Table Of ContentMOLECULAR DETERMINATION OF ARSENATE RESPIRING BACTERIA
IN ESTUARINE SEDIMENTS AND GROUNDWATER
Holly G. Oates
A Thesis Submitted to the
University of North Carolina Wilmington in Partial Fulfillment
of the Requirements for the Degree of
Master of Science
Center for Marine Science
University of North Carolina Wilmington
2007
Approved by
Advisory Committee
Joan Willey Robert Kieber______
Bonkeun Song_____
Chair
Accepted By
Robert Roer_______
Dean, Graduate School
TABLE OF CONTENTS
ABSTRACT..............................................................................................................iii
ACKNOWLEDGEMENTS ........................................................................................iv
DEDICATION...........................................................................................................vi
LIST OF TABLES.....................................................................................................vii
LIST OF FIGURES..................................................................................................viii
CHAPTER 1..............................................................................................................1
Introduction....................................................................................................2
Hypotheses..................................................................................................14
Sources Cited...............................................................................................15
CHAPTER 2............................................................................................................18
Introduction..................................................................................................19
Materials and Methods.................................................................................23
Results.........................................................................................................34
Discussion....................................................................................................45
Sources Cited...............................................................................................49
CHAPTER 3............................................................................................................51
Introduction..................................................................................................52
Materials and Methods.................................................................................57
Results.........................................................................................................68
Discussion....................................................................................................84
Sources Cited...............................................................................................88
CONCLUSION........................................................................................................90
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ABSTRACT
Arsenic is a well-known metalloid and carcinogen with both natural and
anthropogenic origins. Arsenic occurs naturally in the environment as two primary
inorganic forms: arsenate [As(V)] and arsenite [As(III)]. Microbes mediate the presence
and quantity of these two arsenic species through oxidation and reduction processes.
Dissimilatory As(V) respiring bacteria (DARB) contain As(V) respiratory reductase (arrA)
genes and reduce As(V) to As(III), therefore enhancing the solubility of arsenic. Thus,
the detection of the genes encoding As(V) respiratory reductases could interpret the
presence and involvement of DARB in arsenic mobilization. With water samples
collected from two NC wells, and sediment collected from Shem Creek, South Carolina
the presence of DARB using arrA genes as genetic markers were determined and the
As(V) reduction activities were monitored via enrichment culture techniques. Based on
molecular analysis, the DARB in the NC wells are closely related to Geobacter
uraniumreducens while those in Shem Creek sediments are related to
Desulfosporosinus Y5. Anaerobic enrichment cultures were established with NC well
water and Shem Creek sediment to determine the rate of As(V) reduction. The rates for
all of the sites involved showed that the DARB could have a significant impact on the
arsenic levels in the environment. Thus, this study demonstrates the presence and
diversity of DARB in these environments and their contributions to the arsenic
contamination in NC residential drinking water wells, and in the Shem Creek estuary.
iii
ACKNOWLEDGEMENTS
I would first and foremost like to thank my advisor, Bongkeun Song, for all of his
guidance and support over the past two years. I could not have asked for a better
mentor throughout this process of learning how to “do science.” His knowledge of
microbial ecology and molecular techniques continues to amaze and inspire me to work
hard in my scientific endeavors and to always strive for nothing less than perfection. I
will always remember the scientific and life lessons he has taught me and I will carry
them over into everything that I do from here on out in my life.
Also, I would like to thank the members of the microbial ecology lab at UNCW
because if it had not been for them, all of the long hours spent in the lab would not have
been nearly as entertaining. A big holla goes to Oliva Dale, Brian Shirey, Jen Bagwell,
Jennifer Randolph, Dr. Susanna Lopez-Legentil, and the newest member of the lab
Matty Hirsch. All of you are amazing and we have had some funny times over the past
two years. I do not think that I will ever enjoy the people I will work with more than I
have enjoyed working with you all. Love you guys!
This project would not have been possible without the funding provided from NSF
and from the Ahuja Academy Foundation. Also, I would like to thank Chuck Pippen and
Joju Abraham of NCDENR for helping me to obtain the NC well water samples and Guy
Didonato of SC NOAA for allowing me to tag along with him to get the Shem Creek
samples.
I would like to thank many other people at UNCW for their help and friendship
along the way. My committee, Dr. Joan Willey and Dr. Bob Kieber have given me a
great perspective on the environmental chemistry of arsenic and various analyses to
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help improve my research. Also, I would like to thank Dr. Willey individually for a great
marine science chemistry education and for being a great graduate school mentor. I
really appreciate all of your assistance and guidance over the past two years. To my
new found friends at UNCW outside of the microbial ecology lab thanks for all the good
times and study sessions throughout this process. Also, a special thanks to Dr.
Kimberly Mayer for her continued guidance and friendship over the past two years.
I would also like to thank my Mama, Granny Shirley, Grandma Pat, and Grannie
Sallie for their love and support during all of my education milestones including this one.
Nothing has encouraged me more over the years than all of you!
Finally, I would like to thank my husband Will for his patience, support, and love
throughout the years that we have been together. I am so fortunate to have someone
that I can count on to listen while I share my day to day successes and frustrations.
You have been right there with me those late nights at the lab, always helping out any
way that you could. You have pushed me to be the best that I can possibly be in this
and in all aspects of my life and future endeavors. I love you!
v
DEDICATION
I would like to dedicate this thesis to my Granny Shirley who has always taught
me that education is something that no one can take away from you. She has
supported me throughout everything I have ever done and continues to be my model for
the person I want to be when I “grow up.”
vi
LIST OF TABLES
Table Page
1. Levels of arsenic and other metals in NC wells......................................................25
2. Primer sequences used in this study......................................................................26
3. ATCC 1957 Geobacter media components ...........................................................32
4. Summary of abundance and diversity of DARB in NC wells..................................38
5. Levels of metals and PAHs in Shem Creek ...........................................................54
6. Thauera aromatica (TA) media components..........................................................63
7. Summary of diversity of DARB for Shem Creek based on arrA gene detection.....72
8. Rates of As(V) reduction in different enrichment cultures......................................77
9. Test for As(V) reduction in the isolates from Shem Creek .....................................83
vii
LIST OF FIGURES
Figure Page
1. 16S rRNA tree of known As(V) reducing bacteria and archaea................................7
2. Three major microbial pathways which release arsenic ...........................................9
3. As(V) respiratory reductase (arrAB) gene operon..................................................10
4. As(V) respiratory reductase and its biochemical pathway......................................11
5. Map of well water sampling sites in western North Carolina...................................24
6. Phylogenetic tree of arrA genes from the Rushing and Deese wells......................36
7. Rarefaction analysis of arrA genes detected from Rushing and Deese wells.........37
8. As(V) reduction in anaerobic enrichment cultures established with Deese well
water.......................................................................................................................40
9. As(V) reduction in anaerobic enrichment cultures established with Rushing well
water.......................................................................................................................41
10. As(V) reduction in the second anaerobic enrichment cultures established with
Rushing well water .................................................................................................43
11. Map of sampling sites located at Shem Creek, South Carolina..............................58
12. Phylogenetic tree of arrA genes detected from Shem Creek sediment..................69
13. Comparison of the rarefaction analysis of arrA genes at a 2%, 3%, and 5%
similarity detected from Shem Creek sampling sites..............................................71
14. T-RFLP analysis of arrA genes detected from Shem Creek sites 1, 2, and 3.........73
15. As(V) reduction in anaerobic enrichment cultures established with Shem Creek 1
sediment.................................................................................................................75
16. As(V) reduction in anaerobic enrichment cultures established with Shem Creek 2
sediment.................................................................................................................76
17. Benzoate degradation in As(V) reducing enrichment cultures from Shem Creek 1
and 2 ......................................................................................................................79
18. Naphthalene degradation in As(V) reducing enrichment cultures from Shem
Creek 1 and 2.........................................................................................................80
viii
19. As(V) reduction in 1:10 diluted enrichment cultures from Shem Creek 2...............81
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CHAPTER 1: INTRODUCTION TO ARSENIC AND THE PROCESSES INVOLVED IN
THE REDUCTION OF As(V) TO As(III)
Description:presence and involvement of DARB in arsenic mobilization. With water samples
collected from two NC wells, and sediment collected from Shem Creek, South
