Table Of ContentKINETICS OF CALCITE PRECIPITATION BY UREOLYTIC BACTERIA UNDER
AEROBIC AND ANAEROBIC CONDITIONS
by
Stacy Leigh Parks
A thesis submitted in partial fulfillment
of the requirements for the degree
of
Master of Science
in
Chemical and Biological Engineering
MONTANA STATE UNIVERSITY
April 2009
© COPYRIGHT
By
Stacy Leigh Parks
2009
All Rights Reserved
ii
APPROVAL
of a thesis submitted by
Stacy Leigh Parks
This thesis has been read by each member of the thesis committee and has been
found to be satisfactory regarding content, English usage, format, citation, bibliographic
style, and consistency, and is ready for submission to the Division of Graduate Education.
Dr. Robin Gerlach
Approved for the Department of Chemical and Biological Engineering
Dr. Ronald Larsen
Approved for the Division of Graduate Education
Dr. Carl A. Fox
iii
STATEMENT OF PERMISSION TO USE
In presenting this thesis in partial fulfillment of the requirements for a
master’s degree at Montana State University, I agree that the Library shall make it
available to borrowers under rules of the Library.
If I have indicated my intention to copyright this thesis by including a
copyright notice page, copying is allowable only for scholarly purposes, consistent with
“fair use” as prescribed in the U.S. Copyright Law. Requests for permission for extended
quotation from or reproduction of this thesis in whole or in parts may be granted
only by the copyright holder.
Stacy Leigh Parks
April 2009
iv
ACKNOWLEDGMENTS
This project was made possible through funding from the Department of Energy’s
Zero Emissions Research and Technology Project. Thank you to my graduate committee,
Dr. Robin Gerlach, Dr. Alfred Cunningham, Dr. Abigail Richards, and Dr. Andrew
Mitchell. I could not have succeeded without your advice and support. Special thanks to
Adrienne Phillips and Erin Field for teaching me laboratory techniques and answering my
endless questions, as well as to Susan Brumfield for taking some great images for me.
Thank you to everyone at the Center for Biofilm Engineering for creating a great work
environment, especially to Laura Wheeler and Logan Schultz for your support.
v
TABLE OF CONTENTS
1. INTRODUCTION……………………………………………………………………..1
Carbonate Precipitation…………………………………………………………….…..1
Microbial Carbonate Precipitation…………………………………………………......2
Calcite Precipitation by Ureolysis……………………………………………...…3
Biological Applications of Bacterial Ureolysis………………………………………..4
Wastewater Treatment…………………………………………………………….4
Soil Improvement…………………………………………………………………5
Environmental Remediation of Radionuclides……………………………………5
Bacteriogenic Mineral Plugging…………………………………………………..6
Enhanced Oil Recovery………………………………………………………..6
Carbon Sequestration…………………………………………………………..7
Kinetics of Calcite Precipitation by Ureolysis…………..…………………………….8
Variation of Initial Calcium Concentrations………………………………………8
Variation of Initial Cell Concentrations………………………………………….11
Urea Concentration and Temperature Variations………………………………..15
Research Objectives…………………………………..……………………………...20
2. MATERIALS AND METHODS……………………………………………………..21
Bacterial Growth Media and Inoculum Preparation…………………………………..21
Bacterial Strains………………………………………………………………….21
Growth Media……………………………………………………………………21
Aerobic Media………………………………………………………………..22
Anaerobic Media……………………………………………………………..22
Inocula Preparation………………………………………………………………24
Aerobic Inoculum……………………………………………………………24
Anaerobic Inoculum………………………………………………………….25
Experimental Procedure………………………………………………………………25
System Setup and Design………………………………………………………..25
Bacterial Growth………………………………………………………………...26
Plate Counts…………………………………………………………………26
Optical Density………………………………………………………………27
Protein Concentration………………………………………………………..27
pH……………………………………………………………………………….28
Ammonia Concentration………………………………………………………...28
Dissolved Calcium Concentration……………………………………………….29
Imaging……………………………………………………………………………….29
Scanning Electron Microscope (SEM)…………………………………………..29
Transmission Electron Microscope (TEM)……………………………………...30
Kinetics of Calcite Precipitation………………………………………………………31
vi
TABLE OF CONTENTS-CONTINUED
3. RESULTS AND DISCUSSION……………………………………………………...32
Solution Chemistry……………………………………………………………………32
Growth………………………………………………………………………………...38
Activity of Encased Cells……………………………………………………………..41
TEM Imaging………………………………………………..................................41
Diffusion of Urea through Calcite.…….………………………………………….43
Kinetics………………………………………………………………………………..44
Sporosarcina pasteurii……………………………………………………………………44
Bacillus sphaericus 21776………………………………………………………...49
Bacillus sphaericus 21787………………………………………………………...53
Comparison of Kinetics……………………………………………………………….55
Anaerobic Experiments……………………………………………………………….59
Calcite Precipitation Experiments………………………………………………...62
Comparison of Aerobic and Anaerobic Kinetics…………………………………68
4. CONCLUSION……………………………………………………………………….70
Suggestions for Further Studies……………………………………………………….71
APPENDICES…………….……………………………………………………………..72
APPENDIX A: Raw Data Tables…………………………………………………….73
APPENDIX B: Growth Measurement Comparisons…………………………………86
REFERENCES CITED………………..…………………………………………………89
vii
LIST OF TABLES
Table Page
1. Rate coefficients for microbiologically-induced precipitation of CaCO
3
by S. pasteurii at different initial concentrations of CaCl ………......……….10
2
2. Rate coefficients for ammonium production and calcium precipitation....…...14
3. Summary of kinetic parameters for urea hydrolysis, change in saturation
state, calcite precipitation, apparent saturation, and critical
saturation for calcite precipitation at different temperatures………….………20
4. Recipe for Calcite Mineralizing Media……………………………………….21
5. Average pH from triplicate aerobic experiments……………………………...33
6. Summary of kinetic parameters for urea hydrolysis (k ) in experiments
urea
inoculated with S. pasteurii…………………………………………………..46
7. Summary of kinetic parameters for calcite precipitation (k ) in
precip
experiments inoculated with S. pasteurii……………………….……………..48
8. Summary of kinetic parameters for urea hydrolysis (k ) in experiments
urea
inoculated with B. sphaericus 21776…...…………………………………….51
9. Summary of kinetic parameters for calcite precipitation (k ) in
precip
experiments inoculated with B. sphaericus 21776………………….………...52
10. Summary of kinetic parameters for urea hydrolysis (k ) and calcite
urea
precipitation (k ) in experiments inoculated with B. sphaericus 21787….54
precip
11. Summary of kinetic parameters of three ureolytic species…………….……..55
12. Summary of kinetic coefficients and initial growth conditions for
experiments performed for this thesis and those described in
literature review………………………………………………………………56
13. Summary of kinetic parameters for urea hydrolysis (k ) and calcite
urea
precipitation (k ) in anaerobic experiments inoculated with S. pasteurii
precip
in CMM+ and nitrate…………………………………………………………64
viii
LIST OF TABLES-CONTINUED
Table Page
14. Summary of kinetic parameters for urea hydrolysis (k ) and calcite
urea
precipitation (k ) in anaerobic experiments inoculated with S. pasteurii
precip
in CMM+……………………………………………………………………..66
15. Summary of kinetic parameters for urea hydrolysis (k ) in anaerobic
urea
experiments inoculated with S. pasteurii in CMM-………………………….67
16. Summary of kinetic coefficients found in aerobic and anaerobic
experiments inoculated with S. pasteurii……………………………………..69
17. Compiled data from S. pasteurii aerobic experiments in calcium
inclusive media………………………………………………………………74
18. Compiled data from S. pasteurii aerobic experiments in calcium
exclusive media………………………………………………………………75
19. Compiled data from S. pasteurii anaerobic survey experiment in
calcium exclusive media with various TEAs…………………………………76
20. Compiled data from S. pasteurii anaerobic experiments in calcium
inclusive media with NO - as a terminal electron acceptor…………………..77
3
21. Compiled data from S. pasteurii anaerobic experiments in calcium
inclusive media without a terminal electron acceptor………………………..78
22. Compiled data from S. pasteurii anaerobic experiments in calcium
exclusive media with NO - as a terminal electron acceptor………………….79
3
23. Compiled data from B. sphaericus 21776 aerobic experiments in
calcium inclusive media……………………………………………………...80
24. Compiled data from B. sphaericus 21776 aerobic experiments in
calcium exclusive media……………………………………………………...81
25. Compiled data from B. sphaericus 21787 aerobic experiments in
calcium inclusive media……………………………………………………...82
26. Compiled data from B. sphaericus 21787 aerobic experiment in
calcium exclusive media……………………………………………………..83
ix
LIST OF TABLES-CONTINUED
Table Page
27. Compiled data from B. subtilis aerobic experiment in calcium inclusive media…….83
28. Compiled data from sterile control aerobic experiment in calcium
inclusive media……………………………………………………………….………84
29. Compiled data from sterile control aerobic experiment in calcium
exclusive media………………………………………………………………………85
Description:endless questions, as well as to Susan Brumfield for taking some great images . Rate coefficients for microbiologically-induced precipitation of CaCO3 . Dissolved ammonium concentrations over time from hydrolysis of urea (60-90 nm) were cut with a Diatome diamond knife on a Reichert OM-U2.
