for the modeling of the 

column experiments. The focus, therefore, is the adjustment of the reaction parameters in the 

existing model to reproduce the experimental observations.

It should be noted that the field-scale reactive transport model was designed for the suboxic 

Rifle IFRC aquifer conditions, where post-biostimulation oxidation of solid-associated U(IV) has 

not been observed. Since there is no process model support for U(IV) oxidation, the modeling 

scope for the Rifle sediment columns addresses the biostimulation and cessation of influent 

components but not the introduction of elevated concentrations of dissolved oxygen. The 

modeling also does not address columns that have been repacked. This is because of 

uncertainty regarding the alteration of the processes, properties, and conditions of the 

experiments. Accordingly, only the first 193.6, 220.9, and 186.3 days of the columns A, B, and C 

experiments, respectively, are modeled. 

The approach was to first calibrate a set of model parameters for each of the three column 

experiments to make sure that the modeled processes were appropriate and sufficient to 

represent the observed behaviors. After assessing the three calibrated sets of parameters, a 

single set would be identified and applied to all three column experiments. 

The sediments used in the column experiments represent the finer-grained subset of the field 

particle size distribution (Tables 4 and 25). This subset represents the most reactive component 

of the sediments with respect to adsorption. We use the calibration procedure to estimate the 

initial >FeOH site density, which is assumed to control the number of reactive sites in the 

surface complexation modeling.

5.3.2 Challenges

There were two significant challenges: 1) increasing uranium bioreduction rate with time that 

had not been observed in the field, and 2) data limitations and inconsistency between the initial 

component concentrations, influent concentrations, and effluent concentrations, described in 

detail below. 

1) Increasing rate of U(VI) bioreduction. The observation of continuously decreasing aqueous 

U(VI) concentrations in the column experiment after the onset of significant sulfate removal 

indicated increasing U(VI) reactivity over the course of the column biostimulation experiments. 

This was a departure from the field behavior and the existing conceptualization of model 

processes had no mechanism to account for increasing consumption of U(VI) during dominantly 

sulfate reducing conditions. In the conceptualization of modeled processes, the FeRB are 

responsible for both the Fe(III) and U(VI) reduction. It was decided that the reactive transport 
5-11

model would be modified to add Fe(III) reducing bacteria (FeRB) biomass to the goethite and 

layer silicate Fe(III) terminal electron accepting process (TEAP) reactions. This would allow 

FeRB biomass to be incorporated into the U(VI) TEAP rate law. With the growth in FeBR 

biomass concentration, the U(VI) bioreduction rate increases commensurately.

2) Limitations in observed concentrations. The predicted aqueous U(VI) is very sensitive to the 

initial water chemistry and surface complexation sites. While the initial aqueous component 

concentrations in the column are flushed out with the first pore volumes, the initial sorbed 

uranium and Fe(II) concentrations are determined by assuming initial equilibrium with their 

aqueous counterparts. Thus, the initial concentrations of sorbed components are dictated by the 

selection of initial concentrations, as well as the concentration of surface complexation sites. 

Since the Fe(II) and U(VI) dynamics during the experiment are tied to the reservoirs of their 

sorbed species, a consistent set of initial and influent conditions in the context of the reaction 

thermodynamics is necessary to avoid artificial transients. In the absence of initial component 

concentrations, it was found that using the influent water chemistry as the initial condition was 

superior to using the initial effluent concentrations.

Another issue was the variability in the monitored influent concentrations during experiments 

and between experiments. This was most obvious when effluent concentrations were higher 

than influent for acetate, sulfate, and uranium. We chose to honor the effluent values because 

there were more measurements and they were more consistent. Since the influent artificial 

groundwater composition was supposed to be the same for all three columns (with the 

exception of intentional cessation of acetate and uranium in column A, late Fe(II) addition in 

column B, and Fe(II) addition in column C, as well as an inadvertent period of lower Fe(II) and U 

in column C), we chose to use the artificial groundwater composition (Table 1) instead of the 

monitored influent concentrations. This also resolved an issue of sampling coverage for the 

Column C experiment, which was characterized by several long unmonitored periods for the 

influent solution including a 158 day interval between the initial and subsequent measurement of 

acetate and sulfate.

For all three column experiments, the effluent sulfate concentrations were higher than the 

influent concentration (1) before sulfate reducing conditions became dominant, and (2) after 

acetate input ceased in the column A experiment. We infer an uncharacterized sulfate source 

from the column sediments. Since the influent sulfate concentrations were low relative to the 

higher concentrations resulting from the internal source, there is a transient sulfate release of 

indeterminate duration and strength in all three experiments. After about 20 days in all three 

experiments, the sulfate TEAP reaction consumes the bulk of the influent acetate. Thus, there is 

considerable uncertainty in the predicted acetate and sulfate effluent concentrations as the 

model does not account for the internal sulfate source.

5.3.3 Parameter Estimation

Twenty three model parameters were initially targeted for calibration in the biostimulation 

modeling of the Rifle sediment columns: 

• Physical parameters: darcy flux, porosity, dispersivity

• Sulfate TEAP: intrinsic sulfate TEAP reaction rate, initial SRB biomass, decay of SRB 

biomass, max sulfate rate (to control overall rate with biomass and monod terms). Note 

that the TEAP reactions includes biomass production as well as bioreduction.

• Fe(III) TEAP: layer silicate Fe(III) bioreduction rate, goethite bioreduction rate, initial 

FeRB biomass, decay of FeRB biomass

• U(VI) TEAP: aqueous U(VI) bioreduction rate, max uranium rate
5-12

• Surface complexation: initial >FeOH site density, log K for >FeOH + Fe+2 =>FeOFeOH 

+ H+

 

• Iron minerals: siderite reaction rate, FeS reaction rate, sulfide promoted goethite 

dissolution rate

• Major ions: calcite precipitation rate, cation exchange capacity, log Ks for Ca+2, Mg+2, K+, 

Na+

 ion exchange

We initially included Darcy flux, porosity, and dispersivity in the calibration of the reactive 

transport models but found that we could specify the measured Darcy flux and porosity (Table 

23) without significant loss of fidelity. UCODE determined that the calibration was generally 

insensitive to dispersivity and the initial 6 cm value was maintained. The modeled aqueous 

species acetate, Fe(II), U(VI), and sulfate were relatively insensitive to changes in the calcite 

and cation exchange reaction parameters. Consequently, they were omitted from the UCODE 

application, leaving 13 estimated parameters.

Table 23. Hydrologic parameters for columns A, B, and C.

Column A Column B Column C

Darcy Flux (cm/min) 0.00237 0.00237 0.00257

Porosity (-) 0.269 0.286 0.302

Dispersivity (cm) 6.00 6.00 6.00

A two-stage calibration approach was used. The first stage focused on sulfate related 

behaviors: intrinsic sulfate bioreduction rate, initial SRB biomass, decay of SRB biomass, and 

maximum total sulfate bioreduction rate. This is because the sulfate TEAP has the largest 

impact on the acetate and sulfate behavior. In the first stage, calibration of parameters was 

based only on the observed acetate and sulfate concentrations. In the second stage of the 

calibration approach, the calibrated parameters from the first stage were fixed and the rest of 

the parameters selected for estimation were calibrated. In the second stage, only the Fe(II) and 

U(VI) reaction parameters were targeted. To summarize, the approach is to fit the sulfate TEAP, 

then adjust the iron and uranium behaviors. This approach takes advantage of the 

multiprocessor option in UCODE where the sensitivity to each estimated parameter for each 

iteration can be determined through simultaneous HGC simulations.

We used the post-processing executable tecflowchem to create the HGCflowchem.out file that 

is used by UCODE. Weighting factors for the Fe(II) comparisons were lowered by a factor of 

0.03 to adjust for concentrations that were 6 times higher than the other components (i.e., 

weighting is by the inverse square, 6-2 ≈0.03).

5.3.4 Calibration

It was determined that a biomass-dependent U(VI) bioreduction rate would conceptually 

address the increasing removal of U(VI) observed in the column experiments. Since the 

biomass for both the Fe(III) and U(VI) TEAPs are attributed to the FeRB, this required new 

TEAP reactions for both Fe(III) and U(VI):

Phyllosilicate Fe(III)

0.125Ac-+ Fe(III)(ls) = 0.25 HCO3

-

+ 0.9Fe(II)(ls) + 0.1Fe++ + 1.125H+ + 0.0043BM_iron 
5-13

Goethite

0.125Ac- + 0.6FeOOH + 1.155H+ + 0.02NH4

+ = 0.004BM_iron + 0.6 Fe++ + 0.15HCO3-

U(VI)

0.125Ac-+ 0.00113NH4

+ + 0.3875 UO2++ = 0.0113BM_iron + 0.855H+ + 0.1938HCO3-

+ 0.3875UO2(s) 

where, 

(ls) denotes layer silicate

BM_iron is the FeRB biomass concentration based on the molecular formula C5H7O2N. 

New parameters were needed for the initial FeRB biomass, FeRB biomass decay rate, and 

maximum total rate for uranium bioreduction. It should be noted that the conversion of the Fe(III) 

and U(VI) rate laws to biomass dependence requires a much larger intrinsic rate to offset 

multiplication by the biomass concentration.

5.3.5 Initial Simulations

The three column experiments use sediments from a common sample with minor variations in 

packing and hydrologic forcing (Table 2). The influent solution is based on the artificial 

groundwater (AGW) recipe in Table 1 with the following variations: 

• Column A simulation: 

o 111.5 days of AGW

o 35.7 days without acetate

o 35.5 days without both acetate and uranium

• Column B simulation: 

o 210.1 days of AGW

o 10.9 days with the addition of 11.65 uM Fe(II)

• Column C simulation: 

o 135.3 days of AGW with the addition of 13.53 to 11.65 uM Fe(II)

o 23.2 days with low 1.38 uM Fe(II) and low 0.13 to 0.57 uM U(VI)

o 27.8 days with low iron but otherwise standard AGW

The column B experiment was conceptually the simplest (influent concentrations maintained for 

all but the last 11 days of the 221 day biostimulation) and, like column A, had good sampling 

coverage of influent concentrations. The addition of Fe(II) to the influent after 210 days did 

seem to have a subtle effect on the observed acetate, Fe(II), and U(VI) concentrations. The 

column C experiment was characterized by considerably less temporal sampling coverage, with 
5-14

several long unmonitored periods for the influent. For example, after the initial measurement of 

acetate and sulfate, the next measurement was 158 days later. Consequently, we chose to use 

the calibrated reaction parameters from the column B experiment on columns A and C.

Table 24 summarizes the parameters calibrated for column B. Note that the intrinsic U(VI) 

bioreduction rate for the column is four orders of magnitude larger than the field modeling value 

because it is being multiplied by the biomass concentration, which is typically a small value, 

~1E-6 M (Table 24). Nearly all the calibrated reaction parameters represent an increase in 

reactivity over the field-scale modeling. This may be explained to some degree by the use of the 

< 8 mm particle size fraction in the experiments (Table 4), which disproportionately contains the 

most reactive size fractions in the full particle size distribution (Table 25, unpublished data from 

A.L. Ward, Pacific Northwest National Laboratory). In this case, the <8 mm size fraction is 22% 

by weight of the total field sediment size distribution, which implies there could be 5 times higher 

reactivity if it is assumed that the > 8 mm size fraction is largely inert. Interestingly, the

homogeneous sulfate bioreduction reaction also had a higher calibrated reaction rate, 

suggesting a potential linkage to the higher fraction of finer grained sediment. 
5-15

Table 24. Calibrated model parameters for column B

a. Magenta shading indicates parameter value decreased by greater than 25%. Green shading 

indicates parameter value increased by greater than 25%.

b. Parameter increased for biomass-dependent rate. 

Parameter Field Modeling Value Column B Valuea

Intrinsic Crystalline Fe(III) 

Reduction Rate, d-1 1.60E-02 3.80E-03

Intrinsic Sulfate Reduction 

Rate, Md-1 80 2.54E+02

Intrinsic U(VI) Reduction Rate, 

Md-1 2.50E-05 1.02E+00b

Initial Sulfate Reducing 

Bacteria Biomass, M 1.00E-07 2.17E-06

Sulfate Reducing Bacteria 

Decay Rate, d-1 1.50E-02 7.48E-01

Maximum Total Sulfate 

Bioreduction Rate, Md-1 3.26E+02 3.18E+02

Sulfide Promoted Goethite 

Dissolution Rate, M-1d-1 2.08E+02 4.17E+03

FeS(s) Rate, Md-1 5.11E-05 6.07E-02

Initial >FeOH Site 

Concentration, M 3.28E-02 7.67E-02

Initial Fe(III) Reducing Bacteria 

Biomass, M

not included in field reaction 

network 3.51E-07

Fe(III) Reducing Bacteria 

Biomass Decay Rate, d-1

not included in field reaction 

network 2.48E-06

Maximum Total U(VI) 

Bioreduction Rate, Md-1

not included in field reaction 

network 2.36E+00

>FeOH + Fe+2 = H+ + >FeOFeOH 

log K -1.196E+01 -9.84E+00
5-16

Table 25. Particle Size Distribution of Rifle Aquifer

sieve size

(mm)

% of mass % mass finer

256 0.00 100.00

64 41.07 58.93

32 17.17 41.75

16 13.08 28.67

8 7.02 21.64

4 3.84 17.81

2 1.91 15.90

1 2.46 13.45

0.5 7.65 5.79

0.25 2.65 3.14

0.125 1.60 1.55

0.063 0.78 0.77

5.4 Model Simulation Results 

5.4.1 Column B Simulations

During the first ~20 days of all experiments, acetate-driven bioreduction by FeRB results in a 

~70 uM peak in Fe(II) concentrations as well as a ~3 mM decrease in U(VI). Acetate 

consumption is relatively low because of (1) the general efficiency of the TEAP reactions (e.g., 

5-8 moles of Fe(III) reduced for every mole of acetate consumed) and (2) the relatively small 

amounts of metal reduced (e.g., 10 mM U(VI)). From day ~20 to ~50, effluent acetate 

concentrations decrease sharply to ~3 mM before beginning to level off after 50 days (Figure 

46). This generally coincides with the increase in sulfate bioreduction by SRB, which results in 

low effluent sulfate concentrations (1-1.5 mM) after ~50 days. Significant changes in the effluent 

acetate concentrations are thus dictated by the sulfate TEAP reaction, which has ~1:1 acetate 

to sulfate consumption ratio. This explains the similarity in the time-dependent acetate and 

sulfate effluent concentration behaviors. The model used 8 mM for the influent acetate, lower 

than the 10 mM acetate concentration measured in the effluent over the first 22 days of the 

column B experiment. This disparity between the model predicted and measured effluent 

acetate is the result of using the intended AGW recipe influent acetate concentration of 8 mM in 

the model whereas the experimental influent acetate concentration was 10 mM during the first 

70 days of biostimulation of column B and for the entire biostimulation period of column A. The 

experimental acetate influent was 8 mM for the remainder of column B and all of column C. (see 

sections 2.1 and 3.1.2). The modeled effluent acetate generally captures the increasing 

consumption beginning 20 days into the biostimulation experiment (Figure 46). After 50 days, 

the rate of acetate consumption begins to level off leading to an asymptotic value of 2 mM, 

which is generally captured by the model.
5-17

Figure 46. Model simulation column B effluent acetate.

The model adsorbed Fe(II) is increasing during the early buildup of aqueous Fe(II), which 

means the Fe(II) source is from mineral dissolution: Fe(III) bioreduction and/or goethite 

dissolution via biogenic sulfide. The model has two pools of bioavailable Fe(III), goethite and 

phyllosilicate. Since the biogenic Fe(II) from the phyllosilicate Fe(III) is largely reduced in place, 

the principal source of the aqueous Fe(II) is the goethite. As sulfate bioreduction ramps up after 

20 days, Fe(II) and sulfide are consumed in the formation of FeS mineral, putatively 

mackinawite. This results in a fairly rapid and near-complete removal of aqueous Fe(II) (Figure 

47). The addition of 11.65 mM Fe(II) at day 210 is completely consumed in the model result 

compared to the smaller spike that was observed.
5-18

Figure 47. Model simulation column B effluent dissolved iron.

In the model, the decrease in aqueous U(VI) is described to occur primarily by bioreduction. 

This is because the other uranium reactions result in a net desorption over the first 35 days that 

largely depletes the initial pool of adsorbed U(VI). The initial drop in aqueous U(VI), which 

bottoms out after 20 days, coincides with the spike in aqueous Fe(II) concentrations from the 

reductive dissolution of Fe(III) mineral (Figure 48). The dominance of FeRB during the initial 

biostimulation with associated impacts on iron and uranium chemistry are consistent with 

observations from Rifle IFRC field experiments (Anderson et al., 2003; Vrionis et al., 2005; 

Williams et al., 2011). In particular, U(VI) desorption begins to accelerate after 25 days, when 

increasing oxidation of acetate by the SRB significantly elevates the bicarbonate available for 

aqueous complexation with U(VI). This is consistent with the short-term increase in effluent 

aqueous U(VI) concentrations, peaking at 35 days, but still below the influent concentration. 

After 50 days, relatively low adsorbed U(VI) concentrations stabilize with the evolving water 

chemistry. The modeled rate of uranium bioreduction is slowly increasing over the duration of 

the experiment because of the biomass dependent rate law. The drop in observed U(VI) after 

day 210 appears to coincide with the addition of 11.65 mM Fe(II) to the influent. There was no 

similar impact to the modeled U(VI) behavior. This could be due to an uptake mechanism not 

included in the model such as abiotic U(VI) reduction. Similar experiments in the Rifle aquifer 

with the addition of 45 mM Fe(II) had minimal impact on aqueous U(VI) concentrations 

(unpublished, 2012 Rifle IFRC Annual Report).
5-19

Figure 48. Model simulation column B effluent dissolved uranium.

Similar to the modeled acetate behavior, the presence of an uncharacterized internal source of 

sulfate from the column sediments leads to significant underprediction of the sulfate 

concentrations for the first 25 days of the experiment (Figure 49). During this time period, the 

millimolar sulfate concentrations are largely unaffected by the biogeochemical impacts of the 

stimulated FeRB. After this period, the time-dependent sulfate concentrations are a direct 

consequence of the acetate-oxidizing SRB, which accounts for the similarity with the acetate 

behavior (Figure 49). In this case, the biomass-dependent sulfate bioreduction rate becomes 

very high, before leveling off, yielding ~1 mM sulfate concentrations. In the model, there is 

initially a small amount of slow growing SRB that have minimal impact on sulfate 

concentrations. As the SRB biomass increases over time, the bioreduction rate increases 

commensurately until a maximum is reached at ~50 days. It should be noted that the ratelimited microbially-mediated sulfate reduction reactions result in the non-equilibrium coexistence 

of Fe(II), sulfide and sulfate. This behavior is always observed in the field biostimulation 

experiments, underscoring the kinetic control exerted by these TEAP reactions over the redox 

couples.
5-20

Figure 49. Model simulation column B effluent dissolved sulfate. 

5.4.2 Simulation of Column A Effluent using Column B Parameters

We used the reaction parameters calibrated from the column B experimental observations to 

model the column A experiment. The column A experiment is run with the same flow rate, 

influent conditions and sediments as the column B experiments. After 111 days, influent acetate 

is stopped and after 147 days, influent uranium is stopped. Thus, for the first 111 days, the 

model results are the same as for the column B experiment and the comparison with 

experimental observations was also similar (Figure 50). The termination of influent acetate and 

then sulfate did result in model discrepancies, especially for the micromolar Fe(II) and U(VI) 

components. While acetate does behave similarly to Column B, there is a more gradual and 

sustained decrease in acetate concentrations after day 30 until influent acetate is stopped after 

111 days. Unlike the column B experiment, acetate does not level off to a 2 mM concentration. 

Instead it appears that acetate would eventually be completely consumed if the cessation of 

acetate did not occur after 111 days. This implies a continuous increase in the sulfate 

bioreduction rate beyond that in the column B experiment. Acetate concentrations rapidly go to 

zero after acetate is eliminated from the influent solution, which the model reproduces.
5-21

Figure 50. Model simulation column A effluent acetate.

As with the column B experiment, the model does a fairly good job capturing the peak Fe(II) 

concentration just before 20 days (Figure 51). In this case, there is a phase error in the timing of 

the Fe(II) peak arrival as the model is predicting a later breakthrough. The measured column A 

porosity was smaller than column B, 0.269 versus 0.286. A slightly smaller effective porosity 

might account for the observed behavior. For the 50 days prior to the end of biostimulation, 

aqueous Fe(II) is largely diminished by reaction with sulfide, forming an FeS mineral. When 

influent acetate is stopped at 111.5 days, Fe(II) concentrations are below detection, after which 

there is a steady increase in effluent Fe(II) to ~25 mM at the end of the 194 day simulation

(Figure 51) While this behavior has never been observed in the field, ambient Fe(II) 

concentrations of that magnitude (10-50 μM) are typically present at the Rifle site under 

unstimulated conditions. Assuming these concentrations are in quasi-equilibrium with the 

sediments, the absence of Fe(II) in the influent solution creates disequilibria that results in 

desorption and/or dissolution from the sediments. This uncharacterized internal source of Fe(II) 

may have been occurring throughout the experiment but was not detectable because FeS 

solubility was limiting dissolved Fe(II). The cessation of influent U after 147 days has negligible 

impact on the Fe(II) behavior.
5-22

Figure 51. Model simulation column A effluent iron.

While there are some dynamics in the aqueous U(VI) concentrations, there is a general 

increase in U(VI) removal from solution until the influent acetate is stopped after day 111 (Figure 

52). This behavior includes a slight rise in aqueous U(VI) concentrations at day 30 when higher 

bicarbonate concentrations from acetate oxidation in the sulfate TEAP reaction results in U(VI) 

desorption. The uranium behavior under biostimulation is generally captured by the model, but 

concentrations between 70 and 110 days are under predicted (Figure 52). This under prediction

is difficult to explain since, up to this point, columns A and B should have been operating under 

the same conditions. 

The post-biostimulation U(VI) behavior was not captured well by the modeled reaction network. 

Since there has been no evidence for substantial abiotic U(VI) reduction in the Rifle aquifer, the 

rapid drop in observed aqueous U(VI) concentrations when acetate loading stops is more 

consistent with adsorption that has been enhanced by 1) the cessation of bicarbonate 

production from acetate oxidation (i.e., lower bicarbonate concentrations thermodynamically 

favor U(VI) speciation that enhances adsorption), and 2) the availability of surface complexation 

sites vacated during biostimulation via bicarbonate-induced desorption. For the column water 

chemistry, the nominal model specification of >FeOH sites would have to be increased by a 

factor of 50 to match the post-biostimulation uptake. This assumes no abiotic reduction occurs. 

The consequence of increasing the >FeOH sites is that there would be significantly more initial 

U(VI) adsorbed on these sites and desorption during high sulfate reduction would result in a 

massive U(VI) peak after 35 days, which was not observed. The 50 times more surface 

complexation sites required seems excessive since the <4 mm size fraction accounts for 18% of 

the mass of the full particle size distribution (Table 25). The surface area contribution of the <4 
5-23

mm fraction to the bulk sediment is not known. It would appear that the SCM we have been 

using is not consistent with the column geochemistry. One possible explanation is that the 

surface complexation model was developed from experiments on a sediment sample from a 

different part of the Rifle Site than the sediments in the column studies. If the sediments were 

physically and geochemically different, the selected surface complexation reactions and derived 

stability constants for those reactions may not be a good match. There could also be unresolved 

kinetic limitations controlling the uranium in the column studies, or that some other process, 

such as abiotic reduction of U(VI) by sulfide or Fe(II), results in the observed continued uptake, 

as discussed in section 5.1.2. 

As the surface complexation sites are re-saturated with U(VI) after 110 days, the effluent 

concentrations begin to climb ostensibly toward the influent 10 uM U(VI) concentration. This 

process is interrupted when the influent U(VI) is terminated after 147 days and the U(VI) 

concentrations drop to the ~1 uM level, a pseudo-equilibrium driven by the absence of influent 

U(VI).

The comparison of the modeled and observed sulfate behavior prior to stopping the acetate 

loading after 111 days is similar to the column B analysis (Figure 53). As mentioned previously, 

there was an internal sulfate source that initially resulted in ~9 mM effluent sulfate 

concentrations (versus the 6.4 mM influent concentration) that could not be addressed by the 

modeling. The rebound of post-biostimulation (i.e., > 111 days) sulfate concentrations back up 

to the initial concentrations, implied that the initial sulfate release from the sediments was not a 

short transient but more likely continuous over the experiment duration. The uncharacterized 

source, however, is not a part of the modeled reactions. Consequently, the predicted postbiostimulation sulfate concentrations are significantly below the observations.
5-24

Figure 52. Model simulation column A effluent uranium.
5-25

Figure 53. Model simulation column A effluent sulfate.

5.4.3 Simulation of Column C Effluent using Column B Parameters

The column B reactions parameters were next applied to the column C experiment. The flow 

rate in the column C experiment was 8% faster than columns A and B. With the exception of the 

Fe(II) that was added, the influent solution for the column C experiment was based on the same 

artificial groundwater recipe as the columns A and B experiments. Over the first 40 days, the 

three influent Fe(II) measurements were 13.53, 12.52, and 11.65 mM. Because of an error in the 

influent composition from day 135 to 159, the influent U(VI) was as low as 0.13 mM versus the 

10 mM recipe and the only Fe(II) measurement was 1.38 mM. The column C experiment was 

characterized by considerably less temporal sampling coverage, with several long unmonitored 

periods for the influent. For example, after the initial measurement of acetate and sulfate, the 

next measurement was 158 days later.

The acetate behavior is similar to the other experiments in magnitude and timing with no 

obvious impact of the 8% faster flow rate. This might be explained by the pore velocity of 8.5 

cm/min, which is between the 8.3 cm/min for column B and the 8.8 cm/min for column A. In this 

experiment, the effluent acetate measurements over the initial 20 days are only slightly higher 

than the 8 mM influent. After day 40, the model predicted acetate consumption is slightly greater 

than observed, which is stoichiometrically consistent with the slight over prediction of sulfate 

consumption (Figure 54). On day 135, there was a significant decrease in influent U(VI) (2 order 

of magnitude decrease) and Fe(II) (1 order of magnitude decrease). This coincided with an 

observed decrease in acetate, presumably from reaction. The modeled acetate, on the other 
5-26

hand, was relatively unaffected. It’s not clear why acetate consumption would increase when 

influent U(VI) and Fe(II) concentrations were significantly decreased. The model does not have 

a process that would account for enhanced acetate consumption under these conditions.

Figure 54. Model simulation column C effluent acetate.

The predicted column C Fe(II) peak magnitude and timing over the first 40 days was particularly 

good and similar to the other columns (Figure 55). While columns A and B exhibited Fe(II) 

concentrations below detection after the primary peak at 20 days, column C has a few periods 

with ~5 mM concentrations. The modeled Fe(II) is consistent with below detection 

concentrations, primarily due to precipitation of FeS. The observed Fe(II) concentrations 

seemed to be anti-correlated to the changes in influent Fe(II), increasing during the low influent 

Fe(II) period (days 135 to 159) to ~5 mM and decreasing after resumption of the design influent 

Fe(II) concentration.
5-27

Figure 55. Model simulation column C effluent iron.

The observed U(VI) behavior in column C was a significant departure from columns A and B. 

The highest initial effluent U(VI) measurement was 7.8 mM versus 9.6 and 10.6 in columns A 

and B, respectively. From 40 to 100 days the observed U(VI) concentrations in column C were 

~3 mM lower than column A and ~4 mM lower than column B, and significantly over predicted by 

the model (Figure 56). The unplanned drop in influent U(VI) after 135 days is captured by the 

model but the resumption of the design influent U(VI) concentration after 159 days results in 

even lower observed U(VI) concentrations. Column C had continuous ~12 mM influent Fe(II) 

whereas the other columns did not. When column B introduced a similar amount of Fe(II) at the 

end of the experiment, a ~3 mM drop in U(VI) was observed. The model does not have a 

mechanism (e.g., abiotic U(VI) reduction, enhanced U(VI) sorption) that would address the 

enhanced U(VI) removal; consequently, the U(VI) reactivity is significantly under predicted. As 

mentioned previously, a 45 mM Fe(II) injection was performed in the field with no significant 

impact on U(VI) concentrations. More recently, abiotic laboratory studies with up to 1.41 mM 

Fe(II) at pH 7.1 and 400 ppm CO2 (similar conditions to the Rifle site) result in negligible 

additional U(IV) (unpublished, 2012 Rifle IFRC Annual Report).
5-28

Figure 56. Model simulation column C effluent uranium.

The general trends of the observed and modeled sulfate behaviors are similar to the previous 

discussions for columns A and B where the influent 6.4 mM concentrations are considerably 

below the first 20 days of effluent measurements (Figure 57). The sulfate bioreduction is slightly 

over-predicted in the model, by an amount that is approximately stoichiometrically consistent 

with the over prediction of the acetate consumption. The drop in influent U(VI) and Fe(II) at day 

135 coincided with decreasing observed sulfate concentrations. This sulfate concentration drop 

was not restored by the return to design influent Fe(II) and U(VI) after 158.5 days. The model 

has no mechanism that would account for this magnitude of sulfate concentration change from 

transients in trace components. As a result, the predicted sulfate concentrations are unaffected 

by the period of lower Fe(II) and U(VI).
5-29

Figure 57. Model simulation column C effluent sulfate. 

5.5 Discussion of Model Simulations 

Uranium aqueous and surface complexation reactions are very important to the uranium 

behavior during acetate biostimulation. Prior to biostimulation in the field and assuming 

undersaturation of U(VI) minerals, dissolved U(VI) is controlled by adsorption and desorption 

with the majority of U(VI) adsorbed to the sediments. In the modeling of the column 

experiments, 70% of the U(VI) is initially sorbed. Acetate stimulates metal reducing bacteria to 

rapidly catalyze the reduction of U(VI) to solid-associated U(IV), effectively lowering aqueous 

U(VI) concentrations. The microbially-mediated oxidation of acetate leads to the production of 

bicarbonate which complexes with U(VI). U(VI) desorption is thermodynamically favored by the 

lower aqueous U(VI) concentrations and higher alkalinity. Consequently, the pool of initially 

adsorbed U(VI) is rapidly depleted over the initial 40 days of biostimulation. Figure 58 compares 

the adsorbed U(VI) and total solid phase U(IV) over time in the last model grid cell for column A. 

At the end of the three column experiments, the adsorbed U(VI) accounts for 0.15%, 0.16%, 

and 0.24% of the solid associated uranium in columns A, B, and C, respectively. The remainder 

is U(IV). This findings agrees well with the >95% U(IV) measured in column A sediments by 

XANES (Table 9). At the end of the simulated column experiments, the spatial variation from 

inlet to outlet of adsorbed U(VI), U(IV), FeS, elemental S, and phyllosilicate Fe(II) is generally 

small (Table 26). Higher concentrations of U(IV) are predicted near the influent end because 

progressive removal of U(VI) is sufficiently high to limit availability further down the column. The 

predicted trends in solid phase concentrations with position in column are in general agreement 
5-30

with measured distribution of the constituents in sediments recovered after biostimulation 

(Figures 11-19). 

Figure 58. Time-dependent variation in adsorbed U(VI) (top) and U(IV) (bottom) for the grid cell closest to 

the column A effluent. Influent acetate was stopped after 111 days and influent U(VI) was 

stopped after 147 days.
5-31

Table 26. Simulated adsorbed U(VI), U(IV), FeS, elemental S, and phyllosilicate Fe(II) at the end of 

experiments in columns A, B, and C.

Column A (nmol/g) Column B (nmol/g) Column C (nmol/g)

inlet outlet inlet outlet inlet outlet

U(VI) Adsorbed 0.172 0.173 0.460 0.439 0.517 0.499

U(IV) 126 114 353 281 248 212

FeS 1.42E+04 1.48E+04 1.50E+04 1.54E+04 1.85E+04 1.71E+04

S 7.43E+03 7.43E+03 7.73E+03 7.73E+03 8.57E+03 8.58E+03

Fe(II) Layer Silicate 6.37E+02 6.37E+02 6.63E+02 6.63E+02 7.35E+02 7.35E+02

6-1

6 Summary

The primary goal of this study was to evaluate the remobilization of U sequestered during 

biostimulated reduction under conditions in which biostimulation and concomitant U reduction 

extended well into sulfate reduction to enhance precipitation of reduced sulfur phases such as 

iron sulfides. The intent of producing these reduced phases was to test their effect on 

remobilization of the sequestered uranium, either serving as redox buffer by competing for 

dissolved oxygen, or by armoring the reduced uranium. Increased production of iron sulfide 

phases was attempted through addition of ferrous iron in the influent stream throughout 

biostimulation in one column. 

During biostimulation of the ambient microbial population with acetate, the removal of dissolved 

U(VI) from the influent continued over 110 to 330 days of biostimulation, well after the onset of 

sulfate reduction at ~30 days. Dissolved uranium was lowered from the influent 10 mM to <4 mM. 

The uranium sequestered during biostimulation was essentially all U(IV) resulting from the

formation of nano-particulate uraninite that may be associated with organic microbial biomass. 

The sequestered U(IV) may in part be coordinated to C or P in the organic matter. The reduced 

U(IV) formed contiguous coatings on sediment grains with thicknesses of a few 10s of microns

and was in association with S and Fe in some cases. Combined the effluent and solid phase 

measurements from columns during and after the biostimulation stage suggests that stimulation 

and growth of indigenous microbial population in the aquifer sediments from the Rifle site by 

addition of an electron donor and carbon source may be an effective means of removing 

dissolved uranium from contaminated groundwater, consistent with many field and laboratory 

studies.

Determining the stability of the bioreduced uranium in response to changes in groundwater 

chemistry after biostimulation has ceased, such as the return to oxic conditions, is needed to 

evaluate if the biostimulated reduction process would be an effective tool for long term 

remediation of U-contaminated shallow aquifers. Elution of bioreduced U(IV) associated with 

sediments from two columns (A and C) was conducted using AGW at equilibrium with 

atmospheric oxygen. This oxygen level was used to assess the upper limit of dissolved oxygen 

in recharge water. The difference between the two columns was the addition of 15 mM dissolved 

Fe(II) to column C during biostimulation to enhance formation of iron sulfides. 

Release of U during oxic elution was a continuous process over 120 to 140 days (300 to 350 

PV) with dissolved uranium concentrations on the order of 0.8 and 0.2 mM for columns A and C, 

respectively. The effluent concentration from column C decreased over time indicative of a 

decreasing rate of U(IV) oxidation. In contrast, column A effluent [U] was somewhat constant 

over 140 days. The prolonged period of biostimulation and concomitant sulfate reduction 

appears to limit the rate of U(IV) oxidative remobilization. The speciation of U(IV) associated 

with sediments after oxic elution was essentially the same as that measured in samples 

recovered after biostimulation and prior to oxic elution. The limited oxidative remobilization of 

U(IV) in the present column experiments is consistent with the decreased U remobilization 

where sulfate reduction occurred in contrast to large releases of U from columns which did not 

undergo sulfate reduction. Although continued sulfate reduction may cause decrease in 

permeability from precipitation of iron sulfide, the greater apparent stability of the sequestered 

U(IV) provided by the sustained biostimulation should be considered in design of field scale 

remediation efforts.
6-2

Specific Findings

1. The large extent of sequestration of dissolved uranium over the course of the biostimulation 

stage in all three columns indicates that stimulation of the ambient microbial population in 

the Rifle aquifer sediments may be an effective means of remediating dissolved uranium in 

groundwater at contaminated sites. The removal of dissolved uranium, which is in the +6 

oxidation state, occurred predominantly by reduction and subsequent precipitation as U(IV). 

2. The onset of U reduction was coincident with iron reduction, but the extent of U uptake 

increased overall throughout the duration of biostimulation with the greatest uptake 

occurring during sulfate reduction. The increased U removal by reduction may be the result 

of sufficient electron donor availability such that microbial processes are not limited in these 

columns. Thus, competition between sulfate reducing and metal reducing microbes is not 

sufficient to diminish uranium reduction by metal reducing microbes. Uranium reduction also 

could occur by other microbial processes such as reduction by sulfate reducing bacteria, 

which have been shown to reduce uranium directly through enzymatic mechanisms, or by

abiotic processes. 

3. About one third of the total U uptake in column A occurred after the acetate amendment was 

halted at 110 days and sulfate reducing bacterial activity stopped, and likely other microbial 

activity decreased. Because greater than 97% of the total U was determined to be U(IV), the 

observed continued uranium uptake is inferred to occur largely by abiotic reduction 

processes such as by interaction with iron sulfides produced during biostimulation. The 

increase of Fe(II) and reduced S in sediments are consistent with U reduction by 

interactions with iron sulfides. The co-association of U(IV) with Fe and S in some grain 

coatings support this conclusion. 

4. Bioreduction resulted in precipitation and accumulation of U in each column, with between 

62 and 104 μmoles retained on the columns. In all three biostimulation columns, U uptake 

occurred primarily at or near the inlet end of the column. In contrast, iron reduction, as 

evidenced from the increase in extractable Fe(II), was relatively uniform over the entire 

column.

5. The column receiving dissolved Fe(II) in the influent throughout biostimulation (column C) 

had a greater removal rate of U(VI) from the influent as well as better retention of U during 

oxic elution.

6. Sulfate reduction was the dominant TEAP process for oxidation of acetate accounting for 

>75% of electron transfer from acetate oxidation. Acetate was oxidized primarily to CO2. 

Total S increased 5-fold or more in all three columns with fully reduced S, measured as acid 

volatile sulfur (e.g. S-2), contributing 50 to 75% of the total S increase. The remaining 

increase likely includes elemental sulfur and S-1 precipitated as FeS2. The total S increase 

ranged from 50 to 120% of the increase in extractable Fe(II), with AVS equal to 50 to 70% of 

the sediment Fe(II) increase. Small fractionation factors of -0.2 to -5.1‰ δ34S were observed 

indicative of microbial sulfate reduction in systems where sulfate and electron donor are not 

limiting.

7. Increases in extractable Fe(II) were about two orders of magnitude greater than the 

integrated effluent dissolved iron exported from the columns, indicating most of the iron 

reduced was retained within the column either as iron sulfide or as Fe(II) from in-place 

reduction of phyllosilicate Fe(III).
6-3

8. The sequestered U was essentially all in the +4 oxidation state (U(IV)) in sediments 

recovered after the biostimulation period. Fitting of U-EXAFS spectra of column sediments 

are consistent with U(IV) comprised of a mixture of both partially disordered nanoparticulate 

UO2 and non-uraninite U(IV) species associated with biomass, NUSAB. The non-uraninite 

U(IV)-bearing phase may exist separately from the partially polymerized nano-UO2-like 

U(IV). No measurable changes in speciation of U associated with sediments were observed 

as a result of oxic elution.

9. The 30-mm and 2-mm resolution XRF images of thin sections show a limited number of 

grains with distinct coatings of uranium. Sequestered U on sediment grain surfaces formed

near-contiguous coatings ranging from 5 to 30 mm in thickness. Multi-energy 2-mm resolution 

U XANES map, as well as point XANES spectra, shows that U in these coatings is primarily 

U(IV). The U coatings overlay iron phases in some cases. Some U grain coatings have Fe 

and/or S intermixed with U. SEM-BSE images and EDS analysis show areas of high U that 

appear to be forming on the iron sulfide material. These findings are consistent with abiotic 

reduction of U(VI) by iron sulfides. In other grain coatings, little Fe or S was apparent in 

mXRF or SEM images.

10. Fine grained sediments (<63 mm) are abundant (~12% by mass) and contain 3 to 4 times 

higher U concentrations. However, discrete U-bearing particles of this size range were not 

evident in mXRF images. SEM images of U-bearing grain coatings do not appear to be 

comprised of fine grained sediments adhering to larger grains.

11. Microbial analysis of column sediments recovered following biostimulation show that 

Geobacter was higher in abundance than SRB in most samples. Clone analysis showed that 

sequences were not very diverse and were dominated by Geobacteraceae, previously 

described as important in U(VI) reduction in aquifers. Sequences of sulfate reducing 

bacteria also have relatively little diversity and were dominated by Desulfobulbaceae and 

Desulfobacteraceae with Desulfotomaculum and Syntrophobacteraceae present in some 

samples but at lower abundance.

12. Multicomponent biogeochemical reactive transport model simulation of column effluents 

during biostimulation generally described the acetate oxidation, iron, sulfate, and uranium 

reduction for all three columns using parameters derived from simulations of field scale 

biostimulation experiments.

13. The increasing rate of U reduction over the course of biostimulation, previously not observed 

in field experiments, required a rate law with dependency on the growing biomass of metal 

reducing bacteria to simulate column effluent. Continued uranium uptake after cessation of 

acetate was not captured by the model. 

14. Remobilization of uranium during the re-oxidation phase of the experiments was four times 

higher in column A compared to column C with near constant effluent dissolved uranium 

concentrations of about of 0.8 and 0.2 mM for the column A and C, respectively. However, 

even with the greater uranium stability caused by iron sulfide precipitation the effluent 

uranium concentration of approximately 0.2 μM did exceed EPA’s uranium MCL of 30 μg/L 

(0.126 μM). 

15. The U(IV) retained by the sediments and not remobilized was largely still reduced indicating 

that U oxidized during oxic elution was primarily transported out of the columns with little 

U(VI) retained by surface complexation to sediment surfaces such as iron oxides. 

7-1

7 References Cited

Abdelouas, A., Lutze, W., Nuttall, H.E., 1999. Oxidative dissolution of uraninite precipitated on 

Navajo sandstone. J. Contam. Hydrol. 36, 353-375.

Alessi, D.S., Uster, B., Veeramani, H., Suvorova, E.I., Lezama-Pacheco, J.S., Stubbs, J.E., 

Bargar, J.R., Bernier-Latmani, R., 2012. Quantitative separation of monomeric U(IV) from UO2 

in products of U(VI) reduction. Environ. Sci. Technol. 46, 6150-6157.

Anderson, R.T., Rooney-Varga, J.N., Gaw, C.V., Lovley, D.R., 1998. Anaerobic benzene 

oxidation in the Fe(III) reduction zone of petroleum- contaminated aquifers. Environ. Sci. 

Technol. 32, 1222-1229.

Anderson, R.T., Vrionis, H.A., Ortiz-Bernad, I., Resch, C.T., Long, P.E., Dayvault, R., Karp, K., 

Marutzky, S., Metzler, D.R., Peacock, A., White, D.C., Lowe, M., Lovley, D.R., 2003. Stimulating 

the In Situ Activity of Geobacter Species to Remove Uranium from the Groundwater of a 

Uranium-Contaminated Aquifer. Appl. Environ. Microbiol. 69, 5884-5891.

Ankudinov, A.L., Rehr, J.J., 1997. Relativistic calculations of spin-dependent x-ray-absorption 

spectra. Phys. Rev. B 56, R1712-R1715.

Bargar, J.R., Williams, K.H., Campbell, K.M., Long, P.E., Stubbs, J.E., Suvorova, E.I., LezamaPacheco, J.S., Alessi, D.S., Stylo, M., Webb, S.M., Davis, J.A., Giammar, D.E., Blue, L.Y., 

Bernier-Latmani, R., 2013. Uranium redox transition pathways in acetate-amended sediments. 

Proc. Natl. Acad. Sci. 110, 4506-4511.

Bernhard, G., Geipel, G., Reich, T., Brendler, V., Amayri, S., Nitsche, H., 2001. Uranyl(VI) 

carbonate complex formation: Validation of the Ca2UO2(CO3)3(aq.) species. Radiochim. Acta 89, 

511-518.

Bernier-Latmani, R., Veeramani, H., Vecchia, E.D., Junier, P., Lezama-Pacheco, J.S., 

Suvorova, E.I., Sharp, J.O., Wigginton, N.S., Bargar, J.R., 2010. Non-uraninite products of 

microbial U(VI) reduction. Environ. Sci. Technol. 44, 9456-9462.

Bi, Y., Hyun, S.P., Kukkadapu, R., Hayes, K.F., 2013. Oxidative dissolution of UO2 in a 

simulated groundwater containing synthetic nanocrystalline mackinawite. Geochim. 

Cosmochim. Acta 102, 175-190.

Boyanov, M.I., Fletcher, K.E., Kwon, M.J., Rui, X., O'Loughlin, E.J., Laffler, F.E., Kemner, K.M., 

2011. Solution and Microbial Controls on the Formation of Reduced U(IV) Species. Environ. Sci. 

Technol. 45, 8336-8344.

Brooks, S.C., Fredrickson, J.K., Carroll, S.L., Kennedy, D.W., Zachara, J.M., Plymale, A.E., 

Kelly, S.D., Kemner, K.M., Fendorf, S., 2003. Inhibition of bacterial U(VI) reduction by calcium. 

Environ. Sci. Technol. 37, 1850-1858.

Brunk, C.F., Avaniss-Aghajani, E., Brunk, C.A., 1996. A computer analysis of primer and probe 

hybridization potential with bacterial small-subunit rRNA sequences. Appl. Environ. Microbiol. 

62, 872-879.

Burgos, W.D., McDonough, J.T., Senko, J.M., Zhang, G., Dohnalkova, A.C., Kelly, S.D., Gorby, 

Y., Kemner, K.M., 2008. Characterization of uraninite nanoparticles produced by Shewanella 

oneidensis MR-1. Geochim. Cosmochim. Acta 72, 4901-4915.
7-2

Callister, S.J., Wilkins, M.J., Nicoral, C.D., Williams, K.H., Banfield, J.F., VerBerkmoes, N.C., 

Hettich, R.L., N'Guessan, A.L., Mouser, P.J., Elifantz, H., Smith, R.D., Lovley, D.R., Lipton, 

M.S., Long, P.E., 2010. Analysis of Biostimulated Microbial Communities from Two Field 

Experiments Reveals Temporal and Spatial Differences in Proteome Profiles. Environ. Sci. 

Technol. 44, 8897-8903.

Campbell, K.M., Gallegos, T.J., Landa, E.R., 2013. Biogeochemical aspects of uranium 

mineralization, mining, milling, and remediation, In: Seal, R. (Ed.), The Environmental 

Geochemistry of Mineral Deposits, Society of Economic Geology Reviews in Economic 

Geology. 

Campbell, K.M., Veeramani, H., Ulrich, K.-U., Blue, L.Y., Giammar, D.E., Bernier-Latmani, R., 

Stubbs, J.E., Suvorova, E., Yabusaki, S., Lezama-Pacheco, J.S., Mehta, A., Long, P.E., Bargar, 

J.R., 2011. Oxidative dissolution of biogenic uraninite in groundwater at Old Rifle, CO. Environ. 

Sci. Technol. 45, 8748-8754.

Cardenas, E., Wu, W.-., Leigh, M.B., Carley, J., Carroll, S., Gentry, T., Luo, J., Watson, D., Gu, 

B., Ginder-Vogel, M., Kitanidis, P.K., Jardine, P.M., Zhou, J., Criddle, C.S., Marsh, T.L., Tiedje, 

J.M., 2008. Microbial communities in contaminated sediments, associated with bioremediation 

of uranium to submicromolar levels. Appl. Environ. Microbiol. 74, 3718-3729.

Chang, Y., Long, P.E., Geyer, R., Peacock, A.D., Resch, C.T., Sublette, K., Pfiffner, S., 

Smithgall, A., Anderson, R.T., Vrionis, H.A., Stephen, J.R., Dayvault, R., Ortiz-Bernad, I., 

Lovley, D.R., White, D.C., 2005. Microbial Incorporation of 13C-Labeled Acetate at the Field 

Scale: Detection of Microbes Responsible for Reduction of U(VI). Environ. Sci. Technol. 39, 

9039-9048.

Chao, T.T., Zhou, L., 1983. Extraction techniques for selective dissolution of amorphous iron 

oxides from soils and sediments. Soil Sci. Soc. Am. J. 47, 225-232.

Cline, J.D., 1969. Spectrophotometric Determination of Hydrogen Sulfide in Natural Waters. 

Limnol. Oceanogr. 14, 454-458.

Davis, J.A., Meece, D.E., Kohler, M., Curtis, G.P., 2004. Approaches to surface complexation 

modeling of uranium(VI) adsorption on aquifer sediments. Geochim. Cosmochim. Acta 68, 

3621-3641.

Davis, J.A., Curtis, G.P., Wilkins, M.J., Kohler, M., Fox, P., Naftz, D.L., Lloyd, J.R., 2006. 

Processes affecting transport of uranium in a suboxic aquifer. Phys. Chem. Earth 31, 548-555.

Dong, W., Brooks, S.C., 2006. Determination of the formation constants of ternary complexes of 

uranyl and carbonate with alkaline earth metals (Mg2+, Ca2+, Sr 2+, and Ba2+) using anion 

exchange method. Environ. Sci. Technol. 40, 4689-4695.

Dong, W.M., Ball, W.P., Liu, C.X., Wang, Z.M., Stone, A.T., Bai, J., Zachara, J.M., 2005. 

Influence of calcite and dissolved calcium on uranium(VI) sorption to a Hanford subsurface 

sediment. Environ. Sci. Technol. 39, 7949-7955.

Englert, A., Hubbard, S.S., Williams, K.H., Li, L., Steefel, C.I., 2009. Feedbacks Between 

Hydrological Heterogeneity and Bioremediation Induced Biogeochemical Transformations. 

Environ. Sci. Technol 43, 5197-5204.
7-3

Fang, Y., Yabusaki, S.B., Morrison, S.J., Amonette, J.P., Long, P.E., 2009. Multicomponent 

reactive transport modeling of uranium bioremediation field experiments. Geochim. Cosmochim. 

Acta 73, 6029-6051.

Fang, Y.L., Yeh, G.T., Burgos, W.D., 2003. A general paradigm to model reaction-based 

biogeochemical processes in batch systems. Water Res. Research 39(4).

Fang, Y.L., Yabusaki, S.B., Yeh, G.T., 2006. A general simulator for reaction-based 

biogeochemical processes. Comput. Geosci. 32, 64-72.

Fox, P.M., Davis, J.A., Kukkadapu, R., Singer, D.M., Bargar, J., Williams, K.H., 2013. Abiotic 

U(VI) reduction by sorbed Fe(II) on natural sediments. Geochim. Cosmochim. Acta 117, 266-

282.

Fuller, C.C., Van Geen, A., Baskaran, M., Anima, R., 1999. Sediment chronology in San 

Francisco Bay, California, defined by 210Pb, 234Th, 137Cs, and 239,240Pu. Mar. Chem. 64, 7-27.

Gallegos, T.J., Fuller, C.C., Webb, S.M., Betterton, W., 2013. Uranium(VI) interactions with 

mackinawite in the presence and absence of bicarbonate and oxygen. Environ. Sci. Technol.

47, 7357-7364.

Geets, J., Borremans, B., Diels, L., Springael, D., Vangronsveld, J., van der Lelie, D., 

Vanbroekhoven, K., 2006. DsrB gene-based DGGE for community and diversity surveys of 

sulfate-reducing bacteria. J. Microbiol. Meth. 66, 194-205.

Ginder-Vogel, M., Stewart, B., Fendorf, S., 2010. Kinetic and mechanistic constraints on the 

oxidation of biogenic uraninite by ferrihydrite. Environ. Sci. Technol. 44, 163-169.

Gorby, Y.A., Lovley, D.R., 1992. Enzymatic uranium precipitation. Environ. Sci. Technol. 26, 

205-207.

Gu, B., Wu, W.-., Ginder-Vogel, M.A., Yan, H., Fields, M.W., Zhou, J., Fendorf, S., Criddle, C.S., 

Jardine, P.M., 2005. Bioreduction of uranium in a contaminated soil column. Environ. Sci. 

Technol. 39, 4841-4847.

Guillaumont, R., Fanghanel, T., Fugen, J., Grenthe, I., Neck, V., Palmer, D.A., Rand, M.H., 

2003. Chemical Thermodynamics 5. Update on the Chemical Thermodynamics of Uranium, 

Neptunium, Plutonium, Americium, and Technetium. Elsevier, Amsterdam.

Habicht, K.S., Canfield, D.E., 1997. Sulfur isotope fractionation during bacterial sulfate reduction 

in organic-rich sediments. Geochim. Cosmochim. Acta 61, 5351-5361.

Holmes, D.E., O'Neil, R.A., Vrionis, H.A., N'Guessan, L.A., Ortiz-Bernad, I., Larrahondo, M.J., 

Adams, L.A., Ward, J.A., Nicoll, J.S., Nevin, K.P., Chavan, M.A., Johnson, J.P., Long, P.E., 

Lovley, D.R., 2007. Subsurface clade of Geobacteraceae that predominates in a diversity of 

Fe(III)-reducing subsurface environments. ISME Journal 1, 663-677.

Hsieh, Y.P., Yang, C.H., 1989. Diffusion methods for the determination of reduced inorganic 

sulfur species in sediments. Limnol. Ocean. 34, 1126-1130.

Hsieh, Y.-., Chung, S.-., Tsau, Y.-., Sue, C.-., 2002. Analysis of sulfides in the presence of ferric 

minerals by diffusion methods. Chem. Geol. 182, 195-201.

Hua, B., Deng, B., 2008. Reductive immobilization of uranium(VI) by amorphous iron sulfide. 

Environ. Sci. Technol. 42, 8703-8708.
7-4

Hyun, S.P., Davis, J.A., Sun, K., Hayes, K.F., 2012. Uranium(VI) reduction by iron(II) 

monosulfide mackinawite. Environ. Sci. Technol. 46, 3369-3376.

Jimenez-Lopez, C., Romanek, C.S., 2004. Precipitation kinetics and carbon isotope partitioning 

of inorganic siderite at 25°C and 1 atm. Geochim. Cosmochim. Acta 68, 557-571.

Jones, E.J.P., Voytek, M.A., Lorah, M.M., Kirshtein, J.D., 2006. Characterization of a microbial 

consortium capable of rapid and simultaneous dechlorination of 1,1,2,2-tetrachloroethane and 

chlorinated ethane and ethene intermediates. Bioremediation J. 10, 153-168.

Kelly, S.D., Kemner, K.M., Brooks, S.C., 2007. X-ray absorption spectroscopy identifies 

calcium-uranyl-carbonate complexes at environmental concentrations. Geochim. Cosmochim. 

Acta 71, 821-834.

Komlos, J., Kukkadapu, R.K., Zachara, J.M., Jaffé, P.R., 2007. Biostimulation of iron reduction 

and subsequent oxidation of sediment containing Fe-silicates and Fe-oxides: Effect of redox 

cycling on Fe(III) bioreduction. Water Res. 41, 2996-3004.

Komlos, J., Moon, H.S., Jaffé, P.R., 2008a. Effect of sulfate on the simultaneous bioreduction of 

iron and uranium. J. Environ. Qual. 37, 2058-2062.

Komlos, J., Peacock, A., Kukkadapu, R.K., Jaffé, P.R., 2008b. Long-term dynamics of uranium 

reduction/reoxidation under low sulfate conditions. Geochim. Cosmochim. Acta 72, 3603-3615.

Lane, D.J., 1991. 16S/23S rRNA sequencing, In: Stackebrandt, E. and Goodfellow, M. (Eds.), 

Nucleic Acid Techniques in Bacterial Systematics. Wiley, New York, pp. 115-148.

Lee, J.-., Fredrickson, J.K., Kukkadapu, R.K., Boyanov, M.I., Kemner, K.M., Lin, X., Kennedy, 

D.W., Bjornstad, B.N., Konopka, A.E., Moore, D.A., Resch, C.T., Phillips, J.L., 2012. Microbial 

reductive transformation of phyllosilicate Fe(III) and U(VI) in fluvial subsurface sediments. 

Environ. Sci. Technol. 46, 3721-3730.

Li, L., Steefel, C.I., Williams, K.H., Wilkins, M.J., Hubbard, S.S., 2009. Mineral transformation 

and biomass accumulation associated with uranium bioremediation at Rifle, Colorado. Environ. 

Sci. Technol. 43, 5429-5435.

Li, L., Steefel, C.I., Kowalsky, M.B., Englert, A., Hubbard, S.S., 2010. Effects of physical and 

geochemical heterogeneities on mineral transformation and biomass accumulation during 

biostimulation experiments at Rifle, Colorado. J.Contam.Hydrol. 112, 45-63.

Liger, E., Charlet, L., Van Cappellen, P., 1999. Surface catalysis of uranium(VI) reduction by 

iron(II). Geochim. Cosmochim. Acta 63, 2939-2955.

Liu, C.X., Zachara, J.M., Zhong, L.R., Kukkadupa, R., Szecsody, J.E., Kennedy, D.W., 2005. 

Influence of sediment bioreduction and reoxidation on uranium sorption. Environ. Sci. Technol.

39, 4125-4133.

Locock, A.J., Burns, P.C., 2003. The crystal structure of synthetic autunite, 

Ca[(UO2)(PO4)]2(H2O)11. Am. Mineral. 88, 240-244.

Lovley, D.R., Phillips, E.J.P., 1987. Rapid Assay for Microbially Reducible Ferric Iron in Aquatic 

Sediments. Appl. Environ. Microbiol. 53, 1536-1540.

Lovley, D.R., Phillips, E.J.P., Gorby, Y.A., Landa, E.R., 1991. Microbial reduction of uranium. 

Nature 350, 413-416.
7-5

Lovley, D.R., Phillips, E.J.P., 1992. Bioremediation of uranium contamination with enzymatic 

uranium reduction. Environ. Sci. Technol. 26, 2228-2234.

Lovley, D.R., Roden, E.E., Phillips, E.J.P., Woodward, J.C., 1993. Enzymatic iron and uranium 

reduction by sulfate-reducing bacteria. Mar. Geol. 113, 41-53.

Luo, J., Weber, F.-., Cirpka, O.A., Wu, W.-., Nyman, J.L., Carley, J., Jardine, P.M., Criddle, 

C.S., Kitanidis, P.K., 2007a. Modeling in-situ uranium(VI) bioreduction by sulfate-reducing 

bacteria. J. Contam. Hydrol. 92, 129-148.

Luo, J., Wu, W.M., Carley, J., Ruan, C.M., Gu, B.H., Jardine, P.M., Criddle, C.S., Kitanidis, P.K., 

2007b. Hydraulic performance analysis of a multiple injection-extraction well system. J. Hydrol. 

336, 294-302.

Luo, W., Gu, B., 2009. Dissolution and mobilization of uranium in a reduced sediment by natural 

humic substances under anaerobic conditions. Environ. Sci. Technol. 43, 152-156.

Maurer, M., Rittmann, B.E., 2004. Modeling intrinsic bioremediation for interpret observable 

biogeochemical footprints of BTEX biodegradation: The need for fermentation and abiotic 

chemical processes. Biodegradation 15, 405-417.

Mayhew, L.E., Webb, S.M., Templeton, A.S., 2011. Microscale imaging and identification of fe 

speciation and distribution during fluid-mineral reactions under highly reducing conditions. 

Environ. Sci. Technol. 45, 4468-4474.

Mereiter, K., 1982. The crystal structure of Liebigite, Ca2UO2(CO3)3·~11H2O. Tscher. Miner. 

Petrograp. 30, 277-288.

Miletto, M., Williams, K.H., N'Guessan, A.L., Lovley, D.R., 2011. Molecular analysis of the 

metabolic rates of discrete subsurface populations of sulfate reducers. Appl. Environ. Microbiol. 

77, 6502-6509.

Moon, H.S., Komlos, J., Jaffé, P.R., 2007. Uranium reoxidation in previously bioreduced 

sediment by dissolved oxygen and nitrate. Environ. Sci. Technol. 41, 4587-4592.

Moon, H.S., Komlos, J., Jaffé, P.R., 2009. Biogenic U(IV) oxidation by dissolved oxygen and 

nitrate in sediment after prolonged U(VI)/Fe(III)/SO4

2- reduction. J. Contam. Hydrol. 105, 18-27.

Muyzer, G., Stams, A.J.M., 2008. The ecology and biotechnology of sulphate-reducing bacteria. 

Nature Reviews Microbiology 6, 441-454.

O'Loughlin, E.J., Kelly, S.D., Kemner, K.M., 2010. XAFS investigation of the interactions of UVI 

with secondary mineralization products from the bioreduction of FeIII oxides. Environ. Sci. 

Technol. 44, 1656-1661.

Poeter, E.P., Hill, M.C., Banta, E.R., Mehl, S., Christensen, S., 2008. UCODE_2005 and Six 

Other Computer Codes for Universal Sensitivity Analysis, Calibration, and Uncertainty 

Evaluation, U.S. Geological Survey Techniques and Methods 6-A11, U.S. Geological Survey, 

pp. 283.

Poulton, S.W., Krom, M.D., Raiswell, R., 2004. A revised scheme for the reactivity of iron 

(oxyhydr)oxide minerals towards dissolved sulfide. Geochim. Cosmochim. Acta 68, 3703-3715.
7-6

Sani, R.K., Peyton, B.M., Dohnalkova, A., Amonette, J.E., 2005. Reoxidation of reduced 

uranium with iron(III) (Hydr)oxides under sulfate-reducing conditions. Environ. Sci. Technol. 39, 

2059-2066.

Schofield, E.J., Veeramani, H., Sharp, J.O., Suvorova, E., Bernier-Latmani, R., Mehta, A., 

Stahlman, J., Webb, S.M., Clark, D.L., Conradson, S.D., Ilton, E.S., Bargar, J.R., 2008. 

Structure of biogenic uraninite produced by Shewanella oneidensis strain MR-1. Environ. Sci. 

Technol. 42, 7898-7904.

Senko, J.M., Istok, J.D., Suflita, J.M., Krumholz, L.R., 2002. In-situ evidence for uranium 

immobilization and remobilization. Environ. Sci. Technol. 36, 1491-1496.

Senko, J.M., Kelly, S.D., Dohnalkova, A.C., McDonough, J.T., Kemner, K.M., Burgos, W.D., 

2007. The effect of U(VI) bioreduction kinetics on subsequent reoxidation of biogenic U(IV). 

Geochim. Cosmochim. Acta 71, 4644-4654.

Sharp, J.O., Lezama-Pacheco, J.S., Schofield, E.J., Junier, P., Ulrich, K.-U., Chinni, S., 

Veeramani, H., Margot-Roquier, C., Webb, S.M., Tebo, B.M., Giammar, D.E., Bargar, J.R., 

Bernier-Latmani, R., 2011. Uranium speciation and stability after reductive immobilization in 

aquifer sediments. Geochim. Cosmochim. Acta 75, 6497-6510.

Sim, M.S., Ono, S., Donovan, K., Templer, S.P., Bosak, T., 2011. Effect of electron donors on 

the fractionation of sulfur isotopes by a marine Desulfovibrio sp. Geochim. Cosmochim. Acta 75, 

4244-4259.

Singer, D.M., Farges, F., Brown Jr., G.E., 2009. Biogenic nanoparticulate UO2: Synthesis, 

characterization, and factors affecting surface reactivity. Geochim. Cosmochim. Acta 73, 3593-

3611.

Singer, D.M., Chatman, S.M., Ilton, E.S., Rosso, K.M., Banfield, J.F., Waychunas, G.A., 2012. 

Identification of simultaneous U(VI) sorption complexes and U(IV) nanoprecipitates on the 

magnetite (111) surface. Environ. Sci. Technol. 46, 3811-3820.

Stoliker, D.L., Campbell, K.M., Singer, D.M., Kaviani, N., Carey, M., Peck, N.E., Bargar, J.R., 

Kent, D.B., Davis, J.A., 2013. Evaluating Chemical Extraction Techniques for the Determination 

of Uranium Oxidation State in Reduced Aquifer Sediments. Environ. Sci. Technol. 47, 9225-

9332.

Stookey, L.L., 1970. Ferrozine - A new spectrophotometric reagent for iron. Anal. Chem. 42, 

779-781.

Ulrich, K.-U., Ilton, E.S., Veeramani, H., Sharp, J.O., Bernier-Latmani, R., Schofield, E.J., 

Bargar, J.R., Giammar, D.E., 2009. Comparative dissolution kinetics of biogenic and 

chemogenic uraninite under oxidizing conditions in the presence of carbonate. Geochim. 

Cosmochim. Acta 73, 6065-6083.

Ulrich, K.-U., Veeramani, H., Bernier-Latmani, R., Giammar, D.E., 2011. Speciation-dependent 

kinetics of uranium(VI) bioreduction. Geomicrobiol. J. 28, 396-409.

Veeramani, H., Scheinost, A.C., Monsegue, N., Qafoku, N.P., Kukkadapu, R., Newville, M., 

Lanzirotti, A., Pruden, A., Murayama, M., Hochella, M.F., 2013. Abiotic reductive immobilization 

of U(VI) by biogenic mackinawite. Environ. Sci. Technol. 47, 2361-2369. 
7-7

Vrionis, H.A., Anderson, R.T., Ortiz-Bernad, I., O'Neill, K.R., Resch, C.T., Peacock, A.D., 

Dayvault, R., White, D.C., Long, P.E., Lovley, D.R., 2005. Microbiological and geochemical 

heterogeneity in an in situ uranium bioremediation field site. Appl. Environ. Microbiol. 71, 6308-

6318.

Wagner, M., Roger, A.J., Flax, J.L., Brusseau, G.A., Stahl, D.A., 1998. Phylogeny of 

dissimilatory sulfite reductases supports an early origin of sulfate respiration. J. Bacteriol. 180, 

2975-2982.

Wan, J., Tokunaga, T.K., Brodie, E., Wang, Z., Zheng, Z., Herman, D., Hazen, T.C., Firestone, 

M.K., Sutton, S.R., 2005. Reoxidation of bioreduced uranium under reducing conditions. 

Environ. Sci. Technol. 39, 6162-6169.

Ward, B.B., Eveillard, D., Kirshtein, J.D., Nelson, J.D., Voytek, M.A., Jackson, G.A., 2007. 

Ammonia-oxidizing bacterial community composition in estuarine and oceanic environments 

assessed using a functional gene microarray. Environ. Microbiol. 9, 2522-2538.

Webb, S., 2004. SIXPACK: a graphical user interface for XAS analysis using IFEFFIT. Phys. 

Scripta T T115, 1011-1014.

Williams, K.H., Long, P.E., Davis, J.A., Wilkins, M.J., N'Guessan, A.L., Steefel, C.I., Yang, L., 

Newcomer, D., Spane, F.A., Kerkhof, L.J., Mcguinness, L., Dayvault, R., Lovley, D.R., 2011. 

Acetate availability and its influence on sustainable bioremediation of Uranium-contaminated 

groundwater. Geomicrobiol. J. 28, 519-539.

Wilson, T.A., Amirbahman, A., Norton, S.A., Voytek, M.A., 2010. A record of phosphorus 

dynamics in oligotrophic lake sediment. J. Paleolimnol. 44, 279-294.

Wu, T., Shelobolina, E., Xu, H., Konishi, H., Kukkadapu, R., Roden, E.E., 2012. Isolation and 

microbial reduction of Fe(III) phyllosilicates from subsurface sediments. Environ. Sci. Technol.

46, 11618-11626.

Wu, W.-., Carley, J., Gentry, T., Ginder-Vogel, M.A., Fienen, M., Mehlhorn, T., Yan, H., Caroll, 

S., Pace, M.N., Nyman, J., Luo, J., Gentile, M.E., Fields, M.W., Hickey, R.F., Gu, B., Watson, 

D., Cirpka, O.A., Zhou, J., Fendorf, S., Kitanidis, P.K., Jardine, P.M., Criddle, C.S., 2006. Pilotscale in situ bioremedation of uranium in a highly contaminated aquifer. 2. Reduction of U(VI) 

and geochemical control of U(VI) bioavailability. Environ. Sci. Technol. 40, 3986-3995.

Wyckoff, R.W.G., 1963. Crystal Structures, Second Edition, Interscience Publishers, New York, 

New York, pp. 239-244.

Yabusaki, S.B., Fang, Y., Waichler, S.R., Long, P.E., 2010. Processes, properties, and 

conditions controlling in situ bioremediation of uranium in shallow, alluvial aquifers, NUREG/CR7014, Nuclear Regulatory Commission, Office of Nuclear Regulatory Research, Washington, 

DC, pp. 111.

Yabusaki, S.B., Fang, Y., Long, P.E., Resch, C.T., Peacock, A.D., Komlos, J., Jaffe, P.R., 

Morrison, S.J., Dayvault, R.D., White, D.C., Anderson, R.T., 2007. Uranium removal from 

groundwater via in situ biostimulation: Field-scale modeling of transport and biological 

processes. J. Contam. Hydrol. 93, 216-235.

Yeh, G.T., Sun, J.T., Jardine, P.M., Burgos, W.D., Fang, Y., Li, M.H., Siegel, M.D., 2004. 

HYDROGEOCHEM 5.0: a threedimensional model of coupled fluid flow, thermal transport and 
7-8

hydrogeochemical transport through variable saturated conditions – version 5.0, Oak Ridge 

National Laboratory, Oak Ridge, TN, USA.

Zheng, Z.P., Tokunaga, T.K., Wan, J.M., 2003. Influence of calcium carbonate on U(VI) sorption 

to soils. Environ. Sci. Technol. 37, 5603-5608.

Zhong, L., Liu, C., Zachara, J.M., Kennedy, D.W., Szecsody, J.E., Wood, B., 2005. Oxidative 

remobilization of biogenic uranium(IV) precipitates: Effects of iron(II) and pH. J. Environ. Qual. 

34, 1763-1771.

 



UNITED STATES

NUCLEAR REGULATORY COMMISSION

WASHINGTON, DC 20555-0001

------------------

OFFICIAL BUSINESS
NUREG/CR-7178 Uranium Sequestration During Biostimulated Reduction and In Response to the 

Return of Oxic Conditions In Shallow Aquifers